Superinduction of leptin mRNA in mouse hypothalamic neurons Russell E. Brown b , Diane A. Wilkinson a , Paul M.H. Wilkinson a , Syed A. Imran a,c and Michael Wilkinson a,b,c In marked contrast to several other species, including rats and humans, leptin gene expression is undetectable in mouse brain. This unexpected finding may reflect unique energy regulation pathways in the mouse. We investigated possible mechanisms by which leptin (ob) gene expression is suppressed in mouse brain: (a) the possibility that ob mRNA levels might be detectable in vitro through the superinduction of gene expression following protein synthesis inhibition and (b) whether chromatin modification of the ob gene was responsible for this repression. Experiments were conducted on mouse hypothalamic neurons in vitro. Cells were treated with (a) protein synthesis inhibitors: cycloheximide (CHX; 25 lg/ml); puromycin (50 lg/ml); anisomycin (5 lM); (b) trichostatin A (histone deacetylase inhibitor; 500 nM); and (c) 5-aza-2 0 -deoxycytidine (DNA methylation inhibitor; 5 lM). Following the incubations, cells were harvested for the preparation of RNA and ob mRNA was detected using real-time reverse transcription PCR. Protein synthesis inhibitors induced a rapid increase in ob mRNA levels in mouse hypothalamic neurons in vitro. For example CHX stimulation of ob mRNA was detectable at 60 min after treatment and reached a maximum between 4 and 6 h. A dose–response analysis, with concentrations of CHX of 1, 2, 10, 25, and 50 lg/ml, indicated that CHX was already effective at 1.0 lg/ml, with a maximal effect by 25 lg/ml. In contrast, incubation with trichostatin A and 5-aza-2 0 -deoxycytidine had no effect and ob mRNA remained undetectable. These data show that leptin gene expression is superinduced in ob-negative mouse hypothalamic neurons following inhibition of protein synthesis. They confirm that the previously reported absence of leptin mRNA in mouse brain is probably because of an active repressive mechanism, although this may not involve chromatin modification. NeuroReport 23:900–903  c 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2012, 23:900–903 Keywords: 5-aza-2 0 -deoxycytidine, chromatin, cycloheximide, DNA methylation, histone acetylation, hypothalamic neurons, leptin, trichostatin A Departments of a Obstetrics and Gynaecology, b Physiology and Biophysics and c Division of Endocrinology and Metabolism, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Correspondence to Michael Wilkinson, PhD, Department of Obstetrics and Gynaecology, IWK Health Centre, 5850/5980 University Avenue, PO Box 9700, Halifax, NS, Canada B3K 6R8 Tel: + 1 902 876 8126; fax: + 1 902 420 7192; e-mail: mwilk@dal.ca Received 9 July 2012 accepted 14 August 2012 Introduction Leptin, and several other fat-derived hormones (adipo- kines), are expressed and regulated in rat brain, particularly in the hypothalamus (reviewed in Wilkinson et al. [1]. Thus, leptin (ob) mRNA was detectable in human, sheep, pig, xenopus laevis, and tiger salamander brain, as well as in human and rat neuroblastoma cells [1]. Moreover, Eikelis and Esler [2] have reported that leptin is secreted from the human brain. In marked contrast, ob mRNA was undetectable by northern blot analysis in whole mouse brain [3] and by reverse transcription PCR (RT-PCR) in discrete mouse brain regions, including the arcuate nucleus (R.E. Brown and M. Wilkinson; unpub- lished data). The lack of success in detecting ob mRNA in murine brain has considerably influenced current think- ing of the physiology of leptin. All of the central effects of leptin, for example on body weight control and obesity, are assumed to result from circulating fat-derived leptin entering the brain and binding to leptin receptors [4]. The widespread acceptance of such a view, based mainly on mouse data, neglects the findings obtained in other species that suggest that brain-derived leptin has neurotransmitter and neuroprotective properties [1,5–7]. The mouse has attained a highly privileged position in neurobiology such that a variety of readily available, genetically modified mutants with silenced genes (gene knockouts) has provided invaluable insights into many brain mechanisms. Efforts to examine the relevance of brain-derived leptin are therefore not feasible in the mouse, given that the leptin gene is apparently not expressed. Thus, an important question is why ob mRNA remains undetectable in the mouse brain. Evidence from several experimental systems indicates that gene expression can be upregulated by protein synthesis inhibition. For example interleukin-6 mRNA was superinduced by cycloheximide (CHX) in a human breast carcinoma cell line [8], and m-opioid receptor (MOR) mRNA, which is normally undetectable in the murine carcinoma P19 cell line, was significantly elevated by the protein synthesis inhibitors CHX, anisomycin, or puromycin [9]. Chromatin modification has also been 900 Cellular, molecular and developmental neuroscience 0959-4965  c 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/WNR.0b013e3283595709 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.