Volume 25, Issue 5, Pages 1075-1090.e5 (May 2017) A Specific Gut Microbiota Dysbiosis of Type 2 Diabetic Mice Induces GLP-1 Resistance through an Enteric NO-Dependent and Gut-Brain Axis Mechanism  Estelle Grasset, Anthony Puel, Julie Charpentier, Xavier Collet, Jeffrey E. Christensen, François Tercé, Rémy Burcelin  Cell Metabolism  Volume 25, Issue 5, Pages 1075-1090.e5 (May 2017) DOI: 10.1016/j.cmet.2017.04.013 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2017 25, 1075-1090.e5DOI: (10.1016/j.cmet.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Animal Models of High-Fat-Diet-Induced GLP-1 Resistance Mice were fed for 3 months with NCD, HC-HFD, or HFD. (A) I.p. glucose tolerance test (1 g/kg) (n = 6, NCD; n = 36, HC-HFD; n = 24, HFD). (B) i.p. insulin tolerance test (0.75 U/kg). Values are expressed in percentage of initial glycemia (n = 10/group). (C) Weight gain (n = 6, NCD; n = 36, HC-HFD; n = 36, HFD). (D) Plasma insulin concentration (n = 6/group). (E) Plasma portal GLP-1 concentration (n = 6, NCD; n = 24, HC-HFD; n = 24, HFD). (F) Glycemia (n = 6/group) 15 min after an oral glucose challenge (2 g/kg). (G) Plasma insulin fold change (in percentage of vehicle-injected mice) after GLP-1 injections (x axis represented as log of 0, 2.2, 7, 21, 63, and 189 nmol/kg) (n = 5–12/group). (H) Half-maximal effective concentration of GLP-1 (EC50). Values with similar superscript letters are not different, p > 0.05. Lowercase letters are used to compare individual values and capital letters are used to compare curves. The values are compared between NCD and HC-HFD, and HFD-fed mice. Cell Metabolism 2017 25, 1075-1090.e5DOI: (10.1016/j.cmet.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 High-Fat Diet Alters the Enteric Nervous System (A and B) Mice were fed for 3 months with NCD, HC-HFD, or HFD or treated with cisplatin (weekly i.p. injection, during 1 month). (A1) longitudinal muscular myenteric plexus (LMMP) immunohistochemistry; (A2) quantification of the number of HuC/HuD-positive cells, percentage of PGP9.5 and Prph fluorescent area (as neuronal marker) and percentage of S100β fluorescent area (as glial marker) (n = 5–6/group); (B) ileum mRNA concentrations of neuronal markers (PGP9.5 and peripherin-prph) and glial markers (GFAP, S100β) (n = 6, NCD; n = 10, HC-HFD; n = 10, HFD; n = 5, cisplatin-treated mice). Values with similar superscript letter are not different, p > 0.05. The values are compared between NCD and HC-HFD, HFD-fed mice and cisplatin-treated mice. Cell Metabolism 2017 25, 1075-1090.e5DOI: (10.1016/j.cmet.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 High-Fat Diet Alters the Gut-Brain Axis and Induces GLP-1 Resistance (A) In (A1), immunohistochemistry of cFos-positive cells; (A2) quantification of the number of cFos-positive cells per dotted area depicting the nucleus of the tractus solitarius (NTS) and the dorsal motor vagal nucleus (DMNx) in NCD, HC-HFD, and HFD-fed mice stimulated with vehicle or with GLP-1 (7 nmol/kg) (n = 3–5/group). Values with similar superscript letter are not different, p > 0.05. The values are compared between vehicle-injected mice and GLP-1-injected mice. (B) Plasma insulin fold change (in percentage of vehicle-injected mice) after GLP-1 injections (x axis represented as log of 0, 2.2, 7, and 21 nmol/kg) in sham-operated or subdiaphragmatic vagotomy (SDVx)-operated mice (n = 5/group). Values with similar superscript letter are not different, p > 0.05. The values are compared between Sham and SDVx-operated mice. (C) Plasma insulin fold change (in percentage of vehicle-injected mice) after GLP-1 stimulation (7 nmol/kg) in cisplatin- or vehicle-treated mice (weekly i.p. injection, during 1 month, n = 6/group). (D) Plasma insulin fold change (in percentage of vehicle-injected mice) after i.p. and i.v. GLP-1 injections (0, 2.2, 7, and 21 nmol/kg) (n = 3–8/s). Values with similar superscript letter are not different, p > 0.05. The values are compared between vehicle-injected mice and GLP-1-injected mice. Cell Metabolism 2017 25, 1075-1090.e5DOI: (10.1016/j.cmet.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Enteric GLP-1 Sensitivity Requires the Production of NO by Enteric Neurons that Is Impaired in HFD-Fed Mice (A–E) Mice were fed for 3 months with NCD, HC-HFD, or HFD. Ileum mRNA concentrations of (A) GLP-1r and (B) nNOS (n = 15, NCD; n = 20, HC-HFD; n = 25, HFD); (C) Pearson correlation analyses between GLP-1r and nNOS mRNA concentrations in ileum. Values with similar superscript letter are not different, p > 0.05. The values are compared between NCD and HC-HFD-fed, and HFD-fed mice. (D–H) Plasma insulin fold change (percentage of vehicle-injected mice); (D) after GLP-1 (0 and 7 nmol/kg) and nNOS inhibitor injections (L-NAME, 0, 50, 100, and 200 μg/kg) in NCD mice (n = 3–5/group), (E) after L-arginine (0 and 1 g/kg) and Exendin 9 (0 and 1 nmol/mouse) in NCD (n = 5/group), (F) in SDVx-operated mice (n = 5/group), and (G) in HFD-fed mice (n = 5/group), (H) after chronic treatment with L-Arg (0 and 0.5 g/kg/day, during 1 week) and GLP-1 injection (7 nmol/kg) (n = 3/group). Values with similar superscript letter are not different, p > 0.05. The values are compared between all groups. (I) GLP-1-induced NO production by enteric neurons from NCD and HFD mice expressed as percentage of NO from control cells (cells without GLP-1 incubation) (n = 6–18 cells/group). Values with similar superscript letter are not different, p > 0.05. Lowercase letters are used to compare individual values, and capital letters are used to compare curves. The values are compared between neurons from NCD- and HFD-fed mice. Cell Metabolism 2017 25, 1075-1090.e5DOI: (10.1016/j.cmet.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 High-Fat Diet Induces Gut Microbiota Dysbiosis and Modifies the Gut Microbiota Function Mice were fed for 3 months with NCD, HC-HFD, or HFD. (A–C) LDA effect size (LEfSe) cladograms of taxonomic data from 16S rDNA sequence analysis of ileum samples. Pairwise analysis was performed for dietary groups (A) NCD versus HC-HFD, (B) NCD versus HFD, and (C) HC-HFD versus HFD. The cladograms show the taxonomic levels represented by rings with phyla at the innermost ring and genera (identified in the legend) at the outermost ring, and each circle is a member within that level. Taxa at each level are shaded (green, blue, and orange) according to the dietary group in which it is most abundant (p < 0.05; LDA score 2.0). The respective taxonomic cladogram legends are incorporated into the adjacent graphical representation of the percent relative abundance for each genus level differential feature. (D–F) LDA effect size (LEfSe) cladograms of KEGG pathway contributions of predicted metagenomic data for ileum samples. Pairwise analysis was performed for dietary groups (D) NCD versus HC-HFD, (E) NCD versus HFD, and (F) HC-HFD versus HFD. The cladograms show the KEGG pathway hierarchy represented by rings with the consolidated pathway modules (identified in the legend) at the outermost ring, and each circle is a member within that level. KEGG modules are shaded (green, blue, and orange) according to the dietary group in which it is most abundant (p < 0.05; LDA score 2.0). The respective KEGG pathway cladogram legends are incorporated into the adjacent graphical representation of the relative predicted gene count for each differential feature. Cell Metabolism 2017 25, 1075-1090.e5DOI: (10.1016/j.cmet.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Gut Microbiota Dysbiosis Is Responsible for the GLP-1 Resistance (A–D) Germ-free (GF) mice and conventionalized mice with ileum flora from NCD-fed mice (Conv[NCD]) and from HFD-fed mice (Conv[HFD]). (A) Plasma insulin fold change (percentage of vehicle-injected mice) after GLP-1 injection (0 and 7 nmol/kg) (n = 3–8/group), (B) ileum mRNA concentrations of neuronal markers (PGP9.5 and peripherin-prph) and glial markers (GFAP, S100β) (n = 5–15/group); and (C) GLP-1r and (D) nNOS mRNA concentrations in the ileum (n = 5–15/group). Values with similar superscript letter are not different, p > 0.05. The values are compared between GLP-1-untreated mice and GLP-1-treated mice or between control (Ctrl), GF, and all conventionalized mice. (E–H) NCD, HC-HFD, or HFD mice were treated 1 month with antibiotics (Abx); (E) plasma insulin fold change (percentage of vehicle-injected mice) after GLP-1 injection (0 and 7 nmol/kg) (n = 3–8/group); (F) ileum mRNA concentrations of neuronal markers (PGP9.5 and peripherin-prph) and glial markers (GFAP, S100β) (n = 5–10/group); (G) GLP-1r and (H) nNOS mRNA concentrations in the ileum (n = 5–10/group). The values are compared between GLP-1-untreated mice and GLP-1-treated mice or between antibiotic-untreated mice and antibiotic-treated mice. Cell Metabolism 2017 25, 1075-1090.e5DOI: (10.1016/j.cmet.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 The Microbial-Associated Molecular Pattern Receptors NOD2, CD14, and TLR4 Control GLP-1 Sensitivity NOD2, TLR4, and CD14 KO mice were compared to wild-type (WT) mice. (A) Plasma insulin fold change (percentage of vehicle-injected mice) after GLP-1 injection (7 nmol/kg) (n = 3–5/group); (B) ileum mRNA concentrations of neuronal markers (PGP9.5 and peripherin-prph) and glial markers (GFAP, S100β) (n = 5–12/group); (C) GLP-1r and (D) nNOS mRNA concentrations in the ileum (n = 5–12/group). Values with similar superscript letter are not different, p > 0.05. The values are compared between WT and KO mice. GLP-1-induced NO production by enteric neurons from WT mice after incubation with or without (E) MDP (muramyldipeptide) (2 and 20 ng/mL) or (F) LPS (lipopolysaccharide) (10 and 100 μg/mL) and expressed as percentage of NO from control cells (cells without GLP-1, LPS, and MDP incubation) (n = 10–20 cells/group). Values with similar superscript letter are not different, p > 0.05. The values are compared between all conditions. Cell Metabolism 2017 25, 1075-1090.e5DOI: (10.1016/j.cmet.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions