Patients with obstructive sleep apnea (OSA) are at increased risk for cardiovascular diseases (CVDs). Fetuin-A, a novel hepatokine, has been associated with the metabolic syndrome (MetS), insulin resistance, and type 2 diabetes mellitus, all of which are highly prevalent in patients with OSA and associated with increased CVD risk. The goal of this study was to determine whether fetuin-A could be involved in the pathogenesis of CVD risk in patients with OSA, through relations of fetuin-A with MetS components and/or insulin resistance. Overweight or obese, nondiabetic volunteers (n = 120) were diagnosed with OSA by in-laboratory nocturnal polysomnography. Steady-state plasma glucose concentrations derived during the insulin suppression test were used to quantify insulin-mediated glucose uptake; higher steady-state plasma glucose concentrations indicated greater insulin resistance. Fasting plasma fetuin-A and lipoprotein concentrations were measured. Whereas neither the prevalence of MetS nor the number of MetS components was associated with tertiles of fetuin-A concentrations, the lipoprotein components of MetS, triglycerides and high-density lipoprotein cholesterol, increased (p <0.01) and decreased (p <0.05), respectively, across fetuin-A tertiles. Additionally, comprehensive lipoprotein analysis revealed that very low density lipoprotein (VLDL) particles and VLDL subfractions (VLDL 1+2 and VLDL 3 ) were increased across fetuin-A tertiles. In contrast, neither insulin resistance nor sleep measurements related to OSA were found to be modified by fetuin-A concentrations. In conclusion, abnormalities of lipoprotein metabolism, but not MetS or insulin resistance per se, may represent a mechanism by which fetuin-A contributes to increased CVD risk in patients with OSA.
There is substantial evidence that patients with obstructive sleep apnea (OSA) are at increased risk for cardiovascular diseases (CVDs). Pathophysiologic abnormalities in OSA, including oxidative stress, inflammation, endothelial dysfunction, dyslipidemia, and insulin resistance, have been implicated in this pathway, but the relative contribution of these putative mechanisms in mediating CVD risk is not clear. Fetuin-A, a circulating glycoprotein secreted by the liver, may have antiatherosclerotic properties through its ability to inhibit ectopic calcification. Fetuin-A levels have been found to be inversely associated with coronary artery calcification severity in patients without clinically apparent CVD, although the direction of association of fetuin-A with CVD outcomes or mortality has been variable across different populations. At the same time, elevations in fetuin-A have also been linked to the metabolic syndrome (MetS), insulin resistance, and type 2 diabetes mellitus (T2DM), all of which are highly prevalent in patients with OSA and associated with increased CVD risk. The goal of the study was to evaluate whether fetuin-A could be involved in the pathogenesis of CVD risk in patients with OSA and whether the mechanisms could be attributed to the relation of fetuin-A with MetS and/or insulin resistance in patients with OSA. To pursue this goal, we studied 120 overweight or obese patients who had been diagnosed with OSA but without known histories of CVD or T2DM and examined the relations of fetuin-A with individual components of MetS, comprehensive lipid and lipoprotein profiling, and insulin-mediated glucose disposal.
Methods
Eligibility for the study required overweight or obesity (body mass index 25.0 to 40.0 kg/m 2 ) and age 30 to 70 years. Recruitment occurred from July 2010 and July 2014 through local print, online, and radio advertisements and at the Stanford Sleep Medicine Center.
Patients were excluded if they had fasting glucose levels ≥126 mg/dl, histories of T2DM or use of antidiabetic medications, CVD, or kidney or liver diseases or if they had received treatment for OSA. The study protocol was approved by the Stanford Administrative Panels for the Protection of Human Subjects. Informed consent was obtained from all participants.
Full-night polysomnography was performed at the Stanford Sleep Medicine Center according to standard procedures. In addition to characteristic symptoms, a diagnosis of OSA was made by having an apnea-hypopnea index (AHI) ≥5 events/hour. Manual scoring of all polysomnograms was conducted in accordance to the American Academy of Sleep Medicine manual (2007). OSA severity was defined as follows: mild OSA, AHI 5 to 14.9 events/hour; moderate OSA, 15 to 30 events/hour; and severe OSA, >30 events/hour. Additional measurements included minimum and mean oxygen saturation, and oxygen desaturation index (number of events per hour in which a ≥3% decrease in oxygen saturation occurs from baseline).
After a 12-hour fast, patients were admitted to the Stanford Clinical and Translational Research Center. Patients underwent the modified insulin suppression test to quantify insulin-mediated glucose disposal. Participants received intravenous octreotide (0.27 μg/m 2 /min), insulin (32 mU/m 2 /min), and glucose (267 mg/m 2 /min) over 3 hours. During the final 30 minutes, blood was drawn every 10 minutes for measurements of plasma insulin and glucose. These 4 values were averaged to obtain steady-state plasma insulin and steady-state plasma glucose (SSPG) concentrations for each patient. Because steady-state plasma insulin is similar for all subjects, SSPG provides a direct measure of the ability of insulin to mediate disposal of the infused glucose load. Thus, higher SSPG concentrations indicate greater insulin resistance. SSPG is highly correlated with measures derived from the hyperinsulinemic euglycemic clamp (r ≥ −0.87).
Fasting plasma samples were frozen at −80°C until measurements were performed. Plasma fetuin-A levels were quantified by a human fetuin-A sandwich enzyme-linked immunosorbent assay kit (Epitope Diagnostics, San Diego, California) using 2 polyclonal antibodies that bind to different epitopes of human fetuin-A. Intra-assay precision is 4.8% to 5.5% and interassay precision is 5.7% to 6.8%. Lipoprotein analysis was performed using the VAP Test (Atherotech, Inc., Albuquerque, New Mexico), as described previously.
MetS was defined using the “harmonious” criteria, of which 3 of the 5 following components must be met: fasting plasma glucose ≥100 mg/dl (5.56 mmol/L), systolic blood pressure ≥130 mm Hg or diastolic blood pressure ≥ 85 mm Hg, triglycerides ≥150 mg/dl (1.70 mmol/L), and high-density lipoprotein (HDL) cholesterol <40 mg/dl (1.30 mmol/L) in men and <50 mg/dl (1.04 mmol/L) in women. Criteria for abdominal obesity (for subjects in the United States) included waist circumference ≥88 cm in women and ≥102 cm in men.
Statistical analyses were performed using IBM SPSS Statistics version 22.0 (IBM, Armonk, New York).
Fetuin-A and lipoprotein concentrations that were not normally distributed were log transformed before analysis. Patients were categorized into tertiles of fetuin-A. A generalized linear model (analysis of covariance) to adjust for age, gender, race, and body mass index was used to assess for significant associations between groups. Sequential Bonferroni adjustment was used to adjust p values adjusted for multiple pairwise comparisons. Linear trends of lipoprotein concentrations across tertiles of fetuin-A were estimated using analysis of variance. Chi-square tests were performed to compare MetS prevalence, number of MetS components, and low-density lipoprotein density patterns across tertiles of fetuin. A p value <0.05 indicated statistical significance, and all hypothesis testing was 2 tailed.
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