Cohen et al colonized with E coll expressing an active N-acyl serinol synthase to mice colonized with E. coli expressing an NAS point mutant(Extended Data Fig. 8, p. Glu91Ala) that no longer produced N-acyl serinols. In this model the glucose lowering effect of colonization with N- acyl serinol producing E. coli remained significant(Fig. 5f). In the antibiotic treated mice we measured GLP-I and insulin concentrations after glucose gavage. Both hormones were gnificantly increased in the treatment group compared to the control group(Fig. 5 g, h). In all mouse models the observed correlation between hm-NAS gene induction and increased glucose tolerance is similar in magnitude to several studies with small molecule GPri19 agonists including glyburide, an FDA approved therapeutic for diabetes. 24. 2 Discussion ur characterization of human microbial N-acyl amides, together with other investiga of the human microbiota, suggests that host-microbial interactions may rely heavily on many human signaling systems(e.g, neurotransmitters, bioactive lipids, glycans). This is not surprising, as the genomes of the bacterial taxa common to the human gastrointestinal tract(e.g, Bacteroidetes, Firmicutes and Proteobacteria) are often lacking in gene clusters that encode for the production of complex secondary metabolites(e.g, polyketides nonribosomal peptides, terpenes). It appears that biosynthesis of endogenous mammalian produced by the human modest manipulation of primary metabolites. As a result, the structural conservation between metabolites used in host-microbial interactions and endogenous mammalian signaling metabolites may be a common phenomenon in the human microbiome Evolutionarily, the convergence of bacterial and human signaling systems through structurally related GPCR ligands is not unreasonable as GPCRs are thought to have developed in eukaryotes to allow for structurally simple signaling molecules to regulate increasingly complex cellular interactions. 33-33The structural similarities between microbiota-encoded N-acyl amides and endogenous GPCR-active lipids may be indicative of a broader structural and functional overlap among bacterial and human bioactive lipids including other GPCR-active N-acyl amides, eiconasoids(prostaglandins, leukotrienes)and sphingolipids Sphingolipid based signaling molecules may also be common in the human microbiome as prevalent bacterial species are known to synthesize membrane sphingolipids. The GPCRs with which bacterial N-acyl amides were found to interact are all part of the same"lipid-like " GPCR gene family. The potential importance of this GPCR family to the regulation of host-microbial interactions is suggested by their localization to areas of gastrointestinal track enriched in bacteria that are predicted to synthesize GPCr ligands (Extended Data Fig 4). Lipid-like GPCRs have been shown to play roles in disease models that are correlated with changes in microbial ecology including colitis(SIPR4, PTGir PTGER4), obesity(GPRi19), diabetes( GPri19), autoimmunity(G2A) and atherosclerosis (G2A, PTGIR). 9,10, 13, 14 The fact that the expression of an NAS gene in a gastrointestinal nizing bacterium is sufficient to alter host phys between lipid-like GPCRs and their N-acyl amide ligands could be relevant to human physiology and warrants further study. By LCMS analysis we observed most of thecolonized with E. coli expressing an active N-acyl serinol synthase to mice colonized with E. coli expressing an NAS point mutant (Extended Data Fig. 8, p.Glu91Ala) that no longer produced N-acyl serinols. In this model the glucose lowering effect of colonization with N￾acyl serinol producing E. coli remained significant (Fig. 5f). In the antibiotic treated mice we measured GLP-1 and insulin concentrations after glucose gavage. Both hormones were significantly increased in the treatment group compared to the control group (Fig. 5 g, h). In all mouse models the observed correlation between hm-NAS gene induction and increased glucose tolerance is similar in magnitude to several studies with small molecule GPR119 agonists including glyburide, an FDA approved therapeutic for diabetes.24,25 Discussion Our characterization of human microbial N-acyl amides, together with other investigations of the human microbiota, suggests that host-microbial interactions may rely heavily on simple metabolites built from the same common lipids, sugars, and peptides that define many human signaling systems (e.g., neurotransmitters, bioactive lipids, glycans). This is not surprising, as the genomes of the bacterial taxa common to the human gastrointestinal tract (e.g., Bacteroidetes, Firmicutes and Proteobacteria) are often lacking in gene clusters that encode for the production of complex secondary metabolites (e.g., polyketides, nonribosomal peptides, terpenes). It appears that biosynthesis of endogenous mammalian signaling molecules as well as those produced by the human microbiota may rely on the modest manipulation of primary metabolites. As a result, the structural conservation between metabolites used in host-microbial interactions and endogenous mammalian signaling metabolites may be a common phenomenon in the human microbiome. Evolutionarily, the convergence of bacterial and human signaling systems through structurally related GPCR ligands is not unreasonable as GPCRs are thought to have developed in eukaryotes to allow for structurally simple signaling molecules to regulate increasingly complex cellular interactions.33–35 The structural similarities between microbiota-encoded N-acyl amides and endogenous GPCR-active lipids may be indicative of a broader structural and functional overlap among bacterial and human bioactive lipids including other GPCR-active N-acyl amides, eiconasoids (prostaglandins, leukotrienes) and sphingolipids. Sphingolipid based signaling molecules may also be common in the human microbiome as prevalent bacterial species are known to synthesize membrane sphingolipids. 36 The GPCRs with which bacterial N-acyl amides were found to interact are all part of the same “lipid-like” GPCR gene family. The potential importance of this GPCR family to the regulation of host-microbial interactions is suggested by their localization to areas of gastrointestinal track enriched in bacteria that are predicted to synthesize GPCR ligands (Extended Data Fig. 4). Lipid-like GPCRs have been shown to play roles in disease models that are correlated with changes in microbial ecology including colitis (S1PR4, PTGIR, PTGER4), obesity (GPR119), diabetes (GPR119), autoimmunity (G2A) and atherosclerosis (G2A, PTGIR).9,10,13,14 The fact that the expression of an NAS gene in a gastrointestinal colonizing bacterium is sufficient to alter host physiology suggests that the interaction between lipid-like GPCRs and their N-acyl amide ligands could be relevant to human physiology and warrants further study. By LCMS analysis we observed most of the Cohen et al. Page 7 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript