Robin Mesnage

My first project related to fasting involved analyzing gut microbiome data from a study where patients fasted for 10 days. We revealed for the first time how fasting switched the composition of the gut microbiome in a manner similar to what happens in hibernating animals (Mesnage et al., 2019).

We continued investigating how fasting can impact the gut microbiome and whether this can influence human health outcomes of fasting therapies. In 2023, we published a review providing the state of the art on the topic (Ducarmon et al., 2023).

Our latest study on the gut microbiome is the most ambitious (Ducarmon et al., 2024). It provides a high-resolution view of how prolonged fasting impacts the gut microbiome and serum metabolites. Fasting caused extensive remodeling of the gut microbiome, affecting 516 out of 772 bacterial species, and induced a substrate switch from dietary fibers to host glycans. Interestingly, Akkermansia muciniphila, a known mucin forager, remained unaffected. We found that changes in bacterial species during fasting were predictable based on their genomic repertoire of carbohydrate-active enzymes. Fasting also altered 382 out of 721 serum metabolites, including microbiome-derived indole-3-propionic acid, with Oscillibacter species identified as key producers of this crucial metabolite for cardiometabolic health. While the immediate effects of fasting on the microbiome were substantial, they were not long-lasting, unlike the persistent changes seen with antibiotic treatment.

Fasting is one of the most powerful therapeutic interventions to promote healthy longevity. It is a nutritional programme of humans and animals providing body cells with fat as a fuel from the adipose tissue instead of food glucose. Here are highlights of our most impactful studies showing metabolic normalisation following therapeutic fasting:

• Long-term fasting improves cardiovascular risks associated to cholesterol particles (Grundler et al., 2021). LDL cholesterol and triglyceride levels dropped during long­term fasting, with small dense low­density lipoproteins (sdLDL) – the most dangerous particles – showing the most significant reduction.
• Long-term fasting can be used as a complementary therapy for high blood pressure (Grundler et al., 2020). Elevated blood pressure was effectively reduced after an average of 10 days of fasting, as evidenced by a study of 1,610 fasting patients. Among 377 patients who took antihypertensive drugs,the treatment could in most cases either be stopped or reduced during fasting.
• Fasting can boost antioxidant levels and reduce oxidative stress (Grundler et al., 2020 ; Wilhelmi de Toledo et al., 2020) . Oxidative stress is decreased in the blood of patients during 10 days of fasting. This was linked to the retention of uric acid in blood, which does not cause gout in the special metabolic state of fasting, but rather increase antioxidant capacity in blood.
• Long-term fasting provides health improvements in long-COVID patients (Grundler et al., 2023). Long­COVID symptoms like fatigue, breathlessness, muscle and joint pains were reduced in patients fasting at the Buchinger Wilhelmi Clinic. Even less frequent symptoms such as cognitive impairment, smell and taste disorders, showed marked improvements. The findings open a promising avenue to offer hope to millions of individuals worldwide whose lives have been adversely impacted by the effects of COVID­19.
• On the role of fat burning (Grundler et al., 2024). We monitored 1610 participants undergoing the Buchinger Wilhelmi fasting program and found that long-term fasting safely induced ketosis from day 2, with minimal carbohydrate intake modulating but not suppressing ketosis. Patients with stronger ketosis experienced greater reductions in blood glucose, glycated hemoglobin, body weight, waist circumference, and improved antioxidant status.

In 2021, I joined the Buchinger Wilhelmi Clinics as the lead data scientist to work on their most comprehensive research topic conducted so far: the GENESIS study. The first objective of this study was to use MRI technologies to document changes in body composition and the contribution of the main metabolically active tissues (skeletal muscle tissues, adipose tissues, liver, heart, spleen, kidneys, and brain) to the metabolic switch during a 12-day fasting period. We hypothesized that re-expansion and regeneration follow a transient decrease in organ volume during fasting, lasting up to 4 months after food reintroduction.

We showed that fasting preserved muscle integrity and performance, as well as muscle mitochondrial activity, and even resulted in improvements in cardiorespiratory activity.

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This study examined the effects of daily consumption of wild blueberries on cognitive and vascular health in 61 healthy older adults (ages 65-80) over 12 weeks. Participants consumed either wild blueberry powder or a placebo. Results showed significant improvements in cognitive tasks, such as executive function and short-term memory, for those consuming blueberries. Additionally, vascular function improved, with better flow-mediated dilation and a reduction in systolic blood pressure. No changes were observed in cerebral blood flow. The study suggests wild blueberries can benefit cognitive and vascular health, with further investigation needed into the role of (poly)phenol metabolites and the gut microbiome.

People's reactions to food consumption vary due to their gut bacteria, which can be predicted by metabotyping. Foods rich in polyphenols, such as fruits, vegetables, and tea, benefit health, but individual responses differ. Metabotyping clusters people based on their responses to polyphenol intake, like blueberries, into urolithin metabotypes A, B, or 0, with urolithin A offering the most health benefits. These reactions are driven by the gut microbiome, allowing prediction of metabotypes through gut microbiome or urine metabolite profiles. This enables personalized nutrition, potentially normalizing metabolism through tailored dietary interventions.

Health effects of pesticides are not always correctly predicted before products are released for marketing. Laboratory studies are often insufficient to detect effects of pesticides. This is the case for effects on the gut microbiome which are generally not measured in the battery of tests performed to regulate pesticide adverse effects.

Effects of the major herbicide glyphosate on the gut microbiome are heavily debated. Because glyphosate can target the metabolism of gastrointestinal microorganisms in the same manner it targets plants to act as a weedkiller, major concerns have been raised regarding the potential antibiotic effects of glyphosate. However, this had never been tested. We combined high-throughput biology techniques (shotgun metagenomics and metabolomics) to assess effects of glyphosate on the caecum microbiota of Sprague-Dawley rats. (Mesnage et al., 2021). Glyphosate did not act as an antibiotic in the gut. In order for glyphosate to act as an antibiotic, it needs to cause a significant shortage in the production of compounds in bacteria called aromatic amino acids. This is how glyphosate kills plants. More specifically, it interrupts a series of chemical reactions within the shikimate pathway. We reveal for the first time that glyphosate does inhibit the shikimate pathway in the rat gut microbiome. This could have health impact. However, glyphosate likely did not kill bacteria via its impact on the shikimate pathway, because bacteria do not need the shikimate pathway to get the aromatic amino acids they need. Like us, they get them from the food we eat.

Glyphosate’s impact on the shikimate pathway had other consequences. A multitude of small molecules (metabolites) accumulated, or were depleted in the rat gut. The bacterial composition of the gut microbiome also changed, with some bacteria present at higher levels in the glyphosate exposed animals. We have an explanation for this: the glyphosate molecule contains one atom of phosphorus, and it is very likely that this phosphorus atomic bond was used as a source of energy by some bacteria to proliferate in the rat gut.

We evaluated this by measuring if the changes in gut microbiome chemistry caused changes in blood composition. The compounds altered as a consequence of the interruption of the shikimate pathway by glyphosate in the gut were mostly unchanged in blood. However, other chemicals had their levels disrupted by glyphosate, with some indicating that glyphosate caused oxidative stress. This oxidative stress was even more pronounced when we tested a commercial herbicide containing glyphosate called Roundup Bioflow (also known as MON 52276, the representative glyphosate herbicide in the EU).

We performed another study and evaluated if urinary glyphosate excretion associates with changes in faecal microbial community composition and metabolite profiles in 65 adult twins in the UK. This is part of a larger project in which we are comparing the effects of organic and non-organic diets on the composition of the gut microbiota of adult twins from the TwinsUK cohort. This study was done in the UK on 130 individuals (Mesnage et al., 2022). Pyrethroid and/or organophosphorus insecticide residues were found in all urine samples, while the herbicide glyphosate was found in 53% of individuals. Food frequency questionnaires showed that residues from organophosphates were higher with increased consumption of fruit and vegetables. A total of 34 associations between pesticide residue concentrations and faecal metabolite concentrations were detected. Glyphosate excretion was positively associated with an overall increased bacterial species richness, as well as to fatty acid metabolites and phosphate levels.

To further address scientific knowledge gaps, we describe for the first time the effects of glyphosate and a Roundup glyphosate-based herbicide on infant gut microbiota using SHIME technology, which mimics the entire gastrointestinal tract (Mesnage et al., 2022). Roundup and to a lesser extent glyphosate caused an increase in fermentation activity, resulting in acidification of the microbial environment. Ammonium production reflecting proteolytic activities was increased by Roundup exposure. Global metabolomics revealed large scale disturbances in metabolite profiles, including an increased abundance of long chain polyunsaturated fatty acids (n3 and n6). Our results suggested that lactobacilli had their growth stimulated as a result of microenvironment acidification.

Despite growing evidence linking the presence of different categories of fungi in the human gut to observable diseases and health symptoms, no study has investigated if glyphosate can affect these fungal populations. As part of the multifaceted Global Glyphosate Study., which was launched with the aim of providing the most comprehensive evaluation of glyphosate-based herbicides covering long-term toxicity, carcinogenicity and multi-generational effects, we studied changes in bacterial and fungal populations in the caecum of rats exposed prenatally until adulthood (13 weeks after weaning) to three doses of glyphosate (0.5, 5, 50 mg/kg body weight/day), or to the herbicide formulations Roundup Bioflow and RangerPro at the same glyphosate-equivalent doses (Mesnage et al., 2022). Altogether, our data suggested that glyphosate and its formulations caused profound changes in gut microbiome composition by affecting the fitness of major commensals, which in turn reduced competition and allowed opportunistic fungi to grow in the gut.

Despite the widely recognized benefits of organic farming and food consumption, its endorsement by government food standards agencies and the scientific community is generally lacking. One major reason for this is the lack of scientific studies showing direct health benefits of an organic diet.

Our study on the 65 adult twins in the UK performed in collaboration with the group of Pr. Tim Spector originally aimed at testing the effects of organic food consumption. Twins were selected based on their answers to a food frequency questionnaire (FFQ) modified to include a question on organic food consumption. Our aim was to define two groups of individuals, one less likely to be exposed to pesticides than the other because of organic food consumption. A total of 977, mostly female, twin pairs answered questions on organic food dietary intake from the TwinsUK questionnaire. Among these 977 individuals, 65 twin monozygotic twin pairs were found to be discordant for organic food consumption (one twin eats an organic diet whereas the other does not). However, when a new FFQ was performed at the time of urine collection, only 15 pairs were discordant for organic food consumption. The inconsistency in the answers provided to the nutrition questionnaire, convinced us to drop this component of the investigation, as any findings would be deemed as inconclusive.

The observed inconsistency between the results of the FFQ before recruitment and during the study is an important finding, which supports the need for dietary intervention studies to accurately determine the effects of an organic diet on the gut microbiome. An inherent difficulty in studies trying to link the consumption of organic food to health consequences is the number of co-variables or confounding factors, which are a natural part of human life. Organic food consumption is by itself multifactorial and its health effects could be due to a reduced exposure to pesticides and/or antibiotics, or to an increased intake of micronutrients. We addressed this topic in a review (Mesnage et al., 2020), and started the first controlled dietary intervention of the health benefits of an organic diet.

We hypothesised that short term consumption of an organic plant rich diet will lead to higher urinary levels of polyphenols, lower urinary levels of pesticides and improvements in the gut microbiome composition and biomarkers of cardiovascular health in comparison with an equivalent non-organic plant rich diet. To address this hypothesis, we started the ORGAnic and MICrobiome study (ORGAMIC) project and performed the first controlled dietary intervention of the health benefits of an organic diet. This includes comparisons of the effects of an organic verses a non-organic diet on urinary polyphenol and pesticide levels, gut microbiota (bacterial population) and selected biomarkers of cardiovascular health in a group of young healthy individuals. A pilot study was conducted and the results are being analysed.

Since the exposure to a glyphosate formulated product caused a phenotype overlaying those of non-alcoholic fatty liver disease (NAFLD) in rats (Mesnage et al., 2015)(Mesnage et al., 2017), we evaluated if liver cells can be affect by glyphosate and its formulations in vitro. For this purpose, we developed a sensitive assay combining transcriptomics and metabolomics approaches to reveal molecular signatures of NAFLD. First of all, we described the alterations in gene expression profiles and metabolite levels in the human HepaRG liver cell line exposed to the polychlorinated biphenyl 126 (Mesnage et al., 2018a). Exposure to PCB 126 provoked a decrease in polyunsaturated fatty acids as well as an increase in sphingolipid levels, concomitant with a decrease in the activity of genes involved in lipid metabolism. These changes were observed even at the lowest concentration tested. Our study provided the foundation for the development of molecular signatures of fatty liver diseases to rapidly assess chronic toxic effects from exposure to compounds having a direct effect on the liver in a reliable hepatotoxicity model system. The availability of such an in vitro model system can lessen the burden of chemical risk assessment based on in vivo animal experiments.

We then used HepaRG cells to investigate toxic effects of pesticides (Mesnage et al., 2018b). We focused on quizalofop-p-ethyl, isoxaflutole, mesotrione and glyphosate as use of herbicides containing these active principles is predicted to rapidly increase in coming years in an effort to overcome the wide-spread appearance of glyphosate-resistant weeds. Quizalofop-p-ethyl was found to alter lipid metabolism. Glyphosate only caused subtle changes in the gene expression profiles of liver cells. In order to explore the effects of glyphosate in greater depth and detail, we undertook a global metabolome profiling. This revealed a decrease in free long chain fatty acids and polyunsaturated fatty acid levels at the lowest concentration (0.06 μM) of glyphosate, although no effects were detected at the two higher concentrations tested, perhaps suggesting a non-linear dose response. This surprising result will need to be confirmed by additional studies.

My research studies on glyphosate started in 2008 when I joined the group of Pr. Séralini at the University of Caen in France. During this period, my main focus was the comparison of the toxic effects of glyphosate to those of its commercial formulations. I published the first study linking the concentration of ethoxylated surfactants to the toxicity of glyphosate-based herbicides (Mesnage et al., 2013). We tested seven glyphosate formulations. Some were as toxic as glyphosate, and others were approximately 300 times more toxic. We did not stop at the cytotoxicity profiles. We looked beyond the glyphosate label by analysing the composition of the co-formulants by mass spectrometry. This revealed that the toxic effects were linked to the presence of ethoxylated tallowamine surfactants. Although this was not the first study showing the toxicity of these surfactants, it revealed that they were more toxic than other surfactants in other glyphosate-based herbicides sold for the same purposes. So ethoxylated tallowamines were not essential and could be substituted. This observation contributed to the banning of ethoxylated tallowamines from glyphosate-based herbicides in Europe.

In a follow-up study in 2014, I investigated 9 active ingredients. They were selected from herbicides, insecticides, or fungicides of different classes which are used for agricultural or domestic purposes, from the major pesticides used worldwide. I found that 8 formulations out of 9 were up to one thousand times more toxic than their active principles on the three cell lines tested (Mesnage et al., 2014).

In 2021, I put together and led a group of scientists from across the EU to write an article for more transparent data on how pesticides are used on farms. We managed to publish this article in the best journal for ecology from Nature (Mesnage, Straw, et al., 2021). This article triggered debates, some NGOs campaigned to change the regulation, and now it's being reported that the EU will be creating the kind of database we proposed (news article).

I am still pursuing this area of research. Pesticide active ingredients are compared to their commercial formulations in most of the studies I performed to evaluate genotoxicity (Mesnage et al., 2021), endocrine disrupting effects (Mesnage et al., 2017a), obesogenic potential (Biserni et al., 2019) or effects on the gut microbiome (Mesnage et al., 2021). I regularly publish reviews on the topic (Mesnage et al., 2019), to provide clarification of the composition of pesticide formulations based on information available from the scientific literature, patents, industry publications. I also addressed the toxicity of co-formulants in a book on herbicide in concise language appropriate to engage both specialists in the research community and informed persons responsible for legislative, funding, and public health matters in the community at large (Mesnage and Zaller, 2021). I am regularly invited to speak about this topic in scientific conferences and public events, such as at the European Parliament PEST hearing on authorisation of plant protection products by Member States.

The contamination of foodstuffs by glyphosate residues has been linked to its use on crops which have been made tolerant to agricultural applications of Roundup herbicides. When I started my PhD in 2010, the controversy on GM crops was raging. No long-term studies were available to understand if the residues from glyphosate spray on GM plants could be a source of health risk. Repeated-dose 90-day toxicity studies on GM crops fed to rats often produced statistically significant differences in liver and kidney biochemical parameters, but it was not clear whether these differences were due to chance or to early signs of the development of chronic disease. The research group initiated the first study of the long-term toxicity of Roundup in Roundup-tolerant GM maize, that is, sprayed or not sprayed with Roundup.

The first manuscript describing the main findings was published in September 2012 in the journal Food and Chemical Toxicology (Séralini et al, 2012). The study was exploratory and not conceived to conclude with certainty on long-term effects because the pathologies of normal aging introduce background noise that impairs statistical power with 10 animals per group. The strain of rat used, Sprague-Dawley, is particularly prone to tumors. The paper caused an explosion of media coverage because it contained graphic images of rats with large tumors. The media coverage strategy used by Pr. Séralini was heavily criticised and condemned by the scientific community and journalists. This had a strong influence on public opinion about GM foods. The debate unfortunately even led to serious political consequences, with countries such as Kenya and Peru placing a moratorium on GM crops.

One year after publication, the editor-in-chief of the journal requested the raw data. This was examined by a panel of experts, who together with the editor-in-chief decided that the paper should be retracted because “the results presented (while not incorrect) are inconclusive”. Conflicts of interests, confidentiality and censorship in health risk assessment of these products were then discussed (Séralini et al., 2014). The debate continues, as lawsuits have uncovered evidence that Monsanto, the maker of Roundup, engaged with a network of scientists to discredit the study and obtain retraction of the paper. One should bear in mind that the influence of advocate groups is not unilateral. While companies develop strategies to preserve the commercialisation of their pesticides, other advocate groups deploy strategies to ban pesticides. This was the case in the Séralini affair. While Monsanto deployed a strategy to undermine the publication, the network of activists involved in the publication of this study orchestrated a communication operation causing an explosion of media coverage by spreading graphic images of rats with large tumors. Although the Séralini publication didn't provide insights on the toxicity of GM crops because of its inconclusiveness, the series of controversies which became the Séralini affair is a well-documented case of fundamental challenges faced by science in a world increasingly dominated by corporate influence.

We investigated estrogenic effects of bisphenol A (BPA) variants in 3 human breast cancer cell lines (Mesnage et al., 2017b). We found that BPA alternatives are not necessarily less estrogenic than BPA in human breast cancer cells. BPAF, BPB, and BPZ were more estrogenic than BPA. This study was published in the official journal of the Society of toxicology Toxicological Sciences, and highlighted by the Editor-in-Chief of the cover of journal. The EPA issued a press release to ‘highlight the need for testing of replacement chemicals prior to their introduction into commerce to demonstrate that they are safer than the chemical being replaced.’ Our study has subsequently be used as the basis of a new bill by the State of New York.

More recently, Breast Cancer UK has awarded a grant of £100,000 to Dr Michael Antoniou and colleagues Dr Robin Mesnage and Dr Ana Rodriguez-Mateos, King’s College London, to fund a PhD studentship, which will undertake biomonitoring of UK residents to identify levels of bisphenols and evaluate the potential breast cancer initiating and growth promoting activity of bisphenol mixtures.