Search PubMed⌕ Search

Biomedical subjects

P E Declercq

Publications and source records attributed to P E Declercq.

34 records · Page 2Linked to original sources
PubMed1994

The peroxisome proliferator activated receptor regulates malic enzyme gene expression.

A new regulatory element for peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers was found in the promoter of the malic enzyme gene. Similar to previously characterized peroxisome proliferator response elements (PPREs), it consists of a direct repeat of sequences related to the half-site consensus AGGTCA with an interspacing of 1 base pair. Specific binding of PPAR/RXR heterodimers to this element was demonstrated. Furthermore, this sequence conferred ciprofibrate responsiveness of a reporter through the homologous malic enzyme or heterologous thymidine kinase promoters. This PPRE presumably mediates the transcriptional effects of peroxisome proliferators on malic enzyme expression. The presence of a PPRE in the promoter of this lipogenic enzyme suggests a broader function for the PPAR in the regulation of lipid metabolism.

Animals↗
PubMed1994

Regulation of triacylglycerol synthesis in permeabilized rat hepatocytes. Role of fatty acid concentration and diacylglycerol acyltransferase.

Isolated hepatocytes from fed and starved rats, permeabilized with Staphylococcus aureus alpha-toxin, were incubated with increasing concentrations of radiolabelled fatty acids, in the presence of a saturating concentration of 3-GP. Incorporation of label into LPA, PA and DAG was lower in cells from starved rats than in cells from fed rats, apparently reflecting the lower activity of GPAT after starvation. This enzyme approached saturation at high fatty acid levels and determined the overall flux through the esterification pathway. TAG synthesis, however, was the same in both nutritional states and could not be saturated with fatty acid under the given conditions. Taken together with the observed accumulation of DAG, these data suggest that the rate of TAG synthesis is controlled by the fatty acid supply and, more particularly, by the affinity of DGAT for acyl-CoA.

Acyltransferases↗
PubMed1993

Peroxisome proliferators and T3 operate by way of distinct receptors.

Peroxisome proliferators and thyroid hormones have a number of common metabolic effects. The possibility that the signal transduction pathways of both groups of effectors converge at the receptor level was investigated. It was shown that T3, specifically bound to the rat thyroid beta-receptor, was not displaced to a significant extent by ciprofibrate or bezafibrate. No specific binding of T3 to the mouse peroxisome proliferator activated receptor could be demonstrated. In transactivation experiments peroxisome proliferators were unable to activate the thyroid receptor and T3 did not activate a chimeric receptor containing the ligand binding domain of the peroxisome proliferator activated receptor. It is concluded that peroxisome proliferators and thyroid hormone do not cross-react at the level of their nuclear receptors.

Animals↗
PubMed1992

Factors influencing triacylglycerol synthesis in permeabilized rat hepatocytes.

Rat hepatocytes were treated with Staphylococcus aureus alpha-toxin to permeabilize their plasma membrane for low-molecular-mass compounds. During incubation with 1 mM labelled fatty acid, phosphatidate and, less clearly, lysophosphatidate rapidly reached a steady state, whereas labelled diacylglycerol accumulated to some extent, at least in the absence of exogenous CDP-choline. Esterification and oxidation were linearly related to the fatty acid concentration, and there was no indication for saturation with acyl-CoA. However, when permeabilized cells were incubated with labelled sn-glycerol 3-phosphate and 1 mM unlabelled fatty acid, glycerolipid synthesis and the level of esterification intermediates reached a plateau between 0.25 and 0.50 mumol of the triose phosphate/ml. The synthesis of phosphatidylcholine was dependent on addition of CDP-choline. In presence of the latter, diacylglycerol no longer accumulated and triacylglycerol synthesis was suppressed, although the sum of synthesized diacylglycerol, triacylglycerol and phosphatidylcholine remained constant. This indicates that the same pool of diacylglycerol is shared by choline-phosphotransferase and diacylglycerol acyltransferase and that the relative activity of these enzymes depends on the CDP-choline supply. Comparison of the levels of the esterification intermediates with the activity of the respective steps of the pathway reveals that, at a fixed fatty acid concentration, glycerophosphate acyltransferase determines the esterification rate, whereas lysophosphatidate acyltransferase and, at low CDP-choline levels, diacylglycerol acyltransferase approach saturation at elevated sn-glycerol 3-phosphate concentration. There is, however, no indication for a regulatory role of phosphatidate phosphohydrolase in this system. The significance of these findings for the regulation of triacylglycerol synthesis under conditions in vivo is discussed.

3-O-Methylglucose↗
PubMed1992

A specific and inexpensive assay of radiolabeled long-chain acyl-coenzyme A in isolated hepatocytes.

A simple procedure for determining radiolabeled long-chain acyl-CoA levels in isolated rat hepatocytes has been developed. It consists of a classical extraction of long-chain acyl-CoAs after preliminary removal of the excess labeled free fatty acid. A two-step TLC purification ensures elimination of long-chain acylcarnitine, the main interferent in most methods, and other common lipids. The purity of the acyl-CoA was confirmed by a second TLC system and by spectral analysis. Overall recovery was approximately 70%.

Acyl Coenzyme A↗
PubMed1987

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. I. Use of inhibitors.

The effects of various inhibitors of carnitine palmitoyltransferase I were examined in mitochondria from rat liver and skeletal muscle. Three types of inhibitors were used: malonyl-CoA (reversible), tetradecylglycidyl-CoA and three of its analogues (irreversible), and 2-bromopalmitoyl-CoA (essentially irreversible when added with carnitine). Competitive binding studies between labeled and unlabeled ligands together with electrophoretic analysis of sodium dodecyl sulfate-solubilized membranes revealed that in mitochondria from both tissues all of the inhibitors interacted with a single protein. While the binding capacity for inhibitors was similar in liver and muscle (6-8 pmol/mg of mitochondrial protein) the proteins involved were of different monomeric size (Mr 94,000 and 86,000, respectively). Treatment of mitochondria with the detergent, octyl glucoside, yielded a soluble form of carnitine palmitoyltransferase and residual membranes that were devoid of enzyme activity. The solubilized enzyme displayed the same activity regardless of whether carnitine palmitoyltransferase I of the original mitochondria had first been exposed to an irreversible inhibitor or destroyed by chymotrypsin. It eluted as a single activity peak through four purification steps. The final product from both liver and muscle migrated as single band on sodium dodecyl sulfate-polyacrylamide electrophoresis with Mr of approximately 80,000. The data are consistent with the following model. The inhibitor binding protein is carnitine palmitoyltransferase I itself (as opposed to a regulatory subunit). The hepatic monomer is larger than the muscle enzyme. Each inhibitor interacts via its thioester group at the palmitoyl-CoA binding site of the enzyme but also at a second locus that is probably different for each agent and dictated by the chemical substituent on carbon 2. Disruption of the mitochondrial inner membrane by octyl glucoside causes inactivation of carnitine palmitoyltransferase I while releasing carnitine palmitoyltransferase II in active form. The latter is readily purified, is a smaller protein than carnitine palmitoyltransferase I, and has the same molecular weight in liver and muscle. It is insensitive to inhibitors where on or off the mitochondrial membrane.

Acyl Coenzyme A↗
PubMed1985

Interaction of malonyl-CoA and 2-tetradecylglycidyl-CoA with mitochondrial carnitine palmitoyltransferase I.

Malonyl-CoA and 2-tetradecylglycidyl-CoA (TG-CoA) are potent inhibitors of mitochondrial carnitine palmitoyltransferase I (EC 2.3.1.21). To gain insight into their mode of action, the effects of both agents on mitochondria from rat liver and skeletal muscle were examined before and after membrane disruption with octylglucoside or digitonin. Pretreatment of intact mitochondria with TG-CoA caused almost total suppression of carnitine palmitoyltransferase I, with concomitant loss in malonyl-CoA binding capacity. However, subsequent membrane solubilization with octylglucoside resulted in high and equal carnitine palmitoyltransferase activity from control and TG-CoA pretreated mitochondria; neither solubilized preparation showed sensitivity to malonyl-CoA or TG-CoA. Upon removal of the detergent by dialysis the bulk of carnitine palmitoyltransferase was reincorporated into membrane vesicles, but the reinserted enzyme remained insensitive to both inhibitors. Carnitine palmitoyltransferase containing vesicles failed to bind malonyl-CoA. With increasing concentrations of digitonin, release of carnitine palmitoyltransferase paralleled disruption of the inner mitochondrial membrane, as reflected by the appearance of matrix enzymes in the soluble fraction. The profile of enzyme release was identical in control and TG-CoA pretreated mitochondria even though carnitine palmitoyltransferase I had been initially suppressed in the latter. Similar results were obtained when animals were treated with 2-tetradecylglycidate prior to the preparation of liver mitochondria. We conclude that malonyl-CoA and TG-CoA interact reversibly and irreversibly, respectively, with a common site on the mitochondrial (inner) membrane and that occupancy of this site causes inhibition of carnitine palmitoyltransferase I, but not of carnitine palmitoyltransferase II. Assuming that octylglucoside and digitonin do not selectively inactivate carnitine palmitoyltransferase I, the data suggest that both malonyl-CoA and TG-CoA interact with a regulatory locus that is closely juxtaposed to but distinct from the active site of the membrane-bound enzyme.

Acyl Coenzyme A↗
PubMed1984

Rat liver dihydroxyacetone-phosphate acyltransferases and their contribution to glycerolipid synthesis.

Differential and isopycnic centrifugation of rat liver homogenates showed that, besides its established localization in peroxisomes and endoplasmic reticulum, dihydroxyacetone-phosphate acyltransferase is also present in mitochondria. The three activities differed in a number of properties (pH optimum, palmitoyl-CoA and dihydroxyacetone-phosphate dependence, and sensitivity toward N-ethylmaleimide) and are therefore likely associated with three distinct proteins. Glycerol 3-phosphate (5 mM) did not inhibit peroxisomal dihydroxyacetone-phosphate acyltransferase but inhibited the extraperoxisomal activities virtually completely. Peroxisomal dihydroxyacetone-phosphate acyltransferase was located at the inner aspect of the peroxisomal membrane, but the enzyme was not latent. Purified microsomes, from which intact peroxisomes had been removed, were still contaminated with peroxisomal membranes as deduced from the presence of two dihydroxyacetone-phosphate acyltransferase activities: a glycerol 3-phosphate-resistant activity with properties similar to those of peroxisomal dihydroxyacetone-phosphate acyltransferase and a glycerol 3-phosphate-sensitive "true" microsomal dihydroxyacetone-phosphate acyltransferase. We propose that, assayed in the presence of 5mM glycerol 3-phosphate, dihydroxyacetone-phosphate acyltransferase can be used as a marker enzyme for peroxisomal membranes. Such a marker enzyme has not hitherto been available. The differential effect of 5 mM glycerol 3-phosphate on peroxisomal and extraperoxisomal dihydroxyacetone-phosphate acyltransferases enabled us to determine the relative contribution of these activities to overall dihydroxyacetone-phosphate acylation in whole liver homogenates. At near-physiological pH and at near-physiological concentrations of unbound palmitoyl-CoA and of dihydroxyacetone-phosphate plus glycerol 3-phosphate, peroxisomes contributed 50-75%. The remaining percentage was mostly accounted for by the microsomal enzyme. At near-physiological concentrations of glycerol 3-phosphate plus dihydroxyacetone-phosphate, glycerolphosphate acyltransferase contributed 93% and dihydroxyacetone-phosphate acyltransferase 7% to overall glycerolipid synthesis in homogenates. This suggests that the dihydroxyacetone-phosphate pathway is of minor quantitative importance in overall hepatic glycerolipid synthesis but that its main function lies in the synthesis of ether lipids, which have acyldihydroxyacetone-phosphate as obligatory precursor.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyltransferases↗
PubMed1984

Effects of benfluorex and fenofibrate treatment on mitochondrial and peroxisomal marker enzymes in rat liver.

Our results demonstrate that benfluorex at doses that are strongly hypotriglyceridemic does not increase hepatic peroxisomal enzyme activities, whereas fenofibrate at doses that are only slightly hypolipidemic induces a dramatic increase in the activity of these enzymes. Thus, the biochemical approach used in this study reveals that the hypolipidemic drug benfluorex does not belong to the class of hypolipidemic compounds known to induce hepatomegaly, hepatic peroxisome proliferation and hepatocarcinoma in rodents. Morphological studies should confirm the absence of peroxisomal induction.

Animals↗
PubMed1982

Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis.

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. V(max.) of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30-40% in starvation. There was no change in apparent K(m) for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3-0.4 and 0.5-0.65mumol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3mumol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44mumol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids.

Acyltransferases↗
PubMed1982

Glucagon inhibits triacylglycerol synthesis in isolated hepatocytes by lowering their glycerol 3-phosphate content.

Triacylglycerol synthesis by glycogen-depleted hepatocytes from fed rats that have low glycerol 3-phosphate contents was stimulated by the addition of glycerol 3-phosphate precursors. Glucagon decreased triacylglycerol synthesis only when it also lowered glycerol 3-phosphate content. The hyperbolic-like relationship between glycerol 3-phosphate content and rates of triacylglycerol synthesis was identical in the absence or presence of glucagon, indicating that the glucagon effect on triacylglycerol synthesis was not mediated through changes in enzyme activities of the esterification pathway but through changes in cellular glycerol 3-phosphate content.

Animals↗