Regulation of metabolism and diabetes mellitus by tristetraprolin

描述(申请人提供)：糖尿病(DM)是发达国家发病率和死亡率的主要原因，但导致DM发展的分子机制尚不清楚。Tristetraprolin(TTP)是一种串联锌指蛋白，能与靶基因3‘-非翻译区富含AU的元素结合，促进其降解。TTP在炎症领域已被广泛研究，已被证明与肿瘤坏死因子α(肿瘤坏死因子α)基因结合并降解。我们最近的结果表明，糖尿病小鼠肝脏中TTP基因和蛋白水平降低，肝细胞中TTP基因敲除(KD)增加了脂质代谢的主要调节因子--过氧化物酶体增殖物激活受体α(PPAR-α)和葡萄糖利用的负调节因子--丙酮酸脱氢酶-4(PDK4)的基因表达水平。由于TTP KO小鼠出生后不久由于循环中肿瘤坏死因子α水平升高而出现致死性炎症表型，我们获得了缺乏TTP依赖炎症的TTP和肿瘤坏死因子α受体(TnFR1/2)同时系统敲除(KO)的小鼠。我的中心假设是，茶多酚分别通过调节PPARα和PDK4的水平来调节其对肝脏脂肪代谢和葡萄糖利用的影响，并通过其代谢作用来预防糖尿病的发生。在目标1中，我假设TTP KD通过上调PPARα来调节脂质平衡的变化。我将从TTP缺失或不缺失的小鼠中分离出原代肝细胞，并通过RNA测序测量脂质摄取、脂质含量、β-氧化活性和代谢基因表达的变化，以确定TTP对脂质代谢的影响。所有实验将在TTP KO/PPARαKd的设置中重复进行。我还将确定TTP是否通过对PPARα中的3‘-UTRARE进行缺失研究来直接调节PPARα。在目标2中，我假设TTP KD通过上调PDK4来调节葡萄糖动态平衡的变化。我将从TTP缺失或不缺失的小鼠中分离原代肝细胞，并通过RNA测序测量葡萄糖摄取、葡萄糖产生、葡萄糖氧化和糖异生/糖酵解基因表达的变化，以确定TTP对葡萄糖代谢的影响。所有实验将在TTP KO/PDK4KD的设置下重复进行。我还将确定TTP是否通过对PDK4中的3‘-UTR ARE进行缺失研究来直接调节PDK4。最后，在目标3中，我假设TTP介导了对II型糖尿病发展的保护作用。我将让TTP/TNFR1/2 KO和TNFR1/2 KO(对照组)小鼠接受高脂饮食，并从血糖和胰岛素水平方面评估这些小鼠的糖尿病进展情况。这些研究有望促进我们对TTP在糖尿病中的作用的了解，并可能导致新的糖尿病治疗方法的开发。

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 α (TNFα) 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 (PPARα), 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 TNFα, we have obtained mice with simultaneous systemic knockout (KO) of TTP and TNFα 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 PPARα 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 PPARα. 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/PPARα KD. I will also determine whether TTP directly regulates PPARα by performing deletion studies of the 3'-UTR AREs within PPARα. 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.