Cellular Mechanisms for Increased Gluconeogenesis in Type 2 Diabetes

2 型糖尿病糖异生增加的细胞机制

• 批准号: 8762400
• 负责人: VARMAN T SAMUEL
• 金额: --
• 依托单位: VA CONNECTICUT HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8762400
• 关键词: 1,2-diacylglycerol; Abdomen; Accounting; Acyl Coenzyme A; Adipose tissue; Affect; Antisense Oligonucleotides; Binding; Blindness; Blood Glucose; CREB1 gene; Data; Development; Diet; Diglycerides; Enzymes; Fasting; Fatty Liver; Fatty acid glycerol esters; Freezing; Fructose; Gene Expression Profiling; Genetic Transcription; Gluconeogenesis; Glucose; Glucose Plasma Concentration; Glutamine; Glycerol; Glycosylated hemoglobin A; Grant; Hepatic; Human; Hyperglycemia; Insulin; Insulin Resistance; Kidney Failure; Limb structure; Lipids; Lipodystrophy; Liver; Liver Glycogen; Liver diseases; Measures; Messenger RNA; Methods; Modeling; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Operative Surgical Procedures; Pathogenesis; Pathway interactions; Patients; Phosphoenolpyruvate Carboxylase; Proteins; Pyruvate Carboxylase; Rattus; Regulation; Research Personnel; Rodent Model; Role; Sampling; Techniques; Testing; Tissues; Tracer; Transgenic Organisms; Translating; Variant; Veterans; basal insulin; blood glucose regulation; diabetic rat; enzyme activity; fasting plasma glucose; feeding; global health; glucose metabolism; glucose monitor; glucose production; glucose-6-phosphatase; hepatic gluconeogenesis; human data; human subject; improved; in vivo; insulin sensitivity; islet amyloid polypeptide; lipid biosynthesis; lipid metabolism; liver biopsy; mRNA Expression; new therapeutic target; non-alcoholic fatty liver; non-diabetic; novel; overexpression; prevent; promoter; protein expression; sensor; therapeutic target; transcription factor

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.

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。许多人将糖异生作用的增加归因于磷酸烯醇丙酮酸羧激酶 (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，高血糖和葡萄糖生成增加并不增加PEPCK或葡萄糖6-磷酸酶的表达。我们将这些结果转化为人类，结果表明，在控制不佳的 T2D 患者中，空腹高血糖发生时，PEPCK 或 G6Pc 的肝脏表达并未增加。因此，必须有另一种机制来解释 T2D 中糖异生的增加。我们的初步数据现在表明丙酮酸羧化酶表达的增加可能就是这种机制。我们发现 T2D 大鼠模型中 PC 蛋白增加。此外，新的人类数据显示，PC mRNA 变体 2 (PCV2) 和 PC 蛋白（而非 PEPCK 或 G6P）的肝脏表达与非糖尿病受试者中的 HbA1c 密切相关（R=0.80，P<0.001）。该拨款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，我们将评估丙酮酸羧化酶作为潜在的新型治疗靶点，使用特定的反义寡核苷酸（PC ASO）来敲低肝脏和脂肪中的表达。在目标 1 中，我们将确定 T2D 患者中丙酮酸羧化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 PCV2 mRNA、PC 蛋白和 PC 活性的增加有关。我们将从血糖正常和接受选择性腹部手术的 T2D 患者获取肝脏样本。除了其他糖异生酶和潜在变构调节剂的表达/活性之外，这将安全地提供足够数量的肝组织来评估 PC 表达和活性。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。具体来说，我们将评估可使用复杂的“三重示踪剂”同位素方法、关键代谢物通量（例如甘油和谷氨酰胺）的直接定量以及无偏基因表达谱来激活的补偿途径。 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功效将在两种 T2D 模型（ZDF 大鼠和过度表达人胰岛淀粉样多肽的转基因大鼠（HIP 大鼠））中进行测试。在目标 3 中，我们将评估肝脏和脂肪丙酮酸羧化酶的敲低对脂质代谢的影响。我们发现，在高脂肪喂养的大鼠中敲低 PEPCK 可防止肥胖，但会导致非酒精性脂肪肝疾病和肝胰岛素抵抗，类似于轻度脂肪营养不良。相比之下，PC ASO 还可以预防肥胖，但可以减少肝脏脂肪并提高肝脏胰岛素敏感性。我们假设减少 PC 表达，而不是 PEPCK 表达，将减少肝甘油新生，从而减少肝脂质储存。我们将使用复杂的同位素方法量化经 PC ASO 处理的脂肪喂养大鼠的肝脏和脂肪甘油新生，并与 PEPCK ASO 进行比较，并通过 LC-MS/MS 测量脂质代谢物（例如酰基辅酶 A、二酰基甘油）的差异。我们还将量化用 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减少体内 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 患者肝脏糖异生分子调节的理解，并验证丙酮酸羧化酶作为 T2D 和 NAFLD 的新治疗靶点。