Molecular Basis of a New Form of Hyperinsulinism

描述（由申请人提供）：这是一项为期5年的更新资金申请，用于研究儿童先天性高胰岛素血症（HI）新遗传形式的分子基础。近年来，人们对谷氨酸脱氢酶（GDH）功能显性获得性突变引起的高胰岛素血症/高氨血症综合征的发病机制进行了研究。本提案的目的是确定由线粒体脂肪酸氧化酶短链3-羟基酰基辅酶A脱氢酶（SCHAD）的隐性、功能缺失突变引起的HI机制。我们的假设是：（1）SCHAD缺乏症中胰岛素分泌失调是由特定脂肪酸代谢物的积累引起的;（2）与其他通过质膜去极化作用于KATP“触发”胰岛素释放途径的HI疾病不同，SCHAD缺乏症作用于“触发”机制下游的“放大”位点。将使用小鼠SCHAD-/-敲除模型来检验这些假设。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将使用来自SCHAD-/-小鼠和其他HI小鼠模型的灌流分离胰岛，在存在和不存在短链、中链和长链脂肪酸的情况下，定义胰岛素对营养素和格列本脲刺激的反应异常。线粒体脂肪酸氧化中其他步骤的抑制剂将用于测试效应是否对SCHAD底物具有特异性。目的3将使用质谱法测定酰基辅酶A、酰基肉毒碱和游离脂肪酸，以确定分离的SCHAD-/-胰岛中脂肪酸代谢产物的谱。目的4将确定在存在和不存在脂肪酸的情况下，SCHAD缺乏对胰岛细胞溶质钙和线粒体能量对营养刺激的反应的影响。脂肪酸被认为在调节胰腺细胞的胰岛素分泌中起重要作用，然而，其作用机制知之甚少。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受这种疾病影响的儿童的胰岛素分泌，而且还可以阐明这些重要的营养素如何有助于控制正常人和糖尿病患者的胰岛素分泌。

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.