Abstract
Metformin is widely used to surmount insulin resistance (IR) and type 2 diabetes. Evidence indicates that metformin improves insulin resistance associated with gut microbiota, but the underlying mechanism remains unclear. In the present study, metformin effectively improved insulin sensitivity and alleviated liver inflammation and oxidative stress in high-fat diet (HFD)-fed mice. Metabolomics analysis showed that metformin increased tauroursodeoxycholic acid (TUDCA) levels both in intestinal content and liver by reducing the production and activity of bile salt hydrolase (BSH). We further found that TUDCA was able to antagonize with KEAP1 to prevent its binding to Nrf2 and activate Nrf2/ARE pathway, thereby reducing intracellular ROS and improving insulin signaling. Moreover, metformin increased the proportion of Akkermanisia muciniphlia in the HFD-fed mice, while in vitro growth curve test confirmed that it’s TUDCA, not metformin, promoted the proliferation of A. muciniphlia. Subsequently, TUDCA administration could effectively ameliorate insulin resistance, activate hepatic Nrf2/ARE pathways, and increase the abundance of intestinal A. muciniphlia in ob/ob mice. These findings reveal that metformin remodels the gut microbiota, reduces oxidative stress and enhances insulin sensitivity partly due to increasing the production of TUDCA. This provides a novel mechanism by which metformin alleviates diet-induced insulin resistance and improves metabolism.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Abbreviations T2DM, type 2 diabetes mellitus; AMPK, AMP-activated protein kinase; IR, insulin resistance; BA, bile acids; ER, endoplasmic reticulum; BSH, bile salt hydrolase; NASH, nonalcoholic steatohepatitis; ROS, reactive oxygen species; Nrf2, nuclear factor-erythroid-2-related factor 2; KEAP1, Kelch-like ECH-associated protein 1; TUDCA, tauroursodeoxycholic acid; IRS-1, insulin receptor substrate 1; Akt, protein kinase B; 4-HNE, 4-hydroxynonenal; IPGTT, intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test; FFA, free fatty acids; FBG, fasting blood glucose; FINS, fasting serum insulin; HOMA-IR, homeostasis model assessment-estimated insulin resistance; 2-NBDG, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose; NQO1, NAD(P)H:quinone oxidoreductase-1; HO-1, heme oxygenase 1; IL-1β, interleukin-1 β; Il-6, interleukin-6; TNF-α, tumor necrosis factor-α; CYP7a1, cholesterol 7α-hydroxylase; BAAT, bile acid-CoA:amino acid N-acyltransferase; 7α/β-HSDH, 7α/β-hydroxysteroid dehydrogenase.
In this revised manuscript, we supplemented more solid data to support our conclusion, and reorganized the manuscript. The main changes are shown as follows: (1) We verified that TUDCA alleviates PA-induced insulin resistance through the Nrf2/ARE signaling pathway using primary hepatocytes from Nrf2 knockout mice. A molecular docking model of TUDCA with Keap1 was simulated to provide possible active sites of TUDCA binding to Keap1. (2) ob/ob mice were employed to validate the effect of TUDCA in alleviating insulin resistance and lipid accumulation in vivo. We found that, like metformin, TUDCA also activated the Nrf2/ARE signaling pathway and alleviated oxidative stress-induced insulin resistance. In addition, the relative abundance of Akkermansia in the intestine of ob/ob mice was also significantly increased. (3) We enhanced the discussion on the relationship between TUDCA and gut microbiota. Metformin treatment led to a decrease in the relative abundance of Lactobacilli and Bifidobacteria in the intestine of mice, which produce bile salt hydrolase (BSH), a key enzyme in the catabolism of the conjugated bile acid TUDCA. The remodeling of the gut microbiota resulted in a decrease in the amount and activity of BSH in the intestine of mice treated with metformin, which in turn caused an up-regulation of TUDCA level in the intestine, and this tendency was not influenced by metformin itself. Furthermore, accumulated evidence show that metformin alleviates insulin resistance dependent on gut microbiota, which was demonstrated using germ-free mice or antibiotic-treated mice (Li, et al, DOI: 10.3389/fendo.2019.00939; Wu, et al, DOI: 10.1038/nm.4345; Wu, et al, DOI: 10.1016/j.lfs.2019.04.009).