MAMMALIAN SIR2 FUNCTION IN PANCREATIC BETA CELLS

描述(由申请人提供)：我们在这项提案中的长期目标是了解一个新的代谢网络的生理意义，该网络包括作为驱动因素的系统性NAD生物合成和作为调节哺乳动物新陈代谢和衰老的中介的Sirt1。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细胞特异的Sirt1过表达(BESTO)转基因小鼠，证明了Sirt1促进了葡萄糖刺激的b细胞的胰岛素分泌。有趣的是，这种sirt1介导的b细胞功能增强在老年BESTO小鼠中被钝化，部分原因是与年龄相关的NAD生物合成的下降。此外，我们在BESTO和卡路里受限小鼠的胰岛上进行的广泛表达谱表明，CR增强了b细胞中Sirt1的活性，可能是由于在全身水平上增强了NAD的生物合成。这些发现为一种新的令人兴奋的研究途径奠定了基础，该研究涉及NAD生物合成和Sirt1在新陈代谢和衰老调节中的联系。我们先前已经证明，烟酰胺磷酸核糖转移酶(NAMPT)是烟酰胺(维生素B3的一种形式)生物合成NAD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方面发挥重要作用。综上所述，这些发现使我们提出假设，在衰老和热量限制(CR)过程中，NAMPT介导的NAD生物合成的动态变化会在系统水平上导致Sirt1活性的显著变化，从而在多个组织中诱导与年龄相关的和CR反应的代谢变化，如2细胞和肝细胞。为了进一步确定NAMPT介导的NAD生物合成在调节b细胞和肝细胞sirt1活性中的生理学意义，我们将通过从遗传和药物上调控NAMPT和sirt1活性来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nAMPT介导的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、NAMPT和sirt1活性以及b细胞和肝功能在衰老过程中的检测。3)为了分析CR是否促进NAMPT介导的NAD生物合成和Sirt1活性，将在不同的遗传学和药理学模型中分析CR对NAMPT介导的NAD生物合成和Sirt1活性的代谢影响。这些研究应该为这个由全身性NAD生物合成和Sirt1组成的新的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：拟议的研究应该提供以下重要结果：首先，这项研究将确定一个以前未被认识到的代谢网络，该网络在哺乳动物新陈代谢和衰老的调节中发挥关键作用。其次，这项研究将对年龄相关的组织功能变化的机制，特别是胰腺b细胞和肝脏的变化，以及热量限制的抗衰老作用提供见解。最后，这项研究的预期结果将为开发新的预防/治疗与年龄相关的重要代谢并发症的干预措施提供重要的见解，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.