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

Abstract

5-Aminolevulinic acid synthase-1 (ALAS-1) is the first and normally rate-controlling enzyme for hepatic heme biosynthesis. ALAS-1 is highly inducible, especially in liver, in response to changes in nutritional 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 ALAS-1. 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-1, 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 ALAS-1 mRNA and enzyme activity and accumulation of porphyrins by chemicals were used to evaluate heme biosynthesis in LMH cells. Repression of ALAS-1 mRNA and induced activity by exogenous heme (20 μM) was shown to occur in LMH cells as in CELCs. In addition, a synergistic induction of ALAS-1 enzyme 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, 4,6-dioxoheptanoic acid (DHA). This induction of ALAS-1 enzyme 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 ALAS-1. A "glucose effect" to decrease Glut and DHA-induced ALAS-1 enzyme 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 construct containing approximately 9.1 kb of chick ALAS-1 5'- flanking and 5' -UTR region attached to a luciferase/reporter gene (pGcALAS9.1-Luc). Glut (50 μM) and DHA (250 μM) synergistically induced luciferase activity (5-fold) in LMH cells transiently transfected with pGcALAS9.l-Luc. Addition of glucose (11 or 33 mM), in a dose-dependent manner, decreased the Glut+DHA up-regulation of pGcALAS9.1-Luc activity. Gluconeogenic or glycolytic substrates such as fructose, galactose, glycerol and lactate, but not the non-metabolizable sugar sorbitol, also down-regulated pGcALAS9.1-Luc in LMH cells. The cAMP analog 8-CPT-cAMP, augmented Glut induction of ALAS-1, indicating that the glucose effect may be partly mediated by changes in cAMP levels. The remaining studies focused on delineating the synergistic effect of Glut and DHA, and heme-dependent repression of ALAS-1. The 9.1 kb construct was compared with a construct containing the first 3.5 kb (pGcALAS3.5-Luc). The drug and heme effects were shown to be separate as drug induction was present in -3.4 to +0.082 kb region while the heme responsiveness was present in the -9.1 to -3.4 kb region. Using computer sequence analysis, several consensus activator protein-1 (AP-1) sites were found in the 9.1 kb ALAS-1 sequence but no consensus direct repeat (DR)-4 or DR-5 type recognition sequences for nuclear receptors were identified in the drug-responsive 3.5 kb region. Deletion constructs containing +0.082 to -7.6 kb (pGcALAS7.6-Luc) and +0.082 to -6.2 kb (pGcALAS6.3-Luc) cALAS 5'- flanking and 5' - UTR region were generated and tested and pGcALAS6.3-Luc was shown to have heme-dependent repression of basal and Glut and DHA-induced activity. A recently identified 167 bp chick ALAS-1 drug responsive enhancer (DRE) was PCR amplified and inserted upstream of the 9.1 kb (pGcALAS9.1+DRE), a 0.399 kb (+0.082 to -0.317) (pGcALAS0.3+DRE), and pGL3SV40 construct (pGL3SV40+DRE). DRE mediated the up-regulation of pGL3SV40+DRE construct by Glut was ~ 15-30 fold but interestingly only 3.2 and 3.7-fold for pGcALAS9.l +DRE and pGcALAS0.3+DRE constructs, respectively. In summary, in LMH cells drugs up-regulate ALAS-1 through non-DRE element(s) in the first 3.5 kb of ALAS-1 5'-flanking and 5'-UTR region and heme down-regulates ALAS-1 and determines the extent of the drug response through element(s) in the -6.3 to -3.5 kb region of ALAS-1 5'- flanking region.