The Effect Of Endoplasmic Reticulum Stress On De Novo Lipogenesis In HepG2 Cells Induced By Fructose

Non-alcoholic fatty liver disease refers to a series of clinical syndrome characterized by hepatocyte steatosis and fat storage. With the changes of diet and lifestyle, the incidence rate increased in the worldwide. Pathophysiological progress of NAFLD ranges from hepatic steatosis, steatohepatitis to cirrhosis, while most patients only have hepatic steatosis, named as “fatty liver”. Pathogenesis of NAFLD involves: synthesis of lipids, disorder of fatty acid metabolism and transport, insulin resistance, oxidative stress, inflammatory reaction, imbalance of intestinal flora, mitochondrial dysfunction and so on, but the pathological basis of NAFLD is lipid deposition in liver. Excessive free fatty acids accumulated in liver attributed to synthesis of free fatty acid exceeded the use of fatty acid(beta oxidation) and very low density lipoprotein output, leading to the increaseed lipid deposition in the liver. TG synthesized from acetyl coenzyme through hepatic de novo lipogenesis is 5% of all TG usually, but it will increase about 6 times in the abnormal state compared with that in normal state.The endoplasmic reticulum is a large membrane-enclosed cellular organelle in which newly synthesized transmembrane and secretory proteins are folded, assembled and matured. When protein homeostasis is disrupted by environmental stresses or changes in physiological conditions, or misfolded proteins accumulated in the ER lumen under improper functioning called ERS, leading to an ER-originated adaptive signaling response called Unfolded Protein Response(UPR), which including proteinkinase R-like ER kinase(PERK) /eukaryotic translation initiation factor 2-α(e IF-2α) / activating transcription factor-4(ATF-4) pathway, Inositol-requiring enzyme-1(IRE-1) / X-box binding protein-1(XBP-1) pathway and activating transcription factor-6(ATF-6) pathway. There is accumulating evidence implicating that ERS is critically involved in the progression of glucose and lipid metabolism, and ERS may become an important mechanism leading to NAFLD.With high fructose corn syrup(HFCS) was induced into the food processing industry in the last century 70’s, consumption of fructose containing food and soft drink intake increased. Clinical and animal studies suggest that high fructose intake is associated with obesity, dyslipidemia, metabolic syndrome and NAFLD. Because the high fat diet is known to be an important cause leading NAFLD, we researched on rats fed with high fructose and high-fat diet developed lipid deposition in liver and hypertriglyceridemia and confirmed that the short-term(1 weeks) and long-term(16 weeks) of high fructose feeding made the rodents lipid deposition in liver and induced rat mode of NAFLD, promoted two upstream transcription factors sterol regulatory element binding protein-1c(SREBP-1c), carbohydrate response element binding protein(Ch REBP) and 3 key enzymes acetyl coenzyme A carboxylase(ACC), fatty acid synthase(FAS), stearoyl-Co A desaturase-1(SCD-1), accompanied with ERS, while high-fat diet had an opposite effect, indicating that ERS had the effect on lipogenesis of NAFLD. Although previous researches showed that fructose induced NAFLD which was improved by 4-PBA or TUDCA, and ERS inducers made TG increased. Another study showed that knockdown of PERK can avoid mice developing NAFLD induced by high fat, and knockdown of XBP-1 improved the fatty liver, however, there are many interference factors to understand the effect of single ERS pathway on cellular level on NAFLD induced by high fructose. Therefore, in this study Hep G2 cells were cultured in high fructose medium added with ERS inhibitors, then Hep G2 cells were stimulated by ERS inducers to observe the change of cellular lipid content, lipid deposition, and lipogenesis to explore the effect of ERS on de novo lipogenesis. Finally, PERK/e IF-2α/ATF-4 and IRE-1/XBP-1 two pathways were downregulated and upregulated respectively using the method of cell transfection, to evaluate the effects of the single pathway on the lipogenesis. Thus we aim to have a better understanding of the effect of ERS on lipid metabolism induced by high fructose diet at the cellular level in vitro.Part 1 The effect of fructose and palmitic acid incubation of Hep G2 cells on lipogenesisObjective Compare the different effect of fructose and palmitic acid incubation of Hep G2 cells on lipogenesis, and establish high triglyceride model Hep G2 cell stimulated by fructose.Methods Hep G2 cells were cultured in normal medium and different concentrations of fructose(1, 5, 20mmol/L, named as N, F1, F5, F20 group) with different times(12、24、48、72h), then TG levels were detected by GPO-PAP method and lipid deposition were observed by oil red O staining. Then Hep G2 cells were cultured in 20mmol/L fructose(F group) and 0.2mmol/L palmitic acid(PA group), and then two upstream transcription factors SREBP-1c and Ch REBP m RNA expression levels and three downstream key enzymes ACC, FAS and SCD-1 protein expression levels were determined to compare the effect of high fructose and high fat on the synthesis of lipid.Results 1 Level of TG in Hep G2 cells cultured in different concentrations of fructose: the TG level of F20 group was significantly higher than that of N group(P<0.05) after 12 hours with statistically significance, F1 and F5 group has increasing trend without statistically significance(P>0.05); the TG level of F5, F20 group was significantly higher than that of N group(P<0.05 and P<0.01 repectively) after 24 hours, F1 group had an increasing trend, but without statistical significance; the TG level of F5, F20 group after 24 hours was significantly higher than that in N group(all P<0.01) after 48 and 72 hours, F1 group had an increasing trend, but without statistical significance(P>0.05). TG concentration in cells increased with increase of fructose concentration at the same time piont, and the level of TG with same concentration of fructose increased with prolonged intervention time. It showed that TG level reached the highest point in 20 mmol/L fructose after 72 hours. 2 Lipid accumulation of Hep G2 cells cultured with different concentrations of fructose for 72 hours: oil red O staining showed that: red staining was little in N group, got more in F1 group, and reached the most in F20 group. 3 Effect of fructose and palmitic acid on the cellular TG level: compared with N group, the TG level F and PA group increased with statistical significance(P<0.01). 4 Effect of fructose and palmitic acid on upstream regulatory factors and key enzymes in the de novo lipogenesis: Compared with N group, the m RNA expression of Ch REBP of F group had in increase of 1.8 times(P<0.01), without change in PA group, and SREBP-1c in F group had an increase of 5 times, PA group had an increase of 2.4 times, was but lower than F group; The expression of ACC, FAS and SCD-1 protein in F group were higher than PA group(P<0.05), and there is no significance between F20 and PA groups.Conclusion 1 Hep G2 cells cultured by high fructose or high PA both had liver lipid deposition. 2 The extent of lipid deposition was associated with fructose concentration in dose and time-dependent manner. TG levels reached the highest value at the point of 20mmol/L fructose cultured in 72 hours. 3 Expression levels of 2 upstream regulatory factors and 3 key enzymes increased in F group compared with PA group.Part 2 Effect of endoplasmic reticulum stress on de novo lipogenesisObjective In this part, Hep G2 cells were cultured in high fructose medium added with ERS inhibitors 4-phenyl butyric acid(4- PBA) and tauroursodeoxycholate(TUDCA) respectively, and then added with ERS inducers Tunicamycin and Thapsigagin, to observe the effect of endoplasmic reticulum stress on the de novo lipogenesis.Methods ERS inhibitors 4-PBA(20mmol/L, named as 4-PBA group and TUDCA(0.2mmol/L, named as TUDCA group)were added into fructose medium and inducers Tunicamycin(2μg/ml, named as Tm group) and Thapsigagin(600nmol/L, named as Tg group) into normal medium respectively to induce ERS. Determination of the level of TG by GPO-PAP method and oil red O staining were used to observe the cell lipid deposition, finally the application of biological methods in the molecular level to detect the gene levels of upstream transcription factors SREBP-1c, Ch REBP m RNA level and protein levels of the key enzymes ACC, FAS, SCD-1 in the de novo lipogenesis.Results 1 To observe the effect of ERS inhibitors and inducers on ERS: phosphorylation of PERK, e IF-2α, IRE-l and protein of ATF-4, XBP-1 activation increased in Hep G2 cells cultured in F20 group(P<0.01), and p-PERK 、 p-IRE-1/ IRE-1 、 XBP-1s protein levels were decreased after intervention of TUDCA(all P < 0.05), p-e IF-2α/ e IF-2α 、 ATF-4 were decreased after intervention of TUDCA(all P<0.01); p-PERK、p-e IF-2α/ e IF-2α、ATF-4 were decreased after intervention of 4-PBA(all P<0.05), p-IRE-1/ IRE-1、XBP-1s(all P<0.01). These results showed that 2 ERS inhibitors decreased PERK/e IF-2α/ATF-4 and IRE-1/XBP-1 protein level. 2 To observe the effect of ERS inhibitors on Hep G2 cells lipid deposition: TG level were ameliorated after addition with 4-PBA or TUDCA(all P<0.01), and oil red O staining showed less lipid droplets than F20 group. 3 To observe the effect of ERS inhibitors on de novo lipogenesis: SREBP-1c and Ch REBP gene expression levels decreased(P<0.01 and P<0.05), ACC, FAS and SCD-1 protein levels decreased(all P<0.05) in TUDCA group. SREBP-1c and Ch REBP gene expression levels decreased(P<0.01 and P<0.05), ACC protein levels decreased( P<0.01), FAS and SCD-1(all P<0.05) in 4-PBA group. 4 To observe the effect of ERS inducers on ERS: phosphorylation of PERK, e IF-2α, IRE-l and protein of ATF-4, XBP-1 activation increased after separate application of two kinds of ER stress inducers Tm and Tg(all P<0.01). 5 To observe the effect of ERS inducers on Hep G2 cells lipid deposition:TG level of Tg group increased 1.8 folds of N group(all P<0.01), and TG level of Tm group increased 1.8 folds of N group(P<0.05), oil red O staining showed more lipid droplets in Tm and Tg groups. 6 To observe the effect of ERS inducers on de novo lipogenesis: SREBP-1c and Ch REBP gene expression levels incresad(all P<0.01) in Tg group, and FAS protein level incresad(P<0.01), ACC and SCD-1 protein level incresad(all P<0.05). SREBP-1c and Ch REBP gene expression levels and FAS、ACC、SCD-1 protein levels incresad(all P<0.01) in Tg group.Conclusions 1 ERS of Hep G2 cells can be activated by high fructose. ERS inhibitors( 4-PBA and TUDCA) ameliorated TG levels and lipid deposition in Hep G2 cells induced by high fructose, and Upstream transcription factors SREBP-1c, Ch REBP gene expressions and ACC, SCD-1 and FAS protein expressions were increased significantly in high fructose cultured Hep G2 cells, and decreased after application of inhibitors。2 ERS inducers(Tunicmycin and Thapsigargin) increased TG levels and lipid deposition,and Upstream transcription factors SREBP-1c, Ch REBP gene expressions and ACC, SCD-1 and FAS protein expressions increased by inducers Tm and Tg.Part 3 Effect of IRE1/XBP-1 pathway on lipogenesisObjective To observe the effect of IRE1/XBP-1 pathway on liver lipid deposition through determination of upstream regulatory factors and downstream enzymes after we transfected Hep G2 cells with XBP-1 sh RNA and overexpression plasmid.Methods Hep G2 cells cultured in high fructose were transfected with XBP-1s sh RNA firstly, and there were devided into four groups: Untransfection, pc DNA3.1(+), pc DNA3.1(+)-XBP-1u, pc DNA3.1(+)-XBP-1s groups. Then, Hep G2 cells were transfected with XBP-1s overexpression plasmid, XBP-1u overexpression plasmid and negative control plasmid. Determination of TG by GPO-PAP method and oil red O staining were used to observe the cell lipid deposition, finally the application of biological methods, from the molecular level to detect the gene levels of upstream transcription factors SREBP-1c, Ch REBP m RNA and the protein levels of key enzymes ACC, FAS, SCD-1.Results 1 Effect of transfection with XBP-1 sh RNA on XBP-1s: The expression level of XBP-1s decreased after transfection significantly, more than 70%(P < 0.01), showing that the expression level of XBP-1s is suppressed effectively. 2 Effect of transfection with XBP-1 sh RNA on Hep G2 cells lipid deposition: TG decreased 34% after transfection with XBP-1 sh RNA, compared to F group significantly(P<0.01), with oil red O staining showed less lipid droplets. 3 Effect of transfection with XBP-1 sh RNA on lipogenesis: SREBP-1c m RNA decreased after transfection with XBP-1 sh RNA(P<0.01), Ch REBP m RNA had no change. Key enzymes of ACC decreased after transfection with XBP-1 sh RNA(P<0.01), FAS and SCD-1 decreased after transfection with XBP-1 sh RNA(all P<0.05).4 Effect of transfection with pc DNA3.1(-)-XBP1 s plasmid on XBP-1s: The expression level of XBP-1s was increased in pc DNA3.1(-)-XBP1 s group than negative control group(P<0.01), IRE-1 protein phosphorylation level and XBP-1u protein level decreased(P < 0.05). Active XBP-1s was upregulated, and regulated XBP-1u, IRE-1 in negative feedback mode. 5 Effect of transfection with pc DNA3.1(-)-XBP1 s plasmid on lipid deposition: TG increased after transfection, and oil red O showed more lipid droplets. 6 Effect of transfection with pc DNA3.1(-)-XBP1 s plasmid on lipogenesis: SREBP-1c m RNA increased after transfection( P < 0.01), Ch REBP m RNA had no change. Key enzymes of ACC and SCD-1 decreased after transfection with XBP-1 sh RNA(all P<0.01), FAS decreased after transfection with XBP-1 sh RNA(P<0.05).Conclusions 1 The results that liver lipid deposition was ameliorated in Hep G2 cells transfected with XBP-1 Sh RNA, SREBP-1c and ACC, FAS, SCD-1 in the lipogenesis were alleviated, involved that IRE-1/XBP-1 pathway has association with de novo lipogenesis. 2 Liver lipid deposition was induced in XBP1 s overexpression group, accompanied with activated SREBP-1c and ACC, FAS, SCD-1. And SREBP-1c may be an important target of IRE-1/XBP-1 synthesis pathway, and Ch REBP may not be involved in the process.Part 4 Effect of PERK/e IF-2α/ATF-4 pathway on lipogenesisObjective To observe the effect of PERK/e IF-2α/ATF-4 pathway on liver lipid deposition through determination of upstream regulatory factors and downstream enzymes after we transfected Hep G2 cells with ATF-4 si RNA and overexpression plasmid.Methods Hep G2 cells cultured in high fructose were transfected with XBP-1s sh RNA firstly, and there were devided into four groups: N, F, F+NC, F+ ATF-4 si RNA groups. Then, Hep G2 cells were transfected with XBP-1 overexpression plasmid, and there were devided into four groups: Untransfection, NC, ATF-4+ si RNA groups. Determination of TG by GPO-PAP method and oil red O staining were used to observe the cell lipid deposition, finally the application of biological methods, from the molecular level to detect the gene levels of upstream transcription factors SREBP-1c, Ch REBP m RNA and the protein levels of key enzymes ACC, FAS, SCD-1.Results 1 Effect of transfection with ATF-4 si RNA and ATF-4 overexpression plasmid on ATF-4: The expression level of ATF-4 decreased after transfection with ATF-4 si RNA more than 70%, compared to high fructose group and high fructose + negative control group(P<0.01), showing that the expression level of ATF-4 is suppressed effectively. 2 Effect of transfection with ATF-4 si RNA plasmid on lipid deposition: TG decreased after transfection with ATF-4 si RNA, compared to high fructose group and high fructose +negative control group decreased significantly(P<0.01), with oil red O staining showed more lipid droplets. 3 SREBP-1c m RNA decreased after transfection with ATF-4 si RNA, compared to high fructose group and high fructose +negative control group significantly(P<0.01), and Ch REBP m RNA decreased after transfection with ATF-4 si RNA(P < 0.05). Key enzymes of ACC, FAS, SCD-1; they were increased in ATF-4+ group than that in negative control group decreased after transfection with ATF-4 si RNA significantly(all P < 0.01). 4 Effect of transfection with ATF-4 overexpression plasmid on ATF-4: the expression level of ATF-4 was increased after transfection than in negative control group(P<0.01) showing ATF-4 was upregulated. 5 Effect of transfection with ATF-4 overexpression plasmid on lipid deposition: TG increased after transfection, and oil red O showed more lipid droplets. 6 Effect of transfection with ATF-4 overexpression plasmid on lipogenesis: SREBP-1c and Ch REBP m RNA increased after transfection(all P<0.01), and key enzymes of ACC, FAS and SCD-1 decreased after transfection with XBP-1 sh RNA(all P<0.05).Conclusion 1 The results that liver lipid deposition was ameliorated in Hep G2 cells transfected with ATF-4 si RNA, SREBP-1c, Ch REBP and ACC, FAS, SCD-1 in the lipogenesis were alleviated showed that PERK/e IF-2α/ATF-4 has association with de novo lipogenesis. 2 Liver lipid deposition was induced after ATF-4 overexpression, SREBP-1c, Ch REBP and ACC, FAS, SCD-1 in the lipogenesis were activated. 3 SREBP-1c and Ch REBP may both be important targets of PERK/e IF-2α/ATF-4 synthesis pathway.