Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC341
Expression Pattern and Signalling Pathway Alterations in the Brain Concur with the Hypermetabolic Phenotype of Mice Lacking Gnasxl/XLαs
K. Burton1, S. Krechowec1, A. Newlaczyl1, A. Plagge1
1. Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom.
XLαs (XL) is an NH2-terminal splice variant of the stimulatory G-protein α-subunit Gsα. Both are encoded by the imprinted Gnas locus. Similar to Gsα, XL can couple 7-TM receptors to adenylate cyclase in cultured cells. Previously, we generated a knockout (KO) mouse specific for the Gnasxl transcript , which exhibits a lean, hypermetabolic phenotype with elevated sympathetic nervous system (SNS) activity . Changes in phenotype between neonatal and adult stages (e.g. mortality; failure to thrive as neonates vs. healthy but hypermetabolic adults) are not yet fully understood. More recently we established a conditional genetrap KO mouse. Immunohistochemistry and immunofluorescence data were collected to document the changes in expression pattern of XL and determine possible signalling pathways affected by lack of XL in the brain; histological analysis of the LacZ-containing genetrap line revealed new sites of XL expression. A comparison of neonatal and adult brain expression patterns revealed that the lateral hypothalamus (LH), dorsomedial hypothalamus (DMH), arcuate nucleus (Arc), locus coeruleus and ventrolateral medulla express XL in both stages; the laterodorsal tegmental nucleus, hypoglossal and facial nucleus (motor nuclei important for feeding) express XL only in neonates; and expression in the amygdala and preoptic area are only found in adult brain. We undertook colocalisation studies in the brain for XL, neuropeptides and other markers related to regulation of food intake and energy expenditure (EE). Orexin partly colocalised with XL in the LH and DMH (22% orexin neurons XL positive); tyrosine hydroxylase/dopaminergic neurons colocalised with XL in the Arc (60% TH neurons XL positive); and phospho-S6, a component of the leptin signalling pathway, colocalised with XL in the Arc (30% XL neurons pS6 positive). MCH and CRF did not colocalise with XL. Changes in pS6/S6K1 activity in KO Arc neurons are currently being investigated. Analysis of the LacZ-genetrap line revealed that XL is also expressed in spinal cord and peripheral tissues e.g. skeletal muscle, tongue muscle and blood vessel smooth muscle cells at neonatal stages. Expression in muscle tissues, including blood vessel smooth muscle cells is silenced in adult, but spinal cord remains positive for XL. XL expression pattern changes in the brain and peripheral tissues concur with changes in phenotype seen between neonatal and adult mice. pS6 is a good indicator of S6K1 activity, which influences leptin and insulin signalling. A decrease of S6K1 activity in our KOs might explain their leptin sensitivity (Frontera et al in prep). XL colocalises with orexigenic peptides - including orexin and NPY/AGRP neurons (Frontera et al in prep) - in the hypothalamus; however, the function of XL in EE and SNS activity remains elusive.
Where applicable, experiments conform with Society ethical requirements