Myonectin Predicts the Development of Type 2 Diabetes

Myonectin Predicts the Development of Type 2 Diabetes Abstract Context Myonectin has been identified as a myokine, expressed predominantly in skeletal muscle. However, its clinical implications are largely unknown. Objective The aim of this study is to investigate the relationship between myonectin (C1q tumor necrosis factor-α-related protein isoform 15) and type 2 diabetes mellitus (T2DM) in cross-sectional and interventional studies. Design In a separate study, oral glucose tolerance tests, a 45-minute bout of exercise, lipid infusions, and euglycemic-hyperinsulinemic clamps (EHCs) were performed to investigate the association of myonectin with homeostasis model assessment of insulin resistance (HOMA-IR) and T2DM. Circulating myonectin was measured by enzyme-linked immunosorbent assay. Patients One hundred four newly diagnosed T2DM (nT2DM), 109 impaired glucose tolerance (IGT), and 128 healthy individuals were recruited for this study. Results nT2DM and IGT subjects had higher circulating myonectin concentrations than normal subjects (82.3 ± 47.6 and 68.9 ± 46.6 vs. 45.2 ± 23.5 µg/L, P < 0.05 or P < 0.01). It was also found that in nT2DM individuals, circulating myonectin was higher than in IGT subjects. Plasma myonectin correlated positively with waist/hip ratio, percentage of body fat, triglyceride, fasting blood glucose, 2-hour blood glucose after glucose overload, fasting insulin, hemoglobin A1c, and HOMA-IR and negatively with the insulin sensitivity index in all of the study population. Multivariate logistic regression analysis revealed that circulating myonectin levels were significantly correlated with IGT and T2DM. A 45-minute bout of exercise did not change circulating myonectin levels in healthy, young individuals. Circulating myonectin levels were not significantly altered in response to an oral glucose challenge or EHC. In addition, acute elevated free fatty acid levels induced by lipid infusion had no effects on circulating myonectin. Conclusions These data suggest that myonectin may be a useful marker in predicting the development of prediabetes and diabetes. With the rapid increase in prevalence during the last two decades, type 2 diabetes mellitus (T2DM) has become one of the main threats to public health in the world. It has been estimated that >10% of Chinese adults have T2DM (1). T2DM patients have a greater risk for the development of cardiovascular disease (2, 3), with coexistent hypertension further increasing the risk of cardiovascular complications (4). Insulin resistance (IR) has been hypothesized to be the common pathophysiologic factor behind T2DM and hypertension (5). However, its pathogenesis is complex and still unclear. Muscle tissue is an important target tissue of IR and has been recognized as an active endocrine organ producing myokines, such as IL-6 (6), fibroblast growth factor 21 (7), and irisin (8), which modulate insulin sensitivity and thus, may play a role in the pathogenesis of IR. Myonectin, also known as C1q tumor necrosis factor-α-related protein isoform 15, is a myokine and belongs to a family of proteins characterized by an N-terminal signal peptide, a collagen repeat domain, and a C-terminal C1q-like globular domain (9). Myonectin is homologous to adiponectin with respect to domain structure, and it is expressed and secreted primarily by muscle tissues (10). Recently, it has been reported that the expression and plasma levels of myonectin are subject to metabolic control (11). In a mouse study, Seldin et al. (12) found that myonectin expression and high circulating levels were induced by refeeding following an overnight fast. Infusion of recombinant myonectin in vivo lowered circulating levels of free fatty acids (FFAs), in part, by promoting cellular lipid uptake and upregulating the expression of genes involved in lipid uptake (12). Interestingly, it has been shown that circulating myonectin levels are increased when overnight-fasted mice are given a bolus of glucose or emulsified fat (11). Moreover, circulating myonectin levels increased significantly in obese/diabetic animals (12, 13). These reports suggest that myonectin may be a nutrient-regulated cytokine and may have a functional role in IR. Therefore, myonectin seems to be a promising candidate for involvement in the pathogenesis of IR and T2DM. However, there is still a lack of information regarding the relationship among circulating levels of myonectin and IR and diabetes in humans. In the current study, we evaluate circulating levels of myonectin in normal subjects and subjects with impaired glucose tolerance (IGT) and in newly diagnosed T2DM (nT2DM) patients. We also investigate the effects of an oral glucose challenge, hyperinsulinemia, and FFA-induced IR on circulating myonectin in these subjects. Materials and Methods Cross-sectional studies Three hundred forty-one subjects, including 104 nT2DM, 109 IGT, and 128 healthy controls, were recruited to the study. IGT and T2DM were determined according to the World Health Organization diagnostic criteria (14). All individuals with IGT and patients with T2DM were newly diagnosed and were not treated with any hypoglycemic agents, diet control, or physical exercise. Exclusion criteria were patients with type 1 diabetes mellitus and patients with macrovascular or microvascular complications, hypertension, liver cirrhosis, hepatic and renal failure, congestive heart failure, or other major diseases. One hundred twenty-eight age-matched, healthy individuals without any clinical evidence of diseases were recruited from the community or schools through advertisement or routine medical checkups and were used as the controls [normal glucose tolerance (NGT)]. In these individuals, T2DM was excluded by a normal oral glucose tolerance test (OGTT), and the family history of T2DM was also excluded. None of these individuals was taking any medication-related glucose and lipid metabolism, as well as insulin sensitivity. In this study, all premenopausal women had regular menstrual cycles and were studied in the early follicular phase of the menstrual cycle (days 3 to 5 of the cycle). All subjects signed voluntary consent before experiment. The study was approved by the Human Research Ethics Committee of Chongqing Medical University and was registered at the Chinese Clinical Trial Registry chictr.org.cn (CHICTR-OCC-11001422). OGTT To investigate the effects of an oral glucose challenge on circulating myonectin levels in subjects without IR, OGTT was performed in 30 normal, young individuals, including 15 men and 15 women [age: 25 ± 2 years, body mass index (BMI): 21.5 ± 2.1 kg/m2]. In addition, OGTT was performed in all study populations. At 7:00 on the morning of the study days, after a 12-hour overnight fast, all individuals were given a 2-hour, 75-g OGTT, and venous blood was drawn at indicated times (0, 30, 60, and 120 minutes) for measurement of glucose, insulin, or circulating myonectin for 30 young individuals. Euglycemic-hyperinsulinemic clamp The euglycemic-hyperinsulinemic clamps (EHCs) were performed in 30 normal, young individuals, including 15 men and 15 women (age: 25 ± 2 years, BMI: 21.5 ± 2.1 kg/m2), as previously described (15, 16). During the EHCs, regular human insulin (1 mU/kg/min) was infused for 2 hours, and a variable infusion of 20% glucose was administered to maintain plasma glucose at the fasting level. The glucose disposal rate (GDR) was defined as the glucose infusion rate (GIR) during the stable period of the EHC and was related to body weight (M-value). Blood samples for myonectin measurement were obtained at indicated times. The samples were immediately cooled, and plasma was prepared and stored at −80°C until used. Lipid infusion study Lipid infusion was performed in 22 normal, young individuals (11 women and 11 men). These subjects received a 20% intralipid/heparin (0.4 units/kg/min; Pharmacia and Upjohn, Milan, Italy) infusion at a constant rate (1.5 mL/min) for 240 minutes. Two hours after the start of the lipid infusion, a 2-hour EHC was performed as described previously. Blood samples were collected before and at indicated times during lipid infusion. Samples were separated and kept at −80°C for myonectin measurement. Exercise testing procedures A 45-minute bout of treadmill exercise was performed in 12 young subjects (six men and six women; age: 21 to 23 years, BMI: 21.0 ± 1.2 kg/m2). Individuals were excluded if they were taking any medications, presented any contraindications to physical activity, or participated in 20 minutes or more of exercise at least two times per week. After a 12-hour overnight fast, individuals were arranged to ward between 8:00 and 8:30 on the morning of the experiment. A 45-minute bout of exercise was performed in these subjects with 60% of maximal oxygen consumption for 45 minutes. Blood samples were drawn at four time points, including baseline, 45 minutes following a 45-minute bout of exercise, and 60 and 120 minutes at rest after the exercise. Measurements of plasma myonectin Circulating myonectin concentrations were determined in blood samples with a commercial enzyme-linked immunosorbent assay kit following the manufacturer’s protocol (Catalog No. SK00393-19; Aviscera Bioscience, Santa Clara, CA). The limit of detection was 31.25 ng/mL, and intra-assay and interassay variations were <8% and <12%, respectively. Measurements of anthropometric and biochemical parameters The anthropometric determinations and the blood sample collections for biochemicals and other parameters were carried out on the same day. BMI was calculated as weight divided by height squared. Waist circumference and hip circumference were measured, and the waist/hip ratio (WHR) was calculated by the same researcher. Blood pressure (BP) was measured in all individual after they rested at least for 15 minutes. The percentage of body fat (FAT%) was measured by bioelectrical impedance (BIA-101; RJL Systems, Shenzhen, China). Homeostasis model assessment of IR (HOMA-IR) and insulin sensitivity index (ISI) were calculated as previously reported (17, 18). The area under the curve for glucose (AUCglucose) during the OGTT was calculated geometrically following the trapezoidal rule by using a statistical software program (19). Insulin concentrations were measured using chemiluminescence. Hemoglobin A1c (HbA1c) and glucose were measured by the glucose-oxidase method and anion-exchange HPLC, respectively. FFAs, total cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol were measured with a commercial kit. Statistical analysis All statistical analyses were completed with SPSS version 22.0 (SPSS, Armonk, NY). The distribution of data was examined by Kolmogorov-Smirnov test. analysis of variance, a paired t test, or unpaired t test was used for comparison among groups. The relationship between myonectin and other parameters was examined using partial correlation coefficients. We used multivariate regression analyses to assess variables that had associations with circulating myonectin. The Cochran-Armitage trend test was used to analyze the tendency of concentration of circulating myonectin associated with IGT and T2DM. The cutoff points of myonectin concentrations for predicting T2DM and IGT were given by receiver operating characteristics (ROCs) curves. All data were shown as means ± standard deviation, standard error, or median (interquartile range). When compared with the controls, P < 0.05 was considered significant. Results Distribution of circulating myonectin in healthy subjects and characteristics of study participants Fasting myonectin levels were measured in 128 healthy subjects (from 42 to 79 years). Circulating myonectin concentrations in healthy individuals ranged from 41.1 to 49.3 µg/L for the 95% of healthy population. The clinical baseline characteristics of the subgroups (NGT, IGT, and nT2DM) are shown in Table 1. There are no statistically significant differences among IGT, T2DM, and NGT subjects with respect to age, FAT%, total cholesterol, and LDL-C. When compared with the NGT, BMI, WHR, BP, FFA, fasting blood glucose (FBG), 2-hour blood glucose after glucose overload (2h-BG), fasting insulin (FIns), AUCglucose, and HOMA-IR in both IGT and nT2DM groups were significantly increased (P < 0.01 or P < 0.05), whereas high-density lipoprotein cholesterol and ISI were significantly decreased in the nT2DM group (P < 0.01 or P < 0.05). Importantly, circulating myonectin levels were higher in IGT and nT2DM individuals than in control subjects (P < 0.05 or P < 0.01; Fig. 1A). When compared with IGT subjects, T2DM patients had higher circulating myonectin levels (P < 0.05; Fig. 1A). These differences remained significant after adjustment for sex and age. In addition, obese individuals based on Chinese criteria (BMI ≥ 28 kg/m2) had significantly higher myonectin concentrations than lean individuals (BMI < 24 kg/m2) in all study populations (67.7 ± 19.7 vs. 55.9 ± 27.2 µg/L, P < 0.05; Fig. 1B). Significantly higher plasma concentrations of myonectin were also observed in female patients with IGT and nT2DM than in male patients (74.4 ± 53.0 vs. 56.9 ± 24.5 µg/L for IGT; 91.8 ± 60.1 vs. 72.1 ± 25.4 µg/L for nT2DM; both P < 0.05; Fig. 1C). However, no difference was observed in circulating myonectin levels between men and women in the control group (Fig. 1C). Table 1. Main Clinical Features and Circulating Myonectin Levels in the Study Population Variable  NGT (n = 128)  IGT (n = 109)  nT2DM (n = 104)  Men/women  55/73  46/63  50/54  Age, y  54 ± 9  55 ± 11  56 ± 10  BMI, kg/m2  23.5 ± 3.1  24.42 ± 2.98a  24.47 ± 3.17a  FAT%, %  29.49 ± 7.16  31.57 ± 7.84  30.87 ± 9.90  WHR  0.88 ± 0.08  0.91 ± 0.08a  0.90 ± 0.11a  SBP, mm Hg  118.0 ± 13.8  125.3 ± 17.0b  129.2 ± 16.5b  DBP, mm Hg  75.2 ± 9.9  77.6 ± 11.8  81.1 ± 11.6b,c  TC, mmol/L  4.98 ± 0.94  5.22 ± 1.25  5.12 ± 1.08  TG, mmol/L  1.56 ± 1.05  2.03 ± 1.14  2.12 ± 1.47  HDL-C, mmol/L  1.33 ± 0.31  1.40 ± 0.46  1.22 ± 0.41a,d  LDL-C, mmol/L  2.92 ± 0.77  2.97 ± 1.05  3.06 ± 0.90  FFA, µmol/L  0.49 (0.37–0.61)  0.56 (0.44–0.79)b  0.63 (0.45–0.92)b  FBG, mmol/L  5.22 ± 0.46  6.18 ± 1.70b  9.76 ± 4.15b,d  2h-BG, mmol/L  6.21 ± 1.02  8.96 ± 1.46b  18.57 ± 7.49b,d  FIns, mU/L  8.49 ± 3.60  11.97 ± 8.61b  11.64 ± 6.46a,c  2h-Ins, mU/L  57.6 ± 45.8  88.7 ± 55.3b  66.7 ± 58.1d  HbA1c, %  5.63 ± 0.56  5.90 ± 0.68  8.33 ± 2.37b,d  HOMA-IR  1.87 (1.32–2.49)  2.54 (1.93–4.13)b  4.00 (2.70–5.93)b,d  ISI  4.62 (3.02–6.71)  3.39 (2.21–4.41)b  2.32 (1.81–3.40)b,c  AUCglucose  14.9 ± 2.4  19.7 ± 2.8b  34.4 ± 11.1b,d  Myonectin, µg/L  45.2 ± 23.5  68.9 ± 46.6b  82.3 ± 47.6b,c  Myonectin, adjustede  45.2 ± 2.1  68.9 ± 4.5b  82.3 ± 4.6b,c  Variable  NGT (n = 128)  IGT (n = 109)  nT2DM (n = 104)  Men/women  55/73  46/63  50/54  Age, y  54 ± 9  55 ± 11  56 ± 10  BMI, kg/m2  23.5 ± 3.1  24.42 ± 2.98a  24.47 ± 3.17a  FAT%, %  29.49 ± 7.16  31.57 ± 7.84  30.87 ± 9.90  WHR  0.88 ± 0.08  0.91 ± 0.08a  0.90 ± 0.11a  SBP, mm Hg  118.0 ± 13.8  125.3 ± 17.0b  129.2 ± 16.5b  DBP, mm Hg  75.2 ± 9.9  77.6 ± 11.8  81.1 ± 11.6b,c  TC, mmol/L  4.98 ± 0.94  5.22 ± 1.25  5.12 ± 1.08  TG, mmol/L  1.56 ± 1.05  2.03 ± 1.14  2.12 ± 1.47  HDL-C, mmol/L  1.33 ± 0.31  1.40 ± 0.46  1.22 ± 0.41a,d  LDL-C, mmol/L  2.92 ± 0.77  2.97 ± 1.05  3.06 ± 0.90  FFA, µmol/L  0.49 (0.37–0.61)  0.56 (0.44–0.79)b  0.63 (0.45–0.92)b  FBG, mmol/L  5.22 ± 0.46  6.18 ± 1.70b  9.76 ± 4.15b,d  2h-BG, mmol/L  6.21 ± 1.02  8.96 ± 1.46b  18.57 ± 7.49b,d  FIns, mU/L  8.49 ± 3.60  11.97 ± 8.61b  11.64 ± 6.46a,c  2h-Ins, mU/L  57.6 ± 45.8  88.7 ± 55.3b  66.7 ± 58.1d  HbA1c, %  5.63 ± 0.56  5.90 ± 0.68  8.33 ± 2.37b,d  HOMA-IR  1.87 (1.32–2.49)  2.54 (1.93–4.13)b  4.00 (2.70–5.93)b,d  ISI  4.62 (3.02–6.71)  3.39 (2.21–4.41)b  2.32 (1.81–3.40)b,c  AUCglucose  14.9 ± 2.4  19.7 ± 2.8b  34.4 ± 11.1b,d  Myonectin, µg/L  45.2 ± 23.5  68.9 ± 46.6b  82.3 ± 47.6b,c  Myonectin, adjustede  45.2 ± 2.1  68.9 ± 4.5b  82.3 ± 4.6b,c  Data are means ± standard deviation or median (interquartile range). Abbreviations: 2h-Ins, 2-hour plasma insulin after glucose overload; DBP, diastolic BP; FBG, fasting blood glucose; FIns, fasting insulin; HDL-C, high-density lipoprotein cholesterol; SBP, systolic BP; SD, standard deviation; TC, total cholesterol. a P < 0.05 compared with NGT group. b P < 0.01 compared with NGT group. c P < 0.05 compared with IGT group. d P < 0.01 compared with IGT group. e Means ± standard error by general linear model with adjustment of age, sex, and BMI. View Large Figure 1. View largeDownload slide Concentrations of circulating myonectin in study population. (A) Circulating myonectin levels according to NGT, IGT, and nT2DM. (B) Circulating myonectin levels according to BMI (normal weight: BMI <24 kg/m2; obese: BMI ≥28 kg/m2). (C) Circulating myonectin levels according to sex. (D) All factors and stepwise multiple regression analyses of the circulating myonectin in all study population. The circles correspond to the regression coefficients (β), and the error bars indicate the 95% confidence interval (CI) of β. (E) Prevalence of elevated IGT in different tertiles of myonectin: tertile 1 (n = 79), <36.6 µg/L; tertile 2 (n = 63), 36.6 to 60.4 µg/L; tertile 3 (n = 95), >60.4 µg/L. (F) Prevalence of elevated nT2DM in different quartiles of myonectin: tertile 1 (n = 62), <44.6 µg/L; tertile 2 (n = 58), 44.6 to 70.2 µg/L; tertile 3 (n = 112), >70.2 µg/L. Data are means ± SD. *P < 0.05 or **P < 0.01 vs. men, lean subjects, NGT, or tertile 1; ▲P < 0.05 vs. IGT. Ln, Log transformed before analysis; R2, coefficient of determination. Figure 1. View largeDownload slide Concentrations of circulating myonectin in study population. (A) Circulating myonectin levels according to NGT, IGT, and nT2DM. (B) Circulating myonectin levels according to BMI (normal weight: BMI <24 kg/m2; obese: BMI ≥28 kg/m2). (C) Circulating myonectin levels according to sex. (D) All factors and stepwise multiple regression analyses of the circulating myonectin in all study population. The circles correspond to the regression coefficients (β), and the error bars indicate the 95% confidence interval (CI) of β. (E) Prevalence of elevated IGT in different tertiles of myonectin: tertile 1 (n = 79), <36.6 µg/L; tertile 2 (n = 63), 36.6 to 60.4 µg/L; tertile 3 (n = 95), >60.4 µg/L. (F) Prevalence of elevated nT2DM in different quartiles of myonectin: tertile 1 (n = 62), <44.6 µg/L; tertile 2 (n = 58), 44.6 to 70.2 µg/L; tertile 3 (n = 112), >70.2 µg/L. Data are means ± SD. *P < 0.05 or **P < 0.01 vs. men, lean subjects, NGT, or tertile 1; ▲P < 0.05 vs. IGT. Ln, Log transformed before analysis; R2, coefficient of determination. The association of circulating myonectin with anthropometric and biochemical parameters in the study population Next, we investigated the association of plasma myonectin levels with various parameters by using partial correlation analysis. Plasma myonectin correlated positively with WHR, FAT%, TG, FBG, 2h-BG, FIns, HbA1c, and HOMA-IR in all study populations but negatively with ISI (Table 2). All of these correlations remained statistically significant despite adjustments for age, sex, and BMI. Multivariate regression analyses showed that HOMA-IR and LDL-C were independently related factors influencing circulating myonectin levels (Table 2). The multiple regression equation was: Ymyonectin = 3.542 + 0.04 XHOMA-IR + 0.102 XLDL-C (R2 = 0.220, P < 0.01). Table 2. Linear Regression Analysis of Variables Associated With Circulating Myonectin Levels in Study Population Variable  Simple  Multiple  r  P Value  β  P Value  Age, y  0.007  NS      BMI, kg/m2  0.010  NS      FAT%, %  0.123  <0.05      WHR  0.161  <0.01      SBP, mm Hg  0.081  NS      DBP, mm Hg  0.021  NS      TC, mmol/L  0.063  NS      TG, mmol/L  0.142  <0.05      HDL-C, mmol/L  −0.046  NS      LDL-C, mmol/L  0.069  NS  0.102  <0.05  FFA, µmol/L  0.104  NS      FBG, mmol/L  0.296  <0.001      2h-BG, mmol/L  0.362  <0.001      FIns, mU/L  0.123  <0.05      2h-Ins, mU/L  0.076  NS      HbA1c, %  0.253  <0.001      HOMA-IR  0.243  <0.001  0.040  <0.05  AUCglucose  0.357  <0.001      ISI  −0.276  <0.001      Variable  Simple  Multiple  r  P Value  β  P Value  Age, y  0.007  NS      BMI, kg/m2  0.010  NS      FAT%, %  0.123  <0.05      WHR  0.161  <0.01      SBP, mm Hg  0.081  NS      DBP, mm Hg  0.021  NS      TC, mmol/L  0.063  NS      TG, mmol/L  0.142  <0.05      HDL-C, mmol/L  −0.046  NS      LDL-C, mmol/L  0.069  NS  0.102  <0.05  FFA, µmol/L  0.104  NS      FBG, mmol/L  0.296  <0.001      2h-BG, mmol/L  0.362  <0.001      FIns, mU/L  0.123  <0.05      2h-Ins, mU/L  0.076  NS      HbA1c, %  0.253  <0.001      HOMA-IR  0.243  <0.001  0.040  <0.05  AUCglucose  0.357  <0.001      ISI  −0.276  <0.001      In multiple linear regression analysis, values included for analysis were age, sex, BMI, WHR, BP, FBG, insulin, HOMA-IR, FFA, TC, HDL-C, LDL-C, and TG. Abbreviations: HDL-C, high-density lipoprotein cholesterol; NS, not significant; TC, total cholesterol. View Large Multivariate logistic regression analysis revealed that circulating myonectin levels were significantly correlated with IGT and T2DM, even after controlling for anthropometric variables, age, sex, FAT%, BP, and lipid profile (Table 3). When considering all subjects as a whole, regression analyses, including all-factor and stepwise models, showed that the main predictor of circulating myonectin concentrations was HOMA-IR (Fig. 1D). When myonectin concentrations were analyzed by the row mean score difference and the Cochran-Armitage trend test, increasing levels led to a significant linear trend and were independently associated with IGT and T2DM (Supplemental Table 1). In addition, we divided myonectin into three tertiles according to the myonectin concentration of the study population (tertile 1, <36.6 µg/L; tertile 2, 36.6 to 60.4 µg/L; and tertile 3, >60.4 µg/L for IGT; tertile 1, <44.6 µg/L; tertile 2, 44.6 to 70.2 µg/L; and tertile 3, >70.2 µg/L for T2DM), and the odds of having IGT and T2DM were calculated using logistic regression analysis. When myonectin concentrations were in tertiles 2 and 3, the odds ratios of having IGT and T2DM were 3.15 [95% confidence interval (CI), 1.55; 6.41] and 4.24 (95% CI, 2.21; 8.13) for IGT and 18.36 (95% CI, 5.16; 65.31) and 36.81 (95% CI, 10.83; 125.11) for T2DM (vs. tertile 1, all P < 0.01; Fig. 1E and 1F). Finally, the ROC curves of circulating myonectin were performed for predicting IGT and T2DM. The area under the ROC curves was 0.66 (P < 0.01) with a sensitivity of 81% and specificity of 48% for IGT (Fig. 2A) and 0.80 (P < 0.01) with a sensitivity of 92% and specificity of 62% for T2DM (Fig. 2B). The best cutoff values for circulating myonectin to predict IGT and T2DM were 37.6 µg/L and 49.7 µg/L, respectively. Table 3. Association of Plasma Myonectin With IGT and nT2DM in Fully Adjusted Models Model Adjustment  IGT  nT2DM  OR  95% CI  P  OR  95% CI  P  Age  1.022  1.012–1.032  <0.001  1.053  1.037–1.069  <0.001  Age, sex  1.023  1.013–1.033  <0.001  1.055  1.038–1.072  <0.001  Age, sex, BMI  1.024  1.014–1.034  <0.001  1.056  1.039–1.074  <0.001  Age, sex, BMI, WHR  1.023  1.013–1.034  <0.001  1.056  1.039–1.074  <0.001  Age, sex, BMI, WHR, FAT%  1.022  1.012–1.032  <0.001  1.055  1.037–1.074  <0.001  Age, sex, BMI, WHR, FAT%, BP  1.023  1.012–1.033  <0.01  1.059  1.039–1.079  <0.001  Age, sex, BMI, WHR, FAT%, BP, lipid profile  1.034  1.015–1.053  <0.05  1.067  1.042–1.092  <0.001  Model Adjustment  IGT  nT2DM  OR  95% CI  P  OR  95% CI  P  Age  1.022  1.012–1.032  <0.001  1.053  1.037–1.069  <0.001  Age, sex  1.023  1.013–1.033  <0.001  1.055  1.038–1.072  <0.001  Age, sex, BMI  1.024  1.014–1.034  <0.001  1.056  1.039–1.074  <0.001  Age, sex, BMI, WHR  1.023  1.013–1.034  <0.001  1.056  1.039–1.074  <0.001  Age, sex, BMI, WHR, FAT%  1.022  1.012–1.032  <0.001  1.055  1.037–1.074  <0.001  Age, sex, BMI, WHR, FAT%, BP  1.023  1.012–1.033  <0.01  1.059  1.039–1.079  <0.001  Age, sex, BMI, WHR, FAT%, BP, lipid profile  1.034  1.015–1.053  <0.05  1.067  1.042–1.092  <0.001  Results of multivariate logistic regression analysis are presented as the odds ratio (OR); nT2DM and IGT status increases in plasma myonectin. Lipid profile includes total cholesterol, TG, LDL-C, high-density lipoprotein cholesterol, and FFA. View Large Figure 2. View largeDownload slide ROC curve analyses were performed for the prediction of (A) IGT and (B) nT2DM, according to the myonectin levels. Figure 2. View largeDownload slide ROC curve analyses were performed for the prediction of (A) IGT and (B) nT2DM, according to the myonectin levels. The effects of a 45-minute bout of exercise, OGTT, EHC, and FFA-induced IR on circulating myonectin in healthy individuals To investigate which factors regulate circulating myonectin levels invivo, we observed the effects of glucose challenge, EHC, a single exercise, and lipid infusion on circulating myonectin in healthy individuals. In an exercise study, the subjects performed a 45-minute bout of treadmill exercise at 60% of maximal oxygen consumption for 45 minutes and then rested for 120 minutes. As shown in Fig. 3A, after 45 minutes of a 45-minute bout of exercise, the level of myonectin has an increasing trend, but it does not reach statistical significance compared with pre-exercise levels (postexercise: 21.7 ± 5.3 µg/L vs. pre-exercise: 18.2 ± 4.4 µg/L). During the OGTT, in response to oral glucose challenge-induced hyperglycemia and hyperinsulinemia, circulating myonectin levels gradually decreased from 25.1 ± 4.8 µg/L at 0 minutes to 21.1 ± 3.5 µg/L at 30 minutes, then to 18.7 ± 2.8 µg/L at 60 minutes, and finally, to 18.5 ± 3.0 µg/L at 120 minutes but did not reach statistical significance (Fig. 3B). Figure 3. View largeDownload slide Circulating myonectin levels in interventional studies. (A) Circulating myonectin in healthy individuals in response to a 45-minute bout of exercise. (B) Circulating myonectin concentrations in healthy subjects during an OGTT. (C) Time course of circulating myonectin changes in healthy subjects during EHC. (D) Time course of circulating myonectin changes in healthy subjects during lipid infusion combined with EHC. Values are means ± standard error. Figure 3. View largeDownload slide Circulating myonectin levels in interventional studies. (A) Circulating myonectin in healthy individuals in response to a 45-minute bout of exercise. (B) Circulating myonectin concentrations in healthy subjects during an OGTT. (C) Time course of circulating myonectin changes in healthy subjects during EHC. (D) Time course of circulating myonectin changes in healthy subjects during lipid infusion combined with EHC. Values are means ± standard error. To investigate the effect of acute hyperinsulinemia, we next performed EHCs on 30 healthy individuals. During the steady state of EHCs, blood glucose concentrations were clamped at 4.5 to 6 mmol/L, and insulin concentrations were raised from 45.0 ± 12.4 to 347.4 ± 54.2 pmol/L. In response to hyperinsulinemia, the GIR needed to maintain euglycemia (expressed as M-values) rose from 0 to 9.58 ± 2.40 mg/kg/min in these young individuals. Circulating myonectin levels showed a gradual decrease (from 19.9 ± 3.6 µg/L at 0 minutes to 17.3 ± 2.4 µg/L at 80 minutes, to 17.0 ± 2.6 µg/L at 100 minutes, then to 15.5 ± 2.5 µg/L at 110 minutes, and finally, to 14.2 ± 2.0 µg/L at 120 minutes) but did not reach statistical significance (Fig. 3C). In addition, in 30 young subjects, fasting myonectin levels correlated negatively with GDR (r = −0.59, P < 0.05) and M-values (r = −0.57, P < 0.05), whereas during the EHCs, average myonectin levels also correlated negatively with GDR (r = −0.56, P < 0.05). These results further suggest that circulating myonectin is related to IR. To investigate the effect of lipid-induced IR on circulating myonectin in vivo, we investigated the changes of circulating myonectin during lipid infusion combined with an EHC in 22 healthy adolescents. During the steady state of EHC (200 to 240 minutes), GIR in lipid-infused individuals was significantly decreased compared with nonlipid-infused subjects (9.58 ± 2.40 vs. 4.53 ± 1.86 mg/kg/min, P < 0.01), suggesting an acute IR. However, during lipid infusion and EHC, circulating myonectin levels had no change compared with that before lipid infusion (Fig. 3D). Discussion In the current study, we found plasma myonectin concentrations from 41.1 to 49.3 µg/L for most healthy subjects. Of note, the mean levels of myonectin reported by Lim et al. (20) (62.8 ± 14.6 µg/L) were higher compared with ours (45.2 ± 23.5 µg/L) in middle-aged and older individuals. This may be a result of a difference in ethnicity and sex or the difference in sample size (Lim et al., n = 14; ours, n = 128). Furthermore, the difference in assay conditions might be a relevant factor. In this study, we show significantly enhanced plasma myonectin levels in IGT subjects and nT2DM patients compared with normal subjects. Importantly, we demonstrate that circulating myonectin is higher in T2DM patients than in prediabetic (IGT) subjects, indicating a progressive increase of myonectin levels from a prediabetic to diabetic state. Therefore, these results may reveal changes in circulating myonectin over time. In addition, we found that circulating myonectin levels were significantly increased in obese subjects, suggesting that myonectin might act as a circulating biomarker of adiposity and obesity-related metabolic diseases. These results are similar to those observed by Park et al. (13) in obese/diabetic animals. In that study, they found that circulating myonectin levels were increased significantly in ob/ob and db/db mice. Therefore, we speculate that myonectin may have a role in the pathogenesis of IR and T2DM. Although the current study did not allow us to deduce the cause of increased myonectin in IGT and T2DM subjects, we consider that this increase in myonectin in IGT or T2DM subjects might be a defensive response to metabolic stress from resistance to myonectin action. The elevated myonectin levels in IGT and T2DM individuals may be the results of the impairment of myonectin signaling in target tissues and the dysregulation of myonectin synthesis or a response to hyperinsulinemia, hyperglycemia, or cytokines in an IR state. In regression analyses, we found that plasma myonectin levels positively correlated with WHR, FAT%, TG, FBG, 2h-BG, FIns, HbA1c, and HOMA-IR but negatively correlated with ISI in the general population, suggesting that myonectin may play a role in regulating glucose and TG metabolism and be relative to IR. Given the positive relationship between myonectin and blood glucose, insulin, and HbA1c, it is possible that circulating myonectin levels are regulated by either blood glucose or insulin. To address this question, we performed an OGTT (a high-glucose and high-insulin state) to assess the effects of rapidly increasing glucose and insulin levels on circulating myonectin in young individuals. However, in response to the oral glucose challenge-induced hyperglycemia and hyperinsulinemia, circulating myonectin levels showed no change. This result indicated that an acute increase in blood glucose or insulin levels induced by OGTT may have no effect on myonectin release, or the change in circulating myonectin levels was counteracted by the hyperglycemia–hyperinsulinemia interaction. To address this issue, we performed an EHC study (an euglycemic-hyperinsulinemic state) to control blood glucose levels and assess the effect of hyperinsulinemia on circulating myonectin in healthy individuals. We found that short-term hyperinsulinemia in normal subjects failed to change circulating myonectin levels during EHC, suggesting that circulating myonectin may not be regulated by short-term hyperinsulinemia and hyperglycemia. It is generally accepted that metabolic regulation in T2DM and IR subjects is influenced by their diet and physical activity. In fact, physical activity is thought to protect against several types of disease, including cardiovascular disease, T2DM, and certain types of cancer (21). Recently, muscle tissues have been considered to be an endocrine organ that releases some cytokines, termed myokines, which regulate some physiological and metabolic pathways (22). Myonectin has been identified as a myokine, expressed predominantly by muscle tissues (12). To investigate whether myonectin release or secretion is responsive to an alteration in the metabolic state of skeletal muscle after exercise, we observed the effects of a 45-minute bout of exercise on myonectin levels in young individuals. We found that a 45-minute bout of exercise did not result in an increase or decrease in myonectin circulating levels. In animal studies, however, Peterson et al. (23) reported that myonectin protein content in the muscle was elevated with chronic exercise (9 weeks). In addition, contrary to our findings, Lim et al. (20) found that a 10-week exercise training program significantly decreased circulating myonectin levels in healthy women. Although the reason for the discrepancy is not clear, possible explanations include the following: (1) methodological limitations, including different kits used to measure myonectin; (2) that a relatively small number and only healthy women were selected in that study; and (3) the different effects of a single or long-term exercise. Collectively, we consider that the changes of myonectin levels in circulation may represent long-term IR status, and like adiponectin, it may serve as a regulator of insulin sensitivity. Thus, it would be interesting to investigate whether insulin sensitizers, such as thiazolidinediones, could affect myonectin levels as well. Some limitations of this study should be taken into consideration when interpreting our results. (1) Our study population was limited to Chinese subjects. Therefore, our findings may not be directly applicable to all populations. (2) The number of participants was relatively small, and the age of subjects was relatively old. (3) The cross-sectional design limits our ability to infer a causal relationship between circulating myonectin and IR in T2DM patients. (4) Finally, myonectin levels were not a prespecified end point for recruited IGT and T2DM individuals in the study, and measurements of myonectin were made on stored samples. Nonetheless, our data are sufficient to demonstrate associations of circulating myonectin with anthropometric and metabolic parameters and IR in IGT and T2DM subjects. In summary, our data demonstrate that circulating myonectin concentrations progressively increased from prediabetic to diabetic state and were associated with glucose homoeostasis and insulin sensitivity. Circulating myonectin levels were not affected by an oral glucose challenge (a high-glucose and high-insulin state), EHC (an euglycemic-hyperinsulinemic state), or a 45-minute bout of exercise. Thus, our data suggest that myonectin may be a useful marker in the detection of prediabetes and diabetes. However, further studies are needed to clarify the biological mechanisms involving myonectin in the pathogenesis of IR. Abbreviations: 2h-BG 2-hour blood glucose after glucose overload AUCglucose area under the curve for glucose BMI body mass index BP blood pressure CI confidence interval EHC euglycemic-hyperinsulinemic clamp FAT% percentage of body fat FBG fasting blood glucose FFA free fatty acid FIns fasting insulin GDR glucose disposal rate GIR glucose infusion rate HbA1c hemoglobin A1c HOMA-IR homeostasis model assessment of insulin resistance IGT impaired glucose tolerance IR insulin resistance ISI insulin sensitivity index LDL-C low-density lipoprotein cholesterol NGT normal glucose tolerance nT2DM newly diagnosed type 2 diabetes mellitus OGTT oral glucose tolerance test ROC receiver operating characteristic T2DM type 2 diabetes mellitus TG triglyceride WHR waist/hip ratio. Acknowledgments Financial Support: This work was supported by research grants from the National Natural Science Foundation of China (81670755, 81570752), Doctoral Fund of Ministry of Education of China (20105503110002), and Natural Science Foundation Key Project of CQcstc (cstc2012 jjB10022) to G.Y. Clinical Trial Information: chictr.org.cn no. ChiCTR-OCC-11001422 (registered 23 June 2011). Author Contributions: K.L., Q.M., T.Z., X. Liao, C.Z., and X.X. researched data. H.L., H.Z., and Y.J. reviewed and edited the manuscript. L.L. and G.Y. researched data and wrote and edited the manuscript. X. Luo and K.W. analyzed and revised the manuscript. L.L. and G.Y. are the guarantors of this work and as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose. References 1. 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Oxford University Press
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Copyright © 2018 Endocrine Society
ISSN
0021-972X
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1945-7197
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10.1210/jc.2017-01604
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Abstract

Abstract Context Myonectin has been identified as a myokine, expressed predominantly in skeletal muscle. However, its clinical implications are largely unknown. Objective The aim of this study is to investigate the relationship between myonectin (C1q tumor necrosis factor-α-related protein isoform 15) and type 2 diabetes mellitus (T2DM) in cross-sectional and interventional studies. Design In a separate study, oral glucose tolerance tests, a 45-minute bout of exercise, lipid infusions, and euglycemic-hyperinsulinemic clamps (EHCs) were performed to investigate the association of myonectin with homeostasis model assessment of insulin resistance (HOMA-IR) and T2DM. Circulating myonectin was measured by enzyme-linked immunosorbent assay. Patients One hundred four newly diagnosed T2DM (nT2DM), 109 impaired glucose tolerance (IGT), and 128 healthy individuals were recruited for this study. Results nT2DM and IGT subjects had higher circulating myonectin concentrations than normal subjects (82.3 ± 47.6 and 68.9 ± 46.6 vs. 45.2 ± 23.5 µg/L, P < 0.05 or P < 0.01). It was also found that in nT2DM individuals, circulating myonectin was higher than in IGT subjects. Plasma myonectin correlated positively with waist/hip ratio, percentage of body fat, triglyceride, fasting blood glucose, 2-hour blood glucose after glucose overload, fasting insulin, hemoglobin A1c, and HOMA-IR and negatively with the insulin sensitivity index in all of the study population. Multivariate logistic regression analysis revealed that circulating myonectin levels were significantly correlated with IGT and T2DM. A 45-minute bout of exercise did not change circulating myonectin levels in healthy, young individuals. Circulating myonectin levels were not significantly altered in response to an oral glucose challenge or EHC. In addition, acute elevated free fatty acid levels induced by lipid infusion had no effects on circulating myonectin. Conclusions These data suggest that myonectin may be a useful marker in predicting the development of prediabetes and diabetes. With the rapid increase in prevalence during the last two decades, type 2 diabetes mellitus (T2DM) has become one of the main threats to public health in the world. It has been estimated that >10% of Chinese adults have T2DM (1). T2DM patients have a greater risk for the development of cardiovascular disease (2, 3), with coexistent hypertension further increasing the risk of cardiovascular complications (4). Insulin resistance (IR) has been hypothesized to be the common pathophysiologic factor behind T2DM and hypertension (5). However, its pathogenesis is complex and still unclear. Muscle tissue is an important target tissue of IR and has been recognized as an active endocrine organ producing myokines, such as IL-6 (6), fibroblast growth factor 21 (7), and irisin (8), which modulate insulin sensitivity and thus, may play a role in the pathogenesis of IR. Myonectin, also known as C1q tumor necrosis factor-α-related protein isoform 15, is a myokine and belongs to a family of proteins characterized by an N-terminal signal peptide, a collagen repeat domain, and a C-terminal C1q-like globular domain (9). Myonectin is homologous to adiponectin with respect to domain structure, and it is expressed and secreted primarily by muscle tissues (10). Recently, it has been reported that the expression and plasma levels of myonectin are subject to metabolic control (11). In a mouse study, Seldin et al. (12) found that myonectin expression and high circulating levels were induced by refeeding following an overnight fast. Infusion of recombinant myonectin in vivo lowered circulating levels of free fatty acids (FFAs), in part, by promoting cellular lipid uptake and upregulating the expression of genes involved in lipid uptake (12). Interestingly, it has been shown that circulating myonectin levels are increased when overnight-fasted mice are given a bolus of glucose or emulsified fat (11). Moreover, circulating myonectin levels increased significantly in obese/diabetic animals (12, 13). These reports suggest that myonectin may be a nutrient-regulated cytokine and may have a functional role in IR. Therefore, myonectin seems to be a promising candidate for involvement in the pathogenesis of IR and T2DM. However, there is still a lack of information regarding the relationship among circulating levels of myonectin and IR and diabetes in humans. In the current study, we evaluate circulating levels of myonectin in normal subjects and subjects with impaired glucose tolerance (IGT) and in newly diagnosed T2DM (nT2DM) patients. We also investigate the effects of an oral glucose challenge, hyperinsulinemia, and FFA-induced IR on circulating myonectin in these subjects. Materials and Methods Cross-sectional studies Three hundred forty-one subjects, including 104 nT2DM, 109 IGT, and 128 healthy controls, were recruited to the study. IGT and T2DM were determined according to the World Health Organization diagnostic criteria (14). All individuals with IGT and patients with T2DM were newly diagnosed and were not treated with any hypoglycemic agents, diet control, or physical exercise. Exclusion criteria were patients with type 1 diabetes mellitus and patients with macrovascular or microvascular complications, hypertension, liver cirrhosis, hepatic and renal failure, congestive heart failure, or other major diseases. One hundred twenty-eight age-matched, healthy individuals without any clinical evidence of diseases were recruited from the community or schools through advertisement or routine medical checkups and were used as the controls [normal glucose tolerance (NGT)]. In these individuals, T2DM was excluded by a normal oral glucose tolerance test (OGTT), and the family history of T2DM was also excluded. None of these individuals was taking any medication-related glucose and lipid metabolism, as well as insulin sensitivity. In this study, all premenopausal women had regular menstrual cycles and were studied in the early follicular phase of the menstrual cycle (days 3 to 5 of the cycle). All subjects signed voluntary consent before experiment. The study was approved by the Human Research Ethics Committee of Chongqing Medical University and was registered at the Chinese Clinical Trial Registry chictr.org.cn (CHICTR-OCC-11001422). OGTT To investigate the effects of an oral glucose challenge on circulating myonectin levels in subjects without IR, OGTT was performed in 30 normal, young individuals, including 15 men and 15 women [age: 25 ± 2 years, body mass index (BMI): 21.5 ± 2.1 kg/m2]. In addition, OGTT was performed in all study populations. At 7:00 on the morning of the study days, after a 12-hour overnight fast, all individuals were given a 2-hour, 75-g OGTT, and venous blood was drawn at indicated times (0, 30, 60, and 120 minutes) for measurement of glucose, insulin, or circulating myonectin for 30 young individuals. Euglycemic-hyperinsulinemic clamp The euglycemic-hyperinsulinemic clamps (EHCs) were performed in 30 normal, young individuals, including 15 men and 15 women (age: 25 ± 2 years, BMI: 21.5 ± 2.1 kg/m2), as previously described (15, 16). During the EHCs, regular human insulin (1 mU/kg/min) was infused for 2 hours, and a variable infusion of 20% glucose was administered to maintain plasma glucose at the fasting level. The glucose disposal rate (GDR) was defined as the glucose infusion rate (GIR) during the stable period of the EHC and was related to body weight (M-value). Blood samples for myonectin measurement were obtained at indicated times. The samples were immediately cooled, and plasma was prepared and stored at −80°C until used. Lipid infusion study Lipid infusion was performed in 22 normal, young individuals (11 women and 11 men). These subjects received a 20% intralipid/heparin (0.4 units/kg/min; Pharmacia and Upjohn, Milan, Italy) infusion at a constant rate (1.5 mL/min) for 240 minutes. Two hours after the start of the lipid infusion, a 2-hour EHC was performed as described previously. Blood samples were collected before and at indicated times during lipid infusion. Samples were separated and kept at −80°C for myonectin measurement. Exercise testing procedures A 45-minute bout of treadmill exercise was performed in 12 young subjects (six men and six women; age: 21 to 23 years, BMI: 21.0 ± 1.2 kg/m2). Individuals were excluded if they were taking any medications, presented any contraindications to physical activity, or participated in 20 minutes or more of exercise at least two times per week. After a 12-hour overnight fast, individuals were arranged to ward between 8:00 and 8:30 on the morning of the experiment. A 45-minute bout of exercise was performed in these subjects with 60% of maximal oxygen consumption for 45 minutes. Blood samples were drawn at four time points, including baseline, 45 minutes following a 45-minute bout of exercise, and 60 and 120 minutes at rest after the exercise. Measurements of plasma myonectin Circulating myonectin concentrations were determined in blood samples with a commercial enzyme-linked immunosorbent assay kit following the manufacturer’s protocol (Catalog No. SK00393-19; Aviscera Bioscience, Santa Clara, CA). The limit of detection was 31.25 ng/mL, and intra-assay and interassay variations were <8% and <12%, respectively. Measurements of anthropometric and biochemical parameters The anthropometric determinations and the blood sample collections for biochemicals and other parameters were carried out on the same day. BMI was calculated as weight divided by height squared. Waist circumference and hip circumference were measured, and the waist/hip ratio (WHR) was calculated by the same researcher. Blood pressure (BP) was measured in all individual after they rested at least for 15 minutes. The percentage of body fat (FAT%) was measured by bioelectrical impedance (BIA-101; RJL Systems, Shenzhen, China). Homeostasis model assessment of IR (HOMA-IR) and insulin sensitivity index (ISI) were calculated as previously reported (17, 18). The area under the curve for glucose (AUCglucose) during the OGTT was calculated geometrically following the trapezoidal rule by using a statistical software program (19). Insulin concentrations were measured using chemiluminescence. Hemoglobin A1c (HbA1c) and glucose were measured by the glucose-oxidase method and anion-exchange HPLC, respectively. FFAs, total cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol were measured with a commercial kit. Statistical analysis All statistical analyses were completed with SPSS version 22.0 (SPSS, Armonk, NY). The distribution of data was examined by Kolmogorov-Smirnov test. analysis of variance, a paired t test, or unpaired t test was used for comparison among groups. The relationship between myonectin and other parameters was examined using partial correlation coefficients. We used multivariate regression analyses to assess variables that had associations with circulating myonectin. The Cochran-Armitage trend test was used to analyze the tendency of concentration of circulating myonectin associated with IGT and T2DM. The cutoff points of myonectin concentrations for predicting T2DM and IGT were given by receiver operating characteristics (ROCs) curves. All data were shown as means ± standard deviation, standard error, or median (interquartile range). When compared with the controls, P < 0.05 was considered significant. Results Distribution of circulating myonectin in healthy subjects and characteristics of study participants Fasting myonectin levels were measured in 128 healthy subjects (from 42 to 79 years). Circulating myonectin concentrations in healthy individuals ranged from 41.1 to 49.3 µg/L for the 95% of healthy population. The clinical baseline characteristics of the subgroups (NGT, IGT, and nT2DM) are shown in Table 1. There are no statistically significant differences among IGT, T2DM, and NGT subjects with respect to age, FAT%, total cholesterol, and LDL-C. When compared with the NGT, BMI, WHR, BP, FFA, fasting blood glucose (FBG), 2-hour blood glucose after glucose overload (2h-BG), fasting insulin (FIns), AUCglucose, and HOMA-IR in both IGT and nT2DM groups were significantly increased (P < 0.01 or P < 0.05), whereas high-density lipoprotein cholesterol and ISI were significantly decreased in the nT2DM group (P < 0.01 or P < 0.05). Importantly, circulating myonectin levels were higher in IGT and nT2DM individuals than in control subjects (P < 0.05 or P < 0.01; Fig. 1A). When compared with IGT subjects, T2DM patients had higher circulating myonectin levels (P < 0.05; Fig. 1A). These differences remained significant after adjustment for sex and age. In addition, obese individuals based on Chinese criteria (BMI ≥ 28 kg/m2) had significantly higher myonectin concentrations than lean individuals (BMI < 24 kg/m2) in all study populations (67.7 ± 19.7 vs. 55.9 ± 27.2 µg/L, P < 0.05; Fig. 1B). Significantly higher plasma concentrations of myonectin were also observed in female patients with IGT and nT2DM than in male patients (74.4 ± 53.0 vs. 56.9 ± 24.5 µg/L for IGT; 91.8 ± 60.1 vs. 72.1 ± 25.4 µg/L for nT2DM; both P < 0.05; Fig. 1C). However, no difference was observed in circulating myonectin levels between men and women in the control group (Fig. 1C). Table 1. Main Clinical Features and Circulating Myonectin Levels in the Study Population Variable  NGT (n = 128)  IGT (n = 109)  nT2DM (n = 104)  Men/women  55/73  46/63  50/54  Age, y  54 ± 9  55 ± 11  56 ± 10  BMI, kg/m2  23.5 ± 3.1  24.42 ± 2.98a  24.47 ± 3.17a  FAT%, %  29.49 ± 7.16  31.57 ± 7.84  30.87 ± 9.90  WHR  0.88 ± 0.08  0.91 ± 0.08a  0.90 ± 0.11a  SBP, mm Hg  118.0 ± 13.8  125.3 ± 17.0b  129.2 ± 16.5b  DBP, mm Hg  75.2 ± 9.9  77.6 ± 11.8  81.1 ± 11.6b,c  TC, mmol/L  4.98 ± 0.94  5.22 ± 1.25  5.12 ± 1.08  TG, mmol/L  1.56 ± 1.05  2.03 ± 1.14  2.12 ± 1.47  HDL-C, mmol/L  1.33 ± 0.31  1.40 ± 0.46  1.22 ± 0.41a,d  LDL-C, mmol/L  2.92 ± 0.77  2.97 ± 1.05  3.06 ± 0.90  FFA, µmol/L  0.49 (0.37–0.61)  0.56 (0.44–0.79)b  0.63 (0.45–0.92)b  FBG, mmol/L  5.22 ± 0.46  6.18 ± 1.70b  9.76 ± 4.15b,d  2h-BG, mmol/L  6.21 ± 1.02  8.96 ± 1.46b  18.57 ± 7.49b,d  FIns, mU/L  8.49 ± 3.60  11.97 ± 8.61b  11.64 ± 6.46a,c  2h-Ins, mU/L  57.6 ± 45.8  88.7 ± 55.3b  66.7 ± 58.1d  HbA1c, %  5.63 ± 0.56  5.90 ± 0.68  8.33 ± 2.37b,d  HOMA-IR  1.87 (1.32–2.49)  2.54 (1.93–4.13)b  4.00 (2.70–5.93)b,d  ISI  4.62 (3.02–6.71)  3.39 (2.21–4.41)b  2.32 (1.81–3.40)b,c  AUCglucose  14.9 ± 2.4  19.7 ± 2.8b  34.4 ± 11.1b,d  Myonectin, µg/L  45.2 ± 23.5  68.9 ± 46.6b  82.3 ± 47.6b,c  Myonectin, adjustede  45.2 ± 2.1  68.9 ± 4.5b  82.3 ± 4.6b,c  Variable  NGT (n = 128)  IGT (n = 109)  nT2DM (n = 104)  Men/women  55/73  46/63  50/54  Age, y  54 ± 9  55 ± 11  56 ± 10  BMI, kg/m2  23.5 ± 3.1  24.42 ± 2.98a  24.47 ± 3.17a  FAT%, %  29.49 ± 7.16  31.57 ± 7.84  30.87 ± 9.90  WHR  0.88 ± 0.08  0.91 ± 0.08a  0.90 ± 0.11a  SBP, mm Hg  118.0 ± 13.8  125.3 ± 17.0b  129.2 ± 16.5b  DBP, mm Hg  75.2 ± 9.9  77.6 ± 11.8  81.1 ± 11.6b,c  TC, mmol/L  4.98 ± 0.94  5.22 ± 1.25  5.12 ± 1.08  TG, mmol/L  1.56 ± 1.05  2.03 ± 1.14  2.12 ± 1.47  HDL-C, mmol/L  1.33 ± 0.31  1.40 ± 0.46  1.22 ± 0.41a,d  LDL-C, mmol/L  2.92 ± 0.77  2.97 ± 1.05  3.06 ± 0.90  FFA, µmol/L  0.49 (0.37–0.61)  0.56 (0.44–0.79)b  0.63 (0.45–0.92)b  FBG, mmol/L  5.22 ± 0.46  6.18 ± 1.70b  9.76 ± 4.15b,d  2h-BG, mmol/L  6.21 ± 1.02  8.96 ± 1.46b  18.57 ± 7.49b,d  FIns, mU/L  8.49 ± 3.60  11.97 ± 8.61b  11.64 ± 6.46a,c  2h-Ins, mU/L  57.6 ± 45.8  88.7 ± 55.3b  66.7 ± 58.1d  HbA1c, %  5.63 ± 0.56  5.90 ± 0.68  8.33 ± 2.37b,d  HOMA-IR  1.87 (1.32–2.49)  2.54 (1.93–4.13)b  4.00 (2.70–5.93)b,d  ISI  4.62 (3.02–6.71)  3.39 (2.21–4.41)b  2.32 (1.81–3.40)b,c  AUCglucose  14.9 ± 2.4  19.7 ± 2.8b  34.4 ± 11.1b,d  Myonectin, µg/L  45.2 ± 23.5  68.9 ± 46.6b  82.3 ± 47.6b,c  Myonectin, adjustede  45.2 ± 2.1  68.9 ± 4.5b  82.3 ± 4.6b,c  Data are means ± standard deviation or median (interquartile range). Abbreviations: 2h-Ins, 2-hour plasma insulin after glucose overload; DBP, diastolic BP; FBG, fasting blood glucose; FIns, fasting insulin; HDL-C, high-density lipoprotein cholesterol; SBP, systolic BP; SD, standard deviation; TC, total cholesterol. a P < 0.05 compared with NGT group. b P < 0.01 compared with NGT group. c P < 0.05 compared with IGT group. d P < 0.01 compared with IGT group. e Means ± standard error by general linear model with adjustment of age, sex, and BMI. View Large Figure 1. View largeDownload slide Concentrations of circulating myonectin in study population. (A) Circulating myonectin levels according to NGT, IGT, and nT2DM. (B) Circulating myonectin levels according to BMI (normal weight: BMI <24 kg/m2; obese: BMI ≥28 kg/m2). (C) Circulating myonectin levels according to sex. (D) All factors and stepwise multiple regression analyses of the circulating myonectin in all study population. The circles correspond to the regression coefficients (β), and the error bars indicate the 95% confidence interval (CI) of β. (E) Prevalence of elevated IGT in different tertiles of myonectin: tertile 1 (n = 79), <36.6 µg/L; tertile 2 (n = 63), 36.6 to 60.4 µg/L; tertile 3 (n = 95), >60.4 µg/L. (F) Prevalence of elevated nT2DM in different quartiles of myonectin: tertile 1 (n = 62), <44.6 µg/L; tertile 2 (n = 58), 44.6 to 70.2 µg/L; tertile 3 (n = 112), >70.2 µg/L. Data are means ± SD. *P < 0.05 or **P < 0.01 vs. men, lean subjects, NGT, or tertile 1; ▲P < 0.05 vs. IGT. Ln, Log transformed before analysis; R2, coefficient of determination. Figure 1. View largeDownload slide Concentrations of circulating myonectin in study population. (A) Circulating myonectin levels according to NGT, IGT, and nT2DM. (B) Circulating myonectin levels according to BMI (normal weight: BMI <24 kg/m2; obese: BMI ≥28 kg/m2). (C) Circulating myonectin levels according to sex. (D) All factors and stepwise multiple regression analyses of the circulating myonectin in all study population. The circles correspond to the regression coefficients (β), and the error bars indicate the 95% confidence interval (CI) of β. (E) Prevalence of elevated IGT in different tertiles of myonectin: tertile 1 (n = 79), <36.6 µg/L; tertile 2 (n = 63), 36.6 to 60.4 µg/L; tertile 3 (n = 95), >60.4 µg/L. (F) Prevalence of elevated nT2DM in different quartiles of myonectin: tertile 1 (n = 62), <44.6 µg/L; tertile 2 (n = 58), 44.6 to 70.2 µg/L; tertile 3 (n = 112), >70.2 µg/L. Data are means ± SD. *P < 0.05 or **P < 0.01 vs. men, lean subjects, NGT, or tertile 1; ▲P < 0.05 vs. IGT. Ln, Log transformed before analysis; R2, coefficient of determination. The association of circulating myonectin with anthropometric and biochemical parameters in the study population Next, we investigated the association of plasma myonectin levels with various parameters by using partial correlation analysis. Plasma myonectin correlated positively with WHR, FAT%, TG, FBG, 2h-BG, FIns, HbA1c, and HOMA-IR in all study populations but negatively with ISI (Table 2). All of these correlations remained statistically significant despite adjustments for age, sex, and BMI. Multivariate regression analyses showed that HOMA-IR and LDL-C were independently related factors influencing circulating myonectin levels (Table 2). The multiple regression equation was: Ymyonectin = 3.542 + 0.04 XHOMA-IR + 0.102 XLDL-C (R2 = 0.220, P < 0.01). Table 2. Linear Regression Analysis of Variables Associated With Circulating Myonectin Levels in Study Population Variable  Simple  Multiple  r  P Value  β  P Value  Age, y  0.007  NS      BMI, kg/m2  0.010  NS      FAT%, %  0.123  <0.05      WHR  0.161  <0.01      SBP, mm Hg  0.081  NS      DBP, mm Hg  0.021  NS      TC, mmol/L  0.063  NS      TG, mmol/L  0.142  <0.05      HDL-C, mmol/L  −0.046  NS      LDL-C, mmol/L  0.069  NS  0.102  <0.05  FFA, µmol/L  0.104  NS      FBG, mmol/L  0.296  <0.001      2h-BG, mmol/L  0.362  <0.001      FIns, mU/L  0.123  <0.05      2h-Ins, mU/L  0.076  NS      HbA1c, %  0.253  <0.001      HOMA-IR  0.243  <0.001  0.040  <0.05  AUCglucose  0.357  <0.001      ISI  −0.276  <0.001      Variable  Simple  Multiple  r  P Value  β  P Value  Age, y  0.007  NS      BMI, kg/m2  0.010  NS      FAT%, %  0.123  <0.05      WHR  0.161  <0.01      SBP, mm Hg  0.081  NS      DBP, mm Hg  0.021  NS      TC, mmol/L  0.063  NS      TG, mmol/L  0.142  <0.05      HDL-C, mmol/L  −0.046  NS      LDL-C, mmol/L  0.069  NS  0.102  <0.05  FFA, µmol/L  0.104  NS      FBG, mmol/L  0.296  <0.001      2h-BG, mmol/L  0.362  <0.001      FIns, mU/L  0.123  <0.05      2h-Ins, mU/L  0.076  NS      HbA1c, %  0.253  <0.001      HOMA-IR  0.243  <0.001  0.040  <0.05  AUCglucose  0.357  <0.001      ISI  −0.276  <0.001      In multiple linear regression analysis, values included for analysis were age, sex, BMI, WHR, BP, FBG, insulin, HOMA-IR, FFA, TC, HDL-C, LDL-C, and TG. Abbreviations: HDL-C, high-density lipoprotein cholesterol; NS, not significant; TC, total cholesterol. View Large Multivariate logistic regression analysis revealed that circulating myonectin levels were significantly correlated with IGT and T2DM, even after controlling for anthropometric variables, age, sex, FAT%, BP, and lipid profile (Table 3). When considering all subjects as a whole, regression analyses, including all-factor and stepwise models, showed that the main predictor of circulating myonectin concentrations was HOMA-IR (Fig. 1D). When myonectin concentrations were analyzed by the row mean score difference and the Cochran-Armitage trend test, increasing levels led to a significant linear trend and were independently associated with IGT and T2DM (Supplemental Table 1). In addition, we divided myonectin into three tertiles according to the myonectin concentration of the study population (tertile 1, <36.6 µg/L; tertile 2, 36.6 to 60.4 µg/L; and tertile 3, >60.4 µg/L for IGT; tertile 1, <44.6 µg/L; tertile 2, 44.6 to 70.2 µg/L; and tertile 3, >70.2 µg/L for T2DM), and the odds of having IGT and T2DM were calculated using logistic regression analysis. When myonectin concentrations were in tertiles 2 and 3, the odds ratios of having IGT and T2DM were 3.15 [95% confidence interval (CI), 1.55; 6.41] and 4.24 (95% CI, 2.21; 8.13) for IGT and 18.36 (95% CI, 5.16; 65.31) and 36.81 (95% CI, 10.83; 125.11) for T2DM (vs. tertile 1, all P < 0.01; Fig. 1E and 1F). Finally, the ROC curves of circulating myonectin were performed for predicting IGT and T2DM. The area under the ROC curves was 0.66 (P < 0.01) with a sensitivity of 81% and specificity of 48% for IGT (Fig. 2A) and 0.80 (P < 0.01) with a sensitivity of 92% and specificity of 62% for T2DM (Fig. 2B). The best cutoff values for circulating myonectin to predict IGT and T2DM were 37.6 µg/L and 49.7 µg/L, respectively. Table 3. Association of Plasma Myonectin With IGT and nT2DM in Fully Adjusted Models Model Adjustment  IGT  nT2DM  OR  95% CI  P  OR  95% CI  P  Age  1.022  1.012–1.032  <0.001  1.053  1.037–1.069  <0.001  Age, sex  1.023  1.013–1.033  <0.001  1.055  1.038–1.072  <0.001  Age, sex, BMI  1.024  1.014–1.034  <0.001  1.056  1.039–1.074  <0.001  Age, sex, BMI, WHR  1.023  1.013–1.034  <0.001  1.056  1.039–1.074  <0.001  Age, sex, BMI, WHR, FAT%  1.022  1.012–1.032  <0.001  1.055  1.037–1.074  <0.001  Age, sex, BMI, WHR, FAT%, BP  1.023  1.012–1.033  <0.01  1.059  1.039–1.079  <0.001  Age, sex, BMI, WHR, FAT%, BP, lipid profile  1.034  1.015–1.053  <0.05  1.067  1.042–1.092  <0.001  Model Adjustment  IGT  nT2DM  OR  95% CI  P  OR  95% CI  P  Age  1.022  1.012–1.032  <0.001  1.053  1.037–1.069  <0.001  Age, sex  1.023  1.013–1.033  <0.001  1.055  1.038–1.072  <0.001  Age, sex, BMI  1.024  1.014–1.034  <0.001  1.056  1.039–1.074  <0.001  Age, sex, BMI, WHR  1.023  1.013–1.034  <0.001  1.056  1.039–1.074  <0.001  Age, sex, BMI, WHR, FAT%  1.022  1.012–1.032  <0.001  1.055  1.037–1.074  <0.001  Age, sex, BMI, WHR, FAT%, BP  1.023  1.012–1.033  <0.01  1.059  1.039–1.079  <0.001  Age, sex, BMI, WHR, FAT%, BP, lipid profile  1.034  1.015–1.053  <0.05  1.067  1.042–1.092  <0.001  Results of multivariate logistic regression analysis are presented as the odds ratio (OR); nT2DM and IGT status increases in plasma myonectin. Lipid profile includes total cholesterol, TG, LDL-C, high-density lipoprotein cholesterol, and FFA. View Large Figure 2. View largeDownload slide ROC curve analyses were performed for the prediction of (A) IGT and (B) nT2DM, according to the myonectin levels. Figure 2. View largeDownload slide ROC curve analyses were performed for the prediction of (A) IGT and (B) nT2DM, according to the myonectin levels. The effects of a 45-minute bout of exercise, OGTT, EHC, and FFA-induced IR on circulating myonectin in healthy individuals To investigate which factors regulate circulating myonectin levels invivo, we observed the effects of glucose challenge, EHC, a single exercise, and lipid infusion on circulating myonectin in healthy individuals. In an exercise study, the subjects performed a 45-minute bout of treadmill exercise at 60% of maximal oxygen consumption for 45 minutes and then rested for 120 minutes. As shown in Fig. 3A, after 45 minutes of a 45-minute bout of exercise, the level of myonectin has an increasing trend, but it does not reach statistical significance compared with pre-exercise levels (postexercise: 21.7 ± 5.3 µg/L vs. pre-exercise: 18.2 ± 4.4 µg/L). During the OGTT, in response to oral glucose challenge-induced hyperglycemia and hyperinsulinemia, circulating myonectin levels gradually decreased from 25.1 ± 4.8 µg/L at 0 minutes to 21.1 ± 3.5 µg/L at 30 minutes, then to 18.7 ± 2.8 µg/L at 60 minutes, and finally, to 18.5 ± 3.0 µg/L at 120 minutes but did not reach statistical significance (Fig. 3B). Figure 3. View largeDownload slide Circulating myonectin levels in interventional studies. (A) Circulating myonectin in healthy individuals in response to a 45-minute bout of exercise. (B) Circulating myonectin concentrations in healthy subjects during an OGTT. (C) Time course of circulating myonectin changes in healthy subjects during EHC. (D) Time course of circulating myonectin changes in healthy subjects during lipid infusion combined with EHC. Values are means ± standard error. Figure 3. View largeDownload slide Circulating myonectin levels in interventional studies. (A) Circulating myonectin in healthy individuals in response to a 45-minute bout of exercise. (B) Circulating myonectin concentrations in healthy subjects during an OGTT. (C) Time course of circulating myonectin changes in healthy subjects during EHC. (D) Time course of circulating myonectin changes in healthy subjects during lipid infusion combined with EHC. Values are means ± standard error. To investigate the effect of acute hyperinsulinemia, we next performed EHCs on 30 healthy individuals. During the steady state of EHCs, blood glucose concentrations were clamped at 4.5 to 6 mmol/L, and insulin concentrations were raised from 45.0 ± 12.4 to 347.4 ± 54.2 pmol/L. In response to hyperinsulinemia, the GIR needed to maintain euglycemia (expressed as M-values) rose from 0 to 9.58 ± 2.40 mg/kg/min in these young individuals. Circulating myonectin levels showed a gradual decrease (from 19.9 ± 3.6 µg/L at 0 minutes to 17.3 ± 2.4 µg/L at 80 minutes, to 17.0 ± 2.6 µg/L at 100 minutes, then to 15.5 ± 2.5 µg/L at 110 minutes, and finally, to 14.2 ± 2.0 µg/L at 120 minutes) but did not reach statistical significance (Fig. 3C). In addition, in 30 young subjects, fasting myonectin levels correlated negatively with GDR (r = −0.59, P < 0.05) and M-values (r = −0.57, P < 0.05), whereas during the EHCs, average myonectin levels also correlated negatively with GDR (r = −0.56, P < 0.05). These results further suggest that circulating myonectin is related to IR. To investigate the effect of lipid-induced IR on circulating myonectin in vivo, we investigated the changes of circulating myonectin during lipid infusion combined with an EHC in 22 healthy adolescents. During the steady state of EHC (200 to 240 minutes), GIR in lipid-infused individuals was significantly decreased compared with nonlipid-infused subjects (9.58 ± 2.40 vs. 4.53 ± 1.86 mg/kg/min, P < 0.01), suggesting an acute IR. However, during lipid infusion and EHC, circulating myonectin levels had no change compared with that before lipid infusion (Fig. 3D). Discussion In the current study, we found plasma myonectin concentrations from 41.1 to 49.3 µg/L for most healthy subjects. Of note, the mean levels of myonectin reported by Lim et al. (20) (62.8 ± 14.6 µg/L) were higher compared with ours (45.2 ± 23.5 µg/L) in middle-aged and older individuals. This may be a result of a difference in ethnicity and sex or the difference in sample size (Lim et al., n = 14; ours, n = 128). Furthermore, the difference in assay conditions might be a relevant factor. In this study, we show significantly enhanced plasma myonectin levels in IGT subjects and nT2DM patients compared with normal subjects. Importantly, we demonstrate that circulating myonectin is higher in T2DM patients than in prediabetic (IGT) subjects, indicating a progressive increase of myonectin levels from a prediabetic to diabetic state. Therefore, these results may reveal changes in circulating myonectin over time. In addition, we found that circulating myonectin levels were significantly increased in obese subjects, suggesting that myonectin might act as a circulating biomarker of adiposity and obesity-related metabolic diseases. These results are similar to those observed by Park et al. (13) in obese/diabetic animals. In that study, they found that circulating myonectin levels were increased significantly in ob/ob and db/db mice. Therefore, we speculate that myonectin may have a role in the pathogenesis of IR and T2DM. Although the current study did not allow us to deduce the cause of increased myonectin in IGT and T2DM subjects, we consider that this increase in myonectin in IGT or T2DM subjects might be a defensive response to metabolic stress from resistance to myonectin action. The elevated myonectin levels in IGT and T2DM individuals may be the results of the impairment of myonectin signaling in target tissues and the dysregulation of myonectin synthesis or a response to hyperinsulinemia, hyperglycemia, or cytokines in an IR state. In regression analyses, we found that plasma myonectin levels positively correlated with WHR, FAT%, TG, FBG, 2h-BG, FIns, HbA1c, and HOMA-IR but negatively correlated with ISI in the general population, suggesting that myonectin may play a role in regulating glucose and TG metabolism and be relative to IR. Given the positive relationship between myonectin and blood glucose, insulin, and HbA1c, it is possible that circulating myonectin levels are regulated by either blood glucose or insulin. To address this question, we performed an OGTT (a high-glucose and high-insulin state) to assess the effects of rapidly increasing glucose and insulin levels on circulating myonectin in young individuals. However, in response to the oral glucose challenge-induced hyperglycemia and hyperinsulinemia, circulating myonectin levels showed no change. This result indicated that an acute increase in blood glucose or insulin levels induced by OGTT may have no effect on myonectin release, or the change in circulating myonectin levels was counteracted by the hyperglycemia–hyperinsulinemia interaction. To address this issue, we performed an EHC study (an euglycemic-hyperinsulinemic state) to control blood glucose levels and assess the effect of hyperinsulinemia on circulating myonectin in healthy individuals. We found that short-term hyperinsulinemia in normal subjects failed to change circulating myonectin levels during EHC, suggesting that circulating myonectin may not be regulated by short-term hyperinsulinemia and hyperglycemia. It is generally accepted that metabolic regulation in T2DM and IR subjects is influenced by their diet and physical activity. In fact, physical activity is thought to protect against several types of disease, including cardiovascular disease, T2DM, and certain types of cancer (21). Recently, muscle tissues have been considered to be an endocrine organ that releases some cytokines, termed myokines, which regulate some physiological and metabolic pathways (22). Myonectin has been identified as a myokine, expressed predominantly by muscle tissues (12). To investigate whether myonectin release or secretion is responsive to an alteration in the metabolic state of skeletal muscle after exercise, we observed the effects of a 45-minute bout of exercise on myonectin levels in young individuals. We found that a 45-minute bout of exercise did not result in an increase or decrease in myonectin circulating levels. In animal studies, however, Peterson et al. (23) reported that myonectin protein content in the muscle was elevated with chronic exercise (9 weeks). In addition, contrary to our findings, Lim et al. (20) found that a 10-week exercise training program significantly decreased circulating myonectin levels in healthy women. Although the reason for the discrepancy is not clear, possible explanations include the following: (1) methodological limitations, including different kits used to measure myonectin; (2) that a relatively small number and only healthy women were selected in that study; and (3) the different effects of a single or long-term exercise. Collectively, we consider that the changes of myonectin levels in circulation may represent long-term IR status, and like adiponectin, it may serve as a regulator of insulin sensitivity. Thus, it would be interesting to investigate whether insulin sensitizers, such as thiazolidinediones, could affect myonectin levels as well. Some limitations of this study should be taken into consideration when interpreting our results. (1) Our study population was limited to Chinese subjects. Therefore, our findings may not be directly applicable to all populations. (2) The number of participants was relatively small, and the age of subjects was relatively old. (3) The cross-sectional design limits our ability to infer a causal relationship between circulating myonectin and IR in T2DM patients. (4) Finally, myonectin levels were not a prespecified end point for recruited IGT and T2DM individuals in the study, and measurements of myonectin were made on stored samples. Nonetheless, our data are sufficient to demonstrate associations of circulating myonectin with anthropometric and metabolic parameters and IR in IGT and T2DM subjects. In summary, our data demonstrate that circulating myonectin concentrations progressively increased from prediabetic to diabetic state and were associated with glucose homoeostasis and insulin sensitivity. Circulating myonectin levels were not affected by an oral glucose challenge (a high-glucose and high-insulin state), EHC (an euglycemic-hyperinsulinemic state), or a 45-minute bout of exercise. Thus, our data suggest that myonectin may be a useful marker in the detection of prediabetes and diabetes. However, further studies are needed to clarify the biological mechanisms involving myonectin in the pathogenesis of IR. Abbreviations: 2h-BG 2-hour blood glucose after glucose overload AUCglucose area under the curve for glucose BMI body mass index BP blood pressure CI confidence interval EHC euglycemic-hyperinsulinemic clamp FAT% percentage of body fat FBG fasting blood glucose FFA free fatty acid FIns fasting insulin GDR glucose disposal rate GIR glucose infusion rate HbA1c hemoglobin A1c HOMA-IR homeostasis model assessment of insulin resistance IGT impaired glucose tolerance IR insulin resistance ISI insulin sensitivity index LDL-C low-density lipoprotein cholesterol NGT normal glucose tolerance nT2DM newly diagnosed type 2 diabetes mellitus OGTT oral glucose tolerance test ROC receiver operating characteristic T2DM type 2 diabetes mellitus TG triglyceride WHR waist/hip ratio. Acknowledgments Financial Support: This work was supported by research grants from the National Natural Science Foundation of China (81670755, 81570752), Doctoral Fund of Ministry of Education of China (20105503110002), and Natural Science Foundation Key Project of CQcstc (cstc2012 jjB10022) to G.Y. Clinical Trial Information: chictr.org.cn no. ChiCTR-OCC-11001422 (registered 23 June 2011). Author Contributions: K.L., Q.M., T.Z., X. Liao, C.Z., and X.X. researched data. H.L., H.Z., and Y.J. reviewed and edited the manuscript. L.L. and G.Y. researched data and wrote and edited the manuscript. X. Luo and K.W. analyzed and revised the manuscript. L.L. and G.Y. are the guarantors of this work and as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose. References 1. 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