A novel finding associated with hormonal dysregulation
Department of Physiology, College of Medicine, Riyadh, Kingdom of Saudi Arabia, 2Autism Research and Treatment Center, AL-Amodi Autism Research Chair, College of Medicine, Riyadh, Kingdom of Saudi Arabia.
Autism is a neurodevelopmental disorder with unclear pathogenesis. Many clinical observations and hormone studies have suggested the involvement of the neuroprotective hormone ghrelin in autism. The current study aimed to investigate the potential role of ghrelin in autism and to elucidate the associated hormonal dysregulation. This case-control study investigated acyl ghrelin (AG), des-acyl ghrelin (DG), total testosterone (TT), free testosterone (FT), leptin and growth hormone (GH) levels in 31 male children with autism and 28 healthy age and sex-matched controls. Hormone levels were measured in the blood using enzyme-linked immunosorbent assay and chemiluminescence immunoassay kits. AG, DG and GH levels were significantly lower in the autism group than in the control group (p # 0.001, p # 0.005 and p # 0.05, respectively). However, TT, FT and leptin levels were significantly higher in the autism group than in the control group (p # 0.05, p # 0.001 and p # 0.01, respectively). Our results for the first time demonstrate low AG and DG levels in autistic children. Considering the capacity of ghrelin to affect neuroinflammatory and apoptotic processes that are linked to autism, this study suggests a potential role for the hormone ghrelin in the pathogenesis of autism.
Autism spectrum disorders (ASDs) are a group of heterogeneous neurodevelopmental disorders that are classified as pervasive developmental disorders. ASDs are usually characterised by clinical manifestations of delayed or abnormal language development, deficits in social interaction, repetitive behaviours and restricted interest1. The pathogenesis of autism is not completely understood; however, a genetic origin has been recognised, and potential roles for both environmental factors and immune dysfunction have also been reported1. Hormonal dysregulation in autism remains a strong candidate, as a wide range of hormonal abnormalities has been identified in autistic children. This finding indicates the significant involvement of the hypothalamic-pituitary-adrenal axis in the pathophysiology of the disease1.
A significant volume of scientific evidence suggests a possible role for the hormone ghrelin in autism. Ghrelin is a 28-amino acid peptide that stimulates growth hormone (GH) release from the anterior pituitary gland2. Ghrelin has a wide range of physiological functions, and it represents a molecular link between peripheral metabolism and brain cognition. The hippocampus, which is the main target of action for ghrelin in the central nervous system (CNS) and plays an important role in memory and learning3, is also affected in autism4. Moreover, ghrelin plays an important role in synaptogenesis, mainly in the hippocampal area5, and abnormal synaptogenesis in this area has been reported in autism6. Ghrelin has proliferative and anti-apoptotic effects in the CNS, especially during oxygen/glucose deprivation; thus, it may protect the hypothalamus against reactive oxygen species, which have recently been linked to autism7. In addition, ghrelin protects primary cortical neurons from apoptosis induced by glutamate, an amino acid that is elevated in autistic children8.
Ghrelin influences the sleep-wake cycle, and its levels increase in the first hours of sleep in healthy individuals9. Additionally, ghrelin is the most powerful orexigenic peptide and is known to suppress locomotor activity10. Sleep and appetite disturbances and hyperactivity are among the frequent problems facing children with autism11. These observations, taken together, prompted us to hypothesise that ghrelin could be involved in the pathogenesis of autism. It wasn’t clearly distinctive in the literature which of those actions mediated through AG and which were mediated through DG despite AG is considered generally the active form17.
Ghrelin cannot function in isolation from other hormones; for example, in many clinical settings, ghrelin plasma levels are negatively correlated with elevated plasma testosterone levels12, and pre-pubertal testosterone therapy has been shown to be associated with a significant decrease in circulating ghrelin levels in boys13. At the same time, androgens are implicated in the pathophysiology of autism, as hyperandrogenism has been reported in children with autism11. This relationship suggests that ghrelin is prone to suppression by the elevated androgens in children with autism.
Similarly, the hormone leptin represents another potential link between ghrelin and autism, as these hormones have an inverse relationship14,15. Leptin inhibits ghrelin transcription in a dose-dependent manner, thus reducing ghrelin levels16, and leptin levels have been reported to be significantly higher in children with autism14. These elevated leptin levels may be associated with decreased ghrelin levels in autism.
However, ghrelin and GH are strongly connected, as ghrelin is known for its capacity to stimulate the release of GH17. Additionally, in an animal model of induced GH deficiency, gastric ghrelin mRNA levels and circulating ghrelin levels were significantly reduced, suggesting that ghrelin gene expression is influenced by GH status18.
In light of the above-mentioned findings, this study was conducted to elucidate the possible role of ghrelin in autism, which was accomplished by measuring plasma ghrelin levels in autistic boys and healthy age-matched controls. This study also aimed to further elucidate the associated hormonal dysregulation; thus, serum levels of androgens, leptin, and GH were also measured.
Subject selection. This case-control study was conducted on 59 male children: 31 had classic-onset autism, and 28 were age- and sex-matched healthy control children. Their ages ranged from 3 to 8 years (mean 6 SD 5 5.59 6 2.26 years). All of the boys included in the study were pre-pubertal (Tanner stage 1). Boys with autism were recruited from the Autism Research and Treatment Center, College of Medicine, King Saud University, Riyadh, Saudi Arabia. The study was limited to the male sex because of the lack of female patients. The patients in this study fulfilled the criteria for the diagnosis of autism according to the 4th edition of the Diagnostic and Statistical Manual of Mental disorders (DSM4) (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, 4th ed. Washington DC: American Psychiatric Association, 1994.). ADOS (Autism Diagnostic Observation Schedule) and CARS (Childhood Autism Rating Scale) was performed to assign the diagnosis of autism according to DSM4. The patients included in this study had no associated neurological diseases (such as cerebral palsy or tuberous sclerosis) or metabolic disorders (e.g., phenylketonuria). None of the patients have the intellectual disability.
Written consent was obtained from the parents according to the guidelines of the Institutional Review Board (IRB) of the College of Medicine, King Saud University, Riyadh, Saudi Arabia. The IRB approved the study protocol (approval no. E-10-341).
Anthropometric measurements. The height and weight of individuals in the autism and control groups were measured using an electronic scale. The occipitofrontal head circumference, waist circumference, and hip circumference were measured using a meter. The body mass index (BMI) and waist to hip ratio (W/H) were calculated.
Biochemical assays. Venous blood samples (7 mL) were collected between 8:00 and 9:00 am after an overnight fast and were divided immediately as follows: a 3 mL aliquot of blood was dispensed into an EDTA tube containing 30 mL of p- hydroxymercuribenzoic acid (PHMBA) to prevent the degradation of acyl ghrelin (AG). This sample was used to measure both AG and DG. A 4 mL aliquot of blood was dispensed into a plain plastic tube and was used to measure total testosterone (TT), free testosterone (FT), sex hormone-binding globulin (SHBG), leptin and baseline GH. The samples were centrifuged for 10 min at 3 500 r.p.m. followed by the addition of 100 mL of 1 N HCl to the EDTA tube samples, which were further centrifuged for 5 min at 3 500 r.p.m. The blood samples were immediately separated and stored at 280uC until use in the assay, and they were not subjected to freeze/thaw cycles.
Plasma levels of AG and DG were measured using SPI-BIO enzyme-linked immunosorbent assay (ELISA) kits obtained from IBL International GMBH. Baseline serum levels of GH were measured using LIAISONH hGH chemiluminescence immunoassay kits obtained from Diasorin, USA. Serum leptin, TT, FT and SHBG levels were measured using DIAsource ELISA kits (Nivelles, Belgium). All sample measurements were performed in duplicate; thus, two kits per hormone were used.
Statistical analysis. The data were analysed using SPSS Pc1 statistical software (version 18.0). Normality was tested, and the normal distribution of the data was confirmed. The independent samples t-test was used to compare means between the autism and control groups. The correlation between different variables was determined using the Karl Pearson correlation coefficient. Statistical significance was defined as p # 0.05.
Data for the anthropometric measurements and hormonal analysis are presented in Table 1, Table 2 and Table 3 as the mean values 6 standard deviations. As Table 1 shows, the only significant difference between the autistic group and the controls occurred in body weight, which was 18% higher in the autistic group than in the controls (22.7 6 7.9 vs. 19.3 6 4.1 kg; p # 0.05). The mean of body mass Index for children with autism falls between the 50th and 75th percentile which doesn’t indicate obesity.
As Table 2 shows, AG levels were lower in the autistic group than in the controls (116.7 6 50.3 vs. 170.5 6 59.3 pg/mL; p # 0.001). Similarly, DG levels were also lower in autistic individuals compared to the controls (214.4 6 80.5 vs. 299.3 6 139.7 pg/mL; p # 0.005). In the autistic group, TT levels were higher than those in the controls (20.1 6 17.4 vs. 13.1 6 6.7 ng/dL; p # 0.05). Moreover, FT levels were higher in the autistic group than in the controls (90.8 6 77.9 vs. 26.8 6 22.9 pg/dL; p # 0.001). However, there was no significant difference in SHBG levels between the autistic group and the controls (106 6 42.4 vs. 101.8 6 30.1 nmol/L; p50.6). Leptin concentrations were higher in the autistic group compared to the control group (1.4 6 1.3 vs. 0.67 6 0.5 ng/mL; p # 0.01). In autistic children, GH levels were lower compared to the levels in the controls (0.53 6 0.5 vs. 1 6 1.2 ng/mL; p # 0.05).
Table 3 shows the hormone levels in a weight-matched group to exclude the effect of adiposity on ghrelin and leptin levels.
Correlation analysis. Correlation analyses between the measured hormones and different variables in the autism group were performed as follows:
Correlations of AG with different variables: AG had significant negative correlations with age (r520.48, p # 0.007), weight (r520.35, p # 0.05), BMI (r 520.39, p # 0.05), waist circumference (r520.50, p # 0.01), hip circumference (r520.36, p # 0.05)