Free Fatty Acids and Hepatic Insulin Resistance

描述(申请人提供)：肝脏胰岛素抵抗导致内源性葡萄糖生成(EGP)增加是2型糖尿病(T2 DM)发病机制的主要因素。肥胖者特有的血浆游离脂肪酸(FFAs)水平升高已被证实会导致外周(肌肉)和肝脏胰岛素抵抗。近年来的研究大多集中在外周胰岛素抵抗方面。主要由于方法学上的问题，肝脏胰岛素抵抗尚未引起足够的重视。然而，最近已经有几种方法可以非侵入性地测量体内葡萄糖异生(GNG)和糖原分解(GL)的速率，这是EGP的两个组成部分。使用2H2O方法，我们最近在健康志愿者中表明，血浆FFAs的急性升高通过抑制GL的胰岛素抑制而导致肝脏胰岛素抵抗。在当前的应用中，我们计划扩展这些发现。在具体目标1中，我们建议评估健康受试者和轻度和重度T2 DM患者血浆FFAs急性升高对GL/EGP胰岛素抑制的影响的剂量依赖性、持续时间和可能的性别差异。这些研究将涉及在正常血糖-高胰岛素钳夹(正常对照组)或等血糖-高胰岛素钳夹(T2 DM患者)同时输注和不同时输注肝素化脂质(以不同的输注速率)以将血浆FFAs急剧升高到不同水平的情况下，测量GL、GNG和EGP的比率。在特定目标2中，我们建议评估血浆游离脂肪酸长期升高对肝脏胰岛素敏感性的影响。实验方法将是用烟酸类似物Niaspan过夜(12h)降低血浆FFAs，并在第二天早上(在高胰岛素钳夹期间)测定肥胖症患者合并轻度或重度T2 DM时GL/EGP的胰岛素抑制。在具体目标3中，我们将阐述FFA升高导致肝脏胰岛素抵抗的机制。具体地说，我们将检验FFA介导的肝脏胰岛素抵抗与二酰甘油(DAG)在肝脏内的积聚、蛋白激酶C(PKC)的激活、IRS-1/2的丝氨酸增加和酪氨酸磷酸化降低以及PI3激酶对胰岛素的反应减少有关的假说。实验方法是在高胰岛素-正常血糖夹闭期间不同时间间隔处死大鼠，并测定肝脏DAG、PKC活性及其异构体、IRS-1/2、酪氨酸磷酸化和PI3激酶活性。这些研究有望为FFA诱导的肝脏胰岛素抵抗的重要细节和机制提供急需的信息。

DESCRIPTION (provided by applicant): Hepatic insulin resistance resuIting in increased endogenous glucose production (EGP) is a major factor in the pathogenesis of type 2 diabetes (T2DM) Increased plasma levels of free fatty acids (FFAs) which are characteristically seen in obese individuals, have been established to cause peripheral (muscle) as well as hepatic insulin resistance. Most of the research efforts in recent years have focused on peripheral insulin resistance. Mainly due to methodological problems, hepatic insulin resistance has not received much attention. Recently, however, several methods have become available which allow non-invasive measurement of in vivo rates of gluconeogenesis (GNG) and glycogenolysis (GL), the two components of EGP. Using the 2H2O method, we have recently shown in healthy volunteers that acute elevations of plasma FFAs cause hepatic insulin resistance through inhibition of insulin suppression of GL. In the current application, we plan to expand these findings. In Specific Aim 1, we propose to assess dose dependency, duration and possible gender differences of the effects of acute elevation of plasma FFAs on insulin suppression of GL/EGP in healthy subjects and in patients with mild and severe T2DM. These studies will involve measurements of rates of GL, GNG and EGP during euglycemic-hyperinsulinemic clamping (in normal controls) or during isoglycemic-hyperinsulinemic clamping (in patients with T2DM) with and without simultaneous infusion of heparinized lipid (at different rates of infusion) to acutely raise plasma FFAs to different levels. In Specific Aim 2, we propose to evaluate effects of prolonged elevation of plasma FFAs on hepatic insulin sensitivity. The experimental approach will be to lower plasma FFAs overnight (12 h) with Niaspan (a nicotinic acid analog) and to measure insulin suppression of GL/EGP the next morning (during hyperinsulinemic clamping) in obese patients with mild or severe T2DM. In Specific Aim 3, we will address the mechanism by which elevated FFAs cause hepatic insulin resistance. Specifically, we will test the hypothesis that FFA mediated hepatic insulin resistance is associated with intrahepatic accumulation of diacylglycerol (DAG), activation of protein kinase C (PKC), with increased serine and decreased tyrosine phosphorylation of IRS-1/2, and a decrease in PI3 kinase responses to insulin. The experimental approach will be to sacrifice rats at various time intervals during hyperinsulinemic-euglycemic clamping performed with and without lipid/heparin infusions and determine hepatic concentrations of DAG, PKC activity and isoforms, IRS-1/2, tyrosine phosphorylation and PI3 kinase activity. These studies will hopefully provide much needed information relative to important details and mechanisms of FFA induced hepatic insulin resistance.