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PubMed1991

Heterogeneity of response to exercise of rat muscle pyruvate dehydrogenase complex.

Muscle glucose uptake is greatly stimulated by moderate exercise, but full oxidation of the glucose to CO2 depends on the activity of the pyruvate dehydrogenase (PDH) complex. Our aim was to determine how PDH complex in different muscle groups responds to varying periods of moderate exercise. Rats were run on a motor-driven treadmill for 5-30 min and muscle PDH complex activity was determined in heart, diaphragm and red quadriceps muscles after isolation of mitochondria in the presence of inhibitors of PDH complex interconversion. In heart and diaphragm muscle, exercise caused an increase in PDH complex activity after 5 min, but this was followed by a significant decrease in activity as exercise progressed. In red quadriceps muscle, PDH complex activity was reduced after 5 min of exercise and was decreased further as exercise continued. We conclude that increased duration of exercise can lead to reduced PDH complex activity in rat muscles. We propose that this is a consequence of elevated fatty acid oxidation, the products of which stimulate PDH kinase. This implies that increased glycolysis to lactate and increased fatty acid oxidation can simultaneously provide energy for contracting muscle.

Animals↗
PubMed2000

Differential responses to chronic hypoxia and dietary restriction of aerobic capacity and enzyme levels in the rat myocardium.

Chronic exposure of mammals to hypoxia induces a state of anorexia. We aimed to determine the role played by diet restriction in the alterations of myocardial energy metabolism occurring under chronic hypoxia in order to detect the specific effects of hypoxia per se. Adult female rats were exposed to normobaric hypoxia (FiO2 = 0.10) for three weeks; pair-fed rats, kept under normoxic conditions, received the same amount of food as hypoxic rats. The oxidative capacity of myocardial ventricles and some skeletal muscles was evaluated using permeabilized fibers. Several metabolic enzyme activities were measured on extracts from myocardium and soleus. Diet restriction increased the activity of lactate dehydrogenase in both ventricles while it augmented phosphofructokinase and pyruvate kinase activities only in the left ventricle and depressed the respiratory rate in the right ventricle only. Hypoxia per se induced a rise in hexokinase activity in all studied oxidative muscles and a fall of hydroxy-acyl CoA-dehydrogenase activity in both myocardial ventricles. The respiratory rate and the citrate synthase activities were unaffected by hypoxia. We conclude that chronic hypoxia per se leads to specific alterations in myocardial metabolism that could favor the use of exogenous glucose at the expense of free fatty acids without any change in the oxidative capacity.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2001

Repression of cellular anaplerosis as the hypothesized mechanism of gamma-linolenic acid-induced toxicity to tumor cells.

In in vitro cultures, the cell is virtually isolated and can no longer rely on mechanisms for physiological regulation of substrate availability found in tissues. More emphasis is laid on utilization of preponderant substrate in a proposed reciprocal relationship between glycolysis and free fatty acid (FFA) oxidation for energy. Supraphysiological concentrations of gamma-linolenic acid and some other polyunsaturated fatty acids (PUFAs) therefore suppress glycolysis but also inhibit FFA oxidation initiated through a cytochrome P450-mediated epoxidation of PUFA to inhibit fatty acid synthase (FAS) activity. FAS inhibition accumulates malonyl CoA which inhibits carnitine palmitoyl transferase I and prevents FFA oxidation. The cell is starved of energy and anabolic intermediates, leading to decreased proliferation or death for tumor cells. Tumor cells are more vulnerable to this induced toxicity due to possession of specific phenotypes of elevated expression for FAS and pyruvate kinase, type M2, both factors inducing tumor cell apoptosis on inhibition.

Acetyl Coenzyme A↗
PubMed1997

Improved estimation of anaplerosis in heart using 13C NMR.

Anaplerotic enzymes, such as pyruvate carboxylase or malic enzyme, catalyze reactions that fill up the pools of the citric acid cycle (CAC), thereby increasing the total mass of CAC intermediates. Relative anaplerosis (y) denotes the ratio of anaplerotic flux to the flux catalyzed by citrate synthase. We examine conventional methods [C. R. Malloy, A. D. Sherry, and F. M. H. Jeffrey. J. Biol. Chem. 263:6964-6971, 1988; C. R. Malloy, A. D. Sherry, and F. M. H. Jeffrey. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H987-H995, 1990] of measurement of y using 13C-labeled precursors and analysis of [13C]glutamate labeling by nuclear magnetic resonance (NMR) spectroscopy. Through mathematical analysis and computer simulation, we show that isotopic enrichment of the pool of pyruvate that is substrate for anaplerosis will severely decrease the accuracy of estimates of y made with conventional methods no matter how small the mass of the pool of pyruvate. Suppose that the recycling parameter R denotes the fraction of molecules of pyruvate that contain carbons derived from intermediates of the CAC. Each means of estimation of relative anaplerosis in the peer-reviewed literature assumes that R = O, although this assumption has not been confirmed by experiment. We show that conventional formulas, using either fractional enrichments of carbons or isotopomer analysis, actually estimate at most y.(1 - R) instead of y during administration of [2-13C]acetate and unlabeled pyruvate. Using a new formula for estimation of y, we recalculate values of y from the literature and find them approximately 50% too low. We assume that all anaplerosis is via pyruvate and that the difference in isotopic enrichment between cytosolic and mitochondrial malate is negligible.

Animals↗
PubMed1997

Changes in the size and composition of intracellular pools of nonesterified coenzyme A and coenzyme A thioesters in aerobic and facultatively anaerobic bacteria.

Intracellular levels of three coenzyme A (CoA) molecular species, i.e., nonesterified CoA (CoASH), acetyl-CoA, and malonyl-CoA, in a variety of aerobic and facultatively anaerobic bacteria were analyzed by the acyl-CoA cycling method developed by us. It was demonstrated that there was an intrinsic difference between aerobes and facultative anaerobes in the changes in the size and composition of CoA pools. The CoA pools in the aerobic bacteria hardly changed and were significantly smaller than those of the facultatively anaerobic bacteria. On the other hand, in the facultatively anaerobic bacteria, the size and composition of the CoA pool drastically changed within minutes in response to the carbon and energy source provided. Acetyl-CoA was the major component of the CoA pool in the facultative anaerobes grown on sufficient glucose, although CoASH was dominant in the aerobes. Therefore, the acetyl-CoA/CoASH ratios in facultatively anaerobic bacteria were 10 times higher than those in aerobic bacteria. In Escherichia coli K-12 cells, the addition of reagents to inhibit the respiratory system led to a rapid decrease in the amount of acetyl-CoA with a concomitant increase in the amount of CoASH, whereas the addition of cerulenin, a specific inhibitor of fatty acid synthase, triggered the intracellular accumulation of malonyl-CoA. The acylation and deacylation of the three CoA molecular species coordinated with the energy-yielding systems and the restriction of the fatty acid-synthesizing system of cells. These data suggest that neither the accumulation of acetyl-CoA nor that of malonyl-CoA exerts negative feedback on pyruvate dehydrogenase and acetyl-CoA carboxylase, respectively.

Acetyl Coenzyme A↗
PubMed2004

Suppression of hepatic fatty acid synthase by feeding alpha-linolenic acid rich perilla oil lowers plasma triacylglycerol level in rats.

This study was performed to determine the effects of dietary perilla oil, a n-3 alpha-linolenic acid (ALA) source, on hepatic lipogenesis as a possible mechanism of lowering triacylglycerol (TG) levels. Male Sprague-Dawley rats were trained for a 3-hour feeding protocol and fed one of five semipurified diets as follows: 1% (w/w) corn oil control diet, or one of four diets supplemented with 10% each of beef tallow, corn oil, perilla oil, and fish oil. Two separate experiments were performed to compare the effects of feeding periods, 4 weeks and 4 days. Hepatic and plasma TG levels were decreased in rats fed perilla oil and fish oil diets, compared with corn oil and beef tallow diets. The activities of hepatic lipogenic enzymes such as fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase, and malic enzyme were suppressed in the fish oil, perilla oil, and corn oil-fed groups, and the effect was the most significant in the fish oil-fed group. Also, the activities of glycolytic enzymes, glucokinase, and L-pyruvate kinase showed the similar trend as that of lipogenic enzymes. The activity of FAS, the key regulatory enzyme in lipogenesis, was positively correlated with hepatic and plasma TG levels and reduced significantly in the perilla oil-fed group compared with corn oil-fed group. In addition, the FAS activity was negatively correlated with the hepatic microsomal content of EPA and DHA. In conclusion, suppression of FAS plays a significant role in the hypolipidemic effects observed in rats fed ALA rich perilla oil and these effects were associated with the increase of hepatic microsomal EPA and DHA contents.

Animals↗
PubMed2003

Proton-translocating ATP-synthase of Paracoccus denitrificans: ATP-hydrolytic activity.

Tightly coupled membranes of Paracoccus denitrificans catalyze oxidative phosphorylation but are incapable of ATP hydrolysis. The conditions for observation and registration of the venturicidin-sensitive ATPase activity of subbacterial particles derived from this organism are described. The ATP hydrolytic activity does not appear after prolonged incubation in the presence of pyruvate kinase and phosphoenol pyruvate (to remove ADP), EDTA (to remove Mg2+) and/or inorganic phosphate, whereas the activity dramatically increases after energization of the membranes. ATP hydrolysis by -activated ATPase is coupled with electric potential formation. Inorganic phosphate prevents and azide promotes a decline of the enzyme activity during ATP hydrolysis. The addition of uncouplers results in rapid and complete inactivation of ATPase. The -dependent ATPase activity increases upon dilution of the membranes. The results are discussed as evidence for the presence of distinct ATP-synthase and ATP-hydrolase states of F(o)F(1) complex in the coupling membranes (Vinogradov, A. D. (1999) Biochemistry (Moscow), 64, 1219-1229). The proposal is made that part of the free energy released from oxidoreduction in the respiratory chain is used to maintain active conformation of the energy-transducing proteins.

Adenosine Triphosphatases↗
PubMed1986

Nucleotide sequence and deduced amino acid sequence of Escherichia coli pyruvate oxidase, a lipid-activated flavoprotein.

The entire nucleotide sequence of the poxB (pyruvate oxidase) gene of Escherichia coli K-12 has been determined by the dideoxynucleotide (Sanger) sequencing of fragments of the gene cloned into a phage M13 vector. The gene is 1716 nucleotides in length and has an open reading frame which encodes a protein of Mr 62,018. This open reading frame was shown to encode pyruvate oxidase by alignment of the amino acid sequences deduced for the amino and carboxy termini and several internal segments of the mature protein with sequences obtained by amino acid sequence analysis. The deduced amino acid sequence of the oxidase was not unusually rich in hydrophobic sequences despite the peripheral membrane location and lipid binding properties of the protein. The codon usage of the oxidase gene was typical of a moderately expressed protein. The deduced amino acid sequence shares homology with the large subunits of the acetohydroxy acid synthase isozymes I, II, and III, encoded by the ilvB, ilvG, and ilvI genes of E. coli.

Acetolactate Synthase↗
PubMed2006

Intracellular compartmentation of cardiac fibres from rainbow trout and Atlantic cod--a general design of heart cells.

In mammalian cardiomyocytes, mitochondria and adjacent ATPases with participation of creatine kinase (CK) constitute functional compartments with an exchange of ADP and ATP delimited from cytosolic bulk solution. The question arises if this extends to ectothermic vertebrates: their low body temperature and thinner cardiomyocytes with a lower density of membrane structures may reduce the need and structural basis for compartmentation. In saponin-skinned cardiac fibres from rainbow trout and Atlantic cod, we investigated mitochondrial respiration induced by endogenous ADP generated by ATPases and its competition for this ADP with pyruvate kinase (PK) in excess. At low Ca(2+) activity (pCa = 7.0), PK lowered ATP-induced respiration by 40% in trout and 26% in cod. At high Ca(2+) activity (pCa = 5.41), PK had no effect. Additionally, ADP release from the fibres was almost zero but increased drastically upon inhibition of respiration with 1 mM Na-azide. This suggests that fibres are compartmented. PK abolished creatine-stimulated respiration in trout suggesting a less tight coupling of CK to respiration than in mammals. In conclusion, intracellular compartmentation seems to be a general feature of vertebrate cardiomyocytes, whereas the role of CK is unclear, but it seems to be less important for energy transport in species with lower metabolism.

Adenosine Diphosphate↗
PubMed1995

DNase I hypersensitivity sites and nuclear protein binding on the fatty acid synthase gene: identification of an element with properties similar to known glucose-responsive elements.

We have shown previously that fatty acid synthase (FAS) gene expression is positively regulated by glucose in rat adipose tissue and liver. In the present study, we have identified in the first intron of the gene a sequence closely related to known glucose-responsive elements such as in the L-pyruvate kinase and S14 genes, including a putative upstream stimulatory factor/major late transcription factor (USF/MLTF) binding site (E-box) (+ 292 nt to + 297 nt). Location of this sequence corresponds to a site of hypersensitivity to DNase I which is present in the liver but not in the spleen. Moreover, using this information from a preliminary report of the present work, others have shown that a + 283 nt to + 303 nt sequence of the FAS gene can confer glucose responsiveness to a heterologous promoter. The protein binding to this region has been investigated in vitro by a combination of DNase I footprinting and gel-retardation experiments with synthetic oligonucleotides and known nuclear proteins. DNase I footprinting experiments using a + 161 nt to + 405 nt fragment of the FAS gene demonstrate that a region from + 290 nt to + 316 nt is protected by nuclear extracts from liver and spleen. This region binds two ubiquitous nuclear factors, USF/MLTF and the CAAT-binding transcription factor/nuclear factor 1 (CTF/NF1). Binding of these factors is similar in nuclear extracts from liver which does or does not express the FAS gene as observed for glucose-responsive elements in the L-pyruvate kinase and S14 genes. This suggests a posttranslational modification of a factor of the complex after glucose stimulation.

Animals↗
PubMed2001

Serine 187 is a crucial residue for allosteric regulation of Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase.

Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase is sensitive to feedback inhibition by tyrosine. One feedback-insensitive mutant was obtained by in vitro chemical mutagenesis and the mutation was identified as a C-->G mutation at nucleotide 560 causing a Ser(187) to Cys(187) substitution. Replacing Ser(187) with cysteine, tyrosine or phenylalanine by site-directed mutagenesis not only reduced the enzymatic activity but also relieved its feedback inhibition by tyrosine, while Ser(187)Ala exhibited a comparable activity to that of wild-type enzyme and sensitized to allosteric regulation. The His(6)-tagged enzymes were expressed in Escherichia coli and purified to homogeneity by immobilized nickel-ion affinity chromatography. Kinetic analysis showed that tyrosine is a competitive inhibitor of phosphoenol pyruvate, one of the precursors for DAHP biosynthesis.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1992

Contrasting action of short- and long-term adrenaline infusion on dog skeletal muscle glucose metabolism.

There are important differences between the short- and long-term effects of adrenaline on determinants of glucose tolerance. To assess this metabolic adaptation at tissue level, the present study examined the effect of acute and prolonged in vivo elevation of adrenaline on glycogen metabolism and glycolysis in skeletal muscle. Adrenaline (50 ng.kg-1.min-1) was infused for 2 h or 74 h and the results compared with 1 h 0.9% NaCl infusion in six trained dogs. Muscle glycogen content was reduced by long-term adrenaline (161 +/- 17 vs NaCl 250 +/- 24 mumol/g dry weight; p less than 0.05) but not short-term adrenaline (233 +/- 21) indicating a sustained effect of adrenaline on glycogen metabolism. Acutely, glycogen synthase I was reduced (short-term adrenaline 12 +/- 6 vs NaCl 22 +/- 7 mumol glycosyl units.g-1.min-1; p less than 0.05) but returned to normal with prolonged adrenaline infusion (20 +/- 5). In contrast, Km for glycogen phosphorylase alpha was not changed acutely (short-term adrenaline 31 +/- 6 vs NaCl 27 +/- 7 mmol/l inorganic phosphate) but was reduced during long-term infusion (19 +/- 4; p less than 0.05 vs short-term adrenaline). Thus, with short- and long-term adrenaline infusion, there were different enzyme changes, although likely to promote glycogenolysis in both cases. In the glycolytic pathway the substrates glucose 6-phosphate and fructose 6-phosphate did not change significantly and hexokinase was not inhibited. Acutely, phosphofructokinase had reduced Vmax (short-term adrenaline 34 +/- 6 vs NaCl 44 +/- 5 U/g; p less than 0.05) but was still above the maximal operating rate in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1987

Differences in the half-lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity.

The different turnover rates of rat liver mitochondrial enzymes make autophagy unlikely to be the main mechanism for degradation of mitochondria. Although alternatives have been presented, hepatocyte heterogeneity has not been considered. Lighter hepatocytes isolated in a discontinuous Percoll gradient contain more glutamate dehydrogenase (GDH) (half-life 1 day) and a more active autophagic system than heavier hepatocytes. The latter contain more carbamoyl phosphate synthase (CPS) and ornithine carbamoyl transferase (OTC) (half-lives 8 days) but less lysosomal activity. As expected, isolated autophagic vacuoles contain, relative to the mitochondrial content, 3-times less OTC and CPS than GDH, probably reflecting a faster lysosomal engulfment of mitochondria in the light hepatocytes (which contain more GDH). These data may explain some of the half-life differences of the enzymes studied.

Alanine Transaminase↗
PubMed2006

Vitamin K1 accumulation in tobacco plants overexpressing bacterial genes involved in the biosynthesis of salicylic acid.

Phylloquinone (Vitamin K(1)) is an essential component of the photosynthetic electron transfer. As isochorismate is required for the biosynthesis of Vitamin K(1), isochorismate synthase (ICS) activity is expected to be present in all green plants. In bacteria salicylic acid (SA) is synthesized via a two step pathway involving ICS and isochorismate pyruvate lyase (IPL). The effect of the introduction in tobacco plants of the bacterial ICS and IPL genes on the endogenous isochorismate pathway was investigated. Transgenic tobacco plants in which IPL was targeted to the chloroplast suffered severe growth retardation and had low Vitamin K(1) content. Probably because isochorismate was channeled towards SA production, the plants were no longer able to produce normal levels of Vitamin K(1). Transgenic tobacco plants in which the bacterial ICS was present in the chloroplast showed higher Vitamin K(1) contents than wild type plants.

Bacterial Proteins↗
PubMed1994

Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations.

Transgenic mice expressing the P-enolpyruvate carboxykinase (PEPCK)/human insulin chimeric gene have been obtained as a model to study the feasibility of gene therapy for diabetes. These transgenic animals were healthy and normoglycemic and expressed human insulin in a physiologically regulated manner, mainly in the liver. Streptozotocin-treated transgenic mice had high levels of human insulin immunoreactivity in serum and showed a significant decrease (up to 40%) in glycemia compared with streptozotocin-treated control mice. The expression of genes involved in liver glucose metabolism, such as glucokinase, pyruvate kinase, and PEPCK, which is markedly altered by diabetes, was significantly recovered in transgenic mice treated with streptozotocin. In addition, the activity of both glucokinase and glycogen synthase, and the content of glucose 6-phosphate and glycogen, were normal in the liver, even when transgenic animals were treated with diabetogenic doses of streptozotocin. These results constitute an indication in vivo that diabetes gene therapy is possible, by means of the production of insulin in extrapancreatic tissues.

Animals↗