Fig. 2
Glucogenic and orexigenic activities of gonacin in zebrafish (A) Characterization of recombinant zebrafish gonacin protein (rGonacin). Coomassie blue staining (left panel) and immunoblotting (right panel) of bacteria-derived recombinant gonacin. M1, M2: marker proteins, R: rGonacin. (B and C) Serum glucose was measured at indicated times after a single 4 μg dose of intraperitoneal injection of rGonacin or recombinant GFP protein (rGFP, using as the control) into zebrafish that had been subjected to a 72 h fast (B) and 24 h fast (C) before injection. ∗, p < 0.05; ∗∗∗∗, p < 0.0001, compared with control (n = 8 in each group). two-way ANOVA with Bonferroni post-test was used to calculate the p value. (D) Serum insulin levels were measured at 1 h after a single dose of intraperitoneal injection of rGonacin (4 μg) or rGFP in zebrafish (n = 3 in each group). ∗, p < 0.05; Unpaired Student’s t test was used to calculate the p value. (E) Liver glucose content was measured by ELISA at 1 h after a single intraperitoneal injection of rGonacin (4 μg) or rGFP (control) into zebrafish that had been subjected to a 72 h fast (left panel) and 24 h fast (right panel) prior to injection (n = 6 in each group). ∗, p < 0.05, compared with control. Unpaired Student’s t test was used to calculate the p value. (F) The expression of glucogenesis enzymes including fbp1a, pck1, pcxa, and pcxb in the liver after a single intraperitoneal injection of rGonacin (4 μg) or rGFP (control) in vivo. The columns represent fold changes of the relative mRNA levels in the treated group over its respective GFP injection controls (mean ± SEM) ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; n = 5. Unpaired Student’s t test was used to calculate the p value. (G) Food intake was measured at indicated times after a single 4 μg dose of intraperitoneal injection of rGonacin (4 μg) or GFP (control), 4 μL anti-gonacin antibodies (Antibody) or 4 μg rGonacin together with 4 μL antibody (rGonacin+Antibody) in zebrafish that had been subjected to an 18 h fast before injection (n = 6 in each group). (H) Body weight was measured at indicated times after 4 times of intraperitoneal injections of recombinant gonacin or GFP (control) at 3 days intervals in zebrafish. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. two-way ANOVA with Sidak’s multiple comparisons test was used to calculate the p value |