serinol was isolated and purified in the same manner as N-palmitoyl serinol described above and its identity was confirmed by H NMR and HRMS. N-oleoyl serinol was resuspended at 10 mM concentration in DMSO. GLUTag cells were obtained from the Mangelsdorf Lab (University of Texas Southwestern) with permission from Daniel Drucker(Mount SinaI Hospital Toronto). GLUTag cells were grown in DMEM, low glucose, GlutaMAX (ThermoFisher) supplemented with 10% FBS and 1% Penicillin/Streptomycin. Once cells grew to 80% confluence they were harvested and plated 1: I into 24 well culture plates in fresh culture media at 50,000 cells per well. After overnight growth in culture plates, cells were washed twice with Krebs buffer supplemented with 20 HL per ml of DPP4 inhibitor (Millipore). GLUTag cells were incubated for 30 minutes in supplemented Krebs buffer and ompounds were added at 1 uM and 100 uM. Cells were incubated with compounds for 2 hours. Media was then collected, centrifuged at 500xg(4C)for 5 minutes and cell free supernatant was analyzed for GLP-1 level using the Active GLP-1 V2 kit(Mesoscale Discovery ) Data from 2 independent experiments were analyzed together for Figure 5c(N 于9之 6 wells for OEA, N-oleoyl serinol andN=4 wells for 2-OG and DMsO) Colonization of germ-free and antibiotic treated mice with N-acyl serinol producing E coli All experimental procedures were approved by the Animal Care and Use Committee of The Rockefeller University. Germ free C57BL/6 mice were maintained in sterile isolators with Itoclaved food and water in the gnotobiotic Facility of the Mucida laboratory at The Rockefeller University. Wild type C57BL/6 mice were purchased from Jackson Labs. 8- week-old mice were used for all experiments For colonization studies 5 ml of an overnight culture(LB with 50 ug/ml kanamycin)of E coli transformed with pET28c hm-NAS N-acyl serinol synthase( treatment group)or E coli transformed with the empty pET28e vector (control group) was centrifuged at 500 x g for 2 minutes, the supernatant was decanted and the cells were resuspended in 2 ml of sterile PBS. Germ free mice were gavaged with 100 uL of bacterial culture immediately upon removal from sterile isolators. Antibiotic treated mice were given water supplemented with 500 ug/ml ampicillin for 1 week followed by gavage with E. coli clones. Suppression of endogenous microbiota was confirmed prior to gavage by culturing stool on non-selective LB agar and demonstrating no colony formation After colonization mice were housed in specific-pathogen-free conditions and fed with autoclaved water and food. Water was supplemented with 35 ug/ml kanamycin and 25 mM PTG. Fecal pellets from mice were analyzed each week for 3 weeks to confirm colonization by the appropriate bacteria and to check for contamination by plating on LB agar with and without kanamycin 50 ug/ml(Extended Data Fig 7). A single fresh mouse fecal pellet was weighed and resuspended in PBS at a fixed concentration (400 HLper mg). I HL of this suspension was diluted 1: 100 in PBS and plated in duplicate, the average number of colonies per plate(2 plates)was recorded. Plasmids were isolated and restriction mapped from these colonies to confirm the presence of the correct hm-NAS gene insert or lack thereof. Ethyl acetate extracts from broth cultures were also examined as previously and 4(2M, 2F)in the control group. The independent replicate experiment of germ fre 3 5 ssed to confirm the production of N-acyl serinols by bacteria in the tre the first experiment of germ free, 7 mice were studied -3(IM, 2F)in the treatment grou mice also consisted of 7 mice(all female, 3 in the treatment group and 4 in the control group). Mice were all individually caged and at the end of each week food consumption and Nature. Author manuscript; available in PMC 2018 February 28serinol was isolated and purified in the same manner as N-palmitoyl serinol described above and its identity was confirmed by 1H NMR and HRMS. N-oleoyl serinol was resuspended at 10 mM concentration in DMSO. GLUTag cells were obtained from the Mangelsdorf Lab (University of Texas Southwestern) with permission from Daniel Drucker (Mount Sinai Hospital Toronto). GLUTag cells were grown in DMEM, low glucose, GlutaMAX (ThermoFisher) supplemented with 10% FBS and 1% Penicillin/Streptomycin. Once cells grew to 80% confluence they were harvested and plated 1:1 into 24 well culture plates in fresh culture media at 50,000 cells per well. After overnight growth in culture plates, cells were washed twice with Krebs buffer supplemented with 20 µL per ml of DPP4 inhibitor (Millipore). GLUTag cells were incubated for 30 minutes in supplemented Krebs buffer and compounds were added at 1 µM and 100 µM. Cells were incubated with compounds for 2 hours. Media was then collected, centrifuged at 500×g (4 °C) for 5 minutes and cell free supernatant was analyzed for GLP-1 level using the Active GLP-1 V2 kit (Mesoscale Discovery). Data from 2 independent experiments were analyzed together for Figure 5c (N = 6 wells for OEA, N-oleoyl serinol and N = 4 wells for 2-OG and DMSO). Colonization of germ-free and antibiotic treated mice with N-acyl serinol producing E. coli All experimental procedures were approved by the Animal Care and Use Committee of The Rockefeller University. Germ free C57BL/6 mice were maintained in sterile isolators with autoclaved food and water in the Gnotobiotic Facility of the Mucida Laboratory at The Rockefeller University. Wild type C57BL/6 mice were purchased from Jackson Labs. 8- week-old mice were used for all experiments. For colonization studies 5 ml of an overnight culture (LB with 50 µg/ml kanamycin) of E. coli transformed with pET28c:hm-NAS N-acyl serinol synthase (treatment group) or E. coli transformed with the empty pET28c vector (control group) was centrifuged at 500 × g for 2 minutes, the supernatant was decanted and the cells were resuspended in 2 ml of sterile PBS. Germ free mice were gavaged with 100 µL of bacterial culture immediately upon removal from sterile isolators. Antibiotic treated mice were given water supplemented with 500 µg/ml ampicillin for 1 week followed by gavage with E. coli clones. Suppression of endogenous microbiota was confirmed prior to gavage by culturing stool on non-selective LB agar and demonstrating no colony formation. After colonization mice were housed in specific-pathogen-free conditions and fed with autoclaved water and food. Water was supplemented with 35 µg/ml kanamycin and 25 mM IPTG.32 Fecal pellets from mice were analyzed each week for 3 weeks to confirm colonization by the appropriate bacteria and to check for contamination by plating on LB agar with and without kanamycin 50 µg/ml (Extended Data Fig 7). A single fresh mouse fecal pellet was weighed and resuspended in PBS at a fixed concentration (400 µL per 40 mg). 1 µL of this suspension was diluted 1:100 in PBS and plated in duplicate, the average number of colonies per plate (2 plates) was recorded. Plasmids were isolated and restriction mapped from these colonies to confirm the presence of the correct hm-NAS gene insert or lack thereof. Ethyl acetate extracts from broth cultures were also examined as previously discussed to confirm the production of N-acyl serinols by bacteria in the treatment group. In the first experiment of germ free, 7 mice were studied - 3 (1M, 2F) in the treatment group and 4 (2M, 2F) in the control group. The independent replicate experiment of germ free mice also consisted of 7 mice (all female, 3 in the treatment group and 4 in the control group). Mice were all individually caged and at the end of each week food consumption and Cohen et al. Page 12 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript