International Journal of Obesity (2011) 35, 1183–1192 & 2011 Macmillan Publishers Limited All rights reserved 0307-0565/11 www.nature.com/ijo ORIGINAL ARTICLE Combined amylin– treatment lowers blood pressure and adiposity in lean and obese rats

R Seth1, WD Knight1 and JM Overton

Department of Biomedical Sciences and Program in Neuroscience, College of Medicine, Florida State University, Tallahassee, FL, USA

Objective: To examine the cardiovascular effects of combined amylin (AMN) and leptin (LEP) treatment in lean and obese rats. Research design: Rats were instrumented for telemetry and given LEP (300 mgkg–1 day–1), AMN (100 mgkg–1 day–1), AMN þ LEP or vehicle (VEH; 0.9% normal saline) via a subcutaneous mini-osmotic pump for 7 days. The VEH group was subdivided into ad libitum fed and pair-fed to the amount of food AMN þ LEP animals ate daily. Rats were housed in metabolic chambers for analysis of cardiovascular physiology and metabolism. Subjects: Male Fisher 344 Â Brown Norway (FBNF1; Harlan; age ¼ 3–5 months; n ¼ 72) rats were placed on standard rodent chow (LEAN, n ¼ 41) or moderately high-fat diet (OBESE; n ¼ 31) to produce obesity. Results: AMN þ LEP potently reduced food intake (LEAN: 57% OBESE: 59%) and abdominal fat mass (LEAN: 56% OBESE: 41%). Pair-fed rats displayed bradycardia and metabolic suppression. In contrast, AMN þ LEP increased rate and oxygen consumption above levels in LEP or AMN-treated rats. LEP reduced blood pressure in both lean and obese rats but AMN had no effect. LEP-induced reductions in blood pressure were not altered by AMN þ LEP treatment. Thus, AMN þ LEP treatment decreased food intake, body fat and blood pressure in lean and obese rats. Conclusion: We conclude that the potent anti-adiposity actions of AMN þ LEP are due in part to prevention of the bradycardia and metabolic suppression typically observed with negative energy balance. Furthermore, the hypotensive actions of peripheral LEP treatment are observable in spite of the potent AMN þ LEP activation of anorexic and thermogenic mechanisms in the central nervous system. International Journal of Obesity (2011) 35, 1183–1192; doi:10.1038/ijo.2010.262; published online 21 December 2010

Keywords: food intake; metabolism; energy balance; respiratory quotient; telemetry

Introduction while acute infusion of 4 receptor increases blood pressure.8 Clearly, the cardiovascular effects Preclinical and clinical studies have convincingly demon- of anti-obesity drugs must be well understood.9 Although strated a potent and synergistic action of amylin (AMN) and studies have been conducted to understand the anorectic leptin (LEP) in treating obesity.1–4 It is likely that central role of AMN and LEP separately in relation to food intake, nervous system pathways contribute to AMN and LEP there is little information concerning the combined cardio- regulation of appetite and energy expenditure.5 It is now vascular actions of these . well established that these central nervous system pathways Central LEP is sympathoexcitatory and can increase blood also have important actions on the cardiovascular system.6,7 pressure.10,11 However, supraphysiological peripheral doses For example, patients with obesity because of melanocortin are generally required for marginal increases in blood 4 receptor mutations have a low incidence of hypertension pressure, and some studies report that lower levels of LEP either have no effect on or lower blood pressure.12–15 Less is Correspondence: Dr JM Overton, Department of Biomedical Sciences, 1115 W known about the cardiovascular actions of AMN. In general, Call Street, College of Medicine, Florida State University, Tallahassee, FL acute studies have suggested that AMN produces vasodila- 32306, USA. tion via gene-related receptor activa- E-mail: [email protected] tion.16 A recent clinical study reported that chronic 1These authors contributed equally to this work. Received 6 August 2010; revised 20 October 2010; accepted 26 October treatment with synthetic AMN (pramilintide) decreases 2010; published online 21 December 2010 blood pressure in obese human subjects, while subjects Amylin þ leptin lowers blood pressure and body fat R Seth et al 1184 treated with the combination of AMN and sibutramine or ratLEP(PeproTech,Inc.,RockyHill,NJ,USA;300mgkg–1 day–1), phentermine lost more weight, but did not have a beneficial rat AMN (Bachem Inc., Torrance, CA, USA; 100 mgkg–1 day–1), decrease in blood pressure.17 Here, we hypothesized that the both AMN þ LEP or saline vehicle (VEH; 0.9% NaCl, 10 mlh–1). combination of LEP and AMN treatment might produce The VEH group was subdivided into those fed ad libitum (VEH) significant reductions in blood pressure. The goal of this and those that were pair-fed to the amount of food AMN þ LEP study was to determine the cardiovascular and metabolic animals ate daily (PF AMN þ LEP). All animals received their actions of combined LEP and AMN in lean and obese rats. food just before the onset of the dark phase. On the seventh day of infusion, animals were terminated for post-mortem Materials and methods analysis as described below.

Animals and housing Post-mortem dissection and serum analysis Male Fisher 344 Â Brown Norway (FBNF1; Harlan, Indianapolis, Rats were anesthetized with pentobarbital (50 mg kg–1, IN, USA; Age ¼ 3–5 months; n ¼ 72) rats were placed on intraperitoneally). All animals were killed just at the end of standard rodent chow (Labdiet 5001, LabDiets, Richmond, the light cycle. Trunk blood was obtained by decapitation IN, USA; 4.5% fat), and housed individually at Ta ¼ 23 1Cona and blood was stored temporarily at 4 1C for centrifugation. 12-h light–dark cycle. This rat strain was selected because on Blood was spun at 3000 Â g for 15 min at 4 1C; supernatant reaching adulthood, the FBNF1 rat maintains a relatively stable serum was removed and stored at À20 1C for determination plateau of body weight gain when fed ad libitum unlike obesity- of LEP and AMN concentrations. The remaining carcass was prone strains, which display continuous body weight gain.18 dissected for heart, , retroperitoneal, mesenteric and After 2 weeks recovery from transport, 31 FBNF1 rats were epididymal abdominal fat pad weights. LEP hormone placed on a moderately high-fat diet (Research Diets D12266B, analysis was performed using a radioimmunoassay kit for Research Diets, Inc., New Brunswick, NJ, USA; 32% fat) for rat LEP (RL-83K; Linco; St Charles, MO, USA). AMN hormone 5 months to induce obesity. The remaining 41 FBNF1 rats were analysis was done using a radioimmunoassay kit for maintained on standard chow and prepared for study shortly rat/mouse AMN (RK-017-11; Phoenix Pharmaceuticals; Bur- after reaching a stable body weight above 300–350 g. lingame, CA, USA). Both assays were performed according to The apparatus and procedures used for determination of their respective manufacturers’ protocols. energy expenditure and locomotor activity have been described previously.19,20 Briefly, rats were housed in in- dividual polycarbonate 20 l cages each fitted for open circuit Data management respirometry. Oxygen consumption and carbon dioxide During the last hour of the light phase, animals, food and production were continuously measured 23 h day–1. Beneath water were weighed daily. At this time, all data from the each cage is a custom-designed force platform to obtain previous day were transferred for offline analysis. In order to precise quantification of locomotor activity and a telemetry quantify the effects of infusion between groups, these data receiver that collects the transmitted blood pressure data. were expressed as differences from each animal’s average Metabolic and cardiovascular data are monitored continu- baseline values, combined by treatment group, and reported ously and stored for offline analysis. as daily means±s.e.m.

Oxygen consumption (VO2), carbon dioxide production Surgery (VCO2), mean arterial pressure and heart rate (HR) were All FBNF1 rats were implanted with cardiovascular telemetry measured continuously throughout the days of baseline and devices (TA11PA-C40; DSI, St Paul, MN, USA) as described infusion. Respiratory quotient (RQ), an index of substrate 20 previously. After surgery, animals were given subcutaneous utilization, was calculated as the ratio of VCO /VO . Higher –1 2 2 injections of 30 mgkg buprinex analgesic (Henry Schein RQ values indicate increased utilization of carbohydrates Inc., Melville, NY, USA) and intramuscular injections of over fat and vice versa.21 VO was analyzed after normal- –1 2 60 mg kg of cefazolin (Henry Schein Inc.) as a prophylactic. ization to the Kleiber coefficient to account for differences in 1 Rats were then re-housed individually at Ta ¼ 23 C on a 12-h metabolic mass.22–25 In order to quantify the effects of light–dark cycle and their previously assigned diet and infusion between groups, these continuously measured allowed to recover at least 10-day before experimentation. parameters were separated into 12-h dark and 11-h light phase daily means and expressed as differences from each LEP, AMN and AMN þ LEP infusion in lean and obese rats animal’s average baseline values. These ‘delta values’ were After acclimation to metabolic chambers (3–4 days), and combined by treatment group and reported as daily mean- baseline cardiovascular and metabolic data collection (2–3 s±s.e.m. In order to present data in concise format, only days), 7-day osmotic pumps were implanted in the intras- dark phase data for all metabolic and both dark and light capular region under light halothane anesthesia. Baseline phase data for cardiovascular parameters are shown, data representing a 2- to 3-day average for all experimental however, the observed effects were similar in both light groups are displayed in Table 1. Animals received recombinant and dark phases.

International Journal of Obesity Amylin þ leptin lowers blood pressure and body fat R Seth et al 1185 Table 1 Baseline parameters for lean and obese FBNF1 rats

Lean (n ¼ 41) VEH (n ¼ 8) AMN (n ¼ 8) LEP (n ¼ 9) AMN+LEP (n ¼ 8) PF AMN+LEP (n ¼ 8)

Body weight (g) 388±9.2 375±6.2 379±8.4 369±3.0 383±7.0 Caloric intake (kcal day–1) 58.9±2.3 61.6±3.1 62.4±2.0 60.5±2.3 63.1±3.2 Locomotor activity (m) Light 15.7±3.7 12.2±2.9 15.9±2.2 15.9±1.5 15.5±3.4 Dark 37.6±5.9 31.3±7.9 45.2±7.0 45.2±5.6 42±9.8

Adjusted oxygen consumption (ml min–1 kg–0.75) Light 14.2±0.3 14.6±0.3 14.7±0.3 14.5±0.3 14.0±0.1 Dark 18.4±0.4 18.6±0.5 18.3±0.3 18.5±0.3 18.5±0.3

Respiratory quotient (VCO2/VO2) Light 0.89±0.0 0.93±0.0 0.93±0.0 0.94±0.0 0.94±0.0 Dark 0.90±0.0 0.94±0.0 0.95±0.0 0.95±0.0 0.95±0.0

Mean arterial pressure (mm Hg) Light 88±1.1 88±2.4 89±1.8 91±2.0 96±2.0 Dark 96±1.7 94±2.2 97±1.8 96±1.9 101±1.9

Heart rate (b.p.m.) Light 309±5.7 324±10.5 322±4.7 328±2.0 323±2.0 Dark 361±5.2 369±7.6 373±2.2 375±2.4 364±2.4

Obese (n ¼ 31) VEH (n ¼ 6) AMN (n ¼ 6) LEP (n ¼ 6) AMN+LEP (n ¼ 7) PF AMN+LEP (n ¼ 6)

Body weight (g) 441±14.5* 441±12.3* 441±15.5* 437±5.5* 443±9.3* Caloric intake (kcal day–1) 74.2±6.7* 70.2±3.2* 68.2±2.2* 67.2±1.2* 74.1±8.8* Locomotor activity (m) Light 17.1±1.0* 23.5±5.7* 38.6±7.9* 10.7±3.5* 12.7±3.4* Dark 33.7±3.5* 29.6±7.4* 34.3±3.5* 23.6±8.9* 24.0±8.6*

Adjusted oxygen consumption (ml min–1 kg–0.75) Light 13.4±0.2* 13.9±0.2* 13.9±0.3* 13.6±0.2* 13.6±0.4* Dark 16.2±0.3* 17.8±0.2* 17.9±0.4* 16.4±0.5* 16.1±0.5*

Respiratory quotient (VCO2/VO2) Light 0.95±0.0 0.90±0.0 0.90±0.0 0.92±0.0 0.95±0.0 Dark 0.97±0.0 0.95±0.0 0.92±0.0 0.95±0.0 0.96±0.0

Mean arterial pressure (mm Hg) Light 95±1.2* 99±1.1* 105±4.8* 99±5.1* 101±0.3* Dark 101±0.7* 101±0.9* 110±3.8* 102±5.4* 104±0.6*

Heart rate (b.p.m.) Light 340±2.0* 340±3.1* 339±4.2* 324±4.9* 329±1.7* Dark 373±4.5* 372±2.7* 387±3.9* 362±3.7* 365±2.9*

Abbreviations: AMN, amylin; b.p.m., beats per minute; FBNF1, Fisher 344 Â Brown Norway; LEP, leptin; PF, pairfed; VCO2, carbon dioxide production; VEH, vehicle; VO2, oxygen consumption. Table compares baseline values of metabolic and cardiovascular parameters in light and dark phase in lean and obese FBNF1 rats. *Significance (Pp0.05): obese vs lean group.

Statistical analysis Ethical approval Using Sigmastat 3.1 (Systat Software Inc., Chicago, IL, USA), All of the procedures were approved and authorized by the mean differences were analyzed using two-way repeated Animal Care and Use Committee at Florida State University. measures analysis of variance (repeated measure ¼ time (days) Â treatment group) to evaluate effects of drug treatments over time for most dependent variables. Tukey’s Results post hoc tests were used to determine specific days in which treatment groups are significantly different from one Potent inhibition of food intake and prevention of metabolic another. For terminal single-value measurements such as suppression in lean and obese rats serum LEP or fat pad weights, a one-way analysis of variance Animals consuming a 32% fat diet had greater body weight for treatment group and a Tukey’s post hoc test was used. (Table 1), serum LEP and AMN levels (Table 2) and

International Journal of Obesity Amylin þ leptin lowers blood pressure and body fat R Seth et al 1186 Table 2 Serum AMN and LEP values for all the groups

Lean Obese

AMN (pg ml–1) LEP (ng ml–1) AMN (pg ml–1) LEP (ng ml–1)

VEH (n ¼ 6) 66.3±6.8a 3.8±0.5a* VEH (n ¼ 4) 174.0±43.8a 13.3±3.7a,c AMN (n ¼ 6) 516.5±87.7b 1.3±0.2b* AMN (n ¼ 4) 487.9±78.3b 5.7±2.5c LEP (n ¼ 6) 77.7±23.2a 15.1±2.4c* LEP (n ¼ 6) 164.8±34.9a 27.4±9.2b AMN+LEP (n ¼ 7) 544.4±87.4b 15.0±2.7c AMN+LEP (n ¼ 4) 508.8±51.7b 16.4±12.6a,b PF to A+L (n ¼ 6) 102.4±12.9a 1.9±0.4b* PF to A+L (n ¼ 5) 104.7±6.4a 8.2±2.1c

Abbreviations: AMN, amylin; LEP, leptin; VEH, vehicle. Values with different superscript letters are significantly different (Pp0.05) within hormone and within lean and obese cohorts. *Pp0.05 vs obese group of same treatment. Serum LEP and AMN concentration in all the treatments.

Figure 1 Effect of 7-day chronic infusion of LEP (300 mgkg–1 day–1)(a, d), AMN (100 mgkg–1 day–1)(b, e), or AMN þ LEP (c, f) on daily food intake in lean (a–c) and obese (d–f) FBNF-1 rats. Food and body weights were measured every other day for 7 days. Data are presented as delta values of mean±s.e.m. n ¼ 8. Significance (Pp0.05): treatment * vs VEH, $ vs LEP and @ vs AMN.

abdominal body fat (Figure 4) and thus were considered metabolic suppression associated with an even greater

obese. Chronic LEP infusion produced a significant decrease reduction in food intake and maintained VO2 compared in food intake in lean (21%) but not obese rats (Figures 1a with controls (Figures 2c and f). The suppressive effect of and d). Chronic AMN infusion reduced food intake in both such a reduction in food intake is evident in the pair-fed lean (24%) and obese (30%) rats (Figures 1b and e). Further, group to AMN þ LEP-treated rats. infusion of AMN with LEP significantly reduced food intake in both lean (57%) and obese (59%) rats (Figures 1c and f). All the food intake values mentioned above are percentage Synergistic reductions in fat mass with AMN and LEP in lean differences based on 7-day food intake average of VEH- and obese rats treated group. LEP modestly reduced RQ in lean but not obese rats

In lean animals, neither LEP nor AMN maintain VO2 at the (Figures 3a and d). AMN, on the other hand, decreased RQ level of VEH-treated ad lib rats (Figures 2a and b) although in lean rats but only modestly reduced RQ in obese rats

they did prevent the suppression in VO2 associated with pair (Figures 3b and e). In both lean and obese rats, AMN þ LEP feeding (data not shown). In obese animals, there was no produced a sustained decrease in RQ that was significantly

effect of AMN or LEP on VO2 (Figures 2d and e). AMN þ LEP greater than VEH, pair-fed controls, and either treatment in both lean and obese rats completely prevented the alone (Figures 3c and f). In obese animals, abdominal fat

International Journal of Obesity Amylin þ leptin lowers blood pressure and body fat R Seth et al 1187

Figure 2 Effect of 7-day chronic infusion of LEP (a, d), AMN (b, e), or AMN þ LEP (c, f) on daily dark phase VO2 in lean (a–c) and obese (d–f) FBNF1 rats measured every other day for 7 days. Light phase data (not shown) was similar to dark phase data. Data are presented as delta values of mean±s.e.m. Significance (Pp0.05): treatment * vs VEH, # vs pair-fed and $ vs LEP.

Figure 3 Effect of 7-day chronic infusion of LEP (a, d), AMN (b, e), or AMN þ LEP (c, f) on dark phase RQ in lean (a–c) and obese (d–f) FBNF1 rats. Light phase data (not shown) was similar to dark phase data. Data are presented as delta values of mean±s.e.m. Significance (Pp0.05): treatment * vs VEH, # vs pair-fed, $ vs LEP and @ vs AMN.

mass was roughly doubled in VEH-treated animals com- abdominal fat in obese animals (Figure 4). However, pared with lean counterparts (LEAN: 12.1±0.4 g OBESE: AMN þ LEP significantly reduced abdominal fat mass in lean 24.8±1.1 g). In lean animals, LEP and AMN separately and obese rats compared with all groups (Figure 4). (LEAN: reduced total abdominal fat; neither LEP nor AMN reduced (LEP: 9.6±0.4 g AMN: 9.0±1.1 g AMN þ LEP: 4.2±0.5 g PF

International Journal of Obesity Amylin þ leptin lowers blood pressure and body fat R Seth et al 1188 AMN þ LEP: 10.1±0.7 g) OBESE: (LEP: 21.3±1.8 g AMN: 19.2±2.4 g AMN þ LEP: 14.7±1.6 g PF AMN þ LEP: 21.7± 1.3 g)) There were no significant effects of any treatments on heart and kidney weights in these animals.

Cardiovascular effects of AMN and LEP Neither LEP nor AMN increased HR in lean animals in dark phase (Figures 5a and b) or light phase (Figures 6a and b). In obese rats, LEP increased HR but AMN had no effect in dark phase (Figures 5d and e) or light phase (Figures 6d and e). AMN þ LEP significantly increased HR above controls in obese rats only. In both lean and obese animals, AMN þ LEP prevented the bradycardia associated with pair feeding in dark (Figures 5c and f) as well as light phase (Figures 6c and f). LEP reduced blood pressure in both lean and obese rats in dark (Figures 7a and d) and light phase (Figures 8a and d). AMN had no effect on blood pressure in either lean or obese animals in dark (Figures 7b and e) or light (Figures 8b and e) phase. AMN þ LEP reduced blood pressure similar to that observed in LEP-treated animals in dark (Figures 7c and f) and light (Figures 8c and f) phase. These results suggest that reduced blood pressure because of physiological hyperlepti- nemia persists when co-administered with AMN in both lean and obese rats.

Effects of treatments on serum hormone levels Figure 4 Effect of 7-day chronic infusion of LEP, AMN or AMN þ LEP on mesen- The effects of different treatments on the serum concen- teric, retroperitoneal and epididymal fat mass and sum total of all the three fat pads. All the drug treatments are compared with control and pair-fed groups of lean and trations of AMN and LEP are depicted in Table 2. In lean p obese FBNF1 rats. Significance (Pp0.05): treatment * vs VEH; AMN þ LEP vs all. rats, LEP and AMN þ LEP increase serum LEP as expected.

Figure 5 Effect of 7-day chronic infusion of LEP (a, d), AMN (b, e), or AMN þ LEP (c, f) on dark phase HR in lean (a–c) and obese (d–f) FBNF1 rats. Data are presented as delta values of mean±s.e.m. Significance (Pp0.05): treatment * vs VEH, # vs pair-fed and @ vs AMN.

International Journal of Obesity Amylin þ leptin lowers blood pressure and body fat R Seth et al 1189

Figure 6 Effect of 7-day chronic infusion of LEP (a, d), AMN (b, e), or AMN þ LEP (c, f) on light phase HR in lean (a–c) and obese (d–f) FBNF1 rats. Data are presented as delta values of mean±s.e.m. Significance (Pp0.05): treatment * vs VEH, # vs pair-fed and @ vs AMN.

Figure 7 Effect of 7-day chronic infusion of LEP (a, d), AMN (b, e), or AMN þ LEP (c, f) on dark phase mean arterial pressure (MAP) in lean (a–c) and obese (d–f) FBNF1 rats. Data are presented as delta values of mean±s.e.m. Significance (Pp0.05): treatment * vs VEH, # vs pair-fed and @ vs AMN.

AMN treatment reduced serum LEP likely because of Rats consuming a high-fat diet and infused with saline had decreased food intake and fat mass, as seen in pair-fed elevated LEP and AMN level, consistent with increased rats (data not shown). Serum LEP was not significantly abdominal fat mass. LEP treatment in obese animals different between LEP and AMN þ LEP rats. Similarly, serum increased serum LEP above controls despite no effect on AMN was not significantly different between AMN and food intake or fat mass. On the other hand, AMN þ LEP AMN þ LEP rats. treatment did not increase serum LEP above controls

International Journal of Obesity Amylin þ leptin lowers blood pressure and body fat R Seth et al 1190

Figure 8 Effect of 7-day chronic infusion of LEP (a, d), AMN (b, e), or AMN þ LEP (c, f) on light phase mean arterial pressure (MAP) in lean (a–c) and obese (d–f) FBNF1 rats. Data are presented as delta values of mean±s.e.m. Significance (Pp0.05): treatment * vs VEH, # vs pair-fed and @ vs AMN.

possibly because of large reductions in fat mass leading to AMN subside quickly. At the dose of subcutaneous AMN used decreased endogenous LEP production. AMN þ LEP did in this study, we observed a loss of anoretic efficacy within a prevent the drop in serum LEP seen in pair-fed rats. Again, few days of infusion. It is well documented that rats made in obese animals, AMN treatment and AMN þ LEP treatment obese by consuming higher amounts of fat in their diet are raised serum AMN above all other groups but not signifi- resistant to the anorectic actions of LEP.28,29 The mechan- cantly different from one another. isms by which this occurs include both humoral adapta- tions30,31 and inhibitory mechanisms at the cellular and neuronal level.32,33 Our experiments confirm that 5 months Discussion of 32% fat diet-induced resistance to the anorectic effects of LEP. However, the combination treatment with AMN þ LEP 26 Similar to several recent reports, we observed that the circumvented this resistance producing potent reductions combination therapy of AMN þ LEP produced potent reduc- in food intake that after 1 week were greater than the tions in food intake and adiposity in both lean and obese sum of the response produced by individual AMN or rodents. Furthermore, the combination therapy completely LEP treatments. prevented the metabolic suppression associated with weight Throughout the baseline and treatment periods, all rats loss in both lean and obese rats. It is commonly observed studied in these experiments were housed in home cage that caloric restriction-induced metabolic suppression is calorimeters for assessment of energy expenditure. We associated with bradycardia. Here again, we observed that observed no significant thermogenic effects of LEP or AMN the combination therapy effectively prevented this suppres- when given individually; however, pair-fed animals exhib- sion of HR in both lean and obese rats. Finally, we replicated ited reductions in metabolic rate (data not shown). Thus, previous findings indicating subcutaneous LEP infusion either LEP or AMN can prevent caloric restriction-induced lowers blood pressure, while AMN alone did not significantly metabolic suppression. The physiologic responses to mild alter blood pressure. When combined, AMN þ LEP decreased pair feeding are well understood,12 and thus we chose not to blood pressure well below the response observed in pair-fed verify this response for LEP and AMN groups. Rats pair-fed animals suggesting a primary effect rather than a secondary fed to the low levels of food intake produced by AMN þ LEP consequence of weight loss. (Figures 2c and f) displayed potent metabolic suppression. We observed that combination therapy with AMN þ LEP AMN–LEP actions on food intake, energy expenditure and completely prevented this metabolic suppression and tended lipolysis to increase energy expenditure. Chronic AMN decreases food intake in lean and obese We assessed lipolysis by continuously measuring RQ and rats.1,27 However, the anorectic effects of chronically infused by post-mortem fat pad weights after 1 week of therapy.

International Journal of Obesity Amylin þ leptin lowers blood pressure and body fat R Seth et al 1191 AMN produced a transient suppression of RQ in both lean AMN-induced weight loss was associated with the expected and obese rats, while LEP produced a decrease in RQ that was reduction in blood pressure long term and had no effect on only evident in lean rats. When combined, AMN þ LEP HR. When combined with the sympathoexcitatory weight produced a substantial reduction in RQ that was clearly loss drug phentermine, AMN produced greater weight loss, sustained for the week of treatment. As a result, the along with an increase in both HR and blood pressure.17 combination treatment produced substantial, reductions in It appears inevitable that combination therapies will fat pad mass. become common in the treatment of obesity. The results Further studies are warranted to determine the duration of of this study suggest the possibility that AMN þ LEP treat- these appetite-suppressing, thermogenic and lipolytic effects ment may decrease both body fat and blood pressure. and the mechanisms that drive this unabated persistence of If observed in clinical trials, this response would represent negative energy balance. One interpretation of these re- a promising step forward in the effort to develop effective sponses is that the AMN appears to increase LEP sensitivity in drug therapy for obesity without undesired cardiovascular obese animals. It has been reported that AMN and LEP side effects.9 synergistically increase LEP receptor expression in and .34 Chronic AMN þ LEP also reduces expres- sion of lipogenic genes and increases expression of lipolytic Conflict of interest genes in adipose tissue, while either LEP or AMN in monotherapy has no effect.3 AMN þ LEP may also act to The authors declare no conflict of interest. increase LEP signaling in hypothalamic nuclei.34 Within the , AMN suppresses expression of orexigenic normally increased during caloric restric- tion35,36 and upregulates anorectic neuronal signals.1,37 Acknowledgements Interestingly, these signaling pathways overlap with down- stream LEP signaling in the hypothalamus and are impli- This work was supported by funding support from Depart- cated in the anorexigenic and metabolic effects of LEP. ment of Biomedical Science, Florida State University. We Further, the resistance mechanisms such as elevated thank Dr M Freeman, C Fitch-Pye, M M Messina, E Bascom, suppressor of cytokine signaling-3 (Bjorbaek et al.38; Peralta E Stern, M Gierach and D Kopa for their contributions to et al.39) or reduced LEP receptor expression40 by which LEP is the completion of this study. rendered ineffective may be altered by AMN signaling or as a consequence of AMNs anorectic effects.41 Taken together, these findings suggest a potential modulatory role for AMN References in regulation of LEP effects on food intake and adiposity. 1 Roth JD, Hughes H, Kendall E, Baron AD, Anderson CM. Antiobesity effects of the hormone amylin in diet- induced obese rats: effects on food intake, body weight, Cardiovascular effects of LEP persist during AMN co-infusion composition, energy expenditure, and . Endocri- LEP can both increase11,42,43 or decrease12,44,45 arterial nology 2006; 147: 5855–5864. pressure. The pressor responses involve central sympathoex- 2 Roth JD, Roland BL, Cole RL, Trevaskis JL, Weyer C, Koda JE et al. citation46 mediated via arcuate nucleus pathways11 and Leptin responsiveness restored by amylin agonism in diet- 10,47 induced obesity: evidence from nonclinical and clinical studies. downstream melanocortin signaling. We have pre- Proc Natl Acad Sci USA 2008; 105: 7257–7262. viously reported that lower central doses (40–200 ng h–1)of 3 Trevaskis JL, Coffey T, Cole R, Lei C, Wittmer C, Walsh B et al. LEP decrease food intake without increasing blood pres- Amylin-mediated restoration of leptin responsiveness in diet- Endocrinology sure,15 suggesting a differential sensitivity for the anorectic induced obesity: magnitude and mechanisms. 2008; 149: 5679–5687. and sympathetic actions of LEP. Additionally, LEP can act 4 Chan JLRJ, Weyer C. 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