(448d) Alterations in Endothelial Barrier Function Differentially Regulate the Transport of Adiponectin Oligomers

Introduction: Inversely expressed with obesity, the adipokine adiponectin has positive overall insulin-sensitizing and metabolic effects via signaling in various tissues such as liver and skeletal muscle. Adiponectin is unique in its post-translational modifications that form oligomers from the 30kDa monomer up to 540kDa 18-mers found in circulation. These oligomers possess differential efficacy on insulin sensitivity with the high molecular weight (HMW) oligomers, or HMW:Trimer ratio, correlating best with metabolic health. Due to potential transport limitations of HMW adiponectin, we propose that the endothelium presents a transport barrier to adiponectin in a tissue-specific manner, that endothelial barriers are altered with metabolic health, and that endothelial cell function modulates adiponectin’s positive effects.

Materials and Methods: Full-form murine adiponectin was produced from stably transfected HEK293T cells and purified by ion exchange; mammalian cells were necessary for HMW formation. HMW, low molecular weight (LMW), and trimeric adiponectin were purified by gel filtration FPLC. Stokes’ radii and diffusion rates were estimated by dynamic light scattering for each fraction. Murine endothelial cell lines were used in transwell permeability assays to determine the relative transport rates of adiponectin across different monolayers in vitro. IR800 dye-labeled individual fractions were labeled and injected intravenously into several mouse models to assess the role of endothelial barrier function in vivo. To quantify adiponectin circulatory fates with changes in endothelium, mice were analyzed in different metabolic states or treated with isosorbide dinitrate and a PPARγ agonist. Transgenic mice lacking endothelial caveolar transport (Cav1 null) were also used. Serum collected at various time intervals allowed quantification of adiponectin circulatory half-life.

Results and Discussion: Adiponectin oligomers possess Stokes’ radii of approximately 5, 10, and 25nm making physiologic transport phenomena non-trivial events. Ms1 and b.End3 murine cells form tight cell-cell junctions that limit adiponectin transport and permit little passage of the HMW form. The leaky contacts of EOMA cells, however, permit equivalent oligomer transport. Commercially available, bacterially-produced globular adiponectin – smaller than the mammalian trimer – was transported equivalently across all cells highlighting the physiologic relevance of our preparation. Adiponectin clearance from circulation was oligomer size dependent and altered with endothelial dysfunction. In obesity, adiponectin clearance from circulation is reduced, but can be increased with fasting. Acute isosorbide dinitrate treatment increased HMW adiponectin flux with no effect on LMW clearance. Chronic treatment with a PPARγ agonist or caveolin-1 null mice slowed HMW clearance, but hastened LMW uptake. Endothelial dysfunction and the resultant active and passive changes to transendothelial fluxes reduced tissue access to metabolically positive HMW adiponectin.

Conclusions: Access of adiponectin oligomers into tissues of metabolic relevance may be dependent on the endothelial barrier within each tissue. Adiponectin bioavailability for target cells under metabolically challenged conditions is reduced due to reduced transendothelial transport that, in turn, reduces tissue insulin sensitivity. Adiponectin transport into and across these cells modulates the link between adipose tissue health and cardiovascular disease.

Acknowledgement: JMR was supported in part by NIH F32-DK085935