Obesity is one of the most common metabolic disorders and is a risk factor for several pathological conditions including type II diabetes, cardiovascular disease and hypertension. A major contributor to the obese state is a breakdown in the regulatory networks that control the balance between storage and utilization of triglycerides in adipose tissue. Adipocytes, the cells that perform this function, express a plethora of proteins that are involved in regulating glucose and lipid metabolism. Some of the proteins secreted from fat cells act at distant sites (i.e., skeletal muscle and brain) to integrate overall metabolism and regulate energy balance. Most notable among these substances are leptin and TNF-alpha. Leptin, the satiety factor, has been implicated in mechanisms that control fat utilization, while TNF-alpha appears to contribute to insulin resistance associated with obesity related type II diabetes. Expression of these proteins is induced during the differentiation of preadipocytes and is regulated by two main families of adipogenic transcription factors: C/EBPs and PPARs, from which C/EBP-alpha and PPAR-gamma are considered to synergize with one another and regulate the terminal stages of adipogenesis, i.e., insulin-dependent glucose transport and leptin production. The sequential expression and activation of the adipogenic transcription factors is also regulated by extracellular effectors that stimulate a network of intracellular signaling pathways. The goal of this proposal is to determine the role of two of these pathways, PI3-kinase and p42/p44MAP kinase, in regulating adipogenesis.
In Aim 1, we will define the role of these pathways in regulating the C/EBP-alpha and PPAR-gamma genes. Studies will involve blocking the transmission of signals through the PI3-K and p42/p44MAPK pathways using specific inhibitors of each, LY294002 and PD98059, respectively, and analyzing their effect on gene expression.
In Aim 2, we will identify the role of specific mediators of these pathways by ectopic expression of constitutively active p42MAPK/Erk2 and Akt, dominant negative Akt, or MAPK phosphatase-1 in adipogenic cells. The ability of these enzymes to modulate the expression and activity of the C/EBPs and PPAR-gamma will be assessed by Northern and Western blots, EMSA/supershifts and other measures of transcriptional activity.
In Aim 3, we will determine whether the C/EBPs and PPAR-gamma are direct targets of these signaling enzymes. This will involve modifying candidate sites of phosphorylation within these transcription factors using PCR-directed mutagenesis, followed by ectopic expression of the mutant proteins in adipogenic cells to determine how altering the phosphorylation state influences their function during adipogenesis.
