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，肝糖异生的增加是禁食性高血糖的主要原因。许多归因于增加糖异生的糖异生，以增加磷酸烯醇丙酮酸羧基酶（PEPCK）的转录。但是，我们最近的发现挑战了这一教条。我们已经表明，在两个啮齿动物模型中，高血糖和葡萄糖产生增加而没有增加PEPCK或葡萄糖6-磷酸酶的表达，而空腹高血糖。我们将这些结果转化为人类，表明在控制不良的T2D，禁食高血糖的患者中而没有增加PEPCK或G6PC的肝表达。因此，另一种机制必须解释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，新的人类数据表明，在非糖尿病患者中，PC mRNA变异2（PCV2）和PC蛋白（PCV2）和PC蛋白的肝表达与HBA1C密切相关（r = 0.80，p <0.001）。该赠款的具体目的提出的研究将提供有关PC在T2D发病机理中作用的重要新信息。此外，我们将使用特定的反义寡核苷酸（PC ASO）评估丙酮酸羧化酶作为潜在的新型治疗靶标，以敲低肝脏和脂肪的表达。在AIM 1中，我们将确定T2D患者的丙酮酸羧化酶表达和活性是否增加。我们假设T2D患者的禁食性高血症与PCV2 mRNA，PC蛋白和PC活性的增加有关。我们将从正常血糖和T2D患者接受腹部手术的患者中获取肝样品。除了其他糖原酶的表达/活性和潜在的变构修饰剂的表达/活性外，这还将安全提供足够数量的肝组织来评估PC的表达和活性。我们将将这些酶的表达和活性与空腹血糖浓度以及餐前葡萄糖，平均血糖浓度（使用连续葡萄糖监测）和HBA1C联系起来。在AIM 2中，我们将确定丙酮酸羧化酶的敲低如何影响基底和胰岛素刺激肝葡萄糖代谢。具体而言，我们将评估可以使用复杂的“三重跟踪”同位素方法，直接量化关键代谢物通量（例如甘油和谷氨酰胺）以及无偏基因表达谱分析的代码途径。 PC ASO在降低内源性葡萄糖产生和提高胰岛素敏感性方面的功效将在T2D，ZDF大鼠和过表达的人类胰岛多肽（HIP大鼠）的两种模型中测试。在AIM 3中，我们将评估肝和丙酮酸羧化酶对脂质代谢的敲低的影响。我们表明，高脂喂养大鼠中PEPCK的敲低可防止肥胖，但导致非酒精性脂肪肝疾病和肝胰岛素抵抗，类似于轻度的脂肪营养不良。相比之下，PC ASO还可以防止肥胖，但减少了肝脏脂肪并提高了肝胰岛素敏感性。我们假设降低PC表达（而不是PEPCK表达）会降低肝甘油发生，从而减少肝脂质储存。与PEPCK ASO相比，使用PC ASO处理的脂肪喂养大鼠中的复杂同位素方法和测量脂质代谢产物（例如酰基-COA，DIACYL-GLYCEROL）通过LC-MS/MS进行测量，我们将使用PC ASO处理的脂肪喂养大鼠中的复杂同位素方法来量化肝和脂肪甘油生成。我们还将量化用PEPCK ASO或PC ASO处理的果糖喂养大鼠中从头脂肪生成的变化，并评估NAFLD和肝胰岛素抵抗的变化。这些研究将首先量化降低体内PC表达的影响。总而言之，该提案中包含的研究可能会改变我们对T2D患者肝糖异生的分子调节并验证丙酮酸羧化酶作为T2D和NAFLD的新型治疗靶标。