Cell Cycle Regulators in Diabetes and Obesity

糖尿病和肥胖症中的细胞周期调节剂

• 批准号: 7593682
• 负责人: Sushil Rane
• 金额: $ 32.23万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593682
• 关键词: Address; Adipocytes; Adipose tissue; Age; Age-Months; Alleles; Animal Model; Animals; Beta Cell; Biological Assay; Blood specimen; Body Temperature; Body Weight; Body fat; Body measure procedure; Brown Fat; CDKN2A gene; Cell Cycle; Cell Cycle Progression; Cells; Chronic Disease; Color; Conscious; Cyclin-Dependent Kinase 4; Data; Defect; Diabetes Mellitus; Diabetic mouse; Diet; Disease; E2F transcription factors; E2F1 gene; Endocrine Glands; Energy Metabolism; Equilibrium; Event; Exhibits; Family; Fasting; Fatty acid glycerol esters; Fertility; Fostering; Future Generations; Gene Expression; Gene Targeting; Genes; Genotype; Glucose; Gonadal Steroid Hormones; Growth; Growth Factor; Hormones; Hyperglycemia; Hyperinsulinism; Hyperplasia; Immunofluorescence Immunologic; In Vitro; Incidence; Infertility; Inflammatory; Inherited; Insulin; Insulin Resistance; Investigation; Islets of Langerhans; Life Expectancy; Life Style; Liquid substance; Litter Size; Location; Longitudinal Studies; Malignant Neoplasms; Measurement; Measures; Mediating; Metabolic; Metabolic Control; Metabolic Diseases; Metabolism; Modeling; Molecular; Molecular Profiling; Mus; Mutant Strains Mice; Mutation; Numbers; Obesity; Obesity associated disease; Outcome; PPAR gamma; Pancreas; Pathway interactions; Phosphorylation; Plasma; Play; Polymerase Chain Reaction; Prevalence; Production; Public Health; Publishing; Regulation; Rest; Retinoblastoma; Retinoblastoma Protein; Rodent; Role; Site; Societies; Staging; Steroid biosynthesis; Structure of beta Cell of islet; Tail; Testing; Thermogenesis; Time; Tissues; Urine; Weaning; Weight Gain; adipocyte differentiation; blood glucose regulation; cancer risk; cytokine; day; design; embryonic stem cell; energy balance; feeding; insulin sensitivity; insulin sensitivity/resistance; insulin signaling; insulin tolerance; islet; lipid biosynthesis; mouse model; non-diabetic; obesity treatment; precursor cell; response; sedentary; sex; transcription factor; tumor

Study changes in energy balance via changes in adipocyte metabolism and insulin sensitivity. In this proposal we will use mouse models that have mutations in the Cdk4 locus. Cdk4-/- mice are diabetic and lean, and in contrast, Cdk4R24C mice are non-diabetic and heavier. These observations are consistent with the documented, albeit not very well characterized, role of the RB/E2F pathway in adipogenesis. Since Cdk4 is an upstream regulator of the E2F-RB pathway, we hypothesize that Cdk4 activity may regulate adipogenesis. Further, as has been published previously, the effect of Cdk4 on production of insulin, sex hormones and glucose regulation may impact the energy balance in the Cdk4R24C model. Here, we will characterize the energy balance, adiposity and insulin action in the Cdk4 mouse models as follows. Study the expression of Cdk4 in adipocytes at all stages of differentiation of 3T3-L1 preadipocytes. Diet Study and Body Fat Analysis of Cdk4 mutant mice. Mice weaned at 21 days of age will be fed either normal rodent chow or high fat diet and observed for one year. Urine analysis, total fat and lean mass as well as free fluid will be measured that will reveal the differences in white adipose tissue (WAT) and brown adipose tissue (BAT) in the Cdk4 mutant mice on specific diets. Blood samples will be taken from cut tail tips of conscious mice either in the fasting or fed state. Plasma will be collected for measurement of markers of adiposity. We will compare the capacity of Cdk4-/- or Cdk4R24C MEFs to differentiate into adipocytes in vitro in response to hormone stimulation. Histological and immunofluorescence analysis in tissue sections will be performed. Gene expression differences of BAT versus WAT in Cdk4 mutant mice will be evaluated. To assess the alterations in energy expenditure in the Cdk4 mutant mice, we will determine whether the Cdk4-/- and Cdk4R24C mice have abnormal energy expenditure as measured by body temperature. Evaluate the role of obesity and metabolic dysregulation on insulin resistance and cancer. Adipose tissue is not only a site of energy storage, but also an active endocrine organ. We will conduct analyses of the levels of circulating growth and inflammatory factors, and correlate the levels with age, body weight and whole body fat mass. Ultimately, we will assay the incidence of (A) Insulin resistance/sensitivity and changes in insulin signaling and (B) Cancer incidence and location in relation to the age of the animal, level of circulating factors and body fat mass. Insulin resistance, insulin signaling and expression profiling: Alterations in fat mass in Cdk4R24C mice could have an impact on glucose homeostasis. Both fasting glucose and insulin levels will be thus measured in age and sex matched mice. To better determine the insulin sensitivity, we will perform insulin tolerance tests. To measure insulin function, we will perform glucose clearance tests (GTT). Functional analyses of islets will also be performed as usual. If we observe age, or sex, or genotype-dependent alterations in insulin sensitivity we will analyze the underlying molecular mechanisms of the differences in insulin sensitivity. Real-time PCR analysis of a number of genes involved in insulin signaling and metabolic control will be performed. Cdk4R24C mice exhibit increased pancreatic islet mass by 2-3 months of age; exhibit increased levels and function of insulin. Cdk4R24C mice display increased fecundity, produce large size litters and can produce and foster multiple litters. By 6-8 months of age the Cdk4R24C mice display an increased body weight and the first tumors become detectable. Most of the Cdk4R24C mice die between 10 months and 14 months of age, while the rest die before 24 months of age. It is plausible that changes in hormone, metabolic regulators, cytokines and growth factors in the Cdk4R24C mice may influence the energy balance and cancer incidence in this model and we will test this possibility. To this end, Cdk4 mutant mice will be used in a long-term longitudinal study. Mice will be fed either rodent chow or high fat diet. We will conduct analyses to ascertain the contribution of the altered energy balance, hyperinsulinemia, hyperglycemia, and aberrant sex steroidogenesis to the ultimate cancer outcome in these mice.

通过脂肪细胞代谢和胰岛素敏感性的变化研究能量平衡的变化。 在本提案中，我们将使用 Cdk4 基因座发生突变的小鼠模型。 Cdk4-/- 小鼠患有糖尿病且较瘦，相反，Cdk4R24C 小鼠非糖尿病且较重。这些观察结果与记录的 RB/E2F 通路在脂肪形成中的作用一致，尽管还没有很好地表征。由于 Cdk4 是 E2F-RB 途径的上游调节因子，我们假设 Cdk4 活性可能调节脂肪生成。此外，正如之前发表的，Cdk4 对胰岛素产生、性激素和葡萄糖调节的影响可能会影响 Cdk4R24C 模型中的能量平衡。在这里，我们将描述 Cdk4 小鼠模型中的能量平衡、肥胖和胰岛素作用，如下所示。 研究3T3-L1前脂肪细胞分化各阶段脂肪细胞中Cdk4的表达。 Cdk4 突变小鼠的饮食研究和体脂肪分析。 21 天断奶的小鼠将被喂食正常啮齿动物饲料或高脂肪饮食，并观察一年。将测量尿液分析、总脂肪和瘦体重以及游离液体，以揭示特定饮食下 Cdk4 突变小鼠的白色脂肪组织 (WAT) 和棕色脂肪组织 (BAT) 的差异。血液样本将从处于禁食或进食状态的清醒小鼠的尾尖处采集。将收集血浆用于测量肥胖标志物。我们将比较 Cdk4-/- 或 Cdk4R24C MEF 在体外响应激素刺激分化成脂肪细胞的能力。将进行组织切片的组织学和免疫荧光分析。将评估 Cdk4 突变小鼠中 BAT 与 WAT 的基因表达差异。为了评估 Cdk4 突变小鼠能量消耗的变化，我们将确定 Cdk4-/- 和 Cdk4R24C 小鼠是否具有通过体温测量的异常能量消耗。 评估肥胖和代谢失调对胰岛素抵抗和癌症的作用。 脂肪组织不仅是能量储存场所，也是活跃的内分泌器官。 我们将分析循环生长和炎症因子的水平，并将其水平与年龄、体重和全身脂肪量相关联。最终，我们将分析 (A) 胰岛素抵抗/敏感性和胰岛素信号变化的发生率，以及 (B) 癌症发生率和位置与动物年龄、循环因子水平和体脂肪量的关系。 胰岛素抵抗、胰岛素信号传导和表达谱： Cdk4R24C 小鼠脂肪量的变化可能对葡萄糖稳态产生影响。因此，将在年龄和性别匹配的小鼠中测量空腹血糖和胰岛素水平。为了更好地确定胰岛素敏感性，我们将进行胰岛素耐量测试。为了测量胰岛素功能，我们将进行葡萄糖清除率测试（GTT）。胰岛的功能分析也将照常进行。如果我们观察胰岛素敏感性的年龄、性别或基因型依赖性改变，我们将分析胰岛素敏感性差异的潜在分子机制。将对涉及胰岛素信号传导和代谢控制的许多基因进行实时 PCR 分析。 Cdk4R24C 小鼠在 2-3 月龄时胰岛质量增加；表现出胰岛素水平和功能的增加。 Cdk4R24C 小鼠表现出增加的繁殖力，产生大量窝，并且可以产生和培育多窝窝。到 6-8 个月大时，Cdk4R24C 小鼠体重增加，并且第一个肿瘤变得可检测到。大多数Cdk4R24C小鼠在10月龄至14月龄之间死亡，其余的则在24月龄前死亡。 Cdk4R24C 小鼠中激素、代谢调节剂、细胞因子和生长因子的变化可能会影响该模型中的能量平衡和癌症发病率，我们将测试这种可能性。为此，Cdk4突变小鼠将被用于一项长期纵向研究。小鼠将被喂食啮齿动物饲料或高脂肪饮食。我们将进行分析，以确定能量平衡改变、高胰岛素血症、高血糖和异常性类固醇生成对这些小鼠最终癌症结果的影响。