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.

Public Health Relevance

We know that a system of enzymes called the ubiquitin-proteasome pathway influences the ability of PPARto function as the ?master switch? in forming fat cells. We are interested in understanding how this system of enzymes determines PPARprotein 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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
High Priority, Short Term Project Award (R56)
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Cellular Aspects of Diabetes and Obesity Study Section (CADO)
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Haft, Carol R
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Lsu Pennington Biomedical Research Center
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Baton Rouge
United States
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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
Carter, Lauren E; Kilroy, Gail; Gimble, Jeffrey M et al. (2012) An improved method for isolation of RNA from bone. BMC Biotechnol 12:5
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
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