HEPES-buffering of bicarbonate-containing culture medium perturbs lysosomal glucocerebrosidase activity.

Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four N-glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent activity-based probes (ABPs) that covalently label GCase in reaction-based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage-like RAW264.7 cells. Specific attention was paid to the impact of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate-buffered medium. Here, we report how HEPES-buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES-containing medium was also found to reduce maturation of other lysosomal enzymes (α-glucosidase and ß-glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD-patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD.

The Unique Phenotype of Lipid-Laden Macrophages.

Macrophages are key multi-talented cells of the innate immune system and are equipped with receptors involved in damage and pathogen recognition with connected immune response guiding signaling systems. In addition, macrophages have various systems that are involved in the uptake of extracellular and intracellular cargo. The lysosomes in macrophages play a central role in the digestion of all sorts of macromolecules and the entry of nutrients to the cytosol, and, thus, the regulation of endocytic processes and autophagy. Simplistically viewed, two macrophage phenotype extremes exist. On one end of the spectrum, the classically activated pro-inflammatory M1 cells are present, and, on the other end, alternatively activated anti-inflammatory M2 cells. A unique macrophage population arises when lipid accumulation occurs, either caused by flaws in the catabolic machinery, which is observed in lysosomal storage disorders, or as a result of an acquired condition, which is found in multiple sclerosis, obesity, and cardiovascular disease. The accompanying overload causes a unique metabolic activation phenotype, which is discussed here, and, consequently, a unifying phenotype is proposed.

GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice.

The lysosomal storage disease Niemann-Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1-/- mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1-/- liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1-/- liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1-/- liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1-/- hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.

Activity-based probes for functional interrogation of retaining ß-glucuronidases.

Humans express at least two distinct ß-glucuronidase enzymes that are involved in disease: exo-acting ß-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of ß-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states. Unexpectedly, we find that the supposedly inactive HPSE proenzyme proHPSE is also labeled by our ABPs, leading to surprising insights regarding structural relationships between proHPSE, mature HPSE, and their bacterial homologs. Our results demonstrate the application of ß-glucuronidase ABPs in tracking pathologically relevant enzymes and provide a case study of how ABP-driven approaches can lead to discovery of unanticipated structural and biochemical functionality.

Glycoprotein Non-Metastatic Protein B: An Emerging Biomarker for Lysosomal Dysfunction in Macrophages.

Several diseases are caused by inherited defects in lysosomes, the so-called lysosomal storage disorders (LSDs). In some of these LSDs, tissue macrophages transform into prominent storage cells, as is the case in Gaucher disease. Here, macrophages become the characteristic Gaucher cells filled with lysosomes laden with glucosylceramide, because of their impaired enzymatic degradation. Biomarkers of Gaucher cells were actively searched, particularly after the development of costly therapies based on enzyme supplementation and substrate reduction. Proteins selectively expressed by storage macrophages and secreted into the circulation were identified, among which glycoprotein non-metastatic protein B (GPNMB). This review focusses on the emerging potential of GPNMB as a biomarker of stressed macrophages in LSDs as well as in acquired pathologies accompanied by an excessive lysosomal substrate load in macrophages.

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular ß-glucosidases.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading ß-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-ß-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through ß-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.

LIM-only protein FHL2 regulates experimental pulmonary Schistosoma mansoni egg granuloma formation.

LIM-only protein FHL2 is associated with several immune and inflammatory diseases such as arthritis, influenza A virus infection, and lung inflammation. However, the role of FHL2 in macrophage differentiation and in the development of granuloma formation is unknown. Here, we show that expression of FHL2 is induced in mouse bone marrow derived macrophages (BMMs) following stimulation with M2 cytokines such as IL-4 and IL-10. FHL2-knockout (FHL2-KO) BMMs exhibit a proinflammatory M1 phenotype after LPS treatment and display a reduced anti-inflammatory M2 phenotype following IL-4 treatment. Furthermore, thioglycollate-induced migration of macrophages and B cells is enhanced in FHL2-KO mice. To evaluate the importance of FHL2 in the development of pulmonary granuloma formation, FHL2-KO mice were challenged with Schistosoma mansoni eggs. FHL2-KO mice show an enhanced number of granulomas and display decreased expression of Th2 markers and an exacerbated Th1 type of inflammation, characterized by enhanced expression of neutrophil markers and Th1 cytokines. Furthermore, the expression of barrier proteins is reduced in FHL2-KO lung compared to WT. Collectively, these data identify a previously unrecognized role for FHL2 in the pathogenesis of pulmonary granulomatous inflammation, partly through its effect on macrophage polarization, modulation of the Th1/Th2 balance and regulation of permeability in lung.

The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion.

Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury. To further test this, wild-type and S100A9 knockout mice (deficient for S100A8/A9 complex) were subjected to renal I/R. The expression of S100A8/A9 was significantly increased 1 day after I/R and was co-localized with Ly6G (mouse neutrophil marker)-positive cells. These knockout mice displayed similar renal dysfunction and damage and neutrophil influx compared with wild-type mice at this early time point. Interestingly, S100A9 knockout mice displayed altered tissue repair 5 and 10 days post I/R, as reflected by increased renal damage, sustained inflammation, induction of fibrosis, and increased expression of collagens. This coincided with enhanced expression of alternatively activated macrophage (M2) markers, while the expression of classically activated macrophage (M1) markers was comparable. Similarly, S100A9 deficiency affected M2, but not M1 macrophage polarization in vitro. During the repair phase following acute kidney injury, S100A9 deficiency affects M2 macrophages in mice leading to renal fibrosis and damage. Thus, S100A8/A9 plays a crucial part in controlling macrophage-mediated renal repair following I/R.

Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis.

Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

The shifting lipidomic landscape of blood monocytes and neutrophils during pneumonia.

The lipidome of immune cells during infection has remained unexplored, although evidence of the importance of lipids in the context of immunity is mounting. In this study, we performed untargeted lipidomic analysis of blood monocytes and neutrophils from patients hospitalized for pneumonia and age- and sex-matched noninfectious control volunteers. We annotated 521 and 706 lipids in monocytes and neutrophils, respectively, which were normalized to an extensive set of internal standards per lipid class. The cellular lipidomes were profoundly altered in patients, with both common and distinct changes between the cell types. Changes involved every level of the cellular lipidome: differential lipid species, class-wide shifts, and altered saturation patterns. Overall, differential lipids were mainly less abundant in monocytes and more abundant in neutrophils from patients. One month after hospital admission, lipidomic changes were fully resolved in monocytes and partially in neutrophils. Integration of lipidomic and concurrently collected transcriptomic data highlighted altered sphingolipid metabolism in both cell types. Inhibition of ceramide and sphingosine-1-phosphate synthesis in healthy monocytes and neutrophils resulted in blunted cytokine responses upon stimulation with lipopolysaccharide. These data reveal major lipidomic remodeling in immune cells during infection, and link the cellular lipidome to immune functionality.

Microglia in normal appearing white matter of multiple sclerosis are alerted but immunosuppressed.

Little is known about the functional phenotype of microglia in normal appearing white matter (NAWM) of multiple sclerosis (MS), although it may hold valuable clues about mechanisms for lesion development. Therefore, we studied microglia from NAWM obtained post-mortem from controls (n = 25) and MS patients (n = 21) for their phenotype ex vivo and their immune responsiveness in vitro, using a microglia isolation method that omits culture and adherence. By flow cytometry, microglia in MS NAWM displayed elevated CD45 levels and increased size and granularity but were distinct from autologous choroid plexus macrophages by absent or low expression of additional markers, in particular CD206. Flow cytometric analysis of microglia from NAWM of three controls and four MS patients showed alterations in levels of Fc-gamma receptors in MS. In primary microglia from a bigger sample of subjects, analysis of Fc-gamma receptors by quantitative PCR indicated a significant increase in mRNA levels of the inhibitory CD32b isoform in MS NAWM. Despite their changed activation status, microglia from MS NAWM were unresponsive to lipopolysaccharide in vitro. Notably, culture with dexamethasone led to an impaired induction of the inflammation-limiting cytokine CCL18 in microglia from MS NAWM compared with those from control NAWM. Together, these data demonstrate that microglia in MS NAWM are in an alerted state, but display features of immunosuppression. Thus, the activation status of microglia in NAWM of MS patients likely reflects a response to ongoing neuroinflammation, which coincides with upregulation of immunoregulatory molecules to prevent full activation and damage to the vulnerable milieu.

Phenotyping primary human microglia: tight regulation of LPS responsiveness.

Much is still unknown about mechanisms underlying the phenotypical and functional versatility of human microglia. Therefore, we developed a rapid procedure to isolate pure microglia from postmortem human brain tissue and studied their immediate ex vivo phenotype and responses to key inflammatory mediators. Microglia were isolated, along with macrophages from the choroid plexus by tissue dissociation, density gradient separation, and selection with magnetic microbeads. By flow cytometry, microglia were identified by a CD11b(+) CD45(dim) phenotype and a smaller size compared with CD11b(+) CD45(high) macrophages. Interestingly, white matter microglia from donors with peripheral inflammation displayed elevated CD45 levels and increased size and granularity, but were still distinct from macrophages. The phenotype of isolated microglia was further specified by absent surface expression of CD14, CD200 receptor, and mannose receptor (MR, CD206), all of which were markedly expressed by macrophages. Microglia stimulated immediately after isolation with LPS and IFNγ failed to upregulate TNFα or CCR7. Notably, responsiveness to LPS and IFNγ was clearly instigated in microglia after overnight preculture, which coincided with a strong upregulation of CD14. Culture of microglia with IL-4 resulted in the induction of HLA-DR and CCL18 but not MR, whereas culture with dexamethasone did induce MR, in addition to CD163 and CCL18. In conclusion, this study demonstrates phenotypic changes of microglia associated with peripheral inflammation, and reveals tight regulation of responses to LPS and IFNγ as well as distinct microglial responses to IL-4 and glucocorticoids. These findings are of high relevance to studies on human microglia functioning in health and disease.

6-mercaptopurine inhibits atherosclerosis in apolipoprotein e*3-leiden transgenic mice through atheroprotective actions on monocytes and macrophages.

OBJECTIVE: 6-Mercaptopurine (6-MP), the active metabolite of the immunosuppressive prodrug azathioprine, is commonly used in autoimmune diseases and transplant recipients, who are at high risk for cardiovascular disease. Here, we aimed to gain knowledge on the action of 6-MP in atherosclerosis, with a focus on monocytes and macrophages. METHODS AND RESULTS: We demonstrate that 6-MP induces apoptosis of THP-1 monocytes, involving decreased expression of the intrinsic antiapoptotic factors B-cell CLL/Lymphoma-2 (Bcl-2) and Bcl2-like 1 (Bcl-x(L)). In addition, we show that 6-MP decreases expression of the monocyte adhesion molecules platelet endothelial adhesion molecule-1 (PECAM-1) and very late antigen-4 (VLA-4) and inhibits monocyte adhesion. Screening of a panel of cytokines relevant to atherosclerosis revealed that 6-MP robustly inhibits monocyte chemoattractant chemokine-1 (MCP-1) expression in macrophages stimulated with lipopolysaccharide (LPS). Finally, local delivery of 6-MP to the vessel wall, using a drug-eluting cuff, attenuates atherosclerosis in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice (P<0.05). In line with our in vitro data, this inhibition of atherosclerosis by 6-MP was accompanied with decreased lesion monocyte chemoattractant chemokine-1 levels, enhanced vascular apoptosis, and reduced macrophage content. CONCLUSIONS: We report novel, previously unrecognized atheroprotective actions of 6-MP in cultured monocytes/macrophages and in a mouse model of atherosclerosis, providing further insight into the effect of the immunosuppressive drug azathioprine in atherosclerosis.

Glycosphingolipids and insulin resistance.

Glycosphingolipids are structural membrane components, residing largely in the plasma membrane with their sugar-moieties exposed at the cell's surface. In recent times a crucial role for glycosphingolipids in insulin resistance has been proposed. A chronic state of insulin resistance is a rapidly increasing disease condition in Western and developing countries. It is considered to be the major underlying cause of the metabolic syndrome, a combination of metabolic abnormalities that increases the risk for an individual to develop Type 2 diabetes, obesity, cardiovascular disease, polycystic ovary syndrome and nonalcoholic fatty liver disease. As discussed in this chapter, the evidence for a direct regulatory interaction of glycosphingolipids with insulin signaling is still largely indirect. However, the recent finding in animal models that pharmacological reduction of glycosphingolipid biosynthesis ameliorates insulin resistance and prevents some manifestations of metabolic syndrome, supports the view that somehow glycosphingolipids act as critical regulators, Importantly, since reductions in glycosphingolipid biosynthesis have been found to be well tolerated, such approaches may have a therapeutic potential.

Reduction of glycosphingolipid biosynthesis stimulates biliary lipid secretion in mice.

UNLABELLED: Recent reports indicate that glycosphingolipids play an important role in regulation of carbohydrate metabolism. We have shown that the iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM), an inhibitor of the enzyme glucosylceramide synthase, is a potent enhancer of insulin signaling in rodent models for insulin resistance and type 2 diabetes. In this study, we determined whether AMP-DNM also affects lipid homeostasis and, in particular, the reverse cholesterol transport pathway. Treatment of C57BL/6J mice with AMP-DNM for 5 weeks decreased plasma levels of triglycerides and cholesterol by 35%, whereas neutral sterol excretion increased twofold. Secretion of biliary lipid also increased twofold, which resulted in a similar rise in bile flow. This effect was not due to altered expression levels or kinetics of the various export pumps involved in bile formation. However, the bile salt pool size increased and the expression of Cyp7A1 was up-regulated. In vitro experiments using HepG2 hepatoma cell line revealed this to be due to inhibition of fibroblast growth factor-19 (FGF19)-mediated suppression of Cyp7A1 via the FGF receptor. CONCLUSION: Pharmacological modulation of glycosphingolipid metabolism showed surprising effects on lipid homeostasis in C57BL/6J mice. Upon administration of 100 mg AMP-DNM/kg body weight/day, plasma cholesterol and triglyceride levels decreased, biliary lipid secretion doubled and also the endpoint of reverse cholesterol transport, neutral sterol excretion, doubled.

Modulation of glycosphingolipid metabolism significantly improves hepatic insulin sensitivity and reverses hepatic steatosis in mice.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance, and type 2 diabetes. The hyperinsulinemia that occurs as a consequence of insulin resistance is thought to be an important contributor to the development of fatty liver. We have shown that the iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM), an inhibitor of the enzyme glucosylceramide synthase, is a potent enhancer of insulin signaling in rodent models for insulin resistance and type 2 diabetes. The present study was designed to assess the impact of AMP-DNM on insulin levels, liver triglyceride synthesis, and gene expression profile. Treatment of ob/ob mice with AMP-DNM restored insulin signaling in the liver, corrected blood glucose values to levels found in lean mice, and decreased insulin concentration. The expression of sterol regulatory element-binding protein 1c target genes involved in fatty acid synthesis normalized. AMP-DNM treatment significantly reduced liver to body weight ratio and reversed hepatic steatosis, comprising fat as well as inflammatory markers. In addition, AMP-DNM treatment corrected to a large extent the gene expression profile of ob/ob mice livers toward the profile of lean mice. CONCLUSION: Pharmacological lowering of glycosphingolipids with the iminosugar AMP-DNM is a promising approach to restore insulin signaling and improve glucose homeostasis as well as hepatic steatosis.

Lyso-glycosphingolipids: presence and consequences.

Lyso-glycosphingolipids are generated in excess in glycosphingolipid storage disorders. In the course of these pathologies glycosylated sphingolipid species accumulate within lysosomes due to flaws in the respective lipid degrading machinery. Deacylation of accumulating glycosphingolipids drives the formation of lyso-glycosphingolipids. In lysosomal storage diseases such as Gaucher Disease, Fabry Disease, Krabbe disease, GM1 -and GM2 gangliosidosis, Niemann Pick type C and Metachromatic leukodystrophy massive intra-lysosomal glycosphingolipid accumulation occurs. The lysosomal enzyme acid ceramidase generates the deacylated lyso-glycosphingolipid species. This review discusses how the various lyso-glycosphingolipids are synthesized, how they may contribute to abnormal immunity in glycosphingolipid storing lysosomal diseases and what therapeutic opportunities exist.

Increased chitotriosidase activity in serum of leprosy patients: association with bacillary leprosy.

Human phagocyte-specific chitotriosidase is associated with several diseases involving macrophage activation. Since macrophage activation plays an important role in the control of Mycobacterium leprae infection, we studied the association of chitotriosidase with leprosy both in serum and in situ in lesional skin biopsies from patients. Serum samples from 78 Indonesian leprosy patients (39 non-reactional and 39 reactional leprosy patients) and 36 healthy controls (HC) from the same endemic region were investigated. The patients were classified as multibacillary (MB, n=69) or paucibacillary (PB, n=9) based on the bacterial index in slit-skin smears. Thirty-six of the reactional patients had erythema nodosum leprosum (ENL), while only 3 had reversal reaction (RR). Follow-up serum samples after corticosteroid treatment were also obtained from 17 patients with ENL and one with RR. Multibacillary (MB) patients showed increased chitotriosidase activity in serum as compared to paucibacillary (PB) patients and healthy controls. Although no significant difference was observed between reactional and the corresponding non-reactional groups, ENL showed significantly higher chitotriosidase activity as compared to HC. Furthermore, corticosteroid treatment resulted in significant decline of enzyme activity in ENL sera. Chitotriosidase activity correlated with levels of neopterin, another macrophage activation marker, but not with IL-6, IFN-gamma, TNF-alpha and IL-10. Immunohistochemical staining of 6 MB (LL=5, BL=1) lesional skin sections from stored material showed positive staining for chitotriosidase within lipid-laden macrophages suggesting that macrophages are the source of the enzyme detected in serum. Thus, serum chitotriosidase activity is potentially useful in distinguishing MB from PB leprosy and in monitoring response to therapy in ENL.

Adipocytes harbor a glucosylceramide biosynthesis pathway involved in iNKT cell activation.

BACKGROUND: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. RESULTS: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity. CONCLUSION: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes. SIGNIFICANCE: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction. Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes. UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. ß-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion). Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue. Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes.

The Iminosugar AMP-DNM Improves Satiety and Activates Brown Adipose Tissue Through GLP1.

Obesity is taking on worldwide epidemic proportions, yet effective pharmacological agents with long-term efficacy remain unavailable. Previously, we designed the iminosugar N-adamantine-methyloxypentyl-deoxynojirimycin (AMP-DNM), which potently improves glucose homeostasis by lowering excessive glycosphingolipids. Here we show that AMP-DNM promotes satiety and activates brown adipose tissue (BAT) in obese rodents. Moreover, we demonstrate that the mechanism mediating these favorable actions depends on oral, but not central, administration of AMP-DNM, which ultimately stimulates systemic glucagon-like peptide 1 (GLP1) secretion. We evidence an essential role of brain GLP1 receptors (GLP1r), as AMP-DNM fails to promote satiety and activate BAT in mice lacking the brain GLP1r as well as in mice treated intracerebroventricularly with GLP1r antagonist exendin-9. In conclusion, AMP-DNM markedly ameliorates metabolic abnormalities in obese rodents by restoring satiety and activating BAT through central GLP1r, while improving glucose homeostasis by mechanisms independent of central GLP1r.