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.

HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution.

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in cell culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members-TFEB, TFE3 and MITF-from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of MTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in cell culture media due to their potentially confounding effects on experimental results.

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.

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.

Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease.

Impaired function of NPC1 or NPC2 lysosomal proteins leads to the intracellular accumulation of unesterified cholesterol, the primary defect underlying Niemann-Pick type C (NPC) disease. In addition, glycosphingolipids (GSLs) accumulate in lysosomes as well. Intralysosomal lipid accumulation triggers the activation of a set of genes, including potential biomarkers. Transcript levels of Gpnmb have been shown to be elevated in various tissues of an NPC mouse model. We speculated that Gpnmb could serve as a marker for visceral lipid accumulation in NPC disease. We report that Gpnmb expression is increased at protein level in macrophages in the viscera of Npc1nih/nih mice. Interestingly, soluble Gpnmb was also found to be increased in murine and NPC patient plasma. Exposure of RAW264.7 macrophages to the NPC-phenotype-inducing drug U18666A also upregulated Gpnmb expression. Inhibition of GSL synthesis with the glucosylceramide synthase (GCS) inhibitor N-butyl-1-deoxynojirimycin prevented U18666A-induced Gpnmb induction and secretion. In summary, we show that Gpnmb is upregulated in NPC mice and patients, most likely due to GSL accumulation.

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.

Disease causing mutations in the TNF and TNFR superfamilies: Focus on molecular mechanisms driving disease.

The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies comprise multidomain proteins with diverse roles in cell activation, proliferation and cell death. These proteins play pivotal roles in the initiation, maintenance and termination of immune responses and have vital roles outside the immune system. The discovery and analysis of diseases associated with mutations in these families has revealed crucial mechanistic details of their normal functions. This review focuses on mutations causing four different diseases, which represent distinct pathological mechanisms that can exist within these superfamilies: autoimmune lymphoproliferative syndrome (ALPS; FAS mutations), common variable immunodeficiency (CVID; TACI mutations), tumor necrosis factor receptor associated periodic syndrome (TRAPS; TNFR1 mutations) and hypohidrotic ectodermal dysplasia (HED; EDA1/EDAR mutations). In particular, we highlight how mutations have revealed information about normal receptor-ligand function and how such studies might direct new therapeutic approaches.