Yadav Ajay K, Sata Teja Naveen, Verma Daksh, Sah Amrendra K, Mishra Amit K, Hossain Md Musa, Pant Kishor, Venugopal Senthil K
Faculty of Life Sciences and Biotechnology, South Asian University, Akbar Bhawan, Chanakyapuri, New Delhi 110021, India.
The Hormel Institute,University of Minnesota, Austin, MN, 55912 USA.
ILIVER. 2023 Feb 7;2(1):1-9. doi: 10.1016/j.iliver.2023.01.002. eCollection 2023 Mar.
Free fatty acids (FFAs) are one of the important regulators of the progression of nonalcoholic fatty liver disease. The FFAs are shown to modulate the metabolic status of the liver by modulating several cellular pathways in hepatocytes. Here, we elucidated the role of miR-22 in modulating FFAs-mediated gluconeogenesis.
Huh7 and WRL68 cells were transfected with nonspecific miRNA, miR-22 premiRs or anti-miR-22 oligos followed by incubation with palmitic acid, oleic acid, and linoleic acid (300 μM each) for 48 and 72 h after transfection. The expression of miR-22 was performed using real-time polymerase chain reaction and Western blots were performed for SIRT-1, PGC-1α, PEPCK, and glucose-6-phosphatase. Three groups of C57BL/6 mice (6 mice per group) were fed with standard diet, choline sufficient l-amino acid defined diet or choline-deficient l-amino acid defined (CDAA) diet for 6, 18, 32, or 54 weeks. Triglycerides content was measured in the serum. Expression of miR-22 and the protein expression of gluconeogenic enzymes were analyzed in the tissue samples.
Incubation of miR-22-transfected cells with FFAs inhibited the expression of SIRT-1, PGC-1α, PEPCK, and glucose-6-phosphatase, while miR-22 expression was increased. These changes were reversed when the cells were transfected with anti-miR-22 oligos. CDAA-fed mice showed the significant increase in triglycerides content and miR-22 expression, while there was an inhibition of SIRT-1, PGC-1α, PEPCK, and glucose-6-phosphatase expression in CDAA-fed mice.
These data confirm that FFAs inhibited gluconeogenesis via miR-22-mediated inhibition of SIRT-1, which in turn inhibited PGC-1α in hepatic cells.
游离脂肪酸（FFAs）是非酒精性脂肪性肝病进展的重要调节因子之一。研究表明，游离脂肪酸可通过调节肝细胞中的多种细胞途径来调节肝脏的代谢状态。在此，我们阐明了miR-22在调节游离脂肪酸介导的糖异生中的作用。
用非特异性miRNA、miR-22前体或抗miR-22寡核苷酸转染Huh7和WRL68细胞，转染后分别用棕榈酸、油酸和亚油酸（各300μM）孵育48小时和72小时。采用实时聚合酶链反应检测miR-22的表达，并对沉默信息调节因子1（SIRT-1）、过氧化物酶体增殖物激活受体γ共激活因子1α（PGC-1α）、磷酸烯醇式丙酮酸羧激酶（PEPCK）和葡萄糖-6-磷酸酶进行蛋白质免疫印迹分析。将三组C57BL/6小鼠（每组6只）分别喂以标准饮食、胆碱充足的l-氨基酸限定饮食或胆碱缺乏的l-氨基酸限定（CDAA）饮食6、18、32或54周。检测血清中的甘油三酯含量。分析组织样本中miR-22的表达及糖异生酶的蛋白表达情况。
用游离脂肪酸孵育转染了miR-22的细胞会抑制SIRT-1、PGC-1α、PEPCK和葡萄糖-6-磷酸酶的表达，而miR-22的表达则增加。当用抗miR-22寡核苷酸转染细胞时，这些变化会逆转。喂食CDAA的小鼠甘油三酯含量和miR-22表达显著增加，而喂食CDAA的小鼠中SIRT-1、PGC-1α、PEPCK和葡萄糖-6-磷酸酶的表达受到抑制。
这些数据证实，游离脂肪酸通过miR-22介导的对SIRT-1的抑制作用来抑制糖异生，进而抑制肝细胞中的PGC-1α。
