Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage

Abstract

Introduction: Disorders that affect glucose metabolism, namely diabetes mellitus (DM), may favor the development and/or progression of osteoarthritis (OA). Thus far, little is known regarding the ability of chondrocytes to adjust to variations in the extracellular glucose concentration, resulting from hypoglycemia and hyperglycemia episodes, and so, to avoid deleterious effects resulting from deprivation or intracellular accumulation of glucose. The aim of this study was to compare the ability of normal and OA chondrocytes to regulate their glucose transport capacity in conditions of insufficient or excessive extracellular glucose and to identify the mechanisms involved and eventual deleterious consequences, namely the production of reactive oxygen species (ROS).

Methods: Chondrocytes, isolated from normal and OA human cartilage, were maintained in high-density monolayer cultures, in media without or with 10 or 30 mM glucose. Glucose transport was measured as the uptake of 2-deoxy-D-glucose (2-DG). Glucose transporter-1 (GLUT-1) mRNA and protein content were evaluated by real-time RT-PCR and western blot, respectively. ROS production was measured with 2',7'-dichlorodihydrofluorescein diacetate.

Results: Basal and IL-1beta-induced 2-DG uptake, including the affinity (1.066 +/- 0.284 and 1.49 +/- 0.59 mM) and maximal velocity (0.27 +/- 0.08 and 0.33 +/- 0.08 nmol/microg protein/hour), and GLUT-1 content were identical in normal and OA chondrocytes. Glucose deprivation increased 2-DG uptake and GLUT-1 protein both in normal and OA chondrocytes. Exposure to high glucose (30 mM) for 18 or 48 hours decreased those parameters in normal but not in OA chondrocytes. GLUT-1 mRNA levels were unaffected by high glucose, either in normal or OA chondrocytes. The high glucose-induced reduction in GLUT-1 protein in normal chondrocytes was reversed by treatment with a lysosome inhibitor. High glucose induced ROS production, which lasted significantly longer in OA than in normal chondrocytes.

Conclusions: Normal human chondrocytes adjust to variations in the extracellular glucose concentration by modulating GLUT-1 synthesis and degradation which involves the lysosome pathway. Although capable of adjusting to glucose deprivation, OA chondrocytes exposed to high glucose were unable downregulate GLUT-1, accumulating more glucose and producing more ROS. Impaired GLUT-1 downregulation may constitute an important pathogenic mechanism by which conditions characterized by hyperglycemia, like DM, can promote degenerative changes in chondrocytes that can facilitate the progression of OA.

Figure 1

Basal glucose transport and glucose transporter-1 protein in normal and osteoarthritis chondrocytes. (a) 2-Deoxy-D-glucose (2-DG) transport into normal (n = 6) and osteoarthritis (OA) (n = 9) chondrocytes. (b) Concentration dependence of 2-DG influx into normal and OA chondrocytes fitted to the Michaelis–Menten model to determine the affinity and maximal velocity. Each value is the mean ± standard deviation of five independent experiments performed in duplicate. (c) Glucose transporter-1 (GLUT-1) protein normalized to the respective actin band in normal (n = 9) and OA (n = 9) chondrocyte cultures. Bars = mean ± standard deviation.

Figure 2

Stimulation of glucose transport and glucose transporter-1 expression by IL-1β in normal and osteoarthritis chondrocytes. (a) 2-Deoxy-D-glucose (2-DG) transport into normal (n = 4) and osteoarthritis (OA) (n = 5) chondrocytes stimulated or not with IL-1β 30 ng/ml for 48 hours. (b) Glucose transporter-1 (GLUT-1) protein normalized to the respective actin band in normal (n = 4) and OA (n = 9) chondrocyte cultures, stimulated or not with IL-1β 30 ng/ml for 48 hours. Results expressed as the percentage relative to the respective control cells. MW, molecular weight marker. (c) GLUT-1 mRNA levels in normal (n = 3) and OA (n = 3) chondrocyte cultures stimulated or not with IL-1β 30 ng/ml for 12 hours. Results are expressed as the fold increase relative to the respective untreated cells. *P < 0.05 and ***P < 0.001 relative to untreated cells. Bars = mean ± standard deviation.

Figure 3

Modulation of glucose transport by different extracellular glucose concentrations. 2-Deoxy-D-glucose (2-DG) uptake into normal (n = 6) and osteoarthritis (OA) (n = 9) chondrocytes cultured in media with 0 mM, 10 mM (regular glucose medium (RGM)) or 30 mM glucose (high glucose medium (HGM)) for 18 or 48 hours. Results expressed as the percentage relative to the respective control cells maintained in RGM. ***P < 0.001 relative to cells maintained in RGM, ^§§§P < 0.001 between normal and OA chondrocytes exposed to the same glucose concentration for the same period. Bars = mean ± standard deviation.

Figure 4

Modulation of glucose transporter-1 protein content by different extracellular glucose concentrations. Glucose transporter-1 (GLUT-1) protein normalized to the respective actin band in chondrocytes cultured in media with 0 mM, 10 mM (regular glucose medium (RGM)) or 30 mM glucose (high glucose medium (HGM)) for 18 or 48 hours. (a) Normal chondrocytes (n = 4). (b) Osteoarthritis chondrocytes (n = 6). Results expressed as the percentage relative to the respective control cells maintained in RGM. **P < 0.01 and ***P < 0.001 relative to cells maintained in RGM. Bars = mean ± standard deviation.

Figure 5

Regulation of glucose transporter-1 mRNA levels by high glucose. Quantitative real-time RT-PCR analysis of glucose transporter-1 (GLUT-1) mRNA levels in chondrocyte cultures exposed to 30 mM glucose (high glucose medium (HGM)) for 6, 12 or 24 hours or maintained in regular glucose medium (RGM). (a) Normal chondrocytes (n = 3). (b) Osteoarthritis chondrocytes (n = 3). Results expressed as the fold increase relative to the respective control cells maintained in RGM. Bars = mean ± standard deviation.

Figure 6

Roles of the proteasome and the lysosome in mediating high-glucose-induced downregulation of glucose transporter-1 protein. Glucose transporter-1 (GLUT-1) protein content normalized to the respective actin band in normal chondrocytes (n = 3) cultured in regular glucose medium (RGM) or in high glucose medium (HGM, 30 mM) with or without 20 μM chloroquine or 10 μM MG-132 added for the last 6 hours of a total 18-hour incubation period. Results expressed as the percentage relative to untreated cells maintained in RGM. **P < 0.01 relative to cells maintained in RGM, ^§P < 0.01 between glucose 30 mM with or without 20 μM chloroquine. Bars = mean ± standard deviation.

Figure 7

Modulation of reactive oxygen species production by IL-1β and high glucose. (a) Reactive oxygen species (ROS) production in normal and osteoarthritis (OA) chondrocytes treated with or without IL-1β 30 ng/ml for 1 hour (n = 4). (b) ROS production in normal and OA chondrocytes (n = 5) cultured in regular glucose medium (RGM) or in high glucose medium (HGM, 30 mM) for the periods indicated and then loaded with 5 μM 2',7'-dichlorodihydrofluorescein diacetate for 20 minutes at 37°C, as described in Materials and methods. Results expressed as the percentage relative to the respective control cells maintained in RGM. **P < 0.01 and ***P < 0.001 relative to the respective control cells maintained in RGM, ^§§§P < 0.001 between normal and OA chondrocytes exposed to the same glucose concentration for the same period. Bars = mean ± standard deviation.