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Design of a Synthetic Leptin Agonist: Effects on Energy Balance, Glucose Homeostasis, and Thermoregulation¹

Department of Medicine Albany Medical College, Albany, New York 12208

Address all correspondence and requests for reprints to: Patricia Grasso, Ph.D., Department of Medicine, Division of Endocrinology and Metabolism, MC-141, Albany Medical College, Albany, New York 12208. E-mail: grassop{at}mail.amc.edu

	Abstract

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We have previously reported that a synthetic peptide amide corresponding to amino acid residues 116–130 of mouse leptin, LEP-(116–130), reduces body weight gain, food intake, and blood glucose levels in ob/ob and db/db mice. In the present study we show that the activity of LEP-(116–130) resides in a restricted sequence between amino acid residues 116–122. A synthetic peptide corresponding to this sequence (Ser-Cys-Ser-Leu-Pro-Gln-Thr) has been named OB3. Single point D-amino acid substitution was used to study the structure-function relationship of each residue in OB3. D-Amino acid analogs of OB3 were synthesized by the solid phase method, purified to 98+%, and administered (1 mg/day, ip) for 7 days to female C57BL/6J ob/ob mice. The effects of the peptides on body weight gain, food and water intake, glucose homeostasis, and thermoregulation were assessed. In most cases, the efficacy of OB3 on all parameters tested was reduced by substitution of an L-amino acid with its corresponding D-isoform. A statistically significant increase (2.6-fold) in the weight-reducing effect of OB3, however, was observed by inversion of the configuration of the leucine residue at position 4 (Leu-4) of OB3 by substitution with its D-amino acid isoform [D-Leu-4]. Compared with OB3, mice treated with [D-Leu-4]-OB3 consumed 7.9% less food and 16.5% less water. Blood glucose was normalized to levels comparable to those in wild-type control mice within 2 days after initiation of [D-Leu-4]-OB3 treatment. Unlike native leptin, however, neither OB3 nor any of its D-amino acid-substituted analogs had any apparent effect on thermogenesis. Our results indicate that synthetic peptide strategies may be useful in the development of potent and stabile pharmacophores with potential therapeutic significance in the treatment of human obesity and its related metabolic dysfunctions.

	Introduction

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THE STUDY of single gene mutations in rodent models of obesity has led to the identification of a number of proteins, which include but are not limited to leptin (OB) and its receptor, neuropeptide Y, carboxypeptidase E, POMC, and the melanocortin 4-receptor, which play a role in regulating energy balance (1). Based on these animal studies, new pathophysiological pathways have been defined that may contribute to the development of human obesity.

Leptin, the protein product of the ob gene, exerts its influence on food intake, energy expenditure, body weight, and neuroendocrine function through actions on neuronal targets in the hypothalamus (2, 3). The gene for leptin is expressed predominantly by white adipocytes, although leptin synthesis has also been demonstrated in the gastric epithelium and placental trophoblast (4, 5). Plasma leptin concentrations are positively correlated with body mass index and are elevated in obesity (6) and decreased in anorexia nervosa (7). In addition to its effects on energy balance, leptin appears to influence the regulation of FSH, LH, ACTH, cortisol, and GH concentrations (8, 9, 10); to stimulate hematopoiesis (11, 12); and to induce both proliferation of CD4⁺ T cells and cytokine biosynthesis (13).

Mutation of the ob gene results in a syndrome that includes obesity, increased body fat deposition, hyperglycemia, hyperinsulinemia, hypothermia, and impaired thyroid and reproductive function in both male and female homozygous ob/ob obese mice (14). Administration of recombinant leptin to these mice or to normal lean or diet-induced obese mice results in weight loss through reduced food intake and increased energy expenditure (15, 16, 17). Using peripherally administered overlapping synthetic peptides corresponding to the entire sequence of secreted mouse leptin and the ob/ob mouse model, we have shown that the activity of leptin on energy balance and glucose homeostasis resides in a domain toward the C-terminus of the molecule, between amino acid residues 106–140 (18, 19). Most recently, we have discovered that the most potent of these peptides, LEP-(116–130), was effective in reducing body weight gain, food intake, and blood glucose levels in genetically obese db/db mice, suggesting that the effects of this peptide may not be mediated through the long isoform of the leptin receptor (20). Other laboratories have reported that LEP-(116–130) stimulates PRL and LH secretion in rats (21), and enhances proliferative activity in the immature rat adrenal cortex (22).

In the present study we show that the activity of LEP-(116–130) resides in a restricted sequence between amino acid residues 116–122. A synthetic peptide corresponding to this sequence (Ser-Cys-Ser-Leu-Pro-Gln-Thr) has been named OB3 (patent pending). Single point D-amino acid substitution was used to create a more potent peptide analog of OB3, [D-Leu-4]-OB3. The effects of this analog on body weight gain, food and water intake, glucose homeostasis, and thermogenesis in obese female C57BL/6J ob/ob mice are described.

	Materials and Methods

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Peptide synthesis, purification, and characterization
LEP-(116–130), its truncated analogs (Table 1), and D-amino acid-substituted analogs (Table 2) corresponding to residues 116–122 (OB3) of mouse leptin (23) were synthesized on a Rainin model PS3 automated peptide synthesizer (Ridgefield, NJ) by the solid phase method (24). Fluorenylmethoxycarbonyl-protected L- or D-amino acids were used. The peptides were assembled on Rink’s 4,2',4'-dimethyloxyphenol-fluorenylmethoxycarbonyl-aminomethyl)-phenoxy-amide resin (Fisher Scientific, Springfield, NJ). Completed peptide amides were cleaved from the resin with trifluoroacetic acid (84%), using sterile deionized water (4%), ethanedithiol (4%), anisole (4%), and thioanisole (4%) as scavengers. The cleaved peptides were precipitated with anhydrous ether and dried by lyophilization. The peptide amides were purified to 98+% on a Rainin Dynamax preparative column (21.4 mm x 25 cm; C18; 300-A pore diameter). The final peptide products were evaluated for purity by reverse phase liquid chromatography on a Rainin Dynamax analytical column (4.6 mm x 25 cm; C₁₈; 300-A pore diameter) using a linear acetonitrile gradient (0–100%) containing 0.05% trifluoroacetic acid and a flow rate of 1 ml/min. Each peptide amide was represented as a single peak in the chromatographic profile. Fidelity of synthesis was confirmed by mass spectral analysis.

Feeding and weighing schedule. On day 1 of the study and on each day thereafter, a water bottle containing 200 ml water and 200 g pelleted rodent diet (Prolab Rat, Mouse, Hamster 3000, Ralston Purina Co., St. Louis, MO; 22% crude protein, 5% crude fat, 5% fiber, 6% ash, and 2.5% additional minerals) were added to each cage between 0900–1100 h. Food and water remaining after 24 h were measured to the nearest 0.1 g and 0.1 ml, respectively, and the average amount consumed per mouse was calculated (mean ± SEM; n = 6). The mice were weighed once daily between 0900–1100 h on an Acculab V-333 electronic balance (Cole Parmer, Vernon Hills, IL)

Peptide administration. Peptide amides were dissolved in sterile PBS (pH 7.2), and administered daily for 7 days between 1500–1600 h in a single 1 mg/0.2 ml ip injection. Control mice received 0.2 ml PBS (ip) only.

Measurement of blood glucose. Blood was drawn from the tail vein of each mouse 2 h before the onset of the dark period at the beginning of the study and after 2, 4, and 6 days of peptide treatment. The blood was applied to a test strip, and glucose levels were measured with a Glucometer Elite (Bayer Corp., Elkhart, IN) glucose monitor.

Thermoregulatory studies. After 4 and 7 days of peptide treatment, sensitivity to cold was examined by placing the mice, without food or water, in individual cages in a cold room with an ambient temperature of 4 C. Body temperature was measured with a rectal probe every hour for 4 h.

Toxicity
No obvious toxic side-effects, such as reduced activity level or changes in coat quality or stool consistency (diarrhea), were associated with any of the peptides tested in this study. Body temperature, determined by rectal probe, was also unchanged by peptide treatment. All animals appeared healthy throughout the study and were killed at its conclusion by pentobarbitol injection (100 mg/kg BW, ip) by personnel of the Animal Resources Facility.

These animal procedures were reviewed and approved by the animal care and use committee of the Albany Medical College and are in accordance with institutional guidelines.

Statistical analysis
Changes in body weight, and differences in food and water intake, blood glucose levels, and body temperature between peptide-treated and vehicle-injected control mice were analyzed by ANOVA and were considered significant at P < 0.05.

	Results

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Identification of the active epitope in LEP-(116–130)
A series of truncated peptide analogs of LEP-(116–130) corresponding to N-terminal amino acid residues 116–120, 116–121, 116–122, 116–123, and 116–124 (Table 1) were synthesized, purified, and characterized as described in Materials and Methods. LEP-(116–120) was chosen as the lead peptide in these efforts because it represents the overlapping region of LEP-(116–130) and the C-terminus of a second active peptide, LEP-(106–120) (17, 18), immediately upstream of LEP-(116–130). We hypothesized that there might be a conserved domain at the C-terminus of LEP-(106–120) and at the N-terminus of LEP-(116–130), amino acid residues 116–120, which contains an active epitope that gives both peptides their ability to modulate body weight gain in C57BL/6J ob/ob mice.

Female C7BL/6J ob/ob mice were given a single ip injection of vehicle (PBS, pH 7.2), LEP-(116–130), or truncated peptide (1 mg/200 µl PBS) for 7 consecutive days. Daily changes in body weight compared with pretreatment weights of vehicle-injected control and peptide-treated mice are shown in Fig. 1. Mice given vehicle for 7 days increased their initial body weight by 12.5%, whereas mice receiving LEP-(116–130) lost 12.2% of their initial body weight (Fig. 1A). Truncation of LEP-(116–130) to its five (residues 116–120) or six (residues 116–121) N-terminal amino acids substantially reduced its efficacy (data not shown). Mice receiving LEP-(116–122) (OB3) for 7 days, however, were able to resist the increase in body weight seen in vehicle-injected control mice and maintained their initial body weight (Fig. 1B). Addition of Ser¹²³ or Ser¹²³ and Gly¹²⁴ did not improve the efficacy of OB3 (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 1. Effects of seven daily injections (1 mg/day, ip) of LEP-(116–130) and its truncated analog LEP-(116–122) on body weight gain in female C57BL/6J ob/ob mice. The graph shows the changes in body weight (expressed as percentage of the initial weight) in mice treated with vehicle or leptin-related synthetic peptide amide. Each value represents the mean ± SEM change in body weight for a group of six mice.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effects of seven daily injections (1 mg/day, ip) of OB3 and its D-amino acid-substituted analog [D-Leu-4]-OB3 on body weight gain in female C57BL/6J ob/ob mice. The graph shows the changes in body weight (expressed as a percentage of initial weight) in mice treated with vehicle, OB3, or [D-Leu-4]-OB3. Each value represents the mean ± SEM change in body weight for a group of six mice.

View larger version (55K): [in this window] [in a new window]   Figure 3. Effects of seven daily injections (1 mg/day, ip) of OB3 and its D-amino acid-substituted analogs on cumulative food (A) and water (B) intake by female C57BL/6J ob/ob mice. Each bar represents cumulative food and water consumption per mouse (mean ± SEM; n = 6 mice/group).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of cold stress (4 h at 4 C) on thermogenesis in female C57BL/6J wild-type (+/+) and ob/ob mice. Mice were treated with vehicle (0.2 ml/day, ip) for 7 days. On day 8, mice were subjected to cold stress as described in Materials and Methods. Body temperatures were measured with a rectal probe 1, 2, 3, and 4 h later. Each bar and vertical line represent the mean ± SEM core temperature in a group of six mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recombinant leptin has recently entered the arena as a potential pharmacophore for inducing appetite suppression and fat mobilization in humans. This therapy, if as successful in humans as in rodents, may become a very powerful multifaceted approach to weight loss, because leptin not only decreases appetite, but also increases metabolic rate and reduces body fat mass (8, 15). The results of phase I and II clinical trials with recombinant leptin, however, have met with variable success, and undesirable injection site reactions have been reported (27).

Clinical data have indicated that most obese humans are leptin resistant, even though they synthesize 4- to 5-fold more leptin than nonobese humans (28). Thus, the development of small, soluble, leptin-like agonists that can bypass the blood-brain barrier, which has recently been suggested a locus of leptin resistance (6, 29, 30), or can achieve their anorexigenic effects via mechanisms that may be similar to but independent of those of leptin may prove to be a more effective approach to the treatment of human obesity. In light of these observations and our recent discovery that the effects of LEP-(116–130) on energy balance and glucose homeostasis may not require peptide activation of the long form of the leptin receptor (20), efforts to identify the molecular determinants of leptin action and to increase the efficacy of these epitopes take on added importance. To this end, we have been able to define amino acid residues 116–122 (OB3) of mouse leptin as the minimal active epitope in this region of the molecule and to increase the potency of OB3 by inversion of the configuration of the L-leucine residue at position 4 by substitution with its D-isoform.

LEP-(116–130) is a synthetic peptide that has been shown to regulate energy balance and blood glucose levels in ob/ob and db/db mice (18, 19, 20), stimulate PRL and LH secretion in male rats (21), and enhance proliferative activity in rat adrenal cortex (22). In the present study we used a truncation strategy to demonstrate that the active epitope in LEP-(116–130) is composed of amino acid residues 116–122. We have named a synthetic peptide amide corresponding to this epitope OB3 (patent pending). Single point D-amino acid substitution was then used to study the structure-function relationships of each amino acid residue in OB3 and to increase its efficacy.

Our results suggest that the restricted domain represented by OB3 contains a functional epitope that has the ability to mimic at least some of the effects of leptin on energy balance and glucose homeostasis. This finding was not altogether surprising, for there is a growing body of evidence that indicates that the functional epitopes of many protein ligands are much smaller than their structural epitopes (31, 32, 33). Thus, an important goal in peptide research will be to develop strategies for the design of small peptide ligands with specific physical, chemical, and biological properties that will enhance their biological activity and stability.

The design of peptide ligands has generally involved the introduction of conformational constraints into native sequences by techniques that include but are not limited to D-amino acid substitution or cyclization (34). Systematic replacement of L-amino acids by their D-amino acid isoforms can be used to determine the stereostructural requirements of specific residues in a peptide for peptide-receptor interaction and to assess the contributions of certain secondary structural motifs, e.g. -helix or ß-turn, to the bioactivity of the peptide (35). This approach has also been shown to increase peptide resistance to enzymatic hydrolysis and to enhance the properties of biologically active peptides, including receptor binding, functional potency, and duration of action (36, 37, 38, 39). In our laboratory we have recently used this approach to develop a more potent peptide analog related to an active epitope between amino acid residues 32–46 of the glycoprotein hormone common -subunit (40).

Under physiological conditions, most peptides exist as a mixture of more or less well defined interconverting conformers in solution, only some of which are biologically active. Thus, an important strategy in peptide design is to enhance the concentration of a biologically active conformer by introducing conformational constraints into the peptide structure (41). As stated earlier in this report, D-amino acid substitution has been widely used for this purpose (36, 37, 38, 39).

Of the eight D-amino acid-substituted peptide analogs tested in this study, only one analog, [D-Leu-4]-OB3, was more potent (2.6-fold) in reducing body weight gain than native OB3. This analog also had greater anorexigenic activity than OB3 and significantly reduced water intake. The most striking action of [D-Leu-4]-OB3, however, is related to its effects on blood glucose. In contrast to OB3, which maximally reduced blood glucose levels by approximately 100 mg/dl, [D-Leu-4]-OB3 normalized blood glucose to levels seen in nondiabetic wild-type mice within 2 days of peptide treatment. It seems reasonable to suggest that normalization of blood glucose by [D-Leu-4]-OB3 may be physiologically associated with the reduced water consumption observed in mice treated with this peptide by decreasing the polyuria associated with hyperglycemia.

A similar correlation between blood glucose levels and water intake was observed in mice treated with [D-Pro-5]-OB3, although normalization of blood glucose occurred with a different time course, i.e. after 4 days of peptide treatment. The mechanism by which [D-Leu-4]-OB3 and [D-Pro-5]-OB3 exert their antihyperglycemic effects is unclear at the present time and is currently under investigation in our laboratory. A simple decrease in caloric intake, however, is an unlikely sole mechanism given the rapidity with which normalization of serum glucose occurred (2 days). The data presented in this report, however, suggest a possible role for leptin-related peptides in the treatment of diabetes.

Also worthy of note is the observation that, except for [D-Leu-4]-OB3 and [D-Pro-5]-OB3, none of the other D-amino acid-substituted analogs was as effective as OB3 in most of the parameters measured. These results suggest that OB3 contains a sequence that is highly sensitive to changes in stereochemical configuration. Our data indicate that all of the functional groups in the molecule must be aligned in their appropriate spatial positions if the peptide is to achieve maximal biological activity.

The fact that neither OB3 nor any of its D-amino acid-substituted analogs had any effect on thermogenesis, as does native leptin (42), may mean that this region of the molecule is not involved in transducing signals related to the activation of mitochondrial uncoupling proteins. Thus, one could speculate that there may be discrete domains within the leptin molecule that are responsible for its many and varied metabolic effects. If this is the case, our data suggest that the domain represented by OB3 does not participate in the thermoregulatory activity of leptin.

In summary, using a truncation strategy, we have shown that the activity of LEP-(116–130) resides in a restricted domain between amino acid residues 116–122. A synthetic peptide representing this region has been named OB3. D-Amino acid substitution was used to determine the stereospecificity of each residue in OB3 and to create a more potent analog of OB3, [D-Leu-4]-OB3. Our results suggest that synthetic peptide strategies may be useful in the development of potent and stabile pharmacophores with potential therapeutic significance in the treatment of human obesity and its related metabolic dysfunctions.

	Acknowledgments

 
The authors thank Marilyn Brown for her excellent technical assistance.

	Footnotes

 
¹ This work was supported by a grant from the Dr. Willard B. Warring Memorial Fund.

Received February 2, 2000.

	References

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