Absence of Soluble Leptin Receptor in Plasma from dbPas/dbPas and Other db/db Mice*

Cai LiDagger , Ella IoffeDagger , Naseem FidahuseinDagger , Eileen Connolly§, and Jeffrey M. FriedmanDagger §

From the Dagger  Howard Hughes Medical Institute and § The Rockefeller University, New York, New York 10021

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The leptin receptor (Ob-R) is alternatively spliced into at least five different RNAs designated Ob-R(a-e). Ob-R(a-d) predict receptors with a single transmembrane domain, and Ob-Re predicts a secreted form of the receptor. The presence of an ~120-kDa soluble leptin receptor in mouse plasma was confirmed by precipitation with leptin-Sepharose beads followed by immunobloting with anti-leptin receptor antibodies. The soluble leptin receptor is larger than that predicted by the primary sequence. Deglycosylation of the receptor with peptide N:glycosidase F results in a decrease in molecular mass to a size consistent with that of the primary sequence. The secreted receptor was present in plasma from wild type mice but was truncated in plasma from db3J/db3J and absent in dbPas/dbPas plasma. Although db3J/db3J mice are known to have a frameshift mutation at amino acid 625, the basis for the mutation in dbPas/dbPas mice was not known. Further studies indicated that dbPas/dbPas mice carry a duplication of exons 4 and 5 of Ob-R. This mutation introduces a premature stop codon into the protein at amino acid 281. The absence of Ob-R in db3J/db3J and dbPas/dbPas mice confirm the identify of the 120-kDa plasma protein as Ob-Re.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Leptin is an adipocyte hormone that functions as an afferent signal in a negative feedback loop regulating body weight (1). Administration of exogenous leptin to rodents results in a dose-dependent loss of adipose tissue mass (2-7). Leptin exerts its weight-reducing effects via interaction with a receptor, Ob-R, which is localized in the hypothalamus and other tissues (8, 9). Ob-R is a member of the cytokine receptor family and is alternatively spliced. Four of the splice variants, Ob-R(a-d), encode a receptor with a single transmembrane domain and a cytoplasmic region of variable length (9). Ob-Rb encodes a leptin receptor with a long intracytoplasmic region that contains several motifs known to be important for protein-protein interactions and signal transduction (9, 10). One of the splice variants, Ob-Re, does not encode a transmembrane domain and predicts a secreted form of the receptor (11).

The db gene has been shown to be allelic with Ob-R. Three mouse alleles of db are available, each of which leads to severe early onset obesity and diabetes. The C57BL/Ks db/db mutation affects splicing of Ob-R and leads to the specific loss of Ob-Rb RNA (9, 10). db3J/db3J mice carry a frameshift mutation in the amino terminus of the receptor that affects all of the splice forms (11). The nature of these mutations predicts that C57BL/Ks db/db mice should be missing only the Ob-Rb isoform and that 129 db3J/db3J mice should carry mutations of all receptor forms. As the basis for the dbPas/dbPas mutation was not previously known, the affect of this allele on receptor protein(s) could not be predicted.

Assays of the plasma levels of Ob-R protein were made in wild type and mutant mice. Specific antibodies to Ob-R were used to assay for the presence of Ob-Re, the secreted form of the leptin receptor, in plasma from wild type and mutant mice. An ~120-kDa protein corresponding to a glycosylated form of Ob-Re circulates in plasma from wild type and C57BL/Ks db/db mice. The wild type protein was absent in plasma from 129 db3J/db3J mice as well as dbPas/dbPas animals. dbPas/dbPas mice were shown to carry a duplication in the extracellular region of Ob-R that ablates expression of all of the receptor variants.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
Discussion
References

Materials-- Recombinant mouse leptin was obtained from Amgen (Thousand Oaks, CA). Baculovirus cloning vector pMelBac was purchased from Invitrogen (Carlsbad, CA). PNGase F1 was from New England Biolabs, Inc. (Beverly, MA) ob/ob mice, db3J/db3J mice, as well as wild type C57Balb/C mice were from Jackson Labs (Bar Harbor, ME). dbPas/dbPas and lean littermates were from the Pasteur Institute (Paris, France).

Sucrose Gradient Centrifugation of Leptin in Mouse Plasma and PBS-- Continuous sucrose gradient was used for rate zonal centrifugation. Mouse recombinant leptin (Amgen, 50 ng) in a final volume of 100 µl of either PBS or ob/ob serum were loaded onto a linear gradient of 5-20% w/v sucrose in PBS (pH 7.4). The run parameters were 55,000 rpm at 5 °C for 12 h using the SW55 Ti rotor. At the end of the run, 13 fractions of 400 µl each were collected and measured by the Bausch and Lomb hand refractometer to ascertain the sucrose concentration at each separated zone. Fractions were analyzed for leptin using enzyme-linked immunosorbent assay.

Affinity Purification of the Soluble Leptin Receptor-- CNBr-activated Sepharose 4B from Amersham Pharmacia Biotech was coupled to recombinant mouse leptin from Amgen (Thousand Oaks, CA) following manufacturer's instructions. Coupled resin was kept at 4 °C in PBS (Life Technologies, Inc.) as a 50% slurry (v/v) in the presence of 0.02% sodium azide. About 1 mg of leptin was coupled for every ml of Sepharose. To affinity purify the soluble leptin receptor, mouse plasma was first prepared following an eye bleed procedure. Blood was mixed with EDTA to give a final concentration of 2 mM, which was centrifuged for 7 min at 4,000 × g. The resulting supernatant was used directly for leptin binding. In a typical experiment, 12.5 µl of leptin beads was incubated with 100 µl of mouse plasma diluted in 150 µl of PBS. Incubation was allowed overnight at 4 °C, and beads were then washed three times in cold PBS. The pellet was reconstituted in 1× SDS sample buffer and loaded onto an 8% gel. Bound receptor was analyzed with antipeptide antibodies of Ob-R. Peptide antibodies against Ob-R were generated by Research Genetics (Huntsville, AL). Two antibodies were used to detect the receptor: antibody A (corresponding to amino acids 145-158 of mouse Ob-R, amino acid sequences EPLPKNPFKNYDSK) and antibody B (corresponding to amino acids 465-484 of mouse Ob-R, amino acid sequences HRRSLYCPDSPSIHPTSEPK).

Generation of Ob-Re Expression Vector in SF9 Cells-- Ob-Re cDNA was subcloned into the vector pMelBac (Invitrogen, CA), a polyhedrin promoter-based transfer cloning vector of baculovirus. This vector is designed to direct expression of recombinant proteins through the secretory pathway to the extracellular medium. The vector contains a signal sequence for honeybee melittin, which is efficiently secreted by SF9 cells, the host for the baculovirus. To subclone Ob-Re into pMelBac, the endogenous signal sequence of the leptin receptor was not used. It was replaced by the honeybee melittin signal sequence to achieve efficient secretion of the receptor. Ob-R sequence starts at amino acid residue 28 and ends at residue 805 of Ob-Re (GenBankTM accession number U49110) containing 778 amino acids of Ob-Re sequence. Inserts were obtained by PCR using primers that contain restriction sites at both ends for subcloning into pMelBac. Large scale expression of recombinant protein was produced in suspension culture. Supernatant from productive stocks was used directly in the assay for leptin binding. Signal for Ob-R can be obtained directly from 1 µl of total supernatant. In a binding experiment, 15 µl of the supernatant was used for incubation with the leptin-Sepharose.

Deglycosylation of Ob-Re from Baculovirus and Mouse Plasma-- Binding of leptin-Sepharose to mouse plasma and baculovirus supernatant was as described above. At the last wash with PBS, sample was divided into two halves, and pellet was saved for the deglycosylation reaction. Bound protein on leptin-Sepharose beads were denatured by boiling in denaturation buffer for 10 min followed by treatment with or without 1 µl of PNGase F (500 units/µl; New England Biolabs, MA) at 37 °C for 3 h. At the end of the incubation, equal volume of 2× sample buffer was added to each tube, and the entire reaction was loaded onto a 7% SDS-PAGE gel and blotted with anti-receptor antibody A.

Reverse Transcription-PCR Analysis of dbPas/dbPas and Wild Type Mice Hypothalamic First Strand cDNA-- RNA was purified from adult hypothalami of wild type and dbPas/dbPas mice using the Trizol reagent (Life Technologies). 1 µg of total RNA was reverse-transcribed into first-strand cDNA using random primers and SuperScript II Rnase H- reverse transcriptase (Life Technologies, MD). Heat-denatured sample was used directly for reverse transcriptase-PCR analysis. To analyze the mutation of Lepr in dbPas/dbPas mouse, three pairs of primers spanning about 1 kilobase, each overlapping each other, were used to amplify wild type and mutant cDNA. Primer sequences: F1(CL086), ATGATGTGTCAGAAATTCTATG; R1(CL053), AACATCTTGTGTGGTAAAG; F2(CL049), TCAGGAGTCTGGAGTGACTGG; R2(CL005), CTTTTACATCCATGACAAGCG; F3 (CL042), GAGAATAACCTTCAATTCCAGATTC; and R3(CL062), GGTGTGAAATTAACAGTGTTC. Cycle parameters are 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 30 s for a total of 40 cycles. PCR products were purified using Centricon-30 from Amicon (Beverly, MA) by diluting the sample with H2O and spinning at 4 °C until sample contains DNA at a concentration of 0.1 µg/µl. Sequencing of PCR products was performed by the sequencing facility at the Protein DNA Technology Center of The Rockefeller University.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Soluble Leptin Receptor (Ob-Re) Circulates in Mouse Plasma-- Previous data have indicated mouse and human leptin sediment with a higher molecular mass than that predicted of a 16-kDa protein, suggesting that leptin circulates bound to other proteins (12, 13). To confirm this, either human or mouse plasma was loaded on a 5-20% sucrose gradient. The gradient fractions were assayed using enzyme-linked immunosorbent assay (Fig. 1) (12, 13). These data confirmed that a fraction of endogenous human leptin sediments on a sucrose gradient in lower fractions than leptin diluted in PBS (data not shown). When recombinant leptin was added to plasma from ob/ob mice (which are leptin-deficient), its sedimentation was shifted (Fig. 1). The apparent molecular mass of the complex is ~100-kDa (within the limits of resolution of the sucrose gradient). Although the nature of the leptin binding protein(s) was not known, soluble leptin receptor was a possible candidate.


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Fig. 1.   Sucrose gradient sedimentation of recombinant leptin added to plasma from ob/ob mice. The sedimentation of recombinant leptin added to either ob/ob plasma (which are leptin-deficient) or PBS was compared. Leptin added to mouse plasma sediments on a sucrose gradient with a higher apparent molecular mass (~100-kDa) than leptin diluted in PBS standard.

Soluble leptin receptor was detected using a leptin affinity resin and specific anti-receptor antibodies. Recombinant mouse leptin was coupled to CNBr-activated Sepharose 4B and mixed with mouse plasma. After centrifugation, the precipitated proteins were electrophoresed by SDS-PAGE and immunobloted. The blots were developed using either of two antibodies directed against peptides predicted by the Ob-R cDNA sequence. The position of these peptides on the receptor amino acid sequence are shown (Fig. 2A, bottom). Both antibodies detected a band of ~120-kDa in the material precipitated by the leptin-Sepharose. Although a lower molecular weight protein was also detected, this protein was seen after precipitation using bovine serum albumin-Sepharose beads (data not shown). It is likely to be a cross-reactive protein unrelated to Ob-Re.


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Fig. 2.   Soluble leptin receptor circulates in mouse plasma. A, leptin-Sepharose was incubated with mouse plasma or supernatant from SF9 cells infected by a recombinant Ob-Re baculovirus. After precipitation with leptin-Sepharose, the eluted protein was immunoblotted and developed using two different anti-ObR antipeptide antibodies. In both the baculovirus supernatant and mouse plasma, leptin-Sepharose precipitated proteins that were immunoreactive with both anti-receptor antibodies. The baculovirus protein was ~90-kDa versus ~120-kDa for native Ob-Re. Baculo denotes Ob-Re expression construct of the soluble leptin receptor; +/+ denotes plasma that is from wild type mouse. The arrow points to the position of native Ob-Re band. A lower molecular mass (M.W.) protein was also seen. This protein was also detected after precipitation with bovine serum albumin-Sepharose and is nonspecific (data not shown). aa, amino acids. B, binding of soluble leptin receptor to leptin-Sepharose is inhibited by free leptin. The precipitation of baculovirus and mouse Ob-Re was repeated in the presence of a 5-fold excess of recombinant leptin. In both cases, the addition of free leptin inhibited binding of Ob-Re to the leptin-Sepharose resin. The minus and plus signs denote the absence or presence of recombinant leptin added to the binding reaction. C, native Ob-Re and Ob-Re prepared from baculovirus were treated with the glycosidase PNGase F. The minus and plus signs indicate the absence or presence of glycosidase. In both cases, the native protein was truncated by PNGase F and migrated to a similar position (indicated by the arrow).

The size of Ob-Re was larger than that predicted by its primary sequence. This suggested the possibility that Ob-Re is glycosylated in vivo. To test this, the leptin-Sepharose precipitates were imunoblotted with or without PNGase F treatment. PNGase F is an enzyme that removes sugar moieties from glycoproteins. A baculovirus construct expressing the soluble leptin receptor was used as a positive control. The baculovirus supernatant contained immunoreactive material of ~90-kDa that was precipitated by leptin beads (Fig. 2A). The addition of an excess of recombinant leptin inhibited binding of soluble receptor to the leptin-Sepharose beads (Fig. 2B).

The baculovirus receptor protein migrated at a different position from native Ob-Re (~90-kDa versus 120 kDa), possibly due to differences in glycosylation from that of native leptin receptor. To confirm this, mouse plasma and baculovirus supernatant were mixed with leptin-Sepharose beads, and the precipitated proteins were treated with or without PNGase. PNGase F-treated Ob-Re, both from baculovirus and plasma, migrate to a near identical position after SDS-PAGE with an apparent molecular mass of ~75-kDa (Fig. 2C). Several lower molecular mass bands that were seen after the PNGase F treatment are likely the result of partial proteolysis. These data suggest that the glycosylation state of native and baculovirus Ob-Re is different.

Although the 120-kDa soluble leptin receptor was present in plasma from wild type and C57BL/Ks db/db mice, the wild type receptor was absent in db3J/db3J mouse (Fig. 3). This mutation results in truncation of the leptin receptor at amino acid 625 (11). A band of ~95-kDa was seen in plasma from these mice (Fig. 3). In addition, Ob-Re was not detected in this assay in plasma from dbPas/dbPas mice. Several lower molecular mass bands were also visible in all lanes. These bands were also seen when plasma was mixed with bovine serum albumin-Sepharose, suggesting that they are not specific.


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Fig. 3.   Absence of soluble leptin receptor in db/db mutant mice. The precipitation of Ob-R was repeated in the strains of mouse indicated. A soluble receptor of wild type size is absent in plasma from db3J/db3J and dbPas/dbPas mice. In plasma from db3J/db3J mice, a band of ~95-kDa was visible. This is consistent with the presence of a frameshift mutation at codon 625. Ob-Re was not visible in plasma from dbPas/dbPas mice. Several lower molecular mass (M.W.) bands were seen in all of the lanes, including dbPas/dbPas. These bands were also detectable when performing precipitations with bovine serum albumin-Sepharose; they appear to be not specific (data not shown). Baculo, Ob-Re generated in baculovirus; Pas/+, Pas/Pas, 3J/3J, and ob/ob, mice that are lean littermates of dbPas/dbPas, dbPas/dbPas, db3J/db3J, and ob/ob, respectively. 1 µl of total baculovirus supernatant was loaded directly on the gel as a control.

The absence of Ob-Re in dbPas/dbPas mice suggested that the Ob-R mutation in these mice was likely to affect the extracellular region of the receptor. To confirm this possibility, three pairs of primers were prepared, each of which spanned 1 kilobase at the NH2 terminus of Lepr (Fig. 4A). These primers were used to amplify first-strand cDNA from wild type and dbPas/dbPas total hypothalamic RNA. The positions of the primers on the Ob-R cDNA are shown along with the size of the amplification products after electrophoresis on an 0.8% agarose gel (Fig. 4A). PCR products of identical size were amplified from wild type and dbPas/dbPas RNA using primer pairs B and C. However, primer pair A amplified a 1.4-kb product in dbPas/dbPas cDNA versus 1 kb in wild type mice. This PCR product was sequenced using an automatic sequencer (Fig. 4B). The sequence identified a 359-base pair duplication in the mutant cDNA. Based on the published genomic structure, this duplication corresponds to exons 4 and 5 of the leptin receptor (14). The duplication introduces a stop codon one residue after amino acid 281 and is predicted to result in the loss of all Lepr isoforms. This duplication was also confirmed using genomic Southern blotting. When exons 4 and 5 were used as probes, signal intensity was about twice as high in dbPas/dbPas mice versus wild type mice (data not shown). These data indicate that dbPas/dbPas mice have a duplication in the extracellular region of Ob-R that alters the expression of all receptor isoforms.


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Fig. 4.   Duplication of exons 4 and 5 of Ob-R in dbPas/dbPas mouse. A, Reverse transcription-PCR was performed from hypothalamic RNA using primers at the positions indicated below. The three sets of primers that were used spanned the entire extracellular region of Ob-R. Primer set A detected an ~400-base pair larger product in dbPas/dbPas mice relative to wild type (wt) mice. M.W., molecular mass. B, fragment A was sequenced and revealed a 359-base pair insertion corresponding to a duplication of exons 4 and 5. The wild type sequence is bracketed, whereas the duplicated region is underlined. The duplication introduces an in-frame stop codon at amino acid 283.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Leptin circulates in mouse and human plasma either as a free 16-kDa protein or bound to other proteins (12, 13). The identity of these leptin-binding proteins has not been previously elucidated, although a secreted form of the leptin receptor was a logical candidate. The data presented here confirm that soluble leptin receptor is present in mouse plasma. However, the data do not exclude the possibility that proteins other than Ob-Re also bind to leptin.

Several consensus sequences for glycosylation are contained in the Ob-R coding sequence (8). The size of the soluble leptin receptor is larger than that predicted by its amino acid sequence, suggesting that it is glycosylated. The truncation of Ob-Re after PNGase F treatment confirms that it is glycosylated. This conclusion is supported by the observation that after PNGase F treatment, baculovirus and native Ob-Re migrate to the same position by SDS-PAGE. The identify of this 120-kDa band as Ob-Re is also confirmed by the absence of the wild type protein in plasma from dbPas/dbPas and db3J/db3J mice. The db3J/db3J mutation was previously predicted to encode a 625-amino acid secreted protein (11). The observation that Ob-Re is truncated in plasma from its mutant supports this. The mutation in dbPas/dbPas mice is shown here to result from a duplication resulting in the synthesis of a 281-amino acid protein. (Fig. 5). Ob-Re is not detectable in plasma from dbPas/dbPas mice. This suggests that in this mutant, Ob-Re mRNA is not efficiently translated or that its protein may be degraded. Alternatively, the truncated protein may not bind to the leptin-Sepharose.


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Fig. 5.   Rodent mutations at the db locus. The identification of dbPas/dbPas mutation completes the analyses of obese rodents with defects in the leptin receptor. Shown are the structures of the full-length leptin receptor, Ob-Rb, and the mutations in each of the diabetic mice and fatty strains of rats. Solid boxes in the extracellular region represent domains of the receptor that are homologous to the growth hormone receptor, which are thought to bind to leptin. The solid boxes at the intracellular region represent the boxes 1 and 2 motif, which are characteristic of the cytokine family of receptors. aa, amino acids. TMR, transmembrane region. fa/fa, fatty Zucker rat; fak/fak, fatty Koletsky rat.

The observation that leptin-Sepharose resin precipitates Ob-R suggests that soluble receptor may be present in excess relative to leptin in mouse plasma. This is surprising given the observation that only a portion of mouse and human leptin sediments with the apparent molecular mass of free leptin (12, 13). It may be that the leptin-Sepharose displaces leptin already bound to Ob-Re in plasma.

The function of Ob-Re remains to be determined. Soluble forms of other cytokine receptors are also found to circulate in plasma (15). In most cases the circulating receptor acts to chelate the ligand and acts as an inhibitor. This is also possible with leptin. Alternatively, soluble Ob-R could play a role in other aspects of leptin function such as reuptake after filtration by the kidney or in transport across the blood brain barrier (16, 17). Available data indicate that the hypothalamus is an important target of leptin action and that a saturable transport system is responsible for leptin access to its site of action in the brain (18, 19). Further studies will be required to determine whether Ob-Re plays a role in these aspects of leptin's function. The availability of recombinant receptor expressed in baculovirus will facilitate these studies.

It is as yet unclear whether the affinity of leptin for membrane-bound and soluble receptor in vivo is similar. A recent report did show that when the entire extracellular region of Ob-R is expressed in COS7 cells, it binds to leptin with similar affinity as that of the full-length transmembrane receptor (20). It is also not known whether baculovirus Ob-Re binds to leptin with an affinity similar to that of membrane-bound receptor expressed in mammalian cells (21).

With the identification of the dbPas/dbPas mutation, the molecular nature of all the rodent db (and fa) alleles is known. Three of the mutations truncate the receptor in the extracellular region, whereas the db mutation in C57BL/Ks mice is the result of a splicing mutation. This mutation specifically alters splicing of Ob-Rb, highlighting the importance of this receptor variant. Further studies of the other receptor forms (Ob-R(a-d)) will be required to evaluate their functions.

    ACKNOWLEDGEMENTS

We would like to thank Amgen for providing us with recombinant leptin, Dr. Jean-Louis Guenet (Pasteur Institute, Paris) for providing us with dbPas/dbPas mice, Drs. Jeff Winick, Jeff Halaas, and Jason Montez for stimulating discussions.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Howard Hughes Medical Institute, c/o The Rockefeller University, 1230 York Ave., Box 305, New York, NY 10021. Tel: 212-327-8800; Fax: 212-327-7420; E-mail: friedj{at}rockvax.rockefeller.edu.

1 The abbreviations used are: PNGase F, N-glycosidase F; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Lepr, leptin receptor; PBS, phosphate-buffered saline.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J. M. (1994) Nature 372, 425-432[CrossRef][Medline] [Order article via Infotrieve]
  2. Campfield, L. A., Smith, F. J., Guisez, Y., Devos, R., and Burn, P. (1995) Science 269, 546-549[Medline] [Order article via Infotrieve]
  3. Halaas, J. L., Gajiwala, K. S., Maffei, M., Cohen, S. L., Chait, B. T., Rabinowitz, D., Lallone, R. L., Burley, S. K., and Friedman, J. M. (1995) Science 269, 543-546[Medline] [Order article via Infotrieve]
  4. Pelleymounter, M. A., Cullen, M. J., Baker, M. B., Hecht, R., Winters, D., Boone, T., and Collins, F. (1995) Science 269, 540-543[Medline] [Order article via Infotrieve]
  5. Stephens, T. W., Basinski, M., Bristow, P. K., Bue-Valleskey, J. M., Burgett, S. G., Hale, J., Hoffmann, J., Hsiung, H. M., Kriauciunas, A., MacKellar, W., Rosteck, P. R., Jr., Schoner, B., Smith, D., Tinsley, F. C., Zhang, X., and Heiman, M. (1995) Nature 377, 530-532[CrossRef][Medline] [Order article via Infotrieve]
  6. Levin, N., Nelson, C., Gurney, A., Vandlen, R., and de Sauvage, F. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1726-1730[Abstract/Free Full Text]
  7. Halaas, J. L., Boozer, C., Blair-West, J., Fidahusein, N., Denton, D. A., and Friedman, J. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8878-8883[Abstract/Free Full Text]
  8. Tartaglia, L. A., Dembski, M., Weng, X., Deng, N., Culpepper, J., Devos, R., Campfield, L. A., Clark, F. T., Deeds, J., Muir, C., Sanker, S., Moriarty, A., Moore, K. V., Smutko, J. S., Mays, G. G., Woolf, E. A., Monroe, C. A., and Tepper, R. I. (1995) Cell 83, 1263-1271[Medline] [Order article via Infotrieve]
  9. Lee, G. H., Proenca, R., Montez, J. M., Carroll, K. M., Darvishzadeh, J. G., and Lee, J. I. (1996) Nature 379, 632-635[CrossRef][Medline] [Order article via Infotrieve]
  10. Chen, H., Charlat, O., Tartaglia, L. A., Woolf, E. A., Weng, X., Ellis, S. J., Lakey, N. D., Culpepper, J., Moore, K. J., Breitbart, R. E., Duyk, G. M., and Tepper, R. I. (1996) Cell 84, 491-495[Medline] [Order article via Infotrieve]
  11. Lee, G., Li, C., Montez, J., Halaas, J., Darvishzadeh, J., and Friedman, J. M. (1997) Mamm. Genome 8, 445-447[CrossRef][Medline] [Order article via Infotrieve]
  12. Houseknecht, K. L., Mantzoros, C. S., Kuliawat, R., Hadro, E., Flier, J. S., and Kahn, B. B. (1996) Diabetes 45, 1638-1643[Abstract]
  13. Sinha, M. K., Opentanova, I., Ohannesian, J. P., Kolaczynski, J. W., Heiman, M. L., Becker, G. W., Bowsher, R. R., Stephens, T. W., and Caro, J. F. (1996) J. Clin. Invest. 98, 1277-1282[Abstract/Free Full Text]
  14. Chung, W. K., Power-Kehoe, L., Chua, M., Lee, R., and Leibel, R. L. (1996) Genome Res. 6, 1192-1199[Abstract]
  15. Leung, D. W., Spencer, S. A., Cachianes, G., Hammonds, R. G., Collins, C., Henzel, W. J. B., Waters, M. J., and Wood, W. I. (1987) Nature 330, 537-543[CrossRef][Medline] [Order article via Infotrieve]
  16. Cumin, F., Baum, H. P., and Levens, N. (1996) Int. J. Obes. 20, 1120-1126[Medline] [Order article via Infotrieve]
  17. Cumin, F., Baum, H. P., de Gasparo, M., and Levens, N. (1997) Int. J. Obes. 21, 495-504[CrossRef][Medline] [Order article via Infotrieve]
  18. Banks, W. A., Kastin, A. J., Huang, W., Jaspan, J. B., and Maness, L. M. (1996) Peptides 17, 305-311[CrossRef][Medline] [Order article via Infotrieve]
  19. Golden, P. L., Maccagnan, T. J., and Pardridge, W. M. (1997) J. Clin. Invest. 99, 14-18[Abstract/Free Full Text]
  20. Liu, C., Liu, X. J., Barry, G., Ling, N., Maki, R. A., and De Souza, E. B. (1997) Endocrinology 138, 3548-3554[Abstract/Free Full Text]
  21. Devos, R., Guisez, Y., Van der Heyden, J., White, D. W., Kalai, M., Fountoulakis, M., and Plaetinck, G. (1997) J. Biol. Chem. 272, 18304-18310[Abstract/Free Full Text]


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