Forkhead box O (Foxo) transcription factors may be involved in the salutary effect of dietary restriction (DR). This study examined the role of Foxo3 in lifespan extension and cancer suppression in DR mice. Wild-type (WT) and Foxo3-knockout heterozygous ((+/-)) and homozygous ((-/-)) mice were subjected to a 30% DR regimen initiated at 12weeks of age. Control mice were fed adlibitum (AL) throughout the study. In contrast to WT mice, DR did not significantly extend the lifespan of Foxo3(+/-) or Foxo3(-/-) mice. However, DR reduced the prevalence of tumors at death in WT, Foxo3(+/-), and Foxo3(-/-) mice. These results indicate the necessity of Foxo3 for lifespan extension but not cancer suppression by DR. The findings in Foxo3(+/-) mice contrast with those in Foxo1(+/-) mice reported previously by our laboratory suggest differential regulation of cancer and lifespan by DR via Foxo1 and Foxo3.

Calorie restriction (CR), a non-genetic intervention that promotes longevity in animals, may exert anti-aging effects by modulating mitochondrial function. Based on our prior mitochondrial proteome analysis, we focused on the potential roles of cytochrome c oxidase (Cox or Complex IV) subunit 6b1 on formation of mitochondrial supercomplexes comprised of Complex I, III, and IV. Blue native polyacrylamide gel electrophoresis followed by immunoblotting showed that the amount of Cox6b1 and the proportion of high molecular weight supercomplexes (SCs) comprised of Complexes I, III, and IV were increased in the liver of mice subjected to 30 % CR, compared with the liver of mice fed ad libitum. In in vitro experiments, in Cox6b1-overexpressing NIH3T3 (Cox6b1-3T3) cells, Cox6b1 was increased in the SC, III2IV1, and III2IV2 complexes and Cox was concomitantly recruited abundantly into the SC, compared with control (Con)-3T3 cells. The proportions of III2IV1, and III2IV2, relative to IV monomer were also increased in Cox6b1-3T3 cells. Cox6b1-3T3 cells showed increased oxygen consumption rates, Cox activity, and intracellular ATP concentrations, indicating enhanced mitochondrial respiration, compared with Con-3T3 cells. Despite the increased basal level of mitochondrial reactive oxygen species (ROS), cell viability after inducing oxidative stress was greater in Cox6b1-3T3 cells than in Con-3T3 cells, probably because of prompt activation of protective mechanisms, such as nuclear translocation of nuclear factor E2-related factor-2. These in vivo and in vitro studies show that Cox6b1 is involved in regulation of mitochondrial function by promoting the formation of SC, suggesting that Cox6b1 contributes to the anti-aging effects of CR.

Adiponectin (Adipoq), a peptide hormone secreted from the white adipose tissue, may play a role in the anti-aging and/or anti-tumor effects of calorie restriction (CR). We analyzed metabolic traits in Adipoq gene-overexpressing mice fed ad libitumwith a regular diet (RD) or a high-fat diet (HFD), or fed 30% CR of RD initiated at 12weeks of age. Adipoq-RD and -HFDmice at 6 months of age showed reduced blood glucose and insulin concentrations, and thus increased insulin sensitivity, compared with WT mice fed a RD or a HFD. In the epididymal white adipose tissue in Adipoq mice, senescence-like changes such as upregulation of p53 protein and of biomarkers of inflammation, Cd68 and Ccl2 mRNA, were ameliorated compared with WT-RD and WT-HFD mouse tissues. Resistance to stress induced by lipopolysaccharide was also strengthened in Adipoq mice compared with WT mice. These metabolic changes and stress resistance were also noted in the WT-CR mice, suggesting that Adipoq plays a part in the effect of CR. In contrast, in an allograft tumor growth model, tumor growth was not inhibited in Adipoq mice. The present findings suggest that Adipoq plays a part in the anti-aging, but not in the anti-tumor, effects of CR. (C) 2015 Elsevier Inc. All rights reserved.

The beneficial actions of caloric restriction (CR) could be mediated in part by metabolic remodeling of white adipose tissue (WAT). Recently, we suggested that CR for 6 months increased the expressions of proteins involved in de novo fatty acid (FA) biosynthesis in WAT of 9-month-old rats. Herein, we compared the CR-induced chronological alterations of the expression of mRNAs and/or proteins involved in FA biosynthesis in the WAT and liver of rats subjected to CR starting from 3 months of age and their age-matched controls fed ad libitum. The findings suggested that CR was more effective on FA biosynthesis in WAT than in liver. In WAT, CR markedly increased the expressions of mRNAs and/or proteins involved in FA biosynthesis, including sterol regulatory element-binding protein 1c (SREBP1c), a master transcriptional regulator of FA biosynthesis, throughout the experimental period. Interestingly, the CR-enhanced upregulation was temporally attenuated at 5 months of age. CR markedly increased the nuclear phosphorylated form of Akt only at 3.5 months of age. In contrast, CR significantly reduced the expression of leptin at 9 months of age. The CR-induced upregulation was not observed in obese fa/fa Zucker rats homozygous for nonfunctional leptin receptor. Collectively, these data indicate that the V-shaped chronological alterations in WAT are regulated via SREBP1c, which is probably activated by CR duration-dependent modulation of both insulin and leptin signaling.

An orexigenic hormone, neuropeptide Y (NPY), plays a role not only in the hypothalamic regulation of appetite, but also in the peripheral regulation of lipid metabolism. However, the intracellular mechanisms triggered by NPY to regulate lipid metabolism are poorly understood. Here we report that NPY deficiency reduces white adipose tissue (WAT) mass and ameliorates the age-related imbalance of adipose tissue metabolism in mice. Gene expression involved in adipogenesis/lipogenesis was found to decrease, whereas proteins involved in lipolysis increased in gonadal WAT (gWAT) of NPY-knockout mice. These changes were associated with an activated SIRT1- and PPAR gamma-mediated pathway. Moreover, the age-related decrease of de novo lipogenesis in gWAT and thermogenesis in inguinal WAT was inhibited by NPY deficiency. Further analysis using 3T3-L1 cells showed that NPY inhibited lipolysis through the Y1 receptor and enhanced lipogenesis following a reduction in cAMP response element-binding protein (CREB) and SIRT1 protein expression. Therefore, NPY appears to act as a key regulator of adipose tissue metabolism via the CREB-SIRT1 signaling pathway. Taken together, NPY deficiency reduces adiposity and ameliorates the age-related imbalance of adipose tissue metabolism, suggesting that antagonism of NPY may be a promising target for drug development to prevent age-related metabolic diseases.

Knowledge of genes essential for the life-extending effect of dietary restriction (DR) in mammals is incomplete. In this study, we found that neuropeptide Y (Npy), which mediates physiological adaptations to energy deficits, is an essential link between DR and longevity in mice. The lifespan-prolonging effect of lifelong 30% DR was attenuated in Npy-null mice, as was the effect on the occurrence of spontaneous tumors and oxidative stress responses in comparison to wild-type mice. In contrast, the physiological processes activated during adaptation to DR, including inhibition of anabolic signaling molecules (insulin and insulin-like growth factor-1), modulation of adipokine and corticosterone levels, and preferential fatty acid oxidation, were unaffected by the absence of Npy. These results suggest a key role for Npy in mediating the effects of DR. We also provide evidence that most of the physiological adaptations to DR could be achieved in mice without Npy.

The role of the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis in the lifelong caloric restriction (CR)-associated remodeling of white adipose tissue (WAT), adipocyte size, and gene expression profiles was explored in this study. We analyzed the WAT morphology of 6-7-month-old wild-type Wistar rats fed ad libitum (WdAL) or subjected to CR (WdCR), and of heterozygous transgenic dwarf rats bearing an anti-sense GH transgene fed ad libitum (TgAL) or subjected to CR (TgCR). Although less effective in TgAL, the adipocyte size was significantly reduced in WdCR compared with WdAL. This CR effect was blunted in Tg rats. We also used high-density oligonucleotide microarrays to examine the gene expression profile of WAT of WdAL, WdCR, and TgAL rats. The gene expression profile of WdCR, but not TgAL, differed greatly from that of WdAL. The gene clusters with the largest changes induced by CR but not by Tg were genes involved in lipid biosynthesis and inflammation, particularly sterol regulatory element binding proteins (SREBPs)-regulated and macrophage-related genes, respectively. Real-time reverse-transcription polymerase chain reaction analysis confirmed that the expression of SREBP-1 and its downstream targets was upregulated, whereas the macrophage-related genes were downregulated in WdCR, but not in TgAL. In addition, CR affected the gene expression profile of Tg rats similarly to wild-type rats. Our findings suggest that CR-associated remodeling of WAT, which involves SREBP-1-mediated transcriptional activation and suppression of macrophage infiltration, is regulated in a GH-IGF-1-independent manner.

Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.

AMP-activated protein kinase (AMPK) is a key molecule that controls energy homeostasis at cellular and whole body levels. Calorie restriction (CR) may exhibit the anti-aging effect through modulation of AMPK activity. We investigated the hepatic AMPK pathways for gluconeogenesis (the transducer of regulated cyclic adenosine monophosphate response element-binding protein (CREB) 2
CRTC2) and cell growth (mammalian target of rapamycin, mTOR). Male F344 rats at 2.5 months (mo) and 18 mo of age were subjected to 4-mo-long 30% CR
control rats were fed ad libitum (AL) throughout the experiment. Rats were killed 15 min after saline or glucose injection to evaluate activation of signal molecules under transient hyperglycemic and subsequent hyperinsulinemic conditions. Western blot analyses demonstrated a modest reduction of threonine-172-phosphorylated (p)-AMPKα levels and an increment of nuclear CRTC2 in the young CR group as compared with the age-matched AL group. We also confirmed the increased binding of CRTC2 and CREB and up-regulation of gluconeogenic genes (PGC-1α and PEPCK) in the CR group. However, there was no CR-specific alteration in total or phosphorylated mTOR levels. The results suggest down-regulation of hepatic AMPK activity by CR for metabolic adaptation that promotes gluconeogenesis. The effect of CR on mTOR remains elusive. © 2011, Nagasaki University School of Medicine. All rights reserved.

Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulated by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4 alpha (HNF-4 alpha), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4 alpha and its regulator peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 alpha (PGC-1 alpha). SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules. (C) 2010 Elsevier Inc. All rights reserved.

Background: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there.
Methodology/Principal Findings: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K) c was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5K gamma and enhanced PIP5K gamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5K gamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5K gamma, PI(4,5) P-2, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice.
Conclusions/Significance: Our findings indicated that the control of TSH release by the SIRT1-PIP5K gamma pathway is important for regulating the metabolism of the whole body.

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. © 2010 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010.

Calorie restriction (CR), which is thought to be largely dependent on the neuroendocrine system modulated by insulin/insulin-like growth factor-I (IGF-1) 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. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

The hypothalamus is organized as a collection of distinct, autonomously active nuclei that regulate discrete functions, such as feeding activity and metabolism. We used suppression subtractive hybridization (SSH) to identify genes that are enriched in the hypothalamus of the rat brain. We screened a subtractive library of 160 clones, and 4 genes that were predominantly expressed in the hypothalamus, compared to other brain regions. The mRNA for a member of the WD-repeat family of proteins, WDR6, was abundantly expressed in the hypothalamus, and we found that WDR6 interacted with insulin receptor substrate 4 (IRS-4) in the rat brain. Interestingly, WDR6 gene expression in the hypothalamic arcuate nucleus was decreased by caloric restriction, and in growth hormone (GH)-antisense transgenic rats, both of which are associated with an increased life span. Insulin-like growth factor (IGF)-I and insulin treatment increased WDR6 gene expression in mouse hypothalamus-derived GT 1-7 cells. Our results might suggest that WDR6 participates in insulin/IGF-I signaling and the regulation of feeding behavior and longevity in the brain. (C) 2007 Elsevier Inc. All rights reserved.

Immunosenescence or dysregulation of the immune system may accelerate the aging process and shorten the lifespan in animals. Calorie restriction, a well-known nutritional intervention for longevity in laboratory animals, retards immunosenescence and modulates the immunosystem. These effects of CR could contribute partly to extending the lifespan. A transgenic (Tg) dwarf rat strain, in which the growth hormone (GH) axis is selectively suppressed, lived longer and exhibited several phenotypes similar to those in CR rats, suggesting an important role for the GH axis in the effect of CR. Here, we describe the longevity, pathology, thymic and splenic lymphocyte subpopulations and response to endotoxin in Tg rats in comparison with CR rats. The findings support the importance of the GH axis in the effect of CR on the immune system and, in particular, longevity. The Tg rats could be a useful tool to better understand the molecular mechanisms underlying the antiaging effect of CR.

In this review, we discuss the genes and the related signal pathways that regulate aging and longevity by reviewing recent findings of genetic longevity models in rodents in reference to findings with lower organisms. We also paid special attention to the genes and signals mediating the effects of calorie restriction (CR), a powerful intervention that slows the aging process and extends the lifespan in a range of organisms. An evolutionary view emphasizes the roles of nutrient-sensing and neuroendocrine adaptation to food shortage as the mechanisms underlying the effects of CR. Genetic and non-genetic interventions without CR suggest a role for single or combined hormonal signals that partly mediate the effect of CR. Longevity genes fall into two categories, genes relevant to nutrient-sensing systems and those associated with mitochondrial function or redox regulation. In mammals, disrupted or reduced growth hormone (GH)-insulin-like growth factor (IGF)-1 signaling robustly favors longevity. CR also suppresses the GH-IGF-1 axis, indicating the importance of this signal pathway. Surprisingly, there are very few longevity models to evaluate the enhanced anti-oxidative mechanism, while there is substantial evidence supporting the oxidative stress and damage theory of aging. Either increased or reduced mitochondrial function may extend the lifespan. The role of redox regulation and mitochondrial function in CR remains to be elucidated.

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-1 (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. (c) 2008 Elsevier Inc. All rights reserved.

DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin. res once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)-mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.

Insulin/insulin-like growth factor-I (IGF-I) pathways are recognized as critical signaling pathways involved in the control of lifespans in lower organisms to mammals. Caloric restriction (CR) reduces plasma concentration of insulin, growth hormone (GH), and IGF-I. CR retards various age-dependent disorders such as nuerodegenerative diseases and extends lifespan in laboratory rodents. These beneficial effects of CR are partly mimicked in spontaneous or genetically engineered rodent models of reduced insulin and GH/IGF-I axis. Most of these long-living rodents show increased insulin sensitivity; however, recent study has revealed that some other rodents show normal or reduced insulin sensitivity. Thus, increased insulin sensitivity might be not prerequisite for lifespan extension in insulin/GH/IGF-I altered longevity rodent models. These results highlighted that, for lifespan extension, the intracellular signaling molecules of insulin/GH/IGF-I pathways might be more important than actual peripheral or systemic insulin action.

During food shortage, organisms activate defense mechanisms to maximize their chance of survival. At least in part, these responses are triggered by changes in hormonal status and neural status during starvation. The hypothalamus is organized as a collection of distinct autonomously active nuclei and is considered to play crucial roles in these survival responses. To isolate factors involved in these pathways, we carried out suppression subtractive hybridization analyses using complementary DNAs (cDNA) from the hypothalami of fasted and fed rats. We identified four genes, namely ubiquitin-conjugating enzyme E2D 3 (UBE2D3), cAMP-dependent protein kinase C beta subunit (PKC beta), excitatory amino acid carrier 1 (EAAC1), and ferritin heavy polypeptide 1 (Fth1), that were upregulated after a 48-h fast compared to the fed status. According to previous reports, these genes have been implicated in protection against neuronal cell death under various neurodegenerative stresses, such as hvpoxia-ischemia and oxidative stress. Thus, the increased expressions 4 the genes identified in the present study may have protective effects against neural damage that could otherwise result in cell death.

Diminished leptin signaling to the arcuate nucleus of hypothalamus (ARH) may induce calorie restriction (CR)-specific neuroendocrine and metabolic adaptation, which is potentially relevant to the effect of CR. The present study investigated whether restoration of leptin signaling to the ARH could reverse CR-induced alterations in neuropeptide gene expression in rats. Male F344 rats, fed ad libitum or a 30% CR diet from 6 weeks of age, received leptin or vehicle intracerebroventricularly for 14 days via osmotic mini-pumps implanted in the subcutis at 34 weeks of age. The messenger RNA levels were quantified by real-time reverse transcription-polymerase chain reaction using total RNA extracted from microdissected tissues containing the ARH. The results indicated that leptin administration reversed the upregulated expression of neuropeptide Y and agouti-related protein genes in CR rats, suggesting the possibility of a role for the leptin-ARH pathway in the effect of CR. Copyright 2006 by The Gerontological Society of America.

Endonuclease-induced DNA fragmentation is a hallmark of apoptosis. DNase gamma (DNase gamma) was recently identified as one of the endonucleases responsible for apoptotic DNA fragmentation. In this study, immunohistochemistry for DNase gamma was performed on paraffin sections of rodent liver in well-defined models of hepatocyte apoptosis induced by Fas antibody (Fas) or cycloheximide (CHX), and necrosis induced by lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). DNase gamma immunoreactivity was compared with TdT-mediated dUTP nick-end labeling (TUNEL) reactivity. Our results showed TUNEL reactivity in both apoptotic and necrotic hepatocytes. DNase gamma immunoreactivity was not detected during LPS-induced or CCl4-induced hepatocyte necrosis. In contrast, it was evident during CHX-induced, but not Fas-induced, apoptotic DNA fragmentation. These findings suggest that DNase gamma plays an important role in Fas-independent apoptotic DNA fragmentation in hepatocytes.

We applied serial analysis of gene expression (SAGE) to the mouse testis to reveal the global gene expression profile and to identify senescence-dependent changes in that profile. A total of 61,929 SAGE tags, including 19,323 unique tags, were obtained from 3- and 29-month-old BDF1 mice and 14-month-old SAMP1 mice. Genes highly expressed in the testis included those associated with spermatogenesis, protein metabolism, energy metabolism, growth and differentiation, and signal transduction. Testes from old mice of both strains appeared atrophied. Morphological examination of aged testes revealed extremely thin seminiferous epithelia and significantly decreased numbers of spermatids and spermatocytes. Despite the physical deterioration, no gross changes in the gene expression profile were apparent in the testes of old BDF1 mice. However, in 14-month-old SAMP1 mice, protamine 2 gene transcription was approximately 50% lower than in BDF1 mice. This reduction may be associated with the oligozoospermia and early decline in reproductive performance of SAMP1 mice. Our SAGE results are the first quantitative gene expression profile of the mouse testis and provide a reliable transcriptome reference for this organ.

Nijmegen breakage syndrome (NBS) is characterized by radiation hypersensitivity, chromosomal instability, and predisposition to cancer. Nbs1, the NBS protein, forms a tight complex with Mrell and Rad50, and these interactions contribute to proper double-strand break repair. The simian virus 40 (SV40) oncoprotein, large T antigen (T), also interacts with Nbs1, and T-containing cells experience chromosomal hyperreplication in a manner dependent on T/Nbs1 complex formation. A substantial fraction of NBS-deficient fibroblasts reinitiate DNA replication in discrete regions, and wild-type Nbs1 corrects this defect. Similarly, synthesis of an N-terminal Nbs1 fragment induced DNA rereplication and tetraploidy, in NBS-deficient but not NBS-proficient cells. Moreover, SV40 origin-containing DNA hyperreplicated in T-containing NBS-deficient cells by comparison with T-containing, Nbs1-reconstituted derivatives. Thus, Nbs1 suppresses rereplication of cellular DNA and SV40 origin-containing replicons, and T targets Nbs1, thereby enhancing the yield of new SV40 genomes during viral DNA replication.

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. (C) 2002 Elsevier Science Inc. All rights reserved.

Many hormonal signals from peripheral tissues contribute to the regulation of energy homeostasis and food intake. These regulators including leptin, insulin, and ghrelin, modulate the orexigenic and anorexigenic neuropeptide expression in hypothalamic nuclei. The anti-aging effects of caloric restriction have been explained from an evolutional viewpoint of the adaptive response of the neuroendocrine and metabolic response systems to maximize survival during periods of food shortage. In organisms, excess energy is stored in adipose tissues as a triglyceride preparation for such survival situations. Adipose tissue has recently been recognized as an endocrine organ, and leptin, as secreted by adipocyte, seems to be an especially important factor for the adaptive response to fasting and neuroendocrine alterations under caloric restriction. In this review, we discuss the potential involvement of neuroendocrine modulators in longevity and the anti-aging effects of caloric restriction. (C) 2002 Wiley-Liss, Inc.

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.

Apolipoprotein A-II (apoA-II), an apolipoprotein in serum high-density lipoprotein, is a precursor of mouse senile amyloid fibrils. The liver has been considered to be the primary site of synthesis. However, we performed nonradioactive in situ hybridization analysis in tissue sections from young and old amyloidogenic (R1.P1-Apoa2(C)) and amyloid-resistant (SAMR1) mice and revealed that other tissues in addition to the liver synthesize apoA-II. We found a strong hybridization signal in the basal cells of the squamous epithelium and the chief cells of the fundic gland in the stomach, the crypt cells and a small portion of the absorptive epithelial cells in the small intestine, the basal cells of the tongue mucosa, and the basal cells of the epidermis and hair follicles in the skin in both mouse strains. Expression of apoA-II mRNA in those tissues was also examined by RT-PCR analysis. Immunolocalization of apoA-II protein also indicated the cellular localization of apoA-II. ApoA-II transcription was not observed in the heart. Amyloid deposition was observed around the cells expressing apoA-II mRNA in the old R1.P1-Apoa2(C) mice. These results demonstrate that the apoA-II mRNA is transcribed and translated in various extrahepatic tissues and suggest a possible contribution of apoA-II synthesized in these tissues to amyloid deposition.

In mouse senile amyloidosis, apolipoprotein A-II polymerizes into amyloid fibrils (AApoAII) and deposits systemically. Peripheral injection of AApoAII fibrils into young mice induces systemic amyloidosis (Higuchi et at, 1998). We isolated AApoAII amyloid fibrils from the livers of old R1.P1-Apoa2(c) mice and injected them with feeding needles into the stomachs of young R1.P1-Apoa2(c) mice for 5 consecutive days. After 2 months, all mice had AApoAII deposits in the lamina propria of the small intestine. Amyloid deposition extended to the tongue, stomach, heart, and liver at 3 and 4 months after feeding. AApoAII suspended in drinking water also induced amyloidosis. Amyloid deposition was induced in young mice reared in the same cage for 3 months with old mice who had severe amyloidosis. Detection of AApoAII in feces of old mice and induction of amyloidosis by the injection of an amyloid fraction of feces suggested the propagation of amyloidosis by eating feces. Here, we substantiate the transmissibility of AApoAII amyloidosis and present a possible pathogenesis of amyloidosis, ie, oral transmission of amyloid fibril conformation, where we assert that exogenous amyloid fibrils act as templates and change the conformation of endogenous amyloid protein to polymerize into amyloid fibrils.

Mitochondrial DNA (mtDNA) is exclusively inherited maternally and hence could offer a good method for tracing the lineage of mouse strains. We examined the mtDNA sequence of senescence-accelerated mouse (SAM) strains as well as other laboratory strains of inbred mice to deduce the ancestral strain of SAM. Four unique mutations were identified at bases 2256, 10,847, 11,181, and 13,053 in SAM strains. The mutations were not found in other mouse strains including AKR/J, one of the parental strains of SAM. Comparison of the mtDNA sequences also led to the consensus mtDNA sequence of laboratory strains of inbred mice. The seven laboratory strains of common inbred mice showed polymorphisms at base 9348, thymine repeat from base 9818, and adenine repeat from base 9821, and could be classified into five types by combination of the differences. Although we could not identify mouse strains with the same type of mtDNA as SAM in this study, the polymorphisms would provide a promising clue to ascertain the ancestral strain(s) of SAM. The polymorphism in mtDNA could be used to ascertain the genealogy of other mouse strains as well.

Apolipoprotein A-II (apoA-II), the second most abundant apolipoprotein of serum high density lipoprotein, deposits as an amyloid fibril (AApoAII) in old mice. Mouse strains with a high incidence of senile amyloidosis have the type C apoA-II gene (Apoa2(c)), whereas the strains with a low incidence of amyloidosis have the type B apoA-II gene (Apoa2(b)). in this study, to investigate whether the type B apoA-II protein inhibits the extension of amyloid fibrils, we constructed an adenovirus vector bearing the Apoa2(b) cDNA (Adex1CATApoa2(b)), which is expressed under the control of a hepatocyte-specific promoter. The mice were infected with Adex1CATApoa2(b) before induction of amyloidosis by the injection of AApoAII amyloid fibril seeds. Compared with the mice infected with the control virus, amyloid deposition was suppressed significantly in the mice infected with Adex1CATApoa2(b). Fluorometry using thioflavine T also revealed that AApoAII fibril extension was inhibited by the addition of type B apoA-II in vitro. Thus, we propose that Apoa2(b) contributes as an active inhibitor of amyloid fibril extension and overexpression of amyloid-resistant gene variant may be an attractive therapeutic target in amyloidosis.

Amyloidosis refers to a group of diseases characterized by tissue deposition of amyloid fibrils. A single intravenous injection of a very small amount of the native mouse senile amyloid fibrils (AApoAII) induced severe systemic amyloid deposition in young mice having the amyloidogenic apoA-II gene (Apoa2(c)). After AApoAII injection, amyloid deposition occurred rapidly and advanced in an accelerated manner, as observed in spontaneous senile amyloidosis in mice. However, the injection of denatured AApoAII, native apoA-II in high-density lipoprotein (HDL), and denatured apoA-II monomer, which have the same primary structure but without a fibril conformation, did not induce amyloidosis. No amyloid deposition was observed in mice having an amyloid-resistant apoA-II gene (Apoa2(b)) even 3 months after AApoAII injection. Significantly less amyloid deposition was observed in mice having both types of apoA-II genes heterozygously (Apoa2(b/c)). These findings suggest that the nucleation-dependent polymerization found in vitro also occurs in vivo, and that the fibril conformation is required for the injected amyloid fibrils to act as seeds in vivo. Fibril conformation-dependent fibrillization is proposed as a general model of the pathogenesis of various kinds of amyloidosis occurring in vivo; it may be useful in both elucidating the pathogenesis of amyloidosis and developing effective therapeutic modalities to treat this disease.

1.DNA傷害時におけるDbf4/Cdc7のリン酸化の解析(1)DNA傷害によるDbf4のリン酸化とATM/ATRとの関連ATM/ATR過剰発現細胞をもちいてIR(20Gy),UV(50J/m2),HU(1mM,24hrs),etoposide(50μM,24hrs)処理後のDbf4のリン酸化をSDS-PAGEにより解析した。その結果これらのDNA傷害誘導処理とATM/ATRの過剰発現を組み合わせることによりDbf4のリン酸化が増強される可能性を示唆する結果が得られた。(2)ATM/ATRによるDbf4のリン酸化部位の解析Dbf4には7つのATM/ATRによるリン酸化のコンセンサス配列(S/TQ)が存在する。GST融合タンパクを作製し、site-directed mutagenesisによりアラニン変異体を作製しATM/ATRによるリン酸化部位を同定した。その結果、5つのSQ/TQ部位がATM/ATRによるリン酸化の標的である可能性が示唆された。その他の2カ所については継続して解析している。2.Dbf4のリン酸化変異体および抗リン酸化抗体の作製同定した5つのリン酸化部位をアラニン(A ; phospho-negative)またはグルタミン酸(D ; phospho-mimic)に変異させ、表現型解析にもちいるDbf4

カロリー制限(CR)による抗老化・寿命延長作用のメカニズムとして、白色脂肪組織の形質変化(脂肪細胞の小型化、代謝の活性化、炎症の抑制)が重要であり、さらにこの形質変化にsterol regulatory element binding protein (SREBP) 1cが関与している可能性を検証した。CRを行ったSREBP1cノックアウトおよび野生型マウスの解析から、CRによる白色脂肪組織の形質変化のうちSREBP1cにより制御されていると考えられる4つの遺伝子群を同定した。加えて、108週齢現在、自由摂食群、CR群とも、SKOマウスの生存率が抑制されており、またCRに伴う最大体重の減少率がSKOマウスにおいて抑制されているようであった。今後、さらなる検討が必要であるが、本研究成果はCRの作用の一部にSREBP1cが関与している可能性を強く支持するものである

カロリー制限(CR)の抗老化効果におけるNeuropeptide Y (NPY)の役割を実証するために、NPYノックアウト(KO)マウスを自由摂食(AL)、CR下で飼育し、寿命や酸化ストレスに対する耐性をテストした。1) 寿命 : NPY-koマウスの生存率はAL, CRいずれの条件でも野生型マウスよりも低かった。2) 酸化ストレス耐性 : NPY-KOマウスでは、CRの効果(ストレス耐性)が減弱した。以上の結果から、CRの抗老化効果には、NPYが重要な役割を果たしていることが示唆された

我々は、視床下部特異的遺伝子WD-repeat protein 6 (WDR6)を単離した。WDR6は脱リン酸化を制御するホスファターゼと相同性があり、さらにタンパク質-タンパク質の相互作用に重要なWD-repeatモチーフを持っている。本研究により脳においてWDR6がIRS-4(インスリン受容体基質-4)と物理的に相互作用することを明らかにし、WDR6が脳におけるinsulin/IGF-Iシグナルの制御因子であることが示唆された。今後の研究により、認知症などの発症に関連する神経変性に対する治療薬開発等への応用が期待される

カロリー制限(CR)は抗老化・抗酸化作用を誘導し健康寿命を延伸することが様々な生物で報告されている。そのためCRと同様の効果を持つCR模倣物の研究が広く行われている。本研究ではタウリンがCR模倣物として機能し、寿命の延伸にも関わるのではないかと考え解析を行った。培養細胞を用いた解析から、UV照射によるDNA損傷応答が、タウリンの添加によって減弱することが示唆された。この結果は、タウリン処理によって酸化ストレスの誘導が軽減されたことを示唆している。したがって、タウリンが新たなCR模倣物の候補となる可能性があり、今後の更なる検討が必要であると思われる。

　カロリー制限(CR)は、実験動物の老化を遅らせ、寿命を延長させる最も確実な介入方法である。その抗老化作用には、摂食促進の作用を持つニューロペプチドY (NPY)の発現亢進が重要であり、NPYの活性化がCRを行った際の体内シグナル伝達を模倣、または増幅可能なことが示唆されている。そこで本研究では、NPY発現亢進作用が知られている六君子湯に着目し、マウスに六君子湯を混ぜた餌を投与し、酸化ストレスに対する耐性および生体応答を解析した。　その結果、六君子湯がNPYの発現亢進を介し、肝臓ではCRの(1)抗酸化能を上昇させる効果、(2)酸化ストレスに応答し細胞周期を停止させる効果、(3)アポトーシスを誘導する効果、(4)ミトコンドリア機能を改善する効果を模倣または増幅可能なことが示唆された。

　カロリー制限（calorie restriction: CR）は、実験動物の老化を遅らせ、寿命を延長させる最も確実な介入方法である。その抗老化作用に関連するシグナル伝達系はまだ不明な点が多いが、サーチュインと呼ばれる長寿遺伝子の関与が示唆されている。そこで本研究では、サーチュインを活性化させる植物由来成分であるレスベラトロールをマウスの餌に混ぜ、ドキソルビシンによって誘導される酸化ストレスによるDNA損傷に対する保護作用を解析した。　その結果、CRによる脂質代謝関連遺伝子の発現上昇は、ドキソルビシン投与によって抑制され、レスベラトロールを投与されたマウスにおいても同様にそれらの遺伝子の発現上昇は見られなかった。しかし、レスベラトロールを投与したマウスはサーチュイン遺伝子の発現が上昇し、このことが通常食を投与したマウスと比較して酸化ストレス耐性が亢進したことと関連があると示唆された。

　カロリー制限(CR)は、実験動物の老化を遅らせ、寿命を延長させる最も確実な介入方法である。その抗老化作用に関連するシグナル伝達系はまだ不明な点が多いが、酸化ストレスの抑制が重要であることが示唆されている。そこで本研究では、培養細胞に紫外線(UV)を照射して酸化ストレスを誘導し、食品由来成分であるタウリン(Tau)がその酸化ストレスの蓄積を軽減するかを解析した。　その結果、UV処理細胞と比べ、Tau + UV処理細胞では酸化ストレスが抑制されていることが示唆された。タンパク質レベルでは、チェックポイントタンパクであるp-Chk1がUV未処理の細胞に比べ、UV処理細胞において発現が亢進した。タウリン処理はこのChk1のリン酸化を低下させたことからも、タウリンによる酸化ストレス抑制作用が示唆された。これらの結果から、タウリンがCRの抗老化作用を一部模倣することが可能であると示唆された。

　本研究では、タウリンのカロリー制限模倣効果について解析を行った。実験1では4週齢のラットに2週間タウリン含有食を与え、体重、血液生化学などのバイオマーカを解析した。実験2ではマウスにタウリン添加食を与え、3-ニトロプロピオン酸投与による酸化ストレス耐性を解析した。実験1において、血清および肝臓コレステロール濃度はタウリンの添加量に依存して低下した。また、肝臓中性脂肪濃度がタウリンの添加量依存的に低下した。実験2では、タウリン添加によって酸化ストレス誘導後の生存率を上昇させることが示された。これらの結果から、タウリンはカロリー制限模倣物となる可能性を持つと考えられた。

実験動物に対して与える餌の量を制限すること（カロリー制限）、およびインスリンシグナル系の減弱により、寿命の延長や老化関連疾患の発症が抑制されることが知られている。我々はこれまでに、脳の視床下部で高発現している新規遺伝子、WD repeat protein 6 (WDR6)が、インスリンシグナル系の分子として摂食行動やエネルギー代謝などに関連している可能性を明らかにした。本研究では、全身性および脳特異的WDR6ノックアウトマウスの作製を行った。また、得られたマウスの摂食行動など、表現型解析に関する基礎的な研究を行った。　これまでに、Cre-loxPシステムをもちいたWDR6ノックインマウスを作製しており、当該キメラマウスをCreを全身性に発現するCAG-Creマウスと交配させ、ヘテロノックアウトマウスを作製した。その後、ヘテロ同士の交配によりホモノックアウトマウスを作製した。得られたマウスの遺伝子型の分離比は概ね野生型：ヘテロ：ホモ、1:2:1であり、メンデル遺伝の法則に従っていた。また、ホモ同士の交配によっても仔マウスが得られたことから、WDR6ノックアウトマウスは胎生致死でなく、また生殖細胞系にも異常が見られないことが示唆された。脳特異的WDR6ノックアウトマウスは、タモキシフェンによって神経細胞においてのみCreの発現が誘導されるNestin-CreERTマウスとの交配により行った。現在、ハウスの繁殖と交配を継続して行っている。　全身性WDR6ノックアウトマウスの野生型、ヘテロ、ホモの遺伝子判定は、尾部から抽出したゲノムDNAをもちいて行った。得られたマウスの肝臓より、RNAを抽出し、WDR6の遺伝子発現をRT-PCR法により解析した。その結果、RNAレベルにおいても目的とした標的遺伝子組換えが起こっていることが示唆された。今後は、インスリン受容体基質-4(IRS-4)、およびliver kinase B1(LKB1)などの代謝関連タンパク質とWDR6の相互作用を、免疫沈降法をもちいて解析し、WDR6ノックアウトによるタンパク質－タンパク質間の結合への影響を解析する。現在までのところ、若齢においては遺伝子型による摂食行動に対する明確な違いは認められていない。今後も、これらのマウスの飼育を老齢まで継続するとともに、糖・脂質代謝の変化、さらに酸化ストレスなどに対する耐性等を解析する予定である。これらの研究を通じて、WDR6ノックアウトマウスを新たな老化疾患研究モデル動物として確立することを目指す。

インスリンシグナル系による老化制御機構に重要な役割をもつ転写因子複合体の機能解明、およびその活性化分子を明らかにするため、以下の研究を行った。１．In vitroスクリーニング系の確立抗老化に関与する転写因子が結合し、転写を活性化させると示唆されるDNA配列である、DFCR-RE（モチーフ2）を直列に5個結合させ、分泌型アルカリフォスファターゼ(SEAP)の上流に組み込み、さらに選択用のpuromycin耐性遺伝子を導入したコンストラクトを作製した。マウス肝臓由来培養細胞であるTLR2細胞(SV40形質転換細胞)にこのプラスミドをトランスフェクトし、構成的発現細胞株を３系統樹立した。あわせてネガティブコントロール用として、別のDFCR-RE配列（モチーフ5）についても、同様に３系統の細胞株を樹立した。SEAPの発現量をもとに、それぞれ１系統の細胞株を選択し、以下の実験を行った。２．確立した細胞株をもちいたスクリーニング東京大学創薬オープンイノベーションセンターより化合物ライブラリーの提供を受け（36種類）、上記の細胞系をもちいたスクリーニングを行った。36化合物をそれぞれ1μMの濃度で細胞の培地に添加し、0, 24, 48, 72時間後に培養上清を採取し、アッセイキットをもちいてSEAP活性を測定した。この結果をもとに化合物を選択し、さらに以下の解析を行った。３．スクリーニング系の最適化上記の実験で決定したポジティブコントロールとして期待される化合物をもちいて、培養液中に濃度、処理時間を変化させて添加し、培地中に分泌されたSEAPの活性を測定した。その結果、化合物番号５番について期待されるSEAP活性の変化は見られたものの、統計的な有意差は見られなかった。４．HNF-4/PGC-1の結合促進および転写活性化の解析上記の化合物番号５番をもちいた実験について、PGC-1の発現およびHNF-4/PGC-1複合体の形成変化について、それぞれウエスタンブロット、免疫沈降法をもちいて解析した。その結果、PGC-1の発現は化合物の添加に対して濃度依存的な上昇が見られたものの、複合体形成の明らかな増加は見られなかった。以上の結果から、化合物添加前のレポーター発現がより低いコンストラクトが必要であることが示唆された。現在、あらたなレポータープラスミドの作製を行っている。