Circadian Mechanisms of Diabetes Prevention in Aged mice

摘要 热量限制（CR）延长了多个物种的寿命/健康寿命，尽管CR的一个缺点是 合规这导致了旨在延长健康寿命的替代饮食方法。我们建立 一天两次等热量（ITAD）喂养干预，其中食物被分成两个喂养间隔，每个间隔 日，无CR。每天以两次2小时的间隔喂养ITAD小鼠，使得消耗的总卡路里为 与24小时内自由进食（Ad-lib）对照组相似。尽管不存在CR，但ITAD喂养的小鼠仍是 防止糖尿病和衰老代谢综合征。ITAD喂养介导的机制 这些益处仍然是未知的。全昼夜节律分析显示，ITAD喂养导致显著的时间 质量控制途径自噬模式的变化。令人惊讶的是，ITAD小鼠显示出刺激， 在上午11点的第一个进食窗口后立即出现自噬通量，并在下午7点出现显著抑制。而 上午11点的自噬激活增加了脂肪利用，下午7点的自噬抑制与 抑制衰老小鼠中的致衰老基因表达和葡萄糖产生。的机制 ITAD喂养抑制葡萄糖产生尚不清楚。我们最近发表的研究表明， 生物钟蛋白是溶酶体降解的靶点，自噬特异性地降解β 1。我们有 发现，在年轻的Ad-lib喂养小鼠的肝脏中，自噬在下午3点到7点之间受到刺激，导致定时的 降解是一种强大的造血抑制剂。BMP 1与自噬体标记物LC 3结合 LC3-Interacting Region（LIR）急性自噬阻断或其LIR基序的失活 促进β 1积累。由于B_2O_1抑制次生代谢， 并降低血糖。有趣的是，我们的初步数据显示， 每一个都显示出自噬加速了BMP 1的降解，导致持续的细胞再生， 高血糖症虽然自噬通量通常随着年龄的增长而减少，但我们认为， HPLC 1的过度磷酸化，一种被证明可以触发其降解的修饰，有利于其螯合和快速降解。 通过残余自噬降解。从干预的角度来看，我们的初步研究表明， ITAD喂养通过抑制在该时间范围内的自噬来防止在7 pm时的BMP 1水平的损失。我们提出 这种方法可以防止与年龄相关的BMP 1蛋白的下降，使血糖水平正常化， 预防糖尿病。在此前提下，我们假设，年龄相关的核心昼夜节律的过度磷酸化， 阻遏物β 1加速其自噬降解，导致高血糖症和2型糖尿病。我们 提出了使用两种干预措施来维持老年小鼠中的β 1水平，即，ITAD喂养，小 分子被设计来掩盖LIR基序的BMP 1，并阻止其降解，反过来，将抑制 预防老年性糖尿病。在这个建议中，我们将：（1）确定年龄和饮食- 应激相关的蛋白质水平变化和溶酶体降解率的EST 1和额外的核心昼夜节律 （2）确定与年龄相关的修饰是如何加速肝脏中的蛋白质降解和改变肝脏中的蛋白质的; （3）确定ITAD喂养是否通过增加IGF 1逆转年龄相关性糖尿病 水平，和（4）鉴定小分子抑制剂，以阻断LC 1-LC 3相互作用的效力，选择性和 良好的类药性和预防高血糖是老龄小鼠。 重要性：糖尿病相关代谢综合征/2型糖尿病是美国的一个重要健康问题。 在50岁以上的个体中急剧增加到~44%。2型糖尿病的危害 通过影响心血管和脑血管系统、身体活动、视觉和认知。2型 糖尿病被认为易患神经退行性疾病，包括阿尔茨海默氏痴呆症。 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