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. 2020 Oct 8;21(11):e49416. doi: 10.15252/embr.201949416

Figure 3. Prom1 deficiency prevents glucagon‐induced hyperglycemia in vivo and β‐adrenergic receptor‐mediated PKA activation.

Figure 3

  • A–D
    12-week‐old male WT (Prom1 +/+) and KO (Prom1 −/−) mice were fasted for 4 h and intraperitoneally injected with glucagon. (A) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60, and 120 min after glucagon stimulation (200 μg/kg body weight, n = 10/group). (B) Levels of p‐PKA substrates, p‐CREB, CREB, and GAPDH in the liver after a 5 min of glucagon treatment were determined by immunoblotting (2 mg/kg body weight). (C) Relative PKA activities in the liver after a 5 min of glucagon treatment were determined using the PKA assay kit (2 mg/kg body weight, n = 3/group). (D) The cAMP concentration in the liver after a 5 min of gluc, agon treatment was determined using the cAMP assay kit (2 mg/kg body weight, n = 3/group).
  • E, F
    Glucose disposal rates were measured in 12-week‐old male mice using glucose, pyruvate (E), and 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).
  • H–J
    Primary hepatocytes were isolated from 12‐week-old male WT (Prom1 +/+) and KO (Prom1 −/−) mice, serum‐starved for 16 h, and stimulated with isoprenaline. (H) The nuclear localization of p‐CREB after isoprenaline stimulation (10 μM) for 10 min was determined by immunofluorescence staining. Scale bar, 20 μm. (I) Levels of p‐PKA substrates, p‐CREB, CREB, Prom1, and GAPDH after isoprenaline stimulation (10 μM) for 0, 10, or 20 min were determined by immunoblotting. (J) The cAMP concentration after isoprenaline stimulation (10 μM) for 10 min was determined using the cAMP assay kit in the presence of 10 μM IBMX (n = 3/group).
  • K, L
    WT (Prom1 +/+) and KO (Prom1 −/−) mice were fasted for 4 h and intraperitoneally injected with epinephrine (3 μg/10 g). (K) Blood glucose levels (mg/dl) were measured 0, 15, 30, 60, and 120 min after epinephrine stimulation (n = 13 or 14/group). (L) Levels of p‐PKA substrates, p‐CREB, CREB, and GAPDH in the liver 15 min after epinephrine treatment were determined by immunoblotting.
  • M–O
    WT (Prom1 +/+) and KO (Prom1 −/−) mice were fasted for 4 h and subjected to immobilization test. (M) Blood glucose levels (mg/dL) were measured 0, 60, and 120 min after immobilization (n = 10/group). (N) Levels of p‐PKA substrates, p‐CREB, CREB, and GAPDH in the liver after a 30 min of immobilization were determined by immunoblotting. (O) Serum epinephrine level of mice after a 30 min of immobilization was determined (n = 3/control group, 7 or 8/immobilized group).
Data information: Data are presented as mean values ± SEM. Two‐tailed Student's t‐test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, and NS, not significant, P > 0.05. Source data are available online for this figure.