Induction and properties of pyruvate kinase of the cerebral hemisphere of rats of various ages

The induction of pyruvate kinase (ATP: pyruvate 2-O phosphotransferase, EC 2.7.1.40; PK) by estradiol or testosterone in the cerebral hemisphere of male and female rats of different ages was studied. There is a marked decrease in the activity of the enzyme of normal male and female rats with increasing age. Orchiectomy decreases its activity in young and old rats, but not in adult rats. Ovariectomy decreases its activity significantly in all the ages. Estradiol (50μg/100g body wt.) induces its activity in castrated male and female rats, but its effect is greater in female rats. A higher dose of estradiol (100μg/100g body wt.) has age- and sex-dependent induction. Testosterone (50 and 100μg/100g body wt.) has very little effect on its activity in castrated male and female rats of the three ages. The K_m values for PEP and ADP, and the K_t values for ATP and l -phenylalanine for the partially purified enzyme of the cerebral hemisphere of normal young and old rats are the same. Preincubation of the enzyme with l -alanine reverses the l -phenylalanine induced inhibition. The concentration of l -alanine required for this effect is the same for both ages. The concentration of Mg²⁺ required to reverse the inhibitory effect of ATP is the same for young and old rats. Estradiol and testosterone added directly to the incubation mixture do not have any effect on the enzyme activity. These data suggest that the nature of the enzyme molecule does not change with age, but that its induction properties change with age.     

Differential response of rat skeletal muscle glycogen metabolism to testosterone and estradiol.

Although reports on sex steroids have implicated them as promoting protein synthesis and also providing extra strength to the skeletal muscle, it remains unclear whether sex steroids affect glycogen metabolism to provide energy for skeletal muscle functions, since glycogen metabolism is one of the pathways that provides energy for the skeletal muscle contraction and relaxation cycle. The purpose of the current study was to show that testosterone and estradiol act differentially on skeletal muscles from different regions, differentially with reference to glycogen metabolism. To study this hypothesis, healthy mature male Wistar rats (90-120 days of age, weighing about 180-200 g) were castrated (a bilateral orchidectomy was performed to test the significance of skeletal muscle glycogen metabolism in the absence of testosterone). One group of castrated rats was supplemented with testosterone (100 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards). To test whether estradiol has any effect on male skeletal muscle glycogen metabolism 17beta-estradiol (5 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards) was administered to orchidectomized rats. To test whether these sex steroids have any differential effect on skeletal muscles from different regions, skeletal muscles from the temporal region (temporalis), muscle of mastication (masseter), forearm muscle (triceps and biceps), thigh muscle (vastus lateralis and gracilis), and calf muscle (gastrocnemius and soleus) were considered. Castration enhanced blood glucose levels and decreased glycogen stores in skeletal muscle from head, jaw, forearm, thigh, and leg regions. This was accompanied by diminished activity of glycogen synthetase and enhanced activity of muscle phosphorylase. Following testosterone supplementation to castrated rats, a normal pattern of all these parameters was maintained. Estradiol administration to castrated rats did not bring about any significant alteration in any of the parameters. The data obtained suggest a stimulatory effect of testosterone on skeletal muscle glycogenesis and an inhibitory effect on glycogenolysis. Estradiol did not play any significant role in the skeletal muscle glycogen metabolism of male rats.     

Effects of castration and testosterone treatment on the hexobarbital sensitivity in male rats

Castration increases the central nervous system (CNS) sensitivity to hexobarbital as measured by an EEG-threshold method. Testosterone treatment in doses from 1 to 5 mg/kg/day decreases CNS-sensitivity and restores threshold values in castrated rats to a level found in normal young adult male rats.     

Glycolytic enzyme activities in liver, heart and gastrocnemius of Sprague-Dawley and Wistar rats: effect of high sucrose diet

1. Pyruvate kinase, phosphofructokinase, and glucophosphate isomerase activities were measured in liver, heart and gastrocnemius of Wistar and Sprague-Dawley rats. 2. Enzyme activities were significantly lower in tissues of Wistar rats except for pyruvate kinase in gastrocnemius. 3. Sensitivities of pyruvate kinase and phosphofructokinase to inhibition by alanine and citrate differed in these two strains except for pyruvate kinase in gastrocnemius. 4. Phosphofructokinase sensitivity to citrate was greater in the three Wistar tissues. 5. Activities of liver enzymes were more responsive to a high sucrose diet in Wistar rats. 6. Heart pyruvate kinase and phosphofructokinase exhibited modest increases in activity with a high sucrose diet.     

Enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles of hypocaloric rats.

1. The effect of hypocaloric feeding (25% of normal food intake for 21 days) of rats on the enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles was studied. 2. In control and hypocaloric rats the muscle relaxation rates at 100 Hz were 35.76 and 11.38% force loss/10 ms respectively. Control rats exhibited enhanced force of muscle contraction as the frequency of stimulation increased from 10 to 100 Hz, with maximum force being at 100 Hz. Hypocaloric rats exhibited a decrease in the increment of force being exerted at high frequencies, with maintenance of force at lower stimulatory frequencies. 3. In muscles of hypocaloric rats, there were significant decreases in the maximal activities of hexokinase (17.6-37.0%), 6-phosphofructokinase (22.7-34.2%), pyruvate kinase (21.2-36.0%), citrate synthase (34.1-41.5%), oxoglutarate dehydrogenase (29.4-52.4%) and 3-hydroxyacyl-CoA dehydrogenase (26.7-32.1%), whereas the activities of glycogen phosphorylase increased (23.8-43.4%) compared with control values. 4. In soleus-muscle strip preparations of hypocaloric rats, there were significant decreases in the rates of lactate production (28.1%) and glucose oxidation (32.6%) compared with control preparations. 5. Mitochondrial preparations from muscles of hypocaloric rats incubated with various substrates exhibited decreased rates of oxygen uptake compared with control preparations. 6. In muscles of hypocaloric rats (gastrocnemius and soleus), there were significant decreases in the concentrations of glycogen (P less than 0.001) and phosphocreatine (P less than 0.001) and increases in those of pyruvate (P less than 0.001), lactate (P less than 0.001) and ADP (P less than 0.001), whereas those of ATP and AMP remained unchanged. 7. Calculated [lactate]/[pyruvate] and [ATP]/[ADP] ratios exhibited significant increases (P less than 0.05) and decreases (P less than 0.05) in muscles of hypocaloric rats respectively. 8. The results are discussed in relation to the genesis of muscle dysfunction caused by malnutrition.     

Effect of cadmium intoxication on glucose utilization in energy metabolism of muscles

The influence of cadmium intoxication on carbohydrate metabolism in skeletal muscles and liver of the male Wistar rats has been studied. Cadmium was administered as cadmium acetate in a dose of 0.3 mg Cd2+/kg body weight for three months. At the same time the control rats were injected with 0.9% NaCl. The animals were decapitated and samples of their skeletal muscles: the soleus muscle (composed mainly of red slow twitch fibers; ST) the gastrocnemius muscle containing two types of fibers (white fast twitch fibers FTb and red fast twitch fibers, FTa) and the liver were dissected out. In the samples of muscles, liver and serum contents of glycogen, glucose, pyruvate and lactate, as well as activities of hexokinase, pyruvate kinase and lactate dehydrogenase were measured. Intoxication of rats with cadmium for three months resulted in a reduction of glycolytic enzymes in the serum, ST and FTa muscle fibers and in the liver but did not change the activities of glycolytic enzymes in the FTb muscle fibers. The data obtained for the concentrations of glycogen in the liver and skeletal muscles suggest different mechanisms of cadmium influence on glycogen utilization in these organs.     

The influence of testosterone on the alkaline proteolytic activity in rat skeletal muscle

The effects of testectomy and subsequent administration of testosterone propionate on the activity of the alkaline proteinases in rat skeletal muscle were investigated. Castration of the mature rat was followed by a short-term delay in protein accretion in skeletal muscle tissue as measured by the protein/DNA ratio and was paralleled by a 2–3 fold increase in specific activity of the alkaline proteinase(s). This increase of proteolytic activity was equally significant when expressed relative to μg DNA. Although the gain in body weight was significantly lower in the castrated rats, nevertheless the protein/DNA ratio in muscle after 6 weeks approximated the values of sham-operated control rats without normalization of the proteolytic activity.Treatment of the castrated rats with testosterone propoinate resulted in restoring normal levels of previously elevated levels of alkaline proteolytic activity in muscle tissue. The normalization of enzyme activity as well as protein accretion in muscle was dose-dependent. Treatment of the rats with a low dose (0.1 mg/day) of testosterone propionate failed to restore the proteolytic activity, but led to a small increase of the protein/DNA ratio as well as to a progressive increase in body weight. These data indicate a regulatory role of testosterone in the adaptive behaviour of the alkaline proteolytic system in rat skeletal muscle.     

Elevation of tissue coenzyme Q (ubiquinone) and cytochrome c concentrations by endurance exercise in the rat

Six months of enforced and voluntary endurance training of young female Wistar rats resulted in significant decreases of body weight and gastrocnemius muscle wet weight and protein content, and increases in heart weight and protein content, and liver protein content. The coenzyme Q and cytochrome c concentrations of cardiac, gastrocnemius, and deep red region of the vastus lateralis muscles were increased, while small or nonsignificant trends toward increases in cytochrome c and coenzyme Q were seen in kidney, brain, lung, liver, internal + external oblique muscles, and the superficial white region of the vastus lateralis muscle. These results are discussed with regard to several roles for coenzyme Q in cellular function.     

Induction of the self-priming effect of luteinizing hormone releasing hormone during the sexual maturation of the male rat

Pubertal and young adult male rats release more luteinizing hormone (LH) in response to luteinizing hormone releasing hormone (LHRH) if pretreated with LHRH than if pretreated with saline. Immature male rats do not show this self-priming effect. In order to examine the role of acute changes in testicular steroids in this process, immature (29-30 days old) or pubertal (50-51 days old) male rats were castrated or sham operated under ketamine HCl anesthesia. Beginning immediately after completion of the surgery, they were given three priming injections of 10 ng LHRH/100 g body wt or saline at 30-min intervals. Thirty minutes after the third priming injection, a blood sample was obtained by cardiac puncture followed immediately by a challenge injection of 50 ng LHRH/100 g body wt given to both saline and LHRH primed groups. Ten minutes after the challenge injection a final blood sample was obtained by heart puncture. Serum was assayed for LH concentration by radioimmunoassay. Sham-operated pubertal rats showed a typical self-priming effect. Animals pretreated with LHRH released significantly (P less than 0.01) more LH in response to the challenge injection than did rats pretreated with saline. Acute castration also resulted in a significant (P less than 0.001) self-priming effect in pubertal rats. As anticipated, sham castrated immature males did not show a self-priming effect. Acutely castrated immature rats however, showed a significant (P less than 0.05) self-priming effect. These data provide support for the hypothesis that, prior to puberty, increases in testosterone during the priming process inhibit the expression of the self-priming effect.     

Tissue Oxidative Capacity,Fuel Stores and Skeletal Muscle Fatty Acid Composition In Obesity-Prone and Obesity-Resistant Rats

The purpose of the present study was to compare tissue oxidative capacity, skeletal muscle fatty acid composition, and tissue fuel stores in low-fat fed (LFD, 12% of energy from corn oil) male Wistar rats, and in high-fat fed (45% of energy from corn oil) obesity-prone (OP) and obesity-resistant (OR) male Wistar rats. Designation of OP and OR rats was based on body weight gain (upper tertile for OP; lower tertile for OR) after 5 weeks on the high-fat diet. Body weight gain over the 5-week dietary period was 91 ± 9 g in LFD, 98 ± 4 g in OR, and 158 ± 5 g in OP (p<0. 05 vs. LFD and OR). Energy intake over the 5-week dietary period was 3099 ± 101 kcal in LFD, 3185 ± 51 kcal in OR, and 3728 ± 45 kcal in OP (p<0. 05 vs. LFD and OR). Maximal citrate synthase activity (μ. mol^?1min^?1) in the gastrocnemius muscle was not significantly different among groups: 12. 1 ± 2. 4 in LFD, 11. 4 ± 1. 9 in OR and 133 ± 2. 5 in OP rats. Similarly, citrate synthase activity in the heart, 59. 3 ± 7. 2, and liver, 6. 6 ± 0. 4, was also not significantly different among groups. Fatty acid composition of the gastrocnemius muscle was not significantly different among groups. Fasting glycogen levels in the liver, gastrocnemius muscle, and heart were 6. 4 ± 3. 7, 13. 2 ± 2. 3 and 6. 8 ± 1. 9 μmol/g in LFD, 21. 2 ± 5. 1 (p<0. 05 vs. LFD and OP), 10. 4 ± 1. 8 and 5. 9 ± 1. 1 mUmol/g in OR, and 36. 3 ± 4. 8 (p<0. 05 vs. LFD and OR), 10. 2 ± 23 and 53 ± 2. 1 μmol/g in OP rats, respectively. Triglyceride levels were similar among groups in plasma, heart and gastrocnemius muscle, but were significantly (p<0. 05) higher in the liver of OP (15. 5 ± 1. 9 (μmol/g) compared to OR (9. 1 ± 1. 1 μmol/g) and LFD (8. 1 ± 1. 4 μmol/g) rats. These data suggest that susceptibility to dietary obesity, in this rodent model, cannot be explained by differences in tissue oxidative capacity or muscle fatty acid composition.     

Evidence for increased peroxidative activity in muscles from streptozotocin-diabetic rats

The ability of cardiac and skeletal muscles from diabetic rats to metabolize superoxide and hydrogen peroxide was determined by the activities of superoxide dismutase (SOD) and catalase, respectively. Male and female Sprague-Dawley rats, 43 days old, were made diabetic with a single intravenous injection of streptozotocin (70 mg/kg body weight). On the 80th day after injection the blood glucose concentration of these rats was increased fourfold, and the plasma insulin concentration was decreased four- to fivefold compared to controls. Body weights of male diabetic rats were 61% and those of female diabetic rats were 66% of their ad libitum-fed controls. The seven different skeletal muscles examined weighed less in the diabetic rats than in controls of the same age and body weight. The hearts of the diabetic rats weighed more than those of controls of the same age and body weight. Comparison to the body weight controls allowed the distinction of specific effects due to lack of insulin from effects due to retardation in muscle growth. Increased catalase activity in all muscles examined from diabetic rats (plantaris, gastrocnemius, and heart) suggested a response in catalase activity similar to that of starved rats. SOD activity was not altered in the diabetic rat skeletal muscles and erythrocytes, but was somewhat decreased in the heart.     

Age- and sex-related differences in extra-hepatic low-density lipoprotein receptor

To determine whether differences in LDLr behavior in extra-hepatic tissues and whether extra-hepatic receptors could differentially contribute to cholesterol homeostasis under physiological conditions, we evaluated the presence and regulation of LDLr from both a gender and an aging perspective. We used the brain cortex, the gastrocnemius, and the heart ventricle of 3- and 12-month-old male and female rats. We observed a protein decrease of total LDLr in 12-month-old female rat brains that was completely restored by 17-β estradiol treatment. In the gastrocnemius, LDLr accumulates in the skeletal muscle in both male and female aged rats as a precursor probably due to a glycosylation impairment. In the heart, no modifications were observed in either older rats or rats of a specific gender. These data highlight a tissue-specific dysregulation of LDLr that is age- and gender-dependent.     

Isoenzyme spectrum and kinetic properties of pyruvate kinase from the liver of thiamine-deficient rats

Thiamine-deficiency in animals induced by everyday subcutaneous administration of oxythiamine in a dose of 4, 40 and 100 mg/kg of weight for 10 days results in a decrease of the total activity of pyruvate kinase in the liver tissue and does not affect the mentioned index in the kidney and heart tissues. It is shown that as a result of the enzyme fractionation in the column with DEAE-cellulose the total activity of pyruvate kinase in the liver tissue of rats with thiamine-deficiency decreases due to L-isoform while the content of M-isoform remains unchanged. Thiamine deficiency does not affect kinetic characteristics of the L-isoform, extracted from the liver and this shows the absence of changes in the degree of phosphorylation of pyruvate kinase L-isoform under these conditions.     

Effects of repetitive exercise on neonatal rat skeletal muscle oxidative capacity

Since little is known about the training response to exercise in neonatal animals, this study was undertaken to elucidate the potential of oxidative system adaptations in developing skeletal muscle of rats during 50 days of daily treadmill running. The training regimen involved male and female rats (10 days old) initially running 0.1 mph, 0% grade, for 15 min. The program progressed to 1 mph, 25% grade, for 60 min by 50 days of age. At 25 days of age, pyruvate and palmitate oxidative capacity, and citrate synthase activity in red vastus muscle homogenates were elevated in the trained group (T) compared with age- and sex-matched controls (C). These increases were also observed for each subsequent time point tested and occurred in spite of the fact that the peak oxidative capacity of neonatal red vastus muscle was 46% greater than adult values. Further, trained animals tested at 45 days of age responded with a 12% increase in maximal oxygen consumption (Vo2max) compared with controls (P less than 0.05). Assays of muscle phosphofructokinase and of creatine phosphokinase activity conducted at this time point revealed no difference between T and C groups. Collectively, these data suggest that neonatal rats can be successfully trained and that they respond to an endurance-type program qualitatively similarly to adult rats.     

Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration

In young (4-week-old) male and female spontaneously hypertensive (SH) rats, ethanol metabolic rate in vivo and hepatic alcohol dehydrogenase activity in vitro are high and not different in the two sexes. In males, ethanol metabolic rate falls markedly between 4 and 10 weeks of age, which coincides with the time of development of sexual maturity in the rat. Alcohol dehydrogenase activity is also markedly diminished in the male SH rat and correlates well with the changes in ethanol metabolism. There is virtually no influence of age on ethanol metabolic rate and alcohol dehydrogenase activity in the female SH rat. Castration of male SH rats prevents the marked decrease in ethanol metabolic rate and alcohol dehydrogenase activity, whereas ovariectomy has no effect on these parameters in female SH rats. Chronic administration of testosterone to castrated male SH rats and to female SH rats decreases ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in mature males. Chronic administration of oestradiol-17β to male SH rats results in marked stimulation of ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in female SH rats. Chronic administration of ethanol to male SH rats from 4 to 11 weeks of age prevents the marked age-dependent decreases in ethanol metabolic rate and alcohol dehydrogenase activity, but has virtually no effect in castrated rats. In the intoxicated chronically ethanol-fed male SH rats, serum testosterone concentrations are significantly depressed. In vitro, testosterone has no effect on hepatic alcohol dehydrogenase activity of young male and female SH rats. In conclusion, in the male SH rat, ethanol metabolic rate appears to be limited by alcohol dehydrogenase activity and is modulated by testosterone. Testosterone has an inhibitory effect and oestradiol has a testosterone-dependent stimulatory effect on alcohol dehydrogenase activity and ethanol metabolic rate in these animals.     

Sexual dimorphism in N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content in the Harderian gland of Syrian hamsters: changes following gonadectomy

The activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the melatonin content of the Harderian glands of intact and gonadectomized male and female Syrian hamsters were studied. NAT activity in intact male Harderian glands was twice that of the female. Prepubertal or adult castrated males exhibited a decrease in NAT activity to a level comparable to that seen in the female. Testosterone implants in the castrated males led to a recovery of the original male NAT levels. Intact male hamsters had very low levels of Harderian HIOMT activity and melatonin content in comparison with the glands of the females. Prepubertal gonadectomy but not castration of adult males raised the levels of HIOMT activity and the melatonin content to those of the females. Bilateral ovariectomy had no effect on melatonin content, NAT activity, or HIOMT activity in the female hamster Harderian gland.     

Variation in tissue carnitine concentrations with age and sex in the rat

Diabetes, starvation and various hormonal treatments are known to alter drastically carnitine concentrations in the body. Before the mechanisms controlling carnitine metabolism could be determined, it was necessary to establish normal carnitine concentrations in both sexes at different ages. Carnitine was assayed in plasma, liver, heart and skeletal muscle of rats from birth to weaning. The plasma carnitine increased rapidly during the first 2 days after birth. Carnitine in both heart and skeletal muscle increased, whereas liver concentrations declined during the first week of life. A carnitine-free diet containing sufficient precursors for carnitine biosynthesis was fed to weanling rats. Groups of ten male and ten female rats were killed each week for 10 consecutive weeks. Carnitine was determined in plasma, liver, heart, skeletal muscle, urine and epididymis in the male. There was no difference in carnitine concentrations between the sexes at weaning. Plasma, heart and muscle concentrations were higher in adult male rats than in adult females. However, liver carnitine and urinary carnitine concentrations were higher in adult female than in adult male rats. The epididymal carnitine concentration increased very rapidly during 50 to 70 days of age and the differences in carnitine concentrations between the sexes also became apparent during this time. Thus both the age and the sex of the human subject or experimental animal must be considered when investigating carnitine metabolism.     

Some hormonal factors (hypophysectomy, castration and testosterone administration) modifying the course of "compensatory" muscle hypertrophy in the rat.

1. Maximum compensatory hypertrophy of the soleus and plantaris muscle in male rats is attained seven days after tenotomy of the gastrocnemius muscle (39% and 9% respectively). When tenotomy of the gastrocnemius was performed seven days ater hypophysectomy, hypertrophy in these two muscles was aproximately half that found in control animals. 2. After 81-day castration of young male rats the weight of the saleus and plantaris was reduced and hypertrophy following tenotomy of the gastrocneumius muscle did not develop. 3. Chronically castrated rats received testosterone two weeks prior to tenotomy of the gastrocnemius and a week during the muscle hypertrophy phase. Hypertrophy of the soleus in castrated rats which had received testosterone seven days after tenotomy of the gastrocnemius was 25% as compared with muscles of castrated animals. The corresponding value in the plantaris muscle was 10%. 4. These results indicate that even calf muscles of the rat, namely the soleus and plantaris muscles, are significantly affected by testosterone under these conditions, although it is not, as yet, clear whether its action is direct or indirect.     

Hormone induced lordosis and its relation to masculine sexual activity in male rats

Intact and castrated male rats were injected with a single dose of estrogen (75 μg), followed by progesterone (1 mg) or by oil. Intact males showed higher lordosis quotients as compared to castrated males. Progesterone facilitated lordosis behavior in castrated as well as in intact males. An estrogen-progesterone treated group of sexually inactive male rats and of sexually exhausted males showed lower levels of lordosis as compared to the intact sexually active males.     

The effect of sex hormones on the acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in rat liver

The intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity (PEPCK) was investigated in microdissected samples of livers from normal, castrated, castrated and estradiol- or testosterone-treated, and uncastrated and testosterone- or estradiol-treated male and female rats. The total PEPCK activity showed a marked sex dependency, with 1.8 times higher activity in males. The intra-acinar distribution profiles were also sex-dependent. The periportal-to-perivenous gradient was steeper in males. Castration resulted in an approximation of PEPCK activity and its acinar distribution pattern between the sexes due to a reduction in males and an increase in females. Estrogen treatment of castrated males had no further effect on PEPCK activity and its acinar gradient, whereas in ovariectomized animals the activity was reduced to levels near normal. Testosterone treatment of castrated male or female animals led to a marked increase in enzyme activity with a concomitant steepening of the acinar gradient. Administration of estradiol to normal male rats also led to a reduction in activity, together with a change in the acinar activity gradient. Testosterone treatment of normal females resulted in an induction of PEPCK activity which was most prominent in the periportal zone. The most drastic changes were observed in the perivenous zones. In all experiments a periportal-to-perivenous activity gradient persisted thus marking the periportal zone as the area with highest gluconeogenic capacity.