Efforts toward routine islet cell transplantation as a means for reversing type 1 diabetes have been hampered by islet availability as well as allograft rejection. In vitro transdifferentiation of mouse bone marrow (BM)-derived stem (mBMDS) cells into insulin-producing cells could provide an abundant source of autologous cells for this procedure. For this study, we isolated and characterized single cell-derived stem cell lines obtained from mouse BM. In vitro differentiation of these mBMDS cells resulted in populations meeting a number of criteria set forth to define functional insulin-producing cells. Specifically, the mBMDS cells expressed multiple genes related to pancreatic beta-cell development and function (insulin I and II, Glut2, glucose kinase, islet amyloid polypeptide, nestin, pancreatic duodenal homeobox-1 [PDX-1], and Pax6). Insulin and C-peptide production was identified by immunocytochemistry and confirmed by electron microscopy. In vitro studies involving glucose stimulation identified glucose-stimulated insulin release. Finally, these mBMDS cells transplanted into streptozotocin-induced diabetic mice imparted reversal of hyperglycemia and improved metabolic profiles in response to intraperitoneal glucose tolerance testing. These results indicate that mouse BM harbors cells capable of in vitro transdifferentiating into functional insulin-producing cells and support efforts to derive such cells in humans as a means to alleviate limitations surrounding islet cell transplantation.

The islet in type 2 diabetes is characterized by a deficit in beta-cell mass, increased beta-cell apoptosis, and impaired insulin secretion. Also, islets in type 2 diabetes often contain deposits of islet amyloid derived from islet amyloid polypeptide (IAPP), a 37-amino acid protein cosecreted with insulin by beta-cells. Several lines of evidence suggest that proteins with a capacity to develop amyloid fibrils may also form small toxic oligomers that can initiate apoptosis. The amino acid sequence of IAPP in rats and mice is identical and differs from that in humans by substitution of proline residues in the amyloidogenic sequence so that the protein no longer forms amyloid fibrils or is cytotoxic. In the present study, we report a novel rat model for type 2 diabetes: rats transgenic for human IAPP (the HIP rat). HIP rats develop diabetes between 5 and 10 months of age, characterized by an approximately 60% deficit in beta-cell mass that is due to an increased frequency of beta-cell apoptosis. HIP rats develop islet amyloid, but the extent of amyloid was not related to the frequency of beta-cell apoptosis (r = 0.10, P = 0.65), whereas the fasting blood glucose was (r = 0.77, P < 0.001). The frequency of beta-cell apoptosis was related to the frequency of beta-cell replication (r = 0.97, P < 0.001) in support of the hypothesis that replicating cells are more vulnerable to apoptosis than nondividing cells. The HIP rat provides additional evidence in support of the potential role of IAPP oligomer formation toward the increased frequency of apoptosis in type 2 diabetes, a process that appears to be compounded by glucose toxicity when hyperglycemia supervenes.

Diabetic nephropathy is the main cause of end-stage renal disease requiring dialysis in developed countries. In this study, we demonstrated the therapeutic effect of hepatocyte growth factor (HGF) on advanced rather than early diabetic nephropathy using a rat model of streptozotocin-induced diabetes. Early diabetic nephropathy (16 weeks after induction of diabetes) was characterized by albuminuria, hyperfiltration, and glomerular hypertrophy, whereas advanced diabetic nephropathy showed prominent transforming growth factor (TGF)-beta1 upregulation, mesangial expansion, and glomerulosclerosis. An SP1017-formulated human HGF (hHGF) plasmid was administered by intramuscular injection combined with electroporation over a 30-day follow-up in rats with early and advanced diabetic nephropathy. hHGF gene therapy upregulated endogenous rat HGF in the diabetic kidney (rat HGF by RT-PCR was threefold higher than in diabetic rats without therapy). hHGF gene therapy did not improve functional or morphologic abnormalities in early diabetic nephropathy. hHGF gene therapy reduced albuminuria and induced strong regression of mesangial expansion and glomerulosclerosis in advanced diabetic nephropathy. These findings were associated with suppression of renal TGF-beta1 and mesangial connective tissue growth factor (CTGF) upregulation, inhibition of renal tissue inhibitor of metalloproteinase (TIMP)-1 expression, and reduction of renal interstitial myofibroblasts. In conclusion, our results suggest that hHGF gene therapy may be considered as an innovative therapeutic strategy to treat advanced diabetic nephropathy.

There is evidence from animal models of obesity and type 2 diabetes that increased parasympathetic vagal input to the pancreas contributes to hyperinsulinemia. Compared with Caucasians, Pima Indians have a high risk of type 2 diabetes and exhibit marked hyperinsulinemia and elevated plasma levels of pancreatic polypeptide (PP), an islet hormone considered a surrogate marker of parasympathetic nervous system (PNS) drive to the pancreas. To test if hyperinsulinemia in Pima Indians is due to increased vagal input to the beta-cell, we examined the effect of PNS blockade in 17 Caucasian (aged 35 +/- 8 years, body fat 23 +/- 7% [mean +/- SD]) and 17 Pima Indian males (aged 28 +/- 8 years, body fat 29 +/- 5%) with normal glucose tolerance. Each participant underwent four consecutive standardized liquid meal tests (64% carbohydrate, 22% fat, and 14% protein) during which a primed infusion of atropine was administered for 120 min at the following doses: 0, 2.5, 5, and 10 micro g. kg fat-free mass (FFM)(-1). h(-1). Areas under the curve for early (AUC(0-30 min)) and total (AUC(0-120 min)) postprandial insulin and PP secretory responses were calculated. Early postprandial insulin and PP secretory responses were higher in Pima Indians compared with those of Caucasians (both P = 0.01). Secretion of insulin and PP was inhibited by atropine (both P < 0.001). Increasing doses of atropine attenuated the ethnic difference in PP (P = 0.01) but not in early insulin secretory responses (P = 0.6), an effect that was not due to differences in gastric emptying rate (acetaminophen test) and/or circulating glucose. Similar results were observed for total secretory responses. These results confirm that compared with Caucasians, Pima Indians have an exaggerated PNS drive to pancreatic F-cells that secrete PP. However, the hyperinsulinemia of this population does not appear to be due to increased vagal input to pancreatic beta-cells.

Evidence that group VIA cytosolic calcium-independent phospholipase A(2) (iPLA(2)beta) participates in beta-cell signal transduction includes the observations that inhibition of iPLA(2)beta with the bromoenol lactone suicide substrate suppresses glucose-stimulated insulin secretion and that overexpression of iPLA(2)beta amplifies insulin secretory responses in INS-1 insulinoma cells. Immunofluorescence analyses also reveal that iPLA(2)beta accumulates in the perinuclear region of INS-1 cells stimulated with glucose and forskolin. To characterize this phenomenon further, iPLA(2)beta was expressed as a fusion protein with enhanced green fluorescent protein (EGFP) in INS-1 cells so that movements of iPLA(2)beta are reflected by changes in the subcellular distribution of green fluorescence. Stimulation of INS-1 cells overexpressing iPLA(2)beta-EGFP induced greater insulin secretion and punctate accumulation of iPLA(2)beta-EGFP fluorescence in the perinuclear region. To determine the identity of organelles with which iPLA(2)beta might associate, colocalization of green fluorescence with fluorophores associated with specific trackers targeted to different subcellular organelles was examined. Such analyses reveal association of iPLA(2)beta-EGFP fluorescence with the ER and Golgi compartments. Arachidonate-containing plasmenylethanolamine phospholipid species are abundant in beta-cell endoplasmic reticulum (ER) and are excellent substrates for iPLA(2)beta. Arachidonic acid produced by iPLA(2)beta-catalyzed hydrolysis of their substrates induces release of Ca(2+) from ER stores-an event thought to participate in glucose-stimulated insulin secretion.

The beta-isoform of group VIA calcium-independent phospholipase A(2) (iPLA(2)beta) does not require calcium for activation, is stimulated by ATP, and is sensitive to inhibition by a bromoenol lactone suicide substrate. Several potential functions have been proposed for iPLA(2)beta. Our studies indicate that iPLA(2)beta is expressed in beta-cells and participates in glucose-stimulated insulin secretion but is not involved in membrane phospholipid remodeling. If iPLA(2)beta plays a signaling role in glucose-stimulated insulin secretion, then conditions that impair iPLA(2)beta functions might contribute to the diminished capacity of beta-cells to secrete insulin in response to glucose, which is a prominent characteristic of type 2 diabetes. Our recent studies suggest that iPLA(2)beta might also participate in beta-cell proliferation and apoptosis and that various phospholipid-derived mediators are involved in these processes. Detailed characterization of the iPLA(2)beta protein level reveals that beta-cells express multiple isoforms of the enzyme, and our studies involve the hypothesis that different isoforms have different functions.

Hormones produced by adipose tissue play a critical role in the regulation of energy intake, energy expenditure, and lipid and carbohydrate metabolism. This review will address the biology, actions, and regulation of three adipocyte hormones-leptin, acylation stimulating protein (ASP), and adiponectin-with an emphasis on the most recent literature. The main biological role of leptin appears to be adaptation to reduced energy availability rather than prevention of obesity. In addition to the well-known consequences of absolute leptin deficiency, subjects with heterozygous leptin gene mutations have low circulating leptin levels and increased body adiposity. Leptin treatment dramatically improves metabolic abnormalities (insulin resistance and hyperlipidemia) in patients with relative leptin deficiency due to lipoatrophy. Leptin production is primarily regulated by insulin-induced changes of adipocyte metabolism. Dietary fat and fructose, which do not increase insulin secretion, lead to reduced leptin production, suggesting a mechanism for high-fat/high-sugar diets to increase energy intake and weight gain. ASP increases the efficiency of triacylglycerol synthesis in adipocytes leading to enhanced postprandial lipid clearance. In mice, ASP deficiency results in reduced body fat, obesity resistance, and improved insulin sensitivity. Adiponectin production is stimulated by thiazolidinedione agonists of peroxisome proliferator-activated receptor-gamma and may contribute to increased insulin sensitivity. Adiponectin and leptin cotreatment normalizes insulin action in lipoatrophic insulin-resistant animals. These effects may be mediated by AMP kinase-induced fat oxidation, leading to reduced intramyocellular and liver triglyceride content. The production of all three hormones is influenced by nutritional status. These hormones, the pathways controlling their production, and their receptors are promising targets for managing obesity, hyperlipidemia, and insulin resistance.

IGFs are important regulators of pancreatic beta-cell development, growth, and maintenance. Mutations in the IGF genes have been found to be associated with type 2 diabetes, myocardial infarction, birth weight, and obesity. These associations could result from changes in insulin secretion. We have analyzed glucose-stimulated insulin secretion using hyperglycemic clamps in carriers of a CA repeat in the IGF-I promoter and an ApaI polymorphism in the IGF-II gene. Normal and impaired glucose-tolerant subjects (n = 237) were independently recruited from three different populations in the Netherlands and Germany to allow independent replication of associations. Both first- and second-phase insulin secretion were not significantly different between the various IGF-I or IGF-II genotypes. Remarkably, noncarriers of the IGF-I CA repeat allele had both a reduced insulin sensitivity index (ISI) and disposition index (DI), suggesting an altered balance between insulin secretion and insulin action. Other diabetes-related parameters were not significantly different for both the IGF-I and IGF-II gene variant. We conclude that gene variants in the IGF-I and IGF-II genes are not associated with detectable variations in glucose-stimulated insulin secretion in these three independent populations. Further studies are needed to examine the exact contributions of the IGF-I CA repeat alleles to variations in ISI and DI.

Recently published studies of islet cell function reveal unexpected features of glucagon-like peptide-1 (GLP-1) receptor-mediated signal transduction in the pancreatic beta-cell. Although GLP-1 is established to be a cAMP-elevating agent, these studies demonstrate that protein kinase A (PKA) is not the only cAMP-binding protein by which GLP-1 acts. Instead, an alternative cAMP signaling mechanism has been described, one in which GLP-1 activates cAMP-binding proteins designated as cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac). Two variants of Epac (Epac1 and Epac2) are expressed in beta-cells, and downregulation of Epac function diminishes stimulatory effects of GLP-1 on beta-cell Ca(2+) signaling and insulin secretion. Of particular note are new reports demonstrating that Epac couples beta-cell cAMP production to the stimulation of fast Ca(2+)-dependent exocytosis. It is also reported that Epac mediates the cAMP-dependent mobilization of Ca(2+) from intracellular Ca(2+) stores. This is a process of Ca(2+)-induced Ca(2+) release (CICR), and it generates an increase of [Ca(2+)](i) that may serve as a direct stimulus for mitochondrial ATP production and secretory granule exocytosis. This article summarizes new findings concerning GLP-1 receptor-mediated signal transduction and seeks to define the relative importance of Epac and PKA to beta-cell stimulus-secretion coupling.

Intensive therapy for type 1 diabetes results in greater weight gain than conventional therapy. Many factors may predispose to this greater weight gain, including improved glycemic control, genetic susceptibility to obesity, and hypoglycemia. To study this, relationships among family history of type 2 diabetes, frequency of severe hypoglycemia, beta-cell autoantibodies, and weight gain were examined in 1,168 subjects aged > or =18 years at baseline randomized to intensive and conventional therapy groups in the Diabetes Control and Complications Trial. With intensive therapy, subjects with a family history of type 2 diabetes had greater central weight gain and dyslipidemia characterized by higher triglyceride levels and greater cholesterol in VLDLs and intermediate-density lipoproteins compared with subjects with no family history. Neither the frequency of severe hypoglycemia nor positivity to GAD65 and insulinoma-associated protein 2 antibodies was associated with increased weight gain with either intensive or conventional therapy. These data support the hypothesis that increased weight gain with intensive therapy might be explained, in part, by genetic traits.

To assess the role of insulin receptor (IR) substrate (IRS)-2 in insulin action and resistance in the liver, immortalized neonatal hepatocyte cell lines have been generated from IRS-2(-/-), IRS-2(+/-), and wild-type mice. These cells maintained the expression of the differentiated liver markers albumin and carbamoyl phosphate synthetase, as well as bear a high number of IRs. The lack of IRS-2 did not result in enhanced IRS-1 tyrosine phosphorylation or IRS-1-associated phosphatidylinositol (PI) 3-kinase activity on insulin stimulation. Total insulin-induced PI 3-kinase activity was decreased by 50% in IRS-2(-/-) hepatocytes, but the translocation of PI-3,4,5-trisphosphate to the plasma membrane in these cells was almost completely abolished. Downstream PI 3-kinase, activation of Akt, glycogen synthase kinase (GSK)-3 (alpha and beta isoforms), Foxo1, and atypical protein kinase C were blunted in insulin-stimulated IRS-2(-/-) cells. Reconstitution of IRS-2(-/-) hepatocytes with adenoviral IRS-2 restored activation of these pathways, demonstrating that IRS-2 is essential for functional insulin signaling in hepatocytes. Insulin induced a marked glycogen synthase activity in wild-type and heterozygous primary hepatocytes; interestingly, this response was absent in IRS-2(-/-) cells but was rescued by infection with adenoviral IRS-2. Regarding gluconeogenesis, the induction of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase by dibutyryl cAMP and dexamethasone was observed in primary hepatocytes of all genotypes. However, insulin was not able to suppress gluconeogenic gene expression in primary hepatocytes lacking IRS-2, but when IRS-2 signaling was reconstituted, these cells recovered this response to insulin. Suppression of gluconeogenic gene expression in IRS-2-deficient primary hepatocytes was also restored by infection with dominant negative Delta 256Foxo1.

HIV protease inhibitors (PIs) acutely and reversibly inhibit the insulin-responsive glucose transporter Glut 4, leading to peripheral insulin resistance and impaired glucose tolerance. Minimal modeling analysis of glucose tolerance tests on PI-treated patients has revealed an impaired insulin secretory response, suggesting additional pancreatic beta-cell dysfunction. To determine whether beta-cell function is acutely affected by PIs, we assayed glucose-stimulated insulin secretion in rodent islets and the insulinoma cell line MIN6. Insulin release from MIN6 cells and rodent islets was significantly inhibited by the PI indinavir with IC(50) values of 1.1 and 2.1 micro mol/l, respectively. The uptake of 2-deoxyglucose in MIN6 cells was similarly inhibited (IC(50) of 2.0 micro mol/l), whereas glucokinase activity was unaffected at drug levels as high as 1 mmol/l. Glucose utilization was also impaired at comparable drug levels. Insulin secretogogues acting downstream of glucose transport mostly reversed the indinavir-mediated inhibition of insulin release in MIN6 cells. Intravenous infusion of indinavir during hyperglycemic clamps on rats significantly suppressed the first-phase insulin response. These data suggest that therapeutic levels of PIs are sufficient to impair glucose sensing by beta-cells. Thus, together with peripheral insulin resistance, beta-cell dysfunction likely contributes to altered glucose homeostasis associated with highly active antiretroviral therapy.

The pyruvate dehydrogenase complex (PDC) is inactivated in many tissues during starvation and diabetes to conserve three-carbon compounds for gluconeogenesis. This is achieved by an increase in the extent of PDC phosphorylation caused in part by increased pyruvate dehydrogenase kinase (PDK) activity due to increased PDK expression. This study examined whether altered pyruvate dehydrogenase phosphatase (PDP) expression also contributes to changes in the phosphorylation state of PDC during starvation and diabetes. Of the two PDP isoforms expressed in mammalian tissues, the Ca(2+)-sensitive isoform (PDP1) is highly expressed in rat heart, brain, and testis and is detectable but less abundant in rat muscle, lung, kidney, liver, and spleen. The Ca(2+)-insensitive isoform (PDP2) is abundant in rat kidney, liver, heart, and brain and is detectable in spleen and lung. Starvation and streptozotocin-induced diabetes cause decreases in PDP2 mRNA abundance, PDP2 protein amount, and PDP activity in rat heart and kidney. Refeeding and insulin treatment effectively reversed these effects of starvation and diabetes, respectively. These findings indicate that opposite changes in expression of specific PDK and PDP isoenzymes contribute to hyperphosphorylation and therefore inactivation of the PDC in heart and kidney during starvation and diabetes.

Thiazolidinediones exert electrophysiologic effects in noncardiac cells in vitro, but to date there have been no reports of effects on cardiac rhythm. We previously demonstrated that chronic pretreatment with a thiazolidinedione peroxisome proliferator-activated receptor (PPAR)-gamma activator, troglitazone, improves recovery of left ventricular (LV) function and substrate metabolism after ischemia and reperfusion, without causing arrhythmias. In this study, we determined whether similar salutary effects are achieved with acute treatment with troglitazone. Anesthetized pigs underwent 90 min of regional LV ischemia and 90 min of reperfusion. Fifteen pigs were treated with troglitazone (10 mg/kg load, 5 mg. kg(-1). h(-1) infusion i.v.) beginning 1 h before ischemia. Seven pigs received corresponding vehicle. Plasma troglitazone concentration (mean 5 microg/ml) was similar to that achieved in clinical use of this agent. Before ischemia, acute troglitazone treatment had no effect on LV function, electrocardiogram, or substrate utilization. During ischemia or reperfusion, eight pigs in the troglitazone group died of ventricular fibrillation, compared with no pigs in the vehicle group (P < 0.05). Pigs that developed ventricular fibrillation had shorter QT intervals than survivors of either group. Among survivors, neither LV function nor substrate utilization differed between groups. Acute treatment with troglitazone increases susceptibility to ventricular fibrillation during myocardial ischemia and reperfusion. Whether thiazolidinediones have proarrhythmic potential in clinical use requires further investigation.

Catch-up growth is a risk factor for later obesity, type 2 diabetes, and cardiovascular diseases. We show here that after growth arrest by semistarvation, rats refed the same amount of a low-fat diet as controls show 1) lower energy expenditure due to diminished thermogenesis that favors accelerated fat deposition or catch-up fat and 2) normal glucose tolerance but higher plasma insulin after a glucose load at a time point when their body fat and plasma free fatty acids (FFAs) have not exceeded those of controls. Isocaloric refeeding on a high-fat diet resulted in even lower energy expenditure and thermogenesis and increased fat deposition and led to even higher plasma insulin and elevated plasma glucose after a glucose load. Stepwise regression analysis showed that plasma insulin and insulin-to-glucose ratio after the glucose load are predicted by variations in efficiency of energy use (i.e., in thermogenesis) rather than by the absolute amount of body fat or plasma FFAs. These studies suggest that suppression of thermogenesis per se may have a primary role in the development of hyperinsulinemia and insulin resistance during catch-up growth and underscore a role for suppressed thermogenesis directed specifically at catch-up fat in the link between catch-up growth and chronic metabolic diseases.

The metabolic syndrome is a cluster of metabolic and inflammatory abnormalities including obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis. The fatty acid binding proteins aP2 (fatty acid binding protein [FABP]-4) and mal1 (FABP5) are closely related and both are expressed in adipocytes. Previous studies in aP2-deficient mice have indicated a significant role for aP2 in obesity-related insulin resistance, type 2 diabetes, and atherosclerosis. However, the biological functions of mal1 are not known. Here, we report the generation of mice with targeted null mutations in the mal1 gene as well as transgenic mice overexpressing mal1 from the aP2 promoter/enhancer to address the role of this FABP in metabolic regulation in the presence or absence of obesity. To address the role of the second adipocyte FABP in metabolic regulation in the presence and deficiency of obesity, absence of mal1 resulted in increased systemic insulin sensitivity in two models of obesity and insulin resistance. Adipocytes isolated from mal1-deficient mice also exhibited enhanced insulin-stimulated glucose transport capacity. In contrast, mice expressing high levels of mal1 in adipose tissue display reduced systemic insulin sensitivity. Hence, our results demonstrate that mal1 modulates adipose tissue function and contributes to systemic glucose metabolism and constitutes a potential therapeutic target in insulin resistance.

Viral antibody-free BBDR and WF rats never develop spontaneous diabetes. BBDR rats, however, develop autoimmune diabetes after perturbation of the immune system, e.g., by viral infection. We previously identified a disease-susceptibility locus in the BBDR rat, iddm4, which is associated with the development of autoimmune diabetes after treatment with polyinosinic:polycytidylic acid and an antibody that depletes ART2(+) regulatory cells. We have now developed lines of congenic WF.iddm4 rats and report that in an intercross of N5 generation WF.iddm4 rats, approximately 70% of animals either homozygous or heterozygous for the BBDR origin allele of iddm4 became hyperglycemic after treatment to induce diabetes. Fewer than 20% of rats expressing the WF origin allele of iddm4 became diabetic. Testing the progeny of various recombinant N5 WF.iddm4 congenic rats for susceptibility to diabetes suggests that iddm4 is centered on a small segment of chromosome 4 bounded by the proximal marker D4Rat135 and the distal marker D4Got51, an interval of <2.8 cM. The allele at iddm4 has 79% sensitivity and 80% specificity in prediction of diabetes in rats that are segregating for this locus. These characteristics suggest that iddm4 is one of the most powerful non-major histocompatibility complex determinants of susceptibility to autoimmune diabetes described to date.

We investigated whether a polymorphism in codons 22 and 23 of the glucocorticoid (GC) receptor gene [GAGAGG(GluArg) --> GAAAAG(GluLys)] is associated with altered GC sensitivity, anthropometric parameters, cardiovascular risk factors, and sex steroid hormones. In a subgroup of 202 healthy elderly subjects of the Rotterdam Study, we identified 18 heterozygotes (8.9%) for the 22/23EK allele (ER22/23EK carriers). In the highest age group, the number of ER22/23EK carriers was higher (67-82 years, 12.9%) than in the youngest age group (53-67 years, 4.9%; P < 0.05). Two dexamethasone (DEX) suppression tests with 1 and 0.25 mg DEX were performed, and serum cortisol and insulin concentrations were compared between ER22/23EK carriers and noncarriers. After administration of 1 mg DEX, the ER22/23EK group had higher serum cortisol concentrations (54.8 +/- 18.3 vs. 26.4 +/- 1.4 nmol/l, P < 0.0001), as well as a smaller decrease in cortisol (467.0 +/- 31.7 vs. 484.5 +/- 10.3 nmol/l, P < 0.0001). ER22/23EK carriers had lower fasting insulin concentrations (P < 0.001), homeostasis model assessment- insulin resistance (IR) (index of IR, P < 0.05), and total (P < 0.02) and LDL cholesterol concentrations (P < 0.01). Our data suggest that carriers of the 22/23EK allele are relatively more resistant to the effects of GCs with respect to the sensitivity of the adrenal feedback mechanism than noncarriers, resulting in a better metabolic health profile.

Adipocyte factors play a major role in the induction of insulin resistance in skeletal muscle. To analyze this cross-talk, we established a system of co-culture of human fat and skeletal muscle cells. Cells of three muscle donors were kept in co-culture with cells of various fat cell donors, and insulin signaling was subsequently analyzed in myocytes. Insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was completely blocked, with unaltered expression of IRS-1. Troglitazone increased insulin action on IRS-1 phosphorylation, in both the absence and presence of co-culture. Insulin-regulated activation of Akt kinase in the myocytes was significantly reduced after co-culture, with troglitazone restoring insulin action. Addition of tumor necrosis factor (TNF)-alpha (2.5 nmol/l) to myocytes for 48 h reduced IRS-1 expression and inhibited IRS-1 and Akt phosphorylation comparable to the effect of co-culture. Lower doses of TNF-alpha were ineffective. After co-culture, TNF-alpha in the culture medium was below the detection limit of 0.3 pmol/l. A very low level of resistin was detected in the supernatant of myocytes, but not of adipocytes. In conclusion, the release of fat cell factors induces insulin resistance in human skeletal muscle cells; however, TNF-alpha and resistin appear not to be involved in this process.

Adiponectin, the most abundant adipose-specific protein, has been found to be negatively associated with degree of adiposity and positively associated with insulin sensitivity in Pima Indians and other populations. Moreover, adiponectin administration to rodents has been shown to increase insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and also increase whole-body insulin sensitivity. To further characterize the relationship between plasma adiponectin concentration and insulin sensitivity in humans, we examined 1) the cross-sectional association between plasma adiponectin concentration and skeletal muscle IR tyrosine phosphorylation and 2) the prospective effect of plasma adiponectin concentration at baseline on change in insulin sensitivity. Fasting plasma adiponectin concentration, body composition (hydrodensitometry or dual energy X-ray absorptiometry), insulin sensitivity (insulin-stimulated glucose disposal, hyperinsulinemic clamp), and glucose tolerance (75-g oral glucose tolerance test) were measured in 55 Pima Indians (47 men and 8 women, aged 31 +/- 8 years, body fat 29 +/- 8% [mean +/- SD]; 50 with normal glucose tolerance, 3 with impaired glucose tolerance, and 2 with diabetes). Group 1 (19 subjects) underwent skeletal muscle biopsies for the measurement of basal and insulin-stimulated tyrosine phosphorylation of the IR (stimulated by 100 nmol/l insulin). The fold increase after insulin stimulation was calculated as the ratio between maximal and basal phosphorylation. Group 2 (38 subjects) had follow-up measurements of insulin-stimulated glucose disposal. Cross-sectionally, plasma adiponectin concentration was positively associated with insulin-stimulated glucose disposal (r = 0.58, P < 0.0001) and negatively associated with percent body fat (r = -0.62, P < 0.0001) in the whole group. In group 1 plasma adiponectin was negatively associated with the basal (r = -0.65, P = 0.003) and positively associated with the fold increase in IR tyrosine phosphorylation (r = 0.69, P = 0.001) before and after the adjustment for percent body fat (r = -0.58, P = 0.01 and r = 0.54, P = 0.02, respectively). Longitudinally, after adjustment for age, sex, and percent body fat, low plasma adiponectin concentration at baseline was associated with a decrease in insulin sensitivity (P = 0.04). In conclusion, our cross-sectional data suggest a role of physiological concentration of fasting plasma adiponectin in the regulation of skeletal muscle IR tyrosine phosphorylation. Prospectively, low plasma adiponectin concentration at baseline precedes a decrease in insulin sensitivity. Our data indicate that adiponectin plays an important role in regulation of insulin sensitivity in humans.