Background: Cavin-1 expression is critical for the formation of caveolae, exceptionally abundant structure in adipocytes. Results: Adipocytes from cavin-1-null mice are markedly dysfunctional exhibiting decreased insulin-dependent glucose uptake, fatty acid uptake, and lipolysis. Conclusion: Cavin-1/caveolae have major roles in the regulation of adipocyte metabolism. Significance: Fat cell cavin-1/caveolae functions are important for organismal lipid metabolism in mice and men.

Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin secretion from pancreatic ␤ cells, yet does not directly stimulate secretion. The mechanisms underlying this phenomenon are incompletely understood. Here, we report that GLP-1 augments glucose-dependent rises in NAD(P)H autofluorescence in both ␤TC3 insulinoma cells and islets in a manner consistent with post-translational activation of glucokinase (GCK). GLP-1 treatment increased GCK activity and enhanced GCK S-nitrosylation in ␤TC3 cells. A 2-fold increase in S-nitrosylated GCK was also observed in mouse islets. Furthermore, GLP-1 activated a FRET-based GCK reporter in living cells. Activation of this reporter was sensitive to inhibition of nitricoxide synthase (NOS), and incorporating the S-nitrosylationblocking V367M mutation into this sensor prevented activation by GLP-1. GLP-1 potentiation of the glucose-dependent increase in islet NAD(P)H autofluorescence was also sensitive to a NOS inhibitor, whereas NOS inhibition did not affect the response to glucose alone. Expression of the GCK(V367M) mutant also blocked GLP-1 potentiation of the NAD(P)H response to glucose in ␤TC3 cells, but did not significantly affect metabolism of glucose in the absence of GLP-1. Co-expression of WT or mutant GCK proteins with a sensor for insulin secretory granule fusion also revealed that blockade of post-translational GCK S-nitrosylation diminished the effects of GLP-1 on granule exocytosis by ϳ40% in ␤TC3 cells. These results suggest that post-translational activation of GCK is an important mechanism for mediating the insulinotropic effects of GLP-1. Glucagon-like peptide 1 (GLP-1)2 can potentiate glucosestimulated insulin secretion at glucose concentrations that are normally subthreshold, in the 3-5 mM range, but not at glucose concentrations less than that (1-3). In pancreatic ␤ cells, the glucose threshold for insulin secretion is strongly controlled by glucose metabolism and principally limited at the first metabolic step, which is conversion of glucose to glucose 6-phosphate (4). Glucokinase (GCK) activity largely controls the rate of secretion at this step in metabolism because of its weak glucose binding affinity. Half-maximal GCK activity occurs at ϳ8 mM (4, 5), and sufficient glucose metabolism to initiate secretion is observed at ϳ5 mM (6, 7). Therefore, changes in GCK activity are a likely control point for changes in threshold.Secretion at glucose levels that are normally subthreshold is possible if glucose-phosphorylating capacity is artificially modulated through overexpression of exogenous hexokinases (8) or GCK itself (9). Mathematical modeling of the effect of naturally occurring GCK mutations on secretion has also shown a tight relationship between GCK activity and the threshold for insulin secretion (10). Furthermore, mutations that enhance GCK activity reduce the glucose threshold for insulin secretion (11). Thus, there are similarities between the effects of enhancing GCK activity and the effects of incretin hormones on insulin secreti...

Aim: To investigate the effect of the peroxisome proliferator-activator receptor (PPAR)-γ agonist, pioglitazone, on insulin resistance in low-dose streptozotocin and high sucrose-fat diet induced obese rats. Methods: Normal female Wistar rats were injected intraperitoneally with low-dose streptozotocin (STZ, 30 mg/kg) and fed with a high sucrose-fat diet for 8 weeks. Pioglitazone (20 mg/kg) was administered orally to the obese and insulin-resistant rats for 28 d. Intraperitoneal glucose tolerance tests, insulin tolerance tests and gluconeogenesis tests were carried out over the last 14 d. At the end of d 28 of the treatment, serums were collected for biochemical analysis. Glucose transporter 4 (GLUT4) and insulin receptor substrate-1 (IRS-1) protein expression in the liver and skeletal muscle were detected using Western blotting. Results: Significant insulin resistance and obesity were observed in low-dose STZ and high sucrose-fat diet induced obese rats. Pioglitazone (20 mg/kg) treatment significantly decreased serum insulin, triglyceride and free fatty acid levels, and elevated high density lipoprotein-cholesterol (HDL-C) levels. Pioglitazone also lowered the lipid contents in the liver and muscles of rats undergoing treatment. Gluconeogenesis was inhibited and insulin sensitivity was improved markedly. The IRS-1 protein contents in the liver and skeletal muscles and the GLUT4 contents in skeletal muscle were elevated significantly. Conclusion: The data suggest that treatment with pioglitazone improves insulin sensitivity in low-dose STZ and high sucrose-fat diet induced obese rats. The insulin sensitizing effect may be associated with ameliorating lipid metabolism, reducing hyperinsulinemia, inhibiting gluconeogenesis, and increasing IRS-1 and GLUT4 protein expression in insulin-sensitive tissues.

Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic beta-cell; however, the function of this molecular mechanism in regulating the physiology of insulin secretion is not well understood. To more fully understand the function of posttranslational regulation of GCK, we examined two naturally occurring GCK mutations that map to residues proximal to the S-nitrosylated cysteine and cause mild fasting hyperglycemia (maturity-onset diabetes of the young; subtype glucokinase). The kinetics of recombinantly generated GCK-R369P and GCK-V367M were assessed in vitro. The GCK-R369P protein has greatly reduced catalytic activity (relative activity index 0.05 vs. 1.00 for wild type), whereas the GCK-V367M has near normal kinetics (relative activity index 1.26 vs. 1.00 for wild type). Quantitative imaging and biochemical assays were used to assess the effect of these mutants on the metabolic response to glucose, GCK activation, and S-nitrosylation of GCK in betaTC3 insulinoma cells. Expression of either mutant in betaTC3 cells did not affect the metabolic response to 5 mM glucose. However, expression of either mutant blocked the effects of insulin on glucose-stimulated nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate reduction, suggesting defects in posttranslational regulation of GCK. Each of these mutations blocked GCK activation, and prevented posttranslational cysteine S-nitrosylation. Our findings link defects in hormone-regulated GCK S-nitrosylation to hyperglycemia and support a role for posttranslational regulation of GCK S-nitrosylation as a vital regulatory mechanism for glucose-stimulated insulin secretion.

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