Uncoupling Protein-2 45-Base Pair Insertion/Deletion Polymorphism: Is There an Association with Severe Obesity and Weight Loss in Morbidly Obese Subjects?

Abstract

Background:

Uncoupling proteins are attractive candidate genes for obesity and type 2 diabetes mellitus. Our aim was to investigate the potential association of the uncoupling protein-2 (UCP2) 45-bp insertion/deletion (ins/del) polymorphism with obesity, as well as the potential effect of this polymorphism on weight loss variability in severely obese subjects.

Methods:

A total of 158 severely obese subjects (94 without and 64 with metabolic syndrome) and 91 age and sex-matched lean controls were recruited. A subgroup of 124 obese patients participated in a 3-month weight loss program. Anthropometric and metabolic variables were measured. Participants were genotyped for the UCP2 ins/del polymorphism.

Results:

Allelic frequency differed neither between obese subjects and controls (P=0.56), nor between obese subjects with versus without metabolic syndrome (P=0.58). At 3 months, metabolically healthy subjects carrying the insertion allele had significantly greater reduction in body mass index (P=0.029) and fat-free mass (P=0.013) and a borderline significant improvement in the homeostatic model assessment index (P=0.048).

Conclusion:

There is no association of the UCP2 ins/del polymorphism with morbid obesity in our population, but this genotype appears to be linked with a favorable response to dietary changes in metabolically healthy obese subjects.

Introduction

B oth obesity and type 2 diabetes are conditions of impaired energy metabolism. The latter is regulated at the systemic and cellular level by a large variety of enzymatic and nonenzymatic proteins. A family of mitochondrial proteins, called uncoupling proteins (UCPs), plays an important role in generating heat and burning calories by creating a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane, without coupling to any other energy-consuming process.¹ Uncoupling protein-2 (UCP2), a member of this mitochondrial transporter superfamily, is expressed widely, including white adipose tissue, skeletal muscle, and pancreatic islets.² Physiologic studies have shown that UCP2 is critically positioned to maintain fatty acid oxidation and restrict subsequent oxidative damage, allowing sustained mitochondrial oxidative capacity and mitochondrial biogenesis.³ It also negatively regulates insulin secretion and is considered an important link between obesity, β-cell dysfunction, and type 2 diabetes mellitus (T2DM). Hence, today it is being considered a plausible candidate gene for susceptibility to obesity and T2DM.^4,5

The UCP2 insertion/deletion (ins/del) polymorphism is a 45-bp insertion in exon 8 in the 3′-untranslated region of the UCP2 gene that may affect UCP2 mRNA stability, posttranscriptional modification, or translation, all of which could result in altered levels or activity of the mature protein.⁶ This and other polymorphisms have been identified and studied in the UCP2 gene and other UCP family members, but previous association studies with obesity and related metabolic features provided inconsistent results in different populations.^7
–9 Furthermore, variability in weight loss has been observed in obese patients on lifestyle modification response, and it is speculated that genetic effects may play a crucial role.^10
–12

Thus, the aim of the present study was to investigate: (1) The association of the UCP2 45-bp ins/del polymorphism with obesity and related metabolic features and (2) the potential effect of this polymorphism on diet-induced weight loss variability in severely obese Greek subjects.

Methods

A total of 158 severely obese [body mass index (BMI) >40 kg/m²] subjects (65 males, 41.1%; 93 females, 58.9%), designated as the obesity (OB) group, were included. Of these, 94 (59.5%) had no other features of metabolic syndrome and were designated as metabolically healthy but severely obese (MHSO) subjects. The rest were 64 (40.5%) age- and sex-matched subjects with severe obesity and metabolic syndrome according to the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria and designated as the metabolic syndrome group.¹³ A group consisting of 91 age- and sex-matched healthy lean subjects (37 males, 40.6%; 54 females, 59.4%) was used as controls. All patients had normal thyroid function, and none of them had concomitant serious renal, hepatic, or cardiac disease. Overall, 124 patients (74%–59.7% from the MHSO subgroup and 50%–40.3% from of the metabolic syndrome subgroup) agreed to participate in a 3-month outpatient intervention program based on nutritional education, diet (500-kcal deficit diet), and reinforcement for physical exercise, without any pharmacological intervention. Antidiabetic agents or other drugs possibly inducing weight change were not added during the study period.

All obese subjects were consecutively recruited from the Outpatient Clinic of Obesity, Diabetes, and Metabolism in the Second Department of Internal Medicine at Democritus University of Thrace. The study was conducted in accordance with the Helsinki Declaration of Human Rights and all subjects gave their informed consent.

Anthropometric measurements were made with subjects wearing light, indoor clothing and without shoes. The mean of two measurements was recorded for each variable. Body weight was measured to the nearest 0.1 kg on calibrated scales. Body height was measured with a calibrated stadiometer. Waist circumference was measured midway between the iliac crest and the lower costal margin, with the participant wearing only nonrestrictive underwear. Body composition was measured using the Skylark Body Fat analyzer (model BT-905, Gima, Italy). Arterial blood pressure (BP) was evaluated twice (1 min apart) with the subject in the sitting position. The mean value of both systolic and diastolic arterial pressure was considered. Hypertension was defined according to Joint National Committee (JNC) VII criteria.¹⁴

DNA testing was conducted using DNA amplification by polymerase chain reaction. Biochemical measurements were performed in blood samples taken the morning after a 14-h fast, using standard enzymatic methods. Fasting insulin levels were also measured for the estimation of homeostatic model assessment (HOMA) index (HOMA=fasting insulin in mU/L×fasting plasma glucose in mg/dL/405).

Statistical analysis was performed by using standard software (V13.0, SPSS for MS Windows). Variables are presented as mean±standard deviation (SD) unless stated otherwise. Differences in allelic frequencies between the study and control groups were analyzed by the chi-squared test. Changes in anthropometric and metabolic parameters were evaluated and compared between groups using analysis of variance (ANOVA); a paired t-test was used to evaluate the changes in the same group of patients following the 3-month diet and lifestyle program. The nonparametric Wilcoxon rank sum test was used to evaluate differences between two groups. Significance was defined at 5% level (P<0.05).

Results

Clinical characteristics and anthropometric and metabolic parameters of the obese participants according to their genotype are shown in Table 1. Patients with ins/del and ins/ins genotypes (insertion allele carriers) were examined as a group due to the relatively small number of ins/ins homozygotes. No significant association was found between the studied polymorphism and the parameters of the obese phenotype studied. There was also no association between lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides] and the UCP2 ins/del polymorphism in the studied groups (data not shown).

Table 1.

Clinical, Anthropometric, and Basic Metabolic Features of the Obese Participants According to Their Genotype

	OB (n=158)			MHSO (n=94)			Metabolic syndrome (n=64)
	del/del	ins/del and ins/ins	P	del/del	ins/del and ins/ins	P	del/del	ins/del and ins/ins	P
Number	96	62		59	35		37	27
Age (years)	50.0±10.6	50.1±9.8	NS	48.2±10.3	47.1±8.6	NS	53.1±10.9	55.1±7.7	NS
Sex (males %)	41 (42.7%)	24 (38.7%)	NS	24 (40.6%)	14 (40%)	NS	17 (45.9%)	10 (37%)	NS
DM type 2 (history)	—	—	—	—	—	—	32 (86.5%)	17 (80.9%)	NS
AH (history)	—	—	—	—	—	—	21 (65.6%)	13 (61.3%)	NS
BMI	44.5±4.4	43.2±2.5	NS	43.2±2.2	42.2±1.7	NS	46.7±4.7	44.9±3.7	NS
Fat mass (%)	42.2±6.7	43.2±3.6	NS	44.4±5.9	43.3±4.6	NS	41.8±5.9	43±4.4	NS
Waist (cm)	119.3±10	121.3±13	NS	117±13	119±9	NS	123±11	125.3±12	NS
Glucose (mg%)	108.9±26	106.6±46	NS	92±13	95±9	NS	136±43	126±49	NS
Insulin (μIU/mL)	18.9±10.2	17.7±8.2	NS	16.0±8.9	15.4±6.2	NS	23.5±13.2	21.5±10.2	NS
HOMA index	5.1±5.2	4.8±4.2	NS	4.1±3.2	3.9±3.7	NS	6.8±5.9	6.5±4.1	NS

DM, diabetes mellitus; AH, arterial hypertension; BMI, body mass index; HOMA index, homeostatic model assessment index; NS, not significant.

The genotype frequencies, allele frequencies, and odds ratio (OR) estimates for the insertion allele in the OB group, MHSO and metabolic syndrome subgroups, and controls are presented in Table 2. The distribution of the UCP2 ins/del polymorphism in all groups was in Hardy–Weinberg equilibrium. No significant differences of the allelic frequency were found between the OB group, MHSO and metabolic syndrome subgroups, and the control group or between the MHSO and metabolic syndrome subgroups [P=0.56, OR (95% confidence interval, CI) 1.17 (0.68–2.00)]. Although we found a tendency toward a decreased proportion of del/del individuals from controls (65.9%) to MHSO (62.7%) and metabolic syndrome (57.8%), this difference (del/del vs. ins/del+ins/ins) was not statistically significant χ²=1.060, degrees of freedom (df)=2, P=0.589].

Table 2.

Frequencies of the UCP ins/del Genotypes in the Study Group, Subgroups, and Controls

		OB	MHSO	Metabolic syndrome	Controls
Genotypes	del/del	96 (60.8)	59 (62.7)	37 (57.8)	60 (65.9)
	ins/del	55 (34.8)	31 (33.0)	24 (37.5)	27 (24.5)
	ins/ins	7 (4.4)	4 (4.3)	3 (4.7)	4 (3.6)
Alleles	del	247 (78.1)	149 (79.2)	98 (76.5)	147 (80.7)
	ins	69 (21.9)	39 (20.8)	30 (23.5)	35 (19.3)
P ^a		0.56	0.79	0.39
Odds ratio (95% CI)		0.85 (0.54–1.34)	0.91 (0.54–1.51)	0.77 (0.45–1.34)

Versus controls

OB, obesity; MHSO, metabolically healthy but severely obese; CI, confidence interval.

At the 3-month follow-up visit, changes in BMI, fat mass, insulin levels, and HOMA index were recorded, and an analysis with the UCP2 ins/del polymorphism genotypes was carried out. ANOVA analysis showed that changes of BMI and fat-free mass, but not of insulin levels or HOMA index, were statistically significant between the three genotypic groups in MHSO patients (P=0.036, 0.045, 0.71, and 0.56, respectively). Association analysis showed that MHSO patients who carried the insertion allele had significantly greater reduction in BMI (P=0.029) and greater loss of fat free mass (P=0.013), as well as a borderline significant (P=0.048) improvement in HOMA index (but not insulin levels). No other significant association was observed between the measured parameters and the studied polymorphism in this or in the OB and metabolic syndrome groups (Table 3).

Table 3.

Body Mass Index, Fat Mass, Insulin Levels, and Homeostasis Model Assessment Index Changes at Baseline and After 3 Months in Obesity, Metabolically Healthy but Severely Obese, and Metabolic Syndrome Patients Who Participated in the Outpatient Intervention Program According to Genotype

			Baseline	3 Months	P
OB n=124, (del/del=70, ins/del=49, ins/ins=5)	BMI	del/del	44.4±3.1	42.8±3.8	NS
		ins/del and ins/ins	43.6±3.9	41.2±3.5	NS
	Fat mass (%)	del/del	42.9±4.6	41.6±4.1	NS
		ins/del and ins/ins	43.5±3.9	40.1±3.8	NS
	Insulin (μIU/mL)	del/del	20.3±10.3	17.6±11.4	NS
		ins/del and ins/ins	18.7±8.7	16.3±9.1	NS
	HOMA index	del/del	5.1±4.9	4.5±5.0	NS
		ins/del and ins/ins	5.1±4.3	4.3±4.6	NS
MHSO n=74, (del/del=43, ins/del=28, ins/ins=3)	BMI	del/del	43.5±2.5	41.3±4.1	NS
		ins/del and ins/ins	42.6±2.1	39.1±3.2	0.029
	Fat mass (%)	del/del	44.1±4.3	42.7±4.2	NS
		ins/del and ins/ins	43.9±3.8	39.2±3.6	0.013
	Insulin (μIU/mL)	del/del	17.3±10.1	14.5±11.2	NS
		ins/del and ins/ins	16.5±6.2	14.0±8.6	NS
	HOMA index	del/del	4.0±4.3	3.6±4.9	NS
		ins/del and ins/ins	4.0±4.1	3.3±5.1	0.048
Metabolic syndrome n=50, (del/del=27, ins/del=21, ins/ins=2)	BMI	del/del	45.9±4.2	45.0±4.1	NS
		ins/del and ins/ins	45.1±3.6	44.4±3.7	NS
	Fat mass (%)	del/del	41.7±5.2	40.1±5.0	NS
		ins/del and ins/ins	43.1±4.2	41.6±4.1	NS
	Insulin (μIU/mL)	del/del	24.8±11.5	22.4±12.3	NS
		ins/del and ins/ins	22.1±11.1	19.9±10.3	NS
	HOMA index	del/del	6.9±5.3	6.0±5.1	NS
		ins/del and ins/ins	6.8±4.7	5.9±4.2	NS

Matched pairs analysis (signed Wilcoxon rank test).

OB, obesity; BMI, body mass index; NS, not significant; HOMA, homeostasis model assessment; MHSO, metabolically healthy but severely obese.

Discussion

This study examined whether a UCP2 ins/del polymorphism is associated with obesity in severely obese Greek subjects, as well as the potential impact of this polymorphism on weight loss during a lifestyle modification program. UCPs are known to play an important role in the regulation of human energy metabolism.¹⁵

The insertion allele frequency was quite similar to that reported for other Caucasian ethnic groups.^16
–18 The prevalence of the insertion variant did not differ between the obese subjects with or without metabolic syndrome and the control group. Our results are in accordance with studies in Italian,¹⁶ Danish,¹⁷ and French¹⁸ obese subjects, but in contrast with other work that showed a positive correlation between the UCP2 ins/del polymorphism and obesity.^19,20 The discrepancy between these studies may, at least partially, be explained by the assumption that in polygenic diseases like obesity many genes control the phenotype, so that the same phenotype could be caused by variants in different sets of genes.¹⁶

Generally, a major concern with genetic variants exerting only a modest effect is that the population variance of the phenotype could obscure the observable effect of the variant. However, some studies have reported associations with subphenotypes of obesity.⁸ We tried to avoid such heterogeneity of participating subjects by including only subjects with morbid obesity, with the same genetic origin, who either had metabolic syndrome or were metabolically healthy.

We also observed a small but significantly greater reduction in BMI and a greater loss of fat-free mass, as well as a borderline significant improvement in HOMA index among carriers of the insertion allele. Arguably, this result may be ascribed to the regulatory effects of UCP2 on energy homeostasis. Importantly, UCP2 mRNA levels in adipose tissue may be positively related to increased resting metabolic rate.²¹ UCP2 and related genes have also been described to be associated with thermal effect of feeding and 24-h substrate oxidation.^22
–24

Interestingly, Buemann et al.²⁵ have shown that the Val/Ala-55 polymorphism of the UCP2 gene may not only affect basal metabolic rate, but also influence the energy cost of exercise. The same polymorphism also predicts greater weight loss in morbidly obese patients undergoing gastric banding.²⁶ Analogous data suggest that UCP polymorphisms may play a role in the recovery from the overfeeding by regulating substrate oxidation in response to long-term caloric surplus.²⁷ Wang et al.²⁸ have shown that the UCP2 ins/del polymorphism influences fat mass after initiation of maintenance dialysis in patients with chronic renal failure, indicating a role for the regulation of energy metabolism and development of obesity. A possible mechanism that has been proposed is the restriction of ghrelin-induced lipogenesis.²⁹

Dalgaard et al.¹⁷ did not identify any major impact of the ins/del polymorphism in the UCP2 gene on the development of obesity in Danish Caucasian subjects, while Berentzen et al.³⁰ found no interaction between physical activity and the ins/del UCP2 polymorphism or other variants in the UCP2 or UCP3 gene of subsequent weight changes in Danish Caucasian men during a 10-year follow-up. Obviously, the correlation of genetic polymorphisms with energy consumption and expenditure, alongside obesity and related metabolic features, is fascinating, but more data are desirable.

The limitations of the present work may be outlined as follows. The first limitation is the relatively small number of subjects recruited. Because of the small sample sizes there is a low power (20.2%) that implies a relatively high chance of type II errors, i.e., false-negative results. Second, we did not measure basic metabolic rate; therefore, we do not know if the greater reduction in BMI and fat mass of the insertion allele carriers that was observed could be secondary to highest energy expenditure. Moreover, we studied only one polymorphism of the UCP2 gene. Haplotype-based analyses may improve uncertainties or abnormalities introduced by analyses based solely on genotype data. However, such enquiries were beyond the scope of this work. Finally, body composition was assessed using a bioelectrical impedance analyzer (BIA) instrument, a method that has a lot of limitations regarding more powerful methodologies like dual energy X-ray absorptiometry.

In conclusion, our findings suggest that there is no association of the UCP2 ins/del polymorphism with morbid obesity in Greek subjects, with or without metabolic syndrome. However, the UCP2 genotype appeared to be linked with a favorable response to dietary changes. Obviously, the study of the UCP2 ins/del polymorphism is interesting, and additional large studies of well-characterized patients are required to unravel the exact role of this polymorphism in severe obesity.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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