Oral butyrate does not affect innate immunity and islet autoimmunity in individuals with longstanding type 1 diabetes: a randomised controlled trial
The pathophysiology of type 1 diabetes has been linked to altered gut microbiota and more specifically to a shortage of intestinal production of the short-chain fatty acid (SCFA) butyrate, which may play key roles in maintaining intestinal epithelial integrity and in human and gut microbial metabolism. Butyrate supplementation can protect against autoimmune diabetes in mouse models. We thus set out to study the effect of oral butyrate vs placebo on glucose regulation and immune variables in human participants with longstanding type 1 diabetes.
We administered a daily oral dose of 4 g sodium butyrate or placebo for 1 month to 30 individuals with longstanding type 1 diabetes, without comorbidity or medication use, in a randomised (1:1), controlled, double-blind crossover trial, with a washout period of 1 month in between. Participants were randomly allocated to the ‘oral sodium butyrate capsules first’ or ‘oral placebo capsules first’ study arm in blocks of five. The clinical investigator received blinded medication from the clinical trial pharmacy. All participants, people doing measurements or examinations, or people assessing the outcomes were blinded to group assignment. The primary outcome was a change in the innate immune phenotype (monocyte subsets and in vitro cytokine production). Secondary outcomes were changes in blood markers of islet autoimmunity (cell counts, lymphocyte stimulation indices and CD8 quantum dot assays), glucose and lipid metabolism, beta cell function (by mixed-meal test), gut microbiota and faecal SCFA. The data was collected at the Amsterdam University Medical Centers.
All 30 participants were analysed. Faecal butyrate and propionate levels were significantly affected by oral butyrate supplementation and butyrate treatment was safe. However, this modulation of intestinal SCFAs did not result in any significant changes in adaptive or innate immunity, or in any of the other outcome variables. In our discussion, we elaborate on this important discrepancy with previous animal work.
Oral butyrate supplementation does not significantly affect innate or adaptive immunity in humans with longstanding type 1 diabetes.
Netherlands Trial Register: NL4832 (www.trialregister.nl).
Raw sequencing data are available in the European Nucleotide Archive repository (https://www.ebi.ac.uk/ena/browse) under study PRJEB30292.
The study was funded by a Le Ducq consortium grant, a CVON grant, a personal ZONMW-VIDI grant and a Dutch Heart Foundation grant.
KeywordsButyrate Diabetes Microbiota Short-chain fatty acids
C-C chemokine receptor
CXC chemokine receptor
Defective ribosomal product
Iscove’s modified Dulbecco’s medium
Mean fluorescence intensity
Peripheral blood mononuclear cell
Short-chain fatty acid
T regulatory cell
We cordially thank C. Rustemeijer (Amstelland Hospital, Amstelveen, the Netherlands), V. Gerdes (Amsterdam UMC, Amsterdam, the Netherlands), T. Brouwer (OLVG Hospital, Amsterdam, the Netherlands), S. van Dam (OLVG Hospital, Amsterdam, the Netherlands) and J. Hensbergen (Amsterdam UMC, Amsterdam, the Netherlands) for inclusion of participants.
PFdeG, FH, JBLH, BOR and MNi designed the study. SS substantially contributed to the acquisition of data. PFdeG, BOR, MNe, ESGS, FMK, DHvR, TN, SB, GD, HH, MW, BH, JK, EMK, JHML, SI, EL, GMD-T, MD, FAvH, RB and AB contributed to the analysis and/or interpretation of data. PFdeG, BOR and MNi drafted the manuscript. All authors critically revised the manuscript. All authors gave their approval of the final (published) version of the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. PFdeG is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
This study was supported by Le Ducq consortium grant 17CVD01 (to MNi) and CVON grant 2O18.27 (to SB, MNe and MNi). MNi is supported by a personal ZONMW-VIDI grant 2013 (016.146.327) and a Dutch Heart Foundation grant.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
- 1.Patterson CC, Dahlquist GG, Gyürüs E, Green A, Soltész G (2009) Incidence trends for childhood type 1 diabetes in Europe during 1989-2003 and predicted new cases 2005-20: a multicentre prospective registration study. Lancet 373(9680):2027–2033. https://doi.org/10.1016/S0140-6736(09)60568-7 CrossRefPubMedGoogle Scholar
- 6.Samuelsson U, Ludvigsson J (2004) The concentrations of short-chain fatty acids and other microflora-associated characteristics in faeces from children with newly diagnosed type 1 diabetes and control children and their family members. Diabet Med 21(1):64–67. https://doi.org/10.1046/j.1464-5491.2003.01066.x CrossRefPubMedGoogle Scholar
- 8.Russo I, Luciani A, De Cicco P, Troncone E, Ciacci C (2012) Butyrate attenuates lipopolysaccharide-induced inflammation in intestinal cells and Crohn’s mucosa through modulation of antioxidant defense machinery. PLoS One 7(3):e32841. https://doi.org/10.1371/journal.pone.0032841 CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Zimmerman MA, Singh N, Martin PM et al (2012) Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells. Am J Physiol Gastrointest Liver Physiol 302(12):G1405–G1415. https://doi.org/10.1152/ajpgi.00543.2011 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Endesfelder D, Engel M, Davis-Richardson AG et al (2016) Towards a functional hypothesis relating anti-islet cell autoimmunity to the dietary impact on microbial communities and butyrate production. Microbiome 4(1):17. https://doi.org/10.1186/s40168-016-0163-4 CrossRefPubMedPubMedCentralGoogle Scholar
- 34.Velthuis JH, Unger WW, Abreu JRF et al (2010) Simultaneous detection of circulating autoreactive CD8+ T cells specific for different islet cell-associated epitopes using combinatorial MHC multimers. Diabetes 59(7):1721–1730. https://doi.org/10.2337/db09-1486 CrossRefPubMedPubMedCentralGoogle Scholar
- 35.De Baere S, Eeckhaut V, Steppe M et al (2013) Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation. J Pharm Biomed Anal 80:107–115. https://doi.org/10.1016/j.jpba.2013.02.032 CrossRefPubMedGoogle Scholar
- 36.Salonen A, Nikkila J, Jalanka-Tuovinen J et al (2010) Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. J Microbiol Methods 81(2):127–134. https://doi.org/10.1016/j.mimet.2010.02.007 CrossRefPubMedGoogle Scholar
- 37.Ramiro-Garcia J, Hermes GDA, Giatsis C et al (2016) NG-tax, a highly accurate and validated pipeline for analysis of 16S rRNA amplicons from complex biomes [version 1; referees: 2 approved with reservations, 1 not approved]. F1000Res 5:1791. https://doi.org/10.12688/f1000research.9227.1 CrossRefPubMedGoogle Scholar
- 38.Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD (2013) Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol 79(17):5112–5120. https://doi.org/10.1128/AEM.01043-13 CrossRefPubMedPubMedCentralGoogle Scholar
- 40.Chen T, Guestrin C (2016) XGBoost: a scalable tree boosting system. In: Proceedings of the 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining. ACM, New York, NY, USA, pp 785–794Google Scholar
- 52.Korsgren S, Molin Y, Salmela K, Lundgren T, Melhus Å, Korsgren O (2012) On the etiology of type 1 diabetes: a new animal model signifying a decisive role for bacteria eliciting an adverse innate immunity response. Am J Pathol 181(5):1735–1748. https://doi.org/10.1016/j.ajpath.2012.07.022 CrossRefPubMedPubMedCentralGoogle Scholar