In Vivo Evidence for Inverse Agonism of Agouti-Related Peptide in the Central Nervous System of Proopiomelanocortin-Deﬁcient Mice Virginie Tolle1,2 and Malcolm J

Home , Melanotan II, Peptide

ORIGINAL ARTICLE In Vivo Evidence for Inverse Agonism of Agouti-Related Peptide in the Central Nervous System of Proopiomelanocortin-Deﬁcient Mice Virginie Tolle1,2 and Malcolm J. Low1,2,3

OBJECTIVE—Melanocyte-stimulating hormone (MSH) peptides processed from proopiomelanocortin (POMC) regulate energy homeostasis by activating neuronal melanocortin recep- enetic disruption of either mouse or human tor (MC-R) signaling. Agouti-related peptide (AgRP) is a naturally proopiomelanocortin (POMC) causes early-on- occurring MC-R antagonist but also displays inverse agonism at set obesity (1–3), highlighting a major role of constitutively active melanocortin-4 receptor (MC4-R) expressed POMC in the regulation of energy homeostasis. on transfected cells. We investigated whether AgRP functions G POMC is processed posttranslationally into multiple pep- similarly in vivo using mouse models that lack all neuronal MSH, ␤ thereby precluding competitive antagonism of MC-R by AgRP. tides, including the opioid -endorphin and the melanocortins ACTH, ␣-melanocyte-stimulating hormone (␣MSH), RESEARCH DESIGN AND METHODS—Feeding and meta- ␤ ␥ bolic effects of the MC-R agonist melanotan II (MTII), AgRP, and MSH, and MSH. POMC peptides in the central nervous ghrelin were investigated after intracerebroventricular injection system (CNS) are essential in the regulation of energy Ϫ/Ϫ ϩ intake and expenditure as demonstrated in studies using in neural-specific POMC-deficient (Pomc Tg/ ) and global Ϫ Ϫ POMC-deficient (PomcϪ/Ϫ) mice. Gene expression was quanti- compound mutant mice (Pomc / Tg/ϩ) expressing a fied by RT-PCR. Pomc transgene that selectively restored pituitary POMC Ϫ/Ϫ RESULTS—Hyperphagic POMC-deficient mice were more sen- in Pomc mice to produce a neural-selective POMC sitive than wild-type mice to the anorectic effects of MTII. deficiency (4). Lack of ␣MSH is likely the principal cause Hypothalamic melanocortin-3 (MC3)/4-R mRNAs in POMC-defi- of obesity (3,5) due to the loss of agonist signaling at cient mice were unchanged, suggesting increased receptor sen- central melanocortin receptors (MC-R), melanocortin-3 sitivity as a possible mechanism for the heightened anorexia. receptor (MC3-R), and melanocortin-4 receptor (MC4-R), AgRP reversed MTII-induced anorexia in both mutant strains, each of which plays a distinct role in the regulation of demonstrating its ability to antagonize MSH agonists at central energy homeostasis (6–8). MC3/4-R, but did not produce an acute orexigenic response by ␣ itself. The action of ghrelin was attenuated in PomcϪ/ϪTg/ϩ mice, The anorectic actions of centrally administered MSH suggesting decreased sensitivity to additional orexigenic signals. or the synthetic MC3/4-R agonist melanotan II (MTII) However, AgRP induced delayed and long-lasting modifications of (9–11) are blocked by Agouti-related peptide (AgRP), an energy balance in PomcϪ/ϪTg/ϩ, but not glucocorticoid-deficient endogenous MC3/4-R antagonist (12,13), released from PomcϪ/Ϫ mice, by decreasing oxygen consumption, increasing the terminals of neuropeptide Y (NPY)/AgRP arcuate neurons. respiratory exchange ratio, and increasing food intake. In addition to their localization to the same brain regions CONCLUSIONS—These data demonstrate that AgRP can mod- as POMC fibers (14), AgRP nerve terminals send projec- ulate energy balance via a mechanism independent of MSH and tions to neurons that possess MC4-R (15) but are not MC3/4-R competitive antagonism, consistent with either inverse innervated by ␣MSH terminals (14,16). These neuroana- agonist activity at MC-R or interaction with a distinct receptor. tomic findings indicate that AgRP may modulate MC4-R Diabetes 57:86–94, 2008 activity in the absence of endogenous ␣MSH. In vitro data strongly support the ability of AgRP to modulate MC4-R by an inverse agonist mode of action From the 1Center for the Study of Weight Regulation and Associated Disor- (17–20); however, the physiological significance is unre- ders, Oregon Health and Science University, Portland, Oregon; the 2Vollum solved. Modulation of MC4-R constitutive activity may be Institute, Oregon Health and Science University, Portland, Oregon; and the 3Department of Behavioral Neuroscience, Oregon Health and Science Univer- important to maintain long-term energy homeostasis in sity, Portland, Oregon. humans (21). In rodents, the concept of inverse agonism Address correspondence and reprint requests to Virginie Tolle, PhD, has been buttressed by demonstrations that a single injec- UMR549 Inserm, IFR Broca-Ste Anne, 2 ter rue d’Alesia, 75014 Paris, France. E-mail: [email protected]. tion of AgRP induces hyperphagia over several days Received for publication 29 May 2007 and accepted in revised form 26 (22–24), whereas this long-lasting effect cannot be repro- September 2007. duced by synthetic MC4-R antagonists like HS014 or Published ahead of print at http://diabetes.diabetesjournals.org on 1 Octo- ber 2007. DOI: 10.2337/db07-0733. JKC-363 (23,25). Additional information for this article can be found in an online appendix at In the present study, we analyzed the feeding and meta- http://dx.doi.org/10.2337/db07-0733. bolic responses to intracerebroventricular injections of MTII AgRP, Agouti-related peptide; CART, cocaine and amphetamine–related transcript; CNS, central nervous system; CRH, corticotropin-releasing hor- and AgRP in mice deficient in all central MSH peptides. mone; DIO, diet-induced obesity; MC-R, melanocortin receptor; MC3-R, mela- Because responses to melanocortin antagonists appar- nocortin-3 receptor; MC4-R, melanocortin-4 receptor; MSH, melanocyte- ently require the presence of circulating glucocorticoids stimulating hormone; MTII, melanotan II; NPY, neuropeptide Y; PLSD, (26), we compared PomcϪ/Ϫ mice with a global deficiency protected least squares difference; POMC, proopiomelanocortin; RER, respi- Ϫ/Ϫ ratory exchange ratio. of POMC and adrenal insufficiency to Pomc Tg/ϩ mice © 2008 by the American Diabetes Association. with a neural-specific deficiency of POMC but restored 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 glucocorticoids (4). Feeding effects of the orexigenic gut with 18 U.S.C. Section 1734 solely to indicate this fact. peptide ghrelin (27) were also tested.

86 DIABETES, VOL. 57, JANUARY 2008 V. TOLLE AND M.J. LOW

RESEARCH DESIGN AND METHODS A colony of Pomc mutant mice on a hybrid B6;D2;129X1;129S6 genetic background with independently segregating Pomcϩ/Ϫ 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 pur- chased 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 Ϫ0.4 mm, lateral Ϫ1.0 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 PomcϪ/Ϫ mice that required injection with dexamethasone (0.15 ␮g i.p. in 1 ml saline) for 3 days. Feeding and basal metabolic rate. Peptides were injected intracerebrov- entricularly in a volume of 1 ␮l over 1 min using a 33-gauge stainless steel injection cannula extending 0.5 mm below the guide cannula and connected to a1-␮l 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 manufactur- er’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 ␮g RNA were run in duplicate in total reaction volumes of 20 ␮l containing 5 ␮l cDNA, 1ϫ TaqMan Gene Expression Assay, and 1ϫ 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 2Ϫ⌬⌬CT method as previously described (28). Statistical analyses. All data presented are means Ϯ SE. Data were analyzed FIG. 1. Effects of intracerebroventricular injection of MTII on food by repeated-measures ANOVAs or multifactor ANOVAs appropriate for the intake and body weight. Two-hour food intake (A), 24-h food intake design of each experiment with genotype and/or drug as independent vari- (B), and 24-h body weight change (C) after injection of 0.5 nmol MTII ϩ/ϩ ؉ Ϫ/Ϫ ؉ Ϫ/Ϫ ables using Stat View Power PC for Macintosh version 5.0.1 (SAS Institute). in Pomc Tg/ control, Pomc Tg/ , and Pomc mice at the onset of the dark cycle. Repeated-measures ANOVAs showed a significant One-factor ANOVAs were used to follow up significant main effects; post hoc effect of 0.5 nmol–MTII treatment on 2-h food intake in all genotypes ؍ ؍ pairwise comparisons between groups were performed by Fisher’s protected (F2,21 46.6, P < 0.0001) and on 24-h food intake (F2,21 38.3, P < ؍ least squares difference (PLSD) or paired two-tail t tests. P values Ͻ0.05 were 0.0001) and nearly a significant effect on body weight change (F2,21 The effects of 0.5 nmol MTII were prolonged over 24 h .(0.08 ؍ considered significant. 3.4, P .in PomcϪ/ϪTg/؉ and PomcϪ/Ϫ mice but not in Pomcϩ/ϩTg/؉ controls *P < 0.05, **P < 0.01 vs. saline, paired t test analyses. Data are ؍ ؎ RESULTS means SE, n 7–10. Feeding effects of MTII injected at the onset of the dark cycle. A single intracerebroventricular injection In both PomcϪ/ϪTg/ϩ and PomcϪ/Ϫ mice, MTII acutely of 0.5 nmol MTII at the onset of the dark cycle de- decreased food intake by 95%, and in contrast to control creased food intake in control Pomcϩ/ϩTg/ϩ and mutant mice, this effect was sustained with 60 and 54% cumulative PomcϪ/ϪTg/ϩ and PomcϪ/Ϫ mice compared with vehicle- reductions at 24 h (P Ͻ 0.01 vs. saline). Long-term an- treated animals 2 h after the injection (Fig. 1A). In orexia induced by 0.5 nmol MTII was also associated with Pomcϩ/ϩTg/ϩ mice, food intake was inhibited by 67%, and significant weight loss in PomcϪ/ϪTg/ϩ and PomcϪ/Ϫ mice the effect was completely reversed by 24 h (Fig. 1A and B). (P Ͻ 0.01 and 0.05 vs. saline, respectively) (Fig. 1C).

DIABETES, VOL. 57, JANUARY 2008 87 AgRP ACTIONS IN POMC-DEFICIENT MICE

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 Pomcϩ/ϩTg/؉ control, PomcϪ/ϪTg/؉, PomcϪ/Ϫ, and Pomcϩ/Ϫ 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, Post hoc analyses showed that MTII treatment decreased 2-h food intake signiﬁcantly in all genotypes (P < 0.0001 vs. saline) and that .(0.17 ؍ P except for PomcϪ/ϪTg/؉ ,14–7 ؍ AgRP antagonized the anorectic action of MTII (P < 0.0001 MTII plus AgRP vs. MTII). Data are means ؎ SE; n .(3 ؍ mice (n

Feeding effects of co-administered MTII and AgRP. AgRP had short-term orexigenic effects (Fig. 3A; data with To test the ability of AgRP to antagonize MSH agonists at 2 nmol not shown). To test the ability of PomcϪ/Ϫ and MC3/4-R, 0.5 nmol AgRP was injected alone or in combi- PomcϪ/ϪTg/ϩ mice to respond to other orexigenic signals, nation with 0.1 nmol MTII at the onset of the dark cycle. 1 nmol ghrelin was injected during the daytime (Fig. 3B). Although this lower dose of MTII was slightly less potent Ghrelin stimulated 2-h food intake in Pomcϩ/ϩTg/ϩ and, to than 0.5 nmol to reduce feeding 2 h after injection, we a much lesser extent, in PomcϪ/ϪTg/ϩ mice (P Ͻ 0.0001 chose it in combination with AgRP because its anorectic and 0.05 vs. saline, respectively) but had no effect in effects had dissipated in all genotypes at 24 h. There was a PomcϪ/Ϫ mice. significant main effect of the peptide treatments on 2-h Short- and long-term effects of AgRP injected at the food intake but no significant interaction of treatment ϫ onset of the dark cycle on food intake and body genotype (Fig. 2A). Post hoc analyses collapsed across weight. Nighttime injection of 0.5 nmol AgRP, when genotype confirmed that 0.1 nmol MTII alone significantly maximum feeding activity occurs naturally, did not further decreased acute food intake (P Ͻ 0.0001 vs. saline). In increase 2-h food intake in any genotype (Fig. 4A, D, G, contrast, there was no significant main effect of treatment and J). However, hyperphagia was observed 24 h after the or treatment ϫ genotype interaction on 24-h food intake injection of AgRP, was sustained up to 72 h, and was (Fig. 2B). After nighttime injection, when maximum feed- accompanied by body weight gain in Pomcϩ/ϩTg/ϩ mice ing activity is already observed, there was no additive, (Fig. 4B and C). Pomcϩ/Ϫ mice were more sensitive to the short-term orexigenic effect of 0.5 nmol AgRP alone on any long-term effects of AgRP with sustained hyperphagia up genotype. However, AgRP significantly blocked the ano- to 96 h after injection and more pronounced body weight rectic effect of 0.1 nmol MTII in all genotypes 2 h after gain than Pomcϩ/ϩTg/ϩ mice (Fig. 4K and L). Neither co-injection of both peptides (P Ͻ 0.0001, AgRP plus MTII PomcϪ/ϪTg/ϩ nor PomcϪ/Ϫ mice increased their food vs. MTII alone, Fisher PLSD) (Fig. 2A), consistent with a consumption after nighttime injection of 0.5 nmol AgRP competitive antagonist action at the MC3/4-R. (Fig. 4E and H). Furthermore, injection of 0.5 or 2 nmol Short-term–feeding effects of AgRP and ghrelin in- AgRP during the light cycle also did not produce any jected during the light cycle. Administration of 0.5 or 2 long-lasting orexigenic effect in the mutant PomcϪ/ϪTg/ϩ nmol AgRP during the daytime increased 2-h food intake mice, whereas it was orexigenic in Pomcϩ/ϩTg/ϩ control by 160 and 250%, respectively, in Pomcϩ/ϩTg/ϩ mice (P Ͻ mice (data not shown). Similar to Pomcϩ/ϩTg/ϩ controls, 0.05 vs. saline). In PomcϪ/ϪTg/ϩ mice, neither dose of the rate of body weight gain was greater in PomcϪ/ϪTg/ϩ

88 DIABETES, VOL. 57, JANUARY 2008 V. TOLLE AND M.J. LOW

in PomcϪ/ϪTg/ϩ mice (Fig. 5B). Consequently, changes were subtler but more prolonged in PomcϪ/ϪTg/ϩ compared with Pomcϩ/ϩTg/ϩ mice. Analyses performed for the relevant, genotype-specific timeframes showed an increased body weight of 12% over 3 days in Pomcϩ/ϩTg/ϩ (P Ͻ 0.01) and 6% over 6 days in PomcϪ/ϪTg/ϩ (P Ͻ 0.01) mice after AgRP treatment, compared with their initial body weights. In Pomcϩ/ϩTg/ϩ 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 PomcϪ/ϪTg/ϩ 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 PomcϪ/ϪTg/ϩ and PomcϪ/Ϫ mice compared with control Pomcϩ/ϩTg/ϩ mice. Unlike MC3/4-R, levels of AgRP (Fig. 6C), NPY (Fig. 6D), and CART (Fig. 6E) mRNA all differed by genotype. In PomcϪ/Ϫ mice, AgRP expression was significantly decreased by 64%, CART expression was increased by 47%, and NPY expression was unchanged compared with Pomcϩ/ϩTg/ϩ mice. In PomcϪ/ϪTg/ϩ mice, CART expression was increased by 67%, but AgRP and NPY were unchanged compared with Pomcϩ/ϩTg/ϩ. Notably, however, in PomcϪ/ϪTg/ϩ mice, expression of both AgRP and NPY was significantly increased compared with that in the glucocorticoid-deficient PomcϪ/Ϫ mice.

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 DISCUSSION during the light cycle in Pomcϩ/ϩTg/؉ and PomcϪ/ϪTg/؉ mice. B: 2-h food intake after injection of 1 nmol ghrelin during the light cycle in Catabolic effects of MTII were accentuated in obese Pomcϩ/ϩTg/؉, PomcϪ/ϪTg/؉, and PomcϪ/Ϫ mice. Repeated-measures POMC-deficient mice. Both strains of POMC-deficient ؍ ؍ ANOVAs showed a significant effect of 0.5 nmol AgRP (F1,13 7.7, P mice were more sensitive to the short-term anorectic ؍ ؍ 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 action of MT-II than their control siblings. The mechanism AgRP treatment. Pomc؉/؉Tg/؉ and Pomc؊/؊Tg/؉ but not Pomc؊/؊ mice of increased sensitivity to MTII in POMC-deficient mice or responded significantly to ghrelin treatment. *P < 0.05, ***P < 0.0001 other animal models of obesity, such as Zucker rats (fa/fa) for (29), diet-induced obesity (DIO) rats (30), and DIO and 9–6 ؍ vs. saline, paired t test analyses. Data are means ؎ SE, n .for ghrelin treatment 13–6 ؍ AgRP treatment, n ob/ob mice (31) with decreased POMC expression or decreased melanocortin tone (32–34), may involve in- mice injected with 0.5 nmol AgRP than with saline, and creased expression, density, or functional coupling of this difference was significant at 72 h (P Ͻ 0.001) (Fig. 4F). MC-R in response to the chronic absence or decrease of Although a short-lived reduction in food intake and body endogenous melanocortin ligands. There were no signifi- weight was sometimes observed after intracerebroventric- cant differences in the expression of MC4-R or MC3-R ular injections of saline in Pomcϩ/ϩTg/ϩ, Pomcϩ/Ϫ, and to in PomcϪ/ϪTg/ϩ and PomcϪ/Ϫ mice compared with a greater degree in PomcϪ/ϪTg/ϩ mice, food consumption Pomcϩ/ϩTg/ϩ mice, suggesting that either increased MC-R usually rebounded to baseline values within 48 h. In density or signaling is responsible for the heightened contrast, PomcϪ/Ϫ mice had prolonged anorexia and anorectic effect of MTII. However, our quantification did weight loss in response to a saline injection (Fig. 4H and not take into account differential regional expression of I). the MC4-R in various hypothalamic nuclei or extra- Effect of AgRP injected at the onset of the dark cycle hypothalamic expression of MC4-R (15), which are impor- on Vo2 and RER in mice with restricted food access. tant in the regulation of energy homeostasis and have been To test the hypothesis that AgRP may be a long-term shown to mediate the effects of melanocortin agonists and modulator of energy expenditure in PomcϪ/ϪTg/ϩ mice, antagonists and long-term orexigenic actions of AgRP (35). we measured Vo2 and RER during 7 consecutive days Increased sensitivity to MTII could also be due to after the injection of saline or AgRP at the onset of the secondary alterations in the expression of other hypotha- dark cycle (Fig. 5), conditions that induced weight gain lamic orexigenic/anorectic signals. AgRP mRNA was re- in both Pomcϩ/ϩTg/ϩ and PomcϪ/ϪTg/ϩ mice. In most duced in PomcϪ/Ϫ mice, consistent with data reported by Pomcϩ/ϩTg/ϩ mice, as depicted in two representative Coll et al. (36) and supporting the hypothesis that reduced individuals (Fig. 5A), the effects of AgRP were observed up levels of AgRP contributed to the increased response to to 72 h after the injection. In contrast, the onset of AgRP MTII. However, this cannot be the only explanation be- effects was delayed by 24 h but subsequently lasted longer cause AgRP expression was almost normalized in glu-

DIABETES, VOL. 57, JANUARY 2008 89 AgRP ACTIONS IN POMC-DEFICIENT MICE

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 Pomcϩ/ϩTg/؉ control (A–C), PomcϪ/ϪTg/؉ (D–F), PomcϪ/Ϫ (G–I), and Pomcϩ/Ϫ (J–L) 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 Pomcϩ/ϩTg/؉ control and Pomcϩ/Ϫ mice but not in PomcϪ/ϪTg/؉ and PomcϪ/Ϫ mice and on body weight gain in Pomcϩ/ϩTg/؉ controls .8–5 ؍ PomcϪ/ϪTg/؉, and Pomcϩ/Ϫ but not PomcϪ/Ϫ mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline. All data are means ؎ SE, n cocorticoid-replete PomcϪ/ϪTg/ϩ mice, which still displayed anorectic effect of MTII in PomcϪ/ϪTg/ϩ mice, indicating an exaggerated anorectic response to MTII. that the short-term orexigenic effect of AgRP requires the Differential short- and long-term effects of AgRP in presence of ␣MSH and therefore is due to a competitive neural selective POMC-deﬁcient mice. AgRP did not antagonist action at MC3/4-R. Despite reexpression of alter short-term food intake but was able to antagonize the POMC in the pituitary gland, PomcϪ/ϪTg/ϩ mice had

90 DIABETES, VOL. 57, JANUARY 2008 V. TOLLE AND M.J. LOW

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 ϩ/ϩ ؉ Ϫ/Ϫ ؉ (A and B), average 24-h food intake (C and D), average Vo2 (E and F), and average RER (G and H)inPomc 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 (C–F). 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. undetectable levels of ␣MSH in the hypothalamus (4), Furthermore, in a recent study, AgRP was shown to excluding the possibility that ␣MSH from a peripheral exhibit agonistic properties on both MC3-R and MC4-R source leaked into the CNS. Interestingly, Pomcϩ/Ϫ mice expressed in HEK293 cells by inducing arrestin-mediated had stronger feeding responses to both AgRP and MTII endocytosis (38), supporting the inverse agonist hypothe- than control Pomcϩ{/ϩTg/ϩ mice, probably reflecting a sis. However, these alternative signaling properties of gene-dosage effect in the response to melanocortin ago- AgRP have never been directly demonstrated in vivo. Here, nists and antagonists. We previously demonstrated that we show that in addition to its role as a MC3/4-R compet- the hypothalamic content of ␣MSH in Pomcϩ/Ϫ mice is itive antagonist, AgRP is able to modulate energy ho- one-half that of Pomcϩ/ϩTg/ϩ mice (4), indicating that meostasis independently of the presence of the there is less endogenous ␣MSH to antagonize in those endogenous agonist ␣MSH. animals. The mechanism of AgRP-induced weight gain in In vitro studies have clearly demonstrated that AgRP Pomcϩ/ϩTg/ϩ mice involves a long-lasting increase in food acts as a competitive antagonist and as an inverse agonist consumption and RER and a decrease in energy expendi- at MC4R and MC3-R to modulate cAMP levels (17–20,37). ture. In PomcϪ/ϪTg/ϩ mice with ad libitum access to food

DIABETES, VOL. 57, JANUARY 2008 91 AgRP ACTIONS IN POMC-DEFICIENT MICE

؍ 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 ؊⌬⌬CT ؍ mice according to the formula RQ 2 .CT was defined as the threshold cycle of PCR at which amplified product was detected, and 2؊⌬⌬CT represents the fold change in gene expression normalized to 18S and relative to Pomc؉/؉Tg/؉ control mice. ⌬⌬CT 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 .normalized to 18S. *P < 0.05, **P < 0.01, and ***P < 0.0001 by Fisher’s PLSD for the indicated pairwise comparisons (10–6 ؍ RQ (n over the 24-h period, we were not able to measure any intracerebroventricular injections. Our previous data dem- difference in food consumption after AgRP treatment. The onstrated a central dysregulation of the HPA axis in observation that exogenous AgRP can induce weight gain PomcϪ/ϪTg/ϩ mice, characterized by inappropriately high without affecting food consumption in certain physiologi- hypothalamic corticotropin-releasing hormone (CRH) lev- cal conditions (when animals were fed ad libitum in our els (39). Here, we show that CART, another factor in- study) is surprising considering the powerful orexigenic volved in the control of the HPA axis and possessing properties of the peptide. Nevertheless, one should con- anorectic effects (40), is also upregulated in POMC-defi- sider that feeding responses clearly depend on the endog- cient mice. Therefore, elevated CRH and CART tone may enous neuropeptide tone involved in the regulation of counterbalance the feeding effects of AgRP and explain its energy balance. Because PomcϪ/ϪTg/ϩ mice are constitu- apparently subtle orexigenic action in PomcϪ/ϪTg/ϩ mice. tively hyperphagic and have increased mRNA levels for the PomcϪ/Ϫ mice that did not receive glucocorticoid replace- orexigenic peptides AgRP and NPY compared with ment exhibited the most profound catabolic responses to PomcϪ/Ϫ mice, increased daily food consumption over the intracerebroventricular injections of saline but also to heightened baseline may be difficult to detect or to induce. AgRP. The latter results support previous studies showing Although PomcϪ/ϪTg/ϩ mice were still able to respond to that the actions of AgRP on energy balance in adrenalec- the short-term orexigenic effects of ghrelin, which are tomized rats are glucocorticoid dependent (26). Severe partly mediated via increased NPY and AgRP tone dysregulation of their HPA axis due to adrenal insuffi- (27), the percentage of increase was much lower than in ciency (39,41,42) and upregulated CRH expression in the Pomcϩ/ϩTg/ϩ mice, suggesting that they are less sensitive paraventricular nucleus of the hypothalamus (PVH) likely to stimulation by orexigenic signals. Either pharmacolog- explain the paradoxical anorectic response of PomcϪ/Ϫ ical or physiological manipulations to reduce the basal mice. hyperphagia might be useful to reveal a stronger orexi- Independently of the modulation of food intake, AgRP genic response to exogenous AgRP in the mutant mice. also modulates energy expenditure (43,44). We therefore The reduced food consumption of PomcϪ/ϪTg/ϩ and performed indirect calorimetry to determine whether the particularly PomcϪ/Ϫ mice after saline injections indicates weight gain in PomcϪ/ϪTg/ϩ mice could be partly due to a contribution of stress-related feeding responses after decreased Vo2 and/or modification in energy substrate use.

92 DIABETES, VOL. 57, JANUARY 2008 V. TOLLE AND M.J. LOW

Under specific experimental conditions in which access to ated with loss of constitutive activity suggests that modu- food was limited to 16 h each day, a significant but discrete lation of constitutive activity in wild-type receptors by increase in food consumption together with decreased inverse agonists could be important to maintain long-term basal metabolic rate and increased average RER were energy homeostasis in humans (21). Supporting this pos- measured in the mice. Compared with Pomcϩ/ϩTg/ϩ mice, sibility are recent reports that the human MC4-R under- the AgRP responses observed in PomcϪ/ϪTg/ϩ mice were goes ligand-independent cycles of endo- and exocytosis in of smaller amplitude but longer duration with delayed transfected neuroblastoma cells and immortalized hypo- onset, supporting a distinct mechanism of action of AgRP thalamic neurons (48) and that antibodies directed against based on the genotype, which may reflect the lack of an epitope in the NH2-terminal domain of the MC4-R act as short-term antagonist action at MC3/4-R. inverse agonists in cell lines and intact animal models (49). Specific brain targets of these metabolic actions of Assessment of the effects of AgRP in knock-in mouse AgRP in PomcϪ/ϪTg/ϩ mice remain to be determined. The models containing humanized MC4-R with the inactivating central melanocortin system regulates the activities of NH2-terminal mutations or other characterized, constitu- both the sympathetic nervous system and the hypothalam- tively active receptors that respond solely to a synthetic ic-pituitary-thyroid axis, which act in synergy to control ligand (50) would be useful to confirm that the mechanism thermogenesis (45). The presence of AgRP but not ␣MSH of action of AgRP is partly mediated through modulation terminals on some TRH neurons expressing MC4-R sug- of MC4-R constitutive activity. gests that these neurons are good candidates (16). Using MC4-R–deficient mice, several laboratories have indepen- ACKNOWLEDGMENTS dently demonstrated a critical role for MC4-R in maintain- V.T. has received an American Society for Pharmacology ing basal metabolic activity (46,47). However, short- and and Experimental Therapeutics/Merck postdoctoral fel- long-term hyperphagic actions of AgRP were still observed lowship in integrative pharmacology. M.J.L. has received in MC4-R knockout mice, suggesting that MC4-R is not the National Institutes of Health Grant DK-066604. only receptor to mediate effects of the orexigenic peptide. We thank Dr. U. Hochgeschwender for supplying an Interestingly, MC3-R regulates partitioning of fuel stores original breeder pair of Pomcϩ/Ϫ mice and Dr. J. Smart for into fat rather than directly affecting food consumption the construction of the pHalEx2* transgene and original (6,7). In our study, the increased RER after AgRP treat- characterization of the PomcϪ/ϪTg/ϩ mice. Microinjection ment suggests a switch from carbohydrate to fat stores as of the pHalEx2* transgene was performed by the Oregon a source of energy, and this may be mediated through a Health and Science University Transgenic Core Labora- MC3-R mechanism of action. Because the hypothalamic tory. We thank Renee Kruse Bend and Bryce Warren for expression of MC4-R and MC3-R was unchanged in the assistance with breeding of the pHalEx2* colony. RT-PCR mutant mice, we cannot distinguish between the possible experiments were performed at the Oregon Child Health involvement of either receptor to induce the effects of Research Center DNA Sequencing Core in the Center for AgRP. the Study of Weight Regulation at Oregon Health and In conclusion, neuronal-specific POMC-deficient mice ␣ Science University. that lack MSH signaling in the CNS have increased Parts of this study were presented in abstract form at sensitivity to melanocortin agonists and respond with the International Congress of Neuroendocrinology, Pitts- altered kinetics to the feeding and metabolic actions of burgh, Pennsylvania, 19–22 June 2006, and at the 88th AgRP. These data demonstrate that exogenous AgRP can annual meeting of the Endocrine Society, Boston, Massa- modulate energy balance in the CNS independently of chusetts, 24–27 June 2006. 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 REFERENCES PomcϪ/ϪTg/ϩ mice may be relayed by a mechanism 1. Challis BG, Coll AP, Yeo GS, Pinnock SB, Dickson SL, Thresher RR, Dixon involving receptors distinct from MC3 and MC4-R. To J, Zahn D, Rochford JJ, White A, Oliver RL, Millington G, Aparicio SA, Colledge WH, Russ AP, Carlton MB, O’Rahilly S: Mice lacking pro- further test the contribution of each proposed AgRP mode opiomelanocortin are sensitive to high-fat feeding but respond normally to of action, use of additional pharmacological tools is nec- the acute anorectic effects of peptide-YY(3–36). Proc Natl Acad SciUSA essary. If endogenous AgRP actually functions as an 101:4695–4700, 2004 inverse agonist at either MC-R, a pure selective competi- 2. Krude H, Biebermann H, Luck W, Horn R, Brabant G, Gruters A: Severe tive MC-R antagonist should block endogenous AgRP early-onset obesity, adrenal insufficiency and red hair pigmentation caused action and decrease food intake and/or body weight in by POMC mutations in humans. Nat Genet 19:155–157, 1998 3. Yaswen L, Diehl N, Brennan MB, Hochgeschwender U: Obesity in the POMC-deficient mice while having the opposite effect in mouse model of pro-opiomelanocortin deficiency responds to peripheral wild-type mice. Current obstacles to perform or interpret melanocortin. Nat Med 5:1066–1070, 1999 the results of such experiments depend on the availability 4. Smart JL, Tolle V, Low MJ: Glucocorticoids exacerbate obesity and insulin of selective compounds and the possibility that synthetic resistance in neuron-specific proopiomelanocortin-deficient mice. J Clin antagonists may also behave as inverse agonists. Alterna- Invest 116:495–505, 2006 tively, use of small interfering RNA technology to block 5. Tung YC, Piper SJ, Yeung D, O’Rahilly S, Coll AP: A comparative study of the central effects of specific proopiomelancortin (POMC)-derived mela- endogenous AgRP in POMC-deficient mice or breeding of nocortin peptides on food intake and body weight in pomc null mice. AgRP knockout to neural-specific POMC-deficient mice to Endocrinology 147:5940–5947, 2006 create double AgRP/nPOMC knockout mice would be 6. Butler AA, Kesterson RA, Khong K, Cullen MJ, Pelleymounter MA, Dekon- useful approaches to better understand the mechanism of ing J, Baetscher M, Cone RD: A unique metabolic syndrome causes obesity action of the endogenous orexigenic peptide. in the melanocortin-3 receptor-deficient mouse. Endocrinology 141:3518– The physiological significance of the putative inverse 3521, 2000 7. Chen AS, Marsh DJ, Trumbauer ME, Frazier EG, Guan XM, Yu H, agonist action in humans still remains to be determined. A Rosenblum CI, Vongs A, Feng Y, Cao L, Metzger JM, Strack AM, Camacho recent study in obese patients that identified mutations in RE, Mellin TN, Nunes CN, Min W, Fisher J, Gopal-Truter S, MacIntyre DE, the extracellular NH2-terminal domain of MC4-R associ- Chen HY, Van der Ploeg LH: Inactivation of the mouse melanocortin-3

DIABETES, VOL. 57, JANUARY 2008 93 AgRP ACTIONS IN POMC-DEFICIENT MICE

receptor results in increased fat mass and reduced lean body mass. Nat peripheral administration of a melanocortin agonist are more marked in Genet 26:97–102, 2000 genetically obese Zucker (fa/fa) than in lean rats. Endocrinology 143:2277– 8. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berke- 2283, 2002 meier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield 30. Hansen MJ, Ball MJ, Morris MJ: Enhanced inhibitory feeding response to LA, Burn P, Lee F: Targeted disruption of the melanocortin-4 receptor alpha-melanocyte stimulating hormone in the diet-induced obese rat. results in obesity in mice. Cell 88:131–141, 1997 Brain Res 892:130–137, 2001 9. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD: Role of melano- 31. Bluher S, Ziotopoulou M, Bullen JW Jr, Moschos SJ, Ungsunan L, Kokkotou cortinergic neurons in feeding and the agouti obesity syndrome. Nature E, Maratos-Flier E, Mantzoros CS: Responsiveness to peripherally admin- 385:165–168, 1997 istered melanocortins in lean and obese mice. Diabetes 53:82–90, 2004 10. McMinn JE, Wilkinson CW, Havel PJ, Woods SC, Schwartz MW: Effect of 32. Huang XF, Han M, South T, Storlien L: Altered levels of POMC, AgRP and intracerebroventricular alpha-MSH on food intake, adiposity, c-Fos induc- MC4-R mRNA expression in the hypothalamus and other parts of the tion, and neuropeptide expression. Am J Physiol Regul Integr Comp limbic system of mice prone or resistant to chronic high-energy diet- Physiol 279:R695–R703, 2000 induced obesity. Brain Res 992:9–19, 2003 11. Thiele TE, van Dijk G, Yagaloff KA, Fisher SL, Schwartz M, Burn P, Seeley 33. Kim EM, O’Hare E, Grace MK, Welch CC, Billington CJ, Levine AS: ARC RJ: Central infusion of melanocortin agonist MTII in rats: assessment of POMC mRNA and PVN alpha-MSH are lower in obese relative to lean c-Fos expression and taste aversion. Am J Physiol 274:R248–R254, 1998 Zucker rats. Brain Res 862:11–16, 2000 12. Ollmann MM, Wilson BD, Yang YK, Kerns JA, Chen Y, Gantz I, Barsh GS: 34. Korner J, Wardlaw SL, Liu SM, Conwell IM, Leibel RL, Chua SC Jr: Effects Antagonism of central melanocortin receptors in vitro and in vivo by of leptin receptor mutation on Agrp gene expression in fed and fasted lean agouti-related protein. Science 278:135–138, 1997 and obese (LA/N-faf) rats. Endocrinology 141:2465–2471, 2000 13. Pritchard LE, Armstrong D, Davies N, Oliver RL, Schmitz CA, Brennand JC, 35. Hagan MM, Benoit SC, Rushing PA, Pritchard LM, Woods SC, Seeley RJ: Wilkinson GF, White A: Agouti-related protein (83–132) is a competitive Immediate and prolonged patterns of Agouti-related peptide-(83–132)- antagonist at the human melanocortin-4 receptor: no evidence for differ- induced c-Fos activation in hypothalamic and extrahypothalamic sites. ential interactions with pro-opiomelanocortin-derived ligands. J Endocri- Endocrinology 142:1050–1056, 2001 nol 180:183–191, 2004 36. Coll AP, Challis BG, Lopez M, Piper S, Yeo GS, O’Rahilly S: Proopiomela- 14. Haskell-Luevano C, Chen P, Li C, Chang K, Smith MS, Cameron JL, Cone nocortin-deficient mice are hypersensitive to the adverse metabolic effects RD: Characterization of the neuroanatomical distribution of agouti-related of glucocorticoids. Diabetes 54:2269–2276, 2005 protein immunoreactivity in the rhesus monkey and the rat. Endocrinol- 37. Chen M, Celik A, Georgeson KE, Harmon CM, Yang Y: Molecular basis of ogy 140:1408–1415, 1999 melanocortin-4 receptor for AGRP inverse agonism. Regul Pept 136:40–49, 15. Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD: Localization of 2006 the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic 38. Breit A, Wolff K, Kalwa H, Jarry H, Buch T, Gudermann T: The natural control circuits in the brain. Mol Endocrinol 8:1298–1308, 1994 inverse agonist agouti-related protein induces arrestin-mediated endocy- 16. Fekete C, Legradi G, Mihaly E, Huang QH, Tatro JB, Rand WM, Emerson tosis of melanocortin-3 and -4 receptors. J Biol Chem 281:37447–37456, CH, Lechan RM: Alpha-melanocyte-stimulating hormone is contained in 2006 nerve terminals innervating thyrotropin-releasing hormone-synthesizing 39. Smart JL, Tolle V, Otero-Corchon V, Low MJ: Central dysregulation of the neurons in the hypothalamic paraventricular nucleus and prevents fasting- hypothalamic-pituitary-adrenal axis in neuron-specific proopiomelanocor- induced suppression of prothyrotropin-releasing hormone gene expres- tin-deficient mice. Endocrinology 148:647–659, 2007 sion. J Neurosci 20:1550–1558, 2000 40. Stanley SA, Small CJ, Murphy KG, Rayes E, Abbott CR, Seal LJ, Morgan 17. Adan RA, Kas MJ: Inverse agonism gains weight. Trends Pharmacol Sci DG, Sunter D, Dakin CL, Kim MS, Hunter R, Kuhar M, Ghatei MA, Bloom 24:315–321, 2003 SR: Actions of cocaine- and amphetamine-regulated transcript (CART) 18. Chai BX, Neubig RR, Millhauser GL, Thompson DA, Jackson PJ, Barsh GS, peptide on regulation of appetite and hypothalamo-pituitary axes in vitro Dickinson CJ, Li JY, Lai YM, Gantz I: Inverse agonist activity of agouti and and in vivo in male rats. Brain Res 893:186–194, 2001 agouti-related protein. Peptides 24:603–609, 2003 41. Coll AP, Challis BG, Yeo GS, Snell K, Piper SJ, Halsall D, Thresher RR, 19. Haskell-Luevano C, Monck EK: Agouti-related protein functions as an O’Rahilly S: The effects of proopiomelanocortin deficiency on murine inverse agonist at a constitutively active brain melanocortin-4 receptor. adrenal development and responsiveness to adrenocorticotropin. Endo- Regul Pept 99:1–7, 2001 crinology 145:4721–4727, 2004 20. Nijenhuis WA, Oosterom J, Adan RA: AgRP(83–132) acts as an inverse 42. Coll AP, Fassnacht M, Klammer S, Hahner S, Schulte DM, Piper S, Tung YC, agonist on the human-melanocortin-4 receptor. Mol Endocrinol 15:164– Challis BG, Weinstein Y, Allolio B, O’Rahilly S, Beuschlein F: Peripheral 171, 2001 administration of the N-terminal pro-opiomelanocortin fragment 1–28 to 21. Srinivasan S, Lubrano-Berthelier C, Govaerts C, Picard F, Santiago P, PomcϪ/Ϫ mice reduces food intake and weight but does not affect adrenal Conklin BR, Vaisse C: Constitutive activity of the melanocortin-4 receptor growth or corticosterone production. J Endocrinol 190:515–525, 2006 is maintained by its N-terminal domain and plays a role in energy 43. Small CJ, Liu YL, Stanley SA, Connoley IP, Kennedy A, Stock MJ, Bloom homeostasis in humans. J Clin Invest 114:1158–1164, 2004 SR: Chronic CNS administration of Agouti-related protein (Agrp) reduces 22. Hagan MM, Rushing PA, Pritchard LM, Schwartz MW, Strack AM, Van Der energy expenditure. Int J Obes Relat Metab Disord 27:530–533, 2003 Ploeg LH, Woods SC, Seeley RJ: Long-term orexigenic effects of AgRP- 44. Makimura H, Mizuno TM, Mastaitis JW, Agami R, Mobbs CV: Reducing (83–132) involve mechanisms other than melanocortin receptor blockade. hypothalamic AGRP by RNA interference increases metabolic rate and Am J Physiol Regul Integr Comp Physiol 279:R47–R52, 2000 decreases body weight without influencing food intake. BMC Neurosci 23. Kim MS, Rossi M, Abbott CR, AlAhmed SH, Smith DM, Bloom SR: 3:18, 2002 Sustained orexigenic effect of Agouti related protein may be not mediated 45. Fan W, Voss-Andreae A, Cao WH, Morrison SF: Regulation of thermogen- by the melanocortin 4 receptor. Peptides 23:1069–1076, 2002 esis by the central melanocortin system. Peptides 26:1800–1813, 2005 24. Lu XY, Nicholson JR, Akil H, Watson SJ: Time course of short-term and 46. Butler AA, Marks DL, Fan W, Kuhn CM, Bartolome M, Cone RD: Melano- long-term orexigenic effects of Agouti-related protein (86–132). Neurore- cortin-4 receptor is required for acute homeostatic responses to increased port 12:1281–1284, 2001 dietary fat. Nat Neurosci 4:605–611, 2001 25. Kask A, Rago L, Korrovits P, Wikberg JE, Schioth HB: Evidence that 47. Chen AS, Metzger JM, Trumbauer ME, Guan XM, Yu H, Frazier EG, Marsh orexigenic effects of melanocortin 4 receptor antagonist HS014 are medi- DJ, Forrest MJ, Gopal-Truter S, Fisher J, Camacho RE, Strack AM, Mellin ated by neuropeptide Y. Biochem Biophys Res Commun 248:245–249, 1998 TN, MacIntyre DE, Chen HY, Van der Ploeg LH: Role of the melanocortin-4 26. Drazen DL, Wortman MD, Schwartz MW, Clegg DJ, van Dijk G, Woods SC, receptor in metabolic rate and food intake in mice. Transgenic Res Seeley RJ: Adrenalectomy alters the sensitivity of the central nervous 9:145–154, 2000 system melanocortin system. Diabetes 52:2928–2934, 2003 48. Mohammad S, Baldini G, Granell S, Narducci P, Martelli AM, Baldini G: 27. Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams JR, Frazier EG, Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, immortalized hypothalamic neurons. J Biol Chem 282:4963–4974, 2007 Van der Ploeg LH, Howard AD, MacNeil DJ, Qian S: Orexigenic action of 49. Peter JC, Nicholson JR, Heydet D, Lecourt AC, Hoebeke J, Hofbauer KG: peripheral ghrelin is mediated by neuropeptide Y and agouti-related Antibodies against the melanocortin-4 receptor act as inverse agonists in protein. Endocrinology 145:2607–2612, 2004 vitro and in vivo. Am J Physiol Regul Integr Comp Physiol 292:R2151– 28. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using R2158, 2007 real-time quantitative PCR and the 2(ϪDelta Delta C(T)) method. Methods 50. Srinivasan S, Santiago P, Lubrano C, Vaisse C, Conklin BR: Engineering the 25:402–408, 2001 melanocortin-4 receptor to control constitutive and ligand-mediated G(S) 29. Cettour-Rose P, Rohner-Jeanrenaud F: The leptin-like effects of 3-D signaling in vivo. PLoS ONE 2: e668, 2007

94 DIABETES, VOL. 57, JANUARY 2008