Ceramide analog C2-cer induces a loss in insulin sensitivity in muscle cells through the salvage/recycling pathway.

Ceramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes.

Identification of a Gain-of-Function LIPC Variant as a Novel Cause of Familial Combined Hypocholesterolemia.

BACKGROUND: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3, has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family. METHODS: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC-E97G overexpression. RESULTS: Family members carrying the LIPC-E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC-E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC-E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC-E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver. CONCLUSIONS: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3, causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.

Plasma ceramides are associated with MRI-based liver fat content but not with noninvasive scores of liver fibrosis in patients with type 2 diabetes.

BACKGROUND: There is growing evidence that ceramides play a significant role in the onset and progression of non-alcoholic fatty liver disease (NAFLD), a highly prevalent condition in patients with type 2 diabetes associated with hepatic and cardiovascular events. However, the relationship between plasma ceramide levels and NAFLD severity in type 2 diabetes remains unclear. The main purpose of the present study was to investigate whether circulating levels of ceramides in patients with type 2 diabetes are associated with liver steatosis assessed by the highly accurate magnetic resonance imaging proton density fat fraction (MRI-PDFF). The secondary objective was to assess the relationship between plasma ceramides and noninvasive scores of liver fibrosis. METHODS: In this cross-sectional single-center study, plasma concentrations of 7 ceramides were measured by liquid chromatography-mass spectrometry in 255 patients with type 2 diabetes (GEPSAD cohort). Liver fat content was assessed by MRI-PDFF, and noninvasive scores of liver fibrosis (i.e. Fibrosis-4 index, NAFLD Fibrosis Score, FibroTest® and Fibrotic NASH Index) were calculated. A validation cohort of 80 patients with type 2 diabetes was also studied (LIRA-NAFLD cohort). RESULTS: Liver steatosis, defined as a liver fat content > 5.56%, was found in 62.4 and 82.5% of individuals with type 2 diabetes in the GEPSAD and LIRA-NAFLD cohorts, respectively. In GEPSAD, MRI-PDFF-measured liver fat content was positively associated with plasma levels of total ceramides (r = 0.232, p = 0.0002), and 18:0, 20:0, 22:0 and 24:0 ceramides in univariate analysis (p ≤ 0.0003 for all). In multivariate analysis, liver fat content remained significantly associated with total ceramides (p = 0.001), 18:0 (p = 0.006), 22:0 (p = 0.0009) and 24:0 ceramides (p = 0.0001) in GEPSAD, independently of age, diabetes duration, body mass index and dyslipidemia. Overall, similar relationship between plasma ceramides and liver fat content was observed in the LIRA-NAFLD validation cohort. No significant association was found between plasma ceramides and noninvasive scores of fibrosis after adjustment for age in both cohorts. CONCLUSIONS: Plasma ceramide levels are associated with liver steatosis in patients with type 2 diabetes, independently of traditional risk factors for NAFLD. The independent association between plasma ceramides and liver steatosis adds new insights regarding the relationship between ceramides and NAFLD in type 2 diabetes.

Liraglutide reduces plasma dihydroceramide levels in patients with type 2 diabetes.

BACKGROUND: Emerging evidence supports that dihydroceramides (DhCer) and ceramides (Cer) contribute to the pathophysiology of insulin resistance and liver steatosis, and that their circulating concentrations are independently associated with cardiovascular outcomes. Circulating DhCer levels are increased in patients with type 2 diabetes (T2D). On the other hand, the GLP-1 receptor agonist liraglutide reduces major adverse cardiac events, insulin resistance and liver steatosis in T2D patients. The main purpose of the present study was therefore to investigate whether liraglutide decreases circulating levels of DhCer and Cer in T2D patients, which could be a mechanism involved in its cardiometabolic benefits. The secondary purpose was to assess the relationship between liraglutide-induced changes in DhCer/Cer levels and insulin resistance and liver steatosis. METHODS: Plasma concentrations of 11 DhCer and 15 Cer species were measured by a highly-sensitive mass spectrometry system in 35 controls and 86 T2D patients before and after 6 months of liraglutide (1.2 mg/day). Insulin resistance was estimated by the triglyceride-glucose (TyG) index. Liver fat content (LFC) was assessed in 53 patients by proton magnetic resonance spectroscopy. RESULTS: Plasma levels of total DhCer, 7 DhCer and 7 Cer species were increased in T2D patients compared to controls. Liraglutide decreased total DhCer by 15.1% (p = 0.005), affecting 16:0 (p = 0.037), 18:0 (p < 0.0001), 18:1 (p = 0.0005), 20:0 (p = 0.0003), 23:0 (p = 0.005) and 24:1 (p = 0.04) species. Total plasma Cer did not significantly change after liraglutide (p = 0.18), but 5 Cer species decreased significantly, i.e. 18:0 and 18:1 (both p < 0.0001), 19:0 and 24:1 (both p < 0.01) and 26:1 (p = 0.04). In multivariate analysis, the reduction in DhCer after liraglutide was independently associated with the reduction in LFC (p = 0.0005) and in TyG index (p = 0.05). CONCLUSIONS: Liraglutide reduces plasma levels of numerous DhCer and Cer species in T2D patients, which may contribute to the cardiovascular benefit observed in the LEADER trial. The independent association between the decrease in plasma DhCer level with the reduction in LFC and TyG index adds new insights regarding the relationship between DhCer, liver steatosis and insulin resistance. Trial registration ClinicalTrials.gov identifier: NCT02721888.

Comparative Evaluation of Two Paclitaxel-Coated Stents in an Experimental Setting.

INTRODUCTION: Unlike paclitaxel-coated balloons, pre-clinical data comparing different paclitaxel-coated stents (PCSs) are weak. The study objective was to compare the features of the 2 main PCSs: Eluvia® (Boston Scientific, Marlborough, MA) versus ZilverPTX® (Cook Medical, Bloomington, IN). METHOD: Analysis was carried out on 12 pigs divided into 2 groups: Eluvia® (n=6) and ZilverPTX® (n=6). The pigs received the PCS corresponding to their group in each external iliac artery and were paired one by one, to examine 6 different post-implantation timepoints: after 30 minutes, 6 hours, 24 hours, 3 days, 7 days, and 14 days. The paclitaxel concentration measurements and the histological analysis were carried out under blind testing on the plasma, arterial, lymph node, and muscle samples. A linear regression model and Wilcoxon Mann-Whitney test were used to study the variables. RESULTS: The plasma paclitaxel rate decrease over 24 hours after PCS implantation was significantly different between the two groups, expressed by the correlation coefficient 0.19 (0.14-0.23; p<0.001) with an undetectable concentration at the 10th hour for Eluvia® versus 3 days for ZilverPTX®. Significantly higher paclitaxel concentrations with ZilverPTX® PCS were observed in muscle samples at each timepoint: extensor digitorum brevis 3.2 (1.17-5.23; p=0.005), biceps femoris 4.27 (2.27-6.26; p<0.001), semi-tendinosus 3.79 (1.85-5.73; p=0.001), tibialis anterior 3.0 (1.37-4.64; p=0.001), and in the femoral nodes 2.27±1.74 ng/g versus 0.14±0.13 ng/g (p<0.001). Histological analysis revealed a trend for more marked intimal inflammation in the arteries stented with ZilverPTX® (p=0.063), especially after the 7th and 14th days. CONCLUSION: Such a difference in the concentration of paclitaxel in the plasma, muscles, and lymph nodes between the two stents was higher than expected based on differences in device design. The clinical consequences of these results remain to be elucidated, particularly regarding the concerning presence of paclitaxel in muscles and adjacent lymph nodes. CLINICAL IMPACT: This experimental study compares 2 paclitaxel-coated stents. It demonstrates that differences in stent designs and drug features (coatings and concentrations) translate into differences in terms of concentrations of paclitaxel in the plasma, muscles, and lymph nodes. Our results favor the Eluvia® stent over the ZilverPTX® stent, although more studies are required to confirm this conclusion.

High-density lipoprotein infusion protects from acute graft-versus-host disease in experimental allogeneic hematopoietic cell transplantation.

Acute graft-versus-host disease (aGVHD) is a major limitation of the therapeutic potential of allogeneic hematopoietic cell transplantation. Lipopolysaccharides (LPS) derived from intestinal gram-negative bacteria are well-known aGVHD triggers and amplifiers. Here, we explored the LPS metabolism in aGVHD mouse models using an innovative quantification method. We demonstrated that systemic LPS accumulation after transplantation was due, at least partly, to a defect in its clearance through lipoprotein-mediated transport to the liver (i.e., the so-called reverse LPS transport). After transplantation, reduced circulating HDL concentration impaired LPS neutralization and elimination through biliary flux. Accordingly, HDL-deficient (Apoa1tm1Unc ) recipient mice developed exacerbated aGVHD. Repeated administration of HDL isolated from human plasma significantly decreased the mortality and the severity of aGVHD. While the potential role of HDL in scavenging circulating LPS was examined in this study, it appears that HDL plays a more direct immunomodulatory role by limiting or controlling aGVHD. Notably, HDL infusion mitigated liver aGVHD by diminishing immune infiltration (e.g., interferon-γ-secreting CD8+ T cells and non-resident macrophages), systemic and local inflammation (notably cholangitis). Hence, our results revealed the interest of HDL-based therapies in the prevention of aGVHD.

Increased Weight Gain and Insulin Resistance in HF-Fed PLTP Deficient Mice Is Related to Altered Inflammatory Response and Plasma Transport of Gut-Derived LPS.

Bacterial lipopolysaccharides (LPS, endotoxins) are found in high amounts in the gut lumen. LPS can cross the gut barrier and pass into the blood (endotoxemia), leading to low-grade inflammation, a common scheme in metabolic diseases. Phospholipid transfer protein (PLTP) can transfer circulating LPS to plasma lipoproteins, thereby promoting its detoxification. However, the impact of PLTP on the metabolic fate and biological effects of gut-derived LPS is unknown. This study aimed to investigate the influence of PLTP on low-grade inflammation, obesity and insulin resistance in relationship with LPS intestinal translocation and metabolic endotoxemia. Wild-type (WT) mice were compared with Pltp-deficient mice (Pltp-KO) after a 4-month high-fat (HF) diet or oral administration of labeled LPS. On a HF diet, Pltp-KO mice showed increased weight gain, adiposity, insulin resistance, lipid abnormalities and inflammation, together with a higher exposure to endotoxemia compared to WT mice. After oral administration of LPS, PLTP deficiency led to increased intestinal translocation and decreased association of LPS to lipoproteins, together with an altered catabolism of triglyceride-rich lipoproteins (TRL). Our results show that PLTP, by modulating the intestinal translocation of LPS and plasma processing of TRL-bound LPS, has a major impact on low-grade inflammation and the onset of diet-induced metabolic disorders.

Chemical Synthesis, Pharmacokinetic Properties and Biological Effects of JM-00266, a Putative Non-Brain Penetrant Cannabinoid Receptor 1 Inverse Agonist.

Targeting cannabinoid 1 receptors (CB1R) with peripherally restricted antagonists (or inverse agonists) shows promise to improve metabolic disorders associated with obesity. In this context, we designed and synthetized JM-00266, a new CB1R blocker with limited blood-brain barrier (BBB) permeability. Pharmacokinetics were tested with SwissADME and in vivo in rodents after oral and intraperitoneal administration of JM-00266 in comparison with Rimonabant. In silico predictions indicated JM-00266 is a non-brain penetrant compound and this was confirmed by brain/plasma ratios and brain uptake index values. JM-00266 had no impact on food intake, anxiety-related behavior and body temperature suggesting an absence of central activity. cAMP assays performed in CB1R-transfected HEK293T/17 cells showed that the drug exhibited inverse agonist activity on CB1R. In addition, JM-00266 counteracted anandamide-induced gastroparesis indicating substantial peripheral activity. Acute administration of JM-00266 also improved glucose tolerance and insulin sensitivity in wild-type mice, but not in CB1R-/- mice. Furthermore, the accumulation of JM-00266 in adipose tissue was associated with an increase in lipolysis. In conclusion, JM-00266 or derivatives can be predicted as a new candidate for modulating peripheral endocannabinoid activity and improving obesity-related metabolic disorders.

Human cholesteryl ester transfer protein lacks lipopolysaccharide transfer activity, but worsens inflammation and sepsis outcomes in mice.

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

Deletion of lysophosphatidylcholine acyltransferase 3 in myeloid cells worsens hepatic steatosis after a high-fat diet.

Recent studies have highlighted an important role for lysophosphatidylcholine acyltransferase 3 (LPCAT3) in controlling the PUFA composition of cell membranes in the liver and intestine. In these organs, LPCAT3 critically supports cell-membrane-associated processes such as lipid absorption or lipoprotein secretion. However, the role of LPCAT3 in macrophages remains controversial. Here, we investigated LPCAT3's role in macrophages both in vitro and in vivo in mice with atherosclerosis and obesity. To accomplish this, we used the LysMCre strategy to develop a mouse model with conditional Lpcat3 deficiency in myeloid cells (Lpcat3KOMac). We observed that partial Lpcat3 deficiency (approximately 75% reduction) in macrophages alters the PUFA composition of all phospholipid (PL) subclasses, including phosphatidylinositols and phosphatidylserines. A reduced incorporation of C20 PUFAs (mainly arachidonic acid [AA]) into PLs was associated with a redistribution of these FAs toward other cellular lipids such as cholesteryl esters. Lpcat3 deficiency had no obvious impact on macrophage inflammatory response or endoplasmic reticulum (ER) stress; however, Lpcat3KOMac macrophages exhibited a reduction in cholesterol efflux in vitro. In vivo, myeloid Lpcat3 deficiency did not affect atherosclerosis development in LDL receptor deficient mouse (Ldlr-/-) mice. Lpcat3KOMac mice on a high-fat diet displayed a mild increase in hepatic steatosis associated with alterations in several liver metabolic pathways and in liver eicosanoid composition. We conclude that alterations in AA metabolism along with myeloid Lpcat3 deficiency may secondarily affect AA homeostasis in the whole liver, leading to metabolic disorders and triglyceride accumulation.

VEGF-R2/Caveolin-1 Pathway of Undifferentiated ARPE-19 Retina Cells: A Potential Target as Anti-VEGF-A Therapy in Wet AMD by Resvega, an Omega-3/Polyphenol Combination.

Age-related macular degeneration (AMD) is one of the main causes of deterioration in vision in adults aged 55 and older. In spite of therapies, the progression of the disease is often observed without reverse vision quality. In the present study, we explored whether, in undifferentiated ARPE-19 retinal cells, a disruption of the VEGF receptors (VEGF-R)/caveolin-1 (Cav-1)/protein kinases pathway could be a target for counteracting VEGF secretion. We highlight that Resvega®, a combination of omega-3 fatty acids with an antioxidant, resveratrol, inhibits VEGF-A secretion in vitro by disrupting the dissociation of the VEGF-R2/Cav-1 complex into rafts and subsequently preventing MAPK activation. Moreover, DNA ChIP analysis reveals that this combination prevents the interaction between AP-1 and vegf-a and vegf-r2 gene promoters. By these pathways, Resvega could present a potential interest as nutritional complementation against AMD.

Muricholic Acids Promote Resistance to Hypercholesterolemia in Cholesterol-Fed Mice.

BACKGROUND AND AIMS: Hypercholesterolemia is a major risk factor for atherosclerosis and cardiovascular diseases. Although resistant to hypercholesterolemia, the mouse is a prominent model in cardiovascular research. To assess the contribution of bile acids to this protective phenotype, we explored the impact of a 2-week-long dietary cholesterol overload on cholesterol and bile acid metabolism in mice. METHODS: Bile acid, oxysterol, and cholesterol metabolism and transport were assessed by quantitative real-time PCR, western blotting, GC-MS/MS, or enzymatic assays in the liver, the gut, the kidney, as well as in the feces, the blood, and the urine. RESULTS: Plasma triglycerides and cholesterol levels were unchanged in mice fed a cholesterol-rich diet that contained 100-fold more cholesterol than the standard diet. In the liver, oxysterol-mediated LXR activation stimulated the synthesis of bile acids and in particular increased the levels of hydrophilic muricholic acids, which in turn reduced FXR signaling, as assessed in vivo with Fxr reporter mice. Consequently, biliary and basolateral excretions of bile acids and cholesterol were increased, whereas portal uptake was reduced. Furthermore, we observed a reduction in intestinal and renal bile acid absorption. CONCLUSIONS: These coordinated events are mediated by increased muricholic acid levels which inhibit FXR signaling in favor of LXR and SREBP2 signaling to promote efficient fecal and urinary elimination of cholesterol and neo-synthesized bile acids. Therefore, our data suggest that enhancement of the hydrophilic bile acid pool following a cholesterol overload may contribute to the resistance to hypercholesterolemia in mice. This work paves the way for new therapeutic opportunities using hydrophilic bile acid supplementation to mitigate hypercholesterolemia.

RESVEGA, a Nutraceutical Omega-3/Resveratrol Supplementation, Reduces Angiogenesis in a Preclinical Mouse Model of Choroidal Neovascularization.

Age-related macular degeneration (AMD) is an eye disease that is characterized by damage to the central part of the retina, the macula, and that affects millions of people worldwide. At an advanced stage, a blind spot grows in the center of vision, severely handicapping patients with this degenerative condition. Despite therapeutic advances thanks to the use of anti-VEGF, many resistance mechanisms have been found to accentuate the visual deficit. In the present study, we explored whether supplementation with Resvega®, a nutraceutical formulation composed of omega-3 fatty acids and resveratrol, a well-known polyphenol in grapes, was able to counteract laser-induced choroidal neovascularization (CNV) in mice. We highlight that Resvega® significantly reduced CNV in mice compared with supplementations containing omega-3 or resveratrol alone. Moreover, a proteomic approach confirmed that Resvega® could counteract the progression of AMD through a pleiotropic effect targeting key regulators of neoangiogenesis in retina cells in vivo. These events were associated with an accumulation of resveratrol metabolites within the retina. Therefore, a supplementation of omega-3/resveratrol could improve the management or slow the progression of AMD in patients with this condition.

LDL apheresis as an alternate method for plasma LPS purification in healthy volunteers and dyslipidemic and septic patients.

Lipopolysaccharide (LPS) is a key player for innate immunity activation. It is therefore a prime target for sepsis treatment, as antibiotics are not sufficient to improve outcome during septic shock. An extracorporeal removal method by polymyxin (PMX) B direct hemoperfusion (PMX-DHP) is used in Japan, but recent trials failed to show a significant lowering of circulating LPS levels after PMX-DHP therapy. PMX-DHP has a direct effect on LPS molecules. However, LPS is not present in a free form in the circulation, as it is mainly carried by lipoproteins, including LDLs. Lipoproteins are critical for physiological LPS clearance, as LPSs are carried by LDLs to the liver for elimination. We hypothesized that LDL apheresis could be an alternate method for LPS removal. First, we demonstrated in vitro that LDL apheresis microbeads are almost as efficient as PMX beads to reduce LPS concentration in LPS-spiked human plasma, whereas it is not active in PBS. We found that PMX was also adsorbing lipoproteins, although less specifically. Then, we found that endogenous LPS of patients treated by LDL apheresis for familial hypercholesterolemia is also removed during their LDL apheresis sessions, with both electrostatic-based devices and filtration devices. Finally, LPS circulating in the plasma of septic shock and severe sepsis patients with gram-negative bacteremia was also removed in vitro by LDL adsorption. Overall, these results underline the importance of lipoproteins for LPS clearance, making them a prime target to study and treat endotoxemia-related conditions.

IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis.

BACKGROUND & AIMS: Severe alcoholic hepatitis (SAH) is associated with a high risk of infection. The IL-33/ST2 pathway is involved in sepsis control but data regarding its role in alcohol-related liver disease (ALD) are lacking. We aimed to characterize the role of IL-33/ST2 in the polymorphonuclear neutrophils (PMNs) of patients with ALD and SAH. METHODS: Serum and circulating neutrophils were collected from patients with SAH, alcoholic cirrhosis and healthy controls. We quantified IL-33/ST2 pathway activity and CXCR2 at baseline and after exposure to IL-33. We also determined the migration capacity of PMNs. RESULTS: The decoy receptor of IL-33 (soluble ST2 [sST2]) was increased in SAH vs. cirrhosis and controls, demonstrating the defect in this pathway during ALD. The sST2 level was associated with response to treatment, 2-month survival, infection-free survival and probability of infection in SAH. Endotoxemia was weakly correlated with sST2. GRK2, a negative regulator of CXCR2, was overexpressed in PMNs of patients with SAH and cirrhosis and was decreased by IL-33. CXCR2 levels on PMNs were lower in SAH vs. cirrhosis and controls. Treatment with IL-33 partially restored CXCR2 expression in SAH and cirrhosis. PMN migration upon IL-8 was lower in patients with SAH and cirrhosis vs. controls. Treatment with IL-33 partially restored migration in those with SAH and cirrhosis. Interestingly, the migration capacity of PMNs and the response to IL-33 were enhanced in responders to corticosteroids (Lille <0.45) compared to non-responders. CONCLUSION: The IL33/ST2 pathway is defective in SAH and predicts outcome. This defect is associated with decreased CXCR2 expression on the surface of PMNs and lower migration capacity, which can be corrected by IL-33, especially in patients responding to steroids. These results suggest that IL-33 has therapeutic potential for SAH and its infectious complications. LAY SUMMARY: The neutrophils of patients with severe alcoholic hepatitis are associated with a defect in the IL-33/ST2 pathway. This defect is associated with lower migration capacities in neutrophils and a higher probability of getting infected. Administration of IL-33 to the neutrophils at least partly restores this defect and may be effective at reducing the risk of infection in patients with severe alcoholic hepatitis.

Specific shifts in the endocannabinoid system in hibernating brown bears.

In small hibernators, global downregulation of the endocannabinoid system (ECS), which is involved in modulating neuronal signaling, feeding behavior, energy metabolism, and circannual rhythms, has been reported to possibly drive physiological adaptation to the hibernating state. In hibernating brown bears (Ursus arctos), we hypothesized that beyond an overall suppression of the ECS, seasonal shift in endocannabinoids compounds could be linked to bear's peculiar features that include hibernation without arousal episodes and capacity to react to external disturbance. We explored circulating lipids in serum and the ECS in plasma and metabolically active tissues in free-ranging subadult Scandinavian brown bears when both active and hibernating. In winter bear serum, in addition to a 2-fold increase in total fatty acid concentration, we found significant changes in relative proportions of circulating fatty acids, such as a 2-fold increase in docosahexaenoic acid C22:6 n-3 and a decrease in arachidonic acid C20:4 n-6. In adipose and muscle tissues of hibernating bears, we found significant lower concentrations of 2-arachidonoylglycerol (2-AG), a major ligand of cannabinoid receptors 1 (CB1) and 2 (CB2). Lower mRNA level for genes encoding CB1 and CB2 were also found in winter muscle and adipose tissue, respectively. The observed reduction in ECS tone may promote fatty acid mobilization from body fat stores, and favor carbohydrate metabolism in skeletal muscle of hibernating bears. Additionally, high circulating level of the endocannabinoid-like compound N-oleoylethanolamide (OEA) in winter could favor lipolysis and fatty acid oxidation in peripheral tissues. We also speculated on a role of OEA in the conservation of an anorexigenic signal and in the maintenance of torpor during hibernation, while sustaining the capacity of bears to sense stimuli from the environment.

CXCL10 could drive longer duration of mechanical ventilation during COVID-19 ARDS.

BACKGROUND: COVID-19-related ARDS has unique features when compared with ARDS from other origins, suggesting a distinctive inflammatory pathogenesis. Data regarding the host response within the lung are sparse. The objective is to compare alveolar and systemic inflammation response patterns, mitochondrial alarmin release, and outcomes according to ARDS etiology (i.e., COVID-19 vs. non-COVID-19). METHODS: Bronchoalveolar lavage fluid and plasma were obtained from 7 control, 7 non-COVID-19 ARDS, and 14 COVID-19 ARDS patients. Clinical data, plasma, and epithelial lining fluid (ELF) concentrations of 45 inflammatory mediators and cell-free mitochondrial DNA were measured and compared. RESULTS: COVID-19 ARDS patients required mechanical ventilation (MV) for significantly longer, even after adjustment for potential confounders. There was a trend toward higher concentrations of plasma CCL5, CXCL2, CXCL10, CD40 ligand, IL-10, and GM-CSF, and ELF concentrations of CXCL1, CXCL10, granzyme B, TRAIL, and EGF in the COVID-19 ARDS group compared with the non-COVID-19 ARDS group. Plasma and ELF CXCL10 concentrations were independently associated with the number of ventilator-free days, without correlation between ELF CXCL-10 and viral load. Mitochondrial DNA plasma and ELF concentrations were elevated in all ARDS patients, with no differences between the two groups. ELF concentrations of mitochondrial DNA were correlated with alveolar cell counts, as well as IL-8 and IL-1ß concentrations. CONCLUSION: CXCL10 could be one key mediator involved in the dysregulated immune response. It should be evaluated as a candidate biomarker that may predict the duration of MV in COVID-19 ARDS patients. Targeting the CXCL10-CXCR3 axis could also be considered as a new therapeutic approach. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03955887.

Circulating levels of 3-hydroxymyristate, a direct quantification of endotoxaemia in noninfected cirrhotic patients.

BACKGROUND & AIMS: The quantification of lipopolysaccharide (LPS) in biological fluids is challenging. We aimed to measure plasma LPS concentration using a new method of direct quantification of 3-hydroxymyristate (3-HM), a lipid component of LPS, and to evaluate correlations between 3-HM and markers of liver function, endothelial activation, portal hypertension and enterocyte damage. METHODS: Plasma from 90 noninfected cirrhotic patients (30 Child-Pugh [CP]-A, 30 CP-B, 30 CP-C) was prospectively collected. The concentration of 3-HM was determined by high-performance liquid chromatography coupled with mass spectrometry. RESULTS: 3-HM levels were higher in CP-C patients (CP-A/CP-B/CP-C: 68/70/103 ng/mL, P = 0.005). Patients with severe acute alcoholic hepatitis (n = 16; 113 vs 74 ng/mL, P = 0.012), diabetic patients (n = 22; 99 vs 70 ng/mL, P = 0.028) and those not receiving beta blockers (n = 44; 98 vs 72 ng/mL, P = 0.034) had higher levels of 3-HM. We observed a trend towards higher baseline levels of 3-HM in patients with hepatic encephalopathy (n = 7; 144 vs 76 ng/mL, P = 0.45) or SIRS (n = 10; 106 vs 75 ng/mL, P = 0.114). In multivariate analysis, high levels of 3-HM were associated with CP (OR = 4.39; 95%CI = 1.79-10.76) or MELD (OR = 8.24; 95%CI = 3.19-21.32) scores. Patients dying from liver insufficiency (n = 6) during a 12-month follow-up had higher baseline levels of 3-HM (106 vs 75 ng/mL, P = 0.089). CONCLUSIONS: In noninfected cirrhotic patients, 3-HM arises more frequently with impairment of liver function, heavy alcohol consumption, diabetic status, nonuse of beta blockers and a trend towards poorer outcome is also observed. The direct mass measurement of LPS using 3-HM appears reliable to detect transient endotoxaemia and promising to manage the follow-up of cirrhotic patients.

Liraglutide Reduces Postprandial Hyperlipidemia by Increasing ApoB48 (Apolipoprotein B48) Catabolism and by Reducing ApoB48 Production in Patients With Type 2 Diabetes Mellitus.

Objective- Treatment with liraglutide, a GLP-1 (glucagon-like peptide-1) agonist, has been shown to reduce postprandial lipidemia, an important feature of diabetic dyslipidemia. However, the underlying mechanisms for this effect remain unknown. This prompted us to study the effect of liraglutide on the metabolism of ApoB48 (apolipoprotein B48). Approach and Results- We performed an in vivo kinetic study with stable isotopes (D8-valine) in the fed state in 10 patients with type 2 diabetes mellitus before treatment and 6 months after the initiation of treatment with liraglutide (1.2 mg/d). We also evaluated, in mice, the effect of a 1-week liraglutide treatment on postload triglycerides and analysed in vitro on jejunum, the direct effect of liraglutide on the expression of genes involved in the biosynthesis of chylomicron. In diabetic patients, liraglutide treatment induced a dramatic reduction of ApoB48 pool (65±38 versus 162±87 mg; P=0.005) because of a significant decrease in ApoB48 production rate (3.02±1.33 versus 6.14±4.27 mg kg-1 d-1; P=0.009) and a significant increase in ApoB48 fractional catabolic rate (5.12±1.35 versus 3.69±0.75 pool d-1; P=0.005). One-week treatment with liraglutide significantly reduced postload plasma triglycerides in mice and liraglutide, in vitro, reduced the expression of ApoB48, DGAT1 (diacylglycerol O-acyltransferase 1), and MTP (microsomal transfer protein) genes. Conclusions- We show that treatment with liraglutide induces a significant reduction of the ApoB48 pool because of both a reduction of ApoB48 production and an increase in ApoB48 catabolism. In vitro, liraglutide reduces the expression of genes involved in chylomicron synthesis. These effects might benefit cardiovascular health. Clinical Trial Registration- URL: https://www.clinicaltrials.gov . Unique identifier: NCT02721888.

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.