Metabolic effects of neuropeptide Y injected into the sulcal prefrontal cortex

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Brain Research Bulletin, Vol. 24, pp. 363-367. 0 Fwgamon Press plc, 1990.printed in the U.S.A.

Metabolic Effects of Neuropeptide Y Injected Into the Sulcal Prefrontal Cortex IAIN S. MCGREGOR, JO& A. MENl?NDEZ AND DALE M. ATRENS

f)epartment

of

Psychology, The ~niversi~ of Sydney, NSW 2006, A~st~~~ia Received 30 August 1989

McGRBOOR, I. S., J. A. ~~~~ AND D. M. ATRBNS. ~ef~o~ic effects of ~e~r~peFtide Y injected into the su~~~pre~o~3~i cortex. BRAIN RES BULL 24(3) 363-367, 1990.--The metabolic effects of 10, 39 and 156 pmol doses of neuropeptideY (NPY)

injected into the sulcal prefrontal cortex (SIX) were investigatedin an open-circuit calorimeter. The 39 pmol dose produceda large and long lasting increase in respiratoryquotient indicating both increased utilization of carbohydrateas an energy substrate and the synthesis of fat from carbohydrate.The 10 and 39 pmol doses producedan inhibition of energy expenditure that was still evident 24 hours following the 10, but not the 39 pmol, dose. These energy expenditure effects appeared to reflect an inhibition of thermogenesis as they were not systematically related to changes in locomotor activity. In separate tests, 39 pmol NPY reliably enhanced food intake. This combination of effects, nameiy increased carbohydrate util~tion, fat synthesis and food intake with reduced energy expenditure, shows NPY to be a potent anabolic force. In addition, these results indicate both the functional significance of NPY at a cortical level and the important role of the WC in the control of energy balance. Agranular insular cortex Neuropeptide Y NPY Sulcal prefrontal cortex Indirect calorimetry Energy expenditure Tbermogenesis

Metabolism

Respiratory quotient

a.m. and 5:30 p.m. The rats were implanted with 22 gauge cannulae complete with dummy cannulae (Plastics One, Roanoke, VA). The cannulae were aimed at the SPC [coordinates (16): 3.7 mm anterior to bregma, 3.5 mm lateral to the midline and 4.5 mm ventral to the skull surface]. The cannulae placements are illustrated in Fig. 1 for seven of the subjects. The remaining subject failed to complete the study because of a dislodged cannula. The injection sites were all within either the dorsal or ventral agranular insular cortex as described by Krettek and Price (lo), which constitute the two major subregions of the SPC. Two weeks following surgery, metabolic testing commenced in an open-circuit respirometer using methods described at length elsewhere (1, 8, 14). The rats were placed in a clear acrylic cage through which atmospheric air was drawn at slight positive pressure. After passing through the cage, the air was directed through a Perma Pure (PD-750-12PP) permeation drier prior to analysis. The air stream was then split and a sample of 110 ml/mm passed at slight negative pressure (8 KPa) through an Ametek CD-3A carbon dioxide (CO,) analyzer and an Ametek S-3A oxygen (0,) analyzer. The analyzers were calibrated daily with primary gravimetric standards. The testing chamber was placed on a Mettler PE-2000 electronic balance and the unintegrated signal from the strain gauge of the balance recorded every minute by computer to give a record of locomotor activity. From the raw data for 0, consumption and CO2 production, respiratory quotient (RQ = moles CO, produced/moles O2 consumed) and energy expenditure [EE (k.l) = moles 0, X (364 + 113 X RQ)] were computed for each minute of testing. Respiratory quotient is an accurate measure of the metabolic substrate (i.e., carbohydrate, protein or fat) being utilized by the organism (8). Energy expenditure, which is also known as “heat production” [(8) (pp. 125-129 and Appendix 17)] reflects the total energy output of the

NEUROPEPTIDE Y (NPY), a member of the pancreatic polypeptide family, has been shown to be a potent modulator of feeding behaviour and energy balance (4, 14,20, 21). The paraventricular nucleus of the hypothalamus (PVN) is an important site in mediating these effects as injections of NPY into the PVN produce hyperphagia and rapid weight gain (20,21). Recent work in our laboratory using indirect calorimetry has demons~ted that NPY injections into the PVN cause a shift in energy balance towards increased utilization of carbohydrate as an energy substrate (14). These results are consistent with the fact that NPY injections into the PVN also produce specific carbohydrate appetite (21). NPY is widely distributed in the mammalian brain, including the cerebral cortex (5). However, the functional significance of NPY at cortical loci is largely unexplored. NPY-like immunoreactivity is especially prevalent in cortical amas surrounding the rhinal sulcus, including the sulcal prefrontal cortex @PC) (5). The SPC has previously been shown to play an important role in regulating food intake, body weight and energy metabolism. Lesions of the SPC cause acute aphagia (9), chronic weight loss (9) and an increase in metabolic heat production (18), whereas electrical stimulation acutely increases food intake (3), promotes long-term weight gain (13) and inhibits thermogenesis (McGregor er al., unpublished data). We, therefore, reasoned that the NPY innervation of the SPC may mediate functions related to energy balance. Accordingly, the effects on whole body metabolism of microinjections of NPY into the SPC were examined. METHOD

Eight male Wistar rats, weighing from 385-460 grams at the start of testing, were maintained on ad lib access to food and water under a 14-hour light/lo-hour dark cycle (lights off at 8 p.m.). All metabolic testing occurred during the light cycle between 8:30

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time following injection (mins) mm FIG. 2. The effects of 10, 39 and 156 pmol doses of NPY into the SPC on mean respiratory quotient (RQ). The 39 pmol dose produced a significant treatment @
the study. organism achieved through whole body metabolism (7,8). Data for respiratory quotient, energy expenditure and locomotor activity following saline versus NPY injection were compared using two-way ANOVA with repeated measures on the time factor. Each testing session lasted sixty minutes, with a ten-minute calibration period prior to the first measurement. Each rat was subjected to each of four treatments, namely 1) 10 pmol dose of NPY (NPY porcine; Peninsula Lab. Inc., USA) in 0.064 ~1 vehicle (sterile saline; NaCl 0.9%) 2) 39 pmol dose of NPY in 0.25 ~1 vehicle 3) 156 pmol dose of NPY in 1 ~1 vehicle 4) vehicle injection of either 0.064, 0.25 ~1 or 1 ~1 volume counterbalanced across animals. The injections were made over a thirty-second period using a 0.5+1 Hamilton microsyringe and the injector cannula was held in place for a further minute following injection. In the case of the highest NPY dose (156 pmol) two consecutive 0.5 ~1 injections were given, one immediately following the other. No food was available during any of the metabolic tests, but ad lib food was available at all times except when the rats were in the metabolic testing chamber. The order of drug presentation was counterbalanced across rats with at least one week elapsing between each treatment. Each subject was further tested for 60 minutes 24 hours after each treatment. This is because the metabolic (14) and orexigenic (20) effects of certain NPY doses have previously been detected at this duration following injection. A week following the conclusion of metabolic testing, the same subjects were tested for elicited feeding following a single 39 pmol injection of NPY. The rats were placed in clear perspex cages with grid floors in which a weighed amount of standard feed (Allied Rat and Mouse Kubes, Allied Feeds, Rhodes, NSW) was placed on a petri dish. The rats had 72 hours previously been left in these cages for one night with ad lib access to the same food and water in order to habituate them to this testing environment. Testing was conducted in the late afternoon and terminated at least one hour prior to the onset of the dark cycle. Ad lib access to the

same standard feed was available at all times prior to testing. Each animal was tested for food intake over 120 minutes immediately following 1) a 39 pmole dose of NPY or 2) an equivalent injection of sterile saline and 3) no injection (sham treatment). Treatment order was counterbalanced across animals and each test day was separated by a period of 72 hours. Data for food intake were analyzed with the Student’s t-test for paired samples. RESULTS

Respiratory Quotient

It can be seen from Fig. 2, that relative to saline, the 39 pmol injection of NPY produced a large magnitude and statistically significant increase in RQ, with an approximate mean latency of 30 minutes to peak. This latency was variable across subjects, with one rat showing a pronounced elevation of RQ (relative to saline) on the first measurement (10 minutes following injection), and others not peaking until 30 minutes following injection. Similarly the duration of the effect varied, with the RQ of three of the rats still highly elevated (>l) at the end of testing 70 minutes following injection. Every rat showed an RQ of greater than 1 following the 39 pmol injection, while with saline injections no rat displayed an RQ of this magnitude. The mean RQ value following saline injection (0.916+0.009) is typical for the unfasted rat during the light cycle (1,8) indicating no effect of injection per se. Neither the 10 nor the 156 pmol doses of NPY produced a significant effect on RQ. None of the three NPY doses had any effect on RQ measured 24 hours following injection (data not shown). Energy Expenditure and Locomotor Activity The data for energy expenditure and motor activity are presented in Fig. 3. These parameters are considered together as under normal circumstances they are found to closely covary. In cases where changes in energy expenditure occur in the absence of corresponding alterations in motor activity then this reflects alterations in involuntary energy expenditure. Involuntary energy expenditure is also known as thermogenesis (1) or resting metabolic rate (7,8) and reflects the energy output of the organism that

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FIG. 3. The effects of 10 and 39 pmol doses of NPY on mean energy

expenditure (top graph) and mean motor activity (lower graph). Both the 10 and 39 pmol doses produced significant treatment by time effects on energy expenditure @
occurs independently of motor activity. Since the effects of the 156 pmol injection of NPY on activity and energy expenditure were almost identical to saline, only the data for the 10 and 39 pmol injections are presented (Figs. 3 and 4). Analysis showed both doses to have produced significant treatment by time interaction effects on energy expenditure. These results reflect a clear i~ibition of energy expenditure in both groups, seen following an initial lo-minute period of elevation. The locomotor activity data show that this initial lo-minute period of high energy expenditure was associated with elevated motor activity in both cases. The subsequent inhibition of energy expenditure in the 10 pmol condition is especially striking in that up until the final 20 minutes of testing, mean locomotor activity was elevated relative to control conditions whilst energy expenditure was for the most part lower. A similar, although not so

profound trend is evident in the 39 pmol condition. These results indicate the inhibition of involuntary energy expenditure following the 10 and 39 pmol doses.

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(top graph) and mean motor activity (lower graph) measured 24 hours following injection of 10, 39 pmoi NPY or saline. Energy expenditure was significantly decreased at 24 hours following the 10 pmol @
An inhibition of energy expenditure was still evident 24 hours following the 10, but not the 39, pmol dose of NPY (Fig. 4). Although there was also a significant treatment by time effect on motor activity in this case, it can be seen from Fig. 4 that this was not systematically related to the inhibition of energy expenditure. Indeed, motor activity in the 10 pmol condition was somewhat higher than saline for the first 30 minutes of testing and for the final 20 minutes the mean activity in the two conditions was very similar. This suggests that the 10 pmol dose of NPY into the SPC exerts a potent and long lasting inhibition of involuntary energy expenditure. Food

ln~ake

Figure 5 shows that NPY injections into the SPC enhanced food intake. Food intake after 39 pmol NPY was significantly greater than after saline or sham injection. Every animal tested showed eating after NPY injection, although the amount eaten was

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treatment FIG. 5. Mean food intake (+ S.E.M.) following a 39 pm01 dose of NPY compared to saline or sham injection. Food intake was greater following NPY than saline @<0.02) or sham treatments (p
somewhat variable across subjects (range 0.5-6.9 grams) as was the latency of the first eating bout (1 I-60 minutes). DISCUSSION

The present results constitute the first demonstration of clear functional effects of NPY at the cortical level. Until now, the effects of NPY have been studied almost exclusively with hypothalamic or ventricular injections. The high RQ produced by the 39 pmol NPY injections indicates the induction of a metabolic state characterized by an exclusive utilization of carbohydrate as an energy substrate (8). Under conditions of ad lib feeding, rats show a typical RQ in the light cycle of around 0.9, which indicates the mixed utilization of carbohydrates, fats and proteins. Fasting increases the reliance on fat reserves and lowers RQ values to those indicating fat metabolism (i.e., 0.748). The RQ of greater than 1 seen here following 39 pmol NPY indicates not only that the rats were exclusively utilizing carbohydrates, but also that they were synthesizing fat from carbohydrate (8). This raises the possibility that chronic infusions of NPY into the SPC, as with the paraventricular hypothalamus (21), may promote weight gain. In addition, the fact that 39 pmol NPY also produces feeding suggests that the eating induced through electrical stimulation of the SPC (3) may be related to metabolic consequences of the local release of NPY. Such NPY release could produce increased carbohydrate utilization causing decreased levels of circulating glucose and leading to food intake. This possibility is currently under investigation in our laboratory. The metabolic effects of NPY in the SPC are quite different from those obtained in the PVN. The main point of sirn~~ty is that 39 pmol NPY increases RQ when injected into both structures, with a latency of approximately 30 minutes in both cases (14). Additionally, 24 pmol NPY in the PVN enhances food intake (20). However, in contrast to the present results, 10 pm01 NPY in the PVN reduces RQ, whereas higher doses increase RQ with dose-dependent latencies (14). Further, whereas NPY injected into the PVN has no effect on energy expenditure or motor activity over a wide dose range (14), low doses of NPY into the SPC in the present study inhibited energy expenditure in the absence of clearly related effects on motor activity. This latter effect was not wholly unexpected since we have recently discovered that electri-

AND ATRENS

cal or excitatory amino acid stimulation of the SPC also inhibits energy expenditure (McGregor ef al., unpublished data). These data may relate to the previously described role of the SPC in thermoregulation ( 18,19). Spreading depression of the SPC causes a large magnitude rise in heat production and promotes heatseeking behaviour (18). Further, thermosensitive neurones have recently been described in the SPC that modulate the activity of thermoregulatory centres in the hypothalamus (18,19). The present results suggest that NPY may modulate energy expenditure through an influence on SPC thermosensitive neurones that act in concert with hy~~al~ic regulatory mech~isms. Further the i~ibition of the~ogenesis reported here concurs with a recent report of dose-related h~~e~ia produced through in~avent~cul~ injections of NPY (6). The fact that the lowest (IO pmol) dose of NPY produced an inhibition of energy expenditure that was detectable 24 hours later is especially striking. However, as noted above, other long lasting (>22 hour) effects have previously been reported with NPY (14,20). Perhaps the most surprising aspect is that 10, but not 39 or 156 pmol NPY produced an inhibition of energy expenditure at this duration following injection. This suggests that the present study may have investigated the upper limits of the dose effectiveness for NPY into the SPC on energy expenditure. Overall, these results confirm the involvement of the SPC in the control of energy balance. The demons~ation that NPY in the SPC increases carbohydrate utilization, fat synthesis and food intake whilst inhibiting energy expenditure implies a powerful anabolic (i.e., energy conserving) process. However, the exact neural mechanisms underlying these effects remain unclear. The long latency of the effect on RQ produced by the 39 pmol dose, and on energy expenditure produced by the 10 and 39 pmol doses suggests mediation of these effects by either 1) a long latency effect of NPY on SPC neurones indicative of NPY neuromodulation at key receptor sites and/or competition for receptor occupancy between NPY and other endogenous ligands, or 2) an immediate effect of NPY on SPC neurones with a relatively slow subsequent hormonally mediated peripheral effect. As we have recently found that electrical stimulation of the SPC induces almost immediate changes in RQ and energy expenditure (McGregor ef al., unpubIished data), similar to those reported here for NPY, it appears unlikely that the second hypothesis is correct. The effects of other substances, such as glutamic acid and the NMDA antagonist CPP have been reported to occur with long latencies when injected into the frontal cortex (15,17). This suggests that competition at the receptor sites and/or neuromodulatory factors may be responsible for the observed latency of the effects. The SPC is known to have rich subcortical connections with regions central to regulatory and autonomic function, and this has lead to the posterior SPC regions being called “visceral cortex” (11). In addition to its projections to the lateral hypothalamus and basolateral amygdala (2), the SPC projects to the dorsal and lateral portions of the nucleus of the solitary tract (NTS), which receives primary vagal efferents and exercises ~nd~en~l control over basic cardiac, respiratory and autonomic function (11, 12, 17, 22). Interestingly, the PVN also sends highly developed projections to the NTS and adjacent dorsal motor nucleus of the vagus (12,22). Whether the present results reflect SPC influence on these nuclei is an important subject for future research. ACKNOWLEDGEMENTS Research supported by a University of Sydney Postgraduate Scholarship to Iain McGregor and an Australian Research Council grant to Dale Atrens. We are grateful to Dr. Sarah F. Leibowitz (Rockefeller University, NY) for the generous gift of NPY. Dr. Hui Qiang Lin is thanked for his valuable technical assistance.

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