Association of the Polymorphism in Nucleobindin 2 Gene and the Risk of Metabolic Syndrome

Abstract

Objective: Nesfatin-1, originates from the precursor protein nucleobindin 2 (NUCB2) and plays an important role in the development of metabolic syndrome (MetS), including obesity and hypertension. This study aimed to determine whether the 1012C>G polymorphism of NUCB2 gene is correlated with the development of MetS in the Chinese Han population. Materials and Methods: The 1012C>G polymorphism of NUCB2 gene was detected in a population of 326 patients with MetS and 165 healthy subjects. Results: MetS patients showed lower CG and GG genotypes, as well as lower G allele frequencies, compared with healthy subjects. Unconditional logistic regression analysis showed that the GG genotype as well as the G allele were both significantly associated with the decreased risk of developing MetS. In addition, the GG genotype of NUCB2 was significantly correlated with lower levels of waist circumference, body mass index, and fasting plasma glucose in patients with MetS. Conclusions: 1012C>G polymorphism of NUCB2 is correlated with a reduced risk of developing MetS in a Chinese Han population.

Introduction

Metabolic syndrome (MetS) is a cluster of metabolic and cardiovascular disturbances, such as abdominal obesity, hypertension, dyslipidemia, and hyperglycemia. The risk for MetS patients to develop type 2 diabetes, atherosclerosis, and cardiovascular diseases is relatively higher than normal subjects (Jain and Lahiri, 2007). Genetic and environmental factors contribute to the susceptibility for MetS. Recent investigations have focused on the important role of certain genetic polymorphism in the increased susceptibility for MetS (Pollex and Hegele, 2006).

Nucleobindin 2 (NUCB2), containing 396 amino acid residues, is a precursor of nesfatin-1 (residues 1-82), nesfatin-2 (residues 85-163), and nesfatin-3 (residues 166-396) (Oh-I et al., 2006). Nesfatin-1 is a protein highly conserved in humans, rats, and mice (Oh-I et al., 2006). In rats, intracerebroventricular injection of nesfatin-1 resulted in inhibited nocturnal food intake and decreased body weight gain, whereas injection of an antibody-neutralizing nesfatin-1 promoted appetite (Oh-I et al., 2006). Significant differences in plasma nesfatin-1 concentrations were observed in obese subjects and healthy controls (Abaci et al., 2013; Guo et al., 2014). Furthermore, plasma nesfatin-1 levels were significantly higher in hypertension patients than in control groups (Zhao et al., 2015). Recently, genetic investigations have showed that 1012C>G polymorphism of NUCB2 is correlated with obesity and blood pressure (Zegers et al., 2011; Chen et al., 2013; Tragante et al., 2014). Obesity and hypertension are typical characteristics of MetS. Therefore, it is hypothesized that 1012C>G polymorphism of NUCB2 may be associated with the risk of MetS.

Therefore, we aim to determine whether 1012C>G polymorphism of NUCB2 gene is correlated with the risk of MetS in a Chinese Han population.

Materials and Methods

Subjects

A total of 326 subjects with MetS were enrolled in this study. MetS was defined using criteria established in the third report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment in Asian population (Adult Treatment Panel III) as the presence of three or more of the following risk factors (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001): waist circumference (WC) ≥80 cm in women or ≥90 cm in men, triglycerides (TG) ≥1.69 mmol/L, high-density lipoprotein cholesterol (HDL-C) ≤1.29 mmol/L in women or ≤1.03 mmol/L in men, blood pressure ≥130/85 mm Hg, and fasting plasma glucose (FPG) ≥5.6 mmol/L. The control group consisted of 165 subjects who were recruited from individuals attending the routine check up in our hospital. The controls had none of the five criteria of MetS described above. The exclusive criteria were as follows: diabetes mellitus, impaired glucose tolerance, cancer, and any other systemic diseases.

The study was approved by the hospital ethics board, and all patients provided written informed consent.

Measurements

Weight, height, WC, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured. Venous blood was collected after a minimum of 10 h of absolute diet. FPG, TG, serum total cholesterol, HDL-C, and low-density lipoprotein cholesterol (LDL-C) were tested using an auto biochemistry instrument (Hitachi 7170, Tokyo, Japan). Body mass index (BMI) was calculated as weight in kilograms divided by height squared in meters (kg m⁻²).

DNA Genotyping

Blood samples were collected from all subjects. Genomic DNA was extracted from peripheral blood using a DNA extraction kit (Qiagen, Valencia, CA). 1012C>G polymorphism of NUCB2 gene was detected using polymerase chain reaction (PCR) and sequencing method. The primer sequences for genotyping the 1012C>G polymorphism of NUCB2 gene were as follows: 5′-ATCCTAATGACTTTGACCCC-3′ (forward) and 5′-TGAGGAGACATCTTGCAC CAC-3′ (reverse). The PCR was performed with 5 min denaturation at 95°C, followed by 35 cycles of 30 s at 95°C, 45 s at 60°C, and 45 s at 72°C. PCR products were directly sequenced by the Sangon Biotech Company (Shanghai, China).

Statistical analysis

Data are presented as mean ± standard deviation. The normality of distributions of the variables was analyzed by the Kolmogorov-Smirnov test. Clinical variables were compared between MetS patients and control subjects using the Student's t-test or chi-squared (χ²) test. The χ² test was performed to assess the Hardy-Weinberg equilibrium. χ² and p Values for the genotypes of 1012C>G polymorphism in MetS patients and controls, as well as in nonobese and obese MetS subgroups, were calculated using the χ² test. The unconditional logistic regression analysis was utilized to determine the association of the genotype and allele frequencies with the presence of MetS as well as obesity. The variables of different NUCB2 genotypes among MetS patients were analyzed using the one-way ANOVA followed by Tukey's post hoc test to compare the differences among the three groups. Statistical significance was accepted at a level of p < 0.05.

Results

The characteristics of MetS patients and control subjects

The clinical and laboratory parameters of MetS patients and control subjects are shown in Table 1. The MetS group showed higher levels of WC, BMI, SBP, DBP, FPG, TG, LDL-C, and lowered HDL-C level than the control subjects. There were no significant differences in other characteristics between the two groups.

Table 1.

Comparison of Clinical and Laboratory Parameters Between MetS Patients and Control Subjects

Characteristic	MetS group (n = 326)	Control group (n = 165)	p
Age (years)	60.06 ± 8.03	59.55 ± 10.01	0.544
Gender (M/F)	176/150	85/80	0.604
WC (cm)	85.93 ± 10.06	77.90 ± 7.78	<0.001
BMI (kg/m²)	27.02 ± 3.47	24.21 ± 2.45	<0.001
SBP (mm Hg)	143.80 ± 16.63	123.75 ± 14.28	<0.001
DBP (mm Hg)	84.52 ± 10.42	80.67 ± 8.92	<0.001
FPG (mmol/L)	6.10 ± 0.90	5.11 ± 0.39	<0.001
TG (mmol/L)	1.95 ± 1.11	1.08 ± 0.52	<0.001
TC (mmol/L)	5.17 ± 1.12	5.10 ± 0.82	0.490
HDL-C (mmol/L)	1.15 ± 0.26	1.42 ± 0.21	<0.001
LDL-C (mmol/L)	3.38 ± 0.82	3.23 ± 0.54	0.047

Values are given as mean ± standard deviation (SD).

MetS, metabolic syndrome; WC, waist circumference; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FPG, fasting plasma glucose; TG, triglycerides; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

Association of 1012C>G polymorphism of NUCB2 gene with MetS development

The genotype frequencies of 1012C>G polymorphism among the controls were in agreement with the Hardy-Weinberg equilibrium (p = 0.549). MetS patients showed significantly lower CG and GG genotype, as well as G allele frequencies, compared with control subjects (Table 2). As shown in Table 2, the unconditional logistic regression analysis showed that GG genotype as well as G allele was significantly associated with the decreased risk of developing MetS (OR 0.458, 95% CI 0.266-0.791, p = 0.005 and OR 0.672, 95% CI 0.513-0.879, p = 0.004, respectively).

Table 2.

Genotype and Allele Frequencies of the 1012C>G Polymorphism of NUCB2 Gene in MetS Subjects and Control Subjects

	MetS group (n = 326)		Control group (n = 165)			Logistic regression
Genotype	n	%	N	%	p	95% CI	p
CC	136	41.7	51	30.9	0.017	1.00 (reference)
CG	146	44.8	78	47.3		0.702 (0.460-1.072)	0.101
GG	44	13.5	36	21.8		0.458 (0.266-0.791)	0.005
C	418	64.1	180	54.5	0.004	1.00 (reference)
G	234	35.9	150	45.5		0.672 (0.513-0.879)	0.004

NUCB2, nucleobindin 2.

Association of 1012C>G polymorphism of NUCB2 gene with obesity in the MetS group

Chinese classifications of BMI ≥24 for overweight and ≥28 for obesity have been recommended based on the data of the Chinese national survey (Wu et al., 2005). Then, we divided MetS patients into nonobese and obese subgroups and analyzed the association of 1012C>G polymorphism of NUCB2 gene with obesity. Lower GG genotype was observed in obese MetS patients than in nonobese ones (Table 3). However, no significant difference in the allele frequency of 1012C>G polymorphism was found between these two subgroups. The unconditional logistic regression analysis showed that GG genotype was significantly associated with the decreased risk of developing obesity (OR 0.359, 95% CI 0.155-0.832, p = 0.017) (Table 3).

Table 3.

Genotype and Allele Frequencies of the 1012C>G Polymorphism of NUCB2 Gene in Obese and Nonobese MetS Subjects

	Obese (n = 117)		Nonobese (n = 209)			Logistic regression
Genotype	n	%	N	%	p	95% CI	p
CC	52	44.4	84	40.2	0.031	1.00 (reference)
CG	57	48.7	89	42.6		1.035 (0.640-1.672)	0.890
GG	8	6.8	36	17.2		0.359 (0.155-0.832)	0.017
C	161	68.8	257	61.5	0.062	1.00 (reference)
G	73	31.2	161	38.5		0.724 (0.515-1.016)	0.062

Association of 1012C>G polymorphism of NUCB2 gene with clinical parameters

Table 4 presents the characteristics of the different genotypes of the 1012C>G polymorphism of NUCB2 gene in MetS subjects. WC, BMI, and FPG were significantly lower in MetS subjects with GG genotype than those with CC and CG genotypes. However, no significant differences in WC, BMI, and FPG were found between subjects with CC and CG genotypes.

Table 4.

Comparison of Characteristics of Different Genotypes of 1012C>G Polymorphism of NUCB2 Gene in MetS Subjects

Characteristic	CC (n = 136)	CG (n = 146)	GG (n = 44)	p
Age (years)	60.25 ± 8.28	59.65 ± 7.80	60.82 ± 7.45	0.656
Gender (M/F)	74/62	77/69	25/19	0.885
WC (cm)	86.64 ± 10.20	86.31 ± 10.33	82.47 ± 8.06^a,b	0.047
BMI (kg/m²)	27.26 ± 3.73	27.21 ± 3.37	25.64 ± 2.56^a,b	0.017
SBP (mm Hg)	142.85 ± 13.68	143.95 ± 17.84	146.25 ± 20.46	0.496
DBP (mm Hg)	83.90 ± 9.96	85.14 ± 10.72	84.43 ± 10.96	0.608
FPG (mmol/L)	6.13 ± 0.85	6.16 ± 0.94	5.78 ± 0.83^a,b	0.036
TG (mmol/L)	1.93 ± 1.13	1.97 ± 1.15	1.97 ± 0.97	0.928
TC (mmol/L)	5.17 ± 1.15	5.20 ± 1.11	5.09 ± 1.06	0.867
HDL (mmol/L)	1.13 ± 0.24	1.17 ± 0.29	1.17 ± 0.20	0.420
LDL (mmol/L)	3.36 ± 0.89	3.40 ± 0.76	3.37 ± 0.85	0.915

Values are given as mean ± SD.

Significant versus MetS patients with CC genotype.

Significant versus MetS patients with CG genotype.

Discussion

The risk for MetS patients to develop type 2 diabetes, atherosclerosis, and cardiovascular diseases is relatively higher than normal subjects (Jain and Lahiri, 2007). It is essential to find more susceptibility genes for MetS and assess the genetic risk of individuals to develop MetS. Therefore, the subjects with higher risk of developing MetS should perform timely intervention, including changing lifestyle, exercise, and drug treatment such as metformin. The present study demonstrated that 1012C>G polymorphism of NUCB2 was correlated with a reduced risk of developing MetS. This suggests that NUCB2 gene polymorphism could be utilized as a genetic marker to assess the risk of developing MetS. In addition, nesfatin-1 was found to be correlated with MetS. A study performed in obstructive sleep apnea syndrome patients showed that plasma nesfatin-1 levels were significantly lower in the MetS group compared with the non-MetS group (Aksu et al., 2015). However, other studies demonstrated higher plasma nesfatin-1 levels in MetS patients (Mirzaei et al., 2013) and rats (Catak et al., 2014). These findings point to the key role of nesfatin-1/NUCB2 in the development of MetS. However, the exact mechanism of nesfatin-1/NUCB2 in the pathogenesis of MetS is unknown.

Nesfatin-1 is correlated with obesity development. Chronic peripheral administration of nesfatin-1 resulted in a sustained reduction in food intake and modulation of whole-body energy homeostasis in rats (Mortazavi et al., 2015). Central nervous administration of nesfatin-1 increased energy expenditure and reduced food intake in rats (Wernecke et al., 2014). Plasma nesfatin-1 concentrations were significantly decreased in obese subjects compared with healthy controls (Abaci et al., 2013; Guo et al., 2014). Recently, studies have showed that 1012C>G polymorphism of NUCB2 is correlated with obesity (Zegers et al., 2011; Chen et al., 2013). Our finding also demonstrated the association of this polymorphism with obesity in MetS patients. Furthermore, MetS patients with GG genotype had lower WC and BMI levels compared with those with CC and CG genotypes.

Nesfatin-1 also plays an important role in the development of hypertension. Chronic peripheral nesfatin-1 administration increased blood pressure in rats (Ayada et al., 2015). Intravenous application of nesfatin-1 to rats not only increased blood pressure but also impaired the sodium nitroprusside-induced decreases in blood pressure (Yamawaki et al., 2012). In addition, fasting plasma nesfatin-1 levels were significantly higher in hypertension patients than in control groups (Zhao et al., 2015). Tragante et al. (2014) reported that the 1012C>G polymorphism of NUCB2 is correlated with blood pressure. Our study found no significant differences in blood pressure between MetS patients with different genotypes. Our study was conducted in MetS patients and not in healthy subjects. In addition, these two studies, including our investigation, were performed in different ethnic populations. Therefore, the explanation for these conflicting data is unclear but may be attributable to differences in disease advancement or ethnic populations.

Nesfatin-1 serves as a key regulator in glucose metabolism and diabetes development. Nesfatin-1 stimulated insulin secretion from pancreatic β-cells, improved insulin sensitivity, and contributed to energy storage (Nakata and Yada, 2013). Furthermore, plasma nesfatin-1 concentrations were significantly decreased in type 2 diabetes mellitus patients compared with healthy controls (Li et al., 2010; Liu et al., 2014). The current study showed that MetS patients with GG genotype had lower FPG levels compared with those with CC and CG genotypes. This finding suggests the association of 1012C>G polymorphism of NUCB2 gene with glucose metabolism and diabetes.

The potential limitations of these data merit consideration. First, as our study was conducted in a sample of Chinese patients, extrapolation of the data to other ethnic groups should be performed with great caution. Second, the study population was relatively small. Therefore, our results require further investigation with larger sample sizes.

In conclusion, this study revealed that 1012C>G polymorphism of NUCB2 gene was associated with the presence of MetS.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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