Review Reports - Bacterial Metabolites in the Plasma of Type 1 Diabetes Patients: Acetate Levels Are Elevated and Correlate with Glycated Haemoglobin and Para-Cresol Is Associated with Liver Disturbances and Hypertension

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study by Jiménez-Varas et al. is very interesting, as it examines the relationships between SCFAs, MCFAs, and p-cresol with glycemic control, liver dysfunction, and hypertension. The manuscript is suitable for publication.

However, the authors should clarify that serum acetic acid is not solely derived from microbial fermentation but is also produced through endogenous acetogenesis. In patients with diabetes—particularly those with poor glycemic control—endogenous production of acetic acid may be increased. This mechanism could partly explain the observed association between acetic acid levels and HbA1c.

Author Response

The authors should clarify that serum acetic acid is not solely derived from microbial fermentation but is also produced through endogenous acetogenesis. In patients with diabetes—particularly those with poor glycemic control—endogenous production of acetic acid may be increased. This mechanism could partly explain the observed association between acetic acid levels and HbA1c.

We agree with reviewer 1 that endogenous acetogenesis can also contribute to the serum acetic acid levels, although the main contribution to these levels come from microbial fermentation. Thus, we have modified the text in the Discussion section (line 318):

“…disease. Finally, a proportion of circulating SCFA levels may derive from endogenous processes such as acetogenesis; since endogenous production of SCFAs may be increased in patients with T1D, particularly those with poor glycemic control, this mechanism could partly explain the observed association between SCFA levels and HbA1c levels.”

Reviewer 2 Report

Comments and Suggestions for Authors

Technically the research was well done and the manuscript is well written. However, the novelty and originality are not the best. In my opinion, you should modulate some of the enthusiastic comments about your results.

You have written in lines 19-21 and repeated in lines 67-69: “Recent research has explored the potential role of gut microbiota and its metabolites in modulating immune responses and influencing the development of autoimmune diseases like T1D”. This idea has been published by different researchers since at least 10 years ago. Please look for the first references about it.

In lines 224-225 you have written: “Here we described for the first time, at least to the best of our knowledge, that serological AA levels were significantly increased in T1D patients, …”. You are not de first ones. Acetate in serum depends of diet while propionate is related to the blood glucose control in T1D patients. Please see: DOI: 10.1136/bmjopen-2024-096994

Your conclusion: “To develop strategies to restore gut health and immune balance could be essential for the control of T1D”. Since the first studies of gut microbiota in patients with T1D (12 years ago or so) to the last one by Yarmohammadi et al. (2025) it has been published that: “By determining the gut microbiota alterations in patients with diabetes, interventional strategies could be designed to modulate its composition as an adjunctive therapy in diabetes” (doi: 10.1002/edm2.70071).

My suggestion is to do a better revision of the scientific studies about the subject.

Author Response

We agree with reviewer 2 that the possible involvement of short chain fatty acids in T1D have been studied in the last 10 years approximately. However, most of these studies have been performed in feces or in animal models. Circulating metabolites, like serum short chain fatty acids were studied some years laters… but we agree with reviewer that, perhaps the expression “recent research” is more associated with studies performed more recently. Thus, we have modified the text in the Abstract and in the Introduction section (line 77).

We are sorry, but the paper that you cite, entitled: “Sex-specific associations of serum short-chain fatty acids with glycaemic control: an Italian cross-sectional study in adults with type 1 diabetes” only analyze T1D patients, not healthy controls, as it can be read at the beginning of the paper:

Design

Observational study.

Setting

The study was conducted at the diabetes outpatient clinic of Federico II University Teaching Hospital, Naples, Italy.

Population

The study included 198 adults with T1D (100 men and 98 women), aged 18–79 years.

Therefore, they do not compare the levels of T1D patients and healthy controls, as in our study. They found that acetic acid levels are higher in T1D women than in T1D men (Table 1), but they do not say anything about the comparison between T1D patients and healthy controls, since they only include in their study T1D patients.

We agree with the reviewer that short chain fatty acid levels can be modified with the diet, and also with the endogenous production. For that reason we included as a limitation of the study the lack of information about the diet (lines 314-316); we have modified this sentence including the reference cited by the reviewer:

“However, a limitation of the study could be the lack of information about the diet of patients and controls, since diet can modify the composition of the microbiota, and therefore, the circulating metabolites, as it has been previously shown with the AA levels (21).”

We have also included in this paragraph a new sentence regarding the endogenous production of acetate or propionate:

We agree with the reviewer that other authors have performed the same suggestion previously. Thus, we have modified the text (line 329):

“As it has been previously suggested by other authors (27, 37), to develop strategies to restore gut health and immune balance could be useful for the control of T1D.”

Reviewer 3 Report

Comments and Suggestions for Authors

This study was designed to compare plasma bacterial metabolite levels between Type 1 Diabetes (T1D) patients and healthy controls, as well as to correlate these metabolite levels with the patients' demographic, clinical, and analytical data. However, the manuscript is written poorly, and the data presentation is weak. Therefore, I would not recommend this manuscript for publication in IJMS. The specific comments and suggestions are as follows,

Line17-21: “While the exact cause of Type 1 Diabetes (T1D) .............. to immune diseases like T1D" this statement should be revised.” This statement is not clear, it should be revised and concise. The background statement should be to the point.
Line 21: “we designed a study: 1. to compare the....” I suggest the bullets should be deleted and a smooth paragraph of the hypothesis should be made. The same comments should be followed for the results part in the abstract. Similarly, Lines 40-41: “To develop strategies to restore gut microbiota health and immune balance could be essential for the control of T1D”. This should be deleted and replaced with the concluding remarks. The overall abstract should be restructured.
Lines 46-48: “Type 1 diabetes (T1D) is an autoimmune condition ...........leading to little or no insulin production.” This is the general statement that should be cited with the references.
Line 54: “While the exact cause remains unclear, it is widely believed that both genetic. There are a lot of researches about the diabetic study, so this statement should be corrected.
Lines 79-81: “Thus, we performed the current study with the aim to analyze ..............the levels found in a cohort of healthy controls (HC).” This should be revised, its grammatically incorrect.
Ethics Statement: Ethical approval number should be included in this statement.

Author Response

Line17-21: “While the exact cause of Type 1 Diabetes (T1D) .............. to immune diseases like T1D" this statement should be revised.” This statement is not clear, it should be revised and concise. The background statement should be to the point.

Following the suggestion of the reviewer, we have modified the background statement of the Abstract (lines 27-29):

“Type 1 Diabetes (T1D) is thought to result from the interaction of genetic and environmental factors, with different studies highlighting a potential role for the gut microbiota and its metabolites in modulating immune responses and disease development.”

Line 21: “we designed a study: 1. to compare the....” I suggest the bullets should be deleted and a smooth paragraph of the hypothesis should be made. The same comments should be followed for the results part in the abstract. Similarly, Lines 40-41: “To develop strategies to restore gut microbiota health and immune balance could be essential for the control of T1D”. This should be deleted and replaced with the concluding remarks. The overall abstract should be restructured.

Following the suggestion of the reviewer, we have restructured the overall abstract:

INTRODUCTION: Type 1 Diabetes (T1D) is thought to result from the interaction of genetic and environmental factors, with different studies highlighting a potential role for the gut microbiota and its metabolites in modulating immune responses and disease development. We hypothesized that patients with T1D exhibited altered levels of circulating bacterial metabolites compared with healthy controls (HC), and that these metabolite profiles were associated with key demographic, clinical, and analytical features of the disease.

METHODS: A total of 91 T1D patients and 58 HC were recruited. Plasma samples were collected and analyzed with gas chromatography coupled to mass spectrometry for the detection of the metabolites: short-chain fatty acids (SCFA: acetate [AA], propionate [PA], isobutyrate [IBA], butyrate [BA], isovalerate [IVA], valerate [VA] and methyl valerate [MVA]), medium-chain fatty acids (MCFA: hexanoate [HxA] and heptanoate [HpA]) and para-cresol (p-cresol). We also calculated the ratios between the different SCFA with AA.

RESULTS: T1D patients showed significantly higher circulating AA levels than HC, along with reduced PA/AA and IBA/AA ratios, indicating an altered SCFA profile. SCFA diversity was lower in T1D patients, with reduced detection of BA, and total SCFA levels were increased mainly due to elevated AA. AA levels were higher and SCFA ratios lower in women with T1D compared with healthy women, while p-cresol levels were higher in men with T1D than in healthy men. In T1D patients, AA levels positively correlated with HbA1c, whereas PA/AA, IBA/AA, and BA/AA ratios showed negative correlations, particularly in women. MV/AA and non-AA/AA ratios were inversely associated with glucose levels, again mainly in women. p-Cresol levels correlated positively with age and ferritin and were higher in T1D patients with liver dysfunction or hypertension.

CONCLUSIONS: T1D is associated with a marked alteration in circulating gut-derived metabolites, characterized by increased AA levels, particularly in women, and an imbalance in SCFA ratios that correlates with glycemic control. These findings, together with the associations observed for p-cresol and metabolic comorbidities, support a role for the gut microbiota–host metabolic axis in T1D.

Lines 46-48: “Type 1 diabetes (T1D) is an autoimmune condition ...........leading to little or no insulin production.” This is the general statement that should be cited with the references.

The first three sentences of the first paragraph of the introduction section are based on the first reference of the paper (Atkinson MA, Eisenbarth GS, Michels AW. Type 1 diabetes. Lancet. (2014) 383: 69-82). Following the suggestion of the reviewer we have added the number of this reference at the end of the first sentence.

Line 54: “While the exact cause remains unclear, it is widely believed that both genetic. There are a lot of researches about the diabetic study, so this statement should be corrected.

According to the suggestion of the reviewer, we have modified the text of the second paragraph of the Introduction section (lines 64-68):

“Although the precise etiology of T1D is not fully understood, extensive research indicates that both genetic and environmental factors contribute to its development. Genetic predisposition is particularly important, with certain genes, especially those within the human leukocyte antigen (HLA) region, conferring increased susceptibility. In addition, genome-wide association studies (GWAS) have identified more than 50 other loci that influence T1D risk (3-4).”

Lines 79-81: “Thus, we performed the current study with the aim to analyze ..............the levels found in a cohort of healthy controls (HC).” This should be revised, its grammatically incorrect.

Following the comments of the reviewer, we have corrected the sentence:

“Thus, we performed the current study with the aim of analyzing the levels of circulating SCFAs, MCFAs, and p-cresol in a cohort of T1D patients and comparing them with those in a cohort of healthy controls (HC).”

Ethics Statement: Ethical approval number should be included in this statement.

According to reviewer’s suggestion we have added the approval number of our local ethical committee:

“This study was approved by the local Ethic Committee of the Hospital Clínico San Carlos (Comité Ético de Investigación Clínica del Hospital Clínico San Carlos), with the following approval number: C.I. 22/002-E.”

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors carefully revised the manuscript and answered the suggestions and comments, therefore, I would like to accept in the present form.