Figure 3.

The effects of APPL1sv on hepatic adiponectin and insulin signaling. A, suppressing APPL1sv expression enhances adiponectin signaling. Primary mouse hepatocytes infected with adenoviruses encoding either scrambled control shRNA, APPL1sv-shRNA, or APPL1-shRNA were serum-starved for 4 h and then treated with (+) or without (−) 1 μg/ml full-length adiponectin (Ad) for 15 min. Phosphorylation of AMPK at Thr¹⁷² as well as the protein levels of AMPK, APPL1, and APPL1sv were detected by Western blot analysis using specific antibodies as indicated. ERK 1/2 was used as a loading control. B, overexpressing APPL1sv inhibits adiponectin signaling. Mouse hepatocytes transfected with plasmids containing control vector, myc-tagged APPL1sv, or myc-tagged APPL1 were serum-starved for 4 h and treated with (+) or without (−) 1 μg/ml full-length adiponectin for 15 min. Phosphorylation of AMPK at Thr¹⁷² as well as the protein levels of AMPK, myc-tagged APPL1, and myc-tagged APPL1sv were detected by Western blot analysis using specific antibodies as indicated. β-Tubulin was used as a loading control. C, the effect of suppressing APPL1sv expression on insulin signaling. Primary mouse hepatocytes infected with adenoviruses encoding either scrambled control shRNA, APPL1sv-shRNA, or APPL1-shRNA were serum-starved for 4 h and treated with (+) or without (−) 10 nm insulin (Ins) for 5 min. Phosphorylation of Akt at Thr³⁰⁸ as well as the protein levels of Akt, APPL1, and APPL1sv were detected by Western blot analysis using specific antibodies as indicated. β-Tubulin was used as a loading control. D, the effect of overexpressing APPL1sv on insulin signaling. Mouse hepatocytes transfected with plasmids containing control vector, myc-tagged APPL1sv, or myc-tagged APPL1 were serum-starved for 4 h and treated with (+) or without (−) 10 nm insulin for 5 min. Phosphorylation of Akt at Thr³⁰⁸ as well as the protein levels of Akt, myc-tagged APPL1, and myc-tagged APPL1sv were detected by Western blot analysis using specific antibodies as indicated. β-Tubulin was used as a loading control. Statistical significance was determined by two-way ANOVA with Tukey's multiple correction tests. Results are presented as ± S.E. from three independent experiments. *, p < 0.05; NS, no significant difference.