Body weight regulation depends on neuronal signaling by adiposity-related hormones such as insulin and leptin. Activation of receptors for these hormones induces cell signaling via the insulin receptor substrate-phosphatidylinositol 3 OH kinase (IRS-PI3K) pathway, and growing evidence from knock-out models implicates insultin receptor substrate 2 (IRS-2) as a key component of this signal transduction mechanism. As a first step towards the identification of brain areas that utilize IRS-PI3K signaling in the control of energy homeostasis, we used immunohistochemical techniques to investigate the neuronal distribution of IRS-2 protein. Male Wistar rats were perfused with 4% paraformaldehyde in PBS and coronal blocks were embedded in paraffin. Sections (8 microns) were de-waxed and subjected to high-temperature antigen retrieval using a citric acid-based solution in a 99°C water bath. They were blocked using goat normal serum in PBS and incubated with IRS-2 antibody at 4°C overnight. Antibody binding was detected using either a fluorescent secondary antibody or a peroxidase-based system with diaminobenzidine. In the hypothalamus, strong IRS-2 staining was detected chiefly in the arcuate (ARC) and ventromedial (VMN) nuclei and in the medial parvocellular division of the paraventricular nucleus (PVN), whereas the dorsal medial nucleus (DMN) and lateral hypothalamic area (LHA) showed no appreciable staining. Within the ARC, IRS-2 was found to co-localize with alpha-melanocyte stimulating hormone (α-MSH) positive neurons, and seimiquantitative analysis indicated that the majority of α-MSH positive neurons co-localize with IRS-2. In the hindbrain, IRS-2 staining was detected in the medial nucleus of the solitary tract (mNTS) and dorsal motor nucleus of the vagus (DMV) but not in the commissural NTS or the area postrema, although considerable variability was seen across animals in the number of IRS-2 positive cells in the DMV and mNTS. Co-localization studies in the NTS demonstrated the presence of IRS-2 in tyrosine hydroxylase (TH) positive neurons that participate in feeding responses induced by glucoprivation or peripheral satiety signals such as cholecystokinin. Interestingly, IRS-2 positive neurons were also concentrated in the motor nucleus of the hypoglossal nerve. In summary, neurons containing IRS-2 immunoreactivity were identified in both forebrain and hindbrain areas and in cell types that are crucial for the control of food intake and autonomic function. An improved understanding of mechanisms underlying normal and abnormal energy homeostasis may be gained by analysis of the role played by signaling through IRS-2 in these brain areas.
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