Lipoprotein lipase in rat lung. The effect of fasting.

We measured lipoprotein lipase activity in dried defatted preparations of rat lung using doubly labeled chylomicron triglyceride as substrate. The enzyme activity was linear for the first hour of incubation at 37 degrees C, had a pH optimum of 8.1 and was completely inhibited by 0.5 M NaC1. Lungs from fed rats hydrolyzed chylomicron triglyceride at a rate of 13.00 mumoles/g per h; the activity rate was unchanged by fasting 8-72 h. Heparin infusion into isolated lungs caused immediate release of lipoprotein lipase to the venous effluent. The activity released was equivalent to about 10% of total lung lipoprotein lipase activity in both fed and fasted rats. Since the ability to remove blood triglyceride is directly related to the level of lipoprotein lipase activity, these findings indicate that the lung is one of the few tissues able to remove efficiently blood triglyceride during fasting.     

Sex differences in adrenocortical structure and function. XIV. Heparin-releasable liver-lipase like activity in rat adrenals as affected by gonadectomy and testosterone or estradiol replacement

Heparin-releasable liver-lipase like activity was studied in adrenals of intact, gonadectomized and gonadectomized-testosterone or estradiol replaced rats. Besides acid lipase, rat adrenals contain a neutral lipase activity. From both male and female adrenals about 70% of this activity is extractable by heparin containing medium. Activity of this lipase in 8000 g supernatant was higher in female than male adrenals. Orchiectomy of 12 weeks duration increased neutral lipase activity in rat adrenal, an effect reversed to normal level by testosterone replacement. On the other hand ovariectomy had no effect while estradiol replacement resulted in an increased activity of the neutral lipase of rat adrenal gland. The results showed a distinct sex-difference in heparin-releasable liver-lipase like activity of rat adrenals which depends on the inhibitory action of testosterone on the enzyme activity.     

Estradiol increases the secretion of hepatic lipase by rat hepatocyte cultures.

Hepatic lipase (EC 3.1.1.3) is synthesized and secreted by parenchymal hepatocytes and binds to endothelial cells of liver sinusoids. The present study shows that the activity of hepatic lipase secreted by hepatocyte cultures from male rats in increased approx. 6-fold after 10 h culture with 10 microM 17 beta-estradiol. The stimulatory effect of 17 beta-estradiol is biphasic and declines at higher concentrations. In hepatocytes from male rats: progesterone, unlike 17 beta-estradiol, had only a small stimulatory effect when present as the sole hormone and a small inhibitory effect in the presence of 17 beta-estradiol, while testosterone and dexamethasone had no effect. Hepatocyte cultures from female rats had a higher basal rate of hepatic lipase secretion than cells from male rats and showed a smaller stimulation by 17 beta-estradiol. These results suggest that 17 beta-estradiol might regulate the secretion of hepatic lipase by hepatocytes, and presumably the activity of the enzyme at either the endothelial surface of the liver sinusoids or at extrahepatic sites.     

Endogenous plasma lipoprotein lipase activity in fed and fasting rats may reflect the functional pool of endothelial lipoprotein lipase

In this study, a correlation was sought between the circulating lipoprotein lipase activity and nutritional state in the rat. In fed rats, the plasma lipoprotein lipase activity was between 30 and 120 munits/ml, whereas after an overnight fast in restraining cages, the lipoprotein lipase plasma levels were between 280 and 500 munits/ml. The plasma lipoprotein lipase activity was inhibited by a specific high titre goat antiserum to rat lipoprotein lipase. No effect of fasting was seen on the plasma hepatic triacylglycerol lipase. 6 h after fasting, adipose tissue lipoprotein lipase decreased maximally, but plasma lipoprotein lipase was not changed and rose only after 16 h. Thus, it seems that most of the lipoprotein lipase activity in the fasting plasma was related to the 3-fold rise in lipoprotein lipase activity in the heart, which may represent total muscle lipoprotein lipase. The increase in heart lipoprotein lipase was due in part to an increase in the t1/2 of the enzyme from 1.2 to 2.9 h. To determine whether the high plasma levels in the fasting rats might result from impaired clearance of the enzyme by the liver, functional hepatectomy was carried out. 15 min after hepatectomy, plasma lipoprotein lipase rose up to 20-fold in fed and about 6-fold in fasting rats. Lipoprotein lipase activity extracted by the liver was calculated to be 30-60 munits/ml in the fed and 171-247 munits/ml plasma per min in fasting rats. An increase in lipoprotein lipase activity in extrahepatic tissues (heart, lung, kidney, diaphragm and adrenal) occurred 30 min after hepatectomy in fed rats. The increase in heart lipoprotein lipase was due to an increase in heparin-releasable fraction. Since no impairment of hepatic clearance of circulating plasma lipoprotein lipase was found, the high fasting plasma lipoprotein lipase activity may be related to an increase in enzyme synthesis, decreased enzyme turnover and an expansion of the functional pool in tissues such as the heart and probably muscle. The present findings indicate that measurement of endogenous plasma lipoprotein lipase can provide information with respect to the size of the functional pool under normal and pathological conditions.     

The role of glucagon in the regulation of myocardial lipoprotein lipase activity

The role of glucagon in regulating the lipoprotein lipase activities of rat heart and adipose tissue was examined. When starved rats were fed glucose, heart lipoprotein lipase activity decreased while that of adipose tissue increased. Glucagon administration to these animals at the time of glucose feeding prevented the decline in heart lipoprotein lipase activity, but had no effect on the adipose tissue enzyme. When glucagon was administered to fed rats, heart lipoprotein lipase activity increased to levels found in starved animals but there was no change in the adipose tissue enzyme. It is suggested that the reciprocal lipoprotein lipase activities in heart and adipose tissue of fed and starved animals may be regulated by the circulating plasma insulin and glucagon concentrations.     

Intra- and extracellular forms of lipoprotein lipase in adipose tissue.

The location of lipoprotein lipase activity in rat adipose tissue was studied using intact epididymal fat pads, isolated adipocytes, and lipoprotein lipase activity secreted from adipocytes as enzyme sources. The enzyme activities of these preparations were characterized by gel filtration. The method used for isolation of adipocytes had been modified to minimize activation of lipoprotein lipase during the procedures. Extracts of intact adipose tissue separated into two major lipoprotein lipase activity peaks, designated "a" and "b", the "a" fraction representing about 30 (fasted rats) to 50% (fed rats) of the total enzyme activity. An intermediate fraction (designated "i") was frequently observed. Extracts of isolated adipocytes from fed rats contained about 35% and those from fasted rats about 65% of the lipoprotein lipase activity present in intact tissue. The "b" fraction constituted 80--97% of the adipocyte lipoprotein lipase activity. In contrast, the enzyme activity secreted from the adipocytes contained only the "a" and "i" fractions. These data implicate the existance of one intracellular form of lipoprotein lipase (corresponding to the "b" fraction), different from extracellular forms of the enzyme (corresponding to fractions "a" and "i"). A transformation of the intracellular to the extracellular forms appears to occur in conjunction with secretion of enzyme from the fat cell.     

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.     

Increased lipoprotein lipase content in the adipose tissue of suckling and weaning obese Zucker rats.

The aim of this study was to determine whether the increase in lipoprotein lipase activity displayed by the adipose tissue of obese (fa/fa) rats as compared with that of lean (Fa/fa) rats could be ascribed to a change in the content or in the catalytic properties of the enzyme. The question was addressed in rats of two ages: in 7-day-old suckling and in 30-day-old post-weaning pups. Inguinal fat-pads were removed surgically (7 days of age) or after killing (30 days of age), and acetone-extract powders were prepared. The relative quantity of enzyme was assessed by immunotitration using an antiserum raised in goat against purified lipoprotein lipase from rat adipose tissue. The results indicate that increases in enzyme activity in obese animals were strictly paralleled by increases in the amount of enzyme in suckling as well as in post-weaning pups. Moreover, the apparent Km values of lipoprotein lipase for its substrate triacylglycerol were identical in the two genotypes. In conclusion, the genotype-mediated increase in lipoprotein lipase activity in adipose tissue of obese Zucker rats was fully accounted for by an increase in the content of the enzyme. In addition, this work documents the mechanism of the increase in lipoprotein lipase activity during weaning, which is mediated mainly through changes in the adipose-tissue enzyme content.     

Influence of nutritional state on lipoprotein lipase activities in the hypothyroid rat

We have investigated the effects of nutritional state on the lipoprotein lipase activities of the experimentally hypothyroid rat. Both short-term effects (i.e., those of a 24 h fast with and without re-feeding) and long-term effects (due to decreased food intake in hypothyroidism) have been studied. The hypothyroid rats had significantly higher lipoprotein lipase activities of adipose tissue and heart muscle. The effect of hypothyroidism on adipose tissue lipoprotein lipase activities was modified by the nutritional state. In rats studied after 24 h fasting, the hypothyroid group had significantly higher lipoprotein lipase activities than weight-matched, age-matched and pair-fed (i.e., semi-starved) control groups. In rats studied in the re-fed state, the effects of hypothyroidism as such were less evident, since the pair-fed group also demonstrated significantly higher enzyme activities than did the other control groups. We have also studied the lipoprotein lipase activities of different enzyme preparations from adipose tissue. The effects of hypothyroidism were most clearly reflected in an increase of heparin-elutable enzyme activity from adipose tissue, whereas adipocyte lipoprotein lipase activity and the lipoprotein lipase secretion rate from adipocytes were affected to a lesser extent. We conclude that alterations in food intake strongly influence the lipoprotein lipase activities in the hypothyroidism. Our data also imply that the increased lipoprotein lipase activity in the hypothyroid state is due to a decreased degradation of the enzyme, both intra- and extracellularly.     

The site of cartilage matrix degradation.

1. The metabolism of VLD lipoproteins (very-low-density lipoproteins) was studied in intact isolated beating-heart cells and isolated perfused rat heart from starved animals by using [14C]triacylglycerol fatty acid-labelled VLD lipoprotein prepared from rats previously injected with [1-14C]palmitate. 2. 14C-labelled VLD lipoprotein was metabolized by the isolated perfused heart, but was only minimally metabolized by the heart cells unless an exogenous source of lipoprotein lipase was added. 3. Measurements of lipoprotein lipase at pH 7.4 with the natural substrate 14C-labelled VLD lipoprotein indicated that during collagenase perfusion of the heart the enzyme was released into the perfusate, the activity released being proportional to the concentration of collagenase used. Lipoprotein lipase activity in homogenates of hearts that had been perfused with collagenase showed a corresponding loss of activity. 4. At high perfusate concentrations of collagenase, inactivation of the released lipoprotein lipase occurred. 5. Lipoprotein lipase activity was largely undetectable in the homogenate of the isolated heart cells. 6. It is concluded that the lipoprotein lipase responsible for the hydrolysis of VLD lipoprotein triacylglycerol is predominantly located externally to the heart muscle cells and that its release can be facilitated by perfusion of the heart with bacterial collagenase.     

Synthesis of lipoprotein lipase in the liver of newborn rats and localization of the enzyme by immunofluorescence.

In newborn rats, lipoprotein lipase (LPL) activity was higher in the liver than in several other tissues, such as heart, diaphragm or lungs, and accounted for about 3% of total LPL activity in the body. There was no significant correlation between LPL activity in liver and in plasma. Thus transport of the enzyme from extrahepatic tissues was probably not the major source of LPL in liver. To study LPL biosynthesis directly, newborn rats were injected intraperitoneally with [35S]methionine, and LPL was isolated by immunoprecipitation and separation by SDS/polyacrylamide-gel electrophoresis. Radioactivity in LPL increased with a similar time course in all tissues studied, including the liver. Substantial synthesis of LPL was also demonstrated in isolated perfused livers from newborn rats, whereas synthesis was low in livers from adult rats. There was strong LPL immunofluorescence in livers from newborn rats, mainly within sinusoids and along the walls of larger vessels. This labelling disappeared after perfusion with heparin, which indicates that much of the enzyme is in contact with blood and can take part in lipoprotein metabolism.     

Lipoprotein lipase of cultured mesenchymal rat heart cells. IV. Modulation of enzyme activity by VLDL added to the culture medium.

Lipoprotein lipase activity was studied in rat heart cell cultures grown in the presence of 20% fetal calf and horse serum and a medium concentration of triacylglycerol of 0.03 mg/ml. After 6--8 days, when the enzyme activity had reached high levels, the cells were incubated for 24 h in a medium containing 20% serum derived from fasted or fed rats. No change in enzyme activity occurred in the presence of fasted rat serum, but a 50% fall was observed with fed rat serium. When the complete culture medium was supplemented with rat plasma VLDL (0.075--0.75 mg triacylglycerol) a pronounced decrease in lipoprotein lipase activity occurred after 3--5 h of incubation. Similar extent of enzyme fall was observed also in the presence of triacylglycerol-rich lipoproteins isolated from rat plasma after feeding of safflower oil or lard, even though the fatty acid composition of the triacylgylcerol varied markedly. As the addition of VLDL to the culture medium resulted in a lesser fall of heparin releasable than residual activity it seems that there was no direct inhibition of surface bound enzyme activity and that the transport of the enzyme to the cell surface was not affected. These data indicate that addition of VLDL to the culture medium resulted in a fall in enzyme synthesis, while total protein synthesis as determined by incorporation of [3H]leucine, remained unchanged. This inhibition could be reproduced by increasing free fatty acid concentration of the medium, however addition of excess albumin to VLDL-containing medium did not prevent the fall in enzyme activity. The present results obtained with cultured rat hearts cells suggest that in vivo plasma levels of triacylglycerol-rich lipoproteins could modulate the lipoproteins could modulate the lipoprotein lipase activity of the heart.     

The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339.

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.     

Lipoprotein lipase and cholesterol transfer activities of lean and obese Zucker rats.

Adult female lean and obese Zucker rats maintained under standard conditions were used for the estimation of plasma, liver and white adipose tissue (WAT) activity of lipoprotein lipase, plasma and liver hepatic lipase and plasma lecithin-cholesterol acyltransferase. No differences in plasma or tissue levels of lipoprotein lipase between lean and obese rats were detected, but the larger WAT size of the obese rats resulted in higher lipase activity per unit of rat weight. Hepatic lipase levels in plasma were higher in the obese, but in liver, the higher activity was found in lean rats. No significant differences were found for lecithin-cholesterol acyltransferase activity, except when the levels in the HDL fraction were expressed per unit of protein weight, showing lower activity in the obese rats. In conclusion, the essentially maintained enzyme activities in obese rat tissues suggest that they cannot explain the deficient lipoproteins processing of obese rats, and, consequently their dislipidaemia.     

Very low density lipoproteins and lipoprotein lipase in serum of rats deficient in essential fatty acids

Rats fed a diet deficient in essential fatty acids have a low level of serum very low density lipoproteins (VLDL). It was found that after intraperitoneal injection of heparin, deficient rats had a higher level of lipoprotein lipase activity in their plasma than did normal rats. VLDL isolated from serum of normal and deficient rats were compared as substrates for postheparin lipase of rat plasma. There was no significant difference in V(max) between the two preparations of lipoproteins, but the apparent K(m) for lipoproteins from deficient animals was significantly less than that for normal animals. These observations suggest that the low concentration of VLDL in deficient rats may be explained (a) by an increased activity of lipoprotein lipase in the tissues of these animals and (b) by the VLDL of deficient rats being more rapidly hydrolyzed at low concentrations by lipoprotein lipase than VLDL from normal rats.     

Role of lipoprotein lipase in regulating endogenous triacylglycerols in rat heart

Administration of glucagon (10 μg/rat) to the intact animal increased the levels of lipoprotein lipase activity by 92% in the heparin-non-releasable fraction of the heart. At the same time, cardiac levels of triacylglycerols were reduced 47% and free fatty acids were increased about 2-fold. In contrast, the administration of a lower dose of glucagon (0.5 μg/rat) resulted in an 80% reduction in lipoprotein lipase activity in the heparin perfused myocardium. At the same time, triacylglycerols were increased 44% and free fatty acids were decreased 69%. These results provide circumstantial evidence that lipoprotein lipase is involved in the regulation of endogenous triacylglycerols such that higher levels of enzyme activity result in cardiac lipolysis and, conversely, lower levels result in triacylglycerol production.     

Effects of adrenaline on the turnover of lipoprotein lipase in rat adipose tissue

The mechanisms by which adrenaline brings about a reduction in the lipoprotein lipase activity of adipose tissue in vitro were investigated. The incorporation of [3H]leucine into lipoprotein lipase was measured during 1-h pulse incubations of rat epididymal fat bodies that had been preincubated for 4 h in the presence of glucose, insulin and dexamethasone. When adrenaline was added to the incubation medium at the start of the pulse, the incorporation of [3H]leucine was markedly reduced, suggesting that the rate of the enzyme's synthesis had decreased. On the other hand, the degradation of lipoprotein lipase, as measured by the loss of 3H-labelled enzyme protein during pulse-chase incubations of the epididymal fat bodies, was found to be significantly increased by the addition of adrenaline to the incubation medium at the start of the chase period. It is concluded that adrenaline is able both to inhibit the synthesis of lipoprotein lipase and to stimulate its degradation.     

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.     

The lipoprotein lipase of white adipose tissue. Studies on the intracellular distribution of the adipocyte-associated enzyme.

The separation of rat epididymal adipocytes into plasma-membrane, mitochondrial, microsomal and cytosol fractions is described. The fractions, which were characterized by marker-enzyme analysis and electron-micrographic observation, from the cells of fed and 24 h-starved animals were used to prepare acetone/diethyl ether-dried powders for the measurement of lipoprotein lipase activities. The highest specific activities and proportion of recovered lipoprotein lipase activity were found in the plasma-membrane and microsomal fractions. The two fractions from the cells of fed rats showed similar activities and enrichments of the enzyme, these activities being higher than the plasma-membrane and lower than the microsomal activities recovered from the cells of starved animals. Chicken and guinea-pig anti-(rat lipoprotein lipase) sera were prepared, and an indirect labelled-second-antibody cellular immunoassay, using 125I-labelled rabbit anti-(chicken IgG) or 125I-labelled sheep anti-(guinea-pig IgG) antibodies respectively, for the detection of cell-surface enzyme was devised and optimized. The amount of immunodetectable cell-surface lipoprotein lipase was higher for cells isolated from fed animals than for cells from 24 h-starved animals, when either anti-(lipoprotein lipase) serum was used in the assay. The amount of immunodetectable cell-surface lipoprotein lipase fell further when starvation was extended to 48 h. The lipoprotein lipase of plasma-membrane vesicles was shown to be a patent activity and to be immunodetectable in a modification of the cellular immunoassay. Although the functional significance of the adipocyte surface lipoprotein lipase is not known, the possibility of it forming a pool of enzyme en route to the capillary endothelium is advanced.     

Interference with the transport of heparin-releasable lipoprotein lipase in the perfused rat heart by colchicine and vinblastine.

The effect of pretreatment with colchicine or vinblastine on the lipoprotein lipase activity of rat heart was studied. Administration of colchicine or vinblastine 4 h prior to perfusion of the heart caused a very marked reduction in lipoprotein lipase activity released into the perfusate within 1 min of heparin perfusion. At the same time an increase in residual heart lipase occurred so that total lipoprotein lipase content of the heart (heparin releasable plus residual) did not change. The colchicine effect was dose and time dependent; no decrease in heparin-releasable enzyme activity occurred after only 30 min of pretreatment or upon addition of colchicine into the perfusate. These results indicate that colchicine did not impede enzyme synthesis or its release from the cell surface, but may have interfered with the transport of lipoprotein lipase from the site of its synthesis to the endothelial cell surface.