No correlation of plasma cell 1 overexpression with insulin resistance in diabetic rats and 3T3-L1 adipocytes.
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Membrane glycoprotein plasma cell 1 (PC-1) has been shown to be increased in type 2 diabetes and involved in insulin resistance through inhibiting the insulin receptor tyrosine kinase, which was demonstrated using cultured breast cancer cells. However, other reports have shown contradictory results in Chinese hamster ovary cells and in vitro kinase assay. Thus, we considered it necessary to investigate the effect of PC-1 using highly insulin-sensitive cells. Here, we used two of the following approaches: 1) investigating PC-1 expression levels in insulin-responsive tissues in rat models of diabetes and 2) overexpressing PC-1 in 3T3-L1 adipocytes. We found that PC-1 was highly expressed in insulin-responsive tissues, such as liver and adipose tissue, in normal rats. However, high-fat feeding or streptozotocin-induced diabetes did not change its expression levels in liver, adipose tissue, and skeletal muscle. Thus, PC-1 expression levels were not associated with high-fat-diet-induced insulin resistance or hyperglycemia. Although PC-1 was increased in adipose tissue in Zucker fatty rats (protein level, by 50%; mRNA level, by 90%), its expression levels in liver and skeletal muscle, tissues that are more responsible for whole body glucose metabolism than adipose tissue, did not significantly differ from those in normal rats. Next, we overexpressed PC-1 in 3T3-L1 adipocytes using an adenovirus transfection system. PC-1 expression was markedly increased to a level 16-fold greater than that in normal human adipose tissue, which is higher than the previously reported levels in diabetic patients. However, insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1, activation of phosphatidylinositol 3-kinase, and glucose uptake were not affected by PC-1 overexpression. These results strongly suggest that increased PC-1 expression is not causally related to insulin resistance. (+info)
Enhancement by adenosine of insulin-induced activation of phosphoinositide 3-kinase and protein kinase B in rat adipocytes.
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The role of adenosine receptor in regulation of insulin-induced activation of phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B was studied in isolated rat adipocytes. Rat adipocytes are known to spontaneously release adenosine, which in turn binds and stimulates the adenosine A1 receptors on the cells. In the present study, we observed that degradation of this adenosine by adenosine deaminase attenuated markedly the insulin-induced accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), a product of PI 3-kinase. p-Aminophenylacetyl xanthine amine congener (PAPA-XAC), an inhibitor of the adenosine A1 receptor, also inhibited the insulin-induced PtdIns(3,4,5)P3 accumulation. When extracellular adenosine was inactivated by adenosine deaminase, phenylisopropyladenosine, an adenosine A1 receptor agonist, potentiated the insulin-induced accumulation of PtdIns(3,4,5)P3. Insulin-induced activation of protein kinase B, the activity of which is controlled by the lipid products of PI 3-kinase, was also potentiated by adenosine. Prostaglandin E2, another activator of a pertussis toxin-sensitive GTP-binding protein in these cells, potentiated the insulin actions. Thus, the receptors coupling to the GTP-binding protein were found to positively regulate the production of PtdIns(3,4,5)P3, a putative second messenger for insulin actions, in physiological target cells of insulin. (+info)
An adipogenic cofactor bound by the differentiation domain of PPARgamma.
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Ligand activation of the nuclear receptor PPARgamma induces adipogenesis and increases insulin sensitivity, while activation of other PPAR isoforms (-alpha and -delta) induces little or no fat cell differentiation. Expression and activation of chimeras formed between PPARgamma and PPARdelta in fibroblasts has allowed us to localize a major domain of PPARgamma responsible for adipogenesis to the N-terminal 138 amino acids, a region with AF-1 transcriptional activity. Using this region of PPARgamma as bait, we have used a yeast two-hybrid screen to clone a novel protein, termed PGC-2, containing a partial SCAN domain. PGC-2 binds to and increases the transcriptional activity of PPARgamma but does not interact with other PPARs or most other nuclear receptors. Ectopic expression of PGC-2 in preadipocytes containing endogenous PPARgamma causes a dramatic increase in fat cell differentiation at both the morphological and molecular levels. These results suggest that interactions between PGC-2, a receptor isoform-selective cofactor and PPARgamma contribute to the adipogenic action of this receptor. (+info)
Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes.
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Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4. We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4. Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4. In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation. These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation. (+info)
Transcriptional regulation by the gamma5 subunit of a heterotrimeric G protein during adipogenesis.
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The adipocyte enhancer-binding protein (AEBP1) is a novel transcriptional repressor with carboxypeptidase activity. A two-hybrid screen was conducted to identify components of AEBP1 that might be important in regulating its activity. The gamma5 subunit of a heterotrimeric G protein was shown to bind specifically to AEBP1 and to attenuate its transcriptional repression activity. Adipogenic stimulation selectively decreased the Ggamma5 level and enhanced the transcriptional repression activity of AEBP1 during mitotic clonal expansion at the onset of adipogenesis. Thus, the actions of Ggamma5 and AEBP1 are directly linked, which could provide the basis for the regulation of transcription at the onset of differentiation. This report shows that a signal-transducing molecule is involved, by direct protein-protein interaction, in the regulation of transcription during adipogenesis. (+info)
AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes.
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Adenosine 5'-monophosphate-activated protein kinase (AMPK) now appears to be a metabolic master switch, phosphorylating key target proteins that control flux through metabolic pathways of hepatic ketogenesis, cholesterol synthesis, lipogenesis, and triglyceride synthesis, adipocyte lipolysis, and skeletal muscle fatty acid oxidation. Recent evidence also implicates AMPK as being responsible for mediating the stimulation of glucose uptake induced by muscle contraction. In addition, the secretion of insulin by insulin secreting (INS-1) cells in culture is modulated by AMPK activation. The net effect of AMPK activation is stimulation of hepatic fatty acid oxidation and ketogenesis, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipolysis and lipogenesis, stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulation of insulin secretion by pancreatic beta-cells. In skeletal muscle, AMPK is activated by contraction. Type 2 diabetes mellitus is likely to be a disease of numerous etiologies. However, defects or disuse (due to a sedentary lifestyle) of the AMPK signaling system would be predicted to result in many of the metabolic perturbations observed in Type 2 diabetes mellitus. Increased recruitment of the AMPK signaling system, either by exercise or pharmaceutical activators, may be effective in correcting insulin resistance in patients with forms of impaired glucose tolerance and Type 2 diabetes resulting from defects in the insulin signaling cascade. (+info)
Differential regulation of functional responses by beta-adrenergic receptor subtypes in brown adipocytes.
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Brown adipose tissue contains both beta(1)- and beta(3)-adrenergic receptors (beta-ARs), and whereas both receptor subtypes can activate adenylyl cyclase, recent studies suggest that these subtypes have different pharmacological properties and may serve different signaling functions. In this study, primary brown adipocyte cultures were used to determine the role of beta-AR subtypes in mediating lipolysis and uncoupling protein-1 (UCP1) gene expression, elicited by the physiological neurohormone norepinephrine (NE). NE increased both lipolysis and UCP1 mRNA levels in brown adipocyte cultures; the beta(1)-receptor-selective antagonist CGP-20712A strongly antagonized the increase in UCP1 gene expression but had little effect on lipolysis. The beta(3)-receptor-selective agonist CL-316243 (CL) also increased lipolysis and UCP1 mRNA levels, yet CL was more potent in stimulating lipolysis than UCP1 gene expression. NE also increased the phosphorylation of cAMP response element-binding protein (CREB) and perilipin (PL), both of which are protein kinase A substrates that are differentially targeted to the nucleus and lipid droplets, respectively. beta(1)-receptor blockade inhibited NE-stimulated phosphorylation of CREB but not PL. The results suggest that beta-AR subtypes regulate different physiological responses stimulated by NE in brown adipocyte cultures in part by differentially transducing signals to subcellular compartments. (+info)
Effect of diet on fat cell size and hormone-sensitive lipase activity.
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This study was designed to examine the relationship between diet-induced insulin resistance/hyperinsulinemia, fat cell hypertrophy, and hormone-sensitive lipase (HSL) to elucidate whether an attenuated HSL activity leads to obesity. Female Fischer 344 rats were fed either a low-fat, complex-carbohydrate diet or a high-fat, refined-sugar (HFS) diet for 2 wk, 2 mo, or 6 mo. Adipose tissue morphology and HSL activity as well as plasma free fatty acid and glycerol levels were determined at these times. No differences between groups were seen after 2 wk except the previously reported hyperinsulinemia in the HFS animals. At both 2 and 6 mo, the HFS animals demonstrated adipocyte hypertrophy. Basal and stimulated HSL activities and plasma glycerol were significantly elevated in the HFS group. There was a positive correlation between adipocyte size and HSL activity for both basal and stimulated states. These results demonstrate that an attenuated HSL activity is not observed with the onset of insulin resistance/hyperinsulinemia and therefore does not play a role in the development of obesity. (+info)