Dominant gut Prevotella copri in gastrectomised non-obese diabetic Goto-Kakizaki rats improves glucose homeostasis through enhanced FXR signalling

Abstract

Aims/hypothesis: Drug and surgical-based therapies in type 2 diabetes are associated with altered gut microbiota architecture. Here we investigated the role of the gut microbiome in improved glucose homeostasis following bariatric surgery.

Methods: We carried out gut microbiome analyses in gastrectomised (by vertical sleeve gastrectomy [VSG]) rats of the Goto-Kakizaki (GK) non-obese model of spontaneously occurring type 2 diabetes, followed by physiological studies in the GK rat.

Results: VSG in the GK rat led to permanent improvement of glucose tolerance associated with minor changes in the gut microbiome, mostly characterised by significant enrichment of caecal Prevotella copri. Gut microbiota enrichment with P. copri in GK rats through permissive antibiotic treatment, inoculation of gut microbiota isolated from gastrectomised GK rats, and direct inoculation of P. copri, resulted in significant improvement of glucose tolerance, independent of changes in body weight. Plasma bile acids were increased in GK rats following inoculation with P. copri and P. copri-enriched microbiota from VSG-treated rats; the inoculated GK rats then showed increased liver glycogen and upregulated expression of Fxr (also known as Nr1h4), Srebf1c, Chrebp (also known as Mlxipl) and Il10 and downregulated expression of Cyp7a1.

Conclusions: Our data underline the impact of intestinal P. copri on improved glucose homeostasis through enhanced bile acid metabolism and farnesoid X receptor (FXR) signalling, which may represent a promising opportunity for novel type 2 diabetes therapeutics.

Keywords: 16S rDNA; Bile acids; Goto–Kakizaki rat; Microbiome; Type 2 diabetes.

Fig. 1

Outline of experimental design

Fig. 2

Effects of VSG and gut microbiota transfer in gastrectomised GK rats. Changes in body weight (a, f), blood glucose (b) and glucose tolerance (c–e; g–i) in GK rats following VSG or sham operation (a–e), and following inoculation of GK rats with gut microbiota from gastrectomised GK rats or sham controls (f–i). OGTTs (c) were performed following an overnight (16 h) fast, before VSG (Pre Op) and 91 days after VSG (n = 10) or sham operation (n = 9) (c–e). IPGTTs (g) were performed 12 days after caecal microbiota transfer (CMT) from VSG-treated GK rats (n = 5) or sham-operated GK rats (controls; n = 3–4) (g–i). Data are mean ± SEM. The Kruskal–Wallis test was used to analyse glucose tolerance data. The non-parametric Mann–Whitney U test was used to analyse the other variables. *p < 0.05, **p < 0.01, ***p < 0.001 vs sham-operated GK rats (a–e) or GK rats inoculated with microbiota from sham-operated GK rats (g–i). CumG, cumulative glucose levels during the glucose tolerance test; ΔG, cumulative glucose levels above baseline

Fig. 3

Impact of caloric restriction on blood glucose in sham-operated GK rats. Blood glucose was determined ad libitum in a group of sham-operated GK rats (n = 3) 3, 5 and 7 days after the beginning of the pair-feeding experiment and compared with free-fed sham-operated (n = 3) and gastrectomised (VSG; n = 9) GK rats. Non-parametric Mann–Whitney U tests were applied for statistical data analysis. **p < 0.01, ***p < 0.001 vs sham-operated GK rats

Fig. 4

Effects of VSG on gut microbiome architecture in the GK rat. Frequency of 16S rDNA motifs derived from metagenome sequencing was calculated in each caecum and colon sample from GK rats following VSG (n = 10) or sham operation (n = 8) (a). Means of rDNA motif frequencies were calculated for data from caecum, colon and caecum and colon combined in the two rat groups (b). Data are shown for the 84 most abundant motifs. Each line in (a) represents a different motif colour-coded according to its frequency and each colour in (b) represents the relative proportion of each motif; the arrow in (a) and the blue colour in (b) represent V13A7759. Quantitative RT-PCR was carried out to assess enrichment of the motif V13A7759 (P. copri) in GK rats following VSG (n = 6) (c) and GK rats following caecal microbiota transfer (CMT) from VSG-treated GK rats (n = 3) (d); n = 4 for sham/control in both experiments. Details of all rDNA motifs, associated frequencies and statistical differences between the rat groups are given in ESM Table 2. Data are mean ± SEM. Non-parametric Mann–Whitney U tests were applied for statistical analysis of P. copri abundance. *p < 0.05 vs sham-operated GK rats (c) or GK rats inoculated with microbiota from sham-operated GK rats (d)

Fig. 5

Effects of gut microbiota enrichment in P. copri on glucose homeostasis in GK rats. GK rats were either treated with antibiotics permissive to P. copri or remained in antibiotic-free conditions (a–e). A separate group of GK rats was inoculated with P. copri or heat-inactivated P. copri (f–j). Faecal DNA concentration and P. copri abundance were determined in antibiotic-treated (n = 3–6) and control (n = 4–6) rats (a, b). Faecal DNA concentration and P. copri abundance were determined in GK rats treated with a combination of broad spectrum antibiotics prior to inoculation (post antibiotics) (n = 5), and in rats inoculated with P. copri (n = 6) or heat-inactivated P. copri (control) (n = 3–4) (f, g). Glucose tolerance (c–e, h–j) was determined following an IPGTT in overnight fasted (16 h) GK rats 10 days after antibiotic treatment or P. copri inoculation. Data are mean ± SEM. The Kruskal–Wallis test was used to analyse glucose tolerance data. The non-parametric Mann–Whitney U test was used to analyse the other variables. *p < 0.05 vs control. CumG, cumulative glucose levels during the IPGTT; ΔG, cumulative glucose levels above baseline

Fig. 6

Effects of intestinal P. copri enrichment on plasma bile acid concentrations in the GK rat. Mass spectrometry methods were used to determine the plasma concentration of bile acids in GK rats following VSG (n = 8) or sham operation (n = 5) (a–d), in GK rats following caecal microbiota transfer (CMT) from VSG-treated GK rats (n = 5) or sham-operated GK rats (n = 4) (e–h), in GK rats treated with a combination of kanamycin and vancomycin antibiotics (n = 5) or in the absence of antibiotics in controls (n = 4) (i–l), or in GK rats inoculated with P. copri (n = 6) or heat-inactivated P. copri (n = 3) (m–p). Non-parametric Mann–Whitney U tests were used for statistical analysis. Data are mean ± SEM. *p < 0.05, **p < 0.01, ^†p = 0.05, ^‡p = 0.06, ^§p = 0.07 vs the relevant control. BA, bile acid

Fig. 7

Liver gene expression and hepatic triacylglycerol and glycogen storage in treated GK rats and controls. Liver gene expression (a–d) assessed by quantitative RT-PCR and quantification of hepatic triacylglycerol content (e–h) and glycogen storage (i–l) were determined in GK rats following VSG (a, e, i), in GK rats following caecal microbiota transfer (CMT) from VSG-treated GK rats (b, f, j), in GK rats following treatment with P. copri permissive antibiotics (c, g, k) and in GK rats following inoculation with P. copri (d, h, l). n = 4–10 per group. Data are mean ± SEM. Non-parametric Mann–Whitney U tests were used for statistical analysis. *p < 0.05 vs sham/control rats