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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.

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.

Polymorphisms of group VIA calcium-independent phospholipase A2 (iPLA2β orPLA2G6) are positively associated with adiposity, blood lipids, and Type-2 diabetes. Theubiquitously expressed iPLA2β catalyzes the hydrolysis of phospholipids (PLs) to generate a fattyacid and a lysoPL. We studied the role of iPLA2β on PL metabolism in non-alcoholic fatty liverdisease (NAFLD). By using global deletion iPLA2β-null mice, we investigated three NAFLD mousemodels; genetic Ob/Ob and long-term high-fat-diet (HFD) feeding (representing obese NAFLD) aswell as feeding with methionine- and choline-deficient (MCD) diet (representing non-obeseNAFLD). A decrease of hepatic PLs containing monounsaturated- and polyunsaturated fatty acidsand a decrease of the ratio between PLs and cholesterol esters were observed in all three NAFLDmodels. iPLA2β deficiency rescued these decreases in obese, but not in non-obese, NAFLD models.iPLA2β deficiency elicited protection against fatty liver and obesity in the order of Ob/Ob › HFD »MCD. Liver inflammation was not protected in HFD NAFLD, and that liver fibrosis was evenexaggerated in non-obese MCD model. Thus, the rescue of hepatic PL remodeling defect observedin iPLA2β-null mice was critical for the protection against NAFLD and obesity. However, iPLA2βdeletion in specific cell types such as macrophages may render liver inflammation and fibrosis,independent of steatosis protection.

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.

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.

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.

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.

An increase of toxic bile acids such as glycochenodeoxycholic acid occurs during warm ischemia reperfusion causing cholestasis and damage in hepatocytes and intrahepatic biliary epithelial cells. We aim to test antiapoptosis effects of ursodeoxycholyl lysophosphatidylethanolamide under cholestatic induction by glycochenodeoxycholic acid treatment of mouse hepatocytes and hypoxia induction by cobalt chloride treatment of intrahepatic biliary epithelial cancer Mz-ChA-1cell line. Such treatments caused marked increases in apoptosis as evidenced by activation of caspase 3, caspase 8 and poly (ADP-ribose) polymerase-1. Co-treatment with ursodeoxycholyl lysophosphatidylethanolamide significantly inhibited these increases. Interestingly, ursodeoxycholyl lysophosphatidylethanolamide was able to increase expression of antiapoptotic cellular FLICE-inhibitory protein in both cell types. Ursodeoxycholyl lysophosphatidylethanolamide also prevented the decreases of myeloid cell leukemia sequence-1 protein in both experimental systems, and this protection was due to ursodeoxycholyl lysophosphatidylethanolamide’s ability to inhibit ubiquitination-mediated degradation of myeloid cell leukemia sequence-1, and to increase the phosphorylation of GSK-3β. In addition, ursodeoxycholyl lysophosphatidylethanolamide was able to prevent the decreased expression of another antiapoptotic cellular inhibitor of apoptosis 2 in cobalt chloride-treated Mz-ChA-1 cells. Hence, ursodeoxycholyl lysophosphatidylethanolamide mediated cytoprotection against apoptosis during toxic bile-acid and ischemic stresses by a mechanism involving accumulation of cellular FLICE-inhibitory protein, myeloid cell leukemia sequence-1 and cellular inhibitor of apoptosis 2 proteins. Ursodeoxycholyl lysophosphatidylethanolamide may thus be used as an agent to prevent hepatic ischemia reperfusion.

Excess hepatic fat accumulation leads to nonalcoholic steatohepatitis (NASH), a serious threat to health for which no effective treatment is available. However, the mechanism responsible for fatty acid uptake by hepatocytes remains unclear. Using the human hepatocyte-derived tumor cell line HepG2, we found that fatty acid influx is mediated by a heterotetrameric plasma membrane protein complex consisting of plasma membrane fatty acid-binding protein, caveolin-1, CD36, and calcium-independent membrane phospholipase A2 (iPLA2β). Blocking iPLA2β with the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) caused the dissociation of the complex, thereby inhibiting fatty acid influx (IC50 47 μM), and suppressed the synthesis of all subunits through a reduction in lysophosphatidylcholine from 8.0 to 3.5 μmol/mg of protein and corresponding depletion of phosphorylated c-Jun N-terminal kinase. These findings were substantiated by an observed 56.5% decrease in fatty acid influx in isolated hepatocytes derived from iPLA2β-knockout mice. Moreover, steatosis and inflammation were abrogated by UDCA-LPE treatment in a cellular model of NASH. Thus, iPLA2β acts as an upstream checkpoint for mechanisms that regulate fatty acid uptake, and its inhibition by UDCA-LPE qualifies this nontoxic compound as a therapeutic candidate for the treatment of NASH.-Stremmel, W., Staffer, S., Wannhoff, A., Pathil, A., Chamulitrat, W. Plasma membrane phospholipase A2 controls hepatocellular fatty acid uptake and is responsive to pharmacological modulation: implications for nonalcoholic steatohepatitis.

To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines – FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells., Graphical abstract, Highlights • One-step tumorigenic conversion of human epithelial cells does not occur. • Immortalization is a crucial step for tumorigenic conversion. • Immortalized human keratinocytes were used to study carcinogenic potential of Nox1. • Selectable cells with spindle and cobblestone morphology were obtained. • Nox1 regulates intermediate filaments leading to epithelial mesenchymal transition.

FATP4 expression is upregulated in acquired obesity and polymorphisms in the FATP4 gene are associated with increased blood triglycerides (TG). Since non‐alcoholic fatty liver disease (NAFLD) is a manifestation of obesity, it is of interest to understand how FATP4 upon fat overload mediates alterations of lipids that lead to pathogenesis of fatty liver. A model system with FATP4‐overexpressed (FATP4) and control Huh‐7 cell lines is used to determine FATP4 response to palmitate (Pal). By lipidomics methods, cellular phospholipids (PL), neutral lipids TG, free cholesterol (FC), and cholesterol esters (CE) as well as lipoprotein secretion are determined. Without Pal, FATP4 cells show a significant increase of TG, phosphatidylcholine (PC), and the release of TG‐rich and cholesterol‐rich low density lipoproteins. Pal‐treated FATP4 cells show an increase of all PL subclasses except for phosphatidylserine (PS), TG, FC, and CE. By analyses of ratios among PL subclasses and between PL and neutral lipids, it is determined that FATP4 in response to Pal causes sequentially fatty‐acid channeling with a shift from PC and phosphatidylinositol (PI) to neutral lipids and from neutral lipids to PS and phosphatidylethanolamine (PE). This response is concomitant with an activation of CAAT/enhancer binding homologous protein and MAP Kinase JNK. Thus, FATP4 overexpression in response to Pal leads to enhancement of neutral lipids which play a central role in fatty‐acid distribution from PC and PI to PE and PS that led to an alteration of ER and mitochondrial membranes and subsequent stress signalling. Practical Applications: Activation of fatty acids by fatty acid transport proteins (FATP) plays an important role in development of obesity and NAFLD. This work highlights functional role of FATP4 on fatty‐acid distribution among neutral lipids and phospholipids upon fat overload. These changes may contribute to stress signalling in the pathogenesis of NAFLD. Compositional shift among total PL subclasses and neutral lipids.

Fatp4 exhibits acyl-CoA synthetase activity and is thereby able to catalyze the activation of fatty acids for further metabolism. However, its actual function in most tissues remains unresolved, and its role in cellular fatty acid uptake is still controversial. To characterize Fatp4 functions in adipocytes in vivo, we generated a mouse line with adipocyte-specific inactivation of the Fatp4 gene (Fatp4(A-/-)). Under standard conditions mutant mice showed no phenotypical aberrance. Uptake of radiolabeled palmitic and lignoceric acid into adipose tissue of Fatp4(A-/-) mice was unchanged. When exposed to a diet enriched in long chain fatty acids, Fatp4(A-/-) mice gained more body weight compared with control mice, although they were not consuming more food. Pronounced obesity was accompanied by a thicker layer of subcutaneous fat and greater adipocyte circumference, although expression of genes involved in de novo lipogenesis was not changed. However, the increase in total fat mass was contrasted by a significant decrease in various phospholipids, sphingomyelin, and cholesteryl esters in adipocytes. Livers of Fatp4-deficient animals under a high fat diet exhibited a higher degree of fatty degeneration. Nonetheless, no evidence for changes in insulin sensitivity and adipose inflammation was found. In summary, the results of this study confirm that Fatp4 is not crucial for fatty acid uptake into adipocytes. Instead, under the condition of a diet enriched in long chain fatty acids, adipocyte-specific Fatp4 deficiency results in adipose hypertrophy and profound alterations in the metabolism of complex lipids.

A catalytic subunit of NADPH oxidase 1 (Nox1) is implicated to be involved in neoplastic progression in human epithelial cancers. We had previously demonstrated that Nox1 overexpression of immortalized epithelial cells was able to induce the generation of progenitor cells that expressed fetal-type cytokeratins 8 and 18.We aimed to investigate the direct effects and underlying mechanisms of Nox1 on expression of cytokeratin 18 (CK18).Immortalized human epithelial GM16 cells with low CK18 were used in Nox1 overexpression experiments. NuB2 cells with high CK18 were used in Nox1 knockdown experiments. Protein expression of CK18, phosphorylated and ubiquitinated CK18 were analyzed by Western blot.With no effects on the mRNA levels, CK18 protein was increased upon Nox1 overexpression and decreased upon Nox1 knockdown. Treatment with proteasome inhibitor MG132 prevented CK18 degradation and increased CK18 protein indicating translational regulation of CK18. Treatment for NuB2 cells with N-acetyl-L: -cysteine, diphenyleneiodonium, or apocynin decreased CK18 protein levels indicating its regulation involving reactive oxygen species and flavoprotein Nox. It has been known that phosphorylation of CK18 regulates CK18 turnover by ubiquination. Consistently, Nox1 modulated CK18 phosphorylation at ser52. Nox1 knockdown and treatment with diphenyleneiodonium accumulated the levels of ubiquinated CK18 enhancing degradation causing decreased CK18 protein.We demonstrated that Nox1 was able to induce CK18 stabilization by inhibiting CK18 protein degradation in a phosphorylation-dependent manner. CK18 accumulation induced by Nox1 is consistent with the persistence of fetal-type CK18 protein in many epithelial carcinomas.

Eur J Clin Invest 2011 Abstract Introduction The bile acid–phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a promising novel compound with profound hepatoprotective functions in vitro and in vivo. Because of high costs of LPE synthesis from hydrolysis of phosphatidylethanolamide (PE), costs for UDCA-LPE synthesis for in vivo and human use can become quite high. In this study, we evaluated whether ursodeoxycholyl phosphatidylethanolamide (UDCA-PE), which is more cost-effective, could replace UDCA-LPE in terms of protection from hepatocellular injury. Materials and methods Anti-apoptotic and anti-inflammatory properties of UDCA-PE and UDCA-LPE were compared in TNFα/cyclohexamide (CHX)-treated HepG2 cells as well as in a mouse model of d-galactosamine/lipopolysaccharide (Gal/LPS)-induced acute liver injury. Results Ursodeoxycholyl lysophosphatidylethanolamide inhibited TNFα/CHX-induced apoptosis in HepG2 cells in a dose-dependent manner and markedly ameliorated Gal/LPS-mediated fulminant hepatitis in mice. In contrast, UDCA-PE showed weaker hepatoprotective functions at low concentrations, and protection was lost at higher dosage. Analysis of hepatic gene expression showed that both conjugates significantly reduced Gal/LPS-mediated expression of chemoattractants, such as monocyte chemotactic protein 1 (MCP1) and RANTES. These inhibitory effects by UDCA-PE were transient while those by UDCA-LPE were sustained in attenuating expression of inflammatory MCP1 and RANTES expression. Conclusions Our data underline the superiority of UDCA-LPE compared to UDCA-PE in ameliorating acute liver inflammation. This indicates the significance of the lyso-functional group of bile acid conjugate for optimal hepatoprotection and reduction in inflammation in vivo.

Die nichtalkoholische Fettlebererkrankung (NAFLD) und die nichtalkoholische Fettleberhepatitis (NASH) beschreiben atiologisch nicht vollstandig geklarte Krankheitsentitaten, fur die keine gesicherte Therapie vorhanden ist. Dies ist eine medizinische Herausforderung, da ein progredienter Krankheitsverlauf von der einfachen Steatose zur Steatohepatitis mit Entwicklung einer Fibrose bis hin zur Zirrhose und zu eventueller Entstehung eines Leberzellkarzinoms beschrieben ist. Ein Uberangebot von Fettsauren, z. B. bei hochkalorischer Ernahrung oder deren Freisetzung aus dem Fettgewebe, fuhren zur Triglyceridablagerung in den Lipidtropfchen („lipid droplets“) der Hepatozyten. Gesattigte Fettsauren bewirken dort ER-Stress, der uber intrazellulare Signalwege Insulinresistenz, Entzundung und Apoptose induziert (klinisches Bild der NASH). Die dafur verantwortliche intrazellulare Schaltstelle ist das aktivierte JNK1. Die nachgeordnete Deaktivierung von FXR perpetuiert uber Insulinresistenz die Lipidtropfchenakkumulation, unterhalt die Entzundung und fuhrt zur Hyperglykamie. Apoptotischer Zelltod wird durch JNK1 unmittelbar oder durch Freisetzung von TNF-α (verstarkt durch Aktivierung von Entzundungszellen der Leber) mittelbar hervorgerufen. Neben den bekannten Signalkaskaden spielt die Aktivierung von mitochondrialer Phospholipase A2 (iPLA2) mit Freisetzung von Lysophosphatidylcholin bei der Apoptoseinduktion eine entscheidende Rolle. Zukunftige therapeutische Konzepte sollten deshalb auf Vermeidung von ER-Stress, Beibehaltung der FXR-Aktivierung und Hemmung der iPLA2 fokussieren.

Silybin or silymarin extract has been used to treat liver diseases, and has now been entered into clinical trials for cancer treatment. Here, we compared antioxidant and anticancer activities between silybin and its oxidized form 2,3-dehydrosilybin (DHS). With IC50 at three-fold lower concentrations than silybin, DHS inhibited reactive oxygen species generation in glucose-glucose oxidase system and HepG2 cells. Compared with silybin, DHS elicited greater protection against H2O2-induced HepG2 cell death and galactosamine-induced liver injury in vivo. It is known that oxidants induce releases of metalloproteinases (MMP)-2,-9 which are responsible for invasive and metastasis potentials of transformed cells. DHS at 10 microM markedly inhibited MMP-2,-9 releases as well as invasiveness, while silybin at 90 microM had marginal effects. DHS but not silybin at 30 microM induced apoptosis and loss of mitochondrial membrane potentials. LD50 of DHS was five-fold lower than that of silybin. Our data suggest that DHS may be more useful therapeutically than silybin.

Ethanol treatment of immortalised human gingival keratinocytes (IHGK) yields in an epithelium-like (EPI) and fibroblast-like (FIB) phenotype. With respect to the stratified gingival epithelium, putative structural and molecular differences assigning cells to these phenotypes have not, to date, been analysed in a three-dimensional tissue/epithelial context. Therefore, we generated epithelial equivalents (EEs) in organotypic co-cultures of IHGK, EPI and FIB cells for 1 and 2 weeks and conducted protein and gene expression studies on the EEs for epithelial biomarkers including keratin K14, integrin subunits alpha6 and beta1, E-cadherin, and mesenchymal vimentin. As in the EEs of IHGK and EPI, indirect immunofluorescence revealed continuous expression of beta1 integrin in EEs of FIB cells. However, FIB cells exhibited a significant down-regulation in K14 and integrin alpha6 protein and a loss of E-cadherin at week 2, whereas vimentin was increased. FIB EEs were devoid of transcripts for E-cadherin at both time points, although transcription of the other genes remained constant in all phenotypes. Thus, the FIB phenotype exhibited a poor epithelial structure coinciding with disturbances in the expression of epithelial biomarkers and the persistence of mesenchymal vimentin. Transcription analysis revealed post-transcriptional regulation of vimentin in IHGK and EPI and of K14 and alpha6 in FIB cells. Our findings indicate that differences in the epithelial integrity and expression of molecules in EEs allow for the discrimination of EPI and FIB cells. This suggests that FIB cells share features of epithelial-mesenchymal transition and reflect a more progressive stage in epithelial cell transformation.

PLA2G6 or GVIA calcium-independent PLA2 (iPLA2β) is identified as one of the NAFLD modifier genes in humans, and thought to be a target for NAFLD therapy. iPLA2β is known to play a house-keeping role in phospholipid metabolism and remodeling. However, its role in NAFLD pathogenesis has not been supported by results obtained from high-fat feeding of iPLA2β-null (PKO) mice. Unlike livers of human NAFLD and genetically obese rodents, fatty liver induced by high-fat diet is not associated with depletion of hepatic phospholipids. We therefore tested whether iPLA2β could regulate obesity and hepatic steatosis in leptin-deficient mice by cross-breeding PKO with ob/ob mice to generate ob/ob-PKO mice. Here we observed an improvement in ob/ob-PKO mice with significant reduction in serum enzymes, lipids, glucose, insulin as well as improved glucose tolerance, and reduction in islet hyperplasia. The improvement in hepatic steatosis measured by liver triglycerides, fatty acids and cholesterol esters was associated with decreased expression of PPARγ and de novo lipogenesis genes, and the reversal of β-oxidation gene expression. Notably, ob/ob livers contained depleted levels of lysophospholipids and phospholipids, and iPLA2β deficiency in ob/ob-PKO livers lowers the former, but replenished the latter particularly phosphatidylethanolamine (PE) and phosphatidylcholine (PC) that contained arachidonic (AA) and docosahexaenoic (DHA) acids. Compared with WT livers, PKO livers also contained increased PE and PC containing AA and DHA. Thus, iPLA2β deficiency protected against obesity and ob/ob fatty liver which was associated with hepatic fatty-acyl phospholipid remodeling. Our results support the deleterious role of iPLA2β in severe obesity associated NAFLD.

Background and Purpose Chronic hepatic inflammation results in liver fibrosis. As effective anti-fibrogenic agents are lacking, we investigated ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), a synthetic bile acid–phospholipid conjugate with anti-inflammatory and anti-apoptotic properties for tis effects on hepatic fibrogenesis. Experimental Approach To stimulate fibrogenesis, LX2 hepatic stellate cells were cultured with conditioned medium from CL48 liver cells after exposure to stress-inducing conditions – methionine–choline-deficient (MCD) medium or TNFα/cycloheximide (CHX) – with or without UDCA-LPE preincubation. Anti-fibrogenic effects of UDCA-LPE were further studied in CL48 and LX2 cells and in primary human hepatic stellate cells (HHStec) directly exposed to TGF-β1. To test UDCA-LPE in vivo, C57BL/6 mice were fed a MCD diet for 11 weeks followed by 30 mg·kg−1 UDCA-LPE 3× per week for 2.5 weeks. Key Results Expression of α-smooth muscle actin (α-SMA), α1-collagen, vimentin and TGF-β1 was down-regulated by up to 93% by UDCA-LPE in LX-2 cells cultured with conditioned medium. Also, UDCA-LPE inhibited Smad3 phosphorylation in CL48 cells incubated with MCD medium or TNFα/CHX and in LX2 cells exposed to conditioned medium. UDCA-LPE also decreased phosphorylated Smad3 and Smad2 directly induced by TGF-β1. Inhibition of TGF-β1/Smad2/3 signalling with down-regulation of target genes was confirmed in HHStec. In vivo, UDCA-LPE decreased hepatic α-SMA, α1-collagen and TGF-β1 expression and markedly lowered α-SMA protein and collagen deposition in MCD mice. Conclusions and Implications By blocking TGF-β1/Smad2/3 signalling, UDCA-LPE suppressed key mediators of hepatic fibrogenesis. Thus, UDCA-LPE could be suitable for prevention of fibrotic progression of chronic liver disease.