Ethanol‐induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity

Abstract

Lipid droplets (LDs), the organelles central to alcoholic steatosis, are broken down by lipophagy, a specialized form of autophagy. Here, we hypothesize that ethanol administration retards lipophagy by down‐regulating dynamin 2 (Dyn2), a protein that facilitates lysosome re‐formation, contributing to hepatocellular steatosis. Primary hepatocytes were isolated from male Wistar rats fed Lieber–DeCarli control or ethanol (EtOH) liquid diets for 6‐8 weeks. Hepatocytes were incubated in complete medium (fed) or nutrient‐free medium (fasting) with or without the Dyn2 inhibitor dynasore or the Src inhibitor SU6656. Phosphorylated (active) forms of Src and Dyn2 and markers of autophagy were quantified using western blot analysis. Colocalization of LDs with autophagic machinery was determined using confocal microscopy. In hepatocytes from pair‐fed rats, LD breakdown was accelerated during fasting, as judged by smaller LDs and lower triglyceride (TG) content when compared with hepatocytes in complete media. Fasting‐induced TG loss in control hepatocytes was significantly blocked by either SU6656 or Dynasore. Compared with controls, hepatocytes from EtOH‐fed rats had 66% and 40% lower content of phosphorylated Src (pSrc) and phosphorylated Dyn2 (pDyn2), respectively, coupled with a lower rate of fasting‐induced TG loss. This slower rate of fasting‐induced TG loss was blocked in cells coincubated with Dynasore. Microscopic examination of EtOH‐fed rat hepatocytes revealed increased colocalization of the autophagosome marker LC3 on LDs with a concomitant decrease in lysosome marker LAMP1. Whole livers and LD fractions of EtOH‐fed rats exhibited simultaneous increase in LC3II and p62 over that of controls, indicating a block in lipophagy. Conclusion: Chronic ethanol administration slowed the rate of hepatocyte lipophagy, owing in part to lower levels of phosphorylated Src kinase available to activate its substrate, Dyn2, thereby causing depletion of lysosomes for LD breakdown. (Hepatology Communications 2017;1:501–512)

Autophagy in liver diseases: Time for translation?

“…161 An additional mechanism that could underlie reduced autophagy following chronic alcohol exposure is the inactivation of the small guanosine triphosphate Rab7 and reduced dynamin 2 activity, which causes depletion of lysosomes and inhibits hepatocyte lipophagy. 165,166 Finally, inhibition of AMPK by alcohol results in decreased mitochondrial beta-oxidation and increased lipogenesis. 167 Additional anti-inflammatory effects of autophagy in macrophages are likely to reduce cytokine production by macrophages and therefore to block neutrophil recruitment to the liver (Fig.…”

Impaired Rab7 and dynamin2 block fat turnover by autophagy in alcoholic fatty livers

“…It is known that the activity of Dyn2 increased after it is phosphorylated by Src kinase, a non-receptor protein tyrosine kinase. In the second study performed by McNiven and Casey’s group, they utilized similar models to determine the role of Dyn2 in lipophagy after alcohol exposure as showed in the first study (11). They found that the phosphorylated (active) forms of Src and Dyn2 decreased in hepatocytes isolated from chronic alcohol-fed rats compared with the hepatocytes from pair-fed control rats.…”

Lipophagy and Alcohol-Induced Fatty Liver

“…Alcohol-induced fatty liver was reported in humans (Buck, 1948) decades before the metabolic pathways affected by heavy drinking were revealed and well before it was discovered that certain fatty acids are hepatotoxic (Savary et al, 2012). Hepatic fatty acid (and lipid droplet) accumulation after alcohol abuse arises from: (1) accelerated hepatic lipogenesis (You et al, 2002; You and Crabb, 2004); (2) enhanced fatty acid import into the liver from the plasma (Wei et al, 2013); (3) defective secretion of lipoproteins (e.g., very low density lipoproteins VLDLs) from the liver into the plasma, resulting in their hepatic retention (Kharbanda et al, 2009); (4) reduced fatty acid oxidation (FAO) by mitochondria (Fischer et al, 2003); and now, (5) decelerated lipophagy (Rasineni et al, 2017; Schulze et al, 2017a).…”