Mice lacking inhibitory leptin receptor signals are lean with normal endocrine function

Abstract

The adipose-derived hormone, leptin, acts via its receptor (LRb) to convey the status of body energy stores to the brain, decreasing feeding and potentiating neuroendocrine energy expenditure. The failure of high levels of leptin in most obese individuals to promote weight loss defines a state of diminished responsiveness to increased leptin, termed leptin resistance. Leptin stimulates the phosphorylation of several tyrosine residues on LRb to mediate leptin action. We homologously replaced LRb in mice with a receptor with a mutation in one of these sites (Tyr985) in order to examine its role in leptin action and signal attenuation in vivo. Mice homozygous for this mutation are neuroendocrinologically normal, but females demonstrate decreased feeding, decreased expression of orexigenic neuropeptides, protection from high-fat diet-induced obesity, and increased leptin sensitivity in a sex-biased manner. Thus, leptin activates autoinhibitory signals via LRb Tyr985 to attenuate the anti-adiposity effects of leptin, especially in females, potentially contributing to leptin insensitivity in obesity.

Figure 1. Generation of mice expressing LRb^L985.

(A) Cartoon of signaling by LRb showing the activation of specific signals by individual tyrosine phosphorylation (pY) sites. (B) Targeting strategy for generation of the Lepr^L985 allele. Shown is the targeting vector containing the LRb-specific exon 18b with the mutation of Tyr985 (which also generates a novel HindIII site) (exon18b-L985) plus Neo and thymidine kinase (TK) cassettes for positive and negative selection, respectively. Recombination in ES cells replaces the wild-type exon 18b with exon 18b-L985 and the Neo cassette, to mediate physiologic expression of the mutant LRb from the endogenous Lepr gene, as previously described (26). (C) Southern blot using a Lepr locus probe, demonstrating correct targeting of the locus in +/+, l/+, and l/l mice. M, marker. (D) qPCR for hypothalamic LRb mRNA expression in +/+ and l/l mice. (E) Hypothalamic detection of STAT3(PY) in +/+ and l/l mice. Age- and sex-matched animals were treated i.p. with vehicle or leptin for 30 minutes. Brains were processed for immunohistochemical detection of STAT3(PY) in the hypothalamus; representative sections showing the ARC and ventromedial hypothalamic nucleus (VMH) are shown. For reference, position of the third cerebral ventricle (3V) is shown. Original magnification, ×20.

Figure 2. Leanness in l/l mice.

(A and B) Mice of the indicated sex and genotype were weighed weekly from the time of weaning until the age of 8 weeks. No difference between curves by repeated measures ANOVA; *P < 0.05, l/l versus +/+ animals for pairwise comparisons (ANOVA) at indicated age. (C–H) Data regarding adipose tissue weight and hormone levels from 8-week-old female +/+ and l/l mice from A and B. (C–E) Female mice. (F–H) Male mice. All values represent mean ± SEM. *P < 0.05 versus +/+ mice, by ANOVA.

Figure 3. Decreased body weight and feeding in l/l females during HF feeding.

(A–D) C57BL/6 female (A and B) and male (C and D) +/+, l/+, and l/l animals were provided NC (A and C) or HF (B and D) diets from the age of 5 weeks and weighed weekly until 12 weeks of age. All values represent mean ± SEM. ANOVA and repeated measures ANOVA, P = NS, except for ^#P < 0.02 for l/l versus other groups where indicated. (E) Weight gained per female mouse from 5–12 weeks of age on each diet. (F) Food intake for first week of divergent diet for female mice of the indicated genotype. *P < 0.05, **P < 0.01 versus +/+ animals, by ANOVA.

Figure 4. Decreased measures of adiposity and increased hypothalamic leptin action in female l/l animals.

(A and C) Serum leptin and insulin levels from female mice from Figure 3 at 4 and 10 weeks of age. (B) Weight of perigonadal fat pads from animals in Figure 3 at time of sacrifice. (D) At the age of 12 weeks, the ARC was dissected for the detection of STAT3(PY) (representative data shown in inset) and total STAT3 by immunoblotting. The ratio of STAT3(PY) normalized for total STAT3 is plotted for each group of animals. Data are normalized to levels in NC +/+ animals, which were set as 100%. All values represent mean ± SEM. *P < 0.05 versus +/+ animals of same age and diet, by ANOVA.

Figure 5. ARC neuropeptide and Tyr985-interacting protein gene expression in l/l females.

ARC mRNA levels for (A) POMC, (B) AgRP, (C) NPY, (D) SOCS3, and (E) SHP2 from animals in Figure 3. Data are normalized to levels in NC +/+ animals, which were set as 100%. All values represent mean ± SEM. *P < 0.05 versus +/+ animals of same age and diet, by ANOVA.

Figure 6. Increased in vivo leptin sensitivity in l/l mice.

Seven-week-old female +/+ and l/l mice fed NC were injected i.p. twice daily with PBS for 3 days, then with leptin for 3 days, and then with PBS for 2 days. (A) Body weight was measured once per day during the injection period. Day 1 represents the average weight during the initial (first 3 days) PBS injections. Arrows indicate times of leptin injections. (B) Average 24-hour food intake during injection periods. All values represent mean ± SEM. *P < 0.05 and ^#P < 0.001 versus +/+ mice.