Gunilla Bengtsson-Olivecrona

Lipoprotein lipase and hepatic lipase

Knowledge of the molecular structure and interactions of the lipases is expanding rapidly. Lipase activities are regulated by modulation of messenger RNA levels and by incompletely defined post-translational mechanisms. Evidence is growing that lipoprotein lipase acts not only as a hydrolytic enzyme

Phospholipase activity of milk lipoprotein lipase.

Publisher Summary This chapter describes various methods to prepare and handle milk lipoprotein lipase (LpL) and its activator protein and conditions required to be considered in kinetic studies. Bovine milk contains 1–2 mg active LpL per liter, and the enzyme can be purified by simple methods in yields approaching 50%. This has made LpL from bovine milk the most used model enzyme in LpL research. In the lactating mammary gland, LpL has an important role for uptake of lipids from plasma lipoproteins. All the methods to prepare LpL from milk are based on chromatography on heparin-Sepharose, with some different additional steps. The chapter presents a simple method suitable for preparation of LpL for kinetic studies. LpL is also sometimes defined as the salt-sensitive lipase. The enzyme can exert full catalytic activity even in the presence of 1 M NaCl. The effect of salt is not exerted at this level but on the physical state and the stability of the enzyme molecule. For most purposes, NaCl concentrations of 0.05–0.15 M result in optimal activity and stability for kinetic studies.

Distribution of lipoprotein lipase and hepatic lipase between plasma and tissues: effect of hypertriglyceridemia

Lipoprotein lipase and hepatic lipase were measured in rat plasma using specific antisera. Mean values for lipoprotein lipase in adult rats were 1.8-3.6 mU/ml, depending on sex and nutritional state. Values for hepatic lipase were about three times higher. Lipoprotein lipase activity in plasma of newborn rats was 2-4-times higher than in adults. In contrast, hepatic lipase activity was lower in newborn than in adult rats. Following functional hepatectomy there was a progressive increase in lipoprotein lipase activity in plasma, indicating that transport of the enzyme from peripheral tissues to the liver normally takes place. Lipoprotein lipase, but not hepatic lipase, increased in plasma after a fat meal. An even more marked increase, up to 30 mU/ml, was seen after intravenous injection of Intralipid. Plasma lipase activity decreased in parallel with clearing of the injected triacylglycerol. 125I-labeled lipoprotein lipase injected intravenously during the hyperlipemia disappeared somewhat slower from the circulation than in fasted rats, but the uptake was still primarily in the liver. Hyperlipemia, or injection of heparin, led to increased lipoprotein lipase activity in the liver. This was seen even when the animals had been pretreated with cycloheximide to inhibit synthesis of new enzyme protein. These results suggest that during hypertriglyceridemia lipoprotein lipase binds to circulating lipoproteins/lipid droplets which results in increased plasma levels of the enzyme and increased transport to the liver.

Molecular cloning and sequence analysis of cDNA encoding lipoprotein lipase of guinea pig.

We have isolated and sequenced cDNA clones covering the entire coding sequence and short flanking regions of guinea pig lipoprotein lipase. The expression cDNA library used was constructed in lambda gt11 with mRNA derived from adipocytes. The deduced amino acid (aa) sequence starts with a stretch of 17 aa interpreted as a leader peptide. The open reading frame continues with 448 aa residues and ends with a TGA stop codon. Combined with previous data this information allows the assignment of domains in the lipase molecule. A likely candidate for the heparin-binding site is a 9-aa stretch containing five positive charges, analogous to the consensus sequence for receptor-binding sites on apolipoproteins E and B. A previously noted homology to pancreatic lipase is extended. Analysis of polyadenylated RNA from several tissues indicated a high level of expression in adipocytes, heart muscle and mammary gland. No lipoprotein lipase mRNA could be detected in liver. Northern blots revealed three major mRNAs with sizes corresponding to 3.8 kb, 3.3 kb and 2.1 kb, respectively. In adipocytes and heart muscle a fourth mRNA, with an estimated size of 4.5 kb, was also detected. Analysis of genomic DNA by Southern blotting indicated a single gene locus coding for lipoprotein lipase. Hence, modification of the primary transcript seems to be involved in the production of the various mRNAs.

Hepatic and extrahepatic uptake of intravenously injected lipoprotein lipase

Rats were injected intravenously with 125I-labeled bovine lipoprotein lipase. The lipase disappeared within minutes from the blood due to uptake both in the liver (about 50% of the injected dose) and in extrahepatic tissues. Lipase enzyme activity disappeared in parallel to the 125I radioactivity. Thus, there was no inactivation of lipase in the circulating blood. Similar results were obtained when lipoprotein lipase purified from guinea pigs was injected into guinea pigs. Using supradiphragmatic rats we could show that the extrahepatic uptake was saturable and that the amounts of lipase that could be bound far exceeded the amounts of endogenous lipase expected to be present on the endothelium. When the lipase was denatured before injection, its removal in supradiaphragmatic rats became slower, and in intact rats the fraction of the uptake that occurred in extrahepatic tissues was much decreased. It is concluded that recognition by the extrahepatic receptors depends on the native conformation of the lipase. The extrahepatic uptake was strongly impeded by injection of heparin prior to injection of the lipase, and the uptake could to a large extent be reversed by injection of heparin after the lipase. Even after 1 h lipase that had been taken up by extrahepatic tissues reappeared immediately in the blood on injection of heparin. This was true both for enzyme activity and for enzyme radioactivity. Thus, internalization-inactivation-degradation occur only slowly in extrahepatic tissues. It is possible that the extrahepatic binding occurs to the enzymes physiological receptors. The hepatic uptake was not dependent on the native conformation of the lipase, was less sensitive to heparin, could not be reversed by heparin and was not saturable. The enzyme was not rapidly inactivated after uptake; its activity could be detected in liver homogenates even after 1 h. Degradation to acid-soluble products in the liver was relatively slow; the t1/2 for native lipase was about 1 h. In comparison, in parallel experiments asialofetuin was degraded with a t1/2 of about 15 min.

Mouse preheparin plasma contains high levels of hepatic lipase with low affinity for heparin

It was recently noted that newborn mice have much higher lipase activity in plasma than rats or humans, and that most of the activity is due to an enzyme related to the hepatic (heparin-releasable) lipase. Here we report that this lipase is present in plasma of adult mice also. In contrast to the high activity of hepatic lipase, the activity of lipoprotein lipase in plasma was low and similar to that in rats. The source of the plasma lipase was probably the liver, since we could not demonstrate hepatic lipase-like activity in any other organ. When human hepatic lipase was injected into mice, it rapidly disappeared from plasma. Most of the injected lipase located in the liver, and could be released back into circulation by injection of heparin. These results indicate that there are binding sites for hepatic lipase in mouse liver, and suggest that mouse hepatic lipase has an affinity for these sites which is lower than usual. It is currently believed that the endothelial acceptors are heparan-sulfate or similar molecules. Mouse hepatic lipase eluted from heparin-Sepharose at lower salt concentration than rat or human hepatic lipase, demonstrating that it has a relatively low affinity for heparin-like polysaccharides.

Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase

The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and human hepatic lipase was examined in vitro. Chylomicrons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentaenoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the C14-C18 acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eicosatrienoic, docosatrienoic and docosahexanoic acids exhibited an intermediate lipolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 20:4 esters of both tri- and diacylglycerols. Addition of NaCl (final concentration 1 M) during the course of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylglycerol lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 20:4 and 20:5 esters thus exhibit a relative resistance to lipoprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids may accumulate at the surface of the remnant particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase.

New Aspects on Heparin and Lipoprotein Metabolism

Lipoprotein lipase (LPL) and hepatic lipase (HL) are two enzymes which participate in metabolism of plasma lipoproteins. The enzymes are located at vascular surfaces and are released from their binding sites on injection of heparin. In this paper we give a short overview of the structure of the lipases and their role in lipoprotein metabolism. Earlier studies had shown that low molecular weight (LMW) heparin preparations result in lower LPL activities in blood than do corresponding amounts of conventional heparin. Studies with organ perfusion in rats show that the two types of heparin have similar ability to release the lipases from their binding sites in extrahepatic tissues, but that LMW heparin is less effective than conventional heparin in preventing rapid uptake and degradation of LPL by the liver. After injection of heparin the metabolism of triglyceride-rich lipoproteins is initially accelerated, presumably as a result of the high levels of circulating LPL. Then follows a phase when lipoprotein metabolism is slower than normal, perhaps because endothelial LPL has been depleted by accelerated transport to and degradation in the liver.

Release of lipoprotein lipase to plasma by triacylglycerol emulsions. comparison to the effect of heparin

It was previously known that lipoprotein lipase (LPL) activity in plasma rises after infusion of a fat emulsion. To explore the mechanism we have compared the release of LPL by emulsion to that by heparin. After bolus injections of a fat emulsion (Intralipid) to rats, plasma LPL activity gradually rose 5-fold to a maximum at 6-8 min. During the same time the concentration of injected triacylglycerols (TG) decreased by about half. Hence, the time-course for plasma LPL activity was quite different from that for plasma TG. The disappearance of injected 125I-labelled bovine LPL from circulation was retarded by emulsion. This effect was more marked 30 min than 3 min after injection of the emulsion. The data indicate that the release of LPL into plasma is not solely due to binding of the lipase to the emulsion particles as such, but involves metabolism of the particles. Emulsion increased the fraction of labelled LPL found in adipose tissue, heart and the red muscle studied, but had no significant effect on the fraction found in liver. The effects of emulsion were quite different from those of heparin, which caused an immediate release of the lipase to plasma, decreased uptake of LPL in most extrahepatic tissues by 60-95%, and increased the fraction taken up in the liver.

Lipoprotein lipase in liver. Release by heparin and immunocytochemical localization

We have previously demonstrated that infusion of Intralipid to rats causes a pronounced increase of the lipoprotein lipase activity in the liver. In this paper we study where in the liver this lipoprotein lipase is located. When isolated livers from Intralipid-treated rats were perfused with heparin, substantial amounts of lipoprotein lipase were released into the perfusate. The identity of the lipase activity was demonstrated by specific inhibition with antisera to lipoprotein lipase, and to hepatic lipase, respectively, and by separation of the two lipase activities by chromatography on heparin-Sepharose. We have also studied the localization of both enzymes by an immunostaining procedure based on post-embedding incubation of ultrathin tissue sections with specific antibodies which were then visualized using protein A-colloidal gold complexes. There was no marked difference in localization for the two enzymes which were both seen at the luminal side of endothelial cells, at the interdigitations of the space of Disse and inside both hepatocytes and endothelial cells. Thus, lipoprotein lipase is present in the liver in positions similar to where the functional pool of hepatic lipase is located and analogous to where lipoprotein lipase is found in extrahepatic tissues. These results raise the possibility that the enzyme has a functional role in the liver.