Carbohydrate deprivation reduces NADPH-production in fish liver but not in adipose tissue

Little is known about the way in which carnivorous fish such as salmonids mobilise and metabolise dietary carbohydrates, which are essential to lipid metabolism. Thus we have studied changes caused by the absence of dietary carbohydrates to the kinetics and molecular behaviour of the four cellular NADPH-production systems [glucose 6-phosphate dehydrogenase (G6PDH); 6-phosphogluconate dehydrogenase (6PGDH); malic enzyme (ME); and isocitrate dehydrogenase NADP-dependent (NADP-IDH)] in the liver and adipose tissue of rainbow trout (Oncorhynchus mykiss). We used spectrophotometry to study enzyme kinetics and nucleic acid concentrations, and immunoblot analysis to determine specific protein concentrations. The absence of carbohydrate reduced specific enzyme activity, maximum rate and catalytic efficiency by almost 65% in G6PDH and 6PGDH, by more than 50% in ME, and by almost 25% in NADP-IDH but caused no significant changes in the K(m) values or activity ratios in any of these hepatic enzymes. Molecular analysis clearly showed that this kinetic behaviour reflected concomitant changes in intracellular enzyme concentrations, produced by protein-induction/repression processes rather than changes in the activity of pre-existing enzymes. We conclude that the absence of carbohydrates significantly reduces intracellular concentrations of G6PDH, ME and NADP-IDH in trout liver in percentages similar to those recorded for enzyme activity. We found no such variations in the concentrations of any of these enzymes in adipose tissue and no change in the levels of their activity, suggesting that the liver and adipose tissues are subject to different regulation systems with regard to carbohydrates and play distinct roles in lipid metabolism.     

Novel aspects of the activities and subcellular distribution of enzymes of ketone body metabolism in the liver and kidney of the goldfish, Carassius auratus

The metabolic organization of ketone body metabolism of liver and kidney of the goldfish Carassius auratus was assessed by measuring maximal activities, subcellular distribution, and stereoisomer preference of ketone body enzymes. These determinations indicate that the organization of ketone body metabolism in liver and kidney of goldfish differs from that of mammals in some respects. All the enzymes of ketone body metabolism were present in liver and kidney of goldfish, with the exception of hydroxymethylglutaryl-CoA (HMG-CoA) synthetase, which was not detected in liver. Two forms of beta-hydroxybutyrate dehydrogenase (betaHBDH) with different stereospecificity for beta-hydroxybutyrate (D- and L-beta-hydroxybutyrate) were detectable in liver and kidney. All of the ketone body enzymes measured in liver were mainly in the mitochondrial fraction, with the exception of D- and L-betaHBDH, which were cytosolic. In kidney, HMG-CoA synthase, together with HMG-CoA lyase and acetoacetyl CoA thiolase (AcoAT), were found mainly in the mitochondrial fraction. L-betaHBDH was mainly cytosolic in kidney, but by contrast with liver, D-betaHBDH was mainly found in the mitochondria, and SKT was distributed in both the mitochondrial and cytosolic compartments. J. Exp. Zool. 286:434-439, 2000.     

Postnatal changes of some enzymatic activities of energy supplying metabolism in the cortex, inner and outer medulla of the rat kidney

1. In rat kidney cortex, outer and inner medulla the development of activities of seven enzymes was investigated during postnatal ontogeny (10, 20, 30, 60 and 90 days of age). The enzymes were selected in such a manner, as to characterize most of the main metabolic pathways of energy supplying metabolism: hexokinase (glucose phosphorylation, HK), glycerol-3-phosphate dehydrogenase (glycerolphosphate metabolism or shunt, GPDH), triose phosphate dehydrogenase (glycolytic carbohydrate breakdown, TPDH), lactate dehydrogenase (lactate metabolism, LDH), citrate synthase (tricarboxylic acid cycle, aerobic metabolism, CS), malate NAD dehydrogenase (tricarboxylic acid cycle, intra-extra mitochondrial hydrogen transport, MDH) and 3-hydroxyacyl-CoA-dehydrogenase (fatty acid catabolism, HOADH). 2. The renal cortex already differs metabolically from the medullar structures on the 10th day of life. It displays a high activity of aerobic breakdown of both fatty acids and carbohydrates. Its metabolic capacity further increases up to the 30th day of life. 3. The outer medullar structure is not grossly different from the inner medulla on the 10th day of life. Further it differentiates into a highly aerobic tissue mainly able to utilize carbohydrates. It can, however, to some extent, also utilize fatty acids aerobically and produce lactate from carbohydrates anaerobically. 4. The inner medullar structure is best equipped to utilize carbohydrates by anaerobic glycolysis, forming lactate. This feature is already pronounced on the 10th day of life, its capacity increases to some extent during postnatal development, being highest between the 10th and the 60th day of life.     

Effect of hypoxia on carbohydrate metabolism in scorpion tissues

The levels of glycogen, glucose, lactate, as well the activities of ten enzymes of carbohydrates metabolism in brain, liver and white muscle of sea scorpion have been investigated. Metabolite concentrations didn't change in brain and the levels of glycogen and lactate were constant in the rest tissues investigated. Glucose concentration decreased in the liver and increased in muscle. In brain hypoxia decreased the activity of hexokinase and increased one of pyruvate kinase, phosphoglucoisomerase and fructoso-1,6-bisphosphatase. In liver most of the enzymes showed the tendency to decrease of their activities. In muscle the activities of phosphofructokinase and phosphoglucoisomerase decreased. Mechanisms of carbohydrates metabolism regulation under hypoxia are discussed.     

Lipid, ketone body and oxidative metabolism in the African lungfish, Protopterus dolloi following 60 days of fasting and aestivation

The potential importance of lipids and ketone bodies as fuels in the African lungfish, Protopterus dolloi, and the role of oxidative metabolism, were examined under control, fasted and aestivated conditions. In aestivating but not fasting lungfish, the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) (enzymes of oxidative metabolism) showed tissue-specific changes. Significant reductions in CS activity occurred in the kidney, heart, gill and muscle, and in CCO in the liver and kidney tissues. Aestivation, but not fasting, also had a tissue-specific effect on mitochondrial state 3 respiration rates (using succinate as a substrate), with a >50% reduction in the liver, yet no change within muscle mitochondria. There is no indication that enzymes involved in lipid catabolism are up-regulated during periods of fasting or aestivation; however, both 3-hydroxyacyl CoA dehydrogenase (HOAD) and carnitine palmitoyl CoA transferase (CPT) activities were sustained in the liver despite the approximately 42% reduction in CCO activity, potentially indicating lipid metabolism is of importance during aestivation. Lungfish are able to utilize both the d- and l-stereoisomers of the ketone body beta-hydroxybutyrate (beta-HB); however, beta-HB does not appear to be an important fuel source during aestivation or fasting as no changes were observed in beta-HB tissue levels. This study demonstrates that an important aspect of metabolic depression during aestivation in lungfish is the tissue-specific down regulation of enzymes of aerobic metabolism while maintaining the activities of enzymes in pathways that supply substrates for aerobic metabolism.     

Chronic hypoxia-induced alterations of key enzymes of glucose oxidative metabolism in developing mouse liver are mTOR dependent

Hypoxia is a potent regulator of gene expression and cellular energy metabolism and known to interfere with post-natal growth and development. Although hypoxia can induce adaptive changes in the developing liver, the mechanisms underlying these changes are poorly understood. To elucidate some of the adaptive changes chronic hypoxia induces in the developing liver, we studied the expression of the genes of mammalian target of rapamycin (mTOR) signaling and glucose metabolism, undertook proteomic examination with 2D gel-MS/MS of electron transport chain, and determined activities and protein expression of several key regulatory enzymes of glucose oxidative metabolism. To gain insight into the molecular mechanism underlying hypoxia-induced liver metabolic adaptation, we treated a subset of mice with rapamycin (0.5 mg/kg/day) to inhibit mTOR postnatally. Rapamycin-treated mice showed lower birth weight, lower body weight, and liver growth retardation in a pattern similar to that observed in the hypoxic mice at P30. Rapamycin treatment led to differential impact on the cytoplasmic and mitochondrial pathways of glucose metabolism. Our results suggest a decrease in mTOR activity as part of the mechanisms underlying hypoxia-induced changes in the activities of glycolytic and TCA cycle enzymes in liver. Chronic postnatal hypoxia induces mTOR-dependent differential effects on liver glycolytic and TCA cycle enzymes and as such should be studied further as they have pathophysiological implications in hepatic diseases and conditions in which hypoxia plays a role.     

Comparative Evaluation of Carbohydrate Metabolism in Perch (Perca fluviatilis L.) from Water Bodies with Different Contents of Humic Acids

Characteristic features of energy metabolism, related to the adaptation of fish to adverse environmental conditions, were detected in perch (Perca fluviatilis L.) inhabiting the lake with a high humus content, compared to perch from the control lake. We studied a complex of enzymes involved in carbohydrate metabolism and assessed the relationship between metabolic pathways using correlation analysis. The intensity of oxidative metabolism was found to increase in the gills. Activation of energy metabolism in the liver was characterized by an increased consumption of carbohydrates. In addition, the role of the liver-specific lactate dehydrogenase (LDH) isoform D₄ also increased. The activity of enzymes involved in carbohydrate metabolism decreased most strongly in white skeletal muscles, whereas the role of the pentose-phosphate pathway (along with glycolysis) in the production of muscle lactate increased. The adaptation of fish to a high-humus aquatic environment was accompanied by a slight decrease in their fatness.     

Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3‐targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3‐dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3‐targeted sites to chemical acetylation in vitro and fasting‐induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low‐level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues.     

Activation of free fatty acids in subcellular fractions of human skeletal muscle

In human pathology little is known about the activating enzymes for fatty acids of different carbon chain length. In order to have a better insight into disorders of lipid metabolism in human skeletal muscle, we studied the distribution of acyl-CoA synthetases in muscular subcellular fractions. We find that in muscle mainly long chain fatty acids are activated to CoA esters. Distribution of palmityl-CoA synthetase in subcellular fractions compared with marker enzymes suggested that this enzymatic activity is located only in the outer mitochondrial membrane, in contrast to human liver, where this enzyme is also located in the microsomes. In human skeletal muscle we also found low butyryl-CoA formation, which was limited to the mitochondrial matrix. This site of activation implies that short chain fatty acids may not depend on carnitine for their oxidation in the mitochondrial matrix, in contrast to long chain fatty acids activated in the outer mitochondrial membrane.     

Effect of benzylpenicillin and tetracycline on the phagocytic, nonspecific humoral and biochemical activity of uninfected animals

The effect of benzylpenicillin and tetracycline on the activity, intensity and completion of phagocytosis with the leucocytes of the peritoneal exudate, on the activity of lysozyme and beta-lysins of the blood serum, on the content of nucleic acids, the activity of succinate dehydrogenase and total and acid phosphatase in the liver and kidney cells was studied experimentally on noninfected albino mice. The study showed that administration of benzylpenicillin and tetracycline to the animals for 5 days induced a decrease in the activity of the cell and humoral defence and the activity of the above enzymes in the liver and kidney cells. The content of the nucleic acids did not change under the effect of the antibiotics.     

Beta-oxidation of polyunsaturated fatty acids in peroxisomes. Subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase activity in rat liver

The metabolism of the double bonds at the delta 3 position in fatty acids was studied in rat liver. Infusion of delta 3-trans-dodecenoic acid into isolated perfused liver and subcellular fractionation studies showed the presence of both peroxisomal and mitochondrial delta 3,delta 2-enoyl-CoA isomerase activity (EC 5.3.3.8). These findings together with the previous demonstration of peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34) [(1981) J. Biol. Chem. 256, 8259-8262] and D-3-OH-acyl-CoA epimerase (EC 5.1.2.3) [(1985) FEBS Lett. 185, 129-134] activities show that peroxisomes possess all the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids.     

Metabolism of norethynodre, a 19-nor progestin: subcellular localization of enzyme activity

The subcellular distribution of the enzymes involved in the metabolism of norethynodrel (17 alpha-ethynyl-17 beta-hydroxy-estr-5(10)-en-3-one) to the 3alpha and 3beta diols (17 alpha-ethynyl-3alpha (or 3beta-17 beta-dihydroxy-estr-5(10)-ene) and 17 alpha-ethinyl estradiol was studied. The purity of the male rat liver subcellular fractions was evaluated by the use of marker enzymes. Sample sections were viewed by electron microscopy. The data showed that the cytosol fraction contained the highest relative specific activity for the hydroxysteroid dehydrogenases required for the formation of the diols. The cytosol fraction also contained the highest total activity. The enzymes required for the formation of ethinyl estradiol were distributed equally among mitochondrial and microsomal fractions, however, the highest relative specific activity was associated with the heavy microsomal fraction (18,000 g).     

Enzymes of anabolic and catabolic nucleic acid pathways in human hepatomas, in liver of healthy persons, and in liver of patients with cancer of gastrointestinal tract]

The activities of enzymes catalyzing the formation of nucleic acid precursors, nucleoside kinases, as well as of those involved in the degradation of nucleic acids, were studied in nuclei of the liver of healthy persons, human hepatomas and the liver of patients with cancer of gastrointestinal tract. The activities of thymydine kinase and uridine kinase in the human hepatoma nuclear sap were found to increase 40- to 50-fold and 120- to 150-fold, respectively, as compared to those in normal human liver. The activities of DNase and RNase in the fraction of chromatin protein of human hepatomas, on the contrary, decreased almost to zero. As to the liver of patients with cancer of gastrointestinal tract, drastic alterations in the activities of nucleoside kinases and nucleases in the direction characteristic of tumors themselves were observed. This phenomenon is regarded as a manifestation of the systemic effects of the tumor on the host.     

Acyl coenzyme A thioesterase Them5/Acot15 is involved in cardiolipin remodeling and fatty liver development

Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/β-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5(-/-) mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and β-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions.     

Pyrimidine pathways in health and disease

Genetic defects involving enzymes essential for pyrimidine nucleotide metabolism have provided new insights into the vital physiological functions of these molecules in addition to nucleic acid synthesis. Such aberrations disrupt the haematological, nervous or mitochondrial systems and can cause adverse reactions to analogue therapy. Regulation of pyrimidine pathways is also known to be disrupted in malignancies. Nine genetic defects have now been identified but only one is currently treatable. Diagnosis is aided by the accumulation of specific metabolites. Recently, progress has been made in understanding the molecular mechanisms underlying inborn errors of pyrimidine metabolism, together with the key clinical issues and the implications for the future development of novel drugs and therapeutic strategies.     

Topoisomerases in kinetoplastids

Topoisomerases are enzymes that mediate topological changes in DNA that are essential for nucleic acid biosynthesis and for cell survival. The kinetoplastid protozoa, which include pathogenic trypanosomes and Leishmania, have yielded an interesting variety of purified topoisomerase activities as well as several topoisomerase genes. In these parasites, topoisomerases are involved in the metabolism of both nuclear and mitochondrial (kinetoplast) DNA. In this review, Christian Burri, Armette Bodley and Theresa Shapiro summarize what is known about topoisomerases in kinetoplastids, and consider the intriguing possibility that these enzymes may act as valuable antiparasite drug targets.     

Effect of histamine H1-receptor antagonist on the regulation of carbohydrate and lipid metabolism in rat liver

In order to elucidate the role of histamine in the liver, we studied the effect of a histamine H1-receptor antagonist on the carbohydrate and lipid metabolism in the rat liver. The administration of the H1-receptor antagonist decreased significantly the contents of glycogen and malonyl-CoA in the liver. However, it did not affect the levels of serum glucose and free fatty acid. These results suggest that histamine may play a part in the regulation of metabolism of carbohydrates and lipids in the liver.     

Androgenic control of hepatic mitochondrial metabolism in an apoda, Gegenophis carnosus (Beddome).

In vivo administration of testosterone significantly stimulated the activities of cytochrome oxidase, alpha-glycerophosphate dehydrogenase (alpha-GPDH), succinate dehydrogenase (SDH) and adenosine triphosphatase (Mg2+ ATPase), in mitochondria isolated from the liver of G. carnosus. Administration of dehydroepiandrosterone and androstenedione while significantly stimulated the activities of cytochrome oxidase and alpha-GPDH, did not change that of SDH and Mg2+ ATPase. Simultaneous injections of testosterone and actinomycin D or chloramphenicol prevented the testosterone-stimulated activities of all the oxidative enzymes studied. The results clearly document the important stimulatory role of androgens in the regulation of hepatic mitochondrial metabolism in G. carnosus.     

Activity of enzymes of nucleic acid metabolism in rat liver nuclei under induction of RNA synthesis by electric stimulation of hypothalamus]

Electrostimulation of hypothalamus results in an increase of RNA synthesis of the AU-type in liver cells of intact and adrenalectomized rats. No such effect is observed in the animals with denervated liver. An increase in RNA synthesis is accompanied by an increase in the activities of RNA-polymerases II and III and histone hydrolase and a decrease in the activities of RNAse and DNAse. The mechanisms of the effect of the nervous system on the activities of the nucleic acid metabolism enzymes are discussed.     

The effects of Insulin Pre-Administration in Mice Exposed to Ethanol: Alleviating Hepatic Oxidative Injury through Anti-Oxidative,Anti-Apoptotic Activities and Deteriorating Hepatic Steatosis through SRBEP-1c Activation

Alcoholic liver disease (ALD) has become an important liver disease hazard to public and personal health. Oxidative stress is believed to be responsible for the pathological changes in ALD. Previous studies have showed that insulin, a classic regulator of glucose metabolism, has significant anti-oxidative function and plays an important role in maintaining the redox balance. For addressing the effects and mechanisms of insulin pre-administration on ethanol-induced liver oxidative injury, we investigated histopathology, inflammatory factors, apoptosis, mitochondrial dysfunction, oxidative stress, antioxidant defense system, ethanol metabolic enzymes and lipid disorder in liver of ethanol-exposed mice pretreatment with insulin or not. There are several novel findings in our study. First, we found insulin pre-administration alleviated acute ethanol exposure-induced liver injury and inflammation reflected by the decrease of serum AST and ALT activities, the improvement of pathological alteration and the inhibition of TNF-α and IL-6 expressions. Second, insulin pre-administration could significantly reduce apoptosis and ameliorate mitochondrial dysfunction in liver of mice exposed to ethanol, supporting by decreasing caspases-3 activities and the ratio of Bax/Bcl-2, increasing mitochondrial viability and mitochondrial oxygen consumption, inhibition of the decline of ATP levels and mitochondrial ROS accumulation. Third, insulin pre-administration prevented ethanol-mediated oxidative stress and enhance antioxidant defense system, which is evaluated by the decline of MDA levels and the rise of GSH/GSSG, the up-regulations of antioxidant enzymes CAT, SOD, GR through Nrf-2 dependent pathway. Forth, the modification of ethanol metabolism pathway such as the inhibition of CYP2E1, the activation of ALDH might be involved in the anti-oxidative and protective effects exerted by insulin pre-administration against acute ethanol exposure in mice. Finally, insulin pre-administration deteriorated hepatic steatosis in mice exposed to ethanol might be through SRBEP-1c activation. In summary, these results indicated that insulin pre-administration effectively alleviated liver oxidative injury through anti-inflammatory, anti-oxidative and anti-apoptotic activities but also deteriorated hepatic steatosis through SRBEP-1c activation in mice exposed to ethanol. Our study provided novel insight about the effects and mechanisms of insulin on ethanol-induced liver injury.