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. 1997 Jun 24;94(13):7001-5.
doi: 10.1073/pnas.94.13.7001.

Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues

H Fei  1 , H J Okano, C Li, G H Lee, C Zhao, R Darnell, J M Friedman
Affiliations

Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues

H Fei et al. Proc Natl Acad Sci U S A. .

Abstract

Leptin's effects are mediated by interactions with a receptor that is alternatively spliced, resulting in at least five different murine forms: Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd, and Ob-Re. A mutation in one splice form, Ob-Rb, results in obesity in mice. Northern blots, RNase protection assays, and PCR indicate that Ob-Rb is expressed at a relatively high level in hypothalamus and low level in several other tissues. Ob-Ra is expressed ubiquitously, whereas Ob-Rc, -Rd, and -Re RNAs are only detectable using PCR. In hypothalamus, Ob-Rb is present in the arcuate, ventromedial, dorsomedial, and lateral hypothalamic nuclei but is not detectable in other brain regions. These nuclei are known to regulate food intake and body weight. The level of Ob-Rb in hypothalamus is reduced in mice rendered obese by gold thioglucose (GTG), which causes hypothalamic lesions. The obesity in GTG-treated mice is likely to be caused by ablation of Ob-Rb-expressing neurons, which results in leptin resistance.

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Figures

Figure 4
Figure 4
RNase protection assay of mRNA levels of Ob-R in obese mice. The animals used for the preparation of hypothalamic RNAs are indicated across the top. The positions of the free probe and protected bands are indicated on the left. In addition to Ob-Rb probe, β-actin was also probed to the same RNAs in a parallel experiment as a control.
Figure 1
Figure 1
(A) Northern blot analysis of the expression of Ob-R isoforms in mouse tissues. Multiple tissue northern blots (CLONTECH) were hybridized to cDNA probes specific for Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd, Ob-Re, as well as one that recognizes all Ob-R isoforms. Tissue origins are marked on top (Sk. muscle, skeleton muscle). All blots were also probed with human β-actin cDNA to control for RNA loading. The size of the cDNA probes used for hybridization to each Ob-R isoform is indicated on the right. Exposure times for each probe are as follows: common region, −70°C for 3 days; Ob-Ra, −70°C, overnight; Ob-Rb, −70°C for 2 weeks; actin, room temperature for 30 min. (B) RNase protection assay of mRNA levels of Ob-Rb in fat and pancreas. RNase protection assays were performed for white fat, brown fat, pancreatic islets, and whole pancreas. Each sample was assayed in duplicate, and β-actin was used as an internal control in every sample. The protected RNA bands for Ob-Rb and β-actin are indicated by arrows.
Figure 2
Figure 2
(a) In situ hybridization of Ob-Rb in an adult mouse brain. Dark-field photomicrograph showing Ob-Rb antisense (A) and sense strand (C) riboprobe hybridization to continuous coronal sections through hypothalamus. (B) Camera lucida drawing of the region shown in A, which is showing the location of the ventromedial hypothalamic (VMH) nuclei, the arcuate nuclei (Arc N), and the third ventricle (III Vent). (D) High-magnification bright-field photomicrograph of Ob-Rb mRNA hybridization in VMH. (b) In situ hybridization of Ob-Rb in an adult mouse brain coronal section. (A) Dark-field photomicrograph showing Ob-Rb reactivity in the VMH, dorsomedial hypothalamic (DMH) nuclei, and lateral hypothalamic (LH) nuclei. (B) Camera lucida drawing of A showing the location of the VMH, DMH, LH, and III Vent. (c) Dark-field photomicrograph of Ob-R in situ hybridization in an adult mouse brain adjacent to coronal sections through lateral ventricle. A specific antisense riboprobe corresponding to the extracellular region (common probe) (A) and the cytoplasmic region (Ob-Rb-specific probe) (B) of Ob-R cDNA sequence was used for hybridization. Reactivity observed is in the choroid plexus.
Figure 3
Figure 3
Genomic DNA organization of the 3′ end of the Ob-R gene. The genomic structure of the exons that encode amino acids H796–P890 of Ob-Rb is shown (see ref. 9). All other splicing variants of the receptor, Ob-Ra, Rc, Rd, and Re, end within the 13-kb region shown. Each exon is identified at the top with an arrow. Introns are represented by solid lines, with their approximate sizes labeled above (not drawn to scale).
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