Functional changes of the gastric bypass microbiota reactivate thermogenic adipose tissue and systemic glucose control via intestinal FXR-TGR5 crosstalk in diet-induced obesity

Abstract

Background: Bariatric surgery remains the most effective therapy for adiposity reduction and remission of type 2 diabetes. Although different bariatric procedures associate with pronounced shifts in the gut microbiota, their functional role in the regulation of energetic and metabolic benefits achieved with the surgery are not clear.

Methods: To evaluate the causal as well as the inherent therapeutic character of the surgery-altered gut microbiome in improved energy and metabolic control in diet-induced obesity, an antibiotic cocktail was used to eliminate the gut microbiota in diet-induced obese rats after gastric bypass surgery, and gastric bypass-shaped gut microbiota was transplanted into obese littermates. Thorough metabolic profiling was combined with omics technologies on samples collected from cecum and plasma to identify adaptions in gut microbiota-host signaling, which control improved energy balance and metabolic profile after surgery.

Results: In this study, we first demonstrate that depletion of the gut microbiota largely reversed the beneficial effects of gastric bypass surgery on negative energy balance and improved glucolipid metabolism. Further, we show that the gastric bypass-shaped gut microbiota reduces adiposity in diet-induced obese recipients by re-activating energy expenditure from metabolic active brown adipose tissue. These beneficial effects were linked to improved glucose homeostasis, lipid control, and improved fatty liver disease. Mechanistically, these effects were triggered by modulation of taurine metabolism by the gastric bypass gut microbiota, fostering an increased abundance of intestinal and circulating taurine-conjugated bile acid species. In turn, these bile acids activated gut-restricted FXR and systemic TGR5 signaling to stimulate adaptive thermogenesis.

Conclusion: Our results establish the role of the gut microbiome in the weight loss and metabolic success of gastric bypass surgery. We here identify a signaling cascade that entails altered bile acid receptor signaling resulting from a collective, hitherto undescribed change in the metabolic activity of a cluster of bacteria, thereby readjusting energy imbalance and metabolic disease in the obese host. These findings strengthen the rationale for microbiota-targeted strategies to improve and refine current therapies of obesity and metabolic syndrome. Video Abstract Bariatric Surgery (i.e. RYGB) or the repeated fecal microbiota transfer (FMT) from RYGB donors into DIO (diet-induced obesity) animals induces shifts in the intestinal microbiome, an effect that can be impaired by oral application of antibiotics (ABx). Our current study shows that RYGB-dependent alterations in the intestinal microbiome result in an increase in the luminal and systemic pool of Taurine-conjugated Bile acids (TCBAs) by various cellular mechanisms acting in the intestine and the liver. TCBAs induce signaling via two different receptors, farnesoid X receptor (FXR, specifically in the intestines) and the G-protein-coupled bile acid receptor TGR5 (systemically), finally resulting in metabolic improvement and advanced weight management. BSH, bile salt hydrolase; BAT brown adipose tissue.

Keywords: Bile acids; FXR; Gastric bypass; Gut microbiota; TGR5; Taurine metabolism.

Fig. 1

Microbiota depletion impairs weight loss and metabolic improvements resulting from RYGB surgery in diet-induced obesity. a–f Relative body weight change (in %; average body weight (øBW) per group: RYGB(ABx) week (W)0: 385.6 g, W5: 454.3 g; DIO W0: 681.3 g, W5: 762.3 g; RYGB W0: 415.3 g, W5: 437.7 g; # DIO vs. RYGB; * RYGB vs. RYGB(ABx)) (a), final body composition with lean and fat mass (in g), b cumulative energy intake (in kcal) (c), cumulative energy intake of high fat diet (HFD) and standard chow (SC) (in g) (d), energy assimilation (in kJ/24 h) (e), oral glucose-tolerance tests (oGTT) (f), and insulin-sensitivity assessed by insulin-tolerance tests (ITT) (g) in RYGB-operated rats with 35-day antibiotic-treatment (ABx) compared to their respective controls (RYGB-operated rats without ABx). h–k Fasting plasma triglyceride concentrations (in ng/μl) (h), representative images (at least 4 images per group) of liver Oil Red O staining (scale bars 200 μm) with quantification of hepatic lipid accumulation (in %) (i), and relative mRNA expression of hepatic pro- and anti-inflammatory cytokines (j) in RYGB(ABx) compared to RYGB rats. Plasma cytokine concentrations in RYGB(ABx) and RYGB rats (in pg/ml) (k). l-n Representative images of H&E and UCP1 staining in BAT (scale bars 200 μm) (l), total energy expenditure (TEE; in kcal/day) (m) and infrared images of representative RYGB(ABx) or RYGB rats after 6 h of cold-exposure (n). Mouse primary brown adipocytes were treated for 6 h with 50% (vol/vol) of rat serum from RYGB(ABx) or RYGB rats (or with PBS as control). Oxygen consumption rate was measured on a respirometry Seahorse XF Cell Mito Stress Test (Agilent) as described in the “Methods” section (o). Experiments were performed in three independent cohorts. Data are mean ± s.e.m; n = 3–8 animals per group with pooled data from 2 to 3 independent experiments. *,#P < 0.05, **,## P < 0.01, ***,### P <0.001 as assessed by unpaired Student’s t test (for two groups) or two-way ANOVA (multiple groups) with Tukey correction for multiple testing

Fig. 2

RYGB gut microbiota transfer counters adiposity and metabolic disease in conventionally raised HFD-induced obesity. a–e Relative body weight change (in %; øBW per group: DIO W0: 681.3 g, W5: 762.3 g; FMT^RYGB W0: 528 g, W5: 595.2 g; RYGB W0: 415.3 g, W5: 437.7 g; * DIO vs. RYGB; # DIO vs. FMT^RYGB) (a), final body composition with lean and fat mass (in g) (b), cumulative energy intake of HFD and SC (in g) (c), HFD preference (in %) (d) and food efficiency (in %) (e) in HFD-induced obese (DIO) rats 5 weeks after fecal microbiota transfer (FMT) from RYGB-operated rats (FMT^RYGB) compared to DIO controls. f–i Oral glucose-tolerance tests (oGTT) (f), insulin sensitivity assessed by insulin-tolerance tests (ITT) (g), and plasma GLP-1 (in pM) release at 15 min after oral glucose exposure (h) 5 weeks after FMT. Representative immunofluorescent (IF) insulin-stained pancreas sections (scale bars 50 μm) and quantification of pancreatic islet sizes (in %) in FMT^RYGB rats and DIO controls (i). j–m Liver OilRedO staining (scale bars 200 μm) with quantification of hepatic lipid accumulation (in %) (j). Hepatic cytokine gene expression (k), plasma lipid (in ng/μl) (l), and plasma cytokine levels (in pg/ml) (m) in FMT^RYGB rats and DIO controls. Note that for clarity purposes and in order to reduce animal numbers, data from groups DIO and FMT^RYGB have exceptionally also been used in Supplementary Figure S5. Data are mean ± s.e.m; n = 3–8 animals per group with pooled data from 2 to 3 independent experiments. *,# P < 0.05, **,## P < 0.01, ***,### P < 0.001 by unpaired Student’s t test (for two groups) or two-way ANOVA (multiple groups) with Tukey correction for multiple testing

Fig. 3

RYGB microbiota transfer reduces inflammation and enhances lipolysis and thermogenic activity in adipose tissue. a–e H&E staining on sections from epididymal (eWAT) and inguinal (iWAT) white adipose tissue (scale bars 200 μm) 5 weeks after fecal microbiota transfer (FMT) from RYGB-operated rats (FMT^RYGB) compared to their respective controls (DIO rats not receiving RYGB microbiota) (a). Relative mRNA expression of genes involved in lipolysis, fatty acid oxidation, and inflammatory cytokines in eWAT (b, c) and iWAT (d, e). f–i BAT weight (in % of total BW) (f), H&E and UCP1 staining of BAT sections (scale bars 200 μm) (g) and relative mRNA expression of genes involved in fatty acid oxidation and thermogenesis in BAT (h). Electron microscopy of ultra-thin sections of BAT depicting mitochondrial fine structure in DIO vs FMT^RYGB animals (scale bar 1 μm) (i). j–l Relative mRNA expression of genes involved in lipolysis in BAT (j), phosphorylation levels of HSL (p-HSL) (k) and free plasma glycerol levels (in mM) (l). m–r Representative infrared images (m) and corresponding rectal body temperature (in °C) after 6 h of cold-exposure (n). Oxygen consumption (VO₂; in ml/(h*kg)) (o), carbon dioxide production (VCO₂; in ml/(h*kg)) (p), heat production (in kcal/(h*kg)) (q) and locomotor activity (r) of FMT^RYGB and DIO rats (at 5 weeks of FMT treatment). Seahorse analysis of murine adipocytes after pretreatment with rat serum (DIO vs. FMT^RYGB, PBS as control) (s). Data are mean ± s.e.m; n = 3–8 animals per group with pooled data from 2 to 3 independent experiments. *P < 0.05, ** P < 0.01, *** P < 0.001; unpaired Student’s t test

Fig. 4

Modulation of the gut microbiome by RYGB surgery, RYGB microbiota transfer (FMT^RYGB) and RYGB microbiota deletion (RYGB(ABx)) in HFD-induced obesity (DIO). a–c Non-metric multidimensional scaling (NMDS) dissimilarity analysis of 16S rRNA gene profiling data, alpha diversity, and richness based on amplicon sequence variant (ASV)-count (a). Mean relative abundance of bacterial families and phyla (A = Actinobacteria, B = Bacteroidetes, F = Firmicutes, P = Proteobacteria, T = Tenericutes, V = Verrumicrobiota) (b). Significantly altered Taxa (c). Distribution and hierarchical clustering of microbial genera (d). Heatmap of portal vein plasma metabolites of respective groups (e). n = 6–10 animals per group with pooled data from 3 independent experiments; *P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 5

RYGB microbiota transfer modulates intestinal BA receptor signaling and promotes intestinal health in HFD-induced obesity. a–e Concentrations of unconjugated BA (UBA) species (in μM) (a), glycine (G)- (b) and taurine (T)-conjugated BA (CBA) species (in μM) (c), and of total bile acids in plasma (in μM) (d), ratio of total CBA to UBA (e) and hydrophobicity index (f) in plasma from portal vein collected at 5 weeks after fecal microbiota transfer (FMT) from RYGB-operated rats (FMT^RYGB) compared to their respective controls (DIO rats not receiving RYGB microbiota). g–j Relative mRNA expression of FXR target genes in ileum (g), representative immunofluorescent FXR-stained ileal sections (scale bars 100 μm) (h) and immunohistochemistry FGF19-stained ileal sections (scale bars 200 μm) (i), and relative mRNA expression of target genes involved in BA transport in the ileum (j) of FMT^RYGB and DIO animals. k, l Hepatic mRNA expression of genes involved in BA metabolism (k) and FXR target genes (l). m–o Plasma free taurine concentrations (m) and relative mRNA expression of hepatic target genes involved in taurine metabolism (n). Relative mRNA expression (normalized to DIO) of Tgr5 in ileum and additional organs (o). Data are mean ± s.e.m; n = 6–10 animals per group with pooled data from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired two-tailed Student’s t test

Fig. 6

Metaproteomics of intestinal microbiota. a Principal component analysis (PCA) of protein group abundances reveals significant differences (PERMANOVA P = 0.001) in the metaproteomes between treatments. b Significant changes (Kruskal-Wallis test P = 0.0456) in the abundance of choloylglycine hydrolase function (KEGG K01442), known to deconjugate conjugated bile acids, between treatments in the intestinal microbiome was observed, with the post hoc pairwise analysis revealing a significant change between RYGB and DIO (Dunn test, P = 0.0069 (**)), and a trend between DIO and FMT^RYGB (Dunn test, P = 0.0823 (#)). c Microbiome bacteria genera that are significantly associated with the abundance of choloylglycine hydrolase. n = 6–10 animals per group with pooled data from 3 independent experiments

Fig. 7

RYGB microbiota requires intestinal FXR and systemic TGR5 signaling to transfer metabolic health benefits in HFD-induced obesity. a–d Relative mRNA expression of FXR target genes in ileum (a), relative body weight change (in %; øBW per group: FMT(Gly-MCA) W0: 29.3 g, W5: 35.7 g; FMT^RYGB W0: 28.4 g, W5: 31.4 g) (b), and epididymal (eWAT) (c) and inguinal (iWAT) white fat mass (WAT) (d) relative to body weight of HFD-fed mice at 5 weeks of RYGB fecal microbiota transfer (FMT^RYGB) co-treated with Gly-MCA (FMT(Gly-MCA)) compared to FMT control mice without co-treatment with Gly-MCA (FMT^RYGB). e-h Oral glucose-tolerance tests (oGTT) (e) and insulin-sensitivity tests (ITT) (f). OilRedO staining of sections from liver (scale bars 200 μm) (g) and hepatic mRNA expression of FXR target genes (h). i, k Oxygen consumption (VO₂; in ml/(h*kg)) (i), heat (in kcal/(h*kg)) (j) and carbon dioxide production (VCO₂; in ml/(h*kg)) (k). l–o Relative mRNA expression of genes involved in thermogenesis and glucose uptake in BAT (l), H&E and UCP1 staining of sections from BAT (scale bars 200 μm) (m) as well as infrared images (n) and corresponding rectal body temperature (in °C) after 4 h of cold-exposure (o). Data are mean ± s.e.m; n = 5–10 animals per group with pooled data from 2 to 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired two-tailed Student’s t test