Novel Protein Glycan Side-Chain Biomarker and Risk of Incident Type 2 Diabetes MellitusSignificance
Objectives—Enzymatically glycosylated proteins partake in multiple biological processes, including glucose transport and inflammation. We hypothesized that a novel biomarker (GlycA) of N-acetyl methyl groups originating mainly from N-acetylglucosamine moieties of acute-phase glycoproteins is related to incident type 2 diabetes mellitus and compared it with high-sensitivity C-reactive protein.
Approach and Results—In 26 508 initially healthy women free of diabetes mellitus, baseline GlycA and high-sensitivity C-reactive protein were quantified by nuclear magnetic resonance spectroscopy and immunoturbidimetry, respectively. During median follow-up of 17.2 years, 2087 type 2 diabetes mellitus cases occurred. In Cox models with adjustment for age, race, smoking, alcohol, activity, menopausal status, hormone use, family history, and body mass index, quartile 4 versus 1 hazard ratios and 95% confidence intervals were 2.67 (2.26–3.14) for GlycA and 3.93 (3.24–4.77) for high-sensitivity C-reactive protein; both P trend <0.0001. Associations for GlycA and high-sensitivity C-reactive protein were attenuated after additionally adjusting for lipids: 1.65 (1.39–1.95) and 2.83 (2.32–3.44), respectively, both P trend <0.0001, and after mutual adjustment: 1.11 (0.93–1.33; P trend=0.10) and 2.57 (2.09–3.16; P trend<0.0001), respectively.
Conclusions—Our finding of an association between a consensus glycan sequence common to a host of acute-phase reactants and incident type 2 diabetes mellitus provides further support for inflammation in the development of type 2 diabetes mellitus. Additional studies exploring the role of enzymatic glycosylation in the prevention of type 2 diabetes mellitus are warranted.
Enzymatic protein glycosylation represents the most abundant post-translational modification process, with nearly 70% of human plasma proteome bearing a glycan (sugar) structure.1 The significance of glycan attachments is exemplified by their role in modulating biological processes, including cell trafficking, signal transduction, regulation of metabolism, and host pathogen recognition.2 Of clinical importance is that glycan biosynthesis is exquisitely responsive to the cellular milieu,3 and an altered glycosylation pattern may, therefore, reflect the development of disease, hence the interest in studying glycans as early indicators of disease. However, the diagnostic use of glycans is hampered by technological limitations in quantitative glycan analysis due, in part, to their nontemplate-derived nature.3 It is noteworthy to differentiate nonenzymatic glycation products, such as hemoglobin A1c (HbA1c) and advanced glycation end products, from glycans which are indeed different biomolecular species.
Among other techniques, proton nuclear magnetic resonance (NMR) spectroscopy is emerging as a promising high-throughput method for measuring protein glycans.3 Bell et al4 documented the first report of an NMR spectrum common to glycoproteins of acute-phase reactants. Furthermore, their work suggested that glycan modification of acute-phase reactants may be useful for the detection, prognosis, and therapeutic monitoring of tissue damage marked by inflammation.4 LipoScience Inc (Raleigh, NC) recently developed an application for an NMR-derived biomarker referred to as GlycA, which originates from the N-acetyl methyl groups of N-acetylglucosamine residues located on specific glycan branches of plasma proteins mainly from the acute-phase glycoproteins α1-acid glycoprotein, haptoglobin, α1-antitrypsin, α1-antichymotrypsin, and transferrin.5 The proton NMR spectrum to which GlycA was assigned overlaps with the region identified by Bell et al,4 and hence GlycA may be an integrative marker of systemic inflammation.
Low-grade chronic systemic inflammation is known to trigger the development of insulin resistance and β-cell dysfunction.6 The association of circulating levels of inflammatory proteins, in particular acute-phase reactants, with the clinical expression of type 2 diabetes mellitus is also well described in prospective epidemiological studies.7,8 Enzymatic glycosylation modulates the functions of these proteins, and analysis of the ensuing glycans may have important implications for the pathogenesis of type 2 diabetes mellitus. Whether a glycosylation pattern common to a host of acute-phase reactants have potential use as a marker of future risk of type 2 diabetes mellitus is unknown. We, therefore, examined the relation of baseline GlycA, quantified by NMR spectroscopy, with incident type 2 diabetes mellitus, and compared it with high-sensitivity C-reactive protein (hsCRP), a commonly used clinical marker of inflammation. Because metabolic derangements can induce glycan changes relevant to glucose control,9 we also prespecified to assess for effect modification by HbA1c, body mass index (BMI), and smoking status.
Materials and Methods
Materials and methods are available in the online-only Data Supplement.
Baseline Characteristics and Correlations
Mean (SD) age of the study population at baseline was 54.6 (7.1) years. Median (25th–75th percentile) baseline concentrations for GlycA and hsCRP were 368 (325–414) μmol/L and 1.97 (0.79–4.24) mg/L, respectively. Participants with higher levels of GlycA were more likely to be hormone users, postmenopausal, current smokers, and to report a first-degree relative with diabetes mellitus (Table 1). They also had higher BMI, hsCRP, and dyslipidemia; and were less likely to consume alcohol or exercise. HbA1c levels differed minimally across quartiles of GlycA, although achieved statistical significance given the large study sample.
GlycA correlated positively with hsCRP (Spearman correlation coefficient, 0.61; P<0.0001) Metabolic risk indicators also correlated with GlycA: range of coefficients, in increasing magnitude, with HbA1c, interleukin-6 (IL-6), soluble intracellular adhesion molecule-1, BMI, fibrinogen, and triglycerides were 0.21 to 0.46, P for all <0.0001; and with high-density lipoprotein cholesterol (−0.26; P<0.0001). GlycA was not correlated with tumor necrosis factor α receptor type 2, spearman correlation coefficient was 0.02 (P=0.77).
Associations With Incident Type 2 Diabetes Mellitus
During a median follow-up of 17.2 years, a total of 2087 cases of incident type 2 diabetes mellitus occurred. Kaplan–Meier curves for event-free survival diverged according to quartiles of GlycA (log-rank P<0.0001; Figure). In Cox models adjusting for age, race/ethnicity, smoking, alcohol consumption, physical activity, menopausal status, hormone use, family history, BMI, and randomized trial assignments, hazard ratios (HRs) and 95% confidence intervals for type 2 diabetes mellitus across increasing quartiles of GlycA were 1.00, 1.43 (1.20–1.71), 1.90 (1.60–2.24), and 2.67 (2.26–3.14), and for hsCRP: 1.00, 1.86 (1.52–2.28), 2.96 (2.44–3.59), and 3.93 (3.24–4.77); both P trend <0.0001 (Table 2). The associations were attenuated but remained statistically significant after further adjusting for lipids and HbA1c: top versus bottom quartile was 1.43 (1.21–1.69; P trend <0.0001) and 2.43 (2.00–2.96; P trend <0.0001), respectively. In models additionally adjusting for the other biomarker, these HRs became 1.04 (0.87–1.24; P trend=0.35) and 2.31 (1.88–2.85; P trend <0.0001) for GlycA and hsCRP, respectively. Results were similar when the biomarkers were examined per 1 SD. Sensitivity analysis performed after exclusion of type 2 diabetes mellitus cases reported during the first 5 years of follow-up (428 events) yielded similar results.
In stratified models adjusting for risk factors and lipids, the relative risk for GlycA with type 2 diabetes mellitus was somewhat higher for individuals with baseline BMI< versus ≥25 kg/m2: HRs for top versus bottom quartile were 2.20 (1.58–3.13; P trend <0.0001) and 1.37 (1.13–1.67; P trend <0.0001), respectively, P for interaction=0.006. No significant interaction was noted for BMI and hsCRP: corresponding HRs 2.22 (1.57–3.16; P trend <0.0001) and 2.66 (2.06–3.45; P trend <0.0001), respectively, P for interaction=0.61. Both GlycA and hsCRP were significantly associated with the risk of incident type 2 diabetes mellitus within all stratified categories of exercise, although P for interaction were marginally significant (P for interaction=0.03 and 0.04, respectively) Associations of GlycA with type 2 diabetes mellitus was consistent in stratified analyses based on baseline HbA1c and smoking categories, with P for interactions of 0.15 and 0.36, respectively; and similarly for hsCRP (Table 3). A statistically significant interaction was observed between hsCRP and the association of GlycA with incident type 2 diabetes mellitus (P for interaction=0.039), with a borderline significant trend for GlycA to be associated with the risk of type 2 diabetes mellitus only at levels of hsCRP<1 mg/L (P trend=0.054; Table 3). Results were similar when hsCRP was analyses as tertiles. hsCRP remained associated with incident type 2 diabetes mellitus across all tertiles of GlycA (P for interaction=0.13).
Adding GlycA to a model that included age, race, smoking status, alcohol intake, physical activity, menopausal status, hormone replacement use, family history of diabetes mellitus, BMI, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and log triglycerides, resulted in improved metrics of goodness of fit (P for likelihood ratio χ2 ≤0.0001). GlycA did not improve risk discrimination as indicated by the Harrell C-index and the integrated discrimination improvement (Table I in the online-only Data Supplement). However, GlycA yielded a small improvement in category-free net reclassification index of 0.1848 (95% confidence interval, 0.1269–0.2423).
This prospective study of 26 508 apparently healthy women followed for a median of 17.2 years provides the first evidence for the potential role of protein glycan side-chains early in the development of type 2 diabetes mellitus and specifically for GlycA levels as a biomarker for predicting the future risk of type 2 diabetes mellitus. High baseline levels of GlycA, a novel NMR-derived protein glycan biomarker, were associated with a graded increase in the risk of type 2 diabetes mellitus in a pattern similar to that observed for hsCRP. The magnitude of HRs were somewhat greater for hsCRP, and accounting for hsCRP attenuated the GlycA–type 2 diabetes mellitus association, consistent with a potential inflammatory role for GlycA and risk of type 2 diabetes mellitus. The association of GlycA with type 2 diabetes mellitus was significant even among individuals with baseline HbA1c<5% (31 mmol/mol). A quantitative interaction was noted between GlycA and BMI, with greater risk observed in participants with BMI<25 kg/m2.
Evidence for glycan differences in relation to cardiometabolic risk factors as observed in this study is consistent with a previous study, which found significant associations between specific changes in plasma N-glycans and risk factors, including body fat, lipids, and smoking. For instance, hyperlipidemia and smoking were found to correlate with higher levels of galactosylation.10 These results suggest a role for lifestyle changes in modifying the expression of pathogenic glycans. Our finding of a higher GlycA attributable type 2 diabetes mellitus risk in individuals with BMI <25 kg/m2 is intriguing. Recently, Perry and Voight et al11 identified a novel genetic variant whose magnitude of association with type 2 diabetes mellitus was higher in lean (BMI<25 kg/m2) compared with obese (≥30 kg/m2) cases of type 2 diabetes mellitus, also demonstrating that lean compared with obese type 2 diabetes mellitus cases were more enriched with known candidate type 2 diabetes mellitus loci.11 It will, therefore, be of interest to determine whether these genetic variants relate to the expression of the glycosylation machinery (ie, specific glycosidases and glycosyltransferases) responsible for the GlycA signal.
Several proteins, mostly acute-phase reactants, seem to bear the oligosaccharide chains unique to GlycA. Some of the major known contributors to the GlycA signal, including α1-acid glycoprotein, α1-antitrypsin, and transferrin, have been linked to type 2 diabetes mellitus.12–16 A previous analysis of the Atherosclerosis Risk and Communities Study showed that individuals with higher levels of α1-acid glycoprotein had increased risk for developing type 2 diabetes mellitus.12 Related studies have also linked deficiency and impairment of α1-antitrypsin with type 2 diabetes mellitus,14,15 findings that are in support of α1-antitrypsin exerting a regulatory effect on inflammation.17 Transferrin levels were also found to be associated with new onset hyperglycemia, despite being a negative acute-phase reactant.16 One study found that α1-antichymotrypsin, another glycoprotein with major contributions to GlycA, was strongly associated with insulin resistance as determined by postload insulin levels,18 although a cross-sectional study, this report is relevant in the context of insulin exerting a null effect on levels of α1-antichymotrypsin.19 In other words, mechanisms linked to ongoing insulin resistance and not insulin probably account for the observed findings. Furthermore, a set of other acute-phase reactants, including fibrinogen, hsCRP, and IL-6 make negligible contributions to the GlycA signal. As with hsCRP, several prospective studies have consistently shown a positive association between IL-6 and the risk of incident type 2 diabetes mellitus.7,8 Nonacute-phase reactants such as apolipoprotein B also make negligible contribution to the GlycA signal. Thus, the metabolic cluster concomitant with a preponderance of apolipoprotein B may partly account for the observed association between GlycA and type 2 diabetes mellitus as indicated by the attenuation in the magnitude of effect after adjusting for lipids. This study adds to the growing body of literature on the link between inflammation and the pathogenesis of type 2 diabetes mellitus by showing that a unique glycan sequence presumably common to specific glycoforms of acute-phase reactants may serve as a biomarker for the risk of type 2 diabetes mellitus. Nonetheless, despite the temporality of our findings, additional work needs to be done to evaluate if the glycans captured by GlycA are causal with respect to the pathogenesis of diabetes mellitus.
Although the structure of the glycans captured as GlycA is known to contain N-acetyl methyl group protons of the N-acetylglucosamine moieties located on the bi- tri- and tetraantennary branches of specific serum proteins, we do not know the exact carbohydrate structure of the side-chains of the contributing proteins during inflammation and how this may relate to the risk of incident type 2 diabetes mellitus. In this regard, a shift in the glycoform pattern of α1-acid glycoprotein characterized by increased fucosylation has been reported in relation to type 2 diabetes mellitus with similar changes observed on α1-acid glycoprotein isolated from sera of patients with marked inflammation.13 Whether the other glycoproteins contributing to the GlycA signal exhibit similar shifts in their glycan composition in relation to type 2 diabetes mellitus is unknown. Nonetheless, our findings indicate that a consensus glycosylation sequence on these proteins as captured by the GlycA signal may be useful in defining their specific role in promoting the development of type 2 diabetes mellitus through inflammatory pathways.
Enzymatic addition of glycans to proteins occurs through the coordinated expression of glycosidases and glycosyltransferases, which regulate post-translational modifications that alter protein function during metabolism and thus have implication for detecting disease onset. In this regard, the functional integrity of proteins essential to glucose trafficking have been correlated with specific glycosylation patterns, as exemplified in experiments demonstrating that altered glycosylation patterns of insulin signal transduction proteins impair glucose homeostasis and β-islet cell function.9,20 But as yet it is unknown if the glycosylation machinery responsible for the glycan chains unique to GlycA distort the glycosylation pattern needed to preserve normal glucose metabolism. The proposed inflammatory origin of GlycA provides a more plausible mechanistic explanation for the observed association. Increased synthesis of proinflammatory cytokines, including acute-phase reactants is well known to characterize preclinical stages of type 2 diabetes mellitus with further graded increase facilitating disease progression.7 Proof-of-concept studies have also demonstrated the effect of biological agents targeting specific proinflammatory cytokines in improving parameters of glucose control.21,22 From a therapeutic standpoint, GlycA may provide use for evaluating novel strategies targeting the inflammatory network in the prevention of type 2 diabetes mellitus.
Persistent hyperglycemia contribute to impaired insulin responsiveness in target tissues, partly through increased conversion of glucose to donor sugars used in enzymatic glycosylation reactions that render the effector proteins of the insulin signaling cascade defective.23 Sustained hyperglycemia may, therefore, lead to the formation of protein glycans that, in turn, perpetuate insulin resistance. Robustness of our findings among participants with HbA1c in euglycemic range, however, implicate mechanisms other than chronic hyperglycemia. Moreover, results from the sensitivity analysis excluding cases occurring within the first 5 years, that is, potential cases of clinically silent type 2 diabetes mellitus, mitigate against chronic hyperglycemia as an explanation for our observation. These along with the prospective design of this study thus minimizes the influence of reverse causality in the interpretation of our findings. It should also be pointed out that the monotonic pattern of HbA1c across levels of GlycA highlights an important distinction between glycation, which is a consequence of nonenzymatic glycosylation, and enzymatic glycosylation, a different process that is responsible for the glycan conjugation of proteins and other biopolymers.
Limitations of this study warrants discussion. First, our study population was composed of healthy middle-aged and older women, thus our results may not be generalizable to other groups particularly men and women with a different metabolic risk profile. Second, measurements of GlycA were only available at baseline precluding our ability to assess the effect of time-dependent changes in GlycA levels in relation to type 2 diabetes mellitus. Third, we cannot rule out the possibility of undetected type 2 diabetes mellitus at baseline, although our results remained robust in women with HbA1c levels<5% (31 mmol/mol) as well as after excluding events occurring during the first 5 years of follow-up. Finally, we cannot exclude the potential of residual and unmeasured confounding in the interpretation of our findings. Notable strengths of our study include a large sample size, a long prospective follow-up, a systematic ascertainment of incident type 2 diabetes mellitus, and a detailed assessment of cardiometabolic risk factors.
In summary, we found elevated baseline levels of GlycA, a novel protein glycan, to be significantly associated with incident type 2 diabetes mellitus in a cohort of initially healthy middle-aged women. This association was attenuated after adjusting for hsCRP, a clinical biomarker of systemic inflammation, and provides new evidence on the potential role of glycans early in the development of type 2 diabetes mellitus. More research is needed to verify our findings in other populations as well as to investigate the potential of GlycA in the prevention and treatment of type 2 diabetes mellitus.
Sources of Funding
The research for this article was supported by the American Heart Association and by grants HL43851, HL 080467, and CA 47988 from the National Heart, Lung, and Blood Institute and the National Cancer Institute, National Institutes of Health, a charitable gift from the Molino Family Trust and with additional support from U01 HL108630 (Mechanism-Associated Phenotypes for Genetic analyses of Heart, Lung, Blood, and Sleep Diseases [MAPGen for HLBS]). LipoScience Inc supplied the GlycA information at no additional cost. The funding agencies played no role in the design, conduct, data management, analysis, or article preparation related to this study. Dr Akinkuolie was supported by the National Heart, Lung, and Blood Institute (T32 HL007575).
Dr Ridker is listed as a coinventor on patents held by the Brigham and Women’s Hospital that relate to the use of inflammatory biomarkers in cardiovascular disease that have been licensed to AstraZeneca and Seimens. Dr Mora has received institutional research support from AstraZeneca, Atherotech Diagnostics, and National Heart, Lung, and Blood Institute; served as a consultant to Genzyme, Quest Diagnostics, Lilly, Pfizer, and Cerenis Therapeutics. The other authors report no conflicts.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.305635/-/DC1.
- Nonstandard Abbreviations and Acronyms
- body mass index
- hemoglobin A1c
- hazard ratio
- high-sensitivity C-reactive protein
- nuclear magnetic resonance
- Received December 31, 2014.
- Accepted April 7, 2015.
- © 2015 American Heart Association, Inc.
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Several inflammatory proteins have been shown to be associated with the risk of type 2 diabetes mellitus. The biological role of glycans in modulating protein function provides a viable avenue for disease prevention. This study illustrates the potential role of glycans in prognosticating against the risk of type 2 diabetes mellitus by showing that baseline measurements of a unique glycan sequence conjugated to several acute-phase proteins was associated with the risk of developing type 2 diabetes mellitus. Future work defining the biochemical properties of GlycA (the unique glycan sequence investigated in the current study) will provide additional biological information that if targeted, may enhance the diagnostic yield of GlycA. This effort will contribute to the understanding of the link between inflammation and type 2 diabetes mellitus while also serving as a template to initiate the discovery of new therapeutics.