Phenylalanine hydroxylase, a liver-associated enzyme, is induced markedly by glucocorticoids in two permanent rat-hepatoma cell lines. In order to gain evidence that this phenomenon also occurs in vivo, we examined the effect of adrenalectomy and/or hormone supplementation on the levels of phenylalanine hydroxylase in the livers of adult rats: glucocorticoid administration increases, and adrenal ablation reduces, the activity of the hepatic enzyme, and the diminution occurring in the latter instance is entirely prevented by concurrent hormone replacement. These results thus corroborate earlier findings from a single experiment and are consistent with the hypothesis that adrenal corticosteroid hormones participate in modulating phenylalanine-hydroxylase levels within the diploid hepatocyte.

We have examined and characterized the regulation by glucocorticoids of the levels of arginase and argininosuccinate synthetase in two rat hepatoma cell lines (H4-II-E-C3 and MH1C1). Hydrocortisone elevates the activity of both enzymes in a time- and dose-dependent fashion. This effect was blunted markedly by small amounts of ethanol (0.1 to 0.9% [v/v]) and blocked substantially by a high molar excess of the anti-inducer steroid fluoxymesterone. The other optimal inducers dexamethasone and corticosterone were as effective as hydrocortisone in elevating the levels of these enzymes at saturating concentrations. Inhibition of these stimulations by cycloheximide indicated that ongoing cellular protein synthesis was required for both effects, and the admixture of extracts from fully stimulated and basal cells gave no evidence for the existence of direct inhibitors or activators of either enzyme. The results corroborate findings from earlier whole-animal studies and provide evidence for the following conclusions. (i) This stimulation by hydrocortisone of urea-cycle enzymes in the cultured hepatoma cells is mediated by a classical glucocorticoid mechanism involving initial binding to specific cytoplasmic steroid receptors and the eventual accumulation of new enzyme molecules. (ii) These cell lines thus constitute valid experimental models for use in further detailed studies on the molecular mechanism(s) through which glucocorticoids and intermediary metabolites effect a selective modulation of arginase and argininosuccinate-synthetase gene expression in the differentiated mammalian liver.

We have found that the induction of phenylalanine hydroxylase by hydrocortisone and serum in confluent cultures of H4-II-E-C3 rat hepatoma cells is accompanied by an increase in polysomal mRNA specific for phenylalanine hydroxylase, as measured by translation in a cell-free protein-synthesizing system. Thus, the induction is mediated largely, if not entirely, by a pretranslational mechanism, possibly by stimulation of the transcription of the phenylalanine hydroxylase gene.

The protein concentration of crude extracts from cultured cells and of subcellular fractions from rat tissues has been estimated both by the method of Bradford, involving the binding of Coomassie brilliant blue G-250, and by the procedure of Lowry et al. with the same preparation of bovine serum albumin as a common protein standard. Whereas the method of dye binding yielded significantly lower values than did the Lowry procedure, a consistent quantitative relationship existed between the values obtained by the two techniques. Evidence is provided that this discrepancy does not result from the presence in such complex biological mixtures of a class of small peptides, undetected by the method of dye binding, that react with the Lowry reagents.

Measurements of fluorescence polarization in intact diploid skin fibroblasts after exposure to 1,6-diphenyl-1,3,5-hexatriene were used to estimate the fluidity of the lipid phase(s) of cellular membranes. The membrane lipids of cells derived from four patients with homozygous familial hypercholesterolemia were in a more fluid state than those of cells obtained from 13 other individuals of normal and nonrelated mutant genotypes when all cultures were grown on medium with native serum. The only other cell type having membrane lipids of increased fluidity under these conditions was one fibroblast line derived from a patient with the Lesch-Nyhan syndrome. Examination of two additional nonconsanguinous lines of Lesch-Nyhan fibroblasts, however, revealed that an abnormally high level of lipid fluidity was not a common property of the membranes of cells of this genotype. Incubation of cultures in medium containing lipid-depleted serum (virtually devoid of lipoprotein-bound sterol) caused a reversible increase in the fluidity of the membranes of normal cells to values similar to those of the hypercholesterolemic cells, but had no effect on the membranelipid fluidity of the latter. By contrast, exposure of cultures to cholesterol not bound to lipoprotein in serum-free medium resulted in a decrease in the lipid fluidity of the membranes of both normo- and hypercholesterolemic fibroblasts.

Glucose-6-phosphatase (glucose-6-phosphohydrolase and its associated phosphotransferase activities) was determined in brain tissue and in several preparations derived from brain tissue. These included purified capillaries and established cell lines of neuronal or glial origin. Since it has been suggested that glucose-6-phosphatase may be involved in sugar transport, the characteristics of that process were examined in these preparations. The pattern of uptake of 2-deoxy-D-glucose in four cell lines was shown to involve transport of the analog across the cell membrane that was more rapid than the subsequent phosphorylation of the sugar in the intracellular compartment. In the remaining cell lines and in purified capillaries, phosphorylation of 2-deoxy-D-glucose was at least as rapid as uptake. No differences could be found between the cells in these two categories with respect to amount or localization of glucose-6-phosphatase, ability to phosphorylate 3-O-methyl-D-glucose, or ability to phosphorylate extracellular and intracellular 2-deoxy-D-glucose. In the course of these experiments, it was found that there was a rapid efflux of 2-deoxy-D-glucose from cells that had taken up this sugar. The efflux involves a dephosphorylation step catalyzed by intracellular phosphatase that releases free sugar in the cytoplasm. Glucose-6-phosphatase thus probably has no major role in the phosphorylation of glucose in brain cells, but acts in the more conventional sense, i.e. as a phosphohydrolase.

Continued high levels of phenylalanine hydroxylase in cultured H4-II-E-C3 rat hepatoma cells require either serum or glucocorticoids in the culture medium. Upon withdrawal of serum, cellular phenylalanine hydroxylase levels decay exponentially with a half-life of 22 hours for about 60 hours, after which time a low, constant enzyme content persists for at least 96 hours. This decline of phenylalanine hydroxylase is fully reversible; normal enzyme levels are restored in a time- and dosage-dependent fashion upon addition of serum to basal cultures. The serum factor is nondialyzable and moderately heat-stable. The stimulation by serum of the phenylalanine hydroxylas content of basal cultures is blocked by 3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide and requires ongoing cellular protein synthesis. When added to the enzyme-assay mixture in vitro, serum does not alter the phenylalanine hydroxylase activity of extracts from basal cultures. Three lines of evidence suggest that serum contains a nonsteroidal phenylalanine hydroxylase stimulatory components(s): (a) glucocorticoid antagonists inhibit less than one-half of the biological activity of serum; (b) exhaustive extraction of endogenous serum glucocorticoids with charcoal reduces the activity of serum to about one-half of control values; and (c) the stimulatory effects of charcoal reduces the values; and (c) the stimulatory effects of charcoal-extracted serum and hydrocortisone are additive. The phenylalanine hydroxylase stimulatory activities of the charcoal-extracted sera from four mammalian species and from three stages in development in one mammalian species are comparable. A survey of partially purified preparations of a number of known hormones failed to reveal any one capable of elevating the phenylalanine hydroxylas levels of basal cultures in a manner comparable to that of charcoal-extracted serum.

Tyrosine-free modified Ham's F-12 medium supplemented with charcoal-extracted serum permits the exclusive propagation of cultured cells containing detectable levels of phenylalanine hydroxylase. The selective properties of this medium have been verified with two phenylalanine hydroxylase-positive (H4-II-E-C3 and MH1C1) and two phenylalanine hydroxylase-negative (HTC and BRL) cell lines. Although cocultivation experiments revealed that BRL cells could replicate slowly in the selective medium in the presence of sufficient numbers of H4-II-E-C3 cells, the former cells did not survive subcultivation in this medium at low cell inoculum (< 103 cells per 60 × 15-mm Petri dish). This tyrosine-free medium should be particularly effective in selecting for phenylalanine hydroxylase-positive somatic-cell hybrids under conditions of double selection where the phenylalanine hydroxylase-positive parental cell would not survive.

The presence of phenylalanine hydroxylase as a constitutive enzyme in two clonal cell lines (H4-II-E-C3 and MH1C1) derived from rat hepatomas and cultured in vitro is described; eight other clonal cell strains originating from rat or rabbit liver did not contain detectable amounts of this enzyme. The levels of phenylalanine hydroxylase in the two hepatoma cell lines were stimulated by hydrocortisone, corticosterone, dexamethasone or N6-O2'-dibutyryl-3',5'-cyclic adenosine monophosphate. The stimulation of the levels of the enzyme by hydrocortisone in the H4-II-E-C3 line was shown to depend on de novo protein synthesis as it was inhibited by cycloheximide. It is postulated that the hepatoma cell lines are probably good models for studying the factors regulating the expression of the phenylalanine hydroxylase gene.

Mouse L-cells, Clone 929, were adapted to growth on a chemically defined medium, Waymouth's Medium MAP 9541, in the absence of serum. When biotin was excluded in the preparation of this medium, the resulting medium proved to be incapable of sustaining continuous cell proliferation: A biotin requirement was therefore demonstrable in these cells within a three week period.

1. Mammalian cells in culture provide a simple model to study the function of polyunsaturated fatty acids.

The possibility of an inborn error of linoleic acid metabolism or of linoleic acid content in cystic fibrosis (CF) was investigated using fibroblasts from children with CF and from control children. Three experiments were done in which fibroblasts were cultured with 1-14C linoleic acid in media containing: (experiment 1) regular fetal calf serum (FCS); (experiment 2) linoleic acid-supplemented FCS; and (experiment 3) delipidated FCS supplemented with linoleic acid. Radioactivity from 1-14C linoleic acid incorporated into the total lipids was quantitatively similar in CF fibroblasts compared to controls in all three experiments. In the first two experiments the radioactivity incorporated into the phospholipid fraction was slightly higher in CF fibroblasts than in controls, whereas radioactivity incorporated into the neutral lipid fraction of CF fibroblasts was slightly lower than in controls. These differences were not found in experiment 3. No differences in linoleic and arachidonic acid composition were found between CF and control fibroblasts in any of the three experiments. The inability to find major alterations in linoleic acid metabolism or content in fibroblasts from children with CF makes a primary metabolic defect in linoleic acid metabolism unlikely.