Myeloid-specific deletion of group VIA calcium-independent phospholipase A2 induces pro-inflammatory LPS response predominantly in male mice via MIP-1α activation.

Polymorphisms of group VIA calcium-independent phospholipase A2 (PLA2G6) are associated with blood C-reactive protein suggesting its role in inflammation. We showed that myeloid-specific Pla2g6-deficiency in Pla2g6M-/- mice led to exaggerated inflammation and fibrosis in a lean fatty liver model. We here investigated whether these mutants display alteration in immune response after treatment with E. coli lipopolysaccharides (LPS) under acute (a single dose) and persistent (four doses) conditions. Without LPS treatment, male Pla2g6M-/- (but not Flox) mice at 12 months of age exhibited splenomegaly and hepatic necrosis, and ~ 30 % of them exhibited autoimmune hepatitis showing lymphoplasma cells with CD3(+) and CD45R(+) staining. Under acute LPS, male mutants showed an elevation of plasma MIP-1α and immunoglobulinA as well as upregulation of hepatic apoptosis and fibrosis PARP-1, Bax, MCP-1, α-SMA, and collagen I proteins. Their bone-marrow-derived macrophages also showed an elevation of MIP-1α release upon LPS stimulation in vitro. Female mutants under acute LPS showed a moderate increase in plasma KC/CXCL1, MCP-1, and IL10, and they showed no remarkable increase in hepatic fibrosis under acute or persistent LPS. Male mutants under persistent LPS displayed an elevation of aspartate aminotransferase, blood eosinophils, and hepatic apoptosis. Moreover, ~30 % of these mutants exhibited eosinophilic sclerosing portal hepatitis associated with an upregulated protein expression of hepatic CD8α, CD68, eosinophilic cationic protein, and Ly6G. Thus, myeloid-PLA2G6 deficiency led to an autoimmune and LPS-induced inflammatory liver disease via MIP-1α in a male-predominant manner. Our results may be applicable to patients with PLA2G6 mutations who undergo bacterial infection and sepsis.

FATP4 deletion in liver cells induces elevation of extracellular lipids via metabolic channeling towards triglycerides and lipolysis.

Evidence from mice with global deletion of fatty-acid transport protein4 (FATP4) indicates its role on ß-oxidation and triglycerides (TG) metabolism. We reported that plasma glycerol and free fatty acids (FA) were increased in liver-specific Fatp4 deficient (L-FATP4-/-) mice under dietary stress. We hypothesized that FATP4 may mediate hepatocellular TG lipolysis. Here, we demonstrated that L-FATP4-/- mice showed an increase in these blood lipids, liver TG, and subcutaneous fat weights. We therefore studied TG metabolism in response to oleate treatment in two experimental models using FATP4-knockout HepG2 (HepKO) cells and L-FATP4-/- hepatocytes. Both FATP4-deificient liver cells showed a significant decrease in ß-oxidation products by ∼30-35% concomitant with marked upregulation of CD36, FATP2, and FATP5 as well as lipoprotein microsomal-triglyceride-transfer protein genes. By using 13C3D5-glycerol, HepKO cells displayed an increase in metabolically labelled TG species which were further increased with oleate treatment. This increase was concomitant with a step-wise elevation of TG in cells and supernatants as well as the secretion of cholesterol very low-density and high-density lipoproteins. Upon analyzing TG lipolytic enzymes, both mutant liver cells showed marked upregulated expression of hepatic lipase, while that of hormone-sensitive lipase and adipose-triglyceride lipase was downregulated. Lipolysis measured by extracellular glycerol and free FA was indeed increased in mutant cells, and this event was exacerbated by oleate treatment. Taken together, FATP4 deficiency in liver cells led to a metabolic shift from ß-oxidation towards lipolysis-directed TG and lipoprotein secretion, which is in line with an association of FATP4 polymorphisms with blood lipids.

Myeloid-specific fatty acid transport protein 4 deficiency induces a sex-dimorphic susceptibility for nonalcoholic steatohepatitis in mice fed a high-fat, high-cholesterol diet.

Newborns with FATP4 mutations exhibit ichthyosis prematurity syndrome (IPS), and adult patients show skin hyperkeratosis, allergies, and eosinophilia. We have previously shown that the polarization of macrophages is altered by FATP4 deficiency; however, the role of myeloid FATP4 in the pathogenesis of nonalcoholic steatohepatitis (NASH) is not known. We herein phenotyped myeloid-specific Fatp4-deficient (Fatp4M-/-) mice under chow and high-fat, high-cholesterol (HFHC) diet. Bone-marrow-derived macrophages (BMDMs) from Fatp4M-/- mice showed significant reduction in cellular sphingolipids in males and females, and additionally phospholipids in females. BMDMs and Kupffer cells from Fatp4M-/- mice exhibited increased LPS-dependent activation of proinflammatory cytokines and transcription factors PPARγ, CEBPα, and p-FoxO1. Correspondingly, these mutants under chow diet displayed thrombocytopenia, splenomegaly, and elevated liver enzymes. After HFHC feeding, Fatp4M-/- mice showed increased MCP-1 expression in livers and subcutaneous fat. Plasma MCP-1, IL4, and IL13 levels were elevated in male and female mutants, and female mutants additionally showed elevation of IL5 and IL6. After HFHC feeding, male mutants showed an increase in hepatic steatosis and inflammation, whereas female mutants showed a greater severity in hepatic fibrosis associated with immune cell infiltration. Thus, myeloid-FATP4 deficiency led to steatotic and inflammatory NASH in males and females, respectively. Our work offers some implications for patients with FATP4 mutations and also highlights considerations in the design of sex-targeted therapies for NASH treatment.NEW & NOTEWORTHY 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.

Myeloid- and hepatocyte-specific deletion of group VIA calcium-independent phospholipase A2 leads to dichotomous opposing phenotypes during MCD diet-induced NASH.

Polymorphisms of phospholipase A2VIA (iPLA2ß or PLA2G6) are associated with body weights and blood C-reactive protein. The role of iPLA2ß/PLA2G6 in non-alcoholic steatohepatitis (NASH) is still elusive because female iPla2ß-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-/-) and hepatocyte- (LPla2g6-/-) specific PLA2G6 deletion were generated and phenotyped after MCDD feeding. Without any effects on hepatic steatosis, MCDD-fed MPla2g6-/- mice showed further exaggeration of liver inflammation and fibrosis as well as elevation of plasma TNFα, CCL2, and circulating monocytes. Bone-marrow-derived macrophages (BMDMs) from MPla2g6-/- mice displayed upregulation of PPARγ and CEBPα proteins, and elevated release of IL6 and CXCL1 under LPS stimulation. LPS-stimulated BMDMs from MCDD-fed MPla2g6-/- mice showed suppressed expression of M1 Tnfa and Il6, but marked upregulation of M2 Arg1, Chil3, IL10, and IL13 as well as chemokine receptors Ccr2 and Ccr5. This in vitro shift was associated with exaggeration of hepatic M1/M2 cytokines, chemokines/chemokine receptors, and fibrosis genes. Contrarily, MCDD-fed LPla2g6-/- mice showed a complete protection which was associated with upregulation of Ppara/PPARα and attenuated expression of Pparg/PPARγ, fatty-acid uptake, triglyceride synthesis, and de novo lipogenesis genes. Interestingly, LPla2g6-/- mice fed with chow or MCDD displayed an attenuation of blood monocytes and elevation of anti-inflammatory lipoxin A4 in plasma and liver. Thus, PLA2G6 inactivation specifically in myeloid cells and hepatocytes led to opposing phenotypes in female mice undergoing NASH. Hepatocyte-specific PLA2G6 inhibitors may be further developed for treatment of this disease.

Constitutive oxidants from hepatocytes of male iPLA2ß-null mice increases the externalization of phosphatidylethanolamine on plasma membrane.

We have found that group VIA calcium-independent phospholipase A2 (iPLA2ß) 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 iPLA2ß-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 iPLA2ß 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, naïve 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 iPLA2ß deficiency-dependent PE externalization was also not correlated with apoptosis. Thus, constitutive (but not induced) ROS generation in iPLA2ß-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.

FATP4 inactivation in cultured macrophages attenuates M1- and ER stress-induced cytokine release via a metabolic shift towards triacylglycerides.

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.

Methionine- and Choline-Deficient Diet Enhances Adipose Lipolysis and Leptin Release in aP2-Cre Fatp4-Knockout Mice.

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.

The Bile Acid-Phospholipid Conjugate Ursodeoxycholyl-Lysophosphatidylethanolamide (UDCA-LPE) Disintegrates the Lipid Backbone of Raft Plasma Membrane Domains by the Removal of the Membrane Phospholipase A2.

The bile acid-phospholipid conjugate ursodeoxycholyl-lysophosphatidylethanolamide (UDCA-LPE) was shown to have anti-inflammatory, antisteatotic, and antifibrotic properties, rendering it as a drug targeting non-alcoholic steatohepatitis (NASH). On a molecular level, it disrupted the heterotetrameric fatty acid uptake complex localized in detergent-resistant membrane domains of the plasma membrane (DRM-PM). However, its mode of action was unclear. Methodologically, UDCA-LPE was incubated with the liver tumor cell line HepG2 as well as their isolated DRM-PM and all other cellular membranes (non-DRM). The membrane cholesterol and phospholipids were quantified as well as the DRM-PM protein composition by Western blotting. The results show a loss of DRM-PM by UDCA-LPE (50 µM) with a 63.13 ± 7.14% reduction of phospholipids and an 81.94 ± 8.30% reduction of cholesterol in relation to mg total protein. The ratio of phospholipids to cholesterol changed from 2:1 to 4:1, resembling those of non-DRM fractions. Among the members of the fatty acid uptake complex, the calcium-independent membrane phospholipase A2 (iPLA2ß) abandoned DRM-PM most rapidly. As a consequence, the other members of this transport system disappeared as well as the DRM-PM anchored fibrosis regulating proteins integrin ß-1 and lysophospholipid receptor 1 (LPAR-1). It is concluded that UDCA-LPE executes its action by iPLA2ß removal from DRM-PM and consequent dissolution of the raft lipid platform.

Phosphatidylcholine Passes by Paracellular Transport to the Apical Side of the Polarized Biliary Tumor Cell Line Mz-ChA-1.

Phosphatidylcholine (PC) translocation into mucus of the intestine was shown to occur via a paracellular transport across the apical/lateral tight junction (TJ) barrier. In case this could also be operative in biliary epithelial cells, this may have implication for the pathogenesis of primary sclerosing cholangitis (PSC). We here evaluated the transport of PC across polarized cholangiocytes. Therefore, the biliary tumor cell line Mz-ChA-1 was grown to confluency. In transwell culture systems the translocation of PC to the apical compartment was analyzed. After 21 days in culture, polarized Mz-ChA-1 cells revealed a predominant apical translocation of choline containing phospholipids including PC with minimal intracellular accumulation. Transport was suppressed by TJ destruction employing chemical inhibitors and pretreatment with siRNA to TJ forming proteins as well as the apical transmembrane mucin 3 as PC acceptor. Apical translocation was dependent on a negative apical electrical potential created by the cystic fibrosis transmembrane conductance regulator (CFTR) and the anion exchange protein 2 (AE2). It was stimulated by apical application of secretory mucins. The results indicated the existence of a paracellular PC passage across apical/lateral TJ of the polarized biliary epithelial tumor cell line Mz-ChA-1. This has implication for the generation of a protective mucus barrier in the biliary tree.

The Detergent Effect of Mesalazine Interferes with Phosphatidylcholine Binding to Mucin 2.

OBJECTIVES: Therapeutically applied delayed-release phosphatidylcholine (PC) revealed mucosa protection and clinical improvement of ulcerative colitis. However, a recent trial with simultaneous application of delayed-release PC and mesalazine showed lack of efficacy. It is hypothesized that mesalazine acts as detergent to prohibit PC integration into mucus as target compartment, thus preventing topical mucus protection. METHODS: In vitro PC-binding studies with mucin 2 and intestinally differentiated CaCo2 cells as well as outcome analysis of a therapeutic trial with delayed-release PC and additional mesalazine. RESULTS: Choline-containing phospholipids, in particular PC, bind to mucin 2 as main scaffold protein of intestinal mucus to establish a hydrophobic barrier towards microbiota in the intestinal lumen. PC also binds to the apical surface of polarized CaCo2 cells with membrane-anchored mucin 3. Mesalazine removes mucin-bound PC and, thus, reduces transepithelial resistance. A post hoc analysis of patients from a previous multicenter phase IIB trial with delayed-release PC revealed that those without mesalazine showed a PC dose-dependent outcome with regard to achievement of partial and complete remission (p < 0.05 for 1.6 and 3.2 g PC daily) whereas those treated simultaneously with mesalazine showed no PC dose dependency. CONCLUSION: Mesalazine solubilizes PC and, thus, prevents the protective action of therapeutically applied delayed-release PC within mucus.

iPla2ß deficiency in mice fed with MCD diet does not correct the defect of phospholipid remodeling but attenuates hepatocellular injury via an inhibition of lipid uptake genes.

Group VIA calcium-independent phospholipase A2 (iPla2ß) is among modifier genes of non-alcoholic fatty liver disease which leads to non-alcoholic steatohepatitis (NASH). Consistently, iPla2ß 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 iPla2ß-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 iPla2ß-null mice. However, iPla2ß 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, iPla2ß 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, iPla2ß inactivation attenuated hepatocellular injury in MCD-induced NASH with a novel mechanism of lipid uptake inhibition. Taken together, we have shown that iPla2ß 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.

Group VIA phospholipase A2 deficiency in mice chronically fed with high-fat-diet attenuates hepatic steatosis by correcting a defect of phospholipid remodeling.

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 (iPla2ß) 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 iPla2ß-null mice were subjected to chronic feeding with HFD for 6 months. We showed that protection was observed in iPla2ß-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. iPla2ß 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 iPla2ß 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, iPla2ß 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.

Shear-Enhanced Dynamic Adhesion of Lactobacillus rhamnosus GG on Intestinal Epithelia: Correlative Effect of Protein Expression and Interface Mechanics.

The oral uptake of probiotic microorganisms as food additives is one widely used strategy to sustain and improve the homeostasis of intestinal microbiota that protect the intestinal epithelia from attack by pathogenic bacteria. Once delivered to the ileum and colon, probiotics must adhere and form colonies on mucus that coats the surface of intestinal epithelial cells. Although an increasing amount of knowledge about the genetic and molecular level mechanisms of probiotics-mucus interactions has been accumulated, little is known about the physicochemical aspects of probiotics-mucus interactions under physiological shear in intestines. In this study, we established well-defined models of intestinal epithelial cell monolayers based on two major constituents of gut epithelia, enterocytes and goblet cells. First, the formation of a polarized cell monolayer sealed by tight junctions was monitored by transepithelial electrical resistance over time. The establishment of tight junctions and secretion of mucus proteins (mucin) was confirmed by immunofluorescence staining. In the next step, we measured the elasticity of cell monolayer surfaces by indentation using particle-assisted atomic force microscopy. The effective elastic modulus of goblet cell-like cells was 30 times smaller compared to that of enterocyte-like cells, which can be attributed to the secretion of a 3 µm thick mucin layer. As probiotics, we used Lactobacillus rhamnosus GG (LGG), which is one of the most widely used strains as food additives. To investigate the dynamic adhesion of LGG to the intestine model surface, we transferred the epithelial cell monolayer into a microfluidic chamber. A distinct difference in dynamic adhesion between two cell types was observed, which could be attributed to the difference in the mucin expression amount. Remarkably, we found that the dynamic LGG adhesion is enhanced by the increase in shear stress, showing a maximum binding efficiency at 0.3 Pa. Finally, we examined the persistence of LGG adhesion by a stepwise increase in the shear stress exerted on adherent LGG, demonstrating that LGG could withstand high shear stress even beyond that of physiological stress. The obtained results present a large potential to quantitatively understand the influence of engineered foods and probiotics on the homeostasis of microbiota on the surface of intestinal epithelia.

Phospholipase A2 of Microbiota as Pathogenetic Determinant to Induce Inflammatory States in Ulcerative Colitis: Therapeutic Implications of Phospholipase A2 Inhibitors.

BACKGROUND: Attack by commensal microbiota is one component of induction of inflammatory episodes in ulcerative colitis (UC). In UC, the mucus layer is intrinsically devoid of phosphatidylcholine (PC) resulting in low hydrophobicity which facilitates bacterial invasion. Colonic ectophospholipase-carrying bacterial strains are likely candidates to further thinning the PC mucus barrier and to precipitate inflammatory episodes. OBJECTIVE: To evaluate the effect of phospholipase A2 (PLA2) inhibitors on inflammation in a genetic UC mouse model. METHODS: As PLA2 inhibitor, we applied the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) or as control 5% Tween 80 by oral gavage to intestine-specific kindlin 2 knockout mice. RESULTS: Luminal UDCA-LPE reduced the PLA2 activity in stool by 36 ± 8%. Concomitantly no inflammatory phenotype was observed when compared to kindlin 2(-/-) mice not treated with UDCA-LPE. The improvement was documented in regard to stool consistency, calprotectin levels in stool, and macroscopic/endoscopic as well as histologic features of the mucosa. The pattern of colonic microbiota distribution obtained in the UC phenotype mice was reversed by UDCA-LPE to the control mice pattern. CONCLUSION: The inhibition of the bacterial ectophospholipase A2 activity improves mucosal inflammation in a genetic mouse model of UC. It is assumed that the remaining mucus PC shield is better preserved when luminal PLA2 is suppressed.

Genetic Mouse Models with Intestinal-Specific Tight Junction Deletion Resemble an Ulcerative Colitis Phenotype.

BACKGROUND AND AIMS: A key pathogenetic feature of ulcerative colitis [UC] is an intrinsic low mucus phosphatidylcholine[PC] content. Recently, a paracellular transport for PC across tight junctions[TJs] was described, suggesting TJ disturbance as a cause of diminished luminal PC transport. Therefore, we aimed to generate mutant mice with TJ deletion to evaluate whether a UC phenotype developed. METHODS: CL57BL/6 control wild-type mice were compared to mutant mice with tamoxifen-induced villin-Cre-dependent intestinal deletion of kindlin 1 and 2. RESULTS: Electron microscopy of mucosal biopsies obtained from both mutants before overt inflammation following only 2 days of tamoxifen exposure revealed a defective TJ morphology with extended paracellular space and, by light microscopy, expanded mucosal crypt lumina. PC secretion into mucus was reduced by >65% and the mucus PC content dropped by >50%, causing a >50 % decrease of mucus hydrophobicity in both mutants. Consequently, the microbiota was able to penetrate the submucosa. After 3 days of tamoxifen exposure, intestinal inflammation was present in both mutants, with loose bloody stools as well as macroscopic and histological features of colitis. Oral PC supplementation was able to suppress inflammation. By analogy, colonic biopsies obtained from patients with UC in remission also showed a defective epithelium with widened intercellular clefts, and enlarged crypt luminal diameters with functionally impaired luminal PC secretion. CONCLUSIONS: Genetic mouse models with intestinal deletion of kindlin 1 and 2 resulted in TJ deletion and revealed pathophysiological features of impaired PC secretion to the mucus leading to mucosal inflammation compatible with human UC.

The overall fatty acid absorption controlled by basolateral chylomicron excretion under regulation of p-JNK1.

Suppression of fatty acid absorption is one goal to fight obesity. However, the responsible molecular mechanism is poorly understood. Aim of the present study was the search for the key regulator of the overall fatty acid absorption mechanism and its pharmaceutical modulation. As experimental tool we employed the polarized human intestinal tumor derived cell line CaCo2. Here we showed that influx of fatty acids is mediated by an apical heterotetrameric plasma membrane protein complex of which the calcium-independent membrane phospholipase A2 (iPLA2ß) is one constituent. The newly synthesized bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) blocked iPLA2ß, which structurally disrupted the fatty acid-uptake complex. Furthermore, the inhibition of iPLA2ß lead to reduction of cytosolic lysophosphatidylcholine (LPC) production which suppressed p-JNK1, as a central regulator of metabolism. In a concerted action low p-JNK1 levels prohibited synthesis of the members of the fatty acid uptake complex as well as of apolipoprotein B and the connected members of the basolateral vesicular chylomicron excretion machinery, thereby inhibiting cellular lipid excretion. The basolateral chylomicron release was shown to determine the overall fatty acid-absorption capacity as rate limiting step, whereas apical uptake replenishes the cellular stores, enabling continuous transcellular movement of fatty acids. In conclusion, the UDCA-LPE mediated inhibition of p-JNK1 represents a powerful tool to control intestinal absorption of fatty acids and, thus may be employed as a drug to treat obesity.

Phosphatidylcholine passes through lateral tight junctions for paracellular transport to the apical side of the polarized intestinal tumor cell-line CaCo2.

Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1±6.3nmolh(-1)mMPC(-1) was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl(-) and HCO3(-) through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs.

Plasma membrane phospholipase A2 controls hepatocellular fatty acid uptake and is responsive to pharmacological modulation: implications for nonalcoholic steatohepatitis.

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 (iPLA2ß). Blocking iPLA2ß 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 iPLA2ß-knockout mice. Moreover, steatosis and inflammation were abrogated by UDCA-LPE treatment in a cellular model of NASH. Thus, iPLA2ß 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.

FATP4 contributes as an enzyme to the basal and insulin-mediated fatty acid uptake of C2C12 muscle cells.

The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C(2)C(12) muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C(2)C(12) cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.

Evidence of luminal phosphatidylcholine secretion in rat ileum.

BACKGROUND: Intestinal mucus not only facilitates substrate absorption, but also forms a hydrophobic, phosphatidylcholine (PC) enriched, barrier against luminal gut contents. METHODS: For evaluation of the origin of PC in intestinal mucus, we first analyzed the mucus PC in mice with absent biliary phospholipid secretion (mdr2 (-/-) mice) using electrospray ionization (ESI) tandem mass spectroscopy (MS/MS). Second, in situ perfused rat jejunum, ileum and colon were analyzed after i.v. bolus injections of 155 pmol [(3)H]-PC. Additional in vitro experiments were performed with isolated mucosal cells after incubation with the PC precursor [(3)H]-choline. RESULTS: In mdr2 (-/-) mice and control animals no significant quantitative difference in mucus PC was found, indicating that mucus PC is of intestinal and not biliary origin. In situ perfusion studies detected intestinal secretion of [(3)H]-PC, which was stimulated in presence of 2 mM taurocholate (TC). Secretion rates of [(3)H]-PC were highest in ileum (9.0+/-0.8 fmol h(-1)xcm(-1)), lower in jejunum (4.3+/-0.5) and minimal in colon (0.8+/-0.2). It compares to an intestinal secretion of native PC originating to 64% from bile, 9% from jejunum, 28% from ileum, and 1% from colon. Complementary in vitro studies showed 30-min secretion rates for [(3)H]-PC to be highest in enterocytes from ileum (26.5+/-5.3% of intracellular [(3)H]-PC) and jejunum (19.8+/-2.9%), and significantly lower in colonocytes (8.4+/-1.3%). CONCLUSION: PC in the intestinal mucus originates from secretion by ileal and jejunal enterocytes.