CART peptide: central mediator of leptin-induced adipose tissue apoptosis?

Regulatory Peptides 121 (2004) 155 – 162 www.elsevier.com/locate/regpep

CART peptide: central mediator of leptin-induced adipose tissue apoptosis? Yang-Ho Choi a, Mary Anne Della-Fera a,*, ChangLong Li a, Diane L. Hartzell a, Dianne E. Little a, Michael J. Kuhar b, Clifton A. Baile a,c a

Department of Animal and Dairy Science, University of Georgia, 444 Animal Science Complex, Athens, GA 30602-2771, USA b Yerkes Regional Primate Research Center of Emory University, Athens, GA 30329, USA c Department of Food and Nutrition, University of Georgia, Athens, GA 30602-2771, USA Received 19 January 2004; accepted 5 May 2004 Available online 20 June 2004

Abstract Because of connections between CART peptide containing neurons and the sympathetic nervous system (SNS) and the possible role of the SNS in leptin-induced adipose apoptosis, CART may act as a downstream effector of leptin-induced adipose apoptosis. Male Sprague – Dawley rats received continuous intracerebroventricular (i.c.v.) infusion for 4 days of either artificial cerebrospinal fluid (aCSF, 12 Al/day), leptin (15 Ag/ day), or CART55-102 at 2.4 Ag/day (CART2.4) or 9.6 Ag/day (CART9.6). Food intake (FI) was decreased 10.8% for CART2.4, 41.9% for CART9.6 and 33.4% for leptin ( p < 0.05). CART9.6 and leptin reduced meal size and meal number. Body weight (BW) was reduced by CART9.6 (14.6%) and leptin (11.6%) ( p < 0.05), but not by CART2.4. CART9.6 and CART2.4, but not leptin, caused hypothermia, and CART9.6 inhibited physical activity ( p < 0.05). Epididymal, inguinal and retroperitoneal fat pad weights were reduced ( p < 0.05) by both CART treatments and leptin; CART9.6 also reduced gastrocnemius muscle weight (18.1%, p < 0.05). Leptin, but not CART, increased serum free fatty acid concentrations by 31.1% ( p < 0.05) and increased adipose apoptosis by 48% ( p < 0.05). These data show that although leptin and CART55-102 have some similar actions, CART55-102 is probably not a mediator for leptin-induced adipose apoptosis in the brain. D 2004 Elsevier B.V. All rights reserved. Keywords: Feeding behavior; Body weight; Physical activity; Rats; Cerebral ventricular infusion

1. Introduction CART is one of the most abundantly expressed mRNAs in the rat hypothalamus [1], and neuroanatomical studies have shown that CART mRNA is expressed within hypothalamic areas implicated in the CNS control of feeding behavior and metabolism, including the paraventricular (PVN), arcuate and dorsomedial nuclei (DMN), as well as the lateral hypothalamus (LH) [2 – 4]. Although CART receptors have not yet been identified, c-Fos immunoreactivity was increased in the PVN and DMN after intracerebroventricular (i.c.v.) injection of CART, suggesting that these areas are sites of CART action [5].

* Corresponding author. Tel.: +1-7197425263; fax: +1-2082795568. E-mail address: [email protected] (M.A. Della-Fera). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.05.001

A number of studies indicate that CART peptides act centrally to inhibit feeding [6– 8], and CART may be a downstream effector for specific leptin actions in some areas [6,9]. Although CART antibody administered i.c.v. increased food intake in rats, indicating that endogenous CART plays a physiological role in the control of food intake [6,7], no one has thus far demonstrated that CART antibody increases feeding after leptin administration. Therefore, the complete relationship between CART and leptin-induced food intake is not yet clear. Another interesting possibility is that CART mediates leptin’s stimulatory effect on the sympathetic nervous system (SNS). Leptin decreases body weight in part by activating the sympathetic nervous system, resulting in increased thermogenesis and energy expenditure [10]. Both leptin and CART have been shown to increase uncoupling protein levels in adipose tissue after central administration, an effect that is mediated by the SNS [11,12]. Elias et al. [9] showed that

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leptin activates CART neurons in the retrochiasmatic area (RCA) and lateral arcuate nucleus (Arc) that innervate sympathetic preganglionic neurons in the thoracic spinal cord, suggesting that this pathway may contribute to the increased thermogenesis and energy expenditure and decreased body weight observed following leptin administration. Another potential SNS-mediated effect of leptin is an increase in white adipose tissue apoptosis [13,14]. We have shown that leptin-induced apoptosis is not mediated by melanocortin receptors acting downstream of leptin [15]. Thus, CART may act as leptin’s downstream effector for adipose tissue apoptosis. In this experiment, we compared the effects of CART and leptin, administered for 4 days by continuous i.c.v. infusion, on feeding behavior, spontaneous physical activity, body weight and adipose and muscle tissue weight and adipose tissue apoptosis to explore the possibility that CART is a downstream mediator of these leptin effects.

2. Materials and methods 2.1. Animals and diet Forty 8-week-old male Sprague – Dawley rats (250 – 274 g of initial BW) purchased from Harlan (Indianapolis, IN) were housed in a room with 12-h illumination, 22 F 1 jC ambient temperature, and 50% humidity. The rats had ad libitum access to pelleted standard lab chow (5001, PMI Nutritional International, Brentwood, MO) and water throughout the study. All experimental and surgical procedures in this study were approved by the Animal Care and Use Committee for The University of Georgia. 2.2. Peptides and osmotic pumps Both CART peptide (55-102) (Peptides International, Louisville, KY) and recombinant rat leptin (R&D Systems, Minneapolis, MN) were dissolved in an artificial cerebrospinal fluid (aCSF; http://www.alzet.com/faq/ proto2.htm). Alzet osmotic pumps (Model 1007D; 0.5 ul/h) delivered amounts of 2.4 Ag CART/day, 9.6 Ag CART/day and 15 Ag leptin/day. Pumps were primed overnight before implantation. 2.3. Surgical procedures Unilateral lateral ventricular cannulas (model 3280P/ SPC, Plastics One, Roanoke, VA) were surgically implanted as previously described [15]. A tube containing 12 Al of

aCSF (with the caudal end closed) was connected to the side port of each and buried subcutaneously in the intrascapular space of each rat. This volume of aCSF provided a 24-h lag time before treatment solutions were delivered. In order to measure body temperature (BT), a programmable transponder (IPTT-200TM, BioMedic Data Systems, Seaford, DE) was implanted using a needle-syringe type injector. Following surgery, the rats were allowed to recover to presurgical BW, which was approximately 1 week. 2.4. Design and procedures Following recovery rats were randomly assigned to the four treatments groups. Two days after they were transferred to the TSE behavioral monitoring system (see below), individual rats (n = 9 – 10) of each block were implanted with an osmotic pump containing either aCSF, CART2.4 (2.4 Ag/ day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day). Pumps were connected to the icv tubing. Daily body temperature (BT), body weight (BW), food intake (FI), and water intake (WI) were recorded within 3 h after light onset. Treatment solutions were injected for 4 days (Fig. 1). Rats were killed by CO2 asphyxiation and decapitation. Inguinal (iWAT), epididymal (eWAT), and retroperitoneal (rWAT) white adipose tissues, intrascapular brown adipose tissue (BAT), and gastrocnemius muscle (GC) were removed, weighed, flash frozen in liquid nitrogen and stored at 80 jC. 2.5. Feeding behavior and spontaneous activity measurement Rats were individually housed in cages equipped to automatically measure feeding and drinking behavior and spontaneous physical activity (TSE Systems, Bad Homburg, Germany). The InfraMot activity unit registers activity by sensing the body-heat image, i.e. infra-red radiation, and its spatial displacement over time. Rats were removed from the monitoring system for approximately 4 min at the same time each day for BW and BT measurements. 2.6. Gel electrophoresis apoptosis assay Adipose tissue apoptosis was determined as previously described [16]: Two fractions of fragmented and genomic DNA were isolated from fat tissues. Fragmented DNA was run on an agarose gel in order to identify a ladder pattern of internucleosomal DNA degradation that is characteristic of apoptosis. Apoptosis was quantified as the ratio of fragmented- to total-DNA, multiplied by 100.

Fig. 1. Study design.

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Fig. 2. Total apparent food intake (AFI) (g), total net FI (NFI) (g), and total apparent water intake (AWI) (ml) by rats infused i.c.v. for 4 days with aCSF (12 Al), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/ day) via subcutaneous osmotic pumps. a,b,c,d: Means with different letters are significantly different, p < 0.05. Data are means F S.E.M. (n = 8 – 9).

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Fig. 4. Daily apparent water intake (ml; mean F S.E.M.) by rats infused i.c.v. for 4 days with aCSF (12 Al), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) via subcutaneous osmotic pumps. a,b,c: Within a day, means with different letters are significantly different, p < 0.05. (n = 8 – 9).

2.7. Data processing and statistics Total net food intake (NFI) was estimated at the end of the experiment by subtracting the amounts of spillage and leftover from the amount of food initially provided. Because daily spillage was not measured, the total apparent food intake (AFI), which was cumulatively recorded by the TSE Systems, was compared with the total NFI. Spillage was f 2 g per day when equally distributed, and the difference between the total NFI and total AFI was relatively consistent among individual rats. The results of analysis for NFI and AFI showed the same trends. A meal was defined as food intake of z 0.5 g within 5 min. Meals continued until the termination criteria of a meal was met; i.e. no more than 0.1 g of food was eaten in a subsequent 30-min period. Food intake less than 0.5 g, when not part of a meal, was considered ‘‘nibbling’’. Data for meal duration (min), meal size (g), rate of eating during meals (g/min), and inter-meal interval (min) were recorded. Satiety ratio (min/g), an index of the satiety time produced by each gram of food consumed, was computed by dividing post-meal intervals in minutes with the grams of the

Fig. 3. Daily apparent food intake (g; mean F S.E.M.) by rats infused i.c.v. for 4 days with aCSF (12 Al), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/ day), or leptin (15 Ag/day) via subcutaneous osmotic pumps. a,b,c: Within a day, means with different letters are significantly different, p < 0.05.

preceding meal. Hunger ratio (min/g) is the length of the pre-meal interval (min) divided by the post-meal size (g). 2.8. Statistics Meal patterns and activity were analyzed using threeway ANOVA with repeated measures on one factor. All other data were analyzed by two-way ANOVA. Least square means were used to determine significance of differences between means, where appropriate. Data are expressed as least square means F S.E.M., with consideration of significance at p < 0.05.

3. Results 3.1. Food and water intake, body weight and feeding behavior CART2.4, CART9.6 and leptin caused significant reductions in both cumulative AFI and cumulative NFI (Fig. 2).

Fig. 5. Body weight (g; mean F S.E.M.) in rats infused i.c.v. for 4 days with aCSF (12 Al), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) via subcutaneous osmotic pumps. a,b,c: Within a day, means with different letters are significantly different, p < 0.05 (n = 8 – 9).

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Table 1 Meal size, meal number, meal duration and eating rate in rats infused i.c.v. via osmotic pumps containing aCSF (12 Al/day), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) for 4 days

Meal size (g)

Meal number

Meal duration (min)

Eating rate (g/min)

24 h mean Light Dark 24 h total Light Dark 24 h mean Light Dark 24 h mean Light Dark

aCSF

CART2.4

CART9.6

Leptin

3.17 F 0.11a 3.44 F 0.13a 2.90 F 0.14a 7.60 F 0.49a 3.44 F 0.13a 2.90 F 0.14a 11.09 F 0.52a 11.96 F 0.49a 10.22 F 0.52a 0.35 F 0.05 0.36 F 0.04 0.34 F 0.04

3.01 F 0.13a 3.23 F 0.15a 2.78 F 0.14a 6.40 F 0.51ab 3.23 F 0.15a 2.79 F 0.15a 10.11 F 0.59a 10.65 F 0.58a 9.57 F 0.56a 0.41 F 0.06 0.40 F 0.04 0.41 F 0.04

1.86 F 0.18b 1.80 F 0.18b 1.92 F 0.20b 5.10 F 0.54b 1.80 F 0.18b 1.92 F 0.20b 7.06 F 0.80b 6.96 F 0.68b 7.16 F 0.78b 0.48 F 0.08 0.49 F 0.05 0.45 F 0.06

2.08 F 0.13b 1.97 F 0.15b 2.18 F 0.16b 5.30 F 0.47b 1.97 F 0.15b 2.18 F 0.16b 7.48 F 0.62b 6.61 F 0.57b 8.35 F 0.63b 0.41 F 0.06 0.49 F 0.04 0.34 F 0.05

Data are means F S.E.M. abc Means with different letters within each behavioral parameter are different at p < 0.05.

While FI of CART9.6 and leptin groups remained significantly lower by the end of the study, FI of the CART2.4 treated rats was reduced only on day 1 (Fig. 3). Daily apparent water intake (AWI) was decreased by only the CART9.6 and leptin (Fig. 4), and had recovered to normal by day 4. Overall AWI was significantly reduced in CART9.6 and leptintreated groups ( F(3,24) = 6.9, p = 0.002) (Fig. 2). CART9.6 and leptin, but not CART2.4, significantly reduced BW ( p < 0.05) (Fig. 5). At the end of the study, BW for CART9.6 and leptin groups was reduced by 14.7% and 11.5%, respectively ( F(3,26) = 14.31, p < 0.0001). Leptin and CART9.6 treatments decreased meal size, meal number and meal duration without affecting eating rate (Table 1). These effects occurred during both light and dark phases. Leptin and CART9.6 treatments also increased intermeal interval and hunger and satiety ratios (Table 2). No treatment affected nibbling bouts (data not shown). 3.2. Body temperature

followed by a gradual increase to normal temperature. Shivering and increased muscle rigidity were observed in 7 of 9 rats treated with CART9.6. BT of leptin-treated rats remained normal throughout the study (Fig. 6). In contrast, the decrease in BT with CART2.4 treatment was more gradual, reaching significance on day 4 ( p < 0.033). 3.3. Spontaneous physical activity Spontaneous physical activity (SPA) was significantly reduced in CART9.6-treated rats, but not by CART2.4 or leptin (Fig. 7). The effect of CART on SPA was prominent during the dark period and lasted for 2 days. Overall SPA for the dark period ( F(3,26) = 5.12, p = 0.007) and daily total SPA ( F(3,26) = 3.53, p = 0.03) was significantly reduced in the CART9.6 group (Fig. 8). The reduced SPA levels on day 0 was likely a result of the surgical implantation of the osmotic pumps.

Body temperature (BT) was significantly decreased on day 1 in the CART9.6 group ( F(3,26) = 5.1, p = 0.007),

Table 2 Inter-meal interval, satiety ratio (post-meal interval/preceding meal size) and hunger ratio (pre-meal interval/subsequent meal size) in rats infused i.c.v. via osmotic pumps containing aCSF (12 Al/day), CART2.4 (2.4 Ag/ day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) for 4 days aCSF

CART2.4

CART9.6

Leptin

Inter-meal 129.0 F 21.0a 140.1 F 23.7ab 299.2 F 32.2c 164.2 F 25.0b interval (min) Satiety ratio 54.1 F 4.8a 53.1 F 5.4a 87.7 F 6.6b 96.5 F 5.6b (min/g) Hunger ratio 55.5 F 4.6a 58.0 F 5.2a 97.7 F 6.3b 89.9 F 5.3b (min/g) Data are 4-day means F S.E.M. ab Means with different letters within each behavioral parameter are different, p < 0.05.

Fig. 6. Body temperature (jC; mean F S.E.M.) in rats infused i.c.v. for 4 days with aCSF (12 Al), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) via subcutaneous osmotic pumps. a,b: Within a day, means with different letter are significantly different, p < 0.05 (n = 8 – 9).

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Fig. 7. Daily spontaneous physical activity (SPA; mean F S.E.M.) in rats infused i.c.v. for 4 days with aCSF (12 Al), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) via subcutaneous osmotic pumps. a,b: Within a day, means with different letters are significantly different, p < 0.05 (n = 8 – 9).

3.4. Tissue weights and adipose tissue apoptosis Despite the marked loss of body weight in rats treated with CART9.6, its effect on white adipose tissue (WAT) weight was less prominent. Leptin had the greatest effect on WAT weight, followed by CART9.6 and CART2.4 (Fig. 9). BAT weight was slightly but significantly reduced by both leptin and CART9.6. CART9.6 also reduced gastrocnemius (GC) muscle weight ( F(3,26) = 18.2, p < 0.0001). Leptin increased adipose apoptosis of rWAT compared to all other treatments ( p < 0.05). Apoptosis was increased 48% by leptin compared to control. None of the peptide treatments significantly affected apoptosis of eWAT (Table 3). 3.5. Serum free fatty acid, leptin and insulin concentrations Serum free fatty acid concentrations after 4 days of treatment were significantly increased by leptin, but neither CART treatment had any effect (Table 4). Serum insulin

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Fig. 9. Tissue weight (g; mean F S.E.M.) in rats infused i.c.v. for 4 days with aCSF (12 Al), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) via subcutaneous osmotic pumps. BAT, brown adipose tissue; eWAT, epididymal white adipose tissue (WAT); iWAT, inguinal WAT; rWAT, retroperitoneal WAT; GC, gastrocnemius muscle. Columns with different letters are significantly different from one another at p < 0.05. a,b,c: Within a tissue, means with different letter are significantly different, p < 0.05 (n = 8 – 9).

concentration was decreased by CART9.6 and leptin ( p < 0.05).

4. Discussion The primary objective of this study was to determine if CART administered i.c.v. produced effects similar to leptin on feeding behavior, adipose tissue and body weight. Although rats receiving CART9.6 had decreased food intake and reduced body weight, we observed additional behavioral and physiological effects, similar to those reported previously [6,17 –19], including hypothermia, shivering, increased muscle tension and reduced spontaneous physical activity. Rats receiving CART2.4 had reduced total food intake but did not lose body weight. They did not exhibit the marked behavioral effects seen with the higher dose; however, body temperatures decreased gradually and were significantly reduced by day 4. In contrast, rats receiving continuous i.c.v. injection of leptin had normal body temperature and physical activity, and although they ate less and lost weight and fat, gastrocnemius muscle weight was not affected. We also predicted that if CART acted centrally as a downstream mediator of leptin, then it would induce adipose tissue apoptosis, as we have shown for leptin [14,20]. Table 3 Serum concentrations of free fatty acids and insulin in rats infused i.c.v. via osmotic pumps containing aCSF (12 Al/day), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) for 4 days

Fig. 8. Overall, light and dark period spontaneous physical activity (SPA; mean F S.E.M.) in rats infused i.c.v. for 4 days with aCSF (12 Al), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) via subcutaneous osmotic pumps. a,b: Means with different letters are significantly different, p < 0.05 (n = 8 – 9).

Treatment

Free fatty acids mEq/l

Insulin (ng/ml)

aCSF CART2.4 CART9.6 Leptin

297.5 F 28.4a 222.4 F 29.3a 259.8 F 30.7a 389.5 F 26.9b

1.85 F 0.16a 1.41 F 0.16ab 0.99 F 0.15b 0.25 F 0.15c

Data are means F S.E.M. ab Means with different letters within a column are different, p < 0.05.

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Table 4 Adipose tissue apoptosis (%DNA fragmentation) in rats infused i.c.v. via osmotic pumps containing aCSF (12 Al/day), CART2.4 (2.4 Ag/day), CART9.6 (9.6 Ag/day), or leptin (15 Ag/day) for 4 days Fat pad rWAT eWAT

aCSF

CART2.4 x

1.31 F 0.15 1.29 F 0.36

CART9.6 x

1.76 F 0.14 1.14 F 0.55

Leptin x

1.60 F 0.14 1.22 F 0.47

2.20 F 0.28y 1.94 F 0.35

x,y Within a fat pad, means with different letters are different, p < 0.01. Data are means F S.E.M.

This hypothesis was based on evidence pointing to the probability that leptin-induced adipose apoptosis is a result of increased sympathetic stimulation of adipose tissue [10,13,21], and because leptin has been shown to activate CART-containing neurons in the hypothalamus that innervate preganglionic sympathetic neurons in the thoracic spinal cord [9]. We found, however, that adipose tissue apoptosis was significantly increased only by leptin. Our findings suggest that CART may not mediate leptininduced adipose tissue apoptosis but cannot rule out an involvement in leptin’s effects on feeding behavior and body weight. The divergent distribution of CART and leptin receptors could result in some common actions and some independent actions, as well. The genetic evidence that CART can influence body weight in humans is strong. A genetic missense mutation in the CART reading frame in an Italian family results in early onset obesity with a reduction in metabolic rates [22], and a mutation in the CART promoter region in a Japanese population is associated with obesity [23]. Furthermore, mice with CART gene knockout gain significantly more weight on high fat diets than wild type mice [24], and the anatomical distribution of CART in rodents is compatible with a role in feeding [3,4,25 – 28]. Leptin’s regulation of CART expression in relevant areas is also noteworthy [6,29,30]. Other studies in animals, however, have been less compelling, apparently contradicting, and may reflect various aspects of species differences. Although injection of CART into the cerebral ventricles, arcuate or paraventricular nucleus produces a reduction in feeding [6,30 – 32], CART has also been shown to stimulate feeding when injected into the ventromedial hypothalamic nucleus (VMN) or into the arcuate nucleus [33]. In addition, i.c.v. injection of CART has been shown to produce motor abnormalities [6,17,19,33] and a conditioned taste aversion [34]. These effects may reflect regional activities of CART peptides, and one may need to look to the knockouts and genetic findings to obtain evidence for a more global role for CART. At present, the precise site and mechanisms of CART’s role in body weight and feeding is not clear. The data in this study show that not all actions of leptin can be produced by CART and vice versa. However, because of the complex and overlapping neuropeptide systems involved in the control of food intake and regulation of energy balance, CART may be only one player in an orchestra of neuropeptides mediating leptin’s effects in

the brain, particularly since leptin has also been shown to decrease NPY expression and increase POMC expression in the ARC [35,36]. In a previous study, we found that the POMC-derived peptide, aMSH may mediate leptin’s effects on food intake and body weight, but not adipose tissue apoptosis [15]. Thus, both aMSH and CART may only be partial leptin effector peptides. The hypothermia caused by i.c.v. CART administration has not been previously reported and was unexpected, given earlier reports of CART-induced increase in uncoupling protein levels and brown adipose tissue thermogenesis [11,32]. Other peptides have been shown to produce hypothermia after central administration, including orexin a [37], cholecystokinin-8 [38], neuromedin c [39], neurotensin [40], bombesin [41,42] and NPY [37,43,44]. CART has interesting connections with NPY-containing neurons, and these CART-NPY connections could be involved in CART-induced hypothermia and decreased physical activity. Both CART and NPY decrease spontaneous activity and body temperature and increase muscle tone when administered i.c.v. [6,17 –19,45,46]. For NPY these effects occurred predominantly after injection directly into the ARC [44]. Dhillo et al. [47] showed that in hypothalamic explants, CART caused NPY release and NPY induced CART release. Thus, it is possible that interaction between these two neuropeptides is involved in some of the varied responses seen in this study and reported by others. In summary, our findings suggest that CART does not act as a central mediator of leptin-induced adipose tissue apoptosis. Given the complex and site-specific activities of CART, however, it may be premature to draw firm conclusions about CART’s role as a downstream mediator of all of leptin’s actions.

Acknowledgements Supported in part by the Georgia Research Alliance Eminent Scholar endowments held by CAB and MJK, NIDA grant # DA00418 to MJK, and a Georgia Research Alliance Challenge Grant to CAB and MJK.

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