Association of interleukin‐6 polymorphisms with obesity or metabolic traits in young Mexican‐Americans

Introduction

Most genetic association studies for obesity are conducted in older populations where epigenetic effects can result in reduced heritability calculations and distract from investigating true genetic susceptibility 1. Epigenetics may be less influential in younger populations where environment has not yet confounded the genetic contribution to obesity. Studies in obesity‐related inflammation are also generally performed in older age groups, although some studies in adolescents or younger age groups show similar obesity associated elevated levels of pro‐inflammatory cytokines, such as tumour necrosis factor alpha, interleukin‐1 beta and interleukin‐6 (IL6) 2, 3.

Pro‐inflammatory cytokines (e.g. IL6) can influence adipocyte function, lipid metabolism, homeostasis, blood pressure and insulin sensitivity and thus play a major role in the development of diabetes, atherosclerosis and cardiovascular diseases 4. However, IL6 can also be anti‐inflammatory, regenerative and homeostatic by controlling acute‐phase response and modulating glucose and liver metabolism 5, 6.

Interleukin‐6 polymorphisms have been investigated in many populations for associations with various chronic diseases 4. For example, single‐nucleotide polymorphisms (SNPs) rs1800797 (‐597 G/A), rs1800796 (‐572 G/C) and rs1800795 (‐174 G/C), located in the promoter region of IL6, have been shown to be associated with obesity and metabolic traits in different ethnic groups (Table 1). The rs1800797 SNP is associated with type 2 diabetes in German 7 and metabolic syndrome in French 8 populations. The rs1800796 SNP is linked with high insulinogenic index in Danes 10, hyperglycaemia in Mexicans 11 and hypertension, obesity, type 2 diabetes and carotid intima‐medial thickness in Asians 9, 12, 13, 14. These three IL6 promoter SNPs have also been studied as haplotypes (Table 1). Haplotypes generated from the rs1800797, rs1800796 and rs1800795 combinations have been shown to be associated with type 2 diabetes in Danes 10 and hyperglycaemia and obesity in Mexicans 11. The GG haplotype for rs1800797 and rs1800795 has been shown to be associated with type 2 diabetes in Mexicans 15 and Asian Indians 16.

Epidemiological investigations in Mexican‐Americans of South Texas have observed a high prevalence of overweight and obesity (40–50%) 17, 18, 19. Non‐alcoholic fatty liver disease prevalence is also higher in this population, and it is associated with obesity and diabetes 20. Recent studies have observed elevated levels of liver enzymes in young Mexican‐Americans of South Texas with obesity and non‐alcoholic fatty liver disease 19, 20. Based on these observations and the link between IL6 and obesity‐related traits in other populations, this study hypothesized genetic associations between IL6 polymorphisms and obesity or metabolic syndrome in Mexican‐Americans. This is the first study that investigates genetic associations between the IL6 promoter SNPs, rs1800797 and rs1800796, with obesity, insulin resistance and metabolic traits including liver enzymes in a cohort of mostly young (90.2% of cohort population is below 30 years old) Mexican‐Americans of the Rio Grande Valley.

Methods

Results

Participants consisted of 437 Mexican‐Americans: 291 were female, and 146 were male. Anthropometric and clinical characteristics of the population are described in Table 2. Population was mostly young (median age = 17 years, SD = ± 8.26) and with overweight/obesity (52.17%) (Table 4). In Mexican‐Americans, rs1800797 is in complete linkage disequilibrium with rs1800795 (D′ = 1 and r² = 1) and in high linkage disequilibrium (D′ = 1) with rs1800796 but with different allele frequencies resulting in a low r² (r² = 0.08); therefore, rs1800797 and rs1800796 were selected for genotyping and subsequent association analyses. Four hundred and twenty‐nine individuals were successfully genotyped for rs1800797 and 428 for rs1800796. However, complete phenotype data on these individuals varied. Both polymorphisms were in Hardy–Weinberg equilibrium (rs1800797 P = 0.87; rs1800796 P = 0.34) (Table 3).

Minor allele frequencies (MAFs) for both SNPs were compared between subjects with and without obesity phenotypes categorized by BMI and waist circumference. Table 4 describes association of SNPs with BMI. MAFs of rs1800797 and rs1800796 did not differ significantly between the subjects with or without obesity (rs1800797 P = 0.41; rs1800796 P = 0.22). There were no associations found between SNPs and BMI using the additive (rs1800797 P = 0.78; rs1800796 P = 0.60), dominant (rs1800797 P = 0.29; rs1800796 P = 0.09) or recessive (rs1800797 P = 0.87; rs1800796 P = 0.93) models (Table 4). Table 5 describes the association of SNPs with waist circumference. MAF of rs1800797 did not differ significantly between subjects with or without central obesity (P = 0.17), and no associations with waist circumference were found for rs1800797 using the additive (P = 0.79), dominant (P = 0.09) or recessive (P = 0.64) models (Table 5). However, frequency of the rs1800796 minor allele (C‐allele) was significantly higher in subjects with central obesity (P = 0.02; permutation P‐value = 0.04), and a significant association between rs1800796 and waist circumference under the dominant model (CC = CG vs. GG P = 0.01; permutation P ‐value = 0.03) was observed (Table 5).

Associations between rs1800797 and rs1800796 and several metabolic traits were investigated in all subjects, as well as in subjects separated by gender (Table 6). Metabolic traits included systolic and diastolic blood pressure, HOMA‐IR, liver enzymes (AST and ALT), total cholesterol, triglycerides, high‐density and low‐density lipoproteins and glucose. No associations were found between rs1800797 and metabolic traits in all subjects or men and women separately (Table 6). For rs1800796, there were no associations for most of the metabolic traits except for HOMA‐IR, when all subjects were considered and for female subjects separately (P = 0.009 and 0.03, respectively; Table 6). In male subjects, no associations were found between rs1800796 and any metabolic trait.

Plasma IL6, hs‐CRP and liver enzymes (AST and ALT) levels were compared between genotypes for each SNP (Table 7 and Figures 1 and 2). Genotypes were combined because the numbers of the minor allele homozygotes for both SNPs were low compared with other genotypes. For rs1800797, plasma IL6 levels did not differ significantly between A‐allele homozygotes when compared with G‐carriers (AA vs. GG + AG P = 0.48). However, plasma IL6 levels were significantly higher in rs1800797 A‐carriers when compared with G‐allele homozygotes (AA + AG vs. GG P = 0.002) (Table 7 and Figure 1A). For rs1800796, plasma IL6 was significantly lower in C‐allele homozygotes when compared with G‐carriers (CC vs. GG + CG P = 6.6e−07), but it did not differ in C‐carriers when compared with G‐allele homozygotes (CC + CG vs. GG P = 0.33) (Table 7 and Figure 1B).

The inflammation marker, hs‐CRP, did not differ between rs1800797 A‐homozygotes and G‐carriers (AA vs. GG + AG P = 0.48), but levels were significantly lower in A‐carriers when compared with G‐homozygotes (AA + AG vs. GG P = 0.04) (Table 7 and Figure 1A). For rs1800796, hs‐CRP did not differ between C‐homozygotes and G‐carriers (CC vs. GG + CG P = 0.10), but it was significantly higher in C‐carriers when compared with G‐homozygotes (CC + CG vs. GG P = 0.01) (Table 7 and Figure 1B).

Although AST and ALT liver enzymes did not differ significantly when genotypic groups were compared, a modest increase in both enzymes was observed for each SNP's risk allele for obesity and metabolic traits (Table 7 and Figure 2). For rs1800797, higher AST and ALT values were found in G‐carriers (Table 7 and Figure 2A), whereas for rs1800796, higher values were found in C‐carriers (Table 7 and Figure 2B).

Discussion

In this study, association analyses assuming additive, dominant and recessive effects of rs1800797 G‐allele did not show any significant associations with obesity (categorized by BMI and waist circumference) and metabolic traits. The rs1800797 SNP has not been associated with metabolic syndrome and diabetes as much as the rs1800795 SNP 16; however, some studies have reported the contribution of rs1800797 G‐allele to the development of metabolic syndrome and diabetes, but not the A‐allele 7, 8. In this study, despite the lack of statistical significance, frequency of rs1800797 A‐allele was slightly higher among individuals without obesity. Plasma IL6 levels were significantly higher, and hs‐CRP levels were significantly lower in A‐allele carriers than in GG‐homozygotes, suggesting a possible protective effect of rs1800797 A‐allele against obesity and inflammation in this mostly young Mexican‐American cohort.

Findings of this study also demonstrate the possible role of rs1800796 in central obesity and insulin resistance in young Mexican‐Americans. In this study, presence of rs1800796 C‐allele was higher among subjects with increased waist circumference. This result is in accordance with a study performed in non‐Hispanic whites where waist‐to‐hip ratio was significantly higher in postmenopausal women carrying the C‐allele 24. A Caucasian study associated rs1800796 C‐allele with metabolic syndrome, although it did not find an association with waist circumference 25. Also, two studies performed in Caucasians showed associations between rs1800796 and obesity. In one of the reports, the rs1800796 C‐allele was shown to be associated with obesity in children 26, and in the other study, they observed that pregnant women who carry the rs1800796 G‐allele were protected from gestational weight gain 27. However, a study performed in a Dutch cohort observed smaller waist circumference in rs1800796‐CC individuals compared with G‐carriers 28. A Mexican study found that the rs1800797‐A/rs1800796‐G/rs1800795‐C haplotype was associated with lower prevalence of diabetes 29, which suggests that the rs1800796 G‐allele could possibly contribute to the protection against developing type 2 diabetes in Mexicans. A joint analysis including eight studies of mostly Caucasians did not find an association with the rs1800796 C‐allele and diabetes 25, where BMI was included as a covariate instead of waist circumference. The association of rs1800796 C‐allele with risk for obesity and type 2 diabetes has thus been inconsistent across ethnicities. In this study, however, increased waist circumference is associated with the rs1800796 C‐allele, and because waist circumference has been classified as a better predictor of obesity, insulin resistance and type 2 diabetes in Mexican‐Americans 30, it was included as a covariate in the analyses of IL6 SNP associations with metabolic traits. The rs1800796 C‐allele was also found to be associated with higher HOMA‐IR (a surrogate measure of insulin resistance) when analyses were conducted in all subjects (male and female) and in female subjects separately. Thus, this study correlates the rs1800796 C‐allele with increased central obesity and insulin resistance, two important indicators of type 2 diabetes and highly heritable among Mexican‐Americans 31.

Elevated levels of IL6 are typically associated with obesity‐related inflammation and type 2 diabetes in adolescents and adults with obesity 2, 3. One recent study found higher promoter activity of the rs1800796 G‐allele compared with the C‐allele 32. Interestingly, in this study, rs1800796‐CC homozygotes had lower plasma IL6 levels when compared with G‐allele carriers. Additionally, rs1800796 C‐allele carriers were found to express higher hs‐CRP levels, a marker of acute immune response (inflammation) in liver, than G‐allele homozygotes, and liver enzymes AST and ALT trended towards being elevated in rs1800796 C‐carriers. This suggests that in young Mexican‐Americans, rs1800796 C‐allele carriers with lower levels of IL6 have a higher risk for obesity associated inflammation and metabolic disturbances such as insulin resistance and liver inflammation.

There is significant evidence showing that IL6 has anti‐inflammatory, anti‐obesogenic and glucose homeostatic functions. For example, the development of obesity, hepatic insulin resistance, liver inflammation and damage, insulin and glucose intolerance and mitochondrial dysfunctions was observed in high fat diet‐fed IL6‐deficient mice when compared with control mice 33. Another study observed an increase in body fat, body weight, triglycerides, very‐low‐density lipoprotein cholesterol and impaired glucose elimination in mice lacking the IL6 gene 34. In addition, body weight decreased after intraperitoneal and/or intracerebroventricular IL6 injections in IL6 knockout mice 34 and wild‐type rats 35. Disruption of the IL6 anti‐inflammatory signalling in insulin sensitive tissues was present in myeloid cells IL6 receptor inactivated mice, leading to insulin resistance 34. A human study supported the animal studies by observing higher cerebrospinal IL6 levels in subjects without obesity than in subjects with obesity 36. IL6 has been shown to play a homeostatic role in muscle through an acute exercise response. In this response, there is an increase in plasma IL6 levels, IL6 mRNA in adipose tissue and IL6 mRNA and protein content in the exercised skeletal muscle, without causing muscle damage 37. Exercise‐induced IL6 acts as an anti‐inflammatory cytokine by increasing cortisol, lipolysis, catecholamine, IL‐1 receptor antagonist, IL‐10 and CRP (which contributes to the increase of IL‐1RA) to regulate metabolic and energy homeostasis during exercise 37, 38.

A normal homeostatic response of tissues to increased inflammatory factors (caused by elevated reactive oxygen species or hypoxia in expanding adipose tissue) may be an initial acute response of elevated IL6, which in turn up‐regulates anti‐inflammatory molecules (such as cortisol, IL‐1RA, IL‐4 and IL‐10) in insulin sensitive tissues or brain regulating obesity and is associated with metabolic perturbations 36. If this normal acute response of IL6 to inflammation is suppressed or lowered (as in rs1800796‐CC genotypes) in youth, it may lead to unchecked increase in waist circumference and insulin resistance. However, in youths with the rs1800797‐GG genotype, the homeostatic response of IL6 in checking inflammation may be one of the protective factors against metabolic consequences of obesity.

However, IL6 resistance may underlie chronic obesity‐related inflammation with increased plasma levels of IL6 in adolescents and adults. O'Connor et al. 39 were the first one to demonstrate dysfunctional activity of IL‐4, an anti‐inflammatory cytokine, in type 2 diabetes conditions and suggested the development of cytokine resistance to IL‐4 in type 2 diabetes, concluding that impaired suppression of inflammation by anti‐inflammatory cytokines is present in type 2 diabetes. They observed that after IL‐4 treatment, macrophages from obese/diabetic mice failed to express anti‐inflammatory cytokines (IL‐1RA and IL‐1R2), and induction of IRS‐2, mediator of IL‐4 response, was significantly reduced in obese/diabetic macrophages when compared with controls 39. Although IL6 resistance was not studied by O'Connor et al. 39, it is entirely plausible that IL6 resistance is associated with chronic obesity and insulin resistance.

Interleukin‐6 changes its macrophage induction from anti‐inflammatory (M2) macrophages in lean individuals to pro‐inflammatory (M1) macrophages in individuals with obesity 40. In younger individuals with overweight (but not yet with obesity), the initial acute homeostatic IL6 induction of anti‐inflammatory M2 macrophages may help to prevent further increase in adiposity and/or metabolic perturbations. This initial protective M2 response may be absent or weak in rs1800796‐CC genotype carriers, with lower IL6 levels. However, in carriers of rs1800796‐GG genotype, while this phenotype may be protective initially, if obesity/morbid obesity‐associated chronic inflammation develops (with elevated plasma IL6 levels and IL6 resistance), M2 anti‐inflammatory response may no longer be induced by IL6.

In this study, although 52.17% of the subjects had obesity, only 27.6% had insulin resistance. Thus, a relatively metabolically healthy and young population (90.2% below 30 years) in which it may be easier to tease out genetic susceptibility without being influenced by environmental factors such as ageing was studied 1. The study showed that the rs1800796 C‐allele is associated with central obesity and insulin resistance, whereas rs1800797 A‐allele may be protective against obesity in a young Mexican‐American cohort from the Rio Grande Valley. Data from this study support the hypothesis that IL6 plays an anti‐inflammatory homeostatic role in obesity and metabolic diseases in a young Mexican‐American population. The limitations of the study are the small sample size, incomplete metabolic, clinical and IL6/hs‐CRP data on all genotyped samples, as well as lack of genotyping data on additional SNPs. These results will need to be replicated with more SNPs in larger sample sizes and cohorts with more comprehensive measurements to fully substantiate and support the hypothesis proposed. This is the first study investigating genetic associations of IL6 SNPs with obesity, obesity‐related inflammation and metabolic syndrome in an understudied population of Mexican‐Americans from South Texas. Because of the complexity of IL6 regulation in obesity and obesity‐related inflammation, further research needs to be conducted to investigate the myriad cellular and molecular pathways affected by the IL6 polymorphisms.

Conflict of Interest Statement

The authors declared no conflict of interest.

Funding

Research reported in this publication was supported by the National Institute of General Medical Sciences (NIGMS) under award number R25GM065925 and by the National Institute on Minority Health and Health Disparities (NIMHD) under award number P20MD001091 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Supporting information

Boeta‐Lopez, K. , Duran, J. , Elizondo, D. , Gonzales, E. , Rentfro, A. , Schwarzbach, A. E. , and Nair, S. (2018) Association of interleukin‐6 polymorphisms with obesity or metabolic traits in young Mexican‐Americans. Obesity Science & Practice, 4: 85–96. doi: 10.1002/osp4.138.