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Introduction

Gestational diabetes mellitus (GDM) is defined as diabetes during pregnancy and is the most common pregnancy-associated complication (1). Symptoms of GDM include hyperglycemia, glucose intolerance, insulin resistance and fetal maldevelopment (2). A previous study has indicated that GDM may increase the risk of type II diabetes mellitus and cardiovascular disease (3). Studies also suggested that GDM poses a great threat for the fetal and maternal safety during pregnancy (4–6). The International Association of Diabetes in Pregnancy Study Group criteria for GDM have been recommended by World Health Organization (7). To date, various diagnostic methods have been suggested based on the understanding of the molecular mechanisms of the pathogenesis of this disease (8–10). Therefore, investigation of potentially implicated cytokines may be beneficial for diagnosing patients with GDM.

Fibroblast growth factor-21 (FGF-21) is an atypical member of the family of FGFs, and is regarded as a multifunctional cytokine (11). A study has indicated that FGF-21 is also a multifunctional protein predominantly secreted by adipose tissue, pancreas and liver, and has been regarded as a polypeptide with efficacy in the treatment of metabolic disorders (12,13). FGF21 is produced in the liver and has a crucial role in regulating glucose and lipid metabolism, as well as maintaining energy homeostasis; it has been implicated in the regulation of the endocrine metabolism and various chronic diseases occurs via regulated metabolic processes, including glucose and lipid metabolism (14). Wang et al (15) have indicated that the serum concentration of FGF-19 and −21 in maternal patients with gestational diabetes mellitus is associated with insulin resistance, adiponectin and a history of polycystic ovary syndrome. However, the diagnostic value of the serum levels of FGF-21 in gestational diabetes mellitus patients has remained to be fully elucidated.

The present study assessed the potential diagnostic value of human FGF-21 for GDM and its possible association with insulin resistance and glucose tolerance in affected patients. The results demonstrated that the serum levels of FGF-21 were significantly downregulated in GDM patients, which may be associated with glucose and insulin metabolism, and suggest that FGF-21 is a potential diagnostic factor for the diagnosis of GDM.

Materials and methods

Ethical statement

The protocols were approved by Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). Patients and healthy individuals were required to provide written informed consent. The authors had no access to any information that could identify the individual participants during or after data collection.

Patients and healthy volunteers

A total of 50 patients with GDM and 50 age-matched healthy individuals were enrolled in The Third Affiliated Hospital of Sun Yat-sen University between May 2013 and June 2014 in the present study. The mean age was 28.3 and 28.6 years in the GDM group and healthy individuals, respectively. There was a difference in mean BMI values between the GDM patients and healthy individuals. The mean duration of follow-up in the GDM group was 6 months (Table I). The inclusion criteria were as follows: i) Gestation period of 4–8 months; ii) no history of diabetes mellitus, and no presence of renal failure, intake of medications, ischemic heart disease, heart failure or pancreatic disease.

Table I. Characteristics of GDM patients compared with healthy volunteers.

Table I.

Characteristics of GDM patients compared with healthy volunteers.

Parameter	GDM patients	Healthy volunteers
Mean age (years)	28.3 (22.5–35.2)	28.6 (23.2–34.5)
No. of patients	50	50
Follow-up time (months)	6	6
Blood glucose (mmol/l)	9.3±0.9	6.5±0.7a
Blood pressure (mmHg)	124±14	120±16
Insulin concentration (mmol/l)	9.2±1.2	14.9±2.5a
Body mass index (kg/m2)	37.2±12	25.4±12.2a

a P<0.01 vs. GDM group. GDM, gestational diabetes mellitus.

ELISA

The serum levels of FGF-21 in patients with GDM and healthy volunteers were detected using an ELISA kit (cat. no. KA1849; Abnova, Taipei, Taiwan) according to the manufacturer's protocols. Finally, the serum concentration levels of FGF-21 were measured with an ELISA microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from adipocytes using by using an RNAeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. Reverse transcription was performed at 42°C for 2 h in a total of 50 µl containing 50 ng RNA, 2 µl primers, 5 µl dNTP, 2 µl RT-buffer, and 1 µl reverse transcriptase. All of the forward and reverse primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with the following sequences: FGF-21 forward, 5′-CTGCTGGGGGTCTACCAAG-3′ and reverse, 5′-CTGCGCCTACCACTGTTCC-3′; β-actin forward, 5′-CATCTCTTGCTCGAAGTCCA-3′ and reverse, 5′-ATCATGTTTGAGACCTTCAACA-3′. PCR amplification was performed using the following program: Initial denaturation at 94°C for 2 min, followed by 45 cycles of 95°C for 30 sec, annealing at 56°C for 30 sec and 72°C for 10 min. The reaction was performed in a volume of 20 µl containing 50 ng genomic cDNA, 200 µM deoxynucleoside triphosphate, 2.5 units of Taq DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China) and 200 µM primers using PCR (iQ5 Real Time PCR System; Bio-Rad Laboratories, Inc.). Relative mRNA expression levels were calculated via the 2−ΔΔCq method (16). The results are expressed as the ratio of the β-actin control.

Western blot analysis

Adipose cells were isolated from GDM and healthy volunteers as described previously (17) and lysed in radioimmunoprecipitation assay buffer [mammalian protein extraction reagent (PER) for the cells and tissue PER for the tissues; Thermo Fisher Scientific, Inc.] followed by homogenization at 4°C for 0 min. The protein concentration was measured with a bicinchoninic protein assay kit (Thermo Fisher Scientific, Inc.). A total of 10 µg protein extract was electrophoresed on 12.5% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in blocking buffer (5% BSA; Sigma-Aldrich; Merck KGaA) prior to incubation with primary antibodies at 4°C overnight. The following primary rabbit anti-human antibodies were used in the immunoblotting assays: FGF-21 (1:1,000 dilution; cat. no. ab171941) and GAPDH (1:1,500 dilution; cat. no. ab9485; both from Abcam, Cambridge, MA, USA). After the incubation, the membrane was washed three times in Tris-buffered saline containing Tween-20 (TBST) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin monoclonal antibody (cat. no. PV-6001; Zhongshan Goldenbridge-Bio, Beijing, China) for 1 h at 37°C. After three washes in TBST, the membrane was developed using a chemiluminescence assay system (cat no. 17-677; Roche Diagnostics, Basel, Switzerland) and exposed to Kodak film (Eastman-Kodak, Rochester, NY, USA). Densitometric quantification of the immunoblot data was performed by using Quantity-One software 1.20 (Bio-Rad Laboratories, Inc.).

Immunohistochemistry staining

Immunohistochemical analysis was performed as described previously (18). Paraffin-embedded 4-µm in adipose tissue sections were prepared for further analysis. The paraffin sections were incubated with hydrogen peroxide (3%) for 15 min at 37°C and then hydrated in a decreasing series of ethanols. Antigen retrieval was performed using an antigen retrieval kit (cat. no. ab93684; Abcam). Subsequently, 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) was used to block non-specific binding at 37°C for 2 h and tissue sections were incubated with the abovementioned primary antibody to FGF-21 (1:1,000 dilution) at 4°C for 12 h. All sections were washed three times and incubated with the abovementioned HRP-conjugated secondary antibody (1:10,000; cat. no. PV-6001; OriGene Technologies, Inc., Beijing, China) for 12 h at 4°C. Diaminobenzidine (Sigma-Aldrich; Merck KGaA) was incubated for 5 min at 37°C used to visualize the antibodies at room temperature for 10 sec. Positive signals were detected in six random fields of view under an inverted light microscope (Olympus Corporation, Tokyo, Japan) and images were captured.

Glucose tolerance and insulin resistance test

GDM patients and healthy individuals were fasted for 6 h and orally given glucose at a dose of 2.0 g/kg for the glucose tolerance test. The blood glucose concentration was analyzed with an ACCU-CHEK Advantage glucometer (Roche Diagnostics). The glucose tolerance test results were recorded at baseline and after glucose injection (0, 15, 45, 75 and 105 min). For the insulin tolerance test, all participants were intraperitoneally injected insulin at 0.75 U/kg body weight. All participants were intraperitoneally injected with insulin (1 mU/kg) after a 0, 15, 30, 45, 60, 75 and 90 min fast and the blood glucose concentration was measured at baseline and after insulin injection (15, 30, 60, 90 and 120 min).

Statistical analysis

Values are expressed as the mean ± standard error of the mean or median (interquartile range) Data were analyzed by Student's t-test or analysis of variance, followed by Dunnett's post-hoc test using GraphPad Prism software 5.0 (GraphPad Inc., La Jolla, Ca, USA). The differences in regression coefficients between models were compared by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

Serum levels of FGF-21 in GDM patients

First, FGF-21 expression was detected in the serum and cells of GDM patients. As presented in Fig. 1A, the serum levels of FGF-21 were downregulated in patients with GDM compared with those in healthy individuals. Furthermore, FGF-21 protein was downregulated in adipocytes isolated from GDM patients compared with those of healthy volunteers (Fig. 1B). Gene expression analysis also indicated that FGF-21 mRNA levels were downregulated in adipocytes isolated from GDM patients compared with those in healthy volunteers (Fig. 1C). As presented in Fig. 1D, the protein expression levels of FGF-21 in adipocytes were markedly downregulated in patients with GDM compared with those in healthy individuals. Immunohistochemistry indicated that the expression levels of FGF-21 were also decreased in adipose tissue from GDM patients compared with those in healthy individuals (Fig. 1E). These results suggest that FGF-21 is downregulated in serum and adipocytes of patients with GDM.

Figure 1. Analysis serum levels of FGF-21 in GDM patients. (A) Serum levels of FGF-21 in GDM patients and healthy individuals. (B) Protein levels of FGF-21 in adipocytes were downregulated in clinical patients compared with those in healthy volunteers. (C) FGF-21 mRNA expression levels are downregulated in adipocytes of GDM patients compared with those in healthy volunteers. (D) Protein expression levels of FGF-21 were downregulated in patients with GDM in adipocytes compared with those in healthy individuals. (E) Expression levels of FGF-21 in adipose tissue between GDM patients and healthy individuals determined by immunohistochemistry. **P<0.01. FGF, fibroblast growth factor; GDM, gestational diabetes mellitus.

Analysis of glucose and insulin metabolism in GDM patients

Next, the glucose and insulin metabolism was assessed in GDM patients and healthy volunteers. The results indicated that GDM patients presented with a higher blood glucose concentration than healthy volunteers (Fig. 2A). Furthermore, it was observed that insulin levels were slightly decreased in GDM patients (Fig. 2B). It was also revealed that GDM patients had significantly higher glucose intolerance and insulin resistance than healthy individuals (Fig. 2C and D). These results indicate that compared to healthy individuals, GDM patients exhibited disorders in glucose and insulin metabolism.

Figure 2. Analysis of glucose and insulin metabolism in GDM patients. (A) GDM patients had a higher blood glucose concentration than healthy volunteers. (B) Insulin levels were decreased in GDM patients compared with those in healthy volunteers. (C) GDM patients had a higher glucose tolerance than healthy volunteers. (D) GDM patients had a higher insulin resistance than healthy individuals. *P<0.05 and **P<0.01 vs. GDM. GDM, gestational diabetes mellitus.

Association of serum levels of FGF-21 with glucose and insulin metabolism in GDM patients

The correlation of the levels of FGF-21 with parameters of GDM was then assessed. As presented in Fig. 3A, the plasma levels of FGF-21 were positively correlated with the blood glucose levels in GDM patients. Furthermore, the plasma levels of FGF-21 were positively correlated with the concentration of insulin in GDM patients (Fig. 3B). These results indicate that FGF-21 is associated with the serum levels of glucose as well as with the insulin levels in GDM patients.

Figure 3. Analysis of the association of serum levels of FGF-21 with glucose and insulin metabolism in GDM patients. (A) Serum levels of FGF-21 are positively correlated with the concentration of blood glucose in GDM patients. (B) Serum levels of FGF-21 are positively correlated with insulin levels in GDM patients. FGF, fibroblast growth factor; GDM, gestational diabetes mellitus.

Association between serum levels of FGF-21 and prognosis of GDM patients

Finally, the association between serum levels of FGF-21 and the prognosis of GDM patients was analyzed. The results indicated that the serum levels of FGF-21 were upregulated in GDM patients after the end of pregnancy (Fig. 4A). The serum levels of FGF-21 were also associated with the clinical stage of GDM patients (Fig. 4B). These results indicate that FGF-21 may be a prognostic indicator in patients with GDM.

Figure 4. Analysis the correlation between serum levels of FGF-21 and the prognosis of GDM patients. (A) Serum levels of FGF-21 are upregulated in GDM patients with the recovery time progressing (r=0.412). (B) Serum levels of FGF-21 are associated with the clinical stage of GDM patients (r=−0.376). FGF, fibroblast growth factor; GDM, gestational diabetes mellitus.

Discussion

FGF-21 has been reported as a novel hepatoprotective substance (19), and has been identified as a momentous controller and regulator of glucose and lipid metabolism, as well as long-term energy balance (20,21). A study has indicated that attenuation of FGF signaling in mouse β-cells leads to diabetes (22). However, the role of FGF-21 in the diagnosis of GDM has remained to be fully elucidated. The present study investigated the diagnostic value of FGF-21 in patients with suspected GDM. The results indicated that the serum levels of FGF-21 are downregulated in GDM patients. It was demonstrated that the serum levels of FGF-21 are correlated with insulin resistance and glucose tolerance in clinical GDM patients.

Studies have suggested that the effects of FGF-21 on metabolic hormones to regulate energy metabolism are essential for human vascular endothelial cells (23,24). Wang et al (25) have indicated that FGF-21 is positively associated with atrial fibrosis in atrial fibrillation patients with rheumatic heart disease. The present study reported that GDM patients exhibit a disorder in glucose and insulin metabolism compared to healthy individuals. Chen et al (26) have indicated that plasma insulin and Helicobacter pylori outer membrane protein A are independent factors influencing plasma FGF-21 levels, and due to its role in the pathogenesis of insulin resistance and type 2 diabetes mellitus, FGF-21 is a potential diagnostic factor. The present results indicate that the serum levels of FGF-21 are positively correlated with the concentration of blood glucose and insulin metabolism in GDM patients. The results also demonstrated that FGF-21 expression is significantly different between the GDM and healthy group. However, the association between FGF-21 and BMI should be further established in future studies.

FGF-21 alleviates diabetes-associated vascular complications by inhibiting nuclear factor-κB/NACHT, LRR and PYD domains-containing protein 3 inflammasome-mediated inflammation (27). A previous study also suggested that a high dose of polyethylene glycol-conjugated FGF-21 (500 mg/kg) at the start, followed by a low maintenance dose results has favorable effects by controlling the glycolipid metabolic balance, providing a novel method for the management of diabetes (28). The results of the present study indicated that the serum levels of FGF-21 are associated with the prognosis of GDM patients, suggesting that FGF-21 is a potential prognostic marker in patients with GDM. Chen et al (26) have demonstrated that FGF-21 not only increases insulin secretion and insulin content in diabetic islets, but also protects β-cells from apoptosis via the activation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways. The present study reported that the insulin concentration in GDM patients returns to normal levels after the end of the pregnancy (within 3 months). However, the control group was not pregnant, which is a limitation of the present study. Further studies should thus incorporate a pregnant control.

In conclusion, the present study suggests that downregulation of FGF-21 may be associated with the risk of GDM. Of note, the results indicate that FGF-21 may be a diagnostic and prognostic indicator in patients with GDM. However, further studies should be performed in large populations to assess the association between FGF-21 with impaired glucose metabolism.

Acknowledgements

Not applicable.

Funding

The present study was financially supported by the Development of Science and Technology of the Department of Science and Technology of Guangzhou, China (grant no. 201704020170).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

CX designed the present study. ZH, PL and XL performed all experiments and analyzed the data in the present study.

Ethical approval and consent to participate

The protocols were approved by the Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). All patients and healthy individuals were required to provide written informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References