Inhibition of cytokine production by cyclosporine A and G

Cyclosporine G (CsG) is an analogue of CsA that may not have the nephrotoxicity of CsA. Not much is known about the relative immunosuppressive activity of CsG compared with CsA in humans. For this reason we compared the effects of CsA and CsG on in vitro interferon gamma production by mitogen and alloantigen-stimulated peripheral blood mononuclear cells from normal individuals. We also investigated the effects of CsA and CsG on the production of lymphotoxin (LT) and tumor necrosis factor (TNF) activity, as these cytokines have been implicated in transplant rejection. Both CsA and CsG showed very similar dose-dependent inhibition of IFN-G and LT/TNF activity (IC50 CsA for IFN-G = 8.0 ng/ml, for LT/TNF = 9.5 ng/ml; IC50 CsG for IFN-G = 13.0 ng/ml, for LT/TNF = 13.0 ng/ml). Maximum suppression was seen if the drugs were added at culture initiation and suppression was reduced if the drugs were added 24 hr later. Both CsA and CsG showed significant dose- and time-dependent inhibition of IFN-G production in 2 degrees MLC. LT/TNF activity was suppressed by CsA and CsG in 2 degrees MLC, which was also dose- and time-dependent. These results suggest that CsG has an immunosuppressive profile similar to that of CsA and may therefore be useful in clinical situations where CsA nephrotoxicity is a problem.     

Short-term effects of cyclosporine on secretagogue-induced insulin release by isolated islets

Brief exposure (30 min) of isolated islets to 0.5 microgram/ml cyclosporine leads to alterations in the insulin secretory response to selected stimuli. When glucose is used as the secretagogue, cyclosporine slightly stimulates insulin release at substimulatory concentrations of the hexose. The inhibitory effect predominates, however, at stimulatory concentrations of glucose with a threshold at 6 mmol/L glucose and maximal inhibition of 33.5 mmol/L glucose. By contrast, insulin release induced by 17 mmol/L arginine is not affected. Cyclosporine also inhibits by 66% the insulin secretory response to 100 nmol/L phorbol 12-myristate 13-acetate, suggesting that either cyclosporine interferes with phorbol ester action on beta cells or the action site is located beyond the protein kinase C activation. On the other hand ionomycin-stimulated insulin response is also blocked by cyclosporine, indicating that insulin release induced by transient changes in cytosolic Ca++ is also affected. The evidence gathered here suggests that the inhibitory effect of cyclosporine on insulin release is apparent when glucose is used as a fuel stimulant and is reversed following removal of the stimulant. This effect is not reversed by using substances known to activate the protein kinase C or the Ca(++)-dependent branches of the stimulus-secretion coupling system in beta cells, indicating that the site of action of cyclosporine on pancreatic islets might be located in distal steps of the stimulus-secretion coupling of glucose-induced insulin release.     

Impairment of stimulated insulin release from the isolated perfused rat pancreas by cyclosporine pretreatment

Cyclosporine was fed to male Wistar rats in a dose of 5, 10, or 50 mg/kg b.wt. for 7 days, and the effect on insulin secretion from the isolated perfused pancreas was investigated. Dose-dependently plasma insulin and pancreatic insulin content decreased while whole-blood CsA levels increased. An increase in blood glucose was only observed after feeding 50 mg/kg b.wt. CsA resulting in whole-blood CsA levels of 7735 ng/ml. Glucose (20 mM)-stimulated total insulin secretion (ng/50 min) was not affected during feeding 5 mg/kg b.wt. CsA, but was significantly reduced after feeding 10 or 50 mg/kg b.wt. CsA. The biphasic insulin secretion was reduced after 5 mg/kg b.wt. during the initial peak (0-10 min) but not during the second peak (10-50 min), whereas after 10 or 50 mg/kg b.wt. CsA both peaks were markedly reduced. The arginine (20 mM) and the arginine (20 mM)-plus-glucose (20 mM) stimulated insulin secretion was less affected after feeding 10 mg/kg b.wt. CsA than after stimulation with glucose (20 mM) alone. The addition of CsA to the perfusate did not influence glucose-stimulated insulin release from normal rat pancreas. Our results demonstrate a toxic effect of CsA on the pancreatic beta cell that is dose dependent and possibly influences both insulin secretion and biosynthesis.     

Inhibition by cyclosporine of insulin secretion--a beta cell-specific alteration of islet tissue function.

We have reported the potent inhibitory effect of cyclosporine on glucose-induced insulin release in in vitro perfused pancreases. Suppression of both phases of release indicates inhibition of secretion and synthesis. Further studies were performed to examine the effect of high extracellular Ca2+ (4.875 mM). High Ca2+ failed to potentiate release in CsA-treated pancreases, thus we are focusing on the integrity of Ca(2+)-dependent signals in the beta cell. In this study, four groups of pancreases were perfused at 16.7 mM glucose: Control +/- somatostatin (SRIF) and CsA-treated +/- SRIF (60 nM). In control rats, the total 2-hr release decreased 40% with SRIF, from 42.7 +/- 5.5 to 25.5 +/- 3.9 micrograms/300 g body weight (P less than .05). In CsA-treated rats, release decreased 55% with SRIF, from 8.9 +/- 1.1 to 4.0 +/- 0.6 micrograms/300 g body weight. Further, at every time point of these CsA-treated rats, there was approximately 15% greater inhibition by SRIF than in controls. Pancreatic insulin contents were determined, indicating marked depletion of insulin stores in CsA-treated rats (190 +/- 9 vs. 76 +/- 5 micrograms/300 g body weight, P less than .01). Arginine-stimulated secretion of insulin and glucagon was also examined in control and CsA-treated pancreases. CsA exerted no effect on arginine-stimulated glucagon release, yet inhibited insulin approximately 50%. From these studies, we conclude that normal SRIF inhibitory mechanisms must be at least partially intact in CsA-treated pancreases during glucose-induced insulin release, and that CsA inhibition is specific for insulin release, as glucagon stores and arginine-stimulated glucagon release are unaffected by CsA.     

Inhibition of alloantigen presentation by cyclosporine

Cyclosporine (CsA) was examined for its ability to inhibit alloantigen presentation by spleen cells in a primary mixed lymphocyte reaction; by gamma interferon-induced P388.D1 macrophages to an alloreactive T cell clone; and by a B cell lymphoma, B1D.beta to an alloreactive T cell hybridoma. Alloantigen-presenting cells were treated with CsA or its inactive analogs for 2 hr, washed extensively (four times), and added to the T cells. Using this protocol, CsA maximally inhibited allorecognition by the T cells at 1000 ng/ml in all three systems. An HPLC assay for CsA cell failed to detect any significant CsA carryover into the T cell assays. Supernatant transfer experiments also failed to demonstrate CsA carryover in the more sensitive T cell hybridoma assay. These transfer experiments also failed to demonstrate the generation of inhibitory factors during the assay. Northern blot analysis and a cell-surface ELISA failed to observe any decreases in MHC class II induction in/on P388.D1 cells with CsA present during the induction. Due to the lack of detectable (less than 10 ng/ml) CsA carryover, we hypothesize that CsA has a direct effect on the formation of stimulatory MHC class II in our alloreactive systems.     

The effects of transplant mass on insulin release by collagenase-dispersed pancreatic fragments in the diabetic dog

The abnormal glucose tolerance seen in experimental diabetic animals receiving pancreatic transplants has been assumed to be due to a reduced functional islet mass.Collagenase-dispersed autografts were prepared following total pancreatectomy. Six dogs received 100%, and 6 received 50% of the tissue so formed. Blood glucose, lactate, pyruvate, alanine, glycerol, 3-hydroxybutyrate, cholesterol, free fatty acids, insulin, glucagon, and cortisol were determined in the fasting state and following an intravenous glucose load (IVGTT). Glucose clearance (K) was calculated. Fasting euglycemia was achieved and insulin levels were the same in both groups. Similar reductions in K, with elevated fasting levels of glycerol, and 3-hydroxybutyrate, were present in both groups 1 month after transplantation. Glucose-stimulated insulin output was both quantitatively and qualitatively defective with similar levels being achieved in peripheral blood in both groups. This abnormal insulin response exposed elevated levels of free fatty acids and alanine during the IVGTT, although suppression of glucagon was normal in both groups.The results suggest that a simple relationship between transplant mass and insulin secretion does not exist. This may be important in regard to the future clinical application of free transplantation in the treatment of diabetes.

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Resumen Se ha asumido que la anormal tolerancia a la glucosa que se observa en animales experimentales diabéticos recipientes de transplantes pancreáticos se debe a una reducida masa funcional de islotes. El propósito del presente estudio fue el de determinar la factibilidad de producir dos transplantes de un solo páncreas canino y valorar la relación que pudiera existir entre la masa de islotes implantada y su función metabólica.Se prepararon autotransplantes digeridos por colagenasa después de realizada la pancreatectomia. Seis perros recibieron el 100% y seis el 50% de los tejidos así preparados.Glucosa sanguínea, lactato, piruvato, alanina, glicerol, 3-hidroxibutirato, colesterol, ácidos grasos libres, insulina, glucagón y cortisol fueron determinados en ayunas y después de una carga de glucosa intravenosa. También se calculó la depuración de glucosa (K).Se observó euglicemia en ayunas junto con iguales niveles de insulina en los dos grupos de perros. Reducciones similares de K con elevados niveles, en ayunas, de glicerol y de 3-hidroxibutirato fueron aparentes en ambos grupos un mes después del transplante. La secreción de insulina estimulada por glucosa apareció tanto cuantitativa como cualitativamente defectuosa, con niveles similares en la sangre periférica en ambos grupos. Esta respuesta insulínica anormal expuso altos niveles de ácidos grasos libres y de alanina en el curso de la prueba de tolerancia a la carga de glucosa intravenosa, aún cuando la supresión del glucagón apareció normal en los dos grupos.Los resultados sugieren que no existe una relación simple entre la masa del transplante y la secreción de insulina. Este puede ser un hecho de importancia en relación a la futura aplicación clínica de los transplantes libres en el tratamiento de la diabetes.

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Résumé La tolérance anormale au glucose constatée chez les animaux diabétiques transplantés a été attribués à une diminution de la fonction de la masse insulaire.Les autogreffes d'éléments insulaires isolés avaient été préparées après pancréatectomie totale. Six chiens en reçurent la totalité et six la moitié. Le dosage des éléments suivants: glucose, lactate, pyruvate, alanine, glycérol, 3-hydroxyburate, cholestérol, acides gras libres, insuline, glucagon et cortisol a été pratiqué à jeun et après administration intraveineuse d'une quantité déterminée de glucose. La clairance du glucose (K) a été déterminée. L'euglycémie à jeun fut atteinte et les taux d'insuline furent identiques chez les deux groupes d'animaux. Des réductions identiques de la clairance du glucose avec une élévation à jeun du glycérol, du 3-hydroxybutyrate furent constatées dans les deux groupes un mois après la transplantation. Le débit insulinique stimulé par le glucose fut à la fois anormal quantitativement et qualitativement, des taux identiques étant atteints au niveau du sang périphérique dans les deux groupes. Cette réponse insulinique anormale dévoila une augmentation des taux des acides gras libres et de l'alanine lors de la surchage intraveineuse de glucose, bien que la suppression du glucagon fût normale dans les deux groupes.Ces résultats suggèrent qu'une relation simple entre la masse transplantée et la sécrétion insulinique n'existe pas. Ce fait peut être important quand on considère l'application clinique de la transplantation insulaire pour traiter le diabète.

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Presented at the International Association of Endocrine Surgeons at Hamburg, September 1983.

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Supported by the Wellcome Trust and Newcastle Health Authority (Scientific and Research Committee).     

Inhibition of hypothalamic inflammation reverses diet-induced insulin resistance in the liver

Defective liver gluconeogenesis is the main mechanism leading to fasting hyperglycemia in type 2 diabetes, and, in concert with steatosis, it is the hallmark of hepatic insulin resistance. Experimental obesity results, at least in part, from hypothalamic inflammation, which leads to leptin resistance and defective regulation of energy homeostasis. Pharmacological or genetic disruption of hypothalamic inflammation restores leptin sensitivity and reduces adiposity. Here, we evaluate the effect of a hypothalamic anti-inflammatory approach to regulating hepatic responsiveness to insulin. Obese rodents were treated by intracerebroventricular injections, with immunoneutralizing antibodies against Toll-like receptor (TLR)4 or tumor necrosis factor (TNF)α, and insulin signal transduction, hepatic steatosis, and gluconeogenesis were evaluated. The inhibition of either TLR4 or TNFα reduced hypothalamic inflammation, which was accompanied by the reduction of hypothalamic resistance to leptin and improved insulin signal transduction in the liver. This was accompanied by reduced liver steatosis and reduced hepatic expression of markers of steatosis. Furthermore, the inhibition of hypothalamic inflammation restored defective liver glucose production. All these beneficial effects were abrogated by vagotomy. Thus, the inhibition of hypothalamic inflammation in obesity results in improved hepatic insulin signal transduction, leading to reduced steatosis and reduced gluconeogenesis. All these effects are mediated by parasympathetic signals delivered by the vagus nerve.     

Recovery and hypersecretion of insulin and reversal of insulin resistance after withdrawal of short-term cyclosporine treatment.

We have previously demonstrated decreased insulin release and insulin resistance in dogs treated with cyclosporine (20 mg/kg/day). In this study we examine the changes caused by a lower CsA dose and evaluate the reversal of these changes. Six animals were treated for 2 weeks with oral CsA (15 mg/kg/day), after which CsA was discontinued. Glucagon stimulation tests (GST) and euglycemic clamp studies (ECS) were used to evaluate changes in insulin release and insulin resistance. GST were performed before CsA, after 2 weeks of CsA, and 3, 9, and 15 days after discontinuing CsA. ECS were performed before CsA, after 2 weeks of CsA, and 2, 4, 8, and 14 days after discontinuing CsA. The mean serum CsA level after 2 weeks of treatment was 188 +/- 28 ng/ml. GST demonstrated decreased insulin release during CsA with recovery and hypersecretion after CsA withdrawal. ECS showed peripheral insulin resistance during CsA with a rapid recovery and a temporary increase in insulin sensitivity after CsA withdrawal. Comparisons were made with our previous study group given 20 mg/kg/day of CsA. In summary, CsA induces a dose-dependent impairment of glucose homeostasis due to inhibition of insulin release and development of peripheral insulin resistance. Withdrawal of short-term CsA at commonly used therapeutic doses results in reversal of and temporary overcompensation for these changes. CsA withdrawal after long-term treatment results in a slower normalization of the insulin response as compared with after short-term treatment. The hypersecretory reaction of the beta cell may be of help in further investigations of mechanisms of CsA- and FK506-induced inhibition of insulin release.     

Indinavir induces acute and reversible peripheral insulin resistance in rats

The use of HIV protease inhibitors (PIs) has been associated with several metabolic changes, including lipodystrophy, hyperlipidemia, and insulin resistance. The etiology of these adverse effects remains unknown. PIs have recently been found to cause acute and reversible inhibition of GLUT4 activity in vitro. To determine the acute in vivo effects of indinavir on whole-body glucose homeostasis, glucose tolerance tests were performed on PI-na?ve Wistar rats immediately after a single intravenous dose of indinavir. Glucose and insulin levels were significantly elevated in indinavir-treated versus control rats (P < 0.05) during the initial 30 min of the glucose tolerance test. Under euglycemic- hyperinsulinemic clamp conditions, indinavir treatment acutely reduced the glucose infusion rate required to maintain euglycemia by 18 and 49% at indinavir concentrations of 14 and 27 micromol/l, respectively. Muscle 2-deoxyglucose uptake was similarly reduced under these conditions. Restoration of insulin sensitivity was observed within 4 h after stopping the indinavir infusion. Indinavir did not alter the suppression of hepatic glucose output under hyperinsulinemic conditions. These data demonstrate that indinavir causes acute and reversible changes in whole-body glucose homeostasis in rats and support the contribution of GLUT4 inhibition to the development of insulin resistance in patients treated with PIs.     

The effect of peripheral resistance on impedance cardiography measurements in the anesthetized dog

In the vasodilated and septic patient, the impedance method of measuring cardiac output (CO) may underestimate the true value. In this study, we sought to determine whether impedance CO (COIC) measurements are influenced by total peripheral resistance (TPR). In eight anesthetized and ventilated dogs, a high-precision flowprobe was placed on the ascending aorta, and direct CO was measured (CO flowprobe (COFP)). Mean arterial blood pressure was measured from the femoral artery. Simultaneous COIC measurements were made. TPR (mean arterial blood pressure x 80/COFP) was varied over 1-2 h by using infusions of phenylephrine and adrenaline and inhaled halothane. The bias between methods of CO measurement (COIC-COFP) was calculated and compared with TPR by using correlation and regression analysis. A total of 547 pairs of CO measurements were collected from the 8 dogs as TPR was varied. COFP changed by a mean of 190% (range, 89%-425%), and TPR changed by a mean of 266% (range, 94%-580%) during the experiment. The impedance method underestimated CO when TPR was low and overestimated CO when TPR was high. There was a logarithmic relationship between the CO bias and TPR. Correlation coefficients (r) between the CO bias and TPR ranged from 0.46 to 0.89 (P < 0.0001). The bias changed by 0.62 +/- 1.8 L/min, or by 34%, every time TPR halved or doubled. This finding explains the poor agreement between COIC and other methods of CO measurement found in validation studies involving critically ill patients.     

Inhibition of the accessory function of murine macrophages in vitro by cyclosporine

The accessory function of macrophages, which is strictly related to the induction of T cell activation, has been studied to determine whether it is affected by cyclosporine (CsA). Irradiated spleen cells, used as a source of macrophages, were pulsed overnight with beta-galactosidase (GZ) in the presence of CsA. After washing of the pulsed macrophages, cells from a GZ-specific T cell line were added to cultures and 3H-thymidine incorporation was measured 72 hr later. We found that 500 ng/ml CsA present during macrophage pulsing with GZ reduced T cell proliferation to 5%. On the other hand, 100 ng/ml CsA almost completely abrogated the proliferative response when present for the duration of the culture. Similar results were also obtained using antigen-pulsed peritoneal-adherent macrophages to stimulate the T cell line to proliferate, or a T hybridoma clone to produce interleukin-2 (IL-2). The possibility that CsA actually affects interleukin-1 (IL-1) production by macrophages by inhibiting uninvolved T cells could be ruled out. We conclude that CsA-induced inhibition of T cell functions (proliferation and IL-2 production) is partially due to the effect of the drug on the accessory function of macrophages. This immunosuppressive mechanism of action of CsA on macrophages has not been previously described.     

Hyperinsulinemia does not compensate for peripheral insulin resistance in obesity

Based on previous steady-state measures of the biologic activity of insulin, it was thought that postprandial hyperinsulinemia in obesity compensated for insulin resistance. However, we recently demonstrated kinetic defects in insulin action in insulin-resistant nondiabetic obese subjects: activation of insulin-stimulated glucose disposal was slower and deactivation was faster in obese than in normal subjects. In view of these kinetic defects in peripheral insulin action and of the fact that insulin is normally secreted in a phasic manner after meals, we postulated that the hyperinsulinemia of obesity does not compensate for insulin resistance and that the abnormal kinetics of insulin action in obesity are functionally important. To test this hypothesis, oral glucose tolerance tests (OGTTs) were performed in five control (mean age, 33 +/- 2 yr) and five obese (mean age, 41 +/- 5 yr) subjects. All controls had normal glucose tolerance; two obese subjects had normal and three had impaired glucose tolerance. After the results of the OGTTs were available, euglycemic clamp studies were performed in which insulin was infused in a phasic stepped fashion to mimic the rise and fall of mean peripheral insulin levels during the OGTTs. Each subject was clamped at both the "normal" and "obese" OGTT insulin profiles. During the OGTT, glucose and insulin levels were significantly elevated in the obese subjects compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of insulin release from rat pancreatic islets by drugs that are analogues of dopamine

Various synthetic dopamine (DA) analogues have been shown to produce glucose intolerance and inhibit the compensatory increase in serum insulin during an oral glucose tolerance test (OGTT). To investigate the possibility that there is a direct action of dopamine analogues to inhibit glucose-stimulated insulin release from the endocrine pancreas, the following compounds were compared with the effects of epinephrine (EPI) on isolated rat pancreatic islets: apomorphine (APO), pergolide, lergotrile, TL-99 (2-dimethylamino-6,7-dihydroxytetralin), and RDS-127 (2-di-n-propyl-amino-4,7-dimethoxyindane). EPI, TL-99, and pergolide inhibited insulin release in a concentration-dependent fashion (10(-7)-10(-5) M), whereas lergotrile inhibited at 10(-5) M but not at 10(-6) M. RDS-127 and APO were ineffective at 10(-5) M, but produced a greater than 50% inhibition at 2 X 10(-4) M. The potencies of the DA analogues fell into two groups: compounds that are approximately as active as EPI (e.g., TL-99 and pergolide) or compounds that are relatively inactive (e.g., APO, lergotrile, and RDS-127). The inhibitory actions of EPI, TL-99, and pergolide were blocked by the alpha 2-adrenergic receptor antagonist yohimbine, whereas the DA receptor antagonist, sulpiride, had no effect, suggesting an action initiated at alpha 2-adrenergic receptors. Drugs from both groups produced marked glucose intolerance and inhibited the compensatory increase in insulin during an OGTT. Adrenodemedullation blocked the glucose intolerance and inhibition of insulin release caused by RDS-127, whereas these effects of TL-99 were not attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The impact of impaired insulin release and insulin resistance on glucose intolerance after renal transplantation

The current knowledge of the pathogenesis of post-transplant glucose intolerance is sparse. This study was undertaken to assess the relative importance of insulin secretion (ISec) and insulin sensitivity (IS) in the pathogenesis of post-transplant diabetes mellitus (PTDM), impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) after renal transplantation. An oral glucose tolerance test (OGTT) was performed in 167 non-diabetic recipients 10 wk after renal transplantation. Fasting, 1-h and 2-h insulin and glucose levels were used to estimate the insulin secretory response and IS. One year after transplantation 89 patients were re-examined with an OGTT including measurements of fasting and 2 h glucose. Ten weeks after transplantation the PTDM-patients had significantly lower ISec and IS than patients with IGT/IFG, who again had lower ISec and IS than those with normal glucose tolerance (NGT). One year later, a similar difference in baseline ISec was observed between the three groups, whereas baseline IS did not differ significantly. Patients who improved their glucose tolerance during the first year, were mainly characterized by a significantly greater baseline ISec, and they received a significantly higher median prednisolone dose at baseline with a subsequent larger dose reduction during the first year, than the patients who had their glucose tolerance unchanged or worsened. In conclusion, both impaired ISec and IS characterize patients with PTDM and IGT/IFG in the early course after renal transplantation. The presence of defects in insulin release, rather than insulin action, indicates a poor prognosis regarding later normalization of glucose tolerance.     

Primacy of hepatic insulin resistance in the development of the metabolic syndrome induced by an isocaloric moderate-fat diet in the dog

Obesity is highly correlated with insulin resistance and the development of type 2 diabetes. Insulin resistance will result in a decrease in insulin's ability to stimulate glucose uptake into peripheral tissue and will suppress glucose production by the liver. However, the development of peripheral and hepatic insulin resistance relative to one another in the context of obesity-associated insulin resistance is not well understood. To examine this phenomena, we used the moderate fat-fed dog model, which has been shown to develop both subcutaneous and visceral adiposity and severe insulin resistance. Six normal dogs were fed an isocaloric diet with a modest increase in fat content for 12 weeks, and they were assessed at weeks 0, 6, and 12 for changes in insulin sensitivity and glucose turnover. By week 12 of the diet, there was a more than twofold increase in trunk adiposity as assessed by magnetic resonance imaging because of an accumulation in both subcutaneous and visceral fat depots with very little change in body weight. Fasting plasma insulin had increased by week 6 (150% of week 0) and remained increased up to week 12 of the study (170% of week 0). Surprisingly, there appeared to be no change in the rates of insulin-stimulated glucose uptake as measured by euglycemic-hyperinsulinemic clamps throughout the course of fat feeding. However, there was an increase in steady-state plasma insulin levels at weeks 6 and 12, indicating a moderate degree of peripheral insulin resistance. In contrast to the moderate defect seen in the periphery, there was a marked impairment in insulin's ability to suppress endogenous glucose production during the clamp such that by week 12 of the study, there was a complete inability of insulin to suppress glucose production. Our results indicate that a diet enriched with a moderate amount of fat results in the development of both subcutaneous and visceral adiposity, hyperinsulinemia, and a modest degree of peripheral insulin resistance. However, there is a complete inability of insulin to suppress hepatic glucose production during the clamp, suggesting that insulin resistance of the liver may be the primary defect in the development of insulin resistance associated with obesity.