Neuronal deletion of Lepr elicits diabesity in mice without affecting cold tolerance or fertility
Julie E. McMinn,1
Shun-Mei Liu,1
Hong Liu,1
Ioannis Dragatsis,2
Paula Dietrich,2
Thomas Ludwig,3,4
Carol N. Boozer,5,6 and
Streamson C. Chua, Jr.1,5
1Department of Pediatrics, Division of Molecular Genetics, Columbia University College of Physicians and Surgeons, New York, New York; 2Department of Physiology, University of Tennessee, Memphis, Tennessee; 3Department of Anatomy and Cell Biology, 4Institute for Cancer Genetics, and 5Institute of Human Nutrition, and 6St. Luke's-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, New York, New York
Submitted 3 November 2004
; accepted in final form 28 April 2005
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ABSTRACT
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Leptin signaling in the brain regulates energy intake and expenditure. To test the degree of functional neuronal leptin signaling required for the maintenance of body composition, fertility, and cold tolerance, transgenic mice expressing Cre in neurons (CaMKII
-Cre) were crossed to mice carrying a floxed leptin receptor (Lepr) allele to generate mice with neuron-specific deletion of Lepr in
50% (C F/F mice) and
75% (C
17/F mice) of hypothalamic neurons. Leptin receptor (LEPR)-deficient mice (
17/
17) with heat-shock-Cre-mediated global Lepr deletion served as obese controls. At 16 wk, male C F/F, C
17/F, and
17/
17 mice were 13.2 (P < 0.05), 45.0, and 55.9% (P < 0.001) heavier, respectively, than lean controls, whereas females showed 31.6, 68.8, and 160.7% increases in body mass (P < 0.001). Significant increases in total fat mass (C F/F: P < 0.01; C
17/F and
17/
17:P < 0.001 vs. sex-matched, lean controls), and serum leptin concentrations (P < 0.001 vs. controls) were present in proportion to Lepr deletion. Male C
17/F mice had significant elevations in basal serum insulin concentrations (P < 0.001 vs. controls) and were glucose intolerant, as measured by glucose tolerance test (AUC P < 0.01 vs. controls). In contrast with previous observations in mice null for LEPR signaling, C F/F and C
17/F mice were fertile and cold tolerant. These findings support the hypothesis that body weight, adiposity, serum leptin concentrations, and glucose intolerance are proportional to hypothalamic LEPR deficiency. However, fertility and cold tolerance remain intact unless hypothalamic LEPR deficiency is complete.
leptin receptor; Cre recombinase
LEPTIN IS A 16-KDA HORMONE that is required for normal regulation of body composition and ingestive behavior. Leptin is secreted from adipose tissue into the bloodstream and circulates in proportion to overall fat stores. Hyperleptinemia is a common feature of obesity that downregulates leptin receptor (LEPR) signaling, effectively blocking responsiveness to exogenously administered leptin. However, a single injection of leptin administered either centrally or peripherally to normal rodents elicits hypophagia and reduces the size of adipose depots (7). Spontaneous genetic mutations that prevent leptin signaling cause a profound obesity in mice (14), rats (40), and humans (12). Genetic obesity resulting from leptin deficiency in Lepob mice and LEPOB humans is corrected with leptin hormone therapy (7, 19, 23, 35). Leptin signaling deficiency in Lepob/ob mice is also associated with infertility and thermoregulatory deficits that are corrected with intracerebroventricular infusion of leptin (8, 25).
Three membrane-bound LEPR isoforms have been localized to mouse brain: LEPR-A, LEPR-B, and LEPR-C (31). The LEPR-B transcript is generated from 18 coding exons with a janus kinase 2 (JAK2) docking site in exon 17 and a signal transducer and activator of transcription 3 (STAT3) binding site in the terminal exon. These two motifs mediate leptin signaling via the JAK/STAT pathway (5). The obesity of Leprdb mice and Leprfa rats is associated with spontaneous genetic mutations in Lepr (9), and an in vivo tyrosine-to-serine mutation of the STAT3 domain in LEPR-B results in obesity in mice (4), indicating that loss of STAT3 signaling by LEPR-B is sufficient to cause obesity.
LEPR-B predominates in subnuclei of the hypothalamus that regulate body weight, including the arcuate (ARC) and lateral, dorsomedial, and ventromedial hypothalamic nuclei (17). Leptin signaling deficiency in spontaneously obese rodents is associated with chronically elevated expression of the orexigenic neuropeptides agouti-related protein (AGRP) and neuropeptide Y (NPY) in the ARC, accompanied by suppression of anorexigenic neuropeptides, including proopiomelanocortin (POMC). Central dysregulation of these neuropeptides is associated with hyperphagia and weight gain, and intracerebroventricular administration of leptin to Lepob mice restores the neuropeptide profile to that of a lean mouse while reducing food intake and body weight (33, 37). Conversely, leptin-induced anorexia is associated with an inhibition of NPY and AGRP mRNA expression and a concurrent upregulation of POMC mRNA in normal and obese rodents (33, 37). These findings support the hypothesis that leptin action is mediated, at least in part, by ARC neuropeptide signaling pathways.
Previous studies indicate that deficiency in the central expression of LEPR is largely responsible for the dysregulation of energy homeostasis observed in mice with global LEPR mutations (13). However, the question remains how other systems, such as thermoregulatory and reproductive axes, are affected by neuronal leptin signaling deficiency. We (30) previously generated a series of novel Lepr alleles that are substrates for Cre and Flp recombinases. Using these Lepr alleles in combination with a neuron-specific Cre transgene, we generated mice with inactivation of the signal transduction capacity of all membrane-bound LEPR isoforms in neurons(C F/F). To ascertain the degree of Lepr exon 17 excision within the brain, we also developed compound heterozygous mice carrying the neuronal Cre transgene (C
17/F). Our data suggest that adiposity and glucose intolerance increase with increasing degrees of LEPR inactivation. However, fertility and cold tolerance are preserved in this model of partial LEPR deficiency, suggesting that complete or nearly complete LEPR deficiency is necessary for cold sensitivity and infertility to become manifested.
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MATERIALS AND METHODS
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Generation of conditional knockout mice.
Neuron-specific deletion of Lepr was achieved by crossing Leprflox/flox (F/F) mice withC57BL/6129 mice transgenic for Cre recombinase driven by a CaMKII-
promoter (CaMKII
-Cre). The CaMKII
-Cre transgene is widely expressed in forebrain neurons and testes (R1 and L7 lines) (16). Female (CaMKII
-Cre Leprflox/+) progeny (C F/+) were backcrossed to male F/F mice to generate mice with neuron-specific deletion, and the resulting C F/F mice had Lepr deleted in forebrain neurons. Male R1 C F/+ progeny expressed the CaMKII
-Cre transgene in the testes, as verified by
-galactosidase staining in CaMKII
-Cre Rosa26-LacZ mice (Fig. 1A). Testicular expression of Cre recombinase resulted in transmission of a Lepr
17 allele to the progeny of the males. Thus R1 C F/+ males, when mated to F/F females, sired CaMKII
-Cre/+ Lepr
17/flox mice (C
17/F) that were compound heterozygotes for Leprflox and Lepr
17. We have analyzed mice carrying both the R1 and L7 Cre transgenes, and L7 C F/+ males generated C F/F progeny but no C
17/F offspring. Therefore R1 and L7 lines were combined for phenotypic analysis of F/F, C F/+, and C F/F male and female mice, but all C
17/F mice were from the R1 line.
Ablation of exon 17 was achieved by crossing F/F mice to Heat-shock-Cre line 1 transgenic mice maintained on a C57BL/6129 background strain (15) to generate Lepr
17/+ (
17/+) mice. Mice homozygous for the Lepr
17 allele were generated from intercrosses between Lepr
17/+ heterozygotes. For all crosses, progeny were healthy and viable at birth, and genotypes were present at the normal Mendelian frequency.
Animal husbandry.
Mice were housed in Plexiglas cages with corn cob bedding at 22 ± 1°C and a 12:12-h light-dark cycle in the Russ Berrie Medical Science Pavilion barrier facility at Columbia University. Pregnant mice and dams with pups were housed in similar conditions, and pups were weaned on postnatal day 21. Breeders and pups aged 021 days were fed a 21.6% kcal fat chow (5058 Picolab Mouse Diet 20, Purina Mills) and water ad libitum. Offspring were switched to an 11.9% kcal fat chow upon weaning (5053 Picolab Rodent Diet 20, Purina Mills) until they were euthanized, unless otherwise noted. All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Columbia University Health Sciences Division.
PCR verification of mouse genotypes.
DNA was prepared from earclippings, hypothalamus, whole brain, liver, muscle, and adipose tissue and used as a template for PCR with appropriate primers to genotype the transgenic and mutant animals. Multiplex primers were designed to detect relative amounts of Leprflox, Lepr
17, and wild-type Lepr in C F/F x C F/+ crosses (Fig. 1B), with primer 105 (aca ggc ttg aga aca tga aca c), complementary to intronic regions 148 bp downstream of exon 17; primer 106 (gtc tga ttt gat aga tgg tct t), located 358 bp upstream of exon 17; and 65A (AGA ATG AAA AAG TTG TTT TGG GA), which anneals to the 5' end of exon 17. A multiplex primer set was also developed to detect exon 17 deletion in Lepr
17 with primer 105 (described above), primer 103 (TGA GTT CCC TCA TGA TTC TGG), and primer 104 (CAG CCG ACC AAT GCT TAT T) annealing to a sequence between the two loxP sites located 74 bp upstream of exon 17. Cre recombinase transgenes were identified by primer sets amplifying across Cre (Cre-F, TCG TCG GTC CGG GCT GCC AC and Cre-R, TTA GTT ACC CCC AGG CTA AG).
Body weight and food intake.
Twelve- to sixteen-week-old mice from each experimental group were weighed, and body mass was recorded in grams. For food intakes, mice were habituated to individual housing for 2 wk and were acclimated for 1 wk to 10% kcal fat chow (no. D12450B; Research Diets, New Brunswick, NJ). Daily intakes for each genotype were calculated by averaging the 24-h food intake of each mouse over the 3-day experimental period.
Cold challenge.
Core body temperatures of male Lepr+/?, C F/F,C
17/F, and
17/
17 mice were monitored with a rectal probe (Physitemp, Clifton, NJ) prior to cold exposure and then at 20, 40, and 80 min after the initiation of the 0°C challenge, as previously described (27).
Measurement of serum glucose, insulin, leptin, and thyroid function assay.
Blood was collected from the tip of the tail of fed mice between 1400 and 1700 and measured for glucose, insulin, and leptin concentrations, as previously described (27). A thyroid function assay was performed by the Yale Mouse Metabolic Phenotyping Center by using the T-Uptake Enzyme Immunoassay and Thyroxine (T4) Enzyme Immunoassay kits (Diagnostic Reagents, Sunnyvale, CA), using mouse serum. The free thyroxine index (FTI) was calculated using the following equation: FTI = (total T4) x (%Tuptake) (average Tuptake for healthy controls), where average Tuptake = %Tuptake values were obtained from control mice.
Fertility testing.
Fertile wild-type FVB/NJ mice were housed with 4-mo-old
17/
17 and C
17/F mice to ascertain the fertility of the experimental mice. For all matings, one male was housed with one or two females per cage, and the number of litters produced was tabulated for a period of 2 mo.
Body composition analysis.
Mouse carcasses were scanned by dual-energy X-ray absorptiometry (DEXA) using a PIXImus mouse densitometer (Lunar, GE Medical Systems, Waukesha, WI). A tissue calibration scan was performed every 4 h to correct for performance variables over the course of the analysis.
Glucose tolerance tests.
Mice (56 animals per group) were deprived of food overnight (1618 h), after which an intraperitoneal injection of dextrose (1 mg/g) was administered to awake mice. Blood (5 µl) was collected from the tip of the tail before and 20, 40, 60, 90, and 120 min after injection. Blood glucose was measured at each time point using a Glucometer (Glucometer Elite; Bayer, Elkhart, IN).
Histology.
Brown adipose tissue (BAT) was dissected immediately from mice euthanized by carbon dioxide asphyxiation. BAT was fixed in Z-Fix (Anatech, Battle Creek, MI) for 2 days. Tissues were processed and embedded in paraffin. BAT sections (5 µm) were stained with hematoxylin and eosin.
Semiquantitative RT-PCR for LEPR.
RNA was isolated from whole brain and hypothalamic extracts using RNAWiz RNA Isolation Reagent (Ambion, Austin, TX). Total RNA was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA), as previously described (10). LEPR-B cDNA was amplified across exon 17 with primers annealing to exon 16 and 18b (16, TCG ACA AGC AGC AGA ATG AC; 18b, ctg ctg gga cca tct cat c). PCR products were visualized using ethidium bromide staining. Quantification was performed by measuring fluorescence intensity with QuantOne software on an eight-bit camera (Bio-Rad, Hercules, CA). PCR was performed on samples prepared with and without reverse transcriptase.
Real-time PCR for hypothalamic neuropeptide expression.
RNA was isolated from hypothalamic extracts using the RNeasy Mini Kit (Qiagen, Valencia, CA) followed by DNase treatment and removal with a DNA-free Kit (Ambion, Austin, TX). Total RNA was reverse transcribed using SuperScript II reverse transcriptase with oligo(dT)1218 primers and subsequently incubated with RNase H (Invitrogen). The crossing threshold for hypothalamic cDNA was determined using Opticon Monitor v. 2.02.24 software on an Opticon2 Continuous Fluorescence Detector (MJ Research, Boston, MA). NPY, AGRP, POMC, and hypoxanthine-guanine-phosphoribosyltransferase (HPRT) transcripts were amplifed using intron-spanning primers. Crossing thresholds were converted to copy number and normalized to copy number of the HPRT internal standard.
Data analysis.
Data are presented as group mean values (±SE). For experiments with greater than two study groups, comparisons were performed with one-way ANOVA and Newman-Keuls post hoc test, unless otherwise noted. A P value
0.05 between group mean values was considered statistically significant.
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RESULTS
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Deletion of Leprflox in the hypothalamus.
Combination of the neuron-specific Cre transgene (CaMKII
-Cre) with Leprflox should have resulted in neuron-specific deletion of coding exon 17 of Lepr, and a frameshift mutation disrupting the terminal exon (30). Because all membrane-bound isoforms of LEPR contain this exon, deletion of exon 17 resulted in the loss of signal transduction capabilities of all membrane-bound LEPR isoforms. Multiplex PCR amplification of genomic DNA in F/F mice yielded a 250-bp Leprflox band from all tissues examined (Fig. 2A). C F/F mice had an additional 229-bp
17 band present in brain and hypothalamic extracts, representing deletion of coding exon 17 that converts the Leprflox allele to the Lepr
17 allele (Fig. 2B). Peripheral tissues produced an amplicon that corresponded to the Leprflox allele, reflecting the lack of Cre activity within these tissues. The
17 allelic product constituted
50% of the amplified product in both hypothalamus and whole brain.
We also analyzed C
17/F mice, wherein the male parent transmitted a
17 allele, since the CaMKII
-Cre transgene is active within the stem cells that produce sperm (Fig. 1A). Examination of the genomic DNA of C
17/F mice showed two bands representing the two Lepr alleles (Fig. 2C). The lower
17 band was present in all peripheral tissues with the same intensity as the upper Leprflox band, indicating the equal contributions of the two different Lepr alleles. However, in both brain and hypothalamic DNA, the lower
17 band represented
75% of the total amplified product, indicating a greater contribution of the Lepr
17 allele in neuronal tissue than that observed in C F/F mice. The
17 band was the only amplicon present in all tissues examined of
17/
17 mice (Fig. 2D).
Amplification of brain and hypothalamic cDNA from F/F mice with LEPR-B-specific primers (Fig. 3) produced a 354-bp band. The deletion of exon 17 in
17/
17 mice resulted in a smaller band of 279 bp in the tissues of these mice. This is an anticipated result, since the frameshift caused by the deletion of coding exon 17 produces a termination codon only within the native terminal exons. Hypothalamic extracts from C F/F mice showed bands of equal intensity, indicating a 1:1 ratio between Leprflox and Lepr
17 alleles. The LEPR
17 transcript represented
50% of total LEPR-B mRNA in the hypothalamus. Interestingly, there was no amplification of the LEPR
17 transcript in the extrahypothalamic brain. Analysis of Lepr expression in C
17/F mice showed the presence of two LEPR-B transcripts also. In the hypothalamus of C
17/F, the LEPR
17 transcript represented
75% of total LEPR-B mRNA, and in the rest of the brain the LEPR
17 transcript represented
50% of LEPR-B. This analysis indicates that Cre-mediated excision has a greater impact on Lepr expression in C
17/F compared with C F/F mice. No difference was detectable in the total quantities of amplified transcript in mice of all genotypes, suggesting that the LEPR-B isoform of the LEPR
17 transcript is not subject to nonsense-mediated decay.
Body weight and food intake are increased in proportion to hypothalamic Lepr deletion. Neuronal Lepr deletion was proportional to body weight in males and females (Fig. 4, A and B). Because F/+ and
17/+ mice had body weights that were statistically indistinguishable from F/F and F/+ mice with intact Lepr, these mice were grouped together and averaged 34.0 ± 0.7 g for males and 24.7 ± 0.6 g for females (Table 1). At 16 wk of age, male C F/F mice were 13.2% heavier than lean +/? controls (P < 0.05), whereas male C
17/F mice were 45.0% heavier (P < 0.001) and were visibly more obese than the lean mice (Fig. 4C). Male
17/
17 mice were 55.9% heavier than lean controls (Table 1, P < 0.001). Body weights of female mice showed a similar trend as the males. Female C F/F mice averaged a 31.6% increase in body weight compared with lean controls, while female C
17/F mice had a 68.8% increase over controls, and female
17/
17 mice were 160.7% heavier than controls (all P < 0.001 vs. lean mice). Food intake over 24 h was significantly elevated in C
17/F and
17/
17 males only (Table 1, P < 0.01 and P < 0.001, respectively, vs. +/? mice). CaMKII
-Cre-mediated Lepr deletion had no significant effect on food intake in female mice (Table 1).
Adiposity and serum leptin concentrations are positively associated with Lepr deletion. Mean values for absolute fat masses were positively correlated with hypothalamic Lepr deletion within each sex (Fig. 5). Although total fat mass of male +/? mice averaged 8.6 ± 0.5 g, fat mass of male C F/F, C
17/F, and
17/
17 mice was 12.3 ± 1.2, 16.4 ± 1.7, and 26.2 ± 1.9 g (P < 0.01, P < 0.001, and P < 0.001, respectively, vs. lean mice). Similarly, female +/? mice had an average total fat mass of 5.2 ± 0.5 g, whereas fat mass of female C F/F, C
17/F, and
17/
17 mice was 12.0 ± 1.7, 23.4 ± 2.6, and 35.0 ± 5.2 g (P < 0.01, P < 0.001, and P < 0.001, respectively, vs. lean mice). Although the degree of hypothalamic Lepr deletion did not appear to be different between males and females of the same genotypes, body fat percentage was significantly increased in C
17/F and
17/
17 females compared with males of the same genotypes (C
17/F females vs. males: 49.1 vs. 32.1%, P < 0.001;
17/
17 females vs. males: 59.1 vs. 49.7%, P < 0.05). Serum leptin levels were significantly elevated in both male and female C
17/F and
17/
17 mice compared with their respective sex-matched littermates (Table 1, P < 0.001 vs. lean controls). Increased adiposity in females corresponded with a proportional elevation in serum leptin concentrations (Table 1).

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Fig. 5. Total body fat (g) as calculated by dual-energy X-ray absorptometry (DEXA). Mice were killed at 1620 wk of age, exsanguinated, and decapitated. Carcasses were stored at 80°C and were completely thawed immediately before DEXA body composition measurements. Differing superscripts represent significant differences between groups; see text for details.
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Male C
17/F mice are glucose intolerant.
Both male and female C
17/F mice were euglycemic in the fed state, whereas serum glucose levels were elevated to the diabetic range in
17/
17 males (Table 1, P < 0.001 vs. +/? mice) and
17/
17 females (Table 1, P < 0.001 vs. +/? mice). However, when challenged with a 1 mg/kg body wt bolus of intraperitoneal dextrose following an overnight fast, male C
17/F mice showed significant elevations in serum glucose during the time period from 60 to 120 min postinjection relative to lean littermates (Fig. 6), whereas females did not (data not shown). Area under the curve (AUC) was also significantly greater inC
17/F males (P < 0.01).
Male C F/F and C
17/F mice fed ad libitum showed significant increases in serum insulin relative to +/? mice (Table 1, P < 0.05 and P < 0.001, respectively). Despite severe hyperglycemia, male
17/
17 mice had a nonsignificant elevation of serum insulin vs. controls, as individual values varied widely, probably due to loss of
-cells secondarily to chronically elevated blood glucose levels. Female
17/
17 mice maintained significantly elevated insulin concentrations relative to lean controls (Table 1, P < 0.001); however, C F/F and C
17/F females showed no significant differences from controls.
Male C
17/F mice are cold tolerant and have normal BAT morphology and thyroid function.
To investigate the thermoregulatory defect associated with hypothalamic Lepr deletion, C F/F, db/db, and lean male mice were subjected to a cold challenge.
17/
17, C
17/F, and lean control mice were also tested in a separate trial. In agreement with previous reports, the core body temperatures of db/db mice were significantly suppressed relative to lean control mice (7.3 ± 1.2 vs. 0.4 ± 0.8°C, P = 0.0029 by Student's t-test). Similar to db/db mice,
17/
17 mice showed a significant decrease in core body temperature over the 80-min test period (Table 1, P = 0.0366 by Student's t-test). By contrast, C F/F and C
17/F mice maintained core body temperature and were not significantly different from controls after cold exposure. Histological sections of BAT were prepared to investigate the possibility of a thermogenic defect. Brown adipocytes of C F/F mice were virtually indistinguishable from those of lean controls, whereas BAT sections of 20-wk-old C
17/F mice appeared to be infiltrated with white adipocytes (Fig. 7), and adipocytes of C
17/F and
17/
17 mice were enlarged.

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Fig. 7. Fatty infiltration of brown adipose tissue (BAT) due to neuronal LEPR deficiency. BAT was harvested from mice of each genotype and stained using standard methodology. Representative sections are provided for each genotype. Increased adipocyte size was associated with increasing degrees of LEPR signaling deficiency.
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Because leptin signaling regulates the HTA in humans (21) and rodents (29, 34), the FTI was determined for
17/
17, C
17/F, and lean control mice. Average %Tuptake of normal healthy mice was 38.91%, which was used in the denominator to calculate FTI (see MATERIALS AND METHODS). Only obese male
17/
17 mice showed a significant defect in thyroid function compared with lean controls (Table 1, P < 0.01).
Fertility testing.
After 2 mo in mating pairs or trios, four of five male C
17/F mice and five of seven female C
17/F mice produced healthy, viable litters. All F/F mice were fertile. However, similar to db/db and ob/ob mice, the reproductive function of
17/
17 mice of both sexes was grossly impaired, and no litters were generated after 2 mo.
Real-time quantification of hypothalamic NPY, AGRP, and POMC cDNA.
Obese
17/
17 male mice showed a 148.8% increase in hypothalamic NPY mRNA expression (P < 0.01), and
17/
17 females had a significant 54.7% suppression in POMC mRNA (P < 0.01), compared with lean control mice (Table 2). By contrast, hypothalamic mRNA concentrations of NPY, AGRP, and POMC in C
17/F male and female mice were not different from lean controls (Table 2).
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DISCUSSION
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Several previous studies have employed mouse models to test the hypothesis that leptin signaling acts in the central nervous system to regulate body weight. Neuronal overexpression of LEPR-B attenuates the hyperphagia, increased body weight, and adiposity of obese, Lepr-deficient db3J/db3J mice (27), and synapsin-promoter-driven Cre-mediated deletion of neuronal Lepr largely recapitulates the obesity syndrome of mice with global Lepr deletion (13). We have confirmed and extended these previous findings in mice with increased adiposity in proportion to neuronal Lepr deletion.
Fasting increases hypothalamic LEPR mRNA expression and leptin binding in the hypothalamus (2, 3), which is accompanied by increased sensitivity to exogenously administered leptin (3). Because expression of LEPR-B transcripts are therefore indicative of functional leptin signaling, we propose that neuron-specific Cre-mediated deletion of Lepr (C F/F), which resulted in inactivation of
50% of hypothalamic Lepr with an estimated maximum of 50% of hypothalamic cells bearing two defective Lepr
17 alleles (Fig. 8), decreased hypothalamic leptin signaling by
50%. We ascribe this partial deletion effect to mosaic expression of the Cre transgene, mosaicism being a well-documented feature of transgenic mice. C F/F mice were therefore significantly fatter than their +/? littermates, but significantly less fat than Lepr-null
17/
17 mice, due either to incomplete hypothalamic deletion (1) or to the absence of Lepr deletion in key neurons regulating energy homeostasis.
However, male C F/F mice were significantly heavier than male
17/F mice, which also had 50% hypothalamic deletion, suggesting that suppression of leptin signaling did not absolutely correlate with the obesity phenotype. The discrepancy may be explained by considering that cellular distribution of Lepr alleles may have differed between the two genotypes:
17/F mice had one normally functioning allele (Leprflox) and one germline-deleted allele (Lepr
17) in every cell, analogous to Lepr heterozygosity in the db/+ mouse (Fig. 8), whereas C F/F mice are likely to have had two Lepr
17 alleles in Cre-expressing neurons, which we propose comprised
50% of the total neuronal population of the hypothalamus (Fig. 8). We also generated and characterized C
17/F mice, which we presumed had one Lepr
17 allele in each neuron as a result of germline deletion and a second Lepr
17 allele in 50% of hypothalamic neurons expressing Cre (Fig. 8). We propose that the increase in body weight and adiposity in C
17/F vs. C F/F mice was due to this increase in the number of hypothalamic neurons with two defective Lepr alleles.
In addition, we propose that more Lepr deletion may have occurred in the hypothalamus compared with extrahypothalamic brain tissue in the CaMKII
-Cre-expressing mice (Fig. 3, and shown schematically in Fig. 8), since hypothalamic tissue has a higher density of neurons than other parts of the brain. The ratio of hypothalamic LEPR-B mRNA transcripts for the Leprflox and Lepr
17 alleles (Fig. 3) very closely reflected the ratio of alleles in genomic DNA from hypothalamic extracts (Fig. 2, AD), suggesting that a large compensatory increase in LEPR expression from the Leprflox allele did not occur with the loss of LEPR signaling in other cells. Furthermore, the lack of alteration in hypothalamic neuropeptide levels between C
17/F and +/? mice indicates that neurons of the hypothalamus hemizygous for signaling LEPR continued to respond normally to leptin; however, a thorough examination of Lepr deletion on the single-cell level would be necessary to comprehensively assess the effects on hypothalamic neuropeptide levels in the current model.
Most male C
17/F mice had impaired glucose tolerance, wherease female mice of the same genotype were nondiabetic, in contrast to a previous report showing a more pronounced diabetic phenotype in female vs. male mice with neuronal Lepr deletion (13). It is unlikely that differences in LEPR signaling between our male and female C
17/F mice were responsible for the sex-specific phenotypes, because Lepr-null
17/
17 mice, which lacked all LEPR signaling, showed a similar tendency in males for pronounced diabetes and hyperphagia accompanied by reduced adiposity and leptin levels compared with the less diabetic
17/
17 females. Because our finding is in agreement with the observation that db mutations elicit hyperglycemia exclusively in male mice on a mixed-strain background whereas male db-C57BL/6 mice are euglycemic (6), we attribute sex differences in the diabetes and adiposity phenotypes to background effects rather than genotype. Background effects also may have contributed to the lack of phenotype in
17/F vs. +/? mice, which contrasts with previous reports showing increased adiposity and circulating leptin concentrations in mice heterozygous for Lepr (11).
Because body fat accretion was not as great in female C
17/F mice as in female
17/
17 mice, it is possible that the lesser fat accumulation in C
17/F mice prevented infertility. However, a recent study demonstrates that diet-induced obese female DBA/2J mice, with only a 50% increase in adiposity over their lean littermates, exhibit a 60% decrease in natural pregnancy rates (39). The modest accumulation of fat in this model of diet-induced infertility is accompanied by a 95% suppression of LEPR-B transcripts and a 100% increase in NPY transcripts as assayed by real-time PCR in hypothalamic extracts, similar to what we observed in infertile male and female
17/
17 mice, suggesting a hypothalamic rather than peripherally mediated infertility. In further support of NPY's role in maintaining reproductive function, fertility is preserved in several obese mouse models when elevated hypothalamic NPY signaling associated with the Leprdb/db and Lepob/ob phenotypes is significantly attenuated. For instance, LeprS1138 homozygotes, with a single amino acid mutation in the STAT3 signaling portion of LEPR-B, are fertile despite their extreme obesity, which may be attributed to
40% reduced NPY levels compared with db/db mice (4). Similarly, Lepob/ob mice with attenuated NPY signaling due to leptin treatment, or mice resulting from Lepob/ob crosses with NPY- or NPY Y4R-deficient mice, have restored reproductive function (8, 18, 36). However, LeprS1138, Y4r/, and leptin-treated Lepob/ob mice remain incapable of lactation. Therefore, it is likely that the lack of elevated NPY in C
17/F mice prevented the loss of fertility and that the ability of C
17/F mice to lactate, which is also observed in NSE-Rb-Leprdb/db mice, may be attributed to the presence of LEPR signaling in circuits that regulate mammary gland development independently of NPY signaling pathways.
Complete deletion of Lepr may also be required to elicit cold intolerance, which results from abnormal sympathetic nervous system activation and central hypothyroidism (22, 24) secondary to dysregulation of hypothalamic neuropeptides upstream of thyrotropin-releasing hormone (TRH) (20, 28). Previous studies have demonstrated that reduced core body temperature associated with murine leptin deficiency is normalized in NPY-deficient and POMC-overexpressing Lepob/ob mice (18, 32) and that cold intolerance of Lepob/ob mice is attenuated in melanin-concentrating hormone-deficient Lepob/ob mice (38). In our study, neuropeptide levels of NPY, AGRP, and POMC (Table 2) and TRH (data not shown) in C
17/F mice were not significantly different from those in +/? mice. In addition, no impairment of the HTA was observed in C
17/F mice, as measured by FTI (Table 1). These findings contrast with the apparent hypothyroidism and hypothalamic dysregulation of neuropeptides observed in
17/
17 mice, which showed significant suppression of FTI (Table 1) characteristic of mice with leptin-signaling deficiencies (26). Therefore, although histological staining revealed increased fat accumulation in brown adipocytes, we conclude that changes in BAT morphology of C
17/F mice compared with their lean littermates reflect an accumulation of triglycerides due to secondary effects of the obesity syndrome elicited by hypothalamic leptin-signaling deficiency rather than a perturbation of the sympathetic nervous system via the HTA. The normal thermogenic capacity of C F/F and C
17/F mice may have also contributed to a smaller fat mass accretion in these genotypes compared with mice with global Lepr deficiency.
In conclusion, we have generated mice with obesity/diabetes phenotypes that are proportional in their severity to hypothalamic Lepr deletion with respect to energy homeostasis. Partial ablation of LEPR signaling in a fraction of leptin-sensitive neurons can produce obesity and insulin resistance. Our data support the possibility that partial LEPR defects have a subtle effect on neuronal function that has a measurable effect within the context of other neurons that are completely leptin insensitive. However, total loss of LEPR signaling in all relevant neurons may be required to reproduce the cold intolerance and infertility of complete leptin-signaling deficiencies.
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GRANTS
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This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-57621 (S. C. Chua, Jr.), F32-DK-61229 (J. E. McMinn), PO1-DK-26687 (New York Obesity Research Center), and P30-DK-63608 (Columbia University Diabetes and Endocrinology Research Center).
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FOOTNOTES
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Address for reprint requests and other correspondence: S. Chua, Jr., Columbia Univ. College of Physicians and Surgeons, Russ Berrie Medical Science Pavilion, Rm. 627, 1150 St. Nicholas Ave., New York, NY 10032 (e-mail: sc569{at}columbia.edu)
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.
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