Neutral lipid dysregulation of the pancreatic beta-cell

描述：（从申请人的描述扫描）2型糖尿病 糖尿病的特征是慢性高血糖症， 血脂水平升高。本建议的总体目标是 确定长期暴露于高水平脂肪的机制， 在2型糖尿病患者中，脂肪酸（FA）影响胰腺β细胞功能。 以前，我们已经证明，长期暴露于FA损害胰岛素 基因表达只有在高葡萄糖的存在下，这是 与中性脂质合成增加有关。具体目标一：确定 代谢中间产物沿着中性脂质途径产生 合成负责胰岛素分泌和基因损伤 在长期暴露于FA时表达。分离的大鼠胰岛，HIT-T15，和 将β HC-13细胞在存在增加的β HC-13的情况下培养1至7天。 葡萄糖和FA的浓度。药理学工具将用于抑制 或刺激中性脂质合成的每一步，以确定 沿着酯化途径产生的代谢中间体（即， 长链酰基辅酶A、二酰基甘油或三酰基甘油） FA诱导的β细胞功能受损。具体目标二：评估是否 长期暴露于高FA的葡萄糖依赖性有害作用 对β细胞功能的影响是葡萄糖特异性的，这些机制是否 作用是转录的、转录后的或翻译的。β细胞 在实验中，耗竭将与真正的毒性区分开来， 二氮嗪将用于抑制胰岛素释放。葡萄糖特异性 PA效应将通过使用非葡萄糖促分泌素进行研究， 刺激胰岛素分泌和胰岛素基因表达。葡萄糖依赖 FA对胰岛素原生物合成、胰岛素mRNA稳定性和 将评估内源性胰岛素基因转录。FA对 胰岛素启动子活性将在HIT-T15和β HC-13细胞中表征 并使用重组腺病毒在原代胰岛中进行了研究 系统具体目标III：确定高脂喂养是否 高血糖Goto-Kakizaki（GK）大鼠损害胰岛素分泌，胰岛素 生物合成和胰岛素基因表达，以及这些影响是否 通过血糖水平的正常化来预防。GK或对照大鼠将 喂高脂饮食6周，之后胰岛素分泌，胰岛素原 将评估生物合成和胰岛素基因表达。血糖 在GK大鼠中，通过根皮苷给药， 试图防止高脂肪饮食对β细胞的有害影响， 功能这些实验将提供重要的见解， 2型糖尿病的β细胞功能障碍的病理生理学，并有明确的 对治疗这种疾病的影响。

DESCRIPTION: (Scanned from the applicant's description) Type 2 diabetes mellitus is characterized by chronic hyperglycemia and is often associated with elevated plasma lipid levels. The overall objective of this proposal is to ascertain the mechanisms whereby prolonged exposure to elevated levels of fatty acids (FA) affects pancreatic beta-cell function in Type 2 diabetes. Previously, we have demonstrated that prolonged exposure to FA impairs insulin gene expression only in the presence of high glucose, and that this is associated with increased neutral lipid synthesis. Specific Aim I: To identify the metabolic intermediate(s) generated along the pathway of neutral lipid synthesis responsible for the impairment of insulin secretion and gene expression upon prolonged exposure to FA. Isolated rat islets, HIT-T15, and betaHC-l3 cells will be cultured for 1 to 7 days in the presence of increasing concentrations of glucose and FA. Pharmacological tools will be used to inhibit or stimulate each step of neutral lipid synthesis, in order to identify the metabolic intermediate(s) generated along the esterification pathway (i.e., long-chain Acyl-CoA, diacyiglycerols, or triacylglycerols) responsible for the FA-induced impairment of beta-cell function. Specific Aim II: To assess whether the glucose-dependent deleterious effects of prolonged exposure to elevated FA on beta-cell function are glucose-specific, and whether the mechanisms of these effects are transcriptional, post-transcriptional, or translational. beta-cell exhaustion will be distinguished from bona fide toxicity in experiments where diazoxide will be used to inhibit insulin release. The glucose-specificity of PA effects will be investigated by using a non-glucose secretagogue to stimulate insulin secretion and insulin gene expression. The glucose-dependent effects of FA on proinsulin biosynthesis, insulin mRNA stability, and endogenous insulin gene transcription will be assessed. The effects of FA on insulin promoter activity will be characterized in HIT-Tl5 and betaHC-13 cells and also investigated in primary islets using the recombinant adenovirus system. Specific Aim III: To determine whether high-fat feeding in hyperglycemic Goto-Kakizaki (GK) rats impairs insulin secretion, insulin biosynthesis, and insulin gene expression, and whether these effects are prevented by normalization of blood glucose levels. GK or control rats will be fed a high-fat diet for 6 weeks, after which insulin secretion, proinsulin biosynthesis, and insulin gene expression will be assessed. Blood glucose levels will be normalized in GK rats by phloridzin administration, in an attempt to prevent the deleterious effects of high-fat diet on beta-cell function. These experiments will provide important insights into the pathophysiology of beta-cell dysfunction of type 2 diabetes, and have clear implications for the treatment of this disease.