Inverse association between apolipoprotein C-II and cardiovascular mortality: role of lipoprotein lipase activity modulation.

AIMS: Apolipoprotein C-II (ApoC-II) is thought to activate lipoprotein lipase (LPL) and is therefore a possible target for treating hypertriglyceridemia. Its relationship with cardiovascular risk has not been investigated in large-scale epidemiologic studies, particularly allowing for apolipoprotein C-III (ApoC-III), an LPL antagonist. Furthermore, the exact mechanism of ApoC-II-mediated LPL activation is unclear. METHODS AND RESULTS: ApoC-II was measured in 3141 LURIC participants of which 590 died from cardiovascular diseases during a median (inter-quartile range) follow-up of 9.9 (8.7-10.7) years. Apolipoprotein C-II-mediated activation of the glycosylphosphatidylinositol high-density lipoprotein binding protein 1 (GPIHBP1)-LPL complex was studied using enzymatic activity assays with fluorometric lipase and very low-density lipoprotein (VLDL) substrates. The mean ApoC-II concentration was 4.5 (2.4) mg/dL. The relationship of ApoC-II quintiles with cardiovascular mortality exhibited a trend toward an inverse J-shape, with the highest risk in the first (lowest) quintile and lowest risk in the middle quintile. Compared with the first quintile, all other quintiles were associated with decreased cardiovascular mortality after multivariate adjustments including ApoC-III as a covariate (all P < 0.05). In experiments using fluorometric substrate-based lipase assays, there was a bell-shaped relationship for the effect of ApoC-II on GPIHBP1-LPL activity when exogenous ApoC-II was added. In ApoC-II-containing VLDL substrate-based lipase assays, GPIHBP1-LPL enzymatic activity was almost completely blocked by a neutralizing anti-ApoC-II antibody. CONCLUSION: The present epidemiologic data suggest that increasing low circulating ApoC-II levels may reduce cardiovascular risk. This conclusion is supported by the observation that optimal ApoC-II concentrations are required for maximal GPIHBP1-LPL enzymatic activity.

Hepatocyte-specific deletion of adipose triglyceride lipase (adipose triglyceride lipase/patatin-like phospholipase domain containing 2) ameliorates dietary induced steatohepatitis in mice.

BACKGROUND AND AIMS: Increased fatty acid (FA) flux from adipose tissue to the liver contributes to the development of NAFLD. Because free FAs are key lipotoxic triggers accelerating disease progression, inhibiting adipose triglyceride lipase (ATGL)/patatin-like phospholipase domain containing 2 (PNPLA2), the main enzyme driving lipolysis, may attenuate steatohepatitis. APPROACH AND RESULTS: Hepatocyte-specific ATGL knockout (ATGL LKO) mice were challenged with methionine-choline-deficient (MCD) or high-fat high-carbohydrate (HFHC) diet. Serum biochemistry, hepatic lipid content and liver histology were assessed. Mechanistically, hepatic gene and protein expression of lipid metabolism, inflammation, fibrosis, apoptosis, and endoplasmic reticulum (ER) stress markers were investigated. DNA binding activity for peroxisome proliferator-activated receptor (PPAR) α and PPARδ was measured. After short hairpin RNA-mediated ATGL knockdown, HepG2 cells were treated with lipopolysaccharide (LPS) or oleic acid:palmitic acid 2:1 (OP21) to explore the direct role of ATGL in inflammation in vitro. On MCD and HFHC challenge, ATGL LKO mice showed reduced PPARα and increased PPARδ DNA binding activity when compared with challenged wild-type (WT) mice. Despite histologically and biochemically pronounced hepatic steatosis, dietary-challenged ATGL LKO mice showed lower hepatic inflammation, reflected by the reduced number of Galectin3/MAC-2 and myeloperoxidase-positive cells and low mRNA expression levels of inflammatory markers (such as IL-1ß and F4/80) when compared with WT mice. In line with this, protein levels of the ER stress markers protein kinase R-like endoplasmic reticulum kinase and inositol-requiring enzyme 1α were reduced in ATGL LKO mice fed with MCD diet. Accordingly, pretreatment of LPS-treated HepG2 cells with the PPARδ agonist GW0742 suppressed mRNA expression of inflammatory markers. Additionally, ATGL knockdown in HepG2 cells attenuated LPS/OP21-induced expression of proinflammatory cytokines and chemokines such as chemokine (C-X-C motif) ligand 5, chemokine (C-C motif) ligand (Ccl) 2, and Ccl5. CONCLUSIONS: Low hepatic lipolysis and increased PPARδ activity in ATGL/PNPLA2 deficiency may counteract hepatic inflammation and ER stress despite increased steatosis. Therefore, lowering hepatocyte lipolysis through ATGL inhibition represents a promising therapeutic strategy for the treatment of steatohepatitis.

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

BACKGROUND AND AIMS: Lipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear. APPROACH AND RESULTS: Wild-type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two-photon microscopy, and the findings in Bsep KO mice were compared to common bile duct-ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS-induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule-dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice. CONCLUSIONS: Absence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

Bsep/Abcb11 knockout ameliorates Schistosoma mansoni liver pathology by reducing parasite fecundity.

BACKGROUND AND AIMS: Schistosoma mansoni infection is one of the worldwide leading causes of liver fibrosis and portal hypertension. The objective of this study was to evaluate whether polyhydroxylated bile acids (BAs), known to protect mice from the development of acquired cholestatic liver injury, counteract S. mansoni-induced inflammation and fibrosis. METHODS: Adult FVB/N wild type (WT) and Abcb11/Bsep-/- mice were infected with either 25 or 50 S. mansoni cercariae. Eight weeks post infection, effects on liver histology, serum biochemistry, gene expression profile of proinflammatory cytokines and fibrotic markers, hepatic hydroxyproline content and FACS analysis were performed. RESULTS: Bsep-/- mice infected with S. mansoni showed significantly less hepatic inflammation and tendentially less fibrosis compared to infected WT mice. Despite elevated alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in infected Bsep-/- mice, inflammatory cells such as M2 macrophages and Mac-2/galectin-3+ cells were reduced in these animals. Accordingly, mRNA-expression levels of anti-inflammatory cytokines (IL-4 and IL-13) were increased in Bsep-/- mice upon infection. Furthermore, infected Bsep-/- mice exhibited decreased hepatic egg load and parasite fecundity, consequently affecting the worm reproduction rate. This outcome could arise from elevated serum BA levels and lower blood pH in Bsep-/- mice. CONCLUSIONS: The loss of Bsep and the resulting changes in bile acid composition and blood pH are associated with the reduction of parasite fecundity, thus attenuating the development of S. mansoni-induced hepatic inflammation and fibrosis.

Two Single Nucleotide Deletions in the ABCD1 Gene Causing Distinct Phenotypes of X-Linked Adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a rare inborn error of the peroxisomal metabolism caused by pathologic variants in the ATP-binding cassette transporter type D, member 1 (ABCD1) gene located on the X-chromosome. ABCD1 protein, also known as adrenoleukodystrophy protein, is responsible for transport of the very long chain fatty acids (VLCFA) from cytoplasm into the peroxisomes. Therefore, altered function or lack of the ABCD1 protein leads to accumulation of VLCFA in various tissues and blood plasma leading to either rapidly progressive leukodystrophy (cerebral ALD), progressive adrenomyeloneuropathy (AMN), or isolated primary adrenal insufficiency (Addison's disease). We report two distinct single nucleotide deletions in the ABCD1 gene, c.253delC [p.Arg85Glyfs*18] in exon 1, leading to both cerebral ALD and to AMN phenotype in one family, and c.1275delA [p.Phe426Leufs*15] in exon 4, leading to AMN and primary adrenal insufficiency in a second family. For the latter variant, we demonstrate reduced mRNA expression and a complete absence of the ABCD1 protein in PBMC. Distinct mRNA and protein expression in the index patient and heterozygous carriers does not associate with VLCFA concentration in plasma, which is in line with the absence of genotype-phenotype correlation in X-ALD.

Changes in Lipoprotein(a) Levels in People after ST Elevation Myocardial Infarction-The STEMI-Lipids Study.

Lipoprotein(a) (Lp(a)) is considered an independent risk factor for cardiovascular diseases. The plasma concentration of Lp(a) is largely genetically determined but varies over a wide range within the population. This study investigated changes in Lp(a) levels after an acute myocardial infarction. Patients who underwent coronary angiography due to an ST elevation myocardial infarction were enrolled (n = 86), and Lp(a) levels were measured immediately after the intervention, one day, two days, and at a post-discharge follow-up visit at 3 to 6 months after the acute myocardial infarction. Median Lp(a) levels increased from a median of 7.9 mg/dL (3.8-37.1) at hospital admission to 8.4 mg/dL (3.9-35.4) on the following day, then to 9.3 mg/dL (3.7-39.1) on day two (p < 0.001), and to 11.2 mg/dL (4.4-59.6) at the post-discharge follow-up (p < 0.001). Lp(a) levels were the lowest during the acute myocardial infarction and started to increase significantly immediately thereafter, with the highest levels at the post-discharge follow-up. The moderate but significant increase in Lp(a) in people with acute myocardial infarction appears to be clinically relevant on an individual basis, especially when specific Lp(a) cut-off levels are supposed to determine the initiation of future treatment. Hence, a repeated measurement of Lp(a) after myocardial infarction should be performed.

PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence-free survival in HCC patients.

24-Norursodeoxycholic acid reshapes immunometabolism in CD8+ T cells and alleviates hepatic inflammation.

BACKGROUND & AIMS: 24-Norursodeoxycholic acid (NorUDCA) is a novel therapeutic bile acid used to treat immune-mediated cholestatic liver diseases, such as primary sclerosing cholangitis (PSC), where dysregulated T cells including CD8+ T cells contribute to hepatobiliary immunopathology. We hypothesized that NorUDCA may directly modulate CD8+ T cell function thus contributing to its therapeutic efficacy. METHODS: NorUDCA's immunomodulatory effects were first studied in Mdr2-/- mice, as a cholestatic model of PSC. To differentiate NorUDCA's immunomodulatory effects on CD8+ T cell function from its anticholestatic actions, we also used a non-cholestatic model of hepatic injury induced by an excessive CD8+ T cell immune response upon acute non-cytolytic lymphocytic choriomeningitis virus (LCMV) infection. Studies included molecular and biochemical approaches, flow cytometry and metabolic assays in murine CD8+ T cells in vitro. Mass spectrometry was used to identify potential CD8+ T cell targets modulated by NorUDCA. The signaling effects of NorUDCA observed in murine cells were validated in circulating T cells from patients with PSC. RESULTS: NorUDCA demonstrated immunomodulatory effects by reducing hepatic innate and adaptive immune cells, including CD8+ T cells in the Mdr2-/- model. In the non-cholestatic model of CD8+ T cell-driven immunopathology induced by acute LCMV infection, NorUDCA ameliorated hepatic injury and systemic inflammation. Mechanistically, NorUDCA demonstrated strong immunomodulatory efficacy in CD8+ T cells affecting lymphoblastogenesis, expansion, glycolysis and mTORC1 signaling. Mass spectrometry identified that NorUDCA regulates CD8+ T cells by targeting mTORC1. NorUDCA's impact on mTORC1 signaling was further confirmed in circulating PSC CD8+ T cells. CONCLUSIONS: NorUDCA has a direct modulatory impact on CD8+ T cells and attenuates excessive CD8+ T cell-driven hepatic immunopathology. These findings are relevant for treatment of immune-mediated liver diseases such as PSC. LAY SUMMARY: Elucidating the mechanisms by which 24-norursodeoxycholic acid (NorUDCA) works for the treatment of immune-mediated liver diseases, such as primary sclerosing cholangitis, is of considerable clinical interest. Herein, we uncovered an unrecognized property of NorUDCA in the immunometabolic regulation of CD8+ T cells, which has therapeutic relevance for immune-mediated liver diseases, including PSC.

Monoacylglycerol Lipase Inhibition Protects From Liver Injury in Mouse Models of Sclerosing Cholangitis.

BACKGROUND AND AIMS: Monoacylglycerol lipase (MGL) is the last enzymatic step in triglyceride degradation, hydrolyzing monoglycerides into glycerol and fatty acids (FAs) and converting 2-arachidonoylglycerol into arachidonic acid, thus providing ligands for nuclear receptors as key regulators of hepatic bile acid (BA)/lipid metabolism and inflammation. We aimed to explore the role of MGL in the development of cholestatic liver and bile duct injury in mouse models of sclerosing cholangitis, a disease so far lacking effective pharmacological therapy. APPROACH AND RESULTS: To this aim we analyzed the effects of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding to induce sclerosing cholangitis in wild-type (WT) and knockout (MGL-/- ) mice and tested pharmacological inhibition with JZL184 in the multidrug resistance protein 2 knockout (Mdr2-/- ) mouse model of sclerosing cholangitis. Cholestatic liver injury and fibrosis were assessed by serum biochemistry, liver histology, gene expression, and western blot characterization of BA and FA synthesis/transport. Moreover, intestinal FAs and fecal microbiome were analyzed. Transfection and silencing were performed in Caco2 cells. MGL-/- mice were protected from DDC-induced biliary fibrosis and inflammation with reduced serum liver enzymes and increased FA/BA metabolism and ß-oxidation. Notably, pharmacological (JZL184) inhibition of MGL ameliorated cholestatic injury in DDC-fed WT mice and protected Mdr2-/- mice from spontaneous liver injury, with improved liver enzymes, inflammation, and biliary fibrosis. In vitro experiments confirmed that silencing of MGL decreases prostaglandin E2 accumulation in the intestine and up-regulates peroxisome proliferator-activated receptors alpha and gamma activity, thus reducing inflammation. CONCLUSIONS: Collectively, our study unravels MGL as a metabolic target, demonstrating that MGL inhibition may be considered as potential therapy for sclerosing cholangitis.

Hypercortisolism in patients with cholestasis is associated with disease severity.

BACKGROUND: Cholestasis might lead to an impairment of adrenal function as suggested by in vitro and in vivo data as well as by clinical findings. Bile acid and adrenal steroid metabolism not only share the receptors farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor 1 (TGR5), but supraphysiological bile acid levels were found to stimulate steroidogenesis independent of FXR and TGR5. Our previous experimental findings revealed that mice fed bile acids or subjected to common bile duct ligation develop hypercortisolemia. We thus aimed to assess adrenal gland function in patients with cholestasis. METHODS: Adrenal gland function was assessed in 36 patients with cholestasis and in 32 patients without cholestasis by measuring total serum cortisol, adrenocorticotropic hormone (ACTH), as well as the increase of cortisol 20 and 30 min after administration of 1 µg of ACTH. Bile acid levels and bile acid pool composition were determined by high-resolution mass spectrometry. RESULTS: Patients with cholestasis per definition had markedly elevated levels of alkaline phosphatase (AP), bilirubin and serum bile acids. Baseline cortisol and maximum cortisol after ACTH stimulation were significantly higher in patients with cholestasis compared to controls. Increase of cortisol after ACTH stimulation and ACTH did not differ. In the cholestasis group, baseline cortisol correlated with bilirubin but not with AP, total serum bile acids and levels of conjugated and unconjugated bile acid species. Patients with duration of cholestasis < 6 months (n = 30) had significantly higher baseline cortisol levels than those with long standing cholestasis (> 6 months), together with higher bilirubin levels. CONCLUSIONS: We find no evidence of adrenal insufficiency in non-cirrhotic patients with cholestasis. In contrast, patients with cholestasis show hypercortisolism associated with disease severity as mirrored by levels of bilirubin. Lack of ACTH increase in cholestasis suggests a direct effect of cholestasis on adrenals and not on the pituitary gland. Further studies are needed to elucidate the mechanism of cortisol elevation in patients with cholestasis and its clinical significance.

Absence of Adiponutrin (PNPLA3) and Monoacylglycerol Lipase Synergistically Increases Weight Gain and Aggravates Steatohepatitis in Mice.

Altered lipid metabolic pathways including hydrolysis of triglycerides are key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whether adiponutrin (patatin-like phospholipase domain containing protein-3-PNPLA3) and monoacylglycerol lipase (MGL) synergistically contribute to disease progression remains unclear. We generated double knockout (DKO) mice lacking both Mgl and Pnpla3; DKO mice were compared to Mgl-/- after a challenge by high-fat diet (HFD) for 12 weeks to induce steatosis. Serum biochemistry, liver transaminases as well as histology were analyzed. Fatty acid (FA) profiling was assessed in liver and adipose tissue by gas chromatography. Markers of inflammation and lipid metabolism were analyzed. Bone marrow derived macrophages (BMDMs) were isolated and treated with oleic acid. Combined deficiency of Mgl and Pnpla3 resulted in weight gain on a chow diet; when challenged by HFD, DKO mice showed increased hepatic FA synthesis and diminished beta-oxidation compared to Mgl-/-.DKO mice exhibited more pronounced hepatic steatosis with inflammation and recruitment of immune cells to the liver associated with accumulation of saturated FAs. Primary BMDMs isolated from the DKO mice showed increased inflammatory activities, which could be reversed by oleic acid supplementation. Pnpla3 deficiency aggravates the effects of Mgl deletion on steatosis and inflammation in the liver under HFD challenge.

HDL-related biomarkers are robust predictors of survival in patients with chronic liver failure.

BACKGROUND & AIMS: High-density lipoprotein cholesterol (HDL-C) levels are reduced in patients with chronic liver disease and inversely correlate with disease severity. During acute conditions such as sepsis, HDL-C levels decrease rapidly and HDL particles undergo profound changes in their composition and function. We aimed to determine whether indices of HDL quantity and quality associate with progression and survival in patients with advanced liver disease. METHODS: HDL-related biomarkers were studied in 508 patients with compensated or decompensated cirrhosis (including acute-on-chronic liver failure [ACLF]) and 40 age- and gender-matched controls. Specifically, we studied levels of HDL-C, its subclasses HDL2-C and HDL3-C, and apolipoprotein A1 (apoA-I), as well as HDL cholesterol efflux capacity as a metric of HDL functionality. RESULTS: Baseline levels of HDL-C and apoA-I were significantly lower in patients with stable cirrhosis compared to controls and were further decreased in patients with acute decompensation (AD) and ACLF. In stable cirrhosis (n = 228), both HDL-C and apoA-I predicted the development of liver-related complications independently of model for end-stage liver disease (MELD) score. In patients with AD, with or without ACLF (n = 280), both HDL-C and apoA-I were MELD-independent predictors of 90-day mortality. On ROC analysis, both HDL-C and apoA-I had high diagnostic accuracy for 90-day mortality in patients with AD (AUROCs of 0.79 and 0.80, respectively, similar to that of MELD 0.81). On Kaplan-Meier analysis, HDL-C <17 mg/dl and apoA-I <50 mg/dl indicated poor short-term survival. The prognostic accuracy of HDL-C was validated in a large external validation cohort of 985 patients with portal hypertension due to advanced chronic liver disease (AUROCs HDL-C: 0.81 vs. MELD: 0.77). CONCLUSION: HDL-related biomarkers are robust predictors of disease progression and survival in chronic liver failure. LAY SUMMARY: People who suffer from cirrhosis (scarring of the liver) have low levels of cholesterol carried by high-density lipoproteins (HDL-C). These alterations are connected to inflammation, which is a problem in severe liver disease. Herein, we show that reduced levels of HDL-C and apolipoprotein A-I (apoA-I, the main protein carried by HDL) are closely linked to the severity of liver failure, its complications and survival. Both HDL-C and apoA-I can be easily measured in clinical laboratories and are as good as currently used prognostic scores calculated from several laboratory values by complex formulas.

Absence of Bsep/Abcb11 attenuates MCD diet-induced hepatic steatosis but aggravates inflammation in mice.

BACKGROUND: Bile acids (BAs) regulate hepatic lipid metabolism and inflammation. Bile salt export pump (BSEP) KO mice are metabolically preconditioned with a hydrophilic BA composition protecting them from cholestasis. We hypothesize that changes in hepatic BA profile and subsequent changes in BA signalling may critically determine the susceptibility to steatohepatitis. METHODS: Wild-type (WT) and BSEP KO mice were challenged with methionine choline-deficient (MCD) diet to induce steatohepatitis. Serum biochemistry, lipid profiling as well as intestinal lipid absorption were assessed. Markers of inflammation, fibrosis, lipid and BA metabolism were analysed. Hepatic and faecal BA profile as well as serum levels of the BA synthesis intermediate 7-hydroxy-4-cholesten-3-one (C4) were also investigated. RESULTS: Bile salt export pump KO MCD-fed mice developed less steatosis but more inflammation than WT mice. Intestinal neutral lipid levels were reduced in BSEP KO mice at baseline and under MCD conditions. Faecal non-esterified fatty acid concentrations at baseline and under MCD diet were markedly elevated in BSEP KO compared to WT mice. Serum liver enzymes and hepatic expression of inflammatory markers were increased in MCD-fed BSEP KO animals. PPARα protein levels were reduced in BSEP KO mice. Accordingly, PPARα downstream targets Fabp1 and Fatp5 were repressed, while NFκB subunits were increased in MCD-fed BSEP KO mice. Farnesoid X receptor (FXR) protein levels were reduced in MCD-fed BSEP KO vs WT mice. Hepatic BA profile revealed elevated levels of TßMCA, exerting FXR antagonistic action, while concentrations of TCA (FXR agonistic function) were reduced. CONCLUSION: Presence of hydroxylated BAs result in increased faecal FA excretion and reduced hepatic lipid accumulation. This aggravates development of MCD diet-induced hepatitis potentially by decreasing FXR and PPARα signalling.

Sex specificity of kidney markers to assess prognosis in cirrhotic patients with TIPS.

BACKGROUND & AIMS: Renal function assessed by creatinine is a key prognostic factor in cirrhotic patients. However, creatinine is influenced by several factors, rendering interpretation difficult in some situations. This is especially important in early stages of renal dysfunction where renal impairment might not be accompanied by an increase in creatinine. Other parameters, such as cystatin C (CysC) and beta-trace protein (BTP), have been evaluated to fill this gap. However, none of these studies have considered the role of the patient's sex. The present study analysed CysC and BTP to evaluate their prognostic value and differentiate them according to sex. PATIENTS AND METHODS: CysC and BTP were measured in 173 transjugular intrahepatic portosystemic shunt (TIPS)-patients from the NEPTUN-STUDY(NCT03628807) and analysed their relationship with mortality and sex. Propensity score for age, MELD, etiology and TIPS indication was used. RESULTS: Cystatin C and BTP showed excellent correlations with creatinine values at baseline and follow-up. CysC was an independent predictor of overall mortality (HR = 1.66(1.33-2.06)) with an AUC of 0.75 and identified a cut-off of 1.55 mg/L in the whole cohort. Interestingly, CysC was significantly lower in females, also after propensity score matching. In males, the only independent predictor was the creatinine level (HR = 1.54(1.25-1.58)), while in females CysC levels independently predicted mortality (HR = 3.17(1.34-7.52)). CONCLUSION: This study demonstrates for the first time that in TIPS-patients creatinine predicts mortality in males better than in females, whereas CysC is a better predictor of mortality in females. These results may influence future clinical decisions on therapeutic options for example, allocation for liver transplantation in TIPS-patients.

Hypoglycaemia leads to a delayed increase in platelet and coagulation activation markers in people with type 2 diabetes treated with metformin only: Results from a stepwise hypoglycaemic clamp study.

AIMS: To investigate the effect of hypoglycaemia on platelet and coagulation activation in people with type 2 diabetes. MATERIALS AND METHODS: This monocentric, open, single-arm, mechanistic trial included 14 people with established type 2 diabetes (four women, 10 men, age 55 ± 7 years, glycated haemoglobin concentration 51 ± 7 mmol/mol) receiving metformin monotherapy. A stepwise hyperinsulinaemic-hypoglycaemic clamp experiment (3.5 and 2.5 mmol/L, for 30 minutes respectively) was performed, aiming to investigate platelet and coagulation activity during predefined plateaus of hypoglycaemia, as well as 1 day and 7 days later. RESULTS: While platelet activation assessed by light transmittance aggregometry did not significantly increase after the hypoglycaemic clamp procedure, the more sensitive flow cytometry-based measurement of platelet surface activation markers showed hypoglycaemia-induced activation 24 hours (PAC1pos CD62Ppos , PAC1pos CD63Ppos and PAC1pos CD62Ppos CD63pos ; P < .01) and 7 days after the hypoglycaemic clamp (P < .001 for PAC1pos CD63pos ; P < .01 for PAC1pos CD62Ppos and PAC1pos CD62Ppos CD63pos ) in comparison to baseline. Coagulation markers, such as fibrinogen, D-dimer, plasminogen activator inhibitor-1, von Willebrand factor activity and factor VIII, were also significantly increased, an effect that was most pronounced 24 hours after the hypoglycaemic clamp. CONCLUSION: A single event of insulin-induced hypoglycaemia led to an increase in markers of platelet activation and coagulation in people with early stages of type 2 diabetes on metformin therapy. However, the activation occurred with a delay and was evident 24 hours and 7 days after the actual hypoglycaemic episode.

The zinc-finger transcription factor MAZR regulates iNKT cell subset differentiation.

Invariant natural killer T (iNKT) cells represent a subgroup of innate-like T cells and play an important role in immune responses against certain pathogens. In addition, they have been linked to autoimmunity and antitumor immunity. iNKT cells consist of several subsets with distinct functions; however, the transcriptional networks controlling iNKT subset differentiation are still not fully characterized. Myc-associated zinc-finger-related factor (MAZR, also known as PATZ1) is an essential transcription factor for CD8+ lineage differentiation of conventional T cells. Here, we show that MAZR plays an important role in iNKT cells. T-cell lineage-specific deletion of MAZR resulted in an iNKT cell-intrinsic defect that led to an increase in iNKT2 cell numbers, concurrent with a reduction in iNKT1 and iNKT17 cells. Consistent with the alteration in the subset distribution, deletion of MAZR also resulted in an increase in the percentage of IL-4-producing cells. Moreover, MAZR-deficient iNKT cells displayed an enhanced expression of Erg2 and ThPOK, key factors for iNKT cell generation and subset differentiation, indicating that MAZR controls iNKT cell development through fine-tuning of their expression levels. Taken together, our study identified MAZR as an essential transcription factor regulating iNKT cell subset differentiation and effector function.

Apolipoprotein CIII predicts cardiovascular events in patients with coronary artery disease: a prospective observational study.

BACKGROUND: Apolipoprotein CIII (apoCIII) is associated with triglyceride-rich lipoprotein metabolism and has emerged as independent marker for risk of cardiovascular disease. The objective was to test whether apoCIII is regulated postprandially and whether apoCIII concentrations in native and chylomicron-free serum predict future cardiovascular events in patients with stable coronary artery disease (CAD). METHODS: ApoCIII concentrations were measured in native and chylomicron-free serum in the fasting state and after a standardized oral fat load test in 195 patients with stable CAD. Clinical follow-up was 48 months. Chylomicron-free serum was prepared by ultracentrifugation (18,000 rpm, 3 h). The log-rank test and Cox regression analyses were used to investigate the association of apoCIII with recurrent cardiovascular events. RESULTS: Of the 195 patients included, 92 had a cardiovascular event, and 103 did not. 97% were treated with a statin. No significant changes in apoCIII concentration were observed after the oral fat load test. The apoCIII concentration was associated with event-free survival independent of conventional risk factors. This association reached statistical significance only for apoCIII concentration measured in chylomicron-free serum (hazard ratio [95% confidence interval] for apoCIII above the mean: postprandial: 1.67 (1.06-2.29), P = 0.028, fasting: 2.09 (1.32-3.32), P = 0.002), but not for apoCIII concentration measured in native serum (postprandial: 1.47 [0.89-2.43], P = 0.133, fasting: 1.56 [0.95-2.58], P = 0.081). The effects were independent of other risk factors. CONCLUSIONS: ApoCIII concentrations in chylomicron-free serum are independently associated with event-free survival in patients with CAD both in fasting and postprandial state. This findings support considering apoCIII for risk assessment and attempting to test the hypothesis that lowering apoCIII reduces residual cardiovascular risk. TAKE HOME MESSAGE: Apolipoprotein CIII concentration measured in chylomicron-free serum predicts recurrent cardiovascular events in patients with stable coronary artery disease. TRIAL REGISTRATION: The trial which included the participants of this study was registered at https://clinicaltrials.gov (NCT00628524) on March 5, 2008.

Lack of monoacylglycerol lipase prevents hepatic steatosis by favoring lipid storage in adipose tissue and intestinal malabsorption.

Monoacylglycerol lipase (MGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. To examine the role of MGL in hepatic steatosis, WT and MGL KO (MGL-/-) mice were challenged with a Western diet (WD) over 12 weeks. Lipid metabolism, inflammation, and fibrosis were assessed by serum biochemistry, histology, and gene-expression profiling of liver and adipose depots. Intestinal fat absorption was measured by gas chromatography. Primary adipocyte and 3T3-L1 cells were analyzed by flow cytometry and Western blot. Human hepatocytes were treated with MGL inhibitor JZL184. The absence of MGL protected mice from hepatic steatosis by repressing key lipogenic enzymes in liver (Srebp1c, Pparγ2, and diacylglycerol O-acyltransferase 1), while promoting FA oxidation. Liver inflammation was diminished in MGL-/- mice fed a WD, as evidenced by diminished epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80) staining and C-C motif chemokine ligand 2 gene expression, whereas fibrosis remained unchanged. Absence of MGL promoted fat storage in gonadal white adipose tissue (gWAT) with increased lipogenesis and unchanged lipolysis, diminished inflammation in gWAT, and subcutaneous AT. Intestinal fat malabsorption prevented ectopic lipid accumulation in livers of MGL-/- mice fed a WD. In vitro experiments demonstrated increased adipocyte size/lipid content driven by PPARγ. In conclusion, our data uncover that MGL deletion improves some aspects of nonalcoholic fatty liver disease by promoting lipid storage in gWAT and fat malabsorption.

The interrelations between PCSK9 metabolism and cholesterol synthesis and absorption.

Very few studies have investigated the interrelations between proprotein convertase subtilisin/kexin type 9 (PCSK9) metabolism, cholesterol synthesis, and cholesterol absorption. We aimed to address this issue in a large clinical trial of 245 patients with hypercholesterolemia. Serum lipids, PCSK9, lathosterol (cholesterol synthesis marker), campesterol, and sitosterol (cholesterol absorption markers) were measured before and 4-8 weeks after the start of treatment with PCSK9-antibodies (alirocumab or evolocumab). The patients had mean (standard error) LDL-cholesterol and PCSK9 concentrations of 3.87 (0.10) mmol/l and 356 (17) ng/ml, respectively. Eighty-four patients received no lipid-lowering pretreatment, 26 ezetimibe, 38 statins, and 97 ezetimibe + statins. Circulating PCSK9 increased in parallel with the potency of lipid-lowering pretreatment with circulating PCSK9 being highest in the ezetimibe + statin group (P < 0.001). Treatment with PCSK9-antibodies strongly decreased LDL-cholesterol, lathosterol, campesterol, and sitosterol (all P < 0.001) but hardly affected noncholesterol sterol to cholesterol ratios. Lipid-lowering pretreatment was not associated with the effects of PCSK9-antibodies on noncholesterol sterols (all P > 0.05). Summing up, circulating PCSK9 is increased by cholesterol synthesis and absorption inhibitors. Increased PCSK9 expression may partly explain the strong reductions of LDL-cholesterol achieved with PCSK9-antibodies after such pretreatment. On the other hand, treatment with PCSK9-antibodies does not significantly change the balance between cholesterol synthesis and absorption.

Serum concentrations of free fatty acids are associated with 3-month mortality in acute heart failure patients.

Background Plasma free fatty acids (FFA) are higher in heart failure (HF) patients compared to healthy controls. Considering that the extent of FFA elevation in HF might mirror the severity of HF, we hypothesized that the serum levels of FFA may be a useful prognostic indicator for 3-month mortality in acute heart failure (AHF). Methods We analyzed the serum samples of AHF patients obtained at admission to the emergency department. Serum levels of FFA were analyzed using an enzymatic reagent on an automatic analyzer. Results Out of 152 included AHF patients that were originally included, serum samples of 132 patients were available for the quantification of FFA. Of these, 35 (26.5%) died within 3 months of onset of AHF. These patients had significantly higher serum levels of FFA compared to AHF patients who were alive 3 months after onset of AHF. Univariable logistic regression analyses showed a significant positive association of FFA levels with 3-month mortality (odds ratio [OR] 2.76 [95% confidence interval 1.32-6.27], p = 0.010). Importantly, this association remained significant after adjusting for age and sex, as well as for further clinical and laboratory parameters that showed a significant association with 3-month mortality in the univariate analyses. Conclusions We conclude that the admission serum levels of FFA are associated with 3-month mortality in AHF patients. Therefore, measurements of circulating FFA levels may help identifying high-risk AHF patients.