Diabetes Care Volume 40, March 2017 375 Novel Protein Glycan–Derived Carlos Lorenzo,1 Andreas Festa,2 Anthony J. Hanley,3,4 Marian J. Rewers,5 Markers of Systemic Inf lammation Agustin Escalante,1 and Steven M. Haffner1§ and C-Reactive Protein in Relation to Glycemia, Insulin Resistance, and Insulin Secretion Diabetes Care 2017;40:375–382 | DOI: 10.2337/dc16-1569 OBJECTIVE N-acetylglucosamine/galactosamine (GlycA) and sialic acid (GlycB) moieties of glycosylated serum proteins are nonspecific measures of inflammation, but con- clusive data on their relationship with insulin resistance or insulin secretion are missing. Therefore, we aimed to examine the relation of GlycA, GlycB, and C-reactive protein (CRP) to direct measures of insulin sensitivity (insulin sensitivity index [SI]) and insulin secretion (acute insulin response [AIR]). RESEARCH DESIGN AND METHODS PATHOPHYSIOLOGY/COMPLICATIONS This study used cross-sectional analyses and included 1,225 participants with and without type 2 diabetes in the Insulin Resistance Atherosclerosis Study (IRAS). SI and AIR were measured using the frequently sampled intravenous glucose toler- ance test, and GlycA and GlycB were measured using nuclear magnetic resonance spectroscopy. 1Department of Medicine, University of Texas Health Science Center, San Antonio, TX RESULTS 2Eli Lilly and Company, Vienna, Austria 3 GlycA and GlycB had a strong correlation with CRP (r =0.60[P < 0.001] and r = 0.46 Department of Nutritional Sciences and Dalla Lana School of Public Health, University of Tor- [P < 0.001], respectively). In a linear regression model with both GlycA and CRP as onto, Toronto, Ontario, Canada independent variables, GlycA (b31SD,20.04 6 0.02; P < 0.01) and CRP (20.06 6 4Leadership Sinai Centre for Diabetes, Mt. Sinai 0.02; P < 0.001) were independently associated with S even after adjusting for Hospital, Toronto, Ontario, Canada I 5 demographics, smoking, physical activity, plasma glucose, and BMI. However, Barbara Davis Center for Diabetes, University Colorado School of Medicine, Aurora, CO neither CRP nor GlycA had an independent relationship with AIR. Corresponding author: Andreas Festa, andreasfesta@ CONCLUSIONS icloud.com. fl Received 26 July 2016 and accepted 6 December GlycA may complement CRP in evaluating the relationship between in ammation, 2016. glucose tolerance, and insulin resistance. This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/ C-reactive protein (CRP) concentration, a marker of chronic subclinical inflamma- suppl/doi:10.2337/dc16-1569/-/DC1. tion, is related to insulin resistance (1) and has been identified as a risk factor for C.L. and A.F. contributed equally to this study. cardiovascular disease (CVD) (2). CRP concentration may be clinically relevant; it has §Retired. been shown to improve prediction algorithms for the stratification of individuals © 2017 by the American Diabetes Association. according to the risk of future CVD (3). Readers may use this article as long as the work fi is properly cited, the use is educational and not Glycosylation, the most common posttranslational protein modi cation, modu- for profit, and the work is not altered. More infor- lates protein function (4,5). Most acute-phase proteins, released from the liver mation is available at http://www.diabetesjournals during an inflammatory response, are enzymatically glycosylated and circulate .org/content/license. 376 Glycans and Insulin Resistance and Secretion Diabetes Care Volume 40, March 2017 at concentrations high enough to be based studies (the San Antonio Heart place at the University of Southern Cal- measurable via proton nuclear mag- Study and the San Luis Valley Diabetes ifornia (Los Angeles). Plasma insulin netic resonance (NMR) spectroscopy (6,7). Study, respectively). A total of 1,625 indi- concentration was measured using the N-acetylglucosamine/galactosamine viduals were enrolled in the IRAS (56% dextran-charcoal radioimmunoassay. (GlycA) and sialic acid (GlycB) moieties women) from among the 3,416 contacted CRP was measured by in-house ultra- of glycosylated serum proteins are non- (response rate of 48%). The examinations sensitive competitive immunoassay (anti- specificmeasuresofinflammation. Both began in October 1992 and were com- bodies and antigens from Calbiochem), GlycA and GlycB have a strong relation- pleted in April 1994. The IRAS protocol with an interassay coefficient of varia- ship with CRP (7,8). Increased GlycA has was approved by local institutional review tion of 8.9% (1). GlycA and GlycB were been associated with prevalent CVD risk committees, and all participants provided measured using NMR spectroscopy factors including smoking, diabetes, hyper- written informed consent. (LipoScience Inc., Raleigh, NC). GlycA tension, dyslipidemia, and obesity (8). Pro- GlycA and GlycB were measured in and GlycB signals in plasma arise from spectively, GlycA has been associated with 1,489 participants (561 non-Hispanic circulating acute-phase proteins (6,18). incident coronary heart disease and CVD whites, 429 African Americans, and The concentration of glycoproteins re- events independent of conventional risk 499 Hispanics). These participants did sponsible for the GlycA and GlycB signals factors in the Women’sHealthStudyand not differ from those with missing infor- was estimated to be ;13 mg/mL in nor- the Multi-Ethnic Study of Atherosclerosis mation on GlycA and GlycB (n =136)in mal human plasma (18). Fibrinogen, a1- (MESA), respectively (8,9). In the Women’s terms of adiposity, insulin resistance, acid glycoprotein, a1-antichymotrypsin, Health Study, the relation of GlycA to CVD and plasma concentrations of glucose, a1-antitrypsin, haptoglobin, and comple- was comparable to that of CRP (8). How- lipids, and CRP (P . 0.21 for all compar- ment C3 contributed significantly to the ever, the association between GlycA and isons). The present report includes data increase in GlycA in chronic systemic in- CVD was no longer significant after con- from 1,225 participantsd947 individu- flammation (18). Blood samples were trolling for CRP (8). als without diabetes and 278 patients stored at 270°C until analysis (approxi- CRP has been established as a risk factor with type 2 diabetes who were not tak- mately 18 years later). Ritchie et al. (19) for incident type 2 diabetes (10,11). GlycA ing any glucose-lowering drugsdin order already proved the stability of GlycA in also predicts future development of dia- to exclude any potential drug-specific samples stored for more than10 years. betes (12,13), but conclusive data on the confounding on key outcome measures, Fasting and 2-h plasma glucose concen- relation of GlycA and GlycB to insulin re- including markers of inflammation. Thus, trations were used to define categories of sistance or insulin secretion are missing. patients with diabetes were newly diag- glucose tolerance: normal (NGT), fasting It is therefore of interest to determine nosed or were treated with diet and/or glucose ,5.6 mmol/L and 2-h glucose whether the relation of GlycA and GlycB exercise. ,7.8 mmol/L (n = 455); isolated impaired to insulin resistance and insulin secretion fasting glucose (IFG), fasting glucose 5.6– has utility similar or complementary to Measurements 6.9 mmol/L and 2-h glucose ,7.8 mmol/L conventional inflammatory markers such Age, sex, ethnicity, smoking, physical (n = 188); isolated impaired glucose toler- as CRP. Thus, we examined the relation activity, menopausal status, and phar- ance (IGT), fasting glucose ,5.6 mmol/L of GlycA, GlycB, and CRP to measures of macologic treatment (glucose-lowering and 2-h glucose 7.8–11.0 mmol/L (n = insulin resistance and insulin secretion in agents and estrogen and progesterone 99); and IFG/IGT, fasting glucose 5.6– participants of the Insulin Resistance medications) were gathered by trained 6.9 mmol/L and 2-h glucose 7.8–11.0 Atherosclerosis Study (IRAS). In the personnel. Anthropometric measure- mmol/L (n = 205). Diabetes was defined IRAS, a frequently sampled intravenous ments were carried out using standard- as fasting glucose $126 mg/dL and/or glucose tolerance test (FSIGTT) was ad- ized protocols. The IRAS protocol required 2-h glucose $200 mg/dL (n = 278). ministered in all participants to obtain two visits, approximately 4 h each, 1 week HOMA of insulin resistance (HOMA-IR) direct measures of insulin sensitivity apart. Participants were asked before was calculated according to Matthew’s and insulin secretion: the insulin sensitiv- eachvisittofastfor12h,toabstainfrom formula: fasting insulin (mIU/mL) 3 fasting ity index (SI) and acute insulin response heavy exercise and alcohol for 24 h, and to glucose (mmol/L) 4 22.5. Normal weight, (AIR), respectively. refrain from smoking the morning of the overweight, and obesity were defined examination. as BMI ,25, 25–29.9, and $30 kg/m2, RESEARCH DESIGN AND METHODS A 75-g oral glucose tolerance test was respectively. Cigarette smoking was cat- Subjects administered to assess glucose tolerance egorized as nonsmoker and low- and The design and methods of the IRAS have status during the first visit. During the sec- high-degree smokers (0, 1–9, and $10 been described in detail (14). Briefly, the ond visit, insulin sensitivity and insulin se- cigarettes/day, respectively). study was conducted at four clinical cen- cretion were determined using an FSIGTT ters. At centers in Oakland and Los An- (15,16). Insulin sensitivity, expressed as Statistical Analyses geles, California, non-Hispanic whites and the SI, was calculated using mathematical