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PubMed1979

Lipoprotein lipase activity in human adipose tissue during induced hypertriglyceridaemia.

Needle biopsies of adipose tissue and blood samples were obtained before and at short intervals after a "bolus" injection of 10% Intralipid. Lipoprotein lipase activities were measured in acetone--ether extracts of the tissue samples. Levels of serum triglyceride began to fall less than 5 min after the injection of the Intralipid with a half-life of 20 min. During this time interval, no significant changes were observed in the activities of lipoprotein lipase in adipose tissue. A patient with a severe hypertriglyceridaemia (Type V) underwent plasma exchange with a reduction in serum triglyceride levels from 11 to 4 mm/l. There was a parallel fall in adipose tissue lipoprotein lipase activity. We conclude that lipoprotein lipase in adipose tissue is unaltered during experimental hypertriglyceridaemia and that the activity of the enzyme in adipose tissue is probably not reduced as a secondary feature of an elevated plasma triglyceride level.

Adipose Tissue↗

PubMedDate not supplied

Purification and characterization of lipoprotein lipase from human heart.

Human heart lipoprotein lipase was purified by affinity chromatography on heparin-Sepharose 4B. When crude extracts of heart acetone powder were applied to columsn, about 40% of total lipase activity was bound to the gel and then eluted with 1.5 M NaCl. At this stage the eluted enzyme was purified 1900-fold. Disc gel electrophoresis yielded a single protein band corresponding with lipolytic activity. Minimum molecular weight of the protein was 60,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme was highly unstable; however, its activity could be partially stabilized at --20C by bovine serum albumin, glycerol, or ethylene glycol. The activity of the purified enzyme (i) had a pH optimum between 7.8 and 8.0; (ii) required serum for full enzymatic activity; apoC-II could be substituted for serum; (iii) was inhibited by by apoC-I in the presence of activated substrate; (iv) was markedly inhibited by NaCl; and (v) was stimulated by heparin.

Apoenzymes↗

PubMed1993

Characterization and identification of a lipoprotein lipase from Manduca sexta flight muscle.

Lipoprotein lipase (LpL) activity in Manduca sexta flight muscle tissue was measured using in vivo radiolabeled lipophorin as a substrate. LpL hydrolyses diacylglycerol in the low density lipophorin (that occurs during flight) at a higher rate than diacylglycerol in the high density lipophorin (present in the resting insect). LpL has a pH-optimum of 7.5 and is less sensitive to NaCl than mammalian LpL. LpL is inhibited by bovine albumin and chicken ovalbumin. LpL is inhibited by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF), which indicates the presence of an active site serine similar to mammalian LpL. Flight muscle LpL shows affinity for immobilized copper as well as for immobilized heparin. Using radiolabeled DFP, a protein of 37 kDa was identified (after SDS-PAGE) as the DFP-binding protein in a partially purified preparation of LpL. This 37 kDa protein is proposed to be the LpL or a subunit thereof.

Animals↗

PubMedDate not supplied

Lipoprotein lipase immobilization onto porous chitosan beads.

Lipoprotein lipase (LPL) was covalently immobilized onto the surface of porous chitosan beads (ChB) without or with spacers of different lengths. The relative activity (RA) of the immobilized LPL was found to be high toward a small ester substrate, p-nitrophenyl laurate (pNPL). LPL immobilized with spacer gave an almost constant activity, in marked contrast with the immobilized LPL without spacer, whose activity decreased with decreasing surface concentration. Further, the RA of LPL immobilized with spacer was higher than that of LPL immobilized without a spacer. The pH, thermal, and storage stabilities of the immobilized LPL were higher than those of the free one. LPL immobilized directly to the surface of ChB without any spacer gave a higher stability than that immobilized with spacer, in spite of the lower RA. The spacer effect on RA could be explained in terms of the mobility of the immobilized LPL molecule.

Chitin↗

PubMed1987

Antibody against human lipoprotein lipase.

A polyclonal antibody against human lipoprotein lipase (LPL) was prepared. LPL from post-heparin plasma was first purified by heparin Sepharose 4B affinity chromatography. Protein impurities co-eluted with LPL were then eliminated by electrophoresis in the presence of ampholytes. Antithrombin III was identified in this fraction of protein impurities by immunodiffusion against a human antithrombin antiserum, while no antithrombin III could be detected in the purified LPL fraction. Immunodiffusion revealed a single line of precipitation between this antibody and human post-heparin plasma LPL. When pre-incubated with a constant activity of highly purified post-heparin plasma LPL (2.7 mU/75 microliters), an equal volume of the anti-LPL antiserum, either pure or diluted to 1/32 caused complete inhibition of the enzyme activity. Half maximal inhibition was observed at a dilution of approximately 1/200. By using a secondary antibody, it was shown that antiserum inhibited LPL activity by means of its immunoglobulins. This antibody was able to inhibit LPL from human adipose tissue, indicating that human LPL released from endothelial cell membranes has common antigenic determinants with adipose tissue LPL.

Adipose Tissue↗

PubMed1991

Pulse-chase study on lipoprotein lipase in perfused guinea pig heart.

Lipoprotein lipase (LPL) in guinea pig hearts was labeled by perfusion for 15 min with [35S] methionine, followed by chase perfusion with unlabeled medium for less than or equal to 90 min. Suitable conditions for pulse and chase and for immunoprecipitation of LPL were worked out. Differences in labeling between individual hearts were found to be substantial. A technique to take serial biopsies (approximately 20 mg) from the heart ventricle was therefore worked out and validated. Regional differences of LPL labeling within the ventricle were small. There was no evidence for enhanced release of LPL caused by biopsy. When heparin was present throughout the chase, labeled LPL began to appear in the medium after 30 min. Thus LPL can move rapidly from synthesis in parenchymal cells to the endothelial surface and into the medium. After 90 min chase with heparin, approximately equal amounts of LPL (approximately 32%) had been released to the medium, remained in the heart, or had been degraded, respectively. In contrast, no detectable amount of labeled LPL appeared in the medium during 90 min chase without heparin, and a heparin flush at this time brought out only approximately 9% of the pulse-labeled LPL.

Animals↗

PubMed2003

Calreticulin promotes folding/dimerization of human lipoprotein lipase expressed in insect cells (sf21).

Lipoprotein lipase (LPL) is a non-covalent, homodimeric, N-glycosylated enzyme important for metabolism of blood lipids. LPL is regulated by yet unknown post-translational events affecting the levels of active dimers. On co-expression of LPL with human molecular chaperones, we found that calreticulin had the most pronounced effects on LPL activity, but calnexin was also effective. Calreticulin caused a 9-fold increase in active LPL, amounting to about 50% of the expressed LPL protein. The total expression of LPL protein was increased less than 20%, and the secretion rates for active and inactive LPL were not significantly changed by the chaperone. Thus, the main effect was an increased specific activity of LPL both in cells and media. Chromatography on heparin-Sepharose and sucrose density gradient centrifugation demonstrated that most of the inactive LPL was monomeric and that calreticulin promoted formation of active dimers. Higher oligomers of inactive LPL were present in cell extracts, but only monomers and dimers were secreted to the medium. Interaction between LPL and calreticulin was demonstrated, and the effect of the chaperone was prevented by castanospermine, an inhibitor of N-glycan glucose trimming. Our data indicate an important role of endoplasmic reticulum-based chaperones for the folding/dimerization of LPL.

Animals↗

PubMed1983

The effect of the chain length of heparin on its interaction with lipoprotein lipase.

The interactions of bovine milk lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) with the glycosaminoglycans heparin and heparan sulphate were investigated using the technique of fluorescence polarization spectroscopy. The type of complex formed with the enzyme depends on the chain length of the heparin. In 0.05 M NaCl and when the heparin was in molar excess, one heparin chain of Mr 10000-18400 formed a very stable complex with the dimeric protein molecule (the 1:1 complex). With excess protein, weaker interactions produced complexes with higher molecular weights. These two classes of complex were also detected with shorter heparins (Mr 6600-8000), although in these circumstances the more stable complex possessed a heparin:protein dimer ratio of 2:1. In higher salt (0.2 M NaCl) and lower heparin concentrations (less than 6 . 10(-8) M) the weaker class of compound was undetectable and Kd values of 4 . 10(-8) M and 6 . 10(-9) M were assigned to the 2:1 and 1:1 complexes, respectively. Heparan sulphate of Mr 17000 could only form one class of complex. This had a 1:1 stoichiometry and with Kd values of 3 . 10(-8) M and 1.6 . 10(-7) M at 0.05 and 0.2 M NaCl, respectively. The results could be explained if there is a distinct binding region for glycosaminoglycans on each subunit of the dimeric enzyme and a single heparin chain of Mr greater than 10000 can satisfy both sites to form a 1:1 complex. Smaller heparin chains are unable to span the sites and, in order to occupy them, two chains must interact with each enzyme molecule.

Animals↗

PubMed2005

Lysophosphatidic acid-mediated augmentation of cardiomyocyte lipoprotein lipase involves actin cytoskeleton reorganization.

The lipoprotein lipase (LPL)-augmenting property of lysophosphatidylcholine requires the formation of lysophosphatidic acid (LPA) (J Mol Cell Cardiol 37: 931-938, 2004). Given that the actin cytoskeleton has been implicated in regulating cardiomyocyte LPL, we examined whether LPL secretion after LPA involves actin cytoskeleton reassembly. Incubation of myocytes with LPA (1-100 nM) increased basal and heparin-releasable LPL (HR-LPL), an effect that was independent of shifts in LPL mRNA. The influence of LPA on myocyte LPL was reflected at the coronary lumen, with substantial increases of the enzyme at this location. Incubation of myocytes with cytochalasin D not only blocked LPA-induced augmentation of HR-LPL but also abrogated filamentous actin formation. These effects of LPA were likely receptor mediated. Exposure of myocytes to LPA facilitated significant membrane translocation of RhoA and its downstream effector Rho kinase I (ROCK I), and blocking this effect with Y-27632 appreciably reduced basal and HR-LPL activity. Incubation of adipose tissue with LPA also significantly enhanced basal and HR-LPL activity, suggesting that sarcomeric actin likely has a limited role in influencing the LPL secretory function of LPA in the myocyte. Comparable to LPA, hyperglycemia also caused significant membrane translocation of RhoA and ROCK I in hearts isolated from diazoxide-treated animals, effects that were abrogated using insulin. Overall, our data suggest that comparable to hyperglycemia, LPA-induced increases in cardiac LPL occurred via posttranscriptional mechanisms and processes that likely required RhoA activation and actin polymerization. Whether this increase in LPL augments triglyceride deposition in the heart leading to eventual impairment in contractile function is currently unknown.

Actins↗

PubMed2005

Gene therapy for lipoprotein lipase deficiency: working toward clinical application.

Lipoprotein lipase (LPL) deficiency causes hypertriglyceridemia and recurrent, potentially life-threatening pancreatitis. There currently is no adequate treatment for this disease. Previously, we showed that intramuscular administration of an adeno-associated virus serotype 1 (AAV1) vector encoding the human LPL(S447X) variant cDNA (AAV1-LPL(S447X)) normalized the dyslipidemia of LPL-/- mice for more than 1 year. In preparation for a clinical trial, we evaluated the safety and biodistribution of AAV1-LPL(S447X) in wild-type mice and fully characterized six LPL-deficient patients. Toxicological analysis in mice showed that intramuscular administration was well tolerated. Acute inflammatory response markers were transiently increased, and anti- AAV1 antibodies were generated. Histological analyses indicated a dose-dependent reversible spleen hyperplasia, and myositis at the injection sites. Biodistribution data showed short-term vector leakage from injection sites into the circulation, followed by liver-mediated clearance. Persistence of vector DNA was limited to the injected muscle and draining lymph nodes, and spread to reproductive organs was limited. Characterization of LPL-deficient patients showed that all patients presented with hypertriglyceridemia and recurrent pancreatitis. LPL catalytic activity was absent, but LPL protein levels were 20-100% of normal. Myoblasts derived from skeletal muscle biopsies of these patients were efficiently transduced by AAV1-LPL(S447X) and secreted active LPL. These data support the initiation of a clinical trial in LPL-deficient patients, for which regulatory approval has been granted.

Animals↗

PubMed1996

Multiple regulatory steps are involved in the control of lipoprotein lipase activity in brown adipose tissue.

Lipoprotein lipase (LPL) supplies brown adipose tissue with fatty acids for nonshivering thermogenesis. In brown adipose tissue of the Djungarian hamster we studied i) the molecular mechanisms involved in cold-induced stimulation of LPL activity, ii) the adrenergic control of LPL expression, and iii) compared LPL expression in brown and white adipose tissue. i) After 8 h cold exposure we detected a 2-fold increase in LPL activity and protein level in brown adipose tissue, whereas LPL mRNA level remained unchanged. A cold-induced increase (1.5-fold) in LPL activity was observed in brown adipose tissue of hamsters treated with actinomycin D prior to 4 h cold exposure, whereas cycloheximide treatment completely abolished LPL stimulation. Thus, these data suggest that during the initial phase (< 24 h) of cold exposure the stimulation of LPL activity in brown adipose tissue is most likely due to increased translation. In contrast, during prolonged cold exposure, we detected a maximal 7-fold increase in LPL activity and a 2- to 3-fold increase in LPL mRNA level in brown adipose tissue indicating LPL regulation at the pretranslational level. Furthermore, comparison of LPL protein and activity in brown adipose tissue during prolonged (> 24 h) cold exposure provides some evidence that the active fraction of the enzyme pool in brown adipose tissue is increased in response to cold. ii) Surgical denervation and noradrenaline treatment revealed a complex role of the sympathetic innervation in the control of LPL expression in brown adipose tissue. Denervation decreased LPL mRNA level, but increased LPL activity. Noradrenaline treatment stimulated LPL activity to a similar extent as cold exposure. However, cold-induced stimulation of LPL activity was not impaired by denervation. iii) Cold exposure significantly elevated LPL mRNA content of inguinal white adipose tissue, although LPL activity was not affected. Posttranscriptional mechanisms appear to be involved in the tissue specific control of LPL expression.

Adipose Tissue↗

PubMed1986

Comparison of the triacylglycerol hydrolase activity of human post-heparin plasma lipoprotein lipase and hepatic triacylglycerol lipase. A monolayer study.

Interfacial catalysis of hepatic triacylglycerol lipase (H-TGL) and lipoprotein lipase (LpL) isolated from human post-heparin plasma was investigated with mixed monolayers of trioleoylglycerol (TO) and egg phosphatidylcholine. Rates of enzyme catalysis were dependent on surface pressure, substrate concentration, apoC-II (the activator protein for LpL), and cholesteryl oleate (CO). LpL showed a surface pressure optimum between 22 and 24 mN m-1, whereas H-TGL activity decreased at pressures greater than 20 mN m-1. LpL activity was enhanced greater than 10-fold by apoC-II; 1 M NaCl inhibited enzyme activity. ApoC-II, apoC-III, apoA-I, apoA-II, and 1 M NaCl had no effect on H-TGL activity. The substrate (TO) dependency was different for the two lipases. For LpL, there was a marked enhancement of enzyme activity between 2 and 4 mol % TO, whereas for H-TGL, enzyme activity increased linearly between 1 and 10 mol % TO. LpL activity toward monolayers containing 2 mol % TO was enhanced 2.6-fold by the addition of 5 mol % CO; cholesteryl ester had no effect on H-TGL activity. These findings suggest that the two lipolytic enzymes have different interfacial properties, which may have relevance to the rates of hydrolysis of triacylglycerols at a lipoprotein interface.

Heparin↗

PubMed2006

Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase.

Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner. Insulin responsive shedding is not seen in 3T3-L1 fibroblasts. This shedding involves both Ras-MAP kinase and phosphatidylinositol 3-kinase pathways. In response to insulin, adipocytes are known to secrete active lipoprotein lipase, an enzyme that binds to heparan sulfate on the luminal surface of capillary endothelia. Lipoprotein lipase is transported as a stable enzyme from its site of synthesis to its site of action, but the transport mechanism is unknown. Our studies indicate that shed adipocyte syndecans associate with lipoprotein lipase. The shed syndecan ectodomain can stabilize active lipoprotein lipase. These data suggest that syndecan ectodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperones for lipoprotein lipase as it translocates from its site of synthesis to its site of action.

3T3-L1 Cells↗

PubMed1988

Effect of Escherichia coli endotoxin on tissue lipoprotein lipase activities in chickens.

1. Four-week-old broiler chickens were injected intravenously with from 0.01 to 1 mg of E. coli endotoxin/kg body weight or with saline. 2. At all doses used endotoxin markedly depressed food intake and lipoprotein lipase activities in muscle and adipose tissue within 8 h. Heart lipoprotein lipase activity was significantly depressed only at doses of 0.1 mg endotoxin/kg body weight or greater. 3. Treatment of birds with 0.3 mg endotoxin/kg body weight reduced post-heparin lipoprotein lipase activity to 0.13 of that in control birds in 8 h. 4. Endotoxin generally depressed plasma very-low-density lipoprotein concentration. Plasma non-esterified fatty acid concentration was significantly elevated only in birds given 1 mg endotoxin/kg body weight. 5. Fatty acid synthetase activity in the liver of endotoxin-treated birds was significantly lower than in control birds 16 h after administration of endoxin, but not after 8 h. 6. These results show that tissue lipoprotein lipase activity in birds is very responsive to E. coli endotoxin, as in mammals. Hypertriglyceridaemia occurs only occasionally in endotoxin-treated chickens, most probably because of the particularly close relationship between food intake and hepatic lipoprotein synthesis in birds.

Adipose Tissue↗

PubMed1972

Separation of molecular species of lipoprotein lipase from adipose tissue.

When NH(4)OH-NH(4)Cl extracts of adipose acetone powder were applied to agarose gel chromatography columns, two peaks of lipoprotein lipase were eluted. The first activity peak (LPL(a)) was eluted with an elution volume of a protein of molecular weight approximately five times that of the second (LPL(b)). Addition of heparin to the eluted fractions markedly stimulated activity of LPL(a), but suppressed that of LPL(b). Both lipases had the characteristics that distinguish lipoprotein lipase from other tissue lipases: a requirement for serum for substrate activation, inhibition by 1 m NaCl, and an alkaline pH optimum (pH 8.0). It is concluded that these fractions represent two species of lipoprotein lipase.

Adipose Tissue↗

PubMed2003

Lipolysis of triglyceride-rich lipoproteins generates PPAR ligands: evidence for an antiinflammatory role for lipoprotein lipase.

Increased levels of triglyceride-rich lipoproteins provoke lipid accumulation in the artery wall, triggering early inflammatory responses central to atherosclerosis like endothelial adhesion molecule expression. The endogenous mechanisms limiting such reactions remain poorly defined. Lipoprotein lipase (LPL) plays a central role in lipid metabolism by hydrolyzing triglyceride rich lipoproteins and releasing fatty acids. We found that LPL treatment reversed tumor necrosis factor alpha and very low-density lipoprotein (VLDL)-stimulated endothelial vascular cell adhesion molecule 1 (VCAM1) induction and VCAM1 promoter responses, thus recapitulating effects reported with synthetic peroxisome proliferator-activated receptor (PPAR) agonists. In fact, these LPL effects on VCAM1 were absent in endothelial cells isolated from PPAR alpha-deficient mice. This finding suggests a novel antiinflammatory role for LPL. Further studies reveal specificity for PPAR activation through lipolysis in regards to lipoprotein substrate (VLDL >> LDL > HDL), PPAR isoform (PPAR alpha >> PPAR delta > PPAR gamma), and among fatty acid-releasing lipases. These PPAR responses required intact LPL catalytic activity. In vivo, transgenic mice overexpressing LPL had increased peroxisome proliferation, but not in the genetic absence of PPAR alpha. Although human plasma possesses minimal PPAR alpha activation despite containing abundant free fatty acids, marked PPAR alpha activation is seen with human plasma after LPL is added in vitro or systemically released in vivo. These data suggest a previously uncharacterized pathway in which the key lipolytic enzyme LPL can act on circulating lipoproteins to generate PPAR alpha ligands, providing a potentially important link between lipoprotein metabolism and distal PPAR alpha transcriptional effects.

Animals↗

PubMed1977

Clofibrate increases lipoprotein-lipase activity in adipose tissue of hypertriglyceridaemic patients.

Clearance of plasma-triglycerides and activity of lipoprotein lipase in adipose tissue were studied in six hypertriglyceridaemic patients before and after a week of clofibrate therapy (2 g/day). Plasma-triglycerides decreased significantly from 6.85 +/- 1.1 to 2.66 +/- 0.29 mmol/l and triglyceride clearance increased significantly from 1.3 +/- 0.2 to 2.4 +/- 0.4%/min. There were concomitant significant increases in heparin-releasable lipoprotein lipase (95 +/- 16 to 181 +/- 34 nmol free fatty acids/10(6) cells/h) and in extractable lipase (88 +/- 14 to 179 +/- 28 nmol free fatty acids/10(6) cells/h). It is concluded that an important effect of clofibrate may be to increase the levels of adipose-tissue lipoprotein lipase and thereby improve the clearance of plasma-triglycerides.

Adipose Tissue↗

PubMed1997

Regulation of adipose tissue lipoprotein lipase in young and old rats.

OBJECTIVE: Changes in tissue lipoprotein lipase have been reported in several of the metabolic disorders commonly associated with ageing, like insulin resistance, obesity and impaired hormonal balance. We have investigated the effect of normal ageing on the nutritional regulation of lipoprotein lipase (LPL) in rat tissues. MEASUREMENTS: In the first experiment, LPL activity and immunoreactive mass were measured in epididymal and perirenal adipose tissue, and soleus and heart muscle tissue. In the following experiments we focused on epididymal adipose tissue. RESULTS: In young rats (aged 29 d, 87 +/- 5 g), fasting for 24 h decreased LPL activity in epididymal and perirenal adipose tissue to 31% and 51% of fed control, respectively, while LPL mass increased to 146% and 261%, respectively. Consequently, LPL specific activity (activity/mass ratio) decreased to 20% of control. Other tissues studied did not show any large changes in LPL specific activity with the nutritional state. This suggests that the mechanism responsible for the down-regulation of LPL specific activity is specific for adipose tissue. The down-regulation was gradually blunted with increasing age and was non-existent in the old rats (aged 265 d, 564 +/- 14 g). LPL in soleus muscle from young rats was regulated by another mechanism, and was associated with a large increase in LPL activity and mass during fasting (297% and 458% of fed control). Also, this mechanism did not exist in soleus muscle from old rats. Prolonging the fasting period of the old rats to 96 h did not induce the changes in adipose tissue or soleus muscle LPL seen in the young rats. CONCLUSION: The results indicate that the nutritional regulation of LPL in adipose tissue and soleus muscle changes during normal ageing.

Adipose Tissue↗