A role for arcuate cocaine and amphetamine regulated transcript in hyperphagia, thermogenesis, and cold adaptation

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1 The FASEB Journal express article /fj fje. Published online July 18, A role for arcuate cocaine and amphetamine regulated transcript in hyperphagia, thermogenesis, and cold adaptation Wing May Kong,*, Sarah Stanley,*, James Gardiner,* Caroline Abbott,* Kevin Murphy,* Asha Seth,* Ian Connoley, Mohammed Ghatei,* David Stephens, and Stephen Bloom* *Department of Metabolic Medicine, Division of Investigative Sciences, Hammersmith Campus, Faculty of Medicine; and Department of Mathematics, Faculty of Physical Sciences, South Kensington Campus, Imperial College School of Science, Technology and Medicine, London, United Kingdom; Department of Physiology, St. Georges Hospital Medical School, London, United Kingdom. Contributed equally to this paper. Corresponding author: Stephen R. Bloom, Department of Metabolic Medicine, Division of Investigative Sciences, Hammersmith Campus, Faculty of Medicine, Imperial College School of Science, Technology and Medicine, London, W12 0NN, United Kingdom. s.bloom@imperial.ac.uk ABSTRACT We have recently shown that injection of the hypothalamic peptide cocaine and amphetamine regulated transcript (CART) into discrete hypothalamic nuclei stimulates food intake. This stimulation was particularly marked in the arcuate nucleus. Here we show that twice daily intraarcuate injection of 0.2 nmole CART peptide for 7 days was associated with a 60% higher daytime food intake, an 85% higher thermogenic response to the β3 agonist BRL 35135, and a 60% increase in brown adipose tissue UCP-1 mrna. In a separate study, using stereotactically targeted gene transfer, a CART transgene was delivered by using polyethylenimine to the arcuate nucleus of adult rats. Food intake was increased significantly during ad libitum feeding and following periods of food withdrawal and food restriction in CART over-expressing animals. CART over-expressing animals lost 12% more weight than controls following a 24-h fast. Brown adipose tissue uncoupling protein-1 (UCP-1) mrna levels (collected Day 25) were 80% higher in CART over-expressing animals. Finally, by using quantitative in situ hybridization, we found that chronic cold exposure (20 days at 4 o C) increased arcuate nucleus CART mrna by 124%. Together with the orexigenic and thermogenic effects of CART, this finding suggests a role for arcuate nucleus CART in cold adaptation. Key words: CART appetite brown adipose tissue hypothalamus gene transfer cold adaptation C ocaine and amphetamine regulated transcript (CART) was first characterized as a transcript within the rat brain that was up-regulated following administration of psychostimulants (1). CART is widely distributed in the central nervous system (2), and CART mrna and peptide are highly expressed in hypothalamic nuclei involved in the regulation of food intake and brown adipose tissue thermogenesis: the arcuate nucleus,

2 paraventricular nucleus (PVN), the lateral hypothalamus (LH), and the ventromedial hypothalamus (VMH). Interestingly, hypothalamic CART peptide has been found to co-localize with both anorectic and orexigenic neuropeptides, raising the possibility that hypothalamic CART may influence feeding in a number of ways. In the arcuate nucleus over 90% of CART neurons co-express pro-opiomelanocortin (POMC) mrna (3). The POMC product alphamelanocyte stimulating hormone (αmsh) has been shown to be an important anorectic peptide in rodents and humans (4). In the medial LH 95% of CART neurons co-express the orexigenic peptide, melanin-concentrating hormone (MCH) (5). CART has previously been proposed as an endogenous satiety factor. ICV injection of CART (55 102) [nomenclature after Kristensen (6)] causes a dose-dependent reduction in food intake in rats (6, 7). However, we and others have also noted that the reduced food intake following ICV CART (55 102) is associated with marked behavioral abnormalities (6, 8, 9). Feeding behavior is easily affected by non-specific factors (e.g., malaise or stress), and the anorexic effects of ICV CART may be secondary to motor effects. We have recently shown that administration of low doses of CART (55 102) ( nmole) into discrete hypothalamic nuclei (arcuate and VMH) results in increased food intake (9) and is not associated with behavioral abnormalities, suggesting that, in specific hypothalamic nuclei, CART may have a role as an orexigenic peptide. The recently described CART knockout mouse supports the theory of a dual role for CART in the regulation of food intake. The homozygote knockout has an increased food intake on a high-fat diet compared with wild-type littermates in keeping with a role as an anorectic factor. However, the male heterozygote has a significantly reduced food intake compared with wild-type controls, which suggests a potential orexigenic role (10). ICV CART up-regulates brown adipose tissue (BAT) uncoupling protein 1 (UCP-1) mrna in rats (11), possibly by stimulating sympathetic outflow via pre-ganglionic neurons in the thoracic spinal cord (12). In addition, CART terminals synapse with TRH neurons in the PVN. CART treatment produces a dose-dependent increase in neuronal TRH content from primary hypothalamic neuronal cultures (13) and increases TRH release from hypothalamic explants (14). TRH, in turn, can stimulate thermogenesis independent of its effect on TSH and thyroid hormones (15). Cold acclimatization is characterized by hyperphagia and increased BAT thermogenesis. Little is known about the central neurogenic control of cold-induced hyperphagia. Our recent data showing an acute orexigenic effect of hypothalamic CART, together with data showing that CART up-regulates BAT UCP-1, raises the possibility that hypothalamic CART may have a role in cold adaptation. To further characterize the actions of CART, we have investigated the effect of chronic increased arcuate nucleus CART peptide on food intake, body weight, and BAT thermogenesis. We firstly examined the effect of 7 days of repeated intra-arcuate injection of CART (55 102) on food intake and energy balance. To overcome the problems associated with a repeated injection model, we examined the effect of a sustained excess of CART peptide in the arcuate nucleus by using polyethylenimine (PEI)-mediated targeted gene transfer to achieve chronic overexpression of CART cdna in the arcuate nucleus. PEI is a non-viral gene carrier, which has been used to transfect rodent neurons in vivo with both reporter genes and functional genes (16 18) for up to 90 days (19). Finally, the potential involvement of arcuate nucleus CART in cold adaptation was

3 investigated by measuring the effect of 20 days cold exposure on hypothalamic CART mrna expression. MATERIALS AND METHODS Peptides and plasmids For the chronic peptide injection study, CART (55 102) peptide (Peptide Institute Inc., Osaka, Japan) was dissolved in 0.9% saline. A dose of 0.2 nmole was used, as this has been shown to stimulate food intake when injected into the arcuate nucleus (9). The plasmid pcdna-cart expressing full-length CART cdna was produced by using reverse-transcription PCR of total rat hypothalamic RNA and primers corresponding to nucleotides 1 20 and of the CART cdna (Genbank accession number, g563130). The PCR product was cloned into the expression vector pcdna1.1 (amp) (Invitrogen, Leek, The Netherlands), to produce pcdna-cart. The sequence of the insert was confirmed in both directions at least twice. The ability of the pcdna-cart to produce functional CART peptide was tested in vitro in RIN 1056a cells. The secreted product was chromatographically identical to CART (55 102) when eluted by reverse-phase FPLC by using a high-resolution column (Amersham Pharmacia Biotechnology, Uppsala, Sweden). For the gene transfer, plasmid DNA was complexed with 0.1 M PEI by the method of Abdallah et al (19). Briefly, DNA at a concentration of 1 µg/µl in 5% (w/v) glucose was mixed with six equivalents of 0.1 M PEI (average M w ~ 25,000, Aldrich, Poole, Dorset, UK) and vortexed for 30 s. The complexes were incubated at room temperature for 10 min before use. For the gene-transfer studies pcdna1.1 (amp) (without transgene) was used as the control plasmid. Animals Adult, male Wistar rats ( g, A. Tuck, UK) were used in all studies. All animals were maintained in individual cages and, with the exception of the cold-adaptation study, kept under controlled temperature (21 23 o C) and light (12 h light, 12 h dark, lights on at 0700) conditions with ad libitum access to food (RM1 diet, SDS, Witham, UK) and water. All animal procedures undertaken were approved by the British Home Office Animals Scientific Procedures Act Study 1: Repeated intra-arcuate administration of CART (55 102) For intra-arcuate cannulation, animals were implanted with a permanent 26-gauge stainless steel guide cannulae projecting into arcuate nucleus as previously described (20), according to the stereotaxic coordinates of Paxinos and Watson (21). Prior to the start of the study, animals were sham-injected on two occasions to minimize the effects of stress on food intake during the study. On Day 0 of the study, the baseline BAT thermogenic capacity was measured by using the selective β3 agonist BRL (22), given by intraperitoneal (i.p.) injection at a dose of 40 µg/kg. BAT thermogenesis is mediated via β3 receptors (23), and BRL35135 has been shown to up-regulate BAT UCP-1. The core temperature response was measured at 30, 60, and 90 min following injection. Data are presented as the mean thermogenic response over this time. The following day at 0900 h (i.e., in the fed state) animals received either 0.2 nmole CART (55 102)

4 (n=10) or saline (control, n=10) in a volume of 1 µl via a 31-gauge stainless steel injector inserted into the guide cannula. The acute feeding response to intra-arcuate CART was examined by measuring food intake at 1, 2, and 4 h post-cart injection. At 1700 h animals were injected again with 0.2 nmole CART (55 102) or saline and thereafter were injected twice daily (at 0900 and 1700 h) for the following 7 days. Food intake was measured twice daily immediately prior to each injection. Body weight was measured before the morning injection only. Any animal that lost >25 g weight or ate <10 g food on more than one day was considered unwell and was excluded from the study. On Day 7 the thermogenic response to BRL was measured again. The next day animals were killed, and trunk blood was collected for measurement of thyroid hormones and interscapular BAT collected for measurement of UCP-1 mrna. Samples were stored at 70 o C until assayed. Study 2: Chronic overexpression of CART cdna in the arcuate nucleus To overcome the possible problems raised by the use of repeated injections and the relatively short duration of the orexigenic effect of intra-arcuate injected CART (55 102), a targeted genetransfer model was used to further examine the effects of chronic increased CART in the arcuate nucleus. In preliminary studies we confirmed anatomical localization of the CART transgene mrna to the region of the arcuate nucleus by using in situ hybridization (Fig. 1). Using an antisense CART riboprobe, Fig. 1a shows unilateral localization of CART transgene expression in the region of the arcuate nucleus. The positive signal in the sense control (Fig. 1b) corresponds to pcdna-cart plasmid DNA. Effect on food intake and body weight Animal surgical procedures and handling were performed as previously described (14). Under anesthesia, a temporary 26-gauge guide cannula was inserted into the arcuate nucleus as above (Plastics-One, Roanoke, VA). Animals were injected with 1 µg of pcdna-cart/pei (n=9) or pcdna1.1/pei (control, n=10) in a volume of 1 µl infused over 4 min via a 31-gauge steel injector (Plastics One) placed in and projecting 1 mm below the tip of the guide cannula. The cannula and injector were left in position for 5 min to limit back-diffusion and then were slowly withdrawn. After a 7-day recovery period, food intake and body weight were measured daily. On Day 10, post-gene transfer food was withdrawn for 24 h. Five days later (Day 16), when daily food intake had returned to pre-fasting levels for 3 consecutive days, the animals were placed on an 80% food restriction for 5 days. Each animal received 80% of its average daily food intake over the previous 48 h. After 5 days animals were returned to ad libitum rat chow. Water was freely available throughout the dietary manipulations. On day 25, animals were then killed and trunk blood and interscapular BAT was collected as described above. Effect on brown adipose tissue thermogenesis and hypothalamic CART release Under anesthesia, animals were injected with 1 µg of pcdna-cart/pei (n=10) or 1 µg pcdna1.1/pei (control, n=10) via a temporary 26-gauge intra-arcuate cannula as above. BAT thermogenic capacity was measured prior to gene transfer as above. Body weight and food intake were measured from Day 7 post-gene transfer. The thermogenic response to BRL was repeated on Day 9 post gene transfer. The study was ended on Day 10 to minimize the effects of changes in food intake on the parameters measured (in the previous study this was the latest

5 time-point before the effect of CART on food intake became significant). On Day 10, animals were killed by decapitation and the brains were removed for ex vivo measurement of CART release (see below). Trunk blood and BAT were collected as described above. Study 3: Effect of cold exposure on hypothalamic CART mrna Animals were maintained as described above for the other studies except that eight rats were maintained at 4 o C for 21 days and eight controls were kept at 22 o C. Standard rat chow was available ad libitum. Food intake and body weight were measured on alternate days. At the end of the cold exposure, the brains were removed and CART mrna measured by quantitative in situ hybridization. Trunk blood was collected for measurement of serum leptin. Static incubation A static incubation system was used as previously described (14). Rats were killed, the brain was rapidly removed, and a 1.2-mm-thick hypothalamic explant taken parallel to the base of the brain with a vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK); this dimension was chosen to ensure inclusion of the entire arcuate nucleus with minimum encroachment into the PVN and LH (the other major sites of hypothalamic CART peptide). Explants were transferred to artificial cerebrospinal fluid (acsf). The acsf was collected following a 1 h basal incubation and a subsequent 1 h exposure to acsf containing 56 mm potassium chloride and stored at 20 o C until assayed for CART immunoreactivity (CART-IR) and TRH. Explants not showing release above basal values with KCl were not considered viable and were excluded from the analysis. Peptide measurement Plasma thyroid-stimulating hormone (TSH) was measured by radioimmunoassay (RIA), as previously described (24), by using reagents and methods provided by NIDDK and the National Hormone and Pituitary Program (A. Parlow, Harbor University of California, Los Angeles Medical Center). Plasma tetra-iodothyronine (T 4 ) and free tri-iodothyronine (T3) were measured by using a solid-phase RIA (DPC, Los Angeles, CA). CART (55 102) immunoreactivity (CART-IR) content of the acsf was measured by sensitive and specific radioimmunoassay as previously described (25). Plasma leptin was measured by RIA (Linco Research Inc., St. Charles, MO) In situ hybridization In situ hybridization using sense and anti-sense CART riboprobes (corresponding to nucleotides 101 to 411 of CART cdna) was performed on 15 µm cryostat sections, as previously described (26). Every third section was stained for cresyl violet to determine anatomical position. Slides were hybridized overnight at 55 o C with Bq of [ 35 S]CTP labeled probe added to each slide. Subsequently slides were RNase-A treated, washed, ethanol dehydrated, air-dried, and exposed to autoradiographic film (Bio-Max Film MR, Kodak, Hemel Hempstead, Herts, UK) for 7 days. All slides were processed together to ensure uniform hybridization, washing, and development conditions. For Study 3, the resulting images were analyzed densitometrically by

6 using Image software (W. Rashband, NIMH, Bethesda, MD). The values represent integrated optical density expressed as percentage of mean control values. Ribonuclease protection assay for BAT UCP-1 RNA was extracted from frozen interscapular BAT by using Tri-Reagent (Helena Biosciences, Sunderland, UK), according to the manufacturer s protocol. Quantification of UCP-1 mrna was performed by using the Ambion RPA III kit (Ambion Inc., Austin, TX) by using conditions optimized within our laboratory. The UCP-1 riboprobe corresponded to nucleotides 351 to 658 (UCP-1). Rat β-actin (Ambion Inc.) was used as an internal control to correct for RNA loading. Briefly, 0.25 µg RNA was hybridized overnight at 42 o C with Bq of 32 P[CTP]-labeled riboprobe. Reaction mixtures were digested with RNase A/T1, and the protected fragments were precipitated and separated on a 4% polyacrylamide gel. The dried gel was exposed to a phosphorimager screen overnight, and bands were quantified by image densitometry by using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Statistical analysis Values are given as the mean ± SEM. Food and body weight data involving repeated daily measures were analyzed by using 3-way ANOVA using treatment and day as factors and baseline body weight as a co-variate (27). All other data were analyzed by unpaired two-tailed t- test. In Study 2 cumulative four food intake was analyzed as a single measure after each dietary intervention: i) ad lib feeding; ii) 24 food withdrawal; iii) 5 days of food restriction. Statistical significance was taken as P<0.05. RESULTS Study 1: Repeat intra-arcuate administration of CART (55 102) Food intake and body weight Following a single intra-arcuate injection of 0.2 nmole of CART (55 102), food intake was significantly increased in the CART (55 102) treated group at 1 2 h post-injection [0.95 ± 0.23 g (CART) vs. 0.1 ± 0.05 g (saline), P<0.002] and was 500% greater between 1 4 h [1.85 ± 0.4 g (CART) vs ± 0.11 g (saline), P<0.002; Fig. 2a]. In keeping with our previous findings (9), there was no significant difference in food intake between the two groups in the first hour [0.46 ± 0.22 g (CART) vs ± 0.3 g (saline), P=0.4]. Cumulative food intake (0 4 h) was twofold higher in the CART (55 102) group, although this did not reach statistical significance (P=0.08; Fig. 2a). During the 7 days of twice-daily intra-arcuate CART injections, mean daytime food intake was 65% higher in the CART (55 102)-treated group [5.0 ± 0.0 g (CART) vs. 3.0 ± 0.0 g/day (saline), P<0.02], and cumulative daytime food intake was also increased [Day 6 cumulative daytime food intake: 32 ± 2 g (CART) vs. 20 ± 3 g (saline), P<0.0001; Fig. 2b]. No behavioral or movement abnormalities were observed following the CART (55 102) injections. There was no difference in cumulative nighttime food intake between the two groups [93 ± 6 g (CART) vs. 98 ± 5 g (saline)] or in cumulative total daily food intake (Fig. 2c). There was no difference in body weight between the two groups at the start of the study. Seven days treatment

7 with 0.2 nmole CART (55 102) twice daily did not result in any difference in body weight [Day 7; 269 ± 6 g (CART) vs. 266 ± 7 g (saline)]. Brown adipose tissue There was no difference in the baseline thermogenic response to BRL Treatment with CART (55 102) for 7 days resulted in an 85% greater thermogenic response to BRL [1.3 ± 0.2 C (CART) vs. 0.7 ± 0.13 o C (saline), P<0.05; Fig. 3a]. BAT UCP-1 mrna was more than 60% greater in the CART (55 102) animals [33.3± 4.1 (CART) vs ± 4.4 relative units (saline), P<0.05; Fig 3b]. Study 2: Chronic overexpression of CART cdna in the arcuate nucleus Effect on food intake and body weight There was no difference in body weight between the two experimental groups at baseline or at Day 7 post-gene transfer. During 4 days of ad lib feeding (Days 7 10, post-op), cumulative food intake was significantly greater in the CART over-expressing animals [Day 10 cumulative 4 day food intake, mean ± SEM: 135 ± 3 g (pcdna-cart) vs. 125 ± 3 g (control), P<0.02; Fig. 4a], although total daily food intake was not significantly increased between the two groups. Refeeding following a 24-h fast was significantly greater in the pcdna-cart treated animals [cumulative 4-day re-feed: 161 ± 3 g (pcdna-cart) vs. 149 ± 2 g (control), P<0.01; Fig. 3a] as was re-feeding following the 5 days on 80% food restriction [cumulative 4-day re-feed: 161 ± 5 g (pcdna-cart) vs. 149 ± 2 g (control), P<0.05]. Cumulative weight gain was also increased in the CART over-expressing animals [Day 25: 79 ± 3 g (pcdna-cart) vs. 73 ± 3 g (control), P<0.001]. Time was fitted as a factor in the regression model (one level per day) to allow for possible non-linear time-dependent effects. This produced a linear model that fitted very well (R 2 = 0.91). Additionally there was a trend towards increased total body weight in the CART overexpressing group [Day 25: 399 ± 7 g (pcdna-cart) vs. 383 ± 7 g (control), P=0.08]. Considering the effects on BAT thermogenesis shown in Study 1, we were interested to see the effects of food withdrawal and food restriction on body weight. The 24 h fast resulted in significantly greater weight loss in the CART over-expressing animals compared with the control plasmid group [ 27 ± 1 g (pcdna- CART) vs. 24 ± 1 g [control], P<0.01; Fig. 4b). Despite higher daily food consumption, when transferred to 80% food restriction, weight loss was greater in the CART over-expressing animals, although this did not reach statistical significance [weight loss, Day 1 food restriction: 6 ± 1 g (pcdna-cart) vs. 3 ± 1 g (control), P=0.1; Fig. 4b). At the end of the study (Day 25) interscapular BAT UCP-1 mrna (quantified by RPA) was increased by 80% in the pcdna- CART treated animals compared with animals treated with control plasmid [47.3 ± 4.7 relative units (pcdna-cart) vs ± 6.1 relative units (control), P<0.02; Fig. 4c]. Effect on BAT thermogenesis and hypothalamic CART release At Day 0 there was no difference in the thermogenic response to i.p. BRL At Day 9 postgene transfer the mean thermogenic response to BRL in the pcdna-cart treated animals was significantly greater in the pcdna-cart treated group compared with controls [1.5 ± 0.5 o C (pcdna-cart) vs. 0.1 ± 0.5 o C (control) P<0.05].

8 On Day 10 post-gene transfer hypothalami were collected and ex-vivo CART-IR release measured in a static incubation system. Basal CART-IR release was 40% higher in the pcdna- CART treated animals [184 ± 13 fmol/explant (pcdna-cart) vs. 129 ± 19 fmol/explant (control), P<0.01]. Potassium-stimulated CART release was also increased in the pcdna-cart treated animals [319 ± 19 fmol/explant (pcdna-cart) vs. 257 ± 14 fmol/explant (control), P<0.05] consistent with increased hypothalamic CART peptide content (Fig. 5). Histological examination of the remaining brain tissue confirmed that the 1.2-mm medio-basal slices included the entire arcuate nucleus with minimal encroachment into the PVN or LH. Effect of chronic overexpression of CART cdna and repeated administration of CART (55 102) on thyroid hormones Chronic arcuate over-expression of CART cdna for either 10 or 25 days significantly altered the thyroid axis. CART cdna over-expression for 25 days significantly decreased plasma TSH compared with control animals [TSH: 3.5 ± 0.3 ng/ml (pcdna-cart) vs. 4.4 ± 0.3 ng/ml (control plasmid), P<0.05]. In animals over-expressing CART cdna for 10 days, plasma TSH was also significantly reduced compared with controls [TSH: 2.32 ± 0.3 ng/ml (pcdna-cart) vs ± 0.6 ng/ml (control plasmid), P<0.05]. Repeated administration of CART (55 102) also showed a tendency to reduced TSH [TSH: 3.3 ± 0.3 ng/ml (CART) vs. 4.2 ± 0.4 ng/ml (saline), P=0.1]. These changes were accompanied by a trend towards increased T4 in the CART (55 102) treated animals compared with controls (P=0.07; Table 1). Study 3: Effect of cold acclimatization on hypothalamic CART expression There was no difference in body weight between the two groups at the start of the study [241 ± 2 g (4 o C) vs. 241 ± 3 g (22 o C)]. Cumulative weight gain was significantly lower in the coldexposed animals from Day 8 [day 8; 21 ± 4 g (4 o C) vs. 42 ± 4 g (20 o C), P<0.05] and remained so until the last day measured [Day 20; 43 ± 5 g (4 o C) vs. 72 ± 6 g (22 o C), P<0.005; Fig. 6a]. Cumulative food intake was 25% greater (P<0.05) in the cold-exposed group from Day 6 and almost 40% greater by Day 20 [Day 20, 672 ± 24 g (4 o C) vs. 487 ± 8 g (22 o C), P<0.001; Fig. 6b]. On Day 21, brains were collected. With the use of quantitative in situ hybridization, levels of CART mrna in the arcuate nucleus of cold-acclimatized animals were more than double those of the warm-maintained animals [integrated density: 224 ± 50 % (4 o C) vs. 100 ± 21% (22 o C), P<0.05; Fig. 7]. There was no significant difference in CART mrna expression in the PVN between the two groups (P=0.2). Plasma leptin was significantly reduced in the coldacclimatized animals (1.3 ± 0.1 ng/ml vs. 2.8 ± 0.3 ng/ml, P<0.0001). This difference persisted even when differences in body weight were taken into account. DISCUSSION We have shown that chronically increased CART peptide in the arcuate nucleus resulting from either targeted gene transfer or repeated intra-arcuate injections results in increased cumulative food intake. Using the minimally invasive technique of targeted gene transfer, we found that chronically increased CART expression in the arcuate nucleus for 25 days also resulted in increased weight gain. Fasting and food restriction in animals over-expressing CART cdna is associated with increased weight loss compared with controls, which suggests increased energy expenditure. Consistent with this we have also shown that chronically increased CART peptide

9 in the arcuate nucleus results in increased BAT thermogenesis. These results suggest a role for arcuate nucleus CART in cold adaptation. In support of this we have found that chronic cold exposure more than doubles CART mrna levels in the arcuate nucleus. In the first study the orexigenic effect of repeated arcuate injection of CART (55 102) on cumulative daytime food intake persisted throughout the 7 day study period with no evidence of tachyphylaxis. In keeping with our previous results (9), the increase in feeding observed following arcuate injection of CART (55 102) in satiated rats was not apparent until 1 h postinjection. It is possible that CART acts indirectly through the release of another neuropeptide. We have previously shown that CART treatment of medio-basal hypothalamic slices stimulates NPY release and inhibits αmsh release (14). Thus, the orexigenic effect of CART may be through an increase in NPY or inhibition of αmsh. We have shown previously that the orexigenic effect of an acute injection of intra-arcuate CART lasts approximately 4 h. Rats eat 90% of their total daily food intake in the dark phase. During the dark phase the relatively short-lived orexigenic effect of CART ( may have been masked by the much more prolonged normal dark-phase food intake. In addition, repeated twicedaily intra-nuclear injections are relatively invasive and are less well tolerated than repeated ICV injections. This may explain why, despite a robust acute effect on acute food intake, we did not see an effect on total daily food intake or weight gain in contrast with chronic ICV studies by using other orexigenic peptides [e.g., NPY (28), AGRP (29)]. To try to overcome some of the problems associated with repeated intra-nuclear peptide injections, we therefore used targeted gene transfer to produce a sustained increase in arcuate nucleus CART. We found that sustained overexpression of CART in the arcuate nucleus resulted in increased food intake in freely fed rats and when food was returned following food restriction or food withdrawal. Using in situ hybridization we confirmed anatomical localization of the transgene to the arcuate nucleus. Increased CART peptide following PEI-mediated gene transfer was confirmed by significantly greater release of CART-IR from hypothalamic explants from rats injected with the pcdna- CART plasmid compared with controls. We confirmed by histological examination that the 1.2 mm medio-basal slices taken included the entire arcuate nucleus with minimal inclusion of the PVN or LH. Intra-arcuate CART gene transfer was associated with an 8% increase in cumulative food intake, although there was no significant feeding effect on a day-to-day basis. This effect appears relatively modest compared with published reports of continued ICV infusion of other orexigenic peptides (29, 28). Nonetheless such increases, if sustained, are likely to result in weight gain comparable with that seen in human obesity (30). The previously reported ability of ICV CART (55 102) to inhibit feeding has been well documented by both our own laboratory and others. However, this anorectic action is associated with pronounced abnormalities of movement and behavior (6, 8, 9). It is therefore possible that some of the anorectic effects of ICV administered CART (55 102) are indirect. CART has been implicated in extra-hypothalamic reward circuits, and the anorectic effects of CART may be through extra-hypothalamic pathways. ICV administered anti-cart antibodies have been shown to increase nighttime feeding (6). This increased food intake may be due to blockade of extra-hypothalamic circuits. In keeping with this, Aja et al. have recently shown that the behavioral abnormalities and anorectic effect associated with ICV CART (55 102) are greatly

10 reduced if the cerebral aqueduct is plugged and ICV injections of CART peptide are administered rostral to the plug to limit peptide reaching the fourth ventricle. This suggests that the behavioral and anorectic effects are mediated at a hindbrain site and not in the hypothalamus (31). The effect of plugging of the cerebral aqueduct at later time points, when we observed the orexigenic effect of intra-arcuate CART (55 102), was not examined. BAT thermogenesis is an important component of regulated energy expenditure in rodents. It has been shown that ICV administration of CART (55 102) up-regulates BAT UCP-1 mrna. We found that repeated intra-arcuate CART peptide injection increased BAT UCP-1 mrna and the thermogenic response to BRL In view of this we examined the effect of food withdrawal and food restriction (both situations that would be expected to be associated with reduced energy expenditure) in the intra-arcuate CART gene transfer model. Following a 24-h fast, pcdna- CART treated animals lost significantly more weight than the control plasmid group and food restriction was associated with a trend towards greater weight loss in the CART over-expressing group. These findings suggest increased energy expenditure in the CART over-expressing animals. Consistent with this, BAT UCP-1 mrna at Day 25 was 80% higher in the pcdna- CART group. CART may activate thermogenesis by stimulation of sympathetic outflow to BAT (12). The up-regulation of BAT UCP-1 mrna in the 25 day pcdna-cart injected animals may have been influenced by the periods of fasting and food restriction. However, similar upregulation of BAT UCP-1 activity was also seen following repeated intra-arcuate injections of CART peptide (increased mrna and β3 thermogenic response) and in the 10 day pcdna- CART injected animals (increased β3 thermogenic response). Hypothalamic TRH pathways can stimulate thermogenesis independent of effects on TSH and thyroid hormones (15). TRH neurons in the PVN are innervated by CART-IR axons, and CART treatment increase TRH release from primary hypothalamic neuronal cultures (13). Despite the stimulatory effect of CART on hypothalamic TRH, previous groups, including ourselves, have been unable to demonstrate an effect of CART on TSH, which suggests that arcuate nucleus CART activates TRH neurons not involved with thyroid function. Plasma TSH was significantly reduced in pcdna-cart treated animals when compared with control animals. CART may increase sympathetic outflow to the thyroid gland as well as to brown adipose tissue. The thyroid gland has a dense sympathetic innervation, and chronic sympathetic stimulation has been shown to increase serum T3 (32). Such changes could explain our observed reduction in plasma TSH. In contrast to food restriction, the hyperphagia associated with cold acclimatization is associated with increased BAT thermogenesis. Little is known about the central neurogenic control of coldinduced hyperphagia. The hypothalamic feeding peptides that have been studied have opposing effects on food intake and BAT thermogenesis. Thus, ICV-administered NPY stimulates feeding but inhibits BAT thermogenesis, whereas αmsh inhibits food intake and stimulates BAT thermogenesis (33, 34). Hypothalamic NPY mrna levels are unchanged with chronic cold exposure (35, 36). In contrast, we have found that increased CART in the arcuate nucleus increases food intake and stimulates BAT thermogenesis. We found that animals maintained at 4 o C for 21 days had almost double the levels of CART mrna in the arcuate nucleus compared with animals maintained at 22 o C. Interestingly, given the relationship between CART and TRH neurons in the PVN, we found no difference in CART mrna expression in the PVN between the two groups. In the LH, CART co-localizes with the orexigenic peptide MCH and it would be of interest in the future to examine the effect of cold exposure on CART mrna in the LH.

11 Peripheral administration of leptin stimulates CART neurons in the arcuate nucleus (12) and food withdrawal, which is associated with a fall in circulating leptin, is associated with downregulation of CART mrna in the arcuate nucleus (6). Plasma leptin levels in the cold acclimatized animals were less than 50% of those in the animals maintained at 22 o C. Our finding of a twofold increase in arcuate CART mrna expression is therefore unexpected given the reduction in plasma leptin. Our results therefore suggest that cold exposure activates CART by leptin-independent pathways. Interestingly, only 30 40% of CART neurons in the arcuate nucleus express leptin receptors (37). It is therefore possible that the thermogenic and orexigenic effects of intra-arcuate CART are mediated via leptin independent pathways. The effect of intraarcuate CART on energy expenditure may explain the fasting-induced down-regulation of arcuate CART mrna. Increased BAT thermogenesis is likely to be undesirable in situations of food restriction, and therefore it would be logical that arcuate CART would be down-regulated despite its orexigenic action. Our data provide further evidence that, in the arcuate nucleus, CART is an important orexigenic peptide and suggest a possible role for arcuate nucleus CART in cold adaptation and coldinduced hyperphagia in rodents. Recent evidence suggests that CART is also involved in the regulation of energy expenditure and body weight in humans. CART is found in the human hypothalamus in nuclei associated with appetite control (37 39). A systematic study of two common CART polymorphisms in a large human population suggested that CART may influence fat distribution (40). Although adult humans have little brown adipose tissue, a CART mis-sense mutation has recently been described, which was found to co-segregate with severe obesity through three generations of an Italian family and was linked to a reduction in resting energy expenditure (41). It therefore seems likely that the actions of CART on thermogenesis are relevant to the development of obesity in humans. ACKNOWLEDGMENTS W.M.K. is supported by a Wellcome Trust grant. C. A., K. M., and A. S. are supported by the Medical Research Council, United Kingdom. REFERENCES 1. Douglass, J., McKinzie, A. A., and Couceyro, P. (1995) PCR differential display identifies a rat brain mrna that is transcriptionally regulated by cocaine and amphetamine. J. Neurosci. 15, Gautvik, K. M., de Lecea, L., Gautvik, V. T., Danielson, P. E., Tranque, P., Dopazo, A., Bloom, F. E., and Sutcliffe, J. G. (1996) Overview of the most prevalent hypothalamusspecific mrnas, as identified by directional tag PCR subtraction. Proc. Natl. Acad. Sci. USA 93, Elias, C. F., Saper, C. B., Maratos, F. E., Tritos, N. A., Lee, C., Kelly, J., Tatro, J. B., Hoffman, G. E., Ollmann, M. M., Barsh, G. S., et al. (1998) Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J. Comp. Neurol. 402,

12 4. Vaisse, C., Clement, K., Durand, E., Hercberg, S., Guy-Grand, B., and Froguel, P. (2000) Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity. J. Clin. Invest. 106, (see comments) 5. Broberger, C. (1999) Hypothalamic cocaine and amphetamine regulated transcript (CART) neurons: histochemical reelationship to thyrotrophin releasing hormone, melanin concentrating hormone, orexin/hypocretin and neuropeptide Y. Brain Res. 848, Kristensen, P., Judge, M. E., Thim, L., Ribel, U., Christjansen, K. N., Wulff, B. S., Clausen, J. T., Jensen, P. B., Madsen, O. D., Vrang, N., et al. (1998) Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature July 5, Lambert, P. D., Couceyro, P. R., McGirr, K. M., Dall Vechia, S. E., Smith, Y., and Kuhar, M. J. (1998) CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 29, Larsen, P. J., Vrang, N., Petersen, P. C., and Kristensen, P. (2000) Chronic intracerebroventricular administration of recombinant CART(42-89) peptide inhibits and causes weight loss in lean and obese Zucker (fa/fa) rats. Obes. Res. 8, Abbott, C. R., Rossi, M., Wren, A. M., Murphy, K. G., Kennedy, A. R., Stanley, S. A., Zollner, A. N., Morgan, D. G., Morgan, I., Ghatei, M. A., et al. (2001) Evidence of an orexigenic role for cocaine- and amphetamine-regulated transcript after administration into discrete hypothalamic nuclei. Endocrinology 142, Asnicar, M. A., Smith, D. P., Yang, D. D., Heiman, M. L., Fox, N., Chen, Y. F., Hsiung, H. M., and Koster, A. (2001) Absence of cocaine- and amphetamine-regulated transcript results in obesity in mice fed a high caloric diet. Endocrinology 142, Wang, C., Billington, C. J., Levine, A. S., and Kotz, C. M. (2000) Effect of CART in the hypothalamic paraventricular nucleus on feeding and uncoupling protein gene expression. Neuroreport 11, Elias, C. F., Lee, C., Kelly, J., Aschkenasi, C., Ahima, R. S., Couceyro, P. R., Kuhar, M. J., Saper, C. B., and Elmquist, J. K. (1998) Leptin activates hypothalamic CART neurons projecting to the spinal cord. Neuron 21, Fekete, C., Mihaly, E., Luo, L. G., Kelly, J., Clausen, J. T., Mao, Q., Rand, W. M., Moss, L. G., Kuhar, M., Emerson, C. H., et al. (2000) Association of cocaine- and amphetamineregulated transcript-immunoreactive elements with thyrotropin-releasing hormonesynthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting. J. Neurosci. 20, Stanley, S. A., Small, C. J., Murphy, K. G., Rayes, E., Abbott, C. R., Seal, L. J., Morgan, D. G., Sunter, D., Dakin, C. L., Kim, M. S., et al. (2001) Actions of cocaine- and amphetamineregulated transcript (CART) peptide on regulation of appetite and hypothalamo-pituitary axes in vitro and in vivo in male rats. Brain Res. 893,

13 15. Arancibia, S., Rage, F., Astier, H., and Tapia-Arancibia, L. (1996) Neuroendocrine and autonomous mechanisms underlying thermoregulation in cold environment. Neuroendocrinology 64, Martres, M. P., Demeneix, B., Hanoun, N., Hamon, M., Giros, B., Themis, M., Forbes, S. J., Chan, L., Cooper, R. G., Etheridge, C. J., Miller, A. D., Hodgson, H. J. F., Coutelle, C., Goula, D., Benoist, C., Mantero, S., Merlo, G., Levi, G., Demeneix, B. A. (1998) Up- and down-expression of the dopamine transporter by plasmid DNA transfer in the rat brain. Eur. J. Neurosci 10, Boussif, O., Lezoualc, Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA 92, Guissouma, H., Ghorbel, M. T., Seugnet, I., Ouatas, T., and Demeneix, B. A. (1998) Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific. FASEB J. 12, Abdallah, B., Hassan, A., Benoist, C., Goula, D., Behr, J. P., and Demeneix, B. A. (1996) A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum. Gene Ther. 7, Kim, M. S., Rossi, M., Abusnana, S., Sunter, D., Morgan, D. G., Small, C. J., Edwards, C. M., Heath, M. M., Stanley, S. A., Seal, L. J., et al. (2000) Hypothalamic localization of the feeding effect of agouti-related peptide and alpha-melanocyte-stimulating hormone. Diabetes 49, Paxinos, G., and Watson, C. (21998) The Rat Brain.Academic Press. 22. Liu, Y. L., and Stock, M. J. (1995) Acute effects of the beta 3-adrenoceptor agonist, BRL 35135, on tissue glucose utilisation. Br. J. Pharmacol. 114, Zhao, J., Cannon, B., and Nedergaard, J. (1998) Thermogenesis is beta3- but not beta1- adrenergically mediated in rat brown fat cells, even after cold acclimation. Am. J. Physiol. 275, R2002 R Beak, S. A., Small, C. J., Ilovaiskaia, I., Hurley, J. D., Ghatei, M. A., Bloom, S. R., and Smith, D. M. (1996) Glucagon-like peptide-1 (GLP-1) releases thyrotropin (TSH): characterization of binding sites for GLP-1 on alpha-tsh cells. Endocrinology 137, Murphy, K. G., Abbott, C. R., Mahmoudi, M., Hunter, R., Gardiner, J. V., Rossi, M., Stanley, S. A., Ghatei, M. A., Kuhar, M. J., and Bloom, S. R. (2000) Quantification and synthesis of cocaine and amphetamine regulated transcript peptide (79-102)-like immunoreactivity and mrna in rat tissues. J. Endocrinol. 166, Steel, J. H., Hamid, Q., Van, N. S., Jones, P., Denny, P., Burrin, J., Legon, S., Bloom, S. R., and Polak, J. M. (1988) Combined use of in situ hybridisation and immunocytochemistry for

14 the investigation of prolactin gene expression in immature, pubertal, pregnant, lactating and ovariectomised rats. Histochemistry 89, Crowder, M. J., and Hand, D. J. (1990) Analysis of Repeated Measures. Chapman and Hall, London. 28. Catzeflis, C., Pierroz, D. D., Rohner-Jeanrenaud, F., Rivier, J. E., Sizonenko, P. C., and Aubert, M. L. (1993) Neuropeptide Y administered chronically into the lateral ventricle profoundly inhibits both the gonadotropic and the somatotropic axis in intact adult female rats. Endocrinology 132, Small, C. J., Kim, M. S., Stanley, S. A., Mitchell, J. R., Murphy, K., Morgan, D. G., Ghatei, M. A., and Bloom, S. R. (2001) Effects of chronic central nervous system administration of agouti-related protein in pair-fed animals. Diabetes 50, Weigle, D. S. (1994) Appetite and the regulation of body composition. FASEB J. 8, Aja, S., Sahandy, S., Ladenheim, E. E., Schwartz, G. J., and Moran, T. H. (2001) Intracerebroventricular CART peptide reduces food intake and alters motor behavior at a hindbrain site. Am. J. Physiol. 281, R1862 R Scheidegger, K., O'Connell, M., Robbins, D. C., and Danforth, E., Jr. (1984) Effects of chronic beta-receptor stimulation on sympathetic nervous system activity, energy expenditure, and thyroid hormones. J. Clin. Endocrinol. Metab. 58, Haynes, W. G., Morgan, D. A., Djalali, A., Sivitz, W. I., and Mark, A. L. (1999) Interactions between the melanocortin system and leptin in control of sympathetic nerve traffic. Hypertension 33, Billington, C. J., Briggs, J. E., Grace, M., and Levine, A. S. (1991) Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. Am. J. Physiol. 260, R321 R Bing, C., Pickavance, L., Wang, Q., Frankish, H., Trayhurn, P., and Williams, G. (1997) Role of hypothalamic neuropeptide Y neurons in the defective thermogenic response to acute cold exposure in fatty Zucker rats. Neuroscience 80, Bing, C., Frankish, H. M., Pickavance, L., Wang, Q., Hopkins, D. F., Stock, M. J., and Williams, G. (1998) Hyperphagia in cold-exposed rats is accompanied by decreased plasma leptin but unchanged hypothalamic NPY. Am. J. Physiol. 274, R62 R Elias, C. F., Lee, C. E., Kelly, J. F., Ahima, R. S., Kuhar, M., Saper, C. B., and Elmquist, J. K. (2001) Characterization of CART neurons in the rat and human hypothalamus. J. Comp. Neurol. 432, Charnay, Y., Perrin, C., Vallet, P. G., Greggio, B., Kovari, E., and Bouras, C. (1999) Mapping of cocaine and amphetamine regulated transcript (CART) mrna expression in the hypothalamus of elderly human. J. Chem. Neuroanat. 17,

15 39. Edwards, C. M., Abbott, C. R., Sunter, D., Kim, M., Dakin, C. L., Murphy, K. G., Abusnana, S., Taheri, S., Rossi, M., and Bloom, S. R. (2000) Cocaine- and amphetamineregulated transcript, glucagon-like peptide-1 and corticotrophin releasing factor inhibit feeding via agouti-related protein. Brain Res. 866, Challis, B. G., Yeo, G. S., Farooqi, I. S., Luan, J., Aminian, S., Halsall, D. J., Keogh, J. M., Wareham, N. J., and O'Rahilly, S. (2000) The CART gene and human obesity: mutational analysis and population genetics. Diabetes 49, del Giudice, E. M., Santoro, N., Cirillo, G., D'Urso, L., Di Toro, R., and Perrone, L. (2001) Mutational screening of the CART gene in obese children: identifying a mutation (Leu34Phe) associated with reduced resting energy expenditure and cosegregating with obesity phenotype in a large family. Diabetes 50, Received December 4, 2002; accepted May 27, 2003.

16 Table 1 Effect of repeated CART (55 102) intra-arcuate injection (Study 1) and arcuate nucleus CART gene transfer (Studies 2A and 2B) on pituitary and plasma hormones Study 1 Study 2A Study 2B Repeat CART CART/PEI CART/PEI 7 days 25 days 10 days CART Control CART Control (n=7) (n=10) (n=9) (n=10) Control (n=8) CART (n=10) TSH (ng/ml) 4.2 ± ± ± ± 0.3* 3.7 ± ± 0.3* Total T4 (µg/dl) 2.4 ± ± ± ± ± ± 0.2 Free T3 (µg/dl) 2.3 ± ± ± ± ± ± 0.3 *P<0.05

17 Fig. 1 Figure 1. PEI-mediated gene transfer results in localized transgene expression. Rats were injected with 1 µg pcdna-cart via a temporary cannula projecting into the arcuate nucleus. Brains were collected on day 10 post-gene transfer. Representative photomicrographs of in situ hybridization performed on 15 µm sections by using 35 S labeled (a) antisense and (b) sense (control) CART riboprobes. Arrow indicates CART mrna localized unilaterally in arcuate nucleus. Arrowhead indicates unilateral sense riboprobe binding to CART cdna. Arc: arcuate nucleus. Hipp: hippocampus.

18 Fig. 2 Figure 2. Effect of intra-arcuate CART (55-102) injection on food intake. a) Acute administration. Satiated rats were injected with 0.2 nmole CART (55 102) (n=10) or saline (n=10), and food intake measured at 1, 2, and 4 h post-injection. Black bars, CART (55 102) group; white bars, control. Effect of repeated administration on (b) cumulative daytime food intake and (c) cumulative total daily food intake. Rats were injected twice daily with 0.2 nmole of CART (55 102) (=7) or saline (controls, n=8) for 7 days. Closed circles: CART (55 102) treated group. Open triangles: control group. **P<0.01, ***P<0.001 by three-way ANOVA.

19 Fig. 3 Figure 3. Effect of repeated intra-arcuate CART (55 102) on BAT ( a) thermogenesis and (b) UCP-1 mrna. Rats were injected twice daily with 0.2 nmole of CART (55 102) as for Fig. 2b. Thermogenic response (mean change in rectal temperature between 0 90 min) to BRL (40 µg/kg) measured on day 7. UCP-1 mrna quantified by RPA on day 8. Black bars, CART (55 102) group; white bars, controls. *P<0.05 by unpaired t-test.

20 Fig. 4 Figure 4. Effect of chronic arcuate nucleus CART overexpression on (a) 4-day food intake during ad lib feeding and following 24 h food withdrawal (b) 24 h weight loss following 24 h food withdrawal (day 10 post-gene transfer) and 80% food restriction (day 16 post-gene transfer). c) Interscapular BAT UCP-1 mrna levels (collected day 25), mrna quantified by RPA. Rats were injected with pcdna-cart (n=9) or pcdna1.1 (controls, n=10). Black bars, pcdna- CART treated animals; white bars, controls. **P<0.02 by unpaired t-test.

21 Fig. 5 Figure 5. Effect of 10 days CART overexpression in the arcuate nucleus on ex-vivo CART release. Rats were injected with pcdna-cart (n=10) or pcdna1.1 (controls, n=10). Day 10: Brains were collected and CART-IR release measured from 1.2 mm medio-basal hypothalamic slices. Slice viability was confirmed by measurement of potassiumstimulated CART-IR release. Black bars, pcdna-cart group (n=10); white bars, controls (n=8). ** P<0.01, *P<0.05 by unpaired t-test.

22 Fig. 6 Figure 6. Effect of 20 days cold exposure on (a) cumulative weight gain and (b) cumulative food intake. Rats were maintained for 20 days at 4 o C (n=8) or 22 o C (n=8). Closed circles represent 4 C group, open triangles represent 22 C group. **P<0.005, ***P<0.001.

23 Fig. 7 Figure 7. Effect of 20 days cold exposure on arcuate nucleus CART mrna expression. Brains were collected from rats (data shown in Fig. 6) for measurement of CART mrna by quantitative in situ hybridization. Representative photomicrograph of 15 µm brain section from (a) animal maintained at 4 C and (b) from animal maintained at 22 C. c) CART mrna expression measured as optical density and expressed as percentage of mean control levels. Black bar, 4 C group; white bar, 22 C group. Arrowhead: arcuate nucleus. Hipp: hippocampus. IIIv: 3 rd ventricle. *P<0.05 by unpaired t- test.