Lawrence J Mandarino

Interests

Research

Mechanisms of insulin action in skeletal muscle, pathogenesis of type 2 diabetes mellitus, mass spectrometry based proteomics, protein structure and function., cell signaling, post translational modifications of proteins.

Courses

Scholarly Contributions

Chapters

• Mandarino, L. J., & Langlais, P. R. (2019). Proteomic Profiling of Human Skeletal Muscle in Health and Disease. In Cell Omics approaches to understanding muscle biology.

Journals/Publications

• Mandarino, L. J., Chang, P. F., Acevedo, D., & Reyna, S. M. (2023). Triterpenoid CDDO-EA inhibits lipopolysaccharide-induced inflammatory responses in skeletal muscle cells through suppression of NF-κB. Experimental Biology and Medicine, 153537022211391. doi:10.1177/15353702221139188
• Bakhshi, B., Mandarino, L. J., Coletta, D. K., Kohler, L. N., Parra, O. D., Ziller, S. G., & Standage-Beier, C. S. (2022). Tools to Measure Health Literacy among Adult Hispanic Populations with Type 2 Diabetes Mellitus: A Review of the Literature. International Journal of Environmental Research and Public Health. doi:10.3390/ijerph191912551
• Barakati, N., Zapata Bustos, R., Coletta, D. K., Langlais, P. R., Kohler, L. N., Luo, M., Funk, J. L., Willis, W. T., & Mandarino, L. J. (2022). Fuel Selection in Skeletal Muscle Exercising at Low Intensity; Reliance on Carbohydrate in Very Sedentary Individuals. Metabolic Syndrome and Related Disorders.
• Barakati, N., Zapata Bustos, R., Coletta, D. K., Langlais, P. R., Kohler, L. N., Luo, M., Funk, J. L., Willis, W. T., & Mandarino, L. J. (2022). Fuel Selection in Skeletal Muscle Exercising at Low Intensity; Reliance on Carbohydrate in Very Sedentary Individuals. Metabolic syndrome and related disorders.
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  Resting skeletal muscle in insulin resistance prefers to oxidize carbohydrate rather than lipid, exhibiting metabolic inflexibility. Although this is established in resting muscle, complexities involved in directly measuring fuel oxidation using indirect calorimetry across a muscle bed have limited studies of this phenomenon in working skeletal muscle. During mild exercise and at rest, whole-body indirect calorimetry imperfectly estimates muscle fuel oxidation. We provide evidence that a method termed "ΔRER" can reasonably estimate fuel oxidation in skeletal muscle activated by exercise. Completely sedentary volunteers ( = 20, age 31 ± 2 years, V̇O 24.4 ± 1.5 mL O per min/kg) underwent glucose clamps to determine insulin sensitivity and graded exercise consisting of three periods of mild steady-state cycle ergometry (15, 30, 45 watts, or 10%, 20%, and 30% of maximum power) with measurements of whole-body gas exchange. ΔRER, the RER in working muscle, was calculated as (V̇CO2 -V̇CO)/(V̇O - V̇O), from which the fraction of fuel accounted for by lipid was estimated. Lactate levels were low and stable during steady-state exercise. Muscle biopsies were used to estimate mitochondrial content. The rise of V̇O at onset of exercise followed a monoexponential function, with a time constant of 51 ± 7 sec, typical of skeletal muscle; the average O cost of work was about 12 mL O/watt/min, representing a mechanical efficiency of about 24%. At work rates of 30 or 45 watts, active muscle relied predominantly on carbohydrate, independent of insulin sensitivity within this group of very sedentary volunteers. The fraction of muscle fuel oxidation from fat was predicted by power output ( 
• Garcia, L. A., Zapata-Bustos, R., Day, S. E., Campos, B., Hamzaoui, Y., Wu, L., Leon, A. D., Krentzel, J., Coletta, R. L., De Filippis, E., Roust, L. R., Mandarino, L. J., & Coletta, D. K. (2022). Can Exercise Training Alter Human Skeletal Muscle DNA Methylation?. Metabolites, 12(3).
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  Skeletal muscle is highly plastic and dynamically regulated by the body's physical demands. This study aimed to determine the plasticity of skeletal muscle DNA methylation in response to 8 weeks of supervised exercise training in volunteers with a range of insulin sensitivities. We studied 13 sedentary participants and performed euglycemic hyperinsulinemic clamps with basal vastus lateralis muscle biopsies and peak aerobic activity (VO2 peak) tests before and after training. We extracted DNA from the muscle biopsies and performed global methylation using Illumina's Methylation EPIC 850K BeadChip. Training significantly increased peak aerobic capacity and insulin-stimulated glucose disposal. Fasting serum insulin and insulin levels during the steady state of the clamp were significantly lower post-training. Insulin clearance rates during the clamp increased following the training. We identified 13 increased and 90 decreased differentially methylated cytosines (DMCs) in response to 8 weeks of training. Of the 13 increased DMCs, 2 were within the following genes, , and . Of the 90 decreased DMCs, 9 were within the genes , , , , , , , , and . Moreover, pathway analysis showed an enrichment in metabolic and actin-cytoskeleton pathways for the decreased DMCs, and for the increased DMCs, an enrichment in signal-dependent regulation of myogenesis, NOTCH2 activation and transmission, and SMAD2/3: SMAD4 transcriptional activity pathways. Our findings showed that 8 weeks of exercise training alters skeletal muscle DNA methylation of specific genes and pathways in people with varying degrees of insulin sensitivity.
• Mandarino, L. J., Mandarino, L. J., Soltani, L., Soltani, L., Spegman, D. J., Spegman, D. J., Pereira, E., Pereira, E., Molina, P., Molina, P., Mullins, J., Mullins, J., Garcia, D., Garcia, D., Soto, A. A., Soto, A. A., Landes, L., Landes, L., Kohler, L. N., , Kohler, L. N., et al. (2022). Biobanking in Latinos: current status, principles for conduct, and contribution of a new biobank, El Banco por Salud, designed to improve the health of Latino patients of Mexican ancestry with type 2 diabetes. BMJ open diabetes research & care. doi:10.1136/bmjdrc-2021-002709
• McGraw, M. B., Kohler, L. N., Shaibi, G. Q., Mandarino, L. J., & Coletta, D. K. (2022). A performance review of novel adiposity indices for assessing insulin resistance in a pediatric Latino population. Frontiers in pediatrics, 10, 1020901.
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  Body mass index (BMI) percentile or BMI adjusted for age and sex is the most common anthropometric index to monitor and assess obesity in children. However, the ability of BMI to accurately predict insulin resistance (IR) in youth is debated. Determining the best method to noninvasively measure IR in the pediatric population is especially important due to the growing prevalence of type 2 diabetes mellitus (T2DM), which is more likely to develop in people with IR. Therefore, this study analyzed the performance of BMI against newer anthropometric indices in assessing IR in a pediatric Latino identifying sample.
• Parra, O. D., Kohler, L. N., Landes, L., Soto, A. A., Garcia, D., Mullins, J., Molina, P., Pereira, E., Spegman, D. J., Soltani, L., & Mandarino, L. J. (2022). Biobanking in Latinos: current status, principles for conduct, and contribution of a new biobank, El Banco por Salud, designed to improve the health of Latino patients of Mexican ancestry with type 2 diabetes. BMJ open diabetes research & care, 10(3).
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  Underserved Latino communities experience a greater burden of type 2 diabetes mellitus (T2DM) than the general population. Predictors of glycemic control are likely to include both biological/genetic and social determinants of health (SDOH). A variety of approaches have been used with cohorts of Latino patients to study aspects of this health disparity, and those are reviewed briefly here. Such projects range from cohorts that are studies for a primary purpose, for example, to discover genetic variation associated with T2DM or to examine a particular aspect of SDOH that might be involved. Other studies have been conducted more as infrastructure that is broadly based in order to provide a resource that can be used by many investigators to address a variety of questions. From our experience and those of others, we propose a set of principles to ensure that needs of the community are identified and taken into account during the conduct of these studies. As an example of the implementation of these principles, we also describe a new biobank (El Banco), which was designed to improve access to studies designed to improve glycemic control and health in Latinos in partnership with Federally Qualified Health Centers in Arizona.
• Standage-Beier, C. S., Bakhshi, B., Parra, O. D., Soltani, L., Spegman, D. J., Molina, P., Pereira, E., Landes, L., Mandarino, L. J., & Kohler, L. N. (2022). Fruit, Vegetable, and Physical Activity Guideline Adherence and Metabolic Syndrome in El Banco por Salud. Nutrients, 14(9).
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  Adherence to dietary and physical activity recommendations has been associated with reductions in morbidity and mortality. The association between baseline adherence to fruit, vegetable, and physical activity guidelines and metabolic syndrome (MetS) in El Banco por Salud (El Banco) was examined. El Banco is a wellness biobank for Latino individuals affiliated with partnered Federally Qualified Health Centers in southern Arizona. Study participants ( = 972) were 65% female, 62.3% foreign-born, 56.3% obese, 29.2% food insecure, and with an average age of 51.3 years. Adherence scores were developed using baseline questionnaires for fruits and vegetable consumption and self-reported physical activity. Adherence was low in those fully meeting guidelines for fruit, vegetable, and physical activity at 14.6%, 37.5%, and 23.5%, respectively. Roughly 65% ( = 630) had ≥3 cardiometabolic risk factors. Large waist circumference was the most prevalent risk factor at 77.9%. Adherence to physical activity recommendations differed by MetS status with 32.8% without MetS reporting ≥150 min of physical activity per week compared to 18.5% in those with MetS ( < 0.001). There were no significant associations with adherence to any guidelines and MetS in the fully adjusted model. Overall, in this sample guideline adherence was low and the cardiometabolic risk factors prevalence was high.
• Standage-Beier, C. S., Ziller, S. G., Bakhshi, B., Parra, O. D., Mandarino, L. J., Kohler, L. N., & Coletta, D. K. (2022). Tools to Measure Health Literacy among Adult Hispanic Populations with Type 2 Diabetes Mellitus: A Review of the Literature. International journal of environmental research and public health, 19(19).
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  Health literacy (HL) is associated with short- and long-term health outcomes, and this is particularly relevant in Hispanics, who are disproportionally affected by lower HL. Hispanics have become the largest minority population in the United States. Also, Hispanics experience higher burdens of chronic diseases such as type 2 diabetes mellitus (T2DM) than non-Hispanic whites. Thus, effectively choosing culturally appropriate validated instruments that measure a marker found in health assessments should be a serious consideration. Using a systemized approach, we identified and reviewed 33 publications and found eight different HL and numeracy (separate or combined) instruments. We assessed the study designs and instrument structures to determine how HL was measured across these studies. We categorized the results into direct and indirect measurements of HL. The Test of Functional Health Literacy in Adults (TOFHLA) family of HL instruments was favored for direct measures of HL, while the Brief Health Literacy Screen (BHLS) instrument was favored for indirect measures. Despite identified trends in instruments used, more comprehensive measurement tools have been developed but not validated in Hispanic populations. In conclusion, further validation of more comprehensive HL instruments in adult Hispanic populations with T2DM could better assess HL levels and improve health promotion efforts.
• Barakati, N., Stern, J. H., Mandarino, L. J., & Willis, W. T. (2021). Oxidative Phosphorylation K0.5ADP In Vitro Depends on Substrate Oxidative Capacity: Insights from a Luciferase-Based Assay to Evaluate ADP Kinetic Parameters. Biochimica Biophysica Acta-Bioenergetics. doi:doi: 10.1016/j.bbabio.2021.148430
• Coletta, D. K., Hlusko, L. J., Scott, G. R., Garcia, L. A., Vachon, C. M., Norman, A. D., Funk, J. L., Shaibi, G. Q., Hernandez, V., De Filippis, E., & Mandarino, L. J. (2021). Association of EDARV370A with breast density and metabolic syndrome in Latinos. PloS one, 16(10), e0258212.
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  The ectodysplasin receptor (EDAR) is a tumor necrosis factor receptor (TNF) superfamily member. A substitution in an exon of EDAR at position 370 (EDARV370A) creates a gain of function mutant present at high frequencies in Asian and Indigenous American populations but absent in others. Its frequency is intermediate in populations of Mexican ancestry. EDAR regulates the development of ectodermal tissues, including mammary ducts. Obesity and type 2 diabetes mellitus are prevalent in people with Indigenous and Latino ancestry. Latino patients also have altered prevalence and presentation of breast cancer. It is unknown whether EDARV370A might connect these phenomena. The goals of this study were to determine 1) whether EDARV370A is associated with metabolic phenotypes and 2) if there is altered breast anatomy in women carrying EDARV370A. Participants were from two Latino cohorts, the Arizona Insulin Resistance (AIR) registry and Sangre por Salud (SPS) biobank. The frequency of EDARV370A was 47% in the Latino cohorts. In the AIR registry, carriers of EDARV370A (GG homozygous) had significantly (p < 0.05) higher plasma triglycerides, VLDL, ALT, 2-hour post-challenge glucose, and a higher prevalence of prediabetes/diabetes. In a subset of the AIR registry, serum levels of ectodysplasin A2 (EDA-A2) also were associated with HbA1c and prediabetes (p < 0.05). For the SPS biobank, participants that were carriers of EDARV370A had lower breast density and higher HbA1c (both p < 0.05). The significant associations with measures of glycemia remained when the cohorts were combined. We conclude that EDARV370A is associated with characteristics of the metabolic syndrome and breast density in Latinos.
• Finlayson, J., Finlayson, J., Barakati, N., Barakati, N., Langlais, P. R., Langlais, P. R., Funk, J., Funk, J. L., Zapata Bustos, R., Zapata Bustos, R., Coletta, D. K., Coletta, D. K., Luo, M., Luo, M., Willis, W. T., Willis, W. T., Mandarino, L. J., & Mandarino, L. J. (2021). Site-specific acetylation of adenine nucleotide translocase 1 at lysine 23 in human muscle. Analytical biochemistry, 630, 114319.
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  Evidence suggests acetylation of human adenine nucleotide translocase 1 (ANT1) at lysine 23 (Lys23) reduces binding of ADP. Lys23 contributes to the positive charge that facilitates this interaction. This study was undertaken to characterize ANT1 abundance and acetylation by a novel method using small amounts of human skeletal muscle biopsies. Lysates of whole muscle or mitochondria from the same tissue were prepared from needle biopsies of vastus lateralis muscle of healthy volunteers. Lysed proteins were resolved on gels, the section containing ANT1 (surrounding 30 Kd) was excised, digested with trypsin, spiked with labeled unacetylated and acetylated synthetic standard peptides and analyzed by mass spectrometry. Natural logarithm transformation of data linearized ion intensities over a 10-fold range of peptide mass. Coefficients of variation ranged from 7 to 30% for ANT1 abundance and Lys23 acetylation. In three volunteers, ANT1 content was 8.36 ± 0.33 nmol/g wet weight muscle and 0.64 ± 0.05 nmol/mg mitochondria, so mitochondrial content was 13.3 ± 2.4 mg mitochondria per gram muscle. Acetylation of Lys23 averaged 14.3 ± 4.2% and 4.87 ± 1.84% in whole muscle and mitochondria, respectively. This assay makes it possible to assess effects of acetylation on the function of ANT1 in human muscle.
• Garcia, L. A., Day, S. E., Coletta, R. L., Campos, B., Benjamin, T. R., De Filippis, E., Madura, J. A., Mandarino, L. J., Roust, L. R., & Coletta, D. K. (2021). Weight loss after Roux-En-Y gastric bypass surgery reveals skeletal muscle DNA methylation changes. Clinical epigenetics, 13(1), 100.
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  The mechanisms of weight loss and metabolic improvements following bariatric surgery in skeletal muscle are not well known; however, epigenetic modifications are likely to contribute. The aim of our study was to investigate skeletal muscle DNA methylation after weight loss induced by Roux-en-Y gastric bypass (RYGB) surgery. Muscle biopsies were obtained basally from seven insulin-resistant obese (BMI > 40 kg/m) female subjects (45.1 ± 3.6 years) pre- and 3-month post-surgery with euglycemic hyperinsulinemic clamps to assess insulin sensitivity. Four lean (BMI 
• James, J., Zemskova, M., Eccles, C. A., Varghese, M. V., Niihori, M., Barker, N. K., Luo, M., Mandarino, L. J., Langlais, P. R., Rafikova, O., & Rafikov, R. (2021). Single Mutation in the Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells. Arteriosclerosis, thrombosis, and vascular biology, 41(2), 734-754.
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  NFU1 is a mitochondrial iron-sulfur scaffold protein, involved in iron-sulfur assembly and transfer to complex II and LAS (lipoic acid synthase). Patients with the point mutation NFU1 and CRISPR/CAS9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-generated rats develop mitochondrial dysfunction leading to pulmonary arterial hypertension. However, the mechanistic understanding of pulmonary vascular proliferation due to a single mutation in NFU1 remains unresolved. Approach and Results: Quantitative proteomics of isolated mitochondria showed the entire phenotypic transformation of NFU1 rats with a disturbed mitochondrial proteomic landscape, involving significant changes in the expression of 208 mitochondrial proteins. The NFU1 mutation deranged the expression pattern of electron transport proteins, resulting in a significant decrease in mitochondrial respiration. Reduced reliance on mitochondrial respiration amplified glycolysis in pulmonary artery smooth muscle cell (PASMC) and activated GPD (glycerol-3-phosphate dehydrogenase), linking glycolysis to oxidative phosphorylation and lipid metabolism. Decreased PDH (pyruvate dehydrogenase) activity due to the lipoic acid shortage is compensated by increased fatty acid metabolism and oxidation. PASMC became dependent on extracellular fatty acid sources due to upregulated transporters such as CD36 (cluster of differentiation 36) and CPT (carnitine palmitoyltransferase)-1. Finally, the NFU1 mutation produced a dysregulated antioxidant system in the mitochondria, leading to increased reactive oxygen species levels. PASMC from NFU1 rats showed apoptosis resistance, increased anaplerosis, and attained a highly proliferative phenotype. Attenuation of mitochondrial reactive oxygen species by mitochondrial-targeted antioxidant significantly decreased PASMC proliferation.
• Luo, M., Ma, W., Zapata-Bustos, R., Willis, W. T., & Mandarino, L. J. (2021). Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes. Biochemistry and biophysics reports, 26, 100928.
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  VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P 
• Ma, W., Willis, W. T., Luo, M., Mandarino, L. J., & Zapata Bustos, R. (2021). Deletion of Von Willebrand A Domain Containing Protein (VWA8) Raises Activity of Mitochondrial Electron Transport Chain Complexes in Hepatocytes. Biochemical and Biophysical Research Communications.
• Ma, W., Willis, W. T., Luo, M., Mandarino, L. J., & Zapata Bustos, R. (2021). Deletion of Von Willebrand A Domain Containing Protein (VWA8) Raises Activity of Mitochondrial Electron Transport Chain Complexes in Hepatocytes. Biochemical and Biophysical Research Communications. doi:doi: 10.1016/j.bbrep.2021.100928
• Mandarino, L. J., De Filippis, E. A., Grandjean, D., Luo, M., Coletta, D. K., Langlais, P. R., Finlayson, J., & Zapata Bustos, R. (2021). Altered Transcription Factor Expression Responses to Exercise in Insulin Resistance. Frontiers in Physiology.
• Rafikov, R., Rafikova, O., Langlais, P. R., Mandarino, L. J., Luo, M., Barker, N. K., Niihori, M., Varghese, M. V., Eccles, C. A., Zemskova, M., & James, J. (2021). Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells. Arteriosclerosis, Thrombosis, and Vascular Biology.
• Willis, W., Willis, E., Kuzmiak-Glancy, S., Kras, K., Hudgens, J., Barakati, N., Stern, J., & Mandarino, L. (2021). Oxidative phosphorylation KADP in vitro depends on substrate oxidative capacity: Insights from a luciferase-based assay to evaluate ADP kinetic parameters. Biochimica et biophysica acta. Bioenergetics, 1862(8), 148430.
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  The KADP of oxidative phosphorylation (OxPhos) identifies the cytosolic ADP concentration which elicits one-half the maximum OxPhos rate. This kinetic parameter is commonly measured to assess mitochondrial metabolic control sensitivity. Here we describe a luciferase-based assay to evaluate the ADP kinetic parameters of mitochondrial ATP production from OxPhos, adenylate kinase (AK), and creatine kinase (CK). The high sensitivity, reproducibility, and throughput of the microplate-based assay enabled a comprehensive kinetic assessment of all three pathways in mitochondria isolated from mouse liver, kidney, heart, and skeletal muscle. Carboxyatractyloside titrations were also performed with the assay to estimate the flux control strength of the adenine nucleotide translocase (ANT) over OxPhos in human skeletal muscle mitochondria. ANT flux control coefficients were 0.91 ± 0.07, 0.83 ± 0.06, and 0.51 ± 0.07 at ADP concentrations of 6.25, 12.5, and 25 μM, respectively, an [ADP] range which spanned the KADP. The oxidative capacity of substrate combinations added to drive OxPhos was found to dramatically influence ADP kinetics in mitochondria from several tissues. In mouse skeletal muscle ten different substrate combinations elicited a 7-fold range of OxPhos V, which correlated positively (R = 0.963) with KADP values ranging from 2.3 ± 0.2 μM to 11.9 ± 0.6 μM. We propose that substrate-enhanced capacity to generate the protonmotive force increases the OxPhos KADP because flux control at ANT increases, thus KADP rises toward the dissociation constant, KADP, of ADP-ANT binding. The findings are discussed in the context of top-down metabolic control analysis.
• Yang, C., Hallmark, B., Chai, J. C., O'Connor, T. D., Reynolds, L. M., Wood, A. C., Seeds, M., Chen, Y. I., Steffen, L. M., Tsai, M. Y., Kaplan, R. C., Daviglus, M. L., Mandarino, L. J., Fretts, A. M., Lemaitre, R. N., Coletta, D. K., Blomquist, S. A., Johnstone, L. M., Tontsch, C., , Qi, Q., et al. (2021). Impact of Amerind ancestry and FADS genetic variation on omega-3 deficiency and cardiometabolic traits in Hispanic populations. Communications biology, 4(1), 918.
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  Long chain polyunsaturated fatty acids (LC-PUFAs) have critical signaling roles that regulate dyslipidemia and inflammation. Genetic variation in the FADS gene cluster accounts for a large portion of interindividual differences in circulating and tissue levels of LC-PUFAs, with the genotypes most strongly predictive of low LC-PUFA levels at strikingly higher frequencies in Amerind ancestry populations. In this study, we examined relationships between genetic ancestry and FADS variation in 1102 Hispanic American participants from the Multi-Ethnic Study of Atherosclerosis. We demonstrate strong negative associations between Amerind genetic ancestry and LC-PUFA levels. The FADS rs174537 single nucleotide polymorphism (SNP) accounted for much of the AI ancestry effect on LC-PUFAs, especially for low levels of n-3 LC-PUFAs. Rs174537 was also strongly associated with several metabolic, inflammatory and anthropomorphic traits including circulating triglycerides (TGs) and E-selectin in MESA Hispanics. Our study demonstrates that Amerind ancestry provides a useful and readily available tool to identify individuals most likely to have FADS-related n-3 LC-PUFA deficiencies and associated cardiovascular risk.
• Zapata-Bustos, R., Finlayson, J., Langlais, P. R., Coletta, D. K., Luo, M., Grandjean, D., De Filippis, E. A., & Mandarino, L. (2021). Altered Transcription Factor Expression Responses to Exercise in Insulin Resistance. Frontiers in physiology, 12, 649461.
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  Insulin resistant muscle is resistant to gene expression changes induced by acute exercise. This study was undertaken to identify transcription factors that differentially respond to exercise in insulin resistance. Candidate transcription factors were identified from analysis of 5'-untranslated regions (5'-UTRs) of exercise responsive genes and from analysis of the 5'-UTRs of genes coding for proteins that differ in abundance in insulin resistance.
• Brinton, R. D., Finlayson, J., Luo, M., Ma, W., Mandarino, L. J., Sand, Z., Wang, T., Willis, W. T., Mandarino, L. J., Willis, W. T., & Luo, M. (2020). Von Willebrand factor A domain-containing protein 8 (VWA8) localizes to the matrix side of the inner mitochondrial membrane.. Biochemical and biophysical research communications, 521(1), 158-163. doi:10.1016/j.bbrc.2019.10.095
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  VWA8 is a poorly characterized mitochondrial AAA + ATPase protein. The specific submitochondrial localization of VWA8 remains unclear. The purpose of this study was to determine the specific submitochondrial compartment within which VWA8 resides in order to provide more insight into the function of this protein. Bioinformatics analysis showed that VWA8 has a 34 amino acid N-terminal Matrix-Targeting Signal (MTS) that is similar to those in proteins known to localize to the mitochondrial matrix. Experiments in C2C12 mouse myoblasts using confocal microscopy showed that deletion of the VWA8 MTS (vMTS) resulted in cytosolic, rather than mitochondrial, localization of VWA8. Biochemical analysis using differential sub-fractionation of mitochondria isolated from rat liver showed that VWA8 localizes to the matrix side of inner mitochondrial membrane, similar to the inner mitochondrial membrane protein Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETFDH). The results of these experiments show that the vMTS is essential for localization to the mitochondrial matrix and that once there, VWA8 localizes to the matrix side of inner mitochondrial membrane.
• Bustos, R. Z., Coletta, D. K., Filippis, E. A., Grandjean, D., Langlais, P. R., & Mandarino, L. J. (2020).

  166-OR: Lower Response of Connective Tissue Growth Factor (CTGF) to Exercise Characterizes Insulin Resistant Muscle

  . Diabetes, 69(Supplement_1). doi:10.2337/db20-166-or
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  This study was undertaken to identify transcription factors that differentially respond to exercise in insulin resistance. Candidate transcription factors were identified from analysis of 5’-untranslated regions (5’-UTRs) of exercise responsive genes and from analysis of the 5’-UTRs of genes coding for proteins that differ in abundance in insulin resistance. Muscle biopsies were obtained from lean and obese subjects before, 30 min and 240 h. after a single exercise bout. Insulin sensitivity was assessed through glucose clamps. Obese subjects (n=8) were insulin resistant compared to lean (n=12) (7.0 ± 0.95 vs. 11.3 ± 0.8 mg∙kg FFM-1∙min-1, P Disclosure R. Zapata Bustos: None. P.R. Langlais: None. D.K. Coletta: None. E.A. De Filippis: None. D. Grandjean: None. L. Mandarino: None. Funding National Institutes of Health (R01DK047936, DK066483, R01DK094013)
• Hallmark, B., Coletta, D. K., Chilton, F. H., Yang, C., Rich, S. S., Manichaikul, A. W., Mandarino, L. J., Hallmark, B., Coletta, D. K., Chilton, F. H., & Blomquist, S. (2020). Fatty Acid Desaturase Gene‐Induced Omega‐3 Deficiency in Amerindian‐Ancestry Hispanic Populations. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.06389
• Rafikov, R., Coletta, D. K., Mandarino, L. J., & Rafikova, O. (2020). Pulmonary Arterial Hypertension Induces a Distinct Signature of Circulating Metabolites. Journal of clinical medicine, 9(1).
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  Pulmonary arterial hypertension (PAH) is an incurable, progressive disorder, and the early diagnosis and treatment of PAH are associated with increased survival [...].
• Shaibi, G. Q., Kullo, I. J., Singh, D. P., Hernandez, V., Sharp, R. R., Cuellar, I., De Filippis, E., Levey, S., Breitkopf, C. R., Mandarino, L. J., Yang, P., Thibodeau, S. N., & Lindor, N. M. (2020). Returning genomic results in a Federally Qualified Health Center: the intersection of precision medicine and social determinants of health. Genetics in medicine : official journal of the American College of Medical Genetics, 22(9), 1552-1559.
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  This report describes the return of sequencing results to low-income Latino participants recruited through a Federally Qualified Health Center (FQHC). We describe challenges in returning research results secondary to social determinants of health and present lessons learned to guide future genomic medicine implementation studies in low-resource settings.
• Fonseca-Portilla, R., Krell-Roesch, J., Shaibi, G. Q., Caselli, R. J., Mandarino, L. J., Zhang, N., Hentz, J. G., Coletta, D. K., de Filippis, E. A., Dawit, S., & Geda, Y. E. (2019). Brain-Derived Neurotrophic Factor and Its Associations with Metabolism and Physical Activity in a Latino Sample. Metabolic syndrome and related disorders, 17(2), 75-80.
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  Brain-derived neurotrophic factor (BDNF) is associated with body weight and other health conditions but remains understudied in the Latino population. The aim of this study was to examine the associations of BDNF serum levels with body mass index (BMI), physical activity, and the rs6265 nonconservative polymorphism among 349 Latinos aged ≥18 years enrolled in the Arizona Insulin Resistance Registry.
• Jacobs, E. T., Lance, P., Mandarino, L. J., Ellis, N. A., Chow, H. S., Foote, J., Martinez, J. A., Hsu, C. P., Batai, K., Saboda, K., & Thompson, P. A. (2019). Selenium supplementation and insulin resistance in a randomized, clinical trial. BMJ open diabetes research & care, 7(1), e000613.
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  While controversial, observational and randomized clinical trial data implicate the micronutrient selenium (Se) in the development of type 2 diabetes (T2D). The aim of this study was to test the hypothesis that Se supplementation adversely affects pancreatic β-cell function and insulin sensitivity.
• Luo, M., Ma, W., Sand, Z., Finlayson, J., Wang, T., Brinton, R. D., Willis, W. T., & Mandarino, L. J. (2019). Von Willebrand factor A domain-containing protein 8 (VWA8) localizes to the matrix side of the inner mitochondrial membrane. Biochemical and biophysical research communications.
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  VWA8 is a poorly characterized mitochondrial AAA + ATPase protein. The specific submitochondrial localization of VWA8 remains unclear. The purpose of this study was to determine the specific submitochondrial compartment within which VWA8 resides in order to provide more insight into the function of this protein. Bioinformatics analysis showed that VWA8 has a 34 amino acid N-terminal Matrix-Targeting Signal (MTS) that is similar to those in proteins known to localize to the mitochondrial matrix. Experiments in C2C12 mouse myoblasts using confocal microscopy showed that deletion of the VWA8 MTS (vMTS) resulted in cytosolic, rather than mitochondrial, localization of VWA8. Biochemical analysis using differential sub-fractionation of mitochondria isolated from rat liver showed that VWA8 localizes to the matrix side of inner mitochondrial membrane, similar to the inner mitochondrial membrane protein Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETFDH). The results of these experiments show that the vMTS is essential for localization to the mitochondrial matrix and that once there, VWA8 localizes to the matrix side of inner mitochondrial membrane.
• Luo, M., Willis, W. T., Coletta, D. K., Langlais, P. R., Mengos, A., Ma, W., Finlayson, J., Wagner, G. R., Shi, C. X., & Mandarino, L. J. (2019). Deletion of the Mitochondrial Protein VWA8 Induces Oxidative Stress and an HNF4α Compensatory Response in Hepatocytes. Biochemistry, 58(49), 4983-4996.
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  von Willebrand A domain-containing protein 8 (VWA8) is a poorly characterized, mitochondrial matrix-targeted protein with an AAA ATPase domain and ATPase activity that increases in livers of mice fed a high-fat diet. This study was undertaken to use CRISPR/Cas9 to delete VWA8 in cultured mouse hepatocytes and gain insight into its function. Unbiased omics techniques and bioinformatics were used to guide subsequent assays, including the assessment of oxidative stress and the determination of bioenergetic capacity. Metabolomics analysis showed VWA8 null cells had higher levels of oxidative stress and protein degradation; assays of hydrogen peroxide production revealed higher levels of production of reactive oxygen species (ROS). Proteomics and transcriptomics analyses showed VWA8 null cells had higher levels of expression of mitochondrial proteins (electron transport-chain Complex I, ATP synthase), peroxisomal proteins, and lipid transport proteins. The pattern of higher protein abundance in the VWA8 null cells could be explained by a higher level of hepatocyte nuclear factor 4 α (HNF4α) expression. Bioenergetic assays showed higher rates of carbohydrate oxidation and mitochondrial and nonmitochondrial lipid oxidation in intact and permeabilized cells. Inhibitor assays localized sites of ROS production to peroxisomes and NOX1/4. The rescue of VWA8 protein restored the wild-type phenotype, and treatment with antioxidants decreased the level of HNF4α expression. Thus, loss of VWA8 produces a mitochondrial defect that may be sensed by NOX4, leading to an increase in the level of ROS that results in a higher level of HNF4α. The compensatory HNF4α response results in a higher oxidative capacity and an even higher level of ROS production. We hypothesize that VWA8 is an AAA ATPase protein that plays a role in mitochondrial protein quality.
• Mandarino, L. J., Nair, A., Baier, L. J., Wagner, G. R., Finlayson, J., Ma, W., Mengos, A., Langlais, P. R., Coletta, D. K., Willis, W. T., & Luo, M. (2019). Deletion of the Mitochondrial Protein VWA8 Induces Oxidative Stress and an HNF4α Compensatory Response in Hepatocytes.. Biochemistry.
• Mandarino, L. J., Willis, W., Wang, T., Finlayson, J., Sand, Z., Ma, W., Luo, M., & Brinton, R. (2019). Von Willebrand factor A domain-containing protein 8 (VWA8) localizes to the matrix side of the inner mitochondrial membrane. Biochem Biophys Res Commun.
• Going, S. B., Chen, Z., Alexander, G. E., Mandarino, L. J., Wineinger, N. E., Garcia, D. O., Bea, J. W., Raichlen, D. A., & Klimentidis, Y. C. (2018). In response to: ‘Information bias in measures of self-reported physical activity’. Int J Obes (Lond), 42(12), 2064-2065. doi:10.1038/s41366-018-0251-6
• Klimentidis, Y. C., Raichlen, D. A., Bea, J. W., Garcia, D. O., Mandarino, L. J., Alexander, G. E., Chen, Z., & Going, S. B. (2018). Genome-wide association study of habitual physical activity in over 377,000 UK Biobank participants indentifies novel variants and genetic correlations with chronotype and obesity related traits. International Journal of Obesity. doi:10.1038/s41366-018-0120-3
• Klimentidis, Y. C., Raichlen, D. A., Bea, J., Garcia, D. O., Wineinger, N. E., Mandarino, L. J., Alexander, G. E., Chen, Z., & Going, S. B. (2018). Genome-wide association study of habitual physical activity in over 377,000 UK Biobank participants identifies multiple variants including CADM2 and APOE. International journal of obesity (2005), 42(6), 1161-1176.
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  Physical activity (PA) protects against a wide range of diseases. Habitual PA appears to be heritable, motivating the search for specific genetic variants that may inform efforts to promote PA and target the best type of PA for each individual.
• Klimentidis, Y. C., Raichlen, D. A., Bea, J., Garcia, D. O., Wineinger, N. E., Mandarino, L. J., Alexander, G. E., Chen, Z., & Going, S. B. (2018). In response to: 'Information bias in measures of self-reported physical activity'. International journal of obesity (2005), 42(12), 2064-2065.
• Rafikov, R., Mandarino, L. J., Simon, M., Zemskov, E., Langlais, P. R., Desai, A., Nair, V., Srivastava, A., Zemskova, M., McBride, M. L., Williams, E. E., & Rafikova, O. (2018). Hemolysis-induced lung vascular leakage contributes to the development of pulmonary hypertension. American Journal of Respiratory Cell and Molecular Biology.
• Rafikova, O., Williams, E. R., McBride, M. L., Zemskova, M., Srivastava, A., Nair, V., Desai, A. A., Langlais, P. R., Zemskov, E., Simon, M., Mandarino, L. J., & Rafikov, R. (2018). Hemolysis-induced Lung Vascular Leakage Contributes to the Development of Pulmonary Hypertension. American journal of respiratory cell and molecular biology, 59(3), 334-345.
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  Although hemolytic anemia-associated pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH) are more common than the prevalence of idiopathic PAH alone, the role of hemolysis in the development of PAH is poorly characterized. We hypothesized that hemolysis independently contributes to PAH pathogenesis via endothelial barrier dysfunction with resulting perivascular edema and inflammation. Plasma samples from patients with and without PAH (both confirmed by right heart catheterization) were used to measure free hemoglobin (Hb) and its correlation with PAH severity. A sugen (50 mg/kg)/hypoxia (3 wk)/normoxia (2 wk) rat model was used to elucidate the role of free Hb/heme pathways in PAH. Human lung microvascular endothelial cells were used to study heme-mediated endothelial barrier effects. Our data indicate that patients with PAH have increased levels of free Hb in plasma that correlate with PAH severity. There is also a significant accumulation of free Hb and depletion of haptoglobin in the rat model. In rats, perivascular edema was observed at early time points concomitant with increased infiltration of inflammatory cells. Heme-induced endothelial permeability in human lung microvascular endothelial cells involved activation of the p38/HSP27 pathway. Indeed, the rat model also exhibited increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling, there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF-1a during the first 2 weeks of PH regardless of hypoxic conditions. Our data suggest that hemolysis may play a significant role in PAH pathobiology.
• Shaibi, G. Q., Kullo, I. J., Singh, D. P., Sharp, R. R., De Filippis, E., Cuellar, I., Hernandez, V., Levey, S., Radecki Breitkopf, C., Olson, J. E., Cerhan, J. R., Mandarino, L. J., Thibodeau, S. N., & Lindor, N. M. (2018). Developing a Process for Returning Medically Actionable Genomic Variants to Latino Patients in a Federally Qualified Health Center. Public health genomics, 21(1-2), 77-84.
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  To develop a process for returning medically actionable genomic variants to Latino patients receiving care in a Federally Qualified Health Center.
• Tran, L., Kras, K. A., Hoffman, N., Ravichandran, J., Dickinson, J. M., D'Lugos, A., Carroll, C. C., Patel, S. H., Mandarino, L. J., Roust, L., & Katsanos, C. S. (2018). Lower Fasted-State but Greater Increase in Muscle Protein Synthesis in Response to Elevated Plasma Amino Acids in Obesity. Obesity (Silver Spring, Md.), 26(7), 1179-1187.
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  Obesity alters protein metabolism in skeletal muscle, but consistent evidence is lacking. This study compared muscle protein synthesis in adults with obesity and in lean controls in the fasted state and during an amino acid infusion.
• Willis, W. T., Mandarino, L. J., Mielke, C., Miranda-Grandjean, D., Hudgens, J., Langlais, P. R., Zapata Bustos, R., Willis, E. A., De Filippis, E. A., Finlayson, J., Finlayson, J., De Filippis, E. A., Willis, E. A., Zapata Bustos, R., Hudgens, J., Langlais, P. R., Miranda-Grandjean, D., Mielke, C., Mandarino, L. J., & Willis, W. T. (2018). Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.. Archives of Biochemistry and Biophysics.
• Willis, W. T., Willis, E. A., Mandarino, L. J., & Hudgens, J. (2018). KMADP For Oxidative Phosphorylation Depends On Substrate Oxidative Capacity. Biochimica et Biophysica Acta, 1859, e64-e65. doi:10.1016/j.bbabio.2018.09.192
• Day, S. E., Coletta, R. L., Kim, J. Y., Garcia, L. A., Campbell, L. E., Benjamin, T. R., Roust, L. R., De Filippis, E. A., Mandarino, L. J., & Coletta, D. K. (2017). Potential epigenetic biomarkers of obesity-related insulin resistance in human whole-blood. Epigenetics, 1-10.
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  Obesity can increase the risk of complex metabolic diseases, including insulin resistance. Moreover, obesity can be caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are not well defined. Therefore, the identification of novel epigenetic biomarkers of obesity allows for a more complete understanding of the disease and its underlying insulin resistance. The aim of our study was to identify DNA methylation changes in whole-blood that were strongly associated with obesity and insulin resistance. Whole-blood was obtained from lean (n = 10; BMI = 23.6 ± 0.7 kg/m(2)) and obese (n = 10; BMI = 34.4 ± 1.3 kg/m(2)) participants in combination with euglycemic hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing on genomic DNA isolated from the blood. We identified 49 differentially methylated cytosines (DMCs; q < 0.05) that were altered in obese compared with lean participants. We identified 2 sites (Chr.21:46,957,981 and Chr.21:46,957,915) in the 5' untranslated region of solute carrier family 19 member 1 (SLC19A1) with decreased methylation in obese participants (lean 0.73 ± 0.11 vs. obese 0.09 ± 0.05; lean 0.68 ± 0.10 vs. obese 0.09 ± 0.05, respectively). These 2 DMCs identified by obesity were also significantly predicted by insulin sensitivity (r = 0.68, P = 0.003; r = 0.66; P = 0.004). In addition, we performed a differentially methylated region (DMR) analysis and demonstrated a decrease in methylation of Chr.21:46,957,915-46,958,001 in SLC19A1 of -34.9% (70.4% lean vs. 35.5% obese). The decrease in whole-blood SLC19A1 methylation in our obese participants was similar to the change observed in skeletal muscle (Chr.21:46,957,981, lean 0.70 ± 0.09 vs. obese 0.31 ± 0.11 and Chr.21:46,957,915, lean 0.72 ± 0.11 vs. obese 0.31 ± 0.13). Pyrosequencing analysis further demonstrated a decrease in methylation at Chr.21:46,957,915 in both whole-blood (lean 0.71 ± 0.10 vs. obese 0.18 ± 0.06) and skeletal muscle (lean 0.71 ± 0.10 vs. obese 0.30 ± 0.11). Our findings demonstrate a new potential epigenetic biomarker, SLC19A1, for obesity and its underlying insulin resistance.
• Day, S. E., Garcia, L. A., Coletta, R. L., Campbell, L. E., Benjamin, T. R., De Filippis, E. A., Madura, J. A., Mandarino, L. J., Roust, L. R., & Coletta, D. K. (2017). Alterations of sorbin and SH3 domain containing 3 (SORBS3) in human skeletal muscle following Roux-en-Y gastric bypass surgery. Clinical epigenetics, 9, 96.
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  Obesity is a disease that is caused by genetic and environmental factors. However, epigenetic mechanisms of obesity are less well known. DNA methylation provides a mechanism whereby environmental factors can influence gene transcription. The aim of our study was to investigate skeletal muscle DNA methylation of sorbin and SH3 domain containing 3 (SORBS3) with weight loss induced by Roux-en-Y gastric bypass (RYGB).
• Kruse, R., Krantz, J., Barker, N., Coletta, R., Rafikov, R., Luo, M., Hoejlund, K., Mandarino, L. J., & Langlais, P. R. (2017). The CLASP2 Protein Interaction Network in Adipocytes Links CLIP2 to AGAP3, CLASP2 to G2L1, MARK2, and SOGA1, and Identifies SOGA1 as a Microtubule-Associated Protein. Molecular & cellular proteomics : MCP.
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  CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 co-immunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology.
• Langlais, P. R., Langlais, P. R., Mandarino, L. J., Mandarino, L. J., Hoejlund, K., Hoejlund, K., Luo, M., Luo, M., Rafikov, R., Rafikov, R., Coletta, R., Coletta, R., Barker, N., Barker, N., Krantz, J., Krantz, J., Kruse, R., & Kruse, R. (2017). Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein. Molecular & cellular proteomics : MCP, 16(10), 1718-1735. doi:10.1074/mcp.RA117.000011
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  CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 co-immunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology.
• Luo, M., Mengos, A. E., Ma, W., Finlayson, J., Bustos, R. Z., Xiao Zhu, Y., Shi, C. X., Stubblefield, T. M., Willis, W. T., & Mandarino, L. J. (2017). Characterization of the novel protein KIAA0564 (Von Willebrand Domain-containing Protein 8). Biochemical and biophysical research communications, 487(3), 545-551.
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  The VWA8 gene was first identified by the Kazusa cDNA project and named KIAA0564. Based on the observation, by similarity, that the protein encoded by KIAA0564 contains a Von Willebrand Factor 8 domain, KIAA0564 was named Von Willebrand Domain-containing Protein 8 (VWA8). The function of VWA8 protein is almost unknown. The purpose of this study was to characterize the tissue distribution, cellular location, and function of VWA8. In mice VWA8 protein was mostly distributed in liver, kidney, heart, pancreas and skeletal muscle, and is present as a long isoform and a shorter splice variant (VWA8a and VWA8b). VWA8 protein and mRNA were elevated in mouse liver in response to high fat feeding. Sequence analysis suggests that VWA8 has a mitochondrial targeting sequence and domains responsible for ATPase activity. VWA8 protein was targeted exclusively to mitochondria in mouse AML12 liver cells, and this was prevented by deletion of the targeting sequence. Moreover, the VWA8 short isoform overexpressed in insect cells using a baculovirus construct had in vitro ATPase activity. Deletion of the Walker A motif or Walker B motif in VWA8 mostly blocked ATPase activity, suggesting Walker A motif or Walker B motif are essential to the ATPase activity of VWA8. Finally, homology modeling suggested that VWA8 may have a structure most confidently similar to dynein motor proteins.
• Campbell, L. E., Langlais, P. R., Day, S. E., Coletta, R. L., Benjamin, T. R., De Filippis, E. A., Madura, J. A., Mandarino, L. J., Roust, L. R., & Coletta, D. K. (2016). Identification of Novel Changes in Human Skeletal Muscle Proteome After Roux-en-Y Gastric Bypass Surgery. Diabetes, 65(9), 2724-31.
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  The mechanisms of metabolic improvements after Roux-en-Y gastric bypass (RYGB) surgery are not entirely clear. Therefore, the aim of our study was to investigate the role of obesity and RYGB on the human skeletal muscle proteome. Basal muscle biopsies were obtained from seven obese (BMI >40 kg/m(2)) female subjects (45.1 ± 3.6 years) pre- and 3 months post-RYGB, and euglycemic-hyperinsulinemic clamps were used to assess insulin sensitivity. Four age-matched (48.5 ± 4.7 years) lean (BMI
• Coletta, D. K., Campbell, L. E., Weil, J., Kaplan, B., Clarkson, M., Finlayson, J., Mandarino, L. J., & Chakkera, H. A. (2016). Changes in Pre- and Post-Exercise Gene Expression among Patients with Chronic Kidney Disease and Kidney Transplant Recipients. PloS one, 11(8), e0160327.
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  Decreased insulin sensitivity blunts the normal increase in gene expression from skeletal muscle after exercise. In addition, chronic inflammation decreases insulin sensitivity. Chronic kidney disease (CKD) is an inflammatory state. How CKD and, subsequently, kidney transplantation affects skeletal muscle gene expression after exercise are unknown.
• Day, S. E., Coletta, R. L., Kim, J. Y., Campbell, L. E., Benjamin, T. R., Roust, L. R., De Filippis, E. A., Dinu, V., Shaibi, G. Q., Mandarino, L. J., & Coletta, D. K. (2016). Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes. Clinical epigenetics, 8, 77.
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  Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity.
• Kim, J. Y., DeMenna, J. T., Puppala, S., Chittoor, G., Schneider, J., Duggirala, R., Mandarino, L. J., Shaibi, G. Q., & Coletta, D. K. (2016). Physical activity and FTO genotype by physical activity interactive influences on obesity. BMC genetics, 17, 47.
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  Although the effect of the fat mass and obesity-associated (FTO) gene on adiposity is well established, there is a lack of evidence whether physical activity (PA) modifies the effect of FTO variants on obesity in Latino populations. Therefore, the purpose of this study was to examine PA influences and interactive effects between FTO variants and PA on measures of adiposity in Latinos.
• Luo, M., Mengos, A. E., Mandarino, L. J., & Sekulic, A. (2016). Association of liprin β-1 with kank proteins in melanoma. Experimental dermatology, 25(4), 321-3.
• Mandarino, L. J., Langlais, P. R., Campbell, L. E., Day, S. E., Coletta, R. L., Benjamin, T. R., De Filippis, E. A., Madura, J. A., Roust, L. R., & Coletta, D. K. (2016). Identification of Novel Changes in Human Skeletal Muscle Proteome After Roux-en-Y Gastric Bypass Surgery. Diabetes, 65(9), 2724-2731. doi:10.2337/db16-0004
• Shaibi, G., Singh, D., De Filippis, E., Hernandez, V., Rosenfeld, B., Otu, E., Montes de Oca, G., Levey, S., Radecki Breitkopf, C., Sharp, R., Olson, J., Cerhan, J., Thibodeau, S., Winkler, E., & Mandarino, L. (2016). The Sangre Por Salud Biobank: Facilitating Genetic Research in an Underrepresented Latino Community. Public health genomics, 19(4), 229-38.
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  The Sangre Por Salud (Blood for Health; SPS) Biobank was created for the purpose of expanding precision medicine research to include underrepresented Latino patients. It is the result of a unique collaboration between Mayo Clinic and Mountain Park Health Center, a federally qualified community health center in Phoenix, Arizona. This report describes the rationale, development, implementation, and characteristics of the SPS Biobank.
• Tran, L., Hanavan, P. D., Campbell, L. E., De Filippis, E., Lake, D. F., Coletta, D. K., Roust, L. R., Mandarino, L. J., Carroll, C. C., & Katsanos, C. S. (2016). Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit. PloS one, 11(8), e0160057.
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  Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P
• Xie, X., Yi, Z., Sinha, S., Madan, M., Bowen, B. P., Langlais, P., Ma, D., Mandarino, L., & Meyer, C. (2016). Proteomics analyses of subcutaneous adipocytes reveal novel abnormalities in human insulin resistance. Obesity (Silver Spring, Md.), 24(7), 1506-14.
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  To provide a more global view of adipocyte changes in human insulin resistance by proteomics analyses.
• Xu, Q., Hou, Y., Langlais, P., Erickson, P., Zhu, J., Shi, C., Luo, M., Zhu, Y., Xu, Y., Mandarino, L. J., Stewart, K., & Chang, X. (2016). Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival. BMC cancer, 16, 297.
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  Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity.
• Coletta, D. K., Tran, L., Mandarino, L. J., Katsanos, C. S., Coletta, D. K., & Campbell, L. (2015). Skeletal Muscle β-F1-ATPase Translation is Inhibited by Hyperlipidemia-Induced miR-127-5p Expression in Human Obesity. The FASEB Journal, 29(S1). doi:10.1096/fasebj.29.1_supplement.974.6
• Everman, S., Mandarino, L. J., Carroll, C. C., & Katsanos, C. S. (2015). Effects of acute exposure to increased plasma branched-chain amino acid concentrations on insulin-mediated plasma glucose turnover in healthy young subjects. PloS one, 10(3), e0120049.
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  Plasma branched-chain amino acids (BCAA) are inversely related to insulin sensitivity of glucose metabolism in humans. However, currently, it is not known whether there is a cause-and-effect relationship between increased plasma BCAA concentrations and decreased insulin sensitivity.
• Going, S. B., Klimentidis, Y. C., Chen, Z., Raichlen, D. A., Alexander, G. E., Bea, J. W., Mandarino, L. J., Garcia, D. O., Garcia, D. O., Mandarino, L. J., Bea, J. W., Alexander, G. E., Raichlen, D. A., Chen, Z., Klimentidis, Y. C., & Going, S. B. (2017). Genome-wide association study of habitual physical activity in over 277,000 UK Biobank participants indentifies novel variants and genetic correlations with chronotype and obesity related traits. International Journal of Obesity.
• González-García, Z. M., Kullo, I. J., Coletta, D. K., Mandarino, L. J., & Shaibi, G. Q. (2015). Osteocalcin and type 2 diabetes risk in Latinos: a life course approach. American journal of human biology : the official journal of the Human Biology Council, 27(6), 859-61.
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  To examine associations between circulating levels of the bone-derived protein osteocalcin (OC) and type 2 diabetes (T2D) risk in Latino children and adults.
• McLean, C. S., Mielke, C., Cordova, J. M., Langlais, P. R., Bowen, B., Miranda, D., Coletta, D. K., & Mandarino, L. J. (2015). Gene and MicroRNA Expression Responses to Exercise; Relationship with Insulin Sensitivity. PloS one, 10(5), e0127089.
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  Healthy individuals on the lower end of the insulin sensitivity spectrum also have a reduced gene expression response to exercise for specific genes. The goal of this study was to determine the relationship between insulin sensitivity and exercise-induced gene expression in an unbiased, global manner.
• Tran, L., Mandarino, L. J., Lake, D. F., Katsanos, C. S., & Filippis, E. A. (2015). Dyslipidemia Induces Independent Mechanisms Regulating Skeletal Muscle Protein Metabolism and Glucose Balance in Human Obesity. The FASEB Journal, 29(S1). doi:10.1096/fasebj.29.1_supplement.746.3
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  We previously reported perturbed skeletal muscle protein (SMP) metabolism in obese individuals compared to lean controls. Although the precise cause was unclear as obesity is associated with severa...
• Boyle, K. E., Hwang, H., Janssen, R. C., DeVente, J. M., Barbour, L. A., Hernandez, T. L., Mandarino, L. J., Lappas, M., & Friedman, J. E. (2014). Gestational diabetes is characterized by reduced mitochondrial protein expression and altered calcium signaling proteins in skeletal muscle. PloS one, 9(9), e106872.
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  The rising prevalence of gestational diabetes mellitus (GDM) affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM) and obese pregnant women with normal glucose tolerance (ONGT). Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I) subunits (NDUFS3, NDUFV2) and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4) in OGDM (n = 6) vs. ONGT (n = 6). Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (-60-75%) in the OGDM (n = 8) compared with ONGT (n = 10) subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum.
• DeMenna, J., Puppala, S., Chittoor, G., Schneider, J., Kim, J. Y., Shaibi, G. Q., Mandarino, L. J., Duggirala, R., & Coletta, D. K. (2014). Association of common genetic variants with diabetes and metabolic syndrome related traits in the Arizona Insulin Resistance registry: a focus on Mexican American families in the Southwest. Human heredity, 78(1), 47-58.
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  The increased occurrence of type 2 diabetes and its clinical correlates is a global public health issue, and there are continued efforts to find its genetic determinant across ethnically diverse populations. The aims of this study were to determine the heritability of diabetes and metabolic syndrome phenotypes in the Arizona Insulin Resistance (AIR) registry and to perform an association analysis of common single nucleotide polymorphisms (SNPs) identified by GWAS with these traits. All study participants were Mexican Americans from the AIR registry.
• Mandarino, L. J., Coletta, D. K., Miranda, D. N., & Shaibi, G. Q. (2014). Increases in insulin sensitivity among obese youth are associated with gene expression changes in whole blood: Molecular Signatures and Lifestyle in Youth. Obesity, 22(5), 1337-1344. doi:10.1002/oby.20711
• Mielke, C. J., Mandarino, L. J., & Dinu, V. (2014). AMASS: a database for investigating protein structures. Bioinformatics (Oxford, England), 30(11), 1595-600.
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  Modern techniques have produced many sequence annotation databases and protein structure portals, but these Web resources are rarely integrated in ways that permit straightforward exploration of protein functional residues and their co-localization.
• Mielke, C., Lefort, N., McLean, C. G., Cordova, J. M., Langlais, P. R., Bordner, A. J., Te, J. A., Ozkan, S. B., Willis, W. T., & Mandarino, L. J. (2014). Adenine nucleotide translocase is acetylated in vivo in human muscle: Modeling predicts a decreased ADP affinity and altered control of oxidative phosphorylation. Biochemistry, 53(23), 3817-29.
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  Proteomics techniques have revealed that lysine acetylation is abundant in mitochondrial proteins. This study was undertaken (1) to determine the relationship between mitochondrial protein acetylation and insulin sensitivity in human skeletal muscle, identifying key acetylated proteins, and (2) to use molecular modeling techniques to understand the functional consequences of acetylation of adenine nucleotide translocase 1 (ANT1), which we found to be abundantly acetylated. Eight lean and eight obese nondiabetic subjects had euglycemic clamps and muscle biopsies for isolation of mitochondrial proteins and proteomics analysis. A number of acetylated mitochondrial proteins were identified in muscle biopsies. Overall, acetylation of mitochondrial proteins was correlated with insulin action (r = 0.60; P < 0.05). Of the acetylated proteins, ANT1, which catalyzes ADP-ATP exchange across the inner mitochondrial membrane, was acetylated at lysines 10, 23, and 92. The extent of acetylation of lysine 23 decreased following exercise, depending on insulin sensitivity. Molecular dynamics modeling and ensemble docking simulations predicted the ADP binding site of ANT1 to be a pocket of positively charged residues, including lysine 23. Calculated ADP-ANT1 binding affinities were physiologically relevant and predicted substantial reductions in affinity upon acetylation of lysine 23. Insertion of these derived binding affinities as parameters into a complete mathematical description of ANT1 kinetics predicted marked reductions in adenine nucleotide flux resulting from acetylation of lysine 23. Therefore, acetylation of ANT1 could have dramatic physiological effects on ADP-ATP exchange. Dysregulation of acetylation of mitochondrial proteins such as ANT1 therefore could be related to changes in mitochondrial function that are associated with insulin resistance.
• Miranda, D. N., Coletta, D. K., Mandarino, L. J., & Shaibi, G. Q. (2014). Increases in insulin sensitivity among obese youth are associated with gene expression changes in whole blood. Obesity (Silver Spring, Md.), 22(5), 1337-44.
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  Lifestyle intervention can improve insulin sensitivity in obese youth, yet few studies have examined the molecular signatures associated with these improvements. Therefore, the purpose of this study was to explore gene expression changes in whole blood that are associated with intervention-induced improvements in insulin sensitivity.
• Nair, K. S., Abel, E. D., Adler, S. G., Dyck, P. J., Gardner, T. W., Haskins, K. M., Hotamisligil, G., Jensen, M. D., Krook, A., Mandarino, L. J., Mitchell, B. D., Pessin, J. E., Sowers, J. R., Sussel, L., Wareham, N., & Vella, A. (2014). Time to look back and to look forward. Diabetes, 63(4), 1169-70.
• Velgos, S. N., Shaibi, G. Q., Mandarino, L. J., Geda, Y. E., Dawit, S., Champion, M. D., Cevette, N., Caselli, R. J., & Acosta, J. I. (2014). Sanger Sequencing Exons of BDNF Gene Derived from a Latino Sample (P2.144). Neurology, 82.
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  OBJECTIVE: To conduct Sanger sequencing of three targeted exon regions of the Brain Derived Neurotrophic Factor (BDNF) gene within a Latino sample. BACKGROUND: Little is known about BDNF gene sequencing amongst Latinos. DESIGN/METHODS: We obtained blood samples from 357 Latinos (234 females). The mean (SD) age was 41.7 (8.3) and 41.5 (9.4) years for men and women, respectively. Participants included self-identified Latino persons recruited between 2009 and 2011 for a cardiometabolic disease study. Informed consent for future DNA research was obtained. Participants underwent extensive medical evaluation, including a self-administered survey on physical activity. The mean (SD) BMI for those who reported physical exercise was 29.5(4.9), and for those who did not report exercise was 31.9(7.5). In order to determine whether previously identified polymorphisms associated with disease were also carried in the genomes of our population, we conducted Sanger sequencing of three targeted exon regions of the BDNF gene. Custom python scripts were developed to convert raw sequencing reads and quality metrics to trimmed reads which were then aligned to the BDNF reference sequence using the (BWA) package. Polymorphisms were identified and characterized using IGV. RESULTS: A known SNP, (rs2353512) associated with addiction and neuropsychiatric morbidity was found in our sample. Although our sample was overweight or obese, we did not find a well-known polymorphism associated with Obesity (Val66Met [G->A/C->T rs6265 ]). No variations between sequence and reference were observed in region 2 (aln 136-582) or region 3 (aln 25-223). CONCLUSIONS: A well-known polymorphism that is associated with obesity was not observed in our Latino sample, suggesting that sequence variations responsible for the obesity phenotype in our sample are in a region of the gene/genome that we did not sequence. It is also possible that other non-BDNF genomic regions are responsible for the obesity phenotype in our sample. Therefore, future sequencing efforts will focus on whole-genome assessment of our sample. Study Supported by: Mayo Clinic Center for Individualized Medicine Disclosure: Dr. Dawit has nothing to disclose. Dr. Acosta has nothing to disclose. Dr. Champion has nothing to disclose. Dr. Velgos has nothing to disclose. Dr. Shaibi has nothing to disclose. Dr. Mandarino has nothing to disclose. Dr. Cevette has nothing to disclose. Dr. Caselli has received personal compensation in an editorial capacity for Clinical Neurology News. Dr. Geda has nothing to disclose.
• Xie, X., Langlais, P., Zhang, X., Heckmann, B. L., Saarinen, A. M., Mandarino, L. J., & Liu, J. (2014). Identification of a novel phosphorylation site in adipose triglyceride lipase as a regulator of lipid droplet localization. American journal of physiology. Endocrinology and metabolism, 306(12), E1449-59.
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  Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, has long been known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr³⁷² resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. Although it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr³⁷² eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr³⁷² to Ala gave a protein that bound LDs and functioned the same as the wild-type protein. In nonstimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL, whereas the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accord with these findings, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr³⁷² as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL.
• Beeman, S. C., Mandarino, L. J., Georges, J. F., & Bennett, K. M. (2013). Cationized ferritin as a magnetic resonance imaging probe to detect microstructural changes in a rat model of non-alcoholic steatohepatitis. Magnetic resonance in medicine, 70(6), 1728-38.
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  The goal of this work was to detect disease-related microstructural changes to the liver using magnetic resonance imaging. Chronic liver disease can cause microstructural changes in the liver that reduce plasma access to the perisinusoidal space--the site of exchange between the blood plasma and the hepatic parenchyma. The reduced plasma access to the perisinusoidal space inhibits hepatic function and contributes to the ∼30,000 chronic liver disease-related deaths per year.
• Chakkera, H. A., & Mandarino, L. J. (2013). Calcineurin Inhibition and New-Onset Diabetes Mellitus After Transplantation. TRANSPLANTATION, 95(5), 647-652.
• Chakkera, H. A., & Mandarino, L. J. (2013). Calcineurin inhibition and new-onset diabetes mellitus after transplantation. Transplantation, 95(5), 647-52.
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  New-onset diabetes after transplantation independently increases the risk of cardiovascular disease, infections, and graft loss and decreases patient survival. The required balance between insulin sensitivity/resistance and insulin secretion is necessary to maintain normal glucose metabolism. Calcineurin inhibitors are standard immunosuppression drugs used after transplantation and have been implicated in the development of new-onset diabetes after transplantation partially by pancreatic β-cell apoptosis and resultant decrease in insulin secretion. The ability of muscle to take up glucose is critical to blood glucose homeostasis. Skeletal muscle is quantitatively the most important tissue in the body for insulin-stimulated glucose disposal and is composed of diverse myofibers that vary in their properties between healthy and insulin-resistant muscle. Various signaling pathways are responsible for remodeling of skeletal muscle, and among these is the calcineurin/nuclear factor of activated T-cell pathway. The mechanism of action of the calcineurin inhibitors is to bind in a complex with a binding protein to calcineurin and inhibit its dephosphorylation and activation of nuclear factor of activated T cells. In this review, we will provide a detailed discussion of the hypothesis that inhibition of calcineurin in tissues involved in insulin sensitivity/resistance could be at least partially responsible for the diabetogenicity seen with the use of calcineurin inhibitors.
• Hussey, S. E., Sharoff, C. G., Garnham, A., Yi, Z., Bowen, B. P., Mandarino, L. J., & Hargreaves, M. (2013). Effect of exercise on the skeletal muscle proteome in patients with type 2 diabetes. Medicine and science in sports and exercise, 45(6), 1069-76.
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  Exercise training alters protein abundance in the muscle of healthy individuals, but the effect of exercise on these proteins in patients with type 2 diabetes (T2D) is unknown. The aim of this study was to determine how exercise training alters the skeletal muscle proteome in patients with T2D.
• Mandarino, L. J., Vital, V., Coletta, D. K., & Shaibi, G. Q. (2013). The Design and Conduct of a Community-Based Registry and Biorepository: A Focus on Cardiometabolic Health in Latinos: Arizona Insulin Resistance Registry and Repository. Clinical and Translational Science, 6(6), 429-434. doi:10.1111/cts.12114
• Puga, G. M., Meyer, C., Mandarino, L. J., & Katsanos, C. S. (2013). Increased plasma availability of L-arginine in the postprandial period decreases the postprandial lipemia in older adults. Nutrition (Burbank, Los Angeles County, Calif.), 29(1), 81-8.
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  Older adults have exaggerated postprandial lipemia, which increases their risk for cardiovascular disease. We sought to determine the effects of increased plasma L-arginine (L-ARG) availability on the oxidation of ingested fat (enriched with [1,1,1-(13)C]-triolein) and plasma triacylglycerol (TG) concentrations during the postprandial period in older subjects.
• Shaibi, G. Q., Coletta, D. K., Vital, V., & Mandarino, L. J. (2013). The design and conduct of a community-based registry and biorepository: a focus on cardiometabolic health in Latinos. Clinical and translational science, 6(6), 429-34.
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  Latinos are disproportionately impacted by obesity and type 2 diabetes but remain underrepresented in biomedical research. Therefore, the purpose of this project was to develop a research registry and biorepository to examine cardiometabolic disease risk in the Latino community of Phoenix, Arizona. The overarching goal was to establish the research infrastructure that would encourage transdisciplinary research regarding the biocultural mechanisms of obesity-related health disparities and facilitate access to this research for the Latino community.
• Tangen, S. E., Tsinajinnie, D., Nuñez, M., Shaibi, G. Q., Mandarino, L. J., & Coletta, D. K. (2013). Whole blood gene expression profiles in insulin resistant Latinos with the metabolic syndrome. PloS one, 8(12), e84002.
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  Although insulin resistance in skeletal muscle is well-characterized, the role of circulating whole blood in the metabolic syndrome phenotype is not well understood. We set out to test the hypothesis that genes involved in inflammation, insulin signaling and mitochondrial function would be altered in expression in the whole blood of individuals with metabolic syndrome. We further wanted to examine whether similar relationships that we have found previously in skeletal muscle exist in peripheral whole blood cells. All subjects (n=184) were Latino descent from the Arizona Insulin Resistance registry. Subjects were classified based on the metabolic syndrome phenotype according to the National Cholesterol Education Program's Adult Treatment Panel III. Of the 184 Latino subjects in the study, 74 were classified with the metabolic syndrome and 110 were without. Whole blood gene expression profiling was performed using the Agilent 4x44K Whole Human Genome Microarray. Whole blood microarray analysis identified 1,432 probes that were altered in expression ≥ 1.2 fold and P
• Chao, A., Zhang, X., Ma, D., Langlais, P., Luo, M., Mandarino, L. J., Zingsheim, M., Pham, K., Dillon, J., & Yi, Z. (2012). Site-specific phosphorylation of protein phosphatase 1 regulatory subunit 12A stimulated or suppressed by insulin. Journal of proteomics, 75(11), 3342-50.
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  Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. So far, only few specific phosphorylation sites of PP1 regulatory subunit 12A (PPP1R12A) have been shown to regulate the PP1 activity. The effect of insulin on PPP1R12A phosphorylation is largely unknown. Utilizing a mass spectrometry based phosphorylation identification and quantification approach, we identified 21 PPP1R12A phosphorylation sites (7 novel sites, including Ser20, Thr22, Thr453, Ser478, Thr671, Ser678, and Ser680) and quantified 16 of them under basal and insulin stimulated conditions in hamster ovary cells overexpressing the insulin receptor (CHO/IR), an insulin sensitive cell model. Insulin stimulated the phosphorylation of PPP1R12A significantly at Ser477, Ser478, Ser507, Ser668, and Ser695, while simultaneously suppressing the phosphorylation of PPP1R12A at Ser509 (more than 2-fold increase or decrease compared to basal). Our data demonstrate that PPP1R12A undergoes insulin stimulated/suppressed phosphorylation, suggesting that PPP1R12A phosphorylation may play a role in insulin signal transduction. The novel PPP1R12A phosphorylation sites as well as the new insulin-responsive phosphorylation sites of PPP1R12A in CHO/IR cells provide targets for investigation of the regulation of PPP1R12A and the PPP1R12A-PP1cδ complex in insulin action and other signaling pathways in other cell models, animal models, and humans.
• Kim, J. Y., Coletta, D. K., Mandarino, L. J., & Shaibi, G. Q. (2012). Glucose Response Curve and Type 2 Diabetes Risk in Latino Adolescents. DIABETES CARE, 35(9), 1925-1930.
• Kim, J. Y., Coletta, D. K., Mandarino, L. J., & Shaibi, G. Q. (2012). Glucose response curve and type 2 diabetes risk in Latino adolescents. Diabetes care, 35(9), 1925-30.
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  In adults, the shape of the glucose response during an oral glucose tolerance test (OGTT) prospectively and independently predicts type 2 diabetes. However, no reports have described the utility of this indicator in younger populations. The purpose of this study was to compare type 2 diabetes risk factors in Latino adolescents characterized by either a monophasic or biphasic glucose response during an OGTT.
• Langlais, P., Dillon, J. L., Mengos, A., Baluch, D. P., Ardebili, R., Miranda, D. N., Xie, X., Heckmann, B. L., Liu, J., & Mandarino, L. J. (2012). Identification of a role for CLASP2 in insulin action. The Journal of biological chemistry, 287(46), 39245-53.
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  Insulin stimulates the mobilization of glucose transporter 4 (GLUT4) storage vesicles to the plasma membrane, resulting in an influx of glucose into target tissues such as muscle and fat. We present evidence that CLIP-associating protein 2 (CLASP2), a protein previously unassociated with insulin action, is responsive to insulin stimulation. Using mass spectrometry-based protein identification combined with phosphoantibody immunoprecipitation in L6 myotubes, we detected a 4.8-fold increase of CLASP2 in the anti-phosphoserine immunoprecipitates upon insulin stimulation. Western blotting of CLASP2 immunoprecipitates with the phosphoantibody confirmed the finding that CLASP2 undergoes insulin-stimulated phosphorylation, and a number of novel phosphorylation sites were identified. Confocal imaging of L6 myotubes revealed that CLASP2 colocalizes with GLUT4 at the plasma membrane within areas of insulin-mediated cortical actin remodeling. CLASP2 is responsible for directing the distal end of microtubules to the cell cortex, and it has been shown that GLUT4 travels along microtubule tracks. In support of the concept that CLASP2 plays a role in the trafficking of GLUT4 at the cell periphery, CLASP2 knockdown by siRNA in L6 myotubes interfered with insulin-stimulated GLUT4 localization to the plasma membrane. Furthermore, siRNA mediated knockdown of CLASP2 in 3T3-L1 adipocytes inhibited insulin-stimulated glucose transport. We therefore propose a new model for CLASP2 in insulin action, where CLASP2 directs the delivery of GLUT4 to cell cortex landing zones important for insulin action.
• Luo, M., Mengos, A. E., Stubblefield, T. M., & Mandarino, L. J. (2012). High Fat Diet-Induced Changes in Hepatic Protein Abundance in Mice. Journal of proteomics & bioinformatics, 5(3), 60-66.
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  Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance, type 2 diabetes, and dyslipidemia. The purpose of this study was to identify novel proteins and pathways that contribute to the pathogenesis and complications of NAFLD. C57BL/6J male mice were fed a 60% (HFD) or 10% (LFD) high or low fat diet. HFD induced obesity, hepatic steatosis and insulin resistance (euglycemic clamps, glucose infusion rate: LFD 50.5 ± 6.4 vs. HFD 14.2 ± 9.5 μg/ (g·min); = 12). Liver proteins were analyzed by mass spectrometry-based proteomics analysis. Numerous hepatic proteins were altered in abundance after 60% HFD feeding. Nine down-regulated and nine up-regulated proteins were selected from this list for detailed analysis based on the criteria of 1.5-fold difference, consistency across replicates, and having at least 2 spectra assigned. Proteins that decreased in abundance were acyl-coA desaturase-I (SCD-1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), pyruvate kinase isozymes R/L (PKLR), NADP-dependent malic enzyme (ME-1), ATP-citrate synthase (ACL), ketohexokinase (KHK), long-chain-fatty acid-CoA ligase-5 (ACSL-5) and carbamoyl-phosphate synthase-I (CPS-1). Those that increased were KIAA0564, apolipoprotein A-I (apoA-1), ornithine aminotransferase (OAT), multidrug resistance protein 2 (MRP-2), liver carboxylesterase-I (CES-1), aminopeptidase N (APN), fatty aldehyde dehydrogenase (FALDH), major urinary protein 2 (MUP-2) and KIAA0664. KIAA0564 and KIAA0664 proteins are uncharacterized and are novel proteins associated with NAFLD. The decreased abundance of normally highly abundant proteins like FAS and CPS-1 was confirmed by Coomassie Blue staining after bands were identified by MS/MS, and immunoblot analysis confirmed the increased abundance of KIAA0664 after 60% HFD feeding. In conclusion, this study shows NAFLD is characterized by changes in abundance of proteins related to cell injury, inflammation, and lipid metabolism. Two novel and uncharacterized proteins, KIAA0564 and KIAA0664, may provide insight into the pathogenesis of NAFLD induced by lipid oversupply.
• Mandarino, L. J., Puga, G. M., Meyer, C., & Katsanos, C. S. (2012). Postprandial Spillover of Dietary Lipid into Plasma Is Increased with Moderate Amounts of Ingested Fat and Is Inversely Related to Adiposity in Healthy Older Men. The Journal of Nutrition, 142(10), 1806-1811. doi:10.3945/jn.112.162008
• Puga, G. M., Meyer, C., Mandarino, L. J., & Katsanos, C. S. (2012). Postprandial spillover of dietary lipid into plasma is increased with moderate amounts of ingested fat and is inversely related to adiposity in healthy older men. The Journal of nutrition, 142(10), 1806-11.
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  Adverse effects on health mediated by increased plasma FFA concentrations are well established and older individuals are particularly susceptible to these effects. We sought to determine the effects of the amount of dietary fat on increasing the plasma FFA concentrations as a result of "spillover" of dietary fat into the plasma FFA pool during the postprandial period in older men. Healthy, older participants (63-71 y old) were studied in a randomized, crossover design following ingestions of low (LF) and moderate (MF) amounts of [1,1,1-(13)C]-triolein-labeled fat, corresponding to 0.4 and 0.7 g of fat/kg body weight, respectively. Spillover of dietary fatty acids into plasma during the 8-h postprandial period (AUC; mmol L(-1) h) after MF ingestion was 1.2 times greater than that after LF ingestion (2.8 ± 0.4 vs. 1.2 ± 0.1; P < 0.05). The spillover of dietary fatty acids following the MF, but not the LF, ingestion was correlated with the percent body fat (r(s) = -0.89) and percent body fat-free mass (r(s) = 0.94) of the men (P < 0.05). After adjusting to the amount of ingested fat, the spillover of dietary fatty acids in the MF trial was disproportionally higher than that in the LF trial (P < 0.05), but the corresponding postprandial plasma TG responses did not differ between trials. In conclusion, spillover of dietary lipid into plasma is disproportionally increased at higher doses of dietary fat and this response is inversely related to adiposity in healthy men of advanced age.
• Coletta, D. K., & Mandarino, L. J. (2011). Mitochondrial dysfunction and insulin resistance from the outside in: extracellular matrix, the cytoskeleton, and mitochondria. AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM, 301(5), E749-E755.
• Coletta, D. K., & Mandarino, L. J. (2011). Mitochondrial dysfunction and insulin resistance from the outside in: extracellular matrix, the cytoskeleton, and mitochondria. American journal of physiology. Endocrinology and metabolism, 301(5), E749-55.
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  Insulin resistance in skeletal muscle is a prominent feature of obesity and type 2 diabetes. The association between mitochondrial changes and insulin resistance is well known. More recently, there is growing evidence of a relationship between inflammation, extracellular remodeling, and insulin resistance. The intent of this review is to propose a potentially novel mechanism for the development of insulin resistance, focusing on the underappreciated connections among inflammation, extracellular remodeling, cytoskeletal interactions, mitochondrial function, and insulin resistance in human skeletal muscle. Several sources of inflammation, including expansion of adipose tissue resulting in increased lipolysis and alterations in pro- and anti-inflammatory cytokines, contribute to the insulin resistance observed in obesity and type 2 diabetes. In the experimental model of lipid oversupply, an inflammatory response in skeletal muscle leads to altered expression extracellular matrix-related genes as well as nuclear encoded mitochondrial genes. A similar pattern also is observed in "naturally" occurring insulin resistance in muscle of obese nondiabetic individuals and patients with type 2 diabetes mellitus. More recently, alterations in proteins (including α-actinin-2, desmin, proteasomes, and chaperones) involved in muscle structure and function have been observed in insulin-resistant muscle. Some of these cytoskeletal proteins are mechanosignal transducers that allow muscle fibers to sense contractile activity and respond appropriately. The ensuing alterations in expression of genes coding for mitochondrial proteins and cytoskeletal proteins may contribute to the mitochondrial changes observed in insulin-resistant muscle. These changes in turn may lead to a reduction in fat oxidation and an increase in intramyocellular lipid, which contributes to the defects in insulin signaling in insulin resistance.
• Everman, S., Yi, Z., Langlais, P., Mandarino, L. J., Luo, M., Roberts, C., & Katsanos, C. S. (2011). Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit. PloS one, 6(10), e26171.
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  We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R(2) = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase(134-143) peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase(134-143) peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R(2) = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase(134-143) peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase(134-143) peptide.
• Geetha, T., Langlais, P., Luo, M., Mapes, R., Lefort, N., Chen, S. C., Mandarino, L. J., & Yi, Z. (2011). Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1. Journal of the American Society for Mass Spectrometry, 22(3), 457-66.
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  Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.
• Ghosh, S., Lertwattanarak, R., Lefort, N., Molina-Carrion, M., Joya-Galeana, J., Bowen, B. P., Garduno-Garcia, J. d., Abdul-Ghani, M., Richardson, A., DeFronzo, R. A., Mandarino, L., Van Remmen, H., & Musi, N. (2011). Reduction in reactive oxygen species production by mitochondria from elderly subjects with normal and impaired glucose tolerance. Diabetes, 60(8), 2051-60.
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  Aging increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes. It has been proposed that increased reactive oxygen species (ROS) generation by dysfunctional mitochondria could play a role in the pathogenesis of these metabolic abnormalities. We examined whether aging per se (in subjects with normal glucose tolerance [NGT]) impairs mitochondrial function and how this relates to ROS generation, whether older subjects with IGT have a further worsening of mitochondrial function (lower ATP production and elevated ROS generation), and whether exercise reverses age-related changes in mitochondrial function.
• Ghosh, S., Lertwattanarak, R., Lefort, N., Molina-Carrion, M., Joya-Galeana, J., Bowen, B. P., Garduno-Garcia, J., Abdul-Ghani, M., Richardson, A., DeFronzo, R. A., Mandarino, L., Van, R. H., & Musi, N. (2011). Reduction in Reactive Oxygen Species Production by Mitochondria From Elderly Subjects With Normal and Impaired Glucose Tolerance. DIABETES, 60(8), 2051-2060.
• Katsanos, C. S., & Mandarino, L. J. (2011). Protein Metabolism in Human Obesity: A Shift in Focus From Whole-Body to Skeletal Muscle. OBESITY, 19(3), 469-475.
• Katsanos, C. S., & Mandarino, L. J. (2011). Protein metabolism in human obesity: a shift in focus from whole-body to skeletal muscle. Obesity (Silver Spring, Md.), 19(3), 469-75.
• Langlais, P., Yi, Z., & Mandarino, L. J. (2011). The identification of raptor as a substrate for p44/42 MAPK. Endocrinology, 152(4), 1264-73.
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  The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.
• Langlais, P., Yi, Z., Tongchinsub, P., Plummer, E., Meyer, C., Mattern, M., Mapes, R., Mandarino, L. J., Luo, M., Langlais, P. R., Finlayson, J., & Filippis, E. D. (2011). Global IRS-1 phosphorylation analysis in insulin resistance.. Diabetologia, 54(11), 2878-89. doi:10.1007/s00125-011-2271-9
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  IRS-1 serine phosphorylation is often elevated in insulin resistance models, but confirmation in vivo in humans is lacking. We therefore analysed IRS-1 phosphorylation in human muscle in vivo..We used HPLC-electrospray ionisation (ESI)-MS/MS to quantify IRS-1 phosphorylation basally and after insulin infusion in vastus lateralis muscle from lean healthy, obese non-diabetic and type 2 diabetic volunteers..Basal Ser323 phosphorylation was increased in type 2 diabetic patients (2.1 ± 0.43, p ≤ 0.05, fold change vs lean controls). Thr495 phosphorylation was decreased in type 2 diabetic patients (p ≤ 0.05). Insulin increased IRS-1 phosphorylation at Ser527 (1.4 ± 0.17, p ≤ 0.01, fold change, 60 min after insulin infusion vs basal) and Ser531 (1.3 ± 0.16, p ≤ 0.01, fold change, 60 min after insulin infusion vs basal) in the lean controls and suppressed phosphorylation at Ser348 (0.56 ± 0.11, p ≤ 0.01, fold change, 240 min after insulin infusion vs basal), Thr446 (0.64 ± 0.16, p ≤ 0.05, fold change, 60 min after insulin infusion vs basal), Ser1100 (0.77 ± 0.22, p ≤ 0.05, fold change, 240 min after insulin infusion vs basal) and Ser1142 (1.3 ± 0.2, p ≤ 0.05, fold change, 60 min after insulin infusion vs basal)..We conclude that, unlike some aspects of insulin signalling, the ability of insulin to increase or suppress certain IRS-1 phosphorylation sites is intact in insulin resistance. However, some IRS-1 phosphorylation sites do not respond to insulin, whereas other Ser/Thr phosphorylation sites are either increased or decreased in insulin resistance.
• Mandarino, L. J., & Coletta, D. K. (2011). Mitochondrial dysfunction and insulin resistance from the outside in: extracellular matrix, the cytoskeleton, and mitochondria. American Journal of Physiology-Endocrinology and Metabolism, 301(5), E749-E755. doi:10.1152/ajpendo.00363.2011
• Mandarino, L. J., Langlais, P., & Yi, Z. (2011). The Identification of Raptor as a Substrate for p44/42 MAPK. Endocrinology, 152(4), 1264-1273. doi:10.1210/en.2010-1271
• Mandarino, L. J., Langlais, P., Geetha, T., Luo, M., Mapes, R., Lefort, N., Chen, S., & Yi, Z. (2011). Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1. Journal of the American Society for Mass Spectrometry, 22(3), 457-466. doi:10.1007/s13361-010-0051-2
• Mandarino, L., Ghosh, S., Lertwattanarak, R., Lefort, N., Molina-Carrion, M., Joya-Galeana, J., Bowen, B. P., Garduno-Garcia, J. d., Abdul-Ghani, M., Richardson, A., DeFronzo, R. A., Van Remmen, H., & Musi, N. (2011). Reduction in Reactive Oxygen Species Production by Mitochondria From Elderly Subjects With Normal and Impaired Glucose Tolerance. Diabetes, 60(8), 2051-2060. doi:10.2337/db11-0121
• Puga, G. M., Meyer, C., Everman, S., Mandarino, L. J., & Katsanos, C. S. (2011). Postprandial lipemia in the elderly involves increased incorporation of ingested fat in plasma free fatty acids and small (Sf 20-400) triglyceride-rich lipoproteins. American journal of physiology. Endocrinology and metabolism, 301(2), E356-61.
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  In the elderly, the rise in postprandial plasma triglyceride (TG) concentrations is increased, contributing to their increased risk of cardiovascular disease. We sought to determine the incorporation of ingested fat (whipping cream enriched with [1,1,1-(13)C]triolein) into plasma lipids during the postprandial period in six healthy elderly (67 ± 1 yr old) and six healthy young (23 ± 2 yr old) subjects. Blood and expired air samples were taken before and at 2-h intervals during the 8-h postprandial period. As expected, the area under the curve of postprandial plasma TG concentrations was larger in the elderly compared with the young subjects (152 ± 38 vs. 66 ± 27 mg·dl(-1)·h, P < 0.05). The incorporation of [(13)C]oleate in plasma free fatty acids (FFAs) and TG of the small (S(f) = 20-400) triglyceride-rich lipoprotein (TRL) fraction was significantly higher in the elderly compared with the young subjects, resulting in increased postprandial contributions of the ingested lipid to plasma FFAs (41 ± 3 vs. 26 ± 6%, P < 0.05) and the small TRL fraction (36 ± 5 vs. 21 ± 3%, P < 0.05) in elderly. Plasma apoB-100 concentration was higher, whereas the rate of oxidation of the ingested lipid was lower (P < 0.05) in the elderly. We conclude that increased postprandial lipemia in the elderly involves increased contribution of ingested lipid to the plasma small TRLs. This appears to be driven at least in part by increased appearance of the ingested fat as plasma FFA and increased availability of apo B-100 lipoproteins in the elderly.
• Wasserman, D. H., Mcguinness, O. P., Mandarino, L. J., Halseth, A. E., & Bonadonna, R. C. (2011). Regulation of Muscle Glucose Uptake In Vivo. Comprehensive Physiology, 803-845. doi:10.1002/cphy.cp070227
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  The sections in this article are: 1 Methodologies for Measurement of Skeletal Muscle Glucose Uptake 1.1 Theories of Metabolic Control 1.2 Kinetic Theory of Intact Organs 1.3 Arteriovenous Differences 1.4 Muscle Biopsies 1.5 Positron Emission Tomography 1.6 Nuclear Magnetic Resonance Spectroscopy 1.7 Multiple Tracer Dilution Technique 1.8 Measurement of Whole Body Glucose Metabolism 2 Mechanism of Insulin Action at Skeletal Muscle 2.1 Insulin Signaling In Vivo 2.2 Glucose Transport and Phosphorylation in Skeletal Muscle 2.3 Glycogen Synthesis, Glycolysis, and Glucose Oxidation in Skeletal Muscle 2.4 Effect of Insulin on Blood Flow 2.5 Transport of Insulin Across the Endothelial Barrier Time-Limiting Steps in Insulin Action 2.6 Modulation of Insulin-Stimulated Skeletal Muscle Glucose Uptake by Nonesterified Fatty Acid Availability 3 Exercise and Muscle Glucose Uptake 3.1 Mechanism of the Exercise-Induced Increase in Muscle Glucose Uptake 3.2 Modulation of Glucose Uptake by the Working Muscle by the Internal Milieu 3.3 Muscle Glucose Uptake in the Postexercise State 4 Conclusion
• Yi, Z., Sherman, V., Shahani, S., Sathyanarayana, P., Meyer, C., Mandarino, L. J., Hwang, H., Holmes, R. M., Fujiwara, K., Finlayson, J., Filippis, E. D., Dong, L. Q., Christ-roberts, C., Berria, R., & Bajaj, M. (2011). Increased abundance of the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1) in patients with obesity and type 2 diabetes: evidence for altered adiponectin signalling.. Diabetologia, 54(8), 2122-31. doi:10.1007/s00125-011-2173-x
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  The adiponectin signalling pathway is largely unknown, but recently the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1), has been shown to interact directly with adiponectin receptor (ADIPOR)1. APPL1 is present in C2C12 myoblasts and mouse skeletal muscle, but its presence in human skeletal muscle has not been investigated..Samples from type 2 diabetic, and lean and non-diabetic obese participants were analysed by: immunoprecipitation and western blot; HPLC-electrospray ionisation (ESI)-mass spectrometry (MS) analysis; peak area analysis by MS; HPLC-ESI-MS/MS/MS analysis; and RT-PCR analysis of APPL1 mRNA..Immunoprecipitation and western blot indicated a band specific to APPL1. Tryptic digestion and HPLC-ESI-MS analysis of whole-muscle homogenate APPL1 unambiguously identified APPL1 with 56% sequence coverage. Peak area analysis by MS validated western blot results, showing APPL1 levels to be significantly increased in type 2 diabetic and obese as compared with lean participants. Targeted phosphopeptide analysis by HPLC-ESI-MS/MS/MS showed that APPL1 was phosphorylated specifically on Ser(401). APPL1 mRNA expression was significantly increased in obese and type 2 diabetic participants as compared with lean participants. After bariatric surgery in morbidly obese participants with subsequent weight loss, skeletal muscle APPL1 abundance was significantly reduced (p 
• Civitarese, A. E., MacLean, P. S., Carling, S., Kerr-Bayles, L., McMillan, R. P., Pierce, A., Becker, T. C., Moro, C., Finlayson, J., Lefort, N., Newgard, C. B., Mandarino, L., Cefalu, W., Walder, K., Collier, G. R., Hulver, M. W., Smith, S. R., & Ravussin, E. (2010). Regulation of Skeletal Muscle Oxidative Capacity and Insulin Signaling by the Mitochondria! Rhomboid Protease PARL. CELL METABOLISM, 11(5), 412-426.
• Civitarese, A. E., MacLean, P. S., Carling, S., Kerr-Bayles, L., McMillan, R. P., Pierce, A., Becker, T. C., Moro, C., Finlayson, J., Lefort, N., Newgard, C. B., Mandarino, L., Cefalu, W., Walder, K., Collier, G. R., Hulver, M. W., Smith, S. R., & Ravussin, E. (2010). Regulation of skeletal muscle oxidative capacity and insulin signaling by the mitochondrial rhomboid protease PARL. Cell metabolism, 11(5), 412-26.
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  Type 2 diabetes mellitus (T2DM) and aging are characterized by insulin resistance and impaired mitochondrial energetics. In lower organisms, remodeling by the protease pcp1 (PARL ortholog) maintains the function and lifecycle of mitochondria. We examined whether variation in PARL protein content is associated with mitochondrial abnormalities and insulin resistance. PARL mRNA and mitochondrial mass were both reduced in elderly subjects and in subjects with T2DM. Muscle knockdown of PARL in mice resulted in malformed mitochondrial cristae, lower mitochondrial content, decreased PGC1alpha protein levels, and impaired insulin signaling. Suppression of PARL protein in healthy myotubes lowered mitochondrial mass and insulin-stimulated glycogen synthesis and increased reactive oxygen species production. We propose that lower PARL expression may contribute to the mitochondrial abnormalities seen in aging and T2DM.
• Hwang, H., Bowen, B. P., Lefort, N., Flynn, C. R., De Filippis, E. A., Roberts, C., Smoke, C. C., Meyer, C., Højlund, K., Yi, Z., & Mandarino, L. J. (2010). Proteomics analysis of human skeletal muscle reveals novel abnormalities in obesity and type 2 diabetes. Diabetes, 59(1), 33-42.
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  Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here.
• Hwang, H., Bowen, B. P., Lefort, N., Flynn, C. R., De, F., Roberts, C., Smoke, C. C., Meyer, C., Hojlund, K., Yi, Z., & Mandarino, L. J. (2010). Proteomics Analysis of Human Skeletal Muscle Reveals Novel Abnormalities in Obesity and Type 2 Diabetes. DIABETES, 59(1), 33-42.
• Kashyap, S. R., Roman, L. J., Mandarino, L., DeFronzo, R., & Bajaj, M. (2010). Hypoadiponectinemia is closely associated with impaired nitric oxide synthase activity in skeletal muscle of type 2 diabetic subjects. Metabolic syndrome and related disorders, 8(5), 459-63.
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  In vitro studies suggest that adiponectin plays an important role in nitric oxide (NO) generation. We studied the relationship between plasma adiponectin and skeletal muscle nitric oxide synthase (NOS) activity in type 2 diabetic (T2DM) patients.
• Langlais, P., Mandarino, L. J., & Yi, Z. (2010). Label-free Relative Quantification of Co-eluting Isobaric Phosphopeptides of Insulin Receptor Substrate-1 by HPLC-ESI-MS/MS. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 21(9), 1490-1499.
• Langlais, P., Mandarino, L. J., & Yi, Z. (2010). Label-free relative quantification of co-eluting isobaric phosphopeptides of insulin receptor substrate-1 by HPLC-ESI-MS/MS. Journal of the American Society for Mass Spectrometry, 21(9), 1490-9.
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  Intracellular signal transduction is often regulated by transient protein phosphorylation in response to external stimuli. Insulin signaling is dependent on specific protein phosphorylation events, and analysis of insulin receptor substrate-1 (IRS-1) phosphorylation reveals a complex interplay between tyrosine, serine, and threonine phosphorylation. The phospho-specific antibody-based quantification approach for analyzing changes in site-specific phosphorylation of IRS-1 is difficult due to the dearth of phospho-antibodies compared with the large number of known IRS-1 phosphorylation sites. We previously published a method detailing a peak area-based mass spectrometry approach, using precursor ions for peptides, to quantify the relative abundance of site-specific phosphorylation in the absence or presence of insulin. We now present an improvement wherein site-specific phosphorylation is quantified by determining the peak area of fragment ions respective to the phospho-site of interest. This provides the advantage of being able to quantify co-eluting isobaric phosphopeptides (differentially phosphorylated versions of the same peptide), allowing for a more comprehensive analysis of protein phosphorylation. Quantifying human IRS-1 phosphorylation sites at Ser303, Ser323, Ser330, Ser348, Ser527, and Ser531 shows that this method is linear (n = 3; r(2) = 0.85 +/- 0.05, 0.96 +/- 0.01, 0.96 +/- 0.02, 0.86 +/- 0.07, 0.90 +/- 0.03, 0.91 +/- 0.04, respectively) over an approximate 10-fold range of concentrations and reproducible (n = 4; coefficient of variation = 0.12, 0.14, 0.29, 0.30, 0.12, 0.06, respectively). This application of label-free, fragment ion-based quantification to assess relative phosphorylation changes of specific proteins will prove useful for understanding how various cell stimuli regulate protein function by phosphorylation.
• Langlais, P., Yi, Z., Mandarino, L. J., Levin, K., Lefort, N., Langlais, P. R., Højlund, K., Bowen, B. P., & Beck-nielsen, H. (2010). Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle.. Diabetologia, 53(3), 541-51. doi:10.1007/s00125-009-1624-0
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  Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-beta) and determine protein abundance of ATPsyn-beta and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals..Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n = 10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-beta using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps..Seven phosphorylation sites were identified on ATPsyn-beta purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a approximately 30% higher phosphorylation of ATPsyn-beta at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-beta protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-beta by approximately 50% in lean and healthy, but not insulin-resistant, individuals..These data demonstrate that ATPsyn-beta is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-beta together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-beta may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.
• Lefort, N., Glancy, B., Bowen, B., Willis, W. T., Bailowitz, Z., De Filippis, E. A., Brophy, C., Meyer, C., Højlund, K., Yi, Z., & Mandarino, L. J. (2010). Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle. Diabetes, 59(10), 2444-52.
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  The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle.
• Lefort, N., Glancy, B., Bowen, B., Willis, W. T., Bailowitz, Z., De, F., Brophy, C., Meyer, C., Hojlund, K., Yi, Z., & Mandarino, L. J. (2010). Increased Reactive Oxygen Species Production and Lower Abundance of Complex I Subunits and Carnitine Palmitoyltransferase 1B Protein Despite Normal Mitochondrial Respiration in Insulin-Resistant Human Skeletal Muscle. DIABETES, 59(10), 2444-2452.
• Mandarino, L. J., Everman, S., Puga, G. M., Meyer, C., & Katsanos, C. S. (2010). Effects of branched chain amino acids infusion on whole‐body plasma glucose disposal: a pilot study. The FASEB Journal, 24(S1). doi:10.1096/fasebj.24.1_supplement.783.6
• Mandarino, L. J., Willis, W. T., Lefort, N., Glancy, B., Bowen, B., Bailowitz, Z., De Filippis, E. A., Brophy, C., Meyer, C., Højlund, K., & Yi, Z. (2010). Increased Reactive Oxygen Species Production and Lower Abundance of Complex I Subunits and Carnitine Palmitoyltransferase 1B Protein Despite Normal Mitochondrial Respiration in Insulin-Resistant Human Skeletal Muscle. Diabetes, 59(10), 2444-2452. doi:10.2337/db10-0174
• Puga, G. M., Meyer, C., Mandarino, L. J., Katsanos, C. S., & Everman, S. (2010). Effects of branched chain amino acids infusion on whole-body plasma glucose disposal: a pilot study. The FASEB Journal, 24.
• Xie, X., Yi, Z., Bowen, B., Wolf, C., Flynn, C. R., Sinha, S., Mandarino, L. J., & Meyer, C. (2010). Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-Dimensional Gel Electrophoresis and HPLC-ESI-MS/MS. Journal of proteome research, 9(9), 4521-34.
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  Abnormalities in adipocytes play an important role in various conditions, including the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, but little is known about alterations at the protein level. We therefore sought to (1) comprehensively characterize the human adipocyte proteome for the first time and (2) demonstrate feasibility of measuring adipocyte protein abundances by one-dimensional SDS-PAGE and high performance liquid chromatography-electron spray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). In adipocytes isolated from approximately 0.5 g of subcutaneous abdominal adipose tissue of three healthy, lean subjects, we identified a total of 1493 proteins. Triplicate analysis indicated a 22.5% coefficient of variation of protein abundances. Proteins ranged from 5.8 to 629 kDa and included a large number of proteins involved in lipid metabolism, such as fatty acid transport, fatty acid oxidation, lipid storage, lipolysis, and lipid droplet maintenance. Furthermore, we found most glycolysis enzymes and numerous proteins associated with oxidative stress, protein synthesis and degradation as well as some adipokines. 22% of all proteins were of mitochondrial origin. These results provide the first detailed characterization of the human adipocyte proteome, suggest an important role of adipocyte mitochondria, and demonstrate feasibility of this approach to examine alterations of adipocyte protein abundances in human diseases.
• Yi, Z., Sharoff, C. G., Mandarino, L. J., Hussey, S. E., Hargreaves, M., Garnham, A., & Bowen, B. P. (2010). Proteomics Analysis Following Exercise Training in Individuals with T2D Reveals Novel Skeletal Muscle Adaptations. Medicine and Science in Sports and Exercise, 42(10), 16-17. doi:10.1249/01.mss.0000389512.52777.6a
• Hojlund, K., Bowen, B. P., Hwang, H., Flynn, C. R., Madireddy, L., Geetha, T., Langlais, P., Meyer, C., Mandarino, L. J., & Yi, Z. (2009). In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS. JOURNAL OF PROTEOME RESEARCH, 8(11), 4954-4965.
• Højlund, K., Bowen, B. P., Hwang, H., Flynn, C. R., Madireddy, L., Geetha, T., Langlais, P., Meyer, C., Mandarino, L. J., & Yi, Z. (2009). In vivo phosphoproteome of human skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS. Journal of proteome research, 8(11), 4954-65.
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  Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin-resistant muscle using phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO(2). The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines, and 13 phosphotyrosines in at least 2 out of 3 subjects. In addition, 61 ambiguous phosphorylation sites were identified in at least 2 out of 3 subjects. The majority of phosphoproteins detected are involved in sarcomeric function, excitation-contraction coupling (the Ca(2+)-cycle), glycolysis, and glycogen metabolism. Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disk proteins known to be involved in signaling and muscle disorders. These results provide numerous new targets for the investigation of human skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo.
• Lefort, N., Yi, Z., Bowen, B., Glancy, B., De Filippis, E. A., Mapes, R., Hwang, H., Flynn, C. R., Willis, W. T., Civitarese, A., Højlund, K., & Mandarino, L. J. (2009). Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. Journal of proteomics, 72(6), 1046-60.
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  Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small samples of healthy and diseased skeletal muscle.
• Lefort, N., Yi, Z., Bowen, B., Glancy, B., De, F., Mapes, R., Hwang, H., Flynn, C. R., Willis, W. T., Civitarese, A., Hojlund, K., & Mandarino, L. J. (2009). Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. JOURNAL OF PROTEOMICS, 72(6), 1046-1060.
• Mandarino, L. J., Hwang, H., Bowen, B. P., Lefort, N., Flynn, C. R., De Filippis, E. A., Roberts, C., Smoke, C. C., Meyer, C., Højlund, K., & Yi, Z. (2009). Proteomics Analysis of Human Skeletal Muscle Reveals Novel Abnormalities in Obesity and Type 2 Diabetes. Diabetes, 59(1), 33-42. doi:10.2337/db09-0214
• Mandarino, L. J., Langlais, P., Madireddy, L., Højlund, K., Bowen, B. P., Hwang, H., Flynn, C. R., Geetha, T., Meyer, C., & Yi, Z. (2009). In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC−ESI−MS/MS. Journal of Proteome Research, 8(11), 4954-4965. doi:10.1021/pr9007267
• Puga, G. M., Meyer, C., Mandarino, L. J., Katsanos, C. S., Everman, S., & Alexander, J. (2009). L-Arginine infusion attenuates postprandial lipemia in healthy elderly. The FASEB Journal, 23.
• Coletta, D. K., Balas, B., Chavez, A. O., Baig, M., Abdul-Ghani, M., Kashyap, S. R., Folli, F., Tripathy, D., Mandarino, L. J., Cornell, J. E., DeFronzo, R. A., & Jenkinson, C. P. (2008). Effect of acute physiological hyperinsulinemia on gene expression in human skeletal muscle in vivo. AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM, 294(5), E910-E917.
• Coletta, D. K., Balas, B., Chavez, A. O., Baig, M., Abdul-Ghani, M., Kashyap, S. R., Folli, F., Tripathy, D., Mandarino, L. J., Cornell, J. E., Defronzo, R. A., & Jenkinson, C. P. (2008). Effect of acute physiological hyperinsulinemia on gene expression in human skeletal muscle in vivo. American journal of physiology. Endocrinology and metabolism, 294(5), E910-7.
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  This study was undertaken to test the hypothesis that short-term exposure (4 h) to physiological hyperinsulinemia in normal, healthy subjects without a family history of diabetes would induce a low grade inflammatory response independently of glycemic status. Twelve normal glucose tolerant subjects received a 4-h euglycemic hyperinsulinemic clamp with biopsies of the vastus lateralis muscle. Microarray analysis identified 121 probe sets that were significantly altered in response to physiological hyperinsulinemia while maintaining euglycemia. In normal, healthy human subjects insulin increased the mRNAs of a number of inflammatory genes (CCL2, CXCL2 and THBD) and transcription factors (ATF3, BHLHB2, HES1, KLF10, JUNB, FOS, and FOSB). A number of other genes were upregulated in response to insulin, including RRAD, MT, and SGK. CITED2, a known coactivator of PPARalpha, was significantly downregulated. SGK and CITED2 are located at chromosome 6q23, where we previously detected strong linkage to fasting plasma insulin concentrations. We independently validated the mRNA expression changes in an additional five subjects and closely paralleled the results observed in the original 12 subjects. A saline infusion in healthy, normal glucose-tolerant subjects without family history of diabetes demonstrated that the genes altered during the euglycemic hyperinsulinemic clamp were due to hyperinsulinemia and were unrelated to the biopsy procedure per se. The results of the present study demonstrate that insulin acutely regulates the levels of mRNAs involved in inflammation and transcription and identifies several candidate genes, including HES1 and BHLHB2, for further investigation.
• De Filippis, E., Alvarez, G., Berria, R., Cusi, K., Everman, S., Meyer, C., & Mandarino, L. J. (2008). Insulin-resistant muscle is exercise resistant: evidence for reduced response of nuclear-encoded mitochondrial genes to exercise. American journal of physiology. Endocrinology and metabolism, 294(3), E607-14.
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  Mitochondrial dysfunction, associated with insulin resistance, is characterized by low expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and nuclear-encoded mitochondrial genes. This deficit could be due to decreased physical activity or a decreased response of gene expression to exercise. The objective of this study was to investigate whether a bout of exercise induces the same increase in nuclear-encoded mitochondrial gene expression in insulin-sensitive and insulin-resistant subjects matched for exercise capacity. Seven lean and nine obese subjects took part. Insulin sensitivity was assessed by an 80 mU.m(-2).min(-1) euglycemic clamp. Subjects were matched for aerobic capacity and underwent a single bout of exercise at 70 and 90% of maximum heart rate with muscle biopsies at 30 and 300 min postexercise. Quantitative RT-PCR and immunoblot analyses were used to determine the effect of exercise on gene expression and protein abundance and phosphorylation. In the postexercise period, lean subjects immediately increased PGC-1alpha mRNA level (reaching an eightfold increase by 300 min postexercise) and protein abundance and AMP-dependent protein kinase phosphorylation. Activation of PGC-1alpha was followed by increase of nuclear respiratory factor-1 and cytochrome c oxidase (subunit VIc). However, in insulin-resistant subjects, there was a delayed and reduced response in PGC-1alpha mRNA and protein, and phosphorylation of AMP-dependent protein kinase was transient. None of the genes downstream of PGC-1alpha was increased after exercise in insulin resistance. Insulin-resistant subjects have a reduced response of nuclear-encoded mitochondrial genes to exercise, and this could contribute to the origin and maintenance of mitochondrial dysfunction.
• De, F. E., Alvarez, G., Berria, R., Cusi, K., Everman, S., Meyer, C., & Mandarino, L. J. (2008). Insulin-resistant muscle is exercise resistant: evidence for reduced response of nuclear-encoded mitochondrial genes to exercise. AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM, 294(3), E607-E614.
• Højlund, K., Yi, Z., Hwang, H., Bowen, B., Lefort, N., Flynn, C. R., Langlais, P., Weintraub, S. T., & Mandarino, L. J. (2008). Characterization of the human skeletal muscle proteome by one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. Molecular & cellular proteomics : MCP, 7(2), 257-67.
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  Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.
• Yi, Z., Bowen, B. P., Hwang, H., Jenkinson, C. P., Coletta, D. K., Lefort, N., Bajaj, M., Kashyap, S., Berria, R., De Filippis, E. A., & Mandarino, L. J. (2008). Global relationship between the proteome and transcriptome of human skeletal muscle. Journal of proteome research, 7(8), 3230-41.
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  Skeletal muscle is one of the largest tissues in the human body. Changes in mRNA and protein abundance in this tissue are central to a large number of metabolic and other disorders, including, commonly, insulin resistance. Proteomic and microarray analyses are important approaches for gaining insight into the molecular and biochemical basis for normal and pathophysiological conditions. With the use of vastus lateralis muscle obtained from two groups of healthy, nonobese subjects, we performed a detailed comparison of the muscle proteome, obtained by HPLC-ESI-MS/MS, with the muscle transcriptome, obtained using oligonucleotide microarrays. HPLC-ESI-MS/MS analysis identified 507 unique proteins as present in four out of six subjects, while 5193 distinct transcripts were called present by oligonucleotide microarrays from four out of six subjects. The majority of the proteins identified by mass spectrometry also had their corresponding transcripts detected by microarray analysis, although 73 proteins were only identified in the proteomic analysis. Reflecting the high abundance of mitochondria in skeletal muscle, 30% of proteins detected were attributed to the mitochondrion, as compared to only 9% of transcripts. On the basis of Gene Ontology annotations, proteins assigned to mitochondrial inner membrane, mitochondrial envelope, structural molecule activity, electron transport, as well as generation of precursor metabolites and energy, had more corresponding transcripts detected than would be expected by chance. On the contrary, proteins assigned to Golgi apparatus, extracellular region, lyase activity, kinase activity, and protein modification process had fewer corresponding transcripts detected than would be expected by chance. In conclusion, these results provide the first global comparison of the human skeletal muscle proteome and transcriptome to date. These data show that a combination of proteomic and transcriptic analyses will provide data that can be used to test hypotheses regarding the pathogenesis of muscle disorders as well as to generate observational data that can be used to form novel hypotheses.
• Yi, Z., Bowen, B. P., Hwang, H., Jenkinson, C. P., Coletta, D. K., Lefort, N., Bajaj, M., Kashyap, S., Berria, R., De, F., & Mandarino, L. J. (2008). Global relationship between the proteome and transcriptome of human skeletal muscle. JOURNAL OF PROTEOME RESEARCH, 7(8), 3230-3241.
• Bajaj, M., Medina-Navarro, R., Suraamornkul, S., Meyer, C., DeFronzo, R. A., & Mandarino, L. J. (2007). Paradoxical changes in muscle gene expression in insulin-resistant subjects after sustained reduction in plasma free fatty acid concentration. Diabetes, 56(3), 743-52.
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  Lipid oversupply plays a role in developing insulin resistance in skeletal muscle, decreasing expression of nuclear-encoded mitochondrial genes, and increasing extracellular matrix remodeling. To determine if a decrease in plasma lipid content reverses these abnormalities, insulin-resistant subjects with a family history of type 2 diabetes had euglycemic clamps and muscle biopsies before and after acipimox treatment to suppress free fatty acids. Free fatty acids fell from 0.584 +/- 0.041 to 0.252 +/- 0.053 mmol/l (P < 0.001) and glucose disposal increased from 5.28 +/- 0.46 to 6.31 +/- 0.55 mg . kg(-1) . min(-1) (P < 0.05) after acipimox; intramuscular fatty acyl CoA decreased from 10.3 +/- 1.9 to 4.54 +/- 0.82 pmol/mg muscle (P < 0.01). Paradoxically, expression of PGC-1-and nuclear-encoded mitochondrial genes decreased after acipimox, and expression of collagens I and III alpha-subunits (82- and 21-fold increase, respectively, P < 0.05), connective tissue growth factor (2.5-fold increase, P < 0.001), and transforming growth factor-beta1 increased (2.95-fold increase, P < 0.05). Therefore, a reduction in lipid supply does not completely reverse the molecular changes associated with lipid oversupply in muscle. Changes in expression of nuclear-encoded mitochondrial genes do not always correlate with changes in insulin sensitivity.
• Flynn, C. R., Smoke, C. C., Furnish, E., Komalavilas, P., Thresher, J., Yi, Z., Mandarino, L. J., & Brophy, C. M. (2007). Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli. Protein expression and purification, 52(1), 50-8.
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  Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57%+/-4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.
• Luo, M., Langlais, P., Yi, Z., Lefort, N., De Filippis, E. A., Hwang, H., Christ-Roberts, C. Y., & Mandarino, L. J. (2007). Phosphorylation of human insulin receptor substrate-1 at Serine 629 plays a positive role in insulin signaling. Endocrinology, 148(10), 4895-905.
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  The function of insulin receptor substrate-1 (IRS-1) is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, whereas phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of human IRS-1 at Ser(629) was increased by insulin in Chinese hamster ovary cells expressing the insulin receptor (1.26 +/- 0.09-fold; P < 0.05) and L6 cells (1.35 +/- 0.29-fold; P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser(629) revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser(629) in cells was decreased upon treatment with either an Akt inhibitor or by coexpression with kinase dead Akt, whereas Ser(629) phosphorylation was increased by coexpression with constitutively active Akt. In addition, Ser(629) of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser(629) by a Ser(629)Ala mutation resulted in increased phosphorylation of Ser(636), a known negative regulator of IRS-1, without affecting phosphorylation of Tyr(632) or Ser(616). Cells expressing the Ser(629)Ala mutation, along with increased Ser(636) phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol 3'-kinase with IRS-1 and decreased phosphorylation of Akt at Ser(473). Finally, in vitro phosphorylation of a Ser(629)-containing IRS-1 fragment with Akt reduces the subsequent ability of ERK to phosphorylate Ser(636/639). These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser(629), resulting in decreased phosphorylation of IRS-1 at Ser(636) and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and, ultimately, the pathogenesis of type 2 diabetes.
• Luo, M., Langlais, P., Yi, Z., Lefort, N., De, F., Hwang, H., Christ-Roberts, C. Y., & Mandarino, L. J. (2007). Phosphorylation of human insulin receptor substrate-1 at serine 629 plays a positive role in insulin signaling. ENDOCRINOLOGY, 148(10), 4895-4905.
• Maddux, B. A., Chan, A., Filippis, E. A., Mandarino, L. J., & Goldfine, I. D. (2007). IGF-Binding Protein-1 Levels Are Related to Insulin-Mediated Glucose Disposal and Are a Potential Serum Marker of Insulin Resistance: Response to van Haeften et al.. Diabetes Care, 30(6), e54-e54. doi:10.2337/dc07-0385
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  We appreciate the interest of van Haeflen et al. (1) in our article (2) concerning IGF-binding protein (IGFBP)-1 as a maker of insulin resistance in healthy nondiabetic subjects. In contrast to our study, they did not find a strong a correlation between insulin action as determined …
• Mandarino, L. J., Bajaj, M., Medina-Navarro, R., Suraamornkul, S., Meyer, C., & DeFronzo, R. A. (2007). Paradoxical Changes in Muscle Gene Expression in Insulin-Resistant Subjects After Sustained Reduction in Plasma Free Fatty Acid Concentration. Diabetes, 56(3), 743-752. doi:10.2337/db06-0840
• Ostergard, T., Jessen, N., Schmitz, O., & Mandarino, L. J. (2007). The effect of exercise, training, and inactivity on insulin sensitivity in diabetics and their relatives: what is new?. APPLIED PHYSIOLOGY NUTRITION AND METABOLISM-PHYSIOLOGIE APPLIQUEE NUTRITION ET METABOLISME, 32(3), 541-548.
• Pendergrass, M., Bertoldo, A., Bonadonna, R., Nucci, G., Mandarino, L., Cobelli, C., & Defronzo, R. A. (2007). Muscle glucose transport and phosphorylation in type 2 diabetic, obese nondiabetic, and genetically predisposed individuals. American journal of physiology. Endocrinology and metabolism, 292(1), E92-100.
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  Our objectives were to quantitate insulin-stimulated inward glucose transport and glucose phosphorylation in forearm muscle in lean and obese nondiabetic subjects, in lean and obese type 2 diabetic (T2DM) subjects, and in normal glucose-tolerant, insulin-resistant offspring of two T2DM parents. Subjects received a euglycemic insulin (40 mU.m(-2).min(-1)) clamp with brachial artery/deep forearm vein catheterization. After 120 min of hyperinsulinemia, a bolus of d-mannitol/3-O-methyl-d-[(14)C]glucose/d-[3-(3)H]glucose (triple-tracer technique) was given into brachial artery and deep vein samples obtained every 12-30 s for 15 min. Insulin-stimulated forearm glucose uptake (FGU) and whole body glucose metabolism (M) were reduced by 40-50% in obese nondiabetic, lean T2DM, and obese T2DM subjects (all P < 0.01); in offspring, the reduction in FGU and M was approximately 30% (P < 0.05). Inward glucose transport and glucose phosphorylation were decreased by approximately 40-50% (P < 0.01) in obese nondiabetic and T2DM groups and closely paralleled the decrease in FGU. The intracellular glucose concentration in the space accessible to glucose was significantly greater in obese nondiabetic, lean T2DM, obese T2DM, and offspring compared with lean controls. We conclude that 1) obese nondiabetic, lean T2DM, and offspring manifest moderate-to-severe muscle insulin resistance (FGU and M) and decreased insulin-stimulated glucose transport and glucose phosphorylation in forearm muscle; these defects in insulin action are not further reduced by the combination of obesity plus T2DM; and 2) the increase in intracelullar glucose concentration under hyperinsulinemic euglycemic conditions in obese and T2DM groups suggests that the defect in glucose phosphorylation exceeds the defect in glucose transport.
• Yi, Z., Langlais, P., De Filippis, E. A., Luo, M., Flynn, C. R., Schroeder, S., Weintraub, S. T., Mapes, R., & Mandarino, L. J. (2007). Global assessment of regulation of phosphorylation of insulin receptor substrate-1 by insulin in vivo in human muscle. Diabetes, 56(6), 1508-16.
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  Research has focused on insulin receptor substrate (IRS)-1 as a locus for insulin resistance. Tyrosine phosphorylation of IRS-1 initiates insulin signaling, whereas serine/threonine phosphorylation alters the ability of IRS-1 to transduce the insulin signal. Of 1,242 amino acids in IRS-1, 242 are serine/threonine. Serine/threonine phosphorylation of IRS-1 is affected by many factors, including insulin. The purpose of this study was to perform global assessment of phosphorylation of serine/threonine residues in IRS-1 in vivo in humans.
• Yi, Z., Langlais, P., De, F., Luo, M., Flynn, C. R., Schroeder, S., Weintraub, S. T., Mapes, R., & Mandarino, L. J. (2007). Global assessment of regulation of phosphorylation of insulin receptor substrate-1 by insulin in vivo in human muscle. DIABETES, 56(6), 1508-1516.
• Østergård, T., Jessen, N., Schmitz, O., & Mandarino, L. J. (2007). The effect of exercise, training, and inactivity on insulin sensitivity in diabetics and their relatives: what is new?. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme, 32(3), 541-8.
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  Insulin resistance is a hallmark characteristic of type 2 diabetes. However, in healthy first-degree relatives of type 2 diabetics, insulin resistance is often present years before glucose intolerance or diabetes becomes clinically manifest. The mechanisms of insulin resistance involve conditions leading to an increased supply of fatty acids (e.g., high energy intake, obesity) and conditions in which the degradation/oxidation of muscular fatty acids is impaired. Several large-scale studies have documented the fact that increased physical activity can reduce or at least postpone the development of type 2 diabetes, and low physical fitness is a clear independent risk factor for the development of type 2 diabetes. The mechanisms responsible for the improvement in insulin sensitivity after exercise training have been studied extensively, but are not fully understood. This review focuses on insulin resistance in skeletal muscle and, in particular, its relation to changes in aerobic fitness in type 2 diabetics and their first-degree relatives.
• Alvarez, G. E., Meyer, C., Mandarino, L. J., Filippis, E. A., Berria, R., & Alvarez, G. (2006). One exercise (EX) bout increases PPAR-gamma coactivator (PGC)-1 alpha expression in skeletal muscle from healthy and insulin resistant humans. The FASEB Journal, 20(5).
• Berria, R., Wang, L., Richardson, D. K., Finlayson, J., Belfort, R., Pratipanawatr, T., De Filippis, E. A., Kashyap, S., & Mandarino, L. J. (2006). Increased collagen content in insulin-resistant skeletal muscle. American journal of physiology. Endocrinology and metabolism, 290(3), E560-5.
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  Oversupply and underutilization of lipid fuels are widely recognized to be strongly associated with insulin resistance in skeletal muscle. Recent attention has focused on the mechanisms underlying this effect, and defects in mitochondrial function have emerged as a potential player in this scheme. Because evidence indicates that lipid oversupply can produce abnormalities in extracellular matrix composition and matrix changes can affect the function of mitochondria, the present study was undertaken to determine whether muscle from insulin-resistant, nondiabetic obese subjects and patients with type 2 diabetes mellitus had increased collagen content. Compared with lean control subjects, obese and type 2 diabetic subjects had reduced muscle glucose uptake (P
• Civitarese, A. E., Ukropcova, B., Carling, S., Hulver, M., DeFronzo, R. A., Mandarino, L., Ravussin, E., & Smith, S. R. (2006). Role of adiponectin in human skeletal muscle bioenergetics. Cell metabolism, 4(1), 75-87.
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  Insulin resistance is associated with impaired skeletal muscle oxidation capacity and reduced mitochondrial number and function. Here, we report that adiponectin signaling regulates mitochondrial bioenergetics in skeletal muscle. Individuals with a family history of type 2 diabetes display skeletal muscle insulin resistance and mitochondrial dysfunction; adiponectin levels strongly correlate with mtDNA content. Knockout of the adiponectin gene in mice is associated with insulin resistance and low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle. Adiponectin treatment of human myotubes in primary culture induces mitochondrial biogenesis, palmitate oxidation, and citrate synthase activity, and reduces the production of reactive oxygen species. The inhibition of adiponectin receptor expression by siRNA, or of AMPK by a pharmacological agent, blunts adiponectin induction of mitochondrial function. Our findings define a skeletal muscle pathway by which adiponectin increases mitochondrial number and function and exerts antidiabetic effects.
• De Filippis, E., Cusi, K., Ocampo, G., Berria, R., Buck, S., Consoli, A., & Mandarino, L. J. (2006). Exercise-induced improvement in vasodilatory function accompanies increased insulin sensitivity in obesity and type 2 diabetes mellitus. The Journal of clinical endocrinology and metabolism, 91(12), 4903-10.
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  The present study was undertaken to determine whether improved vasodilatory function accompanies increased insulin sensitivity in overweight, insulin-resistant subjects (OW) and type 2 diabetic patients (T2DM) who participated in an 8-wk exercise training regimen.
• De, F. E., Cusi, K., Ocampo, G., Berria, R., Buck, S., Consoli, A., & Mandarino, L. J. (2006). Exercise-induced improvement in vasodilatory function accompanies increased insulin sensitivity in obesity and type 2 diabetes mellitus. JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, 91(12), 4903-4910.
• Maddux, B. A., Chan, A., De Filippis, E. A., Mandarino, L. J., & Goldfine, I. D. (2006). IGF-binding protein-1 levels are related to insulin-mediated glucose disposal and are a potential serum marker of insulin resistance. Diabetes care, 29(7), 1535-7.
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  IGF-binding protein (IGFBP)-1 is negatively regulated by insulin. We determined whether the measurement of IGFBP-1 in serum is a useful marker of insulin resistance.
• Maddux, B. A., Chan, A., Mandarino, L. J., & Goldfine, I. D. (2006). IGF-binding protein-1 levels are related to insulin-mediated glucose disposal and are a potential serum marker of insulin resistance. DIABETES CARE, 29(7), 1535-1537.
• Mandarino, L. J., De Filippis, E., Cusi, K., Ocampo, G., Berria, R., Buck, S., & Consoli, A. (2006). Exercise-Induced Improvement in Vasodilatory Function Accompanies Increased Insulin Sensitivity in Obesity and Type 2 Diabetes Mellitus. The Journal of Clinical Endocrinology & Metabolism, 91(12), 4903-4910. doi:10.1210/jc.2006-1142
• Sriwijitkamol, A., Christ-Roberts, C., Berria, R., Eagan, P., Pratipanawatr, T., DeFronzo, R. A., Mandarino, L. J., & Musi, N. (2006). Reduced skeletal muscle inhibitor of kappaB beta content is associated with insulin resistance in subjects with type 2 diabetes: reversal by exercise training. Diabetes, 55(3), 760-7.
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  Skeletal muscle insulin resistance plays a key role in the pathogenesis of type 2 diabetes. It recently has been hypothesized that excessive activity of the inhibitor of kappaB (IkappaB)/nuclear factor kappaB (NFkappaB) inflammatory pathway is a mechanism underlying skeletal muscle insulin resistance. However, it is not known whether IkappaB/NFkappaB signaling in muscle from subjects with type 2 diabetes is abnormal. We studied IkappaB/NFkappaB signaling in vastus lateralis muscle from six subjects with type 2 diabetes and eight matched control subjects. Muscle from type 2 diabetic subjects was characterized by a 60% decrease in IkappaB beta protein abundance, an indicator of increased activation of the IkappaB/NFkappaB pathway. IkappaB beta abundance directly correlated with insulin-mediated glucose disposal (Rd) during a hyperinsulinemic (40 mU x m(-2) x min(-1))-euglycemic clamp (r = 0.63, P = 0.01), indicating that increased IkappaB/NFkappaB pathway activity is associated with muscle insulin resistance. We also investigated whether reversal of this abnormality could be a mechanism by which training improves insulin sensitivity. In control subjects, 8 weeks of aerobic exercise training caused a 50% increase in both IkappaB alpha and IkappaB beta protein. In subjects with type 2 diabetes, training increased IkappaB alpha and IkappaB beta protein to levels comparable with that of control subjects, and these increments were accompanied by a 40% decrease in tumor necrosis factor alpha muscle content and a 37% increase in insulin-stimulated glucose disposal. In summary, subjects with type 2 diabetes have reduced IkappaB protein abundance in muscle, suggesting excessive activity of the IkappaB/NFkappaB pathway. Moreover, this abnormality is reversed by exercise training.
• Sriwijitkamol, A., Ivy, J. L., Christ-Roberts, C., DeFronzo, R. A., Mandarino, L. J., & Musi, N. (2006). LKB1-AMPK signaling in muscle from obese insulin-resistant Zucker rats and effects of training. American journal of physiology. Endocrinology and metabolism, 290(5), E925-32.
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  AMPK is a key regulator of fat and carbohydrate metabolism. It has been postulated that defects in AMPK signaling could be responsible for some of the metabolic abnormalities of type 2 diabetes. In this study, we examined whether insulin-resistant obese Zucker rats have abnormalities in the AMPK pathway. We compared AMPK and ACC phosphorylation and the protein content of the upstream AMPK kinase LKB1 and the AMPK-regulated transcriptional coactivator PPARgamma coactivator-1 (PGC-1) in gastrocnemius of sedentary obese Zucker rats and sedentary lean Zucker rats. We also examined whether 7 wk of exercise training on a treadmill reversed abnormalities in the AMPK pathway in obese Zucker rats. In the obese rats, AMPK phosphorylation was reduced by 45% compared with lean rats. Protein expression of the AMPK kinase LKB1 was also reduced in the muscle from obese rats by 43%. In obese rats, phosphorylation of ACC and protein expression of PGC-1alpha, two AMPK-regulated proteins, tended to be reduced by 50 (P = 0.07) and 35% (P = 0.1), respectively. There were no differences in AMPKalpha1, -alpha2, -beta1, -beta2, and -gamma3 protein content between lean and obese rats. Training caused a 1.5-fold increase in AMPKalpha1 protein content in the obese rats, although there was no effect of training on AMPK phosphorylation and the other AMPK isoforms. Furthermore, training also significantly increased LKB1 and PGC-1alpha protein content 2.8- and 2.5-fold, respectively, in the obese rats. LKB1 protein strongly correlated with hexokinase II activity (r = 0.75, P = 0.001), citrate synthase activity (r = 0.54, P = 0.02), and PGC-1alpha protein content (r = 0.81, P < 0.001). In summary, obese insulin-resistant rodents have abnormalities in the LKB1-AMPK-PGC-1 pathway in muscle, and these abnormalities can be restored by training.
• Yi, Z., Luo, M., Mandarino, L. J., Reyna, S. M., Carroll, C. A., & Weintraub, S. T. (2006). Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS. Journal of the American Society for Mass Spectrometry, 17(4), 562-567.
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  Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.
• Bajaj, M., Suraamornkul, S., Romanelli, A., Cline, G. W., Mandarino, L. J., Shulman, G. I., & DeFronzo, R. A. (2005). Effect of a sustained reduction in plasma free fatty acid concentration on intramuscular long-chain fatty Acyl-CoAs and insulin action in type 2 diabetic patients. Diabetes, 54(11), 3148-53.
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  To investigate the effect of a sustained (7-day) decrease in plasma free fatty acid (FFA) concentrations on insulin action and intramyocellular long-chain fatty acyl-CoAs (LCFA-CoAs), we studied the effect of acipimox, a potent inhibitor of lipolysis, in seven type 2 diabetic patients (age 53 +/- 3 years, BMI 30.2 +/- 2.0 kg/m2, fasting plasma glucose 8.5 +/- 0.8 mmol/l, HbA 1c 7.5 +/- 0.4%). Subjects received an oral glucose tolerance test (OGTT) and 120-min euglycemic insulin (80 mU/m2 per min) clamp with 3-[3H]glucose/vastus lateralis muscle biopsies to quantitate rates of insulin-mediated whole-body glucose disposal (Rd) and intramyocellular LCFA-CoAs before and after acipimox (250 mg every 6 h for 7 days). Acipimox significantly reduced fasting plasma FFAs (from 563 +/- 74 to 230 +/- 33 micromol/l; P < 0.01) and mean plasma FFAs during the OGTT (from 409 +/- 44 to 184 +/- 22 micromol/l; P < 0.01). After acipimox, decreases were seen in fasting plasma insulin (from 78 +/- 18 to 42 +/- 6 pmol/l; P < 0.05), fasting plasma glucose (from 8.5 +/- 0.8 to 7.0 +/- 0.5 mmol/l; P < 0.02), and mean plasma glucose during the OGTT (from 14.5 +/- 0.8 to 13.0 +/- 0.8 mmol/l; P < 0.05). After acipimox, insulin-stimulated Rd increased from 3.3 +/- 0.4 to 4.4 +/- 0.4 mg x kg(-1) x min(-1) (P < 0.03), whereas suppression of endogenous glucose production (EGP) was similar and virtually complete during both insulin clamp studies (0.16 +/- 0.10 vs. 0.14 +/- 0.10 mg x kg(-1) x min(-1); P > 0.05). Basal EGP did not change after acipimox (1.9 +/- 0.2 vs. 1.9 +/- 0.2 mg x kg(-1) x min(-1)). Total muscle LCFA-CoA content decreased after acipimox treatment (from 7.26 +/- 0.58 to 5.64 +/- 0.79 nmol/g; P < 0.05). Decreases were also seen in muscle palmityl CoA (16:0; from 1.06 +/- 0.10 to 0.75 +/- 0.11 nmol/g; P < 0.05), palmitoleate CoA (16:1; from 0.48 +/- 0.05 to 0.33 +/- 0.05 nmol/g; P = 0.07), oleate CoA (18:1; from 2.60 +/- 0.11 to 1.95 +/- 0.31 nmol/g; P < 0.05), linoleate CoA (18:2; from 1.81 +/- 0.26 to 1.38 +/- 0.18 nmol/g; P = 0.13), and linolenate CoA (18:3; from 0.27 +/- 0.03 to 0.19 +/- 0.02 nmol/g; P < 0.03) levels after acipimox treatment. Muscle stearate CoA (18:0) did not decrease after acipimox treatment. The increase in R(d) correlated strongly with the decrease in muscle palmityl CoA (r = 0.75, P < 0.05), oleate CoA (r = 0.76, P < 0.05), and total muscle LCFA-CoA (r = 0.74, P < 0.05) levels. Plasma adiponectin did not change significantly after acipimox treatment (7.9 +/- 1.8 vs. 7.5 +/- 1.5 microg/ml). These data demonstrate that the reduction in intramuscular LCFA-CoA content is closely associated with enhanced insulin sensitivity in muscle after a chronic reduction in plasma FFA concentrations in type 2 diabetic patients despite the lack of an effect on plasma adiponectin concentration.
• Belfort, R., Mandarino, L., Kashyap, S., Wirfel, K., Pratipanawatr, T., Berria, R., Defronzo, R. A., & Cusi, K. (2005). Dose-response effect of elevated plasma free fatty acid on insulin signaling. Diabetes, 54(6), 1640-8.
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  The dose-response relationship between elevated plasma free fatty acid (FFA) levels and impaired insulin-mediated glucose disposal and insulin signaling was examined in 21 lean, healthy, normal glucose-tolerant subjects. Following a 4-h saline or Liposyn infusion at 30 (n = 9), 60 (n = 6), and 90 (n = 6) ml/h, subjects received a 2-h euglycemic insulin (40 mU . m(-2) . min(-1)) clamp. Basal plasma FFA concentration ( approximately 440 micromol/l) was increased to 695, 1,251, and 1,688 micromol/l after 4 h of Liposyn infusion and resulted in a dose-dependent reduction in insulin-stimulated glucose disposal (R(d)) by 22, 30, and 34%, respectively (all P < 0.05 vs. saline control). At the lowest lipid infusion rate (30 ml/h), insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation, phosphatidylinositol (PI) 3-kinase activity associated with IRS-1, and Akt serine phosphorylation were all significantly impaired (P < 0.05-0.01). The highest lipid infusion rate (90 ml/h) caused a further significant reduction in all insulin signaling events compared with the low-dose lipid infusion (P < 0.05-0.01) whereas the 60-ml/h lipid infusion caused an intermediate reduction in insulin signaling. However, about two-thirds of the maximal inhibition of insulin-stimulated glucose disposal already occurred at the rather modest increase in plasma FFA induced by the low-dose (30-ml/h) lipid infusion. Insulin-stimulated glucose disposal was inversely correlated with both the plasma FFA concentration after 4 h of lipid infusion (r = -0.50, P = 0.001) and the plasma FFA level during the last 30 min of the insulin clamp (r = -0.54, P < 0.001). PI 3-kinase activity associated with IRS-1 correlated with insulin-stimulated glucose disposal (r = 0.45, P < 0.01) and inversely with both the plasma FFA concentration after 4 h of lipid infusion (r = -0.39, P = 0.01) and during the last 30 min of the insulin clamp (r = -0.43, P < 0.01). In summary, in skeletal muscle of lean, healthy subjects, a progressive increase in plasma FFA causes a dose-dependent inhibition of insulin-stimulated glucose disposal and insulin signaling. The inhibitory effect of plasma FFA was already significant following a rather modest increase in plasma FFA and develops at concentrations that are well within the physiological range (i.e., at plasma FFA levels observed in obesity and type 2 diabetes).
• Luo, M., Reyna, S., Wang, L., Yi, Z., Carroll, C., Dong, L. Q., Langlais, P., Weintraub, S. T., & Mandarino, L. J. (2005). Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis: regulatory role of serine 1223. Endocrinology, 146(10), 4410-6.
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  Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation.
• Richardson, D. K., Kashyap, S., Bajaj, M., Cusi, K., Mandarino, S. J., Finlayson, J., DeFronzo, R. A., Jenkinson, C. P., & Mandarino, L. J. (2005). Lipid infusion decreases the expression of nuclear encoded mitochondrial genes and increases the expression of extracellular matrix genes in human skeletal muscle. The Journal of biological chemistry, 280(11), 10290-7.
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  The association between elevated plasma free fatty acid (FFA) concentrations and insulin resistance is well known. Although the cause and effect relationship between FFAs and insulin resistance is complex, plasma FFA is negatively correlated with the expression of peroxisome proliferator activated receptor-gamma cofactor-1 (PGC-1) and nuclear encoded mitochondrial genes. To test whether this association is causal, we infused a triglyceride emulsion (or saline as control) into healthy subjects to increase plasma FFA for 48 h followed by muscle biopsies, microarray analysis, quantitative real time PCR, and immunoblots. Lipid infusion increased plasma FFA concentration from 0.48 +/- 0.02 to 1.73 +/- 0.43 mm and decreased insulin-stimulated glucose disposal from 8.82 +/- 0.69 to 6.67 +/- 0.66 mg/kg.min, both with p < 0.05. PGC-1 mRNA, along with mRNAs for a number of nuclear encoded mitochondrial genes, were reduced by lipid infusion (p < 0.05). Microarray analysis also revealed that lipid infusion caused a significant overexpression of extracellular matrix genes and connective tissue growth factor. Quantitative reverse transcription PCR showed that the mRNA expression of collagens and multiple extracellular matrix genes was higher after the lipid infusion (p < 0.05). Immunoblot analysis revealed that lipid infusion also increased the expression of collagens and the connective tissue growth factor protein. These data suggest that an experimental increase in FFAs decreases the expression of PGC-1 and nuclear encoded mitochondrial genes and also increases the expression of extracellular matrix genes in a manner reminiscent of inflammation.
• Wajcberg, E., Triplitt, C. L., Miyazaki, Y., Mandarino, L. J., Glass, L. C., Filippis, E. A., Defronzo, R. A., Cersosimo, E., & Bajaj, M. (2005). Predictors of improved glycaemic control with rosiglitazone therapy in type 2 diabetic patients: a practical approach for the primary care physician. The British Journal of Diabetes & Vascular Disease, 5(1), 28-35. doi:10.1177/14746514050050010601
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  Objective ‐ To determine predictors of improved glycaemic control in patients with type 2 diabetes mellitus during rosiglitazone therapy using basic clinical parameters that are readily available in daily clinical practice. Research design and methods ‐ Thirty-seven type 2 diabetic patients (men/women = 18/19; age = 54+2 years; diabetes duration = 6+1 years; diet-/sulphonylurea-treated = 24/13) received a 75 g oral glucose tolerance test (OGTT) and determination of body fat before and after rosiglitazone (8 mg/day) for 12 weeks. Results ‐ After rosiglitazone therapy, there were decreases in HbA1C (8.6+0.2 to 7.2+0.2%, p
• Yi, Z., Luo, M., Carroll, C. A., Weintraub, S. T., & Mandarino, L. J. (2005). Identification of phosphorylation sites in insulin receptor substrate-1 by hypothesis-driven high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Analytical chemistry, 77(17), 5693-9.
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  Serine phosphorylation of insulin receptor substrate-1 (IRS-1) can regulate tyrosine phosphorylation of IRS-1 and subsequent insulin signaling. The 182 serine and 60 threonine residues in IRS-1 make position-by-position analysis of potential phosphorylation sites by mutagenesis difficult. Tandem mass spectrometry provides a more efficient way to identify phosphorylated residues in IRS-1. Toward this end, we overexpressed glutathione S-transferase-IRS-1 fusion proteins in E. coli and treated them in vitro with various kinases followed by identification of phosphorylation sites using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Nine phosphorylation sites were detected in the tryptic digests of middle and C-terminal regions of IRS-1 treated with protein kinase A or extracellular signal-regulated kinase 2. Of these sites, five have not previously been detected by any method and provide novel candidates for identification in cells or in vivo.
• Zimmet, P. Z., Walder, K., Trevaskis, J. L., Ravussin, E., Mandarino, L. J., Kissebah, A. H., Kerr-bayles, L., Jowett, J. B., Elliott, K. S., Curran, J. E., Collier, G. R., Civitarese, A. E., Blangero, J., & Bishara, N. (2005). The mitochondrial rhomboid protease PSARL is a new candidate gene for type 2 diabetes.. Diabetologia, 48(3), 459-68. doi:10.1007/s00125-005-1675-9
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  This study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes..We screened for differentially expressed genes in skeletal muscle of Psammomys obesus (Israeli sand rats), and prioritised these on the basis of genomic localisation and bioinformatics analysis for proteins with likely mitochondrial functions..We identified a mitochondrial intramembrane protease, known as presenilins-associated rhomboid-like protein (PSARL) that is associated with insulin resistance and type 2 diabetes. Expression of PSARL was reduced in skeletal muscle of diabetic Psammomys obesus, and restored after exercise training to successfully treat the diabetes. PSARL gene expression in human skeletal muscle was correlated with insulin sensitivity as assessed by glucose disposal during a hyperinsulinaemic-euglycaemic clamp. In 1,031 human subjects, an amino acid substitution (Leu262Val) in PSARL was associated with increased plasma insulin concentration, a key risk factor for diabetes. Furthermore, this variant interacted strongly with age to affect insulin levels, accounting for 5% of the variation in plasma insulin in elderly subjects..Variation in PSARL sequence and/or expression may be an important new risk factor for type 2 diabetes and other components of the metabolic syndrome.
• Adams, J. M., Pratipanawatr, T., Berria, R., Wang, E., DeFronzo, R. A., Sullards, M. C., & Mandarino, L. J. (2004). Ceramide content is increased in skeletal muscle from obese insulin-resistant humans. Diabetes, 53(1), 25-31.
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  Increased intramyocellular lipid concentrations are thought to play a role in insulin resistance, but the precise nature of the lipid species that produce insulin resistance in human muscle are unknown. Ceramides, either generated via activation of sphingomyelinase or produced by de novo synthesis, induce insulin resistance in cultured cells by inhibitory effects on insulin signaling. The present study was undertaken to determine whether ceramides or other sphingolipids are increased in muscle from obese insulin-resistant subjects and to assess whether ceramide plays a role in the insulin resistance of Akt in human muscle. Lean insulin-sensitive and obese insulin-resistant subjects (n = 10 each) received euglycemic-hyperinsulinemic clamps with muscle biopsies basally and after 30, 45, or 60 min of insulin infusion. The rate of glucose infusion required to maintain euglycemia (reflecting glucose uptake) was reduced by >50%, as expected, in the obese subjects at each time point (P < 0.01). Under basal conditions, total muscle ceramide content was increased nearly twofold in the obese subjects (46 +/- 9 vs. 25 +/- 2 pmol/2 mg muscle, P < 0.05). All species of ceramides were increased similarly in the obese subjects; in contrast, no other sphingolipid was increased. Stimulation of Akt phosphorylation by insulin in the obese subjects was significantly reduced after 30 min (0.96 +/- 0.11 vs. 1.84 +/- 0.38 arbitrary units) or 45-60 min (0.68 +/- 0.17 vs. 1.52 +/- 0.26) of insulin infusion (P < 0.05 for both). Muscle ceramide content was significantly correlated with the plasma free fatty acid concentration (r = 0.51, P < 0.05). We conclude that obesity is associated with increased intramyocellular ceramide content. This twofold increase in ceramide may be involved in the decrease in Akt phosphorylation observed after insulin infusion and could theoretically play a role in the reduced ability of insulin to stimulate glucose uptake in skeletal muscle from obese subjects.
• Bajaj, M., & Mandarino, L. J. (2004). Impaired mitochondrial activity and insulin-resistant offspring of patients with type 2 diabetes. The New England journal of medicine, 350(23), 2419-21; author reply 2419-21.
• Bajaj, M., Suraamornkul, S., Kashyap, S., Cusi, K., Mandarino, L., & DeFronzo, R. A. (2004). Sustained reduction in plasma free fatty acid concentration improves insulin action without altering plasma adipocytokine levels in subjects with strong family history of type 2 diabetes. The Journal of clinical endocrinology and metabolism, 89(9), 4649-55.
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  To investigate the effect of a sustained (7-d) decrease in plasma free fatty acid (FFA) concentration in individuals genetically predisposed to develop type 2 diabetes mellitus (T2DM), we studied the effect of acipimox, a potent inhibitor of lipolysis, on insulin action and adipocytokine concentrations in eight normal glucose-tolerant subjects (aged 40 +/- 4 yr, body mass index 26.5 +/- 0.8 kg/m(2)) with at least two first-degree relatives with T2DM. Subjects received an oral glucose tolerance test (OGTT) and 120 min euglycemic insulin clamp (80 mU/m(2).min) with 3-[(3)H] glucose to quantitate rates of insulin-mediated whole-body glucose disposal (Rd) and endogenous (primarily hepatic) glucose production (EGP) before and after acipimox, 250 mg every 6 h for 7 d. Acipimox significantly reduced fasting plasma FFA (515 +/- 64 to 285 +/- 58 microm, P < 0.05) and mean plasma FFA during the OGTT (263 +/- 32 to 151 +/- 25 microm, P < 0.05); insulin-mediated suppression of plasma FFA concentration during the insulin clamp also was enhanced (162 +/- 18 to 120 +/- 15 microm, P < 0.10). Following acipimox, fasting plasma glucose (5.1 +/- 0.1 vs. 5.2 +/- 0.1 mm) did not change, whereas mean plasma glucose during the OGTT decreased (7.6 +/- 0.5 to 6.9 +/- 0.5 mm, P < 0.01) without change in mean plasma insulin concentration (402 +/- 90 to 444 +/- 102 pmol/liter). After acipimox Rd increased from 5.6 +/- 0.5 to 6.8 +/- 0.5 mg/kg.min (P < 0.01) due to an increase in insulin-stimulated nonoxidative glucose disposal (2.5 +/- 0.4 to 3.5 +/- 0.4 mg/kg.min, P < 0.05). The increment in Rd correlated closely with the decrement in fasting plasma FFA concentration (r = -0.80, P < 0.02). Basal EGP did not change after acipimox (1.9 +/- 0.1 vs. 2.0 +/- 0.1 mg/kg.min), but insulin-mediated suppression of EGP improved (0.22 +/- 0.09 to 0.01 +/- 0.01 mg/kg.min, P < 0.05). EGP during the insulin clamp correlated positively with the fasting plasma FFA concentration (r = 0.49, P = 0.06) and the mean plasma FFA concentration during the insulin clamp (r = 0.52, P < 0.05). Plasma adiponectin (7.1 +/- 1.0 to 7.2 +/- 1.1 microg/ml), resistin (4.0 +/- 0.3 to 3.8 +/- 0.3 ng/ml), IL-6 (1.4 +/- 0.3 to 1.6 +/- 0.4 pg/ml), and TNFalpha (2.3 +/- 0.3 to 2.4 +/- 0.3 pg/ml) did not change after acipimox treatment. We concluded that sustained reduction in plasma FFA concentration in subjects with a strong family history of T2DM increases peripheral (muscle) and hepatic insulin sensitivity without increasing adiponectin levels or altering the secretion of other adipocytokines by the adipocyte. These results suggest that lipotoxicity already is well established in individuals who are genetically predisposed to develop T2DM and that drugs that cause a sustained reduction in the elevated plasma FFA concentration may represent an effective modality for the prevention of T2DM in high-risk, genetically predisposed, normal glucose-tolerant individuals despite the lack of an effect on adipocytokine concentrations.
• Bays, H., Mandarino, L., & DeFronzo, R. A. (2004). Role of the adipocyte, free fatty acids, and ectopic fat in pathogenesis of type 2 diabetes mellitus: peroxisomal proliferator-activated receptor agonists provide a rational therapeutic approach. The Journal of clinical endocrinology and metabolism, 89(2), 463-78.
• Christ-Roberts, C. Y., & Mandarino, L. J. (2004). Glycogen synthase: key effect of exercise on insulin action. Exercise and sport sciences reviews, 32(3), 90-4.
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  Exercise improves insulin action in muscle, but the mechanisms are poorly characterized. Despite the notion that increased insulin signaling would accompany improved insulin sensitivity, this is not universally true. Increased activity or expression of other proteins seems to be more important. An increase in activity and expression of glycogen synthase and GLUT4 may be key to the effects of exercise.
• Christ-Roberts, C. Y., Pratipanawatr, T., Pratipanawatr, W., Berria, R., Belfort, R., Kashyap, S., & Mandarino, L. J. (2004). Exercise training increases glycogen synthase activity and GLUT4 expression but not insulin signaling in overweight nondiabetic and type 2 diabetic subjects. Metabolism: clinical and experimental, 53(9), 1233-42.
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  Exercise training improves insulin sensitivity in subjects with and without type 2 diabetes. However, the mechanism by which this occurs is unclear. The present study was undertaken to determine how improved insulin signaling, GLUT4 expression, and glycogen synthase activity contribute to this improvement. Euglycemic clamps with indirect calorimetry and muscle biopsies were performed before and after 8 weeks of exercise training in 16 insulin-resistant nondiabetic subjects and 6 type 2 diabetic patients. Training increased peak aerobic capacity (Vo(2peak)) in both nondiabetic (from 34 +/- 2 to 39 +/- 2 mL O(2)/kg fat-free mass [FFM]/min, 14% +/- 2%, P
• Kashyap, S. R., Belfort, R., Berria, R., Suraamornkul, S., Pratipranawatr, T., Finlayson, J., Barrentine, A., Bajaj, M., Mandarino, L., DeFronzo, R., & Cusi, K. (2004). Discordant effects of a chronic physiological increase in plasma FFA on insulin signaling in healthy subjects with or without a family history of type 2 diabetes. American journal of physiology. Endocrinology and metabolism, 287(3), E537-46.
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  Muscle insulin resistance develops when plasma free fatty acids (FFAs) are acutely increased to supraphysiological levels (approximately 1,500-4,000 micromol/l). However, plasma FFA levels >1,000 micromol/l are rarely observed in humans under usual living conditions, and it is unknown whether insulin action may be impaired during a sustained but physiological FFA increase to levels seen in obesity and type 2 diabetes mellitus (T2DM) (approximately 600-800 micromol/l). It is also unclear whether normal glucose-tolerant subjects with a strong family history of T2DM (FH+) would respond to a low-dose lipid infusion as individuals without any family history of T2DM (CON). To examine these questions, we studied 7 FH+ and 10 CON subjects in whom we infused saline (SAL) or low-dose Liposyn (LIP) for 4 days. On day 4, a euglycemic insulin clamp with [3-3H]glucose and indirect calorimetry was performed to assess glucose turnover, combined with vastus lateralis muscle biopsies to examine insulin signaling. LIP increased plasma FFA approximately 1.5-fold, to levels seen in T2DM. Compared with CON, FH+ were markedly insulin resistant and had severely impaired insulin signaling in response to insulin stimulation. LIP in CON reduced insulin-stimulated glucose disposal (Rd) by 25%, insulin-stimulated insulin receptor tyrosine phosphorylation by 17%, phosphatidylinositol 3-kinase activity associated with insulin receptor substrate-1 by 20%, and insulin-stimulated glycogen synthase fractional velocity over baseline (44 vs. 15%; all P < 0.05). In contrast to CON, a physiological elevation in plasma FFA in FH+ led to no further deterioration in Rd or to any additional impairment of insulin signaling. In conclusion, a 4-day physiological increase in plasma FFA to levels seen in obesity and T2DM impairs insulin action/insulin signaling in CON but does not worsen insulin resistance in FH+. Whether this lack of additional deterioration in insulin signaling in FH+ is due to already well-established lipotoxicity, or to other molecular mechanisms related to insulin resistance that are nearly maximally expressed early in life, remains to be determined.
• Miyazaki, Y., Mahankali, A., Wajcberg, E., Bajaj, M., Mandarino, L. J., & DeFronzo, R. A. (2004). Effect of pioglitazone on circulating adipocytokine levels and insulin sensitivity in type 2 diabetic patients. The Journal of clinical endocrinology and metabolism, 89(9), 4312-9.
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  We examined the effect of pioglitazone (PIO) on circulating adipocytokine levels to elucidate the mechanisms by which thiazolidinediones improve insulin resistance in type 2 diabetes mellitus (T2DM). Twenty-three subjects with T2DM (age 54 +/- 2 yr, body mass index 29 +/- 1 kg/m(2)) were randomly assigned to receive placebo (n = 11) or PIO, 45 mg/d (n = 12), for 4 months. Before and after treatment, subjects received a 75-g oral glucose tolerance test (OGTT); euglycemic insulin clamp (40 mU/m(2).min) with 3-(3)H-glucose; determination of fat mass ((3)H(2)O); and measurement of fasting glucose, free fatty acids (FFAs), leptin, adiponectin, and TNFalpha concentrations. After 4 months of PIO, fasting plasma glucose concentration (Delta = -2.7 mol/liter), mean plasma glucose during OGTT (Delta = -3.8 mol/liter), and hemoglobin A(1c) (Delta = 1.7%) decreased (P < 0.05 vs. placebo) without change in fasting or post-OGTT plasma insulin levels. Fasting FFAs (Delta = 168 micromol/liter) and TNFalpha (Delta = 0.7 pg/ml) concentrations decreased (P < 0.05 vs. placebo), whereas adiponectin (Delta = 8.7 microg/ml) increased (P < 0.01 vs. placebo). Despite the increase in body fat mass (Delta = 3.4 kg) after PIO, plasma leptin concentration did not change significantly. No changes in plasma glucose, FFAs, or adipocytokine levels were observed in placebo-treated subjects. During the insulin clamp, endogenous (hepatic) glucose production decreased (Delta = -2.67 micromol/fat-free mass.min, P < 0.05 vs. placebo), whereas metabolic clearance rate of glucose (MCR) increased (Delta = 0.58 ml/fat-free mass.min, P < 0.05 vs. placebo) after PIO. In all subjects, before and after PIO, the decrease in plasma FFA concentration was correlated with the changes in both endogenous (hepatic) glucose production (r = 0.47, P < 0.05) and MCR (r = -0.41, P < 0.05), whereas the increase in plasma adiponectin concentration was correlated with the change in endogenous (hepatic) glucose production (r = -0.70, P < 0.01) and MCR (r = 0.49, P < 0.05). These results suggest that the direct effects of PIO on adipose tissue to decrease plasma FFA levels and increase plasma adiponectin contribute to the improvements in hepatic and peripheral insulin sensitivity and glucose tolerance in patients with T2DM.
• Richardson, D. K., Ravussin, E., Mandarino, L. J., Kashyap, S., Jenkinson, C. P., Defronzo, R. A., Cusi, K., Civitarese, A. E., Berria, R., Belfort, R., & Bajaj, M. (2004). Adiponectin receptors gene expression and insulin sensitivity in non-diabetic Mexican Americans with or without a family history of Type 2 diabetes.. Diabetologia, 47(5), 816-20. doi:10.1007/s00125-004-1359-x
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  The recent discovery of two adiponectin receptors (AdipoR1 and AdipoR2) will improve our understanding of the molecular mechanisms underlying the insulin-sensitising effect of adiponectin. The aim of this study was to determine for the first time whether skeletal muscle AdipoR1 and/or AdipoR2 gene expression levels are associated with insulin resistance..Using RT-PCR and northern analysis we measured AdipoR1 and AdipoR2 gene expression in skeletal muscle from healthy Mexican Americans with normal glucose tolerance who had (n=8) or did not have (n=10) a family history of Type 2 diabetes..Gene expression profiling indicated that the AdipoR1 and AdipoR2 isoforms are highly expressed in human skeletal muscle, unlike in mice where AdipoR2 expression was highest in the liver, and AdipoR1 was highest in skeletal muscle. In the study subjects, the expression levels of AdipoR1 (p=0.004) and AdipoR2 (p=0.04), as well as plasma adiponectin concentration (p=0.03) were lower in people with a family history of Type 2 diabetes than in those with no family history of the disease. Importantly, the expression levels of both receptors correlated positively with insulin sensitivity (r=0.64, p=0.004 and r=0.47, p=0.048 respectively)..Collectively, these data indicate that both isoforms of the adiponectin receptor play a role in the insulin-sensitising effect of adiponectin.
• Christ-Roberts, C. Y., Pratipanawatr, T., Pratipanawatr, W., Berria, R., Belfort, R., & Mandarino, L. J. (2003). Increased insulin receptor signaling and glycogen synthase activity contribute to the synergistic effect of exercise on insulin action. Journal of applied physiology (Bethesda, Md. : 1985), 95(6), 2519-29.
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  The purpose of this study was to determine the factors contributing to the ability of exercise to enhance insulin-stimulated glucose disposal. Sixteen insulin-resistant nondiabetic and seven Type 2 diabetic subjects underwent two hyperinsulinemic (40 mU x m-2 x min-1) clamps, once without and once with concomitant exercise at 70% peak O2 consumption. Exercise was begun at the start of insulin infusion and was performed for 30 min. Biopsies of the vastus lateralis were performed before and after 30 min of insulin infusion (immediately after cessation of exercise). Exercise synergistically increased insulin-stimulated glucose disposal in nondiabetic [from 4.6 +/- 0.4 to 9.5 +/- 0.8 mg x kg fat-free mass (FFM)-1x min-1] and diabetic subjects (from 4.3 +/- 1.0 to 7.9 +/- 0.7 mg. kg FFM-1x min-1) subjects. The rate of glucose disposal also was significantly greater in each group after cessation of exercise. Exercise enhanced insulin-stimulated increases in glycogen synthase fractional velocity in control (from 0.07 +/- 0.02 to 0.22 +/- 0.05, P < 0.05) and diabetic (from 0.08 +/- 0.03 to 0.15 +/- 0.03, P < 0.01) subjects. Exercise also enhanced insulin-stimulated glucose storage (glycogen synthesis) in nondiabetic (2.9 +/- 0.9 vs. 4.9 +/- 1.1 mg x kg FFM-1x min-1) and diabetic (1.7 +/- 0.5 vs. 4.2 +/- 0.8 mg x kg FFM-1. min-1) subjects. Increased glucose storage accounted for the increase in whole body glucose disposal when exercise was performed during insulin stimulation in both groups; effects of exercise were correlated with enhancement of glucose disposal and glucose storage (r = 0.93, P < 0.001). Exercise synergistically enhanced insulin-stimulated insulin receptor substrate 1-associated phosphatidylinositol 3-kinase activity (P < 0.05) and Akt Ser473 phosphorylation (P < 0.05) in nondiabetic subjects but had little effect in diabetic subjects. The data indicate that exercise, performed in conjunction with insulin infusion, synergistically increases insulin-stimulated glucose disposal compared with insulin alone. In nondiabetic and diabetic subjects, increased glycogen synthase activation is likely to be involved, in part, in this effect. In nondiabetic, but not diabetic, subjects, exercise-induced enhancement of insulin stimulation of the phosphatidylinositol 3-kinase pathway is also likely to be involved in the exercise-induced synergistic enhancement of glucose disposal.
• Kashyap, S., Belfort, R., Gastaldelli, A., Pratipanawatr, T., Berria, R., Pratipanawatr, W., Bajaj, M., Mandarino, L., DeFronzo, R., & Cusi, K. (2003). A sustained increase in plasma free fatty acids impairs insulin secretion in nondiabetic subjects genetically predisposed to develop type 2 diabetes. Diabetes, 52(10), 2461-74.
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  Acute elevations in free fatty acids (FFAs) stimulate insulin secretion, but prolonged lipid exposure impairs beta-cell function in both in vitro studies and in vivo animal studies. In humans data are limited to short-term (< or =48 h) lipid infusion studies and have led to conflicting results. We examined insulin secretion and action during a 4-day lipid infusion in healthy normal glucose tolerant subjects with (FH+ group, n = 13) and without (control subjects, n = 8) a family history of type 2 diabetes. Volunteers were admitted twice to the clinical research center and received, in random order, a lipid or saline infusion. On days 1 and 2, insulin and C-peptide concentration were measured as part of a metabolic profile after standardized mixed meals. Insulin secretion in response to glucose was assessed with a +125 mg/dl hyperglycemic clamp on day 3. On day 4, glucose turnover was measured with a euglycemic insulin clamp with [3-3H]glucose. Day-long plasma FFA concentrations with lipid infusion were increased within the physiological range, to levels seen in type 2 diabetes (approximately 500-800 micromol/l). Lipid infusion had strikingly opposite effects on insulin secretion in the two groups. After mixed meals, day-long plasma C-peptide levels increased with lipid infusion in control subjects but decreased in the FH+ group (+28 vs. -30%, respectively, P < 0.01). During the hyperglycemic clamp, lipid infusion enhanced the insulin secretion rate (ISR) in control subjects but decreased it in the FH+ group (first phase: +75 vs. -60%, P < 0.001; second phase: +25 vs. -35%, P < 0.04). When the ISR was adjusted for insulin resistance (ISRRd = ISR / [1/Rd], where Rd is the rate of insulin-stimulated glucose disposal), the inadequate beta-cell response in the FH+ group was even more evident. Although ISRRd was not different between the two groups before lipid infusion, in the FH+ group, lipid infusion reduced first- and second-phase ISR(Rd) to 25 and 42% of that in control subjects, respectively (both P < 0.001 vs. control subjects). Lipid infusion in the FH+ group (but not in control subjects) also caused severe hepatic insulin resistance with an increase in basal endogenous glucose production (EGP), despite an elevation in fasting insulin levels, and impaired suppression of EGP to insulin. In summary, in individuals who are genetically predisposed to type 2 diabetes, a sustained physiological increase in plasma FFA impairs insulin secretion in response to mixed meals and to intravenous glucose, suggesting that in subjects at high risk of developing type 2 diabetes, beta-cell lipotoxicity may play an important role in the progression from normal glucose tolerance to overt hyperglycemia.
• Miyazaki, Y., He, H., Mandarino, L. J., & DeFronzo, R. A. (2003). Rosiglitazone improves downstream insulin receptor signaling in type 2 diabetic patients. Diabetes, 52(8), 1943-50.
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  Thiazolidinediones (TZDs) improve glycemic control and insulin sensitivity in patients with type 2 diabetes. To determine whether the TZD-induced improvement in glycemic control is associated with enhanced insulin receptor signaling in skeletal muscle, 20 type 2 diabetic patients received a 75-g oral glucose tolerance test (OGTT) and euglycemic insulin (80 mU x m(-2) x min(-1)) clamp with [3-(3)H]glucose/indirect calorimetry/vastus lateralis muscle biopsies before and after 16 weeks of rosiglitazone treatment. Six age-matched nondiabetic subjects served as control subjects. RSG improved fasting plasma glucose (185 +/- 8 to 139 +/- 5 mg/dl), mean plasma glucose during the OGTT (290 +/- 9 to 225 +/- 6 mg/dl), HbA(1c) (8.5 +/- 0.3 to 7.1 +/- 0.3%), insulin-mediated total-body glucose disposal (TGD) (6.9 +/- 0.7 to 9.2 +/- 0.8 mg x kg(-1) fat-free mass x min(-1)) (all P < 0.001), and decreased fasting plasma free fatty acid (FFA) (789 +/- 59 to 656 +/- 50 micro Eq/l) and mean FFA during the OGTT (644 +/- 41 to 471 +/- 35 micro Eq/l) (both P < 0.01). Before RSG treatment, insulin infusion did not significantly increase insulin receptor tyrosine phosphorylation (0.95 +/- 0.10 to 1.08 +/- 0.13 density units; NS) but had a small stimulatory effect on insulin receptor substrate (IRS)-1 tyrosine phosphorylation (1.05 +/- 0.10 to 1.21 +/- 0.12 density units; P < 0.01) and the association of p85 with IRS-1 (0.94 +/- 0.06 to 1.08 +/- 0.06 activity units; P < 0.01). RSG therapy had no effect on basal or insulin-stimulated insulin receptor tyrosine phosphorylation but increased insulin stimulation of IRS-1 tyrosine phosphorylation (1.13 +/- 0.11 to 1.56 +/- 0.17 density units; P < 0.01 vs. prerosiglitazone) and p85 association with IRS-1 (1.00 +/- 0.06 to 1.27 +/- 0.07 activity units; P < 0.05 vs. prerosiglitazone). In control and type 2 diabetic subjects, TGD/nonoxidative glucose disposal correlated positively with the insulin-stimulated increments in IRS-1 tyrosine phosphorylation (r = 0.52/r = 0.57, P < 0.01) and inversely with the plasma FFA concentration during the insulin clamp (r = -0.55/r = -0.53, P < 0.01). However, no significant association between plasma FFA concentrations during the insulin clamp and the increment in either IRS-1 tyrosine phosphorylation or the association of p85 with IRS-1 was observed. In conclusion, in type 2 diabetic patients, rosiglitazone treatment enhances downstream insulin receptor signaling in muscle and decreases plasma FFA concentration while improving glycemic control.
• Patti, M. E., Butte, A. J., Crunkhorn, S., Cusi, K., Berria, R., Kashyap, S., Miyazaki, Y., Kohane, I., Costello, M., Saccone, R., Landaker, E. J., Goldfine, A. B., Mun, E., DeFronzo, R., Finlayson, J., Kahn, C. R., & Mandarino, L. J. (2003). Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1. Proceedings of the National Academy of Sciences of the United States of America, 100(14), 8466-71.
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  Type 2 diabetes mellitus (DM) is characterized by insulin resistance and pancreatic beta cell dysfunction. In high-risk subjects, the earliest detectable abnormality is insulin resistance in skeletal muscle. Impaired insulin-mediated signaling, gene expression, glycogen synthesis, and accumulation of intramyocellular triglycerides have all been linked with insulin resistance, but no specific defect responsible for insulin resistance and DM has been identified in humans. To identify genes potentially important in the pathogenesis of DM, we analyzed gene expression in skeletal muscle from healthy metabolically characterized nondiabetic (family history negative and positive for DM) and diabetic Mexican-American subjects. We demonstrate that insulin resistance and DM associate with reduced expression of multiple nuclear respiratory factor-1 (NRF-1)-dependent genes encoding key enzymes in oxidative metabolism and mitochondrial function. Although NRF-1 expression is decreased only in diabetic subjects, expression of both PPAR gamma coactivator 1-alpha and-beta (PGC1-alpha/PPARGC1 and PGC1-beta/PERC), coactivators of NRF-1 and PPAR gamma-dependent transcription, is decreased in both diabetic subjects and family history-positive nondiabetic subjects. Decreased PGC1 expression may be responsible for decreased expression of NRF-dependent genes, leading to the metabolic disturbances characteristic of insulin resistance and DM.
• Pipek, R., Miyazaki, Y., Mandarino, L. J., & Defronzo, R. A. (2003). Tumor necrosis factor alpha and insulin resistance in obese type 2 diabetic patients.. International journal of obesity and related metabolic disorders : journal of the International Association for the Study of Obesity, 27(1), 88-94. doi:10.1038/sj.ijo.0802187
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  The relationship between basal serum tumor necrosis factor alpha (TNFalpha) levels and peripheral tissue (muscle) sensitivity to insulin was examined in 63 subjects with normal glucose tolerance (NGT), 18 subjects with impaired glucose tolerance (IGT), and 123 patients with type 2 diabetes mellitus (T2DM). The BMI was similar in NGT (28.8+/-0.7 kg/m(2)), IGT (31.1+/-1.0), and T2DM (30.0+/-0.4) groups. The fasting serum TNFalpha concentration in T2DM (4.4+/-0.2 pg/ml) was significantly higher than in NGT (3.1+/-0.2) and IGT (3.4+/-0.2; both P
• Bajaj, M., Berria, R., Pratipanawatr, T., Kashyap, S., Pratipanawatr, W., Belfort, R., Cusi, K., Mandarino, L., & DeFronzo, R. A. (2002). Free fatty acid-induced peripheral insulin resistance augments splanchnic glucose uptake in healthy humans. American journal of physiology. Endocrinology and metabolism, 283(2), E346-52.
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  To investigate the effect of elevated plasma free fatty acid (FFA) concentrations on splanchnic glucose uptake (SGU), we measured SGU in nine healthy subjects (age, 44 +/- 4 yr; body mass index, 27.4 +/- 1.2 kg/m(2); fasting plasma glucose, 5.2 +/- 0.1 mmol/l) during an Intralipid-heparin (LIP) infusion and during a saline (Sal) infusion. SGU was estimated by the oral glucose load (OGL)-insulin clamp method: subjects received a 7-h euglycemic insulin (100 mU x m(-2) x min(-1)) clamp, and a 75-g OGL was ingested 3 h after the insulin clamp was started. After glucose ingestion, the steady-state glucose infusion rate (GIR) during the insulin clamp was decreased to maintain euglycemia. SGU was calculated by subtracting the integrated decrease in GIR during the period after glucose ingestion from the ingested glucose load. [3-(3)H]glucose was infused during the initial 3 h of the insulin clamp to determine rates of endogenous glucose production (EGP) and glucose disappearance (R(d)). During the 3-h euglycemic insulin clamp before glucose ingestion, R(d) was decreased (8.8 +/- 0.5 vs. 7.6 +/- 0.5 mg x kg(-1) x min(-1), P < 0.01), and suppression of EGP was impaired (0.2 +/- 0.04 vs. 0.07 +/- 0.03 mg x kg(-1) x min(-1), P < 0.01). During the 4-h period after glucose ingestion, SGU was significantly increased during the LIP vs. Sal infusion study (30 +/- 2 vs. 20 +/- 2%, P < 0.005). In conclusion, an elevation in plasma FFA concentration impairs whole body glucose R(d) and insulin-mediated suppression of EGP in healthy subjects but augments SGU.
• Bajaj, M., Pratipanawatr, T., Berria, R., Pratipanawatr, W., Kashyap, S., Cusi, K., Mandarino, L., & DeFronzo, R. A. (2002). Free fatty acids reduce splanchnic and peripheral glucose uptake in patients with type 2 diabetes. Diabetes, 51(10), 3043-8.
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  Splanchnic glucose uptake (SGU) plays a major role in the disposal of an oral glucose load (OGL). To investigate the effect of an elevated plasma free fatty acid (FFA) concentration on SGU in patients with type 2 diabetes, we measured SGU in eight diabetic patients (mean age 51 +/- 4 years, BMI 29.3 +/- 1.4 kg/m(2), fasting plasma glucose 9.3 +/- 0.7 mmol/l) during an intravenous Intralipid/heparin infusion and 7-10 days later during a saline infusion. SGU was estimated by the OGL insulin clamp method: subjects received a 7-h euglycemic-hyperinsulinemic clamp (insulin infusion rate = 100 mU x m(-2) x min(-1)), and a 75-g OGL was ingested 3 h after starting the insulin clamp. After glucose ingestion, the steady-state glucose infusion rate during the insulin clamp was decreased appropriately to maintain euglycemia. SGU was calculated by subtracting the integrated decrease in glucose infusion rate during the 4-h period after glucose ingestion from the ingested glucose load (75 g). 3-[(3)H]glucose was infused during the 3-h insulin clamp before glucose ingestion to determine the rates of endogenous glucose production and glucose disappearance (R(d)). Intralipid/heparin or saline infusion was initiated 2 h before the start of the OGL clamp. Plasma FFA concentrations were significantly higher during the OGL clamp with the intralipid/heparin infusion than with the saline infusion (2.5 +/- 0.3 vs. 0.11 +/- 0.02 mmol/l, P < 0.001). During the 3-h insulin clamp period before glucose ingestion, Intralipid/heparin infusion reduced R(d) (4.4 +/- 0.3 vs. 5.3 +/- 0.3 mg x kg(-1) x min(-1), P < 0.01). During the 4-h period after glucose ingestion, SGU was significantly decreased during the intralipid/heparin versus saline infusion (30 +/- 2 vs. 37 +/- 2%, P < 0.01). In conclusion, an elevation in plasma FFA concentration impairs both peripheral and SGU in patients with type 2 diabetes.
• Christ, C. Y., Hunt, D., Hancock, J., Garcia-Macedo, R., Mandarino, L. J., & Ivy, J. L. (2002). Exercise training improves muscle insulin resistance but not insulin receptor signaling in obese Zucker rats. Journal of applied physiology (Bethesda, Md. : 1985), 92(2), 736-44.
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  Exercise training improves skeletal muscle insulin sensitivity in the obese Zucker rat. The purpose of this study was to investigate whether the improvement in insulin action in response to exercise training is associated with enhanced insulin receptor signaling. Obese Zucker rats were trained for 7 wk and studied by using the hindlimb-perfusion technique 24 h, 96 h, or 7 days after their last exercise training bout. Insulin-stimulated glucose uptake (traced with 2-deoxyglucose) was significantly reduced in untrained obese Zucker rats compared with lean controls (2.2 +/- 0.17 vs. 5.4 +/- 0.46 micromol x g(-1) x h(-1)). Glucose uptake was normalized 24 h after the last exercise bout (4.9 +/- 0.41 micromol x g(-1) x h(-1)) and remained significantly elevated above the untrained obese Zucker rats for 7 days. However, exercise training did not increase insulin receptor or insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, phosphatidylinositol 3-kinase (PI3-kinase) activity associated with IRS-1 or tyrosine phosphorylated immunoprecipitates, or Akt serine phosphorylation. These results are consistent with the hypothesis that, in obese Zucker rats, adaptations occur during training that lead to improved insulin-stimulated muscle glucose uptake without affecting insulin receptor signaling through the PI3-kinase pathway.
• Miyazaki, Y., Glass, L., Triplitt, C., Wajcberg, E., Mandarino, L. J., & DeFronzo, R. A. (2002). Abdominal fat distribution and peripheral and hepatic insulin resistance in type 2 diabetes mellitus. American journal of physiology. Endocrinology and metabolism, 283(6), E1135-43.
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  We examined the relationship between peripheral/hepatic insulin sensitivity and abdominal superficial/deep subcutaneous fat (SSF/DSF) and intra-abdominal visceral fat (VF) in patients with type 2 diabetes mellitus (T2DM). Sixty-two T2DM patients (36 males and 26 females, age = 55 +/- 3 yr, body mass index = 30 +/- 1 kg/m2) underwent a two-step euglycemic insulin clamp (40 and 160 mU. m(-2). min(-1)) with [3-3H]glucose. SSF, DSF, and VF areas were quantitated with magnetic resonance imaging at the L(4-5) level. Basal endogenous glucose production (EGP), hepatic insulin resistance index (basal EGP x FPI), and total glucose disposal (TGD) during the first and second insulin clamp steps were similar in male and female subjects. VF (159 +/- 9 vs. 143 +/- 9 cm2) and DSF (199 +/- 14 vs. 200 +/- 15 cm(2)) were not different in male and female subjects. SSF (104 +/- 8 vs. 223 +/- 15 cm2) was greater (P < 0.0001) in female vs. male subjects despite similar body mass index (31 +/- 1 vs. 30 +/- 1 kg/m2) and total body fat mass (31 +/- 2 vs. 33 +/- 2 kg). In male T2DM, TGD during the first insulin clamp step (1st TGD) correlated inversely with VF (r = -0.45, P < 0.01), DSF (r = -0.46, P < 0.01), and SSF (r = -0.39, P < 0.05). In males, VF (r = 0.37, P < 0.05), DSF (r = 0.49, P < 0.01), and SSF (r = 0.33, P < 0.05) were correlated positively with hepatic insulin resistance. In females, the first TGD (r = -0.45, P < 0.05) and hepatic insulin resistance (r = 0.49, P < 0.05) correlated with VF but not with DSF, SSF, or total subcutaneous fat area. We conclude that visceral adiposity is associated with both peripheral and hepatic insulin resistance, independent of gender, in T2DM. In male but not female T2DM, deep subcutaneous adipose tissue also is associated with peripheral and hepatic insulin resistance.
• Miyazaki, Y., Mahankali, A., Matsuda, M., Mahankali, S., Hardies, J., Cusi, K., Mandarino, L. J., & DeFronzo, R. A. (2002). Effect of pioglitazone on abdominal fat distribution and insulin sensitivity in type 2 diabetic patients. The Journal of clinical endocrinology and metabolism, 87(6), 2784-91.
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  We examined the effect of pioglitazone on abdominal fat distribution to elucidate the mechanisms via which pioglitazone improves insulin resistance in patients with type 2 diabetes mellitus. Thirteen type 2 diabetic patients (nine men and four women; age, 52 +/- 3 yr; body mass index, 29.0 +/- 1.1 kg/m(2)), who were being treated with a stable dose of sulfonylurea (n = 7) or with diet alone (n = 6), received pioglitazone (45 mg/d) for 16 wk. Before and after pioglitazone treatment, subjects underwent a 75-g oral glucose tolerance test (OGTT) and two-step euglycemic insulin clamp (insulin infusion rates, 40 and 160 mU/m(2).min) with [(3)H]glucose. Abdominal fat distribution was evaluated using magnetic resonance imaging at L4-5. After 16 wk of pioglitazone treatment, fasting plasma glucose (179 +/- 10 to 140 +/- 10 mg/dl; P < 0.01), mean plasma glucose during OGTT (295 +/- 13 to 233 +/- 14 mg/dl; P < 0.01), and hemoglobin A(1c) (8.6 +/- 0.4% to 7.2 +/- 0.5%; P < 0.01) decreased without a change in fasting or post-OGTT insulin levels. Fasting plasma FFA (674 +/- 38 to 569 +/- 31 microEq/liter; P < 0.05) and mean plasma FFA (539 +/- 20 to 396 +/- 29 microEq/liter; P < 0.01) during OGTT decreased after pioglitazone. In the postabsorptive state, hepatic insulin resistance [basal endogenous glucose production (EGP) x basal plasma insulin concentration] decreased from 41 +/- 7 to 25 +/- 3 mg/kg fat-free mass (FFM).min x microU/ml; P < 0.05) and suppression of EGP during the first insulin clamp step (1.1 +/- 0.1 to 0.6 +/- 0.2 mg/kg FFM.min; P < 0.05) improved after pioglitazone treatment. The total body glucose MCR during the first and second insulin clamp steps increased after pioglitazone treatment [first MCR, 3.5 +/- 0.5 to 4.4 +/- 0.4 ml/kg FFM.min (P < 0.05); second MCR, 8.7 +/- 1.0 to 11.3 +/- 1.1 ml/kg FFM(.)min (P < 0.01)]. The improvement in hepatic and peripheral tissue insulin sensitivity occurred despite increases in body weight (82 +/- 4 to 85 +/- 4 kg; P < 0.05) and fat mass (27 +/- 2 to 30 +/- 3 kg; P < 0.05). After pioglitazone treatment, sc fat area at L4-5 (301 +/- 44 to 342 +/- 44 cm(2); P < 0.01) increased, whereas visceral fat area at L4-5 (144 +/- 13 to 131 +/- 16 cm(2); P < 0.05) and the ratio of visceral to sc fat (0.59 +/- 0.08 to 0.44 +/- 0.06; P < 0.01) decreased. In the postabsorptive state hepatic insulin resistance (basal EGP x basal immunoreactive insulin) correlated positively with visceral fat area (r = 0.55; P < 0.01). The glucose MCRs during the first (r = -0.45; P < 0.05) and second (r = -0.44; P < 0.05) insulin clamp steps were negatively correlated with the visceral fat area. These results demonstrate that a shift of fat distribution from visceral to sc adipose depots after pioglitazone treatment is associated with improvements in hepatic and peripheral tissue sensitivity to insulin.
• Pratipanawatr, T., Cusi, K., Ngo, P., Pratipanawatr, W., Mandarino, L. J., & DeFronzo, R. A. (2002). Normalization of plasma glucose concentration by insulin therapy improves insulin-stimulated glycogen synthesis in type 2 diabetes. Diabetes, 51(2), 462-8.
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  Considerable evidence suggests that skeletal muscle insulin resistance is an inherent feature of type 2 diabetes and contributes to the pathogenesis of the disease. In patients with poorly controlled diabetes, hyperglycemia is thought to produce additional insulin resistance in muscle. The magnitude and nature of hyperglycemia-induced insulin resistance is not known. The purpose of the present study was to determine the biochemical mechanisms responsible for increased insulin-stimulated glucose disposal after the achievement of tight glycemic control with a mixed-split regimen. We performed hyperinsulinemic-euglycemic clamps with indirect calorimetry and vastus lateralis muscle biopsies in eight type 2 diabetic patients who had poor glycemic control (HbA(1c) 10.1%) and again after 3 months of intensive insulin therapy designed to produce near-normoglycemia (HbA(1c) 6.6%). Improved glycemic control increased insulin-stimulated glucose disposal (5.16 +/- 0.32 vs. 3.69 +/- 0.33 mg x kg(-1) x min(-1); P < 0.01); nonoxidative glucose disposal, which primarily reflects glycogen synthesis (2.11 +/- 0.26 vs. 0.90 +/- 0.16 mg x kg(-1) x min(-1); P < 0.01); and glycogen synthase fractional velocity (0.094 +/- 0.017 vs. 0.045 +/- 0.007; P < 0.05). There was no improvement in insulin-stimulated glucose oxidation (3.05 +/- 0.25 vs. 2.79 +/- 0.20 mg x kg(-1) x min(-1)), hexokinase II mRNA expression (increase over basal values), or hexokinase II enzymatic activity (0.51 +/- 0.16 vs. 0.42 +/- 0.18 pmol x min(-1) x microg(-1) protein). All of the increase in insulin-stimulated glucose disposal could be accounted for by increased glycogen synthesis, which is likely attributable to increased activation of glycogen synthase by insulin.
• Pratipanawatr, T., Matsuda, M., Mandarino, L. J., Lee, N. A., Glass, L. C., Defronzo, R. A., & Bressler, P. (2002). Effect of misoprostol (PGE1) on glucose metabolism in type-2-diabetic and control subjects.. Diabetes, obesity & metabolism, 4(3), 195-200. doi:10.1046/j.1463-1326.2002.00203.x
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  In vitro and in vivo studies have demonstrated that prostaglandins of the E series enhance muscle glucose uptake. We examined the effect of acute misoprostol (PGE1) administration on whole body insulin-mediated glucose disposal, as well as the major intracellular pathways of glucose metabolism in type 2 diabetic (n = 10) and non-diabetic (n = 4) subjects. Each subject received two 240-min euglycaemic insulin (40 mU/m2/min) clamp studies with tritiated glucose and indirect calorimetry. During one of the insulin clamp studies, 200 microg of misoprostol was ingested at 90 and 150 min after the start of the insulin infusion. Insulin-mediated total body glucose disposal, glycolysis, glycogenesis and glucose oxidation were similar during the insulin clamp studies performed without and with misoprostol in both the diabetic and non-diabetic groups. These results demonstrate that the acute administration of misoprostol does not enhance insulin-mediated glucose disposal in either type-2-diabetic or non-diabetic subjects.
• Pratipanawatr, T., Pratipanawatr, W., Rosen, C., Berria, R., Bajaj, M., Cusi, K., Mandarino, L., Kashyap, S., Belfort, R., & DeFronzo, R. A. (2002). Effect of IGF-I on FFA and glucose metabolism in control and type 2 diabetic subjects. American journal of physiology. Endocrinology and metabolism, 282(6), E1360-8.
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  The effects of insulin-like growth factor I (IGF-I) and insulin on free fatty acid (FFA) and glucose metabolism were compared in eight control and eight type 2 diabetic subjects, who received a two-step euglycemic hyperinsulinemic (0.25 and 0.5 mU x kg(-1) x min(-1)) clamp and a two-step euglycemic IGF-I (26 and 52 pmol x kg(-1) x min(-1)) clamp with [3-(3)H]glucose, [1-(14)C]palmitate, and indirect calorimetry. The insulin and IGF-I infusion rates were chosen to augment glucose disposal (R(d)) to a similar extent in control subjects. In type 2 diabetic subjects, stimulation of R(d) (second clamp step) in response to both insulin and IGF-I was reduced by approximately 40-50% compared with control subjects. In control subjects, insulin was more effective than IGF-I in suppressing endogenous glucose production (EGP) during both clamp steps. In type 2 diabetic subjects, insulin-mediated suppression of EGP was impaired, whereas EGP suppression by IGF-I was similar to that of controls. In both control and diabetic subjects, IGF-I-mediated suppression of plasma FFA concentration and inhibition of FFA turnover were markedly impaired compared with insulin (P < 0.01-0.001). During the second IGF-I clamp step, suppression of plasma FFA concentration and FFA turnover was impaired in diabetic vs. control subjects (P < 0.05-0.01).
• Miyazaki, Y., Matsuda, M., Mandarino, L. J., Mahankali, S., Mahankali, A., Glass, L. C., Ferrannini, E., Defronzo, R. A., & Cusi, K. (2001). Improved glycemic control and enhanced insulin sensitivity in type 2 diabetic subjects treated with pioglitazone.. Diabetes care, 24(4), 710-9. doi:10.2337/diacare.24.4.710
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  To elucidate the effects of pioglitazone treatment on glucose and lipid metabolism in patients with type 2 diabetes..A total of 23 diabetic patients (age 30-70 years BMI < 36 kg/m2) who being treated with a stable dose of sulfonylurea were randomly assigned to receive either placebo (n = 11) or pioglitazone (45 mg/day) (n = 12) for 16 weeks. Before and after 16 weeks of treatment, all subjects received a 75-g oral glucose tolerance test (OGTT) and hepatic peripheral insulin sensitivity was measured with a two-step euglycemic insulin (40 and 160 mU x min(-1) x m(-2) clamp performed with 3-[3H]glucose and indirect calorimetry HbA1c measured monthly throughout the study period..After 16 weeks of pioglitazone treatment, the fasting plasma glucose (FPG; 184 +/- 15 to 135 +/- 11 mg/dl, P < 0.01), mean plasma glucose during OGTT(293 +/- 12 to 225 +/- 14 mg/dl, P < 0.01), and HbA1c (8.9 +/- 0.3 to 7.2 +/- 0.5%, P < 0.01 ) decreased significantly without change in fasting or glucose-stimulated insulin/C-peptide concentrations. Fasting plasma free fatty acid (FFA; 647 +/- 39 to 478 +/- 49) microEq/l, P < 0.01) and mean plasma FFA during OGTT (485 +/- 30 to 347 +/- 33 microEq/l, P < 0.01) decreased significantly after pioglitazone treatment. Before and after pioglitazone treatment, basal endogenous glucose prodution (EGP) and FPG were strongly correlated (r = 0.67, P < 0.01). EGP during the first insulin clamp step was significantly decreased after pioglitazone treatment (P < 0.05) whereas insulin-stimulated total and nonoxidative glucose disposal during the second insulin clamp was increased (P < 0.01). The change in FPG was related to the change in basal EGP, EGP during the first insulin clamp step, and total glucose disposal during the second insulin clamp step. The change in mean plasma glucose concentration during the OGGTT was strongly related to the change in total body glucose disposl during the second insulin clamp step..These results suggest that pioglitazone therapy in type 2 diabetic patients decreases lasting and postprandial plasma glucose levels by improving hepatic and peripheral (muscle) tissue sensitivity to insulin.
• Osman, A. A., Mandarino, L. J., Ivy, J. L., Hunt, D. G., & Hancock, J. (2001). Exercise training increases ERK2 activity in skeletal muscle of obese Zucker rats.. Journal of applied physiology (Bethesda, Md. : 1985), 90(2), 454-60. doi:10.1152/jappl.2001.90.2.454
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  Acute exercise and training increase insulin action in skeletal muscle, but the mechanism responsible for this effect is unknown. Activation of the insulin receptor initiates signaling through both the phosphatidylinositol (PI) 3-kinase and the mitogen-activated protein kinase [MAPK, also referred to as extracellular signal-regulated kinases (ERK1/2)] pathways. Acute exercise has no effect on the PI3-kinase pathway signaling elements but does activate the MAPK pathway, which may play a role in the adaptation of muscle to exercise. It is unknown whether training produces a chronic effect on basal activity or insulin response of the MAPK pathway. The present study was undertaken to determine whether exercise training improves the activity of the MAPK pathway or its response to insulin in obese Zucker rats, a well-characterized model of insulin resistance. To accomplish this, obese Zucker rats were studied by using the hindlimb perfusion method with or without 7 wk of treadmill training. Activation of the MAPK pathway was determined in gastrocnemius muscles exposed in situ to insulin. Compared with lean Zucker rats, untrained obese Zucker rats had reduced basal and insulin-stimulated activities of ERK2 and its downstream target p90 ribosomal S6 kinase (RSK2). Seven weeks of training significantly increased basal and insulin-stimulated ERK2 and RSK2 activities, as well as insulin stimulation of MAPK kinase activity. This effect was maintained for at least 96 h in the case of ERK2. The training-induced increase in basal ERK2 activity was correlated with the increase in citrate synthase activity. Therefore, 7 wk of training increases basal and insulin-stimulated ERK2 activity. The increase in basal ERK2 activity may be related to the response of muscle to training.
• Pratipanawatr, W., Pratipanawatr, T., Mandarino, L. J., Maezono, K., Jenkinson, C. P., Defronzo, R. A., Cusi, K., Berria, R., & Adams, J. M. (2001). Skeletal muscle insulin resistance in normoglycemic subjects with a strong family history of type 2 diabetes is associated with decreased insulin-stimulated insulin receptor substrate-1 tyrosine phosphorylation.. Diabetes, 50(11), 2572-8. doi:10.2337/diabetes.50.11.2572
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  Normoglycemic subjects with a strong family history of type 2 diabetes are insulin resistant, but the mechanism of insulin resistance in skeletal muscle of such individuals is unknown. The present study was undertaken to determine whether abnormalities in insulin-signaling events are present in normoglycemic, nonobese subjects with a strong family history of type 2 diabetes. Hyperinsulinemic-euglycemic clamps with percutaneous muscle biopsies were performed in eight normoglycemic relatives of type 2 diabetic patients (FH(+)) and eight control subjects who had no family history of diabetes (FH(-)), with each group matched for age, sex, body composition, and ethnicity. The FH(+) group had decreased insulin-stimulated glucose disposal (6.64 +/- 0.52 vs. 8.45 +/- 0.54 mg. kg(-1) fat-free mass. min(-1); P < 0.05 vs. FH(-)). In skeletal muscle, the FH(+) and FH(-) groups had equivalent insulin stimulation of insulin receptor tyrosine phosphorylation. In contrast, the FH(+) group had decreased insulin stimulation of insulin receptor substrate (IRS)-1 tyrosine phosphorylation (0.522 +/- 0.077 vs. 1.328 +/- 0.115 density units; P < 0.01) and association of PI 3-kinase activity with IRS-1 (0.299 +/- 0.053 vs. 0.466 +/- 0.098 activity units; P < 0.05). PI 3-kinase activity was correlated with the glucose disposal rate (r = 0.567, P = 0.02). In five subjects with sufficient biopsy material for further study, phosphorylation of Akt was 0.266 +/- 0.061 vs. 0.404 +/- 0.078 density units (P < 0.10) and glycogen synthase activity was 0.31 +/- 0.06 vs. 0.50 +/- 0.12 ng. min(-1). mg(-1) (P < 0.10) for FH(+) and FH(-) subjects, respectively. Therefore, despite normal insulin receptor phosphorylation, postreceptor signaling was reduced and was correlated with glucose disposal in muscle of individuals with a strong genetic background for type 2 diabetes.
• Printz, R. L., Pratipanawatr, T., Mandarino, L. J., Koval, J., Granner, D. K., Defronzo, R. A., Cusi, K. J., & Ardehali, H. (2001). Exercise increases hexokinase II mRNA, but not activity in obesity and type 2 diabetes.. Metabolism: clinical and experimental, 50(5), 602-6. doi:10.1053/meta.2001.22568
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  Glucose phosphorylation, catalyzed by hexokinase, is the first committed step in glucose uptake in skeletal muscle. Hexokinase II (HKII) is the isoform that is present in muscle and is regulated by insulin and muscle contraction. Glucose phosphorylation and HKII expression are both reduced in obese and type 2 diabetic subjects. A single bout of exercise increases HKII mRNA and activity in muscle from healthy subjects. The present study was performed to determine if a moderate exercise increases HKII mRNA expression and activity in patients with type 2 diabetes. Muscle biopsies were performed before and 3 hours after a single bout of cycle ergometer exercise in obese and type 2 diabetic patients. HKII mRNA and activity and glycogen synthase activity were determined in the muscle biopsies. Exercise increased HKII mRNA in obese and diabetic subjects by 1.67 +/- 0.34 and 1.87 +/- 0.26-fold, respectively (P
• Triplitt, C. L., Miyazaki, Y., Matsuda, M., Mandarino, L. J., Mahankali, S., Mahankali, A., Glass, L. C., Defronzo, R. A., & Cusi, K. (2001). Effect of rosiglitazone on glucose and non-esterified fatty acid metabolism in Type II diabetic patients.. Diabetologia, 44(12), 2210-9. doi:10.1007/s001250100031
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  We aimed to examine the mechanisms by which rosiglitazone improves glycaemic control in Type II (non-insulin-dependent) diabetic patients..Altogether 29 diet-treated diabetic patients were assigned at random to rosiglitazone, 8 mg/day (n = 15), or placebo (n = 14) for 12 weeks. Patients received 75 g OGTT and two-step euglycaemic insulin (40 and 160 mU/m(2)min) clamp with 3-(3)H-glucose, (14)C-palmitate and indirect calorimetry..After 12 weeks, rosiglitazone reduced fasting plasma glucose (195 +/- 11 to 150 +/- 7 mg/dl, p < 0.01), mean plasma glucose (PG) during OGTT (293 +/- 12 to 236 +/- 9 mg/dl, p < 0.01), and HbA1 c (8.7 +/- 0.4 to 7.4 +/- 0.3 %, p < 0.01) without changes in plasma insulin concentration. Basal endogenous glucose production (EGP) declined (3.3 +/- 0.1 to 2.9 +/- 0.1 mg/kg FFM. min, p < 0.05) and whole body glucose metabolic clearance rate increased after rosiglitazone (first clamp step: 2.8 +/- 0.2 to 3.5 +/- 0.2 ml/kg FFM. min, p < 0.01; second clamp step: 6.7 +/- 0.6 to 9.2 +/- 0.8, p < 0.05) despite increased body weight (86 +/- 4 to 90 +/- 4 kg, p < 0.01) and fat mass (33 +/- 3 to 37 +/- 3 kg, p < 0.01). Fasting plasma non-esterified fatty acid (NEFA) (735 +/- 52 to 579 +/- 49 microEq/l, p < 0.01), mean plasma NEFA during OGTT (561 +/- 33 to 424 +/- 35, p < 0.01), and basal NEFA turnover (18.3 +/- 1.5 to 15.5 +/- 1.2 microEq/kg FM. min, p < 0.05) decreased after rosiglitazone. Changes in EPG and mean plasma glucose (PG) during OGTT correlated with changes in basal EGP (r = 0.54; r = 0.58), first EGP (r = 0.36; r = 0.41), first MCR (r = -0.66; r = -0.68), second MCR (r = -0.49; r = -0.54), fasting plasma NEFA (r = 0.53; r = 0.49), and NEFA during OGTT (r = 0.66; r = 0.66)..Rosiglitazone increases hepatic and peripheral (muscle) tissue insulin sensitivity and reduces NEFA turnover despite increased total body fat mass. These results suggest that the beneficial effects of rosiglitazone on glycaemic control are mediated, in part, by the drug's effect on NEFA metabolism.
• Vogt, C., Pratipanawatr, T., Pipek, R., Pijl, H., Matsuda, M., Mandarino, L. J., Kumar, V., Iozzo, P., Defronzo, R. A., & Cusi, K. J. (2001). Physiological hyperinsulinemia impairs insulin-stimulated glycogen synthase activity and glycogen synthesis.. American journal of physiology. Endocrinology and metabolism, 280(5), E712-9. doi:10.1152/ajpendo.2001.280.5.e712
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  Although chronic hyperinsulinemia has been shown to induce insulin resistance, the basic cellular mechanisms responsible for this phenomenon are unknown. The present study was performed 1) to determine the time-related effect of physiological hyperinsulinemia on glycogen synthase (GS) activity, hexokinase II (HKII) activity and mRNA content, and GLUT-4 protein in muscle from healthy subjects, and 2) to relate hyperinsulinemia-induced alterations in these parameters to changes in glucose metabolism in vivo. Twenty healthy subjects had a 240-min euglycemic insulin clamp study with muscle biopsies and then received a low-dose insulin infusion for 24 (n = 6) or 72 h (n = 14) (plasma insulin concentration = 121 +/- 9 or 143 +/- 25 pmol/l, respectively). During the baseline insulin clamp, GS fractional velocity (0.075 +/- 0.008 to 0.229 +/- 0.02, P < 0.01), HKII mRNA content (0.179 +/- 0.034 to 0.354 +/- 0.087, P < 0.05), and HKII activity (2.41 +/- 0.63 to 3.35 +/- 0.54 pmol x min(-1) x ng(-1), P < 0.05), as well as whole body glucose disposal and nonoxidative glucose disposal, increased. During the insulin clamp performed after 24 and 72 h of sustained physiological hyperinsulinemia, the ability of insulin to increase muscle GS fractional velocity, total body glucose disposal, and nonoxidative glucose disposal was impaired (all P < 0.01), whereas the effect of insulin on muscle HKII mRNA, HKII activity, GLUT-4 protein content, and whole body rates of glucose oxidation and glycolysis remained unchanged. Muscle glycogen concentration did not change [116 +/- 28 vs. 126 +/- 29 micromol/kg muscle, P = nonsignificant (NS)] and was not correlated with the change in nonoxidative glucose disposal (r = 0.074, P = NS). In summary, modest chronic hyperinsulinemia may contribute directly (independent of change in muscle glycogen concentration) to the development of insulin resistance by its impact on the GS pathway.
• Mandarino, L. J., & Kelley, D. E. (2000). Fuel selection in human skeletal muscle in insulin resistance: a reexamination.. Diabetes, 49(5), 677-83. doi:10.2337/diabetes.49.5.677
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  For many years, the Randle glucose fatty acid cycle has been invoked to explain insulin resistance in skeletal muscle of patients with type 2 diabetes or obesity. Increased fat oxidation was hypothesized to reduce glucose metabolism. The results of a number of investigations have shown that artificially increasing fat oxidation by provision of excess lipid does decrease glucose oxidation in the whole body. However, results obtained with rodent or human systems that more directly examined muscle fuel selection have found that skeletal muscle in insulin resistance is accompanied by increased, rather than decreased, muscle glucose oxidation under basal conditions and decreased glucose oxidation under insulin-stimulated circumstances, producing a state of "metabolic inflexibility." Such a situation could contribute to the accumulation of triglyceride within the myocyte, as has been observed in insulin resistance. Recent knowledge of insulin receptor signaling indicates that the accumulation of lipid products in muscle can interfere with insulin signaling and produce insulin resistance. Therefore, although the Randle cycle is a valid physiological principle, it may not explain insulin resistance in skeletal muscle.
• Mandarino, L. J., Pratipanawatr, T., Pendergrass, M., Osman, A. A., Mandarino, L. J., Maezono, K., Koval, J., & Cusi, K. (2000). Regulation of MAP kinase pathway activity in vivo in human skeletal muscle.. American journal of physiology. Endocrinology and metabolism, 278(6), E992-9. doi:10.1152/ajpendo.2000.278.6.e992
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  Insulin and exercise potently stimulate glucose metabolism and gene transcription in vivo in skeletal muscle. A single bout of exercise increases the rate of insulin-stimulated glucose uptake and metabolism in skeletal muscle in the postexercise period. The nature of the intracellular signaling mechanisms that control responses to exercise is not known. In mammalian tissues, numerous reports have established the existence of the mitogen-activated protein (MAP) kinase signaling pathway that is activated by a variety of growth factors and hormones. This study was undertaken to determine how a single bout of exercise and physiological hyperinsulinemia activate the MAP kinase pathway. The euglycemic-hyperinsulinemic clamp and cycle ergometer exercise techniques combined with percutaneous muscle biopsies were used to answer this question. In healthy subjects, within 30 min, insulin significantly increased MAP kinase [isoforms p42(MAPK) and p44(MAPK) (ERK1 and ERK2)] phosphorylation (141 +/- 2%, P < 0.05) and activity (177 +/- 5%, P < 0.05), and the activity of its upstream activator MEK1 (161 +/- 16%, P < 0.05). Insulin also increased the activity of the MAP kinase downstream substrate, the p90 ribosomal S6 kinase 2 (RSK2) almost twofold (198 +/- 45%, P < 0.05). In contrast, a single 30-min bout of moderate-intensity exercise had no effect on the MAP kinase pathway activation from MEK to RSK2 in muscle of healthy subjects. However, 60 min of exercise did increase extracellular signal-related kinase activity. Therefore, despite similar effects on glucose metabolism after 30 min, insulin and exercise regulate the MAP kinase pathway differently. Insulin more rapidly activates the MAP kinase pathway.
• Mandarino, L. J., Pratipanawatr, T., Pendergrass, M., Patti, M. E., Osman, A. A., Mandarino, L. J., Maezono, K., Kahn, C. R., Defronzo, R. A., & Cusi, K. (2000). Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle.. The Journal of clinical investigation, 105(3), 311-20. doi:10.1172/jci7535
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  The broad nature of insulin resistant glucose metabolism in skeletal muscle of patients with type 2 diabetes suggests a defect in the proximal part of the insulin signaling network. We sought to identify the pathways compromised in insulin resistance and to test the effect of moderate exercise on whole-body and cellular insulin action. We conducted euglycemic clamps and muscle biopsies on type 2 diabetic patients, obese nondiabetics and lean controls, with and without a single bout of exercise. Insulin stimulation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, as measured by phosphorylation of the insulin receptor and IRS-1 and by IRS protein association with p85 and with PI 3-kinase, was dramatically reduced in obese nondiabetics and virtually absent in type 2 diabetic patients. Insulin stimulation of the MAP kinase pathway was normal in obese and diabetic subjects. Insulin stimulation of glucose-disposal correlated with association of p85 with IRS-1. Exercise 24 hours before the euglycemic clamp increased phosphorylation of insulin receptor and IRS-1 in obese and diabetic subjects but did not increase glucose uptake or PI 3-kinase association with IRS-1 upon insulin stimulation. Thus, insulin resistance differentially affects the PI 3-kinase and MAP kinase signaling pathways, and insulin-stimulated IRS-1-association with PI 3-kinase defines a key step in insulin resistance.
• Miyazaki, Y., Matsuda, M., Mandarino, L. J., Mahankali, A., Defronzo, R., & Cusi, K. (2000). Effect of pioglitazone on glucose metabolism in sulfonylurea-treated patients with type 2 diabetes. Diabetes Research and Clinical Practice, 50, 62. doi:10.1016/s0168-8227(00)81668-x
• Miyazaki, Y., Matsuda, M., Mandarino, L. J., Mahankali, A., Defronzo, R., & Cusi, K. (2000). Effect of pioglitazone on glucose tolerance and insulin sensitivity in diet-controlled type 2 diabetic subjects. Diabetes Research and Clinical Practice, 50, 61-62. doi:10.1016/s0168-8227(00)81667-8
• Miyazaki, Y., Matsuda, M., Mandarino, L. J., Mahankali, S., Mahankali, A., Defronzo, R., & Cusi, K. (2000). Effect of pioglitazone on abdominal fat distribution and insulin sensitivity in patients with type 2 diabetes. Diabetes Research and Clinical Practice, 50, 61. doi:10.1016/s0168-8227(00)81666-6
• Mandarino, L. J., Pendergrass, M., Patti, M. E., Mandarino, L. J., Maezono, K., Koval, J. A., & Defronzo, R. A. (1999). Effects of exercise and insulin on insulin signaling proteins in human skeletal muscle.. Medicine and science in sports and exercise, 31(7), 998-1004. doi:10.1097/00005768-199907000-00012
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  Insulin and exercise independently increase glucose metabolism in muscle. Moreover, exercise training or a prior bout of exercise increases insulin-stimulated glucose uptake in resting skeletal muscle. The present study was undertaken to compare how physiological hyperinsulinemia and moderate intensity aerobic exercise affect the tyrosine phosphorylation state and activity of insulin signaling molecules in healthy, physically inactive volunteers. Subjects had biopsies of the vastus lateralis muscle before and immediately after 30 min of either hyperinsulinemia (euglycemic insulin clamp) or moderate-intensity exercise on a cycle ergometer (approximately 60% of VO2max). Insulin receptor and IRS-1 tyrosine phosphorylation, association of the p85 regulatory subunit of PI 3-kinase with IRS-1, IRS-1 associated PI 3-kinase activity, and glycogen synthase activity were determined in muscle biopsy specimens taken from healthy subjects before and after insulin or exercise. Physiological hyperinsulinemia increased the rate of glucose disposal from 11.4 +/- 1.5 to 25.6 +/- 6.7 micromol x kg(-1) x min(-1) (P < 0.01), insulin receptor and IRS-1 tyrosine phosphorylation (173 +/- 19% and 159 +/- 35% of basal values, respectively, P < 0.05), association of the p85 regulatory subunit of PI 3-kinase with IRS-1 (159 +/- 10%, P < 0.05), and glycogen synthase fractional velocity (136 +/- 11%, P < 0.01). Exercise also increased glucose disposal, from 10.4 +/- 0.5 to 15.6 +/- 1.7 micromol x kg(-1) x min(-1) (P < 0.01) and glycogen synthase fractional velocity (253 +/- 35% of basal, P < 0.01). The exercise-induced increase in glycogen synthase was greater than that due to insulin (P < 0.05). In contrast to insulin, exercise decreased tyrosine phosphorylation of the insulin receptor to 72 +/- 10% of basal values (P < 0.05 vs basal and P < 0.05 vs insulin) and had no effect on IRS-1 tyrosine phosphorylation, or association of p85 with IRS-1. The exercise-induced decreased insulin receptor tyrosine phosphorylation could explain the well-known effect of exercise to enhance the sensitivity of muscle to insulin.
• Matsuda, M., Mandarino, L. J., & Defronzo, R. A. (1999). Synergistic interaction of magnesium and vanadate on glucose metabolism in diabetic rats.. Metabolism: clinical and experimental, 48(6), 725-31. doi:10.1016/s0026-0495(99)90171-3
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  The effect of vanadate (V) alone, magnesium (Mg) alone, and the combination of Mg plus V (MgV) on insulin-mediated glucose disposal and glucose tolerance was investigated in normal and streptozotocin-induced diabetic rats. MgV, magnesium sulfate (MgSO4) and sodium metavanadate (NaV) were added to the drinking water of normal or diabetic rats (approximately 300 g) for 3 weeks. After 3 weeks of V treatment (both MgV and NaV), diabetic rats demonstrated a normal meal tolerance test without any increase in the plasma insulin response. Rats also received a euglycemic insulin clamp (12 mU/kg x min for 120 minutes) with 3-3H-glucose infusion to quantify total body glucose disposal, glycolysis (3H2O production), and glycogen synthesis (total body glucose disposal minus glycolysis). Total glucose disposal was decreased in diabetic versus control rats (29 +/- 2 v 35 +/- 2 mg/kg x min, P < .01) and returned to levels greater than the nondiabetic control values after MgV (41 +/- 2, P < .01). Supersensitivity to insulin was not observed in diabetic rats treated with NaV (34 +/- 1). Glycogen synthesis was increased by both MgV and NaV treatment (23 +/- 21, P < .01 and 18 +/- 1, P < .05 v 14 +/- 2 mg/kg x min) in diabetic rats. A small increase in glycolysis was observed in MgSO4 and MgV rats (18 +/- 1 and 18 +/- 1 v 16 +/- 1, P < .05). NaV alone had no effect on glycolysis. Thus, Mg has a synergistic effect with V to increase muscle glycogen synthesis in diabetic rats. In normal rats, neither MgSO4 nor NaV had any effect on glucose utilization. However, MgV increased glucose disposal to rates that were significantly higher than the rate in untreated control rats (P < .05). Based on these results, MgV is superior to either V alone or Mg alone in improving insulin sensitivity and glycogen synthesis in diabetic rats.
• Mandarino, L. J., Koopmans, S. J., & Defronzo, R. A. (1998). Time course of insulin action on tissue-specific intracellular glucose metabolism in normal rats.. The American journal of physiology, 274(4), E642-50. doi:10.1152/ajpendo.1998.274.4.e642
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  We investigated the time course of insulin action in conscious rats exposed to constant physiological hyperinsulinemia (approximately 100 mU/l) while maintaining euglycemia (approximately 100 mg/dl) for 0, 0.5, 2, 4, 8, or 12 h. [3-3H]glucose was infused to quantitate whole body glucose disposal (rate of disappearance, Rd), glycolysis (generation of 3H2O in plasma), hepatic glucoses production (HGP), and skeletal muscle and liver glycogen synthesis ([3-3H]glucose incorporation into glycogen and time-dependent change in tissue glycogen concentration). The basal Rd, which equals HGP, was 6.0 +/- 0.3 mg.kg-1.min-1. With increased duration of hyperinsulinemia from 0 to 0.5 to 2 to 4 h, Rd increased from 6.0 +/- 0.3 to 21.0 +/- 1.1 to 24.1 +/- 1.5 to 26.6 +/- 0.6 mg.kg-1.min-1 (P < 0.05 for 2 and 4 h vs. 0.5 h). During the first 2 h the increase in Rd was explained by parallel increases in glycolysis and glycogen synthesis. From 2 to 4 h the further increase in Rd was entirely due to an increase in glycolysis without change in glycogen synthesis. From 4 to 8 to 12 h of hyperinsulinemia, Rd decreased by 19% from 26.6 +/- 0.6 to 24.1 +/- 1.1 to 21.6 +/- 1.8 mg.kg-1.min-1 (P < 0.05 for 8 h vs. 4 h and 12 h vs. 8 h). The progressive decline in Rd, in the face of constant hyperinsulinemia, occurred despite a slight increase (8-14%) in glycolysis and was completely explained by a marked decrease (64%) in muscle glycogen synthesis. In contrast, liver glycogen synthesis increased fourfold, indicating an independent regulation of muscle and liver glycogen synthesis by long-term hyperinsulinemia. In the liver, during the entire 12-h period of insulin stimulation, the contribution of the direct (from glucose) and the indirect (from C-3 fragments) pathways to net glycogen formation remained constant at 77 +/- 5 and 23 +/- 5%, respectively. HGP remained suppressed throughout the 12-h period of hyperinsulinemia.
• Mandarino, L. J., Ykijarvinen, H., Vogt, C., Printz, R. L., Pipek, R., Pendergrass, M., Mandarino, L. J., Koval, J., Iozzo, P., Granner, D. K., Defronzo, R. A., & Ardehali, H. (1998). Effects of insulin on subcellular localization of hexokinase II in human skeletal muscle in vivo.. The Journal of Clinical Endocrinology and Metabolism, 83(1), 230-234. doi:10.1210/jc.83.1.230
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  The phosphorylation of glucose to glucose-6-phosphate, catalyzed by hexokinase, is the first committed step in glucose uptake into skeletal muscle. Two isoforms of hexokinase, HKI and HKII, are expressed in human skeletal muscle, but only HKII expression is regulated by insulin. HKII messenger RNA, protein, and activity are increased after 4 h of insulin infusion; however, glucose uptake is stimulated much more rapidly, occurring within minutes. Studies in rat muscle suggest that changes in the subcellular distribution of HKII may be an important regulatory factor for glucose uptake. The present studies were undertaken to determine if insulin causes an acute redistribution of HKII activity in human skeletal muscle in vivo. Muscle biopsies (vastus lateralis muscle) were performed before and at the end of 30 min insulin infusion, performed using the euglycemic clamp technique. Muscle biopsies were subfractionated into soluble and particulate fractions to determine if insulin acutely changes the subcellular ...
• Printz, R. L., O'doherty, R. M., Mandarino, L. J., Koval, J. A., Granner, D. K., Defronzo, R. A., & Ardehali, H. (1998). Regulation of hexokinase II activity and expression in human muscle by moderate exercise.. The American journal of physiology, 274(2), E304-8. doi:10.1152/ajpendo.1998.274.2.e304
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  A single bout of exercise increases the rate of insulin-stimulated glucose uptake and metabolism in skeletal muscle. Exercise also increases insulin-stimulated glucose 6-phosphate in skeletal muscle, suggesting that exercise increases hexokinase activity. Within 3 h, exercise increases hexokinase II (HK II) mRNA and activity in skeletal muscle from rats. It is not known, however, if a single bout of moderate-intensity exercise increases HK II expression in humans. The present study was undertaken to answer this question. Six subjects had percutaneous biopsies of the vastus lateralis muscle before and 3 h after a single 3-h session of moderate-intensity aerobic (60% of maximal oxygen consumption) exercise. Glycogen synthase, HK I, and HK II activities as well as HK I and HK II mRNA content were determined from the muscle biopsy specimens. The fractional velocity of glycogen synthase was increased by 446 +/- 84% after exercise (P < 0.005). Hexokinase II activity in the soluble fraction of the homogenates increased from 1.2 +/- 0.4 to 4.5 +/- 1.6 pmol.min-1.microgram-1 (P < 0.05) but was unchanged in the particulate fraction (4.3 +/- 1.3 vs. 5.3 +/- 1.5). HK I activity in neither the soluble nor particulate fraction changed after exercise. Relative to a 28S rRNA control signal, HK II mRNA increased from 0.091 +/- 0.02 to 0.195 +/- 0.037 (P < 0.05), whereas HK I mRNA was unchanged (0.414 +/- 0.061 vs. 0.498 +/- 0.134, P < 0.20). The increase in HK II activity after moderate exercise in healthy subjects could be one factor responsible for the enhanced rate of insulin-stimulated glucose uptake seen after exercise.
• Ohman, L. E., Mandarino, L. J., Koopmans, S. J., Haywood, J. R., & Defronzo, R. A. (1997). Seven days of euglycemic hyperinsulinemia induces insulin resistance for glucose metabolism but not hypertension, elevated catecholamine levels, or increased sodium retention in conscious normal rats.. Diabetes, 46(10), 1572-8. doi:10.2337/diacare.46.10.1572
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  Epidemiological studies have suggested an association among chronic hyperinsulinemia, insulin resistance, and hypertension. However, the causality of this relationship remains uncertain. In this study, chronically catheterized conscious rats were made hyperinsulinemic for 7 days (approximately 90 mU/l, i.e., threefold over basal), while strict euglycemia was maintained (approximately 130 mg/dl, coefficient of variation < 10%) by using a modification of the insulin/glucose clamp technique. Control rats received vehicle infusion. Baseline mean arterial pressure and heart rate were 125 +/- 5 mmHg and 427 +/- 12 beats/min and remained unchanged during the 7-day infusion of insulin (127 +/- 7 mmHg; 401 +/- 12 beats/min) or vehicle (133 +/- 4 mmHg; 411 +/- 10 beats/min). Baseline plasma epinephrine (88 +/- 15 pg/ml), norepinephrine (205 +/- 31 pg/ml), and sodium balance (0.34 +/- 0.09 mmol) remained constant during the 7-day insulin or vehicle infusion. After 7 days of insulin or vehicle infusion, in vivo insulin action was determined in all rats using a 2-h hyperinsulinemic (1 mU/min) euglycemic clamp with [3-3H]glucose infusion to quantitate whole-body glucose uptake, glycolysis, glucose storage (total glucose uptake minus glycolysis), and hepatic glucose production. Compared with vehicle-treated rats, 7 days of sustained hyperinsulinemia resulted in a reduction (P < 0.01) in insulin-mediated glucose uptake, glucose storage, and glycolysis by 39, 62, and 26%, respectively. Hepatic glucose production was normally suppressed after 7 days of hyperinsulinemia. Neither insulin-stimulated glucose uptake nor glucose storage correlated with blood pressure or heart rate. In conclusion, 7 days of euglycemic hyperinsulinemia induces severe insulin resistance with respect to whole-body glucose metabolism but does not increase blood pressure, catecholamine levels, or sodium retention. This indicates that hyperinsulinemia-induced insulin resistance is not associated with the development of hypertension in rats who do not have a genetic predisposition for hypertension. Because hyperinsulinemia was initiated in normal rats under euglycemic conditions, additional (inherited or acquired) factors may be necessary to observe an effect of hyperinsulinemia and/or insulin resistance to increase blood pressure.
• Ykijarvinen, H., Yki-jarvinen, H., Vogt, C., Virkamaki, A., Pipek, R., Mcclain, D. A., Mandarino, L. J., Makimattila, S., Iozzo, P., Gottschalk, W. K., Defronzo, R. A., & Daniels, M. C. (1997). UDP-N-acetylglucosamine transferase and glutamine: fructose 6-phosphate amidotransferase activities in insulin-sensitive tissues.. Diabetologia, 40(1), 76-81. doi:10.1007/s001250050645
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  Glutamine:fructose 6-phosphate amidotransferase (GFA) is rate-limiting for hexosamine biosynthesis, while a UDP-GlcNAc beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) catalyses final O-linked attachment of GlcNAc to serine and threonine residues on intracellular proteins. Increased activity of the hexosamine pathway is a putative mediator of glucose-induced insulin resistance but the mechanisms are unclear. We determined whether O-GlcNAc transferase is found in insulin-sensitive tissues and compared its activity to that of GFA in rat tissues. We also determined whether non-insulin-dependent diabetes mellitus (NIDDM) or acute hyperinsulinaemia alters O-GlcNAc transferase activity in human skeletal muscle. O-GlcNAc transferase was measured using 3H-UDP-GlcNAc and a synthetic cationic peptide substrate containing serine and threonine residues, and GFA was determined by measuring a fluorescent derivative of GlcN6P by HPLC. O-GlcNAc transferase activities were 2-4 fold higher in skeletal muscles and the heart than in the liver, which had the lowest activity, while GFA activity was 14-36-fold higher in submandibular gland and 5-18 fold higher in the liver than in skeletal muscles or the heart. In patients with NIDDM (n = 11), basal O-GlcNAc transferase in skeletal muscle averaged 3.8 +/- 0.3 nmol/mg.min, which was not different from that in normal subjects (3.3 +/- 0.4 nmol/mg.min). A 180-min intravenous insulin infusion (40 mU/m2.min) did not change muscle O-GlcNAc transferase activity in either group. We conclude that O-GlcNAc transferase is widely distributed in insulin-sensitive tissues in the rat and is also found in human skeletal muscle. These findings suggest the possibility that O-linked glycosylation of intracellular proteins is involved in mediating glucose toxicity. O-GlcNAc transferase does not, however, appear to be regulated by either NIDDM or acute hyperinsulinaemia, suggesting that mass action effects determine the extent of O-linked glycosylation under hyperglycaemic conditions.
• Mandarino, L. J., Kelley, D. E., Jain, A., & Consoli, A. (1996). Interaction of carbohydrate and fat fuels in human skeletal muscle: impact of obesity and NIDDM.. The American journal of physiology, 270(3 Pt 1), E463-70. doi:10.1152/ajpendo.1996.270.3.e463
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  The current study was undertaken to examine the impact that obesity and non-insulin-dependent diabetes mellitus (NIDDM) have on the ability of glucose to stimulate its own uptake and oxidation in muscle. Euglycemic and hyperglycemic clamp experiments were performed with somatostatin infusions so that insulin could be replaced to basal levels or to physiological hyperinsulinemia. Arteriovenous leg balance methods were used to measure the pathways of leg muscle glucose uptake, oxidation, and storage. Percutaneous biopsies of the vastus lateralis muscle were taken to determine the pyruvate dehydrogenase complex or glycogen synthase activities. During basal insulin replacement, obese compared with lean nondiabetic subjects had higher values for glucose uptake, respiratory quotient, and glucose oxidation (all P
• O'doherty, R. M., Sewell, C., Printz, R. L., Osawa, H., O'doherty, R. M., Mandarino, L. J., Kinchington, P., Granner, D. K., Defonzo, R. A., Cusi, K. A., & Consoli, A. (1996). Regulation of hexokinase II and glycogen synthase mRNA, protein, and activity in human muscle (American Journal of Physiology: Endocrinology and Metabolism (October 1995) 269 (E701-E708)). American Journal of Physiology-endocrinology and Metabolism, 270.
• Sewell, C., Printz, R. L., Osawa, H., O'doherty, R. M., Mandarino, L. J., Kinchington, P., Granner, D. K., Defronzo, R. A., Cusi, K. A., & Consoli, A. (1995). Regulation of hexokinase II and glycogen synthase mRNA, protein, and activity in human muscle.. The American journal of physiology, 269(4 Pt 1), E701-8. doi:10.1152/ajpendo.1995.269.4.e701
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  Insulin regulates the activity of key enzymes of glucose metabolism in skeletal muscle by altering transcription or translation or by producing activity-altering modifications of preexisting enzyme molecules. Because of the small size of percutaneous muscle biopsies, these phenomena have been difficult to study in humans. This study was performed to determine how physiological hyperinsulinemia regulates the activities of hexokinase (HK), glycogen synthase (GS), and GLUT-4 in human skeletal muscle in vivo. We determined mRNA abundance, protein content, and activities for these proteins in muscle biopsies before and after a hyperinsulinemic clamp in normal subjects. HK I, HK II, GS, and GLUT-4 were expressed in muscle. HK II accounted for 80% of total HK activity and was increased by insulin from a basal value of 2.11 +/- 0.26 to 3.35 +/- 0.47 pmol.min-1.mg protein-1 (P < 0.05); HK I activity was unaffected. Insulin increased GS activity from 3.85 +/- 0.82 to 6.06 +/- 0.49 nmol.min-1.mg-1 (P < 0.01). HK II mRNA was increased 3.3 +/- 1.3-fold (P < 0.05) by insulin infusion. HK I, GS, and GLUT-4 mRNA and protein were unaffected. Because insulin infusion increased HK II but not GS mRNA, we conclude that HK II and GS may be regulated by insulin by different mechanisms in human skeletal muscle.
• Mandarino, L. J., Hassell, J. R., & Finlayson, J. (1994). High glucose downregulates glucose transport activity in retinal capillary pericytes but not endothelial cells.. Investigative ophthalmology & visual science, 35(3), 964-72.
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  To characterize the properties of the glucose transporters of bovine retinal capillary endothelial cells and pericytes and to determine the effects of increased glucose concentrations on glucose transport activity..Primary cultures of bovine retinal capillary endothelial cells and pericytes were exposed to low and high glucose concentrations, and immunoblot analysis, 14C-3-O-methylglucose transport activity, and cytochalasin B binding assays were used to characterize the glucose transporters..GLUT1, but not GLUT3 or GLUT4 transporter isoforms, was present in plasma membranes isolated from each cell type. The EC50 for glucose transport was similar in endothelial cells and pericytes (3.94 to 0.48 mM versus 2.24 to 0.69 mM) and was consistent with the EC50 previously reported for GLUT1 transporters on other cells, as was the observation that insulin did not acutely stimulate glucose transport in either cell type. The Vmax for glucose transport was greater in pericytes than endothelial cells (71 to 25 versus 14.5 to 0.8 pmol/10 s/g DNA). Exposure of pericytes to 20 mM glucose for 8 days decreased the initial maximal rate of glucose transport by 30%, compared to pericytes cultured in 5 mM glucose (187 to 7 versus 133 to 9 fmol/20 s/g DNA, P < 0.01), but had no effect on glucose transport activity in endothelial cells. Culture in high glucose decreased the apparent amount of immunoreactive pericyte plasma membrane GLUT1 in immunoblots (0.611 to 0.055 versus 1.0 relative density units), decreased the binding of 3H-cytochalasin B to pericyte plasma membranes, and decreased the mRNA level for GLUT1 in pericytes by 25%..High-glucose concentrations downregulate glucose transport activity and GLUT 1 content in retinal capillary pericytes but not in endothelial cells. This effect occurred at a pretranslational level. The selective effects of high-glucose concentrations on retinal capillary pericytes in culture might be related to the selective effects of hyperglycemia on these cells in vivo.
• Kelley, D. E., Mokan, M., & Mandarino, L. J. (1993). Metabolic pathways of glucose in skeletal muscle of lean NIDDM patients.. Diabetes care, 16(8), 1158-66. doi:10.2337/diacare.16.8.1158
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  To characterize the ability of insulin to activate the skeletal muscle metabolic pathways of glucose storage, oxidation, and glycolysis in normal weight patients with NIDDM and nondiabetic volunteer subjects closely matched for age, sex, relative weight, and body composition..Ten patients with NIDDM (body mass index 23.9 +/- 0.74 kg/m2) and 8 nondiabetic volunteer subjects (body mass index 23.4 +/- 0.41 kg/m2) were studied. Leg muscle glucose uptake, non-oxidized glycolysis, glucose oxidation, and glucose storage were determined during euglycemic-hyperinsulinemic clamp experiments using the leg balance technique combined with leg indirect calorimetry. Percutaneous muscle biopsies were obtained to assay insulin stimulation of muscle glycogen synthase activity as a biochemical marker of insulin action..During hyperinsulinemic clamp experiments, leg glucose uptake was equivalent in NIDDM patients and nondiabetic subjects (6.38 +/- 1.14 vs. 6.41 +/- 0.73 mumol.min-1 x 100 ml tissue-1), as were rates of leg glucose oxidation (1.63 +/- 0.25 vs. 2.14 +/- 0.17 mumol.min-1 x 100 ml tissue-1) and leg glucose storage (4.35 +/- 1.10 vs. 3.48 +/- 0.65 mumol.min-1 x 100 ml tissue-1). The combined net balance of lactate and Ala (non-oxidized glycolysis) was lower in NIDDM patients (-0.39 +/- 0.06 vs. -0.79 +/- 0.11 mumol.min-1 x 100 ml tissue-1, P = 0.01). Muscle glycogen synthase was activated to a similar extent during the hyperinsulinemic clamp in NIDDM patients and nondiabetic volunteer subjects, through basal glycogen synthase activity was lower in NIDDM patients. Nondiabetic subjects and NIDDM patients who were withdrawn from sulfonylurea therapy had impaired insulin secretion during a 75-g oral glucose tolerance test, with similar basal levels as nondiabetic subjects (54 +/- 12 vs. 42 +/- 6 pM), but reduced peak insulin levels (126 +/- 30 vs. 468 +/- 102 pM, P < 0.01)..Detailed in vivo and in vitro assessment of insulin regulation of skeletal muscle glucose metabolism in lean NIDDM patients indicates that insulin action is intact in the muscle tissue of these patients.
• Mandarino, L. J., Kelley, D. E., Jain, A., & Consoli, A. (1993). Differential regulation of intracellular glucose metabolism by glucose and insulin in human muscle.. The American journal of physiology, 265(6 Pt 1), E898-905. doi:10.1152/ajpendo.1993.265.6.e898
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  Insulin and glucose stimulate glucose uptake in human muscle by different mechanisms. Insulin has well-known effects on glucose transport, glycogen synthesis, and glucose oxidation, but the effects of hyperglycemia on the intracellular routing of glucose are less well characterized. We used euglycemic and hyperglycemic clamps with leg balance measurements to determine how hyperglycemia affects skeletal muscle glucose storage, glycolysis, and glucose oxidation in normal human subjects. Glycogen synthase (GS) and pyruvate dehydrogenase complex (PDHC) activities were determined using muscle biopsies. During basal insulin replacement, hyperglycemia (11.6 +/- 0.31 mM) increased leg muscle glucose uptake (0.522 +/- 0.129 vs. 0.261 +/- 0.071 mumol.min-1 x 100 ml leg tissue-1, P < 0.05), storage (0.159 +/- 0.082 vs. -0.061 +/- 0.055, P < 0.05), and oxidation (0.409 +/- 0.080 vs. 0.243 +/- 0.085, P < 0.05) compared with euglycemia (6.63 +/- 0.33 mM). The increase in basal glucose oxidation due to hyperglycemia was associated with increased muscle PDHC activity (0.499 +/- 0.087 vs. 0.276 +/- 0.049, P < 0.05). However, the increase in leg glucose storage was not accompanied by an increase in muscle GS activity. During hyperinsulinemia, hyperglycemia (11.9 +/- 0.49 mM) also caused an additional increase in leg glucose uptake over euglycemia (6.14 +/- 0.42 mM) alone (5.75 +/- 1.25 vs. 3.75 +/- 0.58 mumol.min-1 x 100 ml leg-1, P < 0.05). In this case the major intracellular effect of hyperglycemia was to increase glucose storage (5.03 +/- 1.16 vs. 2.39 +/- 0.37, P < 0.05). At hyperinsulinemia, hyperglycemia had no effect on muscle GS or PDHC activity.(ABSTRACT TRUNCATED AT 250 WORDS)
• Simoneau, J. A., Mokan, M., Mandarino, L. J., & Kelley, D. E. (1993). Interaction between glucose and free fatty acid metabolism in human skeletal muscle.. The Journal of clinical investigation, 92(1), 91-8. doi:10.1172/jci116603
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  The mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.
• Sundarraj, N., Mandarino, L. J., Hassell, J. R., Hassell, H. R., & Finlayson, J. (1993). Regulation of fibronectin and laminin synthesis by retinal capillary endothelial cells and pericytes in vitro.. Experimental eye research, 57(5), 609-21. doi:10.1006/exer.1993.1166
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  Retinal capillaries are composed of endothelial cells resting on a basement membrane, in which are embedded pericytes. In diabetes mellitus, the basement membrane becomes thickened, and there is a loss of pericytes. The relative contributions of endothelial cells and pericytes to the synthesis of extracellular matrix proteins which are components of the basement membrane are not well-characterized. To determine how a selective loss of pericytes might affect the composition of retinal capillary basement membranes, we used primary cultures of bovine retinal capillary endothelial cells and pericytes to determine the forms and quantify the amounts of laminin and fibronectin synthesized and secreted by these cell types as well as to determine how high glucose concentrations alter these parameters. Results of ELISAs showed that pericyte cell/matrix layers contained nearly ten times more fibronectin than endothelial cells (288 +/- 24 vs. 34 +/- 5 ng micrograms-1 DNA, P < 0.001), but the amounts of laminin were similar. D-glucose (40 mM) tripled the amount of fibronectin incorporated into the endothelial cell/matrix layer (102 +/- 4 vs. 34 +/- 5 ng micrograms-1 DNA, P < 0.05), but had a lesser effect on pericytes. The non-metabolizable analogue L-glucose, also increased the amount of fibronectin incorporated in both pericyte and endothelial cell/matrix layers. The effects of D- and L-glucose on fibronectin secreted into the medium by both cell types were similar to the effects on incorporation of fibronectin into cell/matrix layers. Glucose had no effect on laminin synthesis. [35S]methionine radiolabeling and immunoprecipitation showed that pericytes and endothelial cells synthesize different forms of fibronectin. Both pericytes and endothelial cells synthesized an A and two B chains of laminin which were of similar apparent size, but the two cell types post-translationally modified the subunits differently. We conclude that pericytes and endothelial cells may contribute different forms and amounts of fibronectin and laminin to the retinal capillary basement membrane, so the preferential loss of pericytes in diabetes could result in basement membrane abnormalities which might lead to endothelial cell dysfunction.
• Mandarino, L. J. (1992). Current hypotheses for the biochemical basis of diabetic retinopathy.. Diabetes care, 15(12), 1892-901. doi:10.2337/diacare.15.12.1892
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  Diabetic retinopathy is one of the leading causes of vision loss in industrialized countries. Despite recent advances, the biochemical basis for the development of this diabetic complication is uncertain. Although retinal circulation is unique in that it is readily observable noninvasively, retinal tissue is extremely difficult to study in humans because of the problems inherent in obtaining fresh, appropriate biopsy material. Moreover, because of the difficulties in working with animal models of diabetic retinopathy, such as the dog, many investigators have turned to cell-culture models, especially those using primary cultures of retinal capillary endothelial cells and pericytes. Diabetic retinopathy involves both morphological and functional changes in the retinal capillaries. Morphological changes include basement membrane thickening and pericyte disappearance; functional changes include one important early change--increased permeability--which may be attributable to endothelial cell changes and basement membrane leakiness. Investigators have described major biochemical changes in cellular signaling pathways, including myo-inositol, inositol phosphates, and DAG metabolism, as well as decreased Na(+)-K(+)-ATPase and increased PKC activity. These defects may be related to the way endothelial cells and pericytes synthesize and interact with the extracellular matrix. Abnormalities in endothelial cell or pericyte interaction with the basement membrane may in turn lead to functional abnormalities, such as increased permeability.
• Mokan, M., Mandarino, L. J., & Kelley, D. E. (1992). Intracellular defects in glucose metabolism in obese patients with NIDDM.. Diabetes, 41(6), 698-706. doi:10.2337/diab.41.6.698
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  Skeletal muscle insulin resistance in obese patients with non-insulin-dependent diabetes mellitus (NIDDM) is characterized by decreased glucose uptake. Although reduced glycogen synthesis is thought to be the predominant cause for this deficit, studies supporting this notion often have been conducted at supraphysiological insulin concentrations in which glucose storage is the overwhelming pathway of glucose disposal. However, at lower, more physiological insulin concentrations, decreased muscle glucose oxidation could play a significant role. This study was undertaken to determine whether, under euglycemic conditions, insulin resistance for leg muscle glucose uptake in NIDDM patients is due primarily to decreased glucose storage or to oxidation. The leg balance technique and leg indirect calorimetry were used under steady-state euglycemic conditions to estimate muscle glucose uptake, storage, and oxidation in eight moderately obese NIDDM patients and eight matched-control subjects. Leg muscle biopsies also were performed to determine whether alterations in muscle pyruvate dehydrogenase or glycogen synthase activities could explain defects in glucose oxidation or storage. At insulin concentrations of approximately 500-600 pM, leg glucose uptake, oxidation, and storage in the NIDDM group (2.03 +/- 0.42, 1.00 +/- 0.13, 0.66 +/- 0.36 mumol.min-1.100 ml-1) were significantly lower (P less than 0.05) than rates in control subjects (5.14 +/- 0.64, 1.92 +/- 0.17, 2.80 +/- 0.54). Pyruvate dehydrogenase and glycogen synthase activities were also decreased, consistent with the in vivo metabolic defects. The average deficit in leg glucose uptake in NIDDM was 3.11 +/- 0.42 mumol.min-1.100 ml-1.(ABSTRACT TRUNCATED AT 250 WORDS)
• Nurjahan, N., Mandarino, L. J., Gerich, J. E., & Consoli, A. (1992). Skeletal muscle is a major site of lactate uptake and release during hyperinsulinemia.. Metabolism: clinical and experimental, 41(2), 176-9. doi:10.1016/0026-0495(92)90148-4
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  During conditions of increased glucose disposal, plasma lactate concentrations increase due to an increase in plasma lactate appearance. The tissue sites of the elevated lactate production are controversial. Although skeletal muscle would be a logical source of this lactate, studies using the limb net balance technique have failed to demonstrate a major change in net lactate output when plasma glucose disposal is increased. Because the limb balance technique underestimates production of a substrate when the limb not only produces but also consumes that substrate, we infused 3-14C-lactate basally and during a hyperinsulinemic euglycemic clamp in seven normal volunteers to determine plasma lactate appearance, forearm lactate fractional extraction, and forearm lactate uptake and release. After 3 hours of hyperinsulinemia, glucose and lactate turnovers increased from basal values of 11.8 +/- 0.13 and 12.2 +/- 0.59 to 32.6 +/- 3.4 and 16.5 +/- 1.07 mumol/(min.kg), accompanied by an increase in plasma lactate from 0.88 +/- 0.07 to 1.16 +/- 0.09 mmol/L (P less than .05). Forearm lactate extraction increased from 27% +/- 2% to 38% +/- 2% (P less than .001), resulting in an increase in forearm lactate uptake from 0.65 +/- 0.09 to 1.18 +/- 0.08 mumol/(min.100 mL tissue) (P less than .001). Although forearm lactate net output decreased during hyperinsulinemia, forearm lactate production increased from 1.04 +/- 0.12 basally to 1.69 +/- 0.13 mumol/(min.100 mL). When forearm data was extrapolated to whole body, muscle could account for 41% +/- 4% of systemic lactate appearance basally and 45% +/- 4% during hyperinsulinemia.(ABSTRACT TRUNCATED AT 250 WORDS)
• Veneman, T., Reilly, J. J., Nurjhan, N., Mitrakou, A., Mandarino, L. J., Kelley, D. E., Jenssen, T., Gerich, J. E., & Consoli, A. (1990). Contribution of impaired muscle glucose clearance to reduced postabsorptive systemic glucose clearance in NIDDM.. Diabetes, 39(2), 211-6. doi:10.2337/diab.39.2.211
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  The reduced postabsorptive rates of systemic glucose clearance in non-insulin-dependent diabetes mellitus (NIDDM) are thought to be the consequence of insulin resistance in peripheral tissues. Although the peripheral tissues involved have not been identified, it is generally assumed to be primarily muscle, the major site of insulin-mediated glucose disposal. To test this hypothesis, we measured postabsorptive systemic and forearm glucose utilization and clearance in 15 volunteers with NIDDM and 15 age- and weight-matched nondiabetic volunteers. Although systemic glucose utilization was increased in NIDDM subjects (14.5 +/- 0.5 vs. 11.2 +/- 0.2 mumol.kg-1.min-1, P less than 0.001), systemic glucose clearance was reduced 1.40 +/- 0.06 vs. 2.13 +/- 0.05 ml.kg-1.min-1, P less than 0.01). Although forearm glucose utilization was increased in NIDDM subjects (0.663 +/- 0.058 vs. 0.411 +/- 0.019 mumol.dl-1.min-1, P less than 0.001), forearm glucose dl-1 clearance was reduced (0.628 +/- 0.044 vs. 0.774 +/- 0.037 ml.L-1.min-1, P less than 0.01). However, extrapolation of forearm data to total-body muscle indicated that impaired clearance reduced muscle glucose disposal by only 61 +/- 21 mumol/min, whereas impaired systemic clearance reduced systemic glucose disposal by 662 +/- 82 mumol/min. Thus, impaired muscle glucose clearance accounted for less than 10% of the reduced systemic glucose clearance in NIDDM subjects. Therefore, we conclude that muscle insulin resistance plays only a minor role in the reduced systemic glucose clearance found in NIDDM in the postabsorptive state and propose that reduced brain glucose clearance is largely responsible.
• Mandarino, L. J., Gerich, J. E., & Campbell, P. J. (1988). Quantification of the relative impairment in actions of insulin on hepatic glucose production and peripheral glucose uptake in non-insulin-dependent diabetes mellitus.. Metabolism: clinical and experimental, 37(1), 15-21. doi:10.1016/0026-0495(88)90023-6
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  In non-insulin-dependent diabetes mellitus (NIDDM), both liver and peripheral tissues are resistant to insulin, but the relative severity and contribution of these abnormalities to fasting hyperglycemia are poorly understood. We, therefore, determined the dose-response characteristics for insulin-mediated suppression of hepatic glucose production (GP) and stimulation of peripheral glucose uptake (GU) in 14 NIDDM subjects and 14 age- and weight-matched nondiabetic volunteers (NV) using the glucose clamp sequential insulin infusion technique along with isotopic estimation of glucose flux. Postabsorptive rates of both GP (94 +/- 7 v 72 +/- 2 mg/M2/min in NV, P less than .01) and GU (88 +/- 5 v 72 +/- 2 in NV, P less than .01) were significantly increased in NIDDM subjects. The ED50 (half-maximally effective plasma insulin concentration) in NIDDM subjects for suppression of GP (64 +/- 14 microU/mL) and stimulation of GU (118 +/- 20 microU/mL were both increased more than twofold above normal (26 +/- 2 and 58 +/- 5 microU/mL, respectively, both P less than .01) and were significantly correlated with one another (r = .68, P less than .01). Although GP could be totally suppressed in the NIDDM subjects, their maximal GU was reduced 30% (287 +/- 20 v 372 +/- 15 mg/m2/min in NV, P less than .01). Nevertheless, at all physiologically relevant plasma insulin concentrations studied, there was comparable impairment in GP and GU responses. Moreover, fasting plasma glucose concentrations in NIDDM subjects were highly correlated with their increased basal rates of GP (r = .81, P less than .005) but not with their reduced GU.(ABSTRACT TRUNCATED AT 250 WORDS)
• Wright, K. S., Mandarino, L. J., Kolterman, O. G., & Beck-nielsen, H. (1988). Decreased activation of skeletal muscle glycogen synthase by mixed-meal ingestion in NIDDM.. Diabetes, 37(4), 436-40. doi:10.2337/diab.37.4.436
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  Glycogen synthase (GS) catalyzes the formation of glycogen in human skeletal muscle, the tissue responsible for disposal of a significant portion of an oral carbohydrate load. Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by fasting and postprandial hyperglycemia in conjunction with reduced rates of insulin-stimulated glucose disposal and storage in peripheral tissues, including muscle. Our objectives in this study were to determine whether ingestion of a mixed meal activates GS in control nondiabetic subjects and whether meal-related GS activation is reduced in NIDDM. To accomplish this, mixed formula meals were administered to 11 NIDDM and 9 age- and weight-matched nondiabetic control subjects. Plasma glucose and insulin values were measured before and for 90 min after meal ingestion. Skeletal muscle biopsies were performed just before and 90 min after meal ingestion for measurement of GS activity. Compared with control subjects, NIDDM subjects had significantly higher postprandial hyperglycemia and reduced postprandial hyperinsulinemia. GS was activated by meal ingestion in control subjects to a significantly greater extent than in NIDDM subjects. In NIDDM subjects, activation of GS was inversely correlated with fasting plasma glucose (r = .69, P less than .05). Therefore, NIDDM is characterized by reduced activation of a key step in the process of muscle glycogen repletion after a meal. Reduced activation of GS by a mixed meal in NIDDM may contribute to the reduced glucose disposal after a meal, thus contributing to the hyperglycemia observed in these subjects.
• Wright, K. S., Verity, L. S., Nichols, J., Mandarino, L. J., Kolterman, O. G., Bell, J. M., & Beck-nielsen, H. (1987). Effects of insulin infusion on human skeletal muscle pyruvate dehydrogenase, phosphofructokinase, and glycogen synthase. Evidence for their role in oxidative and nonoxidative glucose metabolism.. The Journal of clinical investigation, 80(3), 655-63. doi:10.1172/jci113118
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  To determine whether activation by insulin of glycogen synthase (GS), phosphofructokinase (PFK), or pyruvate dehydrogenase (PDH) in skeletal muscle regulates intracellular glucose metabolism, subjects were studied basally and during euglycemic insulin infusions of 12, 30, and 240 mU/m2 X min. Glucose disposal, oxidative and nonoxidative glucose metabolism were determined. GS, PFK, and PDH were assayed in skeletal muscle under each condition. Glucose disposal rates were 2.37 +/- 0.11, 3.15 +/- 0.19, 6.71 +/- 0.44, and 11.7 +/- 1.73 mg/kg X min; glucose oxidation rates were 1.96 +/- 0.18, 2.81 +/- 0.28, 4.43 +/- 0.32, and 5.22 +/- 0.52. Nonoxidative glucose metabolism was 0.39 +/- 0.13, 0.34 +/- 0.26, 2.28 +/- 0.40, and 6.52 +/- 1.21 mg/kg X min. Both the proportion of active GS and the proportion of active PDH were increased by hyperinsulinemia. PFK activity was unaffected. Activation of GS was correlated with nonoxidative glucose metabolism, while activation of PDH was correlated with glucose oxidation. Sensitivity to insulin of GS was similar to that of nonoxidative glucose metabolism, while the sensitivity to insulin of PDH was similar to that of glucose oxidation. Therefore, the activation of these enzymes in muscle may regulate nonoxidative and oxidative glucose metabolism.
• Hayles, A. B., Schwenk, W. F., Rizza, R. A., Mandarino, L. J., Haymond, M. W., Hayles, A. B., & Gerich, J. E. (1986). Familial insulin resistance and acanthosis nigricans. Presence of a postbinding defect.. Diabetes, 35(1), 33-7. doi:10.2337/diab.35.1.33
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  Type A insulin resistance, associated with acanthosis nigricans and menstrual irregularity, has been ascribed to a decreased concentration of insulin receptors. We now report four affected females from one family, a mother and three daughters (including identical twins) who appear to have the type A syndrome. Two of the kindred had no apparent ovarian dysfunction, while the other two had hyperprolactinemia without other findings of polycystic ovary disease, suggesting a genetic disease with variable penetrance. All had normal erythrocyte and monocyte insulin binding. Insulin dose-response studies to assess glucose metabolism and insulin sensitivity were performed in the affected twins. The dose response to insulin was shifted to the right with a decrease in maximal response. These results are consistent with a postbinding defect in insulin action in these patients.
• Olefsky, J. M., Olefsky, J. M., Mandarino, L. J., Kolterman, O. G., Bell, J. M., & Baron, A. D. (1985). Rates of noninsulin-mediated glucose uptake are elevated in type II diabetic subjects.. The Journal of clinical investigation, 76(5), 1782-8. doi:10.1172/jci112169
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  Although insulin is extremely potent in regulating glucose transport in insulin-sensitive tissues, all tissues are capable of taking up glucose by facilitated diffusion by means of a noninsulin-mediated glucose uptake (NIMGU) system. Several reports have estimated that in the postabsorptive state the majority of glucose disposal occurs via a NIMGU mechanism. However, these estimates have been either derived or extrapolated in normal humans. In the present study we have directly measured NIMGU rates in 11 normal (C) and 7 Type II noninsulin-dependent diabetic subjects (NIDDM; mean +/- SE fasting serum glucose, 249 +/- 24 mg/dl). To accomplish this, the serum glucose was clamped at a desired level during a period of insulin deficiency induced by a somatostatin infusion (SRIF, 550 micrograms/h). With a concomitant [3-3H]glucose infusion, we could isotopically quantitate glucose disposal rates (Rd) during basal (basal insulin present) and insulin-deficient (SRIF) conditions. With this approach we found that (a) basal Rd was greater in NIDDM than in C, 274 +/- 31 vs. 150 +/- 7 mg/min, due to elevated hepatic glucose output, (b) NIMGU composes 75 +/- 5% of basal Rd in C and 71 +/- 4% in NIDDM, (c) NIDDMS have absolute basal NIMGU rates that are twice that of C (195 +/- 23 vs. 113 +/- 8 mg/min, P less than 0.05), (d) when C were studied under conditions of insulin deficiency (SRIF infusion) and at a serum glucose level comparable to that of the NIDDM group (250 mg/dl), their rates of NIMGU were the same as that of the NIDDM group (186 +/- 19 vs. 195 +/- 23 mg/min; NS). We conclude that (a) in the postabsorptive state, NIMGU is the major pathway for glucose disposal for both C and NIDDM; (b) for a given glucose level the efficiency of NIMGU (NIMGU divided by serum glucose level) is equal in C and NIDDM, but since basal Rd is elevated in NIDDMs their absolute basal rates of NIMGU are higher; and (c) elevated basal rates of NIMGU in NIDDM may play a role in the pathogenesis of the late complications of diabetes.
• Rizza, R. A., Mandarino, L. J., Gerich, J. E., Genest, J., Baker, B. A., Rizza, R. A., Mandarino, L. J., Gerich, J. E., Genest, J., & Baker, B. A. (1985). Production of insulin resistance by hyperinsulinaemia in man.. Diabetologia, 28(2), 70-5. doi:10.1007/bf00279918
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  It has been proposed that hyperinsulinaemia may cause or exacerbate insulin resistance. The present studies were undertaken to test this hypothesis in man. Glucose utilization, glucose production, and overall glucose metabolism at submaximally and maximally effective plasma insulin concentrations (approximately 80 and approximately 1700 mU/l), and monocyte and adipocyte insulin binding were measured in normal volunteers on two occasions: once after 40 h of hyperinsulinaemia (25-35 mU/l) produced by infusion of insulin and once after infusion of saline (75 mmol/l; plasma insulin approximately 10 mU/l). After 40 h of hyperinsulinaemia, glucose utilization and overall glucose metabolism at submaximally and maximally effective plasma insulin concentrations were both slightly, but significantly, reduced compared with values observed after the infusion of saline (p less than 0.05), whereas glucose production rates were unaffected. Monocyte and adipocyte binding were also unaffected. These results indicate that hyperinsulinaemia of the magnitude observed in insulin resistant states, such as obesity, can produce insulin resistance in man. Assuming that human insulin sensitive tissues possess spare insulin receptors and that monocyte and adipocyte insulin binding accurately reflect insulin binding in insulin-sensitive tissues, the decreased maximal responses to insulin and the lack of change in insulin binding suggest that this insulin resistance occurred at a post-binding site.
• Rizza, R. A., Marsh, H. M., Mandarino, L. J., Haymond, M. W., Gerich, J. E., Dimitriadis, G., Bergman, R. N., & Baker, B. (1985). Effect of thyroid hormone excess on action, secretion, and metabolism of insulin in humans.. The American journal of physiology, 248(5 Pt 1), E593-601. doi:10.1152/ajpendo.1985.248.5.e593
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  To determine the effect of thyroid hormone excess on insulin secretion, metabolism and action in humans, we examined intravenous glucose tolerance, glucose-induced insulin secretion, insulin clearance, monocyte insulin receptor binding, and the dose-response characteristics for the effects of insulin on glucose production, uptake, oxidation, and nonoxidative disposal in 10 normal volunteers for 14 days before and after oral administration of triiodothyronine (T3) in doses that increased plasma T3 to levels observed in spontaneous thyrotoxicosis (P less than 0.001). After T3 postabsorptive plasma glucose (P less than 0.05) and insulin (P less than 0.05) both increased; intravenous glucose tolerance was unaffected, but plasma insulin responses were increased (P less than 0.01); basal glucose production, uptake, and oxidation all increased (all P less than 0.05), whereas nonoxidative glucose disposal was unaffected (P = NS); monocyte insulin receptor binding increased (P less than 0.01) due to increased receptor affinity (P less than 0.05); and receptor number was not significantly altered (P = NS). Insulin clearance was increased. Insulin-induced suppression of glucose production was impaired (Km 22 +/- 3 vs. 37 +/- 7 microU/ml, P less than 0.02); maximal insulin-induced glucose uptake (10.7 +/- 0.6 vs. 13.0 +/- 0.9 mg X kg-1 X min-1, P less than 0.001) and oxidation (3.41 +/- 0.30 vs. 5.34 +/- 0.59 mg X kg-1 X min-1, P less than 0.001) were increased without a significant change in Km. However, submaximal rates of nonoxidative glucose disposal and glucose uptake were inappropriately low for the increased insulin receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)
• Mandarino, L. J. (1984). Insulin and glucagon binding to isolated hepatocytes of Egyptian sand rats (Psammomys obesus): evidence for an insulin receptor defect.. Comparative biochemistry and physiology. A, Comparative physiology, 78(3), 519-23. doi:10.1016/0300-9629(84)90589-9
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  To determine whether a defect in insulin binding could contribute to insulin resistance in Egyptian sand rats (Psammomys obesus), insulin binding to isolated hepatocytes from euglycemic sand rats was compared to that of normal Sprague-Dawley rats (Rattus norvegicus). Because of its potential importance in glucoregulation, glucagon binding to hepatocytes from these species was also measured. Hepatocytes of sand rats exhibit an almost complete lack of insulin receptors compared to hepatocytes from Sprague-Dawley rats, whereas there are numerous high affinity glucagon binding sites on sand rat hepatocytes. The lack of insulin binding to sand rat tissues is sufficient to entirely explain the insulin resistance seen in this species. Glucagon may be primarily responsible for glucose homeostasis in Psammomys obesus.
• Mandarino, L. J., Gottesman, I., & Gerich, J. E. (1984). Use of glucose uptake and glucose clearance for the evaluation of insulin action in vivo.. Diabetes, 33(2), 184-91. doi:10.2337/diab.33.2.184
• Olshan, A., Mandarino, L. J., Hall, L., & Gerich, J. E. (1984). Determination by factor analysis of the relative importance of impaired insulin secretion and insulin resistance in the type ii diabetes. Clinical research, 32(1).
• Rizza, R. A., Mandarino, L. J., Gerich, J. E., Brick, B., & Baker, B. (1984). Influence of changes in insulin receptor binding during insulin infusions on the shape of the insulin dose-response curve for glucose disposal in man.. The Journal of clinical endocrinology and metabolism, 58(2), 392-6. doi:10.1210/jcem-58-2-392
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  To determine the influence of insulin infusions used in dose-response studies on monocyte insulin binding, monocyte insulin binding and glucose disposal were measured in six normal subjects before and at the end of each of four sequential 2-h insulin infusions (0.4, 1.0, 2.0, and 10 mU kg-1 min-1). Monocyte insulin binding was unaltered at the end of the first three infusions (plasma insulin, 31 +/- 2 (SEM), 77 +/- 3, and 184 +/- 10 microU/ml) but was decreased after the last infusion (plasma insulin, 1730 +/- 125 microU/ml) at 0.2 through 10.2 ng/ml insulin concentrations in the binding assay (P less than 0.01). Using a one-site model, this could be ascribed to a decrease in insulin receptor affinity (1.54 +/- 0.26 vs. 2.27 +/- 0.48 X 10(8) M-1, P less than 0.05), whereas in a two-site model this appeared to be due to a decrease in high affinity binding sites (1,868 +/- 228 vs. 2,387 +/- 207, P less than 0.02). Nevertheless, insulin receptor occupancies estimated to occur during the insulin infusions were virtually identical whether preinsulin infusion binding data (745 +/- 72, 1,383 +/- 117, 2,572 +/- 302, and 10,092 +/- 1,708) or binding data at the end of each infusion (702 +/- 56, 1,367 +/- 150, 2,383 +/- 318, and 9,158 +/- 2,023) were used to calculate occupancy. These results indicate that although monocyte insulin binding decreased during dose-response experiments using sequential infusions of insulin, due to the concentrations of insulin at which this occurs this decrease did not alter the shape of the dose-response curve relating glucose disposal to monocyte insulin receptor occupancy.
• Rizza, R. A., Mandarino, L. J., Gerich, J. E., Genest, J., & Baker, B. (1984). Infusion of insulin impairs human adipocyte glucose metabolism in vitro without decreasing adipocyte insulin receptor binding.. Diabetologia, 27(3), 358-63. doi:10.1007/bf00304850
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  To determine whether hyperinsulinaemia can cause insulin resistance in man and, if so, whether this occurs at a receptor or post-receptor site, nine normal volunteers were infused with insulin for 6 h at a rate (2 mU X kg-1 X min-1) which resulted in steady-state plasma insulin concentrations of 140 +/- 13 mU/l and four subjects were infused with saline (0.45%). Isolated adipocytes and monocytes were used as models for studying insulin binding, while adipocytes were also used to study insulin action in vitro. Adipocyte insulin binding did not decrease following infusion of insulin (4.6 +/- 0.5 versus 4.4 +/- 0.4% per 2 X 10(5) cells, before and after, respectively), whereas monocyte insulin binding did (7.2 +/- 0.6 versus 6.2 +/- 0.6% per 10(7) cells, p less than 0.05). Initial rates of adipocyte 3-0-methyl glucose transport were decreased in the absence of insulin (basal) and at submaximally effective (33.3 pmol/l) but not at maximally effective insulin concentrations. At all insulin concentrations and in the absence of insulin, rates of glucose conversion to lipids were decreased more than 50% (p less than 0.05), whereas rates of glucose oxidation were unaffected. This decrease in the rates of conversion of glucose to lipids could not be accounted for by the decrease in rates of glucose transport. These results suggest that hyperinsulinaemia can cause insulin resistance in man and that, at least initially, this occurs at a post-receptor site. Furthermore, the discordant effect of hyperinsulinaemia on monocyte and adipocyte insulin binding indicates that monocyte insulin binding may not always reflect insulin binding in insulin-sensitive tissues.
• Schwenk, W. F., Rizza, R. A., Mandarino, L. J., Haymond, M. W., & Hayles, A. B. (1984). MECHANISM OF DECREASED INSULIN ACTION IN TYPE A FAMILIAL INSULIN RESISTANCE. Pediatric Research, 18(4), 177-177. doi:10.1203/00006450-198404001-00504
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  Type A insulin resistance (associated with acanthosis nigricans and ovarian dysfunction in adolescent females) has been ascribed to decreased insulin receptors or to a postreceptor defect. Four affected females from one family (including a set of twins) with acanthosis nigricans and varying degrees of male habitus, acral hypertrophy, and muscle cramps had normal oral glucose tolerance, but increased fasting (64-170 μU/ml; control 13±1 μU/ml) and peak post-glucose (244-1010 μU/ml; control 90±1 μU/ml) plasma insulins (IRI). To test for a post-receptor defect, the twins underwent euglycemic glucose clamps with plasma IRI varied from 65 to 1600 μU/ml. The insulin dose response curves were shifted to the right with 1/2 max IRI responses (Km) of 135 and 210 μU/ml vs. 72±8 μU/ml in controls. Glucose infusion rates required to maintain euglycemia at 1600 μU/ml (Vmax) were decreased (5.7 and 8.3 vs. 12.0±0.3 mg/kg·min in controls). Decreased insulin action was not due to anti-insulin receptor antibodies or obesity (as assessed by 3H2O). In the one twin in which it was measured, insulin binding to monocytes (7.0% per 107 monocytes) and erythrocytes (3.0 % per 1.6 × 109 RBC's) was less than that observed in controls (10.4±0.8 and 4.8±.5, respectively). Conclusion: Because of a decreased maximal response (Vmax) and sensitivity (Km) to infused insulin, type A insulin resistance may be due to both receptor and post-receptor defects. When a patient with type A insulin resistance is identified, a familial incidence should be sought.
• Tutwiler, G., Tsalikian, E., Rizza, R. A., Marsh, H. M., Mandarino, L. J., Haymond, M. W., Handwerger, B. S., Carney, A. J., Buerklin, E. M., & Bartold, S. (1984). Mechanism of hyperglycemia and response to treatment with an inhibitor of fatty acid oxidation in a patient with insulin resistance due to antiinsulin receptor antibodies.. The Journal of clinical endocrinology and metabolism, 59(4), 658-64. doi:10.1210/jcem-59-4-658
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  Severe hyperglycemia and insulin resistance due to antiinsulin receptor antibodies developed over a period of 3 months in a 50-yr-old insulin-requiring diabetic patient. The hyperglycemia resulted from overproduction of glucose due to excessive rates of glycogenolysis and gluconeogenesis rather than decreased glucose utilization. Treatment with methyl-2-tetradecylglycidate, an inhibitor of fatty acid oxidation, resulted in a decrease in plasma glucose concentration. This was associated with a decrease in the rate of glucose production due to decreases in both gluconeogenesis and glycogenolysis rates, as well as an increase in the respiratory quotient. Plasma glucose concentrations continued to respond to the drug for the next 2 months until the sudden development of terminal hypoglycemia. The hypoglycemic action of the drug is consistent with the existence of an insulin-independent effect of fatty acid oxidation on glucose metabolism in man.
• Zacchei, A. G., Veber, D. F., Vandlen, R. L., Tocco, D. J., Saperstein, R., R, H. D., Perlow, D. S., Paleveda, W. J., Nutt, R. F., Mandarino, L. J., Hoff, D. R., Hirschmann, R., Hall, L. D., Gerich, J. E., Freidinger, R. M., F, H. R., Curley, P. E., Cordes, E. H., Colton, C. D., , Brady, S. F., et al. (1984). A super active cyclic hexapeptide analog of somatostatin.. Life sciences, 34(14), 1371-8. doi:10.1016/0024-3205(84)90009-2
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  The cyclic hexapeptide, cyclo (Pro-Phe-D-Trp-Lys-Thr-Phe), I, has been shown to have the biological properties of somatostatin. We now report structure-activity studies which optimize the potency of this cyclic hexapeptide series with the synthesis of cyclo (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe), II, which is 50-100 times more potent than somatostatin for the inhibition of insulin, glucagon and growth hormone release. The hydroxyl group of tyrosine is seen to lend a 10-fold enhancement to the potency. Potency also is found to be correlated with hydrophobicity. II is found to improve the control of postprandial hyperglycemia in diabetic animals when given in combination with insulin. The analog is found to be quite stable in the blood and in the gastrointestinal tract, but the bioavailability after oral administration is only 1-3%. The biological properties and long duration of II should allow clinical evaluation of the inhibition of glucagon release as an adjunct to insulin in the treatment of patients with diabetes.
• Mandarino, L. J., Gottesman, I., & Gerich, J. E. (1983). Estimation and kinetic analysis of insulin-independent glucose uptake in human subjects.. The American journal of physiology, 244(6), E632-5. doi:10.1152/ajpendo.1983.244.6.e632
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  Using the glucose clamp technique, glucose uptake was determined isotopically in normal human volunteers at plasma glucose concentrations of congruent to 60, 95, and 160 mg/dl during insulin infusions that increased plasma insulin to congruent to 20, 80, and 160 microU/ml. Because glucose uptake was found to be a linear function of plasma insulin at each plasma glucose concentration (r greater than 0.92, P less than 0.01), glucose uptake at 0 plasma insulin was estimated by linear regression analysis. The values thus derived (1.30, 1.62, and 2.59 mg . kg-1 . min-1 for plasma glucose concentrations of 60, 95, and 160 mg/dl, respectively) produced a linear Eadie-Hofstee plot, suggesting that insulin-independent glucose uptake followed Michaelis-Menten kinetics. The Km for glucose uptake at 0 plasma insulin (congruent to 10 mM) was similar to those observed for glucose uptake at the other plasma insulin concentrations studied (congruent to 9-12 mM), but its Vmax was less (5.2 vs. 6.4, 18.5, and 26.8 mg . kg-1 . min-1 for congruent to 20, 80, and 160 U/ml, respectively). These results indicate that in postabsorptive human subjects 75-85% of glucose uptake is noninsulin-mediated and provide additional support for the concept that insulin may increase glucose uptake merely by providing additional transport sites. The method described herein provides an assessment of insulin-independent glucose uptake in vivo that may prove useful in distinguishing between intrinsic defects of the glucose transport system and those due to defects in insulin action.
• Rizza, R. A., Mandarino, L. J., & Gerich, J. E. (1982). Cortisol-induced insulin resistance in man: impaired suppression of glucose production and stimulation of glucose utilization due to a postreceptor detect of insulin action.. The Journal of clinical endocrinology and metabolism, 54(1), 131-8. doi:10.1210/jcem-54-1-131
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  The present studies were undertaken to assess the mechanisms responsible for cortisol-induced insulin resistance in man. The insulin dose-response characteristics for suppression of glucose production and stimulation of glucose utilization and their relationship to monocyte and erythrocyte insulin receptor binding were determined in six normal volunteers after 24-h infusion of cortisol and 24-h infusion of saline. The infusion of cortisol (2 microgram kg-1 min-1) increased the plasma cortisol concentration approximately 4-fold (37 +/- 3 vs. 14 +/- 1 microgram/dl; P less than 0.01) to values observed during moderately severe stress in man. This hypercortisolemia increased postabsorptive plasma glucose (126 +/- 2 vs. 97 +/- 2 mg/dl; P less than 0.01) and plasma insulin (16 +/- 2 vs. 10 +/- 2 microU/ml; P less than 0.01) concentrations and rates of glucose production (2.4 +/- 0.1 vs. 2.1 +/- -0.1 mg kg-1 min-1; P less than 0.01) and utilization (2.5 +/- 0.1 vs. 2.1 +/- 0.1 mg kg-1 min -1; P less than 0.01). Insulin dose-response curves for both suppression of glucose production (half-maximal response at 81 +/- 19 vs. 31 +/ 5 microU/ml; P less than 0.05) and stimulation of glucose utilization (half-maximal response at 104 +/- 9 vs. 64 +/- 7 microU/ml; P less than 0.01) were shifted to the right, with preservation of normal maximal responses to insulin. Neither monocyte nor erythrocyte insulin binding was decreased. However, except at near-maximal insulin receptor occupancy, the action of insulin on glucose production and utilization per number of monocyte and erythrocyte insulin receptors occupied was decreased. These results indicate that the cortisol-induced insulin resistance in man is due to the decrease in both hepatic and extrahepatic sensitivity to insulin. Assuming that insulin binding to monocytes and erythrocytes reflects insulin binding in insulin-sensitive tissues, this decrease in insulin action can be explained on the basis of a postreceptor defect.
• Rizza, R. A., Mandarino, L. J., & Gerich, J. E. (1982). Effects of growth hormone on insulin action in man. Mechanisms of insulin resistance, impaired suppression of glucose production, and impaired stimulation of glucose utilization.. Diabetes, 31(8 Pt 1), 663-9. doi:10.2337/diab.31.8.663
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  The present studies were undertaken to assess the mechanisms responsible for growth hormone-induced insulin resistance in man. The insulin dose-response characteristics for suppression of glucose production and stimulation of glucose utilization and their relationship to monocyte insulin binding were determined in six normal volunteers after 12-h infusion of growth hormone and 12-h infusion of saline. The infusion of growth hormone (2 micrograms . kg-1 . h-1) increased plasma growth hormone nearly threefold (to congruent to 9 ng/ml) within the range observed during sleep and exercise. This increased plasma insulin (14 +/- 1 versus 8 +/- 1 microunits/ml, P less than 0.005) concentrations without significantly altering plasma glucose concentrations or basal rates of glucose production and utilization. Insulin dose-response curves for both suppression of glucose production (half-maximal response at 37 +/- 3 versus 20 +/- 3 microunits/ml, P less than 0.01) and stimulation of glucose utilization (half-maximal response at 98 +/- 8 versus 52 +/- 8 microunits/ml, P less than 0.01) were shifted to the right with preservation of normal maximal responses to insulin. Monocyte insulin binding was unaffected. Thus, except at near maximal insulin receptor occupancy, the action of insulin on glucose production and utilization per number of monocyte insulin receptors occupied was decreased. These results indicate that increases in plasma growth hormone within the physiologic range can cause insulin resistance in man, which is due to decreases in both hepatic and extrahepatic effects of insulin. Assuming that insulin binding to monocytes reflects insulin binding in insulin sensitive tissues, this decrease in insulin action can be explained on the basis of a postreceptor defect.
• Rizza, R. A., Mandarino, L. J., Gottesman, I., & Gerich, J. E. (1982). Insulin increases the Vmax without altering the Km for glucose uptake in man: Evidence that insulin accelerates glucose uptake merely by providing additional transport sites. Clinical research, 30(4).
• Verdonk, C., Rizza, R. A., Mandarino, L. J., Gottesman, I., & Gerich, J. E. (1982). Insulin increases the maximum velocity for glucose uptake without altering the Michaelis constant in man. Evidence that insulin increases glucose uptake merely by providing additional transport sites.. The Journal of clinical investigation, 70(6), 1310-4. doi:10.1172/jci110731
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  The present studies were undertaken to assess the mechanism by which insulin increases glucose uptake in man. Because glucose uptake in most mammalian tissues occurs predominantly by a facilitated transport system that follows Michaelis-Menten kinetics, glucose uptake was measured isotopically in normal volunteers over the physiologic range of plasma glucose and insulin concentrations and was subjected to Lineweaver-Burk and Eadie-Hofstee analysis. With both methods, increases in plasma insulin from 18 microunits/ml to 80 and 150 microunits/ml were found to increase the maximum velocity (Vmax) for glucose uptake nearly three- and fivefold, respectively, (P less than 0.025 and P less than 0.001) without significantly altering the Michaelis constant (Km). Because an increase in the affinity or molecular activity of transport sites or provision of additional transport sites that differed from those present basally should have altered the Km, whereas a mere increase in the number of transport sites would have only increased the Vmax, our results indicate that in man, insulin may increase glucose uptake merely by providing additional transport sites.
• Rizza, R. A., Mandarino, L. J., & Gerich, J. E. (1981). Dose-response characteristics for effects of insulin on production and utilization of glucose in man.. The American journal of physiology, 240(6), E630-9. doi:10.1152/ajpendo.1981.240.6.e630
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  To determine the dose-response characteristics for the effects of insulin on glucose production, glucose utilization, and overall glucose metabolism in normal man, 15 healthy subjects were infused with insulin for 8 h at sequential rates ranging from 0.2 to 5.0 mU.kg-1.min-1; each rate was used for 2 h. Glucose production and utilization were measured isotopically ([3-3H]glucose). Tissue insulin receptor occupancy was estimated from erythrocyte insulin binding. Glucose production was completely suppressed at plasma insulin concentrations of approximately 60 microunits/ml. Maximal glucose utilization (10-11 mg.kg-1.min-1) occurred at insulin concentrations of 200-700 microunits/ml. The concentration of insulin causing half-maximal glucose utilization (55 + 7 microunits/ml) was significantly greater than that required for half-maximal suppression of glucose production (29 +/- 2 microunits/ml, P less than 0.01). Maximal effects of insulin on glucose production and utilization occurred at plasma insulin concentrations causing 11 and 49% insulin receptor occupancy, respectively. The above dose-response relationships indicate that in man 1) glucose production is more sensitive to changes in plasma insulin concentration than is glucose utilization; 2) both hepatic and peripheral tissues may contain "spare" insulin receptors; and 3) relatively minor changes in plasma insulin concentration or insulin receptor function can cause appreciable alterations in glucose metabolism.
• Rizza, R. A., Mandarino, L. J., & Gerich, J. E. (1981). Mechanism and significance of insulin resistance in non-insulin-dependent diabetes mellitus.. Diabetes, 30(12), 990-5. doi:10.2337/diab.30.12.990
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  To determine whether receptor and/or postreceptor abnormalities of insulin action were responsible for insulin resistance in nonobese patients with non-insulin-dependent diabetes mellitus (NIDDM) and to assess the role of insulin resistance in their impaired glucose tolerance, insulin dose-response characteristics, insulin binding to monocytes, and insulin secretion were compared in 10 nonobese patients with NIDDM and six age-weight-matched nondiabetic volunteers. The insulin resistance of the diabetics was characterized by a shift to the right of their insulin dose-response curve (Km 81 +/- 4 microunits/ml vs. 58 +/- 2 microunits/ml in the nondiabetics P less than 0.001) but a normal maximal response to insulin. Although monocyte insulin binding was decreased in the diabetics (P less than 0.01), their response to insulin was appropriate for the number of insulin receptors occupied indicating normal postreceptor function. Insulin secretion was markedly reduced in diabetic subjects (52 +/- 22 vs. 471 +/- 90 microunits . ml-1 . 10 min-1 in the nondiabetic subjects, P less than 0.001) and was more strongly correlated with fasting plasma glucose (r = 0.92, P less than 0.001) and intravenous glucose tolerance (Kivgtt) (r = 0.98, P less than 0.001) than was insulin sensitivity (Km) (r = 0.23, NS, and r = 0.57, P less than 0.05, respectively). We conclude that in nonobese patients with NIDDM, insulin resistance is characterized by a shift to the right of the insulin dose-response curve, which can be accounted for solely by an insulin receptor defect. However, in these patients, impaired insulin secretion rather than insulin resistance appears to be the predominant metabolic abnormality.
• Stenner, D., Nissen, S. L., Mandarino, L. J., Ling, N., Guillemin, R., Gerich, J. E., Esch, F., Brazeau, P., Bohlen, P., & Blanchard, W. (1981). Selective effects of somatostatin-14, -25 and -28 on in vitro insulin and glucagon secretion.. Nature, 291(5810), 76-7. doi:10.1038/291076a0
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  The widely occurring tetradecapeptide somatostatin (SRIF-14) has been variously implicated as a neurotransmitter, a neurohormone, a cybernin (local regulatory factor) and a hormone. In the first isolation of SRIF-14 from hypothalamic extracts and subsequent extracts of other tissues, peptides of higher molecular weight but with similar activity have been noted. Recently two such peptides have been characterized as the 28-amino acid SRIF-28 (from porcine gastro-intestinal tract and porcine and ovine hypothalamus and the 25-amino acid SRIF-25 (from ovine hypothalamus), each of which consists of an N-terminal extension of SRIF-14. We now report that SRIF-28 and SRIF-25 are more potent than SRIF-14 in the inhibition of insulin release, but that SRIF-14 preferentially inhibits glucagon release. This suggests that SRIF-28 and SRIF-25 are not mere biosynthetic precursors of SRIF-14 and that their differential release may be physiologically important.
• Verdonk, C. A., Verdonk, C. A., Service, F. J., Rizza, R. A., Miles, J. M., Mandarino, L. J., Haymond, M. W., & Gerich, J. E. (1981). Pathogenesis of hypoglycemia in insulinoma patients: suppression of hepatic glucose production by insulin.. Diabetes, 30(5), 377-81. doi:10.2337/diab.30.5.377
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  To determine the mechanism by which hyperinsulinemia causes hypoglycemia in insulinoma patients, rates of glucose production and utilization, and circulating levels of insulin, glucagon, alanine, lactate, and glycerol were measured in 6 insulinoma patients during development of fasting hypoglycemia and in 8 normal volunteers studied over an identical interval. Initially, insulinoma patients had a greater plasma insulin (42 +/- 9 versus 15 +/- 1 microunits/ml) and glucagon levels (214 +/- 31 versus 158 +/- 21 pg/ml) than normal subjects, P less than 0.05, but their plasma glucose levels (81 +/- 4 mg/dl) and rates of glucose production and utilization (1.71 +/- 0.08 and 1.74 +/- 0.08 mg/kg . min, respectively) were not significantly different from those of normal subjects (93 +/- 2 mg/dl, 1.93 +/- 0.11, and 1.92 +/- 0.13 mg/kg . min, respectively). During a subsequent 8-h fast, glucose production and glucose utilization decreased in both groups, but more markedly in insulinoma patients. Since glucose utilization exceeded glucose production to a greater extent in insulinoma patients than in normal subjects, plasma glucose decreased to 44 +/- 3 mg/dl in insulinoma patients, but only to 84 +/- 1 mg/dl in normal subjects (P less than 0.001). Glucose utilization in insulinoma patients never exceeded that of normal subjects. These results demonstrate that fasting hypoglycemia in the insulinoma patients is usually due to suppression of glucose production rather than to acceleration of glucose utilization, as is widely thought. A direct effect of insulin on the liver is probably responsible, since circulating levels of gluconeogenic precursors are normal and since plasma glucagon increases during development of hypoglycemia in insulinoma patients.
• Mandarino, L. J., Itoh, M., & Gerich, J. E. (1980). Antisomatostatin gamma globulin augments secretion of both insulin and glucagon in vitro: evidence for a physiologic role for endogenous somatostatin in the regulation of pancreatic A- and B-cell function.. Diabetes, 29(9), 693-6. doi:10.2337/diab.29.9.693
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  To assess the effects of endogenous somatostatin on pancreatic islet A- and B-cell function, isolated rat islets were incubated in antisomatostatin gamma-globulin to bind endogenously released somatostatin, and the insulin and glucagon secretion of these islets was compared with that of islets incubated in gamma-globulin isolated from nonimmune serum. Islets incubated in antisomatostatin gamma-globulin released significantly more insulin at 4, 8, 16, and 32 mM glucose and significantly more glucagon at 8, 16, and 32 mM glucose, P < 0.05-0.005. For glucose-stimulated insulin release the threshold was decreased, the Vmax was increased, but the apparent Km was unaltered; for glucose-suppression of glucagon release the threshold was increased, maximal suppression was decreased, but the apparent Ki was unaltered. The augmentative effect of the antisomatostatin gamma-globulin was most prominent at 4 mM glucose for insulin release and at 8mM glucose for glucagon release, but was not limited to glucose since both insulin and glucagon release stimulated by arginine were also augmented by antisomatostatin gamma-globulin. These results provide evidence that endogenous somatostatin may act as a physiologic local regulator of both insulin and glucagon secretion and that its effect on insulin and glucagon secretion is dependent on the prevailing glucose concentration.
• Patton, G. S., Mandarino, L. J., Itoh, M., Gerich, J. E., & Blanchard, W. (1980). Stimulation of insulin release in the absence of extracellular calcium by isobutylmethylxanthine and its inhibition by somatostatin.. Endocrinology, 106(2), 430-3. doi:10.1210/endo-106-2-430
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  It has been suggested that somatostatin may inhibit insulin release by interfering with pancreatic islet calcium uptake. To further investigate this hypothesis, the effect of somatostatin on insulin release was examined under conditions where islet uptake of calcium would be unlikely to occur. The phosphodiesterase inhibitor, isobutylmethylxanthine (0.75 mM), was found to stimulate biphasic insulin release from rat pancreases perfused in vitro in the absence of added extracellular calcium on a background of 0.3 mM EGTA And 8 mM glucose; these results support previous suggestions that methylxanthine phosphodiesterase inhibitors may stimulate insulin release by increasing islet cytosol free calcium through translocation of bound (stored) intraislet calcium. Somatostatin (1.0 muM) completely inhibited both phases of isobutylmethylxanthine-stimulated insulin release. Since uptake of extracellular calcium by islets was unlikely under the present experimental conditions, these results suggest that somatostatin inhibition of insulin release is probably due to interference with a cAMP-dependent translocation of intraislet calcium, or to interference with some other effects of cAMP or an effect of calcium itself rather than to interference with islet calcium uptake.
• Rizza, R. A., Mandarino, L. J., & Gerich, J. E. (1980). Single day insulin dose-response assessment using sequential infusions of insulin and the glucose clamp technique. Clinical research, 28(1).
• Mandarino, L. J., Itoh, M., & Gerich, J. E. (1979). Endogenous somatostatin modulates both insulin and glucagon secretion. Clinical research, 27(4).
• Rizza, R. A., Mandarino, L. J., Gerich, J. E., & Brick, B. (1979). Effect of feeding and fasting on diurnal variation of insulin binding to circulating human monocytes. Clinical research, 27(4).

Presentations

• Zapata Bustos, R., Langlais, P. R., Coletta, D. K., De Filippis, E. A., Grandjean, D. N., & Mandarino, L. J. (2020, June). Lower Response of Connective Tissue Growth Factor (CTGF) to Exercise Characterizes Insulin Resistant Muscle. American Diabetes Association 80th scientific sessions. Chicago, IL: American Diabetes Association.
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  Rocio Zapata-Bustos, Paul Langlais, Dawn Coletta, Elena A. De Filippis, Danielle Grandjean, Lawrence J. Mandarino Lower Response of Connective Tissue Growth Factor (CTGF) to Exercise Characterizes Insulin Resistant Muscle

Poster Presentations

• Batty, S. R., Roman, M., McCrary, D., Moberly, A. P., Lipinski, A. A., Barker, N. K., Krantz, J., & Langlais, P. R. (2021, January). Identification of a Role for G2L1 in Insulin Action. Undergraduate Biology Research Program.
• Mandarino, L. J., Grandjean, D. N., De Filippis, E. A., Coletta, D. K., Langlais, P. R., & Zapata Bustos, R. (2020, June/Summer). Lower Response of Connective Tissue Growth Factor (CTGF) to Exercise Characterizes Insulin Resistant Muscle. American Diabetes Association 80th scientific sessions. Chicago, IL: American Diabetes Association.
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  Rocio Zapata-Bustos, Paul Langlais, Dawn Coletta, Elena A. De Filippis, Danielle Grandjean, Lawrence J. MandarinoLower Response of Connective Tissue Growth Factor (CTGF) to Exercise Characterizes Insulin Resistant Muscle
• Rafikova, O., Rafikova, O., Rafikova, O., Mandarino, L. J., Mandarino, L. J., Mandarino, L. J., Zemskov, E., Zemskov, E., Zemskov, E., Langlais, P. R., Langlais, P. R., Langlais, P. R., Desai, A., Desai, A., Desai, A., Srivastava, A., Srivastava, A., Srivastava, A., Rafikov, R., , Rafikov, R., et al. (2018, May). Free heme-mediated endothelial barrier dysfunction contributes to the development of pulmonary hypertension. American Thoracic Society.
• Langlais, P. R., & Mandarino, L. J. (2017, November). UA Department of Medicine Quantitative Proteomics Lab. UA College of Medicine Junior Investigators Symposium.
• Langlais, P. R., & Mandarino, L. J. (2017, October). UA Department of Medicine Quantitative Proteomics Lab. UA College of Medicine Innovation Symposium.
• McBride, M. L., Rafikova, O., Williams, E. R., Rafikov, R., Langlais, P. R., Mandarino, L. J., Mandarino, L. J., Langlais, P. R., Williams, E. R., Rafikov, R., McBride, M. L., & Rafikova, O. (2017, June). Inositol Monophosphate 1 (IMPA1) And Rage Interaction: The Role Of Novel Proliferative Pathway In Pulmonary Hypertension. American Heart Association.
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  This is a very first evidence that damage induced RAGE activation is involved in protection from glycolysis-mediated osmotic stress and activation of Akt pathway in PAH by the formation of RAGE/IMPA1 complex.
• Rafikov, R., Srivastava, A., Desai, A., Langlais, P. R., Zemskov, E., Mandarino, L. J., & Rafikova, O. (2017, June). Hemolysis-mediated vascular permeability in lungs contributes to the development of pulmonary hypertension. American Heart Association.
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  Our data indicate that PH patients have increased levels of free Hb in plasma that correlate with disease severity and progression. There is also a significant accumulation of free Hb and depletion of haptoglobin in the sugen/hypoxia rat model. In rats, perivascular edema was observed during first two weeks of PH concomitant with increased infiltration of inflammatory cells. In the cell culture model of HLMVECs, we found that not hemoglobin but free heme-induced endothelial permeability via activation of the p38/HSP27 signaling pathway. Indeed, the rat model also exhibited an increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling; there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF1a during the first two weeks of PAH regardless of hypoxic conditions. We found that heme-mediated effects on endothelium, at least in part, depend on Heme Carrier Protein 1 (HCP-1) and pharmacological inhibition of HCP-1 by sulfasalazine reduced barrier disruptive potential of the heme. Sulfasalazine administration to sugen/hypoxia rats results in attenuation of PH by a reduction in vascular remodeling in the lungs as well as decreasing right heart hypertrophy.