Mutagenesis and homology modeling reveal a predicted pocket of lysophosphatidylcholine acyltransferase 2 to catch Acyl-CoA.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions and promotes several pathological events, including anaphylaxis and neuropathic pain. PAF is biosynthesized by two types of lyso-PAF acetyltransferases: lysophosphatidylcholine acyltransferase 1 (LPCAT1) and LPCAT2, which are constitutive and inducible forms of lyso-PAF acetyltransferase, respectively. Because LPCAT2 mainly produces PAF under inflammatory stimuli, understanding the structure of LPCAT2 is important for developing specific drugs against PAF-related inflammatory diseases. Although the structure of LPCAT2 has not been determined, the crystal structure was reported for Thermotoga maritima PlsC, an enzyme in the same gene family as LPCAT2. Here, we identified residues in mouse LPCAT2 essential for its enzymatic activity and a potential acyl-coenzyme A (CoA)-binding pocket, based on homology modeling of mouse LPCAT2 with PlsC. We also found that Ala115 of mouse LPCAT2 was important for acyl-CoA selectivity. In conclusion, these results predict the three-dimensional (3D) structure of mouse LPCAT2. Our findings have implications for the future development of new drugs against PAF-related diseases.

Multiplex fatty acid imaging inside cells by Raman microscopy.

Visualizing intracellular fatty acids (including free and esterified form) is very useful for understanding how and where such molecules are incorporated, stored, and metabolized within cells. However, techniques of imaging multiple intracellular fatty acids have been limited by their small size, making it difficult to label and track without changing their biological and biophysical characteristics. Here, we present a new method for simultaneously visualizing up to five atomically labeled intracellular fatty acid species. For this, we utilized the distinctive Raman spectra depending on the labeling patterns and created a new, extensible opensource software to perform by-pixel analysis of extracting original spectra from mixed ones. Our multiplex imaging method revealed that fatty acids with more double bonds tend to concentrate more efficiently at lipid droplets. This novel approach contributes to reveal not only the spatial dynamics of fatty acids, but also of any other metabolites inside cells.

Monoacylglycerol lipase deficiency affects diet-induced obesity, fat absorption, and feeding behavior in CB1 cannabinoid receptor-deficient mice.

Excess energy intake causes obesity, which leads to insulin resistance and various other complications of metabolic syndrome, including diabetes, atherosclerosis, dyslipidemia, and nonalcoholic fatty liver disease. Although recent studies have depicted altered lipid metabolism as an underlying feature, the detailed mechanisms are still unclear. Here we describe a possible role in high-fat diet (HFD)-induced obesity for monoacylglycerol lipase (MGL), an enzyme that is also known to hydrolyze the endocannabinoid 2-arachidonoylglycerol in brain. MGL-deficient [MGL-knockout (KO)] mice fed a HFD gained less body weight than wild-type mice and were protected from insulin resistance and hepatic steatosis. Food intake and energy expenditure were not altered in MGL-KO mice, but blood triglyceride levels after oral olive oil gavage were suppressed, indicating a role for MGL in intestinal fat absorption. Experiments with cannabinoid receptor type 1 (CB1)/MGL double-KO mice revealed that these phenotypes may include mechanisms that are independent of CB1-receptor-mediated endocannabinoid functions. We also noted that MGL-KO mice had less preference for HFD over normal chow diet. Oral but not intraperitoneal lipid administration strongly suppressed the appetites of MGL-KO and CB1/MGL double-KO mice, but not of wild-type and CB1-KO mice. Appetite suppression was reversed by vagotomy, suggesting involvement of MGL in the gut-brain axis regulation of appetite. Our results provide mechanistic insights of MGL's role in diet-induced obesity, lipid metabolic disorder, and regulation of appetite.-Yoshida, K., Kita, Y., Tokuoka, S. M., Hamano, F., Yamazaki, M., Sakimura, K., Kano, M., Shimizu, T. Monoacylglycerol lipase deficiency affects diet-induced obesity, fat absorption, and feeding behavior in CB1 cannabinoid receptor-deficient mice.

Limitations of deuterium-labeled internal standards for quantitative electrospray ionization mass spectrometry analysis of fatty acid metabolites.

RATIONALE: The electrospray ionization mass spectrometry (ESI-MS) methodology often shows poor ionization reproducibility in the analysis of biological samples. Therefore, normalization of the measured peak intensities is essential. It is believed that quantitative data with high reproducibility can be obtained by adding a constant amount of an internal standard (IS) material labeled with stable isotopes to each sample, thus allowing the correction of the quantitative value of the target compound by that of the IS. We investigated whether the presence or absence of a labeled IS improves the accuracy of these quantitative values. METHODS: Triple quadrupole MS coupled with liquid chromatography was used to analyze fatty acid metabolites in biological samples as target compounds. Two independent systems were used to provide a measure of reproducibility in two different laboratories. RESULTS: Data having poor reproducibility in the raw peak areas were efficiently normalized using the IS, but, crucially, the IS method using stable isotopes was not always necessary. In some cases, the reproducibility was relatively good even without using the IS. In a contaminant matrix, the MS response behavior of the target compound and its stable isotope-labeled material was complicated. Since ion suppression by matrix contaminants was dependent on the concentration of the target compound, the added amounts of the ISs were also important, Furthermore, an equivalent normalization effect was obtained by using a pooled quality control sample as an external standard, thus obviating the need for labeled IS samples, which are often expensive and sometimes not commercially available. CONCLUSIONS: Our results raise the question as to whether the quantitative method using stable-isotope-labeled ISs is always necessary and beneficial. However, the results obtained in this study cannot be generalized because only fatty acid metabolites were examined using ESI-MS and only a highly substituted deuterium-labeled IS was used.

LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.

Adaption of skeletal muscle to endurance exercise includes PPARδ- and AMP-activated protein kinase (AMPK)/PPARγ coactivator 1α-mediated transcriptional responses that result in increased oxidative capacity and conversion of glycolytic to more oxidative fiber types. These changes are associated with whole-body metabolic alterations including improved glucose handling and resistance to obesity. Increased DHA (22:6n-3) content in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is also reported in endurance exercise-trained glycolytic muscle; however, the DHA-metabolizing enzymes involved and the biological significance of the enhanced DHA content are unknown. In the present study, we identified lysophosphatidic acid acyltransferase (LPAAT)3 as an enzyme that was upregulated in myoblasts during in vitro differentiation and selectively incorporated DHA into PC and PE. LPAAT3 expression was increased by pharmacological activators of PPARδ or AMPK, and combination treatment led to further increased LPAAT3 expression and enhanced incorporation of DHA into PC and PE. Our results indicate that LPAAT3 was upregulated by exercise-induced signaling pathways and suggest that LPAAT3 may also contribute to the enhanced phospholipid-DHA content of endurance-trained muscles. Identification of DHA-metabolizing enzymes in the skeletal muscle will help to elucidate broad metabolic effects of DHA.

Relief from neuropathic pain by blocking of the platelet-activating factor-pain loop.

Neuropathic pain resulting from peripheral neuronal damage is largely resistant to treatment with currently available analgesic drugs. Recently, ATP, lysophosphatidic acid, and platelet-activating factor (PAF) have been reported to play important inductive roles in neuropathic pain. In the present study, we found that pain-like behaviors resulting from partial sciatic nerve ligation (PSL) were largely attenuated by deficiency of lysophosphatidylcholine acyltransferase (LPCAT)2, which is one of the PAF biosynthetic enzymes. By contrast, deficiency of the other PAF biosynthetic enzyme, LPCAT1, did not ameliorate neuropathic pain. With regard to the mechanism of the observed effects, LPCAT2 was detected in wild-type spinal cord microglia, and the absence of LPCAT2 expression precluded spinal PAF expression in LPCAT2-knockout mice. Furthermore, ATP-stimulated PAF biosynthesis in macrophages was decreased by pretreatment with the PAF receptor antagonist ABT-491, indicating the existence of a positive feedback loop of PAF biosynthesis, which we designated the PAF-pain loop. In conclusion, LPCAT2 is a novel therapeutic target for newly categorized analgesic drugs; in addition, our data call for the re-evaluation of the clinical utility of PAF receptor antagonists.-Shindou, H., Shiraishi, S., Tokuoka, S. M., Takahashi Y., Harayama, T., Abe, T., Bando, K., Miyano, K., Kita, Y., Uezono, Y., Shimizu, T. Relief from neuropathic pain by blocking of the platelet-activating factor-pain loop.

Mediator lipidomics by liquid chromatography-tandem mass spectrometry.

Comprehensive quantitative analysis of lipid mediators using liquid chromatography-tandem mass spectrometry is an effective strategy in the elucidation of disease mechanisms; but technically, it has been and is still a great challenge to achieve reliable datasets that cover variety of lipid metabolites contained at trace levels in complex biological matrices. In this opinion article, we introduce our experiences in developing lipid mediator profiling systems, and deliver some comments on limitations of current methodology.

Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have been well analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X-ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br-labeled fatty acids and metabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot-like distribution of Br in the cytoplasm. The current method enabled successful visualization of intracellular Br-labeled fatty acids. Single-element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, which provides a new tool to determine dynamic changes in fatty acids and their derivatives at the single-cell level.-Shimura, M., Shindou, H., Szyrwiel, L., Tokuoka, S. M., Hamano, F., Matsuyama, S., Okamoto, M., Matsunaga, A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Difference between tablet methods and paper questionnaire methods of conducting a survey with community-dwelling elderly.

[Purpose] This study aimed to develop a tablet app that emulates paper questionnaires used in clinical care, and to verify the difference between the utility of tablet survey methods and paper questionnaire methods with elderly people. [Subjects and Methods] A tablet app was developed in the Java language. A questionnaire was provided to 30 community-dwelling elderly people. The subjects were randomly allocated to the group responding on the tablet (tablet group) or that responding to a paper-based questionnaire (questionnaire group). Assessed items included response time to questions, whether or not they had queries regarding the survey, and data input time. For the tablet group, a questionnaire was conducted regarding the operability of the tablet. [Results] There was no difference in response time between the two groups. Significantly more people in the tablet group had queries regarding the survey. Data input time was 426 seconds for the tablet group and 1268 seconds for the questionnaire group. In the survey regarding tablet operability, there were no negative opinions about the visibility of the screen. [Conclusion] Tablets can be used with elderly people to shorten the data input time. The present findings suggested that tablet surveys could be effective for a large-scale investigation.

Interplay between CXCR2 and BLT1 facilitates neutrophil infiltration and resultant keratinocyte activation in a murine model of imiquimod-induced psoriasis.

Psoriasis is an inflammatory skin disease with accelerated epidermal cell turnover. Neutrophil accumulation in the skin is one of the histological characteristics of psoriasis. However, the precise mechanism and role of neutrophil infiltration remain largely unknown. In this article, we show that orchestrated action of CXCR2 and leukotriene B4 receptor BLT1 plays a key role in neutrophil recruitment during the development of imiquimod (IMQ)-induced psoriatic skin lesions in mice. Depletion of neutrophils with anti-Ly-6G Ab ameliorated the disease severity, along with reduced expression of proinflammatory cytokine IL-1ß in the skin. Furthermore, CXCR2 and BLT1 coordinately promote neutrophil infiltration into the skin during the early phase of IMQ-induced inflammation. In vitro, CXCR2 ligands augment leukotriene B4 production by murine neutrophils, which, in turn, amplifies chemokine-mediated neutrophil chemotaxis via BLT1 in autocrine and/or paracrine manners. In agreement with the increased IL-19 expression in IMQ-treated mouse skin, IL-1ß markedly upregulated expression of acanthosis-inducing cytokine IL-19 in human keratinocytes. We propose that coordination of chemokines, lipids, and cytokines with multiple positive feedback loops might drive the pathogenesis of psoriasis and, possibly, other inflammatory diseases as well. Interference to this positive feedback or its downstream effectors could be targets of novel anti-inflammatory treatment.

Establishment of LC-MS methods for the analysis of palmitoylated surfactant proteins.

The surfactant proteins (SPs), SP-B and SP-C, are important components of pulmonary surfactant involved in the reduction of alveolar surface tension. Quantification of SP-B and SP-C in surfactant drugs is informative for their quality control and the evaluation of their biological activity. Western blot analysis enabled the quantification of SP-B, but not SP-C, in surfactant drugs. Here, we report a new procedure involving chemical treatments and LC-MS to analyze SP-C peptides. The procedure enabled qualitative analysis of SP-C from different species with discrimination of the palmitoylation status and the artificial modifications that occur during handling and/or storage. In addition, the method can be used to estimate the total amount of SP-C in pulmonary surfactant drugs. The strategy described here might serve as a prototype to establish analytical methods for peptides that are extremely hydrophobic and behave like lipids. The new method provides an easy measurement of SP-C from various biological samples, which will help the characterization of various experimental animal models and the quality control of surfactant drugs, as well as diagnostics of human samples.

Human FcγRIIA induces anaphylactic and allergic reactions.

IgE and IgE receptors (FcεRI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (FcγRIIIA and FcγRIV) expressed by neutrophils, rather than on IgE and FcεRI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express FcγRIIIA or FcγRIV, but FcγRIIA. We therefore investigated the possible role of FcγRIIA in allergy by generating novel FcγRIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, FcγRIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking FcγRIIA in vivo abolished these reactions. We identified mast cells to be responsible for FcγRIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for FcγRIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after FcγRIIA engagement. IgG and FcγRIIA may therefore contribute to allergic and anaphylactic reactions in humans.

Prostaglandin E2-prostaglandin E receptor subtype 4 (EP4) signaling mediates UV irradiation-induced systemic immunosuppression.

UV radiation induces systemic immunosuppression. Because nonsteroidal anti-inflammatory drugs suppress UV-induced immunosuppression, prostanoids have been suspected as a crucial mediator of this UV effect. However, the identity of the prostanoid involved and its mechanism of action remain unclear. Here, we addressed this issue by subjecting mice deficient in each prostanoid receptor individually or mice treated with a subtype-specific antagonist to UV irradiation. Mice treated with an antagonist for prostaglandin E receptor subtype 4 (EP4), but not those deficient in other prostanoid receptors, show impaired UV-induced immunosuppression, whereas administration of an EP4 agonist rescues the impairment of the UV-induced immunosuppression in indomethacin-treated mice. The EP4 antagonist treatment suppresses an increase in the number of CD4(+)/forkhead box P3-positive (Foxp3(+)) regulatory T cells (Treg cells) in the peripheral lymph nodes (LNs) and dendritic cells expressing DEC205 in the LNs and the skin after UV irradiation. Furthermore, the EP4 antagonist treatment down-regulates UV-induced expression of receptor activator of NF-κB ligand (RANKL) in skin keratinocytes. Finally, administration of anti-RANKL antibody abolishes the restoration of UV-induced immunosuppression by EP4 agonism in indomethacin-treated mice. Thus, prostaglandin E(2) (PGE(2))-EP4 signaling mediates UV-induced immunosuppression by elevating the number of Treg cells through regulation of RANKL expression in the epidermis.

Selective ablation of mast cells or basophils reduces peanut-induced anaphylaxis in mice.

BACKGROUND: Studies with c-kit mutant mast cell (MC)-deficient mice and antibody-mediated depletion of basophils suggest that both MCs and basophils can contribute to peanut-induced anaphylaxis (PIA). However, interpretation of data obtained by using such approaches is complicated because c-kit mutant mice have several phenotypic abnormalities in addition to MC deficiency and because basophil-depleting antibodies can also react with MCs. OBJECTIVE: We analyzed (1) the changes in the features of PIA in mice after the selective and inducible ablation of MCs or basophils and (2) the possible importance of effector cells other than MCs and basophils in the PIA response. METHODS: Wild-type and various mutant mice were orally sensitized with peanut extract and cholera toxin weekly for 4 weeks and challenged intraperitoneally with peanut extract 2 weeks later. RESULTS: Peanut-challenged, MC-deficient Kit(W-sh/W-sh) mice had reduced immediate hypothermia, as well as a late-phase decrease in body temperature that was abrogated by antibody-mediated depletion of neutrophils. Diphtheria toxin-mediated selective depletion of MCs or basophils in Mcpt5-Cre;iDTR and Mcpt8(DTR) mice, respectively, and treatment of wild-type mice with the basophil-depleting antibody Ba103 significantly reduced peanut-induced hypothermia. Non-c-kit mutant MC- and basophil-deficient Cpa3-Cre;Mcl-1(fl/fl) mice had reduced but still significant responses to peanut. CONCLUSION: Inducible and selective ablation of MCs or basophils in non-c-kit mutant mice can significantly reduce PIA, but partial responses to peanut can still be observed in the virtual absence of both cell types. The neutrophilia in Kit(W-sh/W-sh) mice might influence the responses of these mice in this PIA model.

Acute inhibition of diacylglycerol lipase blocks endocannabinoid-mediated retrograde signalling: evidence for on-demand biosynthesis of 2-arachidonoylglycerol.

The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) produced by diacylglycerol lipase α (DGLα) is one of the best-characterized retrograde messengers at central synapses. It has been thought that 2-AG is produced 'on demand' upon activation of postsynaptic neurons. However, recent studies propose that 2-AG is pre-synthesized by DGLα and stored in neurons, and that 2-AG is released from such 'pre-formed pools' without the participation of DGLα. To address whether the 2-AG source for retrograde signalling is the on-demand biosynthesis by DGLα or the mobilization from pre-formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde eCB signalling triggered by Ca(2+) elevation, Gq/11 protein-coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM-188 effectively blocked depolarization-induced suppression of inhibition (DSI), a purely Ca(2+)-dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre-Purkinje cell synapses in the cerebellum OMDM-188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca(2+) elevation and group I metabotropic glutamate receptor (I-mGluR) activation. Moreover, brief OMDM-188 treatments for several minutes were sufficient to suppress both DSI and the I-mGluR-induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2-AG for synaptic retrograde signalling is supplied as a result of on-demand biosynthesis by DGLα rather than mobilization from presumptive pre-formed pools.

Alkylglycerol monooxygenase as a potential modulator for PAF synthesis in macrophages.

Alkylglycerol monooxygenase (AGMO, glyceryl ether monooxygenase) is an enzyme known to catalyze the cleavage of the O-alkyl bond of glyceryl ether lipids. Identification of the gene encoding AGMO was reported recently, however, the involvement of AGMO in modulating cellular lipids has not been reported until now. In this report, we investigate a possible role for AGMO in macrophage platelet-activating factor (PAF) production. AGMO mRNA expression levels decreased with lipopolysaccharide (LPS) treatments in mouse peritoneal macrophages and RAW264.7 cells. Tetrahydrobiopterin-dependent conversion of lyso-PAF to glycerophosphocholine in the microsomal fraction was also reduced in LPS-treated RAW264.7 cells. In the LPS-treated cells, both lyso-PAF and PAF levels increased. Moreover, exogenously expressed AGMO caused a reduction in cellular lyso-PAF and PAF levels in HEK293 cells. Collectively, our results suggest a possible mechanism for AGMO in modulating macrophage PAF production by regulating cellular lyso-PAF levels.

Unveiling Liquid-Crystalline Lipids in the Urothelial Membrane through Cryo-EM.

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

Unveiling Liquid-Crystalline Lipids in the Urothelial Membrane through Cryo-EM.

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this investigation, we employed cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex embedded within the porcine AUM at a resolution of 3.5 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane.

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups.

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.