Abstract
Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty acids, the rabbit liver cells grown on the former media accumulated more lipid than cells grown on the latter media. This difference was shown to be due to the higher concentration of albumin per micro mole of fatty acid in horse serum as compared with rabbit serum. Consequently, the albumin to fatty acid ratio also controls the lipogenic activity of a serum. A linear relationship is presented which relates the cell lipid content to the molar ratio of albumin to free fatty acids and to the absolute concentration of free fatty acids in the medium.
Highlights
Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin
Consistent with such an assumption was the low lipogenic activity of the serum globulins and albumin prepared by the Cohn fractionation procedure in which isopropanol was used as the organic solvent (Table 2)
The results obtained with both (NH4)zso4 fractionation and ultracentrifugation suggested, that albumin rather than the lipoproteins of rabbit serum was responsible for most of its lipogenic activity
Summary
The rabbit liver cell, clone C-15, and the rate liver cell, clone C-1, were isolated in our laboratory [9]; the mouse fibroblast cell, L clone of strain NCTC-2071 [14], was kindly supplied by Dr Virginia Evans of the National Cancer Institute; and the human liver cell isolated by Chang [15] was obtained from Microbiological Associates, Inc., Bethesda, Md. The serum fractions with chloroform-methanol 2: 1 (viv), until the volume of were analyzed for total protein and lipid both before the filtrate reached 10 ml. The composition of the lipid in the various fractions was determined by chemical analysis, silicic acid column chromatography, and silicic acid thin-layer Chromatography as described under Materials and Methods. Lysolecithin was separated from lecithin in butanol-acetic acid-water 80 :20: 20, and in chloroforni-methanol-water 65 :25 :4 (v/v), according to the procedure of Rouser, Galli, Lieber, Blank, and Privett [35]. Reference compounds and their sources were as follows: palmitic acid, tripalmitin, cholesterol, and cholesteryl esters, The Horniel Institute, Austin, Minn. Benzoic acid-l-I4C (Packard Instrument Co., Inc., Downers Grove, Ill.) dissolved in toluene was used as an internal standard to determine the counting efficiency
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