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Cohen et al Mammals produce N-palmitoyl glycine, which differs from commendamide by of the B-hydroxyl and, based on our synthetic N-acyl studies, activates G2A. 19 he absence GPRI19 is the most extensively studied of the GPCRs activated by bacterial ligands we identified (Fig 4) Mechanisms that link endogenous GPRI19 agonists(OEA, 2-OG)to changes in host phenotype are well defined as a result of the exploration of GPri19 as a therapeutic target for diabetes and obesity. 0--3GPR119 agonists are thought to primarily affect glucose homeostasis but also gastric emptying and appetite through both gPr119- dependent hormone release from enteroendocrine cells(GLP-1, GIP, PYY)and pancreatic B-cells(insulin) as well as GPrI19-independent mechanisms including PPARa modulation 9, 16, 24-30 Murine enteroendocrine GLUTag cells have been used as a model system for measuring the ability of potential GPri19 agonists to induce GLP-1 release. When administered to gLUTag cells at equimolar concentrations, microbiota-encoded N-oleoyl serinol or the endogenous ligands oEa and 2-oG induced GLP-I secretion to the same magnitude(Fig. 5c). To provide an orthogonal measurement of GPRi19 activation by M acyl serinols, HEK293 cells were stably transfected with a GPR119 expression construct Both Oea and n-oleoyl serinol increased cellular cAMP concentrations in a gPr119 dependent fashion(Extended Data Fig 5) hm-NAS expression alters blood glucose in mice The functional overlap between endogenous and bacterial metabolites suggested that bacteria expressing microbiota-encoded GPRi19 ligands might elicit host phenotypes that mimic those induced by eukaryotic ligands. Endogenous and synthetic GPRi19 ligands have been associated with changes in glucose homeostasis that are relevant to the etiology and treatment of diabetes and obesity including a study where mice were orally administered bacteria engineered to produce a eukaryotic enzyme that increases endogenous GPR119 ligand(OEA)precursors. 9, 16, 24-27, 31 The metabolic effect of the endogenous GPR119 ligands is believed to occur at the intestinal mucosa as the delivery of OEA intravenously fails to lower blood glucose in mice during an oral glucose tolerance test(OGTT) Consequently, we sought to determine whether mice colonized with bacteria engineered to produce human microbiota N-acyl serinols would exhibit predictable host phenotypes notobiotic mice were colonized with E coli engineered to express the N-acyl serinol synthase gene in an IPTG dependent manner. Control mice were colonized with E. coli containing an empty vector. Based on the number of colony forming units detected in fecal pellets both cohorts of mice were colonized to the same extent(Extended Data Fig. 7).After one week of exposure to IPTG both cohorts were fasted overnight and subjected to an OGTT. At 30 minutes post challenge we observed a statistically significant decrease in blood glucose levels for the group colonized with E. coli expressing the N-acyl serinol synthase gene(Fig. 5d). MS analysis of metabolites present in cecal samples revealed the presence of N-acyl serinols in the treatment cohort but not in the control cohort(Extended Data Fig. 6). After two weeks of withdrawing IPTG from the drinking water we no longer observed a difference in blood glucose between the two cohorts in an OGTT(Fig. 5e).32 To further explore the metabolic phenotype induced by N-acyl serinols we repeated OGTT experiment in an antibiotic treated mouse model. In this study we compared mice Nature. Author manuscript; available in PMC 2018 February 28Mammals produce N-palmitoyl glycine, which differs from commendamide by the absence of the β-hydroxyl and, based on our synthetic N-acyl studies, activates G2A.19 GPR119 is the most extensively studied of the GPCRs activated by bacterial ligands we identified (Fig 4). Mechanisms that link endogenous GPR119 agonists (OEA, 2-OG) to changes in host phenotype are well defined as a result of the exploration of GPR119 as a therapeutic target for diabetes and obesity.20–23 GPR119 agonists are thought to primarily affect glucose homeostasis but also gastric emptying and appetite through both GPR119- dependent hormone release from enteroendocrine cells (GLP-1, GIP, PYY) and pancreatic β-cells (insulin) as well as GPR119-independent mechanisms including PPARα modulation. 9,16,24–30 Murine enteroendocrine GLUTag cells have been used as a model system for measuring the ability of potential GPR119 agonists to induce GLP-1 release. When administered to GLUTag cells at equimolar concentrations, microbiota-encoded N-oleoyl serinol or the endogenous ligands OEA and 2-OG induced GLP-1 secretion to the same magnitude (Fig. 5c). To provide an orthogonal measurement of GPR119 activation by N￾acyl serinols, HEK293 cells were stably transfected with a GPR119 expression construct. Both OEA and N-oleoyl serinol increased cellular cAMP concentrations in a GPR119 dependent fashion (Extended Data Fig 5). hm-NAS expression alters blood glucose in mice The functional overlap between endogenous and bacterial metabolites suggested that bacteria expressing microbiota-encoded GPR119 ligands might elicit host phenotypes that mimic those induced by eukaryotic ligands. Endogenous and synthetic GPR119 ligands have been associated with changes in glucose homeostasis that are relevant to the etiology and treatment of diabetes and obesity including a study where mice were orally administered bacteria engineered to produce a eukaryotic enzyme that increases endogenous GPR119 ligand (OEA) precursors.9,16,24–27,31 The metabolic effect of the endogenous GPR119 ligands is believed to occur at the intestinal mucosa as the delivery of OEA intravenously fails to lower blood glucose in mice during an oral glucose tolerance test (OGTT).26 Consequently, we sought to determine whether mice colonized with bacteria engineered to produce human microbiota N-acyl serinols would exhibit predictable host phenotypes. notobiotic mice were colonized with E. coli engineered to express the N-acyl serinol synthase gene in an IPTG dependent manner. Control mice were colonized with E. coli containing an empty vector. Based on the number of colony forming units detected in fecal pellets both cohorts of mice were colonized to the same extent (Extended Data Fig. 7). After one week of exposure to IPTG both cohorts were fasted overnight and subjected to an OGTT. At 30 minutes post challenge we observed a statistically significant decrease in blood glucose levels for the group colonized with E. coli expressing the N-acyl serinol synthase gene (Fig. 5d). MS analysis of metabolites present in cecal samples revealed the presence of N-acyl serinols in the treatment cohort but not in the control cohort (Extended Data Fig. 6). After two weeks of withdrawing IPTG from the drinking water we no longer observed a difference in blood glucose between the two cohorts in an OGTT (Fig. 5e).32 To further explore the metabolic phenotype induced by N-acyl serinols we repeated the OGTT experiment in an antibiotic treated mouse model. In this study we compared mice Cohen et al. Page 6 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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