Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration.

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca2+-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis.

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

Transcriptional Control of Trpm6 by the Nuclear Receptor FXR.

Farnesoid x receptor (FXR) is a nuclear bile acid receptor that belongs to the nuclear receptor superfamily. It plays an essential role in bile acid biosynthesis, lipid and glucose metabolism, liver regeneration, and vertical sleeve gastrectomy. A loss of the FXR gene or dysregulations of FXR-mediated gene expression are associated with the development of progressive familial intrahepatic cholestasis, tumorigenesis, inflammation, and diabetes mellitus. Magnesium ion (Mg2+) is essential for mammalian physiology. Over 600 enzymes are dependent on Mg2+ for their activity. Here, we show that the Trpm6 gene encoding a Mg2+ channel is a direct FXR target gene in the intestinal epithelial cells of mice. FXR expressed in the intestinal epithelial cells is absolutely required for sustaining a basal expression of intestinal Trpm6 that can be robustly induced by the treatment of GW4064, a synthetic FXR agonist. Analysis of FXR ChIP-seq data revealed that intron regions of Trpm6 contain two prominent FXR binding peaks. Among them, the proximal peak from the transcription start site contains a functional inverted repeat 1 (IR1) response element that directly binds to the FXR-RXRα heterodimer. Based on these results, we proposed that an intestinal FXR-TRPM6 axis may link a bile acid signaling to Mg2+ homeostasis.

Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain.

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.

Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells.

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.

FXR-dependent Rubicon induction impairs autophagy in models of human cholestasis.

BACKGROUND & AIMS: Cholestasis comprises a spectrum of liver diseases characterized by the accumulation of bile acids. Bile acids and activation of the farnesoid X receptor (FXR) can inhibit autophagy, a cellular self-digestion process necessary for cellular homeostasis and regeneration. In mice, autophagy appears to be impaired in cholestasis and induction of autophagy may reduce liver injury. METHODS: Herein, we explored autophagy in human cholestasis in vivo and investigated the underlying molecular mechanisms in vitro. FXR chromatin immunoprecipitation-sequencing and qPCR were performed in combination with luciferase promoter studies to identify functional FXR binding targets in a human cholestatic liver sample. RESULTS: Autophagic processing appeared to be impaired in patients with cholestasis and in individuals treated with the FXR ligand obeticholic acid (OCA). In vitro, chenodeoxycholic acid and OCA inhibited autophagy at the level of autophagosome to lysosome fusion in an FXR-dependent manner. Rubicon, which inhibits autophago-lysosomal maturation, was identified as a direct FXR target that is induced in cholestasis and by FXR-agonistic bile acids. Genetic inhibition of Rubicon reversed the bile acid-induced impairment of autophagic flux. In contrast to OCA, ursodeoxycholic acid (UDCA), which is a non-FXR-agonistic bile acid, induced autophagolysosome formation independently of FXR, enhanced autophagic flux and was associated with reduced Rubicon levels. CONCLUSION: In models of human cholestasis, autophagic processing is impaired in an FXR-dependent manner, partly resulting from the induction of Rubicon. UDCA is a potent inducer of hepatic autophagy. Manipulating autophagy and Rubicon may represent a novel treatment concept for cholestatic liver diseases. LAY SUMMARY: Autophagy, a cellular self-cleansing process, is impaired in various forms of human cholestasis. Bile acids, which accumulate in cholestatic liver disease, induce Rubicon, a protein that inhibits proper execution of autophagy. Ursodeoxycholic acid, which is the first-line treatment option for many cholestatic liver diseases, induces hepatic autophagy along with reducing Rubicon.

Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system.

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.

Nutrient-sensing nuclear receptors coordinate autophagy.

Autophagy is an evolutionarily conserved catabolic process that recycles nutrients upon starvation and maintains cellular energy homeostasis. Its acute regulation by nutrient-sensing signalling pathways is well described, but its longer-term transcriptional regulation is not. The nuclear receptors peroxisome proliferator-activated receptor-α (PPARα) and farnesoid X receptor (FXR) are activated in the fasted and fed liver, respectively. Here we show that both PPARα and FXR regulate hepatic autophagy in mice. Pharmacological activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipid degradation, or lipophagy. This response is lost in PPARα knockout (Ppara(-/-), also known as Nr1c1(-/-)) mice, which are partially defective in the induction of autophagy by fasting. Pharmacological activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in the fasting state, and this response is absent in FXR knockout (Fxr(-/-), also known as Nr1h4(-/-)) mice, which show a partial defect in suppression of hepatic autophagy in the fed state. PPARα and FXR compete for binding to shared sites in autophagic gene promoters, with opposite transcriptional outputs. These results reveal complementary, interlocking mechanisms for regulation of autophagy by nutrient status.

Autophagy in neutrophils.

Autophagy is a highly conserved intracellular degradation and energy-recycling mechanism that contributes to the maintenance of cellular homeostasis. Extensive researches over the past decades have defined the role of autophagy innate immune cells. In this review, we describe the current state of knowledge regarding the role of autophagy in neutrophil biology and a picture of molecular mechanism underlying autophagy in neutrophils. Neutrophils are professional phagocytes that comprise the first line of defense against pathogen. Autophagy machineries are highly conserved in neutrophils. Autophagy is not only involved in generalized function of neutrophils such as differentiation in bone marrow but also plays crucial role effector functions of neutrophils such as granule formation, degranulation, neutrophil extracellular traps release, cytokine production, bactericidal activity and controlling inflammation. This review outlines the current understanding of autophagy in neutrophils and provides insight towards identification of novel therapeutics targeting autophagy in neutrophils.

Structure-function analyses of a stereotypic rheumatoid factor unravel the structural basis for germline-encoded antibody autoreactivity.

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called "stereotypic RFs," are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2-CH3 elbow in the canonical antigen-binding manner involving a large antigen-antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu432-His435 region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system.

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.

TCTP regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality.

If no fertilization occurs for a prolonged time following ovulation, oocytes experience a time-dependent deterioration in quality both in vivo and in vitro due to processes called postovulatory aging. Because the postovulatory aging of oocytes has marked detrimental effects on embryo development and offspring, many efforts have been made to unveil the underlying mechanisms. Here we showed that translationally controlled tumor protein (TCTP) regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality. Spindle dynamics decreased with reduced TCTP level during aging of mouse oocytes. Knockdown of TCTP accelerated the reduction of spindle dynamics, accompanying with aging-related deterioration of oocyte quality. Conversely, overexpression of TCTP prevented aging-associated decline of spindle dynamics. Moreover, the aging-related abnormalities in oocytes were rescued after TCTP overexpression, thereby improving fertilization competency and subsequent embryo development. Therefore, our results demonstrate that TCTP-mediated spindle dynamics play a key role in maintaining oocyte quality during postovulatory aging and overexpression of TCTP is sufficient to prevent aging-associated abnormalities in mouse oocytes.

Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae.

Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm-baculovirus expression vector system (silkworm-BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.

Hepatic FXR/SHP axis modulates systemic glucose and fatty acid homeostasis in aged mice.

The nuclear receptors farnesoid X receptor (FXR; NR1H4) and small heterodimer partner (SHP; NR0B2) play crucial roles in bile acid homeostasis. Global double knockout of FXR and SHP signaling (DKO) causes severe cholestasis and liver injury at early ages. Here, we report an unexpected beneficial impact on glucose and fatty acid metabolism in aged DKO mice, which show suppressed body weight gain and adiposity when maintained on normal chow. This phenotype was not observed in single Fxr or Shp knockouts. Liver-specific Fxr/Shp double knockout mice fully phenocopied the DKO mice, with lower hepatic triglyceride accumulation, improved glucose/insulin tolerance, and accelerated fatty acid use. In both DKO and liver-specific Fxr/Shp double knockout livers, these metabolic phenotypes were associated with altered expression of fatty acid metabolism and autophagy-machinery genes. Loss of the hepatic FXR/SHP axis reprogrammed white and brown adipose tissue gene expression to boost fatty acid usage. CONCLUSION: Combined deletion of the hepatic FXR/SHP axis improves glucose/fatty acid homeostasis in aged mice, reversing the aging phenotype of body weight gain, increased adiposity, and glucose/insulin tolerance, suggesting a central role of this axis in whole-body energy homeostasis. (Hepatology 2017;66:498-509).

A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells.

Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (ß1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans.

Molecular characterization of mitochondrial Zucchini and its relation to nuage-piRNA pathway components in Bombyx mori ovary-derived BmN4 cells.

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with PIWI subfamily proteins, which play an important role in transposon silencing in animal germ cell. The piRNAs biogenesis is divided into two major pathways: primary and secondary, and both pathways are independent of double-stranded RNA-processing enzyme Dicer, which processes the single-stranded RNA transcripts in microRNA (miRNA) and siRNA (small interfering RNA) pathway. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters. Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily protein is conserved among the animals and involved in piRNA biogenesis. Recent studies showed that the Zucchini is an endoribonuclease essential for primary piRNA maturation and production of phased piRNA in secondary piRNA biogenesis of drosophila germ cell. Based on these reports, here we identified and studied the silkworm Zucchini (BmZuc) at subcellular level in ovary-derived BmN4 cell. The silkworm Zuc specifically expressed in germ-related tissues and localized on mitochondria and partially co-localized with perinuclear nuage-piRNA pathway components and nuage marker protein BmVasa. Molecular dissection analyses revealed that the conserved mitochondrial localization sequence, RGV motif, PLDc 2 domain and HKD motif are important for the BmZuc mitochondrial localization. Moreover, the knockdown analyses showed that the piRNA pathway components are independent on BmZuc for their nuage localization, whereas BmZuc depend on piRNA pathway components for the proper localization. Our data provides vital information on mitochondrial BmZuc and its relationship to "nuage" in ovary-derived BmN4 cell.

Characterization of Armitage and Yb containing granules and their relationship to nuage in ovary-derived cultured silkworm cell.

PIWI-interacting RNAs (piRNAs) are a class of endogenous small non-coding RNAs, which are mostly 24-32 nucleotides in length and interact specifically with PIWI subfamily of argonaute proteins. Despite the significant research progress in germ line piRNA pathway, its role in somatic cell is not well known. In Drosophila ovarian somatic cell, maturation of primary piRNA and its loading onto Piwi occurs at perinuclear Yb body. The Armitage (Armi) and Yb proteins are the major components of Yb body and specially expressed in ovarian somatic cell. Based on the reports, here we studied the BmArmi and BmYb in Bombyx mori ovary-derived BmN4 cells expressing BmVasa. In this study, we show that BmArmi and BmYb co-localized with BmVasa at nuage. The helicase domains of BmArmi and BmYb are important for nuage localization. Moreover, RNAi of piRNA components reveal that BmArmi depend on BmAgo3 for nuage localization, and BmArmi and BmYb form cytoplasmic granules independently in the absence of BmVasa. Our results provide evidence that the BmArmi and BmYb coexist with BmVasa and play an important role in perinuclear nuage granules formation in ovary-derived BmN4 cell.

Liver receptor homolog-1 is a critical determinant of methyl-pool metabolism.

UNLABELLED: Balance of labile methyl groups (choline, methionine, betaine, and folate) is important for normal liver function. Quantitatively, a significant use of labile methyl groups is in the production of phosphatidylcholines (PCs), which are ligands for the nuclear liver receptor homolog-1 (LRH-1). We studied the role of LRH-1 in methyl-pool homeostasis and determined its metabolic effects using the methionine and choline-deficient (MCD) diet, which depletes methyl groups and results in a deleterious decrease in the PC-to-phosphatidylethanolamine ratio. We found that MCD diet-fed, liver-specific LRH-1 knockout mice (Lrh-1(-/-) ) do not show the expected decreased methyl-pool and PC/phosphatidylethanolamine ratio and are resistant to the hepatitis and fibrosis normally induced by the diet. Adaptive responses observed in wild-type mice on the MCD diet were also observed in Lrh-1(-/-) mice on a normal diet. This includes reduced expression of the highly active glycine-n-methyltransferase and the biliary phospholipid floppase multidrug-resistance protein 2 (Mdr2/Abcb4), resulting in reduced consumption of methyl groups and biliary PC secretion. In vitro studies confirm that Gnmt and Mdr2 are primary LRH-1 target genes. Additional similarities between hepatic gene expression profiles in MCD diet-fed wild-type and untreated Lrh-1(-/-) mice suggest that methyl-pool deficiency decreases LRH-1 activity, and this was confirmed by in vitro functional results in cells maintained in MCD medium. CONCLUSION: LRH-1 is a novel transcriptional regulator of methyl-pool balance; when the methyl-pool is depleted, decreased LRH-1 transactivation suppresses expression of key genes to minimize loss of labile methyl groups. (Hepatology 2016;63:95-106).

A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.