Adipose tissue plays a central role in controlling energy metabolism within the organism. Its primary function is to store triglycerides during periods of caloric excess and to mobilize these stores as free fatty acids during caloric deprivation. In modern western civilization, individuals usually consume more calories than they expend which can lead to a high incidence of obesity. In humans, obesity is one of the most common metabolic disorders and it is an independent risk factor for several pathological diseases including non-insulin-dependent diabetes mellitus (NIDDM), hypertension and cardiovascular disease. Recent studies suggest that some of these disorders may be linked to a breakdown in the regulatory mechanisms that control the expression of metabolic genes in mature adipocytes. Some progress towards an understanding of these processes has come from studies involving the identification of transcription factors which regulate adipogenesis. Most notable among these factors are members of the family of CCAAT/enhancer binding proteins (C/EBPs) and the peroxisome proliferator activated receptors (PPARs). In the case of the C/EBPs, C/EBPa is expressed in high abundance in a limited number of tissues, including fat and liver, where it plays a central role in regulating energy homeostasis. PPARg2 expression is limited to adipose tissues where it plays a central role in committing multipotential stem cells to the adipogenic lineage, as well as regulating differentiation of preadipocytes into adipocytes. The investigators have recently demonstrated that the conditional ectopic expression of C/EBPb and C/EBP( in multipotential NIH-3T3 fibroblasts induces PPARg expression which in turn stimulates adipogenesis based on the synthesis of triglycerides and their deposition into fat droplets.
The specific aims are designed to define the transcriptional events that control adipogenesis.
Aim 1 will determine the role of C/EBPb, C/EBP( and PPARg in inducing the early commitment phase of adipogenesis which involves the initiation of C/EBPa transcription.
Aim 2 will identify and characterize the regulator elements in the C/EBPa gene and the corresponding nuclear DNA binding proteins that regulate C/EBPa transcription during the differentiation of 3T3-L1 preadipocytes.
Aim 3 will determine the role of PPARg and the C/EBPs in regulating the expression of the mature, terminally differentiated adipocyte. This will involve an analysis of how these transcription factors control the expression of the insulin-dependent glucose transport system which include induction of the glucose transporter 4 (GLUT4), the insulin receptor and the insulin receptor substrate 1 (IRS1). Accordingly, mRNAs and proteins of these markers will be analyzed and the ability of NIH-3T3 cells to facilitate glucose uptake in response to insulin will be assessed. These studies should lead to insights into the molecular mechanisms regulating energy homeostasis and the defects underlying obesity and NIDDM.
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