Pyruvate Kinase. Classes of regulatory isoenzymes in mammalian tissues

El nombre actual de la revista es FEBS Journal: Open Access para artículos anteriores al 2006.-- Autores pertenecientes al extinto Instituto de Enzimología del CSIC.Kinetic studies of the pyruvate kinases of rat and human tissues have led to the identification of three classes of isoenzymes with qualitative differences in regulatory properties. Class L, the major component in liver extracts and a minor component of kidney extracts, shows markedly sigmoidal kinetics with respect to the concentration of phosphoenolpyruvate, allosteric inhibition by ATP and alanine, and activation by fructose 1,6-bisphosphate. Class A, present in adipose tissue, and the major component in kidney and the minor one in liver, shows slightly sigmoidal substrate saturation curves and is allosterically inhibited by alanine and activated by fructose 1,6-bisphosphate. Class M, present in muscle and brain, has none of the above regulatory properties. The activities of the pyruvate kinases of classes L and A respond immediately to changes in the concentration of the effectors alanine and fructose 1,6-bisphosphate. The two regulatory isoenzymes are strongly inhibited by three amino acids: alanine, cysteine, and phenylalanine. Cysteine is the stronger inhibitor for class L, while phenylalanine is the stronger one for class A. The allosteric inhibition is stereospecific for the l-amino acids in contrast with the weak isosteric inhibition of muscle pyruvate kinase by l- or d-alanine as analogue of the product. The allosteric inhibition of pyruvate kinase L by ATP is highly specific in contrast with a rather wide specificity of this enzyme for nucleoside diphosphates as acceptor substrates.The present work was partly supported by a grant from the Fundación Manuel Aguilar. Two of the authors (J. C. and J. E. F.) had fellowship from the Ministerio de Educación y Ciencia.Peer Reviewe

Involvement of both phosphatidylinositol 3-kinase and p44/p42 mitogen-activated protein kinase pathways in the short-term regulation of pyruvate kinase L by insulin

8 pages, 8 figures.Pyruvate kinase L (PK-L) is a key regulatory enzyme of the hepatic glycolytic/gluconeogenic pathway that can be dephosphorylated and activated in response to insulin. However, the signaling cascades involved in this insulin effect have not been established. In this work we have investigated the potential involvement of phosphatidylinositol 3-kinase (PI 3-K) and p44/p42 mitogen-activated protein kinase (MAPK) pathways in the short-term modulation of PK-L by insulin in primary cultures of rat hepatocytes. Wortmannin, at a concentration of 100 nM, caused a marked inhibition of the PI 3-K/protein kinase B pathway, which became complete at 500 nM wortmannin. Likewise, wortmannin at 100 and 500 nM, elicited partial and total inhibitions of insulin-mediated activation of PK-L, respectively. However, this PI 3-K inhibitor also reduced insulin-mediated phosphorylation of p44/p42 MAPK in cultured rat hepatocytes, indicating that both the PI 3-K and MAPK pathways could be involved in PK-L activation by insulin. Three facts appear to reinforce this hypothesis: 1) the selective and complete inhibition of the PI 3-K/protein kinase B pathway by LY294002 (50 microM) was accompanied by a partial blockade of insulin-induced PK-L activation; 2) when signaling through the MAPK cascade was selectively suppressed by the presence of PD98059 (50 microM), a 50% reduction of insulin-induced activation of PK-L was observed; and 3) the effect of PD98059 (50 microM) on PK-L activation was reinforced by the additional presence of 100 nM wortmannin. We also observed that the blockade of p70 S6-kinase by rapamycin did not affect the activation of PK-L by insulin. From these findings it can be concluded that both PI 3-K and MAPK pathways, but not p70 S6-kinase, are involved in the short-term activation of PK-L by insulin in rat hepatocytes.This work was supported by Research Grant 08.6/0018/1998 from the Comunidad Autónoma de Madrid (to J.E.F.) and by predoctoral fellowships from the Fondo de Investigaciones Sanitarias (to B.I. and A.E.G.).Peer reviewe

Modulation of epinephrine-stimulated gluconeogenesis by insulin in hepatocytes isolated from genetically obese (fa/fa) Zucker rats

6 pages, 4 figures, 1 table.Genetically obese (fa/fa) Zucker rats present an impaired response of hepatic glucose production to the inhibition by insulin. In this work, we have investigated the modulation by this hormone of epinephrine-stimulated gluconeogenesis, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/-) littermates. Epinephrine (1 microM) caused a maximal stimulation of [14C]lactate conversion to [14C]glucose in hepatocytes isolated from either obese or lean animals. The stimulation of gluconeogenesis by epinephrine was accompanied by a significant reduction of fructose 2,6-bisphosphate levels, an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, and by a 2-fold increase in the cellular concentrations of cAMP. The presence of insulin in the incubation medium antagonized, in a concentration-dependent manner, the effects of epinephrine. In hepatocytes isolated from lean rats, the reversion caused by insulin was complete, the concentration required for half-maximal insulin action ranging from 0.22 to 0.56 nM. In contrast, in obese rat hepatocytes, insulin only partially blocked epinephrine-mediated effects, and the sensitivity to insulin was 2- to 4-fold lower, as indicated by the corresponding half-maximal insulin action values. Furthermore, insulin (10 nM) almost completely blocked the increase in cAMP levels induced by epinephrine in lean rat hepatocytes, whereas it only provoked a small and nonsignificant reduction of epinephrine-stimulated levels of the cyclic nucleotide in hepatocytes obtained from obese rats.This work was supported by grants from Fondo de Investigación Sanitaria (Grant No. 95/200) and from Boehringer Ingelheim España S.A., Spain. Fellow of the Programa Sectorial de Formación de Profesorado Universitario y Personal Investigador (Spain).Peer reviewe

Inhibition of acid secretion by the nonsteroidal anti-inflammatory drugs diclofenac and piroxicam in isolated gastric glands: analysis of a multifocal mechanism

12 pages, 8 figures, 3 tables.In nonstimulated rabbit gastric glands, acetylsalicylic acid (10-500 microM) and indomethacin (3-300 microM) did not significantly modify the basal rate of acid secretion, whereas diclofenac and piroxicam (10-1,000 microM each) caused a marked and dose-dependent inhibitory effect (EC(50) = 138 and 280 microM, respectively). In gastric glands stimulated by histamine (100 microM), diclofenac also reduced the rate of acid formation in a dose-dependent manner. In contrast, acetylsalicylic acid, indomethacin, and piroxicam exerted a biphasic effect; thus low concentrations (3-100 microM) of these three agents significantly increased the rate of histamine-stimulated acid secretion (10-20% over the corresponding control value) by a cAMP-independent mechanism, whereas higher concentrations reduced the rate of acid formation. With respect to underlying biochemical mechanisms that could mediate inhibitory effects of NSAIDs on gastric acid formation, it was observed that both diclofenac and piroxicam, but not acetylsalicylic acid or indomethacin, decreased the glandular content of ATP, inhibited hydrolytic activity of gastric gland microsomal H(+)-K(+)-ATPase, and reduced the rate of H(+)-K(+)-ATPase-dependent proton transport across microsomal membranes in a dose-dependent manner. Furthermore, diclofenac and piroxicam also significantly increased passive permeability of microsomal membranes to protons. In conclusion, our work shows that diclofenac and piroxicam cause a significant reduction in the rate of basal and histamine-stimulated acid formation in isolated rabbit gastric glands at concentrations that can be attained in the gastric lumen of patients treated with these drugs. Mechanisms involved in these inhibitory effects appear to be multifocal and include different steps of stimulus-secretion coupling.This work was supported by a grant from Boehringer Ingelheim España. M. Salvatella and J. C. Del Valle are fellows of Consejo Superior de Investigaciones Científicas, Spain and Boehringer Ingelheim España, respectively.Peer reviewe

Impairment of H+-K+-ATPase-dependent proton transport and inhibition of gastric acid secretion by ethanol

11 pages, 10 figures, 1 table.Ethanol (1-20% vol/vol) caused a dose-dependent reduction in the basal rate of acid formation in isolated rabbit gastric glands with a calculated EC(50) value of 4.5 +/- 0.2%. Ethanol also reduced ATP levels in isolated gastric glands and in cultured parietal cells (EC(50): 8.8 +/- 0.4% and 8.5 +/- 0.2%, respectively) and decreased both basal and forskolin-stimulated cAMP levels. In studies carried out in gastric gland microsomes, ethanol inhibited the hydrolytic activity of H+-K+-ATPase(EC(50): 8.5 +/- 0.6%), increased passive proton permeability (EC(50): 7.9%), and reduced H+-K+-ATPase-dependent proton transport (EC(50): 3%). Our results show that the inhibition of gastric acid secretion observed at low concentrations of ethanol ( or =7%.This work was supported by a grant from Boehringer Ingelheim España S.A. J. C. Del Valle and R. Andrade are fellows of Boehringer Ingelheim España S.A., M. Salvatella is a fellow of Consejo Superior de Investigaciones Científicas, Spain.Peer reviewe

Sulfonylureas activate glycogen phosphorylase and increase cytosolic free-Ca2+ levels in isolated rat hepatocytes

Without causing significant changes in cellular levels of cyclic adenosine monophosphate (cAMP), the addition of either glibenclamide or gliquidone to isolated rat hepatocytes caused a transient dose- and Ca2+-dependent activation of glycogen phosphorylase. The calculated concentrations corresponding to half-maximal activation were 5 and 2 μmol/L, respectively. In connection with this, it was observed that glibenclamide provoked a dose-dependent increase in cytosolic free-calcium concentration ([Ca2+]i) in Fura-2-loaded hepatocytes. Moreover, the presence of glibenclamide in the incubation medium accelerated the rate of Ca2+ uptake by Ca2+-depleted hepatocytes. These findings suggest that an increase in [Ca2+]i could mediate some of the effects of sulfonylureas in liver metabolism. © 1993.Supported by grants from Fondo de Investigaciones Sanitarias and Dirección General de Investigación Científica y Técnica (DGICYT), Spain.Peer Reviewe

Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) expression is downregulated in poorly differentiated breast invasive ductal carcinoma

Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) is the only known enzyme able to reduce lipid peroxides bound to cell membranes. Moreover it has been involved in apoptosis and can influence intracellular signaling. To investigate the possible relationship between PHGPx and human cancer we have quantified PHGPx expression levels by real-time quantitative PCR and immunohistochemistry in tissue samples of human breast invasive ductal carcinoma from 34 patients compared with their own controls of benign breast tissue. PHGPx expression levels were compared with the clinical and pathological data of these patients. The results showed that PHGPx expression levels are downregulated in poorly differentiated (grade 3) breast invasive ductal carcinoma (P=0.0043). PHGPx expression levels decreased gradually with tumor grade from grade 1 to grade 3. We also found a downregulation of PHGPx in cases that showed p53 accumulation compared with cases without p53 immunostaining (P=0.0011). PHGPx was also downregulated in cases without progesterone receptors (PR) immunostaining compared with cases with PR immunostaining (P=0.0165). Grade 3, p53 immunostaining and absence of PR immunostaining are poor prognostic factors. These results suggest that PHGPx downregulation could be related with a poorer prognosis in breast invasive ductal carcinoma.Peer Reviewe