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A cDNA clone encoding lipoprotein lipase has been isolated from an ovine adipocyte library. Sequencing of this clone has revealed a single open reading frame encoding a 450 amino acid protein. Comparison with known LPL sequences from other species shows a high degree of conservation in the sequence of the protein and in the 5' untranslated region of the DNA sequence.
BACKGROUND: Lipoprotein lipase (LPL) is anchored at the vascular endothelium through interaction with heparan sulfate. It is not known how this enzyme is turned over but it has been suggested that it is slowly released into blood and then taken up and degraded in the liver. Heparin releases the enzyme into the circulating blood. Several lines of evidence indicate that this leads to accelerated flux of LPL to the liver and a temporary depletion of the enzyme in peripheral tissues. RESULTS: Rat livers were found to contain substantial amounts of LPL, most of which was catalytically inactive. After injection of heparin, LPL mass in liver increased for at least an hour. LPL activity also increased, but not in proportion to mass, indicating that the lipase soon lost its activity after being bound/taken up in the liver. To further study the uptake, bovine LPL was labeled with 125I and injected. Already two min after injection about 33 % of the injected lipase was in the liver where it initially located along sinusoids. With time the immunostaining shifted to the hepatocytes, became granular and then faded, indicating internalization and degradation. When heparin was injected before the lipase, the initial immunostaining along sinusoids was weaker, whereas staining over Kupffer cells was enhanced. When the lipase was converted to inactive before injection, the fraction taken up in the liver increased and the lipase located mainly to the Kupffer cells. CONCLUSIONS: This study shows that there are heparin-insensitive binding sites for LPL on both hepatocytes and Kupffer cells. The latter may be the same sites as those that mediate uptake of inactive LPL. The results support the hypothesis that turnover of endothelial LPL occurs in part by transport to and degradation in the liver, and that this transport is accelerated after injection of heparin.
Complete deficiency of lipoprotein lipase (LPL) causes the chylomicronemia syndrome. To understand the molecular basis of LPL deficiency, two siblings with drastically reduced postheparin plasma lipolytic activities were selected for analysis of their LPL gene. We used the polymerase chain reaction to examine the nine coding LPL exons in the two affected siblings and three relatives. DNA sequence analysis revealed a single nucleotide change compared with the normal LPL cDNA: a G----A substitution at nucleotide position 680. This transition caused a replacement of glutamic acid for glycine at amino acid residue 142 of the mature LPL protein. Amino acid sequence comparisons of the region surrounding glycine-142 indicated that it is highly conserved among lipases from different species, suggesting a crucial role of this domain for the LPL structure. Expression studies of the mutant LPL cDNA in COS-7 cells produced normal amounts of enzyme mass. However, the mutated LPL was not catalytically active, nor was it efficiently secreted from the cells. This established that the Gly----Glu substitution at amino acid 142 is sufficient to abolish enzymatic activity and to result in the chylomicronemia syndrome observed in these patients.
A rare case of a 3 month old child with lipoprotein lipase deficiency who presented with bronchopneumonia is reported. After noticing lipaemic serum and lipaemia retinalis, a diagnosis of hyperlipoproteinaemia was considered. Lipoprotein lipase deficiency was confirmed with post heparin lipoprotein lipase enzyme activity estimation.
1. The lipoprotein triglycerides of human plasma can be separated by gas chromatography according to their carbon number into four main groups: C48, C50, C52 and C54. We studied the decomposition of molecular types of triglycerides by posttheparin lipoprotein lipase in vitro. 2. Decomposition of the molecular type C52 does not depend on its concentration. Decomposition of the molecular types C48, C50 and C54 is in a direct correlation to their concentration. 3. The above mechanism assures stability of the triglyceride profile of human plasma, which is species specific and is expressed by the figures: C48--9%, C50--20%, C52--43%, C54--20%, remainder 8%. 4. The plasma triglyceride profile can be altered by dietetic factors. After drinking cream, the triglyceride composition is: C48--12%, C50--22%, C52--37%, C54--14%, remainder 15%. Selective decomposition of molecular types of triglycerides ensures that the profile, after a given time, returns to the original state.
Lipoprotein lipase (LPL) plays a role in lipid usage in skeletal muscle by hydrolyzing plasma triglycerides into fatty acids, which are further utilized for beta-oxidation. Lipid usage is stimulated during fasting, diabetes mellitus and exercise, concomitant with enhanced LPL expression in skeletal muscle. Here we show that the forkhead type transcription factor FKHR is strongly induced in skeletal muscle in fasting mice, in mice with streptozotocin-induced diabetes and in mice after treadmill running. Ectopic expression of FKHR enhanced LPL gene expression in C2C12 muscle cells in culture. These results implicate FKHR as an important modulator of lipid metabolism in skeletal muscle.
The lipoprotein lipase (LPL) activity obtained from the intrapulmonary administration of 2-10 mg of heparin in mice was compared with the same parameter measured for intravenously administered heparin. The doses administered were based on appropriate clinical equivalents. A relatively large dose of intrapulmonary heparin produced a peak LPL activity which was a third of the maximum response obtained from a small dose of i.v. heparin. This was followed by a moderate LPL activity (twice the control level) which persisted for the next 4 days while the response obtained from i.v. administered heparin lasted only 2 h. Both the intrapulmonary and the i.v. administration of heparin produced dose-dependent increases in plasma LPL activity (correlation coefficient r = 0.9). This study indicates that intrapulmonary heparin causes a prolonged antilipemic effect.
Lipoprotein lipase (LPL) was obtained from rat postheparin plasma by chromatographies on heparin-Sepharose and hydroxyapatite. The enzyme was associated with fatty acid ethyl ester synthase (FAEE synthase) as judged by their co-elution profiles and identical profiles of inhibition by diisopropyl fluorophosphate. Only one polypeptide of molecular weight 57,000 in purified LPL fraction was labeled by affinity labeling with [3H]-diisopropyl fluorophosphate. The FAEE synthase activity of LPL was not affected by addition of apolipoprotein C-II. Digestion of the enzyme with trypsin resulted in almost complete loss of the triolein-hydrolyzing activity without change in FAEE synthase activity. The tributyrin-hydrolyzing activity of LPL was also not affected by addition of apolipoprotein C-II or trypsin digestion. On addition at progressively higher concentrations, bovine serum albumin increased FAEE synthesis to a maximum at 2 mg/ml and at higher concentrations inhibited its activity. On incubation of purified LPL with chylomicrons in an ethanol/water mixture, FAEE was formed in the presence of a high concentration of bovine serum albumin. The specific activity of FAEE synthesis from chylomicrons was about 65 times that from oleic acid. Triolein/gum arabic emulsion was used for identification of reaction products. We propose the following mechanism of FAEE formation from chylomicrons by LPL. The enzyme attacks chylomicrons forming an acyl-enzyme intermediate, and during the deacylation process, ethanol binds to fatty acids as an acceptor. These results suggest that LPL contributes to nonoxidative ethanol metabolism (FAEE formation) through degradation of triglyceride-rich lipoproteins such as chylomicrons.
Intravenous injections of anti-lipoprotein lipase serunis quantitatively block the catabolism of very low density lipoprotein (VLDL) and portomicron triglyceride and specifically inhibit triglyceride transport into ovarian follicles. The immunological studies presented provide information on the site of action of lipoprotein lipase (LPL). In the anti-LPL serum-treated animals initial plasma triglyceride accumulation occurs at the time of antiserum injection. This instantaneous inhibition of triglyceride removal provides direct evidence that the functional LPL responsible for VLDL and portomicron triglyceride hydrolysis is located in sites within the plasma compartment readily accessible to immunoglobulins. In vitro immunological studies show that the adipose, heart, ovarian, and liver LPL share common immunological determinants. Biochemical studies on highly purified heart and adipose LPL suggest that these enzymes have identical protein moieties.
Lipoprotein lipase (LPL) is known to be an early marker of adipose cell differentiation. Growth hormone (GH) stimulates in preadipose Ob1771 cells the expression of LPL gene and this effect is mediated at least in part by c-fos protooncogene, the expression of which is transiently activated by a protein kinase C-dependent pathway (Barcellini-Couget et al., Endocrinology, 1993, 132: 55-60). Since GH stimulates the formation of diacylglycerol from phosphatidylcholine independently of Ca2+ mobilization, the role of Ca2+ was studied in regard to LPL gene expression stimulated by GH. The results obtained in the presence of Ca2+ ionophores show that a rise in intracellular free Ca2+ abolishes this expression but has no effect on the expression of various adipose-related genes, including the transient expression of c-fos protooncogene. Therefore, the inability of preadipose cells to mobilize Ca2+ in response to GH, at an early stage of the differentiation process, appears as a prerequisite for the maximal expression of LPL.
OBJECTIVE: The purpose of this study was to evaluate the regulation of lipoprotein lipase activity, protein mass, and messenger ribonucleic acid by estradiol. STUDY DESIGN: Premenopausal women not taking exogenous sex steroids had transdermal 17 beta-estradiol and placebo patches placed in the gluteal region during the early follicular phase of the menstrual cycle. Adipose biopsies were performed from beneath the patches. Adipose tissue lipoprotein lipase activity was determined by a radiometric assay, protein mass was determined by enzyme-linked immunosorbent assay, and messenger ribonucleic acid level was determined by Northern analysis. Comparisons between the treated and placebo sides were analyzed by nonparametric statistics. RESULTS: Adipose tissue from beneath the 17 beta-estradiol patch had significantly decreased lipoprotein lipase activity and extracellular protein mass than did adipose tissue from beneath the placebo patch. There was no difference in lipoprotein lipase messenger ribonucleic acid levels. CONCLUSION: Estrogen decreases lipoprotein lipase activity by a posttranscriptional modification of protein levels. A hypothesis of sex steroid regulation of body fat distribution is proposed.
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.
We examined the lipoprotein lipase (LPL) gene by single strand conformation polymorphism (SSCP) and by restriction fragment length polymorphism (RFLP) analysis in 106 patients with hypertriglyceridemia to screen for novel mutations and to study the contribution of LPL genetic defects in hypertriglyceridemia. We found a single incidence of a homozygous novel nonsense mutation (216G-->A; -14Tryptophan-->stop codon) in exon 1 and 6 cases heterozygous for a single transition (C-->T) at six bp upstream from splicing acceptor site of intron 3. These mutations were not found in 105 normolipidemic controls. The proband homozygous for the nonsense mutation in exon 1, a 74 year old woman, had mild hyperchylomicronemia and her post-heparin plasma showed no LPL protein. However, four heterozygous among family members did not demonstrate hypertriglyceridemia. The frequency of heterozygosity for the C-->T transition in intron 3 was significantly different from that in normolipidemic controls. Therefore, it was suggested that the mutation is involved in hypertriglyceridemia. All of the heterozygotes were men with 4 patients having impaired glucose tolerance or diabetes mellitus. These observations suggest that this polymorphism in intron 3 combined with other as yet undefined factors may be related to hypertriglyceridemia.
The effects of species and plane of nutrition on serum activation of sheep adipose tissue lipoprotein lipase were studied over a range of substrate (triolein) concentrations. Serum, either from two species or from the same species on a different plane of nutrition, had differing effects on adipose tissue lipoprotein lipase activity. Serum from fed sheep was more effective than serum from fed rats in activating sheep adipose tissue lipoprotein lipase at low substrate concentrations. Serum taken from sheep on a restricted plane of nutrition, stimulated adipose tissue lipoprotein lipase activity at physiological substrate concentrations. The increased activity promoted by the factor(s) present in serum would ensure that those tissues (e.g. cardiac and skeletal muscle) which continue to synthesize lipoprotein lipase during fasting or nutritional restriction, are able to assimilate the relatively low amounts of circulating triacylglycerol.
Apolipoprotein CII (apoCII) activates lipoprotein lipase (LPL). Seven residues, located on one face of a model alpha-helix spanning residues 59-75, are fully conserved in apoCII from ten different animal species. We have mutated these residues one by one. Substitution of Ala(59) by glycine, or Thr(62) and Gly(65) by alanine did not change the activation, indicating that these residues are outside the LPL-binding site. Replacement of Tyr(63), Ile(66), Asp(69), or Gln(70) by alanine lowered the affinity for LPL and the catalytic activity of the LPL-apoCII complex. For each residue several additional replacements were made. Most mutants retained some activating ability, but replacement of Tyr(63) by phenylalanine or tryptophan and Gln(70) by glutamate caused almost complete loss of activity. All mutants bound to liposomes with similar affinity as wild-type apoCII, and they also bound with similar affinity to LPL in the absence of hydrolyzable lipids. However, the inactive mutants did not compete with wild-type apoCII in the activation assay. Therefore, we conclude that the productive apoCII-LPL interaction may be dependent on substrate molecules. In summary, our data demonstrate that residues 63, 66, 69, and 70 are of special importance for the function of apoCII, but no single amino acid residue is absolutely crucial.
Lipoprotein lipase (LPL) is important for the delivery of triglyceride fatty acids (TGFA) to a variety of tissues. We have used measurements of heparin-releasable LPL activity, immunohistochemistry, in situ hybridization, and Northern analysis to more fully characterize the location of LPL within the entire central nervous system (CNS) of the rat. Surprisingly, the levels of LPL activity and mRNA in the caudal spinal cord were 5- to 10-times higher than those found in any other area of the brain, levels similar to those found in adipose tissue or skeletal muscle. A number of cell types including neurons in the hippocampus, Purkinje cells of the cerebellum, and cells deep within the cortex were identified as sources of LPL mRNA. LPL protein was found within many vascular structures throughout the CNS, and within Purkinje cells. The strongest immunostaining was around nerve rootlets associated with the caudal spinal cord. Feeding studies were carried out with [14C]oleic acid to see whether CNS LPL functioned in the uptake of TGFA. These studies demonstrated uptake of chylomicron triglyceride fatty acids throughout the CNS. The localization of LPL within these structures suggests that the uptake of triglyceride fatty acids is an integral part of normal lipid metabolism of the central nervous system and may be important in regulating feeding behavior and/or maintaining normal neuronal function.
Circadian rhythms of plasma lipids and lipoproteins, lipoprotein lipase activities and VLDL secretion rates were studied in fed and food-deprived (12 h) male rats after a light/dark synchronization of 14 days. In ad libitum fed rats, a circadian rhythm of plasma triacylglycerol, blood glucose and liver glycogen was clearly identified. A rhythm was also identified for plasma cholesterol, but not phospholipids. The peak of plasma triacylglycerol occurred 2 h after the beginning of the light period (7.00 a.m.), and the nadir, 2 h after the beginning of the dark period (7.00 p.m.). The differences of plasma triacylglycerol at these two circadian stages were even more pronounced in food-deprived rats and were confined to the very-low-density lipoprotein (VLDL) fraction. Plasma post-heparin and heart and muscle lipoprotein lipase activities were 50-100% higher at 7.00 p.m., the time when plasma triacylglycerol were lowest, as compared to 7.00 a.m. Plasma post-heparin hepatic lipase and adipose tissue lipoprotein lipase activities, in contrast, did not change. VLDL secretion rates were somewhat higher at 7.00 a.m. compared to 7.00 p.m., but this difference was not significant. It is concluded that physiological variation of heart and muscle lipoprotein lipase together with small differences of VLDL secretion rates are responsible for normal range oscillations of plasma VLDL triacylglycerol levels.
Increased activity of lipoprotein lipase (LPL) may explain the hypotriglyceridemic effects of fibrates, thiazolidinediones and fatty acids, which are known activators (and/or ligands) of the various peroxisome proliferator-activated receptors (PPARs). Treatment with compounds which activate preferentially PPARalpha, such as fenofibrate, induced LPL expression exclusively in rat liver. In contrast, the antidiabetic thiazolidinedione BRL 49653, a high affinity ligand for PPARgamma, had no effect on liver, but induced LPL expression in rat adipose tissue. In the hepatocyte cell line AML-12, fenofibric acid, but not BRL 49653, induced LPL mRNA, whereas in 3T3-L1 preadipocytes, the PPARgamma ligand induced LPL mRNA levels much quicker and to a higher extent than fenofibric acid. In both the in vivo and in vitro studies, inducibility by either PPARalpha or gamma activators, correlated with the tissue distribution of the respective PPARs: an adipocyte-restricted expression of PPARgamma, whereas PPARalpha was expressed predominantly in liver. A sequence element was identified in the human LPL promoter that mediates the functional responsiveness to fibrates and thiazolidinediones. Methylation interference and gel retardation assays demonstrated that a PPARalpha or gamma and the 9-cis retinoic acid receptor (RXR) heterodimers bind to this sequence -169 TGCCCTTTCCCCC -157. These data provide evidence that transcriptional activation of the LPL gene by fibrates and thiazolidinediones is mediated by PPAR-RXR heterodimers and contributes significantly to their hypotriglyceridemic effects in vivo. Whereas thiazolidinediones predominantly affect adipocyte LPL production through activation of PPARgamma, fibrates exert their effects mainly in the liver via activation of PPARalpha.