COMPARISON OF THE EFFECTS OF INSULIN AND OKADAIC ACID ON PHOSPHOENOLPYRUVATE CARBOXYKINASE GENE-EXPRESSION

Many hormones regulate the rate of synthesis of phosphoenol-pyruvate c arboxykinase (PEPCK), the enzyme that governs the rate-limiting step i n gluconeogenesis. In H4IIE rat hepatoma cells, glucocorticoids, retin oic acid and cyclic AMP (cAMP) increase PEPCK gene transcription where as insulin and phorbol esters have the opposite effect. Insulin and ph orbol esters are dominant as they prevent cAMP- and glucocorticoid-sti mulated PEPCK gene transcription. In contrast, insulin and phorbol est ers both stimulate transcription of gene 33 in the same H4IIE cells, w ith the same time course as seen for their inhibitory effect on PEPCK gene transcription. We now report that the protein phosphatase inhibit or, okadaic acid, mimics the action of insulin and phorbol esters on e xpression of both gene 33 and PEPCK gene in H4IIE cells. Okadaic acid stimulates gene 33 mRNA accumulation whereas it inhibits cAMP- and glu cocorticoid-stimulated PEPCK mRNA accumulation. The effect of okadaic acid on the PEPCK gene is mediated through the PEPCK promoter as, in a cell line, HL1C, stably transfected with a PEPCK-chloramphenicol acet yltransferase (CAT) fusion gene, okadaic acid inhibits cAMP- and gluco corticoid-stimulated CAT expression. Desensitization of the protein ki nase C pathway by exposure to phorbol 12-myristate 13-acetate for 16 h abolishes the subsequent action of the phorbol ester but does not mar kedly affect the inhibition of cAMP- and glucocorticoid-stimulated CAT expression by insulin or okadaic acid. Even though insulin and okadai c acid appear to repress PEPCK gene expression through a pathway initi ally distinct from that used by phorbol esters, transient-transfection studies show that the final target of the action of okadaic acid, ins ulin and phorbol ester is the same DNA element.