Relationship between guanosine triphosphate pathway and tetrahydrobiopterin in gestational diabetes mellitus

Abstract

Purpose

The present study assesses the change in tetrahydrobiopterin (BH4), one of the most important products in the guanosine triphosphate (GTP) pathway and in other parameters that might affect nitric oxide (NO) production, in gestational diabetes mellitus (GDM).

Methods

The study included 100 healthy pregnant women and 100 women diagnosed with GDM. Serum levels of neopterin, BH4 and NO were measured. The levels of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and guanosine triphosphate cyclohydrolase I (GCHI/GTPCH) gene expression were determined.

Results

It was found that diabetes led to an increase in neopterin and NO levels, and a decrease in BH4 levels. A stimulation was observed in eNOS gene expression in the GDM group when compared to the control group, while GCHI levels were found to decrease when compared to the control group. iNOS gene expression was detected in neither the healthy controls nor the patient group.

Conclusions

Decreased NO bioavailability plays an important role in the progression of such macrovascular diseases as diabetes. BH4 levels decrease in diabetes patients, while the increased gene expression of GCHI reverses the diabetes-related BH4 deficiency and allows the endothelial cells to regain their ability to produce NO. Since GCHI is the rate-limiting enzyme in the biosynthesis of BH4, changes in GCHI levels directly affect the BH4 levels and the NO metabolism, leading to an increased risk of macrovascular complications. The significant increase in neopterin levels suggest that this is a potential biomarker for the early diagnosis of GDM.

Introduction

The relationship between GTP pathway and BH4 has been evaluated in several studies, and particularly in studies of neurological diseases. To date, however, the basis of this relationship has not been clearly defined in vascular diseases such as diabetes. Diabetes (diabetes mellitus) is a metabolic disease that is characterized by hyperglycemia, which occurs due to a disturbance in insulin secretion, insulin resistance, or both. Different diabetes subtypes have different underlying mechanisms of insulin resistance and metabolic abnormalities [1]. Diabetes is divided into four subgroups by the American Diabetes Association as type 1, type 2, gestational diabetes and secondary diabetes [2]. The World Health Organization (WHO) reported around 422 million patients with diabetes worldwide and 1.6 million deaths are directly attributed to diabetes each year [3].

Gestational diabetes mellitus is defined as glucose intolerance with onset or first recognition during pregnancy. Although glucose homeostasis is regulated following the completion of pregnancy, there is an increased risk of developing type 2 diabetes in the future [4]. Clinical manifestations of GDM include mainly hyperglycemia, hyperlipidemia, hyperinsulinemia and fetoplacental endothelial dysfunction. GDM is also likely to cause perinatal complications such as abnormal fetal development, macrosomia, neonatal hypoglycemia and neurological disorders [5]. The pathophysiological mechanisms associated with the development of GDM during pregnancy yet to be fully understood [6], although two basic mechanisms have been suggested, being insulin resistance and chronic subclinic inflammation [7].

Neopterin is synthesized from GTP through the GTPCH enzyme induced by interferon gamma (IFN-ɡ), and is a pteridine derivative produced by activated monocytes, macrophages, dendritic cells and endothelial cells [8]. Neopterin has been identified in several body fluids, such as serum, cerebrospinal fluid, synovial fluid, urine and saliva, and there is a positive correlation between increased neopterin levels and the endogenous activation of IFN-ɡ [9–11]. Neopterin production starts three days before the T lymphocyte proliferation reaches the maximum level, and the levels of neopterin elevate about one week before the specific antibodies become detectable. As such, neopterin is believed to have use as an inflammatory marker in the early period [12]. Neopterin concentrations in the body fluids are used as markers in the evaluation of systemic immunity and inflammatory response in diabetes [9, 10].

Guanosine triphosphate cyclohydrolase I is a rate-limiting enzyme that is active in the synthesis of BH4, and is a co-factor in the biosynthesis of catecholamines and NO [8, 13]. NO is a vasoactive molecule synthesized from l-arginine by neuronal (nNOS), iNOS and eNOS nitric oxide synthases. eNOS is the most important of the NO synthase isoforms in vascular endothelium, and plays important roles in the cardiovascular system [14]. The most important of these roles is its regulation of vascular tonus, which depends on the activation of the soluble guanylate cyclase (sGC) produced in the vascular smooth muscle cells. NO produced in endothelial cells is diffused through platelets or in the vascular smooth muscle cell membrane, and catalyzes the conversion of GTP to cyclic guanosine monophosphate (cGMP) by binding to the heme of sGC and forming a metal-nitrosyl addition product. Through this mechanism, platelet reactivity is inhibited and the dilatation of vascular muscles is ensured [13]. The binding of the co-factor BH4 converts the heme iron of eNOS into a high-spin state, and stabilizes the active dimeric form of the enzyme [15]. Accordingly, mechanisms with an effect on the synthesis and consumption of BH4 also affect eNOS [16]. Preventing BH4 deficiency, which is a co-factor of eNOS, contributes to the return of decreased NO levels to their normal state. BH4 and NO production are increased by the overexpression of GTP cyclohydrolase, which is the rate-limiting enzyme in the BH4 synthesis [17, 18].

It is known that hormonal and immunological balance is controlled by the placenta during pregnancy. In some diseases, such as GDM, this balance is observed to be deteriorated. The immune system is suppressed under the control of placenta to prevent the rejection of the fetus during pregnancy. Levels of inflammatory markers, such as neopterin, are elevated in GDM. As a result, insulin resistance becomes more remarkable and the glucose metabolism is impaired. Considering that changes in GTPCH and in the expression of the GCHI gene, which encodes this enzyme to alter NO levels and expressions in the iNOS and eNOS genes, with a direct effect on NO levels, may trigger gestational diabetes, this study assesses changes in the GTP pathway in gestational diabetes.

Materials and methods

Study groups and sample collection

The study was conducted with 100 pregnant women who presented to the Inonu University Turgut Ozal Medical Center Department of Obstetrics and Gynecology and who were diagnosed with GDM, as well as 100 non-GDM pregnant women. The study was granted approval by the Inonu University Clinical Researches Ethics Committee (Approval No. 2017/82). Informed consent was obtained from all patients for the use of their medical data for study purposes. The participants in gestational weeks 24–28, who had not been previously diagnosed with diabetes, were administered a 75 g oral glucose tolerance test (OGTT) in one step, as per the IADPSG criteria [19]. Based on plasma glucose levels, the respondents with fasting plasma glucose level > 92 mg/dL, > 180 mg/dL after 1 hour and > 153 mg/dl after 2 hours constituted the patient group. The healthy control group, in turn, comprised pregnant women in the same gestational week with fasting plasma glucose levels under the specified values recorded in the 75 g OGTT. The exclusion criteria included (i) previous diagnosis of diabetes mellitus; (ii) presence of such secondary cardiovascular diseases as hypertension, hyperlipidemia or coronary artery disease; (iii) smoking, hepatic and renal dysfunction, history of trauma, presence of factors such as recent acute or chronic infection; and (iv) presence of an underlying chronic inflammatory condition such as collagen tissue and inflammatory intestinal diseases. For each participant, detailed information was obtained about risk factors for GDM such as demographic characteristics, age, pre-pregnancy body mass index (BMI = kg/m²), gestational weight gain, history of GDM in previous pregnancies and history of type 2 diabetes in first-degree relatives. Peripheral venous blood samples (1–2 mL) were collected from all of the respondents. The blood samples were centrifuged at 3,500 rpm for 15 minutes at room temperature and the sera were separated for the measurement of neopterin, tryptophan and kynurenine levels. All samples were stored at -20 °C until the time of analysis.

Neopterin, BH4 and NO measurement

The enzyme immunoassay technique was used to determine the serum levels of neopterin, BH4 and NO, and the test procedure was conducted using commercially available enzyme-linked immunosorbent assay (ELISA) kits (DRG Instruments GmbH, Marburg, Germany) in accordance with the manufacturer’s instructions.

Quantification of mRNA expression with relative quantitative real-time PCR

In order to quantify the eNOS, iNOS and GCHI expressions in the blood samples of the patient and control groups, an expression profiling study was performed using Relative Quantitative Real-Time PCR (qRT-PCR).

Statistical analysis

The data obtained from the measurements was analyzed using the IBM SPSS 25.0 software package. For the results derived from the study groups, basic statistical parameters (e.g. mean, median, standard deviation/deviation, and minimum and maximum) were calculated. The data was summarized as mean ± standard deviation, median (min-max) and number (percentage). A Kolmogorov-Smirnov test was used to test the normality of distribution. An Independent Samples t-Test, Yates’ Corrected Chi-Square Test and Kruskal-Wallis Test were used for the statistical analyses. p < 0.05 was accepted as statistically significant.

Results

The control group comprised 100 healthy pregnant women with a mean age of 30.47 ± 5.51 (Min-Max: 18–49) years, and the GDM group consisted of 100 pregnant women with a mean age of 31.78 ± 5.65 (Min-Max: 20–45) years. There was no statistically significant difference in mean age in the two groups (p = 0.101). Advanced age is a risk factor for GDM. According to the American Diabetes Association, those aged > 25 are at greater risk of GDM. In the present study, respondents over the age of 25 accounted for 78% and 86% of the control and diabetes groups, respectively. There was a weak negative correlation between age and neopterin in the control group (p = 0.005), and a weak negative correlation also between the number of pregnancies and tetrahydrobiopterin both in the control group (p = 0.031) and in the patient group (p = 0.031). There was a weak negative correlation between the number of pregnancies and tetrahydrobiopterin in the patient group. Another risk factor for diabetes is miscarriage in previous pregnancies, in which no significant difference was found between the control and patient groups (p = 0.363). There was a history of miscarriage in 29% of the control group, whereas 35% of the diabetes group had miscarried in previous pregnancies. History of diabetes increases the risk of GDM. No significant difference was found in the family history of diabetes between the diabetic and healthy respondents (p = 0.611). Family history of diabetes was positive in 21% and 24% of the healthy and diabetic respondents, respectively, which are close to each other. In terms of gestational week, 82% of the respondents were in weeks 24–28 and 18% were in weeks > 28. A significant (p = 0.038) correlation was found between tetrahydrobiopterin levels and gestational week in the control group. A paired comparison of gestational weeks revealed a significant difference in tetrahydrobiopterin levels between weeks 25–26 and weeks 26–27 (p = 0.007), and weeks 27–28 (p = 0.032). Likewise, tetrahydrobiopterin levels were found to differ between weeks 26–27 and weeks 27–28 (p = 0.026). The demographic characteristics of the study groups are presented in Table 1.

Table 1.

Demographic characteristics of the the study groups

Control		GDM	Total
Age range (year)
< 19	1	-	1
20–25	21	14	35
26–30	32	30	62
31–35	29	27	56
> 35	17	29	46
Abortion
-	71	65	136
1	16	18	34
> 1	13	17	30
Family history of diabetes
+	21	24	45
	79	76	155
Gestational week
24–25	44	24	68
25–26	24	17	41
26–27	12	17	29
27–28	9	17	26
> 28	11	25	36
Pregnancy number
1	28	24	52
2	32	20	52
3	19	25	44
4	15	14	29
> 4	6	17	23

In the control group, OGTT results of the respondents were 78.50 (60–90) mg/dL, 140 (75–175) mg/dL and 121.5 (60–168) mg/dL at hours 0, 1 and 2, respectively. In the GDM group, in turn, the corresponding results were 99 (72–215) mg/dL, 190 (152–241) mg/dL and 160 (102–200) mg/dL. The difference in OGTT results between the groups was found to be significant at hours 0, 1 and 2 (for all, p < 0.001). The respondents with GDM were found to have higher serum levels of neopterin (32.9 nM) than the control group (10.4 nM). Similar to neopterin, nitric oxide levels were found to be higher in the diabetic group (34.4 µM) than in the healthy controls (28.2 µM). Serum levels of BH4 were found to be lower due to diabetes in the patient group (9.6 nM) than in the healthy controls (8.8 nM). The eNOS and GCHI gene expressions were assessed in the blood samples obtained from both the patient and control groups. GCHI gene expression was found to be lower in the GDM patients (1.07) than in the healthy controls (1.27), whereas eNOS gene expression was higher in the GDM group (4.52) than in the control group (3.16). The parameters assessed in the study groups are summarized in Table 2.

Table 2.

Measured parameters in the study groups

	Group	Median	Minimum	Maximum	p
Neopterin (nM)	Control	1.36	0.08	33.95	< 0.001
	GDM	0.65	0.08	24.82
NO (µM)	Control	2.46	0.10	9.17	0.274
	GDM	2.79	0.21	10.24
BH4 (nM)	Control	6.58	0.52	30.12	0.456
	GDM	7.52	1.26	30.14
eNOS	Control	2.62	0.44	8.51	0.918
	GDM	1.95	1.12	8.19
GCHI	Control	0.91	0.44	2.21	0.798
	GDM	1.05	0.52	1.71

Discussion

Due to the decreased bioavailability of NO in diabetes, disorders in NO synthesis play a critical role in the pathogenesis of such microvascular complications as diabetic retinopathy and nephropathy, caused by chronic hyperglycemia in diabetic patients [20]. Some studies have argued that the decreased GCHI expression in endothelial cells is likely to be the primary cause of the BH4 deficiency, and accordingly, the NO formation will be affected in diabetic patients [21, 22]. The authors of these studies thus support the opinion that BH4 supplementation is an applicable therapeutic option for the treatment of diseases leading to changes in NO production [23]. Recent intensive researches have focused on understanding whether or not elevated BH4 levels provide therapeutic benefits in the treatment of cardiovascular diseases setting ground for endothelial dysfunction [24]. The higher levels of neopterin synthesized in the GTP pathway in pregnant women with gestational diabeteswhen compared to healthy pregnant women suggest cellular immune system activation and the induction of neopterin production from GTP in macrophages. However, the decrease in the level of synthesized BH4 in the same pathway suggests that the interim changes should be evaluated in detail.

The present study found increased neopterin levels in the GDM group. Since neopterin is a biomarker of proinflammatory immune conditions, its release is induced in the presence of inflammation. It can thus be concluded that elevated neopterin levels may be linked to the activation of GDM-linked inflammation mechanisms. In pregnancy, the immune system is suppressed under the control of the placenta. In GDM, proinflammatory cytokines like neopterin can be expected to elevate, insulin resistance may become more remarkable and glucose metabolism may be impaired [25, 26]. The release of neopterin from macrophages was found to be induced due to the activation of the cellular immune system, triggered by diabetes. Previous studies aiming to identify the link between nitric oxide and GDM have produced conflicting results. While some researchers have suggested elevated NO levels in diabetes patients [27, 28], others have reported the exact opposite results [29–31]. Studies suggesting that GDM decreases NO levels argue that GDM is characterized by reduced NO bioavailability and associated decreased fetoplacental vasodilatation. Those arguing for the opposite, however, claim that the high serum levels in glucose activate the endothelial cells, and thereby induce the release of NO [32]. In the present study, NO levels were found to be high in the GDM group, leading us to suggest that hyperglycemia is responsible for the high levels of NO. Since BH4 is a key co-factor for eNOS, the serum levels of BH4 reflect the endothelial function [33]. As a result, BH4 deficienciesare observed in conditions associated with impaired endothelial function. The primary enzymes in BH4 synthesis from the endothelial cells in human placental vasculatures are GCHI and 6-pyruvoyltetrahydropterin synthase (PTPS). As pregnancies proceed, the activities of these enzymes decrease, and it is believed that an associated decrease occurs in the functions of BH4 [5]. It has been observed that decreased GCHI levels in human aortic endothelial cells due to hyperglycemia reduce BH4 levels and NO synthesis, and that this is reversed through the overexpression of GCHI [34, 35]. GCHI catalyzes the first and rate-limiting step of the synthesis of BH4, which is a key co-factor in a series of important metabolic enzymes [36]. The present study found decreased GCHI levels associated with diabetes. Decreases in GCHI, which play a role in BH4 synthesis, are a direct cause of BH4 deficiency. In contrast to the decrease in GCHI expression, eNOS levels were observed to increase. Hyperglycemia causes the production of reactive oxygen species (ROS) and reactive nitrogen species through a series of enzymatic processes, and leads to an imbalance in the antioxidant defense system, and such mechanisms are associated with reduced NO bioavailability, and indirectly with eNOS uncoupling [37, 38]. The elevated glucose levels in the fetal endothelial cells of healthy pregnant women have been found to induce eNOS expression. In the present study, eNOS expression was found to be elevated, having been induced by hyperglycemia. Although the mechanisms responsible for GDM have been the subject of recent research, it is believed that such mechanisms are not comparable to the causes of type 2 diabetes, and have yet to be fully clarified. The supplementation of levodopa, 5-hydroxytryptophan or oral calcium folinate is commonly used in clinical practice for neurological diseases associated with GCHI enzyme deficiency. Furthermore, oral BH4 supplementation has been successfully applied in clinical practice to treat diseases with BH4 deficiency, such as hyperphenylalaninemia. Oral BH4 supplementations should be an applicable option in clinical practice also for the treatment of GDM in which the BH4 deficiency is induced. The loss of functional mutations in the GCHI gene can lead to such congenital neurological diseases as dystonia and hyperphenylalaninemia. That said, little is known about the extent to which GCHI and BH4 affect embryonic development in the uterus,or whether the metabolic replacement or supplementation in pregnancy is enough to make up for the genetic GCHI deficiency in the developing embryo. Recent studies have focused on the efficacy of BH4 supplementation in cardiovascular diseases with decreased NO bioavailability.

Conclusions

There have also been a number of preclinical and clinical studies demonstrating a positive association between BH4 supplementation and vascular function, although the efficacy of oral BH4 in cardiovascular diseases in humans is still uncertain. BH4 deficiency, despite being linked with serious outcomes, is treatable, and therefore developing selective screening tests for early detection and establishing the necessary treatment options for cases requiring supplementation would prevent potential complications in the fetus. The findings of the present study suggest that GTP pathway-related biomarkers and the gene expressions related to this pathway may be used for the early detection of GDM.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest concerning this article.