The Ink4a/Arf locus encodes 2 tumor suppressor molecules, p16 INK4a and Arf, which are principal mediators of cellular senescence. To study the links between senescence and aging in vivo, we examined Ink4a/Arf expression in rodent models of aging. We show that expression of p16 INK4a and Arf markedly increases in almost all rodent tissues with advancing age, while there is little or no change in the expression of other related cell cycle inhibitors. The increase in expression is restricted to well-defined compartments within each organ studied and occurs in both epithelial and stromal cells of diverse lineages. The age-associated increase in expression of p16 INK4a and Arf is attenuated in the kidney, ovary, and heart by caloric restriction, and this decrease correlates with diminished expression of an in vivo marker of senescence, as well as decreased pathology of those organs. Last, the age-related increase in Ink4a/Arf expression can be independently attributed to the expression of Ets-1, a known p16 INK4a transcriptional activator, as well as unknown Ink4a/Arf coregulatory molecules. These data suggest that expression of the Ink4a/Arf tumor suppressor locus is a robust biomarker, and possible effector, of mammalian aging.

The Ink4a/Arf locus encodes 2 tumor suppressor molecules, p16 INK4a and Arf, which are principal mediators of cellular senescence. To study the links between senescence and aging in vivo, we examined Ink4a/Arf expression in rodent models of aging. We show that expression of p16 INK4a and Arf markedly increases in almost all rodent tissues with advancing age, while there is little or no change in the expression of other related cell cycle inhibitors. The increase in expression is restricted to well-defined compartments within each organ studied and occurs in both epithelial and stromal cells of diverse lineages. The age-associated increase in expression of p16 INK4a and Arf is attenuated in the kidney, ovary, and heart by caloric restriction, and this decrease correlates with diminished expression of an in vivo marker of senescence, as well as decreased pathology of those organs. Last, the age-related increase in Ink4a/Arf expression can be independently attributed to the expression of Ets-1, a known p16 INK4a transcriptional activator, as well as unknown Ink4a/Arf coregulatory molecules. These data suggest that expression of the Ink4a/Arf tumor suppressor locus is a robust biomarker, and possible effector, of mammalian aging.

Reduced IGF-I/insulin signaling and caloric restriction (CR) are known to extend the life span and delay age-related diseases. To address the interaction of these two interventions, we subjected normal (N) and long-lived GH receptor knockout (GHRKO) mice to CR for 20 months starting at weaning. We also used bovine GH transgenic (bGH Tg) mice, which overexpress GH and are short-lived and insulin resistant, for comparison. Circulating insulin and IGF-I levels were reduced by CR in N animals, whereas GHRKO animals exhibited very low insulin and undetectable IGF-I. Consistently, hepatic Akt phosphorylation was reduced by CR and was very low in GHRKO mice. bGH Tg mice exhibited increased active Akt. The forkhead box O1 (Foxo1) transcription factor was additively increased by CR and GHRKO at the mRNA level. However, Foxo1 protein levels were only elevated in GHRKO mice. The coactivator peroxisome proliferator-activated receptor-gamma coactivator 1alpha was increased at both gene and protein levels in GHRKO mice. N-CR and GHRKO mice also exhibited increased phosphorylated cAMP response element-binding protein and active p38 compared with the N ad libitum-fed mice, and the levels of these proteins were greatly diminished in bGH Tg mice. The protein levels of the deacetylase sirtuin 1 (SIRT1) were elevated in the two CR groups and, unexpectedly, also in bGH Tg mice. These results suggest a major role for the Akt/Foxo1 pathway in the regulation of longevity in rodents. An activated gluconeogenic pathway and increased fat metabolism may be involved in mediating the effects of reduced somatotropic and insulin signaling on longevity. These results also add to the evidence that targeted disruption of the GH receptor/GH-binding protein gene and CR act via overlapping, but distinct, mechanisms.

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Chronic elevation of GH induces resistance to insulin and hyperinsulinemia in both humans and animals, whereas calorie restriction (CR) improves peripheral insulin sensitivity in many species. To investigate the mechanisms that lead to insulin resistance in animals with high levels of GH as well as the mechanisms that might improve insulin sensitivity, we fed GH-overexpressing transgenic mice ad libitum or subjected them to 30% CR. We then assayed the plasma adipocytokines levels related to insulin sensitivity, plasma lipid levels, and tissue triglycerides accumulation and examined adipocyte morphology. Furthermore, we evaluated mRNA expression and protein levels of enzymes or regulators involved in regulating hepatic lipid metabolism. Our results suggest that decreased plasma adiponectin, increased plasma resistin and cholesterol, and elevated levels of TNF-alpha and IL-6 in adipocytes may all contribute to the insulin resistance observed in GH-Tg mice. Increased accumulation of triglycerides and impaired adipocytes differentiation in GH-transgenic mice provide plausible mechanisms for the alterations of adipocytokines. Hepatic and muscle insulin resistance in these mice is probably related to excessive accumulation of fatty acids and their metabolites. An increase in plasma adiponectin and decrease in plasma IL-6, triglycerides, and cholesterol levels in response to CR may improve insulin sensitivity.

To gain insight into the pathways by which caloric restriction (CR) slows aging, gene expression levels were assessed for each of 2,352 genes in the livers of 9-month-old CR and control mice. A total of 352 genes were found to be significantly increased or decreased by CR. The distribution of affected genes among functional classes was similar to the distribution of genes within the test set. Surprisingly, a disruption or knockout of the gene for the GH receptor (GHR-KO), which also produces life extension, had a much smaller effect on gene expression, with no more than 10 genes meeting the selection criterion. There was, however, an interaction between the GHR-KO mutation and the CR diet: the effects of CR on gene expression were significantly lower in GHR-KO mice than in control mice. Of the 352 genes altered significantly by CR, 29 had shown a significant and parallel alteration in expression in a previous study of liver gene expression that compared mice of the long-lived Snell dwarf stock (dw/dw) to controls. These 29 genes, altered both by CR and in dwarf mice, provide a list of biochemical features common to both models of delayed aging, and thus merit confirmation and more detailed study.

Ames dwarf mice are long-lived and insulin sensitive, and have a normal or reduced percentage of body fat. Calorie restriction (CR) is known to improve insulin sensitivity and reduce body fat. The purpose of this study was to evaluate the mechanism of improved insulin sensitivity in the Ames dwarfs and the effects of CR on adipose signaling and metabolism in normal and dwarf mice. Enhanced insulin sensitivity in dwarf mice may be partly due to increased release of adiponectin and the reduced release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Altered levels of adipocytokines might be consequent to the decreased lipid synthesis, plasma triglycerides, and free fatty acid levels. In normal mice, CR improves insulin sensitivity by affecting the release of adipocytokines, and decreasing circulating fatty acid and triglycerides concentrations as well as liver triglyceride accumulation. However, CR may reduce rather than enhance some of the insulin effects in the highly insulin-sensitive dwarf mice.

Resistance to growth hormone, reduced insulin-like growth factor 1 (IGF1) action, and enhanced insulin sensitivity are likely mediators of extended life span and delayed aging process in growth hormone receptor/binding protein knockout (GHR-KO) mice. Fat metabolism and genes involved in fatty acid oxidation are strongly involved in insulin action. Using real-time polymerase chain reaction and western blot we have examined expression of peroxisome proliferator-activated receptors (PPARs) and retinoid X receptor (RXR) genes in the skeletal muscle of normal and GHR-KO mice subjected to 30% caloric restriction. The results indicate that caloric restriction decreased the expression of PPARgamma, PPARalpha, and PPARbeta/delta which would lead to down-regulation of fat metabolism. This suggested metabolic change clearly does not affect whole-body insulin action. These findings suggest that whole-animal insulin sensitivity is not regulated through skeletal muscle insulin action.