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PubMed1986

High-density lipoprotein cholesterol, hepatic lipase and lipoprotein lipase activities in thyroid dysfunction--effects of treatment.

We have investigated the effects of hyper- and hypothyroidism (clinical and subclinical) on lipid metabolism, with special emphasis on serum high-density lipoprotein cholesterol, post-heparin plasma hepatic lipase and lipoprotein lipase activities. In 16 patients with hyperthyroidism, increased post-heparin plasma hepatic lipase activity, decreased serum total cholesterol and serum high-density lipoprotein cholesterol were found while lipoprotein lipase activity and serum triglyceride were normal. In six patients with overt hypothyroidism serum total cholesterol and triglyceride were increased, post-heparin plasma hepatic lipase and lipoprotein lipase were decreased while serum high-density lipoprotein cholesterol was normal. In six patients with subclinical hypothyroidism, serum total cholesterol was increased, serum high-density lipoprotein cholesterol was decreased, while serum triglyceride, post-heparin plasma hepatic lipase and lipoprotein lipase were normal. When the three groups of patients became euthyroid, serum total cholesterol, serum triglyceride, post-heparin plasma hepatic lipase, lipoprotein lipase, and serum high-density lipoprotein cholesterol reverted to normal except for serum high-density lipoprotein cholesterol in the hyperthyroid group which showed no significant change with treatment. A positive correlation was found between serum T3 and post-heparin plasma hepatic lipase while negative correlations were found between serum total cholesterol and serum T3, post-heparin plasma hepatic lipase and serum total cholesterol, lipoprotein lipase and serum triglyceride respectively. Thus in these patients with thyroid dysfunction, significant reversible alterations in serum total cholesterol, triglyceride and high-density lipoprotein cholesterol were found and could be correlated with the observed changes in the activities of hepatic lipase and lipoprotein lipase.

Adult↗

PubMed1998

Cloning, purification and characterisation of the lipase from Staphylococcus epidermidis--comparison of the substrate selectivity with those of other microbial lipases.

On the chromosome of Staphylococcus epidermidis RP62A the lipase gene (gehSE1) is immediately flanked by the icaAA'BC operon, which is involved in biofilm formation. Since lipase production might play a role in staphylococcal skin colonisation as well, we studied the biochemical properties of the staphylococcal lipases more closely. The DNA sequence and the deduced protein sequence revealed that gehSE1 is very similar to the lipase sequence of S. epidermidis strain 9. Like other staphylococcal lipases, gehSE1 is organised as a preproenzyme. The part of gehSE1 coding for the mature lipase was cloned and overexpressed as a fusion protein with an N-terminal histidine tag in Escherichia coli. The lipase was purified to homogeneity using a combination of precipitation techniques, metal-affinity chromatography and gel filtration. Biochemical characterisation showed that this lipase is closely related to the lipase from Staphylococcus aurelis NCTC8530. Both enzymes have a pH optimum around 6, are very stable at low pH, and need calcium as a cofactor for catalytic activity. The preferred substrates are small triacylglycerols, with a maximum activity toward tributyrylglycerol. Comparison of the substrate selectivity with those of other microbial lipases showed that phospholipids are generally poor substrates for lipases. An exception is the lipase from Staphylococcus hyicus, which prefers phospholipids as a substrate, distinguishing this staphylococcal lipase from other microbial lipases. These results are discussed in view of the structure/function relationships of staphylococcal lipases, and the possible involvement of these enzymes in biological processes such as skin colonisation and pathogenesis.

Amino Acid Sequence↗

PubMed1979

Characterization of the extracellular lipase of Bacillus subtilis and its relationship to a membrane-bound lipase found in a mutant strain.

Bacillus subtilis CMK33 is a mutant that is more osmotically fragile than the wild type when it is converted to the protoplast form. The protoplasts of this mutant contain a membrane-bound lipase, which is not found in protoplasts of the wild type. Hydrolysis of the membrane lipid of mutant protoplasts by the lipase is the cause of their fragility. A protein found in the wild type organism specifically inhibits the lipase (Kent, C., and Lennarz, W. J. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2793-2797). This paper reports that cultures of both mutant and wild type cells contain an extracellular lipase which accumulates during the logarithmic phase of growth. The extracellular activity appears to be induced by a component of the growth medium. The membrane-bound lipase of the mutant has been partially purified and its properties have been compared to those of the extracellular lipase of the wild type. Their properties and sensitivity to the wild type inhibitor are similar, which suggests that the two molecules are closely related. The subcellular location of the lipase in the mutant has been investigated and compared to the location of the membrane-bound portion of the lipase inhibitor in the wild type. The lipase is located almost exclusively in the cytoplasmic membrane and not in mesosomal vesicles. In contrast, the lipase inhibitor is located in both types of membranes and is more concentrated in mesosomal vesicles. Under appropriate conditions, the appearance of new extracellular lipase activity in mutant cultures is paralleled by the loss of an equivalent amount of lipase activity from protoplasts prepared from the cells. This suggests that the membrane-bound lipase may be an intermediate in the secretion of the extracellular lipase. Because of the mutation in B. subtilis CMK33, which results in the absence of the lipase inhibitor, this intermediate can be found in protoplasts of the mutant, although it is not detectable in the wild type. Consequently, the mutant may be useful in studies of the mechanism of secretion of exoenzymes by Bacilli.

Bacillus subtilis↗

PubMed1985

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.

Adipose Tissue↗

PubMed1988

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.

Antibodies, Monoclonal↗

PubMed2002

Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C.

Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0 ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (SigmaDiagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36 +/- 0.02 Sigma units/ml, (mean + S.E.M.; n = 16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300 x g for 15 min) and resuspended in NFDSM to a final concentration of 25 x 10(6) spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100 degrees C for 5 min) while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5 degrees C, and stored at 5 degrees C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72 h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100 units/ml but not 10 units/ml of pPL decreased (P < 0.01) total and progressive motility of stored sperm. Heat-inactivated pPL (Treatment 5) did not significantly decrease motility of spermatozoa during storage. Because the lipase activity assayed (Sigma units) and the lipase activity added to cooled semen (pPL units) were not equivalent, pPL was assayed in the Sigma Diagnostic Lipase assay. The relationship between Sigma Units (Y) and pPL units (X) appeared to be a log-linear relationship with log(Y) = -0.912 + 0.007X; R2 = 0.90. Mean lipase activity assayed in stallion seminal plasma was equivalent to approximately 64 pPL units/ml. These data suggest that endogenous lipase activity in stallion seminal plasma may be a factor in the adverse effects of seminal plasma on cooled spermatozoa in some stallions.

Animals↗

PubMed1989

Secretion and degradation of lipoprotein lipase in cultured adipocytes. Binding of lipoprotein lipase to membrane heparan sulfate proteoglycans is necessary for degradation.

Equilibrium-binding data of highly purified 125I-labeled avian lipoprotein lipase to cultured avian adipocytes demonstrate the presence of a class of high affinity binding sites. Analysis of the binding function yielded an association constant of 0.62 x 10(8)M-1 and a maximum binding capacity of 2.1 micrograms/60-mm dish. From a time course of dissociation of 125I-lipoprotein lipase from adipocytes at 4 degrees C, a dissociation rate constant of 6.1 x 10(-5)s-1 was obtained. Pretreatment of cells with heparinase and heparitinase resulted in a quantitative suppression of the high affinity binding component, establishing that lipoprotein lipase is bound to cell surface heparan sulfate proteoglycans. At 37 degrees C, cell surface-bound 125I-lipoprotein lipase is internalized and either degraded or recycled to the medium. The degradation rate constant for 125I-lipoprotein lipase was estimated to be 0.78 h-1. The degradation rate constant was reduced 6-fold when cells were exposed to 100 microM chloroquine, indicating that most of the degradation occurs within the lysosomal compartment. By using cells that had been pulsed with Trans35S-label for 1 h, it was demonstrated that acute treatment with endoglycosidases for up to 1 h resulted in a new lipoprotein lipase secretion rate which was 6-fold higher than that of control cells. Degradation of newly synthesized lipoprotein lipase was essentially blocked 30 min after the initiation of the chase. In other studies it was observed that there were no additive effects of chloroquine and either endoglycosidase or heparin treatment on total lipoprotein lipase levels (intracellular, cell surface, and medium) in adipocyte cultures. These experiments support the hypothesis that the release of lipoprotein lipase from its receptor prevents its internalization and degradation and enhances enzyme efflux from the adipocyte. A new model of lipoprotein lipase secretion in cultured adipocytes is proposed: Newly synthesized lipoprotein lipase is transported to the cell surface where it binds to specific heparan sulfate proteoglycan receptors. The enzyme is either released to the medium or internalized via the receptor, in which case the enzyme is degraded or recycled to the cell surface. Major determinants of enzyme efflux from the cell surface include the number and integrity of receptors, the association constant of the enzyme-receptor complex, and the presence in the medium of competing molecules with high affinity for lipoprotein lipase. In this model, modulation of lipoprotein lipase degradation rate may be a significant mechanism for acute regulation of enzyme efflux independent of changes in the rate of enzyme synthesis.

Adipose Tissue↗

PubMed1996

Inhibition of hepatic lipase by m-aminophenylboronate. Application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases.

Phenylboronates are competitive inhibitors of serine. hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1,1.3). m-Aminophenylboronate inhibited hepatic lipase activity with a Ki value of 55 microM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h-1 mg-1 protein with a total purification factor of 14,400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h-1 mg-1. The purification factor was 23,400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.

Boronic Acids↗

PubMed1994

Effect of protamine on lipoprotein lipase and hepatic lipase in rats.

The polycation protamine impedes the catabolism of triglyceride-rich lipoproteins and this has been suggested to be due to intravascular inactivation of lipoprotein lipase. We have made intravenous injections of protamine to rats and found that both lipoprotein lipase and hepatic lipase activities were released to plasma. The effect of protamine was more short-lived than that obtained by injection of heparin. The release of hepatic lipase by protamine was as effective as the release by heparin, while the amount of lipoprotein lipase released by protamine was only about one-tenth of that released by heparin. This was not due to inactivation of lipoprotein lipase, since injection of an excess of heparin 10 min after injection of protamine released as much lipoprotein lipase activity to plasma as in controls. The results in vivo differed from those obtained in model experiments in vitro. Protamine was able to almost quantitatively release both lipoprotein lipase and hepatic lipase from columns of heparin-agarose. The displacement was dependent on the total amount of protamine that had passed over the column, indicating that it was due to occupation by protamine of all available binding sites. Our results in vivo showed that the binding sites for lipoprotein lipase were not blocked as efficiently as those for hepatic lipase, indicating that the binding structures were not identical. It was concluded that the impaired turnover of lipoproteins by protamine probably was due to prevention of binding of the lipoproteins to endothelial cell surfaces rather than to impaired lipase function.

Animals↗

PubMed1985

Effect of heparin-induced lipolysis on the distribution of apolipoprotein e among lipoprotein subclasses. Studies with patients deficient in hepatic triglyceride lipase and lipoprotein lipase.

In normal subjects, apolipoprotein E (apo E) is present on very low density lipoproteins (VLDL) (fraction I) and on particles of a size intermediate between VLDL and low density lipoproteins (LDL) (fraction II). The major portion of apo E is, however, on particles smaller than LDL but larger than the average high density lipoproteins (HDL) (fraction III). To investigate the possible role of the vascular lipases in determining this distribution of apo E among the plasma lipoproteins, we studied subjects with primary deficiency of either hepatic lipase or of lipoprotein lipase and compared them with normal subjects. Subjects with familial hepatic triglyceride lipase deficiency (n = 2) differ markedly from normal in that fraction II is the dominant apo E-containing group of lipoproteins. When lipolysis of VLDL was enhanced in these subjects upon release of lipoprotein lipase by intravenous heparin, a shift of the apo E from VLDL into fractions II and III was observed. In contrast, apolipoproteins CII and CIII (apo CII and CIII, respectively) did not accumulate in intermediate-sized particles but were shifted markedly from triglyceride rich lipoproteins to HDL after treatment with heparin. In subjects with primary lipoprotein lipase deficiency (n = 4), apo E was confined to fractions I and III. Release of hepatic triglyceride lipase by heparin injection in these subjects produced a shift of apo E from fraction I to III with no significant increase in fraction II. This movement of apo E from large VLDL and chylomicron-sized particles occurred with little hydrolysis of triglyceride and no significant shift of apo CII or CIII into HDL from triglyceride rich lipoproteins. When both lipoprotein lipase and hepatic triglyceride lipase were released by intravenous heparin injection into normal subjects (n = 3), fraction I declined and the apo E content of fraction III increased by an equivalent amount. Either moderate or no change was noted in the intermediate sized particles (fraction II). These data strongly support the hypothesis that fraction II is the product of the action of lipoprotein lipase upon triglyceride rich lipoproteins and is highly dependent on hepatic triglyceride lipase for its further catabolism. In addition, the hydrolysis by hepatic triglyceride lipase of triglyceride rich lipoproteins in general results in a preferential loss of apo E and its transfer to a specific group of large HDL.

Adult↗

PubMed1996

Digestive lipases of the newborn ferret: compensatory role of milk bile salt-dependent lipase.

The amount of mRNA hybridizing to bile salt-dependent lipase and to colipase-dependent lipase probes as well as their translation into active proteins were quantified in the adult and newborn pancreas and lactating mammary gland from the ferret, a species whose milk, similar to that of the human, has bile salt-dependent lipase. The concentration of colipase-dependent lipase mRNA correlated with the amount of activity found in the adult and newborn pancreas, whereas neither mRNA nor activity of this enzyme was detected in the kit pancreas or in the lactating mammary gland. These data indicate that colipase-dependent lipase is actually expressed in adult pancreas and might represent the main lipolytic system in the adult. mRNA hybridizing to the bile salt-dependent lipase probe used in this study were detected in adult and in newborn ferret pancreas as well as in lactating mammary gland. However, the bile salt-dependent lipase activity expressed in the newborn pancreas was very low when compared with the activity expressed either in the mammary gland or in the adult pancreas. These data argue for a compensatory role of milk bile salt-dependent lipase in lipid digestion in the newborn. The hydrolysis of dietary fat might be initiated by preduodenal lipase, the activity of which is only two times lower in the gastric mucosa of the newborn than in the adult ferret. The high concentration of mRNA hybridizing to the bile salt-dependent lipase probe associated with a very poor bile salt-dependent lipase activity and protein suggests either that these mRNA are very unstable or that they are poorly translated into an active pancreatic bile salt-dependent lipase.

Animals↗

PubMed1982

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.

Amino Acids↗

PubMed1992

Lipoprotein lipase and hepatic lipase activities are differentially regulated in isolated hepatocytes from neonatal rats.

Lipoprotein lipase and hepatic lipase are members of the lipase gene family sharing a high degree of homology in their amino acid sequences and genomic organization. We have recently shown that isolated hepatocytes from neonatal rats express both enzyme activities. We show here that both enzymes are, however, differentially regulated. Our main findings are: (i) fasting induced an increase of the lipoprotein lipase activity but a decrease of the hepatic lipase activity in whole liver, being in both cases the vascular (heparin-releasable) compartment responsible for these variations. (ii) In isolated hepatocytes, secretion of lipoprotein lipase activity was increased by adrenaline, dexamethasone and glucagon but was not affected by epidermal growth factor, insulin or triiodothyronine. On the contrary, secretion of hepatic lipase activity was decreased by adrenaline but was not affected by other hormones. (iii) The effect of adrenaline on lipoprotein lipase activity appeared to involve beta-adrenergic receptors, but stimulation of both beta- and alpha 1-receptors seemed to be required for the effect of this hormone on hepatic lipase activity. And (iv), increased secretion of lipoprotein lipase activity was only observed after 3 h of incubation with adrenaline and was blocked by cycloheximide. On the contrary, decreased secretion of hepatic lipase activity was already significant after 90 min of incubation and was not blocked by cycloheximide. We suggest that not only synthesis of both enzymes, but also the posttranslational processing, are under separate control in the neonatal rat liver.

Animals↗

PubMed1991

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.

Animals↗

PubMed1990

Combined lipase deficiency in the mouse. Evidence of impaired lipase processing and secretion.

Newborn combined lipase-deficient (cld) mice have severe hypertriglyceridemia associated with a marked decrease of lipoprotein lipase (LPL) and hepatic lipase (HL) activities. Since the cld mutation and lipase genes reside on separate chromosomes, combined lipase deficiency cannot result from defects occurring within the LPL or HL structural genes. To elucidate the biochemical basis of this trans-acting defect, cld mice were compared to unaffected littermates for changes in lipase mRNA levels, rates of synthesis, and posttranslational processing and secretion. LPL and HL mRNA levels in cld liver and LPL in cld heart were comparable to controls; corresponding lipase synthetic rates were modestly decreased by about 30%. However, these reduced synthetic rates were not lipase-specific, since the rates of apolipoprotein (apo) A-I and apoA-II synthesis in cld liver were similarly decreased. Despite LPL synthetic rates that were 70% of controls, LPL mass in cld postheparin plasma was markedly reduced to only 7% of control values, suggesting that the majority of LPL is not secreted but remains intracellular. Consistent with a lipase secretory defect, neither the LPL nor HL oligomannosyl forms were converted to their respective complex forms in cld tissues, indicating that the lipases had failed to move from the endoplasmic reticulum/cis-Golgi to the medial/trans-Golgi network. In addition, the majority of intracellular LPL was catalytically inactive, since LPL specific activity (units/mg LPL protein) in cld heart, kidney, and brain was reduced 80-97%. In contrast to the severe impairment of lipase posttranslational processing and secretion, cld mouse plasma contained normal levels of another secretory N-linked glycoprotein, adipsin, with its oligosaccharide chains fully processed to the complex form. Thus, the cld mutation appears not to globally disrupt the secretion of all N-linked glycoproteins, but rather selectively impairs LPL and HL at points essential to their normal intracellular transport and secretion.

Animals↗

PubMed1987

Hydrolysis of chylomicron arachidonate and linoleate ester bonds by lipoprotein lipase and hepatic lipase.

Chylomicrons labeled with [3H]arachidonic and [14C]linoleic acid were incubated with bovine milk lipoprotein lipase or rat postheparin plasma, containing both lipoprotein lipase and hepatic lipase. During incubation with bovine lipoprotein lipase, [3H]arachidonic acid was released from chylomicron triacylglycerols at a slower rate than [14C]linoleic acid. Only small amounts of [14C]linoleic acid were found as 1,2(2,3)-diacylglycerols, whereas a transient accumulation as [14C]monoacylglycerols was observed. In contrast, significantly more [3H]arachidonic acid was found as 1,2(2,3)-diacylglycerols than as monoacylglycerols at all time intervals investigated. The initial pattern of triacylglycerol hydrolysis by postheparin plasma was similar to that of bovine lipoprotein lipase. However, in contrast to the results obtained with bovine lipoprotein lipase, little [3H]1,2(2,3)-diacylglycerol accumulated. The addition of antiserum to hepatic lipase increased the amount of 3H found in 1,2(2,3)-diacylglycerols and inhibited the formation of free [3H]arachidonic acid. The antiserum also caused a significant inhibition of the hydrolysis of [3H]-but not of [14C]triacylglycerol. With regard to chylomicron phospholipids, the rate of hydrolysis of [14C]linoleoyl phosphatidylcholine with milk lipoprotein lipase was twofold higher than that of the [3H]arachidonyl phosphatidylcholine. However, the hepatic lipase of postheparin plasma had similar activity towards the two phosphatidylcholine species. Postheparin plasma rapidly hydrolyzed chylomicron 3H-labeled and 14C-labeled phosphatidylethanolamine to the same degree, and lipoprotein lipase similarly hydrolyzed 3H-labeled and 14C-labeled phosphatidylethanolamine at approximately equal rates. Antiserum to hepatic lipase inhibited the postheparin plasma hydrolysis of phosphatidylethanolamine and 3H-labeled phosphatidylcholine by about 60%, but the 14C-labeled phosphatidylcholine by only 27%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized.

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.

Adipose Tissue, Brown↗

PubMed1998

Alkaline lipase from brain: is it the same enzyme as pancreatic lipase from pancreas?

A new alkaline lipase was detected in rat brain and its properties were compared with those of the well-characterized pancreatic lipase and pancreatic lipase-related protein 2. The activity of the alkaline lipase was determined using trioleoylglycerol emulsion at pH 8.0. Subcellular fractions were prepared from brain homogenates by differential centrifugation. Lipase activities of the cytosolic fraction (the supernatant obtained by differential centrifugation of 100,000g) were stimulated by addition of colipase and bile salts and inhibited by addition of an antibody against rat pancreatic lipase. The partially purified enzyme had an isoelectric point of pH 6.8, which was identical to that found for rat pancreatic lipase. The enzyme had interfacial activation and dependence on colipase in the presence of bile salts. The enzyme had no measurable phospholipase A activity. The band produced by the enzyme on SDS-polyacrylamide gel electrophoresis was identical to that of the rat pancreatic lipase when detected by immunoblotting analysis using an antibody against pancreatic lipase. These results show that pancreatic lipase such as alkaline lipase is in rat brain.

Animals↗