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Metabolism and structure of triacylglycerols in rat epididymal fat pad adipocytes determined by 13C nuclear magnetic resonance.

Carbon-13 nuclear magnetic resonance (NMR) methods have been applied to a study of the structure and metabolism of the triacylglycerols from rat epididymal fat pad adipocytes. Complete NMR signal assignments are provided for adipocytes, the extracted triacylglycerols, and methyl esters of the derived fatty acids. 13C NMR yielded rapid, nondestructive, quantitative analysis of the amounts of unsaturation of the fatty acyl chains; in cells from rats given ad libitum access to a standard laboratory diet the predominant fatty acids were found to be palmitate (29.9%), oleate (27.9%), and linoleate (34.1%). These results agreed with gas chromatographic separation of the derived methyl esters of the extracted lipids. Lipid dynamics were examined in situ and showed a substantial restriction of motion of glyceride-glycerol as compared with free glycerol; the nuclear magnetic spin-lattice relaxation times for free glycerol of 2.52 +/- 0.12 (C1,3) and 4.37 +/- 0.21 (C2) s decreased to 0.15 +/- 0.009 and 0.21 +/- 0.013 s, respectively, upon esterification. Segmental motion of the chains, monitored by relaxation time measurements, increased progressively from the alpha-carbon (nT1 = 0.70 s) to the methyl ends of the chains (nT1 = 9.63). The incorporation of C-13-labeled substrates ([1-13C]glucose and [3-13C]lactate) into the glycerol moiety of triacylglycerols was monitored in real time, in the presence of insulin. Lactate (10 mM) inhibited the incorporation of glucose (5.5 mM) into glyceride-glycerol. Lipolysis at the natural abundance level of 13C was measured in the presence of 10 microM isoproterenol. Simultaneous lipogenesis and lipolysis were found to occur in situ and were measured with the aid of [1-13C]glucose and isoproterenol; the labeling pattern of medium glycerol versus extracted triacylglycerols was significantly different from that found using natural abundance glucose. Our results indicate that 13C NMR is a useful new method for the real-time monitoring of lipid structure and metabolism in vivo.

Adipose Tissue↗

[Hepatotoxicity of the lipid components of the lipopolyysaccharides from typhoid bacteria].

Distinct increase in amount of total lipids occurred in mice liver tissue after intraperitoneal administration of typhoid lipids A and B (the latter was nonhydrolytically isolated from typhoid lipopolysaccharides). The alteration referred primarily to neutral lipids, especially to triacyl glycerides, content of which exceeded 3- and more-fold the initial one to the end of a day. The similarity was observed in alteration of triacyl glycerides, phospho- and glycolipids in presence of both lipid preparations. These lipid components resembled intact lipopolysaccharide from typhoid bacteria in their effect on content of lipids in liver tissue. In treatment with these lipid preparations concentration of NEFA was either unaltered or distinctly decreased.

Animals↗

Effect of ethionine on carbohydrate and lipid metabolism.

In ethionine-treated rats, the ATP content of adipose tissue was not decreased whereas in liver it was drastically reduced to about one-fifth of the control level. Shortly after the injection of ethionine, hepatic glycogen was depleted and the blood glucose concentration fell from 120 to 80 mg/100 ml. This was followed by a two- to threefold elevation of the plasma fatty acid level. Hepatic glucose-6-phosphate was decreased and was not elevated by administration of 2 mmoles of glucose unless ATP was partially restored to normal levels. When hepatic ATP was decreased, the disappearance of [(14)C]glucose from the blood and its incorporation into glycogen and glyceride-glycerol and the incorporation of [3-(14)C]pyruvate into glyceride-glycerol were reduced. 6 hr after ethionine injection the plasma triglyceride level fell but there was no significant change in the liver triglyceride concentration, but by 24 hr it had increased markedly. Lipogenesis in adipose tissue was depressed in vivo, possibly due to decreased glycerol-3-phosphate concentrations. A marked decrease of glycerol-3-phosphate in both liver and adipose tissue was noted. Administration of glucose effectively depressed plasma free fatty acid concentration and elevated the glycerol-3-phosphate levels. Ethionine injection to fasted animals further depressed the blood glucose and elevated the plasma free fatty acid level.

Adenosine Triphosphate↗

Lipogenesis from glucose and pyruvate in fat cells from genetically obese rats.

Subcutaneous fat cells were isolated from genetically obese rats and from rats with obesity produced by hypothalamic lesions. Insulin did not augment the oxidation of fatty acids or their synthesis from glucose-1-(14)C or glucose-1-(3)H by fat cells from either group. Radioactivity from pyruvate-3-(14)C was incorporated into fatty acids to the same degree by fat cells from these two groups. The presence of 5 mm glucose in the incubation medium containing fat cells and pyruvate-3-(14)C or aspartate-3-(14)C stimulated the synthesis of fatty acids to a greater extent in cells of genetically obese rats. Fasting, in contrast, reduced the incorporation of radioactivity from pyruvate and glucose into fatty acids by fat cells from the genetically obese animals. In all experiments the fat cells from genetically obese rats converted more radioactivity into glyceride-glycerol relative to CO(2) than did fat cells from hypothalamic obese rats. Parabiosis between one thin and one genetically obese litter mate was performed in three pairs of rats without influencing growth of either rat. Thus in the present studies fat cells from genetically obese rats showed two differences from normal fat cells: they channeled more radioactivity from pyruvate into fatty acids in the presence of glucose, and they uniformly converted more radioactivity into glyceride-glycerol.

Adipose Tissue↗

Molecular weight distributions of milk fat triglycerides from seven species.

The triglyceride compositions of the milk fats of man, dog, guinea pig, cow, sheep, goat, and horse were compared by gas-liquid chromatography of the intact triglycerides and of the butyl esters of the component fatty acids. The milk fats of man, dog, and guinea pig, which were largely made up of long-chain fatty acids, showed a common pattern with major contributions made by the glycerides with 48-54 acyl carbon atoms. The milk fats of cow, sheep, and goat, which were rich in short-chain acids, showed significant proportions of triglycerides with 28-54 acyl carbon atoms. Horse milk, which contains large amounts of medium-chain fatty acids, gave a characteristic triglyceride pattern in the 26-54 carbon atoms range. The experimentally determined distributions of the molecular weights of the triglycerides of all milk fats deviated significantly from the distributions predicted by random association of the fatty acids from a single pool. The data suggest that in all species the milk fat may be formed by a partial resynthesis of preformed glycerides.

Animals↗

The mechanism of the insulin-like effects of ionic zinc.

The insulin-like effects of ionic zinc (Zn2+) were studied in isolated rat adipocytes. Concentrations of Zn2+ between 250 and 1000 microM stimulated 3-O-methylglucose transport and glucose metabolism to CO2, glyceride-fatty acid, and glyceride-glycerol. Selective stimulation of the pentose phosphate cycle was observed since a Zn2+-induced increase in glucose carbon 1 oxidation persisted even when glucose transport was blocked with 50 microM cytochalasin B or when transport was no longer rate-limiting for metabolism at high concentrations of glucose. Enhanced pentose phosphate cycle activity may have been due to a selective inhibition of glutathione reductase by the ion, which was also accompanied by a fall in cellular glutathione content. Zn2+ also inhibited lipolysis stimulated by the beta-adrenergic agent ritodrine in the absence of glucose. The effects of Zn2+ on glucose oxidation and stimulated rates of lipolysis were inhibited by extracellular catalase, indicating that they were largely a result of H2O2 generation. The H2O2 production appeared for the most part to be caused by zinc-catalyzed autoxidation of sulfhydryl groups present on external cell membranes, although involvement of sulfhydryl groups on bovine serum albumin in the buffer could also have contributed. The insulin-like effects of Zn2+ in adipocytes are therefore caused not only by direct effects of the ion on intracellular metabolism but also by indirect effects related to H2O2 generation.

3-O-Methylglucose↗

Apolipoprotein, an intermediate in the processing of the major lipoprotein of the Escherichia coli outer membrane.

A new intermediate (apolipoprotein) in the synthesis of the major lipoprotein of the Escherichia coli outer membrane has been identified. The accumulation of this new form of the lipoprotein was observed when excessive production of lipoprotein was induced or when the membrane fraction containing the prolipoprotein accumulated in the presence of globomycin was incubated at 60 degrees C. The new form of the lipoprotein could be chased into the mature lipoprotein. In addition, from sequential analysis of this new protein by Edman degradation, the NH2 terminus was found to be cysteine, containing a free unmodified amino group and a glyceride-modified sulfhydryl group. These results indicate that this protein is an intermediate in the conversion of glyceride-modified prolipoprotein to the mature lipoprotein. It is believed that the lipoprotein signal peptidase directly cleaves the lipoprotein signal peptide at the peptide bond between the glycine residue at position 20 and the cysteine residue at position 21 of the prolipoprotein. The resulting intermediate, designated here as apolipoprotein, is subsequently acylated at its free amino group to yield the final mature lipoprotein.

Amino Acid Sequence↗

Membrane-bound penicillinases in Gram-positive bacteria.

The penicillinases of Bacillus licheniformis, Bacillus cereus, and Staphylococcus aureus are related in structure and cellular localization to one another more closely than they are to penicillinases from Gram-negative organisms. In the latter, penicillinases are almost exclusively found in the periplasm, while the Gram-positive bacteria retain a substantial proportion as hydrophobic membrane-bound forms. We recently showed (Nielsen, J. B. K., Caulfield, M. P., and Lampen, J. O. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3511-3515) that B. licheniformis membrane attachment was achieved through a glyceride thioether modification identical to that in several Gram-negative outer membrane proteins. We now report that the membrane penicillinases of S. aureus and B. cereus also possess the modification. We do this by demonstrating isotopic labeling of these forms of [3H]palmitate, by showing that they exhibit the same response to the antibiotic globomycin which appears to inhibit processing steps specifically involving the glyceride thioether in Escherichia coli outer membrane proteins, and lastly by isolating glyceryl cysteine sulfone, the oxidation product of the modified cysteine residue. By comparing the modification-susceptible signal sequences of Gram-positive penicillinases and of Gram-negative outer membrane proteins with those of nonmodified Gram-negative penicillinases, we describe in increased detail the structural features within the signal sequence that allow modification and cleavage resulting in membrane anchorage.

Anti-Bacterial Agents↗

[Changes in the cholesterol levels of high density lipoproteins following the intravenous administration of triton WR 1339 to rats (author's transl)].

The aim of the present study was to determine, in the rat, changes in the plasma lipids and lipoprotein levels after single or iterative administration of triton WR 1339 in order to elicit the inverse relation between triglyceride and high density lipoprotein levels in man and animal. 24 hours after the acute intravenous injection of triton WR 1339 increases of cholesterol and glyceride were observed while HDL decreased. These effects are potentiated by iterative intravenous administration of triton. According to Nikkila these variations could be explained by the fact that a part of HDL proceeds from the catabulism of lipoproteins rich in glycerides. At the time of the recovery of epuration process, the HDL regeneration from VLDL that are abondant in the plasma should represent the changes observed. The effects found after iterative intravenous administration of triton, confirm that infusion of the detergent induces in the blood a decrease of the catabolism of lipoproteins rich in triglycerides and at the same time the disappearing of blood HDL.

Animals↗

Role of fatty acid synthesis in the control of insulin-stimulated glucose utilization by rat adipocytes.

A decreased capacity for fatty acid synthesis is associated with a decreased insulin effect on glucose metabolism in large fat cells and fat cells from rats fed a high-fat diet. We have investigated the relationship between these processes by specifically inhibiting fatty acid synthesis with (-)-hydroxycitrate (2.5 mM), an inhibitor of citrate cleavage enzyme, and cerulenin (0.05 mM), an inhibitor of fatty acid synthetase. (-)-Hydroxycitrate and cerulenin decreased maximally insulin-stimulated fatty acid synthesis from [6-(14)C]glucose to 10% and 25% of controls, respectively, while only (-)-hydroxycitrate decreased basal values. Oxidation of [1-(14)C]glucose in the presence of insulin was markedly depressed by each inhibitor. Thus, the percent increase over basal value was decreased from 540% in controls to 151% and 154% by (-)-hydroxycitrate and cerulenin, respectively. In contrast, oxidation of [6-(14)C]glucose was slightly enhanced by both inhibitors. Thus, oxidation of glucose via the pentose shunt was reduced, while Krebs cycle oxidation was unaffected. Basal and insulin-stimulated incorporation of [1-(14)C]glucose and [6-(14)C]glucose into glyceride-glycerol and basal lactate production was unchanged by the inhibition of fatty acid synthesis. Insulin-stimulated lactate production was halved by the inhibition of fatty acid synthesis. Total glucose utilization, as assessed by measuring the disappearance of glucose from the medium, was not detectably changed by inhibiting fatty acid synthesis under basal conditions, but insulin-stimulated values were decreased to 52% and 64% of control by (-)-hydroxycitrate and cerulenin, respectively. This occurred despite the fact that neither agent affected the initial rate of 2-deoxyglucose uptake, or glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase activities. These data therefore provide direct evidence that a limitation of the fatty acid synthetic pathway decreases the ability of insulin to stimulate both pentose shunt glucose oxidation and overall glucose utilization, but not Krebs cycle oxidation or glyceride-glycerol synthesis. The enzymatic capacity of the fat cell for fatty acid synthesis is therefore an important determinant of insulin-stimulated glucose utilization.-Fried, S. K., M. Lavau, and F. X. Pi-Sunyer. Role of fatty acid synthesis in the control of insulin-stimulated glucose utilization by rat adipocytes.

Adipose Tissue↗

Triacylglycerol turnover in large and small rat adipocytes: effects of lipolytic stimulation, glucose, and insulin.

Rates of lipolysis and reesterification were determined under various conditions in adipocytes from epididymal fat pads of old, spontaneously obese rats and compared to cells from younger, leaner animals. No differences were observed in lipolytic responsiveness to several concentrations (2--50 microM) of the beta-adrenergic agent ritodrine compared to cells from younger, leaner rats. The large cells showed diminished rates of lipolysis, reesterification, and glyceride-glycerol synthesis from glucose of a submaximal but not maximal insulin concentrations, probably reflecting decreased large cell receptor numbers. In both cell types reesterification measured in the presence of ritodrine progressively rose with increasing concentrations of glucose in the medium. At each glucose concentration (0.5--25 mM) rates of reesterification were similar in each cell type. When maximal concentrations of insulin (5 nM) were also added, at low glucose concentrations (less than 5 mM) there was a similar increase in reesterification in large and small cells. No insulin effect in either cell type was observed at high concentrations of glucose. Although fatty acid synthesis from glucose in the large cells was markedly diminished, glyceride-glycerol synthesis was well maintained, correlating well with calculated reesterification rates. In fact, reesterification was found to be quantitatively very important in determining total triacylglycerol turnover in both cell types. High rates of reesterification might not only allow maintenance of triacylglycerol stores, but could also increase metabolic sensitivity to changes in hormonal or substrate concentrations.

Adipose Tissue↗

Prolipoprotein signal peptidase in Escherichia coli is distinct from the M13 procoat protein signal peptidase.

We have previously reported a signal peptidase activity in Escherichia coli cell envelope which processes prolipoprotein modified with glyceride (Tokunaga, M., Tokunaga, H., and Wu, H. C. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2255-2259). To ascertain whether the processing enzyme for prolipoprotein is distinct from the signal peptidase for M13 procoat protein purified by Zwizinski and Wickner (Zwizinski, C., and Wickner, W. (1980) J. Biol. Chem. 255, 7973-7977), we have used antibody against purified procoat protein signal peptidase to study the processings of prolipoprotein and M13 procoat protein in vitro. the signal peptidase for modified prolipoprotein remained fully active in solubilized membrane preparations which had been treated with antibody against purified procoat protein signal peptidase whereas the activity towards procoat protein was completely abolished by immunoadsorption. Furthermore, both unmodified and glyceride-modified prolipoprotein were not cleaved by the highly purified signal peptidase preparation provided by Wickner. These data clearly indicate that prolipoprotein signal peptidase is distinct from the M13 procoat protein signal peptidase.

Antigen-Antibody Complex↗

Ethanol induced changes in prostatic lipid profiles of albino rats.

Alcohol induced changes in the ventral and dorsolateral prostatic lipid profiles were studied. The results obtained from the present study indicate that alcohol consumption leads to significant alterations in the ventral and dorsolateral prostatic lipids. An increase in the ventral prostate total cholesterol and total glyceride glycerol was observed, but a reverse trend was seen in total phospholipid. However total lipids remained unaltered. Dorsolateral prostatic total lipids, glyceride glycerol and phospholipids were increased but a decrease in total cholesterol was observed. Withdrawal of ethanol treatment for a period of 30 days brought back most of the changes to normalcy. The abnormalities in lipid profiles of both ventral and dorsolateral prostates may be responsible for the altered semen quality prevalent in chronic alcoholics.

Alcoholism↗

[Metabolic study of 3 lipid emulsions used parenteral feedings. I. Exploration in vivo in the minipig].

The metabolism of three fat emulsions (Lipiphysan, Trive 1000, Intralipid) was compared when administered by intravenous route as complete parenteral nutrition in minipigs during 14 days. With Lipiphysan, an important accumulation of glycerides appeared in liver, spleen and lung whereas the phospholipids content of those tissues was unchanged. The three fat emulsions determined an increase of linoleic acid (18:2) percentages in glycerides and phospholipids of several tissues: liver, heart, lung, spleen and kidney. Moreover, at the level of phosphatidyl-ethanolamine (PE) in liver tissue, the three fat emulsions induced a decrease of some polyunsaturated fatty acids (C20 and C22) percentages. The observed differences in the amount of liver triglycerides between the three fat emulsions seem to be related with the removal mechanisms which appear to be dependent on each fat emulsion. In fact, all tissue lipid analyses showed an increase of 18:2 percentages which agree with an uptake of the different fat emulsions used. The decrease of polyunsaturated fatty acids percentages in PE might be due to an inhibition of elongationdesaturation system or a degradation of those fatty acids.

Animals↗

Lipid transfer from insect fat body to lipophorin: comparison between a mosquito triacylglycerol-rich lipophorin and a sphinx moth diacylglycerol-rich lipophorin.

Two insect lipoproteins, triacylglycerol-rich Aedes aegypti lipophorin and diacylglycerol-rich Manduca sexta lipophorin, were compared in their ability to load neutral lipid from fat body. When fat body of M. sexta was incubated in vitro with [3H]oleic acid, all radiolabeled fatty acids were esterified, predominantly to triacylglycerol. In A. aegypti fat body, however, half of the label remained as free fatty acids. When A. aegypti fat body was radiolabeled with [3H]glycerol, most of the radiolabel was incorporated in triacylglycerol. When either A. aegypti or M. sexta lipophorin was incubated with A. aegypti fat body, labeled with [3H]oleic acid, both lipophorins incorporated mainly radiolabeled free fatty acids, while almost no radiolabeled glycerides were transferred. When the same experiment was performed with A. aegypti fat body, radiolabeled with [3H]glycerol, very little transfer of radiolabeled glycerides was detected. In contrast, when either M. sexta or A. aegypti lipophorin was incubated with M. sexta fat body, both lipophorins incorporated neutral lipids, predominantly diacyglycerol. A. aegypti lipophorin incorporated half the amount of radiolabeled lipid, compared to M. sexta lipophorin. Lipophorins from both species were treated with triacylglycerol lipase of the yeast Candida cylindracea. Although this lipase readily delipidated M. sexta HDLp, it was not able to remove triacylglycerol from A. aegypti HDLp. The data presented suggest that, under the conditions used, lipid transfer from fat body to lipophorin in A. aegypti is not as efficient as in M. sexta.

Aedes↗

[Histological and biochemical studies on the postnatal development of the guinea pig liver (author's transl)].

PROBLEM: In newborn guinea pigs the structural development of the liver was studied with emphasis on changes in the lipid content as related to biochemical parameters. MATERIAL AND METHODS: The livers of 149 guinea pigs (strain PIRBRIGHT) up to an age of 21 days were examined. The mothers were grouped in cages and kept as usual (room temperature 22 degrees C, air humidity 60-70%, natural light-dark rhythm and commercial standard diet ad libitum, sawdust and straw as bedding). Pregnant animals were placed in individual cages being left there together with their offspring until weaning or sacrification. In this period additional uptake of solid diet was allowed to the guinea pigs. The animals were bleeded to death in anesthesia (by head stroke). For histological examination tissue samples from the Lobes sinister lateralis were fixed in Barker's formol or absolute alcohol. The following staining reactions were applied (paraffin embedding or frozen sections): haemalum-eosin stain; PAS-reaction; Sudan III; Sudan black; Best's carmine (for details on the techniques see ROMEIS 1968). In biochemical analysis the total lipids of the liver were determined by the isolation methods reported by FOLCH et al.(1957). Moreover, in modification of the analytical procedures earlier described (SALLMANN 1972 a, b) several fractions of the liver total lipids could be isolated. By dialysis against a rubber membrane a dialysable lipid component (hydrocarbons, triglycerides, incomplete glycerides, cholesterol and free lipid acids) was isolated from the phosphatide fractions. Further separation of the "dialysable" lipids on Florisil columns yielded purified glyceride fractions. The total lipids as well as all components isolated from these were determined gravimetrically after removal of the solvents concerned. In biochemical analysis the livers of fetuses 10-12 days ante partum and of young animals up to 53 days were included. Results (see also table 1): The relative liver weight--related to the body weight reduced for that of the gastrointestinal tract--is highest at birth. It is lowered independent of the development of the gastrointestinal tract within a short period, then it remains fairly constant during the rest of the time of observation. On the 1st day abundant lipids are regularly distributed throughout the whole liver lobule (fig. 1). Beginning with the 3rd day the central parts of the lobules are nearly free from lipids, only in the peripheral zones of the lobules lipid augmentation was still observed. Consequently the marked decrease in liver weight is due to the rapid reduction of lipids within the first 3 days of life. These observations are in accordance with the biochemical findings: about 10 days ante partum noticeable storage of lipids occurs in the liver reaching its peak 2 to 3 days after birth (physiological fatty infiltration of the liver during the last days of intrauterine life). In fetal liver tissue predominantly tryglycerides are augmented.

Animals↗

SOME BIOCHEMICAL CHANGES IN THE GUINEA PIG DURING INFECTION WITH COXIELLA BURNETII.

Paretsky, D. (University of Kansas, Lawrence), C. M. Downs, and C. W. Salmon. Some biochemical changes in the guinea pig during infection with Coxiella burnetii. J. Bacteriol. 88:137-142. 1964.-Guinea pigs infected with Coxiella burnetii, the rickettsial agent of Q fever, were studied for 11 days postinfection. Maximal changes in liver lipids, liver phosphorylase, and uridine diphosphate glucose (UDPG)-glycogen glucosyl-transferase activities occurred 3 to 4 days post-infection. In this period, total liver lipids increased from 1.26 to 5.46 mg/mg of N, with the largest increment in the glyceride fraction. Liver glycogen virtually disappeared by the second day, with no chemically detectable restoration until the eleventh day. A pattern of altered phosphorylase and UDPG-glycogen transglucosylase activities was observed, with maximal phosphorylase and minimal glucosyltransferase activities at the third and fourth days. Histochemical observations confirmed chemical analyses for lipids and glycogen.

Animals↗

LIPIDS OF SARCINA LUTEA. I. FATTY ACID COMPOSITION OF THE EXTRACTABLE LIPIDS.

Huston, Charles K. (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.), and Phillip W. Albro. Lipids of Sarcina lutea. I. Fatty acid composition of the extractable lipids. J. Bacteriol. 88:425-432. 1964.-The extractable lipids of Sarcina lutea were separated into several fractions by a combination of column and thin-layer chromatography. Qualitative and quantitative characterization of the fatty acid content of these lipid fractions was accomplished by means of gas-liquid chromatography and infrared analyses. Of the total extract, the lipids consisted of 2.1% free fatty acids, 51.0% glycerides, and 22.7% complex lipids; they had a fatty acid content with a complete spectrum of carbon numbers from C(8) to C(22). The fatty acids included a large component of branched-acids in addition to the normal straight-chain acids. The branched-acids, comprising 40% of the fatty acids analyzed, constituted a homologous series of iso-acids from C(12) to C(19). Two 18-carbon unsaturates were found cis-9-octadecenoate and cis-11-octadecenoate. A relatively high percentage (20.5%) of the extractable material from S. lutea was found to be hydrocarbon. This material was not further characterized.

Chemical Phenomena↗