A comparison of tissue lipoprotein lipase activities determined in either fresh or defatted tissue preparations

• 1.1. Lipoprotein lipase activities were determined in either fresh aqueous homogenates or homogenates of acetone-diethyl ether dried powders of adipose tissue, heart, skeletal muscle and lung tissue taken from both fed and 24 hr starved rats.
• 2.2. The total tissue enzyme activities detectable in powder preparations were considerably higher than those of fresh preparations in all the tissue except lung.
• 3.3. The identity of the enzyme activity was more clearly demonstrable with homogenates of solvent-dried powders.
• 4.4. The use of both types of preparation in an experiment where rats were injected with either saline or colchicine further demonstrated the advantages of the acetone-diethyl ether-dried tissue preparation in total tissue lipoprotein lipase activity determinations.

     

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.     

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.     

The significance of lipoprotein lipase in rat skeletal muscles.

Lipoprotein lipase was assayed in extracts of acetone-ether powders of rat skeletal muscles. Enzyme activity in soleus had typical characteristics of lipoprotein lipase in other tissues: inhibition by molar NaCl and protamine sulfate and activation by the human apolipoprotein, R-glutamic acid. Activity in muscles with predominantly red fibers (soleus, diaphragm, lateral head of gastrocnemius and anterior band of semitendinosus) was higher than in those with predominantly white fibers (body of gastrocnemius and posterior band of semitendinosus). No effect of a 24 hour fast upon enzyme activity was observed in ten skeletal muscles, but activity decreased substantially in four adipose tissue depots and increased slightly in heart muscle with fasting. Four minutes after intravenous injection of labeled lymph chylomicrons, skeletal muscles with predominantly red fibers incorporated several times more chylomicron triglyceride fatty acids than thos with predominantly white fibers. Estimated lipoprotein lipase activity in total skeletal muscle was about two-thirds that in total adipose tissue of rats fed ad libitum. After a 24 hour fast, total activity in skeletal muscle was about twice that in adipose tissue. These data suggest that a substantial fraction of lipoprotein lipase is in skeletal muscle of rats and that this tissue, especially its red fibers, is an important site of removal of triglycerides from the blood.     

Clearing-factor lipase in adipose tissue. Studies with puromycin and actinomycin

1. When adipose tissue from starved rats is incubated in a medium containing glucose, insulin, heparin and actinomycin (5mug./ml.) the total clearing-factor lipase activity of the system increases at least tenfold over a period of 9hr. In the absence of actinomycin, enzyme activity also increases, but to a lesser extent and for only about 3hr. Some enzyme activity appears in the incubation medium in both the presence and the absence of actinomycin. 2. When the glucose and insulin of the incubation medium are replaced by pyruvate and heparin is omitted, an increase in the total clearing-factor lipase activity in the presence of actinomycin still occurs, but only after a lag of several hours. When only heparin is omitted from the medium, the rise in enzyme activity begins immediately, but there is a shoulder in the time-course curve after a few hours. In the absence of heparin, little enzyme activity appears in the incubation medium. 3. The increases in enzyme activity in the presence of actinomycin are prevented if puromycin (0.5mg./ml.) is present in the incubation medium. 4. Catecholamines and corticotrophin inhibit the increase in enzyme activity caused by actinomycin. 5. The clearing-factor lipase activity of adipose tissue from fed animals declines with a half-life of between 1 and 1.5hr. when the tissue is incubated in the presence of puromycin. The clearing-factor lipase activity of adipose tissue from starved animals is stable under similar circumstances, as is the raised activity found after such tissue has been incubated in the presence of actinomycin. 6. Clearing-factor lipase extracted from adipose tissue of fed animals is less stable in solution than that extracted from the tissue of starved animals after this has been incubated in the presence of actinomycin.     

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 nutrition on subcellular localization of rat fat-cell lipoprotein lipase.

This study supports the possibility for multiple subcellular forms of lipoprotein lipase. 1. The total activity of lipoprotein lipase per g of intact epididymal adipose tissue from fed rats is much higher than that from starved rats. 2. The isolated fat-cells of fed and of starved rats have lipoprotein lipase of almost the same activity per g of fat-pads. The isolated fat-cells of starved rats have a much higher proportion of total activity per g of the intact tissue than do those of fed rats. 3. Under the conditions of homogenization used, only a small proportion of the total activity per g of intact tissue from fed rats was associated with the fat layer which floated to the top of the homogenate during low-speed centrifugation. The different proportions of the specific enzyme activity found in each subcellular fraction are described. 4. Lipoprotein lipase from plasma membranes and microsomal fractions from starved and fed rats was purified by affinity chromatography. 5. The total activity of microsomal lipoprotein lipase per g of intact adipose tissue is enhanced by a normal diet. 6. In intact epididymal adipose tissue from fed rats, the activity per g of tissue of lipoprotein lipase of plasma membranes is much higher than that in the same fraction from starved rats. By contrast, the activities per g of tissue in plasma membranes obtained from starved or from fed rats by collagenase treatment were similar.     

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.     

Clearing-factor lipase in adipose tissue. Factors influencing the increase in enzyme activity produced on incubation of tissue from starved rats in vitro

1. Evidence is presented that the increase in clearing-factor lipase activity that occurs when adipose tissue from starved rats is incubated in a defined medium in vitro is due to an increase in the total enzyme content of the system. It is shown that the clearing-factor lipase activity rises to reach a plateau level where, it is suggested, rates of enzyme synthesis and of enzyme destruction become balanced. 2. The presence of heparin in the incubation medium results in the extraction of part of the clearing-factor lipase originally present in the adipose tissue and this could provide the stimulus for the increase in total enzyme content. 3. Glucose is required in the incubation medium at a very low concentration. It can be replaced by fructose, but not by pyruvic acid, lactic acid, glyceric acid or dihydroxyacetone. 4. Adrenaline and corticotrophin inhibit the increase in enzyme activity when they are present in the incubation medium. 5. The high clearing-factor lipase activity associated with adipose tissue of fed rats is decreased by 50% within 3hr. of the injection of puromycin.     

Clearing-factor lipase in muscle and adipose tissue of pigs

1. Clearing-factor lipase was assayed in acetone-ether-dried powders of heart and adipose tissue of pigs. The enzyme activity in heart was higher than that in adipose tissue. The activity in the outer layer of subcutaneous fat was greater than that in the inner subcutaneous fat and the perirenal fat, which had similar activities. 2. Starvation for 48h, but not for 24h, decreased the activity of the heart enzyme. 3. Starvation for 24h caused a rapid decrease in the activity in all three adipose tissues, but even after 72h of starvation the activity was still highest in the outer subcutaneous fat. 4. Plasma fatty acid, glucose and insulin concentrations were determined in fed and starved pigs. Starvation decreased the plasma insulin concentration and increased the non-esterified fatty acid concentration.     

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 mechanism of heparin stimulation of rat adipocyte lipoprotein lipase

Free fat cells and stromal-vascular cells were prepared from rat adipose tissue by incubation with collagenase. NH(4)OH-NH(4)Cl extracts of acetone-ether powders prepared from fat cells contained lipoprotein lipase activity but extracts of stromal-vascular cells did not. Intact fat cells released lipoprotein lipase activity into incubation medium, but intact stromal-vascular cells did not. The lipoprotein lipase activity of the medium was increased when fat cells were incubated with heparin, and this was accompanied by a corresponding decrease in the activity of subsequently prepared fat cell extracts. Heparin did not release lipoprotein lipase activity from stromal-vascular cells. The lipoprotein lipase activity of NH(4)OH-NH(4)Cl extracts of fat cell acetone powders is increased by the presence of heparin during the assay. This increase is not due to preservation of enzyme activity, but to increased binding of lipoprotein lipase to chylomicrons. Protamine sulfate and sodium chloride have little effect on the binding of lipoprotein lipase to chylomicrons, but they inhibit enzyme activity after binding to substrate has occurred. These inhibitors do, however, inhibit the stimulatory effect of heparin on enzyme-substrate binding.     

Clearing-factor lipase in adipose tissue. Distinction of different states of the enzyme and the possible role of the fat cell in the maintenance of tissue activity

1. Incubation of intact epididymal adipose tissue from fed rats at 37 degrees in an albumin solution at pH7.4 in vitro results in rapid loss of clearing-factor lipase activity until a low activity, stable to prolonged incubation, is attained. The clearing-factor lipase activity of intact tissue from starved rats, which is initially much less than that of tissue from fed rats, is mainly stable to incubation at 37 degrees . 2. Much of the clearing-factor lipase activity of intact epididymal adipose tissue from fed rats is inactivated by collagenase. The enzyme activity of intact tissue from starved rats is not inactivated by collagenase. 3. The clearing-factor lipase activity of fat cells isolated from the epididymal adipose tissue of fed rats is stable to prolonged incubation at 37 degrees . It represents only a small proportion of the total activity of the intact tissue. In starved rats, the isolated fat cells contain a much higher proportion of the activity of the intact tissue. Their activity is also stable at 37 degrees . 4. Incubation of isolated fat cells in a serum-based medium leads to a progressive rise in clearing-factor lipase activity. Actinomycin increases the extent of this rise in activity. No rise in clearing-factor lipase activity occurs when stromal-vascular cells isolated from epididymal adipose tissue are incubated in the medium. 5. The findings indicate that less than 20% of the activity of intact adipose tissue from fed rats is retained when fat cells are isolated from the tissue by collagenase treatment. The activity that is lost could be that which normally functions in the uptake of triglyceride fatty acids by the tissue.     

Influence of age and exercise training on lipid metabolism in Fischer-344 rats

The influence of training on fatty acid and glyceride synthesis by liver and adipose tissue homogenates of young and old Fischer-344 rats was examined. Four groups of rats (10 animals/group) were studied: young untrained, young trained, old untrained, and old trained. Training of each group was for 10 wk at 75% maximal O2 uptake. Young rats were killed at 6 mo of age and old rats were killed at 27 mo of age. Fatty acid synthesis was assessed by measuring the activities of acetyl-CoA carboxylase, fatty acid synthase, ATP citrate-lyase, "malic" enzyme, and glucose-6-phosphate dehydrogenase. Glyceride synthesis was evaluated by determining the rate of incorporation of [14C]glycerol 3-phosphate into lipids. In addition, lipoprotein lipase activity was measured in acetone-ether powders of adipose tissue from the four groups of rats. In liver, training had no effect on fatty acid or glyceride synthesis in either group. However, aging caused a significant decrease in the activities of four of the lipogenic enzymes but had no effect on glyceride synthesis. Training caused an increase in fatty acid synthase and glyceride synthesis in adipose tissue, and aging decreased lipoprotein lipase activity. It was concluded that training enhances the synthetic capacity of lipids by adipose tissue but that aging had a more profound effect in that the activities of the enzymes involved in these processes were lower in the old rats. Furthermore, the decreased activity of lipoprotein lipase in the older rats may explain the higher plasma triglyceride levels that were observed in these animals.     

Lipoprotein lipase activity of rat cardiac muscle. Changes in the enzyme activity during incubations of isolated cardiac-muscle cells in vitro.

1. Isolated cardiac-muscle cells from the hearts of adult rats were shown to retain a high amount of viability during 4 h of incubation when viability was assessed by Trypan Bue stain exclusion and intracellular enzyme leakage. 2. The cells also retained their ability to take up O2 and utilize added substrates over the period of incubation at both 25 and 30 degrees C. 3. When cells from the hearts of fed rats were incubated in a buffered-salts solution at pH 7.4 in the presence of amino acids and heparin, lipoprotein lipase activity in the medium increased progressively. 4. During these incubations the intracellular activity of the enzyme remained constant and the total activity of lipoprotein lipase in the system (cells plus medium) increased by 80% over the 4 h of incubation at 25 degrees C. 5. In the absence of heparin only low amounts of enzyme activity were detectable in the medium and the total lipoprotein lipase activity in the system remained constant. 6. The measurement of lipoprotein lipase activity in either fresh homogenates of the cells or in homogenates of acetone/diethyl ether-dried powders of the cells had no effect on the overall pattern of activity change during the incubations, although as reported previously the total activity detected with acetone/diethyl either-dried preparations was approx. 3-fold higher than with fresh cell homogenates. 7. The observations were compared with published data on lipoprotein lipase activity changes in neonatal heart cell cultures maintained in vitro.     

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.     

The metabolism of very low density lipoprotein and chylomicrons by purified lipoprotein lipase from rat heart and adipose tissue

Crude lipoprotein lipase, extracted from rat adipose tissue or heart acetone-ether powders, was purified about 300 and 350 fold respectively by affinity chromatography. Artifactual increments in the density of very low density lipoprotein, noted after incubation with the crude lipoprotein lipase extract from adipose tissue, were abolished when the purified enzyme was used. Purified enzymes from both tissues showed similar modifications of activity in the presence of activators and inhibitors. The triglyceride moieties of various natural substrates were preferentially hydrolysed in the order Very low density lipoprotein > Serum chylomicrons > Thoracic duct chylomicrons by both enzymes.     

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.     

Lipoprotein lipase activity of rat cardiac muscle. The intracellular distribution of the enzyme between fractions prepared from cardiac muscle and cells isolated from the hearts of fed and starved animals.

1. Subcellular fractions, characterized by using morphological, compositional and enzymic markers, were prepared from rat heart tissue and cells isolated from the hearts of fed and 24 h-starved rats. 2. The lipoprotein lipase activity of fractions from whole tissue and isolated cells was determined in either fresh fractions or in acetone/diethyl ether powders of the fractions. 3. Lipoprotein lipase activity was present in all the fractions from tissue and cells, but was found to be of highest relative specific activity in the microsomal () fractions. 4. In fractions prepared from the isolated cells of hearts from starved rats the proportion of the total lipoprotein lipase present and its relative specific activity in the microsomal fraction were greater than in the equivalent fractions from fed animals. 5. The enhancement of lipoprotein lipase activity as a result of the acetone/diethyl ether powder preparation of fractions was most extensive in the microsomal fractions. 6. Investigation of the microsomal fraction showed that the lipoprotein lipase activity present was in two pools, one of which was within endoplasmic-reticulum vesicles. 7. The observations were consistent with the possibility that the cardiac-muscle cell could be the origin of the lipoprotein lipase activity functional in triacylglycerol uptake by the heart.     

Activity of lipoprotein lipase in thyroidectomized rats

Total plasma postheparin lipolytic activity as well as lipoprotein lipase activity in plasma was higher after heparin injection in thyroidectomized rats than in controls. In contrast, the activity of liver lipase was lower in thyroidectomized rats. Adipose tissue from thyroidectomized rats contained more lipoprotein lipase activity than adipose tissue from controls as measured both in extracts of tissue homogenates and medium from in vitro incubations of tissue pieces. There were no differences between control and hypothyroid rats in the disappearance of intravenously injected 125I-labeled lipoprotein lipase, but when a low dose of heparin was injected before the labeled enzyme, the disappearance of 125I-labeled lipoprotein lipase was more retarded in thyroidectomized rats. The elimination of heparin itself was slightly retarded by thyroidectomy.