Cellular Mechanisms for Increased Gluconeogenesis in Type 2 Diabetes

描述(由申请人提供)： 在T2D患者中，肝脏糖异生增加是空腹高血糖的主要原因。许多人将糖异生的增加归因于磷酸烯醇式丙酮酸羧酸激酶(PEPCK)转录的增加。然而，我们最近的发现挑战了这一教条。我们已经证明，在两种空腹高血糖的啮齿动物模型中，高血糖和葡萄糖产生增加而不增加PEPCK或葡萄糖6-磷酸酶的表达。我们将这些结果翻译到人类身上，结果显示，在T2D控制不佳的患者中，空腹高血糖发生时，肝脏中PEPCK或G6Pc的表达没有增加。因此，另一种机制必须解释T2D糖异生增加的原因。我们的初步数据现在表明，丙酮酸羧基酶的表达增加可能是这种机制。我们发现PC蛋白的增加发生在T2D的大鼠模型中。此外，新的人类数据显示，在非糖尿病受试者中，肝脏中PC mRNA变体-2(PCV2)和PC蛋白的表达与HbA1c密切相关(R=0.8，P&lt；0.001)，而与PEPCK或G6P无关。这项资助的具体目标中提出的研究将为PC在T2D发病机制中的作用提供重要的新信息。此外，我们将使用特定的反义寡核苷酸(PC ASO)来下调肝脏和脂肪中的表达，以评估丙酮酸羧化酶作为潜在的新的治疗靶点。在目标1中，我们将确定T2D患者丙酮酸羧基酶的表达和活性是否增加。我们假设T2D患者的空腹高血糖与PCV2mRNA、PC蛋白和PC活性的增加有关。我们将从血糖正常的患者和接受择期腹部手术的T2D患者身上获取肝脏样本。这将安全地提供足够数量的肝组织来评估PC的表达和活性，以及其他糖异生酶和潜在的变构修饰物的表达/活性。我们将这些酶的表达和活性与空腹血糖浓度、餐前血糖、平均血糖浓度(使用连续血糖监测)和HbA1c联系起来。在目标2中，我们将确定如何敲除丙酮酸羧化酶对基础和胰岛素刺激的肝脏葡萄糖代谢的影响。具体地说，我们将使用复杂的“三重示踪”同位素方法、关键代谢物流量的直接量化(例如甘油和谷氨酰胺)和无偏见的基因表达谱来评估可能被激活的代偿途径。PC ASO在降低内源性葡萄糖产生和改善胰岛素敏感性方面的有效性将在两种T2D模型中进行测试，即ZDF大鼠和高表达人胰岛淀粉样多肽的转基因大鼠(HIP大鼠)。在目标3中，我们将评估肝脏和脂肪丙酮酸羧化酶基因敲除对脂质代谢的影响。我们发现，在高脂饮食的大鼠中，敲除PEPCK可以预防肥胖，但会导致非酒精性脂肪性肝病和肝脏胰岛素抵抗，类似于轻度脂肪营养不良。相比之下，PC ASO还可以预防肥胖，但可以降低肝脏脂肪和改善肝脏的胰岛素敏感性。我们假设，减少PC的表达，而不是PEPCK的表达，会减少肝脏甘油的生成，从而减少肝脏的脂肪储存。我们将使用精密的同位素方法对服用PC ASO和PEPCK ASO的脂肪喂养大鼠的肝脏和脂肪甘油生成进行量化，并通过LC-MS/MS测量脂代谢产物(如酰辅酶A和二酰甘油)的差异。我们还将量化服用PEPCK ASO或PC ASO处理的果糖喂养大鼠的从头脂肪生成的变化，并评估NAFLD和肝脏胰岛素抵抗的变化。这些研究将首次量化体内PC表达减少的影响。综上所述，这项建议中包含的研究可以改变我们对T2D患者肝脏糖异生的分子调控的理解，并验证丙酮酸羧化酶作为T2D和NAFLD的新治疗靶点。

DESCRIPTION (provided by applicant): In patients with T2D, increased hepatic gluconeogenesis is the main cause of fasting hyperglycemia. Many ascribed increased gluconeogenesis to increased transcription of phosphoenolpyruvate carboxykinase (PEPCK). However, our recent findings challenge this dogma. We have shown that hyperglycemia and increased glucose production develop without increased expression of PEPCK or glucose 6-phosphatase in two rodent models with fasting hyperglycemia. We translated these results to humans, showing that in patients with poorly controlled T2D, fasting hyperglycemia developed without increased hepatic expression of PEPCK or G6Pc. Thus, another mechanism must account for increased gluconeogenesis in T2D. Our Preliminary Data now suggests that increased expression of pyruvate carboxylase may be this mechanism. We found that increases in PC protein occur in a rat model of T2D. Moreover, new human data shows that hepatic expression of PC mRNA variant-2 (PCV2) and PC protein, but not PEPCK or G6P, is tightly associated with HbA1c in non-diabetic subjects(R=0.80, P<0.001). The studies proposed in the Specific Aims of this grant will provide important new information on the role of PC in the pathogenesis of T2D. In addition, we will assess pyruvate carboxylase as a potential novel therapeutic target using a specific antisense oligonucleotide (PC ASO) to knockdown expression in liver and fat. In Aim 1, we will determine whether pyruvate carboxylase expression and activity is increased in patients with T2D. We hypothesize that fasting hyperglycemia in patients with T2D will be associated with increases in PCV2 mRNA, PC protein and PC activity. We will obtain liver samples from normoglycemic and patients with T2D undergoing elective abdominal surgery. This will safely provide a sufficient quantity of liver tissue to assess PC expression and activity, in addition to the expression/activity of other gluconeogenic enzymes and potential allosteric modifiers. We will relate the expression and activity of these enzymes to the fasting blood glucose concentration, and also pre-prandial glucose, mean blood glucose concentrations (using continuous glucose monitoring) and HbA1c. In Aim 2, we will determine how knockdown of pyruvate carboxylase affects basal and insulin stimulated hepatic glucose metabolism. Specifically, we will assess the compensatory pathways that may be activated using a sophisticated "triple-tracer" isotopic approach, direct quantification of key metabolite fluxes (e.g. glycerol and glutamine) and unbiased gene expression profiling. The efficacy of PC ASO in lowering endogenous glucose production and improving insulin sensitivity will be tested in two models of T2D, the ZDF rat and a transgenic rat overexpressing human islet amyloid polypeptide (HIP rat). In Aim 3, we will assess the effects of knockdown of hepatic and adipose pyruvate carboxylase on lipid metabolism. We show that knockdown of PEPCK in high-fat fed rats protects against adiposity but leads to nonalcoholic fatty liver disease and hepatic insulin resistance, akin to a mild lipodystrophy. In contrast, PC ASO also protects against adiposity but decreased liver fat and improved hepatic insulin sensitivity. We hypothesize that decreasing PC expression, but not PEPCK expression, will decrease hepatic glyceroneogenesis and, thus hepatic lipid storage. We will quantify hepatic and adipose glyceroneogenesis using sophisticated isotopic methods in fat-fed rats treated with PC ASO in comparison to PEPCK ASO and measuring differences in lipid metabolites (e.g. acyl-CoA's, diacylglycerol) by LC-MS/MS. We will also quantify changes in de novo lipogenesis in fructose-fed rats treated with either PEPCK ASO or PC ASO and assess the changes in NAFLD and hepatic insulin resistance. These studies will the first to quantify the effects of decreasing PC expression in vivo. In summary, the studies contained within this proposal could transform our understanding of the molecular regulation of hepatic gluconeogenesis in patients with T2D and validate pyruvate carboxylase as novel therapeutic target for both T2D and NAFLD.