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In this thesis, three mouse models and cell culture techniques were used to investigate genetic factors leading to glucose intolerance, diabetes or hyperinsulinemia. Loss of nicotinamide nucleotide transhydrogenase (Nnt) function is linked to ROS-mediated uncoupling of mitochondrial metabolism and reduced insulin secretion. The enzymatic activity of Nnt generates mitochondrial NADPH essential for ROS detoxification. However, the exact nature of ROS as well as the antioxidant enzymes involved are still unknown. It was found that MIN6 cells in which Nnt was silenced displayed an increase in mitochondrial H2O2 upon stimulation with both the ROS generator menadione and glucose. Knockdown of GPx1, however, had no effect on mitochondrial H2O2 and was linked to a Ca2+ independent hypersecretion of insulin. Exogenous GSH did not increase the glucose-stimulated rise in [Ca2+]i in Nnt mutant and control islets. This finding substantiates the suggested role of ROS as a signalling molecule in insulin secretion. In contrary to previous studies on MIN6 cells and single β-cells, the glucose-stimulated increase in [Ca2+]i, measured in intact Nnt mutant islets showed no difference compared with control islets. This might indicate differences in single β-cell versus whole islet physiology or be attributable to differences in genetic background. The activating V59M mutation in the KATP channel subunit Kir6.2 causes neonatal diabetes in humans. Transgenic mice constitutively expressing the V59M mutation in their β-cells recapitulated features of the disease phenotype in humans. In vitro studies showed that β-cells exhibited impaired first- and second-phase glucose-dependent insulin secretion. This was paralleled by a complete loss of the initial glucose-dependent rise in [Ca2+]i in V59M islets. However, islets from mice carrying an uninduced Kir6.2-V59M mutation, or mice expressing Cre recombinase, showed no impairment of their [Ca2+]i responses. If expression of the transgene was induced but mice were then treated with the KATP channel blocker glibenclamide to control their diabetes, isolated islets displayed a loss of the initial rise in [Ca2+]i and a reduced sustained increase of [Ca2+]i, which was associated with abrogation of synchronous Ca2+ oscillations in β-cells. In the presence of glibenclamide, both basal Ca2+ levels and insulin secretion from isolated islets were elevated, whereas the glucose-stimulated [Ca2+]i response, synchronicity of Ca2+ oscillations and insulin secretion were restored. Furthermore, Ca2+ imaging revealed that the number of β-cells per islet responding to glucose was similar to control islets, underlining the importance of early treatment with glibenclamide to achieve glycemic control. The E1506K mutation of the SUR1 subunit of the KATP channel causes a reduction in channel activity. It is associated with hyperinsulinism of infancy in early life and leads to the development of glucose intolerance, insulin deficiency and diabetes later in life. Islets isolated from a knock-in mouse expressing the SUR1-E1506K mutation showed enhanced glucose-stimulated insulin secretion from young het E1506K (first- and second-phase) and young hom E1506K islets (basal and first-and second-phase). Old hom E1506K islets exhibited normal basal insulin secretion but first- and second-phase secretion was found to be markedly reduced in comparison to wild-type islets. This was due to a decline in insulin content. Confocal Ca2+ imaging suggested that this was not caused by β-cell loss. Measurements of [Ca2+]i in young het E1506K islets showed spontaneous Ca2+ oscillations in 2 mM glucose, which did not result in an average elevation in [Ca2+]i. However, young hom-E1506K islets displayed vigorous Ca2+ oscillations in 2 mM glucose that led to an average elevation in [Ca2+]i. Depolarisation of E1506K islets with either glucose, tolbutamide or KCl produced a reduced increase in [Ca2+]i compared to wild-type islets indicating either a reduction in Ca2+ influx or an enhanced clearance of Ca2+. Peak voltage-dependent inward currents recorded from β-cells in het-E1506K islets were larger than in wild-type β-cells. Untypically for mouse β-cells, these inward currents contained a Na+ current resulting from a shift in its inactivation towards a more positive membrane potential.

OBJECTIVE: The 10th leading cause of death in the United States is heart disease. Most of the deaths by heart disease has a correlation with an occlusion of the coronary arteries. While diabetes mellitus is currently the 7th leading cause of death, which is a chronic condition that affects more than 37 million people in America. The global epidemic of obesity largely explains the dramatic increase in the incidence and prevalence of type 2 diabetes (T2D) over the past 25 years. Statins are well known drugs to decrease LDL for individuals who suffer from hypercholesterinemia; however, there is also an increased risk of developing diabetes mellitus. An estimation of 10-20 per 10,000 patients per year demonstrated an excess risk of T2D with the long-term use of statin. Here we examine the effects of simvastatin and pitavastatin on pancreatic ß-cell function to determine whether altered insulin secretion may contribute to an increased risk of T2D. METHODS: The experiments were performed using clonal pancreatic ß-cells (INS-1). The cells were grown in 4 mM glucose in RPMI media. Cells were grown for three days before adding the different types of statins: simvastatin and pitavastatin for one day. Then the cells were used to perform the glucose-induced insulin secretion (GSIS) experiment. Insulin secretion and insulin content were assay using a fluorescence-based immunoassay. The study was calculated using Microsoft Excel. Standard variance and standard error were used to assess the difference sets of data. RESULTS: INS-1 cells responded to acute glucose stimulation after chronic culture in both low (4 mM) and high (11 mM) glucose. Secretion from cells cultured at 4 mM glucose was higher than cells cultured at 11 mM glucose at all glucose concentrations tested, characteristic of the effects of glucolipotoxicity (GLT). Insulin content in cells cultured at high glucose was decreased 8.6-fold compared to cells cultured at the more physiological low glucose condition. When normalized to basal secretion cells cultured at high glucose exhibited basal hypersecretion and increased GSIS compared to those in low glucose. Simvastatin (100 nM, 24 hrs) increased basal insulin secretion to a greater extent than Pitavastatin. The effects of pitavastatin on basal insulin secretion were less consistent than seen with simvastatin. Simvastatin was also shown to inhibit GSIS from cells cultured at 4 mM glucose, while pitavastatin increased GSIS. CONCLUSION: Both pitavastatin and simvastatin alter insulin secretion from pancreatic ß-cells. The effect of simvastatin to both increase basal and decrease GSIS, characteristic of GLT suggests pitavastatin may be the statin of choice to reduce the risk of statin-induced T2D.

Insulin secretion plays a crucial role in energy homeostasis. Accordingly, disrupted insulin secretion has been associated with metabolic disorders such as insulin resistance syndrome and diabetes mellitus. Insulin secretion from pancreatic β-cells is tightly regulated by complex ionic mechanisms usually included in a consensus mechanism involving ATP-sensitive K+ channels as the only key player. However, K+ channels solely do not reflect the whole mechanism. Anionic channels also contribute to the machinery of insulin secretion by initiating electrogenic Cl– fluxes in β-cells. The Na+K+2Cl– cotransporter 1 (Nkcc1) and other Cl– transporters participate in the non-equilibrium distribution of Cl– in β-cells. Recent studies from our laboratory and others have demonstrated a potential role of Nkccs in insulin secretion. However, virtually nothing is known regarding the role of Nkcc1 in energy homeostasis. In this study, we unravel part of the physio-pathological consequences that follow the disruption of insulin secretion upon elimination of Nkcc1 from β-cells in mice. Our results suggest that deficient insulin secretion due to altered β-cells Cl– homeostasis impairs satiation responses to feeding leading to nocturnal hyperphagia before the onset of overweight and metabolic complications including, hyperglycemia, hyperinsulinemia, glucose/insulin resistance, steatohepatitis, fat tissue inflammation and obesity. Everything considered, we conclude that loss of β-cell Nkcc1 impairs insulin secretion in vivo impinging long-term metabolic consequences triggered by an early impairment in the feeding behavior. Altogether, our results suggest that elimination of Nkcc1 from β-cells recapitulates, at least in part, the natural progression of metabolic syndrome, a major risk factor for the development of type-2 diabetes.

Congenital hyperinsulinemia is a heterogenous group of disorders resulting in improper insulin secretion from the pancreatic beta cells at birth. Elevated insulin leads to persistent hypoglycemia, increasing the patient’s risk for seizure and many long-term disabilities. ACACB codes for Acetyl-CoA Carboxylase 2 (ACC2), an outer mitochondrial membrane enzyme involved in fatty acid synthesis. Global ACACB knockout mice show continuous fatty acid oxidation and lowered plasma insulin levels when fed a high fat, high carbohydrate diet. Despite this, the role of ACC2 in the pancreatic beta cell has yet to be described. In this study, we have identified five individuals with congenital hyperinsulinemia hypoglycemia and developmental delays. Next generation sequencing found all five individuals have variants of unknown significance in the ACACB gene. Fibroblast samples collected from two affected individuals demonstrated impaired ACC2 enzyme activity and abnormal ACACB gene expression. To examine the mechanism of ACC2 in the pancreatic beta-cells, a conditional pancreatic-specific ACACB knockout mouse model was created. We found conditional pancreatic-specific ACACB knockout results in increased body weights and elevated circulating insulin levels. For the underlying molecular mechanism, we found mutant mice had increased expression of uncoupling protein 2 (UCP2) and mild elevated fatty acid receptor 1 (FFAR1). Our data demonstrates the loss of ACC2 causes congenital hyperinsulinemia in humans. This clinical feature is recapitulated in pancreatic specific conditional knockout mouse model. We propose the ACACB gene is a novel disease-causing gene for congenital hyperinsulinemia hypoglycemia through regulation of insulin secretion in the pancreatic beta cells.

Obesity, a major risk factor in the development of Type 2 Diabetes (T2D), is commonly associated with insulin resistance and hyperinsulinemia. The long accepted view has been that insulin resistance drives hyperinsulinemia; however, there are multiple lines of evidence that hyperinsulinemia can precede and drive insulin resistance. The signals and mechanisms by which chronic excess nutrients promote pancreatic β-cell dysfunction remain poorly understood. This prompted us to define the signaling events that contribute to basal insulin hypersecretion induced by excess glucose. Of particular interest is signaling through mechanistic target of rapamycin complex 1 (mTORC1), a nutrient sensitive kinase complex whose hyperactivation has been shown to promote hyperinsulinemia. Clonal ß-cells (INS-1 cells) with and without mTORC1 inhibition were pre-exposed to physiological (5mM) or excess (11mM) glucose for 4 to 24 hrs. Basal insulin secretion, respiration and metabolites were measured. Pre-exposure to excess glucose resulted in sustained mTORC1 hyperactivation, basal insulin secretion, higher basal respiration and increased maximal respiratory capacity, due to accelerated mitochondrial pyruvate metabolism. Inhibition of mTORC1 reduced basal insulin secretion, basal respiration and maximal respiratory capacity. Moreover, cells challenged with excess glucose had increased levels of glycolysis and TCA cycle intermediates. Our results suggest that hyperactivation of mTORC1 induced by excess glucose results in increased energy demand and in the generation of metabolic factors that can lead to basal insulin hypersecretion. Therefore, targeting mitochondrial pyruvate metabolism and /or mTORC1 signaling could potentially lead to specific therapies to control hyperinsulinemia and diabetes progression.

It is estimated by the World Health Organization (WHO) that 422 million people had diabetes worldwide in 2014, including 30.3 million people in the US. The cost of treating the disease is has tripled from 2003-2013 due to the increased number of patients. One of the genes strongly associated with type 2 diabetes (T2D) is the transcription factor 7 like 2 (TCF7L2). A single nucleotide polymorphism (SNP) of the TCF7L2 results in increased expression of long chain acyl-CoA synthetase 5 (ACSL5) while deletion of this part of the TCF7L2 gene reduces ACSL5 mRNA level. The regulation of ACSL5 gene expression by the high risk TCF7L2 allele highlights the importance of investigating the role of ACSL5 in T2D. ACSL5 is one of a family of enzymes that activates FA to its CoA ester and is required for FA metabolism within cells. Mice lacking this protein have reduced fat mass and are more insulin sensitive. Chronic exposure of clonal pancreatic ß-cells to excess nutrients has been shown to result in increased intrinsic lipid droplets, reduced insulin content, a left-shift in glucose dose-dependent insulin secretion curve characterized by basal insulin hypersecretion (IH) and blunted glucose stimulated insulin secretion (GSIS). We tested the hypothesis that the use of a putative ACSL5 inhibitor (Adipo C) can reduce accumulated lipid droplets, rescue insulin content and reverse the left-shift in glucose dose-dependent insulin secretion curve. INS-1 (823/13) cells were cultured in either 4 mM or 11 mM glucose media representing physiological and excess nutrients environment. Adipo C (10-25 µM) was added to cells to both acutely (2 hrs) and chronically (72 hrs) inhibit ACSL5 activity. Thin layer chromatography with C11 Bodipy fatty acid (BFA) was used to detect acute fatty acid incorporation into neutral lipids. Nile red was used to visualize intrinsic lipid droplets inside cells. Intracellular Ca2+ activity was detected using fura 2. Insulin assay was measured by HTRF. Acute fatty acid incorporation and lipid accumulation were reduced in cells exposed to Adipo C. An Adipo C concentration dependent right shift of glucose dose-dependent insulin release and increased insulin content were observed. 11 mM glucose cells cultured in 25 µM Adipo C showed decreased intracellular Ca2+ activity at 3 mM glucose and increased Ca2+ activity at 12 mM glucose, which are characteristic of cells cultured in 4 mM glucose having reduced lipid stores. These results all indicate possible protective effects on -cells exposed to excess nutrients. Islets of T2D patients who have a physiologically elevated blood glucose level are exposed to a similar excess nutrient environment. Therefore, the results illustrated here warrant further research on Adipo C compound to explore its therapeutic potential on T2D.

Insulin resistance has long been considered to play a crucial role in the pathophysiology of metabolic syndrome that is the leading cause of mortality and morbidity worldwide. Metabolic diseases consist of a group of metabolic abnormalities that increase the risk of health problems, such as type 2 diabetes (T2D) and cardiovascular disease. Nonalcoholic fatty liver disease (NAFLD) is associated with obesity and metabolic syndromes. It is the fastest growing cause of liver dysfunction. Its progressive form nonalcoholic steatohepatitis (NASH) is associated with hepatic fibrosis that can develop into cirrhosis. In addition, there is a growing body of evidence that among risk factors that promote atherosclerosis, metabolic syndrome is a potent predictor of cardiovascular events. Insulin resistance seems to play a major role in the pathophysiology of atherosclerosis in relation with metabolic syndrome. Given that patients with NAFLD/NASH are at a high risk to develop atherosclerosis; these two diseases may share some pathology. However, precise molecular mechanisms underlying the pathogenesis and progression of these diseases are not well understood. Thus studying the molecular link between them would pinpoint sites of more effective pharmacologic interventions. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a protein that is markedly reduced in the liver of patients with NASH, promotes insulin clearance to regulate insulin action. Whether or not CEACAM1 links hyperinsulinemia to NAFLD/NASH and atherosclerosis still needs to be determined. CEACAM1 enhances the rate of uptake of the insulin-receptor complex into the clathrin-coated vesicles of hepatocytes. It also plays a major role in mediating the negative acute effect on fatty acid synthase (Fasn) activity. As such, CEACAM1 regulates insulin and lipid metabolism. Mice with global null mutation of CEACAM1 (Cc1–/–) display hyperinsulinemia resulting from impaired insulin clearance, insulin resistance, steatohepatitis, plaque-like lesions in aortae, and obesity. We herein are highlighting the specific role of hepatic CEACAM1 in regulating insulin and lipid metabolism, hepatic steatosis and altered energy balance. Moreover, liver-based rescuing of CEACAM1 resulted in full normalization of the metabolic phenotype. These data underscore the key role of CEACAM1-dependent hepatic insulin clearance pathways in regulating systemic insulin sensitivity, lipid homeostasis and energy balance. Furthermore, white adipose tissue-derived fatty acids repressed hepatic CEACAM1-dependent regulation of insulin and lipid metabolism and adenoviral-mediated CEACAM1 redelivery countered the adverse metabolic effect of the high-fat diet on insulin resistance, hepatic steatosis, visceral obesity and energy expenditure. These data assign a causative role for lipolysis-driven decrease in hepatic CEACAM1 levels. Furthermore, persistence of hyperinsulinaemia and insulin resistance in Cc1-/- mice after blocking lipolysis with nicotinic acid demonstrates that these metabolic abnormalities arise independent of visceral obesity and that they are the cause rather than the consequence of lipolysis. We show here that hepatic CEACAM1 deficiency, acting through hyperinsulinemia and systemic insulin resistance promotes a state of lipid-dependent vascular dysfunction, alters endothelial cell function, contributing to atherosclerosis development in a cell-nonautonomous fashion. In addition, we are showing that the cell autonomous metabolic and fibrogenic effect of specifically deleting CEACAM1 from hepatic stellate cells (HSC) leads to their activation. We also have identified a novel role of exenatide, a glucagon-like peptide-1 receptor agonist, on insulin clearance by inducing CEACAM1 expression in parallel to insulin secretion to prevent chronic hyperinsulinemia and subsequent increase in hepatic de novo lipogenesis and NAFLD. Exenatide also prevented diet-induced pro-fibrogenesis and hepatocellular injury in a CEACAM1-dependent mechanism. The physiologic implication of increased insulin extraction in the face of elevated insulin secretion by exenatide is to maintain homeostatic plasma insulin levels, an essential determinant of insulin sensitivity and lipid homeostasis. Hence, our studies demonstrate that the CEACAM1-dependent mechanism of hyperinsulinemia-induced insulin resistance plays a critical role in the development of NAFLD/NASH and atherosclerosis. This finding is of clinical importance because it suggests that the metabolic syndrome is not only a collection of abnormalities that should be considered and treated independently. A defect in insulin singling links those diseases since it acts on both glucose and cholesterol metabolisms. These data suggest that finding and curbing the molecular pathways that lead the insulin resistance associated with metabolic syndrome will add a new dimension to experimental therapeutics in this area.

Pancreatic beta-cells regulate blood glucose by secreting insulin in response to nutrients. The development of Type 2 Diabetes (T2D) is characterized by elevated insulin secretion in the fasted state and a failure to adequately respond to nutrient influx, particularly glucose. Current dogma states that insulin resistance is the initiating event in the development of T2D, with compensation by beta-cells necessary to maintain glucose homeostasis. An alternative model, which will be a central theme throughout this thesis, is that hypersecretion of insulin is the initiating and sustaining event in the development of T2D. The underlying cause of insulin hypersecretion is unclear. Determining this is important in order to test this alternative model as a viable target for prevention and treatment of T2D. Because of the association between obesity and hyperinsulinemia, we hypothesized that exposure of the β-cell to high levels of nutrients stimulates insulin hypersecretion. We found that chronic incubation of β-cells in high glucose and/or oleate, which mimics nutrient conditions in obesity, lowered the half-maximal response for glucose to stimulate insulin secretion. The degree of the left-shift correlated with lipid stores. We determined that heightened sensitivity of granule exocytosis to Ca2+ was driving this left-shift. Thus glucose, while not necessarily abnormal in obesity, may cause hypersecretion of insulin due to altered sensitivity of the β-cell to this secretagogue. Iron stores are increased in obesity and are predictive of T2D development. We found that iron acutely stimulated both basal and glucose-stimulated insulin secretion (GSIS) in a reactive oxygen species dependent manner. Interestingly, iron did not increase insulin secretion via Ca2+ influx. Thus, both iron and glucose/oleate induce insulin hypersecretion via an aspect of the triggering pathway that is not Ca2+, the putative triggering signal. Previous work in our laboratory documented that exogenous mono-oleoyl-glycerol, an endogenous lipid signaling molecule and food additive, increases basal insulin secretion. We found that inhibition of monoacylglycerol lipase, which increases cellular monacylglycerol species, reduced GSIS, possibly via a reduction in long-chain CoA. Collectively, our works supports the hypothesis that chronic exposure to high nutrient levels drives insulin hypersecretion in obesity.

The prevalence of obesity has risen substantially throughout the world in recent decades. Nonsurgical treatments for reducing adiposity remain paramount to diminishing obesity and its associated comorbidities. Conjugated linoleic acid has received considerable attention due to its ability to reduce body weight and adiposity in a number of species. The mechanisms responsible for adipose depletion by conjugated linoleic acid remain controversial and are not completely understood. The first objective of this research was to determine if conjugated linoleic acid altered adipose phenotype by inducing adaptive thermogenesis and increasing oxidative capacity. In this study, conjugated linoleic acid dramatically reduced weight gain and adipose mass. Additionally, conjugated linoleic acid induced the expression of numerous genes involved in adaptive thermogenesis in epididymal white adipose tissue. Lipid droplet morphology was unchanged by conjugated linoleic acid treatment, although mitochondrial density was increased in epididymal white adipose tissue. Unexpectedly, whole body oxygen consumption and body temperatures were reduced and unchanged by conjugated linoleic acid treatment, respectively. We attributed these observations to a significant reduction in brown adipose tissue with a concomitant compensatory response in epididymal white adipose tissue. Our data suggest that significant reductions in brown adipose tissue by conjugated linoleic acid initiates white adipose adaptive thermogenesis as a means to maintain body temperature. Although conjugated linoleic acid reduces body weight and adipose mass, it also paradoxically induces lipodystrophy-associated disorders including hepatic steatosis, hyperglycemia, hyperinsulinemia, and insulin resistance. The second objective of this research was to determine if hepatic steatosis induced by conjugated linoleic acid would predict for hepatic diacylglycerol accumulation and increased membrane-associated protein kinase C epsilon protein. This study revealed that in addition to increased hepatic triacylglycerol, conjugated linoleic acid increased hepatic diacylglycerol concentration and membrane-associated protein kinase C epsilon. Diacylglycerol concentration positively predicted for fasting hyperglycemia and hyperinsulinemia. The increase in diacylglycerol concentration was also associated with increased hepatic gluconeogenic gene expression in conjugated linoleic acid-treated animals. This study suggested that conjugated linoleic acid initiates the pathophysiology responsible for hepatic insulin resistance. The objective of the third study was to determine if hepatic diacylglycerol accumulation and increased membrane-associated protein kinase C epsilon by conjugated linoleic acid would initiate hepatic insulin resistance. In this study, conjugated linoleic acid induced hyperglycemia and hyperinsulinemia while increasing hepatic lipid deposition. Hepatic diacylglycerol and membrane-associated protein kinase C epsilon were also significantly increased by conjugated linoleic acid treatment. Hyperinsulinemic-euglycemic clamp studies revealed that conjugated linoleic acid caused hepatic insulin resistance, as evidenced by increased endogenous glucose production. Unexpectedly, white adipose tissue glucose disposal was increased by conjugated linoleic acid treatment, whereas skeletal muscle glucose disposal was unchanged. Collectively, these studies demonstrate that conjugated linoleic acid increases epididymal white adipose tissue oxidative capacity, although this alteration is not sufficient to abrogate lipodystrophy-associated disorders that consequently lead to hepatic insulin resistance.

Impaired hepatic insulin clearance causes hyperinsulinemia and secondary insulin resistance, which may progress to involve various components of metabolic syndrome. The carcinoembryonic-related cell adhesion molecule 1 (CEACAM1) has been shown to promote insulin clearance, downregulate the mitogenic action of insulin, and limit lipogenesis in the early hours of refeeding. Mice with liver-specific Ceacam1 inactivation (L-SACC1) or with global null mutation (Cc1–/–), exhibit impairment of insulin clearance and hyperinsulinemia, which causes insulin resistance. Since the lack of CEACAM1 correlates with insulin resistance, and regulates insulin action in liver, an important diet-responsive organ, we proposed that reduction of CEACAM1 is implicated in the pathogenesis of diet-induced insulin resistance; and that increasing hepatic levels of CEACAM1 would be protective against metabolic abnormalities associated with metabolic syndrome. Therefore we examined CEACAM1 levels in an animal model of metabolic syndrome and non-alcoholic steatohepatitis (NASH), the low aerobic capacity runner (LCR) rats, in comparison to high aerobic capacity runner (HCR) rats. We found that in response to caloric restriction by 30% over a period of 2-3 months profound improvements in insulin sensitivity and reversal of hepatic inflammation, oxidative stress and fibrosis. Caloric restriction exerts these effects along with increases in fasting levels of CEACAM1 in liver. Additionally we examined the effect of high-fat diet on wild-type mice and on a transgenic mouse with liver-specific overexpression of rat CEACAM1 (L-CC1). We found that an early event associated with high-fat feeding is repression of CEACAM1 by a PPARalpha-mediated mechanism, and that this leads to impaired insulin clearance prior to the development of a pro-inflammatory state. Transgenic overexpression of CEACAM1 in liver prevents hyperinsulinemia and insulin resistance, and limits visceral obesity and the metabolic response to high-fat intake.

Obesity afflicts more than 30% of the U.S. population and is a major risk factor for the development of hypertension, type II diabetes, and cardiovascular disease. Studies in humans and animals indicate that obesity is associated with increased sympathetic outflow to the vasculature and kidneys. One mechanism postulated to underlie the increase in sympathetic nerve activity (SNA) in obesity is hyperinsulinemia. Little is known regarding the central circuitry underlying elevated SNA and arterial blood pressure (ABP) during hyperinsulinemia and obesity or if sympathoexcitatory circuits are still responsive to insulin in obesity. Hyperinsulinemic-euglycemic clamps elevate SNA to the hind limb vasculature in lean rodents but obesity is associated with resistance to the peripheral and anorexic effects of insulin. Therefore, the first aim was to determine whether diet-induced obesity causes development of insulin resistance in the central circuits mediating SNA. The sympathoexcitatory response to insulin was still intact in diet-induced obese rats indicating a role for insulin in the elevation in SNA and ABP in obesity. The second aim of this project was to identify the specific receptors in the rostral ventrolateral medulla (RVLM) that mediate the elevated SNA during hyperinsulinemia. The RVLM provides basal sympathetic tone and maintains baseline ABP. Glutamate is the major excitatory neurotransmitter and glutamate receptors of the RVLM are known to mediate multiple forms of hypertension. Blockade of RVLM NMDA-specific glutamatergic receptors reverses the increased lumbar SNA associated with hyperinsulinemia. In contrast, blockade of angiotensin II type 1 or melanocortin receptors in the RVLM had no effect on the sympathoexcitatory response to insulin. The goal of the third aim was to identify the cellular mechanisms within RVLM that mediate the elevated SNA and ABP in diet-induced obesity. Blockade of RVLM glutamate receptors reversed the elevated ABP and lumbar SNA associated with diet-induced obesity while it had no effect on rats on a low fat diet or those resistant to weight gain on the high fat diet. Similar to the findings during hyperinsulinemia, blockade of RVLM angiotensin II type 1 or melanocortin receptors had no effect on lumbar SNA or ABP during diet-induced obesity.

Maternai peripheral hyperinsulinemia and fetal hyperinsulinism that occurs as a conséquence of the treatment of diabetic pregnancy hâve been associated with both gross and microscopic, cellular changes. Thèse include an apparent hyperplacentation and fetal macrosomia and trophoblastic morphofunctional pathology, which may contribute to increased morbidity and mortality rates. Reports hâve indicated that fetal and placental macrosomia does not always resuit from maternai hyperglycemia and that insulin m a y stimulate cellular prolifération in vitro. A family of growth factors known as the Insulin-like Growth Factors (IGFs) hâve been identified and shown to share insulin's biological activities. Thèse, too, are thought to play an important rôle in the growth and development of the fétus. Due to the structural similarities, insulin and the IGFs m a y bind with each other's receptors to elicit a response. The aim of this study was to investigate and compare the effects of insulin and the IGFs on the prolifération and differentiation of cells isolated from the human placenta.

BACKGROUND: Adjustments of carbohydrate intake and oxidation occur in both normal-weight and overweight individuals. Nevertheless, the contribution of carbohydrates to the accumulation of fat through either reduction of fat oxidation or stimulation of fat synthesis in obesity remains poorly investigated. OBJECTIVE: The objective of this study was to assess the postprandial metabolic changes and the fractional hepatic de novo lipogenesis (DNL) induced by a high-carbohydrate, low-fat meal in lean and overweight young men. DESIGN: A high-carbohydrate, low-fat meal was administered to 6 lean and 7 overweight men after a 17.5-h fast. During the fasting and postprandial periods, energy expenditure (EE), macronutrient oxidation, diet-induced thermogenesis, and serum insulin, glucose, triacylglycerol, and fatty acids were measured. To determine DNL, [1-13C]sodium acetate was infused and the mass isotopomer distribution analysis method was applied. RESULTS: After intake of the high-carbohydrate meal, the overweight men had hyperinsulinemia and higher fatty acid and triacylglycerol concentrations than did the lean men. The overweight group showed a greater EE, whereas there was no significant difference in carbohydrate oxidation between the groups. Nevertheless, the overweight men had a marginally higher protein oxidation and a lower lipid oxidation than did the lean men. DNL was significantly higher before and after meal intake in the overweight men and was positively associated with fasting serum glucose and insulin concentrations. Furthermore, postprandial DNL was positively correlated with body fat mass, EE, and triacylglycerol. CONCLUSION: After a high-carbohydrate, low-fat meal, overweight men had a lower fat oxidation and a higher fractional hepatic fat synthesis than did lean men.