Gut microbe-derived extracellular vesicles induce insulin resistance, thereby impairing glucose metabolism in skeletal muscle

Youngwoo Choi, Yonghoon Kwon, Dae-Kyum Kim, Jinseong Jeon, Su Chul Jang, Taejun Wang, Minjee Ban, Min-Hye Kim, Seong Gyu Jeon, Min-Sun Kim, Cheol Soo Choi, Young-Koo Jee, Yong Song Gho, Sung Ho Ryu, Yoon-Keun Kim, Youngwoo Choi, Yonghoon Kwon, Dae-Kyum Kim, Jinseong Jeon, Su Chul Jang, Taejun Wang, Minjee Ban, Min-Hye Kim, Seong Gyu Jeon, Min-Sun Kim, Cheol Soo Choi, Young-Koo Jee, Yong Song Gho, Sung Ho Ryu, Yoon-Keun Kim

Abstract

Gut microbes might influence host metabolic homeostasis and contribute to the pathogenesis of type 2 diabetes (T2D), which is characterized by insulin resistance. Bacteria-derived extracellular vesicles (EVs) have been suggested to be important in the pathogenesis of diseases once believed to be non-infectious. Here, we hypothesize that gut microbe-derived EVs are important in the pathogenesis of T2D. In vivo administration of stool EVs from high fat diet (HFD)-fed mice induced insulin resistance and glucose intolerance compared to regular diet (RD)-fed mice. Metagenomic profiling of stool EVs by 16S ribosomal DNA sequencing revealed an increased amount of EVs derived from Pseudomonas panacis (phylum Proteobacteria) in HFD mice compared to RD mice. Interestingly, P. panacis EVs blocked the insulin signaling pathway in both skeletal muscle and adipose tissue. Moreover, isolated P. panacis EVs induced typical diabetic phenotypes, such as glucose intolerance after glucose administration or systemic insulin injection. Thus, gut microbe-derived EVs might be key players in the development of insulin resistance and impairment of glucose metabolism promoted by HFD.

Figures

Figure 1. Insulin resistance and glucose intolerance…
Figure 1. Insulin resistance and glucose intolerance are induced by stool EVs isolated from HFD-fed mice.
(A) An experimental protocol for isolating stool EVs in HFD- and RD-fed mice. (B) Transmission electron microscopic (TEM) images of stool EVs (hEV vs. rEV). (C) Western blot data of insulin signaling molecules in L6 myotubes after treating stool EVs (hEV or rEV), with or without insulin (Ins). (D) 2-Deoxy[14C]glucose uptake in L6 myotubes in response to stool EVs (hEV or rEV), with or without Ins treatment. *P < 0.05. (E) GLUT4 translocation to the membrane of L6 myotubes in response to stool EVs (hEV or rEV), with or without Ins. *P < 0.05. (F) Change of mouse body weight at the indicated day after the oral administration of stool EVs (hEV or rEV) (n = 8 mice per group). (G) Phosphorylation of IRS1 and AKT in skeletal muscle from hEV- or rEV-administered mice. *P < 0.05.
Figure 2. Stool metagenomic analysis indicates that…
Figure 2. Stool metagenomic analysis indicates that HFD increases the composition of EVs derived from phylum Proteobacteria, including Pseudomonas panacis.
For all figures, stools were isolated from mice after 12 weeks of HFD or RD. (A) Relative abundance (% of total 16S rDNA gene sequences) of gut microbes and gut microbe-derived EVs at the phylum level. (B) Principal component analysis (PCA) of bacteria and stool EVs. Note the change of EVs composition is greater than that of bacterial composition, since EVs composition is separated with principal component 1. (C) Four filters were used to select the most essential species, in which EVs were significantly changed after HFD, and then P. cedrina and P. panacis were selected. (D) Heatmap plot of stool bacteria and bacterial EVs at the species level. Note that the only species occupying more than 1% in at least one sample were included.
Figure 3. Characterization of EVs derived from…
Figure 3. Characterization of EVs derived from P. cedrina and P. panacis.
(A) Transmission electron microscopy (TEM) image of P. cedrina EVs and P. panacis EVs. (B) Size of P. cedrina EVs and P. panacis EVs. (C) SDS-PAGE analysis of P. cedrina whole cell lysate (WCL), P. cedrina EVs, P. panacis whole cell lysate (WCL), and P. panacis EVs. (D) Western blot data (left panel) and ELISA assay (right panel) of lipid A in P. cedrina EVs and P. panacis EVs.
Figure 4. P. panacis EVs interfere with…
Figure 4. P. panacis EVs interfere with insulin signaling in both myotubes and adipocytes, and impair glucose uptake in myotubes.
(A) Immunoblot analysis of the insulin signaling molecules in L6 myotubes after the treatment of LPS, P. cedrina EVs, or P. panacis EVs, with or without insulin (Ins). (B) Werstern blot data of the insulin signaling molecules in 3T3-L1 adipocytes after the application of LPS, P. cedrina EVs, or P. panacis EVs, with or without Ins. *P < 0.05 vs. P. cedrina EVs. (C) 2-Deoxy[14C]glucose uptake in L6 myotubes in response to LPS, P. cedrina EVs, or P. panacis EVs, with or without Ins. *P < 0.05. (D) GLUT4 translocation to the membrane of L6 myotubes after the treatment of LPS, P. cedrina EVs, or P. panacis EVs, with or without Ins. *P < 0.05.
Figure 5. P. panacis EVs induce insulin…
Figure 5. P. panacis EVs induce insulin resistance and diabetic phenotypes in RD-fed mice.
For all figures, LPS, P. cedrina EVs, and P, panacis EVs were administered orally to mice once every 2 days for 4 weeks. All mice were fed with RD. (A) Phosphorylation of IRS1 and AKT in skeletal muscle after the last injection. *P < 0.05. (B) Immunoblot analysis of insulin signaling molecules in the adipose tissue after the last application. *P < 0.05. (C) Glucose tolerance test (GTT). GTT was performed in mice 12 h fasting after the last application (n = 4 mice per group). *P < 0.05 vs. the other groups. (D) Insulin tolerance test (ITT). ITT was performed in mice 6 h fasting after the last application (n = 5 mice per group). *P < 0.05 vs. the other groups.
Figure 6. Absorption and distribution of P.…
Figure 6. Absorption and distribution of P. panacis bacteria and P. panacis EVs after oral administration.
(A) In vivo fluorescent whole body image of P. panacis bacteria and P. panacis EVs trafficking in C57BL/6J mice before and after the administration by gavage after overnight fasting (upper panel). Lower panel indicates the images of insulin-sensitive organs (liver, adipose tissue, and skeletal muscle), which were extracted from mice 12 h after the oral administration. *P < 0.05, **P < 0.01. (B) Immunohistochemistry using P. panacis EVs-reactive polyclonal antibodies. Images were taken from the large intestine 10 minutes after the surgical injection of P. panacis bacteria and P. panacis EVs. P. panacis EVs-reactive antibodies (green dots indicated by white arrows) are seen in the lamina propria (LP) of the large intestine. Lu: intestinal lumen. (C) In vivo two-photon image of the large intestine before and 10 minutes after the luminal administration of P. panacis EVs. EVs (green dots indicated by white arrows) are observed inside the blood vessels in the intestinal lamina propria. Scale bar = 25 μm. (D) Immunohistochemistry using P. panacis EVs-reactive polyclonal antibodies. P. panacis EVs-reactive antibodies (green dots indicated by white arrows) are observed in liver, adipose tissue, and skeletal muscle, extracted from mice 12 h after the oral administration of P. panacis EVs. Scale bar = 25 μm.

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