Multiorgan Systems Study Reveals Igfbp7 as a Suppressor of Gluconeogenesis after Gastric Bypass Surgery.

Roux-en-Y gastric bypass (RYGB) surgery reduces weight in obese patients. A marked decrease in blood glucose levels occurs before weight loss; however, key molecules that improve the glycemic profile remain largely unknown. Using a murine RYGB surgery model, we performed multiorgan proteomics and bioinformatics to monitor the proteins and molecular pathways that change in this early glycemic response. Multiplexed proteomic kinetics data analysis revealed that the Roux limb, biliopancreatic limb, liver, and pancreas each exhibited unique temporal and molecular responses to the RYGB surgery. In addition, protein-protein network analysis indicated that the changes to the microbial environment in the intestine may play a crucial role in the beneficial effects of RYGB surgery. Furthermore, insulin-like growth factor binding protein 7 (Igfbp7) was identified as an early induced protein in the Roux limb. Known secretory properties of Igfbp7 prompted us to further investigate its role as a remote organ regulator of glucose metabolism. Igfbp7 overexpression decreased blood glucose levels in diet-induced obese mice and attenuated gluconeogenic gene expression in the liver. Secreted Igfbp7 appeared to mediate these beneficial effects. These results demonstrate that organs responded differentially to RYGB surgery and indicate that Igfbp7 may play an important role in improving blood glucose levels.

LPA-producing enzyme PA-PLA1α regulates hair follicle development by modulating EGFR signalling.

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA-producing enzyme, phosphatidic acid-selective phospholipase A(1)α (PA-PLA(1)α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA(6)). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle development. PA-PLA(1)α-deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA-PLA(1)α was co-localized with TACE, TGFα and tyrosine-phosphorylated EGFR in the IRS. In PA-PLA(1)α-deficient hair follicles, cleaved TGFα and tyrosine-phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA-PLA(1)α enzyme induced P2Y5- and TACE-mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA-PLA(1)α-LPA-P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE-TGFα-EGFR pathway, thus underscoring the physiological importance of LPA-induced EGFR transactivation.

TGFα shedding assay: an accurate and versatile method for detecting GPCR activation.

A single-format method to detect multiple G protein-coupled receptor (GPCR) signaling, especially Gα(12/13) signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-α (TGFα) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase-tagged TGFα (AP-TGFα) and its release into conditioned medium. AP-TGFα shedding response occurred almost exclusively downstream of Gα(12/13) and Gα(q) signaling. Relying on chimeric Gα proteins and promiscuous Gα(16) protein, which can couple with Gα(s)- and Gα(i)-coupled GPCRs and induce Gα(q) signaling, we used the TGFα shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as Gα(12/13)-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα(12/13)-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.

Antifungal profile against Candida auris clinical isolates of tyroscherin and its new analog produced by the deep-sea-derived fungal strain Scedosporium apiospermum FKJ-0499.

A new antifungal compound, named N-demethyltyroscherin (1), was discovered from the static fungal cultured material of Scedosporium apiospermum FKJ-0499 isolated from a deep-sea sediment sample together with a known compound, tyroscherin (2). The structure of 1 was elucidated as a new analog of 2 by MS and NMR analyses. The absolute configuration of 1 was determined by chemical derivatization. Both compounds showed potent in vitro antifungal activity against clinically isolated Candida auris strains, with MIC values ranging from 0.0625 to 4 µg ml-1.

A combination strategy of a semisynthetic macrolide, 5-O-mycaminosyltylonolide with polymyxin B nonapeptide for multi-drug resistance P. aeruginosa.

The emergence and spread of antimicrobial resistant pathogens continue to threaten our ability to combat several infections. Among them, Pseudomonas aeruginosa (P. aeruginosa) poses a major threat to human health. P. aeruginosa has intrinsic resistance to many antibiotics due to the impermeability of its outer membrane and a resistance-nodulation-cell division type multidrug efflux pump system. Therefore, only limited therapeutic drugs are effective against the pathogen. To address this problem, we have recently discovered an overlooked anti- P. aeruginosa compound, 5-O-mycaminosyltylonolide (OMT) from the Omura Natural Compound library using an efflux pump deletion P. aeruginosa mutant strain, YM64. In this report, we aim to demonstrate the promising potential of OMT for as a novel anti- P. aeruginosa compound and performed combination assays of OMT with polymyxin B nonapeptide, an example of a permeabilizing agent, against multi-drug resistant P. aeruginosa clinical isolates.

An efflux pump deletion mutant enabling the discovery of a macrolide as an overlooked anti-P. aeruginosa active compound.

Antimicrobial resistance is a serious, worldwide problem. Pseudomonas aeruginosa (P. aeruginosa) is the pathogen that poses a major threat to human health. However, resistance-nodulation-cell division type multidrug efflux pump systems defend P. aeruginosa from many antibiotics. Therefore, only limited therapeutic drugs are available. In this regard, we screened overlooked anti- P. aeruginosa compounds from the Omura Natural Compound library using an efflux pump deletion P. aeruginosa mutant strain, YM64, which led us to find a semisynthetic macrolide, 5-O-mycaminosyltylonolide, whose anti- P. aeruginosa activity against a standard laboratory adapted strain, PAO1, was enhanced by an efflux pump inhibitor, phenylalanine-arginine beta-naphthylamide.

Rediscovery of Tetronomycin as a Broad-Spectrum and Potent Antibiotic against Drug-Resistant Gram-Positive Bacteria.

Tetronomycin (1), first isolated from a cultured broth of Streptomyces sp. by Juslen et al. in 1974, is a polycyclic polyether compound. However, the biological activity of 1 has not been thoroughly examined. In this study, we found that 1 exhibits more potent antibacterial activity than two well-known antibacterial drugs (vancomycin and linezolid) and is effective against several drug-resistant clinical isolates including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. Furthermore, we reassigned the 13C NMR spectra of 1 and performed a preliminary structure-activity relationship study of 1 to synthesize a chemical probe for target identification, which implied different targets based on its ionophore activity.

Surface loops of extracellular phospholipase A(1) determine both substrate specificity and preference for lysophospholipids.

Members of the pancreatic lipase family exhibit both lipase activity toward triacylglycerol and/or phospholipase A(1) (PLA(1)) activity toward certain phospholipids. Some members of the pancreatic lipase family exhibit lysophospholipase activity in addition to their lipase and PLA(1) activities. Two such enzymes, phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)) and phosphatidic acid (PA)-selective PLA(1)α (PA-PLA(1)α, also known as LIPH) specifically hydrolyze PS and PA, respectively. However, little is known about the mechanisms that determine their substrate specificities. Crystal structures of lipases and mutagenesis studies have suggested that three surface loops, namely, ß5, ß9, and lid, have roles in determining substrate specificity. To determine roles of these loop structures in the substrate recognition of these PLA(1) enzymes, we constructed a number of PS-PLA(1) mutants in which the three surface loops are replaced with those of PA-PLA(1)α. The results indicate that the surface loops, especially the ß5 loop, of PA-PLA(1)α play important roles in the recognition of PA, whereas other structure(s) in PS-PLA(1) is responsible for PS preference. In addition, ß5 loop of PS-PLA(1) has a crucial role in lysophospholipase activity toward lysophosphatidylserine. The present study revealed the critical role of lipase surface loops, especially the ß5 loop, in determining substrate specificities of PLA(1) enzymes.

Autotaxin regulates vascular development via multiple lysophosphatidic acid (LPA) receptors in zebrafish.

Autotaxin (ATX) is a multifunctional ecto-type phosphodiesterase that converts lysophospholipids, such as lysophosphatidylcholine, to lysophosphatidic acid (LPA) by its lysophospholipase D activity. LPA is a lipid mediator with diverse biological functions, most of which are mediated by G protein-coupled receptors specific to LPA (LPA1-6). Recent studies on ATX knock-out mice revealed that ATX has an essential role in embryonic blood vessel formation. However, the underlying molecular mechanisms remain to be solved. A data base search revealed that ATX and LPA receptors are conserved in wide range of vertebrates from fishes to mammals. Here we analyzed zebrafish ATX (zATX) and LPA receptors both biochemically and functionally. zATX, like mammalian ATX, showed lysophospholipase D activity to produce LPA. In addition, all zebrafish LPA receptors except for LPA5a and LPA5b were found to respond to LPA. Knockdown of zATX in zebrafish embryos by injecting morpholino antisense oligonucleotides (MOs) specific to zATX caused abnormal blood vessel formation, which has not been observed in other morphant embryos or mutants with vascular defects reported previously. In ATX morphant embryos, the segmental arteries sprouted normally from the dorsal aorta but stalled in midcourse, resulting in aberrant vascular connection around the horizontal myoseptum. Similar vascular defects were not observed in embryos in which each single LPA receptor was attenuated by using MOs. Interestingly, similar vascular defects were observed when both LPA1 and LPA4 functions were attenuated by using MOs and/or a selective LPA receptor antagonist, Ki16425. These results demonstrate that the ATX-LPA-LPAR axis is a critical regulator of embryonic vascular development that is conserved in vertebrates.

LPA and its analogs-attractive tools for elucidation of LPA biology and drug development.

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) is a simple phospholipid but displays an intriguing cell biology that is mediated via interactions with both G-protein-coupled seven transmembrane receptors (GPCRs) and nuclear hormone receptors. So far, seven GPCRs (LPA(1-5) and recently reported GPR87/LPA(6) and P2Y5/LPA(7)) and a nuclear hormone receptor, PPARgamma, have been identified. LPA is predominantly produced in blood and a plasma enzyme, autotaxin, is involved in its production. Recent gene manipulating studies of these proteins have shown that LPA is involved in both pathological and physiological states including brain development, neuropathy pain, implantation, protection against radiation-induced intestinal injury and blood vessel formation. In addition, lipids similar to LPA, such as sphingosine 1-phosphate (S1P) and 2-arachidonylglycerol (2-AG), share common cellular signaling pathways with LPA and are now considered as promising targets of human therapy including immunosuppressant and anti-obesity drugs. Thus, LPA is now one of the most attractive targets for prevention and treatment of various diseases. Receptor-selective antagonists and agonists as well as inhibitors of LPA producing enzymes are undoubtedly useful. Recognition of the ligand, LPA, by each receptor seems to be quite different, as LPA species with various fatty acids at either the sn-1 or sn-2 position of the hydroxy residue activate each receptor quite differently. In the last decade a series of LPA analogs in which the sn-1 or sn-2 hydroxy, acyl chain, glycerol and phosphate group are modified have been created and evaluated by several laboratories. Here we review recent advances in the development of LPA-receptor targeted compounds (agonists and antagonists) and anti-autotaxin inhibitors.

ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation.

The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA1 signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA1 signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through ß1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA1 axis is a key regulator of cartilage formation.