Differential expression of circulating microRNAs in diabetic and healthy lean cats q Stefanie N. Fleischhacker a , Stefan Bauersachs b , Astrid Wehner a , Katrin Hartmann a , Karin Weber a,⇑ a Centre for Clinical Veterinary Medicine, Clinic of Small Animal Medicine, Ludwig Maximilian University Munich, Veterinaerstrasse 13, 80539 Munich, Germany b Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig Maximilian University Munich, Feodor Lynen Strasse 25, 81377 Munich, Germany article info Article history: Accepted 25 March 2013 Available online xxxx Keywords: Diabetes mellitus Feline MicroRNA abstract MicroRNAs (miRNAs) regulate gene expression and play a role in the pathogenesis of human type 2 dia- betes mellitus. This study investigated whether miRNA expression profiles differ between healthy and diabetic cats. Total RNA was extracted from sera of healthy lean cats, newly diagnosed diabetic cats and cats in diabetic remission. Microarrays representing 1079 mouse miRNA targets were used to mea- sure miRNA expression in serum samples from eight healthy lean and seven newly diagnosed diabetic cats; 227 distinct miRNAs could be detected. Nineteen miRNAs were differentially expressed in newly diagnosed diabetic cats compared to healthy lean cats, with a false discovery rate of 10%. Hierarchical cluster analysis of these 19 miRNAs grouped healthy lean and newly diagnosed diabetic cats into separate clusters. After correction for multiple testing, only miR-122 and miR-193b reached statistical significance (P < 0.05), with a false discovery rate of 1%. Specific quantitative real-time PCR assays for three target miRNAs (miR-122, miR-193b and miR-483  ) were applied to four samples from each of the three groups. miR-122 expression was >40-fold higher in newly diagnosed diabetic cats compared to healthy lean cats and cats in diabetic remission, whereas miR-193b showed >14-fold higher expression. MiR-483  was expressed sixfold higher in newly diagnosed diabetic cats compared to both other groups. Ó 2013 Elsevier Ltd. All rights reserved. Introduction Diabetes mellitus (DM) is one of the most common metabolic disorders in humans and cats. In cats, 0.5–2% of the population are diabetic (Lederer et al., 2009) and the majority of cats develop type 2 diabetes mellitus (T2DM), which is characterised by inap- propriate insulin secretion and/or insulin resistance of insulin tar- get tissues. If treatment is provided in the early stages of disease, diabetic remission can be achieved (Marshall et al., 2009). MicroRNAs (miRNAs) are endogenous, short, non-coding RNAs that negatively regulate gene expression at the post-transcrip- tional level. They are found in almost all tissues and body fluids, and a high stability in serum has been reported (Lagos-Quintana et al., 2002; Chen et al., 2008). miRNAs have been associated with the development of T2DM by affecting glucose homeostasis, insu- lin secretion, insulin gene expression, maintenance of normal pan- creatic a- and b-cell mass and insulin signalling in insulin-sensitive tissues (Dehwah et al., 2012). Circulating miRNAs are potential diagnostic and prognostic bio- markers and have been used as a sensitive, non-invasive diagnostic tool for early disease detection in human malignancies (Chen et al., 2012; Kanemaru et al., 2011; Anindo and Yaqinuddin, 2012). In diabetic rats and humans, changes in miRNA expression have been detected in blood samples, pancreatic islets and insulin-sensitive tissues (Karolina et al., 2011; Kong et al., 2011). Expression of miR- NAs is altered at different stages of diabetes progression (Gallagher et al., 2010; Zampetaki et al., 2010; Karolina et al., 2011). The avail- ability of blood-based biomarkers to diagnose T2DM before clinical signs appear, to assess b-cell function or to predict disease progress would be beneficial for feline diabetes therapy and prognosis. The aims of this study were to investigate whether the diabetic state of cats is reflected in serum miRNA expression profiles. Expression of three miRNAs was determined in the serum of newly diagnosed diabetic cats, healthy cats and cats in diabetic remission. Materials and methods Animals Serum samples were collected from 20 privately owned cats presented to the Clinic of Small Animal Medicine of the Ludwig Maximilian University Munich, Ger- many. These animals were classified as either healthy lean (n = 8), diabetic (n = 8) or diabetic in remission (n = 4). Cats were considered to be healthy when history, com- plete physical examination, complete blood count and serum biochemistry revealed no abnormal findings. Healthy cats were presented to the clinic for annual health checks. Cats were classified as lean if their body condition score was 3–5 on a 9- 1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2013.03.027 q Preliminary results were presented at the American College of Veterinary Internal Medicine (ACVIM) 2012 ACVIM Forum, New Orleans, Louisiana, USA, 30 May to 2 June 2012. ⇑ Corresponding author. Tel.: +49 89 21801672. E-mail address: karin.weber@lmu.de (K. Weber). The Veterinary Journal xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl Please cite this article in press as: Fleischhacker, S.N., et al. Differential expression of circulating microRNAs in diabetic and healthy lean cats. The Veter- inary Journal (2013), http://dx.doi.org/10.1016/j.tvjl.2013.03.027