The human gut microbiota contributes to type-2 diabetes non-resolution 5-years after Roux-en-Y gastric bypass

Abstract

Roux-en-Y gastric bypass (RYGB) is efficient at inducing drastic albeit variable weight loss and type-2 diabetes (T2D) improvements in patients with severe obesity and T2D. We hypothesized a causal implication of the gut microbiota (GM) in these metabolic benefits, as RYGB is known to deeply impact its composition. In a cohort of 100 patients with baseline T2D who underwent RYGB and were followed for 5-years, we used a hierarchical clustering approach to stratify subjects based on the severity of their T2D (Severe vs Mild) throughout the follow-up. We identified via nanopore-based GM sequencing that the more severe cases of unresolved T2D were associated with a major increase of the class Bacteroidia, including 12 species comprising Phocaeicola dorei, Bacteroides fragilis, and Bacteroides caecimuris. A key observation is that patients who underwent major metabolic improvements do not harbor this enrichment in Bacteroidia, as those who presented mild cases of T2D at all times. In a separate group of 36 patients with similar baseline clinical characteristics and preoperative GM sequencing, we showed that this increase in Bacteroidia was already present at baseline in the most severe cases of T2D. To explore the causal relationship linking this enrichment in Bacteroidia and metabolic alterations, we selected 13 patients across T2D severity clusters at 5-years and performed fecal matter transplants in mice. Our results show that 14 weeks after the transplantations, mice colonized with the GM of Severe donors have impaired glucose tolerance and insulin sensitivity as compared to Mild-recipients, all in the absence of any difference in body weight and composition. GM sequencing of the recipient animals revealed that the hallmark T2D-severity associated bacterial features were transferred and were associated with the animals' metabolic alterations. Therefore, our results further establish the GM as a key contributor to long-term glucose metabolism improvements (or lack thereof) after RYGB.

Keywords: Microbiota; bacteroides; bariatric surgery; clustering; diabetes remission; fecal matter transplantation; obesity; relapse; roux-en-Y gastric bypass; type-2 diabetes.

Figure 1.

Categorization and long-term evolution of T2D severity after RYGB (n = 100). (a) Patients’ PCA projection according to their T2D severity at all time points. ● represent patients’ baseline position, while ▲ and ■ indicate their position at 1- and 5-years post-RYGB, respectively. The arrows represent the intensity and the directionally of each variable’s contribution to the projection. (b) Long-term trajectories of T2D severity, and association with long-term T2D remission, which was defined according to the ADA’s criteria. 5y-, 5-years; DR, T2D remission; Rep+, good responders; Rep-, poor responders; RYGB, Roux-en-Y gastric bypass; T2D, type-2 diabetes.

Figure 2.

T2D severity after RYGB is associated with specific GM signatures (n = 99). (a) Proportion of explained GM variation (dbRDA based on genus-level Bray-Curtis dissimilarity matrix). (b) Left: Relative class abundances representing at least 0.1% of the total ecosystem (n = 13) across T2D severity clusters. Right: Cliff’s delta effect size estimates, 95% confidence intervals and Welch Two-Samples t-tests p-values across T2D severity clusters and taxonomic classes representing at least 0.1% of the total ecosystem (n = 13). (c) Genus-level community PCoA ordination of Bray-Curtis dissimilarities. Arrows represent the 6 genera contributing the most to the ordination (R² > 0.5). (d) Cliff’s delta effect size estimates, 95% confidence intervals and Welch Two-Samples t-tests p-values across T2D severity clusters and bacterial species representing at least 0.1% of the total ecosystem (n = 95). #-noted differences remain significant when controlled for metformin intake and cluster transition. (e) Heatmap of Spearman’s rank correlations between the abundance of the 16 species presented in Figure D, and 5-years clinical variables (*: p < .05; **: q < 0.1). 5y, 5-years; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; BMI, body mass index; HDL, high density lipoprotein; PCoA, Principal Coordinate Analysis; RYGB, Roux-en-Y gastric bypass; T2D, type-2 diabetes.

Figure 3.

Metabolic alterations were transferred upon fecal matters transfers (n = 52). (a) Experimental design. (b) Body (●), fat (■) and lean (▲) mass evolutions throughout the follow-up. (c) Daily food intake. (d) Daily fecal excretion of calories. (e) Daily caloric absorption. (f) Oral glucose tolerance test (dose: 2 g/kg) performed 10 weeks after the inoculation. (g) AUC of the glycemia measured during the OGTT. (h) Insulin levels quantified in plasma collected during the OGTT. (i) HOMA-IR index. (j) QUICKI index. (k-m) Liver, epididymal and inguinal adipose tissues masses. Numbers within the dots on boxplots represent the donor’s number according to Table 2. AT, adipose tissue; AUC, area under the curve; FMT, fecal microbiota transfer; HFHS, high-fat high-sucrose; OGTT, oral glucose tolerance test; MRI, magnetic resonance imaging; PEG, polyethylene glycol; r-, recipient.

Figure 4.

The transfer of bacteria associated with T2D severity induces alterations of the recipients’ metabolic phenotype (n = 52). (a) Overall proportion of donor’s species detected in the recipient animals (proportion in green). (b) Proportion of GM variation (dbRDA based on genus-level Bray-Curtis matrix) explained by recipient animals’ groups and body weight. (c) Left: Relative class abundances across recipients’ clusters. Right: Cliff’s delta effect size estimates, 95% confidence intervals and Welch Two-Samples t-tests p-values across recipients’ clusters and taxonomic classes representing at least 0.1% of the total ecosystem (n = 10). (d) Cliff’s delta effect size estimates, 95% confidence intervals and Welch Two-Samples t-tests p-values between r-Mild and r-Severe animals across bacterial species representing at least 0.1% of the total ecosystem (n = 75 species in total). Bolded species remain significant when considering pseudoreplication. (e) Heatmap of Spearman’s rank correlations between the abundance of the 15 species presented in Figure C, and animals’ phenotypic variables (.: p < .1, *: p < .05; **: q < 0.1; FDR correction; #-noted relations remain significant (p < .05) when pseudoreplication is accounted for).