Age- and training-related changes in the collagen metabolism of rat skeletal muscle

The effects of ageing and life-long endurance training on the collagen metabolism of skeletal muscle were evaluated in a longitudinal study. Wistar rats performed treadmill running 5 days a week for 2 years. The activities of collagen biosynthesis enzymes, prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, were highest in the muscles of the youngest animals, decreased up to the age of 2 months and from then on remained virtually unchanged. The enzyme activity in young animals was higher in the slow collagenous soleus muscle than in the rectus femoris muscle. The enzyme activity in the soleus muscle was higher for older trained rats than older untrained rats. The relative proportion of type I collagen increased and that of type III collagen decreased with age, suggesting a more marked contribution by type I collagen to the age-related accumulation of total muscular collagen. The results show that collagen biosynthesis decreases with maturation and that life-long endurance training maintains a higher level of biosynthesis in slow muscles.     

Differential effect of fasting on IGF-BPs in serum of young and adult rats and its implication to impaired skin GAG content

During fasting or aging of animals there is a decreased content of skin glycosaminoglycans (GAGs). It has been found that the skin of adult rats contains about 60% of GAGs found in the skin of young animals. Fasting of both groups of animals (young and adult) resulted in decrease of GAG content. However, GAG content in the skin of fasted young rats decreased by 30% and in fasted adult rats by 15% only, compared to fed animals, respectively. The mechanism for the phenomena is not known. We considered insulin-like growth factor-I (IGF-I) as a potential candidate involved in regulation of GAG biosynthesis in both experimental models of animals. Adult rat sera were found to contain about 75% of IGF-I recovered from young rat sera. Fasting of both groups of animals resulted in dramatic decrease in serum IGF-I levels to about 50% of initial values. Since IGF-I activity and IGF-I serum half-life depends on the level of specific IGF-binding proteins (IGFBPs) we determined (i) relationship between main groups of IGFBPs, namely high molecular weight binding proteins (HMWBPs) and low molecular weight binding proteins (LMWBPs) and (ii) the amounts of IGF-I bound to respective proteins in the sera of all experimental animals. Control young rat serum was found to contain about 90% of HMWBPs and about 10% of LMWBPs as determined by ligand binding assay. In contrast, control adult rat serum contained about 60% of HMWBPs and about 40% of LMWBPs. Fasting of both groups of animals resulted in significant increase in serum levels of LMWBPs. Control young rat serum was found to contain about 8% IGF-I bound to LMWBPs while serum of control adult rats contained 18% IGF-I bound to these proteins. In sera of fasted young animals however, about 75% of the bound IGF-I was recovered from LMWBPs (about 60% of total serum IGF-I) while in sera of fasted adult animals only about 56% of the bound IGF-I was recovered from LMWBPs (about 50% of total serum IGF-I). Evidence was provided that during fasting of both groups of animals there is a significant decrease in serum BP-3 and dramatic increase in serum BP-1 concentrations, compared to respective controls. However, the concentration of BP-1 in serum of fasted young rats was increased by about 60 fold while in serum of fasted adult rats only by about 10 fold, compared to respective control animals. Negative correlation between skin GAG content and LMWBPs derived IGF-I during fasting of young (r = - 0.943, p < 0.001) and adult ( r = - 0.571, p < 0.01) rats was found.The data presented suggest that the effects of aging and fasting on decreased skin GAG content may be due to induction of LMWBPs that are known to (i) inhibit IGF-I dependent function and (ii) increase clearance of IGF-I from circulation. However, the effects of fasting are distinct in respect to young and adult rats suggesting that mechanisms involved in regulation of IGF-I bioactivity during aging are more complex that during fasting.     

Aging-related hyperinflammation in endotoxemia is mediated by the α2A-adrenoceptor and CD14/TLR4 pathways

AimsSepsis is a major cause of morbidity and mortality in the elderly population. In prior studies, we have shown that in vivo, the inflammatory response in aged animals is exaggerated as compared to young animals and that this response likely accounts for the increased morbidity and mortality. Part of this uncontrolled inflammatory response in sepsis is due to the innate immune response. However, recent studies have shown that the pathogenesis of sepsis is much more complex. The adrenergic autonomic nervous system is now thought to play a key role in modulating the inflammatory response in sepsis. In this study, we hypothesize that not only is the innate immune response enhanced in response to lipopolysaccharide (LPS) in aged animals, but that the adrenergic nervous system also plays a role in the release of excess inflammatory cytokines.Main methodsMale Fischer-344 rats (young: 3 months; aged: 24 months) were used. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS, 15 mg/kg BW). Splenic tissues were harvested and mRNA and protein were extracted. The protein expression of CD14 and TLR4, key mediators of LPS in the innate response, as well as alpha-2A adrenergic receptor (α_2A-AR) and phosphodiesterase 4D (PDE4D), as the means by which the autonomic nervous system exerts its effects were analyzed.Key findingsSplenic tissue concentrations of α_2A-AR, PDE4D, CD14, and TLR4 were significantly increased in septic aged rats as compared to aged sham rats and septic young rats. The increased expression of α_2A-AR in septic aged rats was further confirmed by immunohistochemical staining of splenic tissues.SignificanceThese data support the hypothesis that not only is the innate immune response increased in aged animals during sepsis, but that there is also an upregulated response of the adrenergic autonomic nervous system that contributes to excess proinflammatory cytokine release.     

Oral supplementation of 2′-fucosyllactose during lactation improves memory and learning in rats

Human milk oligosaccharides have been proposed to exert beneficial effects on brain development. During the last decades, most of the studies have focused on the evaluation of sialylated structures but recent experiments have also tested fucosylated oligosaccharides, i.e. 2′-fucosyllactose (2′-FL). The present study aimed to determine whether oral 2′-FL has an effect on the development of newborn brain, contributing to enhance cognitive skills later in life. Rat pups received an oral supplementation of 2′-FL (2′-FL group) or water (control group) during the lactation period. Thereafter, animals were maintained on a rodent standard diet. Rats (n = 12 rats/group) were evaluated twice, at age 4–6 weeks and again at age 1 year, using classical behavioral tests. In vivo long-term potentiation (LTP) was also performed at the same ages (n = 10 rats/group). Both groups showed similar behavior when the animals were assessed just after weaning (age 4–6 weeks), although the 2′-FL group seemed to perform slightly better in Morris Water Maze. At age 1 year, 2′-FL rats performed significantly better in the Novel Object Recognition and Y maze paradigms, when compared to controls. In addition, LTP was more intense and longer lasting in the rats supplemented with 2′-FL than in control animals, both in young and adult animals. Oral administration of 2′-FL exclusively during lactation enhanced cognitive abilities, not only in childhood but also in adulthood.     

Dietary selenium requirements based on tissue selenium concentration and glutathione peroxidase activities in old female rats

Dietary nutrient requirements for older animals have been studied far less than have requirements for young growing animals. To determine dietary selenium (Se) requirements in old rats, we fed female weanling rats a Se-deficient diet (0.007 μg Se/g) or supplemented rats with graded levels of dietary Se (0–0.3 μg Se/g) as Na₂SeO₃ for 52 weeks. At no point did Se deficiency or level of Se supplementation have a significant effect (P>0.05) on growth. To determine Se requirements, Se response curves were determined for 7 Se-dependent parameters. We found that minimum dietary Se requirements in year-old female rats were at or below 0.05 μg Se/g diet based on liver Se, red blood cell glutathione peroxidase (Gpx1) activity, plasma Gpx3 activity, liver and kidney Gpx1 activity, and liver and kidney Gpx4 activity. In conclusion, this study found that dietary Se requirements in old female rats were decreased at least 50% relative to requirements found in young, rapidly growing female rats. Collectively, this indicates that the homeostatic mechanisms related to retention and maintenance of Se status are still fully functional in old female rats.     

Fever and behavioral thermoregulation in young and old rats

At standard laboratory ambient temperatures (T(a)) of 20-24 degrees C, peripheral injections of lipopolysaccharide (LPS) reliably produce fever in young rats. In contrast, old rats may show a blunted fever, no fever, or even hypothermia after LPS. In the present study we hypothesized that old rats might use behavioral thermoregulation to help them develop a fever. Young and old rats were implanted with temperature transmitters. At least 1 wk postoperatively they were placed in a thermally graded alleyway (T(a) 10-40 degrees C). On the third and sixth day they were taken out of the gradient, placed at an T(a) of 23 degrees C, injected intraperitoneally with LPS or saline, and left at 23 degrees C for 3 h. At the end of that time, all young rats had become febrile, whereas the old rats had not. When the rats were replaced in the thermal gradient, the young animals continued to develop a fever that was similar to fever in young rats left at 23 degrees C. The old animals chose significantly warmer positions in the thermal gradient than did the young animals and only then became febrile. Although there was a tendency for the young rats to prefer higher T(a) after LPS than after saline, these differences were not significant. However, the differences in the old rats were significant. These results suggest that the LPS had increased the thermal set point in the old rats, but they could develop febrile responses only at the warm T(a) they selected.     

The active avoidance reaction of laboratory rats: differences between experiments carried out in the phase of motor activity and inactivity

The influence of two phases of the circadian cycle (motor activity and motor inactivity) on the rate of acquisition and extinction of an active avoidance reaction was studied in 35-day-old male laboratory rats reared in cages (with limited social contacts), in young reared from the age of 15 days in communities (with the broad social contacts typical of this species) and in adult males reared in cages. A difference was found between the results of experiments carried out in the morning (during the motor inactivity period) and in the early evening (at the outset of the motor activity period) in both young and adult animals. The factor deciding whether acquisition or extinction was influenced depended on the mode of life. In animals reared in cages, inhibition was influenced; extinction was elaborated faster in the evening in adult animals and juvenile young were capable of 100% extinction only in the evening (in the morning only 50%). Community young achieved 100% extinction in both cases. In young rats which lived in a community from the 15th day, acquisition was influenced (it was achieved faster in the evening). The correlation between the rate of acquisition and extinction in cage-bred adult and young rats was negative if the experiments were carried out in the morning and was positive in evening experiments on young animals. In community-bred young it was positive in both cases.     

Ventricular myosin from young and adult animals with respect to the thyroid state

Studies were conducted to analyze the effect of the thyroid hormone on ventricular myosin during ontogenesis of mice, rats and rabbits. Hypothyroidism was induced in mice and rats by administering propylthiouracyl in drinking water. Rabbits were made hyperthyroid by chronic administration of thyroxine. The change in the thyroid state of rats and rabbits influenced young and adult animals differently depending on whether V1 or V3 was the major ventricular isomyosin form present. Measurements of Ca2+-ATPase activity of myosins from young and old control animals and from animals with changed thyroid state showed that hypothyroidism in rats is associated with a greater decrease of myosin ATPase in young rats which contain V1 isomyosin only, when compared with old rats which contain a preponderance of V3 isomyosin and less of the V1 form. In rabbits, ATPase activity of ventricular myosin was more elevated after thyroxine administration in adult rabbits, which contain V3 isomyosin only, than in young rabbits in which myosin consists of V1 and V3 isomyosins. Ventricular myosins of young and adult mice did not differ in their ATPase activity and the treatment of mice with propylthiouracyl had only slight effect on myosin ATPase. It can be concluded based on these results that the hypothesis concerning hypothyroidism inducing transformation of V1 into V3 isomyosin does not hold generally.     

Effect of repeated stress and administration of phenobarbital on the lymphoid tissue and on protein metabolism in the lymphocytes of infant and adult rats

Further study of the response to chronic stress stimulation in the early postnatal phase showed that the i.p. injection of physiological saline (stress stimulation) induced lymphopenia, a 50% decrease in the incorporation of 3H-leucine into isolated lymphocytes and a decrease in the weight of the thymus in 7-day-old male rats. No such changes were observed in adult animals. If repeated doses of phenobarbital were administered to stressed young rats, however, lymphopenia did not occur and the rate of the incorporation of 3H-leucine into isolated lymphocytes was not different from the control value; the protein content of the lymphocytes was significantly raised, however. In adult animals, phenobarbital increased the rate of incorporation of 3H-leucine into the lymphocytes. The repeated administration of phenobarbital reduced the weight of the thymus in both young and adult animals, but a decrease in spleen weight was recorded only in the young animals. A single i.p. injection of ACTH or dexamethasone caused lymphopenia and slowed down the incorporation of 3H-leucine into the lymphocytes of both young and adult animals. The results show that the striking decrease observed in the rate of the liver metabolism of corticosterone in suckling young rats not injured by repeated stress stimulation is accompanied by significant changes in the lymphoid tissue.     

Aging-related decreases in hepatic mitochondrial and cytosolic δ-aminolevulinic acid synthase during experimental porphyria

The basal- and allylisopropylacetamide-induced activities of the first enzyme of heme biosynthesis, δ-aminolevulinic acid synthase (ALAS) were measured in hepatic mitochondria and cytosol of young, adult, and aged Fisher 344 rats. The total cellular ALAS activity induced by allylisopropylacetamide decreased 67% with age. The specific activity of mitochondrial ALAS in normal and induced animals decreased with aging when assayed in whole or broken mitochondria. The levels of ALAS which accumulated in the cytosol after allylisopropylacetamide administration were proportionally greater in both the young and senescent than in the mature animals. During aging, no evidence for a fragile population of mitochondria in either normal or induced animals was observed suggesting that mitochondrial matrix proteins are not released during homogenization. The hepatic mitochondrial content decreased during aging when calculated using both a membrane-bound marker enzyme cytochrome oxidase and a matrix marker enzyme citrate synthase and was unaffected by allylisopropylacetamide treatment. This reduced mitochondrial content further diminishes the level of functional ALAS available in the liver during senescence. This study confirms the age-dependent decrease in mitochondria ALAS in normal and induced animals and also suggests an age-related change in the process by which cytosolic ALAS is translocated into the mitochondria.     

The aging Leydig cell: 1. Testosterone and adenosine 3',5'-monophosphate responses to gonadotropin stimulation in rats

Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells.     

Effect of ascorbic acid deficiency on the in vivo synthesis of carnitine

The effects of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and kidney carnitine levels were not affected by the deficiency, but scorbutic animals had 50% less carnitine in heart and skeletal muscle than control animals. Labeled carnitine precursors, 6-N-tri-methyl-L-lysine and 4-N-trimethylaminobutyrate, both of which require ascorbate for their enzymatic hydroxylation, were injected into the vena cava of control, pair-fed and scorbutic animals. The distribution of isotope in compounds present in the liver and kidney after 1 h was determined. The uptake of trimethyllysine by the liver was less than 2% in 1 h, while the kidney took up approx. 20% of the 14C. Control and pair-fed animals converted trimethyllysine to kidney trimethylaminobutyrate 8--10 times as well as did scorbutic animals. Trimethylaminobutyrate hydroxylase, present in the liver but almost absent from the kidney, converted nearly all of substrate taken up by the liver to carnitine in both the scorbutic and control animals.     

The effect of administration of estradiol and testosterone on body growth of young male rats.

The influence of estradiol and testosterone on body growth of young male Wistar rats was investigated. In the first experiment, estradiol was given to intact ad libitum fed male rats at 32, 37 and 42 days of age. Moreover, two untreated groups of animals were used: one was fed restrictedly according to the food intake of animals receiving estradiol and another was fed ad libitum. The animals were sacrificed at 47 days of age. Both untreated groups of animals achieved significantly higher body weight and length of tibia than estradiol treated animals. Also the growth of the tail of untreated animals was more intensive than that of estradiol treated animals. In the second experiment, estradiol was given to intact ad libitum fed male rats at 30, 35 and 45 days of age. Moreover, testosterone was given to a half of these animals at 45, 50 and 55 days of age. The animals were sacrificed at 60 days of age. Administration of testosterone significantly increased the growth of the tail and tibia in comparison to the animals which did not receive testosterone after estradiol administration. The results of the present study show that the inhibitory effect of estradiol on body growth of young male rats is not only the result of decreased food intake and that testosterone can improve the skeletal growth of male rats altered by previously given estradiol.     

Effect of silicoorganic compounds on protein biosynthesis in granulation-fibrous tissue]

The authors studied the effect of propoxysilatran (POS) on the biosynthesis of the main connective tissue biopolymeres. The use of POS in the form of 0.5 and 2.0% ointments on lanolin-vaseline base caused stimulation of cellular proliferation in the granulation fibrous tissue developing in the open skin defects of albino rats. Stimulation of cellular proliferation in these animals was accompanied by increase of collagen biosynthesis and of noncollagen proteins. In concentrations of 10(-3)--10(-4) M POS caused intensification of collagen biosynthesis (formation of peptide-bound nondialyzed 14C-oxyproline) in vitro in the cartilage tissue of chick embryos. Thus, silatrans are biologically-active substances producing a regulating effect on the course of the reparative-proliferative processes in the connective tissue.     

Stimulation of hepatic phosphatidylcholine biosynthesis in rats fed a high cholesterol and cholate diet correlates with translocation of CTP: phosphocholine cytidylyltransferase from cytosol to microsomes

A new model system for the study of phosphatidylcholine biosynthesis is presented. Young rats were fed a diet that contained 5% cholesterol and 2% cholate. After 6 days there was a 2-fold increase in the concentration of plasma phospholipid (243 mg/dl compared to 132 mg/dl for control animals) and a 3-fold increase in the concentration of plasma phosphatidylcholine. The rate of phosphatidylcholine biosynthesis was measured after injection of [Me-3H]choline into the portal veins. The incorporation of tritium into choline, phosphocholine and betaine by liver was similar for experimental and control animals, whereas there was a 3-fold increased incorporation into phosphatidylcholine of the cholesterol/cholate-fed rats. The activities of the enzymes of phosphatidylcholine biosynthesis in cytosol and microsomes were assayed. The only change detected was in the cytosolic and microsomal activities of CTP: phosphocholine cytidylyltransferase which were increased more than 2-fold in specific activity. When total cytidylyltransferase activity per liver was determined, a dramatic translocation of the enzyme to microsomes was observed. The control livers had 24% of the cytidylyltransferase activity associated with microsomes, whereas this value was 61% in the livers from cholesterol/cholate-fed rats. When the cytosolic cytidylyltransferase was assayed in the presence of phospholipid, the enzyme was stimulated several-fold and the difference in specific activity between control and cholesterol/cholate-fed rats was abolished. The increased activity in cytosol appears to be the result of a 2-fold increase in the amount of phospholipid in the cytosol from cholesterol/cholate-fed rats. The data strongly support the hypothesis that the special diet stimulates phosphatidylcholine biosynthesis by causing a translocation of the cytidylyltransferase from cytosol to microsomes where it is activated.     

Influence of ethosuximide on metrazol-induced seizures during ontogenesis in rats

Effects of metrazol (pentylenetetrazole and ethosuximide were studied in male albino rats aged 7, 12, 18 and 90 days. The 18-day-old rats exhibited the highest sensitivity to metrazol. CD50s in the remaining three age groups were nearly the same. Ethosuximide was reliably effective against metrazol only in adult rats; in young animals it did not significantly change CD50s. Metrazol induced in ethosuximide-pretreated young rats either modified (long-lasting minimal seizures in 18-day-old animals) or new seizure patterns (minimal seizures in 7- and 12-day-old rats).     

Thyroxine-induced cardiomegaly in rats of different age

In the present study the effect of thyroxine treatment on the development of cardiomegaly was compared in young (10-day-old) and adult (12-week-old) rats. L-thyroxine was administered subcutaneously in a dose of 1 mg per kg b.w. for 5 days. In young thyroxine-treated rats the heart weight increased by 79% in comparison with the control rats. The number of blood capillaries and muscle fibres per mm2 remained unchanged. The concentration of hydroxyproline was even lower than in control animals. The number of 3H-thymidine-labelled muscle cell nuclei was significantly higher both in the left and right ventricles of thyroxine treated rats. The density of capillaries and muscle fibres was significantly lower in adult rats than in the group of young animals. In adult thyroxine-treated animals the heart weight was higher by 36%, the number of capillaries and muscle fibres as well as the concentration of hydroxyproline was unchanged. Thyroxine induced significant increase in the number of DNA synthesizing nuclei of muscle cells in the left ventricle while the change in the right ventricular myocardium was not statistically significant. The present data indicate that a hyperplastic response of cardiac muscle cells to thyroxine occurs in both ventricles of young rats and also in the left ventricle of adult animals.     

Mucin of the rat submaxillary gland: influence of the animal's age and sex on the composition of its glucidic fraction

Mucin was isolated from submaxillary glands of young (30 and 45 days) and adult (100 days) male and female rats; it was purified first by fractional precipitation with ethanol and then by zone electrophoresis at pH 6.0 on Pevikon. The mucin contains N-acetylhexosamines (N-acetylglucosamine and N-acetylgalactosamine, 3:2), galactose and sialic acid. The total sugar content of the glycoprotein of adult rats is about twice that of the young animals, while the galactose content remains almost unchanged. Incorporation of [¹⁴C]glucosamine into the rat submaxillary gland mucin starts to increase considerably at the moment of sexual maturity and reaches a constant level (90 days); the increase in biosynthesis velocity is higher in males than in females.     

Sinusoidal electromagnetic field of 50 hz helps in retaining calcium in tibias of aged rats

Effect of 50Hz sinusoidal electromagnetic field (SEMF) on normal bone physiology was evaluated in young and old female and male Wistar rats. Exposure to SEMF resulted in increased 45Ca retention in tibias of aged animals only. Levels of serum calcium in young female and male rats were significantly less than in respective aged rats. These were further decreased after 4 weeks of SEMF exposure. SEMF exposure did not change the serum calcium levels in aged rats, and inorganic phosphates in young and aged animals. Similarly, the levels of tartrate resistant acid and alkaline phosphatase were significantly decreased in young rats, whereas the levels remained unchanged in aged rats of either sex. The results revealed that SEMF of 1mT can prevent bone calcium loss due to aging in animals.