Lifespan Extension Despite Greatly Elevated Insulin and Body Fat

描述（由申请人提供）：来自饮食限制Lepob （ob/ob）突变体C57BL/6J小鼠的新数据-我们的DR- ob模型-挑战了饮食限制（DR）的益处需要胰岛素敏感性，低胰岛素和低脂肪的范式。DR- ob小鼠有严重的胰岛素抵抗、高胰岛素水平和高肥胖，但与胰岛素敏感、低胰岛素和低肥胖的饮食限制对照组（DR +/?）一样长寿。该模型将用于测试产生DR有益作用的三类机制：1)胰岛素，IGF-1， mTOR和营养信号转导途径，2)燃料利用途径，3)脂肪组织功能。在6、12和22月龄时，将五组小鼠进行比较：DR- ob、部分限制性ob/ob （partial DR- ob）、自由喂养ob/ob （AL- ob）、限制饮食和自由喂养遗传对照（DR +/?）和（AL +/?）。DR- ob和DR +/?之间的信号转导、燃料利用和脂肪组织标志物差异将确定候选机制，与低胰岛素和低肥胖相关，但不增加寿命。DR- ob和DR +/？，并且与DR- ob、部分DR- ob和AL-OB的寿命相关，将指定DR可能增加寿命的候选机制，稍后将进一步测试。目前的项目是独特的，因为它区分了dr相关的低胰岛素血症和瘦弱的特征，这些特征不会延长寿命。这项工作将通过测试以下假设来指定一系列候选机制，包括那些对耐药介导的延长寿命至关重要的机制：目的1。在DR- ob小鼠中，mTOR、amp激酶和胰岛素/IGF-1信号转导通路的关键分支从胰岛素抵抗、高胰岛素血症和肥胖的典型模式转变为DR和延长寿命的典型模式。这些途径的关键中间体将在肝脏和腓肠肌中进行测试，使用western blots来比较蛋白质池和位点特异性磷酸化水平。目标2。在DR- ob小鼠中，燃料利用途径在延长寿命所需的临界点或之前从典型的代谢综合征模式转变为典型的DR模式，减少线粒体自由基的产生。确定这一点的体内试验包括短期空腹血糖和胰岛素水平、葡萄糖耐量、胰岛素敏感性、糖异生、糖原分解、β -氧化、葡萄糖利用、脂质利用和24小时呼吸交换比。合作者将测试自由基对肝脏和肌肉线粒体的电子传递链蛋白质的损伤。目标3。在DR- ob小鼠中，脂肪组织的关键方面反映了DR +/？而不是AL-OB小鼠的肥胖状态。将确定以下内容：身体组成和皮下/内脏脂肪组织分布（使用计算机断层扫描成像）；循环脂肪因子（脂联素、抵抗素、IL-6和tnf - α）；游离脂肪酸、甘油三酯和胆固醇的循环和组织水平；以及肠系膜和皮下脂肪组织中脂肪细胞的大小分布。

DESCRIPTION (provided by applicant): New data from diet restricted Lepob (ob/ob) mutant C57BL/6J mice-our DR-OB model-challenge the paradigm that benefits of diet restriction (DR) require insulin sensitivity, low insulin, and low adiposity. DR-OB mice have severe insulin resistance, high insulin levels, as well as high adiposity, but live as long as diet restricted controls (DR +/?) that are insulin sensitive with low insulin and low adiposity. This model will be used to test three categories of mechanisms proposed to produce the beneficial effects of DR: 1) the insulin, IGF-1, mTOR, and nutrient signaling transduction pathways, 2) fuel utilization pathways, 3) adipose tissue function. Five groups of mice will be compared at 6, 12, and 22 months of age: DR-OB, partially restricted ob/ob (partial DR-OB), ad lib fed ob/ob (AL-OB), diet restricted and ad lib fed genetic controls (DR +/?) and (AL +/?). Signal transduction, fuel utilization, and adipose tissue markers that differ between DR-OB and DR +/? will identify candidate mechanisms that correlate with low insulin and low adiposity but do not increase lifespan. Markers that are similar in DR-OB and DR +/?, and that correlate with lifespan in DR-OB, partial DR-OB and AL-OB, will specify candidate mechanisms by which DR may increase lifespan, which will be further tested later. The current project is unique in that it distinguishes features of DR-associated hypoinsulinemia and leanness that extend lifespan from features that do not. This work will specify the set of candidate mechanisms comprising those critical for DR-mediated, extended lifespan by testing the following hypotheses: Aim 1. That, in DR-OB mice, critical branches of the mTOR, AMP-kinase, and insulin/IGF-1 signal transduction pathways shift from patterns typical of insulin resistance, hyperinsulinemia and obesity to patterns typical of DR and extended longevity. Key intermediates of these pathways will be tested in liver and gastrocnemius muscle, using western blots to compare protein pool and site-specific phosphorylation levels. Aim 2. That, in DR-OB mice, fuel utilization pathways shift-at or before a critical point necessary to extend lifespan-from patterns typical of metabolic syndrome to patterns typical of DR that reduce mitochondrial free radical production. In vivo tests to identify this point will include short-term fasting glucose and insulin levels, glucose tolerance, insulin sensitivity, gluconeogenesis, glycogenolysis, beta-oxidation, glucose utilization, lipid utilization, and 24-hr respiratory exchange ratio. Collaborators will test free radical damage to proteins of the electron transport chains for liver and muscle mitochondria. Aim 3. That, in DR-OB mice, critical aspects of adipose tissue reflect the lean state of DR +/? mice rather than the obese state of AL-OB mice. The following will be determined: body composition and subcutaneous/visceral fat tissue distribution (using computerized tomography imaging); circulating adipokines (adiponectin, resistin, IL-6, and TNF-alpha); circulating and tissue levels of free fatty acids, triglyceride and cholesterol; and fat cell size distribution in mesenteric and subcutaneous fat tissue. PUBLIC HEALTH RELEVANCE: In the US, the continuing increase in childhood and adult obesity is expected to increase such ailments as type 2 diabetes, hypertension, and metabolic syndrome as the population ages. This study will suggest treatments to retard deleterious changes with age, especially those related to high insulin levels and adiposity. Effects of DR that extend life spans in both normal control and ob/ob mice in this study are likely targets for interventions to retard aging and extend healthy lifespan in human beings.