Hepatic Rictor Knockout Mouse Model and the Metabolic Syndrome

伴随着肥胖和糖尿病的平行流行，我们的医疗保健系统的负担越来越大，原因是高血糖(肾病、神经病变、视网膜病变和血管疾病)和高脂血症(动脉粥样硬化和心血管疾病)的并发症。胰岛素抵抗是肥胖、糖尿病和代谢综合征及其许多相关并发症的常见病理生理学因素。肝脏是葡萄糖和脂肪代谢的中心，胰岛素起着关键的调节作用。肝脏葡萄糖产生增加导致高血糖；而极低密度脂蛋白(VLDL)-甘油三酯(TG)产生增加导致动脉粥样硬化(TGS升高，小而密的低密度脂蛋白(LDL)和降低的高密度脂蛋白(HDL))。由此产生的高血糖和血脂异常可归因于肝脏中胰岛素作用的改变。肝脏的“选择性”胰岛素抵抗的特征是胰岛素不能抑制糖异生(FOXO1调节受损)，同时继续刺激新生脂肪生成和极低密度脂蛋白-甘油三酯(VLDL-TG)分泌(SREBP1c调节完整)。因此，选择性胰岛素抵抗是继而发生高血糖和高脂血症的潜在机制；然而，所涉及的分子缺陷尚不清楚。我假设“选择性的”胰岛素抵抗是由于AKT的部分激活，这是由于AKT上的丝氨酸473(S473)和苏氨酸308(T308)的磷酸化位点解偶联所致；S473的磷酸化丢失导致AKT无法磷酸化FOXO1并抑制关键的糖异生酶的表达，而部分激活的AKT(仅T308)足以磷酸化并抑制GSK3，导致SREBP1c激活和高甘油三酯血症。我将通过研究一个小鼠模型来验证这一假设，在这个小鼠模型中，mTORC2调节蛋白Rictor在肝细胞中被遗传缺失，导致S473磷酸化受损，而T308磷酸化完整。我将验证Rictor在肝脏中的遗传缺失、基因剂量对Rictor基因表达的特异性影响以及Rictor/mTORC2功能(AKT磷酸化)。正常血糖钳夹和示踪稀释技术将被用来量化胰岛素抑制肝脏葡萄糖产生的能力。高甘油三酯血症将通过使用泰洛沙普和放射性标记的脂质研究来评估，以量化极低密度脂蛋白-甘油三酯的产生和清除速度，以及对脂蛋白谱(极低密度脂蛋白、低密度脂蛋白、高密度脂蛋白和载脂蛋白B)的影响。肝脏脂肪变性的可能性将通过肝脏脂肪定量来评估。

Concomitant with the parallel obesity and diabetes epidemics, an increasing burden on our healthcare system is due to the complications of hyperglycemia (nephropathy, neuropathy, retinopathy and vascular disease) and hyperlipidemia (atherosclerosis and cardiovascular disease). Insulin resistance is a common contributor to the pathophysiology of obesity, diabetes and metabolic syndrome, and many of their associative complications. The liver is central to glucose and lipid metabolism and insulin plays a key regulatory role. Elevated hepatic glucose production contributes to hyperglycemia; whereas, increased very low-density lipoproteins (VLDL)- Triglyceride (TG) production contributes to atherogenic dyslipidemia (elevated TGs, small and dense low- density lipoproteins (LDL) and reduced high-density lipoproteins (HDL)). The resultant hyperglycemia and dyslipidemia are attributed to altered insulin action in the liver. "Selective" insulin resistance in liver is characterized by inability of insulin to suppress gluconeogenesis (impaired FOXO1 regulation) while continuing to stimulate de novo lipogenesis and VLDL-TG secretion (intact regulation of SREBP1c). Thus, selective insulin resistance is a potential mechanism by which hyperglycemia and hyperlipidemia can ensue; however, the molecular defect involved is not known. I hypothesize that "selective" insulin resistance is due to partial AKT activation that results from the uncoupling of Serine473 (S473) and Threonine308 (T308) phosphorylation sites on AKT; loss of S473 phosphorylation results in the failure of AKT to phosphorylate FOXO1 and suppress expression of key gluconeogenic enzymes, whereas partially activated AKT (only T308) is sufficient to phosphorylate and inhibit GSK3, leading to SREBP1c activation and hypertriglyceridemia. I will test this hypothesis by studying a mouse model in which the mTORC2 regulatory protein, rictor, has been genetically deleted in hepatocytes leading to impaired S473 phosphorylation with intact T308 phosphorylation. I will validate genetic loss of rictor in the liver, gene dosage specific effects of rictor gene expression and rictor/mTORC2 function (AKT phosphorylation). Euglycemic clamp and tracer dilution techniques will be employed to quantify the ability of insulin to suppress hepatic glucose production. Hypertriglyceridemia will be assessed by utilizing tyloxapol and radiolabeled lipid studies to quantify the rate of VLDL-TG production and clearance, and effects on the lipoprotein profile (VLDL, LDL, HDL and apoB). The possibility of hepatic steatosis will be assessed by liver lipid quantification.