Background: A high consumption of fructose leads to hepatic steatosis. About 20-30% of triglycerides are synthesized via de novo lipogenesis. Some studies showed that endoplasmic reticulum stress (ERS) is involved in this process, while others showed that a lipotoxic environment directly influences ER homeostasis. Here, our aim was to investigate the causal relationship between ERS and fatty acid synthesis and the effect of X-box binding protein-1 (XBP-1), one marker of ERS, on hepatic lipid accumulation stimulated by high fructose.
Methods: HepG2 cells were incubated with different concentrations of fructose. Upstream regulators of de novo lipogenesis (i.e., carbohydrate response element-binding protein [ChREBP] and sterol regulatory element-binding protein 1c [SREBP-1c]) were measured by polymerase chain reaction and key lipogenic enzymes (acetyl-CoA carboxylase [ACC], fatty acid synthase [FAS], and stearoyl-CoA desaturase-1 [SCD-1]) by Western blotting. The same lipogenesis-associated factors were then evaluated after exposure of HepG2 cells to high fructose followed by the ERS inhibitor tauroursodeoxycholic acid (TUDCA) or the ERS inducer thapsigargin. Finally, the same lipogenesis-associated factors were evaluated in HepG2 cells after XBP-1 upregulation or downregulation through cell transfection.
Results: Exposure to high fructose increased triglyceride levels in a dose- and time-dependent manner and significantly increased mRNA levels of SREBP-1c and ChREBP and protein levels of FAS, ACC, and SCD-1, concomitant with XBP-1 conversion to an active spliced form. Lipogenesis-associated factors induced by high fructose were inhibited by TUDCA and induced by thapsigargin. Triglyceride level in XBP-1-deficient group decreased significantly compared with high-fructose group (4.41 ± 0.54 μmol/g vs. 6.52 ± 0.38 μmol/g, P < 0.001), as mRNA expressions of SREBP-1c (2.92 ± 0.46 vs. 5.08 ± 0.41, P < 0.01) and protein levels of FAS (0.53 ± 0.06 vs. 0.85 ± 0.05, P = 0.01), SCD-1 (0.65 ± 0.06 vs. 0.90 ± 0.04, P = 0.04), and ACC (0.38 ± 0.03 vs. 0.95 ± 0.06, P < 0.01) decreased. Conversely, levels of triglyceride (4.22 ± 0.54 μmol/g vs. 2.41 ± 0.35 μmol/g, P < 0.001), mRNA expression of SREBP-1c (2.70 ± 0.33 vs. 1.00 ± 0.00, P < 0.01), and protein expression of SCD-1 (0.93 ± 0.06 vs. 0.26 ± 0.05, P < 0.01), ACC (0.98 ± 0.09 vs. 0.43 ± 0.03, P < 0.01), and FAS (0.90 ± 0.33 vs. 0.71 ± 0.02, P = 0.04) in XBP-1s-upregulated group increased compared with the untransfected group.
Conclusions: ERS is associated with de novo lipogenesis, and XBP-1 partially mediates high-fructose-induced lipid accumulation in HepG2 cells through augmentation of de novo lipogenesis.
X-盒结合蛋白-1在高果糖诱导的HepG2细胞脂质从头合成中的作用研究摘要背景：高果糖易导致肝脏的脂质沉积，其中20-30%的甘油三酯来源于脂质从头合成。有的研究证实内质网应激(Endoplasmic reticulum stress，ERS)参与脂质沉积的过程，而有的研究发现高脂环境可诱发ERS。因此本研究的目的旨在深入探讨ERS和脂质合成之间的关系，及ERS的重要因子X-盒结合蛋白-1（X-box binding protein-1，XBP-1）在高果糖诱发的脂质沉积中的作用。 方法：首先收集不同浓度的果糖干预处理后的HepG2细胞，应用PCR的方法测定其脂质从头合成的上游调控因子碳水化合物反应元件结合蛋白(carbohydrate response element-binding protein，ChREBP) 和（sterol regulatory element-binding protein 1c，SREBP-1c），应用Western方法测定了脂质从头合成的三个关键酶乙酰-CoA 羧化酶（acetyl-CoA carboxylase，ACC）、脂肪酸合酶（fatty acid synthase，FAS）、硬脂酰-CoA 去饱和酶（stearoyl-CoA desaturase-1，SCD-1）。然后HepG2 细胞中加入ERS诱导剂（thapsigargin），或是ERS抑制剂牛磺熊去氧胆酸 （tauroursodeoxycholic acid，TUDCA）加入到高果糖培养的HepG2 细胞中，再通过上述方法测定脂质从头合成的相关因子的表达水平。最后，采用细胞转染技术分别上调和下调XBP-1后，测定脂质从头合成的相关因子的表达。 结果： 高果糖以时间和浓度依赖性增加细胞内脂质沉积，增加细胞内SREBP-1c和ChREBP的mRNA的表达水平，及FAS、ACC和SCD-1 的蛋白表达水平，并促进了XBP-1转化为活性形式。而且这一过程可以被ERS的抑制剂TUDCA所抑制，相反，ERS的诱导剂却促进脂质从头合成。下调XBP-1组甘油三酯的水平较高果糖组明显下降(4.41±0.54 μmol/g vs. 6.52±0.38 μmol/g，P<0.001)，且脂质从头合成的上游调控因子SREBP-1c的mRNA (2.92±0.46 vs. 5.08±0.41，P<0.01)和FAS(0.53±0.06 vs. 0.85±0.05，P=0.01)、SCD-1(0.65±0.06 vs. 0.90±0.04，P=0.04) ACC(0.38±0.03 vs. 0.95±0.06，P<0.01)的蛋白水平明显下降。与之相反的是上调XBP-1s组的甘油三酯的水平较未转染组却明显升高(4.22±0.54 μmol/g vs. 2.41±0.35 μmol/g，P<0.001)，且上游调控因子SREBP-1c的mRNA (2.70±0.33 vs. 1.00±0.00，P<0.01) 和SCD-1(0.93±0.06 vs. 0.26±0.05，P<0.01)、ACC(0.98±0.09 vs. 0.43±0.03，P<0.01)、FAS(0.90±0.33 vs. 0.71±0.02，P=0.04)的蛋白表达水平明显增加。 结论：ERS与脂质从头合成有关，且XBP-1在高果糖诱导的HepG2细胞的脂质沉积的发挥着一定的作用，可能是与上调了脂质从头合成有关。.
Keywords: Endoplasmic Reticulum Stress; Fatty Liver; Lipogenesis; X-Box Binding Protein-1.