Obesity is associated with development of metabolic syndrome, non-insulin dependent diabetes mellitus (NIDDM), and cardiovascular diseases such as hypertension. Adipocytes play a central role in the physiological consequences of the energy imbalance inherent to obesity. Formation of adipocytes depends on the peroxisome proliferator-activated receptor gamma (PPAR), a protein that functions as the master switch in regulating the production of other proteins needed for lipid and carbohydrate metabolism in adipocytes. PPAR is also the cellular target of a commonly prescribed class of anti-diabetic drugs, the thiazolidinediones (TZDs). These drugs alter PPAR activity and PPAR protein levels, an indication that understanding the link between PPAR activity and PPAR protein stability may offer new insights into how obesity contributes to NIDDM. PPAR stability in adipocytes is regulated by enzymes of the ubiquitin proteasome pathway, a highly selective signaling pathway that targets proteins to the proteasome by tagging the protein with multiple ubiquitin polypeptides. Our preliminary studies also show that the region of PPAR responsible for binding TZDs contains a signal for binding to ubiquitin and that a functioning ubiquitin system is necessary for TZDdependent regulation of PPAR activity in adipocytes. Using siRNA-based screening, we identified an ubiquitin ligase, Siah2 that regulates PPAR protein levels in adipocytes. Siah2 expression is increased during adipocyte formation and is abundant in adipose tissue. Moreover, Siah2 is required for insulin-stimulated glucose uptake in 3T3-L1 adipocytes, potentially linking modulation of PPAR protein stability by the ubiquitin system to regulation of insulin sensitivity. We hypothesize that Siah2-dependent regulation of PPAR protein levels is a determinant of PPAR activity and insulin signaling in adipocytes. Our goal is to provide mechanistic insight into the role of Siah2 in controlling the relationship between the ubiquitin proteasome pathway, PPAR activity, and insulin sensitivity in adipocytes.
In specific aim 1, we will use 3T3-L1 adipocytes to determine if Siah2 regulates the ubiquitin proteasome-dependent degradation of PPAR and TZDdependent activation of PPAR via direct interaction with PPAR.
In specific aim 2, we will use 3T3-L1 adipocytes and murine models to examine the role of Siah2 in adipogenesis and insulin signaling in adipocytes.
Specific aim 3 will use a murine model of obesity to focus on the effect of diet-induced obesity and insulin resistance on Siah2 expression and ubiquitin proteasome activity in adipose tissue. The proposed study will provide new insight into how PPAR activity is regulated in adipocytes. Our long-term goal is to define the mechanisms underlying the role of PPAR in glucose and lipid metabolism and to identify potential novel therapeutic targets in the treatment of obesity and type 2 diabetes through studies of ubiquitin proteasome regulation of PPAR activity and expression in adipocytes.
We know that a system of enzymes called the ubiquitin-proteasome pathway influences the ability of PPARto function as the ?master switch? in forming fat cells. We are interested in understanding how this system of enzymes determines PPARprotein stability and influences PPAR?s function in fat cells. These studies are relevant to understanding the relationship between adipocyte biology and obesity-related disorders such as diabetes and hypertension.
| Kilroy, Gail; Carter, Lauren E; Newman, Susan et al. (2015) The ubiquitin ligase Siah2 regulates obesity-induced adipose tissue inflammation. Obesity (Silver Spring) 23:2223-32 |
| Floyd, Z Elizabeth; Stephens, Jacqueline M (2012) Controlling a master switch of adipocyte development and insulin sensitivity: covalent modifications of PPAR?. Biochim Biophys Acta 1822:1090-5 |
| Carter, Lauren E; Kilroy, Gail; Gimble, Jeffrey M et al. (2012) An improved method for isolation of RNA from bone. BMC Biotechnol 12:5 |
| Kilroy, Gail E; Zhang, Xiaoying; Floyd, Z Elizabeth (2009) PPAR-gamma AF-2 domain functions as a component of a ubiquitin-dependent degradation signal. Obesity (Silver Spring) 17:665-73 |
