Age-associated increases in the activity of multiple caspases in Fisher 344 rat organs

As organisms age, an increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling positive cells has been observed in a variety of tissues and cell types. However, whether this represents the increase of apoptosis has not been validated on molecular level. In this study we examined the endogenous activity of caspases that are known to be responsible for the execution of caspase-dependent apoptosis as a function of age in rat liver, lung, and spleen. We demonstrate that the extent of apoptosis in rat liver increases late during the aging process (i.e. 23-27 month) as indicated by the activation of executioner caspases-3, -6, and -7. We also found that the activity of caspase-3, -6, and -7 increased drastically in rat lung and spleen at late stages of aging. Despite reports that the level of Fas mRNA increases with age in rat liver and that Fas system regulates liver homeostasis, we did not detect activation of caspase-8, a key mediator of Fas-induced apoptosis, in aged liver. We also observed increased activities of two caspases, caspase-2 and caspase-9, which are involved in mitochondrion-mediated apoptosis in livers isolated from old rats, and found that hepatocytes isolated from old animals (>23 month) are more sensitive to oxidative stress that targets the mitochondria compared to those isolated from young (6 month) animals. Lastly, we demonstrate that the level of cytochrome c is lower in liver from old animals, probably as a result of expeditious degradation following its release into cytosol. Collectively, our results demonstrate that aging is associated with an increase in the activity of multiple caspases, suggesting that the extent of apoptosis increases as organs age. In the case of rat liver, this increase in caspase activation is more likely associated with the mitochondrial (i.e. intrinsic) pathway rather than the Fas-mediated caspase-8 (extrinsic) pathway of apoptosis.     

Estrogen increases galanin immunoreactivity in hyperplastic prolactin-secreting cells in Fisher 344 rats.

Galanin is a widely distributed regulatory peptide which modulates the pituitary secretion of PRL and GH. Estrogen administration strongly stimulates galanin gene expression in the rat anterior pituitary. In adult female Fischer 344 rats, estrogen also induces hyperplasia of lactotropes. We used immunocytochemical analysis to assess the effects of estrogen on galanin-like immunoreactivity (Gal-IR) in the rat pituitary and hypothalamus during sc diethylstilbestrol (DES) implantation and after its removal at 30 days. In the anterior pituitary, DES implantation increased the portion of Gal-IR-containing cells from less than 2% in the control rats to 18.3% after 3 days of DES and 36% after 30 days. These changes paralleled the lactotrope hyperplasia exhibited in response to DES exposure. Ten and 30 days after removal of the DES capsules, the percentage of Gal-IR-containing cells in the anterior pituitary decreased to 6.3% and 1.5%, respectively. Colocalization studies revealed that Gal-IR-containing cells were predominantly lactotropes. Immunoelectron microscopy demonstrated that Gal-IR was concentrated in the Golgi region of these hyperplastic lactotropes and suggests that little of the synthesized galanin is secreted. The distribution of Gal-IR in the hypothalamus, median eminence, and neurohypophysis was unaffected by DES treatment. These data demonstrate that galanin is synthesized by hyperplastic pituitary lactotropes of Fischer 344 rats and that peptide accumulation is dependent on the presence of circulating estrogens. In contrast, neuronal galanin synthesis in the hypothalamus does not appear to be regulated by estrogen.     

Effects of aging and calorie restriction of Fischer 344 rats on hepatocellular response to proliferative signals

It is well established that the proliferative potential of the liver declines with aging. Epidermal growth factor (EGF)-stimulated DNA synthesis is reduced in hepatocytes from aged rats relative to young rats, and this reduction correlates with diminished activation of the extracellular signal-regulated kinase (ERK) pathway and lower phosphorylation of the EGF receptor on residue Y1173. Calorie restriction (CR) can increase rodent life span and retard many age-associated declines in physiologic function, but its influence on cell proliferation is unknown. Here, we investigated the effects of long-term CR on proliferation of hepatocytes derived from young and aged rats following in vitro stimulation with either low-dose hydrogen peroxide or EGF. CR reduced the proliferative response of hepatocytes derived from young hosts, but long-term CR was associated with enhanced proliferation in aged cells relative to that of ad libitum (AL)-fed animals. ERK activation mirrored the effects of CR on proliferation, in that young CR cells exhibited lower ERK activation than young AL cells, but old CR cells showed higher ERK activation than old AL cells. Finally, a decline in EGF receptor phosphorylation on Y1173, which normally occurs with aging, was absent in cells of old hosts maintained on long-term CR, supporting the view that alterations in this early signaling event underlie the age-related decline in proliferative potential in rat hepatocytes.     

Liver DT-diaphorase activity increases with age and dietary restriction in Lobund-Wistar rats

Effects of a 30 percent (%) dietary restriction on liver DT-diaphorase activity toward various substrates were studied in male Lobund-Wistar (L-W) rats throughout the life span. DT-diaphorase catalyzes the reduction of quinones to hydroquinones, thus playing a major role in protecting cells against oxidative stress manifested by quinines. Eighty rats were divided into 2 groups and fed ad libitum or a 30% calorie-restricted diet. Eight rats in each group were killed at 6, 12, 18, 24 and 30 months of age. DT-diaphorase activity in liver cytosol was determined. The specific activity (per mg protein) of the enzyme in the ad libitum group toward 1,4-naphthoquinone (NQ) increased with age from 6 to 30 months, while those toward 2,6-dichlorophenol-indophenol (DCPIP), 2-methyl-1,4-naphthoquinone (MNQ) and ubiquinone₀ (UQ₀) increased with age from 18 to 30 months. The specific activity of the enzyme toward all four substrates in the restricted group did not change between 6 and 18 months, then increased with age from 18 to 30 months. When the enzyme activity was calculated on a per gram body weight basis or a per gram liver basis, the age-associated patterns of the ad libitum and restricted groups were similar to those on per mg protein basis. Dietary restriction increased the activity of DT-diaphorase on per mg protein basis, per gram body weight basis, or per gram liver basis, at almost all age points. The results suggest that dietary restriction contributes to the delaying of the aging process by decreasing oxidative stress exerted by quinones through increased DT-diaphorase activity.     

Low-level caloric restriction rescues proteasome activity and Hsc70 level in liver of aged rats

Proteasome activity is known to decrease with aging in ad libitum (AL) fed rats. Severe caloric restriction (CR) significantly extends the maximum life-span of rats, and counteracts the age-associated decrease in liver proteasome activities. Since few investigations have explored whether lower CR diets might positively counteract the age associated decrease in proteasome activity, we then investigated the effects of a mild CR regimen on animal life-span, proteasome content and function. In addition, we addressed the question whether both CR regimens might also affect the expression of Hsc70 protein, a constitutive chaperone reported to share a role in the function of proteasome complex and in the repair of proteotoxic damage, and whose level decreased during aging. In contrast to severe CR, mild CR had a poor effect on life-span; however, it better counteracted the decrease of proteasome activities. Both regimens, however, maintain Hsc70 in liver of old rats at level comparable to that of young rats. Interestingly, the effects of aging and CRs on liver proteasome enzyme activities did not appear to be associated with parallel changes in the amount of proteasome proteins suggesting that the quality (molecular activity of the enzymes) rather than the quantity are likely to be modified with age. In conclusion, the results presented in this work show that a mild CR can have beneficial effects on liver function of aging rats because is adequate to counteract the decrease of proteasome function and Hsc70 chaperone level.     

Glial glucocorticoid receptors in aged Fisher 344 (F344) and F344/Brown Norway rats

Glucocorticoid receptors (GR) regulate glial function, and changes in astrocyte gene expression are implicated in age-related pathology. We evaluated changes in astroglial GR expression in two strains of rats – Fisher 344 (F344; 4, 12 and 24 months) and F344/Brown Norway strain (F344/BN; 4, 12 and 30 months). In both strains basal levels of corticosterone were higher in the oldest groups of rats. Age-related increases in GR (+) astrocytes but not the percent of astrocytes expressing GR were observed in the hippocampus CA1 region in F344 rats. Age-related decreases in CA1 GR (+) astrocytes and the percentage of GR (+) astrocytes were observed in the F344/BN strain only. Similar strain-specific changes were observed in the dentate gyrus. In the hypothalamic paraventricular nucleus: (1) F344 rats exhibited significant decreases in the overall number of glial profiles with age, (2) F344/BN rats exhibited decreases in the numbers of GR (+) astrocytes with aging and (3) the proportion of GR (+) astrocytes decreased in older F344/BN, but not F344 rats. Overall, the data demonstrate age- and strain-related alterations in GR astrocytic expression that may explain unique phenotypic differences in brain function observed in both strains.     

Repeat mild heat shock increases dermal fibroblast activity and collagen production

Repeat mild heat shock (RMHS) has been shown to have anti-aging effects on cellular and biological processes within human dermal fibroblasts. We have investigated the potential of an abridged mild heat shock regime to impact upon the functional properties of human dermal fibroblasts derived from three donors (male, 12 years; female, 22 years; female, 65 years). For each donor mild heat shock increased the rate of contraction of fibroblast-containing collagen gels and increased the de novo synthesis of collagen. Thus, hormetic mechanisms are proposed to provide functional anti-aging benefits to skin cells.     

Probiotics inhibit nuclear factor-kappaB and induce heat shock proteins in colonic epithelial cells through proteasome inhibition

BACKGROUND AND AIMS: The extent and severity of mucosal injury in inflammatory bowel diseases are determined by the disequilibrium between 2 opposing processes: reparative and cytoprotective mechanisms vs. inflammation-induced injury. Probiotics may provide clinical benefit by ameliorating colitis; however, their mechanisms of action remain largely unknown. Our objective was to investigate microbial-epithelial interactions that could explain the beneficial therapeutic effects of probiotics. METHODS: The effect of VSL#3-conditioned media on the nuclear factor-kappaB pathway in young adult mouse colonic epithelial cells was assessed by using monocyte chemoattractant protein-1 enzyme-linked immunosorbent assays; IkappaBalpha, IkappaBbeta, and p105 immunoblot analysis; and nuclear factor-kappaB luciferase reporter gene and proteasome assays. Effects on heat shock proteins were determined by electrophoretic mobility shift assay and immunoblot for heat shock proteins 25 and 72 in young adult mouse colonic cells. Cytoprotection against oxidant injury was determined by chromium 51 release and filamentous and globular actin assays. RESULTS: VSL#3 produces soluble factors that inhibit the chymotrypsin-like activity of the proteasome in gut epithelial cells. Proteasome inhibition is an early event that begins almost immediately after exposure of the epithelial cells to the probiotic-conditioned media. In addition, these bacteria inhibit the proinflammatory nuclear factor-kappaB pathway through a mechanism different from the type III secretory mechanisms described for other nonpathogenic enteric flora. They also induce the expression of cytoprotective heat shock proteins in intestinal epithelial cells. CONCLUSIONS: The resulting inhibition of nuclear factor-kappaB and increased expression of heat shock proteins may account for the anti-inflammatory and cytoprotective effects reported for probiotics and may be a novel mechanism of microbial-epithelial interaction. These effects seem to be mediated through the common unifying mechanism of proteasome inhibition.     

The effect of aging on vasopressin system in Fisher 344 rats

Vasopressin (VP) was measured by RIA in the plasma, neurohypophysis (NH) and hypothalamus (HT) of young (2 months), adult (12 months) and old (30 months) male Fisher 344 rats, ten of each age. Plasma VP concentration was significantly lower in old compared to adult and to young rats. VP content in the NH expressed per mg weight was similar in all three groups, while the hypothalamic VP content was decreased in the aged rats. This suggests that reduced synthesis and release and/or increased degradation of VP occurs in aged rats. These data are in an agreement with our previous results obtained with Sprague-Dawley rats indicating that these differences are not strain-related. In a pilot experiment, we further studied VP release from isolated perifused NH of young and old rats. Two glands of each age were simultaneously perifused in individual microchambers with medium TC 199 and subsequently stimulated by Locke's medium containing 56 mM K+. Both, the initial and the basal VP release from the NH of the old rats, as well as the response to high K+, were about a half that of the young rats. Thus, a decreased VP release may contribute to the findings of lower plasma VP concentration in aged rats.     

Cholesterol synthesis in tissues of young and old Fisher 344 and Sprague-Dawley rats

Slices of liver, kidney, spleen and colon from young (45-day) or old (18-month) Fisher 344 and Sprague-Dawley rats were used to measure cholesterol synthesis from sodium (1-¹⁴C) acetate. Cholesterogenesis is reduced with age. Kidney and colon slices from young Fisher 344 rats synthesized more cholesterol that their Sprague-Dawley counterparts. Synthesis of cholesterol by tissues from old rats was similar in the two strains.     

Influence of aging and calorie restriction on MAPKs activity in rat kidney

Mitogen-activated protein kinases (MAPK), which include the extracellular signal-related kinases (ERK), the c-Jun N-terminal kinases (JNK), and the p38 MAPK, are important regulatory proteins by which a wide variety of extracellular signals are transduced into intracellular sites. Recent studies reported that mitogenic signal transduction in various cell types are exquisitely sensitive to reactive oxygen species (ROS) and the celluar redox status. In the present study, we investigated the activation of MAPK activity by aging and calorie restriction (CR) in rat kidneys isolated from Fischer 344 rats, ages 6, 12, 18, and 24 months fed ad libitum (AL) and CR diets. Results showed that the aging process strongly enhanced all three of the MAPK activities studied, ERK, JNK, and p38 MAPK, in parallel to increased ROS status. In contrast, we observed CR to markedly suppress the age-related activation of MAPKs. Based on these data, we concluded that an age-related increase in MAPK activity is associated with increased ROS, which was effectively suppressed by the anti-oxidative action of CR.     

Similar metabolic responses to calorie restriction in lean and obese Zucker rats

Calorie restriction (CR), which is thought to be largely dependent on the neuroendocrine system modulated by insulin/insulin-like growth factor-I (IGF-I) 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.     

Short-term caloric restriction and regulatory proteins of apoptosis in heart,skeletal muscle and kidney of Fischer 344 rats

Long-term caloric restriction reduces oxidative stress, increases mean and maximum lifespan in rodents and tends to enhance apoptosis, particularly in the liver. We investigated the effect of short-term (2 months) caloric restriction (40% reduction) in 6-month-oldmale Fischer 344 rats on various indicators of apoptosis (caspase-3, -7, -12, the inhibitor of apoptosis protein XIAP and cytoplasmic histone-associated DNA fragments)in the post-mitotic heart and gastrocnemius muscle, and the kidney that contains mitotic cells. Short-term caloric restriction significantly reduced body mass (30%), gastrocnemius muscle mass (22%), heart mass (25%) and kidney mass (32%) compared toad libitum controls. The levels of procaspase-3 in gastrocnemius muscle and caspase-3in kidney were significantly lower in the caloric restricted than in the ad libitumfed group. While caloric restriction did not alter DNA fragmentation levels(indicative of apoptosis), differences did exist amongst tissues with significantly elevated levels of fragmentation in the kidney compared to the heart and gastrocnemius muscle and significantly higher levels in the heart compared to gastrocnemius muscle. No differences were observed between groups in the levels of procaspase-7 or -12 or in XIAP (an endogenous inhibitor of apoptosis, particularly of caspase-3 and -7) in any tissue. The active forms of caspase-7 and -12 were present only in the kidney. These findings suggest that while the rate of apoptosis was higher in the kidney, which contains mitotic cells, compared to the post-mitotic heart and gastrocnemius muscle, short-term caloric restriction did not enhance the apoptosis rate in any tissue measured. This revised version was published online in July 2006 with corrections to the Cover Date.     

Resistance training increases heat shock protein levels in skeletal muscle of young and old rats

Heat shock proteins (HSP) HSP72, HSC70 and HSP25 protein levels and mRNA levels of HSP72 genes (Hsp72-1, Hsp72-2, Hsp72-3) and HSC70 were examined in tibialis anterior muscles from young and old rats following 4.5 weeks of heavy resistance exercise. Young (3 months) (n=10) and old (30 months) (n=9) rats were subjected to 14 sessions of electrically evoked resistance training using stretch-shortening contractions of the left limb that activated the dorsiflexor muscle group, including the tibialis anterior muscle, while the right side served as the intra-animal control. Muscle wet weight of the left tibialis anterior increased by 15.6% in young animals compared to the untrained right side, while the aged rats demonstrated no significant hypertrophy based on muscle wet weight. There were no differences in mRNA expression between the control and experimental muscles in either the old or the young animals for any of the four genes examined. On the other hand, HSP72 levels as determined by Western blots were significantly (p<0.01) higher (968.8 and 409.1%) in the trained as compared to the contralateral control muscle in young and old animals, respectively. HSP25 expression was increased significantly (p<0.01) by training in muscles of young rats (943.1%) and old rats (420.3%). Moreover, there was no training by age interaction for HSP72, while a significant age and training by age effects were found in muscles for HSP25. There was no change in HSC70 protein expression in response to the training intervention in either age group. SOD-1 enzyme level increased by 66.6% in the trained muscles of the young rats, while this enzyme was 33% lower in trained muscles compared to the untrained control side in old rats. Moreover, a significant (p<0.05) training by age interaction was found for SOD-1 enzyme levels. This study suggests that fast contracting muscles in young and old animals are capable of increasing HSP expression in response to high intensity contractile stress. Furthermore, the data are consistent with the hypothesis that higher levels of oxidative stress in muscles of old animals limit HSP levels and/or function in response to high intensity contractile stress.     

Effect of pre-induction of heat shock proteins on indomethacin-induced small-intestinal lesion in rats

Systemic hyperthermia induces the synthesis of heat shock proteins (HSPs) in several organs. However, the mechanism of induction and the functions of HSPs in the small-intestinal mucosa have not been established. We examined the expression of HSPs in the small-intestinal mucosa after systemic hyperthermia, and evaluated the cytoprotective function of preinduced HSPs on experimentally induced mucosal damage. HSP expression was investigated by Western blot and densitometric analysis before and after hyperthermia (42.5°C; 20min). Expression of a 72-kDa heat shock protein (HSP72) and a 73-kDa heat shock protein (HSP73), both of which are endogenous cytoprotectants in vitro significantly increased, peaking 6–9h after hyperthermia, without any pathologic alterations, whereas the expression of a 60-kDa heat shock protein (HSP60) did not increase. To investigate the influence of pre-induction of HSPs on small-intestinal damage, rats received indomethacin (10mg/kg; orally) with or without pre-treatment with hyperthermia. Small-intestinal damage caused by indomethacin was not influenced by pre-induction of HSP72 and HSP73. We demonstrated that systemic hyperthermia induced HSP72 and HSP73, although pre-induction of these proteins did not have a cytoprotective function in the smallintestinal damage caused by indomethacin.     

Expression and immunolocalization of heat shock proteins in the healing of gastric ulcers in rats

BACKGROUND: Heat shock proteins are induced when cells are subjected to noxious stimuli. They afford cytoprotection and increase the resistance of the tissue to damage. However, their roles in the healing of gastric ulcers have not been well established. In this study, the expression and immunolocalization of three heat shock proteins (HSPs); namely inducible HSP70 (iHSP70), HSP47, and HSP32 during ulcer healing were investigated in rats with gastric ulcer. METHODS: Gastric ulcers (kissing ulcers) were induced by luminal application of acetic acid solution. Gastric tissue samples were obtained from the ulcer base, ulcer margin, and non-ulcerated area around the ulcer margin at different time intervals after ulcer induction. The protein levels and distributions of HSPs were analyzed with Western blotting and immunohistochemical methods, respectively. RESULTS: It was found that all HSPs were expressed in normal, non-ulcerated, and gastric ulcer tissues. HSP32 was elevated during inflammation (1-8 days after ulcer induction), while HSP47 expression was exacerbated at the ulcer base and margin during ulcer healing (3-12 days). Decreased expression of iHSP70 was observed at the ulcer base immediately after ulcer induction, but returned to normal level by the end of the healing stage (8-12 days). Inducible HSP70 was found distributed in the gastric glands and injured tissues in the inflamed areas. Wide distribution of HSP47 was detected in granulation tissues and collagen producing cells, while HSP32 was localized in the gastric glands and inflammatory cells. CONCLUSIONS: The findings indicate that iHSP70, HSP47, and HSP32 play different roles during ulcer healing. HSP32 seems to act as an inflammatory defensive factor, and HSP47 as a collagen-specific molecular chaperon contributing significantly to gastric ulcer healing. However, the role of iHSP70 in the ulcer healing process is still undefined.     

Locomotor activity in female rhesus monkeys: assessment of age and calorie restriction effects

As a component of a long-term, longitudinal study of aging in this primate model, the objective of the current experiment was to assess age and diet effects on locomotor activity in a cross-sectional analysis. By attaching a motion detection device to the home cage, locomotor activity was monitored over a week in a group (N = 47) of female rhesus monkeys (Macaca mulatta) 6-26 yrs of age. About half these monkeys composed a control group fed a nutritionally fortified diet near ad libitum levels, whereas an experimental group had been fed the same diet at levels 30% less than comparable control levels for approximately 5 yrs prior to testing. Among control monkeys, a marked age-related decline in activity was noted when total activity was considered and also when diurnal and nocturnal periods of activity were analyzed separately. When comparing activity levels between control and experimental groups, only one significant diet effect was noted, which was in the youngest group of monkeys (6-8 yrs of age) during the diurnal period. Monkeys in the experimental group exhibited reduced activity compared to controls. Body weight was not consistently correlated to activity levels. In some older groups, heavier monkeys tended to show greater activity, but in younger groups the opposite pattern was observed.     

Age-related changes of muscarinic cholinergic receptor subtypes in the striatum of Fisher 344 rats

Striatum expresses a cholinergic system involved in the regulation of its activity and changes in striatal cholinergic receptors may be related to cognitive impairment. This study has investigated muscarinic cholinergic M1-M5 receptor subtype expression in striatum of Fischer 344 rats aged 6 (young), 15 (adult) and 22 months (senescent) to assess the contribution of different muscarinic cholinergic receptor subtypes in age-related changes of striatal cholinergic neurotransmission. Western blot analysis revealed the expression of the M1-M5 muscarinic receptor subtytpes in the striatum of rats of the three age groups investigated. Both radioligand binding assay and light microscope autoradiography showed in young rats a M4>M1>M2>M3>M5 rank order of receptor density. With the exception of M1 receptor, the density of which is similar in the dorsal (motor) and ventral (limbic) striatum, other receptor subtypes were more abundant in ventral than in dorsal striatum. M1 receptor expression was unchanged between young and adult rats and decreased in senescent animals both in dorsal and ventral striatum. In dorsal striatum M2 and M5 receptor expression did not show age-related changes, whereas in ventral striatum it was slightly decreased in adult rats compared to young or senescent cohorts. M3 receptor expression did not show age-related modifications, whereas a progressive age-related decrease of M4 receptor was found, both in dorsal and ventral striatum. These data indicate a heterogeneous response to age of different muscarinic receptor subtypes. Striatal cholinergic markers are thought to correlate with cognitive impairment in aged rats. In view of this, the identification of age-related changes of striatal muscarinic receptor subtypes may contribute to develop cholinergic strategies to counter cholinergic neurotransmission changes occurring with aging.