Chicken ovalbumin upstream promoter-transcription factor II, a new partner of the glucose response element of the L-type pyruvate kinase gene, acts as an inhibitor of the glucose response.
(17/1157)
Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP. The DNA regulatory sequence responsible for mediating glucose and cyclic AMP responses, called glucose response element (GlRE), consists of two degenerated E boxes spaced by 5 base pairs and is able to bind basic helix-loop-helix/leucine zipper proteins, in particular the upstream stimulatory factors (USFs). From ex vivo and in vivo experiments, it appears that USFs are required for correct response of the L-PK gene to glucose, but their expression and binding activity are not known to be regulated by glucose. A genetic screen in yeast has allowed us to identify a novel transcriptional factor binding to the GlRE, i.e. the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Binding of COUP-TFII to the GlRE was confirmed by electrophoretic mobility shift assays, and COUP-TFII-containing complexes were detectable in liver nuclear extracts. Neither abundance nor binding activity of COUP-TFII appeared to be significantly regulated by diets. In footprinting experiments, two COUP-TFII-binding sites overlapping the E boxes were detected. Overexpression of COUP-TFII abrogated the USF-dependent transactivation of an artificial GlRE-dependent promoter in COS cells and the glucose responsiveness of the L-PK promoter in hepatocytes in primary culture. In addition, a mutated GlRE with increased affinity for USF and very low affinity for COUP-TFII conferred a dramatically decreased glucose responsiveness on the L-PK promoter in hepatocytes in primary culture by increasing activity of the reporter gene in low glucose condition. We propose that COUP-TFII could be a negative regulatory component of the glucose sensor complex assembled on the GlRE of the L-PK gene and most likely of other glucose-responsive genes as well. (+info)
Negative cyclic AMP response elements in the promoter of the L-type pyruvate kinase gene.
(18/1157)
L-type pyruvate kinase gene expression is modulated by hormonal and nutritional conditions. Here, we show by transient transfections in hepatocytes in primary culture that both the glucose response element and the contiguous hepatocyte nuclear factor 4 (HNF4) binding site (L3) of the promoter were negative cyclic AMP (cAMP) response elements and that cAMP-dependent inhibition through L3 requires HNF4 binding. Another HNF4 binding site-dependent construct was also inhibited by cAMP. However, HNF4 mutants whose putative PKA-dependent phosphorylation sites have been mutated still conferred cAMP-sensitive transactivation of a L3-dependent reporter gene. Overexpression of the CREB binding protein (CBP) increased the HNF4-dependent transactivation but this effect remained sensitive to cAMP inhibition. (+info)
Alteration of gene expression for glycolytic enzymes in aerobic and ischemic myocardium.
(19/1157)
The purpose of this report was to describe mRNA abundance for the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and pyruvate dehydrogenase in ischemic and adjacent aerobic myocardium. Mechanical, metabolic, and mRNA data were acquired in a pig model of regulated coronary flow using extracorporeal perfusion. Trials of coronary hypoperfusion included sustained and intermittent exposures of acute ischemia with or without reperfusion. These were compared with a chronic 4-day model of partial coronary stenosis. In ischemic tissues, levels of mRNA, normalized by mRNA for beta-actin, were increased over control values for GAPDH (range 2.7- to 4.6-fold), pyruvate kinase (2.9-fold), and pyruvate dehydrogenase (2.1-fold). It is of interest that increases in mRNA levels over control values were also observed in adjacent aerobic heart muscle from intervention hearts, including 3.6- to 4.5-fold elevations in message for GAPDH and a 2.1-fold increase in signal for pyruvate dehydrogenase. Augmentation in mRNA abundance occurred in as short a time as 40 min of ischemia and was maintained for as long as 4 days in partial coronary stenosis. Whether the former time was of an interval sufficient to affect protein production is problematic, but the latter time was ample to influence enzyme concentration, which may in turn have regulated glycolysis in this condition. (+info)
Allometric scaling of RNA, DNA, and enzyme levels: an intraspecific study.
(20/1157)
The activities of oxidative and glycolytic enzymes show body size-dependent relationships across a wide variety of taxa; however, the mechanistic basis remains unknown. We sampled white epaxial muscle from rainbow trout (Oncorhynchus mykiss) spanning a 100-fold range in body mass. We measured activities of enzymes from aerobic and anaerobic metabolic pathways, RNA [total RNA and mRNA, pyruvate kinase (PK), citrate synthase (CS), and MyoD mRNA], and total DNA. Total RNA and DNA showed a biphasic relationship with body size, with a break point occurring after fish reached 1 yr of age. In contrast, total RNA/total DNA was constant across the entire size range. Neither CS activity nor CS mRNA levels scaled with body mass. PK activity and PK mRNA levels increased in parallel in yearling fish only (r(2) = 0.91, P < 0.01). This suggests that although PK expression is transcriptionally regulated in yearlings, the molecular mechanisms regulating expression change with growth and age. This was supported by a positive correlation between MyoD and PK mRNA levels (r(2) = 0.17, P < 0.05). (+info)
Paradoxical inhibition of c-myc-induced carcinogenesis by Bcl-2 in transgenic mice.
(21/1157)
Here, we investigated changes in apoptosis during tumor progression by analyzing the effect of coexpressing various antiapoptotic genes on the multistage process of c-myc-induced hepatocarcinogenesis in transgenic mice. Whereas continuous c-myc gene overexpression in the liver led to cellular hepatocarcinoma, the coexpression of the bcl-2 gene inhibited the emergence of liver tumors, by inhibiting a pretumoral phase characterized by increased proliferation and apoptosis. This antioncogenic effect was specific to Bcl-2 and was not shared by other antiapoptotic genes such as bcl-xL and a dominant negative form of p53. Thus, we have shown that Bcl-2 can have a tumor suppressor effect in vivo on c-myc-induced hepatocarcinogenesis during the emergence of neoplastic foci. (+info)
Subunit dissociation and inactivation of pyruvate kinase by hydrostatic pressure oxidation of sulfhydryl groups and ligand effects on enzyme stability.
(22/1157)
The effect of hydrostatic pressure on the stability of tetrameric rabbit muscle pyruvate kinase was investigated by enzyme activity measurements, size-exclusion chromatography, circular dichroism and fluorescence spectroscopies. Under nonreducing conditions, enzyme activity was irreversibly inhibited by increasing pressure and was completely abolished at 350 MPa. Inhibition was dependent on the concentration of pyruvate kinase, indicating that it was related to pressure-induced subunit dissociation. Size-exclusion chromatography of pressurized samples confirmed a decrease in the proportion of tetramers and an increase in monomers relative to native samples. Addition of dithiothreitol immediately following pressure release led to full recovery of both enzyme activity and of native tetramers. Furthermore, no irreversible inhibition of pyruvate kinase was observed if pressure treatment was carried out in the presence of dithiothreitol. These data suggest that pressure-dissociated monomers undergo conformational changes leading to oxidation of sulfhydryl groups, which prevents correct refolding of native tetramers on decompression. These conformational changes are relatively subtle, as indicated by the lack of significant changes in far-UV circular dichroism and intrinsic fluorescence emission spectra of previously pressurized samples. The effects of various physiological ligands on the pressure stability of pyruvate kinase were also investigated. A slight protection against inhibition was observed in the simultaneous presence of K+, Mg2+ and ADP. Both phosphoenolpyruvate and the allosteric inhibitor, phenylalanine, caused marked stabilization against pressure, suggesting significant energy coupling between binding of these ligands and stabilization of the tetramer. (+info)
Tay-Sachs' and Sandhoff's diseases: the assignment of genes for hexosaminidase A and B to individual human chromosomes.
(23/1157)
The techniques of somatic cell genetics have been used to establish the linkage relationships of loci coding for two forms (A and B) of hexosaminidase (EC 3.2.1.30; 2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase) and to determine whether a structural relationship exists between these forms. In a series of human-mouse hybrid cell lines, hexosaminidase A and B segregated independently. Our results and those reported by other investigators are used to analyze the proposed structural models for hexosaminidase. We have also been able to establish a syntenic relationship between the gene locus responsible for the expression of hexosaminidase A and those responsible for mannosephosphate isomerase and pyruvate kinase-3 and to assign the gene for hexosaminidase B to chromosome 5 in man. There is thus a linkage between specific human autosomes and enzymes implicated in the production of lipid storage diseases. (+info)
Sterol response element-binding protein 1c (SREBP1c) is involved in the polyunsaturated fatty acid suppression of hepatic S14 gene transcription.
(24/1157)
Polyunsaturated fatty acids (PUFA) suppress hepatic lipogenic gene transcription through a peroxisome proliferator activated receptor alpha (PPARalpha)- and cyclooxygenase-independent mechanism. Recently, the sterol response element-binding protein 1 (SREBP1) was implicated in the nutrient control of lipogenic gene expression. In this report, we have assessed the role SREBP1 plays in the PUFA control of three hepatic genes, fatty acid synthase, L-pyruvate kinase (LPK), and the S14 protein (S14). PUFA suppressed both the hepatic mRNA(SREBP1) through a PPARalpha-independent mechanism as well as SREBP1c nuclear content (nSREBP1c, 65 kDa). Co-transfection of primary hepatocytes revealed a differential sensitivity of the fatty acid synthase, S14, and LPK promoters to nSREBP1c overexpression. Of the three promoters examined, LPK was the least sensitive to overexpressed nSREBP1c. Promoter deletion and gel shift analyses of the S14 promoter localized a functional SREBP1c cis-regulatory element to an E-box-like sequence ((-139)TCGCCTGAT(-131)) within the S14 PUFA response region. Although overexpression of nSREBP1c significantly reduced PUFA inhibition of S14CAT, overexpression of other factors that induced S14CAT activity, such as steroid receptor co-activator 1 or retinoid X receptor alpha, had no effect on S14CAT PUFA sensitivity. These results suggest that PUFA regulates hepatic nSREBP1c, a factor that functionally interacts with the S14 PUFA response region. PUFA regulation of nSREBP1c may account for the PUFA-mediated suppression of hepatic S14 gene transcription. (+info)