- Open Access
Beneficial reward-to-risk action of glucosamine during pathogenesis of osteoarthritis
- Yeon-Ho Kang†1,
- Sujeong Park†1,
- Chihyun Ahn1,
- Jinsoo Song1,
- Dongkyun Kim1 and
- Eun-Jung Jin1, 2Email author
© Kang et al. 2015
- Received: 24 May 2015
- Accepted: 22 September 2015
- Published: 31 October 2015
The Erratum to this article has been published in European Journal of Medical Research 2016 21:3
Glucosamine is widely used to improve the symptoms and to delay the structural progression of osteoarthritis. However, its efficacy in osteoarthritis has been controversial and its underlying mechanism of action remains unclear. The aim of this study was to investigate the effects of glucosamine and the underlying mechanisms in human chondrocytes.
Chondrocytes from normal human articular cartilage were treated with glucosamine (10–100 mM). Subsequently, cell death was analyzed by Annexin V staining and FACS and mitochondrial function was studied by measuring the mitopotential. Peroxisomal function was analyzed by BODIPY staining, and gene expression of PMP70 and acyl-CoA oxidase 1, by real-time PCR. Total lipids were analyzed by gas chromatography/mass spectrometry. Autophagy activation was determined by western blotting of beclin and light chain 3B. Autophagosome formation was analyzed by introduction of green fluorescent protein (GFP) LC3, and pexophagy was determined by introduction of mRFP-EGFP-SKL plasmids.
Treatment of chondrocytes with glucosamine exerts exposure time-dependent dual effects on apoptosis/autophagy. Short time exposure of glucosamine to chondrocytes activated autophagy, pexophagy, and peroxidation. On the other hand, long time exposure of glucosamine had opposite effects, namely accumulation of very long chain fatty acids and peroxisomal dysfunction.
We highlight the dual role of glucosamine in apoptosis/autophagy in human chondrocytes depending on exposure time. Although further research is required to fully understand the dual effects of glucosamine, dosage and duration of glucosamine treatment are clear contributing factors towards the line of beneficial reward-to-risk action.
- Short time exposure
- Long time exposure
- Beneficial reward-to-risk action
Osteoarthritis (OA), one of the most disabling arthritic conditions, affects not only cartilage, but also induces metabolic and structural modifications of the subchondral bone and the synovial membrane . Despite the increasing number of OA patients, to date no cure for this disease has been found. Currently, the management of OA consists mostly of symptom management, i.e., the reduction of pain and improvement of joint function with conventional treatment methods such as the use of analgesics or non-steroidal anti-inflammatory drugs [2, 3]. Recently, the failure of these conventional treatments has given rise to the increasing use of symptomatic slow-acting drugs for OA (SYSADOA)-based therapies.
One of the commonly used SYSADOAs is glucosamine . Glucosamine, an amino monosaccharide, is an indispensable component of chondroitin sulfate and keratin sulfate, which are the main glycosaminoglycans (GAG) in the cartilage [5, 6]. Glucosamine has been used in the supplementary treatment of OA, although its effects on the extracellular matrix (ECM) metabolism in chondrocytes remain controversial [7, 8]. In chondrocyte pellet culture, glucosamine acts as a suppressor of interleukin-1 (IL-1)-induced prostaglandin E2 synthase and stimulates proteoglycan synthesis [9–11]. Administration of glucosamine in a chymopapain-induced damaged rabbit joint  and murine OA model  resulted in increased cartilage matrix. Moreover, the investigators reported mild protection efficacy of glucosamine in human articular cartilage, including the reduction of surface fibrillation, loss of safranin O staining, and loss of sulfated glycosaminoglycans . In 2013, Caramés et al. demonstrated the therapeutic efficacy of glucosamine in OA by providing evidence that it activated autophagy by inhibiting the AKT/FoxO3/mammalian target of the rapamycin pathway . They suggested that a specific form of autophagy, mitophagy, could remove the damaged mitochondria frequently observed in OA cartilage.
On the other hand, in 2014, Jiang et al. highlighted the negative effect of autophagic cell death in articular chondrocytes . They observed that high concentrations of glucosamine had a negative effect on cell viability, possibly due to autophagic cell death.
To date, a dual role for glucosamine has been implied, i.e., a potentially desired chondro-protective effect and an undesired negative effect. Therefore, in studying the effects of glucosamine, particular attention should be paid to the regulatory signals and molecules responsible for these dual functions. Here, we attempt to elucidate the underlying regulatory mechanisms that may determine the switch between these two possible opposing functions.
Although chondrocytes reportedly produce only 25 % of their total ATP via oxidative phosphorylation (OXPHOS) , recent studies have demonstrated that, in humans, OA chondrocytes demonstrated lower activity of mitochondrial respiratory chain complexes I, II, and III than normal chondrocytes . In addition, this mitochondrial dysfunction may further affect the pathophysiological features of OA, such as cartilage degradation, via reduced chondrocyte biosynthesis and growth, cartilage matrix calcification, and increased chondrocyte apoptosis and inflammatory responses. Moreover, inflammatory cytokines such as TNF-α and IL-1β decrease ATP levels and mitochondrial membrane potential . Taken together, these reports suggest that mitochondrial damage may play a significant role in the pathogenesis of OA. Recently, we suggested the possible functional interconnection between mitochondria and peroxisome , i.e., peroxisomal dysfunction may trigger mitochondrial dysfunction in human articular chondrocytes. However, these functional interconnections during OA pathogenesis remain poorly understood and need to be further characterized. In the present study, we investigated the dual functions of glucosamine in human articular chondrocytes and assessed whether peroxisomal dysfunction may play a role in regulating these glucosamine-induced beneficial and disadvantageous functions.
Cell line and cell culture
Human chondrocytes derived from normal human articular cartilage were purchased from Cell Applications (San Diego, CA). Cells were cultured in chondrocyte growth medium (Cell Applications). Cells were maintained in a humidified incubator at 37 °C with 5 % CO2. To check the differential ability of chondrocytes, pellet cultures were applied and stained with Alcian blue and the RNA levels of type II collagen and aggrecan were analyzed before the experiments. For this study, cells were treated with 10, 30, 50 and 100 mM d-(+)-glucosamine hydrochloride for 2 h (short time exposure) or 24 h (long time exposure) unless exposure time is indicated, respectively (Sigma-Aldrich, St. Louis, MO, dissolved in growth media).
Pellet culture of chondrocytes
For differentiation, alginate beads with encapsulated human chondrocytes (Cell applications, San Diego, CA, USA) were grown in a chondrocyte differentiation medium (Cell applications, San Diego, CA, USA) for 2 weeks. Briefly, human chondrocyte were suspended at a density of 4 × 106 per milliliters in a 1.2 % solution of sterile alginate in 0.15 M NaCl. The cell suspension was slowly expressed through a 22-gauge needle and dropped into a 102-mM CaCl2 solution. The beads with approximately 40,000 cells/bead (diameter 3 mm) were allowed to polymerize for 10 min and washed twice with 0.15 M NaCl, followed by two washes in DMEM/F12. The beads were then transferred to medium and cultured at 37 °C in a humidified atmosphere of 5 % CO2. Alcian blue staining was used to detect chondrocyte nodule formation after 2 weeks of alginate beads culture. Alginate beads were rinsed with PBS and fixed in 4 % paraformaldehyde in PBS for 20 min. Alginate Beads were washed with PBS three times and stained in 1 % Alcian blue in 0.1 N HCl for 8 h at room temperature. Alginate Beads were de-stained in 0.1 N HCl two times and stored in water for image capture.
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated using RNAiso Plus (TaKaRa, Japan) according to the manufacturer’s protocol. Aliquots of total RNA (1 μg) from each sample were reverse-transcribed into cDNA according to the instructions of the PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa, Japan). Quantitative real-time PCR (qRT-PCR) was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). PCR were prepared and heated to 95 °C for 2 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at specific Tm for 15 s, and extension at 72 °C for 20 s. Quantification of the PCR signals was achieved by comparing the cycle threshold value (C t) of the gene of interest with the C t value of the reference gene GAPDH. The following oligonucleotides were used as primers: MMP13: 5′-TTGCAGAGCGCTACCTGAGATCAT-3′, antisense, 5′-TTTGCCAGTCACCT CTAAGCCGAA-3′, sense; ADAMTS4: 5′-CGCTTTGCTTCACTGAGTAGAT-3′, antisense, 5′-CTGTTAGCAGGTAGCGCTTTAG-3′, sense; PMP70: 5′- CCAGTTGGGTCATATCCT TGAA-3′, antisense, 5′- CTTGCCATCGCC ATTCTTTG-3′, sense; LC3A: 5′- ACAGCATGG TGAGTGTGTC-3′, antisense, 5′- GGGAGGCGTAGACCATATAGA-3′, sense; LC3B: 5′- GCCTTCTTCCTGTTGGTGAA-3′, antisense, 5′- TGGGAGGCATAGACCATGTA-3′, sense; CASP1: 5′-GGCAGGCCTGGA TGATGA-3′, antisense, 5′-ATACCAAGAACT GCCCAAGTTTG-3′, sense, CASP3: 5′-GCGCCCTGGCAGCAT-3′, antisense, 5′-GCCTACAGCCCATTTCTCCAT-3′ sense, CASP9: 5′-AACAGCATTAGCGACCCTAAGC-3′ antisense, 5′-AGCAGTGGGCTCAC TCTGAAG-3′, sense, FAS: 5′-CCAGCATGG TTGTTGAGCAA-3′ antisense, 5′-ACCCGCTCAGTACGGAGTTG-3′, sense, COMP: 5′-TCACAAGCATCTCCCACAAA-3′ antisense, 5′-GACAGTGATGGCGATGGTATAG-3′ sense, ACAN: 5′-TCGAGGGTGT AGCGTGTAGAGA-3′ antisense, 5′-TCGAGGACA GCGAGGCC-3′ sense, GAPDH: 5′-GATCATCAGCAATGCCTCCT-3′, antisense, 5′-TGTGGTCATG AGTCCT TCCA-3′, sense.
Annexin V and viability cell assays
The apoptotic and necrotic cell populations were analyzed by Muse Cell Analyzer (Merck Millipore), which is a miniaturized fluorescent flow cytometer. Analyses were performed using specific fluorescent dyes Muse Annexin V and Dead Cell Kit (Merck Millipore). Briefly, both floating and adherent treated cells were collected, centrifuged at 300×g for 5 min, and suspended in phosphate buffered saline (PBS). Aliquots of 100 μL of cell suspension were added to 100 μL of diluted Muse Annexin V and Dead Cell reagent and incubated for 20 min at room temperature (RT). Subsequently, cells were analyzed and the percentage of early or late apoptotic cells was determined in accordance with the Millipore guidelines.
Mitochondrial membrane potential assay
The mitochondrial depolarization state of cells was analyzed by Muse Cell Analyzer (Merck Millipore). We simultaneously measured changes in the mitochondrial membrane potential by assay kit (Merck Millipore). Briefly, both floating and adherent treated cells were collected, centrifuged at 300×g for 5 min, and suspended in cell culture medium. Briefly, 100 μL aliquots of cell suspension were first added to 95 μL of diluted Muse Mitopotential dye and mixed for 20 min at 37 °C before incubating with 5 μL of 7-AAD reagent dye at RT. After 5 min, the cell suspensions were analyzed and the percentages of live, depolarized, and dead cells were determined in accordance with the Millipore guidelines.
BODIPY 493/503 or BODIPY 665/676 (Molecular Probes, Carlsbad, CA, USA), was diluted in PBS at a concentration of 1 mg/mL. Following fixation with 4 % paraformaldehyde (PFA) for 10 min and staining with 4,6-diamidino-2-phenylindole (DAPI) for identification of nuclei, the samples were washed 3 times in PBS for 10 min and stained with BODIPY. The samples were mounted in VECTASHIELD (Vector Laboratories), covered with glass cover slips, and digital images were obtained with an Olympus Fluoview FV100 confocal laser scanning microscope under epifluorescent optics.
Tandem fluorochrome pexophagy assay
Human chondrocytes were transfected with the mRFP-EGFP-SKL plasmid. Two days after the first transfection, the cells were cultured for another 24 h in the presence of lysosomal inhibitors, 120 μM leupeptin (Sigma-Aldrich), and 2 μM E-64 (Enzo Life Sciences). After incubation, the cells were washed in PBS and fixed with 4 % PFA in PBS for 15 min at RT.
LC3-positive vesicle formation
Human chondrocytes were transfected with LC3-GFP plasmid. Transfection efficiency of the plasmid was assessed visually (>60 % and identical for all wells). After 2 days, the cells were starved for 3 h in the presence of 20 μM chloroquine (InvivoGen) and permeabilized with 0.025 % digitonin in PBS for 5 min to wash out the cytosolic LC3 protein and enrich for the vesicle-associated LC3. Subsequently, the cells were washed in PBS and fixed with 4 % PFA in PBS for 15 min at RT.
Gas chromatography/mass spectrometry
Total lipids were extracted using a chloroform/methanol (2:1 v/v) mixture. The extracted lipids were separated on a Sep-Pak Silica Cartilage column (Waters, Milford, MA, USA) and transmethylated with 0.5 M CH3ONa in methanol by heating in a sealed tube at 70 °C for 1 h under nitrogen. The fatty acid methyl esters were extracted with hexane. Subsequent gas chromatography–mass spectrometry (GC–mass) analysis was performed according to the standard protocol. Briefly, the sample was injected into a Finnigan MAT-8430 mass spectrometer connected to an HP-5890 gas chromatography (Finnigan MAT, Bremen, Germany) equipped with a DB-5 capillary column.
Apoptosis and autophagy profiling PCR array
Apoptosis and autophagy profiling was conducted using the RT2 Profiler PCR Array Kit (QIAGEN, GmbH, Hilden, Germany), which included specific primers for apoptosis (PAHS-012Z) or autophagy (PAHS-084Z). Each profiling array was performed according to the manufacturer’s instructions. cDNA was synthesized with the RT2 First Strand Kit (Qiagen, #330401) and qRT-PCR was performed using the RT2 qPCR SYBR Green Fluor qPCR Master Mix (Qiagen, #330512) according to the manufacturer’s instructions. Data were analyzed and gene expression data visualized using GenEx (Weihenstephan, Germany).
Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, containing 150 mM NaCl, 1 % Nonidet P-40, 0.1 % SDS, 0.1 % deoxycholic acid, 10 mM NaF, 10 mM Na4P2O7, 0.4 mM Na3VO4, and protease inhibitors) for 30 min on ice. The total protein content of the cells was determined by BCA Protein Acid Reagent (bicinchoninic acid) (Pierce Biotechnology Inc., Rockford, MN, USA). Proteins (30 μg) were separated by 10 % polyacrylamide gel electrophoresis containing 0.1 % SDS and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Uppsala, Sweden). The membranes were incubated for 1 h at room temperature in a blocking buffer (20 mM Tris–HCl, 137 mM NaCl, pH 8.0, containing 0.1 % Tween and 3 % skimmed dry milk), and probed with antibodies against Beclin, LC-3B and GAPDH (Cell Signaling, Beverly, MA, USA). The blots were developed with an HRP-conjugated secondary antibody and reacted proteins were visualized using an electrochemiluminescence (ECL) system (GE Healthcare Life Sciences).
High-dose glucosamine induces pathological characteristics of OA
High-dose glucosamine induced peroxisome dysfunction and accumulation of very long chain fatty acids (VLCFA)
Lipidomics of glucosamine-treated chondrocytes
Glucosamine 10 mM
Glucosamine 50 mM
rac Methadone N-oxide
Octadecanoic acid 2,3-dihydroxypropyl ester
1-Dodecanol, 2-octyl- eicosane
Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester
9,12-Octadecadienoic acid (Z,Z)-, methyl ester
2-Propenoic caid, tridecyl ester
Pentafluoropropionic acid tetradecyl ester
2-Ethylhexyl methyl isophthalate
Heptafluorobutyric acid, n-tetradecyl ester
Fumaric acid, 2-ethylhexyl hexyl ester
2-Propenoic acid, tridecyl ester
Propanoic acid, 3-mercapto-, dodecyl ester
Sulfurous acid, dodecyl 2-propyl ester
Propanoic acid,3-mercapto-, dodecyl ester
2-Propenoic acid oxybis(methyl-2,1-ethanediyl) ester
Decane, 2, 3, 5-trimethyl
4-Ethoxybenzoic acid ethyl ester
Carbonochloridic acid, decyl ester
High-dose glucosamine stimulates autophagic cell death
Glucosamine is the main component of proteoglycans, and the major non-collagenous cartilage-specific proteoglycan of the intervertebral disc is aggrecan. In the clinic, glucosamine has been used in the treatment of OA, although the application has not been widely accepted and remains controversial [7, 8]. In this study, we demonstrated that glucosamine could act differently depending on its dosage and period of treatment. Exposure of chondrocytes to high-dose glucosamine induced lipid accumulation, possibly through suppression of PMP70 and ACOX1, and resulted in the stimulation of cell death, thus indicating a regulatory role for glucosamine in peroxisomal function. Since the peroxisome is involved in lipid metabolism, including β-oxidation of fatty acids and transfer of oxidized lipid to mitochondria for utilization in energy metabolism [23–25], peroxisomal dysfunction could be a trigger for OA-induced chondrocyte death.
One of the typical characteristics in OA pathogenesis is chondrocyte apoptosis in cartilage, but the underlying regulatory mechanisms have not been well studied. Recently, nitric oxide (NO) and reactive oxygen species (ROS) have been suggested to be key factors in mediating chondrocyte apoptosis through mitochondrial dysfunction such as damage of mitochondrial DNA . Impairment of mitochondrial events have been known to be involved in chondrocyte apoptosis, including reduction of the mitochondrial transmembrane protein complex IV, decrease of mitochondrial membrane potentials and release of cytochrome c . In the present report, we observed that long-term exposure to glucosamine induced impairment of mitochondrial membrane potentials, suggesting the involvement of glucosamine in the regulation of mitochondrial function during OA pathogenesis.
Recently, other and our group have provided evidence for an interconnection between cellular organelles, particularly between the mitochondria and the peroxisome. Peroxisome dysfunction hindered mitochondrial function and vice versa. Likewise, we also found in this study that glucosamine induced dysfunction of mitochondria as well as that of the peroxisome in human chondrocytes. Long-term exposure to glucosamine induced the accumulation of VLCFA and this could be responsible for chondrocyte apoptosis. Recent studies showed a correlation between VLCFA accumulation and cell death as a result of the silencing of peroxisomal transporters and altered mitochondrial function . ABCD-1 deficiency induced apoptosis in oligodendrocytes and led to astrocytic inflammatory responses and loss of myelin [29, 30]. In addition, VLCFA depletion is known to result in constitutive activation of autophagy , indicating that VLCFAs serve to dampen the amplitude of autophagy. VLCFA accumulation induced by long-term exposure to glucosamine could be responsible for suppression of autophagic responses in OA chondrocytes. This suggests that the functional integrity of peroxisomes in the regulation of VLCFA has important implications for autophagy and cell homeostasis.
Further, we described, for the first time, a direct correlation between glucosamine and pexophagy. Even though glucosamine is widely used to treat or prevent OA in humans, the effects of treatment on cartilage degeneration are controversial. We showed that glucosamine exhibits a concentration- and exposure time-dependent dual action in the regulation of chondrocyte survival and apoptosis. Recent research on an association between glucosamine and the PI3K/AKT/mTOR signaling pathway has elicited controversial reports. Caramés et al. had demonstrated that the mTOR pathway was inhibited by glucosamine in chondrocytes . In contrast, Shintani et al. found that the mTOR pathway did not participate in glucosamine-induced autophagy in HeLa and COS7 cells . Here, short exposure of glucosamine activated autophagic responses (referred to as short time response), whereas long exposure of glucosamine inhibited autophagic response, particularly pexophagy and led to impairment of peroxidation (referred to as long time response). Taken together, these results indicate that depending on the period of exposure, glucosamine may act as both a suppressor and inducer of apoptosis and autophagy.
Collectively, our data demonstrated the role of glucosamine in autophagy and showed a direct correlation between glucosamine and peroxisomal function. We also highlighted a dual role of glucosamine in apoptosis and autophagy in human chondrocytes. Glucosamine exerts exposure time-dependent dual effects on apoptosis and autophagy. Although further research is required to fully understand the relationship between dosage and exposure time to glucosamine, the differential opposing effects of glucosamine indicate that dosage and duration of treatment may be a determining factor in the line of beneficial reward-to-risk action in OA therapy.
E-JJ designed and performed the experiments, analyzed the data, and wrote the paper. JS, DK, and YK performed experiments and analyzed data. All authors read and approved the final manuscript.
This works was supported by National Research Foundation (NRF) of Korea Grant funded by the Korean Governments by the Korea government (MSIP) [2013R1A1A2011999], [NRF-2013R1A2A2A01067194], and [2011-0030130]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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