Fig 2: Induction of PEPCK, cyclic AMP and gluconeogenesis by AG-205. (A) Western blot analysis and quantification of PGRMC1 and PEPCK in Control, Glucagon (GCG), and AG-205 group. GCG (100 µM, 2 h) and AG-205 (10 µg/ml, 18 h) were treated in DMEM-low glucose medium (50 mg/dl, w/o FBS, 1% Penicillin/Streptomycin, 1 nM dexamethasone). Values represent means ± SD. *p < 0.05 versus Control. (B) Western blot analysis and quantification of PGRMC1 and PEPCK in control versus AG-205 group. AG-205 (0.1, 1, 5, and 10 µg/ml) was treated in DMEM-low glucose medium (50 mg/dl, w/o FBS, 1% Penicillin/Streptomycin, 1 nM dexamethasone) for 18 h. ß-actin was used as an internal control. Values represent means ± SD. *p < 0.05 versus control. (C) Glucose production was measured by glucose peroxidase assay. After AG-205 pre-treatment (10 µg/ml) for 48 h, cells were incubated with glucose production buffer (sodium pyruvate 2 mM, dexamethasone 1 nM, w/o glucose) for 2 h. Values represent means ± SD. *p < 0.05 versus control. (D) Western blot analysis and quantification of PGRMC1 and PEPCK in 0 h versus 3, 6, 12, 24, 36, 48, and 72 h. AG-205 (10 µg/ml) was treated in DMEM-low glucose medium (50 mg/dl, w/o FBS, 1% Penicillin/Streptomycin, 1 nM dexamethasone) for indicated hours. ß-actin was used as an internal control. Values represent means ± SD. *p < 0.05 vesus 0 h. (E) Cyclic AMP (cAMP) levels of indicated hours. AG-205 (10 µg/ml) was treated in DMEM-low glucose medium. Data were normalized to loading cell number, 5000 cells per well. Values represent means ± SD. *p < 0.05 versus 0 h. All experiments were repeated at least 3 times.

Fig 3: Exercise mediated induction of PEPCK and PDK4.The exercise-mediated induction of PEPCK and PDK4 is attenuated in eWAT from IL-6-/- deficient mice. Data are presented as means + SE for 10–12 animals per group and are expressed as fold increases in gene expression compared to the sedentary control group of the same genotype. *P <0.05 compared to WT.

Fig 4: Treatment with CL 316,243 increases select lipolytic and glyceroneogenic enzymes in white adipose tissue in an IL-6-dependent manner. WT and whole-body IL-6-/- mice were injected with CL (1.0 mg/kg bw) or an equivalent volume of sterile saline once per day for five consecutive days and changes in PEPCK, ATGL, FAS, ACC, and PPAR? determined in epididymal adipose tissue (eWAT) (A) and inguinal adipose tissue (iWAT) (B). Data are presented as mean + SEM for seven samples/group. Representative blots are shown in C. *Significantly different than saline treated in the same genotype, #significantly different than CL treated in WT, P < 0.05. The vertical bar in C indicates the separation of noncontiguous bands from the same gel. Please note that the PPAR? antibody that was used detects both PPAR?1 (lower band) and PPAR?2 (upper band).

Fig 5: Hepatic PEPCK (A), G6Pase 36 kDa (B), G6Pase 40.5 kDa (C), PDK2 (D) PDK4 (E) protein content and representative blots (F) in Floxed and IL-6 MKO mice after 16 weeks on Chow, HFD or HFD with exercise training (n = 9–10). Values are mean ± SE. *Significantly different from Chow within given genotype (P < 0.05); ¤Significantly different from HFD within given genotype (P < 0.05); #Significantly different from Floxed mice within given intervention (P < 0.05).