Leptin-derived peptides that stimulate food intake and increase body weight following peripheral administration

Regulatory Peptides 170 (2011) 24–30

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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p

Leptin-derived peptides that stimulate food intake and increase body weight following peripheral administration Graham L. Barrett ⁎, Tim Naim, Jennifer Trieu Department of Physiology, University of Melbourne, Parkville 3010, Australia

a r t i c l e

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Article history: Received 29 September 2009 Received in revised form 29 April 2011 Accepted 10 May 2011 Available online 24 May 2011 Keywords: Obesity Appetite Hormone structure and function Peptide hormone receptor

a b s t r a c t We previously showed that peptides containing leptin sequences 1–33 or 61–90 are taken up by the rat brain. We now report the effects of these peptides on food intake and body weight in mature rats. Peptides were infused intravenously for 4 weeks, using Alzet minipumps. Dosages were 20 μg/kg/day in experiment I, and 60 μg/kg/day in experiment 2. In experiment 1, female rats receiving peptides 1–33 and 61–90 each underwent an approximate doubling of the weight gain of control rats. These peptides also increased food intake in female rats. Peptide 15–32, which has a lesser degree of brain uptake, gave a smaller weight gain. Peptide 83–108, which is not taken up by the brain, had no effect on weight gain or food intake. Similar results were obtained in experiment 2. In male rats, however, none of the peptides caused significant changes in food intake or body weight. This was at least partly due to the fact that all male rats underwent vigorous weight increases. We conclude that peptides 1–33 and 61–90 acted as leptin antagonists, stimulating food intake and body weight increases, at least in female rats. These peptides may lead to clinical applications in conditions such as anorexia and cachexia. © 2011 Published by Elsevier B.V.

1. Introduction Leptin is one of a small number of peptide and protein signaling molecules that are known to be taken up across the blood-brain barrier. Since leptin is produced by adipocytes, and some of its most important signaling actions are on the brain, brain uptake is essential to its normal functioning. Leptin acts on hypothalamic neurons to reduce food intake and body weight, while leptin receptors are also found in many other brain regions [1]. Leptin receptors also occur on many cells in peripheral tissues, where leptin has modulatory roles on reproductive, endocrine and immune system function [2–5]. The leptin brain uptake mechanism is saturable and has a rather low capacity [6–8]. Indeed, brain uptake may be an important limiting factor that restricts the effectiveness of leptin in regulation of body weight. The majority of human obese patients have high leptin levels [9], yet their obesity persists. When leptin levels are raised further by giving exogenous leptin, the effect on body weight has been disappointing [10]. This is the phenomenon of leptin resistance, in which inadequate brain uptake may be a key factor [7,11,12].

Abbreviations: HPLC, High Pressure liquid Chromatography; PBS, Phosphate buffered saline; SEM, Standard Error of the Mean. ⁎ Corresponding author at: Department of Physiology, University of Melbourne, Victoria, 3010, Australia. Tel.: + 61 3 8344 5869; fax: + 61 3 8344 5818. E-mail address: [email protected] (G.L. Barrett). 0167-0115/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.regpep.2011.05.004

We previously screened leptin peptide fragments for their brain uptake ability. We found that peptide sequences 1–33 and 61–90, and some shorter peptides within these regions, displayed good brain uptake [13]. We therefore decided to investigate the in vivo effects of these peptides on regulation of body weight and food intake. These effects are the subject of the present report. Peptides 1–33 and 61–90, which display brain uptake on a par with leptin itself, both contain important receptor binding sequences. Leptin itself contains 3 receptor binding sites; site II is almost solely responsible for high affinity binding [14,15], while site III, and to a lesser extent site I, are crucial for receptor activation [15]. Binding site II is formed by the adjacent, anti-parallel helices A and C, which consist of residues 2 to 26 and 71 to 94, respectively [16]. Peptides 1–33 and 61–90 correspond approximately to these helices. Indeed, virtually all of the leptin residues that have been determined to be necessary for high affinity receptor binding are found within the regions spanned by these two peptides [14]. This observation, that peptides with the highest brain uptake also contain the residues necessary for receptor binding, strengthens the evidence that the leptin receptor is involved in brain uptake [13]. Since peptides 1–33 and 61–90 each contain important receptorbinding residues, we undertook in vivo experiments to look for antagonistic or agonistic actions. We expected the peptides to be able to access hypothalamic receptors when administered peripherally, since they have good brain uptake. Leptin itself causes dramatic weight loss when administered to obese, leptin-deficient ob/ob mice [17–19], and also decreases food intake and body weight in normal (non-leptin-deficient) mice [20,21] and rats [22–24], albeit to a milder

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degree. Conversely, leptin antagonists, usually made by site-specific mutagenesis of the binding site III receptor activation site, have been shown to be orexigenic agents, capable of stimulating food intake and increasing body weight in normal mice [25–28]. Our investigation was undertaken using normal Sprague–Dawley rats, approximately 3 months of age at the commencement of the treatment. Rats with ad libitum food access increase body weight throughout most of their life-span, but this age was chosen because weight gain is substantially slower than in younger rats. Male and female rats were tested separately. Peptides were infused intravenously for 4 weeks, using osmotic minipumps, and body weight and food intake were monitored daily. We chose to infuse for 4 weeks to allow both acute and chronic effects to be detected. As controls, some rats were infused with phosphate-buffered saline (PBS), and some rats received leptin peptide 83–108, which we have shown not to be taken up by the brain [13]. We also tested peptide 15–32, which was shown to have an intermediate level of brain uptake. Peptides 1–33 and 61–90 were found to produce increased food intake and weight gain in female rats, while peptide 15–32 produced a similar but smaller effect. In male rats, however, the same peptides failed to increase food intake and weight gain to a statistically significant extent. This was due, at least in part, to the masking effect of the large background weight increases that occurred in male rats during the experimental period. The results are consistent with antagonist actions by leptin peptides that are able to be taken up by the brain, at least in female rats.

2. Methods 2.1. Study design Two studies were performed. In each study, peptides or PBS were administered intravenously for 28 days, using Alzet osmotic minipumps. All peptides were infused at a constant rate of 20 μg/kg/day in the first study, and 60 μg/kg/day in the second study. Body weight was measured daily in both studies, and food intake was measured daily in the second study. Sprague–Dawley rats were used, and male and female rats were analysed separately. There were 5–6 rats in most treatment groups, the variable number being due to the exclusion of several rats due to catheter displacement or incomplete infusion (see under Peptide Infusions). Rats were assigned to each treatment group in such a way as to make average starting weights identical, or very close to it, across the groups. Rats were housed in groups of 2 or 3 per cage in experiment 1, and in individual cages in study 2. They were maintained on a 12 hour light:dark schedule, and allowed water and rat chow ad libitum. At the commencement of each study, the female Sprague–Dawley rats were between 12 and 15 weeks old. Their average starting weight was 289 g in study 1, and 284 g in study 2. The male rats were between 11 and 13 weeks old. Their average starting weight was 380 g for study 1, and 387 g for study 2. Animals were treated as humanely as possible at all times, and the study was approved by the University of Melbourne Animal Ethics Committee.

2.2. Peptides Leptin peptides 1–33, 15–32, 61–90 and 83–108 were used. The numbering is based on the sequence of mature human leptin, excluding the pro sequence. Peptides were synthesized by Ezbiolab (Westfield, IN, USA) and Mimotopes Pty Ltd (Clayton, Australia), and were HPLC purified by the suppliers. All peptides had structure and purity confirmation by HPLC and mass spectrometry. We also checked peptide integrity by HPLC, prior to loading the osmotic minipumps. Recombinant murine leptin was obtained from Phoenix Pharmaceuticals (Burlingame, CA, USA).

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2.3. Peptide infusions Alzet 2004 minipumps (Cupertino, CA, USA) were used to deliver the test solutions continuously for 28 days, at 0.25 μl per hour. Minipumps were filled with 200 μl of the appropriate peptide solutions in PBS (pH 7.4), or with PBS alone. Prior to filling, solutions were filter-sterilized through 0.2 μm syringe filters. Rats were anaesthetized with Xylazil (6 mg/kg) and Ketamine (100 mg/kg), and the jugular vein was exposed and catheterized with a polyethylene catheter. The catheter was connected to an Alzet 2004 minipump which had been primed before the operation to ensure prompt onset of infusion after implantation. The catheter was routed subcutaneously to the back of the neck, and the minipump was implanted subcutaneously behind the base of the neck. After 28 days, rats were anaesthetized again, and the Alzet minipumps were removed and checked to ensure complete delivery of their contents. In the few cases in which pump emptying was incomplete, or there was detachment of displacement of the catheter, the rats were excluded from the study. All rats remained apparently healthy throughout the experiment, and there did not appear to be any toxic effects from the peptides or minipump insertion. 2.4. Body weight and food intake Rats were weighed daily throughout each 28 day experimental period. In the second study, food intake was also monitored. To assess food intake, rats were housed in individual cages. 400 grams of standard rat chow pellets (4.6% fat content, Specialty Feeds, Glen Forrest, Western Australia) were added to each hopper at 10 am daily, and the remainder of the previous day's allotment was removed and weighed. The amount consumed was used to calculate the amount of food consumed per day per rat. Rats were allowed free access to water throughout the study. 2.5. Statistical analysis The effects of peptides on body weight were analysed by a mixed design two-way ANOVA with repeated measures, with peptide treatment and time as the variable factors. Repeated measures were used because data was gathered on each rat over multiple time-points. This was followed by post-hoc individual comparisons using Tukey's test. All analyses were performed using the Minitab 15 software package (State College PA, USA). The daily food intake data were analysed by two-way ANOVA with repeated measures, with peptide treatment and time as the variable factors. The food intake data were further analysed by subjecting each time-point to one-way ANOVA, with peptide treatment as the variable factor. Food intake data were additionally presented as averages over a weekly period, with the aim of reducing crowding and revealing overall trends. The weekly averages were analysed in the same way as the daily intake. 3. Results 3.1. Leptin administration To verify the experimental model, as well as provide a basis for comparison for the effects of the peptides, we tested the effects of 4 weeks intravenous infusion of leptin. Control rats had an average initial weight of 251 g, and were given PBS by osmotic minipump. The leptin group had an average starting weight of 255 g. The concentration of leptin in the minipumps was adjusted to provide a constant daily dosage of 125 μg/kg/day, based on their starting weight. After 4 weeks the average weight of the PBS group increased to 282 g. The mean weight increase of this group was 31 ± 4.7 g (Fig. 1). The group that received leptin lost weight in the first week, but thereafter gained weight. Their mean weight after 4 weeks was 270 g, and the mean weight increase was 15± 4.1 g. The effect of leptin was significantly

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Fig. 1. The effect of constant intravenous leptin infusion on body weight gain in female rats. Leptin was infused at 125 μg/kg/day for 28 days. Body weight gain was greater in control rats, which received PBS infusions, than in the leptin-treated rats, throughout the duration of the experiment. The effect of leptin was statistically significant (p b 0.001, 2-way ANOVA with repeated measures). * The difference was statistically significant at each of the 4 time points (p b 0.05, 2-way ANOVA and Tukey's post-hoc test). Results are means ± sem. N = 4 for both groups.

different to the controls (F1,6 = 17, p b 0.001, 2-way ANOVA with repeated measures). Moreover, the effects were statistically significant at each of the 4 time points (p b 0.05, Tukey's post-hoc pairwise comparisons). 3.2. Peptide experiment 1 Peptides 15–32, 61–90 and 1–33 were administered at 20 μg/kg/day, based on the initial weight, continuously for 28 days. In female rats, all 3 of the peptide groups exhibited greater average weight gain than the PBS group, evident at 2, 3 and 4 weeks (Fig. 2A). After 4 weeks, the greatest weight gain occurred with peptide 1–33; these rats gained an average of 45.7 ± 9.4 g in the 28-day period, more than double the weight gain of rats receiving PBS (20.3 ± 5.1 g). Peptide 61–90 caused almost as much weight gain (42 ± 6.4 g). Peptide 15–32, which has less brain uptake capability than the other two peptides, caused a smaller weight increase, 28.3 ± 4.4 g. To assess the significance of weight gain differences over the whole experiment, 2-way ANOVA with repeated measures was used. This confirmed a significant effect of treatment on weight gain (F3,15 = 4.9, p b 0.05). Tukey's posthoc analysis indicated that peptides 1–33 and 61–90, but not peptide 15–32, had greater effects on weight gain than PBS. At 2 weeks and 4 weeks, the weight gains in the peptide 1–33 and 61–90 groups were significantly greater than in the PBS group (p b 0.05, Tukey's post-hoc pairwise comparisons). Obviously enough, there was a significant effect of time (F3,15 = 44, p b 0.001) in the experiment. The ANOVA did not, however, reveal any interaction between treatment and time. In male rats, peptides 15–32, 61–90 and 1–33 each resulted in greater average weight gains than PBS, but the differences were not significant (Fig. 2B). This was due, at least partly, to the large weight gain of controls; male rats receiving PBS showed a consistent weight increase of about 20 g per week in this experiment, much greater than the 5 g per week average in females. After 4 weeks, PBS rat weight increased by 86.1 ± 12.5 g. A weight gain of 108.5 ± 23.3 g was achieved with peptide 1–33, and a weight gain of 105 ± 15.3 g was achieved with peptide 61–90. A weight gain of 96.5 ± 20 g was achieved with peptide 15–32.

Fig. 2. Body weight gain during experiment 1, in which peptides were infused at 20 μg/ kg/day for 28 days. Results are means ± sem. A. Females: Throughout the experiment, all 3 peptide groups recorded greater average weight increases than the PBS group. In an overall analysis of the entire 4 week study, body weight increases in rats receiving peptides 1–33 and 61–90 were significantly greater than in those receiving PBS (p b 0.05, two-way ANOVA with repeated measures, and Tukey's post-hoc test). * At 2 weeks and 4 weeks, the weight gains in the peptide 1–33 and 61–90 groups were significantly greater than in the PBS group (p b 0.05, Tukey's post-hoc pairwise comparisons). N = 5 for all groups except peptide 1–33 (N = 4). B. Males: There were no significant differences in weight gain between any of the treatment groups (twoway ANOVA with repeated measures). N = 8 for PBS, N = 6 for leptin 61–90, N = 5 for leptin 15–32, and N = 4 for leptin1–33.

108, a peptide previously shown to exhibit little or no brain uptake [13]. For peptides 1–33, 61–90 and 15–32, the degree of weight gain in female rats was essentially the same at 60 μg/kg/day as at 20 μg/kg/day (Fig. 3A). Female rats receiving leptin 61–90 had a 4-week weight gain of 46.8 ± 3 g, compared to 42± 6.4 g in experiment 1. This was significantly higher than the weight gain in rats receiving PBS (26 ± 3.7 g, p b 0.05). Leptin 1–33 caused a weight gain of 41± 11.2 g, and leptin 15–32 caused a weight gain of 38± 5.3 g, but these failed to reach significant differences compared to the PBS group. The weight gain with peptide 83–108, which is not taken up by the brain, differed very little from the weight gain with PBS, throughout the treatment period. Administration of leptin peptides to male rats at 60 μg per day did not result in any significant changes in weight gain compared to PBS treatment (Fig. 3B). Treatment with leptin 1–33 caused a weight gain of 139 ± 16 g, whereas Leptin 15–32 gave a weight gain of 134 ± 8.6 g, leptin 83–108 gave a weight gain of 130 ± 10 g, and PBS gave a weight gain of 117 ± 11 g. Leptin 61–90 was not given to males in this experiment. 3.4. Food intake

3.3. Peptide experiment 2 Peptides were administered by the same means, and for the same duration, as in experiment 1, but at a 3-fold higher dose, i.e., 60 μg/kg/ day. As an additional control, a group of rats received leptin peptide 83–

Food intake was monitored during experiment 2, in which animals were housed individually and peptides were infused at 60 μg/kg/day. Food intake was measured and plotted daily, so each point in Figs. 4A (females) and 4B (males) represents the average food intake during

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icance. We therefore analysed each day's data using one-way ANOVA. In this analysis, leptin 61–90 caused a significantly greater food intake than the PBS group on each of days 4 and 5 (p b 0.05). Leptin 1–33 resulted in significantly higher food intake than PBS on day 4 only. Food intake was also presented graphically as the average daily intake during each of the 4 weeks of the experiment (bar graphs in Fig. 4A and B). Surprisingly, no significant differences were evident, even by one-way ANOVA. This was probably a result of the marked

Fig. 3. Body weight gain during experiment 2, in which peptides were infused at 60 μg/ kg/day for 28 days. Peptide 83–108 is represented by the horizontally striped bar. Data were analyzed by two-way ANOVA with repeated measures, followed by post-hoc Tukey's comparisons. Results are means ± sem. A. Females: A significant body weight increase, relative to rats receiving PBS, occurred only in the group receiving peptide 61– 90. *p b 0.05, with respect to the PBS group. N = 5 for all groups except peptide 83–108 (N = 4) and peptide 1–33 (N = 4). B. Males: There were no significant differences in weight gain between any of the treatment groups. N = 5 for all groups.

the preceding 24 h. In female rats, analysis of the entire data set by two-way ANOVA, did not reveal any significant effects of peptide or time on food intake. Nevertheless, inspection of the data reveals that leptin 1–33 and leptin 61–90 both resulted in consistently higher values for daily food intake than PBS between days 3 and 17 (Fig. 4A). Clearly, the large number of data points and the very small numerical changes in food intake militated against achieving statistical signif-

Fig. 4. Food intake during experiment 2, with peptides infused at 60 μg/kg/day. Food intake was measured daily, and the female (A) and male (B) results are presented separately. The data is presented in two forms. In each of A and B, the upper tracings show the daily food intake on each day of the 28-day infusion. Error bars were deleted from these tracings to reduce clutter and improve clarity In the bar graphs, the same data is plotted as the average daily intake during each week. Data were analyzed by two-way ANOVA with repeated measures, and subsequently by performing separate one-way ANOVAs on each day of the experiment. A. Female rats were treated with PBS, peptide 1–33 or peptide 61–90. To assist clarity, the results for peptide 15–32 and peptide 83–108 are not shown. Two-way ANOVA did not reveal any significant effect of peptide treatment. Subsequently, one-way ANOVA was performed. Asterisks above the graphs indicate significant differences between the peptide 61–90 and PBS groups, which occurred on days 4 and 5 (p b 0.05, one-way ANOVA). The asterisk below the graphs indicates significant difference between the peptide 1–33 and PBS groups, which occurred on day 4 only (p b 0.05). Standard errors of the mean were between 0.3 and 1.6 g for all of the daily intake means. In average intake over a whole week, however, there were no significant differences between any of the treatment groups. N = 5 for PBS and peptide 61–90, N = 4 for peptide 1–33. B. Male rats were treated with PBS, peptide 1–33 or peptide 15–32. Peptide 83–108 has been omitted for the sake of clarity. There were no significant differences between any of the treatments. Standard errors of the mean were between 0.3 and 1.9 g for all of the daily intake means. N = 5 for all male groups.

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daily fluctuation by individuals, which tended to even out their weekly averages. In male rats, there were no significant differences in food intake between treatment groups at any stage of the experiment. Inspection of Fig. 4B shows, nevertheless, that the peptide 1–33 group had higher average food intake than the PBS group on the majority of days. 4. Discussion The present study follows from our previous report identifying leptin-derived peptides that could be taken up into the brain [13]. The peptides found to be capable of brain uptake contained sequences that have been mapped as receptor-interacting sequences, consistent with a role for the leptin receptor in brain uptake. Since we were confident of brain uptake, we decided to investigate the effects of peripheral administration of these peptides. An additional reason for testing the peptides in vivo, rather than characterizing their in vitro properties, was our concern that in vitro effects of leptin peptides do not always predict in vivo effects. This is discussed in greater depth below. In the present report, several leptin peptides produced increased food intake and/or weight gain when administered intravenously over a 4 week period. The peptides with the greatest brain uptake produced the greatest weight gain. Peptides 1–33 and 61–90, given by continuous infusion, both doubled the normal weight gain in female rats over a 4 week period. Peptide 15–32, which has a lesser degree of brain uptake, produced a lesser degree of weight increase. Peptide 83–108, having no brain uptake capability, similarly had no effect on weight gain. None of the peptides produced a decrease in weight, or a diminution of the normal weight gain. The likely interpretation is that peptides 1–33 and 61–90 are antagonists, blocking the action of leptin on its receptors. It is well established that leptin acts via receptors on hippocampal arcuate neurons to reduce food intake and hence body weight. Leptin peptides 1–33 and 61–90 both have good brain uptake, and it is likely that they act as antagonists on leptin receptors on arcuate neurons. The same is probably true of leptin 15–32, although its brain uptake is lower. An alternative possibility is that the peptides act as antagonists on receptor-mediated leptin transport across the Blood-Brain-Barrier. The food intake data in females indicates that the peptides have significant effects on food intake, but this could be explained by either mechanism, i.e.; by blocking leptin uptake into the brain, or by blocking leptin action at the arcuate neuron receptors. We are presently unable to distinguish between these two possible modes of action. In males, no statistically significant change in weight gain was recorded, although average weight gain was higher with peptides 15–32, 61–90 and 1–33 than with PBS. Indeed, peptides 61–90 and 1–33 produced weight changes of similar magnitude in females and males; In experiment 1, peptide 61–90 caused an additional weight gain of 21.7 g in female rats and an additional gain of 18.9 g in male rats (these weight gains are expressed relative to PBS controls); peptide 1–33 caused an additional weight gain of 25.3 g in female rats, and an additional gain of 22.4 g in male rats. However, male rats underwent a large background weight increase. In experiment 1, male rats receiving PBS gained 86.1 ± 12.5 g over the 4 week experiment, whereas the female PBS group gained just 20.3 ± 5.1 g. In experiment 2, male rats receiving PBS gained 117 ± 10.8 g over the 4 week experiment, whereas the female PBS group gained just 26 ± 3.7 g. Thus, the statistical significance of any peptide-induced weight increase in males may have been lost against the much larger underlying weight gain and its proportionally large individual variation. Our purpose in choosing mature 300 g female and 400 g male rats was to investigate the effects of peptides on body weight in an environment where body weight was stable, or relatively stable. We achieved this aim in the case of female rats, but were caught somewhat unawares by the vigorous weight increases in males. We found that, in rats of about 3 months of age, it is much easier to detect

body weight increases in females than in males, due to the former's relatively stable body weight baseline. We cannot conclude that leptin had no effect on body weight in males, because the average values were consistently highest in males receiving peptide 1–33. With regard to food intake in males, leptin peptides similarly failed to bring about significant increases. Inspection of Fig. 4B indicates, nevertheless, that on the majority of days after day 3, food intake was higher in rats receiving peptide 1–33 and peptide 15–32 than in rats receiving PBS. Visually, the initial response to peptide 1–33 in males resembled the female response, with a pronounced increase in food intake during the initial 6 days. In the absence of significant differences, however, the response to peptide 1–33 in males remains inconclusive. Despite the obscuring effect of rapid weight gain in males, the results imply that the leptin peptides have stronger effects in females than in males. This, in turn, suggests that leptin itself has stronger effects in females than in males. The presence of such a sexual dichotomy is, in fact, amply supported by the literature. Female rats exhibit more pronounced feeding suppression than male rats, in response to leptin [29], and it is known that the obesity to leptin relationship is more pronounced in females than males [30,31]. The basis for these effects is unclear. The most obvious candidate would be an interaction between estrogen and leptin. This is unlikely, however, since estrogen fluctuations throughout the ovarian cycle do not effect leptin-dependent eating behavior [29]. In summary, the lack of statistically significant effects in males may be a result of two factors; there appears to be greater sensitivity to leptin (and thus to peptide leptin antagonists) in females; the rapid underlying weight gain in males also tended to obscure the effects of the peptides on body weight. Leptin is a member of the 4-helical cytokine family and, like other members, contains 3 receptor binding sites. Binding sites I and III, both important for receptor activation, are located at the C- and N-termini, respectively, of helix D [15]. Residues within the loop between helices A and B, between positions 36 and 43, also contribute to receptor activation, but there is disagreement as to whether they contribute to site I [32] or site III [33]. The critical residues of binding site II, the site responsible for high affinity binding, are located in the regions 9–20 and 75–86, within helices A and C respectively [14]. The assertion that site II is most important for binding affinity, whereas site III is essential for receptor activation, is supported by experiments showing that mutagenesis of site III ablates effector activity while having little or no effect on receptor binding affinity [14,15]. The apparent antagonist role of peptides 1–33 and 61–90 is consistent with their localization to binding site II; this binding site confers the ability to occupy but not activate the receptor, which are the ideal attributes of a competitive antagonist. A number of previous studies of leptin peptides have been carried out, some employing peptides comparable to those in the present study. Gonzalez and co-workers tested a peptide corresponding to helix C, consisting of residues 70–95 [34]. This peptide was shown to bind the receptor with high-affinity (Ki ≈ 60 pM), albeit with an atypical binding curve, and to inhibit leptin in an in vitro assay [34]. Another group undertook in vitro studies on a slightly shorter helix C peptide, consisting of residues 70–89, and found it to bind to the receptor with high affinity and to be both antagonist and partial agonist [35]. A further group tested peptide 71–94 in an in vitro assay system, finding it to have agonist activity [36]. In the latter study, the peptide was not tested in the presence of leptin, so antagonist effects were not examined. Thus, three in vitro studies of decidedly similar helix C peptides have found them to act as an agonist [36], strong antagonist [34] and mixed antagonist and partial agonist [35], respectively. The reports of agonist activity in peptides 70–89 and 71–94 are a little surprising, given the established importance of site III for receptor activation [15]. Gonzales and coworkers also tested peptide 70–95 in vivo, showing it to act as an antagonist in mammary cancer cells and endometrium [37,38].

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Peptide 61–90, used in the present study, contains all of the receptor-interacting residues present in leptin 70–95, described above, since residues 91–95 do not participate in binding to the receptor [14,15]. It would therefore be expected, like peptide 70–95, to bind to the receptor with high affinity. The effects of peptide 61–90 on food intake and body weight are consistent with the predominantly antagonist actions reported for peptides 70–95 and 70–89. Despite the reports of helix C peptides with agonist activity in vitro, it is clearly the antagonist activity that predominates in the case of peptide 61–90 in vivo. Peptide 1–33 also increased weight gain and appeared to be a leptin antagonist. This peptide contains the helix A component of binding site II, and would be expected to share the properties of helix C peptides, of binding to the receptor while having a poor efficacy at activating it. This is in keeping with the work of Gonzalez and co-workers, who found that peptide 3–34 acts as an antagonist in female reproductive tissue [38]. In an earlier study, however, Samson and co-workers identified peptide 1–34 as a candidate leptin agonist, showing that it caused suppression of food intake, but not loss of body weight, after intra-cerebro-ventricular administration [39]. We note that this peptide was given centrally and at a much higher dosage than in our study, factors which may have contributed to the difference in results. In the present study, peptide 15–32 also increased weight gain and food intake and appeared to be an antagonist, albeit a weaker one than peptide 1–33. Leptin antagonists have considerable therapeutic potential. The ability to stimulate appetite and weight gain would be beneficial in disorders such as cachexia and anorexia. In addition, leptin has effects on other systems, as described in the introduction. Due to its actions on the immune system and autoimmunity [40,41], leptin has been proposed to play a role in the pathogenesis of conditions such as multiple sclerosis [42], rheumatoid arthritis [43] and atherosclerosis [5,44]. Thus, the future role of leptin antagonists may be quite broad. Much work has already been done on developing leptin antagonists, mostly recombinant muteins of the full-length leptin protein. By mutating key residues in site III, the main activating site, leaving site II intact, it has been possible to produce competitive antagonists with normal or nearnormal binding affinities [15,26,33]. Peptides, nevertheless, have several undeniable advantages over proteins. They are cheaper to produce in quantity, usually have no post-translational modification issues, and are less difficult to prepare to the specifications required for human administration. Importantly, they have size advantages for penetrating the blood brain barrier. We believe that the favorable brain uptake properties of the peptides in the present study allowed them to be effective at reasonable dosages, with peripheral administration. In future work, we plan to investigate the actions of leptin peptide antagonists in a variety of disease models. Acknowledgements We thank Circadian Technologies Ltd of Melbourne, Australia, for the financial support, and in particular the suggestions and encouragement of Leon Serry, former CEO. References [1] Harvey J, Ashford ML. Leptin in the CNS: much more than a satiety signal. Neuropharmacology 2003;44:845–54. [2] Bluher S, Mantzoros CS. Leptin in humans: lessons from translational research. Am J Clin Nutr 2009;89:991S–7S. [3] Farooqi IS, O'Rahilly S. Leptin: a pivotal regulator of human energy homeostasis. Am J Clin Nutr 2009;89:980S–4S. [4] Friedman JM. Leptin at 14 y of age: an ongoing story. Am J Clin Nutr 2009;89: 973S–9S. [5] Peelman F, Waelput W, Iserentant H, Lavens D, Eyckerman S, Zabeau L, Tavernier J. Leptin: linking adipocyte metabolism with cardiovascular and autoimmune diseases. Prog Lipid Res 2004;43:283–301. [6] Banks WA. The many lives of leptin. Peptides 2004;25:331–8.

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