Norman S. Wolf

Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.

Metformin improves healthspan and lifespan in mice

Metformin is a drug commonly prescribed to treat patients with type 2 diabetes. Here we show that long-term treatment with metformin (0.1% w/w in diet) starting at middle age extends healthspan and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimics some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced LDL and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increases AMP-activated protein kinase activity and increases antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation. Our results indicate that these actions may contribute to the beneficial effects of metformin on healthspan and lifespan. These findings are in agreement with current epidemiological data and raise the possibility of metformin-based interventions to promote healthy aging.

Enhanced cell proliferation and biosynthesis mediate improved wound repair in refed, caloric-restricted mice

Aged mice that have undergone long-term caloric-restriction (CR) have improved health and enhanced longevity in comparison to aged mice that are ad libitum-fed (AL). However, caloric-restriction does not benefit the impaired wound healing of aged mice. To test the hypothesis that CR mice have the capacity for enhanced wound repair, but require a short-term period of additional nutrient intake to show this advantage, we assessed wound healing in CR mice that had been refed (RF) an ad libitum diet for 4 weeks prior to wounding. Two strains of AL young (Y AL) (4-6 months), AL middle-aged (M AL) (15-17 months), and three different, matched cohorts of old mice (O) (30-33 months): O AL, O CR, and O RF were studied. Two full-thickness 4 mm diameter punch biopsy skin wounds were created on the dorsum of each mouse. Animals were sacrificed and wounds were harvested at 1,2,3,5, and 7 days post-wounding. Repair of wounds was slower in O AL and O CR mice compared to Y AL and M AL animals. In contrast, the O RF mice healed similarly to that of the Y AL and M AL mice, as assessed by measures of wound area and histologic criteria. O RF mice demonstrated enhanced synthesis of type I collagen mRNA in comparison to O AL and O CR mice. A greater number of endothelial cells and fibroblasts at the wound edge of the O RF mice exhibited replication in vivo as measured by uptake of BrdU. O RF mice had higher levels of insulin-like binding protein 3 (IGFBP-3). Furthermore, fibroblasts derived from the explant of the punch biopsy of O CR mouse skin revealed enhanced proliferation and contraction in vitro, in comparison to fibroblasts from the O AL mice. In conclusion, O RF mice demonstrate an enhanced capacity to undergo wound repair in comparison to O AL mice. This effect appears to be mediated, in part, by enhanced cell proliferation, contraction, and collagen biosynthesis. In addition, short-term refeeding induced an increase in the serum level of IGFBP-3, the major binding protein for IGF-1. These data confirm that cells from O CR animals have a preserved proliferative, biosynthetic, and contractile capacity, but that an adequate source of nutrients is necessary to demonstrate this advantage in wound healing.

Cellular proliferation potential during aging and caloric restriction in rhesus monkeys (Macaca mulatta)

Caloric restriction (CR) is the most successful method of extending both median and maximal lifespans in rodents and other short‐lived species. It is not yet clear whether this method of life extension will be successful in longer‐lived species, possibly including humans; however, trials in rhesus monkeys are underway. We have examined the cellular proliferative potential of cells from CR and AL (ad libitum fed) monkey skin cells using two different bioassays: colony size analysis (CSA) of dermal fibroblasts isolated and cloned directly from the skin and beta‐galactosidase staining at pH 6.0 (BG‐6.0) of epidermal cells in frozen sections of skin. Decreases in both proliferative markers occurred with age, but no differences were observed between CR and AL animals. Skin biopsies were obtained from AL and CR rhesus monkeys from two different aging colonies, one at the National Institute on Aging (NIA) and one at the University of Maryland‐Baltimore (UMB). These biopsies were used as a source of tissue sections and cells for two biomarkers of aging assays. The CR monkeys had been maintained for 9–12 years on approximately 70% of the caloric intake of control AL animals. In the CSA studies, the fraction of small clones increased significantly and the fraction of large clones decreased significantly with increasing age in AL monkeys. The frequency of epidermal BG‐6.0 staining cells increased with age in older (>22 years) AL monkeys, but most predominately in those of the UMB colony, which were somewhat heavier than the NIH AL controls. Old monkeys on CR tended to have fewer BG‐6.0‐positive cells relative to old AL‐derived epidermis, but this effect was not significant. These results indicate that cellular proliferative potential declined with age in Macaca mulatta, but was not significantly altered by CR under these conditions. Although these experiments are consistent with an absence of effect of CR on monkey skin cell proliferative potential, we have found in previous experiments with mice that a longer duration of CR (as a fraction of total lifespan) was needed to demonstrate CR‐related improvement in clone size in mice. Further studies on the now mid‐aged monkeys will be needed as their age exceeds 20 years to conclusively rule out an effect of CR on proliferative potential of skin cells from these primates. J. Cell. Physiol. 180:123–130, 1999.

Disruption of protein kinase A in mice enhances healthy aging

Mutations that cause a reduction in protein kinase A (PKA) activity have been shown to extend lifespan in yeast. Loss of function of mammalian RIIβ, a regulatory subunit of PKA expressed in brain and adipose tissue, results in mice that are lean and insulin sensitive. It was therefore hypothesized that RIIB null (RIIβ−/−) mice would express anti-aging phenotypes. We conducted lifespan studies using 40 mutant and 40 wild type (WT) littermates of equal gender numbers and found that both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the 80% lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). There was no difference in either median or 80% lifespan in female genotypes. WT mice of both genders became increasingly obese with age, while mutant mice maintained their lean phenotype into old age. Adiposity was found to correlate with lifespan for males only. 50% of male mice between 30 and 35 g, corresponding to about 5% body fat, for either genotype lived over 1000 days. No male mouse outside of this weight range achieved this lifespan. During their last month of life, WT mice began losing weight (a total of 8% and 15% of body weight was lost for males and females, respectively), but RIIβ−/− male mice maintained their lean body mass to end of life. This attenuation of decline was not seen in female mutant mice. Old male mutant mice were insulin sensitive throughout their life. Both genders showed modestly lower blood glucose levels in old mutants compared to WT. Male mutants were also resistant to age-induced fatty liver. Pathological assessment of tissues from end of life male mutant mice showed a decrease in tumor incidence, decreased severity of renal lesions, and a trend towards a decrease in age-related cardiac pathology. These findings help establish the highly conserved nature of PKA and suggest that disruption of PKA affects physiological mechanisms known to be associated with healthy aging.

Dissecting the hematopoietic microenvironment. II. The kinetics of the erythron of the sl/sld mouse and the dual nature of its anemia.

An analysis of the S1/S1d mouse for ferrokinetics and fate of peripheral red blood cells has shown the cause of its anemia to be dual in nature. While the S1/S1d produces red cells at a slightly greater rate than its normal littermate, its bone marrow and spleen appear to be operating near their maximal capacity and will reduce their output if anemic stress is partly relieved. the cause of the moderately high level of erythropoiesis in the S1/S1d is a mean daily loss of 2.5–3.0% of its total blood volume via the intestinal tract.

DISSECTING THE HEMATOPOIETIC MICROENVIRONMENT. I. STEM CELL LODGMENT AND COMMITMENT, AND THE PROLIFERATION AND DIFFERENTIATION OF ERYTHROPOIETIC DESCENDANTS IN THE Sl/Sld MOUSE

The anemic Sl/Sld mouse and its normal (+/+) congenic control were used to explore the possibility of stromal control over four phases of erythropoiesis: CFU lodgment, commitment of multipotent stem cells to the erythropoietic line, proliferation of stem cells and their descendants, and the differentiation of those descendants into successively more mature forms. Lodgment was found to be the same in the Sl/Sld as in the normal mouse, but commitment, although characteristically different for spleen compared to the bone marrow, was subnormal. The stimulus to proliferate, as measured by spleen colony size and cell type content, was even more reduced. It is suggested that the direct control of differentiation into more mature cells may not be under stromal control.

DNA double-strand breaks in mouse kidney cells with age

A Biojector device fitted with a CO2 cartridge was used to prepare single cellsuspensions from kidneys of 12-month-(middle-aged) and 24-month-old (old) C57Bl/6mice. Microgel electrophoresis of DNA fromthese cells revealed a modest but significant7.3% increase (P = 0.04) in DNA double-strand breaks in old mice. This increase is equivalent to the DNA damage induced by 0.1 Gray of X-rays (5 double-strand breaks) in kidney cells of 10-month-old mice, as determined by a standard calibration curve. Greater DNA damage with aging was also positively correlated with higher levels of pathology in the kidneys.

Hormone levels and cataract scores as sex-specific, mid-life predictors of longevity in genetically heterogeneous mice

Serum levels of thyroxine (T4), leptin, and insulin-like growth factor-I (IGF-I), as well as cataract severity, were evaluated as predictors of life span in a population of genetically heterogeneous mice (UM-HET3). Long life span was predicted by low levels of leptin at age 4 months in females, and by low levels of IGF-I at age 15 months and high levels of T4 at age 4 months, in males. Cataract severity at either 18 or 24 months was also a significant predictor of life span in females only, but in contrast to what has been reported in human studies, relatively severe cataract was correlated with longer life span. Additional work is needed to evaluate the role of these hormones as potential modulators of the aging process, and to resolve the conflicting data obtained for cataract severity as a predictor of life span.

The effect of long-term caloric restriction on function of T-cell subsets in old mice

The effect of caloric restriction (from weaning to old age) on CD3-stimulated CD4+ and CD8+ lymphocyte proliferation and calcium mobilization was examined. Young ad libitum (ad lib) fed, old ad lib fed, old calorically restricted, and old calorically restricted mice which were fed ad lib during the last 6 weeks of their life (restricted/refed) were compared in both BDF1 [(C57BL/6 x DBA/2)F1] and C57BL/6 mice. Proliferation of CD4+ cells was lower in old ad lib animals than in young animals; this difference was not seen in CD8+ cells. Those CD4+ cells which did proliferate in old ad lib animals underwent similar cell cycle progression as young cells. In calorically restricted and calorically restricted/refed animals, CD4+ cell proliferation was similar to the young animals, and CD8+ cells showed a higher proliferative capacity than cells from either young or old ad lib mice. Differences in proliferative capacity were not correlated with alterations in transmembrane signaling efficiency as peak [Ca2+]i was reduced in both T-cell subsets in all groups of old mice relative to young mice. Additionally, reduced [Ca2+]i was observed in the CD8+ subset for which there was no deficit in proliferation, and the enhanced proliferation seen in old restricted and old restricted/refed mice did not manifest as increased [Ca2+]i mobilization. The percentage of CD4+ cells from both mouse strains was reduced in all groups of old mice compared with young mice, while the percentage of CD8+ cells was generally similar in young and all groups of old mice. Our studies would suggest that lifelong caloric restriction of mice prevents the age-associated decrease in T-cell proliferative capacity but that the enhanced proliferation of these cells is not due to increased efficiency of transmembrane signaling.