(a–e) The livers of 2-month-old non-fasted male Flox and LIRKO mice were subjected to metabolic profiling (a) and microarray analysis (b); all metabolites and genes with an adjusted P value <0.05 are shown. Alternatively, hepatic gene expression (c) was measured using real-time PCR and protein levels (d) were measured by western blotting whole-cell lysates. Plasma TMAO levels (e) were measured using LC/MS. Data represent the mean and s.e.m.; n=5–11; *P<0.05 (Student's t-test). (f–h) Gene expression was measured in primary hepatocytes from male rats (f,g) or mice (h) after 6 h of stimulation with insulin (f), glucagon (g) or dexamethasone (h). Average or representative results of two to four independent experiments are shown; *P<0.05 (Student's t-test); n=3 replicates per condition.

(a–k) Four- to six-week-old male Flox and LIRKO mice were treated with control (Con) or FMO3 ASO for 7 weeks and killed in the non-fasted state. Hepatic gene expression (a,f,h,i,j,k) was measured by real-time PCR, and in (h) expressed as a heat map, with each column representing data from a single mouse. The data are row-normalized with red and blue representing high and low expression, respectively. Protein levels were measured by western blotting whole-cell lysates (b,g) or nuclear fractions (i). (c) TMAO was measured in plasma collected at the time of sacrifice using LC/MS. Glucose (d) and insulin (e) tolerance testing were performed after 5 weeks of ASO treatment. Data represent the mean and s.e.m.; n=5–7; *P<0.05 (Student's t-test) LIRKO versus Flox mice treated with the control ASO; ^#P<0.05 (Student's t-test) control versus FMO3 ASO treatment of LIRKO mice. (l–o) Primary mouse hepatocytes were infected with control adenovirus or adenovirus expressing SREBP-2 (l–n) or miR-182 (o). Alternatively, shRNA expression plasmids were transfected into H2.35 hepatoma cells (p). Gene expression was measured by real-time PCR (m,n). Data represent mean and s.e.m.; *P<0.05 (Student's t-test); n=3 wells per condition; results are representative of three independent experiments. (l,o,p) Whole-cell lysates were subjected to western blotting (l,o,p) and quantification (o,p right panels). (o,p) Data represent the mean and s.e.m. of three independent experiments; *P<0.05 (Student's t-test). Representative images are shown in l and the left panels of o,p. (q–t) Eight- to ten-week-old male LIRKO mice were fed a chow diet with or without supplementation of lovastatin and ezetimibe (L+E) for 1 week and euthanized in the non-fasted state. Hepatic gene expression (q,r,t) was measured by real-time PCR. Hepatic FoxO1 protein (s) was measured by western blotting whole-cell lysates. Data represent the mean and s.e.m.; n=5–7; ^#P<0.05 (Student's t-test).

At 4 to 6 weeks of age, male Flox and LIRKO mice were placed on an atherogenic Paigen diet, treated with control (Con) or FMO3 ASO for 16 weeks, and killed in the non-fasted state. Hepatic gene expression (a) was measured by real-time PCR. Hepatic protein levels (b,f) were measured by western blotting whole-cell lysates. (c,e) Plasma taken at the time of sacrifice was used to measure TMAO (c) or pooled (n=5–7 mice per group) and subjected to FPLC analysis (e). Total cholesterol (d) was measured after 12 weeks of ASO treatment and a 4-h fast. (g,h) Abdominal aortas were dissected and stained with Oil-Red-O (representative images shown in g). Lesion area was quantified and expressed as a percentage of the whole aorta (h). In the above in vivo experiments, data represent the mean and s.e.m.; n=5–13; *P<0.05 (Student's t-test) LIRKO mice treated with the control ASO versus Flox mice; ^#P<0.05 (Student's t-test) control ASO versus FMO3 ASO-treated LIRKO mice.

(a–h) Eight- to ten–week-old male mice were killed in the non-fasted state. (a–d) Streptozotocin (STZ)-treated mice were compared with their vehicle-treated controls (Veh) and (e–h) ob/ob mice were compared with their wild-type controls (Con); all mice were killed in the non-fasted state. (i–m) Six-week-old male ob/ob mice were treated with control (Con) or FMO3 ASO for 4 weeks and were killed in the non-fasted state. Hepatic gene expression (b,c,f,g,i,k,l) was measured using real-time PCR, and protein levels (d,h,j) were measured by western blotting whole-cell lysates. Glucose tolerance testing (m) was performed after 3 weeks of ASO treatment. Data represent the mean and s.e.m.; n=5–8; *P<0.05 (Student's t-test) vehicle versus STZ treated (a to d), control versus ob/ob mice (e to h), control ASO-treated ob/ob mice versus FMO3 ASO-treated ob/ob mice (i to m). (n–o) Two- to three-month-old male and female Flox and LIRKO mice were killed in the non-fasted state. Hepatic gene expression (n) was measured using real-time PCR, and protein levels (o) were measured by western blotting whole-cell lysates. (p–s) Four- to six-week-old female Flox and LIRKO mice were treated with control (Con) or FMO3 ASO for 7 weeks and killed in the non-fasted state. Hepatic gene expression (p,r) was measured by real-time PCR, and protein levels were measured by western blotting whole-cell lysates (q). Glucose tolerance testing (s) was performed after 3 weeks of ASO treatment. Data represent the mean and s.e.m.; n=5; *P<0.05 (Student's t-test) Flox versus LIRKO mice with control ASO treatment; ^#P<0.05 (Student's t-test) control versus FMO3 ASO-treated mice of the same genotype (blue # is for Flox mice and red # is for LIRKO mice). (t,u) Liver biopsies were taken from age and sex-matched patients undergoing bariatric surgery (Obese) or other abdominal surgeries (Controls), and FMO3 expression was measured by real-time PCR (u); biochemical and physiological data from these patients are shown (t). Data represent the mean and s.e.m., as described in Methods. P values were determined by the Mann–Whitney test for continuous variables and by the χ² test for categorical variables (t) or the Student's t-test (u).