A Specific Gut Microbiota Dysbiosis of Type 2 Diabetic Mice Induces GLP-1 Resistance through an Enteric NO-Dependent and Gut-Brain Axis Mechanism

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1 Article A Specific Gut Microiot Dysiosis of Type 2 Dietic Mice Induces GLP-1 Resistnce through n Enteric NO-Dependent nd Gut-Brin Axis Mechnism Grphicl Astrct Authors Estelle Grsset, Anthony Puel, Julie Chrpentier, Xvier Collet, Jeffrey E. Christensen, Frnçois Tercé, Rémy Burcelin Correspondence remy.urcelin@inserm.fr In Brief Grsset et l. ddress the issue of GLP-1 resistnce in dietes therpy. They identify set of ileum cteri, ssocited with pthwys involved in mino cid metolism nd trnsport, which counterct GLP-1-induced nitric oxide production y enteric neurons involved in mediting insulin secretion nd gstric emptying. Highlights d Enteric GLP-1 ctivtes NO production y enteric neurons tht is impired in T2D d d d Gut microiot dysiosis induces enteric neuropthy Gut microiot dysiosis is responsile for the GLP-1 resistnce A specific gut microiot rchitecture suggested excessive mino cid metolism Grsset et l., 217, Cell Metolism 25, My 2, 217 ª 217 Elsevier Inc.

2 Cell Metolism Article A Specific Gut Microiot Dysiosis of Type 2 Dietic Mice Induces GLP-1 Resistnce through n Enteric NO-Dependent nd Gut-Brin Axis Mechnism Estelle Grsset, 1,2 Anthony Puel, 1,2 Julie Chrpentier, 1,2 Xvier Collet, 1,2 Jeffrey E. Christensen, 1,2 Frnçois Tercé, 1,2 nd Rémy Burcelin 1,2,3, * 1 Institut Ntionl de l Snté et de l Recherche Médicle (INSERM), 3124 Toulouse, Frnce 2 Université Pul Stier (UPS), Unité Mixte de Recherche (UMR) 148, Institut des Mldies Métoliques et Crdiovsculires (I2MC), Tem 2: Intestinl Risk Fctors, Dietes, Dyslipidemi, Hert Filure, F Toulouse, Cedex 4, Frnce 3 Led Contct *Correspondence: remy.urcelin@inserm.fr SUMMARY Glucgon-like peptide-1 (GLP-1)-sed therpies control glycemi in type 2 dietic (T2D) ptients. However, in some ptients the tretment must e discontinued, defining stte of GLP-1 resistnce. In niml models we identified specific set of ileum cteri impiring the GLP-1-ctivted gutrin xis for the control of insulin secretion nd gstric emptying. Using prediction lgorithms, we identified cteril pthwys relted to mino cid metolism nd trnsport system modules ssocited to GLP-1 resistnce. The conventionliztion of germ-free mice demonstrted their role in enteric neuron iology nd the gut-rin-periphery xis. Altogether, insulin secretion nd gstric emptying require functionl GLP-1 receptor nd neuronl nitric oxide synthse in the enteric nervous system within euiotic gut microiot environment. Our dt open novel route to improve GLP-1-sed therpies. INTRODUCTION The glucgon-like peptide-1 (GLP-1) is n insulinotropic hormone secreted y enteroendocrine L cells in response to mel nd plys key role in glucose control. It regultes insulin nd glucgon secretions, gstric emptying, food intke, nd lood flow (Holst, 27). Through the prcrine mode of ction, GLP-1 inds loclly to its receptor (GLP-1r), expressed y the enteric nervous system (ENS) nd the vgus nerve (Richrds et l., 214) tht ctivtes the gut-rin-periphery xis. The enteric hormonl signl is trnsmitted to peripherl tissues tht engge the ove physiologicl functions (Burcelin et l., 29). Through second endocrine mode of ction, GLP-1 triggers distnt cells, notly the cells from the Lngerhns islets (Drucker et l., 1987; Frill et l., 23; Richrds et l., 214). For oth neurons nd cells, G proteins, denylte cyclse, camp, EPAC, PKA, CREB, PKC, Akt, ERK, nitric oxide (NO), nd NO synthse (NOS) re the min signling molecules responsile for GLP-1 intrcellulr ction (Cou et l., 211; Ding nd Zhng, 212; Drucker et l., 1987; Pujds et l., 215; Quoyer et l., 21). Bsed on these importnt fetures, the ntive GLP-1 hs een the sis for the tretment of type 2 dietes (T2D) in which two strtegies hve een designed to overcome the short hlf-life of the ctive peptide GLP rpidly degrded y the uiquitously distriuted dipeptidyl peptidse 4 (DPP4) (Hnsen et l., 1999). They involve GLP-1r gonists resistnt to the DPP4 nd DPP4 inhiitors (Dlle et l., 213). Although this process efficiently reduces HA1c y.8% 1.2% over period of 3 6 months, it is currently oserved in clinicl prctice tht not ll T2D ptients rech therpeutic trget in response to GLP-1-sed therpies, i.e., to HA1c lower thn 6.5%. Furthermore, some of them, due to lck of efficcy, even hve to discontinue their tretment, defining stte of GLP-1 resistnce (Knop et l., 212; Muscelli et l., 28). The mechnisms responsile for GLP-1 unresponsiveness could e relted to lipo-glucotoxicity (Duc et l., 213; Hodson et l., 213; Ten Kulve et l., 216; Xu et l., 27), utonomic neuropthy (Lee et l., 212; Loinet et l., 215), nd gut microiot dysiosis (B ckhed et l., 24; Gill et l., 26; Ley et l., 25). Regrding the ltter hypothesis, we nd others notly demonstrted the role plyed y the proinflmmtory cteril determinnt lipopolyscchrides (LPSs) nd peptidoglycns on inflmmtion-induced insulin resistnce (Amr et l., 211; Cni et l., 27; Denou et l., 215; Schertzer et l., 211). Therefore, we explored the role plyed y gut microiot on the control of GLP-1 ction. We generted two mouse models of T2D chrcterized y different signtures of gut microiot dysiosis nd different extents of GLP-1 responsiveness. We then quntified the ction of GLP-1 on gut-rin-regulted insulin secretion, gstric emptying, nd food intke. We identified tht specific set of ileum cteri, ssocited with pthwys involved in mino cid metolism nd trnsport system modules, induced GLP-1 resistnce, ltering the function of the GLP-1r nd the NO production y the neurons from the ENS. RESULTS Animl Models of High-Ft Diet-Induced GLP-1 Resistnce To identify whether gut microiot regultes GLP-1 responsiveness in vivo, we hve set up two mouse models with different Cell Metolism 25, , My 2, 217 ª 217 Elsevier Inc. 175

3 A B C Glycemi (mg/dl) Intrperitonel glucose tolernce test HC- 5 c c 25 c A c B c C Time (min) Plsm portl GLP-1 (pm) % of initil glycemi Intrperitonel insulin tolernce test HC A c 5 B C , HC- Time (min) D E F Plsm insulin (ng/ml) c HC- Glucose orl stimultion Glycemi (mg/dl) Weight gin (g) HC- HC- Figure 1. Animl Models of High-Ft-Diet- Induced GLP-1 Resistnce Mice were fed for 3 months with, HC-, or. (A) I.p. glucose tolernce test (1 g/kg) (n = 6, ; n = 36, HC-; n = 24, ). (B) i.p. insulin tolernce test (.75 U/kg). Vlues re expressed in percentge of initil glycemi (n = 1/group). (C) Weight gin (n = 6, ; n = 36, HC-; n = 36, ). (D) Plsm insulin concentrtion (n = 6/group). (E) Plsm portl GLP-1 concentrtion (n = 6, ; n = 24, HC-; n = 24, ). (F) Glycemi (n = 6/group) 15 min fter n orl glucose chllenge (2 g/kg). (G) Plsm insulin fold chnge (in percentge of vehicle-injected mice) fter GLP-1 injections (x xis represented s log of, 2.2, 7, 21, 63, nd 189 nmol/kg) (n = 5 12/group). (H) Hlf-mximl effective concentrtion of GLP-1 (EC 5 ). Vlues with similr superscript letters re not different, p >.5. Lowercse letters re used to compre individul vlues nd cpitl letters re used to compre curves. The vlues re compred etween nd HC-, nd -fed mice. G Plsm insulin fold chnge HC Ctrl log [GLP-1], nmol/kg gut microiot dysiosis (Everrd et l., 214; Gridou et l., 215; Serino et l., 212). A first set of mice fed high-crohydrte high-ft diet (HC-) is oese nd dietic (Figures 1A 1C nd S2A S2C). The second one, fed high-ft ut crohydrte-free diet (), is dietic ut remined mostly len. Following the phenotyping of the mouse models nd to study the impct of GLP-1 on insulin secretion, we dministered glucose orlly, since it is y fr more efficient thn when dministered i.p. We oserved tht, t the 15 min time point fter n orl glucose chllenge, plsm insulin concentrtion ws twice s high in HC--fed oese dietic mice, while it ws lower in -fed len dietic mice when compred with -fed mice (Figure 1D). Importntly, t the sme time point portl vein plsm GLP-1 concentrtion ws three to four times higher in oth dietic models when compred to -fed mice (Figure 1E), while the glycemi were lmost similr (Figures 1F nd S2D), suggesting different GLP-1 responsiveness. To esti- A B C Increse of insulin concentrtion induced y GLP-1 Bsl stte of insulin concentrtion 176 Cell Metolism 25, , My 2, 217 H Clculted from dose-response curve EC5 (nmol/kg) HC- mte GLP-1 responsiveness precisely, we nlyzed the impct on glucoseinduced insulin secretion using incresing doses of GLP-1. To void the confounding effect of endogenously relesed GLP-1, we dministered glucose i.v. while GLP-1 ws injected i.p., which llows similr glycemic profiles etween groups (Figure S1A). The dt show tht the mximl fold chnge of insulin secretion in nd HC--fed mice ws otined t the dose of 7 nmol/kg of GLP-1, while doses ove 21 nmol/kg were required for the -fed mice (Figures 1G nd S2E). We quntified the concentrtion of portl vein GLP-1 following GLP-1 injections nd found tht doses rnging from 2 to 21 nmol/kg induced plsm GLP-1 concentrtion within the physiologicl rnge of 5 12 pm, which is similr to wht is oserved following mel. The clculted EC 5 ws two nd four times higher for the HC-- nd the -fed mice, respectively, thn for the -fed mice (Figure 1H). The mximl effect ws 312, 287, nd 548 times over sl in, HC-, nd, respectively, shifting the dose-response curve to the right nd therefore witnessing stte of GLP-1 resistnce, s lso shown (Figure S2F). This phrmcologicl nlysis further confirmed stte of resistnce to GLP-1 in oth dietic models, lthough to different extent. We lso determined whether GLP-1 unresponsiveness ffected gstric emptying nd food intke. Some degree of GLP-1 resistnce ws detected in oth dietic models, since the plsm concentrtions of cetminophen remined elevted fter GLP-1 dministrtion (Figures S2G nd S2H). GLP-1

4 resistnce lso ffected the -fed mice, since the mount of food intke during the feeding period (drkness for 6 hr) nd during refeeding ws incresed when compred to -fed mice (Figures S2I nd S2J), while incresing doses of GLP-1 only modestly reduced energy intke in oth -fed mice when compred to -fed mice (Figure S2K). Altogether, this first set of dt demonstrtes tht the -fed mice were GLP-1 resistnt to glucose-induced insulin secretion, gstric emptying, nd food intke, while in the HC--fed mice GLP-1 ction ws only mildly impired. Furthermore, the orl use of DPP4i t dose mostly inhiiting the enteric nd not the systemic enzyme ctivity (Wget et l., 211) suggests tht the loction of GLP-1 resistnce could e restricted to the enteric re. High-Ft Diet Alters the Enteric Nervous System to Brin Axis nd Induces GLP-1 Resistnce To further demonstrte tht the ENS could e trget of the GLP-1 resistnce, we then quntified y immunohistochemistry the numer of HuC/HuD, PGP9.5, nd prph (peripherin)-positive cells, i.e., enteric neurons (Stenkmp-Strhm et l., 213), nd oserved tht the GLP-1-resistnt -fed mice were chrcterized y reduced numer of enteric neurons (Figures 2A1 nd 2A2) defining neuropthy. The numer of S1-positive cells i.e., glil cells, s descried (Kouridis et l., 215), remined norml, showing tht ffects neurons only. To comfort the reduction of the numer of enteric neurons in the fed len dietic mice, we quntified the concentrtions of ileum mrna encoding for neuronl proteins, i.e., PGP9.5, xonl protein; i.e., prph nd glil proteins; nd i.e., GFAP nd S1. PGP9.5 mrna concentrtion ws significntly reduced in oth niml models, while tendency ws oserved regrding the reduction of prph mrna concentrtion (Figure 2B). GFAP nd S1 mrna concentrtions remined unchnged (Figure 2B). The impct of the diet ws restricted to neurons from the ENS, since the mrna concentrtion encoding neuronl nd glil proteins from the nodose gnglion remined similr in ll groups (Figure S3A). To demonstrte tht the gut-rin xis ws functionlly ffected, we then performed functionl iossy. We recorded the numer of cfos-positive cells in the rin stem in response to the dministrtion of GLP-1 into the peritonel cvity t the ctive dose, i.e., 7 nmol/kg. This dose is the lowest one triggering the mximl response to GLP-1-induced insulin secretion in control mice (Figures 1G nd S2E). The hindrin medull includes the nucleus of the trctus solitrius (NTS) where primry viscerl fferents end. It lso includes the dorsl motor nucleus (DMNx) where pregnglionic motor neurons innervting the gstrointestinl trct re locted. The dt show tht the numer of cfos-positive neurons increses in response to GLP-1 in mice, ut not in HC- nd -fed mice (Figures 3A1 nd 3A2), rguing for functionl impirment of the GLP-1-dependent gut-rin communiction in response to ft diet. We further vlidted this oservtion y ssessing GLP-1 sensitivity in -fed mice following sudiphrgmtic vgotomy (SDVx). This procedure increses GLP-1 resistnce, since the plsm insulin concentrtion ws lower in the SDVx mice when compred to the shm mice nd while the plsm GLP-1 concentrtion incresed in the former mice (Figures S3B S3D). We further confirmed this oservtion when the mice were stimulted with GLP-1. GLP-1-induced insulin secretion t the physiologicl dose of 7 nmol/kg ws reduced y the vgotomy procedure, further demonstrting the importnce of functionl gut-rin xis s the mode of ction of GLP-1 (Figures 3B nd S3E). Interestingly, vgotomy induced GLP-1 resistnce of gstric emptying s well (Figures S3F nd S3G), while, conversely, energy intke during the feeding period or in response to refeeding ws similr etween shm nd SDVx-operted mice in response to GLP-1 (Figures S3H S3J). Eventully, we induced enteric neuropthy y treting control mice with cispltin, s descried (Ver et l., 211). This reduced the numer of HuC/HuD, PGP9.5, nd prph-positive enteric cells, i.e., the neurons (Figures 2A1 nd 2A2) s well s the corresponding mrna concentrtions (Figure 2B). In such conditions, the fold chnge of insulin secretion over control induced y GLP-1 in response to i.v. glucose ws reduced (Figure 3C). To demonstrte tht the ction of i.p. GLP-1 in mice ws specific to the gut-rin xis rther thn to the cell, we performed GLP-1 dose response y dministering the peptide i.v. In such conditions glucose-induced insulin secretion ws not engged, suggesting tht the gut- cell xis is more sensitive thn the cells themselves to GLP-1 on insulin secretion (Figure 3D). Then in ll mouse models we used glyenclmide to directly stimulte insulin secretion without recruiting glucose nd GLP-1 signling. The dt show tht in dietic mice only insulin secretion ws impired, suggesting reduced cell insulin secretory cpcity (Figure S3K) which could e either intrinsic to the cell or due to the impired gut- cell xis. To distinguish etween oth hypotheses, we studied the ction of glyenclmide on vgotomized mice nd showed tht in such conditions insulin secretion ws lso impired, demonstrting the mjor role of the vgus nerve- cell xis (Figure S3L). Altogether, the impirment of GLP-1 physiologicl ctions such s insulin secretion nd gstric emptying induced y vgotomy or y cispltin-induced enteric neuropthy mimicked wht ws oserved in response to, i.e., the GLP-1-resistnt dietic len mouse model. The next question ws relted to the identifiction of the moleculr mechnism impired in dietic mice tht trnsmit the enteric GLP-1 signl to the rin nd then the cell. Enteric GLP-1 Sensitivity Requires the Production of NO y Enteric Neurons nd Is Impired in -Fed Mice To identify the enteric neuron moleculr mechnisms responsile for GLP-1 resistnce, we first quntified the concentrtion of GLP-1r mrna throughout the intestinl trck of the different mouse models. In -fed mice, the concentrtion of the GLP-1r mrna ws reduced in oth the jejunum nd ileum s well s the left nodose gnglion from the dietic mice when compred to controls, while it remined unchnged in the duodenum nd the colon (Figures 4A, S4A, nd S4B). These results could explin GLP-1 resistnce s it mimics wht ws oserved in GLP-1r KO mice regrding insulin secretion, gstric emptying, nd food intke (Figures S4C S4E). It is noteworthy tht recent dt show tht the expression of the GLP-1r gene is restricted in enteric neurons within the intestine nd in the left nodose gnglion (Richrds et l., 214). As we nd others hve previously shown tht GLP-1 signling involves the ctivtion of nnos in neurons (Cou et l., 211; Ding nd Zhng, 212; Rotondo et l., 211), nd tht the Cell Metolism 25, , My 2,

5 A1 A2 S1 Prph PGP9.5 HuC/HuD B HC- Cispltin # HuC/HuD positive cells/mm PGP9.5 fluorescent re (%) Prph fluorescent re (%) S1 fluorescent re (%) , HC- Cispltin, HC- Cispltin, HC- Cispltin HC- Cispltin PGP9.5 mrna (AU) HC-, Cispltin Prph mrna (AU) HC- Cispltin GFAP mrna (AU) HC- Cispltin S1 mrna (AU) HC-,, Cispltin Figure 2. High-Ft Diet Alters the Enteric Nervous System (A nd B) Mice were fed for 3 months with, HC-, or or treted with cispltin (weekly i.p. injection, during 1 month). (A1) longitudinl musculr myenteric plexus (LMMP) immunohistochemistry; (A2) quntifiction of the numer of HuC/HuD-positive cells, percentge of PGP9.5 nd Prph fluorescent re (s neuronl mrker) nd percentge of S1 fluorescent re (s glil mrker) (n = 5 6/group); (B) ileum mrna concentrtions of neuronl mrkers (PGP9.5 nd peripherin-prph) nd glil mrkers (GFAP, S1) (n = 6, ; n = 1, HC-; n = 1, ; n = 5, cispltin-treted mice). Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween nd HC-, -fed mice nd cispltin-treted mice. GLP-1r mrna concentrtion is minly locted in nnos positive enteric neurons (Amto et l., 21; Richrds et l., 214), we first mesured the nnos mrna concentrtion. We oserved decresed concentrtion in the ileum tht ws even more mrked in the left nodose gnglion of the -fed GLP-1-resistnt mice (Figures 4B nd S4F). In HC- mice, we oserved decrese 178 Cell Metolism 25, , My 2, 217

6 A1 A2 # cfos positive cells/nts-dmnx re HC- Vehicle 4 GLP HC- Vehicle B Plsm insulin fold chnge Shm SDVx Ctrl log [GLP-1], nmol/kg GLP-1 of nnos mrna concentrtion in left nodose gnglion only (Figures 4B nd S4F). Liner regression nlyses etween nnos nd GLP-1r mrna concentrtions showed strong correltion in oth the ileum nd the nodose gnglion, suggesting cusl reltionship (Figures 4C nd S4G). Altogether, the mrna concentrtion nlyses suggested tht -induced GLP-1 resistnce might e linked to n impired GLP-1r expression nd the corresponding induction of NO production through nnos signling. To further demonstrte this hypothesis, we treted non-dietic mice with different doses of the nnos inhiitor (L-NAME) (Figure S1B). This loss-of-function procedure prtilly reduced GLP-1-induced insulin secretion in mice (Figure 4D). Similrly, the NOS inhiitor ltered GLP-1-induced gstric emptying inhiition, ut not GLP-1-control of food intke (Figures S4H nd S4I). Then, using orl dministrtion of L-rginine, sustrte of nnos (Figure S1C), in gin-of-function experiment we could increse glucose-induced insulin secretion (Figure 4E). It ws specific to GLP-1 ction, since it could e reversed using the GLP-1 receptor ntgonist exendin9 (Figure 4E) s well s y vgotomy (Figure 4F). Similr results were otined on gstric emptying (Figures S4J nd S4L), while no chnge ws oserved on food intke (Figure S4K). On -fed mice, glucose-induced insulin secretion could e incresed y n cute tretment with L-rginine tretment C Plsm insulin fold chnge Vehicle GLP-1 NCl Cispltin AP DMNx D Plsm insulin fold chnge NTS nmol/kg 2,2nmol/kg 7nmol/kg 21nmol/kg c, IP GLP-1,c IV GLP-1 Figure 3. High-Ft Diet Alters the Gut-Brin Axis nd Induces GLP-1 Resistnce (A) In (A1), immunohistochemistry of cfos-positive cells; (A2) quntifiction of the numer of cfospositive cells per dotted re depicting the nucleus of the trctus solitrius (NTS) nd the dorsl motor vgl nucleus (DMNx) in, HC-, nd -fed mice stimulted with vehicle or with GLP-1 (7 nmol/kg) (n = 3 5/group). Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween vehicle-injected mice nd GLP-1-injected mice. (B) Plsm insulin fold chnge (in percentge of vehicle-injected mice) fter GLP-1 injections (x xis represented s log of, 2.2, 7, nd 21 nmol/ kg) in shm-operted or sudiphrgmtic vgotomy (SDVx)-operted mice (n = 5/group). Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween Shm nd SDVx-operted mice. (C) Plsm insulin fold chnge (in percentge of vehicle-injected mice) fter GLP-1 stimultion (7 nmol/kg) in cispltin- or vehicle-treted mice (weekly i.p. injection, during 1 month, n = 6/group). (D) Plsm insulin fold chnge (in percentge of vehicle-injected mice) fter i.p. nd i.v. GLP-1 injections (, 2.2, 7, nd 21 nmol/kg) (n = 3 8/s). Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween vehicle-injected mice nd GLP-1-injected mice. (Figure 4G), suggesting n improved GLP-1 sensitivity. We were le to vlidte this hypothesis using chronic 7-dy tretment with L-rginine, since glucose-induced insulin secretion in response to GLP-1 ws enhnced when compred to sline tretment dietic mice (Figure 4H). Similrly, gstric emptying ws stimulted y L-rginine tretment (Figures S4M), which ws not oserved on food intke, since this function does not require the gut-rin xis (Figure S4N). To scertin tht GLP-1 directly increses NO production from enteric neurons, we incuted incresing doses of GLP-1 with primry culture of enteric neurons from nd -fed GLP-1-resistnt mice. The enteric neurons from the -fed mice were drmticlly resistnt to GLP-1- induced NO production (Figure 4I). Altogether, we show here tht enteric NO production is signling molecule triggered y GLP-1 on enteric neurons to control metolic fetures impired in -fed mice. Mechnisms responsile for this impirment could e relted to the gut microiot dysiosis nd ssocited hyperglycemi. Gut Microiot Dysiosis Is Responsile for the GLP-1 Resistnce Here we tested the hypothesis of the role plyed y gut microiot dysiosis in GLP-1 resistnce. To identify the corresponding cteril tx, we performed trgeted 16S sequencing of the ileum microiot, since GLP-1-producing cells re mostly locted in the ileum of the mouse intestine. The liner discriminnt nlysis (LEfSe) demonstrted significntly different txonomic Cell Metolism 25, , My 2,

7 A GLP-1r mrna (AU) Ileum HC- B nnos mrna (AU) Ileum HC- C nnos mrna (AU) Ileum r 2 =.1774 p< GLP-1r mrna (AU) D E F Plsm insulin fold chnge GLP-1 L-NAME -,,, Plsm insulin fold chnge L-Arg Ex Plsm insulin fold chnge cute L-Arg SDVx - + G Plsm insulin fold chnge cute L-Arg - + H Plsm insulin fold chnge chronic - + L-Arg GLP I GLP-1 induced NO production (% of control) Ctrl Enteric Neurons log[glp-1], nm A B Figure 4. Enteric GLP-1 Sensitivity Requires the Production of NO y Enteric Neurons tht Is Impired in -Fed Mice (A E) Mice were fed for 3 months with, HC-, or. Ileum mrna concentrtions of (A) GLP-1r nd (B) nnos (n = 15, ; n = 2, HC-; n = 25, ); (C) Person correltion nlyses etween GLP-1r nd nnos mrna concentrtions in ileum. Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween nd HC--fed, nd -fed mice. (D H) Plsm insulin fold chnge (percentge of vehicle-injected mice); (D) fter GLP-1 ( nd 7 nmol/kg) nd nnos inhiitor injections (L-NAME,, 5, 1, nd 2 mg/kg) in mice (n = 3 5/group), (E) fter L-rginine ( nd 1 g/kg) nd Exendin 9 ( nd 1 nmol/mouse) in (n = 5/group), (F) in SDVx-operted mice (n = 5/group), nd (G) in -fed mice (n = 5/group), (H) fter chronic tretment with L-Arg ( nd.5 g/kg/dy, during 1 week) nd GLP-1 injection (7 nmol/kg) (n = 3/group). Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween ll groups. (I) GLP-1-induced NO production y enteric neurons from nd mice expressed s percentge of NO from control cells (cells without GLP-1 incution) (n = 6 18 cells/group). Vlues with similr superscript letter re not different, p >.5. Lowercse letters re used to compre individul vlues, nd cpitl letters re used to compre curves. The vlues re compred etween neurons from - nd -fed mice. signtures specific for ech dietry group (Figures 5A 5C). First, the reltive undnce of Lctocilli ws drmticlly reduced in the ileum of the -fed GLP-1-resistnt mice, while it ws modestly reduced in the HC--fed GLP-1-sensitive mice (Figures 5B nd 5C). Liner regression nlyses lso showed tht the reltive undnce of Lctocilli ws positively correlted with 18 Cell Metolism 25, , My 2, 217

8 A B C D E F Figure 5. High-Ft Diet Induces Gut Microiot Dysiosis nd Modifies the Gut Microiot Function Mice were fed for 3 months with, HC-, or. (A C) LDA effect size (LEfSe) cldogrms of txonomic dt from 16S rdna sequence nlysis of ileum smples. Pirwise nlysis ws performed for dietry groups (A) versus HC-, (B) versus, nd (C) HC- versus. The cldogrms show the txonomic levels represented y rings with phyl t the innermost ring nd gener (identified in the legend) t the outermost ring, nd ech circle is memer within tht level. Tx t ech level re shded (green, lue, nd ornge) ccording to the dietry group in which it is most undnt (p <.5; LDA score 2.). The respective txonomic cldogrm legends re incorported into the djcent grphicl representtion of the percent reltive undnce for ech genus level differentil feture. (D F) LDA effect size (LEfSe) cldogrms of KEGG pthwy contriutions of predicted metgenomic dt for ileum smples. Pirwise nlysis ws performed for dietry groups (D) versus HC-, (E) versus, nd (F) HC- versus. The cldogrms show the KEGG pthwy hierrchy represented y rings with the consolidted pthwy modules (identified in the legend) t the outermost ring, nd ech circle is memer within tht level. KEGG modules re shded (green, lue, nd ornge) ccording to the dietry group in which it is most undnt (p <.5; LDA score 2.). The respective KEGG pthwy cldogrm legends re incorported into the djcent grphicl representtion of the reltive predicted gene count for ech differentil feture. Cell Metolism 25, , My 2,

9 the ileum GLP-1r nd nnos mrna concentrtions (Figure S5A). Significnt correltions with ileum GLP-1r nd nnos expression were lso oserved up to the phylum txonomic level for Firmicutes (dt not shown). Second, the reltive undnce of the cteril txonomic orders for Bcteroidles, Burkholderiles, Clostridiles, nd TM7 (including severl unclssified under respective clsses) were incresed in the dietic mice when compred to controls (Figures 5A 5C). Liner regression nlyses lso showed strong negtive correltion for the reltive undnce of cteril fmily Porphyromondcee (Bcteroidles order) with ileum GLP-1r nd nnos mrna concentrtions (Figure S5A). To explore the iologicl impct of the diet-ltered microiomes, we generted the predicted metgenome using PICRUSt (Lngille et l., 213). A totl of 5,684 KEGG orthologs were ssigned using the complete 16S sequence dtset for, HC-, nd ileum smples. Following ssocition with the KEGG module hierrchy, 798 nd 654 unique KOs were ssigned to 212 metolic pthwys nd 165 structurl complex modules, respectively. Anlysis of vrince (ANOVA, p <.5) identified 22 metolic pthwy genes nd 186 structurl complex genes which were differentilly enriched in t lest one of the dietry groups. Hierrchl liner discriminnt nlysis (LEfSe) of metolic pthwys from PICRUSt-predicted metgenomes showed tht the frequency of the cteril genes involved in the metolism of nucleotides nd mino cids ws incresed in the dietic mice (Figures 5D 5F). Venn nlyses of pthwy modules differentilly represented s compred to the mice indicted tht there re 25 common pthwys enriched in oth the HC- nd diet groups (Figure 5F), while 19 pthwy modules were uniquely enriched in the mice. In prticulr, significnt gene enrichment ws demonstrted for pthwy modules involved in the metolism of severl mino cids including rginine, histidine, leucine, lysine, proline, tryptophn, nd tyrosine (Figures 5D 5F; Tle S1). Also, genes for ssimiltory nitrte reduction (nitrte /mmoni, KEGG module M531) were significntly enriched in the group mice (Tle S1). Anlysis of predicted structurl complexes indicted generl enrichment in components relted to metllic ction, iron-siderophore, nd vitmin B12 trnsport in dietic mice when compred to controls (Figures S5B S5D). Venn nlyses of structurl complex modules differentilly represented s compred to the mice indicted tht there re 25 common structurl fetures enriched in oth the HC- nd diet groups (Figures S5B S5D), while nine pthwy modules were uniquely enriched in the mice. In prticulr, significnt gene enrichment ws demonstrted for genes involved in structurl components for glutmine nd puttive ABC trnsport systems (Tle S2). To further demonstrte the cusl role of gut microiot on insulin secretion, we quntified GLP-1 resistnce in germ-free (GF) mice. The dt showed tht the sence of microiot prevented GLP-1-induced insulin secretion, demonstrting strong stte of GLP-1 resistnce (Figures 6A, S6A, nd S6B). This impirment ws ssocited with lck of ctivtion of the gutrin xis, since the numer of cfos-positive cells in the rin stem could not e incresed y GLP-1 i.p. dministrtion (Figures S6C1 nd S6C2). This impirment might lso e due to reduced cell glucose sensitivity. Therefore, to test this hypothesis, we treted the GF mice with glyenclmide nd showed tht glucose-induced secretion could not e stimulted. This result demonstrted tht, in ddition to the gut- cell xis, the cells lone could e glucose unresponsive in the sence of gut microiot (Figure S6D). We focused our ttention on the ENS sensitivity to GLP-1 y studying the production of NO y enteric neurons from GF mice. We showed tht NO production could not e stimulted y GLP-1, demonstrting tht gut microiot is required feture for the ctivtion of the gut-rin xis y GLP-1 through the production of NO (Figure S6E). The GLP-1-resistnt stte in GF mice cn e linked to decresed of enteric neuron (PGP9.5, prph) nd glil cell (S1) mrna in the ileum (Figure 6B). Furthermore, the concentrtion of GLP-1r ws drmticlly reduced in GF mice when compred to conventionl mice (Figure 6C), trend which ws lso oserved for the concentrtion of nnos mrna (Figure 6D). We vlidted the role of gut microiot on these fetures y conventionlizing the GF mice with the ileum microiot from -fed or -fed mice. After ileum microiot trnsplnt, the GLP-1 sensitivity to glucose-induced insulin secretion ws completely reversed in the -conventionlized mice, while it remined impired in the -conventionlized mice (Figure 6A). The mrna concentrtion of PGP9.5 nd the enteric GLP-1r ws incresed only in the -conventionlized mice when compred to GF mice (Figures 6B 6D), which ws not the cse for the glil cell-relted mrna nd nnos (Figures 6B 6D). As finl demonstrtion of the cusl role of gut microiot on the control of GLP-1 sensitivity, we treted the - nd -fed mice with lrge-spectrum ntiiotics for 1 month. This procedure induced strong GLP-1 resistnce in fed mice (Figure 6E), which ws oserved t different doses of GLP-1 (Figure S6B), showing tht euiotic microiot is required for GLP-1 sensitivity. Conversely, GLP-1 resistnce of -fed mice ws reversed when the dysiotic microiot were eliminted y the ntiiotic tretment (Figure 6E). This hd functionl impct on the ENS, since NO production could e induced y GLP-1 from neurons of dietic mice when treted with ntiiotics (Figure S6F). This result ws linked to the impct of ntiiotics on neuron-specific protein PGP9.5 nd glil-specific protein S1 mrna concentrtions, which were incresed following ntiiotic tretment (Figure 6F). While the ntiiotics reduced the corresponding gene expression in -fed mice, the tretment incresed the gene expression in HDF-fed mice (Figure 6F). Similr results were otined for the GLP-1r nd for the nnos mrna concentrtions in the ileum (Figures 6G nd 6H). This lst set of dt strongly supports the notion tht euiotic gut microiot enhnces GLP-1 sensitivity while dysiotic microiot reduces it. The impct of ntiiotics ws extended to the control of gstric emptying y GLP-1 (Figures S6G nd S6H). This set of dt further reinforces the importnce of the ileum microiot on the GLP-1-dependent gut-rin xis. The control of food intke in ft-enricheddiet-fed mice, which is not under the control of the GLP-1- dependent gut-rin xis, remined unffected y ntiiotics (Figures S6I nd S6J). 182 Cell Metolism 25, , My 2, 217

10 A B Plsm insulin fold chnge Ctrl Vehicle GLP-1 GF Conv() Conv() PGP9.5 mrna (AU), 1.,.5. Ctrl GF Conv() Conv() Prph mrna (AU) Ctrl GF Conv() Conv() GFAP mrna (AU) 2. 1.,,.5. Ctrl GF Conv() Conv() S1 mrna (AU) 2. 1.,,.5. Ctrl GF Conv() Conv() C D GLP-1r mrna (AU) 2., Ctrl GF Conv() Conv() nnos mrna (AU) Ctrl GF Conv() Conv() E F Plsm insulin fold chnge Vehicle 4 GLP Ax + Ax PGP9.5 mrna (AU) Vehicle Ax Prph mrna (AU) GFAP mrna (AU) S1 mrna (AU) G H GLP-1r mrna (AU) nnos mrna (AU) Figure 6. Gut Microiot Dysiosis Is Responsile for the GLP-1 Resistnce (A D) Germ-free (GF) mice nd conventionlized mice with ileum flor from -fed mice (Conv[]) nd from -fed mice (Conv[]). (A) Plsm insulin fold chnge (percentge of vehicle-injected mice) fter GLP-1 injection ( nd 7 nmol/kg) (n = 3 8/group), (B) ileum mrna concentrtions of neuronl mrkers (PGP9.5 nd peripherin-prph) nd glil mrkers (GFAP, S1) (n = 5 15/group); nd (C) GLP-1r nd (D) nnos mrna concentrtions in the ileum (n = 5 15/ group). Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween GLP-1-untreted mice nd GLP-1-treted mice or etween control (Ctrl), GF, nd ll conventionlized mice. (E H), HC-, or mice were treted 1 month with ntiiotics (Ax); (E) plsm insulin fold chnge (percentge of vehicle-injected mice) fter GLP-1 injection ( nd 7 nmol/kg) (n = 3 8/group); (F) ileum mrna concentrtions of neuronl mrkers (PGP9.5 nd peripherin-prph) nd glil mrkers (GFAP, S1) (n = 5 1/group); (G) GLP-1r nd (H) nnos mrna concentrtions in the ileum (n = 5 1/group). The vlues re compred etween GLP-1-untreted mice nd GLP-1-treted mice or etween ntiiotic-untreted mice nd ntiiotic-treted mice. Altogether, chnges in ileum microiot were cuslly influencing GLP-1 responsiveness through the ctivtion of the gut-rin xis nd notly the stimultion of insulin secretion. While the role of Lctocillus species in the control of insulin secretion ws ddressed, their role on GLP-1 sensitivity remins to e further understood. The Microil-Associted Moleculr Pttern Receptors NOD2, CD14, nd TLR4 Control GLP-1 Sensitivity The mechnisms ensuring norml GLP-1 physiology nd gut-rin xis control could e relted to the role plyed y the microil-ssocited moleculr pttern receptors NOD1, NOD2, TLR4, nd CD14 (Cni et l., 27, 28; Denou et l., Cell Metolism 25, , My 2,

11 A Plsm insulin fold chnge GLP-1-7nmol/kg WT NOD2KO Plsm insulin fold chnge GLP-1-7nmol/kg WT TLR4KO Plsm insulin fold chnge GLP-1-7nmol/kg WT CD14KO B PGP9.5 mrna (AU) WT NOD2KO CD14KO TLR4KO Prph mrna (AU) WT NOD2KO CD14KO TLR4KO S1 mrna (AU) WT NOD2KO CD14KO TLR4KO GFAP mrna (AU) WT NOD2KO CD14KO TLR4KO C D E F GLP-1r mrna (AU) WT NOD2KO CD14KO TLR4KO nnos mrna (AU) WT NOD2KO CD14KO TLR4KO GLP-1 induced NO production (% of control) Enteric Neurons GLP-1 GLP-1+MDP (2) GLP-1 +MDP (2) GLP-1 induced NO production (% of control) Enteric Neurons GLP-1 GLP-1+LPS (1) GLP-1 + LPS (1) Figure 7. The Microil-Associted Moleculr Pttern Receptors NOD2, CD14, nd TLR4 Control GLP-1 Sensitivity NOD2, TLR4, nd CD14 KO mice were compred to wild-type (WT) mice. (A) Plsm insulin fold chnge (percentge of vehicle-injected mice) fter GLP-1 injection (7 nmol/kg) (n = 3 5/group); (B) ileum mrna concentrtions of neuronl mrkers (PGP9.5 nd peripherin-prph) nd glil mrkers (GFAP, S1) (n = 5 12/group); (C) GLP-1r nd (D) nnos mrna concentrtions in the ileum (n = 5 12/ group). Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween WT nd KO mice. GLP-1-induced NO production y enteric neurons from WT mice fter incution with or without (E) MDP (murmyldipeptide) (2 nd 2 ng/ml) or (F) LPS (lipopolyscchride) (1 nd 1 mg/ml) nd expressed s percentge of NO from control cells (cells without GLP-1, LPS, nd MDP incution) (n = 1 2 cells/group). Vlues with similr superscript letter re not different, p >.5. The vlues re compred etween ll conditions. 215; Prjpti et l., 214). The dt show tht GLP-1-induced insulin secretion ws drmticlly reduced in NOD2, TLR4, or CD14 KO when compred to WT mice (Figure 7A). However, in the KO mice the mrna of the GLP-1r ws incresed when compred to WT mice (Figure 7C), while the mrna encoding for nnos remined unchnged (Figure 7D). PGP9.5, prph, GFAP, nd S1 in the ileum remined similr to WT mice (Figure 7B), suggesting tht the impired cteril signling is upstrem to the ENS. Furthermore, gstric emptying ws lso impired in ll KO mice (Figure S7A), while food intke remined unchnged (Figure S7B). To functionlly ddress the role of cteril determinnt on the control of GLP-1 sensitivity, we studied the impct of MDP (Murmyldipeptide, NOD2 gonist) nd LPS (Lipopolyscchrides, CD14/TLR4 gonist) on NO production y enteric neurons. The dt show tht GLP-1-induced NO production ws enhnced y high doses of oth microil-ssocited moleculr ptterns (Figures 7E nd 7F). This set of dt further reinforces the role plyed y gut microiot on the control of GLP-1-induced insulin secretion nd gstric emptying inhiition. We here dd to the knowledge tht PRR signling is importnt for the control of GLP-1 signling. DISCUSSION Here we identified in different mouse models of T2D tht specific set of ileum cteri induces GLP-1 unresponsiveness 184 Cell Metolism 25, , My 2, 217

12 of enteric neurons. Gut microiot dysiosis hmpers GLP-1- induced NO production y enteric neurons tht prevent n efficient ctivtion of the gut-rin to periphery xis for the control of insulin secretion nd gstric emptying, ut not food intke. To identify puttive mechnisms responsile for chnges in GLP-1 function on the glucose control, s descried in clinic, we took dvntge of two different niml models tht we nd others previously chrcterized from metolic nd gut microiot dysiosis stndpoints (Burcelin et l., 22; Everrd et l., 213; Gridou et l., 215; Turnugh et l., 28). Briefly, mice fed HC- re oese nd dietic, wheres mice fed re dietic ut remined minly len, lthough the ltter were chrcterized y slight increse of ft mss nd decrese of len mss. The differences in ody weight etween models re most likely due to differentil impct of lipogenesis on insulin secretion leding to vrious plsm insulin concentrtions nd whole-ody metolic rte, i.e., insulin resistnce. Chnges in insulin nd metolic rtes ffect oth lipogenesis nd lipolysis, leding to chnges in ody weight, s descried (Burcelin et l., 22). Of note, the len -fed mice could lso e considered ketogenic, since they were fed crohydrte-free diet nd chrcterized with low plsm insulin concentrtion. However, the plsm ketone ody -hydroxy utyrte concentrtions (.97 ±.18 versus 1. ±.13 mm in controls nd -fed mice, respectively) nd even liver lipid content were similr etween groups, suggesting tht the heptic ketogenic pthwy ws not minly triggered in this mouse model, s we previously descried (Gridou et l., 215). In such niml models, we here show tht response to orl glucose insulin secretion ws reduced when compred to control nd HC--fed mice. This ws not relted to reduced glucose-induced GLP-1 secretion, since portl vein plsm GLP-1 concentrtion ws drmticlly incresed in ll mouse models of T2D. Our oservtion ws previously reported in -fed mice (Yng et l., 216), nd we here confirm this result. The mjor novel oservtion ws tht despite the excessive plsm portl GLP-1 concentrtion of oth niml models, glucose-induced insulin secretion ws efficiently triggered in HC- oese dietic mice only. Therefore, stte of GLP-1 resistnce ws suspected to e responsile for this oservtion. To demonstrte this hypothesis, we showed tht in experiments where incresing doses of GLP-1 were dministered in control nd dietic mice the dose-response curve ws shifted to the right in the ltter group of mice. The hlf-mximl effect of GLP-1 ws otined for doses higher in the dietic mice thn in controls, demonstrting tht such mice were resistnt to exogenous GLP-1. The quntifiction of portl vein plsm GLP-1 concentrtion showed tht GLP-1 resistnce ws pronounced for physiologicl concentrtions. It ws more drmtic in - thn in HC--fed mice. The resistnce to GLP-1 could e locted t different levels: the cell itself, the enteric or vgus nervous systems, or even the rin, which could rely the neurl ction of GLP-1 towrd the cell. To evlute the corresponding role of ech GLP-1 trget site, we first ssessed the efficcy of the cell to secrete insulin. In response to sulfonylure dministrtion, insulin secretion ws lower in oth models of dietes when compred to controls, showing tht the cell response ws impired nd suggesting tht it could e site of GLP-1 unresponsiveness. In ddition to the cell GLP-1 resistnce, we explored the enteric or vgus nerve system s puttive site of GLP-1 resistnce. The dministrtion of DPP4i orlly nd t dose 5 times lower thn wht is currently used to systemticlly inhiit the DPP4 llows inhiition of the DPP4 mostly in the ENS, rther thn in the systemic lood, s previously reported (Wget et l., 211). Such conditions increse the enteric ction of GLP-1 on the gut-rin xis rther thn on the cell in the control mice, while it ws drmticlly impired in the T2D mice. In ddition, to specificlly impir the ctivtion of the gut-rin- cell xis, we performed vgotomy in control mice nd showed tht in response to physiologicl doses of GLP-1, plsm insulin secretion ws drmticlly reduced. This hs lso een shown in cispltin-treted mice, n niml model of enteric neuropthy (Ver et l., 211). As functionl iossy of the gut-rin xis, we quntified the numer of cfos-expressing cells in the rin in response to the physiologicl dose of GLP-1 nd found tht they were reduced in dietic mice when compred to controls. Altogether, these first sets of experiments demonstrted tht the i.p. dministrtion of GLP-1 t physiologicl dose triggers the gut-rin xis for the control of insulin secretion, which is impired in T2D mice. Importntly, in our experimentl condition in T2D mice, GLP-1 resistnce ws most likely not only relted to the impired response of the cell, since in control non-dietic mice the i.v. dministrtion of physiologicl doses of GLP-1 did not induce insulin secretion. Therefore, n impired cell GLP-1 response ws not t ply in our experimentl procedure where GLP-1 ws dministered i.p. t physiologicl doses. This lso shows tht physiologicl doses of GLP-1 when dministered i.v. cnnot directly recruit cells. This could e due in prt to the rpid degrdtion of the i.v.-injected GLP-1 y the circulting systemic DPP4. Lrger doses re currently required, s demonstrted cliniclly, to stimulte cell insulin secretion. Altogether, the i.p. dministrtion of physiologicl doses of GLP-1 triggers the gut-rin cell xis, which is the trget site of GLP-1 most impired in the dietic mice. However, we cnnot rule out the possiility tht the rin trget site ws not involved in GLP-1 unresponsiveness preventing the triggering of glucose-induced insulin secretion, s we previously showed (Knuf et l., 25, 28). The impired gut-rin xis could lso prevent cell GLP-1 sensitivity, s numerous neuropeptides relesed y pncretic terminl nerve endings could enhnce camp concentrtions fvoring glucose nd GLP-1 sensitivity. Altogether, our dt show tht -induced dietes is chrcterized y stte of GLP-1 resistnce tht ffects insulin secretion nd gstric emptying. This conclusion could reconcile oservtions in the clinic where the tretment with GLP-1r gonists hs to e discontinued over the course of the tretment due to the lck of efficcy. The moleculr mechnisms responsile for GLP-1 resistnce in the -fed T2D mice could e primrily relted to the reduced expression of the GLP-1r. This is supported y the oservtion tht the concentrtion of the corresponding mrna ws decresed y 5% in ENS of the ileum, the jejunum, nd the nodose gnglion s the receptor is uniquely expressed in this cell type (Richrds et l., 214). The downregultion nd the desensitiztion of the GLP-1r (Bggio et l., 24; Widmnn Cell Metolism 25, , My 2,

13 et l., 1997) hve een proposed s mechnisms reducing GLP-1 ction, s shown y the reduction of the production of secondry messengers such s camp, nd NO in different cells (Cou et l., 211; Ding nd Zhng, 212; Drucker et l., 1987; Pujds et l., 215; Quoyer et l., 21). In niml models of T2D or in dietic ptients, reduced numer of GLP-1r t the surfce of cells nd hypothlmic neuronl cells hs een oserved tht ws induced y hyperglycemi nd hyperlipidemi (Burcelin et l., 29; Hodson et l., 213; Ten Kulve et l., 216; Xu et l., 27; Yng et l., 216; Younn nd Rshed, 27). A downstrem messenger molecule of interest in the signling of the GLP-1r is NO (Ding nd Zhng, 212; Ishii et l., 214; Rotondo et l., 211). We here oserved tht the mrna concentrtions of oth the GLP-1r nd the nnos in the ileum were tightly correlted in our cohort of controls nd dietic mice, suggestive of common role. As function iossy in primry culture of enteric neurons, GLP-1-induced NO production ws impired in cells from -fed dietic mice when compred to controls, further confirming the stte of GLP-1 resistnce tht ffects the ENS. In vivo GLP-1 unresponsiveness could e mimicked in control mice when mice were treted with nnos inhiitors such s L-NAME, while GLP-1 resistnce could e reversed in dietic mice when mice were treted with NO donor such s L-rginine. The effect ws specific to the GLP-1r nd recruits the vgus nerve nd gut-rin xis, since the GLP-1r ntgonist exendin9 nd vgotomy locked GLP-1-induced insulin secretion. Interestingly, GLP-1 resistnce lso ffected gstric emptying, which could e similrly reversed in dietic mice y L-rginine tretment nd induced in control mice y L-NAME, while the control of food intke y GLP-1 remins mostly unffected y these molecules. Insulin secretion nd gstric emptying re tightly dependent upon functionl gut-rin-periphery xis (Holst, 27; Krieger et l., 215, 216). In our niml models, we oserved indices of enteric neuropthy such s the reduced numer of enteric neurons in the -fed mice when compred to controls. This reduction ws specific to neurons, s the numer of glil cells remins similr in ll groups of mice. Inducing enteric neuropthy in control mice using cispltin (Ver et l., 211) could mimic GLP-1 resistnce, further showing the importnce of functionl ENS on the control of GLP-1 ction. Our conclusions re comforted y few dt from the literture, which showed tht in humns the NO-donor L-rginine improves mel-induced insulin secretion when dministered orlly in helthy T2D ptients nd dietic mice (Ozek et l., 29; Tng et l., 213). Therefore, novel therpeutic strtegy for the tretment of T2D could emerge nd enefit ptients notly when GLP-1-sed therpies re filing t inclusion or overtime during the tretment. The phrmcologicl trgeting of the ENS nd, hence, the ctivtion of the gut-rin-periphery xis for the control of numerous functions such s GLP-1-induced insulin secretion nd gstric emptying re promising in the light of the present results. The origins of the moleculr impirment of GLP-1 ction on the ENS could e numerous. The discovery of the mjor impct of gut microiot on metolic disese, of its diversity, nd tht T2D ptients re chrcterized y gut microiot dysiosis opens new knowledge opportunities out the mechnisms of GLP-1 resistnce (B ckhed et l., 24; Cni et l., 27, 28; Ley et l., 25). To ddress this hypothesis, we first show tht GF mice re drmticlly GLP-1 resistnt to the control of insulin secretion, suggesting n importnt role of the microiot. We confirmed this hypothesis in control mice when treted with ntiiotics for 1 month showing tht some cteri from the gut microiot were responsile for GLP-1 sensitivity. The expression of the GLP-1r nd nnos ws reduced in the ENS. Conversely, the tretment of gut microiot dysiosis y ntiiotics reversed GLP-1 resistnce s well s the expression of the GLP-1r nd the nnos mrnas within the ENS. Altogether, our dt show eneficil role of some gut cteri while others re deleterious for the iology of the ENS. It ws shown tht GF mice re chrcterized y enteric nd neuronl trophy nd tht conventionliztion restores the neuronl function (McVey Neufeld et l., 215). We lso oserved stte of neuropthy in T2D mice s evidenced y the reduced numer of neurons nd specific neuronl mrkers of the ENS showing the importnce of microiot on the development of the gut-rin xis, s reported in other instnces (Ocho-Repárz nd Ksper, 216). We here showed the cusl role of gut microiot euiosis or dysiosis on the eneficil or deleterious effect on GLP-1 sensitivity, respectively, in GF conventionlized mice, since the trnsfer of ileum mucosl microiot progrmmed GLP-1-induced insulin secretion. Conventionliztion with euiotic microiot specificlly trgets the ENS, since the expression of PGP9.5, ut not of mrkers of glil cells, ws triggered. The dysiotic microiot ws unle to do so, suggesting tht microes specificlly present in the ileum microiot from control mice were responsile. The sequencing of the ileum microiot from the different mouse models shows numerous differences including n incresed frequency of Porphyromondcee, Clostridicee, Peptostreptococcee, Burkholderice, nd TM7 fmilies nd reduced frequency of Lctocillcee in the -fed GLP-1-resistnt mice. The prediction of metolic pthwys using PICRUSt nlysis suggested tht n incresed metolism of multiple mino cids (notly rginine, histidine, leucine, lysine, proline, tryptophn, nd tyrosine) could e involved. In ddition, pthwy components relted to ssimiltory nitrte reduction with mmoni production were highly enriched in the -fed GLP-1-resistnt mice. Severl structurl fetures relted to metl/iron cquisition were lso predicted to e incresed in the ileum microiot from GLP-1-resistnt mice. This comes in ddition to previous studies, which hve focused their ttention on the production of SCFA notly issued from the cpcity of Lctocillus to ferment complex crohydrte into SCFA (Cni et l., 27; De Vdder et l., 214; Nøhr et l., 213; Perry et l., 216; Tolhurst et l., 212). However, we ruled out this hypothesis, since chnges in SCFA concentrtions oserved in preiotic-treted mice were not ssocited with GLP-1 responsiveness (dt not shown), s descried previously (Gridou et l., 215). A second mechnism could e the reduction of PRR signling (such s NOD2) s consequence of reduced levels of peptidoglycns from grm-positive cteri such s the Lctocillus in -fed GLP-1-resistnt mice. We explored this second possiility nd showed tht the control of GLP-1 ction on insulin secretion nd gstric emptying ws drmticlly impired in the NOD2, CD14, nd TLR4 KO mice, showing the importnce of cteril recognition for GLP-1 sensitivity. NOD2 recognizes peptidoglycn motifs from cteril cell wlls tht consist of N-cetylglucosmine nd N-cetylmurmic cid. Precisely, NOD2 receptor cn sense intrcellulr MDP, 186 Cell Metolism 25, , My 2, 217