Haruyoshi Yamaza

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.

Cryopreserved dental pulp tissues of exfoliated deciduous teeth is a feasible stem cell resource for regenerative medicine.

Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25–30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine.

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.

The distribution of BrdU- and TUNEL-positive cells during odontogenesis in mouse lower first molars

This study investigated the minute distribution of both proliferating and non-proliferating cells, and cell death in the developing mouse lower first molars using 5-bromo-2′-deoxyuridine (BrdU) incorporation and the terminal deoxynucleotidyl transferase-mediated deoxyuridine-5′-triphosphate (dUTP)-biotin nick end labeling (TUNEL) double-staining technique. The distribution pattern of the TUNEL-positive cells was more notable than that of the BrdU-positive cells. TUNEL-positive cells were localized in the following six sites: (1) in the most superficial layer of the dental epithelium during the initiation stage, (2) in the dental lamina throughout the period during which tooth germs grow after bud formation, (3) in the dental epithelium in the most anterior part of the antero-posterior axis of the tooth germ after bud formation, (4) in the primary enamel knot from the late bud stage to the late cap stage, (5) in the secondary enamel knots from the late cap stage to the late bell stage, and (6) in the stellate reticulum around the tips of the prospective cusps after the early bell stage. These peculiar distributions of TUNEL-positive cells seemed to have some effect on either the determination of the exact position of the tooth germ in the mandible or on the complicated morphogenesis of the cusps. The distribution of BrdU-negative cells was closely associated with TUNEL-positive cells, which thus suggested cell arrest and the cell death to be essential for the tooth morphogenesis.

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.

SIRT1 regulates thyroid-stimulating hormone release by enhancing PIP5Kγ activity through deacetylation of specific lysine residues in mammals

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)γ was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kγ and enhanced PIP5Kγ enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kγ knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kγ, PI(4,5)P2, 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-PIP5Kγ pathway is important for regulating the metabolism of the whole body.

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.

Position-independent human β-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken β-globin insulator

AbstractThe position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken β-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human β-globin locus control region (LCR), the human β-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken β-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human β-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse βmaj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human β-globin gene ranged from 51.6% to 765.6% of that in the mouse βmaj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human β-globin gene ranged from 52.8% to 58.3% of that in the mouse βmaj-globin gene. These results indicate that the insulator functioned dramatically to reduce the variability of transgene expression due to the position effect. This insulator-rAAV vector system holds promise to provide a constant level of transgene expression for gene therapy, regardless of the insertion sites on the chromosome.