Group VIA calcium-independent phospholipaseA2 (iPLA2β or PLA2G6) is a homeostatic enzyme involved in basal glycerophospholipid metabolism. The mutations in the PLA2G6 gene lead to heterogenous neurodegenerative disorders. Global PLA2G6 inactivation in iPLA2β-null mice exhibited liver fibrosis and intestinal atrophy when they reached an advanced age at 20–22 months old. Here, we analyzed the phenotypes of iPLA2β-null mice which happened to be exposed to natural pathogens in our animal facility. Compared with wild-type, male iPLA2β-null mice at 9–14 months of age exhibited reduced body, liver, and subcutaneous fat weights concomitant with decreased hepatic triacylglycerol and decreased expression of de novo lipogenesis genes. Hepatocytes from male mutants were sensitive to apoptosis induced by palmitic acid. Male but not female mutants displayed attenuation of hepatic lipid synthesis; however hepatic fibrosis was increased in mutants of both sexes. Hepatic apoptosis was also increased in mutants of both sexes, and they were susceptible to endotoxin-induced liver injury. Hence, global PLA2G6 inactivation combined with natural infection accelerates progression of chronic liver disease in both male and female mice with male-biased alteration of hepatocellular glycerolipid metabolism.

BACKGROUND AND AIMS:Polymorphisms of group VIA calcium-independent phospholipase A2 (iPLA2β or PLA2G6) are associated with Type-2 diabetes, blood lipids and inflammation. Global deficiency in iPLA2β-null mice elicited protection against hepatic steatosis but not hepatic inflammation after high-fat diet (HFD) feeding. We aimed to determine whether HFD-induced phenotypes could be affected by PLA2G6 deficiency specifically in myeloid cells and hepatocytes. METHODS:Male control Pla2g6flox/flox, myeloid-(Pla2g6M-/-) and hepatocyte-(Pla2g6Hep-/-) specific Pla2g6-deficient mice were subjected to chow or HFD feeding for 6 months. The contents of phospholipids, white blood cell counts, plasma cytokines and metabolic parameters were quantified. Hepatic inflammation, lymphopoiesis and fibrosis were evaluated by histology, immunohistochemistry, Western blot and qRT-PCR. RESULTS:Increased levels of phospholipids were observed in bone marrow-derived macrophages and livers from chow-fed Pla2g6M-/- and Pla2g6Hep-/- mice, respectively. After HFD feeding, Pla2g6M-/- mice displayed a further increase in hepatic recruitment of granulocytes and lymphocytes, plasma cytokines/lipids, liver inflammation/fibrosis as well as metabolic parameters including plasma lipoproteins, plasma/liver lipopolysaccharides, liver triglycerides/non-esterified free fatty acids, plasma insulin/leptin and HOMA-IR. These metabolic parameters were further increased in HFD-fed Pla2g6Hep-/- mice; however, they were protected from hepatic programmed cell death and inflammatory fibrosis with attenuation of plasma lipids and cytokines. Remarkably, these metabolic parameters were also increased in both mutants under chow. CONCLUSION:Myeloid- and hepatocyte-PLA2G6 deficiency elicited aggravation and protection against HFD-induced hepatic inflammation, respectively. However, PLA2G6 deficiency in both cell types exacerbated insulin resistance. PLA2G6 inactivation specifically in hepatocytes may provide a potential therapy option to alleviate diet-induced liver inflammation.

Genetic PLA2G6 variants are associated with C-reactive protein in humans. Myeloid-specific PLA2G6-deficient (Pla2g6M-/-) mice show increased hepatic myeloperoxidase and recruitment of granulocytes in response to lipopolysaccharide (LPS). We hypothesized that Pla2g6M-/- mice could be protected from acetaminophen (APAP) hepatotoxicity whereby neutrophils, eosinophils, and alternatively activated macrophages are reportedly protective. Herein, Pla2g6M-/- mice treated with 300 mg/kg APAP for 24 h showed attenuated hepatic necrosis and plasma cytokines, and with elevated levels of Ly6Clo in peripheral blood mononuclear cells and plasma lipoxin A4. Remarkably, bone-marrow-derived macrophages (BMDMs) from untreated Pla2g6M-/- mice exhibited elevated baseline expression of cPLA2α, NOX2, Rac1, Arg-1, phospho-MLKL, and iNOS protein, which was exacerbated by LPS in vitro. APAP administration preconditioned Pla2g6M-/- BMDMs for further activation of enzymes involving in phagocytosis (Rac1 and phospho-MLKL) and eicosanoids (COX2 and A15LOXB). Pla2g6M-/- BMDMs showed an increased release of pro-resolution lipid mediators lipoxin A4, PGE2, and 15d-PGJ2, which was further elevated by LPS in vitro or APAP in vivo. Phagocytic gene signatures (myeloperoxidase and NOX2) were also upregulated in livers of untreated and APAP-treated Pla2g6M-/- mice. APAP protection in Pla2g6M-/- mice was associated with increased proportion of neutrophils (Ly6G), eosinophils (eosinophilic cationic protein), and M2 macrophages (CD206) in/at the portal tract and central vein as determined by immunohistochemistry. Thus, myeloid-specific PLA2G6 deficiency preconditioned macrophages for eicosanoid and phagocytic pathways rendering protection against APAP hepatotoxicity. Our results may be applicable to patients with PLA2G6 mutations, and PLA2G6 inhibition specifically in myeloid cells may represent a new strategy to alleviate APAP poisoning.

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 Pla2g6(M-/-) 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 Pla2g6(M-/-) (but not Flox) mice at 12 months of age exhibited splenomegaly and hepatic necrosis, and similar to 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-1 alpha and immunoglobulinA as well as upregulation of hepatic apoptosis and fibrosis PARP-1, Bax, MCP-1, alpha-SMA, and collagen I proteins. Their bone-marrow-derived macrophages also showed an elevation of MIP-1 alpha 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, similar to 30 % of these mutants exhibited eosinophilic sclerosing portal hepatitis associated with an upregulated protein expression of hepatic CD8 alpha, CD68, eosinophilic cationic protein, and Ly6G. Thus, myeloid-PLA2G6 deficiency led to an autoimmune and LPS-induced inflammatory liver disease via MIP-1 alpha in a male-predominant manner. Our results may be applicable to patients with PLA2G6 mutations who undergo bacterial infection and sepsis.

Fatty acid transport protein (FATP)4 was thought to mediate intestinal lipid absorption, which was disputed by a study using keratinocyte-Fatp4-rescued Fatp4(-/-) mice. These knockouts when fed with a Western diet showed elevated intestinal triglyceride (TG) and fatty acid levels. To investigate a possible role of FATP4 on intestinal lipid processing, ent-Fatp4 (KO) mice were generated by Villin-Cre-specific inactivation of the Fatp4 gene. We aimed to measure circulating and intestinal lipids in control and KO mice after acute or chronic fat intake or during aging. Remarkably, ent-Fatp4 mice displayed an approximately 30% decrease in ileal behenic, lignoceric, and nervonic acids, ceramides containing these FA, as well as, ileal sphingomyelin, phosphatidylcholine, and phosphatidylinositol levels. Such decreases were concomitant with an increase in jejunal cholesterol ester. After a 2-wk recovery from high lipid overload by tyloxapol and oral-lipid treatment, ent-Fatp4 mice showed an increase in plasma TG and chylomicrons. Upon overnight fasting followed by an oral fat meal, ent-Fatp4 mice showed an increase in plasma TG-rich lipoproteins and the particle number of chylomicrons and very low-density lipoproteins. During aging or after feeding with a high-fat high-cholesterol (HFHC) diet, ent-Fatp4 mice showed an increase in plasma TG, fatty acids, glycerol, and lipoproteins as well as intestinal lipids. HFHC-fed KO mice displayed an increase in body weight, the number of lipid droplets with larger sizes in the ileum, concomitant with a decrease in ileal ceramides and phosphatidylcholine. Thus, enterocyte FATP4 deficiency led to a metabolic shift from polar to neutral lipids in distal intestine rendering an increase in plasma lipids and lipoproteins. NEW & NOTEWORTHY Enterocyte-specific Fatp4 deficiency in mice increased intestinal lipid absorption with elevation of blood lipids during fasting and aging, as well as after an acute oral fat-loading or chronic HFHC feeding. Lipidomics revealed that knockout mice displayed a shift from very long-chain to long-chain fatty acids, and from polar to neutral lipids, predominantly in the ileum. Thus, FATP4 may have a physiological function in the control of blood lipids via metabolic shifts in distal intestine.

Background Polymorphisms of PLA2G6 are associated with elevation of serum C-reactive protein, and PLA2G6 mutations lead to neurodegeneration and Parkinson’s disease. Age-related abnormalities including hepatic fibrosis have been reported in global Pla2g6-null mice. We recently showed that myeloid-specific Pla2g6-deficient mice exhibited NASH susceptibility (BBA 2023). We therefore evaluated whether these mutants would exhibit inflammatory fibrogenesis during aging.

FATP4 deficiency in BMDMs and Kupffer cells led to increased proinflammatory response. Fatp4 M−/− 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.

Evidence from mice with global deletion of fatty-acid transport protein4 (FATP4) indicates its role on β-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 β-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 β-oxidation towards lipolysis-directed TG and lipoprotein secretion, which is in line with an association of FATP4 polymorphisms with blood lipids.

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. In GASL2021 abstract, we reported that female mice with myeloid-specific PLA2G6 deletion showed exacerbated hepatic fibrosis after feeding with methionine- and choline-deficient diet (MCDD). Herein, female mice with hepatocyte- (LPla2g6−/−) specific PLA2G6 deletion were phenotyped after feeding with chow or MCDD for 3.5 weeks. LPla2g6−/−mice fed with chow displayed an attenuation of blood monocytes and elevation of anti-inflammatory/pro-resolution lipoxin A4 in plasma and liver. Compared with chow, MCDD-fed mutants showed a greater elevation of hepatic lipoxin A4. Consistently, MCDD-fed LPla2g6−/−mice showed attenuated levels of plasma alanine aminotransferase, plasma non-esterified fatty acids, blood monocytes, and hepatic triglycerides. Hepatic steatosis protection was associated with upregulation of PPARalpha and attenuated hepatic expression of PPARgamma, fatty-acid uptake, triglyceride synthesis, and de novo lipogenesis genes. LPla2g6−/−mice were also protected against MCDD-induced hepatic fibrosis associated with attenuated expression of IL-6, TNF-alpha, CD115, Collagen1alpha1, Collagen4alpha1, alpha-SMA, TIMP1, TGFbeta-1, vimentin, CHOP, p-JNK, as well as the reduction of sirus-red, alpha-SMA, and cleaved caspase 3 (+) staining. Thus, PLA2G6 inactivation specifically in myeloid cells and hepatocytes led to opposing phenotypes in female mice undergoing NASH. Mechanistically, PLA2G6 inactivation in hepatocytes may induce the mobilization of arachidonate which becomes the substrate of 12/15-lipoxygenases activated by MCDD, thus resulting in the elevation of protective lipoxin A4. Hepatocyte-specific PLA2G6 inhibitors may be further developed for treatment of this 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−/−) 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.

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.

Background PLA2g6 mutations lead to neurodegeneration and Parkinson’s disease and polymorphisms of PLA2g6 are associated with increase of serum C-reactive protein. Hepatic inflammation was exaggerated in PLA2g6-null mice during autoimmune hepatitis and NASH. The role of PLA2g6 in macrophages (MΦ) is still elusive. Here, we studied the response of MΦ-specific PLA2g6 deficient (KO) mice to E.Coli lipopolysaccharide (LPS) treatment.

Background Increased expression of adipose Fatty Acid Transport Protein 4 (FATP4) is observed in obese individuals. FATP4 mutations are associated with blood triglycerides (TG) and insulin resistance. FATP4 deficiency may cause metabolic risks. We have reported an increase in secreted TG, glycerol, and non-esterified free fatty acids (NEFA) in liver-specific Fatp4-deficient (L-FATP4-/-) mice fed with high-sugar and high-fat diets. These lipids were also elevated in FATP4-deficient HepG2 (HepKO) cells. Here, we investigated the mechanisms for these increases.

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.

Background Polymorphisms of iPLA2β are associated with an increase of serum C-reactive protein suggesting its role in inflammation. It is known that iPla2β regulates monocyte differentiation and monocyte migration to sites of inflammation, however, its role in NASH is still elusive. We previously reported that female iPla2β-null mice fed with methionine- and choline-deficient diet (MCDD) displayed opposing phenotypes; on one hand attenuation of liver enzymes and liver fatty acids but on the other hand exacerbation of liver fibrosis (BBA 2019, 1864, 677). Here, we examined whether macrophage-specific iPla2β deletion has any effects on NASH induced by MCDD.

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, 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 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.

Background Mutations of fatty acid transport protein 4 (FATP4) cause ichthyosis prematurity syndrome (IPS) characterized by skin lesions and associated with eosinophilia and allergies. We hypothesized that FATP4 may have biological functions in immune cells, particularly macrophages, as M2 macrophages are known to mediate allergic inflammation. Therefore, lysM-Cre Fatp4 deficient (KO) mice were generated and subjected to high-fat/high-cholesterol (HFHC) diet as a model of non-alcoholic steatohepatitis (NASH) and chronic stress.

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 fatty acid transport protein 4 (FATP4) are associated with blood lipoproteins and insulin resistance. As FATP4 is localized in intracellular organelles and highly expressed in mouse small intestine, Fatp4 role in fat absorption in mice is still controversial. As our recent studies have suggested Fatp4 role in triglyceride (TAG) metabolism, we here investigated intestinal fat absorption and blood TAG-chylomicrons (CM) in villin-Cre specific inactivation of the Fatp4 gene with exon 3 deletion (entFatp4KO mice).