Abstract
SUMMARYSecretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 μmol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes.
Highlights
Alcoholic liver disease (ALD) is a leading cause of liver-related deaths in the United States (Paula et al, 2010)
The authors expanded and refined a protocol that they previously developed to induce ALD in zebrafish larvae. They use this model to address two important questions relevant to human ALD: (1) is ethanol metabolism required for the hepatic defects, and (2) is oxidative stress involved in secretory pathway defects and steatosis caused by ethanol? They report that alcohol-exposed larvae exhibit hepatic damage that is marked by changes in hepatic gene expression, hepatic stellate cell (HSC) activation, formation of reactive oxygen species, secretory pathway dysfunction, induction of the unfolded protein response, and steatosis
Blocking ethanol metabolism using 4-methylpyrazole and chlormethiazole, which inhibit the major metabolic enzymes required for ethanol metabolism in mammals, completely reversed alcohol-induced steatosis and reduced secretory pathway dysfunction in hepatocytes and HSC activation
Summary
Alcoholic liver disease (ALD) is a leading cause of liver-related deaths in the United States (Paula et al, 2010). Both chronic and binge drinking cause mitochondrial dysfunction (Mantena et al, 2008), secretory pathway stress (Ji, 2012) and lipid accumulation in hepatocytes (i.e. steatosis) (Rubin and Lieber, 1968). These cellular responses are largely attributed to the toxic byproducts of ethanol metabolism by hepatocytes and are central to ALD pathophysiology.
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