Prom1 deficiency prevents glucagon- induced hyperglycemia in vivo and β-adrenergic receptor-mediated PKA activation.Figure 312-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (A) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (200 µg/kg body weight, n = 10/group). 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (B) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting (2 mg/kg body weight). 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (C) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit (2 mg/kg body weight, n = 3/group). 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (D) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit (2 mg/kg body weight, n = 3/group). Glucose disposal rates were measured in 12-week-old male mice using glucose, pyruvate (E) tolerance tests (n = 10 mice/group). Glucose disposal rates were measured in 12-week-old male mice using insulin (F) tolerance tests (n = 10 mice/group). G. Levels of p-AKT, AKT, p-ERK, ERK, IRβ and GAPDH were determined by immunoblotting after insulin stimulation (10 nM). Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (H) The nuclear localization of p-CREB after isoprenaline stimulation (10 μM) for 10 minutes was determined by immunofluorescence staining. Scale bar, 20 μm. Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (I) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH after isoprenaline stimulation (10 μM) for 0, 10 or 20 minutes were determined by immunoblotting. Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (J) The cAMP concentration after isoprenaline stimulation (10 μM) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX (n = 3/group). WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (K) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after epinephrine stimulation WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (L) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting. M-O. WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (M) Blood glucose levels (mg/dL) were measured 0, 60 and 120 minutes after immobilization (n = 10/group). WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (N) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 30 minutes-immobilization were determined by immunoblotting. WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (O) Serum epinephrine level of mice after a 30 minutes-immobilization was determined (n = 3/control group, 7 or 8/immobilized group).