The

The Veterinary Journal 169 (2005) 216–222

Veterinary Journalwww.elsevier.com/locate/tvjl

Differential regulation of the GLUT1 and GLUT3 glucosetransporters 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-DD-[2, 6-3H] glucose and on the expression levels of

GLUT1 and GLUT3.

Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-a (100 ngmL�1), IL-1b (100 ngmL�1), IGF-I

(20 ngmL�1), TGF-b (20 ngmL�1) and insulin (12.5 lgmL�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.

� 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-

* 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 � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tvjl.2004.01.026

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 aprecursor 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.

T. Phillips et al. / The Veterinary Journal 169 (2005) 216–222 217

The facilitated transport of glucose and glucose-derived compounds across the chondrocyte membrane

represents the rate-limiting step in glucose metabolism

(Shikhman et al., 2001a) and is therefore essential for

chondrogenesis and the functional integrity of articu-

lating joints. Mammalian cells transport glucose across

the plasma membrane by facilitated diffusion through

the glucose transporters (or GLUTs) which belong to

family of over fourteen membrane proteins collectivelyknown as the glucose GLUT/SLC2A family of glucose/

polyol transporters (Joost and Thorens, 2001; Joost

et al., 2002; Wu and Freeze, 2002). Recent reports sug-

gest that chondrocytes express multiple isoforms of

the GLUT/SLC2A family, including the GLUT1 and

GLUT3 proteins (Ohara et al., 2001; Shikhman et al.,

2001a; Mobasheri et al., 2002a). Anabolic endocrine

factors such as insulin-like growth factor I (IGF-I)stimulate glucose uptake in human chondrocytes (Rich-

ardson et al., 2003).

Studies from our group (Richardson et al., 2003) and

one other laboratory (Shikhman et al., 2001a) suggest

that net glucose transport in human chondrocytes is also

stimulated by pro-inflammatory cytokines such as inter-

leukin 1 beta (IL-1b), interleukin 6 (IL-6) and tumour

necrosis factor alpha (TNF-a). There is also evidence tosuggest that the GLUT1 protein is a stress inducible

glucose transporter in human chondrocytes and is sig-

nificantly upregulated in cells under the influence of pro-

inflammatory cytokines (Shikhman et al., 2001a).

Therefore, enhanced glucose transport and glycolysis by

articular chondrocytes may be important pre-requisites

for cartilage degradation in degenerative diseases of ar-

ticulating joints. However, it is not yet known if the in-dividual GLUT isoforms expressed in equine articular

chondrocytes respond to growth factors and cytokines in

a similar way to human chondrocytes. It is also not

known if the stimulated glucose uptake in response to

these physiologically relevant mediators is the result of de

novo synthesis and upregulation of glucose transporters.

The primary objective of this study was to test a novel

hypothesis that may implicate chondrocyte glucosetransporters in osteoarticular disorders thus establishing

a link between chondrocyte bioenergetics, nutrient status

and joint disease. The hypothesis tested was that glucose

transporters in equine chondrocytes are differentially

regulated by growth factors and pro-inflammatory cy-

tokines whose pathophysiological and bioenergetic ef-

fects may exacerbate the phenotypic and molecular

alterations observed in degenerative joint disease. Ac-cordingly, the aim of the investigation was to determine

the effects of IL-1b, TNF-a, IGF-I and transforming

growth factor beta (TGF-b) on (1) the net glucose uptake

capacity of equine articular chondrocytes in monolayer

culture and (2) the expression of the GLUT1 andGLUT3

glucose transporter protein isoforms in equine articular

chondrocytes in alginate culture.

2. Materials and methods

2.1. Chemicals

All chemicals used in this study were molecular biol-

ogy grade and purchased from Sigma/Aldrich. 2-Deoxy-

DD-[2, 6-3H] glucose was from Amersham Biosciences,

protein assay kits were obtained from Bio-Rad and an-

tibodies were purchased from Chemicon International.

2.2. Cartilage source

Normal equine articular cartilage was obtained from

the stifle joints of three horses (age range: 5–21 years)

euthanased for clinical reasons at the Philip Leverhulme

Large Animal Hospital, University of Liverpool. The

study was conducted with ethical approval in strict ac-cordance with local guidelines (none of these three

horses were euthanased for the purpose of this study).

2.3. Chondrocyte isolation and culture

Equine cartilage shavings were rinsed with phos-

phate-buffered saline (PBS), cut into small slices and

incubated overnight with type I collagenase (EC 3.4.24.3from Clostridium histolyticum) (approximately 100 col-

lagen digestion units mL�1) in Dulbecco’s modified

Eagles medium (DMEM) supplemented with 1000

mgL�1 glucose, 10% fetal calf serum and 1% antibiotic/

antimycotic solution. The filtered cell suspension was

washed three times in fresh DMEM and the cells

counted on a haemocytometer; cell viability was deter-

mined by trypan blue dye exclusion and was usually 95%or higher. The cells (2� 106 cells/mL) were cultured in

alginate beads as previously described (Mobasheri et al.,

2002a; Richardson et al., 2003).

Two different culture protocols were used in this

study for the following reason: 2-deoxy-DD-[2,6-3H] glu-

cose uptake experiments can only be performed with

monolayer cultured chondrocytes as the final washes in

non-radioactive medium cannot be performed rapidlywith alginate beads. Cells used for uptake studies were

not passaged more than three times to prevent chon-

drocyte dedifferentiation and phenotypic instability.

Chondrocytes encapsulated and maintained in alginate

culture were used for Western blot analyses as this

method allows investigators to maintain equal cell

numbers thus ensuring equal protein loading on SDS–

PAGE.

2.4. Deoxy-DD-[2,6-3H] glucose uptake

Net glucose transport was determined by measuring

the uptake of non-metabolizable 2-deoxy-DD-[2,6-3H]

glucose into equine chondrocytes in 24-well plates in the

presence and absence of the glucose transport inhibitor

218 T. Phillips et al. / The Veterinary Journal 169 (2005) 216–222

cytochalasin B (1 lM). Most of the uptake experimentsdescribed were performed with chondrocytes stimulated

for 24 h with TNF-a (100 ngmL�1), IL-1b (100

ngmL�1), IGF-I (20 ngmL�1), TGF-b (20 ngmL�1)

and insulin (12.5 lgmL�1). Selected experiments were

carried to study the short-term effect of insulin on 2-

deoxyglucose uptake by pre-incubating chondrocytes

for 15 min with insulin (12.5 lgmL�1). The insulin used

in this study was derived from the bovine species. All thecytokines and growth factors used in this study were

human recombinants.

Prior to the commencement of the assay, the chon-

drocyte cultures were rinsed three times with PBS and the

uptake of the radiolabelled 2-deoxyglucose (1 lCi mL�1

culture medium) was assayed for 35 min at 20 �C in

modified DMEM lacking glucose, pyruvate and serum.

The 24-well plates were washed three times with ice-coldPBS and the chondrocytes lysed with a cell lysis solution

consisting of 0.5% sodium dodecyl sulphate and 0.5%

Triton X-100 in PBS. Aliquots (450 lL) of the cell lysis

solution were mixed with 3.55 mL of NACS104 aqueous

scintillation cocktail (Amersham Biosciences) and

counted in a scintillation counter. The remaining 50 lLaliquots of the cell lysis solution were used to determine

total cell protein content using a Bio-Rad detergentcompatible (DC) protein assay kit and 2-deoxyglucose

uptake was normalized to total cell protein. All uptake

experiments were carried out in triplicate and repeated

under identical conditions at least three times and the

data are presented as percentage change in total 2-deoxy-

DD-[2,6-3H] glucose uptake.

2.5. SDS–PAGE and western blot analysis

Briefly, chondrocytes from alginate cultures were

rinsed in PBS and proteins were extracted with lysis

buffer (50 mM Tris/HCl, pH 7.2, 150 mM NaCl, l% (v/v)

Triton X-100, 1 mM sodium orthovanadate, 50 mM

sodium pyrophosphate, 100 mM sodium fluoride, 0.01%

(v/v) aprotinin, 4 lg/mL pepstatin A, 10 lg/mL leupep-

tin, 1 mM PMSF) on ice for 30 min. After adjustment oftotal protein concentration using a Bio-Rad DC protein

assay kit, samples were separated by SDS–PAGE on

12% minigels under reducing conditions. Separated

proteins were transferred onto nitrocellulose membranes

(Sigma) and incubated in blocking buffer (5% (w/v)

skimmed milk powder in PBS/0.1% Tween 20) for 1 h at

room temperature (RT). Membranes were incubated

with polyclonal primary antibodies to GLUT1 andGLUT3 (Chemicon International) diluted 1 in 1000 in

blocking buffer for 1 h at RT, washed three times with

blocking buffer, and incubated with secondary goat anti-

rabbit antibody conjugated with alkaline phosphatase

for 30 min at RT. Membranes were rinsed in blocking

buffer and washed three times in 0.1 M Tris pH 9.5,

containing 0.05 M MgCl2 and 0.1 M NaCl. Specific an-

tigen-antibody complexes were visualised by incubatingwith premixed nitro blue tetrazolium (NBT) in con-

junction with 5-bromo-4-chloro-3-indolyl phosphate

(BCIP) as precipitating agent for alkaline phosphatase.

2.6. Statistical analysis

The results are expressed as the means� SE of a

representative experiment performed in triplicate. Themeans were compared using Student’s t test assuming

equal variances. P < 0:05 was considered statistically

significant.

2.7. Image analysis

Quantification of Western blots was carried out using

Scion Image for Windows (version 4.0.2 http://www.scioncorp.com/) based on NIH Image for Macintosh.

The GLUT1 and GLUT3 immunoblots were scanned in

128 shades of grey. Thresholding was used to segment

scanned blots into objects of interest displayed in black

(i.e. GLUT bands) and background displayed in white

based on the grey levels in the original blot.

3. Results

We found that phenotypically stable equine articular

chondrocytes were capable of transporting 2-deoxy-DD-

[2,6-3H] glucose, which is consistent with our previous

observations in human chondrocytes and provides, for

the first time evidence for the presence of functional

glucose transporters in equine articular chondrocytes.

3.1. 2-Deoxy-DD-[2,6-3H] glucose uptake is not signifi-

cantly influenced by insulin

Equine chondrocytes in 24-well plates were stimulated

with bovine insulin for up to 15 min (short term) at room

temperature or at least 24 h (long term) at 37 �C before

measuring net 2-deoxy-DD-[2,6-3H] glucose uptake. Up-take into untreated (control) chondrocytes was consid-

ered to be 100%. The specificity of uptake was determined

by addition of cytochalasin B at the point of assay initi-

ation.We found that short-term or long-term stimulation

with bovine insulin did not affect 2-deoxyglucose uptake

in a statistically significant manner (Fig. 1). Cytochalasin

B inhibited 2-deoxyglucose transport in control and in-

sulin stimulated cultures (P < 0:025). Short-term stimu-lation with insulin (15 min) resulted in a small but

statistically insignificant (P ¼ 0:22) increase in net 2-

deoxy-DD-[2,6-3H] glucose uptake. Long-term incubation

with insulin (24 h) also had no stimulatory effect on

glucose uptake (P ¼ 0:15). These observations suggest

that the insulin-responsive GLUT4 isoform is either not

expressed in equine articular chondrocytes or that its

**

*

P = 0.22

P = 0.15%

of

2-D

eoxy

-D-[

2,6-

3 H]

Glu

cose

Up

take

0

25

50

75

100

125

150

175

Control Control + Cyt B Insulin(Short Term)

Insulin(Short Term)

+ Cyt B

Insulin(Long Term)

Insulin(Long Term)

+ Cyt B

Fig. 1. 2-Deoxy-DD-[2,6-3H] glucose uptake is not significantly influenced by insulin stimulation of equine chondrocytes. Cells were stimulated with

insulin for 15 min (short term) at room temperature or 24 h (long term) at 37 �C. 2-deoxyglucose uptake into untreated (control) cells was considered

to be 100%. Specificity of uptake was determined cytochalasin B treatment during the assay period. Neither short-term nor long-term stimulation

with insulin resulted in a significant change in 2-deoxyglucose uptake. There was no significant increase in 2-deoxyglucose uptake following short

term insulin stimulation (P ¼ 0:22). Long-term stimulation with insulin resulted in a decrease in 2-deoxyglucose uptake (P ¼ 0:15). * Denotes a

significant difference (P < 0:025) between control and experimental groups.

T. Phillips et al. / The Veterinary Journal 169 (2005) 216–222 219

plasma membrane abundance is too low to affect netglucose uptake in response to insulin treatment in equine

articular chondrocytes.

3.2. Cytokine and endocrine stimulation of 2-deoxy-[2,6-3H] glucose uptake

Uptake of 2-deoxy-[2,6-3H] glucose was significantly

increased in equine articular chondrocytes pre-stimu-lated with IGF-I, TGF-b, TNF-a and IL-1b (Fig. 2). In

these experiments baseline 2-deoxyglucose uptake was

measured in un-stimulated (control) chondrocytes and

Fig. 2. Uptake of 2-deoxy-[2,6-3H] glucose by equine articular chondrocytes

glucose uptake in un-stimulated chondrocytes (control cells) was considere

* Denotes a significant difference between control and experimental groups

the value represented as 100%. Treatment with IGF-I,TGF-b, IL-1b and TNF-a resulted in increased 2-de-

oxyglucose uptake by over 65% compared to control

values (P < 0:025).

3.3. GLUT1 expression is upregulated by pro-inflamma-

tory cytokines and growth factors but only IGF-I up-

regulates GLUT3

We observed that the effects of growth factors and

pro-inflammatory cytokines on the individual GLUT

protein isoforms (i.e. GLUT1 and GLUT3) was not the

stimulated with IGF-I, TGF-b, TNF-a and IL-1b. Baseline 2-deoxy-

d as 100%. Error bars indicate standard errors of the means (n ¼ 3).

(P < 0:025).

220 T. Phillips et al. / The Veterinary Journal 169 (2005) 216–222

same despite the fact that the uptake of 2-deoxy-[2,6-3H]glucose was significantly increased in equine articular

chondrocytes pre-stimulated with IGF-I, TGF-b, TNF-

a and IL-1b. Western blot analysis of equine chondro-

cyte cell lysates demonstrated that equine chondrocytes

expressed the GLUT1 and GLUT3 proteins with ap-

parent molecular weights of between 55 and 60 kDa.

Previous studies have revealed similar molecular weights

for GLUT1 and GLUT3 in human chondrocytes.Stimulation of alginate cultures of equine chondrocytes

with IGF-I, TGF-b, TNF-a and IL-1b all resulted in

upregulation of GLUT1 (P < 0:05). In the case of

GLUT3, a similar upregulation was measured for cul-

tures exposed to IGF-I (P < 0:05). However TGF-bappeared to reduce the levels of GLUT3 protein and

TNF-a and IL-1b had no significant effect on the

abundance of GLUT3. Note that the same cells exhib-ited a large increase in 2-deoxyglucose uptake in re-

sponse to TGF-b treatment which suggests that while

GLUT3 was down-regulated in response to this growth

factor, GLUT1 (and possibly other GLUT isoforms)

may have been activated or significantly up-regulated,

resulting in an increase in net 2-deoxyglucose transport.

4. Discussion

The major findings of this study are summarized as

follows: (1) equine chondrocytes express functional glu-

cose transporters; (2) the uptake of 2-deoxy-DD-[2,6-3H]

glucose into equine chondrocytes is consistent with a fa-

cilitated, GLUT-mediated process for glucose transport;

(3) short-term insulin treatment resulted in a statisticallyinsignificant increase in the uptake of 2-deoxyglucose but

long-term insulin treatment had no effect on 2-deoxy-

glucose uptake; (4) IGF-I, IL-1b, TGF-b and TNF-a all

resulted in a significant (over 65%) increase in glucose

uptake compared to basal levels; (5) IGF-I stimulation of

equine chondrocytes over a period of 24 h resulted in

upregulation ofGLUT1andGLUT3proteins; (6) TGF-bapparently reduced the levels of GLUT3; (7) TNF-aand IL-1b stimulation upregulated GLUT1 expression

but down-regulated GLUT3 supporting the hypothesis

that growth factors and pro-inflammatory cytokines dif-

ferentially regulate the GLUT1 and GLUT3 glucose

transporters.

Articular cartilage is an avascular tissue in which

oxygen tensions are generally very low (Otte, 1991;

Mobasheri et al., 2002b). In the absence of oxidativephosphorylation, chondrocytes endure hypoxic condi-

tions by generating ATP by glycolytic breakdown of

glucose (Mobasheri et al., 2002a). Hypoxia generally in-

duces expression of several important genes including

vascular endothelial growth factor (VEGF), glucose

transporter-1 (GLUT1; SLC2A1) and glucose trans-

porter-3 (GLUT3; SLC2A3) (Semenza, 1999). Evidence

from other cell types in the literature suggests that ele-vated glucose uptake stimulated by pro-inflammatory

cytokines is accompanied by upregulation of GLUT

transporter proteins. In response to pro-inflammatory

cytokines a number of genes are activated including

VEGF and GLUT1 (Pufe et al., 2001; Shikhman et al.,

2001a; Richardson et al., 2003) resulting in the upregu-

lation of the proteins encoded by these genes in chon-

drocytes. These proteins are key mediators of nutrientand energy provision in degenerative conditions such as

osteoarthritis, which is particularly relevant to the equine

species; the glucose transporters provide the means for

accelerated glucose transport and enhanced substrate

utilization in response to stimulation by pro-inflamma-

tory, catabolic cytokines, including TNF-a, IL-6 and IL-

1b (Shikhman et al., 2001a; Shikhman et al., 2001b;

Richardson et al., 2003) and to chemokines such as con-nective tissue activating peptide III (Ku Tai et al., 1992).

Studies in chondrocytes from the growth plates of

rodents (Wang et al., 1999; Ohara et al., 2001) and in

human Meckel’s cartilage (Ishizeki et al., 2002) have

shown that the insulin-responsive GLUT4 isoform is

expressed and functional in these cells. However, we and

others have previously reported on the absence of in-

sulin-responsive glucose transporters in mature humanarticular chondrocytes (Shikhman et al., 2001a; Moba-

sheri et al., 2002a). The results presented in this study

(Fig. 1) support our earlier observations in human

chondrocytes (Richardson et al., 2003) and confirm that

insulin-responsive glucose transporters are not ex-

pressed in fully developed equine articular chondro-

cytes, and that insulin does not acutely increase glucose

transport in these cells. In the presence of high levels ofserum, chondrocytes from Meckel’s cartilage have been

shown to change phenotype and differentiate into

adipocytes (Ishizeki et al., 2002). There are no published

reports that suggest articular chondrocytes are capable

of undergoing a similar transdifferentiation.

The cytokines and growth factors used in this study

were human recombinants. This was due to the absence

of suitable equine counterparts. The observed differ-ences in the magnitude of cytokine and growth factor

stimulated 2-deoxyglucose transport (Fig. 2) is likely to

be due to the lower biological activity of human re-

combinant cytokines and growth factors on equine

chondrocytes. The magnitude of the changes we ob-

served in glucose uptake in response to IGF-I, IL-1band TNF-a was similar to the values previously reported

by our group (Richardson et al., 2003). However, theaugmented 2-deoxyglucose uptake was lower than val-

ues reported by another group for human chondrocytes

(Shikhman et al., 2001a).

In response to long-term TGF-b stimulation we

observed increased 2-deoxyglucose transport and up-

regulation of GLUT1 but GLUT3 appeared to be

down-regulated (Fig. 3). Studies in the kidney have

Fig. 3. Evidence for growth factor and cytokine regulation of GLUT1 and GLUT3 expression in equine articular chondrocytes. Western blot analysis

demonstrated that equine chondrocytes expressed GLUT1 and GLUT3. IGF-I stimulation resulted in upregulation of GLUT1 and GLUT3. TGF-bstimulation upregulated GLUT1 but down-regulated GLUT3. TNF-a and IL-1b stimulation resulted in upregulation of GLUT1 but did not affect

GLUT3. TGF-b also reduced the levels of the GLUT3 protein. * Denotes a significant difference (P < 0:05); ns denotes a statistically insignificant

difference.

T. Phillips et al. / The Veterinary Journal 169 (2005) 216–222 221

shown that TGF-b stimulates glucose transport by en-hancing GLUT1 expression in mesangial cells (Inoki

et al., 1999), which confirms our observations. TGF-balso stimulates glucose transport by increasing GLUT1

expression in fibroblasts (Kitagawa et al., 1991). The

expression of GLUT3 has not been studied in response

to TGF-b stimulation (no published reports in PubMed)

making it difficult to compare our results with published

information in the literature.Elevated glucose transport and upregulation of

GLUT proteins may be an early and sustained event in

inflammatory processes in equine articular cartilage.

The increased glucose uptake observed in equine chon-

drocytes stimulated with pro-inflammatory cytokines

and growth factors appears to be an important com-

ponent of the chondrocyte response to both anabolic

and catabolic mediators and is likely to be due to themutual dependency of catabolic and anabolic pathways

on regulated glucose transport (Mobasheri et al.,

2002b). However, the GLUT isoforms appear to re-

spond differently to different stimuli. GLUT2 and

GLUT4 proteins have not been detected by immuno-

histochemical methods in equine articular cartilage (A.

Mobasheri, unpublished observations) supporting pre-

vious findings in human chondrocytes (Shikhman et al.,2001a; Mobasheri et al., 2002a). Further studies are

needed to determine if other GLUT isoforms identified

in chondrocytes (i.e. GLUT5, GLUT9 and GLUT12)

(Ohara et al., 2001; Shikhman et al., 2001a; Macheda

et al., 2002; Richardson et al., 2003) are involved in the

chondrocyte responses to growth factors and cytokines.

Arthropathies are a major cause of poor welfare in thehorse and the physiology and nutrition of articular car-

tilage are key issues in this process. Cartilage degrada-

tion in equine osteoarthritis has been shown to be

stimulated by pro-inflammatory cytokines and accom-

panied by apoptosis (Clegg and Mobasheri, 2003; Kim

et al., 2003) and elevatedmatrixmetalloprotease secretion

(particularly MMP-2 and MMP-9; Clegg and Carter,

1999). The data presented here suggests that growthfactors can stimulate net glucose uptake by chondro-

cytes. Furthermore, pro-inflammatory cytokines also

stimulate increased uptake of glucose in order to provide

the metabolic energy required for extracellular matrix

degradation. However, growth factors and pro-inflam-

matory cytokines have differential effects on the indi-

vidual GLUT isoforms; most up-regulate GLUT1

expression but GLUT3 is only up-regulated by IGF-I. Aclearer understanding of cytokine and endocrine regu-

lated transport systems in chondrocyte physiology may

lead to identification of key molecules or biochemical

pathways involved in the nutritional regulation of

cartilage matrix integrity.

Acknowledgements

We would like to thank the University of Liverpool

Research Development Fund for funding this work. T.

Phillips would like to acknowledge the support of Pro-

fessor David J. Back (Department of Pharmacology and

Therapeutics, University of Liverpool). This study was

222 T. Phillips et al. / The Veterinary Journal 169 (2005) 216–222

also funded by a short-term fellowship granted to I.Ferraz from the Servicio de Reumatologia, Hospital

Universitario de Canarias, in Santa Cruz de Tenerife.

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