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）突变型C57 BL/6 J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、低胰岛素和低肥胖的范式。DR-OB小鼠具有严重的胰岛素抵抗、高胰岛素水平以及高肥胖，但与饮食限制对照（DR +/？）低胰岛素和低脂肪的胰岛素敏感型。该模型将用于测试提出的产生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。将在6、12和22月龄时比较5组小鼠：DR-OB、部分限制ob/ob（部分DR-OB）、自由进食ob/ob（AL-OB）、饮食限制和自由进食遗传对照（DR +/？）和（AL +/？）。DR-OB和DR +/？之间不同的信号转导、燃料利用和脂肪组织标志物将确定与低胰岛素和低肥胖相关但不延长寿命的候选机制。DR-OB和DR +/？中相似的标志物，并且与DR-OB、部分DR-OB和AL-OB中的寿命相关，将指定DR可以增加寿命的候选机制，这将在以后进一步测试。目前的项目是独一无二的，因为它区分了DR相关的低胰岛素血症和消瘦的特征，这些特征可以延长寿命。这项工作将指定一组候选机制，包括那些关键的DR介导的，延长寿命，通过测试以下假设：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检测，使用蛋白质印迹法比较蛋白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代谢综合征的典型模式转变为减少线粒体自由基产生的DR的典型模式。确定这一点的体内试验将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、新生血管生成、糖原分解、β-氧化、葡萄糖利用、脂质利用和24小时呼吸交换率。合作者将测试自由基对肝脏和肌肉线粒体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。将测定以下内容：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、β-内酰胺酶、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;肠系膜和皮下脂肪组织中的脂肪细胞大小分布。 公共卫生相关性：在美国，随着人口老龄化，儿童和成人肥胖的持续增加预计将增加2型糖尿病、高血压和代谢综合征等疾病。这项研究将提出治疗方法，以延缓随着年龄的有害变化，特别是那些与高胰岛素水平和肥胖有关的变化。在这项研究中，DR延长正常对照和ob/ob小鼠寿命的作用可能是延缓衰老和延长人类健康寿命的干预措施的目标。

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.