Cosmo G. Mackenzie

Regulation of cell lipid metabolism and accumulation III. The lipid content of mammalian cells and the response to the lipogenic activity of rabbit serum

Abstract Total lipid was isolated from five mammalian cell lines maintained on a chemically defined medium plus horse serum. The lipid was separated quantitatively into four fractions by silicic acid chromatography and the results expressed as μg cell lipid per μg cell protein. The polar lipid constituent was in all cases a function of the protein content according to the relationship, polar lipid=0.154 × protein. The S. E. of the constant was ±0.003. The levels of the free cholesterol and hydrocarbon plus sterol ester fractions, while much smaller, were also approximately the same in all cells. Significant differences were observed, however, in the cell triglyceride to protein ratios which ranged from 0.01 to 0.05. Cells with triglyceride to protein ratios of 0.03 or more invariably contained large numbers of cytoplasmic lipid-rich particles. Substitution of rabbit serum for horse serum in the medium had no marked effect on the lipid pattern of three of the cell lines but caused a five- to tenfold increase in the triglyceride content of the rabbit liver cell and the L cell without comparable changes in their other lipid fractions. The effect of rabbit serum on the triglyceride content of these two cells was completely reversible. The lipogenic activity of rabbit serum was independent of the triglyceride and total lipid concentrations of the medium, the growth rate, and the population density. The lipogenic factor(s) was nondialyzable and was neither inhibited nor augmented by horse serum. It is concluded that a quantitative relationship exists between the polar lipid and protein constituents of a variety of mammalian cells when grown under identical environmental conditions, and that the triglyceride and total lipid content of these cells is determined by their ‘genetic’ constitution and the chemical nature of the external environment.

Fatty acid ester turnover: a control factor in triacylglycerol and lipid-rich particle accumulation in cultured mammalian cells.

Summary1.Experiments with albumin-bound [1-14C] palmitic acid, in which fatty acid synthesis was repressed, have shown that the larger accumulation of triacylglycerol and lipid-rich particles in a representative high-lipid cell, a rabbit liver cell clone, as compared to a representative low-lipid cell, a HeLa cell clone, was due primarily to the faster turnover of lipid in the HeLa cell.2.Thus in short-term experiments, the specific activities of the following lipid fractions were twice as high in the HeLa as in the rabbit liver cells: total cell lipid, phospholipid, triacyglycerol, and the palmitic acid isolated from the latter fractions. The palmitic acid content of the cell lipid, as well as its oxidation to CO2, was approximately the same in the two cell lines.3.In long-term experiments, the specific activity of the total cell lipid of the rabbit cell approached that of the HeLa cell. Two-thirds of the triacyglycerol palmitic acid in both cell lines was derived from the albumin-bound [1-14C]palmitic acid of the medium. Concomitantly, two-thirds and two-fifths of the phospholipid palmitic acid in the HeLa and rabbit cells, respectively, were derived from this same source.4.In chase experiments employing these heavily labeled cells, the % secretion of radioactive lipid, especially fatty acids derived from triacylglycerol, was substantially higher in the HeLa than in the rabbit liver cells. Despite the differences in turnover rates, the results indicate that in both cell lines the fatty acids of the lipid-rich particles were in dynamic equilibrium with the albumin-bound fatty acids of the serum.

Differential labeling of triglycerides and polar lipids of cultured mammalian cells by albumin-bound [1-14C] fatty acids of serum

SummaryRabbit liver cells were cultured in medium containing serum whose albumin-bound fatty acids were labeled with [1-14C] palmitic or oleic acid of determined specific activity. After 7 to 500 fold increases in cell mass, the cell lipid was extracted and fractionated by silicic acid column chromatography. The triglyceride and polar lipid fractions were saponified and their constituent fatty acids, in the form of methyl esters, were separated and isolated by gas chromatography and their specific activities determined. Based on their14C content, approximately three-quarters of the palmitic and oleic acids of the accumulated triglycerides, which constituted half of the cell lipid, were derived from their counterparts in the albumin-bound fatty acids of the medium. In the case of the structural polar lipids, approximately only one-half of the palmitic and oleic acids were derived from their albumin-bound counterparts. Since the presence of serum in the medium completely represses thede novo synthesis of fatty acids in cultured mammalian cells, it is concluded that an appreciable portion of the polar lipid fraction is derived from the complex lipids of the serum lipoproteins, or their partial hydrolysis products. Based on these considerations, a function of serum lipoproteins is to act as precursors of a portion of the cells structural lipids, or constituent parts thereof.Within the cell, [1-14C] palmitic acid was converted to radioactive stearic, oleic, and palmitoleic acids. [1-14C] oleic acid, however, was neither reduced nor converted in detectable amounts to polyenoic fatty acids. Comparison of the rabbit serum albumin-bound fatty acids with the fatty acids of the cells complex lipids showed that the latter contained lower concentrations of C16:0 and higher concentrations of C18:0 and C20:4 fatty acids than did the albumin. Also, within the cell, C16:0 was higher in the accumulated triglycerides whereas C18:0 and C20:4 were higher in the polar lipids. Concentrations of C18:1 and C18:2 did not differ greatly in the two fractions, but the small amount of C18:3 was confined almost entirely to triglycerides.

[141] Sarcosine dehydrogenase and dimethylglycine dehydrogenase (rat liver; monkey liver)☆

Publisher Summary This chapter discusses the assay, purification, and properties of sarcosine dehydrogenase and dimethylglycine dehydrogenase. Assay of the sarcosine and dimethylglycine dehydrogenase employs phenazine methosulfate and 2, 6-dichlorophenolindophenol (DCPIP). Sarcosine dehydrogenase and dimethylglycine dehydrogenase are found in the liver mitochondria of the rat, pig, rabbit, guinea pig, pigeon, and chicken. These enzyme activities are also present in microorganisms. Sarcosine dehydrogenase does not oxidize glycine, serine, monomethylaminoethanol, dimethylaminoethanol, choline, betaine, and threonine. Sarcosine dehydrogenase, but not dimethylglycine dehydrogenase, from mammalian liver mitochondria is inhibited reversibly and competitively by methoxyacetate and related acetates. The absorption spectra of the oxidized form of the purified enzymes show a major peak at 405–415 m μ and a broad shoulder in the region of 430–460 m μ . With sodium phosphate or pyrophosphate buffers of ionic strength 0.1, the pH optima of the sarcosine dehydrogenase and dimethylglycine dehydrogenase in the phenazine methosulphate (PMS)–DCPIP assay system are 8 and 8.5–9, respectively. The purified preparations of both the sarcosine and dimethylglycine dehydrogenases can be lyophilized, and the dry powders are stable for at least a week when stored at -4°.

Vitamin E Activity of Alpha-Tocopherylhydroquinone and Muscular Dystrophy

Summary and Conclusions The disuc-cinate of α-tocopherylhydroquinone has been tested for antisterility activity by daily intravenous injections in pregnant vit. E-deficient rats. This antidystrophic compound was found to be devoid of the antisterility activity associated with α-tocopherol. Moreover, the injection of dystrophic rabbits with large curative doses of α-tocopherylhydroquinone; or its disuccinate, did not lead to the appearance of tocopherol in the serum. Both of these observations indicate that α-tocopherylhydroquinone is not converted to α-tocopherol to a significant extent in the body. It appears therefore that α-tocopherylhydroquinone is an antidystrophic factor or vitamin per se. Alpha-tocopherol, on the other hand, is the antisterility vitamin. It may possess antidystrophic activity as such, or alternatively, it may only be the provitamin for α-tocopherylhydroquinone. The separation of antidystrophic from antisterility activity is discussed with relation to the function of vit. E in muscular dystrophy.

Prolongation of Consciousness in Anoxia of High Altitude by Glucose.

Conclusions 1. The preflight administration of a single dose of glucose in water to individuals on a normal diet produced a significant increase in the resistance to unconsciousness from anoxia at 27,000 and 30,000 ft. 2. The protection afforded flying personnel by the glucose solution was greater 30 to 50 minutes following its administration than after an interval of 60 to 80 minutes. 3. At 27,000 ft. the mean duration of consciousness was increased by approximately 40%, or more than one minute, by the preflight ingestion of glucose solution. 4. Vitamin C, either alone or in conjunction with glucose, had no demonstrable effect on the duration of consciousness.

Regulation of Cell Lipid Metabolism and Accumulation. VII. Increase by Glycerol of the Polar Lipid and Triglyceride Content of Cultured Cells

Summary Glycerol, at the concentrations used in cell freezing and storage procedures, causes a large increase in the lipid content of cultured mammalian cells. Column chromatography on silicic acid of the isolated lipid indicates that the increase is due primarily to increases in polar lipids and triglycerides. The homologues of glycerol, ethylene glycol and erythritol, also increase cell lipid.

[55] Electron transfer flavoprotein

Publisher Summary This chapter discusses Electron Transfer Flavoprotein (ETF). The quantitative determination of ETF in the presence of excess sarcosine and sarcosine dehydrogenase is based on the measurement of the rate of reduction of dichlorophenolindophenol (DCPIP). Several chromatographic procedures for purifying the ETF of rat liver mitochondria are presented. The two preparative methods most commonly employed on ETF are those beginning with the osmotic supernatant, followed by chromatography on either equilibrated or unequilibrated DEAE-cellulose. The supernatant fraction obtained from centrifugation of the sonically irradiated mitochondria is referred as the “sonic supernatant.” The ETF in the mitochondria is located in this fraction. The ETF, in the 60-80% ammonium sulfate fractions, is stable for about a week when these fractions are stored as wet precipitates at –18°. The activity of the purified ETF, similar to that of the original 60-80% ammonium sulfate fraction, can be stimulated with the addition of FAD. ETF is located in liver mitochondria, and is absent from other fractions of the liver cell. The richest sources of the enzyme found are rat, pig, and monkey liver. The visible absorption spectrum of rat liver ETF shows a maximum at 410 mμ and a broad shoulder at 450 mμ. The only substrates found for ETF are the substrate-reduced sarcosine and dimethylglycine dehydrogenases and the acyl-eoenzyme A dehydrogenases.