Triacylglycerol Lipase Activity Research Articles

Effects of nutritional state, insulin, and glucagon on lipid mobilization in rainbow trout, Oncorhynchus mykiss

The effects of nutritional state, insulin, and glucagon on lipid mobilization were determined in rainbow trout, Oncorhynchus mykiss. In nutritional state experiments, fish were either fed continuously (except 24 to 36 hr prior to experimentation) with commercial trout chow or fasted for 4 weeks. Lipase activity in liver tissue isolated from fasted fish and cultured for 5 hr was greater than that in tissue isolated from fed fish and cultured. The presence of glucose (5.55 m M) in the incubation medium accentuates lipolytic activity in both liver and adipose tissue. Hormone response was assessed both in vivo and in vitro. Salmon insulin was injected into anesthetized fish (fed continuously except 24 hr prior to injections) in 10 μl of saline/g body weight; final hormone dose was 100 ng/g body weight. Tissue and plasma were sampled 1 and 3 hr after injection. Insulin resulted in depressed plasma FA concentration and reduced hepatic triacylglycerol lipase activity. In vitro effects of hormones were evaluated by incubating liver and adipose tissue pieces in Hanks-MEM. Glucagon (bovine/porcine) directly stimulated lipid breakdown in both liver and adipose tissue. These actions were manifested by enhanced FA and glycerol released into the culture medium and by elevated triacylglycerol lipase activity. Insulin (bovine) generally appeared antilipolytic as this agent inhibited glucagon-stimulated lipase activity and glucagon-stimulated FA release. Furthermore, insulin (in the presence of glucose) reduced net lipolysis, as indicated by glycerol release, compared to control cultures. These results indicate that nutritional state and glucose are important modulators of lipid mobilization and that glucagon and insulin act directly on lipid storage sites to coordinate lipolysis in rainbow trout.

Stimulation of the activity and mRNA level of hepatic triacylglycerol lipase by triiodothyronine in HepG2 cells

We have studied the effects of triiodothyronine (T 3) on the production of hepatic triacylglycerol lipase (HTGL) in the human hepatocellular carcinoma cell line, HepG2, by measuring its activity and mRNA levels. The HTGL activity released into the medium by heparin, increased after the addition of T 3 in a both time- (27% increase after 24 and 75% increase after 48 h) and dose-dependent manner (maximum activity with over 0.2 μg/ml of T 3 in the medium). Messenger RNA levels of HTGL in cells incubated with T 3 for 24 and 48 h were increased by 33% and 98% compared to those of the control. These results suggest that the production of HTGL may be regulated by thyroid hormone at the level of gene expression.

Carbachol stimulation of triacylglycerol lipase activity in pancreatic acinar cells

Carbachol (CCh) reduced the levels of [ 3H]arachidonic acid in triacylglycerol (TG) of pancreatic acinar cells. In cells prelabeled with [ 14C]glycerol, CCh reduced [ 14C]TG and increased [ 14C]diacylglycerol levels. Using [ 3H]triolein as exogenous substrate, CCh enhanced TG lipase activity 3-fold in a particulate fraction derived from intact acinar cells. These results portray a mechanism for generating diacylglycerol and arachidonic acid in exocrine pancreas involving agonist stimulation of TG hydrolysis.

Lipoprotein lipase in heart and myocytes: Characteristics with intralipid as substrate

1. 1. Intralipid is a suitable substrate for measuring lipoprotein lipase activity in the presence of other triacylglycerol lipases in heart and myocytes. 2. 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. 3. Both heparin-releasable and non-releasable lipoprotein lipase fractions had similar K m values for Intralipid and a similar pattern of inhibition by high density lipoprotein but different responses to heparin. 4. 4. Isoproterenol did not alter lipoprotein lipase activity in cardiac myocytes.

Annexin II inhibits calcium-dependent phospholipase A 1 and lysophospholipase but not triacyl glycerol lipase activities of rat liver hepatic lipase

A member of the annexin family (the heterotetrameric annexin II 2pl 1 2 complex purified from porcine intestinal epithelium) was tested for its ability to affect different calcium-dependent intrinsic lipolytic activities of rat liver hepatic lipase (HL). Whereas annexin II in the presence of calcium failed to interfere with HL triacyl glycerol lipase (EC 3.1.1.3) activity, it inhibited HL phospholipase A 1 (EC 3.1.1.32) and lysophospholipase (EC 3.1.1.5) activities. Inhibition could be overcome by increasing the substrate concentration. Under phospholipase A 1 assay conditions, annexin II did not bind to the purified HL enzyme. These results therefore suggest that only inhibitor/substrate interactions lead to inhibition of HL phospholipase A 1 and lysophospholipase activities, an obviously general mechanism of phospholipase inhibition by annexins. Possible implications of HL inhibition in vivo by annexins are discussed.

Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring(14)C-oleic acid release from(14)C-triolein.(14)C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200-400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40-43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that Vmax=0.016 nmol/h/mg protein and that Km=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg(2+). These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.

Effect of platelet‐activating factor on lipoprotein lipase and blood lipids

We investigated the effect of platelet-activating factor (PAF) and of the PAF specific antagonist CV-6209 on plasma lipid metabolism, and particularly on post-heparin plasma lipolytic activity in male Wistar rats. Lipoprotein lipase (LPL) activity was enhanced by intravenous injection of PAF before intravenous injection of heparin when the PAF dose was low (0.2 micrograms/kg). PAF activated hepatic triacylglycerol lipase (HTGL) activity dose-dependently. Plasma triacylglycerols (TG) significantly decreased with the activation of LPL and/or HTGL. Plasma total cholesterol (TC) and phospholipid (PL) levels decreased at a low dose of PAF (0.2 micrograms/kg), but increased when higher doses were used. The PAF antagonist CV-6209 partially reversed the PAF induced effects on HTGL, TC and PL.

Acute effects of exercise on triacylglycerol metabolism in fasted, untrained rats treated with nadolol

The current study investigated the early response of some determinants of triacylglycerol (TG) metabolism to acute exercise in rats that were treated or not with the nonselective β-adrenergic blocker, nadolol (25 mg·kg b.wt. -1·day -1 for 30 days). Measurements of hepatic TG secretion rate (HTGSR), postheparin plasma hepatic TG lipase (HTGL) activity, and that of lipoprotein lipase (LPL) in postheparin plasma, heart, vastus lateralis muscle (VLM), white (WAT) and brown (BAT) adipose tissues were carried out in untrained rats at rest or immediately after a 1 h run on treadmill (22 m·min -1, 0° grade). All animals were in the fasted state. Exercise reduced serum TG levels (46% below resting levels) and doubled those of nonesterified fatty acids. HTGSR was enhanced (+28%) by exercise while HTGL activity was not modified. The decrease in postheparin plasma LPL activity (-24%) caused by exercise was consistent with a reduction in enzyme activity in WAT (-32%), BAT (-45%) and heart (-25%). One h of treadmill running did not influence LPL activity in VLM. β-Adrenergic blockade did not affect any of the variables of lipid metabolism, except for a slight decrease in specific activity of LPL in the heart (-14%). This study demonstrates that absolute variations in HTGSR and in LPL activity cannot account for the acute fall in serum TG levels caused by moderate exercise, in fasted, untrained rats. In addition, these results show that the β-adrenergic pathway is either not involved in, or not necessary for, the latter effects of exercise on TG metabolism.

Effect of simvastatin (MK-733) on plasma triacylglycerol levels in rats

The effect of simvastatin (MK-733), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, on plasma triacylycerol (TG) levels was studied in rats. Dietary administration of MK-733 (0.055%, w/w) for 7 days significantly (P < 0.05) reduced plasma TG levels by 30.6% associated with a 44.3% significant (P < 0.01) reduction in very low density lipoprotein TG (VLDL-TG) as compared to those in the concurrent control rats. Clofibrate (0.08%, w/w) also significantly (P < 0.05) decreased plasma TG levels by 26.1%. MK-733 did not affect the tricylglycerol secretion rate (TGSR) during 0–1.5 hr after administration of Triton WR-1339, but reduced it by 33.9% during 1.5–3.0 hr. Clofibrate also decreased TGSR during 1.5–3.0 hr. MK-733 increased lipoprotein lipase (LPL) activity in epididymal adipose tissue and thigh muscle by 36.3 and 55.0% respectively. MK-733 significantly (P < 0.05) increased LPL activity in the post-heparin plasma by 21.5%, although it did not affect hepatic triacylglycerol lipase (H-TGL) activity. Clofibrate did not affect LPL activity in the tissues or LPL and H-TGL activities in the post-heparin plasma. It is considered that the mechanism of plasma TG-lowering effect of MK-733 is the removal of VLDL-TG by an increase in LPL activity in the tissues as well as a decrease in the TGSR.

Lipoprotein lipase and hepatic triacylglycerol lipase activities in peripheral and skeletal muscle lymph.

We studied the interstitial fluid concentration of two lipid-metabolizing enzymes (lipoprotein lipase and hepatic triacylglycerol lipase) to determine their importance in interstitial modification of filtered lipoproteins. Despite the use of a very sensitive lipase assay (1 nmol of fatty acid release/ml/hr), lipase activities in plasma and in peripheral and skeletal muscle lymph from control dogs were below the sensitivity of our assay. After heparin injection, hepatic triacylglycerol lipase and lipoprotein lipase activities in plasma were similar. However, the postheparin hepatic triacylglycerol lipase activities in peripheral and skeletal muscle lymph were only 1.4% and 1.1%, respectively, those of plasma. This concentration is considerably less than the lymph concentration of albumin, which has a similar size to the lipases but has a lymph concentration of 30% to 40% of plasma. Lipoprotein lipase activity in peripheral lymph and skeletal muscle lymph was 2.7% and 4.8%, respectively, of plasma activity. Since lipoprotein lipase has a similar size as hepatic triacylglycerol lipase, the disproportionate amount of lipoprotein lipase in lymph as compared to hepatic triacylglycerol lipase could be due to heparin crossing the capillary endothelium and displacing lipoprotein lipase from peripheral cells. Injection of radioactive heparin confirmed that it does cross into the interstitial space in sufficient concentrations to displace lipase from peripheral cells. We conclude that most of the lipase found in lymph after heparin injection is derived from peripheral cells and not from plasma. Furthermore, hepatic triacylglycerol lipase does not play a role in high density lipoprotein remodeling in interstitial fluid. Therefore, it seems likely that the considerable remodeling of high density lipoprotein that we found previously results from its interaction with peripheral cells.

Dibutyryl cAMP-induced Increases in triacylglycerol lipase activity in developing L8 myotube cultures

Triacylglycerol (TG) lipase activity, with an alkaline pH optimum, has been identified in the cellular fraction of L8 myotube cultures. This TG lipase activity was stimulated by serum and inhibited by NaCl and protamine sulfate. These characteristics have been classically described for lipoprotein lipase. It was possible to increase the activity of this TG lipase three- to five-fold by incubating the cells with dibutyryl cAMP. Maximal enzyme activity was observed 16 h following the addition of 10-100 microM dibutyryl cAMP to the cultured cells. Enzyme activity returned to control levels 24 h after removal of the nucleotide from the culture medium. Serum-sensitive alkaline TG lipase activity was also identified in five other myotube preparations of cultured muscle cells. The highest levels of activity were found in rat skeletal muscle primary, H9, and L6 cell types. The finding that dibutyryl cAMP is an effective inducer of alkaline TG lipase activity provides us with a valuable model to investigate mechanisms regulating synthesis, compartmentalization, and transport of lipoprotein lipase in muscle.

Triacylglycerol metabolism in rat skeletal muscle after exercise

The temporal relationships between triacylglycerol (TG) content and TG lipase activity in slow-twitch (STR) and fast-twitch red (FTR) muscles were determined in rats during recovery from a 2-h swim. Immediately after the exercise, plasma free fatty acid (FFA) was elevated and glycogen concentrations were decreased. TG content was decreased 40% in STR muscle and reduced 45% in FTR muscle. The TG concentration of STR muscle increased in a linear fashion throughout recovery so that control levels were reached within the first 24 h after exercise. TG lipase activity of STR muscle was elevated 36% above control immediately after the swim and continued to increase to 84% above control 24 h after the work. In STR muscle there was a net synthesis of TG, while lipase activity was elevated above that measured in muscle of control rats. TG content of FTR muscle remained 45% below control throughout the first 24 h of recovery, and TG lipase activity increased from 26% (P greater than 0.05) greater than control immediately after exercise to threefold above control 24 h after work. All parameters returned to control levels by 48 h of recovery. These data indicated that a net TG synthesis occurs in STR muscle when lipolytic activity is elevated. In FTR muscle, however, a gradual increase in TG lipase activity that occurs during the first 24 h of recovery accompanies a TG concentration well below the control level throughout this same time frame.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure et histochimie des glandes salivaires sublinguales du dromadaire (&lt;em&gt;Camelus dromedaríus&lt;/em&gt;)

Morphometric, histological and histochemical studies were carried out on the sublingual salivary glands of the Arabian camel (Camelus dromedarius). The glands are of the tubulo-acinar type and consist of many lobules that are composed of two types of cells, mucoserous and seromucous. The mucoserous cells form the main secretory units of the gland but seromucous cells are much more seldom and form associated acini. The former cells secrete and elaborate large quantities of neutral mucosubstances, sialomucins and little sulphomucins while only the apical portion of the latter cells shows weak to moderate activity for neutral and acid mucosubstances. The histoenzymological tests employed here detected a considerable activity of alkaline phosphatase, succinic dehydrogenase, aminopeptidase and non-specific esterases, but weak activities of cytochrome oxidase, peroxidase and no activities of triacylglycerol lipase, beta-glucoronidase and amylase. The functional significance of these findings is discussed.

Separation and differential activation of rat liver cytosolic cholesteryl ester hydrolase, triglyceride lipase and retinyl palmitate hydrolase by cholestyramine and protein kinases

Cholesteryl ester hydrolase (CEH), triacylglycerol lipase (TGL) and retinyl palmitate hydrolase (RPH) were measured in 104,000 x g supernatants from rat liver under optimal conditions for measurement of cytosolic CEH. Similar levels of hydrolytic activity were seen with oil droplet dispersions of cholesteryl oleate, trioleoylglycerol and retinyl palmitate. No cytosolic TGL activity was seen with substrate presented in the triton-albumin emulsion used for measurement of lipoprotein lipase-like TGL associated with hepatic plasma membrane. Cytosolic CEH, TGL and RPH were differentially partially purified by both ammonium sulfate precipitation and anion exchange fast protein liquid chromatography (FPLC). Of the tree activities, only CEH was stimulated by cholestyramine feeding and by activators of protein kinases A and C. All three activities were inhibited by alkaline phosphatase treatment, although to different degrees. It is concluded that these activities are catalyzed by at least three differentially regulated enzymes with a high degree of specificity for their respective substrates.

Metabolism of pyrenedecanoic acid in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and from a patient with multisystemic lipid storage myopathy

A lymphoid cell line has been established from a patient with multisystemic lipid storage myopathy and showed a major triacylglycerol storage, whereas the content of other neutral lipids and phospholipids was in the normal range. The metabolism of the triacylglycerols has been investigated in this lymphoid cell line from multisystemic lipid storage myopathy as well as in control cells through pulse-chase experiments using 10-(1-pyrene)decanoic acid (P10), a fluorescent fatty acid derivative, as precursor. After 1 h incubation, the uptake of P10 was not significantly different in multisystemic lipid storage myopathy and control lymphoid cells. The amount of fluorescent lipids synthesized by the lymphoid cells was proportional to the concentration of P10 in the culture medium. After 24 h incubation, at any extracellular concentration of P10, the content of P10-labelled triacylglycerols was much higher in multisystemic lipid storage myopathy cells than in controls. Chase experiments showed an impairment in the rate of degradation of biosynthesized triacylglycerols in multisystemic lipid storage myopathy lymphoblasts compared to controls with time of chase (the ratio P10-triacylglycerols/P10-phospholipids increased in mutant cells while it decreased in normal cells). Elsewhere, no enzyme deficiency of the neutral triacylglycerol lipase activity, has been found in multisystemic lipid storage myopathy lymphoid cells.

Hormones and triacylglycerol metabolism under normoxic and ischemic conditions.

Fatty acids, the preferred substrate in normoxic myocardium, are derived from either exogenous or endogenous triacylglycerols. The supply of exogenous fatty acids is dependent of the rate of lipolysis in adipose tissue and of the lipoprotein lipase activity at the coronary vascular endothelium. A large part of the liberated fatty acids is reesterified with glycerol-3-phosphate and converted to triacylglycerols. Endogenous lipolysis and lipogenesis are intracellular compartmentalized multienzyme processes of which individual hormone-sensitive steps have been demonstrated in adipose tissue. The triacylglycerol lipase is the rate-limiting enzyme of lipolysis and glycerol-3-phosphate acyltransferase and possibly phosphatidate phosphohydrolase are the rate-limiting enzymes of lipogenesis. The hormonal regulation of both processes in heart is still a matter of dispute. Triacylglycerol lipase activity in myocardial tissue has two intracellular sources: 1. the endoplasmic reticular and soluble neutral lipase, and 2. the lysosomal acid lipase. Studies in our laboratory have indicated that whereas lipolysis is enhanced during global ischemia and anoxia, overall lipolytic enzyme activities in heart homogenates were not altered. In addition we were unable to demonstrate alterations in tissue triacylglycerol content and glycerol-3-phosphate acyltransferase activity under these conditions. Lipolysis, is subject to feedback inhibition by product fatty acids. Therefore all processes leading to an increased removal of fatty acids from the catalytic site of the lipase will stimulate lipolysis. These studies will be reviewed. In addition, studies from our department have demonstrated the capacity of myocardial lysosomes to take up and degrade added triacylglycerol-particles in vitro. Such a process, stimulated by Ca2+ and stimulated by acidosis, offers another physiological target for hormone actions.

Cardiac triacylglycerol content and lipase activity during recovery from exercise.

Female rats swam for 2-h to determine the temporal relationship between triglyceride (TG) repletion and TG lipase activity in the heart during recovery from exercise. Immediately after the exercise, plasma free fatty acids (FFA) had increased from a resting value of 0.44 +/- 0.04 to 0.84 +/- 0.04 mM. Heart TG concentration was reduced 75%, whereas the glycogen level was decreased 34% below control. TG lipase activity was elevated 33% above control activity. One hour after the end of the exercise, lipolytic activity was still 26% above control and did not return to the resting level until the 4th h of recovery. The cardiac TG concentration was back to control levels by the 2nd h after the swim. Plasma FFA concentrations remained elevated during the first 4 h of recovery and were back to the control level by h 8. Cardiac glycogen was "supercompensated" during recovery h 1 and 2 and returned to the preexercise level by h 4. These data indicate that TG is being synthesized in the heart while lipolytic enzyme activity is elevated above control levels. This points out that the rate of TG synthesis is in excess of the hydrolysis. Since plasma FFA concentrations are elevated during periods of augmented TG synthesis, substrate availability, namely plasma FFA, may play a key role in regulating the size of the intracellular TG pool.

Determination of triacylglycerol lipase activity using carbon‐13‐labeled triacylglycerols and nuclear magnetic resonance spectroscopy: Evidence that hepatic lipase hydrolyzes medium‐chain triacylglycerols

Triacylglycerol lipase activity was studied using glycerol [1-13C]trioctanoate mixed with postheparin rat plasma. 13C NMR spectroscopy demonstrated triacylglycerol hydrolysis into free fatty acids with no difference at the 1,3 or 2 glycerol positions. There was no inhibition by high sodium concentration, consistent with lipase of hepatic origin.

Metabolic changes in coho and chinook salmon resulting from acute insufficiency in pancreatic hormones

Acute deficiency in pancreatic peptides (insulin, somatostatin-25, glucagon, and glucagon-like peptide) was invoked for 9-12 hr in coho, Oncorhynchus kisutch, and chinook, O. tshawytscha, salmon by administration of specific antisera raised against purified salmon hormones. Insulin-deficient fish were hyperglycemic, had diminished glycogen content in the liver (Plisetskaya et al., '88a, elevated liver triacylglycerol lipase activity, and higher concentration of plasma triiodothyronine (T3) compared to a control group of fish injected with nonspecific rabbit serum. After immunoneutralization of somatostatin-25, fish remained normoglycemic, with higher liver glycogen content, decreased lipase activity, and elevated plasma levels of insulin, while the levels of T3 declined. The induced deficiency in glucagon family peptides led to comparatively smaller changes: liver glycogen content was increased after anti-glucagon-like peptide (aGLP) injection and transient hyperglycemia was apparent following anti-glucagon (aGLU) administration. Circulating levels of insulin remained unaffected for at least 9 hr following aGLU and aGLP treatments. The velocity of pyruvate kinase at 2.5 mM phosphoenolpyruvate (V2.5) was depressed, especially after the combined administration of aGLU + aGLP. The effectiveness of immunoneutralization experiments was greatly dependent on the particular stage of the fish life cycle. Antisera against fish pancreatic peptides proved to be a suitable tool in the studies of hormonal regulation of fish metabolism.

Lipoprotein lipase in rat heart—II. influence of apolipoproteins and nutritional factors on tri- di- and monoacylglycerol lipase activities in post-heparin effluents

1. 1. The in vitro effects of serum and apolipoproteins (apo), and the influence of the nutritional state of the animals were compared on triacylglycerol lipase (TAGL), diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL) activities in post-heparin effluent from rat heart. 2. 2. Serum and apoC-II stimulated DAGL and MAGL 3-fold less than TAGL, the activity that measures lipoprotein lipase (LPL). 3. 3. The preexisting nutritional state of the heart, that strongly modulated LPL, did not influence DAGL and MAGL. 4. 4. ApoA-I, apoC-I, apoC-III 1 and apoC-III 2 did not stimulate LPL and counteracted its stimulation by apoC-II; MAGL, and not DAGL, was inhibited by apoA-I and apoC-I, an effect reversed by apoC-II. 5. 5. TAGL, DAGL and MAGL appeared to act as a single physiological unit, although differing in functional details; MAGL displayed the greatest dissimilarity.