Loss Of Dpr1 Impairs Autophagic Lipolysis Via Akt/mTOR Signaling In Liver Regeneration

Liver regeneration is an extremely complex biological process relating to multi-cells,multi-cytokines,and multi-signal pathways.The study of liver regeneration process originated from the 19 th century.Although a few key processes of liver regeneration were revealed,there are still many unsolved problems.At the early phase of liver regeneration,the liver will undergo steatosis and the current study revealed that steatosis is helpful for providing sufficient energy,but the mechanism of steatosis is still unknown.The lipid droplets in the cells are transported to the lysosomes by autophagy,and are decomposed into free fatty acids in the lysosome.The free fatty acids enter the mitochondria,complete ?-oxidation and produce large amounts of ATP.Autophagy is crucial for normal liver cell in the lipid droplet degradation process.At the same time,studies have shown that DPR1 can regulate the function of Vps34-Beclin1-Atg14 L complex during autophagy formation in the nervous system.In the recent study,we found that a large amount of lipid accumulation in hepatocytes after hepatectomy happen in Dpr1 knockout mice,and the duration was prolonged obviously.These findings suggest that the loss of Dpr1 in hepatocytes may affect hepatic fat metabolism.Our result implied that the liver from Dpr1 knockout mice after hepatectomy displayed LC3 reduction and p62 accumulation,which indicating Dpr1 played a key role in the process of autophagy.Primary hepatocytes from Dpr1 knockout mice were treated with oleic acid in vitro and it found that autophagy in the knockout group was low and the lipid droplets in the cells were accumulated,which indicating that Dpr1 participates in the process of autophagic degradation of lipid droplets.Finally,autophagy was significantly increased in the knockout mice after treated with autophagy-inducing agents in vivo and in vitro,indicating that Dpr1 is involved in the upstream part of autophagy,and detection of Akt-mTOR signaling pathway-related molecular revealed significant activation indicating that Dpr1 inhibits the activation of this signaling pathway by inhibiting the phosphorylation of Akt and promote the production of autophagy.In conclusion,the aim of this study was to reveal that DPR1 can promote the autophagy of liver cells by inhibiting the phosphorylation of Akt,thus accelerating the degradation rate of lipid droplets in liver cells,providing sufficient energy for liver regeneration and accelerating liver regeneration.?Methods?1.The middle lobe and left lateral lobe of mice under anesthesia were resected.Respectively,after 0,6,12,24,48,72,120,168 hours,mice were sacrificed.The weight of the body and liver was calculated.Fresh tissue,frozen tissue,paraffin tissue and blood samples of the liver were collected.2.Primary liver cells were isolated by two-step perfusion of liver.Primary hepatocytes were isolated by gradient centrifugation and cultured in vitro.3.The proliferation-associated molecules were detected by Western blotting and immunohistochemistry.4.The morphological changes of the liver were observed by H&E stain.5.The cell size of the two groups of mice was observed by immunofluorescence staining.6.The lipid accumulation in the liver was detected by oil red and Nile red stain.7.Autophagy and lipid accumulation were measured by immunoblotting,electron microscopy and RFP-GFP-LC3 double-labeled adenovirus.?Result?1.The mortality of Dpr1 knockout mice after 2/3 liver resection was significantly higher than that of the control group.Liver damage was more severe than the control group.Although liver recovery rate of Dpr1 knockout mice was faster than the control group,there was no significant difference in the liver proliferation in the both two group.2.In the early stage of regeneration,the volume of hepatocyte in Dpr1 knockout group was significantly larger than that in control group.3.The lipid content of Dpr1 knockout group was significantly higher than the control group.4.The expression of autophagy-related protein in Dpr1 knockout group was significantly lower than that in control group.5.In vitro,autophagy of Dpr1 knock-out hepatocytes was lower than that of the control group,and the lipid content of Dpr1 knockout group was higher than that of the control group.6.Detection of autophagy signal pathway m TOR and Akt found that it was activated in the Dpr1 knockout group.7.In vivo and vitro,after given mTOR inhibitors-Rapamycin,RAD001 and Akt inhibitor-MK2206,after treatment,Dpr1 knockout mice significantly increased the level of autophagy and lipid accumulation was alleviated.?Conclusion?Transient accumulation of fat will appear in the early process of liver regeneration.Studies have shown that it related to the energy supply.Under normal circumstances,autophagy can degrade intracellular lipid droplets by endocytosis.Our study implied that autophagy is involved in both lipid accumulation and degradation in the process of liver regeneration.And DPR1 regulated the process of autophagy through the Akt-mTOR signaling pathway,which promoting the degradation of lipid.