Primary Culture Of Hepatocytes From Grouper (Epinephelus Coioides) And Effect Of Cortisol On The Metabolism Of Hepatocytes

1.Study on the isolation and primary culture of hepatocytes from liver of grouper(Epinephelus coioides)In order to explore the stable and reliable methods for isolation and primary culture of hepatocytes from grouper(Epinephelus coioides),we selected the hepatocytes of grouper and then cultivated under different conditions.Grouper hepatocytes were isolated by tissue separation and trypsin(0.25% with EDTA)digestion.The hepatocytes could be separated and purified by density gradient centrifugation.The harvested hepatocytes were then suspended in DMEM/F-12,M199,or L-15 medium(cultured with 5% CO2).The yield was determined by a hemocytometer.The viability was assessed by Trypan blue exclusion test.Proliferation of hepatocytes was tested by MTT assay.Function of hepatocytes was examined according to the levels of lactic acid dehydrogenase(LDH),albumin(ALB),and urea nitrogen(BUN)of supernatant at different times,respectively.Trypsin digestion method was better than tissue culture.Using 0.25% warm trypsin digestion,the cell yield was 1.6×108 cells per g(liver weight)and the viability was more than 95%.The cells growth better were cultured in L-15 medium than which were cultured in DMEM/F-12 and M199 medium.The liver function index showed that lactic acid dehydrogenase(LDH)significantly decreased(P<0.05),urea nitrogen(BUN)and albumin(ALB)significantly increased(P<0.05)during 48-72 h with a strong proliferation.This study indicates that the best method of isolation was pancreatin digestion and the best medium was L-15.After 48 h to 72 h in culture,the growth and metabolism of hepatocytes were increased.2.Effect of cortisol on viability and function of primary hepatocyte of grouper(Epinephelus coioides)In the present study,in order to identify the effect of cortisol on liver cells viability and function,the hepatocytes isolated from grouper were cultured with cortisol.Hepatocytes were exposed to cortisol at concentrations of 0,1,10,100,1000 nmol/L for 24 hours,and then proliferation of hepatocytes was tested by MTT assay.Function of the hepatocytes was examined by measuring activities of glutamate transaminase(ALT),aspartate aminotransferase(AST),lactic acid dehydrogenase(LDH)and the levels of urea nitrogen(BUN),albumin(ALB)in the supernatant,respectively.The results indicated that high concentration of cortisol(100?1000 nmol/L)showed significantly increased(P<0.05)the activities or levels of ALT,AST,LDH and BUN.The 1000 nM cortisol group was significantly decreased the viability of hepatocytes(P<0.05).These rsults demonstrated that high concentration of cortisol negatively effected the function and activity of hepatocytes.3.Effects of cortisol on carbohydrate and lipid metabolism in primary cultured hepatocytes from grouper(Epinephelus coioides)In the present study,freshly hepatocytes were isolated from grouper(Epinephelus coioides),cultured and subjected to different cortisol levels(0,100 and 1000 nM)for 24 h and 36 h.Medium glucose concentrations,glycogen content,pyruvate content and triglyceride(TG)content,activity and mRNA expression of key enzymes involved in carbohydrate and lipid metabolism,as well as mRNA levels of key transcription factor related to lipid metabolism,including phosphoenolpyruvate carboxykinase(PECK),glucose-6-phosphatase(G6Pase),glycogen synthase(GSase),fatty acid synthase(FAS),lipoprotein lipase(LPL),hormone sensitive lipase(HSL)and peroxisomeproliferator-activatedreceptor ?(PPAR?),were assessed at 24 h and 36 h,respectively.The results showed that cortisol significantly increased the medium glucose concentrations,but significantly decreased the pyruvate content and TG content(P<0.05),both at 24 h and 36 h.The time of exposure to cortisol were showed no significant effect on medium glucose concentrations,but significantly decreased the pyruvate content and TG content(P>0.05).At 24 h,Cortisol treatments showed no significant effect on glycogen content(P>0.05),however,at 36 h,cortisol significantly decreased glycogen content(P<0.05).With the time of exposure to cortisol extending,glycogen content was significantly decreased(P<0.05).Cortisol significantly increased the activities of PEPCK and G6 Pase,and significantly decreased the activities of MDH and ICD(P<0.05),however,there was no significant difference in activity of GSase(P>0.05).At 24 h,Cortisol treatments showed no significant effect on the activity of PK(P>0.05),but at 36 h,cortisol significantly increased g the activity of PK(P<0.05).The time of exposure to cortisol significantly decreased activity of G6 Pase,and significantly increased activity of PK(P<0.05),however,there was no significant effect on the activities of MDH and ICD(P>0.05).Cortisol significantly increased the PEPCK and G6 Pase mRNA expression(P<0.05),both at 24 h and 36 h.At 24 h,Cortisol significantly increased the mRNA expression of PK(P<0.05),but at 36 h,cortisol showed no significant effect on the activity of PK(P>0.05).With the time of exposure to cortisol extending,G6 Pase and PK mRNA expression were significantly increased(P<0.05),however,MDH mRNA expression showed no significant differences(P>0.05).Both at 24 h and 36 h,cortisol significantly decreased the FAS and PPAR? mRNA expression,and significantly increased the LPL and HSL mRNA expression(P<0.05).The time of exposure to cortisol significantly decreased LPL mRNA expression(P<0.05),and showed no significant effect on FAS,PPAR? and HSL mRNA expression(P>0.05).The present study demonstrates that cortisol enhanced gluconeogenic capacity,and exert a inhibition on lipogenesis and stimulation on lipolysis of the primary cultured hepatocytes from grouper.