Association of serum fetuin-B with insulin resistance and pre-diabetes in young Chinese women: evidence from a cross-sectional study and effect of liraglutide

Research population

A total of 304 young women were enrolled in this study (Fig. 1), including 80 newly diagnosed IR patients (IR-NG group), 97 individuals with impaired glucose tolerance (IGT group), and 127 healthy women (NGT group). The individuals were recruited from the outpatient department of Endocrinology or from daily physical examination, the Second Affiliated Hospital of Chongqing Medical University from January 2017 to December 2019. IGT was diagnosed according to the American Diabetes Association (ADA) (American Diabetes, 2012). The presence of pre-diabetes was defined by the occurrence of IGT at 2-hours oral glucose tolerance (2h-OGTT). M-value <6.28 was considered to have IR (Wei-Gang et al., 2009). Individuals with M-value <6.28 and normal fasting blood glucose (FBG) levels were included in the IR-NG group. All IGT and IR patients were newly diagnosed without any medication or lifestyle intervention. The exclusion criteria included type 1 diabetes mellitus (T1DM), T2DM, hypertension, and other important organ diseases such as heart, liver, and kidney. NGT group had normal blood glucose [FBG <5.1 mmol; 2-h blood glucose post-glucose load (2-h BG) <7.8 mmol], no family history of diabetes and hypertension, no chronic diseases, and medication recently. All subjects signed informed consent before the experiment. This study was conducted according to the declaration of Helsinki and approved by the ethics committee of the Second Affiliated Hospital of Chongqing Medical University (2014 Colombo Review No. (72)). Human research was registered at the Chinese Clinical Trial Registry (registered 23 June 2011), chictr.org.cn (CHICTR-OCC-11001422).

Anthropometric and biochemical measurements

After fasting for 10–12 h, all subjects were given a physical examination and biochemical tests by professionals. The physical examination included body weight, blood pressure (BP), waist circumference (WC), and body fat content (Fat %). Biochemical parameters included total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), free fatty acid (FFA), HbA1c, FBG, 2-hour postprandial blood glucose (2h-BG), fasting insulin (FIns), and 2-hour postprandial insulin (2h- INS), as previously reported (Xu et al., 2020).

Determination of serum fetuin-B concentration

Serum fetuin-B concentration was determined by an ELISA kit (RayBiotech, Inc. Norcross, GA, USA). The detection limit for fetuin-B was 4.0 ng/ml. Intra- and inter-assay variations (CV) were 10% and 12%, respectively. This method has high sensitivity, good specificity, and no obvious cross-reaction.

OGTT and euglycemic-hyperinsulinemic clamp test (EHC)

After 10–12 h of fasting overnight, all subjects were given 75g glucose orally and the OGTT test was performed at 8:00 AM (American Diabetes, 2012). During the OGTT, blood samples were collected for the measurements of blood glucose, insulin, and serum fetuin-B at 0, 30, 60, and 120 min. The EHC was performed in all subjects. The operation process was as previously reported (Xu et al., 2020). During the EHC, human insulin (1mU/kg/min) was continuously infused for 2 h. At the same time, the infusion rate of the 20% glucose was adjusted according to the blood glucose levels, tested every 15 min, to maintain blood glucose at the basic level (4.5−5.5 mmol/L). In the steady-state of the clamp, GIR was equal to glucose disposal rate (GDR), and M-value was related to GIR and body weight. Blood samples were collected at 0, 80, 100, and 120 min for the determination of fetuin-B and insulin. Blood samples were centrifuged, and serum was separated and stored at −80 °C for subsequent analysis.

GLP-1RA intervention

A total of 26 overweight/obese women with IR participated in GLP-1RA intervention for 24 weeks. These women were enrolled for intervention with lira due to ovarian dysfunction and refractory menstrual problems. Liar, a GLP-1RA, was injected subcutaneously once a day. The dose increased from 0.6 mg to 1.8 mg. Inclusion criteria included age of 18–35 and BMI of 25–35 kg/m². Exclusion criteria included the family history of thyroid tumor, the history of medullary thyroid carcinoma, severe gastrointestinal disease, acute pancreatitis, and pregnancy. Those subjects did not use any drugs in the last 3 months. Physical examination and biochemical tests, OGTT, and EHC tests were performed before treatment, 12 and 24 weeks after treatment. All subjects signed informed consent before treatment.

Calculation formula

BMI = body weight/height 2, WHR = WC/hip circumference (HC), HOMA2-IR was calculated by the software (HOMA calculator v2.2.2) (Wallace, Levy & Matthews, 2004), M-value = GIR/body weight, and M-value <6.28 is considered to have IR (Wei-Gang et al., 2009).

Statistical analysis

SPSS version 24.0 software (SPSS 2.0, Chicago, IL) was used for statistical analysis. Data were expressed as mean ±  SD or median. Variables that are not normally distributed were converted logarithmically. Analysis of variance was used for comparison among groups, and Fisher’s Least Significant Difference (LSD) test was used for comparison between two groups. The relationship between serum fetuin-B and other variables was evaluated by linear regression analysis with controlled covariates. Multiple linear regression analysis was used to identify independent factors associated with serum fetuin-B. Multiple logistic regression analysis was conducted to understand the association of serum fetuin-B with IR and IGT. ROC curves were constructed to evaluate the sensitivity and specificity of serum fetuin-B in predicting IR and IGT.

We calculated tolerance and variance inflation factor (VIF) values to evaluate multicollinearity between variables. A tolerance <0.1 and VIF >10 is considered indicative of multicollinearity. Compared with the control group, p <0.05 was considered to be statistically significant.

Serum fetuin-B levels and biochemical parameters in the study population

Table 1 shows the clinical biochemical indexes and circulating fetuin-B levels in each group. We found that BMI, Fat %, WC, BP, TG, TC, LDL, HbA1c, FBG, 2 h-BG, FIns, 2 h-INS, HOMA2-IR, the area under the curve of glucose (AUCg) and insulin (AUCi) in IR-NG and IGT groups were significantly higher than those in the control group (all p < 0.01), while HDL-C and M-values were lower (p < 0.01). In addition, FFA, 2h-BG, 2h-Ins, and AUCg in the IGT group were significantly higher than those in the IR-NG group (p < 0.05 or p < 0.01). serum fetuin-B levels in IGT and IR-NG groups were significantly higher than those in normal controls (p < 0.05 or p < 0.01, Table 1 and Fig. 2A). fetuin-B levels were higher in the IGT group than those in the IR-NG group (Fig. 2A, p <  0.05). Furthermore, serum fetuin-B levels in obese/overweight women (BMI ≥ 24 kg/m²) were significantly higher than those in normal controls (7.63 ± 0.38 vs. 5.95 ±  0.32 mg/L, p <  0.01) (Fig. 2B). The relationship between serum fetuin-B and IR was analyzed by combing IR-NG and IGT individuals into the IR group. The results showed that serum fetuin-B levels were significantly correlated with IR (Table S2).

Association of serum fetuin-B with clinical and biochemical indexes in young women

In all study subjects, serum fetuin-B levels were positively correlated with obesity and IR- related parameters (BMI, fat%, WC, TG, FIns, 2h-INS, AUCi, AUCg, HOMA2-IR, and M-value) (all p < 0.01), but not with TC, HDL-C, LDL-C and FFA (Table 2). After controlling age and Fat%, serum fetuin-B levels were still significantly positively correlated with WC, FIns, HOMA2-IR, and M-value (p < 0.05 or p < 0.01, Table 2). Multiple regression analysis showed that M-value was an independent factor affecting serum fetuin-B (Fig. 2C, Table 2). The regression equation is Y_fetuin−B = 9.231 − 0.379X M-value.

Association of serum fetuin-B with IR and IGT in women

By multiple logistic regression analysis, we found that serum fetuin-B levels were significantly correlated with the occurrence of IR-NG and IGT (IR-NG: OR, 1.13; 95% CI [1.04–1.22]; p <  0.01; IGT: OR, 1.20; 95% CI [1.09–1.27], p <  0.01). After control of age, Fat %, BMI, WC, BP, and TG, serum fetuin-B levels were still correlated with IGT (p <  0.05 or p <  0.01, Table S1), while by controlling age only, fetuin-B was correlated with the occurrence of IR in women (p <  0.01, Table S2).

To further analyze the association of serum fetuin-B with IR-NG and IGT, we divided serum fetuin-B concentrations into four quartiles in NGT and IR-NG groups (Quartile 1, 0.85−2.12 mg/L; Quartile 2, 2.13−6.35 mg/L; Quartile 3, 6.36−8.61 mg/L; Quartile 4, >8.61 mg/L), and in NGT and IGT groups (Quartile 1, 0.85−3.39 mg/L; Quartile 2, 3.40−6.69 mg/L; Quartile 3, 6.70−8.99 mg/L; Quartile 4, >8.99 mg/L). As shown in Figs. 2D and 2E, the risk of IGT and IR increased with the increase of serum fetuin-B concentration.

ROC curve analysis

To evaluate the predictive value of serum fetuin-B levels for IGT and IR, ROC curves were drawn. The results showed that the area under the ROC curve for IGT (AUC _IGT) was 0.67, sensitivity 79%, and specificity 56%. The best cut-off value of fetuin-B for predicting IGT was 4.45 mg/L (Fig. S1A). AUC_IR was 0.63, sensitivity 72%, and specificity 51%. The best cut-off value of fetuin-B for predicting IR was 5.44 mg/L (Fig. S1B).

Effect of the OGTT and EHC on serum fetuin-B levels in women

To further explore the regulatory factors of serum fetuin-B level, we performed the OGTT and EHC experiments. During the OGTT, serum fetuin-B levels in the NGT group were significantly increased (Fig. 3A) and reached the peak at 30 min (11.42 ±  1.0 mg/L vs. 3.42 ±  0.57 mg/L, p < 0.01), but no change in both IR-NG and IGT groups. The areas under the curve of serum fetuin-B (AUC fetuin-B) in the IGT and IR-NG groups were significantly higher than those in the control group (Fig. 3B). During the EHC experiment, as expected, the M-values in IR-NG and IGT groups were significantly lower than those in the NGT group (4.72 ±  0.21 and 3.86 ±  0.39 vs. 9.98 ±  0.53 mg/kg/min; p < 0.01; Fig. 3C). In the IGT group, serum fetuin-B levels increased gradually following the stimulation of hyperinsulinemia (from 6.29 ±  0.99 mg/L to 10.18 ±  1.30 mg/L) (Fig. 3D). Interestingly, there was no significant change in serum fetuin-B levels in both IR-NG and NGT groups during the EHC, suggesting that serum fetuin-B levels were regulated by insulin in different glucose tolerance populations.

Effect of GLP-1RA treatment on serum fetuin-B level in obese individuals

Twenty-six overweight/obese women with IR (BMI, 28.4 ±  0.6 kg/m2) were treated with a Liar for 24 weeks. The changes in general clinical and biochemical indexes before and after treatment are shown in Table 3. After the treatment, obesity, and lipid metabolism-related indicators (BMI, Fat %, WC and TG), glucose metabolism and IR-related indicators (FBG, FIns, HbA1c, HOMA2-IR) were significantly improved compared with those before treatment (p <  0.01 or p < 0.05; Table 3). In addition, Liar treatment significantly decreased the M-values of the EHC in these obese women, which further indicates the improvement of IR (Fig. 4A). Importantly, serum fetuin-B levels were significantly reduced after the 24 weeks of GLP-1RA intervention in these obese women with IR (from 8.08 ±  1.77 mg/l to 6.75 ±  1.57 mg/l, p < 0.05; Fig. 4B).