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Endocrinology, doi:10.1210/en.2006-1389
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Endocrinology Vol. 148, No. 4 1550-1560
Copyright © 2007 by The Endocrine Society

Role of the Central Melanocortin Circuitry in Adaptive Thermogenesis of Brown Adipose Tissue

Adriana Voss-Andreae, Jonathan G. Murphy, Kate L. J. Ellacott, Ronald C. Stuart, Eduardo A. Nillni, Roger D. Cone and Wei Fan

Center for the Study of Weight Regulation and Associated Disorders (A.V.-A., J.G.M., K.L.J.E., R.D.C., W.F.) and Vollum Institute, Oregon Health and Sciences University, Portland, Oregon 97239; and Division of Endocrinology (R.C.S., E.A.N.), Brown Medical School/Rhode Island Hospital, Providence, Rhode Island 02903

Address all correspondence and requests for reprints to: Roger D. Cone or Wei Fan, Center for the Study of Weight Regulation and Associated Disorders and Vollum Institute, Oregon Health and Sciences University, Portland, Oregon 97239-3098. E-mail: cone{at}ohsu.edu or fanw{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The central melanocortin 4 receptor (MC4R) plays a critical role in energy homeostasis, although little is known regarding its role in the regulation of adaptive thermogenesis of brown adipose tissue (BAT). Here we show using retrograde transsynaptic tracing with attenuated pseudorabies virus coupled with dual-label immunohistochemistry that specific subsets of MC4R-expressing neurons in multiple nuclei of the central nervous system known to regulate sympathetic outflow polysynaptically connect with interscapular BAT (IBAT). Furthermore, we show that MC4R–/– and agouti-related peptide-treated mice are defective in HF diet-induced up-regulation of uncoupling protein 1 in IBAT. Additionally, MC4R–/– mice exposed to 4 C for 4 h exhibit a defect in up-regulation of uncoupling protein 1 levels in IBAT. Our results provide a neuroanatomic substrate for MC4R regulating sympathetically mediated IBAT thermogenesis and demonstrate that the MC4R is critically required for acute high-fat- and cold-induced IBAT thermogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MELANOCORTIN-4 RECEPTOR NULL (MC4R–/–) mice exhibit a reduced basal oxygen consumption (1) and gain more weight than wild-type control animals when placed on a moderate-fat diet due to a combination of increased food intake and a profound defect in acute diet-induced thermogenesis (DIT) (2). The mechanisms underlying the MC4R–/– mouse’s inability to coordinate energy intake with energy expenditure remain largely unexplained.

Brown adipose tissue (BAT) is one of the major thermogenic organs in rodents and human newborns used for adaptive thermogenesis in response to environmental challenges, such as diet and cold. BAT is highly vascularized and richly innervated by the sympathetic nervous system (SNS), and brown adipocytes contain a high density of large mitochondria that express the unique GDP-binding protein uncoupling protein 1 (UCP1) on their inner membranes. UCP1 disassociates, or uncouples, the respiratory chain from ATP production and dissipates the resulting oxidation energy as heat. BAT thermogenesis is triggered and regulated primarily by sympathetic nerve activity as well as thyroid hormone activity in BAT. The thermogenic capacity of BAT is largely due to UCP1, which is under the tight control of the synergistic interactions of norepinephrine (NE) and T3. Upon sympathetic stimulation, NE released from sympathetic nerve terminals acts on the adrenergic receptors, including the ß3-receptors on the surfaces of mature brown adipocytes to increase intracellular cAMP levels. The NE-induced elevation in cAMP activates hormone-sensitive lipase, which stimulates the lipolysis of triglyceride droplets, thereby increasing the levels of intracellular free fatty acids. The elevated levels of free fatty acids in turn activate UCP1 and initiate heat production (3). The NE-induced elevation in cAMP increases UCP1 gene expression to increase the thermogenic capacity of BAT through phosphorylation of the transcription factor cAMP response element binding protein. In addition, the NE-induced increase in cAMP activates the type 2 iodothyronine deiodinase in BAT, which converts intracellular T4 to T3. It is required for intracellular T3 acting on the nuclear thyroid hormone receptor-{alpha} to maintain the normal adrenergic responsiveness of the brown adipocytes and acting on the thyroid hormone receptor-ß to up-regulate UCP1 gene expression (4, 5).

DIT was initially defined by studies showing that consumption of a high-fat (HF) diet induces recruitment of interscapular BAT (IBAT), accompanied by a large increase in heat production (6, 7). These studies first proposed the idea that adaptive nonshivering thermogenesis, a form of thermogenesis found in BAT, may play a role in resistance to obesity. In addition to DIT, one of the most important functions of BAT in smaller mammals and human newborns is to sustain normal body temperature during chronic cold exposure, and it is the only site of cold-acclimatization-recruited nonshivering thermogenesis (3, 8, 9).

Chronic or acute central administration of the melanocortin agonist Melanotan II or {alpha}-MSH leads to an increase, whereas the antagonist agouti-related protein (AgRP) leads to a decrease in oxygen consumption, IBAT sympathetic nerve activity, and UCP1 expression levels, respectively (10, 11, 12, 13, 14, 15, 16, 17, 18). In addition, mice with postembryonic ablation of AgRP neurons have increased UCP1 expression in addition to reduced total body fat and plasma insulin (19), indicating that the melanocortin system is involved in mediating IBAT-induced thermogenesis. The MC4R also appears to mediate at least part of leptin’s activation of IBAT through stimulation of UCP1 (1, 20, 21, 22). Although the MC4R is expressed in numerous brain regions, including areas known to regulate autonomic activity, thermogenesis, and thermoregulation, such as the hypothalamic paraventricular nucleus (PVH), the dorsomedial hypothalamus (DMH), the raphe pallidus (RPa), etc. (23, 24, 25), the neuronal circuitry used by the central melanocortin system to regulate BAT thermogenesis has not been well defined, and it remains unknown whether the central melanocortin system mediates the alteration of UCP1 expression of BAT under physiological conditions known to activate BAT, such as an increase in dietary fat or cold exposure.

Attenuated pseudorabies virus (PRV) is a powerful tool that has been used extensively for defining multisynaptic circuitry in a number of systems and is transported exclusively in a time-dependent retrograde transsynaptic fashion (26). To elucidate the neuronal circuitry and the potential physiological roles of MC4R signaling in the regulation of adaptive thermogenesis of BAT, we first sought to map the polysynaptic pathways between IBAT- and MC4R-expressing regions in the brain and spinal cord, using PRV retrograde transsynaptic tracing in MC4R-green fluorescent protein (GFP) transgenic mice. We then used alteration of physiological conditions that stimulate UCP1 expression to ascertain whether the central melanocortin system is involved in mediating regulation of UCP1 levels in IBAT. In this study, we have combined neuroanatomical, physiological, and pharmacological approaches to examine the circuitry through which the central melanocortin system may regulate IBAT thermogenesis and demonstrate that MC4R–/– mice have a defect in their ability to up-regulate UCP1 in response to acute increased dietary fat and short-term cold exposure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All animal treatments and procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Oregon Health and Sciences University (OHSU).

PRV study
Animal maintenance.
MC4R-GFP mice (kindly provided by Dr. Jeffrey Friedman and Dr. Hongyan Liu, Rockefeller University, New York, NY) were bred in-house and maintained in a standard 12-h light, 12-h dark cycle with ad libitum access to food (Laboratory Rodent Diet, Purina 5001; PMI Nutrition International, Brentwood, MO) and water.

PRV injections.
Work with active attenuated PRV was conducted in a biosafety level 2 containment facility dedicated exclusively to these experiments, and the health of the animals was carefully monitored through the postinoculation period. Under full anesthesia with ketamine hydrochloride, MC4R-GFP mice received a series of injections with attenuated PRV (PRV-Bablue, kindly provided by Dr. Lynn Enquist, Princeton University, Princeton, NJ, and grown by Dr. Todd Wisner and Dr. David Johnson, OHSU, Portland, OR) into the left and right upper site of the exposed IBAT (2 x 109 pfu/ml in a total of 200 nl per injection at six injection sites per IBAT pad) using a 30-gauge needle connected to a Hamilton syringe (27, 28, 29, 30). Injections were made carefully under magnification and the injection site subsequently cleaned with saline-soaked swabs and the skin sewn closed. The time course of infection was empirically determined by carefully observing the pattern of infection at exactly 2-, 3-, 4-, and 5-d survival times. The animals were then killed under deep anesthesia with ketamine hydrochloride and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde-borate fixative (pH 9.5) through the left ventricle of the heart. Brains and spinal cords were removed and postfixed for 2 h in 4% paraformaldehyde-borate and overnight in a 20% sucrose solution at 4 C before being processed for immunohistochemistry visualization.

Fluorescence immunohistochemistry.
To determine which PRV-immunoreactive (IR) neurons coexpress MC4R-GFP, distinct fluorophores were used for PRV and GFP. Immunohistochemistry was performed using standard procedures. Briefly, postfixed brains and spinal cords were blocked, sliced into 30-µm coronal sections on a freezing-stage sledge microtome, and collected into eight serially ordered sets of sections. Sections were incubated at 4 C overnight in 0.02 M potassium PBS (KPBS; pH 7.4) containing 2% normal donkey serum and 0.4% Triton X-100 (LKPBS), followed by incubation with guinea-pig polyclonal antibody against PRV (1:5000; National Institute of Allergy and Infectious Diseases, Bethesda, MD) and a rabbit polyclonal antibody against GFP (1:10,000; Molecular Probes, Eugene, OR) in LKPBS for 72 h at 4 C. Sections were then rinsed several times in KPBS over a 1-h period followed by incubation with Cy3-conjugated donkey anti-guinea pig IgG (1:2000; Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexafluor 488-conjugated donkey antirabbit IgG (H+L) (1:1000; Molecular Probes, Eugene, OR) in KPBS containing 0.4% Triton-X-100 for 1 h at room temperature. Sections were then rinsed thoroughly in KPBS. All sections were mounted onto gelatin-coated slides, air dried, and coverslipped with mounting media (Biomeda, Foster City, CA). Immunofluorescence was visualized by using a Zeiss Axioplan photomicroscope with a filter set for visualization of Alexafluor 488 (excitation range, 425–525 nm; emission range, 500–600 nm) and Cy3 (excitation range, 500–560 nm; emission range, 560–650 nm). PRV-infected neurons are identified with red fluorescence, and MC4R-expressing neurons are recognized by green fluorescence. Images were overlaid using Adobe Photoshop, and double-labeled neurons are presented as yellow. High-magnification analysis was used to determine whether overlapping yellow images were due to colocalization in the same neuron or overlap of independently labeled neurons.

UCP1 study
Animal maintenance.
Male MC4R–/– mice (F10, bred in-house, 7.5 wk of age, individually housed after weaning at ~4 wk) and age-matched male C57BL/6J strain mice as wild-type controls (Jackson Laboratories, Bar Harbor, ME) had ad libitum access to Laboratory Rodent Diet (Purina 5001 from PMI Nutrition International, composed of 12% fat, 28.0% protein, and 49% carbohydrate by calories; 3.04 kcal/g metabolizable energy) and water.

HF diet.
MC4R–/– and wild-type mice were switched to a HF diet (Research Diets, New Brunswick, NJ; 60% fat calories, 20% protein, 20% carbohydrate; 5.24 kcal/g) for 48 h before being euthanized and IBAT immediately dissected as described below. Control animals were maintained on standard low-fat (LF) chow (Purina 5001 from PMI Nutrition International, composed of 12% fat, 28.0% protein, 49% carbohydrate by calories; 3.04 kcal/g metabolizable energy) for the experimental period.

Cold exposure.
The 5.5- to 6.5-wk-old MC4R–/– and wild-type mice (for the 5-d cold experiment) and 7- to 8-wk-old mice (for the 4-h cold experiment) were transferred from the colony room into chambers set at 4 or 24 C. All animals were maintained in the same 12-h light, 12-h dark cycle with ad libitum access to food (Purina 5001 from PMI Nutrition International) and water. After 4 h or 5 d, animals were euthanized and immediately dissected as described below.

Thyroid hormone measurements.
Blood was collected into serum-collecting tubes (Becton Dickinson, Franklin Lakes, NJ) immediately after euthanasia. Blood samples were briefly spun down at 4 C, and the serum was transferred to fresh tubes. The serum was then frozen in liquid nitrogen and kept frozen until assays were performed. Assays of total T3 and total T4 were performed using RIAs and antibodies previously described (31, 32) using the same serum volumes in duplicate. The sensitivities of the T3 and T4 assays were 12.5 ng/dl and 1.2 µg/dl, and the intra- and interassay variabilities were approximately 5–7 and 10–11%, respectively.

Protein extraction and Western blot.
Both IBAT pads were removed in each animal and any visible surrounding white adipose tissue carefully dissected out and discarded. The IBAT pads were immediately placed in homogenization solution in 10 mM Tris-HCl, 2% SDS, 1 mM phenylmethanesulfonyl fluoride (pH 6.8) containing protease inhibitors (Complete EDTA-free protease inhibitor cocktail; Roche Diagnostics, Alameda, CA), cut into small pieces, and briefly sonicated. Samples were passed through a 0.45-µm MCE syringe filter (Millipore, Billerica, MA) and snap-frozen in liquid nitrogen until ready to process further. Bicinchoninic acid protein assay reagent (Pierce Biotechnology, Rockford, IL) was used to determine protein concentrations, and 20 µg of each sample was resolved alongside a protein standard (Precision Plus Dual Color; Bio-Rad, Hercules, CA) with 12% SDS-PAGE and electrotransferred to an Immobilon-FL polyvinylidene fluoride membrane (Millipore). The membrane was blocked with Aquablock (EastCoast Bio, North Berwick, ME) and then incubated overnight 1:100 with guinea-pig antihuman UCP1 antibody, cross-reactive with mouse (Linco Research, St. Charles, MO) and 1:2500 with antibody specific to mouse tubulin E7 ascites (Developmental Studies Hybridoma Bank, University of Iowa) diluted in Aquablock with 0.1% Tween. Subsequent immunoblotting was performed in accordance with the Odyssey near-infrared imaging platform system (LI-COR Biosciences, Lincoln, NE), and data were captured using the Odyssey scanner. The data using the Odyssey system was desensitized with the system software and further processed and normalized using Microsoft Excel.

Quantitative RT-PCR.
The following protocol was performed using RNase-free equipment and solutions to minimize RNA degradation. RNA was extracted from IBAT using TRIzol according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). After DNase I treatment (Amp Grade, 1 U/µl; Invitrogen), cDNA was generated from 1 µg RNA using random hexamer priming and Maloney murine leukemia virus reverse transcriptase (Invitrogen) according to the manufacturer’s instructions.

Initially, standard PCR was performed with the IBAT cDNA and the following primers to amplify a fragment of UCP1: mouse UCP1 (GenBank U63418) forward position 409–428, 5'-GTG AAG GTC AGA ATG CAA GC-3', and reverse position 586–605, 5'-AGG GCC CCC TTC ATG AGG TC-3' (33). To 1 µl BAT cDNA we added 0.4 µl primer mix (each primer at 50 pmol/µl), 12.5 µl 2x PCR Master Mix [containing Taq DNA polymerase (recombinant; 0.05 U/µl) in reaction buffer, 0.4 mM dNTPs (0.4 mM each of dATP, dCTP, dGTP, and dTTP), 4 mM MgCl2; Fermentas Inc., Hanover, MD] and 10.1 µl water. The cycling conditions were as follows: denaturing at 95 C for 3 min followed by 40 cycles of denaturing at 95 C for 1 min, annealing at 60 C for 1 min, polymerization at 72 C for 1 min, and after 40 cycles, a final polymerization step at 72 C for 5 min. The resulting 197-bp UCP1 fragment was run out on a 1% low-melt agarose gel and isolated from the gel using a QIAquick PCR purification kit according to the manufacturer’s protocol (QIAGEN Inc., Valencia, CA).

Quantitative PCR was performed using the following conditions (per reaction): 2 µl 10x PCR buffer, 0.6 µl MgCl2 (preoptimized for the given primers, 2.5 mM final concentration), 5 µl primer mix (1.5 mM final concentration of each UCP1 primer), 0.4 µl dNTPs (10 mM; containing equimolar concentrations of dATP, dCTP, dGTP, and dTTP), 0.2 µl Sybr green (50x; Applied Biosystems, Foster City, CA), and 0.4 µl Platinum Taq DNA polymerase (Invitrogen). Gel-isolated UCP1 cDNA was added at 1:10, 1:100, 1:1000, 1:10,000, and 1:100,000 to make a standard curve. Water was added to make the final reaction volume 20 µl. The same reaction conditions were also used for the amplification of the housekeeping gene 18S for quantitation (forward 5'-CCG CAG CTA GGA ATA ATG GA-3' and reverse 5'-CCC TCT TAA TCA TGG CCT CA-3') and water as a negative/no-template control. Each reaction was run in duplicate on a fluorescence temperature cycler (Opticon Continuous Fluorescence Detector; MJ Research Inc., South San Francisco, CA) using the following conditions based on optimization for 18S: 95 C for 2 min followed by 40 cycles of denaturing at 95 C for 15 sec, annealing at 58.6 C for 30 sec, and a plate read after each cycle. Final melting curves were calculated with a start temperature of 75 C, an ending temperature of 95 C, and a 1 C temperature increment. Standard curves, which allow for the determination of the relative concentration of RNA in the samples, were calculated by plotting the log of the input RNA against the critical threshold cycle, the cycle in which the fluorescence signal of the sample is greater than the baseline threshold. Amplification carried out in the fluorescence temperature cycler was verified by electrophoresis on 1.5% ethidium-bromide-stained agarose gels to make sure the size of the amplified fragments matched the calculated fragment size for UCP1 (197 bp).

Intracerebroventricular (icv) cannulations and injections.
Surgical procedures were performed using sterile technique. Adult wild-type C57BL/6J mice were deeply anesthetized with a combination of ketamine hydrochloride and isoflurane and implanted with a 25-gauge stainless steel guide cannula with obdurator stylet into the lateral cerebral ventricle under stereotaxic control (coordinates were –0.6 mm relative to bregma, midline, and 4.75 mm below the surface of the skull). The cannula was secured to the skull with dental cement. Animals were individually housed and allowed to recover for 1 wk with ad libitum access to standard LF chow (Purina 5001 from PMI Nutrition International, composed of 12% fat, 28.0% protein, and 49% carbohydrate by calories; 3.04 kcal/g metabolizable energy). After recovery, 1 nmol/injection·d AgRP (83–132) in a 2-µl volume or 2 µl saline was injected over 1 min using a polyethylene 0.28-mm diameter tubing attached to a Hamilton microsyringe. The injector was left in place for another minute before removal. At this point, the LF chow was switched to the HF chow (Research Diets; 60% fat calories, 20% protein, 20% carbohydrate; 5.24 kcal/g) for the remainder of the 48-h experiment, and 24 h after the first injection, a second identical dose was injected. The dose of AgRP was chosen based on efficacy in previous studies in which icv injection of 1 nmol AgRP (83–132) significantly stimulated food intake (34). The animals were euthanized, and IBAT was removed 24 h after the second injection as described above. The position of the cannula was verified at the end of the experiment by injection of India ink and dissection of the brain. UCP1 levels in IBAT were assessed by quantitative PCR and Western blotting as described above.

Statistical methods
Results are presented as mean ± SE (SEM). Data sets were analyzed for significance using a one-way and two-way ANOVA followed by Bonferroni post hoc testing on PRISM software (GraphPad, San Diego, CA). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regions in the central nervous system (CNS) coexpressing MC4R-GFP-IR and PRV-IR after inoculation of IBAT
Twelve male MC4R-GFP mice were injected with the attenuated PRV-Bablue. We did not observe as much variability in the extent of infection within each survival interval as reported in other studies (27, 30, 35). Distribution patterns of PRV-infected neurons at varying infection times (2, 3, 4, and 5 d) allowed us to determine the order of labeling throughout the spinal cord and CNS and were very similar to those seen in previous studies performed in Sprague Dawley rats (30, 35). Two control mice that underwent a sympathectomy did not show any signs of infection in the brain or spinal cord at 5 d post injection, indicating that the virus was infecting the brain specifically via the sympathetic nerves projecting to IBAT (data not shown). The number of coexpressing neurons in the spinal cord and brain regions at different postinfection durations are summarized in Table 1Go.


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  		TABLE 1. Temporal distribution of PRV-infected neurons coexpressing MC4R

 
Although many areas throughout the spinal cord and brain were singly labeled for either MC4R or PRV, our analysis focused on areas in which colocalization was observed. For all calculations, consecutive sections from a 1:8 set of sections per animal were analyzed and the results for three animals for each infection time averaged. The spinal cords of animals killed at 2 and 3 d post inoculation were sectioned coronally. At 2 d post injection (n = 3), PRV infection was predominantly restricted to the spinal cord. Colocalization of MC4R and PRV was seen in the intermediolateral cell column (IML) in the thoracic spinal cord (coexpression in 58% of the PRV-infected neurons and 45% of MC4R-expressing neurons) (Table 1Go, d 2, and Fig. 1Go, A–C).


Figure 1
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  		FIG. 1. Photomicrograph illustrating specific subset of MC4R-GFP-IR neurons are coexpressed with PRV-IR in the IML of thoracic spinal cord (A–C), the brainstem RPa (D–F), and the hypothalamic PVH (G–I). A, PRV-infected neurons (red) in the IML of spinal cord 2 d after injection into IBAT; B, same section as A, depicting MC4R-expressing neurons (green); C, overlap of A and B, depicting neurons coexpressing PRV and MC4R (yellow); D, PRV-infected neurons (red) in the RPa 4 d after injection into IBAT; E, same section as D, depicting MC4R-expressing neurons (green); F, overlap of D and E, depicting neurons coexpressing PRV and MC4R (yellow); G, PRV-infected neurons (red) in the PVH 4 d after injection into IBAT; H, same section as D, depicting MC4R-expressing neurons (green); I, overlap of G and H, depicting neurons coexpressing PRV and MC4R (yellow). Scale bar, 50 µm.

 
At 3 d post injection (n = 3), colocalization of MC4R and PRV was seen in the thoracic spinal cord and in two regions in the brain (Table 1Go, d 3), the RPa (coexpression in 40% of all PRV-infected neurons and 25% of MC4R-expressing neurons in the RPa) and the gigantocellular reticular nucleus (Gi; coexpression in 41% of the PRV-infected neurons and 26% of MC4R-expressing neurons). Additionally, two of the animals began to show colocalization of PRV and MC4R in the PVH dorsal parvicellular (dpv) subdivision, and one animal began to show colocalization in the rostroventrolateral medulla (RVLM).

At 4 d post injection (n = 3), colocalization of MC4R and PRV began to become more widespread. Colocalization was still apparent in the RPa, Gi, and dpv PVH as well as in several new regions (Table 1Go, d 4) including the periaqueductal gray, ventrolateral part (four colocalized neurons, making up 15% of all PRV-infected neurons and 25% of all MC4R neurons) and the medial nucleus of the solitary tract (coexpression in 42% of all PRV-infected neurons and 56% of all MC4R-expressing neurons). In the PVH, coexpression in all the animals was observed by d 4, as well as a large increase in the number of PRV-infected neurons, primarily in the dpv, ventromedial parvocellular PVH, and lateral parvicellular PVH (coexpression in 20% of all PRV-infected neurons and 42% of all MC4R neurons in the PVH) (Fig. 1Go, G–I). The number of singly labeled PRV neurons and the number of colocalized neurons increased from d 2 in the RVLM (coexpression in 12% of all PRV-infected neurons and 12% of all MC4R-expressing neurons) and in the RPa (coexpression in 47% of all PRV-infected neurons and 90% of all MC4R-expressing neurons) (Fig. 1Go, D–F).

At 5 d post injection (n = 3), colocalization of MC4R and PRV was seen in all the same regions as in d 4 with small increases in the number of PRV-infected neurons in those areas (Table 1Go, d 5). From d 4 to 5, the most substantial increase seen in the number of PRV-infected and colocalized cells was in the PVH (coexpression in 30% of all PRV-infected neurons and 72% of all MC4R-expressing neurons). There were also increases in the Gi (coexpression in 8% of all PRV-infected neurons and 50% of all MC4R neurons) and the RVLM (coexpression in 18% of all PRV-infected neurons and 25% of all MC4R neurons). The first colocalized neurons were detected in the lateral hypothalamus at d 5, and although small in number, they were observed in all animals (coexpression in 10% of all PRV-infected neurons and 22% of all MC4R-expressing neurons). The first colocalized cells were also detected in the caudal part of the pontine reticular nucleus (coexpression in 12% of all PRV-infected neurons and 17% of all MC4R-expressing neurons), the laterodorsal tegmental nucleus (coexpression in 73% of all PRV-infected neurons and 40% of all MC4R neurons), the DMH (coexpression in 9% of all PRV-infected neurons and 30% of all MC4R neurons), and the locus ceruleus (coexpression in 18% of all PRV-infected cells and 31% of all MC4R neurons).

MC4R–/– mice exhibit attenuated UCP1 expression in IBAT in response to a HF diet
The data described above suggest the melanocortin circuitry is directly involved in regulation of IBAT. To determine the potential physiological roles of MC4R signaling in the regulation of adaptive thermogenesis of BAT, we first used a genetic approach to examine the role of MC4R signaling in mediating the regulation of UCP1 levels in IBAT in response to an acute HF diet challenge.

Age-matched male wild-type and MC4R–/– mice that were raised on a LF diet (Purina 5001 from PMI Nutrition International, containing 23.0% protein, 4.5% fat, and 49% carbohydrate with 3.04 kcal/g metabolizable energy) were either continued on the LF diet or were placed for 48 h on a HF diet (Research Diets; 60% fat calories, 20% protein, and 20% carbohydrate with 5.24 kcal/g metabolizable energy). The food intake and body weight of these animals were then measured, and UCP1 expression in IBAT was detected using Western blot technique.

After 48 h on the HF diet, a one-way ANOVA reveals that the relative UCP1 protein levels in HF-fed wild-type animals were 4.3 ± 0.8-fold higher (n = 11) than in LF-fed wild-type animals (n = 12, P < 0.001; Fig. 2AGo). In contrast, relative UCP1 protein levels in MC4R–/– mice that were fed LF (1.5 ± 0.3; n = 10) or HF (1.0 ± 0.1; n = 12) diets were not significantly different from each other or from wild-type animals fed a LF diet. By two-way ANOVA, the effect of genotype (P = 0.001) and diet (P = 0.0014) on UCP1 levels as well as the interaction between the two (P < 0.0001) were significant.


Figure 2
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  		FIG. 2. Lean male MC4R–/– and AGRP-treated mice have attenuated UCP1 protein levels in response to a HF diet. UCP1 (32 kDa) is normalized with respect to tubulin (55 kDa) for each animal, and the four sets of animals within each experiment are further normalized to either LF-fed wild-type (A) or saline-treated (B) mice. Representative Western blot results for two animals from each group are shown above the bar graphs. A, UCP1 protein levels are induced 4-fold by a HF diet in wild-type mice. After a HF diet, MC4R–/– mice (n = 12) do not exhibit the increase in UCP1 levels (n = 11). UCP1 levels are the same in MC4R–/– (n = 10) and wild-type mice (n = 12) fed a LF diet. B, UCP1 protein levels are induced 2.5-fold by a HF diet in wild-type mice treated with icv saline. After a HF diet, mice treated icv with AgRP (n = 13) do not exhibit the increase in UCP1 levels found in saline-treated mice (n = 14). UCP1 levels are the same in AgRP-treated (n = 11) and saline-treated (n = 11) mice fed a LF diet. By one-way ANOVA: *, P < 0.05; ***, P < 0.0001.

 
AgRP treatment attenuates UCP1 protein levels in response to a high-fat (HF) diet
To exclude the possibility that the block of UCP1 up-regulation shown above results from a developmental defect caused by the MC4R knockout, we next repeated the above protocol but used a pharmacological approach in place of a genetic one. Age-matched male wild-type mice were fed a LF or HF diet while being centrally treated with AgRP (83–132) or saline for 2 d. As expected from previous studies, third ventricle AgRP-treated mice consumed more food in grams or calories than saline controls (data not shown).

By one-way ANOVA, AgRP-treated animals fed a HF diet had similar UCP1 levels (0.6 ± 0.1; n = 13) to LF saline (1.0 ± 0.1; n = 11) and LF AgRP (0.9 ± 0.1; n = 11) but significantly lower relative levels of UCP1 protein than the HF-fed saline-treated control group (2.8 ± 0.8; n = 14; P < 0.05; Fig. 2BGo). By two-way ANOVA, the effect of diet alone on UCP1 levels was not significant (P = 0.09), but the effect of drug treatment (P = 0.01) and the drug treatment by diet interaction (P = 0.03) were significant.

Up-regulation of UCP1 by the central melanocortin system in response to an acute HF diet does not appear to be mediated by the MC4R-hypothalamus-pituitary-thyroid (HPT) axis
In rodents, thyroid hormones act synergistically with the SNS to regulate UCP1 expression (36). Because the melanocortin system can modulate both sympathetic outflow to the IBAT (13, 17) and the function of the HPT axis (37), we tested whether the MC4R-mediated up-regulation of UCP1 expression in response to a HF diet involves the HPT axis. We first measured plasma T3 and T4 levels in wild-type and MC4R–/– mice in response to an acute HF diet challenge.

By one-way ANOVA, total T3 levels were modestly, yet significantly, elevated in both HF-fed wild-type mice (105.8 ± 6.5 ng/dl; n = 11; P < 0.01) and MC4R–/– mice (110.5 ± 6.2 ng/dl; n = 12; P < 0.05) compared with LF-fed wild-type (74.5 ± 2.6 ng/dl; n = 12) and MC4R–/– mice (85.0 ± 7.6 ng/dl; n = 8) (Fig. 3AGo). By two-way ANOVA, the effect of diet on total T3 levels was significant (P < 0.0001), although there was no effect of genotype on T3 levels (P = 0.21) or a genotype by diet interaction (P = 0.62).


Figure 3
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  		FIG. 3. MC4R–/– and AGRP-treated mice have normally elevated total T3 serum hormone levels in response to a 2-d HF diet. A, Total T3 levels are elevated in MC4R–/– (n = 12) and wild-type (n = 11) mice fed a HF diet compared with MC4R–/– (n = 8) and wild-type (n = 12) mice fed a LF diet; B, total T3 levels are elevated in HF-fed AgRP-treated (n = 8) and saline-treated (n = 8) mice compared with AgRP-treated (n = 7) and saline-treated (n = 6) mice fed a LF diet; C, total T4 levels are reduced, but not significantly, in MC4R–/– (n = 4) and wild-type mice (n = 8) fed a HF diet compared with MC4–/– (n = 6) and wild-type (n = 9) mice fed a LF diet. By one-way ANOVA: *, P < 0.05; **, P < 0.01.

 
In a separate experiment using a pharmacological approach to block the MC4R, total T3 levels were also significantly elevated (P < 0.0001) in both HF-fed AgRP-treated (98.1 ± 8.2; n = 8) (P < 0.05) and saline-treated (117.3 ± 6.2; n = 8) (P < 0.01) mice compared with LF-fed AgRP-treated (73.0 ± 3.9; n = 7) and saline-treated (78.6 ± 5.5; n = 6) mice (Fig. 3BGo). By two-way ANOVA, the effect of drug treatment (P = 0.06) and the drug treatment by diet interaction (P = 0.3) were not significant, whereas the effect of diet was significant (P < 0.0001).

By one-way ANOVA, total T4 levels in MC4R–/– and wild-type mice were not significantly different between any two individual groups (Fig. 3CGo). However, by two-way ANOVA, total T4 levels were significant for diet (P = 0.01) with HF-fed animals having lower T4 levels (LF wild-type, 3.4 ± 0.2 µg/dl, n = 9; LF MC4R–/–, 3.3 ± 0.3 µg/dl, n = 6) than LF-fed animals (HF wild-type, 2.9 ± 0.2 µg/dl, n = 8; HF MC4R–/–, 2.6 ± 0.2 µg/dl, n = 4) but not significantly different between the groups for genotype (P = 0.28) or genotype by diet interaction (P = 0.72).

Effect of cold exposure on UCP1 levels in MC4R–/– and wild-type mice
To test the hypothesis that the MC4R is involved in regulating IBAT thermogenesis in response to cold exposure, we examined whether MC4R–/– mice exposed to 4 C had a defect in IBAT function compared with wild-type mice. IBAT function was assessed by measuring UCP1 protein levels using Western blotting and UCP1 mRNA levels using real time RT-PCR.

Age-matched lean male and female wild-type and MC4R–/– mice were maintained at room temperature (24 C) or exposed to cold (4 C) for 4 h. After 4 h, the relative UCP1 levels in cold-exposed wild-type mice (1.9 ± 0.1, n = 8) were significantly higher than for cold-exposed MC4R–/– mice (1.0 ± 0.1, n = 9; P < 0.01) or wild-type (1 ± 0.1, n = 8; P < 0.001) and MC4R–/– (1.1 ± 0.2, n = 8; P < 0.01) mice kept at room temperature (Fig. 4AGo). By two-way ANOVA, the effect of cold exposure on UCP1 (P = 0.006), the effect of genotype on UCP1 (P = 0.013), and the cold exposure by genotype interaction (P = 0.002) were significant.


Figure 4
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  		FIG. 4. Lean male MC4R–/– mice fail to increase UCP1 protein levels in response to 4 h of cold exposure (A) but are able to up-regulate UCP1 protein and mRNA levels after a 5-d cold exposure (B and C). UCP1 protein (32 kDa) and mRNA levels are normalized with respect to tubulin (55 kDa) or 18S, respectively, for each animal, and the four sets of animals within each experiment are further normalized to 24 C wild-type mice. Representative Western blot results for two animals from each group are shown above the bar graphs in A and B. A, After exposure to 4 C for 4 h, 7- to 8-wk-old MC4R–/– mice (n = 9) do not exhibit the almost 2-fold increase in UCP1 protein levels observed in age-matched wild-type mice (n = 8). UCP1 levels are the same in MC4R–/– (n = 9) and wild-type mice at 24 C (n = 8). B, After exposure to 4 C for 5 d, 5.5- to 6.5-wk-old MC4R–/– mice (n = 4) up-regulate UCP1 levels, as do age-matched wild-type mice (n = 4). UCP1 levels are the same in MC4R–/– (n = 4) and wild-type mice (n = 4) after 5 d at 24 C. C, UCP1 mRNA levels are increased almost 2-fold in wild-type mice (n = 5) and in MC4R–/– mice after 5 d at 4 C (n = 5) when compared with control wild-type (n = 5) and MC4R–/– (n = 5) mice at 24 C. By one-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

 
In another experiment, age-matched lean male wild-type and MC4R–/– were placed at room temperature (24 C) or exposed to cold (4 C) for 5 d. After 5 d of cold exposure, using one-way ANOVA, MC4R–/– mice (3.2 ± 0.1, n = 4) had an increase in their relative UCP1 levels compared with MC4R–/– mice (1.4 ± 0.2, n = 4) at room temperature (P < 0.001), similar to the increase observed between wild-type mice at cold exposure (2.1 ± 0.2, n = 4) and wild-type mice kept at room temperature (1 ± 0.1, n = 4; P < 0.01) (Fig. 4BGo). MC4R–/– mice with cold exposure had an even larger increase in UCP1 levels compared with wild-type mice under the same conditions (P < 0.01). By two-way ANOVA, the effect of cold exposure on UCP1 levels (P < 0.0001) and the effect of genotype on UCP1 levels were significant (P = 0.0005). However, the effect of cold exposure on genotype (P = 0.03) was not significant. (1.0 ± 0.1; P < 0.05) and MC4R–/– mice (0.9 ± 0.1; P < 0.05) kept at room temperature, respectively. By two-way ANOVA, the effect of temperature on UCP1 mRNA levels was significant (P = 0.0002), whereas the effect of genotype (P = 0.55) and the temperature by genotype interaction (P = 0.83) were not significant (Fig. 4CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neural pathways from the central melanocortin circuitry to IBAT
The specific retrograde transsynaptic progression of the attenuated PRV-Bablue is time dependent, thus providing information on the hierarchical order of multisynaptic circuits. According to previously published results, rats surviving 2–3 d after PRV injection into their IBAT exhibit labeling of second-order neurons, 3–4 d post injection labels third-order neurons, and 4–5 d post injection labels fourth-order neurons (30, 35). The sites of PRV infection in the brain and spinal cord resulting from IBAT injection and the rate of progression were very similar to those found in previous studies (30, 35), enabling us to compare our PRV-Bablue labeling results in mice with previous studies done in rats. In addition, it has been previously shown in rats that supraspinal labeling is specific for the input to IBAT adipocytes and not blood vessels (30, 35). In this study, we focus principally on those PRV-infected areas that also express MC4R. Transsynaptic tracing by injection of attenuated PRV-Bablue into the IBAT of MC4R-GFP transgenic mice, coupled with dual-labeling immunohistochemical techniques, reveals a specific subset of dual-labeled MC4R-GFP/PRV-IR neurons in multiple nuclei at various levels of the CNS. These nuclei, which contain varying degrees of dual-labeled neurons, are found throughout the CNS, including the IML of the spinal cord, brainstem RPa, nucleus of the solitary tract, midbrain and hypothalamus PVH, DMH, and lateral hypothalamus. These data provide the first evidence for potential action sites of MC4R signaling in the regulation of sympathetic outflow to IBAT and suggest that a complex network of MC4R-expressing neurons in multiple nuclei at various levels of the CNS may participate in the regulation or modulation of sympathetically mediated IBAT thermogenesis (Fig. 5Go). Our data, together with previous studies demonstrating that central administration of melanocortin agonists and antagonists cause an increase or decrease, respectively, in UCP1 expression levels or IBAT sympathetic nerve activity (11, 12, 13, 14, 15, 16, 17, 18), provide strong evidence in support of the hypothesis that central MC4R signaling plays a direct role in the regulation of IBAT thermogenesis.


Figure 5
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  		FIG. 5. Schematic diagram showing MC4R-expressing areas in the mouse brain infected at different intervals after PRV injection into IBAT (green, 2 d; brown, 4 d; red, 5 d; hatched red, some coexpression observed at 5 d). A semiquantitative description of the coexpression of MC4R-GFP-IR and PRV-IR in each area is presented in Table 1Go. HF diet activates MC4R signaling in the neural circuitry controlling IBAT to induce a sympathetically mediated increase in UCP1 expression in IBAT. LC, Locus ceruleus; LH, lateral hypothalamus; LPB, lateral parabrachial nucleus; LTD, laterodorsal tegmental nucleus; NTS, nucleus of the solitary tract; PAG, periaqueductal gray; PRN, pontine reticular nucleus; POA, preoptic area.

 
Central MC4R signaling is critically required in diet-induced UCP1 up-regulation in IBAT
Basal and total oxygen consumption in MC4R–/– mice, in contrast to wild-type C57BL/6J mice, is not up-regulated when animals are fed a moderate-fat diet (2). Our results demonstrate that MC4R–/– and AgRP-treated wild-type mice have a defect in their ability to up-regulate UCP1 protein expression in IBAT in response to a 2-d HF diet. These data indicate that central MC4R signaling is critically required for acute diet-induced thermogenesis in IBAT and suggest that attenuated IBAT thermogenic function could account for at least part of the decrease in basal oxygen consumption and increased obesity observed in MC4R–/– animals fed a HF diet. Interestingly, we found that AgRP treatment in mice does not affect UCP1 levels when they are fed a normal LF diet, which differed from previous studies in rats showing that AgRP treatment increases (37) or decreases (15) UCP1. These varying results may be due to the different methodologies, such as species and size of animal used as well as time course, dose, and method used for AgRP administration. However, it is clear from our results that AgRP-treated mice, in contrast to saline-treated control mice, were unable to increase UCP1 expression when switched from a LF to a HF diet.

Putative mechanisms underlying MC4R signaling-dependent up-regulation of UCP1 in response to a HF diet
UCP1 expression is tightly regulated by a complex synergistic interaction between the SNS and the thyroid hormones T4 and T3 (36, 38). UCP1 expression relies on functional T3-response elements and cAMP response element binding protein motifs in the UCP1 gene upstream enhancer region (38, 39, 40). BAT contains abundant type II T4 5'-deiodinase, which catalyzes the conversion of T4 to the more biologically active and potent T3 and which is activated by the SNS (41). TRH neurons in the PVH express MC4R and are densely innervated by {alpha}-MSH-containing and AgRP-containing axon terminals in both rats and humans (42, 43, 44, 45). Furthermore, the melanocortin system has also been implicated in the regulation of the HPT axis (37, 42, 46), and a chronic increase in dietary fat has been shown to increase both the IBAT NE turnover in rodents (47) and serum T3 levels (48, 49). These studies led to our hypothesis that perhaps there may be a defect in the MC4R-HPT axis that contributes to the inability of MC4R–/– mice to up-regulate UCP1 expression in BAT in response to a HF diet. Contrary to our original hypothesis, MC4R–/– and AgRP-treated mice exhibit the same increase in total T3 hormone levels as wild-type controls when switched from a LF to a HF diet. Interestingly, we also found that total T4 levels in high-fat-fed MC4R–/– and wild-type mice are decreased, which is likely related to the observed increases in T3, because T4 is converted to T3. Overall, these results suggest that the central melanocortin-mediated regulation of the HPT axis may not be involved in the DIT-induced up-regulation of UCP1, although the melanocortin system may mediate part of the leptin-induced secretion of TRH from hypothalamic explants (50). Our study cannot completely rule out the possibility that the HPT axis is involved in mediating the melanocortin system’s effects on UCP1 regulation. It is, for instance, possible that wild-type mice have higher free T3 levels (the more biologically active form) than MC4R–/– mice and that we did not detect this difference by measuring only the total T3, which is comprised mostly of the less active plasma protein-bound T3. However, free T4 and T3 are generally considered to be in equilibrium with the protein-bound hormones in both plasma and tissue, and thus, total serum thyroid hormone levels are thought to be reflective of the relative free thyroid hormone levels. Our data suggest, therefore, that the most likely mechanism underlying the MC4R–/– animal’s inability to up-regulate UCP1 in IBAT is a defect in sympathetic outflow to IBAT in response to a HF diet. Our previous study reported that after 24 h on a moderate-fat diet, wild-type mice display comparable NE turnover in IBAT to that of wild-type mice on a LF diet, suggesting that changes in sympathetic activity in IBAT is not involved in DIT in this model (2). However, it remains quite possible that a difference was not detected because of the limited time frame in which the experiment was performed. In contrast, multiple studies have found that a HF diet does lead to increases in UCP1 levels (14, 51, 52, 53, 54, 55, 56, 57), and at least one study demonstrated an increased NE turnover in the IBAT and heart of rats in response to a 5-d increase in dietary fat or sucrose in rodents (47). Furthermore, the other previous observation (58) that BAT of sympathectomized mice failed to respond by increases in [3H]GDP binding to isolated mitochondria and uncoupling protein concentration when animals were offered a palatable HF dietary supplement supports our current hypothesis. Additional studies are needed to clarify the likely role of the central melanocortin system in regulation of sympathetic outflow to BAT.

Effect of cold exposure on the melanocortin system’s regulation of UCP1
UCP1 plays a central role in sustaining normal body temperatures of rodents during cold exposure. Our finding that MC4R–/– mice exposed to 4 C for 4 h were unable to up-regulate UCP1 expression indicates that MC4R signaling is also critically required for an acute cold-induced IBAT thermogenesis and provides additional evidence that MC4R–/– mice do indeed have some defect in their ability to induce nonshivering thermogenesis. However, after a 5-d cold exposure, MC4R–/– mice regain their ability to induce UCP1. One interpretation could be that the melanocortin system is involved in, or necessary, only for the short-term regulation of UCP1 and that other compensatory mechanisms are activated upon longer periods of cold exposure that do not require central MC4R signaling. It is also possible that animals may differentially regulate UCP1 in response to cold and dietary fat such that even though BAT recruitment in MC4R–/– mice may remain intact during long-term exposure to cold temperatures, they do not have the ability to recruit BAT in response to increased dietary fat. The mechanisms underlying the differences in the melanocortin system’s ability to regulate UCP1 during short-term and long-term cold exposure remain to be understood.

In summary, using specific polysynaptic retrograde tracing from IBAT coupled with dual-labeling immunocytochemistry, we show coexpression of MC4R-GFP-IR and PRV-IR in many hypothalamic and brainstem regions known to play an important role in nonshivering thermogenesis, demonstrating an anatomical link between the central melanocortin system and the sympathetically mediated regulation of IBAT thermogenic function. Additionally, using both genetic and pharmacological antagonism, we show that intact MC4R signaling is required to appropriately up-regulate UCP1 in response to physiological stimuli, such as a HF diet and short-term cold exposure. Our results that MC4R–/– and AgRP-treated mice on a HF diet have a similar increase in their total serum T3 levels to wild-type controls also suggests that the MC4R-mediated up-regulation of UCP1 in response to a HF diet may be independent of the central HPT axis. Our data showing that UCP1 is up-regulated normally in MC4R–/– mice after longer periods of cold exposure suggest that melanocortin system-mediated nonshivering thermogenesis may be differentially regulated by diet and cold or alternatively that compensatory mechanisms may develop in these animals after long-term cold exposure. This study provides a neuroanatomic and physiological basis for the regulation of nonshivering thermogenesis by central melanocortin system and furthers our insight into the mechanisms by which the central melanocortin signaling maintains energy homeostasis by matching changes in energy intake with energy expenditure.


   Acknowledgments
 
We gratefully acknowledge Dr. Jeffrey Friedman and Dr. Hongyan Liu for kindly providing the MC4R-GFP mice, Dr. Lynn Enquist for kindly providing us with PRV-Bablue, and Drs. Todd Wisner and David Johnson for their generous help in growing the virus. We also thank Ilia G. Halatchev, Julian Voss-Andreae, Ken Gruber, and Paul Kievit for their help.


   Footnotes
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Grants DK62179 to W.F., DK70332 to R.D.C., and DK58148 and NS045231 to E.A.N., and The Wellcome Trust Fellowship 068303 to K.L.J.E.

Disclosure Summary: A.V.-A., J.G.M., K.L.J.E., R.C.S., E.A.N., and W.F. have nothing to declare. OHSU and R.D.C. have equity interests in Orexigen, and R.D.C. has an equity interest in Neurocrine Biosciences, Inc.

First Published Online December 28, 2006

Abbreviations: AgRP, Agouti-related protein; BAT, brown adipose tissue; CNS, central nervous system; DIT, diet-induced thermogenesis; DMH, dorsomedial hypothalamic nucleus; dpv, dorsal parvicellular; GFP, green fluorescent protein; Gi, gigantocellular reticular nucleus; HF, high-fat; HPT, hypothalamus-pituitary-thyroid; IBAT, interscapular brown adipose tissue; icv, intracerebroventricular; IML, intermediolateral cell column; IR, immunoreactive or immunoreactivity; KPBS, 0.02 M potassium PBS; LF, low-fat; MC4R, melanocortin 4 receptor; NE, norepinephrine; PRV, pseudorabies virus; PVH, paraventricular hypothalamic nucleus; RPa, raphe pallidus; RVLM, rostroventrolateral medulla; SNS, sympathetic nerve system; UCP1, uncoupling protein 1.

Received October 12, 2006.

Accepted for publication December 15, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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