Pancreatic polypeptide infusions reduce food intake in Prader-Willi syndrome

Peptides,Vol. 14, 10p.497-503, 1993

0196-9781/93 $6.00 + .00 Copyright© 1993PergamonPressLtd.

Printed in the USA.

Pancreatic Polypeptide Infusions Reduce Food Intake in Prader-Willi Syndrome G A R Y G. B E R N T S O N , * t I W I L L I A M B. ZIPF,t:]: T H O M A S M. O'DORISIO,:]:§ J A M E S A. H O F F M A N ¶ A N D R O N A L D E. C H A N C E ¶

*Department of Psychology, Ohio State University, Columbus, OH 43210, ?Department of Pediatrics, Ohio State University, Columbus, OH 43210, ~.Children's Hospital, Columbus, OH 43205, #Internal Medicine, Ohio State University, Columbus, OH 43210, and ¶Lilly Research Laboratories, Indianapolis, IN 46285 Received 28 August 1992 BERNTSON, G. G., W. B. ZIPF, T. M. O'DORISIO, J. A. HOFFMAN AND R. E. CHANCE. Pancreaticpolypeptideinfusions reducefood intake in Prader-Willi syndrome. PEPT1DES 14(3) 497-503, 1993.--Prader-Willi syndrome is characterized by dramatic hyperphagia and morbid obesity, and is associated with a deficiency in basal and meal-stimulated serum pancreatic polypeptide (PP) levels. Intravenous infusions of pancreatic polypeptide (90 rain, 50 pmol/kg/h) restored normal serum PP levels, and a regimen of morning and afternoon PP infusions was found to significantlyreduce food intake in Prader-Willi subjects. Food intake was evaluated in a 60-rain free-feedingtest that shows high reliability and validity. Basal food intake during saline infusions was striking (~60 chicken sandwich quarters), and this intake was reduced overall by ~ 12% during PP infusions. This reduction was apparent only for female subjects, and may have reflected enhanced satiation rather than an overall suppression of food intake. No differenceswere apparent across subjects, in either basal food intake or the PP-related decrease in food intake, in the presence or absence of the widely recognized chromosomal marker for this syndrome [deletion of 15(ql 1-q13)]. More specific gene defects as recently reported in these subjects, however, suggest that the Prader-Willi syndrome may represent an important model for the study of food intake regulation. Pancreatic polypeptide

Prader-Willi syndrome

Food intake

PRADER-WILLI syndrome (PWS) is characterized by infant hypotonia, hypogonadism, craniofacial dysmorphism, short stature, as well as childhood-onset hyperphagia and morbid obesity (6,17). Obesity is a characteristic reported for 100% of the patients in the large clinical reviews (3,17), and appears to arise from both metabolic factors and an abnormal appetite (5,19). The abnormal appetite is especially apparent in the delayed satiation of these subjects. While the initial food intake of PWS children does not appreciably differ from weight-matched obese controls, PWS subjects show a considerably slower satiation than obese controls (50). The etiology of the hyperphagia of PWS is unknown. PraderWilli subjects, however, have been consistently shown to be severely deficient in basal and meal-stimulated pancreatic polypeptide, which does not appear to be secondary to obesity (44,52,53). Deficits in pancreatic polypeptide (PP) secretion have also been reported in non-PWS obesity (13,20), although PP deficiencies are not a consistent feature in the latter population (24,35,36,49). Pancreatic polypeptide has been suggested to be among a growing number of peptidergic satiety factors (2,32,33). Like

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PWS patients, genetically obese mice (ob/ob and NZO) have been reported to show low levels of PP release (12,23). Pancreatic polypeptide infusions in dogs and genetically obese mice has been reported to reduce food intake, body weight, or weight gain (12,31,40). Moreover, PP has two important attributes of an expected satiety factor. First, the magnitude of the postprandial PP response has been shown to be correlated with the size of the meal ingested (10). Secondly, the magnitude of PP release by oral, gastric, and intestinal stimuli is proportional to the degree of satiation associated with that site (26). Finally, a role of PP in appetite abnormalities is consistent with the report that PP is significantly elevated in anorexia nervosa (45). In view of the potential role of PP as a satiety agent, and the striking deficiency of this peptide in PWS, we previously examined the effects of acute PP infusions on food intake in PWS patients (51). While PP failed to yield significant satiety effects, several features of that study suggested the need for a further examination of this issue. First, the infusion dosage employed in that study led to abnormally high levels of circulating PP. Secondly, we employed only a single acute test, and it is possible that potential appetite effects may emerge over time. Finally,

Requests for reprints should be addressed to Gary G. Berntson, 48 Townshend Hall, Ohio State University, Columbus, OH 43212.

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anecdotal reports from parents suggested the possibility of a delayed effect on food intake of the subjects. Consequently, we have further examined the potential effects of PP infusions on food intake in PWS patients. The present protocol varied from the earlier study in two principal ways: a) the infusion dosage was decreased to more closely approximate normal blood levels of PP, and b) repeated PP infusions were administered over 2 days. METHOD

Subjects A total of 16 PWS subjects were identified for the present study. Subjects were diagnosed by the Pediatric Genetics and Birth Defects Clinics of Ohio State University (Children's Hospital), or by other medical centers who referred patients for evaluation. Of these 16 subjects, one was withdrawn from the study by the parents, another failed to receive the scheduled PP infusion, and a final subject had only incomplete data. Thus, 13 subjects completed the study. All subjects were obese, while none had clinical symptoms of glucose intolerance and all had normal fasting serum glucose values. Chromosomal studies revealed that 7 of the 13 subjects had an interstitial deletion of 15(ql 1-ql 3), which has been reported to occur in approximately 50% of children with PWS (4,16,28). All studies were approved by the Biomedical Sciences Institutional Review Board of Children's Hospital and Ohio State University, and were performed only after obtaining informed consent from the parents as well as assent of the child.

Drugs and Assays Bovine pancreatic polypeptide (18) was supplied by Lilly Research Laboratories (Ronald E. Chance). The full physiologic activity of PP is found in the C-terminal hexapeptide, which is identical in human and bovine PP (30), and bovine PP has been shown to have expected biological effects in man (14). The peptide was supplied as a sterilized lyophilized powder after purification (0.5 mg/vial). The infusion was in saline at a dose of 50 pmol/kg/h. Our previous study (51) had indicated that this regimen of PP infusions causes no physical effects or changes in routine serum chemistries, insulin, C peptide, glucagon, or cortisol when evaluated at the end of the 90-rain PP infusion. This is in general accord with a previous study of bovine PP infusions in humans, which reported only a decrease in pancreatic output of trypsin and bilirubin (14). The serum assays for PP were performed in the core laboratory of the GCRC at University Hospital by an established specific double-antibody RIA as previously described (53). In brief, a rabbit antibovine antiserum is used at a dilution of 1: 750,000. HPLC-purified ~25I-labeledbovine PP was used for the tracer and highly purified human PP antigen was used for the standard. The antiserum and standard were generously supplied by Lilly Research Laboratories, Indianapolis. The GIP, somatostatin, glucagon, insulin, vasoactive intestinal polypeptide, cholecystokinin, and secretin do not cross-react in this assay. The sensitivity of the assay is 30 pg/ml plasma, the interassay variability is 15%, and the intra-assay variability is 5%, between 1585% total binding. Charcoal-stripped plasma was used for the standard curve to account for nonspecific plasma protein effects.

Procedure The appetite tests were those previously described and standardized in PWS and non-PWS obese children (50). Each test was 60 min in duration, during which water and a plate of stan-

dard chicken salad sandwich quarters (30 kcal with 50% carbohydrate, 30% fat, and 20% protein) were continuously available. Sandwich quarters were presented on a plate in front of the subject and were replaced (from a concealed cooler) as they were ingested. Testing began at least 24 h after admission to the Clinical Research Center. During testing, the subjects were isolated (in their clinic room) from extraneous stimuli and activities, and were informed that they could eat as much as they desired. They were required to remain at the meal table for the full hour even if they finished eating, although they were allowed to go to the bathroom facility within the room. The number of sandwich quarters consumed each minute was recorded by the nurse attendant, who was instructed not to engage in conversation and to avoid eye contact with the subject. Questions were to be answered with a yes, no, or by as brief a response as possible. A total of four feeding tests were given on successive days, two with saline infusions and two with PP infusions. In the previous study, PP infusions and feeding tests were given in the morning. Some parents reported a reduction in food intake at the evening meal following the final morning PP infusion test (after which they were discharged). Because of the possibility of a delayed or cumulative effect, experimental procedures of the present study were modified. First, the feeding tests were administered in the afternoon (1700-1800 h) rather than the morning. Second, to capitalize on potential delayed effects, subjects received two infusions (saline or PP) each day, one in the morning (0800-0930 h) and one in the afternoon (1630-1800 h). Finally, to further maximize potential cumulative effects and minimize carryover between treatments, the PP infusion tests were given on successive days with the order of treatment (AABB) counterbalanced across subjects. No formal feeding test was given in the morning, although the subjects received a standard 300 kcal breakfast at 0830. In all cases, the breakfast meal was ingested within 15 min. No other food was given until the afternoon feeding tests. Thirty minutes prior to each morning and afternoon session, intravenous lines were placed in each arm, one for infusion and one for blood sampling for PP assays. Serum samples were obtained immediately prior to each infusion, and again after 30 min and at the conclusion of the 90-min infusion sessions. Pancreatic polypeptide assays from the morning sessions provided measures of basal and meal-stimulated PP levels, and the effectiveness of the PP infusions in raising serum PP levels. Preinfusion PP levels also allowed examination of endogenous changes in PP levels over the course of the day, and an evaluation of potential long-term effects of the morning PP infusions on PP levels in the afternoon.

Data Analysis The overall data analysis was by repeated measures analysis of variance (ANOVA) according to a 2 (drug treatment: saline vs. PP) X 4 (15-min time blocks) X 2 (replications) design. Further exploratory analyses were based on parametric t-tests or Pearson's correlation coefficients, and the nonparametric chisquare test. Analyses of PP levels were also evaluated by ANOVA. Separate analyses were run to address specific issues or contrasts. Basal levels and meal-stimulated PP levels were evaluated from the morning sessions on saline days before and after the morning meal. The effectiveness of the PP infusions in raising serum PP levels was evaluated by comparing PP levels before and after (90 min) PP infusions, and by contrasting postinfusion PP levels on saline vs. PP days. Preinfusion PP levels also allowed examination of endogenous changes in PP levels over the course

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FIG. 1. Serum pancreatic polypeptide levels in Prader-Willi subjects. Basal values represent the PP levels during saline control days, obtained prior to infusions and breakfast in the morning, and prior to infusions and feeding tests in the late afternoon. The single am value (left bar) represents the data from all 13 subjects, while the paired am and pm values represent data from a subset of 10 subjects for whom both values were available. Infusion values were also obtained in the morning to avoid confound from the variable food intake during the afternoon feeding tests. The pre bar represents the preinfusion data from 11 subjects over all test days. The sal bar depicts the data from the same 11 subjects after the 90-rain infusion of saline and the 300 kcal breakfast, and the pp bar represents the corresponding data after PP infusions.

of the day (saline conditions), and the effects of the morning PP infusions on PP levels in the afternoon (afternoon PP on saline vs. PP infusion days). RESULTS

Serum Pancreatic Polypeptide Levels Basal serum PP levels, and PP levels after the 90-min infusions, are illustrated in Fig. 1. All subjects had a patent infusion line (a criterion for inclusion), although in some cases the second IV line for blood sampling could not be established, or lost patency. Consequently, the data presented in Fig. 1, and analyzed below, are based on subjects for which appropriate values were available. As illustrated in Fig. 1 (left bar), all subjects uniformly demonstrated low basal PP levels, measured prior (0800) to the first morning meal and before the start of infusions. Pancreatic polypeptide levels were below detection limits (31 pg/ml) on at least some test days for 11 of the 13 subjects, and in these cases the data value was taken as the detection limit for purposes of analysis. Serum PP levels increased somewhat over the course of the day, as evidenced by the higher PP levels obtained prior to the (1700) afternoon tests (see Fig. 1, middle bars). While modest (morning mean on vehicle infusion days = 59.2 +_ 16.6 SEM; afternoon mean = 196.6 ___60.3), this increase was highly significant, F(I, 9) = 10.4, p < 0.01. No residual effects of the morning PP infusions were apparent on afternoon serum PP levels, since serum PP levels prior to the afternoon tests were equivalent on vehicle and PP infusion days (vehicle infusion days = 196.6 + 60.3; PP infusion days = 243.1 + 76.4). The morning infusions were begun at 0800 h, and a standard (300 cal) breakfast was given at 0830 h, with blood samples taken before infusions and at the end of the infusion 90 min later (0930). As illustrated by the left bars of Fig. 1, serum PP levels were increased somewhat after the breakfast under saline infusion conditions, although the significance level was marginal,

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F(I, 10) = 4.67, p = 0.054. Blood PP levels, however, were dramatically increased by the PP infusions. Serum PP levels after PP infusion were significantly greater than both preinfusion levels, F( 1, 11) = 10.49, p < 0.01, and levels after saline infusions, F( 1, 11 ) = 8.14, p < 0.02. Equivalent serum PP levels were also obtained after PP infusions in the afternoon tests (mean postinfusion serum PP levels; morning = 1283.1 +_ 392.4 SEM; afternoon = 1205.4 +__161.8). Thus, the infusion protocols achieved the desired experimental manipulation of serum PP levels. As evidenced by the large standard errors, however, considerable variation was apparent in the maximal PP levels achieved, across both subjects and test days. Figure 2 illustrates the time course of rise in serum PP, as revealed by subset of nine subjects who had PP data available for each of the 0-, 30-, and 90-min time points. As is apparent, PP infusions resulted in a progressive increase in serum PP over the 90-min infusion period. Serum PP levels were significantly elevated over basal values within 30 min of the start of PP infusions, F(1, 8) = 15.04, p < 0.005. Serum PP levels continued to rise, however, and the values obtained at 90 min were significantly elevated over the 30-min levels, F(1, 8) = 52.3, p < 0.001.

Food Intake As illustrated in Fig. 3, food consumption over the 60-min feeding tests was striking. During saline control tests, subjects ingested an average of 59.8 _+ 5.51 quarter sandwiches. The reliability of the feeding test was documented by the high correlation between the results of the two (replicate) feeding tests each subject received under each experimental condition (r = 0.80, p < 0.001). Further indication of the reliability of the feeding test is indicated by the similar intakes (within 10%) obtained in PWS patients over the present and prior studies (50,51). In addition to its face validity, the validity of the feeding test is documented by its sensitivity to satiation effects throughout the sessions (see Fig. 3) and its sensitivity to caloric preload observed in a previous study (51).

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FIG. 3. Food intake during the 60-min afternoon free-feeding tests for all 13 subjects during saline and PP (50 pmol/kg/h) infusions. Data points represent mean food intake values (in quarter sandwiches) and error bars depict the standard errors of the means.

As illustrated in Fig. 3, infusions of pancreatic polypeptide significantly decreased food intake in the formal eating tests. Results were evaluated by an analysis of variance according to a 2 (drug treatment) × 4 ( 15-min time blocks) X 2 (replications) design. The A N O V A revealed a significant main effect of time block, related to the overall decline in food intake throughout the 60-min test, F(3, 36) = 59.21, p < 0.001. A main effect of drug treatment reflected the reduced food intake during pancreatic polypeptide infusions, F(I, 12) = 4.64, p < 0.05. While significant, the overall drug effect was modest. Mean intake during the 60-rain eating tests under vehicle conditions was 59.8 _+ 5.3 sandwich quarters, compared to 52.7 + 4.7 during PP infusions. This reflected a reduction of 7.2 + 3.3 sandwich quarters, or approximately 12%. Pancreatic polypeptide had little effect on the initial rate of eating (first time block in Fig. 3), but appeared to enhance satiation over the course of the session. Although the drug treatment x time block interaction was not significant, the A N O V A design tested the absolute difference in food intake across the blocks, independent of the declining basal intakes over blocks. When the reduction in food intake associated with PP is expressed as a ratio of the basal (vehicle control) values, the differences across blocks are notable. Pancreatic polypeptide infusions yielded less than a 2% reduction in the first time block, and a 27% reduction in the last block. Subject characteristics and PP effects. Further analyses revealed an apparent gender interaction in the effects of PP, since only female subjects responded to PP with a decrease in food intake (see Fig. 4). While the number of male subjects was small (n = 3), there was no overlap in the effects of PP between males and females. The mean change in food intake under the PP condition for the 10 female subjects was - 1 0 . 8 + 3.6 sandwich quarters, compared to +4.8 + 2.1 for the three male subjects. Despite the small sample sizes, gender differences were so consistent as to be significant by a t-test, t( 11 ) = 2.3, p < 0.05. Given the small sample sizes, this difference was also analyzed by the nonparametric chi-square test for goodness of fit on the proportions of subjects who increased or decreased food intake under the PP condition. These proportions (9:1 for females, 0:3 for males) were again highly significant, x2(l) = 8.78, p < 0.01. Sex differences in the effect of PP on food intake did not appear to

be an artifact of gender differences in physical or clinical characteristics, since similar values were obtained across sex in basal food intake (mean for females = 59.8, range = 24.5-93.0; mean for males = 60.0, range = 34.5-79.5), percentage of ideal body weight (mean for females = 220, range = 123-315; mean for males = 188, range = 172-204), or age (mean for females = 23.3, range = 4.3-32.0; mean for males = 16.2, range = 11.521.0). Nor did males appreciably differ from group averages on either basal serum PP levels (mean = 44, range = 31-71) or maximal serum PP levels after PP infusions (mean = 989, range = 908-1062). Seven of the 13 subjects evidenced the chromosomal marker [deletion of 15(q 11-q 13)] as described for approximately 50% of Prader-Willi patients (28). As discussed above, however, all

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PANCREATIC PP INFUSIONS IN PRADER-WILLI SYNDROME

patients showed the classical clinical features of Prader-Willi syndrome. Consistent with the clinical features, the presence or absence of the chromosomal marker was not associated with appreciable differences in the percentage of ideal body weight (mean for subjects with chromosomal deletion = 198, range = 123-279; mean for remaining subjects = 228, range = 187315), basal food intake (mean with chromosomal deletion = 66.2, range 39.0-93.0, mean for remaining subjects = 52.3, range = 24.5-74.0), nor the decrease in food intake during PP infusions (mean with chromosomal deletion = -5.1, range = - 3 0 . 0 to +3.0; mean for remaining subjects = -9.5, range = - 2 9 . 0 to +9.0). Additional exploratory analyses failed to identify significant independent correlations between the PP effect on food intake and the subject variables of age (r = -0.46), weight (r = 0.09), percent ideal body weight (r = 0.00), or basal food intake (r --0.47). DISCUSSION

Results of the present study are consistent with previous report of low basal and meal-stimulated serum pancreatic polypeptide levels in subjects with Prader-Willi syndrome (44,52,53). The present study also confirms our previous finding (51) that PP infusions (50 pmol/kg/h) can dramatically increase serum PP, with a gradual rise occurring over the course o f a 90-min infusion period to a level (> 1200 pg/ml) beyond normal meal-stimulated values [ ~ 6 0 0 pg/ml; see (52,53)]. The results also provide new information on PP regulation in PWS. As expected, basal pretest assays on saline and PP days reveal that PP infusions in the morning have no significant effect on basal PP levels in the afternoon. However, while PP levels are generally low in PWS patients, basal pretest morning and afternoon PP levels on saline days reveal a significant elevation in PP during the course of the day. The magnitude of this increase (~fourfold) approximates the PP-releasing effects of a meal (300 kcal) in these PraderWilli subjects. The present results reveal that the increase in serum PP levels with infusion of bovine PP was associated with a significant reduction in food intake of PWS subjects. This finding is consistent with reports that PP infusions can reduce food intake, body weight, or weight gain in dogs and genetically obese mice (12,31,40). A reduction in food intake by PP, however, is not universally reported in animals. Additional studies have either failed to observe a decrease in feeding after PP administration or observed a reduction only after high nonphysiological doses (2,42,43). We previously failed to observe a significant effect of acute PP infusions on food intake in PWS subjects. The basis of this discrepancy is unclear, since some procedural differences were implemented in the present study. First, the present regimen entailed two infusion sessions per day (morning and afternoon) with a feeding test in the afternoon, while our earlier study employed only a single daily (morning) session. Secondly, in the present study PP infusions were given over two successive days (AABB order), rather than alternate days (ABAB order), in order to capitalize on delayed or cumulative effects. The present data do not allow a definitive evaluation of the relative contribution of these factors or other factors. The two infusions/day regimen, however, may have been more important than the PP infusions over successive days, since no significant effects of infusion day were apparent. While the effect of PP infusions on food intake in the present study was significant, the overall effect was modest ( ~ 12%) and limited to female subjects (who showed an overall reduction in food intake of ~ 17%). This sex difference did not appear to be

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related to obvious gender differences in subject characteristics such as age, body weight, percent ideal body weight, chromosomal status, or basal (saline) food intake. While statistically significant, the small number of male subjects in the present study renders the finding of gender differences merely suggestive. What remains clear is that exogenous restoration of serum PP deficiencies in PWS subject does not normalize food intake, even for female subjects. This should not be surprising, given the complexity of controls over food intake, together with the likely interactions of multiple factors. Pancreatic polypeptide deficiencies in PWS may be more of a marker than the fundamental cause of food intake disturbances. Indeed, PWS has been suggested to be characterized by a widespread endocrinopathy possibly associated with hypothalamic dysfunction (44). Pancreatic polypeptide release is regulated by both humoral and autonomic controls. A number of endogenous peptides can enhance PP release, including CCK, GIP, and bombesin (1,25). Pancreatic polypeptide release is also stimulated by vagal activity, while sympathetic actions are predominantly inhibitory via a receptors but may also have facilitatory effects via/3 receptors (22,25,27). These multiple neural and hormonal controls appear to be differentially sensitive to distinct dimensions of food intake. Thus, CCK antagonists can reduce PP release to a meal or to systemic CCK, but do not appreciably effect the PP-releasing effects of sham feeding or intracerebroventricular CCK infusions (21,22,25,29,34). In contrast, parasympathetic blockade or vagotomy blocks PP release stimulated by sham feeding or intracerebroventricular CCK, but has lesser effects on the PP-releasing effects of an ingested meal or systemic CCK administration (22,34). Both hormonal and autonomic controls of PP may be disrupted in PWS. In addition to potential direct effects of PP on food intake, these regulatory dysfunctions may reveal more fundamental disturbances in systems regulating hunger and food intake. The putative satiety agent CCK (39) has potent central and peripheral PP-releasing actions (1,22,25,34). While systemic release of CCK does not appear to be grossly abnormal in PWS, and may have a relatively normal PP-releasing effect in these patients (44), little information is available on central CCK in PWS. Prader-Willi syndrome patients also have a documented deficiency in an additional PP-releasing peptide, GIP (52). Analysis of the origins of disturbances in food intake and body weight regulation of Prader-Willi syndrome is complicated by the apparent multiplicity of metabolic, endocrinological, physiological, and neurological anomalies in these subjects. An added complexity relates to parallel behavioral, cognitive, and neuropsychological disturbances that are common in these patients (7,11,15,47). While fluoxetine and fenfluramine have been reported to facilitate weight control in Prader-Willi patients, these agents also yielded significant improvements in more general aspects of behavior (9,37). Thus, it is difficult to disambiguate the direct effects of these agents on appetite from changes in food intake that may be secondary to effects on behavioral compliance and impulse control. In view of the multiple anomalies of the Prader-Willi syndrome, it is our belief that fundamental insights into food intake disturbances in this syndrome will await more systematic, hypothesis-driven research. Multiple neuroanatomical-neurochemical systems have been implicated in food intake and body weight regulation, and these systems may differentially impact on specific food preferences, patterns of food intake, or responsiveness to metabolic signals. Specific determinants of food motivation, including deprivation and taste preferences, have been investigated in PWS patients (41,50). We need a much more comprehensive picture of the features and determinants of

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ingestive behavior of these patients, however, which may lead to testable inferences or hypotheses concerning underlying neurobiological mechanisms. While PWS patients appear to prefer sweet tastes (41), for example, it is not clear if their feeding disturbance is related to specific classes of macronutrients. Additionally, a more comprehensive perspective on the diverse neurological, endocrinological, and physiological features of Prader-Willi syndrome may reveal a more basic etiology that can organize the multiple features of this syndrome. Recent developments hold considerable promise in this regard. Deletions of the proximal arm of chromosome 15(q I 1-q 13) have been recognized in a substantial subset of Prader-Willi patients (4,16,28). A more recent study has documented that the gene encoding the 133 subunit of the GABAA receptor maps onto this region (46). This gene defect could hold an important key to understanding the multiplicity of anomalies of this syndrome. The widespread distribution of G A B A receptor systems and the multiplicity of regulatory functions mediated by GABA may offer an important organizing basis for the diversity of anomalies in Prader-Willi syndrome. In this regard, it is noteworthy that the hypothalamus has been implicated in autonomic control, neuroendocrine function, as well as metabolic and food

intake regulation. It is also of interest that the ~3 subunit is disproportionately represented in hypothalamic GABA receptors (48). In view of these considerations, it is possible that deficiencies in the r3 G A B A receptor subunit may underlie many of the diverse manifestations of Prader-Willi syndrome. While G A B A / benzodiazepine agonists or antagonists are known to exert potent effects on food intake in normal subjects (8,38), little data are available for Prader-Willi patients. In summary, the present results are consistent with the hypothesis that food intake abnormalities of PWS subjects are related in part to disturbances in PP release. Multiple neural, endocrinological, and behavioral dysfunctions, however, likely contribute to the striking alteration in food intake in these subjects. ACKNOWLEDGEMENTS This research was supported by grants from Children's Hospital Research Foundation, Columbus, OH, and from the Melinda Gabelman Prader-Willi Fund, Cincinnati, OH. Additional support and laboratory assays were provided by the General Clinical Research Center of Ohio State University Hospitals. We wish to thank Lisa Raskin and Annette Fieldstone for assistance.

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