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.

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.

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.

Induction of Sphk1 activity in obese adipose tissue macrophages promotes survival.

During obesity, adipose tissue macrophages (ATM) are increased in concert with local inflammation and insulin resistance. Since the levels of sphingolipid (SLs) in adipose tissue (AT) are altered during obesity we investigated the potential impact of SLs on ATMs. For this, we first analyzed expression of SL metabolizing genes in ATMs isolated from obese mice. A marked induction of sphingosine kinase 1 (Sphk1) expression was observed in obese ATM when compared to lean ATM. This induction was observed in both MGL-ve (M1) and MGL1+ve (M2) macrophages from obese WAT. Next, RAW264.7 cells were exposed to excessive palmitate, resulting in a similar induction of Sphk1. This Sphk1 induction was also observed when cells were treated with chloroquine, a lysosomotropic amine impacting lysosome function. Simultaneous incubation of RAW cells with palmitate and the Sphk1 inhibitor SK1-I promoted cell death, suggesting a protective role of Sphk1 during lipotoxic conditions. Interestingly, a reduction of endoplasmic reticulum (ER) stress related genes was detected in obese ATM and was found to be associated with elevated Sphk1 expression. Altogether, our data suggest that lipid overload in ATM induces Sphk1, which promotes cell viability.

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.

Elevation of glycoprotein nonmetastatic melanoma protein B in type 1 Gaucher disease patients and mouse models.

Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL18, which are established markers for human Gaucher cells. In Gaucher mice, gpNMB is also produced by Gaucher cells. Correction of glucocerebrosidase deficiency in mice by gene transfer or pharmacological substrate reduction reverses gpNMB abnormalities. In conclusion, gpNMB acts as a marker for glucosylceramide-laden macrophages in man and mouse and gpNMB should be considered as candidate biomarker for Gaucher disease in treatment monitoring.

Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard.

We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of quantitation. Caution is warranted with determination of plasma S1P levels. Earlier it was reported that S1P is elevated in plasma of Fabry disease patients. We investigated this with the improved quantification. No clear conclusion could be drawn for patient plasma samples given the lack of uniformity of blood collection and plasma preparation. To still obtain insight, plasma and tissues were identically collected from α-galactosidase A deficient Fabry mice and matched control animals. No significant difference was observed in plasma S1P levels. A significant 2.3 fold increase was observed in kidney of Fabry mice, but not in liver and heart. Comparative analysis of S1P in cultured fibroblasts from normal subjects and classically affected Fabry disease males revealed no significant difference. In conclusion, accurate quantification of S1P in biological materials is feasible by mass spectrometry using the internal standards (13)C5 C18-S1P or C17-S1P. Significant local increases of S1P in the kidney might occur in Fabry disease as suggested by the mouse model.

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.

Lysosomal stress in obese adipose tissue macrophages contributes to MITF-dependent Gpnmb induction.

In obesity, adipose tissue (AT) contains crown-like structures where macrophages surround nonviable adipocytes. To understand how AT macrophages (ATMs) contribute to development of insulin resistance, we examined their character in more detail. In silico analysis of F2 mouse populations revealed significant correlation between adipose glycoprotein nonmetastatic melanoma protein B (Gpnmb) expression and body weight. In obese mice and obese individuals, Gpnmb expression was induced in ATMs. Cultured RAW264.7 cells were used to obtain insight into the mechanism of Gpnmb regulation. Gpnmb was potently induced by lysosomal stress inducers, including palmitate and chloroquine, or Torin1, an inhibitor of mammalian target of rapamycin complex 1 (mTORC1). These stimuli also provoked microphthalmia transcription factor (MITF) translocation to the nucleus, and knockdown of MITF by short hairpin RNA indicated its absolute requirement for Gpnmb induction. In agreement with our in vitro data, reduced mTORC1 activity was observed in isolated ATMs from obese mice, which coincided with increased nuclear MITF localization and Gpnmb transcription. Aberrant nutrient sensing provokes lysosomal stress, resulting in attenuated mTORC1 activity and enhanced MITF-dependent Gpnmb induction. Our data identify Gpnmb as a novel marker for obesity-induced ATM infiltration and potentiator of interleukin-4 responses and point toward a crucial role for MITF in driving part of the ATM phenotype.

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.

Obesity activates a program of lysosomal-dependent lipid metabolism in adipose tissue macrophages independently of classic activation.

Obesity activates a complex systemic immune response that includes the recruitment of macrophages and other immune cells to key metabolic tissues. Current models postulate that obesity and excess lipids classically activate macrophages, polarizing them toward an M1 (inflammatory) state. Little is known about noninflammatory functions of adipose tissue macrophages (ATMs). Here, we show that the expansion of adipose tissue (AT) across models of obesity induces a program of lysosome biogenesis in ATMs and is associated with lipid catabolism but not a classic inflammatory phenotype. This program is induced by factors produced by AT and is tightly coupled to lipid accumulation by ATMs. Inhibition of ATM lysosome function impairs lipid metabolism and increases lipid content in ATMs and reduces whole AT lipolysis. These data argue that ATMs contribute quantitatively to the development of obesity-induced inflammation but also serve an important role in lipid trafficking independent of their inflammatory phenotype.

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.

The prolactin receptor is expressed in macrophages within human carotid atherosclerotic plaques: a role for prolactin in atherogenesis?

Atherosclerotic vascular disease is the consequence of a chronic inflammatory process, and prolactin has been shown to be a component of the inflammatory response. Additionally, recent studies indicate that prolactin contributes to an atherogenic phenotype. We hypothesized that this may be the result of a direct effect of prolactin on atherogenesis through activation of the prolactin receptor. Human carotid atherosclerotic plaques were obtained from patients by endarteriectomies. The mRNA of prolactin receptor, but not of prolactin, was detected in these atherosclerotic plaques by quantitative real-time PCR. In situ hybridization confirmed the expression of the prolactin receptor in mononuclear cells. Analysis at the protein level using immunohistochemistry and immunoelectron microscopy revealed that the prolactin receptor was abundantly present in macrophages near the lipid core and shoulder regions of the plaques. Our findings demonstrate that the prolactin receptor is present in macrophages of the atherosclerotic plaque at sites of most prominent inflammation. We therefore propose that prolactin receptor signaling contributes to the local inflammatory response within the atherosclerotic plaque and thus to atherogenesis.

Curdlan-mediated regulation of human phagocyte-specific chitotriosidase.

Human phagocyte-specific chitotriosidase is part of innate immunity and shows anti-fungal activity towards chitin-containing fungi. We investigated the effect of stimulation of the C-type lectin receptor dectin-1 by beta-1,3-glucan (curdlan) on chitotriosidase expression and release by human phagocytes. We observed that curdlan triggers chitotriosidase release from human neutrophils. In addition, we show that curdlan impairs chitotriosidase induction in monocytes. Finally, curdlan temporarily induces chitotriosidase in enzyme-expressing monocyte-derived macrophages, followed by reduction of chitotriosidase expression after prolonged stimulation. These data on regulation of phagocyte-specific chitotriosidase following curdlan recognition support an important role of chitotriosidase in the elimination of chitin-containing pathogens.

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.

Differential expression of the EGF-TM7 family members CD97 and EMR2 in lipid-laden macrophages in atherosclerosis, multiple sclerosis and Gaucher disease.

The members of the epidermal growth factor (EGF)-transmembrane (TM)7 family of adhesion class G-protein coupled receptors are abundantly expressed by cells of the myeloid lineage. A detailed investigation of their expression by functional subsets of activated macrophages is still lacking. Therefore, we determined the expression of CD97, EGF module-containing mucin-like receptor (EMR)2 and EMR3 by monocyte-derived macrophages experimentally polarized in vitro. This was compared to three types of disease-associated lipid-laden macrophages displaying an alternatively activated phenotype in situ. Polarization in vitro towards classically activated M1 versus alternatively activated M2 extremes of macrophage activation did not result in a congruent regulation of EGF-TM7 receptor mRNA and protein except for a down-regulation of CD97 by IL-10. In contrast, macrophages handling lipid overload in vivo displayed differences in the expression of CD97 and EMR2. While foamy macrophages in atherosclerotic vessels expressed both CD97 and EMR2, foam cells in multiple sclerosis brain expressed CD97, but only little EMR2. Foam cell formation in vitro by oxidized LDL and myelin did not affect CD97 or EMR2 expression. Gaucher spleen cells accumulating glucosylceramide expressed very high levels of CD97 and EMR2. These findings indicate that complex cellular expression programmes rather than activation modes regulate the expression of EGF-TM7 receptors in macrophages.

Reducing glycosphingolipid content in adipose tissue of obese mice restores insulin sensitivity, adipogenesis and reduces inflammation.

Adipose tissue is a critical mediator in obesity-induced insulin resistance. Previously we have demonstrated that pharmacological lowering of glycosphingolipids and subsequently GM3 by using the iminosugar AMP-DNM, strikingly improves glycemic control. Here we studied the effects of AMP-DNM on adipose tissue function and inflammation in detail to provide an explanation for the observed improved glucose homeostasis. Leptin-deficient obese (Lep(Ob)) mice were fed AMP-DNM and its effects on insulin signalling, adipogenesis and inflammation were monitored in fat tissue. We show that reduction of glycosphingolipid biosynthesis in adipose tissue of Lep(Ob) mice restores insulin signalling in isolated ex vivo insulin-stimulated adipocytes. We observed improved adipogenesis as the number of larger adipocytes was reduced and expression of genes like peroxisome proliferator-activated receptor (PPAR) gamma, insulin responsive glucose transporter (GLUT)-4 and adipsin increased. In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM. As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN). In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals.

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.