Effects Of ChREBP On Glucose And Lipid Metabolism In L02 Cell Line

Background and ObjectiveNAFLD has increased rapidly in China in the last decade due to the pandemic of diabetes and obesity. NAFLD can coexist with type 2 diabetes, and type 2 diabetes mellitus may increase the prevalence and the severity of NAFLD significantly, but the exact mechanisms remain unclear. In 2001, Yamashita et al[1] discovered a transcription factor binding on the ChoRE sequence of LPK gene promoter, which named as carbohydrate response element binding protein (ChREBP). ChREBP belongs to basic helix-loop-helix /leucine zipper (bHLH-ZIP) transcription factor family[2]. Recent studies have found that glucose metabolism regulates glycolysis and fat synthesis enzyme gene expression by way of ChREBP, independently insulin signaling pathway. For better understanding the relations of hyperglycemia and NAFLD, we investigated the role of ChREBP in hepatocyte fatty degeneration induced by high glucose concentration.Methods1. L02 cells were cultured with 1640 medium containing 10% fetal bovine serum and passaged.2. Experiment groups: group G1(11mmol/L glucose medium, control), group G2(18mmol/L glucose medium), group G3(25mmol/L glucose medium).3. The accumulation of lipid droplets and the content of triglyceride in L02 cells were observed by oil red O staining and biochemical assays respectively.4. The nuclear translocation of ChREBP was observed by immunofluorescence.5. The expression of LPK, SREBP-1c mRNA and FAS, LXRαprotein were detected by RT-PCR and Western blot respectively.Results1. Steatosis models in L02 cell were established successfully. Accompanied with increase of glucose and extension of time, steatosis hepatocyte was more aggravated.1) Accompanied with increase of glucose and extension of time, TG content in L02 cell increased. Compared with the G1 group, TG content increased in G2 and G3 group markedly at 48 hours (3.18±0.25, 6.62±0.95 vs 1.32±0.28, P=0.0010 and P=0.0008), and the TG content in G3 group increased more significantly.2) Only small lipid droplets were observed in G1 group. There were more visible orange lipids accumulations in G2 and G3 groups, and with extension of time, lipids accumulations increased more significantly. According to the TG content determination and oil red O staining in L02 cells, we found that hepatocyte fatty degeneration was more significant in G3 group, so we selected 25mmol/L as the observation concentration of high glucose for subsequent experiments.2. Translocation of ChREBP from cytoplasm to nucleus in groups.ChREBP mainly located in cytoplasm in control group. Compared with control group, ChREBP in high glucose group translocated from cytoplasm to nucleus gradually after L02 cells were cultured in high glucose for 3 hours, and the translocations of ChREBP from cytoplasm to nucleus were significantly increased at 6 and 12 hours, about 20%. ChREBP in nucleus at 24 hours decreased gradually.3. Expression of LPK and SREBP-1c mRNA in groups.Compared with control group, expression of LPK mRNA in high glucose group was increased at 6, 12, 24, 36 hours (P =0.0066,P =0.0003,P =0.0118,P =0.0376), and peaked at 12 hours, then decreased gradually. And Compared with control group, expression of SREBP-1c mRNA in high glucose group were not significantly different(P >0.05).4. Expression of FAS and LXRαprotein in groups.Compared with control group, expression of FAS protein in high glucose group was increased at 24, 48 hours (P =0.0030, P =0.0001), and with the prolonging of the culture time, FAS protein in high glucose group increased more significantly. And Compared with control group, expression of LXRαprotein in high glucose group were not significantly different(P >0.05).Conclusion1. High glucose may induce hepatocyte fatty degeneration by way of ChREBP-LPK-FAS with its metabolism.2. High glucose and its metabolism can not induce hepatocyte fatty degeneration by way of LXRα- SREBP-1c -FAS.