Relationship between the hsa miR 150-5p and FTO gene expression in white subcutaneous adipose tissue with overweight/ obesity, lipid profi le and glycemia

: The overweight population is growing in the world, and the search for obesity-associated mechanisms is important for a better understanding of this disease. Few studies with the FTO gene and miRs show how they associate to obesity and how they can impact this disease. The aim of this study was to verify the relationship between the FTO gene and the hsa-miR-150-5p expression with overweight/obesity, lipid profi le, and fast blood glucose. Men and women (18 years older or above), with body mass index ≥ 18.5 kg/m 2 , were enrolled in the present study and the FTO gene and hsa-miR-150-5p expression, biochemical parameters of blood and anthropometric measurements were analyzed. The results highlight that the FTO gene expression is associated to obesity ( p 0.029), LDL-C ( p 0.02) and fasting blood glucose ( p 0.02), but not with triglycerides ( p 0.69), total cholesterol ( p 0.21), and HDL-C ( p 0.24). The hsa-miR-150-5p is not associated to obesity ( p 0.84), triglycerides ( p 0.57), total cholesterol ( p 0.51), HDL-C ( p 0.75), LDL-C ( p 0.32), and fasting blood glucose ( p 0.42). The FTO gene expression is related to obesity, LDL-C and blood fasting glucose, representing a good molecular marker for obesity.


INTRODUCTION
According to the World Health Organization. (2016), there are more than 1.9 billion adults and more than 340 million children and adolescents with overweight or obesity. In a simple manner, obesity results from the unbalance between energetic intake and energetic spend (Cui & Chen 2017). However, other factors are behind this disease, such as genetics, cultural, and psychologic issues (Upadhyay et al. 2018).
The FTO gene (fat mass and obesityassociated gene) is placed on chromosome 16 (16q12.2) and was first described in a mouse model with fused toes and thymus hyperplasia. It was demonstrated to be highly expressed during embryonic development and was associated with programmed cell death, craniofacial development, and left-right asymmetry (Peters et al. 1999). However, in humans, the trisomy of the 16 chromosome (the region in which the FTO gene is located) was associated with mental retardation, obesity, dysmorphic facies, and several bilateral clinodactyly of some fi ngers (Stratakis et al. 2000). After the first studies, some more appeared and reported association with humans obesity. It is highly expressed in the brain, but the gene expression in other tissues and which obesity trait it is related remains unclear (Locke et al. 2015, Gulati et al. 2013.
MiRs are small, endogenous RNAs of approximately 22 nucleotides that act avoiding mRNA translation, mediated by the complementarity of the miR (from nucleotide 2 to 8) with the 3' untranslated region (UTR) of the mRNA target (Treiber et al. 2019). MiRs are conserved in many different species, which suggest that they have an important function in the organism physiology, participating in crucial cellular process and diseases (Liz & Esteller 2016, Mens & Ghanbari 2018, Kinser & Pincus 2019. Among the miRs, the miR-150-5p is known as a circulating miRNA, being involved in inflammatory processes, lipoprotein profile, and vascular system (Ying et al. 2016, Desgagné et al. 2017, Desjarlais et al. 2017. However, the expression of miRs can be tissue-specific. Thus, it is necessary to know how they are associated with metabolic processes and diseases in a tissue-specific manner, to permit their use as diagnostic biomarkers or therapeutic targets in complex diseases in which they have not been used.
Thus, to the best of our knowledge, miRs and the FTO gene expression with BMI, lipid profile and glycemia in white adipose tissue from obese individuals have not been well investigated and the few studies in literature present controversial results. Hence, the purpose of the present study was to compare the hsa-miR-150-5p and FTO gene expression between eutrophic and overweight/obesity individuals to investigate the relationship between the hsa-miR-150-5p and FTO gene expression with obesity, lipid profile and glycemia; and verify a possible relationship between the hsa-miR-150-5p and FTO gene expression.

Study sample
Men and women, above the age of 18 years (42.14 ± 12.29 years) with overweight (25 ≤ BMI ≤ 29.9 kg/m 2 ) or obesity (BMI ≥ 30 kg/m 2 ) (n=15) were enrolled in this study. Eutrophic individuals (18.5 ≤ BMI ≤ 24.9 kg/m 2 ), control group (men and women n=5), were also invited (26.4 ± 2.07 years). Prior to the study beginning, all the procedures were explained and if the participants had any inquiries, they enlightened -all the participants assigned a consent form. The present study was approved by the ethical committee from the School of Physical Education and Sport of Ribeirão Preto (CAAE 37573114.6.0000.5659) and by the Hospital das Clinicas of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (CAAE 37573114.6.3001.5440).

Clinical analyses
The participants were submitted to the following analyses: body mass and height with a balance for the weight (precision of 50g) coupled to a stadiometer (precision of 1mm) (Welmy W200ALCD), waist circumference (umbilicus level) and body fat (bioimpedance -Maltron BF-906). Prior to the analyses, they were informed not to consume alcohol 24h earlier or caffeine 4h before, not exercise, and to consume 2 or 4 glasses of water 2 hours before until the test performance (De Lima Augustemak et al. 2008). Fast blood glucose, triglycerides, total cholesterol, high-density cholesterol (HDL-C), low-density cholesterol (LDL-C) were evaluated from venous blood after 12h fasting (Silva et al. 2007). In addition, the International Physical Activity Questionnaire (IPAQ) and the Food Consumption Markers Questionnaire were utilized to measure the level of physical activity in the last week and the ingestion frequency of 10 groups of foods in the last seven days, respectively. If the food consumption marker presents a positive result, means that the group ingests health food instead of unhealthy food and if this questionnaire presents negative result, means the opposite (Silva et al. 2007, Damé et al. 2011.

Biopsy
White subcutaneous adipose tissue was collected with a supraumbilical incision of 2 cm under local anesthesia, by an experienced doctor. Approximately 0.5 g of tissue was collected, immediately stored in liquid nitrogen and taken to the laboratory for posterior analysis. The participants were oriented to not perform physical activities, to have breakfast until 8 a.m., and fasting until this minor surgery time (midday) at the collect day.

Selection of the microRNA of interest
To select a microRNA to investigate we imputed in the mirDB (http://mirdb.org/) the access number of the FTO gene from NCBI (ID: 79068) and a list of 78 miRs were generated as output. We considered a score of 80 as a cutting limit and from 17 miRs that attended the criterion score, we selected the hsa-miR-150-5p with a score of 85. To confirm the selection of this miR for posterior analysis we confirmed a hypothetical interaction of this miR with the 3' UTR of the FTO mRNA using the RNAhybrid (http://bibiserv. techfak.uni-bielefeld.de/rnahybrid) and we observed a hybridization free energy (ΔG) of -28.9 Kcal/mol (Rehmsmeier et al. 2004, Wong & Wang 2014. The hsa-miR-150-5p was select to further study due to their low free energy, as low as better, and to their vestige of relationship with energy metabolism, metabolic associated diseases and lipid metabolism, below mentioned in the discussion.

Statistical analysis
Sample size calculation was calculated considering the FTO gene expression change from 1.0 to 2.0 (arbitrary unit; main variable in the study), a standard deviation of 0.6 (data from a pilot study), with a power of 80%, and α of 0.05 -the obtained sample size was at least five individuals per group. To reach the proposed objectives we utilized the following statistical tests: The Pearson's correlation coefficient; multiple regression analysis; Student's t-test, and linear regression analysis. The utilized software were SAS (version 9.2) and Prism (version 5.0), and the statistical significance value considered was p ≤ 0.05.

RESULTS
To characterize the differences between the groups, we compared the anthropometrical measures, physical activity level and the food consumption markers between them (Table I).
The overweight/obesity group presented higher values compared to the eutrophic group for body mass index (BMI) (34.24 ± 1.46 kg/m 2 vs 21.36 ± 1.05 kg/m 2 ), higher percentage of body fat (48.44 ± 1.98% vs 28.82 ± 1.43), higher waist circumference (98.58 ± 2.42 cm vs 71.00 ± 3.69 cm) and lower level of high intensity physical activity (0.7 ± 2.67 min/week vs 24.00 ± 53.67 min/week). However, the groups are comparable for walking level, moderate physical activity, and in the food consumption markers.
Aiming to respond how were the FTO gene expression and the hsa-miR-150-5p expression profile in eutrophic and obesity individuals, we measured the FTO gene expression (Figure 1) and the hsa-miR-150-5p expression ( Figure 2) in both groups. We could not observe a difference in FTO gene expression between the eutrophic and overweight/obese individuals. When we performed the comparison between the groups with the hsa-miR-150-5p expression, we did not observe any difference either.
To respond if the FTO gene expression and the hsa-miR-150-5p expression were related to obesity, lipid profile, and glycemia, we first performed a Pearson's correlation. We could demonstrate that the FTO gene expression was correlated to BMI (r = 0.50, p 0.029) and LDL-C (r = -0.54, p 0.038) (Table II), but for the hsa-miR-150-5p we did not find any correlation with obesity, lipid profile and glycemia (Table III).
Based on the results obtained in the correlation analysis, we performed a multiple regression analysis with the aim of demonstrating which variables were related to FTO gene expression and the hsa-miR-150-5p. We could observe a relationship of the FTO gene expression with LDL-C (β = -0.047 ± 0.016, p 0.02) and fasting glucose (β = 0. 114 ± 0.04, p 0.02) (Table IV). When we performed the same analysis with the miR-150-5p, we could not observe the relationship among the tested variables (Table V).
Lastly, aiming to respond if the FTO was related to the miR-150-5p expression, we performed a linear regression between FTO gene expression and hsa-miR-150-5p (Figure 3) -no relationship was found.

DISCUSSION
The present study demonstrated that FTO gene expression is related to BMI, LDL-C and fasting glucose, while the hsa-miR-150-5p expression is not related to obesity-related variables. In addition, there were no differences in the FTO and hsa-miR-150-5p expression in eutrophic and overweight/obese individuals. No relationship was found between FTO and the hsa-miR-150-5p expression.
We demonstrated the relationship of the   There is finite information about the FTO metabolic view, however, some mechanisms are proposed. Gulati et al. (2013) proposed that the FTO would act as an amino acid sensor. In embryonic fibroblast cells from FTO Knockout mice, they demonstrated reduced mTORC1 (mechanistic target of rapamycin complex 1) activation, a related protein to amino acid availability, suggesting that under low amino acid availability the FTO gene would be negative regulated, making the cell less sensitive to amino acids, which result in mTORC1 metabolic pathway impaired, increasing autophagy. Wu et al. (2010) proposed that FTO would act epigenetically as a transcriptional coactivator, recruiting DNA demethylase, regulating gene expression, while Lin et al. (2014) prosed an increase in CREB (cAMP response element binding) phosphorylation FTO overexpression dependent, positively regulating NPY (neuropeptide Y) and BDNF (Brain-derived neurotrophic factor), which would regulate the food intake.
Concerning the hsa-miR-150-5p expression, we did not observe relationship to any obesityrelated variable studied and no differences in the expression level between eutrophic and overweight/obesity individuals. MicroRNAs can regulate several genes, and they can be involved in several cellular processes in a tissue or moment specific way. Dahlmans et al . (2017), studying mitochondrial metabolism in skeletal muscle, demonstrated that the hsa-miR-150-5p positively regulates the mitochondrial metabolismonce the mitochondrial function is impaired, it is related to diabetes type II and insulin resistance. In addition, the authors compared the expression level of this miRNA in individuals with different levels of physical capacity and demonstrated a higher expression level in athletes when compared to overweight and obese patients. Ying et al. (2016), in a study with knockout mice for the miR-150, demonstrated that this miRNA was involved in the regulation of genes that would act on β-cells receptors modulating macrophages and T cells activation and affecting the adipocyte, with consequent alteration in inflammation, glycemic and insulin profile. On the other hand, Chou et al. (2014) demonstrated that the decrease in the miR-150 processing machinery increased the expression of genes involved in the remodeling of white adipose tissue to brown adipose tissue, suggesting that this microRNA is involved in lipid metabolism.
Finally, an important limitation of our study should be mentioned -the number of participants.

CONCLUSION
The results obtained in our study can corroborate and sum with the finite studies that seek to   Adjusted by the level of physical activity and food ingest quality; Data are presented as estimative ± standard deviation, p-value and confidence interval; *p ≤ 0.05. Adjusted by the level of physical activity and food ingest quality; Data are presented as estimative ± standard deviation, p-value and confidence interval. find genetic markers related to obesity. Thus, we conclude that the FTO gene expression is moderately related to BMI, LDL, and blood fasting glucose, and we suggest it as a molecular marker for obesity. However, more studies regarding genes and miRs association with obesity seem necessary, since it is a complex disease and results from a vast number of factors.