RESUMEN
BACKGROUND: Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. METHODS: The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low K(m) aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I alcohol dehydrogenase (ADH) (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. RESULTS: The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the ß1 and ß2 ADHs was constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing ß2 ADH plus ALDH2. CONCLUSION: The activity of the human ß2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady-state acetaldehyde concentration in hepatocytes during ethanol metabolism.
Asunto(s)
Acetaldehído/metabolismo , Alcohol Deshidrogenasa/metabolismo , Depresores del Sistema Nervioso Central/metabolismo , Etanol/metabolismo , Isoenzimas/metabolismo , Hígado/metabolismo , Alcohol Deshidrogenasa/genética , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Alelos , Animales , Carcinoma Hepatocelular , Línea Celular Tumoral , Células HeLa , Hepatocitos/metabolismo , Humanos , Isoenzimas/genética , Masculino , NAD/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: AMP-dependent protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) alpha facilitate fatty acid oxidation. We have shown that treatment of hepatoma cells with ethanol or feeding ethanol-containing diets to mice inhibited both PPARalpha and AMPK activity. Importantly, WY-14,643 reversed the development of fatty liver in alcohol-fed mice. Whether WY-14,643, a PPARalpha agonist, has any effects on AMPK is not known. The aim of this study was to investigate the effect of WY-14,643 on AMPK activity. METHODS: The effect of WY-14,643 on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3). The effect of WY-14,643 on upstream kinases of AMPK, PKC-zeta/LKB1, intracellular AMP:ATP ratio, oxidative stress, and AMPK gene expression were studied. RESULTS: Treatment of the H4IIEC3 cells with WY-14,643 for 24h led to 60% increase in the phosphorylation of AMPK. The effect of WY-14,643 on AMPK phosphorylation is PKC-zeta/LKB1 independent. WY-14,643 did not alter the levels of intracellular AMP:ATP ratio and it did not increase the levels of reactive oxygen species at 24-h of treatment. WY-14,643-induced AMPK alpha subunit expression by 2- to 2.5-fold, but there was no change in AMPKalpha subunit protein at 24h. The effect of WY-14,643 on AMPK phosphorylation did not altered by the presence of an NADPH oxidase inhibitor. CONCLUSIONS: WY-14,643 induced AMPKalpha subunit phosphorylation and the activity of the enzyme. This was associated with induction of AMPKalpha1 and alpha2 mRNA, but the mechanism for this activation is uncertain.