Cellular Mechanisms for Increased Gluconeogenesis in Type 2 Diabetes

描述（由申请人提供）： 在T2D患者中，肝再生增加是空腹高血糖的主要原因。许多人将增加的细胞生成归因于磷酸烯醇丙酮酸羧激酶（PEPCK）的转录增加。然而，我们最近的发现挑战了这一教条。我们已经证明，在两种空腹高血糖啮齿动物模型中，高血糖和葡萄糖产生增加，而PEPCK或葡萄糖6-磷酸酶表达不增加。我们将这些结果转化为人类，表明在控制不佳的T2D患者中，空腹高血糖症的发生没有增加PEPCK或G6Pc的肝脏表达。因此，另一种机制必须解释T2D中增加的血管生成。我们的初步数据现在表明，丙酮酸羧化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大鼠模型中。此外，新的人体数据显示，非糖尿病受试者中PC mRNA变体-2（PCV 2）和PC蛋白（而非PEPCK或G6 P）的肝脏表达与HbA1c密切相关（R = 0.80，P <0.001）。本补助金的具体目标中提出的研究将提供有关PC在T2D发病机制中作用的重要新信息。此外，我们将评估丙酮酸羧化酶作为一个潜在的新的治疗目标，使用特定的反义寡核苷酸（PC ASO）敲低表达的肝脏和脂肪。在目标1中，我们将确定丙酮酸羧化酶的表达和活性是否在T2D患者中增加。我们假设T2D患者的空腹高血糖与PCV 2 mRNA、PC蛋白和PC活性增加相关。我们将从血糖正常和接受择期腹部手术的T2D患者中获取肝脏样本。这将安全地提供足够量的肝组织，以评估PC表达和活性，以及其他致炎酶和潜在变构调节剂的表达/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相关。在目标2中，我们将确定丙酮酸羧化酶的敲低如何影响基础和胰岛素刺激的肝脏葡萄糖代谢。具体而言，我们将评估补偿途径，可能会被激活使用一个复杂的“三重示踪剂”同位素方法，直接定量的关键代谢物通量（如甘油和谷氨酰胺）和公正的基因表达谱。将在两种T2D模型（ZDF大鼠和过表达人胰岛淀粉样多肽的转基因大鼠（HIP大鼠））中测试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功效。在目标3中，我们将评估敲低肝脏和脂肪丙酮酸羧化酶对脂质代谢的影响。我们发现，敲低PEPCK在高脂喂养大鼠防止肥胖，但导致非酒精性脂肪肝疾病和肝脏胰岛素抵抗，类似于轻度脂肪代谢障碍。相比之下，PC ASO也可以防止肥胖，但可以减少肝脏脂肪并改善肝脏胰岛素敏感性。我们假设PC表达的降低，而不是PEPCK表达的降低，将降低肝甘油生成，从而降低肝脂质储存。我们将在用PC ASO治疗的脂肪喂养大鼠中使用复杂的同位素方法定量肝脏和脂肪甘油生成，并与PEPCK ASO进行比较，并通过LC-MS/MS测量脂质代谢物（例如酰基-CoA、二酰基甘油）的差异。我们还将定量用PEPCK ASO或PC ASO治疗的果糖喂养大鼠中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降低体内PC表达的影响。总之，该提案中包含的研究可以改变我们对T2D患者肝脏再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.