(A) Fasting glucose and (B) glucose tolerance test at 1-week following VHL disruption in the liver (Vhl^LivKO). (C) Insulin stimulated AKT phosphorylation and (D) qPCR analysis of G6pase and Pepck mRNA in PH treated with 100 nM Wortmannin for 2-hours. (E) Insulin tolerance test at 1-week following tamoxifen treatment. (F and G) qPCR analysis in the livers of fed or overnight fasted mice. (H) Glucagon tolerance test at 1-week after VHL disruption. (I) Serum insulin and (J) serum glucagon assessed at 1-week following VHL disruption. Each bar represents the mean± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 compared to Vhl^F/F. #p < 0.05, ##p < 0.01 compared to Fed. Four to five mice were used per group and experiments with PH were done in triplicates and repeated at least three times.

(A) Pyruvate tolerance test at 1-week after disruption of VHL in liver (Vhl^LivKO). (B) G6pase activity and (C) hepatic glycogen content assessed in Vhl^F/F and Vhl^LivKO mice. *p < 0.05, **p < 0.01, ***p < 0.001 compared to Vhl^F/F. (D) De novo gluconeogenesis in Vehilcle (Veh), substrate (S) and substrate+glucagon (S+G) treated cells. (E) qPCR analysis for HIF2α target gene, Epo mRNA and glucagon induction of G6pase and Pepck in PH. (F) Western blot analysis of G6Pase and PEPCK in PH treated with 100 nM glucagon for 6-hours. Glucagon-induced G6pase expression in PH pre-treated with (G) 50 nM Wortmannin for 2-hours or (H) 50 nM LY294002 or 50 nM MK-2206 for 1-hour. (I) Glucose production in PH pretreated for 1-hour with or without 50 nM Wortmannin or 50 nM LY294002 or 50 nM MK-2206. ***p < 0.001 compared Vhl^F/F. # p < 0.05, ###p < 0.001 compared to Vehicle. $p<0.05, $$p < 0.001, $$$p < 0.0001 compared to substrate alone. (J) G6pase promoter luciferase assay in PH. Luciferase values were normalized to protein content. (K) qPCR analysis of G6pase and Pepck in the PH treated with 10 nM dexamethasone for 2-hours. pCREB assessed (L) in vivo and (M) in PH. Each bar represents the mean value ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 compared to Vhl^F/F. #p < 0.05, ## p < 0.01, ###p < 0.001 compared to Vehicle. Four to five mice were used per group and the experiments with PH were done in triplicate and repeated at least three times.

(A) Blood glucose, (B) serum insulin levels (top panel) and serum glucagon levels (bottom panel) assessed at 1-week following tamoxifen treatment. (C) qPCR for G6pase and Pepck mRNA in the livers of overnight fasted mice. *p < 0.05 compared to Vhl^LivKO. #p < 0.05 compared to Fed. (D) Glucose tolerance, (E) Insulin tolerance, (F) glucagon tolerance and (G) pyruvate tolerance test in Vhl^LivKO and Vhl/Hif2^LivKO mice. Five to eight mice were assessed per group. *p < 0.05, **p < 0.01 compared to Vhl^F/F. #p < 0.001 compared Vhl^LivKO. (H) G6pase and Pepck mRNA expression and (I) pCREB in glucagon treated PH. *** p < 0.001 compared to Vhl^LivKO; #, p < 0.05 compared to Vehicle; ###, p < 0.001 compared to Vehicle. Each bar represents the mean value ± S.E.M. Experiments with PH were done in triplicate and repeated at least three times.

(A) In vivo imaging of livers and (B) liver tissue luciferase assays in mice fasted overnight or refed for 30-, 60- and 120-minutes. *p < 0.05 compared to fasted. Three mice were assessed per group. (C) Western blot for HIF2α and (D) co-immunostaining for pimonidazole adducts and HIF2α in the overnight fasted and 30 or 60 minutes refed livers. Five mice were assessed per group. (E) qPCR analysis of Hif2α mRNA in hepatocytes. (F) Glucose and (G) glucagon tolerance performed in 6-week-old WT and Hif2α^LivKO mice. (H) Glucose levels monitored in overnight fasted and refed mice. *p < 0.05 compared to Hif2α^F/F. (I) Western blot analysis for pCREB and pAKT in WT and Hif2α^LivKO mice fasted overnight and refed for 30-minutes to 2-hours. Six mice were assessed per group.

(A) Glucagon stimulated pCREB in PH pre-treated with phosphatase inhibitor okadaic acid (200 nM OKA) for 2-hours. (B) PKA activity and (C) Western blot analysis for phospho-PKA (p-PKA) substrates and (D) cAMP levels assessed in PH treated with glucagon. Western blot analysis for pCREB and qPCR for G6pase mRNA (bottom panel) in PH treated with (E) 20 μM forskolin or (F) 200 μM 8-br-cAMP for 2-hours. (G) PDE activity assessed by 5′-AMP levels in PH isolated at 1-week following VHL disruption. (H) Glucagon-stimulated pCREB, (I) G6pase expression and (J) cAMP levels in PH pretreated with 1 μM IBMX for 1-hour. (K) Glucagon-stimulated pCREB, G6pase and Pepck (L) mRNA or (M) protein expression (glucagon treatment for 6-hours) in PH pretreated for 16-hours with Rolipram (Rp, 1 μM), Glaucine (Gl, 1 μM), Ibudilast (Ib, 1 μM), and CP80,633 (Cp, 1 μM). PH were treated again with respective PDE inhibitors 30 minutes before glucagon treatment. Experiment with PH were done in triplicates and repeated at least twice. Each bar represents the mean value ± S.E.M. *p < 0.05, **p < 0.05, ***p < 0.001 compared to Vhl^F/F. # p < 0.05, ###p < 0.001, ####p < 0.0001 compared to Vehicle, $p < 0.05, $$$p < 0.001 are IBMX treated compared to their untreated controls. ^^^p < 0.001 compared to no inhibitor treatment.

(A) qPCR analysis of G6pase in PH pre-treated with inhibitors or activators for two-hours and then with glucagon for additional 2-hours (V-Veh; G- Glucagon; G+T- Glucagon + treatments). (B and C) pERK levels assessed in livers and PH of Vhl^LivKO mice. pERK levels, (D) Glucagon induced pCREB and (E) qPCR analysis of G6pase mRNA in PH pre-treated with 2.5 μM GSK1120212 and 10 μM PD98059 and10 μMFR180204 for 16-hours. Each bar represents the mean value ± S.E.M. ***p < 0.001 compared to Vhl^F/F. ###p < 0.001 compared to no glucagon. $p < 0.05, $$$p < 0.001 compared to without inhibitor treatment. (F) Glucose production in PH pre-treated with GSK for 16-hours. Each bar represents the mean value ± S.E.M. ##p < 0.001 compared to no substrate or glucagon. ^p < 0.05, ^^p <0.01 compared to substrate and glucagon treated, *p <0.05 compared to Vhl^F/F $p < 0.05, $$$p < 0.001 compared to without inhibitor treatment. Glucagon stimulated (G) pCREB and (H) Pepck and G6pase mRNA expression in WT PH infected with Ad-GFP or Ad-MEK-ERK-LA virus for 48-hours. ***p < 0.001 compared to Ad-GFP. ####p < 0.001 compared to no glucagon. (I) PDE activity in PH infected with Ad-GFP or Ad-MEK-ERK-LA virus for 48-hours. Five to six mice were used per group and experiments with PH were done in triplicates and repeated at least twice.

(A) Glucose, (B) insulin, (C) glucagon tolerance test performed in HIF2α^F/F and HIF2α^LivKO mice fed a 60% HFD for 6-weeks. *p < 0.05 compared to HIF2α^F/F. (D) Blood glucose monitored in STZ treated mice. (E) qPCR analysis for G6pase and Pepck mRNA in livers of STZ treated mice. *p < 0.05 compared to Vhl^F/F, ***p < 0.001 compared to Vhl^F/F. Five to seven mice assessed per group. (F) Blood glucose monitored in Vhl^LivKO mice injected i.p. with MK-2206 (50mg/kg body weight). *p < 0.05 compared to mice before treatment, #p < 0.05 compared to Vhl^F/F. Each bar represents the mean value ± S.E.M. Six mice to eleven mice were assessed per group. (G) Schematic in which HIF2α-mediated increase in ERK signaling represses glucagon-PKA-CREB signaling through increased PDE-dependent hydrolysis of cAMP in liver.