Specific conditions for enhancement of lipoprotein lipase activity by platelets.

Homogenates of human blood platelets, but not of red blood cells, have been found to stimulate lipoprotein lipase activity only when assayed against an emulsified triglyceride substrate sonicated for a short period of time. The degree of stimulation was inversely related to the time of sonication of the substrate. Using chylomicrons as substrate no stimulation of lipoprotein lipase activity by platelet homogenate was seen.     

Triacylglycerol lipase activities of cultured rat L6 myoblasts

The utilization of exogenous triacylglycerol by fusing and non-fusing rat L6 myoblasts grown in culture was investigated. Although small quantities of triacylglycerol were accumulated by both cell lines during an incubation of 2 h, no evidence could be found for the presence of lipoprotein lipase, either in the cells or released into the medium. Cell homogenate studies confirmed the absence of lipoprotein lipase but revealed the presence of an acid lipase having a pH optimum at 4.6. Acid lipase activity was mainly associated with a 15 000 g pellet and was capable of hydrolysing triolein at maximum velocity in the millimolar range. Unlike lipoprotein lipase, acid lipase was strongly inhibited by serum and preliminary investigations suggest that the inhibitory component of serum is located amongst the higher density lipoproteins. It is likely that the acid lipase is of lysosomal origin and is responsible for the hydrolysis of internalized triacylglycerol for subsequent utilization by the cell.     

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.     

Effect of low doses of ethanol on platelet function in long-life abstainers and moderate-wine drinkers

In vitro, high concentrations of ethanol (EtOH) reduce platelet aggregation. Less is known about the effect of low EtOH doses on platelet function in a selected human population of long-life abstainers and low moderate-wine drinkers to avoid rebound effect of EtOH on platelet aggregation. Results of our experiments suggest that moderate-wine drinkers have higher levels of high density lipoprotein (HDL) than long-life abstainers while fibrinogen levels are unchanged. Furthermore, platelets obtained from these individuals do not differ in their response when stimulated by agonists such as AA and collagen. The effect of in vitro exposure of low doses of EtOH has been studied in PRP and in washed platelets. EtOH (0.1-10 mM) inhibits platelet aggregation induced by collagen at its ED50 while is ineffective when aggregation was triggered by U-46619 and by 1 microM adenosine diphosphate (ADP). 5-10 mM EtOH partially reduces the second wave of aggregation induced by 3 microM ADP. 0.1-10 mM EtOH dose-dependently lowers the aggregation induced by AA at its ED50 but it is less effective at ED75 of AA. The antiaggregating effect of EtOH on aggregation induced by AA is unchanged by inhibitor of nitric oxide synthase. In addition, 10 mM EtOH reduces thromboxane (Tx) formation. In washed platelets, 1-10 mM EtOH partially inhibits platelet aggregation induced by thrombin. In washed resting platelets, 10 mM EtOH does not change the resting [Ca++]i while significantly reduces the increase in [Ca++]i triggered by AA. The results of ex vivo experiments have demonstrated that wine increases the HDL. However, this observation may or may not influence the response of platelets to agonists. Results of our studies demonstrate that low doses of alcohol reduces platelet function.     

RHC 80267 does not inhibit the diglyceride lipase pathway in intact platelets

RHC 80267 inhibits diglyceride lipase activity in microsomes from canine platelets (1). Chau and Tai (2) reported that RHC 80267 prevents the transient accumulation of monoglyceride in thrombin-stimulated human platelets, while leaving arachidonate release unimpaired. In contrast, we find that while the drug inhibits both diglyceride lipase (I₅₀=15 μM) and monoglyceride lipase (I₅₀=11 μM) activities in platelet microsomes, it is ineffective when added to intact platelets. The transient intermediates in the diglyceride lipase pathway, 1,2-diglyceride and 2-monoglyceride, both accumulated after thrombin stimulation of intact platelets treated with RHC 80267, and arachidonate release was not inhibited. We conclude that RHC 80267 cannot be used to evaluate the diglyceride lipase pathway in intact platelets.     

Activation of myocardial neutral triglyceride lipase and neutral cholesterol esterase by cAMP-dependent protein kinase

Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.     

Effect of cod liver oil supplementation on plasma lipids, lipoproteins, lipase activity and platelet aggregation in normotensive and hypertensive volunteers

No significant change in plasma levels of total cholesterol (TC), triglycerides, phospholipids, very-low-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), lipase activity and TC/HDL-C ratio could be observed in both normotensive and hypertensive individuals after cod liver oil supplementation. Measure of platelet aggregation rates did not also show any significant change after cod liver oil ingestion in both normotensive and hypertensive individuals. The results suggest that supplementation of normal diets with 600 mg cod liver oil per day for 50 days neither affects plasma lipids, lipoproteins and lipase activity nor affects platelet aggregation in both normotensive and hypertensive individuals.     

Isolation of homogeneous phospholipase A2 from human platelets

Phospholipase A₂ from human blood platelets has been purified to homogeneity by treatment of the platelet homogenate with H₂SO₄, followed by affinity chromatography of the extract. The platelet phospholipase is a soluble, heat-stable protein of MW 44000. The purified enzyme inhibits ADP-induced platelet aggregation.     

Retention of glucose by N-linked oligosaccharide chains impedes expression of lipoprotein lipase activity: effect of castanospermine.

The effect of castanospermine (CSTP), an inhibitor of glucosidase I, on processing, activity, and secretion of lipoprotein lipase was studied in 3T3-L1 adipocytes. Processing was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endoglycosidase H (endo H)-digested subunits of lipoprotein lipase from cells incubated 1-2 h with [35S]methionine. Lipoprotein lipase in untreated cells consisted of two groups of subunits, M(r) = 55,000-58,000 and M(r) = 53,000-55,000. The heavier subunits were endo H-resistant, whereas the others were either totally or partially endo H-sensitive. The lipase secreted by untreated cells contained primarily endo H-resistant subunits. Immunofluorescent studies showed that lipoprotein lipase accumulated in Golgi in untreated cells. CSTP, 100 micrograms/ml for 18 h, decreased intracellular lipase activity by 80% and decreased secretion of lipase activity by 91%. Most of the lipase subunits in CSTP-treated cells were totally endo H-sensitive with M(r) = 57,000, some were partially endo H-sensitive, and a trace was endo-H resistant. Totally endo H-sensitive subunits in CSTP-treated cells had a M(r) 2,000-4,000 larger than that in untreated cells, indicating impaired trimming of sugar residues from oligosaccharide chains of the lipase in CSTP-treated cells. The small amount of lipase secreted by CSTP-treated cells consisted primarily of partially endo H-sensitive subunits, with one sensitive and one resistant chain per subunit. Immunofluorescent studies showed that lipoprotein lipase was excluded from Golgi in CSTP-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation

To assess the possibility that hydrolysis of the platelet surface thrombin substrate, glycoprotein V, is a necessary step in thrombin-induced platelet activation, thrombin-catalyzed hydrolysis of glycoprotein V was correlated with thrombin-induced platelet activation. Hydrolysis of tritium-labeled glycoprotein V on washed human platelets was measured by the appearance of a labeled supernatant fragment, and platelet activation was measured as secretion of ATP. Hydrolysis of glycoprotein V was linear with respect to both thrombin concentration and time of incubation. The extent of platelet activation was correlated with the rate of hydrolysis but not with the amount hydrolyzed. Maximum platelet activation could be obtained with thrombin treatments resulting in hydrolysis of as little as 4% of glycoprotein V per min. Glycoprotein V was partially removed from platelets by pretreatment with either platelet calcium-dependent protease or chymotrypsin. The rate of thrombin-catalyzed hydrolysis of the remaining glycoprotein V from these pretreated platelets was as little as 1.5% the rate from control platelets, but there was no impairment of the extent of platelet activation. Thus, these protease-pretreated platelets compared with control platelets showed a different correlation of glycoprotein V hydrolysis with platelet activation. Glycoprotein V was also partially removed by pretreatment of prostacyclin-inhibited platelets with thrombin. After removal of thrombin and prostacyclin, these platelets were desensitized to subsequent activation by thrombin. Incubation of desensitized platelets with nonsaturating levels of thrombin led to less than 25% of the activation seen with control platelets but to a slightly greater hydrolysis of glycoprotein V. Thus, the desensitization to thrombin was not due to loss of ability of the activating thrombin to hydrolyze glycoprotein V. These results do not exclude a role for glycoprotein V as a component of the platelet thrombin receptor, but they indicate that there is no simple relationship between thrombin-induced hydrolysis of glycoprotein V and platelet activation.     

Plasma lipoproteins affect platelet malondialdehyde and thromboxane B2 production

Platelet interaction with plasma lipoproteins was studied using gel-filtered platelets free of plasma constituents and purified lipoproteins. On incubation of gel-filtered platelets with plasma lipoproteins at 30 degrees C for 30 min, 100 micrograms of protein/ml of very-low as well as low-density lipoprotein caused 10% increment in platelet aggregation and [14C]serotonin release in parallel to elevation of around 15% of malondialdehyde and thromboxane B2 production. High-density lipoprotein showed the opposite effect and reduced platelet aggregation as well as thromboxane B2 synthesis by 17 and 32%, respectively. Lipoprotein-deficient plasma enhanced platelet function. Preincubation of the platelet suspension with prostacyclin did not prevent the effect of the lipoproteins on the in vitro platelet response as well as on the platelet prostaglandin pathway. Our results suggest that the formation of thromboxane B2 and malondialdehyde is influenced by plasma lipoproteins and that these, in turn, affect platelet aggregation and the release reaction. The possible significance of these results to platelet function in hyperlipidemic patients is discussed.     

Rapid activation of human platelets by low concentrations of low-density lipoprotein via phosphatidylinositol cycle

The interaction of low-density lipoprotein (LDL) with the human platelet was investigated with regard to saturable high-affinity binding, shape change, cytosolic free Ca2+ concentration, phosphatidylinositol (PtdIns) turnover, and thromboxane B2 biosynthesis. The experiments show that LDL, at a concentration approximately 100 times lower than in plasma, causes platelet activation concomitantly with stimulation of the PtdIns cycle and thromboxane B2 formation, similarly to other activators of platelets. The effects of LDL were inhibited by high-density lipoprotein. The results suggest that activation of platelets by low concentrations of LDL may play a role in pathophysiological conditions and that platelet can serve as a model for studying the influence of LDL on various target cells.     

Native and modified low-density-lipoprotein interaction with human platelets in normal and homozygous familial-hypercholesterolaemic subjects.

The binding of low-density lipoproteins (LDL) as well as LDL modified by cyclohexanedione (CHD-LDL) to gel-filtered platelets (GFP) and its effect on platelet function were studied in normal and in homozygous familial hypercholesterolaemic (HFH) subjects. Only normal-derived LDL could significantly compete with normal 125I-labelled LDL for binding to normal platelets. When GFP from normal subjects were incubated with normal LDL at concentrations of 25-200 micrograms of protein/ml, platelet aggregation in the presence of thrombin (0.5 i.u./ml) was increased by 65-186%. CHD-LDL, at similar concentrations, caused the opposite effect and decreased platelet aggregation by 26-47%. Both LDL and CHD-LDL (100 micrograms/ml) from HFH patients, when incubated with normal GFP, caused a significant reduction in platelet aggregation (33 and 50% respectively). When HFH-derived platelets were used, both patient LDL and CHD-LDL (but not the normal lipoprotein) could markedly compete with the patient 125I-labelled LDL for binding to the platelets. LDL and CHD-LDL (100 micrograms/ml) from normal subjects decreased aggregation of HFH-platelets by 52 and 85% respectively, while corresponding concentrations of LDL derived from HFH subjects (HFH-LDL) and CHD-LDL derived from HFH subjects (CHD-HFH-LDL) increased platelet aggregation by 165 and 65% respectively. The present results support the following conclusions: platelet activation by LDL in normal subjects is through the arginine-rich apoprotein-binding site; more than one binding site for LDL exists on platelets; under certain circumstances, LDL binding can cause a reduction in platelet activity; specificity for LDL binding to the platelets resides in different regions of the lipoprotein in HFH and in normal subjects. We have thus suggested a model for LDL-platelet interaction in normal and in HFH subjects.     

The functional status of lipoprotein lipase in rat liver

1. Acetone-dried powders of liver and heart tissues from rats given a high-carbohydrate diet or a fat meal were assayed for lipoprotein lipase activity. Heart tissue showed typical lipoprotein lipase activity, whereas none was detected in liver by the usual assay procedures. 2. When mixed acetone-dried powders were prepared from heart plus liver, there was a marked suppression of the expected activity, indicating that an inhibitor was present in the liver. This inhibition was partially overcome in the presence of relatively large amounts of heparin. 3. Lipoprotein lipase was also detected in liver alone when large quantities of heparin were added to the assay system. 4. No increase in lipoprotein lipase activity in either liver or heart was detected when rats were given a fat meal. 5. It is concluded that the liver of the rat contains lipoprotein lipase that is normally present in an inactive state. The results imply that a heparinase is the agent responsible for the inactivation. 6. The significance of the non-functional status of lipoprotein lipase in the liver is discussed. The results support the view that direct hydrolysis of plasma triglycerides by the liver is not a significant physiological process.     

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.     

Insulin-mediated modifications of myocardial lipoprotein lipase and lipoprotein metabolism

Recirculating organ perfusion in vitro was conducted with hearts from control rats, animals given a single dose of streptozotocin (65 mg/kg) 48 h earlier, and streptozotocin-treated rats administered insulin (5 units), 2 h prior to organ perfusion. During 45-min perfusions, the lipolysis of very low density lipoprotein (VLDL) triglyceride was significantly less in hearts from diabetics than in controls (41.9 +/- 7.3% of control). This was associated with significant reductions in heparin-releasable (functional) lipoprotein lipase and tissue lipoprotein lipase of perfused hearts. The decreases in VLDL triglyceride metabolism and the levels of myocardial lipoprotein lipase were completely reversed by treatment of diabetic rats with insulin 2 h prior to study. Similar improvement of VLDL triglyceride metabolism and increases in myocardial lipoprotein lipase activity were observed in hearts from diabetic rats by direct addition of 100 milliunits/ml of insulin to the recirculating perfusion media. Under these conditions, the increase in both fractions of lipoprotein lipase in response to insulin was completely inhibited, and utilization of VLDL triglyceride was partially inhibited by pre-perfusion with cycloheximide for 10 min. The data derived from either VLDL triglyceride lipolysis in organ perfusion or direct measurement of myocardial lipoprotein lipase demonstrate a direct effect of insulin on myocardial lipoprotein lipase activity, and suggest that the response to insulin may be due in part to effects on protein synthesis.     

From low-density lipoprotein to platelet activation

There is a strong correlation between the level of plasma low-density lipoprotein (LDL) and death by cardiovascular disease (CVD). As a main carrier of cholesterol, a high low-density lipoprotein concentration stimulates atherogenesis by its capacity to become oxidized and to become endocytosed by macrophages in the vessel wall forming cholesterol-rich plaques that are sites for arterial occlusion. New evidence points at a second role of low-density lipoprotein in increasing cardiovascular disease-risk. Contact with low-density lipoprotein induces platelet hypersensitivity to agonists that initiate platelet functions thereby enhancing adhesion, aggregation and secretion of granule contents. The signalling pathways that mediate the priming of platelets by native and oxidized low-density lipoprotein have now been characterized.     

Measurement of two plasma triglyceride lipases by an immunochemical method: studies in patients with hypertriglyceridemia.

Postheparin plasma lipolytic activity consists of two hydrolytic activities, hepatic triglyceride lipase and lipoprotein lipase. These two enzymes were separated and partially purified by means of ammonium sulfate precipitation and affinity chromatography using Sepharose with covalently linked heparin and concanavalin A, respectively. Antibodies were produced against hepatic triglyceride lipase and they did not cross react with lipoprotein lipase. Optimal conditions for selective precipitation of hepatic lipase and specific measurement of these two lipases were investigated. This method was applied to the study of 15 patients with hypertriglyceridemia and 8 patients with familial lecithin-cholesterol-acyltransferase deficiency of whom 6 also had a marked elevated plasma triglyceride concentration. All patients had normal values of hepatic plasma lipase. All 8 patients with Type I and 2 of 4 patients with Type V hyperlipoproteinemia had lipoprotein lipase activities that were markedly reduced. The patients with Type III hyperlipoproteinemia and all 8 patients with lecithin-cholesterol-acyltransferase deficiency also had normal lipoprotein lipase values. These studies emphasize the necessity for differentiating between triglyceride lipase activity of hepatic and extrahepatic origin in evaluating patients with impaired triglyceride metabolism.