Endocannabinoid system seems to be hyperactivated resulting in enhanced endocannabinoid levels in circulation and visceral adipose tissue in obesity. The cannabinoid receptor CB1 is expressed not only in brain but also in adipose tissue and skeletal muscle. Few studies in vitro suggest that CB1 activation leads to enhanced glucose uptake in adipocytes. However, CB1 activation effect on glucose transporter protein GLUT4 expression and possible transcriptional factors involved remain completely unknown. Moreover, CB1 actions on NF-kB and SREBP in adipocytes have not yet been described. Thus, the aim of the present project is to verify in adipocytes 3T3-L1 and in adipose tissue of glutamate monosodium induced-obese mice the cannabinoid receptor CB1 modulation of GLUT4 expression and NF-kB and SREBP possible involvement. For this, 3T3-L1 adipocytes will be treated with a selective CB1 receptor agonist (arachidonoyl 2'-chloroethylamide) and/or selective CB1 receptor antagonist (AM251) for 24 and 48 hours. For the transcriptional factors investigation, cells will be treated previously with NF-kN inhibitor (SN50), and/or SREBP inhibitor (25-hydroxycholesterol). In parallel, control and obese mice will be treated with a selective CB1 antagonist (rimonabant) for 10, 20 and 30 days to verify the best treatment period for amelioration of whole-body insulin resistance, which will be evaluated by Intravenous Insulin Tolerance Test. Epidydimal adipose tissue and treated cells will be analysed for: (1) GLUT4 gene expression, by Northern blotting, (2) GLUT4 protein expression, by Western blotting, (3) SREBP-1c gene expression, by semi-quantitative RT-PCR, and (4) NF-kB and SREBP-1c activation on GLUT4 gene promoter, by electrophoretic mobility shift assay. Thus, the present project intends to determine the role of CB1 receptor on GLUT4 expression and its contribution to insulin resistance in adipose tissue.
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