Effects of castration, dietary fat and adipose tissue sites on adipocyte plasma-membranes cyclic AMP phosphodiesterase activity in the pig.

1. cAMP Phosphodiesterase activity and kinetic parameters (Km and Vmax) were measured in subcutaneous and perirenal adipocyte plasma membranes from Large White male and castrated pigs. The animals were fed a control low fat diet or a sunflower diet enriched with linoleic acid (C18:2 n-6). 2. Phosphodiesterase activity, low Km and Vmax were lowered by castration. 3. In animals fed the sunflower diet, phosphodiesterase activity decreased without affecting either Km or Vmax. 4. Phosphodiesterase activity was higher in perirenal sites than in subcutaneous ones, particularly in male pigs. This may be explained by a lower Km or a higher cAMP phosphodiesterase affinity to cAMP in perirenal sites. 5. Theophylline was a potent inhibitor of phosphodiesterase activity principally in perirenal sites. 6. The intermediate role of cAMP phosphodiesterase in adenylate cyclase activity and lipolytic processes is discussed.     

Clearing-factor lipase in muscle and adipose tissue of pigs

1. Clearing-factor lipase was assayed in acetone-ether-dried powders of heart and adipose tissue of pigs. The enzyme activity in heart was higher than that in adipose tissue. The activity in the outer layer of subcutaneous fat was greater than that in the inner subcutaneous fat and the perirenal fat, which had similar activities. 2. Starvation for 48h, but not for 24h, decreased the activity of the heart enzyme. 3. Starvation for 24h caused a rapid decrease in the activity in all three adipose tissues, but even after 72h of starvation the activity was still highest in the outer subcutaneous fat. 4. Plasma fatty acid, glucose and insulin concentrations were determined in fed and starved pigs. Starvation decreased the plasma insulin concentration and increased the non-esterified fatty acid concentration.     

Gender differences and time course of castration-induced changes in porphyrins, indoles, and proteins in the Harderian glands of the Syrian hamster.

Sexual differences and the effects of orchidectomy were determined for porphyrin and melatonin concentrations and for the activities of the enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase, which synthesize melatonin from serotonin, in the Harderian glands of the Syrian hamster. Porphyrin concentrations in intact males were about 1/400th those of intact females. Castration for 1 week increased male Harderian porphyrin concentrations 10-fold; by 3 weeks, castrated male porphyrin levels were 140 times those of control values. N-Acetyltransferase activity in intact male Harderian glands was about 4 times that of females. Castration led to a drop in N-acetyltransferase activity to female levels within 2 weeks. Hydroxyindole-O-methyltransferase activity was 7 times higher in females than in males and castration had no effect on male Harderian hydroxyindole-O-methyltransferase activity. Neither gender nor castration influenced Harderian melatonin concentrations. Soluble proteins in Harderian glands from male and female hamsters and from male hamsters castrated for 1 and 4 weeks were examined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The gel profiles revealed several differences among the protein distribution in male and female gland lysates. Orchidectomy led to a female protein pattern within 4 weeks.     

Effects of starvation, refeeding, and fat feeding on adipocyte ghost adenyl cyclase activity

Basal adenyl cyclase activity and its response to epinephrine and glucagon were studied in isolated adipocyte ghosts obtained from fed, starved, refed, and fat-diet-adapted rats. Epinephrine stimulation of adenyl cyclase was significantly increased in fasted rats, but the glucagon response did not change. Rats fasted for 48 hr and refed a high carbohydrate, low fat diet for 48 or 96 hr showed no differences from chow-fed animals in either basal or hormone-stimulated adenyl cyclase activity. Rats adapted to a high fat, low carbohydrate diet showed an initial and transitory increase in basal activity but a progressive loss of epinephrine- and glucagon-stimulated enzyme activities. The loss in hormone responsiveness correlated well with a decrease in hormone-stimulated lipolysis of fat pads and was associated with a significant increase in fat cell diameter.     

Testicular involvement in peripubertal gonadotropin levels and accessory sex organ weight of male hamsters

This study evaluates the influence of testicular secretions during development in male hamsters on peripubertal gonadotropin levels. Castration or sham operations were performed on the day of birth (Day 1), Day 5, 10, or 20 of life. Repeated plasma samples on Days 20-60 at 10-day intervals were taken via orbital sinus puncture. Castrated animals received a subcutaneous testosterone capsule on Day 60 and were killed on Day 70. In addition, seminal vesicles and ventral prostate weights were taken in all animals at Day 70. Castrated animals, regardless of day of castration, had higher gonadotropin levels and suppressed sexual accessory organ weights. Animals castrated on the day of birth had lower luteinizing hormone (LH) levels than animals castrated on other days. Castration on Day 10 resulted in lower follicle stimulating hormone (FSH) levels. Males castrated on Day 20 were most sensitive to the negative feedback effect of testosterone on LH secretion, while Day 10 castrates had elevated FSH levels after testosterone exposure. Sexual accessory weights also differed depending upon the day of castration. Results point out the importance of testicular secretions on the developmental processes as well as the differing ages at which various systems may be influenced.     

Effects of oestrogen on adenylate cyclase system and glucose output in rat liver.

Effects of chronic oestrogen treatment on catecholamine- and glucagon-sensitive adenylate cyclase activity and glucose output in hepatocytes of castrated male rats were studied. In hepatocytes from male intact or castrated rats, the beta-adrenergic agonist isoprenaline did not stimulate adenylate cyclase activity and glycogenolysis, but glucagon markedly stimulated all these activities. Treatment of castrated animals with 17 beta-oestradiol for 7 days led to the appearance of beta-adrenergic-stimulated increases in both cyclic AMP generation and glucose output. The basal, glucagon- or fluoride-stimulated activities of adenylate cyclase of hepatic membranes prepared from oestrogen-treated rats were similar to those of control animals. Treatment with oestrogen did not influence the number or affinity of beta-adrenergic receptors. In hepatic plasma membranes from control rats, GTP failed to decrease the affinity of beta-adrenergic receptors for agonists, whereas the GTP-induced shift was apparently observed in those from oestrogen-treated animals. These results suggest that oestrogen is able to facilitate the coupling of hepatic beta-adrenergic receptors to the enzyme by increasing the effectiveness of receptor-guanine nucleotide regulation.     

Effects of castration and androgen treatment on androgen-receptor levels in rat skeletal muscles.

The effects of castration and dihydrotestosterone (DHT) treatment on levels of skeletal muscle androgen receptor (AR) were examined in three groups of adult male rats: 1) intact normal rats, 2) rats castrated at 16 wk of age, and 3) rats castrated at 16 wk of age and given DHT for 1 wk starting at week 17. All animals were killed at 18 wk of age. Castration caused a decrease (P < 0.05) in the weights of the levator ani and bulbocavernosus muscles. The administration of DHT to the castrated rats increased (P < 0.05) the weights of the levator ani and bulbocavernosus muscles. Castration caused a significant downregulation of AR levels in the bulbocavernosus (P < 0.05) but had no significant effect on AR levels in the levator ani muscle. DHT administration to the castrated group upregulated AR levels in the bulbocavernosus and levator ani muscles. The plantaris muscle did not significantly (P > 0.05) change for any of the treatments. These findings suggest that the effects of castration and androgen replacement differentially affect skeletal muscle mass and AR levels.     

Comparison of the effect of castration on the development of postural and non-postural muscles of mice.

The effect of castration on the development of muscle mass of postural and non-postural muscles was studied in 18 male mice (9 castrated, 9 uncastrated). Results obtained indicated that the castrated males grew faster and were bigger in body size and weight at maturity than the intact males. The bigger body size of castrated males was not due to larger muscle mass but was probably due to increased subcutaneous fat deposition. Atrophy of muscles usually observed following castration was significantly greater in the non-postural (biceps brachii) muscle of the forelimb as compared to the postural (triceps brachii) muscle of the forelimb. Conversely, the amount of reduction in muscle mass was similar in both postural (soleus) and non-postural (tibialis cranialis) muscles of the hindlimb.     

Changes of norepinephrine levels, tyrosine hydroxylase and dopamine-beta-hydroxylase activities after castration and testosterone treatment in vas deferens of adult rats

Norepinephrine levels and tyrosine hydroxylase and dopamine-beta-hydroxylase activities have been used to evaluate the effect of castration and testosterone treatment on the sympathetic innervation of the adult vas deferens. Castration was followed by a decrease in both norepinephrine content and tyrosine hydroxylase activity, even though the changes were not concomitant. Treatment of castrated animals with testosterone reversed the effect of castration on organ weight and norepinephrine content, but only a short-lasting increase in tyrosine hydroxylase activity occurred at the beginning of testosterone treatment. In contrast, the testosterone-induced recovery of norepinephrine content observed at this time was accompanied by a marked increase in dopamine-beta-hydroxylase activity. The results suggest that in rat vas deferens, norepinephrine levels are under androgenic control and that this regulation mainly involves changes in dopamine-beta-hydroxylase activity rather than a modulation of tyrosine hydroxylase.     

Traceability of grass feeding in beef: terpenes, 2,3-octanedione and skatole accumulation in adipose tissue of young bulls

The development of analytical methods to verify the production system of meat products requires the identification of biomarkers that can trace the product's origin, and secondly the factors that govern the deposition of these markers in animal tissue need to be defined. In this study, 2,3-octanedione, skatole and terpenes were selected as biomarkers, and their deposition was investigated in bull calves reared under three different strategies. All of the animals were reared indoors until approximately 150 days of age. They were suckled twice a day by their mothers, and both calves and cows had free access to cocksfoot hay. Then the first two groups of animals were kept indoors, suckled by their mothers twice a day and received either cocksfoot hay (HL) or freshly cut-green herbage (GL) and a limited quantity of concentrate. The third group of calves (PH) was kept on pasture with their mothers and offered concentrate ad libitum. The pasture supporting the PH animals was highly diversified, containing several terpene-rich plant species, whereas the herbage for the GL animals contained no species known to be aromatic. Perirenal and subcutaneous adipose tissues were analysed for volatile compounds. The perirenal fat was found to be more responsive to the treatment and a more reliable substrate than the subcutaneous adipose tissue. Higher levels of 2,3-octanedione (P < 0.05) were found in PH and GL than in HL fat (6.56, 6.51 and 5.77 area arbitrary units, respectively, in perirenal fat), confirming the ability of this molecule to trace green herbage feeding. Skatole was detected in the perirenal and subcutaneous fat of all the animals. Animals receiving high concentrate level (PH group) presented lower (P < 0.05) skatole values (5.83 area arbitrary units in perirenal fat) than animals receiving low concentrate level (HL and GL groups, 6.23 and 6.71 area arbitrary units, respectively, in perirenal fat). Terpenoids, and especially sesquiterpenes, were found at higher levels and diversities in the PH than in the GL and HL animals. Two monoterpenoids allowed group discrimination considering perirenal or subcutaneous fat without distinction, whereas 11 and 5 sesquiterpenoids from perirenal and subcutaneous fat, respectively, allowed it.     

Lipoprotein lipase and lipogenic enzyme activities in adipose tissue from rats fed different lipid sources

This work was designed to study the effect of different lipid sources on the activities of lipoprotein lipase and lipogenic enzymes in adipose tissue from rats fedad libitum or energy-controlled diets. Male Wistar rats were fed diets containing 40% of energy as fat (olive oil, sunflower oil, palm oil or beef tallow), for 4 wk. Underad libitum feeding no differences were found among dietary fat groups in final body weight, adipose tissue weights and total body fat. Under energy-controlled feeding, despite isoenergetic intake, rats fed the beef tallow diet gained significantly less weight than rats fed the other three diets. Beef tallow fed rats showed the lowest values for adipose tissue weights and total body fat. When rats had free access to food no effect of dietary lipid source on lipogenic enzyme activities was found. In contrast, under energy-controlled feeding rats fed the beef tallow diet showed significantly higher activities of glucose-6-phosphate dehydrogenase and fatty acid synthase than rats fed the other three diets. Heparin-releasable lipoprotein lipase activity in perirenal and subcutaneous adipose tissues was not different among rats fed olive oil, safflower oil, palm oil or beef tallow. When comparing both adipose tissue anatomical locations, significantly higher activities were found in subcutaneous than in perirenal fat pad independently of dietary fat. In conclusion, under our experimental protocol, lipogenesis in rat adipose tissue does not seem to be affected by dietary fat type.     

The effect of ammonium ion concentration on the activity of adenylate cyclase in various rat tissues in vitro.

The effects of three different ammonium ion concentrations (0.143; 1.43; 14.3 mmol .1(-1)) on adenylate cyclase activities in liver, muscle, fat and brain tissue in vitro were studied in the rat. At physiological levels, ammonium ions reduced adenylate cyclase activity in liver and fat by about 30%, did not affect enzyme activity in muscle tissue but increased adenylate cyclase activity in brain by 40%. Similar effects were observed at higher ammonium ion concentrations though there were no statistically significant correlations between ammonium ion concentration and the degree of change of enzyme activity.     

Changes in enzyme activities of thymidine kinase and 5'-nucleotidase for dTMP during hormonal regeneration of seminal vesicles of mice.

An increase of thymidine kinase [EC 2.7.1.21] activity and decrease of 5'-nucleotidase [EC 3.1.3.5] activity for dTMP were found during hormonal regeneration of the seminal vesicles by daily or single administration of testosterone propionate into mice castrated 2 weeks previously. Actinomycin D injected on day 0 of testosterone treatment completely inhibited both the increase of thymidine kinase and the decrease of 5'-nucleotidase. When injected on day 2, actinomycin D decreased thymidine kinase activity below the control level and 5'nucleotidase activity was not restored to the normal level. The activity of 5'-nucelotidase in a mixed sample, in which seminal vesicles of castrated mice and those of testosterone-treated mice were homogenized together, was intermediate between the activities determined separately. This indicates the absence of any inhibitor of 5'nucleotidase in the regenerating vesicles. Changes in total activity of 5'nucleotidase and total protein content in extracts during various treatments showed that the decrease in specific activity of 5'-nucleotidase in the first 2 days of testosterone treatment was not due to inhibition of enzyme activity but to dilution of the enzyme with other proteins which increased in content more rapidly than 5'-nucleotidase.     

The influence of changes in the phospholipid pattern of intact fibroblasts on the activities of four membrane-bound enzymes.

Human skin fibroblasts, grown to confluency in the presence of 32P for random labelling of the phospholipids, showed upon 24 h incubation in the presence of either 8 mM L-serine or 4 mM ethanolamine an increased content of phosphatidylserine (150% of control cells) or phosphatidylethanolamine (116% of control cells), respectively. Concomitantly the phosphatidylcholine correspondingly decreased. Upon cell harvesting and gentle enzyme preparation the base-treated cells demonstrated a significantly higher unstimulated, fluoride- and thyrotropin-stimulated activity of adenylate cyclase. The activities of total ATPase, ouabain-sensitive ATPase, 5'-nucleotidase and gamma-glutamyltransferase remained unaltered. When subjecting enzyme preparations from fibroblasts to ultrasonication the activity of adenylate cyclase decreased progressively with energy applied, whereas the activities of the other enzymes were unaltered ((K+ + Na+)-ATPase, 5'-nucleotidase) or even increased (Mg2+-ATPase, gamma-glutamyltransferase). The results have a bearing upon the regulatory function of the phospholipid microenvironment of membrane-bound enzymes.     

Influence of the diet and grazing on adipose tissue lipogenic activities and plasma leptin in steers

The objectives of the two experiments were to determine the respective effects and interactions of diet type (grass v. maize diets) and physical activity (grazing v. zero grazing) on lipogenic enzyme activities and adipose cell size in subcutaneous, perirenal and intermuscular adipose tissues and on plasma metabolites and hormones in Charolais steers. After weaning, the steers were assigned to two (Experiment 1, n = 24) or three (Experiment 2, n = 24) groups, with steers in Experiment 1 grazed grass or indoors maize-silage-fed and steers in Experiment 2 grazed grass, indoors cut grass- or indoors maize-silage-fed. Both experiments lasted for 23 months. All grass-fed animals were fed grass silage during the two winter seasons. During the two summer seasons, steers fed on grass were rotationally grazed on a perennial rye-grass pasture while steers fed on cut grass were fed indoors on freshly cut grass alone. Steers fed on maize silage were fed maize silage indoors during the entire experiment. All animals were reared for similar body weight and growth rates and slaughtered at the same age (31 to 32 months). Activities of lipogenic enzymes were significantly lower in the three adipose tissue sites of steers fed cut grass compared with maize silage, although there were less-marked effects in intermuscular adipose tissue. Plasma insulin and glucose concentrations were also lower in steers fed cut grass whereas plasma leptin concentration was similar. As body fat content was not affected by nutritional treatment, it is suggested that the decrease in potential lipogenic activity was associated with the nature of the diet and not to differences in available net energy. In other respects, grazed grass compared with eating cut grass did not affect lipogenic enzyme activities but decreased plasma leptin concentrations in the older steers and increased plasma non-esterified fatty acids and glucose concentrations without affecting adipose tissue weight and adipose cell size.     

Effect of orchidectomy and testosterone substitution on enzyme activities and DNA content in rat liver and epididymal fat

Orchidectomy of rats resulted in increased concentration and whole organ amount of DNA both in the epididymal fat pad and liver. Liver hexokinase (HK) and phosphofructokinase (PFK) activities were raised after orchidectomy, but were normalized by testosterone substitution. Several glycolytic enzymes, and fumarase and aspartate aminotransferase were increased by orchidectomy in epididymal fat. Most of the enzyme changes tended to normalize after testosterone administration. Activities of NADPH generating enzymes were increased after orchidectomy both in liver and epididymal fat. When related to DNA, several enzyme activities in both tissues fell following castration. However, liver HK, PFK and NADPH generating enzymes, as well as epididymal fat HK and isocitrate dehydrogenase were elevated after castration also when related to DNA. The results suggest that the influence of testosterone on cell proliferation is organ-specific. The observed enzyme alterations after orchidectomy might partly explain fat accumulation and hyperlipoproteinemia encountered in castrates.     

Effect of castration and testosterone on norepinephrine storage and on the release of [3H]norepinephrine from rat vas deferens

Norepinephrine and dopamine-β-hydroxylase, used as noradrenergic vesicle markers, were found to be decreased in the rat vas deferens 10 days after castration. Five days of testosterone administration to castrated animals increased the enzyme activity over that of controls but did not modify norepinephrine content. In tissue fractions obtained by differential centrifugation, the highest activities of the noradrenergic markers appeared in the vesicular fraction of controls and in the soluble fraction of castrated animals. Testosterone reversed the effect of castration: it increased dopamine-β-hydroxylase activity in the vesicular and soluble fractions, while norepinephrine increased only in the vesicular fraction. Results obtained after continuous sucrose gradient centrifugation of vesicular fractions suggested that these changes principally affected the number of light noradrenergic vesicles while testosterone increased the number of vesicles reduced by castration. Hormonal manipulations also modified some functional properties of nerve endings: norepinephrine depletion after transmural stimulation in the presence of tetraethylammonium, as well as the release of the neurotransmitter, were decreased after castration. These effects were reversed by testosterone. The results suggest a modulatory effect of testosterone on the norepinephrine storage system and on the functional properties of the adrenergic innervation of vas deferens.     

Effects of castration and hormone replacement on male sexual behavior and pattern of expression in the brain of sex-steroid receptors in BALB/c AnN mice

Little is known about the hormonal regulation of sexual behavior and about the pattern of expression in the brain of sex-steroid receptors in the BALB/c AnN strain of mice (Mus musculus). In this study, 8-week old male BALB/c AnN mice were castrated and the temporal course of decline of sexual behavior was studied, as well as the effects of daily treatment with either testosterone propionate (TP), estradiol benzoate (EB), or dihydrotestosterone propionate (PDHT). Castration resulted in rapid decline of sexual behavior, in both control or vehicle-treated mice. TP maintained full sexual behavior of castrated mice, while PDHT or EB did not have this effect. The expression of ER-alpha dropped nearly 50% after castration, and this pattern remained in TP or PDHT-treated mice, while EB increased the ER-alpha mRNA levels to almost the same values as in intact control mice. The same pattern was found when ER-beta mRNA levels were analyzed. The expression of the PR-A/B gene in the different brain regions in intact mice and after castration, or among the differently treated mice, showed significant differences between normal and castrated mice at all times in all brain regions studied, with the exception of the frontal cortex. Castration reduced the expression of AR by 10-fold, as compared to intact control mice, while TP or PDHT treatment returned its expression to the same levels as in intact control mice, in all brain areas studied. The changes are more prominent in POA-HIP than in HYP and CF. These results demonstrated a rapid decline of sexual behavior in this strain of mice after castration, and show that only TP was able to maintain male sexual behavior, with no correlation with the pattern of expression of sex hormone receptors in specific areas of the mouse brain.     

Interaction of androgens and prolactin on prostatic enzymes of the pyruvate-malate cycle involved in lipogenesis in castrated mature monkey, Macaca radiata.

Effects of androgens, prolactin (Prl) and bromocriptine (Br) on the specific activities of prostatic (caudal and cranial) enzymes of the pyruvate-malate cycle were studied in castrated mature bonnet monkeys. Castration decreased the activity of NADP+ isocitrate dehydrogenase (ICDH), ATP citrate lyase, malate dehydrogenase (MDH), malic enzyme and fatty acid synthase (FAS). Administration of testosterone propionate (TP)/dihydrotestosterone (DHT) increased the activities of all these enzymes in both lobes. Malate dehydrogenase maintained normal activity. Prl also had a stimulatory effect on the enzymes and was further enhanced when Prl was given in combination with TP/DHT. Unlike Prl, bromocriptine treatment inhibited all the enzymes in both lobes. Thus, prolactin was found to have a direct as well as a synergistic effect with androgens on enzymes of the pyruvate-malate cycle in the prostate of castrated mature monkeys.