Correlation of the Homeostasis Model Assessment Index and Adiponectin , Leptin and Insulin Levels to Body Mass Index-Associated Gene Polymorphisms in Adolescents

Objectives
This study aimed to describe correlations between glucose, insulin and adipokine levels and the homeostasis model assessment (HOMA) index with regards to the presence/absence of fat mass and obesity-associated (FTO) rs9939609 and peroxisome proliferator-activated receptor (PPAR)-y rs1801282 single nucleotide polymorphisms (SNPs) as indicators of body mass index in adolescents.


Methods
This cross-sectional study was conducted between September and December 2016 in Toluca, Mexico. A total of 71 students between 14-18 years old were included. Various anthropometric and laboratory measurements were collected, including lipid profile, glucose, insulin and adipokine levels and HOMA index. The degree of association between variables was evaluated with regards to the presence/absence of the SNPs.


Results
Leptin levels were significantly higher among female students (P = 0.001), although adiponectin levels did not differ significantly (P = 0.060). There were significant positive correlations between insulin levels and HOMA index with FTO (r = 0.391; P = 0.007 and r = 0.413; P = 0.005, respectively) and PPARγ (r = 0.529; P = 0.007 and r = 0.537; P = 0.007, respectively) SNPs. Leptin showed a significant positive correlation in the presence of PPARγ (r = 0.483; P = 0.007) or in the absence of both SNPs (r = 0.627; P = 0.039). However, adiponectin was significantly negatively correlated in the presence of FTO, either alone (r = -0.333; P = 0.024) or in combination with PPARγ (r = -0.616; P = 0.043).


Conclusion
The presence of FTO and/or PPARγ SNPs might be related to a genetic predisposition to metabolic syndrome.


O
besity is a multifactorial disease determined by a complex interaction between genetic and environmental factors.Previous research has associated an obesogenic phenotype with the presence of specific polymorphisms also linked to the development of insulin resistance, diabetes mellitus and metabolic syndrome. 1Moreover, pancreatic function may be altered by mutations that cause changes in the activity or expression of proteins involved in the regulation of basal energy expenditure; the latter phenomenon is explained in part by the various mechanisms of oxidative phosphorylation.Although the effect of each individual gene or combination of different genetic variants on metabolism and thermogenesis is uncertain, this may explain why certain individuals have a greater tendency to develop metabolic disorders. 2n recent decades, efforts have intensified in the search for single nucleotide polymorphisms (SNPs) causing observed changes in the signalling pathways that regulate metabolic processes, leading to failures in energy homeostasis and predisposing individuals to diabetes and obesity. 3Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that function as transcription factors.These receptors are present in different metabolic pathways related to energy homeostasis, in addition to the lipid route.So far, three PPAR isoforms have been identified; the PPARγ isoform, which is highly expressed in white adipose tissue, is involved in lipid storage and energy dissipation and is recognised to be the master regulator of adipogenesis. 4he fat mass and obesity-associated (FTO) gene is composed of nine exons on chromosome 16 (16q12.2). 5 It was the first gene to be associated with obesity in genomewide association studies. 6Frayling et al. observed that adults with the FTO rs9939609 SNP had a 1.67-fold increased risk of obesity and weighed an average of 3-4 kg more than those without this polymorphism. 7n Mexico, genomic studies have demonstrated several genetic ancestries for the local population. 8However, this implies that there may be discrepancies in the role of some SNPs in the presence of obesity-related conditions, such as metabolic syndrome.This study aimed to identify correlations between glucose, insulin and adipokine levels and the homeostasis model assessment (HOMA) index with regards to the presence or absence of FTO rs9939609 and PPARy rs1801282 SNPs as indicators of body mass index (BMI) in Mexican adolescents.

Methods
This cross-sectional study was conducted from September to December 2016 at the Autonomous University of Mexico State (UAEM) in Toluca, Mexico.All adolescents between 14-18 years old attending the Lic.Adolfo López Mateos Preparatory School in Toluca were invited to participate in the study.The exclusion criteria included a history of smoking, being pregnant or having been diagnosed with a chronic or acute disease.The sample size was calculated as follows: 9 where n is the infinite population of available participants (981 adolescents), z is the 95% confidence level (1.96), p is the estimated percentage of the studied polymorphisms in the total population (30%), ε is the margin of error set at 11% with a replacement rate of 9%, n' is the required finite sample size and N is the population size.Based on this, the required sample size was set at 68 adolescents.
A body composition monitor was used to measure the subjects' weight (BC-533 InnerScan Body Composition Monitor ® , Tanita Corp., Tokyo, Japan), while a standard stadiometer was used to measure height.BMI was calculated as weight in kg divided by height in m 2 .According to gender-and age-specific World Health Organization BMI classifications for adolescents, the participants were categorised as either normal, overweight (≥1 standard deviation [SD] of the z score) or obese (≥2 SD of the z score). 10Waist and hip circumferences were measured using a fibreglass tape to the nearest 0.1 cm and waist-to-height and waist-to-hip ratios calculated accordingly.Blood pressure was checked by auscultation using a sphygmomanometer with an appropriately sized cuff.Systolic and diastolic hypertension was determined according to the criteria for high blood pressure in children and adolescents from the National High Blood Pressure Education Program Working Group. 11fter a fasting period of 8 hours, 3 mL of venous blood were drawn from participants and collected into BD Vacutainer ® tubes (Becton, Dickinson and Co., Franklin Lakes, New Jersey, USA).Using enzymatic methods, concentrations of glucose, uric acid, total cholesterol, -The current study found that certain adipokines were significantly correlated with FTO and PPARγ expression.

Application to Patient Care -Determining the presence or absence of FTO and PPARy SNPs among adolescents might help in the design and implementation of more
intensive obesity prevention strategies in this population.
high-density lipoproteins (HDLs), low-density lipoproteins (LDLs) and triglycerides were determined according to the manufacturer's instructions for each reagent assay kit (Randox Laboratories Ltd., Crumlin, County Antrim, UK).The participants' thyroid profiles were measured by radioimmunoassay, whereas adiponectin, insulin and leptin levels were determined using the 900 series Invitrogen ® enzyme-linked immunosorbent assay (Thermo Fisher Scientific Inc., Pittsburgh, Pennsylvania, USA).One sample remained frozen at -70 °C until DNA extraction.The atherogenic index of plasma (AIP) was calculated as total cholesterol divided by HDL.The HOMA index for insulin resistance was calculated as follows: 12 where fPI is fasting plasma insulin and fPG is fasting plasma glucose.A diagnosis of metabolic syndrome was based on the criteria of the International Diabetes Federation (IDF) and adjusted for age. 13A extraction was performed using the MagNA Pure LC 2.0 Instrument and MagNA Pure LC DNA Isolation Kit 1 (Roche Diagnostics GmbH, Manheim, Germany).Results were quantified using the N60 Nano-Photometer ® (Implen GmbH, Munich, Germany), reporting the concentration (in μg/mL) and purity (the ratio of the absorbance of 260/280 nm).Genotyping was performed by polymerase chain reaction analysis in a Life ECO ® thermal cycler (Bioer Technology Co. Ltd., Hangzhou, China).The primers and conditions for each polymorphism are listed in Table 1.14 Oligonucleotides were designed using the PrimerQuest ® web tool (Integrated DNA Technologies Inc., Skokie, Illinois, USA) and synthesised at the Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Morelos, Mexico.The Basic Local Alignment Search Tool ® (National Library of Medicine, Bethesda, Maryland, USA) was used to verify the correct hybridisation.The final products were visualised in 2% agarose gel, stained with ethidium bromide and digitally documented using an ultraviolet transilluminator system (Gel Logic 212 Pro, Carestream Health, Rochester, New York, USA) [Figure 1].Sequencing was performed at the National Institute of Genomic Medicine in Mexico City to confirm the detection of polymorphisms as per previously described methods.14 Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), Version 19 (IBM Inc., Armonk, New York, USA).Descriptive continuous data were presented as means and SDs while qualitative variables were expressed as percentages.Either the Student's t-test or Mann-Whitney U test were used, depending on whether the variables were normally distributed.Using Pearson's correlation coefficient, the degree of association between glucose, insulin and adipokine levels, AIP and HOMA index were evaluated in two different settings, the first being the absence of either SNP and the second being the presence of either or both SNPs.Multivariate linear modelling was performed to establish the possible effect of the presence of polymorphisms and gender on lipid profile.A linear regression model weighted by gender was used to determine whether very-low-density lipoprotein (VLDL), LDL, HDL and triglyceride levels were predictors of HOMA index.A linear regression analysis was also used fPI × fPG HOMA index = 22.5

Results
A total of 71 adolescents were included in the study, of which 40 (56.3%) were female and 31 (43.7%) were male.The mean age was 15.7 ± 0.7 years and the mean BMI was 24.5 ± 3.8 kg/m 2 .Based on the IDF criteria, 21.1% of the subjects had metabolic syndrome. 13Nevertheless, 56 adolescents (78.8%) were for at least one diagnostic criterion of metabolic syndrome, with high triglyceride levels being most frequently observed (52%).In terms of family history, 14 (19.7%) and 16 (22.5%)adolescents had a mother or father, respectively, with metabolic disease.Interesting, 39 adolescents (54.9%) had relatives affected by chronic disease; however, none of these individuals had been diagnosed with hypothyroidism.
According to gender, there was a statistically significant difference among the participants in terms of weight, height, waist-to-hip ratio and cholesterol, HDL, leptin and uric acid levels (P <0.050 each) [Table 2].Age and leptin levels showed a non-parametric distribution.Although FTO SNPs were more frequent among females, this difference was not statistically significant (χ 2 = 2.4671; P = 0.116).Similarly, there was a non-significant increase in PPARγ SNP frequency among males (χ 2 = 1.2884;P = 0.256).There was no difference according to gender in terms of the presence of both SNPs (χ 2 = 0.2819; P = 0.595) [Table 3].According to a linear regression analysis weighted by gender, VLDL, HDL, LDL and triglyceride levels significantly influenced HOMA index (P ≤0.001).Similar results were observed among adolescents with FTO or PPARγ SNPs (P = 0.002) as well as those without PPARγ polymorphisms (P = 0.035).Overall, the PPARγ SNP was present in 25 adolescents (35.2%) and the FTO SNP was present in 46 (64.8%), including 11 cases (15.5%) in which both PPARγ and FTO SNPs were present.Neither SNP was present in 11 individuals (15.5%).Insulin levels and HOMA index were statistically correlated with the presence of FTO (P = 0.007 and 0.005, respectively), PPARγ (P = 0.007 each) and both SNPs combined (P = 0.039 and 0.029, respectively).While leptin showed positive significant correlation in the presence of PPARγ (r = 0.483; P = 0.014) and in the absence of either SNP (r = 0.627; P = 0.039), adiponectin was significantly negatively correlated with FTO, either alone (r = −0.333;P = 0.024) or in combination with PPARγ (r = −0.616;P = 0.043) [Table 4].A multivariate linear model showed that the presence of the SNPs were not determinants of BMI and lipid profile.However, the effect of gender was significant (P <0.050), particularly in the setting of both SNPs together (P = 0.021) [Table 5].

Discussion
Worldwide, various studies of different ethnic populations have shown conflicting results regarding the relationship between body weight and FTO and PPARγ polymorphisms.For example, the PPARγ Pro12Ala rs1801282 polymorphism has been associated with obesity and insulin resistance among Asian Indians; however, in a Chinese sample, it was reported to be a protective factor against type 2 diabetes and obesity. 15,16uang et al. found that the presence of the rs2282440-SDC3T/T genotype alongside the rs1801282 PPARγ2 carrier genotype was strongly associated with obesity. 17n the other hand, Queiroz et al. found that PPARγ rs1801282 conferred a higher risk of altered insulin levels and HOMA index for insulin resistance among overweight adolescents. 18To some extent, the latter observation is consistent with the results of the present study.
A previous study demonstrated that circulating lipids in Mexican children modified the association between the PPARγ2 rs1801282 genotype and insulin resistance. 19n the current study, significant associations were noted between HOMA index and the presence of FTO or PPARγ polymorphisms, taking into account the role of lipids and weighted by gender.In Asiatic populations, FTO has been associated with an increased risk of obesity and type 2 diabetes. 20Saldaña-Alvarez et al. found that FTO SNPs made differential contributions to obesity risk, supporting the hypothesis that mechanisms involving these variants are gender-dependent and that these changes may contribute to disease development. 21lthough the researchers evaluated different FTO SNPs (rs1121980, rs17817449, rs3751812, rs9930506 and rs17817449) to that of the present study (rs9939609), such findings confirm the possibility that gender is a risk  factor for obesity. 17Similarly, these results are supported by those of Huđek et al., who found a statistically significant positive correlation between frequencies of high-risk FTO genotypes in obese women (AA rs9939609, CC rs1421085 and GG rs17817449). 22az-Anzaldúa et al. reported that differences in mean BMI levels among Mexican patients with bipolar disorder were explained by the presence of FTO rs8050136 and rs9939609 genotypes. 23Another study showed that the locus of the FTO gene was significantly associated with increased BMI in indigenous Mexican populations. 24Muñoz-Yáñez et al. found that FTO rs9939609 polymorphisms were associated with obesityrelated traits, including elevated BMI, cholesterol and LDL levels, tricep skinfolds and an increased waist circumference and waist-to-height ratio. 25In the present study, an extremely high percentage of adolescents had FTO SNPs (64.8%).Given a population of approximately nine million Mexicans aged 15-20 years old, there are an estimated 5,830,200 young people with FTO rs9939609 polymorphisms and, therefore, a corresponding risk of obesity. 26uch findings are alarming for the local healthcare system.Fortunately, despite a genetic predisposition influenced by polymorphisms, obesity is a modifiable condition, even with a positive genetic profile risk score. 27n the PREDIMED-NAVARRA randomised trial, PPARγ Pro12Ala rs1801282 carriers exhibited lower telomere shortening compared to those with the Pro/Pro genotype after five years of adherence to a Mediterraneanstyle diet. 28Unfortunately, the molecular effects of a traditional Mexican diet have yet to be fully elucidated.
In the current study, mean HDL levels were significantly higher among females.However, this may be because the mean level for males was below recommendations reported for the Mexican population. 29In addition, female students also had significantly higher leptin levels.Although leptin is a significant predictor of carotid intima media thickness, previous research has confirmed that men suffer from higher cardiovascularrelated mortality compared to women. 30,31Such findings undermine the epidemiological evidence for utilising leptin as a prognostic tool for cardiovascular disease in adolescents.
As in previous research, females in the current study also demonstrated significantly higher cholesterol levels. 32A strong correlation has been established between cholesterol and visceral adipose tissue as quantified by dual-energy X-ray absorptiometry. 33Unfortunately, this technique was not utilised in the present study.Finally, the frequency of metabolic disease among the parents of the studied adolescents was lower than expected; instead, students' relatives were found to have a high rate of chronic disease.However, it is unclear whether these individuals had lipid-related abnormalities.
It is worth noting that various researchers have recommended lowering BMI cut-off values for over- weight and obese categories in Mexican and Asiatic populations. 34,35Thus, if a BMI of ≥27 kg/m 2 were considered to indicate obesity, 31% of the adolescents in the current study would have been considered obese.
Males were significantly heavier than females; this was accordingly reflected by slight elevations in glucose and insulin levels and HOMA index.However, these differences were not significant, possibly due to similarities in BMI in both genders.Therefore, further studies are recommended including other variables which could influence BMI in adolescents, such as physical activity and dietary habits. 36Another limitation of this study was the small sample size; nevertheless, investigations regarding associations between polymorphisms and clinical parameters for metabolic syndrome or obesity can be clinically significant in small samples. 37

Conclusion
The results of this study suggest significant positive correlations between insulin levels and HOMA index with BMI-related FTO rs9939609 and/or PPARγ rs180-1282 polymorphisms.Future epidemiological studies are recommended to determine the net causative effect of such gene variants so as to halt the development of a pandemic of obesity-related health problems.Although the main causes of obesity, diabetes and metabolic syndrome are lifestyle factors, the role of such polymorphisms could help to explain why some individuals have a greater tendency to develop metabolic disorders and are more resistant to weight control interventions than others.
c o n f l i c t o f i n t e r e s t The authors declare no conflicts of interest.The preliminary version of this study was submitted as a M.Sc.thesis to the UAEM in 2016.This version is available on the university website.

Figure 1 :
Figure 1: Agarose gel electrophoresis image of polymerase chain reaction amplification products showing the presence of a fat mass and obesity-associated (FTO) polymorphism.Lane one contains the molecular marker, lanes 2-8 contain amplified FTO gene DNA fragments, lane nine contains the positive control and lane 10 contains the negative control.

f u n d i n g
This study was funded with the aid of a grant from the Mexican Ministry of Education (grant #PROMEP/ 2013/CA-186/103105/13/9057).a c k n o w l e d g e m e n t s

Table 1 :
14imers and polymerase chain reaction conditions for the studied polymorphisms14Correlation of the Homeostasis Model Assessment Index and Adiponectin, Leptin and Insulin Levels to Body Mass Index-Associated Gene Polymorphisms in Adolescents This study was approved by the Institutional Review Board of the Medical Sciences Research Center at UAEM (#2015/14).All subjects and their parents/guardians provided informed consent prior to participation in the study.All of the study procedures were in line with the ethical standards of the revised Declaration of Helsinki as well as the Mexican Standard for the Implementation of Projects of Research for Health in Humans (#NOM-012-SSA3-2012).
e294 | SQU Medical Journal, August 2018, Volume 18, Issue 3 to test BMI as the dependent variable and age as the independent variable according to gender and weighted by the presence of polymorphisms.A P value of ≤0.050 was considered statistically significant.

Table 2 :
Anthropometric and laboratory variables according to gender among adolescents in Toluca, Mexico (N = 71)

Table 4 :
Correlations between glucose, insulin and adipokine levels and homeostasis model assessment index with body mass index-related polymorphisms among adolescents in Toluca, Mexico (N = 71) FTO = fat mass and obesity-associated; PPAR = peroxisome proliferator-activated receptor; HOMA = homeostasis model assessment; AIP = atherogenic index of plasma.*Including the 11 adolescents with both polymorphisms.

Table 3 :
Frequency of body mass index-related polymorphisms according to gender among adolescents in Toluca, Mexico (N = 71)Correlation of the Homeostasis Model Assessment Index and Adiponectin, Leptin and Insulin Levels to Body Mass Index-Associated Gene Polymorphisms in Adolescents e296 | SQU Medical Journal, August 2018, Volume 18, Issue 3

Table 5 :
Multivariate linear model for body mass indexrelated polymorphisms and gender as determinants for body mass index and lipid profile