Role of Autophagy in Alcohol-induced Adipose Atrophy and Liver Injury

Alcoholic liver disease (ALD) is a worldwide health issue claiming 2 million lives per year. The pathogenesis of ALD is characterized by steatosis, alcoholic hepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. There is no ideal treatment other than liver transplantation for late stage of ALD, and novel treatment targets especially for early stage of ALD are still needed. In recent years, increasing evidence implicates the role of adipose-liver axis in in the pathogenesis of ALD. Chronic alcohol consumption leads to increased lipolysis, adipose tissue atrophy, and proinflammatory adipokine secretion. However, the mechanisms of how adipose tissue is affected by alcohol and contributes to ALD are largely unknown. In this study, I found that chronic-plus-binge alcohol treatment, a model resulting in liver injury and steatosis, led to smaller adipocytes and decreased adipose tissue mass. Mechanistically, I found that chronic-plus-binge alcohol inhibited mammalian target of rapamycin (mTOR)/Akt signaling pathways and enhanced autophagy degradation in epididymal adipose tissue. Although the adipose-specific autophagy-related gene 5 knockout (A-Atg5 KO) mice with adipose autophagy deficiency did not present adipose atrophy at basal condition, they were resistant to alcohol-induced adipose tissue atrophy. Moreover, A-Atg5 KO mice had increased adipocytes with multilocular lipid droplets in subcutaneous white adipose tissue. Interestingly, A-Atg5 KO mice were more resistant to alcohol-induced liver injury, though they still developed alcohol-induced liver steatosis. Alcohol metabolism and reactive oxygen species generation in liver was not affected in A-Atg5 KO mice. While the chronic-plus-binge alcohol increased liver mRNA levels of several proinflammatory genes in both wild-type and A-Atg5 KO mice, A-Atg5 KO mice had significantly decreased induction of Ccl2. Compared with wild-type mice, A-Atg5 KO mice had similar serum lipids (triglycerides, free fatty acids, free glycerol), but had higher basal levels of adiponectin and fibroblast growth factor 21 (FGF21). Additionally, chronic-plus-binge alcohol did not induce inflammation or cell death in white adipose tissues. I next used cultured 3T3-L1 preadipocytes to further determine the role of autophagy in adipocyte differentiation and how adipogenesis is affected by alcohol. I found that there was increased autophagy degradation along with increased levels of mitochondria proteins during preadipocyte adipogenesis. Using fluorescence microscopy, I found that elongated, sharp mitochondria signal became diffused and enhanced in mature adipocytes. Electron microscopy analysis revealed that the number of elongated mitochondria with clear cristae structure largely disappeared while electron-dense vacuoles containing undegraded cellular components frequently appeared during adipogenesis. Long-term cotreatment using chloroquine, a lysosome inhibitor that blocks autophagy degradation, sufficiently blocked 3T3-L1 adipogenesis. Intriguingly, in chloroquine-treated cells typical mitochondria still decreased while the electron-dense vacuoles containing undegraded cellular components further increased. Moreover, long-term ethanol or acetaldehyde treatment did not inhibit morphological changes or lipid droplet accumulation during adipogenesis in 3T3-L1 cells. Interestingly, short-term ethanol but not acetaldehyde treatment induced autophagic flux, and long-term ethanol but not acetaldehyde treatment increased mitochondria protein levels. The change in mitochondria morphology during adipogenesis was not affected by long-term ethanol or acetaldehyde treatment. These data suggest that autophagy is required for the proper adipogenesis of cultured 3T3-L1 cells, which is associated with dynamic mitochondrial remodeling. In summary, I characterized the effect of chronic-plus-binge alcohol treatment on adipose morphology, mTOR signaling and autophagy in mice. I demonstrated that chronic-plus-binge alcohol inhibited mTOR and increased autophagic flux and adipose tissue atrophy in mice. I further demonstrated that mice with chronic adipose tissue autophagy deficiency were more resistant to alcohol-induced adipose atrophy. These data indicate that autophagy activation contributes to the adipose dysfunction induced by alcohol. In addition, I also demonstrated that A-Atg5 KO mice were more resistant to alcohol-induced liver injury likely due to the increased secretion of adiponectin and FGF21 at the basal levels of A-Atg5 KO mice. These data thus support an important role of adipose-liver axis in the pathogenesis of ALD. These studies further support the notion that targeting adipose tissue autophagy may be helpful in improving alcohol-induced liver injury