Abstract
Adiponectin is a multifunctional adipokine that circulates as several oligomeric complexes in the blood stream. However, the molecular basis that regulates the production of the adiponectin oligomers remains largely elusive. We have shown previously that several conserved lysine residues (positions 68, 71, 80, and 104) within the collagenous domain of adiponectin are modified by hydroxylation and glycosylation (Wang, Y., Xu, A., Knight, C., Xu, L. Y., and Cooper, G. J. (2002) J. Biol. Chem. 277, 19521-19529). Here, we investigated the potential roles of these post-translational modifications in oligomeric complex formation of adiponectin. Gel filtration chromatography revealed that adiponectin produced from mammalian cells formed trimeric, hexameric, and high molecular weight (HMW) oligomeric complexes. These three oligomeric forms were differentially glycosylated, with the HMW oligomer having the highest carbohydrate content. Disruption of hydroxylation and glycosylation by substitution of the four conserved lysines with arginines selectively abrogated the intracellular assembly of the HMW oligomers in vitro as well as in vivo. In type 2 diabetic patients, both the ratios of HMW to total adiponectin and the degree of adiponectin glycosylation were significantly decreased compared with healthy controls. Functional studies of adiponectin-null mice revealed that abrogation of lysine hydroxylation/glycosylation markedly decreased the ability of adiponectin to stimulate phosphorylation of AMP-activated protein kinase in liver tissue. Chronic treatment of db/db diabetic mice with wild-type adiponectin alleviated hyperglycemia, hypertriglyceridemia, hepatic steatosis, and insulin resistance, whereas full-length adiponectin without proper post-translational modifications and HMW oligomers showed substantially decreased activities. Taken together, these data suggest that hydroxylation and glycosylation of the lysine residues within the collagenous domain of adiponectin are critically involved in regulating the formation of its HMW oligomeric complex and consequently contribute to the insulin-sensitizing activity of adiponectin in hepatocytes.
Highlights
In addition to its metabolic effects, adiponectin possesses antiatherogenic, anti-inflammatory, and hepatoprotective functions
Adiponectin derived from Escherichia coli, which is not glycosylated, formed only low molecular weight (LMW) and middle molecular weight (MMW) oligomers, but lacked the capacity to further assemble into the high molecular weight (HMW) oligomeric complexes
The carbohydrate contents of the HMW adiponectin were significantly higher than those of the MMW and LMW oligomeric complexes. These results suggest that the post-translational modifications might be important for the production of the HMW oligomeric complex of adiponectin
Summary
T2DM, type 2 diabetes mellitus; AMPK, AMP-activated protein kinase; LMW, low molecular weight; MMW, middle molecular weight; HMW, high molecular weight; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzymelinked immunosorbent assay; TG, triglyceride; ADN-K/R, adiponectin with the four lysines mutated to arginines. Weight reduction or treatment with the insulin-sensitizing drug rosiglitazone preferentially elevates the HMW form of adiponectin without significant effects on the other two oligomeric complexes [30, 37]. Two rare missense mutations (G84R and G90S) identified in T2DM patients can form trimers and hexamers, but lack the capacity to form the HMW complex [36]. These data collectively suggest that impaired formation of the HMW adiponectin might be causally associated with insulin resistance and T2DM. We investigated the potential roles of these post-translational modifications in regulating the oligomeric complex formation of adiponectin in cell culture systems, animal models, and human subjects
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