Molecular Basis of a New Form of Hyperinsulinism

描述(由申请人提供)：这是一项为期5年的新的资金申请，用于研究儿童先天性高胰岛素血症(HI)新的遗传形式的分子基础。近年来，高胰岛素/高氨血症综合征的发病机制主要集中在谷氨酸脱氢酶功能突变的显性获得。本研究的目的是探讨线粒体脂肪酸氧化酶短链3-羟基酰辅酶A脱氢酶(SCHAD)的隐性功能缺失所致缺氧缺血性脑病的发病机制。我们的假设是(1)Schad缺乏症的胰岛素分泌失调是由一种特定的脂肪酸代谢物(S)积聚引起的；(2)与其他HI疾病不同的是，Hi障碍通过质膜去极化作用于KATPKATP型“触发”胰岛素释放途径，而Schad缺乏症则作用于“触发”机制下游的一个“放大”部位(S)。这些假说将使用小鼠Schad-/-基因敲除模型进行检验。目的1将确定Schad-/-小鼠在体内对禁食和葡萄糖负荷的反应，以及这些反应是否被高脂饮食改变。目的2将利用来自Schad-/-小鼠和其他HI小鼠模型的灌流分离胰岛，确定在存在和不存在短、中、长链脂肪酸的情况下，胰岛素对营养和格列本脲刺激的反应的异常。线粒体脂肪酸氧化的其他步骤的抑制剂将被用来测试是否对Schad底物有特定的影响。AIM 3将使用质谱仪测定分离的Schad-/-胰岛中脂肪酸代谢物的分布，以测量酰基-COAs、酰基-肉毒碱和游离脂肪酸。目的4研究在存在和不存在脂肪酸的情况下，Schad缺乏对胰岛细胞内钙和线粒体对营养刺激的能量反应的影响。脂肪酸被认为在调节胰腺细胞的胰岛素分泌方面起着重要作用，然而，其作用机制却知之甚少。Schad缺乏症提供了一个独特的自然实验，不仅可以阐明脂肪酸如何干扰患有这种疾病的儿童的胰岛素分泌，而且还可以阐明这些重要的营养物质如何有助于控制正常和糖尿病人类的胰岛素分泌。

DESCRIPTION (provided by applicant): This is a request for 5 years of renewed funding to study the molecular basis of novel genetic forms of congenital hyperinsulinism (HI) in children. Previous years focused on the mechanisms of the hyperinsulinism / hyperammonemia syndrome associated with dominant, gain of function mutations of glutamate dehydrogenase (GDH). The goal of this proposal is to determine the mechanisms of HI caused by recessive, loss of function mutations of the mitochondrial fatty acid ¿-oxidation enzyme, short-chain 3-hydroxy acyl-CoA dehydrogenase (SCHAD). Our hypotheses are (1) that the dysregulation of insulin secretion in SCHAD deficiency is caused by an accumulation of a specific fatty acid metabolite(s); and (2) that, unlike other HI disorders which act on the KATP "triggering" pathway of insulin release via plasma membrane depolarization, SCHAD deficiency acts on an "amplification" site(s) downstream of the "triggering" mechanism. These hypotheses will be examined using a mouse SCHAD-/- knockout model. Aim 1 will determine the responses of SCHAD-/- mice to fasting and glucose loading in vivo and whether these responses are altered by a high fat diet. Aim 2 will define the abnormalities in insulin responses to nutrient and glyburide stimulation in the presence and absence of short, medium, and long-chain fatty acids using perifused isolated islets from SCHAD-/- mice and other HI mouse models. Inhibitors of other steps in mitochondrial fatty acid oxidation will be used to test whether effects are specific to SCHAD substrates. Aim 3 will determine the profiles of fatty acid metabolites in isolated SCHAD-/- islets using mass spectrometry to measure acyl-CoAs, acyl-carnitines, and free fatty acids. Aim 4 will determine the effects of SCHAD deficiency on islet cytosolic calcium and mitochondrial energy responses to nutrient stimuli in the presence and absence of fatty acids. Fatty acids are considered to play important roles in regulating insulin secretion by pancreatic ¿-cells, however, their mechanisms of action are poorly understood. SCHAD deficiency provides a unique "experiment of nature" for elucidating not only how fatty acids disturb insulin secretion in children affected with this disorder, but also how these important nutrients contribute to the control of insulin secretion in normal and diabetic humans.