GDE5/Gpcpd1 activity determines phosphatidylcholine composition in skeletal muscle and regulates contractile force in mice.

Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline and glycerol 3-phosphate; this study aimed to elucidate its physiological function in vivo. Heterozygous whole-body GDE5-deficient mice reveal a significant GPC accumulation across tissues, while homozygous whole-body knockout results in embryonic lethality. Skeletal muscle-specific GDE5 deletion (Gde5 skKO) exhibits reduced passive force and improved fatigue resistance in electrically stimulated gastrocnemius muscles in vivo. GDE5 deficiency also results in higher glycolytic metabolites and glycogen levels, and glycerophospholipids alteration, including reduced levels of phospholipids that bind polyunsaturated fatty acids (PUFAs), such as DHA. Interestingly, this PC fatty acid compositional change is similar to that observed in skeletal muscles of denervated and Duchenne muscular dystrophy mouse models. These are accompanied by decrease of GDE5 expression, suggesting a regulatory role of GDE5 activity for glycerophospholipid profiles. Furthermore, a DHA-rich diet enhances contractile force and lowers fatigue resistance, suggesting a functional relationship between PC fatty acid composition and muscle function. Finally, skinned fiber experiments show that GDE5 loss increases the probability of the ryanodine receptor opening and lowers the maximum Ca2+-activated force. Collectively, GDE5 activity plays roles in PC and glucose/glycogen metabolism in skeletal muscle.

Capillary electrophoresis using triple layer modified capillary facilitating salivary ion analyses: Application to search for potential stress markers induced by cold pressure test.

In this study, we introduce a novel approach for the analysis of salivary ions using capillary electrophoresis (CE) with a triple-layer coated capillary. The capillary is sequentially coated with cationic silylating reagents, poly(vinylsulfonate), and polybrene to form a custom designed surface that effectively inhibits adsorption of protein matrix on the capillary inner wall and allows for reproducible ion analysis. For the CE with capacitively coupled contactless conductivity detection, we used suitable background electrolytes (BGEs) for salivary ion analysis. Anions were separated using a mixture of 2-(N-morpholino)ethanesulfonic acid and l-arginine, and cations were separated using that with 18-crown-6. This setup enabled rapid separation, within 4 min, together with sensitive detection. We quantified nine common anions and five cations typically found in saliva samples using this CE method, both before and after a cold pressure test (CPT, a standard stress test). The CE system demonstrated consistent ion separation across 30 consecutive measurements without requiring capillary replacement. Notably, the salivary ion balance remained predominantly anion-rich, regardless of the CPT. Cold water exposure induced greater variation in the total anion concentration than in the total cation concentration. Further analysis using multiple regression analysis revealed strong relationships between nitrate and nitrite, formate and phosphate, and potassium and nitrate, before and after the CPT. Notably, potassium and nitrate ions exhibited variations in response to stress. These results provided a method for assessing salivary ion composition and insights into the potential of salivary ions as biomarkers for stress.

Inactivation of ackA and pta Genes Reduces GlpT Expression and Susceptibility to Fosfomycin in Escherichia coli.

Fosfomycin is used to treat a variety of bacterial infections, including urinary tract infections caused by Escherichia coli. In recent years, quinolone-resistant and extended-spectrum ß-lactamase (ESBL)-producing bacteria have been increasing. Because fosfomycin is effective against many of these drug-resistant bacteria, the clinical importance of fosfomycin is increasing. Against this background, information on the mechanisms of resistance and the antimicrobial activity of this drug is desired to enhance the usefulness of fosfomycin therapy. In this study, we aimed to explore novel factors affecting the antimicrobial activity of fosfomycin. Here, we found that ackA and pta contribute to fosfomycin activity against E. coli. ackA and pta mutant E. coli had reduced fosfomycin uptake capacity and became less sensitive to this drug. In addition, ackA and pta mutants had decreased expression of glpT that encodes one of the fosfomycin transporters. Expression of glpT is enhanced by a nucleoid-associated protein, Fis. We found that mutations in ackA and pta also caused a decrease in fis expression. Thus, we interpret the decrease in glpT expression in ackA and pta defective strains to be due to a decrease in Fis levels in these mutants. Furthermore, ackA and pta are conserved in multidrug-resistant E. coli isolated from patients with pyelonephritis and enterohemorrhagic E. coli, and deletion of ackA and pta from these strains resulted in decreased susceptibility to fosfomycin. These results suggest that ackA and pta in E. coli contribute to fosfomycin activity and that mutation of these genes may pose a risk of reducing the effect of fosfomycin. IMPORTANCE The spread of drug-resistant bacteria is a major threat in the field of medicine. Although fosfomycin is an old type of antimicrobial agent, it has recently come back into the limelight because of its effectiveness against many drug-resistant bacteria, including quinolone-resistant and ESBL-producing bacteria. Since fosfomycin is taken up into the bacteria by GlpT and UhpT transporters, its antimicrobial activity fluctuates with changes in GlpT and UhpT function and expression. In this study, we found that inactivation of the ackA and pta genes responsible for the acetic acid metabolism system reduced GlpT expression and fosfomycin activity. In other words, this study shows a new genetic mutation that leads to fosfomycin resistance in bacteria. The results of this study will lead to further understanding of the mechanism of fosfomycin resistance and the creation of new ideas to enhance fosfomycin therapy.

Estimation of the Depletion Layer Thickness in Silicon Nanowire-Based Biosensors from Attomolar-Level Biomolecular Detection.

Silicon nanowire (SiNW) biosensors have attracted a lot of attention due to their superior sensitivity. Recently, the dependence of biomolecule detection sensitivity on the nanowire (NW) width, number, and doping density has been partially investigated. However, the primary reason for achieving ultrahigh sensitivity has not been elucidated thus far. In this study, we designed and fabricated SiNW biosensors with different widths (10.8-155 nm) by integrating a complementary metal-oxide-semiconductor process and electron beam lithography. We aimed to investigate the detection limit of SiNW biosensors and reveal the critical effect of the 10-nm-scaled SiNW width on the detection sensitivity. The sensing performance was evaluated by detecting antiovalbumin immunoglobulin G (IgG) with various concentrations (from 6 aM to 600 nM). The initial thickness of the depletion region of the SiNW and the changes in the depletion region due to biomolecule binding were calculated. The basis of this calculation are the resistance change ratios as functions of IgG concentrations using SiNWs with different widths. The calculation results reveal that the proportion of the depletion region over the entire SiNW channel is the essential reason for high-sensitivity detection. Therefore, this study is crucial for an indepth understanding on how to maximize the sensitivity of SiNW biosensors.

PPARα activation partially drives NAFLD development in liver-specific Hnf4a-null mice.

HNF4α regulates various genes to maintain liver function. There have been reports linking HNF4α expression to the development of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis. In this study, liver-specific Hnf4a-deficient mice (Hnf4aΔHep mice) developed hepatosteatosis and liver fibrosis, and they were found to have difficulty utilizing glucose. In Hnf4aΔHep mice, the expression of fatty acid oxidation-related genes, which are PPARα target genes, was increased in contrast to the decreased expression of PPARα, suggesting that Hnf4aΔHep mice take up more lipids in the liver instead of glucose. Furthermore, Hnf4aΔHep/Ppara-/- mice, which are simultaneously deficient in HNF4α and PPARα, showed improved hepatosteatosis and fibrosis. Increased C18:1 and C18:1/C18:0 ratio was observed in the livers of Hnf4aΔHep mice, and the transactivation of PPARα target gene was induced by C18:1. When the C18:1/C18:0 ratio was close to that of Hnf4aΔHep mouse liver, a significant increase in transactivation was observed. In addition, the expression of Pgc1a, a coactivator of PPARs, was increased, suggesting that elevated C18:1 and Pgc1a expression could contribute to PPARα activation in Hnf4aΔHep mice. These insights may contribute to the development of new diagnostic and therapeutic approaches for NAFLD by focusing on the HNF4α and PPARα signaling cascade.

Characterization of recombinant murine GDE4 and GDE7, enzymes producing lysophosphatidic acid and/or cyclic phosphatidic acid.

GDE4 and GDE7 are membrane-bound enzymes that exhibit lysophospholipase D activities. We found that GDE7 produced not only lysophosphatidic acid (LPA) but also cyclic phosphatidic acid (cPA) from lysophospholipids by a transphosphatidylation reaction. In contrast, GDE4 produced only LPA. The analysis of substrate specificity showed that 1-alkyl-lysophosphospholipids were preferred substrates for both enzymes rather than 1-alkyl-lysophospholipids and 1-alkenyl-lysophospholipids. Among the various lysophospholipids with different polar head groups that were tested, lysophosphatidylglycerol and lysophosphatidylserine were preferred substrates for GDE4 and GDE7, respectively. The detailed analysis of the dependency of the enzyme activities of GDE4 and GDE7 on divalent cations suggested multiple divalent cations were bound in the active sites of both enzymes. Taken together, these results suggest the possibility that GDE7 functions as a cPA-producing enzyme in the body.

Simultaneous capillary electrophoresis of anions and cations in a single injection using an anion exchanger-modified capillary for determination of salivary ions in combination with statistical analyses.

This paper describes the simultaneous capillary electrophoresis (CE) of anions and cations using an anion exchange-modified capillary, which was prepared by chemical coating with a cationic silylating reagent, and its application to saliva analysis. The CE method provides high-throughput (5 min for a single sample injection) analysis by generating a high-velocity electroosmotic flow at pH 3.0-3.5. The detection limits at a signal-to-noise ratio of 3 ranged from 1.2 to 18 µM for anions and 1.0 to 2.7 µM for cations. The relative standard deviations for the migration times and peak areas of analytes (n = 4) ranged from 0.05% to 0.40% and 0.94% to 4.7%, respectively. The CE system was used to analyze 11 common ions in saliva samples collected from long-distance runners and sedentary university students before and after running for a set distance or a set time. Interestingly, the SCN- concentrations decreased in the saliva samples of all 14 athletes and 16 sedentary students after running. Furthermore, when the concentrations of the analyzed ions were compared with that of cortisol as a typical stress marker by multiple regression analysis, SCN- and NO3- in saliva samples from the two subject groups strongly correlated with cortisol levels, as determined by an electrochemiluminescence immunoassay. This study improves our knowledge of both the analytical methodology for CE and statistical methods for identifying common ions that could be used as physical stress markers.

Design and Fabrication of Silicon Nanowire-Based Biosensors with Integration of Critical Factors: Toward Ultrasensitive Specific Detection of Biomolecules.

As critical factors affecting the sensing performance of silicon nanowire (SiNW) biosensors, the structure, functional interface, and detection target were analyzed and designed to improve sensing performance. For an improved understanding of the dependence of sensor structure on sensitivity, a simple theoretical analysis was proposed to predict the sensitivity of biosensors with different SiNW types, widths, and doping concentrations. Based on the theoretical analysis, a biosensor integrating optimized critical factors was designed and fabricated. Optimizations focusing on the following aspects are considered: (1) employing n-type SiNW and controlling the impurity doping concentration of SiNW at approximately 2 × 1016-6 × 1016 atoms/cm3 to obtain a suitable charge density, (2) minimizing the SiNW width to 16.0 nm to increase the surface area-to-volume ratio, (3) using a native oxide layer on SiNW as a gate insulator to transport the captured charge molecules closer to the SiNW surface, (4) modifying the SiNW surface by 2-aminoethylphosphonic acid coupling to form a high-density self-assembled monolayer for enhancing the stability bound molecules, and (5) functionalizing the SiNW with ovalbumin molecules for specifically capturing the target immunoglobulin G (IgG) molecules. The sensing performance was evaluated by detecting IgG with concentrations ranging from 6 aM to 600 nM and control experiments. The SiNW biosensor revealed ultrahigh sensitivity and specific detection of target IgG with a measured limit of detection of 6 aM. The integration of the critical SiNW biosensor factors provides a significant possibility of a rapid and ultrasensitive diagnosis of diseases at their early stages.

CD8+ regulatory T cells are critical in prevention of autoimmune-mediated diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic ß-cells are destroyed. Intestinal helminths can cause asymptomatic chronic and immunosuppressive infections and suppress disease in rodent models of T1D. However, the underlying regulatory mechanisms for this protection are unclear. Here, we report that CD8+ regulatory T (Treg) cells prevent the onset of streptozotocin -induced diabetes by a rodent intestinal nematode. Trehalose derived from nematodes affects the intestinal microbiota and increases the abundance of Ruminococcus spp., resulting in the induction of CD8+ Treg cells. Furthermore, trehalose has therapeutic effects on both streptozotocin-induced diabetes and in the NOD mouse model of T1D. In addition, compared with healthy volunteers, patients with T1D have fewer CD8+ Treg cells, and the abundance of intestinal Ruminococcus positively correlates with the number of CD8+ Treg cells in humans.

The role of autonomously secreted PGE2 and its autocrine/paracrine effect on bone matrix mineralization at the different stages of differentiating MC3T3-E1 cells.

Bone is consisted of osteoblast-linage cells, bone-forming cells in various differentiation stages. However, it is not fully understood how communicate and interact these cells immigrated from bone marrow. In this study, we showed that prostaglandin E2 (PGE2) had a role in autonomous modification of matrix mineralization in osteoblastic cell line, MC3T3-E1, and interactions across the cells in different differentiation stages. Analysis using LC-MS/MS and inhibitors showed the autonomous secretion of PGE2 among the prostanoids in differentiation stages and that depend on COX-2, a key enzyme for production of PGE2. Treatment with inhibitors of PGE2 receptors and COX-2 indicated that secreted PGE2 regulates matrix mineralization in an autocrine/paracrine manner. In addition, we showed that the expression profile of PGE2 receptors (EP1-EP4) and PGE2 effects on matrix mineralization derived from it changed during cell differentiation. Treatment with inhibitors of PGE2 signaling in the early differentiation stage of MC3T3-E1 cells induced significant changes in matrix mineralization several days after. Stimulation with the extracts from culture medium of the matured cells including PGE2 and co-culture with the matured cells secreting PGE2 significantly promoted matrix mineralization of the early stage cells, in contrast, treatment with inhibitor of COX-2 and PGE2 receptors failed to do so. These results support that PGE2 plays important roles in the interaction system of osteoblast-linage cells in bone tissue to regulate matrix mineralization reflecting condition of bone-forming cells, that is, population and maturation.

Regulation of MC3T3-E1 differentiation by actin cytoskeleton through lipid mediators reflecting the cell differentiation stage.

Actin cytoskeleton is reported to be related in various functions of osteoblast, bone-forming cell. However the function of actin cytoskeleton in osteoblasts is not fully understood, since bone formation is derived from extracellular interactions of functional proteins produced from osteoblasts, including osteocalcin (Ocn), and it is a result of closely and complex organized sequence of biochemical events. In this study, we showed that actin cytoskeleton of MC3T3-E1 cells functioned in recognition of cell condition and regulation of extracellular matrix mineralization, bone formation. Maturation of MC3T3-E1 cells by 14 days of culture reduced F-actin filaments, while induced expression of Ocn mRNA known as late stage differentiation marker and matrix mineralization, terminal stage of cell differentiation. The disruption of actin cytoskeleton with Cyto D in immature MC3T3-E1 cells significantly increased expression of Ocn mRNA in 24 h. Both PTX-induced inhibition of signal transduction through GPCRs and celecoxib-induced suppression of lipid mediators in immature MC3T3-E1 cells reduced actin filaments and suppressed matrix mineralization. Furthermore, addition of lipid mediators extracted from culture mediums of differentiated MC3T3-E1 cells by Bligh-Dyer method induced actin cytoskeleton reorganization and matrix mineralization change in MC3T3-E1 cells. Taken together, our data suggest that actin cytoskeleton of MC3T3-E1 cells regulates activation of developmental pathway reflecting cell differentiation stages through lipid mediators. The function we identified is important for bone formation tightly regulated by mechanical stress, since actin cytoskeleton is also known as a mechanosensor of osteoblasts.

GDE5 inhibition accumulates intracellular glycerophosphocholine and suppresses adipogenesis at a mitotic clonal expansion stage.

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTß), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.

Enzymatic characterization of recombinant rat DDHD2: a soluble diacylglycerol lipase.

DDHD2 has been reported to exhibit phospholipase A1, triacylglycerol (TG) lipase and diacylglycerol (DG) lipase activities. However, the detailed enzymatic properties of DDHD2 have not yet been elucidated. In the current study, the substrate specificity of DDHD2 towards DG, TG and phosphatidic acid (PA) has been examined using highly purified recombinant rat DDHD2 (rDDHD2) with a liquid chromatography mass spectrometer. The k cat/Km value for DG (18:0/20:4) was much higher than those for TG (18:1/18:1/18:1), and PA (18:0/20:4) in the presence of sodium deoxycholate. The enzyme activity of rDDHD2 towards DG (18:0/20:4) was highest among all of the substrates tested. In addition, rDDHD2 was highly specific to DG substrates with a polyunsaturated fatty acid at their sn-2 position. The levels of 2-arachidonoylglycerol (2-AG) in CHO cells were quantified by gas chromatography-tandem mass spectrometry, showing that CHO cells expressing recombinant rDDHD2 contained higher levels of 2-AG when cells were treated with a monoacylglycerol lipase inhibitor, URB602. These results therefore support the idea that DDHD2 functions as a DG lipase in vivo and produces 2-AG.

Protein purification and cloning of diacylglycerol lipase from rat brain.

Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia.

New members of the mammalian glycerophosphodiester phosphodiesterase family: GDE4 and GDE7 produce lysophosphatidic acid by lysophospholipase D activity.

The known mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) hydrolyze glycerophosphodiesters. In this study, two novel members of the mammalian GP-PDE family, GDE4 and GDE7, were isolated, and the molecular basis of mammalian GP-PDEs was further explored. The GDE4 and GDE7 sequences are highly homologous and evolutionarily close. GDE4 is expressed in intestinal epithelial cells, spermatids, and macrophages, whereas GDE7 is particularly expressed in gastro-esophageal epithelial cells. Unlike other mammalian GP-PDEs, GDE4 and GDE7 cannot hydrolyze either glycerophosphoinositol or glycerophosphocholine. Unexpectedly, both GDE4 and GDE7 show a lysophospholipase D activity toward lysophosphatidylcholine (lyso-PC). We purified the recombinant GDE4 and GDE7 proteins and show that these enzymes can hydrolyze lyso-PC to produce lysophosphatidic acid (LPA). Further characterization of purified recombinant GDE4 showed that it can also convert lyso-platelet-activating factor (1-O-alkyl-sn-glycero-3-phosphocholine; lyso-PAF) to alkyl-LPA. These data contribute to our current understanding of mammalian GP-PDEs and of their physiological roles via the control of lyso-PC and lyso-PAF metabolism in gastrointestinal epithelial cells and macrophages.

Nicotine exposure alters human vascular smooth muscle cell phenotype from a contractile to a synthetic type.

OBJECTIVE: Cigarette smoking is a known risk factor for arteriosclerosis. In atheromatous plaques, vascular smooth muscle cells (VSMCs) display a phenotype that is different from the contractile type under normal conditions. Nicotine is the major pharmacological agent in cigarette smoke. However, any direct effect of nicotine on VSMCs remains uncertain. Because nicotine promotes VSMC migration, its phenotype may change due to nicotine. APPROACH AND RESULTS: We used human aorta primary smooth muscle cells (HuAoSMCs), differentiated with transforming growth factor-ß, to investigate changes in the protein levels of differentiation markers and in the activity of mitogen-activated protein kinases (MAPKs) after exposure to 0.1 µM of nicotine for 48 h. After nicotine exposure, the protein levels of myosin II 10 (2.93-fold) and ß-actin (1.66-fold), synthetic type markers, were increased. In contrast, the levels of the contractile type markers, myosin II 11 (0.63-fold), high-molecular-weight caldesmon (0.40-fold) and SM22 (0.66-fold), which concern differentiated VSMC, were decreased. Moreover, nicotine exposure induced enhanced activation of p38 MAPK (1.30-fold) and extracellular signal-regulated kinase (1.91-fold). These results indicated that the phenotype of HuAoSMCs had changed to a synthetic-like type because of nicotine exposure. Thus, nicotine is one factor that can alter protein expression of differentiation markers in VSMCs. Besides, the increase of intracellular Ca(2+) levels suggested that these effects of nicotine were mediated through nicotinic acetylcholine receptors. CONCLUSION: Nicotine has already been reported to promote VSMC migration from the tunica media to atheromatous plaques in the vascular intima. This phenomenon may occur because nicotine directly induces VSMC transformation from contractile type to synthetic-like type via nicotinic acetylcholine receptors and G protein-coupled receptors.

Combination of postmortem mass spectrometry imaging and genetic analysis reveals very long-chain acyl-CoA dehydrogenase deficiency in a case of infant death with liver steatosis.

CASE HISTORY: A 3-month-old infant was found dead in his bed. A postmortem computed tomography (CT) scan suggested fatty attenuation in the liver parenchyma, but no other potentially fatal changes were found. To clarify the cause of death, a medicolegal autopsy was carried out. AUTOPSY FINDINGS: Internal examination confirmed the presence of liver steatosis as well as hepatomegaly. There were no other significant findings including encephalitis or brain edema. MASS SPECTROMETRY ANALYSIS: To clarify the mechanism underlying lipid accumulation in the liver, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) analysis was conducted. This indicated a significant accumulation of C14:1 acylcarnitine in the liver of the deceased, suggesting very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. GENETIC ANALYSIS: To find the cause of the VLCAD deficiency, genetic analysis of the responsible gene, acyl-CoA dehydrogenase, very long chain (ACADVL), was performed. This revealed two novel mutations that may have accounted for the disease. CONCLUSION: A combination of these data revealed that the liver steatosis in this case might have been caused by VLCAD deficiency based on genetic mutations of ACADVL. Thus, the deceased might have been vulnerable to energy crisis and sudden infant death. The present findings show that MALDI-IMS analysis as well as genetic analysis can be useful for elucidating the cause of death.

The emerging physiological roles of the glycerophosphodiesterase family.

The glycerophosphodiester phosphodiesterases are evolutionarily conserved proteins that have been linked to several patho/physiological functions, comprising bacterial pathogenicity and mammalian cell proliferation or differentiation. The bacterial enzymes do not show preferential substrate selectivities among the glycerophosphodiesters, and they are mainly dedicated to glycerophosphodiester hydrolysis, producing glycerophosphate and alcohols as the building blocks that are required for bacterial biosynthetic pathways. In some cases, this enzymatic activity has been demonstrated to contribute to bacterial pathogenicity, such as with Hemophilus influenzae. Mammalian glyerophosphodiesterases have high substrate specificities, even if the number of potential physiological substrates is continuously increasing. Some of these mammalian enzymes have been directly linked to cell differentiation, such as GDE2, which triggers motor neuron differentiation, and GDE3, the enzymatic activity of which is necessary and sufficient to induce osteoblast differentiation. Instead, GDE5 has been shown to inhibit skeletal muscle development independent of its enzymatic activity.

A novel glycerophosphodiester phosphodiesterase, GDE5, controls skeletal muscle development via a non-enzymatic mechanism.

Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5DeltaC471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5DeltaC471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.