Fasting activates neuropeptide Y neurons in the arcuate nucleus and the paraventricular nucleus in the rhesus macaque

Molecular Brain Research 113 (2003) 133–138 www.elsevier.com / locate / molbrainres

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Fasting activates neuropeptide Y neurons in the arcuate nucleus and the paraventricular nucleus in the rhesus macaque Kevin L. Grove a , *, Peilin Chen a,b , Frank H. Koegler a , Andrew Schiffmaker a , M. Susan Smith a,b , Judy L. Cameron a,b a

Division of Neuroscience, Oregon National Primate Research Center, 505 NW 185 th Avenue, Beaverton, OR 97006 -5384, USA b Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239, USA Accepted 3 March 2003

Abstract It is well accepted that neuropeptide Y (NPY) plays a pivotal role in the regulation of food intake and energy homeostasis in the rodent, with NPY neurons in the arcuate nucleus (ARH) being thought of as the major contributor to the complex central feeding circuitry. Recent data from our group also indicate that NPY is important in the regulation of energy homeostasis in the nonhuman primate (NHP); exogenous NPY administration into the 3rd ventricle is a potent stimulator of food intake in the male rhesus macaque. The purpose of this study was to determine if NPY neurons in the rhesus macaque respond to a metabolic challenge, induced by 48 h of fasting, in a manner similar to that seen in the rodent. NPY mRNA was detected in hypothalamic sections from 48-h fasted or fed rhesus monkeys by in situ hybridization, using a [ 35 S]UTP-labeled riboprobe specific for human NPY. Not surprisingly, NPY mRNA was abundant in the ARH of the NHP; however, of great interest was the expression of NPY mRNA in neurons within the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON). This raised the question as to whether all of these populations of NPY neurons are sensitive to changes in energy availability. Indeed, NPY expression in the ARH and PVH was significantly elevated in response to fasting; however, no significant change was detected in the SON. These data indicate that the NPY neurocircuitry involved in the regulation of food intake is more complex in the NHP than in rodents.  2003 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Neuroendocrine regulation: other Keywords: Hypothalamus; PVH; ARH; Feeding; Nonhuman primate; NPY

The hypothalamic circuitry that controls appetite and energy expenditure is complex and involves numerous neurotransmitter and neuropeptide systems, many of which function in parallel to either increase or decrease energy availability (see reviews: Refs. [17,20,22,25,27]). However, the neuropeptide Y (NPY) system is considered as one of the most abundant and potent modulators of energy homeostasis in the rodent brain. In the normal adult rodent, NPY neurons in the arcuate nucleus (ARH) are the major source of NPY that is involved in the regulation of food *Corresponding author. Tel.: 11-503-690-5380; fax: 11-503-6905384. E-mail address: [email protected] (K.L. Grove).

intake and energy balance. Because the ARH has a reduced blood brain barrier, the NPY neurons are in an excellent position to sense peripheral signals of energy balance (i.e. leptin, insulin and ghrelin) and transmit that signal to other important feeding centers (i.e. paraventricular nucleus of the hypothalamus, PVH). Our group has previously demonstrated that many of the neuronal systems that have been described as potent modulators of food intake in the rodent are also critical in the regulation of feeding in the nonhuman primate (NHP). These include a-melanocyte stimulating hormone (aMSH) and agouti related protein (AGRP) [12], modulators of MC3 / 4 receptors, leptin [24] and NPY [15]. In fact, doses of NPY (1 mg) that stimulate feeding in rodents [4,23] are

0169-328X / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0169-328X(03)00093-7

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similarly potent when given intracerebroventricularly (i.c.v.) in the monkey, in spite of the large differences in body weight and brain size [15], indicating that the NPY receptor system in the monkey is extremely sensitive. Furthermore, we have demonstrated that proopiomelanocortin (POMC) neurons in the ARH are sensitive to subtle changes in energy balance, because they are inhibited in animals missing a single meal [12]. These data indicate that like the rodent, ARH neurons (NPY/ AGRP and aMSH) in the primate are likely to play a critical role in transmitting peripheral signals of energy balance to important feeding centers within the hypothalamus and other areas of the brain. However, preliminary data indicate that, unlike the normal adult rodent, the NHP displays NPY mRNA expression in neurons in several hypothalamic regions, including the PVH and supraoptic nucleus (SON) as well as the expected expression within the ARH. The purpose of the present study was to determine if NPY mRNA expression within these different hypothalamic regions of the rhesus macaque is affected by a metabolic challenge induced by 48 h of fasting. A 48-h fast was chosen, as our previous work in the NHP has shown that significant metabolic and neuroendocrine responses are apparent in male rhesus monkeys after this duration of fasting [1,2,10,12,18,19,26], including changes in circulating levels of insulin, glucose, ketone bodies, cortisol, thyroid hormone, leptin, growth hormone and luteinizing hormone. Eight adult male rhesus monkeys, housed individually in stainless steel cages, were maintained in temperature (2462 8C) and humidity regulated rooms. Animals were maintained on a 12:12-h light / dark cycle, with lights on at 07:00 h. Prior to the testing period, animals had ad libitum access to drinking water and were fed two meals per day to simulate natural meal patterns. High-protein monkey chow biscuits (no. 5047, jumbo size, Purina Mills, St. Louis, MO), weighing approximately 16.5 g each (3.11 metabolizable cal / g), were provided at 09:00 h (seven biscuits) and 15:00 h (12 biscuits) daily (975 cal / day). One half piece of fresh fruit (half of a medium red apple) was provided daily with the morning meal. Uneaten biscuits were removed before each meal. Monkeys were randomly assigned to one of two groups: (1) fed group (n54, 12.162.3 years of age; 11.861.8 kg) that were killed at 13:00 h without missing any meals (4 h after the morning meal), and (2) a 48-h fasted group (n54, 10.262.3 years of age; 9.360.6 kg) that were killed at 13:00 h after missing two 09:00 h meals and two 15:00 h meals. All animal procedures were approved by the Oregon National Primate Research Center Institutional Animal Care and Use Committee and were performed according to federal guidelines. The animals were perfused with fixative as previously described [12]. Briefly, animals were sedated with ketamine HCl (10 mg / kg, i.m.) at approximately 12:30 h and then deeply anesthetized with sodium pentobarbital

(.30 mg / kg, i.v.). Each animal was transcardially perfused with 0.9% NaCl containing 2% sodium nitrite, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The brains were quickly removed, and hypothalamic tissue blocks were cut and immersed in the same fixative for 2 h at 4 8C and then saturated in 20% glycerol. The tissue was then frozen and stored at 280 8C until sectioned. In situ hybridization for NPY mRNA was performed similarly to that previously described [8,12]. Briefly, coronal hypothalamic sections (25 mm) were collected in a 1-in-4 series using a freezing microtome. The sections were stored in cryoprotectant (30% sucrose, 30% polyethylene glycol, buffered with NaPO 4 , pH 7.2) at 220 8C. Sections were mounted on slides (Superfrost / Plus, Fisher Scientific, Pittsburgh, PA) and dried. The sections were then postfixed in 4% paraformaldehyde (pH 7.4 in 0.1 M NaPO 4 ), rinsed in phosphate buffer, incubated with proteinase K (5 mg / ml), and then treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). The sections were washed in 23 SSC, dehydrated through a graded series of alcohols, delipidated in chloroform and rehydrated through a second series of alcohols and air dried. A human-specific NPY cRNA probe was transcribed from a 500 bp cDNA clone in a pGEM4 vector with 25% of the UTP being labeled with 35 S (New England Nuclear Life Sciences, Boston, MA). The specific activity of the probe ranged from 6 to 7310 8 dpm / mg. A saturating concentration of 0.3 mg / ml per kb of the probe diluted in hybridization buffer (50% formamide, 6.25% dextran sulfate, 0.7% Ficoll, 0.7% polyvinyl pyrrolidone) was used. The sections were hybridized with the cRNA probes for approximately 16 h in a moist chamber at 55 8C. After incubation, the slides were washed in 43 SSC, RNase A at 37 8C, and in 0.13 SSC at 60 8C. Slides were then dehydrated through a graded series of alcohols and dried. Slides were then dipped in Kodak NBT2 emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 in 600 mM ammonium acetate and placed in light-tight boxes containing desiccant and stored at 4 8C for 10–14 days. The slides were developed and counter stained with cresyl violet, and the distribution of the silver grains analyzed by darkfield microscopy. Images of silver grain distribution were captured under darkfield illumination using a Micromax Digital Camera (Princeton Instruments) connected to a Nikon microscope (Labphoto-2) with a Plan Apo 20X (NA 0.75) objective. The Metamorph Imaging system (Universal Imaging Corp., West Chester, PA, USA) was used to process the images and brightness, and contrast levels of the digital images were adjusted with Photoshop (Adobe Systems, San Jose, CA, USA). Slides were analyzed for silver grain density using Optimus Imaging software (Media Cybernetics, Silver Springs, MO). An individual brain section was captured by a CCD camera (Cohu High Performance CCD Camera).

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The silver grain density (representing [ 35 S]NPY cRNA labeling) was measured using a sampling box that encompassed the entire region of interest. This sampling box was held constant for all sections and animals. Measurements were taken bilaterally through the complete rostralcaudal extent of the PVH, SON and ARH, as determined by histological analysis of the sections. Some sections were not measured because of tears or folds in the region of interest. Background labeling was determined by placing the sampling box over the ventromedial hypothalamic nucleus, a region known not to contain NPY gene expression. Background labeling was then subtracted from densitometric values of labeling in the ARH, PVH and SON. To best represent the relative level of cRNA labeling and to give the mean value for an individual animal, the densitometric values from anatomically matched sections (across all animals) were averaged (PVH, 10; SON, 10; ARH, 15). The detection of NPY mRNA by in situ hybridization was divided into two assays because of the large number of slides. Therefore, to eliminate inter-assay variability, the data was normalized to the average value of the control animals for each individual assay. The arcuate NPY mRNA expression data from one fed animal was eliminated from the study because of damage to this region

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during necropsy. The NPY expression in PVH and SON were unaffected by this damage, therefore this data was included in the analysis. This resulted in an n53 for the ARH and an n54 for the PVH and SON in fed animals. The data are represented as the mean6S.E.M. Differences between the groups for each brain region were determined with a t-test, with the level of significance set at P,0.05. In the normally-fed adult rhesus macaque hypothalamus, NPY mRNA was present not only in the ARH (Fig. 1), but surprisingly was also abundant in the SON of some animals (Fig. 3) as well as low amounts in the PVH (Fig. 2). In animals that were fasted for 48 h, NPY mRNA expression significantly increased by 5406160% in the ARH compared to fed controls (P50.024; Fig. 1). The high variability in ARH-NPY expression in the fasted animals was due to increased NPY mRNA expression that ranged from 214% to 718% compared to the mean for the fed animals. NPY mRNA expression also significantly increased by 214630% in the PVH in response to fasting (P50.021; Fig. 2). In contrast, fasting did not significantly alter NPY mRNA expression in the SON, rather there was a nonsignificant decrease in the average levels (Fig. 3). Specifically, two of the four fasted animals had nearly undetectable levels of NPY mRNA in the SON, while one animal displayed normal levels (110% compared to fed

Fig. 1. Neuropeptide Y mRNA expression in the ARH of fed and fasted rhesus monkeys. The figures are digital images of silver grains, representing [ 35 S]NPY cRNA probe labeling, under darkfield illumination. The white dashed boxes in the images in the first column represent the approximate area shown in the second column. The numbers in the upper right corner indicate the approximate anterior–posterior coordinates, relative to bregma, of the digital images (according to the rhesus macaque brain atlas of Paxinos et al. [16]). 3V, third ventricle. White bars represent 100 mm.

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Fig. 2. Neuropeptide Y mRNA expression in the PVH of fed and fasted rhesus monkeys. The figures are digital images of silver grains, representing [ 35 S]NPY cRNA probe labeling, under darkfield illumination. The white dashed boxes in the images in the first column represent the approximate area shown in the second column. The numbers in the upper right corner indicate the approximate anterior–posterior coordinates, relative to bregma, of the digital images (according to the rhesus macaque brain atlas of Paxinos et al. [16]). 3V, third ventricle. White bars represent 100 mm.

controls) and one animal displayed moderate levels (68% compared to fed controls). All four of the control animals displayed detectable levels of NPY gene expression in the SON. The level of NPY expression within the SON for an individual animal did not correlate with NPY expression within the ARH or PVH; indicating that the variability in NPY mRNA expression was not due to quality of the tissue. The present study demonstrates that like the rodent, the NHP displays an abundance of NPY gene expression within neurons in the ARH. However, unlike the normal

adult rat, NPY mRNA is also present within neurons in the SON and in relatively low levels in the PVH in the NHP. Although the major source of NPY within the hypothalamus of the adult rodent comes from the ARH, NPY gene expression has been reported in the PVH in the rat during specific stages of postnatal development and in the monosodium glutamate lesioned animal [7,11,21], and in the SON in response to severe dehydration [14]. However, NPY is not normally expressed in these regions in the adult rodent. This clear difference between the rodent and NHP indicates that the neurocircuitry that controls food

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Fig. 3. Neuropeptide Y mRNA expression in the SON of the rhesus monkey. The figures are digital images of silver grains, representing [ 35 S]NPY cRNA probe labeling, under darkfield illumination. (A) and (B) represent NPY expression in the SON from two different animals. NPY expression in the SON did not correlate with levels of expression in the PVH or the ARH, nor did it significantly change in response to fasting. OT, optic tract. White bars represent 100 mm.

intake and energy balance in the primate is likely to be more complex. Of interest, our group previously reported an abundance of NPY mRNA expression within the ARH of newborn rhesus monkeys [8]; however, NPY mRNA was not detected within the PVH or SON of these neonatal monkeys. The animals in that study were not fasted and therefore, NPY expression may simply have been undetectable under those conditions. It is possible that longer exposure times may have revealed low levels of expression within the PVH, or that NPY expression within the PVH and SON are not activated until later stages of postnatal development. El Majdoubi et al. [6] has also characterized NPY gene expression within the hypothalamus of the juvenile and pubertal NHP; while they did report NPY expressing cells dorsal to the ARH, they did not identify the exact location of these neurons. It is yet unknown what cell type(s) expresses NPY in the SON and PVH. Although NPY mRNA expression is present in large neurons within the SON, and is found in the PVH predominantly in the lateral magnocellular portion, it remains to be determined if NPY is colocalized within magnocellular oxytocinergic or vasopressinergic neurons. The presence of multiple populations of hypothalamic NPY neurons in the NHP also raised the question as to whether they are similarly regulated and all involved in the regulation of food intake and energy balance. Like the rodent, ARH-NPY gene expression was significantly increased in response to fasting. However, PVH-NPY neurons also increased gene expression by 214% in response to the 48-h fast, indicating that the adult primate contains a population of PVH-NPY neurons that are also responsive to metabolic challenge. However, it is difficult to determine, as it is for all fasting studies, if the PVH- and ARH-NPY neurons are responding directly to the physiological negative energy balance or simply responding to the psychological stress of fasting. In the rodent, NPY is

involved in the activation of the HPA axis [5,9]. Furthermore, ARH-NPY neurons are responsive to glucocorticoids [3,13]. And, our previous studies in rhesus monkeys fasted for 24–48 h have shown that the monkey has a number of metabolic changes (such as a decrease in plasma levels of glucose, insulin, leptin, thyroid hormone, and an increase in ketone bodies and growth hormone), including an increase in cortisol and ACTH, in response to this metabolic challenge [1,2,10,18,19,26]. These changes are consistent with those reported in rodent models, suggesting that the peripheral feedback mechanism to the brain in response to the negative energy balance is similar in the rodent and NHP. What is different between these two models is the central processing of this information, namely that in the NHP NPY neurons in both the PVH and ARH are activated. In contrast, NPY expression in the SON was unaffected by fasting, suggesting that this population is not involved in the regulation of energy balance and independent of a putative psychological stress induced by fasting. More likely, SON-NPY neurons may be involved in fluid homeostasis, as has been suggested for the rodent [14]. In the present study, the animals had free access to water throughout the fasting period. It remains to be determined if other peripheral regulators of energy balance, such as leptin, insulin and ghrelin, regulate NPY expression not only in the ARH, but in the PVH as well. Compounds that function to inhibit appetite through suppression of the NPY-ergic system may have access to ARH-NPY neurons, which are outside the blood brain barrier, but not to PVH-NPY neurons, which are inside the barrier. It is important to investigate not only the afferent regulators of PVH-NPY neurons, but the downstream targets as well. It is possible that ARH-NPY and PVH-NPY neurons in the primate may have different roles in the intricate neurocircuitry that regulates energy homeo-

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stasis, i.e. increasing appetite vs. decreasing energy expenditure.

Acknowledgements This work was supported by National Institutes of Health grants HD18185, RR00163 and DK55819.

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