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FATP4 deficiency in BMDMs and Kupffer cells led to increased proinflammatory response. Fatp4M−/− mice displayed thrombocytopenia, splenomegaly, and elevated liver enzymes. In response to HFHC feeding, male mutants were prone to hepatic steatosis, whereas female mutants showed exaggerated fibrosis. Our study provides insights into a sex-dimorphic susceptibility to NASH by myeloid-FATP4 deficiency.

Fatty acid (FA) metabolism is a series of processes that provide structural substances, signalling molecules and energy. Ample evidence has shown that FA uptake is mediated by plasma membrane transporters including FA transport proteins (FATPs), caveolin-1, fatty-acid translocase (FAT)/CD36, and fatty-acid binding proteins. Unlike other FA transporters, the functions of FATPs have been controversial because they contain both motifs of FA transport and fatty acyl-CoA synthetase (ACS). The widely distributed FATP4 is not a direct FA transporter but plays a predominant function as an ACS. FATP4 deficiency causes ichthyosis premature syndrome in mice and humans associated with suppression of polar lipids but an increase in neutral lipids including triglycerides (TGs). Such a shift has been extensively characterized in enterocyte-, hepatocyte-, and adipocyte-specific Fatp4-deficient mice. The mutants under obese and non-obese fatty livers induced by different diets persistently show an increase in blood non-esterified free fatty acids and glycerol indicating the lipolysis of TGs. This review also focuses on FATP4 role on regulatory networks and factors that modulate FATP4 expression in metabolic tissues including intestine, liver, muscle, and adipose tissues. Metabolic disorders especially regarding blood lipids by FATP4 deficiency in different cell types are herein discussed. Our results may be applicable to not only patients with FATP4 mutations but also represent a model of dysregulated lipid homeostasis, thus providing mechanistic insights into obesity and development of fatty liver disease.

Polymorphisms of phospholipase A2VIA (iPLA2β or PLA2G6) are associated with body weights and blood C-reactive protein. The role of iPLA2β/PLA2G6 in non-alcoholic steatohepatitis (NASH) is still elusive because female iPla2β-null mice showed attenuated hepatic steatosis but exacerbated hepatic fibrosis after feeding with methionine- and choline-deficient diet (MCDD). Herein, female mice with myeloid- (MPla2g6

We have found that group VIA calcium-independent phospholipase A2 (iPLA2β) has specificity for hydrolysis of phosphatidylethanolamine (PE) in mouse livers. Phospholipids (PLs) are transported to plasma membrane and some PLs including PE are externalized to maintain membrane PL asymmetry. Here we demonstrated that hepatocytes of iPLA2β-null (KO) mice showed an increase in PE containing palmitate and oleate. We aimed to examine whether externalization of PE on the outer leaflets could be affected by iPLA2β deficiency and its modulation by reactive oxygen species (ROS) or apoptosis. As duramycin has high affinity to PE, we used duramycin conjugated with biotin (DLB) and streptavidin 488 as a probe for detection of externalized PE. Compared to WT, naive KO hepatocytes showed an increase in both PE externalization and ROS generation. These events were observed in male but not in female KO mice. Hydrogen peroxide or menadione treatment enhanced PE externalization to the same extent for both male/female WT and KO hepatocytes. By indirect immunofluorescence, DLB-streptavidin staining was observed as small punctuated spots on the cell surface of menadione-treated KO hepatocytes. Unlike the reported PS externalization, CD95/FasL treatment did not lead to any increase in PE externalization, and iPLA2β deficiency-dependent PE externalization was also not correlated with apoptosis. Thus, constitutive (but not induced) ROS generation in iPLA2β-deficient hepatocytes leads to PE externalization observed only in male mice. Such PE externalization may imply detrimental effects regarding further oxidation of PE fatty acids and the binding with pathogens on the outer leaflets of hepatocyte plasma membrane.

Group VIA phospholipase A2 (iPLA2β) play diverse biological functions in epithelial cells and macrophages. Global deletion in iPLA2β-null (KO) mice leads to protection against hepatic steatosis in non-alcoholic fatty liver disease, in part, due to the replenishment of the loss of hepatocellular phospholipids. As the loss of phospholipids also occurs in hepatocellular carcinoma (HCC), we hypothesized that global deletion in KO mice may lead to protection against HCC. Here, HCC induced by diethylnitrosamine (DEN) was chosen because DEN causes direct injury to the hepatocytes. Male wild-type (WT) and KO mice at 3–5 weeks of age (12–13 mice/group) were subjected to a single intraperitoneal treatment with 10 mg/kg DEN, and mice were killed 12 months later. Analyses of histology, plasma cytokines, and gene expression were performed. Due to the low-dose DEN used, we observed a liver nodule in 3 of 13 WT and 2 of 12 KO mice. Only one DEN-treated WT mouse was confirmed to have HCC. DEN-treated KO mice did not show any HCC but showed suppressed hepatic expression of cell-cycle cyclinD2 and BCL2 as well as inflammatory markers IL-1β, IL-10, and VCAM-1. Notably, DEN-treated KO mice showed increased hepatic necrosis and elevated levels of plasma lactate dehydrogenase suggesting an exacerbation of liver injury. Thus, global iPLA2β deficiency in DEN-treated mice rendered HCC protection by an induction of cell-cycle arrest. Our results suggest the role of iPLA2β inhibition in HCC treatment.

A defect of hepatic remodeling of phospholipids (PL) is seen in non-alcoholic fatty liver disease and steatohepatitis (NASH) indicating pivotal role of PL metabolism in this disease. The deletion of group VIA calcium-independent phospholipase A2 (iPla2β) protects ob/ob mice from hepatic steatosis (BBAlip 1861, 2016, 440-461), however its role in high-fat diet (HFD)-induced NASH is still elusive. Here, wild-type and iPla2β-null mice were subjected to chronic feeding with HFD for 6 months. We showed that protection was observed in iPla2β-null mice with an attenuation of diet-induced body and liver-weight gains, liver enzymes, serum free fatty acids as well as hepatic TG and steatosis scores. iPla2β deficiency under HFD attenuated the levels of 1-stearoyl lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylinositol (LPI) as well as elevation of hepatic arachidonate, arachidonate-containing cholesterol esters and prostaglandin E2. More importantly, this deficiency rescued a defect in PL remodeling and attenuated the ratio of saturated and unsaturated PL. The protection by iPla2β deficiency was not observed during short-term HFD feeding of 3 or 5 weeks which showed no PL remodeling defect. In addition to PC/PE, this deficiency reversed the suppression of PC/PI and PE/PI among monounsaturated PL. However, this deficiency did not modulate hepatic PL contents and PL ratios in ER fractions, ER stress, fibrosis, and inflammation markers. Hence, iPla2β inactivation protected mice against hepatic steatosis and obesity during chronic dietary NASH by correcting PL remodeling defect and PI composition relative to PC and PE.

Fatty acid transport protein 4 (FATP4) belongs to a family of acyl-CoA synthetases which activate long-chain fatty acids into acyl-CoAs subsequently used in specific metabolic pathways. Patients with FATP4 mutations and Fatp4-null mice show thick desquamating skin and other complications, however, FATP4 role on macrophage functions has not been studied. We here determined whether the levels of macrophage glycerophospholipids, sphingolipids including ceramides, triacylglycerides, and cytokine release could be altered by FATP4 inactivation. Two in vitro experimental systems were studied: FATP4 knockdown in THP-1-derived macrophages undergoing M1 (LPS + IFNγ) or M2 (IL-4) activation and bone marrow-derived macrophages (BMDMs) from macrophage-specific Fatp4-knockout (Fatp4M−/−) mice undergoing tunicamycin (TM)-induced endoplasmic reticulum stress. FATP4-deficient macrophages showed a metabolic shift towards triacylglycerides and were protected from M1- or TM-induced release of pro-inflammatory cytokines and cellular injury. Fatp4M−/− BMDMs showed specificity in attenuating TM-induced activation of inositol-requiring enzyme1α, but not other unfolded protein response pathways. Under basal conditions, FATP4/Fatp4 deficiency decreased the levels of ceramides and induced an up-regulation of mannose receptor CD206 expression. The deficiency led to an attenuation of IL-8 release in THP-1 cells as well as TNF-α and IL-12 release in BMDMs. Thus, FATP4 functions as an acyl-CoA synthetase in macrophages and its inactivation suppresses the release of pro-inflammatory cytokines by shifting fatty acids towards the synthesis of specific lipids.

Background Hereditary hemochromatosis (HH) is an autosomal recessive genetic disorder with increased intestinal iron absorption and therefore iron Overload. iron overload leads to increased levels of toxic non-transferrin bound iron which results in oxidative stress and lipid peroxidation. The impact of iron on lipid metabolism is so far not fully understood. The aim of this study was to investigate lipid metabolism including lipoproteins (HDL, LDL), neutral (triglycerides, cholesterol) and polar lipids (sphingo- and phospholipids), and PNPLA3 polymorphism (rs738409/I148M) in HH. Methods We conducted a cohort study of 54 subjects with HH and 20 healthy subjects. Patients were analyzed for their iron status including iron, ferritin, transferrin and transferrin saturation and serum lipid profile on a routine follow-up examination. Results HH group showed significantly lower serum phosphatidylcholine (PC) and significantly higher phosphatidylethanolamine (PE) compared to healthy control group. The ratio of PC/PE was clearly lower in HH group indicating a shift from PC to PE. Triglycerides were significantly higher in HH group. No differences were seen for HDL, LDL and cholesterol. Hepatic steatosis was significantly more frequent in HH. PNPLA3 polymorphism (CC vs. CG/GG) did not reveal any significant correlation with iron and lipid parameters including neutral and polar lipids, grade of steatosis and fibrosis. Conclusion Our study strengthens the hypothesis of altered lipid metabolism in HH and susceptibility to nonalcoholic fatty liver disease. Disturbed phospholipid metabolism may represent an important factor in pathogenesis of hepatic steatosis in HH.

The epidermis-specific lipid acylceramide plays a pivotal role in the formation of the permeability barrier in the skin; abrogation of its synthesis causes the skin disorder ichthyosis. However, the acylceramide synthetic pathway has not yet been fully elucidated: Namely, the acyl-CoA synthetase (ACS) involved in this pathway remains to be identified. Here, we hypothesized it to be encoded by FATP4/ACSVL4, the causative gene of ichthyosis prematurity syndrome (IPS). In vitro experiments revealed that FATP4 exhibits ACS activity toward an ω-hydroxy fatty acid (FA), an intermediate of the acylceramide synthetic pathway. Fatp4 knockout (KO) mice exhibited severe skin barrier dysfunction and morphological abnormalities in the epidermis. The total amount of acylceramide in Fatp4 KO mice was reduced to ∼10% of wild-type mice. Decreased levels and shortening of chain lengths were observed in the saturated, nonacylated ceramides. FA levels were not decreased in the epidermis of Fatp4 KO mice. The expression levels of the FA elongase Elovl1 were reduced in Fatp4 KO epidermis, partly accounting for the reduction and shortening of saturated, nonacylated ceramides. A decrease in acylceramide levels was also observed in human keratinocytes with FATP4 knockdown. From these results, we conclude that skin barrier dysfunction observed in IPS patients and Fatp4 KO mice is caused mainly by reduced acylceramide production. Our findings further elucidate the molecular mechanism governing acylceramide synthesis and IPS pathology.

The roots of Trigonostemon reidioides, Thai medicinal plant, have long been used as an antidote, laxative, and antiasthmatic, and also used as folk remedy for relieving inflammatory symptoms from poisonous insect and snake bites as well as abscesses and sprains. Here, we studied anti-inflammatory effects of a major diterpenoid named trigonoreidon B (TR-B) isolated from T. reidioides roots in lipopolysaccharide (LPS)-activated RAW264.7 macrophages and D-galactosamine (D-GalN)/LPS-induced inflammatory liver injury in mice. RAW264.7 cells were treated with TR-B or other available minor diterpenoids, and cell viability was determined by AlamarBlue. The levels of inflammatory mediators were determined by nitrite assay, ELISA, and luminescence. NF-κB nuclear translocation was investigated by indirect immunofluorescence. Expression levels were determined by real-time PCR and Western blotting. Transaminases and caspase activities were determined by using assay kits. Our results showed that TR-B was able to suppress PI3K/Akt activation and inflammatory induction in LPS-activated macrophages. These events were concomitant with TR-B's ability to hamper activated generation of reactive oxygen species, nitric oxide, prostaglandin E2, and cytokines as well as NF-κB p65 nuclear translocation. In an in vivo model of inflammatory liver injury, an administration of TR-B protected mice from D-GalN/LPS-induced liver injury by suppressing the elevation of serum TNF-α, transaminase activities, and hepatocyte apoptosis as well as an improvement of liver histopathology. During protection against liver damage, TR-B also prevented the loss of Akt phosphorylation. Collectively, the results of this present study suggested that TR-B exerted an anti-inflammatory effect via attenuating macrophage-mediated inflammation and inflammatory liver injury in vivo. TR-B may represent a promising lead compound for anti-inflammatory drug development.

Ageing is a major risk factor for various forms of liver and gastrointestinal (GI) disease and genetic background may contribute to the pathogenesis of these diseases. Group VIA phospholipase A2 or iPLA2β is a homeostatic PLA2 by playing a role in phospholipid metabolism and remodeling. Global iPLA2β-/- mice exhibit aged-dependent phenotypes with body weight loss and abnormalities in the bone and brain. We have previously reported the abnormalities in these mutant mice showing susceptibility for chemical-induced liver injury and colitis. We hypothesize that iPLA2β deficiency may sensitize with ageing for an induction of GI injury. Male wild-type and iPLA2β-/- mice at 4 and 20-22months of age were studied. Aged, but not young, iPLA2β-/-mice showed increased hepatic fibrosis and biliary ductular expansion as well as severe intestinal atrophy associated with increased apoptosis, pro-inflammation, disrupted tight junction, and reduced number of mucin-containing globlet cells. This damage was associated with decreased expression of intestinal endoplasmic stress XBP1 and its regulator HNF1α, FATP4, ACSL5, bile-acid transport genes as well as nuclear receptors LXRα and FXR. By LC/MS-MS profiling, iPLA2β deficiency in aged mice caused an increase of intestinal arachidonate-containing phospholipids concomitant with a decrease in ceramides. By the suppression of intestinal FXR/FGF-15 signaling, hepatic bile-acid synthesis gene expression was increased leading to an elevation of secondary and hydrophobic bile acids in liver, bile, and intestine. In conclusions, ageing sensitized by iPLA2β deficiency caused a decline of key intestinal homeostatic genes resulting in the development of GI disease in a gut-to-liver manner.

Increased activation of CD95/Fas by Fas ligand in viral hepatitis and autoimmunity is involved in pathogenesis of fulminant hepatitis and liver failure. We designed a bile-acid phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE with LPE containing oleate at the sn-1) as a hepatoprotectant that was shown to protect against fulminant hepatitis induced by endotoxin. We herein further assessed the ability of UDCA-LPE to prevent death receptor CD95/Fas-induced fulminant hepatitis. C57BL/6 mice were intravenously administered with CD95/Fas agonistic monoclonal antibody (Jo-2) with or without 1 h pretreatment with 50 mg/kg UDCA-LPE. Jo-2 administration caused massive hepatocyte damage as seen by histology, and this was associated with a significant decrease in hepatic phosphatidylcholine (PC), lysoPC, and lysophosphatidylethanolamine levels. By histology, UDCA-LPE pretreatment improved hepatocyte damage and restored the loss of these phospholipids in part by a mechanism involving an inhibition of cytosolic phospholipaseA2 expression. Accordingly, Jo-2 treatment increased hepatic expression of cleaved caspase 8, caspase 3, and poly (ADP-Ribose) polymerase-1, and on the other hand decreased that of anti-apoptotic cellular FLICE-inhibitory protein. UDCA-LPE pretreatment was able to reverse all these changes. Moreover, UDCA-LPE attenuated inflammatory response by lowering the levels of Jo-2-induced proinflammatory cytokines TNF-α, IL-6, and IL-1β in liver and serum. UDCA-LPE was also able to decrease the levels of stimulated Th1/Th17 cytokines in Jo-2-primed isolated splenocytes. Taken together, UDCA-LPE exhibited potent anti-inflammatory effects against CD95/Fas-induced fulminant hepatitis.

Phytochemical investigation of Trigonostemon reidioides roots led to the isolation of fourteen compounds. These included six new diterpenoids, trigonoreidons A−F (1−6), together with eight known diterpenoids 7−14. The structures of the new compounds were elucidated by spectroscopic techniques and the absolute configuration at the asymmetric carbon was determined by the modified Mosher's method. The structure of trigonoreidon B (2) was confirmed by X-ray crystallographic analysis. The known compounds were identified by comparison of the spectroscopic and physical data with those of reported values. The isolated compounds were evaluated for antimycobacterial activity against Mycobacterium tuberculosis. Among the compounds that exhibited antimycobacterial activity, the diterpenoids rediocide C (12) and rediocide G (14) were the most active compounds, with the MIC value of 3.84 μM.

The Pregnane X (PXR), Vitamin D (VDR) and Farnesoid X (FXR) nuclear receptors have been shown to be receptors of bile acids controlling their detoxification or synthesis. Chenodeoxycholic (CDCA) and lithocholic (LCA) acids are ligands of FXR and VDR, respectively, whereas 3-keto and acetylated derivates of LCA have been described as ligands for all three receptors. In this study, we hypothesized that oxidation or acetylation at position 3, 7 and 12 of bile acids DCA (deoxycholic acid), LCA, CA (cholic acid), and CDCA by detoxification enzymes or microbiome may have an effect on the interactions with bile acid nuclear receptors. We employed reporter gene assays in HepG2 cells, the TR-FRET assay with recombinant PXR and RT-PCR to study the effects of acetylated and keto bile acids on the nuclear receptors activation and their target gene expression in differentiated hepatic HepaRG cells. We demonstrate that the DCA 3,12-diacetate and CA 3,7,12-triacetate derivatives are ligands of PXR and DCA 3,12-diacetate induces PXR target genes such as CYP3A4, CYP2B6 and ABCB1/MDR1. In conclusion, we found that acetylated DCA and CA are potent ligands of PXR. Whether the acetylated bile acid derivatives are novel endogenous ligands of PXR with detoxification or physiological functions should be further studied in ongoing experiments.

Polymorphisms of GVIA calcium-independent PLA2 (iPLA2β) implicate its role in plasma lipids, type-2 diabetes, and nonalcoholic fatty liver disease (NAFLD). iPLA2β is thought as a target for NAFLD therapy. We investigated the role of hepatic phospholipids in three NAFLD models: genetic Ob/Ob mice, long-term high-fat diet (HFD)-fed mice (representing obese NAFLD), and mice fed with methionine-choline-deficient (MCD) diet (representing nonobese NAFLD). A defect in hepatic fatty-acyl remodeling of phosphatidylcholine (PC) was observed in these three NAFLD models. By using iPLA2β-null mice, our profiling results revealed that the defect in PC remodeling was rescued in obese NAFLD associated with fatty liver and obesity protection. The rescue of PC remodeling and steatosis protection was not observed in MCD-fed mice. Our results indicate a critical role of hepatic PC for protection by iPLA2β inactivation, suggesting that iPLA2β inhibitors may be used as a therapy for obese but not nonobese NAFLD.

Scope Inadequate intake of choline commonly leads to liver diseases. Methionine- and choline-deficient diets (MCDD) induce fatty liver in mice which is partly mediated by triglyceride (TG) lipolysis in white adipose tissues (WATs). Because Fatp4 knockdown has been shown to increase adipocyte lipolysis in vitro, here, the effects of MCDD on WAT lipolysis in aP2-Cre Fatp4-knockout (Fatp4A-/- ) mice are determined. Methods and results Isolated WATs of Fatp4A-/- mice exposed to MCD medium show an increase in lipolysis, and the strongest effect is noted on glycerol release from subcutaneous fat. Fatp4A-/- mice fed with MCDD for 4 weeks show an increase in serum glycerol, TG, and leptin levels associated with the activation of hormone-sensitive lipase in subcutaneous fat. Chow-fed Fatp4A-/- mice also show an increase in serum leptin and very-low-density lipoproteins as well as liver phosphatidylcholine and sphingomyelin levels. Both chow- and MCDD-fed Fatp4A-/- mice show a decrease in serum ketone and WAT sphingomyelin levels which supports a metabolic shift to TG for subsequent WAT lipolysis CONCLUSIONS: Adipose Fatp4 deficiency leads to TG lipolysis and leptin release, which are exaggerated by MCDD. The data imply hyperlipidemia risk by a low dietary choline intake and gene mutations that increase adipose TG levels.

Interleukin-1β (IL-1β) is a potent, pro-inflammatory cytokine of the innate immune system that plays an essential role in host defense against infection. However, elevated circulating levels of IL-1β can cause life-threatening systemic inflammation. Hence, mechanisms controlling IL-1β maturation and release are of outstanding clinical interest. Secretory leukocyte protease inhibitor (SLPI), in addition to its well-described anti-protease function, controls the expression of several pro-inflammatory cytokines on the transcriptional level. In the present study, we tested the potential involvement of SLPI in the control of ATP-induced, inflammasome-dependent IL-1β maturation and release. We demonstrated that SLPI dose-dependently inhibits the ATP-mediated inflammasome activation and IL-1β release in human monocytic cells, without affecting the induction of pro-IL-1β mRNA by LPS. In contrast, the ATP-independent IL-1β release induced by the pore forming bacterial toxin nigericin is not impaired, and SLPI does not directly modulate the ion channel function of the human P2X7 receptor heterologously expressed in Xenopus laevis oocytes. In human monocytic U937 cells, however, SLPI efficiently inhibits ATP-induced ion-currents. Using specific inhibitors and siRNA, we demonstrate that SLPI activates the calcium-independent phospholipase A2β (iPLA2β) and leads to the release of a low molecular mass factor that mediates the inhibition of IL-1β release. Signaling involves nicotinic acetylcholine receptor subunits α7, α9, α10, and Src kinase activation and results in an inhibition of ATP-induced caspase-1 activation. In conclusion, we propose a novel anti-inflammatory mechanism induced by SLPI, which inhibits the ATP-dependent maturation and secretion of IL-1β. This novel signaling pathway might lead to development of therapies that are urgently needed for the prevention and treatment of systemic inflammation.

Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a synthetic bile acid-phospholipid conjugate with profound hepatoprotective and anti-fibrogenic functions in vitro and in vivo. Herein, we aimed to demonstrate the inhibitory effects of UDCA-LPE on pro-fibrogenic integrin signalling. UDCA-LPE treatment of human embryonic liver cell line CL48 and primary human hepatic stellate cells induced a non-classical internalization of integrin &beta, 1 resulting in dephosphorylation and inhibition of SRC and focal adhesion kinase (FAK). Signalling analyses suggested that UDCA-LPE may act as a heterobivalent ligand for integrins and lysophospholipid receptor1 (LPAR1) and co-immunoprecipitation demonstrated the bridging effect of UDCA-LPE on integrin &beta, 1 and LPAR1. The disruption of either the UDCA-moiety binding to integrins by RGD-containing peptide GRGDSP or the LPE-moiety binding to LPAR1 by LPAR1 antagonist Ki16425 reversed inhibitory functions of UDCA-LPE. The lack of inhibitory functions of UDCA-PE and UDCA-LPE derivatives (14:0 and 12:0, LPE-moiety containing shorter fatty acid chain) as well as the consistency of the translocation of UDCA-LPE and integrins, which co-fractionated with LPE but not UDCA, suggested that the observed UDCA-LPE-induced translocation of integrins was mediated by LPE endocytic transport pathway.

Fatty acid transport protein4 (FATP4) is upregulated in acquired and central obesity and its polymorphisms are associated with blood lipids and insulin resistance. Patients with FATP4 mutations and mice with global FATP4 deletion exhibit skin abnormalities characterized as ischthyosis prematurity syndrome (IPS). Cumulating data have shown that an absence of FATP4 increases the levels of cellular triglycerides (TG). However, FATP4 role and consequent lipid and TG metabolism in the hepatocyte is still elusive. Here, hepatocyte-specific FATP4 deficient (Fatp4L−/−) mice were generated. When fed with chow, these mutant mice displayed no phenotypes regarding blood lipids. However when fed low-fat/high-sugar (HS) or high-fat/high-sugar (HFS) for 12 weeks, Fatp4L−/− mice showed a significant increase of plasma TG, free fatty acids and glycerol when compared with diet-fed control mice. Interestingly, Fatp4L−/− mice under HS diet had lower body and liver weights and they were not protected from HFS-induced body weight gain and hepatic steatosis. Male mutant mice were more sensitive to HFS diet than female mutant mice. Glucose intolerance was observed only in female Fatp4L−/− mice fed with HS diet. Lipidomics analyses revealed that hepatic phospholipids were not disturbed in mutant mice under both diets. Thus, hepatic FATP4 deletion rendered an increase of blood lipids including glycerol indicating a preferential fatty-acid channeling to TG pools that are specifically available for lipolysis. Our results imply a possible risk of hyperlipidemia as a result of abnormal metabolism in liver in IPS patients with FATP4 mutations who consume high-sugar diets.

Group VIA calcium-independent phospholipase A2 (iPla2β) is among modifier genes of non-alcoholic fatty liver disease which leads to non-alcoholic steatohepatitis (NASH). Consistently, iPla2β deletion protects hepatic steatosis and obesity in genetic ob/ob and obese mice chronically fed with high-fat diet by replenishing the loss of hepatic phospholipids (PL). As mouse feeding with methionine- and choline-deficient (MCD) diet is a model of lean NASH, we tested whether iPla2β-null mice could still be protected since PL syntheses are disturbed. MCD-diet feeding of female wild-type for 5 weeks induced hepatic steatosis with a severe reduction of body and visceral fat weights concomitant with a decrease of hepatic phosphatidylcholine. These parameters were not altered in MCD-fed iPla2β-null mice. However, iPla2β deficiency attenuated MCD-induced elevation of serum transaminase activities and hepatic expression of fatty-acid translocase Cd36, fatty-acid binding protein-4, peroxisome-proliferator activated receptorγ, and HDL-uptake scavenger receptor B type 1. The reduction of lipid uptake genes was consistent with a decrease of hepatic esterified and unesterified fatty acids and cholesterol esters. On the contrary, iPla2β deficiency under MCD did not have any effects on inflammasomes and pro-inflammatory markers but exacerbated hepatic expression of myofibroblast α-smooth muscle actin and vimentin. Thus, without any rescue of PL loss, iPla2β inactivation attenuated hepatocellular injury in MCD-induced NASH with a novel mechanism of lipid uptake inhibition. Taken together, we have shown that iPla2β mediates hepatic steatosis and lipotoxicity in hepatocytes in both obese and lean NASH, but elicits exacerbated liver fibrosis in lean NASH likely by affecting other cell types.

Background: Non-alcoholic fatty liver disease (NAFLD) is associated with inefficient macro- and micronutrient metabolism, and alteration of circulating phospholipid compositions defines the signature of NAFLD. This current study aimed to assess the pattern of serum phospholipids in the spectrum of NAFLD, and its related comorbidities and genetic modifications. Methods: 97 patients with diagnosed NAFLD were recruited at a single center during 2013–2016. Based on histological and transient elastography assessment, 69 patients were divided into non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver (NAFL) subgroups. 28 patients served as healthy controls. Serum phospholipids were determined by liquid-chromatography mass spectrometry (LC-MS/MS). Results: The total content of phosphatidylcholine (PC) and sphingomyelin in the serum was significantly increased in NAFL and NASH patients, compared to healthy controls. In addition, serum lysophospatidylethanolamine levels were significantly decreased in NAFL and NASH individuals. Circulating PC species, containing linoleic and α-linolenic acids, were markedly increased in NAFLD patients with hypertension, compared to NAFLD patients without hypertension. The pattern of phospholipids did not differ between NAFLD patients with diabetes and those without diabetes. However, NAFLD patients with hyperglycemia (blood glucose level (BGL) >100 mg/dL) exhibited significantly a higher amount of monounsaturated phosphatidylethanolamine than those with low blood glucose levels. In addition, NAFLD patients with proven GG-genotype of PNPLA3, who were at higher risk for the development of progressive disease with fibrosis, showed lower levels of circulating plasmalogens, especially 16:0, compared to those with CC- and CG-allele. Conclusions: Our extended lipidomic study presents a unique metabolic profile of circulating phospholipids associated with the presence of metabolic risk factors or the genetic background of NAFLD patients.

Background: Non-alcoholic fatty liver disease (NAFLD) is associated with inefficient macro- and micronutrient metabolism, and alteration of circulating phospholipid compositions defines the signature of NAFLD. This current study aimed to assess the pattern of serum phospholipids in the spectrum of NAFLD, and its related comorbidities and genetic modifications. Methods: 97 patients with diagnosed NAFLD were recruited at a single center during 2013–2016. Based on histological and transient elastography assessment, 69 patients were divided into non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver (NAFL) subgroups. 28 patients served as healthy controls. Serum phospholipids were determined by liquid-chromatography mass spectrometry (LC-MS/MS). Results: The total content of phosphatidylcholine (PC) and sphingomyelin in the serum was significantly increased in NAFL and NASH patients, compared to healthy controls. In addition, serum lysophospatidylethanolamine levels were significantly decreased in NAFL and NASH individuals. Circulating PC species, containing linoleic and α-linolenic acids, were markedly increased in NAFLD patients with hypertension, compared to NAFLD patients without hypertension. The pattern of phospholipids did not differ between NAFLD patients with diabetes and those without diabetes. However, NAFLD patients with hyperglycemia (blood glucose level (BGL) >100 mg/dL) exhibited significantly a higher amount of monounsaturated phosphatidylethanolamine than those with low blood glucose levels. In addition, NAFLD patients with proven GG-genotype of PNPLA3, who were at higher risk for the development of progressive disease with fibrosis, showed lower levels of circulating plasmalogens, especially 16:0, compared to those with CC- and CG-allele. Conclusions: Our extended lipidomic study presents a unique metabolic profile of circulating phospholipids associated with the presence of metabolic risk factors or the genetic background of NAFLD patients.

Proinflammation can predispose the body to autoimmunity and cancer. We have reported that iPLA2β−/− mice are susceptible to autoimmune hepatitis and colitis. Here we determined whether cytokine release by immune cells could be affected by iPLA2β deficiency alone or combined with CD95/FasL-antibody treatment in vivo. We also determined whether cancer risk could be increased in aged mutant mice. Immune cells were isolated from 3-month old male WT and iPLA2β−/− mice, and some were injected with anti-CD95/FasL antibody for 6 h. Kupffer cells (KC) or splenocytes and liver lymphocytes were stimulated in vitro by lipopolysaccharide or concanavalinA, respectively. Whole-body iPLA2β deficiency caused increased apoptosis in liver, spleen, and mesenteric lymph node (MLN). KC from mutant mice showed suppressed release of TNFα and IL-6, while their splenocytes secreted increased levels of IFNγ and IL-17a. Upon CD95/FasL activation, the mutant KC in turn showed exaggerated cytokine release, this was accompanied by an increased release of IFNγ and IL-17a by liver lymphocytes. Aged iPLA2β−/− mice did not show follicular MLN lymphoma commonly seen in aged C57/BL6 mice. Thus, iPLA2β deficiency renders M1- and Th1/Th17-proinflammation potentially leading to a reduction in age-related MLN lymphoma during aging.

Chronic bowel disease can co-exist with severe autoimmune hepatitis (AIH) in an absence of primary sclerosing cholangitis. Genetic background may contribute to this overlap syndrome. We previously have shown that the deficiency of iPLA2β causes an accumulation of hepatocyte apoptosis, and renders susceptibility for acute liver injury. We here tested whether AIH induction in iPLA2β-null mice could result in intestinal injury, and whether bile acid metabolism was altered. Control wild-type (WT) and female iPLA2β-null (iPLA2β−/−) mice were intravenously injected with 10mg/kg concanavalinA (ConA) or saline for 24h. ConA treatment of iPLA2β−/− mice caused massive liver injury with increased liver enzymes, fibrosis, and necrosis. While not affecting WT mice, ConA treatment of iPLA2β−/− mice caused severe duodenal villous atrophy concomitant with increased apoptosis, cell proliferation, globlet cell hyperplasia, and endotoxin leakage into portal vein indicating a disruption of intestinal barrier. With the greater extent than in WT mice, ConA treatment of iPLA2β−/− mice increased jejunal expression of innate response cytokines CD14, TNF-α, IL-6, and SOCS3 as well as chemokines CCL2 and the CCL3 receptor CCR5. iPLA2β deficiency in response to ConA-induced AIH caused a significant decrease in hepatic and biliary bile acids, and this was associated with suppression of hepatic Cyp7A1, Ntcp and ABCB11/Bsep and upregulation of intestinal FXR/FGF15 mRNA expression. The suppression of hepatic Ntcp expression together with the loss of intestinal barrier could account for the observed bile acid leakage into peripheral blood. Thus, enteropathy may result from acute AIH in a susceptible host such as iPLA2β deficiency.

Background Hepatic fat accumulation with disturbed lipid homoeostasis is a hallmark of nonalcoholic fatty liver disease (NAFLD). The bile acid phospholipid conjugate Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a novel anti-inflammatory agent with hepatoprotective effects in murine high-fat-diet (HFD)-induced NAFLD. The aim of this work was to study changes in the hepatic lipidome due to UDCA-LPE. Materials and methods High fat diet mouse model, mass spectometry, RT-PCR. Results Hepatic lipid extracts of HFD mice were analysed by mass spectrometry. The results determined higher levels of total, saturated, mono- and diunsaturated fatty acids (FA) in HFD mice, which were decreased by UDCA-LPE predominantly by the reducing the most abundant FA species palmitic acid and oleic acid. Unlike other FA species, levels of long-chain polyunsaturated fatty acids (LCPUFA), which are composed of arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were increased in HFD mice upon UDCA-LPE treatment, mainly due to elevated hepatic ARA pools. Analysis of hepatic phospholipids species showed a decrease in total phosphatidylcholine (PC), especially monounsaturated PC (PUFA-PC) levels in HFD mice. Loss of total PC was reversed due to UDCA-LPE by increasing hepatic PUFA-PC pools. Gene expression analysis showed that UDCA-LPE upregulated PPARα, a key transcriptional regulator of fatty acid oxidation, as well as downstream target genes CPT1α and AOX, which are crucially involved in mitochondrial and peroxisomal fatty acid oxidation. Conclusion UDCA-LPE modulates defective fatty acid metabolism during experimental NAFLD thereby restoring altered lipid profiles in addition to its pronounced anti-inflammatory effects. Thus, UDCA-LPE may be a promising drug candidate for the management of NAFLD.

Fatty acid transport protein (FATP) 4 is a minor FATP in the liver but it has some activity towards palmitate 16:0 (Pal). We here chose FATP4 as a representative model enzyme for acyl-CoA synthetases (ACSs), and FATPs to determine whether Pal activation would lead to apoptosis and alteration in lipid metabolism. By using FATP4 overexpressed (FATP4) Huh-7 cells, we showed that FATP4 was localized in the endoplasmic reticulum (ER) and mitochondria of FATP4 cells. FATP4 cells were more responsive to Pal than the control GFP cells in increasing palmitoyl-CoA and oleoyl-CoA activities as well as apoptosis by ~2-3 folds. The lipoapoptosis susceptibility by FATP4 was coupled with the increased JNK, PUMA, caspase3, PARP-1 activation as well as Rac-1-mediated cytoskeletal reorganization, and decreased insulin sensitivity. This was associated with increased contents of neutral lipids and significant alteration in composition of phospholipids and sphingolipids including increased lysophosphatidylcholine (LPC), ceramide, and hexosylceramide, as well as an increase of saturated:polyunsaturated fatty acid ratio in LPC and PC, but a decrease of this ratio in phosphatidylethanolamine pool. By use of ceramide synthase inhibitors, our results showed that FATP4-sensitized lipoapoptosis was not mediated by ceramides. Moreover, FATP4 expression was increased in fatty livers in vivo. Thus, our model system has provided a clue that Pal activation FATP4 triggers hepatocellular apoptosis via altered phospholipid composition and steatosis by acylation into complex lipids. This may be a redundant mechanism for other ER-localizing ACSs and FATPs in the liver, and hence their involvement in the development of fatty liver disease.

The ischemia and reperfusion (I/R) injury that occurs during liver transplantation causes severe complications leading to transplantation failure. We have designed a cytoprotective agent, ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), which promotes the survival of cultured hepatocellular cell lines and inhibits apoptosis and inflammation in the in vivo models of liver injury. Here, we show that UDCA-LPE increased the viability of mouse hepatocytes by activating the Akt/glycogen synthase kinase 3β survival signaling pathways. We further tested whether UDCA-LPE could protect hepatic I/R injury in mice by clamping liver lobes of C57/BL6 mice for 90 min of ischemia followed by unclamping and reperfusion for 2 h. Two regimens for UDCA-LPE treatment were carried out; with a single dose of 100 mg/kg UDCA-LPE intraperitoneally injected 30 min prior to ischemia and a double dose of 50 mg/kg UDCA-LPE given 30 min prior to ischemia and just prior to reperfusion. Using histology and liver enzyme determination, we observed that hepatic I/R caused significant hepatic necrosis, which was decreased in UDCA-LPE-treated mice undergoing I/R. Ursodeoxycholyl LPE concomitantly protected against I/R-induced apoptosis (cleaved caspase 3, cleaved poly[ADP-ribose] polymerase 1), inflammation (IL-1β, CD11b, chemokine ligands 2 and 3, chemokine receptor 2), and portal fibrogenesis (α-smooth muscle actin, plasminogen activator inhibitor 1), as determined by Western blot, quantitative real-time polymerase chain reaction, and immunohistochemical analyses. The protection by UDCA-LPE was found to be better in the double-dose than in the single-dose regimen. Thus, UDCA-LPE promoted the survival of mouse hepatocytes and protected against hepatic I/R injury and thus may be of therapeutic use in liver transplantation settings.