Functional Polymorphism Located in MMP-9 Gene Promoter Is Strongly Associated with Obesity

Abstract

The adipose tissue expansion is accompanied by remodeling of extracellular matrix performed by matrix metalloproteinases (MMPs). Higher plasma and tissue MMP-9 levels are found in obese; therefore, we evaluated if the functional C⁻¹⁵⁶²T polymorphism (rs3918242) located in promoter region of the MMP-9 gene is associated with obesity in women. We studied 112 lean and 114 obese women. Plasma MMP-9 and tissue inhibitor of MMP-9 (TIMP)-1 were measured using enzyme-linked immunosorbent assay. We found different genotype frequencies between lean and obese women (p=0.008), prevailing T-allele in obese (2.3-fold). However, although obese women present higher levels of plasma MMP-9, lack of modulation by the polymorphism was found (all p>0.05). Our findings suggest that C⁻¹⁵⁶²T polymorphism may contribute to pathogenetic mechanisms involved in the development of obesity in women.

Introduction

O besity is strongly associated to elevated risk of cardiovascular morbi-mortality development, including stroke, congestive heart failure, and myocardial infarction (Kenchaiah et al., 2002). The expansion of adipose tissue and enlargement of fat cells is accompanied by a remodeling of the stromal matrix performed mainly by matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) (Halberg et al., 2008). Particularly important, the MMP-9 has been found at elevated concentration in obese subjects (Laimer et al., 2005; Garvin et al., 2008) and at present several studies have been demonstrating the participation of MMP-9 in a number of pathological conditions (Mun-Bryce and Rosenberg, 1998; Loftus et al., 2000; Mun-Bryce et al., 2004; Lacchini et al., 2010a,b). Therefore, it is possible that genetic polymorphisms in the MMP-9 gene may be associated with obesity. The polymorphism C⁻¹⁵⁶²T (rs3918242) located in promoter region modulates transcriptional activity of MMP-9 gene, being associated with some cardiovascular diseases (Peters et al., 1999; Pollanen et al., 2001). To our knowledge no study has addressed the association of this polymorphism with adult obesity. Therefore, this study aimed at comparing the genotype and allelic frequencies of the functional C⁻¹⁵⁶²T polymorphism between lean and obese adult women. In addition, we evaluated the impact of this variation on plasmatic MMP-9 levels.

Patients and Methods

Approval for use of human subjects was obtained from the Institutional Review Board at the Santa Casa de Belo Horizonte, Brazil, and subjects gave informed consent. One hundred fourteen obese women were recruited from Department of Obesity, Center of Medical Specialities of Minas Gerais, Brazil, and 112 lean women were recruited from local community. Women present body mass index (BMI) > 30 kg/m²; was considered obese and BMI > 18.5 and 24.9 kg/m²; lean according with the guidelines of the Brazilian Association the Study of Obesity and the Metabolic Syndrome. Type 2 diabetes was defined as fasting plasma glucose ≥ 126 mg/dL, confirmed by repeating the test on another day or under hypoglycemic medication and insulin. Blood pressure (BP) measurement was taken three times from all volunteers with subject in the seated position with the use of an automatic BP device. The individuals were considered hypertensive if their systolic or diastolic BP was > 140 or > 90 mmHg, respectively, or were on antihypertensive therapy. At the time of clinic attendance, venous blood samples were collected into standard Vacutainer tubes containing ethylenediaminetetraacetic acid. Subjects with acute diseases or chronic diseases, except hypertension and type 2 diabetes mellitus (T2DM), were excluded from the study. The tubes were immediately centrifuged at 2000 g for 10 min at room temperature, and plasma samples were stored at −80°C until used to measure plasma MMP-9 and TIMP-1 concentrations. Genomic DNA was extracted from the cellular component of 1 mL whole blood by a salting method and stored at −20°C until analyzed.

Glucose concentrations and lipid parameters were determined with routine enzymatic methods using commercial kits (Katal Biotechnology Industry and Trade Ltd.). Concentrations of MMP-9 and TIMP-1 were measured in plasma using a commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer's instructions. Women were genotyped for C⁻¹⁵⁶²T polymorphism by restriction fragment length polymorphism (RFLP), as described earlier (Palei et al., 2010). A second method to confirm the genotypes was performed in 30 samples (15 lean and 15 obese) using high-resolution melting assay with HRM primers, Type-It HRM PCR kit (Qiagen), and Eco Real-Time PCR System (Illumina). All genotypes obtained from RFLP were confirmed by HRM genotyping.

All the results were expressed as mean±standard deviation. The clinical and biochemical characteristics of studied groups were compared by the unpaired Student t-test or one-way analysis of variance followed by the Bonferroni multiple comparisons test. Contingency table-based analyses were used for comparisons of nominal variables, and Chi-square test for trend was used to evaluate the distribution of genotypes and allele frequencies between groups. A value of p<0.05 was considered statistically significant. Our study has 88% power to detect an effect with a relative risk of 2.3 in the group of obese and lean controls under a dominant genetic model.

Results

Table 1 shows clinical and biochemical parameters of the 226 subjects enrolled in the present study. As expected, obese women present higher BMI, waist circumference, waist-to-hip ratio, hypertensives, and diabetics than lean women. Moreover, we found higher MMP-9 levels and MMP-9/TIMP-1 ratio in obese women than in lean group (p<0.05). Importantly, when we excluded women with comorbidities (hypertension and T2DM) we observed significant reduction of MMP-9 and TIMP-1 levels in this group compared to all obese, although MMP-9/TIMP-1 ratio present similar values.

Table 1.

Clinical and Biochemical Parameters of Studied Groups

			Without comorbidities
	All lean	All obese	Lean	Obese
N	112	114	85	42
Age (years)	41.3±11.7	43.5±11.1	33.7±11.5	37.7±9.0^a
BMI (kg/m²)	20.9±3.2	47.4±7.1^b	20.6±2.2	42.8±6.8^a,c
WC (cm)	68.1±4.2	124.8±14.3^b	67.8±3.9	121.3±14.2^c
WHR	0.75±0.02	0.96±0.09^b	0.74±0.02	0.93±0.09^c
HT (%)	23	33	—	—
HT+T2DM (%)	1	30^b	—	—
Tot Chol. (mg/dL)	132.4±27.4	181.5±36.9^b	121.4±17.4	171.8±27.9^c
LDL Chol. (mg/dL)	94.4±27.5	114.0±33.8^b	82.3±17.5	109.1±31.8^c
HDL Chol. (mg/dL)	59.2±9.6	45.4±9.7^b	61.7±9.2	49.1±8.4^c
TGs (mg/dl)	106.3±27.9	128.7±67.2^b	100.6±24.0	110.6±58.6
FG (mg/dL)	86.2±14.8	104.1±36.6^b	84.9±12.1	87.7±10.1
MMP-9 (ng/mL)	312.1±276.7	563.2±356.5^b	282.6±153.5	402.5±249.9^a,c
TIMP-1 (ng/mL)	425.9±149.4	485.2±293.5	431.0±120.2	342.8±93.6^a,c
MMP-9/TIMP-1 ratio	0.79±0.7	1.29±0.83^b	0.71±0.5	1.32±0.97^c

Values are mean±standard deviation.

p<0.05 versus all obese.

p<0.05 versus all lean.

p<0.05 versus lean.

BMI, body mass index; WC, waist circumference; WHR, waist-to-hip ratio; HT, hypertension; T2DM, type 2 diabetes mellitus; Tot Chol., total cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides; FG, fasting glucose; MMP, matrix metalloproteinase; TIMP-1, tissue inhibitor of MMP-9.

The genotypes and alleles frequencies of C/T polymorphism are present in Table 2. No deviation from Hardy–Weinberg equilibrium was observed. The first analysis included all lean and obese women, whereas the second one took into consideration only women without comorbidities. Interestingly, we observed important differences in the overall distribution of C/T genotypes (p=0.008) and allele (p=0.059) frequencies comparing all lean versus all obese women. Similar results were observed excluding the comorbidities, with exception of CT genotype that increase about 45% in both group (lean and obese). Importantly, subjects carrying T variant were 2.2-fold more frequent in obese than in lean women. In addition, despite obese women present higher plasmatic levels of MMP-9 compared to control, without a compensatory increase of TIMP-1, a lack of modulation of these levels by polymorphism was observed, as well as on MMP-9/TIMP-1 ratio (data not shown).

Table 2.

Genotypes and Alleles Frequencies of the C⁻¹⁵⁶²T Polymorphism Analyzed in Lean and Obese Women

				MMP-9 levels (ng/mL)					MMP-9 levels (ng/mL)
	All lean	All obese	p-Value	Lean	Obese	Lean	Obese	p-Value	Lean	Obese
Genotype
CC (%)	89	74	0.008	368±134	548±373	85	67	0.003	313±154	418±285
CT (%)	10	23		293±168	477±371	15	33		247±157	315±77
TT (%)	1	3		315	621±608	—	—		—	—
Allele
C (%)	94	86	0.059	—	—	93	84	0.046	—	—
T (%)	6	14		—	—	7	16		—	—

Discussion

This study was the first to evaluate the association between the single nucleotide polymorphism (SNP) C⁻¹⁵⁶²T located in promoter region of the MMP-9 gene and obesity. The findings show meaningful prevalence of T variant in the obese group, suggesting an important effect on weight gain in T-carriers. Interestingly, an in vitro study demonstrated that T allele is associated with increased MMP-9 expression (Zhang et al., 1999) due of the loss of a nuclear repressor protein binding site when the T variant is present. In addition, despite obese women present higher plasmatic levels of MMP-9 compared to control, absence of modulation by the polymorphism was found. This may suggest a particular effect of this variant and consequently of MMP-9 expression in situ (adipose tissue) that is not reflected in circulating MMP-9 levels, or it is possible that an impact of polymorphism on these levels may be observed temporally during tissue expansion.

Knowing that clinical conditions and medications, such as hypertension, diabetes, cardiovascular disease, use of statins, and smokers (Garvin et al., 2008; Gerlach et al., 2009; Goncalves et al., 2009; Jacob-Ferreira et al., 2010), may interfere on MMPs levels, we performed analysis in a women subgroup without medication or comorbidities, and the results were similar to those reported including all women. Interestingly, a previous study showed a sexual dimorphism of some angiogenic and anti-angiogenic biomarkers (Silha et al., 2005), supporting our choice to evaluate only women.

The clinical association of this functional polymorphism with obesity susceptibility may consolidate the relevance of MMP-9 for adipose tissue expansion. The mechanisms underlying this association may be related to matrix-bound vascular endothelial growth factor mobilized by MMP-9 that indirectly induces angiogenesis (Bergers et al., 2000). In addition, a study performed with adipocyte culture demonstrates that MMP-9 is released and increased during adipocytes differentiation, which may be inhibited by drugs that reduce MMP-9 activity or by antibodies that neutralize the MMP-9 (Bouloumie et al., 2001). This suggests that the extracellular matrix remodeling by MMP-9 may be an important regulator of adipocyte differentiation that may contribute to the obesity development.

Although of the MMP-9 gene presents several polymorphisms, the analysis of others variants or haplotypes may compromise the statistical analysis due to low power, even though we are conscious about the importance of haplotype analysis as demonstrated previously (Belo et al., 2012). Therefore, the option of C/T variant was supported by functional and several clinical studies reporting association (Zhang et al., 1999; Palei et al., 2010). Genotype and allele frequencies in lean women showed similar results reported previously in the Brazilian population (Lacchini et al., 2010) and in databases (Hapmap and Seattle SNPs).

In conclusion, the current study demonstrates for the first time an important impact of MMP-9 polymorphism on obesity susceptibility in women. Moreover, our data suggest that MMP-9 polymorphisms may contribute to the development of cardiovascular complications and cancers associated with obesity and, moreover, indicate a possibility of new therapeutic approaches in subjects carrying T-allele.

Footnotes

Acknowledgments

This study was funded by the Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG-Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and IEP-SCBH—Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte.

Disclosure Statement

The authors declare no conflict of interest.

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