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Sulfatide (3-O-sulfogalactosylceramide, SM4) is a glycosphingolipid, highly multifunctional and particularly enriched in the myelin sheath of neurons. The role of sulfatide has been implicated in various biological fields such as the nervous system, immune system, host-pathogen recognition and infection, beta cell function and haemostasis/thrombosis. Thus, alterations in sulfatide metabolism and production are associated with several human diseases such as neurological and immunological disorders and cancers. The unique lipid-rich composition of myelin reflects the importance of lipids in this specific membrane structure. Sulfatide has been shown to be involved in the regulation of oligodendrocyte differentiation and in the maintenance of the myelin sheath by influencing membrane dynamics involving sorting and lateral assembly of myelin proteins as well as ion channels. Sulfatide is furthermore essential for proper formation of the axo-glial junctions at the paranode together with axonal glycosphingolipids. Alterations in sulfatide metabolism are suggested to contribute to myelin deterioration as well as synaptic dysfunction, neurological decline and inflammation observed in different conditions associated with myelin pathology (mouse models and human disorders). Body fluid biomarkers are of importance for clinical diagnostics as well as for patient stratification in clinical trials and treatment monitoring. Cerebrospinal fluid (CSF) is commonly used as an indirect measure of brain metabolism and analysis of CSF sulfatide might provide information regarding whether the lipid disruption observed in neurodegenerative disorders is reflected in this body fluid. In this review, we evaluate the diagnostic utility of CSF sulfatide as a biomarker for neurodegenerative disorders associated with dysmyelination/demyelination by summarising the current literature on this topic. We can conclude that neither CSF sulfatide levels nor individual sulfatide species consistently reflect the lipid disruption observed in many of the demyelinating disorders. One exception is the lysosomal storage disorder metachromatic leukodystrophy, possibly due to the genetically determined accumulation of non-metabolised sulfatide. We also discuss possible explanations as to why myelin pathology in brain tissue is poorly reflected by the CSF sulfatide concentration. The previous suggestion that CSF sulfatide is a marker of myelin damage has thereby been challenged by more recent studies using more sophisticated laboratory techniques for sulfatide analysis as well as improved sample selection criteria due to increased knowledge on disease pathology., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sulfatides (STs) in cerebrospinal fluid (CSF), as well as magnetic resonance imaging (MRI)-detected white matter hyperintensities (WMHs), may reflect demyelination. Here, we investigated the diagnostic utility of CSF ST levels in the subcortical small vessel type of dementia (SSVD), which is characterized by the presence of brain WMHs., Objective: To study the diagnostic utility of CSF ST levels in SSVD., Methods: This was a mono-center, cross-sectional study of SSVD (n = 16), Alzheimer's disease (n = 40), mixed dementia (n = 27), and healthy controls (n = 33). Totally, 20 ST species were measured in CSF by liquid chromatography-mass spectrometry (LC-MS/MS)., Results: CSF total ST levels, as well as CSF levels of hydroxylated and nonhydroxylated ST species, did not differ across the study groups. In contrast, CSF neurofilament light chain (NFL) levels separated the patient groups from the controls. CSF total ST level correlated with CSF/serum albumin ratio in the total study population (r = 0.64, p < 0.001) and in all individual study groups. Furthermore, CSF total ST level correlated positively with MRI-estimated WMH volume in the total study population (r = 0.30, p < 0.05), but it did not correlate with CSF NFL level., Conclusion: Although there was some relation between CSF total ST level and WMH volume, CSF ST levels were unaltered in all dementia groups compared to the controls. This suggests that CSF total ST level is a poor biomarker of demyelination in SSVD. Further studies are needed to investigate the mechanisms underlying the marked correlation between CSF total ST level and CSF/serum albumin ratio.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Several biomarkers including proteins and lipids have been reported in MS cerebrospinal fluid (CSF), reflecting different aspects of the pathophysiology particularly of relapsing-remitting MS (RRMS). Sulfatide, abundant in the myelin sheath and a proposed target for autoimmune attack in MS, has been reported altered in MS CSF. Here, we investigated the potential of CSF sulfatide and its isoforms as biomarkers in MS. A highly sensitive and quantitative mass spectrometry method was employed to determine levels of sulfatide isoforms in CSF from RRMS and progressive MS (PMS) patients, and healthy donors (HD). We demonstrate that levels of total CSF sulfatide and C24:1, C26:1, and C26:1-OH isoforms were significantly increased in PMS compared with RRMS patients and HD, while C23:0-OH was significantly decreased in CSF from PMS patients compared to the other two groups. Multivariate discriminant analysis showed that CSF sulfatide isoform pattern in PMS patients was distinct and non-overlapping with that of RRMS patients and HD. Sulfatide levels did not correlate with tested biomarkers or clinical parameters. The results suggest that CSF sulfatide isoform levels may be used to discriminate the phenotype of MS and might play a role in the progression of the disease., (© 2018 International Society for Neurochemistry.)

Sulfatides (STs) are a group of glycosphingolipids that are highly expressed in brain. Due to their importance for normal brain function and their potential involvement in neurological diseases, development of accurate and sensitive methods for their determination is needed. Here we describe a high-throughput oriented and quantitative method for the determination of STs in cerebrospinal fluid (CSF). The STs were extracted using a fully automated liquid/liquid extraction method and quantified using ultra-performance liquid chromatography coupled to tandem mass spectrometry. With the high sensitivity of the developed method, quantification of 20 ST species from only 100 μl of CSF was performed. Validation of the method showed that the STs were extracted with high recovery (90%) and could be determined with low inter- and intra-day variation. Our method was applied to a patient cohort of subjects with an Alzheimer's disease biomarker profile. Although the total ST levels were unaltered compared with an age-matched control group, we show that the ratio of hydroxylated/nonhydroxylated STs was increased in the patient cohort. In conclusion, we believe that the fast, sensitive, and accurate method described in this study is a powerful new tool for the determination of STs in clinical as well as preclinical settings., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

The endogenous glycosphingolipid sulfatide is a ligand for CD1d-restricted type II natural killer T (NKT) lymphocytes. Through the action of these cells,sulfatide treatment has been shown to modulate the immune response in mouse models for autoimmune diseases, infections and tumour immunity. Sulfatide exists naturally in different organs including the pancreas, where sulfatide colocalizes with insulin within the Langerhans islet b-cells, targets for the immune destruction in type 1 diabetes (T1D). Human T1D patients, but not patients with type 2 diabetes nor healthy individuals, have autoantibodies against sulfatide in serum, suggesting that sulfatide induces an immune response in the natural course of T1D in humans. Here, we investigate sulfatide as an autoantigen and a modulator of autoimmune disease in the murine model forT1D, the non-obese diabetic (NOD) mice. We demonstrate that aged NOD mice displayed serum autoantibody reactivity to sulfatide; however, this reactivity did not correlate with onset of T1D. Repeated administration of sulfatide did not result in an increase in serum reactivity to sulfatide. Moreover, a multidose sulfatide treatment of female NOD mice initiated at an early (5 weeks of age),intermediate (8 weeks of age) or late (12 weeks of age) phase of T1D progression did not influence the incidence of disease. Thus, we demonstrate that a fraction of NOD mice develop autoantibody reactivity to sulfatide; however, we fail to demonstrate that sulfatide treatment reduces the incidence of T1D in this mouse strain.

Natural killer T (NKT) lymphocytes are implicated in the early response to microbial infection. Further, sulfatide, a myelin self-glycosphingolipid, activates a type II NKT cell subset and can modulate disease in murine models. We examined the role of NKT cells and the effect of sulfatide treatment in a murine model of Staphylococcus aureus sepsis. The lack of CD1d-restricted NKT cells did not alter survival after a lethal inoculum of S. aureus. In contrast, sulfatide treatment significantly improved the survival rate of mice with S. aureus sepsis, accompanied by decreased levels of tumor necrosis factor alpha and interleukin-6 in the blood. The protective effect of sulfatide treatment depended on CD1d but not on type I NKT cells, suggesting that activation of type II NKT cells by sulfatide has beneficial effects on the outcome of S. aureus sepsis in this model.

A proportion of healthy siblings of multiple sclerosis (MS) patients have an oligoclonal immunological reaction in their cerebrospinal fluid (CSF) termed the "MS oligoclonal trait". The CSF levels of the major myelin glycosphingolipid sulfatide and serum antibodies against the glycosphingolipids sulfatide and galactosylceramide were recently reported to be increased in MS patients. We studied the levels of these substances in pairs of 46 patients and their 46 healthy siblings and 50 unrelated healthy blood donors (HBD). The sulfatide concentration in CSF was assayed by thin layer chromatography and immunostaining, and the concentration of galactosylceramide by densitometry after thin layer chromatography. Anti-glycosphingolipid antibody levels were assayed by ELISA. In the healthy siblings, the CSF sulfatide concentrations were markedly increased (p<0.001, age adjusted p=0.025), and the serum IgM anti-GalCer antibodies were increased in healthy siblings compared with HBD (p=0.02). The increased sulfatide or antibody levels did not co-segregate with the "MS oligoclonal trait" or the HLA-DR15 phenotype. In conclusion, a proportion of healthy siblings of MS patients have increased CSF sulfatide and anti-glycosphingolipid antibody levels, which may, analogous to the "MS oligoclonal trait", constitute an "MS glycosphingolipid endophenotype". Endophenotypes could potentially simplify the genetics of complex disorders., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sulfatide-reactive CD1d-restricted natural killer T (NKT) lymphocytes belong to the type II NKT cell subset with diverse TCRs, and have been found to regulate experimental auto-immune encephalomyelitis, tumor immunity, and experimental hepatitis in murine models. NKT cells can be activated by self-lipids presented by CD1d, manifested as autoreactivity. The identity of most of these self-lipids remains unknown. By isolating lipids from a CD1d-expressing, highly stimulatory antigen presenting cell, we identified isoforms of β-glucosylceramide (GlcCer), with sphingosine and fatty acid chain lengths of C24:0 and C16:0, that activated a sulfatide-reactive type II NKT cell hybridoma. A screen of structurally related glycosphingolipids demonstrated β-galactosylceramide (GalCer) as another ligand, and further, that the lysoforms were the most potent isoform of the glycosphingo-lipid ligands, followed by isoforms with a long fatty acid chain of C24. Thus, the same type II NKT cell was activated by several ligands, namely sulfatide, GlcCer, and GalCer. However, CD1d-dependent reactivity to antigen presenting cells lacking all GlcCer-based glycosphingolipids, or all glycosphingolipids, was maintained. This suggests that other endogenous, nonglycosphingolipid, lipid ligands contribute to steady-state autoreactivity by type II NKT cells., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The glycosphingolipid sulfatide (SO(3)-3Galbeta1Cer) is a demonstrated ligand for a subset of CD1d-restricted NKT cells, which could regulate experimental autoimmune encephalomyelitis, a murine model for multiple sclerosis, as well as tumor immunity and experimental hepatitis. Native sulfatide is a mixture of sulfatide isoforms, i.e. sulfatide molecules with different long-chain bases and fatty acid chain lengths and saturation. Here, we demonstrate that sulfatide-specific CD1d-restricted murine NKT hybridomas recognized several different sulfatide isoforms. These included the physiologically relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet beta-cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation of the long fatty acid (C24:0), resulted in reduced stimulatory capacity, and fatty acid hydroxylation abolished the response. Moreover, sulfatide was not responsible for the natural autoreactivity toward splenocytes by XV19 T hybridoma cells. Our results reveal a promiscuity in the recognition of sulfatide isoforms by a CD1d-restricted NKT-cell clone, and suggest that sulfatide, a major component of the myelin sheet and pancreatic beta-cells, is one of several natural ligands for type II CD1d-restricted NKT cells.

Background: The glycosphingolipid sulfatide has previously been found in several mammalian tissues, but information on the uptake of exogenously administered sulfatide in different organs in vivo is limited. In pancreatic beta cells, sulfatide has been shown to be involved in insulin processing and secretion in vitro. In this study, we examined the uptake of exogenously administered sulfatide and its distribution to the pancreatic beta cells. This might encourage future studies of the function(s) of sulfatide in beta cell physiology in vivo. Radioactive sulfatide was given orally to mice whereafter the uptake of sulfatide in the gastrointestinal tract and subsequent delivery to the pancreas was examined. Sulfatide uptake in pancreas was also studied in vivo by i.p. administration of radioactive sulfatide in mice, and in vitro in isolated rat islets. Isolated tissue/islets were analysed by scintillation counting, autoradiography and thin-layer chromatography-ELISA., Results: Sulfatide was taken up in the gastrointestinal tract for degradation or further transport to other organs. A selective uptake of short chain and/or hydroxylated sulfatide fatty acid isoforms was observed in the small intestine. Exogenously administered sulfatide was found in pancreas after i.p, but not after oral administration. The in vitro studies in isolated rat islets support that sulfatide, independently of its fatty acid length, is endocytosed and metabolised by pancreatic islets., Conclusion: Our study supports a selective uptake and/or preservation of sulfatide in the gastrointestinal tract after oral administration and with emphasises on pancreatic sulfatide uptake, i.p. administration results in sulfatide at relevant location.

Sulfatide (3'-sulfo-beta-galactosyl ceramide) is a glycosphingolipid present in mammalians in various fatty acid isoforms of which the saturated 16 carbon-atom length (C16:0) is more abundant in pancreatic islets than in neural tissue, where long-chain sulfatide isoforms dominate. We previously reported that sulfatide isolated from pig brain inhibits glucose-induced insulin secretion by activation of ATP-sensitive K+ channels (K(ATP) channels). Here, we show that C16:0 sulfatide is the active isoform. It inhibits glucose-stimulated insulin secretion by reducing the sensitivity of the K(ATP) channels to ATP. (The half-maximal inhibitory concentration is 10.3 and 36.7 micromol/l in the absence and presence of C16:0 sulfatide, respectively.) C16:0 sulfatide increased whole-cell K(ATP) currents at intermediate glucose levels and reduced the ability of glucose to induce membrane depolarization, reduced electrical activity, and increased the cytoplasmic free Ca2+ concentration. Recordings of cell capacitance revealed that C16:0 sulfatide increased Ca2+-induced exocytosis by 215%. This correlated with a stimulation of insulin secretion by C16:0 sulfatide in intact rat islets exposed to diazoxide and high K+. C24:0 sulfatide or the sulfatide precursor, beta-galactosyl ceramide, did not affect any of the measured parameters. C16:0 sulfatide did not modulate glucagon secretion from intact rat islets. In betaTC3 cells, sulfatide was expressed (mean [+/-SD] 0.30 +/- 0.04 pmol/microg protein), and C16:0 sulfatide was found to be the dominant isoform. No expression of sulfatide was detected in alphaTC1-9 cells. We conclude that a major mechanism by which the predominant sulfatide isoform in beta-cells, C16:0 sulfatide, inhibits glucose-induced insulin secretion is by reducing the K(ATP) channel sensitivity to the ATP block.

Mammalian tissues express beta-isoforms of glycosphingolipids and, among these, sulfatide (sulphated galactosylceramide) is present in the beta cells, and it is here that the short fatty acid chain (C16) isoform is predominately found. In vitro studies have shown that sulfatide preserves insulin crystals and facilitates insulin monomerisation under certain biochemical conditions. It also activates beta cell potassium channels and moderates insulin secretion. Anti-sulfatide antibodies are seen in type 1 diabetes, and immunological presentation of glycosphingolipids by the non-classical CD1 molecules has recently been reported. It is via this mechanism that alpha-galactosylceramide and sulfatide are able to influence the innate immune system and inhibit autoimmunity, possibly through regulatory natural killer T cells. Administration of sulfatide substantially reduces the incidence of diabetes in non-obese diabetic mice and prevents antigen-induced experimental autoimmune encephalomyelitis in wild-type mice. Sulfatide has specific anti-inflammatory properties, increasing the number of CD3+CD25+ regulatory T cells and reducing production of several cytokines, including TNF-alpha. Patients with type 2 diabetes have low serum concentrations of sulfatide, and some animal models of type 2 diabetes have low pancreatic expression of C16:0 sulfatide; administration of this increases insulin secretion and improves first-phase insulin response in Zucker fatty rats. Glycosphingolipids in general, and sulfatide in particular, appear relevant to both type 1 and type 2 diabetes.

Aims: The glycosphingolipid sulfatide (sulfated galactosyl-ceramide) increases exocytosis of beta-cell secretory granules, activates K(ATP)-channels and is thereby able to influence insulin secretion through its presence in the islets. A closely related compound, sulfated lactosylceramide (sulf-lac-cer), is present in the islets during fetal and neonatal life when, as in Type 2 diabetes, insulin is secreted autonomically without the usual first phase response to glucose. The aim was to examine whether serum concentrations of these glycolipids are associated with Type 2 diabetes., Methods: A case-control study, comprising 286 women and 283 men, was designed using a population-based sample of patients with Type 2 diabetes and a population survey., Results: Low serum concentrations of sulfatide were associated with Type 2 diabetes, independent of traditional risk factors for diabetes in a sex-specific analysis: odds ratio (OR) 2.1 (95% confidence interval 1.1, 3.9) in men, and 2.3 (1.2, 4.3) in women, comparing the lowest and the highest tertiles. Type 2 diabetes was also associated with detectable amounts of sulf-lac-cer in serum: OR 1.7 (0.9, 3.4) in men, and 7.6 (3.8, 15.2) in women. After adjustment for confounding from other diabetes risk factors, these associations remained basically unchanged. The connections between sulfatide and Type 2 diabetes, and sulf-lac-cer and Type 2 diabetes were independent of each other. Insulin resistance (HOMA-IR) was negatively correlated with sulfatide concentration and positively correlated with sulf-lac-cer (both P < 0.0001, independently)., Conclusions: We report a new, robust and highly significant independent association between Type 2 diabetes and serum concentrations of sulfatide in both sexes, and sulf-lac-cer in females. The associations were also independent of other known diabetes risk factors.

Background: Sulfatide is present in the secretory granules of beta cells and has been shown, in vitro, to be involved in insulin processing and secretion. Of particular interest is one of the major sulfatide isoforms in the beta cells, the C16:0 fatty acid isoform, which has been shown to be involved in insulin crystal preservation in vitro. The aim of this study was to investigate the ability of C16:0 fatty acid isoform of sulfatide to affect insulin secretion and/or action and glycaemic control in the adipogenic 'prediabetic' Zucker rat., Methods: The C16:0 sulfatide was administered to Zucker rats for 10 weeks, and fasting levels of plasma insulin and glucose were measured as well as levels after an intravenous (i.v.) glucose load. In addition, the sulfatide expression, examined by thin-layer chromatography-enzyme-linked immunosorbent assay and mass spectrometry, in the pancreas of C16:0 sulfatide-treated Zucker rats was compared to controls., Results: The in vivo treatment of Zucker rats with C16:0 sulfatide resulted in significantly elevated glucose-stimulated insulin secretion (60-80% increase, p < 0.05), without significant changes in glucose tolerance. The treatment was associated with an ameliorated first-phase insulin response (3-4-fold, p = 0.009, 0.016) and a 60% increase of pancreatic sulfatide content (p = 0.001), possible by an uptake of C16:0 sulfatide. The fasting hyperinsulinaemia and blood glucose levels were unchanged., Conclusions: The treatment with C16:0 sulfatide elevates glucose-stimulated insulin secretion and enhances sulfatide content in the pancreas of Zucker rats., (Copyright 2004 John Wiley & Sons, Ltd.)

Sulfatide (3'-sulfogalactosyl-ceramide) is a glycosphingolipid mainly located in the nervous system, but has also been found in the islets of Langerhans. Previous studies have suggested that sulfatide is involved in insulin processing and secretion. In this study, sulfatide expression and metabolism in pancreas and isolated islets of the type II diabetes models, ob/ob- and db/db mouse, was investigated using TLC-ELISA, metabolic labelling and electron microscopy. As in non-diabetic Lewis rat and human pancreas, sulfatide was located in secretory granules of the beta cells. However, the type II diabetic animal models and their background strains had an altered sulfatide expression, involving the lack of the C16:0 sulfatide fatty acid isoform, compared to non-diabetic Lewis rat, BALB/c mouse and human pancreatic tissue, in which the two dominating pancreatic sulfatide isoforms C16:0 and C24:0 are expressed. Correspondingly, in isolated ob/ob islets, sulfatide synthesis excluded the production of C16:0 sulfatide. Insulin administration to ob/ob mouse, which lowers beta cell activity, resulted in significantly increased sulfatide expression in pancreas (p=0.0003), but still no expression of the C16:0 sulfatide isoform. In vitro, the C16:0 sulfatide was shown to be the isomer involved in the preservation of insulin crystals. Thus, it is hypothesized that the selection of sulfatide isomers in pancreas might be a genetic factor contributing to disease development in type II diabetic animal models.

Previous studies have shown that sulfatide is present and functionally involved in beta cells, and that anti-sulfatide antibodies (ASA) exist during development of type I diabetes mellitus. To further explore the possible role of sulfatide in type I diabetes, developmental expression was examined in human pancreas and in pancreas of the type I diabetes models BB rat and NOD mouse compared to Lewis rat and BALB/c mouse, respectively. Sulfatide was not only expressed in adult pancreas, but also in human fetal and rodent neonatal pancreas, i.e., during the growing period of the immunological self. Sulfatide had a different expression pattern in human beings and rodents, concerning both the amounts of sulfatide and expression during development. There was no change in the sulfatide fatty acid isoform expression during development. The pancreatic expression of another sulfated glycosphingolipid, sulfated lactosylceramide, indicated that this molecule is a potential fetal/neonatal marker, which was further expressed in the type I diabetic models. In conclusion, these findings give further support to the possibility that sulfatide is a relevant autoantigen in type I diabetes and that sulfated lactosylceramide might function as a potential risk factor for disease development, at least in the animal models., (Copyright 2003 Wiley-Liss, Inc.)

Previous studies using pancreas from various mammals and freshly isolated islets from rat pancreas have provided evidence supporting possible involvement of the glycosphingolipid sulfatide in insulin processing and secretion. In this study, sulfatide expression and metabolism in the beta cell line RINr1046-38 (RIN-38), commonly used as a model for beta cell functional studies, were investigated and compared with previous findings from freshly isolated islets. RIN-38 cells expressed similar amounts (2.7 +/- 1.1 nmol/mg protein, n = 19) of sulfatide as isolated rat islets and also followed the same metabolic pathway, mainly through recycling. Moreover, in agreement with findings in isolated islets, the major species of sulfatide isolated from RIN-38 cells contained C16:0 and C24:0 fatty acids. By applying subcellular isolations and electron microscopy and immunocytochemistry techniques, sulfatide was shown to be located to the secretory granules, the plasma membrane and enriched in detergent insoluble microdomains. In the electron microscopy studies, Sulph I staining was also associated with mitochondria and villi structures. In conclusion, RIN-38 cells might be an appropriate model, as a complement to isolated islets where the amount of material often limits the experiments, to further explore the role of sulfatide in insulin secretion and signal transduction of beta cells.