Monoacylglycerol lipase activity in cardiac myocytes

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100,000 X g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100,000 X g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 microM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 microM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA greater than free fatty acids greater than fatty acyl carnitine.     

Characterization of triacylglycerol hydrolase activities in isolated myocardial cells from rat heart.

Triacylglycerol (TG) hydrolase activities were characterized in myocytes isolated from rat hearts. Acid hydrolase activity with a pH optimum of 5 could be measured in myocyte homogenates, and the subcellular distribution suggested that this activity originated in lysosomes. Lipoprotein lipase (LPL) was also present in myocyte homogenates, as evidenced by TG hydrolase activity that was stimulated by serum and apolipoprotein CII, and inhibited by apolipoprotein CIII2, high ionic strength (NaCl and MgCl2, I = 1 M) and antibodies to LPL. Serum-independent neutral (pH 7.5) TG hydrolase activity was less sensitive to inhibition by 1 M-NaCl, by antibodies to LPL and by preincubation at 40 degrees C than was serum-stimulated hydrolase activity. Furthermore, there were modest but significant differences in the subcellular distribution of the serum-independent and serum-stimulated hydrolase activities. Hydrolase activities in myocyte homogenates could be solubilized by 7.2 mM-deoxycholate. Acid hydrolase activity was recovered in the unbound fraction after heparin-Sepharose chromatography, whereas LPL was bound to the affinity column and was eluted by 0.9-1.2 M-NaCl. Approximately one-third of the serum-independent TG hydrolase activity was not bound to the heparin-Sepharose affinity column. This unbound TG hydrolase activity had a pH optimum of 7 and was stimulated by 50 mM-MgCl2, but not by serum and was resistant to inhibition by high ionic strength (1 M-NaCl), to preincubation at 40 degrees C for 2 h, and by antibodies to LPL. It is concluded that, in addition to an acid lysosomal TG hydrolase and LPL, myocytes from rat heart contain a serum-independent TG hydrolase with unique characteristics.     

Long term incubation of cardiac myocytes with oleic acid and very-low density lipoprotein reduces heparin-releasable lipoprotein lipase activity

An exogenous [³H]triolein emulsion was hydrolyzed by intact cardiac myocytes with functional LPL located on the cell surface. This surface-bound LPL could be released into the medium when cardiac myocytes were incubated with heparin. Incubation of cardiac myocytes with VLDL, or the products of TG breakdown, oleic acid or 2-monoolein, did not increase LPL activity in the medium. However, incubation of cardiac myocytes with either VLDL or oleic acid for > 60 min did reduce heparin-releasable LPL activity. In the heart, this inhibitory effect of FFA could regulate the translocation of LPL from its site of synthesis in the cardiac myocyte to its functional site at the capillary endothelium.Abbreviations LPL lipoprotein lipase - TG triacylglycerol - FFA free fatty acids - VLDL very-low density lipoprotein     

Secretion of lipoprotein lipase from myocardial cells isolated from adult rat hearts

Summary Heparin (5 U/ml) induced the release of LPL into the incubation medium of cardiac myocytes isolated from adult rat hearts. The secretion of LPL occurred in two phases: a rapid release (5–10 min of incubation with heparin) that was independent of protein synthesis followed by a slower rate of release that was inhibited by cycloheximide. The rapid release of LPL induced by heparin likely occurs from sites that are at or near the cell surface. LPL secretion could also be stimulated by heparan sulfate and dermatan sulfate, but not by hyaluronic acid, chondroitin sulfate or keratan sulfate. Heparin-releasable LPL activity measured in short-term incubations represented a large fraction (40–50%) of the initial LPL activity associated with myocytes, but the fall in cellular LPL activity following heparin was less than the amount of LPL activity secreted into the incubation medium. This discrepancy was not due to latency of LPL in the pre-heparin cell homogenates, but in part could be due to a three-fold greater affinity of the heparin-released enzyme for substrate as compared to LPL in post-heparin myocyte homogenates.Abbreviations LPL lipoprotein lipase     

Triacylglycerol lipase activity in the rabbit renal medulla

Although the renal medulla is rich in triacylglycerols, the lipolysis of these intracellular triacylglycerols by a renomedullary triacylglycerol lipase has not been directly demonstrated. The present study demonstrates triacylglycerol lipase activity localized in the particulate subcellular fractions of rabbit renal medullae. Renomedullary triacylglycerol lipase activity, as determined by the hydrolysis of [14C]triolein to [14C]oleic acid, was observed to have a pH optimum of 5.8. Addition of cAMP/ATP/magnesium acetate resulted in an 80% activation of crude homogenate triacylglycerol lipase activity; addition of exogenous cAMP-dependent protein kinase resulted in a further activation of lipolysis. 3 mM CaCl2 had no effect on basal triacylglycerol lipase activity. 1 M NaCl did not inhibit lipolysis, suggesting that the lipase activity measured was not due to lipoprotein lipase. Endogenous renomedullary triacylglycerols were hydrolysed by a lipase in the 100,000 X g pellet of renomedullary homogenates, resulting in the release of free fatty acids including arachidonic and adrenic acids. Dispersed renomedullary cells were prepared to monitor hormone-sensitive triacylglycerol lipase activity in intact cells. Addition of 10 microM forskolin and 10 microM epinephrine resulted in 8-fold and 50-fold increases in triacylglycerol lipase activity, respectively, as defined by release of free glycerol from the cells. These studies demonstrate that a cAMP-dependent hormone-sensitive triacylglycerol lipase is present in the renal medulla, and is responsible for the hydrolysis of renomedullary triacylglycerols.     

The activity and properties of an acidic triacylglycerol lipase from adult and fetal rat lung

Triacylglycerol lipase with maximal activity at pH 5 was present in adult and fetal lung. The activity was inhibited by serum concentrations used to measure lipoprotein lipase and by 0.5 M NaCl. The activity in homogenates from fetal lung was about 40% of the activity in adult lung homogenates. The activity increased to 80% of the adult levels during the first 24–48 h following birth. Acidic triacylglycerol lipase was present in all subcellular fractions from adult lung. However, the major amount of activity appeared to be associated with lysosomes. Fetal lung contained significantly more activity in the cytosolic fraction compared to the adult. The reaction produced free fatty acids (65%), 1,2(2,3)-diacylglycerol (22%) and 2-monoacylglycerol (12%). Minimal amounts of 1,3-diacylglycerol and 1(3)-monoacylglycerol were formed. Diacylglycerol lipase and monoacylglycerol hydrolase activities at pH 5 were independently determined and both were higher than the triacylglycerol lipase activity. The subcellular distribution of diacylglycerol lipase and monoacylglycerol hydrolase differed from that of triacylglycerol lipase. Overall, the results indicated that the lung has considerable intracellular lipase activity and therefore could readily hydrolyze intracellular triacylglycerol to free fatty acids. The reaction also produced significant amounts of 1,2-diacylglycerol which suggests that triacylglycerol could be a direct source of diacylglycerol for phospholipid synthesis.     

Regulation of lipoprotein lipase activity in cardiac myocytes from control and diabetic rat hearts by plasma lipids.

The objective of this investigation was to test the hypothesis that the diabetes-induced reduction in lipoprotein lipase activity in cardiac myocytes may be due to hypertriglyceridemia. Administration of 4-aminopyrazolopyrimidine (50 mg/kg) to control rats for 24 h reduced plasma triacylglycerol levels and increased the heparin-induced release of lipoprotein lipase into the incubation medium of cardiac myocytes. The acute (3-5 days) induction of diabetes by streptozotocin (100 mg/kg) produced hypertriglyceridemia and reduced heparin-releasable lipoprotein lipase activity in cardiac myocytes. Treatment of diabetic rats with 4-aminopyrazolopyrimidine resulted in a fall in plasma triacylglycerol content and increased heparin-releasable lipoprotein lipase activity. Administration of Triton WR-1339 also resulted in hypertriglyceridemia, but the heparin-induced release of lipoprotein lipase from control cardiac myocytes was not reduced in the absence of lipolysis of triacylglycerol-rich lipoproteins. Treatment with Triton WR-1339 did, however, increase the heparin-induced release of lipoprotein lipase from diabetic cardiac myocytes. Preparation of cardiac myocytes with 0.9 mM oleic acid resulted in a decrease in both total cellular and heparin-releasable lipoprotein lipase activities. These results suggest that the diabetes-induced reduction in heart lipoprotein lipase activity may, at least in part, be due to an inhibitory effect of free fatty acids, derived either from lipoprotein degradation or from adipose tissue lipolysis, on lipoprotein lipase activity in (and (or) release from) cardiac myocytes.     

Effects of hormones, fasting and diabetes on triglyceride lipase activities in rat heart and liver

Male rats were fasted for 3 days, subjected to streptozotocin-diabetes or injected with L-thyroxine, Kenacort-A40 (corticosteroid) and Synacthen (ACTH). Cardiac heparin-releasable lipoprotein lipase (LPL) activity was increased after fasting, experimental diabetes and all hormone treatments. Cardiac neutral lipase activity was decreased during diabetes and enhanced in the fasted state and by L-thyroxine, corticosteroid and ACTH administration. The close correlation between vascular LPL and tissue neutral lipase with cardiac triglyceride content is in agreement with the contention that tissue neutral lipase is similar to LPL (Hülsmann, Stam and Breeman 1982). Myocardial acid lipase activity was reduced during diabetes and L-thyroxine treatment, increased during fasting and corticosteroid administration and not affected by short-term ACTH treatment. Hepatic acid lipase activity was increased during fasting, diabetes and by L-thyroxine and reduced after corticosteroid and ACTH treatment. The alkaline liver lipase activity was depressed by fasting, experimental diabetes, corticosteroid and ACTH treatment, whereas L-thyroxine induced a slight increase in enzyme activity. The possible mechanism underlying the observed changes in acid, neutral, alkaline, and LPL activities in heart and liver are discussed.     

Characterization of mono-, di- and triacylglycerol lipase activities in the isolated rat heart

The lipolytic activities of heart tissue towards full and partial acylglycerols were characterized. Tissue lysosomal, acid lipase activity (pH 4.8) was inhibited by high salt, protamine sulfate, NaF, MgATP, Triton X-100, serum and the esterase-inhibitor diethylparanitrophenyl phosphate. The tissue neutral triacylglycerol lipase activity (pH 7.4) was recovered predominantly in the microsomal and soluble fractions and exhibited essentially identical properties towards activators (serum, apolipoprotein C-II) and reagents (NaCl, Triton X-100, NaF, MgATP and diethylparanitrophenyl phosphate) relative to vascular lipoprotein lipase, except for protamine sulfate which increased the serum-stimulated neutral triacylglycerol lipase activity. Triacylglycerol hydrolysis at acid pH was incomplete, whereas at neutral pH full hydrolysis occurred. Myocardial mono- and diacylglycerol lipase activities, with pH optima of 8.0 and 7.4, respectively, were recovered in the microsomal fraction. They differed immunologically from neutral lipase and lipoprotein lipase and did not bind to heparin-Sepharose 4B. They were kinetically different, partially inhibited by NaCl and differentially affected by protamine sulfate. NaF, Triton X-100 and diethylparanitrophenyl phosphate. Our data suggest that endogenous hydrolytic activity against full and partial acylglycerols is mediated by separate enzymes.     

The presence and role of hormone-sensitive lipase in heart muscle.

Hormone-sensitive lipase (HSL) catalyses the initial, rate-limiting, reaction in adipose-tissue lipolysis. Hormone-stimulated lipolytic activity has also been observed in the heart, where endogenous triacylglycerol is the major energy store. However, the identity of the intracellular lipase responsible has yet to be established. We have partially purified a neutral lipase from bovine heart muscle and compared its properties with those of HSL from bovine adipose tissue. The heart lipase has the same subunit Mr as HSL, is immunoprecipitated by antiserum raised against purified HSL and is phosphorylated by cyclic AMP-dependent protein kinase, apparently at the same site as HSL (as judged by h.p.l.c. of tryptic phosphopeptides). Phosphorylation of the heart lipase was found to result in increased enzyme activity, demonstrating the lipase's potential to respond to hormonal stimuli. The heart lipase was shown to be present in myocytes by its immunoprecipitation from homogenates of rat myocytes by anti-HSL antiserum. These findings are consistent with the conclusion that HSL is responsible for intracellular lipolysis in heart.     

Lipoprotein lipase and hepatic lipase activities in a hypercholesterolaemic (RICO) strain of rat. Effect of dietary cholesterol.

Hepatic lipase (HL) and lipoprotein lipase (LPL) were assayed in heparinized plasma from male normocholesterolaemic (SW) and genetically hypercholesterolaemic (RICO) rats. Both strains were fed on either a semi-purified control diet or the same diet enriched with 0.5% or 1% cholesterol. HL activity was similar in both groups of rats fed on the control diet. LPL activity was found to be significantly lower in RICO rats (35% decrease, P less than 0.05). Feeding with a high-cholesterol diet led to a decrease in HL activity (15-23%) in both groups of rats but no change was detected in LPL activity, which remained consistently lower in the RICO rats. Thus, with the control diet, LPL activity is lower in RICO rats but presumably is not rate-limiting for their triacylglycerol clearance, given the normal triacylglycerol levels present. After cholesterol feeding, however, the lower LPL activity may become rate-limiting together with the decrease in HL activity, as in these circumstances hypertriacylglycerolaemia was evident and the hypercholesterolaemia of this strain was further increased.     

Lipoprotein lipase in heart and myocytes: characteristics with intralipid as substrate.

1. Intralipid is a suitable substrate for measuring lipoprotein lipase activity in the presence of other triacylglycerol lipases in heart and myocytes. 2. Triacylglycerol lipase activity in heart and myocytes was increased 10-fold in the presence of serum at pH 7.4 and 8.1. The serum-stimulated activity in myocytes was 95% inhibited by saturating concentrations of antiserum to lipoprotein lipase. 3. Both heparin-releasable and non-releasable lipoprotein lipase fractions had similar Km values for Intralipid and a similar pattern of inhibition by high density lipoprotein but different responses to heparin. 4. Isoproterenol did not alter lipoprotein lipase activity in cardiac myocytes.     

Lipoprotein lipase activities in heart muscle of streptozotocin-induced diabetic rats

Triglyceride lipase (TGL) activities in the homogenates of the rat heart muscle were studied. TGL activity per mg protein of heart muscle was the highest in heart muscle homogenate utilizing 2.1 M glycine buffer, pH 8.3 among the assays investigated. The effects of NaCl, serum and heparin on TGL activities in heart muscle homogenates indicated the characteristics of lipoprotein lipase (LPL). Twelve-hour fasting increased heart muscle LPL activity, while enzyme activities in 48 hour- and 72 hour-fasted rats were lower than those in fed rats. LPL activities in heart muscle homogenates in streptozotocin (STZ)-induced diabetic rats either 3 days or 4 weeks after STZ injection, were decreased significantly as compared with those of control rats.     

Membrane-bound lipoprotein lipase on human monocyte-derived macrophages: localization by immunocolloidal gold technique

Macrophages from both rodent and human sources have been shown to produce lipoprotein lipase (LPL), the enzyme activity of which can be measured in culture media and in cellular homogenates. The studies reported here show the presence of LPL on the surface of human monocyte-derived macrophages. An inhibitory monoclonal antibody to human LPL was used for cellular and immunoelectron microscopy studies. This antibody is a competitive inhibitor of LPL hydrolysis of triacylglycerol but does not inhibit LPL hydrolysis of a water-soluble substrate, p-nitrophenyl acetate. Furthermore, when postheparin plasma was mixed with monoclonal antibody prior to gel filtration on 6% agarose, the LPL activity eluted with the lipoproteins and was not inhibited by the antibody. These studies suggest that the antibody recognized the lipid/lipoprotein binding site of the LPL molecule. Membrane-bound LPL was demonstrated on human monocyte-derived macrophages using colloidal gold-protein A to detect the monoclonal antibody to LPL. The surface colloidal gold was randomly distributed with a surface density of 56,700 gold particles per cell. Control cells cultured in heparin-containing media (10 units/ml) or cells reacted with anti-hepatic triacylglycerol lipase monoclonal IgG or nonimmune mouse IgG did not exhibit membrane binding of protein A-gold. Macrophages were incubated with control and monoclonal anti-LPL IgGs and 125I-labeled anti-mouse IgG F(ab')2. Heparin-releasable membrane-bound anti-LPL antibody was demonstrated. These studies demonstrate the presence of LPL on the surface of human monocyte-derived macrophages, such that the LPL is oriented with its lipid-binding portion (recognized by this antibody) exposed. Membrane-associated LPL may be important in the interaction and subsequent uptake of lipid and lipoproteins by macrophages and in the generation of atherosclerotic foam cells.     

Adipose, muscle and lung tissue lipoprotein lipase activities in young streptozotocin treated rats

Lipoprotein lipase (LPL) activity was studied in adipose, muscle and lung tissues of post-weanling rats 48 and 96 hours after experimentally induced diabetes by streptozotocin administration. Weight gain was reduced, and blood glucose level increased about 3-4 fold above the control level as an indication of the diabetic state. LPL activity in brown and white adipose tissues decreased in diabetic rats to 10-30% of the control level. In soleus muscle the LPL activity was slightly enhanced 96 hours after the streptozotocin injection. In cardiac muscle the LPL activity was markedly increased already 48 hours after the administration of streptozotocin and the increase remained significant until 96 hours. There was in the pulmonary tissue also an increase of LPL activity of diabetic rats, although this was significant only 96 hours after streptozotocin treatment. The results suggest marked tissue specific variation in the LPL activity. Moreover, tissue responses to experimentally induced diabetes vary. In adipose tissue the decrease in the LPL activity suggests that lipid transport to adipocytes is decreased while an increase in skeletal and cardiac muscles and in lung tissue proposes that their lipid utilization is enhanced.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes

A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g . cm-3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19-1.22 g . cm-3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.     

Effect of improved diabetes control on the expression of lipoprotein lipase in human adipose tissue.

Patients with diabetes commonly manifest hypertriglyceridemia along with decreased adipose tissue lipoprotein lipase (LPL) activity, and improved diabetes control tends to reverse these abnormalities. To better understand the mechanism of regulation of LPL in diabetes, 11 diabetic patients (3 type I, 8 type II) were brought under improved glycemic control, and adipose tissue LPL gene expression was assessed by performing paired fat biopsies. Six of the 11 patients attained improved control with insulin, with a decrease in glycohemoglobin (glyc Hgb) from 13.8 +/- 0.9 to 10.4 +/- 0.6%; 5 patients attained improved control with glyburide (glyc Hgb fell from 14.2 +/- 2.4 to 8.8 +/- 0.6%), and together they demonstrated a lowering of serum triglycerides and total cholesterol. No changes were observed in HDL cholesterol. Improved diabetes control resulted in a significant increase in LPL activity in both the heparin-releasable (HR) and extractable (EXT) fractions of adipose tissue, as well as in LPL immunoreactive mass. The change in LPL activity with improved control was variable, and showed a positive correlation with the HDL levels prior to treatment (r = 0.74, P less than 0.02). When adipose tissue was pulse-labeled with [35S]methionine, there was an increase in isotope incorporation into LPL after treatment, indicating an increase in LPL synthetic rate. However, improved diabetes control resulted in no significant change in LPL mRNA levels. Thus, improved glycemic control resulted in an increase in LPL activity which correlated with each patient's basal high density lipoprotein. This increase in LPL activity was accompanied by an increase in LPL immunoreactive mass, and an increase in LPL synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)