Autophagy regulates turnover of lipid droplets via ROS-dependent Rab25 activation in hepatic stellate cell

Abstract

Activation of hepatic stellate cells (HSCs) is a pivotal event in liver fibrosis, characterized by dramatic disappearance of lipid droplets (LDs). Although LD disappearance has long been considered one of the hallmarks of HSC activation, the underlying molecular mechanisms are largely unknown. In this study, we sought to investigate the role of autophagy in the process of LD disappearance, and to further examine the underlying mechanisms in this molecular context. We found that LD disappearance during HSC activation was associated with a coordinate increase in autophagy. Inhibition or depletion of autophagy by Atg5 siRNA impaired LD disappearance of quiescent HSCs, and also restored lipocyte phenotype of activated HSCs. In contrast, induction of autophagy by Atg5 plasmid accelerated LD loss of quiescent HSCs. Importantly, our study also identified a crucial role for reactive oxygen species (ROS) in the facilitation of autophagy activation. Antioxidants, such as glutathione and N-acetyl cysteine, significantly abrogated ROS production, and in turn, prevented autophagosome generation and autophagic flux during HSC activation. Besides, we found that HSC activation triggered Rab25 overexpression, and promoted the combination of Rab25 and PI3KCIII, which direct autophagy to recognize, wrap and degrade LDs. Down-regulation of Rab25 activity, using Rab25 siRNA, blocked the target recognition of autophagy on LDs, and inhibited LD disappearance of quiescent HSCs. Moreover, the scavenging of excessive ROS could disrupt the interaction between autophagy and Rab25, and increase intracellular lipid content. Overall, these results provide novel implications to reveal the molecular mechanism of LD disappearance during HSC activation, and also identify ROS-Rab25-dependent autophagy as a potential target for the treatment of liver fibrosis.

Hepatic stellate cells as key target in liver fibrosis

“…LXRs balance lipid stores in HSCs through Rab18, a retinoid responsive lipid droplet protein [303]. Autophagy regulates turnover of lipid droplets via ROS-dependent Rab25 activation in HSC [304]. Alcohol dehydrogenases (ADHs) oxidize retinol to retinaldehyde, which is metabolized to retinoic acid.…”

Breaking fat: The regulation and mechanisms of lipophagy

“…TRAPP complexes are known to be involved in the autophagic process [52]; therefore, future investigations will be useful in determining whether Rab18-TRAPP interactions play any defined role in the selective process of lipophagy. Other Rabs, such as Rab25, have recently been suggested to participate in the autophagic turnover of retinyl ester-enriched LDs from hepatic stellate cells (HSCs) [53]. Generation of ROS during the process of HSC activation was found to result in an increase in Rab25 expression.…”

Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation

“…Endogenous neutral lipids are thought to traffic to the lysosomal compartment by the autophagy pathway. An active autophagy pathway has indeed been described in activated mouse HSCs (13)(14)(15). To investigate the involvement of the autophagy pathway in rat HSCs, we made use of the autophagy marker protein LC3B, which can be detected as two isoforms; one represents the cytosolic LC3B-I and the other one represents LC3B-II, which is conjugated with phosphatidylethanolamine and is present on autophagosomes.…”

“…These findings are in line with similar results described recently by Grumet et al (34). The REs are most likely delivered to the lysosomes by the autophagy pathway known to be active in activated HSCs (13)(14)(15) (Fig. 8).…”

“…This suggests the existence of another lipolysis pathway (9). In mouse HSCs, lipid breakdown was shown to be partially mediated by a lipophagic pathway, as inhibition of autophagy increased the amount of LDs (13)(14)(15). Because inhibition of autophagy was shown to impair HSC activation in mice and this effect could be partially reversed by the addition of exogenous FAs, it was suggested that LD breakdown is required to fulfill the energy demands of HSCs during activation (14).…”