Activation of Atypical PKC by Insulin and Other Agonists

胰岛素和其他激动剂对非典型 PKC 的激活

• 批准号: 7454306
• 负责人: ROBERT V FARESE
• 金额: $ 32.54万
• 依托单位: ROSKAMP INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7454306
• 关键词: 1-Phosphatidylinositol 3-Kinase; 2,4-thiazolidinedione; AICA ribonucleotide; Address; Adipocytes; Agonist; Binding; Cell membrane; Cells; Clinical; Exercise; Gene Targeting; Generations; Insulin; Insulin Resistance; Knock-out; Knockout Mice; Liver; Location; Mediating; Membrane Microdomains; Methods; Mus; Muscle; Muscle Cells; Non-Insulin-Dependent Diabetes Mellitus; Obesity; PIK3CG gene; PTK2B gene; Pathway interactions; Phosphatidic Acid; Phospholipase D; Phosphotransferases; Play; Protein Kinase; Proto-Oncogene Proteins c-akt; Recombinants; Relative (related person); Reporting; Role; Signal Transduction; Testing; Thiazolidinediones; Tissues; Transgenic Mice; Tyrosine; Viral; embryonic stem cell; glucose output; glucose transport; mutant; protein kinase C lambda; size; src Homology Region 2 Domain

DESCRIPTION (provided by applicant): Atypical PKC (aPKC) activation appears to be required for increases in glucose transport, and, most importantly, this activation is defective in obesity and type 2 diabetes. Thus, it is critical to elucidate mechanisms used by insulin, exercise and other agents to activate aPKCs. Although initial findings suggest that IRS-1 is required for insulin activation of aPKCs, thiazolidinediones (TZDs) were reported to activate aPKCs and glucose transport in 3T3/L1 adipocytes via CbI/PI3K, independently of IRS-1/2. Importantly, insulin activates CbI/PI3K to the same extent as TZDs in 3T3/L1 adipocytes, and, while this is small relative to IRS-1/PI3K, initial findings suggest that CbI/PI3K is required for activation of aPKCs and glucose transport by insulin. Accordingly, IRS-1 and Cbl may be co-required for PI3K and aPKC activation in these and other cells. To examine this possibility (a) Cbl mutants will be expressed in 3T3/L1 adipocytes and L6 myocytes to see if pYXXM motifs in Cbl are required for activation of the SH2 domain of p85/PI3K, aPKCs and glucose transport; and (b) mice and brown adipocytes in which IRS-1 or IRS-2 has been knocked out will be used to see if IRS-1/2 are required for activation of aPKCs and glucose transport in muscle and adipocytes. Concerning exercise, we have reported that exercise and AICAR activate aPKCs, AICAR uses ERK and phospholipase D (PLD) to activate aPKCs, and aPKCs are required for AICAR-stimulated glucose transport. To see if aPKCs are required for exercise-stimulated glucose transport, we will use transgenic mice that express kinase-inactive aPKCs or muscle-specific aPKC knockout mice. The see if AICAR uses PYK2 to sequentially activate ERK, PLD, aPKCs and glucose transport, we will use viral-mediated expression of mutant forms of these signaling factors. Finally, the hypothesis that insulin-stimulated glucose transport is dependent on aPKCs will be definitively tested in adipocytes and myocytes derived from embryonic stem cells in which PKC-lambda has been knocked out by recombinant methods.

描述（由申请人提供）： 非典型 PKC (aPKC) 激活似乎是葡萄糖转运增加所必需的，最重要的是，这种激活在肥胖和 2 型糖尿病中是有缺陷的。因此，阐明胰岛素、运动和其他药物激活 aPKC 的机制至关重要。尽管初步研究结果表明胰岛素激活 aPKC 需要 IRS-1，但据报道噻唑烷二酮 (TZD) 可通过 CbI/PI3K 激活 3T3/L1 脂肪细胞中的 aPKC 和葡萄糖转运，与 IRS-1/2 无关。重要的是，胰岛素激活 CbI/PI3K 的程度与 3T3/L1 脂肪细胞中的 TZD 相同，虽然相对于 IRS-1/PI3K 而言较小，但初步研究结果表明，CbI/PI3K 是激活 aPKC 和葡萄糖转运所必需的。胰岛素。因此，IRS-1和Cbl可能是这些细胞和其他细胞中PI3K和aPKC激活共同需要的。为了检查这种可能性 (a) Cbl 突变体将在 3T3/L1 脂肪细胞和 L6 肌细胞中表达，以观察 Cbl 中的 pYXXM 基序是否是激活 p85/PI3K、aPKC 和葡萄糖转运的 SH2 结构域所必需的； (b) IRS-1或IRS-2已被敲除的小鼠和棕色脂肪细胞将用于观察肌肉和脂肪细胞中aPKC的激活和葡萄糖转运是否需要IRS-1/2。关于运动，我们报道了运动和 AICAR 激活 aPKC，AICAR 使用 ERK 和磷脂酶 D (PLD) 激活 aPKC，并且 aPKC 是 AICAR 刺激的葡萄糖转运所必需的。为了了解运动刺激的葡萄糖转运是否需要 aPKC，我们将使用表达激酶失活 aPKC 的转基因小鼠或肌肉特异性 aPKC 敲除小鼠。为了看看 AICAR 是否使用 PYK2 依次激活 ERK、PLD、aPKC 和葡萄糖转运，我们将使用这些信号因子的突变形式的病毒介导表达。最后，胰岛素刺激的葡萄糖转运依赖于 aPKC 的假设将在源自胚胎干细胞的脂肪细胞和肌细胞中得到明确的测试，其中 PKC-lambda 已通过重组方法被敲除。