|Zeba Haque*1, Farzana Yasmeen2 and Darakhshan J Haleem3
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Introduction: The prevalence of obesity and depression is increasing at an alarming rate. The state of obesity and perception of job stress is different in both genders. The circulating levels of stress related hormones cortisol, serotonin and leptin are attenuated in stress.
Objective: To investigate variations in the coding regions of leptin gene in relation to stress perception and other biochemical and physiological parameters of stress perception.
Method: With verbal and written consent, twenty working women were identified from 5 corporate sections of local banks in Karachi. Stress perception was measured by standardized questionnaire. Depending on stress scores the data were classified as no/minimal, mild, moderate and severe stress groups. Changes in appetite were determined by visual analogue scale for hunger and desire to eat during stress. The chemical analytes were measured by ELISA. Leptin gene was sequenced using 3130 Genetic Analyzer of (ABI) Applied Biosystems using BigDye Terminator 3.1 Sequencing Kit protocols at Centralized Science Laboratory University of Karachi. One way ANOVA was applied to analyze data. Result: The BMI and VAS for hunger and desire to eat increased significantly with the severity of stress (p<0.05). Although circulating levels of leptin and cortisol also increased with stress perception (p<0.05) but a decrease in cortisol levels was observed in the group with severe stress perception. Plasma serotonin decreased with increase in stress perception. A single nucleotide polymorphism was found in 5’ flanking region of exon 2 of leptin gene compared to the reference gene fragment retrieved from gene bank with no minimal perception of stress. The guanosine at position 15719 in the reference fragment was replaced by cytidine. No variation was found in the DNA sequences of exon 2 of women with mild, moderate or severe perception of stress. No variation was found in the segment of exon 3 of leptin gene. Conclusion: The consistent finding of a G15719C variant on the 5’ flanking region of exon 2 of LEP in the group of women who claimed no/minimal stress suggest a role of this gene in stress induced obesity and other physiological reactions to stress.
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|Modernization, persistent urge to compete with the high living standards, sedentary lifestyles and availability of low cost high calorie diet has led the human race to a consistent state of stress and weight gain. Daily life events, the stress at job and even the buzzes from personal electronic gadgets play a role in stress response by an individual . A persistent state of stress may lead to depression and anxiety . Perception of stress is different in both genders. Earlier we observed  that women perceive a higher degree of stress perception relative to the men working in same environment . Men tend to have a greater BMI in moderate stress compared to women who exhibit increased appetite and tend to have higher than normal BMI both in moderate and severe stress.|
|The prevalence of obesity and depression is increasing at an alarming rate in modern societies of both developed and developing countries . Although a strong correlation exists between depression and obesity, extreme weight loss with anorexia nervosa is also observed in significant number of depressed patients . The basis of disparity of eating and stress coping behaviors in depression remains largely unexplained.|
|The adipocyte hormone leptin administration in ob/ob mice resulted in weight reduction and increased energy expenditure . On the other hand human obesity is also associated with high circulating leptin  suggesting its resistance at receptor level. Evidence of both hyper and hypophagia leading to derangements in body weight during depression provides a conceptual understanding of leptin involvement in stress related behavioral changes [9,10, 11].|
|Hypercortisolism is a feature of both obesity and depression (12). Dallman et al,  reported a blunted adrenal glucocoricoid release in obesity. Humans with major depression show decreased sensitivity to glucocorticoids  and increased cortisol levels in blood . Plasma cortisol decreases the HPA axis activity by negative feedback mechanism, decreasing corticotropic hormone (CRH) in the hypothalamus. A dysregulated CRH is reported in depression  with a blunted ACTH and normal cortisol response to CRH stimulation . The biological effect of leptin in stress remains ambiguous. Leptin administration to starvation and restraint stressed animals, normalized the increases in ACTH and corticosterone levels due to post-stress hyperactivity of HPA axis . Glucocorticoids increase the leptin expression and release while leptin adds to the negative feedback loop regulating HPA axis .|
|Serotonin (5-HT) has also been identified as the food intake and body weight modulator. 5-HT reuptake inhibitors are used as weight reducing agents . Serotonin transporter protein and leptin receptor genes co-express in dorsal raphe nuclei . Reduced expression of 5-HT transporter protein in leptin deficient mice  and increased content and metabolism of 5-HT in forebrain by leptin [23, 24] further explain that leptin exerts a positive effect on central 5-HT metabolism. All these data suggest the existence of an interaction between leptin and serotonin in modulation of appetite and stress perception.|
|Leptin gene is located at chromosome 7q31.3 [Accession number NG_007450.1]. It has 23351 base pairs . The variations of leptin receptor gene have been identified and these result in the development of the leptin resistance leading to a state of hyperleptinemia and obesity . To best of our knowledge the variations in the leptin gene are not correlated with the perception and coping capabilities during the stress.|
Aims & Objectives
|Previous observations of increased leptin, cortisol and decreased serotonin with increasing stress perception both in men and women and the antianxiety/depressant effects of injected leptin to restrained rats evident from the reversal of stress induced behavioral changes, led us to make an attempt to investigate the possible interactions of the variations in the coding regions of the obesity gene and stress responses leading to behavioral (eating) disorders and chemical (plasma leptin, serotonin, cortisol levels) changes, resulting in clinical depression/anxiety disorders and obesity.|
Material and Method
|Subjects: Adult working women were identified from the corporate section of five branches of a local bank in Karachi. All the women were not suffering from any chronic, metabolic or nutritional disease. After obtaining the verbal and written consent the women were asked to fill a questionnaire to evaluate the perception of stress . Stress perception was calculated as described earlier in general analytical methods. Subjective appetite was measured using visual analogue scale (VAS) method . The anthropometric measures were taken to calculate BMI. 3 ml of blood was collected in plain tube to collect serum after centrifugation to estimate serum leptin levels. Another 5 ml of blood was collected in heparinized tube to determine serotonin and cortisol and DNA sequence analysis.|
|The chemical variables were determined by ELISA. The kit for serum leptin  and serotonin  were obtained from BioSource Europe S.A. The kit for cortisol  was obtained from The Equipar Diagnostics.|
|DNA Sequencing Protocol: The genomic DNA was isolated from white blood cells (WBC) using phenol chloroform method . Samples were subjected to PCR amplification of the target sequence in an Applied Biosystems Thermal Cycler 2720 with 5’- TTTGACGGGATGGTAGCCAG-3’, and 5’- AGAGTGGAGCCCTGTGCTTT-3’ as forward and reverse primers exon 2 respectively and 5’- TCACCTGGGTGCAGGATACAA-3’ and 5’GGGTCTTATGCCTTTGGAAGAG-3’ forward and reverse primers for exon 3 respectively. The primers were designed for exon 2 and 3 sequences of the leptin gene, using primer designing website [https://www.yeastgenome.org/cgi-bin/webprimer]. Amplified products were confirmed on gel electrophoresis and then cleaned up enzymatically using ExoSAP. The purified product was quantified by taking absorbance at 260nm using UV spectrophotometer and 100ng of the sample DNA was used for analysis of the sequence of target fragment of gene using 3130 Genetic Analyzer of (ABI) Applied Biosystems using BigDye Terminator 3.1 Sequencing Kit protocols at Centralized Science Laboratory University of Karachi.|
Gene Analysis, Discovery and Characterization of “novel SNP”.
|The sequences were identified as fragments of human leptin by using blast (33). The partial leptin gene fragments containing exon 2 (425bp) and exon 3 (668bp) of each group (n=5 each) were assembled using DNA star software. The reference sequence of leptin gene was retrieved from the gene bank . The artifactual and low signal peaks responses at the electrophrogram were removed using staden package software. The mapped positions with up to two mismatches were recorded.|
Statistical Analysis of Blood Parameters.
|One way analysis of variance (ANOVA) was applied to evaluate the effect of stress perception score on behavioral (Appetite, VAS for hunger and VAS for desire to eat), physical (BMI) and chemical (Plasma leptin, cortisol and serotonin) parameters. Tuckeys test was applied as a post-hoc test on the data. The differences in the genotype frequencies of LEP for Groups for various stress perception was evaluated using Fisher’s exact test if expected values of 20% of cells were smaller than 5.|
|Adult women volunteers participated in the study with an average age of 34.53 ± 11.74 years ranging from 29 – 59 years. One way ANOVA showed that perception of stress significantly affects the BMI (F3,19=4.82, p<0.05) (Fig-1). Post-hoc analysis showed significant increase in the BMI of the women who perceived severe stress (32.61±2.54) compared to no/minimal (24.36 ±0.86) stressed women. The BMI of the women with mild (26.71 ± 0.96) or moderate (28.03 ± 1.35) perception of stress were not found significantly different from the other groups. Fig-2 A and B show the behavioral changes related to appetite effected by stress. Data analyzed with one way ANOVA revealed a significant difference of the VAS values of hunger (F3,19=13.70, p<0.01) and desire to eat (F3,19=8.812, p<0.01) values. Post-hoc analysis revealed that moderately and severely stressed women had significantly higher values of hunger and desire to eat compared to the women with no/minimal and mild stress perception. Severely stressed women also showed significantly higher VAS for desire to eat compared to mildly stressed women. A significant effect of stress perception was noted on appetite (F3,19=7.60, p<0.01). Posthoc analysis showed the appetite of moderately and severely stressed women was significantly higher compared to no/minimal stress perception group. The chemical variables plasma leptin and cortisol are shown in Fig-3. One way ANOVA showed a significant effect of stress perception of circulating levels of leptin (F3,19=30.47, p<0.01) and cortisol (F3,19=27.50, p<0.01). Post-hoc analysis showed that plasma leptin levels were significantly greater in moderately and severely stressed women compared to the women with no/minimal (p<0.001) and mild stress perception (p<0.01). Severely stressed women also showed significantly higher concentrations of circulating leptin compared to women with moderate stress (p<0.01). The plasma cortisol levels were significantly higher in women with mild and moderate stress perception (p<0.01) compared to no/minimal stress group. Strikingly the women who reported severe stress perception had significantly lesser concentration of circulating cortisol compared to mild (p<0.05) and moderate (p<0.001) stress perception group. Fig-4 depicts the graphical presentation of plasma serotonin concentrations in the women with various levels of stress perception. One-way ANOVA showed a significant effect of stress perception on plasma serotonin concentrations (F3,19=9.624, p<0.01). Post-hoc analysis of the data revealed significantly lower plasma serotonin levels in the women with moderate and severe stress perception compared to no/minimal stress perception groups. The sequence of fragment of leptin gene including protein coding regions exon (Ex) 2 and 3 are given in table 1 and 2 respectively. A single nucleotide polymorphism was found in 5’ flanking region of exon 2 of leptin gene compared to the reference gene fragment retrieved from gene bank with no minimal perception of stress within the same job environment. The guanosine at position 15719 in the reference fragment was replaced by cytidine in our samples. No variation was found in the DNA sequences of EXON 2 of women with mild perception of stress.|
|Six hundred and fifty to six hundred and seventy base pairs of linear DNA fragment of leptin gene including exon 3 were sequenced. No variation was found in the any of the samples belonging to various groups of stress perception. One way ANOVA was applied to analyze the consequences of presence of a SNP G15719C at the 5’ flanking region of exon 2 on parameters of obesity and stress. It exerts a significant effect on levels of stress perception (F1,19= 27.0, p<0.01), change of appetite during stress perception (F1,19= 12.69,p<0.01), VAS for hunger (F1,19= 7.95, p<0.05) and VAS for desire to eat (F1,19= 8.21, p<0.05). The serum leptin levels were also significantly affected by the presence of G15719C variant (F1,19= 5.67, p<0.05), plasma cortisol (F1,19= 5.34,p<0.05) and serotonin (F1,19= 11.76, p<0.05). The body weight (F1,19= 5.164, p<0.05) also significantly affected by the presence of G15719C variant.|
|The adipocyte hormone leptin has been reported to have an antianxiety/depression activity . The antianxiety effects of leptin are functionally similar to the antidepressant targeting serotonergic system  suggesting the interaction of peripheral leptin expression with central 5-HT actions. The coexistence of obesity and depression indicates that there is some common factor that regulates the mood swings, behavioral changes and body weight gain. Leptin plays a role in cognitive processes, learning and memory by activation of receptors in limbic areas of brain including hippocampus . Human obesity is associated with the higher circulating leptin  resulting in leptin resistance. In the present study we found that severely stressed women are obese consistent with the previous findings . An impaired CNS action of leptin is reported in depression with obesity . The appetite along with the VAS for hunger and desire to eat was also increased in the women with perception of severe stress.|
|Stress perception is marked with HPA axis activation and high circulating cortisol levels . The increase in corticotrophic hormone (CRH) enhances appetite along with the release of adrenocorticotropic hormone (ACTH). The glucocorticoid induced hyperphagia and increase in body weight has also been reported . Our results confirm that the women who perceive moderate stress exhibit higher levels of cortisol (Fig-3) and an increased VAS for hunger and desire to eat. Cortisol exerts stimulatory effect on leptin synthesis and secretion while leptin in turn inhibits the secretion of cortisol via feedback mechanism acting centrally . Our results showed the reciprocal relation of both circulating cortisol and leptin in the women who perceive severe stress suggesting the effect of higher leptin on cortisol secretion and decrease in their stress coping capabilities.|
|The exposure to acute stress is associated with high central 5-HT levels. The levels are decreased in repeated stress due to adaptation response . The peripheral 5-HT is synthesized in GIT chromaffin cells, released in blood and stored in platelets. Peripheral 5-HT is implicated in aggregation of platelets. Reduced platelet 5-HT concentrations have been suggested as probable biomarkers in insomnia and alcoholism . The reduction in plasma serotonin levels with the severity of stress perception in our results suggests the possible marker of stress perception. The circulating 5-HT reduces leptin secretion via its receptors on the adipocytes  indicating a role of peripheral 5-HT in leptin expression/secretion. A reduction in circulating 5-HT associated with larger leptin levels and severity of stress perception might be the cause of increased appetite exhibited by the women who perceive higher level of stress and increased weight gain.|
|Present study provides the first evidence of the impact of a single nucleotide polymorphism G15719C on the 5’ flanking region of the second exon of leptin gene, on the smaller perception of stress in working women population of Pakistan. Leptin gene deficiency (ob/ob) in mice is associated with morbid obesity . Although many have reported genetic contribution in rodent obesity [46, 47, 48] mutations in leptin gene are rare in obese humans . Mammes et al. [50, 51] demonstrated that the G-2548A variant in the promoter of LEP was associated with decrease in BMI of overweight women. Apart from the link with obesity leptin gene polymorphisms are also linked to the various other disease phenotypes . The rarity of LEP mutations led the researchers to find out the variations in 5’ flanking and promoter regions of leptin gene [53, 54]. Several silent mutations have been described . Many variations have been identified highly polymorphic 5’ flanking region of LEP of obese women population in USA, but the association was is clear due to low variability of the coding regions . Our results also did not differ in the coding regions (exon 2 and 3) compared to the reference sequence retrieved from the gene bank. The interesting finding in our result was the variation of a single nucleotide G15719C in 5’ flanking region of exon 2 of the women who perceive no/minimal stress in the same job environment as the other groups. Although the Leptin molecule is translated from three exons, exon-1 codes for signal sequence required for the entrance of leptin into the endoplasmic reticulum. The exon 2 and 3 contribute to the amino acids in mature leptin molecules. It might be suggested that the G15719C variant is associated for the lesser perception of stress (p<0.01). This group also exhibited lesser VAS for hunger and desire to eat (p<0.01) as well as had lesser alleviations of the circulating levels of leptin, cortisol and serotonin (p<0.05). The BMI within the normal limits along with other findings suggest the well regulated metabolic loops required for coping capabilities and behavioral changes during stress. Smaller sample size and cross sectional study design were the limitations for the establishment of G15719C variant on LEP as a causal effect of less perception of stress.|
|Present study therefore hypothesize that the consistent finding of a G15719C variant on the 5’ flanking region of exon 2 of LEP in the group of women who perceive no/minimal stress, is involved in stress induced increases in BMI and other physiological reactions to stress.|
Conflict of Interest
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