Molecular Basis of a New Form of Hyperinsulinism

描述（由申请人提供）：这是一份为期5年的续期资金申请，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。前几年的研究重点是高胰岛素血症/高氨血症综合征与谷氨酸脱氢酶（GDH）显性功能突变相关的机制。本提案的目的是确定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HI的机制。我们的假设是：(1)SCHAD缺乏症中胰岛素分泌失调是由一种特定脂肪酸代谢物的积累引起的；(2)与其他HI疾病通过质膜去极化作用于胰岛素释放的KATP“触发”途径不同，SCHAD缺陷作用于“触发”机制下游的“扩增”位点。这些假设将通过小鼠SCHAD-/-敲除模型进行检验。目的1将确定SCHAD-/-小鼠对体内禁食和葡萄糖负荷的反应，以及这些反应是否会被高脂肪饮食改变。目的2将使用SCHAD-/-小鼠和其他HI小鼠模型的胰岛，确定在存在或不存在短链、中链和长链脂肪酸的情况下，胰岛素对营养物质和格列本脲刺激的异常反应。线粒体脂肪酸氧化其他步骤的抑制剂将用于测试是否对SCHAD底物有特异性影响。目的3将使用质谱法测量酰基辅酶a、酰基肉碱和游离脂肪酸，确定分离的SCHAD-/-胰岛中脂肪酸代谢物的谱。目的4将确定在脂肪酸存在和不存在的情况下，SCHAD缺乏对胰岛胞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，其作用机制尚不清楚。SCHAD缺乏症提供了一个独特的“自然实验”，不仅阐明了脂肪酸如何干扰患有这种疾病的儿童的胰岛素分泌，而且还阐明了这些重要的营养物质如何有助于控制正常和糖尿病人的胰岛素分泌。

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.