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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

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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 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) pep- tides 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 melano- cortins 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 previ- ously 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.
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