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. 2001 Oct;108(8):1113-21.
doi: 10.1172/JCI13914.

Selective deletion of leptin receptor in neurons leads to obesity

P Cohen  1 , C Zhao, X Cai, J M Montez, S C Rohani, P Feinstein, P Mombaerts, J M Friedman
Affiliations

Selective deletion of leptin receptor in neurons leads to obesity

P Cohen et al. J Clin Invest. 2001 Oct.

Abstract

Animals with mutations in the leptin receptor (ObR) exhibit an obese phenotype that is indistinguishable from that of leptin deficient ob/ob mice. ObR is expressed in many tissues, including brain, and the relative importance of leptin's effects on central versus peripheral sites has not been resolved. To address this, we generated mice with neuron-specific (ObR(SynI)KO) and hepatocyte-specific (ObR(Alb)KO) disruption of ObR. Among the ObR(SynI)KO mice, the extent of obesity was negatively correlated with the level of ObR in hypothalamus and those animals with the lowest levels of ObR exhibited an obese phenotype. The obese mice with low levels of hypothalamic ObR also show elevated plasma levels of leptin, glucose, insulin, and corticosterone. The hypothalamic levels of agouti-related protein and neuropeptide Y RNA are increased in these mice. These data indicate that leptin has direct effects on neurons and that a significant proportion, or perhaps the majority, of its weight-reducing effects are the result of its actions on brain. To explore possible direct effects of leptin on a peripheral tissue, we also characterized ObR(Alb)KO mice. These mice weigh the same as controls and have no alterations in body composition. Moreover, while db/db mice and ObR(SynI)KO mice have enlarged fatty livers, ObR(Alb)KO mice do not. In summary, these data suggest that the brain is a direct target for the weight-reducing and neuroendocrine effects of leptin and that the liver abnormalities of db/db mice are secondary to defective leptin signaling in the brain.

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Figures

Figure 1
Figure 1
LoxP targeting of the ObR locus. Gene targeting was used to insert loxP sites on either side of the first coding exon of ObR. (a) Restriction maps (from top to bottom) of the genomic locus, targeting vector, homologous recombinant, and the type II and type I deletion alleles. Probe 1, located outside the targeting construct, was used to screen for homologous recombinants. Probe 2, located in the second coding exon, distinguishes the endogenous allele, homologous recombinants, and type I (deletion of the first coding exon) and II (deletion of the targeting cassette leaving loxP sites on either side of the first coding exon) deletions. A, AflII; B, BamHI; Bg, BglII; H, HindIII; N, NcoI. (b) Southern blot analysis of NcoI-digested genomic DNA from ES cell clones using probe 1. The endogenous allele (Wt) and homologous recombinants (HR) migrated at the predicted sizes. (c) Southern blot analysis of Hind-III digested genomic DNA using probe 2. The endogenous allele (Wt), homologous recombinant (HR), and type II deletion were detected from ES cell DNA. ObRflox/+ mice were generated from the type II deletion and crossed to adenovirus EIIA-Cre mice. The type I deletion was detected in tail DNA from progeny derived from this cross. All alleles migrated at the predicted sizes. (d) Body weight at 8 weeks of age of ObRΔ/Δ and littermate control (ObRΔ/+) mice. Data represent the mean ± SEM of at least nine animals of each genotype and gender. *P < 0.02; **P < 0.001; unpaired Student’s t test.
Figure 2
Figure 2
Cre-mediated recombination specifically in the brain of ObRSynIKO mice. (a) The breeding strategy that was used to generate ObRSynIKO mice and littermate controls. ObRAlbKO mice (described below) were generated using the same strategy. (b) Genomic DNA was prepared from several tissues from ObRflox/+, SynI-Cre(+) mice and were PCR-amplified using primers flanking the first coding exon of ObR. The locations of the primers are shown on the schematic below the gel. In tissues expressing the Cre recombinase, exon 1 is excised and a single loxP site remains, generating an ObRΔ allele. While primers 1 and 3 can amplify a product from the ObRΔ allele, in the ObRflox or ObR+ (wild-type) DNA, the primers are separated by a distance too great for amplification to occur. As a control, primers 1 and 2 were used to amplify both the ObRflox allele and the ObR+ alleles from all tissues. The ObR+ allele produces a slightly smaller product due to the absence of loxP sequences.
Figure 3
Figure 3
ObR RNA levels. (a) The distribution of body weight and percent body fat for male and female ObRSynIKO mice at 16 weeks of age. Those mice with less than 15% of ObR RNA (see b) are shown in yellow. (b) Expression levels were determined using Taqman real-time PCR with the ABI Prism 7700 Sequence Detection System. The locations of primers and fluorescent probe are as indicated. Primers were derived from sequences in the 5′ untranslated region and the second coding exon. The fluorescent probe is located within the first coding exon 1. When this exon is deleted, no signal is generated. As a control for input amount, each cDNA sample was also amplified using primers and a probe for cyclophilin. Data were analyzed with ABI Sequence Detector software, and the levels of ObR were normalized to cyclophilin. Levels are represented as the percentage of the levels in ObRflox/+, SynI-Cre(+) and ObRflox/+, SynI-Cre(–) wild-type mice. Data represent the mean ± SEM. At least 13 animals were analyzed for each genotype. P values for comparisons between genotypes are indicated.
Figure 4
Figure 4
Obesity in a subset of ObRSynIKO mice. Significant obesity was evident in those ObRSynIKO mice that had 15% or less ObR RNA in the hypothalamus than did wild-type mice. (a) Weight curves of male and female ObRSynIKO mice (filled squares), heterozygote littermates (open squares), and ObR-null mice (filled triangles). Mice were weighed weekly from 5 weeks of age. Data represent the mean ± SEM of four male and two female ObRSynIKO mice, seven male and nine female heterozygote littermates, and six male and five female ObR-null mice. For both sexes, ObRSynIKO versus heterozygotes, P < 0.05 at all ages; ObRSynIKO versus ObR-null, P < 0.05 at all ages. (b) Expression levels of AGRP, NPY, POMC, and CART were determined by Taqman and are expressed as normalized to cyclophilin. Levels were measured in ObRSynIKO, heterozygotes (HET), and ObR-nulls. (n = 5 for heterozygotes, n = 6 for knockouts, and n = 3 for null mice.)
Figure 5
Figure 5
Normal body weight and liver phenotype in ObRAlbKO mice. (a) Schematic of Albumin-Cre transgene. (b) Genomic DNA was prepared from several tissues from ObRflox/+, Alb-Cre(+) mice and was PCR-amplified using primers flanking the first coding exon of ObR as described in Figure 2b. (c) Weight curves of male and female ObRAlbKO mice (filled squares) and heterozygote littermates (open squares). Mice were weighed weekly from 5 weeks of age. Data represent the mean ± SEM of 9 male and 11 female ObRAlbKO mice and 23 male and 23 female heterozygote littermates. For both sexes, ObRAlbKO versus heterozygotes, P values were not significant at all ages. (d) Photographs of freshly dissected livers from representative mice with the given genotypes. (e) Liver triglycerides (milligrams of triglyceride per grams of liver) were determined for ObRAlbKO mice with less than 30% of wild-type ObR RNA and heterozygote controls. Data represent the mean ± SEM of six male and five female ObRAlbKO mice, five male and five female heterozygote controls, and six male and three female ObR-null mice. For both sexes, P < 0.05 for ObRAlbKO and heterozygotes relative to ObR null.
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