Oscar K. Reiss

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.

Acute and chronic hypokalemia sensitize the isolated heart to hypoxic injury

We examined the effects of acute and/or chronic hypokalemia on responses to 30 min of hypoxia and recovery in the isolated, perfused heart model. We found that both acute hypokalemia and chronic hypokalemia impaired contractility [expressed as maximum slope of pressure increase over time (dP/d t): 501 ± 49 and 529 ± 48 vs. 1,302 ± 118 mmHg/s, P < 0.01] and recovery of ATP concentrations (determined with31P NMR spectroscopy: 30 ± 6 and 40 ± 10 vs. 67 ± 5% initial, P < 0.05) at 30 min of recovery. Moreover, the combination of acute hypokalemia and chronic hypokalemia had additive effects (dP/d t 166 ± 15 mmHg/s and ATP 21 ± 7% initial, both P < 0.01). We also measured cytosolic calcium with surface fluorescence spectroscopy after indo 1 loading. Acute hypokalemia and acute hypokalemia + chronic hypokalemia increased cytosolic calcium (averaged throughout the cardiac cycle) during and after hypoxia (390- to 460-nm ratio at 30 min of recovery: 0.46 ± 0.07 and 0.65 ± 0.07 vs. 0.18 ± 0.03, P < 0.01), whereas control and chronic hypokalemia hearts had only small changes with hypoxia and recovery. Finally, when we examined mitochondria isolated from hearts perfused under experimental conditions, we found that chronic hypokalemia-alone mitochondria and chronic hypokalemia + acute hypokalemia mitochondria had marked impairment of state 3 respiration compared with control hearts (52 ± 13 and 50 ± 9 vs. 128 ± 10 natm ⋅ min-1 ⋅ mg protein-1 with succinate as substrate, P < 0.01), whereas acute hypokalemia mitochondria demonstrated only subtle changes. These data suggest that both acute hypokalemia and chronic hypokalemia impair cardiac responses to hypoxia. The mechanism may involve impairment of calcium metabolism, but cytosolic calcium alterations do not explain all of the metabolic and functional effects of acute hypokalemia and chronic hypokalemia in the setting of hypoxia.We examined the effects of acute and/or chronic hypokalemia on responses to 30 min of hypoxia and recovery in the isolated, perfused heart model. We found that both acute hypokalemia and chronic hypokalemia impaired contractility [expressed as maximum slope of pressure increase over time (dP/dt): 501 +/- 49 and 529 +/- 48 vs. 1,302 +/- 118 mmHg/s, P < 0.01] and recovery of ATP concentrations (determined with 31P NMR spectroscopy: 30 +/- 6 and 40 +/- 10 vs. 67 +/- 5% initial, P < 0.05) at 30 min of recovery. Moreover, the combination of acute hypokalemia and chronic hypokalemia had additive effects (dP/dt 166 +/- 15 mmHg/s and ATP 21 +/- 7% initial, both P < 0.01). We also measured cytosolic calcium with surface fluorescence spectroscopy after indo 1 loading. Acute hypokalemia and acute hypokalemia + chronic hypokalemia increased cytosolic calcium (averaged throughout the cardiac cycle) during and after hypoxia (390- to 460-nm ratio at 30 min of recovery: 0.46 +/- 0.07 and 0.65 +/- 0.07 vs. 0.18 +/- 0.03, P < 0.01), whereas control and chronic hypokalemia hearts had only small changes with hypoxia and recovery. Finally, when we examined mitochondria isolated from hearts perfused under experimental conditions, we found that chronic hypokalemia-alone mitochondria and chronic hypokalemia + acute hypokalemia mitochondria had marked impairment of state 3 respiration compared with control hearts (52 +/- 13 and 50 +/- 9 vs. 128 +/- 10 natm.min-1.mg protein-1 with succinate as substrate, P < 0.01), whereas acute hypokalemia mitochondria demonstrated only subtle changes. These data suggest that both acute hypokalemia and chronic hypokalemia impair cardiac responses to hypoxia. The mechanism may involve impairment of calcium metabolism, but cytosolic calcium alterations do not explain all of the metabolic and functional effects of acute hypokalemia and chronic hypokalemia in the setting of hypoxia.

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.

Effects of fine carbonaceous particles containing high and low unpaired electron spin densities on lungs of female mice

The negative impacts on human health that accompany inhalation of atmospheric particles are documented in numerous epidemiologic studies, but the effect of specific chemical properties of the particles is generally unknown. We developed and employed technology for generating inhalable aerosols of carbonaceous air pollution particles that have specific physical and chemical properties. We find that inhaling particles with greater unpaired electron spin (free radical) densities stimulates greater lung inflammatory and oxidative stress responses. Cultured alveolar macrophages take up more particles of greater free radical content, develop mitochondrial abnormalities, and release more leukotriene B(4) (LTB(4)) than alveolar macrophages exposed to lesser free-radical-containing particles in vitro. Mice exposed to high free radical particles in vivo also develop mitochondrial abnormalities in alveolar macrophages and increased oxidative stress, which is reflected by increases in lung nitrotyrosine staining and lung lavage nitrogen oxide levels compared with those of lesser free radical density. These results provide insight for the unexplained geographic differences and have implications for fossil fuel combustion conditions and the impact of fine particles on health and disease.

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.

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.