Metformin Protects against LPS-Induced Intestinal Barrier Dysfunction by Activating AMPK Pathway

Metformin not only regulates energy metabolism but also participates in many cellular processes. In this study, we investigated the effect of metformin on lipopolysaccharide (LPS)-induced intestinal barrier damage. We found that LPS treatment decreased the expression of tight junction proteins and caused a proinflammatory response and oxidative stress in the intestine. Interestingly, metformin treatments attenuated LPS-induced intestinal barrier damage, inflammation, and oxidative stress. We found that metformin improved the expression of intestinal tight junction proteins (ZO1, occludin, and Claudin1) that were reduced by LPS stimulation. Moreover, metformin alleviated LPS-induced NF-κB phosphorylation, promoted Nrf2 nuclear translocation, and increased the expression of the antioxidative genes (HO-1 and NQO-1), leading to reduced intestinal ROS content. Mechanistically, we found that metformin protects against LPS-induced intestinal barrier dysfunction by activating AMPK. These results reveal the potential of metformin as an effective therapy for treating intestinal diseases

miR-133a and miR-128 target the 3′ UTR of Prdm16 in HEK293 cells.

<p>(A–B) qPCR analysis of miR-1, miR-206, miR-133a, miR-128 and Prdm16, for asWAT and ingWAT of wildtype mice. (C) Putative miRNA target sites within the 3 ′UTR of <i>Prdm16</i>. (D–E) Luciferase assay. Plasmids carrying luciferase gene linked to 3′ UTR of <i>Prdm16</i> were cotransfected to HEK293 cells, along with control miRNA, miR-133a (D) or miR-128 (E) at indicated doses. Luciferase activity was measure at 48h post-transfection and normalized. N = 4, *P<0.05, **P<0.01.</p

Knockdown of miR-133a leads to browning of WAT and improves body insulin sensitivity in vivo.

<p>Wildtype (WT; miR-133a1^+/+a2^+/+) and miR-133a knockdown (KO; <i>miR-133a1^−/−a2^+/−</i>) mice were used. (A) Representative image of inguinal WAT isolated from WT and KO mice. (B) Western blot image showing relative expression of UCP1 and β-actin in WT and KO ingWAT. (C–D) H&E staining of WT (C) and KO (D) ingWAT. (E–F) immunohistostaining of UCP1 of WT (E) and KO (F) ingWAT. UCP1 signal is in brown and cell membrane is counter stained in blue. Scale bar = 50 µm. (G) Blood glucose levels during IP-GTT test (n = 4 pairs of mice). (H) Blood glucose levels after IP insulin injection, normalized to initial glucose measurement as 100% (n = 6 pairs of mice). *P<0.05, **P<0.01.</p

Mutation of miR-133a predispose white preadipocytes to become adaptive beige adipocytes upon differentiation.

<p>qPCR analysis of relative gene expression in inguinal WAT tissue (n = 3 pairs) and cultured WAT adipocytes from SVF (n = 4 pairs) of wildtype (WT) and miR-133a knockdown (KO; <i>miR-133a1^−/−a2^+/−</i>) mice. (A) miR-133a in ingWAT, (B) BAT marker genes in ingWAT, (C) beige adipocyte marker genes, <i>CD137</i>, <i>Tmem26</i> and <i>Tbx1</i>, in ingWAT. (D) <i>Ucp1</i> and (E) other BAT marker gene expression in adipocytes differentiated from stromal vascular cells of ingWAT. *P<0.05, **P<0.01.</p

Genetic ablation of <i>miR-133a</i> promotes the browning and thermogenic gene program in SAT but not BAT.

<p>miR-133a has two alleles, miR-133a1 and miR-133a2. The miR-133a-dKO mice (miR-133a1^−/−; miR-133a2^−/−) were obtained by intercrossing mice with the genotype of miR-133a1^−/−; miR-133a2^+/−. The mice (miR-133a1^−/−; miR-133a2^−/−) were used as a control for the qPCR analysis of BAT, asWAT and ingWAT. (A) miR-133a levels in BAT, (B) brown marker expression in BAT, (C) thermogenic gene program in BAT. (D) miR-133a levels in asWAT, (E) brown marker expression in asWAT, (F) thermogenic gene program in asWAT. (G) miR-133a levels in ingWAT, (H) brown marker expression in ingWAT, (I) thermogenic gene program in ingWAT. The brown markers include <i>Prdm16</i>, <i>Ucp1</i> and <i>Cidea</i>; the thermogenic genes include <i>Cox8b</i>, <i>Hsl</i>, <i>Atgl</i> and <i>Cpt2</i>. N = 3, *P<0.05, **P<0.01.</p

Downregulation of miR-133a with upregulation of <i>Prdm16</i> along brown adipocyte commitment and differentiation.

<p>(A) In aP2-Cre/mTmG mouse model, aP2 derived cells show green fluorescence (mG⁺) and non-aP2 derived cells show red fluorescence (mT⁺). The stromal-vascular fraction (SVF) of BAT was sorted based on fluorescence and the freshly sorted cells were used for qPCR analysis of (B) myogenic markers, (C) adipogenic markers and (D) adipogenic miRNAs. (E) Strategy for isolating adipose progenitor cells (APC) by FACS. (F) Relative expression of miR-113a, miR-206 and miR-128 in freshly sorted APC and mature adipocytes collected from the floating fraction of collagenase digested BAT. (G–K) Relative expression of BAT related genes in freshly sorted APC and mature adipocytes. N = 3–5, *P<0.05, **P<0.01.</p

Knockdown of miR-133a promotes the activity of cold-inducible thermogenesis gene program in vivo.

<p>qPCR analysis of relative expression of genes in inguinal WAT tissue after 5 day exposure of wildtype (WT) and miR-133a knockdown (KO; <i>miR-133a1^−/−a2^+/−</i>) mice at 4°C. (A) miR-133a, (B) <i>Ucp1</i>, (C) other brown adipocyte markers, <i>Prdm16, Pgc1α, Pparγ2, Cidea</i>, (D) mitochondria genes <i>Cox8b</i> and <i>Cpt2</i>, and lipolysis genes <i>Hsl</i> and <i>Atgl</i>. N = 4–6, *P<0.05, **P<0.01.</p

miR-133a inhibits brown adipocyte biogenesis of BAT progenitors.

<p>(A) Strategies for electroporation of miRNAs or LNAs to cultured BAT APCs, followed by induction and differentiation for 4 days. (B–D) qPCR analysis of miR-133a and the brown markers in the control and miR-133 overexpressed BAT adipocytes. (E–F) qPCR analyses of Prdm16 and BAT specific genes in adipocytes overexpressing control miRNA, miR-133a with control retrovirus and miR-133a with Prdm16-overexpressing retrovirus. (G) qPCR analysis of BAT marker gene expression after miR-133a was inhibited by LNAs. N = 3, *P<0.05, **P<0.01, ***P<0.001.</p