Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes Toby Phillips, Ivan Ferraz 1 , Susan Bell, Peter D. Clegg, Stuart D. Carter, Ali Mobasheri * Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UK Accepted 16 January 2004 Abstract Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extra- cellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-D-[2, 6- 3 H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-a (100 ng mL 1 ), IL-1b (100 ng mL 1 ), IGF-I (20 ng mL 1 ), TGF-b (20 ng mL 1 ) and insulin (12.5 lg mL 1 ) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-b, IL-1b and TNF-a resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflam- matory cytokines and anabolic endocrine factors. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cartilage; Chondrocyte; Glucose transport; GLUT; Cytokine; Growth factor 1. Introduction Recent research suggests that osteoarticular disorders in humans and veterinary species may be directly linked to obesity and may therefore have nutritional and en- docrine abnormalities at the root of their pathogenesis. Glucose is an essential energy source for mammalian cells and in articular cartilage glucose plays a pivotal role in the physiology of the chondrocytes by driving the extracellular matrix biosynthetic machinery of this un- ique cell type (Wang et al., 1999; Mobasheri et al., 2002b). Glucose is also a major energy source and a precursor for the synthesis of glycosaminoglycans (Mobasheri et al., 2002a). Despite this realisation, there is limited published information about the molecular mechanisms responsible for nutrient transport across the chondrocyte membrane and their regulation by growth factors and pro-inflammatory cytokines. The Veterinary Journal 169 (2005) 216–222 The Veterinary Journal www.elsevier.com/locate/tvjl * Corresponding author. Tel.: +44-151-794-4284; fax: +44-151-794- 4243. E-mail address: a.mobasheri@liverpool.ac.uk (A. Mobasheri). 1 Present Address: Servicio de Reumatologia, Hospital Universitario de Canarias, Tenerife, La Cuesta, Santa Cruz de Tenerife and Departamento de Bioqu  ımica y Biolog  ıa Molecular, Universidad de La Laguna, Tenerife, Spain. 1090-0233/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.01.026