Kolluri S1, Sadlon Tj2, Bonkovsky H1
1University of Connecticut Health Science Center, Farmington, CT, USA, 2 University of Adelaide, Australia
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 many drugs that induce cytochrome P-450 and/or affect heme synthesis. The critical biochemical abnormality of the acute porphyrias is an uncontrolled up-regulation of ALAS-1 in the liver. High intakes of glucose or other metabolizable sugars and intravenous heme are the cornerstones of therapy of acute attacks of these types of hepatic porphyrias. Both glucose and heme are known to repress the uncontrolled over-expression of hepatic ALAS-1, although their molecular mechanisms of action have not been characterized. In previous work, we and others showed that the LMH hepatoma cell line is extraordinarily useful for study of heme metabolism, including regulation of ALAS-1. In this work, we investigated the effects of glucose and heme in LMH cells transfected with ALAS-1 promoter-luciferase (Luc) reporter constructs. Treatment for 16 h with the barbiturate-like drug, glutethimide (Glut, 50 μM), and the inhibitor of heme synthesis, 4,6-dioxoheptanoic acid (DHA, 250 μM), produced a synergistic (5-fold) up-regulation of ALAS-1 promoter-reporter activity in LMH cells transiently transfected with a construct containing 9.1 Kb of the ALAS-1 5΄flanking region and the ALAS-1 5΄untranslated region (UTR) attached to the luciferase (pGL3 Basic) reporter gene (Fig 1). Addition of glucose (11 or 33 mM), in a dose-dependent manner, decreased the Glut+DHA up-regulation of pGcALAS9.1-Luc activity (Fig. 1). Exogenous heme (20 μM) repressed basal luciferase activity (4-fold) and had further and an additive effect on glucose repression and completely abrogated the induction by Glut and DHA (Fig. 2). The glucose analog 2-deoxyglucose, which cannot be metabolized to glucose, significantly augmented the induction by Glut and DHA and abrogated the glucose effect on ALAS-1, indicating that the glucose needs to be metabolized to exert its effect on ALAS-1. Metabolizable sugars such as fructose, galactose, glycerol and lactate, but not the non-metabolizable sugar sorbitol, also down-regulated ALAS-1 in LMH cells. In conclusion, these results establish that both heme and glucose or other metabolizable sugars lead to down-regulation of hepatic ALAS-1 by acting directly on hepatocytes through a mechanism that requires the first 9.1 Kb of the 5΄- regulatory region of the ALAS-1 gene. These effects were observed in the absence of insulin, glucagon, other hormones or serum. Our focus is to perform fine mapping and detailed characterization of the 5΄-flanking region to identify cis-acting elements that mediate these repressive effects on ALAS-1 gene expression.