Fig. 2. Nicotine-induced activation of intestinal AMPKα-SMPD3 axis in NAFLD progression.

a, Representative western blot of ileal AMPK signaling in nonsmokers (n = 30) and smokers with low nicotine (n = 14) or high nicotine (n = 16) contents in terminal ileum mucosa biopsies. Prkaa1^fl/fl and Prkaa1^ΔIE mice (SPF) were administrated an HFHCD plus nicotine water for 20 weeks (n = 8 mice/group). b, Liver weight–to–body weight ratios. c, Liver TG content. d, e, Serum ALT (d) and AST (e) levels. f, Representative H&E staining (left), Oil Red O staining (middle) and Sirius Red staining (right) of liver sections (n = 4 mice/group, 3 images/mouse). Scale bar, 100 μm. g, Quantification of ileal ceramide profiles between Prkaa1^fl/fl (WT, n = 12) and Prkaa1^ΔIE (KO, n = 11) mice administrated an HFHCD plus nicotine water for 20 weeks. h, Volcano map shows all phosphorylated sites of ceramide metabolism-related proteins identified from phosphorylated proteomics analysis of the ileal epithelia of Prkaa1^fl/fl (WT) and Prkaa1^ΔIE (KO) mice administrated an HFHCD plus nicotine water for 20 weeks (n = 5 mice/group). P values calculated by two-sided Student’s t-test. i, The assessment of AMPK activation and SMPD3 protein levels in ileal organoids treated with control, nicotine or nicotine plus compound C (CC, AMPK inhibitor) for 12 h. j, Co-IP of AMPKα1 with SMPD3. Constructs encoding Flag-tagged SMPD3 were transfected into SW480 cells, and then treated with nicotine for 4 h. k, Mass spectrometry analysis of the phosphorylation intensity at S209 on SMPD3 with or without nicotine treatment for 24 h. l, In vitro phosphorylation of WT or S209A mutant SMPD3 (86–655) by AMPK(α1/β1/γ2) with or without CC treatment. Data are the means ± s.e.m. b, c, e, Two-tailed Student’s t-test. d, g, Two-tailed Mann-Whitney U-test. Experiments in i-l were performed three times independently.