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

Meilian Liu,Fresnida J. Ramos,Hak Joo Lee,Xiaoban Xin,Feng Liu,Hongzhi Chen,Xuming Mao,Lily Q. Dong,Chintan K. Kikani,Ruihua Xiang,Changhua Wang

doi:10.1074/jbc.m109.010355

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 Yin-Yang regulator of adiponectin signaling and mediate the cross-talk between adiponectin and insulin signaling pathways in muscle cells.

Highlights

We have recently identified APPL1 as a signaling protein immediately downstream of adiponectin receptors and positively mediates adiponectin signaling in muscle cells [6]
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]
We show that APPL2 negatively regulates adiponectin signaling in muscle cells (Fig. 7A)

Summary

EXPERIMENTAL PROCEDURES

Adiponectin, and Antibodies—The cDNAs of fulllength and truncations of human APPL1 and mouse APPL2 were generated by PCR and subcloned into the mammalian expression vectors pcDNA3.1 (Myc-tagged), pBEX (HAtagged), pFLAG-CMV2 (FLAG-tagged), or pCMV-3Tag-2 (Myc-tagged), respectively. AdipoR1, APPL1, and APPL2 in cell lysates were detected by Western blot analysis with the antibodies specific to the proteins as indicated. 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. 20 mM ␤-glycerophosphate, 10 mM sodium fluoride, 2 mM Myocytes—The findings that APPL1 and APPL2 interact with sodium orthovanadate, 2 mM EDTA, 1.0% Igepal (a nonionic, AdipoR1 via different mechanisms suggest that the two isonondenaturing detergent), 10% glycerol, 2 mM phenylmethyl- forms may exert distinct functions in mediating adiponectin sulfonyl fluoride, 1 mM magnesium chloride, 1 mM calcium signaling To test this hypothesis, we examined the effects of chloride, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin.

RESULTS

DISCUSSION