Serine Hydroxymethyltransferase 2 Regulates Hepatocytes Proliferation In Liver Regeneration

Backgroud: Ischemia reperfusion injury(IRI)and immune rejection during decoyed donor liver transplantation(DDLT)will lead to massive hepatocyte injury and necrosis.So liver regeneration(LR)is of great importance after surgery so as to restore physiological function.Although living donor liver transplantation(LDLT)has minimized IRI,however,the graft volume is too small.In order to meet the needs of the recipents,hepatocytes must rapidly proliferate to achieve a suitable hepatic/weight ratio.For donors,regeneration after hepatectomy is the only way to restore normal physiological function.Therefore,is an important basis for liver function recovery in patients after LT.Serine hydroxymethyl-transferase 2(SHMT2)is a key enzyme contrls the metabolism of glycine.Glycine is reported to affect the activity of Akt-m TOR pathway which regulates cell proliferation and survival.In this study,we explored the feasibility of promoting LR through SHMT2 and identify the molecular mechanisms through both in vitro and in vivo perspectives via mouse primary hepatocytes and classical LR models.Methods:1.C57 mice were used to establish classic LR models with 2/3 partial hepaectomy(PH).Liver samples were collected 48 h after surgery and total RNA was extracted.The sample transcriptome was detected by Mouse Genome 430 2.0 Assay microarray,and the bioinformatics analysis of the results was performed to identify the key differentially expressed genes(DEGs)and signaling pathways during the proliferative phase of LR.2.The primary hepatocytes of C57 mice were isolated and cultured.The cells were cultured with different concentrations of glycine after starvation.To determine the effect of glycine on hepatocytes proliferation and Akt-m TOR pathway activity,western-blot analysis(WB)and cell proliferation(Ed U)assay were applied.To determine whether SHMT2 regulated cell proliferation via Akt-m TOR pathway,hepatocytes overexpressed with SHMT2 were cultured in a glycine free medium with or without adding Akt inhibitor before analysis.To determine whether SHMT2 regulated Akt-m TOR pathway activity via glycine,hepatocytes downregulated with SHMT2 were cultured with glycine-free medium and a rescue group with glycine was designed.3.Before constructing the LR models,mice were transfected with adenovirus associated virus(AAV)to down-regulate the expression of SHMT2 in hepatocytes.Live tissues and whole blood were collected at designated time spots after PH.WB was applied to detect the Akt-m TOR pathway activity.To detect the impact of SHMT2 during the process of LR,serological enzymes and immunohistochemistry were used in all samples collected at different time points after PH.Results:1.A total number of 367 DEGs were screened at 48 h after PH.Gene ontology(GO)analysis indicated that DEGs were mainly enriched in cell cycle,DNA synthesis,mitosis,nucleotide binding and serine/threonine protein kinase activity;enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway suggested that DEGs were mainly enriched in cell cycle,glycine/serine/threonine metabolism and serine/threonine protein kinase activity.Important pathways such as p53 signaling and bile acid secretion were also included.Protein-protein interaction(PPI)network analysis showed that most of the proteins regulated by DEGs had an interaction relationship and the top 5 clustering of this PPI network were obtained by Cytoscape.2.Different concentrations of glycine led to the activation of Akt-m TOR pathway and increase of DNA synthesis in hepatocytes.Overexpression of SHMT2 enhanced cell proliferation,intracellular glycine level and the activity of Akt-m TOR pathway,however,this proliferative effect was blocked by the administration of Akt inhibitor.Clearance of intracellular glycine down-regulated the activity of Akt-m TOR pathway and DNA synthesis.Inhibition of SHMT2 expression further enhanced the negative effect induced by glycine elimination.Exogenous glycine addition rescued the cells from the negative effects caused by SHMT2 down-regulation.3.In the proliferation phase of LR,SHMT2 expression,Akt-mTOR pathway activity and tissue glycine level were significantly higher than the control group.However,by down-reulation of SHMT2,hepatocytes proliferation and liver function were dampened and eventually leading to a reduced liver volume in the late stage of LR,which could be accounted for the decreased activity of Akt-m TOR pathway.Conclusions:1.The proliferation phase of LR is a complex process regulated by multiple genes,while serine/threonine protein kinase activity and glycine/serine/threonine metabolism play important roles.2.Glycine can regulate hepatocyte proliferation through the Akt-mTOR pathway,and the effects induced by overexpression of SHMT2 on hepatocyte proliferation via Akt-m TOR pathway is mediated by glycine.3.The expression of SHMT2 during LR proliferative phase is beneficial to hepatocyte replication.Inhibition of SHMT2 will lead to early liver function damage and decreased regenerative liver volume.