Intracellular Trafficking and Secretion of Adiponectin Is Dependent on GGA-coated Vesicles

Silvia Mora,Linglin Xie,Daniel Boyle,Philipp E. Scherer,Jeffrey E. Pessin,Daniel Sanford

doi:10.1074/jbc.m511313200

Abstract

Adiponectin (Acrp30) is an insulin-sensitizing hormone produced and secreted exclusively by adipose tissue. Confocal fluorescent microscopy demonstrated the colocalization of adiponectin with the Golgi membrane markers p115, beta-COP, and the trans-Golgi network marker, syntaxin 6. Treatment of cells with brefeldin A redistributed adiponectin to the endoplasmic reticulum where it colocalized with the chaperone protein BIP and inhibited secretion of adiponectin demonstrating a requirement for a functional Golgi apparatus for adiponectin release. Confocal fluorescent microscopy also demonstrated a colocalization of endogenous adiponectin with that of expressed GGA1myc (Golgi-localizing gamma-adaptin ear homology ARF-binding protein) but with no significant overlap between adiponectin and the GGA2myc or GGA3myc isoforms. Consistent with confocal fluorescent microscopy, transmission electron microscopy demonstrated the colocalization of GGA1 with adiponectin. Although GGA1 did not directly interact with the adiponectin protein, the adiponectin enriched membrane compartments of adipocyte were precipitated by a GST-GGA1 cargo binding domain (VHS) fusion protein but not with a GST-GGA2 VHS or GST-GGA3 VHS fusion proteins. Moreover, co-expression of adiponectin with a GGA1 dominant-interfering mutant (GGA1-VHS GAT domain) resulted in a marked inhibition of adiponectin secretion in both 3T3L1 adipocytes and HEK293 cells, whereas no inhibition was detected with the truncated mutants GGA2-VHSGAT or GGA3-VHSGAT. Moreover, co-expression of wild type GGA1 with adiponectin enhanced secretion of adiponectin. Interestingly, leptin secretion was unaffected by neither the wild type form or GGA1 mutant. Taken together these data demonstrate that the trafficking of adiponectin through its secretory pathway is dependent on GGA-coated vesicles.

Highlights

Adipocytes (4 –7) and functions in vivo as an insulin sensitizer (8 –10), reducing glucose production by the liver [11] and enhancing fatty acid oxidation in skeletal muscle [10] through the activation of two distinct receptor isoforms [12]
Based upon the central role that adiponectin plays in the control of metabolism, energy homeostasis, and insulin sensitivity, we elected to study the secretory membrane trafficking pathways that are responsible for the release of this adipokine from adipocytes
Confocal microscopy studies in 3T3L1 adipocytes showed that adiponectin was mostly distributed to the perinuclear region, with some punctuate staining throughout the cytoplasm

Summary

Introduction

Adipocytes (4 –7) and functions in vivo as an insulin sensitizer (8 –10), reducing glucose production by the liver [11] and enhancing fatty acid oxidation in skeletal muscle [10] through the activation of two distinct receptor isoforms [12]. A dominantinterfering mutant of the GGA1 protein (GGA1 VHS GAT domain) was able to block both traffic of the GLUT4 glucose transporter to its insulin-sensitive intracellular compartment and secretion of adiponectin but did not affect leptin secretion. These data suggest that adiponectin (but not leptin) secretion is dependent on GGA proteins and suggest that GGA adaptors participate in the regulation of selective adipokine trafficking in adipocytes.

Results

Conclusion