Microbiota-modulated CART+ enteric neurons autonomously regulate blood glucose - PubMed (original) (raw)

Microbiota-modulated CART+ enteric neurons autonomously regulate blood glucose

Paul A Muller et al. Science. 2020.

Abstract

The gut microbiota affects tissue physiology, metabolism, and function of both the immune and nervous systems. We found that intrinsic enteric-associated neurons (iEANs) in mice are functionally adapted to the intestinal segment they occupy; ileal and colonic neurons are more responsive to microbial colonization than duodenal neurons. Specifically, a microbially responsive subset of viscerofugal CART+ neurons, enriched in the ileum and colon, modulated feeding and glucose metabolism. These CART+ neurons send axons to the prevertebral ganglia and are polysynaptically connected to the liver and pancreas. Microbiota depletion led to NLRP6- and caspase 11-dependent loss of CART+ neurons and impaired glucose regulation. Hence, iEAN subsets appear to be capable of regulating blood glucose levels independently from the central nervous system.

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Conflict of interest statement

Competing interests: The authors declare no competing financial interests.

Figures

Fig. 1.. TRAP-seq profiling of iEANs reveals anatomical region–specific differences.

_Snap25_RiboTag SPF mice were analyzed in (A) to (E). (A) Whole-mount immunofluorescence (IF) of duodenum, ileum, and colon myenteric plexus using anti-neuronal nuclear (ANNA-1, green) and anti-HA (red) antibodies. Scale bars are 50 mm. (B) Principal components analysis (PCA) of neuronal translatomes from DRG, NG, CG-SMG, duodenum, ileum, and colon. (C) Gene ontology (GO) pathways of differentially expressed genes (DEGs) [log2 fold change (FC) > 1, and adjusted P value (_P_adj) < 0.05] enriched in ileum versus indicated ganglia. GPCR, G protein–coupled receptor; TCA cycle, tricarboxylic acid cycle. (D) PCA of neuronal translatomes from duodenum, ileum, and colon. (E) Volcano plots of DEGs between myenteric iEANs. Pink dots represent all intestine neuronal immunoprecipitation (IP)–enriched transcripts; colored dots represent DEGs of interest between each pair of intestine segments. Sample numbers are indicated in parentheses. (F) Number of total iEANs in different gut regions. (G to J) Numbers and percentages of (G) NPY+, (H) SST+, (I) CART+, and (J) FST+ myenteric iEANs in different gut regions. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; for numbers of iEANs in (F) to (J), Brown-Forsythe and Welch analysis of variance (ANOVA) with Dunnett’s T3 multiple comparisons test was performed; for percentages of iEANs in (G) to (J), Kruskal-Wallis test with Dunn’s multiple comparisons test was performed. Error bars indicate SD.

Fig. 2.. Microbiota affect iEAN translatomes in a compartmentalized manner.

(A) AdipoClear light-sheet images of ileum and colon of GF or SPF C57BL/6J mice using anti-TUJ1 antibody. Scale bars for ileum and GF colon are 200 μm; scalebar for SPF colon is 100 μm. (B) PCA of GF and SPF iEANs from duodenum, ileum, and colon. (C and D) Volcano plots of (C) duodenum, ileum, and colon iEAN DEGs between GF and SPF mice and (D) differentially expressed “duodenum signature” transcripts in ileum and colon iEANs of GF and SPF mice. Pink dots represent all intestine neuronal IP-enriched transcripts; colored dots and numbers represent significantly differentially expressed transcripts (log2 FC > 1, and _P_adj < 0.05). Sample numbers are indicated in parentheses. (E) GO pathways of DEGs enriched in SPF versus GF samples. In (B) to (E), _Snap25_RiboTag mice were analyzed. (F and G) Numbers of SST+ and CART+ iEANs in (F) ileum and (G) colon of GF mice, exGF mice, and GF mice colonized with exGF feces (exGF FC). (H) Total iEANs in ileum (left) and colon (right) of GF, exGF, and exGF FC mice. **P < 0.01, ***P < 0.001, and ****P <0.0001; for numbers of SST+ iEANs in (F) and (G), CART+ iEANs in (F), and total iEANs in (H), one-way ANOVA with Tukey’s multiple comparisons test was performed; for numbers of CART+ iEANs in (G), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test was performed. Error bars indicate SD.

Fig. 3.. iEAN loss after antibiotic treatment is mediated by NLRP6 and caspase 11.

(A) Myenteric iEAN numbers in duodenum, ileum, and colon of C57BL/6J and _Casp11_−/− mice treated with antibiotics (vancomycin, ampicillin, metronidazole, and neomycin, referred to collectively as Abx) or Splenda. (B and C) Ileal myenteric iEAN numbers for (B) Snap25_Δ_Nlrp6 or (C) Snap25_Δ_Casp11 mice on Abx. (D) Ileal myenteric iEAN numbers for C57BL/6J SPF mice after 4 weeks on Abx or Splenda, or Abx followed by Splenda for 2 weeks each (Switch). (E) Ileal myenteric iEAN numbers for C57BL/6J SPF mice on metronidazole (Metro), ampicillin (Amp), vancomycin (Vanco), neomycin (Neo), or Splenda. (F) CART+ myenteric iEAN numbers in duodenum, ileum, and colon of C57BL/6J and _Casp11_−/− mice on Abx or Splenda. (G and H) CART+ ileal myenteric iEAN numbers for (G) Snap25_Δ_Nlrp6 and control or (H) Snap25_Δ_Casp11 and control mice on Abx. *P < 0.05, **P < 0.01, and ****P < 0.0001; for (A) and (F), two-way ANOVA with Tukey’s multiple comparisons test was performed; for (B), (D), (E), and (G), one-way ANOVA with Tukey’s multiple comparisons test was performed. Error bars indicate SD.

Fig. 4.. CART+ iEANs in the distal intestine are viscerofugal and glucoregulatory.

(A and B) Whole-mount IF image of the (A) ileum myenteric plexus (MP) and (B) CG-SMG of _Cart_Cre+ injected with AAVrg-FLEX-tdTomato into (A) ileum and (B) duodenum, ileum, and colon. (C and D) Whole-mount IF image of the ileum MP of _Cart_Cre+ mice injected with AAV9-hSyn-DIO-hM3Dq-mCherry into the ileum, (C) stained for CART (green) and mCherry (red) and (D) 3 hours after C21 administration, stained for mCherry (red) and cFos (green). (E) Food consumption at night 2 hours (left) and 4 hours (right) after C21 administration in _Cart_Cre+ and _Cart_Cre− mice injected with AAV9 as in (C). (F) Blood glucose levels after C21 administration (left) and area under curve (AUC) analysis (right) in fed _Cart_Cre+ and _Cart_Cre− (control) mice injected with AAV9-hSyn-DIO-hM3DqmCherry or control AAV9-hSyn-DIO-mCherry into ileum and colon. (G and H) Plasma (G) insulin and (H) glucagon levels after C21 administration in _Cart_Cre+ and _Cart_Cre− mice injected with AAV9-hSyn-DIO-hM3Dq-mCherry into ileumand colon. Scale bars in (A) to (D) are 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; for (E) and (F), two-tailed unpaired Student’s t test was performed; for (G), two-way (left) or one-way ANOVA (right) with Tukey’s multiple comparisons test was performed. Error bars indicate SD.

Fig. 5.. CART+ viscerofugal neurons are polysynaptically connected to the liver and pancreas through the CG-SMG.

(A and B) Whole-mount IF image of (A) CG-SMG and (B) ileum MP of _Cart_Cre+ mice injected with AAV9-hSyn-DIO-hM3Dq-mCherry into ileum and colon. Scale bars are 100 μm. (C) Whole-mount IF image of colon (left) and ileum MP (right) of SPF mice injected with PRV-GFP (pancreas) and PRV-RFP (liver). Scale bars are 50 mm. (D) (Left) cFos (green) and mCherry (red) expression and (right) number of cFos+ neurons in the CG-SMG of _Cart_Cre+ mice injected with AAV as in (A), 3 hours after C21 injection. Scale bar is 50 mm. (E and G) CTB-AF647+ neuron numbers in duodenum, ileum, and colon (labeled D, I, and C, respectively) of (E) C57BL/6J or (G) Casp11 −/− mice treated with Splenda or antibiotics for 2 weeks after CTB injection into the CG-SMG. (F and H) CTB-AF647+ CART+ neuron numbers in the ileum of (F) C57BL/6J or (H) Casp11 −/− mice treated with Splenda or antibiotics for 2 weeks after CTB injection into the CG-SMG. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; for (D) and comparisons between Splenda and Abx [(E) to (H)], two-tailed Student’s unpaired t test was performed; for Splenda group comparisons between gut segments [(E) and (G)], one-way ANOVA with Tukey’s multiple comparisons test was performed. Error bars indicate SD.

Fig. 6.. Control of blood glucose is microbiota- and CART+ iEAN- dependent.

(A to G) Blood glucose levels of fasted (A) C57BL/6J, _Casp11_−/−, and _Casp1/11_−/− mice treated with Abx or Splenda; (B) GF, exGF B6, and _Casp11_−/− mice; (C) C57BL/6J mice on ampicillin, neomycin, or Splenda; (D) GF, exGF FC, and _Casp11_−/− FC mice; (E) Snap25_Δ_Nlrp6 mice on Abx; and (F) Snap25_Δ_Casp11 mice on Abx. In (G), an intraperitoneal pyruvate tolerance test (IP-PTT) of fasted C57BL/6J mice on Abx or Splenda is presented, with blood glucose curves shown on the left and AUC analysis shown on the right. (H and I) IP-PTT AUC analysis of fasted (H) GF, GF FC, and C57BL/6J mice housed in bioexclusion isolator cages, and (I) C57BL/6J and _Casp11_−/− mice on Abx or Splenda. (J and K) IP-PTT AUC analysis of fasted (J) Snap25_Δ_Nlrp6 and control or (K) Snap25_Δ_Casp11 and control mice on Abx. (L to N) _Cart_Cre+ mice injected with AAV5-DTA into ileum and colon. Shown are (L) fasted (left) or fed (right) blood glucose levels, (M) fasted (left) or fed (right) plasma insulin levels, and (N) fasted IP-PTT blood glucose curves (left) and AUC analysis (right). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; for (G) and (L) to (N), two-tailed unpaired Student’s t test was performed; for (B) to (E), (H), and (I), one-way ANOVA with Tukey’s multiple comparisons test was performed; for (A) and (J), two-way ANOVA with Tukey’s multiple comparisons test was performed. Error bars indicate SD.

Comment in

Regional ENS effects from gut microbiota.
Dickson I. Dickson I. Nat Rev Gastroenterol Hepatol. 2020 Nov;17(11):650. doi: 10.1038/s41575-020-00367-4. Nat Rev Gastroenterol Hepatol. 2020. PMID: 32913301 No abstract available.

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Microbiota-modulated CART+ enteric neurons autonomously regulate blood glucose - PubMed (original) (raw)