Figures and data
The changes in gut microbiota in spontaneous T2DM macaques.
(A) Alpha diversity estimates (Shannon index) between T2DM and control groups (ns, not significant, two-tailed t-test, n=8).
(B) Alpha diversity estimates (Simpson index) between T2DM and control groups (ns, not significant, two-tailed t-test, n=8).
(C) Principal Coordinate Analysis (PCoA) (n=8).
(D) Differential analysis of gut microbial composition in T2DM and control groups (n=8).
(E) LEfSe analysis between T2DM and control groups (n=8).
(F) Differential analysis of gut microbial function in T2DM and control groups (n=5). The pathways with red color were associated with T2DM and inflammation. Error bar is mean with ± standard deviation (s.d.).
(G) Differential analysis of gut microbial CAZy enzyme in T2DM and control groups (n=5). CBMs: carbohydrate-binding module (p=0.022, two-tailed t-test); GTs: Glycosyl Transferases (p=0.013, two-tailed t-test); PLs: Polysaccharide Lyases (p=0.017, two-tailed t-test); AA: Auxiliary activity enzymes (ns, not significant, two-tailed t-test); GH: Glycoside hydrolases (ns, not significant, two-tailed t-test); CE: Carbohydrate esterases (p=0.039, two-tailed t-test). For all boxplots: centre lines, upper and lower bounds show median values, 25th and 75th quantiles; upper and lower whiskers show the largest and smallest non-outlier values. In c, ellipses represent the 95% confidence intervals.
Physiological and biochemical parameters of Control and T2DM group
The alterations of fecal metabolites and gene expression in spontaneous T2DM macaques.
(A) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plots based on the metabolic profiles.
(B) Volcano plots of metabolomics (p<0.05, two-tailed t-test).
(C) Fecal metabolites with significant differences between T2DM and control groups (VIP>1, p<0.05, two-tailed t-test).
(D) Enrichment analysis of the differentially abundant pathways between T2DM and control groups (p<0.05, two-tailed t-test).
(E) Non-metric multidimensional scaling (NMDS) analysis between T2DM and control groups (p=0.019, two-tailed t-test).
(F) Volcano plots of DEGs (log fold change≥1, p<0.05, two-tailed t-test).
(G) The GO and KEGG pathway enrichment analyses (p<0.05, two-tailed t-test).
(H) Weighted Gene Co-Expression Network Analysis (WGCNA).
(I) Venn analysis between hub genes and DEGs. In A and E, ellipses represent the 95% confidence intervals. Data shown are from 8 individuals per group.
LCFAs accumulation and inflammation occurred in spontaneous T2DM macaques.
(A-E) The contents of SFA (A, p=0.038), MUFA (B), PUFA (C), N3 (D), and N6 (E) in plasma (ns, not significant, two-tailed t-test).
(F) The univariate analysis by two-tailed t-test, error bar is mean with ± s.d.
(G) The multidimensional analysis by VIP value (VIP>1).
(H-J) The contents of serum inflammatory cytokines, including IL-1β (H, p=0.032), TNF-α (I) and IL-6 (J) (ns, not significant, two-tailed t-test).
(K) Correlation analysis between DEGs, differential metabolites, and differential microbes using Spearman rank correlation (|r|>0.5, adj p<0.05). For all boxplots: centre lines, upper and lower bounds show median values, 25th and 75th quantiles; upper and lower whiskers show the largest and smallest non-outlier values. Data shown are from 7 individuals per group.
blood routine examination of Control and T2DM group
The FMT and high PA diet mice developed pre-T2DM characteristics.
(A) Experimental scheme of FMT and high PA diet treatment.
(B-H) Metabolic analysis, including the trend of FPG within 120 days (B), FPG (C, p=0.0003), OGTT (D), AUC of OGTT (E, p=0.028), FPI (F, p=0.007), ITT (G), and body weight change (H) on day 120.
(I-J) The contents of TC (I, p=0.005) and TG (J, p=0.041) in serum on day 120.
(K and L) Representative H-E staining images of liver (K) and pancreas (L). For all boxplots: centre lines, upper and lower bounds show median values, 25th and 75th quantiles; upper and lower whiskers show the largest and smallest non-outlier values. Significance was determined using one-way ANOVA. In d, g, and h: *p<0.05, **p<0.01. Data shown are from 4-6 individuals per group.
The PA accumulation required the specific gut microbiota.
(A-C) Total PA contents in serum (A, p=0.013), ileum (B, p=0.016) and feces (C, p=0.014) on day 120.
(D) Quantitative RT-PCR for Cd36 transcripts in ileum on day 120 (p=0.049).
(E) The content of IL-17A in ileum on day 120 (p=0.027). For all boxplots: centre lines, upper and lower bounds show median values, 25th and 75th quantiles; upper and lower whiskers show the largest and smallest non-outlier values. Significance was determined using one-way ANOVA. Data shown are from 3-4 individual macaques per group.
(F) NMDS analysis (p=0.001, one-way ANOVA), ellipses represent the 95% confidence intervals.
(G and H) LEfSe analysis between FTPA and control groups (G), FT and control groups (H). Data shown are from 4 individuals per group.
(I) Specific gut microbiota structure promoted the absorption of excess PA by regulating the expression of IL-17A and Cd36, leading to the LCFAs accumulation and insulin resistance.
Integration of multi-omics results.
(A) Insulin resistance, fatty acid oxidation disorders, LCFAs accumulation and inflammation occurred in spontaneous T2DM macaques.
(B) Incomplete mitochondrial LCFAs β oxidation. The expression levels of fatty acid metabolism-related genes HADHB and ACSM3 were downregulated in spontaneous T2DM macaques, which could lead to accumulation of acylcarnitine, including l-propionylcarnitine, hexanoyl-l-carnitine, (r)-butyrylcarnitine, and isovaleryl-l-carnitine.
(C) Gut inflammation. The decrease of Lactobacillus sp. likely caused the reduction of serotonin and indole-3-acetaldehyde, which promotes the expansion of PA producer Erysipelotrichacea and ultimately led to PA accumulation. Both Erysipelotrichacea and Ruminococcus gnavus promote the development of inflammation. Accumulation of PA and inflammation are important factors in the development of T2DM.
(D) Accumulation of PA promoted the development of insulin resistance. In the PA-mTORC1-Akt pathway, the changes of RAP1A, SESTRIN3, and IRS1 expression promoted the development of insulin resistance in spontaneous T2DM macaques. The increase of PA promoted the development of T2DM by up-regulating the NF-κB signaling pathway.
Primers of RT-PCR
RT-PCR reaction components
