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Effect of prior nutritional status on the activity of lipogenic enzymes in primary monolayer cultures of rat hepatocytes.

ABSTRACT: Effect of prior nutritional status of the animal on the activity of lipogenic enzymes and the fatty acid content of cultured hepatocytes was investigated. Hepatocytes were isolated from rats that were starved for 24 h ('starved') or continuously fed ('fed'), or starved for 48 h and then re-fed for 48 h ('re-fed') with a carbohydrate-rich fat-free diet, and maintained as monolayer cultures for 96 h in a serum-free glucose-rich medium (Waymouth's MB752/1) supplemented with insulin, dexamethasone and tri-iodothyronine. The fatty acid content and the activities of acetyl-CoA carboxylase, fatty acid synthase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were determined initially at 3 h after plating and then every 24 h. Initially the activities of all the four enzymes were highest in hepatocytes isolated from the re-fed rats and lowest in those from the starved rats. With time in culture, the activity of all these enzymes increased severalfold (2-5, depending on the enzyme under consideration) in hepatocytes isolated from fed and starved rats, whereas there was a severalfold (2-5) decrease in the activity of these enzymes in hepatocytes isolated from re-fed rats. The initial fatty acid content of the hepatocytes from re-fed rats was 2-3 times that in the other two groups of hepatocytes. The fatty acid content seemed to increase in all three groups of hepatocytes during the 96 h in culture, but these apparent increases were not statistically significant.

SUBMITTER: Kelley DS

PROVIDER: S-EPMC1146652 | biostudies-other | 1986 Apr

REPOSITORIES: biostudies-other

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Project description:We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7 alpha-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most predominant bile acid in rat bile, to the culture medium suppressed cholesterol 7 alpha-hydroxylase activity and mRNA time- and dose-dependently. The decrease in enzyme activity paralleled the changes in mRNA. Maximal suppression of cholesterol 7 alpha-hydroxylase mRNA (-91%) and enzyme activity (-89%) was observed after a 16 h incubation period with 50 microM taurocholic acid. The declines in mRNA and enzyme caused by taurocholic acid were tightly coupled and followed first-order kinetics with a half-life of 4 h. Transcriptional activity, as assessed with nuclear run-on assays, was decreased by 44% at 50 microM taurocholic acid. Mass production of bile acids (chenodeoxycholic acid and beta-muricholic acid) was inhibited to a similar extent as the cholesterol 7 alpha-hydroxylase when different concentrations of taurocholic acid were used, giving maximal inhibition (-81%) at 50 microM taurocholic acid. Glycocholic acid and unconjugated cholic acid were equally effective as taurocholic acid in suppressing cholesterol 7 alpha-hydroxylase mRNA. The more hydrophobic bile acids (chenodeoxycholic acid and deoxycholic acid) showed profound suppression of the cholesterol 7 alpha-hydroxylase mRNA by 85% and 75% respectively, whereas the other trihydroxy bile acids in rat bile, alpha- and beta-muricholic acid, were not or only marginally active. We conclude that rat bile acids, in particular the more hydrophobic ones, in concentrations commonly observed in portal blood, exert negative feedback control at the level of cholesterol 7 alpha-hydroxylase mRNA in cultured rat hepatocytes through a direct effect on the hepatocytes, and that down-regulation of transcription is only one of the mechanisms involved in this regulation.

Project description:Amino acid and K(+) transport during development has been investigated in hepatocyte monolayer cultures with either alpha-amino[1-(14)C]isobutyrate or (86)Rb(+) used as a tracer for K(+). Parenchymal cells from neo- and post-natal rat livers have been isolated by an improved non-perfusion technique [Bellemann, Gebhardt & Mecke (1977)Anal.Biochem.81, 408-415], and the resulting hepatocyte suspensions purified from non-hepatocytes before inoculation. In the presence of Na(+) (Na(+)-dependent component), the rates of amino acid uptake in neonatal hepatocytes were markedly enhanced compared with cells from 30-day-old rats. When Na(+) was replaced by choline (Na(+)-independent component) the accumulation of alpha-aminoisobutyrate was decreased and it was not affected by the age of the animals. Kinetic analysis of Na(+)-dependent alpha-aminoisobutyrate transport revealed the existence of a high-affinity low-K(m) component (K(m)0.91mm) with a V(max.) of 2.44nmol/mg of protein per 4min, which later declined gradually with progressive development. Rates of Rb(+) transport were concomitantly enhanced in neonatal hepatocytes and thereafter declined with postnatal age. The increased Rb(+) influx was effectively inhibited by ouabain and reflected elevated activity of the electrogenic Na(+)/K(+)-pump during early stages of development. Kinetic evaluation of the enhanced rates of Rb(+) uptake indicates multiple and co-operative binding sites of the enzyme involved in the Rb(+) uptake, and the transport system is positively co-operative (the Hill coefficient h is >1.0). In short, amino acid transport in neonatal rat hepatocytes is increased as a result of an existing low-K(m) component for the Na(+)-dependent alpha-aminoisobutyrate uptake, which endows the hepatocytes with a high capability for concentrating amino acids at low ambient values. The concomitant enhancement of K(+) transport reflects changes in the electrochemical gradient for Na(+) across the hepatocellular membrane and, along with this, presumably alterations in the membrane potential; the latter might be the driving force for the enhanced alpha-aminoisobutyrate transport in the alanine-preferring system during postnatal age.

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Effect of prior nutritional status on the activity of lipogenic enzymes in primary monolayer cultures of rat hepatocytes.

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