Since I mentioned in my first post about N-acetyl glucosamine (GlcNAc), I thought I should expand on it. Glycan chains, aka sugar chains, are structures made from monosaccharides (simple sugars) like glucose, galactose, and N-acetylglucosamine, that link together to form chains that attach to proteins or lipids. On proteins they form glycoproteins and on lipids, glycolipids. Adding glycans to proteins or lipids is glycosylation, a common post-translational modification (PTM) that gives the cell more functional diversity. High N-glycan branching promotes binding to galectins, sugar-binding proteins. Different galectin-glycoprotein interactions on the cell surfaces form a type of lattice that can simultaneously control movement, clustering, and/or endocytosis of multiple receptors and transporters to control signaling, cell growth, differentiation, and death.
Inflammation is a biological response caused by injury, either infectious, traumatic, toxic, or autoimmune to tissues. Acute inflammation is a controlled process that ends by restoring homeostasis, but persistent and dysregulated inflammation turns chronic. Chronic inflammation changes physiological and biochemical systems, mostly due to secreted proinflammatory cytokines (e.g., TNFα, IL-2, IL-17, IFN-γ) that get released excessively in inflammation. They alter N-glycosylation by changing substrate synthesis pathways and glycosyltransferase (GT) expression needed to biosynthesize N-glycans (i.e., GTs add glycans to proteins).
This affects N-glycan structure height, antennae, composition, and ligand epitopes. Basically they alter N-glycosylation on key cells involved in the inflammatory response, like endothelial adhesion molecules, innate and adaptive immune cells, and immunoglobulins, for proinflammatory functions.
Galectins are glycan-binding proteins that interact with GTs. They bind to carbohydrates (sugars), and affect T cell activation and immune responses. Galectin-1 (Gal-1) can induce T cell death, Gal-3 can suppress T cell responses, and Gal-9 can modulate T cell recruitment. Glycan structures are affected by glycan-modifying enzyme activity and substrate availability.
Glycan-modifying enzyme Golgi Beta-1,6-N-acetylglucosaminyltransferase V (MGAT5) catalyzes tetra-antennary N-linked glycan biosynthesis in T cells. At the genetic level, several MGAT5 single-nucleotide polymorphisms (SNPs) reduce its expression in T cell glycosylation changes in IBD, COPD, and MS. A very interesting aspect is that in chronic viral infection, like EBV, IL-10 induced MGAT5 expression in CD8+ T cells forms the Gal-3 lattice and increases the antigen activation threshold. This is usually anti-inflammatory, but in viral infection it lets the pathogen rapidly replicate, causing persistent chronic inflammation. (I’ll have to make a post soon about the role of EBV infections in MS as it”s very significant). Mgat5 deletion mildly reduces N-glycan branching, but Mgat1 deletion completely blocks it.
(MGAT1, or N-acetylglucosaminyltransferase I, initiates formation of complex and hybrid N-glycans. It transfers GlcNAc from UDP-GlcNAc to a high mannose N-glycan structure on proteins to create the first branch point, crucial for cellular function and development).
(UDP-GlcNAc (Uridine diphosphate N-acetylglucosamine) is an activated form of GlcNAc, a monosaccharide derivative of glucose).
IL-2 reduces N-glycan branching in resting T cells, but increases it in activated T cells. IL-2 has a ∼250x higher affinity for UDP-GlcNAc than MGAT5, so increased Mgat1 in activated T cells inhibits UDP-GlcNAc for N-glycan branching by Mgat5. N-glycan branching reduces when immune cells deactivate following GlcNAc induced elevations in N-glycan branching. So, if T cells never deactivate?? Vitamin D3 also raises N-glycan branching and a clinical trial of oral Vitamin D3 in MS shows reduced N-glycan branching in resting T cells. Another example of low vitamin D affects in MS.
The hexosamine biosynthetic pathway (HBP) is the main source of UDP-GlcNAc for N-glycan branching. De novo UDP-GlcNAc synthesis converts fructose-6-phosphate to glucosamine-6-phosphate by the rate-limiting enzyme glutamine-fructose-6-phosphate transaminase (GFPT). The final step needs glutamine to convert GFPT to UDP-GlcNAc.
However, inflammatory cells switch metabolism from oxidative phosphorylation to glycolysis and glutaminolysis when activated during inflammation that takes up the fructose-6-phosphate and glutamine needed to make UDP-GlcNAc. These drive proinflammatory cell differentiation over regulatory cells, that keeps N-glycan branching reduced.
The above information shows GlcNAc is the rate-limiting metabolite for N-glycan branching and some aspects regulating its availability. Extracellular GlcNAc can enter cells via macropinocytosis, and supplementing GlcNAc can inhibit pro-inflammatory T-cell responses in inflammatory demyelination models by enhancing N-glycan branching. It helps change N-glycosylation to decrease inflammation and neurodegeneration, and increase myelination. (However, Google AI says the NGT1 transporter transfers GlcNAc Into cells and macropinocytosis is less efficient and needs much higher concentrations. Like I said in the first post, one answer creates three questions).
GlcNAc can cross the BBB for N-glycan biosynthesis, which interact with galectins to co-regulate various glycoproteins clustering/signaling/endocytosis.
The increased N-glycan branching can suppress inflammatory demyelination by T and B cells, and trigger stem/progenitor cell remyelination.
Studies show GlcNAc and N-glycan branching can trigger oligodendrogenesis from OPCs by inhibiting platelet-derived growth factor receptor-α (PDGFRa) cell endocytosis. (Endocytosis is when a receptor is internalized in a cell so that it can’t be activated). Platelet-derived growth factor–AA (PDGFAA) is critical for oligodendrogenesis, and its receptor, PDGFRα, expresses in oligodendrocyte precursor cells (OPCs). The increased cell surface PDGFRα expression triggers OPC differentiation and likely also affects other cell surface receptors/transporters in OPCs to drive myelination. Subtle changes in N-glycan branching can greatly impact oligodendrocyte (OL) differentiation from NSCs in vitro.
N-glycan branching also reduces integrin clustering so OPCs can travel to demyelination sites and helps cells uptake glucose by stimulating glucose transporter surface retention. Glucose transporter 1 (GLUT1) in OLs transfer lactate to axons and can increase glucose supply to the glycolytic pathway in OLs.
N-glycan branching can suppress T cell activation through T-cell receptors, inhibit pro-inflammatory TH1 and TH17 differentiation, and enhance anti-inflammatory Treg cell differentiation.
In B cells, N-glycan branching can dose dependently reduce pro-inflammatory innate responses by titrating decreases in TLR4 and TLR2 surface expression via endocytosis to inhibit APC activity, while promoting adaptive responses via B-cell receptors.
Oral GlcNAc can cross the BBB and is metabolized to UDP-GlcNAc by CNS cells and is secreted at sufficient levels in breast milk to raise UDP-GlcNAc in brains of suckling pups to drive primary myelination. Breastfed newborns consume ∼0.5–1.5 g of GlcNAc per day or ∼100–300 mg/kg/day for a 5-kg infant. Similar to the ∼160 mg/kg/day dose used to promote myelination in adult mice. GlcNAc isn’t in commercial baby formula and breastfed infants display increased myelination and cognitive function vs formula-fed infants.
A study finds WM lesion volume correlates with lower HexNAc serum levels, while lesion count does not. As active MS has similar serum HexNAc levels to inactive MS, GlcNAc must affect permanent demyelination rather than acute inflammatory demyelination.
(HexNAc can refer to either N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc))
Two studies show significantly lower GlcNAc amounts in progressive multiple sclerosis (PMS) blood serum compared to healthy controls (HCs) or even relapsing remitting multiple sclerosis (RRMS). One finds mean serum level plus its stereoisomers (HexNAc) is 710 nM in HCs and slightly reduced in RsRRMS patients, 682 nM, while PMS patients are further reduced at 548 nM. The other finds RRMS patients with a mean level of 709 nM and PMS patients at 405 nM. Lower GlcNAc and HexNAc serum levels correlate with more neurodegeneration.
The low endogenous GlcNAc seen in PMS causes remyelination failure, or at least partly considering all the other factors inhibiting it, therefore it’s linked to a progressive disease course, clinical disability, and MRI neurodegeneration.
So, oral GlcNAc can be used to reduce pro-inflammatory T-cell responses, pro-inflammatory innate B-cell activity, and myelin repair. (Hope so, I’m taking a tablespoon every morning, roughly 10 to 12 grams, seems helpful)
In a study, MS patients take 6 g or 12 g GlcNAc daily for 4 weeks. Serum IFNγ from TH1 cells and IL-17 from TH17 cells are elevated in MS compared to HCs. Oral GlcNAc reduces serum IFNγ, IL-6, and IL-17 at 12 g, but not at 6 g. Inflammatory cytokines reduce mostly in the last 2 weeks on GlcNAc therapy, as it first impacts N-glycan branching, then reduces pro-inflammatory cytokines, and persists in the washout period. Enhanced myelination by GlcNAc depends on time, ∼2x greater at 4 weeks compared to 1 week.
IL-10 is thought to protect in MS, but levels likely elevate as a compensatory response to lessen ongoing chronic CNS inflammation. Oral GlcNAc at 12 g, but not 6 g reduces serum IL-10, matching reduced IFNγ, IL-6, and IL-17.
Neurofilament light chain (NFL) only expresses in neurons and is released by cell injury. Serum neurofilament light chain (sNfL) is used as a biomarker for acute neuroinflammation, therapy response, and to predict long-term (> 15 years) progression. High baseline sNfL indicates active axon injury. Ocrelizumab B cell depletion only reduces sNfL in PMS with high baseline levels. As 12 g, but not 6 g of GlcNAc impacts TH1 and TH17 cytokines, only 12 g of GlcNAc can significantly reduces sNfL in those with high baseline sNfL.
Other MS DMTs, like ocrelizumab, can’t reduce serum IFNγ or IL-17 levels. They act in the periphery to inhibit T and/or B cell responses and don’t directly target CNS inflammation, suggesting reduced inflammatory cytokines and sNfL are from GlcNAc crossing the BBB to target chronic active CNS inflammation. EDSS in both 6 g and 12 g improves in about 30% after just 4 weeks on GlcNAc, but inflammatory markers only improve by 12 g GlcNAc.
As immune cells and NSCs/OPCs are targeted separately by GlcNAc, lower doses may be fine for NSCs/OPCs, with a higher dose for residual inflammation.
A different study reports GlcNAc amounts needed to raise N-glycan branching in vivo are much lower than needed in vitro. GlcNAc can enter cells by macropinocytosis, thus time and membrane turnover rates influence how much GlcNAc is needed to raise N-glycan branching. Short-term in vitro studies need high amounts to raise intracellular GlcNAc levels when primary cells are still viable. Cells can be exposed to GlcNAc over a longer time period in vivo, so lower concentrations can raise N-glycan branching. Macropinocytosis rates are much higher in vivo than in vitro. (Will have to see how NGT1 transporter is involved in GlcNAc uptake into cells)
GlcNAc is highly safe in humans. Breastfed infants consume a lot, and large IV doses (20 g and 100 g) in humans shows no toxicity or alterations in blood glucose or insulin. Oral GlcNAc (3–6 g/day) was used in children with IBD for ∼2 years without toxicity and/or side effects.
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Sy, M., Newton, B.L., Pawling, J. et al. N-acetylglucosamine inhibits inflammation and neurodegeneration markers in multiple sclerosis: a mechanistic trial. J Neuroinflammation 20, 209 (2023). https://doi.org/10.1186/s12974-023-02893-9
Brandt AU, Sy M, Bellmann-Strobl J, Newton BL, Pawling J, Zimmermann HG, Yu Z, Chien C, Dörr J, Wuerfel JT, Dennis JW, Paul F, Demetriou M. Association of a Marker of N-Acetylglucosamine With Progressive Multiple Sclerosis and Neurodegeneration. JAMA Neurol. 2021 Jul 1;78(7):842-852. https://doi.org/10.1001/jamaneurol.2021.1116. PMID: 33970182; PMCID: PMC8111565.
Radovani B and Gudelj I (2022) N-Glycosylation and Inflammation; the Not-So-Sweet Relation. Front. Immunol. 13:893365. https://doi.org/10.3389/fimmu.2022.8933
Cvetko A, Kifer D, Gornik O, Klarić L, Visser E, Lauc G, Wilson JF, Štambuk T. Glycosylation Alterations in Multiple Sclerosis Show Increased Proinflammatory Potential. Biomedicines. 2020 Oct 13;8(10):410. https://doi.org/10.3390/biomedicines8100410. PMID: 33065977; PMCID: PMC7599553.
N-acetylglucosamine drives myelination by triggering oligodendrocyte precursor cell differentiation Sy, Michael et al. Journal of Biological Chemistry, Volume 295, Issue 51, 17413 – 17424
