APPL2 interacts with Adiponectin receptors. A, protein expression of APPLs and AdipoR1 were detected in mouse brain (B), liver (Li), muscle (M), fat (F), heart (H), kidney (K), spleen (S), and pancreas (P) by Western blot analysis with antibodies specific for APPL1, APPL2, and AdipoR1. 30 μg of protein in tissue homogenates was loaded in each lane. B, APPL2 interacts with adiponectin receptors in vitro. GST or GST-APPL2 fusion protein was incubated with cell lysates of C2C12 myotubes. Endogenous AdipoR1 and AdipoR2 associated with recombinant GST-APPL2, and their protein levels were detected by Western blot analysis with antibodies specific for AdipoR1 and AdipoR2, respectively. The GST fusion proteins were visualized by Coomassie Blue staining (right panel). C, APPL2 interacts with AdipoR1 in C2C12 myotubes. Serum-starved C2C12 myotubes were treated with or without globular adiponectin (Ad, 1 μg/ml, 10 min). Endogenous APPL1 or APPL2 was immunoprecipitated. Co-immunoprecipitated endogenous AdipoR1 and the protein expression levels of AdipoR1, APPL1, and APPL2 in cell lysates were detected by Western blot analysis with the antibodies specific to the proteins as indicated. D, graphic presentation of effect of adiponectin on the interaction between APPLs and AdipoR1 shown in C. Error bars represents mean ± S.E. (n = 3). *, p < 0.05. E, APPL2 interacts with AdipoR1 through its BAR domain. HA-tagged AdipoR1 and FLAG-tagged APPL2 or APPL2 (ΔBAR) mutant were co-expressed in C2C12 myoblasts. APPL2 or APPL2 (ΔBAR) mutants were immunoprecipitated by anti-FLAG antibody. Co-immunoprecipitated AdipoR1 and the protein expression levels of AdipoR1 and APPL2 in cell lysates were detected by anti-HA or anti-FLAG-antibodies as indicated. The results shown in Fig. 1 are representative of three independent experiments with similar findings.

Adiponectin and metformin regulate the subcellular translocation of APPLs. A, translocation of APPL1 in response to adiponectin and metformin stimulation. C2C12 myocytes overexpressing Myc-tagged APPL1 were serum-starved for 6 h and treated with or without globular adiponectin (Ad, 1 μg/ml) for 10 min or metformin (500 μm) for 45 min. The localization of APPL1 was detected by immunofluorescence staining with Myc antibody (green). The cell nuclei were stained with DAPI (blue). The scale bars represent 50 μm. B, translocation of APPL2 in response to adiponectin and metformin stimulation. C2C12 myocytes overexpressing Myc-tagged APPL2 were serum-starved for 6 h and treated with or without globular adiponectin (Ad, 1 μg/ml) for 10 min or metformin (500 μm) for 45 min. The localization of APPL2 was detected by immunofluorescence staining with Myc antibody (green). The cell nuclei were stained with DAPI (blue). The scale bars represent 50 μm. C, graphic representation of percentage of cells with APPLs localizing on membrane as shown in A and B. Error bars represent mean ± S.E. (n = 3). * indicates p < 0.05.

Biological functions of APPL2 in muscle cells. A, suppression of APPL2 expression enhances adiponectin-stimulated glucose uptake in C2C12 myotubes. Scramble control or APPL2-suppressed C2C12 myotubes were serum-starved for 4 h and treated with or without globular adiponectin (Ad, 1 μg/ml) for 1 h. Glucose uptake was measured as described under “Experimental Procedures.” The graph represents the fold of glucose uptake increase. * indicates p < 0.05 (n = 6). B, suppression of APPL2 expression enhances adiponectin-stimulated fatty acid oxidation in C2C12 myotubes. Scramble control or APPL2-suppressed C2C12 myotubes were serum-starved for 2 h and treated with or without globular adiponectin (Ad, 1 μg/ml) for 1.5 h. Fatty acid oxidation was measured as described under “Experimental Procedures.” The graph represents the fold of increased fatty oxidation. * indicates p < 0.05 (n = 6).

APPL isoforms play Yin-Yang regulatory roles in adiponectin signaling and adiponectin-insulin cross-talk. A, roles of APPL isoforms in adiponectin signaling. Under the basal condition, APPL2 occupies the N terminus of AdipoR1 and sequesters APPL1 in cytosol by forming an APPL1-APPL2 heterodimer, thus inhibits adiponectin signaling. Upon adiponectin stimulation, APPL2 dissociates from AdipoR1 and releases APPL1, facilitating the recruitment of APPL1 onto AdipoR1, which is a critical step for adiponectin signaling. B, role of APPL isoforms in the cross-talk between adiponectin and insulin pathways. Under the basal condition, APPL2 sequesters APPL1 and prevents the interaction of APPL1 with the components in insulin signaling. Upon adiponectin stimulation, APPL2 releases APPL1, facilitating the interaction of APPL1 with components in insulin signaling. Meanwhile, AMPK is activated, which in turn prevents the inhibitory effect of S6K on insulin signaling.

APPL2 inhibits adiponectin signaling in C2C12 myocytes. A, overexpression of APPL2 inhibits adiponectin-induced AMPK activation. C2C12 myoblasts overexpressing Myc-tagged APPL1, APPL2, or APPL2 (BAR) were serum-starved for 4 h and treated with or without 1 μg/ml globular adiponectin (Ad) for 20 min. Phosphorylation of AMPK at Thr-172 and ACC at Ser-79 as well as their protein levels were detected by Western blot analysis using specific antibodies as indicated. B, overexpression of APPL2 inhibits adiponectin-induced p38 MAPK activation. C2C12 myoblasts overexpressing Myc-tagged APPL1, APPL2, or APPL2 (BAR) were serum-starved for 4 h and treated with or without 1 μg/ml globular adiponectin (Ad) for 10 min. Phosphorylation of p38 MAPK at Thr-180 and Tyr-182 and the total p38 MAPK protein were detected by Western blot analysis using specific antibodies as indicated. C, suppression of APPL2 expression enhances AMPK activity. Scramble control or APPL2-suppressed C2C12 myotubes were serum-starved for 6 h and treated with or without 1 μg/ml globular adiponectin (Ad) for 20 min. Phosphorylation of AMPK at Thr-172 as well as the total AMPK protein was detected by Western blot analysis using specific antibodies as indicated. Equal loading of protein was determined by Western blot analysis using anti-β-tubulin antibody. D, suppression of APPL2 expression enhances p38 MAPK activity. Scramble control or APPL2 suppressed C2C12 myotubes were serum-starved for 6 h and treated with or without 1 μg/ml globular adiponectin (Ad) for 10 min. Phosphorylation of p38 MAPK at Thr-180 and Tyr-182 as well as the total p38 MAPK protein were detected by Western blot analysis using specific antibodies as indicated. Equal loading of protein was determined by Western blot analysis using anti-β-tubulin antibody. The results shown in Fig. 2 are representative of three independent experiments with similar findings.

APPL2 forms heterodimer with APPL1 and interrupts the APPL1-AdipoR1 interaction. A, adiponectin induces dissociation of APPL1-APPL2 heterodimer. C2C12 myoblast overexpressing Myc-tagged APPL1 and HA-tagged APPL2 were serum-starved for 6 h and treated with or without globular adiponectin (Ad, 1 μg/ml) for the indicated time. APPL2 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitated APPL1 as well as the protein expression levels of APPL2 and APPL1 in cell lysates were detected by Western blot analysis with anti-HA or anti-Myc antibody as indicated. B, metformin treatment leads to dissociation of APPL1-APPL2 heterodimer. C2C12 myoblast-overexpressing Myc-tagged APPL1 and HA-tagged APPL2 were serum-starved for 6 h and treated with or without metformin (500 μm) for the indicated times. APPL2 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitated APPL1 as well as the protein expression levels of APPL2 and APPL1 in cell lysates were detected by Western blot analysis with anti-HA or anti-Myc antibody as indicated. C, suppression of APPL2 expression enhances APPL1-AdipoR1 interaction. Scramble control or APPL2-suppressed C2C12 myotubes were serum-starved for 6 h and treated with or without globular adiponectin (Ad, 1 μg/ml) for 20 min. Endogenous APPL1 was immunoprecipitated with anti-APPL1 antibody. Co-immunoprecipitated AdipoR1 as well as the protein expression levels of AdipoR1, APPL1, and APPL2 in cells were detected by Western blot analysis with the antibodies specific to these proteins as indicated. D, overexpression of APPL2 interrupts APPL1-AdipoR1 interaction. C2C12 myocytes overexpressing HA-tagged AdipoR1, Myc-tagged APPL1, and different doses of HA-tagged APPL2 were serum-starved for 6 h and treated with or without globular adiponectin (Ad, 1 μg/ml) for 10 min. APPL1 was immunoprecipitated with anti-Myc antibody. Co-immunoprecipitated AdipoR1 as well as the protein expression levels of AdipoR1, APPL1, and APPL2 in cell lysates were detected by Western blot analysis with anti-Myc or anti-HA antibody as indicated. E, effect of APPL2 expression suppression on the translocation of APPL1 in response to adiponectin stimulation. Scramble control or APPL2-suppressed C2C12 myocytes overexpressing Myc-tagged APPL1 were serum-starved for 6 h and treated with or without globular adiponectin (Ad, 1 μg/ml) for 10 min. The localization of APPL1 was detected by immunofluorescence staining with Myc antibody (green). The cell nuclei were stained with DAPI (blue). The scale bars represent 20 μm. F, graphic representation of percentage of cells with APPL1 localizing on membrane as shown in E. Error bars represent mean ± S.E. (n = 3). * indicates p < 0.05. The results shown in Fig. 4 are representative of three independent experiments with similar findings.

APPL2 blocks the insulin sensitization effects of adiponectin. A, overexpression of APPL2 inhibits the insulin sensitization effect of adiponectin. C2C12 myoblasts overexpressing FLAG-tagged full-length or BAR domain truncated form of APPL2 were serum-starved for 6 h and pretreated with or without globular adiponectin (Ad, 1 μg/ml) for 10 min followed with insulin (Ins, 100 nm) for 5 min. Phosphorylation of Akt (T308) as well as the protein levels of Akt and FLAG-tagged APPL2 was detected by Western blot analysis with specific antibodies as indicated. B, suppression of APPL2 expression enhances the insulin sensitization effect of adiponectin. Scramble control and APPL2-suppressed C2C12 myotubes were serum-starved for 16 h and pretreated with or without globular adiponectin (Ad, 1 μg/ml) for 15 min followed with insulin (Ins, 10 nm) for 5 min. Phosphorylation of Akt (Thr308) as well as the protein levels of Akt, APPL2, and β-tubulin were detected by Western blot analysis with specific antibodies as indicated. These results are representative of three independent experiments with similar findings.