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Hyperleptinemia, Leptin Resistance, and Polymorphic Leptin Receptor in the New Zealand Obese Mouse¹

Institut für Pharmakologie und Toxikologie der RWTH (M.I., W.B., H.-G.J.), Aachen; and Diabetesforschungsinstitut (L.H.), Düsseldorf, Germany

Address all correspondence and requests for reprints to: Dr. H. G. Joost, Institut für Pharmakologie und Toxikologie, Medizinische Fakultät der RWTH Aachen, Wendlingweg 2, D-52057 Aachen, Germany.

	Abstract

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New Zealand Obese (NZO) mice exhibit a polygenic syndrome of hyperphagia, obesity, hyperinsulinemia, and hyperglycemia similar to that observed in young diabetes mutant mice on the C57BLKS/J background (C57BLKS/J-Lepr^db/Lepr^db). Here we show that in NZO this syndrome is accompanied by a marked elevation of the leptin protein in adipose tissue and serum. The promoter region and the complementary DNA of the ob gene of NZO mice, including its 5'-untranslated region, are identical with the wild-type sequence (C57BL, BALB/c), except that the transcription start is located 5 bp upstream of the reported site. In contrast to C57BLKS/J^+/+ and C57BL/6J-Lep^ob/Lep^ob mice, NZO mice failed to respond to recombinant leptin (7.2 µg/g) with a reduction of food intake. Leptin receptor messenger RNA as detected by PCR appears as abundant in hypothalamic tissue of NZO mice as in tissue from lean mice. Ten nucleotide polymorphisms are found in the complementary DNA of the leptin receptor, resulting in two conservative substitutions (V541I and V651I) in the extracellular part of the receptor and one nonconservative substitution (T1044I) in the intracellular domain between the presumed Jak and STAT binding boxes. However, these mutations are also present in the related lean New Zealand Black strain (body fat at 9 weeks: New Zealand Black, 6.2 ± 1.3%; NZO, 17.0 ± 1.7%). Thus, the polymorphic leptin receptor seems to play only a minor, if any, role in the obesity and hyperleptinemia of the NZO mouse. It is suggested that the main defect in NZO is located distal from the leptin receptor or at the level of leptin transport into the central nervous system.

	Introduction

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NEW ZEALAND Obese (NZO) mice originated from a mixed colony of agouti mice selected for spontaneous obesity from F12 to F17, and the genotype was fixed by continuous inbreeding (1). They exhibit a polygenic syndrome of hyperphagia, obesity, insulin resistance, and hyperglycemia similar to that of young C57BLKS/J-Lepr^db/Lepr^db mice (previous nomenclature: C57BLKS/J-db/db) (2). Recently, it was shown that the obesity of the NZO mice is paralleled by a marked increase in the expression of leptin messenger RNA (mRNA) in adipose tissue (3). This finding suggests that either an aberrant leptin protein or resistance to leptin contributes to the development of obesity and insulin resistance.

The positional cloning of the gene responsible for morbid obesity in the C57BL/6J-Lep^ob/Lep^ob mouse (previous nomenclature: C57BL/6J-ob/ob) has revealed that adipose tissue secretes a protein, subsequently designated leptin, that controls food intake and thermogenesis (4). Lack of leptin in this mutant strain is responsible for excessive overeating, obesity, and secondary metabolic alterations, e.g. a marked insulin resistance. A similar syndrome is caused by a defective leptin receptor in the C57BLKS/J-Lepr^db/Lepr^db mouse and the Zucker rat (5, 6, 7). Furthermore, it has been shown that recombinant leptin reduces food intake and normalizes thermogenesis when given to C57BL/6J-Lep^ob/Lep^ob mice (8, 9, 10). Thus, it is generally accepted that leptin is the crucial mediator in the feedback control among adipose tissue mass, the central regulation of feeding behavior, and energy expenditure (11).

Data from obese patients indicate that leptin mRNA and serum levels are increased in approximate proportion with the body mass index (12). This increase in leptin levels appears to reflect a reduced sensitivity to the hormone, as in none of the patients was an aberrant sequence of leptin found. With the exception of the nonobese nondiabetic mouse (13), increased levels of leptin mRNA have also been found in obese rodents with polygenic obesity, e.g. the mildly obese Sprague-Dawley rat (3), the Otsuka Long-Evans Tokushima fatty (OLETF) rat (14), and NZO and KK mice (3, 15). It was assumed, therefore, that in the majority of obese rodents and also in morbidly obese humans, resistance to leptin is the crucial parameter of the disease.

The discovery of leptin and its receptor has raised several important questions concerning the pathogenesis of various forms of obesity, the relation between leptin and insulin resistance, and the genes involved in polygenic syndromes of morbid obesity. Because of the polygenic basis of their obesity and their marked insulin resistance, we consider the NZO strain an ideal animal model to use in the search for additional obesity genes. Here we show that neither an aberrant ob gene nor the polymorphic leptin receptor can be fully responsible for the elevated serum leptin levels and the failure of exogenous hormone to reduce food intake.

	Materials and Methods

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Animals and leptin treatment
C57BLKS/J-Lepr^db/Lepr^db and NZO-Hl mice were bred in the Diabetesforschungsinstitut (Düsseldorf, Germany). New Zealand Black (NZB) and C57BL/6J-Lep^ob/Lep^ob mice were purchased from Harlan Co. (Borchen, Germany). All animals were fed a standard laboratory diet; they had free access to food and water. On each day of the treatment schedule, food was withdrawn at 0800 h and returned 1 h after the sc injection of recombinant leptin (PeproTech, Rocky Hill, NJ) at 1400 h. Food consumption was measured 6 and 18 h after leptin administration. The Principles of Laboratory Animal Care (NIH publication 85–23, revised 1985) were followed, and approval for the treatment protocol was obtained from the ethical committee for animal experimentation of the Regierungspräsidium Düsseldorf.

Preparation of RNA and complementary DNA (cDNA)
Animals were killed by decapitation, and sc, perirenal, and gonadal fat pads were dissected. Midbrain sections comprising the hypothalamic area were dissected, and all samples were immediately frozen in liquid nitrogen. Samples of adipose tissue were homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) in guanidine thiocyanate (4 M; supplemented with 7% mercaptoethanol), and RNA was isolated by centrifugation on a cesium chloride cushion (5.88 M; 28,000 rpm for 29 h at 20 C in an SW40 rotor). Hypothalamic tissue (30 mg) was homogenized in a Potter-Elvehjem homogenizer, and total RNA was isolated with the RNeasy kit from Qiagen (Hilden, Germany). First strand cDNA was synthesized from total RNA with a kit from Pharmacia (Freiburg, Germany).

PCR, rapid amplification of cDNA ends (RACE), and PCR cloning
The total coding region of the ob gene was amplified by PCR with primers derived from the published sequence (accession no. U18812; upstream primer, 5'-AAG ATC CCA GGG AGG AAA-3'; reverse primer, 5'-CTG GTG GCC TTT GAA ACT-3'). The 5'-untranslated region was amplified by the RACE procedure with a kit from Life Technologies (Gaithersburg, MD). Eight overlapping cDNA fragments of the leptin receptor b (5, 6, 16) and the promoter region of the ob gene (17, 18) were amplified with primers derived from the published sequences (accession no. of LepR, U46135 and U49107; accession no. of ob promoter, U52147 and S81087; primer sequences on request). PCR products were separated on agarose and subcloned into the SmaI site of pUC19 (Sureclone kit, Pharmacia). Plasmid DNA was prepared and sequenced in both directions. Mismatches were confirmed by a second PCR with cDNA from a different animal. For quantitative assessment of the PCR products, [-³²P]deoxy-CTP was added, and the reaction was stopped after 20, 25, and 30 cycles.

Primer extension
Primer extension analysis was performed with a kit from Promega (Madison, WI) with an end-labeled oligonucleotide corresponding to nucleotides 153–178 of the ob cDNA (accession U18812). Processing of the samples was performed according to the technical bulletin of the manufacturer. A sequencing reaction of the longest RACE clone with the same oligonucleotide was used as standard for determination of the size of the product.

Northern blot analysis
Samples of total RNA (15 µg) were separated and hybridized as described previously (3) with probes generated by random oligonucleotide priming (19).

Ribonuclease protection assay
Ribonuclease protection assays were performed with a kit from Ambion (Austin, TX) according to the protocol provided by the manufacturer. The probe corresponding with the sequence of LepRb (2628–3075 bp) was constructed by PCR and subcloned into pBS-SK (Stratagene, Heidelberg, Germany).

Preparation of adipose tissue extracts and assay of leptin
Samples of epididymal or perirenal adipose tissue were homogenized with a Potter-Elvehjem homogenizer in ice-cold lysis buffer [20 mM Tris (pH 7.4), 150 mM sodium chloride, 0.2 mM phenylmethylsulfonylfluoride, and 1% Triton X-100] and centrifuged for 30 min (2000 rpm, 4 C). Specific antiserum (2.5 µl) raised against recombinant leptin (9) was added to 250 µl of the lysates (1 mg total protein), and the samples were incubated for 2 h at 4 C. Immunocomplexes were adsorbed to protein A-Sepharose (Pharmacia), washed three times with buffer containing 20 mM Tris (pH 7.4), 150 mM sodium chloride, and 0.1% Triton X-100 and twice with the same buffer containing 0.05% Triton X-100, and eluted with Laemmli’s sample buffer. The samples were separated by SDS-PAGE and transferred to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) with a tank blot apparatus (Pharmacia). The membranes were blocked overnight by incubation in a buffer containing 10 mM Tris (pH 7.5), 100 mM sodium chloride, and 0.1% Tween-20 and were incubated for 2 h at room temperature with the antileptin antiserum at a dilution of 1:100. Washing and detection of bound antibodies with [¹²⁵I]protein A (Amersham-Buchler, Braunschweig, Germany) was performed as previously described (20).

Immunoprecipitation and assay of leptin in serum
Samples of specific antileptin antiserum (2.5 µl) were adsorbed to protein A-Sepharose (100 µl packed beads) by an overnight incubation in 250 µl PBS buffer. To remove -globulins, serum from normal and obese animals was preincubated with protein A-Sepharose. The stripped serum was added to Sepharose beads loaded with specific serum and incubated for 2 h. The beads were separated by centrifugation and washed three times with PBS buffer. Immunocomplexes were eluted, separated by SDS-PAGE, and probed as described in the preceding paragraph.

Other assays
Serum immunoreactive insulin was assayed with RIA kits from Pharmacia. Purified rat insulin (Novo Research Institute, Bagsvaerd, Denmark) was used as standard. Blood glucose was determined by an automated glucose oxidase method (Care Diagnostica, Voerde, Germany).

	Results

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Obesity, hyperglycemia, and hyperinsulinemia in NZO mice compared with the C57BLKS/J strain
As the genotype of NZO mice is fixed by continuous inbreeding, metabolically normal controls with an identical genetic background are not available. We have, therefore, compared the obese NZO mice with obese C57BLKS/J-Lepr^db/Lepr^db mice and their lean litter mates (C57BLKS/J^+/+). In some experiments, NZB mice were used for comparison (e.g. leptin receptor sequence). NZB separated from the line leading to NZO at F3 (1). Table 1 characterizes the NZO strain with respect to body weight, hyperinsulinemia, and hyperglycemia. Compared with lean C57BLKS/J^+/+ mice, NZO were markedly overweight, similar to obese C57BLKS/J-Lepr^db/Lepr^db mice (Table 1). Furthermore, the time course of weight gain (not shown) exhibited an essentially parallel increase in age-matched NZO and Lepr^db/Lepr^db mice during the dynamic phase of their obesity. It should be noted, however, that age-matched NZO mice were longer and had a lower percentage of body fat, and thus a lower degree of obesity, than Lepr^db/Lepr^db mice (see also Table 3). Like the Lepr^db/Lepr^db mice, NZO mice showed a marked hyperglycemia and hyperinsulinemia at 4 months of age (Table 1).

View larger version (41K): [in this window] [in a new window]   Figure 1. Levels of leptin mRNA (A) and protein (B and C) in adipose tissue (A and B) or serum (C) of C57BLKS/J+/+, C57BLKS/J-Leprdb/Leprdb, and NZO mice. A, Total mRNA was isolated from adipose tissue of lean (C57BLKS/J+/+) or obese (C57BLKS/J-Leprdb/Leprdb, NZO) mice, separated, blotted onto nylon membranes, and probed with radiolabeled leptin cDNA as described in Materials and Methods. The position of ribosomal RNA is marked on the left margin. B, Adipose tissue from the indicated animals was homogenized in lysis buffer, and leptin was isolated by immunoprecipitation as described. The immunoprecipitates were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with the same antiserum. Bound -globulins were detected with protein A. Standards of recombinant leptin were run in parallel. C, Leptin was isolated by immunoprecipitation from serum obtained from the indicated animals, and the immunoprecipitates were assayed as described in B.

Similar results were obtained when the leptin protein was immunoprecipitated from serum of the different mouse strains. As illustrated in Fig. 1C, levels of leptin were markedly elevated in serum from Lepr^db/Lepr^db and NZO mice.

cDNA sequence of leptin in NZO mice
To test the possibility whether an aberrant sequence of leptin contributes to the obese phenotype of the NZO mouse, a fragment comprising the coding region of the ob gene was amplified from RNA of NON mice by PCR. The nucleotide sequence (not shown) of this fragment is identical with that of the published wild-type sequence (4). In addition, we amplified the 5'-untranslated region with the RACE procedure, because it was conceivable that a reduced leptin synthesis, in addition to a reduced action, is contributing to the obesity syndrome. PCR products were isolated (Fig. 2), which started 1–5 bp upstream from the published transcription initiation site (17, 18). In the primer extension assay, a transcript starting at -4 was detected (Fig. 2). The sequence of the PCR products included the 5'-untranslated region of the ob gene from NZO and was identical with that of the wild-type sequence reported by other groups (4, 17, 18).

View larger version (6K): [in this window] [in a new window]   Figure 2. Transcription initiation site of the NZO ob gene. The transcription initiation site of the ob gene of NZO was determined by RACE and primer extension as described in Materials and Methods. Vertical arrows represent the 5'-terminus of each of four analyzed RACE clones and of the initiation site determined by primer extension. The published transcription start is located at nucleotide +1. The sequence of exon 1 is given in capital letters; lowercase letters symbolize the sequences of the 5'-flanking region and of intron 1. The sequence of promoter region (until nucleotide -274), the remaining 5'-untranslated region, and the coding region were determined by RACE and PCR cloning (sequence not shown) and were identical with the reported wild-type sequence.

Lack of effect of recombinant leptin in NZO mice on 12-h food intake
Based on the finding that levels of immunoreactive leptin were markedly elevated in serum of NZO mice, we assumed that the animals were resistant to leptin. However, the possibility was not excluded that the endogenous hormone was inactive because of an aberrant posttranslational modification. With such a defect, NZO mice should be as sensitive to exogenous leptin as mice lacking leptin (C57BL/6J-Lep^ob/Lep^ob). Thus, we treated NZO, Lep^ob/Lep^ob and +/+ mice with recombinant leptin and monitored 12-h food intake. In initial experiments (not shown), injections of both vehicle and leptin reduced food intake in NZO mice, whereas in Lep^ob/Lep^ob mice only leptin was active. Thus, NZO mice appeared to be particularly sensitive to the stress of the injection. In subsequent experiments (Fig. 3), the animals were adapted to the experimental procedure with daily injections of saline over a period of 4 days. As illustrated in Fig. 3 (top panel), leptin in doses of 0.8–7.2 µg/g failed to significantly reduce the food consumption of NZO mice. In contrast, even the lowest dose (0.8 µg/g) produced an effect in Lep^ob/Lep^ob mice. In lean C57BL^+/+ mice, the highest dose (7.2 µg/g) produced a somewhat smaller effect than in the Lep^ob/Lep^ob mice, as was anticipated from the previous reports (8, 9, 10).

View larger version (19K): [in this window] [in a new window]   Figure 3. Lack of effect of recombinant leptin on 12-h food intake of NZO mice. The animals (C57BLKS/J+/+, C57BL/6J-Lepob/Lepob, and NZO) received one daily injection of the indicated dose of recombinant leptin or vehicle on 3 subsequent days. Food consumption was monitored over the whole treatment period; control values (open bars) represent food consumption during the 3 days before treatment. Data are the mean ± SD of four animals. Asterisks indicate the significance (P < 0.05, by two-sided t test) of the differences between leptin and vehicle treatments.

View larger version (71K): [in this window] [in a new window]   Figure 4. PCR amplification of the intracellular domain of the leptin receptor and restriction fragmentation with BspHI. Oligonucleotide primers deduced from the published sequence of the receptor were used to amplify a fragment of the receptor comprising its intracellular domain with the Thr/Ile exchange of NZO (A). A portion of the PCR products was subjected to a digestion with BspHI (B). cDNA isolated from midbrain or genomic DNA (NZC) was used as template. The identities of the PCR products were confirmed by sequencing.

View larger version (36K): [in this window] [in a new window]   Figure 5. Comparison of leptin receptor mRNA levels in midbrain of NZO and lean BLKS/J+/+ mice. A, RNA was isolated from mouse midbrain, and cDNA was synthesized as described in Materials and Methods. The PCR was run with oligonucleotide primers specific for the LepRa (4) under conditions providing a proportional relationship between LepRa cDNA and PCR product (25 cycles) in the presence of tracer deoxy-CTP. PCR products were separated by PAGE and visualized by autoradiography. +/+, C57BLKS/J+/+ mice. B, Ribonuclease protection assay of LepRb mRNA. RNA from mouse midbrain was hybridized with a riboprobe corresponding to 446 bp of the Leprb mRNA and digested with ribonuclease. The reaction products were separated by denaturing PAGE and autoradiographed. Note that the hybridized riboprobe runs with a somewhat lower electrophoretic mobility than the corresponding DNA marker (HinfI-digested phage X174).

	Discussion

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The present data indicate that the sequence of leptin in NZO is identical with that of the wild-type sequence, and that immunoreactive leptin is abundant in the serum of NZO mice. It appeared conceivable, however, that an aberrant posttranslational processing of the protein has rendered it inactive. In this case, one would expect a phenotype similar to that of BL/6J-Lep^ob/Lep^ob, in that the animals are very sensitive to recombinant leptin. However, exogenous leptin was inactive in NZO up to a dose of 7.2 µg/g, which is 9 times higher than the dose reducing food intake in Lep^ob/Lep^ob mice. We consider this finding indirect evidence that the endogenous leptin in NZO is normal, and that the main defect of NZO mice is resistance to leptin. It should be noted that the present data do not allow us to compare the severity of the defect with that in Lepr^db/Lepr^db, as higher doses of leptin might overcome the resistance in NZO. Furthermore, we cannot exclude the possibility that the leptin resistance observed here reflects a defect in the transport of leptin into the central nervous system.

The sequence of the leptin receptor of NZO mice differed from that of the wild-type receptor (BLKS/J^+/+) by three amino acid substitutions. Two substitutions (Val/Ile) in the extracellular part of the receptor were conservative, and it is doubtful that they alter the function of the protein. The third, nonconservative substitution, Thr/Ile, is located in the large intracellular domain of the leptin receptor b isoform, which has been shown to activate transcription via a Jak/STAT pathway (23). The exchange is located between the presumed Jak and STAT binding boxes in a region of the protein, with little similarity to other class I cytokine receptors. The leptin receptor might require a polar amino acid in position 1044, as the human sequence contains an asparagine instead of threonine in this position (16). Thus, it is conceivable that the mutation of Thr¹⁰⁴⁴ to Ile affects the signal transduction of the receptor. However, we found that NZB mice, a related, lean strain, carried the same LepRb allele as the NZO strain. NZB mice are clearly lean, and it is safe to conclude that the Thr/Ile mutation does not disrupt the function of the leptin receptor to a degree observed with the Lepr^db and the Lepr^fa (former nomenclature: fa) mutation. Thus, the polymorphic leptin receptor alone cannot produce obesity and can be only a minor, if any, contributor of obesity in NZO. Therefore, we suggest that the main defect producing obesity in NZO mice is located distal from the leptin receptor or at the level of leptin transport into the central nervous system.

	Acknowledgments

 
We thank Dr. E. Leiter for communicating results before publication. Contributions of each author: M.I., all assays, cloning, sequencing, and animal experimentation; W.B., conceptual and methodological contributions; L.H., breeding of mouse strains, conceptual input, and animal experimentation; and H.G.J., concepts and writing of paper. The skillful technical assistance of Ms. Angela Schraven and Ms. Susanne Breitwieser is gratefully acknowledged.

	Footnotes

 
¹ This work was supported by the Deutsche Forschungsgemeinschaft (SFB 351/C5) and the Bundesministerium für Bildung, Forschung, und Technologie.

Received February 27, 1997.

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