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Investigation of interplay between multiple determinants influencing response to metformin: search for reliable predictors for efficacy of type 2 diabetes therapy



Project Title: „Investigation of interplay between multiple determinants influencing response to metformin: search for reliable predictors for efficacy of type 2 diabetes therapy”

Funding: European Regional Development Fund (ERDF), Measure “Support for applied research”

Project Nr.:

Period: 1st January 2017 – 31st December 2019

Project costs: 648 235,48 EUR 

Principle Investigator: Dr. biol., Assoc. Prof. J. Kloviņš 


The objective of the project is to explore underinvestigated factors characterizing response to metformin with the aim to identify reliable biomarkers of efficacy and tolerability of metformin in the treatment of type 2 diabetes (T2D). One of the main activities of the project will be dedicated to the evaluation of the correlation between metformin related changes in taxonomic and functional composition of gut microbiome and response to the treatment. We will test the hypothesis that gut microbiome contributes to glucose lowering effects of metformin and the concomitant decrease in its taxonomic diversity gives rise to pathogens responsible for gastrointestinal side effects. This will be achieved by integrating data from metagenome and 16S rRNA sequencing of gut microbiome from healthy volunteers and T2D patients before and after metformin use. The same cohorts will be used to identify novel epigenetic and RNA expression signatures in humans associated with the use of metformin, that could be used to establish clinically relevant biomarkers. To understand the causality of associations observed in humans we will perform in depth characterization of metformin affected microbiome and novel pathways in mouse models. Apart from that these experiments will give an opportunity to assess the potential benefits of metformin treatment in combination with probiotics/prebiotics to prevent undesirable changes in the gut microbiome.

Proposed project will improve our understanding of mechanisms underlying efficacy and intolerance of metformin treatment. Firstly, planned activities will help to clarify the role of gut microbiome in response to metformin. Secondly, it will provide insight in epigenetic markers and their functional impact in the context of metformin use. Experiments in mice will bring the studies one step further giving an opportunity to interfere with the negative effects of metformin. Obtained information will assist improvement of algorithms for prediction of treatment outcome as well as support development of tools for increasing efficacy and tolerance of metformin treatment.

Information published: 02.01.2017.

Progress of the project

1 January 2017 - 31 March 2017

During the first quarter of the project implementation we have initiated recruitment of potential participants as well as collection of samples for characterisation of metformin induced variation in taxonomic composition of gut microbiome and determination of epigenetic profiles in white blood cells. Concurrently algorithms for high throughput sequencing data processing, including data filtering, quality control, adjustment for cofactors and other aspects of data analysis, have been under development to fit specific longitudinal design of the research project.

Information published: 31.03.2017.

Progress of the project

1 April 2017 - 30 June 2017

During the second quarter of the research implementation we have continued with selection of project specific cohorts (healthy participants, patients newly diagnosed with type 2 diabetes (T2D) and patients with a T2D duration >3 years) and collection of biosamples for investigation of factors modifying efficiency and tolerance of metformin therapy. To characterise metformin induced variation in taxonomic composition of gut microbiome, analysis of 16S rRNS gene amplicon libraries with high throughput sequencing has been initiated. Concurrently we have started sample collection and preparation for evaluation of variation in epigenetic profiles in white blood cells during treatment with metformin. To ensure high quality of data interpretation algorithms for high throughput sequencing data processing (data filtering, quality control, adjustment for cofactors, etc.) have been developed to match longitudinal design of the research project. First reports on the progress of the research project have been given in two international conferences (19th European Congress of Endocrinology, 20.-23.05; EASD-SGGD meeting "Genetics of diabetes and its role in precision medicine", 11.-13.05).

Information published: 30.06.2017.


Progress of the project

1 July 2017 - 30 September 2017

For investigation of factors interacting with metformin treatment in diabetics recruitment of patients (newly diagnosed patients and patients with a T2D duration >3 years) in collaboration with endocrinologists has been continued. Amplicon libraries of 16S rRNS gene V3 region derived from healthy metformin users have been processed and grouped forming in-house database for proceeding with statistical analyses. To evaluate how changes in taxonomic composition are reflected in functional profile of gut microbiome, we have started preparation of metagenomic libraries for high throughput sequencing analysis and optimisation of respective data processing workflows. Apart from investigation of interplay between gut microbiome and metformin we have continued with analysis of epigenetic variation in white blood cells of metformin users. Epigenetic profiles have been derived from 12 healthy persons taking metformin. Concurrently selection of biological candidate genes has been initiated for identification of metabolic pathways mediating effects of metformin treatment.

Information published: 29.09.2017.


Progress of the project

1 October 2017 - 31 December 2017

We have proceeded with high throughput sequencing metagenome analysis of gut microbiome within the group of healthy, non-diabetic volunteers as well as adapted data processing workflows for specific needs of the ongoing project. Concurrently investigation of metformin affected methylation profiles has revealed 11 differentially methylated gene regions in white blood cells from healthy metformin users. Ongoing analysis of metabolic pathways represented by differentially methylated genes of particular interest indicates towards the potential role they may play in mediating metformin effects for example on cell metabolism. For validation of obtained results we continue patient recruitment in cohorts comprising patients with different duration of anti-diabetic medicamentous therapy (newly diagnosed, treatment naive patients and patients with a T2D duration > 3 years recruited from T2D cohort of longitudinal study of anti-diabetic treatment as well as using LGDB resources).

Information published: 02.01.2018.


Progress of the project

1 January 2018 - 31 March 2018

 For assessment of functional consequences of metformin induced shift in taxonomic composition of gut microbiome in healthy persons, NGS based shotgun metagenomic sequencing is in progress. Recently global DNA methylation analysis has been completed within the same cohort including healthy participants revealing potential epigenetic targets of metformin. Most of the top-ranked differentially methylated loci comprise genes such as CAMKK1, ADAM8 and POFUT2 representing signalling pathways relevant to the known therapeutic effects of metformin like improvement of energy homeostasis, anti-inflammatory and anti-cancer effects. At the same time we have started genome wide RNA profiling in white blood cells to investigate effects of metformin on gene expression that may provide novel information on metformin action at the molecular level as well as highlight potential biomarkers. To investigate metformin effects on diabetic background cohorts comprising patients with different duration of anti-diabetic medicamentous therapy (newly diagnosed, treatment naive patients and patients with T2D duration > 3 years) have been recruited. Recently analysis of taxonomic composition of gut microbiota in diabetic metformin users has been initiated.

We also have launched experiments in animal models for in detail characterisation of interaction between gut microbiome and metformin, as well as evaluation of wide spectrum of treatment effects on the organism. Currently high-fat feeding phase of the experiment to induce insulin resistance is in progress.

 Information published: 29.03.2018.


Progress of the project

1 April 2018 – 30 June 2018

Previously we have demonstrated metformin induced variation in taxonomic profile of healthy non-diabetic gut microbiome. Ongoing analysis of the metagenomic sequencing data will allow assessment of the impact of observed shift in proportions of several taxonomic units on functions of the gut microbiome. Metformin treatment associated taxonomic and functional changes in the gut microbiome are investigated also in the cohorts comprising more than 250 patients with T2D. These studies are not only allowing evaluation of overlap of gut microbiome related markers between healthy persons and diabetics, the major target group of the medicament, but also providing opportunity for investigation of metformin effects on the dysbiotic gut microbiome associated with specific phenotypes. Considering pleiotropic action of metformin, concurrently studies of metformin intake associated changes in gene expression are carried out by genome wide RNA profiling in white blood cells donated by healthy persons. In detail characterisation of functional significance of observed correlations in metformin target tissues are carried out in animal models.

Information published: 29.06.2018.


Progress of the project

1 July 2018 – 30 September 2018

To evaluate metformin-induced changes in the taxonomic and functional gut microbiome profiles of healthy individuals, database of metagenomic sequences has been created and data statistical analysis is being carried out. A comprehensive integrative analysis of the interaction between gut microbiome taxonomic composition and the functional profile has been started, and an analysis of the microbiome changes in diabetic metformin users is continuing. Sequencing of blood sample transcriptome from healthy individuals has been finished and data analysis is being carried out to identify metformin-induced differentially expressed genes and their associated pathways. Based on the DNA methylation analysis results in healthy individuals, candidate genes are being selected and methodology for targeted validation of methylation profiles is being developed with aim to test them in the previously selected cohorts of T2D patients with variable duration of diabetes and length of metformin therapy. Experiments in mice models for a detailed assessment of the wide spectrum of metformin effects are ongoing. Part of the animals have been sacrificed, and metformin target tissue samples have been collected for the purpose of determining the changes in gene regulation and the effect of metformin therapy on the gut microbiome.

Information published 28.09.2018.


Progress of the project

1 October 2018 – 31 December 2018

The integrative analysis of the taxonomical and functional profile of the gut microbiome is being optimized, as well as additional analysis of the indicators characterizing the obtained interactions are performed. The profiling of RNA expression in white blood cells derived from healthy metformin users has been completed, and an integrative analysis of DNA methylation profiles and gene expression changes has begun. A catalogue with genes whose expression changes is associated with metformin therapy has been created, and a 16S rRNA sequence database that characterizes gut microbiome composition in T2D patients has been created. Set of the selected T2D patient DNA samples is being prepared for replication study of the results obtained from methylation analysis in healthy individuals. Various gut microbiome and metformin target tissue samples have been collected from the mice models involved in animal experiments to evaluate the role of metformin therapy on wide range of biological aspects. The processing of gut microbiome samples so to obtain the necessary data has been initiated.

 Information published 28.12.2018.


Progress of the project

1 January 2019 – 31 March 2019

Publication on identified metformin-induced changes in intestinal microbiome has been prepared. In addition, analysis of the contribution of gut microbiome to metformin pharmacokinetics has been started. In order to clarify the functional significance of differentially methylated DNA regions resulting from the administrations of metformin, an integrative analysis of the global DNA methylation and gene expression profiles of healthy individuals is performed. DNA methylation data of blood cells collected from type 2 diabetes patients are currently being processed. Continuing the study in animal models, microbiome analysis has been performed in faecal and intestinal biospecimens collected from mice. Mice-derived metformin target tissue are also used for the ongoing gene expression analysis.

Information published 29.03.2019.


Progress of the project

1 April 2019 – 30 June 2019

A draft for the publication of identified metformin-induced changes in the human gut microbiome has been prepared. Integrative analysis of DNA methylation and gene expression has been performed, 3 genes identified, whose expression level changes correlate with DNA methylation levels after metformin administration. Evaluation of DNA methylation profiles of T2D patients' blood cells has been completed, analysis of the obtained data is performed, and differentially methylated DNA loci associated with metformin efficacy have been identified. In parallel, a metformin pharmacokinetic study is being conducted to collect 24-hour urine samples from metformin users. An RNA-Seq based transcriptome analysis in target organs of metformin has been performed using tissue samples collected in animal experiments conducted within the project. Shotgun metagenome sequencing has been performed in the samples of the animal intestinal microbiome samples, analysis of the obtained data is being performed.

Information published 28.06.2019.


Progress of the project

1 July 2019 – 30 September 2019

Integrative analysis of pharmacokinetic data and taxonomic composition of the gut microbiome has been performed. Detailed analysis of various biomarkers identified during the study has been initiated to predict the potential efficacy or side effects of metformin therapy. Metagenome sequencing has been performed using fecal samples obtained from animals before the beginning of metformin therapy and after ten weeks-long treatment. Analysis of the acquired data is being performed.  Preparation of a draft for the publication describing the results obtained in animal studies is in progress.

Information published 30.09.2019.


Progress of the project

1 October 2019 – 31 December 2019

We have identified a list of gene expression-based biomarkers for the prediction of metformin response in type 2 diabetes patient cohort, and a manuscript describing these transcriptome alterations is currently being prepared. Moreover, the correlation analysis between DNA methylation and gene expression levels has provided four functionally relevant loci among affected epigenetic patterns. Manuscript on identification of metformin induced changes in gut microbiome taxonomic and functional profiles both in healthy individuals and newly diagnosed diabetes patients is also submitted for publication. Sex-related differences in metformin response regarding metabolic measurements and affected taxa was observed in mouse model of diabetes and manuscript describing metformin-induced changes in gut microbiome composition in high fat diet-induced type 2 diabetes mouse model of both sexes is submitted for publication in peer reviewed journal. Finally, based on gathered methylation, transcriptome and microbiome data obtained during the implementation of the project, algorithm for the prediction of metformin response in type 2 diabetes patients is developed.

Information published 30.12.2019.

Mājas lapas izstrādi finansēja ERAF aktivitātes projekts Nr. 2010/0196/2DP/ "Latvijas biomedicīnas pētījumu integrācija Eiropas zinātnes telpā".