Huang T H, Li C F, Qiu L W, Liao S T, Mei Z C
Department of Gastroenterology, the Second Affiliated Hospital, Chongqing 400010, China.
Department of Infectious Disease, the Second Affiliated Hospital, Chongqing 400010, China.
Zhonghua Gan Zang Bing Za Zhi. 2019 Jun 20;27(6):450-456. doi: 10.3760/cma.j.issn.1007-3418.2019.06.012.
To observe the effect of differentiated mature adipocytes on hepatic steatosis and aquaporin-9 (AQP9) expressions in HepG2 cells and further explore its possible mechanism of action. Human preadipocytes were cultured and differentiated to full maturity. HepG2 cells were co-cultured with non-differentiated adipocytes and differentiated mature adipocytes for 48 h, and then labeled as control group and experimental group. Oil red O staining and intracellular triglyceride content were performed on co-cultured HepG2 cells and simultaneous changes in phosphatidylinositol 3-kinase (PI3K) - serine/threonine kinase (Akt) signaling pathway, and AQP9 mRNA and protein levels were detected. The experimental group was co-cultured with recombinant human insulin-like growth factor-I (IGF-I), with the addition of 100ng/ml PI3K-Akt pathway agonist, labeled as experimental group + IGF-I group. The activation of PI3K-Akt pathway was verified by Western blotting (WB). The expression of AQP9 was detected by RT-q PCR and WB. The recombinant lentivirus LV-AQP9 or empty-loaded virus LV-PWPI was transfected with HepG2 cells by recombinant lentiviral transfection tecnique, and labeled as HepG2-AQP9 and HepG2-PWPI. The transfection efficiency was assessed by confocal laser scanning microscopy and RT-qPCR and WB detected the change of AQP9 expression level after virus transfection. Afterwards, the stable over-expressed HepG2-AQP9 cells and the empty-loaded HepG2-PWPI cells were co-cultured with differentiated mature adipocytes for 48h, and labeled as HepG2-AQP9 co-culture group, and then intracellular triglyceride content were detected with Oil red O staining. Finally, IGF-I was added to the HepG2-AQP9 co-culture group, which was recorded as HepG2-AQP9 co-culture + IGF-I group. Intracellular triglyceride content was detected with Oil red O staining, and WB verified PI3K-Akt signaling pathway activation and changes in AQP9 mRNA and protein levels. A t-test was used to compare the two independent samples. The intracellular lipid droplets and triglyceride content (0.052 ± 0.005) in the experimental group was increased significantly than the control group (0.033 ± 0.003) (= 5.225,= 0.006), suggesting that adipocyte co-culture had induced steatosis in HepG2 cells. RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (3.615 ± 0.330) and protein levels (0.072 ± 0.005) in the experimental group were significantly higher than the control group (= 13.708, 11.225,= 0.005, < 0.001). WB results showed that the expression level of phosphorylated Akt (p-Akt) protein (0.116±0.003) in the experimental group was significantly lower than the control group (0.202 ± 0.003) (= 27.136,< 0.001). The total Akt protein was constant, and the p-Akt/total Akt (0.182 ± 0.017)was significantly lower than the control group (0.327 ± 0.019) (= 2.431,= 0.001), suggesting that adipocyte co-culture had inhibited PI3K- Akt signaling pathway in HepG2 cells and up-regulated the expression level of AQP9. WB results indicated that the expression level of p-Akt protein (0.194 ± 0.021) in the experimental group + IGF-I group was significantly higher than the experimental group (0.132 ± 0.003) (= 5.082,= 0.007). The total Akt protein was constant, and the p-Akt/total Akt (0.281 ± 0.009) was significantly higher than the control group (0.184 ± 0.132) (= 10.311,< 0.001). Simultaneously, RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (0.327 ± 0.347) and protein levels (0.042 ± 0.004) in the experimental group + IGF-I group were significantly lower than the experimental group (= 33.573, 5.598,< 0.001, 0.005), suggesting that adipocyte co-culture had possibility to regulate the expression level of AQP9 through the PI3K-Akt pathway. Confocal laser microscopy analysis showed that the transfection efficiency was more than 90%. RT-q PCR and WB results indicated that the expression levels of AQP9 mRNA and protein levels (0.373 ± 0.221) in HepG2-AQP9 group were significantly higher than HepG2-PWPI group (=14.953, 28.931,= 0.002 and 0.000), suggesting that the stable overexpression of AQP9 cell line was successfully constructed. The intracellular lipid droplets and triglyceride content in HepG2-AQP9 co-culture group was significantly increased (= 5.478, 5.369,= 0.005) than HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+ IGF-I group, suggesting that the increased expression of AQP9 had promoted HepG2 steatosis in co-cultured adipocytes. WB results showed the expression levels of p-Akt protein (0.168 ± 0.006) and p-Akt/total Akt (0.265±0.009) in HepG2-AQP9 co-culture + IGF-1 group was significantly increased (= 16.311, 8.769,< 0.001) than HepG2-AQP9 co-culture group, while the expression levels of AQP9 mRNA (0.327 ± 0.034) and protein (0.375 ± 0.025) was significantly decreased (= 33.573, 9.146,< 0.001 and 0.001). Adipocytes co-culture can induce steatosis in HepG2 cells, and may participate in inhibiting PI3K-Akt signaling pathway to upregulate the expression of AQP9 in steatotic HepG2 cells.
观察分化成熟脂肪细胞对HepG2细胞肝脂肪变性及水通道蛋白9（AQP9）表达的影响，并进一步探讨其可能的作用机制。培养人前脂肪细胞并使其分化至完全成熟。将HepG2细胞与未分化脂肪细胞和分化成熟脂肪细胞共培养48小时，然后分别标记为对照组和实验组。对共培养的HepG2细胞进行油红O染色和细胞内甘油三酯含量测定，并同时检测磷脂酰肌醇3激酶（PI3K）-丝氨酸/苏氨酸激酶（Akt）信号通路以及AQP9 mRNA和蛋白水平的变化。实验组与重组人胰岛素样生长因子-I（IGF-I）共培养，并添加100ng/ml的PI3K-Akt通路激动剂，标记为实验组+IGF-I组。通过蛋白质免疫印迹法（WB）验证PI3K-Akt通路的激活情况。采用逆转录-定量聚合酶链反应（RT-qPCR）和WB检测AQP9的表达。通过重组慢病毒转染技术将重组慢病毒LV-AQP9或空载病毒LV-PWPI转染HepG2细胞，分别标记为HepG2-AQP9和HepG2-PWPI。通过共聚焦激光扫描显微镜评估转染效率，并通过RT-qPCR和WB检测病毒转染后AQP9表达水平的变化。之后，将稳定过表达的HepG2-AQP9细胞和空载的HepG2-PWPI细胞与分化成熟脂肪细胞共培养48小时，标记为HepG2-AQP9共培养组，然后用油红O染色检测细胞内甘油三酯含量。最后，向HepG2-AQP9共培养组中添加IGF-I，记录为HepG2-AQP9共培养+IGF-I组。用油红O染色检测细胞内甘油三酯含量，并用WB验证PI3K-Akt信号通路激活情况以及AQP9 mRNA和蛋白水平的变化。采用t检验比较两个独立样本。实验组细胞内脂滴和甘油三酯含量（0.052±0.005）显著高于对照组（0.033±0.003）（t=5.225，P=0.006），表明脂肪细胞共培养诱导了HepG2细胞脂肪变性。RT-qPCR和WB结果显示，实验组AQP9 mRNA表达水平（3.615±0.330）和蛋白水平（0.072±0.005）显著高于对照组（t=13.708，11.225，P=0.005，<0.001）。WB结果显示，实验组磷酸化Akt（p-Akt）蛋白表达水平（0.116±0.003）显著低于对照组（0.202±0.003）（t=27.136，P<0.001）。总Akt蛋白恒定，p-Akt/总Akt（0.182±0.017）显著低于对照组（0.327±0.019）（t=2.431，P=0.001），表明脂肪细胞共培养抑制了HepG2细胞中PI3K-Akt信号通路并上调了AQP9的表达水平。WB结果表明，实验组+IGF-I组p-Akt蛋白表达水平（0.194±0.021）显著高于实验组（0.132±0.003）（t=5.082，P=0.007）。总Akt蛋白恒定，p-Akt/总Akt（0.281±0.009）显著高于对照组（0.184±0.132）（t=10.311，P<0.001）。同时，RT-qPCR和WB结果表明，实验组+IGF-I组AQP9 mRNA表达水平（0.327±0.347）和蛋白水平（0.042±0.004）显著低于实验组（t=33.573，5.598，P<0.001，0.005），表明脂肪细胞共培养可能通过PI3K-Akt通路调节AQP9的表达水平。共聚焦激光显微镜分析显示转染效率超过90%。RT-qPCR和WB结果表明，HepG2-AQP9组AQP9 mRNA和蛋白水平（0.373±0.221）显著高于HepG2-PWPI组（t=14.953，28.931，P=0.002和0.000），表明成功构建了AQP9稳定过表达细胞系。HepG2-AQP9共培养组细胞内脂滴和甘油三酯含量显著高于HepG2-PWPI共培养组和HepG2-AQP9共培养+IGF-I组（t=5.478，5.369，P=0.005），表明AQP9表达增加促进了共培养脂肪细胞中HepG2的脂肪变性。WB结果显示，HepG2-AQP9共培养+IGF-1组p-Akt蛋白表达水平（0.168±0.006）和p-Akt/总Akt（0.265±0.009）显著高于HepG2-AQP9共培养组（t=16.311，8.769，P<0.001），而AQP9 mRNA表达水平（0.327±0.034）和蛋白（0.375±0.025）显著降低（t=33.573，9.146，P<0.001和0.001）。脂肪细胞共培养可诱导HepG2细胞脂肪变性，并可能通过抑制PI3K-Akt信号通路上调脂肪变性HepG2细胞中AQP9的表达。
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