Chronic Repression of mTOR Complex 2 Induces Changes in the Gut Microbiota of Diet-induced Obese Mice

Abstract

Alterations in the gut microbiota play a crucial role in host physiology and metabolism; however, the molecular pathways underlying these changes in diet-induced obesity are unclear. Mechanistic target of rapamycin (mTOR) signaling pathway is associated with metabolic disorders such as obesity and type 2 diabetes (T2D). Therefore, we examined whether changes in the regulation of mTOR signaling induced by diet (a high-fat diet [HFD] or normal-chow diet) and/or therapeutics (resveratrol [a specific inhibitor of mTOR complex 1] or rapamycin [an inhibitor of both mTOR complex 1 and 2]) altered the composition of the gut microbiota in mice. Oral administration of resveratrol prevented glucose intolerance and fat accumulation in HFD-fed mice, whereas rapamycin significantly impaired glucose tolerance and exacerbated intestinal inflammation. The abundance of Lactococcus, Clostridium XI, Oscillibacter, and Hydrogenoanaerobacterium increased under the HFD condition; however, the abundance of these species declined after resveratrol treatment. Conversely, the abundance of unclassified Marinilabiliaceae and Turicibacter decreased in response to a HFD or rapamycin. Taken together, these results demonstrated that changes in the composition of intestinal microbiota induced by changes in mTOR activity correlate with obese and diabetic phenotypes.

Figure 1. Resveratrol, but not rapamycin, mitigates HFD-induced obesity.

(A) Effects of resveratrol or rapamycin treatment on temporal changes in the body weight (BW) of NCD- or HFD-fed mice over 8 weeks. (B) BW, (C) adiposity index (AI), and (D) epididymal fat pad weight measured after 8 weeks of resveratrol or rapamycin treatment (E) Food intake (FI) by the resveratrol- or rapamycin-treated groups. Data are expressed as the mean ± SEM (n = 5 per group). F- and p-values are from two-way ANOVA after Bonferroni’s post hoc test. ^*P < 0.05, ^**P < 0.005.

Figure 2. Resveratrol improves, whereas rapamycin impairs, glucose homeostasis.

(A,B) Blood glucose levels (repeated measures two-way ANOVA after Bonferroni’s post hoc test, F = 13.88; P < 0.005 for interaction, F = 186.0; P < 0.005 for time, F = 92.14; P < 0.005 for diet, ^*P < 0.05; ^**P < 0.005 compared with NCD-CT, ^#P < 0.05; ^##P < 0.005 compared with HFD-CT) and (C) area under the curve (AUC) during the glucose tolerance test (GTT) (n = 5 per group). (D) Fasting blood glucose levels (n = 10 per group). (E,F) Blood glucose levels (repeated measures two-way ANOVA after Bonferroni’s post hoc test, F = 3.33; P = 0.06 for interaction, F = 19.65; P < 0.005 for time, F = 5.63; P = 0.08 for diet, ^*P < 0.05; ^**P < 0.005 compared with NCD-CT) and (G) AUC during the insulin tolerance test (ITT) (n = 3 per group). (H) Fasting serum insulin levels (n = 5 per group). (I) HOMA2 indices and (J) QUICKI were calculated from fasting glucose and insulin levels (n = 5 per group). (K) The rate constant for plasma glucose disappearance (K_ITT) during the insulin tolerance test (ITT) (n = 3 per group). Mice were overnight-fasted (16 h) before the GTT and ITT. Data are expressed as the mean ± SEM. F- and p-values are from two-way ANOVA after Bonferroni’s post hoc test. ^*P < 0.05, ^**P < 0.005.

Figure 3. Changes in the faecal bacterial community following resveratrol or rapamycin treatment.

Bacterial communities were clustered using unweighted UniFrac distance-based principal coordinates analysis (PCoA). (A) Principal coordinate (PC) 1 versus PC2 and (B) PC1 versus PC3. The percentage variation in the plotted PC is indicated on the axes. (C) Bar charts showing the relative abundance (%) of different bacterial genera in the different diet and treatment groups. Each group of mice is represented by a different symbol or bar on the x axis of the graph, and each spot or column indicates one sample (n = 5 per group).

Figure 4

Marked differences in the relative abundance of gut bacterial genera in the different diet and treatment groups. Effect of (A,B) resveratrol or (C,D) rapamycin on the relative abundance of 7 or 10 bacterial genera, and hierarchical clustering analysis of these bacterial profiles based on the Manhattan distance, were examined in NCD- and HFD-fed mice. Data are expressed as the mean ± SEM (n = 5 per groups). F- and p-values are from two-way ANOVA after Bonferroni’s post hoc test (Supplementary Table S2). ^*P < 0.05 and ^**P < 0.005 compared with NCD-CT. ^#P < 0.05 and ^##P < 0.005 compared with HFD-CT.

Figure 5

Pearson’s r correlation coefficients heat maps showing the association between metabolic markers and the abundance of specific bacterial genera after (A) resveratrol or (B) rapamycin treatment. Given the large number of correlation tests performed, a significance threshold of P < 0.005 was used, which is indicated by ‘+’.