Kai-Lin Lee

Insulin enhances transcription of the tyrosine aminotransferase gene in rat liver.

The mechanism of insulin-mediated induction of tyrosine aminotransferase in rat liver was investigated using a cloned cDNA probe. The level of aminotransferase mRNA increases about fourfold following administration of the hormone. This induced mRNA accumulation does not require de novo protein synthesis. Nuclear runoff transcription assays in isolated liver nuclei demonstrate that insulin has a rapid and time-dependent stimulatory effect on aminotransferase gene transcription. The magnitude of enhanced transcription can fully account for the increase in the mRNA. We conclude that the induction of tyrosine aminotransferase in rat liver by insulin is primarily a consequence of a selective increase in the rate of transcription of the aminotransferase gene.

Regulation of yolk protein synthesis in amphibian liver: II. Elevation of ribonucleic acid synthesis by estrogen

Abstract Changes in RNA synthesis during the course of induction of yolk protein synthesis were determined in the livers of Xenopus laevis males given estrogen. Increased synthesis of rapidly labeled nuclear RNA precedes the changes in protein synthesis induced by the hormone. Base composition analyses of this RNA suggest an initial synthesis of uridylic acid-rich RNA followed by increased synthesis of ribosomal RNA. No significant changes in leucyl-, methionyl-, or seryl-tRNAs were detected, and phosphoserine was not found in seryl-tRNA formed either in vivo or in vitro.

Turnover of tyrosine transaminase in cultured hepatoma cells after inhibition of protein synthesis.

Abstract Addition of cycloheximide to hydrocortisone-induced cell cultures of the Reuber (H-35) hepatoma results in an immediate decrease in the levels of tyrosine transaminase (L-tyrosine-2-oxoglutarate aminotransferase, E.C.2.6.1.5). The half-life of the enzyme is about 1.6 hr during the first hour after treatment; however, the rate of inactivation decreases such that the enzyme has a half-life of about 10 hr between 4 and 9 hr after addition of the inhibitor. The cells are functionally viable after 3 hr treatment with cycloheximide and retain their capacity both to inactivate tyrosine transaminase and to have the synthesis of this enzyme reinduced by hydrocortisone after removal of the inhibitor. The loss of 14 C-labeled tyrosine transaminase in H-35 cells is blocked by cycloheximide after a l-hr period of normal turnover. Puromycin also blocks the turnover of the enzyme in the interval between 4 and 6 hr after addition of the inhibitor.

Induction of alanine transaminase by adrenal steroids in cultured hepatoma cells

Abstract The capacity of adrenal steroid hormones to accelerate synthesis of alanine transaminase ( L -alanine-2-oxoglutarate aminotransferase, E. C. 2.6. 1.2) in cultured cells of the H-35 hepatoma was investigated. The transaminase of these cells was immunologically identical to the rat liver enzyme. By use of an isotopic- immunochemical method, it was found that transaminase synthesis was increased 150%, 24 hr after hydrocortisone was added to the medium. We conclude that the induction of this enzyme by glucocorticoids in vivo is due to direct action of the hormones of hepatic cells. Lactic dehydrogenase levels were un changed by the steroids tested. The responses of alanine transaminase to various concentrations of hydrocortisone and to modifications of the steroid structure were like those of tyrosine transaminase in H-35 cells, except that synthesis of the latter enzyme is stimulated to a much larger extent.

Properties of tyrosine aminotransferase from rat liver

Abstract A series of sequential chromatographic procedures which yield essentially homogeneous tyrosine aminotransferase ( l -tyrosine:2-oxglutarate aminotransferase, EC 2.6.1.5) from rat livers is described. Analysis of the purified enzyme indicates that its molecular weight is about 100,000, and that it consists of two subunits of identical mass and charge, each bearing one functional site for reaction with pyridoxal phosphate.

Turnover of Gene Products in the Control of Gene Expression

The role of turnover of messenger RNAs and proteins in determining the steadystate levels of intracellular enzymes was analyzed by comparing the parameters governing levels of tyrosine aminotransferase (TAT) and alanine aminotransferase (AAT) of rat liver. Programming of AAT is characterized by low rates of synthesis and turnover of both mRNA and enzyme. TAT mRNA is produced much faster but this product, like the enzyme, undergoes rapid turnover, resulting in a low steadystate level of this enzyme. Programming of TAT expression appears to be designed for regulation. (Accepted for publication 12 October 1981)

Insulin increases transcription of rat gene 33 through cis-acting elements in 5'-flanking DNA

Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, we have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequences in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme. This expression was increased by insulin treatment in a fashion resembling the effect of this hormone on transcription of the native gene. In vitro transcription assays in nuclear extracts also revealed increased transcription of the chimeric plasmids when the extracts were prepared from insulin-treated rat hepatoma cells. The results demonstrate that induction by insulin is mediated by cis-acting nucleotide sequences located between bp -480 to +27 relative to the tsp.

Reconstitution of aminotransferases in vivo and their metabolic turnover

Abstract Rat liver tyrosine aminotransferase and alanine aminotransferase are similar enzymes in most properties, but they differ markedly in their ease of coenzyme dissociation and rate of metabolic turnover. Dissociation of coenzyme does not determine rate of turnover ( K.L. Lee, P. L. Darke, and F. T. Kenney, 1977 , J. Biol. Chem. 252 , 4958–4961), but these parameters may reflect structural properties of the enzymes which determine both. To explore this possibility we studied these enzymes in livers of rats fed a pyridoxine-deficient diet in which both enzymes were largely in apoenzyme form. This form of alanine aminotransferase, not previously characterized, was identified as an immunologically cross-reactive material which was converted to active enzyme when extracts were incubated with pyridoxal phosphate in vitro . This apoenzyme behaved like the active holoenzyme in chromatographic and electrophoretic analyses but was more sensitive than the holoenzyme to heat, low pH, or proteolysis by trypsin or chymotrypsin. Relative rates of reconstitution of the two holoenzymes in vivo after injection of pyridoxine were determined as a measure of conformational stability of the two enzymes as they exist in the intracellular environment. Restoration of the tyrosine aminotransferase holoenzyme was completed within 30 to 45 min, but that of the alanine enzyme required 8 h. These results suggest that tyrosine aminotransferase in vivo is a relaxed structure which facilitates both coenzyme dissociation and rapid metabolic turnover, whereas alanine aminotransferase assumes a taut structure resistant to both dissociation and degradative processes.

Changes in hepatic differentiation following treatment of rat fetuses with 5-azacytidine☆

Rat fetuses of 20 days gestational age were treated in utero with 5-azacytidine. Within 14 to 18 h after treatment several significant changes in the fetal livers were observed, including a dramatic maturation of hepatocyte morphology with little alteration in hematopoietic elements. Assessment of mRNA levels by hybridization to cloned cDNAs, together with other measures of gene expression, established that the change in hepatocyte morphology was associated with strong activation of expression of genes normally activated later in development, including those coding for the liver enzymes tyrosine aminotransferase and phosphoenolcarboxykinase and a gene of unknown specificity that is regulated in liver much like the aminotransferase. Rates of transcription of two of these genes, measured in isolated nuclei, were significantly increased after 5-azacytidine treatment. Expression of alpha-fetoprotein, normally declining during the perinatal period of development, was reactivated following treatment with the drug, while albumin expression was somewhat enhanced. For the most part the changes observed reflect temporal advancement of events normally programmed to occur later in differentiation of the liver. These changes appear to be the consequence of multiple effects of 5-azacytidine, including enhanced gene transcription and stabilization of gene products.

HORMONAL MECHANISMS IN REGULATION OF GENE EXPRESSION.

Research on the question of how genes are regulated in mammalian cells has accelerated rapidly in the last decade, with much attention being paid to systems in which gene expression is regulated by hormones. Virtually all the hormones have been implicated in this facet of regulation, including those acting via cyclic AMP as intracellular mediator; indeed, there is now firm evidence that cyclic AMP acts to regulate synthesis of specific enzymes in mammalian cells (1,2) as well as in bacteria (3,4). Our recent work, however, has involved hormones which appear to act independently of cyclic AMP, at least in the experimental system we employ. We shall review here some of our recent observations on the mechanisms involved in the stimulation of synthesis of a specific enzyme by the steroid hormone, hydrocortisone, as well as by the polypeptide hormone, insulin. As might be anticipated by the difference in structure of these hormones, we conclude that their mechanisms of enzyme induction are quite different, the steroid acting via a transcriptional mechanism and insulin clearly acting at some post-transcriptional stage of enzyme synthesis. Details of experimental approaches not fully described here can be found in our earlier publications on this work (4–7).