Toshimitsu Komatsu

Life span extension by reduction of the growth hormone-insulin-like growth factor-1 axis: relation to caloric restriction.

A reduced growth hormone (GH)‐insulin‐like growth factor (IGF)‐1 axis is associated with an extension of lifespan in laboratory rodents. Several phenotypes of such animal models resemble those induced by caloric restriction (CR). Using a transgenic male Wistar rat model whose GH‐IGF‐1 axis was moderately suppressed by overexpression of the antisense GH transgene (tg), we elucidated a relationship between the effects of a reduced GH‐IGF‐1 axis and CR for some biomarkers of aging, lifespan, and pathologies. Heterozygous (tg/−) rats fed ad libitum (AL) had a dwarf phenotype similar to that of control nontransgenic (−/−) rats subjected to 30% CR from 6 wk of age. Both the reduced GH‐IGF‐1 axis and CR extended lifespan to a similar extent, although the effect of CR seemed to be greater. There was an additive effect of CR to lifespan extension when tg/− rats were subjected to CR. Pathologic analyses indicated that the preventive effect of CR on selected diseases was greater than that of the reduced GH‐IGF‐1 axis. The present study suggests that CR affects aging and longevity by mechanisms other than suppression of the GH‐IGF‐1 axis, although CR might exhibit its effects partly through the reduced GH‐IGF‐1 axis.

Life Span Extension by Reduction in Growth Hormone-Insulin-Like Growth Factor-1 Axis in a Transgenic Rat Model

The longer life span in dwarf mice suggests that a reduction in the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis retards aging and extends the life span in mammals. We tested this hypothesis in a transgenic strain of rats whose GH gene was suppressed by an anti-sense GH transgene. Male rats homozygous for the transgene (tg/tg) had a reduced number of pituitary GH cells, a lower plasma concentration of IGF-1, and a dwarf phenotype. Heterozygous rats (tg/-) had an intermediate phenotype in plasma IGF-1, food intake, and body weight between tg/tg and control (-/-) rats. The life span of tg/tg rats was 5 to 10% shorter than -/- rats. In contrast, the life span of tg/- rats was 7 to 10% longer than -/- rats. Pathological analysis suggested that neoplasms caused earlier death in tg/tg rats; in contrast, tg/- rats had reduced nonneoplastic diseases and a prolonged life span. Immunological analysis revealed a smaller population and lower activity of splenic natural killer cells in tg/tg rats. The results of the present study support the hypothesis, but suggest that there is an optimal level of the GH-IGF-1 axis to maximize survival in mammals.

FoxO1 is involved in the antineoplastic effect of calorie restriction

The FoxO transcription factors may be involved in the antiaging effect of calorie restriction (CR) in mammals. To test the hypothesis, we used FoxO1 knockout heterozygotic (HT) mice, in which the FoxO1 mRNA level was reduced by 50%, or less, of that in wild‐type (WT) mouse tissues. The WT and HT mice were fed ad libitum (AL) or 30% CR diets from 12 weeks of age. Aging‐ and CR‐related changes in body weight, food intake, blood glucose, and insulin concentrations were similar between the WT and HT mice in the lifespan study. The response to oxidative stress, induced by intraperitoneal injection of 3‐nitropropionic acid (3‐NPA), was evaluated in the liver and hippocampus at 6 months of age. Several of the selected FoxO1‐target genes for cell cycle arrest, DNA repair, apoptosis, and stress resistance were up‐regulated in the WT‐CR tissues after 3‐NPA injection, while the effect was mostly diminished in the HT‐CR tissues. Of these gene products, we focused on the nuclear p21 protein level in the liver and confirmed its up‐regulation only in the WT‐CR mice in response to oxidative stress. The lifespan did not differ significantly between the WT and HT mice in AL or CR conditions. However, the antineoplastic effect of CR, as indicated by reduced incidence of tumors at death in the WT‐CR mice, was mostly abrogated in the HT‐CR mice. The present results suggest a role for FoxO1 in the antineoplastic effect of CR through the induction of genes responsible for protection against oxidative and genotoxic stress.

Manipulation of caloric content but not diet composition, attenuates the deficit in learning and memory of senescence-accelerated mouse strain P8

Calorie restriction (CR) is an experimental intervention in laboratory animals that attenuates age-associated increases in morbidity, mortality, and functional impairment. It is characterized by mild ketosis, hypoinsulinemia and hypoglycemia. In this study, we examined whether metabolic simulation of CR by a diet of isocaloric ketogenic or hypoinsulinemic diets ameliorated the learning and memory deficit in a strain of senescence-accelerated prone mice (SAMP8), a mouse model of age-dependent impairments in learning and memory. Male SAMP8 mice were fed high carbohydrate (CHO), high fat (FAT), or high protein (PRO) diets after weaning, and calorie intake was adjusted to 95% (sub ad libitum, sAL) or 70% (CR) of the mean calorie intake of control mice. At 28 weeks of age, we found CR ameliorated the performance defects of SAMP8 mice in a passive avoidance task. Neither FAT nor PRO diets affected performance of the task when fed sAL level, although a diet of these compositions partially mimicked the serum parameters of CR mice. These results suggest restriction of calorie intake is important for the prevention of learning and memory deficits, and that the simulation of serum changes induced by CR is not sufficient to prevent the cognitive defects of SAMP8 mice.

Down-regulation of AMP-activated protein kinase by calorie restriction in rat liver

AMP-activated protein kinase (AMPK) may act as a key enzyme for metabolic adaptation to calorie restriction (CR) or reduced growth hormone (GH)-insulin-like growth factor (IGF)-1 signaling, an experimental intervention for lifespan extension in animals. We investigated the protein levels of AMPKalpha and a downstream enzyme, acetyl-CoA carboxylase (ACC), by immunoblotting of liver and quadriceps femoris muscle (QFM) extracts from 6-month-old wild-type (W) and GH-suppressed transgenic (Tg) Wistar rats fed ad libitum (AL) or 30% CR diets from 6weeks of age. A modified alternate-day feeding regimen for CR yielded a fed-fasted cycle in CR rats, and therefore the effects of overnight fasting in W-AL rats were also evaluated. CR decreased threonine-172-phosphorylated AMPKalpha (p-AMPKalpha; an activated form) levels in the liver, whereas the CR-fed-fasted cycle or overnight fasting did not significantly affect the p-AMPKalpha level. In the QFM, the p-AMPKalpha level was slightly elevated in the CR-fasted phase, but greatly increased in the AL-fasted phase. Suppression of GH did not affect the p-AMPKalpha level. The phosphorylated-ACC levels did not alter in parallel with the p-AMPKalpha level, particularly in the liver. The present results suggest that CR down-regulates the AMPK activity in the liver on a long-term basis.

A transgenic dwarf rat model as a tool for the study of calorie restriction and aging

We have previously reported a long-lived transgenic dwarf rat model, in which the growth hormone (GH)-insulin like growth factor (IGF)-1 axis was selectively suppressed by overexpression of antisense GH transgene. Rats heterozygous for the transgene (tg/-) manifest phenotypes similar to those in calorie-restricted (CR) rats. To further characterize the transgenic rat in comparison with CR rats, the present study evaluated glucose and insulin tolerance in tg/- and control Wistar (-/-) rats at 6-9 months of age. Rats were fed ad libitum (AL) or 30% CR from 6 weeks of age. In CR rats, glucose disposal after glucose load was facilitated without any significant surge of serum insulin, and insulin tolerance test also indicated increased insulin sensitivity. In transgenic rats, similar findings were observed after glucose and insulin load, and CR in tg/- rats further facilitated glucose disposal during glucose and insulin tolerance tests. These findings suggest the presence of both common and separate mechanisms regulating the glucose-insulin system between CR and the reduced GH-IGF-1 axis paradigms. The transgenic rat model is, therefore, a useful one for studies of CR and aging.

Modulation of oxidative phosphorylation machinery signifies a prime mode of anti-ageing mechanism of calorie restriction in male rat liver mitochondria

Mitochondria being the major source and target of reactive oxygen species (ROS) play a crucial role during ageing. We analyzed ageing and calorie restriction (CR)-induced changes in abundance of rat liver mitochondrial proteins to understand key aspects behind the age-retarding mechanism of CR. The combination of blue-native (BN) gel system with fluorescence Difference Gel Electrophoresis (DIGE) facilitated an efficient analysis of soluble and membrane proteins, existing as monomers or multi-protein assemblies. Changes in abundance of specific key subunits of respiratory chain complexes I, IV and V, critical for activity and/or assembly of the complexes were identified. CR lowered complex I assembly and complex IV activity, which is discussed as a molecular mechanism to minimize ROS production at mitochondria. Notably, the antioxidant system was found to be least affected. The GSH:GSSG couple could be depicted as a rapid mean to handle the fluctuations in ROS levels led by reversible metabolic shifts. We evaluated the relative significance of ROS generation against quenching. We also observed parallel and unidirectional changes as effect of ageing and CR, in subunits of ATP synthase, cytochrome P450 and glutathione S-transferase. This is the first report on such ‘putatively hormetic’ ageing-analogous effects of CR, besides the age-retarding ones.

Similar metabolic responses to calorie restriction in lean and obese Zucker rats

Calorie restriction (CR), which is thought to be largely dependent on the neuroendocrine system modulated by insulin/insulin-like growth factor-I (IGF-I) and leptin signaling, decreases morbidity and increases lifespan in many organisms. To elucidate whether insulin and leptin sensitivities are indispensable in the metabolic adaptation to CR, we investigated the effects of CR on obese Zucker (fa/fa) rats and lean control (+/+) rats. CR did not fully improve insulin resistance in (fa/fa) rats. Nonetheless, CR induced neuropeptide Y (NPY) expression in the hypothalamic arcuate nucleus and metabolism related gene expression changes in the liver in (fa/fa) rats and (+/+) rats. Up-regulation of NPY augmented plasma corticosterone levels and suppressed pituitary growth hormone (GH) expression, thereby modulating adipocytokine production to induce tissue-specific insulin sensitivity. Thus, central NPY activation via peripheral signaling might play a crucial role in the effects of CR, even in insulin resistant and leptin receptor deficient conditions.

Pituitary growth hormone suppression reduces resistin expression and enhances insulin effectiveness: Relationship with caloric restriction

Caloric restriction (CR) retards various age-dependent disorders, increases lifespan, and improves insulin activity in laboratory animals. Recently, adipocytes were found to act together as an active endocrine organ that produces various hormones called adipocytokines. The peripheral and central activities of these adipocytokines have been suggested to mediate the anti-aging effects of CR. Here, we tested this notion by analyzing the effect of CR and suppression of growth hormone/insulin-like growth factor-I (GH/IGF-I) axis on the expression of resistin, adiponectin, and adipsin genes by rat white adipose tissue (WAT). We found that CR and GH/IGF-I suppression markedly downregulated resistin gene expression. We also found plasma resistin levels correlated positively with pituitary GH mRNA expression levels. Our observations suggest that CR reduces resistin expression and increases insulin effectiveness in a GH/IGF-I-dependent manner. The beneficial effects of CR and GH/IGF-I suppression appear to be mediated, at least in part, by changes in glucose metabolism that result from reductions in plasma resistin levels.

Effects of caloric restriction on gene expression in the arcuate nucleus

Neuroendocrine alterations that repress energy-costly physiologic processes such as reproduction and growth and induce stress responses, might underlie the antiaging effect of caloric restriction (CR). Neurons in the arcuate nucleus of the hypothalamus (ARH) might have a pivotal role in these neuroendocrine alterations. We investigated the effects of CR on gene expression of neuropeptide Y (NPY), proopiomelanocortin (POMC), growth hormone-releasing hormone (GHRH), somatostatin (SRIH), and cyclophilin (CP) in the ARH in male F344 rats at 6 months of age. Rats were fed ad libitum or a 30% caloric restricted diet with a modified alternate-days feeding regimen from 6 weeks of age. Reverse transcription-polymerase chain reaction (RT-PCR) methods were used to quantify mRNA levels over multiple time points during the 12-h/12-h dark/light cycle over a 2-days feeding cycle. The present study demonstrated that CR increased NPY-mRNA levels, but decreased POMC, GHRH, and CP mRNA levels differentially over the feeding cycle. The SRIH level was not significantly affected by CR. The present results support the neuroendocrine hypothesis of CR.