Volume 23, Issue 2, Pages 324-334 (February 2016) Gut Commensal E. coli Proteins Activate Host Satiety Pathways following Nutrient- Induced Bacterial Growth  Jonathan Breton, Naouel Tennoune, Nicolas Lucas, Marie Francois, Romain Legrand, Justine Jacquemot, Alexis Goichon, Charlène Guérin, Johann Peltier, Martine Pestel-Caron, Philippe Chan, David Vaudry, Jean-Claude do Rego, Fabienne Liénard, Luc Pénicaud, Xavier Fioramonti, Ivor S. Ebenezer, Tomas Hökfelt, Pierre Déchelotte, Sergueï O. Fetissov  Cell Metabolism  Volume 23, Issue 2, Pages 324-334 (February 2016) DOI: 10.1016/j.cmet.2015.10.017 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Metabolism 2016 23, 324-334DOI: (10.1016/j.cmet.2015.10.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Effects of Repeated Nutrient Supply on E. coli Growth In Vitro and Proteomic Analysis (A and B) Dynamics of E. coli K12 bacterial growth during nine regular provisions of MH medium. (C–E) Proteins were extracted during the last growth cycle as indicated by arrows during the Exp phase (Exp, a) and the Stat phase (Stat, b). Representative images of Coomassie brilliant blue-stained 2D gels of cytoplasmic proteins extracted from E. coli K12 in Exp (D) and in Stat (E) phases; MW, molecular weight. (F) Number of E. coli proteins increased in each growth phase in relation to their catabolic or anabolic properties. (G) Effects of bacterial proteins on ATP production in vitro from different food substrates. (H–J) ClpB protein levels ± SEM in two growth phases was analyzed by western blot (H and I) and ELISA (J). OD, optical density. Student’s t test, ∗p < 0.05. Cell Metabolism 2016 23, 324-334DOI: (10.1016/j.cmet.2015.10.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Effects of Intestinal Infusions of Nutrients and E. coli Proteins in Rats (A and B) Bacterial growth dynamics after colonic instillations of MH medium or water, and (B) plasma ClpB before and during instillations. (C) Detection of ClpB DNA in rat feces and its correlation with plasma ClpB levels before instillations. (D–G) Effects of colonic infusion of E. coli proteins from Exp and Stat growth phases on concentrations of ClpB in colonic mucosa (D) and plasma (E), and plasma concentrations of GLP-1 (F) and PYY (G), before (T0) and 20 min after infusions (T20). (D) Student’s t test, ∗p < 0.05; (F and G), ANOVA, p = 0.02 and p = 0.0004, respectively, Tukey’s post-test ∗p < 0.05 and ∗∗∗p < 0.001. All data are shown as mean ± SEM. Cell Metabolism 2016 23, 324-334DOI: (10.1016/j.cmet.2015.10.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Food Intake after Acute Injections of E. coli Proteins in Rats (A and B) Food intake after i.p. injections of membrane (A) and cytoplasmic (B) fractions of E. coli proteins from Exp and Stat growth phases or PBS as a control (Contr) in rats during refeeding after overnight food restriction. (C) Two hour food intake in free-feeding rats injected with total E. coli proteins before the onset of the dark phase. (A) ANOVA p = 0.04, Tukey’s post-test Contr. versus Stat., ∗p < 0.05, Student’s t test Contr. versus Stat. $p = 0.01. (B) Student’s t tests, Exp. versus Contr. and versus Stat. $p < 0.05. (C) ANOVA p = 0.05, Tukey’s post-test Contr. versus Stat., ∗p < 0.05 and Student’s t test Exp. versus Stat. $p = 0.04. All data are shown as mean ± SEM. Cell Metabolism 2016 23, 324-334DOI: (10.1016/j.cmet.2015.10.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Effects of E. coli Proteins on c-Fos Expression in the Brain Immunohistochemical detection of c-Fos (green) in the ARC (A–C), VMN (G–I), and CeA (K–N) 2 hr after i.p. injections of Exp. and Stat. E. coli proteins in rats. Double staining with β-endorphin (β-end, red) in the ARC (A–C) and with CGRP in the CeA (red, K–N) including a confocal image (N). c-Fos-positive cell number in the ARC (D), VMN (J), and CeA (O). Percentage of β-end activated cells in the ARC (E) and their correlation with food intake (F). Correlation between number of c-Fos cells in the CeA and food intake (P). (D and J) ANOVA p < 0.05, Tukey’s post-test, ∗p < 0.05. (E) ANOVA, p = 0.006, Tukey’s post-tests ∗p < 0.05 and ∗∗p < 0.01. (O) ANOVA, p = 0.0003, Tukey’s post-tests, ∗∗p < 0.01 and ∗∗∗p < 0.001. All data are shown as mean ± SEM. Cell Metabolism 2016 23, 324-334DOI: (10.1016/j.cmet.2015.10.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Effects of Chronic Injections of E. coli Proteins in Mice (A and B) Effects of bidiurnal injections during 6 days, of E. coli proteins from Exp and Stat growth phases or PBS as a control (Contr) in mice on body weight (A), food intake (B), meal size (C), and meal number (D). Total mean food intake was also analyzed separately in light (E) and dark (F) periods. The satiety ratio, expressed as postmeal interval (s) × 1,000 versus food (g) consumed in the preceding meal, at day 1 (G) and day 6 (H). (A and B) Two-way RM ANOVA, Bonferroni post-tests Contr. versus Stat., ∗∗∗p < 0.001 and ∗∗p < 0.01. (C) Student’s t test Contr. versus Stat. #p < 0.05. (E and F) K-W test p < 0.002, Dunn’s post-tests, ∗∗∗p < 0.001 and M-W test #p < 0.05. (G) ANOVA p = 0.005, Tukey’s post-test, ∗∗p < 0.01. (H) Student’s t test, $p < 0.05. All data are shown as mean ± SEM. Cell Metabolism 2016 23, 324-334DOI: (10.1016/j.cmet.2015.10.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Effect of ClpB on Electrical Activity of ARC POMC Neurons POMC-eGFP neuron (arrow) visualized with 60× objective under infrared (A) and fluorescent light (B) during patch-clamp recording. Action potential frequency expressed in percent of change versus basal level, mean ± SEM (C). Representative cell-attached recording of a POMC-eGFP neuron activated by ClpB (1 nM), thick black bar above the trace. Washout, 10 min. RM ANOVA p < 0.05, Bonferroni post-tests ∗p < 0.05,∗∗p < 0.01. Cell Metabolism 2016 23, 324-334DOI: (10.1016/j.cmet.2015.10.017) Copyright © 2016 Elsevier Inc. Terms and Conditions