The anabolic effects of glucosamine were primarily thought to be attributable to its capacity for providing building blocks for the synthesis of GAGs by chondrocytes [8
]. Other effects have also been reported, however, such as its anti-catabolic potency, seen by its inhibition of the expression and/or activity of catabolic enzymes such as phospholipase A2
, matrix metalloproteinases or aggrecanases [11
]. Another study confirmed the potency of glucosamine to inhibit the expression and activity of aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS -5)) in transiently transfected cell lines [13
]. The authors suggested that the symptomatic and functional effects of glucosamine would be justified by the fact that glucosamine interferes with the matrix metalloproteinases responsible for proteoglycan degradation in OA.
Various properties were demonstrated for glucosamine in the three main tissues involved in OA, cartilage, synovial membrane and subchondral bone. The main potencies were shown in articular chondrocytes, where glucosamine was demonstrated to reverse the deleterious effects of IL-1β [11
]. In rat and human chondrocytes, the glucosamine effect was shown to occur through the inhibition of NF-κB signaling [16
]. The reversal of the effects of IL-1β in human chondrocytes also inhibited inflammatory enzymes, such as the inducible form of nitric oxide synthase and cyclooxygenase-2 [18
]. This effect of glucosamine was further detailed by Imagawa and co-workers [19
], who were able to demonstrate that glucosamine prevents the demethylation of specific CpG sites in the IL-1β promoter, consequently preventing the expression of IL-1β.
A recent pharmacoproteomic study revealed that glucosamine (10 mM) differentially regulates the pattern of expression of IL-1β-induced proteins in human articular chondrocytes [20
]. The proteins affected by glucosamine are mainly involved in the signal transduction pathways, redox and stress response, protein synthesis and protein folding. In addition, glucosamine increased the expression of the GRP78 chaperone protein. This observation supports the reported anti-inflammatory effect of glucosamine. This study also compared glucosamine to chondroitin sulfate; these compounds produced different patterns of protein modification when tested alone or in combination [20
]. A synergistic effect for the modification of superoxide dismutase expression was demonstrated when cells were exposed to both compounds, implying a potent effect on oxidative stress in addition to the modulation of energy production and metabolic pathways produced by chondroitin sulfate.
The pro-anabolic effects of glucosamine were demonstrated in both human chondrocytes and synovial cells, where glucosamine was shown to induce the production of hyaluronic acid (HA) and to directly enter the GAG biosynthetic pathway (that is, for the production of HA, keratan sulfate and sulfated GAGs) [21
]. The authors also proposed that the chondroprotective effect of glucosamine results from the modulation of enzymes responsible for HA synthesis.
The potential of glucosamine to induce HA production in the synovial membrane was previously suggested in a study that used synovial explants [22
]. In addition, cationic glucosamine derivatives were shown to produce an anti-inflammatory effect through the inhibition of mitogen-activated protein kinase signaling pathways in lipopolysaccharide-stimulated macrophages [23
Glucosamine sulfate was also shown to be effective in human OA osteoblasts [24
]. Glucosamine increased the osteoprotegrin/receptor activator of nuclear factor kappa-B ligand (RANKL) ratio and reduced bone resorption. This effect was increased when glucosamine was used in combination with chondroitin sulfate.
It is important to point out that these studies were performed in different culture systems, with various formulations and concentrations of glucosamine. The details of the culture systems and the formulations are provided in Table . The concentrations used in the in vitro
studies were 'super physiological' - in some cases up to 2,000 times higher than the maximal concentration that can realistically be achieved in plasma (10 μM) after oral administration of 1,500 mg of glucosamine sulfate in human subjects. Furthermore, some of these studies compared the effects of two formulations of glucosamine in order to provide evidence for the superiority of one or another [21
]. It was proposed that the differences, if they truly existed, might contribute to the different results observed in clinical trials with various glucosamine formulations. For example, glucosamine sulfate was shown to be a stronger inhibitor of gene expression than glucosamine hydrochloride [25
]. Both formulations were commercially available from a lab supplier. The same group compared glucosamine hydro-chloride to N-acetylglucosamine [22
] and observed no effect of N-acetylglucosamine on HA production whereas glucosamine hydrochloride appeared to modulate this parameter. The same conclusion was reached in the study by Igarashi and colleagues [21
]. In contrast, another study compared the effect of native glucosamine and N-acetylglucosamine on the metabolic activity of human articular chondrocytes [26
]. Both compounds exhibited different potencies on glucose transport, and GAG and HA synthesis. Indeed, N-acetylglucosamine appeared to accelerate the facilitated glucose uptake and increase both GAG and HA synthesis, suggesting that N-acetyl-glucosamine may be more efficient than native glucosamine.
Principal characteristics of the in vitro studies testing glucosamine
Glucosamine has also been tested in combination with chondroitin sulfate in vitro
. Some of the results have already been discussed above [20
]. The effect of combinations of glucosamine and chondroitin was also reported by Chan and colleagues [27
]. Glucosamine hydrochloride was tested in combination with chondroitin sulfate on bovine cartilage explants. The combination was shown to inhibit both inflammatory and catabolic intermediates and was slightly superior to glucosamine alone [27
Finally, some authors have studied the contribution of exogenous glucosamine to the synthesis of chondroitin sulfate in human articular chondrocytes in culture [31
]. They concluded that exogenous glucosamine cannot stimulate the synthesis of chondroitin sulfate. Furthermore, they showed that glucose can increase endogenous glucosamine to reach concentrations superior to those achieved after oral administration.
In conclusion, many of the in vitro
investigations carried out so far have been performed using high concentrations of glucosamine - concentrations that are unlikely to be achieved in plasma with oral doses of the drug. Several authors have proposed that the therapeutic doses used did not allow the identification of proteoglycan synthesis as a mechanism of action of glucosamine [31
]. Therefore, extrapolation of the in vitro
data to the in vivo
situation should be done with great caution.