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Department of Nutrition, The University of Tennessee, Knoxville, TN 37996 and * The Jackson Laboratory, Bar Harbor, ME 04609
3To whom correspondence should be addressed. E-mail: jhkim{at}utk.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • coisogenic • congenic • Lepr • mice • mutations
Leptin is a peptide hormone secreted by adipocytes and functions as a negative feedback signal in regulating body weight through reducing food intake and stimulating energy expenditure (1 ). Both leptin transcription and serum levels of leptin protein are affected by body fat mass and feeding status; up-regulated by increased body fat mass and excessive food intake; and down-regulated during weight loss and fasting (2 ). Leptin deficiency causes a marked hyperphagia and morbid obesity in mammals including mice and humans (1 ). Administration of recombinant leptin in a leptin-deficient animal model, the Lepob homozygous mouse, as well as in human patients with leptin deficiency, suppresses food intake and stimulates energy expenditure, ultimately reducing body fat mass (1 ,3 ). Furthermore, a significant leptin effect on reducing fat mass has been demonstrated in both normal and diet-induced obese mice (2 ).
The available data suggest that the hypothalamus is an important site for leptin action, and leptin signaling is mediated by its specific receptor, the leptin receptor (LEPR) (1 ). The role of the LEPR in leptin action has been well demonstrated by the lack of response to recombinant leptin in Leprdb homozygous mice deficient for the leptin receptor.
Mutations in the leptin receptor gene (Lepr) cause severe early onset obesity, insulin resistance and chronic hyperglycemia in rodents (4 ,5 ) and in humans (6 ), primarily by preventing leptin signaling to the hypothalamic satiety center (7 ).
The LEPR is a single membrane-spanning receptor of the class I cytokine receptor family (8 ), and five distinct, alternatively spliced forms (a–e) have been identified in mice (9 ). The b isoform (or long form) has the longest intracellular domain, containing janus kinase (JAK) and signal transducer and activator of transcription (STAT) interaction motifs, and has the ability to activate STAT proteins (10 ,11 ). Studies in vitro have demonstrated that the Lepr b form can activate leptin signal transduction through JAK and STAT proteins upon ligand binding and dimerization (1 ). The src homology protein tyrosine phosphatases 2 (SHP-2) and suppressor of cytokine signaling (SOCS)-3 have been implicated in down-regulating leptin signals (1 ). The e isoform lacks a transmembrane domain and is found in the circulation and may act as a leptin carrier (12 ). The functions of the Lepr a, c and d forms remain to be defined. Despite the rapid progress that has been made since the discovery of leptin, many details of the leptin signal transduction pathway remain to be fully elucidated.
Mutations within the Lepr, including Leprdb, Leprdb-3J, Leprdb-Pas and Leprdb-NCSU (in mice), Leprfa and Leprfa-k (in rats), and in humans have been reported (6 ,13 –19 ), and identification of the molecular bases of these mutations has provided important insights into the cellular mechanism of LEPR function (4 ,5 ,9 ,10 ,12 ,14 ,20 –26 ).
Three new mutations of Lepr, designated Leprdb-rtnd, Leprdb-dmpg and Leprdb-rlpy, respectively, arose spontaneously in mice at The Jackson Laboratory. The aims of this study were to determine the molecular bases for these new Lepr mutations and to characterize the new mutant stocks, with respect to obesity and diabetes. This study provides new information to the current understanding of Lepr and new mouse models of obesity and diabetes.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The obese mouse deviants initially named rotund, dumpling and rolypoly were identified in the CBA/J (CBA), the B10.D2-H8b (57N)/Sn (B10) and the NU/J strains, respectively at The Jackson Laboratory. Upon realizing the phenotypic similarities, we conducted a test for allelism by mating heterozygous mutants with C57BL/6J (B6)-Leprdb/+ and B6-Lepob/+ mice. All new mutations produced obese F1 progeny when mated with B6-Leprdb/+. Thereby, we concluded that rotund, dumpling and rolypoly were alleles of Leprdb and designated them Leprdb-rtnd, Leprdb-dmpg and Leprdb-rlpy, respectively.
Because breeding was not prosperous in the original strains, Leprdb-rtnd and Leprdb-dmpg were backcrossed onto B6, on which several obesity mutations including Leprdb, Lepob and Ay exist. N5F1 progeny of B6.CBA-Leprdb-rtnd/db-rtnd and B6.B10-Leprdb-dmpg/db-dmpg were used for the physiological characterization in this study. In the case of Leprdb-rtnd, (B6xCBA)F1-Leprdb-rtnd/+ hybrid mice were also backcrossed onto the CBA strain in an attempt to recover the original background, creating a congenic line of CBA.B6CBA-Leprdb-rtnd. For this line, N5F1 progeny were also used for the physiological characterization. Leprdb-rlpy mutation was maintained in the original strain (coisogenic). Because mice homozygous for these mutations are infertile, the colonies were maintained by heterozygous mating. All mice ate the NIH-mouse/rat diet with 40 g/kg fat (PMI Nutrition International, St. Louis, MO), ad libitum, with free access to water (HCl acidified, pH 2.8–3.2) in a temperature- and humidity-controlled room with a 12-h light and dark cycle. All animal studies in this work were carried out with the approval of The Jackson Laboratory Animal Care and Use Committee. Mice were killed by CO2 asphyxiation.
Southern blot analysis
High molecular weight genomic DNA was isolated from spleens (27
), and 4–10 µg of DNA was digested overnight with restriction enzymes BamHI, EcoRI or PvuII (Roche, Indianapolis, IN) according to the manufacturer’s instructions. Restriction fragments were separated by electrophoresis on 0.7% agarose gels, transferred to Hybond N+ (Amersham, Piscataway, NJ) by capillary action in an alkaline buffer and hybridized with random primed (Amersham) 
-32P-labeled Lepr cDNA probe. The probes were obtained by reverse transcription–polymerase chain reaction (RT-PCR) by use of various primer pairs derived from the long form of the mouse Lepr cDNA (Table 1): F12/R6 (the first 588 amino acids), F2/R7 (exons 2–3), F7/R9 (exons 3–4), F11/R10 (exons 6–7) and F6/R6 (exons 7–9). The blots were washed in 2x SSC/0.1% SDS and in 0.5x SSC/0.1% SDS at 65°C for 20 min each and then exposed to X-ray film at -70°C.
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Total RNA was extracted from whole brains by use of the RNeasy kit (Qiagen, Valencia, CA) and treated with DNAse I (Roche) according to the manufacturer’s protocol. The RNA (10 µg) was reverse-transcribed with SUPERSCRIPT RT (Gibco BRL, Carlsbad, CA) by use of oligo d(T)12–18 as primer and digested with RNAse H according to the manufacturer’s instructions. The single-strand cDNA was diluted with water (1:5, v/v), and 2 µL of the diluate was used to amplify Lepr cDNA by use of the Expand Long Template PCR System (Roche). The full-length coding sequence was amplified by use of the primer pairs F1/R5 and F3/R4 (Table 1).
PCR products were first electrophoresed on a 1.2% agarose gel. Bands of interest were excised from the gel, and DNA fragments were isolated (Clontech, Palo Alto, CA). The gel-purified PCR products were directly sequenced with primers originally used to amplify the PCR products and with nested primers when needed. Sequencing was carried out either automatically with fluorescent tags (Applied Biosystems, Foster City, CA) or manually by use of 
-33P by cycle sequencing (Epicentre, Madison, WI).
Semiquantitative RT-PCR
For estimating Lepr expression, the single-strand cDNA diluents obtained from the whole brains of Leprdb-rtnd/db-rtnd or wild type mice as described above were amplified with primers (F10/R8) spanning exons 3 and 4 of the Lepr, by use of Taq polymerase. The mouse ß-actin was amplified with a pair of primers 5'-GTGGGCCGCTCTAGGCACCAA-3' and 5'-CTCTTTGATGTCACGCACGATTTC-3' (28
) as the control. PCR was performed in various numbers of amplification cycles (15, 20, 23, 26, 29 and 32 cycles), to ensure the linear range of amplification kinetics. Cycling conditions were as follows: 2 min 95°C initial denaturation, 5 s at 94°C, 10 s at 55°C and 40 s at 72°C. PCR products were electrophoresed, transferred to Hybond N+, hybridized with the original PCR primer labeled with -32P and exposed to X-ray film. The intensity of the bands was quantitated by use of Image Quant data analysis (Molecular Dynamics, Sunnyvale, CA).
Rapid amplification of 5'-cDNA ends (5'-RACE)
Double-stranded cDNA was synthesized from brain total RNA of the Leprdb-dmpg mutant by use of the SUPERSCRIPT Choice System (Gibco BRL) following the manufacturer’s instructions. The double-stranded cDNA was blunt-ended by use of T4 DNA ligase, and adaptor (Marathon Adaptor, Clontech) was then ligated overnight at 16°C. The 5'-RACE products were amplified by adaptor and reverse priming with a specific primer derived from the Lepr cDNA (R10, Table 1). The PCR products were sequenced directly or after subcloning into pBlueScript II SK.
Genomic PCR
The fragment containing the Leprdb-rtnd mutation was amplified from genomic DNA of wild type and Leprdb-rtnd homozygous mice with primer pairs of F14 and R8 by Expand Long Template PCR System (Roche). The PCR products were sequenced directly or after subcloning into the TA vector (Invitrogen, Carlsbad, CA).
Plasma glucose and insulin levels
Blood was drawn from the orbital sinus of nonfasted mice between 0730 and 1030 h in heparinized capillary tubes, and plasma was obtained by centrifugation (1200 x g) at 4°C. Plasma glucose levels were determined with a commercial colorimetric assay (Sigma, St. Louis, MO). Plasma insulin levels were determined by use of a Rat Insulin RIA kit (Linco Research, St. Charles, MO).
Histological examination of the pancreas
Mice were killed by CO2 asphyxiation. Pancreases were dissected, placed in Bouin’s fixative and paraffin embedded, and ß-cells were stained with aldehyde-fuchsin. The sections were then counterstained with hematoxylin and eosin.
Statistical analysis
ANOVA implemented in StatView 4.5 for Macintosh (Abacus Concepts, Berkeley, CA) were used to compare gender- and age-matched lean and obese mice in each group (Table 2). All values are expressed as means ± SEM. Differences of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Leprdb-rtnd. Southern blots prepared from Leprdb-rtnd/db-rtnd and wild type DNA were probed with the cDNA fragment corresponding to the first 588 amino acids of Lepr (Fig. 1A ). There were no size differences in the BamHI, EcoRI and PvuII restriction fragments in digested DNA from wild type and Leprdb-rtnd/db-rtnd mice, indicating that the mutation was not a gross genomic deletion, insertion or rearrangement.
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The RT-PCR product corresponding to the N-terminus of Lepr (amplified with F1/R5) was smaller in size in Leprdb-dmpg/db-dmpg mice than that in wild type mice (Fig. 2 B). Sequencing analysis of the RT-PCR product revealed a deletion of exons 1 to 6 in Leprdb-dmpg mutant mice (not shown). We observed a similar result in the sequences of the 5'-RACE product in Leprdb-dmpg/db-dmpg mice (not shown). To define whether the RT-PCR results represent an additional splice variant, we hybridized multiple genomic Southern blots prepared from Leprdb-dmpg/db-dmpg and wild type mice with overlapping cDNA fragments corresponding to the extracellular domain of the Lepr (Fig. 4 ). We observed the complete absence of restriction fragments hybridizing to probes corresponding to exons 2–4 of the receptor (Fig. 4 A, B). In contrast, only some of the restriction fragments were absent when probed with exons 6–7 (Fig. 4 C), indicating that this probe crossed the deletion boundary. An adjacent probe spanning exons 7–9 showed the wild type pattern in Leprdb-dmpg mutant mice (Fig. 4 D). Therefore, it appears that the Leprdb-dmpg mutation is a large genomic deletion including exons 2 through 6 in the Lepr. The extreme GC-rich nature of the 5' end of the Lepr has so far prevented the localization of the proximal deletion breakpoint by cDNA and genomic sequencing. However, the microsatellite marker most closely flanking the Lepr proximally, D4Mit176, was not deleted (not shown).
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Physiological characterization
Physiological data obtained from congenic mice for Leprdb-rtnd and for Leprdb-dmpg and from coisogenic mice for Leprdb-rlpy are summarized in Table 2. Both female and male mice homozygous for Leprdb-rtnd, for Leprdb-dmpg and for Leprdb-rlpy mutations became morbidly obese, weighing 43–53 g at 12 wk of age, compared to 22–35 g for the controls. With the exception of B6.CBA-Leprdb-rtnd/db-rtnd females, they also developed hyperglycemia that was accompanied by hyperinsulinemia compared to the respective gender- and age-matched controls.
In addition to the normoglycemia shown in B6.CBA-Leprdb-rtnd/db-rtnd female mice, males homozygous for Leprdb-rtnd manifested milder hyperglycemia in the B6 than in the CBA backgrounds (P = 0.008, 12 wk). This milder hyperglycemia was associated with higher plasma insulin levels (P = 0.0002) in the B6 than in the CBA. This diabetes resistance effect of the B6 genome, however, was not observed in B6.B10-Leprdb-dmpg/db-dmpg mice.
In all four stocks of mice, obese mice had enlarged pancreatic islets compared to lean controls, likely to compensate for hyperglycemia in these mice (Fig. 5 ). Along with this enlargement, B6.CBA-Leprdb-rtnd/db-rtnd obese mice had well-granulated and vascularized pancreatic islets (Fig. 5 A), unlike the other three obese stocks.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All three mutations result in a morbid obesity syndrome with various degrees of diabetes depending on the genetic background. A characteristic fat deposition (apple shaped) begins at 3 to 4 wk of age in all three mutants. At 4 to 5 wk of age, all mutants exhibit mild hyperglycemia, with the highest plasma glucose levels in Leprdb-rlpy homozygous mice (not shown). At this young age, plasma insulin levels of these mutant mice are also 10–100 times higher than controls (not shown). This early onset of hyperglycemia preceded by hyperinsulinemia was previously reported in Leprdb homozygous mice (29 ).
Leprdb-rtnd contains one nucleotide deletion, introducing stop codon 17 residues after amino acid 191. Leprdb-dmpg is a large deletion in the extracellular domain of the receptor, including the first ligand binding site, and Leprdb-rlpy is a complete deletion of the receptor. These three mutations are predicted to result in the loss of all Lepr isoforms. It cannot be excluded at this point that additional genes are also deleted in Leprdb-dmpg and in Leprdb-rlpy. However, the fact that the microsatellite markers most closely flanking Lepr are not deleted in either mutation may exclude this possibility. Furthermore, neither mutation showed any unusual phenotypic alterations, other than those found in known Lepr mutants. The nature of the mutations in each of the reported Lepr alleles in animals and in humans is summarized in Figure 6 . The Leprdb mutation produces a truncated long form of the receptor without affecting other isoforms, and fails to activate STAT3 in the hypothalamus (9 ,4 ,10 ,20 ). The Leprfa mutation results in a single amino acid substitution (Q269P) in the extracellular domain and affects cell surface expression of the receptor (5 ,22 ). Leprdb-3J, Leprdb-Pas and Leprfa-k produce a frameshift, exon duplication and nonsense mutation in the extracellular domain, respectively (12 ,22 –25 ).
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Differential expression of the diabetic phenotype depending on host genetic background has been observed in various Lepr mutations in mice. Leprdb homozygous mice, for instance, exhibit severe hyperglycemia in the C57BLKS/J (BKS) background but mild diabetes accompanied by marked hyperinsulinemia in the B6 background, (32 –34 ). Among other known Lepr mutations, the "BKS-type" hyperglycemia is observed in CD-1-Leprdb-NCSU and in CBA/Lt-Leprdb homozygous mice (CBA/Lt is a subline separated from CBA/LsJ, which is a subline separated from CBA/J in 1978) (17 ,35 ,36 ). On the other hand, 129/J-Leprdb-3J and DW-Leprdb-pas homozygous mice show the "B6-type" mild diabetes or normoglycemia (14 ,16 ,17 ). In rats, Leprfa homozygotes develop hyperglycemia in the Wistar Kyoto strain but not in the 13M strain (37 ). Collectively, these data imply that diabetes produced by Lepr mutations depends on the interaction of the Lepr with the host background modifier genes rather than reflecting allelic effects. Therefore, identification of the interacting host genes may contribute to our understanding the etiology of diabetes associated with Lepr mutations. Studies of genetic modifiers are much more feasible in mice than in humans, where in most cases family sizes are too small to map modifier loci, much less to positionally clone them; in contrast, our congenic mice may be useful for carrying out genetic crosses to map and identify these modifier genes.
In humans, thus far, only one case of a null mutation in the LEPR has been reported in a consanguineous family of Kabilian origin (HD family) (6
). The HD mutation is a G 
A base substitution in the splice donor site of exon 16 and is predicted to encode a truncated LEPR lacking both the transmembrane and intracellular domains (6
). Three sisters homozygous for this mutation are morbidly obese with body mass indexes of 71.5, 65.5 and 52.5 kg/m2, respectively, at 13 and 19 y of age (6
). Because of the few mutations reported thus far, the influence of genetic background on the phenotype of human LEPR mutations is difficult to assess. However, two of the three patients developed only very mild hyperglycemia and one normoglycemia despite a seemingly severe LEPR mutation, suggesting compensatory influences by the genetic background.
Changes in blood glucose levels are typically associated with morphological changes of the pancreatic islets in diabetic patients. As blood glucose levels increase, degranulation of ß-cells (detected by aldehyde-fuchsin negative staining) increases. The increased degranulation implies hypersecretion of insulin by the ß-cells. With disease progression, ß-cells become hypertrophied, followed by atrophy accompanied by loss of glycemic control. However, depending on genetic backgrounds, dramatic differences in pancreatic morphology are observed in Lepr mutations. In BKS-Leprdb obese mice, the ß-cells are extensively degranulated and atrophied with age (32 ). B6-Leprdb, B6-Leprdb-2J and 129/J-Leprdb-3J obese mice, however, contain ß-cells well stained with aldehyde-fuchsin, an indication of rapid replication and synthesis of insulin, and maintain hypertrophy as well as hyperplasia through life (14 ,30 ,32 ). This hyperactivity of the pancreas may in part be responsible for the mild hyperglycemia with marked hyperinsulinemia observed in these mutants. B6.CBA-Leprdb-rtnd obese mice exhibit hyperplastic and hypertrophied islets with well-granulated ß-cells (Fig. 5) . In contrast, B6.B10-Leprdb-dmpg obese mice exhibit heavily degranulated ß-cells but with hyperplasia and hypertrophy (Fig. 5) . CBA.B6CBA-Leprdb-rtnd obese mice have degranulated and hypertrophied ß-cells, and NU/J-Leprdb-rlpy obese mice show additional atrophic changes including fibrosis (Fig. 5) . Hypertrophied and degranulated pancreatic ß-cells are also seen in WKY-Leprfa obese rats, suggesting that the pancreas in these models is in the active insulin synthesizing and secreting state (37 ).
Lepr mutations are rare in human obesity, and lead to rapid onset, morbid obesity. Diet-induced obesity, on the other hand, develops rather gradually, at various levels and at a later age in both humans and mice (38 ). Leptin resistance/insensitivity, however, is commonly observed in diet-induced obesity in animals and humans (39 ). Furthermore, there appears to be some degree of interaction between Lepr regulation and consumption of a high fat diet [i.e., Lepr mRNA expression is down-regulated in the hypothalamus of rats by long-term high fat feeding (40 )]. Accordingly, understanding LEPR functions will contribute to unraveling the integrated pathogenic networks of human obesity, and identifying the nature of various Lepr mutations will provide important information for understanding the biological functions of LEPR.
In summary, we have discovered three new mouse mutations of Lepr: Leprdb-rtnd, Leprdb-dmpg and Leprdb-rlpy. These mutations have not been detected previously either in animals or in humans but have in common with all known Lepr mutations that they represent drastic alterations of the LEPR protein. We propose that these alleles encode a truncated LEPR or lack of LEPR, producing a null state for Lepr with respect to the obesity phenotype. The new congenic stocks for Leprdb-rtnd and for Leprdb-dmpg will be useful in elucidating the effects of genetic background on the Lepr mutations. The coisogenic Leprdb-rlpy/db-rlpy mice, which completely lack the entire Lepr, may be useful as a control in testing the specificity of antibodies to LEPR.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the American Heart Association Postdoctoral Fellowship Award 9820036T (J.H.K.) and National Institutes of Health Grant DK-46977 (J.K.N.). Institutional shared services were supported by National Cancer Institute Cancer Center Grant CA-34196 (T.J.L.).
4 Abbreviations used: JAK, janus kinase; LEPR, leptin receptor; 5'-RACE, rapid amplification of 5'-cDNA ends; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TM, transmembrane.
Manuscript received 10 December 2002. Initial review completed 2 January 2003. Revision accepted 27 January 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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