Leptin Receptor Long-form Splice-variant Protein Expression in Neuron Cell Bodies of the Brain and Co-localization with Neuropeptide Y mRNA in the Arcuate Nucleus

Abstract

SUMMARYReduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.

Adipocyte-selective Reduction of the Leptin Receptors Induced by Antisense RNA Leads to Increased Adiposity, Dyslipidemia, and Insulin Resistance

“…2A indicate that the two different leptin receptor antibodies detected the same long and/or short forms of leptin receptors from the hypothalamic and WAT samples on Western blot assays. The sizes of the long and short forms of leptin receptors detected here are consistent with those observed in previous studies (21,(27)(28)(29). The identification of OB-Rb was confirmed by further comparing the immunoreactive bands of the wild-type brain with those of db/db hypothalamic and brain extracts because the db/db mice do not express OB-Rb but maintain the expression of short forms of OBR (4) (Fig.…”

Subcellular Localization and Internalization of the Four Human Leptin Receptor Isoforms

“…The amounts of 35 S-labeled leptin receptor paralleled the total number of leptin binding sites (Fig. 3), with HLR-5 having the most labeled protein and HLR-67 the least (data not shown).…”

“…This result is supported by other studies on the localization of endogenous receptor in brain. It has been shown that the endogenous long isoform of the leptin receptor is found predominantly within the cell bodies of rat neurons, not at the cell surface (35,36). In addition, immunohistochemical localization studies using an antibody that recognizes all leptin receptor isoforms showed intracellular immunoreactivity in brain, including intense intracellular staining of the choroid plexus epithelium, where it is thought that most of the receptor is the shortest isoform (HLR-5) (36,37).…”

Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity

“…We recently showed that OB-R internalizes via a clathrin-mediated pathway and is transported to lysosomes where it is degraded. Most OB-Rs (endogenously expressed or transfected) are localized in intracellular membranes (16)(17)(18)(19)(20)(21)28). Importantly, these receptors are fully functional in terms of ligand binding (19,20).…”

“…Possible mechanisms underlying this pathological state, termed leptin resistance, are impaired leptin bioavailability and transport across the blood-brain barrier, up-regulation of negative feed-back regulators of OB-R signaling, and defects in OB-R trafficking and signaling (13)(14)(15). Importantly, at steady state, most OB-R (endogenously expressed and transfected) are in intracellular membranes (16)(17)(18)(19)(20)(21) and are fully functional in terms of ligand binding (19,20). However, they are unable to participate in the functional response, as leptin does not penetrate the cell.…”