Glucosamine (GlcN) is a derivative of cellular glucose metabolism and is also a component of glycosaminoglycans, hyaluronic acid, and proteoglycans in the cartilage matrix that form the ends of bones. Exogenous GlcN primarily exists in two forms, D-GlcN hydrochloride (HCl) and GlcN sulfate [¹]. Both forms are immediately ionized into GlcN, and therefore D-GlcN∙HCl and GlcN sulfate are largely redundant [²]. In Japan, GlcN sulfate is a medical agent but not used as a nutritional supplement. In the Glucosamine/Chondroitin Arthritis Intervention Trial (GAIT), GlcN∙HCl supplements were used, and based on that, we chose the same form for our study [³].

GlcN is useful for the treatment of joint diseases in both humans and animals [⁴,⁵]. The bioavailability of GlcN has been reported to be 26% in humans [⁶], 19% in rats [⁷], 12% in dogs [⁸], and 2%–6.1% in horses [⁹,¹⁰,¹¹]. The maximum drug concentration time (Tmax) after intravenous injection of GlcN in rats was 2 h [¹²], while Tmax after oral administration in dogs was 1 h [¹³]. These results suggest species-specific differences in GlcN absorption and metabolism.

Glycosaminoglycans (GAGs) and proteoglycans are found at high concentrations in cartilage. The results of our previous studies suggested that oral administration of GlcN∙HCl (1 g per head per day) led to regeneration of both GAGs and proteoglycans [¹⁴]. We also found that it protected cartilage in experimentally induced osteoarthritis by inhibition of type II collagen degradation and enhancement of type II collagen synthesis in articular cartilage [¹⁵].

In the GAIT study, GlcN was orally administered for 24 weeks [³]. However, there have been no studies investigating the relationship between long-term oral administration of amino monosaccharides and amino acid synthesis. The aim of this study was to examine the metabolism of amino acids in dogs after oral administration of GlcN∙HCl for five weeks.

Glucosamine is a widely used dietary supplement for promoting joint health. There have been concerns that oral GlcN supplementation at usual doses may adversely affect glucose metabolism in subjects with impaired glucose tolerance. However, a recent report showed that GlcN had no effects on fasting blood glucose levels, glucose metabolism, or insulin sensitivity at any oral dose in healthy subjects, in those with diabetes, and in those with impaired glucose tolerance [¹⁷].

In this study, GlcN was not detected in the plasma the day after stopping oral administration of GlcN∙HCl (day 36), which is in agreement with the results of our previous study [¹³]. That report revealed that amino acid levels in the plasma changed 1 h after oral GlcN∙HCl administration and that the metabolomics profile could change within a day. The observed changes in metabolomic profiles in the current study were definitely related to GlcN∙HCl administration because all other life cycle variables, including diet, were constant. Because the plasma levels of GlcN increased slightly just after feeding, we assume that GlcN did not denature. However, in the future we need to investigate the stability of GlcN∙HCl in food.

In this study, levels of N-acetylglucosamine (GlcNAc) remained nearly unchanged before and after administration of GlcN∙HCl. Changes in GlcNAc levels were evaluated, but our measurement technique was not able to quantify its concentration in the plasma because of the lack of a standard substance. We assume that the administration of GlcN∙HCl did not affect the metabolism of GlcNAc. In future studies, the concentration of GlcNAc should be quantified using high performance liquid chromatography.

No prior reports have described the increases of succinic acid, malic acid, and fumaric acid resulting from oral administration of GlcN. It is known that some parasites and cancer cells depend on fumarate respiration using a reverse reaction involving succinate dehydrogenase, producing succinate as a byproduct [¹⁸,¹⁹]. Under conditions of glucose depletion and severe hypoxia, ATP is synthesized without oxygen by fumarate respiration. The active use of fumarate respiration after oral administration of GlcN∙HCl provides a possible explanation of the accumulation of fumaric acid observed in this study.

During glycolysis, the levels of lactic acid and pyruvic acid increased after administration of GlcN∙HCl. Lactic acid is a predominant source of carbon atoms for glucose synthesized by gluconeogenesis. Accumulated lactic acid is recycled by the liver via the Cori cycle to form glucose; however, this is an energy-consuming process. Yalamanchi et al. [²⁰] showed that lactic acid significantly increased transforming growth factor-beta (TGF-β) peptide expression, receptor expression, and functional activity. TGF-β is a cytokine with numerous functions related to wound healing, including recruitment of fibroblasts and macrophages, stimulation of collagen production, and formation of new capillaries [²¹,²²]. Cartilage regeneration might therefore result from its increase.

During fasting, muscles break down protein, and amino acids are released into circulation. In the present study, the levels of hydroxyproline and alanine after oral administration of GlcN∙HCl were significantly higher than levels prior to administration, with alanine higher to a greater extent. Alanine can be directly converted to pyruvic acid and used as a substrate for gluconeogenesis (via the alanine cycle) [²³], which could possibly explain the increase in plasma concentrations of pyruvic acid we found.

Glucose depletion might result from hexosamine-induced insulin resistance [²⁴]. GlcN taken up by cells is phosphorylated into GlcN-6-phosphate by hexokinase, which then bypasses GFAT to enter the hexosamine pathway [²⁵]. It is then converted to uridine diphosphate-GlcNAc. Elevated levels of GlcNAc inhibit insulin-stimulated glucose uptake (via GLUT4 translocation) and glycogen synthesis [²⁴]. GlcNAc also serves as a donor substrate for the synthesis of nucleotides, glycosyl phosphatidylinositol anchors, complex protein glycosylation, and O-linked-β-N-acetylglucosamine (O-GlcNAc) protein modification. O-GlcNAc modification of cytosolic and nuclear proteins occurs via the action of O-GlcNAc transferase [²⁴]. This post-translational modification enhances the transcription of TGF-β and plasminogen activator inhibitor-1 [²⁴,²⁶]. Similarly, TGF-β induced by O-GlcNAc might stimulate collagen production.

In this study, accelerated fumarate respiration and elevated plasma levels of lactic acid and alanine were observed after oral administration of GlcN∙HCl. These results indicate that oral administration of GlcN∙HCl induced anaerobic respiration and starvation in cells and further suggest that lactic acid and O-GlcNAc could potentially induce TGF-β production. We hypothesize that these conditions induced by oral administration of GlcN∙HCl promote cartilage regeneration as shown in the schematic diagram in Figure 3. However, further studies are required to evaluate the expression of TGF-β.

We thank Kazuki Watanabe for providing advice on data interpretation.