Yin-Yang Regulation of Adiponectin Signaling by APPL Isoforms in Muscle Cells

Abstract

APPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells.Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 expression by RNAi significantly enhances adiponectin-stimulated glucose uptake and fatty acid oxidation. In addition to targeting directly to and competing with APPL1 in binding with the adiponectin receptors, APPL2 also suppresses adiponectin and insulin signaling by sequestrating APPL1 from these two pathways. In addition to adiponectin, metformin also induces APPL1-APPL2 dissociation. Taken together, our results reveal that APPL isoforms function as an integrated YinYang regulator of adiponectin signaling and mediate the crosstalk between adiponectin and insulin signaling pathways in muscle cells.Adiponectin, an adipocyte-secreted hormone that regulates energy homeostasis and insulin sensitivity, has been shown to be a promising therapeutic drug target for the treatment of type 2 diabetes (1-3). Adiponectin binds to its membrane receptors (AdipoR1 and AdipoR2) 3 and regulates lipid and glucose metabolism by activating downstream signaling molecules, such as AMP-activated protein kinase (AMPK), p38 MAP kinase (MAPK), and PPAR␣, in the muscle and liver (1, 4). Activation of AMPK by adiponectin reduces S6 kinase-mediated IRS-1 serine phosphorylation and increases IRS-1 tyrosine phosphorylation thus sensitizes insulin signaling in C2C12 myotubes (5), suggesting a direct cross-talk between the adiponectin and insulin signaling pathways.We have recently identified APPL1 (adaptor protein-containing PH domain, PTB domain, and leucine zipper motif) as a signaling protein immediately downstream of adiponectin receptors and positively mediates adiponectin signaling in muscle cells (6). This adaptor protein was previously shown to interact with the catalytic subunit of PI 3-kinase (p110) and Akt, which are two key kinases in the PI 3-kinase pathway downstream of the insulin receptor (7). The interaction between APPL1 and Akt is required for insulin-stimulated GLUT4 translocation (8) and for controlling Akt substrate selectivity (9). It has been shown that APPL1-potentiated Akt activity to suppress androgen receptor transactivation in prostate cancer cells (10). APPL1 has also been suggested to function as an adaptor protein in regulating follicle-stimulated hormone (FSH)-mediated PI 3-kinase/ Akt signaling pathway (11, 12). Our results showed that APPL1 binds directly to the intracellular part of the adiponectin receptors and positively mediates adiponectin signaling to the AMPK and p38 MAPK pathways, leading to increased glucose uptake and fatty acid oxidation in muscle cells (6). In addition, we found that APPL1 plays a critical ro...

RNA-Binding Protein PTB and MicroRNA-221 Coregulate AdipoR1 Translation and Adiponectin Signaling

“…Indeed, it was shown that the two adiponectin receptors, AdipoR1 and AdipoR2, are capable of homoand hetero-dimerization, and changes in their cellular ratio/amounts lead to alterations in AMPK phosphorylation (44). Activation of adiponectin signaling was also observed in C2C12 myotubes under depletion of adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2), an AdipoR1-binding protein that acts as a negative regulator of adiponectin signaling (45). This study argued that under basal conditions, APPL2 interacts with AdipoR1 and inhibits downstream phosphorylation of AMPK and other signaling molecules activated by AdipoR1, while adiponectin treatment stimulates APPL2 disassociation from AdipoR1, thus leading to its activation.…”

Globular adiponectin induces LKB1/AMPK-dependent glucose uptake via actin cytoskeleton remodeling

“…Indeed, the regulation of glucose transport by adiponectin in skeletal muscle is dependent on the interaction of APPL1 with AdipoR (Mao et al 2006, 2009a, Zhou et al 2009, Xin et al 2011, leading to the subsequent activation of AMPK (Tomas et al 2002, Yamauchi et al 2002, Tsao et al 2003, Ceddia et al 2005, Mao et al 2006, Zhou et al 2009). Adiponectin-induced phosphorylation and activation of AMPK are primarily mediated through the cytosolic translocation of LKB1, which is mediated by PKC-z (Deepa et al 2011), and the interaction of LKB1 with APPL1, which localizes it to the cytosol (Zhou et al 2009, Fang et al 2010.…”

Wogonin enhances intracellular adiponectin levels and suppresses adiponectin secretion in 3T3-L1 adipocytes

“…Western blot was performed as our previously described protocol [6,7,28,29]. Briefly, 3T3-L1 adipocytes were lysed in lysis buffer containing 50 mmol/L HEPES, pH 7.6, 150 mmol/L NaCl, 1% Triton X-100, 10 mmol/L NaF, 20 mmol/L sodium pyrophosphate, 20 mmol/L β-glycerol phosphate, 1 mmol/L sodium orthovanadate, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mmol/L phenylmethanesulfonyl fluoride.…”

“…tors AdipoR1 or/and AdipoR2, adiponectin stimulates downstream events including activation of AMPK and p38 MAPK leading to an increase of glucose uptake and fat oxidation in muscle, and a reduction of fatty acid uptake and hepatic gluconeogenesis in liver [3][4][5][6][7][8]. Decreased adiponectin levels have been implicated with insulin-resistant states such as obesity and type 2 diabetes in both humans and animal models [1][2][3][4].…”