(251a) Effects of the Cellular Microenvironment and Enzyme Inhibitions On Adipocyte Metabolism

Obesity is the most lethal preventative disease in the United States. Obesity results from a chronic imbalance in caloric intake and expenditure. The excess calories are stored mainly as lipids (triglycerides), leading to an expansion of body fat or adipose tissue through increases in fat cell (adipocyte) size and number. One approach to controlling body fat could be to intervene in the metabolic processes of the adipose tissue that directly contribute to lipid accumulation and cell growth. This approach requires a systematic investigation of adipose tissue-specific metabolism, preferably in isolation from confounding systemic influences. We have previously assembled a three dimensional (3-D) co-culture model of the adipose tissue using type I collagen gel as the scaffolding material. This model included three cell types: human umbilical vein endothelial cells (HUVECs), 3T3-L1 preadipocytes and adipocytes derived from these preadipocytes through in vitro differentiation. This co-culture significantly enhanced lipid accumulation of adipocytes as well as proliferation and organization of endothelial cells. The goal of the present study is to determine the impact of the 3-D co-culture on the metabolism of adipocytes. To this end, we compared the metabolite and flux profiles of adipocytes treated with metabolic enzyme inhibitors in 3-D co-culture, 3-D mono-culture and 2-D mono-culture.

Results of metabolic flux analysis (MFA) showed that the co-culture with endothelial cells significantly up-regulated the adipocytes' overall metabolic activity. For all time points, normalization with respect to the glucose uptake flux substantially reduced the number of significantly different reaction fluxes, suggesting that the flux differences between the mono-culture and co-culture mostly reflected a general up-regulation of glucose metabolism. Compared to 2-D mono-culture, the 3-D mono-culture significantly increased adipocyte lipid accumulation and metabolic activity, with quantitatively larger differences between pathways in and around the TCA cycle and lipogenesis relative to other pathways. Significant differences remained even after normalizing for increased glucose uptake in the 3-D culture. There were significant increases in the normalized fluxes of FFA synthesis (17.0-fold) and TG synthesis (15.8-fold). These estimates indicate that the increase in lipid accumulation in the 3-D culture reflects a redistribution of carbon flux into lipogenesis via the TCA cycle. Taken together, the metabolite and flux profiles obtained in this study clearly demonstrated that adipocytes exhibit distinct metabolic phenotypes depending on their culture environment. Our findings underscore the need to study adipocyte metabolism in well-defined and physiologically meaningful microenvironments. Ongoing work investigates the impact of targeted metabolic enzyme inhibitions in 3-D co-culture.