Gut bacterium Intestinimonas butyriciproducens improves host metabolic health: evidence from cohort and animal intervention studies

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Short chain fatty acids are among the most known microbial metabolites that signal to the host. Intestinimonas butyriciproducens is a prevalent commensal bacterium that has a unique capability of converting dietary fructoselysine to butyrate and acetate and has a completed fructoselysine catabolic pathway. Dietary fructoselysine is an abundant Amadori product formed in foods during processing and is part of food products rich in dietary advanced glycation end products which can be potentially toxic. Therefore, understanding the role of this bacterium and fructoselysine metabolism in metabolic health is highly relevant. Results: We accessed associations of I. butyriciproducens with metabolic risk biomarkers via both strain and functional levels using a human cohort characterized by fecal metagenomic analysis. We observed that the level of the bacterial strain as well as fructoselysine fermentation genes were reversely associated with BMI, triglycerides, HbA1c and fasting insulin levels. We also investigated degradation capacity of fructoselysine within the Intestinimonas genus using a culture dependent approach and observed that I. butyriciproducens as a key player in the butyrogenic fructoselysine metabolism in the gut. To explore the function of I. butyriciproducens on host metabolism, we employed the diet-induced obesity mouse model to mimic the human metabolic syndrome. Oral supplementation of I. butyriciproducens counteracted body weight gain, hyperglycemia as well as adiposity. Moreover, within the inguinal white adipose tissue, bacterial administration reduced inflammation and promotes pathways involved in browning and insulin signaling. The observed effects are attributable to the formation of the short-chain fatty acids butyrate and acetate from dietary fructoselysine, as their plasma levels were significantly augmented by the bacterial strain, thereby contributing to systemic effects of the bacterial treatment. Conclusions: I. butyriciproducens ameliorates host metabolism in the context of obesity and may thus be a good candidate for new microbiota-therapeutic approaches to prevent or treat metabolic diseases. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Diet, the human microbiome and host genetics are determinants of metabolic status, partially through the production of metabolites via fermentation of dietary components by the gut microbiota [ 1 , 2 ]. Understanding the metabolism of dietary ingredients by the microbiome is key to mediate the effects of the human microbiome on host metabolism. Hence, mechanistic studies on the interactions between dietary components, the microbiome and the host are urgently needed. The question regarding the ability of the gut microbiota to metabolize dietary compounds not present in raw foods but formed by food processing is a great interest: are they harmful xenobiotics or human microbiota is able to metabolize them for good? Fructoselysine is an abundant Amadori product formed via the non-enzymatic reaction between a reducing sugar and amino acids in foods during thermal processing and storage. The formation of Amadori compounds partially blocks amino acids from absorption, hence reducing their bioavailability to the host [ 3 ]. It is estimated that daily intake of Amadori products is around 0.5–1 g of which, in healthy subjects, the large majority reaches the large intestine for further digestion [ 4 ]. Only a few intestinal bacteria have been reported to metabolize fructoselysine. Strains marketed as probiotics, mainly Bifidobacterium and Lactobacillus spp. have been reported to utilize fructoselysine and only generated glucose moiety but not lysine [ 5 ]. E. coli is able to degrade fructoselysine and use the released glucose for growth in vitro [ 6 ] while Collinsella intestinalis can metabolize fructoselysine to acetate, formate both in vitro and in vivo [ 7 ]. All these strains failed to use the liberated lysine moiety for growth. Intestinimonas butyriciproducens showed exceptional metabolic features being able to use both liberated glucose and lysine moiety from fructoselysine breakdown to form butyrate via two separate routes [ 8 ]. Fructoselysine conversion to butyrate is highly desired. Butyrate serves as an energy source for colonocytes but also suppresses inflammation in various tissues [ 9 – 11 ], involving in regulation of insulin release via gut hormone stimulation [ 10 , 12 ]. This is in line with numerous reports on depletion of butyrate-producing species in diabetic and obese subjects and supplementation of these species provided metabolic benefits to the host [ 13 – 15 ]. Therefore, it is of great interest to explore the therapeutic potential of I. butyriciproducens in improving metabolic health via its unique capability of converting fructoselysine to butyrate. In this study, we investigated the associations between butyrogenic Intestinimonas butyriciproducens and fructoselysine pathway genes and metabolic risk biomarkers in subjects with metabolic compromising conditions compared to healthy subjects in the Swedish impaired glucose tolerance (IGT) cohort (n = 1011) [ 16 ]. Subsequently, we isolated and studied the capacity and genomic made-up of fructoselysine fermentation by four human Intestinimonas isolates. We later administered Intestinimonas butyriciproducens GL3 isolate to diet-induced obesity mice to explore the impact on obesity and host metabolism and to achieve a proof-of-concept study in human. Results Intestinimonas butyriciproducens and fructoselysine fermentation were reduced in subjects with high metabolic risks. To investigate whether Intestinimonas butyriciproducens associates with host metabolic health, we employed the metagenomics data from a Swedish cohort, comprising 1,011 individuals with prediabetes and treatment-naïve T2D and healthy relatives [16]. When comparing the rarefied read abundances of I. butyriciproducens in subjects with different glycemic status raging from normal glucose tolerance to type 2 diabetes, we observed that the relative abundance of I. butyriciproducens was significantly reduced in groups with impaired fasting glucose (IFG, p = 0.034), impaired glucose tolerance (IGT, p = 0.02), combined glucose intolerance (CGI, p = 0.021) and type 2 diabetes (T2D, P = 0.055) as compared to low-risk normal glucose tolerance (IrNGT) (Fig. 1A). To further explore associations of I. butyriciproducens with metabolic risk factors, we correlated the relative abundance of identified fructoselysine pathway genes [8] to various metabolic biomarkers (Fig. 1B). Most of the individual fructoselysine pathway genes were negatively associated with metabolic risks, except genes involved in fructoselysine degradation including fructoselysine kinase (AF_949) and fructosamine deglycase (AF_00951). This can be explained by the fact that these two genes were also present in the genomes of some pathogenic strains including E. coli and Salmonella [6, 17]. Nevertheless, these bacteria are not capable of fermenting lysine or producing butyrate. To investigate the relations of Intestinimonas butyriciproducens with metabolic risks via its unique capacity to convert fructoselysine to butyrate, we studied the associations between the total abundance of fructoselysine pathway genes and metabolic markers. We found significant negative correlations between the fructoselysine pathway relative abundance and body mass index (BMI, rho=-0.15; p = 7.4e-07), triglycerides (rho=-0.19; p = 1.0e-09), glycated hemoglobin (HbA1c, rho=-0.10; p = 0.002) and fasting insulin (rho=-0.16; p = 2.4e-07) (Fig. 1D-F), which is in line with the observed reduced abundance of I. butyriciproducens in individuals with T2D or impaired glucose tolerance. Intestinimonas as a key player in the butyrogenic fructoselysine metabolism in the gut To explore the capacity of fructoselysine fermentation by Intestinimonas , we next performed a conventional cultivation technique using a selective medium on agar plate to isolate Intestinimonas species from human stool from three healthy volunteers. We obtained three Intestinimonas isolates and subsequently sequenced 16S rRNA genes as well as genomes to enable further genomic and physiological characterization. To determine the taxonomic relationship of these isolates, a phylogenetic tree was made using 16S rRNA gene sequences of three Intestinimonas isolates (strain GL3, IY4 and AS-BT) and other closely related species in Clostridium cluster IV and cluster XIVa (Fig. 2A). While 16S rRNA genes of three strains IY4 and GL3 were highly similar (> 99.7%) to Intestinimonas butyriciproducens type strain and previously isolated Intestinimonas strain AF211, 16S rRNA gene of strain AS-BT was only 95% similar to that of the type of strain, suggesting strain AS-BT likely represents a new species of Intestinimonas genus. Nevertheless, all three Intestinimonas isolates were able to grow and convert fructoselysine and lysine to butyrate and acetate with a similar degradation rate (Fig. 2B-E) and have complete fructoselysine pathway genes in their genomes (Supplementary table 1). Interestingly, we found that strain AS-BT did not have a complete vitamin B12 pathway in the genome, whereas three other strains had (Supplementary table 2), indicating the potential capacity of strain GL3 and IY4 to synthesize pseudovitamin B12 as strain AF211 [18]. As antibiotic resistance is highly relevant for gut bacteria, we also determined the minimum inhibitory concentration (MIC) of various antibiotics in four strains using Etest (Supplementary table 3). AS-BT clearly had a different MIC profile as compared to the other three strains which may be attributed to its distant taxonomic position of this strain from three other Intestinimonas isolates. In general, Intestinimonas isolates were sensitive to teicoplanin, chloramphenicol, vancomycin, cefotaxime, and oxacillin, which are inhibitors of cell wall synthesis in Gram-positive bacteria, but they showed relatively high MIC values with ciprofloxacin and sulfamethoxazole, however, no gene has been attributed to resistance mechanisms for these two antibiotics. Although some vancomycin resistance genes (vanB, vanW, vanS and vanR) were detected in the genomes (Supplementary table 4), those genes were not sufficient to form a complete operon to confer vancomycin resistance [19]. Two out of four strains were resistant against erythromycin at a very high level while all four strains were found to be resistant to tetracycline at different levels (from 2 µg/ml to 16 µg/ml). Notably, no tetracycline resistance gene tetW was found in the genome of GL3 whereas tetW was present in all genomes from three other Intestinimonas strains. In contrast, there was a large number of gene copies for efflux pumps found in all Intestinimonas genomes with the highest number of 27 pumps from GL3 isolate which may be an important mechanism to eliminate toxic compounds. Intestinimonas butyriciproducens limits body weight gain in mice fed a high-fat diet To explore the effects of Intestinimonas butyriciproducens GL3 strain (from now referred as IB) on systemic host metabolism, we used the murine diet-induced obesity (DIO) model [20]. C57BL/6J mice were fed a high-fat diet (HFD) or matching low-fat diet (LFD) for 13 weeks and subjected to oral gavage 3 times a week of placebo or IB. Importantly, HFD contains significantly higher levels of protein-bound fructoselysine as compared to a regular chow-diet (Supplementary Fig. 1; 128.3 versus 567.5 mg/100gr protein, p = < 0.0001), making HFD an excellent source of dietary fructoselysine for the production of butyrate by IB Although we did not observe changes in food intake between placebo and IB-treated mice, bacterial administration resulted in significantly less body weight gain in the HFD-fed mice as compared to placebo treatment after 13 weeks (Fig. 3A; 15.1 g versus 11.5 g, p = 0.038). The difference in body weight between placebo and IB groups became apparent after 11 weeks suggesting that the bacterium affects the host only after a prolonged treatment and/or the bacterial treatment becomes effective when mice develop exacerbated adiposity/metabolic dysregulation. Notably, the bacterial supplementation did not affect body weight in the LFD-fed mice. As butyrate-producing bacteria have been linked to insulin sensitivity in both human and mice studies, we next assessed blood glucose levels in fasting state and following intraperitoneal insulin injection. As expected HFD-feeding increased the fasting glucose levels and bacterial supplementation significantly lowered fasting glucose concentrations (Fig. 3B; 11.3 mM versus 9.4 mM glucose, p = 0.035). During the insulin-tolerance test, blood glucose levels of IB-treated mice were constantly lower than in placebo mice, suggesting an amelioration of insulin sensitivity (Fig. 3C). Analysis of the area under the curve (AUC) showed a trend toward reduced glucose rate over time (Fig. 3D; AUC of 48.41 versus 42.08, p = 0.057) upon bacterial administration. Intestinimonas butyriciproducens counteracts the fat accumulation and promotes lipid degradation and browning processes in inguinal white adipose tissue Analysis of white adipose tissues (WAT) revealed a reduction in subcutaneous/inguinal white adipose tissue (iWAT) in IB-treated mice on HFD (2.43 versus 1.58% BW, p = 0.023), while the proportion of visceral/epididymal white adipose tissue (eWAT) relative to total body weight was unchanged between placebo and IB groups (Fig. 3E, F). Since IB intake led to a reduction in the proportion of IWAT tissue, we next assessed the expression of key metabolic genes in iWAT. We found that the expression of genes involved in mitochondria metabolism and, specifically, fatty acid oxidation was strongly upregulated following IB treatment in HFD-fed mice. Indeed, IB supplementation reduced the HFD-induced expression of Ppargc1a4 (0.007108 versus 0.01577, p = 0.037), Ppara (0.009667 versus 0.03057 p = 0.025), and Cpt1a (0.1497 versus 0.3879, p = 0.017) to levels similar to the ones observed in LFD-treated mice (Fig. 4A-C). In contrast, the expression rates of the browning markers were downregulated upon HFD-feeding but augmented by oral IB treatment (Fig. 4D-H). In fact, compared to placebo, bacterial supplementation in obese mice resulted in a significant enhanced expression of the browning markers Ucp1 (0.009592 versus 0.01908, p = 0.043), Cidea (0.08147 versus 0.2776, p = 0.023), Prdm16 (0.04065 versus 0.1585, p = 0.0023), Dio2 (0.03754 versus 0.2598, p = 0.054) and Tbx1 (0.04912 versus 0.1597, p = 0.015). Overall, these data suggest that the reduced accumulation of iWAT is driven by increased energy expenditure in IB-treated mice. Intestinimonas butyriciproducens constrains white adipose tissue inflammation and loss of insulin sensitivity in obesity As WAT is an important modulator of systemic insulin sensitivity [21] and IB intake augmented glucose uptake after insulin injection, we next measured the intra-iWAT expression of Irs2 (insulin receptor substrate 2), which mediates the cytoplasmic signaling of insulin [22]. Expression of Irs2 was markedly diminished in iWAT of obese mice, however in line with the amelioration of insulin resistance, IB treatment led to an enhanced expression of IRS2 both at gene (Fig. 5A; 0.146 versus 0.3123, p = 0.0029) and protein levels (Fig. 5B; 0.314 versus 0.8455, p = 0.0086). Since the development of insulin resistance and adipose tissue inflammation are intertwined, we subsequently investigated the iWAT production of relevant pro-inflammatory cytokines in obese mice. This revealed that intestinal IB exerted anti-inflammatory effects as IB treatment significantly reduced the intra-WAT secretion of tumor necrosis factor (TNF)-α (0.5837 versus 0.3859, p = 0.0048), interleukin (IL)-6 (1.003 versus 0.6825, p = 0.0053) and IL-1β (0.4915 versus 0.3886, p = 0.0165) (Fig. 5D-F). In addition, staining for the F4/80 macrophage marker showed that macrophage recruitment was diminished, albeit not significant, in IB-treated mice (Fig. 5G), possibly indicating that immune infiltrating cells as well as parenchymal cells were the source of the detected pro-inflammatory cytokines. Oral intake of Intestinimonas butyriciproducens results in higher plasma SCFA levels without affecting intestinal barrier function Finally, to understand the distal effects of intestinal IB, we determined the circulating levels of SCFAs. Plasma levels of butyrate and acetate were significantly increased upon IB treatment as compared to placebo in the obese mice (butyrate: 0.9817 versus 1.12 µM, p = 0.0367; acetate: 374.3 versus 664.3 µM, p = 0.0068) but not in lean mice, while the concentrations of propionate were unchanged (Fig. 6A-C). In addition, although the plasma concentrations of fructoselysine were similar between the groups, the butyrate/fructoselysine ratio was increased in the HFD-fed mice receiving IB (p = 0.059), underscoring the active conversion of dietary fructoselysine into butyrate (Fig. 6D, E). To exclude that the observed changes in gut-derived metabolites were due to alterations in intestinal barrier function, we also examined the small intestine and colonic expression of genes encoding for tight-junction molecules (claudin-4, occludin and zona occludens-1). The results showed that IB did not affect barrier integrity as expression levels were comparable between treatment groups (Supplementary Fig. 2A-F). Altogether, these findings underscore that the systemic effects of IB, observed in obese mice, are mediated by produced butyrate and acetate, which were subsequently absorbed into the systemic circulation. The lack of plasma SCFA changes in mice fed LFD suggests that the IB-mediated production of butyrate and acetate may have derived from the higher fructoselysine content in HFD (Supplementary Fig. 1). Discussion Amadori product fructoselysine is abundantly present in cooked foods but only a small portion of protein-bound lysine Amadori products can be absorbed in the gut [ 3 ], hence funneling the majority to the lower gastrointestinal tract for microbial use. We previously demonstrated that I. butyriciproducens uniquely ferments fructoselysine to butyrate via lysine and acetyl-CoA pathway simultaneously [ 8 ]. In the present study, we reported three additional Intestinimonas isolates from human subjects, all of which have the capacity to convert fructoselysine to butyrate and harbor all genes for a complete fructoselysine pathway (Fig. 2 ). This pinpoints that Intestinimonas is a key player in intestinal fructoselysine fermentation to butyrate and I. butyriciproducens is the most abundant species in the human gut which is in good agreement with our previous report [ 18 ]. In addition, we isolated a new Intestinimonas species (AS-BT isolate) which shared a high commonality of metabolic features with I. butyriciproducens except the capacity to synthesize pseudovitamin B12, an essential cofactor of lysine-5,6-aminomutase, a key protein involved in lysine/fructoselysine fermentation [ 23 ]. Intriguingly, we found that the abundance of both I. butyriciproducens and fructoselysine pathway genes were reversely associated with various metabolic markers in a (pre)diabetic cohort (n = 1011 subjects), suggesting a reduced capacity of the microbiome to convert fructoselysine to butyrate in cardiovascular and metabolically compromised subjects compared to healthy individuals. The reduced capacity of the human microbiota to convert fructoselysine to beneficial butyrate may lead to a higher accumulation of fructoselysine and lower levels of colonic butyrate, both of which are undesired. While butyrate is required for healthy colon, high fructoselysine level may facilitate its pH driven conversion to advanced glycation end products (AGEs) the level of which has been associated with aging, atherosclerosis and diabetes [ 24 – 27 ]. We also observed that the prevalence of Intestinimonas and fructoselysine fermentation genes are also associated with feeding modes in infants [ 28 ]. In contrast, butyrate or short chain fatty acids in general have been reported as an important component in controlling body weight and insulin sensitivity [ 12 ]. This association from the cohort study is well in line with the results obtained from the in vivo study which discloses that administration of I. butyriciproducens GL3 strain (IB) exerts multiple metabolic benefits in diet-induced obesity. Indeed, bacterial supplementation led to significantly decreased body weight gain, iWAT accumulation and fasting glucose levels in obese mice. Moreover, upon insulin administration, blood glucose levels remained lower in the IB-treated mice as compared to placebo-treatment. These effects were accompanied by increased IRS2 expression, enhanced expression of genes crucial for lipid catabolism as well as browning and reduced inflammation in IWAT of IB-treated obese mice. Lastly, bacterial administration resulted in a higher concentration of circulating butyrate and acetate. Most of the observed IB-induced metabolic benefits in vivo are likely resulting from the higher rate of SCFA production executed by this commensal bacterium. Indeed, previous reports have shown that sodium butyrate administration limits gain-weight, and improves glucose kinetics, insulin sensitivity, energy expenditure as well as mitochondrial function in DIO murine models [ 29 – 31 ]. Similarly, oral administration of sodium acetate was sufficient to counteract adiposity, ameliorate insulin resistance and boost energy expenditure and oxidative metabolism in DIO mice [ 30 ]. The short chain fatty acid butyrate and acetate exert pleiotropic effects on host by serving as an energy source (accounting for approximately 10% of the caloric requirement in humans), functioning as histone-deacetylase inhibitors and signaling through host G-protein–coupled receptors (GPR) 41, 43 [ 32 ] and 109A/43 [ 33 ]. Besides their protective effects against adiposity, SCFAs are also known anti-inflammatory molecules [ 9 , 34 – 36 ]. In line, we found that the administration of the butyrate-producing I. butyriciproducens strain significantly reduced the production of TNF-α, IL-6 and IL-1β in iWAT. These effects are likely driven by SCFA-mediated inhibition of both the nuclear factor–κB (NF-κB) signaling and NLRP3 inflammasome activation [ 37 – 40 ]. Notably, both pathways are critical in the induction of meta-inflammation and obesity-induced insulin resistance [ 41 , 42 ]. Accordingly, we observed that IB intake led to significantly lower fasting blood glucose levels and a trend toward enhanced glucose disposal upon insulin injection. Hence, the IB-driven improvement in glucose homeostasis may result from the SCFA inhibitory effects on local inflammation in WAT and thus maintenance of functioning insulin signaling in adipocytes. In line, we reported that the iWAT expression of IRS2 was markedly increased upon IB administration as compared to placebo treatment. This effect is supported by previous reports disclosing the ability of butyrate to enhance the expression of the signaling molecules IRS1 and IRS2 [ 43 ], thereby facilitating insulin signaling. Tissue inflammation and insulin-resistance are tightly linked to exacerbated intracellular lipid accumulation. In this regard, the transcriptional changes observed in iWAT upon bacterial treatment indicate an induction of fatty acid beta-oxidation through PGC1-alpha and PPAR-alpha pathways given the marked upregulation of the genes encoding for these master transcriptional regulators of mitochondria biogenesis and oxidative metabolism [ 44 ]. Moreover, the expression of Cpt1a, encoding for the key rate-limiting enzyme in fatty acid oxidation (FAO) [ 45 ], was significantly upregulated in iWAT of IB-treated obese mice, whereas the relative iWAT weight was reduced, underscoring that increased FAO resulted in less adipocyte hypertrophy [ 46 ]. The increased circulating levels of SCFAs mediated by administered bacterial strain, are likely the underlying cause of these protective effects; indeed, both in vitro and in vivo butyrate has been shown to activate PGC1alpha pathway and FAO by enhancing CPT1 activity [ 47 – 49 ] as well as reduce adipocyte expansion [ 50 ]. Overall, these anti-lipogenic effects of I. butyriciproducens may contribute to diminished WAT inflammation. In support of this concept, butyrate was shown to suppress inflammatory responses in the context of adipocyte-macrophage interactions through inhibition of lipolysis [ 51 ]. In line with the ability of butyrate to promote energy expenditure in vivo , the transcriptional signature of iWAT in IB-treated mice is indicative of WAT browning/beiging. The latter is characterized by an augmented expression of thermogenesis-related genes, which are typically expressed by brown adipose tissue, a reduction in lipid accumulation and increased energy expenditure [ 52 ]. Compared to visceral (epididymal) WAT, subcutaneous (inguinal) iWAT is more susceptible to browning due to the higher expression of browning markers UCP1, Cidea, and Pdrm16, as well as the beige markers Tbx1 and P2rx5 [ 53 ], possibly indicating that iWAT is more responsive to external stimuli, such as bacterial metabolites. Overall, the described effects on iWAT and the plausible WAT browning may explain the reduction in body weight gain in IB-treated obese mice, as stimulation of WAT browning may increase total body energy expenditure and promote fat reduction [ 54 , 55 ]. Lastly, the observations that elevated plasma SCFA levels were found only in obese mice but not lean mice following bacterial intake and the lack of differences in all clinical parameters measured between placebo- and IB-treated lean mice underscore that the bacterial SCFA production is driven by the diet. In fact, HFD contains a higher amount of fructoselysine compared to regular chow-diet, therefore the protective effects of I. butyriciproducens can be more prominent in DIO mice, likely due to a higher rate of fructoselysine-to-butyrate conversion. Moreover, the anti-inflammatory effects of I. butyriciproducens could be attributed, at least in part, to the utilization of a critical precursor, fructoselysine, of α-dicarbonyls and AGEs, which are increased in type 2 diabetes, and associated with diabetic complications as they instigate oxidative stress and pro-inflammatory cytokine release [ 56 , 57 ], [ 58 , 59 ]. In light of the fact that mice were sacrificed after 6 hours of fasting, thus during fructoselysine-depletion, the significant, yet mild, increase in circulating butyrate is probably an underestimation of the circulating levels present during fed-state upon bacterial administration. Overall, this study highlights the important role of the gut microbiota in the regulation of host physiology and particularly host metabolism. Notably, our findings support the development of microbiome-targeting approaches for the prevention or amelioration of metabolic disorders, such as obesity and type 2 diabetes. Conclusions In summary, we found that Intestinimonas plays a key role in the conversion of dietary fructoselysine to butyrate in the gut and the abundance of Intestinimonas butyriciproducens as well as fructoselysine pathway genes were reversely correlated with multiple risk biomarkers in a cohort study. In vivo, Intestinimonas butyriciproducens counteracts adiposity, ameliorates glucose metabolism and tissue inflammation by converting dietary fructoselysine to butyrate and acetate in DIO mice. Material and methods Metagenomics analysis in the Swedish IGT cohort The rarefied abundance levels of metagenomics species (CAG00017), annotate as Intestinimonas butyriciproducens , and 33 KEGG orthologies (KOs) involved in the fructoselysine metabolism were obtained from a previous study aiming to characterize the gut microbial changes in prediabetes and diabetes based on the Swedish IGT cohort (n=1,011)[16]. The relative differences of IB were then compared across individuals with distinct glucose intolerance levels versus the healthy control group. To examine the potential importance of fructoselysine metabolism to glucose intolerance, the relative abundances of each KO and/or the whole pathway (based on aggregated sum values of all KOs) were associated with 12 common clinical variables, such as the levels of fasting glucose, insulin, HbA1c, and triglycerides, indicative of the T2D status, respectively. The study was approved by the IRB of Sahlgrenska Hospital, Gothenburg University and all subjects provided written informed consent. Isolation of Intestinimonas from human stool Fresh fecal samples were collected in 15 ml falcon tubes containing anaerobic phosphate buffer (pH7) and later stored in 25% glycerol in 5 ml anaerobic bottles kept at -80 o C freezer. 0.5 ml of these fecal slurries was transferred to 10 ml anaerobic bicarbonate-buffered mineral salt medium (CP medium) containing 40mM lysine as energy and carbon source to enrich lysine-fermenting bacteria. The headspace was filled with CO 2 /N 2 (1:4) at 1.5 atm and incubation was at 37 o C. Subsequently, the enrichment cultures were transferred two more times in the same medium before being plated on YCFA agar medium containing 40 mM lysine as substrate (YCFA_L). Single colonies were picked and plated at least 3 times on the same medium which resulted in an axenic culture. The purity of the strains, designated as strain GL3, AS-BT and IY4 was confirmed by 16S rRNA gene sequencing and microscopy. The strains were routinely maintained in YCFA_L medium at 37 °C. 16S gene sequences of three isolates and strain AF211 were aligned with the multiple sequence aligner SINA [60] and merged with the Silva SSU Ref database (release 111). A phylogenetic tree of three isolates and Intestinimonas AF211 and closely related strains was constructed in the ARB software package (v. 6) by an algorithm [61]. Enrichment medium was done in anaerobic bicarbonate-buffered mineral salt medium (CP medium) [62] consisting of (l −1): 0.53 g Na2HPO4 . 2H2O, 0.41 g KH2PO4, 0.3 g NH4Cl, 0.11 g CaCl2 . 2H2O, 0.10 g MgCl2 . 6H2O, 0.3 g NaCl, 4.0 g NaHCO3 and 0.48 g Na2S . 9H2O as well as alkaline and acid trace elements (each 1 ml l −1) and vitamins (0.2 ml l −1) [62]. The alkaline trace element solution contained the following (mM): 0.1 Na2SeO3, 0.1 Na2WO4, 0.1 Na2MoO4 and 10 NaOH. The acid trace element solution was composed of the following (mM): 7.5 FeCl2, 1 H3BO4, 0.5 ZnCl2, 0.1 CuCl2, 0.5 MnCl2, 0.5 CoCl2, 0.1 NiCl2 and 50 HCl. The vitamin solution had the following composition (g l −1): 0.02 biotin, 0.2 niacin, 0.5 pyridoxine, 0.1 riboflavin, 0.2 thiamine, 0.1 cyanocobalamin, 0.1 p-aminobenzoic acid and 0.1 pantothenic acid. YCFA medium (l −1): 10 g soy peptone, 10 g yeast extract, 4 g NaHCO 3 , 2.7g sodium acetate, 4.5g K 2 HPO 4 , 0.7g KH 2 PO 4 , 0.9 g NH 4 Cl, 0.9 g NaCl, 0.09 MgSO 4 , 0.09 CaCl 2 , 1 ml vitamin solution (1mg biotin, 1mg cobalamin, 3mg PABA, 5mg folic acid, 15mg pyridoxamine in 100 ml H 2 O), 1ml resazurine (0.5 g/l), 0.5g L-cysteine. In case of agar medium, 10g noble agar (DIFCO) was added to YCFA liquid before autoclave. The agar medium was then brought inside an anaerobic chamber and poured on agar plates. Those plates were then left slightly open for 30min till the agar got dried. The plates were kept for a maximum a week in the chamber before use. All streaking and plating were performed in the anaerobic chamber while the plate incubation was done in anaerobic jars filled with N 2 /CO 2 in the gas phase by a gas exchange machine. Genome sequencing and fructoselysine pathway gene analysis Strain GL3, IY4 and AS-BT were cultivated in 50ml YCFA_L liquid medium for an overnight at 37 o C. The bacterial cells were harvested at the late exponential phase by centrifuging at 4700 rpm at 4 o C. The cell pellets were used for DNA extraction using MasterPure™ Gram Positive DNA Purification Kit (Epicentre) according to the manufacturer’s instructions. After checking the quality on a Nanodrop, 30µl of high-quality DNA solution were send in dry ice to GATC for draft genomes using Illumina sequencing technology. Draft genome assemblies were constructed using the MyPro assembly pipeline [63]. Raw reads were quality checked using FastQC [64]. Reads were trimmed and subsampled to a total coverage of 100X (50X for forward reads, 50X for reverse reads), then assembled using 4 different assembly tools: VelvetOptimiser [65], Edena [66], SOAPdenovo [67] and SPAdes [68]. The resulting contigs were ordered with r2cat [69] using the Intestinimonas butyriciproducens AF211 genome (CP011307) as a reference and overlapping contigs merged resulting in the final genome assembly. Genome assemblies were then annotated using RAST [70]. The annotation was done by RAST server [70]. Functional prediction of proteins was verified manually by BLASTing the amino acid sequences in Pfam [71], Brenda, Interpro [72] and Uniprot databases. In addition, the screening of antibiotic resistance genes was done using ABRICATE against the NCBI, ARG-ANNOT, ResFinder and VFDB databases. Fructoselysine growth experiment Conversion of fructoselysine was tested in CP medium containing 5 mM fructoselysine (provided by TRC, North York, Canada). The inoculum was 2.5% from active cultures of strain GL3, IY4, AS-BT and AF211 for the growth test. All strains were pre-cultured in YCFA containing 20mM lysine. The bacterial cultures were sampled during the growth up to 48 hours for substrate and end metabolite measurements as described below. Antibiotic resistance profile The E-test was done to identify minimal inhibitory concentrations (MICs) according to the manufacturer's protocol (bioMérieux, France). Both strains were pre-grown in RCM broth (overnight cultures) and 50 µl was spread on RCM agar plates (1.5% w/v agar) until the agar surface was dry and the liquid was absorbed by the agar. Two E-test strips were used per antibiotic and considered as duplicates. Antibiotics tested included ciprofloxacin, cefotaxime, erythromycin, oxacillin, teicoplanin, tetracycline, tobramycin, vancomycin and sulfamethoxazole. The concentration range was 0.016–256 µg/ml for chloramphenicol, oxacillin, tetracycline, tobramycin and vancomycin, and 0.016–32 µg/ml for ciprofloxacin, cefotaxime, erythromycin, teicoplanin and sulfamethoxazole. MIC values were recorded directly from the strips after 24 h, 48 h and rechecked after 4 days. As tetW gene was detected in the genomes of most Intestinimonas strains and some of Intestinimonas strains were found to be resistant in erythromycin from the Etest, tetracycline and erythromycin were selected to perform the MIC test in liquid according to EFSA guideline. The test was done in 10ml YCFA medium containing lysine as substrate in anaerobic bottles filled with CO 2 /N 2 (1:4) at 1.5 atm. The concentration of tetracycline was 2-fold reduction in each bottle from 256µg/ml to 1µg/ml. The 256 ug/ml tetracycline bottle was prepared in 20 ml growth medium by adding 1ml of tetracycline filter sterilized stock solution (5.12mg/ml) to 19 ml complete medium. This medium was then serial diluted two-fold to get concentrations of 128, 64, 32, 16, 8, 4, 2 and 1 ug/ml. All these bottles were inoculated 2% with an overnight culture. The growth was monitored by OD measurement at 24h, 48h, 72h and 96h. Bottles without tetracycline were used as a positive control. MIC test for erythromycin in liquid was done in the same way by replacing tetracycline by erythromycin. Animal studies All animal studies were approved by the Institute Ethical Committee. C57BL6/J male mice were purchased from Charles River at the age of 4 weeks, fed a regular chow diet and kept under regular 12h/12h light/dark cycles. To determine the impact of IB on whole-body metabolism, male mice were randomized in 4 groups (N=12) receiving 3 times/week 2x10 9 CFU Intestinimonas butyriciproducens GL3 or placebo solution (anaerobic PBS) by oral gavage and fed ad libitum a low-fat diet (LFD, 10%kcal from fat, Research Diets, D12450Ji) or high-fat diet (HFD, 60%Kcal from fat, Research Diets, D12492i). Mice were fed LFD/HFD for 13 weeks and IB/placebo supplementation started one week before switching to special diets. To avoid cage-effects on the microbiota composition, 3 mice were housed in one cage. Body weight and food consumption were monitored once a week. At the end of the study, insulin-tolerance test (ITT) was performed in obese mice on HFD: after 6 hour-fasting, mice received an intraperitoneal injection of insulin (0.5U/kg); blood glucose levels were assessed by tail prick using glucometer strips at 0, 15, 30, 60 minutes post-injection. To avoid unnecessary discomfort and suffering due to potential hypoglycemic events, lean mice fed a LFD were not subjected to ITT. Mice were sacrificed under anesthesia (5% isoflurane, O 2 flow of 2 L/minute), blood was collected by cardiac puncture, harvested organs were stored in formalin (for later paraffin-embedding) and snap-frozen in liquid-nitrogen. White-adipose tissues were weighed immediately after collection. Protein bound fructoselysine in high fat diet and low fat diet Protein bound fructoselysine in the two diet was indirectly quantified through furosine concentration according to the method of Troise et al. [73] by using a Nexera U-HPLC system coupled with a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan). In brief, a 0.5 g aliquot of each chow was added to 4 mL of HCl (7.4 M) and then incubated at 110 °C, for 20 h. After filtering, 400 µL of the hydrolysate suspension was dried under vacuum using a rotary evaporator and reconstituted in 400 µL of 80% aqueous acetonitrile along with d4-furosine as internal standard (final concentration 200 µg/l). For separation of furosine and its internal standard, a core-shell Kinetex HILIC column (2.6 µm, 2.1 mm × 100 mm, Phenomenex) thermostated at 30 °C, with a flow rate of 0.4 mL/min was used. The mobile phases consisted of 0.1% formic acid (solvent A), 0.1% formic acid in acetonitrile (solvent B), and 50 mmol/L ammonium formate (solvent C). The gradient was as follows (t in [min]/[%B]): (0.0/80), (3.5/40), (6.5/40), with 4.5 min for equilibration, while solvent C was kept at 10%. Positive ionization multiple reaction monitoring (MRM) mode was used; the spray voltage was 4.0 kV and the collision energies (CE) were the following (in bold quantifier ions for furosine): furosine ( m/z 255.3 à130, 84 , CE: 12 and 18), furosine-d4 ( m/z 259 à 134, 88 CE: 12 and 15). Profile data were acquired and analyzed through LabSolutions (Shimadzu Corporation). Targeted liquid chromatography tandem mass spectrometry of mouse cecum and plasma samples Fructoselysine, lysine and SCFAs in CP medium, mice caecum and mice plasma were analysed by liquid chromatography high resolution tandem mass spectrometry (LC-MS/MS) by means of a Vanquish Core LC system interfaced to an Exploris 120, hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Because of target analytes concentration and matrix effect, SCFA in mice plasma underwent a different procedure including 3-nitrophenylhydrazine (3-NPH) derivatization. For fructoselysine and lysine quantification in CP medium, samples were diluted in a solution acetonitrile/water (50:50, v/v) according to the linearity range used for calibration curve. For fructoselysine quantification in caecum and plasma, analytical protocol was adapted from Wolf et al. [7], with minor modifications. Briefly, 20 µL of plasma or 20 µL of caecum supernatants were diluted in ice cold methanol (ratio 1:3). Suspensions were centrifuged at 12700 rpm for 10 min, 4°C and 50 µL were dried under vacuum in a centrifugal evaporator (Savant, Thermo Fisher Scientific). Dried samples were resuspended in a solution consisting of 50% acetonitrile in water. Lysine and its Amadori compound were separated at 35°C through a zwitterionic sulfobetaine column (Atlantis Premier BEH, Z-HILIC, 100 x 2.1, 1.7 µm, Waters, Etten-Leur, the Netherlands) with the following gradient of solvent B (minutes/%B): (0/5), (1/5), (2/50), (6/50). Mobile phases consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B) and the flow rate was 0.2 mL/min. Heated electrospray (H-ESI) interface parameters were as follows: static spray voltage 3.3 kV, ion transfer tube and vaporizer temperature were both at 280 °C; sheath gas flow and auxiliary gas flow were 30 and 5 arbitrary units. The analyzer resolution was set at 60000 (FWHM at m/z 200), fructoselysine and lysine were identified and quantified in product ion scan positive mode screening the precursor ions (C 12 H 24 N 2 O 7 [M+H] + 309.1656, for fructoselysine and C 6 H 14 N 2 O 2 [M+H] + 147.1128 for lysine) with an increasing normalized collision energies set at 25, 50 and 60% to improve fragmentation pattern and screening the product ions, monitoring for both fructoselysine and lysine the characteristic fragment ion at m/z 84.0808. For product ion scan mode, Orbitrap resolution was set at 15000 (FWHM at m/z 200) and the quadrupole resolution was set at 1. A linear calibration curve was built in the range 100-10000 nM and concentration reported in mM was measured through standard addition technique by using CP medium, caecum or plasma as blank samples. For acetate, butyrate and isobutyrate separation in CP growth experiments, supernatants were directly diluted in o-phosphoric acid (0.5 % final concentration in water, 1:10, v:v), while for mice caecum content, samples were centrifuged at 4 °C, 12000 rpm for 15 min and supernatants diluted 1:10 v/v in 0.5% o-phosphoric acid. Samples were centrifuged at 12000 rpm before transferring clear supernatants to glass vial. Analytes were separated through a graphite column thermostated at 40°C (Hypercarb, 100 x 1.0, 1.7 µm, Thermo Fisher Scientific) with the following gradient of solvent B (minutes/%B): (0/0), (2/0), (6/75), (8/75). Mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) and the flow rate was 0.1 mL/min. H-ESI parameters were as follows: static spray voltage 3.2 kV, ion transfer tube and vaporizer temperature were both at 280 °C; sheath gas flow and auxiliary gas flow were 35 and 7 arbitrary units. The analyzer resolution was set at 60000 (FWHM at m/z 200), working in the scan range 50-350. Acetate, butyrate and isobutyrate were preliminary identified in full MS to evaluate effective separation of the two C4:0 isomers. Acetate and butyrate were quantified in full MS scan positive ion mode screening the two precursor ions (C 2 H 4 O 2 [M+H] + 61.0284 and C 4 H 8 O 2 [M+H] + 89.0597) with a mass accuracy below 3 ppm. A linear calibration curve was built in the range 0.5-10 mM by using acetate and butyrate as internal standard and concentration reported in mM. For fructoselysine and SCFA analytical procedures in product ion scan mode and in full MS acquisition mode, profile data were collected using Xcalibur 4.5 (Thermo Fisher Scientific, Waltham, MA) and fragmentation spectra were recorded by using Free Style software (v. 1.8, Thermo Fisher Scientific, Waltham, MA). EASY-IC with fluoranthene in positive ion mode (m/z 202.0777 [M]+) was used to improve mass accuracy in both full scan and product ion scan mode. Analytical performance robustness, sensitivity, reproducibility, repeatability, linearity, accuracy, carry over and matrix effects were evaluated by following the procedures previously reported by Troise and coworkers through authentic analytical standard according to an in-house procedure developed in Trace Finder environment (v. 5.1, Thermo Fisher Scientific, Waltham, MA) encompassing identification of isotopic distribution, elemental composition, mass accuracy below 3 ppm for precursors and product ions and number of scan points higher than 8. SCFA concentration in mouse plasma samples were quantified according to the procedure detailed by Garcia-Rivera et al [74] with minor modifications. Briefly, 10 µL of plasma were spiked with 1 µL of SCFA internal standard mix including 13 C 2 -acetate, 13 C 3 -propionate and 13 C 4 -butyrate (final concentration 0.1 mM for each carbon labelled compound). Upon protein precipitation with the addition of 60 µL of 75:25 methanol: water (v/v) solution, samples were mixed with 60 µL of 3-NPH (200 mM) and 10 µL of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, 120 mM in 6% pyridine). Samples were incubated at room temperature (22°C) for 45 min under gentle shaking in an orbital shaker. Derivatization reaction was stopped upon the addition of 10 µL quinic acid (200 mM) and incubation under shaking at room temperature for 15 min. Samples were centrifuged at 15000 rpm for 5 min at 4°C and supernatants diluted up to 1 mL with 10:90 methanol:water solution (v/v). Before transferring to glass vial, samples were centrifuged again at 4°C, 5 min, 15000 rpm. Hydrazone derivative quantitation was achieved by a U-HPLC system (Ultimate 3000 RS, Thermo Fisher Scientific) interfaced to a linear ion trap hybrid Orbitrap high resolution mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scinetific). Chromatographic separation included a reversed phase C18 column thermostated at 40°C (Kinetex C18 PS, 100 x 2.1 mm, 2.6 µm, Phenomenex, Torrance, CA) with the following gradient of solvent B (minutes/%B): (0/5), (5/5), (12.3/35), (13.3/85), (14/99), (16/99). Mobile phases consisted of water (solvent A) and acetonitrile (solvent B) and the flow rate was 0.2 mL/min. LC stream was interfaced to an electrospray ion source (ESI) working in negative ion mode scanning the ion in the m/z range 100-400; resolution was set at 30000 (FWHM at m/z 200), capillary temperature was 300°C, while sheath and auxiliary gases were set at 25 and 15 arbitrary units. Analytes profile data in full MS mode were collected using Xcalibur 2.1 (Thermo Fisher Scientific). Calibration curve was performed by internal standard technique in the linearity range 1-1000 µM by using the same procedure detailed above for plasma samples. Analytical performances for the three procedures are detailed in Supplementary Table 5. RNA isolation RNA was isolated from iWAT tissues, which were stored at -80˚C until analysis, using standard RNA isolation protocol. In short, biopsies were mixed with 1ml TriPure (Roche) and homogenized using a ceramic beads homogenizer. After adding 0.2ml chloroform to 1ml Tripure solution, samples were centrifuged (15 min, 12000 x g, 4˚C). The aqueous phase was transferred and mixed with 0.5ml isopropanol and centrifuged (15 min, 12000 x g, 4˚C). afterwards the pellets were resuspended in 1ml of 70% ethanol and centrifuged (15 min, 7500 x g, 4˚C). RNA was eluted in 20 µl RNAse free water. RNA concentrations were measured using the NanoDrop 1000 (Thermo Scientific). Real Time quantitative polymerase chain reaction (RT-qPCR) 1mg of RNA was converted to cDNA with SensiFAST cDNA synthesis kit (Meridian Bioscience) according to the manufacturer’s instructions. qPCR was performed on a CFX Opus 384 PCR machine (BioRad) using SensiFAST SYBR No-ROX Green (Meridian Bioscience).Gene expression was normalized with the expression of the housekeeping gene Hprt ; relative gene expression was calculated with the “delta Ct” method and shown as 2ˆ- delta Ct. All primers were manufactured by Sigma-Aldrich; primer sequences are provided in Supplementary Table 6. Western blotting iWAT tissues were lysated in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (cOmplete™ Protease inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails, Sigma) using a ceramic beads homogenizer. After centrifugation, the lipid layer was removed and protein concentration was determined by BCA assay (Thermo Fisher Scientific). b-mercaptoethanol was added as a reducing agent to sample lysates. Samples were loaded on 4-12% NuPage Bis-Tris polyacrylamide gels (Invitrogen). Proteins were transferred to PVDF membranes (BioRad), which were blocked in 5% milk in TBS-T (Tris Buffered Saline – Tween-20). Membranes were incubated overnight a 4˚C with primary antibodies: rabbit antibodies anti-IRS2 (insulin receptor substrate 2, L1326, Cell Signaling, 1:1000) and mouse anti-beta-actin (Genetex, 1:10000). Horseradish peroxidase (HRP)-conjugated secondary antibodies (polyclonal goat anti-rabbit IgG or monoclonal goat anti-mouse IgG, Dako, 1:3000) were incubated for 1 hour at room temperature. HRP activity was visualized with peroxidase substrate for enhanced chemiluminescence and imaged with ChemiDoc MP Imaging System (BioRad). Densitometric quantification analysis was performed using the Image J software. All protein levels were normalized to the loading control (b-actin). ELISA Sections of frozen iWAT tissues were cut and homogenized in 400 µl RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (cOmplete™ Protease inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails, Sigma) using a ceramic beads homogenizer. Protein concentration was determined with BCA assay (Thermo Fisher Scientific). The tissue concentrations of TNF-α, IL-6 and IL-1β cytokines were quantified using respectively the mouse TNFalpha/IL-6 DuoSet ELISA (R&D Systems) and ELISA MAX™ Deluxe Set Mouse IL-1β (BioLegend) according to the manufacturers’ protocol. The absorbance was measured at an optical density (OD) of 450 nm and 570 nm using the Tunable Microplate Reader VersaMax (Molecular Devices, USA). The cytokine levels were normalized for protein concentrations. Histology and Immunohistochemical staining Tissues were fixed in 10% buffered formalin overnight, dehydrated in 70% ethanol and embedded in paraffin. Sections of 4 µm were cut and stained for macrophage marker F4/80. Formalin-fixed paraffin-embedded (FFPE) sections were deparaffinized in 100% xylene and rehydrated in ethanol (100%, 96% and 70%) and H2O, followed by block of endogenous peroxidase in 3% H 2 O 2 methanol for 20 minutes and heat-induced epitope retrieval (HIER) in citrate buffer pH 6.0 at 100 °C for 10 min. iWAT sections were incubated with the following antibodies: FITC anti-mouse F480 (BioLegend) diluted 1:5000 for 2 hours at room temperature, rabbit anti-FITC (Bio-Rad) diluted 1:1000 for 1 hour at room temperature, finally BrightVision Poly-HRP-conjugates goat anti-rabbit IgG (ImmunoLogic) diluted 1:2 for 30 minutes at room temperature. Staining was visualized with 3,3’Diaminobenzidine (DAB) kit (Sigma Aldrich) and counterstaining was performed using hematoxylin. Per sections, ten pictures were captured at random using a Leica MC170 HD stand-alone microscope camera (Danaher Corporation, USA). Subsequently, analysis of the digital images was conducted using Image-J software. Statistical analysis of the in vivo data Statistical differences between the placebo and IB-treated groups were assessed using Mann Whitney test, and the differences were considered statistically significant with P values < 0.05. Declarations Ethics approval and consent to participate The human work protocol was approved by the Ethics Review Board in Gothenburg as previously indicated [16]. All animal experiments were conducted according to the ‘Guide to the Care and Use of Experimental Animals’ approved by the Ethics Committee on Animal Care and Use in Academisch Medisch Centrum, the Netherlands. Fresh stools for bacterial isolation work were collected from two healthy donors of whom informed consents were obtained following Good Clinical Practice. Consent for publication Not applicable Funding The work is partially funded by the Euro-Trans-Bio grant HBC.2017.0100 for the DM Prevent-project. TPNB is supported by an NWO VENI grant and Amsterdam UMC bridging grant. ER and NR are supported by an NWO VIDI grant and AUMC Starter grant (appointed to ER). MN is supported by a personal ZONMW-VICI grant 2020 (09150182010020) and an ERC-Advanced Grant 2023 FATGAP (101141346). ADT, SDP and AS research was partially funded by the National Recovery and Resilience Plan, mission 4, component 2, investment 1.3, call n. 341/2022 of Italian Ministry of University and Research funded by the European Union - NextGenerationEU for the project “ON Foods - Research and innovation network on food and nutrition Sustainability, Safety and Security - Working ON Foods”, project PE00000003, concession decree n. 1550/2022, CUP B83C22004790001. Availability of data and materials The human metagenomic data from the human cohort have been deposited in EGA as previously published [16] Acknowledgements We thank prof. Willem M. DeVos and Dr. Jos Seegers for valuable discussions for this work. Contributions TPNB and ER conceptualized and designed the study. HW did metagenomic analysis. TPNB performed culturing, isolation and genomic analysis. ER and NR, SH, JA performed the animal work, performed ex vivo measurement and analyzed the data. ADT, VF, AS and SDP performed extraction and measurements of fructoselysine and SCFA in cecum content and plasma samples. SG produced the bacteria for the animal work. ER, NR and TPNB are major contributors in writing the manuscript. All authors read and approved the final manuscript. Competing Interests M.N is co-founder and member of the Scientific Advisory Board of Caelus Pharmaceuticals and Advanced Microbial Interventions, the Netherlands. 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Hao, F., et al., Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation. Proceedings of the National Academy of Sciences, 2021. 118 (22): p. e2014681118. Gao, Z., et al., Butyrate improves insulin sensitivity and increases energy expenditure in mice. Diabetes, 2009. 58 (7): p. 1509-17. Hong, J., et al., Butyrate alleviates high fat diet-induced obesity through activation of adiponectin-mediated pathway and stimulation of mitochondrial function in the skeletal muscle of mice. Oncotarget, 2016. 7 (35): p. 56071-56082. Aguilar, E.C., et al., Sodium butyrate modulates adipocyte expansion, adipogenesis, and insulin receptor signaling by upregulation of PPAR-γ in obese Apo E knockout mice. Nutrition, 2018. 47 : p. 75-82. Ohira, H., et al., Butyrate attenuates inflammation and lipolysis generated by the interaction of adipocytes and macrophages. J Atheroscler Thromb, 2013. 20 (5): p. 425-42. Thyagarajan, B. and M.T. Foster, Beiging of white adipose tissue as a therapeutic strategy for weight loss in humans. Horm Mol Biol Clin Investig, 2017. 31 (2). Zuriaga, M.A., J.J. Fuster, N. Gokce, and K. Walsh, Humans and Mice Display Opposing Patterns of “Browning” Gene Expression in Visceral and Subcutaneous White Adipose Tissue Depots. Frontiers in Cardiovascular Medicine, 2017. 4 . Shabalina, I.G., et al., UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic. Cell Rep, 2013. 5 (5): p. 1196-203. Yoneshiro, T., et al., Recruited brown adipose tissue as an antiobesity agent in humans. J Clin Invest, 2013. 123 (8): p. 3404-8. Maasen, K., et al., Habitual Intake of Dietary Dicarbonyls is Associated with Greater Insulin Sensitivity and Lower Prevalence of Type 2 Diabetes: The Maastricht Study. Am J Clin Nutr, 2023. 118 (1): p. 151-161. Wu, Y., et al., The metabolism and transformation of casein-bound lactulosyllysine in vivo: Promoting dicarbonyl stress and the formation of advanced glycation end products accompanied by systemic inflammation. Food Chem, 2024. 444 : p. 138681. Singh, V.P., A. Bali, N. Singh, and A.S. Jaggi, Advanced glycation end products and diabetic complications. Korean J Physiol Pharmacol, 2014. 18 (1): p. 1-14. Yamagishi, S., et al., Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes. Biochim Biophys Acta, 2012. 1820 (5): p. 663-71. Pruesse, E., J. Peplies, and F.O. Glöckner, SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics, 2012. 28 (14): p. 1823-1829. Ludwig, W., et al., ARB: a software environment for sequence data. Nucleic Acids Research, 2004. 32 (4): p. 1363-1371. Stams, A.J.M., J.B. Van Dijk, C. Dijkema, and C.M. Plugge, Growth of syntrophic propionate-oxidizing bacteria with fumarate in the absence of methanogenic bacteria. Applied and Environmental Microbiology, 1993. 59 (4): p. 1114-1119. Liao, Y.-C., et al., MyPro: A seamless pipeline for automated prokaryotic genome assembly and annotation. Journal of Microbiological Methods, 2015. 113 : p. 72-74. Andrews, S. FastQC A Quality Control tool for High Throughput Sequence Data . Available from: citeulike-article-id:11583827 http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Zerbino, D.R. and E. Birney, Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Research, 2008. 18 (5): p. 821-829. Hernandez, D., et al., De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer. Genome Research, 2008. 18 (5): p. 802-809. Luo, R., et al., SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience, 2012. 1 (1): p. 1-6. Bankevich, A., et al., SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. Journal of Computational Biology, 2012. 19 (5): p. 455-477. Husemann, P. and J. Stoye, r2cat: synteny plots and comparative assembly. Bioinformatics, 2010. 26 (4): p. 570-571. Aziz, R., et al., The RAST server: Rapid annotations using subsystems technology. BMC Genomics, 2008. 9 (1): p. 75. Finn, R.D., et al., Pfam: the protein families database. Nucleic Acids Research, 2014. 42 (D1): p. D222-D230. Hunter, S., et al., InterPro in 2011: new developments in the family and domain prediction database. Nucleic Acids Research, 2012. 40 (10): p. 4725. Troise, A.D., A. Fiore, M. Wiltafsky, and V. Fogliano, Quantification of Nε-(2-Furoylmethyl)-L-lysine (furosine), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL) and total lysine through stable isotope dilution assay and tandem mass spectrometry. Food Chem, 2015. 188 : p. 357-64. García-Rivera, M.A., et al., Identification and validation of small molecule analytes in mouse plasma by liquid chromatography-tandem mass spectrometry: A case study of misidentification of a short-chain fatty acid with a ketone body. Talanta, 2022. 242 : p. 123298. Additional Declarations Competing interest reported. M.N is co-founder and member of the Scientific Advisory Board of Caelus Pharmaceuticals and Advanced Microbial Interventions, the Netherlands. However, none of these possible conflicts of interest bear direct relations to the outcomes of this specific study. The other authors declare no competing interests. Supplementary Files SupplementaryFiguresAndTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4364001","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":301195016,"identity":"dee55e66-a52a-4461-b82c-43abfa3c9bd2","order_by":0,"name":"Elena Rampanelli","email":"","orcid":"","institution":"Amsterdam UMC","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Rampanelli","suffix":""},{"id":301195017,"identity":"0304f924-4c62-4c1c-a52d-d853be50f99b","order_by":1,"name":"Nadia Romp","email":"","orcid":"","institution":"Amsterdam UMC","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Romp","suffix":""},{"id":301195018,"identity":"dc94562c-44d4-438b-9d63-8d57e97bbc0e","order_by":2,"name":"Antonio Dario Troise","email":"","orcid":"","institution":"National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"Dario","lastName":"Troise","suffix":""},{"id":301195019,"identity":"92f0061b-cb37-4fb7-ace0-bc317b5b1cca","order_by":3,"name":"Jakshana Ananthasabesan","email":"","orcid":"","institution":"Amsterdam UMC","correspondingAuthor":false,"prefix":"","firstName":"Jakshana","middleName":"","lastName":"Ananthasabesan","suffix":""},{"id":301195020,"identity":"19846389-7484-4e20-8421-e0d540ddc9a2","order_by":4,"name":"Hao Wu","email":"","orcid":"","institution":"Fudan 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12:05:06","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4364001/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4364001/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56358357,"identity":"afc8abca-1bf2-451b-ab61-a6d3d271f195","added_by":"auto","created_at":"2024-05-13 06:55:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":457654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReverse associations between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIntestinimonas butyriciproducens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and fructoselysine fermentation genes and metabolic biomarkers in the human cohort. (A\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSignificant reduction of \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ein IFG, IGT, CGI, and T2D groups as compared to\u003cstrong\u003e \u003c/strong\u003eirNGT control based on\u003cstrong\u003e \u003c/strong\u003erarefied reads of \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e in a Swedish prediabetes cohort (n=1011; Spearman correlation). (\u003cstrong\u003eB\u003c/strong\u003e) metagenomic analysis reveals negative associations of individual FL pathway genes to various metabolic biomarkers (Wilcox rank-sum test). −P \u0026lt; 0.1; *P \u0026lt; 0.05; +P \u0026lt; 0.01; #P \u0026lt; 0.001. \u003cstrong\u003eC- F\u003c/strong\u003e: Significant reverse associations between FL pathway gene abundance with BMI (P = 7.4e-07) (\u003cstrong\u003eC\u003c/strong\u003e), Triglycerides (P = 1e-09) (\u003cstrong\u003eD\u003c/strong\u003e), HbA1c (P = 0.002) (\u003cstrong\u003eE\u003c/strong\u003e) and fasting insulin (P = 2.4e-07) (\u003cstrong\u003eF\u003c/strong\u003e), respectively.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4364001/v1/e0da30af6a4a4cbbb39a7de1.jpg"},{"id":56358355,"identity":"010171cc-cccf-4e4c-a4e6-d401f3321ffa","added_by":"auto","created_at":"2024-05-13 06:55:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":315756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIntestinimonas \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eisolates and the conversion of fructoselysine. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) a phylogenetic tree based on 16S rRNA genes of 4 \u003cem\u003eIntestinimonas\u003c/em\u003e human isolates and \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e DSM26588\u003csup\u003eT\u003c/sup\u003e and closely related species. Bar represents 10% sequence divergence (\u003cstrong\u003eB\u003c/strong\u003e) fructoselysine conversion by \u003cem\u003eIntestinimonas\u003c/em\u003e strain GL3; IY4, AS-BT and AF211. All data are presented as mean values ± SD (n=2 biological replicates).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4364001/v1/7dad489dc7999d22151619b2.jpg"},{"id":56358358,"identity":"8c4a2292-175c-4bd0-a813-321c6450bba8","added_by":"auto","created_at":"2024-05-13 06:55:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":250769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiet-induced obesity murine model: systemic effects of \u003c/strong\u003e\u003cem\u003eIntestinimonas butyriciproducens \u003c/em\u003eGL3 (IB)\u003cstrong\u003e. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Body weight gain, shown as a difference in body weight compared to baseline. (\u003cstrong\u003eB\u003c/strong\u003e) Fasting blood glucose levels assessed at the end of the study. (\u003cstrong\u003eC, D\u003c/strong\u003e) Intraperitoneal insulin tolerance test (IPITT), (\u003cstrong\u003eC\u003c/strong\u003e) blood glucose levels before and after insulin injection, (\u003cstrong\u003eD\u003c/strong\u003e) area under the curve (AUC) of glucose excursion during IPITT (mM*minute). (\u003cstrong\u003eE, F\u003c/strong\u003e) Epididymal and inguinal WAT weight, shown as a percentage of total body weight. All data are presented as mean values ± SEM (N=12/group). *P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4364001/v1/2b4267f5a3d4f1fa34d55238.jpg"},{"id":56358356,"identity":"d07e2033-b20c-4a0d-be93-6f075090fb53","added_by":"auto","created_at":"2024-05-13 06:55:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":324636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInguinal white adipose tissue: gene expression after placebo-/IB-treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA-H\u003c/strong\u003e) Expression of genes encoding for proteins involved in (\u003cstrong\u003eA-C\u003c/strong\u003e) mitochondrial oxidative metabolism and fatty acid oxidation and in (\u003cstrong\u003eD-H\u003c/strong\u003e) WAT browning. Data shown as 2^-dCt\u003cstrong\u003e.\u003c/strong\u003e Data are shown as mean ± SEM (N=12/group). *P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4364001/v1/b357452ff78f57d04dd9c28b.jpg"},{"id":56358361,"identity":"f876c0ae-f549-4110-af6d-8771492c1d81","added_by":"auto","created_at":"2024-05-13 06:55:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":200316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInguinal white adipose tissue: insulin receptor and cytokine levels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Expression of mRNA transcripts of Irs2 gene in iWAT tissues of HFD-fed mice. (\u003cstrong\u003eB\u003c/strong\u003e) IRS2 protein expression levels in iWAT tissues of HFD-fed mice. IRS2 levels normalized to beta-actin levels. (\u003cstrong\u003eD-F\u003c/strong\u003e) Pro-inflammatory cytokine levels detected in lysate of iWAT tissues from obese mice and normalized to protein concentrations. (\u003cstrong\u003eG\u003c/strong\u003e) Macrophage infiltration of iWAT, determined by immunohistochemistry staining on FFPE section. Data shown as a percentage of positive areas on total areas analyzed. All data are presented as mean values ± SEM (N=12/group). *P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4364001/v1/e766c79b5c340fc8b303c265.jpg"},{"id":56358360,"identity":"c77552f0-68ca-4a75-9063-2a296992932f","added_by":"auto","created_at":"2024-05-13 06:55:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":224062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirculating levels of bacterial SCFA and dietary fructoselysine. \u003c/strong\u003e(\u003cstrong\u003eA-D\u003c/strong\u003e) Plasma concentrations of butyrate, acetate, propionate and fructoselysine in mice. (\u003cstrong\u003eE\u003c/strong\u003e) Ratio calculated with plasma concentrations of butyrate / fructoselysine. All data are shown as mean values ± SEM (N=12/group). *P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4364001/v1/44e686cb1763664a5a05f9f4.jpg"},{"id":57007636,"identity":"359008be-de22-42c5-9088-3f1a336b8d08","added_by":"auto","created_at":"2024-05-23 10:25:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2884659,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4364001/v1/007f425d-edd7-439c-968f-687a4ed665fe.pdf"},{"id":56358359,"identity":"4391f2b0-d1a8-4726-9be3-d60212872064","added_by":"auto","created_at":"2024-05-13 06:55:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":207279,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresAndTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4364001/v1/597dffb5533be10895e5a587.docx"}],"financialInterests":"Competing interest reported. M.N is co-founder and member of the Scientific Advisory Board of Caelus Pharmaceuticals and Advanced Microbial Interventions, the Netherlands. However, none of these possible conflicts of interest bear direct relations to the outcomes of this specific study. The other authors declare no competing interests.","formattedTitle":"Gut bacterium Intestinimonas butyriciproducens improves host metabolic health: evidence from cohort and animal intervention studies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiet, the human microbiome and host genetics are determinants of metabolic status, partially through the production of metabolites via fermentation of dietary components by the gut microbiota [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Understanding the metabolism of dietary ingredients by the microbiome is key to mediate the effects of the human microbiome on host metabolism. Hence, mechanistic studies on the interactions between dietary components, the microbiome and the host are urgently needed. The question regarding the ability of the gut microbiota to metabolize dietary compounds not present in raw foods but formed by food processing is a great interest: are they harmful xenobiotics or human microbiota is able to metabolize them for good?\u003c/p\u003e \u003cp\u003eFructoselysine is an abundant Amadori product formed via the non-enzymatic reaction between a reducing sugar and amino acids in foods during thermal processing and storage. The formation of Amadori compounds partially blocks amino acids from absorption, hence reducing their bioavailability to the host [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is estimated that daily intake of Amadori products is around 0.5\u0026ndash;1 g of which, in healthy subjects, the large majority reaches the large intestine for further digestion [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Only a few intestinal bacteria have been reported to metabolize fructoselysine. Strains marketed as probiotics, mainly \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e spp. have been reported to utilize fructoselysine and only generated glucose moiety but not lysine [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e is able to degrade fructoselysine and use the released glucose for growth \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] while \u003cem\u003eCollinsella intestinalis\u003c/em\u003e can metabolize fructoselysine to acetate, formate both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. All these strains failed to use the liberated lysine moiety for growth. \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e showed exceptional metabolic features being able to use both liberated glucose and lysine moiety from fructoselysine breakdown to form butyrate via two separate routes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Fructoselysine conversion to butyrate is highly desired. Butyrate serves as an energy source for colonocytes but also suppresses inflammation in various tissues [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], involving in regulation of insulin release via gut hormone stimulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This is in line with numerous reports on depletion of butyrate-producing species in diabetic and obese subjects and supplementation of these species provided metabolic benefits to the host [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, it is of great interest to explore the therapeutic potential of \u003cem\u003eI. butyriciproducens\u003c/em\u003e in improving metabolic health via its unique capability of converting fructoselysine to butyrate.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the associations between butyrogenic \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e and fructoselysine pathway genes and metabolic risk biomarkers in subjects with metabolic compromising conditions compared to healthy subjects in the Swedish impaired glucose tolerance (IGT) cohort (n\u0026thinsp;=\u0026thinsp;1011) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Subsequently, we isolated and studied the capacity and genomic made-up of fructoselysine fermentation by four human \u003cem\u003eIntestinimonas\u003c/em\u003e isolates. We later administered \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e GL3 isolate to diet-induced obesity mice to explore the impact on obesity and host metabolism and to achieve a proof-of-concept study in human.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIntestinimonas butyriciproducens\u003c/strong\u003e \u003cstrong\u003eand fructoselysine fermentation were reduced in subjects with high metabolic risks.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e associates with host metabolic health, we employed the metagenomics data from a Swedish cohort, comprising 1,011 individuals with prediabetes and treatment-na\u0026iuml;ve T2D and healthy relatives [16]. When comparing the rarefied read abundances of \u003cem\u003eI. butyriciproducens\u003c/em\u003e in subjects with different glycemic status raging from normal glucose tolerance to type 2 diabetes, we observed that the relative abundance of \u003cem\u003eI. butyriciproducens\u003c/em\u003e was significantly reduced in groups with impaired fasting glucose (IFG, p\u0026thinsp;=\u0026thinsp;0.034), impaired glucose tolerance (IGT, p\u0026thinsp;=\u0026thinsp;0.02), combined glucose intolerance (CGI, p\u0026thinsp;=\u0026thinsp;0.021) and type 2 diabetes (T2D, P\u0026thinsp;=\u0026thinsp;0.055) as compared to low-risk normal glucose tolerance (IrNGT) (Fig. 1A). To further explore associations of \u003cem\u003eI. butyriciproducens\u003c/em\u003e with metabolic risk factors, we correlated the relative abundance of identified fructoselysine pathway genes [8] to various metabolic biomarkers (Fig. 1B). Most of the individual fructoselysine pathway genes were negatively associated with metabolic risks, except genes involved in fructoselysine degradation including fructoselysine kinase (AF_949) and fructosamine deglycase (AF_00951). This can be explained by the fact that these two genes were also present in the genomes of some pathogenic strains including \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e [6, 17]. Nevertheless, these bacteria are not capable of fermenting lysine or producing butyrate. To investigate the relations of \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e with metabolic risks via its unique capacity to convert fructoselysine to butyrate, we studied the associations between the total abundance of fructoselysine pathway genes and metabolic markers. We found significant negative correlations between the fructoselysine pathway relative abundance and body mass index (BMI, rho=-0.15; p\u0026thinsp;=\u0026thinsp;7.4e-07), triglycerides (rho=-0.19; p\u0026thinsp;=\u0026thinsp;1.0e-09), glycated hemoglobin (HbA1c, rho=-0.10; p\u0026thinsp;=\u0026thinsp;0.002) and fasting insulin (rho=-0.16; p\u0026thinsp;=\u0026thinsp;2.4e-07) (Fig. 1D-F), which is in line with the observed reduced abundance of \u003cem\u003eI. butyriciproducens\u003c/em\u003e in individuals with T2D or impaired glucose tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntestinimonas\u003c/strong\u003e \u003cstrong\u003eas a key player in the butyrogenic fructoselysine metabolism in the gut\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the capacity of fructoselysine fermentation by \u003cem\u003eIntestinimonas\u003c/em\u003e, we next performed a conventional cultivation technique using a selective medium on agar plate to isolate \u003cem\u003eIntestinimonas\u003c/em\u003e species from human stool from three healthy volunteers. We obtained three \u003cem\u003eIntestinimonas\u003c/em\u003e isolates and subsequently sequenced 16S rRNA genes as well as genomes to enable further genomic and physiological characterization. To determine the taxonomic relationship of these isolates, a phylogenetic tree was made using 16S rRNA gene sequences of three \u003cem\u003eIntestinimonas\u003c/em\u003e isolates (strain GL3, IY4 and AS-BT) and other closely related species in Clostridium cluster IV and cluster XIVa (Fig. 2A). While 16S rRNA genes of three strains IY4 and GL3 were highly similar (\u0026gt;\u0026thinsp;99.7%) to \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e type strain and previously isolated \u003cem\u003eIntestinimonas\u003c/em\u003e strain AF211, 16S rRNA gene of strain AS-BT was only 95% similar to that of the type of strain, suggesting strain AS-BT likely represents a new species of \u003cem\u003eIntestinimonas\u003c/em\u003e genus. Nevertheless, all three \u003cem\u003eIntestinimonas\u003c/em\u003e isolates were able to grow and convert fructoselysine and lysine to butyrate and acetate with a similar degradation rate (Fig. 2B-E) and have complete fructoselysine pathway genes in their genomes (Supplementary table 1). Interestingly, we found that strain AS-BT did not have a complete vitamin B12 pathway in the genome, whereas three other strains had (Supplementary table 2), indicating the potential capacity of strain GL3 and IY4 to synthesize pseudovitamin B12 as strain AF211 [18].\u003c/p\u003e\n\u003cp\u003eAs antibiotic resistance is highly relevant for gut bacteria, we also determined the minimum inhibitory concentration (MIC) of various antibiotics in four strains using Etest (Supplementary table 3). AS-BT clearly had a different MIC profile as compared to the other three strains which may be attributed to its distant taxonomic position of this strain from three other \u003cem\u003eIntestinimonas\u003c/em\u003e isolates. In general, \u003cem\u003eIntestinimonas\u003c/em\u003e isolates were sensitive to teicoplanin, chloramphenicol, vancomycin, cefotaxime, and oxacillin, which are inhibitors of cell wall synthesis in Gram-positive bacteria, but they showed relatively high MIC values with ciprofloxacin and sulfamethoxazole, however, no gene has been attributed to resistance mechanisms for these two antibiotics. Although some vancomycin resistance genes (vanB, vanW, vanS and vanR) were detected in the genomes (Supplementary table 4), those genes were not sufficient to form a complete operon to confer vancomycin resistance [19]. Two out of four strains were resistant against erythromycin at a very high level while all four strains were found to be resistant to tetracycline at different levels (from 2 \u0026micro;g/ml to 16 \u0026micro;g/ml). Notably, no tetracycline resistance gene tetW was found in the genome of GL3 whereas tetW was present in all genomes from three other \u003cem\u003eIntestinimonas\u003c/em\u003e strains. In contrast, there was a large number of gene copies for efflux pumps found in all \u003cem\u003eIntestinimonas\u003c/em\u003e genomes with the highest number of 27 pumps from GL3 isolate which may be an important mechanism to eliminate toxic compounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntestinimonas butyriciproducens\u003c/strong\u003e \u003cstrong\u003elimits body weight gain in mice fed a high-fat diet\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the effects of \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e GL3 strain (from now referred as IB) on systemic host metabolism, we used the murine diet-induced obesity (DIO) model [20]. C57BL/6J mice were fed a high-fat diet (HFD) or matching low-fat diet (LFD) for 13 weeks and subjected to oral gavage 3 times a week of placebo or IB. Importantly, HFD contains significantly higher levels of protein-bound fructoselysine as compared to a regular chow-diet (Supplementary Fig. 1; 128.3 versus 567.5 mg/100gr protein, p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), making HFD an excellent source of dietary fructoselysine for the production of butyrate by IB\u003c/p\u003e\n\u003cp\u003eAlthough we did not observe changes in food intake between placebo and IB-treated mice, bacterial administration resulted in significantly less body weight gain in the HFD-fed mice as compared to placebo treatment after 13 weeks (Fig.\u0026nbsp;3A; 15.1 g versus 11.5 g, p\u0026thinsp;=\u0026thinsp;0.038). The difference in body weight between placebo and IB groups became apparent after 11 weeks suggesting that the bacterium affects the host only after a prolonged treatment and/or the bacterial treatment becomes effective when mice develop exacerbated adiposity/metabolic dysregulation. Notably, the bacterial supplementation did not affect body weight in the LFD-fed mice.\u003c/p\u003e\n\u003cp\u003eAs butyrate-producing bacteria have been linked to insulin sensitivity in both human and mice studies, we next assessed blood glucose levels in fasting state and following intraperitoneal insulin injection. As expected HFD-feeding increased the fasting glucose levels and bacterial supplementation significantly lowered fasting glucose concentrations (Fig.\u0026nbsp;3B; 11.3 mM versus 9.4 mM glucose, p\u0026thinsp;=\u0026thinsp;0.035). During the insulin-tolerance test, blood glucose levels of IB-treated mice were constantly lower than in placebo mice, suggesting an amelioration of insulin sensitivity (Fig.\u0026nbsp;3C). Analysis of the area under the curve (AUC) showed a trend toward reduced glucose rate over time (Fig.\u0026nbsp;3D; AUC of 48.41 versus 42.08, p\u0026thinsp;=\u0026thinsp;0.057) upon bacterial administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntestinimonas butyriciproducens\u003c/strong\u003e \u003cstrong\u003ecounteracts the fat accumulation and promotes lipid degradation and browning processes in inguinal white adipose tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of white adipose tissues (WAT) revealed a reduction in subcutaneous/inguinal white adipose tissue (iWAT) in IB-treated mice on HFD (2.43 versus 1.58% BW, p\u0026thinsp;=\u0026thinsp;0.023), while the proportion of visceral/epididymal white adipose tissue (eWAT) relative to total body weight was unchanged between placebo and IB groups (Fig.\u0026nbsp;3E, F).\u003c/p\u003e\n\u003cp\u003eSince IB intake led to a reduction in the proportion of IWAT tissue, we next assessed the expression of key metabolic genes in iWAT. We found that the expression of genes involved in mitochondria metabolism and, specifically, fatty acid oxidation was strongly upregulated following IB treatment in HFD-fed mice. Indeed, IB supplementation reduced the HFD-induced expression of \u003cem\u003ePpargc1a4\u003c/em\u003e (0.007108 versus 0.01577, p\u0026thinsp;=\u0026thinsp;0.037), \u003cem\u003ePpara\u003c/em\u003e (0.009667 versus 0.03057 p\u0026thinsp;=\u0026thinsp;0.025), and \u003cem\u003eCpt1a\u003c/em\u003e (0.1497 versus 0.3879, p\u0026thinsp;=\u0026thinsp;0.017) to levels similar to the ones observed in LFD-treated mice (Fig. 4A-C). In contrast, the expression rates of the browning markers were downregulated upon HFD-feeding but augmented by oral IB treatment (Fig. 4D-H). In fact, compared to placebo, bacterial supplementation in obese mice resulted in a significant enhanced expression of the browning markers \u003cem\u003eUcp1\u003c/em\u003e (0.009592 versus 0.01908, p\u0026thinsp;=\u0026thinsp;0.043), \u003cem\u003eCidea\u003c/em\u003e (0.08147 versus 0.2776, p\u0026thinsp;=\u0026thinsp;0.023), \u003cem\u003ePrdm16\u003c/em\u003e (0.04065 versus 0.1585, p\u0026thinsp;=\u0026thinsp;0.0023), \u003cem\u003eDio2\u003c/em\u003e (0.03754 versus 0.2598, p\u0026thinsp;=\u0026thinsp;0.054) and \u003cem\u003eTbx1\u003c/em\u003e (0.04912 versus 0.1597, p\u0026thinsp;=\u0026thinsp;0.015). Overall, these data suggest that the reduced accumulation of iWAT is driven by increased energy expenditure in IB-treated mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntestinimonas butyriciproducens\u003c/strong\u003e \u003cstrong\u003econstrains white adipose tissue inflammation and loss of insulin sensitivity in obesity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs WAT is an important modulator of systemic insulin sensitivity [21] and IB intake augmented glucose uptake after insulin injection, we next measured the intra-iWAT expression of \u003cem\u003eIrs2\u003c/em\u003e (insulin receptor substrate 2), which mediates the cytoplasmic signaling of insulin [22]. Expression of Irs2 was markedly diminished in iWAT of obese mice, however in line with the amelioration of insulin resistance, IB treatment led to an enhanced expression of IRS2 both at gene (Fig. 5A; 0.146 versus 0.3123, p\u0026thinsp;=\u0026thinsp;0.0029) and protein levels (Fig. 5B; 0.314 versus 0.8455, p\u0026thinsp;=\u0026thinsp;0.0086).\u003c/p\u003e\n\u003cp\u003eSince the development of insulin resistance and adipose tissue inflammation are intertwined, we subsequently investigated the iWAT production of relevant pro-inflammatory cytokines in obese mice. This revealed that intestinal IB exerted anti-inflammatory effects as IB treatment significantly reduced the intra-WAT secretion of tumor necrosis factor (TNF)-\u0026alpha; (0.5837 versus 0.3859, p\u0026thinsp;=\u0026thinsp;0.0048), interleukin (IL)-6 (1.003 versus 0.6825, p\u0026thinsp;=\u0026thinsp;0.0053) and IL-1\u0026beta; (0.4915 versus 0.3886, p\u0026thinsp;=\u0026thinsp;0.0165) (Fig. 5D-F). In addition, staining for the F4/80 macrophage marker showed that macrophage recruitment was diminished, albeit not significant, in IB-treated mice (Fig. 5G), possibly indicating that immune infiltrating cells as well as parenchymal cells were the source of the detected pro-inflammatory cytokines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOral intake of\u003c/strong\u003e \u003cstrong\u003eIntestinimonas butyriciproducens\u003c/strong\u003e \u003cstrong\u003eresults in higher plasma SCFA levels without affecting intestinal barrier function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, to understand the distal effects of intestinal IB, we determined the circulating levels of SCFAs. Plasma levels of butyrate and acetate were significantly increased upon IB treatment as compared to placebo in the obese mice (butyrate: 0.9817 versus 1.12 \u0026micro;M, p\u0026thinsp;=\u0026thinsp;0.0367; acetate: 374.3 versus 664.3 \u0026micro;M, p\u0026thinsp;=\u0026thinsp;0.0068) but not in lean mice, while the concentrations of propionate were unchanged (Fig.\u0026nbsp;6A-C). In addition, although the plasma concentrations of fructoselysine were similar between the groups, the butyrate/fructoselysine ratio was increased in the HFD-fed mice receiving IB (p\u0026thinsp;=\u0026thinsp;0.059), underscoring the active conversion of dietary fructoselysine into butyrate (Fig.\u0026nbsp;6D, E).\u003c/p\u003e\n\u003cp\u003eTo exclude that the observed changes in gut-derived metabolites were due to alterations in intestinal barrier function, we also examined the small intestine and colonic expression of genes encoding for tight-junction molecules (claudin-4, occludin and zona occludens-1). The results showed that IB did not affect barrier integrity as expression levels were comparable between treatment groups (Supplementary Fig.\u0026nbsp;2A-F).\u003c/p\u003e\n\u003cp\u003eAltogether, these findings underscore that the systemic effects of IB, observed in obese mice, are mediated by produced butyrate and acetate, which were subsequently absorbed into the systemic circulation. The lack of plasma SCFA changes in mice fed LFD suggests that the IB-mediated production of butyrate and acetate may have derived from the higher fructoselysine content in HFD (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAmadori product fructoselysine is abundantly present in cooked foods but only a small portion of protein-bound lysine Amadori products can be absorbed in the gut [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], hence funneling the majority to the lower gastrointestinal tract for microbial use. We previously demonstrated that \u003cem\u003eI. butyriciproducens\u003c/em\u003e uniquely ferments fructoselysine to butyrate via lysine and acetyl-CoA pathway simultaneously [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the present study, we reported three additional \u003cem\u003eIntestinimonas\u003c/em\u003e isolates from human subjects, all of which have the capacity to convert fructoselysine to butyrate and harbor all genes for a complete fructoselysine pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This pinpoints that \u003cem\u003eIntestinimonas\u003c/em\u003e is a key player in intestinal fructoselysine fermentation to butyrate and \u003cem\u003eI. butyriciproducens\u003c/em\u003e is the most abundant species in the human gut which is in good agreement with our previous report [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, we isolated a new \u003cem\u003eIntestinimonas\u003c/em\u003e species (AS-BT isolate) which shared a high commonality of metabolic features with \u003cem\u003eI. butyriciproducens\u003c/em\u003e except the capacity to synthesize pseudovitamin B12, an essential cofactor of lysine-5,6-aminomutase, a key protein involved in lysine/fructoselysine fermentation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntriguingly, we found that the abundance of both \u003cem\u003eI. butyriciproducens\u003c/em\u003e and fructoselysine pathway genes were reversely associated with various metabolic markers in a (pre)diabetic cohort (n\u0026thinsp;=\u0026thinsp;1011 subjects), suggesting a reduced capacity of the microbiome to convert fructoselysine to butyrate in cardiovascular and metabolically compromised subjects compared to healthy individuals. The reduced capacity of the human microbiota to convert fructoselysine to beneficial butyrate may lead to a higher accumulation of fructoselysine and lower levels of colonic butyrate, both of which are undesired. While butyrate is required for healthy colon, high fructoselysine level may facilitate its pH driven conversion to advanced glycation end products (AGEs) the level of which has been associated with aging, atherosclerosis and diabetes [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We also observed that the prevalence of \u003cem\u003eIntestinimonas\u003c/em\u003e and fructoselysine fermentation genes are also associated with feeding modes in infants [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In contrast, butyrate or short chain fatty acids in general have been reported as an important component in controlling body weight and insulin sensitivity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This association from the cohort study is well in line with the results obtained from the \u003cem\u003ein vivo\u003c/em\u003e study which discloses that administration of \u003cem\u003eI. butyriciproducens\u003c/em\u003e GL3 strain (IB) exerts multiple metabolic benefits in diet-induced obesity. Indeed, bacterial supplementation led to significantly decreased body weight gain, iWAT accumulation and fasting glucose levels in obese mice. Moreover, upon insulin administration, blood glucose levels remained lower in the IB-treated mice as compared to placebo-treatment. These effects were accompanied by increased IRS2 expression, enhanced expression of genes crucial for lipid catabolism as well as browning and reduced inflammation in IWAT of IB-treated obese mice. Lastly, bacterial administration resulted in a higher concentration of circulating butyrate and acetate.\u003c/p\u003e \u003cp\u003eMost of the observed IB-induced metabolic benefits \u003cem\u003ein vivo\u003c/em\u003e are likely resulting from the higher rate of SCFA production executed by this commensal bacterium. Indeed, previous reports have shown that sodium butyrate administration limits gain-weight, and improves glucose kinetics, insulin sensitivity, energy expenditure as well as mitochondrial function in DIO murine models [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Similarly, oral administration of sodium acetate was sufficient to counteract adiposity, ameliorate insulin resistance and boost energy expenditure and oxidative metabolism in DIO mice [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe short chain fatty acid butyrate and acetate exert pleiotropic effects on host by serving as an energy source (accounting for approximately 10% of the caloric requirement in humans), functioning as histone-deacetylase inhibitors and signaling through host G-protein\u0026ndash;coupled receptors (GPR) 41, 43 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and 109A/43 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Besides their protective effects against adiposity, SCFAs are also known anti-inflammatory molecules [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In line, we found that the administration of the butyrate-producing \u003cem\u003eI. butyriciproducens\u003c/em\u003e strain significantly reduced the production of TNF-α, IL-6 and IL-1β in iWAT. These effects are likely driven by SCFA-mediated inhibition of both the nuclear factor\u0026ndash;κB (NF-κB) signaling and NLRP3 inflammasome activation [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Notably, both pathways are critical in the induction of meta-inflammation and obesity-induced insulin resistance [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Accordingly, we observed that IB intake led to significantly lower fasting blood glucose levels and a trend toward enhanced glucose disposal upon insulin injection. Hence, the IB-driven improvement in glucose homeostasis may result from the SCFA inhibitory effects on local inflammation in WAT and thus maintenance of functioning insulin signaling in adipocytes. In line, we reported that the iWAT expression of IRS2 was markedly increased upon IB administration as compared to placebo treatment. This effect is supported by previous reports disclosing the ability of butyrate to enhance the expression of the signaling molecules IRS1 and IRS2 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], thereby facilitating insulin signaling.\u003c/p\u003e \u003cp\u003eTissue inflammation and insulin-resistance are tightly linked to exacerbated intracellular lipid accumulation. In this regard, the transcriptional changes observed in iWAT upon bacterial treatment indicate an induction of fatty acid beta-oxidation through PGC1-alpha and PPAR-alpha pathways given the marked upregulation of the genes encoding for these master transcriptional regulators of mitochondria biogenesis and oxidative metabolism [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, the expression of Cpt1a, encoding for the key rate-limiting enzyme in fatty acid oxidation (FAO) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], was significantly upregulated in iWAT of IB-treated obese mice, whereas the relative iWAT weight was reduced, underscoring that increased FAO resulted in less adipocyte hypertrophy [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The increased circulating levels of SCFAs mediated by administered bacterial strain, are likely the underlying cause of these protective effects; indeed, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e butyrate has been shown to activate PGC1alpha pathway and FAO by enhancing CPT1 activity [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] as well as reduce adipocyte expansion [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Overall, these anti-lipogenic effects of \u003cem\u003eI. butyriciproducens\u003c/em\u003e may contribute to diminished WAT inflammation. In support of this concept, butyrate was shown to suppress inflammatory responses in the context of adipocyte-macrophage interactions through inhibition of lipolysis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn line with the ability of butyrate to promote energy expenditure \u003cem\u003ein vivo\u003c/em\u003e, the transcriptional signature of iWAT in IB-treated mice is indicative of WAT browning/beiging. The latter is characterized by an augmented expression of thermogenesis-related genes, which are typically expressed by brown adipose tissue, a reduction in lipid accumulation and increased energy expenditure [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Compared to visceral (epididymal) WAT, subcutaneous (inguinal) iWAT is more susceptible to browning due to the higher expression of browning markers UCP1, Cidea, and Pdrm16, as well as the beige markers Tbx1 and P2rx5 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], possibly indicating that iWAT is more responsive to external stimuli, such as bacterial metabolites. Overall, the described effects on iWAT and the plausible WAT browning may explain the reduction in body weight gain in IB-treated obese mice, as stimulation of WAT browning may increase total body energy expenditure and promote fat reduction [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLastly, the observations that elevated plasma SCFA levels were found only in obese mice but not lean mice following bacterial intake and the lack of differences in all clinical parameters measured between placebo- and IB-treated lean mice underscore that the bacterial SCFA production is driven by the diet. In fact, HFD contains a higher amount of fructoselysine compared to regular chow-diet, therefore the protective effects of \u003cem\u003eI. butyriciproducens\u003c/em\u003e can be more prominent in DIO mice, likely due to a higher rate of fructoselysine-to-butyrate conversion. Moreover, the anti-inflammatory effects of \u003cem\u003eI. butyriciproducens\u003c/em\u003e could be attributed, at least in part, to the utilization of a critical precursor, fructoselysine, of α-dicarbonyls and AGEs, which are increased in type 2 diabetes, and associated with diabetic complications as they instigate oxidative stress and pro-inflammatory cytokine release [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In light of the fact that mice were sacrificed after 6 hours of fasting, thus during fructoselysine-depletion, the significant, yet mild, increase in circulating butyrate is probably an underestimation of the circulating levels present during fed-state upon bacterial administration.\u003c/p\u003e \u003cp\u003eOverall, this study highlights the important role of the gut microbiota in the regulation of host physiology and particularly host metabolism. Notably, our findings support the development of microbiome-targeting approaches for the prevention or amelioration of metabolic disorders, such as obesity and type 2 diabetes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we found that \u003cem\u003eIntestinimonas\u003c/em\u003e plays a key role in the conversion of dietary fructoselysine to butyrate in the gut and the abundance of \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e as well as fructoselysine pathway genes were reversely correlated with multiple risk biomarkers in a cohort study. \u003cem\u003eIn vivo, Intestinimonas butyriciproducens\u003c/em\u003e counteracts adiposity, ameliorates glucose metabolism and tissue inflammation by converting dietary fructoselysine to butyrate and acetate in DIO mice.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003eMetagenomics analysis in the Swedish IGT cohort\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rarefied abundance levels of metagenomics species (CAG00017), annotate as \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e, and 33 KEGG orthologies (KOs) involved in the fructoselysine metabolism were obtained from a previous study aiming to characterize the gut microbial changes in prediabetes and diabetes based on the Swedish IGT cohort (n=1,011)[16]. The relative differences of IB were then compared across individuals with distinct glucose intolerance levels versus the healthy control group. To examine the potential importance of fructoselysine metabolism to glucose intolerance, the relative abundances of each KO and/or the whole pathway (based on aggregated sum values of all KOs) were associated with\u0026nbsp;12 common clinical variables, such as the levels of fasting glucose, insulin, HbA1c, and triglycerides, indicative of the T2D status, respectively. The study was approved by the IRB of Sahlgrenska Hospital, Gothenburg University and all subjects provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of \u003cem\u003eIntestinimonas\u0026nbsp;\u003c/em\u003efrom human stool\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh fecal samples were collected in 15 ml falcon tubes containing anaerobic phosphate buffer (pH7) and later stored in 25% glycerol in 5 ml anaerobic bottles kept at -80 \u003csup\u003eo\u003c/sup\u003eC freezer. 0.5 ml of these fecal slurries was transferred to 10 ml anaerobic bicarbonate-buffered mineral salt medium (CP medium) containing 40mM lysine as energy and carbon source to enrich lysine-fermenting bacteria. The headspace was filled with CO\u003csub\u003e2\u003c/sub\u003e/N\u003csub\u003e2\u003c/sub\u003e (1:4) at 1.5 atm and incubation was at 37\u003csup\u003eo\u003c/sup\u003eC. Subsequently, the enrichment cultures were transferred two more times in the same medium before being plated on YCFA agar medium containing 40 mM lysine as substrate (YCFA_L). Single colonies were picked and plated at least 3 times on the same medium\u0026nbsp;which resulted in an axenic culture. The purity of the strains, designated as strain GL3, AS-BT and IY4 was confirmed by 16S rRNA gene sequencing and microscopy. The strains were routinely maintained in YCFA_L medium at 37 °C. 16S gene sequences of three isolates and strain AF211 were aligned with the multiple sequence aligner SINA\u0026nbsp;[60]\u0026nbsp;and merged with the Silva SSU Ref database (release 111). A phylogenetic tree of three isolates and \u003cem\u003eIntestinimonas\u003c/em\u003e AF211 \u0026nbsp;and closely related strains was constructed in the ARB software package (v. 6) by an algorithm\u0026nbsp;[61].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEnrichment medium was done in anaerobic bicarbonate-buffered mineral salt medium (CP medium)\u0026nbsp;[62]\u0026nbsp;consisting of (l\u0026nbsp;−1): 0.53 g Na2HPO4 . 2H2O, 0.41 g KH2PO4, 0.3 g NH4Cl, 0.11 g CaCl2 . 2H2O, 0.10 g MgCl2 . 6H2O, 0.3 g NaCl, 4.0 g NaHCO3\u0026nbsp;and 0.48 g Na2S . 9H2O as well as alkaline and acid trace elements (each 1 ml l\u0026nbsp;−1) and vitamins (0.2 ml l\u0026nbsp;−1)\u0026nbsp;[62]. The alkaline trace element solution contained the following (mM): 0.1 Na2SeO3, 0.1 Na2WO4, 0.1 Na2MoO4\u0026nbsp;and 10 NaOH. The acid trace element solution was composed of the following (mM): 7.5 FeCl2, 1 H3BO4, 0.5 ZnCl2, 0.1 CuCl2, 0.5 MnCl2, 0.5 CoCl2, 0.1 NiCl2\u0026nbsp;and 50 HCl. The vitamin solution had the following composition (g l\u0026nbsp;−1): 0.02 biotin, 0.2 niacin, 0.5 pyridoxine, 0.1 riboflavin, 0.2 thiamine, 0.1 cyanocobalamin, 0.1 p-aminobenzoic acid and 0.1 pantothenic acid.\u003c/p\u003e\n\u003cp\u003eYCFA medium (l\u0026nbsp;−1): 10 g soy peptone, 10 g yeast extract, 4 g NaHCO\u003csub\u003e3\u003c/sub\u003e, 2.7g sodium acetate, 4.5g K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.7g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.9 g NH\u003csub\u003e4\u003c/sub\u003eCl, 0.9 g NaCl, 0.09 MgSO\u003csub\u003e4\u003c/sub\u003e, 0.09 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 ml vitamin solution (1mg biotin, 1mg cobalamin, 3mg PABA, 5mg folic acid, 15mg pyridoxamine in 100 ml H\u003csub\u003e2\u003c/sub\u003eO), 1ml resazurine (0.5 g/l), 0.5g L-cysteine. In case of agar medium, 10g noble agar (DIFCO) was added to YCFA liquid before autoclave. The agar medium was then brought inside an anaerobic chamber and poured on agar plates. Those plates were then left slightly open for 30min till the agar got dried. The plates were kept for a maximum a week in the chamber before use. All streaking and plating were performed in the anaerobic chamber while the plate incubation was done in anaerobic jars filled with N\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e in the gas phase by a gas exchange machine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequencing and fructoselysine pathway gene analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrain GL3, IY4 and AS-BT were cultivated in 50ml YCFA_L liquid medium for an overnight at 37\u003csup\u003eo\u003c/sup\u003eC. The bacterial cells were harvested at the late exponential phase by centrifuging at 4700 rpm at 4\u003csup\u003eo\u003c/sup\u003eC. The cell pellets were used for DNA extraction using MasterPure™ Gram Positive DNA Purification Kit (Epicentre) according to the manufacturer’s instructions. After checking the quality on a Nanodrop, 30µl of high-quality DNA solution were send in dry ice to GATC for draft genomes using Illumina sequencing technology. Draft genome assemblies were constructed using the MyPro assembly pipeline\u0026nbsp;[63]. Raw reads were quality checked using FastQC\u0026nbsp;[64]. Reads were trimmed and subsampled to a total coverage of 100X (50X for forward reads, 50X for reverse reads), then assembled using 4 different assembly tools: VelvetOptimiser\u0026nbsp;[65], Edena\u0026nbsp;[66], SOAPdenovo\u0026nbsp;[67]\u0026nbsp;and SPAdes\u0026nbsp;[68].\u0026nbsp;The resulting contigs were \u0026nbsp;ordered with r2cat\u0026nbsp;[69]\u0026nbsp;using the \u003cem\u003eIntestinimonas butyriciproducens\u0026nbsp;\u003c/em\u003eAF211 genome (CP011307) as a reference and overlapping contigs merged resulting in the final genome assembly. Genome assemblies were then annotated using RAST\u0026nbsp;[70].\u0026nbsp;The annotation was done by RAST server\u0026nbsp;[70]. Functional prediction of proteins was verified manually by BLASTing the amino acid sequences in Pfam\u0026nbsp;[71], Brenda, Interpro\u0026nbsp;[72]\u0026nbsp;and Uniprot databases. In addition, the screening of antibiotic resistance genes was done using ABRICATE against the NCBI, ARG-ANNOT, ResFinder and VFDB databases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFructoselysine growth experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConversion of fructoselysine was tested in CP medium containing 5 mM fructoselysine (provided by TRC, North York, Canada). The inoculum was 2.5% from active cultures of strain GL3, IY4, AS-BT and AF211 for the growth test. All strains were pre-cultured in YCFA containing 20mM lysine. The bacterial cultures were sampled during the growth up to 48 hours for substrate and end metabolite measurements as described below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiotic resistance profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe E-test was done to identify minimal inhibitory concentrations (MICs) according to the manufacturer's protocol (bioMérieux, France). Both strains were pre-grown in RCM broth (overnight cultures) and 50 µl was spread on RCM agar plates (1.5% w/v agar) until the agar surface was dry and the liquid was absorbed by the agar. Two E-test strips were used per antibiotic and considered as duplicates. Antibiotics tested included ciprofloxacin, cefotaxime, erythromycin, oxacillin, teicoplanin, tetracycline, tobramycin, vancomycin and sulfamethoxazole. The concentration range was 0.016–256 µg/ml for chloramphenicol, oxacillin, tetracycline, tobramycin and vancomycin, and 0.016–32 µg/ml for ciprofloxacin, cefotaxime, erythromycin, teicoplanin and sulfamethoxazole. MIC values were recorded directly from the strips after 24 h, 48 h and rechecked after 4 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs tetW gene was detected in the genomes of most \u003cem\u003eIntestinimonas\u003c/em\u003e strains and some of \u003cem\u003eIntestinimonas\u003c/em\u003e strains were found to be resistant in erythromycin from the Etest, tetracycline and erythromycin were selected to perform the MIC test in liquid according to EFSA guideline. The test was done in 10ml YCFA medium containing lysine as substrate in anaerobic bottles filled with CO\u003csub\u003e2\u003c/sub\u003e/N\u003csub\u003e2\u003c/sub\u003e (1:4) at 1.5 atm. The concentration of tetracycline was 2-fold reduction in each bottle from 256µg/ml to 1µg/ml. The 256 ug/ml tetracycline bottle was prepared in 20 ml growth medium by adding 1ml of tetracycline filter sterilized stock solution (5.12mg/ml) to 19 ml complete medium. This medium was then serial diluted two-fold to get concentrations of 128, 64, 32, 16, 8, 4, 2 and 1 ug/ml. All these bottles were inoculated 2% with an overnight culture. The growth was monitored by OD measurement at 24h, 48h, 72h and 96h. Bottles without tetracycline were used as a positive control. MIC test for erythromycin in liquid was done in the same way by replacing tetracycline by erythromycin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were approved by the Institute Ethical Committee. C57BL6/J male mice were purchased from Charles River at the age of 4 weeks, fed a regular chow diet and kept under regular 12h/12h light/dark cycles. To determine the impact of IB on whole-body metabolism, male mice were randomized in 4 groups (N=12) receiving 3 times/week 2x10\u003csup\u003e9\u003c/sup\u003e CFU \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e GL3 or placebo solution (anaerobic PBS) by oral gavage and fed ad libitum a low-fat diet (LFD, 10%kcal from fat, Research Diets, D12450Ji) or high-fat diet (HFD, 60%Kcal from fat, Research Diets, D12492i). Mice were fed LFD/HFD for 13 weeks and IB/placebo supplementation started one week before switching to special diets. To avoid cage-effects on the microbiota composition, 3 mice were housed in one cage. Body weight and food consumption were monitored once a week. At the end of the study, insulin-tolerance test (ITT) was performed in obese mice on HFD: after 6 hour-fasting, mice received an intraperitoneal injection of insulin (0.5U/kg); blood glucose levels were assessed by tail prick using glucometer strips at 0, 15, 30, 60 minutes post-injection. To avoid unnecessary discomfort and suffering due to potential hypoglycemic events, lean mice fed a LFD were not subjected to ITT. Mice were sacrificed under anesthesia (5% isoflurane, O\u003csub\u003e2\u003c/sub\u003e flow of 2 L/minute), blood was collected by cardiac puncture, harvested organs were stored in formalin (for later paraffin-embedding) and snap-frozen in liquid-nitrogen. White-adipose tissues were weighed immediately after collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein bound fructoselysine in high fat diet and low fat diet\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein bound fructoselysine in the two diet was indirectly quantified through furosine concentration according to the method of Troise et al.\u0026nbsp;[73]\u0026nbsp;by using a Nexera U-HPLC system coupled with a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan). In brief, a 0.5 g aliquot of each chow was added to 4 mL of HCl (7.4 M) and then incubated at 110 °C, for 20 h. After filtering, 400 µL of the hydrolysate suspension was dried under vacuum using a rotary evaporator and reconstituted in 400 µL of 80% aqueous acetonitrile along with d4-furosine as internal standard (final concentration 200 µg/l). For separation of furosine and its internal standard, a core-shell Kinetex HILIC column (2.6 µm, 2.1 mm × 100 mm, Phenomenex) thermostated at 30 °C, with a flow rate of 0.4 mL/min was used. The mobile phases consisted of 0.1% formic acid (solvent A), 0.1% formic acid in acetonitrile (solvent B), and 50 mmol/L ammonium formate (solvent C). The gradient was as follows (t in [min]/[%B]): (0.0/80), (3.5/40), (6.5/40), with 4.5 min for equilibration, while solvent C was kept at 10%. Positive ionization multiple reaction monitoring (MRM) mode was used; the spray voltage was 4.0 kV and the collision energies (CE) were the following (in bold quantifier ions for furosine): furosine (\u003cem\u003em/z\u003c/em\u003e 255.3\u0026nbsp;à130, \u003cstrong\u003e84\u003c/strong\u003e, CE: 12 and 18), furosine-d4 (\u003cem\u003em/z\u003c/em\u003e 259\u0026nbsp;à\u0026nbsp;134, 88 CE: 12 and 15). Profile data were acquired and analyzed through LabSolutions (Shimadzu Corporation).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeted liquid chromatography tandem mass spectrometry of mouse cecum and plasma samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFructoselysine, lysine and SCFAs in CP medium, mice caecum and mice plasma were analysed by liquid chromatography high resolution tandem mass spectrometry (LC-MS/MS) by means of a Vanquish Core LC system interfaced to an Exploris 120, hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Because of target analytes concentration and matrix effect, SCFA in mice plasma underwent a different procedure including 3-nitrophenylhydrazine (3-NPH) derivatization. For fructoselysine and lysine quantification in CP medium, samples were diluted in a solution acetonitrile/water (50:50, v/v) according to the linearity range used for calibration curve. For fructoselysine quantification in caecum and plasma, analytical protocol was adapted from Wolf et al.\u0026nbsp;[7], with minor modifications. Briefly, 20 µL of plasma or 20 µL of caecum supernatants were diluted in ice cold methanol (ratio 1:3). Suspensions were centrifuged at 12700 rpm for 10 min, 4°C and 50 µL were dried under vacuum in a centrifugal evaporator (Savant, Thermo Fisher Scientific). Dried samples were resuspended in a solution consisting of 50% acetonitrile in water. Lysine and its Amadori compound were separated at 35°C through a zwitterionic sulfobetaine column (Atlantis Premier BEH, Z-HILIC, 100 x 2.1, 1.7 µm, Waters, Etten-Leur, the Netherlands) with the following gradient of solvent B (minutes/%B): (0/5), (1/5), (2/50), (6/50). Mobile phases consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B) and the flow rate was 0.2 mL/min. Heated electrospray (H-ESI) interface parameters were as follows: static spray voltage 3.3 kV, ion transfer tube and vaporizer temperature were both at 280 °C; sheath gas flow and auxiliary gas flow were 30 and 5 arbitrary units. The analyzer resolution was set at 60000 (FWHM at \u003cem\u003em/z\u003c/em\u003e 200), fructoselysine and lysine were identified and quantified in product ion scan positive mode screening the precursor ions (C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e [M+H]\u003csup\u003e+\u003c/sup\u003e 309.1656, for fructoselysine and C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [M+H]\u003csup\u003e+\u003c/sup\u003e 147.1128 for lysine) with an increasing normalized collision energies set at 25, 50 and 60% to improve fragmentation pattern and screening the product ions, monitoring for both fructoselysine and lysine the characteristic fragment ion at \u003cem\u003em/z\u003c/em\u003e 84.0808. For product ion scan mode, Orbitrap resolution was set at 15000 (FWHM at m/z 200) and the quadrupole resolution was set at 1. A linear calibration curve was built in the range 100-10000 nM and concentration reported in mM was measured through standard addition technique by using CP medium, caecum or plasma as blank samples. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor acetate, butyrate and isobutyrate separation in CP growth experiments, supernatants were directly diluted in o-phosphoric acid (0.5 % final concentration in water, 1:10, v:v), while for mice caecum content, samples were centrifuged at 4 °C, 12000 rpm for 15 min and supernatants diluted 1:10 v/v in 0.5% o-phosphoric acid. Samples were centrifuged at 12000 rpm before transferring clear supernatants to glass vial. Analytes were separated through a graphite column thermostated at 40°C (Hypercarb, 100 x 1.0, 1.7 µm, Thermo Fisher Scientific) with the following gradient of solvent B (minutes/%B): (0/0), (2/0), (6/75), (8/75). Mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) and the flow rate was 0.1 mL/min. H-ESI parameters were as follows: static spray voltage 3.2 kV, ion transfer tube and vaporizer temperature were both at 280 °C; sheath gas flow and auxiliary gas flow were 35 and 7 arbitrary units. The analyzer resolution was set at 60000 (FWHM at \u003cem\u003em/z\u003c/em\u003e 200), working in the scan range 50-350. Acetate, butyrate and isobutyrate were preliminary identified in full MS to evaluate effective separation of the two C4:0 isomers. Acetate and butyrate were quantified in full MS scan positive ion mode screening the two precursor ions (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [M+H]\u003csup\u003e+\u003c/sup\u003e 61.0284 and C\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [M+H]\u003csup\u003e+\u003c/sup\u003e 89.0597) with a mass accuracy below 3 ppm. A linear calibration curve was built in the range 0.5-10 mM by using acetate and butyrate as internal standard and concentration reported in mM. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor fructoselysine and SCFA analytical procedures in product ion scan mode and in full MS acquisition mode, profile data were collected using Xcalibur 4.5 (Thermo Fisher Scientific, Waltham, MA) and fragmentation spectra were recorded by using Free Style software (v. 1.8, Thermo Fisher Scientific, Waltham, MA). EASY-IC with fluoranthene in positive ion mode (m/z 202.0777 [M]+) was used to improve mass accuracy in both full scan and product ion scan mode. Analytical performance robustness, sensitivity, reproducibility, repeatability, linearity, accuracy, carry over and matrix effects were evaluated by following the procedures previously reported by Troise and coworkers through authentic analytical standard according to an in-house procedure developed in Trace Finder environment (v. 5.1, Thermo Fisher Scientific, Waltham, MA) encompassing identification of isotopic distribution, elemental composition, mass accuracy below 3 ppm for precursors and product ions and number of scan points higher than 8. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSCFA concentration in mouse plasma samples were quantified according to the procedure detailed by Garcia-Rivera et al\u0026nbsp;[74]\u0026nbsp;with minor modifications. Briefly, 10 µL of plasma were spiked with 1 µL of SCFA internal standard mix including \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e-acetate, \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e3\u003c/sub\u003e-propionate and \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e4\u003c/sub\u003e-butyrate (final concentration 0.1 mM for each carbon labelled compound). Upon protein precipitation with the addition of 60 µL of 75:25 methanol: water (v/v) solution, samples were mixed with 60 µL of 3-NPH (200 mM) and 10 µL of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, 120 mM in 6% pyridine). Samples were incubated at room temperature (22°C) for 45 min under gentle shaking in an orbital shaker. Derivatization reaction was stopped upon the addition of 10 µL quinic acid (200 mM) and incubation under shaking at room temperature for 15 min. Samples were centrifuged at 15000 rpm for 5 min at 4°C and supernatants diluted up to 1 mL with 10:90 methanol:water solution (v/v). Before transferring to glass vial, samples were centrifuged again at 4°C, 5 min, 15000 rpm. Hydrazone derivative quantitation was achieved by a U-HPLC system (Ultimate 3000 RS, Thermo Fisher Scientific) interfaced to a linear ion trap hybrid Orbitrap high resolution mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scinetific). Chromatographic separation included a reversed phase C18 column thermostated at 40°C (Kinetex C18 PS, 100 x 2.1 mm, 2.6 µm, Phenomenex, Torrance, CA) with the following gradient of solvent B (minutes/%B): (0/5), (5/5), (12.3/35), (13.3/85), (14/99), (16/99). Mobile phases consisted of water (solvent A) and acetonitrile (solvent B) and the flow rate was 0.2 mL/min. LC stream was interfaced to an electrospray ion source (ESI) working in negative ion mode scanning the ion in the \u003cem\u003em/z\u003c/em\u003e range 100-400; resolution was set at 30000 (FWHM at \u003cem\u003em/z\u003c/em\u003e 200), capillary temperature was 300°C, while sheath and auxiliary gases were set at 25 and 15 arbitrary units. Analytes profile data in full MS mode were collected using Xcalibur 2.1 (Thermo Fisher Scientific). Calibration curve was performed by internal standard technique in the linearity range 1-1000 µM by using the same procedure detailed above for plasma samples. Analytical performances for the three procedures are detailed in Supplementary Table 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was isolated from iWAT tissues, which were stored at -80˚C until analysis, using standard RNA isolation protocol. In short, biopsies were mixed with 1ml TriPure (Roche) and homogenized using a ceramic beads homogenizer. After adding 0.2ml chloroform to 1ml Tripure solution, samples were centrifuged (15 min, 12000 x g, 4˚C). The aqueous phase was transferred and mixed with 0.5ml isopropanol and centrifuged (15 min, 12000 x g, 4˚C). afterwards the pellets were resuspended in 1ml of 70% ethanol and centrifuged (15 min, 7500 x g, 4˚C). RNA was eluted in 20 µl RNAse free water. RNA concentrations were measured using the NanoDrop 1000 (Thermo Scientific).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal Time quantitative polymerase chain reaction (RT-qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1mg of RNA was converted to cDNA with SensiFAST cDNA synthesis kit (Meridian Bioscience) according to the manufacturer’s instructions. qPCR was performed on a CFX Opus 384 PCR machine (BioRad) using SensiFAST SYBR No-ROX Green (Meridian Bioscience).Gene expression was normalized with the expression of the housekeeping gene \u003cem\u003eHprt\u003c/em\u003e; relative gene expression was calculated with the “delta Ct” method and shown as 2ˆ- delta Ct. All primers were manufactured by Sigma-Aldrich; primer sequences are provided in Supplementary Table 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eiWAT tissues were lysated in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (cOmplete™ Protease inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails, Sigma) using a ceramic beads homogenizer. After centrifugation, the lipid layer was removed and protein concentration was determined by BCA assay (Thermo Fisher Scientific).\u0026nbsp;b-mercaptoethanol was added as a reducing agent to sample lysates. Samples were loaded on 4-12% NuPage Bis-Tris polyacrylamide gels (Invitrogen). Proteins were transferred to PVDF membranes (BioRad), which were blocked in 5% milk in TBS-T (Tris Buffered Saline – Tween-20). Membranes were incubated overnight a 4˚C with primary antibodies: rabbit antibodies anti-IRS2 (insulin receptor substrate 2, L1326, Cell Signaling, 1:1000) and mouse anti-beta-actin (Genetex, 1:10000). Horseradish peroxidase (HRP)-conjugated secondary antibodies (polyclonal goat anti-rabbit IgG or monoclonal goat anti-mouse IgG, Dako, 1:3000) were incubated for 1 hour at room temperature. HRP activity was visualized with peroxidase substrate for enhanced chemiluminescence and imaged with ChemiDoc MP Imaging System (BioRad). Densitometric quantification analysis was performed using the Image J software. All protein levels were normalized to the loading control (b-actin).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSections of frozen iWAT tissues were cut and homogenized in 400 µl RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (cOmplete™ Protease inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails, Sigma) using a ceramic beads homogenizer. Protein concentration was determined with BCA assay (Thermo Fisher Scientific). The tissue concentrations of TNF-α, IL-6 and IL-1β cytokines were quantified using respectively the mouse TNFalpha/IL-6 DuoSet ELISA (R\u0026amp;D Systems) and ELISA MAX™ Deluxe Set Mouse IL-1β (BioLegend) according to the manufacturers’ protocol. The absorbance was measured at an optical density (OD) of 450 nm and 570 nm using the Tunable Microplate Reader VersaMax (Molecular Devices, USA). The cytokine levels were normalized for protein concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology and Immunohistochemical staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were fixed in 10% buffered formalin overnight, dehydrated in 70% ethanol and embedded in paraffin. Sections of 4 µm were cut and stained for macrophage marker F4/80. Formalin-fixed paraffin-embedded (FFPE) sections were deparaffinized in 100% xylene and rehydrated in ethanol (100%, 96% and 70%) and H2O, followed by block of endogenous peroxidase in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e methanol for 20 minutes and heat-induced epitope retrieval (HIER) in citrate buffer pH 6.0 at 100 °C for 10 min. iWAT sections were incubated with the following antibodies: FITC anti-mouse F480 (BioLegend) diluted 1:5000 for 2 hours at room temperature, rabbit anti-FITC (Bio-Rad) diluted 1:1000 for 1 hour at room temperature, finally BrightVision Poly-HRP-conjugates goat anti-rabbit IgG (ImmunoLogic) diluted 1:2 for 30 minutes at room temperature. Staining was visualized with 3,3’Diaminobenzidine (DAB) kit (Sigma Aldrich) and counterstaining was performed using hematoxylin. Per sections, ten pictures were captured at random using a Leica MC170 HD stand-alone microscope camera (Danaher Corporation, USA). Subsequently, analysis of the digital images was conducted using Image-J software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis of the \u003cem\u003ein vivo\u003c/em\u003e data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical differences between the placebo and IB-treated groups were assessed using Mann Whitney test, and the differences were considered statistically significant with P values \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human work protocol was approved by\u0026nbsp;the Ethics Review Board in Gothenburg as previously indicated\u0026nbsp;[16]. All animal experiments were conducted according to the \u0026lsquo;Guide to the Care and Use of Experimental Animals\u0026rsquo; approved by the Ethics Committee on Animal Care and Use in Academisch Medisch Centrum, the Netherlands.\u003c/p\u003e\n\u003cp\u003eFresh stools for bacterial isolation work were collected from two healthy donors of whom informed consents were obtained following Good Clinical Practice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work is partially funded by the Euro-Trans-Bio grant HBC.2017.0100 for the DM Prevent-project. TPNB is supported by an NWO VENI grant and Amsterdam UMC bridging grant. ER and NR are supported by an NWO VIDI grant and AUMC Starter grant (appointed to ER). MN is supported by a personal ZONMW-VICI grant 2020 (09150182010020) and an ERC-Advanced Grant 2023 FATGAP (101141346). ADT, SDP and AS research was partially funded by the National Recovery and Resilience Plan, mission 4, component 2, investment 1.3, call n. 341/2022 of Italian Ministry of University and Research funded by the European Union - NextGenerationEU for the project \u0026ldquo;ON Foods - Research and innovation network on food and nutrition Sustainability, Safety and Security - Working ON Foods\u0026rdquo;, project PE00000003, concession decree n. 1550/2022, CUP B83C22004790001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human metagenomic data from the human cohort have been deposited in EGA as previously published\u0026nbsp;[16]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank prof. Willem M. DeVos and Dr. Jos Seegers for valuable discussions for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTPNB and ER conceptualized and designed the study. HW did metagenomic analysis. TPNB performed culturing, isolation and genomic analysis. ER and NR, SH, JA performed the animal work, performed ex vivo measurement and analyzed the data. ADT, VF, AS and SDP performed extraction and measurements of fructoselysine and SCFA in cecum content and plasma samples. SG produced the bacteria for the animal work. ER, NR and TPNB are major contributors in writing \u0026nbsp;the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.N is co-founder and member of the Scientific Advisory Board of Caelus Pharmaceuticals and Advanced Microbial Interventions, the Netherlands. However, none of these possible conflicts of interest bear direct relations to the outcomes of this specific study. The other authors declare no competing interests. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFujisaka, S., et al., \u003cem\u003eDiet, Genetics, and the Gut Microbiome Drive Dynamic Changes in Plasma Metabolites.\u003c/em\u003e Cell Rep, 2018. \u003cstrong\u003e22\u003c/strong\u003e(11): p. 3072-3086.\u003c/li\u003e\n\u003cli\u003eCorbin, K.D., et al., \u003cem\u003eHost-diet-gut microbiome interactions influence human energy balance: a randomized clinical trial.\u003c/em\u003e Nature Communications, 2023. \u003cstrong\u003e14\u003c/strong\u003e(1): p. 3161.\u003c/li\u003e\n\u003cli\u003eErbersdobler, H.F. and V. 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Fogliano, \u003cem\u003eQuantification of N\u0026epsilon;-(2-Furoylmethyl)-L-lysine (furosine), N\u0026epsilon;-(Carboxymethyl)-L-lysine (CML), N\u0026epsilon;-(Carboxyethyl)-L-lysine (CEL) and total lysine through stable isotope dilution assay and tandem mass spectrometry.\u003c/em\u003e Food Chem, 2015. \u003cstrong\u003e188\u003c/strong\u003e: p. 357-64.\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Rivera, M.A., et al., \u003cem\u003eIdentification and validation of small molecule analytes in mouse plasma by liquid chromatography-tandem mass spectrometry: A case study of misidentification of a short-chain fatty acid with a ketone body.\u003c/em\u003e Talanta, 2022. \u003cstrong\u003e242\u003c/strong\u003e: p. 123298.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4364001/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4364001/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe human gut microbiome strongly influences host metabolism via fermentation of dietary components to metabolites that allow communication with peripheral tissues. Short chain fatty acids are among the most known microbial metabolites that signal to the host. \u003cem\u003eIntestinimonas butyriciproducens\u003c/em\u003e is a prevalent commensal bacterium that has a unique capability of converting dietary fructoselysine to butyrate and acetate and has a completed fructoselysine catabolic pathway. Dietary fructoselysine is an abundant Amadori product formed in foods during processing and is part of food products rich in dietary advanced glycation end products which can be potentially toxic. Therefore, understanding the role of this bacterium and fructoselysine metabolism in metabolic health is highly relevant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe accessed associations of \u003cem\u003eI. butyriciproducens\u003c/em\u003e with metabolic risk biomarkers via both strain and functional levels using a human cohort characterized by fecal metagenomic analysis. We observed that the level of the bacterial strain as well as fructoselysine fermentation genes were reversely associated with BMI, triglycerides, HbA1c and fasting insulin levels. We also investigated degradation capacity of fructoselysine within the \u003cem\u003eIntestinimonas\u003c/em\u003e genus using a culture dependent approach and observed that \u003cem\u003eI. butyriciproducens\u003c/em\u003e as a key player in the butyrogenic fructoselysine metabolism in the gut. To explore the function of \u003cem\u003eI. butyriciproducens \u003c/em\u003eon host metabolism, we employed the diet-induced obesity mouse model to mimic the human metabolic syndrome. Oral supplementation of \u003cem\u003eI. butyriciproducens \u003c/em\u003ecounteracted body weight gain, hyperglycemia as well as adiposity. Moreover, within the inguinal white adipose tissue, bacterial administration reduced inflammation and promotes pathways involved in browning and insulin signaling. The observed effects are attributable to the formation of the short-chain fatty acids butyrate and acetate from dietary fructoselysine, as their plasma levels were significantly augmented by the bacterial strain, thereby contributing to systemic effects of the bacterial treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003e\u003cem\u003eI. butyriciproducens\u003c/em\u003e ameliorates host metabolism in the context of obesity and may thus be a good candidate for new microbiota-therapeutic approaches to prevent or treat metabolic diseases.\u003c/p\u003e","manuscriptTitle":"Gut bacterium Intestinimonas butyriciproducens improves host metabolic health: evidence from cohort and animal intervention studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-13 06:55:13","doi":"10.21203/rs.3.rs-4364001/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"37b56e70-5abb-4a68-9a00-bf8da4e52362","owner":[],"postedDate":"May 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-23T10:17:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-13 06:55:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4364001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4364001","identity":"rs-4364001","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}