Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

Use of the glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol as a probe to investigate hypertrehalosemic hormone-mediated change in carbohydrate and lipid metabolism in the cockroach, Periplaneta americana

The glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DIA) was used to investigate the action of hypertrehalosemic hormone (HTH) on carbohydrate, neutral lipid, and phospholipid metabolism in the hemolymph and fat body of the cockroach, Periplaneta americana. DIA blocked the hypertrehalosemic and hyperglycemic effects of HTH, as well as the decrease in hemolymph neutral lipid and phospholipid normally induced by HTH. DIA diminished the accumulation of neutral lipid in the fat body formed under the influence of HTH and partly blocked the decrease in fat body phospholipid evoked by HTH. The increased incorporation of (14)C-glucose into fat body triacylglycerol in the presence of HTH was decreased by more than two-thirds when DIA was coinjected with the hormone. The results suggest that glucose derived from hemolymph trehalose is an important contributor to the formation of the glycerol backbone of newly formed triacylglycerol in the fat body.     

Overexpression of rat long chain acyl-coa synthetase 1 alters fatty acid metabolism in rat primary hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.     

Glycerol metabolism in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozocinin-diabetic rats. With glucose in the incubation medium, incorporation of exogenous [1,3-14C]glycerol into disaturated phosphatidylcholine, total phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) was increased 4-fold in cells from diabetic rats. In the absence of glucose, glycerol incorporation was 5-fold greater than in its presence in cells from normal animals, but was further increased 2.2-fold in cells from diabetic rats. Insulin treatment of diabetic rats returned all incorporation rates to control values. The increased glycerol incorporation rates were not due to differences in either phospholipid turnover or the size of the glycerol 3-phosphate precursor pool. Kinetic analysis of glycerol entry into the acid-soluble cell fraction indicated that glycerol transport occurred largely by simple diffusion, and was not rate limiting for its entry into lipids. Glycerol entry into the total lipid fraction was saturable, reaching a Vmax of 48 pmol/micrograms DNA per h in normal cells and 120 pmol/micrograms DNA per h in cells from diabetic rats, with no change in the Km (0.31 mM). While glycerol oxidation was reduced 23% in cells from diabetic rats in the presence of glucose and by 44% in the absence of glucose, glycerol kinase activity in sonicates of cells from diabetic animals was increased 210% and was reversed by in vivo insulin treatment. These results suggest that glycerol utilization in type II pneumocytes is a hormonally regulated function of both glycerol oxidation and glycerol phosphorylation.     

Phospholipid Transfer Protein Plays a Major Role in the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in Mouse Primary Hepatocytes

In this study, we tested the hypothesis that phospholipid transfer protein (PLTP) is a plausible mediator of phospholipid (PL) transfer to the N-terminal 1000 residues of apoB (apoB:1000) leading to the initiation of apoB-containing lipoprotein assembly. To this end, primary hepatocytes from wild type (WT) and PLTP knock-out (KO) mice were transduced with adenovirus-apoB:1000 with or without co-transduction with adenovirus-PLTP, and the assembly and secretion of apoB:1000-containing lipoproteins were assessed. PLTP deficiency resulted in a 65 and 72% reduction in the protein and lipid content, respectively, of secreted apoB:1000-containing lipoproteins. Particles secreted by WT hepatocytes contained 69% PL, 9% diacylglycerol (DAG), and 23% triacylglycerol (TAG) with a stoichiometry of 46 PL, 6 DAG, and 15 TAG molecules per apoB:1000. PLTP absence drastically altered the lipid composition of apoB:1000 lipoproteins; these particles contained 46% PL, 13% DAG, and 41% TAG with a stoichiometry of 27 PL, 10 DAG, and 23 TAG molecules per apoB:1000. Reintroduction of Pltp gene into PLTP-KO hepatocytes stimulated the lipidation and secretion of apoB:1000-containing lipoproteins by ∼3-fold; the lipid composition and stoichiometry of these particles were identical to those secreted by WT hepatocytes. In contrast to the WT, apoB:1000 in PLTP-KO hepatocytes was susceptible to intracellular degradation predominantly in the post-endoplasmic reticulum, presecretory compartment. Reintroduction of Pltp gene into PLTP-KO hepatocytes restored the stability of apoB:1000. These results provide compelling evidence that in hepatocytes initial recruitment of PL by apoB:1000 leading to the formation of the PL-rich apoB-containing initiation complex is mediated to a large extent by PLTP.     

Conformational changes in human urokinase induced by a specific reduction of disulfide bond in Cys-194-Cys-222 associated with exhibition of enzymatic activity

The mechanism of action of hepatic triacylglycerol lipase (EC 3.1.1.3) was examined by comparing the hydrolysis of a water-soluble substrate, tributyrin, with that of triolein by hepatic triacylglycerol lipase purified from human post-heparin plasma. The hydrolyzing activities toward tributyrin and triolein were coeluted from heparin-Sepharose at an NaCl concentration of 0.7 M. The maximal velocity of hepatic triacylglycerol lipase (Vmax) for tributyrin was 17.9 mumol/mg protein per h and the Michaelis constant (Km) value was 0.12 mM, whereas the Vmax for triolein was 76 mumol/mg per h and the Km value was 2.5 mM. The hydrolyses of tributyrin and triolein by hepatic triacylglycerol lipase were inhibited to similar extends by procainamide, NaF, Zn2+, Cu2+, Mn2+, SDS and sodium deoxycholate. Triolein hydrolysis was inhibited by the addition of tributyrin. Triolein hydrolysis was also inhibited by the addition of dipalmitoylphosphaidylcholine vesicles. In contrast, the additions of triolein emulsified with Triton X-100 and dipalmitoylphosphatidylcholine vesicles enhanced the rate of tributyrin hydrolysis by hepatic triacylglycerol lipase. In the presence of dipalmitoylphosphatidylcholine, the Vmax and Km values of hepatic triacylglycerol lipase for tributyrin were 41 mumol/mg protein per h and 0.12 mM, respectively, indicating that the enhancement of hepatic triacylglycerol lipase activity for tributyrin by dipalmitoylphosphatidycholine vesicles was mainly due to increase in the Vmax. The enhancement of hepatic triacylglycerol lipase activity for tributyrin by phospholipid was not correlated with the amount of tributyrin associated with the phospholipid vesicles. On Bio-Gel A5m column chromatography, glycerol tri[1-14C]butyrate was not coeluted with triolein emulsion, and hepatic triacylglycerol lipase activity was associated with triolein emulsion even in the presence of 2 mM tributyrin. These results suggest that hepatic triacylglycerol lipase has a catalytic site for esterase activity and a separate site for lipid interface recognition, and that on binding to a lipid interface the conformation of the enzyme changes, resulting in enhancement of the esterase activity.     

Oxidation affects the regulation of hepatic lipid synthesis by chylomicron remnants

The effects of native and oxidized chylomicron remnants on lipid synthesis in normal and oxidatively stressed liver cells were investigated using MET murine hepatocytes (MMH cells), a nontransformed mouse hepatocyte cell line that maintains a highly differentiated hepatic phenotype in culture. Lipid synthesis was determined by measuring the incorporation of [(3)H]oleate into cholesteryl ester, triacylglycerol, and phospholipid by the cells. The formation of cholesteryl ester and phospholipid was decreased by chylomicron remnants in a dose-dependent manner, while triacylglycerol synthesis was increased. Exposure of MMH cells to mild oxidative stress by incubation with CuSO(4) (2.5 microM) for 24 h led to significantly increased incorporation of [(3)H]oleate into triacylglycerol and phospholipid, but not cholesteryl ester, in the absence of chylomicron remnants. In the presence of the lipoproteins, however, similar effects to those found in untreated cells were observed. Oxidatively modified chylomicron remnants prepared by incubation with CuSO(4) (10 microM, 18 h, 37 degrees C) did not influence cholesteryl ester or phospholipid synthesis in MMH cells, but had a similar effect to that found with native remnants on triacylglycerol synthesis. These findings show that hepatic lipid metabolism is altered by exposure to mild oxidative stress and by lipids from the diet delivered to the liver in chylomicron remnants, and these effects may play a role in the development of atherosclerosis.     

Implications of the simultaneous occurrence of hepatic glycolysis from glucose and gluconeogenesis from glycerol.

Glycolysis from [6-(3)H]glucose and gluconeogenesis from [U-(14)C]glycerol were examined in isolated hepatocytes from fasted rats. A 5 mm bolus of glycerol inhibited phosphorylation of 40 mm glucose by 50% and glycolysis by more than 60%, and caused cellular ATP depletion and glycerol 3-phosphate accumulation. Gluconeogenesis from 5 mm glycerol was unaffected by the presence of 40 mm glucose. When nonsaturating concentrations of glycerol (< 200 microm) were maintained in the medium by infusion of glycerol, cellular ATP concentrations remained normal. The rate of uptake of infused glycerol was unaffected by 40 mm glucose, but carbohydrate synthesis from glycerol was inhibited 25%, a corresponding amount of glycerol being diverted to glycolytic products, whereas 10 mm glucose had no inhibitory effect on conversion of infused glycerol into carbohydrate. Glycerol infusion depressed glycolysis from 10 mm and 40 mm glucose by 15 and 25%, respectively; however, the overall rates of glycolysis were unchanged because of a concomitant increase in glycolysis from the infused glycerol. These studies show that exposure of hepatocytes to glucose and low quasi-steady-state concentrations of glycerol result in the simultaneous occurrence, at substantial rates, of glycolysis from glucose and gluconeogenesis from the added glycerol. We interpret our results as demonstrating that, in hepatocytes from normal rats, segments of the pathways of glycolysis from glucose and gluconeogenesis from glycerol are compartmentalized and that this segregation prevents substantial cross-over of phosphorylated intermediates from one pathway to the other. The competition between glucose and glycerol implies that glycolysis and phosphorylation of glycerol take place in the same cells, and that the occurrence of simultaneous glycolysis and gluconeogenesis may indicate channelling within the cytoplasm of individual hepatocytes.     

Lipid synthesis in permeabilized cultured rat hepatocytes

Hepatic lipid synthesis was verified and studied in lysolecithin-permeabilized cultured rat hepatocytes and compared to that of intact liver cells. Triacylglycerol synthesis in permeabilized cells incubated in the presence of glycerol 3-phosphate and long chain fatty acids approached that of intact hepatocytes. Similarly, phosphatidylcholine synthesis in permeable cells incubated in the presence of exogenous CDP-choline was similar to that of intact hepatocytes and at the expense of microsomal neutral lipid synthesis. Phosphatidic acid accumulation in lysolecithin-permeabilized liver cells was remarkably increased as compared to that of intact cells, and its synthesis was mostly accounted for by the activity of mitochondrial glycerol-3-phosphate acyltransferase. Mitochondrial-generated phosphatidate was found to migrate to the endoplasmic reticulum, thus establishing a novel lipid esterification pathway which begins in mitochondrial glycerol 3-phosphate acylation and results in microsomal triacylglycerol and phospholipid synthesis. The free access of permeabilized liver cells to substrates and modulators of lipid synthesis, while maintaining an overall synthetic pattern similar to that of intact hepatocytes, makes them a system of choice for studying hepatic lipid synthesis in general and the microsomal/mitochondrial distribution of fluxes in particular.     

Effect of Glycerol Deprivation on the Phospholipid Metabolism of a Glycerol Auxotroph of Staphylococcus aureus

A study of the effects of glycerol deprivation on the content and metabolism of the phospholipids of a glycerol auxotroph of Staphylococcus aureus showed that (i) there was an increase in the proportions of lysylphosphatidylglycerol (LPB) and a concomitant decrease in the proportion of phosphatidylglycerol. The total phospholipid content per sample and the proportion of cardiolipin did not change, but the phosphatidic acid increased transiently and then fell to pretreatment levels. (ii) The loss of (32)P from the lipids during the chase in a pulse-chase experiment was essentially the same in phosphatidylglycerol, cardiolipin, and phosphatidic acid during glycerol deprivation or growth in the presence of glycerol. LPG lost half the radioactivity in slightly more than two doubling times when grown with glycerol. In the absence of glycerol, (32)P accumulated in LPG for about 20 min and then stopped, after which time there was no apparent turnover. (iii) During glycerol deprivation, the initial (32)P incorporation decreased sixfold compared to that of the control with glycerol. The initial incorporation into LPG decreased only 2.5-fold, whereas that of PG decreased 45-fold. (iv) During glycerol deprivation, the free fatty acid content increased from 1.2 to 12.5% of the total extractable fatty acids and then slowly decreased. The increase was largely iso- and anti-iso-branched 21-carbon-atom fatty acids. In glycerol-supplemented cultures, the major fatty acids were branched 14- to 18-carbon fatty acids. The decrease in longer chain free fatty acids after 60 min represented their esterification into lipids. (v) During glycerol deprivation ribonucleic acid synthesis and cell growth continued for 40 min and protein synthesis continued for 90 min. Then synthesis and growth stopped. (vi) After the addition of glycerol to glycerol-deprived cells, (32)P and (14)C-glycerol were incorporated into the phospholipids without lag; ribonucleic acid, protein synthesis, and cell growth began after a 5- to 10-min lag at the pretreatment rate. The initial rate of lipid synthesis after the addition of glycerol was three times greater than the growth rate. This rapid rate continued for about 25 min until the lipid content and proportions of LPG and phosphatidylglycerol were restored.     

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm,Spodoptera litura

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.     

Isolation, Composition, and Structure of Membrane of Listeria monocytogenes

The plasma membrane of Listeria monocytogenes strain 42 was prepared by osmotic lysis of protoplasts with tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.2, containing MgCl₂ and glucose, followed by washing with NaCl and MgCl₂ in Tris buffer. Electron microscopy showed that the preparation was not contaminated with cytoplasmic material. The membrane preparation was composed of 55 to 60% protein, 1.5% ribonucleic acid, 0.1% deoxyribonucleic acid, 1.3 to 2.3% carbohydrate, 0.17 to 0.38% amino sugar, 0.2 to 0.4% rhamnose, 3.5 to 4.0% phosphorus, 10.5 to 12.0% nitrogen, and 30 to 35% lipid. Amino acid composition of the washed membrane showed some variation from that of the whole cells. Sulfur-containing amino acids were not present in the membrane hydrolysate. The membrane carbohydrate contained glucose, galactose, ribose, and arabinose. The membrane lipid was 80 to 85% phospholipid and 15 to 20% neutral lipid. The lipid contained 2.3 to 3.0% phosphorus, 2.5 to 3.0% carbohydrate, and a very small amount of nitrogen (0.2 to 0.3%). The phospholipid was of the phosphatidyl glycerol type. Electron micrographs of the washed membrane showed three layers. The outer and inner layers varied in thickness from 25 to 37 A and the middle layer from 20 to 25 A. The total thickness varied between 85 and 100 A. These preparations contained many vesicles which stained heavily with lead citrate. Some vesicles were also attached to the protoplast ghosts in the form of extrusions or intrusions, or both. Membrane preparations obtained by lysis of protoplasts in the absence of MgCl₂ were fragmented and contained less lipid (20 to 22%) and ribonucleic acid (0.3 to 0.5%) than preparations prepared with MgCl₂.     

Effects of dexamethasone and insulin on the synthesis of triacylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes.

Rat hepatocytes in monolayer culture were preincubated for 19 h with 1 microM-dexamethasone, and the incubation was continued for a further 23 h with [14C]oleate, [3H]glycerol and 1 microM-dexamethasone. Dexamethasone increased the secretion of triacylglycerol into the medium in particles that had the properties of very-low-density lipoproteins. The increased secretion was matched by a decrease in the triacylglycerol and phosphatidylcholine that remained in the hepatocytes. Preincubating the hepatocytes for the total 42 h period with 36 nM-insulin decreased the amount of triacylglycerol in the medium and in the cells after the final incubation for 23 h with radioactive substrates. However, insulin had no significant effect on the triacylglycerol content of the cell and medium when it was present only in the final 23 h incubation. Insulin antagonized the effects of dexamethasone in stimulating the secretion of triacylglycerol from the hepatocytes, especially when it was present throughout the total 42 h period. The labelling of lysophosphatidylcholine in the medium when hepatocytes were incubated with [14C]oleate and [3H]glycerol was greater than that of phosphatidylcholine. The appearance of this lipid in the medium, unlike that of triacylglycerol and phosphatidylcholine, was not stimulated by dexamethasone, or inhibited by colchicine. However, the presence of lysophosphatidylcholine in the medium was decreased when the hepatocytes were incubated with both dexamethasone and insulin. These findings are discussed in relation to the control of the synthesis of glycerolipids and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by the liver, particularly in relation to the interactions of glucocorticoids and insulin.     

Differential incorporation of docosahexaenoic and arachidonic acids by the yolk sac membrane of the avian embryo

During avian development, lipoproteins derived from yolk lipid are assembled in the yolk sac membrane (YSM) for secretion into the embryonic circulation. To investigate how yolk polyunsaturated fatty acids, essential for the development of certain tissues, are distributed among the lipid classes of the lipoproteins, pieces of YSM were incubated in vitro with [14C]arachidonic and [14C]docosahexaenoic acids (DHA). There was a marked difference in the partitioning of these two precursors among the lipid classes of the tissue. Of the radioactivity incorporated into total lipid from [14C]-arachidonic acid during 1 h of incubation, 67.3% was esterified as phospholipid and 29.5% as triacylglycerol. In contrast, only 14.6% of the label incorporated from [14C]-DHA was esterified as phospholipid, whereas 73.2% was recovered in triacylglycerol. This pattern of differential partitioning was observed at all time points and across a 20-fold range of fatty acid concentrations. There was no evidence for conversion of the radioactive arachidonic and DHAs to other fatty acids prior to incorporation into tissue lipids. It is suggested that the selective incorporation of yolk-derived DHA into the triacylglycerol of secreted lipoproteins represents part of a mechanism for directing this polyunsaturate to particular embryonic tissues.     

The influence of acclimation temperature on the lipid composition of the larval lamprey, Petromyzon marinus, depends on tissue and lipid class

This study was designed to examine the effect of thermal acclimation on the lipid composition of fat depot organs the liver and kidneys of larval sea lamprey, Petromyzon marinus. We found that 21 °C-acclimated larvae possessed lower total lipid amounts in the liver (39% lower) and kidneys (30% lower) than 13 °C-acclimated larvae. Relatively lower lipid contents in the liver and kidneys of 21 °C-acclimated lamprey primarily resulted from a reduction in stored lipid reserve, triacylglycerol, but not the structural lipid, phospholipid. Compared to 21 °C-acclimated larvae, 13 °C-acclimated larvae were found to possess fewer saturated fatty acids (SFAs) and more unsaturated fatty acids (USFAs) in renal triacylglycerol and phospholipid classes, while there were no significant differences in the SFAs and USFAs of hepatic triacylglycerol, phospholipid, cholesteryl ester, fatty acid, and monoacylglycerol classes. Fewer SFAs, found in the kidney triacylglycerol of 13 °C-acclimated lamprey, were due to lower 12:0 and 14:0 fatty acids, but those in the renal phospholipid class were characterized by fewer 14:0, 15:0, and 16:0 fatty acids. More USFAs in renal triacylglycerol, as indicated by a higher unsaturation index, primarily resulted from higher polyunsaturated fatty acids (18:2ω6, 18:3ω3, and 18:4ω3); whereas, in the renal phospholipid class, this was a result of higher monoenes (18:1, 20:1, and 22:1ω9) and ω3 polyunsaturated fatty acids (18:4ω3). These data suggest that the influence of thermal acclimation on the lipid composition of lamprey fat depot organs depends on tissue and lipid class.     

Lipid metabolism by rat lung in vitro. Effect of starvation and re-feeding on utilization of [U-14C]glucose by lung slices

1. The incorporation of [U-(14)C]glucose into several lipid components of lung and liver slices, and the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ;malic' enzyme (EC 1.1.1.40) and NADP-isocitrate dehydrogenase (EC 1.1.1.42) of the cell cytosol were examined in normal, starved and re-fed rats. 2. Lipogenesis and the activities of these enzymes in liver were decreased markedly in rats starved for 72h. Re-feeding starved rats on a fat-free diet for 72h resulted in the well documented hyperlipogenic response in liver, particularly in its ability to convert glucose into neutral lipid, and increased activities of glucose 6-phosphate dehydrogenase, ;malic' enzyme and 6-phosphogluconate dehydrogenase to values approx. 700, 470 and 250% of controls respectively. 3. Approx. 70% of the total label in lung lipids was present in the phospholipid fraction. Hydrolysis of lung phospholipids revealed that lipogenesis from glucose was considerable, with approx. 40% of the total phospholipid radioactivity present in the fatty acid fraction. 4. Incorporation of glucose into total lung lipids was decreased by approx. 40% in lung slices of starved rats and was returned to control values on re-feeding. Although phospholipid synthesis from glucose was decreased in lung slices of starved rats, the decrease proportionally was greater for the fatty acid fraction (approx. 50%) as compared with the glycerol fraction (approx. 25%). 5. The activities of lung glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-isocitrate dehydrogenase were not affected by the dietary alterations. ;Malic' enzyme activity was not detected in lung cytosol preparations. 6. The results are discussed in relation to the surface-active lining layer (surfactant) of the lung.     

Inactivation of microsomal triglyceride transfer protein impairs the normal redistribution but not the turnover of newly synthesized glycerolipid in the cytosol, endoplasmic reticulum and Golgi of primary rat hepatocytes.

The requirements for microsomal triglyceride transfer protein (MTP) during the turnover and transfer of glycerolipids from intracellular compartments into secretory very low-density lipoprotein (VLDL) were studied by pre-labelling lipids with [(3)H]glycerol and [(14)C]oleate in primary cultures of rat hepatocytes. The intracellular redistribution of pre-labelled glycerolipids was then compared at the end of subsequent chase periods during which the MTP inhibitor BMS-200150 was either present or absent in the medium. Inhibition of MTP resulted in a decreased output of VLDL triacylglycerol (TAG) and a delayed removal of labelled TAG from the cytosol and from the membranes of the smooth endoplasmic reticulum (SER), the cis- and the trans-Golgi. Inactivation of MTP did not decrease the bulk lipolytic turnover of cellular TAG as reflected by changes in its [(3)H]glycerol:[(14)C]oleate ratios. However, a larger proportion of the resultant TAG fatty acids was re-esterified and remained with the membranes of the various subcellular fractions rather than emerging as VLDL. The effects of BMS-200150 on the pattern of phospholipid (PL) mechanism and redistribution suggested that inhibition of MTP prevented the normal lipolytic transfer of PL-derived fatty acids out of the SER, cis- and trans-Golgi membrane pools. Finally, changes in the (14)C specific radioactivities of the cytosolic and membrane pools of TAG suggested that inhibition of MTP prevented a normal influx of relatively unlabelled fatty acids into these pools during the chase period.     

Modulation of (Na+,K+)-ATPase activity by the lipid bilayer examined with dansylated phosphatidylserine

The fluorescent probe 8-(dimethylamino)naphthalene-1-sulfonylphosphatidylserine (Dns-PS) was incorporated into purified lamb kidney Na+- and K+-stimulated adenosinetriphosphatase (EC 3.6.1.3) [(Na+,K+)-ATPase] by using a purified phospholipid exchange protein. Phospholipase C was used to reduce phospholipid content. Up to 40% of the phospholipid could be hydrolyzed with only 10% inhibition of the (Na+,K+)-ATPase, but when 67% of the phospholipid was hydrolyzed, the enzyme was inhibited 53%. To examine the effect of protein on the phospholipid bilayer, the fluorescent parameters of the probe incorporated into the enzyme preparation were contrasted with the same parameters for the probe incorporated into the total lipid extract of the preparation. The polarization of fluorescence of the probe in the lipid extract was 0.118 while in the enzyme preparation it was 0.218. This reflected a decrease in fluidity of the glycerol region of the phospholipid bilayer which was mediated by the protein. This effect increased as the phospholipid content of the (Na+,K+)-ATPase preparation was reduced so that with maximal phospholipid reduction the polarization of fluorescence was 0.262. The protein caused a decrease in the transition temperature from gel to fluid states of the bilayer detected by polarization of the probe. The midpoint temperature transition of the enzyme preparation decreased from 33 degrees C when all phospholipids were present to 20 degrees C when 67% of the phospholipids were hydrolyzed. This decrease was not observed for the lipid extract of these samples. A direct correlation between the (Na+,K+)-ATPase specific activity and the polarization of fluorescence of Dns-PS was found. The reduction in phospholipid content did not affect the steady-state level of phosphorylation of the enzyme by ATP but did affect the rate of dephosphorylation which would require conformational changes of the enzymes. The data showed that the fluidity of the phospholipid bilayer can modulate the activity of the (Na+,K+)-ATPase.     

Effects of ethynyloestradiol on the metabolism of [1-14C]-oleate by perfused livers and hepatocytes from female rats

Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.     

Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.