The Mechanical Forces On Glycogen Metabolism In Hepatocellular Cell

Background and objectivePortal hypertension is the central driver of complications in patients with chronic liver diseases and cirrhosis,the most common causes is the obstruction of hepatic sinus and sinus is mainly block,the formation of false flocculus made the original blood circulation pathways blocked or diverted,pressure vessels,limiting blood flow.In addition,the hepatic sinus and sinus blockage make the lymph circulation blocked,and the blood exchange decreases,which further increases the pressure.This process is closely related to the mechanical microenvironment of the liver,but the mechanical biological mechanism is still unclear.Blood glucose balance is one of the most important metabolic balances of the human body while liver is the main organ for carbohydrate metabolism and blood glucose balance maintenance.In patients with end-stage liver disease combined with portal hypertension,the liver has a disorder in the metabolic function of glycogen and causes blood glucose disorder.Although there are many researches on liver glycogen metabolism disorder,for mechanical factors such as extracellular pressure are still far from enough..In this study,we applied the extracellular pressure to normal hepatocytes and hepatocellular carcinoma cells by modifying the cell incubation device,by which simulating the mechanical microenvironment,and tried to discover the effects of pressure on Hepatic glucose metabolism.Therefore,we could find how the pressur regulating glucose metabolism in hepatocytes.The current study contains three section:?1?Effect of extracellular pressure on hepatic glucose metabolism in hepatocytes.?2?Increasing pressures suppress glycogen synthesis through enhance phosphorylation of Y216 in Gsk3?.?3?Increasing pressures suppress glycogen synthesis through activating p53/Pten pathwayMethod1.The effect of extracellular pressure on the content of glycogen in hepatocytes.1.1Cell culture and pressure loadingThe human hepatocyte cell line HL7702 and hepatocellular carcinoma cell line HepG2were cultured in high-glucose Dulbecco's modified Eagle's medium supplemented with 10%FBS and 1%penicillin/streptomycin.Cells grew in a humidified atmosphere of 5%CO2 at37?in Cell incubator.The cells were seeded in 24-well plates at a density of 1×10⁴ cells/well.After culturing24hs,the cells were treated with pressure loading.The pressure-loading apparatus was set up as described previously with some modifications.To generate pressure,compressed helium gas was released into the chamber.The pressure was confirmed by a sphygmomanometer through atube connected to the outlet.Cells used for experiments were placed in the chamber inside the incubator.After 24 hours'incubation,the cultured cells were exposed to pressure from 0 to 15mmHg for 24 hours for further experiments.1.3 Glycogen analysis assayAfter pressure loading,the cells would be treated as the protocol of Glycogen PAS Staining Kit.At last,the cells were examined under a fluorescence microscope to capture the images.For the glycogen concentration analysis assay.the cells were treated with treated with Glycogen Content Detection Kit.Finally,the absorbance at 620 nm was recorded with SpectraMax M5.2.The effect of mechanical factors on glycogen metabolism.After the pressure loading?10mmHg,24hr?,total RNA and protein were drawn from HepG2 and HL7702.We used to detect the mRNA and protein expressions of GSY1?GSK3??G6PC3 and Agl by Real-time PCR and Western blot analysis.3.Rna-seq sequencing the whole genome of hepatoma cells after stress loading.For RNA-seq,after the pressure loading?10mmHg,24hr?and the extraction of HepG2and HL7702,500 ng extracted total RNA of each sample was used for library construction.Libraries were constructed using Truseq RNA Sample Preparation Kit V2.Adapters with index sequences were attached to the libraries.After the average length of the libraries was confirmed,the concentration of each library was adjusted to 10 nM.Sequencing was performed for 100 bp by using HiSeq2500,with the single-read method.Mapping,data normalization,and statistical analyses were performed using CLC Genomics Workbench.For Volcano plotting,we averaged gene expression levels over the replicates for each cell line after normalization and compared the cell lines by calculating the FC.FC was defined as the ratio between the averages and we determined P values for each gene expressed.Volcano plots use FC as the xaxis and the P value as the y-axis.We defined significantly changed genes as those with thresholds ofFC>2 and P<0.01.Gene set enrichment analysis?GSEA?was performed at http://www.broadinstitute.org/gsea/.4.Study on the mechanism of glycogen synthesis inhibition of hepatocellular carcinoma by mechanical factors.After the pressure loading?10mmHg,24hr?,total RNA and protein were drawn from HepG2 and HL7702.We used to detect the mRNA and protein expressions of p53,PTEN and Akt by Real-time PCR and Western blot analysis.5.Statistical AnalysisData are represented as mean±SD;n=3.*,**and***indicate p<0.05,p<0.01,and p<0.001,respectively.Result1.Increasing pressures decrease hepatocellular glycogen concentration.To evaluate the effects of extracellular pressure on hepatocellular glycogen metabolism,PAS assay was used to analyze intracellular glycogen concentration of HepG2 cells which were treated with different pressures?0,5,10 and 15 mmHg?for 24 hours.When the pressure increased from 0 mmHg?Control?to 15 mmHg,the glycogen concentration was gradually decreased.When the pressure reached 15 mmHg,the glycogen concentration was not considerably decreased compared to 10 mmHg,indicating that the effect of pressure reached saturation.And,similar effect was also observed in normal hepatic cell line HL-7702.To confirmed this result,we detected the glycogen concentration with Glycogen Content Detection Kit.When the pressure increased,the glycogen concentration gradually decreased in HepG2 and HL-7702 cells.The results indicated that certain pressures decreased hepatocellular glycogen concentration,which may be related to the decrease of glycogen synthase activity.2.Increasing pressures suppress glycogen synthesis through inhibiting phosphorylation of GS and Gsk3?We detected GS and Gsk3?expression in HepG2 and HL-7702 cells treated with different pressures.Previous data showed that hepatocytes'glycogen was significantly reduced when the pressure reached 10 mmHg,and there was not considerably different compared to 15 mmHg.So we used 0 mmHg and 10 mmHg pressures for further study.We evaluated their expression with qRT-PCR.After treating with 0 mmHg and 10 mmHg pressures for 24 hrs,the expression levels remained unchanged.It is reported that p-S641GS is the activated isoform and is closely associated with GS.Therefore,we detected the protein levels of GS and p-S⁶⁴¹GS via western blotting.The total protein level of GS remained unchanged,however,the p-S⁶⁴¹GS significantly decreased under 10 mmHg.While the phosphorylation modification of p-Y²¹⁶GSK3?was increased.It can activate the activity of GSK3?.These results suggested that mechanical pressures suppress glycogen synthesis through increasing phosphorylation of GSK3?at Y²¹⁶.Increasing pressures activate p53/PTEN pathway.To further explore the mechanism of pressure on glycogen synthesis,we analyzed the genome-wide gene expression changes in HepG2 cells treated with different pressures?0and 10 mmHg?for 24 hours by RNA-seq assay.The gene expression of 10 mmHg was different from 0 mmHg.Of the 13,456 mapped genes in HepG2 cell,we identified 2,253differentially expressed genes including 949 down-and 1,304 up-regulated genes.To identify the effect on cell signaling,we analyzed the data with GSEA.The expression of genes in the regulation of glycogen metabolism process was not changed significantly,which is consistant with our previous results.Interestingly,the genes in the regulation of execution phase of apoptosis were significantly up-regulated.The expression of p53 and PTEN were greatly increased.These data imply that increasing pressures inhibit hepatocellular cell glycogen synthesis via activating p53/PTEN pathway.Increasing pressures suppress glycogen synthesis through activating p53/Pten pathway.It is reported that PTEN and p53 involved in the regulation of glycogen synthesis.Next,we detected the expression of p53and PTEN in HepG2 and HL-7702 cells which were incubated in different pressures?0 and 10 mmHg?for 24 hours.When the pressure was increased,the mRNA levels of p53 and PTEN were also increased.However,the mRNA level of Akt was not significantly changed.And the similar effect was also observed in their protein levels.P53 can inhibit Akt activity by inhibiting its phosphorylation.We found that the phosphorylated Akt was reduced significantly when the pressure increased,which can increasing expression of p-Y²¹⁶Gsk.These data further confirmed that pressure regulates the glycogen synthesis by regulating the p53/PTEN pathway.Conclusions:1.Increasing pressures can decrease the hepatocellular glycogen concentration through suppressing glycogen synthesis.2.The mechanical pressures suppress glycogen synthesis through increasing phosphorylation of Gsk3?at Y²¹⁶.3.The further study imposed that the p53/PTEN pathway is involved into the mechanics suppress glycogen synthesis in hepatocellular cells.