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)来刺激肌肉和脂肪细胞的葡萄糖转运和肝脏的脂肪合成。肌肉特异性敲除一个PKC-lambda等位基因(Het-Mlambda KO)会减少葡萄糖的转运，从而导致糖耐量受损、胰岛素抵抗、腹型肥胖、肝骨病和高脂血症，即代谢综合征(MS)，最终导致2型糖尿病(T2 DM)。Het-M KO小鼠的高胰岛素血症异常激活肝脏类固醇受体元件受体结合蛋白-1c(SREBP-1c)和核因子B，由此引起的脂质和细胞因子的产生增加可能导致胰岛素抵抗，这可能是最近在老年Het-Mlambda KO小鼠中观察到的常规和新型PKC(α，B2，epsilon，theta)的肝脏活动增加所加剧的，我们还产生了脂肪细胞(A)特异性的PKC-lambda KO(Alambda KO)小鼠，并且在分离的脂肪细胞中葡萄糖的运输减少。然而，与Mlambda KO小鼠不同的是，Alambda-KO小鼠身材瘦削，糖耐量正常，血清胰岛素和血脂水平正常，钳夹研究显示，由于磷酸烯醇式丙酮酸羧酸激酶(PEPCK)表达减少，肝脏对胰岛素的敏感性增加，肝脏葡萄糖输出受到抑制。我们推测，Alambda-KO脂肪细胞中葡萄糖转运的减少限制了甘油-PO4用于脂肪合成的可能性，由此导致的瘦弱可能是通过低脂血症，下调了肝脏PEPCK的表达，从而提供了“代谢保护”。肝脏特异性PKC-lambda KO(Llambda KO)还通过减少肝脏SREBP-1c和NFb的激活，以及通过减少PEPCK和葡萄糖-6-磷酸酶(G6Pase)的空腹依赖表达来保护脂质和葡萄糖的动态平衡。因此，通过腺病毒介导的激酶失活的aPKC或我们新开发的PKC-lambda抑制剂的特异性抑制肝脏aPKC，可以降低SREBP-1c/NFB的胰岛素激活和PEPCK/G6Pase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转导和胰岛素抵抗。我们还产生了完全缺失一个PKC-lambda等位基因(Het-TBlambda KO)的小鼠，正如预期的那样，肌肉和脂肪组织中的葡萄糖运输减少；然而，这些小鼠出人意料地在肌肉、脂肪和肝脏组织中对胰岛素受体和Akt以及aPKC的激活很差。尽管存在这些信号缺陷，但除了轻度糖耐量异常和高胰岛素血症外，Het-TBlambda KO小鼠不会患上MS或T2 DM。我们推测，在Het-TB KO中，肝脏PKC-lambda的部分缺失通过降低SREBP-1c、NFB、PEPCK和G6Pase的活性/表达来提供“代谢保护”。在特定的目标1中，为了确定AlambdaKO小鼠瘦身和代谢保护的机制，我们将检查：脂肪细胞中的造脂和脂解过程；导致瘦身的因素；高脂喂养期间肝脏造脂、炎症和糖异生途径的变化；以及低蛋白血症在减少肝脏糖异生中的作用。在特定的目标2中，我们将测试肝脏PKC-lambda的部分缺失通过补充肝脏aPKC而对Het-TB KO小鼠提供代谢保护的想法。在特定的目标3中，我们将检测Het-Mlambda KO小鼠组织中传统和新型PKC活性随年龄的变化，并将这些变化与这些小鼠进行性糖尿病时胰岛素信号的损害相关联。