Lawrence J Mandarino
- Professor, Medicine
- Division Chief, Division of Endocrinology
- Director, UAHS Center for Disparities in Diabetes/Obesity-Metabolism
- Member of the Graduate Faculty
- Ph.D. Physical Anthropology
- Arizona State University, Tempe, Arizona, United States
- Enzyme Adaptation and Diabetes in Pima Indians
- M.A. Anthropology
- Arizona State University, Tempe, Arizona, United States
- B.A. Mathematics
- Arizona State University, Tempe, Arizona, United States
- University of Arizona College of Medicine (2016 - Ongoing)
- Mayo Medical School (2010 - 2016)
- Arizona State University, Tempe, Arizona (2004 - 2016)
- University of Texas Health Science Center (1992 - 2004)
- University of Pittsburgh, Pittsburgh, Pennsylvania (1986 - 1992)
- UC San Diego (1983 - 1986)
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.
DissertationCTS 920 (Spring 2022)
DissertationCTS 920 (Fall 2021)
Individualized Science WritingCTS 585 (Fall 2021)
DissertationCTS 920 (Spring 2021)
Honors ThesisECOL 498H (Spring 2020)
ResearchCTS 900 (Spring 2020)
Honors ThesisECOL 498H (Fall 2019)
ResearchCTS 900 (Fall 2019)
ResearchCTS 900 (Spring 2019)
ResearchCTS 900 (Fall 2018)
Honors Independent StudyPSIO 399H (Spring 2018)
- 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.More infoThe 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., Barakati, N., Langlais, P. R., Funk, J., Zapata Bustos, R., Coletta, D. K., Luo, M., Willis, W. T., & Mandarino, L. J. (2021). Site-specific acetylation of adenine nucleotide translocase 1 at lysine 23 in human muscle. Analytical biochemistry, 630, 114319.More infoEvidence 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.More infoThe 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.More infoNFU1 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.More infoVWA8 (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
- 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.More infoThe 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.More infoLong 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.More infoInsulin 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.
- 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).More infoPulmonary 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.More infoThis 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.More infoBrain-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.More infoWhile 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.More infoVWA8 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.More infovon 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.More infoPhysical 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.
- Mandarino, L. J., Willis, W. T., 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., Willis, W. T., & Mandarino, L. J. (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.
- 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.More infoAlthough 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.More infoTo 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.More infoObesity 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.
- 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.More infoObesity 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.More infoObesity 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.More infoCLASP2 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.RA117.000011More infoCLASP2 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.More infoThe 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.More infoThe 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.More infoDecreased 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.More infoObesity 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.More infoAlthough 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.
- 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.More infoThe 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.More infoOur 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.More infoTo 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.More infoImmunomodulatory 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.
- 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.More infoPlasma 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.More infoTo 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.More infoHealthy 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.
- 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.More infoThe 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.More infoThe 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.
- Mielke, C. J., Mandarino, L. J., & Dinu, V. (2014). AMASS: a database for investigating protein structures. Bioinformatics (Oxford, England), 30(11), 1595-600.More infoModern 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.More infoProteomics 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.More infoLifestyle 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.
- 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.More infoAdipose 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.More infoThe 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.More infoNew-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.More infoExercise 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.
- 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.More infoOlder 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.More infoLatinos 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.More infoAlthough 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.More infoProtein 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.More infoIn 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.More infoInsulin 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.More infoNonalcoholic 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.
- 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.More infoAdverse 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.More infoInsulin 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.More infoWe 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.More infoProtein-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.More infoAging 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.More infoThe 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.
- 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.More infoIn 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.
- 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.More infoType 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.More infoInsulin 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.More infoIn 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.More infoIntracellular 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.
- 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.More infoThe 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.
- 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.More infoAbnormalities 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.
- 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.More infoProtein 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.More infoMitochondria 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.
- 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.More infoThis 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.More infoMitochondrial 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.More infoChanges 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.More infoSkeletal 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.More infoLipid 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.More infoProtein-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.More infoThe 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.
- 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.More infoOur 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.More infoResearch 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.More infoInsulin 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.
- 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.More infoOversupply 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.More infoInsulin 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.More infoThe 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.More infoIGF-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.
- 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.More infoSkeletal 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.More infoAMPK 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.More infoSerine/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.More infoTo 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.More infoThe 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.More infoInsulin 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.More infoThe 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.
- 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.More infoSerine 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.
- 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.More infoIncreased 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.More infoTo 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.More infoExercise 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.More infoExercise 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.More infoMuscle 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.More infoWe 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.
- 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.More infoThe 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.More infoAcute 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.More infoThiazolidinediones (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.More infoType 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.
- 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.More infoTo 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.More infoSplanchnic 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.More infoExercise 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.More infoWe 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.More infoWe 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.More infoConsiderable 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., 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.More infoThe 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).
- 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., Mandarino, L. J., Langlais, P. R., Langlais, P. R., Mandarino, L. J., 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.More infoThis 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.More infoOur 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.