ALCOHOL INTAKE, TUMOUR NECROSIS FACTOR-{alpha}, LEPTIN AND CRAVING: FACTORS OF A POSSIBLY VICIOUS CIRCLE?

F. Kiefer,*, H. Jahn, M. Schick and K. Wiedemann

Department of Psychiatry, University Hospital of Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany

Received 28 February 2002; in revised form 26 April 2002; accepted 26 April 2002


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aims: Since the appetite-regulating peptide leptin was recently found to be highly correlated with both craving for alcohol and lifetime ethanol intake, the aim of our study was to test the hypothesis whether tumour necrosis factor-{alpha} (TNF-{alpha}) might be the factor that links alcohol intake with elevated leptin levels. Methods: TNF-{alpha}, leptin, and alcohol craving were assessed in male alcohol addicts at the onset of alcohol withdrawal and in matched controls. Results: Increased leptin plasma levels in alcohol addicts correlated significantly with an enhanced secretion of TNF-{alpha}, which was itself related to the duration of alcohol misuse. Conclusions: Since leptin was shown to be associated with alcohol craving, a possible vicious circle is suggested, including the components: alcohol intake, increase of TNF-{alpha}, enhanced leptin secretion, enhanced alcohol craving, and consecutively increased alcohol intake.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neuropeptide leptin links adipose stores with hypothalamic centres and serves as an endocrine signal involved in the regulation of appetite (Campfield et al., 1995Go; Halaas et al., 1995Go; Pelleymounter et al., 1995Go). Additionally, leptin has been shown to alter the gene expression of corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC) in the hypothalamus, suggesting a role both in regulating the hypothalamic–pituitary–adrenocortical (HPA) stress hormone axis and possibly in the endorphinergic modulation of the drug reward system (Inui, 1999Go). Both systems are altered specifically in patients suffering from alcoholism, and we recently reported significantly increased leptin plasma levels at the onset of alcohol withdrawal, which were highly correlated with self-rated craving for alcohol (Kiefer et al., 2001bGo). Moreover, Nicolás et al. (2001) found plasma leptin elevated in active alcoholics and significantly related to lifetime ethanol consumption. The longer the process of addiction progresses, the higher the leptin plasma levels, and the more frequent craving for the drug.

Based on these findings, two questions seem relevant: (1) does leptin itself mediate alcohol craving in addicts; (2) what might be the pathophysiological mechanism that links alcohol intake with increased leptin plasma levels in normal weighted alcohol addicts?

Regarding question (1), we tested the hypothesis whether application of exogenous leptin modulates voluntary alcohol consumption in mice. We found free-choice ethanol consumption in mice habituated to ethanol to be significantly elevated after intraperitoneal (i.p.) injection of a 1 mg/kg dose of leptin, indicating a positive alcohol-deprivation effect (Kiefer et al., 2001cGo). We suggested that leptin may enhance motivation for alcohol consumption in habituated mice after alcohol withdrawal.

To answer question (2), we have now conducted a study of whether TNF-{alpha} might represent the link that connects alcohol intake with increased leptin plasma levels. This focus on TNF-{alpha} is based upon the rationale that TNF-{alpha} and TNF-{alpha}-inducible leptin mRNA levels have been shown to be increased after chronic intake of ethanol in rats independently of calorie consumption (Lin et al., 1998Go). White adipose tissue was proposed to be an important source of cytokines in non-obese subjects and was suggested to be a target for ethanol's actions. Moreover, since leptin levels have been reported to be directly increased after injection of TNF-{alpha} (Barbier et al., 1998Go), a pathway including alcohol intake, TNF-{alpha} increase, and leptin increase seemed possible. To test this hypothesis, we studied TNF-{alpha} secretion, leptin levels and craving in a sample of alcohol addicts with a well-documented history of alcoholism. Since craving in alcoholics is regularly detectable during alcohol withdrawal, we investigated the sample during an inpatient detoxification and abstention programme. Additionally, we chose to measure plasma concentrations of cortisol to identify mutual interactions with the HPA axis activity.


    SUBJECTS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
A consecutive sample of 30 male alcoholic inpatients (mean age ± SD: 41.0 ± 8.3 years, weight: 70.0 ± 8.0 kg) admitted to the psychiatric department for detoxification purposes was included in the study on the day following admission, 15–25 h after last alcohol intake. Subjects were primary alcoholics according to Schuckit et al. (1994) without any psychiatric comorbidity or substance abuse other than alcohol or nicotine. Patients fulfilled at least six of nine diagnostic criteria for dependence according to DSM-IV (American Psychiatric Association, 1994Go). All patients were off medication prior to the study, including neuroleptics, antidepressants, benzodiazepines, antihypertensive or hypoglycaemic drugs for at least 3 months. The following data were gathered for the present investigation: body weight, years since symptoms of alcohol-related problems first occurred, number of inpatient detoxifications, amount of alcohol consumed daily, tobacco consumed, family history of alcoholism, and routine laboratory parameters. Patient characteristics are presented in Table 1Go.


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Table 1. Patient sample data
 
For comparison, 30 mentally and physically healthy age-matched male volunteers (age: 39 ± 8.4 years, weight: 75 ± 8.6 kg) were included in the study. These subjects had no history of substance misuse or dependence and had abstained from alcoholic beverages for >=72 h.

Drug screening for benzodiazepines, opioids, cannabinoids, cocaine and amphetamines revealed negative results in all patients and controls. None of the patients or controls showed any symptoms of inflammatory, cardiac, endocrine, renal or hepatic disease, except of nutrient fatty liver disease in the inpatient sample.

The study protocol was in accordance with the Declaration of Helsinki (1964) and was approved by the Local Ethics Committee. All subjects gave written informed consent prior to the investigation.

Testing procedures
Blood samples (9 ml each) for determination of plasma TNF-{alpha}, plasma leptin, plasma cortisol levels by radioimmunoassay (RIA), and routine laboratory parameters were drawn at 09:00, 15–25 h after the last alcohol intake. Basic data were obtained with a structured interview. Craving was assessed with the German version of the Obsessive–Compulsive Drinking Scale (OCDS) (Anton et al., 1996Go; Mann and Ackermann, 2000Go) including three 100 mm visual analogue scales (VAS) concerning mean and maximum craving and its frequency.

Assay procedures
Blood samples were drawn into EDTA-coated tubes, cooled, and immediately centrifuged; plasma was collected in aliquots stored at –80°C until analysis.

TNF-{alpha} concentrations were analysed using a commercially available RIA kit with the coated tube technique (DRG Diagnostics, Mountainside, NY, USA). The detection limit was 3.0 ng/ml plasma; intra- and interassay coefficients of variation (CVs) were <9%.

For measuring leptin, a human leptin RIA kit (Linco, St Louis, MO, USA) was used. The detection limit was 0.25 ng/ml plasma; intra- and interassay CVs for levels of 4.9 and 15.7 ng/ml were <8.5%. Plasma cortisol concentrations were analysed using a commercially available RIA kit with the coated tube technique (ICN Biomedicals, Carson, CA, USA). The detection limit was 0.3 ng/ml plasma; intra- and interassay CVs for 20 and 40 ng/ml levels were <7%. Laboratory parameters of alcohol misuse were measured using standard clinical chemistry analyses.

Data analysis
All data are expressed as means ± SD. For the statistical analysis of group effects regarding hormone levels, analysis of variance followed by post hoc Bonferroni's correction was applied. Whenever a significant F-ratio was detected, means were compared using independent-sample t-tests. Normal distribution of data was tested with the Kolmogoroff–Smirnov test. Correlations were analysed using Pearson's correlation coefficients. Statistical significance was accepted at P < 0.05.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TNF-{alpha}, leptin and cortisol
In patients, TNF-{alpha} was significantly elevated compared to controls (16.5 ± 10.8 vs 7.8 ± 6.0 pg/ml; P < 0.05). Leptin plasma levels in patients were also shown to be increased significantly compared to controls (7.7 ± 3.3 vs 4.0 ± 2.3 pg/ml; P < 0.05). TNF-{alpha} and leptin plasma levels correlated significantly in patients (r = 0.40; P < 0.03). Whereas cortisol plasma levels were elevated in patients compared to controls (248.5 ± 60 vs 172 ± 74 ng/ml; P < 0.01), both TNF-{alpha} and leptin were without any relation to plasma cortisol, suggesting that elevated plasma TNF-{alpha} and leptin plasma levels were largely independent from an activation of the pituitary–adrenocortical axis. No significant relationship between plasma TNF-{alpha}, leptin, and cortisol was detected in the control subjects.

Associations with alcohol-related data and craving
TNF-{alpha} was highly associated with the duration of alcohol misuse (years since first alcohol-related problems occurred; r = 0.43; P < 0.02) but without any relation to self-rated alcohol craving (Fig. 1Go). However, leptin plasma levels correlated significantly with alcohol craving measured with the VAS for maximal craving (66.6 ± 35.4; r = 0.32, P < 0.05). Despite the significant correlation of maximal craving with other craving measures (mean craving: r = 0.70; P < 0.001; craving frequency: r = 0.59; P < 0.001; OCDS sum score: r = 0.48; P < 0.01), the correlation of leptin with mean craving (53.3 ± 35.3), craving frequency (48.9 ± 40.0), or the OCDS sumscore (30.4 ± 7.9) did not reach statistical significance. There was no association of TNF-{alpha}, leptin or cortisol with the number of inpatient detoxifications, mean amount of alcohol consumed daily during the last months prior to admission or tobacco consumption (data not shown). Plasma cortisol was without any relationship to duration of alcoholism or craving.



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Fig. 1. Graphical summary of the results. Left box: patient sample (n = 30). Double arrows ({leftrightarrow}) show significant positive correlations (Pearson's correlation coefficient). Right box: control sample (n = 30). For group comparisons between tumour necrosis factor-{alpha} (TNF-{alpha}) and leptin plasma levels, independent-sample t-tests were applied. Values are means ± SD.

 
Clinical chemistry parameters
According to prior results, no association of routine laboratory parameters, especially liver enzymes ({gamma}-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase) and mean corpuscular volume with TNF-{alpha}, leptin, and cortisol levels was observed.


    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presented data suggest that increased leptin plasma levels in alcoholics might result from an alcohol-induced enhanced expression of TNF-{alpha} that seems to be directly related to the duration of alcohol consumption. Nicolás et al. (2001) recently reported an association of lifetime alcohol intake and elevated plasma leptin levels in alcoholics, but without reporting the pathophysiological mechanism of this association. Our data support results from pre-clinical studies showing both an association of alcohol intake with increased TNF-{alpha} (Lin et al., 1998Go) and a releasing effect of TNF-{alpha} on leptin (Barbier et al., 1998Go). Hence, our data suggest that TNF-{alpha} might be the factor responsible for increased plasma leptin levels in patients suffering from alcoholism.

In two independent studies, an initial study with 20 alcoholics (Kiefer et al., 2001bGo) and a replication study with 78 alcoholics (Kiefer et al., 2001aGo), our group found elevated plasma leptin levels at the onset of withdrawal to be significantly correlated with self-rated craving. Moreover, in alcohol-habituated mice, the application of leptin (1 g/kg i.p.) resulted in an increased free-choice alcohol intake after a 3-day period of alcohol withdrawal (Kiefer et al., 2001cGo). This positive so-called ‘alcohol-deprivation effect’ reflects an accepted model for testing addictive behaviour and craving in animals (Spanagel and Holter, 1999Go). Interactions of leptin with reinforcing brain functions that could explain the effects of leptin on craving have been reported by Fulton et al. (2000). They showed that the effectiveness of a rewarding electrical stimulation was attenuated by intracerebroventricular infusion of leptin. From their point of view, this reflects the comparative process believed to underlie behavioural allocation (Maffei et al., 1995Go) with reducing food reward, while enhancing the value of competing behaviours. We think this might explain the positive association of increased alcohol craving with elevated plasma leptin in our alcoholic sample. In accordance with earlier studies on pharmacological effects on alcohol craving (Kiefer et al., 2001aGo,bGo), the measure ‘maximum craving’ seemed to be most sensitive for the detection of short-term variations during acute alcohol withdrawal. In contrast to the measures ‘mean craving’, ‘frequency of craving’ and the OCDS sum score, maximum craving describes neither a mean score nor cumulative measures of 1 week, but one maximal measure experienced during 1 week. However, there was no direct association between TNF-{alpha} and craving, suggesting that the TNF-{alpha}-mediated leptin increase might be the behaviour modulator of this pathway. Taken together, we found evidence that TNF-{alpha} might mediate an increase of plasma leptin after chronic alcohol intake. Additionally, leptin was shown to be associated with appetite for alcohol and alcohol craving. Alcohol craving, defined as an urge to consume alcohol, has been associated with an enhanced probability of renewed alcohol intake (Anton, 1999Go). Hence, a possible vicious circle including the components alcohol intake, increase of TNF-{alpha}, enhanced leptin secretion, enhanced alcohol craving, and consecutively increased alcohol intake, is suggested. Further research should establish the validity of this hypothesis and may help to understand the neurobiological mechanisms of origin and maintenance of alcoholism serving as a gateway for the development of new pharmacological strategies in the therapy of addiction.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
American Psychiatric Association (1994) Diagnostic and Statistical Manual of Mental Disorders, 4th edn. American Psychiatric Press, Washington, DC.

Anton, R. F. (1999) Alcohol craving — a renaissance. Alcoholism: Clinical and Experimental Research 23, 1289–1295.[ISI][Medline]

Anton, R. F., Moak, D. H. and Latham, P. K. (1996) The obsessive compulsive drinking scale: a new method of assessing outcome in alcoholism treatment studies. Archives of General Psychiatry 53, 225–231.[Abstract]

Barbier, M., Cherbut, C., Aube, A. C., Blottiere, H. M. and Galmiche, J. P. (1998) Elevated plasma leptin concentrations in early stages of experimental intestinal inflammation in rats. Gut 43, 783–790.[Abstract/Free Full Text]

Campfield, L. A., Smith, F. J., Guisez, Y., Devos, R. and Burn, P. (1995) Recombinant mouse ob protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269, 546–549.[ISI][Medline]

Fulton, S., Woodside, B. and Shizgal, P. (2000) Modulation of brain reward circuitry by leptin. Science 287, 125–128.[Abstract/Free Full Text]

Halaas, J. L., Gajiwala, K. S., Maffei, M., Cohen, S. L., Chait, B. T., Rabinowitz, D., Lallone, R. L., Burley, S. K. and Friedman, J. M. (1995) Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269, 543–546.[ISI][Medline]

Inui, A. (1999) Feeding and body-weight regulation by hypothalamic neuropeptides — mediation of the actions of leptin. Trends in Neurosciences 22, 62–67.[ISI][Medline]

Kiefer, F., Jahn, H., Jaschinski, M., Holzbach, R., Wolf, K., Naber, D., Wiedemann, K. (2001a) Leptin: a modulator of alcohol craving? Biological Psychiatry 49, 782–787.[ISI][Medline]

Kiefer, F., Jahn, H., Kellner, M., Naber, D. and Wiedemann, K. (2001b) Leptin as a possible modulator of craving for alcohol. Archives of General Psychiatry 58, 509–510.[Free Full Text]

Kiefer, F., Jahn, H., Wolf, K., Kampf, P., Knaudt, K. and Wiedemann, K. (2001c) Free-choice alcohol consumption in mice after application of the appetite regulating peptide leptin. Alcoholism: Clinical and Experimental Research 25, 787–789.[ISI][Medline]

Lin, H. Z., Yang, S. Q., Zeldin, G. and Diehl, A. M. (1998) Chronic ethanol consumption induces the production of tumor necrosis factor-alpha and related cytokines in liver and adipose tissue. Alcoholism: Clinical and Experimental Research 22, 231–237.[ISI][Medline]

Maffei, M., Halaas, J., Ravussin, E., Pratley, R. E., Lee, G. H., Zhang, Y., Fei, H., Kim, S., Lallone, R. and Ranganathan, S. (1995) Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nature Medicine 1, 1155–1161.[ISI][Medline]

Mann, K. and Ackermann, K. (2000) Die OCDS-G: Psychometrische Kennwerte der deutschen Version der Obsessive Compulsive Drinking Scale. Sucht 2, 90–100.

Nicolás, J. M., Fernández-Solà, J., Fatló, F., Batáller, R., Sacanella, E., Tobías, E., Badía, E. and Estruch, R. (2001) Increased circulating leptin levels in chronic alcoholism. Alcoholism: Clinical and Experimental Research 25, 83–88.[ISI][Medline]

Pelleymounter, M. A., Cullen, M. J., Baken, M. B., Hecht, R., Winters, D., Boone, T. and Collins, F. (1995) Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269, 540–543.[ISI][Medline]

Schuckit, M. A., Irwin, M. and Smith, T. L. (1994) One-year incidence rate of major depression and other psychiatric disorders in 239 alcoholic men. Addiction 89, 441–445.[ISI][Medline]

Spanagel, R. and Holter, S. M. (1999) Long-term alcohol self-administration with repeated alcohol deprivation phases: an animal model of alcoholism? Alcohol and Alcoholism 34, 231–243.[Abstract]