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CYP gene expressions in obesity-associated metabolic syndrome
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CYP gene expressions in obesity-associated metabolic syndrome Suzan Tabur a,∗, Serdar Oztuzcu b, Elif Oguz c, Seniz Demiryürek d, Hasan Dagli b, Belgin Alasehirli e, Mesut Ozkaya a, Abdullah T. Demiryürek e a
Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey b Department of Medical Biology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey c Department of Medical Pharmacology, Faculty of Medicine, Harran University, Sanliurfa, Turkey d Department of Physiology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey e Department of Medical Pharmacology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey Received 25 August 2015 ; received in revised form 28 January 2016; accepted 2 March 2016
KEYWORDS Cytochrome P450; Gene expression; Metabolic syndrome; Obesity; Polymerase chain reaction
Summary Purpose: The contribution of cytochrome P450 (CYP) gene expressions in metabolic syndrome (MetS) has not been elucidated, and was the aim of this study. Methods: A total of 51 MetS patients and 41 healthy controls with similar age and sex were included to this study. mRNA from blood samples was extracted, and realtime polymerase chain reaction was performed for gene expressions using a dynamic array system. Results: We observed marked suppressions in CYP2A6 (p = 0.0123), CYP4F2 (p = 0.0005), CYP3A5 (p = 0.0003), and CYP17A1 (p < 0.0001) gene expressions in MetS patients. Conclusions: This is the first study to provide evidence that depressed expressions of CYP2A6, CYP4F2, CYP3A5, and CYP17A1 genes may play a role in MetS. © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author at: Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, University of Gaziantep, 27310 Gaziantep, Turkey. Tel.: +90 342 3606060/76156; fax: +90 342 3601617. E-mail address: [email protected] (S. Tabur). http://dx.doi.org/10.1016/j.orcp.2016.03.001 1871-403X/© 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tabur S, et al. CYP gene expressions in obesity-associated metabolic syndrome. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.03.001
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Introduction The metabolic syndrome (MetS) is a constellation of cardiovascular and metabolic risk factors including abdominal obesity, hyperglycemia, dyslipidemia, and high blood pressure [1]. MetS is also a public health problem predicting higher rates of type 2 diabetes and cardiovascular disease. The incidence of the MetS has been increasing worldwide in parallel with an increase in overweight and obesity [2]. The pathogenesis of the MetS is not fully understood, but insulin resistance and central obesity are thought to play the key role [1]. The MetS has a significant genetic component. Genetic studies have provided so far only limited evidence for a common genetic background of the MetS [3]. Epigenetic changes such as DNA methylation and histone modification lead to changes in gene expression and cellular phenotypes, which are stable through cell divisions without altering the DNA sequence [4]. Small non-coding RNAs, microRNAs, regulating gene expression and translation of mRNAs to proteins, represent additional factors likely to play an important role in the pathogenesis of the MetS [3]. Cytochromes P450 (CYP) is gene superfamily consisting of a large and diverse group of heme proteins involved in the metabolism of numerous xenobiotics and endogenous substrates. In human, 57 functional CYP genes have been identified [5]. The majority of human P450 families are constituted of enzymes that act primarily on important classes of endogenous compounds, notably steroids, bile acids, vitamins, and fatty acids. Using a systems biology approach, Sookoian and Pirola [6], identified 50 genes that were significantly related to the risk of metabolic syndrome components. Two of these genes were reported to be CYP11A1 and CYP11B1. It is known that hormonal factors can influence the expression of CYP genes [7]. However, contribution of the CYP gene expressions to the MetS is unknown. Therefore, the aim of this study was to investigate the possible role of CYP gene expressions in MetS.
Materials and methods Study populations A total of 92 unrelated Turkish subjects, 51 with MetS and 41 non-MetS controls evaluated at Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey were recruited into this study. The study was approved by the local Ethics Committee. Written informed consent prior to participation in the study was obtained from patients
and healthy volunteers according to the Declaration of Helsinki. The diagnosis of the MetS was done by clinicians according to the National Cholesterol Education Program Adult Treatment Panel III criteria thatis an acceptable and well-recognized criterion for MetS diagnosis [1,8]. A MetS diagnosis was made when a subject fulfilled three of the following five criteria: waist circumference ≥102 cm in men and ≥88 cm in women, triglyceride (TG) ≥150 mg/dl or treatment of dyslipidemia, high density lipoprotein (HDL) cholesterol <40 mg/dl in men and <50 mg/dl in women or treatment of dyslipidemia, systolic/diastolic blood pressure ≥130/85 mm Hg or antihypertensive treatment, and fasting blood glucose ≥100 mg/dl or treatment of type 2 diabetes. Patients who had a history of percutaneous coronary intervention within 6 months or coronary artery bypass surgery within 1 year were excluded. Other exclusion criteria included patients who had heart failure, cardiomyopathy, or valvular heart diseases. All sex and age-matched controls were healthy and had no symptoms of MetS. All the control subjects were from the same geographical area with a similar socioeconomic and ethnic background. The socioeconomic background was identified by the information obtained from the patient and the controls through a health questionnaire. All the patients and controls were in the middle-income status. Exclusion criteria for selecting the control subjects were presence of coronary artery disease, peripheral occlusive arterial disease, coagulopathy, vasculitis, autoimmune disease, severe kidney and hepatic diseases, cancer, and pregnancy.
cDNA synthesis and gene expression Venous blood samples were drawn from each subject after ≥8 h or overnight fasting. To confirm the gene expression of cellular signaling structures in blood, mRNA was isolated from leukocytes by using -mercaptoethanol, and stored at −80 ◦ C until use. cDNA was produced with the Qiagen miScript Reverse Transcription Kit according to manufacturer’s protocol. PCR was performed by BioMark HD system (Fluidigm, South San Francisco, CA, USA) with CYP1A1, CYP1A2, CYP2A6, CYP3A4, CYP3A5, CYP3A7, CYP11A1, CYP17A1, CYP19A1, CYP21A2, CYP24A1, CYP1B1, CYP11B2, CYP2B6, CYP27B1, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP4F2, CYP2J2 primers, and beta-actin (ACTB, housekeeping gene). We screened 23 CYP genes for expression study. Data were analyzed
Please cite this article in press as: Tabur S, et al. CYP gene expressions in obesity-associated metabolic syndrome. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.03.001
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CYP gene expressions in MetS Table 1
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Baseline demographic and clinical characteristics of MetS patients and controls. Patients (n = 51)
Controls (n = 41)
44.21 ± 12.26
45.96 ± 11.92
0.4926
10 (19.6) 41 (80.4) 7 (13.7) 41.51 ± 8.17 114.21 ± 13.65 131.52 ± 15.77 86.74 ± 13.42 12 (23.5) 121.52 ± 46.32 6.98 ± 1.69 199.55 ± 40.78 130.39 ± 32.56 39.77 ± 8.21 172.73 ± 66.82 1.92 ± 4.85 23.31 ± 16.78 5.68 ± 3.92
7 (17.1) 34 (82.9) 4 (9.8) 22.31 ± 3.75 85.32 ± 7.59 116.61 ± 8.95 76.83 ± 7.23 — 83.51 ± 9.87 5.17 ± 0.48 150.43 ± 18.77 98.47 ± 10.87 44.92 ± 7.48 123.87 ± 25.72 0.28 ± 0.17 22.49 ± 9.36 2.07 ± 0.39
0.7938
a
Age (years) Gender Male (n, %) Female (n, %) Alcohol intake BMI (kg/m2 )a Waist circumference (cm)a Systolic BP (mm Hg)a Diastolic BP (mm Hg)a Diabetes mellitus (n, %) Fasting plasma glucose (mg/dl)a HbA1c (%)a Total cholesterol (mg/dl)a Low density lipoprotein cholesterol (mg/dl)a High density lipoprotein cholesterol (mg/dl)a Triglyceride (mg/dl)a High-sensitive C-reactive protein Insulin (pmol/l)a HOMA-IRa
p value
0.7489 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0025 <0.0001 <0.0001 0.7804 <0.0001
a Data are mean ± S.D. BMI, body mass index. BP, blood pressure. Fasting glucose and insulin plasma levels are used to calculate homeostasis model assessment of insulin resistance (HOMA-IR).
using the 2−Ct method, according to the formula: Ct = CtCYP − CtACTB , where Ct = threshold cycle.
Statistical analyses Results are expressed as the mean ± S.D. or percentage. Gene expression data are presented as mean ± SEM. For comparisons of the differences between mean values of two groups, the unpaired Student’s t test was used. Fisher’s exact test was used for calculation of the significance of differences in categorical data. The Mann—Whitney U test was performed to compare gene expression data. Statistical analysis was performed using GraphPad Instat version 3.05 (GraphPad Software Inc., San Diego, CA, USA). All probability values were based on two-tailed tests. p values less than 0.05 were considered to be statistically significant.
Results Demographic and clinical characteristics of the study population are presented in Table 1. The prevalences of cardiovascular risk factors, including hypertension, fasting glucose levels, lipid profiles, body mass index (BMI), and waist circumference, are shown in Table 1 for the control and MetS subjects. Compared with the control group, the average age, gender, alcohol intake and insulin levels in the MetS group were similar. BMI, waist
circumference, blood pressure, fasting glucose, HbA1c, total cholesterol, LDL cholesterol, TG, highsensitive C-reactive protein levels, and HOMA-IR were all greater among MetS subjects, and HDL cholesterol levels were lower among MetS subjects. All of the patients were obese with high BMI (41.51 ± 8.17 kg/m2 ). Of the MetS patients, 23.5% was diagnosed with type 2 diabetes (Table 1). Gene expression analysis showed that CYP2A6, CYP4F2, CYP3A5, and CYP17A1 mRNA contents in leukocytes were markedly depressed in MetS patients when compared to the control groups (Fig. 1, p < 0.05). No marked changes in gene expressions were noted in other 19 CYP genes studied (p > 0.05).
Discussion The results of the present study showed that CYP2A6, CYP4F2, CYP3A5, and CYP17A1 gene expressions were markedly suppressed in MetS. To the best of our knowledge, this is the first study to evaluate the CYP gene expressions in MetS. The CYP2A6 is an important hepatic enzyme that metabolizes approximately 3% of therapeutic drugs [9]. So, it is considered that CYP2A6 is a relatively minor drug-metabolizing enzyme in the liver, with narrow substrate specificity. The regulation mechanism of CYP2A6 expression is not fully understood, but available data suggest that several nuclear receptors including pregnane X
Please cite this article in press as: Tabur S, et al. CYP gene expressions in obesity-associated metabolic syndrome. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.03.001
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Figure 1 Comparison of the peripheral blood mRNA CYP2A6, CYP4F2, CYP3A5, and CYP17A1 expressions in healthy controls (n = 41, open bars) and in patients with metabolic syndrome (n = 51, solid bars). Values are given as mean ± SEM, *p = 0.0123, p = 0.0005, p = 0.0003, and p < 0.0001 values were obtained for CYP2A6, CYP4F2, CYP3A5, and CYP17A1, respectively.
receptor and glucocorticoid receptor are involved in its regulation. This enzyme also participates in the biotransformation of several endogenous compounds such as retinoid acids and steroids [9]. Functional significance of low CYP2A6 expression in MetS is not known, but it may impair the biotransformation of steroids. The mechanisms underlying depressed CYP2A6 gene in MetS are currently unknown, but it may be related to variability in the expression of nuclear transcription factors. CYP2A6 gene mRNA levels have been shown to significantly correlate with mRNA levels of the nuclear constitutive androstane receptor and hepatic nuclear factor 4-␣ [10]. CYP4F2 catalyzes the synthesis of 20hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. 20-HETE has both vasoconstrictive and natriuretic effects [11,12]. It has been shown that patients with MetS have significantly elevated plasma and urinary 20-HETE levels [13]. A report by Laffer et al. [14] showed a negative correlation between urinary 20-HETE levels and circulating insulin in essential hypertension with obesity. It has been also reported that there is a negative correlation between urinary 20-HETE and BMI in obese men and women with essential hypertension [14]. Moreover, cyclooxygenase-2 dependent metabolism of 20-HETE increases adiposity and adipocyte hypertrophy in mesenchymal stem cell-derived adipocytes [15]. In the present study, we have observed depressed gene expression of CYP4F2 in MetS patients. Taken together, these data may suggest that decreased CYP4F2mediated 20-HETE formation or cyclooxygenase-2 dependent 20-HETE metabolism play a role in MetS development.
Since CYP3A5 is 83% homogenous to CYP3A4, it is believed that the substrate specificity of CYP3A5 is similar to that of CYP3A4; but some differences in catalytic properties have been found [16,17]. CYP3A4 and CYP3A5 are involved in the metabolism of more than half of all currently used drugs [18]. CYP3A5 is involved in the synthetic modification of endogenous steroid hormones including the metabolism of cortisol to 6--hydroxycortisol [18]. The CYP3A5 enzyme is also expressed in the kidney and has been implicated in renal sodium reabsorption and blood pressure regulation [19]. Decreased CYP3A5 expression seen in this study may contribute altered drug metabolism or hypertension in MetS patients. CYP17A1, which has both 17␣-hydroxylase and 17,20-lyase activities, determines the final products in steroid hormone biosynthesis and plays an important role in cell homeostasis [20]. The 17-hydroxylase activity of CYP17A1 is required for the production of the glucocorticoid cortisol, whereas the 17,20-lyase activity leads to androgens (and in turn estrogens) [21]. The equilibrium between sex hormones and glucocorticoids may be a critical element in the timing of the manifestation of metabolic syndrome-related pathologies [22]. Underexpression of seen CYP17A1 gene in the present study may imply that depressed CYP17A1 can contribute to the development of MetS. The major limitation of this study is the small number of individuals used for expression profiling. Therefore, large population studies are necessary to confirm these results. In conclusion, our data suggest that depressed CYP2A6, CYP4F2, CYP3A5, and CYP17A1 gene expressions may play a role MetS development. This study provides novel insights into mechanisms of MetS. Our findings show that CYP gene may have a role in pathogenesis of MetS, but further studies are required to validate these results.
Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this article. No funding was received for this study.
Ethical statement We have read and have abided by the statement of ethical standards for manuscripts submitted to the Obesity Research & Clinical Practice.
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References [1] Grundy SM, Brewer Jr HB, Cleeman JI, Smith Jr SC, Lenfant C. American Heart Association; National Heart, Lung, and Blood Institute. Definition of metabolic syndrome: Report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Circulation 2004;109:433—8. [2] Grundy SM. Metabolic syndrome pandemic. Arterioscler Thromb Vasc Biol 2008;28:629—36. [3] Stanˇ cáková A, Laakso M. Genetics of metabolic syndrome. Rev Endocr Metab Disord 2014;15:243—52. [4] Wolffe AP, Guschin D. Review: chromatin structural features and targets that regulate transcription. J Struct Biol 2000;129:102—22. [5] Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM, Nebert DW. Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 2004;14:1—18. [6] Sookoian S, Pirola CJ. Metabolic syndrome: from the genetics to the pathophysiology. Curr Hypertens Rep 2011;13:149—57. [7] Monostory K, Dvorak Z. Steroid regulation of drugmetabolizing cytochromes P450. Curr Drug Metab 2011;12:154—72. [8] Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001;285:2486—97. [9] Di YM, Chow VD, Yang LP, Zhou SF. Structure, function, regulation and polymorphism of human cytochrome P450 2A6. Curr Drug Metab 2009;10:754—80. [10] Wortham M, Czerwinski M, He L, Parkinson A, Wan YJ. Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines interindividual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver. Drug Metab Dispos 2007;35:1700—10.
[11] Wu CC, Gupta T, Garcia V, Ding Y, Schwartzman ML. 20-HETE and blood pressure regulation: clinical implications. Cardiol Rev 2014;22:1—12. [12] Alvarellos ML, Sangkuhl K, Daneshjou R, Whirl-Carrillo M, Altman RB, Klein TE. PharmGKB summary: very important pharmacogene information for CYP4F2. Pharmacogenet Genomics 2015;25:41—7. [13] Tsai IJ, Croft KD, Mori TA, Falck JR, Beilin LJ, Puddey IB, et al. 20-HETE and F2-isoprostanes in the metabolic syndrome: the effect of weight reduction. Free Radic Biol Med 2009;46:263—70. [14] Laffer CL, Laniado-Schwartzman M, Nasjletti A, Elijovich F. 20-HETE and circulating insulin in essential hypertension with obesity. Hypertension 2004;43:388—92. [15] Kim DH, Puri N, Sodhi K, Falck JR, Abraham NG, Shapiro J, et al. Cyclooxygenase-2 dependent metabolism of 20HETE increases adiposity and adipocyte enlargement in mesenchymal stem cell-derived adipocytes. J Lipid Res 2013;54:786—93. [16] Daly AK. Significance of the minor cytochrome P450 3A isoforms. Clin Pharmacokinet 2006;45:13—31. [17] de Wildt SN, Kearns GL, Leeder JS, van den Anker JN. Cytochrome P450 3A: ontogeny and drug disposition. Clin Pharmacokinet 1999;37:485—505. [18] MacPhee IA. Pharmacogenetic biomarkers: cytochrome P450 3A5. Clin Chim Acta 2012;413:1312—7. [19] Kreutz R, Zuurman M, Kain S, Bolbrinker J, de Jong PE, Navis G. The role of the cytochrome P450 3A5 enzyme for blood pressure regulation in the general Caucasian population. Pharmacogenet Genomics 2005;15:831—7. [20] Gilep AA, Sushko TA, Usanov SA. At the crossroads of steroid hormone biosynthesis: the role, substrate specificity and evolutionary development of CYP17. Biochim Biophys Acta 2011;1814:200—9. [21] Yoshimoto FK, Auchus RJ. The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1). J Steroid Biochem Mol Biol 2015;151:52—65. [22] Alemany M. Do the interactions between glucocorticoids and sex hormones regulate the development of the metabolic syndrome? Front Endocrinol (Lausanne) 2012;3:27.
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Obesity Research & Clinical Practice (2016) xxx, xxx—xxx
SHORT REPORT
CYP gene expressions in obesity-associated metabolic syndrome Suzan Tabur a,∗, Serdar Oztuzcu b, Elif Oguz c, Seniz Demiryürek d, Hasan Dagli b, Belgin Alasehirli e, Mesut Ozkaya a, Abdullah T. Demiryürek e a
Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey b Department of Medical Biology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey c Department of Medical Pharmacology, Faculty of Medicine, Harran University, Sanliurfa, Turkey d Department of Physiology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey e Department of Medical Pharmacology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey Received 25 August 2015 ; received in revised form 28 January 2016; accepted 2 March 2016
KEYWORDS Cytochrome P450; Gene expression; Metabolic syndrome; Obesity; Polymerase chain reaction
Summary Purpose: The contribution of cytochrome P450 (CYP) gene expressions in metabolic syndrome (MetS) has not been elucidated, and was the aim of this study. Methods: A total of 51 MetS patients and 41 healthy controls with similar age and sex were included to this study. mRNA from blood samples was extracted, and realtime polymerase chain reaction was performed for gene expressions using a dynamic array system. Results: We observed marked suppressions in CYP2A6 (p = 0.0123), CYP4F2 (p = 0.0005), CYP3A5 (p = 0.0003), and CYP17A1 (p < 0.0001) gene expressions in MetS patients. Conclusions: This is the first study to provide evidence that depressed expressions of CYP2A6, CYP4F2, CYP3A5, and CYP17A1 genes may play a role in MetS. © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author at: Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, University of Gaziantep, 27310 Gaziantep, Turkey. Tel.: +90 342 3606060/76156; fax: +90 342 3601617. E-mail address: [email protected] (S. Tabur). http://dx.doi.org/10.1016/j.orcp.2016.03.001 1871-403X/© 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tabur S, et al. CYP gene expressions in obesity-associated metabolic syndrome. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.03.001
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S. Tabur et al.
Introduction The metabolic syndrome (MetS) is a constellation of cardiovascular and metabolic risk factors including abdominal obesity, hyperglycemia, dyslipidemia, and high blood pressure [1]. MetS is also a public health problem predicting higher rates of type 2 diabetes and cardiovascular disease. The incidence of the MetS has been increasing worldwide in parallel with an increase in overweight and obesity [2]. The pathogenesis of the MetS is not fully understood, but insulin resistance and central obesity are thought to play the key role [1]. The MetS has a significant genetic component. Genetic studies have provided so far only limited evidence for a common genetic background of the MetS [3]. Epigenetic changes such as DNA methylation and histone modification lead to changes in gene expression and cellular phenotypes, which are stable through cell divisions without altering the DNA sequence [4]. Small non-coding RNAs, microRNAs, regulating gene expression and translation of mRNAs to proteins, represent additional factors likely to play an important role in the pathogenesis of the MetS [3]. Cytochromes P450 (CYP) is gene superfamily consisting of a large and diverse group of heme proteins involved in the metabolism of numerous xenobiotics and endogenous substrates. In human, 57 functional CYP genes have been identified [5]. The majority of human P450 families are constituted of enzymes that act primarily on important classes of endogenous compounds, notably steroids, bile acids, vitamins, and fatty acids. Using a systems biology approach, Sookoian and Pirola [6], identified 50 genes that were significantly related to the risk of metabolic syndrome components. Two of these genes were reported to be CYP11A1 and CYP11B1. It is known that hormonal factors can influence the expression of CYP genes [7]. However, contribution of the CYP gene expressions to the MetS is unknown. Therefore, the aim of this study was to investigate the possible role of CYP gene expressions in MetS.
Materials and methods Study populations A total of 92 unrelated Turkish subjects, 51 with MetS and 41 non-MetS controls evaluated at Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey were recruited into this study. The study was approved by the local Ethics Committee. Written informed consent prior to participation in the study was obtained from patients
and healthy volunteers according to the Declaration of Helsinki. The diagnosis of the MetS was done by clinicians according to the National Cholesterol Education Program Adult Treatment Panel III criteria thatis an acceptable and well-recognized criterion for MetS diagnosis [1,8]. A MetS diagnosis was made when a subject fulfilled three of the following five criteria: waist circumference ≥102 cm in men and ≥88 cm in women, triglyceride (TG) ≥150 mg/dl or treatment of dyslipidemia, high density lipoprotein (HDL) cholesterol <40 mg/dl in men and <50 mg/dl in women or treatment of dyslipidemia, systolic/diastolic blood pressure ≥130/85 mm Hg or antihypertensive treatment, and fasting blood glucose ≥100 mg/dl or treatment of type 2 diabetes. Patients who had a history of percutaneous coronary intervention within 6 months or coronary artery bypass surgery within 1 year were excluded. Other exclusion criteria included patients who had heart failure, cardiomyopathy, or valvular heart diseases. All sex and age-matched controls were healthy and had no symptoms of MetS. All the control subjects were from the same geographical area with a similar socioeconomic and ethnic background. The socioeconomic background was identified by the information obtained from the patient and the controls through a health questionnaire. All the patients and controls were in the middle-income status. Exclusion criteria for selecting the control subjects were presence of coronary artery disease, peripheral occlusive arterial disease, coagulopathy, vasculitis, autoimmune disease, severe kidney and hepatic diseases, cancer, and pregnancy.
cDNA synthesis and gene expression Venous blood samples were drawn from each subject after ≥8 h or overnight fasting. To confirm the gene expression of cellular signaling structures in blood, mRNA was isolated from leukocytes by using -mercaptoethanol, and stored at −80 ◦ C until use. cDNA was produced with the Qiagen miScript Reverse Transcription Kit according to manufacturer’s protocol. PCR was performed by BioMark HD system (Fluidigm, South San Francisco, CA, USA) with CYP1A1, CYP1A2, CYP2A6, CYP3A4, CYP3A5, CYP3A7, CYP11A1, CYP17A1, CYP19A1, CYP21A2, CYP24A1, CYP1B1, CYP11B2, CYP2B6, CYP27B1, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP4F2, CYP2J2 primers, and beta-actin (ACTB, housekeeping gene). We screened 23 CYP genes for expression study. Data were analyzed
Please cite this article in press as: Tabur S, et al. CYP gene expressions in obesity-associated metabolic syndrome. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.03.001
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CYP gene expressions in MetS Table 1
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Baseline demographic and clinical characteristics of MetS patients and controls. Patients (n = 51)
Controls (n = 41)
44.21 ± 12.26
45.96 ± 11.92
0.4926
10 (19.6) 41 (80.4) 7 (13.7) 41.51 ± 8.17 114.21 ± 13.65 131.52 ± 15.77 86.74 ± 13.42 12 (23.5) 121.52 ± 46.32 6.98 ± 1.69 199.55 ± 40.78 130.39 ± 32.56 39.77 ± 8.21 172.73 ± 66.82 1.92 ± 4.85 23.31 ± 16.78 5.68 ± 3.92
7 (17.1) 34 (82.9) 4 (9.8) 22.31 ± 3.75 85.32 ± 7.59 116.61 ± 8.95 76.83 ± 7.23 — 83.51 ± 9.87 5.17 ± 0.48 150.43 ± 18.77 98.47 ± 10.87 44.92 ± 7.48 123.87 ± 25.72 0.28 ± 0.17 22.49 ± 9.36 2.07 ± 0.39
0.7938
a
Age (years) Gender Male (n, %) Female (n, %) Alcohol intake BMI (kg/m2 )a Waist circumference (cm)a Systolic BP (mm Hg)a Diastolic BP (mm Hg)a Diabetes mellitus (n, %) Fasting plasma glucose (mg/dl)a HbA1c (%)a Total cholesterol (mg/dl)a Low density lipoprotein cholesterol (mg/dl)a High density lipoprotein cholesterol (mg/dl)a Triglyceride (mg/dl)a High-sensitive C-reactive protein Insulin (pmol/l)a HOMA-IRa
p value
0.7489 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0025 <0.0001 <0.0001 0.7804 <0.0001
a Data are mean ± S.D. BMI, body mass index. BP, blood pressure. Fasting glucose and insulin plasma levels are used to calculate homeostasis model assessment of insulin resistance (HOMA-IR).
using the 2−Ct method, according to the formula: Ct = CtCYP − CtACTB , where Ct = threshold cycle.
Statistical analyses Results are expressed as the mean ± S.D. or percentage. Gene expression data are presented as mean ± SEM. For comparisons of the differences between mean values of two groups, the unpaired Student’s t test was used. Fisher’s exact test was used for calculation of the significance of differences in categorical data. The Mann—Whitney U test was performed to compare gene expression data. Statistical analysis was performed using GraphPad Instat version 3.05 (GraphPad Software Inc., San Diego, CA, USA). All probability values were based on two-tailed tests. p values less than 0.05 were considered to be statistically significant.
Results Demographic and clinical characteristics of the study population are presented in Table 1. The prevalences of cardiovascular risk factors, including hypertension, fasting glucose levels, lipid profiles, body mass index (BMI), and waist circumference, are shown in Table 1 for the control and MetS subjects. Compared with the control group, the average age, gender, alcohol intake and insulin levels in the MetS group were similar. BMI, waist
circumference, blood pressure, fasting glucose, HbA1c, total cholesterol, LDL cholesterol, TG, highsensitive C-reactive protein levels, and HOMA-IR were all greater among MetS subjects, and HDL cholesterol levels were lower among MetS subjects. All of the patients were obese with high BMI (41.51 ± 8.17 kg/m2 ). Of the MetS patients, 23.5% was diagnosed with type 2 diabetes (Table 1). Gene expression analysis showed that CYP2A6, CYP4F2, CYP3A5, and CYP17A1 mRNA contents in leukocytes were markedly depressed in MetS patients when compared to the control groups (Fig. 1, p < 0.05). No marked changes in gene expressions were noted in other 19 CYP genes studied (p > 0.05).
Discussion The results of the present study showed that CYP2A6, CYP4F2, CYP3A5, and CYP17A1 gene expressions were markedly suppressed in MetS. To the best of our knowledge, this is the first study to evaluate the CYP gene expressions in MetS. The CYP2A6 is an important hepatic enzyme that metabolizes approximately 3% of therapeutic drugs [9]. So, it is considered that CYP2A6 is a relatively minor drug-metabolizing enzyme in the liver, with narrow substrate specificity. The regulation mechanism of CYP2A6 expression is not fully understood, but available data suggest that several nuclear receptors including pregnane X
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Figure 1 Comparison of the peripheral blood mRNA CYP2A6, CYP4F2, CYP3A5, and CYP17A1 expressions in healthy controls (n = 41, open bars) and in patients with metabolic syndrome (n = 51, solid bars). Values are given as mean ± SEM, *p = 0.0123, p = 0.0005, p = 0.0003, and p < 0.0001 values were obtained for CYP2A6, CYP4F2, CYP3A5, and CYP17A1, respectively.
receptor and glucocorticoid receptor are involved in its regulation. This enzyme also participates in the biotransformation of several endogenous compounds such as retinoid acids and steroids [9]. Functional significance of low CYP2A6 expression in MetS is not known, but it may impair the biotransformation of steroids. The mechanisms underlying depressed CYP2A6 gene in MetS are currently unknown, but it may be related to variability in the expression of nuclear transcription factors. CYP2A6 gene mRNA levels have been shown to significantly correlate with mRNA levels of the nuclear constitutive androstane receptor and hepatic nuclear factor 4-␣ [10]. CYP4F2 catalyzes the synthesis of 20hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. 20-HETE has both vasoconstrictive and natriuretic effects [11,12]. It has been shown that patients with MetS have significantly elevated plasma and urinary 20-HETE levels [13]. A report by Laffer et al. [14] showed a negative correlation between urinary 20-HETE levels and circulating insulin in essential hypertension with obesity. It has been also reported that there is a negative correlation between urinary 20-HETE and BMI in obese men and women with essential hypertension [14]. Moreover, cyclooxygenase-2 dependent metabolism of 20-HETE increases adiposity and adipocyte hypertrophy in mesenchymal stem cell-derived adipocytes [15]. In the present study, we have observed depressed gene expression of CYP4F2 in MetS patients. Taken together, these data may suggest that decreased CYP4F2mediated 20-HETE formation or cyclooxygenase-2 dependent 20-HETE metabolism play a role in MetS development.
Since CYP3A5 is 83% homogenous to CYP3A4, it is believed that the substrate specificity of CYP3A5 is similar to that of CYP3A4; but some differences in catalytic properties have been found [16,17]. CYP3A4 and CYP3A5 are involved in the metabolism of more than half of all currently used drugs [18]. CYP3A5 is involved in the synthetic modification of endogenous steroid hormones including the metabolism of cortisol to 6--hydroxycortisol [18]. The CYP3A5 enzyme is also expressed in the kidney and has been implicated in renal sodium reabsorption and blood pressure regulation [19]. Decreased CYP3A5 expression seen in this study may contribute altered drug metabolism or hypertension in MetS patients. CYP17A1, which has both 17␣-hydroxylase and 17,20-lyase activities, determines the final products in steroid hormone biosynthesis and plays an important role in cell homeostasis [20]. The 17-hydroxylase activity of CYP17A1 is required for the production of the glucocorticoid cortisol, whereas the 17,20-lyase activity leads to androgens (and in turn estrogens) [21]. The equilibrium between sex hormones and glucocorticoids may be a critical element in the timing of the manifestation of metabolic syndrome-related pathologies [22]. Underexpression of seen CYP17A1 gene in the present study may imply that depressed CYP17A1 can contribute to the development of MetS. The major limitation of this study is the small number of individuals used for expression profiling. Therefore, large population studies are necessary to confirm these results. In conclusion, our data suggest that depressed CYP2A6, CYP4F2, CYP3A5, and CYP17A1 gene expressions may play a role MetS development. This study provides novel insights into mechanisms of MetS. Our findings show that CYP gene may have a role in pathogenesis of MetS, but further studies are required to validate these results.
Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this article. No funding was received for this study.
Ethical statement We have read and have abided by the statement of ethical standards for manuscripts submitted to the Obesity Research & Clinical Practice.
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Please cite this article in press as: Tabur S, et al. CYP gene expressions in obesity-associated metabolic syndrome. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.03.001