Combined metabolic activators therapy ameliorates liver fat in nonalcoholic fatty liver disease patients

Abstract Nonalcoholic fatty liver disease (NAFLD) refers to excess fat accumulation in the liver. In animal experiments and human kinetic study, we found that administration of combined metabolic activators (CMAs) promotes the oxidation of fat, attenuates the resulting oxidative stress, activates mitochondria, and eventually removes excess fat from the liver. Here, we tested the safety and efficacy of CMA in NAFLD patients in a placebo‐controlled 10‐week study. We found that CMA significantly decreased hepatic steatosis and levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine, whereas found no differences on these variables in the placebo group after adjustment for weight loss. By integrating clinical data with plasma metabolomics and inflammatory proteomics as well as oral and gut metagenomic data, we revealed the underlying molecular mechanisms associated with the reduced hepatic fat and inflammation in NAFLD patients and identified the key players involved in the host–microbiome interactions. In conclusion, we showed that CMA can be used to develop a pharmacological treatment strategy in NAFLD patients.


19th Jul 2021 1st Editorial Decision
Thank you for submitting your work to Molecular Systems Biology. We have now heard back from two of the three reviewers who agreed to evaluate your manuscript. Unfortunately, after a series of reminders, we did not manage to obtain a report from Reviewer #4. In the interest of time, and since the recommendations of the other two reviewers are quite similar, I prefer to make a decision now rather than further delaying the process. As you will see from the reports below, the reviewers acknowledge the potential interest of the study. They raise however a series of concerns, which we would ask you to address in a major revision.
Since the reviewers' recommendations are rather clear, there is no need to reiterate all the points listed below. Some of the key issues that would need to be addressed are the following: -The gender and age of the participants need to be considered in the data analysis.
-Reviewer #3 is concerned about the smaller number of controls compared to the study group, which needs to be justified, and limitations in this regard need to be discussed.
-In light of the concerns of Reviewer # 3, we would ask you to edit the manuscript to make sure that the microbiome part is sufficiently clear and easily accessible to the general audience of Molecular Systems Biology.
All other issues raised by the reviewers need to be satisfactorily addressed as well. As you may already know, our editorial policy allows in principle a single round of major revision and it is therefore essential to provide responses to the reviewers' comments that are as complete as possible.
On a more editorial level, we would ask you to address the following issues: Reviewer #1: In the present work, Zeybel et al. have studied the efficacy of combined metabolic activators (CMA) in nonalcoholic fatty liver disease (NAFLD) patients in a placebo-controlled study. The results of plasma metabolites, proteins and gut and oral microbiome are compared between CMA and placebo groups at Day 14 and 70. The study has some interesting findings and potential for treatment strategy in NAFLD patients. Some of the concerns related to the methods and results in the study are: Major points: 1. Of the 31 individuals who participated in this study, 24 were male and 7 were female. Did the authors observe any sex-specific changes in the CMA and placebo group? Some of the metabolites reported in this study, such as those that are part of tryptophan metabolism, are affected by sex of the individual. How do the authors justify that the results observed in the CMA and placebo group is not masked by the male population in this study? 2. For the statistical analysis, the authors have adjusted for weight loss in the linear model. Why wasn't sex and age considered for adjusting along with the weight loss? Both sex and age does have a major influence on the metabolic changes observed in the individuals. It will be good to show how the trend changes when the calculations are adjusted for age, sex and weight loss.
3. For the intervention study, why were the patients in treatment group given a single dose of CMA for 2 weeks and then two doses for the next 56 days? Did increasing the dose after 14 days have an influence in the results observed for patients who took CMA compared to the placebo group? 4. For the inflammatory protein markers measured in this study, can the authors comment whether these observed proteins (CD8A, CSF-1, CCL23, FGF-21, and oncostatin-M (OSM)) are associated with increasing pro-or anti-inflammation response in the treated and placebo groups? 5. Did CMA treatment have an influence on the bile acid levels in the individuals? Considering bile acids have an important role in the progression of NAFLD, it is worthwhile to focus on the primary and secondary bile acids in the treatment and placebo group. Dataset S6 has some primary bile acids that are significant in the treated group.
6. Are the authors planning a follow-up study of these individuals beyond 70 days of treatment? Does CMA treatment have a permanent effect on the oxidation of fats and mitochondrial function? As the treatment was stopped after 70 days, did the treated individuals revert back to the original state?
Minor points: 1. The authors should mention in the methods section when was the blood drawn done (time of the day) and whether it was fasted blood measurements or not. Also, when was the stool sample collected for these individuals? These details are currently lacking in the manuscript.

Reviewer #3:
Zeybel et al., describe how Combined metabolic activators (CMA) act as reducers of liver fat in NAFLD patients. The paper is interesting with potential non-harmful impact for treatment of NAFLD in the future. It drives from previous studies from the Mardinglu group. Here, the authors performed a small clinical study with patients treated with a combination of CMA for 70 days. They provide a thorough analysis, particularly of metabolome and microbiome, in the treated and the control groups. The acquisition of data is appropriate, also the data analysis. The paper is a bit lengthy in the microbiome part since there are no clear conclusions. The paper is not always easy to follow. Some major facts are missing in the main part and might have been overlooked in the comprehensive supplementary files. Some figures that would facilitate the reader to follow the story should be produced. Major comments: 1. A drawback of the study is its small size and the number of controls smaller compared to the study group (2 : 1). Good study design requires the control group to be at least of the same size or larger compared to the treated group. The authors should justify why they believe that the 2 : 1 ratio (treated : control) would be sufficient for this study.

Detailed point-by-point response to the reviewer comments
The authors would like to thank the reviewers for their time and expert comments and the editor for providing us with the opportunity to resubmit our manuscript to the journal following corrections and further changes as requested. We have re-written the text to address the points raised by the reviewers. Our answers to reviewers' questions can be found in blue in the text below. We hope that the reviewers and editors agree that the manuscript has been markedly improved as a result of these changes and that it is now suitable for publication.
To support our findings in our study, we also added results from our recently completed clinical trials related to COVID-19, Alzheimer's Disease and Parkinson's Disease. The results of plasma metabolites, proteins and gut and oral microbiome are compared between CMA and placebo groups at Day 14 and 70. The study has some interesting findings and potential for treatment strategy in NAFLD patients. Some of the concerns related to the methods and results in the study are: Major points: 1. Of the 31 individuals who participated in this study, 24 were male and 7 were female. Did the authors observe any sex-specific changes in the CMA and placebo group? Some of the metabolites reported in this study, such as those that are part of tryptophan metabolism, are affected by sex of the individual. How do the authors justify that the results observed in the CMA and placebo group is not masked by the male population in this study?.

Response:
We appreciate the reviewer's comment regarding overrepresentation of male participants in the study. Generally speaking, NAFLD prevalence is at least two times higher in men than women 1 , however, male subjects were even more predominant in our study. We believe that this was at least partly due to the exclusion of women who are planning pregnancy, breastfeeding, and reluctance to use contraception during the study. Although we attempted to analyse sex-specific changes in metabolite levels, the sample size for female subjects were rather small (3 in CMA group and 4 in placebo), statistical analysis would have been generated with a low statistical power. This is the limitation of this study that we have now highlighted in the manuscript as shown below. We have taken this comment on board and we will ensure the fair female/male recruitment in the design of the phase 2b/phase 3 study. 2. For the statistical analysis, the authors have adjusted for weight loss in the linear model. Why wasn't sex and age considered for adjusting along with the weight loss? Both sex and age does have a major influence on the metabolic changes observed in the individuals. It will be good to show how the trend changes when the calculations are adjusted for age, sex and weight loss.

Response:
We very much appreciate this point. We realized that we missed an important explanation in the statistical analysis section, that we performed this analysis in a pairwise manner. That was done to avoid the effect of individual variations, since we were comparing each subject to themself in different timepoints. So with this paired analysis, adding sex and age in the model will not make any difference in the results since these variables were constant for the same subject, unlike weight variables that changed between visits for each subject. We have added the clarification about the paired analysis in the statistical analysis section.

'The calculation was done by pairing each subject from different visits and adjusted for weight loss in a linear model using the limma package in R (v4.0.2)'
3. For the intervention study, why were the patients in the treatment group given a single dose of CMA for 2 weeks and then two doses for the next 56 days? Did increasing the dose after 14 days have an influence in the results observed for patients who took CMA compared to the placebo group?
Response: We determined the safe dose by considering the findings in the non-clinical animal and phase-1 human studies as well as the existing literature of the constituents of the CMA 1,2 . In order to limit and monitor the potential adverse reactions in NAFLD patients (due to the potential differences in target distribution and pharmacokinetics of the drug between healthy volunteers and patients), we used a lower starting dose that is expected to have a minimal pharmacological effect and is safer to use. Later, we increased the doses (twice a day) and continued screening the patients during the study. 4. For the inflammatory protein markers measured in this study, can the authors comment whether these observed proteins (CD8A, CSF-1, CCL23, FGF-21, and oncostatin-M (OSM)) are associated with increasing pro-or anti-inflammation response in the treated and placebo groups? Response: We appreciate this comment. Generally speaking, these proteins are pro-inflammatory but FGF-21 might have anti-inflammatory properties in this context. A long acting FGF-21 analogue attenuated hepatic inflammation in animal models of liver disease 1 . These proteins are induced by a variety of environmental or metabolic stimuli, play crucial roles in the response to these challenges. As example: 1) CD8 has been well known to be expressed in cytotoxic T cells to recognize and kill infected cells. CD8, usually composed of one CD8α and one CD8β chain 2 ; 2) CSF-1 is a key differentiation, growth and survival factor for monocytes/macrophages and osteoclasts 3 ; 3) CCL23 has been reported to be expressed in activated monocytes and dendritic cells and is a biomarker for some inflammatory diseases 4 ; 4) Fibroblast growth factor 21 (FGF21) levels are increased by fasting, fibrate treatment, obesity, and type 2 diabetes 5 ; 5) Oncostatin M is a member of the IL-6 family of cytokines that induces a key endothelial cell function, initiation of the inflammatory response 6 . 5. Did CMA treatment have an influence on the bile acid levels in the individuals? Considering bile acids have an important role in the progression of NAFLD, it is worthwhile to focus on the primary and secondary bile acids in the treatment and placebo group. Dataset S6 has some primary bile acids that are significant in the treated group.
Response: In total, we measured 32 metabolites belonging to bile acid metabolism. As shown in the summary table of Dataset S6 below, only 2 of them (taurochenodeoxycholate and glycoursodeoxycholate) were significantly increased between Day 70 vs Day 0 in CMA group than placebo. There are also a few significant metabolites both in placebo and drug groups on Day 14 and Day 70 (yellow marked). However, we concluded that these findings require further validation in a larger clinical trial as well as a longer treatment period. Response: Authors are currently planning a Phase 2b trial with a longer period of (36 weeks) treatment. We agree that adding an additional lag phase visit for investigating durability of treatment response is an excellent idea.
Minor points: 1. The authors should mention in the methods section when was the blood drawn done (time of the day) and whether it was fasted blood measurements or not. Also, when was the stool sample collected for these individuals? These details are currently lacking in the manuscript.

Response:
We thank the review for this comment. Fasting blood sampling was performed in the morning (8.00 am -10.00 am). Stool collection kits were provided to the patients and advised to collect stool samples in the last 24 hours of the visit. We have now clarified this in the Method and Extended View Appendix section.
Reviewer #3: Zeybel et al., describe how Combined metabolic activators (CMA) act as reducers of liver fat in NAFLD patients. The paper is interesting with potential non-harmful impact for treatment of NAFLD in the future. It drives from previous studies from the Mardinglu group. Here, the authors performed a small clinical study with patients treated with a combination of CMA for 70 days. They provide a thorough analysis, particularly of metabolome and microbiome, in the treated and the control groups. The acquisition of data is appropriate, also the data analysis. The paper is a bit lengthy in the microbiome part since there are no clear conclusions. The paper is not always easy to follow. Some major facts are missing in the main part and might have been overlooked in the comprehensive supplementary files. Some figures that would facilitate the reader to follow the story should be produced. Major comments: 1. A drawback of the study is its small size and the number of controls smaller compared to the study group (2 : 1). Good study design requires the control group to be at least of the same size or larger compared to the treated group. The authors should justify why they believe that the 2:1 ratio (treated: control) would be sufficient for this study.

Response:
We agree with the reviewer that the sample size is not large. However, we followed patients longitudinally and performed comprehensive multi-omic analysis (proteomics, metabolomics and oral/gut metagenomics) and clinical evaluation at the same time point. With the encouraging results of this phase 2 study, we are planning to conduct phase 2b and 3 clinical trials with larger patient cohorts.
It is not uncommon to perform a 2:1 ratio of treatment and placebo in NAFLD clinical trials 1,2 . We also chose a 2:1 allocation and recruited more patients in the drug group for several reasons. These can be listed as: 1) gaining experience about the efficacy of the drug, 2) better evaluation of in-cohort changes between different time points, and 3) gathering more information about the side effects. 2. It is not clear how the patients were monitored during the 70-day protocol -I understood they were living at home and returning to the clinics for routine investigations connected to the study. This is a crucial piece of information that needs to be visible. If patients were treated in an outpatient clinic setting, this can be the reason for additional variation of the microbiome.
Response: Some of the visits changed as "optional" on the study protocol due to COVID-19 pandemics and local restrictions. Clinical trial nurses performed weekly telephone visits and gathered crucial clinical information including adverse events and assessed compliance to the medications. Patients (both treatment arms) were seen three times by dieticians and received advice for healthy diet. Despite these efforts and presence of a placebo arm we cannot rule out the effect of diet on microbiome.
3. I could not find the information how were patients dispersed by gender. Were they only males? If they were of both sexes it should be explained that both gender groups were combined in statistical analyses.

Response:
We thank the reviewer for this comment. We recruited 24 males and 7 females for this study. In general, NAFLD prevalence is at least two times higher in men than women 1 . The male subjects were more predominant in this study because we excluded the women who are planning pregnancy, breastfeeding, and reluctance to use contraception during the study according to the study protocol. We have now clarified this in the Methods section.
4. CMA: The composition should be visible in the main paper. If it is a part of IP protection it has to be disclosed.

Response:
The composition of CMA has been provided in the main manuscript method section (under Randomization, Interventions, and Follow-up subtitle) as well as in the supplementary appendix. Please see the paragraph below.
"CMA treatment was given for 70 days after the initial diagnosis of high hepatic fat by MRI-PDFF. Patients in the treatment group took one dose of CMA (3.73 g L-carnitine tartrate, 1 g nicotinamide riboside, 12.35 g serine, and 2.55 g N-acetyl-l-cysteine) daily for the first 14 days (after dinner) and two doses daily for the next 56 days (after breakfast and dinner). Further information is provided in the Supplementary Appendix." 5. From the biochemical standpoint there is a statistically significant difference between the control and treated groups in multiple metabolites, some of them not described in detail in the paper. Could the authors explain why i. e. the changes in blood lipid composition was not studies deeper? Response: We thank the reviewer for the guidance. Although abnormal lipid metabolism is characteristic of NAFLD, this is often demonstrated at hepatic lipidomics level rather than plasma lipid changes.
Certain lipids mediate inflammatory pathways leading to oxidative stress and may contribute to disease progression. Interestingly, a few metabolites in fatty acid metabolism significantly altered, which 2R,3R-dihydroxybutyrate were decreased both on Day 14 and Day 70 in CMA group (p= 0,00007 and p= 0,0000006 respectively) while an increase of 3-hydroxybutyrate (BHBA) on Day 14 (p= 0,02). Landmark lipids of diacylglycerol and dihydroceramide levels were not altered throughout the study. We observed some alterations in phosphatidylcholine and phosphatidylethanolamine along with ceramide species; however, as we had not measured hepatic levels of these lipids, we concluded that the effect of CMA on hepatic lipid composition needs further hepatic lipid studies.
6. The authors disclose that the fat content of the liver was evaluated by the MRI method and no biopsies were taken. A clinician should judge whether the MRI fat data from table 1 are sufficient to declare in the manuscript title that CMAs reduce liver fat. Certainly, the patients have lost weight. Is this only the effect of CMA? Another clinical trial in biopsy proven NASH is needed to delineate the effects of CMA on hepatic injury and inflammation.

Response:
We thank the reviewer for the comment. Recent clinical studies demonstrated that Magnetic Resonance Imaging-derived proton density fat fraction (MRI-PDFF) can accurately measure hepatic fat content to assess treatment response in phase-2 NASH trials 1 . MRI-PDFF-based hepatic fat measurement shows excellent correlation with biopsy-based steatosis assessment 2 . However, we and regulatory authorities agree with the reviewer that there is a need to perform a biopsy proven Phase 3 NASH study to investigate the effect of supplementation on inflammation or steatohepatitis. 7. Safety issues: it is not clear whether the side effects described are linked to CMA or are certainly not linked to the treatment.

Response:
We do not have evidence to claim these adverse events are directly related to treatment. However, larger clinical trials with a longer treatment might provide evidence for the matter. Of note, we recently published a phase 2 and 3 clinical trial to evaluate the efficacy of CMA in COVID-19 patients. In a total of 397 participants, side effects were seen only in 4 (1%) of them 1 . All had a mild rash or skin changes and decided to complete the study without changing the dose.
8. Certain weight loss combinations of drugs can have also detrimental effect in human health. Authors should better explain why lowering uric acid and creatinine is certainly beneficial for patients.
Response: Uric acid is a natural waste product from the digestion of foods that contain purines. Hyperuricemia is frequently associated wıth a number of clinical conditions including gout, hyperuricemic nephropathy and coronary artery disease 1 . Research has also shown a link between high uric acid levels and type 2 diabetes, high blood pressure, and fatty liver disease 2 . There is some evidence to suggest that patients with high creatinine levels in the blood may be more at risk for either developing kidney damage or for kidney damage that they already have getting worse 3 . However, there is limited data and larger studies are required to study the effect of creatinine lowering therapy on kidney protection. common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; are tests one-sided or two-sided? are there adjustments for multiple comparisons? exact statistical test results, e.g., P values = x but not P values < x; definition of 'center values' as median or average; definition of error bars as s.d. or s.e.m.
1.a. How was the sample size chosen to ensure adequate power to detect a pre-specified effect size? 1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria preestablished?
3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe.
For animal studies, include a statement about randomization even if no randomization was used.
4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
Is there an estimate of variation within each group of data? NA The sample pair with missing value was removed in a pairwise comparison.
Yes, patient information (patient number, date of birth, initials) was entered into the web-based randomization system, and the randomization codes were entered into the electronic case report form.

Manuscript Number: MSB-2021-10459
Yes, we have described the test and the significance level we used in the figure legends. Moreover, the complete results of statistical analysis can be found in the corresponding supplementary files.
Yes, we followed the data transformation based on the test requirement. For example, limma requires a log transformation before calculation when we compared the differnece across time points within each group. In addition, we used one-way ANOVA to test the difference between different groups, it is non-parametric without the requirement for specific distribution.
We used paired analysis when we compared the difference across different time points, so this is not calculated.

NA
Yes, the randomization procedure is blind for the investigator. NA 1. Data the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.
The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
We included 31 NAFLD pateints in our statistical analysis, including 20 samples of NAFLD patients and 11 samples of placebo group. Since the sample size of each group is higher than 10, the statistical power should be enough. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.

definitions of statistical methods and measures:
a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).

Reporting Checklist For Life Sciences Articles (Rev. June 2017)
This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal's authorship guidelines in preparing your manuscript.