Further studies of the ethanol-induced changes in pancreatic lipid metabolism

Previous in vitro studies have shown that ethanol increased de novo triglyceride synthesis in the rat pancreas. The present study extends these observations on the effects of ethanol on pancreatic lipid metabolism. Ethanol significantly stimulated [1-¹⁴C]acetate incorporation into pancreatic lipids at concentrations as low as 0.068 mM, as well as at 3.4 and 34 mM. This suggests that known metabolic pathways of ethanol oxidation are not involved in these changes. Ethanol also stimulated the incorporation of [1-¹⁴C]oleate into pancreatic triglyceride and cholesteryl ester, but not into phospholipids. These changes were less marked than those obtained with [1-¹⁴C]acetate. Furthermore, incorporation of [1-¹⁴C]palmitate into pancreatic lipid was affected even less by ethanol. Thus, ethanol-induced changes in pancreatic lipid metabolism are unlikely to be due to fatty acid esterification alone.     

Lymphocyte activation: rapid changes in the phospholipid metabolism of plasma membranes during stimulation

Lymph node lymphocytes from normal or BCG-sensitized rabbits were stimulated by phytohemagglutinin (PHA) or purified protein derivative of tuberculin (PPD). After incubation of intact lymphocytes with [¹⁴C]oleate, the cells were disrupted and microsomes (consisting of plasma membrane and endoplasmic reticulum) were isolated. In some experiments the microsomal fraction was separated into plasma membrane and endoplasmic reticulum by dextran gradient centrifugation. After 10 min microsomes from PHA-stimulated lymphocytes had incorporated 3 times more [¹⁴C]oleate into lecithin than microsomes from controls. With PPD as antigen, after 1 h incubation a significant increase in the incorporation of [¹⁴C]oleate into microsomal lecithin was detected. The stimulation index with respect to [¹⁴C]oleate uptake remained constant in both cases when the lymphocytes were cultured for longer times. Approximately 90 % of the [¹⁴C]oleate incorporated into microsomal lecithin was recovered in the plasma membrane which exhibited the highest stimulation. The results are discussed in relation to possible biochemical events underlying the triggering process of lymphocytes.     

Altered Phospholipid Metabolism in Escherichia coli Accompanying Killing by Disrupted Granulocytes

The effect of bactericidal concentrations of disrupted rabbit granulocytes and of partially purified granulocyte fractions on phospholipid metabolism by Escherichia coli has been investigated. Previous studies in this laboratory have shown that, during and after killing of E. coli by granulocytes, bacterial macromolecular synthesis continues. Similarly, despite almost complete loss of viability within 15 min, incorporation of [1-¹⁴C]palmitate, [2-¹⁴C]glycerol, and [1-¹⁴C]acetate into E. coli phospholipids, in the presence of granulocyte preparations, remains the same as in control E. coli populations for at least 1 h. Incorporation of [1-¹⁴C]oleate into E. coli phospholipids is actually stimulated during the first 60 min of incubation in the presence of granulocyte preparations (more than twofold at 30 min and 40% at 60 min). With all labeled lipid precursors, bactericidal granulocyte preparations cause a relative increase in the labeling of E. coli cardiolipin, with a corresponding drop in labeled phosphatidyl-glycerol. Labeled lyso-compounds accumulate in the presence of granulocyte preparations when [1-¹⁴C]palmitate, but not when [1-¹⁴C]oleate is the labeled precursor. Since oleate occurs mainly in the 2-acyl position of E. coli phospholipids, whereas at least 50% of palmitate occurs in the 1 position, it appears that a phospholipase A₂ acts on the E. coli phospholipids. These various effects are also seen when E. coli are exposed to highly purified granulocyte preparations that possess potent bactericidal and phospholipase A₂ activities. We speculate that this phospholipase A₂ in the granulocyte preparations stimulates oleate but not palmitate incorporation by initiating increased turnover of the fatty acid in the 2-acyl position of E. coli phospholipids, causing formation of 1-acyl lyso-compounds likely to be preferentially reacylated with unsaturated fatty acids.     

Early changes in the phospholipid metabolism of lymphocytes following stimulation with phytohemagglutinin and with lysolecithin

Following stimulation of rabbit lymphocytes with phytohemagglutinin (PHA) [¹⁴C]oleic acid incorporation was augmented into phospholipids but not into neutral fats. The main phospholipid into which oleate was incoporated was lecithin. Stimulation of lymphocytes with lysolecithin in short-time experiments also increased the incorporation of oleate into lecithin. The distribution of the labeled fatty acids in the newly formed lecithin molecules was determined using snake venom phospholipase A. Of the incorporated oleate 80 % was found in position 1 of the glycerol moiety both in unstimulated and stimulated lymphocytes. In addition to natural [1-acyl]-lysolecithin a sythetic non-metabolized lysolecithin analogue also effected lymphocyte stimulation, suggesting that exogenous lysolecithin acts not as a substrate for lysolecithin-acyltransferase. Microsomal membranes of normal and stimulated lymphocytes were examined for enzyme ectivities involved in the incorporation of long-chain fatty acids into phospholipids especially (a) fatty acid: CoA-ligase, (b) lysolecithin-acyltransferase, (c) phospholipase A and (d) lysophospholipase. After 3 h of cultivation with PHA, fatty acid: CoA-ligase (which is rate-limiting for the incorporation of oleate into phospholipids) and phospholipase A were activated. Lysolecithin produced only an activation of phospholipase A supporting the idea that oleate is incorporated by reacylation of endogenously formed [2-acyl]-lysolecithin and consistent with our findings that de novo synthesis of phospholipids, as measured by [¹⁴C]choline uptake, is low during the early phase of stimulation.     

Effect of hypertension on the in vitro incorporation of [1-14C]-oleate into phospholipid and cholesterol ester in the aortae of normal-fed and cholesterol-fed rabbits

Summary The effect of prior hypertension on the in vitro incorporation of [1-¹⁴C]-oleate into phospholipid and cholesterol ester in aortae from cholesterolfed and normal fed rabbits was studied.Incorporation of [1-¹⁴C]-oleate into phospholipid was not increased in aortae from either hypertensive normal-fed or hypertensive cholesterol-fed rabbits when compared to the appropriate normotensive controls.In the normal-fed rabbits, incorporation of [1-¹⁴C]-oleate into cholesterol ester was increased by hypertension in all aortic regions. In cholesterol-fed rabbits cholesterol esterification was found to be proportional to the intimal cholesterol concentration, irrespective of the prior blood pressure or the particular aortic region studied.It is concluded that the increased lipid synthesis in atherosclerotic vessels from hypertensive rabbits is a consequence of the increased lipid accumulation produced by hypertension and not the result of hypertension directly stimulating arterial wall metabolism.     

Incorporation of Choline,Serine, Ethanolamine and Inositol into Phospholipids of Isolated Rat Mast Cells

The incorporation of labelled phospholipid precursors, [Me-¹⁴C]-choline, L-[3-¹⁴C]-serine, [2-¹⁴C]-ethanol-amine and [2-³H]-myoinositol into the phospholipids of isolated rat mast cells was studies. The label from the different precursors were found to be essentially associated with compounds with the t.l.c.-motility of the respective phospholipids. Whereas the incorporation of [Me-¹⁴C]-choline and L-[3-¹⁴C]-serine showed evidence of saturation, the incorporation of [2-¹⁴C]-ethanolamine was linear with time (2 h) and it was not saturated by increasing the concentration from 0.07 mM to 2.07 mM. The incorporation of [2-³H]-myoinositol was stimulated by Ca²⁺ (1 mM) or Mg²⁺ (1 mM), while the incorporation of the other precursors was stimulated only in the presence of both Ca²⁺ and Mg²⁺ (1 mM). Antimycin A (1 μM), an inhibitor of the respiratory chain, significantly inhibited the incorporation of [Me-¹⁴C]-choline, L-[3-¹⁴C]-serine and [2-³H]-myoinositol but not that of [2-¹⁴C]-ethanolamine. The experimental system used might be a useful model for studies on the turnover of membrane phospholipids during histamine release     

RNA-nascent DNA complexes in Ehrlich ascites tumor cells in vivo

When mice bearing Ehrlich ascites tumors were given a single intraperitoneal injection of [5-³H]uridine, the ascites cells incroporated radioactivity into DNA for 4 hr, while incorporation into total cellular RNA ceased after 20–30 min. Nascent DNA, isolated by Cs₂SO₄ isopycnic centrifugation 30 min, 4 hr or 50 hr after [5-³H]uridine injection contained RNA fragments. Treatments with hydroxyurea, an inhibiton hibitor of DNA synthesis, reduced the incorporation of [5-³H]uridine into the DNA-RNA complex to 43% of controls. Treatment with actinomycin D, an inhibitor of DNA-dependent RNA synthesis, abolished the incorporation of [5-³H]uridine into the DNA-RNA complex. Finally, when DNA was pulse-labeled with [¹⁴C]deoxythymidine and then denatured by heat-treatment, it banded in a region with a higher density than that of reference DNA. However, if treated with alkali, it banded at the density of reference DNA, again suggesting the presence of RNA fragments in nascent DNA. The length of the RNA chains in this DNA-RNA complex was estimated to be in the order of 70 nucleotides.     

Lipid metabolism in focal areas of normal-fed and cholesterol-fed pig aortas

The in vitro uptake and incorporation into various lipid fractions of [1-¹⁴C]oleic acid and [³²P]phosphate was investigated in focal areas of differing permeability in pig aortic intima and media identified in vivo by Evans blue dye uptake. In normal-fed pigs, no difference in the concentration of total cholesterol between blue and white areas of either intima or media was observed. Most of the oleic acid taken up by the aorta in the normal-fed pig was incorporated into phospholipid (primarily lecithin) and triglyceride with little incorporation into cholesteryl ester. The uptake and incorporation of the [1-¹⁴C]oleic acid into each of the lipid fractions studied was similar for blue and white areas of both intima and media. After 6 wk and 16 wk of cholesterol feeding, the concentration of cholesterol in the intima, but not in the media, of the blue areas were significantly greater than that in the corresponding white areas. In the 6-wk cholesterol-fed aortas, the uptake of ¹⁴C-labeled oleic acid and its incorporation into phospholipid, triglyceride and particularly into cholesteryl ester were all significantly increased in the blue areas of the intima compared with the corresponding white areas. This increase paralleled the increase in cholesterol concentration shown for these areas. No difference in uptake or incorporation of oleic acid into any of the lipid fractions studies occurred between blue and white areas from the 6-wk cholesterol-fed pig aortic media. After 16 wk of cholesterol feeding, there was no demonstrable difference between blue and white areas for either the intima or media with respect to the uptake of ¹⁴C-labeled oleic acid and its incorporation into lipid.With ³²P-labeled phosphate as precursor, no evidence for differing phospholipid synthesis in the blue and white areas of the normal-fed pig aortic intima or media could be obtained. When 6-wk cholesterol-fed pig aortic intima was studied, significant increases in sphingomyelin synthesis in blue compared to white areas were present. A trend towards increased incorporation into lecithin, phosphatidyl inositol and phosphatidyl ethanolamine and reduced incorporation of phosphatidyl inositol relative to lecithin was present in the blue compared with the white areas in the intima, but these differences were not statistically significant. No difference in [³²P]phosphate incorporation into phospholipid between the blue and white areas of the media was apparent for any of the phospholipid fractions.     

Retardation of protein synthesis in rat foetal liver on inhibiting rate of histamine formation

1. The rate of protein synthesis, as gauged by the incorporation of [¹⁴C]leucine, has been determined in vitro in foetal and 5-day-old rat liver, tissues with respectively high and low histidine decarboxylase activity.

2. Incorporation of [¹⁴C]leucine was considerably faster in foetal liver than in liver of the young.

3. Following inhibition of histamine formation by α-methyl histidine, a specific inhibitor of histidine decarboxylase, the rate of incorporation of [¹⁴C]leucine was substantially diminished in foetal but not in liver of the young.

4. The retarded incorporation of [¹⁴C]leucine consequent on inhibition of endogenous histamine formation could not be restored by extracellular histamine, i.e. by adding histamine to foetal liver tissue.

5. On complete inhibition of protein synthesis by puromycin in foetal and 5-day-old rat liver the rate of histamine formation was not affected.

6. The present observations support the view of endogenous histamine formation, `nascent histamine', as an essential part of the metabolic machinery in some rapidly growing tissues.

     

The effect of herpesvirus infection and 2-deoxy-D-glucose on glycosphingolipids in BHK-21 cells.

The effect of Herpes simplex virus (HSV) infection and 2-deoxy-d-glucose (dGlc) on the content of radiolabeled glycosphingolipids was studied in BHK-21 cells, after a 16 hr pulse with D-[3-³H]serine and [1-¹⁴C]palmitate. HSV infection caused a stimulated incorporation of the labeled precursors into total glycosphingolipids. The content of newly synthesized hematosides was reduced by 50% and newly synthesized ceramides, ceramide monohexosides, and ceramide dihexosides was increased two-to-threefold, suggesting a simplification of the glycolipid pattern. Treatment of infected and uninfected cells with 10 mM dGLc decreased radioactivity in all glycosphingolipids, whereas the ratio of cerarnide to its glycosylated derivatives increased. Glycosylation of endogenous ceramide was studied in vitro using microsomal fractions and UDP-[³H]glucose as the sugar donor. UDP-glucose:ceramide-glucosyltransferase activity was higher in microsomal fractions from HSV-infected cells than in preparations from controls, and treated cells had higher activities than untreated controls. Infection and dGlc treatment increased the number or accessibility of glucose acceptor sites. Thus, dGlc inhibits glycolipid synthesis in control and HSV-infected cells by blocking glycosylation of ceramide; the inhibition of glycolipid synthesis may have a role in virion assembly.     

Biosynthesis of various sterols, sterol esters, and squalene from [14C]mevalonate by normal swine intima and media in vitro: a comparative study.

The incorporation of dl-[2-¹⁴C]mevalonic acid into lipids of normal swine aorta was examined in vitro. Intima and media of intact arterial segments synthesized squalene and cholesterol and a variety of other C₂₇ sterols as well as C₃₀ sterols. Relative to the intima, squalene synthesis in the media of intact arterial segments was twice as great, while C₂₇ sterol synthesis was only half that of the intima; these differences were minimized when intima and media were separated prior to incubation. The esterification of de novo synthesized C₂₇ sterols, but not C₃₀ sterols, occurred in both intima and media of intact arterial segments and was significantly greater (P < 0.05) in the media. The fatty-acid pattern of the newly synthesized sterol esters, however, did not conform to the fatty-acid pattern usually found in normal arterial tissue in that a higher proportion of the esterified fatty acids were identified as tetraenes by argentation chromatography of the arterial sterol esters. The data suggest that de novo synthesized sterols enter a separate metabolic pool for esterification with fatty acids.     

Ether lipid synthesis from enantiomeric medium-chain and long-chain O-alkyl-sn-glycerols in Leishmania donovani promastigotes

A medium-chain O-alkylglycerol, 1-O-[1′-¹⁴C]dodecyl-sn-glycerol, has been found to be incorporated into plasmenyl ethanolamine by Leishmania donovani promastigotes as revealed by radio gas—liquid chromatography; however, the ether bond of the administered O-alkyl-glycerol was cleaved extensively as judged from the occurrence of radioactive acyl moieties. The labelling pattern produced by the radioactive ‘natural’ 1-O-octadecyl-sn-glycerol was similar though the latter served as a slightly better substrate for plasmalogens. Experiments with the enantiomeric 3-O-alkyl-sn-glycerols in comparison revealed that these were poor substrates for plasmalogen synthesis, although they were taken up in identical amounts and cleaved even to a higher extent. Therefore, we conclude the 1-O-alkyl-sn-glycerols were utilized directly for plasmenyl ethanolamine synthesis. The incorporation of the dodecyl residue into plasmenyl ethanolamine did not affect the multiplication and shape of cells.     

Labeled Citrate Metabolism in Bone Fractures and Impaired Innervation

Changes in ¹⁴C incorporation into regenerate after bone fracture and impairment of mandibular innervation, and injection of [3-¹⁴C]cytrate corresponded to the stages of reparative osteogenesis: after 1 week ¹⁴C incorporation in the cellular-fibrous callus surpassed its release, after 2 weeks the rates of ¹⁴C incorporation and release in the chondroid callus become similar, and after 4 weeks the release of the label predominated in the primary bone callus. Denervation reduced ¹⁴C incorporation into regenerate, which impaired bone remodeling. Citrate in the bones is characterized by high metabolic activity.     

Studies on methylene chloride-induced fatty liver.

The fatty liver induced by methylene chloride inhalation in the guinea pig was investigated by studying the effects of this solvent on several pathways of hepatic lipid metabolism. Inhalation of 5200 ppm (18,000 mg/m³) methylene chloride for 6 hr resulted in a 2.5-fold increase in hepatic triglyceride. Electron microscopy revealed the hepatocytes following exposure to be of normal appearance except for the presence of numerous lipid droplets in the cell periphery. No changes were evident by light microscopy.Serum free fatty acid levels were unaffected by exposure. The incorporation of palmitic acid into triglyceride by liver slices was also unchanged by methylene chloride. These results suggest that increased triglyceride synthesis rates were not responsible for the fatty liver. Exposure resulted in a 63% decrease in serum triglyceride, suggesting a defect in triglyceride secretion as a cause of the fatty liver. This defect does not appear to be due to decreased hepatic protein synthesis, in as much as incorporation of [1-¹⁴C]leucine into hepatic proteins was unaltered, or by decreased availability of phospholipids, because hepatic phospholipids actually increased with triglyceride. Liver slices from exposed animals incubated with control guinea pig serum containing [1-¹⁴C]palmitic acid were able to incorporate [¹⁴C] into triglyceride and then secrete triglyceride into the medium at control rates. In toto, the results may suggest that methylene chloride inhalation leads to the depletion or alteration of some component present in serum necessary for the maintenence of normal hepatic triglyceride secretion.     

A reevaluation of the status of cholesterol in erythrocytes infected by Plasmodium knowlesi and P. falciparum

The cholesterol synthesis of rhesus monkey erythrocytes parasitized by Plasmodium knowlesi and human erythrocytes infected by P. falciparum, as measured by incorporation of [1-¹⁴C]acetate and ³H₂O, was almost undetectable, concordant with very low levels of measurable 3-hydroxy-3-methyl glutaryl-CoA reductase activity. In addition, both types of infected cells exchanged cholesterol with the plasma at the same rate as uninfected cells. The data do not exclude the possibility of cholesterol transfer from uninfected to infected cells.     

Mechanism of renal necrosis induced by bromobenzene or chlorobenzene

Treatment of mice or rats with a single intraperitoneal dose of [¹⁴C]-bromobenzene or [¹⁴C]-chlorobenzene produced necrosis of the proximal convoluted renal tubules within 24–48 hr. The development of renal necrosis was associated with the covalent binding of substantial amounts of radiolabeled material to kidney proteins, and autoradiograms revealed that most of the covalently bound material was localized within the necrotic tubular cells. Prior inhibition of [¹⁴C]-bromobenzene metabolism in vivo with piperonyl butoxide blocked both the necrosis and the binding, suggesting that the necrosis and binding are caused by a toxic metabolite. Studies on the metabolism and covalent binding of [¹⁴C]-bromobenzene in hepatic and renal microsomes in vitro were compatible with the interpretation that the renal necrosis was caused by a metabolite formed in the liver and transported by the circulation to binding sites in the renal tubules.     

Fatty acid synthesis in the arrested rabbit heart during ischemia

In rabbit hearts arrested by a carnosine-buffered cardioplegic solution the incorporation of [1-¹⁴C]acetate into lipids was investigated. After 10–20 and 60 min of ischemia the radioactivities of phospholipids, mono-, di-, and triacylglycerols, acyl-CoA, acylcarnitine, and free fatty acids were determined. In the first period of ischemia mainly acylcarnitine was labelled (ca. 50%) and only 25% of [¹⁴C]-activity was found in phospholipids which showed the lowest specific activity of all lipid classes. After 60 min of ischemia the percentage of total radioactivity of acylcarnitine and phospholipids was decreased, whereas that of neutral lipids was increased to more than 50%. During the increase of total radioactivity the relative specific activity of all lipids decreased except that of triacylglycerols. Only fatty acids up to chain lengths of 16 carbon atoms were labelled. Lauric and myristic acid had high specific activities. These results indicated de novo synthesis of fatty acids accumulating in triacylglycerols during ischemia of the arrested heart.     

Influence of cyclosporine A on protein synthesis in rat liver

The influence of the immunosuppressive agent cyclosporine A on protein synthesis was investigated in rat liver in vivo and in vitro. Incorporation of [14C]leucine into total protein by microsomes was inhibited 36% in the presence of cyclosporine A. Treatment of rats with cyclosporine A also resulted in decreased protein synthesis by microsomes isolated from the same animals. This inhibition was dependent on the dose administered and the duration of treatment. The inhibitory factor in this latter case appeared to be associated with the supernatant fraction. Decreased in vivo incorporation of [35S]methionine into proteins of microsomal membranes and peroxisomes, but not into microsomal luminal proteins was also observed after treatment of rats with cyclosporine A. On SDS-polyacrylamide gel electrophoresis certain of the microsomal membrane and peroxisomal proteins displayed decreased labeling. Furthermore, cyclosporine A treatment decreased the inductive effects of phenobarbital, clofibrate, and phthalate ester on microsomal and peroxisomal enzymes. It is suggested that certain of the toxic effects of cyclosporine A are exerted through inhibition of cellular protein synthesis.     

Effect of beta-aminopropionitrile on lipid metabolism by bone cell cultures: biochemistry and morphology

The effects of β-aminopropionitrile (BAPN) on [¹⁴C]acetate monitored by bone cells derived from newborn rat calvaria were examined by biochemical techniques and light and electron microscopic procedures. The effects of BAPN depends upon both concentration and time of exposure to the lathyrogen. BAPN tends to stimulate the incorporation of [¹⁴C]acetate into most lipids at lathyrogen concentrations up to 40 mM. At 100 mM the incorporation is significantly reduced. Prolonging the time of exposure generally enhances lipid synthesis. The major exception is the reduced synthesis of cholesterol esters. At BAPN concentrations higher than 5 mM, vesicles, which appear to be dilated endoplasmic reticulum, are formed in the cell cytoplasm. Further, the BAPN treated cells do not appear to adhere to each other in the same manner as untreated cells suggesting changes in the cell glycocalyx. This study demonstrates BAPN induced alterations in lipogenesis in cells which have the potential to calcify. These alterations suggest an increase in cell membrane area giving rise to the vesicles and dilated endoplasmic reticulum.     

Tryptophan-induced stimulation of hepatic ornithine decarboxylase activity in the rat

The effect of a single tube feeding of l-tryptophan on hepatic ornithine decarboxylase (ODC) activity in rats was investigated. The levels of ODC activity in the livers of control and experimental rats were assayed in vitro by measuring the release of ¹⁴CO₂ from DL-[1-¹⁴C]ornithine. Single tube feedings of varying levels of l-tryptophan ($\text{2.5–30 mg}\text{100 g}$ body wt) to overnight-fasted rats 1 hr before sacrifice exhibited increases in the hepatic ODC activities. l-Tryptophan ($\text{30 mg}\text{100 g}$ body wt) tube fed to overnight-fasted rats $\text{1}\text{6}$ to 12 hr before sacrifice induced hepatic ODC activities which were significantly elevated beginning at 1 hr and peaking at 2 hr (6.5-fold increase over controls). In vitro [¹⁴C]leucine incorporation into protein using hepatic microsomes of tryptophan-treated rats was significantly increased at 1 hr in comparison with that of controls. The tryptophan-induced stimulation of hepatic ODC activity was not affected by prior adrenalectomy but was abolished by pretreatment with cycloheximide. These studies demonstrate that a single feeding of l-tryptophan can significantly enhance in the rat the activity of ODC, a key enzyme in the biosynthesis of polyamines.