Orexin neurons are stimulated by conditions that are glucoprivic, suggesting that orexin signaling may be increased during nutritional duress. We have previously shown that injection of orexin A (OxA) into the rostral lateral hypothalamic area (rLHa) robustly and dose-dependently increases feeding behavior. Thus we hypothesized that exogenous administration of orexin A would induce a greater feeding response after acute food deprivation or perceived caloric duress achieved through 2-deoxyglucose (2DG) administration. To test our hypothesis, male Sprague-Dawley rats implanted with internal guide cannulas directed to the rLHa were exposed to varying degrees of food deprivation (0, 3, 12, 24 h) and 2DG (200 mg/kg) before intra-rLHa OxA (500 pmol) infusion. We also performed a dose-response study using graded doses of OxA (0, 31.25, 125, and 500 pmol) in fed and 24-h fasted rats. OxA administration in conjunction with the highest level of prior food deprivation (24 h) resulted in the greatest feeding response (above baseline means; 0 h deprivation: 1.9 ± 0.6; 24 h deprivation: 4.4 ± 0.8; P = 0.0034) and showed a dose-dependent enhancement of feeding. Additionally, 2DG administration before OxA administration resulted in a significantly higher feeding response (above baseline means: 2DG = 1.8 ± 0.5; OxA = 1.8 ± 0.4; 2DG + OxA = 5.1 ± 0.6; P < 0.0001). These data support the hypothesis that orexin signaling may be important in modulating the feeding network under times of nutritional duress.
- lateral hypothalamus
orexin a and orexin b (also known as hypocretin 1 and 2) are 33 and 28 amino acid residues, respectively, produced from a common peptide precursor (9, 34). The orexins are produced by neurons located perifornically in the caudal aspect of the lateral hypothalamus. Orexin A has two disulfide bridges, while orexin B is linear, and both peptides have amidated carboxy terminals (34). The two orexin receptors (OX1R and OX2R) belong to the g-protein coupled superfamily of receptors. OX2R, thought to be or Gi- or Gq-coupled, has nearly equal affinity for both peptides, whereas OX1R, believed to be Gq-coupled, has a much greater affinity for orexin A than for orexin B (34, 37, 43). The orexin neurons demonstrate markedly diffuse projections throughout the central nervous system, corresponding with their receptor distribution (1, 7–9, 27, 29, 40).
Orexin signaling likely modulates arousal and sleep thresholds via connectivity to hindbrain nuclei (14, 29). It is thought that orexin signaling at these nuclei and in the ventro-lateral preoptic area forms a means by which arousal can be positively influenced (35). Diurnal variations in cerebroventricular levels of orexin support this role in that levels are high during the active phase of the rat circadian cycle and low during the resting phase (12). In line with this hypothesis of orexin's function within the ascending arousal system, the human condition of narcolepsy coincides with selective loss of orexin neurons and low-orexin cerebrospinal fluid levels (41).
The first published accounts of orexin behavioral effects linked the orexins to the modulation of feeding (34). Orexin cell bodies reside within what was classically considered the “feeding center” (2), and thus orexins were named for the Greek root for appetite (orexis). This hypothesis for orexin function was further supported by behavioral evidence where orexin A (orexin B has a less consistent record of inducing feeding) infused intraventricularly, or in discrete hypothalamic nuclei, resulted in feeding in rats and mice (11, 16, 22). Additionally, orexin receptors are expressed in many hypothalamic nuclei important for the regulation of feeding behavior, and interconnectivity between orexin, neuropeptide Y (NPY), and proopiomelanocortin (POMC) neurons has been demonstrated (1, 17). Peripheral administration of an orexin receptor antagonist SB334867A inhibits feeding (15). However, some evidence suggests that orexin A may modulate feeding behavior only under conditions of nutritional duress. Orexin neurons are activated by 48-h food deprivation and when blood glucose concentrations are dramatically lowered (4, 13, 26).
In line with these data, we previously reported that there was increased sensitivity to orexin A when rats were placed on a restricted diet (42). Furthermore, we hypothesized that increased sensitivity may be related to changes in glucose levels inherent to the restricted-feeding paradigm. Thus, in the present study, we tested the hypothesis that modifying metabolic status either behaviorally via food deprivation or pharmacologically with 2-deoxyglucose would increase sensitivity to orexin A-induced feeding. These data shed light on the broader theory that orexin signaling may be particularly tuned to modulating feeding behavior under conditions of caloric challenge.
MATERIALS AND METHODS
Fifty-five male Sprague-Dawley rats weighing 250–275 g (Harlan, Madison, WI) at the beginning of the experiment were housed in individual hanging wire cages, with water available ad libitum via a continuous dipper at the back of the cage, and rat chow (Harlan Teklad 8604) available ad libitum unless otherwise specified via a standard hopper located at the front of the cage. Animals were maintained in a temperature-controlled (22°C) room on a 12:12-h light-dark cycle with lights on at 0400. All experiments had local Institutional Animal Care and Use Committee approval.
One week after arrival, rats underwent stereotaxic surgery for the implantation of guide cannulas (26 g, Plastics One, Roanoke, VA) directed 0.5 mm dorsal to the rLHA using coordinates defined by Paxinos and Watson (28). Animals were anesthetized with a combination of intramuscular ketamine (90 mg/kg) and xylazine (15 mg/kg). The following stereotaxic coordinates were used: dorsal/ventral = −7.3, medial/lateral = −1.9, anterior/posterior to bregma = −2.2; with the incisor bar set at −3.3 mm to maintain a level skull. Animals received subcutaneous Flunixin (2.5 mg/kg) as a postoperative analgesic. After surgery and between injections, a dummy stylet was placed in the guide cannula that extended to the tip of the cannula. Correct cannula placement was confirmed with postmortem histological verification. Five rats were excluded from the final analysis, four because of incomplete data, and one because of a misplaced cannula.
Injections were made over 30 s with an injector (33 g, Plastics One) that extended 1 mm beyond the tip of the guide cannula. Injectors were left in place for an additional 15 s to allow for diffusion of injectate. Animals were handled daily, and at least three mock injections were carried out before experimental manipulations. During mock injections, animals were handled exactly as they would be during an experimental injection, and the dummy cannula was removed and replaced. Thus, during experimental manipulations, animals were already habituated to the procedure. Specificity of injections to the intended site has been verified by past experiments, where positive behavioral response correlated with the distance of the infusion from these specific coordinates (42).
Rats (n = 19) were injected intraparenchymally via rLHA cannulas with orexin A (500 pmol) or vehicle [0.5 μl artificial cerebrospinal fluid (aCSF)] in a counter-balanced design 2 h after the onset of the dark cycle. With this design, animals received both treatments, and both treatments were represented (roughly equally) on each experimental day. The dose of orexin A was chosen based on our previous work, indicating that it produces a reliable feeding response (39, 42). Injections were preceded by varying levels of food-deprivation (0, 3, 12, and 24 h) with at least 72 h between experimental manipulations. Chow intake was monitored 1, 2, and 4 h after injections by manually weighing chow hoppers on a digital scale and correcting the difference for the weight of food spillage.
The most robust feeding response to orexin A was seen after 24-h food deprivation. To evaluate the relative dose-response relationship between orexin A and the state of deprivation, in a separate group of animals (n = 13) orexin A (0, 31.25, 125, and 500 pmol) was injected after 24-h food deprivation or no deprivation. Injections were carried out as described in experiment 1a, and the experimental conditions (dose and state of deprivation) were counterbalanced across treatment days. Food intake was evaluated 1 and 2 h after injection. At least 72 h elapsed between treatment days.
Rats (n = 18) were injected subcutaneously with 2-DG (200 mg/kg, Chemicon, Temecula, CA) or vehicle (saline) 1 h after the onset of the dark cycle. Chow was immediately removed. One hour after subcutaneous injections, orexin A (500 pmol) or vehicle (aCSF) was injected intra-rLHA. Chow was returned after rLHA injections, and food intake was monitored as above. Injections took place according to a Latin square design, such that each treatment was given each experimental day, and each animal received each treatment. Thus, on each experimental day, the following four treatments were administered (subcutaneously+intra-rLHA): 1) saline+aCSF, 2) saline+orexin A, 3) 2DG+aCSF, and 4) 2DG+orexin A.
Three-way ANOVA with deprivation level (0, 3, 12, and 24 h), time after injection (0–1, 1–2, and 2–4 h), and treatment (vehicle or orexin A) as between subjects factors and food intake as the dependent variable revealed a main effect for each factor (deprivation level: F3,360 = 56.9, P < 0.0001; time: F2,360 = 331, P < 0.0001; treatment: F1,360 = 8.2, P = 0.004). On the basis of these findings, separate repeated-measures ANOVA, using treatment (vehicle or orexin A) as the within-subjects factor and chow consumption (grams) as the dependent variable for each time point after infusion (0–1 h, 1–2 h, and 2–4 h) within each degree of deprivation (0, 3, 12, and 24 h), revealed a main effect of treatment for the first hour of feeding (0–1 h) after injection for all degrees of deprivation (data represented as means ± SE): 0 h deprivation (MV = 0.7 ± 0.3, MOxA = 2.7 ± 0.5, F1,15 = 11.0, P = 0.0047), 3 h deprivation (MV = 8.1 ± 0.5, MOxA = 10.9.4 ± 0.9, F1,15 = 4.5, P = 0.049), 12-h deprivation (MV = 6.8 ± 0.4, MOxA = 9.6 ± 0.6, F1,15 = 24.1, P < 0.0001), and 24-h deprivation (MV = 10.0 ± 0.5, MOxA = 13.7 ± 0.9, F1,15 = 12.5, P = 0.0025). There was no effect of treatment during other time points (1–2 and 2–4 h) during any level of deprivation. Thus feeding postinjection was significantly augmented by orexin A after each degree of deprivation, and this was limited to acute augmentation during the first hour (Fig. 1).
Repeated-measures ANOVA with the data represented as grams above baseline (GAB; each animal's vehicle response subtracted from their individual orexin A response) revealed a main effect of treatment (0, 3, 12, and 24 h food deprivation) during the 0–1 h postinfusion time point (0 h: M = 2.0 ± 0.6, 3 h: M = 1.9 ± 0.8, 12 h: M = 2.9 ± 0.6, 24 h: M = 4.2 ± 0.8; F1,15 = 18.2, P = 0.028). A Fishers paired least significant difference (PLSD) post hoc analysis revealed a significant difference between 24 h GAB and 0 or 3 h deprivation (24 h vs. 0 h GAB, P = 0.011) (24 h vs. 3 h GAB, P = 0.008, Fig. 2).
Orexin A dose-dependently augmented feeding after 24-h food deprivation (Fig. 3). A two-way repeated-measures ANOVA with dose (31.25, 125, and 500 pmol OxA) and deprivation level (24 h fast or fed ad libitum) as between subjects factors and food intake (GAB) as the dependent variable, revealed a main effect of dose (P = 0.03) and deprivation level (P = 0.05) during the 0–1 h postinfusion time point (fasted condition: M31.25 = 1.0 ± 0.6, M125 = 1.8 ± 0.7, M500 = 3.2 ± 0.7; fed condition: M31.25 = 0.3 ± 0.6, M125 = 0.04 ± 0.7, M500 = 0.8 ± 0.7). Unpaired t-tests comparing feeding response above baseline for doses both within each deprivation level and between each deprivation level demonstrated significant differences between GAB 500 pmol fasted and 31.25 pmol fasted (P = 0.03) and 500 pmol fasted and all doses in the fed condition (for 31.25, 125, and 500 pmol; P = 0.006, P = 0.0028, and P = 0.028, respectively). There was no significant effect of treatment on chow intake (grams above baseline) during the second hour after infusion after either level of deprivation.
Orexin A alone, 2DG alone, and orexin A and 2DG together significantly augmented feeding 1 h after intra-rLHA infusions (Fig. 4A) and between 2–4 h (data not shown). A repeated-measures ANOVA with treatment [vehicle-vehicle (V-V), orexin A+vehicle (OxA+V), 2DG+vehicle (2DG+V), and 2DG+orexin A (2DG+OxA)] as the within-subjects factor and chow consumption as the dependent variable revealed a main effect of treatment (F3,54 = 27.8, P < 0.0001) the first hour following orexin A infusion. A Fishers PLSD post hoc analysis revealed a significant difference between the V-V (MV-V = 2.5 ± 0.5) treatment and all other treatments (MV+OxA = 4.3 ± 0.5, P = 0.002; M2DG+V = 4.3 ± 0.5, P = 0.0027; M2DG+OxA = 7.6 ± 0.6, P < 0.0001; Fig. 3A), as well as the 2DG+orexin A treatment and Orexin A+V (P < 0.0001) and 2DG+V (P < 0.0001; Fig. 3A). Using the same statistical analysis as above, a main effect of treatment was revealed during the 2–4 postinjection time point (F3,54 = 4.3, P = 0.009; data not shown). The decline in feeding during the 2–4 h period after orexin A and 2DG treatments, likely represents a compensatory response to the acute overeating during the 0–1 h time period after injection.
A repeated-measures ANOVA, with the data represented as GAB (equal to each subject's individual V-V value subtracted from the individual response to other treatments), revealed amain effect of treatment (F2,63 = 19.3, P < 0.0001, Fig. 4B). A Fisher's PLSD post hoc analysis revealed a significant difference between 2DG+orexin A (M2DG+OxA = 5.1 ± 0.6) and V+orexin A (MV+OxA = 1.8 ± 0.4, P < 0.0001) or 2DG+V (M = 1.8 ± 0.5, P < 0.0001) treatment groups.
We investigated the hypothesis that orexin A modulates feeding behavior during metabolic challenge. As one approach, we examined the ability of orexin A to augment feeding under experimental conditions that lower blood glucose concentrations. A greater feeding response was seen after food deprivation (Figs. 1–3) or before administration of 2DG (Fig. 4). Orexin A dose-dependently enhanced feeding to a greater extent following a 24-h fast (Fig. 3). Thus, not only was the presentation of a metabolic challenge permissive for orexin A-induced feeding, but it further amplified the feeding response. These data provide support for the hypothesis that orexin A modulates the feeding network during nutritional duress.
There are different mathematical methods of determining a feeding “response,” and each method may result in seemingly different conclusions, unless one considers the subtleties involved in interpretation of biological responses. As a percentage of “total chow consumed,” the amount that orexin A induces feeding above baseline (response to vehicle) decreases with increasing deprivation. However, analyzing the data in this manner is deceptive because the interpretation of food intake above that observed during an existing large caloric load is different than that in animals consuming a small caloric load. For example, an increase of 2 GAB of 1 g is a 200% increase. A 2-g increase over a baseline of 10 g is only 20%. Thus, as a percentage, it would seem that the induced feeding in the first example is far greater than that in the second example. However, as the amount of chow eaten in a specific period of time increases, it becomes increasingly difficult to augment feeding, since interoceptive satiety cues are much higher in animals that have consumed a large caloric load, and gastric fill is limited. Therefore, an observation of increased feeding above an already high baseline is indicative of a powerful consummatory drive. Thus statistical analysis of the value “grams above baseline” may be a more relevant measure of consummatory drive than a percentage increase.
Previous studies have shown that orexin A delivered directly into hypothalamic parenchyma results in feeding behavior (11, 39). We have postulated that the greatest feeding response is seen following administration of orexin A to the rLHA (42). Thus, although the functional neuroanatomy of this aspect of the LH is not clearly defined, we chose to investigate orexin A action at this site. This region of the LH is rostro-caudally at the level of the paraventricular nucleus of the hypothalamus (PVN), dorsolateral to the fornix, and ventromedial to the internal capsule. It does not appear as though the feeding response following orexin A in this site is due to diffusion to the PVN, as injections directly into the PVN yields less robust feeding responses (39).
Unfortunately, little is known about this aspect of the LH. As a whole, the LH is a large and heterogeneously populated region and receives projections from the nucleus of the solitary tract, hypothalamic nuclei, and other hind-brain nuclei. There are multiple ascending neuronal pathways traversing through the lateral hypothalamus (LH; 2). It is likely that this rostral portion of the LH receives these projections as well. Efferent projections from this site, to our knowledge, are not specifically known. We previously demonstrated that orexin A infused into this site results in increased c-Fos immunohistochemical staining [an indicator of cellular activity; Fos-like immunoreactivity (Fos-LI)] in several nuclei important to feeding behavior (24). Thus this portion of the LH likely has connectivity with many aspects of the feeding network, including the arcuate nucleus, dorsomedial nucleus, PVN, and the nucleus of the solitary tract. Additionally, this discrete area showed increased Fos-LI after muscimol injection in the nucleus accumbens, suggesting interconnectivity and a role for this site in the hedonics of appetite (38). Thus the rLHA is likely interconnected with other nuclei important for the regulation of feeding behavior.
Blood glucose concentrations are relatively constant, yet fluctuations in blood glucose concentrations may be an important part of appetite regulation (20). Within the LH and other areas of the hypothalamus, there are numerous glucose-sensing neurons, some glucose inhibited (stimulated by falling glucose levels) and some glucose excited (stimulated by increasing glucose). Orexin A stimulates glucose-inhibited neurons and inhibits glucose-excited neurons (21, 36). Furthermore, falling blood glucose levels may stimulate orexin neurons, as there is increased orexin mRNA after a 48-h fast, and increases in Fos-LI in orexin neurons after insulin or 2DG challenges (4–6).
To further test the theory of glucose involvement, we sought to pharmacologically deprive neurons of glucose. To this end, the nonmetabolizable glucose competitor 2DG was administered subcutaneously 1 h before orexin A administration. The dose of 2DG used (200 mg/kg) reliably increases feeding when peripherally administered (30). GAB feeding response was significantly greater after injection of both 2DG and orexin A, compared with either orexigenic agent alone (Fig. 3). 2DG acts within the central nervous system to induce hyperphagia, yet local glucopenia, induced via 2DG infused into the LH, does not result in hyperphagia (3, 23, 31, 32). Thus the enhanced response to orexin A that occurred in the present study may be due to changes in targets downstream from the injection site.
The same mechanism that stimulates orexin neurons during times of extreme nutritional challenge may be responsible for the enhanced feeding response to orexin A after 24-h food deprivation or 2DG. In the present study, 24-h food deprivation resulted in a dose-dependent and significantly greater feeding response (above baseline) compared with 0 or 3 h of deprivation (Fig. 2). These data are consistent with our previous findings in which rats on a restricted diet responded robustly to orexin A (5.4 GAB; Ref. 42). Whether sensitization occurs at the site of infusion or elsewhere in the signaling pathway is not directly apparent in the current study. However, during caloric challenge, in conjunction with or because of increased orexin signaling, the feeding network may be more receptive to orexin A. Together, the deprivation and 2DG data provide support for the hypothesis that orexin A is particularly proficient at modulating the feeding network under severe caloric challenge.
NPY pathways may be involved in this process. When subthreshold doses of intracerebroventricular NPY and orexin A are injected simultaneously, feeding is significantly enhanced (33), suggesting that orexin A- and NPY-responsive pathways interact to influence feeding behavior. Injecting orexin A into this portion of the LH or intracerebroventricularly increases Fos-LI in the arcuate nucleus (24, 44), the primary site of NPY-producing neurons. Administration of antagonists for the Y1 receptor has been shown to block orexin A-induced feeding (18, 19, 44). The demonstration of reciprocal neuronal connectivity between orexin neurons and NPY neurons in the arcuate nucleus provides a neuroanatomical basis for these findings (17). Furthermore, orexin A directly stimulates NPY neurons in the arcuate nucleus and inhibits POMC neurons in the arcuate nucleus (25).
This set of experiments supports the hypothesis that orexin signaling may play a larger modulatory role in feeding behavior during times of caloric challenge. This may be in part due to changes in blood glucose concentrations. As demonstrated here, during times of caloric need (food-deprivation or 2DG-induced), exogenous orexin A-induced feeding may be enhanced. In agreement with what others have proposed, orexins may be acting at extra-hypothalamic sites to increase arousal and within the hypothalamus to increase appetite, both of which would be fundamentally important for survival (10). Furthermore, data suggesting that orexin signaling is important in regulating a particular aspect of behavior may be influenced by the metabolic status of the animal at the time of experimental manipulation.
This work was supported by the Department of Veterans Affairs, the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57573, and the Minnesota Craniofacial Training Program National Institute of Dental and Craniofacial Research T32 DE-07288–8.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 the American Physiological Society