W.C. Hülsmann

On the metabolic function of heparin-releasable liver lipase

Abstract Intravenous administration of specific antibody against heparin-releasable liver lipase (liver lipase) induced a 75% inhibition of the enzyme activity in situ . Administration of the antibody resulted in an increase of high density lipoprotein (density range 1.050–1.13 g/ml; HDL2) phospholipid levels (20% after 1 h; 54% after 4 h). Short-term (1 h) treatment with antibody had no significant effect on any of the other lipoprotein components. After long-term (4 h) treatment the free cholesterol level of HDL2 and all components in the very low density lipoprotein (VLDL) + intermediate density lipoprotein (IDL) fraction were elevated (1.5–2.0 fold). In the low density lipoprotein (LDL) fraction only the phospholipid level was affected (increased by 72%). All lipid components in the HDL3 fraction were decreased by the antibody treatment, but this decrease was only statistically significant for the cholesterolesters. The rate of removal of iodine-labeled high density lipoprotein (HDL) and LDL from serum was not affected by the antibody treatment. These results suggest that liver lipase may promote phospholipid removal in vivo and show that a lowering of liver lipase in situ has profound consequences for serum lipoprotein metabolism.

Distribution of L- and M-type pyruvate kinase between parenchymal and Kupffer cells of rat liver

Abstract 1. 1.|Parenchymal cells were isolated from rat liver by using EDTA + lysozyme or citrate. These cells contained only the L-type pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40). 2. 2.|Kupffer cells were isolated from rat liver by using pronase. The Kupffer cell preparation showed a ratio of M-type to L-type pyruvate kinase 10–20 times higher than the ratio in a total liver homogenate, suggesting that Kupffer cells only (probably) contain the M-type pyruvate kinase. 3. 3.|The results obtained suggest that gluconeogenesis is confined to the parenchymal cells of rat liver.

Binding of liver lipase to parenchymal and non-parenchymal rat liver cells.

Summary The present paper describes the in vitro binding of liver lipase partially purified from postheparin rat serum to isolated liver cells. The binding of liver lipase to non-parenchymal liver cells was 100 times more, per mg cell protein, than to parenchymal cells. The in vitro bound lipase to non-parenchymal cells was largely (80%) releasable by heparin. The presented data suggest that in vivo the heparin-releasable liver lipase is mainly (85–92%) located at the surface of non-parenchymal cells.

Secretion of triacylglycerol hydrolase activity by isolated parenchymal rat liver cells.

More than 80% of the neutral triacylglycerol hydrolase activity of rat liver can be released from the liver by heparin [ 11. Isolation of parenchymal or nonparenchymal liver cells with collagenase results in the loss of > 90% total neutral lipase activity, present in the liver before collagenase perfusion [2,3] . This and the releasability of the enzyme by heparin indicate that the major part of the enzyme is located at the outer surface of the cells present in liver tissue. Such a localization suggests a role of the lipase in extracellular lipid metabolism, e.g.,in the hydrolysis of serum lipid, but a definite role of the lipase in lipid metabolism has not been proven yet. A major problem in the study on the function of the lipase is the lack of lipase activity in isolated liver cells. This study was undertaken to see whether the lipase activity could be restored in isolated parenchymal cells. Incubation of isolated parenchymal cells for 4 h at 37°C did not increase the cell-associated lipase activity to a considerable extent. When rat serum was present in the incubation mixture, a time-dependent increase in lipase activity was found in the medium, but not in the cells. The lipase activity in the medium could be inhibited almost completely (> 95%) by an antibody raised against heparin-releasable liver lipase isolated from post-heparin rat serum, Secretion of lipase activity into the medium and the activity in the cells were found to be inhibited when the protein synthesis inhibitor, cycloheximide, was present during incubation of the cells. 2. Methods and materials

The activation and oxidation of octanoate and palmitate by rat skeletal muscle mitochondria

Abstract 1. 1. Rat skeletal muscle mitochondria can oxidize octanoate just as well as palmitate. The oxidation of both fatty acids is strongly carnitine dependent, which indicates that the fatty acid CoA ester formation (fatty acid activation) is localized on the outer membrane. 2. 2. The palmitoyl-CoA synthetase and octanoyl-CoA synthetase activities were measured in these mitochondria. Palmitoyl-CoA synthetase was inhibited by octanoate while the octanoyl -CoA synthetase was inhibited by palmitate. Both inhibitions seem to be of the competitive type. These results suggest that both fatty acids are activated by the same enzyme. 3. 3. Octanoyl-CoA synthetase in rat skeletal muscle mitochondria was strongly (3 to 4 times) stimulated by the addition of skeletal muscle cytosol or by the addition of a high concentration of salt.

The localization of palmitoyl-CoA: Carnitine palmitoyltransferase in rat liver

1. The distribution of palmitoyl-CoA:carnitine palmitoyltransferase has been studied in subcellular fractions of rat liver. By using two different estimations for the enzyme activity and by differential centrifugation and linear sucrose density gradient centrifugation, the enzyme is shown to be localized both in mitochondria and microsomes. 2. The mitochondrial palmitoyl-CoA: carnitine palmitoyltransferase is localized in the inner membrane plus matrix fraction. 3. During palmitate oxidation by isolated mitochondria, in the presence of a physiological concentration of carnitine, palmitoylcarnitine accumulates. From this and experiments with sonicated mitochondria, it is concluded that the capacities of long-chain fatty acid activation and of palmitoyl-CoA:carnitine palmitoyltransferase in vitro by far exceed the capacity of fatty acid oxidation.

Dual localization and properties of ATP-dependent long-chain fatty acid activation in rat liver mitochondria and the consequences for fatty acid oxidation

Abstract 1. Direct evidence is given for the existence of two ATP-dependent palmitoyl-CoA synthesizing enzymes, localized in different compartments of the rat-liver mitochondrion. 2. About 90% of the total activity of rat liver mitochondria is localized in the mitochondrial outer membrane and about 10% in the inner membrane-matrix compartment. 3. The two enzyme systems show different apparent K m s for fatty acid and ATP, and different apparent K i s for AMP and adenosine. 4. The inner membrane-matrix enzyme, in contrast to the outer membrane and the microsomal enzyme, is strongly inhibited by octanoate. 5. Comparison of the kinetics of the two ATP-dependent long-chain fatty acid-activating enzymes with the kinetics of long-chain fatty acid oxidation shows, that during fatty acid oxidation at low concentrations of palmitate or oleate, in the presence of carnitine, the outer membrane acyl-CoA synthetase is operating. In the absence of carnitine and at high concentrations of long-chain fatty acids the activation reaction occurs in the inner membrane-matrix compartment of the mitochondrion. 6. The long-chain fatty acid concentration needed for half-maximal velocity of fatty acid oxidation by isolated rat liver mitochondria is about 1 μM in the presence of carnitine and 100–200 μM in the absence of carnitine.

Two interconvertible forms of L-type pyruvate kinase from rat liver.

Abstract 1. 1. Reduced L-type pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from rat liver can be converted into an oxidized form by incubation with oxidized mercaptoethanol and oxidized glutathione. This interconversion can be completely reversed by incubation with reduced mercaptoethanol. 2. 2. The kinetic and allosteric properties of the reduced and oxidized forms are described. 3. 3. The results are discussed in view of a possible regulation of the enzyme.

Dietary fatty acids and myocardial function

It is widely recognized that dietary polyunsaturated fatty acids (PUFAs) and cholesterol can profoundly influence the development of atherosclerotic plaques in coronary vessels, which may lead to myocardial infarction. The possibility that dietary fatty acids may also directly influence cardiac function has received less attention. We therefore reviewed the evidence of the effects of dietary fatty acids, in particular n-3 and n-6 PUFAs, on myocardial phospholipid fatty acid composition and cardiovascular performance. Heart organelles appear to incorporate uncommon fatty acids like 22:1 and trans- 18:1. Diets enriched with 22:1 induce myocardial lipidosis. N-9, n-6 and n-3 families compete among membrane C20 and C22 acids. Several studies have dealt with the relation between diet-induced changes of cardiac membrane (sarcolemma, sarcoplasmic reticulum and mitochondria) phospholipids and membrane function. In view of the variety of diets used and of the membrane functions studied, the results do not permit equivocal interpretation. Several investigators have reported an altered stress response of the heart due to a change of PUFAs in the diet. In rats fed with a low 18:2n-6/18:3n-3 ratio combined with relatively low amounts of saturated fatty acids, a high incidence of myocardial lesions has been observed. Pigs are less sensitive but more susceptible to the development of vitamin E deficiency, when the dietary PUFA content is high. Increased contractility and coronary flow rate have been reported for Langendorff-perfused hearts of rats fed 18:2n-6-rich diets. The effects on coronary flow rate are possibly related to alterations in eicosanoid synthesis, which may also contribute to the reduction by n-6 or n-3 PUFAs in infarct size, magnitude of recovery of function and suppression of reperfusion arrhythmias following release of a coronary artery ligation. On the other hand, increased peroxidation of membrane lipids, due to their high content of n-3 PUFA, may be deleterious.

On hepatic and extrahepatic postheparin serum lipase activities and the influence of experimental hypercortisolism and diabetes on these activities.

This paper shows that the palmitoyl-CoA hydrolase activity of postheparin serum of the rat is mainly derived from the liver. The identity of this activity with the heparin-releasable hepatic triacylglycerol hydrolase activity is established. The consequences of the different substrate specificities of the hepatic and extrahepatic enzymes for the measurement of the overall postheparin serum lipase activity are discussed. Treatment of the rats with either a corticosteroid or with streptozotocin was found to lower the lipolytic activity from the liver and to enhance the extrahepatic activity. Also in human postheparin serum, palmitoyl-CoA hydrolase activity is shown to behave identical with hepatic triacylglycerol hydrolase activity. The possible function of the liver in the serum triacylglycerol metabolism is discussed in connection with the proposed mechanism for the role of extrahepatic lipoprotein lipase in atherogenesis.