CENTRAL/PERIPHERAL TARGETS FOR METABOLIC ACTIONS OF LEPTIN

这项建议的总体目标是了解瘦素快速增强胰岛素敏感性的分子机制，而不是依赖于它对食物摄入量和体重的影响。我们将在外周组织水平上研究中枢神经系统介导和直接作用的贡献。在目标1中，我们将确定胰岛素和瘦素是否共享共同的细胞内信号转导途径。静脉注射。在大鼠体内应用瘦素，我们将检验瘦素的胰岛素增敏作用涉及瘦素激活和胰岛素激活的信号转导通路之间协同作用的汇聚这一假设。我们将在体内确定胰岛素靶组织(骨骼肌、BAT、Wat肝脏)中胰岛素受体和IRSS的磷酸化以及PI3、MAPK和STAT1、STAT的激活的瘦素激活的时间进程。我们将研究瘦素和胰岛素的潜在相加作用。在目标2中，我们将检验这一假设，即瘦素早期激活信号通路的结果是直接影响靶组织的水平，但后来的影响也涉及中枢神经系统的调节。我们将在一段时间内测量静脉注射或侧脑室注射瘦素后下丘脑信号通路的激活情况。我们将确定骨骼肌交感神经切除(BT)后的效果。我们将确定脂肪细胞和肌肉中哪些信号通路在体外被激活。在目标3中，我们将检验瘦素的一些生物学作用直接在脂肪细胞水平上发挥作用的假设。我们将利用aP2启动子/增强子在脂肪中选择性表达瘦素受体的转基因db/db小鼠。我们将确定仅在脂肪细胞中恢复长型瘦素受体是否会改变脂肪细胞的生理、葡萄糖稳态或胰岛素的作用。在im4中，我们将检验特定的下丘脑核团参与瘦素的胰岛素增敏作用的假设。我们将向DMH、VMH和PVH微量注射瘦素，并通过正糖钳夹测量葡萄糖的处置和体内特定肌肉和脂肪库的葡萄糖摄取。因此，我们将绘制胰岛素敏感区。这些研究将提供关于瘦素直接和中枢神经系统介导的机制的重要信息，瘦素通过这些机制提高胰岛素的敏感性，并可能导致治疗肥胖症和糖尿病的新途径。

The overall goal of this proposal is to understand the molecular mechanisms underlying the effect of leptin to rapidly enhance insulin sensitivity independent of its effects on food intake and body weight. We will investigate the contribution of CNS-mediated and direct effects at the level of peripheral tissues. In Aim 1 we will determine whether insulin and leptin share common intracellular signal transduction pathways. With i.v. administration of leptin in rats, we will test the hypothesis that the insulin- sensitizing effects of leptin involve convergence of synergism between leptin-activated and insulin-activated signal transduction pathways. We will determine the time course for leptin activation of phosphorylation of the insulin receptor and IRSs and activation of PI3 kinase, MAPK kinase and Stat1 and Stat in insulin target tissues (skeletal muscle, BAT, WAT liver) in vivo. We will investigate potential additive effects of leptin and insulin. In Aim 2 we will test the hypothesis that early activation of signaling pathways by leptin results from direct effects the level of the target tissues but later effects involve CNS mediation as well. We will measure activation of signaling pathways in the hypothalamus after iv or ICV leptin over a time course. We will determine which effects are present after sympathectomy of skeletal muscle or BT. We will determine which signaling pathways are activated in vitro in adipocytes and muscle. In Aim 3 we will test the hypothesis that some of the biological actions of leptin are exerted directly at the level of the adipocyte. We will generate transgenic db/db mice expressing the leptin receptor selectively in fat using the aP2 promoter/enhancer. We will determine whether restoration of long form leptin receptors exclusively in adipocytes alters adipocyte physiology, glucose homeostasis, or insulin action. In im 4 we will test the hypothesis that specific hypothalamic nuclei are involved in the insulin-sensitizing effects of leptin. We will microinject leptin into the DMH, VMH and PVH and measure glucose disposal by euglycemic clamp and glucose uptake into specific muscles and adipose depots in vivo. Thus, we will map the regions of insulin sensitivity. These studies will yield important information about the direct and CNS-mediated mechanisms by which leptin enhances insulin sensitively and may lead to new avenues for treatment of obesity and diabetes.