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APPL1 potentiates insulin sensitivity by facilitating the binding of IRS1/2 to the insulin receptor.

Ryu J, Galan AK, Xin X, Dong F, Abdul-Ghani MA, Zhou L, Wang C, Li C, Holmes BM, Sloane LB, Austad SN, Guo S, Musi N, DeFronzo RA, Deng C, White MF, Liu F, Dong LQ - Cell Rep (2014)

Bottom Line: However, the mechanism of IRS1/2 recruitment to the IR remains elusive.Here, we identify adaptor protein APPL1 as a critical molecule that promotes IRS1/2-IR interaction.The interaction between APPL1 and IR depends on insulin- or adiponectin-stimulated APPL1 phosphorylation, which is greatly reduced in insulin target tissues in obese mice. appl1 deletion in mice consistently leads to systemic insulin resistance and a significant reduction in insulin-stimulated IRS1/2, but not IR, tyrosine phosphorylation, indicating that APPL1 sensitizes insulin signaling by acting at a site downstream of the IR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.

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Ser401 Phosphorylation Is Essential for the Promoting Effect of APPL1 on Insulin Signaling(A) C2C12 myotubes were serum starved and treated with insulin (10 nM) for the indicated times. Phosphorylation of APPL1 and Akt were detected by western blot analysis with the specific antibodies indicated.(B) C57BL/6 mice were fasted and injected with saline or insulin (0.5 U/kg of body weight, 3 min). The phosphorylation of APPL1 and Akt and their protein expression in muscle tissues were detected by western blot analysis. n = 9 per group.(C) Normal chow (NC) and high-fat diet (HFD)-fed male C57BL/6 mice were fasted overnight and treated with or without insulin (0.5 U/kg of body weight, 3 min). The phosphorylation of APPL1 and APPL1 protein in muscle tissues was detected by western blot analysis. Phosphorylation of IRβ, IRS1, and IRS2 was detected after immunoprecipitation with indicated antibodies, respectively. Bar graphs represent the ratios of insulin-stimulated phosphorylation over their total protein levels. n = 4 per group.(D) APPL1-suppressed C2C12 myoblasts were transfected with myc-tagged and RNAi-resistant wild-type (WT), S401A, or S401D mutants of APPL1. The cells were serum starved and treated with or without 100 nM insulin (3 min). WT and mutants of APPL1 were immunoprecipitated with an anti-myc antibody. Bar graphs represent the binding of IRβ, IRS1, or IRS2 to APPL1 protein. Data are mean ± SEM from three independent experiments.All results are presented as mean ± SEM. p values were calculated using the Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figure S4.
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Figure 6: Ser401 Phosphorylation Is Essential for the Promoting Effect of APPL1 on Insulin Signaling(A) C2C12 myotubes were serum starved and treated with insulin (10 nM) for the indicated times. Phosphorylation of APPL1 and Akt were detected by western blot analysis with the specific antibodies indicated.(B) C57BL/6 mice were fasted and injected with saline or insulin (0.5 U/kg of body weight, 3 min). The phosphorylation of APPL1 and Akt and their protein expression in muscle tissues were detected by western blot analysis. n = 9 per group.(C) Normal chow (NC) and high-fat diet (HFD)-fed male C57BL/6 mice were fasted overnight and treated with or without insulin (0.5 U/kg of body weight, 3 min). The phosphorylation of APPL1 and APPL1 protein in muscle tissues was detected by western blot analysis. Phosphorylation of IRβ, IRS1, and IRS2 was detected after immunoprecipitation with indicated antibodies, respectively. Bar graphs represent the ratios of insulin-stimulated phosphorylation over their total protein levels. n = 4 per group.(D) APPL1-suppressed C2C12 myoblasts were transfected with myc-tagged and RNAi-resistant wild-type (WT), S401A, or S401D mutants of APPL1. The cells were serum starved and treated with or without 100 nM insulin (3 min). WT and mutants of APPL1 were immunoprecipitated with an anti-myc antibody. Bar graphs represent the binding of IRβ, IRS1, or IRS2 to APPL1 protein. Data are mean ± SEM from three independent experiments.All results are presented as mean ± SEM. p values were calculated using the Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figure S4.

Mentions: APPL1 contains several potential phosphorylation sites including Ser151, Ser401, Ser427, and Ser430 (Gant-Branum et al., 2010) that may be subjected to insulin- or adiponectin-stimulated phosphorylation. Among these potential phosphorylation sites, Ser401 is highly conserved in APPL1 among different species, and this residue is absent in the corresponding location of its isoform APPL2 (Figure S4A); the latter does not interact with the IR (data not shown). To determine the potential roles of APPL1 phosphorylation, we generated a phosphospecific antibody to Ser401 of APPL1 (Figure S4B). By western blot analysis using this antibody, we found that APPL1 phosphorylation at Ser401 is rapidly stimulated by insulin in C2C12 cells (Figure 6A) and in mouse skeletal muscle tissues (Figure 6B). The insulin-stimulated APPL1 Ser401 phosphorylation was significantly reduced in insulin target tissues of mice fed a high-fat diet compared to mice fed with normal chow, which was associated with an impaired PI3K signaling pathway (Figures 6C, S4C, and S4D). Together, these results indicate a correlation between APPL1 phosphorylation at Ser401 and insulin sensitivity in vivo.


APPL1 potentiates insulin sensitivity by facilitating the binding of IRS1/2 to the insulin receptor.

Ryu J, Galan AK, Xin X, Dong F, Abdul-Ghani MA, Zhou L, Wang C, Li C, Holmes BM, Sloane LB, Austad SN, Guo S, Musi N, DeFronzo RA, Deng C, White MF, Liu F, Dong LQ - Cell Rep (2014)

Ser401 Phosphorylation Is Essential for the Promoting Effect of APPL1 on Insulin Signaling(A) C2C12 myotubes were serum starved and treated with insulin (10 nM) for the indicated times. Phosphorylation of APPL1 and Akt were detected by western blot analysis with the specific antibodies indicated.(B) C57BL/6 mice were fasted and injected with saline or insulin (0.5 U/kg of body weight, 3 min). The phosphorylation of APPL1 and Akt and their protein expression in muscle tissues were detected by western blot analysis. n = 9 per group.(C) Normal chow (NC) and high-fat diet (HFD)-fed male C57BL/6 mice were fasted overnight and treated with or without insulin (0.5 U/kg of body weight, 3 min). The phosphorylation of APPL1 and APPL1 protein in muscle tissues was detected by western blot analysis. Phosphorylation of IRβ, IRS1, and IRS2 was detected after immunoprecipitation with indicated antibodies, respectively. Bar graphs represent the ratios of insulin-stimulated phosphorylation over their total protein levels. n = 4 per group.(D) APPL1-suppressed C2C12 myoblasts were transfected with myc-tagged and RNAi-resistant wild-type (WT), S401A, or S401D mutants of APPL1. The cells were serum starved and treated with or without 100 nM insulin (3 min). WT and mutants of APPL1 were immunoprecipitated with an anti-myc antibody. Bar graphs represent the binding of IRβ, IRS1, or IRS2 to APPL1 protein. Data are mean ± SEM from three independent experiments.All results are presented as mean ± SEM. p values were calculated using the Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figure S4.
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Figure 6: Ser401 Phosphorylation Is Essential for the Promoting Effect of APPL1 on Insulin Signaling(A) C2C12 myotubes were serum starved and treated with insulin (10 nM) for the indicated times. Phosphorylation of APPL1 and Akt were detected by western blot analysis with the specific antibodies indicated.(B) C57BL/6 mice were fasted and injected with saline or insulin (0.5 U/kg of body weight, 3 min). The phosphorylation of APPL1 and Akt and their protein expression in muscle tissues were detected by western blot analysis. n = 9 per group.(C) Normal chow (NC) and high-fat diet (HFD)-fed male C57BL/6 mice were fasted overnight and treated with or without insulin (0.5 U/kg of body weight, 3 min). The phosphorylation of APPL1 and APPL1 protein in muscle tissues was detected by western blot analysis. Phosphorylation of IRβ, IRS1, and IRS2 was detected after immunoprecipitation with indicated antibodies, respectively. Bar graphs represent the ratios of insulin-stimulated phosphorylation over their total protein levels. n = 4 per group.(D) APPL1-suppressed C2C12 myoblasts were transfected with myc-tagged and RNAi-resistant wild-type (WT), S401A, or S401D mutants of APPL1. The cells were serum starved and treated with or without 100 nM insulin (3 min). WT and mutants of APPL1 were immunoprecipitated with an anti-myc antibody. Bar graphs represent the binding of IRβ, IRS1, or IRS2 to APPL1 protein. Data are mean ± SEM from three independent experiments.All results are presented as mean ± SEM. p values were calculated using the Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figure S4.
Mentions: APPL1 contains several potential phosphorylation sites including Ser151, Ser401, Ser427, and Ser430 (Gant-Branum et al., 2010) that may be subjected to insulin- or adiponectin-stimulated phosphorylation. Among these potential phosphorylation sites, Ser401 is highly conserved in APPL1 among different species, and this residue is absent in the corresponding location of its isoform APPL2 (Figure S4A); the latter does not interact with the IR (data not shown). To determine the potential roles of APPL1 phosphorylation, we generated a phosphospecific antibody to Ser401 of APPL1 (Figure S4B). By western blot analysis using this antibody, we found that APPL1 phosphorylation at Ser401 is rapidly stimulated by insulin in C2C12 cells (Figure 6A) and in mouse skeletal muscle tissues (Figure 6B). The insulin-stimulated APPL1 Ser401 phosphorylation was significantly reduced in insulin target tissues of mice fed a high-fat diet compared to mice fed with normal chow, which was associated with an impaired PI3K signaling pathway (Figures 6C, S4C, and S4D). Together, these results indicate a correlation between APPL1 phosphorylation at Ser401 and insulin sensitivity in vivo.

Bottom Line: However, the mechanism of IRS1/2 recruitment to the IR remains elusive.Here, we identify adaptor protein APPL1 as a critical molecule that promotes IRS1/2-IR interaction.The interaction between APPL1 and IR depends on insulin- or adiponectin-stimulated APPL1 phosphorylation, which is greatly reduced in insulin target tissues in obese mice. appl1 deletion in mice consistently leads to systemic insulin resistance and a significant reduction in insulin-stimulated IRS1/2, but not IR, tyrosine phosphorylation, indicating that APPL1 sensitizes insulin signaling by acting at a site downstream of the IR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.

Show MeSH
Related in: MedlinePlus