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. 2010 Jan;24(1):229-39.
doi: 10.1210/me.2009-0133. Epub 2009 Oct 23.

Sialic acid modification of adiponectin is not required for multimerization or secretion but determines half-life in circulation

Ayanthi A Richards  1 , Michelle L Colgrave, Jialiang Zhang, Julie Webster, Fiona Simpson, Elaine Preston, Donna Wilks, Kyle L Hoehn, Matthew Stephenson, Graeme A Macdonald, John B Prins, Gregory J Cooney, Aimin Xu, Jonathan P Whitehead
Affiliations

Sialic acid modification of adiponectin is not required for multimerization or secretion but determines half-life in circulation

Ayanthi A Richards et al. Mol Endocrinol. 2010 Jan.

Abstract

Adiponectin is an adipocyte-secreted, insulin-sensitizing hormone the circulating levels of which are reduced in conditions of insulin resistance and diabetes. Previous work has demonstrated the importance of posttranslational modifications, such as proline hydroxylation and lysine hydroxylation/glycosylation, in adiponectin oligomerization, secretion, and function. Here we describe the first functional characterization of adiponectin sialylation. Using a variety of biochemical approaches we demonstrated that sialylation occurs on previously unidentified O-linked glycans on Thr residues of the variable domain in human adiponectin. Enzymatic removal of sialic acid or its underlying O-linked sugars did not affect adiponectin multimer composition. Expression of mutant forms of adiponectin (lacking the modified Thr residues) or of wild-type adiponectin in cells defective in sialylation did not compromise multimer formation or secretion, arguing against a structural role for this modification. Activity of desialylated adiponectin was comparable to control adiponectin in L6 myotubes and acute assays in adiponectin(-/-) mice. In contrast, plasma clearance of desialylated adiponectin was accelerated compared with that of control adiponectin, implicating a role for this modification in determining the half-life of circulating adiponectin. Uptake of desialylated adiponectin by isolated primary rat hepatocytes was also accelerated, suggesting a role for the hepatic asialoglycoprotein receptor. Finally, after chronic administration in adiponectin(-/-) mice steady-state levels of desialylated adiponectin were lower than control adiponectin and failed to recapitulate the improvements in glucose and insulin tolerance tests observed with control adiponectin. These data suggest an important role for sialic acid content in the regulation of circulating adiponectin levels and highlight the importance of understanding mechanisms regulating adiponectin sialylation/desialylation.

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Figures

Fig. 1.
Fig. 1.
Detection of adiponectin sialylation using MAL II and 2DE. Conditioned serum-free medium from SGBS human adipocytes and HEK cells stably expressing human adiponectin was subjected to neuraminidase treatment (Neu) before analysis by (A) reducing, denaturing SDS-PAGE and dual immunoblotting with anti-adiponectin (ADN, green) and the MAL II lectin (red) or (B) 2DE followed by immunoblotting for adiponectin. Blots are representative of at least three independent experiments.
Fig. 2.
Fig. 2.
Characterization of adiponectin sialylation. A, WT adiponectin and a mutant (K1-5R; lacking all five glycosylated lysine residues in the collagenous and variable domains) were subjected to neuraminidase treatment before analysis by reducing, denaturing SDS-PAGE, and Western blotting. B, WT adiponectin was either untreated, treated with neuraminidase (Neu), treated with neuraminidase followed by O-glycosidase (O-Glyc), or treated with O-glycosidase alone as indicated before analysis by SDS-PAGE and Western blotting. C, SGBS adiponectin was either untreated or treated with neuraminidase alone (Neu) or with neuraminidase followed by O-glycosidase (O-Glyc) before analysis by SDS-PAGE under nonreducing, non-heat-denaturing conditions and subsequent Western blotting. D, WT adiponectin was treated as above and analyzed by velocity sedimentation on 5–20% sucrose gradients. E, HEK-secreted adiponectin and human serum adiponectin were fractionated on 5–20% sucrose gradients to obtain LMW and HMW multimer fractions and analyzed by 2DE analysis and Western blotting. Data are representative of two to three independent experiments. H, Hexamers; T, trimers.
Fig. 3.
Fig. 3.
Sialic acid modification is not required for multimer formation or secretion of human or mouse adiponectin. A, WT and Lec2 CHO cells were transiently transfected with human (Hu) or mouse (Mo) adiponectin, and secreted adiponectin was analyzed by SDS-PAGE and Western blotting. B, Velocity sedimentation analysis of human adiponectin secreted from WT and Lec2 CHO. C, 2DE of WT and Lec2 samples. Data are representative of three independent experiments. H, Hexamers; T, trimers.
Fig. 4.
Fig. 4.
Identification of novel O-glycosylated sialylated Thr residues in human adiponectin. A, FLAG-tagged headless (Stalk) and globular head (Glob) truncated proteins were either untreated or treated with neuraminidase before 2DE and Western blotting for the FLAG tag. B, 2DE of Thr to Ala mutants of human adiponectin secreted from transfected CHO cells. C, SDS-PAGE and Western blotting of the same samples. D, Velocity sedimentation analysis of secreted WT and T20-22A adiponectin. E, Sequence alignment of the adiponectin variable domain from various mammals is shown and reveals limited conservation of the modified sequence/residues (shaded). Data are representative of two to three independent experiments. H, Hexamers; T, trimers.
Fig. 5.
Fig. 5.
Sialic acid is a terminal modification occurring immediately before secretion. A, HEK cell lines stably expressing HA-tagged WT human adiponectin or the K1–5R mutant (lacking the five known glycosylated lysines in the variable and collagenous domains) were metabolically labeled for 2 h before replacement of the radiolabeled amino acids with an excess of unlabeled amino acids and continued culture for increasing periods of ‘chase’. Lysates and media were harvested and immunoprecipitated with HA antibody before analysis by SDS-PAGE and autoradiography. B, 2DE and Western blotting of lysates, with and without Brefeldin A (BFA) treatment, and medium from CHO cells stably expressing human adiponectin. Data are representative of two independent experiments.
Fig. 6.
Fig. 6.
Acute administration of desialylated adiponectin: accelerated clearance but unaltered activity. A, Male Wistar rats were administered iv with either desialylated (Desia-ADN) or control (Con-ADN) [125I]adiponectin and blood samples were taken from a jugular cannula at various time points after infusion as described in Materials and Methods. TCA-precipitable radioactivity in the serum samples was measured and normalized to the total radioactivity administered (n =5 desialylated and n = 4 control; *, P < 0.001 for the difference in exponential rate constant for disappearance of TCA-precipitable radioactivity from the plasma for control and desialylated adiponectin). Serum samples were also analyzed by SDS-PAGE and autoradiography to confirm the loss of [125I]adiponectin (inset). B, Uptake of control or desialylated [125I]adiponectin by primary rat hepatocytes. Cells were incubated in serum-free culture medium containing the radioligands for various periods of time before washing and harvesting of lysates for measurement of radioactivity (n = 3; *, P < 0.05 Con-ADN vs. Desia-ADN). C, Differentiated L6 rat myotubes expressing HA-GLUT4 were serum starved for 2 h before stimulation with control or desialylated adiponectin for 20 min. Cells were fixed and adiponectin-stimulated GLUT4 translocation to the plasma membrane was assessed (n = 4; *, P < 0.05 Basal vs. Con-ADN or Desia-ADN). D, Male adiponectin−/− mice were starved overnight before administration of control or desialylated recombinant mouse adiponectin-FLAG (50 μg/mouse) or saline by tail vein injection (*, P < 0.05; Con-ADN vs. Desia-ADN). E, Liver lysates from mice killed at 30 min after injection were analyzed by Western blotting for total and phospho-AMPK (pAMPK; Thr172). Note that the circulating concentrations of adiponectin in mice administered with untreated or desialylated adiponectin were 8.3 ± 1.4 mg/liter and 7.7 ± 1.0 mg/liter, respectively (n = 4; P > 0.05).
Fig. 7.
Fig. 7.
Chronic administration of desialylated adiponectin: reduced steady-state levels and metabolic effects. High-fat-fed male adiponectin−/− mice were chronically administered (by osmotic pump) with control (Con-ADN, n = 6) and desialylated (Desia-ADN, n = 5) adiponectin preparations (1.0 mg/kg body weight · d) or saline for a period of 2 wk. A, Serum adiponectin levels were measured every 3 d (*, P < 0.05; **, P < 0.01). GTT (panel B) and ITT (panel C) were conducted on d 10 and d 13, respectively, and panel D shows the area under the curves (*, P < 0.05; **, P < 0.01 vs. saline; #, P < 0.05 vs. Con-ADN by ANOVA).
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