Lipid metabolism in human platelets: I. Evidence for a complete fatty acid synthesizing system

Extracts from human platelets contain the enzymes of de novo fatty acid biosynthesis. The pattern of incorporation of acetate-1-(14)C into fatty acids by intact platelets indicates that these enzymes function in platelets. The level of acetyl-coenzyme A (CoA) carboxylase activity in extracts of platelets from normal subjects is 0.036 +/-0.01 mmumole of malonyl-CoA formed per min per mg of protein and that of fatty acid synthetase is 0.075 +/-0.016 mmumole of malonyl-CoA utilized per min per mg of protein. Thus, platelets are the only formed elements of the blood capable of de novo fatty acid synthesis. The capacity of platelets to synthesize fatty acids is similar to human liver based on enzyme activity per milligram of soluble protein.Acetyl-CoA carboxylase was purified 16-fold from platelet extracts, and this partially purified enzyme was compared to enzyme from rat liver. The two enzymes were similar with respect to requirements, substrate affinities, pH profile of activity, inhibition by malonyl-CoA, and aggregation in the presence of citrate. Thus, while fatty acid synthesis may serve a different function in platelets than in liver, the properties of acetyl-CoA carboxylase from these tissues are alike.The levels of the enzymes of fatty acid synthesis were significantly higher in platelets from splenectomized subjects than in controls. Acetyl-CoA carboxylase levels were 0.086 +/-0.027 mmumole of malonyl-CoA formed per min per mg of protein, and fatty acid synthetase levels were 0.151 +/-0.039 mmumole of malonyl-CoA utilized per min per mg of protein. These changes in the enzymes of fatty acid synthesis occurred promptly after splenectomy with peak values being reached within 7-10 days.     

The enzyme glutathione peroxidase in arachidonic acid metabolism of human platelets

We investigated the possible regulatory role of glutathione peroxidase on thromboxane formation by reducing peroxides in platelets. Experiments carried out in platelet lysates demonstrated that the burst of the arachidonate metabolism was accompanied by a simultaneous burst of hydrogen transfer from glutathione to peroxides, catalyzed by endogenous glutathione peroxidase. The burst of hydrogen transfer was partially inhibited by acetylsalicylate concurrently with the complete inhibition of malondialdehyde formation, thus suggesting that the hydrogen acceptor peroxides were derived in part from the cyclooxygenase pathway. Moreover, increasing glutathione peroxidase activity by adding purified enzyme to the incubation media decreases thromboxane formation. Intact platelets, stimulated with arachidonic acid or thrombin, produced malondialdehyde and thromboxane in amounts roughly inversely related to the endogenous glutathione peroxidase activity. In contrast, no correlation was observed between glutathione peroxidase activity and agonist-induced platelet aggregation. Our experiments suggest that in normal platelets, glutathione peroxidase controls thromboxane formation.     

Effects of anti-convulsants on fatty acid metabolism in human serum

The effects of diphenylhydantoin and phenobarbital on lipids metabolism were studied by quantification of lipids in human serum with GC-MS. The ratios of palmitic acid to stearic, oleic and linoleic acids decreased in patients on diphenylhydantoin therapy. Increases of free palmitic, stearic and oleic acids concentrations as well as higher cholesterol ester levels were observed in patients receiving either diphenylhydantoin or phenobarbital. With respect to fatty acids composition of cholesterol esters and phosphatidyl-choline, higher concentrations of palmitic, oleic and linoleic acids were observed in patients on diphenylhydantoin and phenobarbital therapy. Although the concentration of palmitic acid in serum phosphatidylcholine remained the same for all groups, a marked decrease in stearic, oleic and linoleic acids concentrations in serum phosphatidylcholine was observed in diphenylhydantoin treated group, and oleic acid in phenobarbital treated group. These results suggested the relationship to lecithin-cholesterol acyltransferase activity by these drugs.     

Citrate metabolism by human platelets

As part of a study on the utilization of substrates by platelets in defined media the metabolism of citrate was measured, since citrate is a common anticoagulant of nearly all such media, and is also an intermediate of oxidative metabolism. Human platelets transferred from plasma to an artificial medium by gel filtration, were incubated with [¹⁴C]citrate at 22 °C and labelled carbon dioxide produced was measured during short-term incubations of 2 h. Citrate (1 m m ) was oxidized to carbon dioxide at low (0.3 nmol per 10⁹ platelets h⁻¹) but significant rates, and the oxidation was decreased by the presence of an alternative substrate (acetate) in the medium. There was, however, no significant conversion of citrate to glycogen. It was calculated that under normal storage conditions of platelet concentrates for transfusion purposes, the amount of citrate used cannot decrease citrate concentrations sufficiently to bring about platelet activation.     

Free fatty acid metabolism of the human heart at rest

Myocardial substrate metabolism was studied in 13 subjects at the time of diagnostic cardiac catheterization by means of palmitic acid-(14)C infusion with arterial and coronary sinus sampling. Two subjects were considered free of cardiac pathology and all, with one exception, demonstrated lactate extraction across the portion of heart under study. Data for this single lactate-producing subject were treated separately.The fractional extraction of (14)C-labeled free fatty acids (FFA) (44.4+/-9.5%) was nearly twice that of unlabeled FFA (23.2+/-7.8%) and raised the possibility of release of FFA into the coronary sinus. FFA uptake, based on either the arterial minus coronary sinus concentration difference or the FFA-(14)C fractional extraction, was directly proportional to the arterial FFA concentration. Gas-liquid chromatography failed to demonstrate selective handling of any individual FFA by the heart. Fractional oxidation of FFA was 53.5+/-12.7%, accounting for 53.2+/-14.4% of the heart's oxygen consumption while nonlipid substrates accounted for an additional 30.0+/-17.3%. Determinations of both labeled and unlabeled triglycerides suggested utilization of this substrate by the fasting human heart.Direct measurement of FFA fractional oxidation as well as FFA uptake, exclusive of possible simultaneous FFA release, would appear necessary in studies concerned with human myocardial FFA metabolism.     

Targeting fatty acid metabolism in glioblastoma

Glioblastoma (GBM) is a primary tumor of the brain defined by its uniform lethality and resistance to conventional therapies. There have been considerable efforts to untangle the metabolic underpinnings of this disease to find novel therapeutic avenues for treatment. An emerging focus in this field is fatty acid (FA) metabolism, which is critical for numerous diverse biological processes involved in GBM pathogenesis. These processes can be classified into four broad fates: anabolism, catabolism, regulation of ferroptosis, and the generation of signaling molecules. Each fate provides a unique perspective by which we can inspect GBM biology and gives us a road map to understanding this complicated field. This Review discusses the basic, translational, and clinical insights into each of these fates to provide a contemporary understanding of FA biology in GBM. It is clear, based on the literature, that there are far more questions than answers in the field of FA metabolism in GBM, and substantial efforts should be made to untangle these complex processes in this intractable disease.     

Interaction between glucose and free fatty acid metabolism in human skeletal muscle.

The mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.     

Energy substrate metabolism in fresh and stored human platelets

The latent capacity of human platelets for oxidizing several important energy-yielding substrates has been revealed by hypoosmolaric incubation conditions.The data show that the human platelet has a considerable capacity to oxidize both glucose and long-chain fatty acids. Long-chain fatty acids appear to rank favorably with glucose as a potential energy substrate. In a number of mammalian tissues, (-)-carnitine serves to regulate the rate at which long-chain fatty acids are oxidized. Evidence was obtained which suggests that (-)-carnitine functions in a similar role in the platelet.After storage of human platelets at 4 degrees C for 24 hr, the oxidative capacity for glucose was reduced by approximately 25% and for long-chain fatty acids by almost 50%. Investigation of the component parts of the metabolic pathways indicated that a marked decrease in the capacity of the Krebs cycle could be responsible for the decrement in energy substrate oxidation.     

Utilization of long-chain free fatty acids by human platelets

There was a rapid net uptake of free fatty acid (FFA) by human platelets when long-chain FFA, bound to human serum albumin, were incubated with platelet suspensions. Results from experiments in which both palmitate and albumin were labeled indicated that the fatty acid dissociated from the protein during uptake. Much of the FFA taken up by the platelet in short-term incubations remained in unesterified form, i.e., it was recovered as platelet FFA. As the incubation continued, increasing amounts of FFA were oxidized to CO(2) and incorporated into platelet lipid esters, particularly lecithin. Essentially all of the fatty acid that was incorporated into the platelet FFA fraction was released rapidly from the cells when they were exposed to a medium containing FFA-free albumin. The magnitude of uptake into the platelet FFA fraction was similiar at 0 degrees and 37 degrees C. Likewise, the rate and magnitude of FFA release from the platelet were similar at 0 degrees and 37 degrees C. Therefore, it is likely that both FFA uptake and FFA release occur by energy-independent mechanisms. The major effect of increasing the FFA concentration of the incubation medium was increased fatty acid uptake into the platelet FFA fraction. Similar results occurred when platelets were incubated in human plasma containing increasing amounts of added palmitate. At a given extracellular FFA concentration, considerably more of the saturated fatty acids, palmitate and stearate, were taken up as platelet FFA than either oleate or linoleate.     

Altered lipid metabolism in human platelets after primary aggregation

Thrombin and poly-l-lysine alter the incorporation of acetate, glycerol, and fatty acids into the lipids of washed human platelets. Both aggregating agents decrease the incorporation of acetate into all lipid classes other than free fatty acids. Similarly, glycerol incorporation into complex lipids is impaired by both thrombin and polylysine. Thrombin caused marked depression of the incorporation of palmitic acid into both lecithin and triglycerides. By contrast it enhanced the incorporation of oleic acid into lecithin, but not into triglycerides. The data suggest that the process of primary platelet aggregation is associated with a defect in the assembly of complex lipids.     

Regulation of Plasma fatty acid metabolism.

Although adipose tissue serves a crucial function in energy storage, excess adipose tissue--that is, obesity--is often associated with diabetes and cardiovascular disease. A common thread in the weave of complications is increased plasma concentrations of fatty acids. In the present review, we have focused on two specific points that relate to obesity: (i) What are the metabolic consequences of increased free fatty acid concentrations? and (ii) What are the physiological factors that are involved in the regulation of fatty acid uptake or release from adipose tissue? We have tried to emphasize new factors that act as hormones on adipose tissue and in so doing regulate the net concentration of circulating free fatty acids.     

Stimulation of glucose metabolism in human blood cells by inhibitors of carnitine-dependent fatty acid transport

According to a well accepted hypothesis, increased fatty acid oxidation can lead to hyperglycaemia by stimulating gluconeogenesis and reducing glycolysis. Therefore, inhibitors of fatty acid metabolism should cause hypoglycaemia by inhibiting gluconeogenesis and activating glycolysis. Various substances were tested to validate this hypothesis with regard to glucose oxidation in human mononuclear leukocytes and thrombocytes. 2-(3-Methyl-cinnamyl-hydrazono)-propionate, an inhibitor of the carnitine acyltransfer system was found to cause hypoglycaemia in whole animals and to inhibit gluconeogensis in the perfused guinea pig liver, while the acetyl-CoA/CoASH ratio was decreased. This substance stimulated the metabolism of glucose to CO2 in human mononuclear leukocytes and especially in platelets. This effect could be potentiated if concanavalin A and 2-(3-methyl-cinnamyl-hydrazono)-propionate were applied simultaneously. Under these conditions, however, fatty acid oxidation was no longer inhibited. From these results, it can be concluded that the activation of glucose oxidation by 2-(3-methyl-cinnamyl-hydrazono)-propionate is independent of its effect on fatty acid metabolism. Other inhibitors of fatty acid metabolism which were also investigated behaved similarly.     

Role of GTP-binding proteins in phospholipid metabolism in human platelets]

Phosphoinositide-specific phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG), which act as second messengers. Substantial evidence has strongly suggested that a putative G-protein (s), Gp, regulates PLC activity in human platelets. Recently, the molecular mechanism of PLC activity regulation was clarified as to two types of enzymes, PLC-gamma and PLC-beta. In this chapter, the regulatory mechanisms of the PLCs via a Gp or tyrosine kinase is summarized, and the involvement of some G-protein in the regulation of other phospholipases, phosphatidylcholine-specific PLC, phospholipase A2 and phospholipase D, is also discussed.     

Isolation and effects of some garlic components on platelet aggregation and metabolism of arachidonic acid in human blood platelets

Effects of aqueous extract of garlic, of materials extracted in two organic solvents, viz., ether and chloroform in succession, and of some fractions obtained after TLC of the aqueous extract were examined on platelet aggregation induced by several aggregating agents. Their effects were also investigated on the formation of thromboxane and lipoxygenase products from endogenous arachidonic acid in intact platelets. The aqueous extract inhibited aggregation induced by all aggregating agents and so did the materials extracted in two organic solvents. Only two fractions obtained from TLC of the aqueous garlic extract were examined for effects on epinephrine- and arachidonic acid (AA)-induced aggregation; they were found to be antiaggregatory. The material extracted in ether (MEE) inhibited the incorporation of labelled AA into platelets in platelet-rich plasma. Garlic extracts (MEE and material extracted in chloroform, MEC) at higher dosage inhibited the degradation of platelet phospholipids and reduced the formation of thromboxane (TxB2) and lipoxygenase-derived products from labelled platelets. The two organic extracts at low dosage, while not affecting the degradation of platelet phospholipids, inhibited the cyclooxygenase and lipoxygenase enzymes. A concomitant increase in the amount of released AA was observed in the treated platelets. Similar effects in relation to dosage were observed with the aqueous extract of garlic.     

Effect of homocysteine on arachidonic acid release in human platelets

BACKGROUND: It has been suggested that homocysteine is implicated in the risk of atherosclerosis and thrombosis. The pathogenic mechanism has not been clarified, but oxygen-free species produced by the homocysteine metabolism and auto-oxidation could have a role. DESIGN: We have studied the effect of homocysteine on arachidonic acid release in human platelets. Two important products of arachidonic acid metabolism - thromboxane B2 (TXB2) and reactive oxygen species (ROS) - have been assayed. RESULTS: Results indicate that homocysteine induces arachidonic acid release that is partially inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA). Platelet incubation with homocysteine significantly increases basal levels of TXB2 and ROS. The effect is time- and dose-dependent. The TXB2 formation is strictly correlated with the arachidonic acid release. Moreover, ROS accumulation is largely inhibited by ETYA and partially reduced by diphenyleneiodonium (DPI), suggesting the involvement both of enzymes metabolising arachidonic acid (cyclooxygenase, lipooxygenase, cytochrome P450 monooxygenase) and of NAD(P)H oxidase. CONCLUSION: Homocysteine induces oxidative stress in human platelets in vitro. The unbalance in platelet redox-state and the increased TXB2 formation may generate hyperactivation, contributing to a thrombogenic state leading to cardiovascular diseases.