Peptides, Vot. 12, pp. 251-255. © Pergamon Press plc, 1991. Printed in the U.S.A. 0196-9781/91 $3.00 + .00

Acute Fenfluramine Administration Reduces Neuropeptide Y Concentrations in Specific Hypothalamic Regions of the Rat:

Possible Implications for the Anorectic Effect of Fenfluramine

P A U L A R O G E R S , P A U L I N E E. M c K I B B I N A N D G A R E T H W I L L I A M S l

Department o f Medicine, The University o f Liverpool, PO Box 147, Liverpool L69 3BX, United Kingdom

R e c e i v e d 12 S e p t e m b e r 1990

ROGERS, P., P. E. McKIBBIN AND G. WILLIAMS. Acute fenfluramine administration reduces neuropeptide Y concentrations in specific h)Tothalamic regions of the rat: Possible implications for the anorectic effect of fenfluramine. PEPTIDES 12(2) 251- 255, 1991.--Neuropeptide Y (NPY), a powerful central appetite stimulant, coexists in several hypothalamic areas with serotonin, which suppresses feeding. This study investigated the effect of acute administration of the serotonergic drug, fenfluramine, on NPY concentrations in microdissected hypothalamic nuclei. Adult male Wistar rats were given fenfluramine (10 mg/kg, n = 7) or saline (n = 7), intraperitoneally 1 h before darkness. Food was presented immediately before darkness and the rats were killed during the first 4 h of darkness. Fenfluramine injection significantly reduced food intake. In fenfluramine-injected animals, NPY levels in the ventromedial and dorsomedial nuclei and in the lateral hypothalamic and lateral preoptic areas were significantly lower than in sa- line-injected controls. The ventromedial and dorsomedial nuclei and the lateral hypothalamic area are sites which mediate the hyper- phagic action of centrally injected NPY. Selective NPY changes in specific nuclei after fenfluramine injection suggest functional interaction between NPYergic and serotonergic systems, and may indicate that NPY is involved in mediating the anorectic effect of serotonergic agents.

Feeding behavior Neuropeptide Y Serotonin Fenfluramine Hypothalamus Rats

THERE is increasing evidence that neuropeptide Y (NPY) is im- portant in the regulation of appetite and energy balance. NPY is a 36 amino acid peptide (23) which is highly concentrated in the hypothalamus and particularly in specific regions implicated in the control of feeding behavior, notably the paraventricular and dorsomedial nuclei (3, 4, 19). When injected into these areas or the ventromedial nucleus and lateral hypothalamic area, NPY powerfully stimulates both eating and drinking and induces sus- tained hyperphagia and obesity if given in the longer term (5, 13, 14, 20, 22). NPY-induced hyperphagia is selective for carbohy- drate-rich foods (20). Hypothalamic NPY concentrations change under conditions of altered food intake and energy balance. For example, NPY levels are increased in the lateral hypothalamic area after the onset of darkness (11), the main cue for eating in rodents, and in the central hypothalamus (26) and specific nuclei of food-restricted rats (17); the increase in NPY levels in the paraventricular nucleus after starvation is reversed by refeeding (17). Hypothalamic NPY concentrations are also increased, par-

IRequests for reprints should be addressed to Dr. Gareth Williams.

ticularly in NPY-sensitive appetite-regulating areas, in insulin- deficient diabetic rats (18, 25, 27, 29) which show striking hyperphagia with a preference for carbohydrates (8). Hypotha- lamic levels of NPY mRNA are also increased in diabetic rats, especially in the arcuate nucleus which is the main source of the NPY-containing projection to the paraventricular nucleus (15,24). These observations are consistent with the hypothesis that hypo- thalamic NPYergic activity is increased in diabetes and starvation and suggest that NPY may mediate the hyperphagia which is characteristic of these conditions and which could be viewed as a homeostatic response to counteract weight loss (28). NPY in the hypothalamus may therefore act to maintain nutritional state (13,28).

However, the overall significance of hypothalamic NPY and its possible interactions with other neurotransmitter systems which may regulate appetite are unknown. Serotonin is a powerful in- hibitor of eating when injected into the paraventricular nucleus and other central hypothalamic areas, and selectively reduces car- bohydrate intake (9,10). NPY- and serotonin-immunoreactive nerve

251

252 ROGERS, McKIBBIN AND WILLIAMS

fibers are closely related within several hypothalamic areas (6,7), raising the possibility that the two systems may interact function- ally to modulate appetite. Fenfluramine, structurally related to amphetamine, enhances serotonergic activity and inhibits eating in rodents and man (10,30). This study aimed to examine possi- ble interactions between hypothalamic NPYergic and serotonergic systems by measuring NPY concentrations in individual hypotha- lamic nuclei of the rat following acute administration of fenflur- amine.

METHOD

Experimental Protocol

Male Wistar rats weighing approximately 250 g (obtained from Charles River UK Ltd., Margate, Kent, UK) were caged singly and habituated to a 12:12 h light:dark cycle with lights off at 1900 h. Ambient temperature was maintained at 22°C. Food was provided immediately before darkness for 10 days before sacri- fice and water was freely available throughout.

The study examined the first few hours of the dark phase, when food intake is normally maximal and the anorectic effect of fenfluramine would be seen most clearly. One group of rats (n = 7) was given an intraperitoneal injection of D,L-fenfluramine (Sigma Ltd., Poole, UK), 10 mg/kg in 0.2 ml isotonic saline, 1 h before lights out. A control group (n = 7) received an equal volume of isotonic saline injected at the same time. Animals from each group were killed alternately at approximately 20-minute intervals during the 4 h after darkness. This procedure was car- ried out under a low-intensity red photographic safe-light to avoid even momentary reexposure to bright light which could disturb circadian rhythmicity. All animals were killed by CO 2 inhalation (within 45 seconds) and immediately exsanguinated by cardiac puncture. Plasma was separated and stored at - 4 0 ° C until as- sayed for insulin and glucose concentrations. Intake of food from its presentation at the onset of darkness until the time of killing was measured for each animal.

Hypothalamic Dissection

The brains were rapidly removed and a frontal slice including the hypothalamus was excised between the mammillary bodies and optic chiasm. This was immediately transferred to ice-cold isotonic saline and cut into 330-500 Ixm sections using a vibrat- ing microtome. Individual hypothalamic regions and nuclei were then punched out with a blunt-ended 19-gauge needle as previ- ously described (25) and boiled in 0.5 M acetic acid for 10 min to extract NPY. The hypothalamic areas studied were: medial and lateral preoptic area, paraventricular nucleus, ventromedial nu- cleus, dorsomedial nucleus, lateral hypothalamic area and the re- gion at the base of the third ventricle which included the arcuate nucleus together with the median eminence. The entire dissection procedure took 15 minutes for each animal.

Extracts were frozen at - 4 0 ° C and later assayed for NPY us- ing a radioimmunoassay (2) which employed ~25I-labeled NPY (Amersham International Plc, Amersham, UK), NPY antiserum (Cambridge Research Biochemicals, Cambridge, UK) and syn- thetic porcine NPY as standard (Bachem Inc., Saffron Walden, UK). The assay sensitivity was 10 frnol/tube and the within-assay coefficient of variation was 4.2%. NPY concentrations in hypo- thalamic extracts were expressed as fmol/~g protein, protein con- centrations in the extract being measured by the Coomassie Blue micromethod (Pierce and Warriner, Chester, UK).

Chromatographic Methods

To characterize NPY-like immunoreactivity in hypothalamic

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FIG. 1. Effect of fenfluramine (black columns) and saline injection (emp- ty columns) on food intake in individual rats after the onset of darkness.

tissue, extracts from several hypothalamic areas were pooled from 4 rats in each group and subjected to high performance liquid chromatography (HPLC) using a LiChrospher-300 RP-8 10-1xm column (250 × 4 mm) (Merck: BDH, Poole, UK). The column was eluted at a flow rate of 1 ml per min and equilibrated at 20% acetonitrile in water containing 5% trifluoroacetic acid (1% TFA in water). UV absorbance was monitored at 280 nm at a sensitiv- ity of 0.01 absorbance units for full-scale deflection. Following the addition of the sample, a linear gradient was established from 20°7o rising to 48% acetonitrile in water containing 5% TFA over 20 minutes. Porcine NPY (Bachem Inc., Saffron Walden, UK) was used as standard. Fractions collected at 1-min intervals were lyophilized, reconstituted in assay buffer and assayed for NPY as described above (2).

Plasma Glucose and Insulin Assays

Plasma glucose concentrations were measured using an au- toanalyzer (glucose oxidase method) and plasma insulin using a radioimmunoassay kit (Novo-Nordisk Diagnostics Limited, Cam- bridge, UK) for which the within-assay coefficient of variation was 4.9%.

Statistical Analysis

Statistical analysis of the data consisted of two-way analysis of variance for main effects, followed by Bonferroni's multiple range test (16). Single comparisons were made by two-sample Student's t-tests. A significance level of p<0.05 was selected. Data are presented throughout as mean_+ SEM.

RESULTS

Effects of Fenfluramine on Food Intake, Blood Glucose and Insulin Levels

Figure 1 shows that fenfluramine significantly reduced food intake in individual animals as compared with saline-injected controls. Food intake was minimal in each fenfluramine-treated rat between the presentation of food (at lights out) and the end of the experiment. Overall, mean food intake was reduced from 6 .9±1 .8 g in the saline-injected animals to 0 .9±0 .1 g (p<0.001) in those given fenfluramine.

Blood glucose concentrations were not significantly different between the saline- and fenfluramine-injected groups. Plasma in- sulin levels were significantly greater in the saline-injected con-

FENFLURAMINE REDUCES HYPOTHALAMIC NPY 253

TABLE 1

PLASMA GLUCOSE AND INSULIN LEVELS AND FOOD CONSUMPTION IN FENFLURAMINE- AND SALINE-INJECTED GROUPS OF RATS

Fenfluramine Saline

n= 7 7 Plasma glucose 9.1 • 0.5 10.2 +_ 1.1

concn. (mmol/1) Plasma insulin 67 _ 14" 250 +- 74

concn. (pmol/l) Food consumption 0.9 +-- 0.1+ 6.9 -- 1.8

(g/rat)

Data are mean _+ SEM. Differences between groups: *p<0.05; lp<O.O01.

trois than in fenfluramine-treated animals (p<0.05) (Table 1).

Effect of Fenfluramine on Hypothalamic NPY Levels

Using HPLC, the porcine NPY standards eluted after 23 min- utes at 48% acetonitrile (Fig. 2). The dominant peak of NPY-like immunoreactivity in the hypothalamic extracts from both experi- mental groups eluted in the position of the NPY standard, con- firming the specificity of the NPY antiserum (Fig. 2).

NPY levels (expressed as fmol/l~g protein) in the seven hypo- thalamic regions are shown in Fig. 3. Two-way analysis of vari- ance revealed a significant group effect, F(1,84)= 5.16, p<0.05,

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FIG. 2. NPY-like immunoreactivity (NPY-like IR) in 300-p,1 aliquots of HPLC fractions of pooled hypothalamic tissue extract from (a) saline-in- jected and (b) fenfluramine-injected rats, The elution position of synthetic porcine NPY standard is shown by the vertical arrow.

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hypothalamic area

• fenfluramine

[ ] saline

FIG. 3. NPY concentrations in individual hypothalamic regions in the sa- line-injected group (empty columns) and the fenfluramine-injected ani- mals (black columns). The regions are: medial preoptic area (MPO), lateral preoptic area (LPO), paraventricular nucleus (PVN), lateral hypothalamic area (LHA), ventromedial nucleus (VMH), dorsomedial nucleus (DMH) and the arcuate nucleus/median eminence complex (ARC/ME). Error bars represent SEM. Statistical significance of differences between groups: *p<0.05; **p<0.02; ***p<0.01.

and a significant difference between nuclei, F(6,84)=37.59, p<0.01. Further analysis using Bonferroni's multiple range test showed that NPY levels were significantly lower (p<0.05) in the fenfluramine-treated group than in the saline-injected controls. When NPY levels were examined in the seven individual hypo- thalamic nuclei, fenfluramine-treated rats showed significant 20- 30% decreases in NPY concentrations in the lateral preoptic area (p<0.02), ventromedial nucleus (p<0.02), the dorsomedial nu- cleus (p<0.05) and the lateral hypothalamic area (p<0.01), as compared with saline-injected controls.

DISCUSSION

This acute study has demonstrated that a single injection of fenfluramine which significantly reduced food intake also de- creased NPY concentrations in several specifc hypothalamic re- gions. These were the lateral preoptic area, the ventromedial and dorsomedial nuclei and the lateral hypothalamic area. Of these, the ventromedial and dorsomedial nuclei and the lateral hypotha- lamic area are implicated in appetite control (9) and also mediate the hyperphagic action of NPY injected centrally (13, 14, 20, 21). The fall in NPY concentrations in the lateral hypothalamic area following fenfluramine injection is particularly striking, as we have shown that NPY levels in this region increase signifi- cantly after the start of the dark phase [(17); see below], which coincides with increased feeding activity in normal rats. The lat- eral preoptic area, in which NPY levels were also reduced follow- ing fenfluramine injection, is not specifically implicated in the regulation of feeding behavior. Although manipulation of food intake can affect hypothalamic NPY synthesis and tissue levels of the peptide (17), the widespread fall in hypothalamic NPY con- centrations induced by fenfluramine cannot simply be attributed to reduced food intake in these animals, for two main reasons. Firstly, the study of food restriction by Sahu et al. (17) did not find any changes in NPY concentrations in any of the hypotha- lamic regions which they examined (medial preoptic area; paraventricular, ventromedial, dorsomedial and arcuate nuclei; median eminence) after 48 h of complete starvation; significant increases in the paraventricular nucleus and other regions were only seen after 72 h of food deprivation. The second piece of ev- idence comes from our own study of NPY concentrations in indi-

254 ROGERS, McKIBBIN AND WILLIAMS

vidual hypothalamic nuclei before and after the onset of darkness (11). Rats examined up to 5 h after darkness showed increased NPY concentrations in the lateral hypothalamic area only, as compared with rats sacrificed at the end of the light phase. This specific increase was observed in two separate groups of rats, which were either given or not given food at the onset of dark- ness. There were no differences between fed and unfed rats in NPY levels in any of the hypothalamic regions examined, indi- cating that, over this time period at least, the presence or absence of food does not alter hypothalamic NPY concentrations.

The possible interactions between serotonin and NPY in the modulation of appetite are particularly intriguing in view of their close anatomical relationship in the hypothalamus (6,7) and the striking carbohydrate specificity of their effects on food intake (9, 10, 20). However, these changes in tissue NPY levels cannot be related with certainty to alterations in NPY turnover or NPYergic activity in these areas, as reduced levels could be due to reduced synthesis, increased local release, increased local breakdown or a combination of these processes. In view of the relatively rapid changes (<5 h), changes in local release and/or breakdown seem more likely than a reduction in NPY synthesis which, in the hy- pothalamus, occurs predominantly in the arcuate nucleus (24), in which no alterations in NPY concentrations were found in this study. Studies of hypothalamic NPY mRNA levels may help to distinguish these possibilities.

The functional importance of these localized NPY concentra- tion changes is also uncertain. As fenfluramine, an anorectic agent, reduces levels of NPY, a powerful inducer of feeding, it is tempting to speculate that these changes should logically rep- resent reduced NPYergic activity, and perhaps that fenfluramine exerts its appetite-suppressing action by inhibiting the action of NPY. This possibility might apply primarily in the lateral hypo- thalamic area and dorsomedial nuclei, where injected NPY and

serotonin exert their respective effects on feeding (9, 10, 14, 20). It may be relevant that high doses of fenfluramine are known to reduce brain concentrations of norepinephrine (10), which is co- localized with NPY in several hypothalamic areas (19) and also has a stimulatory effect, albeit less potent than NPY, on food in- take (9,10).

Another consequence of the possible interaction between fen- fluramine and hypothalamic NPY may have been the impairment of insulin release in the fenfluramine-treated group as compared with the saline-injected controls (Table 1), as central NPY injec- tion is known to stimulate insulin secretion (1,12). Alternatively, reduced insulin release may simply be due to the greatly dimin- ished food intake in the fenfluramine-treated group, although blood glucose levels did not differ significantly from those of the saline-injected controls.

In summary, this study has demonstrated acute reductions in NPY concentrations in specific hypothalamic areas, including those thought to regulate appetite, following acute fenfluramine injection. This observation points to possible interactions between serotonergic and NPYergic systems which may serve to regulate feeding in rats and is further evidence that NPY may have a phys- iological role in controlling appetite and energy balance.

ACKNOWLEDGEMENTS

We are grateful to the British Diabetic Association and the Mersey Regional Health Authority for financial support of Dr. Pauline McKibbin and to Servier Laboratories UK for additional funding; to Dr. Richard Venn (Pain Relief Foundation Laboratory, Liverpool) for his invaluable guidance and assistance with the HPLC characterizations; to Mr. Peter Hynes, Miss Linda Horan and their colleagues for their conscientious care of the animals; to Dr. David Percy for help with the statistical analyses; and to Miss Luine Weir for typing the manuscript.

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