Control of liver carbohydrate and fatty acid metabolism by cell volume.
Explore the source record for details and available documents.
Acetyl-CoA Carboxylase↗
Biomedical subjects
L Hue
Publications and source records attributed to L Hue.
Flunarizine and cinnarizine inhibit mitochondrial complexes I and II: possible implication for parkinsonism.
Cinnarizine and flunarizine are piperazine derivatives with calcium antagonist and anticonvulsant properties and are used widely in the treatment of vertigo and circulatory disorders. They have been implicated recently in the aggravation, or even the induction, of parkinsonism in elderly patients. Because the aetiology of parkinsonism has been suggested as having a mitochondrial component, we have investigated the effects of both compounds on mitochondrial respiration and on the activities of the individual respiratory chain complexes. In intact mitochondria from rat liver, both drugs inhibited respiration rates, with substrates entering at Complex I (glutamate/malate) and Complex II (succinate). These effects could be explained by potent inhibitions (Ki 3-10 microM) of both complexes. Complex I is inhibited at a site near the ubiquinone-binding site, which is not competitive with respect to ubiquinone, whereas the inhibition of Complex II is apparently caused by competition with ubiquinone. Furthermore, the inhibition of NADH oxidation by flunarizine in submitochondrial particles caused an NADH-dependent generation of superoxide. These inhibitory properties of both compounds could be significant factors in the aggravation or induction of parkinsonism in elderly patients, in whom mitochondrial function already may be impaired.
Animals↗
Mechanism of activation of liver acetyl-CoA carboxylase by cell swelling.
The activation of hepatic glycogen synthase by the amino-acid-induced cell swelling has been attributed to the stimulation of [glycogen-synthase]-phosphatase resulting from an increase in the intracellular content in glutamate and aspartate, and a decrease in intracellular Cl-, which is a compensatory response to cell swelling [Meijer, A. J., Baquet, A., Gustafson, L., van Woerkom, G. M. & Hue, L. (1992) J. Biol. Chem. 267, 5823-5828]. Here we studied whether the activation of acetyl-CoA carboxylase by cell swelling could be explained by the same mechanism. The activation of endogenous or purified acetyl-CoA carboxylase was measured in gel-filtered liver extracts or cytosols. No activation could be observed under basal conditions but a fivefold stimulation was obtained with concentrations of glutamate (20-25 mM) found in hepatocytes incubated with glutamine. A similar stimulation was also observed with other dicarboxylic acids such as malonate and succinate, or with metal ions like Mg2+, Ca2+ and Mn2+ (10 mM). The addition of 50-100 mM Cl- was found to inhibit the activation of acetyl-CoA carboxylase by some 20-30%. Mg2+ was also found to stimulate the activation of the endogenous glycogen synthase. The glutamate-stimulated and Mg(2+)-stimulated activation of glycogen synthase and acetyl-CoA carboxylase was unaffected by 10 microM inhibitor-2, a specific inhibitory protein of protein phosphatase-1, but could be nearly completely blocked by the phosphatase inhibitor microcystin-LR. Our data suggest that the amino-acid-induced activation of acetyl-CoA carboxylase and glycogen synthase in the liver occurs by a common ionic mechanism.
Acetyl-CoA Carboxylase↗
Cloning and expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine heart.
Distinct 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) cDNAs were cloned from bovine heart, showing that PFK-2/FBPase-2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000-M(r) isozyme. In B2 mRNA, exon 15 encodes four more residues than in B1. In B4 mRNA, exon 15 encodes six more residues than in B1, but exon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000-M(r) isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK-2 activity.
Amino Acid Sequence↗
Rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the kinase domain by site-directed mutagenesis.
Sequence alignment and modeling of the 2-kinase domain of the liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, on 6-phosphofructo-1-kinase from Bacillus stearothermophilus and Escherichia coli (Bazan, J. F., Fletterick, R. J., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646) suggested that Cys-160 of the 2-kinase would correspond to Asp-127 of the 1-kinase, which acts as a general base catalyst. We have studied the validity of this alignment by site-directed mutagenesis of residues in the 2-kinase domain of skeletal muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Cys-160 was mutated to Asp or Ser. Two adjacent residues, Glu-157 and Asp-162, either of which could act as a general base catalyst, were mutated to Ala. Asp-162 corresponds to Asp-129 in the bacterial 1-kinase, which is also essential for catalysis and might bind Mg2+. None of these mutations significantly decreased the Vmax of the 2-kinase, suggesting that the mutated amino acids are not essential for catalysis and therefore do not play the same role as Asp-127 and Asp-129 in the bacterial 1-kinase. Mutation of Glu-157 and Asp-162 to alanine had no effect on the kinetic parameters of the bifunctional enzyme, indicating that these two negatively charged residues are not involved in catalysis and substrate binding.
Amino Acid Sequence↗
Role of fructose 2,6-bisphosphate in the control of heart glycolysis.
The aim of this work was to study whether changes in fructose 2,6-bisphosphate concentration are correlated with variations of the glycolytic flux in the isolated working rat heart. Glycolysis was stimulated to different extents by increasing the concentration of glucose, increasing the workload, or by the addition of insulin. The glycolytic flux was measured by the rate of detritiation of [2-3H]- and [3-3H]glucose. Under all the conditions tested, an increase in fructose 2,6-bisphosphate content was observed. The glucose- or insulin-induced increase in fructose 2,6-bisphosphate content was related to an increase in the concentration of fructose 6-phosphate, the substrate of 6-phosphofructo-2-kinase. An increase in the workload correlated with a 50% decrease in the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate. Similar changes in Km have been observed when purified heart 6-phosphofructo-2-kinase was phosphorylated in vitro by the cyclic AMP-dependent protein kinase or by the calcium/calmodulin-dependent protein kinase. Since the concentration of cyclic AMP was not affected by increasing the workload, it is possible that the change in Km of 6-phosphofructo-2-kinase, which was found in hearts submitted to a high load, resulted from phosphorylation by calcium/calmodulin protein kinase; other possibilities are not excluded. Anoxia decreased the external work developed by the heart, stimulated glycolysis and glycogenolysis, but did not increase fructose 2,6-bisphosphate.
Animals↗
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification, kinetics and immunological properties.
Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55,800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53,900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53,300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mumol.l-1).(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Fructose 2,6-bisphosphate and the control of glycolysis by growth factors, tumor promoters and oncogenes.
Tumor and proliferating cells maintain a high glycolytic rate even under aerobic conditions. The discovery of fructose 2,6-bisphosphate, a potent stimulator of glycolysis, has prompted a re-investigation of this phenomenon. Rat hepatoma cells and fibroblasts stimulated by mitogens or transformed by the Rous sarcoma virus carrying the v-src oncogene were used as models. The results indicate that in established lines of hepatoma cells the biochemical properties of the bifunctional enzyme, PFK-2/FBPase-2, involved in the synthesis and degradation of fructose 2,6-bisphosphate, differ from those of the enzyme from normal liver. In addition, the stimulation of glycolysis induced by phorbol esters and pp60v-src can be explained by an increase in the concentration of fructose 2,6-bisphosphate and an activation of PFK-2. The mechanism of stimulation involves the transcription of a gene whose product activates PFK-2 or is a distinct PFK-2 isozyme. Finally, mercaptopurines were found to block fructose 2,6-bisphosphate synthesis in vitro and in lymphocytes and lymphoblastic cells. In these cells, this resulted in an inhibition of glycolysis.
Animals↗
Mammalian 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a bifunctional enzyme that controls glycolysis.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Effects of hypo- and hyperthyroidism on the complex I activity in rat heart mitochondria.
Explore the source record for details and available documents.
Animals↗
Role of fructose 2,6-bisphosphate in the control of glycolysis. Stimulation of glycogen synthesis by lactate in the isolated working rat heart.
Fructose 2,6-bisphosphate (Fru-2,6-P2) is the most potent stimulator of 6-phosphofructo-1-kinase (PFK-1), a key enzyme of glycolysis. We studied whether this regulator is involved in the changes of glycolysis that can be induced experimentally in the isolated working rat heart. The glycolytic flux was assessed by the rate of detritiation of [2-3H]- and [3-3H]glucose, by lactate output and by the changes in glycogen content. A 20-40% increase in Fru-2,6-P2 content was observed when glycolysis was stimulated by increasing either the workload (by increasing both preload and afterload) or the concentration of glucose (from 2 to 11 mM), or by adding 7 microM insulin. Anoxia decreased the external work developed by the heart, stimulated glycolysis by activating glycogenolysis, but did not increase Fru-2,6-P2. The increase of Fru-2,6-P2 content observed after insulin, high workload or glucose load might be related to a stimulation of glucose transport, and/or an activation of 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of Fru-2,6-P2. Addition to the perfusate of 0.5 to 10 mM lactate, which is a preferred substrate for the heart, with pyruvate in a 10:1 ratio, induced a dose-dependent inhibition of the glycolytic flux through PFK-1, with a maximal inhibition of 75% at 5 mM lactate. The accumulation of hexose 6-phosphates without change in fructose 1,6-bisphosphate and triose-phosphates concentrations confirmed that the inhibition of glycolysis was mainly exerted on PFK-1. This inhibition resulted from a doubling of the citrate concentration, an inhibitor, and from 75% decrease in Fru-2,6-P2. Despite the inhibition of glycolysis, glucose phosphorylation was barely affected by lactate, suggesting a change in glucose metabolism. Indeed, lactate induced a dose-dependent increase in glycogen content, which doubled at 5 mM lactate, reaching the level obtained after addition of 7 microM insulin. Increased glycogen synthesis was explained by the accumulation of UDP glucose, the substrate, and glucose 6-phosphate, a stimulator of glycogen synthase. We conclude that, during aerobiosis, Fru-2,6-P2 can be regarded as a glycolytic signal which is switched on by glucose availability, workload and insulin, and which is switched off by the availability of alternative oxidative substrates such as lactate. The latter also controls glucose metabolism by diverting glucose from glycolysis to glycogen synthesis.
Animals↗
Molecular forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expressed in rat skeletal muscle.
The rat cDNA for the muscle-type (M) isozyme of 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) contains two putative translation initiation sites. To determine whether the M isozyme expressed in rat skeletal muscle corresponds to the short (PFK2M-sf) or the long (PFK2M-lf) isoform, we have expressed them in Escherichia coli. A third construction was also expressed in which the second ATG codon was deleted (PFK2M-lf delta ATG) to ensure that initiation started at the first ATG. The properties of these recombinant proteins were compared with those of the PFK-2/FBPase-2 present in rat skeletal muscle and liver. The recombinant proteins displayed PFK-2 and FBPase-2 activities and the M(r) values of the subunits measured by SDS-polyacrylamide gel electrophoresis were compatible with the calculated ones. The purified recombinant lf form contained not only the expected lf band (54,500 M(r)) but also the sf band (52,000 M(r)), indicating that the expression system could synthesize the long and the short isoforms from the same mRNA. The kinetic properties of the recombinant sf form were not different from those of the rat muscle enzyme. By contrast, lf delta ATG PFK-2 displayed a higher Km for its substrates and a lower Vmax. Immunoblotting with an antibody directed against the long isoform revealed a 54,500 M(r) band both in the lf and the lf delta ATG recombinant, but no band in rat skeletal muscle extracts. In these extracts, one band of 52,000 and a minor one of 54,500 M(r) were detected by an anti PFK-2/FBPase-2 antibody. The 54,500 M(r) band was recognized by an antibody directed against the L isozyme, suggesting that a small amount of the latter is expressed in skeletal muscle. Thus, the M isozyme differs from the L isozyme by replacement of the first 32 amino acids of the L isozyme by an unrelated nonapeptide.
Amino Acid Sequence↗
Cell shrinkage follows, rather than mediates, the short-term effects of glucagon on carbohydrate metabolism.
The initial effects of glucagon on glycogen breakdown in isolated hepatocytes were found to be independent of cell volume and, when it occurred, cell shrinkage followed rather than mediated the glycogenolytic effect of glucagon. Similar conclusions could be drawn for the effect of glucagon on glycolysis/gluconeogenesis and for the antagonistic effect of insulin on glucagon action.
Animals↗
Evidence for new phosphorylation sites for protein kinase C and cyclic AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was phosphorylated by incubation with [gamma-32P]MgATP and cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). After digestion with chymotrypsin, the phosphorylation sites for the two protein kinases were identified by peptide mapping, and microsequencing. Evidence for new phosphorylation sites for PKA (Ser-483) and PKC (Ser-84 and Ser-466) was obtained.
Amino Acid Sequence↗
Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by phenylglyoxal. Evidence for essential arginine residues.
Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine-specific reagent, phenylglyoxal, irreversibly inactivated both 6-phosphofructo-2-kinase and fructose-6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6-bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic-AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme.
Amino Acid Sequence↗
The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing.
Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.
Amino Acid Sequence↗
Activation of 6-phosphofructo-2-kinase by pp60v-src is an indirect effect.
6-Phosphofructo-2-kinase (PFK-2) catalyses the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), a potent stimulator of glycolysis. In chick-embryo fibroblasts, PFK-2 activity and Fru-2,6-P2 concentration increase upon transformation by Rous sarcoma virus. We show here that the increase in PFK-2 activity required more than 2 h after shifting fibroblasts infected with a thermosensitive mutant of Rous sarcoma virus from the restrictive to the permissive temperature. Pretreatment of the cells with actinomycin D prevented this increase in PFK-2 activity, suggesting a requirement for RNA synthesis. However, the increase in PFK-2 activity did not correspond to an increase in immunoprecipitable PFK-2. Moreover, the thermostability of PFK-2 and the affinity of this enzyme for its substrate fructose 6-phosphate were increased upon transformation by Rous sarcoma virus. Staurosporine, an inhibitor of protein kinase C, prevented the increase in PFK-2 activity brought about by the shift to the permissive temperature. This, together with a comparison of the effects of phorbol esters on PFK-2 activity, suggests that pp60v-src stimulates, via protein kinase C, the transcription of a gene whose products is a distinct PFK-2 isoenzyme or a protein that activates PFK-2.
Alkaloids↗
Mechanism of activation of liver glycogen synthase by swelling.
The mechanism linking the stimulation of liver glycogen synthesis to swelling induced either by amino acids or hypotonicity was studied in hepatocytes, in gel-filtered liver extracts, and in purified preparations of particulate glycogen to which glycogen-metabolizing enzymes are bound. High concentrations of KCl, but not of potassium glutamate, were found to inhibit glycogen synthesis in permeabilized hepatocytes. Similarly, physiological concentrations (30-50 mM) of Cl- ions were also found to inhibit synthase phosphatase in vitro, whereas 10-20 mM Cl- ions, a concentration found in swollen hepatocytes, did not inhibit synthase phosphatase. Synthase phosphatase activity was more sensitive to inhibition by Cl- ions at low (0.1%) than at high (1%) concentrations of glycogen. By contrast, 10 mM glutamate and aspartate, a concentration observed in hepatocytes incubated with glutamine or proline, stimulated synthase phosphatase in vitro. Therefore, it is proposed that the fall in intracellular Cl- concentration as well as the increase in intracellular glutamate and aspartate concentrations, that are observed in swollen hepatocytes in the presence of amino acids, are responsible, at least in part, for the stimulation of synthase phosphatase and, hence, of glycogen synthesis.
Animals↗