T-lymphocyte cholecystokinin-8 and beta-endorphin concentrations in eating disorders: I. Anorexia nervosa

ELSEVIER Psychiatry Research 59 (1995) 43-50

PSYCHIATRY

RESEARCH

T-lymphocyte cholecystokinin-8 and beta-endorphin concentrations in eating disorders: I. Anorexia nervosa

Francesca Brambilla* a, Mirella Brunettab, Angela Peironeb, Giampaolo Pernab, Paola Sacerdotec, Barbara Manfredic, Albert0 E. Paneraic

Centro di Psiconeuroendocrinoiogia, Ospedaie Psiehiatrico Pini, Milan 20161. Italy

bDipartimento di Scienze Neuropsichiche. Istituto Scientifico S. Raffaele, Milan. Italy ‘Dipartimento di Farmacologia Universitb. Milan, Italy

Received 17 May 1994; revision received 8 May 1995; accepted 26 June 1995

Abstract

Baseline concentrations of cholecystokinin-8 (CCK-8) and /3-endorphin (&EP) were measured in T-lymphocytes from 33 restricting patients with anorexia nervosa (AN-R), 23 bingindpurging patients with anorexia nervosa (AN- BP), and 24 healthy volunteers. CCK-8 basal values were significantly lower and /3-EP values significantly higher in AN-R and AN-BP patients than in normal volunteers. Levels of the peptides were measured three more times during a Cmonth combined cognitive-behavioral/psychopharmacological treatment (nortriptyline or fluoxetine in AN-R, fluoxetine or amineptine in AN-BP). CCK-8 values fluctuated (nonsignificantly) during each treatment, while /SEP values decreased (to a significant degree only in fluoxetine-treated AN-R patients).

Keywork Anorexia nervosa; Peptides; Nortriptyline; Fluoxetine; Amineptine

1. Introduction

Impaired secretion of the neurotransmitters, neuropeptides, neurohormones, and peripheral hormones that regulate eating behavior may play a pathogenetic role in the development of anorexia nervosa (AN). Hunger, satiety, and food choice are severely altered in AN, with hunger being generally increased, with satiety being decreased or increased, and with aversion for fat and prefer-

* Corresponding author, Piazza Grahdi 3, Milan0 20127, Italy. Tel: +39 3 68 3017420; Fax: +39 2 70122889.

ence for glucose (Owen et al., 1985; Nakai et al., 1987; Drewnowski et al., 1992; Rolls et al., 1992; Sunday et al., 1992; Halmi et al., 1993; Simon et al., 1993). In experimental animals, centrally and peripherally secreted cholecystokinin-8 (CCK-8) stimulates satiety, preferentially inhibiting lipid and glucose consumption, while /3-endorphin (/3- EP) stimulates hunger and preference for fat and glucose, and increases the rewarding aspects of food intake (Morley et al., 1985; Leibowitz, 1986; Morley, 1989; Hetherington et al., 1991; Beck, 1992; Bednar et al., 1992; Cooper et al., 1992). The influence that the two peptides exert physiological-

0165-1781/95/.W9.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0165-1781(95)02664-I

44 F. Brambilla et al. /Psychiatry Research 59 (1995) 43-W

ly on eating behavior suggests that their alteration may be involved in the development of AN. Data in the literature about this, however, are rather scanty and controversial. Cerebrospinal fluid (CSF) concentrations of CCK-8 are normal (Gerner and Yamata, 1982) and plasma levels after a test meal are normal or higher than normal (Harty et al., 1991; Geracioti et al., 1992). Opioid- like activity (OLA) is increased in CSF of anorectic patients, but &EP levels have been found to be both normal and decreased, and dynorphin At_* to be normal, while plasma &EP levels have been normal in some studies and increased in others (Gerner and Sharp, 1982; Kaye et al., 1982, 1987; Brambilla et al., 1985, 1991; Melchior et al., 1988; Lesem et al., 1991).

In a preliminary investigation of AN patients, we studied the concentrations of CCK-8 and fl-EP in T-lymphocytes, a peripheral compartment that has been observed in animal and human studies to mimic brain neuronal secretion and regulation (Kavelaars et al., 1990; Panza et al., 1991; Sacer- dote et al., 1991a). We measured basal levels of the two peptides in a group of subjects under drug-free conditions and then longitudinally during combined cognitive-behavioral/psychopharmacological therapy. The aim of the study was to deter- mine whether the concentrations of the two peptides were correlated with the eating disorder, or with specific aspects of it, and whether biochemi- cal and psychopathological parameters could be modified concomitantly by the combined therapy, thus suggesting the existence of a correlation between the two compartments. Finally, by giving antidepressant drugs that act selectively on dif-

ferent central regulatory systems, we wanted to define, tentatively, the specific effects of each neurotransmitter on pathological eating behavior and in parallel on T-lymphocyte CCK-8 and P-EP concentrations.

2. Methods

Fifty-six anorectic women, 33 restricted (AN-R) and 23 binge eating and purging (AN-BP), and 24 healthy women selected from the hospital staff for comparison, entered the study. All of them gave informed consent. Table 1 presents demographic data for the subjects.

The diagnoses of AN-R and AN-BP were made with the Structured Clinical Interview for DSM- HI, Upjohn version (Spitzer et al., 1983), accord- ing to DSM-III-R and DSM-IV diagnostic criteria (American Psychiatric Association, 1987, 1994). Four of the AN patients had present or past co- morbidity with major depression. The healthy subjects received the same structured interview to exclude present or past psychopathology. Other criteria for exclusion from the study were en- docrinopathies and metabolic disorders, obesity or recent weight loss for controls, and chronic diseases, especially infections and immunopath- ologies. None of the patients had ever been treated with psychopharmacological drugs before our study, and some of them had had brief periods (a few months) of psychodynamic treatments of various types without significant amelioration of their disorders. They were all outpatients of our Center for Eating Disorders. CCK-8 and /3-EP concentrations were measured in T-lymphocytes

Table I Demographic data of patients with anorexia nervosa and healthy comparison subjects (mean f SD)

Age (years) Duration of illness Duration of ame- Body mass index

(years) norrhea (years)

Anorexia nervosa, restricting type (n = 33)

Anorexia nervosa, hinging/purging type (n = 23)

Healthy subjects (n = 24)

12-35 0.4-10 0.3-9 10-17.2 (21.4 zt 5.3) (3.4 f 3) (2.3 f 2.2) (14.8 f 2.4) 17-43 0.3-13 O-12 12.1-18.0 (23.1 f 6.8) (4.6 zt 3.9) (1.9 f 3.3) (17.5 f 3.3) 14-42 19. I-24.6 (22.4 f 6.9) (22.4 + 2)

F. Brambilla et al. /Psychiatry Research 59 (1995) 43-50 45

of all the patients and healthy subjects under drug- free conditions and then in 35 of the patients after 1, 2, and 4 months of therapy. The other 21 patients did not receive the pharmacological therapy, because they could not, for personal reasons, be regularly supervised as required by the protocol.

Treatment of the 35 patients included cognitive- behavioral therapy, with one interview/week for 4 months, administered by a team of three therapists who were trained and supervised by the same person. Nutritional counseling followed the guidelines given by the dietitian of the Institute. The concomitant psychopharmacological therapy also lasted 4 months, and the patients were ran- domly divided into four therapeutic groups. Group 1 included seven AN-R patients treated with 75 mg/day of nortriptyline, p.o.; group 2 included 15 AN-R patients, treated with 60 mg/day of fluoxetine, p.o.; group 3 included six AN-BP patients, treated with 60 mg/day of fluoxetine oral- ly; and group 4 included seven AN-BP patients, treated with 300 mgday of amineptine, p.o_ The choice of different antidepressants for each group was dictated by their relative selectivity for one neurotransmitter system, by knowledge of the specific effects of each neurotransmitter on eating behavior, and by reports in the literature on the impairment of each neurotransmitter in eating disorders (Halmi et al., 1978; Kaye et al., 1984a, 1984b, 1985; Pirke et al., 1988). Nortriptyline is mainly a noradrenergic agonist, and noradrenalin (NA) stimulates hunger and carbohydrate preference, which is why we administered it only to AN- R subjects. Fluoxetine is a serotonergic agonist: since serotonin (5-hydroxytryptamine; 5-HT) stimulates satiety and inhibits carbohydrate and lipid consumption, we administered it to AN-R and AN-BP subjects because both may have reduced satiety. Finally, amineptine is a dopaminergic agonist, and dopamine (DA) inhibits hunger, which is why we gave it only to AN- BP subjects (Wurtman et al., 1979; Leibowitz et al., 1984, 1986; Morley and Blundell, 1988). The three neurotransmitters modulate CCK-8 and P- EP secretion, with NA and 5-HT stimulating both peptides and DA stimulating CCK-8 and inhibiting &EP (Hollt et al., 1982; Morley, 1982; Meyer et al., 1983; Pettibone and Mueller, 1981;

Abu Samra et al., 1984; Petraglia et al., 1984; Mar- tin et al., 1986; Holsboer, 1988; Cooper et al., 1992).

Levels of symptomatology of the patients were monitored with the Eating Disorder Inventory (EDI; Garner et al., 1983), which was administered by trained staff members before therapy began and again after 1, 2, and 4 months of treatment. Assessments of symptomatology were concomitant with measurements of P-EP and CCK-8 concentrations. Body mass index (BMI) was also measured at baseline, and then after 1, 2, and 4 months of therapy.

For the study of the two peptides, patients and comparison subjects came to our day hospital in the morning, after 12 h of fasting and 1 h of bed rest. A butterfly needle was inserted at 08:30 h into a forearm vein and kept patent by saline infusion. At 09:OO h, heparinized blood was drawn for the CCK-8 and /3-EP assays, and the blood specimens were processed immediately.

For the CCK-8 assay, peripheral blood mononuclear cells were separated by gradient sedimen- tation of whole blood on Ficoll Paque (Pharmacia). Cells were resuspended in 1 ml of 0.1 acetic acid, homogenized in a blade homogenizer, and the supernatant was frozen until assayed. CCK-8 was measured by radioimmunoassay with C-terminal-directed antibodies for the sulfated fragment 26-33 of CCK. The antiserum was rais- ed in the rabbit against the synthetic peptide 26-33 sulfated, and shows 100% reactivity with the nonsulfated octapeptide and with gastrin. It should be mentioned that the ratio between the sulfated and the nonsulfated forms of CCK has been reported to be 50150 and that gastrin is not present in lymphocytes (Sacerdote et al., 1991b). Interassay and intra-assay coefficients of variation were 8% and 1 l%, respectively. There was no cross-reactivity with P-EP, leu- and met- enkephalin, substance P, somatostatin, thyrotropin-releasing hormone, corticotropin- releasing hormone, neurotensin, vasopressin, bombesin, vasoactive intestinal peptide, insulin, follicle stimulating hormone, luteinizing hormone, prolactin, growth hormone, the cytokines interleukin-la and interleukin-10, and tumor necrosis factor. The radioimmunoassay procedure

46 F. Brambilla et al. /Psychiatry Research 59 (1995) 43-50

Table 2 3. Results Basal cholecystokinin-8 and /3-endorphin concentrations in

lymphocytes Baseline CCK-8 concentrations were significantly lower and those of &EP significantly higher in AN-R and AN-BP patients than in comparison subjects, but they did not differ in the two eating disorders (Table 2). During the treatments, CCK-8 values fluctuated (Table 3). Repeated measures ANOVA did not reveal significant results. Repeated measures ANCOVA indicated the ab- sence of a significant effect of time, group, or time x group interaction in fluoxetine- or nortripytline-treated AN-R patients, while in fluoxetine- or amineptine-treated AN-BP patients there were significant effects of time (F = 3.3; df = 3,29; P = 0.03), group (F = 11.6; df = 1,9; P = 0.008) and time x group (F = 3.6; df = 3,29; P = 0.003).

Normal AN-R AN-BN subjects (n = 33) (n = 23)

(n = 24)

Mean SD Mean SD Mean SD

Cholecystokinin-8 47.7 21.3 28.0a 18.1 24.9b I I.5

(pg/lO cells)

B-Endorphin 25.0 11.9 39.9c 29.2 57.1d 55.2

(p&l0 cells)

Note. Patients vs. normal subjects, by analysis of variance:

aP = 0.02; bP = 0.007; CP = 0.003; dP = 0.001.

has been previously validated (Salerno et al., 1983). The concentrations of P-EP were measured, after extraction of the peptide as reported above for CCK-8, by radioimmunoassay with C- terminal-directed antibody specific for P-EP (Panerai et al., 1983). Interassay and intra-assay coefficients of variation were 8% and 1 l%, respectively. With this method there is no cross-reactivity with met- or leu-enkephalin, substance P, CCK, or cytokines, but there is 100% cross-reaction with fl- lipotropin which, however, is practically absent in lymphocytes. All the assays were run by the same trained technician, who used the same batch of every reagent and the same equipment to minimize intra-assay variability.

Data were analyzed statistically by one-way analysis of variance (ANOVA), by ANOVA for repeated measures, and by analysis of covariance (ANCOVA) for repeated measures.

Levels of /I-EP showed a trend toward a pro- gressive decrease in most of the patients, which was significant only in fluoxetine-treated AN-R patients (time: F = 4.8; df = 3, 33; P = 0.007, repeated measures ANOVA) (Table 4). in nortriptyline- or floxetine-treated AR-N patients, repeated measures ANCOVA did not show a significant effect of group, but showed a significant effect of time (F = 6.1; df = 3, 59; P = 0.01) or time x group interaction (F = 4.8; df = 3, 59; P = 0.005). Amineptine- or fluoxetine-treated AN- BP patients did not show a significant effect of time, group, or group X time interaction.

BMI increased significantly and progressively in nortriptyline- or fluoxetine-treated AN-R patients (time: F = 7.4; df = 3, 18; P = 0.002 and F = 16.7; df = 3, 33; P = 0.009, ANOVA for repeated mea-

Table 3

Concentrations of cholecystokinin-8 (pg/lO cells) in T-lymphocytes during treatments

Patients Baseline

Mean SD

Month 1

Mean SD

Month 2

Mean SD

Month 4

Mean SD

Anorexia nervosa, restricting type Nortriptyline-treated (n = 7)

Fluoxetine-treated (n = 15)

Anorexia nervosa. hinging/purging fype Fluoxetine-trated (n = 6)

Amineptine-treated (n = 7)

30.4 21.8 31.3 21.6 41.7 30.0 20.2 14.2

24.4 11.9 33.0 23.9 32.5 19.0 29.4 17.6

18.1 9.1 16.0 7.0 14.6 II.0 19.1 4.2

25.3 13.7 59.4 31.6 24.2 13.3 27.5 2.8

F. Brambilia et al. /Psychiatry Research 59 (1995) 43-50

Table 4 Concentrations of fl-endorphins (pg/lO cells) in T-lymphocytes during treatment

Patients Baseline Month I

Mean SD Mean SD

Month 2 Month 4

Mean SD Mean SD

Anorexia nervosa, restricting type Nortriptyline-treated (n = 7) Fluoxetine-treated (n = IS)

Anorexia nervosa. hinging/purging type Fluoxetine-treated (n = 6) Amineptine-treated (n = 7)

58.6 43.4 26. I 11.0 24.2 13.5 26.4 21.3 27.7 13.6 37.1 25.9 22.9 18.8 12.5 9.3

75.8 78.8 55.8 30.0 34.6 38.0 30.5a 17.4 30.3 20.8 30.6 13.8 25.8 13.6 29.8 8.3

Note. Repeated measures analysis of variance: “F= 4.8; df= 3, 3; P = 0.007

sures), while no significant change was found in AN-BP patients, whose pretreatment BMI had been borderline-normal (Table 5). Examination of the psychopathological data revealed that EDI scores fluctuated throughout the therapies, but repeated measures ANOVA did not reveal signili- cant results in any of the groups. Repeated measures ANCOVA did not find significant cor- relations between CCK-8 or &EP values and EDI scores.

4. Discussion

These preliminary data reveal that in AN-R and AN-BP patients basal concentrations of CCK-8 in T-lymphocytes are lower than normal while those of &EP are higher than normal. These abnormalities might mirror similar ones in the central nervous system (Kavelaars et al., 1990; Panza et al., 1991; Sacerdote et al., 1991a), thus possibly

Table 5 Body mass index values during treatment

reflecting one pathogenetic factor in the modulation of anorexic symptomatology.

The lower than normal basal values of CCK-8 in our patients might be the basis for the impaired satiety that often characterizes the disease. Reports in the literature about CCK-8 concentrations in AN patients are not in agreement with our results, since some found normal values of the peptide in CSF and others found normal or increased concentrations in plasma after a test meal (Gerner and Yamada, 1982; Harty et al., 1991; Geracioti et al., 1992). The difference between these data and ours cannot be explained on the basis of selection of patients, which was similar in all the studies. It might, however, reflect the different compartment examined and type of assay used. Even though CCK-8 content in T-lymphocytes has been sug- gested to mimic that of cerebral neurons, it does not necessarily parallel levels of CCK-8 in the cen-

Patients Baseline

Mean SD

Month I

Mean SD

Month 2

Mean SD

Month 4

Mean SD

Anorexia nervosa, restricting type Nortriptyline-treated (n = 7) Fluoxetine-treated (n = 15)

Anorexia nervosa, binginglpurging type Fluoxetine-treated (n = 6) Amineptine-treated (n = 7)

15.8 1.9 16.9 I.8 17.4 1.9 18.3’ 1.7 14.7 1.4 15.3 1.8 15.9 1.9 16.3b 2.6

18.7 4.0 19.1 3.6 19.6 3.2 18.6 6.2 18.3 3.3 17.6 2.9 17.6 2.8 17.7 2.6

Note. Repeated measures analysis of variance: aF= 7.4; df= 2, 18; P = 0.002. bF = 16.7; dJ= 3, 3; P = 0.009.

48 F. Brambilla et al. /Psychiatry Research 59 ( 1995) 43-50

tral nervous system or levels in plasma, which relate only to peripheral secretion.

The high basal concentrations in &EP of our AN-R and AN-BP patients might parallel the increased hunger and glucose preference that characterize these subjects, which may be related to increased opioid secretion. The data in the literature are again conflicting, with reports of normal, increased, and decreased concentrations of the opioid in CSF or plasma of AN patients; again, the differences between studies may reflect the same factors discussed above for CCK-8 values (Gerner and Sharp, 1982; Brambilla et al., 1985, 1991; Kaye et al., 1987; Melchior et al., 1988; Lesem et al., 1991).

The finding that in our patients /3-EP values were higher than normal and that, in parallel, CCK-8 values were lower than normal is interesting, since it has been reported that the two peptides reciprocally inhibit each other’s secretion in experimental animals and men (Basso et al.. 1981; Micevych et al., 1985).

While the pretreatment data of our patients suggest that the abnormal concentrations of CCK-8 and P-EP in T-lymphocytes, by mimicking similar alterations in the central nervous system, might be related to the alterations of hunger and satiety of AN, those obtained during the pharmacological studies are more difficult to interpret. Since NA and 5-HT have been reported to stimulate the secretion of /3-EP and CCK-8 whereas DA has been reported to stimulate CCK-8 and inhibit &EP secretion (Hiillt et al., 1982; Meyer and Krauss, 1983; Abu-Samra et al., 1984; Martin et al., 1986), we expected that treatment with the mainly NA- stimulating drug nortriptyline and with the mainly 5-HT-stimulating drug fluoxetine would increase the concentrations of the two peptides, and that treatment with amineptine, a mainly DA- stimulating drug, would increase CCK-8 concentrations and decrease P-EP concentrations in our AN patients (H611t et al., 1982; Morley, 1982; Chang et al., 1983; Meyer et al., 1983; Pettibone and Mueller, 1981; Petraglia et al., 1984; Holsboer, 1988; Abu-Samra et al., 1984; Martin et al., 1986; Cooper et al., 1992). Instead, even though increases of CCK-8 and decreases of &EP values in individual patients were observed to occur during

the treatments, often in parallel with improvements in EDI scores and BMI, changes were not always statistically significant for both the bio- chemical and the psychopathological data. This may be due to the fact that the groups were small and the standard deviations high; in a larger popu- lation, it is possible that the increases in CCK-8 concentrations and the decreases in @-EP concentrations observed during treatment would have at- tained statistical significance and would have been correlated with improvements in psychopathology.

The observation that fluoxetine increased concentrations of CCK-8 in AN-BP patients and decreased concentrations of &EP in AN-R patients more significantly than did nortriptyline or amineptine is interesting. Even though the hormo- nal changes did not covary with EDI items in the two groups of patients, fluoxetine has been reported in the literature to improve both anorexia and bulimia nervosa (Freeman and Hampson, 1987; Hudson and Pope, 1987; Trygstadt, 1990; Goldstein and Wilson, 1994; W.H. Kaye, personal communication).

In conclusion, our data reveal that the study of CCK-8 and /3-EP concentrations in T-lymphocytes may be a useful tool for investigation of the peptide pathology in the course of mental disorders. The parallel impairments of peptide levels and of the specific symptoms of AN at baseline might suggest that the alterations of the two peptides in T-lymphocytes, which might mirror those in brain neurons, might be involved in the pathogenesis of AN for both the restricting and the binge- eating/purging forms of the disorder.

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T-lymphocyte cholecystokinin-8 and beta-endorphin concentrations in eating disorders: I. Anorexia nervosa