Gao Jie, Zhang Manqian, Zu Xingwang, Gu Xue, Hao Erwei, Hou Xiaotao, Bai Gang
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300353, China.
Guangxi Key Laboratory of Efficacy Study on Chinese Materia Medica, Guangxi Collaborative Innovation Center for Research on Functional Ingredients of Agricultural Residues, Guangxi University of Chinese Medicine, Nanning 530200, China.
Chin Herb Med. 2023 Apr 12;15(3):398-406. doi: 10.1016/j.chmed.2022.11.005. eCollection 2023 Jul.
Phenolic acids widely exist in the human diet and exert beneficial effects such as improving glucose metabolism. It is not clear whether phenolic acids or their metabolites play a major role . In this study, caffeic acid (CA) and ferulic acid (FA), the two most ingested phenolic acids, and their glucuronic acid metabolites, caffeic-4'--glucuronide (CA4G) and ferulic-4'--glucuronide (FA4G), were investigated.
Three insulin resistance models were established by using TNF-α, insulin and palmitic acid (PA) in HepG2 cells, respectively. We compared the effects of FA, FA4G, CA and CA4G on glucose metabolism in these models by measuring the glucose consumption levels. The potential targets and related pathways were predicted by network pharmacology. Fluorescence quenching measurement was used to analyze the binding between the compounds and the predicted target. To investigate the binding mode, molecular docking was performed. Then, we performed membrane recruitment assays of the AKT pleckstrin homology (PH) domain with the help of the PH-GFP plasmid. AKT enzymatic activity was determined to compare the effects between the metabolites with their parent compounds. Finally, the downstream signaling pathway of AKT was investigated by Western blot analysis.
The results showed that CA4G and FA4G were more potent than their parent compounds in increasing glucose consumption. AKT was predicted to be the key target of CA4G and FA4G by network pharmacology analysis. The fluorescence quenching test confirmed the more potent binding to AKT of the two metabolites compared to their parent compounds. The molecular docking results indicated that the carbonyl group in the glucuronic acid structure of CA4G and FA4G might bind to the PH domain of AKT at the key Arg-25 site. CA4G and FA4G inhibited the translocation of the AKT PH domain to the membrane, while increasing the activity of AKT. Western blot analysis demonstrated that the metabolites could increase the phosphorylation of AKT and downstream glycogen synthase kinase 3β in the AKT signaling pathway to increase glucose consumption.
In conclusion, our results suggested that the metabolites of phenolic acids, which contain glucuronic acid, are the key active substances and that they activate AKT by targeting the PH domain, thus improving glucose metabolism.
酚酸广泛存在于人类饮食中，并发挥如改善葡萄糖代谢等有益作用。目前尚不清楚酚酸或其代谢产物是否起主要作用。在本研究中，对两种摄入最多的酚酸——咖啡酸（CA）和阿魏酸（FA）及其葡萄糖醛酸代谢产物咖啡酸 - 4'- - 葡萄糖醛酸苷（CA4G）和阿魏酸 - 4'- - 葡萄糖醛酸苷（FA4G）进行了研究。
分别使用肿瘤坏死因子 - α、胰岛素和棕榈酸（PA）在HepG2细胞中建立三种胰岛素抵抗模型。通过测量葡萄糖消耗水平，比较FA、FA4G、CA和CA4G对这些模型中葡萄糖代谢的影响。采用网络药理学预测潜在靶点和相关途径。利用荧光猝灭测量分析化合物与预测靶点之间的结合。为研究结合模式，进行了分子对接。然后，借助PH - GFP质粒对AKT普列克底物蛋白同源（PH）结构域进行膜募集分析。测定AKT酶活性以比较代谢产物与其母体化合物之间的作用效果。最后，通过蛋白质免疫印迹分析研究AKT的下游信号通路。
结果表明，CA4G和FA4G在增加葡萄糖消耗方面比其母体化合物更有效。通过网络药理学分析预测AKT是CA4G和FA4G的关键靶点。荧光猝灭试验证实，与母体化合物相比，这两种代谢产物与AKT的结合更强。分子对接结果表明，CA4G和FA4G葡萄糖醛酸结构中的羰基可能在关键的Arg - 25位点与AKT的PH结构域结合。CA4G和FA4G抑制AKT PH结构域向膜的转位，同时增加AKT的活性。蛋白质免疫印迹分析表明，这些代谢产物可增加AKT信号通路中AKT和下游糖原合酶激酶3β的磷酸化，从而增加葡萄糖消耗。
总之，我们的结果表明，含有葡萄糖醛酸的酚酸代谢产物是关键活性物质，它们通过靶向PH结构域激活AKT，从而改善葡萄糖代谢。
