Luc J. Debeer

Hepatic fatty acid oxidation and ketogenesis after clofibrate treatment

The effect of clofibrate treatment on hepatic ketogenic capacity was studied in rats. Ketogenesis from octanoate and oleate was increased 2- and 4,5-fold, respectively, in hepatocytes from fed, treated rats. In contrast to controls ketogenic rates did not increase upon starvation. While ketogenesis from oleate was higher in fed, treated animals than in fasted controls, endogenous ketogenesis was lower and increased upon starvation. Ketogenesis from octanoate and oleate was stimulated approx. 2-fold in homogenates from treated animals. Labeled pyruvate and succinate oxidation was unaltered. [1-14C]Oleate oxidation was severely inhibited by cyanide, both in homogenates from controls and treated animals. Clofibrate caused a 3-fold increase in hepatic carnitine levels. Catalase and glutamate dehydrogenase activities were also increased by the drug. Cytochrome c oxidase did not change. Despite their increased ketogenic capacity hepatocytes from treated rats esterified as much oleate as controls. The increased oxidation was matched by an increased oleate uptake. Plasma ketones were increased 2-fold in fasted, treated animals. Plasma free fatty acids were unaffected. It is concluded that the enhanced ketogenic capacity induced by clofibrate is the result of an increase in mitochondrial beta-oxidation, an increase in the activity of carnitine palmitoyltransferase and possibly of the observed increases in hepatic carnitine content and fatty acid uptake.

Subcellular distribution of coenzyme A: evidence for a separate coenzyme A pool in peroxisomes

Summary Subcellular fractions enriched in peroxisomes, that were prepared by differential centrifugation, contained more free and total (free plus esterified) CoA than could be accounted for by mitochondrial contamination. Separation of peroxisomes and mitochondria by isopycnic centrifugation of fractions enriched in peroxisomes resulted in a bimodal distribution of free and total CoA in which both peak fractions coincided with the fractions that contained most of the peroxisomal and mitochondrial marker enzyme activities respectively. These results indicate that peroxisomes have a separate CoA-pool. This CoA appears to be available for the thiolase reaction during peroxisomal β-oxidation of fatty acids but not for the activation of fatty acids by the peroxisomal acyl-CoA synthetase.

Lipolysis of hepatic triacylglycerol stores

Our knowledge concerning the mechanism of hepatic triacylglycerol synthesis and secretion has made considerable progress over the last few years and general patterns of control are beginning to emerge [l]. Yet, very little is known about the breakdown of hepatic triacylglycerols. Studies on the control and the physiological role of hepatic lipolysis have been hampered by the fact that it was unknown which liver lipase was responsible for the hydrolysis of hepatic triacylglycerol stores (hepatic lipolysis). Throughout the text the terms lipolytic activity and lipase activity refer to triacylglycerol lipase activity (EC 3.1.1.3). Net lipolysis is defined as the decrease in triacylglycerol stores, measured either chemically or by the disappearance of radioactivity from triacylglycerols prelabelled with radioactive fatty acids. Consequently, net lipolysis is the balance of triacylglycerol breakdown (which is termed lipolysis per se) and triacylglycerol synthesis; in the case of prelabelled triacylglycerols the latter process means reesterification of labelled fatty acids. phospholipids [lo]. Monoacylglycerols produced from trior diacylglycerols are not processed further by the lysosomal lipase [7,10] but may be hydrolysed by a microsomal monoacylglycerol hydrolase [ 111. Lipolytic activities occurring in cytosol, microsomes and plasma membranes display an alkaline pH optimum of 8-9.5 and exhibit an identical response to inhibitors such as NaCl and protamine sulfate. The highest specific activity is found in plasma membranes. Hence, the possibility has been raised that the various lipolytic activities represent the plasma membrane enzyme that is partially released during homogenization and fractionation [6,7].

Effect of sulfonylureas on triglyceride metabolism in the rat liver: possible role of the lysosomes in hepatic lipolysis.

It has been suggested previously that chlorpropamide and other hypoglycemic sulfonylureas interfere with hepatic triglyceride breakdown. Since ketogenesis from endogenous hepatic lipid stores is a measure of hepatic triglyceride hydrolysis, ketogenesis derived from endogenous lipids as well as ketogenesis derived from exogenously added isotopic oleate was determined in isolated hepatocytes from fasted rats in an attempt to identify the nature of the direct effects of sulfonylureas on hepatic lipid metabolism. Ketogenesis from endogenous lipids was inhibited by 1 mM chlorpropamide, while ketone production from exogenous oleate did not change. The effect of chlorpropamide on hepatic triglyceride metabolism was further studied in the isolated perfused liver of normal rats in the presence of a continuous [3H]oleate infusion and in isolated liver cells incubated in the presence of [3H]oleate. In liver perfusion experiments, 1 mM chlorpropamide enhanced the incorporation of tritium into triglycerides (but not other lipid classes) and increased both liver triglyceride content and triglyceride secretion. Using isolated cells similar effects could be demonstrated at 0.5 mM chlorpropamide. Chlorpropamide, tolbutamide, and carbutamide, all of which inhibited endogenous ketogenesis in isolated liver cells, also inhibited lysosomal triglyceride lipase activity in rat liver homogenates. The drugs were not inhibitory towards alkaline lipase activity. Demethylglycodiazin (2-benzolsulfonamid--5-(beta-hydroxyethoxy)-pyrimidin), which did not inhibit endogenous ketogenesis in isolated liver cells, did not affect lysosomal lipase activity. The lysosomotropic drug chloroquine was markedly antiketogenic when tested in liver cells. The reduction in endogenous ketogenesis, the enhanced accumulation of liver triglycerides, and the stimulation of hepatic triglyceride output by chlorpropamide are ascribed to an interference of the drug with hepatic triglyceride breakdown. The present results also suggest that the lysosomes play a significant role in hepatic lipolysis.

Glycerol-3-phosphate content and triacylglycerol synthesis in isolated hepatocytes from fed and starved rats

For a given load of fatty acids the perfused liver or isolated hepatocytes from fed rats esterify a greater proportion of the incoming fatty acids than do similar preparations from starved rats. In the latter oxidation is increased relative to esterification [l-3]. Since esterification and oxidation constitute a branch-point, the nutritional and hormonal control of esterification and oxidation can theoretica~y be exerted either on one arm of the branch-point or in reciprocal manner on both arms simultaneously. Recently, evidence has been offered that fatty acid oxidation is controlled by the hepatic levels of carnitine and especially of maionyl-CoA, a potent inhibitor of carnitine palmitoyltransferase I [4]. It is not clear, however, whether there exists a separate control on esterification. The moderate decrease in the activity of glycerol3-phosphate acyltransferase that is observed in the starved state 15-71, has been proposed as a possible factor regulating esterification. The availability of glycerol3-phosphate has been considered as another regulatory factor [ 1,7-l 11. However,a wide range of overlapping glycerol-3-phosphate concentrations has been reported for fed and starved animals [2,10,12-151. Moreover, interpretation of the data is complicated by the fact that the concentration range of glycerol-3-phosphate which might be regulatory in the intact hepatocyte, is not known. Here, the intracellular concentration range within which glycerol-3-phosphate exerts a regulatory function, was determined. 2. MateriaIs and methods

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.

Metabolic effects of hypoglycemic sulfonylureas. 3. Effect of chlorpropamide on energy metabolism and ketogenesis in the isolated perfused rat liver.

Abstract The effects of 1 and 5 mM chlorpropamide were studied in the isolated perfused liver from fasted rats in the absence of exogenous substrate. 1 mM chlorpropamide caused a slight, but not significant, decrease in ATP content and in ATP/ADP ratio in the liver; 5 mM chlorpropamide caused a significant decrease in ATP, total adenine nucleotides and ATP/ADP ratio and an increase in ADP, AMP and inorganic phosphate content. The redox states of both cytosol and mitochondria, as measured by the lactate/pyruvate and β-hydroxybutyrate/acetoacetate ratios, respectively, were unaffected. However, there was a significant and dose dependent decrease in acetoacetate and β-hydroxybutyrate content. Livers perfused with 1 mM chlorpropamide showed a slight, but not significant, increase in oxygen uptake; 5 mM chlorpropamide had a biphasic effect: an initial stimulation, followed by a progressive inhibition of oxygen uptake. Ketone body production measured in the perfusion medium was inhibited by 1 and 5 mM chlorpropamide. No influenee could be seen on ureogenesis. The measured rates of oxygen uptake by the liver compared with the changes in oxygen uptake calculated from the reduced ketogenesis indicate a mixed effect of chlorpropamide on the perfused liver; (1) a decreased oxidation of endogenous triglycerides and (2) an increased oxidation of acetyl coenzyme A through the tricarboxylic acid cycle. The present findings suggest that chlorpropamide acts on the perfused liver as an uncoupling agent and as an inhibitor of the hepatic triglyceride lipase. (Glycerol ester hydrolase, EC 3.1.1.3.) Both effects could be related.

Metabolic effects of hypoglycemic sulfonylureas—IV: Interference of sulfonylureas with mitochondrial oxidative phosphorylation☆

Abstract The effects of various hypoglycemic and non-hypoglycemic sulfonylureas on succinate oxidation by rat liver mitochondria have been studied. At low succinate concentration, respiration is initially stimulated and then inhibited by all the sulfonylureas tested, except carbutamide and the compound P571. Both phenomena are dose-dependent. The respiration inhibition can be reversed by increasing the succinate concentration, but not by adding ATP plus oligomycin. Respiratory control disappears progressively in the presence of increasing concentrations of sulfonylureas, including carbutamide and P571, mainly through inhibition of state 3 respiration. This inhibition can also be lowered by increasing the succinate concentration. The effect of sulfonylureas on respiration is oligomycin-insensitive. 2,4-Dinitrophenol has the same effect as the sulfonylureas under the same experimental conditions. It is concluded that the sulfonylureas studied, except carbutamide and P571, exert a dinitrophenol-like effect on mitochondrial respiration, and that, like dinitrophenol, all sulfonylureas, including carbutamide and P571, interfere with mitochondrial substrate uptake. A relationship between the observed effects and the clinical activity of these drugs is discussed.

Metabolic effects of hypoglycemic sulfonylureas—V: In vitro effects of sulfonylureas on mitochondrial adenosine triphosphatase activity

Abstract The ATPase (ATP phosphohydrolase, EC 3.6.1.4) stimulating activity of sulfonylureas has been examined. Sulfonylureas stimulate ATPase to variable extents, and this stimulation is oligomycin-sensitive. They are less active than 2,4-dinitrophenol and their relative stimulating activity parallels their uncoupling potency on oxidative phosphorylation. Carbutamide, which in itself does not stimulate ATPase, inhibits ATPase stimulation by other sulfonylureas. Experiments with combinations of chlorpropamide and P594, two active compounds, suggest that P594 penetrates more readily into mitochondria and is a stronger uncoupling agent. All sulfonylureas inhibit ATPase stimulated by optimal concentrations of dinitrophenol. Possible implications of these observations with regard to the metabolic effects of sulfonylureas are discussed.

Metabolic effects of hypoglycemic sulfonylureas-VI. Effects of chlorpropamide and carbutamide on ketogenesis and on mitochondrial redox state in the isolated perfused rat liver.

Abstract The effect of 5 mM chlorpropamide and 5 mM carbutamide on fatty acid oxidation in the perfused rat liver was studied. Chlorpropamide as well as carbutamide inhibited endogenous lipid oxidation to ketone bodies. Chlorpropamide had no effect on ketogenesis from exogenously added octanoate, but inhibited ketone body production from a single dose of oleate. Ketogenesis from a continuous oleate infusion was reduced by chlorpropamide during the first 15 min of infusion but was unaffected during the next 45 min studied. Carbutamide did not decrease ketogenesis during oleate infusion. The uptake of octanoate and oleate remained unchanged in the presence of both drugs. Chlorpropamide caused a stimulation of oxygen consumption in the absence of fatty acid substrates but was unable to alter oleate-stimulated respiration. Chlorpropamide, but not carbutamide, stimulated oligomycin-inhibited respiration although to a lesser degree than 2,4-dinitrophenol. Krebs cycle flux during oleate infusion was slightly stimulated by chlorpropamide. Mitochondrial redox state, as measured by the β-hydroxybutyrate/acetoacetate ratio in the perfusate, was markedly lowered by chlorpropamide in the octanoate and oleate experiments. The lactate/pyruvate ratio was unaffected by chlorpropamide. Carbutamide did not produce any change compared with control experiments. From these experiments it is concluded that chlorpropamide and carbutamide inhibit endogenous lipid oxidation by interfering with hepatic triglyceride lipase activity. Changes in oxygen uptake and in mitochondrial redox state caused by chlorpropamide are attributed to the uncoupling activity of this drug.