Expression of lipoprotein lipase gene in combined lipase deficiency

The expression of the gene for lipoprotein lipase (LPL) was studied in brown adipose tissue and the liver of combined lipase deficient (cld/cld) and unaffected mice. The mRNA specific for LPL was detected in both animals. Although the size of LPL mRNA in cld mice was similar to that of unaffected mice, the mRNA concentration in affected animals was higher than in unaffected animals. We also studied the LPL gene mutation in cld mice by Southern blot analysis. No restriction fragment length polymorphisms were observed after digestion with 16 endonucleases. These data indicate that there is no gene insertion or deletion, but do not exclude the possibility of point mutation in the LPL structural gene. However, the present results agree with the hypothesis that the genetic defect in cld is not due to a mutation in the LPL structural gene, but instead involves the defective post-translational processing of LPL or defective cellular function affecting transport and secretion of this enzyme group.     

Appraisal of hepatic lipase and lipoprotein lipase activities in mice

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.     

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.     

Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family.

A large family is reported with familial hepatic triglyceride lipase (HTGL) deficiency and with the coexistence of reduced lipoprotein lipase (LPL) similar to the heterozygote state of LPL deficiency. The proband was initially detected because of hypertriglyceridemia and chylomicronemia. He was later demonstrated to have beta-VLDL despite an apo E3/E3 phenotype and the lack of stigmata of type III hyperlipoproteinemia. The proband had no HTGL activity in postheparin plasma. Two of his half-sisters had very low HTGL activity (39 and 31 nmol free fatty acids/min/ml; normal adult female greater than 44). His son and daughters had decreased HTGL activity (normal male and preadolescent female greater than 102), which would be expected in obligate heterozygotes for HTGL deficiency. Low HTGL activity was associated with LDL particles which were larger and more buoyant. Several family members, including the proband, had reduced LPL activity and mass less than that circumscribed by the 95% confidence-interval ellipse for normal subjects and had hyperlipidemia similar to that described in heterozygote relatives of patients with LPL deficiency. All the sibs with hyperlipidemia had a reduced LPL activity and mass, while subjects with isolated reduced HTGL (with normal LPL activity) had normal lipid phenotypes. Analysis of genomic DNA from these subjects by restriction-enzyme digestion revealed no major abnormalities in the structure of either the HTGL or the LPL gene. Compound heterozygotes for HTGL and LPL deficiency show lipoprotein physiological characteristics typical for HTGL deficiency, while their variable lipid phenotype is typical for LPL deficiency.     

Linkage of low-density lipoprotein size to the lipoprotein lipase gene in heterozygous lipoprotein lipase deficiency.

Small low-density lipoprotein (LDL) particles are a genetically influenced coronary disease risk factor. Lipoprotein lipase (LpL) is a rate-limiting enzyme in the formation of LDL particles. The current study examined genetic linkage of LDL particle size to the LpL gene in five families with structural mutations in the LpL gene. LDL particle size was smaller among the heterozygous subjects, compared with controls. Among heterozygous subjects, 44% were classified as affected by LDL subclass phenotype B, compared with 8% of normal family members. Plasma triglyceride levels were significantly higher, and high-density lipoprotein cholesterol (HDL-C) levels were lower, in heterozygous subjects, compared with normal subjects, after age and sex adjustment. A highly significant LOD score of 6.24 at straight theta=0 was obtained for linkage of LDL particle size to the LpL gene, after adjustment of LDL particle size for within-genotype variance resulting from triglyceride and HDL-C. Failure to adjust for this variance led to only a modest positive LOD score of 1.54 at straight theta=0. Classifying small LDL particles as a qualitative trait (LDL subclass phenotype B) provided only suggestive evidence for linkage to the LpL gene (LOD=1. 65 at straight theta=0). Thus, use of the quantitative trait adjusted for within-genotype variance, resulting from physiologic covariates, was crucial for detection of significant evidence of linkage in this study. These results indicate that heterozygous LpL deficiency may be one cause of small LDL particles and may provide a potential mechanism for the increase in coronary disease seen in heterozygous LpL deficiency. This study also demonstrates a successful strategy of genotypic specific adjustment of complex traits in mapping a quantitative trait locus.     

Compound heterozygosity for mutant hepatic lipase in familial hepatic lipase deficiency

In a kindred with three hyperlipidemic subjects who had premature atherosclerosis and complete deficiency of hepatic lipase activity, we had previously identified a novel structural hepatic lipase gene variant. We now report the identification of three more hepatic lipase gene mutations in this family and demonstrate that compound heterozygosity for two hepatic lipase mutations (designated S267F and T383M) underlies hepatic lipase deficiency.     

Molecular genetics of human lipoprotein lipase deficiency

Lipoprotein lipase (LPL) hydrolysis the triglyceride core of circulating chylomicrons and very-low-density lipoprotein, and modulates the levels and lipid composition of low and high density lipoproteins. Worldwide, more than 20 mutations in the LPL gene have been identified in patients with familial LPL deficiency. Most of these mutations are clustered in the region encoded by exons 4, 5 and 6 which forms the proposed catalytic domain of LPL. In French Canadians who have the highest reported frequency for LPL deficiency, three common mutations in the LPL gene have been identified which account for approximately 97% of mutant genes in this group. Simple DNA-based tests for the detection of all these mutations have been developed for the screening for carriers of LPL deficiency. This will facilitate further studies of phenotypic expression in heterozygous carriers and assessment of the risk of atherosclerosis in these individuals.     

Function of hepatic triglyceride lipase in lipoprotein metabolism

Rat hepatic triglyceride lipase (H-TGL) was purified from liver tissue extracts by affinity chromatography on Sepharose 4B with covalently linked heparin. The purified rat H-TGL exhibited the properties previously described for this enzyme. Enzyme protein was injected into rabbits for anti-H-TGL antibody production. Antirat-H-TGL did not react against rat lipoprotein lipase (LPL) but inhibited H-TGL-activity both in vitro and in vivo greater than 90%. These antibodies were injected into rats and lipoprotein analyses were performed over a 36-hr period. It could be shown that inactivation of H-TGL by anti-H-TGL gamma-globulins in vivo led to an increase in total triglyceride concentration from 70 mg/dl to 230 mg/dl due to an increase in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) triglycerides 4 hr after antibody injection; a marked increase in high density lipoprotein (HDL) phospholipid concentration was observed with almost no change in HDL-cholesterol and HDL-triglycerides. This study documents the ability of antirat-H-TGL-gamma-globulins to inhibit H-TGL in vitro and in vivo. Furthermore, the inhibition of triglyceride removal in vivo demonstrated that this enzyme together with LPL is responsible for the catabolism of VLDL-triglyceride.     

Lipoprotein metabolism in hepatic lipase deficiency: studies on the turnover of apolipoprotein B and on the effect of hepatic lipase on high density lipoprotein

Hepatic lipase deficiency produces significant distortion in the plasma lipoprotein profile. Particles with reduced electrophoretic mobility appear in very low density lipoprotein (VLDL). Intermediate density lipoprotein (IDL) increases markedly in the circulation and plasma low density lipoprotein (LDL) levels fall. At the same time there is a mass redistribution within the high density lipoprotein (HDL) spectrum leading to dominance in the less dense HDL2 subfraction. The present study examines apolipoprotein B turnover in a patient with hepatic lipase deficiency. The metabolism of large and small very low density lipoproteins was determined in four control subjects and compared to the pattern seen in the patient. Absence of the enzyme did not affect the rate at which large very low density lipoproteins were converted to smaller particles within this density interval (i.e., of VLDL). However, subsequent transfer of small very low density lipoproteins to intermediate density particles was retarded by 50%, explaining the abnormal accumulation of VLDL in the patient's plasma. Despite this, intermediate density particles accumulated to a level 2.4-times normal because their subsequent conversion to low density lipoprotein has been almost totally inhibited. Consequently, the plasma concentration of low density lipoprotein was only 10% of normal. On the basis of these observations, hepatic lipase appears to be essential for the conversion of small very low density and intermediate density particles to low density lipoproteins. The pathways of direct plasma catabolism of these species were not affected by the enzyme defect. In vitro studies were performed by adding purified hepatic lipase to the patient's plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a lipoprotein lipase cofactor-binding site by chemical cross-linking and transfer of apolipoprotein C-II-responsive lipolysis from lipoprotein lipase to hepatic lipase

To localize the regions of lipoprotein lipase (LPL) that are responsive to activation by apoC-II, an apoC-II peptide fragment was cross-linked to bovine LPL. Following chemical hydrolysis and peptide separation, a specific fragment of LPL (residues 65-86) was identified to interact with apoC-II. The fragment contains regions of amino acid sequence dissimilarity compared with hepatic lipase (HL), a member of the same gene family that is not responsive to apoC-II. Using site-directed mutagenesis, two sets of chimeras were created in which the two regions of human LPL (residues 65-68 and 73-79) were exchanged with the corresponding human HL sequences. The chimeras consisted of an HL backbone with the suspected LPL regions replacing the corresponding HL sequences either individually (HLLPL-(65-68) and HLLPL-(73-79)) or together (HLLPLD). Similarly, LPL chimeras were created in which the candidate regions were replaced with the corresponding HL sequences (LPLHL-(77-80), LPLHL-(85-91), and LPLHLD). Using a synthetic triolein substrate, the lipase activity of the purified enzymes was measured in the presence and absence of apoC-II. Addition of apoC-II to HLLPL-(65-68) and HLLPL-(73-79) did not significantly alter their enzyme activity. However, the activity of HLLPLD increased approximately 5-fold in the presence of apoC-II compared with an increase in native LPL activity of approximately 11-fold. Addition of apoC-II to LPLHL-(77-80) resulted in approximately 10-fold activation, whereas only approximately 6- and approximately 4-fold activation of enzyme activity was observed in LPLHL-(85-91) and LPLHLD, respectively. In summary, our results have identified 11 amino acid residues in the N-terminal domain of LPL (residues 65-68 and 73-79) that appear to act cooperatively to enable substantial activation of human LPL by apoC-II.     

Effects of heparin infusion on plasma lipoproteins in subjects with lipoprotein lipase deficiency: Evidence for a role of hepatic endothelial lipase in the metabolism of high-density lipoprotein subfractions in man

The role of heparin-releasable hepatic endothelial lipase (HL) in human plasma lipoprotein metabolism was investigated by examining the effects of intravenous infusion of heparin (180 units/kg over 2 h) in 8 subjects with primary extrahepatic lipoprotein lipase deficiency. In addition to reducing the triglyceride concentration in very low-density lipoproteins, heparin-induced release of HL reduced the phopholipid and protein concentrations in the HDL₂ subclass of high-density lipoprotein (by 28% and 36% respectively, mean values) and simultaneously increased the HDL₃ phospholipid concentration (by 23%), providing the first in vivo evidence for a function of HL in the interconversion of the major HDL subfractions in man.     

Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase. Reactivity with monoclonal antibodies to hepatic triglyceride lipase

Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.     

Hypertriglyceridemia associated with decreased post-heparin plasma hepatic triglyceride lipase activity in hypoxic rats

Exposure of sated rats to 45% N2 in air for 5h increased serum triglyceride levels by 212% over the levels in normoxic rats. This increase in triglyceride levels was accompanied by a decrease in plasma triglyceride hydrolase activity after intravenous injection of heparin. Further fractionation of the activity by inhibition of lipoprotein lipase indicated that the low triglyceride hydrolase activity is mainly due to a reduction in hepatic triglyceride lipase, which is inversely correlated with the serum triglyceride level. The hypoxic exposure decreased the arterial blood [acetoacetate]/[beta-hydroxybutyrate] ratio in the sated rats, which is believed to reflect the oxidation-reduction state in hepatic mitochondria, but did not affect the level of serum enzymes indicative of tissue damage. On the other hand, triglyceride levels did not change during hypoxic exposure in fasted rats. Thus, hypertriglyceridemia in sated rats following exposure to hypoxia may result from impaired removal of circulating triglycerides by hepatic triglyceride lipase located in the sinusoidal surface of the liver.     

Relationship of decreased hepatic lipase activity and lipoprotein abnormalities to essential fatty acid deficiency in cystic fibrosis patients

Polyunsaturated fatty acids are known to affect plasma lipids and lipoproteins but there is no information on the effect of essential fatty acid (EFA) deficiency on lipoprotein composition. The purpose of this study was to characterize lipoproteins from 17 cystic fibrosis (CF) patients in relationship to their EFA status (eicosatrienoic/arachidonic acid ratio) and compare them with those of 10 healthy siblings (SIB) and of 10 unrelated controls. In 7 EFA-deficient (EFAD) and 10 EFA-sufficient (EFAS) patients, hypocholesterolemia was associated with a decrease of HDL-cholesterol and of LDL-cholesterol which was more marked in the EFAD group. Similarly, although triglyceride enrichment of VLDL, LDL, HDL2, and HDL3 with a concomitant reduction of cholesteryl esters from all particles except HDL2 was observed in both CF groups, it was more sizable in the EFAD patients. These changes led to an increase in the particle size of VLDL, LDL, and HDL2 whereas the distribution of HDL3 was skewed to smaller particles. Alterations in the apoprotein composition of particles were greater in EFAD than in EFAS. A decrease of total postheparin lipolytic activity was observed in the two groups of CF patients as well as in siblings. It was entirely accounted for by hepatic lipase (mumol FFA/ml per h) which was more severely diminished in EFAD (2.8 +/- 0.6) than in EFAS (4.4 +/- 0.7) and SIB (5.1 +/- 0.5). Although the two groups of CF children differed in terms of growth, severity of malabsorption, and vitamin E status, these data suggest that disturbance of lipoprotein concentration, composition, size, and metabolism (hepatic lipase) may be in part related to EFA deficiency. Further studies are necessary to explore the effect of EFA deficiency on hepatic lipase activity.     

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.     

Effects of threonine-poor apolipoproteins on post-heparin plasma hepatic triacylglycerol lipase and lipoprotein lipase

Whole-irradiated rabbit pre-heparin plasma had an important inhibitory effect on hepatic triacylglycerol lipase and lipoprotein lipase activities, whereas control rabbit pre-heparin plasma slightly inhibited hepatic triacylglycerol lipase activity at a high concentration and enhanced lipoprotein lipase activity. As some apolipoproteins were known to modulate these two lipolytic enzymes, the inhibitory effects of irradiated rabbit plasma were investigated in apolipoproteins. Three apolipoproteins, with isoelectric points of about 6.58, 6.44 and 6.12, characterized by their low content in threonine (threonine-poor apolipoproteins) were produced in high concentrations in rabbit VLDL and HDL after irradiation. The effects of these apolipoproteins on control rabbit post-heparin plasma hepatic triacylglycerol lipase and extrahepatic lipoprotein lipase were studied. Threonine-poor apolipoproteins substantially inhibited the hepatic triacylglycerol lipase activity and enhanced the apolipoprotein C-II-stimulated activity of lipoprotein lipase. The amounts of these apolipoproteins in triacylglycerol-rich lipoprotein particles may determine the lipolytic activity of lipoprotein lipase and hepatic triacylglycerol lipase in triacylglycerol hydrolysis. The existence of another inhibitor of lipoprotein lipase remains to be determined.     

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

Measurement of lipoprotein lipase and hepatic triacylglycerol lipase in post-heparin plasma of the cynomolgus monkey

Conditions for measurement of the lipolytic activities, lipoprotein lipase and hepatic triacylglycerol lipase in cynomolgus monkey postheparin plasma are described. The two activities are separable by heparin-Sepharose chromatography. Goat anti-human hepatic triacylglycerol lipase serum inhibits monkey hepatic triacylglycerol lipase activity and allows direct measurement of lipoprotein lipase in post-heparin plasma. While both human and homologous serum can be used as a source of activator apolipoprotein, homologous serum produces a much greater activation.