Figure 6. Asprosin Uses the cAMP Second-Messenger System and Activates PKA in the Liver.

(A) Liver cAMP level was measured 15 min after a single 30-mg dose of subcutaneous recombinant asprosin or GFP in mice that had been subjected to a 2-hr fast prior to injection (n = 6 in each group).

(B) Liver PKA activity was measured in mice from (A).

(C) Immunoblot analysis for phosphorylated PKA catalytic subunit or for phosphorylated serine/threonine PKA substrate was performed on liver lysates from mice in (A).

(D) Hepatocyte cAMP level was measured 10 min after incubating mouse primary hepatocytes with 50 nM recombinant asprosin, 1 hr following isolation of cells from WT mice, without plating the cells.

(E) Hepatocyte PKA activity was measured in samples from (D).

(F) Hepatocyte PKA activity was measured upon 2 hr of incubation of mouse primary hepatocytes with 0, 4, 8, 16, 32, 64, 138, 275, 550, or 1,100 nM recombinant asprosin or GFP, 1 hr following isolation of cells from WT mice, without plating the cells.

(G) Media glucose accumulation was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, with or without a G protein inhibitor (Suramin) (5 mM), 1 hr following isolation of cells from WT mice, without plating the cells.

(H) Hepatocyte PKA activity was measured in samples from (G).

(I) Media glucose accumulation was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, with or without a competitive antagonist of cAMP-induced activation of PKA (cAMPS-Rp) (200 μM), 1 hr following isolation of cells from WT mice, without plating the cells.

(J) Media glucose accumulation was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, or 10 μg/ml glucagon, with or without a non-competitive antagonist of the glucagon receptor (L168,049) (1 μM) 1 hr following isolation of cells from WT mice, without plating the cells.

(K) The same analysis was performed as in (J) using 100 μM epinephrine, with or without an antagonist of the β-adrenergic receptor (propranolol) (100 μM). The r GFP and r asprosin controls are common for (J) and (K).

(L) Hepatocyte PKA activity was measured 2 hr after incubating mouse primary hepatocytes with 50 nM recombinant asprosin or GFP, with vehicle or 10 mg/l insulin, 1 hr following isolation of cells from WT mice, without plating the cells.

(M) Hepatocyte glucose production was measured in samples from (L).

Data are represented as the mean ± SEM.