Abstract—Many individuals with schizophrenia also suffer from metabolic syndrome (MetS), which is a major risk factor for the development of cardiovascular disroders associated with a heavy burden of disease, as well as with premature death of patients. This study investigated the expression of 7 genes potentially important for the development of metabolic syndrome. QuantiGene Plex 2.0 technology was used to measure how 7 studied genes (DRD3, GHRL, FTO, LEPR, INSIG2, GSTP1, and ABCB1 (MDR1)) were expressed in leukocytes in 60 recently admitted patients with schizophrenia who had been on treatment with antipsychotic drugs. The preliminary results of our study show a change in the expression of the FTO gene in schizophrenic males with metabolic disorders, however, further studies are needed to determine the role of disturbances in the expression of this gene in the development of the metabolic syndrome in patients with schizophrenia.
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INTRODUCTION
The incidence of metabolic syndrome (MetS) in patients with schizophrenia receiving antipsychotic therapy ranges from 28 to 46% [1–3], and obesity, from 16.4 to 48.9% [4, 5]. MetS is associated with cardiovascular factors that increase the risk of premature death in patients with mental disorders. Hormonal regulators of metabolism and the genes encoding them can provide information on pathogenesis and are considered as potential biomarkers of susceptibility to the development of metabolic syndrome and lipid metabolism disorders [6, 7].
The dopamine receptor genes encoding proteins that ensure adequate transmission of neurotransmitter signals in the ascending dopaminergic pathways from the midbrain are studied quite well. These pathways connect fiber regions that play important roles in motivation, controlled thinking, and the reward system, the latter also playing a significant role in regulating satiety and hunger [8]. Antipsychotic drugs work in these areas, including by blocking D3 receptors, reducing dopamine levels and therefore reducing psychotic symptoms. However, they can also lead to cognitive exhaustion and metabolic changes [9]. The DRD3 gene encodes a protein that has different isoforms as a result of alternative splicing. The D2 receptor gene (DRD2) may also be involved in the pathogenesis of metabolic disorders in patients with schizophrenia. The functional polymorphism rs1799732 of the DRD2 gene was shown to be associated with drug-induced metabolic syndrome in women [10]. There is also evidence of the influence of the DRD3 gene on changes in the level of triglycerides, one of the components of the metabolic syndrome, in patients with mental disorders [11].
Ghrelin is a 28 amino acid peptide hormone that is produced primarily in the stomach. It is a hormonal regulator and has many biological effects, the most famous of which is increased hunger [12–14]. Interestingly, ghrelin is also involved in the circadian regulation of homeostatic nutrition [15], and the peripherally secreted hormone is involved in hedonic-motivated food cravings [16]. The GHRL gene encodes a protein that is cleaved to produce two peptides (ghrelin and obestatin). Ghrelin is secreted in two forms: less than 10% is acylated and binds to growth hormone secretagogue receptor 1a (GHSR1a). The residue is not acylated and reacts with a receptor that is yet unknown.
The protein encoded by the FTO gene (associated with fat mass and obesity) can be considered one of the most important enzymes that removes N6-methyladenosine residues from the RNA molecule [17, 18]. Although the FTO gene encodes an RNA demethylase, this is probably not its primary role in obesity. An animal model of FTO-deficient mice was studied: the mice exhibited postnatal growth retardation and decreased food intake with a decrease in adipose tissue [19, 20]. Consistent with this, overexpression of FTO induces an increase in adipose tissue [21].
Currently, there are various literature data indicating the association of polymorphisms of the leptin receptor gene (LEPR) with weight gain and drug-induced metabolic syndrome [13, 22, 23]. Additionally, LEPR has previously been associated with increased weight and BMI (body mass index) in patients with psychiatric disorders [24]. It has been suggested that the short isoform LEPRa is responsible for the transport of leptin across the BBB (blood-brain barrier), and the long isoform LEPRb is the receptor responsible for signal transduction in hypothalamic neurons [25]. Leptin is one of the main hormonal signaling agents from adipose tissue to other tissues, including the central nervous system [26]. It is mainly produced by mature adipocytes of white adipose tissue based on the expression of the LEP gene. Previously, we studied four polymorphisms of this gene and found that the genotypes and alleles of the rs3828942 variant have a significant association with the development of MS [23].
The insulin-inducing gene (INSIG2) encodes a 225 amino acid residue protein that plays an important role in inhibiting the synthesis of cholesterol and other lipids in adipocytes [27]. INSIG2 plays important regulatory roles in adipocyte differentiation, cholesterol homeostasis, lipogenesis, and glucose metabolism [28].
Glutathione-S-transferase P (GSTP) belongs to a superfamily of enzymes that catalyze the conjugation reaction of reduced glutathione with various xenobiotics, including carcinogens, antibiotics, neuroleptics, and products of the oxidative process [29, 30]. There is evidence that an imbalance between reactive oxygen species and antioxidants may be associated with insulin resistance in both mice and humans [31, 32]. It has been suggested that oxidative stress may also be associated with the pathogenesis of MetS [33]. Thus, functional polymorphisms of the GSTP1 gene can be considered as potential genetic markers of a risk factor for the development of obesity and antipsychotic-induced MS.
The P-glycoprotein (P-gp) transporter, encoded by the ATP-binding gene (ABCB1), also known as the multidrug resistance gene (MDR1), is a transmembrane protein expressed on the blood–brain barrier. It transports invading chemicals back from the brain into the bloodstream, and ABCB1 is therefore often considered a pharmacogenetic marker of response to therapy and the development of side effects [34–37].
Despite the fact that there are quite a lot of association studies between genetic polymorphisms and metabolic syndrome in the literature, there are no studies on the expression of genes associated with the development of obesity and metabolic syndrome in schizophrenia. In this regard, the purpose of our pilot study is to assess gene expression in peripheral blood leukocytes of schizophrenia patients with and without metabolic syndrome.
MATERIALS AND METHODS
The study included 60 patients of Russian nationality with schizophrenia, living in the Siberian region, who were hospitalized in the clinics of the Mental Health Research Institute, Tomsk National Research Medical Center. The main criteria for including patients in the study: verified diagnosis of schizophrenia (F20 according to ICD-10), age 18–60 years, informed consent of the patient, and absence of organic pathology and severe somatic disorders leading to organ failure. Antipsychotic and concomitant therapy received at the time of examination, as well as previous therapy during the previous six months, were assessed. The total antipsychotic load was brought to uniformity in terms of chlorpromazine equivalent (CPE). All patients were taking second-generation antipsychotics (risperidone, olanzapine, quetiapine). To make a diagnosis of MS, the criteria proposed by the International Diabetes Federation (IDF, 2005) were chosen.
Blood was taken from the subjects in the morning on an empty stomach. RNAgard Blood Tubes with RNAgard Blood reagent (Biomatrica, United States) were used for RNA isolation. Determination of the expression of 7 studied genes (DRD3, GHRL, FTO, LEPR, INSIG2, GSTP1, and ABCB1 (MDR)) and 3 housekeeping genes (GAPDH, POLR2A, and RPS200) was carried out using QuantiGene Plex 2.0 technology (Thermo Fisher Scientific, United States). The QuantiGene Plex assay enables multiplex measurement of gene expression by combining branched DNA signal amplification with xMAP technology to detect multiple analytes in a small amount of sample using fluorescent magnetic beads. The resulting fluorescence signal depends on the individual color of the magnetic microspheres, which we detected on multiplex analyzers Magpix and Luminex 200 (Luminex, United States) (Center for Common Use “Medical Genomics,” Tomsk National Research Center). The signal is recorded as median fluorescence intensity (MFI) and is proportional to the number of target RNA molecules present in the sample. The obtained MFI results were normalized in accordance with the housekeeping genes taken as reference. Housekeeping genes (HKG) are genes that are expressed in almost all tissues and cells at a relatively constant level and function everywhere, at all stages of the life cycle of an organism. Normalization was performed by subtracting the mean background signal for each gene, dividing by the geometric mean of the signal obtained for the three reference genes, and multiplying by 100%.
Statistical data processing was performed using SPSS Statistics (version 23, for Windows). The data were checked for compliance with the normal distribution law using the Shapiro–Wilk test. For independent variables that did not correspond to a normal distribution, the significance of differences was determined using the Mann–Whitney U test with calculation of the median and quartiles (Me [Q1; Q3]). For data corresponding to the law of normal distribution, significant differences were determined by Student’s t test with calculation of the mean and standard deviation (SD). The chi-square test was used to analyze categorical variables. Differences were considered statistically significant at p less than 0.05.
RESULTS AND DISCUSSION
A comprehensive clinical and laboratory examination was carried out on 60 patients with paranoid schizophrenia who had been receiving antipsychotic treatment for a long time. The patients were divided into two groups: 45 subjects without metabolic disorders and 15 subjects with MS. Table 1 presents the main demographic and clinical characteristics of the studied groups of patients.
Patients with metabolic syndrome are significantly older, have had schizophrenia for a longer period of time, and have higher values of body mass index and waist circumference compared to patients without metabolic syndrome.
When assessing the expression of the studied genes in patients with schizophrenia with MS and without metabolic disorders, no significant differences were revealed (Table 2).
In further analysis of the results, the study sample was divided depending on the gender of the patients (Tables 3 and 4). In female patients with schizophrenia, no significant differences were detected in the expression levels of the studied genes, while in male patients significant changes were observed.
Men with MS have a higher level of FTO gene expression compared to male patients without MS (p = 0.041) (Table 4).
This pilot study was the first to evaluate the expression of 7 genes potentially important for the development of metabolic syndrome in peripheral blood leukocytes in patients with schizophrenia who received long-term antipsychotic drugs. Leukocytes seem to be an accessible, adequate, and promising model and can be used to study processes such as the development of metabolic disorders or, for example, the processes of immunoinflammation with the possibility of assessing the functions of genes that are significant in each case expressed in the periphery (and not just associations of polymorphisms). The FTO gene is associated with fat mass and obesity. Increased expression of this gene in male patients with metabolic syndrome may be of interest and may be important in the development of metabolic imbalance in psychiatric disorders.
The FTO gene is a relatively recently discovered gene associated with obesity and is expressed in various tissues of the human body, with particularly high expression in the brain. FTO is involved in a number of biological processes, including not only obesity but also brain function [38]. In particular, recent studies have shown that FTO, which is an N6-methyladenosine (m6A) demethylase, can influence brain function through modification of m6A mRNA, demonstrating a potential role of the FTO gene in schizophrenia through the regulation of m6A modification of dopamine-related genes [39].
Post-genome-wide functional analysis revealed common genetic pathways between metabolic and psychiatric disorders, and identified 86 differentially expressed genes that were located in different regions of the brain and peripheral blood in type 2 diabetes and psychiatric disorders, with the central genes of the identified cross-over network being FTO and TCF7L2 [38].
In our previous works, we investigated the relationship of gene polymorphisms associated with metabolic syndrome and obesity [7, 23, 40]. Thus, it was found that polymorphic variants (rs9939609, rs1421085, rs3751812, and rs8050136) of the FTO gene show a significant association with body mass index in 517 patients with schizophrenia who received antipsychotic drugs and trends for an association with waist circumference (reflecting central obesity).
Our study was the first to reveal the gender specificity of FTO gene expression in peripheral blood cells in patients with schizophrenia and metabolic syndrome. A number of other studies performed on patients with non-mental pathology also demonstrated sex differences in the carriage of polymorphisms and gene expression, for example, an association of rs1075440 in the FTO gene with plasma leptin levels in men was identified [41], and associations of FTO gene polymorphisms with body mass index were also identified in men [42].
Limitations of this study are as follows: the fact that blood was drawn for gene expression assessment in the first days of admission to the clinic, but we did not take into account the influence of previous maintenance antipsychotic treatment that patients received throughout the duration of the disease, as well as patient compliance. In addition, groups with and without MS differ in age (the risk of MS increases with age) and duration of the disease (the likelihood of developing MS is higher as the duration of taking antipsychotic drugs increases). The results obtained are preliminary and require further verification due to existing limitations; a prospective study of first-episode patients treated with the same antipsychotic drug seems most promising.
CONCLUSIONS
Pilot results assessing gene expression in the peripheral blood of patients with schizophrenia revealed higher expression levels of the FTO gene associated with fat mass and obesity in male patients with metabolic disorders compared to men without MS. Further study of the molecular genetic factors in the development of MS and the mechanisms of the influence of antipsychotics on metabolic parameters is necessary in order to assess the risk of metabolic disorders and implement a personalized approach to the therapeutic tactics of patients.
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ACKNOWLEDGMENTS
The authors express their gratitude to Irina Andreevna Mednova for her assistance in determining the biochemical parameters necessary for the criteria for diagnosing metabolic syndrome.
Funding
The study was carried out as part of a state assignment, research topic: “Biopsychosocial Mechanisms of Pathogenesis and Clinical Polymorphism, Adaptive Potential, and Predictors of Therapy Effectiveness in Patients with Mental and Behavioral Disorders in the Siberian Region,” state registration number 122020200054-8.
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ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The study protocol, according to which patients were selected, was approved by the Biomedical Ethics Committee of the Research Institute of Mental Health of the Tomsk National Medical Research Center (No. 101 of June 13, 2017).
Informed consent. The study was conducted taking into account ethical standards and the mandatory signing of informed consent by all participants. Study participants could refuse to participate at any time for any reason (in accordance with the Declaration of Helsinki) or be excluded subject to exclusion criteria.
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Boiko, A.S., Paderina, D.Z., Mikhalitskaya, E.V. et al. Pilot Study of the DRD3, GHRL, FTO, LEPR, INSIG2, GSTP1, and ABCB1 Gene Expression in Peripheral Blood Leukocytes in Schizophrenic Patients with Metabolic Syndrome. Neurochem. J. 18, 29–35 (2024). https://doi.org/10.1134/S1819712424010045
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DOI: https://doi.org/10.1134/S1819712424010045