Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process.
Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.
D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis*; D000368:Aged; D000502:Allylisopropylacetamide / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media; D004790:Enzyme Induction / Q000187:drug effects; D006418:Heme / Q000096:biosynthesis; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008858:Microscopy, Phase-Contrast; D010477:Perfusion; D011166:Porphyrins / Q000096:biosynthesis*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014284:Triiodothyronine / Q000494:pharmacology
D003470:Culture Media; D007328:Insulin; D011166:Porphyrins; D014284:Triiodothyronine; D000502:Allylisopropylacetamide; D006418:Heme; D000624:5-Aminolevulinate Synthetase; D006854:Hydrocortisone