**8. Caloric restriction and life span**

Fasting and caloric restriction have received so much attention because of their relationship to longevity. Fasting and caloric restriction to the extent that they do not lead to malnutrition are associated with longevity from nematodes to vertebrates, and are effective against aging and age-related diseases. For example, it has already been known for 80 years that restricting food intake prolongs lifespan in rats and mice [31]. Studies in monkeys showed that feeding a calorie-restricted diet from a young age inhibits the development of obesity, type 2 diabetes, cardiovascular disease, and cancer, and delays the onset of sarcopenia, senile deafness, and brain atrophy [31]. Although the molecular mechanisms by which caloric restriction maintains health and extends lifespan are not yet fully understood, various factors have been identified to be involved, including FGF21, insulin and IGF-1 signaling, mTOR, AMPK, SIRTs, NAD+ , FOXO, heat shock factor (HSF), and nuclear factorerythroid 2-related factor 2 (NRF2) [31].

FOXO is a DNA-binding transcriptional regulator, which promotes the expression of a group of genes involved in DNA repair, autophagy, antioxidant activity, stress tolerance, and cell proliferation [31]. NRF2 and HSF1 are thought to be involved in the maintenance of protein homeostasis and cell structure through induction of antioxidant and drug-metabolizing enzymes, thus extending lifespan [32].

mTOR, first found in bacteria collected from Easter Island soil, is a serine/ threonine kinase that is activated by a variety of factors and regulates a wide

range of biological processes. mTOR is activated by insulin and IGF-1 signaling, intracellular nutrients such as amino acids and glucose, and energy states as well as oxygen and stresses to regulate cell proliferation, metabolism, nutrition, and environmental stresses [33]. It was also shown that inhibition of mTOR signaling prolongs lifespan in yeast, *C. elegans*, Drosophila, and mouse, but the mechanism is not fully understood [32, 33]. mTOR forms two distinct protein complexes, mTORC1 and mTORC2, each of which phosphorylates different target substrates. While mTORC1 is strongly inhibited by rapamycin, mTORC2 is less affected [32]. Therefore, much remains to be learned about biological functions of mTORC2.

mTORC1 is activated by growth factors such as IGF-1, stresses, energy states, oxygen, and amino acids [33]. mTORC1 promotes ribosome biosynthesis and protein synthesis in the muscles, and inhibits ketone body production in the liver. In addition, it promotes adipocyte differentiation through activation of sterol regulatory element binding protein 1 (SREBP1) and PPARγ, and increases the number and the size of pancreatic β-cells. Furthermore, mTORC1 suppresses autophagy, enhances glycolytic gene expression, and regulates mitochondrial oxidative metabolism. Suppression of mTOR signaling by caloric restriction, methionine-restricted diet, and rapamycin is thought to be involved in longevity, presumably through the complex regulatory mechanisms described above [32].

mTORC2 is activated by insulin, IGF-1, and leptin through PI3K and other pathways, and then phosphorylates Akt, serum- and glucocorticoid-induced protein kinase 1 (SGK1), and protein kinase C (PKC) family members. Akt and SGK1 further phosphorylate FOXO1, which results in nuclear export of FOXO1 and repression of the expression of gluconeogenesis-related genes [32, 33]. Thus, mTORC2 is involved in the regulation of energy metabolism, but its role in longevity is still unclear.

SIRTs are known as "longevity genes," and forced expression of SIRTs extends lifespan in yeast, *C. elegans*, and Drosophila [34]. SIRTs are NAD<sup>+</sup> -dependent deacetylases. Since NAD<sup>+</sup> and NADH levels are strongly related to fatty acid oxidation, ketone body production, and gluconeogenesis, SIRTs are thought to be involved in lifespan extension by fasting. Although the molecular mechanisms of longevity are not fully understood, it was reported that SIRT1 and AMPK activates PGC-1α, and that overexpression of SIRT1, SIRT3, and SIRT6 results in enhanced genomic stability, suppression of NF- κB signaling, and improvement of metabolic homeostasis via histone deacetylation [31]. In addition, Sir2.1 of *C. elegans* activates DAF-16, a FOXO-type transcription factor [34].

Insulin and IGF-1 signals function as a sensor of nutritional states, and are suppressed by fasting and caloric restriction. During fasting, IGF-1 signaling is repressed by FGF21 as described earlier. On the other hand, the mechanism of suppression of IGF-1 signaling by caloric restriction is complex and may differ among species. In humans, it was reported that protein intake is more important than total caloric intake for the suppression of IGF-1 signaling [31]. Insulin and IGF-1 signals have been shown to be involved in longevity, but its mechanism remains unclear. Genomewide association studies (GWAS) showed an association between insulin and IGF-1 signaling and human lifespan, and also suggested that Akt, FOXO, PI3K, and SGK signaling pathways are involved in longevity [35]. Treatment of epithelial cells with serum from patients with Laron syndrome, which is characterized by refractoriness to GH, increases the expression of the superoxide dismutase 2 (SOD2) and decreases the expression of mTOR [36]. In addition, FOXO transcription factors are upregulated in the absence of IGF-1 signaling [36]. These studies suggest that insulin and IGF-1 signaling suppresses stress resistance responses. In *C. elegans*, mutations of the daf-2 gene that suppresses the activity of DAF-2, a homolog of the insulin/IGF-1 receptor, increase lifespan through the inhibition of the PI3K-phosphoinositide-dependent

kinase (PDK)-Akt pathway [34]. In Snell mice with mutations in pituitary-specific positive transcription factor-1 (PIT-1) and Ames mice with mutations in the gene encoding the regulator of PIT-1, their lifespan increased by up to 68% compared to control mice [36]. In these mice, in addition to GH, thyroid stimulating hormone (TSH) and prolactin levels were low. However, the observations that lifespan is extended in GH receptor-deficient mice and in mice lacking the gene encoding pregnancy-associated plasma protein A (PAPP-A), an activator of IGF-1 [36], support the idea that insulin and IGF-1 signaling contributes to longevity.
