Nicotine (Nic) perfusion induces IR and lower adiposity. (a) IPGTT, insulin release, and ITT; n = 10–11 each. (b,c) Hyperinsulinemic-euglycemic (H-E) clamp. Glucose infusion rate (GIR), hepatic glucose production (HGP), the rate of the disappearance (R_d) and the glucose metabolic index (R_g) in tibialis anterior and soleus (b) or epididymal white adipose tissue (eWAT, c); n = 9 each. (d) Body weight changes; n = 11 each. (e) Representative MRI images (n = 4 each) for fat distribution, fat mass and lean mass (n = 11 each). Scale bars, 1 cm. (f) Representative eWAT sections (n = 10 sections each) and quantification of adipocyte diameter (Scale bars, 50 µm, n = 10 each), and temperature of brown adipose tissue (BAT) area and rectal temperature (n = 10 each) with representative infrared thermal images (Scale bars, 1 cm). (g) Serum FFA after overnight fasting or insulin perfusion at 40 min during ITT (n = 10 each), and the linear regression between fat mass and serum FFA levels during insulin perfusion (n = 10 each). (h) In vivo lipolysis. (n = 8 each). (i) In vitro lipolysis assay of isolated adipocytes; n = 7 each. (j) Respiratory quotients (RQ, VCO₂/VO₂) (left) and the average data (right). n = 10 each. Significance determined by one-way ANOVA with repeated measures for the inter-assay evaluations (a,b,d,h), Student’s t-test (c,e,f,j), one-way ANOVA with Bonferroni’s post-hoc test (g,i) and *P < 0.05. All values are means ± SEM.

Lipolysis inhibition blocks nicotine-induced IR and adiposity reduction. Wild-type mice were treated with Nic, vehicle (Veh) or Nic + acipimox (Nic + Acipi). (a) Body weight changes; n = 8–9 each. (b) Fat mass and lean mass; n = 8–9 each. (c) Representative H&E-stained WAT sections (n = 10 sections each) and the diameters of adipocytes in WAT (Scale bar, 100 µm); n = 8–9 each. (d) Serum FFA levels in insulin-perfused mice (at 40 min during ITT); n = 8 each. (e) The respiratory quotients (RQ, VCO₂/VO₂) and food intake (feed) during light and dark cycles, as indicated by the white and gray areas (average data in the right panels); n = 6 each. (f,h) IPGTT, insulin release, ITT data; n = 8–9 each. (i) H-E clamp (n = 8 each). Glucose infusion rate (GIR), hepatic glucose production (HGP), the rate of the disappearance (R_d), and the glucose metabolic index (R_g) in tibialis anterior and soleus muscles or eWAT. (j) Irs1 and Akt signaling in muscle, liver and WAT; n = 8 each. Significance determined by one-way ANOVA with repeated measures for the inter-assay evaluations (a,f,g,h), one-way ANOVA with Bonferroni’s post-hoc test (b,c,d,e,i), *P < 0.05 (Veh vs. Nic), ^#P < 0.05 (Nic vs. Nic + Acpi. All values are means ± SEM.

Deletion of AMPKα2, but not AMPKα1, blocks nicotine-induced IR and inhibition of weight gain in mice. Nic or Veh was perfused by minipump in wild type (WT), Prkaa1^−/−, or Prkaa2^−/− mice. (a–c) IPGTT, insulin release, and ITT in mice; n = 8–10 each. (d) Body weight changes; n = 8–10 each. (e) Representative images of WAT sections (n = 10 sections each) analyzing cell sizes and the adipocyte diameter (Scale bar, 100 µm); n = 8 each. (f) Serum FFA levels after overnight fasting (Fasted) or insulin perfusion (at 40 min during ITT); n = 8 each. Significance determined by one-way ANOVA with repeated measures for the inter-assay evaluations (a,b,c,d), Student’s t-test (e), one-way ANOVA with Bonferroni’s post-hoc test (f) and *P < 0.05. All values are means ± SEM.

Adipose AMPKα2 is required for the nicotine-dependent inhibitory effects on weight gain and insulin signaling in mice. Nic or Veh was perfused in control (Con, black), Prkaa1^Δad (brown) and Prkaa2^Δad (purple) mice. (a) Phosphorylated AMPK at Thr172 (pAMPK) and phosphorylated ACC (pACC) expression in WAT; n = 8 each. (b) Genotypes of adipose-specific knockout mice and the expressions of AMPKα, AMPKα1, and AMPKα2 in isolated adipocytes; n = 8 each. (c) Body weight in mice perfused with Veh or Nic; n = 8–9 each. (d) Representative H&E-stained WAT sections (n = 10 section each) and the diameters of adipocytes in WAT (Scale bar, 50 µm); n = 8 each. (e) Serum FFA levels after overnight fasting (Fasted) or insulin perfusion at 40 min during ITT; n = 8 each. (f) IPGTT in mice treated with Veh or Nic; n = 8–9 each. (g) Irs1, pIrs1-Ser307 (pIrs1), pAkt-Ser473 (pAkt), p38, and JNK signaling in WAT of mice injected with saline or insulin (1 unit kg⁻¹); n = 8 each. Significance determined by Student’s t-test (a,d), one-way ANOVA with repeated measures for the inter-assay evaluations (c,f), one-way ANOVA with Bonferroni’s post-hoc test (e,g) and *P < 0.05. All values are means ± SEM.

Nicotine treatment results in greater pMKP1-Ser334 levels and subsequent degradation through AMPK. (a) MKP1 expression (top) and its serine (Ser) /threonine (Thr) phosphorylation (bottom) in WAT from Veh- or Nic-treated mice. Gapdh was detected as a loading control; n = 8 each. (b) AMPKα2-MKP1binding in MEF-derived adipocytes; n = 5 each. (c) AMPK activation and MKP1 serine phosphorylation in nicotine-treated isolated adipocytes; n = 4 each. (d) MKP1 phosphorylation in HEK293 cells transfected with vector, GFP-tagged WT MKP1 (WT) or the indicated site-directed mutants of MKP1; n = 4 each. (e) The indicated peptides (the SAMS peptide and its mutant, SAMSmu) or peptides spanning the indicated amino acid residues of MKP1; n = 3 each. (f) MKP1 ubiquitination in HEK293 cells transfected with WT MKP1 (WT) or MKP1-S334A; n = 4 each. (g) MKP1 protein expression in MG132-treated isolated adipocytes; n = 5 each. (h) The subcellular localization of AMPKα1/2, p38 and MKP1 in adipocytes treated with or without nicotine. LDH (lactate dehydrogenase), cytoplasmic marker; H3 (histone 3), nuclear marker; n = 5 each. (i) Insulin signaling (western blot, top) and lipolysis (bottom) in Nic- or Veh-treated MEF-derived adipocytes transfected with adenovirus GFP (Ad-GFP) or MKP1 (Ad-MKP1).; n = 5 each. (j) Detection of pMKP1-S334 in WAT from the indicated strains in Nic- or Veh-treated mice (top) and its quantitation (bottom). Beta-actin was detected as a loading control; n = 6 each. Significance determined by one-way ANOVA with Bonferroni’s post-hoc test (a,i,j) and *P < 0.05. All values are means ± SEM.

MKP1 reduction is required for nicotine-mediated IR and lipolysis. (a–g) WT and Mkp1^−/− mice were treated with Veh or Nic. (a) Body weight changes and fat mass in mice treated with Veh or Nic; n = 7–8 each. (b) In vivo lipolysis. Plasma glycerol concentration measured at 0, 15, 60, 120 min; n = 6 each. (c,d) IPGTT (c) and ITT (d) data in mice; n = 7–8 each. (e,f) The glucose infusion rate (GIR) (e) and the hepatic glucose production (HGP), the rate of the disappearance (R_d) and in tibialis anterior and soleus muscles or eWAT the glucose metabolic index (R_g) (f) during a hyperinsulinemic-euglycemic clamp (n = 6 each). (g) MKP1, pIrs1- Ser307, Irs1 and p38 signaling in WAT of mice of the indicated genotype; n = 6 each. (h) AchRα7 expression in isolated adipocytes, hepatocytes, β-cells, and myocytes after treatment with Nic or Veh; n = 6 each. (i) Detection of pAMPK, AMPK, MKP1, and Irs1 expression in WAT in smokers and nonsmokers; n = 12 each. (j) Oral glucose tolerance test (OGTT) (left), the insulin levels during OGTT (right) and the areas under the curve (AUC); n = 12 each. Significance determined by one-way ANOVA with Bonferroni’s post-hoc test (a,f,g,j), one-way ANOVA with repeated measures for the inter-assay evaluations (b,c,d,e), Student’s t-test (h,i), *P < 0.05 Veh vs.Nic, and ^#P < 0.05 WT vs. Mkp1^−/− mice. All values are means ± SEM.