High-resolution identification of human adiponectin oligomers and regulation by pioglitazone in type 2 diabetic patients Ellene H. Mashalidis a , David B. Briggs a , Mowei Zhou a , Ashley M. Vergara a , Jimmy J. Chhun a , Ronald K. Ellsworth a , Rebecca M. Giron a , Jennifer Rood b , George A. Bray b , Steven R. Smith b,1 , Vicki H. Wysocki a , Tsu-Shuen Tsao a,⇑ a Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85724, USA b Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA article info Article history: Received 11 July 2012 Received in revised form 3 February 2013 Accepted 7 February 2013 Available online 21 February 2013 Keywords: Adiponectin Adipokine Type 2 diabetes Oligomerization Electrophoresis Thiazolidinedione abstract Adiponectin is an adipokine with insulin-sensitizing, anti-inflammatory, and cardiac protective actions. It homo-oligomerizes into trimers, hexamers, and higher molecular weight (HMW) species, which are not fully characterized. We describe high-resolution separation of adiponectin oligomers under native condi- tions in polyacrylamide gel coupled with methods for producing standards to provide facile and accurate identification of the oligomers. Using these procedures, adiponectin trimers in human and rodent plasma were found to migrate as two distinct populations. Distributions of these two populations are linearly proportional in plasma from type 2 diabetic patients before (R 2 = 0.903, P < 0.001) and after (R 2 = 0.960, P < 0.0001) 12 weeks of treatment with pioglitazone as well as from control subjects (R 2 = 0.891, P < 0.0001). In addition, HMW adiponectin could be separated into three distinct oligomers: nonamer (9mer), dodecamer (12mer), and the previously characterized octadecamer (18mer). Plasma concentra- tions of all oligomers increased on pioglitazone treatment, with the largest fold increase being observed in 9mers and 12mers compared with baseline. Increasing concentrations of adiponectin during oligomer- ization in vitro led to a disproportionate increase in 18mers. The difference between in vivo and in vitro observations suggests that higher total adiponectin protein concentration contributes to pioglitazone’s ability to enhance HMW adiponectin levels, but additional factors likely affect oligomer assembly or turn- over independently. Ó 2013 Elsevier Inc. All rights reserved. Adiponectin is a hormone produced mainly by adipocytes with positive or protective effects on insulin action, cardiac functions, and vasculature [1–5]. High levels of adiponectin are consistently associated with decreased risk for type 2 diabetes across many studies [6], whereas low levels of circulating adiponectin are asso- ciated with insulin resistance, coronary artery disease, and obesity [7–9]. Treatment with the thiazolidinedione (TZD) 2 class of insulin- sensitizing compounds results in elevated blood adiponectin [10]. Circulating adiponectin concentration has been proposed as a bio- marker for metabolic disorders and glycemic control improvement [11,12]. As a result, it is important to understand the mechanisms that govern the concentration of adiponectin in circulation. In rodents and humans, adiponectin circulates as several homo- oligomeric species. The smallest mature adiponectin oligomer is a homo-trimer that consists of a glycosylated collagen-like triple helical domain [7,13,14] and a globular head domain with a highly hydrophobic inner core formed by the interface of all three mono- mers [15]. The hexamer (6mer) is composed of two trimers ar- ranged in a head-to-head, tail-to-tail orientation [16]. Larger adiponectin oligomeric species are readily observed in serum [13,17–19]. These species are collectively and commonly referred to as higher molecular weight (HMW) adiponectin, a term that re- flects the paucity of information on their biochemical composition. The predominant HMW species has been determined to consist of 18mers [20,21]. Although the level of circulating adiponectin is closely associ- ated with insulin sensitivity, the degree of association appears to be stronger with HMW adiponectin than with total adiponectin [22–29]. In particular, the S a index, defined as the ratio of HMW to the sum of HMW and lower molecular weight (LMW) abundance in sucrose velocity gradient [22], has been shown to 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.02.008 ⇑ Corresponding author. Fax: +1 520 626 3644. E-mail address: tsushuen@email.arizona.edu (T.-S. Tsao). 1 Current address: Translational Research Institute for Metabolism and Diabetes, Sanford Burnham Medical Research Institute, Orlando, FL 32804, USA. 2 Abbreviations used: TZD, thiazolidinedione; HMW, higher molecular weight; LMW, lower molecular weight; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; SEC, size exclusion chromatography; PBS, phosphate-buffered saline; FPG, fasting plasma glucose; Fx, fraction; ANOVA, analysis of variance; HSD, honestly significant difference. Analytical Biochemistry 437 (2013) 150–160 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio