AMPK is a serine-threonine kinase which regulates cellular metabolism and has an essential role in activating glucose transport during hypoxia and ischemia. The mechanisms responsible for AMPK stimulation of glucose transport are uncertain, but may involve interaction with other signaling pathways or direct effects on glucose transporter (GLUT) vesicular trafficking. One potential downstream mediator of AMPK signaling is the nitric oxide pathway.
The aim of this study was to examine the extent to which AMPK mediates glucose transport through activation of the nitric oxide signaling pathway in isolated heart muscles. Incubation with 1 mM 5-amino-4-imidazolecarboxamide ribofuranoside (AICAR) activated AMPK (P<0.01), stimulated glucose uptake (P<0.05) and translocation of the cardiomyocyte glucose transporter GLUT4 to the cell surface (P<0.05). AICAR treatment increased phosphorylation of endothelial nitric oxide synthase (eNOS) by ~1.8- fold (P<0.05). eNOS, but not nNOS, co-immunoprecipitated with both the alpha2 and alpha1 AMPK catalytic subunits in heart muscle. Nitric oxide donors also increased glucose uptake and GLUT 4 translocation (P<0.05). Inhibition of NOS with L-NAME and LNMMA, reduced AICAR-stimulated glucose uptake by 21 +/- 3% (P<0.05) and 25 +/- 4% (P<0.05) respectively. Inhibition of guanylate cyclase with ODQ and LY83583, reduced AICAR-stimulated glucose uptake by 31 +/- 4% (P<0.05) and 22 +/- 3% (P<0.05) respectively, as well as GLUT4 translocation to the cell surface (P<0.05). Taken together, these results indicate that activation of the nitric oxide/guanylate cyclase pathway contributes to, but is not the sole mediator of AMPK-stimulation of glucose uptake and GLUT4 translocation in heart muscle. UCN-01 infused for 72 h by continuous intravenous infusion induced insulin (INS) resistance during Phase-I clinical trials. To understand the mechanism for this observation, we examined the effect of UCN-01 on insulin-stimulated glucose transport activity using 3-O-methylglucose in isolated rat adipose cells. UCN-01 inhibits glucose transport activity in a dose-dependent manner at all insulin concentrations. At the clinically relevant concentration of 0.25 mM UCN-01, glucose transport is inhibited 66, 29, and 26% at insulin concentrations of 10, 50, and 100K mU/ml respectively, thus shifting the dose-response curve to the right. Increasing concentrations of UCN-01 up to 2.5 mM progressively shift the insulin dose-response curve even further. As Akt is known to mediatte in part action initiated at the insulin receptor, we also studied the effect of UCN-01 on Akt activation in whole-cell homogenates of these cells. Decreased glucose transport activity directly parallels decreased Akt Thr308 phosphorylation in both an insulin and UCN-01 dose-dependent manner, while Akt Ser473 phosphorylation is inhibited only at the lowest insulin concentration, and then only modestly. UCN-01 also inhibits insulin-induced Thr308, but not Ser473, phosphorylation of Akt associated with the plasma membranes (PM) and low-density microsomes (LDM), and inhibits translocation of GLUT4 from LDM to PM as expected from the glucose transport activity measurements. These data suggest that UCN-01 induces clinical insulin resistance by blocking Akt activation and subsequent GLUT4 translocation in response to insulin, and this effect appears to occur by inhibiting Thr308 phosphorylation even in the face of almost completely unaffected Ser473 phosphorylation. To elucidate the roles of adipose tissue and skeletal muscle in the early development of insulin resistance, we characterized gene expression profiles of isolated adipose cells and skeletal muscle of non-diabetic insulin-resistant first-degree relatives of type 2 diabetic patients using oligonucleotide microarrays. About 600 genes and expressed sequence tags, which displayed a gene expression pattern of cell proliferation, were differentiaally expressed in the adipose cells. The differentially expressed genes in the skeletal muscle were mostly related to the cellular signal transduction and transcriptional regulation. To verify the microarray findings, we studied expression of genes participating in adipogenesis. The expression of Wnt signaling genes, WNT1, FZD1, DVL1, GSK3beta, beta-catenin, and TCF1, and adipogenic transcription factors, C/EBPalpha and beta and delta, PPARgamma, and SREBP-1, was reduced in the adipose tissue. The expression of adipose-specific proteins related to terminal differentiation, such as adiponectin and aP2, was reduced both in the adipose tissue and in the adipose cells isolated from portions of the biopsies. The adipose cells were enlarged in the insulin-resistant relatives and the cell size inversely correlated with the expression of the Wnt signaling genes, adiponectin, and aP2. Our findings suggest that insulin resistance is associated with an impaired adipogenesis.
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