Cell Cycle Regulators in Diabetes and Obesity

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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.