Effect of adrenalectomy and dexamethasone treatment on testicular enzymes involved in carbohydrate metabolism

The effect of adrenalectomy on specific activities of testicular hexokinase, 6-phosphofructokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glycogen concentration have been studied. A general increase in specific activities of all enzymes was seen after adrenalectomy in rats of all ages studied. However, lactate dehydrogenase activity and glycogen concentration in pre-pubertal rats alone were depleted with no alteration in pubertal and adult animals. This was accompanied by increased prolactin titres, but gonadotrophins and testosterone were unaltered. Dexamethasone treatment returned all enzyme activities and hormonal profiles to normal.     

Effect of Adrenalectomy and Corticosterone Replacement on Epididymal Carbohydrate Metabolism - Studies on Mature Male Rats

The effect of adrenalectomy and corticosterone replacement on epididymal enzymes involved in obligatory steps of glycolysis and pentose phosphate pathway were studied along with serum hormonal profiles. Adrenalectomy was found to elevate serum prolactin while the gonadotropins and testosterone were unaltered. In caput epididymal tissue enzymes of the pentose phosphate pathway. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were increased after adrenalectomy. However, in corpus epididymal tissue the key enzymes viz. hexokinase, 6-phosphofructokinase and pyruvatekinase of the glycolytic pathway were elevated leaving the pentose phosphate pathway unaffected. Adrenalectomy was also found to favour glycolysis of the epididymal spermatozoa. The possible direct effect of prolactin is discussed to explain the enzymatic changes in epididymis. Corticosterone replacement was found to maintain the enzyme activities along with serum prolactin and corticosterone at control levels. In conclusion, it is suggested that the adrenalectomy induced changes in enzyme activities could be due to the direct effect of prolactin.     

Effect of dexamethasone on testicular enzymes of the Embden-Meyerhof and pentose-phosphate pathways

The influence of dexamethasone on the specific activities of testicular enzymes involved in the Embden-Meyerhof and pentose-phosphate pathways was studied in pre-pubertal, pubertal and adult rats. All of the enzymes showed a decrease in specific activity after dexamethasone treatment, an effect which was most drastic in pre-pubertal animals. After cessation of treatment, the specific activity of all the enzymes reverted to normal levels, except for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the pre-pubertal group.     

Specific effect of the thyroid on testicular enzymes involved in carbohydrate metabolism

The effect of thyroidectomy (Tx) on some key enzymes involved in glycolysis and the HMP-shunt pathway were studied in the testis of pre-pubertal, pubertal and adult rats. Hexokinase (HK) and phosphofructokinase (PFK) did not show any significant change in activity after Tx. However, pyruvate kinase (PK) activity was reduced after Tx in the testis of pre-pubertal animals although in pubertal and adult rats it was not affected. Both glucose-6-phosphate dehydrogenase (G6P-DH) and 6-phosphogluconate dehydrogenase (6-PG-DH) activities were reduced after Tx and thyroxine (T4) replacement brought both enzyme activities back towards normal. The possible effect of the thyroid on testicular steroidogenesis acting through the HMP-shunt is discussed.     

Influence of hypothyroidism on epididymal enzymes involved in carbohydrate metabolism Studies in pubertal and adult rats

The influence of thyroidectomy on key epididymal enzymes of the Embden-Meyerhof and pentose phosphate pathway have been studied in pubertal and adult animals in relation to the serum hormone profile. Age related differences in the response of epididymal segments were observed with respect to hexokinase activity, although the other 2 key enzymes of the Embden-Meyerhof pathway (6-PFK and PK) were suppressed in all regions of the epididymis in both pubertal and adult rats. The enzymes involved in the pentose phosphate pathway (G-6-PDH and 6-PGDH) remained unaltered. The serum hormone profile revealed that while FSH and testosterone titres were reduced, LH and Prl were unaltered. Replacement of T4 in thyroidectomized animals maintained serum hormone levels and the activities of the enzymes studied at control levels. It is inferred that thyroid hormones may be one part of a complex mechanism that controls carbohydrate metabolism in the epididymis.     

Epididymal carbohydrate metabolism in experimental hypercorticosteronism: studies on mature male rats

Corticosterone induced changes in serum hormonal profiles and the key enzymes involved in glycolytic and pentose phosphate pathways were studied in caput, corpus and cauda epididymides of mature male rats (200–250 g body weight). Corticosterone (3.5 mg/100 g body weight sc. for 20 days) treatment was found to depress serum testosterone and prolactin while the gonadotrophins were unaltered. Enzymes of both the glycolytic and pentose phosphate pathways were significantly decreased in caput epididymidis. But in corpus and cauda epididymides only the glycolytic enzymes were reduced. Withdrawal of treatment (for 20 days), resulted in restoration of the glycolytic enzymes to normalcy. The serum hormonal profiles were also found to be within the normal range. The pentose phosphate pathway in caput epididymidis showed a significant increase in enzyme activities following withdrawal of treatment. From the present investigation it is clear that hypercorticosteronism had a definite influence on epididymal enzymes involved in carbohydrate metabolism.     

Enzymes of glucose metabolism and of the citrate cleavage pathway in adipose tissue of normal and diabetic subjects.

Enzyme activities operative in glucose degradation and citrate cleavage pathway were studied in the adipose tissue of twenty-four patients with adult-onset diabetes and normal body weight, aged 59+/-9 years, and twenty-four matched controls. In normal tissue, type II (heat-inactivated) hexokinase moderately predominated over type I (heat-resistant). 6-Phosphofructokinase had an extremely low activity, which was by far the lowest among the ten glycolytic enzyme activities investigated, and which therefore might greatly limit the glycolytic rate. The level of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating) was elevated above that occurring in other tissues. This, especially if considered together with the low 6-phosphofructokinase activity, would suggest a major role of pentose cycle in glucose degradation. Of the citrate cleavage pathway enzymes, ATP citrate-lyase, although having a lower activity than malate dehydrogenase and malate dehydrogenase (decarboxylating) (NADP), was readily measurable, which contrasts with previous data by others. This finding is consistent with the occurrence of lipogenetic capacity in human adipose tissue. In diabetic tissue, there was a decreased activity, both on a protein and on a wet-weight basis, of enzymes concerned with the glucose entry into metabolic pathways, namely hexokinase (both type I and, especially, type II) and pentose cycle dehydrogenases, as well as of pyruvate kinase. This could be connected with the defective glucose utilization by adipose tissue in diabetes. Beside the above-mentioned dehydrogenases, malate dehydrogenase (decarboxylating) (NADP) was also diminished. The reduction of these NADPH-forming enzymes, which supply reducing equivalents for fatty acid synthesis, would suggest a depressed lipogenesis.     

Prolactin and Leydig cell steroidogenic enzymes in the bonnet monkey (Macaca radiata)

The effect of treatment with prolactin or bromocryptine on testicular steroidogenesis and serum hormone levels were studied in immature and mature bonnet monkeys. Leydig cells alone showed the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in normal immature and mature monkeys. Administration of prolactin increased the activity of 3 beta-HSD and 17 beta-HSD in Leydig cells from mature monkeys, and also increased the serum levels of testosterone. Bromocryptine treatment induced exactly the opposite effect. These changes occurred in the absence of any change in serum gonadotrophin levels. In immature monkeys, prolactin and bromocryptine had no significant effect. These results suggest a direct stimulatory effect of prolactin on testicular steroidogenesis in mature monkeys.     

Effects of androgens, prolactin and bromocriptine on seminal vesicular enzymes of the pyruvate malate cycle involved in lipogenesis in castrated mature monkeys, Macaca radiata

The interaction of androgens and prolactin, the major factors regulating the male accessory sex organs, on the specific activity of seminal vesicular enzymes of the pyruvate/malate cycle were studied in castrated mature monkeys. Castration decreased the activity of these enzymes, including NADP+ isocitrate dehydrogenase, ATP citrate lyase, malate dehydrogenase, malic enzyme and fatty acid synthase. Testosterone propionate (TP)/dihydrotestosterone given as replacement to castrates increased the activity of all these enzymes, except for malate dehydrogenase. Prolactin restored normal activity of ATP citrate lyase, malic enzyme and fatty acid synthase but not of isocitrate dehydrogenase and malate dehydrogenase (MDH). Prolactin had a specific control over MDH. Moreover, when prolactin was combined with androgens a further stimulatory influence was observed on fatty acid synthase activity. In order to prove the direct influence of prolactin on enzymes of the pyruvate/malate cycle, bromocriptine was administered and this inhibited all of the enzymes. Thus prolactin was found to have a direct, as well as a synergistic, action with androgens on enzymes of the pyruvate/malate cycle in the seminal vesicles of monkeys.     

Glycolytic enzyme activities in bovine hard dental tissue at different ages

The activity of the following enzymes was determined by improved spectrophotometrical methods in inely pulverized young and adult bovine hard dental tissue extracts: hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase, pyruvate kinase, lactate dehydrogenase. The enzymes were solubilized from the calcified matrix by crushing the dentine at the temperature of liquid nitrogen. This technique led to substantial improvement in the extraction of many enzymes. The influence of the extracting fluids of enzyme activities was also examined. All the enzymes investigated were present in bovine dentine; the activities are consistent with a fast flux through the Embden-Meyerhof-pathway. Well-defined quantitative proportions between the maximal activities of functional correlated enzymes were observed, as had previously been reported in a number of quite different organisms and tissues. Most glycolytic activities increased on ageing; hexokinase, phosphofructokinase and aldolase were constant; lactate dehydrogenase showed a dramatic fall. A similar difference in aging pattern has been displayed by these enzymes in other avascular tissues.     

Evaluation of the usefulness of enzymatic diagnosis of myocardial infarction in patients with acute arterial occlusion of the lower extremities

The serum activities of aspartate aminotransferase, lactate dehydrogenase, creatine kinase and estimated creatine kinase isoenzyme MB (CK-B) were investigated in 12 patients before and after revascularization of ischaemic lower extremities. All patients suffered from sudden lower limb arterial occlusion and underwent embolectomy through a small arteriotomy in the groin. The median serum activity of all four enzymes was elevated before surgery and further increased during the first 24-48 h after revascularization. Median serum activity of aspartate aminotransferase, creatine kinase and lactate dehydrogenase were continuously elevated 7 days after the operation. A high relative CK-B activity coincided in one patient with the development of electrocardiographic evidence of acute myocardial infarction. It is concluded that any of these four enzymes should be used with caution in the diagnosis of acute myocardial infarction before, during or after operation in patients who have sustained prolonged ischaemia of the lower extremities.     

Mechanism responsible for inactivation of skeletal muscle pyruvate dehydrogenase complex in starvation and diabetes.

Regulation of the activity of the pyruvate dehydrogenase complex in skeletal muscle plays an important role in fuel selection and glucose homeostasis. Activation of the complex promotes disposal of glucose, whereas inactivation conserves substrates for hepatic glucose production. Starvation and diabetes induce a stable increase in pyruvate dehydrogenase kinase activity in skeletal muscle mitochondria that promotes phosphorylation and inactivation of the complex. The present study shows that these metabolic conditions induce a large increase in the expression of PDK4, one of four pyruvate dehydrogenase kinase isoenzymes expressed in mammalian tissues, in the mitochondria of gastrocnemius muscle. Refeeding starved rats and insulin treatment of diabetic rats decreased pyruvate dehydrogenase kinase activity and also reversed the increase in PDK4 protein in gastrocnemius muscle mitochondria. Starvation and diabetes also increased the abundance of PDK4 mRNA in gastrocnemius muscle, and refeeding and insulin treatment again reversed the effects of starvation and diabetes. These findings suggest that an increase in amount of this enzyme contributes to hyperphosphorylation and inactivation of the pyruvate dehydrogenase complex in these metabolic conditions. It was further found that feeding rats WY-14,643, a selective agonist for the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), also induced large increases in pyruvate dehydrogenase kinase activity, PDK4 protein, and PDK4 mRNA in gastrocnemius muscle. Since long-chain fatty acids activate PPAR-alpha endogenously, increased levels of these compounds in starvation and diabetes may signal increased expression of PDK4 in skeletal muscle.     

Effects of luteinizing hormone, follicle-stimulating hormone, and epidermal growth factor on expression and kinase activity of cyclin-dependent kinase 5 in Leydig TM3 and Sertoli TM4 cell lines

We examined the effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and epidermal growth factor (EGF) on the expression and kinase activity of cyclin-dependent kinase 5 (Cdk5) in Leydig TM3 and Sertoli TM4 cell lines. Hormonal regulation of the expression and activity of Cdk5 by using normal and hypophysectomized rat testes was also investigated to elucidate its role. Cdk5 levels and kinase activity were significantly elevated in TM3 cells that were grown in the presence of 7.5% serum, EGF, or LH and were associated with an increase in testosterone production compared with controls. These increases were accompanied by an increase in proliferation of TM3 cells after treatment with serum or EGF but not with LH suggest that Cdk5 may be involved in cellular differentiation that is induced with LH treatment. In contrast, the presence of neither serum, EGF, nor FSH had a significant effect on Cdk5 activity levels in the Sertoli TM4 cell line, and there was no correlation with proliferative activity or transferrin levels. A significant decrease in Cdk5 expression and activity were noted in rat testis after hypophysectomy compared with normal rat testis and is associated with a simultaneous decrease in testosterone and transferrin levels. Immunohistochemical analysis revealed that Cdk5 was strongly expressed in the nuclei and cytoplasm of Leydig cells, Sertoli cells, spermatogonia, and peritubular cells of normal adult rat testis. After hypophysectomy, the pattern of Cdk5 staining differed markedly from that in normal rat testis and a profound reduction in staining of Cdk5 was observed in each tubule. Our results suggest that LH and EGF influence and modulate Cdk5 expression and activity in Leydig TM3 cells and may, conceivably, be involved in signal transduction cascades that are initiated by hormones or growth factors. Cdk5 in Sertoli TM4 cells is likely to possess some constitutive functions that are not affected by the cells' proliferation state. Moreover, Cdk5 is probably involved in the constitutive and hormonally stimulated activities of the rat testis, in addition to its involvement in cell proliferation.     

In chronic uremia, insulin activates receptor kinase but not pyruvate dehydrogenase.

We studied in vivo biochemical effects of insulin in the skeletal muscle of chronically uremic and control rats. The rate of disappearance of blood glucose (determined with a short intravenous test) was reduced by 38% in uremia (p less than 0.05). Intraperitoneal treatment with insulin plus glucose for 30 min caused a 3-fold increase in the activity of insulin receptor tyrosine kinase in the skeletal muscle of both rat groups. Conversely, pyruvate dehydrogenase activity increased by 115% in controls but only by 26% in uremics (p less than 0.01). Exercise (swimming for 30 min) increased muscle pyruvate dehydrogenase activity approximately 2-fold in both groups of animals. These experiments show that in uremic rats, insulin binds normally to its muscle receptors and adequately activates receptor tyrosine kinase but fails to activate an otherwise responsive pyruvate dehydrogenase.     

Glycolytic enzyme activities are decreased during acute rejection in transplanted rat hearts

Metabolic alterations have been characterized in various heart diseases. However, no data are available concerning metabolic changes during acute rejection episodes. Heterotopic heart transplantations in rats were done using Lewis rats as donors and recipients as a control group. The rejection group included Brown-Norway rat donors to Lewis rat recipients. Nonoperated hearts were also studied. Enzyme activities were determined for phosphofructokinase, pyruvate kinase, and lactate dehydrogenase. There were no alterations in the control group compared to nonoperated hearts. However, the rejection cohort of hearts showed decreased glycolytic enzymes. Although lactate dehydrogenase maintained similar levels compared to the control group, phosphofructokinase showed only 50% activity, and pyruvate kinase showed less than 10% of the activity compared with controls. These results suggested that metabolic alterations in rejected hearts differ from other cardiomyopathies.     

Studies on liver cell injury in obstructive jaundice: activities of key enzymes responsible for carbohydrate metabolism in rat after bile duct ligation

Key enzyme activities of carbohydrate metabolism were evaluated as sensitive indicators of liver cell injury of rats. Hexokinase and glucose 6-phosphatase distribution in the acinus of the liver was also studied histochemically with the following results: The activities of liver-specific enzymes, such as glucose 6-phosphatase, fructose 1.6-diphosphatase, glucokinase and pyruvate kinase Type L, were decreased. While the activities of nonspecific enzymes to liver, such as hexokinase, glucose 6 phosphate dehydrogenase, phosphofructokinase and pyruvate kinase Type M2 were increased. The decrease in glucokinase activity was marked and was found as early as one day after bile duct ligation. Isozymes of HK, I, II and III, all increased to the same extents. Increased hexokinase activity was found in the centrolobular area, where the decreased activity of glucose 6-phosphatase was observed. These changes in the key enzyme activities indicated the presence of a hepatocyte injury caused by bile duct ligation.     

Chronic hyperinsulinemia in the fetal rhesus monkey: effects on hepatic enzymes active in lipogenesis and carbohydrate metabolism.

Hyperinsulinemia was produced in fetal rhesus monkeys for 21 days in the last third of gestation by subcutaneous pork insulin injected at 19 U a day. Plasma insulin concentrations in treated fetuses (N = 4) were 3525 microU/ml. There was no difference in paired pre- and post-treatment fetal plasma glucose concentration. Activity of the hepatic enzymes that promote glucose utilization (glucokinase and hexokinase) and glycolysis (phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase) was unaffected. Similarly, glycogen metabolism enzymes (active and inactive synthase and phosphorylase) were unaltered. Two gluconeogenic enzymes (PEPCK and glucose-6-phosphatase) were diminished in the treated group compared with controls. Fetal hyperinsulinemia enhanced lipogenic and NADPH-producing enzyme activities, as evidenced by a twofold increase in fatty acid synthase and in citrate cleavage enzyme activity. Malic enzyme was absent. Hyperinsulinemia with euglycemia (1) increases the activity of enzymes that participate in lipogenesis, (2) decreases some of those controlling gluconeogenesis, and (3) has no effect on the enzymes of glycolysis.     

Energy metabolism changes of renal cortex in a rat model of progressive kidney disease and its mechanisms

Objective To explore the changes of renal cortical energy metabolism and its related molecular mechanisms in rats with progressive kidney disease. Methods A rat model of 5/6 nephrectomy was established as the model of progressive nephropathy. Rats were divided into surgical group (5/6Nx group) and sham-operated group (Sham group). Respectively, the rats were sacrificed at 1 week and 12 weeks after completing the model, and their blood, urine sample and kidney specimens were collected. Blood urea nitrogen, serum creatinine and 24 h urine protein were used to evaluate the renal function. Pathological changes in renal tissue were detected by PAS staining and Sirius red staining. The renal cortical energy metabolites were made quantitative analysis by liquid chromatography-mass spectrometry-based targeted metabolomics. The mRNA expressions of inflammatory cytokines (IL-6, IL-1β), fibrosis factors (fibronectin, collagen-1), glycolytic and tricarboxylic acid (TCA) cycle related enzymes were confirmed by real-time PCR. The protein expressions of fibrotic proteins (fibronectin, collagen-1), silent information regulator 1 (SIRT-1) and liver kinase B1 (LKB1) were tested by Western blotting. Results Compared with those in Sham group, the renal function indexes increased, the renal tissue pathological damage was obvious, the mRNA expressions of renal cortical inflammatory and fibrosis factors increased, and fibrotic proteins also increased in 5/6Nx group rats at 1 week and 12 weeks (all P＜0.05), meanwhile, kidney damage worsened over time. Compared with those in Sham group, in the renal cortex of 5/6Nx group glycolytic metabolite lactate, the TCA cycle metabolites (citrate, isocitrate, oxaloacetate) and the oxidized phosphorylation metabolite reduced coenzymeⅠ were up-regulated (all P＜0.05), but adenosine triphosphate (ATP) was no change at 1 week, then the abnormal metabolites increased further at 12 weeks, such as the down-regulation of pyruvate, oxidized coenzyme Ⅰ and ATP (all P＜0.05). The pentose phosphate pathway metabolites (reduction and oxidized coenzyme Ⅱ) shows no statistical significant difference in the two group (all P＞0.05). Compared with those in Sham group, in the 5/6Nx group the mRNA expressions of glycolytic enzyme hexokinase 2 and lactate dehydrogenase a were up-regulated in the renal cortex at 1 week, whereas the mRNA expressions of pyruvate dehydrogenase α, pyruvate dehydrogenase β and succinate dehydrogenase of the TCA cycle related enzymes were down-regulated (all P＜0.05). Meanwhile, renal abnormal metabolic enzyme mRNA expressions were further increased in the 5/6Nx group at 12 weeks. The protein levels of SIRT-1 and LKB1 were not significantly different in the renal cortex of two group rats at 1 week, while SIRT-1 and LKB1 levels decreased in 5/6Nx group than those in Sham group at 12 weeks (all P＜0.05). Conclusions During the progression of nephropathy, rats accompanied with renal fibrosis and inflammatory have energy metabolism changes in the renal cortex which accompanies. The features of metabolic changes are manifested as enhanced glycolysis and decreased oxidative phosphorylation, which is aggravated gradually. Its mechanism is related to the inhibition of energy-regulating proteins LKB1 and SIRT-1.     

Effect of total hepatectomy on selected cerebral substrates and enzymes of the glycolytic pathways and Krebs cycle.

Posthepatectomy coma was produced in 13 dogs and the cerebrums were biopsied for analysis of concentrations of glucose, glucose-6-phosphate, dihydroxyacetone-phosphate, phosphoenolpyruvate, pyruvate, lactate, citrate, alpha-ketogulutarate, fumarate, malate, oxaloacetate, adenosinetriphosphate, ammonia, and glutamine as well as for activities of glucokinase, phosphofructokinase, pyruvate kinase, isocitrate dehydrogenase, glutamate dehydrogenase, malate dehydrogenase, and malic enzyme. There were no differences from normal in the brain glycolytic substrate concentrations. Four of the Krebs cycle substrates were significantly reduced, but not differently than in dogs sedated for 24 hours. The glycolytic pathway, Krebs cycle, and related enzyme activities were not significantly altered. Cerebral adenosine triphosphate concentration was unchanged but the concentrations of ammonia and glutamine increased threefold.