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Decreased galanin serum levels are associated with alcohol-craving during withdrawal
Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 568–572
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Decreased galanin serum levels are associated with alcohol-craving during withdrawal Annemarie Heberlein a,b,⁎, Marc Muschler a, Helge Frieling a,b, Bernd Lenz b, Julia Wilhelm a,b, Michael Gröschl c, Johannes Kornhuber b, Stefan Bleich a,b, Thomas Hillemacher a,b a b c
Center for Addiction Research, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, Germany Department of Psychiatry and Psychotherapy, University Hospital Erlangen, Germany Department of Pediatrics, University Hospital Erlangen, Germany
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Article history: Received 21 November 2010 Received in revised form 18 December 2010 Accepted 22 December 2010 Available online 31 December 2010 Keywords: Alcohol craving Alcohol dependence Alcohol withdrawal Galanin Neurotrophic growth factors
a b s t r a c t Background: The hypothalamic galanin expression has been associated with increased intake of carbohydrates and fats in preclinical studies. The appetite stimulating effect of galanin is thought to underlie the positive association between alcohol consumption and hypothalamic galanin expression observed in preclinical studies. Methods: In this pilot study we investigated alterations in galanin serum levels (33 male patients) in alcoholdependent patients during alcohol withdrawal (days 1, 7 and 14) in comparison to healthy controls (19 male controls). In order to assess the putative association between appetite regulation, galanin serum levels and alcohol consumption we additionally investigated the serum levels of insulin, glucose and triglycerides. Results: The galanin serum levels on day 1 of alcohol withdrawal were significantly reduced in the alcoholdependent patients (T = − 3.302, p = 0.002) and increased significantly from day 1 to day 14 of alcohol withdrawal (F = 6.437, p = 0.002). We found a significant negative association between the galanin serum levels and alcohol craving measured by the Obsessive Compulsive Drinking Scale (OCDS) (r=−0.449, p=0.009) and the obsessive subscale of the OCDS (r = − 0.521, p = 0.002) on day 1 of alcohol withdrawal. There was no association between the galanin serum levels and the parameters of energy homeostasis (triglycerides, cholesterol, insulin, and glucose) investigated. Conclusions: Acute alcohol withdrawal was associated with decreased galanin serum levels in this pilot study. There was no association between the galanin serum levels and the parameters of energy homeostasis. Further research of galanin serum levels in active drinkers will be necessary to clarify the putative association between galanin serum levels, appetite regulation and alcohol consumption. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Galanin is a regulatory neuropeptide that is known to be widely distributed within the nervous system and in the gut. Centrally, galanin has been described to interact with dopaminergic, serotonergic and Abbreviations: AAT, aspartate aminotransferase; ALAT, alanine aminotransferase; AUDIT-C, Alcohol Use Disorder Identification Test — Alcohol Consumption Questions; BAC, Breath Alcohol Concentration; BDI, Beck's Depression Inventory; BMI, body mass index; CIWA-Ar, Clinical Institute Withdrawal Assessment for Alcohol; DI, daily intake of alcohol in grams; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders; GGT, gamma glutamyl transferase; ICD-10, International Classification of Diseases; LC, leukocyte count; N.A., not available; OCDS, Obsessive and Compulsive Drinking Scale; SD, standard deviation; SESA, Severity Scale of Alcohol Dependence; STAI-I, State Anxiety, State and Trait Anxiety Inventory; STAI-II, Trait Anxiety, State and Trait Anxiety Inventory; TC, thrombocyte count; YD, years of drinking. ⁎ Corresponding author. Center for Addiction Research, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, Carl-Neuberg-Straße 1, D-30625 Hanover, Germany. Tel.: +49 176 1 532 8235. E-mail address: [email protected] (A. Heberlein). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.12.021
noradrenergic neurotransmission (Leibowitz et al., 2003; Sevcik et al., 1993). Modulation of dopaminergic neurotransmission by galanin is thought to underlie reduced behavioural responses following treatment with several non-caloric addictive substances like morphine or amphetamines as demonstrated in animal studies investigating either galanin overexpression or intracerebral galanin treatment (Pierce and Kumaresan, 2006; Tsuda et al., 1998). Contrasting to the antiaddictive effects of galanin regarding these non-caloric addictive substances a positive association between the central galanin expression and alcohol consumption has been reported in preclinical studies. In particular, central microinjection of galanin (Lewis et al., 2004; Rada et al., 2004) was observed to increase voluntary alcohol self-administration. Moreover, alcohol consumption has been associated with an increase of galanin mRNA expression (Leibowitz et al., 2003) indicating a stimulatory feedback loop between alcohol intake and the central galanin expression. This positive association between the central galanin expression and alcohol consumption is thought to
A. Heberlein et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 568–572
be based on the appetite stimulating function of galanin. Preclinical evidence shows that galanin increases food intake, in particular the intake of carbohydrates and fats, via the activation of hypothalamic neuronal circuits (Kyrkouli et al., 1990). Consistently, clinical studies report an association between circulating galanin levels, obesity (Poritsanos et al., 2009) and circulating triglycerides (Plaisier et al., 2009). In particular, it has been suggested that there is a positive feedback-loop between the hypothalamic galanin expression and alcohol consumption, which is stimulated by alcohol induced increase of peripheral triglyceride levels (Leibowitz, 2007). Consistent with the impact of galanin in energy homeostasis the hypothalamic galanin expression was found to be inhibited by insulin in diabetic as well as in nondiabetic rats (Tang et al., 1997; Wang and Leibowitz, 1997). Although in humans a putative association between the galanin serum levels and the symptomatology of alcohol dependence and alcohol withdrawal has not been investigated yet, there are study results that show associations between polymorphisms of the galanin gene and alcohol dependence (Belfer et al., 2006, 2007). In respect with these study results we investigated galanin serum levels in alcohol-dependent patients undergoing alcohol withdrawal and compared them with galanin serum levels obtained from healthy controls. Moreover, we aimed to assess a putative association between the appetite regulatory function of galanin and alcohol craving in the alcohol dependent patients. Therefore we investigated associations between peripheral blood levels of galanin in the alcohol-dependent patients, insulin, triglyceride levels and alcohol craving measured by the Obsessive Compulsive Drinking Scale (OCDS). 2. Materials and methods The present pilot study was part of a large prospective research project (Studies in Neuroendocrinology and Neurogenetics in Alcoholism (NENA), Heberlein et al., 2010) that was approved by the local Ethics Committee of the University of Erlangen-Nuremberg. The investigation was conducted in accordance with the Declaration of Helsinki of 1975 (revised in 2008). Each participant gave written informed consent. Because animal studies suggest gender differences regarding the regulation of galanin (Leibowitz et al., 2007) we included only male patients in the study. In total we investigated galanin serum levels of 33 male patients suffering from alcohol dependence according to ICD-10 and DSM-IV after admission for detoxification treatment (Clinic for Psychiatry, Psychotherapy and Psychosomatics, Obermain, Kutzenberg, Germany). Patients suffering from axis-one diagnoses other than alcohol and nicotine dependence were excluded from the study. Smokers (28 smokers) and non-smokers (5 non-smokers) were included in the group of patients. Further exclusion criteria were substance abuse apart from
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alcohol or nicotine (controlled for by drug urine screening), overweight (defined by age related body mass index), diabetes mellitus, diseases of the gastrointestinal tract, severe neurological diseases like cerebral ischemia, cerebral haemorrhage and epilepsy, and cardiovascular and renal diseases. All patients included in this study were treated with clomethiazole and oxcarbazepine (Martinotti et al., 2007) in order to prevent alcohol withdrawal symptoms. The dosage increase or decrease was adjusted in response to the severity of individual withdrawal symptoms. There was no further psychopharmacological treatment. All patients underwent a detailed physical examination, routine laboratory testing (including liver enzyme levels, blood count, glucose, and triglyceride levels) and additional measurements of serum insulin and serum cortisol. As a control group we enrolled randomly selected healthy male persons that did not suffer from any current somatic disease (19 male controls). Smokers (4 controls) and non-smokers (15 controls) were included in the control group. Controls were screened for mental diseases by a structured clinical interview. Controls were screened for alcohol dependence and abuse using the CAGE questionnaire (Mayfield et al., 1974) and the Alcohol Use Disorder Identification Test — Alcohol Consumption Questions (AUDIT-C) (Saunders et al., 1993). Controls received no psychopharmacological treatment. Controls were negative for alcohol abuse, alcohol dependence and any other mental disease according to ICD-10 or DSM-IV. There was no significant difference regarding sociodemographic characteristics in both groups (see Table 1 for details). Patients and controls were instructed to fast overnight. Fasting blood samples were taken directly on admission between 8 and 10 a. m. All blood samples were centrifuged and stored at − 80 °C immediately after collection. The galanin serum levels, serum insulin levels and serum glucose were investigated on day 1, day 7 and day 14 of alcohol withdrawal and were compared to the serum levels of the healthy control group. Galanin serum levels were assessed using the enzyme-linked immunosorbent assay (ELISA) (E1084Hu, USCN Life Science & Technology Co., Wuhan, China). Insulin serum levels were assessed by enzyme immuno assay (DSL 10-1600, Beckman Coulter Inc., Krefeld, Germany). The sample concentrations in each plate were calculated according to standard curves and dilution factors. Bodily symptoms of alcohol withdrawal like alterations in pulse frequency, blood pressure and body temperature were assessed directly before blood was taken on day 1, day 7 and day 14. In addition, cortisol serum levels were assessed by ELISA (DRG EI-1887, DRG Instruments GmbH, Marburg, Germany). Additional data like age, body mass index (BMI), years of drinking (YD) and daily intake of alcohol in grams (DI) were obtained in a structured interview.
Table 1 Characteristics of the alcohol dependent patients in comparison with the healthy controls.
Age (years) BMI Years of drinking Daily alcohol consumption (in g) SESA BAC
Galanin serum levels (pg/mL) OCDS BDI STAI-I STAI-II
Alcohol dependent patients (n = 33) Mean +/− SD
Healthy control group (n = 19) Mean +/− SD
42.91 +/− 7.34 22.97 +/− 2.49 9.09 +/− 7.19 202.50 +/− 98.23 55.63 +/− 18.77 1.00 +/− 1.04
41.11 +/− 19.58 23.74 +/− 3.16 N.A. N.A. N.A. N.A.
Day 1
Day 7
Day 14
12.94 +/− 9.03 19.42 +/− 7.39 17.03 +/− 9.53 48.33 +/− 12.54 48.70 +/− 11.30
33.25 +/− 57.08 10.59 +/− 6.99 9.35 +/− 9.27 37.55 +/− 13.12
29.74 +/− 17.71 9.34 +/− 6.73 5.86 +/− 7.89 36.64 +/− 11.62
81.11 +/− 137.92 N.A. 3.89 +/− 4.67 33.89 +/− 7.10 33.32 +/− 9.38
Legend: BMI: body mass index, SESA: Severity Scale of Alcohol Dependence, BAC: Breath Alcohol Concentration, OCDS: Obsessive and Compulsive Drinking Scale, STAI: State–Trait Anxiety Inventory, BDI: Beck's Depression Inventory, and N.A.: not available.
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Intensity of alcohol craving was measured by the Obsessive and Compulsive Drinking Scale and its obsessive and compulsive subscales (OCDS, Anton et al., 1995). The severity of alcohol dependence was assessed by the SESA questionnaire and its subscales (John et al., 2003). Data regarding affective symptoms were collected by the Beck's Depression Inventory (BDI, Beck et al., 1961) and the State and Trait Anxiety Inventory (STAI, Spielberger et al., 1970). The severity of alcohol withdrawal was measured by the Clinical Institute Withdrawal Assessment for Alcohol (CIWA-Ar) (Sullivan et al., 1989). Psychometric measurements of affective symptoms of alcohol withdrawal were taken once a day on day 1, day 7 and day 14. 2.1. Statistical analysis Hypothesis of the normal distribution of galanin serum levels was rejected by means of the Kolmogorov–Smirnov test. Therefore the galanin serum levels were ln-transformed in order to reach normal distribution. Correlations were calculated by Pearson's correlation coefficient. Differences in galanin serum levels were compared in the alcohol-dependent patients versus the controls using the t-test for independent samples. Alterations of the galanin serum levels during alcohol withdrawal were assessed using analysis of variance (repeatedmeasures ANOVA). Associations between the psychometric dimensions of alcohol withdrawal and the galanin serum levels were obtained by multiple regression analysis. Ln-transformed galanin serum levels were set as a dependent variable whereas the scores obtained in the OCDS and its obsessive subscale were set as independent variables. The statistical tests were performed applying a significance level of α b 0.05. Data were analyzed employing PASW Statistics 18.0 and Graph Pad Prism™ 5.0 (Graph Pad Software Inc., San Diego, CA). 3. Results Galanin serum levels were not significantly associated with age (r = −0.049, p = 0.787), self-reported duration of alcohol dependence (r = − 0.018, p = 0.920), self-reported amount of daily alcohol consumption in alcohol-dependent patients (r = −0.037, p = 0.839) and body mass index (BMI, r = 0.007, p = 0.968). 3.1. Alterations in alcohol-dependent patients compared to healthy controls The galanin serum levels were significantly reduced in the alcoholdependent patients on day 1 of alcohol withdrawal compared to the healthy control group (T = − 3.302, p = 0.002, see Fig. 1). The galanin serum levels increased significantly during alcohol withdrawal (F = 6.437, p = 0.002). Galanin serum levels on day 1 were not associated with blood levels of parameters that are typically altered in alcohol-dependent patients like gamma-glutamyltransferase (GGT) (r= 0.103, p = 0.568), aspartate-aminotransferase (AST) (r = −0.090, p = 0.617), alanineaminotransferase (ALT) (Spearman's rho= −0.010, p = 0.956), thrombocyte count (r = −0.226, p = 0.213) and the mean cellular erythrocyte volume (MCV) (Spearman's rho= 0.052, p = 0.772). 3.2. Association between galanin serum levels and parameters of energy homeostasis The decreased galanin serum levels on day 1 were not significantly associated with the levels of serum glucose (r = −0.222, p = 0.214), serum insulin (r = 0.054, p = 0.767), the triglyceride levels (r = 0.196, p = 0.273) and the serum cholesterol (r = 0.259, p = 0.146).
Fig. 1. Galanin serum levels of the alcohol dependent patients during alcohol withdrawal in comparison to the galanin serum levels of the healthy control group. The galanin serum levels of the alcohol dependent patients on day 1 of withdrawal are significantly decreased (left y axis) compared to the galanin serum levels of the healthy control group (right y axis, p = 0.002).
3.3. Association with bodily withdrawal symptoms There was no association between the decreased galanin serum levels on day 1 and systolic (r = − 0.054, p = 0.764) and diastolic (r = −0.137, p = 0.447) blood pressures, body temperature (r = −0.123, p = 0.502) and heart frequency (r = − 0.149, p = 0.407). There was no association between blood levels of cortisol and the galanin serum levels on day 1 (r = −0.008, p = 0.967). Neither the cortisol levels nor blood pressure and heart rate were associated with the OCDS total score or its subscales (data not shown). There was no association between the investigated parameters of alcohol withdrawal, the OCDS and its subscales and the galanin serum levels on day 7 and day 14 (data not shown). 3.4. Associations with psychometric dimensions of alcohol withdrawal The galanin serum levels on day 1 were significantly negatively associated with the total score of the OCDS (r = − 0.487, p = 0.006) and its obsessive subscale (r = − 0.517, p = 0.003). There was no significant association between the compulsive subscale of the OCDS and the decreased galanin serum levels on day 1 (r = −0.266, p = 0.134). Regression analysis (dependent variable: ln-transformed galanin serum levels, and independent variables: the total score of the OCDS and the score of the obsessive subscale of the OCDS) confirmed the association between the galanin serum levels and the total score of the OCDS and the score of its obsessive subscale on day 1 (F = 6.169, p = 0.006). Controlling the association between the galanin serum levels, the OCDS total score and its obsessive subscale for alcohol withdrawal intensity (total score of the Clinical Withdrawal Assessment for Alcohol (CIWA), systolic and diastolic blood pressures, heart rate, and cortisol serum levels) and the severity of alcohol dependence (SESA total score and anamnestic alcohol intake) did not change the results (data not shown). The galanin serum levels were not associated with cortisol serum levels, affective symptoms of alcohol withdrawal measured by the STAI-I, STAI-II, and the BDI nor with the psychometric intensity of alcohol withdrawal measured by the CIWA (data not shown). 4. Discussion In this study the galanin serum levels were significantly reduced in the alcohol-dependent patients on day 1 of alcohol withdrawal. The decreased galanin serum levels increased significantly during alcohol withdrawal. The galanin serum levels of the patients did not differ significantly from control levels in the time period from days 7 to 14 (see Fig. 1). With respect to the preclinical study results suggesting a positive feedback loop between the hypothalamic galanin expression
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and alcohol consumption (Leibowitz, 2007; Rada et al., 2004) and clinical study results reporting about positive associations between the galanin plasma levels and obesity (Baranowska et al., 1997), we expected increased galanin serum levels in the alcohol dependent patients reflecting a putative association between the appetite regulatory function of galanin and alcohol consumption. Contrasting to our expectations we found the galanin serum levels to be significantly decreased in the alcohol dependent patients. Preclinical study results show that central galanin expression decreases following treatment with the opioid antagonist naloxone (Leibowitz et al., 2003). These results suggest that the decrease of ethanol stimulated dopamine release may be causally related to a decrease of central galanin expression. Therefore, the decreased galanin serum levels found on day 1 of alcohol withdrawal in this study may be caused by central alterations in dopaminergic neurotransmission and the subsequent decrease of central galanin expression due to alcohol withdrawal. In respect with the lack of study results proofing a direct association between peripheral and central galanin expressions also increased receptor binding of the circulating galanin may explain the decreased galanin serum levels observed in this study. Galanin is reported to inhibit gastric acid secretion (Kisfalvi et al., 2000) and to modulate the motility of the gastrointestinal tract (Balint et al., 2001). Gastrointestinal symptoms like increased motility of the gastrointestinal tract are typical for patients actually withdrawing from alcohol. Therefore besides central alterations in galanin expression increased binding to peripheral galanin receptors may as well explain the decrease of galanin serum levels observed in this study. The reduced galanin serum levels on day 1 of withdrawal were negatively associated with the score obtained in the OCDS. Preclinical studies show a positive association between alcohol intake and the hypothalamic galanin expression (Lewis et al., 2004; Rada et al., 2004). In particular, a regulatory loop between alcohol consumption, circulating triglycerides and hypothalamic galanin expression was reported (Leibowitz, 1998). Moreover, a negative association between circulating insulin levels and the hypothalamic galanin expression was noted in preclinical and clinical studies underlining the involvement of hypothalamic galanin in the regulation of pancreatic peptides (Brodish et al., 1994; Tang et al., 1997). In respect with these study results we expected a positive association between craving for alcohol and the galanin serum levels. Moreover, we suggested an association between the galanin serum levels and the parameters of energy homeostasis investigated reflecting the appetite regulatory function of galanin. Though, contrasting to the results obtained in preclinical studies the galanin serum levels were not associated with the investigated triglyceride levels. Moreover, we found no association between the galanin serum levels and the circulating glucose and insulin levels in the alcohol dependent patients. Therefore the alterations of the galanin serum levels observed in this study and in particular the negative association between the galanin serum levels and the obsessive subscale of the OCDS may be independent from the appetite regulatory function of galanin. The negative association between the galanin serum levels and the OCDS and its obsessive subscale remained significant although controlled for withdrawal intensity measured psychometrically by the CIWA as well as for bodily withdrawal symptoms (blood pressure, heart rate, body temperature and serum cortisol levels). Therefore clinical withdrawal symptoms cannot explain the negative association between the OCDS and the galanin serum levels on day 1 of alcohol withdrawal found in this study. Although the neurobiological mechanisms that underlie alcohol craving have not been fully investigated, obsessive craving is thought to be based on disturbances in serotonergic neurotransmission (Verheul et al., 1999). In contrast, compulsive craving is defined by uncontrolled drug seeking behaviour and uncontrolled drug intake (Robinson and Brewer, 2008). Regarding this concept craving
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associated with the appetite regulatory function of galanin would be expected to be compulsive rather than obsessive. Consistent with the lack of an association between alcohol withdrawal symptoms, the serum triglyceride, the serum glucose and the serum galanin levels, we found a strong relationship between the decreased galanin serum levels and the obsessive subscale of the OCDS only. Galanin is known to closely interact with further neurotransmitters. For example, co-localization of galanin and serotonin has been reported in the dorsal raphe nuclei as well as with noradrenalin in the locus coeruleus (Ögren et al., 2010). Moreover, galanin is known to be involved in the regulation of the central release of serotonin and noradrenaline, thereby affecting emotional processes like anxiety and depression (Ögren et al., 2010). Preclinical study results show an inhibitory effect of galaninergic neurotransmission on the serotonergic neurons in the dorsal raphe nuclei as well as on the noradrenergic neurons in the locus coeruleus (Hökfelt and Tatemoto, 2008). Therefore disturbances in serotonergic neurotransmission during early alcohol withdrawal may underlie the negative association between the obsessive subscale of the OCDS and alcohol craving observed in this pilot study, although the underlying neurobiological mechanisms cannot be uncovered by the data obtained. This pilot study was limited in the following ways. We investigated serum levels of galanin only in 33 male patients and only during early alcohol withdrawal. Moreover, patients investigated in our study received various doses of oxcarbazepine and clomethiazole depending on the severity of their individual withdrawal symptoms. The known effects of these medications on the peripheral blood levels of neurotrophic growth factors limit the explanatory power of the results obtained in this pilot study. Follow-up studies of galanin blood levels in active drinkers without psychotropic medication are warranted in order to investigate the putative association between galanin serum levels, appetite regulation and alcohol consumption in alcohol dependent patients. We also included smokers and nonsmokers in our research. Comparisons of the differences between smokers and non-smokers showed no significant difference between the galanin serum levels of alcohol-dependent patients who were actively smoking (28 patients) and those of the non-smokers included in our study (5 patients). Though, follow-up studies should be based on samples of all-smokers or all-non-smokers to avoid invalid results. 5. Conclusions In conclusion, we found decreased galanin serum levels in alcohol dependent patients during early alcohol withdrawal. The reduced galanin serum levels were associated with increased alcohol craving measured by the OCDS. Alterations in the galanin serum levels were neither associated with psychometric dimensions of alcohol withdrawal (i.e. depression and anxiety) nor with parameters of energy homeostasis (i.e. triglycerides, glucose, and insulin) nor with bodily withdrawal symptoms (i.e. heart rate, blood pressure, body temperature, and cortisol serum levels). Further research will be necessary in order to interpret the preliminary results obtained in this pilot study correctly. In particular, studies are warranted that investigate the relationship between peptides involved in energy homeostasis and the galanin serum levels in alcohol dependent patients not currently withdrawing from alcohol. References Anton RF, Moak DH, Latham P. The Obsessive Compulsive Drinking Scale: a self-rated instrument for the quantification of thoughts about alcohol and drinking behavior. Alcohol Clin Exp Res 1995;19:92–9. Balint A, Feher E, Kisfalvi Jr I, Mate M, Zelles T, Vizi ES, et al. Functional and immunocytochemical evidence that galanin is a physiological regulator of human jejunal motility. J Physiol Paris 2001;95:129–35. Baranowska B, Wasilewska-Dziubinska E, Radzikowska M, Plonowski A, Roguski K. Neuropeptide Y, galanin, and leptin release in obese women and in women with anorexia nervosa. Metabolism 1997;46:1384–9.
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Decreased galanin serum levels are associated with alcohol-craving during withdrawal Annemarie Heberlein a,b,⁎, Marc Muschler a, Helge Frieling a,b, Bernd Lenz b, Julia Wilhelm a,b, Michael Gröschl c, Johannes Kornhuber b, Stefan Bleich a,b, Thomas Hillemacher a,b a b c
Center for Addiction Research, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, Germany Department of Psychiatry and Psychotherapy, University Hospital Erlangen, Germany Department of Pediatrics, University Hospital Erlangen, Germany
a r t i c l e
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Article history: Received 21 November 2010 Received in revised form 18 December 2010 Accepted 22 December 2010 Available online 31 December 2010 Keywords: Alcohol craving Alcohol dependence Alcohol withdrawal Galanin Neurotrophic growth factors
a b s t r a c t Background: The hypothalamic galanin expression has been associated with increased intake of carbohydrates and fats in preclinical studies. The appetite stimulating effect of galanin is thought to underlie the positive association between alcohol consumption and hypothalamic galanin expression observed in preclinical studies. Methods: In this pilot study we investigated alterations in galanin serum levels (33 male patients) in alcoholdependent patients during alcohol withdrawal (days 1, 7 and 14) in comparison to healthy controls (19 male controls). In order to assess the putative association between appetite regulation, galanin serum levels and alcohol consumption we additionally investigated the serum levels of insulin, glucose and triglycerides. Results: The galanin serum levels on day 1 of alcohol withdrawal were significantly reduced in the alcoholdependent patients (T = − 3.302, p = 0.002) and increased significantly from day 1 to day 14 of alcohol withdrawal (F = 6.437, p = 0.002). We found a significant negative association between the galanin serum levels and alcohol craving measured by the Obsessive Compulsive Drinking Scale (OCDS) (r=−0.449, p=0.009) and the obsessive subscale of the OCDS (r = − 0.521, p = 0.002) on day 1 of alcohol withdrawal. There was no association between the galanin serum levels and the parameters of energy homeostasis (triglycerides, cholesterol, insulin, and glucose) investigated. Conclusions: Acute alcohol withdrawal was associated with decreased galanin serum levels in this pilot study. There was no association between the galanin serum levels and the parameters of energy homeostasis. Further research of galanin serum levels in active drinkers will be necessary to clarify the putative association between galanin serum levels, appetite regulation and alcohol consumption. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Galanin is a regulatory neuropeptide that is known to be widely distributed within the nervous system and in the gut. Centrally, galanin has been described to interact with dopaminergic, serotonergic and Abbreviations: AAT, aspartate aminotransferase; ALAT, alanine aminotransferase; AUDIT-C, Alcohol Use Disorder Identification Test — Alcohol Consumption Questions; BAC, Breath Alcohol Concentration; BDI, Beck's Depression Inventory; BMI, body mass index; CIWA-Ar, Clinical Institute Withdrawal Assessment for Alcohol; DI, daily intake of alcohol in grams; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders; GGT, gamma glutamyl transferase; ICD-10, International Classification of Diseases; LC, leukocyte count; N.A., not available; OCDS, Obsessive and Compulsive Drinking Scale; SD, standard deviation; SESA, Severity Scale of Alcohol Dependence; STAI-I, State Anxiety, State and Trait Anxiety Inventory; STAI-II, Trait Anxiety, State and Trait Anxiety Inventory; TC, thrombocyte count; YD, years of drinking. ⁎ Corresponding author. Center for Addiction Research, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, Carl-Neuberg-Straße 1, D-30625 Hanover, Germany. Tel.: +49 176 1 532 8235. E-mail address: [email protected] (A. Heberlein). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.12.021
noradrenergic neurotransmission (Leibowitz et al., 2003; Sevcik et al., 1993). Modulation of dopaminergic neurotransmission by galanin is thought to underlie reduced behavioural responses following treatment with several non-caloric addictive substances like morphine or amphetamines as demonstrated in animal studies investigating either galanin overexpression or intracerebral galanin treatment (Pierce and Kumaresan, 2006; Tsuda et al., 1998). Contrasting to the antiaddictive effects of galanin regarding these non-caloric addictive substances a positive association between the central galanin expression and alcohol consumption has been reported in preclinical studies. In particular, central microinjection of galanin (Lewis et al., 2004; Rada et al., 2004) was observed to increase voluntary alcohol self-administration. Moreover, alcohol consumption has been associated with an increase of galanin mRNA expression (Leibowitz et al., 2003) indicating a stimulatory feedback loop between alcohol intake and the central galanin expression. This positive association between the central galanin expression and alcohol consumption is thought to
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be based on the appetite stimulating function of galanin. Preclinical evidence shows that galanin increases food intake, in particular the intake of carbohydrates and fats, via the activation of hypothalamic neuronal circuits (Kyrkouli et al., 1990). Consistently, clinical studies report an association between circulating galanin levels, obesity (Poritsanos et al., 2009) and circulating triglycerides (Plaisier et al., 2009). In particular, it has been suggested that there is a positive feedback-loop between the hypothalamic galanin expression and alcohol consumption, which is stimulated by alcohol induced increase of peripheral triglyceride levels (Leibowitz, 2007). Consistent with the impact of galanin in energy homeostasis the hypothalamic galanin expression was found to be inhibited by insulin in diabetic as well as in nondiabetic rats (Tang et al., 1997; Wang and Leibowitz, 1997). Although in humans a putative association between the galanin serum levels and the symptomatology of alcohol dependence and alcohol withdrawal has not been investigated yet, there are study results that show associations between polymorphisms of the galanin gene and alcohol dependence (Belfer et al., 2006, 2007). In respect with these study results we investigated galanin serum levels in alcohol-dependent patients undergoing alcohol withdrawal and compared them with galanin serum levels obtained from healthy controls. Moreover, we aimed to assess a putative association between the appetite regulatory function of galanin and alcohol craving in the alcohol dependent patients. Therefore we investigated associations between peripheral blood levels of galanin in the alcohol-dependent patients, insulin, triglyceride levels and alcohol craving measured by the Obsessive Compulsive Drinking Scale (OCDS). 2. Materials and methods The present pilot study was part of a large prospective research project (Studies in Neuroendocrinology and Neurogenetics in Alcoholism (NENA), Heberlein et al., 2010) that was approved by the local Ethics Committee of the University of Erlangen-Nuremberg. The investigation was conducted in accordance with the Declaration of Helsinki of 1975 (revised in 2008). Each participant gave written informed consent. Because animal studies suggest gender differences regarding the regulation of galanin (Leibowitz et al., 2007) we included only male patients in the study. In total we investigated galanin serum levels of 33 male patients suffering from alcohol dependence according to ICD-10 and DSM-IV after admission for detoxification treatment (Clinic for Psychiatry, Psychotherapy and Psychosomatics, Obermain, Kutzenberg, Germany). Patients suffering from axis-one diagnoses other than alcohol and nicotine dependence were excluded from the study. Smokers (28 smokers) and non-smokers (5 non-smokers) were included in the group of patients. Further exclusion criteria were substance abuse apart from
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alcohol or nicotine (controlled for by drug urine screening), overweight (defined by age related body mass index), diabetes mellitus, diseases of the gastrointestinal tract, severe neurological diseases like cerebral ischemia, cerebral haemorrhage and epilepsy, and cardiovascular and renal diseases. All patients included in this study were treated with clomethiazole and oxcarbazepine (Martinotti et al., 2007) in order to prevent alcohol withdrawal symptoms. The dosage increase or decrease was adjusted in response to the severity of individual withdrawal symptoms. There was no further psychopharmacological treatment. All patients underwent a detailed physical examination, routine laboratory testing (including liver enzyme levels, blood count, glucose, and triglyceride levels) and additional measurements of serum insulin and serum cortisol. As a control group we enrolled randomly selected healthy male persons that did not suffer from any current somatic disease (19 male controls). Smokers (4 controls) and non-smokers (15 controls) were included in the control group. Controls were screened for mental diseases by a structured clinical interview. Controls were screened for alcohol dependence and abuse using the CAGE questionnaire (Mayfield et al., 1974) and the Alcohol Use Disorder Identification Test — Alcohol Consumption Questions (AUDIT-C) (Saunders et al., 1993). Controls received no psychopharmacological treatment. Controls were negative for alcohol abuse, alcohol dependence and any other mental disease according to ICD-10 or DSM-IV. There was no significant difference regarding sociodemographic characteristics in both groups (see Table 1 for details). Patients and controls were instructed to fast overnight. Fasting blood samples were taken directly on admission between 8 and 10 a. m. All blood samples were centrifuged and stored at − 80 °C immediately after collection. The galanin serum levels, serum insulin levels and serum glucose were investigated on day 1, day 7 and day 14 of alcohol withdrawal and were compared to the serum levels of the healthy control group. Galanin serum levels were assessed using the enzyme-linked immunosorbent assay (ELISA) (E1084Hu, USCN Life Science & Technology Co., Wuhan, China). Insulin serum levels were assessed by enzyme immuno assay (DSL 10-1600, Beckman Coulter Inc., Krefeld, Germany). The sample concentrations in each plate were calculated according to standard curves and dilution factors. Bodily symptoms of alcohol withdrawal like alterations in pulse frequency, blood pressure and body temperature were assessed directly before blood was taken on day 1, day 7 and day 14. In addition, cortisol serum levels were assessed by ELISA (DRG EI-1887, DRG Instruments GmbH, Marburg, Germany). Additional data like age, body mass index (BMI), years of drinking (YD) and daily intake of alcohol in grams (DI) were obtained in a structured interview.
Table 1 Characteristics of the alcohol dependent patients in comparison with the healthy controls.
Age (years) BMI Years of drinking Daily alcohol consumption (in g) SESA BAC
Galanin serum levels (pg/mL) OCDS BDI STAI-I STAI-II
Alcohol dependent patients (n = 33) Mean +/− SD
Healthy control group (n = 19) Mean +/− SD
42.91 +/− 7.34 22.97 +/− 2.49 9.09 +/− 7.19 202.50 +/− 98.23 55.63 +/− 18.77 1.00 +/− 1.04
41.11 +/− 19.58 23.74 +/− 3.16 N.A. N.A. N.A. N.A.
Day 1
Day 7
Day 14
12.94 +/− 9.03 19.42 +/− 7.39 17.03 +/− 9.53 48.33 +/− 12.54 48.70 +/− 11.30
33.25 +/− 57.08 10.59 +/− 6.99 9.35 +/− 9.27 37.55 +/− 13.12
29.74 +/− 17.71 9.34 +/− 6.73 5.86 +/− 7.89 36.64 +/− 11.62
81.11 +/− 137.92 N.A. 3.89 +/− 4.67 33.89 +/− 7.10 33.32 +/− 9.38
Legend: BMI: body mass index, SESA: Severity Scale of Alcohol Dependence, BAC: Breath Alcohol Concentration, OCDS: Obsessive and Compulsive Drinking Scale, STAI: State–Trait Anxiety Inventory, BDI: Beck's Depression Inventory, and N.A.: not available.
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Intensity of alcohol craving was measured by the Obsessive and Compulsive Drinking Scale and its obsessive and compulsive subscales (OCDS, Anton et al., 1995). The severity of alcohol dependence was assessed by the SESA questionnaire and its subscales (John et al., 2003). Data regarding affective symptoms were collected by the Beck's Depression Inventory (BDI, Beck et al., 1961) and the State and Trait Anxiety Inventory (STAI, Spielberger et al., 1970). The severity of alcohol withdrawal was measured by the Clinical Institute Withdrawal Assessment for Alcohol (CIWA-Ar) (Sullivan et al., 1989). Psychometric measurements of affective symptoms of alcohol withdrawal were taken once a day on day 1, day 7 and day 14. 2.1. Statistical analysis Hypothesis of the normal distribution of galanin serum levels was rejected by means of the Kolmogorov–Smirnov test. Therefore the galanin serum levels were ln-transformed in order to reach normal distribution. Correlations were calculated by Pearson's correlation coefficient. Differences in galanin serum levels were compared in the alcohol-dependent patients versus the controls using the t-test for independent samples. Alterations of the galanin serum levels during alcohol withdrawal were assessed using analysis of variance (repeatedmeasures ANOVA). Associations between the psychometric dimensions of alcohol withdrawal and the galanin serum levels were obtained by multiple regression analysis. Ln-transformed galanin serum levels were set as a dependent variable whereas the scores obtained in the OCDS and its obsessive subscale were set as independent variables. The statistical tests were performed applying a significance level of α b 0.05. Data were analyzed employing PASW Statistics 18.0 and Graph Pad Prism™ 5.0 (Graph Pad Software Inc., San Diego, CA). 3. Results Galanin serum levels were not significantly associated with age (r = −0.049, p = 0.787), self-reported duration of alcohol dependence (r = − 0.018, p = 0.920), self-reported amount of daily alcohol consumption in alcohol-dependent patients (r = −0.037, p = 0.839) and body mass index (BMI, r = 0.007, p = 0.968). 3.1. Alterations in alcohol-dependent patients compared to healthy controls The galanin serum levels were significantly reduced in the alcoholdependent patients on day 1 of alcohol withdrawal compared to the healthy control group (T = − 3.302, p = 0.002, see Fig. 1). The galanin serum levels increased significantly during alcohol withdrawal (F = 6.437, p = 0.002). Galanin serum levels on day 1 were not associated with blood levels of parameters that are typically altered in alcohol-dependent patients like gamma-glutamyltransferase (GGT) (r= 0.103, p = 0.568), aspartate-aminotransferase (AST) (r = −0.090, p = 0.617), alanineaminotransferase (ALT) (Spearman's rho= −0.010, p = 0.956), thrombocyte count (r = −0.226, p = 0.213) and the mean cellular erythrocyte volume (MCV) (Spearman's rho= 0.052, p = 0.772). 3.2. Association between galanin serum levels and parameters of energy homeostasis The decreased galanin serum levels on day 1 were not significantly associated with the levels of serum glucose (r = −0.222, p = 0.214), serum insulin (r = 0.054, p = 0.767), the triglyceride levels (r = 0.196, p = 0.273) and the serum cholesterol (r = 0.259, p = 0.146).
Fig. 1. Galanin serum levels of the alcohol dependent patients during alcohol withdrawal in comparison to the galanin serum levels of the healthy control group. The galanin serum levels of the alcohol dependent patients on day 1 of withdrawal are significantly decreased (left y axis) compared to the galanin serum levels of the healthy control group (right y axis, p = 0.002).
3.3. Association with bodily withdrawal symptoms There was no association between the decreased galanin serum levels on day 1 and systolic (r = − 0.054, p = 0.764) and diastolic (r = −0.137, p = 0.447) blood pressures, body temperature (r = −0.123, p = 0.502) and heart frequency (r = − 0.149, p = 0.407). There was no association between blood levels of cortisol and the galanin serum levels on day 1 (r = −0.008, p = 0.967). Neither the cortisol levels nor blood pressure and heart rate were associated with the OCDS total score or its subscales (data not shown). There was no association between the investigated parameters of alcohol withdrawal, the OCDS and its subscales and the galanin serum levels on day 7 and day 14 (data not shown). 3.4. Associations with psychometric dimensions of alcohol withdrawal The galanin serum levels on day 1 were significantly negatively associated with the total score of the OCDS (r = − 0.487, p = 0.006) and its obsessive subscale (r = − 0.517, p = 0.003). There was no significant association between the compulsive subscale of the OCDS and the decreased galanin serum levels on day 1 (r = −0.266, p = 0.134). Regression analysis (dependent variable: ln-transformed galanin serum levels, and independent variables: the total score of the OCDS and the score of the obsessive subscale of the OCDS) confirmed the association between the galanin serum levels and the total score of the OCDS and the score of its obsessive subscale on day 1 (F = 6.169, p = 0.006). Controlling the association between the galanin serum levels, the OCDS total score and its obsessive subscale for alcohol withdrawal intensity (total score of the Clinical Withdrawal Assessment for Alcohol (CIWA), systolic and diastolic blood pressures, heart rate, and cortisol serum levels) and the severity of alcohol dependence (SESA total score and anamnestic alcohol intake) did not change the results (data not shown). The galanin serum levels were not associated with cortisol serum levels, affective symptoms of alcohol withdrawal measured by the STAI-I, STAI-II, and the BDI nor with the psychometric intensity of alcohol withdrawal measured by the CIWA (data not shown). 4. Discussion In this study the galanin serum levels were significantly reduced in the alcohol-dependent patients on day 1 of alcohol withdrawal. The decreased galanin serum levels increased significantly during alcohol withdrawal. The galanin serum levels of the patients did not differ significantly from control levels in the time period from days 7 to 14 (see Fig. 1). With respect to the preclinical study results suggesting a positive feedback loop between the hypothalamic galanin expression
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and alcohol consumption (Leibowitz, 2007; Rada et al., 2004) and clinical study results reporting about positive associations between the galanin plasma levels and obesity (Baranowska et al., 1997), we expected increased galanin serum levels in the alcohol dependent patients reflecting a putative association between the appetite regulatory function of galanin and alcohol consumption. Contrasting to our expectations we found the galanin serum levels to be significantly decreased in the alcohol dependent patients. Preclinical study results show that central galanin expression decreases following treatment with the opioid antagonist naloxone (Leibowitz et al., 2003). These results suggest that the decrease of ethanol stimulated dopamine release may be causally related to a decrease of central galanin expression. Therefore, the decreased galanin serum levels found on day 1 of alcohol withdrawal in this study may be caused by central alterations in dopaminergic neurotransmission and the subsequent decrease of central galanin expression due to alcohol withdrawal. In respect with the lack of study results proofing a direct association between peripheral and central galanin expressions also increased receptor binding of the circulating galanin may explain the decreased galanin serum levels observed in this study. Galanin is reported to inhibit gastric acid secretion (Kisfalvi et al., 2000) and to modulate the motility of the gastrointestinal tract (Balint et al., 2001). Gastrointestinal symptoms like increased motility of the gastrointestinal tract are typical for patients actually withdrawing from alcohol. Therefore besides central alterations in galanin expression increased binding to peripheral galanin receptors may as well explain the decrease of galanin serum levels observed in this study. The reduced galanin serum levels on day 1 of withdrawal were negatively associated with the score obtained in the OCDS. Preclinical studies show a positive association between alcohol intake and the hypothalamic galanin expression (Lewis et al., 2004; Rada et al., 2004). In particular, a regulatory loop between alcohol consumption, circulating triglycerides and hypothalamic galanin expression was reported (Leibowitz, 1998). Moreover, a negative association between circulating insulin levels and the hypothalamic galanin expression was noted in preclinical and clinical studies underlining the involvement of hypothalamic galanin in the regulation of pancreatic peptides (Brodish et al., 1994; Tang et al., 1997). In respect with these study results we expected a positive association between craving for alcohol and the galanin serum levels. Moreover, we suggested an association between the galanin serum levels and the parameters of energy homeostasis investigated reflecting the appetite regulatory function of galanin. Though, contrasting to the results obtained in preclinical studies the galanin serum levels were not associated with the investigated triglyceride levels. Moreover, we found no association between the galanin serum levels and the circulating glucose and insulin levels in the alcohol dependent patients. Therefore the alterations of the galanin serum levels observed in this study and in particular the negative association between the galanin serum levels and the obsessive subscale of the OCDS may be independent from the appetite regulatory function of galanin. The negative association between the galanin serum levels and the OCDS and its obsessive subscale remained significant although controlled for withdrawal intensity measured psychometrically by the CIWA as well as for bodily withdrawal symptoms (blood pressure, heart rate, body temperature and serum cortisol levels). Therefore clinical withdrawal symptoms cannot explain the negative association between the OCDS and the galanin serum levels on day 1 of alcohol withdrawal found in this study. Although the neurobiological mechanisms that underlie alcohol craving have not been fully investigated, obsessive craving is thought to be based on disturbances in serotonergic neurotransmission (Verheul et al., 1999). In contrast, compulsive craving is defined by uncontrolled drug seeking behaviour and uncontrolled drug intake (Robinson and Brewer, 2008). Regarding this concept craving
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associated with the appetite regulatory function of galanin would be expected to be compulsive rather than obsessive. Consistent with the lack of an association between alcohol withdrawal symptoms, the serum triglyceride, the serum glucose and the serum galanin levels, we found a strong relationship between the decreased galanin serum levels and the obsessive subscale of the OCDS only. Galanin is known to closely interact with further neurotransmitters. For example, co-localization of galanin and serotonin has been reported in the dorsal raphe nuclei as well as with noradrenalin in the locus coeruleus (Ögren et al., 2010). Moreover, galanin is known to be involved in the regulation of the central release of serotonin and noradrenaline, thereby affecting emotional processes like anxiety and depression (Ögren et al., 2010). Preclinical study results show an inhibitory effect of galaninergic neurotransmission on the serotonergic neurons in the dorsal raphe nuclei as well as on the noradrenergic neurons in the locus coeruleus (Hökfelt and Tatemoto, 2008). Therefore disturbances in serotonergic neurotransmission during early alcohol withdrawal may underlie the negative association between the obsessive subscale of the OCDS and alcohol craving observed in this pilot study, although the underlying neurobiological mechanisms cannot be uncovered by the data obtained. This pilot study was limited in the following ways. We investigated serum levels of galanin only in 33 male patients and only during early alcohol withdrawal. Moreover, patients investigated in our study received various doses of oxcarbazepine and clomethiazole depending on the severity of their individual withdrawal symptoms. The known effects of these medications on the peripheral blood levels of neurotrophic growth factors limit the explanatory power of the results obtained in this pilot study. Follow-up studies of galanin blood levels in active drinkers without psychotropic medication are warranted in order to investigate the putative association between galanin serum levels, appetite regulation and alcohol consumption in alcohol dependent patients. We also included smokers and nonsmokers in our research. Comparisons of the differences between smokers and non-smokers showed no significant difference between the galanin serum levels of alcohol-dependent patients who were actively smoking (28 patients) and those of the non-smokers included in our study (5 patients). Though, follow-up studies should be based on samples of all-smokers or all-non-smokers to avoid invalid results. 5. Conclusions In conclusion, we found decreased galanin serum levels in alcohol dependent patients during early alcohol withdrawal. The reduced galanin serum levels were associated with increased alcohol craving measured by the OCDS. Alterations in the galanin serum levels were neither associated with psychometric dimensions of alcohol withdrawal (i.e. depression and anxiety) nor with parameters of energy homeostasis (i.e. triglycerides, glucose, and insulin) nor with bodily withdrawal symptoms (i.e. heart rate, blood pressure, body temperature, and cortisol serum levels). Further research will be necessary in order to interpret the preliminary results obtained in this pilot study correctly. In particular, studies are warranted that investigate the relationship between peptides involved in energy homeostasis and the galanin serum levels in alcohol dependent patients not currently withdrawing from alcohol. References Anton RF, Moak DH, Latham P. The Obsessive Compulsive Drinking Scale: a self-rated instrument for the quantification of thoughts about alcohol and drinking behavior. Alcohol Clin Exp Res 1995;19:92–9. Balint A, Feher E, Kisfalvi Jr I, Mate M, Zelles T, Vizi ES, et al. Functional and immunocytochemical evidence that galanin is a physiological regulator of human jejunal motility. J Physiol Paris 2001;95:129–35. Baranowska B, Wasilewska-Dziubinska E, Radzikowska M, Plonowski A, Roguski K. Neuropeptide Y, galanin, and leptin release in obese women and in women with anorexia nervosa. Metabolism 1997;46:1384–9.
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