Atypical PKC Knockout Models: Effect on Glucose and Lipid Homeostasis

非典型 PKC 敲除模型：对血糖和脂质稳态的影响

• 批准号: 8330406
• 负责人: ROBERT V FARESE
• 金额: --
• 依托单位: JAMES A. HALEY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8330406
• 关键词: 1-Phosphatidylinositol 3-Kinase; Accounting; Address; Adenoviruses; Adipocytes; Adipose tissue; Alleles; B-Lymphocytes; Binding Proteins; Carbohydrates; Cardiovascular Diseases; Cardiovascular system; Central obesity; Clinical; Defect; Development; Disease; Elements; Embryo; Enzymes; Fasting; Fatty acid glycerol esters; Gluconeogenesis; Glucose; Glucose Intolerance; Glycerol; Health; Hepatic; Homeostasis; Hyperglycemia; Hyperinsulinism; Hyperlipidemia; Impairment; Inflammatory; Insulin; Insulin Receptor; Insulin Resistance; Islets of Langerhans; Knock-out; Knockout Mice; Leptin; Lipids; Liver; Mediating; Medical center; Metabolic; Metabolic syndrome; Metabolism; Modeling; Mus; Muscle; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Pathogenesis; Pathway interactions; Patients; Phenotype; Phosphoenolpyruvate Carboxylase; Phosphotransferases; Population; Process; Production; Protein Kinase C Alpha; Protein Kinase C Inhibitor; Reporting; Rest; Role; Serum; Signal Transduction; Sterols; Testing; Thinness; Tissues; Veterans; adenoviral-mediated; age related; atypical protein kinase C; blood glucose regulation; cytokine; diabetic; drug development; feeding; glucose output; glucose production; glucose tolerance; glucose transport; glucose-6-phosphatase; hepatic gluconeogenesis; impaired glucose tolerance; improved; inhibitor/antagonist; insulin receptor substrate 1 protein; insulin sensitivity; insulin signaling; knockout gene; novel; prevent; protein kinase C lambda; receptor; receptor binding; recombinase

DESCRIPTION (provided by applicant): Insulin stimulates glucose transport in muscle and adipocytes, and lipid synthesis in liver by activating atypical protein kinase C (aPKC) and Akt (PKB). Muscle-specific knockout of one PKC-lambda allele (Het-Mlambda KO) diminishes glucose transport, thus causing impaired glucose tolerance, insulin resistance, abdominal obesity, hepatosteatosis, and hyperlipidemia, i.e., a "metabolic syndrome" (MS), and ultimately type 2 diabetes mellitus (T2DM). Hyperinsulinemia in Het-M KO mice inordinately activates hepatic sterol receptor element receptor binding protein-1c (SREBP-1c) and NF B, and resultant increases in lipid and cytokine production presumably contribute to insulin resistance, and this is probably abetted by recently observed increases in hepatic activities of conventional and novel PKCs (alpha,B2,epsilon,theta ) in older Het-Mlambda KO mice, We have also generated adipocyte (A)-specific PKC-lambda KO (Alambda KO) mice and glucose transport is diminished in isolated adipocytes. However, unlike Mlambda KO mice, Alambda -KO mice are lean and have normal glucose tolerance, normal serum levels of insulin and lipids, and clamp studies reveal increases in hepatic insulin sensitivity and suppression of hepatic glucose output, owing to decreased expression of phosphoenolpyruvate carboxykinase (PEPCK). We postulate that diminished glucose transport in Alambda -KO adipocytes limits glycerol-PO4 availability for fat synthesis, and resulting leanness, possibly via hypoleptinemia, downregulates hepatic PEPCK expression and thereby confers "metabolic protection". Liver-specific PKC-lambda KO (Llambda KO) also protects lipid and glucose homeostasis by diminishing activation of hepatic SREBP-1c and NF B, and by diminishing fasting-dependent expression of PEPCK and glucose-6-phosphatase (G6Pase). Thus, specific inhibition of hepatic aPKC by adenoviral-mediated expression of kinase-inactive aPKC, or our newly developed PKC-lambda inhibitors, diminishes insulin activation of SREBP-1c/NF B, and fasting expression of PEPCK/G6Pase, thereby markedly improving obesity, hyperlipidemia, hyperglycemia, insulin signaling in muscle, and insulin resistance in obese/T2DM mice. We have also generated mice with total body loss of one PKC-lambda allele (Het-TBlambda KO) and as expected glucose transport in muscle and adipose tissues is diminished; however, these mice unexpectedly have poor activation of the insulin receptor and Akt, as well as aPKC, in muscle, adipose and liver tissues. Despite these signaling defects, aside from mild glucose intolerance and hyperinsulinemia, Het-TBlambda KO mice do not develop MS or T2DM. We postulate that partial loss of hepatic PKC-lambda in Het-TB KO confers "metabolic protection" by diminishing activity/expression of SREBP-1c, NF B, PEPCK and G6Pase. In Specific Objective 1, to define mechanisms underlying leanness and metabolic protection in AlambdaKO mice, we will examine: lipogenic and lipolytic processes in adipocytes; factors that contribute to leanness; alterations in hepatic lipogenic, inflammatory and gluconeogenic pathways during high-fat feeding; and, the role of hypoleptinemia in diminishing hepatic gluconeogenesis. In Specific Objective 2, we will test the idea that the partial loss of hepatic PKC-lambda confers metabolic protection in Het-TB KO mice by replenishing hepatic aPKC. In Specific Objective 3, we will examine age-dependent alterations in activities of conventional and novel PKCs in tissues of Het-Mlambda KO mice and correlate these alterations with impairments in insulin signaling as these mice become progressively diabetic.

描述（由申请人提供）： 胰岛素通过激活非典型蛋白激酶C（aPKC）和Akt（PKB）刺激肌肉和脂肪细胞中的葡萄糖转运以及肝脏中的脂质合成。肌肉特异性敲除一个PKC-λ等位基因（Het-Mlambda KO）减少葡萄糖转运，从而引起葡萄糖耐量受损、胰岛素抵抗、腹部肥胖、脂肪肝和高脂血症，即，“代谢综合征”（MS），最终是2型糖尿病（T2 DM）。Het-M KO小鼠高胰岛素血症过度激活肝固醇受体元件受体结合蛋白-1c（SREBP-1c）和NF B，以及由此产生的脂质和细胞因子产生的增加可能有助于胰岛素抵抗，并且这可能是最近观察到的常规和新型PKC的肝脏活性增加所助长的（α、B2、ε、θ）在老年Het-Mlambda KO小鼠中，我们还产生了脂肪细胞（A）特异性PKC-λ KO（Alambda KO）小鼠，并且分离的脂肪细胞中葡萄糖转运减少。然而，与Mlambda KO小鼠不同，Alambda-KO小鼠是瘦的，具有正常的葡萄糖耐量、正常的胰岛素和脂质血清水平，并且钳夹研究揭示了由于磷酸烯醇丙酮酸羧激酶（PEPCK）的表达降低，肝胰岛素敏感性增加和肝葡萄糖输出抑制.我们假设Alambda-KO脂肪细胞中葡萄糖转运减少限制了脂肪合成的甘油-PO 4可用性，并且可能通过低肽血症导致的消瘦下调了肝脏PEPCK表达，从而赋予“代谢保护”。肝脏特异性PKC-λ KO（Llambda KO）还通过减少肝脏SREBP-1c和NF B的激活，以及减少PEPCK和葡萄糖-6-磷酸酶（G6 β）的空腹依赖性表达来保护脂质和葡萄糖稳态。因此，通过腺病毒介导的激酶失活的aPKC的表达或我们新开发的PKC-λ抑制剂特异性抑制肝aPKC，减少了SREBP-1 c/NF B的胰岛素活化和PEPCK/G6 β的空腹表达，从而显著改善肥胖/T2 DM小鼠的肥胖、高脂血症、高血糖、肌肉中的胰岛素信号传导和胰岛素抵抗。我们还产生了具有一个PKC-λ等位基因（Het-TBlambda KO）的全身损失的小鼠，并且如预期的，肌肉和脂肪组织中的葡萄糖转运减少;然而，这些小鼠在肌肉、脂肪和肝组织中出乎意料地具有胰岛素受体和Akt以及aPKC的不良活化。尽管存在这些信号传导缺陷，但除了轻度葡萄糖耐受不良和高胰岛素血症外，Het-TBlambda KO小鼠不会发生MS或T2 DM。我们假设Het-TB KO中肝PKC-λ的部分缺失通过降低SREBP-1c、NF B、PEPCK和G6 β的活性/表达而赋予“代谢保护”。在具体目标1中，为了确定AlambdaKO小鼠的瘦和代谢保护的潜在机制，我们将研究：脂肪细胞中的脂肪生成和脂解过程;促成瘦的因素;高脂喂养期间肝脏脂肪生成、炎症和脂肪生成途径的改变;以及低肽血症在减少肝脏脂肪生成中的作用。在具体目标2中，我们将测试肝脏PKC-λ的部分损失通过补充肝脏aPKC赋予Het-TB KO小鼠代谢保护的想法。在具体目标3中，我们将检查Het-Mlambda KO小鼠组织中常规和新型PKC活性的年龄依赖性变化，并将这些变化与胰岛素信号传导障碍相关，因为这些小鼠逐渐患糖尿病。