MAMMALIAN SIR2 FUNCTION IN PANCREATIC BETA CELLS

描述（由申请人提供）：我们在本提案中的长期目标是了解由系统性NAD生物合成作为驱动因素和Sirt1作为介质组成的新型代谢网络在哺乳动物代谢和衰老调节中的生理意义。我们特别关注Sirt1在胰腺b细胞中的作用，并通过分析胰腺β细胞特异性Sirt1过表达（BESTO）转基因小鼠，证明Sirt1促进葡萄糖刺激的b细胞胰岛素分泌。有趣的是，这种sirt1介导的b细胞功能增强在老年BESTO小鼠中被减弱，部分原因是NAD生物合成的年龄相关下降。此外，我们在BESTO和热量限制小鼠的胰岛中进行了广泛的表达谱分析，表明CR增强了b细胞中Sirt1的活性，可能是由于在系统水平上增强了NAD的生物合成。这些发现为研究NAD生物合成与Sirt1在代谢和衰老调节中的联系开辟了一条令人兴奋的新途径。我们之前已经证明，烟酰胺磷酸核糖基转移酶（Nampt）是烟酰胺（维生素B3的一种形式）NAD生物合成途径中的限速酶，在调节Sirt1活性和葡萄糖刺激的胰岛素分泌中发挥重要作用。总之，这些发现使我们提出了一个假设，即衰老和热量限制（CR）过程中nampt介导的NAD生物合成的动态改变导致Sirt1活性在系统水平上发生显著变化，从而在多种组织（如2细胞和肝细胞）中诱导年龄相关和CR响应性代谢变化。为了解决这一假设，我们提出以下具体目标：1)为了进一步确定Nampt介导的NAD生物合成对b细胞和肝细胞Sirt1活性调控的生理意义，将通过遗传学和药理学方法对Nampt和Sirt1活性进行调控，检测原代胰岛和肝细胞中NAD含量、Sirt1靶基因表达和生理功能；2)研究Nampt介导的b细胞和肝细胞中NAD生物合成和Sirt1活性是否随着年龄的增长而下降，Nampt蛋白水平，3)为了分析CR是否增强了b细胞和肝细胞中Nampt介导的NAD生物合成和Sirt1活性，我们将在各种遗传和药理模型中分析CR对Nampt介导的NAD生物合成和Sirt1活性的代谢影响。这些研究应该为这种由系统性NAD生物合成和Sirt1组成的新型代谢网络在人类年龄相关并发症中的生理意义和治疗应用提供新的见解。公共卫生相关性：拟议的研究应提供以下重要结果：首先，本研究将确定一个以前未被认识的代谢网络，该网络在哺乳动物的代谢和衰老调节中起关键作用。其次，本研究将深入了解年龄相关的组织功能变化机制，特别是胰腺b细胞和肝脏，以及热量限制的抗衰老作用。最后，本研究的预期结果将为开发新的预防/治疗干预措施提供重要的见解，以治疗重要的年龄相关代谢并发症，如人类糖耐量受损和2型糖尿病。

DESCRIPTION (provided by applicant): Our long-term objectives in this proposal are to understand the physiological significance of a novel metabolic network comprised of systemic NAD biosynthesis as a driver and Sirt1 as a mediator in the regulation of metabolism and aging in mammals. We have focused particularly on the role of Sirt1 in pancreatic b cells and demonstrated that Sirt1 promotes glucose-stimulated insulin secretion in b cells by analyzing pancreatic beta cell-specific Sirt1-overexpressing (BESTO) transgenic mice. Interestingly, this Sirt1-mediated enhancement of b cell function is blunted in aged BESTO mice, partly due to an age-associated decline in NAD biosynthesis. Furthermore, our extensive expression profiling performed in islets from BESTO and calorically restricted mice suggests that CR enhances Sirt1 activity in b cells, possibly due to augmented NAD biosynthesis at a systemic level. These findings set the stage for a novel avenue of exciting research concerning the connection between NAD biosynthesis and Sirt1 in the regulation of metabolism and aging. We have previously demonstrated that nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the NAD biosynthetic pathway from nicotinamide (a form of vitamin B3), plays an important role in the regulation of Sirt1 activity and glucose-stimulated insulin secretion in 2 cells. Together, these findings led us to the hypothesis that a dynamic alteration in Nampt-mediated NAD biosynthesis in aging and caloric restriction (CR) causes significant changes in Sirt1 activity at a systemic level and thereby induces age-associated and CR-responsive metabolic changes in multiple tissues, such as 2 cells and hepatocytes. To address this hypothesis, we propose the following specific aims: 1) To further establish the physiological significance of Nampt-mediated NAD biosynthesis in the regulation of Sirt1 activity in b cells and hepatocytes, NAD content, Sirt1 target gene expression, and physiological functions will be examined in primary islets and hepatocytes by manipulating Nampt and Sirt1 activities genetically and pharmacologically, 2) to investigate whether Nampt-mediated NAD biosynthesis and Sirt1 activity decline with age in b cells and hepatocytes, Nampt protein levels, Nampt and Sirt1 activities, and b cell and hepatic functions will be examined in the process of aging, and 3) to analyze whether CR augments Nampt-mediated NAD biosynthesis and Sirt1 activity in b cells and hepatocytes, the metabolic effects of CR on Nampt-mediated NAD biosynthesis and Sirt1 activity will be analyzed in various genetic and pharmacological models. These studies should provide new insight into the physiological significance and therapeutic applications of this novel metabolic network comprised of systemic NAD biosynthesis and Sirt1 for age-associated complications in humans. Public Health Relevance: The proposed study should provide the following important outcomes: First, this study will identify a previously unrecognized metabolic network that plays a critical role in the regulation of metabolism and aging in mammals. Second, this study will provide insights into the mechanisms for age-associated changes in tissue function, especially in pancreatic b cells and liver, and anti-aging effects of caloric restriction. Lastly, the anticipated outcome of this study will provide important insight into the development of new preventive/therapeutic interventions for important age-associated metabolic complications, such as impaired glucose tolerance and type 2 diabetes in humans.