Role of autophagy in Dynamic Remodeling of Cultured Mouse Hepatocytes

Abstract

Primary culture of hepatocytes is a valuable tool to study mechanisms involved in liver metabolic, endocrine and secretory functions. However, freshly isolated hepatocytes dedifferentiate and lose many features of hepatocyte including the transporters and metabolic enzymes in the course of primary culture. Here, we investigated the role and mechanisms of autophagy in the dynamic remolding of long‐term cultured mouse hepatocytes. Mouse hepatocytes were freshly isolated from wild type mice and liver‐specific Atg5 as well as Drp1 knockout mice and cultured for different time points from 2 hours to 7 days. Autophagy and mitophagy flux were determined using the tandem GFP‐mCherry‐LC3 and Cox8‐GFP‐mCherry. We found that hepatocytes gradually lost their typical cuboidal morphology and displayed fibroblast like morphology, which was accompanied by increased stress fibers and highly fused mitochondrial network formation in the course of culture. These mitochondrial dynamic changes were associated with decreased major mitochondrial fusion proteins Mfn1/2 and fission protein Drp1. The levels of hepatic cytochrome P450 enzymes (such as 2E1 and 2B10) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) markedly decreased, suggesting loss of hepatic metabolic features of drug and glucose metabolism in cultured hepatocytes. GFP‐mCherry‐LC3 puncta analysis and Cox8‐GFP‐mCherry analysis revealed that basal autophagic flux and mitophagy increased as early as day 2 and continue to increase in dedifferentiated hepatocytes during the culture. This mitophagy was independent of Parkin, as the levels of Parkin were dramatically decreased after 12 hours culture in hepatocytes. Mitophagy was markedly blunted in cultured Drp1 knockout hepatocytes, suggesting that elongated mitochondrial may be protective against autophagic degradation. Importantly, the levels of P450 enzymes, Parkin and GADPH remained higher in Atg5 knockout hepatocytes compared with wild type hepatocytes during the culture, suggesting the autophagy may be involved in the degradation of these metabolic enzymes. In conclusion, these findings indicate that autophagy is important for the remodeling of cultured hepatocytes and manipulation of autophagy process may be helpful to maintain some of the hepatocyte features during the culture.