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，而不依赖于其对食物摄入和体重的影响。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Aim 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。通过在大鼠体内静脉注射瘦素，我们将验证瘦素的胰岛素增敏效应涉及瘦素激活和胰岛素激活信号转导途径之间协同作用的收敛的假设。我们将在体内测定瘦素激活胰岛素受体和IRSs磷酸化以及胰岛素靶组织（骨骼肌、BAT、WAT肝）中PI3激酶、MAPK激酶、Stat1和Stat的激活时间过程。我们将研究瘦素和胰岛素的潜在叠加效应。在Aim 2中，我们将验证瘦素信号通路的早期激活是由靶组织水平的直接影响引起的，但后期的影响也涉及中枢神经系统的调解。我们将在一段时间内测量iv或ICV瘦素后下丘脑信号通路的激活。我们将确定骨骼肌交感神经切除术或BT后存在哪些影响。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Aim 3中，我们将检验瘦素的一些生物作用直接施加于脂肪细胞水平的假设。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。我们将确定脂肪细胞中长形瘦素受体的恢复是否会改变脂肪细胞的生理、葡萄糖稳态或胰岛素的作用。在第4章中，我们将检验特定的下丘脑核参与瘦素的胰岛素增敏作用的假设。我们将微量注射瘦素到DMH， VMH和PVH中，并通过正糖钳和葡萄糖摄取到体内特定肌肉和脂肪库来测量葡萄糖的处理。因此，我们将绘制胰岛素敏感性区域。这些研究将提供关于瘦素增强胰岛素敏感性的直接和中枢神经系统介导机制的重要信息，并可能为肥胖和糖尿病的治疗开辟新的途径。

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.