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MTII administered peripherally reduces fat without invoking apoptosis in rats
Physiology & Behavior 79 (2003) 331 – 337
MTII administered peripherally reduces fat without invoking apoptosis in rats Yang-Ho Choi, ChangLong Li, Diane L. Hartzell, Ji Lin, Mary Anne Della-Fera, Clifton A. Baile* Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602-2771, USA Department of Food and Nutrition, University of Georgia, Athens, GA 30602-2771, USA Received 28 December 2002; received in revised form 4 March 2003; accepted 28 April 2003
Abstract The melanocortin (MC) system in the brain is believed to be an important downstream effector of leptin signaling; interference with MC functioning results in severe obesity. Melanotan II (MTII), an MC3/4-receptor agonist, produces similar behavioral and metabolic outcomes to those observed after leptin treatments, which enhance apoptosis in specific fat depots. To determine whether MTII also mediates adipose apoptosis induced by leptin treatment, two groups of rats (n = 8) received MTII (2 mg/kg, ip) or saline (2 ml/kg) once daily for 4 days and had free access to food and water, and a third group was injected with saline and pair-fed (PF) to MTII treated rats. Food intake, water intake, body temperature, and body weight were measured daily. MTII reduced food and water intake and body weight gain ( P < .05) and decreased body temperature compared to PF and saline-treated control groups. Retroperitoneal white adipose tissue (WAT) mass and epididymal WAT mass were reduced 46.3% and 21.1%, respectively ( P < .05), after MTII, but not after PF, compared with the saline control rats. Both MTII(25.0%) and PF (33.3%)-treated rats had decreased brown fat weight ( P < .05), whereas muscle mass remained unchanged. Free fatty acid concentrations in serum were not different between MTII and control groups, but increased by 56.4% in PF group. DNA fragmentation assay did not support a role for MTII as an apoptotic signal in any of the fat tissues tested. These results show that in addition to reducing food intake and inhibiting body weight gain, intraperitoneal administration of MTII reduces fat mass, most likely by accelerated lipid mobilization, but not by apoptosis. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Food intake; Body weight; Body temperature; Adipose apoptosis; Lipolysis; Melanocortin; Melanotan II; Leptin; Free fatty acids; Insulin
1. Introduction The proopiomelanocortin (POMC) gene encodes for various peptides, such as the melanocortin peptides a-, b-, and g-melanocyte stimulating hormone (MSH), as well as adrenocorticotropin and b-endorphin. a-MSH functions as a primary ligand on its receptors. Two of the melanocortin (MC) receptors, MC3-R and MC4-R, are predominantly located in the brain and play a key role in food intake and body weight regulation [1,2]. Antagonism or stimulation of the MC system results in increase or decrease, respectively, in food intake in several experimental models [3], and null mutations in the POMC gene cause obesity in humans and * Corresponding author. 444 Animal Science Complex, University of Georgia, Athens, GA 30602-2771, USA. Tel.: +1-706-542-4094; fax: +1706-542-7925. E-mail address: [email protected] (C.A. Baile).
mice [4,5]. Furthermore, mice that lack specific melanocortin receptors (MC3-R or MC4-R) also develop obesity [6– 8]. The MC system exerts a variety of endocrine and metabolic effects. Treatments with melanotan II (MTII), a synthetic agonist of MC3/4-R, decreased circulating leptin and insulin concentrations and increased oxygen consumption in rats fed a highly palatable diet [9], as well as in dietinduced obese mice [10]. MTII dose-dependently suppressed food intake, body weight, and respiratory quotient in both Zucker obese and lean rats, but the obese rats were more sensitive to MTII’s inhibitory effect on body weight [11]. In contrast, antagonism of the MC system using SHU9119 for 11 days increased daily food intake, body weight, and total fat mass and increased serum concentrations of corticosterone, insulin, and leptin [12]. The MC system has functional interactions with leptin. Leptin selectively activated POMC neurons within the arcuate nucleus via MC3-R and MC4-R [13 – 15]. Food intake
0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0031-9384(03)00118-5
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reduction induced by intracerebroventricular injection of leptin was completely prevented by co-injection of the MC3/4-R antagonist SHU9119 in male Sprague – Dawley rats [16], and leptin’s effects were significantly attenuated in obese MC4-R-deficient mice but not in non-obese MC4-Rdeficient mice [17]. Furthermore, intraperitoneal administration of MTII reduced food intake and body weight in both lean and obese Zucker rats, with stronger effects in the latter [18] and in C57BL/6J mice in a dose-dependent manner [10]. Thus, these findings support the contention that the anorexic effect of leptin is mediated at least in part by the MC system in the brain. One of the distinctive actions of leptin is its stimulatory effect on apoptosis in adipose tissues [19] while sparing lean body mass [8]. Although there are numerous similarities in leptin and MTII actions [18], the effect of the MC system on apoptosis in fat tissues has not been previously investigated. In the present study, rats were used to test the hypothesis that intraperitoneal injection of MTII for 4 days would induce apoptosis in adipose tissues in rats.
2. Materials and methods 2.1. Animals Twenty eight male Sprague –Dawley rats (200 –224 g) purchased from Harlan, (Indianapolis, IN) were individually housed in hanging wire-mesh-stainless steel cages in a room with controlled lighting (lights on 0600– 1800 h and off 1800– 0600 h), 22 ± 1 C ambient temperature, and 50% humidity. They had free access to ground PMI Rat Diet 5012 (PMI Nutritional International, Richmond, IN) and water from bottles throughout the study. All procedures were approved by the Animal Care and Use Committee for the University of Georgia. 2.2. Adaptation Three days after delivery, the rats were acclimated to the experimental procedures. During this period, all rats were injected intraperitoneally once a day with saline (2 ml/kg) using 1-ml syringes with 3/8-in. 26G needles. Four days later, each rat was subcutaneously implanted in the scapular region with a programmable transponder (IPTT-200TM, BioMedic Data Systems, Seaford, DE) to measure body temperature. Two days before the trial was initiated, food intake and water intakes were added to these daily measurements. 2.3. Peptide preparation MTII (Cat. #68-1-30; American Peptide, Sunnyvale, CA) was diluted in 0.9% saline (Phoenix Pharmaceutical, St. Joseph, MO) at a concentration of 1 mg/ml, based on the manufacturer’s stated protein content.
2.4. Experimental design and procedure On day 10, 24 rats were selected, blocked by body weight and randomly assigned to three treatment groups (n = 8). Saline (2 ml/kg) or MTII (2 mg/2 ml/kg) were administered as intraperitoneal bolus injections to the two groups once daily at 1700 h for 4 successive days. The dose of MTII was based on that used in previous studies [18]. The pair-fed (PF) group was also injected with saline (2 ml/kg rat). A calculated amount of food was given to the PF rats based on the intake of the MTII-treated rats from the previous 24 h. The following calculation was used to determine the amount of food provided to the PF rats: the average food intake divided by the average body weight 24 h after MTII injection multiplied by the individual body weight of rats assigned within this experiment [20]. The PF rats received the daily allotment of food at 1800 h when dark phase started. 2.5. Tissue collection Five days after the initiation of treatment, a final body temperature, food intake, water intake, and body weight measurements were recorded between 0800 and 0900 h, and the rats were euthanized by CO2. The intrascapular brown adipose tissue (iBAT), white adipose tissues (WAT; retroperitoneal [rWAT], epididymal [eWAT], and inguinal [iWAT] fat pads), and the gastrocnemius muscle (GC) were collected. Both left and right sides of each fat pad and muscle were pooled, weighed, frozen in liquid nitrogen, and stored at 80 C until assayed. 2.6. Blood chemical assay Serum concentrations of insulin and leptin were determined by RIA according to the manufacturer’s instruction (Linco Research, St. Charles, MO). Serum free fatty acid (FFA) concentrations were assayed using an NEFA C kit (Cat. #: 994-75409; Wako, Richmond, VA). 2.7. Gel electrophoresis apoptosis assay Apoptosis was assayed in two ways: DNA isolated from fat tissues was separated into two fractions: fragmented and genomic DNA. First, the fragmented DNA was run on an agarose gel in order to identify a ladder pattern of internucleosomal DNA degradation that is characteristic of apoptosis [19]. Second, apoptosis was quantified as the ratio of fragmented to total DNA, multiplied by 100 [21]. Briefly, approximately 150 mg of the epididymal, inguinal, or retroperitoneal WATs were homogenized in lysis buffer (10 mM Tris – HCl, pH 8.0; 10 mM EDTA, pH 8.0; 0.5% Triton X100) and centrifuged at 14,000 g for 15 min to separate fragmented DNA from genomic DNA. The supernatant, containing fragmented DNA, was extracted with phenol – chloroform – isoamyl alcohol (25:24:1), and the DNA was
Y.-H. Choi et al. / Physiology & Behavior 79 (2003) 331–337
Fig. 1. Changes in food intake in response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Food intake was measured at 1700 h on days 1 – 4 and at 0900 h on day 5. Arrow indicates treatments. Means with different letters are significantly different at P .05. Data show mean ± S.E.M. (n = 8).
precipitated by adding polyacryl carrier (Molecular Research Center, Cincinnati, OH) and ethanol. Genomic (nonfragmented) DNA was extracted from the pellet with DNAzol and the polyacryl carrier. DNA in each fraction was quantified by the PicoGreen method (Molecular Probes, Eugene, OR), and fluorescence was measured using a SpectroMax Gemini (Molecular Devices). 2.8. Statistics Data were analyzed by a two-way ANOVA with repeated measures for behavioral data and a one-way ANOVA for tissue and apoptosis data sets, followed by post hoc mean
Fig. 2. Changes in water intake in response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Water intake was measured at 1700 h on days 1 – 4 and at 0900 h on day 5. Arrow indicates treatments. Means with different letters are significantly different at P .05. Data show mean ± S.E.M. (n = 8).
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Fig. 3. Changes in body weight in response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Body weight was measured at 1700 h on days 1 – 4 and at 0900 h on day 5. Arrow indicates treatments. Means with different letters are significantly different at P .05. * P=.0525. Data show mean ± S.E.M. (n = 8).
comparisons between groups. Data are expressed as mean ± S.E.M. and considered significant at P .05.
3. Results As shown in Fig. 1, MTII administration resulted in a large reduction in food intake [ F(2,21) = 73.39, P=.0001]. Food intake was consistent over the 4 days except on the 5th day, during which it was lower because the mealtime interval was shortened to 16 h. On this day, the MTII group ate 7.7 g less than saline control ( P < .05).
Fig. 4. Changes in body temperature (C) in response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/ kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Body temperature was measured 1 and 2 h after injections via transponders implanted subcutaneously; results shown were obtained on day 1. Arrow indicates treatments. Means with different letters are significantly different at P .05. Data show mean ± S.E.M. (n = 8).
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Table 1 Changes in body temperature (C) at 1 and 2 h after intraperitoneal injections of MTII (2 mg/kg) to male Sprague – Dawley rats once daily for 4 daysa,b Treatment
Day 1
Day 2
0h
1h
2h
0h
1h
2h
Saline Pair-fed MTII
37.7 ± 0.1 37.7 ± 0.1 37.7 ± 0.1
38.2 ± 0.1a 38.0 ± 0.1a 37.2 ± 0.3b
38.2 ± 0.1a 38.2 ± 0.2a 37.3 ± 0.2b
37.7 ± 0.1 38.1 ± 0.1 37.9 ± 0.1
38.0 ± 0.1 38.3 ± 0.2 38.0 ± 0.1
38.2 ± 0.1a 38.6 ± 0.1b 38.2 ± 0.1a
Day 3
Saline Pair-fed MTII a b
Day 4
0h
1h
2h
0h
1h
2h
37.9 ± 0.2 38.3 ± 0.1 37.9 ± 0.1
37.9 ± 0.1 38.3 ± 0.1 37.8 ± 0.2
38.4 ± 0.1ab 38.6 ± 0.1a 38.2 ± 0.1b
38.1 ± 0.1a 37.5 ± 0.1b 38.0 ± 0.1a
38.1 ± 0.1 38.3 ± 0.1 37.9 ± 0.2
38.5 ± 0.1 38.1 ± 0.1 38.4 ± 0.1
Means with different letters within a column are different at P < .05. Data are mean ± S.E.M. (n = 8 each treatment).
Total water intake was significant reduced by MTII [ F(2,21) = 4.56, P=.023]. Water intake after MTII treatment was reduced compared with that after saline except for day 4,
but daily water intake in the PF group was variable (Fig. 2). Total water intake-to-food intake ratio (%) was 157.1 ± 9.7 for saline, 191.6 ± 28.9 for PF, and 187.6 ± 21.6 for MTII. Body weight was decreased in both MTII and PF groups (Fig. 3). Consistent with this, overall ANOVA showed a significant treatment effect [ F(2,21) = 3.68, P=.043], day effect [ F(4,84) = 26.0, P=.0001] and treatment day interaction [ F(4,84) = 35.7, P=.0001]. On day 5, body weight of the saline-treated rats was 28.8 g greater than that of the MTII and 24.6 g greater than that of the PF-treated rats, respectively ( P < .05). Body temperature was unexpectedly reduced after MTII administration on day 1 ( P < .05), followed by inconsistent changes over the next 3 days (Fig. 4 and Table 1). In contrast, body temperature was increased in both PF and saline groups after saline injection. Overall, ANOVA revealed a significant treatment effect at 1 h [ F(2,21) = 7.57, P=.004] and 2 h [ F(2,21) = 5.35, P=.014]. The temperature measured in the morning of the last day was not incorporated into these analyses due to the different time of measurement, but it was not different from that measured at 1700 h on previous days. Compared to saline-treated rats (0.81 ± 0.07 g), rWAT mass was reduced by 46.3% (0.43 ± 0.06 g; P < .05) in MTII-treated rats, but not in PF rats (15.4%; 0.68 ± 0.10 g; P>.05), (Fig. 5C). In contrast, a smaller but significant reduction in eWAT mass was seen in MTII- (21.1%, 1.78 ± 0.09 g, P < .05) but not in PF-treated rats (6.1%, Table 2 lnsulin, leptin, and free fatty acid concentrations in serum collected from rats treated with MTII (2 mg/kg, ip), pair-fed, or saline for 4 days
Fig. 5. Tissue response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Tissues were collected between 16 and 18 h after the last injections of MTII. (A) Epididymal white adipose tissue (WAT), (B) inguinal WAT, (C) retroperitoneal WAT, (D) intrascapular brown adipose tissue (BAT), (E) total WAT, and (F) gastrocnemius muscle. Means with different letters are significantly different at P .05. Data show mean ± S.E.M. (n = 8).
Treatment
Insulin (ng/ml)
Leptin (ng/ml)
Free fatty acids (meq/l)
MTII Pair-fed Saline
1.04 ± 0.10a 1.13 ± 0.10a 1.75 ± 0.15b
ND ac 0.27 ± 0.13a 1.43 ± 0.22b
232.7 ± 20.0a 394.2 ± 40.2b 252.1 ± 24.6a
Data are means ± S.E.M.; n = 8 rats/treatment. ab Treatment means within a column denoted with different letters are different, P < .05. c ND: below the detection limit (0.5 ng/ml).
Y.-H. Choi et al. / Physiology & Behavior 79 (2003) 331–337
2.12 ± 0.09 g, P>.05), compared with saline-treated rats (2.26 ± 0.08 g). iBAT mass was reduced in both MTII (25.0%, 0.29 ± 0.02 g) and PF (33.3%, 0.26 ± 0.02 g) groups ( P < .05), compared with saline (0.38 ± 0.02 g), but there were no significant treatment effects for either iWAT or muscle mass ( P>.05) (Fig. 2). Serum insulin [ F(2,21) = 10.25, P < .001] and leptin [ F(2,21) = 26.43, P < .0001] concentrations of rats were reduced by both MTII and PF treatments compared with those of rats receiving saline treatment (Table 2). Serum leptin concentrations in leptin-treated rats were below the detection limit of 0.5 ng/ml. The mean concentrations of both serum insulin and leptin of MTII and PF groups of rats were not different. Serum FFA concentrations were increased only in the PF group of rats compared with those from both the MTII and control groups of rats [ F(2,21) = 8.90, P < .001]. To test the hypothesis that the mechanism for reduction in WAT mass included apoptosis, the three tissues were analyzed for DNA fragmentation, an indicator of apoptosis [19]. There were no differences in percent fragmented DNA in any WAT tissue: eWAT [ F(2,21) = 0.61, P=.55], iWAT
Fig. 6. Percent fragmented DNA in the epididymal white adipose tissue (eWAT) (A), inguinal WAT (iWAT) (B), and retroperitoneal WAT (rWAT) (C) collected from rats injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Data show mean ± S.E.M. (n = 8 except for MTII of iWAT and rWAT, where n = 7).
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[ F(2,20) = 0.59, P=.57], and rWAT [ F(2,20) = 0.57, P=.57] (Fig. 6A –C), respectively.
4. Discussion MTII has been shown to have a consistently strong inhibitory effect on food intake, following either central [10,11,22– 25] or peripheral administration [10,18], or a combination of both [24]. In the present study, a large reduction in food intake and body weight gain was observed in rats treated with MTII or PF. However, although body weights of PF- and MTII-treated rats were not different, MTII treatment resulted in significantly greater reductions in eWAT and rWAT mass compared to pair-feeding. Because of previous findings of adipose tissue apoptosis following central [19] or peripheral injection [26] of leptin, and studies indicating that a-MSH acts as a downstream effector of some of leptin’s central effects [14,15,27], we hypothesized that the reduction in WAT mass following MTII treatment might be a result of increased adipocyte apoptosis. To test this hypothesis, we quantitated the percent of fragmented DNA in the three white fat depots (epididymal, inguinal, and retroperitoneal) as an indicator of apoptosis [21,28]. Our results showed that MTII administered intraperitoneally failed to induce apoptosis in fat tissues. Although it is possible that the time at which tissues were collected had passed a peak of apoptosis, in previous studies in which centrally administered leptin was shown to induce adipose apoptosis, tissue collection was carried out 24 h after the last leptin treatment [19,29]. In the present study, we collected tissues 16 –19 h after the last treatments of MTII. Thus, it is unlikely that the time interval is the reason we failed to observe apoptosis. The results of this study suggest that the MC signaling that governs food intake and body weight is independent of the leptin signal cascade in the brain that leads to apoptosis in adipose tissues. The reduced adipose tissue mass in the fat pads and the lack of evidence of increased apoptosis in our MTII-treated rats suggest that the reduction of adipose tissue mass was a result of lipolysis alone. In analyzing blood samples taken 19 h after the last intraperitoneal injection, we found that MTII treatment prevented the expected increase in serum FFA concentrations resulting from the reduced food intake, as was observed in the PF rats. In contrast, neither leptin nor insulin concentrations were different between MTII and PF groups. Consistent with our findings, a recent study reported no increase of FFA concentrations in blood samples 6 h after the last intraperitoneal injection of MTII [18]. These results are in contrast, however, to results reported from both lean and ob/ob mice sampled 1 h following intraperitoneal injections of an MSH analog, [Ac – Cys4, D-Phe7, Cys10] a-MSH [30]. Following this shorter interval after the injections in mice, FFA concentrations were increased in serum. The increased FFA concentrations occurring shortly
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after the MSH analog treatment may have activated mechanism(s) that result in increased FFA utilization, thus leading to reduced FFA concentrations compared with those in PF rats. This is supported by the demonstration of normal mRNA expression for uncoupling protein-1, -2, and -3 in BAT of MTII-treated rats, whereas in PF rats, uncoupling protein mRNA expression was significantly suppressed [18]. Furthermore, rats treated with MTII had normal mRNA expression in liver and WAT of carnitine palmitoyltransferase-1, the enzyme responsible for transferring longchain fatty acids into the mitochondria, whereas PF rats showed a reduction in expression of this key enzyme [18]. This clearly supports the contention that there is a continuation of energy utilization from fat tissues despite reduced body weight and the lack of the expected increased serum FFA concentration. We conclude, therefore, that our findings support those of previous studies showing that peripheral treatment with MTII, a-MSH, or its analogues resulted in strong lipolytic effects in mice and rats [18,30]. Our observation of a hypothermic rather than hyperthermic effect of MTII was unexpected. Intracerebroventricular administration of MTII resulted in an increase in abdominal temperature in Sprague – Dawley rats [23] and increased rectal temperature in ob/ob mice [24]. However, in these studies, MTII was administered centrally, and the times and methods of temperature measurement were different from those in our study. These methodological differences may account for the difference in results between our study and theirs. It should also be noted that in addition to a strong lipolytic effect, systemic a-MSH has been shown to have a strong antipyretic effect in endotoxin-treated rats. Because this effect was blocked by central administration of SHU9119, an MC3/4-R antagonist [31], the suppressive effect on body temperature appears to have been mediated by a specific MC receptor. The finding that MTII treatments inhibited water consumption is consistent with previous findings in rats injected intracerebroventricularly with MTII [22,23], but differs from the response of ob/ob mice to MTII [24]. In the mouse study both intraperitoneal and intracerebroventricular injections of MTII failed to inhibit 10-day cumulative water consumption [24]. Whether this is due to a species difference or to an absence of leptin in these mice, is unknown. In conclusion, the present study shows that in contrast to the effects of either centrally or peripherally administered leptin on adipose apoptosis, peripherally administered MTII reduces the mass of specific WAT depots without causing apoptosis. These findings, therefore, suggest that it is unlikely that a-MSH is a CNS downstream effector for leptin-induced adipose apoptosis.
Acknowledgements The authors acknowledge the technical assistance of Sherry Hulsey and Brent Jackson. This study was supported
in part by the Georgia Research Alliance Eminent Scholar endowment held by CAB.
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MTII administered peripherally reduces fat without invoking apoptosis in rats Yang-Ho Choi, ChangLong Li, Diane L. Hartzell, Ji Lin, Mary Anne Della-Fera, Clifton A. Baile* Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602-2771, USA Department of Food and Nutrition, University of Georgia, Athens, GA 30602-2771, USA Received 28 December 2002; received in revised form 4 March 2003; accepted 28 April 2003
Abstract The melanocortin (MC) system in the brain is believed to be an important downstream effector of leptin signaling; interference with MC functioning results in severe obesity. Melanotan II (MTII), an MC3/4-receptor agonist, produces similar behavioral and metabolic outcomes to those observed after leptin treatments, which enhance apoptosis in specific fat depots. To determine whether MTII also mediates adipose apoptosis induced by leptin treatment, two groups of rats (n = 8) received MTII (2 mg/kg, ip) or saline (2 ml/kg) once daily for 4 days and had free access to food and water, and a third group was injected with saline and pair-fed (PF) to MTII treated rats. Food intake, water intake, body temperature, and body weight were measured daily. MTII reduced food and water intake and body weight gain ( P < .05) and decreased body temperature compared to PF and saline-treated control groups. Retroperitoneal white adipose tissue (WAT) mass and epididymal WAT mass were reduced 46.3% and 21.1%, respectively ( P < .05), after MTII, but not after PF, compared with the saline control rats. Both MTII(25.0%) and PF (33.3%)-treated rats had decreased brown fat weight ( P < .05), whereas muscle mass remained unchanged. Free fatty acid concentrations in serum were not different between MTII and control groups, but increased by 56.4% in PF group. DNA fragmentation assay did not support a role for MTII as an apoptotic signal in any of the fat tissues tested. These results show that in addition to reducing food intake and inhibiting body weight gain, intraperitoneal administration of MTII reduces fat mass, most likely by accelerated lipid mobilization, but not by apoptosis. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Food intake; Body weight; Body temperature; Adipose apoptosis; Lipolysis; Melanocortin; Melanotan II; Leptin; Free fatty acids; Insulin
1. Introduction The proopiomelanocortin (POMC) gene encodes for various peptides, such as the melanocortin peptides a-, b-, and g-melanocyte stimulating hormone (MSH), as well as adrenocorticotropin and b-endorphin. a-MSH functions as a primary ligand on its receptors. Two of the melanocortin (MC) receptors, MC3-R and MC4-R, are predominantly located in the brain and play a key role in food intake and body weight regulation [1,2]. Antagonism or stimulation of the MC system results in increase or decrease, respectively, in food intake in several experimental models [3], and null mutations in the POMC gene cause obesity in humans and * Corresponding author. 444 Animal Science Complex, University of Georgia, Athens, GA 30602-2771, USA. Tel.: +1-706-542-4094; fax: +1706-542-7925. E-mail address: [email protected] (C.A. Baile).
mice [4,5]. Furthermore, mice that lack specific melanocortin receptors (MC3-R or MC4-R) also develop obesity [6– 8]. The MC system exerts a variety of endocrine and metabolic effects. Treatments with melanotan II (MTII), a synthetic agonist of MC3/4-R, decreased circulating leptin and insulin concentrations and increased oxygen consumption in rats fed a highly palatable diet [9], as well as in dietinduced obese mice [10]. MTII dose-dependently suppressed food intake, body weight, and respiratory quotient in both Zucker obese and lean rats, but the obese rats were more sensitive to MTII’s inhibitory effect on body weight [11]. In contrast, antagonism of the MC system using SHU9119 for 11 days increased daily food intake, body weight, and total fat mass and increased serum concentrations of corticosterone, insulin, and leptin [12]. The MC system has functional interactions with leptin. Leptin selectively activated POMC neurons within the arcuate nucleus via MC3-R and MC4-R [13 – 15]. Food intake
0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0031-9384(03)00118-5
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reduction induced by intracerebroventricular injection of leptin was completely prevented by co-injection of the MC3/4-R antagonist SHU9119 in male Sprague – Dawley rats [16], and leptin’s effects were significantly attenuated in obese MC4-R-deficient mice but not in non-obese MC4-Rdeficient mice [17]. Furthermore, intraperitoneal administration of MTII reduced food intake and body weight in both lean and obese Zucker rats, with stronger effects in the latter [18] and in C57BL/6J mice in a dose-dependent manner [10]. Thus, these findings support the contention that the anorexic effect of leptin is mediated at least in part by the MC system in the brain. One of the distinctive actions of leptin is its stimulatory effect on apoptosis in adipose tissues [19] while sparing lean body mass [8]. Although there are numerous similarities in leptin and MTII actions [18], the effect of the MC system on apoptosis in fat tissues has not been previously investigated. In the present study, rats were used to test the hypothesis that intraperitoneal injection of MTII for 4 days would induce apoptosis in adipose tissues in rats.
2. Materials and methods 2.1. Animals Twenty eight male Sprague –Dawley rats (200 –224 g) purchased from Harlan, (Indianapolis, IN) were individually housed in hanging wire-mesh-stainless steel cages in a room with controlled lighting (lights on 0600– 1800 h and off 1800– 0600 h), 22 ± 1 C ambient temperature, and 50% humidity. They had free access to ground PMI Rat Diet 5012 (PMI Nutritional International, Richmond, IN) and water from bottles throughout the study. All procedures were approved by the Animal Care and Use Committee for the University of Georgia. 2.2. Adaptation Three days after delivery, the rats were acclimated to the experimental procedures. During this period, all rats were injected intraperitoneally once a day with saline (2 ml/kg) using 1-ml syringes with 3/8-in. 26G needles. Four days later, each rat was subcutaneously implanted in the scapular region with a programmable transponder (IPTT-200TM, BioMedic Data Systems, Seaford, DE) to measure body temperature. Two days before the trial was initiated, food intake and water intakes were added to these daily measurements. 2.3. Peptide preparation MTII (Cat. #68-1-30; American Peptide, Sunnyvale, CA) was diluted in 0.9% saline (Phoenix Pharmaceutical, St. Joseph, MO) at a concentration of 1 mg/ml, based on the manufacturer’s stated protein content.
2.4. Experimental design and procedure On day 10, 24 rats were selected, blocked by body weight and randomly assigned to three treatment groups (n = 8). Saline (2 ml/kg) or MTII (2 mg/2 ml/kg) were administered as intraperitoneal bolus injections to the two groups once daily at 1700 h for 4 successive days. The dose of MTII was based on that used in previous studies [18]. The pair-fed (PF) group was also injected with saline (2 ml/kg rat). A calculated amount of food was given to the PF rats based on the intake of the MTII-treated rats from the previous 24 h. The following calculation was used to determine the amount of food provided to the PF rats: the average food intake divided by the average body weight 24 h after MTII injection multiplied by the individual body weight of rats assigned within this experiment [20]. The PF rats received the daily allotment of food at 1800 h when dark phase started. 2.5. Tissue collection Five days after the initiation of treatment, a final body temperature, food intake, water intake, and body weight measurements were recorded between 0800 and 0900 h, and the rats were euthanized by CO2. The intrascapular brown adipose tissue (iBAT), white adipose tissues (WAT; retroperitoneal [rWAT], epididymal [eWAT], and inguinal [iWAT] fat pads), and the gastrocnemius muscle (GC) were collected. Both left and right sides of each fat pad and muscle were pooled, weighed, frozen in liquid nitrogen, and stored at 80 C until assayed. 2.6. Blood chemical assay Serum concentrations of insulin and leptin were determined by RIA according to the manufacturer’s instruction (Linco Research, St. Charles, MO). Serum free fatty acid (FFA) concentrations were assayed using an NEFA C kit (Cat. #: 994-75409; Wako, Richmond, VA). 2.7. Gel electrophoresis apoptosis assay Apoptosis was assayed in two ways: DNA isolated from fat tissues was separated into two fractions: fragmented and genomic DNA. First, the fragmented DNA was run on an agarose gel in order to identify a ladder pattern of internucleosomal DNA degradation that is characteristic of apoptosis [19]. Second, apoptosis was quantified as the ratio of fragmented to total DNA, multiplied by 100 [21]. Briefly, approximately 150 mg of the epididymal, inguinal, or retroperitoneal WATs were homogenized in lysis buffer (10 mM Tris – HCl, pH 8.0; 10 mM EDTA, pH 8.0; 0.5% Triton X100) and centrifuged at 14,000 g for 15 min to separate fragmented DNA from genomic DNA. The supernatant, containing fragmented DNA, was extracted with phenol – chloroform – isoamyl alcohol (25:24:1), and the DNA was
Y.-H. Choi et al. / Physiology & Behavior 79 (2003) 331–337
Fig. 1. Changes in food intake in response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Food intake was measured at 1700 h on days 1 – 4 and at 0900 h on day 5. Arrow indicates treatments. Means with different letters are significantly different at P .05. Data show mean ± S.E.M. (n = 8).
precipitated by adding polyacryl carrier (Molecular Research Center, Cincinnati, OH) and ethanol. Genomic (nonfragmented) DNA was extracted from the pellet with DNAzol and the polyacryl carrier. DNA in each fraction was quantified by the PicoGreen method (Molecular Probes, Eugene, OR), and fluorescence was measured using a SpectroMax Gemini (Molecular Devices). 2.8. Statistics Data were analyzed by a two-way ANOVA with repeated measures for behavioral data and a one-way ANOVA for tissue and apoptosis data sets, followed by post hoc mean
Fig. 2. Changes in water intake in response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Water intake was measured at 1700 h on days 1 – 4 and at 0900 h on day 5. Arrow indicates treatments. Means with different letters are significantly different at P .05. Data show mean ± S.E.M. (n = 8).
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Fig. 3. Changes in body weight in response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Body weight was measured at 1700 h on days 1 – 4 and at 0900 h on day 5. Arrow indicates treatments. Means with different letters are significantly different at P .05. * P=.0525. Data show mean ± S.E.M. (n = 8).
comparisons between groups. Data are expressed as mean ± S.E.M. and considered significant at P .05.
3. Results As shown in Fig. 1, MTII administration resulted in a large reduction in food intake [ F(2,21) = 73.39, P=.0001]. Food intake was consistent over the 4 days except on the 5th day, during which it was lower because the mealtime interval was shortened to 16 h. On this day, the MTII group ate 7.7 g less than saline control ( P < .05).
Fig. 4. Changes in body temperature (C) in response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/ kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Body temperature was measured 1 and 2 h after injections via transponders implanted subcutaneously; results shown were obtained on day 1. Arrow indicates treatments. Means with different letters are significantly different at P .05. Data show mean ± S.E.M. (n = 8).
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Table 1 Changes in body temperature (C) at 1 and 2 h after intraperitoneal injections of MTII (2 mg/kg) to male Sprague – Dawley rats once daily for 4 daysa,b Treatment
Day 1
Day 2
0h
1h
2h
0h
1h
2h
Saline Pair-fed MTII
37.7 ± 0.1 37.7 ± 0.1 37.7 ± 0.1
38.2 ± 0.1a 38.0 ± 0.1a 37.2 ± 0.3b
38.2 ± 0.1a 38.2 ± 0.2a 37.3 ± 0.2b
37.7 ± 0.1 38.1 ± 0.1 37.9 ± 0.1
38.0 ± 0.1 38.3 ± 0.2 38.0 ± 0.1
38.2 ± 0.1a 38.6 ± 0.1b 38.2 ± 0.1a
Day 3
Saline Pair-fed MTII a b
Day 4
0h
1h
2h
0h
1h
2h
37.9 ± 0.2 38.3 ± 0.1 37.9 ± 0.1
37.9 ± 0.1 38.3 ± 0.1 37.8 ± 0.2
38.4 ± 0.1ab 38.6 ± 0.1a 38.2 ± 0.1b
38.1 ± 0.1a 37.5 ± 0.1b 38.0 ± 0.1a
38.1 ± 0.1 38.3 ± 0.1 37.9 ± 0.2
38.5 ± 0.1 38.1 ± 0.1 38.4 ± 0.1
Means with different letters within a column are different at P < .05. Data are mean ± S.E.M. (n = 8 each treatment).
Total water intake was significant reduced by MTII [ F(2,21) = 4.56, P=.023]. Water intake after MTII treatment was reduced compared with that after saline except for day 4,
but daily water intake in the PF group was variable (Fig. 2). Total water intake-to-food intake ratio (%) was 157.1 ± 9.7 for saline, 191.6 ± 28.9 for PF, and 187.6 ± 21.6 for MTII. Body weight was decreased in both MTII and PF groups (Fig. 3). Consistent with this, overall ANOVA showed a significant treatment effect [ F(2,21) = 3.68, P=.043], day effect [ F(4,84) = 26.0, P=.0001] and treatment day interaction [ F(4,84) = 35.7, P=.0001]. On day 5, body weight of the saline-treated rats was 28.8 g greater than that of the MTII and 24.6 g greater than that of the PF-treated rats, respectively ( P < .05). Body temperature was unexpectedly reduced after MTII administration on day 1 ( P < .05), followed by inconsistent changes over the next 3 days (Fig. 4 and Table 1). In contrast, body temperature was increased in both PF and saline groups after saline injection. Overall, ANOVA revealed a significant treatment effect at 1 h [ F(2,21) = 7.57, P=.004] and 2 h [ F(2,21) = 5.35, P=.014]. The temperature measured in the morning of the last day was not incorporated into these analyses due to the different time of measurement, but it was not different from that measured at 1700 h on previous days. Compared to saline-treated rats (0.81 ± 0.07 g), rWAT mass was reduced by 46.3% (0.43 ± 0.06 g; P < .05) in MTII-treated rats, but not in PF rats (15.4%; 0.68 ± 0.10 g; P>.05), (Fig. 5C). In contrast, a smaller but significant reduction in eWAT mass was seen in MTII- (21.1%, 1.78 ± 0.09 g, P < .05) but not in PF-treated rats (6.1%, Table 2 lnsulin, leptin, and free fatty acid concentrations in serum collected from rats treated with MTII (2 mg/kg, ip), pair-fed, or saline for 4 days
Fig. 5. Tissue response to MTII. Rats were injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 successive days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Tissues were collected between 16 and 18 h after the last injections of MTII. (A) Epididymal white adipose tissue (WAT), (B) inguinal WAT, (C) retroperitoneal WAT, (D) intrascapular brown adipose tissue (BAT), (E) total WAT, and (F) gastrocnemius muscle. Means with different letters are significantly different at P .05. Data show mean ± S.E.M. (n = 8).
Treatment
Insulin (ng/ml)
Leptin (ng/ml)
Free fatty acids (meq/l)
MTII Pair-fed Saline
1.04 ± 0.10a 1.13 ± 0.10a 1.75 ± 0.15b
ND ac 0.27 ± 0.13a 1.43 ± 0.22b
232.7 ± 20.0a 394.2 ± 40.2b 252.1 ± 24.6a
Data are means ± S.E.M.; n = 8 rats/treatment. ab Treatment means within a column denoted with different letters are different, P < .05. c ND: below the detection limit (0.5 ng/ml).
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2.12 ± 0.09 g, P>.05), compared with saline-treated rats (2.26 ± 0.08 g). iBAT mass was reduced in both MTII (25.0%, 0.29 ± 0.02 g) and PF (33.3%, 0.26 ± 0.02 g) groups ( P < .05), compared with saline (0.38 ± 0.02 g), but there were no significant treatment effects for either iWAT or muscle mass ( P>.05) (Fig. 2). Serum insulin [ F(2,21) = 10.25, P < .001] and leptin [ F(2,21) = 26.43, P < .0001] concentrations of rats were reduced by both MTII and PF treatments compared with those of rats receiving saline treatment (Table 2). Serum leptin concentrations in leptin-treated rats were below the detection limit of 0.5 ng/ml. The mean concentrations of both serum insulin and leptin of MTII and PF groups of rats were not different. Serum FFA concentrations were increased only in the PF group of rats compared with those from both the MTII and control groups of rats [ F(2,21) = 8.90, P < .001]. To test the hypothesis that the mechanism for reduction in WAT mass included apoptosis, the three tissues were analyzed for DNA fragmentation, an indicator of apoptosis [19]. There were no differences in percent fragmented DNA in any WAT tissue: eWAT [ F(2,21) = 0.61, P=.55], iWAT
Fig. 6. Percent fragmented DNA in the epididymal white adipose tissue (eWAT) (A), inguinal WAT (iWAT) (B), and retroperitoneal WAT (rWAT) (C) collected from rats injected intraperitoneally with either saline (Sal) (2 ml/kg) or MTII (2 mg/kg) at 1700 h once daily for 4 days and fed ground chow ad libitum (Sal & MTII) or pair-fed (PF). Data show mean ± S.E.M. (n = 8 except for MTII of iWAT and rWAT, where n = 7).
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[ F(2,20) = 0.59, P=.57], and rWAT [ F(2,20) = 0.57, P=.57] (Fig. 6A –C), respectively.
4. Discussion MTII has been shown to have a consistently strong inhibitory effect on food intake, following either central [10,11,22– 25] or peripheral administration [10,18], or a combination of both [24]. In the present study, a large reduction in food intake and body weight gain was observed in rats treated with MTII or PF. However, although body weights of PF- and MTII-treated rats were not different, MTII treatment resulted in significantly greater reductions in eWAT and rWAT mass compared to pair-feeding. Because of previous findings of adipose tissue apoptosis following central [19] or peripheral injection [26] of leptin, and studies indicating that a-MSH acts as a downstream effector of some of leptin’s central effects [14,15,27], we hypothesized that the reduction in WAT mass following MTII treatment might be a result of increased adipocyte apoptosis. To test this hypothesis, we quantitated the percent of fragmented DNA in the three white fat depots (epididymal, inguinal, and retroperitoneal) as an indicator of apoptosis [21,28]. Our results showed that MTII administered intraperitoneally failed to induce apoptosis in fat tissues. Although it is possible that the time at which tissues were collected had passed a peak of apoptosis, in previous studies in which centrally administered leptin was shown to induce adipose apoptosis, tissue collection was carried out 24 h after the last leptin treatment [19,29]. In the present study, we collected tissues 16 –19 h after the last treatments of MTII. Thus, it is unlikely that the time interval is the reason we failed to observe apoptosis. The results of this study suggest that the MC signaling that governs food intake and body weight is independent of the leptin signal cascade in the brain that leads to apoptosis in adipose tissues. The reduced adipose tissue mass in the fat pads and the lack of evidence of increased apoptosis in our MTII-treated rats suggest that the reduction of adipose tissue mass was a result of lipolysis alone. In analyzing blood samples taken 19 h after the last intraperitoneal injection, we found that MTII treatment prevented the expected increase in serum FFA concentrations resulting from the reduced food intake, as was observed in the PF rats. In contrast, neither leptin nor insulin concentrations were different between MTII and PF groups. Consistent with our findings, a recent study reported no increase of FFA concentrations in blood samples 6 h after the last intraperitoneal injection of MTII [18]. These results are in contrast, however, to results reported from both lean and ob/ob mice sampled 1 h following intraperitoneal injections of an MSH analog, [Ac – Cys4, D-Phe7, Cys10] a-MSH [30]. Following this shorter interval after the injections in mice, FFA concentrations were increased in serum. The increased FFA concentrations occurring shortly
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after the MSH analog treatment may have activated mechanism(s) that result in increased FFA utilization, thus leading to reduced FFA concentrations compared with those in PF rats. This is supported by the demonstration of normal mRNA expression for uncoupling protein-1, -2, and -3 in BAT of MTII-treated rats, whereas in PF rats, uncoupling protein mRNA expression was significantly suppressed [18]. Furthermore, rats treated with MTII had normal mRNA expression in liver and WAT of carnitine palmitoyltransferase-1, the enzyme responsible for transferring longchain fatty acids into the mitochondria, whereas PF rats showed a reduction in expression of this key enzyme [18]. This clearly supports the contention that there is a continuation of energy utilization from fat tissues despite reduced body weight and the lack of the expected increased serum FFA concentration. We conclude, therefore, that our findings support those of previous studies showing that peripheral treatment with MTII, a-MSH, or its analogues resulted in strong lipolytic effects in mice and rats [18,30]. Our observation of a hypothermic rather than hyperthermic effect of MTII was unexpected. Intracerebroventricular administration of MTII resulted in an increase in abdominal temperature in Sprague – Dawley rats [23] and increased rectal temperature in ob/ob mice [24]. However, in these studies, MTII was administered centrally, and the times and methods of temperature measurement were different from those in our study. These methodological differences may account for the difference in results between our study and theirs. It should also be noted that in addition to a strong lipolytic effect, systemic a-MSH has been shown to have a strong antipyretic effect in endotoxin-treated rats. Because this effect was blocked by central administration of SHU9119, an MC3/4-R antagonist [31], the suppressive effect on body temperature appears to have been mediated by a specific MC receptor. The finding that MTII treatments inhibited water consumption is consistent with previous findings in rats injected intracerebroventricularly with MTII [22,23], but differs from the response of ob/ob mice to MTII [24]. In the mouse study both intraperitoneal and intracerebroventricular injections of MTII failed to inhibit 10-day cumulative water consumption [24]. Whether this is due to a species difference or to an absence of leptin in these mice, is unknown. In conclusion, the present study shows that in contrast to the effects of either centrally or peripherally administered leptin on adipose apoptosis, peripherally administered MTII reduces the mass of specific WAT depots without causing apoptosis. These findings, therefore, suggest that it is unlikely that a-MSH is a CNS downstream effector for leptin-induced adipose apoptosis.
Acknowledgements The authors acknowledge the technical assistance of Sherry Hulsey and Brent Jackson. This study was supported
in part by the Georgia Research Alliance Eminent Scholar endowment held by CAB.
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