Risk of glycemic disorder in elderly women adjusted by anthropometric parameters and cytokine genotypes

Abstracts

OBJECTIVE: The objective of the present study was to examine the association of glucose intolerance and type-2 diabetes mellitus with the -174 G > C and -308 G > A allelic variations of IL-6 and TNF-α, respectively, through anthropometric measurements and age strata. METHODS: This is a cross-sectional study using data from 285 community dwelling elderly women who underwent physical, biochemical, and genetic examinations. RESULT: Genotype-unadjusted analysis revealed that the risk of glucose intolerance and diabetes in elderly women with elevated BMI was 1.71 and 2.82 times higher, respectively, whereas age and conicity index did not show predictive value. Prevalence ratios for glucose intolerance and diabetes across allelic variants of IL-6 and TNF-α did not associate IL-6 with unbalanced glucose levels, despite adjustment for BMI, age, and conicity index. Conversely, carriers of the TNF-α A allele were 2.06 and 5.58 times more likely to exhibit glucose intolerance and diabetes, respectively, compared to GG homozygotes. CONCLUSION: Our results suggest that bearing the A allele of the -308 G > A polymorphism of TNF-α predisposes to anthropometric measure-sensitive impaired glucose metabolism in older women.

Elderly health; insulin resistance; cytokines; polymorphism, genetic; anthropometry

OBJETIVO: Analisar a associação da intolerância à glicose e do diabetes mellitus tipo 2 com as variações alélicas -174 G > C e -308 G > A de IL-6 e TNF-α, respectivamente, à luz de indicadores antropométricos e faixa etária. MÉTODOS: Trata-se de um estudo transversal com dados obtidos de 285 mulheres idosas da comunidade, submetidas a exames físicos, bioquímicos e genéticos. RESULTADOS: Análise não ajustada para genótipos revelou que idosas com IMC elevado apresentaram risco 1,71 e 2,82 vezes maior para intolerância à glicose e diabetes, respectivamente, enquanto faixa etária e índice de conicidade não apresentaram qualquer valor preditivo. Razões de prevalência para intolerância à glicose e diabetes conforme variantes alélicas de IL-6 e TNF-α não associam genótipos de IL-6 com desregulação glicêmica, a despeito de ajustes para IMC, idade e índice de conicidade. Por outro lado, portadores do alelo A de TNF-α apresentaram 2,06 e 5,58 vezes mais chance de intolerância à glicose e diabetes, respectivamente, comparadas a homozigotas GG no estrato com IMC < 27 kg/m². CONCLUSÃO: Os resultados sugerem que o alelo A do polimorfismo -308 G > A de TNF-α predispõe a distúrbios do metabolismo glicêmico em mulheres idosas de um modo sensível a medidas antropométricas.

Idoso; resistência à insulina; citocinas; polimorfismo genético; antropometria

ORIGINAL ARTICLE

Risk of glycemic disorder in elderly women adjusted by anthropometric parameters and cytokine genotypes

Aparecido Pimentel Ferreira^I; Cristiane Batisti Ferreira^II; Vinícius Carolino Souza^III; Audrey Cecília Tonet Furioso^IV; Juliana Oliveira Toledo^V; Clayton Franco Moraes^VI; Cláudio Córdova^VII; Otávio Tolêdo Nóbrega^VIII

^IPhD in Physical Education; Professor and Coordinator of the Interdisciplinary Research Nucleus of ICESP/Faculdades Promove; Professor and Leader of the Exercise Physiology and Health Study Group, Universidade Paulista (UNIP), DF, Brazil

^IIBSc in Pharmacy; Researcher of the Interdisciplinary Research Nucleus of ICESP/Faculdades Promove de Brasília, Brasília, DF, Brazil

^IIIBSc in Biology; MSc Student, Postgraduate Program in Health Sciences, UnB; Technician of the Immunogentology Laboratory of Universidade Católica de Brasília (UCB), Brasília, DF, Brazil

^IVMSc in Gerontology; Postgraduate Program in Gerontology, Universidade Católica de Brasília; Postgraduate Program in Medical Sciences, Universidade de Brasília (UnB), Brasília, DF, Brazil

^VMSc in Gerontology; Postgraduate Program in Gerontology, UCB; PhD Student, Postgraduate Program in Health Sciences, UnB; Professor, Faculdade Lourdes Santana - LS, Brasília, DF, Brazil

^VIMSc in Gerontology; Postgraduate Program in Gerontology, UCB, Brasília, DF, Brazil

^VIIPhD in Physical Education; Postgraduate Program in Physical Education and Health of UCB, Brasília, DF, Brazil

^VIIIPhD in Molecular Pathology; Professor of the Postgraduate Program in Health Sciences of UnB, Brasília, DF, Brazil

Correspondence to

SUMMARY

OBJECTIVE: The objective of the present study was to examine the association of glucose intolerance and type-2 diabetes mellitus with the -174 G > C and -308 G > A allelic variations of IL-6 and TNF-α, respectively, through anthropometric measurements and age strata.

METHODS: This is a cross-sectional study using data from 285 community dwelling elderly women who underwent physical, biochemical, and genetic examinations.

RESULTS: Genotype-unadjusted analysis revealed that the risk of glucose intolerance and diabetes in elderly women with elevated BMI was 1.71 and 2.82 times higher, respectively, whereas age and conicity index did not show predictive value. Prevalence ratios for glucose intolerance and diabetes across allelic variants of IL-6 and TNF-α did not associate IL-6 with unbalanced glucose levels, despite adjustment for BMI, age, and conicity index. Conversely, carriers of the TNF-α A allele were 2.06 and 5.58 times more likely to exhibit glucose intolerance and diabetes, respectively, compared to GG homozygotes.

CONCLUSION: Our results suggest that bearing the A allele of the -308 G > A polymorphism of TNF-α predisposes to anthropometric measure-sensitive impaired glucose metabolism in older women.

Keywords: Elderly health; insulin resistance; cytokines; polymorphism, genetic; anthropometry.

Introduction

The increase in the number of people over 60 years of age is a visible phenomenon in almost every country. In Brazil, the last census indicates the possibility that the number of elderly people is above 30 million¹, representing almost 13% of the population². It is known that aging exposes people to higher prevalence of chronic diseases, such as type 2 diabetes mellitus, which affects 17.4% of the population from 60 to 69 years of age, according to the census of 1986 and 1988³. Its high incidence rate, added to the different clinical complications resulting from macro- and microvascular disruption, make this disease a public health problem.

Among anthropometric variables that commonly predict endocrine-metabolic disorders, the body mass index (BMI) and conicity index (C index) should be emphasized^4,5. Despite the imminent relationship between age and deregulation of glycemic levels, primary and contributing causes for changes in this aspect of human metabolism are still debatable. Etiologic possibilities currently discussed involve aspects related to lifestyle due to inadequate diet and physical inactivity, factors closely related to obesity⁶, as well as endocrine deficiency secondary to exhaustion of β cells and/or idiopathic humoral interferences in glucose uptake by cells^7,8, with possible allele-specific contributions from important inflammatory mediators^9-12.

The objective of the present study was to analyze the association between BMI, C index, and age with glucose intolerance phenotypes and type 2 diabetes mellitus in elderly women, considering the influence of classical allelic variations of IL-6 and TNF-α genes.

Methods

This is an analytical transversal study with data from 285 elderly females (> 60 years) recruited consecutively from the community to participate in the study conducted from 2005 to 2008, whose protocol was approved by the institutional ethics on research committee. Every participant signed an informed consent. Participants were not on nutritional follow-up nor did they practice regular exercises before laboratorial tests and anthropometric measurements were performed.

Physical and biochemical exams

Patients were wearing light clothes during the physical exam. Height was measured using a manual stadiometer placed on the wall, with patients barefoot and erect on a flat and rigid surface with arms along their body. Body mass index (BMI) was obtained by dividing body mass (in kg) by the square height (in m2), while the C index was estimated to be 9.17 times the ratio between waist circumference (m) and the square root of the ratio between body mass (kg) and height (m).

Biochemical evaluation consisted of 12-hour fasting glucose serum levels using Roche^® (USA) reagents. The reference value (> 126 mg/dL) recommended by the American Diabetes Association¹³ or current use of oral hypoglycemic agent or insulin was used to classify type 2 diabetes. Glucose intolerance was defined as glucose levels > 100 mg.dL^-1 or presence of diabetes, according to recommendations of the International Diabetes Federation^14,15.

Laboratorial analysis

To determine IL-6 alleles, a region of 628 bp of the gene containing a single nucleotide polymorphism (SNP) -174 G > C of the promoter region (rs1800795) was amplified using a pair of oligonucleotides: 5'-GAA CAC AGA AGA ACT CAG ATG ACT GG-3' (forward) and 5'- AGG AGT TCA TAG CTG GGC TCC TGG AG-3' (reverse). Variability of the TNF-α gene was investigated by amplifying a fragment of 327 bp containing the -308 SNP G > A (rs1800629) of the promoter region of the gene, using oligonucleotides A (5'-CCT CAA GCC TGC CAC CAA GC-3'; forward) and B (5'-TCC TCC CTG CTC CGA TTC CG-3'; reverse). Each individual reaction was composed of: 100 ng DNA. 10 mM Tris-HCL pH 9.2; 25 mM KCL; 1.5 mM MgCl₂; 0.2 mM dNTP; 20 pmol of each oligonucleotide; 0.5 μg ovalbumin; and 1 unit of Taq DNA polymerase (Phoneutria, Minas Gerais, Brazil) in a final volume of 50 μL. After a hot start at 80ºC for 1 minute and a initial denaturation step at 94ºC for 2 minutes, each amplification cycle consisted of 40 seconds at 94ºC, 45 s at 64ºC, and 50 s at 72ºC repeated 36 times and finalized by an extension step at 72ºC for 5 minutes. Each PCR product was sequenced in the ABI PRISM 3130 DNA analyzer system (Applied Biosystems, Foster City, USA) using the oligonucleotide 5'-GCC TCA GAG ACA TCT CCA GTC C-3', for IL-6 products, and oligonucleotide A, for TNF-α products. Each sequence obtained was examined using the biostatistics software package Staden (MRC, Cambridge, United Kingdom) and confirmed by visual inspection.

Statistical analysis

Data on sample characterization are presented as mean and standard deviation or relative frequency. General prevalence of glucose intolerance and diabetes was calculated for each stratum (age < 65 years and > 65 years, BMI < 27 kg/m2 and > 27 kg/m2, conicity index < 1.25 and > 1.25), as well as the prevalence of IL-6 and TNF-α genotypes in each stratum. The Chi-square test was used to identify frequency differences among strata. Prevalence ratios (PR) of glucose intolerance and diabetes were calculated as dichotomies in elected strata (lower stratum = 0 and upper stratum = 1). Additionally, dummy variables were created for comparison among strata, assuming the lower stratum (= 0) as the reference, of each study variable for IL-6 and TNF polymorphisms, for IL-6 (CG = 0 and GC and CC = 1), for TNF (GA and AA = 0 and GG = 1) and, when the genes were analyzed together (GG, for IL-6, and GA and AA, for TNF = 0 and the remaining combinations = 1). Differences were analyzed by the Mantel- Hanzel test. The confidence interval of 95% was used. Data were analyzed by the statistical software Stata, version 7.0.

Results

Table 1 shows the characteristics of the study population, including age profile, anthropometric data, and representative variables of glycemic metabolism in elderly women who participated in the study.

Considering the mean values of age, BMI, and C index, we decided to use dichotomization of the sample (age < 65 years or > 65 years, BMI < 27 kg/m² or > 27 kg/m², conicity index < 1.25 or > 1.25) in subsequent analysis. Thus, statistics comparing the prevalence of glucose intolerance and type 2 diabetes among groups are presented in Table 2. In this analysis, age and C index were not predictive of glucose intolerance or diabetes. However, the group of elderly women with BMI > 27 kg/m² was 1.71 and 2.82 times more likely to present glucose intolerance and diabetes, respectively.

When prevalence ratios of glucose intolerance and diabetes were analyzed, according to variants of IL-6 and TNF-α genes, adjusted for BMI, age, and C index, it was noticed that neither IL-6 genotype was a risk or protection factor for glycemic deregulation (Table 3). On the other hand, carriers of the TNF-α allele A were 2.06 and 5.58 times more likely to have glucose intolerance and diabetes, respectively, when compared to GG homozygous in the strata of people with BMI < 27 kg/m² (Table 4).

Discussion

In the present study, glucose intolerance and diabetes were observed in 27.6% and 16.8% of all cases investigated, respectively. The prevalence of glucose intolerance and diabetes did not differ among the different strata for age, C index, or IL-6 or TNF-α genotypes. However, it differed between BMI strata (p = 0.031), with glucose intolerance and diabetes significantly higher in the group with BMI higher than 27 kg/m2. Subsequent analyses were adjusted to cancel the influence of the BMI, being observed that prevalence of glucose intolerance or diabetes did not show significant variation according to IL-6 genotypic category. Similar analysis performed for TNF-α genotypic categories showed different results, in which carriers of the TNF-α A allele had more chances of presenting glucose intolerance and diabetes. However, this finding was observed only in the stratum below the threshold stratification established for the study (BMI < 27 kg/m2). This result was most likely due to the fact that excessive adiposity superseded the effects of an isolated gene on the pathophysiology of insulin resistance, masking the contribution of the TNF-α A allele on glycemic disorders, making it evident only in strata of body composition with a tendency to eutrophy¹⁶.

However, one cannot deny the hypothesis of the TNF-α A allele being an explanation in cases of insulin resistance and diabetes. To this end, González-Sanches et al.¹⁷, in a population-based epidemiologic study, analyzed the interaction among both genes, some cardiovascular risk factors, and biochemical disorders in more than 800 Caucasian patients ages 35 to 74 years. They observed that the -308 G > A SNP of the TNF-α gene can be related to a higher prevalence of insulin resistance and incidence of type 2 diabetes mellitus, regardless of insulin resistance, BMI, and waist-hip relationship.

Nicaud et al.¹² investigated the role of -308 G > A SNP of the TNF-α gene in insulin resistance and they observed the association between the A allele with type 2 diabetes. Studies have suggested that the TNF-α -308A/G gene SNP is related with serum levels of the respective mediator because the A allele seems to predispose higher transcriptional levels of the gene, producing increased serum concentrations compared to the allele G^18,19. According to the evidence that different cardiovascular risk factors (including hypertension, dyslipidemias, and insulin resistance) are intensified by circulating levels of TNF-α, regardless of gender and age stratum^20-23, allele A is more common among patients with metabolic disorders common in the elderly^12,16,24.

It has been reported in the literature that TNF-α has an important role in insulin resistance, interfering with the insulin signaling pathway¹². The mechanism of induction of insulin resistance by this cytokine is probably due to induction of phosphorylation of the insulin receptor substrate 1 (IRS-1), decreasing its affinity for the insulin receptor, attenuating signal transmission by compromising phosphorylation of the receptor to tyrosine^25-27. The impossibility to determine circulating levels of the inflammatory mediators investigated represents a limitation of the present study. Besides, performing this study with elderly women implies that caution should be taken when extrapolating the results to other gender or age group.

Summarizing, non-adjusted analysis for genotype showed that elderly women with elevated BMI had 1.71 and 2.82 times greater risk of developing glucose intolerance and diabetes, respectively, while age group and C index did not show any predictive value. Apart from this association, our results suggest that the presence of the A allele of -308 G > A TNF-α gene predisposes to the development of glycemic disorders sensitive to the context, since glucose intolerance and type 2 diabetes phenotypes are not influenced by any of the genotypes being investigated among individuals with BMI compatible with obesity.

Acknowledgements

Our acknowledgements to the Hospital da Universidade Católica de Brasília and Laboratório SABIN for providing conditions to perform this study, and to professor Soraia Figueiredo Carmo for the orthographic review.

References

• ¹
  Instituto Brasileiro de Geografia e Estatística. 2009. [citado 16 fev 2009]. Disponível em: http://www.ibge.gov.br/home/estatistica/populacao/projecao_da_populacao/piramide/piramide.shtm
  » link
• 2. Nobrega OT, Faleiros VP, Telles JL. Gerontology in the developing Brazil: achievements and challenges in public policies. Geriatr Gerontol Int 2009;9:135-9.
• 3. Mathias TA, Jorge MH. Diabetes mellitus na população idosa em municípios da Região Sul do Brasil: um estudo da mortalidade e morbidade hospitalar. Arq Bras Endocrinol Metabol 2004;48:505-12.
• 4. Ferreira AP, Nobrega Ode T, Franca NM. Association of body mass index and insulin resistance with metabolic syndrome in Brazilian children. Arq Bras Cardiol 2009;93:147-53.
• 5. Pitanga FJG, Lessa I. Sensibilidade e especificidade do índice de conicidade como discriminador do risco coronariano de adultos em Salvador, Brasil. Rev Bras Epidemiol 2004;7:259-69.
• 6. Hawley JA, Houmard JA. Introduction-preventing insulin resistance through exercise: a cellular approach. Med Sci Sports Exerc 2004;36:1187-90.
• 7. Ivy JL. Muscle insulin resistance amended with exercise training: role of GLUT4 expression. Med Sci Sports Exerc 2004;36:1207-11.
• 8. Dela F, Ploug T, Handberg A, Petersen LN, Larser JJ, Mikines KJ et al. Physical training increases muscle GLUT4 protein and mRNA in patients with NIDDM. Diabetes 1994;43:862-5.
• 9. Beránek M, Kanková K, Benes P, Izakovicováet-Hollá L, Znojil V, Hájek D et al Polymorphism R25P in the gene encoding transforming growth factor-beta (TGF-beta1) is a newly identified risk factor for proliferative diabetic retinopathy. Am J Med Genet 2002;109:278-83.
• 10. Huth C, Heid IM, Vollmert C, Gieger D, Grallert H, Wolford JK et al IL6 gene promoter polymorphisms and type 2 diabetes: joint analysis of individual participants data from 21 studies. Diabetes 2006;55:2915-21.
• 11. Tonet AC, Karnikowski M, Moraes CF, Gomes L, Karnikowski MG, Córdova C et al. Association between the -174 G/C promoter polymorphism of the interleukin-6 gene and cardiovascular disease risk factors in Brazilian older women. Braz J Med Biol Res 2008;41:47-53.
• 12. Nicaud V, Raoux S, Poirier O, Cambien F, O'Reilly DS, Tiret L. The TNF alpha/G-308A polymorphism influences insulin sensitivity in offspring of patients with coronary heart disease: the European Atherosclerosis Research Study II. Atherosclerosis 2002;161:317-25.
• 13. Diagnosis and classification of diabetes mellitus. Diabetes Care 2004;27(Suppl 1):S5-S10.
• 14. Hanley AJ, Karter AJ, Williams K, Festa A, D'Agostino RB Jr, Wagenlnech T et al. Prediction of type 2 diabetes mellitus with alternative definitions of the metabolic syndrome: the Insulin Resistance Atherosclerosis Study. Circulation 2005;112:3713-21.
• ¹⁵
  International Diabetes Federation. 2005. [citado 18 fev 2009]. Disponível em: http://www.idf.org/metabolic_syndrome
  » link
• 16. Pihlajamaki J, Ylinen M, Karhapaa P, Vauhkonen I, Laakso M. The effect of the -308A allele of the TNF-alpha gene on insulin action is dependent on obesity. Obes Res 2003;11:912-7.
• 17. González-Sánchez JL, Martínez-Calatrava MJ, Martínez-Larrad MT, Zabena C, Fernández-Pérez C, Laakso M et al Interaction of the -308G/A promoter polymorphism of the tumor necrosis factor-alpha gene with single-nucleotide polymorphism 45 of the adiponectin gene: effect on serum adiponectin concentration in Spanish population. Clin Chem 2006;52:97-103.
• 18. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci USA 1997;94:3195-9.
• 19. Fernandez-Real JM. Genetic predispositions to low-grade inflammation and type 2 diabetes. Diabetes Technol Ther 2006;8:55-66.
• 20. Bennet AM, van Maarle MC, Hallqvist J, Morgenstern R, Frostegard J, Wiman B et al. Association of TNF-alpha serum levels and TNFA promoter polymorphisms with risk of myocardial infarction. Atherosclerosis 2006;187:408-14.
• 21. Skoog T, Dichtl W, Boquist S, Skoglund-Andersson C, Karpe F, Tang R et al Plasma tumour necrosis factor-alpha and early carotid atherosclerosis in healthy middle-aged men. Eur Heart J 2002;23:376-83.
• 22. Lechleitner M, Herold M, Dzien-Bischinger C, Hoppichler F, Dzien A. Tumour necrosis factor-alpha plasma levels in elderly patients with Type 2 diabetes mellitus-observations over 2 years. Diabet Med 2002;19:949-53.
• 23. Pickup JC, Chusney GD, Thomas SM, Burt D. Plasma interleukin-6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes. Life Sci 2000;67:291-300.
• 24. Herrmann SM, Ricard S, Nicaud V, Mallet C, Arveiler D, Evans A et al. Polymorphisms of the tumour necrosis factor-alpha gene, coronary heart disease and obesity. Eur J Clin Invest 1998;28:59-66.
• 25. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993;259:87-91.
• 26. Hotamisligil GS, Peraldi P, Budavari A, Ellis R, White MF, Spiegelman BM. IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance. Science 1996;271:665-8.
• 27. Rui L, Aguirre V, Kim JK, Shulman GI, Lee A, Corbould A et al Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest 2001;107:181-9.

Publication Dates

• Publication in this collection
  14 Oct 2011
• Date of issue
  Oct 2011

History

• Received
  14 Apr 2011
• Accepted
  30 June 2011