Obesity is a result of a chronic imbalance between energy expen- diture and energy uptake, which results in the cumulative storage of fat in both the subcutaneous and visceral adipose depots.. The morbidities associated with obesity are well described and include dyslipidaemia, insulin resistance, type 2 diabetes (T2DM), hypertension and cardiovascular disease, all of which contribute to increased mortality. Increases in adipose tissue arise as a consequence of both hyperplasia (increase in fat cell number) and hypertrophy (increase in fat cell size). Mature adipocytes secrete adipokines such as leptin, adiponectin, resistin, PAI-1, IL-6, MCP-1 and TNF-±. The high production of some cytokines, is surely the most important component in obesity that is associated with a set of low-grade chronic inflammation, which predisposes to insulin resistance and development of type 2 diabetes mellitus and increased cardiovascular risk. This project aims to determine the effect of the saturated (palmitic acid [PA, C16: 0) and poly unsaturated (docosahexaenoic acid [DHA, C22: 6 n-3] fatty acid on the expression of adipokines in primary culture of stromal vascular derived cells extracted from subcutaneous and visceral adipose tissue. It is proposed to analyze the differential response between the two fatty pads, using mesenchymal multipotent cells present in these deposits, that will be differentiated (in vitro) into adipocytes. Therefore, subcutaneous adipose pads (inguinal) and visceral (epididymal) will be taken from Swiss mice for isolation of stromal vascular cells. These cells will be cultured in DMEM medium until confluence; differentiation will be induced by addition of the adipogenic cocktail for 48 h. After 8 days of differentiation, the adipocytes will be treated with such fatty acids, for additional 24 hours. The cells will be used for cytotoxicity studies of the fatty acids by flow cytometry (in order to determine the optimal concentrations for this model), and for adipokines and protein (inflammatory and of insulin signaling pathway) such as leptin, adiponectin, resistin, PAI-1, IL-6, MCP-1, IL-10, IL-1², TNF-±, JNK, IKK-², PKC, IRS-1, AKT, IR² and GLUT4) studies of expression by real time RT-PCR and/or enzyme immunoassay and/or western blotting.
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