Leptin (ob gene) of the South African clawed frog Xenopus laevis

Abstract

Leptin, the protein product of the obese (ob) gene, is a type-I cytokine hormone secreted by fat that is integral to food intake regulation and influences almost every physiological system in juvenile and adult mammals. Since the identification of leptin in the mouse in 1994, biologists have searched for orthologous genes in other species with limited success. In this article, we report the identification and functional characterization of leptin and leptin receptor (LR) in Xenopus. Despite low amino acid sequence similarity to mammalian leptins ( approximately 35%) the frog protein has a nearly identical predicted tertiary structure and can activate the frog and mouse LRs in vitro. We showed that recombinant frog leptin (rxLeptin) is a potent anorexigen in frogs, as it is in mammals, but this response does not develop until midprometamorphosis. However, during early prometamorphosis, exogenous rxLeptin induced growth and development of the hind limb, where LR mRNA is expressed. The rxLeptin also stimulated cell proliferation in cultured hind limbs from early prometamorphic tadpoles, as measured by [(3)H]thymidine uptake. These findings are evidence that leptin can influence limb growth and differentiation during early development. Furthermore, the isolation and characterization of leptin and its receptor in a nonamniote provides an essential foundation for elucidating the structural and functional evolution of this important hormone.

Fig. 1.

Molecular characterization of frog leptin. (A) The predicted genomic structure of the frog ob gene is conserved with human. Dark shading shows coding regions. (B) Amino acid alignment of vertebrate leptins. Arrowheads show conserved cysteines. GenBank accession nos. are as follows: X. laevis, AY884210; human, NP000221; rat, BAA08296; chicken, AF012727; pufferfish (Tetraodon), AB193549. (C) Ribbon diagrams of predicted tertiary structures of X. laevis, rat, and pufferfish leptins (SWISS–MODEL automated protein homology-modeling server; based on human leptin Protein Data Bank structure file 1AX8.pdb).

Fig. 2.

Molecular and functional characterization of frog leptin receptor. (A) Predicted gene structure of the frog obr gene (xobr) and comparison with the human obr gene (hobr). The frog obr gene spans 87.3 kb. (B) Neighbor-joining phylogenetic tree of amino acid sequences of leptin receptors and related cytokine receptor genes. We used the Align X module within vector nti suite (v. 5.5; Informax, Bethesda) to conduct the analysis. GenBank accession numbers are provided in supporting information. (C) rxLeptin activates the mouse and the frog LR in transient transfection assays. COS-7 cells were transfected with a STAT-3-responsive luciferase reporter plasmid (GAS) or with both GAS plus pcDNA3.1-mLR (mouse leptin receptor) (Left) or pcDNA3.1-xLR (frog leptin receptor; separate experiments) (Right), then exposed to different doses of rxLeptin or human recombinant leptin (hrLeptin). Bars represent means ± SEM (n = 4–6 per treatment; a representative experiment is shown, and each experiment was repeated three times). Asterisks indicate significant differences between leptin treated and untreated controls (Fisher’s least significant difference test; P < 0.05). RLU, relative light units.

Fig. 3.

Expression of leptin and LR mRNAs in the frog. (A) The tissue distribution in juvenile frogs of leptin and LR mRNAs was analyzed by quantitative RT-PCR (see Methods). Leptin and LR mRNA levels were normalized to the expression of the ribosomal protein L8 gene (rpL8). (B) The developmental expression of leptin mRNA in X. laevis was analyzed by semiquantitative RT-PCR (see Methods).

Fig. 4.

Effects of i.c.v. injection of rxLeptin on time spent foraging in early prometamorphic or midprometamorphic S. hammondii tadpoles and on meal size in X. laevis juveniles. Letters indicate Duncan’s pairwise differences among treatments (P < 0.05).

Fig. 5.

Effects of rxLeptin injections on hind-limb growth and development in early prometamorphic S. hammondii tadpoles. (A) Mean ± SEM hind-limb length divided by body length and Gosner stage before and after rxLeptin injections (i.p. every other day for 7 days; n = 8 per treatment). Letters indicate Duncan’s pairwise differences among treatments (P < 0.05). (B) Mean ± SEM hind-limb length divided by body length and Gosner stage of tadpoles fed or food-deprived (FD) and given 2 μg of rxLeptin i.p. every other day for 6 days (n = 10). Letters indicate significant differences between experimental groups (Tukey’s multiple comparisons test; P < 0.05). (C) Representative hind limbs of tadpoles given saline or rxLeptin injections as described above. (D) RT-PCR analysis of leptin and LR expression in two independent hind-limb samples from X. laevis NF stage 56 (four hind limbs pooled per sample). rpL8, ribosomal protein L8; C, water control; L, DNA ladder.