Our long-term objective is to identify the molecular mechanism(s) responsible for delayed aging and prolonged longevity of animals exposed to caloric restriction (CR). We will approach this goal by examining interactions of CR and longevity assurance genes (LAGs) in a mammalian system. Will help identify the unique, intersecting and additive effects of CR and dwarfism upon aging to provide an inferential basis to determine which candidate and novel mechanisms specifically relate to aging in these models. This experimental design is unique and powerful. The proposed domains of phenotypic analysis combine careful study of standard candidates and exploratory use of microarrays. Hormone therapy is proposed as an experimental manipulation to further test the hypotheses. The concept is promising, but the design requires additional controls (revise and add to budget). The environment, investigators and model background provide high confidence for an informative study. Loss-of-function mutations at the Prop1 and Pit1 loci, as well as targeted disruption of the growth hormone receptor (GHR)/binding protein gene, delay aging and greatly prolong life in the mouse. Phenotypic characteristics of these novel models of delayed aging overlap with many of the well-documented effects of CR. We have recently demonstrated that in one of these models, the Ames dwarf (Prop1df) mouse, CR may extend life beyond the limits achievable with CR or dwarfism alone. We believe that studying interaction of CR with different LAGs is a powerful approach for identifying those changes in gene expression and in physiological characteristics that are consistently associated with prolonged longevity. These changes can be viewed as potential mechanisms and/or biomarkers of delayed aging. In the proposed studies, we will determine whether CR increases life span in Snell dwarf, in GHR knock out (GHR-KO) mice, and in Ames dwarf mice given replacement therapy with thyroid hormone. We will also examine ad libitum (AL) fed and CR dwarfs at 2.0, 2.5 and 3.0 years of age to determine whether extension of life span in Ames dwarfs by 30% CR is associated with delay of aging as assessed by tests of cognitive and immune function. To identify mechanisms of CR action in long-lived mice, we will examine the effects of 6 months of CR on gene expression in the liver and muscle, on plasma glucose, insulin, and corticosterone levels, on activity of antioxidant enzymes, and on body temperature in dwarf and GHR-KO, as compared to normal mice. Gene expression will be assessed by microarrays as in our recent studies of the effects of Ames dwarfism on age-related changes in hepatic gene expression, and results of particular interest will be verified by Northern analysis. The results will be used to determine how CR and LAGs interact to delay aging, and to identify putative mechanisms of action of CR at the molecular and organismal levels. In future studies, we will verify involvement of these mechanisms in the control of normal and delayed aging and examine their potential in designing interventions that prolong health span.
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