Search

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.

The nature of the lipases released into the circulation during low level continuous infusion of heparin (1 unit/ml total parenteral nutrition) and after bolus heparin injection (10 units/kg) was investigated in a group of 11 low birth weight infants (gestational age 27-34 wk, and postnatal age of 7-26 days) receiving total parenteral nutrition with Intralipid (0.5 g/kg). Hepatic lipase and extra-hepatic lipoprotein lipase were differentiated with the aid of an antibody specific for human hepatic lipase. The data show that continuous low level heparin infusion leads to a constant baseline postheparin lipolytic activity of 0.77 +/- 0.18 mumol free fatty acids released per milliliter serum per hour. Bolus heparin injection leads to peak lipolytic activity levels of 3.77 +/- 0.46 mumol free fatty acids per milliliter serum per hour, 10 min after injection. About two-thirds of the total postheparin lipolytic activity was of the hepatic type during low level continuous infusion or after bolus injection of heparin.

Conditions for extraction and assay of hepatic type (H-type) lipase from rat ovaries were studied. An alkaline buffer with protease inhibitors and detergents gave the most efficient extraction. The specificity of the assays was ascertained using antiserum to H-type lipase from heparin perfusates of rat livers. H-type lipase activity was determined in ovarian compartments during pseudopregnancy (1-13 days) as well as during the ensuing period of luteal regression (Day 17). The activity was low in the luteal compartment immediately after ovulation, increased 6-fold to a maximum between Day 5 and Day 8 and then decreased again. This is similar to previously known changes in blood flow. There was a significant correlation between luteal H-type lipase activity and plasma progesterone regardless of luteal age. In contrast, neither the activity in the remainder of the ovary nor the activity in plasma changed during the luteal phase or correlated to plasma progesterone. Injection of heparin at the height of the luteal cycle (Day 8) caused a pronounced decrease in luteal lipase and in plasma progesterone. These studies suggest that the H-type lipase activity has an important role in luteal steroidogenesis, probably to facilitate uptake of cholesterol from lipoproteins.

Human hepatic triglyceride lipase (HTGL), purified from plasma obtained after heparin injection, markedly enhanced the anti-Xa clotting activity of normal plasma. This was shown to be caused by direct inhibition of factor Xa clotting activity by HTGL, although the amidolytic activity of factor Xa was unaffected. Preincubation of factor Xa with CaCl2 and phospholipid reduced the rate of inhibition of HTGL, indicating that phospholipid-binding sites may be involved. Heparin, and low-affinity heparin, reduced the anti-Xa activity of HTGL, suggesting that heparin and factor Xa compete for the same binding sites on the lipase molecule. These results suggest that at least part of the enhanced anti-Xa clotting activity observed after injection of heparin and heparin analogues is caused by the release of HTGL. This release could contribute toward the anticoagulant and antithrombotic actions of these drugs.

Heparin can dissociate lipoprotein lipase from casein micelles, and addition of heparin enhances lipolysis in bovine but not in caprine milk. Heparin shortened the lag-time for binding of lipoprotein lipase to milk fat globules and for lipolysis. Heparin counteracted the inhibitory effects of skim milk on binding of lipase and on lipolysis. Heparin stimulated lipolysis in all bovine milk samples when added before cooling and in spontaneously lipolytic milk samples also when added after cooling. Heparin enhanced lipolysis of isolated milk fat globules. Hence, its effect is not solely due to dissociation of lipoprotein lipase from the casein micelles. Cooling of goat milk caused more marked changes in the distribution of lipase than cooling of bovine milk; the fraction of added 125I-labeled lipase that bound to cream increased from about 8 to 60%. In addition, caprine skim milk caused less inhibition of lipolysis than bovine skim milk. These observations provide an explanation for the high degree of cold storage lipolysis in goat milk. Heparin had only small effects on the distribution of lipoprotein lipase in caprine milk, which explains why heparin has so little effect on lipolysis in caprine milk. The distribution of 35S-labeled heparin in bovine milk was studied. In warm milk less than 10% bound to the cream fraction, but when milk was cooled, binding of heparin to cream increased to 45%.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of lipoprotein lipase among cream, casein, and milk serum can be evaluated by addition of a trace amount of 125I-labeled lipoprotein lipase to milk. Radioactive lipase was distributed in parallel to endogenous lipase under several conditions. In some milk samples, binding of lipase to cream increased when the milk was cooled. Correlation was good between bound labeled lipase and degree of cold-induced lipolysis in corresponding milk samples. Binding of lipase to cream or to casein was not saturable by addition of two-to threefold more lipase than is normally present in milk. In milk with a relatively high fraction of lipase bound to cream, a correspondingly lower fraction was associated with casein, whereas the fraction of lipase in milk serum was similar in all milk samples. Cold-induced binding of lipoprotein lipase to cream was not fully reversed when the milk was warmed again. Heparin released lipase from casein and increased the amount of lipase bound to cream after cooling.

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 enzyme's 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.

To determine whether an apolipoprotein-free artificial triacylglycerol emulsion can substitute for VLDL in studying cholesterol ester-triacylglycerol exchange processes between triacylglycerol-rich lipoproteins and cholesterol ester-rich lipoproteins, we used Intralipid to modify human plasma LDL. Intralipid was incubated with LDL in the presence of lipoprotein-poor plasma (d greater than 1.21 g/ml) at 37 degrees C. Intralipid served as an acceptor for cholesterol ester and as a donor of triacylglycerol, modifying the low-density lipoproteins so that triacylglycerol became the major core lipid in the particle - the contribution of cholesterol ester to LDL mass decreased from 38% to 18%, while that of triacylglycerol increased from 4.9% to 26%. On lipolysis most added LDL triacylglycerol (59-72%) was hydrolyzed, resulting in a smaller particle than the "native' LDL particle with net loss of cholesterol ester. Incubation of LDL with the original Intralipid emulsion resulted in modified LDL with a high relative weight of phospholipid (27.7%). On removal of excess phospholipid from Intralipid and incubation of the resultant "washed' Intralipid with LDL, the relative weight of phospholipid in modified LDL decreased to 20%, which was similar to that observed after incubation of LDL with VLDL. We demonstrate that artificial triacylglycerol emulsion can indeed substitute for VLDL in neutral lipid exchange processes, and further confirm that transfer of core cholesterol ester and triacylglycerol occurs independently of the apolipoproteins present in triacylglycerol-rich lipoproteins and LDL.

A structural homology between lipoprotein lipase, pancreatic lipase and hepatic lipase is known and indicates that all three lipases are members of a common protein family. Lipoprotein lipase and pancreatic lipase utilize small protein co-factors, apolipoprotein C-II and co-lipase, respectively, but comparisons reveal no homology between the co-factor molecules. Hence, they do not show the same relationship as their target enzymes. Neither do screenings detect any extensive similarities between lipoprotein lipase, serine hydrolases, or apolipoproteins. Scannings against data bank proteins show that a 105-residue segment of lipoprotein lipases exhibits a 35-40% residue identity with a sub-region of Drosophila vitellogenins. One fifth of the conserved amino acid residues (8 of 40) are glycine, a pattern which is typical of distantly related forms of protein families. This supports a true relationship between large segments of Drosophila vitellogenins and lipases. Physiological and functional aspects of the vitellogenin/lipoprotein lipase similarities are given. The region concerned is entirely within the N-terminal domain of lipoprotein lipase and constitutes the segment where the similarity to hepatic and pancreatic lipases is most pronounced. Within this lipase region a 10-residue putative lipid-binding site exists for which further similarities have been found to the otherwise not closely related lingual/gastric lipases, prokaryotic lipases and lecithin-cholesterol acyltransferase. Another segment in lipoprotein lipase, where the heparin-binding site has been mapped, exhibits a correlation between strength of heparin binding and extent of basic residues among members of the lipase family. It further exhibits weak similarities with the 'Zn-finger' DNA-binding segment of steroid hormone receptors and may indicate convergence in a binding interaction. Thus, a functional subdivision of lipoprotein lipase into different segments can be distinguished.

Methods to quantitate factors in milk relevant to cold-induced lipolysis are described. Skim milk was incubated with milk fat globules isolated from a pool of normal milk and a fixed amount of purified lipoprotein lipase. The release of fatty acids in 24 h at 4 degrees C was determined. Most skim milk samples inhibited lipolysis, but the effect varied greatly. Skim milk from milk prone to spontaneous lipolysis was less inhibitory than skim milk from normal milk. In general, both the casein and the serum fractions of skim milk inhibited lipolysis. However, variation was greater in the effects of individual samples of milk serum. In a few extreme cases, with samples from milk with spontaneous lipolysis, the serum fraction actually stimulated lipolysis. Globules were isolated and then incubated with skim milk from normal milk and a fixed amount of purified lipoprotein lipase. This gave a measure of accessibility to lipolysis of milk fat globules in normal skim milk. There was a considerable variation in propensity toward lipolysis between milk fat globules from individual milk samples. Milk showing different levels of lipolysis obtained from five cows revealed that skim milk inhibition of lipolysis and the propensity of milk fat globules toward lipolysis were characteristic for each cow.

Hydrolysis by lipoprotein lipase in capillaries of the adipose tissue is the step that determines the rate at which fatty acids from lipoprotein triacylglycerols are transported into the tissue for storage. Like many other key enzymes, adipose tissue lipoprotein lipase can be regulated both in amount and in activity. The amount of active lipoprotein lipase at the capillary endothelium is under hormonal control. The enzyme molecule itself carries several functional sites. Three of these sites, for interaction with lipid interfaces, with activator protein, and with fatty acids, regulate the action of the enzyme's active site. Another, independent, site on the molecule anchors it to cell surface heparan sulfate chains and thus holds it in place at the endothelium. Together these properties allow for a fine-tuned regulation of the lipoprotein lipase reaction so that lipids are transferred into the adipose tissue at the right rate, at the right time, and in the right place.

Extraction of membrane proteins from milk fat globules by GuHCl or by MgCl2 made the lipids more accessible to lipolysis by added lipoprotein lipase. The increase in lipolysis paralleled the loss of membrane proteins and was continuous up to 2.5 M GuHCl, which was the highest concentration used. About twice as much protein was extracted with 2.5 M GuHCl as with buffer only. The amount of protein lost was about 50% of total milk fat globule protein. Lipolysis of milk fat globules was inhibited by addition of the extracted protein. The extracted proteins also reduced lipolysis when added to whole milk. More protein was needed to inhibit lipolysis of milk fat globules treated with GuHCl compared with globules treated with buffer only. The inhibition by a given amount of protein decreased if more milk fat globules were used. Protein extracted with MgCl2 had similar effects as those extracted with GuHCl. The major components extracted with MgCl2 migrated in the 40 to 50-kdalton region on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration chromatography, two protein fractions were obtained, which inhibited lipolysis more efficiently than the total extract. As has previously been found for inhibition of lipolysis by skim milk, the amount of extracted protein needed to inhibit lipolysis varied between preparations of milk fat globules. Milk with propensity to cold-induced ("spontaneous") lipolysis was normalized by addition of extracted proteins.