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Effects of acute metabolic stress on the dopaminergic and pituitary–adrenal axis activity in patients with schizophrenia, their unaffected siblings and controls
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Schizophrenia Research 100 (2008) 206 – 211 www.elsevier.com/locate/schres
Effects of acute metabolic stress on the dopaminergic and pituitary–adrenal axis activity in patients with schizophrenia, their unaffected siblings and controls Jerome Brunelin a,b,⁎, Thierry d'Amato a , Jim van Os b,c , Alain Cochet a , Marie-Françoise Suaud-Chagny a , Mohamed Saoud a a
b
Université de Lyon, Lyon, F-69003, France; Université Lyon 1, Lyon, EA4166; CH Le Vinatier, Bron, F-69677, France; Institut Fédératif des Neurosciences de Lyon (IFNL), Hôpital neurologique, Bron, F-69394, France Department of Psychiatry and Neuropsychology, South Limburg Mental Health Research and Teaching Network, EURON, Maastricht University, PO BOX 616 (DRT 10), 6200 MD Maastricht, The Netherlands. c Division of Psychological Medicine, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK. Received 12 September 2007; received in revised form 6 November 2007; accepted 9 November 2007 Available online 21 December 2007
Abstract A genetically mediated abnormal sensitivity to stress is thought to play a role in the onset, exacerbation and relapse of schizophrenia. In a double blind, placebo-controlled crossover study, peak increases in plasma ACTH (ΔACTH) and homovanillicacid, a dopamine metabolite, (ΔHVA) following exposure to a metabolic stressor(2DG) were studied in unaffected siblings of patients with schizophrenia (n = 15), their patient relatives (n = 15) and healthy controls (n = 14). Siblings showed a stress response (both ΔACTH and ΔHVA) that was significantly greater compared to controls and significantly less pronounced compared to patients. The results suggest that the genetic risk for schizophrenia may be characterized by an enhanced sensitivity to stress. © 2007 Elsevier B.V. All rights reserved. Keywords: Schizophrenia; Vulnerability; 2DG, Stress
1. Introduction An enhanced response to stress mediated by activation of the Hypothalamo–Pituitary–Adrenocortical (HPA) axis is thought to play an important role in the onset, exacerbation and relapse of schizophrenia. According to the dopamine (DA) hypothesis of schizophrenia, psy-
⁎ Corresponding author. EA 4166, Service du Pr d'Amato, CH le vinatier, 95 bd Pinel 69677 Bron cedex France. Tel.: +33 4 37 91 55 65. E-mail address: [email protected] (J. Brunelin). 0920-9964/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2007.11.009
chotic symptoms are associated with increases in dopamine signalling. This hypothesis is supported by an increase in amphetamine-induced DA release observed in patients with schizophrenia (Laruelle et al., 1996). Thus, enhanced responses to stress mediated by HPA and DA is associated with schizophrenia (for review, see Phillips et al., 2006). In experimental studies, a direct and objective way to study the effects of stress is exposure to metabolic stress using intravenous infusion of the glucose analogue 2-deoxy-D-glucose (2DG). This metabolic stress paradigm has been found to produce robust activation of the
J. Brunelin et al. / Schizophrenia Research 100 (2008) 206–211
HPA axis (Breier et al., 1993; Marcelis et al., 2004). Moreover, it strongly affects central and peripheral dopamine function (Breier et al., 1993; Adler et al., 2000), as well as the plasma levels of homovanillic acid (HVA), a breakdown product of dopamine as well as noradrenaline (Breier et al., 1993; Marcelis et al., 2004; Brunelin et al., 2007). It is unlikely that stress is a sufficient cause for the onset of schizophrenia. More likely is a scenario of geneenvironment interaction, according to which some genes increase the risk for schizophrenia by affecting the sensitivity to psychosis-inducing environmental risk factors (for review see van Os and Marcelis 1998). A useful paradigm to study gene-environment interactions is experimental exposure to stress followed by measurement of the physiological response in individuals who are genetically at risk, such as the unaffected siblings of patients, who on average share 50% of their genes. The value of vulnerability markers in unaffected siblings is classically intermediate between controls and patients (for review see Gur et al., 2007). In a previous study, unaffected siblings of patients with schizophrenia displayed an HVA response to metabolic stress (2DG) that was intermediate to patients and controls (Brunelin et al., 2007), similar to the results reported by Marcelis and coworkers (2004), albeit statistically inconclusive in the latter study. The present study is an extension of this previous work, extending both the sample size and the hypotheses tested. In the current study, we wished to repeat examination of the effect of metabolic stress (2DG administration) on plasma HVA in the unaffected siblings of patients with schizophrenia, and, for the first time, also examine the effect of metabolic stress on the HPA axis, measuring adrenocorticotropic hormone (ACTH) and cortisol. It was hypothesized that unaffected siblings would display a
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response to metabolic stress that was intermediate to that of patients and controls. In addition, it was hypothesised that HVA and HPA responses would be associated with each other, representing indicators of a general stress response. 2. Methods and material 2.1. Subjects Fifteen remitted outpatients with a DSM IV diagnosis of schizophrenia, 15 of their first-degree non psychotic relatives and 14 frequency-matched healthy subjects were included in a double-blind, placebo-controlled crossover study (Table 1). The sample represents an extension of 5 new subjects per group compared to previously published work. The study was approved by the standing ethics committee. All participants gave their written informed consent after a detailed description of the study. All patients received second-generation antipsychotic drugs (mean dose of 587 (SD = 628) equivalent mg chlorpromazine per day according to Woods, 2003; mean illness duration 7.4 (SD = 6.0). years, mean number of hospitalisations 4.8 (SD = 4.6)) with no dose change throughout the study period (mean same treatment duration 2 (SD = 0.4) years). Outpatients were recruited in the professorial unit of the psychiatric hospital, which serves a normal catchment area (Pr. Dalery's university service of psychiatry). Firstdegree relatives were all unaffected brothers or sisters of their included sibling patient and had no past or current history of psychosis or of substance abuse. Control subjects were recruited by the Clinical Investigation Centre of Lyon (Cardiologic Hospital, Lyon, France). They also had no personal or familial history of psychosis or of substance abuse. Past and current histories of psychosis in
Table 1 Socio-demographical and clinical characteristics of sample and baseline measures
n Age years Sex (F/M) Years of full time education years Weight kg PANSS positive subscale PANSS negative subscale PANSS general psychopathology ACTH baseline ng/mL HVA baseline ng/mL Cortisol Baseline nmol/mL
Patients with schizophrenia
Unaffected siblings
Healthy controls
p-value⁎
15 28.6 (7.5) 5/10 11.1 (2.4) 78.7 (13.9) 12.8 (4.9) 16.8 (5.7) 30.6 (10.6) 19.8 (8.7) 16.1 (5.5) 391.0 (105.3)
15 28.5 (7.5) 8/7 11.6 (2.8) 66.6 (14.0) – – – 12.6 (5.6) 16.2 (15.1) 397.3 (189.4)
14 29.1 (7.2) 8/6 12.5 (1.8) 78.0 (14.7) – – – 16.6 (7.4) 13.5 (7.7) 541.8 (303.1)
0.93 0.28 0.27 0.06 – – – 0.04 0.16 0.28
Results are give in mean (SD). ⁎Kruskal–Wallis ANOVA H(2,44); Sex: Chi2; SD = standard deviation.
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changes in plasma measures (hereafter: ΔACTH, Δcortisol, ΔHVA) were compared between the 3 groups using Kruskal–Wallis ANOVA, followed by post-hoc U Mann– Whitney tests. Main effect of the 2DG condition compared to the placebo effect was calculated using a one-sample student t test with peak ACTH, peak cortisol and peak HVA. Pearson correlations were used to investigate the association between ΔACTH and ΔHVA. 3. Results 3.1. Sample
Fig. 1. 2DG-induced minus placebo-induced peak changes in plasma ACTH in patients with schizophrenia, unaffected siblings and controls; in ng/mL. Lines indicate means. Kruskal Wallis (H(2,44) = 12.073, p= 0.002).
siblings and controls were assessed using clinical interview and the Mini International Neuropsychiatric Interview semi-standardized evaluation (MINI version 4.4). 2.2. Method All subjects were in good physical health, as evidenced by physical examination one week before the procedure, thyroid function test, Complete Blood Count, urinalysis, HIV and HVB antibody test, toxicology screen and ECG. The day before the procedure, participants were admitted at 18:00 to the Clinical Investigation Centre of Lyon (France) and received a standardised diner at 20:00. On the morning of the procedure, subjects had fasted and refrained from alcohol, tobacco, caffeine and physical activity. All participants received double-blind administration of placebo and 2DG (40 mg/kg, 5 min bolus at 9:00 am) in randomized order separated by a period of 2 weeks. Plasma ACTH, cortisol and HVA were assessed twice before the start of the 2DG/placebo infusion (baseline values), and five times again after infusion (T + 20, T + 40, T + 60, T + 120 and T + 150 min). Blood samples were analysed using High Performance Liquid Chromatography (HPLC). 2.3. Analyses After correction for baseline values (i.e. baseline = 0 for all subjects), 2DG-induced minus placebo-induced peak
There were no large or significant differences between patients with schizophrenia, unaffected siblings and comparison subjects in age (H(2,44) = 0.125; p = 0.93), sex (Chi2 = 2.552; p = 0.28), and years of fulltime education (H(2,44) = 2.570; p = 0.27); there was a suggestive difference in weight (H(2,44) = 5.650; p = 0.06). Further characteristics are given in Table 1. Similarly, baseline HVA (H(2,44) = 3.575; p = 0.16) and cortisol (H(2,44) = 2.554; p = 0.27) measures were comparable between the groups; no crossover sequence effect was observed (H(1,44) = 3.314; p = 0.67 for ACTH peak; H(1,44) = 0.049; p = 0.82 for HVA and H(1,44) = 1.521; p = 0.22 for cortisol). A significant group difference in ACTH baseline level was observed (H(2,44) = 6.116; p = 0.04; Table 1). 3.2. ACTH, HVA and cortisol responses Patients with schizophrenia (t = 3.636; p = 0.003), unaffected siblings (t = 2.269; p = 0.04) and comparison subjects (t = 1.686; p = 0.1) all showed increases in plasma ACTH levels following 2DG exposure (Fig. 1; Table 2). In siblings, the ΔACTH was intermediate to the values observed in patients and controls. A significant group effect on ΔACTH was observed (H2,44 = 12.073; p = 0.002). Table 2 2DG-induced minus placebo-induced peak (Δ) changes in plasma in each group Patients with Unaffected schizophrenia siblings
Healthy controls
p value⁎
ΔACTH 38.0 (40.5) 21.6 (36.9) 5.9 (8.5) 0.002 (ng/mL) ΔHVA 8.5 (5.5) 3.9 (3.9) 1.9 (4.2) 0.001 (ng/mL) Δcortisol 205.0 (228.7) 217.7 (179.5) 109.8 (122.0) 0.3 (nmol/mL) Results are give in mean (SD). ⁎Kruskal–Wallis ANOVA.
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Post-hoc U tests showed that ΔACTH in siblings tended to differ from that of patients (U = 68.0; Z = 1.847; p = 0.06) and was significantly different compared to controls (U = 59.5; Z = 1.985; p = 0.04). ΔACTH in patients was significantly higher than that in controls (U = 30.0; Z = 3.277; p = 0.001). The group effect as well as post hoc differences was comparable when 2 outliers in the sibling and patient groups (2 brothers) were excluded from the analysis (effect group: H(2;42) = 11.370; p = 0.003; siblings versus patients: U = 54.0; Z = 2.023; p = 0.04; siblings versus controls: U = 59.5; Z = −1.771; p = 0.07). Patients with schizophrenia (t = 5.917; p b 0.001), unaffected siblings (t = 3.884; p = 0.02) and control subjects (t = 2.596; p = 0.02) all demonstrated increases in plasma HVA levels after 2DG challenge compared with placebo condition (Fig. 2; Table 2). In siblings, ΔHVA was intermediate to the values observed in patients and controls. A significant group effect was observed (H(2,44) = 13.466; p = 0.001). In addition, post-hoc tests showed that ΔHVA in siblings was significantly lower than ΔHVA observed in the patients (U = 56; Z = 2.434; p = 0.01) and higher than ΔHVA observed in the controls (U = 58; Z = −2.051; p = 0.04). Patients with schizophrenia (t = 3.472; p = 0.004), unaffected siblings (t = 4.669; p b 0.001) and control subjects (t = 3.369; p = 0.005) all demonstrated increases in plasma cortisol level after 2DG infusion. Analyses of Δcortisol data revealed no significant group effect (H(2,44) = 2.140; p = 0.3; Table 2).
Fig. 2. 2DG-induced minus placebo-induced peak changes in plasma HVA in patients with schizophrenia, unaffected siblings and controls; in ng/mL. Lines indicate means. Kruskal Wallis: (H(2,44) = 13.466, p = 0.001).
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There was a significant correlation between ΔACTH level and ΔHVA (r = 0.54; p = 0.0001) across groups. 4. Discussion 4.1. Summary of findings As previously reported and in line with the initial hypotheses, siblings of patients with a diagnosis of schizophrenia displayed exaggerated ACTH (Elman et al., 1998) and HVA (Breier et al., 1993; Brunelin et al., 2007) responses to 2DG, siblings showing values that were intermediate to those of patients and controls (Marcelis et al., 2004; Brunelin et al., 2007). However, as previously reported (Elman et al., 1998), no difference between the groups was observed in Δcortisol, although patients and siblings had numerically greater values. The fact that the cortisol measure was not sensitive to group effects may be explained by a ceiling effect of cortisol reactivity in response to acute stress (Crowley et al., 1993). 4.2. ACTH and HVA stress response To the best of our knowledge, this is the first time that an increased ACTH response to stress in unaffected siblings of patients with schizophrenia has been reported. An abnormal HVA response to stress in siblings compared to controls was also reported by Marcelis and colleagues (2004), who showed directionally similar differences albeit not statistically significant, suggesting that a type II error may have occurred. The greater sensitivity of the current study may be explained by the fact that patient and siblings groups were related, whereas in the Marcelis study they were mostly from different families. In addition, Marcelis and colleagues also included parents of patients, in whom the risk to develop schizophrenia is less densely distributed. This result is also in accordance with those reported by MyinGermeys and colleagues (2005) who reported, in an observational study measuring behavioural sensitisation, that subjects at genetic risk displayed increased behavioural sensitivity to daily life stress. The evidence suggests that enhanced reactivity to stress co-segregates with psychosis and therefore may be considered as a biological vulnerability marker. Given the fact that expression of vulnerability for schizophrenia takes on the form of continuous intermediary phenotypes such as cerebral grey matter volume and continuous cognitive ability, our results are line showing slight shifts in continuous distributions of ΔACTH and ΔHVA values in patients and, to a lesser degree, siblings compared to controls.
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4.3. Neurobiological mechanisms According to the vulnerability model of schizophrenia, an accurate marker of schizophrenia vulnerability should detect ill, stabilized or recovered patients with schizophrenia as well as unaffected individuals at high risk of schizophrenia. Thus, the correlated alterations in 2DG-induced ACTH and HVA responses in the unaffected sibling group in response to a 2DG challenge may be a biological marker of genetic vulnerability to schizophrenia. Given the amount of overlap in values between patients, siblings and controls, the findings are of aetiopathogenic rather than diagnostic importance. It has been reported that lower volumes of total cerebral grey and white matter (Marcelis et al., 2006) as well as the prefrontal cortex (Breier et al., 1993) in schizophrenia are associated with stress response dysregulation. The release of cortisol and its neurotoxicity for the hippocampus and prefrontal cortex (McEwen and Magarinos, 1997) may result in cognitive deficits (Mizoguchi et al., 2000), that have also been considered as a marker for genetic risk of schizophrenia (Lencz et al., 2006; for review see Brewer et al., 2006). Although traditional neurodevelopmental models of psychosis suggest that early life trauma damages the hippocampus resulting in heightened vulnerability towards the development of psychosis, more recent evidence suggests that changes to the medial temporal lobes and possibly the hippocampus occur close to the onset of a full-blown psychotic episode (Pantelis et al., 2003). A subtle brain process may be continuing through the first few years of a schizophrenic illness causing greater than the normal adult cortical deterioration (DeLisi et al., 1997; Thompson et al., 2001). Meta analytic (Wright et al., 2000; Boos et al., 2007) studies have suggested that hippocampal volume reduction in schizophrenia as well as impairment in cognitive skills are associated with hippocampal functioning such as memory, attention and problem-solving (Aleman et al., 1999). Indeed, cognitive function, specifically hippocampus-dependent verbal memory, is a strong predictor of functional outcome in schizophrenia (Green, 1996). Post-mortem studies in patients with schizophrenia reported reduced cell size in the hippocampus as well as abnormal apical dendrites on pyramidal cells in the subiculum (Rosoklija et al., 2000), findings that could be consistent with the known effects of stress and cortisol on apical dendrites (McEwen and Magarinos, 1997). In support of this idea, there have been reports of progressive cognitive deficits, including deficits in explicit memory (Cosway et al., 2000), and hippocampal volume reduction (Lawrie et al., 1999) in the time period from the prodrome to the onset of psychosis. Thus, ac-
cording to our findings, one can hypothesize that hyperreactivity of the HPA axis in response to stress in unaffected siblings could accelerate alterations in neuronal integrity, which in turn may contribute to alterations in subcortical circuits regulating dopaminergic response to stress. The findings support the hypothesis that alterations in cortico-subcortical dopaminergic connections affect psychosis susceptibility through an altered, illness-related stress response. Further studies are needed to investigate the relationship between volumes of grey/white matter, the hippocampus, HPA and dopaminergic reactivity to stress and cognitive performance in schizophrenia and in unaffected siblings. Moreover, even if plasma HVA is assumed to be an indicator of central dopaminergic activity (Amin et al., 1992), these relationships have to be confirmed directly. Role of the funding source Funding for this study was provided by a regional PHRC 2002 & CSR 2003; they had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Contributors M Saoud, T d'Amato & J van Os designed the study; M Saoud & T d'Amato wrote the protocol. J Brunelin, MF Suaud-Chagny & A Cochet managed the literature searches and analyses. J Brunelin and M Saoud undertook the statistical analysis and J Brunelin & J van Os wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest None.
Acknowledgements The authors thank the nurse team of Clinical Investigation Centre (CIC; Pr F. Gueyffier, Dr C. Cornu); JM Cottet-Emard; RM CottetEmard, AVouillarmet and B Claustrat for blood samples dosages; Dr P. Martin for 2DG preparation; G Pranal for English corrections. This study was supported by a regional grant: PHRC 2002 and by CSR 2003 CH Le Vinatier.
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Effects of acute metabolic stress on the dopaminergic and pituitary–adrenal axis activity in patients with schizophrenia, their unaffected siblings and controls Jerome Brunelin a,b,⁎, Thierry d'Amato a , Jim van Os b,c , Alain Cochet a , Marie-Françoise Suaud-Chagny a , Mohamed Saoud a a
b
Université de Lyon, Lyon, F-69003, France; Université Lyon 1, Lyon, EA4166; CH Le Vinatier, Bron, F-69677, France; Institut Fédératif des Neurosciences de Lyon (IFNL), Hôpital neurologique, Bron, F-69394, France Department of Psychiatry and Neuropsychology, South Limburg Mental Health Research and Teaching Network, EURON, Maastricht University, PO BOX 616 (DRT 10), 6200 MD Maastricht, The Netherlands. c Division of Psychological Medicine, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK. Received 12 September 2007; received in revised form 6 November 2007; accepted 9 November 2007 Available online 21 December 2007
Abstract A genetically mediated abnormal sensitivity to stress is thought to play a role in the onset, exacerbation and relapse of schizophrenia. In a double blind, placebo-controlled crossover study, peak increases in plasma ACTH (ΔACTH) and homovanillicacid, a dopamine metabolite, (ΔHVA) following exposure to a metabolic stressor(2DG) were studied in unaffected siblings of patients with schizophrenia (n = 15), their patient relatives (n = 15) and healthy controls (n = 14). Siblings showed a stress response (both ΔACTH and ΔHVA) that was significantly greater compared to controls and significantly less pronounced compared to patients. The results suggest that the genetic risk for schizophrenia may be characterized by an enhanced sensitivity to stress. © 2007 Elsevier B.V. All rights reserved. Keywords: Schizophrenia; Vulnerability; 2DG, Stress
1. Introduction An enhanced response to stress mediated by activation of the Hypothalamo–Pituitary–Adrenocortical (HPA) axis is thought to play an important role in the onset, exacerbation and relapse of schizophrenia. According to the dopamine (DA) hypothesis of schizophrenia, psy-
⁎ Corresponding author. EA 4166, Service du Pr d'Amato, CH le vinatier, 95 bd Pinel 69677 Bron cedex France. Tel.: +33 4 37 91 55 65. E-mail address: [email protected] (J. Brunelin). 0920-9964/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2007.11.009
chotic symptoms are associated with increases in dopamine signalling. This hypothesis is supported by an increase in amphetamine-induced DA release observed in patients with schizophrenia (Laruelle et al., 1996). Thus, enhanced responses to stress mediated by HPA and DA is associated with schizophrenia (for review, see Phillips et al., 2006). In experimental studies, a direct and objective way to study the effects of stress is exposure to metabolic stress using intravenous infusion of the glucose analogue 2-deoxy-D-glucose (2DG). This metabolic stress paradigm has been found to produce robust activation of the
J. Brunelin et al. / Schizophrenia Research 100 (2008) 206–211
HPA axis (Breier et al., 1993; Marcelis et al., 2004). Moreover, it strongly affects central and peripheral dopamine function (Breier et al., 1993; Adler et al., 2000), as well as the plasma levels of homovanillic acid (HVA), a breakdown product of dopamine as well as noradrenaline (Breier et al., 1993; Marcelis et al., 2004; Brunelin et al., 2007). It is unlikely that stress is a sufficient cause for the onset of schizophrenia. More likely is a scenario of geneenvironment interaction, according to which some genes increase the risk for schizophrenia by affecting the sensitivity to psychosis-inducing environmental risk factors (for review see van Os and Marcelis 1998). A useful paradigm to study gene-environment interactions is experimental exposure to stress followed by measurement of the physiological response in individuals who are genetically at risk, such as the unaffected siblings of patients, who on average share 50% of their genes. The value of vulnerability markers in unaffected siblings is classically intermediate between controls and patients (for review see Gur et al., 2007). In a previous study, unaffected siblings of patients with schizophrenia displayed an HVA response to metabolic stress (2DG) that was intermediate to patients and controls (Brunelin et al., 2007), similar to the results reported by Marcelis and coworkers (2004), albeit statistically inconclusive in the latter study. The present study is an extension of this previous work, extending both the sample size and the hypotheses tested. In the current study, we wished to repeat examination of the effect of metabolic stress (2DG administration) on plasma HVA in the unaffected siblings of patients with schizophrenia, and, for the first time, also examine the effect of metabolic stress on the HPA axis, measuring adrenocorticotropic hormone (ACTH) and cortisol. It was hypothesized that unaffected siblings would display a
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response to metabolic stress that was intermediate to that of patients and controls. In addition, it was hypothesised that HVA and HPA responses would be associated with each other, representing indicators of a general stress response. 2. Methods and material 2.1. Subjects Fifteen remitted outpatients with a DSM IV diagnosis of schizophrenia, 15 of their first-degree non psychotic relatives and 14 frequency-matched healthy subjects were included in a double-blind, placebo-controlled crossover study (Table 1). The sample represents an extension of 5 new subjects per group compared to previously published work. The study was approved by the standing ethics committee. All participants gave their written informed consent after a detailed description of the study. All patients received second-generation antipsychotic drugs (mean dose of 587 (SD = 628) equivalent mg chlorpromazine per day according to Woods, 2003; mean illness duration 7.4 (SD = 6.0). years, mean number of hospitalisations 4.8 (SD = 4.6)) with no dose change throughout the study period (mean same treatment duration 2 (SD = 0.4) years). Outpatients were recruited in the professorial unit of the psychiatric hospital, which serves a normal catchment area (Pr. Dalery's university service of psychiatry). Firstdegree relatives were all unaffected brothers or sisters of their included sibling patient and had no past or current history of psychosis or of substance abuse. Control subjects were recruited by the Clinical Investigation Centre of Lyon (Cardiologic Hospital, Lyon, France). They also had no personal or familial history of psychosis or of substance abuse. Past and current histories of psychosis in
Table 1 Socio-demographical and clinical characteristics of sample and baseline measures
n Age years Sex (F/M) Years of full time education years Weight kg PANSS positive subscale PANSS negative subscale PANSS general psychopathology ACTH baseline ng/mL HVA baseline ng/mL Cortisol Baseline nmol/mL
Patients with schizophrenia
Unaffected siblings
Healthy controls
p-value⁎
15 28.6 (7.5) 5/10 11.1 (2.4) 78.7 (13.9) 12.8 (4.9) 16.8 (5.7) 30.6 (10.6) 19.8 (8.7) 16.1 (5.5) 391.0 (105.3)
15 28.5 (7.5) 8/7 11.6 (2.8) 66.6 (14.0) – – – 12.6 (5.6) 16.2 (15.1) 397.3 (189.4)
14 29.1 (7.2) 8/6 12.5 (1.8) 78.0 (14.7) – – – 16.6 (7.4) 13.5 (7.7) 541.8 (303.1)
0.93 0.28 0.27 0.06 – – – 0.04 0.16 0.28
Results are give in mean (SD). ⁎Kruskal–Wallis ANOVA H(2,44); Sex: Chi2; SD = standard deviation.
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changes in plasma measures (hereafter: ΔACTH, Δcortisol, ΔHVA) were compared between the 3 groups using Kruskal–Wallis ANOVA, followed by post-hoc U Mann– Whitney tests. Main effect of the 2DG condition compared to the placebo effect was calculated using a one-sample student t test with peak ACTH, peak cortisol and peak HVA. Pearson correlations were used to investigate the association between ΔACTH and ΔHVA. 3. Results 3.1. Sample
Fig. 1. 2DG-induced minus placebo-induced peak changes in plasma ACTH in patients with schizophrenia, unaffected siblings and controls; in ng/mL. Lines indicate means. Kruskal Wallis (H(2,44) = 12.073, p= 0.002).
siblings and controls were assessed using clinical interview and the Mini International Neuropsychiatric Interview semi-standardized evaluation (MINI version 4.4). 2.2. Method All subjects were in good physical health, as evidenced by physical examination one week before the procedure, thyroid function test, Complete Blood Count, urinalysis, HIV and HVB antibody test, toxicology screen and ECG. The day before the procedure, participants were admitted at 18:00 to the Clinical Investigation Centre of Lyon (France) and received a standardised diner at 20:00. On the morning of the procedure, subjects had fasted and refrained from alcohol, tobacco, caffeine and physical activity. All participants received double-blind administration of placebo and 2DG (40 mg/kg, 5 min bolus at 9:00 am) in randomized order separated by a period of 2 weeks. Plasma ACTH, cortisol and HVA were assessed twice before the start of the 2DG/placebo infusion (baseline values), and five times again after infusion (T + 20, T + 40, T + 60, T + 120 and T + 150 min). Blood samples were analysed using High Performance Liquid Chromatography (HPLC). 2.3. Analyses After correction for baseline values (i.e. baseline = 0 for all subjects), 2DG-induced minus placebo-induced peak
There were no large or significant differences between patients with schizophrenia, unaffected siblings and comparison subjects in age (H(2,44) = 0.125; p = 0.93), sex (Chi2 = 2.552; p = 0.28), and years of fulltime education (H(2,44) = 2.570; p = 0.27); there was a suggestive difference in weight (H(2,44) = 5.650; p = 0.06). Further characteristics are given in Table 1. Similarly, baseline HVA (H(2,44) = 3.575; p = 0.16) and cortisol (H(2,44) = 2.554; p = 0.27) measures were comparable between the groups; no crossover sequence effect was observed (H(1,44) = 3.314; p = 0.67 for ACTH peak; H(1,44) = 0.049; p = 0.82 for HVA and H(1,44) = 1.521; p = 0.22 for cortisol). A significant group difference in ACTH baseline level was observed (H(2,44) = 6.116; p = 0.04; Table 1). 3.2. ACTH, HVA and cortisol responses Patients with schizophrenia (t = 3.636; p = 0.003), unaffected siblings (t = 2.269; p = 0.04) and comparison subjects (t = 1.686; p = 0.1) all showed increases in plasma ACTH levels following 2DG exposure (Fig. 1; Table 2). In siblings, the ΔACTH was intermediate to the values observed in patients and controls. A significant group effect on ΔACTH was observed (H2,44 = 12.073; p = 0.002). Table 2 2DG-induced minus placebo-induced peak (Δ) changes in plasma in each group Patients with Unaffected schizophrenia siblings
Healthy controls
p value⁎
ΔACTH 38.0 (40.5) 21.6 (36.9) 5.9 (8.5) 0.002 (ng/mL) ΔHVA 8.5 (5.5) 3.9 (3.9) 1.9 (4.2) 0.001 (ng/mL) Δcortisol 205.0 (228.7) 217.7 (179.5) 109.8 (122.0) 0.3 (nmol/mL) Results are give in mean (SD). ⁎Kruskal–Wallis ANOVA.
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Post-hoc U tests showed that ΔACTH in siblings tended to differ from that of patients (U = 68.0; Z = 1.847; p = 0.06) and was significantly different compared to controls (U = 59.5; Z = 1.985; p = 0.04). ΔACTH in patients was significantly higher than that in controls (U = 30.0; Z = 3.277; p = 0.001). The group effect as well as post hoc differences was comparable when 2 outliers in the sibling and patient groups (2 brothers) were excluded from the analysis (effect group: H(2;42) = 11.370; p = 0.003; siblings versus patients: U = 54.0; Z = 2.023; p = 0.04; siblings versus controls: U = 59.5; Z = −1.771; p = 0.07). Patients with schizophrenia (t = 5.917; p b 0.001), unaffected siblings (t = 3.884; p = 0.02) and control subjects (t = 2.596; p = 0.02) all demonstrated increases in plasma HVA levels after 2DG challenge compared with placebo condition (Fig. 2; Table 2). In siblings, ΔHVA was intermediate to the values observed in patients and controls. A significant group effect was observed (H(2,44) = 13.466; p = 0.001). In addition, post-hoc tests showed that ΔHVA in siblings was significantly lower than ΔHVA observed in the patients (U = 56; Z = 2.434; p = 0.01) and higher than ΔHVA observed in the controls (U = 58; Z = −2.051; p = 0.04). Patients with schizophrenia (t = 3.472; p = 0.004), unaffected siblings (t = 4.669; p b 0.001) and control subjects (t = 3.369; p = 0.005) all demonstrated increases in plasma cortisol level after 2DG infusion. Analyses of Δcortisol data revealed no significant group effect (H(2,44) = 2.140; p = 0.3; Table 2).
Fig. 2. 2DG-induced minus placebo-induced peak changes in plasma HVA in patients with schizophrenia, unaffected siblings and controls; in ng/mL. Lines indicate means. Kruskal Wallis: (H(2,44) = 13.466, p = 0.001).
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There was a significant correlation between ΔACTH level and ΔHVA (r = 0.54; p = 0.0001) across groups. 4. Discussion 4.1. Summary of findings As previously reported and in line with the initial hypotheses, siblings of patients with a diagnosis of schizophrenia displayed exaggerated ACTH (Elman et al., 1998) and HVA (Breier et al., 1993; Brunelin et al., 2007) responses to 2DG, siblings showing values that were intermediate to those of patients and controls (Marcelis et al., 2004; Brunelin et al., 2007). However, as previously reported (Elman et al., 1998), no difference between the groups was observed in Δcortisol, although patients and siblings had numerically greater values. The fact that the cortisol measure was not sensitive to group effects may be explained by a ceiling effect of cortisol reactivity in response to acute stress (Crowley et al., 1993). 4.2. ACTH and HVA stress response To the best of our knowledge, this is the first time that an increased ACTH response to stress in unaffected siblings of patients with schizophrenia has been reported. An abnormal HVA response to stress in siblings compared to controls was also reported by Marcelis and colleagues (2004), who showed directionally similar differences albeit not statistically significant, suggesting that a type II error may have occurred. The greater sensitivity of the current study may be explained by the fact that patient and siblings groups were related, whereas in the Marcelis study they were mostly from different families. In addition, Marcelis and colleagues also included parents of patients, in whom the risk to develop schizophrenia is less densely distributed. This result is also in accordance with those reported by MyinGermeys and colleagues (2005) who reported, in an observational study measuring behavioural sensitisation, that subjects at genetic risk displayed increased behavioural sensitivity to daily life stress. The evidence suggests that enhanced reactivity to stress co-segregates with psychosis and therefore may be considered as a biological vulnerability marker. Given the fact that expression of vulnerability for schizophrenia takes on the form of continuous intermediary phenotypes such as cerebral grey matter volume and continuous cognitive ability, our results are line showing slight shifts in continuous distributions of ΔACTH and ΔHVA values in patients and, to a lesser degree, siblings compared to controls.
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4.3. Neurobiological mechanisms According to the vulnerability model of schizophrenia, an accurate marker of schizophrenia vulnerability should detect ill, stabilized or recovered patients with schizophrenia as well as unaffected individuals at high risk of schizophrenia. Thus, the correlated alterations in 2DG-induced ACTH and HVA responses in the unaffected sibling group in response to a 2DG challenge may be a biological marker of genetic vulnerability to schizophrenia. Given the amount of overlap in values between patients, siblings and controls, the findings are of aetiopathogenic rather than diagnostic importance. It has been reported that lower volumes of total cerebral grey and white matter (Marcelis et al., 2006) as well as the prefrontal cortex (Breier et al., 1993) in schizophrenia are associated with stress response dysregulation. The release of cortisol and its neurotoxicity for the hippocampus and prefrontal cortex (McEwen and Magarinos, 1997) may result in cognitive deficits (Mizoguchi et al., 2000), that have also been considered as a marker for genetic risk of schizophrenia (Lencz et al., 2006; for review see Brewer et al., 2006). Although traditional neurodevelopmental models of psychosis suggest that early life trauma damages the hippocampus resulting in heightened vulnerability towards the development of psychosis, more recent evidence suggests that changes to the medial temporal lobes and possibly the hippocampus occur close to the onset of a full-blown psychotic episode (Pantelis et al., 2003). A subtle brain process may be continuing through the first few years of a schizophrenic illness causing greater than the normal adult cortical deterioration (DeLisi et al., 1997; Thompson et al., 2001). Meta analytic (Wright et al., 2000; Boos et al., 2007) studies have suggested that hippocampal volume reduction in schizophrenia as well as impairment in cognitive skills are associated with hippocampal functioning such as memory, attention and problem-solving (Aleman et al., 1999). Indeed, cognitive function, specifically hippocampus-dependent verbal memory, is a strong predictor of functional outcome in schizophrenia (Green, 1996). Post-mortem studies in patients with schizophrenia reported reduced cell size in the hippocampus as well as abnormal apical dendrites on pyramidal cells in the subiculum (Rosoklija et al., 2000), findings that could be consistent with the known effects of stress and cortisol on apical dendrites (McEwen and Magarinos, 1997). In support of this idea, there have been reports of progressive cognitive deficits, including deficits in explicit memory (Cosway et al., 2000), and hippocampal volume reduction (Lawrie et al., 1999) in the time period from the prodrome to the onset of psychosis. Thus, ac-
cording to our findings, one can hypothesize that hyperreactivity of the HPA axis in response to stress in unaffected siblings could accelerate alterations in neuronal integrity, which in turn may contribute to alterations in subcortical circuits regulating dopaminergic response to stress. The findings support the hypothesis that alterations in cortico-subcortical dopaminergic connections affect psychosis susceptibility through an altered, illness-related stress response. Further studies are needed to investigate the relationship between volumes of grey/white matter, the hippocampus, HPA and dopaminergic reactivity to stress and cognitive performance in schizophrenia and in unaffected siblings. Moreover, even if plasma HVA is assumed to be an indicator of central dopaminergic activity (Amin et al., 1992), these relationships have to be confirmed directly. Role of the funding source Funding for this study was provided by a regional PHRC 2002 & CSR 2003; they had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Contributors M Saoud, T d'Amato & J van Os designed the study; M Saoud & T d'Amato wrote the protocol. J Brunelin, MF Suaud-Chagny & A Cochet managed the literature searches and analyses. J Brunelin and M Saoud undertook the statistical analysis and J Brunelin & J van Os wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest None.
Acknowledgements The authors thank the nurse team of Clinical Investigation Centre (CIC; Pr F. Gueyffier, Dr C. Cornu); JM Cottet-Emard; RM CottetEmard, AVouillarmet and B Claustrat for blood samples dosages; Dr P. Martin for 2DG preparation; G Pranal for English corrections. This study was supported by a regional grant: PHRC 2002 and by CSR 2003 CH Le Vinatier.
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