HORMONAL SIGNALING AND PREADIPOCYTE DIFFERENTIATION

描述：肥胖是最常见的代谢紊乱之一，是一种 几种病理情况的危险因素，包括II型糖尿病， 心血管疾病和高血压。导致肥胖状态的主要因素 是控制平衡的监管网络的崩溃 脂肪组织中甘油三酯的储存和利用。脂肪细胞，即 执行这一功能的细胞，表达大量的蛋白质，这些蛋白质是 参与调节糖脂代谢。一些蛋白质 脂肪细胞分泌的物质作用于远处的部位(即骨骼肌和大脑) 统筹整体代谢，调节能量平衡。最值得注意的是 这些物质是瘦素和肿瘤坏死因子-α。瘦素，饱腹感的因素，已经被 与控制脂肪利用的机制有关，而肿瘤坏死因子-α的出现 导致与肥胖相关的II型胰岛素抵抗 糖尿病。这些蛋白的表达是在分化过程中诱导的 前体脂肪细胞，受两个主要的成脂转录家族调控 因素：C/EBPS和PPAR，从中考虑C/EBP-α和PPAR-伽马 相互协同，调控脂肪生成的末期， 即胰岛素依赖的葡萄糖转运和瘦素的产生。顺序号 成脂转录因子的表达和激活也是 由刺激细胞内网络的细胞外效应器调节 信号通路。这项提案的目标是确定两个人的角色 在这些通路中，PI3-激酶和p42/p44MAP激酶在调节 成脂作用。在目标1中，我们将定义这些通路在调控中的作用 C/EBP-α和PPAR-γ基因。研究将涉及阻止 通过PI3-K和p42/p44MAPK通路的信号传递 LY294002和PD98059各自的特异性抑制剂，并分析 它们对基因表达的影响。在目标2中，我们将确定 这些途径的特异性介体通过构件性异位表达 活化的p42MAPK/ERK2和Akt，显性阴性Akt，或MAPK磷酸酶-1 成脂细胞。这些酶调节表达和表达的能力 C/EBPS和PPAR-γ的活性将由Northern和Western评估 印迹、EMSA/超移位和其他转录活性的测量。在AIM 3，我们将确定C/EBPS和PPAR-伽马是否是 这些信号酶。这将涉及修改以下候选站点 用聚合酶链式反应指导这些转录因子的磷酸化 诱变，然后异位表达突变蛋白。 成脂细胞确定磷酸化状态的改变如何影响 它们在脂肪形成过程中的作用。

DESCRIPTION: Obesity is one of the most common metabolic disorders and is a risk factor for several pathological conditions including type II diabetes, cardiovascular disease and hypertension. A major contributor to the obese state is a breakdown in the regulatory networks that control the balance between storage and utilization of triglycerides in adipose tissue. Adipocytes, the cells that perform this function, express a plethora of proteins that are involved in regulating glucose and lipid metabolism. Some of the proteins secreted from fat cells act at distant sites (i.e., skeletal muscle and brain) to integrate overall metabolism and regulate energy balance. Most notable among these substances are leptin and TNF-alpha. Leptin, the satiety factor, has been implicated in mechanisms that control fat utilization, while TNF-alpha appears to contribute to insulin resistance associated with obesity related type II diabetes. Expression of these proteins is induced during the differentiation of preadipocytes and is regulated by two main families of adipogenic transcription factors: C/EBPs and PPARs, from which C/EBP-alpha and PPAR-gamma are considered to synergize with one another and regulate the terminal stages of adipogenesis, i.e., insulin-dependent glucose transport and leptin production. The sequential expression and activation of the adipogenic transcription factors is also regulated by extracellular effectors that stimulate a network of intracellular signaling pathways. The goal of this proposal is to determine the role of two of these pathways, PI3-kinase and p42/p44MAP kinase, in regulating adipogenesis. In Aim 1, we will define the role of these pathways in regulating the C/EBP-alpha and PPAR-gamma genes. Studies will involve blocking the transmission of signals through the PI3-K and p42/p44MAPK pathways using specific inhibitors of each, LY294002 and PD98059, respectively, and analyzing their effect on gene expression. In Aim 2, we will identify the role of specific mediators of these pathways by ectopic expression of constitutively active p42MAPK/Erk2 and Akt, dominant negative Akt, or MAPK phosphatase-1 in adipogenic cells. The ability of these enzymes to modulate the expression and activity of the C/EBPs and PPAR-gamma will be assessed by Northern and Western blots, EMSA/supershifts and other measures of transcriptional activity. In Aim 3, we will determine whether the C/EBPs and PPAR-gamma are direct targets of these signaling enzymes. This will involve modifying candidate sites of phosphorylation within these transcription factors using PCR-directed mutagenesis, followed by ectopic expression of the mutant proteins in adipogenic cells to determine how altering the phosphorylation state influences their function during adipogenesis.