Free Fatty Acids and Hepatic Insulin Resistance

描述（由申请人提供）：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（T2DM）发病的主要因素。血浆游离脂肪酸（FFAs）水平升高是肥胖个体的特征，已被证实可引起外周（肌肉）和肝脏胰岛素抵抗。近年来的研究主要集中于外周胰岛素抵抗。由于研究方法的问题，肝脏胰岛素抵抗并没有得到足够的重视。然而，最近已经有几种方法可以用于无创测量体内糖异生（GNG）和糖原溶解（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发现血浆FFAs的急性升高通过抑制胰岛素对GL的抑制而引起肝脏胰岛素抵抗。在目前的应用中，我们计划扩大这些发现。在具体目标1中，我们建议评估健康受试者和轻度和重度T2DM患者血浆游离脂肪酸急性升高对胰岛素抑制GL/EGP影响的剂量依赖性、持续时间和可能的性别差异。这些研究将包括测量血糖、GNG和EGP在正常对照（正常对照组）或在血糖、高胰岛素夹紧（T2DM患者）期间的血糖、GNG和EGP的比率，同时和不同时输注肝素化脂质（以不同的输注速率），以急性提高血浆FFAs到不同的水平。在特异性目标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。实验方法将是使用Niaspan（一种烟酸类似物）降低血浆ffa过夜（12小时），并在第二天早上（高胰岛素夹紧期间）测量轻度或重度T2DM肥胖患者的GL/EGP胰岛素抑制。在Specific Aim 3中，我们将探讨FFAs升高引起肝脏胰岛素抵抗的机制。具体来说，我们将验证FFA介导的肝脏胰岛素抵抗与肝内二酰基甘油（DAG）的积累、蛋白激酶C （PKC）的激活、IRS-1/2的丝氨酸磷酸化增加和酪氨酸磷酸化减少以及PI3激酶对胰岛素的反应减少有关的假设。实验方法将是在不同的时间间隔内牺牲大鼠进行高胰岛素-正血糖钳夹，有或没有脂质/肝素输注，并测定肝脏DAG浓度，PKC活性和同工型，IRS-1/2，酪氨酸磷酸化和PI3激酶活性。这些研究有望为FFA诱导肝脏胰岛素抵抗的重要细节和机制提供更多所需的信息。

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.