Birth Weight In Relation To Obesity And Diabetes Gene Expression In Healthy Infants; A Case- Control Study

Background: Since obesity and diabetes are a prevalent phenomenon in the world, recognizing the factors affecting these two conditions can be effective in modifying them. We decided to investigate the expression of obesity and diabetes genes in infants with birth weight lower than 2500 g. Methods: The current case- control study was performed on 215 healthy infants aged 5-6 months which referred to health and treatment centers of Kermanshah. After measuring weight and height of the infant, the values were compared with the diagram of WHO to assure being healthy and well-grown and infants who were healthy, were selected to the study. 78 infants was in case group and 137 infants was in control group. 5 cc of intravenous blood was collected from all infants. Blood samples were collected in Ethylenediaminetetraacetic acid (EDTA) coated vials to evaluate the expression of MC4R, MTNR1B, PTEN, ACACB, PPAR-γ, PPAR-α, (NRXN3 (NTRK2), (PCSK1), (A2BP1), (TMEM18), (LXR), (BDNF), (TCF7L2), (FTO) and (CPT1A) genes. The data was analysed by Chi square, Mann-Whitney U and Spearman tests. Results: An inverse signicant correlation between birth weight and obesity and diabetes genes including MTNR1B, NTRK2, PCSK1, and PTEN genes (r= -0.221, -0.235, -0.246, and -0.418, respectively). In addition, the expression level of the mentioned genes in the LBW infant was signicantly up-regulated than the normal weight infants (P= 0.001, 0.007, 0.001, and <0.001, respectively). The expression level of PPAR-a gene had signicantly positive correlation with the birth weight (r= 0.19, P= 0.005). The expression level of PPAR-a gene in the normal weight infants was signicantly up-regulated than the LBW infants (P= 0.049). Conclusion: Expression level of MTNR1B, NTRK2, PCSK1, and PTEN genes up-regulated in the LBW infants, however, the expression level of PPAR-a gene was signicantly down regulated in the LBW infants compared to the infants with normal birth weight.


Introduction
Low birth weight (LBW) is enhanced short-term and long-term consequences such as infant mortality, cognitive disorders, growth failure, and is associated with obesity and diabetes in adulthood [1,2]. According to de nition by World Health Organization, LBW infants refers to infants whom their birth weight is less than 2500 grams [3]. Approximately 15-20% of births are related to LBW infants which annually accounts for about 20 million births [4].
Some studies have found a U-shaped or J-shaped association between birth weight and obesity in adulthood, although these ndings have been contradictory [5,6]. On the other hand, it has been suggested that LBW can increase the risk of developing diabetes in adulthood [5]. Since obesity and diabetes are a prevalent phenomenon in the world, recognizing the factors affecting these two conditions can be effective in modifying them [7,8].
Both environmental and genetic factors are involved in the pathogenesis of obesity and diabetes [8][9][10].
Among the genes involved in the etiology of obesity and diabetes can be named Melanocortin 4 receptor (MC4R), Melatonin Receptor 1B (MTNR1B), Peroxisome proliferator-activated receptor gamma (PPAR-γ), Phosphatase and tensin homolog (PTEN), Acetyl-CoA Carboxylase Beta (ACACB), and peroxisome proliferator-activated receptor-α (PPAR-α), MC4R gene encodes melanocortin receptor, MC 4 protein, a Gprotein receptor that high it's expression is related to body fat distribution and energy intake in children. MTNR1B gene is located on chromosome 11q21 and synthesis melatonin receptor 2 in which this gene is related to all diabetes types including diabetes mellitus, type 1 diabetes, and gestational diabetes [11,12]. In addition, down-regulated of PTEN and ACACB genes can decrease blood sugar and subsequently prevent diabetes and obesity PTEN is a phosphatase which plays role in signaling pathway and suppression of tumor [10,11,13]. ACACB is a biotin dependent enzyme which catalyzes irreversible carboxylation of acetyl CoA to manolyl CoA and is effective in obesity and diabetes by reducing fatty acids oxidation and increasing of insulin resistance [14,15]. Peroxisome proliferative activating receptors (PPARs) are part of the nuclear hormone receptors [16]. Up-regulated of expression level of PPAR-γ regulates the secretion of adipose tissue hormones and reduces insulin resistance [17]. In addition, PPAR-α, which is expressed mostly in tissues with a high level of fatty acid catabolism, therefore up-regulated of PPAR-α is involved in reducing obesity and body fat [18]. Since the epidemiologic studies showed that birth weight can play role in incidence of the obesity and diabetes, this study aimed to investigate the expression of obesity and diabetes genes in infants with birth weight lower than 2500 g.

Methods And Materials
The current case control study was performed on healthy infants aged 5-6 months which referred to health and treatment centers of Kermanshah. Sample size was calculated based on the weight mean of children in study by Zarrati et al. [19] using the sample size formula for case studies with 90% power and 95% con dence in each group of 45 infants. Inclusion criteria were healthy infants, lack of metabolic diseases, not using medications in last month, breastfeeding or using infant milk (formula), not initiating supplementary nutrition, healthy parent and lack of metabolic disease of their parents. Based on birth weight, we considered infants with birth weight less than 2500 g in the case group and weight between 2500-4000 g in the control group [3]. For more reassurance, we entered 80 infants in the case group and twice as many in the control group, which due to the fact that the information of some samples was incomplete, nally 78 infants remained in the control group and 138 infants in the control group.

Ethical consideration
At rst, the process of the study was completely explained for the parents, and informed written consent was obtained from their parents. This study was approved by the ethics committee of Kermanshah University of medical science (ethical number: IR.KUMS.RES.1397.081).

Anthropometry
Firstly the health information of infant including term or preterm, birth height, weight, and head circumference, and the food kind was recorded in the questionnaire. After that, the infants' height was measured by tape and in supine position and the infants weight was measured with the least cloth and without diaper. After measuring weight and height of the infant, the values were compared with the diagram of WHO to assure being healthy and well-grown and infants who were healthy, were selected to the study.

Results
In this present study, 137 infant and 78 infant were enrolled in the control and case studied groups, respectively. In term of gender of the infants, food kind of the infants, their mother age, their mother weight before pregnancy, their mother and father BMI were not seen any difference between two studied groups. The mean of weight in control and case groups were 3.18 ± 0.27 and 1.79 ± 0.48 kg, respectively (P < 0.001). All characteristics of infants are presented in Table 2. In current study, we found that there was an inverse signi cant correlation between birth weight and obesity and diabetes genes including MTNR1B, NTRK2, PCSK1, and PTEN genes (r= -0.221, -0.235, -0.246, and − 0.418, respectively). In addition, the expression level of the mentioned genes in the LBW infant was signi cantly up-regulated than the normal weight infants (P = 0.001, 0.007, 0.001, and < 0.001, respectively). (Table 3) P2 was obtained Mann-Whitney U. Table 3, we observed that the expression level of PPAR-a gene had signi cantly positive correlation with the birth weight (r = 0.19, P = 0.005). The expression level of PPAR-a gene in the normal weight infants was signi cantly up-regulated than the LBW infants (P = 0.049).

Furthermore in
Liner regression showed a signi cant negative association between the birth weight and the expression level of obesity and diabetes genes including MTNR1B, NTRK2, PCSK1, and PTEN genes, as well as, a signi cant positive association between the birth weight and the expression level of PPAR-a gene. No signi cant association was seen between the birth weight and other studied expression level of obesity and diabetes genes. (Fig. 1)

Discussion
This present case-control study demonstrated that the normal birth weight had preventive role on upregulated expression level of MTNR1B, NTRK2, PCSK1, and PTEN genes, while the expression level of PPAR-a gene was signi cantly down regulated in the LBW infants compared to the infants with normal birth weight. LBW infants are at higher risk for obesity, neurodevelopment failure, cardiovascular disease, and other metabolic disorders [20]. Birth weight is considered as a suitable indicator for the quality of fetal growth and a predictor of health throughout life [20,21]. To best our knowledge, this current study was evaluated the association between birth weight and obesity and diabetes gene expression in healthy infants.
Our ndings indicated that the birth weight was associated with up-regulated the expression level of MTNR1B. Holzapfel et al. [22] observed that the MTNR1B was associated with diabetes in children and adolescents. Another study by Lianget al. [23] showed that maternal MTNR1B genotype involved in the etiology of childhood obesity. Recently, the MTNR1B has been shown to increase the risk of obesity and type 2 diabetes [22,24]. It is also highly expressed in retinal cells, pancreas and especially pancreatic islet cells. Melatonin is a neurohormone secreted by pineal gland which can adjust the circadian rhythm and the circadian rhythm regulates the insulin level. However, melatonin secretion is impaired in diabetics [24].
We also indicated up-regulated the expression level of the NTRK2 with increasing of the birth weight. In study by Metrustry et al. variants in the NTRK2 gene and birth weight were examined. This study was showed that this gene was highly expressed in LBW twins [25]. NTRK2 is located on 9q21.33. NTRK2 encodes a member of the neurotrophic tyrosine receptor kinase (NTRK) family that is a membrane-bound receptor for BDNF and regulates energy balance downstream of MC4R. Also it involved (involves) in MAPK pathway and cell differentiation [26]. Mutations of NTRK2 have been associated with obesity and eating behavior [25,27].
In current study the expression level of the PCSK1 was up-regulated in the LBW infants. Ruiz-Narváez et al. showed that LBW plays a role the expression level of the PCSK1 by disrupting central nervous system mechanisms and increases obesity in adulthood [28]. PCSK1 which is located on 5q15 encodes a prohormone convertase 1/3 (PC 1/3) that involved in pro-insulin processing under in uencing of TCF7L2 [29]. As well as PCSK1 is involved in the processing of pro-opiomelanocortin, proglucagon, proGnRH and proTRH. In addition, PCSK1 variants are associated with extreme obesity, impaired glucose tolerance, and poly cystic ovarian syndrome. Rare mutations in PCSK1 cause childhood obesity, impaired prohormone processing and abnormal glucose homeostasis with increasing proinsulin concentrations [28,30].
In present study, another up-regulated the expression level of gene was related to the PTEN gene. In study by Li et al. [31] the high expression level of gene PTEN was associated with increase of insulin resistance. Although the gene was rst identi ed as a tumor suppressor, it has recently been shown to be important with its antagonistic function in the insulin signaling cascade and is involved in glucose metabolism [32]. PTEN is a phosphatase which plays role in signaling pathway and suppression of tumor in which is able to suppress phosphatidylinositol 3-kinase (PI3K) signaling [10,11,13]. Since activation of PI3K is essential for insulin performance, PTEN is known to be effective in development of insulin resistance by reducing PI3K [33].
On the other hand, we observed that the expression level of PPAR-a gene was signi cantly down regulated in the LBW infants. Laleh et al. [34] demonstrated that the high expression level of PPAR-a suppressed appetite in obesity. Priego et al. [35] showed that the higher expression level of PPAR-α gene in infants is associated with a lower risk of overweight. PPAR-a is a group of legend activated nuclear receptors that are mainly expressed in tissues that are vital for fatty acid metabolism, such as the liver, kidney, and heart, where they play an important role in regulating transcription of fatty acid metabolism, lipid homeostasis, and regulation of obesity [36]. During times of starvation and energy depletion, the PPAR-α increases fatty acids beta-oxidation. Animal study showed that PPAR-a de ciency was related with obesity and dyslipidemia [18].

Limitations
This current study was the rst case-control study on birth weight and obesity and diabetes gene expression in healthy infants. However, it suffered from some limitations. The sample size was small due to economic constraints. Second, although we controlled many infants and maternal factors between the two groups, there was difference between the two groups because many infants in the control group were preterm in term of pregnancy weight gain and current weight infants.

Conclusions
In conclusion, this present study re ected that the expression level of MTNR1B, NTRK2, PCSK1, and PTEN genes up-regulated in the LBW infants, however, the expression level of PPAR-a gene was signi cantly down regulated in the LBW infants compared to the infants with normal birth weight. Informed consent: Written informed consent was obtained from all parents after explaining the purpose of the study.
Consent for publication: not applicable.
Availability of data and materials: The datasets used and/or analyzed during the current study will be available from the corresponding author on reasonable request.
Competing interests: On behalf of all co-authors, the corresponding author states that there is no con ict of interest.
Funding: This study was supported by Kermanshah University of Medical Science (Grant No: 97155).
Author contributions: SM and SC contributed in conception and design of the research; SC and AM contributed to data collection; SM contributed to the acquisition, the analysis, and the interpretation of the data; SM and AM contributed to draft the manuscript. All authors are in agreement with the manuscript and declare that the content has not been published elsewhere 3 . Tang Y, Vanlandingham MM, Wu Y, Beland FA, Olson GR, Fang J-L. Role of peroxisome proliferatoractivated receptor alpha (PPARα) and PPARα-mediated species differences in triclosan-induced liver toxicity. Arch Toxicol. 2018;92(11):3391-402. Figure 1