Novel insights into nutrient-dependent regulation of beta cell proliferation

功能性β细胞质量下降和随后无法维持足够的血糖控制是1型和2型糖尿病的标志。创新的治疗方法旨在保留和恢复糖尿病中的功能性β细胞群;然而，安全地扩增β细胞群的策略仍有待确定。使β细胞群适应胰岛素需求增加状态的主要机制是通过调节β细胞复制。因此，人们对理解调节β细胞复制的机制有相当大的兴趣，目的是发现新的治疗靶点以促进β细胞再生。来自我们实验室的初步未发表证据表明，NAD+依赖性细胞质脱乙酰酶Sirtuin 2（SIRT 2）在啮齿动物和人类β细胞中充当促有丝分裂信号传导的营养依赖性调节剂。在人类胰岛中使用小鼠遗传和抑制剂方法，我们发现SIRT 2活性的丧失在高血糖条件下刺激β细胞增殖和β细胞质量扩增。我们还获得了通过操纵NAD+可用性来模拟营养状态变化的证据，其以与SIRT 2依赖性反应一致的方式调节β细胞增殖。由于细胞内NAD+水平随葡萄糖可用性而波动，我们假设SIRT 2将β细胞增殖与葡萄糖代谢偶联。此外，我们已经发现SIRT 2通过抑制MAPK信号传导来抑制β细胞增殖，并且全身性高血糖症中的SIRT 2抑制促进β细胞增殖，同时保护β细胞免于激活内质网（ER）应激反应下游的促凋亡信号传导。在这个提议中，我们将探索SIRT 2如何调节β细胞中的促有丝分裂信号以及ER应激反应。为了实现这一目标，我们将追求三个目标。在目标1中，我们将采用小鼠遗传方法和人类胰岛实验来确定葡萄糖和营养状态如何影响β细胞增殖的SIRT 2依赖性调节。在这里，我们将研究NAD代谢，细胞能量稳态AMPK的主要调节因子的活性，SIRT 2活性和β细胞增殖之间的联系，以获得对β细胞中将营养物质可用性与增殖相结合的信号级联的机制见解。为了理解SIRT 2如何调节细胞内信号传导以影响β细胞中葡萄糖诱导的增殖和凋亡反应，在目的2中，我们将采用蛋白质组学以及体外和体内方法鉴定SIRT 2在β细胞增殖调节中的下游效应物。最后，在目的3中，我们将检查SIRT 2抑制对体内人β细胞增殖和功能的影响，并探索不同促有丝分裂信号传导途径的组合靶向是否可以增强SIRT 2抑制的促增殖作用。总之，根据该提案进行的实验将揭示β细胞如何将营养线索转化为促有丝分裂信号，并为开发安全增加糖尿病患者β细胞质量的药理学策略铺平道路。

A decline in functional β-cell mass and subsequent inability to maintain adequate glycemic control are hallmarks of both type 1 and type 2 diabetes. Innovative therapeutic approaches are aimed at preserving and restoring functional β-cell mass in diabetes; however, strategies to safely expand β-cell mass remain to be identified. The predominant mechanism for adapting β-cell mass to states of increased insulin demand is through modulation of β-cell replication. Therefore, there has been considerable interest in understanding the mechanisms that regulate β-cell replication with the goal of discovering new therapeutic targets to promote β-cell regeneration. Preliminary unpublished evidence from our laboratory suggests that the NAD+-dependent cytoplasmic deacetylase Sirtuin 2 (SIRT2) acts as a nutrient-dependent regulator of mitogenic signaling in rodent and human β-cells. Using mouse genetic and inhibitor approaches in human islets, we found that loss of SIRT2 activity stimulates β-cell proliferation and β-cell mass expansion under hyperglycemic conditions. We have also obtained evidence that mimicking nutrient state changes by manipulating NAD+ availability regulates β-cell proliferation in a manner consistent with SIRT2-dependent responses. Since intracellular NAD+ levels fluctuate with glucose availability, we hypothesize that SIRT2 couples β-cell proliferation to glucose metabolism. Furthermore, we have found that SIRT2 inhibits β-cell proliferation by dampening MAPK signaling and that SIRT2 inhibition in systemic hyperglycemia promotes β-cell proliferation, while protecting β-cells from activating pro-apoptotic signaling downstream of the endoplasmic reticulum (ER) stress response. In this proposal, we will explore how SIRT2 regulates mitogenic signaling as well as ER stress responses in β-cells. To accomplish this, we will pursue three Aims. In Aim 1 we will employ mouse genetic approaches and experiments in human islets to determine how glucose and nutrient state affect SIRT2-dependent regulation of β-cell proliferation. Here, we will investigate links between NAD metabolism, activity of the master regulator of cellular energy homeostasis AMPK, SIRT2 activity, and β-cell proliferation to gain mechanistic insight into the signaling cascades that couple nutrient availability to proliferation in β-cells. To understand how SIRT2 modulates intracellular signaling to affect glucose-induced proliferative and apoptotic responses in β-cells, in Aim 2, we will identify the downstream effectors of SIRT2 in the regulation of β-cell proliferation, employing proteomic as well as in vitro and in vivo approaches. Finally, in Aim 3, we will examine the effects of SIRT2 inhibition on human β-cell proliferation and function in vivo and explore whether combinatorial targeting of different mitogenic signaling pathways can augment pro-proliferative effects of SIRT2 inhibition. Together, experiments under this proposal will uncover how β-cells translate nutrient cues into mitogenic signals as well as pave the way for developing pharmacological strategies to safely increase β-cell mass in humans with diabetes.