Cellular mechanisms for increased gluconeogenesis in type 2 diabetes mellitus: the role of lipid induced pyruvate carboxylase acetylation in increasing hepatic gluconeogenic capacity.

2型糖尿病中糖异生增加的细胞机制：脂质诱导的丙酮酸羧化酶乙酰化在增加肝糖异生能力中的作用。

• 批准号: 9240860
• 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 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9240860
• 关键词: Accounting; Acetyl Coenzyme A; Acetyl-CoA Carboxylase; Acetylation; Acetyltransferase; Adipose tissue; Affect; Antisense Oligonucleotides; Biopsy Specimen; Blindness; Cell Culture Techniques; Cell Line; Chronic; Coenzyme A; Data; Deacetylase; Development; Diabetes Mellitus; Enzymes; Fasting; Fatty Acids; Fatty Liver; Fatty acid glycerol esters; Funding; Genetic Transcription; Gluconeogenesis; Glucose-6-Phosphate; Glycosylated hemoglobin A; Hepatic; Hepatocyte; Human; Hyperglycemia; Hyperlipidemia; In Vitro; Insulin Resistance; Kidney Failure; Limb structure; Link; Lipase; Lipids; Lipolysis; Liver; Lysine; Messenger RNA; Metabolic; Metabolic Diseases; Metabolic syndrome; Mitochondria; Mitochondrial Matrix; Modeling; Morbidity - disease rate; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Pathway interactions; Patients; Phosphoenolpyruvate Carboxylase; Process; Proteins; Pyruvate Carboxylase; Rattus; Regulation; Research Personnel; Rodent; Rodent Model; Role; Serum; Site; Source; Steatohepatitis; Transaminases; Translating; Ubiquitination; Veterans; Weight Gain; Work; bariatric surgery; cohort; controlled release; diabetes mellitus therapy; diabetic; diabetic patient; experimental study; fasting glucose; fatty acid oxidation; glucose production; glucose-6-phosphatase; hepatic gluconeogenesis; improved; in vivo; inhibitor/antagonist; ketogenic diet; knock-down; lipid metabolism; liver biopsy; mRNA Expression; mortality; mutant; nonalcoholic steatohepatitis; novel; novel therapeutics; oxidation; oxidative damage; prevent; protein expression

Type 2 diabetes (T2D) affects 1 in 6 veterans and is the leading cause of blindness, renal failure and non-traumatic loss of limb. Increased hepatic gluconeogenesis is the main cause of fasting hyperglycemia and contributes to postprandial hyperglycemia. Many attribute the increase in gluconeogenesis to increased transcription of phosphoenolpyruvate carboxykinase and glucose 6- phosphate. However, previous studies by this lab demonstrated that hyperglycemia develops in humans with T2D and rodents with hyperglycemia without increases in PEPCK mRNA or protein expression. Knockdown of PEPCK does not affect fasting glucose concentration or rates of glucose production. In short, PEPCK expression does not appreciably impact hepatic gluconeogenesis. In searching for alternate explanations accounting for the increases in gluconeogenesis, hepatic pyruvate carboxylase (PC) protein was observed to closely relate with HbA1c in humans (R=0.80, P<0.01). Hepatic PC protein content is also increased in chronically fat-fed rodents. Similar increases in PC protein expression are seen with prolonged fasting and ketogenic diets, conditions with increased β- oxidation. These changes occurred without changes in PC mRNA. Moreover, decreasing PC expression in a variety of rodent models demonstrated that PC expression, unlike PEPCK expression, regulates glucose production. Decreasing PC expression also improved multiple metabolic insults associated with overfeeding, including weight gain, hepatic steatosis, insulin resistance and hyperlipidemia. Preliminary data suggest that this increase in PC protein content is associated with an increase in lysine acetylation of PC with a reciprocal decrease in PC ubiquitination. The overarching hypothesis for these studies is that increased acetyl CoA promotes lysine acetylation of PC which decreases ubiquitination of PC leading to an increase in protein content and increases the gluconeogenic capacity of the liver. The studies described in this proposal will explore the underlying mechanisms for, and metabolic impact of, increased hepatic PC protein. The studies in Aim 1 will establish the mechanism of lysine acetylation of PC. Specifically, whether fatty acids are the source of the acetyl group attached to PC and whether increasing or decreasing lipid oxidation leads to similar changes in lysine acetylation and PC protein content using a variety of in vitro, in vivo rodent studies where we manipulate cellular lipolysis and acetyl CoA concentrations. In Aim 2, experiments will assess whether lysine acetylation occurs via a non-enzymatic process, identify the specific lysine sites are acetylated with HFF, determine the impact of specific sites on protein activity and stability using lysine mutants (using KQ mutants) and determine whether PC ubiquitination occurs within the mitochondria and impact protein stability. Finally, in Aim 3, we will translate these findings to humans. We will use human cell culture to establish whether lipolysis and lipid oxidation regulated PC lysine acetylation and ubiquitination and identify the specific lysine residues that are involved. Moreover, we will assess hepatic PC lysine acetylation and protein content in a cohort of normoglycemic and diabetic patients undergoing bariatric surgery. This will allow us to establish the extent to which PC KAc and protein content regulate glycemia, insulin resistance and possibly even steatohepatitis. Together these studies will establish the mechanisms underlying the increases in PC protein content and establish how the increases in the gluconeogenic capacity of the liver and potentially contribute to the development of the metabolic syndrome and T2D.

2型糖尿病（T2 D）影响六分之一的退伍军人，是失明，肾衰竭和糖尿病的主要原因。 非创伤性肢体缺失肝硬化是禁食的主要原因 高血糖症并导致餐后高血糖症。许多人认为， 磷酸烯醇式丙酮酸羧激酶和葡萄糖6- 磷酸盐然而，该实验室以前的研究表明，高血糖症的发展， 患有T2 D的人类和患有高血糖症但PEPCK mRNA或蛋白质未增加的啮齿类动物 表情敲低PEPCK不影响空腹血糖浓度或血糖速率 生产简而言之，PEPCK表达不会明显影响肝脏的新生。在 寻找替代解释，解释肝内异生、肝丙酮酸 羧化酶（PC）蛋白与HbA 1c密切相关（R=0.80，P<0.01）。 肝脏PC蛋白含量也增加，在长期脂肪喂养的啮齿动物。PC的类似增长 蛋白质表达与长期禁食和生酮饮食有关，β- 氧化这些变化在PC mRNA没有变化的情况下发生。此外，降低PC 在多种啮齿动物模型中的表达表明，与PEPCK表达不同， 调节葡萄糖的产生。降低PC表达也改善了多种代谢损伤 与过度喂养有关，包括体重增加，肝脏脂肪变性，胰岛素抵抗和 高脂血症初步数据表明，PC蛋白含量的增加与 PC的赖氨酸乙酰化增加，而PC泛素化相应减少。总体 这些研究的假设是，增加的乙酰辅酶A促进PC的赖氨酸乙酰化， 降低PC的泛素化，导致蛋白质含量增加，并增加 肝脏的致炎能力。本提案中描述的研究将探讨潜在的 肝PC蛋白增加的机制和代谢影响。目标1中的研究将 建立了PC赖氨酸乙酰化的反应机理。具体来说，脂肪酸是否是 连接到PC上的乙酰基以及增加或减少脂质氧化是否会导致类似的 使用各种体外、体内啮齿动物研究的赖氨酸乙酰化和PC蛋白含量变化 在那里我们操纵细胞脂解和乙酰辅酶A浓度。在目标2中，实验将 评估赖氨酸乙酰化是否通过非酶促过程发生，确定特定的赖氨酸位点 用HFF乙酰化，使用 赖氨酸突变体（使用KQ突变体），并确定PC泛素化是否发生在 线粒体和影响蛋白质稳定性。最后，在目标3中，我们将把这些发现转化为人类。 我们将使用人类细胞培养，以确定是否脂解和脂质氧化调节PC赖氨酸 乙酰化和泛素化，并确定所涉及的特定赖氨酸残基。而且我们 将评估血糖正常和糖尿病患者队列中的肝PC赖氨酸乙酰化和蛋白质含量 接受减肥手术的患者。这将使我们能够确定PC KAc和 蛋白质含量调节胰岛素抵抗，甚至脂肪性肝炎。综合这些 研究将建立PC蛋白含量增加的机制，并确定如何 肝脏致炎能力的增加，并可能有助于发展 代谢综合征和T2 D的风险。