Fibroblasts from long-lived mutant mice exhibit increased autophagy and lower TOR activity after nutrient deprivation or oxidative stress

Previous work has shown that primary skin-derived fibroblasts from long-lived pituitary dwarf mutants resist the lethal effects of many forms of oxidative and nonoxidative stress. We hypothesized that increased autophagy may protect fibroblasts of Pit-1(dw/dw) (Snell dwarf) mice from multiple forms of stress. We found that dwarf-derived fibroblasts had higher levels of autophagy, using LC3 and p62 as markers, in response to amino acid deprivation, hydrogen peroxide, and paraquat. Fibroblasts from dwarf mice also showed diminished phosphorylation of mTOR, S6K, and 4EBP1, consistent with the higher levels of autophagy in these cells after stress. Similar results were also observed in fibroblasts from mutant mice lacking growth hormone receptor (GHRKO mice) after amino acid withdrawal. Our results suggested that increased autophagy, regulated by TOR-dependent processes, may contribute to stress resistance in fibroblasts from long-lived mutant mice.     

Augmented autophagy pathways and MTOR modulation in fibroblasts from long-lived mutant mice

Fibroblasts from long-lived pituitary dwarf mutants, including Snell dwarf, Ames dwarf and the growth hormone receptor knockout (GHRKO) mice, are resistant in culture to multiple forms of lethal stress. We found that fibroblasts from Snell dwarf and GHRKO mice are more susceptible than control cells to autophagy induced by amino acid withdrawal or by oxidative stress. We also found evidence for lower MTOR function in dwarf cells under conditions that induce autophagy, consistent with the evidence that increased autophagy requires lower TOR activity. Our results provide new hints about the connections between autophagy and aging in long-lived mutants with alterations in GH-IGF1 levels, and suggest a role for hyperactive autophagy in the resistance of cells from these mice to lethal stresses.     

Mechanisms of stress resistance in Snell dwarf mouse fibroblasts: Enhanced antioxidant and DNA base excision repair capacity,but no differences in mitochondrial metabolism

Dermal fibroblasts from long-lived Snell dwarf mice can withstand a variety of oxidative and non-oxidative stressors compared to normal littermate controls. Here, we report differences in the levels and activities of intracellular antioxidant and DNA repair enzymes between normal and Snell dwarf mice fibroblasts cultured under a variety of conditions, including: 3% and 20% ambient O₂; the presence and absence of serum; and the addition of an exogenous oxidative stress. The only significant difference between normal and dwarf cells cultured in complete medium, at 20% O₂, was an approximately 40% elevation of glutathione peroxidase (GPx) activity in the mutant cells. Serum deprivation elicited increases in GPx in both genotypes, but these activities remained higher in dwarf mouse cells. Dwarf mouse cells deprived of serum and challenged with exposure to paraquat or hydrogen peroxide showed a generally greater upregulation of catalase and DNA base excision repair enzymes. As these toxins can interact with mitochondria to increase mitochondrial ROS production, we explored whether there were differences in mitochondrial metabolism between normal and dwarf mouse cells. However, neither mitochondrial content nor the apparent mitochondrial membrane potential differed between genotypes. Overall, the results suggest that superior hydrogen peroxide metabolism and a marginally greater DNA base excision repair capacity contribute to the stress resistance phenotype of Snell dwarf mouse fibroblasts.     

Fibroblasts from long-lived Snell dwarf mice are resistant to oxygen-induced in vitro growth arrest

Snell dwarf mice live longer than controls, and show lower age-adjusted rates of lethal neoplastic diseases. Fibroblast cells from adult dwarf mice are resistant to the lethal effects of oxidative and nonoxidative stresses, including the carcinogen methyl methanesulfonate. We now report that dwarf-derived fibroblasts are slow to enter the stage of growth arrest induced by culturing normal cells under standard culture conditions at 20% O(2). Dwarf cells cultured at 20% O(2) resemble control cells cultured at 3% O(2) not only in their delayed growth arrest, but also in their rapid growth rates and resistance to both oxidative and nonoxidative forms of cytotoxic stress. Levels of the heat-shock protein HSP-70 respond to serum withdrawal and stress only in control cells, showing that intracellular signals are blunted in dwarf-derived cells. These data suggest a model in which stable epigenetic changes induced in skin fibroblasts by the hormonal milieu of the Snell dwarf lead to resistance to multiple forms of injury, including the oxidative damage that contributes to growth arrest in vitro and neoplasia in intact mice.     

Altered oxidative stress response of the long-lived Snell dwarf mouse

Several single gene mutations in mice that increase the murine life span have been identified, including the Pit-1 mutation which results in the Snell dwarf (Pit1(dw/dw)), however, the biological mechanism of this life-span extension is still unclear. Based on studies that show oxidative stress plays an important role in the aging process, we hypothesized that the increased longevity seen in Snell dwarf mice may result from a resistance to oxidative stress. We report that Snell dwarf mice respond to oxidative stress induced by 3-NPA differently than their wild type littermates. This altered response results in diminished activation of the MEK-ERK kinase cascade and virtually no phosphorylation of c-Jun at Ser63 in dwarf mice after 3-NPA treatment, despite a robust phosphorylation of Ser63 in wild type mice. We propose that this altered management of oxidative stress in dwarf mice is partially responsible for the increased longevity in Snell dwarf mice.     

Requirement of the Escherichia coli dnaK gene for thermotolerance and protection against H2O2

Thermotolerance in Escherichia coli is induced by exposing cells to a brief heat shock (42 degrees C for 15 min). This results in resistance to the lethal effect of exposure to a higher temperature (50 degrees C). Mutants defective in the recA, uvrA and xthA genes are more sensitive to heat than the wild-type. However, after development of thermotolerance these mutants are like the wild-type in their heat sensitivity. This suggests that thermotolerance is an inducible response capable of protecting cells from the lethal effects of heat, independently of recA, uvrA and xthA. Thermotolerance does not develop in a dnaK mutant. In addition, the dnaK mutant is sensitive to heat and H2O2, but is resistant to UV irradiation. This implies that the E. coli heat-shock response includes a mechanism that protects cells from heat and H2O2, but not from UV.     

Factors Contributing to the Poor Myelination in the Brain of the Snell Dwarf Mouse

Abstract: Conventional histological examination of the pituitary does not distinguish Snell dwarf mutants (dw/dw) from their normal littermates (+/?) in the neonatal stage. However, immunohistochemical examination of pituitaries of litters born to heterozygous Snell parents revealed that in approximately 25% of the glands examined, the number of positive cells was very low in the neonatal stage. We attempted to delineate the events resulting in the poor myelination in the brain of the Snell dwarf mouse, and to devise an immunohistochemical method for identifying the mutant neonate. Differences in the brain weights of the dw/dw and +/? mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in CNPase activity was found to be suppressed in the cerebrum and brain stem throughout the developmental stage, but not in the other parts of the brain. The yield of isolated myelin decreased by 58% in the mutant mouse, but CNPase activity was equivalent to that of control myelin. Differences in DNA content per cerebrum from the dw/dw and +/? mice first became apparent on the 10th day of age. Henceforth, the dw/dw mice showed no further increase, although the +/? mice continued to increase. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about 50% of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the mutant cerebrum is a hypomyelination due to reduced oligodendroglial proliferation caused by lack of circulating growth hormone.     

Low utilization of circulating glucose after food withdrawal in Snell dwarf mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.     

Skin-derived fibroblasts from long-lived species are resistant to some, but not all, lethal stresses and to the mitochondrial inhibitor rotenone

Fibroblast cell lines were developed from skin biopsies of eight species of wild-trapped rodents, one species of bat, and a group of genetically heterogeneous laboratory mice. Each cell line was tested in vitro for their resistance to six varieties of lethal stress, as well as for resistance to the nonlethal metabolic effects of the mitochondrial inhibitor rotenone and of culture at very low glucose levels. Standard linear regression of species-specific lifespan against each species mean stress resistance showed that longevity was associated with resistance to death induced by cadmium and hydrogen peroxide, as well as with resistance to rotenone inhibition. A multilevel regression method supported these associations, and suggested a similar association for resistance to heat stress. Regressions for resistance to cadmium, peroxide, heat, and rotenone remained significant after various statistical adjustments for body weight. In contrast, cells from longer-lived species did not show significantly greater resistance to ultraviolet light, paraquat, or the DNA alkylating agent methylmethanesulfonate. There was a strong correlation between species longevity and resistance to the metabolic effects of low-glucose medium among the rodent cell lines, but this test did not distinguish mice and rats from the much longer-lived little brown bat. These results are consistent with the idea that evolution of long-lived species may require development of cellular resistance to several forms of lethal injury, and provide justification for evaluation of similar properties in a much wider range of mammals and bird species.     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

VCAM‐1 upregulation accompanies muscle remodeling following resistance‐type exercise in Snell dwarf (Pit1dw/dw) mice

Snell dwarf mice (Pit1^dw/dw) exhibit deficiencies in growth hormone, prolactin, and thyroid stimulating hormone. Besides being an experimental model of hypopituitarism, these mice are long‐lived (>40% lifespan extension) and utilized as a model of slowed/delayed aging. Whether this longevity is accompanied by a compromised quality of life in terms of muscular performance has not yet been characterized. In this study, we investigated nontrained and trained muscles 1 month following a general validated resistance‐type exercise protocol in 3‐month‐old Snell dwarf mice and control littermates. Nontrained Snell dwarf gastrocnemius muscles exhibited a 1.3‐fold greater muscle mass to body weight ratio than control values although muscle quality, maximum isometric torque normalized to muscle mass, and fatigue recovery were compromised. For control mice, training increased isometric torque (17%) without altering muscle mass. For Snell dwarf mice, isometric torque was unaltered by training despite decreased muscle mass that rendered muscle mass to body weight ratio comparable to control values. Muscle quality and fatigue recovery improved twofold and threefold, respectively, for Snell dwarf mice. This accompanied a fourfold increase in levels of vascular cell adhesion molecule‐1 (VCAM‐1), a mediator of progenitor cell recruitment, and muscle remodeling in the form of increased number of central nuclei, additional muscle fibers per unit area, and altered fiber type distribution. These results reveal a trade‐off between muscle quality and longevity in the context of anterior pituitary hormone deficiency and that resistance‐type training can diminish this trade‐off by improving muscle quality concomitant with VCAM‐1 upregulation and muscle remodeling.     

Induction of chromosomal aberrations in active oxygen-generating systems. II. A study with hydrogen peroxide-resistant cells in culture

Cells hyper-resistant to hydrogen peroxide (H2O2) were obtained from a Chinese hamster cell line (CHL) by repeated treatments with H2O2 at stepwise increasing concentrations. A clonal line (R-8) was approximately 10 times more resistant to H2O2 than the parental cells, and retained its resistance for about 2 months in normal medium. However, with further passages after the completion of the present study, the elevated resistance gradually decreased. Although the concentration of H2O2 required to induce chromosomal aberrations in 50% of treated cells was about 10 times higher in R-8 than in the parental cells, there were no distinct differences between the cells in the induction of chromosomal aberrations by 3 alkylating agents (N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea and mitomycin C). The catalase activity of R-8 was 10-fold in comparison with the parental cells, but no obvious differences were seen in the activities of superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase. Therefore, the elevated H2O2-resistance seemed to be associated with the enhanced catalase activity. The induction of chromosomal aberrations in two O2- generating systems--xanthine oxidase plus hypoxanthine (XO + HX), and paraquat--was compared between R-8 cells and the ordinary CHL cells. XO + HX produced chromosomal aberrations in the parental cells but not in the R-8, while paraquat induced almost the same level of aberrations in both cell lines. This finding suggests that different active oxygens are responsible for the induction of aberrations in these two O2- generating systems, i.e., H2O2 in XO + HX and O2- in paraquat.     

Heightened induction of proapoptotic signals in response to endoplasmic reticulum stress in primary fibroblasts from a mouse model of longevity

Previous work from our laboratory has shown that primary fibroblasts from long-lived Snell dwarf mice display a higher sensitivity to the lethal effects of endoplasmic reticulum (ER) stressors, such as thapsigargin, than cells from normal mice. Here we show that thapsigargin induces higher expression of CHOP, enhanced cleavage of caspase-12, higher caspase-3 activity, and increased phosphorylation of c-JUN, all indicators of enhanced apoptosis, in dwarf fibroblasts. Dwarf and normal fibroblasts show no genotypic difference in up-regulating BiP, GRP94, and ERp72 proteins after exposure to thapsigargin. However, dwarf fibroblasts express lower basal levels of a number of putative XBP1 target genes including Armet, Edem1, Erdj3, p58(IPK) and Sec61a1, as well as Ire1α itself. Furthermore, when exposed to thapsigargin, dwarf fibroblasts display attenuated splicing of Xbp1, but similar phosphorylation of eIF2α, in comparison to normal fibroblasts. These data support the notion that IRE1/XBP1 signaling is set at a lower level in dwarf fibroblasts. Diminished Xbp1 splicing in dwarf-derived fibroblasts may tilt the balance between prosurvival and proapoptotic signals in favor of apoptosis, thereby leading to higher induction of proapoptotic signals in these cells and ultimately their increased sensitivity to ER stressors. These results, together with recent findings in Caenorhabditis elegans daf-2 mutants, point to a potential interplay between insulin/IGF-1 signals and unfolded protein response signaling.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O(2) and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O(2) exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway

Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals. Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination. These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks. The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells. Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells. The fda- mutants were slightly more resistant to UV light and H2O2. Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.     

Chronic low dose exposure to hydrogen peroxide changes sensitivity of V79 cells to different damaging agents

Chinese hamster V79 cells were repeatedly exposed to a low dose of hydrogen peroxide (H2O2) over several weeks and then exposed to H2O2, cisplatin or ultraviolet (UV) light. Cell killing was examined by colony formation, following these treatments. It was seen that cells conditioned by multiple low doses of H2O2 showed resistance to killing in case of H2O2 and cisplatin but the sensitivity to UV light was same as the control cells. Apoptosis was also determined in these cells after the same treatments. UV light failed to induce apoptosis in both conditioned and in control cells, but in case of cells treated with H2O2 and with cisplatin, there was less apoptosis in the conditioned cells compared to the control cells. From our observation we can say that the enhanced survival of cells after treatment with H2O2 or cisplatin could be due to inhibition of apoptosis.     

Fibroblasts from long‐lived species of mammals and birds show delayed,but prolonged,phosphorylation of ERK

Fibroblasts from long‐lived mutant mice show diminished phosphorylation of the stress‐activated protein kinases ERK1/2 after exposure to peroxide, cadmium, or paraquat. We have now evaluated the kinetics of ERK phosphorylation in fibroblasts from long‐lived and short‐lived species of mammals and birds in response to stress by cadmium or hydrogen peroxide. Fibroblasts from the shorter‐lived species of rodents and birds showed rapid induction of ERK phosphorylation, with a decline to basal level within 60 min. In contrast, cells from longer‐lived species showed slower and more prolonged activation of ERK phosphorylation. These results suggest that fibroblasts from long‐lived species may be less susceptible to the early phases of damage from cadmium or peroxide and suggest that altered kinetics of ERK activity may contribute to their stress resistance properties.     

Essential role of the czc determinant for cadmium, cobalt and zinc resistance in Gluconacetobacter diazotrophicus PAl 5

The mechanisms of cadmium, cobalt and zinc resistance were characterized in the plant-growth-promoting bacterium Gluconacetobacter diazotrophicus PAl 5. The resistance level of the wild-type strain was evaluated through the establishment of minimum inhibitory concentrations (MIC) of the soluble compounds CdCl2·H2O, CoCl2·6H2O and ZnCl2. Gluconacetobacter diazotrophicus PAl 5 was resistant to high concentrations of Cd, Co and Zn, with MICs of 1.2, 20 and 20 mM, respectively. Screening of an insertion library from transposon EZ-Tn5 in the presence of ZnO revealed that the mutant GDP30H3 was unable to grow in the presence of the compound. This mutant was also highly sensitive to CdCl2·H2O, CoCl2·6H2O and ZnCl2. Molecular characterization established that the mutation affected the czcA gene, which encodes a protein involved in metal efflux. In silico analysis showed that czcA is a component of the czcCBARS operon together with four other genes. This work provides evidence of the high tolerance of G. diazotrophicus PAl 5 to heavy metals and that czc is a determinant for metal resistance in this bacterium.