The effect of fasting on the utilization of chylomicron triglyceride fatty acids in relation to clearing factor lipase (lipoprotein lipase) releasable by heparin in the perfused rat heart

Hearts from rats that have been starved for 10 or 24 hr oxidize (14)C-labeled chylomicron triglyceride fatty acids perfused through them at a higher rate than do hearts from rats in the fed state. Starvation for such periods increases the total clearing factor lipase activity of the heart. It is suggested that most of this increase may be accounted for by a rise in that portion of the total enzyme activity of the tissue that is released on perfusion with heparin. In rats starved for 48 hr, removal of this portion by heparin preperfusion reduces the capacity of the heart to oxidize (14)C-labeled chylomicron triglyceride fatty acids perfused subsequently by more than 80%. It is concluded that correlations between triglyceride fatty acid utilization and clearing factor lipase activity in the heart should be sought only with that portion of the total enzyme activity which is released from the intact organ by heparin.     

Effect of fasting on the clearing factor lipase (lipoprotein lipase) activity of fresh and defatted preparations of rat heart muscle

The clearing factor lipase (lipoprotein lipase) activities of homogenates of fresh tissue and of acetone-ether powders have been compared in hearts from fed and starved rats. The activity of the enzyme measured in homogenates of acetone-ether powders is generally higher than that in homogenates of the fresh tissue. Activation is due to an effect of the acetone-ether treatment on enzyme which is associated with the tissue residue in fresh tissue homogenates. A similar activation occurs when the tissue residue is treated with deoxycholate. When rats are fasted, a marked increase in the clearing factor lipase activity of the heart occurs. Peak activities are reached after 10-24 hr, and thereafter the activity falls slowly. This pattern of activity is observed in homogenates of fresh tissue and of acetone-ether powders. The activity of clearing factor lipase in diaphragm muscle also increases in rats starved for 8 or 24 hr. The importance of the change in muscle clearing factor lipase activity on fasting in relation to triglyceride fatty acid utilization by this tissue is emphasized.     

The significance of changes in tissue clearing-factor lipase activity in relation to the lipaemia of pregnancy

1. The concentration of triglyceride fatty acid in the plasma of the pregnant rat rises to a maximum 2-4 days before parturition. Thereafter there is a rapid decline in the concentration to near normal values at parturition. 2. A similar increase occurs in animals fed on a diet low in fat. There is no increase in food consumption at the time when the triglyceride fatty acid concentration in the plasma is at its peak. 3. Rates of entry of triglyceride fatty acid into the blood during pregnancy have been estimated from the rate of accumulation of triglyceride in the plasma of animals injected with a non-ionic detergent, Triton. A progressive increase occurs in the entry rate as the body weight increases throughout pregnancy. Expressed per constant body weight, the entry rate does not change significantly. 4. Adipose-tissue clearing-factor lipase activity is low at the time when the plasma triglyceride fatty acid concentration is raised. Activity of the enzyme in heart, lung and diaphragm is unchanged. 5. It is suggested that the ;lipaemia of pregnancy' may be due to diminished uptake of triglyceride fatty acids by adipose tissue, and, further, that the disappearance of the lipaemia may be due to increased uptake of triglyceride fatty acids by the mammary gland.     

Studies on the mechanism of hypertriglyceridemia in the genetically obese Zucker rat

The possibility that impaired removal of lipoprotein triglyceride from the circulation may be a participating factor in the hypertriglyceridemia of the obese Zucker rat was examined. We found no significant differences in the heparin-released lipoprotein lipase (LPL) activities of the adipose tissue, skeletal muscle, and heart (expressed per gram of tissue) from the lean and obese Zucker rats. Furthermore, the kinetic properties of adipose tissue and heart LPL from the lean and obese rats were similar, indicating that the catalytic efficiency of the enzyme was unaltered in the obese animals. The postheparin plasma LPL activities of lean and obese rats were also similar. However, the postheparin plasma hepatic triglyceride lipase (H-TGL) activity in the obese rats was elevated. The higher activity of H-TGL could not alleviate the hypertriglyceridemia in these animals. Since hypertriglyceridemia in the obese rats could also be due to the hepatic production of triglyceride-rich lipoproteins which are resistant to lipolysis, we therefore isolated very low density lipoproteins (VLDL) from lean and obese rat liver perfusates and examined their degradation by highly purified human milk LPL. Although certain differences were observed in hepatic VLDL triglyceride fatty acid composition, the kinetic patterns of LPL-catalyzed triglyceride disappearance from lean and obese rat liver perfusate VLDL were similar. The isolated liver perfusate VLDL contained sufficient apolipoprotein C-II for maximum lipolysis. These results indicate that impaired lipolysis is not a contributing factor in the genesis of hypertriglyceridemia in the genetically obese Zucker rat. The hyperlipemic state may be attributed to hypersecretion of hepatic VLDL and consequent saturation of the lipolytic removal of triglyceride-rich lipoproteins from the circulation.     

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.     

Effect of an anti-lipoprotein lipase serum on plasma triglyceride removal.

Anti-lipoprotein lipase sera injected intravenously in roosters blocked quantitatively the catabolism of very low density lipoprotein (VLDL) triglyceride. Antibodies were produced in rabbits immunized with highly purified lipoprotein lipase (LPL, glycerol ester hydrolase, E C 3.1.1.3) prepared from chicken adipose tissue. Following anti-LPL serum injection there was a linear increase in plasma triglyceride concentration. The rate of entry of triglyceride in plasma was estimated from the rate of triglyceride accumulation in the plasma of animals injected with anti-LPL serum, or from the disappearance curve of biologically labelled VLDL. In instances where both measurements were conducted in the same animals there was very close agreement between the two procedures. Inhibition of VLDL triglyceride catabolism of anti-LPL serum provided a way to characterize newly secreted VLDL that exhibited a broad spectrum of particle sizes with a median of 625 A degrees. They contained 76.2 +/- 1.2% triglyceride and had a high ratio of free to ester cholesterol (2.46 +/- 0.45). In control VLDL samples there was 46.1% triglyceride, and the ratio of free to ester cholesterol was 1.19. The complete inhibition of triglyceride removal by an antiserum prepared against adipose tissue LPL demonstrates that the NaCl-inhibited, serum-activated lipase prepared by affinity chromatography on heparin-Sepharose and concanavalin A-Sepharose columns is the enzyme responsible in vivo for the catabolism of VLDL triglyceride. Further, the kinetics of triglyceride accumulation in the plasma provide evidence that the site of degradation of VLDL triglyceride is within the plasma compartment.     

Rapid preparation of a relatively stable, partially purified lipoprotein lipase from perfused rat heart

Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or postheparin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37 degrees C and retained 75% of its activity after 10 days at 4 degrees C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.     

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Changes in fat content and some characteristics of lipolytic activity during pregnancy and lactation in mouse mammary gland.

The amount of free fatty acid in the mouse mammary gland continuously increased throughout pregnancy and lactation, while the amount of triglyceride which had been stored in the gland rapidly decreased after parturition. Higher lipolytic activity in the gland was observed in pregnancy than in nonpregnant and lactating animals. The optimum pH of the activities before and after parturition were about 6 and 7, respectively, and the activities did not decrease at high ionic strength in contrast to the ion dependent inactivation described in lipoprotein lipase. Incubation of the enzyme extract of the lactating mouse mammary gland at 50 degrees C for 10 min led to a remarkable increase in the lipolytic activity measured at pH 6.0, suggesting the existence of either an inactive form of the lipase whose optimum pH is 6.0 or some heat sensitive inhibitor(s) or inactivator(s) of the enzyme in the lactating mammary gland. The triglyceride stored in the gland in pregnancy will be consumed within the first 3rd days after parturition, and the lipases play an important role in the decomposition of the triglyceride.     

The clearing-factor lipase activity of isolated fat-cells.

1. When fat-cells are isolated from the epididymal adipose tissue of 24h-starved rats and incubated at 25 degrees C in the presence of dialysed serum, glucose, insulin, amino acids and heparin, the total clearing-factor lipase acitivity of the incubation system increases progressively over a period of several hours. 2. All of the increase in activity is accounted for by the appearance of enzyme in the appearance of enzyme in the incubation medium and the fat-cell activity does not change significantly. Cycloheximids, at a concentration that prevents protein synthesis, does not affect the appearance of enzyme in the incubation medium, but the fat-cell enzyme activity is decreased in its presence. 3. The magnitude of the increase in total clearing factor lipase activity is unaffected by the omission of heparin from the medium. However, less enzyme is extracted in tis absence and the fat-cell activity increases. Cycloheximide again only affects the rise in cell activity and does not alter the activity in the incubation medium. 4. When serum in the incubation medium is replaced by casein, the distribution of enzyme between the cells and the medium is changed, but the magnitudes of the increases in total enzyme activity are similar. 5. These characteristics of the clearing-factor lipase response of isolated fat-cells differ in several respects from those observed earlier with intact adipose tissue from 24h-starved rats (Robinson & Wing, 1971; Cryer et al., 1973). The differences could be due, in part, to changes in the relative amounts of two different molecular forms of the enzyme that occur during the isolation of the fat-cells.     

Effects of glycerol on human adipose tissue triglyceride lipase activity

Glycerol fully protects the human adipose tissue triglyceride lipase against the denaturing effects of high and low temperatures. Under such protection, storage of crude preparations at -10 degrees C or incubation at 50 degrees C resulted in a 1.5-3-fold increase of the measured lipase activity. This increase was shown to be related to enzyme newly released from tissular microparticles present in the samples. Advantage may be taken of these observations to improve greatly the conditions of extraction and storage of this lipase activity.     

Effects of heparin-induced lipolytic activity on the structure of rat high-density lipoprotein

Following its secretion into the plasma compartment, the high-density lipoprotein (HDL) is presumed to be acted upon by both soluble enzymes, such as lecithin:cholesterol acyltransferase (LCAT), and membrane-associated enzymes, such as lipoprotein lipase and hepatic lipase. Rats were injected intravenously with heparin to release membrane-associated lipolytic activities into the circulation and the collected plasma was incubated overnight at 37 degrees C in the presence or absence of an LCAT inhibitor or an inhibitor of lipoprotein lipase (1 M NaCl). It was observed that lipoprotein lipase accounted for most of the triglyceride hydrolase activity in the heparin-treated plasma, and that the heparin-releasable activities caused an increase in HDL density but no measurable change in particle size when LCAT was inhibited. Heparin treatment caused about a 60% decrease in plasma triacylglycerol during the interval between injection of heparin and blood collection. Although this caused marked compositional changes in the d less than 1.063 g/ml lipoproteins, no changes were observed in the lipid composition or apoprotein distribution in the HDL. Subsequent incubation for 18 h at 37 degrees C produced marked increases in the apoE content of HDL from heparin-treated plasma even when LCAT was inhibited. Time-course studies showed that in the presence of an LCAT inhibitor there was considerable conversion of phosphatidylcholine to lysophosphatidylcholine in heparin-treated plasma, and that this activity was diminished by 1 M NaCl, but that no phospholipolysis was observed in control plasma. By contrast, both heparin-treated and control plasma possessed substantial triglyceride hydrolase activity. The concurrent action of lipases and LCAT was observed to reduce the maximum level of cholesterol esterification which could be achieved in the absence of lipase activity. It is concluded that changes in HDL particle size are mainly attributable to LCAT, but that lipase activities, which are either free in rat plasma or releasable by heparin, play a role in restructuring the phospholipid moiety and altering the protein composition of the HDL, especially with respect to apoE, a potential ligand to cellular receptors.     

Effects of insulin, glucocorticoids and adrenaline on the activity of rat adipose-tissue lipoprotein lipids.

The lipoprotein lipase activity of epididymal fat-bodies from starved rats was measured during incubations at 37 degrees C in vitro. Protein synthesis independent activation of the enzyme, previously observed during incubations at 25 decrease C, also occurs at 37 degrees C. Protein-synthesis-dependent increases in the activity of the enzyme occur in the presence of insulin and are markedly potentiated by glucocorticoids. The effects on the activity of the enzyme of insulin alone, or in the presence of glucocorticoids, are correlated with its effects on total protein synthesis in the tissue. Adrenaline antagonizes the increase in activity of the enzyme brought about by insulin and abolishes the potentiation of insulin action by glucocorticoids. These changes may be due, at least in part, to its stimulation of inactivation of the enzyme in the tissue. It is suggested that changes in adipose-tissue lipoprotein lipase activity that occur with changes in nutritional status in vivo result from the combined effects of changes in plasma insulin and glucocorticoid concentrations.     

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.     

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.     

Lipid metabolism in endotoxic rats: decrease in hepatic triglyceride lipase activity

Studies were conducted to investigate the effect of E. coli endotoxin administration on hepatic triglyceride lipase (H-TGL) activity in rats, since H-TGL activity is known to behave differently from lipoprotein lipase (LPL) activity in various situations. Plasma triglyceride and free fatty acid concentrations were markedly elevated in animals after injection of endotoxin. Cholesterol and phospholipids were also increased significantly. Lipoprotein analysis by ultracentrifugation showed that the most pronounced increase of lipoproteins was in the VLDL and IDL fractions. Triglyceride lipase activities in post-heparin plasma were markedly decreased. A selective assay for H-TGL activity using a specific antibody revealed that this enzyme as well as LPL is significantly decreased (26% of control) in endotoxic animals. Thus, the increase of VLDL and IDL appears to result from the decrease of both of LPL and H-TGL.     

Photoperiodic regulation of body mass, food intake, hibernation, and reproduction in intact and castrated male European hamsters,Cricetus cricetus

A group of sexually active male European hamsters were raised either in short-photoperiod conditions (SP; LD 8:16) or in long-photoperiod conditions (LP; LD 16:8) from their capture at the end of the hibernation period. Another group of hamsters was castrated in April and gonadectomized animals were maintained in SP and cold (7 degrees C) or in a succession of SP and LP plus cold. Another group, castrated in May or in September and raised in LP conditions, was transferred in September to SP conditions and cold. 1. Normal hamsters raised in continuous SP or LP apparently did not show signs of rhythmic behavior, except possibly in gonadal activity. 2. Body weight increased continuously, plasma testosterone levels oscillated between 1.5 and 2.5 ng/ml, and animals raised in SP and in cold did not enter hibernation. 3. Similar results were also found in castrated animals kept in SP conditions and cold. 4. The sequence LP-SP induced a decrease in food intake and body weight and a decrease in plasma testosterone levels and triggered entry into hibernation in both intact and castrated animals. 5. After 6 months continuously in SP and with exposure to cold spontaneous recrudescence in food intake and body weight occurred and hibernation ended in both intact and castrated animals. 6. In normal animals a spontaneous increase in plasma testosterone levels was observed. 7. In both normal and gonadectomized animals the phase of refractoriness could be broken by exposure to LP conditions. 8. The critical photoperiod lies between 15 and 15.5 h. These results demonstrate that the European hamster is a photoperiodic species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of lipoprotein lipase from pig myocardium.

1. Lipoprotein lipase was purified from pig myocardium by a two-step purification procedure involving (a) the formation of an enzyme-substrate complex and (b) affinity chromatography on Sepharose which contained covalently linked heparin. The purified enzyme gave in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis one main band with an apparent molecular weight of 73 000. The enzyme, which was purified 70 000-fold, had a specific activity of 860 mumol of unesterified fatty acid liberated/h per mg of protein. 2. The purified enzyme hydrolysed [14C]triolein emulsions in the absence of added cofactors but its activity was increased fivefold by adding normal human serum. Of the low-density lipoprotein apoproteins only apolipoprotein CII could be substituted for serum in activating the enzyme. This lipase had maximum activity at 0.05-0.15 M-NaCl. Heparin increased the activity of the purified enzyme twofold at low concentrations, but high concentrations inhibited. The triglyceride lipase of pig myocardium thus resembles lipoprotein lipase purified from adipose tissue and from plasma, but is clearly different from pig hepatic triglyceride lipase.     

Effect of temperature on lipoprotein lipase and lipogenic enzyme activities in brown adipose tissue of hypophysectomized rats

It has been shown that the same modifications on the composition of brown adipose tissue (BAT) which are normally induced following cold stimulation are also observed in hypophysectomized rats acclimated either at 28 degrees C or 15 degrees C. To test the possibility of BAT stimulation in hypophysectomized rats, we have determined some enzymatic activities known to modulate the energy supply to that organ. Seven week old Long-Evans rats were hypophysectomized. Three weeks later, they were exposed to either 28 degrees C or 15 degrees C ambient temperature for five or six weeks. Hypophysectomized rats were compared to age matched or weight matched controls. Total lipoprotein lipase activity (LPL) (triglyceride uptake) was enhanced in BAT of 28 degrees C hypophysectomized rats compared to controls. Cold acclimation led to a large increased activity. Total LPL activity was comparable in BAT of hypophysectomized and control rats. Total malic enzyme and glucose-6-phosphate dehydrogenase activities (in situ lipogenesis) were doubled in BAT of 28 degrees C hypophysectomized compared to controls. A large enhancement was observed in BAT of either 15 degrees C control or 15 degrees C hypophysectomized rats. Among the studied organs (liver, white adipose tissue, heart, BAT) hypophysectomy promotes the three enzyme activities only in BAT. These variations were discussed with relation to the effect of hypophysectomy on brown adipose tissue at 15 degrees C and 28 degrees C.