Sean W Limesand

Interests

Research

Perinatal Biology, Fetal Endocrinology, Islet Biology, and Developmental Origins of Health and DiseaseDisease emphasis: Diabetes Mellitus and Intrauterine Growth Restriction

Teaching

Endocrinology; Reproduction; Physiology

Courses

Scholarly Contributions

Chapters

• Yates, D., Chen, X., & Limesand, S. (2011). Effects of environment on placental function and fetal development. In Environmental Physiology of Livestock. Ames, Iowa: John Wiley & Sons.
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  Editor(s): Collier, RJ

Journals/Publications

• Blomberg, J., Luna Ramirez, R. I., Goyal, D., Limesand, S. W., & Goyal, R. (2023). Sexual dimorphic gene expression profile of perirenal adipose tissue in ovine fetuses with growth restriction. Frontiers in physiology, 14, 1179288.
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  Worldwide, fetal growth restriction (FGR) affects 7%-10% of pregnancies, or roughly 20.5 million infants, each year. FGR increases not only neonatal mortality and morbidity but also the risk of obesity in later life. Currently, the molecular mechanisms by which FGR "programs" an obese phenotype are not well understood. Studies demonstrate that FGR females are more prone to obesity compared to males; however, the molecular mechanisms that lead to the sexually dimorphic programming of FGR are not known. Thus, we hypothesized that FGR leads to the sexually dimorphic programming of preadipocytes and reduces their ability to differentiate into mature adipocytes. To test the hypothesis, we utilized a maternal hyperthermia-induced placental insufficiency to restrict fetal growth in sheep. We collected perirenal adipose tissue from near-term (∼140 days gestation) male and female FGR and normal-weight fetal lambs ( = 4 to 5 in each group), examined the preadipocytes' differentiation potential, and identified differential mRNA transcript expression in perirenal adipose tissue. Male FGR fetuses have a lower cellular density (nuclei number/unit area) compared to control male fetuses. However, no difference was observed in female FGR fetuses compared to control female fetuses. In addition, the ability of preadipocytes to differentiate into mature adipocytes with fat accumulation was impaired in male FGR fetuses, but this was not observed in female FGR fetuses. Finally, we examined the genes and pathways involved in the sexually dimorphic programming of obesity by FGR. On enrichment of differentially expressed genes in males compared to females, the Thermogenesis KEGG Pathway was downregulated, and the Metabolic and Steroid Biosynthesis KEGG pathways were upregulated. On enrichment of differentially expressed genes in male FGR compared to male control, the Steroid Biosynthesis KEGG Pathway was downregulated, and the PPAR Signaling KEGG pathway was upregulated. No pathways were altered in females in response to growth restriction in perirenal adipose tissue. Thus, the present study demonstrates a sexually dimorphic program in response to growth restriction in sheep fetal perirenal adipose tissue.
• Castillo-Salas, C. A., Luna-Nevárez, G., Reyna-Granados, J. R., Luna-Ramirez, R. I., Limesand, S. W., & Luna-Nevárez, P. (2023). Molecular markers for thermo-tolerance are associated with reproductive and physiological traits in Pelibuey ewes raised in a semiarid environment. Journal of thermal biology, 112, 103475.
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  Pelibuey sheep exhibit reproductive activity through the year, but warm weather lowers their fertility and demonstrates physiological limitations of environmental heat stress. Single nucleotide polymorphisms (SNPs) associated with heat stress tolerance in sheep have been reported previously. The objective was to validate the association of seven thermo-tolerance SNP markers with reproductive and physiological traits in Pelibuey ewes raised in a semiarid region. Pelibuey ewes were assigned to a cool (January 1st- March 31st; n = 101) or warm (April 1st- August 31st; n = 104) experimental group. All ewes were exposed to fertile rams and assessed for pregnancy diagnosis 90 days later; lambing day was reported at birth. These data served to calculate the reproductive traits of services per conception, prolificacy, days to estrus, days to conception, conception rate and lambing rate. Rectal temperature, rump/leg skin temperature and respiratory rate were measured and reported as physiological traits. Blood samples were collected and processed to extract DNA, which was genotyped using the TaqMan allelic discrimination method and qPCR. A mixed effects statistical model was used to validate associations between SNP genotypes and phenotypic traits. The SNPs rs421873172, rs417581105 and rs407804467 were confirmed as markers associated with reproductive and physiological traits (P 
• Goyal, D., Limesand, S. W., & Goyal, R. (2023). Vascular Stem Cells and the Role of B-Raf Kinase in Survival, Proliferation, and Apoptosis. International journal of molecular sciences, 24(8).
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  Neovascularization is an essential process in organismal development and aging. With aging, from fetal to adult life, there is a significant reduction in neovascularization potential. However, the pathways which play a role in increased neovascularization potential during fetal life are unknown. Although several studies proposed the idea of vascular stem cells (VSCs), the identification and essential survival mechanism are still not clear. In the present study, we isolated fetal VSCs from the ovine carotid artery and identified the pathways involved in their survival. We tested the hypothesis that fetal vessels contain a population of VSCs, and that B-Raf kinase is required for their survival. We conducted viability, apoptotic, and cell cycle stage assays on fetal and adult carotid arteries and isolated cells. To determine molecular mechanisms, we conducted RNAseq, PCR, and western blot experiments to characterize them and identify pathways essential for their survival. Results: A stem cell-like population was isolated from fetal carotid arteries grown in serum-free media. The isolated fetal VSCs contained markers for endothelial, smooth muscle, and adventitial cells, and formed a de novo blood vessel ex vivo. A transcriptomic analysis that compared fetal and adult arteries identified pathway enrichment for several kinases, including B-Raf kinase in fetal arteries. Furthermore, we demonstrated that B-Raf- Signal Transducer and Activator of Transcription 3 (STAT3)-Bcl2 is critical for the survival of these cells. Fetal arteries, but not adult arteries, contain VSCs, and B-Raf-STAT3-Bcl2 plays an important role in their survival and proliferation.
• Luna-Ramirez, R. I., Kelly, A. C., Anderson, M. J., Bidwell, C. A., Goyal, R., & Limesand, S. W. (2023). Elevated Norepinephrine Stimulates Adipocyte Hyperplasia in Ovine Fetuses With Placental Insufficiency and IUGR. Endocrinology, 165(1).
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  Prevailing hypoxemia and hypoglycemia in near-term fetuses with placental insufficiency-induced intrauterine growth restriction (IUGR) chronically increases norepinephrine concentrations, which lower adrenergic sensitivity and lipid mobilization postnatally, indicating a predisposition for adiposity. To determine adrenergic-induced responses, we examined the perirenal adipose tissue transcriptome from IUGR fetuses with or without hypercatecholaminemia. IUGR was induced in sheep with maternal hyperthermia, and hypercatecholaminemia in IUGR was prevented with bilateral adrenal demedullation. Adipose tissue was collected from sham-operated control (CON) and IUGR fetuses and adrenal-demedullated control (CAD) and IUGR (IAD) fetuses. Norepinephrine concentrations were lower in IAD fetuses than in IUGR fetuses despite both being hypoxemic and hypoglycemic. In IUGR fetuses, perirenal adipose tissue mass relative to body mass was greater compared with the CON, adrenal-demedullated control, and IAD groups. Transcriptomic analysis identified 581 differentially expressed genes (DEGs) in CON vs IUGR adipose tissue and 193 DEGs in IUGR vs IAD adipose tissue. Integrated functional analysis of these 2 comparisons showed enrichment for proliferator-activated receptor signaling and metabolic pathways and identified adrenergic responsive genes. Within the adrenergic-regulated DEGs, we identified transcripts that regulate adipocyte proliferation and differentiation: adipogenesis regulatory factor, C/CCAAT/enhancer binding protein α, and sterol carrier protein 2. DEGs associated with the metabolic pathway included pyruvate dehydrogenase kinase 4, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4, IGF-binding proteins (IGFBP-5 and IGFBP-7). Sex-specific expression differences were also found for adipogenesis regulatory factor, pyruvate dehydrogenase kinase 4, IGFBP5, and IGFBP7. These findings indicate that sustained adrenergic stimulation during IUGR leads to adipocyte hyperplasia with alterations in metabolism, proliferation, and preadipocyte differentiation pathways.
• Luna-Ramirez, R. I., Limesand, S. W., Goyal, R., Pendleton, A. L., Rincón, G., Zeng, X., Luna-Nevárez, G., Reyna-Granados, J. R., & Luna-Nevárez, P. (2023). Blood Transcriptomic Analyses Reveal Functional Pathways Associated with Thermotolerance in Pregnant Ewes Exposed to Environmental Heat Stress. Genes, 14(8).
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  Environmental heat stress triggers a series of compensatory mechanisms in sheep that are dependent on their genetic regulation of thermotolerance. Our objective was to identify genes and regulatory pathways associated with thermotolerance in ewes exposed to heat stress. We performed next-generation RNA sequencing on blood collected from 16 pregnant ewes, which were grouped as tolerant and non-tolerant to heat stress according to a physiological indicator. Additional samples were collected to measure complete blood count. A total of 358 differentially expressed genes were identified after applying selection criteria. Gene expression analysis detected 46 GO terms and 52 KEGG functional pathways. The top-three signaling pathways were p53, RIG-I-like receptor and FoxO, which suggested gene participation in biological processes such as apoptosis, cell signaling and immune response to external stressors. Network analysis revealed , , , , and as over-expressed genes with high regulatory potential. A co-expression network involving the immune-related genes , and was detected in lymphocytes and monocytes, which was consistent with hematological findings. In conclusion, transcriptomic analysis revealed a non-viral immune mechanism involving apoptosis, which is induced by external stressors and appears to play an important role in the molecular regulation of heat stress tolerance in ewes.
• Pendleton, A. L., Limesand, S. W., & Goyal, R. (2023). In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus. Journal of visualized experiments : JoVE.
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  The ability of an organism to maintain a constant blood flow to the brain in response to sudden surges in systemic blood pressure (BP) is known as cerebral autoregulation (CAR), which occurs in the carotid artery. In contrast to full-term neonates, preterm neonates are unable to reduce the cerebral blood flow (CBF) in response to increased systemic BP. In preterm neonates, this exposes the fragile cerebral vessels to high perfusion pressures, leading to their rupture and brain damage. Ex vivo studies using wire myography have demonstrated that carotid arteries from near-term fetuses constrict in response to the activation of adrenergic alpha1 receptors. This response is blunted in the preterm fetus. Thus, to examine the role of alpha1-AR in vivo, presented here is an innovative approach to determine the effects of drugs on a carotid arterial segment in vivo in an ovine fetus during the developmental progression of gestation. The presented data demonstrate the simultaneous measurement of fetal blood flow and blood pressure. The perivascular delivery system can be used to conduct a long-term study over several days. Additional applications for this method could include viral delivery systems to alter the expression of genes in a segment of the carotid artery. These methods could be applied to other blood vessels in the growing organism in utero as well as in adult organisms.
• Zhao, W., Kelly, A. C., Luna-Ramirez, R. I., Bidwell, C. A., Anderson, M. J., & Limesand, S. W. (2023). Decreased Pyruvate but Not Fatty Acid Driven Mitochondrial Respiration in Skeletal Muscle of Growth Restricted Fetal Sheep. International journal of molecular sciences, 24(21).
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  Fetuses with intrauterine growth restriction (FGR) have impaired oxidative and energy metabolism, with persistent consequences on their postnatal development. In this study, we test the hypothesis that FGR skeletal muscle has lower mitochondrial respiration rate and alters the transcriptomic profiles associated with energy metabolism in an ovine model. At late gestation, mitochondrial oxygen consumption rates (OCRs) and transcriptome profiles were evaluated in the skeletal muscle collected from FGR and control fetuses. The ex vivo mitochondrial OCRs were reduced < 0.01) in permeabilized FGR soleus muscle compared to the control muscle but only with pyruvate as the metabolic substrate. Mitochondrial OCRs were similar between the FGR and control groups for palmitoyl-carnitine (fatty acid-driven) or pyruvate plus palmitoyl-carnitine metabolic substrates. A total of 2284 genes were differentially expressed in the semitendinosus muscle from growth restricted fetuses (false discovery rate (FDR) ≤ 0.05). A pathway analysis showed that the upregulated genes (FGR compared to control) were overrepresented for autophagy, HIF-1, AMPK, and FOXO signaling pathways (all with an FDR < 0.05). In addition, the expression of genes modulating pyruvate's entry into the TCA cycle was downregulated, whereas the genes encoding key fatty acid oxidation enzymes were upregulated in the FGR muscle. These findings show that FGR skeletal muscle had attenuated mitochondrial pyruvate oxidation, possibly associated with the inability of pyruvate to enter into the TCA cycle, and that fatty acid oxidation might compensate for the attenuated energy metabolism. The current study provided phenotypic and molecular evidence for adaptive deficiencies in FGR skeletal muscle.
• Camacho, L. E., Davis, M. A., Kelly, A. C., Steffens, N. R., Anderson, M. J., & Limesand, S. W. (2022). Prenatal Oxygen and Glucose Therapy Normalizes Insulin Secretion and Action in Growth Restricted Fetal Sheep. Endocrinology.
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  Placental insufficiency (PI) lowers fetal oxygen and glucose concentrations, which disrupts glucose-insulin homeostasis and promotes fetal growth restriction (FGR). To date, prenatal treatments for FGR have not attempted to correct the oxygen and glucose supply simultaneously. Therefore, we investigated whether a five-day correction of oxygen and glucose concentrations in PI-FGR fetuses would normalize insulin secretion and glucose metabolism. Experiments were performed in near-term FGR fetal sheep with maternal hyperthermia-induced PI. Fetal arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction and glucose was infused into FGR fetuses (FGR-OG). FGR-OG fetuses were compared to maternal air insufflated, saline-infused fetuses (FGR-AS) and control fetuses. Prior to treatment, FGR fetuses were hypoxemic and hypoglycemic and had reduced glucose-stimulated insulin secretion (GSIS). During treatment, oxygen, glucose, and insulin concentrations increased, and norepinephrine concentrations decreased in FGR-OG fetuses, whereas FGR-AS fetuses were unaffected. On treatment day 4, glucose fluxes were measured with euglycemic and hyperinsulinemic-euglycemic clamps. During both clamps, rates of glucose utilization and production were greater in FGR-AS than FGR-OG fetuses, while glucose fluxes in FGR-OG fetuses were not different than control rates. After five-days of treatment, GSIS increased in FGR-OG fetuses to control levels and their ex vivo islet GSIS was greater than FGR-AS islets. Despite normalization in fetal characteristics, GSIS, and glucose fluxes, FGR-OG and FGR-AS fetuses weighed less than controls. These findings show that sustained, simultaneous correction of oxygen and glucose normalized GSIS and whole-body glucose fluxes in PI-FGR fetuses after the onset of FGR.
• Camacho, L. E., Davis, M. A., Kelly, A. C., Steffens, N. R., Anderson, M. J., & Limesand, S. W. (2022). Prenatal Oxygen and Glucose Therapy Normalizes Insulin Secretion and Action in Growth-Restricted Fetal Sheep. Endocrinology, 163(6).
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  Placental insufficiency (PI) lowers fetal oxygen and glucose concentrations, which disrupts glucose-insulin homeostasis and promotes fetal growth restriction (FGR). To date, prenatal treatments for FGR have not attempted to correct the oxygen and glucose supply simultaneously. Therefore, we investigated whether a 5-day correction of oxygen and glucose concentrations in PI-FGR fetuses would normalize insulin secretion and glucose metabolism. Experiments were performed in near-term FGR fetal sheep with maternal hyperthermia-induced PI. Fetal arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction and glucose was infused into FGR fetuses (FGR-OG). FGR-OG fetuses were compared with maternal air insufflated, saline-infused fetuses (FGR-AS) and control fetuses. Prior to treatment, FGR fetuses were hypoxemic and hypoglycemic and had reduced glucose-stimulated insulin secretion (GSIS). During treatment, oxygen, glucose, and insulin concentrations increased, and norepinephrine concentrations decreased in FGR-OG fetuses, whereas FGR-AS fetuses were unaffected. On treatment day 4, glucose fluxes were measured with euglycemic and hyperinsulinemic-euglycemic clamps. During both clamps, rates of glucose utilization and production were greater in FGR-AS than FGR-OG fetuses, while glucose fluxes in FGR-OG fetuses were not different than control rates. After 5 days of treatment, GSIS increased in FGR-OG fetuses to control levels and their ex vivo islet GSIS was greater than FGR-AS islets. Despite normalization in fetal characteristics, GSIS, and glucose fluxes, FGR-OG and FGR-AS fetuses weighed less than controls. These findings show that sustained, simultaneous correction of oxygen and glucose normalized GSIS and whole-body glucose fluxes in PI-FGR fetuses after the onset of FGR.
• Limesand, S. W., & Goyal, R. (2022). Epigenetic Modifications Guide Maturational Processes in Rat Pancreatic Islets. Endocrinology, 163(1).
• Liu, T., Li, R., Luo, N., Lou, P., Limesand, S. W., Yang, Y., Zhao, Y., & Chen, X. (2022). Hepatic Lipid Accumulation and Dysregulation Associate with Enhanced Reactive Oxygen Species and Pro-Inflammatory Cytokine in Low-Birth-Weight Goats. Animals : an open access journal from MDPI, 12(6).
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  Occurrence of low birth weight (LBW) is a major concern in livestock production, resulting in poor postnatal growth, lowered efficiency of feed utilization, and impaired metabolic health in adult life. In the southwest region of China, birth weight of indigenous strains of goats varies seasonally with lower weights in summer and winter, but the metabolic regulation of the LBW offspring is still unknown. In this study, by comparing LBW goats to normal birth weight group, we examined hepatic lipid content in association with regulatory mechanisms. Histological studies showed higher microvesicular morphology in the liver of LBW goats in accompany with a significantly higher level of hepatic free fatty acids, total triglycerides, and cholesterols. Lipid metabolism impairment, increased oxidative stress, and inflammation were observed by transcriptome analysis. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation further demonstrated lipid peroxidation, antioxidant pathway, and pro-inflammatory response involved in the hepatic lipid dysregulation from LBW group. Therefore, dysregulations of hepatic lipid metabolism, including fatty acid biosynthesis and degradation, lipid transportation, and oxidative stress, played important roles to contribute the lipid accumulation in LBW goats. Moreover, due to impaired antioxidant capacity, the oxidative damage could interact with persisting pro-inflammatory responses, leading to a higher risk of liver injury and metabolic syndromes in their adult life.
• Posont, R. J., Most, M. S., Cadaret, C. N., Marks-Nelson, E. S., Beede, K. A., Limesand, S. W., Schmidt, T. B., Petersen, J. L., & Yates, D. T. (2022). Primary myoblasts from intrauterine growth-restricted fetal sheep exhibit intrinsic dysfunction of proliferation and differentiation that coincides with enrichment of inflammatory cytokine signaling pathways. Journal of animal science, 100(8).
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  Intrauterine growth restriction (IUGR) is linked to lifelong reductions in muscle mass due to intrinsic functional deficits in myoblasts, but the mechanisms underlying these deficits are not known. Our objective was to determine if the deficits were associated with changes in inflammatory and adrenergic regulation of IUGR myoblasts, as was previously observed in IUGR muscle. Primary myoblasts were isolated from IUGR fetal sheep produced by hyperthermia-induced placental insufficiency (PI-IUGR; n = 9) and their controls (n = 9) and from IUGR fetal sheep produced by maternofetal inflammation (MI-IUGR; n = 6) and their controls (n = 7). Proliferation rates were less (P < 0.05) for PI-IUGR myoblasts than their controls and were not affected by incubation with IL-6, TNF-α, norepinephrine, or insulin. IκB kinase inhibition reduced (P < 0.05) proliferation of control myoblasts modestly in basal media but substantially in TNF-α-added media and reduced (P < 0.05) PI-IUGR myoblast proliferation substantially in basal and TNF-α-added media. Proliferation was greater (P < 0.05) for MI-IUGR myoblasts than their controls and was not affected by incubation with TNF-α. Insulin increased (P < 0.05) proliferation in both MI-IUGR and control myoblasts. After 72-h differentiation, fewer (P < 0.05) PI-IUGR myoblasts were myogenin+ than controls in basal and IL-6 added media but not TNF-α-added media. Fewer (P < 0.05) PI-IUGR myoblasts were desmin+ than controls in basal media only. Incubation with norepinephrine did not affect myogenin+ or desmin+ percentages, but insulin increased (P < 0.05) both markers in control and PI-IUGR myoblasts. After 96-h differentiation, fewer (P < 0.05) MI-IUGR myoblasts were myogenin+ and desmin+ than controls regardless of media, although TNF-α reduced (P < 0.05) desmin+ myoblasts for both groups. Differentiated PI-IUGR myoblasts had greater (P < 0.05) TNFR1, ULK2, and TNF-α-stimulated TLR4 gene expression, and PI-IUGR semitendinosus muscle had greater (P < 0.05) TNFR1 and IL6 gene expression, greater (P < 0.05) c-Fos protein, and less (P < 0.05) IκBα protein. Differentiated MI-IUGR myoblasts had greater (P < 0.05) TNFR1 and IL6R gene expression, tended to have greater (P = 0.07) ULK2 gene expression, and had greater (P < 0.05) β-catenin protein and TNF-α-stimulated phosphorylation of NFκB. We conclude that these enriched components of TNF-α/TNFR1/NFκB and other inflammatory pathways in IUGR myoblasts contribute to their dysfunction and help explain impaired muscle growth in the IUGR fetus.
• Davis, M. A., Davis, M. A., Camacho, L. E., Camacho, L. E., Pendleton, A. L., Pendleton, A. L., Antolic, A. T., Antolic, A. T., Luna-Ramirez, R. I., Luna-Ramirez, R. I., Kelly, A. C., Kelly, A. C., Steffens, N. R., Steffens, N. R., Anderson, M. J., Anderson, M. J., Limesand, S. W., & Limesand, S. W. (2021). Augmented glucose production is not contingent on high catecholamines in fetal sheep with IUGR. The Journal of endocrinology, 249(3), 195-207.
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  Fetuses with intrauterine growth restriction (IUGR) have high concentrations of catecholamines, which lowers the insulin secretion and glucose uptake. Here, we studied the effect of hypercatecholaminemia on glucose metabolism in sheep fetuses with placental insufficiency-induced IUGR. Norepinephrine concentrations are elevated throughout late gestation in IUGR fetuses but not in IUGR fetuses with a bilateral adrenal demedullation (IAD) at 0.65 of gestation. Euglycemic (EC) and hyperinsulinemic-euglycemic (HEC) clamps were performed in control, intact-IUGR, and IAD fetuses at 0.87 of gestation. Compared to controls, basal oxygen, glucose, and insulin concentrations were lower in IUGR groups. Norepinephrine concentrations were five-fold higher in IUGR fetuses than in IAD fetuses. During the EC, rates of glucose entry (GER, umbilical + exogenous), glucose utilization (GUR), and glucose oxidation (GOR) were greater in IUGR groups than in controls. In IUGR and IAD fetuses with euglycemia and euinsulinemia, glucose production rates (GPR) remained elevated. During the HEC, GER and GOR were not different among groups. In IUGR and IAD fetuses, GURs were 40% greater than in controls, which paralleled the sustained GPR despite hyperinsulinemia. Glucose-stimulated insulin concentrations were augmented in IAD fetuses compared to IUGR fetuses. Fetal weights were not different between IUGR groups but were less than controls. Regardless of norepinephrine concentrations, IUGR fetuses not only develop greater peripheral insulin sensitivity for glucose utilization but also develop hepatic insulin resistance because GPR was maintained and unaffected by euglycemia or hyperinsulinemia. These findings show that adaptation in glucose metabolism of IUGR fetuses are independent of catecholamines, which implicate that hypoxemia and hypoglycemia cause the metabolic responses.
• Jones, A. K., Rozance, P. J., Brown, L. D., Lorca, R. A., Julian, C. G., Moore, L. G., Limesand, S. W., & Wesolowski, S. R. (2021). Uteroplacental nutrient flux and evidence for metabolic reprogramming during sustained hypoxemia. Physiological reports, 9(18), e15033.
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  Gestational hypoxemia is often associated with reduced birth weight, yet how hypoxemia controls uteroplacental nutrient metabolism and supply to the fetus is unclear. This study tested the effects of maternal hypoxemia (HOX) between 0.8 and 0.9 gestation on uteroplacental nutrient metabolism and flux to the fetus in pregnant sheep. Despite hypoxemia, uteroplacental and fetal oxygen utilization and net glucose and lactate uptake rates were similar in HOX (n = 11) compared to CON (n = 7) groups. HOX fetuses had increased lactate and pyruvate concentrations and increased net pyruvate output to the utero-placenta. In the HOX group, uteroplacental flux of alanine to the fetus was decreased, as was glutamate flux from the fetus. HOX fetuses had increased alanine and decreased aspartate, serine, and glutamate concentrations. In HOX placental tissue, we identified hypoxic responses that should increase mitochondrial efficiency (decreased SDHB, increased COX4I2) and increase lactate production from pyruvate (increased LDHA protein and LDH activity, decreased LDHB and MPC2), both resembling metabolic reprogramming, but with evidence for decreased (PFK1, PKM2), rather than increased, glycolysis and AMPK phosphorylation. This supports a fetal-uteroplacental shuttle during sustained hypoxemia whereby uteroplacental tissues produce lactate as fuel for the fetus using pyruvate released from the fetus, rather than pyruvate produced from glucose in the placenta, given the absence of increased uteroplacental glucose uptake and glycolytic gene activation. Together, these results provide new mechanisms for how hypoxemia, independent of AMPK activation, regulates uteroplacental metabolism and nutrient allocation to the fetus, which allow the fetus to defend its oxidative metabolism and growth.
• Jones, A. K., Wang, D., Goldstrohm, D., Brown, L. D., Rozance, P. J., Limesand, S. W., & Wesolowski, S. R. (2021). Tissue specific responses that constrain glucose oxidation and increase lactate production with the severity of hypoxemia in fetal sheep. American journal of physiology. Endocrinology and metabolism.
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  Fetal hypoxemia decreases insulin and increases cortisol and norepinephrine concentrations and may restrict growth by decreasing glucose utilization and altering substrate oxidation. Specifically, we hypothesized that hypoxemia would decrease fetal glucose oxidation and increase lactate and pyruvate production. We tested this by measuring whole-body glucose oxidation and lactate production, and molecular pathways in liver, muscle, adipose, and pancreas tissues of fetuses exposed to maternal hypoxemia for 9 days (HOX) compared with control fetal sheep (CON) in late gestation. Fetuses with more severe hypoxemia had lower whole-body glucose oxidation rates, and HOX fetuses had increased lactate production from glucose. In muscle and adipose tissue, expression of the glucose transporter GLUT4 was decreased. In muscle, pyruvate kinase (PKM) and lactate dehydrogenase B (LDHB) expression was decreased. In adipose tissue, LDHA and lactate transporter (MCT1) expression was increased. In liver, there was decreased gene expression of PKLR and MPC2 and phosphorylation of PDH, and increased LDHA gene and protein abundance. LDH activity, however, was decreased only in HOX skeletal muscle. There were no differences in basal insulin signaling across tissues, nor differences in pancreatic tissue insulin content, beta cell area, or genes regulating beta cell function. Collectively, these results demonstrate coordinated metabolic responses across tissues in the hypoxemic fetus that limit glucose oxidation and increase lactate and pyruvate production. These responses may be mediated by hypoxemia induced endocrine responses including increased norepinephrine and cortisol, which inhibit pancreatic insulin secretion resulting in lower insulin concentrations and decreased stimulation of glucose utilization.
• Li, R., Huang, H., Limesand, S. W., & Chen, X. (2021). Pancreatic Islets Exhibit Dysregulated Adaptation of Insulin Secretion after Chronic Epinephrine Exposure. Current issues in molecular biology, 43(1), 240-250.
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  Chronic adrenergic stimulation is the dominant factor in impairment of the β-cell function. Sustained adrenergic exposure generates dysregulated insulin secretion in fetal sheep. Similar results have been shown in Min6 under the elevated epinephrine condition, but impairments after adrenergic removal are still unknown and a high rate of proliferation in Min6 has been ignored. Therefore, we incubated primary rats' islets with half maximal inhibitory concentrations of epinephrine for three days, then determined their insulin secretion responsiveness and related signals two days after removal of adrenaline via radioimmunoassay and qPCR. Insulin secretion was not different between the exposure group (1.07 ± 0.04 ng/islet/h) and control (1.23 ± 0.17 ng/islet/h), but total islet insulin content after treatment (5.46 ± 0.87 ng/islet/h) was higher than control (3.17 ± 0.22 ng/islet/h, < 0.05), and the fractional insulin release was 36% ( < 0.05) lower after the treatment. Meanwhile, the mRNA expression of Gαs, Gαz and Gβ1-2 decreased by 42.8% 19.4% and 24.8%, respectively ( < 0.05). Uncoupling protein 2 (Ucp2), sulphonylurea receptor 1 (Sur1) and superoxide dismutase 2 (Sod2) were significantly reduced (38.5%, 23.8% and 53.8%, < 0.05). Chronic adrenergic exposure could impair insulin responsiveness in primary pancreatic islets. Decreased G proteins and Sur1 expression affect the regulation of insulin secretion. In conclusion, the sustained under-expression of Ucp2 and Sod2 may further change the function of β-cell, which helps to understand the long-term adrenergic adaptation of pancreatic β-cell.
• Luna-Nevárez, G., Pendleton, A. L., Luna-Ramirez, R. I., Limesand, S. W., Reyna-Granados, J. R., & Luna-Nevárez, P. (2021). Genome-wide association study of a thermo-tolerance indicator in pregnant ewes exposed to an artificial heat-stressed environment. Journal of thermal biology, 101, 103095.
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  Environmental heat stress negatively influences sheep production in warm semi-arid regions. An animal's ability to tolerate warm weather is difficult to measure naturally due to environmental variability and genetic variation between animals. In this study we developed a thermo-tolerance indicator (TTI) to define heat stress tolerance in pregnant sheep in a controlled environment. Next, we performed a genome-wide association study (GWAS) to identify genomic regions and target genes associated with thermo-tolerance in sheep. Pregnant Columbia-Rambouillet crossbred ewes (n = 127) were heat-stressed inside a climate-controlled chamber for 57 days by increasing the temperature-humidity index to ≥30. Rectal temperature (RT) and feed intake (FI) data were collected daily and used for the predictive TTI analysis. After the tenth day of heat stress, the regression analyses revealed that FI was stable; however, when the ewe's RT exceeded 39.8 °C their FI was less than thermo-tolerant ewes. This average predicted temperature was used to classify each ewe as heat stress tolerant (≤39.8 °C) and non-heat stress tolerant (>39.8 °C). A GWAS analysis was performed and genomic regions were compared between heat stress tolerant and non-tolerant ewes. The single-marker genomic analysis detected 16 single nucleotide polymorphisms (SNP) associated with heat stress tolerance (P 
• Pendleton, A. L., Wesolowski, S. R., Regnault, T. R., Lynch, R. M., & Limesand, S. W. (2021). Dimming the Powerhouse: Mitochondrial Dysfunction in the Liver and Skeletal Muscle of Intrauterine Growth Restricted Fetuses. Frontiers in endocrinology, 12, 612888.
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  Intrauterine growth restriction (IUGR) of the fetus, resulting from placental insufficiency (PI), is characterized by low fetal oxygen and nutrient concentrations that stunt growth rates of metabolic organs. Numerous animal models of IUGR recapitulate pathophysiological conditions found in human fetuses with IUGR. These models provide insight into metabolic dysfunction in skeletal muscle and liver. For example, cellular energy production and metabolic rate are decreased in the skeletal muscle and liver of IUGR fetuses. These metabolic adaptations demonstrate that fundamental processes in mitochondria, such as substrate utilization and oxidative phosphorylation, are tempered in response to low oxygen and nutrient availability. As a central metabolic organelle, mitochondria coordinate cellular metabolism by coupling oxygen consumption to substrate utilization in concert with tissue energy demand and accretion. In IUGR fetuses, reducing mitochondrial metabolic capacity in response to nutrient restriction is advantageous to ensure fetal survival. If permanent, however, these adaptations may predispose IUGR fetuses toward metabolic diseases throughout life. Furthermore, these mitochondrial defects may underscore developmental programming that results in the sequela of metabolic pathologies. In this review, we examine how reduced nutrient availability in IUGR fetuses impacts skeletal muscle and liver substrate catabolism, and discuss how enzymatic processes governing mitochondrial function, such as the tricarboxylic acid cycle and electron transport chain, are regulated. Understanding how deficiencies in oxygen and substrate metabolism in response to placental restriction regulate skeletal muscle and liver metabolism is essential given the importance of these tissues in the development of later lifer metabolic dysfunction.
• Davis, M. A., Camacho, L. E., Anderson, M. J., Steffens, N. R., Pendleton, A. L., Kelly, A. C., & Limesand, S. W. (2020). Chronically elevated norepinephrine concentrations lower glucose uptake in fetal sheep. American journal of physiology. Regulatory, integrative and comparative physiology, 319(3), R255-R263.
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  Fetal conditions associated with placental insufficiency and intrauterine growth restriction (IUGR) chronically elevate plasma norepinephrine (NE) concentrations. Our objective was to evaluate the effects of chronically elevated NE on insulin-stimulated glucose metabolism in normally grown, non-IUGR fetal sheep, which are independent of other IUGR-related reductions in nutrients and oxygen availability. After surgical placement of catheters, near-term fetuses received either a saline (control) or NE intravenous infusion with controlled euglycemia. In NE fetuses, plasma NE concentrations were 5.5-fold greater than controls, and fetal euglycemia was maintained with a maternal insulin infusion. Insulin secretion was blunted in NE fetuses during an intravenous glucose tolerance test. Weight-specific fluxes for glucose were measured during a euinsulinemic-euglycemic clamp (EEC) and a hyperinsulinemic-euglycemic clamp (HEC). Plasma glucose and insulin concentrations were not different between groups within each clamp, but insulin concentrations increased 10-fold between the EEC and the HEC. During the EEC, rates of glucose uptake (umbilical uptake + exogenous infusion) and glucose utilization were 47% and 35% lower ( < 0.05) in NE fetuses compared with controls. During the HEC, rates of glucose uptake were 28% lower ( < 0.05) in NE fetuses than controls. Glucose production was undetectable in either group, and glucose oxidation was unaffected by the NE infusion. These findings indicate that chronic exposure to high plasma NE concentrations lowers rates of net glucose uptake in the fetus without affecting glucose oxidation rates or initiating endogenous glucose production. Lower fetal glucose uptake was independent of insulin, which indicates insulin resistance as a consequence of chronically elevated NE.
• Jackson, I. J., Puttabyatappa, M., Anderson, M., Muralidharan, M., Veiga-Lopez, A., Gregg, B., Limesand, S., & Padmanabhan, V. (2020). Developmental programming: Prenatal testosterone excess disrupts pancreatic islet developmental trajectory in female sheep. Molecular and cellular endocrinology, 518, 110950.
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  Prenatal testosterone (T)- treated female sheep manifest juvenile insulin resistance, post-pubertal increase in insulin sensitivity and return to insulin resistance during adulthood. Since compensatory hyperinsulinemia is associated with insulin resistance, altered pancreatic islet ontogeny may contribute towards metabolic defects. To test this, pregnant sheep were treated with or without T propionate from days 30-90 of gestation and pancreas collected from female fetuses at gestational day 90 and female offspring at 21 months-of-age. Uterine (maternal) and umbilical (fetal) arterial blood insulin/glucose ratios were determined at gestational day 90. The morphological and functional changes in pancreatic islet were assessed through detection of 1) islet hormones (insulin, glucagon) and apoptotic beta cells at fetal day 90 and 2) islet hormones (insulin, glucagon and somatostatin), and pancreatic lipid and collagen accumulation in adults. At gestational day 90, T-treatment led to maternal but not fetal hyperinsulinemia, decrease in pancreatic/fetal weight ratio and alpha cells, and a trend for increase in beta cell apoptosis in fetal pancreas. Adult prenatal T-treated female sheep manifested 1) significant increase in beta cell size and a tendency for increase in insulin and somatostatin stained area and proportion of beta cells in the islet; and 2) significant increase in pancreatic islet collagen and a tendency towards increased lipid accumulation. Gestational T-treatment induced changes in pancreatic islet endocrine cells during both fetal and adult ages track the trajectory of hyperinsulinemic status with the increase in adult pancreatic collagen accumulation indicative of impending beta cell failure with chronic insulin resistance.
• Luna-Nevarez, G., Kelly, A. C., Camacho, L. E., Limesand, S. W., Reyna-Granados, J. R., & Luna-Nevarez, P. (2020). Discovery and validation of candidate SNP markers associated to heat stress response in pregnant ewes managed inside a climate-controlled chamber. Tropical animal health and production, 52(6), 3457-3466.
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  Sheep production in desert environments during summer is challenging due to heat stress which reduces feed intake, growth, and fertility. Despite warm conditions, some ewes are able to maintain a normal performance suggesting the existence of genetic bases underlying heat tolerance. Our objective was to discover and validate genetic markers associated with thermo-tolerance in pregnant ewes exposed to warm environmental conditions. Using a well-defined model laboratory of heat stress in sheep, pregnant Columbia-Rambouillet crossbred ewes (n = 100) were examined. Following acclimation to the laboratory at thermo-neutral conditions, heat stress was induced in ewes by increasing the temperature-humidity index in a control environmental chamber during mid-gestation. Feed intake, water consumption, and rectal temperature were recorded daily and used to establish the heat stress tolerance index (HSTI) for each ewe. Rectal temperature was a predictor (P 
• Pendleton, A. L., Antolic, A. T., Kelly, A. C., Davis, M. A., Camacho, L. E., Doubleday, K., Anderson, M. J., Langlais, P. R., Lynch, R. M., & Limesand, S. W. (2020). Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction. American Journal of Physioly - Endocrinology & Metabolism.
• Pendleton, A. L., Antolic, A. T., Kelly, A. C., Davis, M. A., Camacho, L. E., Doubleday, K., Anderson, M. J., Langlais, P. R., Lynch, R. M., & Limesand, S. W. (2020). Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction. American journal of physiology. Endocrinology and metabolism, 319(1), E67-E80.
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  Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower hindlimb oxygen consumption rates (OCRs), indicating depressed mitochondrial oxidative phosphorylation capacity in their skeletal muscle. We hypothesized that OCRs are lower in skeletal muscle mitochondria from IUGR fetuses, due to reduced electron transport chain (ETC) activity and lower abundances of tricarboxylic acid (TCA) cycle enzymes. IUGR sheep fetuses ( = 12) were created with mid-gestation maternal hyperthermia and compared with control fetuses ( = 12). At 132 ± 1 days of gestation, biceps femoris muscles were collected, and the mitochondria were isolated. Mitochondria from IUGR muscle have 47% lower State 3 (Complex I-dependent) OCRs than controls, whereas State 4 (proton leak) OCRs were not different between groups. Furthermore, Complex I, but not Complex II or IV, enzymatic activity was lower in IUGR fetuses compared with controls. Proteomic analysis ( = 6/group) identified 160 differentially expressed proteins between groups, with 107 upregulated and 53 downregulated mitochondria proteins in IUGR fetuses compared with controls. Although no differences were identified in ETC subunit protein abundances, abundances of key TCA cycle enzymes [isocitrate dehydrogenase (NAD) 3 noncatalytic subunit β (IDH3B), succinate-CoA ligase ADP-forming subunit-β (SUCLA2), and oxoglutarate dehydrogenase (OGDH)] were lower in IUGR mitochondria. IUGR mitochondria had a greater abundance of a hypoxia-inducible protein, NADH dehydrogenase 1α subcomplex 4-like 2, which is known to incorporate into Complex I and lower Complex I-mediated NADH oxidation. Our findings show that mitochondria from IUGR skeletal muscle adapt to hypoxemia and hypoglycemia by lowering Complex I activity and TCA cycle enzyme concentrations, which together, act to lower OCR and NADH production/oxidation in IUGR skeletal muscle.
• Beede, K. A., Limesand, S. W., Petersen, J. L., & Yates, D. T. (2019). Real supermodels wear wool: summarizing the impact of the pregnant sheep as an animal model for adaptive fetal programming. Animal frontiers : the review magazine of animal agriculture, 9(3), 34-43.
• Goyal, D., Limesand, S. W., & Goyal, R. (2019). Epigenetic responses and the developmental origins of health and disease. The Journal of endocrinology, 242(1), T105-T119.
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  Maternal and paternal factors influence offspring development and program its genome for successful postnatal life. Based on the stressors during gestation, the pregnant female prepares the fetus for the outside environment. This preparation is achieved by changing the epigenome of the fetus and is referred to as 'developmental programming'. For instance, nutritional insufficiency in utero will lead to programming events that prepare the fetus to cope up with nutrient scarcity following birth; however, offspring may not face nutrient scarcity following birth. This discrepancy between predicted and exposed postnatal environments are perceived as 'stress' by the offspring and may result in cardiovascular and metabolic disorders. Thus, this developmental programming may be both beneficial as well as harmful depending on the prenatal vs postnatal environment. Over the past three decades, accumulating evidence supports the hypothesis of Developmental Origin of Health and Disease (DOHaD) by the programming of the fetal phenotype without altering the genotype per se. These heritable modifications in gene expression occur through DNA methylation, histone modification and noncoding RNA-associated gene activation or silencing, and all are defined as epigenetic modifications. In the present review, we will summarize the evidence supporting epigenetic regulation as a significant component in DOHaD.
• Jones, A. K., Rozance, P. J., Brown, L. D., Goldstrohm, D. A., Hay, W. W., Limesand, S. W., & Wesolowski, S. R. (2019). Sustained hypoxemia in late gestation potentiates hepatic gluconeogenic gene expression but does not activate glucose production in the ovine fetus. American journal of physiology. Endocrinology and metabolism, 317(1), E1-E10.
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  Fetal hypoxemia is associated with pregnancy conditions that cause an early activation of fetal glucose production. However, the independent role of hypoxemia to activate this pathway is not well understood. We hypothesized that fetal hypoxemia would activate fetal glucose production by decreasing umbilical glucose uptake and increasing counter-regulatory hormone concentrations. We induced hypoxemia for 9 days with maternal tracheal N gas insufflation to reduce maternal and fetal arterial Po by ~20% (HOX) compared with fetuses from ewes receiving intratracheal compressed air (CON). At 0.9 of gestation, fetal metabolic studies were performed ( = 7 CON, 11 HOX). Umbilical blood flow rates, net fetal oxygen and glucose uptake rates, and fetal arterial plasma glucose concentrations were not different between the two groups. Fetal glucose utilization rates were lower in HOX versus CON fetuses but not different from umbilical glucose uptake rates, demonstrating the absence of endogenous glucose production. In liver tissue, mRNA expression of gluconeogenic genes 0.01) and ( = 0.06) were six- and threefold greater in HOX fetuses versus CON fetuses. Increased fetal norepinephrine and cortisol concentrations and hepatic and expression were inversely related to fetal arterial Po. These findings support a role for fetal hypoxemia to act with counter-regulatory hormones to potentiate fetal hepatic gluconeogenic gene expression. However, in the absence of decreased net fetal glucose uptake rates and plasma glucose concentrations, hypoxemia-induced gluconeogenic gene activation is not sufficient to activate fetal glucose production.
• Kelly, A. C., & Limesand, S. W. (2019). Classic solutions to a modern problem: exercise training improves metabolic disorders in offspring from fathers on a high fat diet. The Journal of physiology, 597(1), 9-10.
• Kelly, A. C., Smith, K. E., Purvis, W. G., Min, C. G., Weber, C. S., Cooksey, A. M., Hasilo, C., Paraskevas, S., Suszynski, T. M., Weegman, B. P., Anderson, M. J., Camacho, L. E., Harland, R. C., Loudovaris, T., Jandova, J., Molano, D. S., Price, N. D., Georgiev, I. G., Scott, W. E., , Manas, D. M., et al. (2019). Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling. Transplantation, 103(1), 160-167.
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  All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS.
• Limesand, S. W. (2019). Gestational Diabetes-Induced Programming of Pancreatic Islets. Endocrinology, 160(9), 2117-2118.
• Limesand, S. W., Thornburg, K. L., & Harding, J. E. (2019). 30th anniversary for the Developmental Origins of Endocrinology. The Journal of endocrinology.
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  This special issue for the Journal of Endocrinology celebrates the 30th anniversary of David Barker's seminal findings that led to the scientific field of the Developmental Origins of Health and Disease (DOHaD). In 1989, Barker and colleagues reported that low birth weight and weight at one year, proxies for fetal growth restriction, were related to an individual's risk for developing hypertension and cardiovascular heart disease. Barker's initial epidemiological studies also demonstrated that low birth weight was predictive of later glucose intolerance, Type 2 Diabetes, and other metabolic-related diseases. As the developmental origins concept developed, the number of epidemiological studies continued to expand globally, consistently demonstrating the higher risk of developing chronic degenerative diseases if one was born small. In this thematic issue on the early origins of disease, there are a series of review articles and research papers that capture the impact of early events on endocrine systems, as major mechanisms underlying Barker's original observations. Importantly, over the past 30 years as the DOHaD concept has become widely accepted, we have seen it applied to an ever expanding breadth of human health problems. This expansion is evidenced by the growth of new model systems and establishment of new causal relationships in neuroendocrinology, reproductive systems, obesity, and behavior. The importance the DOHaD concept, its continued evolution, and new underlying mechanism are captured in the articles of this issue dedicated to Professor Barker's legacy.
• Pendleton, A. L., Humphreys, L. R., Davis, M. A., Camacho, L. E., Anderson, M. J., & Limesand, S. W. (2019). Increased pyruvate dehydrogenase activity in skeletal muscle of growth-restricted ovine fetuses. American journal of physiology. Regulatory, integrative and comparative physiology, 317(4), R513-R520.
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  Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower fractional rates of glucose oxidation and greater gluconeogenesis, indicating lactate shuttling between skeletal muscle and liver. Suppression of pyruvate dehydrogenase () activity was proposed because of greater pyruvate dehydrogenase kinase (PDK) 4 and PDK1 mRNA concentrations in IUGR muscle. Although PDK1 and PDK4 inhibit PDH activity to reduce pyruvate metabolism, PDH protein concentrations and activity have not been examined in skeletal muscle from IUGR fetuses. Therefore, we evaluated the protein concentrations and activity of PDH and the kinases and phosphatases that regulate PDH phosphorylation status in the semitendinosus muscle from placenta insufficiency-induced IUGR sheep fetuses and control fetuses. Immunoblots were performed for PDH, phosphorylated PDH (E1α), PDK1, PDK4, and pyruvate dehydrogenase phosphatase 1 and 2 (PDP1 and PDP2, respectively). Additionally, the PDH, lactate dehydrogenase (LDH), and citrate synthase (CS) enzymatic activities were measured. Phosphorylated PDH concentrations were 28% lower (P < 0.01) and PDH activity was 67% greater (P < 0.01) in IUGR fetal muscle compared with control. PDK1, PDK4, PDP1, PDP2, and PDH concentrations were not different between groups. CS and LDH activities were also unaffected. Contrary to the previous speculation, PDH activity was greater in skeletal muscle from IUGR fetuses, which parallels lower phosphorylated PDH. Therefore, greater expression of PDK1 and PDK4 mRNA did not translate to greater PDK1 or PDK4 protein concentrations or inhibition of PDH as proposed. Instead, these findings show greater PDH activity in IUGR fetal muscle, which indicates that alternative regulatory mechanisms are responsible for lower pyruvate catabolism.
• Yates, D. T., Camacho, L. E., Kelly, A. C., Steyn, L. V., Davis, M. A., Antolic, A. T., Anderson, M. J., Goyal, R., Allen, R. E., Papas, K. K., Hay, W. W., & Limesand, S. W. (2019). Postnatal β2 adrenergic treatment improves insulin sensitivity in lambs with IUGR but not persistent defects in pancreatic islets or skeletal muscle. The Journal of physiology, 597(24), 5835-5858.
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  Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity. Although whole-body glucose clearance is normal, 1-month-old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation. Impaired glucose-stimulated insulin secretion in IUGR lambs is due to lower intra-islet insulin availability and not from glucose sensing. We investigated adrenergic receptor (ADR) β2 desensitization by administering oral ADRβ modifiers for the first month after birth to activate ADRβ2 and antagonize ADRβ1/3. In IUGR lambs ADRβ2 activation increased whole-body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies. IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose-stimulated insulin secretion and insulin-stimulated glucose oxidation, providing new insights into potential mechanisms for this risk.
• Hart, N. J., Weber, C., Papas, K. K., Limesand, S. W., Vagner, J., & Lynch, R. M. (2018). Multivalent activation of GLP-1 and Sulfonylurea receptors modulates β-cell Second Messenger Signaling and Insulin Secretion. American journal of physiology. Cell physiology.
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  Linking two pharmacophores that bind different cell surface receptors into a single molecule can enhance cell-targeting specificity to cells that express the complementary receptor pair. In this report, we developed and tested a synthetic multivalent ligand consisting of glucagon-like peptide-1 (GLP-1) linked to glibenclamide (Glb) for signaling efficacy in β-cells. Expression of receptors for these ligands, as a combination, are relatively specific to the β-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca, although Ca responses were significantly depressed compared to the monomeric Glb. Moreover, GLP-1/Glb increased glucose-stimulated insulin secretion in a dose-dependent manner. However, unlike the combined monomers, GLP-1/Glb did not augment insulin secretion at non-stimulatory glucose concentrations in INS 832/13 β-cells or human islets of Langerhans. These data suggest that linking two binding elements into a single bivalent ligand, such as GLP-1 and Glb, can provide a unique functional agent targeted to β-cells.
• Hill, M. G., Kelly, A. C., Reed, K. L., & Limesand, S. W. (2018). Plasma markers of physiological stress in human fetuses will intrauterine growth restriction and abnormal umbilical artery Doppler velocimetry. American Journal of Clinical and Experimental Obstetrics and Gynecology, 5(1), 1-7.
• Kelly, A. C., Bidwell, C. A., Chen, X., Macko, A. R., Anderson, M. J., & Limesand, S. W. (2018). Chronic Adrenergic Signaling Causes Abnormal RNA Expression of Proliferative Genes in Fetal Sheep Islets. Endocrinology, 159(10), 3565-3578.
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  Intrauterine growth restriction (IUGR) increases the risk of developing diabetes in later life, which indicates developmental programming of islets. IUGR fetuses with placental insufficiency develop hypoxemia, elevating epinephrine and norepinephrine (NE) concentrations throughout late gestation. To isolate the programming effects of chronically elevated catecholamines, NE was continuously infused into normally grown sheep fetuses for 7 days. High plasma NE concentrations suppress insulin, but after the NE infusion was terminated, persistent hypersecretion of insulin occurred. Our objective was to identify differential gene expression with RNA sequencing (RNAseq) in fetal islets after chronic adrenergic stimulation. After determining the NE-regulated genes, we identified the subset of differentially expressed genes that were common to both islets from NE fetuses and fetuses with IUGR to delineate the adrenergic-induced transcriptional responses. A portion of these genes were investigated in mouse insulinoma (Min6) cells chronically treated with epinephrine to better approximate the β-cell response. In islets from NE fetuses, RNAseq identified 321 differentially expressed genes that were overenriched for metabolic and hormone processes, and the subset of 96 differentially expressed genes common to IUGR islets were overenriched for protein digestion, vitamin metabolism, and cell replication pathways. Thirty-eight of the 96 NE-regulated IUGR genes changed similarly between models with functional enrichment for proliferation. In Min6 cells, chronic epinephrine stimulation slowed proliferation and augmented insulin secretion after treatment. These data establish molecular mechanisms underlying persistent adrenergic stimulation in hyperfunctional fetal islets and identify a subset of genes dysregulated by catecholamines in IUGR islets that may represent programming of β-cell proliferation capacity.
• Kelly, A. C., Camacho, L. E., Pendarvis, K., Davenport, H. M., Steffens, N. R., Smith, K. E., Weber, C. S., Lynch, R. M., Papas, K. K., & Limesand, S. W. (2018). Adrenergic receptor stimulation suppresses oxidative metabolism in isolated rat islets and Min6 cells. Molecular and cellular endocrinology.
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  Insulin secretion is stimulated by glucose metabolism and inhibited by catecholamines through adrenergic receptor stimulation. We determined whether catecholamines suppress oxidative metabolism in β-cells through adrenergic receptors. In Min6 cells and isolated rat islets, epinephrine decreased oxygen consumption rates compared to vehicle control or co-administration of epinephrine with α2-adrenergic receptor antagonist yohimbine. Epinephrine also decreased forskolin-stimulated oxygen consumption rates, indicating cAMP dependent and independent actions. Furthermore, glucose oxidation rates were decreased with epinephrine, independent of the exocytosis of insulin, which was blocked with yohimbine. We evaluated metabolic targets through proteomic analysis after 4 h epinephrine exposure that revealed 466 differentially expressed proteins that were significantly enriched for processes including oxidative metabolism, protein turnover, exocytosis, and cell proliferation. These results demonstrate that acute α2-adrenergic stimulation suppresses glucose oxidation in β-cells independent of nutrient availability and insulin exocytosis, while cAMP concentrations are elevated. Proteomics and immunoblots revealed changes in electron transport chain proteins that were correlated with lower metabolic reducing equivalents, intracellular ATP concentrations, and altered mitochondrial membrane potential implicating a new role for adrenergic control of mitochondrial function and ultimately insulin secretion.
• Limesand, S. W., Camacho, L. E., Kelly, A. C., & Antolic, A. T. (2018). Impact of thermal stress on placental function and fetal physiology. Animal Reproduction, 15(Supplement 1), 886-898. doi:DOI: 10.21451/1984-3143-AR2018-0056
• Posont, R. J., Beede, K. A., Limesand, S. W., & Yates, D. T. (2018). Changes in myoblast responsiveness to TNFα and IL-6 contribute to decreased skeletal muscle mass in intrauterine growth restricted fetal sheep. Translational animal science, 2(Suppl 1), S44-S47.
• Smith, K. E., Purvis, W. G., Davis, M. A., Min, C. G., Cooksey, A. M., Weber, C. S., Jandova, J., Price, N. D., Molano, D. S., Stanton, J. B., Kelly, A. C., Steyn, L. V., Lynch, R. M., Limesand, S. W., Alexander, M., Lakey, J. R., Seeberger, K., Korbutt, G. S., Mueller, K. R., , Hering, B. J., et al. (2018). In vitro characterization of neonatal, juvenile, and adult porcine islet oxygen demand, β-cell function, and transcriptomes. Xenotransplantation, 25(6), e12432.
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  There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted.
• Benjamin, J. S., Culpepper, C. B., Brown, L. D., Wesolowski, S. R., Jonker, S. S., Davis, M. A., Limesand, S. W., Wilkening, R. B., Hay, W. W., & Rozance, P. J. (2017). Chronic anemic hypoxemia attenuates glucose-stimulated insulin secretion in fetal sheep. American journal of physiology. Regulatory, integrative and comparative physiology, 312(4), R492-R500.
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  Fetal insulin secretion is inhibited by acute hypoxemia. The relationship between prolonged hypoxemia and insulin secretion, however, is less well defined. To test the hypothesis that prolonged fetal hypoxemia impairs insulin secretion, studies were performed in sheep fetuses that were bled to anemic conditions for 9 ± 0 days (anemic, n = 19) and compared with control fetuses (n = 15). Arterial hematocrit and oxygen content were 34% and 52% lower, respectively, in anemic vs. control fetuses (P < 0.0001). Plasma glucose concentrations were 21% higher in the anemic group (P < 0.05). Plasma norepinephrine and cortisol concentrations increased 70% in the anemic group (P < 0.05). Glucose-, arginine-, and leucine-stimulated insulin secretion all were lower (P < 0.05) in anemic fetuses. No differences in pancreatic islet size or β-cell mass were found. In vitro, isolated islets from anemic fetuses secreted insulin in response to glucose and leucine as well as control fetal islets. These findings indicate a functional islet defect in anemic fetuses, which likely involves direct effects of low oxygen and/or increased norepinephrine on insulin release. In pregnancies complicated by chronic fetal hypoxemia, increasing fetal oxygen concentrations may improve insulin secretion.
• Boehmer, B. H., Limesand, S. W., & Rozance, P. J. (2017). The impact of IUGR on pancreatic islet development and β-cell function. The Journal of endocrinology, 235(2), R63-R76.
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  Placental insufficiency is a primary cause of intrauterine growth restriction (IUGR). IUGR increases the risk of developing type 2 diabetes mellitus (T2DM) throughout life, which indicates that insults from placental insufficiency impair β-cell development during the perinatal period because β-cells have a central role in the regulation of glucose tolerance. The severely IUGR fetal pancreas is characterized by smaller islets, less β-cells, and lower insulin secretion. Because of the important associations among impaired islet growth, β-cell dysfunction, impaired fetal growth, and the propensity for T2DM, significant progress has been made in understanding the pathophysiology of IUGR and programing events in the fetal endocrine pancreas. Animal models of IUGR replicate many of the observations in severe cases of human IUGR and allow us to refine our understanding of the pathophysiology of developmental and functional defects in islet from IUGR fetuses. Almost all models demonstrate a phenotype of progressive loss of β-cell mass and impaired β-cell function. This review will first provide evidence of impaired human islet development and β-cell function associated with IUGR and the impact on glucose homeostasis including the development of glucose intolerance and diabetes in adulthood. We then discuss evidence for the mechanisms regulating β-cell mass and insulin secretion in the IUGR fetus, including the role of hypoxia, catecholamines, nutrients, growth factors, and pancreatic vascularity. We focus on recent evidence from experimental interventions in established models of IUGR to understand better the pathophysiological mechanisms linking placental insufficiency with impaired islet development and β-cell function.
• Camacho, L. E., Chen, X., Hay, W. W., & Limesand, S. W. (2017). Enhanced insulin secretion and insulin sensitivity in young lambs with placental insufficiency-induced intrauterine growth restriction. American journal of physiology. Regulatory, integrative and comparative physiology, 313(2), R101-R109.
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  Intrauterine growth restriction (IUGR) is associated with persistent metabolic complications, but information is limited for IUGR infants. We determined glucose-stimulated insulin secretion (GSIS) and insulin sensitivity in young lambs with placental insufficiency-induced IUGR. Lambs with hyperthermia-induced IUGR (n = 7) were compared with control lambs (n = 8). GSIS was measured at 8 ± 1 days of age, and at 15 ± 1 days, body weight-specific glucose utilization rates were measured with radiolabeled d-glucose during a hyperinsulinemic-euglycemic clamp (HEC). IUGR lambs weighed 23% less (P < 0.05) than controls at birth. Fasting plasma glucose and insulin concentrations were not different between IUGR and controls for either study. First-phase insulin secretion was enhanced 2.3-fold in IUGR lambs compared with controls. However, second-phase insulin concentrations, glucose-potentiated arginine-stimulated insulin secretion, and β-cell mass were not different, indicating that IUGR β-cells have an intrinsic enhancement in acute GSIS. Compared with controls, IUGR lambs had higher body weight-specific glucose utilization rates and greater insulin sensitivity at fasting (1.6-fold) and hyperinsulinemic periods (2.4-fold). Improved insulin sensitivity for glucose utilization was not due to differences in skeletal muscle insulin receptor and glucose transporters 1 and 4 concentrations. Plasma lactate concentrations during HEC were elevated in IUGR lambs compared with controls, but no differences were found for glycogen content or citrate synthase activity in liver and muscle. Greater insulin sensitivity for glucose utilization and enhanced acute GSIS in young lambs are predicted from fetal studies but may promote conditions that exaggerate glucose disposal and lead to episodes of hypoglycemia in IUGR infants.
• Chen, X., Kelly, A. C., Yates, D. T., Macko, A. R., Lynch, R. M., & Limesand, S. W. (2017). Islet adaptations in fetal sheep persist following chronic exposure to high norepinephrine. The Journal of endocrinology, 232(2), 285-295.
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  Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion after chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. After an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129 ± 1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were performed again on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P 
• Harris, S. E., De Blasio, M. J., Davis, M. A., Kelly, A. C., Davenport, H. M., Wooding, F. B., Blache, D., Meredith, D., Anderson, M., Fowden, A. L., Limesand, S. W., & Forhead, A. J. (2017). Hypothyroidism in utero stimulates pancreatic beta cell proliferation and hyperinsulinaemia in the ovine fetus during late gestation. The Journal of physiology, 595(11), 3331-3343.
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  Thyroid hormones are important regulators of growth and maturation before birth, although the extent to which their actions are mediated by insulin and the development of pancreatic beta cell mass is unknown. Hypothyroidism in fetal sheep induced by removal of the thyroid gland caused asymmetric organ growth, increased pancreatic beta cell mass and proliferation, and was associated with increased circulating concentrations of insulin and leptin. In isolated fetal sheep islets studied in vitro, thyroid hormones inhibited beta cell proliferation in a dose-dependent manner, while high concentrations of insulin and leptin stimulated proliferation. The developing pancreatic beta cell is therefore sensitive to thyroid hormone, insulin and leptin before birth, with possible consequences for pancreatic function in fetal and later life. The findings of this study highlight the importance of thyroid hormones during pregnancy for normal development of the fetal pancreas.
• Kelly, A. C., Bidwell, C. A., McCarthy, F. M., Taska, D. J., Anderson, M. J., Camacho, L. E., & Limesand, S. W. (2017). RNA Sequencing Exposes Adaptive and Immune Responses to Intrauterine Growth Restriction in Fetal Sheep Islets. Endocrinology, 158(4), 743-755.
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  The risk of type 2 diabetes is increased in children and adults who exhibited fetal growth restriction. Placental insufficiency and intrauterine growth restriction (IUGR) are common obstetrical complications associated with fetal hypoglycemia and hypoxia that reduce the β-cell mass and insulin secretion. In the present study, we have defined the underlying mechanisms of reduced growth and proliferation, impaired metabolism, and defective insulin secretion previously established as complications in islets from IUGR fetuses. In an IUGR sheep model that recapitulates human IUGR, high-throughput RNA sequencing showed the transcriptome of islets isolated from IUGR and control sheep fetuses and identified the transcripts that underlie β-cell dysfunction. Functional analysis expanded mechanisms involved in reduced proliferation and dysregulated metabolism that include specific cell cycle regulators and growth factors and mitochondrial, antioxidant, and exocytotic genes. These data also identified immune responses, wnt signaling, adaptive stress responses, and the proteasome as mechanisms of β-cell dysfunction. The reduction of immune-related gene expression did not reflect a change in macrophage density within IUGR islets. The present study reports the islet transcriptome in fetal sheep and established processes that limit insulin secretion and β-cell growth in fetuses with IUGR, which could explain the susceptibility to premature islet failure in adulthood. Islet dysfunction formed by intrauterine growth restriction increases the risk for diabetes.
• Limesand, S. W., & Rozance, P. J. (2017). Fetal adaptations in insulin secretion result from high catecholamines during placental insufficiency. The Journal of physiology, 595(15), 5103-5113.
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  Placental insufficiency and intrauterine growth restriction (IUGR) of the fetus affects approximately 8% of all pregnancies and is associated with short- and long-term disturbances in metabolism. In pregnant sheep, experimental models with a small, defective placenta that restricts delivery of nutrients and oxygen to the fetus result in IUGR. Low blood oxygen concentrations increase fetal plasma catecholamine concentrations, which lower fetal insulin concentrations. All of these observations in sheep models with placental insufficiency are consistent with cases of human IUGR. We propose that sustained high catecholamine concentrations observed in the IUGR fetus produce developmental adaptations in pancreatic β-cells that impair fetal insulin secretion. Experimental evidence supporting this hypothesis shows that chronic elevation in circulating catecholamines in IUGR fetuses persistently inhibits insulin concentrations and secretion. Elevated catecholamines also allow for maintenance of a normal fetal basal metabolic rate despite low fetal insulin and glucose concentrations while suppressing fetal growth. Importantly, a compensatory augmentation in insulin secretion occurs following inhibition or cessation of catecholamine signalling in IUGR fetuses. This finding has been replicated in normally grown sheep fetuses following a 7-day noradrenaline (norepinephrine) infusion. Together, these programmed effects will potentially create an imbalance between insulin secretion and insulin-stimulated glucose utilization in the neonate which probably explains the transient hyperinsulinism and hypoglycaemia in some IUGR infants.
• Smith, K. E., Kelly, A. C., Min, C. G., Weber, C. S., McCarthy, F. M., Steyn, L. V., Badarinarayana, V., Stanton, J. B., Kitzmann, J. P., Strop, P., Gruessner, A. C., Lynch, R. M., Limesand, S. W., & Papas, K. K. (2017). Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans. Transplantation, 101(11), 2705-2712.
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  Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived β cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high β cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention.
• Brown, L. D., Davis, M., Wai, S., Wesolowski, S. R., Hay, W. W., Limesand, S. W., & Rozance, P. J. (2016). Chronically Increased Amino Acids Improve Insulin Secretion, Pancreatic Vascularity, and Islet Size in Growth-Restricted Fetal Sheep. Endocrinology, 157(10), 3788-3799.
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  Placental insufficiency is associated with reduced supply of amino acids to the fetus and leads to intrauterine growth restriction (IUGR). IUGR fetuses are characterized by lower glucose-stimulated insulin secretion, smaller pancreatic islets with less β-cells, and impaired pancreatic vascularity. To test whether supplemental amino acids infused into the IUGR fetus could improve these complications of IUGR we used acute (hours) and chronic (11 d) direct fetal amino acid infusions into a sheep model of placental insufficiency and IUGR near the end of gestation. IUGR fetuses had attenuated acute amino acid-stimulated insulin secretion compared with control fetuses. These results were confirmed in isolated IUGR pancreatic islets. After the chronic fetal amino acid infusion, fetal glucose-stimulated insulin secretion and islet size were restored to control values. These changes were associated with normalization of fetal pancreatic vascularity and higher fetal pancreatic vascular endothelial growth factor A protein concentrations. These results demonstrate that decreased fetal amino acid supply contributes to the pathogenesis of pancreatic islet defects in IUGR. Moreover, the results show that pancreatic islets in IUGR fetuses retain their ability to respond to increased amino acids near the end of gestation after chronic fetal growth restriction.
• Camacho, L. E., Yates, D. T., Allen, R. E., & Limesand, S. W. (2016). Reduced Insulin-Stimulated Glucose Uptake in Skeletal Muscle Strips from Intrauterine Growth Restricted Lambs.. REPRODUCTIVE SCIENCES, 23, 312A-312A.
• Camacho, L. E., Yates, D. T., Davenport, H. M., Allen, R. E., & Limesand, S. W. (2016). Decreased Satellite Cell Proliferation Rates Contribute to Small Fibers in the Semitendinosus Muscle of Intrauterine Growth Restricted Lambs.. REPRODUCTIVE SCIENCES, 23, 316A-317A.
• Davis, M., Brown, L., Wai, S., Wesolowski, S., Limesand, S., & Rozance, P. (2016). Chronic Amino Acid Supplementation Increases Beta-Cell Mass, Islet Area, and Pancreatic Vascularization in Growth Restricted Fetal Sheep.. REPRODUCTIVE SCIENCES, 23, 150A-150A.
• Hay Jr., W. W., Brown, L. D., Rozance, P. J., Wesolowski, S. R., & Limesand, S. W. (2016). Challenges in nourishing the intrauterine growth-restricted foetus - Lessons learned from studies in the intrauterine growth-restricted foetal sheep. ACTA PAEDIATRICA, 105(8), 881-889.
• Hay, W. W., Brown, L. D., Rozance, P. J., Wesolowski, S. R., & Limesand, S. W. (2016). Challenges in nourishing the intrauterine growth-restricted foetus - Lessons learned from studies in the intrauterine growth-restricted foetal sheep. Acta paediatrica (Oslo, Norway : 1992), 105(8), 881-9.
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  Previous attempts to improve growth and development of the intrauterine growth-restricted (IUGR) foetus during pregnancy have not worked or caused harm. Our research identifies tissue-specific mechanisms underlying foetal growth restriction and then tests strategies to improve growth and ameliorate many of the metabolic problems before the infant is born. The goal of our studies is to reduce the impact of foetal growth restriction at critical stages of development on the lifelong complications of IUGR offspring.
• Hill, M. G., Kelly, A. C., Camp, S. M., Reed, K. L., Limesand, S. W., & Garcia, J. (2016). Increased Glucagon and Decreased Visfatin Concentrations in the Cord Blood of Fetuses from Type I Diabetic Patients.. REPRODUCTIVE SCIENCES, 23, 140A-140A.
• Kelly, A., Bidwell, C., Camacho, L., McCarthy, F., & Limesand, S. (2016). Transcriptome Expression Profiles Identify Increased Metabolic Capacity in Adipose Tissue from Fetal Sheep with Intrauterine Growth Restriction.. REPRODUCTIVE SCIENCES, 23, 225A-226A.
• Kelly, A., Bidwell, C., Chen, X., Anderson, M., & Limesand, S. (2016). Identifying the Adrenergic Component of Intrauterine Growth Restriction in Fetal Pancreatic beta-Cells.. REPRODUCTIVE SCIENCES, 23, 94A-94A.
• Macko, A. R., Yates, D. T., Chen, X., Shelton, L. A., Kelly, A. C., Davis, M. A., Camacho, L. E., Anderson, M. J., & Limesand, S. W. (2016). Adrenal Demedullation and Oxygen Supplementation Independently Increase Glucose-Stimulated Insulin Concentrations in Fetal Sheep With Intrauterine Growth Restriction. ENDOCRINOLOGY, 157(5), 2104-2115.
• Yates, D. T., Cadaret, C. N., Beede, K. A., Riley, H. E., Macko, A. R., Anderson, M. J., Camacho, L. E., & Limesand, S. W. (2016). Intrauterine growth-restricted sheep fetuses exhibit smaller hindlimb muscle fibers and lower proportions of insulin-sensitive Type I fibers near term. AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY, 310(11), R1020-R1029.
• Yates, D. T., Cadaret, C. N., Beede, K. A., Riley, H. E., Macko, A. R., Anderson, M. J., Camacho, L. E., & Limesand, S. W. (2016). Intrauterine growth-restricted sheep fetuses exhibit smaller hindlimb muscle fibers and lower proportions of insulin-sensitive Type I fibers near term. American journal of physiology. Regulatory, integrative and comparative physiology, 310(11), R1020-9.
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  Intrauterine growth restriction (IUGR) reduces muscle mass and insulin sensitivity in offspring. Insulin sensitivity varies among muscle fiber types, with Type I fibers being most sensitive. Differences in fiber-type ratios are associated with insulin resistance in adults, and thus we hypothesized that near-term IUGR sheep fetuses exhibit reduced size and proportions of Type I fibers. Placental insufficiency-induced IUGR fetuses were ∼54% smaller (P < 0.05) than controls and exhibited hypoxemia and hypoglycemia, which contributed to 6.9-fold greater (P < 0.05) plasma norepinephrine and ∼53% lower (P < 0.05) plasma insulin concentrations. IUGR semitendinosus muscles contained less (P < 0.05) myosin heavy chain-I protein (MyHC-I) and proportionally fewer (P < 0.05) Type I and Type I/IIa fibers than controls, but MyHC-II protein concentrations, Type II fibers, and Type IIx fibers were not different. IUGR biceps femoris muscles exhibited similar albeit less dramatic differences in fiber type proportions. Type I and IIa fibers are more responsive to adrenergic and insulin regulation than Type IIx and may be more profoundly impaired by the high catecholamines and low insulin in our IUGR fetuses, leading to their proportional reduction. In both muscles, fibers of each type were uniformly smaller (P < 0.05) in IUGR fetuses than controls, which indicates that fiber hypertrophy is not dependent on type but rather on other factors such as myoblast differentiation or protein synthesis. Together, our findings show that IUGR fetal muscles develop smaller fibers and have proportionally fewer Type I fibers, which is indicative of developmental adaptations that may help explain the link between IUGR and adulthood insulin resistance.
• Andrews, S. E., Brown, L. D., Thorn, S. R., Limesand, S. W., Davis, M., Hay, W. W., & Rozance, P. J. (2015). Increased adrenergic signaling is responsible for decreased glucose-stimulated insulin secretion in the chronically hyperinsulinemic ovine fetus. Endocrinology, 1(1), 367-76.
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  Insulin may stimulate its own insulin secretion and is a potent growth factor for the pancreatic β-cell. Complications of pregnancy, such as diabetes and intrauterine growth restriction, are associated with changes in fetal insulin concentrations, secretion, and β-cell mass. However, glucose concentrations are also abnormal in these conditions. The direct effect of chronic fetal hyperinsulinemia with euglycemia on fetal insulin secretion and β-cell mass has not been tested. We hypothesized that chronic fetal hyperinsulinemia with euglycemia would increase glucose-stimulated insulin secretion (GSIS) and β-cell mass in the ovine fetus. Singleton ovine fetuses were infused with iv insulin to produce high physiological insulin concentrations, or saline for 7-10 days. The hyperinsulinemic animals also received a direct glucose infusion to maintain euglycemia. GSIS, measured at 133 ± 1 days of gestation, was significantly attenuated in the hyperinsulinemic fetuses (P < .05). There was no change in β-cell mass. The hyperinsulinemic fetuses also had decreased oxygen (P < .05) and higher norepinephrine (1160 ± 438 vs 522 ± 106 pg/mL; P < .005). Acute pharmacologic adrenergic blockade restored GSIS in the hyperinsulinemic-euglycemic fetuses, demonstrating that increased adrenergic signaling mediates decreased GSIS in these fetuses.
• Davis, M. A., Macko, A. R., Steyn, L. V., Anderson, M. J., & Limesand, S. W. (2015). Fetal Adrenal Demedullation Lowers Circulating Norepinephrine and Attenuates Growth Restriction but not Reduction of Endocrine Cell Mass in an Ovine Model of Intrauterine Growth Restriction. Nutrients, 7(1), 500-16.
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  Placental insufficiency is associated with fetal hypoglycemia, hypoxemia, and elevated plasma norepinephrine (NE) that become increasingly pronounced throughout the third trimester and contribute to intrauterine growth restriction (IUGR). This study evaluated the effect of fetal adrenal demedullation (AD) on growth and pancreatic endocrine cell mass. Placental insufficiency-induced IUGR was created by exposing pregnant ewes to elevated ambient temperatures during mid-gestation. Treatment groups consisted of control and IUGR fetuses with either surgical sham or AD at 98 days gestational age (dGA; term = 147 dGA), a time-point that precedes IUGR. Samples were collected at 134 dGA. IUGR-sham fetuses were hypoxemic, hypoglycemic, and hypoinsulinemic, and values were similar in IUGR-AD fetuses. Plasma NE concentrations were ~5-fold greater in IUGR-sham compared to control-sham, control-AD, and IUGR-AD fetuses. IUGR-sham and IUGR-AD fetuses weighed less than controls. Compared to IUGR-sham fetuses, IUGR-AD fetuses weighed more and asymmetrical organ growth was absent. Pancreatic β-cell mass and α-cell mass were lower in both IUGR-sham and IUGR-AD fetuses compared to controls, however, pancreatic endocrine cell mass relative to fetal mass was lower in IUGR-AD fetuses. These findings indicate that NE, independently of hypoxemia, hypoglycemia and hypoinsulinemia, influence growth and asymmetry of growth but not pancreatic endocrine cell mass in IUGR fetuses.
• Hill, M., Kelly, A. C., Reed, K. L., & Limesand, S. W. (2015). 359: Fetal growth restriction and markers of metabolic dysfunction and inflammation. American Journal of Obstetrics and Gynecology, 212(1), S188-S189. doi:10.1016/j.ajog.2014.10.405
• Limesand, S. W. (2015). Insights Into the Progression of β-Cell Dysfunction Caused by Preterm Birth. Endocrinology, 156(10), 3494-5.
• Rozance, P. J., Anderson, M., Martinez, M., Fahy, A., Macko, A. R., Kailey, J., Seedorf, G. J., Abman, S. H., Hay, W. W., & Limesand, S. W. (2015). Placental insufficiency decreases pancreatic vascularity and disrupts hepatocyte growth factor signaling in the pancreatic islet endothelial cell in fetal sheep. Diabetes, 64(2), 555-64.
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  Hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGFA) are paracrine hormones that mediate communication between pancreatic islet endothelial cells (ECs) and β-cells. Our objective was to determine the impact of intrauterine growth restriction (IUGR) on pancreatic vascularity and paracrine signaling between the EC and β-cell. Vessel density was less in IUGR pancreata than in controls. HGF concentrations were also lower in islet EC-conditioned media (ECCM) from IUGR, and islets incubated with control islet ECCM responded by increasing insulin content, which was absent with IUGR ECCM. The effect of ECCM on islet insulin content was blocked with an inhibitory anti-HGF antibody. The HGF receptor was not different between control and IUGR islets, but VEGFA was lower and the high-affinity VEGF receptor was higher in IUGR islets and ECs, respectively. These findings show that paracrine actions from ECs increase islet insulin content, and in IUGR ECs, secretion of HGF was diminished. Given the potential feed-forward regulation of β-cell VEGFA and islet EC HGF, these two growth factors are highly integrated in normal pancreatic islet development, and this regulation is decreased in IUGR fetuses, resulting in lower pancreatic islet insulin concentrations and insulin secretion.
• Smith, R., Limesand, S. W., Kelly, A. C., Dibase, S., & Davenport, H. M. (2015). Adrenaline mediated metabolic and functional quiescence protects insulin producing cells from hypoxia. Endocrine Abstracts, 38. doi:10.1530/endoabs.38.p207
• Steyn, L. V., Ananthakrishnan, K., Anderson, M. J., Patek, R., Kelly, A., Vagner, J., Lynch, R. M., & Limesand, S. W. (2015). A Synthetic Heterobivalent Ligand Composed of Glucagon-Like Peptide 1 and Yohimbine Specifically Targets β Cells Within the Pancreas. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging.
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  β Cell specificity for a heterobivalent ligand composed of glucagon-like peptide-1 (GLP-1) linked to yohimbine (GLP-1/Yhb) was evaluated to determine its utility as a noninvasive imaging agent.
• Chen, X., Green, A. S., Macko, A. R., Yates, D. T., Kelly, A. C., & Limesand, S. W. (2014). Enhanced insulin secretion responsiveness and islet adrenergic desensitization after chronic norepinephrine suppression is discontinued in fetal sheep. American Journal of Physiology - Endocrinology and Metabolism, 306(1), E58-E64.
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  Abstract: Intrauterine growth-restricted (IUGR) fetuses experience prolonged hypoxemia, hypoglycemia, and elevated norepinephrine (NE) concentrations, resulting in hypoinsulinemia and β-cell dysfunction. Previously, we showed that acute adrenergic blockade revealed enhanced insulin secretion responsiveness in the IUGR fetus. To determine whether chronic exposure to NE alone enhances β-cell responsiveness afterward, we continuously infused NE into fetal sheep for 7 days and, after terminating the infusion, evaluated glucose-stimulated insulin secretion (GSIS) and glucose-potentiated arginine-induced insulin secretion (GPAIS). During treatment, NE-infused fetuses had greater (P < 0.05) plasma NE concentrations and exhibited hyperglycemia (P < 0.01) and hypoinsulinemia (P < 0.01) compared with controls. GSIS during the NE infusion was also reduced (P < 0.05) compared with pretreatment values. GSIS and GPAIS were approximately fourfold greater (P < 0.01) in NE fetuses 3 h after the 7 days that NE infusion was discontinued compared with age-matched controls or pretreatment GSIS and GPAIS values of NE fetuses. In isolated pancreatic islets from NE fetuses, mRNA concentrations of adrenergic receptor isoforms (α1D, α2A, α2C, and β1), G protein subunit-αi-2, and uncoupling protein 2 were lower (P < 0.05) compared with controls, but β-cell regulatory genes were not different. Our findings indicate that chronic exposure to elevated NE persistently suppresses insulin secretion. After removal, NE fetuses demonstrated a compensatory enhancement in insulin secretion that was associated with adrenergic desensitization and greater stimulus-secretion coupling in pancreatic islets. © 2014 the American Physiological Society.
• Hart, N. J., Chung, W. J., Weber, C., Ananthakrishnan, K., Anderson, M., Patek, R., Zhang, Z., Limesand, S. W., Vagner, J., & Lynch, R. M. (2014). Hetero-bivalent GLP-1/glibenclamide for targeting pancreatic β-cells. ChemBioChem, 15(1), 135-145.
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  Abstract: G protein-coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell-specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin-secreting pancreatic β-cells are the sulfonylurea-1 (SUR1) and the glucagon-like peptide-1 (GLP-1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP-1 (7-36 GLP-1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to β-cells, by using fluorescence microscopy or time-resolved saturation and competition binding assays, respectively. Once the ligand binds to β-cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium-labelled GLP-1, likely a result of cooperative binding to the complementary receptors on the βTC3 cells. The high-affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for β-cell targeting in vivo. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Kelly, A. C., Steyn, L. V., Kitzmann, J. P., Anderson, M. J., Mueller, K. R., Hart, N. J., Lynch, R. M., Papas, K. K., & Limesand, S. W. (2014). Function and expression of sulfonylurea, adrenergic, and glucagon-like peptide 1 receptors in isolated porcine islets. Xenotransplantation, 21(4), 385-91.
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  The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P < 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion.
• Yates, D. T., Clarke, D. S., Macko, A. R., Anderson, M. J., Shelton, L. A., Nearing, M., Allen, R. E., Rhoads, R. P., & Limesand, S. W. (2014). Myoblasts from intrauterine growth-restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres. The Journal of physiology, 592(Pt 14), 3113-25.
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  Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P < 0.05) than controls and had smaller (P < 0.05) carcasses and semitendinosus myofibre areas. IUGR semitendinosus muscles had similar percentages of pax7-positive nuclei and pax7 mRNA but lower (P < 0.05) percentages of myogenin-positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer (P < 0.05) proliferating myoblasts (PNCA-positive-pax7-positive) than controls (44 ± 2% vs. 52 ± 1%). Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer (P < 0.05) myoD-positive cells than controls and replicated 20% less (P < 0.05) during a 2 h BrdU pulse. IUGR myoblasts also replicated less (P < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P < 0.05) with IUGR FSS-supplemented media compared to control FSS-supplemented media. In differentiation-promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin-positive nuclei after 5 days and formed similar-sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofibre size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR newborn offspring.
• Chen, X., Green, A. S., Macko, A. R., Yates, D. T., Kelly, A. C., & Limesand, S. W. (2013). Enhanced Insulin Responsiveness and Islet Adrenergic Desensitization after Discontinuing Chronic Norepinephrine Suppression in Fetal Sheep. American journal of physiology. Endocrinology and metabolism.
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  Intrauterine growth restricted (IUGR) fetuses experience prolonged hypoxemia, hypoglycemia, and elevated norepinephrine (NE) concentrations, resulting in hypoinsulinemia and β-cell dysfunction. We previously showed that acute adrenergic blockade revealed enhanced insulin secretion responsiveness in the IUGR fetus. To determine if chronic exposure to NE alone enhances β-cell responsiveness afterward, we continuously infused NE into fetal sheep for 7 days and, after terminating the infusion, evaluated glucose-stimulated insulin secretion (GSIS) and glucose-potentiated arginine-induced insulin secretion (GPAIS). During treatment, NE-infused fetuses had greater (P < 0.05) plasma NE concentrations, and exhibited hyperglycemia (P < 0.01) and hypoinsulinemia (P < 0.01) compared to controls. GSIS during the NE infusion also was reduced (P < 0.05) compared to pre-treatment values. GSIS and GPAIS were ~4-fold greater (P < 0.01) in NE fetuses three hours after discontinuing the 7 days NE infusion compared to age-matched controls or pre-treatment GSIS and GPAIS values of NE fetuses. In isolated pancreatic islets from NE fetuses, mRNA concentrations of adrenergic receptor isoforms (α1D, α2A, α2C, and β1), G-protein subunit αi-2, and uncoupling protein 2 were lower (P < 0.05) compared to controls, but β-cell regulatory genes were not different. Our findings indicate that chronic exposure to elevated NE persistently suppresses insulin secretion. After removal, NE fetuses demonstrated a compensatory enhancement in insulin secretion that was associated with adrenergic desensitization and greater stimulus-secretion coupling in pancreatic islets.
• Gadhia, M. M., Maliszewski, A. M., O'Meara, M. C., Thorn, S. R., Lavezzi, J. R., Limesand, S. W., Hay Jr., W. W., Brown, L. D., & Rozance, P. J. (2013). Increased amino acid supply potentiates glucose-stimulated insulin secretion but does not increase β-cell mass in fetal sheep. American Journal of Physiology - Endocrinology and Metabolism, 304(4), E352-E362.
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  PMID: 23211516;PMCID: PMC3566506;Abstract: Amino acids and glucose acutely stimulate fetal insulin secretion. In isolated adult pancreatic islets, amino acids potentiate glucose-stimulated insulin secretion (GSIS), but whether amino acids have this same effect in the fetus is unknown. Therefore, we tested the effects of increased fetal amino acid supply on GSIS and morphology of the pancreas. We hypothesized that increasing fetal amino acid supply would potentiate GSIS. Singleton fetal sheep received a direct intravenous infusion of an amino acid mixture (AA) or saline (CON) for 10-14 days during late gestation to target a 25-50% increase in fetal branched-chain amino acids (BCAA). Early-phase GSIS increased 150% in the AA group (P < 0.01), and this difference was sustained for the duration of the hyperglycemic clamp (105 min) (P < 0.05). Glucose-potentiated arginine-stimulated insulin secretion (ASIS), pancreatic insulin content, and pancreatic glucagon content were similar between groups. β-Cell mass and area were unchanged between groups. Baseline and arginine-stimulated glucagon concentrations were increased in the AA group (P < 0.05). Pancreatic α-cell mass and area were unchanged. Fetal and pancreatic weights were similar. We conclude that a sustained increase of amino acid supply to the normally growing lategestation fetus potentiated fetal GSIS but did not affect the morphology or insulin content of the pancreas. We speculate that increased β-cell responsiveness (insulin secretion) following increased amino acid supply may be due to increased generation of secondary messengers in the β-cell. This may be enhanced by the paracrine action of glucagon on the β-cell. © 2013 the American Physiological Society.
• Gadhia, M. M., Maliszewski, A. M., O'Meara, M. C., Thorn, S. R., Lavezzi, J. R., Limesand, S. W., Hay, W. W., Brown, L. D., & Rozance, P. J. (2013). Increased amino acid supply potentiates glucose-stimulated insulin secretion but does not increase β-cell mass in fetal sheep. American journal of physiology. Endocrinology and metabolism, 304(4).
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  Amino acids and glucose acutely stimulate fetal insulin secretion. In isolated adult pancreatic islets, amino acids potentiate glucose-stimulated insulin secretion (GSIS), but whether amino acids have this same effect in the fetus is unknown. Therefore, we tested the effects of increased fetal amino acid supply on GSIS and morphology of the pancreas. We hypothesized that increasing fetal amino acid supply would potentiate GSIS. Singleton fetal sheep received a direct intravenous infusion of an amino acid mixture (AA) or saline (CON) for 10-14 days during late gestation to target a 25-50% increase in fetal branched-chain amino acids (BCAA). Early-phase GSIS increased 150% in the AA group (P < 0.01), and this difference was sustained for the duration of the hyperglycemic clamp (105 min) (P < 0.05). Glucose-potentiated arginine-stimulated insulin secretion (ASIS), pancreatic insulin content, and pancreatic glucagon content were similar between groups. β-Cell mass and area were unchanged between groups. Baseline and arginine-stimulated glucagon concentrations were increased in the AA group (P < 0.05). Pancreatic α-cell mass and area were unchanged. Fetal and pancreatic weights were similar. We conclude that a sustained increase of amino acid supply to the normally growing late-gestation fetus potentiated fetal GSIS but did not affect the morphology or insulin content of the pancreas. We speculate that increased β-cell responsiveness (insulin secretion) following increased amino acid supply may be due to increased generation of secondary messengers in the β-cell. This may be enhanced by the paracrine action of glucagon on the β-cell.
• Hart, N. J., Chung, W. J., Weber, C., Ananthakrishnan, K., Anderson, M., Patek, R., Zhang, Z., Limesand, S. W., Vagner, J., & Lynch, R. M. (2013). Hetero-bivalent GLP-1/Glibenclamide for Targeting Pancreatic β-Cells. Chembiochem : a European journal of chemical biology.
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  G protein-coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell-specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin-secreting pancreatic β-cells are the sulfonylurea-1 (SUR1) and the glucagon-like peptide-1 (GLP-1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP-1 (7-36 GLP-1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to β-cells, by using fluorescence microscopy or time-resolved saturation and competition binding assays, respectively. Once the ligand binds to β-cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium-labelled GLP-1, likely a result of cooperative binding to the complementary receptors on the βTC3 cells. The high-affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for β-cell targeting in vivo.
• Limesand, S. W., Rozance, P. J., Macko, A. R., Anderson, M. J., Kelly, A. C., & Hay Jr., W. W. (2013). Reductions in insulin concentrations and β-cell mass precede growth restriction in sheep fetuses with placental insufficiency. American Journal of Physiology - Endocrinology and Metabolism, 304(5), E516-E523.
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  PMID: 23277186;PMCID: PMC3602661;Abstract: In pregnancy complicated by placental insufficiency (PI) and intrauterine growth restriction (IUGR), the fetus near term has reduced basal and glucose-stimulated insulin concentrations and reduced β-cell mass. To determine whether suppression of insulin concentrations and β-cell mass precedes reductions in fetal weight, which would implicate insulin deficiency as a cause of subsequent IUGR, we measured basal and glucose-stimulated insulin concentrations and pancreatic histology at 0.7 gestation in PI fetuses. Placental weights in the PI pregnancies were 40% lower than controls (265 ± 26 vs. 442 ± 41 g, P< 0.05), but fetal weights were not different. At basal conditions blood oxygen content, plasma glucose concentrations, and plasma insulin concentrations were lower in PI fetuses compared with controls (2.5 ± 0.3 vs. 3.5 ± 0.3 mmol/l oxygen, P< 0.05; 1.11 ± 0.09 vs. 1.44 ± 0.12 mmol/l glucose; 0.12 ± 0.01 vs. 0.27 ± 0.02 ng/ml insulin; P< 0.05). During a steady-state hyperglycemic clamp (~2.5 ± 0.1 mmol/l), glucose-stimulated insulin concentrations were lower in PI fetuses than controls (0.28 ± 0.02 vs. 0.55 ± 0.04 ng/ml; P< 0.01). Plasma norepinephrine concentrations were 3.3-fold higher (P< 0.05) in PI fetuses (635 ± 104 vs. 191 ± 91 pg/ml). Histological examination revealed less insulin area and lower β-cell mass and rates of mitosis. The pancreatic parenchyma was also less dense (P< 0.01) in PI fetuses, but no differences were found for pancreatic progenitor cells or other endocrine cell types. These findings show that hypoglycemia, hypoxemia, and hypercatecholaminemia are present and potentially contribute to lower insulin concentrations and β-cell mass due to slower proliferation rates in early third-trimester PI fetuses before discernible reductions in fetal weight. © 2013 the American Physiological Society.
• Limesand, S. W., Rozance, P. J., Macko, A. R., Anderson, M. J., Kelly, A. C., & Hay, W. W. (2013). Reductions in insulin concentrations and β-cell mass precede growth restriction in sheep fetuses with placental insufficiency. American journal of physiology. Endocrinology and metabolism, 304(5).
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  In pregnancy complicated by placental insufficiency (PI) and intrauterine growth restriction (IUGR), the fetus near term has reduced basal and glucose-stimulated insulin concentrations and reduced β-cell mass. To determine whether suppression of insulin concentrations and β-cell mass precedes reductions in fetal weight, which would implicate insulin deficiency as a cause of subsequent IUGR, we measured basal and glucose-stimulated insulin concentrations and pancreatic histology at 0.7 gestation in PI fetuses. Placental weights in the PI pregnancies were 40% lower than controls (265 ± 26 vs. 442 ± 41 g, P < 0.05), but fetal weights were not different. At basal conditions blood oxygen content, plasma glucose concentrations, and plasma insulin concentrations were lower in PI fetuses compared with controls (2.5 ± 0.3 vs. 3.5 ± 0.3 mmol/l oxygen, P < 0.05; 1.11 ± 0.09 vs. 1.44 ± 0.12 mmol/l glucose; 0.12 ± 0.01 vs. 0.27 ± 0.02 ng/ml insulin; P < 0.05). During a steady-state hyperglycemic clamp (~2.5 ± 0.1 mmol/l), glucose-stimulated insulin concentrations were lower in PI fetuses than controls (0.28 ± 0.02 vs. 0.55 ± 0.04 ng/ml; P < 0.01). Plasma norepinephrine concentrations were 3.3-fold higher (P < 0.05) in PI fetuses (635 ± 104 vs. 191 ± 91 pg/ml). Histological examination revealed less insulin area and lower β-cell mass and rates of mitosis. The pancreatic parenchyma was also less dense (P < 0.01) in PI fetuses, but no differences were found for pancreatic progenitor cells or other endocrine cell types. These findings show that hypoglycemia, hypoxemia, and hypercatecholaminemia are present and potentially contribute to lower insulin concentrations and β-cell mass due to slower proliferation rates in early third-trimester PI fetuses before discernible reductions in fetal weight.
• MacKo, A. R., Yates, D. T., Chen, X., Green, A. S., Kelly, A. C., Brown, L. D., & Limesand, S. W. (2013). Elevated plasma norepinephrine inhibits insulin secretion, but adrenergic blockade reveals enhanced β-cell responsiveness in an ovine model of placental insufficiency at 0.7 of gestation. Journal of Developmental Origins of Health and Disease, 4(5), 402-410.
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  Abstract: In pregnancies complicated by placental insufficiency (PI), fetal hypoglycemia and hypoxemia progressively worsen during the third trimester, which increases circulating norepinephrine (NE). Pharmacological adrenergic blockade (ADR-block) at 0.9 gestation revealed that NE inhibits insulin secretion and enhanced β-cell responsiveness in fetuses with PI-induced intrauterine growth restriction (IUGR). NE concentrations in PI fetuses at 0.7 gestation were threefold greater compared with age-matched controls, but the levels were similar to near-term controls. Therefore, our objective was to determine whether elevations in plasma NE concentrations inhibit insulin secretion and produce compensatory β-cell responsiveness in PI fetuses at 0.7 gestation. Fetal insulin was measured under basal, glucose-stimulated insulin secretion (GSIS) and glucose-potentiated arginine-stimulated insulin secretion (GPAIS) conditions in the absence and presence of an ADR-block. Placental weights were 38% lower (P < 0.05) in PI fetus than in controls, but fetal weights were not different. PI fetuses had lower (P < 0.05) basal blood oxygen content, plasma glucose, insulin-like growth factor-1 and insulin concentrations and greater plasma NE concentrations (891 ± 211 v. 292 ± 65 pg/ml; P < 0.05) than controls. GSIS was lower in PI fetuses than in controls (0.34 ± 0.03 v. 1.08 ± 0.06 ng/ml; P < 0.05). ADR-block increased GSIS in PI fetuses (1.19 ± 0.11 ng/ml; P < 0.05) but decreased GSIS in controls (0.86 ± 0.02 ng/ml; P < 0.05). Similarly, GPAIS was 44% lower (P < 0.05) in PI fetuses than in controls, and ADR-block increased (P < 0.05) GPAIS in PI fetuses but not in controls. Insulin content per islet was not different between treatments. We conclude that elevations in fetal plasma NE suppress insulin concentrations, and that compensatory β-cell stimulus-secretion responsiveness is present before IUGR. © 2013 Cambridge University Press and the International Society for Developmental Origins of Health and Disease.
• Macko, A. R., Yates, D. T., Chen, X., Green, A. S., Kelly, A. C., Brown, L. D., & Limesand, S. W. (2013). Elevated plasma norepinephrine inhibits insulin secretion, but adrenergic blockade reveals enhanced β-cell responsiveness in an ovine model of placental insufficiency at 0.7 of gestation. Journal of developmental origins of health and disease, 4(5).
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  In pregnancies complicated by placental insufficiency (PI), fetal hypoglycemia and hypoxemia progressively worsen during the third trimester, which increases circulating norepinephrine (NE). Pharmacological adrenergic blockade (ADR-block) at 0.9 gestation revealed that NE inhibits insulin secretion and enhanced β-cell responsiveness in fetuses with PI-induced intrauterine growth restriction (IUGR). NE concentrations in PI fetuses at 0.7 gestation were 3-fold greater compared to age-matched controls but the levels were similar to near-term controls. Therefore, our objective was to determine whether elevations in plasma NE concentrations inhibit insulin secretion and produce compensatory β-cell responsiveness in PI fetuses at 0.7 gestation. Fetal insulin was measured under basal, glucose-stimulated (GSIS), and glucose potentiated arginine-stimulated (GPAIS) conditions in the absence and presence of an ADR-block. Placental weights were 38% lower (P
• Penrod, L. V., Allen, R. E., Rhoads, M. L., Limesand, S. W., & Arns, M. J. (2013). Oxytocin stimulated release of PGF2α and its inhibition by a cyclooxygenase inhibitor and an oxytocin receptor antagonist from equine endometrial cultures. Animal reproduction science, 139(1-4).
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  Uterine inflammation results in a poor uterine environment and early embryonic loss in the mare due to an inhibition of maternal recognition of pregnancy caused from increased prostaglandin F2α (PGF2α). Oxytocin binds to endometrial cell receptors to activate prostaglandin synthesis. An oxytocin receptor antagonist (Atosiban) and a cyclooxygenase inhibitor (indomethacin) both decrease PGF2α production. The aim of this study was to evaluate the in vitro effects of Atosiban and indomethacin on equine uterine prostaglandin secretion. Equine endometrial explants were harvested on day two of behavioral estrus. Endometrial explant cultures were challenged with oxytocin (250nM) and PGF2α concentrations were measured over time. Explants were also cultured with Atosiban and indomethacin for 6h to determine the influence on PGF2α secretion. When endometrial explants were challenged with oxytocin, PGF2α concentrations were greater (P
• Penrod, L. V., Allen, R. E., Turner, J. L., Limesand, S. W., & Arns, M. J. (2013). Effects of oxytocin, lipopolysaccharide (LPS), and polyunsaturated fatty acids on prostaglandin secretion and gene expression in equine endometrial explant cultures. Domestic animal endocrinology, 44(1).
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  Increased secretion of prostaglandin F(2)α (PGF(2)α) within the uterus because of uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence PG production in many species, although the effects on the mare remain unknown. The present study aimed to determine fatty acid uptake in equine endometrial explants and evaluate their influence on PG secretion and expression of enzymes involved in PG synthesis in vitro. Equine endometrial explants were treated with 100 μM arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid and then challenged with oxytocin (250 nM) or lipopolysaccharide (LPS; 1 μg/mL). Production of PGF(2)α and PG E(2) (PGE(2)) was measured, and mRNA expression of enzymes involved in PG synthesis was determined with quantitative real-time PCR. Media concentrations of PGF(2)α and PGE(2) were higher (P < 0.0001) from endometrial explants challenged with oxytocin or LPS compared with controls despite which fatty acid was added. Only DHA lowered (P < 0.0001) media concentrations of PGF(2)α and PGE(2) from explants. Endometrial explants stimulated with oxytocin had increased expression of PG-endoperoxide synthase 1 (PTGS1; P < 0.02), PG-endoperoxide synthase 2 (PTGS2; P < 0.001), PG F(2)α synthase (PGFS; P < 0.01), PG E(2) synthase (PGES; P < 0.01), and phospholipase A(2) (PLA(2); P < 0.005) compared with controls and regardless of fatty acid treatment; whereas stimulation with LPS increased expression of PTGS2 (P < 0.004), PGFS (P < 0.03), PGES (P < 0.01), and PLA(2) (P < 0.01) compared with controls and regardless of fatty acid treatment. Treatment with PUFAs, specifically DHA, can influence PG secretion in vitro through mechanisms other than enzyme expression.
• Chen, X., Rozance, P. J., Hay Jr., W. W., & Limesand, S. W. (2012). Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas. Experimental Biology and Medicine, 237(5), 524-529.
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  PMID: 22581814;PMCID: PMC3404611;Abstract: Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P, 0.01), 76% (P, 0.05), 76% (P, 0.05) and 52% (P, 0.01) lower, respectively, compared with control fetuses. Conversely, insulin-like growth factor binding protein 2 (IGFBP-2) mRNA and protein concentrations were 2.25- and 1.2-fold greater (P, 0.05) in the IUGR pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P, 0.05), and insulin mRNA was 56% less (P, 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication. © 2008 Society for Experimental Biology and Medicine.
• Chen, X., Rozance, P., Hay, J. W., & Limesand, S. (2012). Insulin-like Growth Factor and Fibroblast Growth Factor Expression Profiles in Growth Restricted Fetal Sheep Pancreas. Experimental Biology and Medicine, 237(5), 524-529.
• Frost, M. S., Zehri, A. H., Limesand, S. W., Hay Jr., W. W., & Rozance, P. J. (2012). Differential effects of chronic pulsatile versus chronic constant maternal hyperglycemia on fetal pancreatic β-cells. Journal of Pregnancy, 2012.
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  PMID: 23133755;PMCID: PMC3486011;Abstract: Constant maternal hyperglycemia limits, while pulsatile maternal hyperglycemia may enhance, fetal glucose-stimulated insulin secretion (GSIS) in sheep. However, the impact of such different patterns of hyperglycemia on the development of the fetal β-cell is unknown. We measured the impact of one week of chronic constant hyperglycemia (CHG, n=6) versus pulsatile hyperglycemia (PHG, n=5) versus controls (n=7) on the percentage of the fetal pancreas staining for insulin (β-cell area), mitotic and apoptotic indices and size of fetal β-cells, and fetal insulin secretion in sheep. Baseline insulin concentrations were higher in CHG fetuses (P < 0.05) compared to controls and PHG. GSIS was lower in the CHG group (P < 0.005) compared to controls and PHG. PHG β-cell area was increased 50 (P < 0.05) compared to controls and CHG. CHG β-cell apoptosis was increased over 400 (P0.05) compared to controls and PHG. These results indicate that late gestation constant maternal hyperglycemia leads to significant β-cell toxicity (increased apoptosis and decreased GSIS). Furthermore, pulsatile maternal hyperglycemia increases pancreatic β-cell area but did not increase GSIS, indicating decreased β-cell responsiveness. These findings demonstrate differential effects that the pattern of maternal hyperglycemia has on fetal pancreatic β-cell development, which might contribute to later life limitation in insulin secretion. © 2012 Mackenzie S. Frost et al.
• Frost, M. S., Zehri, A. H., Limesand, S. W., Hay, W. W., & Rozance, P. J. (2012). Differential effects of chronic pulsatile versus chronic constant maternal hyperglycemia on fetal pancreatic β-cells. Journal of pregnancy, 2012.
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  Constant maternal hyperglycemia limits, while pulsatile maternal hyperglycemia may enhance, fetal glucose-stimulated insulin secretion (GSIS) in sheep. However, the impact of such different patterns of hyperglycemia on the development of the fetal β-cell is unknown. We measured the impact of one week of chronic constant hyperglycemia (CHG, n = 6) versus pulsatile hyperglycemia (PHG, n = 5) versus controls (n = 7) on the percentage of the fetal pancreas staining for insulin (β-cell area), mitotic and apoptotic indices and size of fetal β-cells, and fetal insulin secretion in sheep. Baseline insulin concentrations were higher in CHG fetuses (P < 0.05) compared to controls and PHG. GSIS was lower in the CHG group (P < 0.005) compared to controls and PHG. PHG β-cell area was increased 50% (P < 0.05) compared to controls and CHG. CHG β-cell apoptosis was increased over 400% (P < 0.05) compared to controls and PHG. These results indicate that late gestation constant maternal hyperglycemia leads to significant β-cell toxicity (increased apoptosis and decreased GSIS). Furthermore, pulsatile maternal hyperglycemia increases pancreatic β-cell area but did not increase GSIS, indicating decreased β-cell responsiveness. These findings demonstrate differential effects that the pattern of maternal hyperglycemia has on fetal pancreatic β-cell development, which might contribute to later life limitation in insulin secretion.
• Frost, M., Zehri, A., Limesand, S., Hay, J. W., & Rozance, P. (2012). Differential effects of chronic pulsatile vs. chronic constant maternal hyperglycemia on fetal pancreatic beta-cells. Journal of Pregnancy.
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  Article ID 812094, 2012
• Green, A. S., Chen, X., Macko, A. R., Anderson, M. J., Kelly, A. C., Hart, N. J., Lynch, R. M., & Limesand, S. W. (2012). Chronic pulsatile hyperglycemia reduces insulin secretion and increases accumulation of reactive oxygen species in fetal sheep islets. Journal of Endocrinology, 212(3), 327-342.
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  PMID: 22182602;PMCID: PMC3516619;Abstract: Children from diabetic pregnancies have a greater incidence of type 2 diabetes. Our objective was to determine if exposure to mild-moderate hyperglycemia, by modeling managed diabetic pregnancies, affects fetal β-cell function. In sheep fetuses, β-cell responsiveness was examined after 2 weeks of sustained hyperglycemia with 3 pulses/day, mimicking postprandial excursions, and compared to saline-infused controls (n=10). Two pulsatile hyperglycemia (PHG) treatments were studied: mild (mPHG, n=5) with +15% sustained and +55% pulse; and moderate (PHG, n=10) with +20% sustained and +100% pulse. Fetal glucose-stimulated insulin secretion and glucose-potentiated arginine insulin secretion were lower (P
• Green, A., Chen, X., Macko, A., Anderson, M., Kelly, A., Hart, N., Lynch, R., & Limesand, S. (2012). Chronic Pulsatile Hyperglycemia Reduces Insulin Secretion and Increases Accumulation of Reactive Oxygen Species in Fetal Sheep Islets. Journal of Endocrinology, 212(3), 327-42.
• Limesand, S. W., Yates, D., Macko, A., Nearing, M., Chen, X., Rhoads, P., & Limesand, S. W. (2012). Developmental Programming in Response to Intrauterine Growth Restriction Impairs Myoblast Function and Skeletal Muscle Metabolism. Journal of Pregnancy.
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  Article ID 631038, 2012
• Limesand, S., Chen, X., Rozance, P. J., Hay, W. W., & Limesand, S. W. (2012). Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas. Experimental biology and medicine (Maywood, N.J.), 237(5).
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  Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P < 0.01), 76% (P < 0.05), 76% (P < 0.05) and 52% (P < 0.01) lower, respectively, compared with control fetuses. Conversely, insulin-like growth factor binding protein 2 (IGFBP-2) mRNA and protein concentrations were 2.25- and 1.2-fold greater (P < 0.05) in the IUGR pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P < 0.05), and insulin mRNA was 56% less (P < 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication.
• Limesand, S., Green, A. S., Chen, X., Macko, A. R., Anderson, M. J., Kelly, A. C., Hart, N. J., Lynch, R. M., & Limesand, S. W. (2012). Chronic pulsatile hyperglycemia reduces insulin secretion and increases accumulation of reactive oxygen species in fetal sheep islets. The Journal of endocrinology, 212(3).
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  Children from diabetic pregnancies have a greater incidence of type 2 diabetes. Our objective was to determine if exposure to mild-moderate hyperglycemia, by modeling managed diabetic pregnancies, affects fetal β-cell function. In sheep fetuses, β-cell responsiveness was examined after 2 weeks of sustained hyperglycemia with 3 pulses/day, mimicking postprandial excursions, and compared to saline-infused controls (n = 10). Two pulsatile hyperglycemia (PHG) treatments were studied: mild (mPHG, n = 5) with +15% sustained and +55% pulse; and moderate (PHG, n = 10) with +20% sustained and +100% pulse. Fetal glucose-stimulated insulin secretion and glucose-potentiated arginine insulin secretion were lower (P < 0.05) in PHG (0.86 ± 0.13 and 2.91 ± 0.39  ng/ml plasma insulin) but not in mPHG fetuses (1.21 ± 0.08 and 4.25 ± 0.56  ng/ml) compared to controls (1.58 ± 0.25 and 4.51 ± 0.56  ng/ml). Islet insulin content was 35% lower in PHG and 35% higher in mPHG vs controls (P < 0.01). Insulin secretion and maximally stimulated insulin release were also reduced (P < 0.05) in PHG islets due to lower islet insulin content. Isolated PHG islets also had 63% greater (P < 0.01) reactive oxygen species (ROS) accumulation at 11.1  mmol/l glucose than controls (P < 0.01), but oxidative damage was not detected in islet proteins. PHG fetuses showed evidence of oxidative damage to skeletal muscle proteins (P < 0.05) but not insulin resistance. Our findings show that PHG induced dysregulation of islet ROS handling and decreased islet insulin content, but these outcomes are independent. The β-cell outcomes were dependent on the severity of hyperglycemia because mPHG fetuses had no distinguishable impairments in ROS handling or insulin secretion but greater insulin content.
• Limesand, S., Yates, D. T., Macko, A. R., Chen, X., Green, A. S., Kelly, A. C., Anderson, M. J., Fowden, A. L., & Limesand, S. W. (2012). Hypoxaemia-induced catecholamine secretion from adrenal chromaffin cells inhibits glucose-stimulated hyperinsulinaemia in fetal sheep. The Journal of physiology, 590(Pt 21).
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  Abstract  Hypoxaemia elicits adrenergic suppression of fetal glucose-stimulated hyperinsulinaemia. We postulate that this effect is mediated by catecholamines, exclusively, from fetal adrenal chromaffin cells. To investigate this hypothesis, square-wave hyperglycaemic clamp studies were performed under normoxaemic (26 ± 0.9 mmHg) and hypoxaemic (14 ± 0.3 mmHg) steady-state conditions in near-term fetal sheep that had undergone either surgical sham or bilateral adrenal demedullation (AD), values mentioned are ± SEM. Under normoxaemic conditions plasma noradrenaline concentrations were lower in AD fetuses than in sham-operated fetuses (457 ± 122 versus 1073 ± 103 pg ml(-1), P < 0.05). Plasma insulin concentrations were not different at euglycaemia between shams (0.46 ± 0.07 ng ml(-1)) and AD fetuses (0.44 ± 0.04 ng ml(-1)) and increased (P < 0.05) with hyperglycaemia in both groups although to a lesser extent in AD fetuses (0.94 ± 0.19 ng ml(-1)) compared to shams (1.31 ± 0.15 ng ml(-1); P < 0.05). Hypoxaemia increased plasma adrenaline (26-fold) and noradrenaline (5-fold) in shams but elicited no change in AD fetuses. Under hypoxaemic conditions, euglycaemic plasma insulin concentrations were reduced (P < 0.05) in both sham and AD fetuses to 0.30 ± 0.05 ng ml(-1) and 0.27 ± 0.01 ng ml(-1) respectively, and the insulin response to hyperglycaemia was abolished in shams but not affected in AD fetuses (0.33 ± 0.06 versus 0.73 ± 0.02 ng ml(-1), P < 0.05). Hypoxaemia also induced hyperlactacaemia and hypocarbia to a greater extent in shams than in AD fetuses, indicating that catecholamines potentiate reductions in oxidative metabolism independently of insulin. These findings demonstrate that the fetal adrenal chromaffin cells are the source for acute hypoxaemia-induced elevations in fetal plasma catecholamines and suppression of glucose-stimulated hyperinsulinaemia, but other factors reduce plasma insulin at euglycaemia.
• Limesand, S., Yates, D. T., Macko, A. R., Nearing, M., Chen, X., Rhoads, R. P., & Limesand, S. W. (2012). Developmental programming in response to intrauterine growth restriction impairs myoblast function and skeletal muscle metabolism. Journal of pregnancy, 2012.
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  Fetal adaptations to placental insufficiency alter postnatal metabolic homeostasis in skeletal muscle by reducing glucose oxidation rates, impairing insulin action, and lowering the proportion of oxidative fibers. In animal models of intrauterine growth restriction (IUGR), skeletal muscle fibers have less myonuclei at birth. This means that myoblasts, the sole source for myonuclei accumulation in fibers, are compromised. Fetal hypoglycemia and hypoxemia are complications that result from placental insufficiency. Hypoxemia elevates circulating catecholamines, and chronic hypercatecholaminemia has been shown to reduce fetal muscle development and growth. We have found evidence for adaptations in adrenergic receptor expression profiles in myoblasts and skeletal muscle of IUGR sheep fetuses with placental insufficiency. The relationship of β-adrenergic receptors shifts in IUGR fetuses because Adrβ2 expression levels decline and Adrβ1 expression levels are unaffected in myofibers and increased in myoblasts. This adaptive response would suppress insulin signaling, myoblast incorporation, fiber hypertrophy, and glucose oxidation. Furthermore, this β-adrenergic receptor expression profile persists for at least the first month in IUGR lambs and lowers their fatty acid mobilization. Developmental programming of skeletal muscle adrenergic receptors partially explains metabolic and endocrine differences in IUGR offspring, and the impact on metabolism may result in differential nutrient utilization.
• Yates, D. T., Chen, X., & Limesand, S. W. (2012). Environmental Heat Stress Impairs Placental Function, Fetal Growth and Development, and Postnatal Performance in Livestock. Environmental Physiology of Livestock, 209-228.
• Yates, D. T., MacKo, A. R., Nearing, M., Chen, X., Rhoads, R. P., & Limesand, S. W. (2012). Developmental programming in response to intrauterine growth restriction impairs myoblast function and skeletal muscle metabolism. Journal of Pregnancy, 2012.
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  PMID: 22900186;PMCID: PMC3415084;Abstract: Fetal adaptations to placental insufficiency alter postnatal metabolic homeostasis in skeletal muscle by reducing glucose oxidation rates, impairing insulin action, and lowering the proportion of oxidative fibers. In animal models of intrauterine growth restriction (IUGR), skeletal muscle fibers have less myonuclei at birth. This means that myoblasts, the sole source for myonuclei accumulation in fibers, are compromised. Fetal hypoglycemia and hypoxemia are complications that result from placental insufficiency. Hypoxemia elevates circulating catecholamines, and chronic hypercatecholaminemia has been shown to reduce fetal muscle development and growth. We have found evidence for adaptations in adrenergic receptor expression profiles in myoblasts and skeletal muscle of IUGR sheep fetuses with placental insufficiency. The relationship of β-adrenergic receptors shifts in IUGR fetuses because Adrβ2 expression levels decline and Adrβ1 expression levels are unaffected in myofibers and increased in myoblasts. This adaptive response would suppress insulin signaling, myoblast incorporation, fiber hypertrophy, and glucose oxidation. Furthermore, this β-adrenergic receptor expression profile persists for at least the first month in IUGR lambs and lowers their fatty acid mobilization. Developmental programming of skeletal muscle adrenergic receptors partially explains metabolic and endocrine differences in IUGR offspring, and the impact on metabolism may result in differential nutrient utilization. © 2012 D. T. Yates et al.
• Yates, D. T., Macko, A. R., Chen, X., Green, A. S., Kelly, A. C., Anderson, M. J., Fowden, A. L., & Limesand, S. W. (2012). Hypoxaemia-induced catecholamine secretion from adrenal chromaffin cells inhibits glucose-stimulated hyperinsulinaemia in fetal sheep. Journal of Physiology, 590(21), 5439-5447.
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  PMID: 22907052;PMCID: PMC3515829;Abstract: Hypoxaemia elicits adrenergic suppression of fetal glucose-stimulated hyperinsulinaemia. We postulate that this effect is mediated by catecholamines, exclusively, from fetal adrenal chromaffin cells. To investigate this hypothesis, square-wave hyperglycaemic clamp studies were performed under normoxaemic (26 ± 0.9 mmHg) and hypoxaemic (14 ± 0.3 mmHg) steady-state conditions in near-term fetal sheep that had undergone either surgical sham or bilateral adrenal demedullation (AD), values mentioned are ± SEM. Under normoxaemic conditions plasma noradrenaline concentrations were lower in AD fetuses than in sham-operated fetuses (457 ± 122 versus 1073 ± 103 pg ml-1, P < 0.05). Plasma insulin concentrations were not different at euglycaemia between shams (0.46 ± 0.07 ng ml-1) and AD fetuses (0.44 ± 0.04 ng ml-1) and increased (P < 0.05) with hyperglycaemia in both groups although to a lesser extent in AD fetuses (0.94 ± 0.19 ng ml-1) compared to shams (1.31 ± 0.15 ng ml-1; P < 0.05). Hypoxaemia increased plasma adrenaline (26-fold) and noradrenaline (5-fold) in shams but elicited no change in AD fetuses. Under hypoxaemic conditions, euglycaemic plasma insulin concentrations were reduced (P < 0.05) in both sham and AD fetuses to 0.30 ± 0.05 ng ml-1 and 0.27 ± 0.01 ng ml-1 respectively, and the insulin response to hyperglycaemia was abolished in shams but not affected in AD fetuses (0.33 ± 0.06 versus 0.73 ± 0.02 ng ml-1, P < 0.05). Hypoxaemia also induced hyperlactacaemia and hypocarbia to a greater extent in shams than in AD fetuses, indicating that catecholamines potentiate reductions in oxidative metabolism independently of insulin. These findings demonstrate that the fetal adrenal chromaffin cells are the source for acute hypoxaemia-induced elevations in fetal plasma catecholamines and suppression of glucose-stimulated hyperinsulinaemia, but other factors reduce plasma insulin at euglycaemia. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.
• Yates, D., Macko, A., Chen, X., Green, A., Kelly, A., Anderson, M., Fowden, A., & Limesand, S. (2012). Hypoxaemia-induced catecholamine secretion from adrenal chromaffin cells inhibits glucose-stimulated hyperinsulinaemia in fetal sheep. Journal of Physiology, 590, 5439-47.
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  (Pt 21)
• Brown, L. D., Green, A. S., Limesand, S. W., & Rozance, P. J. (2011). Maternal amino acid supplementation for intrauterine growth restriction. Frontiers in Bioscience - Scholar, 3 S(2), 428-444.
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  PMID: 21196387;PMCID: PMC3181118;Abstract: Maternal dietary protein supplementation to improve fetal growth has been considered as an option to prevent or treat intrauterine growth restriction. However, in contrast to balanced dietary supplementation, adverse perinatal outcomes in pregnant women who received high amounts of dietary protein supplementation have been observed. The responsible mechanisms for these adverse outcomes are unknown. This review will discuss relevant human and animal data to provide the background necessary for the development of explanatory hypotheses and ultimately for the development therapeutic interventions during pregnancy to improve fetal growth. Relevant aspects of fetal amino acid metabolism during normal pregnancy and those pregnancies affected by IUGR will be discussed. In addition, data from animal experiments which have attempted to determine mechanisms to explain the adverse responses identified in the human trials will be presented. Finally, we will suggest new avenues for investigation into how amino acid supplementation might be used safely to treat and/or prevent IUGR.
• Green, A. S., Macko, A. R., Rozance, P. J., Yates, D. T., Chen, X., Hay Jr., W. W., & Limesand, S. W. (2011). Characterization of glucose-insulin responsiveness and impact of fetal number and sex difference on insulin response in the sheep fetus. American Journal of Physiology - Endocrinology and Metabolism, 300(5), E817-E823.
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  PMID: 21343544;PMCID: PMC3093975;Abstract: GSIS is often measured in the sheep fetus by a square-wave hyperglycemic clamp, but maximal β-cell responsiveness and effects of fetal number and sex difference have not been fully evaluated. We determined the dose-response curve for GSIS in fetal sheep (0.9 of gestation) by increasing plasma glucose from euglycemia in a stepwise fashion. The glucose- insulin response was best fit by curvilinear third-order polynomial equations for singletons (y = 0.018x3 - 0.26x2 + 1.2x - 0.64) and twins (y = -0.012x3 + 0.043x2 + 0.40x - 0.16). In singles, maximal insulin secretion was achieved at 3.4 ± 0.2 mmol/l glucose but began to plateau after 2.4 ± 0.2 mmol/l glucose (90% of maximum), whereas the maximum for twins was reached at 4.8 ± 0.4 mmol/l glucose. In twin (n = 18) and singleton (n = 49) fetuses, GSIS was determined with a square-wave hyperglycemic clamp >2.4 mmol/l glucose. Twins had a lower basal glucose concentration, and plasma insulin concentrations were 59 (P < 0.01) and 43% (P < 0.05) lower in twins than singletons during the euglycemic and hyperglycemic periods, respectively. The basal glucose/insulin ratio was approximately doubled in twins vs. singles (P < 0.001), indicating greater insulin sensitivity. In a separate cohort of fetuses, twins (n = 8) had lower body weight (P < 0.05) and β-cell mass (P < 0.01) than singleton fetuses (n = 7) as a result of smaller pancreata (P < 0.01) and a positive correlation (P < 0.05) between insulin immunopositive area and fetal weight (P < 0.05). No effects of sex difference on GSIS or β-cell mass were observed. These findings indicate that insulin secretion is less responsive to physiological glucose concentrations in twins, due in part to less β-cell mass. © 2011 the American Physiological Society.
• Limesand, S. W., Green, A., Macko, A., Rozance, P., Yates, D., Chen, X., Hay, J. W., & Limesa, S. (2011). Characterization of glucose-insulin responsiveness and impact of fetal number and gender on insulin response in the sheep fetus. American Journal of Physiology-Endocrinology and Metabolism, 300(5), E817-23.
• Limesand, S. W., Yates, D., , C., Ross, T., Hallford, D., Carter, B., & Limesan, S. (2011). Effects of bacterial lipopolysaccharide injection on white blood cell counts, hematological parameters, and serum glucose, insulin, and cortisol conce. Journal of Animal Science, 89(12), 4286-93.
• Limesand, S. W., Yates, D., Green, A., & Limesan, S. (2011). Catecholamines Mediate Multiple Fetal Adaptations during Placental Insufficiency that Contribute to Intrauterine Growth Restriction: Lessons from Hype. Journal of Pregnancy.
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  Article ID 740408
• Limesand, S., Green, A. S., Macko, A. R., Rozance, P. J., Yates, D. T., Chen, X., Hay, W. W., & Limesand, S. W. (2011). Characterization of glucose-insulin responsiveness and impact of fetal number and sex difference on insulin response in the sheep fetus. American journal of physiology. Endocrinology and metabolism, 300(5).
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  GSIS is often measured in the sheep fetus by a square-wave hyperglycemic clamp, but maximal β-cell responsiveness and effects of fetal number and sex difference have not been fully evaluated. We determined the dose-response curve for GSIS in fetal sheep (0.9 of gestation) by increasing plasma glucose from euglycemia in a stepwise fashion. The glucose-insulin response was best fit by curvilinear third-order polynomial equations for singletons (y = 0.018x(3) - 0.26x(2) + 1.2x - 0.64) and twins (y = -0.012x(3) + 0.043x(2) + 0.40x - 0.16). In singles, maximal insulin secretion was achieved at 3.4 ± 0.2 mmol/l glucose but began to plateau after 2.4 ± 0.2 mmol/l glucose (90% of maximum), whereas the maximum for twins was reached at 4.8 ± 0.4 mmol/l glucose. In twin (n = 18) and singleton (n = 49) fetuses, GSIS was determined with a square-wave hyperglycemic clamp >2.4 mmol/l glucose. Twins had a lower basal glucose concentration, and plasma insulin concentrations were 59 (P < 0.01) and 43% (P < 0.05) lower in twins than singletons during the euglycemic and hyperglycemic periods, respectively. The basal glucose/insulin ratio was approximately doubled in twins vs. singles (P < 0.001), indicating greater insulin sensitivity. In a separate cohort of fetuses, twins (n = 8) had lower body weight (P < 0.05) and β-cell mass (P < 0.01) than singleton fetuses (n = 7) as a result of smaller pancreata (P < 0.01) and a positive correlation (P < 0.05) between insulin immunopositive area and fetal weight (P < 0.05). No effects of sex difference on GSIS or β-cell mass were observed. These findings indicate that insulin secretion is less responsive to physiological glucose concentrations in twins, due in part to less β-cell mass.
• Limesand, S., Victory, K., Burd, R., Fribley, A., Sittadjody, S., Arnett, D., Klein, R. R., & Limesand, S. W. (2011). Head and neck tumor cell radiation response occurs in the presence of IGF1. Journal of dental research, 90(3).
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  Radiation therapy for head and neck cancer results in severe secondary side-effects in salivary glands. We previously demonstrated that the administration of IGF1 preserves or restores salivary gland function following radiation. Based on these findings, we propose to study the effect of IGF1 on human head and neck carcinoma cells. Head and neck tumor cells treated with radiation have significant reductions in tumor cell survival, as measured by MTT and crystal violet assays, regardless of IGF1 pre-treatment. Head and neck squamous carcinoma cell xenografts treated with concurrent radiation+IGF1 also exhibit significant tumor growth delay; however, growth rates are elevated compared with those in irradiated xenografts. In contrast, administration of IGF1 after radiation treatment has no effect on tumor xenograft growth rates. Analysis of these data suggests that localized delivery may be required for concurrent therapy to prevent secondary side-effects of radiotherapy, while post-therapy administration of IGF1 could be considered for the restoration of salivary function.
• Limesand, S., Yates, D. T., Green, A. S., & Limesand, S. W. (2011). Catecholamines mediate multiple fetal adaptations during placental insufficiency that contribute to intrauterine growth restriction: lessons from hyperthermic sheep. Journal of pregnancy, 2011.
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  Placental insufficiency (PI) prevents adequate delivery of nutrients to the developing fetus and creates a chronic state of hypoxemia and hypoglycemia. In response, the malnourished fetus develops a series of stress hormone-mediated metabolic adaptations to preserve glucose for vital tissues at the expense of somatic growth. Catecholamines suppress insulin secretion to promote glucose sparing for insulin-independent tissues (brain, nerves) over insulin-dependent tissues (skeletal muscle, liver, and adipose). Likewise, premature induction of hepatic gluconeogenesis helps maintain fetal glucose and appears to be stimulated by both norepinephrine and glucagon. Reduced glucose oxidation rate in PI fetuses creates a surplus of glycolysis-derived lactate that serves as substrate for hepatic gluconeogenesis. These adrenergically influenced adaptive responses promote in utero survival but also cause asymmetric intrauterine growth restriction and small-for-gestational-age infants that are at greater risk for serious metabolic disorders throughout postnatal life, including obesity and type II diabetes.
• Yates, D. T., Green, A. S., & Limesand, S. W. (2011). Catecholamines mediate multiple fetal adaptations during placental insufficiency that contribute to intrauterine growth restriction: lessons from hyperthermic sheep.. Journal of pregnancy, 2011, 740408-.
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  PMID: 21773031;PMCID: PMC3135098;Abstract: Placental insufficiency (PI) prevents adequate delivery of nutrients to the developing fetus and creates a chronic state of hypoxemia and hypoglycemia. In response, the malnourished fetus develops a series of stress hormone-mediated metabolic adaptations to preserve glucose for vital tissues at the expense of somatic growth. Catecholamines suppress insulin secretion to promote glucose sparing for insulin-independent tissues (brain, nerves) over insulin-dependent tissues (skeletal muscle, liver, and adipose). Likewise, premature induction of hepatic gluconeogenesis helps maintain fetal glucose and appears to be stimulated by both norepinephrine and glucagon. Reduced glucose oxidation rate in PI fetuses creates a surplus of glycolysis-derived lactate that serves as substrate for hepatic gluconeogenesis. These adrenergically influenced adaptive responses promote in utero survival but also cause asymmetric intrauterine growth restriction and small-for-gestational-age infants that are at greater risk for serious metabolic disorders throughout postnatal life, including obesity and type II diabetes.
• Yates, D. T., Löest, C., Ross, T. T., Hallford, D. M., Carter, B. H., & Limesand, S. W. (2011). Effects of bacterial lipopolysaccharide injection on white blood cell counts, hematological variables, and serum glucose, insulin, and cortisol concentrations in ewes fed low- or high-protein diets. Journal of Animal Science, 89(12), 4286-4293.
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  PMID: 21788428;PMCID: PMC3319785;Abstract: Bacterial lipopolysaccharide endotoxins (LPS) elicit inflammatory responses reflective of acute bacterial infection. We determined if feeding ewes high-CP (15.5%) or low-CP (8.5%) diets for 10 d altered inflammatory responses to an intravenous bolus of 0 (control), 0.75 (L75), or 1.50 (L150) μg of LPS/ kg of BW in a 2 × 3 factorial arrangement of treatments (n = 5/treatment). Rectal temperatures, heart and respiratory rates, blood leukocyte concentrations, and serum cortisol, insulin, and glucose concentrations were measured for 24 h after an LPS bolus (bolus = 0 h). In general, rectal temperatures were greater (P ≤ 0.05) in control ewes fed high CP, but LPS increased (P ≤ 0.05) rectal temperatures in a dose-dependent manner at most times between 2 and 24 h after the bolus. Peak rectal temperatures in L75 and L150 occurred 4 h after the bolus. A monophasic, dose-independent increase (P ≤ 0.023) in serum cortisol occurred from 0.5 to 24 h after the bolus, with peak cortisol at 4 h. Serum insulin was increased (P ≤ 0.016) by LPS in a dose-dependent manner from 4 to 24 h after the bolus. Insulin did not differ between control ewes fed high- and low- CP diets but was greater (P < 0.001) in L75 ewes fed low CP compared with high CP and in L150 ewes fed high CP compared with low CP. Increased insulin was not preceded by increased serum glucose. Total white blood cell concentrations were not affected (P ≥ 0.135) by LPS, but the neutrophil and monocyte fractions of white blood cells were increased (P ≤ 0.047) by LPS at 12 and 24 h and at 24 h after the bolus, respectively, and the lymphocyte fraction was increased (P = 0.037) at 2 h and decreased (P ≤ 0.006) at 12 and 24 h after the bolus. Red blood cell and hemoglobin concentrations and hematocrit (%) were increased (P ≤ 0.022) by LPS at 2 and 4 h after the bolus. Rectal temperatures and serum glucose were greater (P ≤ 0.033) in ewes fed a high-CP diet before LPS injection, but these effects were lost at and within 2.5 h of the bolus, respectively. Feeding high-CP diets for 10 d did not reduce inflammation in ewes during the first 24 h after LPS exposure but may benefit livestock by preventing acute insulin resistance when endotoxin exposure is mild. © 2011 American Society of Animal Science. All rights reserved.
• Chen, X., Fahy, A. L., Green, A. S., Anderson, M. J., Rhoads, R. P., & Limesand, S. W. (2010). β2-Adrenergic receptor desensitization in perirenal adipose tissue in fetuses and lambs with placental insufficiency-induced intrauterine growth restriction. Journal of Physiology, 588(18), 3539-3549.
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  PMID: 20643771;PMCID: PMC2988517;Abstract: Placental insufficiency-induced intrauterine growth restriction (IUGR) fetuses have chronic hypoxaemia and elevated plasma catecholamine concentrations. In this study, we determined whether adrenergic responsiveness becomes desensitized in the perirenal adipose tissue of IUGR fetuses and lambs by measuring adrenergic receptor (AR) mRNA and protein levels. We also tested the ability of adrenaline to mobilize non-esterified fatty acids (NEFAs) in young lambs. Perirenal adipose tissue was collected from IUGR and control fetuses at 133 days of gestational age (dGA) and lambs at 18 days of age (dA) β2-AR mRNA concentrations were 59% and 74% lower (P < 0.05) in IUGR fetuses and lambs compared to controls, respectively, which also resulted in lower protein levels (P < 0.05). No treatment differences were detected for α1A-, α1B-, α1D-, α2A-, α2B-, α2C-, β1- and β3-AR expression. mRNA concentrations were also determined for hormone sensitive lipase (HSL), perilipin (lipid droplet-associated protein), and two adipokines, leptin and adiponectin. Adiponectin and HSL were not different between treatments at either age. Compared to controls, perilipin and leptin mRNA concentrations were lower (P < 0.05) in IUGR fetuses but not in lambs. Because of the β2-AR results, we challenged a second cohort of lambs with exogenous adrenaline at 21 dA. The ability of adrenaline to mobilize NEFA was 55 ± 15% lower (P < 0.05) in IUGRs than controls. Collectively, our findings indicate that elevated catecholamine exposure in utero causes desensitization of adipose tissue by down-regulation of β2-AR, and this persists in lambs. This impairment in adrenergic stimulated lipolysis might partially explain early onset obesity in IUGR offspring. © 2010 The Authors. Journal compilation © 2010 The Physiological Society.
• Green, A. S., & Limesand, S. W. (2010). Remembering development - epigenetic responses to fetal malnutrition. Journal of Physiology, 588(9), 1379-1380.
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  PMID: 20436037;PMCID: PMC2876789;
• Green, A. S., Rozance, P. J., & Limesand, S. W. (2010). Consequences of a compromised intrauterine environment on islet function (Journal of Endocrinology (2010) 205 (211-224)). Journal of Endocrinology, 206(3), 335-.
• Green, A. S., Rozance, P. J., & Limesand, S. W. (2010). Consequences of a compromised intrauterine environment on islet function. JOURNAL OF ENDOCRINOLOGY, 205(3), 211-224.
• Green, A. S., Rozance, P. J., & Limesand, S. W. (2010). Consequences of a compromised intrauterine environment on islet function. Journal of Endocrinology, 205(3), 211-224.
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  PMID: 20223861;PMCID: PMC3526069;Abstract: Low birth weight is an important risk factor for impaired glucose tolerance and diabetes later in life. One hypothesis is that fetal β-cells inherit a persistent defect as a developmental response to fetal malnutrition, a primary cause of intrauterine growth restriction (IUGR). Our understanding of fetal programing events in the human endocrine pancreas is limited, but several animal models of IUGR extend our knowledge of developmental programing in β-cells. Pathological outcomes such as β-cell dysfunction, impaired glucose tolerance, and diabetes are often observed in adult offspring from these animal models, similar to the associations of low birth weight and metabolic diseases in humans. However, the identified mechanisms underlying β-cell dysfunction across models and species are varied, likely resulting from the different methodologies used to induce experimental IUGR, as well as from intraspecies differences in pancreas development. In this review, we first present the evidence for human β-cell dysfunction being associated with low birth weight or IUGR. We then evaluate relevant animal models of IUGR, focusing on the strengths of each, in order to define critical periods and types of nutrient deficiencies that can lead to impaired β-cell function. These findings frame our current knowledge of β-cell developmental programing and highlight future research directions to clarify the mechanisms of β-cell dysfunction for human IUGR. © 2010 Society for Endocrinology.
• Leos, R. A., Anderson, M. J., Chen, X., Pugmire, J., Anderson, K. A., & Limesand, S. W. (2010). Chronic exposure to elevated norepinephrine suppresses insulin secretion in fetal sheep with placental insufficiency and intrauterine growth restriction. AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM, 298(4), E770-E778.
• Leos, R. A., Anderson, M. J., Chen, X., Pugmire, J., Anderson, K. A., & Limesand, S. W. (2010). Chronic exposure to elevated norepinephrine suppresses insulin secretion in fetal sheep with placental insufficiency and intrauterine growth restriction. American Journal of Physiology - Endocrinology and Metabolism, 298(4), E770-E778.
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  PMID: 20086198;PMCID: PMC2853210;Abstract: In this study, we examined chronic norepinephrine suppression of insulin secretion in sheep fetuses with placental insufficiency-induced intrauterine growth restriction (IUGR). Glucose-stimulated insulin secretion (GSIS) was measured with a square-wave hyperglycemic clamp in the presence or absence of adrenergic receptor antagonists phentolamine (α) and propranolol (β). IUGR fetuses were hypoglycemic and hypoxemic and had lower GSIS responsiveness (P ≤ 0.05) than control fetuses. IUGR fetuses also had elevated plasma norepinephrine (3,264 ± 614 vs. 570 ± 86 pg/ml; P ≤ 0.05) and epinephrine (164 ± 32 vs. 60 ± 12 pg/ml; P ≤ 0.05) concentrations. In control fetuses, adrenergic inhibition increased baseline plasma insulin concentrations (1.7-fold, P ≤ 0.05), whereas during hyperglycemia insulin was not different. A greater (P ≤ 0.05) response to adrenergic inhibition was found in IUGR fetuses, and the average plasma insulin concentrations increased 4.9-fold at baseline and 7.1-fold with hyperglycemia. Unlike controls, basal plasma glucose concentrations fell (P ≤ 0.05) with adrenergic antagonists. GSIS responsiveness, measured by the change in insulin, was higher (8.9-fold, P ≤ 0.05) in IUGR fetuses with adrenergic inhibition than controls (1.8-fold, not significant), showing that norepinephrine suppresses insulin secretion in IUGR fetuses. Strikingly, in IUGR fetuses, adrenergic inhibition resulted in a greater GSIS responsiveness, because β-cell mass was 56% lower and the maximal stimulatory insulin response tended (P < 0.1) to be higher than controls. This persistent norepinephrine suppression appears to be partially explained by higher mRNA concentrations of adrenergic receptors α1D, α2A, and α2B in a cohort of fetuses that were naïve to the antagonists. Therefore, norepinephrine suppression of insulin secretion was maintained, in part, by upregulating adrenergic receptor expression, but the β-cells also appeared to compensate with enhanced GSIS. These findings may begin to explain why IUGR infants have a propensity for increased glucose requirements if norepinephrine is suddenly decreased after birth. Copyright © 2010 the American Physiological Society.
• Limesand, S., Chen, X., Fahy, A. L., Green, A. S., Anderson, M. J., Rhoads, R. P., & Limesand, S. W. (2010). β2-Adrenergic receptor desensitization in perirenal adipose tissue in fetuses and lambs with placental insufficiency-induced intrauterine growth restriction. The Journal of physiology, 588(Pt 18).
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  Placental insufficiency-induced intrauterine growth restriction (IUGR) fetuses have chronic hypoxaemia and elevated plasma catecholamine concentrations. In this study, we determined whether adrenergic responsiveness becomes desensitized in the perirenal adipose tissue of IUGR fetuses and lambs by measuring adrenergic receptor (AR) mRNA and protein levels. We also tested the ability of adrenaline to mobilize non-esterified fatty acids (NEFAs) in young lambs. Perirenal adipose tissue was collected from IUGR and control fetuses at 133 days of gestational age (dGA) and lambs at 18 days of age (dA). β(2)-AR mRNA concentrations were 59% and 74% lower (P < 0.05) in IUGR fetuses and lambs compared to controls, respectively, which also resulted in lower protein levels (P < 0.05). No treatment differences were detected for α(1A)-, α(1B)-, α(1D)-, α(2A)-, α(2B)-, α(2C)-, β(1)- and β(3)-AR expression. mRNA concentrations were also determined for hormone sensitive lipase (HSL), perilipin (lipid droplet-associated protein), and two adipokines, leptin and adiponectin. Adiponectin and HSL were not different between treatments at either age. Compared to controls, perilipin and leptin mRNA concentrations were lower (P < 0.05) in IUGR fetuses but not in lambs. Because of the β(2)-AR results, we challenged a second cohort of lambs with exogenous adrenaline at 21 dA. The ability of adrenaline to mobilize NEFA was 55 ± 15% lower (P < 0.05) in IUGRs than controls. Collectively, our findings indicate that elevated catecholamine exposure in utero causes desensitization of adipose tissue by down-regulation of β(2)-AR, and this persists in lambs. This impairment in adrenergic stimulated lipolysis might partially explain early onset obesity in IUGR offspring.
• Limesand, S., Green, A. S., & Limesand, S. W. (2010). Remembering development - epigenetic responses to fetal malnutrition. The Journal of physiology, 588(Pt 9).
• Limesand, S., Green, A. S., Rozance, P. J., & Limesand, S. W. (2010). Consequences of a compromised intrauterine environment on islet function. The Journal of endocrinology, 205(3).
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  Low birth weight is an important risk factor for impaired glucose tolerance and diabetes later in life. One hypothesis is that fetal beta-cells inherit a persistent defect as a developmental response to fetal malnutrition, a primary cause of intrauterine growth restriction (IUGR). Our understanding of fetal programing events in the human endocrine pancreas is limited, but several animal models of IUGR extend our knowledge of developmental programing in beta-cells. Pathological outcomes such as beta-cell dysfunction, impaired glucose tolerance, and diabetes are often observed in adult offspring from these animal models, similar to the associations of low birth weight and metabolic diseases in humans. However, the identified mechanisms underlying beta-cell dysfunction across models and species are varied, likely resulting from the different methodologies used to induce experimental IUGR, as well as from intraspecies differences in pancreas development. In this review, we first present the evidence for human beta-cell dysfunction being associated with low birth weight or IUGR. We then evaluate relevant animal models of IUGR, focusing on the strengths of each, in order to define critical periods and types of nutrient deficiencies that can lead to impaired beta-cell function. These findings frame our current knowledge of beta-cell developmental programing and highlight future research directions to clarify the mechanisms of beta-cell dysfunction for human IUGR.
• Limesand, S., Leos, R. A., Anderson, M. J., Chen, X., Pugmire, J., Anderson, K. A., & Limesand, S. W. (2010). Chronic exposure to elevated norepinephrine suppresses insulin secretion in fetal sheep with placental insufficiency and intrauterine growth restriction. American journal of physiology. Endocrinology and metabolism, 298(4).
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  In this study, we examined chronic norepinephrine suppression of insulin secretion in sheep fetuses with placental insufficiency-induced intrauterine growth restriction (IUGR). Glucose-stimulated insulin secretion (GSIS) was measured with a square-wave hyperglycemic clamp in the presence or absence of adrenergic receptor antagonists phentolamine (alpha) and propranolol (beta). IUGR fetuses were hypoglycemic and hypoxemic and had lower GSIS responsiveness (P < or = 0.05) than control fetuses. IUGR fetuses also had elevated plasma norepinephrine (3,264 +/- 614 vs. 570 +/- 86 pg/ml; P < or = 0.05) and epinephrine (164 +/- 32 vs. 60 +/- 12 pg/ml; P < or = 0.05) concentrations. In control fetuses, adrenergic inhibition increased baseline plasma insulin concentrations (1.7-fold, P < or = 0.05), whereas during hyperglycemia insulin was not different. A greater (P < or = 0.05) response to adrenergic inhibition was found in IUGR fetuses, and the average plasma insulin concentrations increased 4.9-fold at baseline and 7.1-fold with hyperglycemia. Unlike controls, basal plasma glucose concentrations fell (P < or = 0.05) with adrenergic antagonists. GSIS responsiveness, measured by the change in insulin, was higher (8.9-fold, P < or = 0.05) in IUGR fetuses with adrenergic inhibition than controls (1.8-fold, not significant), showing that norepinephrine suppresses insulin secretion in IUGR fetuses. Strikingly, in IUGR fetuses, adrenergic inhibition resulted in a greater GSIS responsiveness, because beta-cell mass was 56% lower and the maximal stimulatory insulin response tended (P < 0.1) to be higher than controls. This persistent norepinephrine suppression appears to be partially explained by higher mRNA concentrations of adrenergic receptors alpha(1D), alpha(2A), and alpha(2B) in a cohort of fetuses that were naïve to the antagonists. Therefore, norepinephrine suppression of insulin secretion was maintained, in part, by upregulating adrenergic receptor expression, but the beta-cells also appeared to compensate with enhanced GSIS. These findings may begin to explain why IUGR infants have a propensity for increased glucose requirements if norepinephrine is suddenly decreased after birth.
• Limesand, S., Mitchell, G. C., Fillinger, J. L., Sittadjody, S., Avila, J. L., Burd, R., & Limesand, S. W. (2010). IGF1 activates cell cycle arrest following irradiation by reducing binding of ΔNp63 to the p21 promoter. Cell death & disease, 1.
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  Radiotherapy for head and neck tumors often results in persistent loss of function in salivary glands. Patients suffering from impaired salivary function frequently terminate treatment prematurely because of reduced quality of life caused by malnutrition and other debilitating side-effects. It has been previously shown in mice expressing a constitutively active form of Akt (myr-Akt1), or in mice pretreated with IGF1, apoptosis is suppressed, which correlates with maintained salivary gland function measured by stimulated salivary flow. Induction of cell cycle arrest may be important for this protection by allowing cells time for DNA repair. We have observed increased accumulation of cells in G2/M at acute time-points after irradiation in parotid glands of mice receiving pretreatment with IGF1. As p21, a transcriptional target of the p53 family, is necessary for maintaining G2/M arrest, we analyzed the roles of p53 and p63 in modulating IGF1-stimulated p21 expression. Pretreatment with IGF1 reduces binding of ΔNp63 to the p21 promoter after irradiation, which coincides with increased p53 binding and sustained p21 transcription. Our data indicate a role for ΔNp63 in modulating p53-dependent gene expression and influencing whether a cell death or cell cycle arrest program is initiated.
• Cole, L., Anderson, M., Antin, P. B., & Limesand, S. W. (2009). One process for pancreatic beta-cell coalescence into islets involves an epithelial-mesenchymal transition. JOURNAL OF ENDOCRINOLOGY, 203(1), 19-31.
• Jeckel, K. M., Limesand, S. W., & Anthony, R. V. (2009). Specificity protein-1 and -3 trans-activate the ovine placental lactogen gene promoter. Molecular and Cellular Endocrinology, 307(1-2), 118-124.
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  PMID: 19389461;PMCID: PMC2700009;Abstract: The proximal promoter (-383/+16) of the ovine placental lactogen (oPL) gene provides trophoblast-specific expression in vitro. Footprint 6 (FP6; -319/-349) lies within this region, and transfection of two-base pair mutations across FP6 into BeWo cells identified potential binding sites for CCAAT-enhancer binding protein (CEBP) and specificity proteins (Sp). Transfection of CEBP dominant negative or over-expression constructs did not impact transactivation of the proximal promoter. However, Sp1 and Sp3 over-expression constructs increased (p ≤ 0.05) transactivation. Additionally, Sp1 and Sp3 short-hairpin RNA constructs reduced (p ≤ 0.01) transactivation of the proximal promoter. In EMSA supershift assays, Sp1 and Sp3 antibodies were able to inhibit migration of the complexes formed with nuclear extracts from BeWo cells and ovine chorionic binucleate cells (oBNC). Furthermore, Southwestern analysis of oBNC nuclear extracts identified a nuclear protein corresponding with Sp3, identified by Western analysis. In conclusion, these results indicate that Sp1 and Sp3 are capable of interacting with FP6 of the oPL gene proximal promoter and function to enhance its transactivation. © 2009 Elsevier Ireland Ltd. All rights reserved.
• Limesand, S. W., Rozance, P. J., Brown, L. D., & Hay Jr., W. W. (2009). Effects of chronic hypoglycemia and euglycemic correction on lysine metabolism in fetal sheep. American Journal of Physiology - Endocrinology and Metabolism, 296(4), E879-E887.
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  PMID: 19190258;PMCID: PMC2670627;Abstract: In this study, we determined rates of lysine metabolism in fetal sheep during chronic hypoglycemia and following euglycemic recovery and compared results with normal, age-matched euglycemic control fetuses to explain the adaptive response of protein metabolism to low glucose concentrations. Restriction of the maternal glucose supply to the fetus lowered the net rates of fetal (umbilical) glucose (42%) and lactate (36%) uptake, causing compensatory alterations in fetal lysine metabolism. The plasma lysine concentration was 1.9-fold greater in hypoglycemic compared with control fetuses, but the rate of fetal (umbilical) lysine uptake was not different. In the hypoglycemic fetuses, the lysine disposal rate also was higher than in control fetuses due to greater rates of lysine flux back into the placenta and into fetal tissue. The rate of CO2 excretion from lysine decarboxylation was 2.4-fold higher in hypoglycemic than control fetuses, indicating greater rates of lysine oxidative metabolism during chronic hypoglycemia. No differences were detected for rates of fetal protein accretion or synthesis between hypoglycemic and control groups, although there was a significant increase in the rate of protein breakdown (P < 0.05) in the hypoglycemic fetuses, indicating small changes in each rate. This was supported by elevated muscle specific ubiquitin ligases and greater concentrations of 4E-BP1. Euglycemic recovery after chronic hypoglycemia normalized all fluxes and actually lowered the rate of lysine decarboxylation compared with control fetuses (P < 0.05). These results indicate that chronic hypoglycemia increases net protein breakdown and lysine oxidative metabolism, both of which contribute to slower rates of fetal growth over time. Furthermore, euglycemic correction for 5 days returns lysine fluxes to normal and causes an overcorrection of lysine oxidation. Copyright © 2009 the American Physiological Society.
• Limesand, S., Limesand, S. W., Cole, L., Anderson, M., & Antin, P. B. (2009). One process for pancreatic beta-cell coalescence into islets involves an epithelial-mesenchymal transition. The Journal of Endocrinology, 203(1).
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  Islet replacement is a promising therapy for treating diabetes mellitus, but the supply of donor tissue for transplantation is limited. To overcome this limitation, endocrine tissue can be expanded, but this requires an understanding of normal developmental processes that regulate islet formation. In this study, we compare pancreas development in sheep and human, and provide evidence that an epithelial-mesenchymal transition (EMT) is involved in beta-cell differentiation and islet formation. Transcription factors know to regulate pancreas formation, pancreatic duodenal homeobox factor 1, neurogenin 3, NKX2-2, and NKX6-1, which were expressed in the appropriate spatial and temporal pattern to coordinate pancreatic bud outgrowth and direct endocrine cell specification in sheep. Immunofluorescence staining of the developing pancreas was used to co-localize insulin and epithelial proteins (cytokeratin, E-cadherin, and beta-catenin) or insulin and a mesenchymal protein (vimentin). In sheep, individual beta-cells become insulin-positive in the progenitor epithelium, then lose epithelial characteristics, and migrate out of the epithelial layer to form islets. As beta-cells exit the epithelial progenitor cell layer, they acquire mesenchymal characteristics, shown by their acquisition of vimentin. In situ hybridization expression analysis of the SNAIL family members of transcriptional repressors (SNAIL1, -2, and -3; listed as SNAI1, -2, -3 in the HUGO Database) showed that each of the SNAIL genes was expressed in the ductal epithelium during development, and SNAIL-1 and -2 were co-expressed with insulin. Our findings provide strong evidence that the movement of beta-cells from the pancreatic ductal epithelium involves an EMT.
• Rozance, P. J., Limesand, S. W., Barry, J. S., Brown, L. D., & Hay Jr., W. W. (2009). Glucose replacement to euglycemia causes hypoxia, acidosis, and decreased insulin secretion in fetal sheep with intrauterine growth restriction. Pediatric Research, 65(1), 72-78.
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  PMID: 18704001;PMCID: PMC2678008;Abstract: Nutritional interventions for intrauterine growth restriction (IUGR) have raised concerns for fetal toxicity, the mechanisms of which are unknown. Most of these attempts did not aim to normalize fetal metabolic conditions. Therefore, we used a model of IUGR to determine whether normalization of fetal hypoglycemia for 2 wks would be tolerated and increase insulin concentrations and pancreatic β-cell mass. IUGR fetuses received either a direct saline infusion (Sal, the control group) or a 30% dextrose infusion (Glu) to normalize glucose concentrations. Neither insulin concentrations (0.11 ± 0.01 Glu vs. 0.10 ± 0.01 ng/mL Sal) nor β-cell mass (65.2 ± 10.3 Glu vs. 74.7 ± 18.4 mg Sal) changed. Glucose stimulated insulin secretion (GSIS) was lower in the Glu group. Glu fetuses became progressively more hypoxic: O 2 content 1.4 ± 0.5 Glu vs. 2.7 ± 0.4 mM Sal, p < 0.05. Partial pressure of carbon dioxide (Paco2) (53.6 ± 0.8 Glu vs. 51.6 ± 0.8 Sal, p < 0.05) and lactate (7.74 ± 3.82 Glu vs. 2.47 ± 0.55 mM Sal, p < 0.0001) were greater and pH lower (7.275 ± 0.071 Glu vs. 7.354 ± 0.003 Sal, p < 0.01) in the Glu group. We conclude that correction of fetal hypoglycemia is not well tolerated and fails to increase insulin concentrations or ß-cell mass in IUGR fetuses. Copyright © 2008 International Pediatric Research Foundation, Inc.
• Hiscox, A. M., Stone, A. L., Limesand, S., Hoying, J. B., & Williams, S. K. (2008). An islet-stabilizing implant constructed using a preformed vasculature. TISSUE ENGINEERING PART A, 14(3), 433-440.
• Rozance, P. J., Limesand, S. W., Barry, J. S., Brown, L. D., Thorn, S. R., LoTurco, D., R., T., Friedman, J. E., & Hay Jr., W. W. (2008). Chronic late-gestation hypoglycemia upregulates hepatic PEPCK associated with increased PGC1α mRNA and phosphorylated CREB in fetal sheep. American Journal of Physiology - Endocrinology and Metabolism, 294(2), E365-E370.
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  PMID: 18056789;PMCID: PMC3857025;Abstract: Hepatic glucose production is normally activated at birth but has been observed in response to experimental hypoglycemia in fetal sheep. The cellular basis for this process remains unknown. We determined the impact of 2 wk of fetal hypoglycemia during late gestation on enzymes responsible for hepatic gluconeogenesis, focusing on the insulin-signaling pathway, transcription factors, and coactivators that regulate gluconeogenesis. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNA increased 12-fold and 7-fold, respectively, following chronic hypoglycemia with no change in hepatic glycogen. Chronic hypoglycemia decreased fetal plasma insulin with no change in glucagon but increased plasma cortisol 3.5-fold. Peroxisome proliferator-activated receptor-γ coactivator-1α mRNA and phosphorylation of cAMP response element binding protein at Ser133 were both increased, with no change in Akt, forkhead transcription factor FoxO1, hepatocyte nuclear factor-4α, or CCAAT enhancer binding protein-β. These results demonstrate that chronic fetal hypoglycemia triggers signals that can activate gluconeogenesis in the fetal liver. Copyright © 2008 the American Physiological Society.
• Limesand, S. W., Rozance, P. J., Smith, D., & Hay Jr., W. W. (2007). Increased insulin sensitivity and maintenance of glucose utilization rates in fetal sheep with placental insufficiency and intrauterine growth restriction. AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM, 293(6), E1716-E1725.
• Rozance, P. J., Limesand, S. W., Zerbe, G. O., & Hay Jr., W. W. (2007). Chronic fetal hypoglycemia inhibits the later steps of stimulus-secretion coupling in pancreatic β-cells. American Journal of Physiology - Endocrinology and Metabolism, 292(5), E1256-E1264.
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  PMID: 17213478;Abstract: We measured the impact of chronic late gestation hypoglycemia on pancreatic islet structure and function to determine the cause of decreased insulin secretion in this sheep model of fetal nutrient deprivation. Late gestation hypoglycemia did not decrease pancreas weight, insulin content, β-cell area, β-cell mass, or islet size. The pancreatic islet isolation procedure selected a group of islets that were larger and had an increased proportion of β-cells compared with islets measured in pancreatic sections, but there were no morphologic differences between islets isolated from control and hypoglycemic fetuses. The rates of glucose-stimulated pancreatic islet glucose utilization (126.2 ± 25.3 pmol glucose·islet -1·h-1, hypoglycemic, vs. 93.5 ± 5.5 pmol glucose·islet-1·h-1, control, P = 0.47) and oxidation (10.5 ± 1.7 pmol glucose·islet -1·h-1, hypoglycemic, vs. 10.6 ± 1.6 pmol glucose·islet-1·h-1, control) were not different in hypoglycemic fetuses compared with control fetuses. Chronic late gestation hypoglycemia decreased insulin secretion in isolated pancreatic islets by almost 70% in response to direct nonnutrient membrane depolarization and in response to increased extracellular calcium entry. β-Cell ultrastructure was abnormal with markedly distended rough endoplasmic reticulum in three of the seven hypoglycemic fetuses studied, but in vitro analysis of hypoglycemic control islets showed no evidence that these changes represented endoplasmic reticulum stress, as measured by transcription of glucose regulatory protein-78 and processing of X-box binding protein-1. In conclusion, these studies show that chronic hypoglycemia in late gestation decreases insulin secretion by inhibiting the later steps of stimulus-secretion coupling after glucose metabolism, membrane depolarization, and calcium entry. Copyright © 2007 the American Physiological Society.
• Barry, J. S., Davidsen, M. L., Limesand, S. W., Galan, H. L., Friedman, J. E., R., T., & Hay Jr., W. W. (2006). Developmental changes in ovine myocardial glucose transporters and insulin signaling following hyperthermia-induced intrauterine fetal growth restriction. Experimental Biology and Medicine, 231(5), 566-575.
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  PMID: 16636305;Abstract: Developmental changes in ovine myocardial glucose transporters and insulin signaling following hyperthermia-induced intrauterine fetal growth restriction (IUGR) were the focus of our study. Our objective was to test the hypothesis that the fetal ovine myocardium adapts during an IUGR gestation by increasing glucose transporter protein expression, plasma membranebound glucose transporter protein concentrations, and insulin signal transduction protein concentrations. Growth measurements and whole heart tissue were obtained at 55 days gestational age (dGA), 90 dGA, and 135 dGA (term = 145 dGA) in fetuses from control (C) and hyperthermic (HT) pregnant sheep. Additionally, in 135 dGA animals, arterial blood was obtained and Doppler ultrasound was used to determine umbilical artery systolic (S) and diastolic (D) flow velocity waveform profiles to calculate pulsatility (S - D/mean) and resistance (S - D/S) indices. Myocardial Glut-1, Glut-4, insulin signal transduction proteins involved in Glut-4 translocation, and glycogen content were measured. Compared to age-matched controls, HT 90-dGA fetal body weights and HT 135-dGA fetal weights and gross heart weights were lower. Heart weights as a percent of body weights were similar between C and HT sheep at 135 dGA. HT 135-dGA animals had (i) lower fetal arterial plasma glucose and insulin concentrations, (ii) lower arterial blood oxygen content and higher plasma lactate concentrations, (iii) higher myocardial Glut-4 plasma membrane (PM) protein and insulin receptor β protein (IRβ) concentrations, (iv) higher myocardial glycogen content, and (v) higher umbilical artery Doppler pulsatility and resistance indices. The HT ovine fetal myocardium adapts to reduced circulating glucose and insulin concentrations by increasing plasma membrane Glut-4 and IRβ protein concentrations. The increased myocardial Glut-4 PM and IRβ protein concentrations likely contribute to or increase the intracellular delivery of glucose and, together with the increased lactate concentrations, enhance glycogen synthesis, which allows for maintained myocardial growth commensurate with fetal body growth. Copyright © 2006 by the Society for Experimental Biology and Medicine.
• Limesand, S. W., Rozance, P. J., Zerbe, G. O., Hutton, J. C., & Hay Jr., W. W. (2006). Attenuated insulin release and storage in fetal sheep pancreatic islets with intrauterine growth restriction. Endocrinology, 147(3), 1488-1497.
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  PMID: 16339204;Abstract: We determined in vivo and in vitro pancreatic islet insulin secretion and glucose metabolism in fetuses with intrauterine growth restriction (IUGR) caused by chronic placental insufficiency to identify functional deficits in the fetal pancreas that might be caused by nutrient restriction. Plasma insulin concentrations in theIUGRfetuses were 69% lower at baseline and 76% lower after glucose-stimulated insulin secretion (GSIS). Similar deficits were observed with arginine-stimulated insulin secretion. Fetal islets, immunopositive for insulin and glucagon, secreted insulin in response to increasing glucose and KCl concentrations. Insulin release as a fraction of total insulin content was greater in glucose-stimulated IUGR islets, but the mass of insulin released per IUGR islet was lower because of their 82% lower insulin content. A deficiency in islet glucose metabolism was found in the rate of islet glucose oxidation at maximal stimulatory glucose concentrations (11 mmol/liter). Thus, pancreatic islets from nutritionally deprived IUGR fetuses caused by chronic placental insufficiency have impaired insulin secretion caused by reduced glucose-stimulated glucose oxidation rates, insulin biosynthesis, and insulin content. This impaired GSIS occurs despite an increased fractional rate of insulin release that results from a greater proportion of releasable insulin as a result of lower insulin stores. Because this animal model recapitulates the human pathology of chronic placental insufficiency and IUGR, the β-cell GSIS dysfunction in this model might indicate mechanisms that are developmentally adaptive for fetal survival but in later life might predispose offspring to adult-onset diabetes that has been previously associated with IUGR. Copyright © 2006 by The Endocrine Society.
• Rozance, P. J., Limesand, S. W., & Hay Jr., W. W. (2006). Decreased nutrient-stimulated insulin secretion in chronically hypoglycemic late-gestation fetal sheep is due to an intrinsic islet defect. American Journal of Physiology - Endocrinology and Metabolism, 291(2), E404-E411.
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  PMID: 16569758;Abstract: We measured in vivo and in vitro nutrient-stimulated insulin secretion in late gestation fetal sheep to determine whether an intrinsic islet defect is responsible for decreased glucose-stimulated insulin secretion (GSIS) in response to chronic hypoglycemia. Control fetuses responded to both leucine and lysine infusions with increased arterial plasma insulin concentrations (average increase: 0.13 ± 0.05 ng/ml leucine; 0.99 ± 0.26 ng/ml lysine). In vivo lysine-stimulated insulin secretion was decreased by chronic (0.37 ± 0.18 ng/ml) and acute (0.27 ± 0.19 ng/ml) hypoglycemia. Leucine did not stimulate insulin secretion following acute hypoglycemia but was preserved with chronic hypoglycemia (0.12 ± 0.09 ng/ml). Isolated pancreatic islets from chronically hypoglycemic fetuses had normal insulin and DNA content but decreased fractional insulin release when stimulated with glucose, leucine, arginine, or lysine. Isolated islets from control fetuses responded to all nutrients. Therefore, chronic late gestation hypoglycemia causes defective in vitro nutrient-regulated insulin secretion that is at least partly responsible for diminished in vivo GSIS. Chronic hypoglycemia is a feature of human intrauterine growth restriction (IUGR) and might lead to an islet defect that is responsible for the decreased insulin secretion patterns seen in human IUGR fetuses and low-birth-weight human infants. Copyright © 2006 the American Physiological Society.
• Limesand, S. W., Jensen, J., Hutton, J. C., & Hay Jr., W. W. (2005). Diminished β-cell replication contributes to reduced β-cell mass in fetal sheep with intrauterine growth restriction. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 288(5 57-5), R1297-R1305.
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  PMID: 15650129;Abstract: Human fetuses with severe intrauterine growth restriction (IUGR) have less pancreatic endocrine tissue and exhibit β-cell dysfunction, which may limit β-cell function in later life and contribute to their increased incidence of noninsulin-dependent diabetes mellitus. Three factors, replication, apoptosis, and neoformation, contribute to fetal β-cell mass. We studied an ovine model of IUGR to understand whether nutrient deficits lead to decreased rates of fetal pancreatic β-cell replication, increased rates of apoptosis, or lower rates of differentiation. At 90% of term gestation, IUGR fetal and pancreatic weights were 58% and 59% less than pair-fed control, respectively. We identified a selective impairment of β-cell mass compared with other pancreatic cell types in IUGR fetuses. Insulin and insulin mRNA contents were less than other pancreatic endocrine hormones in IUGR fetuses, as were pancreatic insulin positive area (42%) and β-cell mass (76%). Pancreatic β-cell apoptosis was not different between treatments. β-cell capacity for cell cycling, determined by proliferating cell nuclear antigen (PCNA) immunostaining, was not different between treatment groups. However, the percentage of β-cells actually undergoing mitosis was 72% lower in IUGR fetuses. These results indicate that in utero nutrient deficits decrease the population of pancreatic β-cells by lengthening G1, S, and G2 stages of interphase and decreasing mitosis near term. Diminished β-cell mass in IUGR infants at birth, if not adequately compensated for after birth, may contribute to insufficient insulin production in later life and, thus, a predisposition to noninsulin-dependent diabetes. Copyright © 2005 the American Physiological Society.
• Wallace, J. M., Regnault, T. R., Limesand, S. W., Hay Jr., W. W., & Anthony, R. V. (2005). Investigating the causes of low birth weight in contrasting ovine paradigms. Journal of Physiology, 565(1), 19-26.
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  PMID: 15774527;PMCID: PMC1464509;Abstract: Intrauterine growth restriction (IUGR) still accounts for a large incidence of infant mortality and morbity worldwide. Many of the circulatory and transport properties of the sheep placenta are similar to those of the human placenta and as such, the pregnant sheep offers an excellent model in which to study the development of IUGR. Two natural models of ovine IUGR are those of hyperthermic exposure during pregnancy, and adolescent overfeeding, also during pregnancy. Both models yield significantly reduced placental weights and an asymmetrically growth-restricted fetus, and display altered maternal hormone concentrations, indicative of an impaired trophoblast capacity. Additionally, impaired placental angiogenesis and uteroplacental blood flow appears to be an early defect in both the hyperthermic and adolescent paradigms. The effects of these alterations in placental functional development appear to be irreversible. IUGR fetuses are both hypoxic and hypoglycaemic, and have reduced insulin and insulin-like growth factor-1 (IGF-1), and elevated concentrations of lactate. However, fetal utilization of oxygen and glucose, on a weight basis, remain constant compared with control pregnancies. Maintained utilization of these substrates, in a substrate-deficient environment, suggests increased sensitivities to metabolic signals, which may play a role in the development of metabolic diseases in later adult life. © The Physiological Society 2005.
• Wooding, F. B., Fowden, A. L., Bell, A. W., Ehrhardt, R. A., Limesand, S. W., & Hay, W. W. (2005). Localisation of glucose transport in the ruminant placenta: Implications for sequential use of transporter isoforms. Placenta, 26(8-9), 626-640.
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  PMID: 16085042;Abstract: The facilitative glucose transporters 1 and 3 are the major routes for glucose transport across placental membranes. Using light and electron microscope immunocytochemistry on acrylic sections this study shows a similar pattern of expression from mid to late pregnancy in all four ruminants examined [cow, deer, ewe and goat]. GT1 and GT3 are localised on different membrane layers of the synepitheliochorial placental barrier and glucose must utilise both isoforms sequentially to pass from the maternal to fetal circulations. It is suggested that this arrangement is designed to support the high glucose utilisation by the multilayered placenta in the ruminant. © 2004 Elsevier Ltd. All rights reserved.
• Limesand, S. W., Jeckel, K. M., & Anthony, R. V. (2004). Purα, a Single-Stranded Deoxyribonucleic Acid Binding Protein, Augments Placental Lactogen Gene Transcription. Molecular Endocrinology, 18(2), 447-457.
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  PMID: 14645500;Abstract: Placental lactogen (PL) is thought to alter maternal metabolism to increase the pool of nutrients available for the fetus and to stimulate fetal nutrient uptake. The ovine (o) PL gene is expressed in chorionic binucleate cells (oBNC) and cis-elements located within the proximal promoter (-124 to +16 bp) are capable of trophoblast-specific expression in human (BeWo) and rat (Rcho-1) choriocarcinoma cells. Protein-DNA interactions were identified with oBNC nuclear extracts, and mutational analysis of these regions revealed a previously undefined cis-element from -102/-123 bp that enhances promoter activity in BeWo cells but not Rcho-1 cells. Characterization of this region identified the nucleotide sequence CCAGCA (-105/ -110; o110) as the responsible cis-acting element. Southwestern analysis with this element identified a binding protein with an apparent Mr of approximately 41,000. Expression screening of an ovine placental cDNA library identified six homologous cDNAs, which shared identity with human (97%) and mouse (95%) Purα, a single-stranded DNA binding protein. The Purα-o110 interaction was confirmed by electrophoretic mobility-supershift assays with oBNC and BeWo extracts but was absent with Rcho-1 extracts. Furthermore, overexpression of ovine Purα enhanced transactivation of the oPL gene proximal promoter in both choriocarcinoma cell lines through this novel cis-element. This study identified a previously undefined cis-element, which interacts with Purα to augment PL gene transcription.
• Limesand, S. W., Regnault, T. R., & Hay Jr., W. W. (2004). Characterization of glucose transporter 8 (GLUT8) in the ovine placenta of normal and growth restricted fetuses. Placenta, 25(1), 70-77.
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  PMID: 15013641;Abstract: Facilitated glucose transporters (GLUTs) in the chorionic epithelium are primary conduits for glucose delivery to placental and fetal tissues. The objective of this study was to characterize GLUT8 in the ovine placenta and determine if differences in mRNA and protein concentrations occur in an ovine model of intrauterine growth restriction (IUGR). A GLUT8 partial mRNA was generated, which shares 95 per cent identity with bovine GLUT8 nucleotide sequence. Northern hybridization identified a 2.1 kilobase transcript. GLUT8 mRNA concentrations normalized to β-actin mRNA concentrations increased during late gestation. Western immunoblots with an affinity-purified anti-mouse GLUT8 antiscrum detected GLUT8 in late gestation ovine placenta plasma membranes. GLUT8 was immunolocalized to the chorionic epithelial layer and uterine epithelial cells from mid to late gestation. GLUT8 mRNA and protein concentrations at 135 days gestational age were decreased by 34.8 per cent and 21.8 per cent, respectively (P
• Limesand, S. W., & Hay Jr., W. W. (2003). Adaptation of ovine fetal pancreatic insulin secretion to chronic hypoglycaemia and euglycaemic correction. Journal of Physiology, 547(1), 95-105.
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  PMID: 12562941;PMCID: PMC2342612;Abstract: Fetal pancreatic adaptations to relative hypoglycaemia, a characteristic of intra-uterine growth restriction, may limit pancreatic β-cell capacity to produce and/or secrete insulin. The objective of this study was to measure β-cell responsiveness in hypoglycaemic (H) fetal sheep and ascertain whether a 5 day euglycaemic recovery period would restore insulin secretion capacity. Glucose-stimulated insulin secretion (GSIS) was measured in euglycaemic (E) control fetuses, fetuses made hypoglycaemic for 14 days, and in a subset of 14-day hypoglycaemic fetuses returned to euglycaemia for 5 days (R fetuses). Hypoglycaemia significantly decreased plasma insulin concentrations in H (0.13 ± 0.01 ng ml-1) and R fetuses (0.11 ± 0.01 ng ml-1); insulin concentrations returned to euglycaemic control values (0.30 ± 0.01 ng ml-1) in R fetuses (0.29 ± 0.04 ng ml-1) during their euglycaemic recovery period. Mean steady-state plasma insulin concentration during the GSIS study was reduced in H fetuses (0.40 ± 0.07 vs. 0.92 ± 0.10 ng ml-1 in E), but increased (P < 0.05) in R fetuses (0.73 ± 0.10 ng ml-1) to concentrations not different from those in the E group. Nonlinear modelling of GSIS showed that response time was greater (P < 0.01) in both H (15.6 ± 2.8 min) and R (15.4 ± 1.5 min) than in E fetuses (6.3 ± 1.1 min). In addition, insulin secretion responsiveness to arginine was reduced by hypoglycaemia (0.98 ± 0.11 ng ml-1 in H vs. 1.82 ± 0.17 ng ml-1 in E, P < 0.05) and did not recover (1.21 ± 0.15 ng ml-1 in R, P < 0.05 vs. E). Thus, a 5 day euglycaemic recovery period from chronic hypoglycaemia reestablished GSIS to normal levels, but there was a persistent reduction of β-cell responsiveness to glucose and arginine. We conclude that programming of pancreatic insulin secretion responsiveness can occur in response to fetal glucose deprivation, indicating a possible mechanism for establishing, in fetal life, a predisposition to type 2 diabetes.
• Anthony, R. V., Limesand, S. W., & Jeckel, K. M. (2001). Transcriptional regulation in the placenta during normal and compromised fetal growth. Biochemical Society Transactions, 29(2), 42-48.
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  PMID: 11356124;Abstract: The placenta synthesizes a number of cytokines and growth factors that are involved in the establishment, maintenance or regulation of pregnancy. Included are interferons, placental lactogens, other members of the growth hormone/prolactin gene family, leptin, and an array of angiogenic growth factors. While their roles in pregnancy differ, in their absence pregnancy is either lost or compromised. Therefore an understanding of the cell-specific transcriptional regulation of these genes is imperative if we are ever to alter their expression to benefit pregnancy progression. Our understanding of transcriptional regulation in the placenta is still in its infancy, and there appears to be considerable divergence in the transcriptional regulation of these genes between species, as well as between the various cytokine genes being examined. For example, while there are some commonalities in the regulation of human, rodent and ruminant placental lactogens, there are differences that require the study of placental lactogen gene regulation across species. However, one common theme that is emerging with the angiogenic growth factors, such as vascular endothelial growth factor and the angiopoietins, is the transcriptional control of these genes by oxygen tension within the placenta. Examination of transcriptional regulation in normal and compromised pregnancies will provide additional insight in this area.
• Limesand, S. W., & Anthony, R. V. (2001). Novel activator protein-2α splice-variants function as transactivators of the ovine placental lactogen gene. European Journal of Biochemistry, 268(8), 2390-2401.
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  PMID: 11298758;Abstract: Activator protein-2 (AP-2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expressed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP-2α was localized in ovine chorionic epithelial cells by immunohistochemistry and a 3-kb transcript was identified by Northern hybridization. Four nearly full-length AP-2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP-2 mRNA expressed in the ovine placenta shares identity with human AP-2α, but variations in the predicted N-terminus were observed, and three unique AP-2α splice-variants were identified. Expression of AP-2α variants in HepG2 cells, devoid of endogenous AP-2, indicates that enhancement through the AP-2 element in the oPL gene minimal promoter was variant dependent. RNA transcripts for all of the ovine AP-2α splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2α transcript, which shares identity to Xenopus AP-2α, was expressed in BeWo cells. Immunoblot analysis confirmed AP-2α variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP-2α variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2α splice-variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells.
• Liang, R., Limesand, S. W., & Anthony, R. V. (1999). Structure and transcriptional regulation of the ovine placental lactogen gene. European Journal of Biochemistry, 265(3), 883-895.
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  PMID: 10518781;Abstract: Ovine placental lactogen (oPL), a member of the growth hormone/prolactin gene family, is produced by chorionic binucleate cells at the maternal-fetal interface, and is thought to modulate metabolic processes and enhance fetal growth. We have determined that the oPL gene contains five exons and four introns, and the transcriptional start site was mapped 91 bp 5' of the initiation codon (AUG). An additional 4.5 kb of 5'-flanking sequence was sequenced and used for transient transfection analysis in human (BeWo) and rat (Rcho-1) choriocarcinoma cell lines to examine trophoblast cell-specific activity. Trophoblast cell-specific transactivation of the reporter gene was conferred by the proximal 1.1 kb of oPL gene 5'-flanking sequence. Transfection of deletion constructs derived from the 1.1 kb of 5'-flanking sequence resulted in varying profiles of transactivation between the two choriocarcinoma cell lines, but maximal activation in both cell lines resided within the proximal 383 bp of oPL gene 5'-flanking sequence. DNase I protection analysis using ovine chorionic binucleate cell nuclear protein, identified 19 footprints within the 1.1-kb sequence, six of which are located within the 383-bp region. Electrophoretic mobility-shift assays and mutational analysis identified two functional GATA (-67, -102) sequences as transactivators of the oPL gene. However, a previously undefined element (GAGGAG) residing at -338 and -283 is required for full transactivation, and mutation of either significantly reduces reporter activity. In addition, an AP-2 site (-58) and an E-box (-163) were identified and may coordinate oPL transactivation. Transcriptional regulation of human and rodent PL genes has been previously characterized, and our results indicate that tissue-specific regulation of oPL expression may result from cis-acting elements in common with human and rat genes expressed within the placenta. However, our data indicate that regulation of oPL also results from novel cis-acting elements.
• Cerdá, J., Calman, B. G., LaFleur Jr., G. J., & Limesand, S. (1996). Pattern of vitellogenesis and follicle maturational competence during the ovarian follicular cycle of Fundulus heteroclitus. General and Comparative Endocrinology, 103(1), 24-35.
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  PMID: 8812325;Abstract: The patterns of vitellogenesis and follicle maturational competence were examined across the semilunar spawning cycle of Fundulus heteroclitus. Daily egg collection showed spawning cyclicity in six experimental groups, with a mean period between spawnings of 14.9 ± 0.3 days, indicated by the nonlinear regression sine-curve matching analysis. Each cycle was then dated from Day - 7 to Day 7, with Day 0 as the peak egg-collection day. Females from each group were sampled on selected days during two to three consecutive spawning cycles, and these days were each chronologically given a temporal relation to Day 0 to pool the data into a composite. The analysis of the size-frequency distribution of ovarian follicles ≤0.5-mm diameter across the composite revealed a constant recruitment of small follicles (0.5- to 0.7-mm diameter) into vitellogenesis, which was supported by the continuous presence of vitellogenin (Vtg) 1 mRNA in the liver of the females. The plasma levels of Vtg were also essentially constant across the cycle, except for a progressive decrease from Day -7 through Day 3 that could be related to a more active Vtg uptake by a dominant population of follicles up to 1.7 mm in diameter. A second and more selective recruitment of full-grown follicles (1.3- to 1.4- mm diameter) toward maturation was noted at Days -5, -4, which appeared associated with high plasma levels of estradiol-17β. However, the responsiveness of those follicles undergoing oocyte maturation in vitro after gonadotropin and maturation-inducing steroid (MIS), 17,20β-dihydroxy-4- pregnen-3-one, stimulation dramatically declined from Days -1, 0, 1 to Days 4, 5, concomitantly with an increase of the population of the largest follicles (1.8- to 2.1-mm diameter) in the ovary. These findings extend previous observations on the process of follicular recruitment in F. heteroclitus and indicate that full-grown follicles may be recruited into maturation by a mechanism that modulates the oocyte sensitivity to the MIS.
• Hsiao, S., Limesand, S. W., & Wallace, R. A. (1996). Semilunar follicular cycle of an intertidal fish: The Fundulus model. Biology of Reproduction, 54(4), 809-818.
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  PMID: 8924500;Abstract: In their habitat, Fundulus heteroclitus (Cyprinodontidae) spawn against a variety of specific substrates that become available only at spring tides; they thus exhibit a semilunar reproductive cyclicity. In the laboratory at 28 ± 0.6°C and 14L:10D, this cyclicity is free running and can be observed by means of daily egg collection and frequent ovarian sampling. Daily egg collection cycles gathered from three different spawning groups were pooled chronologically; the cycles were determined by nonlinear regression sine- curve matching and so were dated from Days -6 to +6, with Day 0 as the peak egg collection day. Ovaries were sampled on Days -6, -4, -2, -1, +1, +2, and +4. The pooled data indicate that early-maturing follicles (1.3- to 1.6-mm diameter) are recruited from a constant reserve of vitellogenic follicles (0.7- to 1.2-mm diameter) and become abundant early in the cycle but are depleted during Days -6 to +4 by the formation of late-maturing follicles and ovulated eggs. A midcycle peak of ovulated eggs in the ovary corresponds to the egg collection peak. This progression of follicles followed by spawning decreases the total count of the follicles (≥ 0.7-mm diameter) and eggs in the ovary by 52% (from 454 to 219 per 10 g of female weight). The semilunar follicular cycle in this Fundulus model thus includes 1) an early-cycle follicle recruitment, 2) an early- and midcycle follicle maturation, and 3) a midcycle egg ovulation and spawning. This follicular cycle also proceeds in fish deprived of a spawning substrate. A female in the wild would thus execute the follicular cycling in a timely sequence, in anticipation of each recurring spring tide, whether or not a natural substrate becomes available. We conclude that F. heteroclitus provides a useful nonmammalian model for the study of cyclic reproductive activity in the laboratory.

Presentations

• Tabsh, K. K., Pendleton, A. L., Barker, N. K., Langlais, P. R., Limesand, S. W., & Hill, M. G. (2020, March/Spring). Confirmation of Inflammatory Biomarkers from Maternal Plasma in Women with Intrahepatic Cholestasis of Pregnancy. Society for Reproductive Investigation 67th Annual Scientific Meeting.
• Antolic, A. T., Pendleton, A. L., Kelly, A. C., Anderson, M. J., Davis, M. A., Bidwell, C. A., Camacho, L. E., & Limesand, S. W. (2019, March). Greater STAT3 Phosphorylation and Enhanced Mitochondrial Respiration in the Left Ventricle of Growth Restricted Ovine Fetuses. Society for Reproductive Investigation 66th Annual Scientific Meeting. Paris, France: Society for Reproductive Investigation.
• Limesand, S. W. (2019, August). Development of insulin resistance and tissue-specific differences. Aspen-Snowmass Perinatal Biology Symposium 2019. Snowmass Village CO: American Society Of Animal Science.
• Limesand, S. W. (2019, March). Oxygen and Glucose Correction Improves Insulin Secretion in Sheep Fetuses with Placental Insufficiency. Mini Symposium: Developing Interventions That Address IUGR. Paris France: Society for Reproductive Investigation.
• Limesand, S. W. (2019, September). Perinatal Development of Glucose Intolerance through Tissue-Specific Programming. Seminar Series for the Department of Basic Medical Sciences, University of Arizona College of Medicine – Phoenix. Phoenix Arizona: Department of Basic Medical Sciences, University of Arizona College of Medicine – Phoenix.
• Luna Ramirez, R. I., Kelly, A. C., Bidwell, C. A., Davis, M. A., Camacho, L. E., Anderson, M. J., & Limesand, S. W. (2019, March). Norepinephrine Spares Adipose Tissue in Ovine Fetuses Complicated with Placental Insufficiency.. Society for Reproductive Investigation 66th Annual Scientific Meeting. Paris, France: Society for Reproductive Investigation.
• Pendleton, A. L., Davis, M. A., Kelly, A. C., Camacho, L. E., Anderson, M. J., & Limesand, S. W. (2019, March). Decreased Complex 1 Expression and Impaired Mitochondrial Function in Growth Restricted Ovine Fetuses with Placental Insufficiency. Society for Reproductive Investigation 66th Annual Scientific Meeting. Paris, France: Society for Reproductive Investigation.
• Camacho, L. E., Davis, M. A., Steffens, N. R., Kelly, A. C., & Limesand, S. W. (2018, March). Oxygen and Glucose Correction Improves Insulin Secretion in Sheep Fetuses with Placental Insufficiency. Society for Reproductive Investigation, 65th Annual Meeting. San Diego, CA: Reproductive Sciences 25(1):S-082.
• Kelly, A. C., Davis, M. A., Camacho, L. E., Davenport, H., Steffens, N. R., & Limesand, S. W. (2018, March). Loss of Uncoupling Protein 2 Explains Hyper-Secretion Following Persistent Adrenergic Receptor Signaling in Fetal Sheep Islets. Reproductive Investigation 65th Annual Scientific Meeting. San Diego, CA: Reproductive Sciences 25(1):O-061.
• Limesand, S. W. (2018, March). Perinatal programming of glucose homeostasis in sheep fetuses with placental insufficiency. Northeast American Society of Animal Science 2018 Annual Meeting. University of Connecticut, Storrs CT: ASAS.
• Limesand, S. W. (2018, November). Catecholamines Regulate Fetal Adaptation during Placental Insufficiency. Departmental Seminar Series; Brigham Young University. Provo, UT: Department of Physiology and Developmental Biology, Brigham Young University.
• Camacho, L. E., & Limesand, S. W. (2017, Fall). Oxygen and glucose correction improves insulin secretion in sheep fetuses with placental insufficiency.. Perinatal Research Society Annual Meeting. Atlanta GA: Perinatal Research Society.
• Camacho, L. E., Davis, M. A., Steffens, N. R., Kelly, A. C., & Limesand, S. W. (2017, March). Oxygen and glucose supplementation reduces stress in growth restricted sheep fetuses.. Reproductive Sciences, 24(Supplement):73A, Abstract: O-059Society of Reproductive Investigation.
• Pendleton, A., Smith, R. M., Camaacho, L. E., & Limesand, S. W. (2017, Fall). Impaired satellite cell differentiation and regulation in lambs with intrauterine growth restriction. The Arizona Physiology Society Annual Meeting. Northern Arizona University, Flagstaff AZ: AZAPS.
• Limesand, S. W., Davis, M., Smith, K., Korbutt, G., & Papas, K. (2015, Nov). . Stimulation of porcine beta-cell proliferation is inhibited with hypoxic culture conditions.. International Pancreas and Islet Transplant Association,. Melbourne, Australia..
• Lynch, R., Steyn, L., Yosten, G., Ananthakrishnan, K., Limesand, S. W., & Papas, K. (2015, Mar). High Specificity Multivalent Contrast Agents Targeted to Beta Cells.. Symposium on Beta Cell Imaging, BetaTrain Consortium and DiabImage..
• Smith, K., Kelly, A., Weber, C., Min, C., Smith, B., McCarthy, F., Steyn, L., Badarinarayana, V., Strop, P., Lynch, R., Limesand, S. W., & Papas, K. (2015, Nov). Acute ischemic exposure leads to functional deficits and a transcriptome enriched in inflammatory and hypoxic pathways in human islets.. International Pancreas and Islet Transplant Association,. Melbourne, Australia..
• Smith, K., Kelly, A., Weber, C., Min, C., Smith, B., McCarthy, F., Steyn, L., Badarinarayana, V., Strop, P., Lynch, R., Limesand, S. W., & Papas, K. (2015, Nov). Impaired function, viability, and enhanced inflammatory and hypoxia-related signaling and the impact of temperature following acute ischemic exposure in human islets.. Transplantation Science Symposium..
• Smith, K., Steyn, L., Weber, C., Min, C., Purvis, W., Stanton, J., Burachek, A., Salama, B., Korbutt, G., Lynch, R., Limesand, S. W., & Papas, K. (2015, Nov). Direct comparison of adult, juvenile, and neonatal porcine islets in vitro reveals age dependent differences in viability and function.. International Pancreas and Islet Transplant Association. Melbourne, Australia..
• Steyn, L., Davis, M., Min, C., Smith, K., Purvis, W., Stanton, J., Loudovaris, T., Weegman, B., Mueller, K., Kitzman, J., Avgoustiniatos, E., Tempelman, L., Limesand, S. W., & Papas, K. (2015, Nov). In Situ Oxygen Delivery to Islets within an Implantable Immunoisolating Device In Vivo Enhances Viability and Function.. International Pancreas and Islet Transplant Association,. Melbourne, Australia..
• Steyn, L., Smith, K., Min, C., Purvis, W., Stanton, J., Davis, M., Mueller, K., Kitzmann, J., Weegman, B., Avgoustiniatos, E., Tempelman, L., Limesand, S. W., & Papas, K. (2015, Nov). Increasing Islet Density within an Immunoisolation Device has a Detrimental Impact on Viability In Vivo.. International Pancreas and Islet Transplant Association,. Melbourne, Australia..
• Limesand, S. W., Kelly, A. C., Bidwell, C. A., & McCarthy, F. M. (2014, June). Inflammatory state of pancreatic islets in intrauterine growth restricted fetuses. 34th Annual Meeting of American Society for Reproductive Immunology.
• Lynch, R., Ananthakrishnan, K., Weber, C., Hart, N., Limesand, S. W., & Lynch, R. (2014, Spring). Targeting glucagon like peptide-1 and α2 adrenergic receptor combination using GLP-1/Yohimbine to achieve β-cell specific targeting and therapy.. FASEB J.
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  1095.19
• Lynch, R., Weber, C., Hart, N., Limesand, S. W., Xu, L., Morse, D., Gillies, R., & Vagner, J. (2014, Spring). Multivalent cell specific therapeutics for treatment of metabolic disorders.. FASEB J.
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  71. Lynch, R.M. C. Weber, N. Hart, S.W. Limesand, L. Xu,, R. Gillies, J. Vagner (2014). 1108.1.
• Rozance, P., Anderson,, M. J., Martinez, M., Fahy, A., Macko, A. R., & Limesand, S. W. (2014, Summer). Intrauterine Growth Restriction Decreases Pancreatic Vascularity and Disrupts Hepatic Growth Factor Signaling between the Endothelial Cell and ß-Cell in Fetal Sheep.. Diabetes 74nd Scientific Session.
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  73. ,, , , and S.W. Limesand (2014). Abstract Book, 1668-P.
• Limesand, S. W. (2013, March). Exposure to heat stress during pregnancy impairs metabolism in the ruminant fetus. Midwest Animal Science Meeting. Des Moines IA: Midwest Animal Science.
• Limesand, S. W. (2013, March). Insulin secretion and sensitivity in intrauterine growth restricted sheep fetuses. Departmental Seminar. West Lafayette, Indiana: University of Purdue.
• Limesand, S. W. (2013, October). Adrenergic desensitization in IUGR fetuses. Bill and Melinda Gates Foundation, Healthy Growth Workshop. Rio de Janeiro, Brazil: Gates Foundation.
• Limesand, S. W., Penrod, L. V., Ananthakrishnan, K., Patek, R., Vagner, J., & Lynch, R. M. (2013, April). A heterobivalent ligan comprised of GLP-1 and Yohimbine specifically targets pancreatic beta-cells in vivo. NIDDK Workshop: Imaging the Pancreatic Beta-Cell. Bethesda, MD: NIDDK.
• Papas, K. K., Mueller, K. R., Penrod, L. V., Davis, M., Kitzmann, J. P., Gruessner, S. G., Suszynski, T., Jie, T., Tempelman, L., Limesand, S. W., & Avgoustiniatos, E. S. (2013, September). Macroencapsulated human islet viability is drastically reduced in vivo as the number of islets per device is increased.. 14th World Congress of the International Pancreas and Islet Transplantation Association. Monterey, CA, USA.
• Penrod, L. V., Ananthakrishnan, K., Patek, R., Vagner, J., Lynch, R. M., & Limesand, S. W. (2013, June). A heterovalent ligand containing GLP-1 and yohimbine specifically targets beta-cells in vivo. American Diabetes Association Annual Meeting. Chicago, IL: ADA.
• Yates, D. T., Macko, A. R., & Limesand, S. W. (2013, August). Intrinsic deficiencies in myoblast proliferation results in less differentiated myoblasts and smaller myofibers in IUGR fetal sheep. Aspen Perinatal Biology Symposium.
• Limesand, S. W. (2012). Early Life Origins of Insulin Resistance and Type 2 Diabetes. The Australian Early Origins of Hypertension Workshop. Adelaide, Australia.
• Limesand, S. W. (2012). Healthy Growth Workshop. Bill and Melinda Gates Foundation, Healthy Growth Workshop. Ottawa, Ontario Canada: Bill and Melinda Gates Foundation.
• Limesand, S. W. (2012). Ruminant Stress: Effects of environment on fetal programming in ruminants. ADSA-AMPA-ASAS-CSAS-WSASAS Joint Annual Meeting: WSASAS Symposium. Phoenix, Arizona.
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  Abstract #810 in Journal of Animal Sciences Volume 90 Supplement 3, Page 719
• Limesand, S. W. (2012, June). Healthy Growth Workshop. Healthy Growth Workshop. Washington, DC: Bill and Melinda Gates Foundation.
• Penrod, ., Ananthakrishnan, L., Patek, R., Vagner, J., Lynch, R., & Limesand, S. (2012). Biodistribution and Specific Targeting of Pancreatic beta-cells with a Heterobivalent GLP-1/ Yohimbine Ligand. Western Regional Islet Study Group Meeting (WRISG). Seattle, WA.
• Limesand, S. W. (2011). Fetal islet adaptations to intrauterine growth restriction: a role for the adrenergic system. Department of Obstetrics and Gynecology. Denver, CO.
• Limesand, S. W. (2011). Role of Catecholamines in IUGR Fetuses. Aspen Perinatal Biology Symposium: Interaction of Maternal, Placental, and Fetal Systems in Perinatal Development. Aspen, Colorado.

Poster Presentations

• Limesand, S. W., Langlais, P. R., Anderson, M. J., Camacho, L. E., Kelly, A. C., Davis, M. A., & Pendleton, A. L. (2020, March/Spring). Decreased Mitochondrial Complex I Activity in Skeletal Muscle of Growth Restricted Ovine Fetuses with Placental Insufficiency. Society for Reproductive Investigation 67th Annual Scientific Meeting.
• Davis, M. A., Camacho, L. E., Pendleton, A. L., Anderson, M. J., Steffens, N. R., & Limesand, S. W. (2019, March). Promotion of Glucose Utilization and Production Rates is Independent of Norepinephrine in Intrauterine Growth Restricted Ovine Fetuses. Society for Reproductive Investigation 66th Annual Scientific Meeting. Paris, France: Society for Reproductive Investigation.
• Davis, M. A., Camacho, L. E., Steffens, N. R., & Limesand, S. W. (2019, March). Adrenal Demedullation Lowers Placental Glucose Uptake Resulting in Higher Rates of Fetal Glucose Utilization and Production during Hyperinsulinemia. Society for Reproductive Investigation 66th Annual Scientific Meeting. Paris, France: Society for Reproductive Investigation.
• Gonzalez, M. G., Anderson, M. J., Camacho, L. E., Limesand, S. W., & Hill, M. G. (2019, March). Collagen, Fibrillin, and Elastin Gene Expression in Pregnancies Complicated by Pregnancy Induced Hypertensive Disorders and Spontaneous Preterm Birth.. Society for Reproductive Investigation 66th Annual Scientific Meeting. Paris, France: Society for Reproductive Investigation.
• Kelly, A. C., Bidwell, C. A., & Limesand, S. W. (2019, March). Comparative RNA Sequencing Reveals Common Responses in Metabolic Tissues Following Intrauterine Growth Restriction in Fetal Sheep. Society for Reproductive Investigation 66th Annual Scientific Meeting. Paris, France: Society for Reproductive Investigation.
• Pendleton, A. L., Humphreys, L. R., Davis, M. A., Camacho, L. E., Anderson, M. J., & Limesand, S. W. (2019, March). Normal Pyruvate Dehydrogenase Activity in Skeletal Muscle of Growth Restricted Ovine Fetuses. Society for Reproductive Investigation 66th Annual Scientific Meeting. Paris, France: Society for Reproductive Investigation.
• Camacho, L. E., Davis, M. A., Steffens, N. R., Kelly, A. C., Anderson, M. J., & Limesand, S. W. (2018, March). Oxygen and Glucose Correction Reestablished Insulin-Stimulated Glucose Utilization Rates in Sheep Fetuses with Placental Insufficiency.. Society for Reproductive Investigation, 65th Annual Meeting. San Diego: Reproductive Sciences T-081.
• Davis, M. A., Camacho, L. E., Anderson, M. J., Steffens, N. R., Kelly, A. C., & Limesand, S. W. (2018, March). Chronically Elevated Norepinephrine Concentrations Impair Glucose Uptake in Sheep Fetuses. Society for Reproductive Investigation, 65th Annual Meeting. San Diego, CA: Reproductive Sciences 25(1):S-062.
• Gonzalez, M. G., Kelly, A. C., Anderson, M. J., Bogan, R. L., Craig, Z. R., & Limesand, S. W. (2018, March). Phthalate Exposure Lowers Viability of Choriocarcinoma Cells. Society for Reproductive Investigation 65th Annual Scientific Meeting. San Diego, CA: Reproductive Sciences 25(1);T-147.
• Jones, A. K., Hay, W. W., Brown, L. D., Rozance, P. J., Limesand, S. W., & Wesolowski, S. R. (2018, Fall). Late gestation hypoxia increases norepinephrine and gluconeogenic expression to potentiate hepatic glucose production in fetal sheep.. DOHaD US North Carolina.
• Pendleton, A. L., Smith, R. M., Camacho, L. E., Anderson, M. J., Allen, R. E., & Limesand, S. W. (2018, March). Enhanced Skeletal Muscle Satellite Cell Differentiation and Altered TGFβ Signaling in Lambs with Intrauterine Growth Restriction. Reproductive Investigation 65th Annual Scientific Meeting. San Diego, CA: Reproductive Sciences 25(1):F-065.
• Hill, M., Kelly, A., Camp, S., Reed, K., Limesand, S. W., & Garcia, J. (2016, Mar). Increased glucagon and decreased visfatin concentrations in the cord blood of fetuses from Type I diabetic patients.. 36th Annual Meeting, The Pregnancy Meeting, Society for Maternal-Fetal Medicine.
• Kelly, A. C., Davenport, H., Taska, D. J., Camacho, L. E., Bidwell, C., Allen, R. E., & Limesand, S. W. (2017, March). β-Oxidation compensates for impaired glucose metabolism in skeletal muscle from intrauterine growth restricted sheep fetuses.. Reproductive Science 24(Supplement):200A, Abstract: F-098.. Orlando FL: Society for Reproductive Investication.
• Steyn, L., Jandova, J., Min, C., Molano, D., Price, N., Purvis, W., Loudovaris, T., Limesand, S. W., & Papas, K. (2017, Spring). N.D. Price, J.B. Stanton, W.G. Purvis, T. Loudovaris, S.W. Limesand, and K.K. Papas (2017) Increasing Islet Packing Density within Immunoisolation Devices Increases Cell Death and Alters Molecular Profiles Post-transplant.. International Pancreas and Islet Transplant Association.
• Benjamin, J., Culpepper, C., Brown, L., Thorn, S., Jonker, S., Davis, M., Limesand, S. W., Wilkening, R., Hay, W., & Rozance, P. (2015, Spring). Attenuated glucose stimulated insulin secretion following chronic hypoxemia is due to an intrinsic islet defect in fetal sheep. Western Society for Pediatric Research.
• Camacho, L., & Limesand, S. W. (2015, Nov). Enhanced insulin secretion and insulin action in young lambs with intrauterine growth restriction. SfE/BES.
• Hill, M., Kelly, A., Reed, K., & Limesand, S. W. (2015, Feb). Fetal growth restriction and markers of metabolic dysfunction and inflammation. Society for Maternal-Fetal Medicine.
• Kelly, A., Bidwell, C., Chen, X., McCarthy, F., & Limesand, S. W. (2015, Nov). Transcriptional Profiles Explain Insulin Hypersecretion Following Chronic Adrenergic Stimulation in Fetal Sheep Islets. SfE/BES2015.
• Kelly, A., Hill, M., Reed, K., & Limesand, S. W. (2015, Nov). Expression of Cytokines in Placenta from Pregnancies Complicated by Intrauterine Growth Restriction with Abnormal Umbilical Artery Doppler Indices. SfE/BES2015. Edinburgh, Scotland.
• Kelly, A., Smith, K., Badarinarayana, V., Steyn, L., Strop, P., Limesand, S. W., & Papas, K. (2015, Nov). Transcriptome Profiles of Human Pancreatic Islets Following Transient Ischemia Identify Inflammatory and Cell Death Responses. SfE/BES2015.
• Kelly, A., Smith, R., Dibase, S., Davenport, H., & Limesand, S. W. (2015, Nov). Adrenaline Mediated Metabolic and Functional Quiescence Protects Insulin Producing Cells From Hypoxia. SfE/BES2015. Edinburgh, Scotland.
• Smith, K., Steyn, L., Weber, C., Min, C., Purvis, W., Stanton, J., Burachek, A., Salama, B., Korbutt, G., Lynch, R., Limesand, S. W., & Papas, K. (2015, Nov). In vitro comparison of adult, juvenile, and neonatal porcine islets reveals age dependent resistance to ischemia. International Pancreas and Islet Transplant Association. Melbourne, Australia.
• Weegman, B., Einstein, S., Purvis, W., Steyn, L., Stanton, J., Kitzmann, J., Mueller, K., Limesand, S. W., Suszynski, T., Firpo, M., Graham, M., Janecek, J., Eberly, L., Garwood, M., Papas, K., Weegman, B., Einstein, S., Purvis, W., Steyn, L., , Stanton, J., et al. (2015, May). Non-Invasive Monitoring of Hypoxia and Viability Assessment of Implanted High-Cell-Density Tissue-Engineered Grafts. The Nineteenth Annual Hilton Head Regenerative Medicine Workshop.
• Limesand, S. W., Kelly, A., McCarthy, F., & Bidwell, C. (2014, March). Altered Genome Expression in Intrauterine Growth Restricted Fetal Islets.. Reproductive Sciences.
• Rozance, P., Wei, S., Davis, M., Thorn, S. R., Limesand, S. W., Hay Jr., W. W., & Brown, L. D. (2014, summer). Chronically Increased Amino Acids Improve Glucose-stimulated Insulin Secretion in Growth Restricted Fetal Shee. Diabetes 74nd Scientific Session.
• Ananthakrishnan, K., Penrod, L., Hart, N., Weber, C., Anderson, M., Vagner, J., Limesand, S. W., & Lynch, R. (2013, April). Multivalent GLP-1/Yhb for targeted therapy and imaging of pancreatic beta-cells.. FASEB J27:1154.18.
• Hart, N., Vagner, J., Weber, C., Ananthakrishnan, K., Limesand, S. W., & Lynch, R. M. (2013, April). Therapeutic potential of a bi-functional GLP-1/Glibenclamide targeted to the pancreatic beta-cell.. FASEB J27:729.11.
• Kelly, A., Penrod, L., Anderson, M., Kitzmann, J., Hart, N., Mueller, K., Lynch, R. M., Limesand, S. W., & Papas, K. K. (2013, September). Sulfonylurea receptor, glucagon-like peptide 1 receptor, and adrenergic receptor α2A are expressed and functional in adult porcine islets. 14th World Congress of the International Pancreas and Islet Transplantation Association.
• Kelly, A., Penrod, L., Kitzmann, J., Mueller, K., Papas, K. K., & Limesand, S. W. (2013, September). Epinephrine culture creates metabolic quiescence that leads to enhanced insulin secretion in isolated porcine islets and Min6.. 14th World Congress of the International Pancreas and Islet Transplantation Association.
• Macko, A. R., Chen, X., Anderson, M., Kelly, A., & Limesand, S. W. (2013, March). Elevated fetal plasma norepinephrine suppresses insulin and alters fetal metabolism prior to growth restriction in an ovine model of placental insufficiency. Reproductive Sciences 20(3):F-075.
• Macko, A. R., Yates, D. T., Kelly, A., Anderson, M., & Limesand, S. W. (2013, March). Effects of fetal adrenal demedullation at 0.7 gestation on fetal growth and metabolism at 0.9 gestation in an ovine model of placental insufficiency and intrauterine growth restriction.. Reproductive Sciences, 20(3):F-076..
• Ananthakrishnan, K., Hart, N., Weber, C., Vagner, J., Hart, N., Chung, W., Weber, C., Vagner, J., Limesand, S., Limesand, S., de, S. C., Lynch, R., & Lynch, R. (2012, April). 2012 Experimental Biology. San Diego, California.
• Chen, X., Yates, D., Macko, A., Kelly, A., & Limesand, S. (2012, March). Chronic Norepinephrine Exposure Desensitizes Fetal Sheep Islets and Enhances Insulin Secretion. Society for Gynecological Investigation. San Diego, California.
• Hart, N., Ananthikrishnan, K., Vagner, J., Chung, W., Weber, C., Limesand, S., & Lynch, R. (2012, June). American Diabetes Annual Meeting. Philadelphia, PA.
• Hart, N., Vagner, J., Chung, W., Weber, C., Limesand, S., Silva, C., & Lynch, R. (2012, April). Specific targeting of pancreatic -cells for imaging and therapy using multivalent targeting of receptor combinations. 2012 Experimental Biology. San Diego, California.
• Herway, C., Chen, X., Anderson, M., Reed, K., & Limesand, S. (2012, March). The Presence of Mitochondrial Progesterone Receptors in the Amniochorionic Membranes. Society of Gynecological Investigation. San Diego, California.
• Kelly, A., Yates, D., Green, A., & Limesand, S. (2012, June). Epinephrine Acutely Suppresses Oxidative Metabolism in Isolated Rat Islets and Pancreatic Beta Cell Lines. American Diabetes Annual Meeting. Philadelphia, PA.
• Macko, A., Chen, X., Kelly, A., Anderson, M., Yates, D., & Limesand, S. (2012, March). Adrenal demedullation improves glucose stimulated insulin secretion in feral sheep with placental insufficiency-induced IUGR. Society for Gynecological Investigation. San Diego, California.
• Rozance, P., Klopatek, S., Lavezzi, J., & Limesand, S. (2012, June). Chronic Fetal Hypoglycemia Lowers Glucose Stimulated Insulin Concentrations but Increases Vascular Density in Pancreatic Islets. American Diabetes Annual Meeting. Philadelphia, PA.

Others

• Limesand, S. W. (2016, July). Title: Perinatal Programming of Insulin Responsiveness after Hyperthermia-induced Placental Insufficiency.. 12. Physiology 2016 - Joint meeting of The Physiological Society and the American Physiological Society.
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  -Stress during pregnancy- Physiological consequences for intrauterine developmentDublin, Ireland July 29, 2016
• Limesand, S. W. (2016, June). Perinatal Programming of Insulin Secretion Responsiveness in Sheep Fetuses with Intrauterine Growth Restriction.. Reproductive and Developmental Sciences Program.
  More info

  Michigan State University, East LancingJune 9, 2016.
• Limesand, S. W. (2016, October). Catecholamines Regulate Fetal Adaptation during Placental Insufficiency. 21. Biotechnology and Life Sciences Center, University of Nebraska-Lincoln.
• Benjamin, J. S., Culpepper, C. B., Brown, L., Thorn, S., Jonker, S., Davis, M., Limesand, S., Wilkening, R., Hay, W. W., & Rozance, P. (2015, JAN). ATTENUATED GLUCOSE STIMULATED INSULIN SECRETION FOLLOWING CHRONIC HYPOXEMIA IS DUE TO AN INTRINSIC ISLET DEFECT IN FETAL SHEEP. JOURNAL OF INVESTIGATIVE MEDICINE.
• Andrews, S., Brown, L., Thorn, S., Davis, M., Limesand, S., & Hay, W. (2014, MAR). Increased Adrenergic Signaling Is Responsible for Decreased Glucose-Stimulated Insulin Secretion in the Chronically Hyperinsulinemic Ovine Fetus. REPRODUCTIVE SCIENCES.
• Benjamin, J., Culpepper, C., Brown, L., Thorn, S., Jonker, S., Limesand, S., Wilkening, R., Hay, W., & Rozance, P. (2014, JAN). CHRONIC FETAL HYPOXEMIA ATTENUATES GLUCOSE STIMULATED INSULIN SECRETION IN FETAL SHEEP. JOURNAL OF INVESTIGATIVE MEDICINE.
• Papas, K. K., Steyn, L., Davies, M., Loudovaris, T., Limesand, S., Tempelman, L., & Avgoustiniatos, E. S. (2014, DEC 1). Enhanced Oxygen Supply to Immunoisolated Islets In vivo Enables their Viability and Function at Tissue Like Densities. TISSUE ENGINEERING PART A.