Upregulation of Heme Pathway Enzyme ALA Synthase-1 by Glutethimide and 4,6-Dioxoheptanoic Acid and Downregulation by Glucose and Heme: A Dissertation

Aminolevulinic acid synthasel (ALASI) is the first and normally rate-controlling enzyme for hepatic heme biosynthesis. ALASl is highly inducible , especially in liver, in response to changes in nutrtional status , and to drugs that induce cytochrome P-450. The critical biochemical abnormality of the acute porphyrias , a group of disorders of heme synthesis, is an uncontrolled up-regulation of ALASI. High intakes of glucose or other metabolizable sugars and intravenous heme are the cornerstones of therapy for acute attacks of porphyrias and both repress the over-expression ALAS, although their mechanisms of action have not been fully characterized. In this work, the chick hepatoma cell line, LMH, was characterized with respect to its usefulness in studies of heme biosynthesis and compared with chick embryo liver cells (CELCs), a widely used model for studies of heme metabolism. The inducibility of ALASI mRA and enzyme activity and accumulation of porphyrins by chemicals were used to evaluate heme biosynthesis in LMH cells. Repression of ALASl mRA and induced activity by exogenous heme (20 IlM) was shown to occur in LMH cells as in CELCs. In addition, a synergistic induction of ALASl enzye activity was observed in LMH cells , as shown previously in CELCs , by treatment with a barbiturate-like chemical , Glutethimide (Glut), in combination with an inhibitor of heme synthesis , 4 dioxoheptanoic acid (DHA). This induction of ALASl enze activity is analogous to what occurs in patients with acute hepatic porphyrias and LMH cells were used to further characterize effects of Glut, DHA, glucose, and heme on ALASA "glucose effect" to decrease Glut and DHA-induced ALASenzyme activity was obtained in LMH cells and CELCs in the absence of serum or hormones. This "glucose effect" was further characterized in LMH cells using a constrct containing approximately 9. 1 kb of chick ALASl S' flanking and S' -UTR region attached to a luciferase/reporter gene (pGcALAS9. Luc). Glut (SO IlM) and DHA (2S0 IlM) synergistically induced luciferase activity (S-fold) in LMH cells transiently transfected with pGcALAS9.1-Luc. Addition of glucose (11 or 33 mM), in a dose-dependent manner, decreased the Glut+DHA up-regulation ofpGcALAS9.1-Luc activity. Gluconeogenic or glycolytic substrates such as frctose , galactose glycerol and lactate, but not the non-metabolizable sugar sorbitol , also down-regulated pGcALAS9. Luc in LMH cells. The cAMP analog 8-CPTcAMP, augmented Glut induction of ALAS, indicating that the glucose effect may be partly mediated by changes in cAMP levels.