Differentiated serum levels of Krüppel-like factors 2 and 4, sP-selectin, and sE-selectin in patients with gestational diabetes mellitus

Abstract Objectives This study aims to determine serum levels of human Krüppel-like factors (KLFs), sP-selectin and sE-selectin and establish correlations between them in patients with gestational diabetes mellitus (GDM). Methods Twenty-six GDM patients aged between 22 and 35 years and 25 healthy pregnant women aged between 23 and 34 years were recruited. Maternal serum levels of KLF2, KLF4, and their target proteins sP-selectin, sE-selectin were measured using enzyme-linked immunosorbent assays at 24–28 weeks of gestation. Results Women with GDM had significantly lower serum KLF2 than controls. However, the differences in levels of serum KLF4 between the control and GDM groups were not significant. Additionally, elevated serum sP-selectin and sE-selectin were found in the GDM group and not in the healthy group. Importantly, we also found that serum KLF2 levels were negatively correlated with indicators of glucose metabolism, including insulin, fasting blood glucose, 1-h oral glucose tolerance test, and glycated hemoglobin. Conclusion We conclude that (i) serum KLF2 might be indicative of GDM risk, and (ii) sP-selectin and sE-selectin were increased in GDM patients.


Introduction
Gestational diabetes mellitus (GDM) is a significant complication of pregnancy. In recent years, with changes in lifestyle and dietary habits, the morbidity of diabetes in China increases annually. Furthermore, with the implementation of the two-child policy, there is an increase in the number of aged pregnant women. Considering other factors, including increased resting time, decreased activity, and increased food intake, the population of GDM patients increased during the last years. The prevalence rate of GDM reported in different countries varies due to race, detection methods, and diagnostic criteria. The prevalence worldwide is approximately 7-14% [1], with 5-8.5% reported in China alone [2]. Severe complications can result from GDM, including preeclampsia, infection, macrosomia, neonatal respiratory distress syndrome (RDS), hypoglycemia, hydramnios, as well as a significantly increased risk of premature delivery and cesarean delivery [3]. Furthermore, a higher risk of developing type 2 diabetes mellitus (T2DM) in the future is reported in GDM patients [4,5]. Clinical screening of differentially expressed factors in the serum of GDM patients is of great significance for the early diagnosis and prevention of GDM.
The Kruppel-like transcription factors (KLFs) family is a class of nuclear transcription factors with zinc-finger structures and 17 subtypes, which are involved in regulating a variety of cellular physiological processes, including proliferation, differentiation, growth, development, cardiovascular, digestive, respiratory, blood and immune systems [6]. Mreich et al. [7] found that the KLF4 level was significantly reduced in proximal tubular cells of human kidneys under high glucose stimulation, leading to the upregulation of inflammatory proteins, such as migration inhibitory factor (MIF) and monocyte chemoattractant protein (MCP-1). MIF and MCP-1 levels can be repressed in high glucose-stimulated proximal tubular cells by the overexpression of KLF4, thereby reducing inflammation in diabetic nephropathy.
Lee et al. reported that KLF2 is as an essential regulator of endothelial function in diabetes [8]. Furthermore, Yue et al. [9] found that the protective role of liraglutide, a glucagon-like peptide-1 receptor agonist, was closely associated with the expression of KLF2. Among adhesion molecules, the selectin family has attracted much attention in diabetes investigations. Selectins play an important role in the pathophysiological processes of various vascular complications in diabetes, such as atherosclerosis, thrombosis, and microvascular injury [10]. Gokularishnam et al. [11] found that an increased plasma P-selectin level accompanied the increased level of insulin in insulin-resistant patients. Matsumoto et al. [12] reported that the level of serum E-selectin in diabetic patients with macrovascular and microvascular lesions was significantly higher than in patients without vascular lesions. However, the functions of KLF2, KLF4, sP-selectin, and sE-selectin in GDM remain unclear. The present study investigates the relative expressions of these factors to explore their potential role as biomarkers of GDM.

Studied population
The pregnant women who underwent elective cesarean section in the department of Obstetrics of Hanchuan People's Hospital, Hubei Province (China), from May 2019 to January 2022, were recruited to participate in the study, including 25 healthy pregnant women (controls) and 26 GDM patients. GDM was diagnosed when the fasting plasma glucose level was equal or exceeded 5.1 mmol/L. Women were excluded when the fasting plasma glucose was lower than 4.4 mmol/L. Pregnancies with fasting glucose between 4.4 and 5.1 mmol/L underwent a 75-g oral glucose tolerance test (OGTT). Under this circumstance, a diagnosis of GDM was verified when at least one glucose value was elevated (fasting glucose ≥ 5.1 mmol/L, 1-h OGTT ≥ 10.0 mmol/L, or 2-h OGTT ≥ 8.5 mmol/L). Pregnant women were excluded if they were diagnosed with cardiovascular diseases, cancer, or any other major illness.

Laboratory tests
Prior to testing, all pregnant women were required to fast for 12 h, and 5 ml of blood was collected and placed in the biochemical and heparin tube for 30 min. After centrifugation at 3000 r/min for 10 min, the serum and plasma were respectively achieved. The automatic biochemical analyzer (Cedex Bio, Roche, Switzerland) was utilized to measure the laboratory parameters. Serum hemoglobin, hematocrit, white blood cells (WBC), platelet count, triglycerides (TG), total cholesterol (T-chol), high-density lipoprotein-cholesterol (HDL-c), and low-density lipoprotein-cholesterol (LDL-c) were measured. The value of fasting blood glucose (FBG) was measured using the glucose oxidase method [13]. After administering glucose for 1-h and 2-h, the values of the 1-h oral glucose tolerance test (OGTT) and the 2-h OGTT were measured using the glucometer (Roche, Switzerland) [14].
The serum level of insulin (Abcam, USA), glycated hemoglobin (Abcam, USA), KLF2 (Shanghai Xingyi Biotechnology Co., LTD, China), KLF4 (Abcam, USA), serum P-selectin (sP-selectin, Abcam, USA), and serum E-selectin (eP-selectin, Abcam, USA) were determined using the ELISA methods. Serum obtained from each woman was placed into the 96-well plate, followed by incubating with the conjugate reagents for 90 min. Procedures were followed according to manufacturers. The absorbance at 450 nm was detected with a microplate reader (BMG LABTECH, Germany).

Statistical analysis
Data were expressed as mean ± SD. The Shapiro-Wilk test was used for the test of normality distribution. Data with a normal distribution were compared with the student's t test. Data without normal distribution were compared with the Mann-Whitney-U test. The Pearson correlation analysis was used to verify the correlation between KLF2, KLF4, and related factors. Statistical significance was determined when the value of p was lower than .05.

Clinical features and blood parameters
The average age in the GDM group was 29.85 ± 5.62, the gestational week was 25.0 ± 3.6 and the BMI value was 29.36 ± 2.53 kg/ m 2 . The average age in the control group was 28.21 ± 4.25, the gestational week was 24.5 ± 4.5 and the BMI value was 25.11 ± 2.36 kg/m 2 . No significant difference was observed in the average age and gestational week between the control and the GDM group. The BMI in the GDM group was significantly higher than that in the control group. The characteristics of the patients are shown in Table 1. There were no significant differences in hemoglobin, hematocrit, platelet count, triglycerides, T-chol, HDL-c, and LDL-c (Table 1) values between the GDM and control groups. However, the average concentration of the white blood cells (WBCs) was significantly higher (12.18 × 10 3 / mL) in the GDM women than that in the control group (10.65 × 10 3 /mL).

Glucose metabolism was impaired in GDM patients
The pathological state of diabetes in GDM patients was evaluated using glucose metabolism parameters ( Table 2). The average values of fasting blood glucose (FBG) in the control and GDM groups were 85.98 and 119.85 mg/dL, respectively. The 1-h OGTT value was dramatically increased from 125.06 to 182.33 mg/dL in GDM patients, likewise, that of 2-h OGTT was greatly elevated from 104.6 to 158.21 mg/dL. Moreover, the average insulin levels in the control and GDM groups were 9.5 and 16.42 μIU/ mL, respectively. The HOMA-IR value was promoted from 2.01 to 4.63, and that of HbA1c increased from 4.25 to 5.65 in the  GDM group. These data show that the glucose metabolism in GDM patients was impaired.

Serum KLF2, KLF4, P-selectin and E-selectin levels
The average serum KLF2 level in GDM patients (33.56 ± 5.62 μg/L) was significantly lower than in the control group (67.33 ± 11.08 μg/L; Table 3). There was no significant difference in serum KLF4 levels between GDM patients and the control women ( Table 3). As shown in Table 3, the average levels of P-selectin were significantly higher in GDM patients (88.26 ng/ mL ng/mL) than in normal pregnant women (52.13 ± 6.08 ng/ mL). The E-selectin levels were also higher in pregnant women with GDM (751.05 ± 137.65 ng/mL) as compared to normal pregnant women (412.16 ± 83.6 ng/mL; Table 3). BMI-corrected statistical analysis has been performed to suggest a significant difference (Table 3). Finally, we did the statistical analysis of the correlation between KLF2 and indicators of glucose metabolism. Serum levels of KLF2 were negatively correlated with FBG, 1-h OGTT, 2-h OGTT, Insulin, HOMA-IR, and HbA1c, and the correlation factor R values were −0.361, −0.23, −0.314, −0.425, −0.18, −0.43, respectively (p < .05).

Discussion
Decreased insulin sensitivity and insulin resistance are important pathological inducers of GDM. Studies have shown that HOMA-IR is the basis of diabetes diagnosis, which refers to the decrease of glucose uptake and utilization ability of insulin by target organs [15]. Consistent with previous observations, significantly elevated insulin levels and HOMA-IR values were observed in GDM patients in the present study. These data on glucose metabolism parameters suggest that typical pathological changes were observed in recruited GDM patients in the present study. P-selectin (CD62P) is a highly glycosylated glycoprotein, also known as transmembrane single chain glycoprotein, which belongs to the selectin family together with E-selectin and L-selectin [16]. In recent years, with the development of molecular biology, the structure, function, and pathophysiological effects of P-selectin have been extensively studied. Yngen et al. [17] believed that insulin resistance contributed to the elevated expression of P-selectin, and its high expression of P-selectin further aggravated insulin resistance and platelet activation. E-selectin (CD62E) belongs to the human leukocyte differentiation antigen family and is a 110 kD transmembrane glycoprotein expressed on vascular endothelial cells and activated by cytokines. The complexity of E-selectin structure determines the transience and specificity of its function [18]. Several studies have demonstrated that the serum levels of various adhesion molecules, including sE-selectin, in patients with type 2 diabetes are significantly higher than those in the general population [19,20]. However, the functions of sP-selectin and sE-selectin in GDM remain uncertain. Our data reveal that the sP-selectin and sE-selectin levels were elevated in GDM patients. However, no direct correlation between sP-selectin or sE-selectin and parameters of glucose metabolism was observed, suggesting the functions of sP-selectin and sE-selectin in GDM need to be further identified using more patient samples.
KLF4 has been reported to be closely related to the development of diabetic complications, especially diabetic nephropathy [21]. However, whether KLF4 functions as an important regulator in GDM is not precisely known. We found that the serum levels of KLF4 were not significantly altered in GDM patients. KLF2 is an essential transcription factor that maintains endothelial homeostasis, and its target genes are involved in the anti-inflammatory, antithrombotic, antioxidative, antiproliferative, and vascular integrity maintenance effects of endothelial cells [22,23]. High glucose inhibition of endothelial cell KLF2 may be a mechanism of endothelial dysfunction in diabetes [24]. The role of KLF2 in GDM remains unknown. We found that KLF2 was lower in GDM patients, and its expression was negatively associated with the parameters of glucose metabolism, suggesting that the low levels of KLF2 might be a risk factor for the development of GDM. In future work, the function and regulatory mechanism of KLF2 in GDM will be further explored and identified in GDM animal models. Taken together, our data suggest that serum KLF2 might be indicative of GDM.
We have to address the limitations of the current study. Firstly, we only included women who had C-sections, and those who underwent natural labor were excluded from this study. By limiting our inclusion criteria to C-section patients only, we were able to select better-matched samples for both control and GDM patients. However, the tradeoff of this inclusion criteria is that our cohort does not represent the general pregnant women. Secondly, we only recruited a relatively small number of patients, therefore, the findings in this study need to be validated in future large number-observational studies or clinical trials on pregnant women. Table 3. The levels of serum Krüppel-like factor 2, Krüppel-like factor 4, P-selectin and e-selectin for both women with GdM (n = 26) and healthy controls (n = 25

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Funding
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