Evidence of a physiological role for neuropeptide Y in ventromedial hypothalamic lesion-induced hyperphagia
Post on 19-Aug-2016
ELSEVIER Brain Research 690 (1995) 275-278
Evidence of a physiological role for neuropeptide Y in ventromedial hypothalamic lesion-induced hyperphagia
Michael G. Dube a, Pushpa S. Kalra b, William R. Crowley c, Satya P. Kalra ~'* a Department ofNeuroscience, PO Box 100244, University of Florida College of Medicine, Gainesville, FL 32610-0244, USA
b Department of Physiology, PO Box 100244, University of Florida College of Medicine, Gainest,ille, FL 32610-244, USA c Department of Pharmacology, University of Tennessee College of Medicine, Memphis, TN 38163, USA
Accepted 23 May 1995
We evaluated the role of neuropeptide Y (NPY), a potent endogenous orexigenic signal, in the ventromedial hypothalamic (VMH) lesion-induced hyperphagia in rats. To produce hyperphagia and excessive weight gain, adult female rats received bilateral electrolytic or sham lesions in the VMH. Concurrently, a permanent intracerebroventricular cannula was implanted in the third ventricle of the brain. After a recovery period, these rats were passively immunized against NPY to evaluate the role of endogenous NPY on hyperphagia. The results showed that intraventricular administration of NPY antibodies abolished the hyperphagia in VMH-lesioned rats. These revelations are in agreement with the notion that altered hypothalamic NPY release or action may underlie the hyperphagia and excessive weight gain seen in response to structural damage in the VMH.
Keywords. Neuropeptide Y; Hyperphagia; VMH lesion; Immunoneutralization
Gaining insight into the neural mechanism responsible for hyperphagia and obesity has a long history of investi- gation [1-3,9]. One of the experimental models exten- sively studied to understand the role of the hypothalamus in hyperphagia and attendant body weight gain is the ventromedial hypothalamic (VMH)-lesioned rat. Bilateral indiscriminate structural damage to the VMH by elec- trolytic and radiofrequency lesions leads to rapid onset of hyperphagia and excessive body weight gain [1,9,13,16,22,26]. The postoperative hyperphagia and obe- sity syndrome in the VMH-lesioned rats has a temporal aspect; an early dynamic phase followed by a later static phase that is characterized by marked differences in eating patterns and body weight gain [9,16,22]. Various investiga- tions have implicated alterations in metabolic and sympa- thetic nervous system activities and changes in blood hormone levels as causal factors in this VMH-lesion-in- duced syndrome of hyperphagia and excessive body weight gain [2,3,13,16,26].
Recent studies show that neuropeptide Y (NPY) pro-
* Corresponding author. Fax: (1) (904) 392-8347.
0006-8993/95/$09.50 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00644-3
duced in the arcuate nucleus (ARC) [4,8] and released in the paraventricular nucleus (PVN) of the hypothalamus, may play an important role in the control of normal feeding as well as hyperphagia in the experimentally-in- duced diabetic rat [7,10,12,19-21]. We have reported re- cently that passive immunization of rats against NPY by continuous infusion of NPY-antibody into the third ventri- cle of the brain, markedly suppressed 24 h food intake, thereby suggesting a physiological role for NPY in regula- tion of the daily food intake pattern . These observations led us to hypothesize that hyperphagia and the attendant increase in body weight gain in VMH-lesioned rats may be a consequence of disturbances in the function of NPY in the hypothalamus. To test this hypothesis, in the current study we examined the effects of passive immunization against NPY on feeding in VMH-lesioned rats.
Adult female Sprague-Dawley rats (Hsd:Sprague Daw- ley SD; Harlan Sprague-Dawley Inc., Indianapolis, IN) weighing 200-225 g upon arrival in the laboratory were used. Animals were maintained under controlled light (lights on 05.00-19.00 h) and temperature (21-24C) con- ditions with ad libitum access to Purina lab chow pellets and water. Following approximately one week of adapta- tion to the laboratory, surgery was performed under sodium
276 M.G. Dube et aL /Brain Research 690 (1995) 275-278
pentobarbital anesthesia (40 mg/kg, i.p.). Animals were mounted in a stereotaxic instrument with the nosebar 3.3 mm below the interaural line. A stainless steel monopolar electrode, positioned 6.4 mm anterior to the interaural line and 0.6 mm lateral to the midline, was lowered to the base of the brain and then raised 0.5 mm . To lesion the VMH, rats received a 2.5 mAmp anodal current for 15 sec between the electrode and the ear bars. The electrode was then raised and the procedure was repeated on the other side of the midline. In sham-operated animals, the elec- trode was lowered, but no current was passed. Following the lesion or sham lesion procedures the animals remained in the stereotaxic instrument and the nosebar was adjusted to 5.0 mm above the interaural line for the placement of a permanent stainless steel cannula in the third cerebroven- tricle using a method previously described . Animals were then allowed to recover for 12-26 days before the experiment. Rats had ad libitum access to Purina lab chow pellets and water throughout. Body weight was monitored to confirm successful placement of the lesion. Addition- ally, rats were handled and underwent mock intraventricu- lar (i.v.t.) injections during the recovery period. A five day experiment was begun for lesioned rats that exhibited excessive weight gain and for sham rats (weight gain at least 3 times the sham mean) matched for post-surgical interval. On day 0 of the experiment, we first determined the amount of food intake by each rat during a two hour period (13.00-15.00 h) in the middle of the lights-on phase. This was done by placing preweighed food pellets in their home cage and weighing remaining pellets and any spillage 2 h later. On the following day (day 1) the rats were not disturbed. On the next day (day 2), with the rats remaining in their home cages, the i.v.t, cannula was opened and efflux of CSF was observed to confirm pa- tency of the third ventricle cannula. Rats were then in- jected with 3/xl undiluted anti-NPY immunoglobulin (IgG) at 09.00, 11.00 and 13.00 h. After the last injection (13.00 h), food in the home cage was replaced with preweighed food pellets to measure food intake over the next 2 h. On day 3, food intake was measured as on day 0 to determine whether the experimental procedures on day 2 had affected normal food intake in these rats. We observed that food intake on day 3 was similar to the baseline intake deter- mined on day 0 (mean food intake: lesioned rats 5.5 + 1.0 g; sham rats 1.4 + 0.2 g; P < 0.05; t-test). On the follow- ing day (day 4), normal rabbit serum (NRS) IgG was injected into the third cerebroventricle of all rats at 09.00, 11.00 and 13.00 h and 2 h food intake was again moni- tored as on day 2. At the end of the experiment, the animals were anesthetized with an overdose of pento- barbital and perfused intracardially with formol saline. Brains were removed, stored in formol saline and histolog- ically processed to verify site of the VMH lesions.
The NPY antiserum was prepared as previously de- scribed . This NPY antiserum is used in radioimmunoas- says at a final dilution of 1:40,000. It displays 100%
cross-reactivity with the related pancreatic polypeptide pYY and less than 1% cross-reactivity with rPP, luteiniz- ing hormone releasing hormone, growth hormone releasing hormone, vasoactive intestinal polypeptide, galanin, neu- ropeptide K, neurotensin, Met-enkephalin, Leu-enkephalin, fl-endorphin, dynorphin A, dynorphin B, and somatostatin. Since pYY is not found in the hypothalamus, this NPY antiserum is suitable for hypothalamic administration. The anti-NPY IgG was purified with the aid of an Econo-Pac Serum IgG Purification Kit (#732-2027, Bio-Rad Labora- tories, Hercules, CA). IgG was also purified from NRS for controls (NRS IgG). The purified IgG solutions were lyophilized for storage and reconstituted with normal saline on experimental days in a volume equal to the volume of serum from which the IgG was derived.
Data are presented as mean _+ S.E.M. The food intake data were analyzed by two-way ANOVA's for repeated measures to compare treatment groups (lesion and sham) and treatments (baseline x anti-NPY IgG NRS IgG). This was followed by Fisher's PLSD test for individual paired comparisons. Lesion and sham groups were com- pared using t-tests. Only differences with P < 0.05 were considered significant.
On day 0, body weight gain in VMH-lesioned rats was significantly higher (101 _+ 5.2 g; n = 5) than in sham-le- sioned rats (25.7_+ 6.2 g; n = 6) over the same time interval. Whereas no discernible tissue damage was appar- ent in the sham-lesioned rats, extensive neural disruption in the VMH was seen in the VMH-lesioned rats; however, the ARC and PVN showed little sign of damage (Fig. 1). As shown in Fig. 2, the VMH-lesioned rats consumed significantly greater amounts of food as compared to sham controls (P < 0.01) during the 2 h of baseline observation on day 0. After three injections of anti-NPY IgG on day 2, food intake in sham control rats was unaffected, but the striking hyperphagia in VMH-lesioned rats was abolished. Food intake during the 2 h period was significantly sup- pressed in VMH-lesioned rats as compared to their intake on day 0. The hyperphagic response in VMH-lesioned rats was again apparent on day 4 after injection of control NRS IgG; food intake was in the range measured on day 0 for these rats.
Thus, as expected [1,9,13,22], we observed that destruc- tion of the VMH produced hyperphagia and increased body weight gain. Since immunoneutralization of endoge- nous NPY by anti-NPY IgG drastically reduced the food intake, it implies that NPY participates in the hyperphagic response in these rats. In light of the observation that immunoneutralization of endogenous NPY by anti-NPY IgG infusion intraventricularly also suppressed nighttime feeding , we infer that an imbalance in NPY function may underlie hyperphagia in the VMH-lesioned rats. Since the bulk of current evidence suggests that NPY release and action in the PVN plays a major role in the central regulation of ingestive behavior [7,10-12,21,23,24], we conclude that destruction of neural elements in the VMH,
M.G. Dube et al. / Brain Research 690 (1995) 275-278 277
including the fibers of passage, results in either increased release of NPY or altered action at receptive sites in the PVN. Whether this altered NPY function is a consequence of loss of an inhibitory network impinging upon the NPY system remains to be determined [1-3,9,16]. A compre- hensive study of the disruption at the cellular and molecu- lar levels induced by these VMH lesions is underway. Recently, some behavioral effects were observed after NPY antibody injection into the hypothalamus of mice during their recovery from anesthesia . In our study, we failed to observe any behavioral changes after NPY antibody injection into the third cerebroventricle. Addition- ally, it was of interest to observe that passive immunoneu- tralization against NPY failed to completely abolish food intake not only in the VMH-lesioned but also in sham-le- sioned rats. Similarly, passive immunization against NPY failed to completely suppress daily food intake . It is possible that either the intracerebroventricular route of administration of anti-NPY IgG is not optimally effective or that our anti-NPY IgG failed to completely block endogenous NPY. It is also likely that in the absence of NPY, other orexigenic messenger molecules such as galanin and opioids [6,15,17], may stimulate feeding, albeit to a much lesser degree.
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278 M.G. Dube et al. // Brain Research 690 (1995) 275-278
duced hyperphagia. Overall, the current results together with similar evidence in diabetic rats [14,20,21] support the hypothesis that disturbances in the function of NPY may be one of the key factors underlying hyperphagia in these two experimental models.
Supported by a grant from the National Institutes of Health (DK 37273). Thanks are due to Ms. Sally Mc- Donell for secretarial assistance.
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