Regulation of metabolism and diabetes mellitus by tristetraprolin

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DESCRIPTION (provided by applicant): Diabetes mellitus (DM) is a major cause of morbidity and mortality in the developed world, but the molecular mechanisms that lead to the development of DM are not well understood. Tristetraprolin (TTP) is a tandem zinc finger protein that binds to AU-rich elements in the 3'-untranslated region (3'-UTR) of target mRNA molecules, and promotes their degradation. TTP has been extensively studied in the field of inflammation, where it has been shown to bind to and degrade tumor necrosis factor ? (TNF??) mRNA. Our recent results suggest that TTP mRNA and protein levels are reduced in diabetic murine livers, and that TTP knockdown (KD) in hepatocytes increases the mRNA levels of peroxisome proliferator-activated receptor alpha (PPAR??), a major regulator of lipid metabolism, and pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose utilization. Because TTP KO mice develop a lethal inflammatory phenotype shortly after birth due to elevated circulating levels of TNF??, we have obtained mice with simultaneous systemic knockout (KO) of TTP and TNF?? receptors (TNFR1/2) which lack TTP-dependent inflammation. My central hypothesis is that TTP mediates its effects on hepatic lipid metabolism and glucose utilization by modulating the levels of PPAR?? and PDK4, respectively, and that TTP protects against the development of DM through its metabolic effects. In Aim 1, I hypothesize that TTP KD mediates changes in lipid homeostasis through up regulation of PPAR??. I will isolate primary hepatocytes from mice with or without TTP deletion and determine the effects of TTP on lipid metabolism by measuring changes in lipid uptake, lipid content, beta- oxidation activity, and metabolic gene expression by RNA sequencing. All experiments will be repeated in the setting of TTP KO/PPAR?? KD. I will also determine whether TTP directly regulates PPAR?? by performing deletion studies of the 3'-UTR AREs within PPAR??. In Aim 2, I hypothesize that TTP KD mediates changes in glucose homeostasis through up regulation of PDK4. I will isolate primary hepatocytes from mice with or without TTP deletion and determine the effects of TTP on glucose metabolism by measuring changes in glucose uptake, glucose production, glucose oxidation, and gluconeogenic/glycolytic gene expression by RNA sequencing. All experiments will be repeated in the setting of TTP KO/PDK4 KD. I will also determine whether TTP directly regulates PDK4 by performing deletion studies of the 3'-UTR AREs within PDK4. Finally, in Aim 3, I hypothesize that TTP mediates protective effects against the development of type II diabetes mellitus. I will subject TTP/TNFR1/2 KO and TNFR1/2 KO (control) mice to high-fat diet and assess the progression of diabetes in these mice in terms of blood glucose and insulin levels. These lines of investigation promise to advance our knowledge of the role of TTP in diabetes, and may potentially lead to the development of novel diabetic therapies.