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The synthesis of lipids from [1-14C]acetate by human venous endothelium in tissue culture.

Endothelial cells from human umbilical cords were harvested, using a trypsin technique, grown in tissue culture and lipid synthesis studied using [1-14C]acetate as a precursor. Radiosubstrate was incorporated into fatty acids, mono-, di- and triglycerides, cholesterol esters and phospholipids. Radioactivity was also present in the culture medium in the mono-and diglyceride fractions and in the phospholipids running with the solvent front.

Acetates↗

Lipid transport in the avian species. Part I. Isolation and characterization of apolipoproteins and major lipoprotein density classes of male turkey serum.

(1) Lipoproteins from the serum of male turkeys maintained on a normal diet were separated by sequential preparative ultracentrifugation into VLDL (d less than 1.006 g/ml), LDL (d = 1.006-1.063 g/ml), HDL (d = 1.063-1.21 g/ml) and VHDL (d greater than 1.21 g/ml). Lipoprotein density classes were characterized by analytical ultracentrifugation, agarose electrophoresis, immunodiffusion and immunoelectrophoresis, and by quantitative determination of protein, lipids and individual phosphatides. (2) HDL were the major density class representing 75% of the total lipoprotein content, LDL accounted for approximately 20% and VLDL for only 3-5% of the total lipoproteins. (3) VLDL were characterized by a relatively low content of glyceride (34%). Cholesterol esters were the major lipid (38%) of LDL, and the phospholipids (26%) of HDL. Glycerides of all major density classes consisted of equal amounts of triglycerides and diglycerides. (4) Phosphatidylcholine was the major phosphatide in all density classes. The composition of phosphatides was very similar in the VLDL and LDL, but it was different in the HDL. The ratio of phosphatidylcholine/sphingomyelin was higher in HDL than in VLDL and LD. (5) Immunological and electrophoretic studies showed that all three major density classes consisted of two lipoprotein families designated, in analogy to the human serum lipoprotein system [1], as LP-A and LP-B. The exception was HDL3 (d = 1.125-1.21 g/ml) which contained only LP-A. (6) ApoB was insoluble in aqueous buffers but could be solubilized after reduction and carboxymethylation. No C- or N-terminal amino acids were released by the usual chemical methods. The carbohydrate moiety of ApoB contained mannose, galactose and galactosamine. (7) ApoA consisted of a non-identical polypeptides designated in analogy to the human polypeptides as A-I and A-II. A-I was the major ApoA polypeptide and had a molecular weight of about 27,000. This polypeptide contained no half cystine, and the aspartic acid as the N-terminal and alanine as the C-terminal amino acids. A-II had a molecular weight of about 10,000, contained no half cystine and had alanine as the C-terminal amino acid. A-II showed no N-terminal amino acid by either dansylation, dinitrophenylation or Edman's procedure. Neither A-I nor A-II contained neutral sugars or hexosamines. (8) Concentrations of polypetides analogous to human ApoC, ApoD and "arginine-rich" polypeptide, if present, were too low for their unequivocal chemical characterization.

Animals↗

In vitro clearance of chylomicron triglycerides containing (omega-3) eicosapentaenoate.

Rat mesenteric lymph chylomicrons, containing triglycerides enriched with either [14C]oleic acid or [14C]eicosapentaenoic acid, were prepared by ultracentrifugation of lymph samples collected for 6 h after a single duodenal infusion of an emulsion containing 0.3 mmol of either fatty acid. After determination of protein and of total fatty acid content and composition, enriched chylomicrons were suspended in Krebs-bicarbonate buffer. Non-working hearts were perfused in a recirculating system for 45 min using the enriched chylomicron preparations. At 15 min intervals during perfusion, the media were assayed for total radioactivity, 14CO2 and 14C-labeled fatty acids associated with triglycerides, unesterified fatty acids, phospholipids, mono- and diglycerides. After perfusion, the hearts were extracted and assayed for total lipid radioactivity and isotope distribution among heart lipid fractions. With this membrane-supported lipoprotein lipase system, clearances of chylomicron triglycerides containing either fatty acid were identical, as were the myocardial uptakes of the fatty acids and oxidations to 14CO2. Furthermore, except for a significantly greater incorporation of eicosapentaenoate into myocardial phospholipids, tissue isotope distributions of the two labeled fatty acids were also the same. These studies suggest that at least the initial phases of peripheral clearance of chylomicrons enriched in omega-3 fatty acids is as efficient as with those containing oleate.

Animals↗

Fetal aortic cholesterol concentration and metabolism: relationship to plasma cholesterol and potential role of placental factors.

The relationship of fetal plasma cholesterol levels to aortic free and esterified cholesterol concentration was determined in the rabbit during gestation. Plasma cholesterol levels (mg/dl) in early fetuses were markedly high and decreased at term. While aortic free cholesterol content increased with fetal age, cholesteryl ester concentration did not change significantly and no pathological accumulation was evident. This occurred despite high fetal aortic cholesterol esterification activity noted in earlier studies. We evaluated the potential effect of rabbit placental extracts on lipid metabolism and cellular proliferation in fetal aortic explants to explore the role of placental factors in affecting lipid metabolism. Labeled [14C]oleate and [3H]thymidine were used to investigate the rates of incorporation of (a) oleate into lipids, and (b) thymidine into DNA, respectively. Placental extracts at term (but not from 22 days gestation) significantly decreased oleate incorporation (P less than 0.05) into cholesteryl esters, phospholipids and diglycerides. This effect of placental extracts was noted both in absence or presence of serum in the culture medium, and was predominantly found in fraction of Mr greater than 100,000. [3H]Thymidine incorporation (dpm/g protein) into DNA was significantly decreased (P less than 0.01) by placental extracts. These studies suggest that placental extracts contain factor(s) influencing fetal aortic lipid metabolism in culture.

Animals↗

Mediation by GTP gamma S and Ca2+ of inositol trisphosphate generation in rat heart membranes.

The possibility, that a GTP-binding protein is involved in the transducing mechanism leading to the formation of inositol trisphosphate (InsP3) in heart was explored in rat heart ventricles. Accordingly, a crude membrane fraction was isolated from 3[H] inositol prelabelled rat heart ventricles. When incubated with the non-hydrolysable GTP analogues GTP gamma S and GMP-PNP, it produced InsP3 in a time- and concentration-dependent manner. GDP beta S and the aminoglycoside antibiotic neomycin were effective inhibitors of this activation. In the absence of GTP gamma S or GMP-PNP, no such formation occurred with Ca2+ concentration from 10 nM to 1 microM but formation tripled in relation to the control level when Ca2+ concentration was raised from 1 microM to 100 microM. GTP gamma S increased the Ca2+ sensitivity of InsP3 production towards more physiologically relevant concentrations occurring during diastole (100 nM). These findings strongly suggest the presence in heart of a particulate Ca2(+)-dependent phospholipase C, whose activity is regulated by guanine nucleotides. This Ca2(+)-dependent phospholipase C observed in a cell free system was evidenced also in a multicellular system when altering the free Ca2+ concentrations around the physiological range. The results support the possibility that the enzyme might be activated during each cardiac cycle and thus produce two potential activators of cardiac contraction, namely InsP3 and diglycerides.

Animals↗

Myotonic muscular dystrophy. Calcium-dependent phosphatidate metabolism in the erythrocyte membrane.

It has been suggested that the erythrocytes of myotonic dystrophy (MyD) patients have a decreased calcium-stimulated phosphatidic acid (PA) accumulation. This could be the result of a defect in the calcium-stimulated hydrolysis of the polyphosphoinositides (calcium-dependent phosphodiesterase) or in the subsequent formation of PA from its precursors (diacylglycerol kinase). In vitro assays were established for both enzymes in erythrocyte membranes. Calcium-dependent phosphodiesterase activity was assayed with both endogenous 32P-labeled erythrocyte diphosphoinositide and triphosphoinositide and with the same phospholipids isolated from rat brain. No significant differences in activity were found between MyD patients and normal controls with either method of substrate preparation. No difference in diglyceride kinase activity was found between ghosts prepared from MyD patients and normal controls. Thus, there were no differences in either of the membrane-associated enzymes of phosphatidic acid metabolism.

Calcium↗

Differences in Ca2+ mobilization induced by alpha-adrenergic agonist and phosphatidic acid in cultured hepatocytes.

In an attempt to elucidate the relationship between phosphatidylinositol breakdown and alpha-adrenergic responses, effects of phosphatidic acid and phosphatidylinositol related metabolites on Ca2+ mobilization and glucose output in cultured hepatocytes were examined. Norepinephrine induced the net 45Ca2+ efflux from preloaded cells and stimulated glucose output via alpha-adrenergic receptor stimulation, whereas phosphatidic acid caused 45Ca2+ uptake to cells and did not stimulate glucose output. Myo-inositol-monophosphate, diglyceride and arachidonic acid, which are released by phosphatidylinositol breakdown, had no effect on 45Ca2+ efflux and glucose output in cells. These results suggest that phosphatidic acid and phosphatidylinositol related metabolites can not mimic the alpha-adrenergic actions in cultured hepatocytes.

Animals↗

Phospholipase D in cell signalling and its relationship to phospholipase C.

Phospholipases C and D are phosphodiesterases which act on phospholipid head groups. Although the presence of these enzymes in living organisms has long been known, it is only recently that their role in cell signal transduction has been appreciated. The new developments on phospholipases D (PLD) are especially noteworthy, since these enzymes catalyze a novel pathway for second messenger generation. In a variety of mammalian cell systems, several biological or chemical agents have recently been shown to stimulate PLD activity. Depending on the system, activation of PLD has been suggested to be either dependent on, or independent of, Ca2+ and protein kinase C. PLD primarily hydrolyses phosphatidylcholine (PC) but phosphatidylinositol and phosphatidylethanolamine have also been reported as substrates. Different forms of endogenous PLD may also exist in cells. Exogenous addition of PLD causes alterations in cellular functions. In many instances, Ca2+ mobilizing agonists may stimulate both PLC and PLD pathways. Interestingly, several metabolites of these two enzymes are second messengers and are common to both pathways (e.g. phosphatidic acid, diglyceride). This has raised the issue of the interrelationship between these pathways. The regulation of either PLC or PLD by cellular components, e.g. guanine nucleotide binding proteins or protein kinases, is under intense investigation. These recent advances are providing novel information on the significance of phospholipase C and D mediated phospholipid turnover in cellular signalling. This review highlights some of these new discoveries and emerging issues, as well as challenges for future research on phospholipases.

Animals↗

The effects of exercise on lipogenic enzyme activity and glyceride synthesis by liver homogenates of diabetic rats.

The purpose of this study was to determine if exercise ameliorates the elevated levels of triglycerides in diabetic rats and also to determine if the capacity of liver to synthesize glycerides correlates with changes in plasma triglyceride levels. Forty female rats were divided into four groups: sedentary control, sedentary diabetic, exercised control, and exercised diabetic. Diabetes was induced by intravenous injection of alloxan (40 mg/kg), and control rats were sham-dosed with physiologic saline. All rats remained sedentary in their cages for the first week after the injections. The exercised groups were exercised for seven consecutive days for 2 h/d at 20 m/min (0 grade). All rats were killed 24 hours after the last exercise bout. Blood glucose levels were significantly higher in the diabetic group than the nondiabetic counterparts, but exercise did not affect glucose levels in either the diabetic or nondiabetic groups. Exercise, however, significantly lowered plasma triglyceride and free fatty acid levels in both diabetic and nondiabetic rats. The activities of the five enzymes involved in fatty acid synthesis were all depressed in the diabetic rats compared to controls, and exercise had no effect on the activities of the enzymes. The capacity of liver to synthesize total lipids, diglycerides, or triglycerides was not different in the diabetic rats from that of nondiabetic rats, and exercise did not change that. Only phospholipid synthesis from glycerol-3-phosphate was affected by diabetes. It is concluded that exercise ameliorates the elevations in plasma triglyceride levels that result from diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Fasting and diabetes mellitus elicit opposite effects on agonist-stimulated prostacyclin synthesis by the rat aorta.

The effects of prolonged fasting and experimental nonketonuric diabetes on rat aortic prostacyclin (PGl2) synthesis were compared. Whereas fasting (for 48 hours or longer) resulted in a marked increase in trauma-, adrenaline-, and U46619-stimulated aortic PGI2 synthesis, prolonged experimental (streptozotocin-induced) nonketonuric diabetes caused a marked decrease in aortic PGI2 synthesis stimulated by the above agonists. Arachidonic acid (AA)-stimulated aortic PGI2 synthesis in fasted and diabetic rats, however, was not different from that in controls. The reduction in adrenaline- and U46619-stimulated, but not AA-induced, PGI2 synthesis in the diabetic rat suggests that the diminished production of PGI2 in diabetes may be due to diminished phospholipase A2 (or of the phospholipase C-diglyceride lipase system) activity, diminished AA stores, or both. The opposite effects of prolonged fasting and diabetes on aortic PGI2 synthesis suggest that caution should be exercised when comparing the metabolic consequences of starvation with those of diabetes.

Animals↗

Incorporation of hydroxyeicosatetraenoic acids into cellular lipids of adrenal glomerulosa cells: inhibition of aldosterone release by 5-HETE.

Metabolites of arachidonic acid appear to be involved in the regulation of aldosterone secretion. Adrenal cells metabolize arachidonic acid to several products including hydroxyeicosatetraenoic acids (HETEs). Since HETEs may be incorporated into the membrane lipids in some cells, we investigated whether HETEs were incorporated into lipids of adrenal glomerulosa cells and tested the influence of incorporation on aldosterone secretion. Cells were incubated with [3H] -arachidonic acid, -5-HETE, -12-HETE, -15-HETE or -LTB4. The cellular lipids were extracted and analyzed by TLC. Arachidonic acid was incorporated into all of the cell lipids with greatest accumulations in phospholipids (22%), cholesterol esters (50%), and triglycerides (21%). Uptake was maximal by 30 min. 5-HETE was incorporated into diglycerides and monoglycerides but not into phospholipids or other neutral lipids. The uptake followed a similar temporal pattern as arachidonic acid. 12-HETE was incorporated to a small extent into phospholipids, predominantly phosphatidylcholine. Neither 15-HETE or LTB4 were associated with cellular lipids. Angiotensin increased the uptake of 5-HETE and arachidonic acid into phosphatidylinositol/phosphatidylserine without altering uptake into the other lipids. When cells were pretreated with 5-HETE and washed to remove the unesterified HETE, basal aldosterone release as well as release stimulated by angiotensin, potassium and ACTH were significantly reduced. 15-HETE, which is not incorporated into cellular lipids, was without effect on aldosterone secretion. These studies indicate that 5-HETE may be incorporated into the cellular lipids of adrenal cells and may modulate steroidogenesis.

Aldosterone↗

Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity.

Phosphoinositide breakdown in response to thrombin stimulation of human platelets results in the formation of the calcium-mobilizing messenger molecules inositol 1,4,5-trisphosphate and inositol 1,2-cyclic-4,5-trisphosphate and of diglyceride, which activates protein kinase C. We find that protein kinase C phosphorylates and thereby increases the activity of inositol 1,4,5-trisphosphate 5'-phosphomonoesterase, a phosphatase that hydrolyzes these molecules to inert compounds. The 5'-phosphomonoesterase phosphorylated using [gamma-32P]ATP comigrates on SDS-polyacrylamide gels with a protein (40 kd) phosphorylated rapidly in response to thrombin stimulation of 32PO4-labeled platelets. Peptide maps of proteolytic digests of these two phosphorylated proteins indicate that they are the same. We propose that platelet Ca2+ mobilization is regulated by protein kinase C phosphorylation of the inositol 1,4,5-trisphosphate 5'-phosphomonoesterase. These results explain the observation that phorbol ester treatment of intact human platelets results in decreased levels of inositol trisphosphate and decreased Ca2+ mobilization upon subsequent thrombin addition.

Blood Platelets↗

Effects of ionophore A23187 and Ba++ ions on labelling of phospholipids in rat pancreatic islets.

Ionophore A23187, either in the presence or absence of added Ca2+ or Mg2+, caused a marked accumulation of [32P]-phosphatidic acid in pancreatic islets pre-labelled with 32 Pi. A similar effect was observed following the addition of 4 mM Ba2+ ions in the absence of added Ca2+. Neither agent caused a significant modification of labelling in other lipid fractions, although there was a persistent trend towards reduced labelling of phosphatidylcholine and phosphatidylethanolamine. Ionophore A23187 also potentiated the incorporation of 3H-glycerol into phosphatidic acid and reduced the incorporation of this precursor into phosphatidylcholine. In islets pre-labelled with 3H-glycerol and subsequently exposed to A23187 or Ba2+, no significant changes were observed in label associated with either phospholipids or neutral glycerolipids. These results suggest that ionophore A23187 and Ba2+ ions can divert the synthesis of phospholipids resulting in increased formation of phosphatidic acid at the expense of non-acidic phospholipids, principally phosphatidylcholine. We tentatively suggest that this effect may be the result of inhibition by Ca2+ of the breakdown of phosphatidic acid to diglyceride, an enzymic step which may regulate the relative amounts of acidic and neutral phospholipids.

Animals↗

Impact of pesticides on lipid metabolism in the freshwater catfish, Clarias batrachus, during the vitellogenic phase of its annual reproductive cycle.

Specimens of either sex of the freshwater catfish Clarias batrachus were exposed to safe and sublethal concentrations of an organochlorine, gamma-BHC (2 and 8 ppm), and an organophosphorus compound, malathion (1 and 4 ppm), for 4 weeks during the vitellogenic phase of their annual reproductive cycle. The effects on total lipid and its various fractions, viz., free fatty-acids, monoglycerides, diglycerides, triglycerides, phospholipids, free cholesterol, and esterified cholesterol, were studied in the liver, plasma, gonads, and muscle. Except for elevated liver lipid in the male in response to malathion, no significant change in total lipid could be observed following pesticide exposure. However, various lipid fractions responded differently to two concentrations of the pesticides. Both pesticides affected the metabolism of nonpolar and less polar lipids alike. Malathion inhibited only mobilization of hepatic phospholipid to gonads but not its hepatic biosynthesis, whereas gamma-BHC reduced its synthesis in the liver as well. These pesticides seemed to restrict the conversion of esterified cholesterol into free cholesterol without affecting the biosynthesis of cholesterol as such. In both sexes, esterification of free fatty acids to acyl glycerides and their mobilization from liver to gonads seemed to be restricted as a result of pesticides action.

Animals↗

Impact of malathion and gamma-BHC on lipid metabolism in the freshwater female catfish, Heteropneustes fossilis.

Female Heteropneustes fossilis were exposed to sublethal concentrations of malathion (5 and 20 microliters liter-1) and gamma-BHC (4 and 16 micrograms liter-1) for 4 weeks during different phases of their annual reproductive cycle. The impact of these pesticides on free fatty acids (FFA), monoglycerides (MG), diglycerides (DG), triglycerides (TG), phospholipids (PL), free cholesterol (CF), and esterified cholesterol (CE) in the liver, plasma, and ovary was assessed. During the preparatory phase both the pesticides reduced the levels of all the hepatic and ovarian lipids with elevated hepatic CE. During the prespawning phase these pesticides decreased all the lipids in the liver, plasma, and ovary with the elevation of hepatic FFA and CE. During the spawning phase a reduction of hepatic MG and CF with a decreased level of plasma and ovarian levels of FFA and PL was recorded, whereas ovarian levels of TG and CE were elevated in response to both the pesticides. During postspawning and resting phases all the hepatic lipids were reduced with the elevation of CE in response to the exposure. These pesticides also restricted their mobilization to the ovary. Cholesterol biosynthesis seemed unaffected but the hydrolysis of CE to CF was adversely affected during preparatory and prespawning phases which is a period of sex steroid hormone biosynthesis.

Animals↗

Modulatory actions of ovine luteinizing hormone-releasing hormone and Mystus gonadotropin on gamma-BHC-induced changes in lipid levels in the freshwater catfish, Heteropneustes fossilis.

Female specimens of Heteropneustes fossilis were exposed to a sublethal concentration of gamma-BHC (16 mg/liter) for 4 weeks during the preparatory and prespawning phases of their annual reproductive cycle. The effect of gamma-BHC on free fatty acids (FFA), monoglycerides (MG), diglycerides (DG), and triglycerides (TG) was monitored. gamma-BHC in general was inhibitory to lipogenesis during both of the reproductively active phases as judged by decreased levels of FFA, MG, DG, and TG. Lower doses of ovine luteinizing hormone-releasing hormone (oLH-RH) and Mystus gonadotropin (mGTH) were ineffective in modulating the impact of gamma-BHC; however, their higher doses effectively revoked the impact of gamma-BHC. From the evidence obtained here, it is concluded that gamma-BHC suppressed the action of oLH-RH and mGTH and affected lipogenesis in this species.

Animals↗

Prostaglandin and thromboxane biosynthesis.

We describe the enzymological regulation of the formation of prostaglandin (PG) D2, PGE2, PGF2 alpha, 9 alpha, 11 beta-PGF2, PGI2 (prostacyclin), and thromboxane (Tx) A2 from arachidonic acid. We discuss the three major steps in prostanoid formation: (a) arachidonate mobilization from monophosphatidylinositol involving phospholipase C, diglyceride lipase, and monoglyceride lipase and from phosphatidylcholine involving phospholipase A2; (b) formation of prostaglandin endoperoxides (PGG2 and PGH2) catalyzed by the cyclooxygenase and peroxidase activities of PGH synthase; and (c) synthesis of PGD2, PGE2, PGF2 alpha, 9 alpha, 11 beta-PGF2, PGI2, and TxA2 from PGH2. We also include information on the roles of aspirin and other nonsteroidal anti-inflammatory drugs, dexamethasone and other anti-inflammatory steroids, platelet-derived growth factor (PDGF), and interleukin-1 in prostaglandin metabolism.

Amino Acid Sequence↗

Lipid composition of metacestodes of Taenia taeniaeformis and lipid changes during growth.

A lipid analysis was performed on developing metacestodes of Taenia taeniaeformis removed from the livers of rats at times varying from 3 to 35 weeks post infection. Lipid accounted for 7-21% of the dry weight of the parasites. The highest proportions were found at the earlier stages. The distribution was as follows; neutral lipid 27-45%; glycolipid 5-11%; and phospholipid 50-61%. The major neutral lipid was cholesterol, and minor neutral lipids were sterol esters, triglycerides, diglycerides and monoglycerides. Hydrocarbons were present throughout development, but in the highest amounts at the earlier stages. Five different glycolipids were found, all of which were identified as glycosphingolipids. An increase in the proportion of more complex glycolipids was noted as parasites grew older. Ten different phospholipids were identified, with the major components being phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Other phospholipids were: lysophosphatides, phosphatidylinositol, phosphatidic acid, diphosphatidylglycerol, sphingomyelin, and an unknown phospholipid component. Changes in the relative amounts of the two major phospholipids were found when the early and late stages were compared. Two lipids found throughout development were identified as glycosylated dolichol phosphates, and they comprised between 1 and 3% of the total phospholipid fraction. Nineteen fatty acids were detected, and the fatty acid distribution for each lipid class at each stage was determined. Seven major fatty acids were common to each. These were: hexadecanoic, octadecanoic, oleic, linoleic, arachidonic, docosanoic, and docosahexaenoic.

Animals↗