Multi-omics investigation of spontaneous T2DM macaque emphasizes gut microbiota could up-regulate the absorption of excess palmitic acid in the T2DM progression

Reviewer #1 (Public review):

Summary:

The authors sought to identify the relationships between gut microbiota, lipid metabolites and the host in type 2 diabetes (T2DM) by using spontaneously developed T2DM in macaques, considered among the best human models.

Strengths:

The authors compared comprehensively the gut microbiota, plasma fatty acids between spontaneous T2DM and the control macaques, verifying the results with macaques in a high-fat diet-fed mice model.

Comments on revisions:

The authors responded to the comments raised, and the manuscript has been improved.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

The authors tried to identify the relationships between gut microbiota, lipid metabolites and the host in type 2 diabetes (T2DM) by using spontaneously developed T2DM in macaques, considered among the best human models.

Strengths:

The authors compared comprehensively the gut microbiota, plasma fatty acids between spontaneous T2DM and the control macaques, and tried verified the results with macaques in high-fat diet-fed mice model.

Weaknesses:

The observed multi-omics on macaques can be done on humans, which weakens the conclusion of the manuscript, unless the observation/data on macaques could cover during the onset of T2DM that would be difficult to obtain from humans.

Regarding the metabolomic analysis on fatty acids, the authors did not include the results obtained form the macaque fecal samples which should be important considering the authors claimed the importance of gut microbiota in the pathogenesis of T2DM. Instead, the authors measured palmitic acid in the mouse model and tried to validate their conclusions with that.

In murine experiments, palmitic acid-containing diet were fed to mice to induce diabetic condition, but this does not mimic spontaneous T2DM in macaques, since the authors did not measure in macaque feces (or at least did not show the data from macaque feces of) palmitic acid or other fatty acids; instead, they assumed from blood metabolome data that palmitic acid would be absorbed from the intestine to affect the host metabolism, and added palmitic acid in the diet in mouse experiments. Here involves the probable leap of logic to support their conclusions and title of the study.

In addition, the authors measured omics data after, but not before, the onset of spontaneous T2DM of macaques. This can reveal microbiota dysbiosis driven purely by disease progression, but does not support the causative effect of gut microbiota on T2DM development that the authors claims.

We are sorry for misunderstanding your point and failing to address your question regarding macaque fecal metabolomics in our previous response. Our study performed untargeted metabolomics on macaque feces and indeed detected the differential metabolite palmitic acid (PA) content, which showed an obvious decrease in T2DM macaques compared with the control (Table 1). However, the difference in PA level between the two groups was not significant (p = 0.17). It may be attributed to the limitation of untargeted metabolomics methodology in absolute quantitative analysis. In addition, we found many other long-chain fatty acids were down-regulated in the T2DM macaque feces (Table 1). Such results are consistent with our observation in murine experiments. We examined PA levels in the feces, ileum, and serum in mice and found that PA level was significantly decreased in fecal samples but increased in the ileum and serum. These findings demonstrated that without the transplantation of gut microbiota, the ileum could not absorb the PA effectively even at a high concentration of ingested PA. Only mice receiving fecal microbiota transplants from T2DM macaques and fed a high-PA diet showed a significant increase in the ileum and serum alongside a decrease in fecal PA concentration. Both the macaque metabolomics and mice experiment results suggest that gut microbiota mediated the absorption of excess PA in the ileum leading to the accumulation of PA in the serum. In the revised manuscript, we added the results of all differential metabolites in Table S2.

Author response table 1.

Table 1. Differential analysis of palmitic acid and other fatty acids from fecal untargeted metabolomics in macaques.

Regarding the causative effect of gut microbiota on T2DM development, we agree with the reviewer that the omics data were obtained after, but not before, the onset of spontaneous T2DM macaques, the microbiota dysbiosis is probably driven by disease progression. For this reason, we have changed the title of our manuscript and some of our conclusions, which can be found in our response below.

Reviewer #1 (Recommendations for the authors):

As described above, the data presented does not support the notion that gut microbiota change in T2DM macaques promote the disease - rather it showed the outcome of the disease progression. In addition, the involvement of palmitic acid absorption was only shown in mice but not in macaques. Therefore, the authors should change their title and conclusions to more precisely reflect their observation.

According to your suggestion, we changed the title and the conclusion to make them more precise and avoid emphasizing the causative effect of gut microbiota on T2DM. The new title is “Multi-omics investigation of spontaneous T2DM macaque emphasizes gut microbiota could up-regulate the absorption of excess palmitic acid in the T2DM progression”. We also revised the wording of the results and conclusions to acknowledge the limitation of our study, “We also revealed the specific structure of gut microbiota that promoted T2DM development by regulating the absorption of excess PA in mice, providing experimental evidence for the functional role of gut microbiota in T2DM pathogenesis.” (Lines 122-125), “In particular, concentrations of PA, palmitoleic acid, and oleic acid were significantly higher in the T2DM group than control group (p<0.05 and VIP>1). The concentration of PA in the plasma of T2DM macaques increased, while the concentration of palmitic acid in the feces decreased (Figures 3F and G, Table S2).” (Lines 228-233), and “Our study confirms the functional role of gut microbiota and PA in the T2DM progression. The microbiota composition, specifically higher abundance of R. gnavus (current name: M. gnavus) and Coprococcus sp., and lower abundance of Treponema, F. succinogenes, Christensenellaceae, and F16, promoted the absorption of excess PA which is important for the development of T2DM. However, in this study, such microbial alterations were detected in macaques after they had developed the disease of T2DM instead of before or onset of T2DM, the causative effect of gut microbiota and their action mechanism on the development of T2DM is worth further investigation.” (Lines 450-458).