Molecular characterization and partial cDNA cloning of facilitative glucose transporters expressed in human articular chondrocytes; stimulation of 2-deoxyglucose uptake by IGF-I and elevated MMP-2 secretion by glucose deprivation.
OBJECTIVE
Recent evidence suggests that human chondrocytes express several facilitative glucose transporter (GLUT) isoforms and also that 2-deoxyglucose transport is accelerated by cytokine stimulation. The aim of the present investigation was to determine if human articular chondrocytes express any of the recently identified members of the GLUT/SLC2A gene family and to examine the effects of endocrine factors, such as insulin and IGF-I on the capacity of human chondrocytes for transporting 2-deoxyglucose.


METHODS
PCR, cloning and immunohistochemistry were employed to study the expression of GLUT/SLC2A transporters in normal human articular cartilage. The uptake of 2-deoxyglucose was examined in monolayer cultured immortalized human chondrocytes following stimulation with TNF-alpha, insulin and IGF-I. Levels of MMP-2 were assessed by gelatin zymography following glucose deprivation of alginate cultures.


RESULTS
Using PCR we detected transcripts for eight glucose transporter isoforms (GLUTs 1, 3, 6, 8, 9, 10, 11 and 12) and for a fructose transporter (GLUT5) in human articular cartilage. Expression of GLUT1, GLUT3 and GLUT9 proteins in normal human articular cartilage was confirmed by immunohistochemistry. The uptake of 2-deoxyglucose was dependent on time and temperature, inhibited by cytochalasin B and phloretin, and significantly accelerated in chondrocyte cultures stimulated with IGF-I. However, 2-deoxyglucose uptake was unaffected by short and long-term insulin treatment, which ruled out a functional role for insulin-sensitive GLUT4-mediated glucose transport. Furthermore, secretion of MMP-2 was increased in alginate cultures deprived of glucose.


CONCLUSIONS
The data supports a critical role for glucose transport and metabolism in the synthesis and degradation of cartilage.
Richardson
S
S
Connective Tissue Research Group, Department of Veterinary Preclinical Sciences, Faculty of Veterinary Science, University of Liverpool, UK.
Neama
G
G
Phillips
T
T
Bell
S
S
Carter
S D
SD
Moley
K H
KH
Moley
J F
JF
Vannucci
S J
SJ
Mobasheri
A
A
D015153:Blotting, Western / Q000379:methods; D002358:Cartilage, Articular / Q000378:metabolism; D002460:Cell Line; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D003571:Cytochalasin B / Q000494:pharmacology; D004270:DNA, Circular / Q000235:genetics*; D003847:Deoxyglucose / Q000493:pharmacokinetics*; D005947:Glucose / Q000493:pharmacokinetics; D006133:Growth Substances / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007328:Insulin / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D010693:Phloretin / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D020033:Protein Isoforms; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004270:DNA, Circular; D006133:Growth Substances; D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; D009004:Monosaccharide Transport Proteins; D020033:Protein Isoforms; D014409:Tumor Necrosis Factor-alpha; C065700:myotrophin; D003571:Cytochalasin B; D003847:Deoxyglucose; D020778:Matrix Metalloproteinase 2; D005947:Glucose; D010693:Phloretin