Circadian Mechanisms of Diabetes Prevention in Aged mice

摘要 卡路里限制(CR)延长了多种物种的寿命/健康寿命，尽管CR的一个缺点是很差 合规性。这导致了旨在延长健康寿命的替代饮食方法。我们已经确立了 一种一天两次等卡路里(ITAD)的喂食干预，其中食物被分成两个喂食间隔 一天，没有CR。ITAD小鼠每天以两次2小时的间隔喂养，因此消耗的总卡路里是 与随机对照(Ad-lib)在24小时内吃的食物相似。尽管没有CR，但喂饲ITAD的小鼠 预防糖尿病和衰老的代谢综合征。Itad摄食的调节机制 这些好处仍不得而知。全昼夜节律分析显示，伊塔德喂食会导致明显的短暂 质量控制途径自噬模式的转变。令人惊讶的是，ITAD小鼠表现出刺激 上午11点第一个摄食窗口后立即出现自噬通量，晚上7点显著抑制而当 上午11点的自噬激活增加了脂肪的利用，晚上7点自噬的抑制与 抑制衰老小鼠糖异生基因的表达和葡萄糖的产生。这一机制通过它 喂食ITAD抑制葡萄糖产生的作用尚不清楚。我们最近发表的研究表明，核心 Clock蛋白是溶酶体降解的目标，而自噬特异性地降解CRY1。我们有 发现自噬在下午3点到7点在喂食Ad-lib的幼鼠的肝脏中被刺激，导致定时的 降解糖异生的强效抑制剂CRY1.75。Cry1与自噬体标记物Lc3结合 通过其LC3-相互作用区(LIR)基序。急性自噬阻断或其LIR基序失活 促进CRY1的积累。由于CRY1抑制糖异生，因此它的积累受阻 糖异生，降低血糖。有趣的是，我们的初步数据显示，衰老和肥胖的小鼠 它们都表现出通过自噬加速CRY1的降解，导致持续的糖异生和 高血糖症。虽然自噬通量通常会随着年龄的增长而减少，但我们认为与年龄相关的 CRY1的过度磷酸化，一种被证明触发其降解的修饰，有利于其隔离和快速 通过残留的自噬进行降解。从干预的角度来看，我们的初步研究表明， 在晚上7点，ITAD通过抑制自噬来防止CRY1水平的下降。我们建议 防止CRY1蛋白年龄相关性下降的方法将使血糖水平正常化，并 预防糖尿病。在这个前提下，我们假设与年龄相关的核心昼夜节律的过度磷酸化 抑制物CRY1加速其自噬降解，导致高血糖和2型糖尿病。我们 建议使用两种干预措施来维持老龄小鼠的CRY1水平，即Itad喂养和小鼠 旨在掩盖CRY1的LIR基序并阻止其降解的分子，反过来将抑制 糖异生和预防年龄相关的糖尿病。在这项建议中，我们将：(1)确定年龄和饮食- CRY1和附加核心昼夜节律蛋白水平和溶酶体降解率的应激相关变化 肝脏中的蛋白质；(2)确定CRY1的年龄相关修饰如何加速其降解和改变 糖异生；(3)确定Itad喂养是否通过增加CRY1逆转年龄相关性糖尿病 水平，和(4)确定小分子抑制剂，以阻断CRY1-LC3相互作用的效力，选择性和 具有良好的类药物特性，可预防老年小鼠高血糖。 意义：年龄相关代谢综合征/2型糖尿病在美国是一个重要的健康问题 在50岁以上的人中，这一比例急剧增加到~44%。2型糖尿病影响健康跨度 通过影响心脑血管系统、体力活动、视力和认知。类型2 糖尿病被认为是神经退行性疾病的易感因素，包括阿尔茨海默氏症。 1

Abstract Caloric restriction (CR) extends life/healthspan in multiple species, although one drawback of CR is poor compliance. This has led to alternative dietary approaches aimed at healthspan extension. We have established an isocaloric twice-a-day (ITAD) feeding intervention wherein food is partitioned into two feeding intervals each day, without CR. ITAD mice are fed at two 2-hour intervals each day such that total calories consumed are similar to those eaten by ad-libitum (Ad-lib) controls in 24 hours. Despite absence of CR, ITAD-fed mice are protected against diabetes and metabolic syndrome of aging. The mechanisms by which ITAD feeding mediates these benefits remain unknown. Circadian-wide analyses revealed that ITAD feeding causes marked temporal shifts in patterns of the quality control pathway autophagy. Surprisingly, ITAD mice show stimulation of autophagy flux immediately after the 1st feeding window at 11am and marked suppression at 7pm. While autophagy activation at 11am increases fat utilization, suppression of autophagy at 7pm associates with inhibition of gluconeogenic gene expression and glucose production in aging mice. The mechanism by which ITAD feeding suppresses glucose production is not known. Our recently published work has shown that core clock proteins are targets for lysosomal degradation, and that autophagy specifically degrades CRY1. We have found that autophagy is stimulated from 3pm to 7pm in livers from young Ad-lib-fed mice, leading to the timed- degradation of CRY1¾a robust inhibitor of gluconeogenesis. CRY1 binds to the autophagosome marker LC3 through its LC3-Interacting Region (LIR) motifs. Acute autophagy blockage or inactivation of its LIR motifs promotes CRY1 accumulation. Since CRY1 suppresses gluconeogenesis, its accumulation blocks gluconeogenesis and lowers blood glucose. Interestingly, our preliminary data reveal that aged and obese mice each display accelerated degradation of CRY1 by autophagy, leading to sustained gluconeogenesis and hyperglycemia. Although autophagy flux typically decreases with age, we propose that age-related hyperphosphorylation of CRY1, a modification shown to trigger its degradation, favors its sequestration and rapid degradation by residual autophagy. From an interventional stand-point, our preliminary studies indicate that ITAD feeding prevents the loss of CRY1 levels at 7pm by suppressing autophagy at this time-frame. We propose that approaches preventing the age-related decline in CRY1 protein will normalize blood glucose levels and prevent diabetes. On this premise, we hypothesize that age-related hyperphosphorylation of the core circadian repressor CRY1 accelerates its autophagic degradation leading to hyperglycemia and type 2 diabetes. We propose the use of two interventions to maintain CRY1 levels in aged mice, i.e., ITAD feeding, and small molecules designed to mask LIR motifs of CRY1 and block its degradation that, in turn, will suppress gluconeogenesis and prevent age-related diabetes. In this proposal, we will: (1) Determine the age and dietary- stress-related changes in protein levels and lysosomal degradation rates of CRY1 and additional core circadian proteins in liver; (2) Determine how age-related modifications to CRY1 accelerates its degradation and alters gluconeogenesis; (3) Determine whether ITAD feeding reverses age-associated diabetes by increasing CRY1 levels, and (4) Identify small molecule inhibitors to block CRY1-LC3 interaction with potency, selectivity and favorable drug-like properties and prevent hyperglycemia is aged mice. Significance: Age-related metabolic syndrome/type 2 diabetes is a significant health problem in the United States that increases sharply to ~44% in individuals older than 50 years. Type 2 diabetes impacts healthspan through effects on cardiovascular and cerebrovascular systems, physical activity, vision, and cognition. Type 2 diabetes is thought to predispose to neurodegenerative disease, including Alzheimer’s dementia. 1