In vitro model of adipose tissue metabolism

To improve the physiological relevance of current in vitro adipose tissue models, it is imperative to develop co-culture systems that include the different cell types of the adipose tissue.;The first part of this thesis investigated the growth and differentiation of 3T3-L1 adipocytes and human umbilical vein endothelial cells (HUVEC) in three-dimensional (3D) co-culture with collagen gel as the extracellular matrix. The co-culture improved the growth and differentiation of both the adipocytes and endothelial cells. These effects were abolished by treating the co-culture with a blocking antibody for the vascular endothelial cell growth factor receptor 2 (VEGFR2). These results demonstrated that the synergistic effects of the co-culture depended on paracrine interactions mediated by secretion of VEGF from the adipocytes and activation of the VEGFR2 pathway in the endothelial cells.;The second part of this thesis applied metabolic flux analysis to characterize the metabolism of adipocytes in 3D co-culture and mono-culture. The co-culture with endothelial cells significantly up-regulated the metabolic activity of the adipocytes compared to the mono-culture. The up-regulation was broadly distributed across most of the major metabolic pathways. Even without the endothelial cells, the 3D setting significantly increased the metabolic activity as determined from a comparison with two dimensional (2D) mono-culture. In addition to generally enhancing metabolism, the 3D culture also redistributed the fluxes, favoring lipogenesis over glycolysis. Taken together, these data demonstrated that the cellular microenvironment significantly affects the metabolism of adipocytes.;The third part of this thesis developed a gradient generating microfluidic system for long-term culture and adipogenic differentiation of preadipocytes. An adipogenic cocktail gradient was used to establish a spatially defined co-culture of adipocytes and preadipocytes. Transient application of the cocktail gradient, followed by basal medium treatment showed a biphasic induction of differentiation. The two phases of differentiation correlated with a spatial gradient in adipocyte size. This finding provided the first in vitro data supporting the size-dependent release of preadipocyte differentiation factors by enlarged adipocytes.;In conclusion, this thesis developed a powerful set of new in vitro adipose tissue models to enable controlled studies on the paracrine interactions involved in the expansion of the adipose tissue.