Nicolas Coant

Ex vivo effects of high‐density lipoprotein exposure on the lipopolysaccharide‐induced inflammatory response in patients with severe cirrhosis

High‐density lipoproteins (HDL) are known to neutralize lipopolysaccharide (LPS). Because patients with cirrhosis have lower HDL levels, this may contribute to LPS‐induced ex vivo monocyte overproduction of proinflammatory cytokines. However, the effects of HDL on cytokine production by monocytes from patients with cirrhosis have never been studied. The aim of this study was to determine the effects of HDL on LPS‐induced proinflammatory cytokine production in whole blood and isolated monocytes from patients with severe cirrhosis and controls. Plasma levels of HDL and cytokines were determined. The effects of reconstituted HDL (rHDL) on LPS‐induced cytokine production in whole blood were assessed by cytokine array and on tumor necrosis factor alpha (TNF‐α) and interleukin‐10 (IL‐10) production in isolated monocytes. Plasma HDL levels were significantly lower in patients with cirrhosis than in controls. Plasma levels of TNF‐α and IL‐6 were significantly higher in patients with cirrhosis than in controls. Incubation of rHDL with whole blood prevented LPS‐induced TNF‐α and IL‐6 overproduction in patients with cirrhosis. LPS‐induced TNF‐α production and CD14 expression were significantly more marked in cirrhotic monocytes than in control monocytes, and both decreased significantly after rHDL incubation. LPS‐induced down‐regulation of scavenger receptor, class B, type I (SR‐BI) expression was abolished in cirrhotic monocytes. Conclusions: This study shows that rHDL abolishes the LPS‐induced overproduction of proinflammatory cytokines in whole blood from patients with severe cirrhosis. These results were confirmed in isolated monocytes from these patients. This suggests that administration of rHDL might be a useful strategy for the treatment of cirrhosis to limit LPS‐induced cytokine overproduction. (HEPATOLOGY 2009;49:175‐184.)

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.

Hippocampal lipoprotein lipase regulates energy balance in rodents

Brain lipid sensing is necessary to regulate energy balance. Lipoprotein lipase (LPL) may play a role in this process. We tested if hippocampal LPL regulated energy homeostasis in rodents by specifically attenuating LPL activity in the hippocampus of rats and mice, either by infusing a pharmacological inhibitor (tyloxapol), or using a genetic approach (adeno-associated virus expressing Cre-GFP injected into Lpl (lox/lox) mice). Decreased LPL activity by either method led to increased body weight gain due to decreased locomotor activity and energy expenditure, concomitant with increased parasympathetic tone (unchanged food intake). Decreased LPL activity in both models was associated with increased de novo ceramide synthesis and neurogenesis in the hippocampus, while intrahippocampal infusion of de novo ceramide synthesis inhibitor myriocin completely prevented body weight gain. We conclude that hippocampal lipid sensing might represent a core mechanism for energy homeostasis regulation through de novo ceramide synthesis.

Glucolipotoxicity Impairs Ceramide Flow from the Endoplasmic Reticulum to the Golgi Apparatus in INS-1 β-Cells

Accumulating evidence suggests that glucolipotoxicity, arising from the combined actions of elevated glucose and free fatty acid levels, acts as a key pathogenic component in type II diabetes, contributing to β-cell dysfunction and death. Endoplasmic reticulum (ER) stress is among the molecular pathways and regulators involved in these negative effects, and ceramide accumulation due to glucolipotoxicity can be associated with the induction of ER stress. Increased levels of ceramide in ER may be due to enhanced ceramide biosynthesis and/or decreased ceramide utilization. Here, we studied the effect of glucolipotoxic conditions on ceramide traffic in INS-1 cells in order to gain insights into the molecular mechanism(s) of glucolipotoxicity. We showed that glucolipotoxicity inhibited ceramide utilization for complex sphingolipid biosynthesis, thereby reducing the flow of ceramide from the ER to Golgi. Glucolipotoxicity impaired both vesicular- and CERT-mediated ceramide transport through (1) the decreasing of phospho-Akt levels which in turn possibly inhibits vesicular traffic, and (2) the reducing of the amount of active CERT mainly due to a lower protein levels and increased protein phosphorylation to prevent its localization to the Golgi. In conclusion, our findings provide evidence that glucolipotoxicity-induced ceramide overload in the ER, arising from a defect in ceramide trafficking may be a mechanism that contributes to dysfunction and/or death of β-cells exposed to glucolipotoxicity.

Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells

Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.

Neutral ceramidase: Advances in mechanisms, cell regulation, and roles in cancer

Extensive research conducted in the last three decades has identified the roles for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) as key regulators of cellular homeostasis, growth and death. One of the major groups of enzymes in the ceramide pathway, ceramidases, converts ceramide into sphingosine and fatty acids, with sphingosine being further metabolized to S1P. Thus, these enzymes play important roles in the network controlling the functions associated with these bioactive sphingolipids. Among the family of ceramidases, neutral ceramidase (nCDase), which is named according to its optimal pH for catalytic activity, has received increased attention in the last decade. The goal of this review is to provide a brief background on bioactive sphingolipids and the ceramidases. We then describe more recent advances on nCDase, specifically the resolution of its crystal structure and understanding its roles in cell biology and physiology.

PO21 La gluco-lipotoxicité inhibe le transport des céramides entre le réticulum endoplasmique et l’appareil de Golgi dans les cellules beta pancréatiques

Introduction Au cours du diabete de type 2, les effets deleteres des acides gras satures sur la survie des cellules beta pancreatiques est potentialisee par l’hyperglycemie. Cette action deletere combinee des acides gras et du glucose sur les cellules beta est connue sous le terme de gluco-lipotoxicite. Les mecanismes moleculaires impliques dans les effets deleteres des acides gras comme le palmitate inclus en particulier la synthese de novo des ceramides. L’augmentation des taux de ceramides dans le reticulum endoplasmique (RE) peut etre due a la fois a une augmentation de la biosynthese des ceramides et/ou a une diminution de leur utilisation. Au cours de cette etude, nous avons donc recherche si la glucolipotoxicite pouvait reguler le trafic des ceramides entre le RE et l’appareil de Golgi dans les cellules beta Materiels et methodes Le metabolisme de la 3H-Sphingosine, precurseur de la biosynthese des sphingolipides a ete mesure dans la lignee beta pancreatique INS-1 traitee avec du palmitate et des concentrations variables de glucose. Le transport des C5-Bodipy-ceramides entre le RE et l’appareil de Golgi a ete mesure par microscopie a fluorescence. Resultats La gluco-lipotoxicite inhibe l’utilisation des ceramides pour la biosynthese des sphingolipides complexes, comme la sphingomyeline. De facon interessante, la gluco-lipotoxicite inhibe le transport vesiculaire des ceramides mais egalement celui medie par la proteine CERT. L’inhibition du transport vesiculaire semble etre due a une inhibition de la voie Akt alors que l’inhibition de CERT est mediee par la diminution de son expression et sa phosphorylation. Enfin, l’inhibition de l’expression de CERT potentialise l’apoptose des cellules beta induite par la gluco-lipotoxicite. Conclusion Nos resultats mettent en evidence que la gluco-lipotoxicite induit une accumulation de ceramides en bloquant le transport des ceramides entre le RE et l’appareil de Golgi. De facon importante, ce dernier mecanisme semble contribuer aux effets deleteres de la gluco-lipotoxicite.