In Vivo Evidence for Inverse Agonism of Agouti-Related Peptide in the Central Nervous System of Proopiomelanocortin-Deficient Mice

  1. Virginie Tolle12 and
  2. Malcolm J. Low123
  1. 1Center for the Study of Weight Regulation and Associated Disorders, Oregon Health and Science University, Portland, Oregon
  2. 2Vollum Institute, Oregon Health and Science University, Portland, Oregon
  3. 3Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon
  1. Address correspondence and reprint requests to Virginie Tolle, PhD, UMR549 Inserm, IFR Broca-Ste Anne, 2 ter rue d’Alesia, 75014 Paris, France. E-mail: virginie.tolle{at}broca.inserm.fr

Abstract

OBJECTIVE—Melanocyte-stimulating hormone (MSH) peptides processed from proopiomelanocortin (POMC) regulate energy homeostasis by activating neuronal melanocortin receptor (MC-R) signaling. Agouti-related peptide (AgRP) is a naturally occurring MC-R antagonist but also displays inverse agonism at constitutively active melanocortin-4 receptor (MC4-R) expressed on transfected cells. We investigated whether AgRP functions similarly in vivo using mouse models that lack all neuronal MSH, thereby precluding competitive antagonism of MC-R by AgRP.

RESEARCH DESIGN AND METHODS—Feeding and metabolic effects of the MC-R agonist melanotan II (MTII), AgRP, and ghrelin were investigated after intracerebroventricular injection in neural-specific POMC-deficient (Pomc−/−Tg/+) and global POMC-deficient (Pomc−/−) mice. Gene expression was quantified by RT-PCR.

RESULTS—Hyperphagic POMC-deficient mice were more sensitive than wild-type mice to the anorectic effects of MTII. Hypothalamic melanocortin-3 (MC3)/4-R mRNAs in POMC-deficient mice were unchanged, suggesting increased receptor sensitivity as a possible mechanism for the heightened anorexia. AgRP reversed MTII-induced anorexia in both mutant strains, demonstrating its ability to antagonize MSH agonists at central MC3/4-R, but did not produce an acute orexigenic response by itself. The action of ghrelin was attenuated in Pomc−/−Tg/+ mice, suggesting decreased sensitivity to additional orexigenic signals. However, AgRP induced delayed and long-lasting modifications of energy balance in Pomc−/−Tg/+, but not glucocorticoid-deficient Pomc−/− mice, by decreasing oxygen consumption, increasing the respiratory exchange ratio, and increasing food intake.

CONCLUSIONS—These data demonstrate that AgRP can modulate energy balance via a mechanism independent of MSH and MC3/4-R competitive antagonism, consistent with either inverse agonist activity at MC-R or interaction with a distinct receptor.

Genetic disruption of either mouse or human proopiomelanocortin (POMC) causes early-onset obesity (13), highlighting a major role of POMC in the regulation of energy homeostasis. POMC is processed posttranslationally into multiple peptides, including the opioid β-endorphin and the melanocortins ACTH, α-melanocyte-stimulating hormone (αMSH), βMSH, and γMSH. POMC peptides in the central nervous system (CNS) are essential in the regulation of energy intake and expenditure as demonstrated in studies using compound mutant mice (Pomc−/−Tg/+) expressing a Pomc transgene that selectively restored pituitary POMC in Pomc−/− mice to produce a neural-selective POMC deficiency (4). Lack of αMSH is likely the principal cause of obesity (3,5) due to the loss of agonist signaling at central melanocortin receptors (MC-R), melanocortin-3 receptor (MC3-R), and melanocortin-4 receptor (MC4-R), each of which plays a distinct role in the regulation of energy homeostasis (6,7,8).

The anorectic actions of centrally administered αMSH or the synthetic MC3/4-R agonist melanotan II (MTII) (911) are blocked by Agouti-related peptide (AgRP), an endogenous MC3/4-R antagonist (12,13), released from terminals of neuropeptide Y (NPY)/AgRP arcuate neurons. In addition to their localization to the same brain regions as POMC fibers (14), AgRP nerve terminals send projections to neurons that possess MC4-R (15) but are not innervated by αMSH terminals (14,16). These neuroanatomic findings indicate that AgRP may modulate MC4-R activity in the absence of endogenous αMSH.

In vitro data strongly support the ability of AgRP to modulate MC4-R by an inverse agonist mode of action (1720); however, the physiological significance is unresolved. Modulation of MC4-R constitutive activity may be important to maintain long-term energy homeostasis in humans (21). In rodents, the concept of inverse agonism has been buttressed by demonstrations that a single injection of AgRP induces hyperphagia over several days (2224), whereas this long-lasting effect cannot be reproduced by synthetic MC4-R antagonists like HS014 or JKC-363 (23,25).

In the present study, we analyzed the feeding and metabolic responses to intracerebroventricular injections of MTII and AgRP in mice deficient in all central MSH peptides. Because responses to melanocortin antagonists apparently require the presence of circulating glucocorticoids (26), we compared Pomc−/− mice with a global deficiency of POMC and adrenal insufficiency to Pomc−/−Tg/+ mice with a neural-specific deficiency of POMC but restored glucocorticoids (4). Feeding effects of the orexigenic gut peptide ghrelin (27) were also tested.

RESEARCH DESIGN AND METHODS

A colony of Pomc mutant mice on a hybrid B6;D2;129X1;129S6 genetic background with independently segregating Pomc+/− and pHalEx2* Tg alleles was established as described previously (4). Mice were maintained under controlled temperature and photoperiod (12-h light, 12-h dark; lights on at 7:00 a.m.) with free access to water and chow (4.5% fat, 20% protein, 6% fiber, and 3.4 kcal/g; PicoLab Rodent Diet 20; PMI Nutrition International, St. Louis, MO). Experimental procedures were approved by the Institutional Animal Care and Use Committee and followed Public Health Service guidelines.

Peptides.

MTII, hAgRP (83–132), mAgRP (82–131), and rGhrelin were purchased from Phoenix Pharmaceuticals (Mountain View, CA) and dissolved in physiological saline.

Intracerebroventricular cannulation.

Mice were anesthetized with 2% Avertin. Twenty-six–gauge stainless steel guide cannulae cut 2.5 mm below the pedestal (Plastics One, Roanoke, VA) were implanted stereotaxically into the right lateral ventricle (posterior −0.4 mm, lateral −1.0 mm, relative to bregma), secured to the skull using cap screws (Small Parts) and dental cement, and occluded with stainless steel dummy obturators. Mice were then housed individually for 7–10 days of recovery without specific treatment, except for the Pomc−/− mice that required injection with dexamethasone (0.15 μg i.p. in 1 ml saline) for 3 days.

Feeding and basal metabolic rate.

Peptides were injected intracerebroventricularly in a volume of 1 μl over 1 min using a 33-gauge stainless steel injection cannula extending 0.5 mm below the guide cannula and connected to a 1-μl Hamilton syringe with polyethylene tubing. Mice were returned to their home cage, and the remaining food in containers on the cage floor was weighed at different time intervals. Basal metabolic rate was determined by indirect open-circuit calorimetry (Oxymax; Columbus Instruments) as previously described (4). After 3 days of chamber habituation, six measurements were recorded daily from each mouse at 60-min intervals. Individual basal oxygen consumption (Vo2) levels were established by averaging the two lowest Vo2 measurements. Respiratory exchange ratios (RERs) were recorded as the molar ratio of Vco2 to Vo2, and a daily average value was calculated.

Experimental design.

Male and female mice were used in all experiments. Peptides or saline were administered between 11:00 a.m. and 12:00 p.m. for daytime experiments and between 6:00 and 7:00 p.m. (lights off at 7:00 p.m.) for nighttime experiments. Mice were randomized into different groups based on their 24-h food intake before experiments and received each dose of peptide and saline in a counterbalanced order. Detailed designs for each experiment and the number of animals per group are provided in the online appendix (available at http://dx.doi.org/10.2337/db07-0733).

Real-time RT-PCR.

Mice were decapitated between 9:00 and 11:00 a.m., and hypothalami were dissected on ice, harvested in TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA), and extracted according to the manufacturer's directions. PCRs were performed on an ABI Prism 7300 Sequence Detection System instrument (Perkin-Elmer Applied Biosystems, Foster City, CA) using TaqMan Gene Expression Assays containing a set of sequence-specific primers and a 6-FAM dye-labeled TaqMan MGB probe for MC4-R, MC3-R, AgRP, NPY, or cocaine and amphetamine–related transcript (CART) and the TaqMan endogenous control 18S. cDNA samples obtained from reverse transcription of 1 μg RNA were run in duplicate in total reaction volumes of 20 μl containing 5 μl cDNA, 1× TaqMan Gene Expression Assay, and 1× TaqMan Universal Master Mix. Thermal cycling conditions included an initial denaturation step at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Real-time PCR data were analyzed with the 2−ΔΔCT method as previously described (28).

Statistical analyses.

All data presented are means ± SE. Data were analyzed by repeated-measures ANOVAs or multifactor ANOVAs appropriate for the design of each experiment with genotype and/or drug as independent variables using Stat View Power PC for Macintosh version 5.0.1 (SAS Institute). One-factor ANOVAs were used to follow up significant main effects; post hoc pairwise comparisons between groups were performed by Fisher's protected least squares difference (PLSD) or paired two-tail t tests. P values <0.05 were considered significant.

RESULTS

Feeding effects of MTII injected at the onset of the dark cycle.

A single intracerebroventricular injection of 0.5 nmol MTII at the onset of the dark cycle decreased food intake in control Pomc+/+Tg/+ and mutant Pomc−/−Tg/+ and Pomc−/− mice compared with vehicle-treated animals 2 h after the injection (Fig. 1A). In Pomc+/+Tg/+ mice, food intake was inhibited by 67%, and the effect was completely reversed by 24 h (Fig. 1A and B). In both Pomc−/−Tg/+ and Pomc−/− mice, MTII acutely decreased food intake by 95%, and in contrast to control mice, this effect was sustained with 60 and 54% cumulative reductions at 24 h (P < 0.01 vs. saline). Long-term anorexia induced by 0.5 nmol MTII was also associated with significant weight loss in Pomc−/−Tg/+ and Pomc−/− mice (P < 0.01 and 0.05 vs. saline, respectively) (Fig. 1C).

Feeding effects of co-administered MTII and AgRP.

To test the ability of AgRP to antagonize MSH agonists at MC3/4-R, 0.5 nmol AgRP was injected alone or in combination with 0.1 nmol MTII at the onset of the dark cycle. Although this lower dose of MTII was slightly less potent than 0.5 nmol to reduce feeding 2 h after injection, we chose it in combination with AgRP because its anorectic effects had dissipated in all genotypes at 24 h. There was a significant main effect of the peptide treatments on 2-h food intake but no significant interaction of treatment × genotype (Fig. 2A). Post hoc analyses collapsed across genotype confirmed that 0.1 nmol MTII alone significantly decreased acute food intake (P < 0.0001 vs. saline). In contrast, there was no significant main effect of treatment or treatment × genotype interaction on 24-h food intake (Fig. 2B). After nighttime injection, when maximum feeding activity is already observed, there was no additive, short-term orexigenic effect of 0.5 nmol AgRP alone on any genotype. However, AgRP significantly blocked the anorectic effect of 0.1 nmol MTII in all genotypes 2 h after co-injection of both peptides (P < 0.0001, AgRP plus MTII vs. MTII alone, Fisher PLSD) (Fig. 2A), consistent with a competitive antagonist action at the MC3/4-R.

Short-term–feeding effects of AgRP and ghrelin injected during the light cycle.

Administration of 0.5 or 2 nmol AgRP during the daytime increased 2-h food intake by 160 and 250%, respectively, in Pomc+/+Tg/+ mice (P < 0.05 vs. saline). In Pomc−/−Tg/+ mice, neither dose of AgRP had short-term orexigenic effects (Fig. 3A; data with 2 nmol not shown). To test the ability of Pomc−/− and Pomc−/−Tg/+ mice to respond to other orexigenic signals, 1 nmol ghrelin was injected during the daytime (Fig. 3B). Ghrelin stimulated 2-h food intake in Pomc+/+Tg/+ and, to a much lesser extent, in Pomc−/−Tg/+ mice (P < 0.0001 and 0.05 vs. saline, respectively) but had no effect in Pomc−/− mice.

Short- and long-term effects of AgRP injected at the onset of the dark cycle on food intake and body weight.

Nighttime injection of 0.5 nmol AgRP, when maximum feeding activity occurs naturally, did not further increase 2-h food intake in any genotype (Fig. 4A, D, G, and J). However, hyperphagia was observed 24 h after the injection of AgRP, was sustained up to 72 h, and was accompanied by body weight gain in Pomc+/+Tg/+ mice (Fig. 4B and C). Pomc+/− mice were more sensitive to the long-term effects of AgRP with sustained hyperphagia up to 96 h after injection and more pronounced body weight gain than Pomc+/+Tg/+ mice (Fig. 4K and L). Neither Pomc−/−Tg/+ nor Pomc−/− mice increased their food consumption after nighttime injection of 0.5 nmol AgRP (Fig. 4E and H). Furthermore, injection of 0.5 or 2 nmol AgRP during the light cycle also did not produce any long-lasting orexigenic effect in the mutant Pomc−/−Tg/+ mice, whereas it was orexigenic in Pomc+/+Tg/+ control mice (data not shown). Similar to Pomc+/+Tg/+ controls, the rate of body weight gain was greater in Pomc−/−Tg/+ mice injected with 0.5 nmol AgRP than with saline, and this difference was significant at 72 h (P < 0.001) (Fig. 4F). Although a short-lived reduction in food intake and body weight was sometimes observed after intracerebroventricular injections of saline in Pomc+/+Tg/+, Pomc+/−, and to a greater degree in Pomc−/−Tg/+ mice, food consumption usually rebounded to baseline values within 48 h. In contrast, Pomc−/− mice had prolonged anorexia and weight loss in response to a saline injection (Fig. 4H and I).

Effect of AgRP injected at the onset of the dark cycle on Vo2 and RER in mice with restricted food access.

To test the hypothesis that AgRP may be a long-term modulator of energy expenditure in Pomc−/−Tg/+ mice, we measured Vo2 and RER during 7 consecutive days after the injection of saline or AgRP at the onset of the dark cycle (Fig. 5), conditions that induced weight gain in both Pomc+/+Tg/+ and Pomc−/−Tg/+ mice. In most Pomc+/+Tg/+ mice, as depicted in two representative individuals (Fig. 5A), the effects of AgRP were observed up to 72 h after the injection. In contrast, the onset of AgRP effects was delayed by 24 h but subsequently lasted longer in Pomc−/−Tg/+ mice (Fig. 5B). Consequently, changes were subtler but more prolonged in Pomc−/−Tg/+ compared with Pomc+/+Tg/+ mice.

Analyses performed for the relevant, genotype-specific timeframes showed an increased body weight of 12% over 3 days in Pomc+/+Tg/+ (P < 0.01) and 6% over 6 days in Pomc−/−Tg/+ (P < 0.01) mice after AgRP treatment, compared with their initial body weights. In Pomc+/+Tg/+ mice, increased daily food consumption, increased average daily RER, and decreased Vo2 were observed within 24 h after the injection (0–24 h) and were sustained up to 72 h after the injection (24–72 h) (Fig. 5C, E, and G). In contrast, none of the parameters was modified within 24 h after AgRP injection (0–24 h) in Pomc−/−Tg/+ mice, but the delayed effects on food intake, Vo2, and RER were significant between 24 and 72 h and persisted up to 168 h (Fig. 5D, F, and H).

MC3/4-R, AgRP, NPY, and CART gene expression in the hypothalamus.

Expression levels of MC4-R (Fig. 6A) and MC3-R (Fig. 6B) from the whole hypothalamus were unchanged in Pomc−/−Tg/+ and Pomc−/− mice compared with control Pomc+/+Tg/+ mice. Unlike MC3/4-R, levels of AgRP (Fig. 6C), NPY (Fig. 6D), and CART (Fig. 6E) mRNA all differed by genotype. In Pomc−/− mice, AgRP expression was significantly decreased by 64%, CART expression was increased by 47%, and NPY expression was unchanged compared with Pomc+/+Tg/+ mice. In Pomc−/−Tg/+ mice, CART expression was increased by 67%, but AgRP and NPY were unchanged compared with Pomc+/+Tg/+. Notably, however, in Pomc−/−Tg/+ mice, expression of both AgRP and NPY was significantly increased compared with that in the glucocorticoid-deficient Pomc−/− mice.

DISCUSSION

Catabolic effects of MTII were accentuated in obese POMC-deficient mice.

Both strains of POMC-deficient mice were more sensitive to the short-term anorectic action of MT-II than their control siblings. The mechanism of increased sensitivity to MTII in POMC-deficient mice or other animal models of obesity, such as Zucker rats (fa/fa) (29), diet-induced obesity (DIO) rats (30), and DIO and ob/ob mice (31) with decreased POMC expression or decreased melanocortin tone (32,33,34), may involve increased expression, density, or functional coupling of MC-R in response to the chronic absence or decrease of endogenous melanocortin ligands. There were no significant differences in the expression of MC4-R or MC3-R in Pomc−/−Tg/+ and Pomc−/− mice compared with Pomc+/+Tg/+ mice, suggesting that either increased MC-R density or signaling is responsible for the heightened anorectic effect of MTII. However, our quantification did not take into account differential regional expression of the MC4-R in various hypothalamic nuclei or extra-hypothalamic expression of MC4-R (15), which are important in the regulation of energy homeostasis and have been shown to mediate the effects of melanocortin agonists and antagonists and long-term orexigenic actions of AgRP (35).

Increased sensitivity to MTII could also be due to secondary alterations in the expression of other hypothalamic orexigenic/anorectic signals. AgRP mRNA was reduced in Pomc−/− mice, consistent with data reported by Coll et al. (36) and supporting the hypothesis that reduced levels of AgRP contributed to the increased response to MTII. However, this cannot be the only explanation because AgRP expression was almost normalized in glucocorticoid-replete Pomc−/−Tg/+ mice, which still displayed an exaggerated anorectic response to MTII.

Differential short- and long-term effects of AgRP in neural selective POMC-deficient mice.

AgRP did not alter short-term food intake but was able to antagonize the anorectic effect of MTII in Pomc−/−Tg/+ mice, indicating that the short-term orexigenic effect of AgRP requires the presence of αMSH and therefore is due to a competitive antagonist action at MC3/4-R. Despite reexpression of POMC in the pituitary gland, Pomc−/−Tg/+ mice had undetectable levels of αMSH in the hypothalamus (4), excluding the possibility that αMSH from a peripheral source leaked into the CNS. Interestingly, Pomc+/− mice had stronger feeding responses to both AgRP and MTII than control Pomc+{/+Tg/+ mice, probably reflecting a gene-dosage effect in the response to melanocortin agonists and antagonists. We previously demonstrated that the hypothalamic content of αMSH in Pomc+/− mice is one-half that of Pomc+/+Tg/+ mice (4), indicating that there is less endogenous αMSH to antagonize in those animals.

In vitro studies have clearly demonstrated that AgRP acts as a competitive antagonist and as an inverse agonist at MC4R and MC3-R to modulate cAMP levels (1720,37). Furthermore, in a recent study, AgRP was shown to exhibit agonistic properties on both MC3-R and MC4-R expressed in HEK293 cells by inducing arrestin-mediated endocytosis (38), supporting the inverse agonist hypothesis. However, these alternative signaling properties of AgRP have never been directly demonstrated in vivo. Here, we show that in addition to its role as a MC3/4-R competitive antagonist, AgRP is able to modulate energy homeostasis independently of the presence of the endogenous agonist αMSH.

The mechanism of AgRP-induced weight gain in Pomc+/+Tg/+ mice involves a long-lasting increase in food consumption and RER and a decrease in energy expenditure. In Pomc−/−Tg/+ mice with ad libitum access to food over the 24-h period, we were not able to measure any difference in food consumption after AgRP treatment. The observation that exogenous AgRP can induce weight gain without affecting food consumption in certain physiological conditions (when animals were fed ad libitum in our study) is surprising considering the powerful orexigenic properties of the peptide. Nevertheless, one should consider that feeding responses clearly depend on the endogenous neuropeptide tone involved in the regulation of energy balance. Because Pomc−/−Tg/+ mice are constitutively hyperphagic and have increased mRNA levels for the orexigenic peptides AgRP and NPY compared with Pomc−/− mice, increased daily food consumption over the heightened baseline may be difficult to detect or to induce. Although Pomc−/−Tg/+ mice were still able to respond to the short-term orexigenic effects of ghrelin, which are partly mediated via increased NPY and AgRP tone (27), the percentage of increase was much lower than in Pomc+/+Tg/+ mice, suggesting that they are less sensitive to stimulation by orexigenic signals. Either pharmacological or physiological manipulations to reduce the basal hyperphagia might be useful to reveal a stronger orexigenic response to exogenous AgRP in the mutant mice.

The reduced food consumption of Pomc−/−Tg/+ and particularly Pomc−/− mice after saline injections indicates a contribution of stress-related feeding responses after intracerebroventricular injections. Our previous data demonstrated a central dysregulation of the HPA axis in Pomc−/−Tg/+ mice, characterized by inappropriately high hypothalamic corticotropin-releasing hormone (CRH) levels (39). Here, we show that CART, another factor involved in the control of the HPA axis and possessing anorectic effects (40), is also upregulated in POMC-deficient mice. Therefore, elevated CRH and CART tone may counterbalance the feeding effects of AgRP and explain its apparently subtle orexigenic action in Pomc−/−Tg/+ mice. Pomc−/− mice that did not receive glucocorticoid replacement exhibited the most profound catabolic responses to intracerebroventricular injections of saline but also to AgRP. The latter results support previous studies showing that the actions of AgRP on energy balance in adrenalectomized rats are glucocorticoid dependent (26). Severe dysregulation of their HPA axis due to adrenal insufficiency (39,41,42) and upregulated CRH expression in the paraventricular nucleus of the hypothalamus (PVH) likely explain the paradoxical anorectic response of Pomc−/− mice.

Independently of the modulation of food intake, AgRP also modulates energy expenditure (43,44). We therefore performed indirect calorimetry to determine whether the weight gain in Pomc−/−Tg/+ mice could be partly due to decreased Vo2 and/or modification in energy substrate use. Under specific experimental conditions in which access to food was limited to 16 h each day, a significant but discrete increase in food consumption together with decreased basal metabolic rate and increased average RER were measured in the mice. Compared with Pomc+/+Tg/+ mice, the AgRP responses observed in Pomc−/−Tg/+ mice were of smaller amplitude but longer duration with delayed onset, supporting a distinct mechanism of action of AgRP based on the genotype, which may reflect the lack of short-term antagonist action at MC3/4-R.

Specific brain targets of these metabolic actions of AgRP in Pomc−/−Tg/+ mice remain to be determined. The central melanocortin system regulates the activities of both the sympathetic nervous system and the hypothalamic-pituitary-thyroid axis, which act in synergy to control thermogenesis (45). The presence of AgRP but not αMSH terminals on some TRH neurons expressing MC4-R suggests that these neurons are good candidates (16). Using MC4-R–deficient mice, several laboratories have independently demonstrated a critical role for MC4-R in maintaining basal metabolic activity (46,47). However, short- and long-term hyperphagic actions of AgRP were still observed in MC4-R knockout mice, suggesting that MC4-R is not the only receptor to mediate effects of the orexigenic peptide. Interestingly, MC3-R regulates partitioning of fuel stores into fat rather than directly affecting food consumption (6,7). In our study, the increased RER after AgRP treatment suggests a switch from carbohydrate to fat stores as a source of energy, and this may be mediated through a MC3-R mechanism of action. Because the hypothalamic expression of MC4-R and MC3-R was unchanged in the mutant mice, we cannot distinguish between the possible involvement of either receptor to induce the effects of AgRP.

In conclusion, neuronal-specific POMC-deficient mice that lack αMSH signaling in the CNS have increased sensitivity to melanocortin agonists and respond with altered kinetics to the feeding and metabolic actions of AgRP. These data demonstrate that exogenous AgRP can modulate energy balance in the CNS independently of MC3/4-R competitive antagonism and strongly support an inverse agonist mode of action for AgRP in vivo. However, it is a possibility that long-lasting AgRP actions in Pomc−/−Tg/+ mice may be relayed by a mechanism involving receptors distinct from MC3 and MC4-R. To further test the contribution of each proposed AgRP mode of action, use of additional pharmacological tools is necessary. If endogenous AgRP actually functions as an inverse agonist at either MC-R, a pure selective competitive MC-R antagonist should block endogenous AgRP action and decrease food intake and/or body weight in POMC-deficient mice while having the opposite effect in wild-type mice. Current obstacles to perform or interpret the results of such experiments depend on the availability of selective compounds and the possibility that synthetic antagonists may also behave as inverse agonists. Alternatively, use of small interfering RNA technology to block endogenous AgRP in POMC-deficient mice or breeding of AgRP knockout to neural-specific POMC-deficient mice to create double AgRP/nPOMC knockout mice would be useful approaches to better understand the mechanism of action of the endogenous orexigenic peptide.

The physiological significance of the putative inverse agonist action in humans still remains to be determined. A recent study in obese patients that identified mutations in the extracellular NH2-terminal domain of MC4-R associated with loss of constitutive activity suggests that modulation of constitutive activity in wild-type receptors by inverse agonists could be important to maintain long-term energy homeostasis in humans (21). Supporting this possibility are recent reports that the human MC4-R undergoes ligand-independent cycles of endo- and exocytosis in transfected neuroblastoma cells and immortalized hypothalamic neurons (48) and that antibodies directed against an epitope in the NH2-terminal domain of the MC4-R act as inverse agonists in cell lines and intact animal models (49). Assessment of the effects of AgRP in knock-in mouse models containing humanized MC4-R with the inactivating NH2-terminal mutations or other characterized, constitutively active receptors that respond solely to a synthetic ligand (50) would be useful to confirm that the mechanism of action of AgRP is partly mediated through modulation of MC4-R constitutive activity.

FIG. 1.

Effects of intracerebroventricular injection of MTII on food intake and body weight. Two-hour food intake (A), 24-h food intake (B), and 24-h body weight change (C) after injection of 0.5 nmol MTII in Pomc+/+Tg/+ control, Pomc−/−Tg/+, and Pomc−/− mice at the onset of the dark cycle. Repeated-measures ANOVAs showed a significant effect of 0.5 nmol–MTII treatment on 2-h food intake in all genotypes (F2,21 = 46.6, P < 0.0001) and on 24-h food intake (F2,21 = 38.3, P < 0.0001) and nearly a significant effect on body weight change (F2,21 = 3.4, P = 0.08). The effects of 0.5 nmol MTII were prolonged over 24 h in Pomc−/−Tg/+ and Pomc−/− mice but not in Pomc+/+Tg/+ controls. *P < 0.05, **P < 0.01 vs. saline, paired t test analyses. Data are means ± SE, n = 7–10.

FIG. 2.

Feeding effects of intracerebroventricular injection of AgRP on the anorectic response to MTII. Two-hour food intake (A) and 24-h food intake (B) after administration of saline, 0.5 nmol AgRP alone, 0.1 nmol MTII alone, or co-administration of 0.1 nmol MTII and 0.5 nmol AgRP at the onset of the dark cycle in Pomc+/+Tg/+ control, Pomc−/−Tg/+, Pomc−/−, and Pomc+/− mice. Repeated-measures ANOVAs showed a significant effect of the treatments on 2-h food intake in all genotypes (F3,29 = 18.5, P < 0.0001) that was not observed after 24 h (F3,29 = 1.7, P = 0.17). Post hoc analyses showed that MTII treatment decreased 2-h food intake significantly in all genotypes (P < 0.0001 vs. saline) and that AgRP antagonized the anorectic action of MTII (P < 0.0001 MTII plus AgRP vs. MTII). Data are means ± SE; n = 7–14, except for Pomc−/−Tg/+ mice (n = 3).

FIG. 3.

Short-term feeding effects of intracerebroventricular injection of AgRP and ghrelin. A: 2-h food intake after injection of AgRP 0.5 nmol during the light cycle in Pomc+/+Tg/+ and Pomc−/−Tg/+ mice. B: 2-h food intake after injection of 1 nmol ghrelin during the light cycle in Pomc+/+Tg/+, Pomc−/−Tg/+, and Pomc−/− mice. Repeated-measures ANOVAs showed a significant effect of 0.5 nmol AgRP (F1,13 = 7.7, P = 0.0016) and of 1 nmol ghrelin injection (F2,33 = 12.3, P = 0.0013). Pomc+/+Tg/+ but not Pomc−/−Tg/+ mice responded significantly to AgRP treatment. Pomc+/+Tg/+ and Pomc−/−Tg/+ but not Pomc−/− mice responded significantly to ghrelin treatment. *P < 0.05, ***P < 0.0001 vs. saline, paired t test analyses. Data are means ± SE, n = 6–9 for AgRP treatment, n = 6–13 for ghrelin treatment.

FIG. 4.

Short- and long-term effects of AgRP on food intake and body weight. Two-hour food intake (A, D, G, and J), daily 24-h food intake (B, E, H, and K), and body weight change (C, F, I, and L) over a period of 96 h after a single injection of 0.5 nmol AgRP at the onset of the dark cycle in Pomc+/+Tg/+ control (AC), Pomc−/−Tg/+ (DF), Pomc−/− (GI), and Pomc+/− (JL) mice. Baseline corresponds to 24-h food intake measured in noninjected animals. Paired t test (saline vs. AgRP) applied on individual genotypes showed a significant effect of 0.5 nmol AgRP on 24-h food intake in Pomc+/+Tg/+ control and Pomc+/− mice but not in Pomc−/−Tg/+ and Pomc−/− mice and on body weight gain in Pomc+/+Tg/+ controls, Pomc−/−Tg/+, and Pomc+/− but not Pomc−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline. All data are means ± SE, n = 5–8.

FIG. 5.

Long-term effects of AgRP on body weight, food intake, Vo2, and RER in mice with restricted access to food. Representative body weights (A and B), average 24-h food intake (C and D), average Vo2 (E and F), and average RER (G and H) in Pomc+/+Tg/+ control and Pomc−/−Tg/+ mice housed for 6–8 h during the daytime in oxymax chambers without access to food and water. Each animal received saline and 0.5 nmol AgRP in a randomized manner at the onset of the dark cycle (indicated by plain arrows), and parameters were recorded up to 168 h after the injection. In addition, all animals received an injection of saline 2 days before the injection of the drug (indicated by dotted arrows). Data marked as “basal saline” were the mean values of these 2 days. Body weight data are individual values of two representative animals per genotype (A and B). Gray shades highlight the timeframe of the effect of AgRP on body weight (A and B), which is different in Pomc+/+Tg/+ control (3 days) and Pomc−/−Tg/+ mice (6 days). All other data are means ± SE, n = 7–8 (CF). Paired t tests showed a significant effect of AgRP on food intake, Vo2, and RER. *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline.

FIG. 6.

Relative quantification (RQ) of MC4-R, MC3-R, AgRP, NPY, and CART gene expression in the hypothalamus. MC4-R mRNA (F2,21 = 0.040, P = 0.961) (A), MC3-R mRNA (F2,19 = 1.048, P = 0.3701) (B), AgRP mRNA (F2,19 = 13.759, P = 0.0002) (C), NPY mRNA (F2,19 = 4.605, P = 0.0234) (D), and CART mRNA (F2,19 = 4.516, P = 0.0249) (E) levels in Pomc−/−Tg/+ and Pomc−/− mice compared with Pomc+/+ Tg/+ control mice according to the formula RQ = 2−ΔΔCT. CT was defined as the threshold cycle of PCR at which amplified product was detected, and 2−ΔΔCT represents the fold change in gene expression normalized to 18S and relative to Pomc+/+Tg/+ control mice. ΔΔCT was calculated as follows: (CT, MC4-R − CT,18S)Pomc−/−Tg/+ − (CT, MC4-R − CT,18S)Pomc+/+Tg/+. Data are the means (horizontal bars) and scattergrams of all individual RQ (n = 6–10) normalized to 18S. *P < 0.05, **P < 0.01, and ***P < 0.0001 by Fisher's PLSD for the indicated pairwise comparisons.

Acknowledgments

V.T. has received an American Society for Pharmacology and Experimental Therapeutics/Merck postdoctoral fellowship in integrative pharmacology. M.J.L. has received National Institutes of Health Grant DK-066604.

We thank Dr. U. Hochgeschwender for supplying an original breeder pair of Pomc+/− mice and Dr. J. Smart for the construction of the pHalEx2* transgene and original characterization of the Pomc−/−Tg/+ mice. Microinjection of the pHalEx2* transgene was performed by the Oregon Health and Science University Transgenic Core Laboratory. We thank Renee Kruse Bend and Bryce Warren for assistance with breeding of the pHalEx2* colony. RT-PCR experiments were performed at the Oregon Child Health Research Center DNA Sequencing Core in the Center for the Study of Weight Regulation at Oregon Health and Science University.

Parts of this study were presented in abstract form at the International Congress of Neuroendocrinology, Pittsburgh, Pennsylvania, 19–22 June 2006, and at the 88th annual meeting of the Endocrine Society, Boston, Massachusetts, 24–27 June 2006.

Footnotes

  • Published ahead of print at http://diabetes.diabetesjournals.org on 1 October 2007. DOI: 10.2337/db07-0733.

    Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-0733.

    The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Accepted September 26, 2007.
    • Received May 29, 2007.

REFERENCES

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