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Caloric restriction and genetic disruption of growth hormone signaling have been shown to counteract aging in mice. The effects of these interventions on aging are examined through age-dependent survival or through the increase in age-dependent mortality rates on a logarithmic scale fitted to the Gompertz model. However, these methods have limitations that impede a fully comprehensive disclosure of these effects. Here we examine the effects of these interventions on murine aging through the increase in age-dependent mortality rates on a linear scale without fitting them to a model like the Gompertz model. Whereas these interventions negligibly and non-consistently affected the aging rates when examined through the age-dependent mortality rates on a logarithmic scale, they caused the aging rates to increase at higher ages and to higher levels when examined through the age-dependent mortality rates on a linear scale. These results add to the debate whether these interventions postpone or slow aging and to the understanding of the mechanisms by which they affect aging. Since different methods yield different results, it is worthwhile to compare their results in future research to obtain further insights into the effects of dietary, genetic, and other interventions on the aging of mice and other species.

Mutations causing decreased somatotrophic signaling are known to increase insulin sensitivity and extend life span in mammals. Caloric restriction and every other day (EOD) dietary regimens are associated with similar improvements to insulin signaling and longevity in normal mice; however, these interventions fail to increase insulin sensitivity or life span in growth hormone receptor knockout (GHRKO) mice. To investigate the interactions of the GHRKO mutation with caloric restriction and EOD dietary interventions, we measured changes in the metabolic parameters oxygen consumption (VO2) and respiratory quotient produced by either long-term caloric restriction or EOD in male GHRKO and normal mice. GHRKO mice had increased VO2, which was unaltered by diet. In normal mice, EOD diet caused a significant reduction in VO2 compared with ad libitum (AL) mice during fed and fasted conditions. In normal mice, caloric restriction increased both the range of VO2 and the difference in minimum VO2 between fed and fasted states, whereas EOD diet caused a relatively static VO2 pattern under fed and fasted states. No diet significantly altered the range of VO2 of GHRKO mice under fed conditions. This provides further evidence that longevity-conferring diets cause major metabolic changes in normal mice, but not in GHRKO mice.

Mammalian life span can be extended by both calorie restriction (CR) and mutations that diminish somatotropic signaling. Sirt1 is a mediator of many effects of CR in mammals, but any role in controlling somatotropic signaling has not been shown. Since the somatotropic axis is controlled by the brain, we created mice lacking Sirt1 specifically in the brain and examined the impacts of this manipulation on somatotropic signaling and the CR response. These mutant mice displayed defects in somatotropic signaling when fed ad libitum, and defects in the endocrine and behavioral responses to CR. We conclude that Sirt1 in the brain is a link between somatotropic signaling and CR in mammals.

Most mutations that delay aging and prolong lifespan in the mouse are related to somatotropic and/or insulin signaling. Calorie restriction (CR) is the only intervention that reliably increases mouse longevity. There is considerable phenotypic overlap between long-lived mutant mice and normal mice on chronic CR. Therefore, we investigated the interactive effects of CR and targeted disruption or knock out of the growth hormone receptor (GHRKO) in mice on longevity and the insulin signaling cascade. Every other day feeding corresponds to a mild (i.e. 15%) CR which increased median lifespan in normal mice but not in GHRKO mice corroborating our previous findings on the effects of moderate (30%) CR on the longevity of these animals. To determine why insulin sensitivity improves in normal but not GHRKO mice in response to 30% CR, we conducted insulin stimulation experiments after one year of CR. In normal mice, CR increased the insulin stimulated activation of the insulin signaling cascade (IR/IRS/PI3K/AKT) in liver and muscle. Livers of GHRKO mice responded to insulin by increased activation of the early steps of insulin signaling, which was dissipated by altered PI3K subunit abundance which putatively inhibited AKT activation. In the muscle of GHRKO mice, there was elevated downstream activation of the insulin signaling cascade (IRS/PI3K/AKT) in the absence of elevated IR activation. Further, we found a major reduction of inhibitory Ser phosphorylation of IRS-1 seen exclusively in GHRKO muscle which may underpin their elevated insulin sensitivity. Chronic CR failed to further modify the alterations in insulin signaling in GHRKO mice as compared to normal mice, likely explaining or contributing to the absence of CR effects on insulin sensitivity and longevity in these long-lived mice.

Calorie restriction (CR) improves insulin sensitivity and increases life span in normal but not in long-lived growth hormone-resistant knockout (GHRKO) mice. In this study, we examined interactive effects of GH resistance and long-term CR on cardiac insulin action. GHRKO mice exhibited marked increases in the insulin-induced phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Akt, and ERK1/2 along with elevated insulin-stimulated IRS-1-associated regulatory subunit of phosphatidylinositol 3-kinase in the heart. These changes were associated with elevated protein levels of IR, IRS-1, and Akt and with a down-regulation of cardiac glucose transporter 4 (GLUT4). In normal mice, CR induced an important increase in the phosphorylation of cardiac Akt without elevation of Akt protein, reaching activation levels similar to those seen in GHRKO mice. This change may be cardioprotective and thus contribute to increased longevity in response to CR. Interestingly, the insulin signaling cascade in the heart of GHRKO mice was unaffected by CR.

The hypothalamic-pituitary-gonadal (HPG) axis and the somatotropic axis are influenced by nutritional factors. Calorie restriction (CR) extends lifespan but suppresses both the HPG and the somatotropic axes. Since most CR studies use a fairly severe (40%-60%) reduction of calorie intake, we hypothesized that a milder CR (20%) might not be deleterious to reproduction in male mice. To test this hypothesis, we evaluated the effects of 20% CR on testicular testosterone content and on testicular expression of genes that are relevant to testicular function and reproductive competence, including insulin-like growth factor-I, cytochrome P450 aromatase (Cyp19a1), androgen receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, cytochrome P450c17 and 3-beta-hydroxysteroid dehydrogenase/isomerase. To relate CR effects to the activity of the somatotropic axis, we have used growth hormone-resistant GHR knockout mice as well as transgenic mice overexpressing GH. Mild CR did not affect testosterone levels in testis homogenates and had little effect on expression of the examined genes in the reproductive organs. Altered activity of the GH/insulin-like growth factor-1 axis had a major impact on the parameters analyzed. The results also suggest that expression of several key genes involved in the control of testicular function is preserved under conditions of mild CR and encourage speculation that mild regimens of CR can produce longevity benefits without impairing reproduction.

The somatotropic axis, the hypothalamic-pituitary-gonadal axis and the nutritional status are deeply interrelated in mammals. Calorie restriction (CR) prolongs lifespan, but usually at some cost to reproduction. Interestingly, many of the physiological characteristics of animals with interruption in the somatotropic axis are shared by CR animals. The level of CR in most studies is 30-60%. We tested if a milder (20%) CR would promote health benefits without inhibiting reproduction in four types of mice with altered somatotropic axis: Ames dwarfs, GHR-KO, and PEPCK-bGH and MT-bGH transgenics. Fertility was not altered by CR in any of the examined groups. Mild CR did not affect final body weights or relative reproductive organ weights; did not alter plasma levels of glucose, insulin, IGF-I, testosterone, progesterone or estradiol; and did not influence hepatic expression of genes related to longevity. Altered activity of the GH/IGF-I axis in the different mice models studied had a major impact on the parameters analyzed. This preliminary study encourages speculation that mild regimens of CR can produce health and longevity benefits without the "costs" of impaired reproductive potential.

Growth hormone receptor-deficient (GHRKO) mice are long-lived and have reduced insulin-like growth factor (IGF)-1 and insulin levels and enhanced insulin sensitivity thus resembling the phenotype of animals subjected to calorie restriction (CR). In contrast to its effects in normal mice, CR does not improve insulin sensitivity or increase longevity in GHRKO males. In an attempt to identify mechanisms underlying this differential response to CR, effects of CR on the expression of insulin-related genes were compared in GHRKO and normal mice. In addition to changes detected in both genotypes, and responses unique to GHRKO mice, the levels of Akt2 and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1alpha) were increased and levels of phosphorylated c-Jun N-terminal kinase (JNK)1 were reduced in response to CR only in normal mice. These changes may be related to mechanisms of improving insulin sensitivity and life expectancy.

Hypopituitary Ames dwarf mice and growth-hormone-resistant (growth hormone receptor knockout, GHRKO) mice have reduced plasma levels of insulin-like growth factor 1 and insulin, enhanced insulin sensitivity and a remarkably increased life span. This resembles the phenotypic characteristics of genetically normal animals subjected to dietary restriction (DR). Interestingly, DR leads to further increases in insulin sensitivity and longevity in Ames dwarfs but not in GHRKO mice. It was therefore of interest to examine the effects of DR on the expression of insulin-related genes in these two types of long-lived mutant mice. The effects of DR partially overlapped but did not duplicate the effects of Ames dwarfism or GHR deletion on the expression of genes related to insulin signaling and cell responsiveness to insulin. Moreover, the effects of DR on the expression of the examined genes in different insulin target organs were not identical. Some of the insulin-related genes were similarly affected by DR in both GHRKO and normal mice, some were affected only in GHRKO mice and some only in normal animals. This last category is of particular interest since genes affected in normal but not GHRKO mice may be related to mechanisms by which DR extends longevity.

Reduced intake of nutrients [calorie restriction (CR)] extends longevity in organisms ranging from yeast to mammals. Mutations affecting somatotropic, insulin, or homologous signaling pathways can increase life span in worms, flies, and mice, and there is considerable evidence that reduced secretion of insulin-like growth factor I and insulin are among the mechanisms that mediate the effects of CR on aging and longevity in mammals. In the present study, mice with targeted disruption of the growth hormone (GH) receptor [GH receptor/GH-binding protein knockout (GHRKO) mice] and their normal siblings were fed ad libitum (AL) or subjected to 30% CR starting at 2 months of age. In normal females and males, CR produced the expected increases in overall, average, median, and maximal life span. Longevity of normal mice subjected to CR resembles that of GHRKO animals fed AL. In sharp contrast to its effects in normal mice, CR failed to increase overall, median, or average life span in GHRKO mice and increased maximal life span only in females. In a separate group of animals, CR for 1 year improved insulin sensitivity in normal mice but failed to further enhance the remarkable insulin sensitivity in GHRKO mutants. These data imply that somatotropic signaling is critically important not only in the control of aging and longevity under conditions of unlimited food supply but also in mediating the effects of CR on life span. The present findings also support the notion that enhanced sensitivity to insulin plays a prominent role in the actions of CR and GH resistance on longevity.

Blockade of growth hormone (GH), decreased insulin-like growth factor-1 (IGF1) action and increased insulin sensitivity are associated with life extension and an apparent slowing of the aging process. We examined expression of genes involved in insulin action, IR, IRS1, IRS2, IGF1, IGF1R, GLUT4, PPARs and RXRs in the hearts of normal and GHR-/- (KO) mice fed ad libitum or subjected to 30% caloric restriction (CR). CR increased the cardiac expression of IR, IRS1, IGF1, IGF1R and GLUT4 in normal mice and IRS1, GLUT4, PPARalpha and PPARbeta/delta in GHR-KO animals. Expression of IR, IRS1, IRS2, IGF1, GLUT4, PPARgamma and PPARalpha did not differ between GHR-KO and normal mice. These unexpected results suggest that CR may lead to major modifications of insulin action in the heart, but high insulin sensitivity of GHR-KO mice is not associated with alterations in the levels of most of the examined molecules related to intracellular insulin signaling.

Growth hormone receptor/binding protein knockout (GHR-KO) mice live approximately 40% longer than their normal siblings do. These mice have dramatically reduced plasma levels of insulin-like growth factor 1 (IGF1) and enhanced insulin sensitivity. We examined the expression level of peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors family genes in the livers of normal and GHR-KO mice fed ad libitum or subjected to long-term 30% caloric restriction (CR). The levels of PPARgamma and PPARalpha messenger RNA and proteins and the levels of retinoid X receptors messenger RNA were elevated in long-lived GHR-KO mice as compared to normal mice with no major effect of CR in either genotype. These findings suggest that enhanced insulin sensitivity of GHR-KO mice may be related to the altered actions of PPARs family members in the liver. The results also indicate that CR may increase insulin sensitivity through a different mechanism.

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.

Growth hormone receptor/binding protein knockout (GHR-KO) mice are characterized by resistance to growth hormone (GH), reduced insulin like growth factor 1 (IGF1) levels and enhanced insulin sensitivity and markedly increased lifespan. Findings in these and other long-lived mutant mice, and in normal animals subjected to caloric restriction (CR) indicate that insulin signaling is importantly involved in the control of longevity. We have examined the mRNA expression level of genes involved in insulin/IGF1 action in the skeletal muscle and liver of normal and GHR-KO mice fed ad libitum or subjected to long term 30% CR. The levels of IR, IRS1, IRS2, GLUT4 and IGF1 message in the skeletal muscle were reduced by CR in both normal and GHR-KO mice. In the liver, the results indicate that in GHR-KO mice mRNA expression of genes related to early steps of insulin signaling is up-regulated in the liver but not in the muscle. The results also show that improved insulin sensitivity in response to CR is not due to increased mRNA expression of the above genes in either normal or GHR-KO animals.

Ames dwarf mutant mice are long-lived, hypoinsulinemic and hypoglycemic and exhibit enhanced sensitivity to injected insulin. Their phenotypic characteristics show many similarities to animals subjected to caloric restriction (CR) but Ames dwarf mice are not CR mimetics. Reducing daily food intake by 30% prolongs longevity in both normal and Ames dwarf mice. In the present study, the animals were subjected to a different type of CR, every other day feeding (EOD). Using real-time PCR, we have examined the expression of genes related to insulin signaling in the liver of normal and dwarf mice after 9 months of EOD. The results indicate that EOD produces some changes in the insulin and IGF1 signaling pathways, and that these changes are consistent with EOD increasing insulin sensitivity.

In this study, we analyzed the effects of long-term (14 months) caloric restriction (CR) on the first steps of the insulin signaling system in skeletal muscle of normal mice. CR induced a significant decrease in serum insulin and glucose levels, indicating an enhancement of insulin sensitivity. CR reduced the in vivo insulin-induced phosphorylation of the insulin receptor substrate (IRS)-1 by 27%, but this difference was not significant (p =.298). CR reduced insulin receptor (IR) abundance by 34% from the ad libitum values, but this difference did not reach significance (p =.246). The abundance of the p85 regulatory subunit of PI3K and glucose transporter 4 was unaltered after CR. However, IRS-1 abundance was significantly increased by 42% in muscle of mice exposed to CR. These findings indicate that the CR-induced improvement of insulin action in mice is not related to changes in glucose transporter 4, the p85 regulatory subunit of PI3K, or IR abundance in skeletal muscle but might be related to an increase in IRS-1 abundance in this tissue.

Long-lived Ames dwarf mice share many phenotypic characteristics with animals subjected to caloric restriction (CR) but they are not CR mimetics. CR prolongs longevity in both normal and Ames dwarf mice. Using real-time polymerase chain reaction and western blot, we have examined the expression of genes related to insulin signaling in the liver of normal and dwarf mice subjected to 30% CR. The results revealed divergent responses of dwarf and normal animals to CR raising an interesting possibility that CR may affect longevity of normal and dwarf mice by different mechanisms. Moreover, effects of dwarfism on the expression of the examined genes differed from the effects of CR, thus adding to the evidence that these long-lived mutants are not CR mimetics., (Copyright 2004 The Gerontological Society of America)

The interaction of longevity-conferring genes with longevity-conferring diets is poorly understood. The growth hormone receptor gene-disrupted (GHR-KO) mouse is long lived; and this longevity is not responsive to 30% caloric restriction, in contrast to wild-type animals from the same strain. To determine whether this may have been limited to a particular level of dietary restriction, we subjected GHR-KO mice to a different dietary restriction regimen, an intermittent fasting diet. The intermittent fasting diet increased the survivorship and improved insulin sensitivity of normal males, but failed to affect either parameter in GHR-KO mice. From the results of two paradigms of dietary restriction, we postulate that GHR-KO mice would be resistant to any manner of dietary restriction; potentially due to their inability to further enhance insulin sensitivity. Insulin sensitivity may be a mechanism and/or a marker of the lifespan extending potential of an intervention. [ABSTRACT FROM AUTHOR]