Serum metabolite profiling yields insights into health promoting effect of A. muciniphila in human volunteers with a metabolic syndrome

Abstract

Reduction of A. muciniphila relative abundance in the gut microbiota is a widely accepted signature associated with obesity-related metabolic disorders. Using untargeted metabolomics profiling of fasting plasma, our study aimed at identifying metabolic signatures associated with beneficial properties of alive and pasteurized A. muciniphila when administrated to a cohort of insulin-resistant individuals with metabolic syndrome. Our data highlighted either shared or specific alterations in the metabolome according to the form of A. muciniphila administered with respect to a control group. Common responses encompassed modulation of amino acid metabolism, characterized by reduced levels of arginine and alanine, alongside several intermediates of tyrosine, phenylalanine, tryptophan, and glutathione metabolism. The global increase in levels of acylcarnitines together with specific modulation of acetoacetate also suggested induction of ketogenesis through enhanced β-oxidation. Moreover, our data pinpointed some metabolites of interest considering their emergence as substantial compounds pertaining to health and diseases in the more recent literature.

Keywords: A. muciniphila; acylcarnitines; amino-acids; human; ketone bodies; metabolic syndrome; metabolomic; obesity; prediabetes.

Figure 1.

Differential and common metabolomic effects of both treatments relative to the control group. (a) Study design of the Microbes4U© intervention. 52 obese or overweight individuals diagnosed with a pre-diabetic state and a metabolic syndrome were recruited and started the intervention. After three months of supplementation, blood samples were collected from 32 individuals. (b-c) Volcano plots depicting the metabolites which most notably changed following 3 months of supplementation with pasteurized (b) or alive A. muciniphila (c), under comparison with the placebo interventional effect. Labeled points represent metabolites for which the results of the Mann–Whitney U test comparing deltas of the treatment groups versus the placebo group were significant. Metabolites marked in red, exhibiting positive value for ‘mean difference from placebo’ are compounds that increased significantly following treatment with A. muciniphila. Metabolites marked in blue, exhibiting negative value for ‘mean difference from placebo’ are compounds that decreased significantly following treatment with A. muciniphila. The compounds which did not satisfy the condition of p < .05 for the univariate Mann–Whitney U test appear in yellow, and are not labeled. For readability, metabolites whose global delta exceeded the absolute value of 4, and for which the – result of the Mann–Whitney U test was not significant, were omitted from the plot. Metabolites for which the – log₁₀Pvalue was below 0.5 were also discarded from the plot. The two dash lines correspond to p-value cutoffs of 0.05 and 0.01, respectively. See also Table S1 for the detailed list of the metabolites, with corresponding value and pathway

Figure 2.

A. muciniphila negatively modulated circulating levels of several amino acids-derived metabolites that correlated with hepatic dysfunction Qualitative changes in phenylalanine, and tyrosine (a), and tryptophan (b) metabolites in plasma following the intervention. Intermediate metabolites which were not assayed are shown in gray and written in a smaller font. The routes including several steps and intermediaries not shown are represented by a dashed line. Enzymes are omitted. Compounds shown in red had a percentage of detection below 100% in our cohort. The trio of boxes below each metabolite represents the three groups, from left to right, given placebo, pasteurized, and alive A. muciniphila. Matched-pairs t-tests were performed on median scaled, log-transformed data, to verify changes from baseline (intragroup changes). Boxes with filled background mark metabolites that showed significant changes (p ≤ 0.05). The color represents the mean change from baseline value per group: red and green indicate a significant mean increase or decrease following intervention for the corresponding metabolite, respectively. Mann–Whitney U tests were performed to compare the differential values of both treated groups versus the placebo group (intergroup changes). Light red – and light green-shaded cells indicate 0.05 < p-value<0.10. The box was left emptied when the test was not significant. The direction of the arrow below both intervention groups indicates whether the metabolite had globally increased or decreased compared to the placebo effect. When the Mann–Whitney U test was significant (p ≤ 0.05), the arrow was colored according to the direction of the global change (red: increase, green, decrease). Light red – and light green-shaded cells indicate 0.05< pvalue<0.10. (c) Baseline Spearman’s correlation matrix between plasmatic hepatic enzymes and metabolites of the tyrosine and phenylalanine metabolism in the Microbes4U© cohort (n = 52). Negative correlations are colored in shades of blue and positive correlations in red. *: p < .05; **: p < .01; *** : p < .001

Figure 3.

A. muciniphila administration was associated with a positive modulation of acylcarnitine-circulating levels. (a) Bars represent the mean difference from placebo of the relative plasma concentrations of measured acylcarnitines for the groups given pasteurized and alive A. muciniphila. The color indicates the direction of the global delta. (b) Principal component analysis of variables for acylcarnitines and direction of vector for acetoacetate if applied to the acylcarnitine variables plot. (c) Scatterplot showing the linear relation between first principal components and acetoacetate

Figure 4.

A. muciniphila’s cross-talk with ketogenesis, lipid metabolism and glycolysis related metabolites. (a) Spearman’s rank correlation matrix of A. muciniphila and plasma metabolites belonging to ketone bodies metabolism, TCA cycle, and lipid metabolism, expressed as differential value (delta) (*P < .05). (b) Correlation Network map illustrating the Spearman’s correlation matrix. Metabolites that were highly correlated are brought together. The positioning of the metabolites was calculated by multidimensional scaling of the absolute values of the correlations. Gradient color, distance, and thickness of the lines were applied to metabolites nodes depending on coefficients of correlation. Negative and positives correlations are colored in shades of red and blue, respectively. Abbreviations: Akk, A. muciniphila; BHB, 3-hydroxybutyrate; NEFA, non-esterified fatty acids; X3.HHC, 3-hydroxhexanoylcarnitine

Figure 5.

Overview of the mechanism proposed for the A. muciniphila-mediated metabolomic switch toward β-oxidation and ketogenesis. In physiological conditions, overnight fast is associated with high lipolytic rates to increase the availability of substrates for β – oxidation. This results in a substantial efflux of non-esterified fatty acids and esterified carnitine in the plasma. Entry of acyl-CoA into the mitochondria is facilitated by the acylcarnitine transport system, while part of the generated acylcarnitines are released in the plasma. Fatty acids are then broken down into Acetyl-coA through β-oxidation in the mitochondria. Administration of A. muciniphila in insulin-resistant overweight individual turndowned glycolysis and amplified the aforementioned pathway and triggered a switch toward the use of Acetyl-CoA for ketogenesis rather than toward the TCA cycle. In turn, elevated levels of ketones bodies, alongside gluconate, may contribute to reduce the redox state, favoring the activity of CACT. The thicker the arrow the stronger the corresponding pathway was triggered. Elements and arrows in pink reflect known facts from literature relative to A. muciniphila action. Increase and decrease metabolites are shown in blue and green, respectively. CACT, Carnitine-acylcarnitine translocase; CPT1α, carnitine palmitoyl‐transferase I; G6P, Glucose-6-phosphatase; PPARα, peroxisome proliferator-activated receptor alpha; TCA, tricarboxylic acid cycle