Oral Anaerobutyricum soehngenii augments glycemic control in type 2 diabetes

Summary This randomized, double-blind, placebo-controlled trial investigated the impact of 14-day Anaerobutyricum soehngenii L2-7 supplementation on postprandial glucose levels in 25 White Dutch males with type 2 diabetes (T2D) on stable metformin therapy. The primary endpoint was the effect of A. soehngenii versus placebo on glucose excursions and variability as determined by continuous glucose monitoring. Secondary endpoints were changes in ambulatory 24-h blood pressure, incretins, circulating metabolites and excursions of plasma short-chain fatty acids (SCFAs) and bile acids upon a standardized meal. Results showed that A. soehngenii supplementation for 14 days significantly improved glycemic variability and mean arterial blood pressure, without notable changes in SCFAs, bile acids, incretin levels, or anthropometric parameters as compared to placebo-treated controls. Although well-tolerated and effective in improving glycemic control in the intervention group, further research in larger and more diverse populations is needed to generalize these findings.


A B
Suppl. Figure 8: Tax bar plots of fecal bacterial groups at Phylum (A) and Genus (B) level in the placebo group (week 0 and week 4) and in the intervention group (week 0 and week 4).Related to figure 3. Outcome measures: The primary endpoint is the effect of E. hallii versus placebo on (postprandial) glucose excursions as determined by a wearable CGM glucose sensor during 14 days after the start of the intervention.Secondary endpoints are changes in plasma metabolites and glucose/lipids upon standardized meal in relation to changes of E. hallii and other microbiota as well as plasma/fecal SCFA in fecal samples.Also Daily dietary intake will be monitored during the course of the study by https://mijn.voedingscentrum.nl/nl/eetmeter.

Sample Size:
We hypothesize that a peak difference in postprandial glucose excursions using CGM is seen between type 2 diabetes patients using placebo + metformin (13.0 mmol/l) vs E hallii + metformine (10.0 mmol/l) with a SD of 2.5 mmol/l; using unpaired t-test with 0.05 two-sided significance levels and 80% power, we will need 12 subjects per group to detect a significant difference.Of note, all power calculations were performed with an online power calculation (www.biomath.info/power/).Based on the data retrieved from this intervention study, we will apply the prediction algorithm by Zeevi et al for validation 1 .We will include 24 males with type 2 diabetes treated with metformin.All will receive 2 weeks treatment with either E. hallii or 10% glycerol placebo.
Nature and extent of the burden and risks associated with participation, benefit and group relatedness: The total duration of this study is 4 weeks and participants will visit the AMC four (screening, run in, randomization and end of study visit) times.All participants are required to fill out food diaries three days per week and are required to collect feces at baseline, week 2 and week 4 of the study.Furthermore, subjects will undergo a mixed meal test (MMT) before and after the intervention with blood sampling during 2 hours from a placed venflon.Afterwards, subjects are provided with a disposable continuous glucose sensor (FreeStyle Libre) 1 , which will be worn for 14 days before and 14 days after the intervention to monitor (postprandial) glucose excursions both short term and long term, with this Free Style glucose sensor no additional blood glucose measurements with finger pricks need to be done.Also, subjects receive an blood pressure monitor to ambulatory measure their blood pressure during 24 hours before and after treatment.In total subjects will spent approximately 6 hours in the AMC (screening visit, 2x 2 hours for the MMT plus CGM at baseline, randomization and after intervention) and we will collect 240 ml blood (at baseline, week 2 and week 4) in total.

INTRODUCTION AND RATIONALE
The prevalence of type 2 diabetes is expected to rise to 33% of the adult population in 2050 2 .
The pathophysiology of this disease is complex, involving both environmental (dietary) and genetic factors affecting altered intestinal microbiota composition.The development of culture-independent approaches, using high-throughput metagenomic sequencing via 16S rRNA 3 , has drastically increased the knowledge of the gut microbiome, now linking any disturbances in it, both in human and animal models, to the pathophysiology of metabolic diseases such as obesity and type 2 diabetes mellitus (T2DM) 4 6 .Transplantation of lean healthy microbiota in subjects with insulin resistance showed an significantly increased insulin sensitivity and an increased abundance of butyrate-producing bacteria in the gut 7 .In this pilot study we identified a specific increase in the butyrate-producer Eubacterium hallii in small intestinal biopsies of human obese and insulin resistant subjects upon lean donor fecal transplantation (see figure 1).E. hallii is an anaerobic, Gram-positive, catalase-negative bacterium belonging to the clostridial cluster XIVa of the phylum Firmicutes and is present in both murine as human feces 8,9 .It is a butyrate-producing species, but in contrast to other well-known human isolates such as Roseburia and Faecalibacterium spp. that produce butyrate from monosaccharides, E. hallii has the capacity to produce butyrate from lactate and acetate in an acid environment (pH 1-2) as found in the small intestine 9 .This makes it very plausible that this bacterial strain can effectively survive in low pH environments.Moreover, E. hallii has the capability to convert a potentially damaging acid (e.g.lactic acid) into other short chain fatty acid butyrate, which is known to exert beneficial effects on glucose metabolism 9 .
We recently published an animal study in which we studied the effect of E. hallii treatment on metabolism in mice 10 .We found that increasing dosage of daily E. hallii treatment was safe and did not induce adverse effects.Moreover we observed a dose dependent effect of E.   In contrast with 16S sequencing (that showed no differences in E hallii concentrations), by using strain specific shotgun sequencing of the whole bacterial genome, we did find a significant correlation between E Hallii relative abundance and improvement in insulin sensitivity (Rd) with the largest effect in the highest dose group of 10e9/day (high) E. hallii once daily (r= 0.4, p<0.05 ) (Fig 4).Based on the fact that we administered daily E hallii in a drink, we thus postulate that in the group of responders, E. hallii is more likely to pass the stomach and thus exert beneficial effects/engraft in the (small) intestinal microbiota and improve metabolism.In this regard, it is interesting to note that DM2 subjects on metformin treatment have increased levels of lactate in their feces 12 .Since E hallii uses intestinally produced lactate to produce butyrate that is thought to be the beneficial compound driving the effects on insulin sentivity, we hypothesize that adding E hallii to metformin treatment in subjects with DM2 may improve their glycemic control.This as the produced lactate by metformin treatment potentially could be converted by E hallii bacterial strains into the more metabolically beneficial SCFA butyrate.Thus, in the current study we propose to test the effect of daily oral E. hallii treatment in 10e9/ml dose (duration 14 days) in males with type 2 diabetes treated with stable dosages of metformin (2 -3dd 500mg or 2 dd 850mg once daily).
Furthermore, a recent study showed that the amount of faecal butyrate-producing bacteria, such as eubacterium hallii, has a favorable effect on blood pressure regulation (Menni C, Eur Heart Journ 2018).In this regard, we will measure the 24-hour blood pressure of all participants before and after 2 weeks of treatment with eubactecterium hallii or placebo in order to be able to determine any improvement after treatment.
In order to test the efficacy of 2 weeks oral E. hallii treatment on (postprandial) glucose excursions subjects are asked to wear a subcutaneous continuous glucose monitor (CGM), which measures interstitial fluid glucose on certain time points, during seven days.CGMs estimates blood glucose levels with high accuracy 1 .
Thus, the goal of this study is to see if there is any biological effect of E. hallii on the small intestinal microbiota, and (short and long term) glucose metabolism, so that we can see if oral E. hallii is a viable option to improve treatment efficacy.This hypothesis is in line with a recent study that suggested that (postprandial) glucose metabolism as determined by a disposable continuous glucose meter (CGM) can be directly linked to gut microbiota composition 1 .

OBJECTIVES
In this randomized, double-blind, placebo-controlled single center study we propose to study the effect of 14 days oral E. hallii treatment on (postprandial) glucose excursions in subjects with stable metformin monotherapy treated type 2 diabetes.
Primary objective: The primary endpoints are (postprandial) glucose excursions determined by a wearable continuous glucose monitor (CGM) during the 14 days of intervention as well as upon 2h after standardized mixed meal test in relation to SCFA levels in faeces.
Dietary intake will be monitored during the course of the study by online dietary lists.

Secondary objective:
Secondary endpoints are changes in plasma metabolites and glucose/lipids upon standardized meal in relation oral E. hallii treatment on fecal microbiota composition 12 plasma/fecal SCFA and plasma metabolites collected at baseline, week 2 and week 4. Also Daily dietary intake will be monitored during the course of the study by https://mijn.voedingscentrum.nl/nl/eetmeter

STUDY DESIGN
This is a randomized double blind placebo-controlled trial in which we aim to test the effect of 14 days oral E. hallii treatment.During the run in phase subjects will continue their stable dosage of metformin (2 -3dd 500mg or 2 dd 850mg once daily) and their glucose levels will be determined by continuous glucose measurements.Afterwards, subjects will be randomised to either oral E hallii treatment or placebo during 2 weeks (see figure 5): Male Caucasian patients with type 2 diabetes treated with stable dosages of metformin (2 3 dd 500mg or 2 dd 850mg) will be recruited via advertisements in local magazines and at the outpatient clinic.The EMDM2 trial will be executed as follows: Screening A screening visit will be performed in the AMC, inclusion and exclusion criteria will be verified and, after oral and written explanation of the study, informed consent will be obtained.
Medical history will be recorded and physical examination will take place including body weight, height, waist and hip circumference and blood pressure.The use of proton pomp inhibitors and antibiotics in the past 3 months are exclusion criteria.When a potential subject is eligible, appointments will be made for all experiments to be completed during the study.

Run in phase (baseline visit), first and second study day
The preparation for the first study day starts at home with the recording of dietary habits for the duration of the study with an online dietary booklet (https://mijn.voedingscentrum.nl/nl/eetmeter).If necessary (due to for instance long travel distance), the screeningsvisit and the baselinevisit can be combined.After an overnight fast, subjects will visit our clinical research unit (08.30h) and bring a fresh morning fecal sample.A standardized mixed meal test (Nutridrink) will be performed at randomization and after the intervention period, with blood sampling from a placed venflon in the cubital vein during 2 hours.Also, subjects are provided with a disposable continuous glucose sensor (FreeStyle Libre) 1 , which will be worn for 14 days before and 14 days after the intervention to monitor (postprandial) glucose excursions both short term and long term after the intervention.Also, they receive an blood pressure monitor to ambulatory measure their blood pressure during 24 hours before and after treatment.Moreover, subjects will be asked to collect a fresh fecal sample the next morning (t=24h) at both time points (see figure 5 for the study overview).

STUDY POPULATION 4.1 Population
Male Caucasian subjects with type 2 diabetes treated with stable metformin monotherapy will be recruited by local newspaper advertisements.

Inclusion criteria
In order to be eligible to participate in this study, patients must meet all of the following criteria: Caucasian males 21 to 69 years-old diagnosed with type 2 diabetes using oral metformin on a stable dose (i.e.no changes in the last three months), 2 -3dd 500mg or 2 dd 850mg once daily no other medication use

Exclusion criteria
The following conditions are exclusion criteria for participation in this study:

Smoking
Alcohol abuse (>12 to 15 g of alcohol per day) History of cardiovascular event (myocardial infarction or pacemaker implantation)

Cholecystectomy
Use of any medication other than metformin, including insulin, proton pump inhibitors (PPI as this influences intestinal microbiota composition) 6 , oral anticoagulants and/or oral antibiotics in the past three months (Expected) prolonged compromised immunity (e.g.due to recent cytotoxic chemotherapy or HIV-infection with a CD4 count < 240) Excessive weight loss of >10% in the last months or have overt untreated GI disease/ abnormal bowel habits.
Levels of plasma aspartate aminotransferase and alanine aminotransferase 2.5 times or more the upper limit of the normal range

Sample size calculation
We hypothesize that a peak difference in postprandial glucose excursions using CGM is seen between type 2 diabetes patients using placebo + metformin (13.0 mmol/l) vs E hallii + metformin (10.0 mmol/l) with a SD of 2.5 mmol/l; using unpaired t-test with 0.05 two-sided significance levels and 80% power, we will need 12 subjects per group to detect a significant difference.Of note, all power calculations were performed with an online power calculation (www.biomath.info/power/).Based on the data retrieved from this intervention study, we will apply the prediction algorithm by Zeevi et al for validation 1 .
We will include 24 males with type 2 diabetes treated with metformin.All will receive 2 weeks treatment with either E. hallii or 10% glycerol placebo.
overview MPN of QA samples  4).

Use of co-intervention
As a placebo 10ml of 10% glycerol will be used.Also metformin tablets will be used, but these are part of standard of care medication of all participating DM2 subjects.

Randomisation, blinding and treatment allocation
Subjects and treating physicians are blinded for treatment group.Randomisation of subjects will be performed by a randomisation list produced by the clinical pharmacy of the AMC.In order to prevent the distribution of participants with different doses of metformin distributed unevenly between the placebo and the intervention group, the pharmacy will apply stratification.Two groups will be made, namely one with low dose metformin (1000mg daily) and one with high dose metformin (1500mg or 1600mg daily), and these will be randomized in blocks of two.Vials with E.hallii suspension and placebo (10% glycerol) vials look similar.To maintain the blinding of the subject and investigators, the labels will be removed from the vials at the AMC pharmacy.Vials will be labelled with subject specific information prior to delivery to the study physician.

Mixed meal test (MMT)
At randomisation and 2 weeks after treatment with either E. hallii or placebo, a 2-hour standardized mixed meal test (nutridrink) will be performed after an overnight fast.For mixed meal tolerance test a venflon will be placed in the cubital vein.The mixed meal test will be a liquid solution with a standardized amount of nutrients (nutridrink 250 cc), which has to be consumed in 5 minutes and blood samples will be taken during the test 13 .

Continuous glucose monitoring (CGM)
Subjects will have an implanted Freestyle Libre monitor (http://abbottnextfrontier.com/freestyle-libre)subcutaneously that will be used for 14 days before and after the intervention to measure interstitial glucose levels 1 .Their glucose levels will then be continuously monitored during week 1 and 2 and week 2 and 3.
Subjects are not required to perform finger pricks in order to calibrate the monitor and can perform all normal activities whilst wearing a monitor.

24-hour ambulatory blood pressure measurements
An automatic ambulatory blood pressure monitor (Spacelabs 90207, Spacelabs In., Redmond, Washington, USA) is placed on the patient with the cuff on the non-dominant arm unless there is a 20/10 mmHg difference between arms in which case, the arm with the higher reading is used.Systolic and diastolic blood pressure and heart rate are recorded over at least 24 h.The arm should be held still and at heart level during measurement.An exception is made when a measurement is being taken while a patient is engaged in certain activities, such as driving.Recordings are programmed for every 30 minutes during the day (07.00 22.00) and 60 minutes at night (22.00 to 07.00).These times are adjusted accordingly if the subject works at night.After 24 hours from the start of monitoring, the subject turns off and removes the monitor.All studies are performed with the monitor display switched off, to avoid anticipation of the blood pressure readings by the patients.The ambulatory blood pressure monitoring study is only accepted when at least 70% of all measurements are successful.Mean 24-hours, daytime and night-time ambulatory SBP, DBP and heart rate will be reported.

24h fecal samples
Three times 2x 24h fecal samples will be collected by the subjects at baseline, week 2 and week 4 in order to study the gut microbiota.Patients will be collecting their stools in the provided materials (wearing gloves) and store it in the fridge (3-4ºC) until their visit.
Samples will be transported to the AMC on icepacks.

Withdrawal of individual subjects
Subjects can leave the study at any time for any reason if they wish to do so without any consequences.The investigator can decide to withdraw a subject from the study for urgent medical reasons.

Replacement of individual subjects after withdrawal
Withdrawn subjects will be replaced during the recruitment period in order to attain the desired sample size.
6.6 Follow-up of subjects withdrawn from treatment Withdrawn subjects will be followed for SAEs until 3 months after inclusion.

Temporary halt for reasons of subject safety
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study if there is sufficient ground that continuation of the study will jeopardise subject health or safety.The sponsor will notify the accredited METC without undue delay of a temporary halt including the reason for such an action.The study will be suspended pending a further positive decision by the accredited METC.The investigator will take care that all subjects are kept informed.Adverse events are defined as any undesirable experience occurring to a subject during a clinical trial, whether or not considered related to the investigational drug.All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded.

Serious adverse events (SAEs)
A serious adverse event is any untoward medical occurrence or effect that results in death; is life threatening (at the time of the event); -requires hospitalisation or prolongation of existing hospitalisation; results in persistent or significant disability or incapacity; is a congenital anomaly or birth defect; or any other important medical event that did not result in any of the outcomes listed above due to medical or surgical intervention but could have been based upon appropriate judgement by the investigator.
An elective hospital admission will not be considered as a serious adverse event.
All SAEs will be reported to the accredited METC that approved the protocol, according to the requirements of that METC.

Regulation statement
The study will be conducted according to the principles of the Declaration of Helsinki (Fortalezea Brazil, October 2013) and in accordance with the Medical Research Involving Human Subjects Act (WMO).

Recruitment and consent
Male patients with type 2 diabetes treated with metformin will be recruited via advertisements in local magazines and at the outpatient clinic.Inclusion and exclusion criteria will be verified and, after oral and written explanation of the study, informed consent will be obtained.

Compensation for injury
The sponsor/investigator has a liability insurance which is in accordance with article 7, subsection 6 of the WMO.
The sponsor (also) has an insurance which is in accordance with the legal requirements in The insurance applies to the damage that becomes apparent during the study or within 4 years after the end of the study.

Incentives
All participants will receive a financial compensation of 300 euro and a refund for travel costs.

ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 10.1 Handling and storage of data and documents
The principle investigator will maintain a signature list of appropriately qualified persons to documents that support the data collected from each subject and all study documents, and will treat these data with confidentially according to the Dutch Personal Data Protection Act.Data will be entered directly in the eCRF, also verification of the eligibility criteria, medication use and adverse events are directly entered in the eCFR.A subject identification code list will be drawn, and per subject data will b unique code.The subject will be identified by this number for the duration of the trial.All the participating investigators have access to the source data.Blood samples for analysis and storage will be labelled with this code and visit number.Codes cannot be retraced to the corresponding subject without the identification code list.All essential documentation and human materials will be retained by the institution.Study documents will be archived for 15 years.

Amendments
application, or to the protocol or any other supporting documentation, that is likely to affect to a significant degree: the safety or physical or mental integrity of the subjects of the trial; the scientific value of the trial; the conduct or management of the trial; or the quality or safety of any intervention used in the trial.
All substantial amendments will be notified to the METC and to the competent authority.
Non-substantial amendments will not be notified to the accredited METC and the competent authority, but will be recorded and filed by the sponsor.For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups Outcomes and estimation 17a For each primary and secondary outcome, results for each group, and the estimated effect size and its precision (such as 95% confidence interval)

Figure 5 :Suppl. Figure 7 :
Effect of 14 days placebo (A) and A. soehngenii (B) supplementation on insulin levels, placebo (C) and A. soehngenii (D) supplementation on glucagon-like peptide 1 (GLP-1) levels and (E) Effect of placebo or A. soehngenii on ΔHOMA-IR following 14 days of supplementation Related to figure1.Fecal lactic acid and butyric acid levels expressed as delta concentration within the placebo and intervention group Related to figure 1.

Figure 1 .
Figure 1.Increased Eubacterium Hallii levels in small intestinal biopsies of insulin resistant subjects 6 weeks after being treated with allogenic (lean donor) feces.
hallii on improved insulin sensitivity (ITT fig2a) in correspondence with fecal E. hallii levels (fig2b).Furthermore, we found significant changes in hepatic as well as small intestinal genes involved in bile acid and glucose metabolism (fig2c) including a reduction in FXR expression, which is associated with improved insulin sensitivity and resulted in less hepatic triglycerides and obesity in mice11 .

Figure 2 .
Figure 2. Results of dosefinding study of daily E.Hallii treatment on a) insulin sensitivity (as assessed by insulin tolerance test), b) concentrations of Eubacterium hallii in feces and c) small intestinal genes involved in bile acid metabolism upon 10E9 E hallii.FXR denotes farnesoid X receptor, a gene central in bile acid and glucose metabolism

Figure 3 .
Figure 3. Amount of metabolic responders upon E halii treatment (upper panel) as well as dose dependent in increase of fecal E hallii L2-7 levels compared to endogenous strains (left panel: low, middle panel: middle dose and right panel: high dose).

Figure 4 .
Figure 4. Linear correlation between Rd change (peripheral insulin sensitivity on y-axis0 and changes in fecal E halli L2-7 concentrations (as expressed by relative abundance on x-axis) in 27 metabolic syndrome subjects.A significant linear correlation was found between increase in Rd and increase in fecal E hallii levels (r=0.4,P<0.05) when pooling all groups.

Figure 5 .
Figure 5. Study overview of the EMDM2 trial

Figure 4 .
Figure 4. Viability (MPN on Ya-xis) during last years (x axis from 2014 to 2017) for E hallii 10e9/ml vial (blue bar) stored at AMC dept of clinical pharmacy.

7. 2
AEs, SAEs and SUSARs 7.2.1 Adverse events (AEs) E. hallii treatment from the same batch has been given in our previous study, the DIME trial (METC 2014_285).No adverse events were expected and none did.The possible complications are cited in the written patient information and are expected to be mild.

7 12b 7 Results 12 13b 12 14b
assessing outcomes) and how 11b If relevant, description of the similarity of interventions NA Statistical methods 12a Statistical methods used to compare groups for primary and secondary outcomes Methods for additional analyses, such as subgroup analyses and adjusted analyses Participant flow (a diagram is strongly recommended) 13a For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome For each group, losses and exclusions after randomisation, together with reasons 12 Recruitment 14a Dates defining the periods of recruitment and follow-up Why the trial ended or was stopped 12 Baseline data 15 A table showing baseline demographic and clinical characteristics for each group 12 Numbers analysed 16 to investigate the effect of 14 days once daily oral Eubacterium hallii (E.hallii) treatment on postprandial glucose levels in relation to SCFA levels in feces in patients with type 2 diabetes treated with metformin.
Suppl.Figure9Shows filtered dichotomized clustered SNP profiles of different subjects at different time points.Related to figure3.SUMMARY Objective: monotherapyIntervention: Subjects will be given oral 10 ml E. hallii suspension with a total concentration of 10e9 cells/ml in 10% glycerol or 10ml 10 % glycerol only (both produced by NIZO and stored at Dept of Clinical Pharmacy AMC) once daily during 14 days.Standardized mixed meal test and 14 days wearing of a glucose sensor (CGM Freestyle libre) to measure effect on (postprandial) glucose levels.
We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration for important clarifications on all the items.If relevant, we also recommend reading CONSORT extensions for cluster randomised trials, non-inferiority and equivalence trials, non-pharmacological treatments, herbal interventions, and pragmatic trials.Additional extensions are forthcoming: for those and for up to date references relevant to this checklist, see www.consort-statement.org. *