Effects of glucagon and dibutyryl adenosine 3', 5'-cyclic monophosphate on oxidative desaturation of fatty acids in the rat.

The present work was undertaken to study the effect of anti-insulinic and glycogenolytic factors on the oxidative desaturation of fatty acids. The effects of glucagon and dibutyryl cyclic AMP on the desaturation of linoleic acid to gamma-linolenic acid, alpha-linolenic acid to octadeca-6,9,12,15-tetraenoic acid, stearic acid to oleic acid, and eicosa-8,11,14-trienoic acid to eicosa-5,8,11,14-tetraenoic acid by rat liver microsomal preparations were investigated. Fasted rats had low desaturating activity, but refeeding a fat-free diet enhanced the activity. Administration of glucagon or dibutyryl cyclic AMP abolished the increase of the 6-desaturase activity elicited by refeeding. However, a similar effect on the 9-desaturase and 5-desaturase activity was not observed. The relationship between these effects and glucose metabolism is discussed.     

Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity

Epidermal growth factor (EGF), a mitogen for renal proximal tubule cells, activated the hexose monophosphate (HMP) shunt in renal proximal tubule cells (Stanton, R. C., and Seifter, J. L. (1988) Am. J. Physiol. 254, C267-C271). We therefore evaluated the effect of EGF on the HMP shunt enzymes glucose 6-phosphate dehydrogenase (G6PD, the rate-limiting enzyme) and 6-phosphogluconate dehydrogenase. Rat renal cortical cells (RCC) were incubated with either EGF or platelet-derived growth factor (PDGF) and then assayed for G6PD and 6-phosphogluconate dehydrogenase activities. EGF and PDGF increased G6PD activity by 25 and 27% respectively. Although phorbol myristate acetate (PMA), ionomycin, PMA + ionomycin, and 8-bromo-cyclic AMP had no significant effect on the activity, a 5-min preincubation with PMA potentiated the activation of G6PD by PDGF. Growth factor activation of G6PD was also seen in a fibroblast and epithelial cell line. None of the agents affected 6-phosphogluconate dehydrogenase activity in the RCC or in the cell lines. Further exploration into a possible mechanism for G6PD activation revealed that growth factors caused release of G6PD from a structural element within the cell. Streptolysin O permeabilization of RCC did not cause significant release of G6PD. However, within 1 min of addition of EGF or PDGF to permeabilized cells, G6PD was released into the cell supernatant. The nonhydrolyzable analog of GTP, guanosine 5'-O-(thiotriphosphate), caused a similar release of G6PD. Preincubation with pertussis toxin or guanyl-5'-yl thiophosphate inhibited the PDGF but not the EGF effect. Although the data do not establish a definitive proof linking G6PD release and G6PD activation, these results suggest that they are related. Thus, growth factor stimulation of the HMP shunt likely occurs by a novel mechanism associated with release of bound G6PD.     

The association of lysosomal enzymes with nuclei during enzyme induction in rat liver.

Male rats of the Holtzman strain were fasted for 3 days and refed a diet high in carbohydrate (68.9%). The induction of liver glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was monitored for up to 48 h after refeeding. Induction occurred by 24 h, and by 48 h, 4.2- and 1.5-fold increases were observed for glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, compared with that of livers of pellet-fed rats. After refeeding, lysosomes increased in fragility as judged by an increased release of acid phosphatase activity during standard homogenization. Fragility was greatest 3 h after refeeding, but normal fragility was observed 24 h after refeeding. Nuclei were isolated from the liver samples before and after refeeding. Those isolated just before refeeding revealed small latent acid phosphatase activity (4–6%). However, after refeeding the carbohydrate-rich diet, a transient and significant (P < 0.01) increase in the latent activity occurred that was maximal (20%) at 1 h, returning to normal by 24 h. Cross-mixing the 800g nuclear pellet from livers of animals starved for 3 days with the 800g supernatant fraction from livers of animals refed the carbohydrate-containing diet did not alter the nuclear lysosomal-free (overt) or latent (detergent-released) enzyme activity. Similarly, mixing the 800g nuclear pellet from livers of animals refed for 1 h with the 800g supernatant fraction from livers of animals starved for 3 days, but not refed, did not change the nuclear lysosomalfree or latent enzyme activity. Purified nuclei, further washed in hypotonic buffer, lost acid phosphatase activity, but those isolated from livers of rats refed for 1 h retained 10% of the enzyme latency, whereas all latency was lost from those isolated from uninduced rats. A second lysosomal enzyme, β-galactosidase, became associated with the nuclei with the same temporal pattern as for acid phosphatase. However, no variation in nuclear content of cytosolic lactate dehydrogenase occurred as a result of feeding the high-carbohydrate diet to starved rats. When similarly starved rats were refed a diet high in lipid and carbohydrate-free, no induction of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was observed. Lysosomes were not temporarily fragile and purified nuclei did not exhibit increased latency of acid phosphatase activity. Though the evidence presented does not establish a direct correlation between lysosome migration to nuclei as a required function in enzyme induction, the temporal and specific nature of the phenomenon support the hypothesis that liver lysosomal enzymes participate in early signals in the induction of enzymes of lipogenesis.     

Glycolytic cancer cells lacking 6-phosphogluconate dehydrogenase metabolize glucose to induce senescence

We show that knockdown of 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway (PPP) inhibits growth of lung cancer cells by senescence induction. This inhibition is not due to a defect in the oxidative PPP per se. NADPH and ribose phosphate production are normal in 6PGD knockdown cells and shutdown of PPP by knockdown of glucose-6-phosphate dehydrogenase (G6PD) has little effect on cell growth. Moreover, 6PGD knockdown cells can proliferate when the PPP is bypassed by using fructose instead of glucose in medium. Significantly, G6PD knockdown rescues proliferation of cells lacking 6PGD, suggesting an accumulation of growth inhibitory glucose metabolics in cells lacking 6PGD. Therefore, 6PGD inhibition may provide a novel strategy to treat glycolyic tumors such as lung cancer.     

On lysosomal fragility and induction of liver hexose-monophosphate dehydrogenases in the fasted-refed rat.

Young adult male rats were fasted for 3 days, then fed a glucose-rich diet, ad libitum. At the end of the fasting period, the specific activity of liver glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was decreased to 60% of control (nonfasted) levels. After 24 to 72 h of refeeding, the specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increased seven- and twofold, respectively. During the fasting period, the liver lysosome fragility increased, as judged by increased release of bound acid phosphatase and β-N-acetylglucosammidase activity during standard homogenization. Three hours after feeding a carbohydrate-rich diet, a further increase in liver lysosomal fragility was observed that returned to control values prior to the induction of the dehydrogenases. Similarly, the susceptibility of liver lysosomes from fasted rats to increased fragility by the intraperitoneal injection of glucose or galactose was also observed. Prior starvation was not a requisite for labilization of lysosomal membranes by injected glucose, but induction of the pentose phosphate shunt dehydrogenase was not observed.In a group of 6-week old male rats fed a commercial pellet diet throughout, the injection of insulin caused no change in liver lysosomal fragility, though hypoglycemia resulted. Similar animals made diabetic by treatment with Streptozotocin and diabetic rats given insulin, showed no change in liver lysosmal fragility based on the percentage of free to total activities of β-N-acetylglucosaminidase, β-glucuronidase, β-galactosidase, and Cathespin D. However, when adult female rats were fasted for 24 h, then injected with sufficient insulin to produce hypoglycemia, liver lysosomal fragility, based on the release of β-N-acetylglucosaminidase during homogenization, increased nearly threefold. These studies demonstrate that stimulated lysosomal fragility can be initiated by refeeding fasted animals a carbohydrate-rich diet, by intraperitoneal injections of fasted rats with glucose or galactose, or by administering insulin alone to fasted rats. However, hyperglycemia induced by diabetogenic doses of Streptozotocin, or hypoglycemia induced in well-fed animals by insulin injection failed to elicit an enhanced liver lysosomal fragility. Whether induction of the enzymes of lipogenesis by rat liver is dependent upon a prior lysosomal membrane labilization remains to be determined.     

Differential effects of glucagon on induction of rat liver glucose-6-phosphate dehydrogenase by liver injury and dietary change.

Glucagon effectively prevented the increase in glucose-6-phosphate dehodrogenase activity of rat liver following the administration of a glucose-casein mixture without altering the amount of the diet consumed. However, the increase of the enzyme level in carbon tetrachloride-injured rat liver was virtually insensitive to glucagon. The results obtained gave further evidence for the difference between these two induction mechanisms of glucose-6-phosphate dehydrogenase.     

Inactivation of glyceraldehyde-3-phosphate dehydrogenase of human malignant cells by methylglyoxal

The effect of methylglyoxal on the activity of glyceraldehyde-3-phosphate dehydrogenase (GA3PD) of several normal human tissues and benign and malignant tumors has been tested. Methylglyoxal inactivated GA3PD of all the malignant cells (47 samples) and the degree of inactivation was in the range of 25-90%, but it had no inhibitory effect on this enzyme from several normal cells (24 samples) and benign tumors (13 samples). When the effect of methylglyoxal on other two dehydrogenases namely glucose 6-phosphate dehydrogenase (G6PD) and L-lactic dehydrogenase (LDH) of similar cells was tested as controls it has been observed that methylglyoxal has some inactivating effect on G6PD of all the normal, benign and malignant samples tested, whereas, LDH remained completely unaffected. These studies indicate that the inactivating effect of methylglyoxal on GA3PD specifically of the malignant cells may be a common feature of all the malignant cells, and this phenomenon can be used as a simple and rapid device for the detection of malignancy.     

Investigation of the Effects of Some Phenolic Compounds on the Activities of Glucose‐6‐Phosphate Dehydrogenase and 6‐Phosphogluconate Dehydrogenase from Human Erythrocytes

Polyphenols are the important compounds that have various bioactivities. They constitute vital active agents of not only daily diet but also natural medicines that are used traditionally. It is generally considered that they are safe because they are natural. In some conducted studies, different negative effects of these compounds were mentioned. Twelve phenolic compounds have been assayed to determine the effect of inhibition on glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activity. For in vitro studies, the enzymes were purified from human erythrocytes using 2′,5′‐ADP Sepharose 4B affinity chromatography. Naringenin, caffeic acid, ellagic acid, ferulic acid, and sinapic acid against two enzymes, hesperidin and polydatin, only on G6PD activity and chrysin solely against 6PGD showed inhibitory effect. Chlorogenic acid, p‐coumaric acid, and syringic acid did not exhibit an effect on the activity of the two enzymes.     

Regulation of cyclic GMP, cyclic AMP and lactate dehydrogenase by putative neurotransmitters in the C6 rat glioma cell line.

In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by l-propranolol, suggesting mediation by a β-receptor. Phentolamine potentiated the NE-increased cAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of α-adrenergic receptors in C6 cells. Carbamylcholine decreased the levels of both cyclic nucleotides, with hexamethonium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catecholamine-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% and decreased cyclic AMP levels 36%. Calcium- and magnesium-free media inhibited the norepinephrine-induced levels of cyclic GMP and cyclic AMP respectively.     

Purification and Characterization of the Main Isozyme of Polyphenol Oxidase in Mung Bean (Vigna mungo) Seedlings

The induction of NADPH-generating enzymes by polychlorinated biphenyls (PCB) in rats was investigated. The administration of PCB to rats for 3 and 14 days increased the activities of malic enzyme (ME, EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.44) about 2-fold above the control level in the liver. Hepatic mRNA levels of ME, G6PD, and 6PGD, except for G6PD mRNA of the 14-day group, were also elevated to the same degree as the enzyme activities in PCB-treated rats. In rats fed a PCB-containing diet for 1 day, the hepatic mRNA levels of ME and G6PD were elevated prior to the induction of enzyme activity. In the kidney, lung, spleen, heart, and testis, the mRNA levels of ME, G6PD, and 6PGD were not affected by PCB. The induction of hepatic NADPH-generating enzymes would imply an increased demand of NADPH in the liver of rats fed with a PCB-containing diet.     

Stimulation by 6-N,2'-O-dibutyryladenosine 3' :5'-cyclic monophosphate and glucocorticoids of phosphoenolpyruvate carboxykinase in the isolated perfused rat liver

Dibutyryl cyclic AMP stimulated the activity of phosphoenolpyruvate carboxykinase in perfused livers of rats, fed on a low-protein diet, linearly over a 6h period. The enzyme activity was also significantly elevated by dexamethasone, the effect being considerably lower than that of the cyclic nucleotide. Since the time-course of phosphoenolpyruvate carboxykinase activity in response to dibutyryl cyclic AMP resembled that observed after dibutyryl cyclic AMP injection into intact animals, it is suggested that induction of the enzyme in vivo is due to a direct action of the cyclic nucleotide on the liver. Combined administration of dibutyryl cyclic AMP and glucocorticoids did not lead to an additive increase of liver phosphoenolpyruvate carboxykinase activity, either in vivo or in the perfused organ.     

Control by amino acids of the activity of system A-mediated amino acid transport in isolated rat hepatocytes.

The effect of amino acids, in concentrations corresponding to those found in the portal vein of rats given a high-protein diet, was investigated on the activity of system A amino acid transport in hepatocytes from fed rats. Amino acids counteracted the induction of system A by insulin or glucagon. This effect was observed at all concentrations of hormones tested, up to 1 microM. Amino acids did not affect the basal cyclic AMP concentration in hepatocytes, or the large rise in cyclic AMP elicited by glucagon. The reversal of system-A induction was observed at relatively low concentration of amino acids, corresponding to plasma values reported in rats given a basal diet. Amino acids were separately tested: substrates of system A were particularly efficient, but so were glutamine and histidine. Non-metabolizable substrates of system A, such as 2-aminoisobutyrate, were also inhibitory, suggesting that a part of the effect of amino acids is independent of their cellular metabolism. Provision of additional energy substrates such as lactate and oleate did not affect induction of system A or the inhibitory effects of amino acids. Thus amino acids do not act by serving as an energy source and by maintaining the integrity of hepatocytes. Inhibition of mRNA synthesis by actinomycin practically abolished the effect of amino acids on the induction of system A by glucagon. The results suggest that amino acids may promote the synthesis of protein(s) affecting the activity of system A either directly at the carrier unit or at an intermediate stage of its emergence.     

Metabolic effects of ethanol in the rat liver.

The NADPH is one of the cofactors in ethanol metabolism. The aim of the study was to investigate the effect of ethanol on a NADPH generating enzyme (G6P-DH) and on some metabolic parameters of the liver. After a 2-day starvation period rats were fed a lipid free diet for three days. During this refeeding period the animals were divided into three groups; they received a single daily dose of 4 g per kg b.w. ethanol, isocaloric aqueous glucose solution or water by gastric tube. In response to ethanol the activity of hepatic G6P-DH decreased. The amount of triglyceride remained unchanged, certain changes occurred in the fatty acid composition of total lipid. The liver glycogen content was elevated. In female rats treated with ethanol the activity of glucose-6-phosphatase increased.     

The effects of glucose and cyclic GMP on RNA synthesis and nuclear morphology in starved rats.

Feeding rats in diet high in glucose has been demonstrated to inhibit the induction of many enzymes, block the action of glucocorticoids, and, in general, appears to result in decreased cyclic AMP activity. We found that glucose feeding depresses both messenger RNA (mRNA) and non-mRNA synthesis. Electron microscopic examination of the nucleus revealed that glucose feeding decreases the granular component of liver cell nucleoli. It only slightly decreases liver cyclic AMP levels, but produces a sixfold elevation in levels of the cyclic AMP antagonist, cyclic GMP. Administration of bromocyclic GMP, like glucose feeding, depresses mRNA synthesis, but does not simulate the effect of the carbohydrate on nuclear morphology. In addition, glucose feeding halves liver inorganic phosphate and triples ATP levels. Phosphorylation of nuclear proteins, however, remains unaltered. Despite the antagonism between glucose feeding and glucocorticoid activity, the former compound did not change the binding of dexamethasone to liver nuclei.     

Erythrophagocytosis by macrophages: suppression of heme oxygenase by cyclic AMP.

On incubation of peritoneal macrophages with antibody-coated radiolabeled erythrocytes, a reproducible fraction of the erythrocytes was phagocytized and heme oxygenase was induced. Addition of cyclic AMP, dbcyclic AMP, or theophylline to the incubation medium suppressed the substrate-mediated induction of heme oxygenase in a dose-related manner but did not impair the rate or extent of erythrophagocytosis. A similar effect was produced by epinephrine, norepinephrine, isoproterenol, and prostaglandins, which generate endogenous cyclic AMP by stimulating the adenyl cyclase system. Propanolol completely blocked the suppressive effect of epinephrine, while phentolamine was ineffective. In contrast to the cyclic adenosine nucleotide, cyclic GMP probably slightly enhanced the substrate-mediated induction of heme oxygenase and partly reversed the suppressive effect of cyclic AMP. Cyclic adenosine nucleotides, prostaglandin, and theophylline significantly reduced the incorporation of labeled uridine or leucine into RNA and protein of erythrophagocytic macrophages, but failed to impair the uptake of these precursors by the phagocytizing cells. These compounds also reduced the conversion of [1-¹⁴C] glucose to ¹⁴CO₂ by the incubated macrophages, whereas ¹⁴CO₂ formation was enhanced by epinephrine. None of these effects was reversible by addition of insulin or by glucose supplementation, which is in sharp contrast to the suppressive effect of glucocorticoids on heme oxygenase induction.     

Metabolic responses to prolonged starvation, food restriction, and refeeding in the brown trout, Salmo trutta: oxidative stress and antioxidant defenses

The effects of long-term starvation and food restriction (49 days), followed by refeeding (21 days) have been studied with respect to antioxidant defense in the liver and gills (branchial tissues) of the brown trout, Salmo trutta. Malondialdehyde levels in both tissues increased in parallel with starvation and food restriction and these values did not return to normal after the refeeding period. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in liver and gills increased during the 49 days of starvation, but glucose-6-phosphate dehydrogenase (G6PD) activities decreased. Glutathione S-transferase (GST) activity decreased in the liver at the 49th day of starvation, but increased in the branchial tissues. Some of the antioxidant enzyme activities (such as hepatic GST and branchial G6PD) returned to control values of fed fish after the refeeding period, but others (e.g. hepatic SOD and branchial GPx) did not return to normal values. In conclusion, our study indicates that total or partial food deprivation induces oxidative stress in brown trout.     

Utilization of gluconate by Escherichia coli. A role of adenosine 3'':5''-cyclic monophosphate in the induction of gluconate catabolism.

1. Cultures of Escherichia coli growing on gluconate use both gluconate and glucose when glucose is added. 2. Glycerol-grown cells adapt to gluconate utilization even in media containing glucose as well as gluconate. 3. The rates of gluconate utilization by cells growing on a mixture of glucose and gluconate, and the specific activities of the gluconate uptake system and of gluconate kinase, are greater if adenosine 3':5'-cyclic monophosphate (cyclic AMP) is present in the medium than in its absence. 4. Growth on media containing gluconate and cyclic AMP is accompanied by the formation of methyl glyoxal and pyruvate, and progressive inhibition of growth. 5. A mutant devoid of adenylate cyclase activity (cya) grew well on glucose in the absence of exogenous cyclic AMP but grew only poorly on gluconate; neither the gluconate uptake system nor gluconate kinase was adequately induced. The addition of cyclic AMP promoted growth on gluconate and facilitated the induction of proteins required for gluconate catabolism. 6. Phage Pl-mediated transduction of cya+ into the cya-mutant also restored the wild-type phenotype in its ability to adapt to gluconate utilization.     

Metabolism of cyclic adenosine 3'',5''-monophosphate and induction of tryptophanase in Escherichia coli.

The relationship between cyclic adenosine 3',5'-monophosphate (cyclic AMP) metabolism and the induction of tryptophanase and beta-galactosidase was studied in several strains of Escherichia coli grown with succinate, acetate, glycerol, or glucose as the carbon source. No consistent relationship between the intracellular concentration of cyclic AMP in the several strains cultured and the various carbon sources was discerned. In E. coli K-12-1 the induction of tryptophanase was found to vary in the order: succinate greater than acetate greater than glycerol greater than glucose, and that of beta-galactosidase was found in the order: glycerol greater than acetate greater than succinate greater than glucose. Rate of accumulation of cyclic AMP in the culture filtrate was in the order: succinate greater than acetate greater than glycerol greater than glucose. The addition of glycerol to E. coli K-12-1 grown in acetate caused inhibition of tryptophanase and slight inhibition of accumulation of extracellular cyclic AMP. These same conditions caused beta-galactosidase induction to be stimulated. The addition of exogenous cyclic AMP to cultures grown with four different carbon sources had an effect characteristic for each of the two enzymes studied as well as each individual carbon source. The results suggest that there are control elements distinct from cyclic AMP and its receptor protein which respond to the catabolic situation of the cell.