Insufficiency of Janus kinase 2-autonomous leptin receptor signals for most physiologic leptin actions

Abstract

Objective: Leptin acts via its receptor (LepRb) to signal the status of body energy stores. Leptin binding to LepRb initiates signaling by activating the associated Janus kinase 2 (Jak2) tyrosine kinase, which promotes the phosphorylation of tyrosine residues on the intracellular tail of LepRb. Two previously examined LepRb phosphorylation sites mediate several, but not all, aspects of leptin action, leading us to hypothesize that Jak2 signaling might contribute to leptin action independently of LepRb phosphorylation sites. We therefore determined the potential role in leptin action for signals that are activated by Jak2 independently of LepRb phosphorylation (Jak2-autonomous signals).

Research design and methods: We inserted sequences encoding a truncated LepRb mutant (LepRb(Delta65c), which activates Jak2 normally, but is devoid of other LepRb intracellular sequences) into the mouse Lepr locus. We examined the leptin-regulated physiology of the resulting Delta/Delta mice relative to LepRb-deficient db/db animals.

Results: The Delta/Delta animals were similar to db/db animals in terms of energy homeostasis, neuroendocrine and immune function, and regulation of the hypothalamic arcuate nucleus, but demonstrated modest improvements in glucose homeostasis.

Conclusions: The ability of Jak2-autonomous LepRb signals to modulate glucose homeostasis in Delta/Delta animals suggests a role for these signals in leptin action. Because Jak2-autonomous LepRb signals fail to mediate most leptin action, however, signals from other LepRb intracellular sequences predominate.

FIG. 1.

Generation of mice expressing LepRb^Δ65. A: HEK293 cells were transfected with plasmids encoding the indicated LepR isoforms, made quiescent overnight, incubated in the absence (−) or presence (+) of leptin (625 ng/ml) for 15 min before lysis, and immunoprecipitated with αJak2 (24). Immunoprecipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the indicated antibodies. The figures shown are typical of multiple independent experiments. B: Diagram of gene-targeting strategy to replace wild-type exon 18b with that encoding the COOH-terminally truncated LepRb^Δ65. C: Southern blotting of control (wild-type) and correctly targeted (D/+, C1, C2) Lepr^Δ65 ES lines, using a Lepr-specific probe. M indicates marker lane. D: Image of gel electrophoresis of Lepr-specific RT-PCR products from hypothalamic mRNA of five wild-type and five Δ/Δ animals.

FIG. 2.

Similar hyperphagia and obesity in Δ/Δ and db/db mice. Wild-type (■), db/db (○), and Δ/Δ (▴) mice of the indicated age (n = 8–10 per genotype) were weaned at 4 weeks and body weight (A and B) and food intake (C and D) were monitored weekly from 4 to 8 weeks of age. Food intake represents cumulative food intake over the time course. Data are plotted as means ± SEM. *P < 0.05 compared with wild type (WT) by one-way ANOVA and Tukey post-test.

FIG. 3.

ARC neuropeptide expression and AgRP neuron c-fos-IR in wild-type (WT), db/db, and Δ/Δ mice. A–C: mRNA was prepared from the hypothalami of 10- to 11-week-old male mice. Quantitative PCR was used to determine (A) Pomc, (B) Npy, and (C) Agrp mRNA levels (n = 14–19 per genotype). D: c-fos-IR in AgRP neurons of ad libitum–fed wild-type, db/db, and Δ/Δ animals. All mouse groups were bred onto a background expressing LacZ under the AgRP promoter, enabling the identification of AgRP neurons by staining for β-gal. Representative images showing immunofluorescent detection of c-fos (top), β-gal (middle), and merged c-fos/β-gal (bottom). E: Quantification of double-labeled c-fos/AgRP-IR neurons. Double-labeled AgRP neurons are plotted as percentage of total AgRP neurons ± SEM. A–C and E: *P < 0.05 compared with wild type; †P < 0.05 compared with db/db by one-way ANOVA with Tukey post-test. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Reduced numbers and proliferation of splenic T-cells in Δ/Δ and ob/ob but not s/s or l/l mice. Spleens were isolated from the indicated genotypes of male mice, separated using autoMACS, and counted for (A) total splenocytes and (B) CD4⁺ cells using a flow cytometer (n = 7–22 per genotype). Data are plotted as means ± SEM; *P < 0.05 compared with wild type by one-way ANOVA and Tukey post-test. C: For proliferation assays, CD4⁺CD25⁻ naive T-cells were isolated by autoMACS (n = 4–7 per genotype), incubated in the presence of bone marrow–derived dendritic cells from C57BL/6J mice and stimulated with anti-CD3e. Incorporation of H³-thymidine (1 mCi/well) by proliferating cells was measured during the last 6 h of culture. Proliferation is expressed as a percentage of a paired wild-type sample analyzed concurrently (dashed line) and is plotted as mean ± SEM; *P < 0.05 by one-way ANOVA and Tukey post-test.

FIG. 5.

Delayed onset of hyperglycemia in Δ/Δ compared with db/db mice. A–D: Blood glucose was determined for ad libitum–fed (A and B) or fasted (5 h) (C and D) animals of the indicated genotype (wild type [WT], ■; db/db, □; and Δ/Δ, ▴) and sex at the indicated ages (n = 8–12 per genotype). E and F: Serum was collected from mice of the indicated genotype and sex at the indicated ages (n = 8–10 per genotype), and insulin content was determined by ELISA. All panels: Data are plotted as means ± SEM; *P < 0.05, db/db compared with Δ/Δ at the indicated time points by one-way ANOVA and Tukey post-test.