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In Vivo Effects of Leptin-Related Synthetic Peptides on Body Weight and Food Intake in Female ob/ob Mice: Localization of Leptin Activity to Domains Between Amino Acid Residues 106–140¹

Department of Medicine (D.W.L.) and Department of Biochemistry and Molecular Biology (P.G., D.W.L.), Albany Medical College, Albany, New York 12208

Address all correspondence and requests for reprints to: Dr. Daniel W. Lee, Division of Endocrinology and Metabolism A-44, Albany Medical College, Albany, New York 12208.

	Abstract

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In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106–167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg ip injections of LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106–120), LEP-(116–130), or LEP-(126–140) were most pronounced during the first week of peptide treatment. Within 7 days, mice receiving these peptides lost 12.3%, 13.8%, and 9.8%, respectively, of their initial body weights. After 28 days, mice given vehicle alone, LEP-(136–150), LEP-(146–160), or LEP-(156–167) were 14.7%, 20.3%, 25.0%, and 24.8% heavier, respectively, than they were at the beginning of the study. Mice given LEP-(106–120) or LEP-(126–140) were only 1.8% and 4.2% heavier, respectively, whereas mice given LEP-(116–130) were 3.4% lighter. Food intake by mice receiving LEP-(106–120), LEP-(116–130), or LEP-(126–140), but not by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167), was reduced by 15%. The results of this study indicate 1) that leptin activity is localized, at least in part, in domains between residues 106–140; 2) that leptin-related peptides have in vivo effects similar to those of native leptin; and 3) offer hope for development of peptide analogs of leptin having potential application in human or veterinary medicine.

	Introduction

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THE MOUSE ob gene and its human homologue have been cloned (1). Leptin, the product of this gene, is a 167-amino acid plasma protein that is synthesized in adipose tissue and acts as a blood-borne hormone responsible for weight maintenance (2, 3, 4). In C57BL/6J ob/ob mice, a single base mutation of the ob gene at codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated, inactive form of leptin (5). A phenotype that includes obesity, increased body fat deposition, hyperglycemia, hyperinsulinemia, hypothermia, and impaired thyroid and reproductive functions is associated with this mutation (6, 7).

Ob messenger RNA (mRNA) expression has been shown to be increased in genetically obese mice (1), rats made obese by ventromedial hypothalamic lesions (8), and obese humans (9). Peripheral administration of low doses of recombinant leptin to hormone-deficient ob/ob mice corrects their hyperglycemia, hyperinsulinemia, and hypothermia, whereas higher doses normalize food intake and body weight (3, 4, 5, 10). Central administration of the ob protein inhibits feeding in ob/ob mice, and studies confirming the presence of leptin receptors in the brain recently have been reported (10, 11). Neuropeptide Y neurons in the hypothalamic arcuate nucleus may be also be involved in leptin action (12, 13).

In humans, the ob gene is expressed exclusively in adipose tissue and codes for a protein that is 84% homologous to mouse leptin (14, 15). To date, no deleterious mutations and only one single-base polymorphism has been detected in the human ob gene (16). Possible linkage of extreme obesity to markers flanking the human ob gene, however, has recently been suggested (17, 18).

Expression of the Ob gene in humans is highly correlated with body fat and body mass index, with greater expression observed in obese than in normal-weight individuals (19). Leptin concentrations in the serum of obese individuals have been found to be approximately 4-fold higher than in normal-weight subjects. A similar correlation between leptin concentrations and ob mRNA levels in adipose tissue of obese individuals has also been made (20). Taken together, these observations suggest that most obese humans are resistant to endogenously circulating leptin.

In this report, we present data that demonstrate for the first time that leptin-related synthetic peptide amides have the ability to regulate body weight and food intake in genetically obese mice. Our results suggest that alternate therapies utilizing synthetic peptide analogs of leptin for the treatment of obesity may be feasible, and offer hope for development of more potent peptide analogs with potential application in human or veterinary medicine.

	Materials and Methods

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Peptide synthesis, purification, and characterization
Six overlapping peptide amides (Table 1) corresponding to residues 106–167 of mouse leptin (1) were synthesized by Quality Controlled Biochemicals (QCB, Hopkinton, MA) using the fluorenylmethoxycarbonyl-protection scheme. The peptide amides were purified to greater than 95% and evaluated by reversed-phase HPLC QCB. Each peptide amide was represented by a single peak. Fidelity of synthesis was confirmed by mass spectral analysis QCB.

Feeding and weighing schedule. On day 1 of the study, and on each day thereafter, 200 g pelleted rodent diet was added to the hopper in each cage between 0900–1100 h. Food remaining after 24 h was weighed to the nearest 0.1 g, and the average amount of food consumed per animal was calculated (mean ± SEM, n = 6). The mice were allowed water ad libitum throughout the study.

The mice were weighed once daily between 0900–1100 h using a standard mouse balance (Taconic Farms, Germantown, NY).

Peptide administration. Peptide amides were dissolved in sterile, pH 7.2, or Ringer’s solution and administered daily between 0900–1100 h in a single 1-mg/0.2-ml ip injection for 28 days. Control mice received 0.2 ml PBS only.

Euthanasia. No apparent toxic side effects, e.g. lethargy, diarrhea, change in coat quality, were associated with any of the peptides used in this study. All animals remained healthy throughout its course, and were euthanized at its conclusion by pentobarbitol injection (100 mg/kg body weight, 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
Differences in body weight and food intake between peptide-treated mice and vehicle-injected control mice were analyzed by Dunnett’s Multiple Range Test and were considered significant when P < 0.01.

	Results

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Rationale for design of leptin-related peptides
It is known that in genetically obese mice, a nonsense mutation in the ob gene changes the coding sequence for arginine (Arg)-105 in normal leptin to a stop codon. The resulting mRNA is translated into a truncated, inactive form of leptin (1). These observations suggest that leptin activity may be localized, at least in part, toward the C-terminus of the protein in domain(s) distal to Arg-105. To test this hypothesis, we prepared a series of six overlapping synthetic peptide amides (Table 1) corresponding to residues 106–167 of mouse leptin (Fig. 1) and examined their effects on body weight and food intake in female ob/ob mice.

View larger version (10K): [in this window] [in a new window]   Figure 1. Primary structure of mouse leptin as predicted by ob cDNA isolated from a mouse white fat complementary DNA library (1). The overlapping regions of the synthetic peptide amides used in this study are indicated.

View larger version (40K): [in this window] [in a new window]   Figure 2. Effects of daily injections (1 mg/g body weight, ip) of leptin-related peptide amides on body weight in female ob/ob mice. Graph shows changes in body weight (in grams) from pretreatment weight for obese female ob/ob mice treated with vehicle or leptin-related peptide amides. Each value represents mean change in body weight for a group of six mice. Mean pretreatment weight for each group is shown in Table 3. Mean body weight of mice given LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) different from vehicle-injected control group when compared by Dunnett’s multiple range test.

View larger version (16K): [in this window] [in a new window]   Figure 3. Individual body weights of female ob/ob mice given daily injections (1 mg/g body weight, ip) of LEP-(106–120), LEP-(116–130), or LEP-(126–140) peptide amide for 7 or 28 days. Each point represents change in body weight (in grams) from pretreatment weight of each mouse (n = 6) on day 7 (A) and day 28 (B) of study.

To be certain that the observed effects of leptin-related peptides on body weight were specific, a group of mice (n = 6) was injected daily with a similar concentration of CaM-(17–31), a peptide amide 15 amino acids long corresponding to residues 17–31 of calmodulin, having no sequence similarity to leptin. Body weight and food intake by mice receiving CaM-(17–31) were not significantly different from mice injected with vehicle alone (data not shown).

Effects of leptin-related peptide amides on food intake
Cumulative food intake (grams per mouse in each group, n = 6) throughout the study is shown in Fig. 4. Although significant weight loss compared with vehicle-injected control mice occurred in mice given LEP-(106–120), LEP-(116–130), or LEP-(126–140) peptide amides during the first 7 days of the study (Fig. 2 and 3A), there was no statistically significant difference in their food consumption compared with control mice during that time. Food consumption by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) peptide amides was also similar to that of vehicle-injected control mice.

View larger version (40K): [in this window] [in a new window]   Figure 4. Cumulative effects of leptin-related peptide amides on food consumption by female ob/ob mice. After first 7 days of treatment, mice injected daily (1 mg/g body weight, ip) with LEP-(106–120), LEP-(116–130), or LEP-(126–150) peptide amide consumed significantly (P < 0.01) less food than vehicle-injected control mice or mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) peptide amide. Each bar represents cumulative food consumption per mouse (mean ± SEM, n = 6 mice/group) after 7, 14, 21, and 28 days of treatment. *, Food consumption significantly (P < 0.01) less than vehicle-injected control mice.

	Discussion

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Significant advances in defining the molecular mechanisms that integrate energy intake, energy expenditure, and energy storage in adipose tissue have recently been made. Currently, a growing body of evidence suggests involvement of circulating signals that are produced in adipose tissue, are proportional to its mass, and act on the brain to regulate feeding behavior and energy expenditure. Discovery of the ob gene (1), its product leptin (3, 4, 10), and a cerebral receptor for leptin (11, 21, 22) have broadened our understanding of obesity and may lead to the development of new treatments for this complex and potentially debilitating disorder.

A number of animal models have been used to study obesity. Two distinct mutations of the ob gene have been demonstrated in homozygous ob/ob mice. One of these mutants, SM/Ckc-+^Dacob^2J/ob^2J, expresses no leptin mRNA (2), whereas the other, C57BL/6J, overexpresses by 20-fold a mRNA species which encodes a truncated, inactive form of the protein (1). The obese phenotype in both strains of ob/ob mice has been attributed to a deficiency in active leptin. No similar mutations, however, have been detected in the human ob gene (14, 23).

A number of laboratories have shown that treatment of ob/ob mice, normal lean, or diet-induced obese mice with recombinant leptin results in weight loss through reduced food intake and increased energy expenditure (3, 4, 5, 10, 12). In the present study, we examined the effects of synthetic peptide amides corresponding to restricted domains of the leptin sequence on food intake and body weight in female C57BL/6J ob/ob mice that lack endogenously circulating active leptin. Based on our previous studies with FSH and FSH receptor-related synthetic peptides (24, 25, 26), we hypothesized that the entire sequence of leptin might not be required for its action, and that smaller peptides encompassing one or more active domains within its primary structure may be sufficient to induce satiety and stimulate weight loss in this animal model.

Our data suggest the presence of such domains between residues 106–140 of leptin, and indicate that synthetic peptides representing these domains have the ability to mimic, although at a lower potency, the effects of recombinant leptin on body weight and food intake in this animal model. In the original leptin study by Halaas et al. (3), on which our study was based, daily ip injections of approximately 300 µg (18.75 nmol) of recombinant leptin reduced body weight by approximately 40% in 33 days and stabilized food intake at 40% of vehicle-injected control mice after 4 days. Our most active peptide, LEP-(116–130), given daily at 1 mg (640.6 nmol), caused a 3.43% weight loss and reduced food intake by 15% after 28 days. The decreased potency of our peptide was not surprising, however, given the lower affinity most peptides have for receptors when compared with intact protein ligands (27).

Interestingly, the greatest activity observed in this study was elicited by LEP-(106–120) and LEP-(116–130) peptide amides, suggesting that the overlapping residues in both peptides, Ser-116, Cys-117, Ser-118, Leu-119, and Pro-120 are important to leptin action and may comprise an active site in this region of the molecule. Studies with truncated peptides that will allow us to define this site with even greater precision are currently in progress.

Worthy of note is the observation that LEP-(126–140) peptide amide, also shown to restrict weight gain and reduce food intake in this study, contains Pro at position 128, Ser at position 130, and Leu at position 131. These residues may be functionally homologous to Ser-116, Leu-119, and Pro-120 in LEP-(106–120) and LEP-(116–130) peptide amides and may contribute to the activity of LEP-(126–140) peptide amide. The observed inability of LEP-(136–150), LEP-(146–150), or LEP-(156–167) peptide amide to modulate food intake or weight gain suggests the absence of active sites in this region of the protein.

Full activity of leptin, however, appears to involve sites proximal to residue 105, as recently suggested by Samson et al. (28), as well as sites between residues 106–140. Although these investigators found that a cyclic peptide corresponding to residues 116–167 of mouse leptin was without activity in their assay system, the discrepancy between these results and our own may be explained by the size of the peptide tested. The inactive peptide (116–167) included residues 136–167, a region that our data suggest is without activity. Thus, it is possible that the presence of these residues, as well as the cyclization used to conformationally constrain the peptide, may have locked the larger peptide into a conformation less favorable for receptor interaction and masked the activity of the smaller region. Mapping the N-terminus of leptin with smaller overlapping peptides via strategies similar to that used in this study, will allow us to make this determination with certainty.

We considered the possibility that the weight loss seen in mice given LEP-(106–120), LEP-(116–130), or LEP-(126–140) might have been related to some as yet unidentified toxic effect of these peptides. Several lines of evidence, however, suggest otherwise. First, we noted no ill effects of these peptides on the appearance or behavior of these mice. Their coat quality, stools, and activity level appeared similar to vehicle-injected control mice. Second, as noted earlier, we observed that the weight-reducing effect of these peptides was most pronounced during the first week of administration. If this were accompanied by some toxic effect, this effect would be expected to become more pronounced as the study progressed. This was not the case; the mice appeared healthy for the entire 4 weeks of the study, and until they were euthanized 14 days after its conclusion. Third, the effects of the peptides on body weight were apparent before any significant changes in food intake were observed. This suggests that the mice given these peptides were more metabolically active after peptide administration. Such would not be the case if the peptides were somehow toxic. Taken together, these observations indicate that the weight loss induced by LEP-(106–120), LEP-(116–130), and LEP-(126–140) was not the result of a nonspecific toxic effect associated with continued peptide administration.

The results of this study demonstrate for the first time that peripherally administered synthetic peptides corresponding to restricted domains within the primary structure of leptin are biologically active and have the ability to alter feeding behavior and energy balance in female obese ob/ob mice. Extension of these initial studies to other obese and nonobese animal models, together with an examination of their effects on thermoregulation, glucose and insulin concentrations, body fat deposition, thyroid, and reproductive functions will further elucidate the therapeutic potential of biologically active peptide analogs of leptin.

	Footnotes

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

Received October 15, 1996.

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