Metformin exerts glucose-lowering action in high-fat fed mice via attenuating endotoxemia and enhancing insulin signaling

Abstract

Aim: Accumulating evidence shows that lipopolysaccharides (LPS) derived from gut gram-negative bacteria can be absorbed, leading to endotoxemia that triggers systemic inflammation and insulin resistance. In this study we examined whether metformin attenuated endotoxemia, thus improving insulin signaling in high-fat diet fed mice.

Methods: Mice were fed a high-fat diet for 18 weeks to induce insulin resistance. One group of the mice was treated with oral metformin (100 mg·kg(-1)·d(-1)) for 4 weeks. Another group was treated with LPS (50 μg·kg(-1)·d(-1), sc) for 5 days followed by the oral metformin for 10 d. Other two groups received a combination of antibiotics for 7 d or a combination of antibiotics for 7 d followed by the oral metformin for 4 weeks, respectively. Glucose metabolism and insulin signaling in liver and muscle were evaluated, the abundance of gut bacteria, gut permeability and serum LPS levels were measured.

Results: In high-fat fed mice, metformin restored the tight junction protein occludin-1 levels in gut, reversed the elevated gut permeability and serum LPS levels, and increased the abundance of beneficial bacteria Lactobacillus and Akkermansia muciniphila. Metformin also increased PKB Ser473 and AMPK T172 phosphorylation, decreased MDA contents and redox-sensitive PTEN protein levels, activated the anti-oxidative Nrf2 system, and increased IκBα in liver and muscle of the mice. Treatment with exogenous LPS abolished the beneficial effects of metformin on glucose metabolism, insulin signaling and oxidative stress in liver and muscle of the mice. Treatment with antibiotics alone produced similar effects as metformin did. Furthermore, the beneficial effects of antibiotics were addictive to those of metformin.

Conclusion: Metformin administration attenuates endotoxemia and enhances insulin signaling in high-fat fed mice, which contributes to its anti-diabetic effects.

Figure 1

Metformin improves glucose metabolism and attenuates endotoxemia. Mice were fed an LFD or HFD for 18 weeks, and then a set of HFD-fed mice were treated with metformin for another 4 weeks. (A) Body weight; (B) Glucose tolerance test (IPGTT); (C) Pyruvate tolerance test (IPPTT); (D) Serum LPS contents; (E) Serum D-xylose contents; (F) Western blot analysis of gut occludin-1 contents; (G) Quantitative real-time PCR analysis of gut lactobacillus. Mean±SEM. n=4–6. ^*P<0.05, ^**P<0.01 vs LFD. ^#P<0.05, ^##P<0.01 vs HFD.

Figure 2

LPS injection blocks the beneficial effects of metformin on glucose metabolism and insulin signaling. The mice were fed an LFD or HFD for 18 weeks, and then a set of HFD-fed mice were treated without (HM) or with a subcutaneous injection of LPS (HML) for 5 d, and then both groups continued to receive metformin treatment for another 10 d. (A) glucose tolerance test (IPGTT); (B) pyruvate tolerance test (IPPTT); (C) HOMA IR; (D) liver weight; (E) weight of epididymal fat pads; Western blot analysis of insulin-stimulated PKB Ser473, AMPK T172, IκBα and p-eIF2α levels in the liver (F) or muscle (G), with GAPDH serving as a loading control. Right panels show the protein levels, represented by optical density scanning, corresponding to the left panels. Mean±SEM. n=3–6. ^*P<0.05, ^**P<0.01 vs LFD. ^#P<0.05, ^##P<0.01 vs HFD. ^$P<0.05 vsHM.

Figure 3

LPS exerts opposite effects to those of metformin on tissue MDA levels, Nrf2 function and PTEN levels. The mice were treated as described in Figure 2, and MDA contents in the liver (A) and muscle tissue (B) (left panels) and in their mitochondrial fractions (right panels) were measured. Western blot analysis of total and nuclear Nrf2, NQO-1, PTEN, and HO-1 in liver (C) and muscle (D) tissues (left panels). Right panels show the protein levels, represented by optical density scanning, corresponding to the left panels. Mean±SEM. n=4–6. ^*P<0.05, ^**P<0.01 vs LFD. ^#P<0.05 vs HFD. ^$P<0.05 vsHM.

Figure 4

Antibiotic treatment has similar effects to those of metformin in improving glucose metabolism and insulin signaling. The mice were fed an LFD or HFD for 18 weeks, and then a set of HFD-fed mice were treated with a combination of antibiotics for another 7 d and then continually treated without (HA) or with metformin (HMA) in their drinking water for 4 weeks. (A) Glucose tolerance test (IPGTT); (B) Liver weight; (C) Weight of epididymal fat pads; Western blot analysis of insulin-stimulated PKB Ser473, AMPK T172, IκBα and p-eIF2α levels in the liver (D) and muscle (E), with GAPDH serving as a loading control. Right panels show the protein levels, represented by optical density scanning, corresponding to the left panels. Mean±SEM. n=4–6. ^*P<0.05, ^**P<0.01 vs LFD. ^#P<0.05 vs HFD. ^$P<0.05 vsHM.

Figure 5

Antibiotic treatment shows similar effects to those of metformin in reducing MDA levels, activating Nrf2 function and repressing PTEN expression in the liver and muscle. The mice were treated as described in Figure 4 and the MDA contents in the liver (A) and muscle tissue (B) (left panels) and in their mitochondrial fractions (right panels) were measured. Western blot analysis of PTEN, total and nuclear Nrf2 and HO-1 in liver (C) and muscle (D) tissues (left panels). Right panels show the protein levels, represented by optical density scanning, corresponding to the left panels. Mean±SEM. n=4–6. ^*P<0.05, ^**P<0.01 vs LFD. ^#P<0.05 vs HFD. ^$P<0.05 vs HM.