[Effects of warming on physicochemical property of Cunninghamia lanceolata branch and leaf litter in subtropical plantation]. / 增温对亚热带杉木枝和叶凋落物理化性质的影响.

At the regional scale, substrate properties are the key factors driving litter decomposition rate. In this study, soil temperature was increased by buried heating cables to explore the impacts of climate warming on the physical and chemical properties in branch and leaf of Cunninghamia lanceolata litter. The results showed that after 5 years of soil warming (4 ℃), the contents of nitrogen (N), phosphorus (P) and water-soluble substance in branch litter increased by 35.2%, 40.8% and 7.6%, while that in leaf litter increased by 41.2%, 45.9% and 5.9%, respectively. The contents of carbon (C), cellulose and C/N in branch litter decreased by 5.1%, 11.6% and 28.8%, and in leaf litter decreased by 5.3%, 11.3% and 33.3%, respectively. Soil warming led to 29.8% increase in specific leaf area (SLA) and 40.7% decrease in tensile strength (LTS) of leaf litter. However, warming did not affect lignin content and pH value in both branch and leaf litter. 13C NMR and infrared spectrum analysis showed that the contents of amino acids, polysaccharides, polyphenols and aliphatic compounds in litter changed significantly after warming. Warming effect differed between litter organs, in that polysaccharides increased significantly only in leaf litter and the increase of amino acids in branch litter was greater than that in leaf litter. Overall, soil warming significantly changed the physical and chemical properties in C. lanceolata branch and leaf litter, which might accelerate the decomposition rate at the initial stage due to the increase of N, P contents and the decrease of LTS, but might decelerate the decomposition rate at the later stage due to an increase of complex polymers content in the litter.

Lysophospholipid acylation modulates plasma membrane lipid organization and insulin sensitivity in skeletal muscle.

Aberrant lipid metabolism promotes the development of skeletal muscle insulin resistance, but the exact identity of lipid-mediated mechanisms relevant to human obesity remains unclear. A comprehensive lipidomic analysis of primary myocytes from individuals who were insulin-sensitive and lean (LN) or insulin-resistant with obesity (OB) revealed several species of lysophospholipids (lyso-PLs) that were differentially abundant. These changes coincided with greater expression of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme involved in phospholipid transacylation (Lands cycle). Strikingly, mice with skeletal muscle-specific knockout of LPCAT3 (LPCAT3-MKO) exhibited greater muscle lysophosphatidylcholine/phosphatidylcholine, concomitant with improved skeletal muscle insulin sensitivity. Conversely, skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) promoted glucose intolerance. The absence of LPCAT3 reduced phospholipid packing of cellular membranes and increased plasma membrane lipid clustering, suggesting that LPCAT3 affects insulin receptor phosphorylation by modulating plasma membrane lipid organization. In conclusion, obesity accelerates the skeletal muscle Lands cycle, whose consequence might induce the disruption of plasma membrane organization that suppresses muscle insulin action.

Metabolic Effects of Selective Deletion of Group VIA Phospholipase A2 from Macrophages or Pancreatic Islet Beta-Cells.

To examine the role of group VIA phospholipase A2 (iPLA2ß) in specific cell lineages in insulin secretion and insulin action, we prepared mice with a selective iPLA2ß deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2ß-KO), or in insulin-secreting ß-cells (ß-Cell-iPLA2ß-KO), respectively. MØ-iPLA2ß-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2ß control mice after consuming a high-fat diet (HFD). MØ-iPLA2ß-KO mice exhibited normal glucose-stimulated insulin secretion (GSIS) in vivo and from isolated islets ex vivo compared to controls. Male MØ-iPLA2ß-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. In contrast, ß-cell-iPLA2ß-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further when introduced to a HFD. ß-Cell-iPLA2ß-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls. ß-Cell-iPLA2ß-KO mice also exhibited an enhanced insulin responsivity compared to controls. These findings suggest that MØ iPLA2ß participates in HFD-induced deterioration in glucose tolerance and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, ß-cell iPLA2ß plays a role in GSIS and also appears to confer some protection against deterioration in ß-cell functions induced by a HFD.

N-acyl-O-phosphocholineserines: structures of a novel class of lipids that are biomarkers for Niemann-Pick C1 disease.

Niemann-Pick disease type C1 (NPC1) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, there is an urgency to improve diagnostics and monitor therapeutic efficacy with biomarkers. In this study, we sought to define the structure of an unknown lipid biomarker for NPC1 with [M + H]+ ion at m/z 509.3351, previously designated as lysoSM-509. The structure of N-palmitoyl-O-phosphocholineserine (PPCS) was proposed for the lipid biomarker based on the results from mass spectrometric analyses and chemical derivatizations. As no commercial standard is available, authentic PPCS was chemically synthesized, and the structure was confirmed by comparison of endogenous and synthetic compounds as well as their derivatives using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PPCS is the most abundant species among N-acyl-O-phosphocholineserines (APCS), a class of lipids that have not been previously detected in biological samples. Further analysis demonstrated that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.

PexRAP Inhibits PRDM16-Mediated Thermogenic Gene Expression.

How the nuclear receptor PPARγ regulates the development of two functionally distinct types of adipose tissue, brown and white fat, as well as the browning of white fat, remains unclear. Our previous studies suggest that PexRAP, a peroxisomal lipid synthetic enzyme, regulates PPARγ signaling and white adipogenesis. Here, we show that PexRAP is an inhibitor of brown adipocyte gene expression. PexRAP inactivation promoted adipocyte browning, increased energy expenditure, and decreased adiposity. Identification of PexRAP-interacting proteins suggests that PexRAP function extends beyond its role as a lipid synthetic enzyme. Notably, PexRAP interacts with importin-ß1, a nuclear import factor, and knockdown of PexRAP in adipocytes reduced the levels of nuclear phospholipids. PexRAP also interacts with PPARγ, as well as PRDM16, a critical transcriptional regulator of thermogenesis, and disrupts the PRDM16-PPARγ complex, providing a potential mechanism for PexRAP-mediated inhibition of adipocyte browning. These results identify PexRAP as an important regulator of adipose tissue remodeling.

Fatty acid synthesis configures the plasma membrane for inflammation in diabetes.

Dietary fat promotes pathological insulin resistance through chronic inflammation. The inactivation of inflammatory proteins produced by macrophages improves diet-induced diabetes, but how nutrient-dense diets induce diabetes is unknown. Membrane lipids affect the innate immune response, which requires domains that influence high-fat-diet-induced chronic inflammation and alter cell function based on phospholipid composition. Endogenous fatty acid synthesis, mediated by fatty acid synthase (FAS), affects membrane composition. Here we show that macrophage FAS is indispensable for diet-induced inflammation. Deleting Fasn in macrophages prevents diet-induced insulin resistance, recruitment of macrophages to adipose tissue and chronic inflammation in mice. We found that FAS deficiency alters membrane order and composition, impairing the retention of plasma membrane cholesterol and disrupting Rho GTPase trafficking-a process required for cell adhesion, migration and activation. Expression of a constitutively active Rho GTPase, however, restored inflammatory signalling. Exogenous palmitate was partitioned to different pools from endogenous lipids and did not rescue inflammatory signalling. However, exogenous cholesterol, as well as other planar sterols, did rescue signalling, with cholesterol restoring FAS-induced perturbations in membrane order. Our results show that the production of endogenous fat in macrophages is necessary for the development of exogenous-fat-induced insulin resistance through the creation of a receptive environment at the plasma membrane for the assembly of cholesterol-dependent signalling networks.

Skeletal Muscle Phospholipid Metabolism Regulates Insulin Sensitivity and Contractile Function.

Skeletal muscle insulin resistance is an early defect in the development of type 2 diabetes. Lipid overload induces insulin resistance in muscle and alters the composition of the sarcoplasmic reticulum (SR). To test the hypothesis that skeletal muscle phospholipid metabolism regulates systemic glucose metabolism, we perturbed choline/ethanolamine phosphotransferase 1 (CEPT1), the terminal enzyme in the Kennedy pathway of phospholipid synthesis. In C2C12 cells, CEPT1 knockdown altered SR phospholipid composition and calcium flux. In mice, diet-induced obesity, which decreases insulin sensitivity, increased muscle CEPT1 expression. In high-fat diet-fed mice with skeletal muscle-specific knockout of CEPT1, systemic and muscle-based approaches demonstrated increased muscle insulin sensitivity. In CEPT1-deficient muscles, an altered SR phospholipid milieu decreased sarco/endoplasmic reticulum Ca(2+) ATPase-dependent calcium uptake, activating calcium-signaling pathways known to improve insulin sensitivity. Altered muscle SR calcium handling also rendered these mice exercise intolerant. In obese humans, surgery-induced weight loss increased insulin sensitivity and decreased skeletal muscle CEPT1 protein. In obese humans spanning a spectrum of metabolic health, muscle CEPT1 mRNA was inversely correlated with insulin sensitivity. These results suggest that high-fat feeding and obesity induce CEPT1, which remodels the SR to preserve contractile function at the expense of insulin sensitivity.

Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy.

Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that contributes to abnormal Ca(2+) homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition.

Reduced efficiency of sarcolipin-dependent respiration in myocytes from humans with severe obesity.

OBJECTIVE: Sarcolipin (SLN) regulates muscle energy expenditure through its action on sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump. It is unknown whether SLN-dependent respiration has relevance to human obesity, but whole-transcriptome gene expression profiling revealed that SLN was more highly expressed in myocytes from individuals with severe obesity (OB) than in lean controls (LN). The purpose of this study was to examine SLN-dependent cellular respiratory rates in LN and OB human muscles. METHODS: Primary myocytes were isolated from muscle biopsy from seven LN and OB Caucasian females. Cellular respiration was assessed with and without lentivirus-mediated SLN knockdown in LN and OB myocytes. RESULTS: SLN mRNA and protein abundance was greater in OB compared to LN cells. Despite elevated SLN levels in wild-type OB cells, respiratory rates among SLN-deficient cells were higher in OB compared to LN. Obesity-induced reduction in efficiency of SLN-dependent respiration was associated with altered sarcoplasmic reticulum phospholipidome. CONCLUSIONS: SLN-dependent respiration is reduced in muscles from humans with severe obesity compared to lean controls. Identification of the molecular mechanism that affects SLN efficiency might lead to interventions that promote an increase in skeletal muscle energy expenditure.

Group VIA phospholipase A2 mitigates palmitate-induced ß-cell mitochondrial injury and apoptosis.

Palmitate (C16:0) induces apoptosis of insulin-secreting ß-cells by processes that involve generation of reactive oxygen species, and chronically elevated blood long chain free fatty acid levels are thought to contribute to ß-cell lipotoxicity and the development of diabetes mellitus. Group VIA phospholipase A2 (iPLA2ß) affects ß-cell sensitivity to apoptosis, and here we examined iPLA2ß effects on events that occur in ß-cells incubated with C16:0. Such events in INS-1 insulinoma cells were found to include activation of caspase-3, expression of stress response genes (C/EBP homologous protein and activating transcription factor 4), accumulation of ceramide, loss of mitochondrial membrane potential, and apoptosis. All of these responses were blunted in INS-1 cells that overexpress iPLA2ß, which has been proposed to facilitate repair of oxidized mitochondrial phospholipids, e.g. cardiolipin (CL), by excising oxidized polyunsaturated fatty acid residues, e.g. linoleate (C18:2), to yield lysophospholipids, e.g. monolysocardiolipin (MLCL), that can be reacylated to regenerate the native phospholipid structures. Here the MLCL content of mouse pancreatic islets was found to rise with increasing iPLA2ß expression, and recombinant iPLA2ß hydrolyzed CL to MLCL and released oxygenated C18:2 residues from oxidized CL in preference to native C18:2. C16:0 induced accumulation of oxidized CL species and of the oxidized phospholipid (C18:0/hydroxyeicosatetraenoic acid)-glycerophosphoethanolamine, and these effects were blunted in INS-1 cells that overexpress iPLA2ß, consistent with iPLA2ß-mediated removal of oxidized phospholipids. C16:0 also induced iPLA2ß association with INS-1 cell mitochondria, consistent with a role in mitochondrial repair. These findings indicate that iPLA2ß confers significant protection of ß-cells against C16:0-induced injury.

Screening method to evaluate point-of-care human chorionic gonadotropin (hCG) devices for susceptibility to the hook effect by hCG ß core fragment: evaluation of 11 devices.

BACKGROUND: The predominant hCG variant in urine, hCG ß core fragment (hCGßcf), has been demonstrated to cause false-negative results in qualitative point-of-care (POC) hCG devices. This is a major concern for healthcare professionals using POC pregnancy tests. We developed a screening method to evaluate qualitative POC hCG devices for their susceptibility to inhibition by hCGßcf. Using this method, we evaluated the performance of 11 commonly used devices. METHODS: A wide range of purified hCG and hCGßcf concentrations were mixed and tested on 2 POC devices. By use of those results, a screening method was defined and 9 additional POC devices were evaluated. Two solutions containing (a) 500 pmol/L (171 IU/L) intact hCG with 0 pmol/L hCGßcf and (b) 500 pmol/L intact hCG with 500 000 pmol/L hCGßcf were used to screen all POC devices. RESULTS: The OSOM and Cen-Med Elite devices were found to be most susceptible to false-negative results due to hCGßcf. The BC Icon 20 and the Alere were the least susceptible. The remaining 7 were moderately affected. Devices that gave the strongest signal with hCGßcf alone were those that were least likely to show a hook effect. CONCLUSIONS: The screening method put forth here can be used by device users and manufacturers to evaluate POC devices for inhibition by hCGßcf. Of 11 devices evaluated, only 2 have been identified that exhibit minimal to no susceptibility to hCGßcf.

Insulin-regulated protein palmitoylation impacts endothelial cell function.

OBJECTIVE: Defects in insulin signaling are associated with abnormal endothelial cell function, which occurs commonly in cardiovascular disease. Targets of insulin signaling in endothelial cells are incompletely understood. Protein S-palmitoylation, the reversible modification of proteins by the lipid palmitate, is a post-translational process relevant to cell signaling, but little is known about the role of insulin in protein palmitoylation. APPROACH AND RESULTS: To test the hypothesis that insulin alters protein palmitoylation in endothelial cells, we combined acyl-biotin exchange chemistry with stable isotope labeling by amino acids in cell culture to perform quantitative proteomic profiling of human endothelial cells. We identified ≈380 putative palmitoylated proteins, of which >200 were not known to be palmitoylated; ≈10% of the putative palmitoylated proteins were induced or suppressed by insulin. Of those potentially affected by insulin, <10 have been implicated in vascular function. For one, platelet-activating factor acetylhydrolase IB subunit gamma (PAFAH1b3; not previously known to be palmitoylated), we confirmed that insulin stimulated palmitoylation without affecting PAFAH1b3 protein abundance. Chemical inhibition of palmitoylation prevented insulin-induced angiogenesis in vitro; knockdown of PAFAH1b3 had the same effect. PAFAH1b3 knockdown also disrupted cell migration. Mutagenesis of cysteines at residues 56 and 206 prevented palmitoylation of PAFAH1b3, abolished its capacity to stimulate cell migration, and inhibited its association with detergent-resistant membranes, which are implicated in cell signaling. Insulin promoted the association of wild-type PAFAH1b3 with detergent-resistant membranes. CONCLUSIONS: These findings provide proof of principle for using proteomics to identify novel insulin-inducible palmitoylation targets relevant to endothelial function.

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα.

Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status.

Muscle lipogenesis balances insulin sensitivity and strength through calcium signaling.

Exogenous dietary fat can induce obesity and promote diabetes, but endogenous fat production is not thought to affect skeletal muscle insulin resistance, an antecedent of metabolic disease. Unexpectedly, the lipogenic enzyme fatty acid synthase (FAS) was increased in the skeletal muscle of mice with diet-induced obesity and insulin resistance. Skeletal muscle-specific inactivation of FAS protected mice from insulin resistance without altering adiposity, specific inflammatory mediators of insulin signaling, or skeletal muscle levels of diacylglycerol or ceramide. Increased insulin sensitivity despite high-fat feeding was driven by activation of AMPK without affecting AMP content or the AMP/ATP ratio in resting skeletal muscle. AMPK was induced by elevated cytosolic calcium caused by impaired sarco/endoplasmic reticulum calcium ATPase (SERCA) activity due to altered phospholipid composition of the sarcoplasmic reticulum (SR), but came at the expense of decreased muscle strength. Thus, inhibition of skeletal muscle FAS prevents obesity-associated diabetes in mice, but also causes muscle weakness, which suggests that mammals have retained the capacity for lipogenesis in muscle to preserve physical performance in the setting of disrupted metabolic homeostasis.

Liver fatty acid binding protein (L-Fabp) modulates murine stellate cell activation and diet-induced nonalcoholic fatty liver disease.

UNLABELLED: Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes, also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP(-/-) mice contain fewer LDs than wild-type (WT) HSCs, and exhibit up-regulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP(-/-) mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP(-/-) and WT mice a high-fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP(-/-) mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP(-/-) mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. CONCLUSION: L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. (Hepatology 2013).

Group VIB Phospholipase A(2) promotes proliferation of INS-1 insulinoma cells and attenuates lipid peroxidation and apoptosis induced by inflammatory cytokines and oxidant agents.

Group VIB Phospholipase A(2) (iPLA(2)γ) is distributed in membranous organelles in which ß-oxidation occurs, that is, mitochondria and peroxisomes, and is expressed by insulin-secreting pancreatic islet ß-cells and INS-1 insulinoma cells, which can be injured by inflammatory cytokines, for example, IL-1ß and IFN-γ, and by oxidants, for example, streptozotocin (STZ) or t-butyl-hydroperoxide (TBHP), via processes pertinent to mechanisms of ß-cell loss in types 1 and 2 diabetes mellitus. We find that incubating INS-1 cells with IL-1ß and IFN-γ, with STZ, or with TBHP causes increased expression of iPLA(2)γ mRNA and protein. We prepared INS-1 knockdown (KD) cell lines with reduced iPLA(2)γ expression, and they proliferate more slowly than control INS-1 cells and undergo increased membrane peroxidation in response to cytokines or oxidants. Accumulation of oxidized phospholipid molecular species in STZ-treated INS-1 cells was demonstrated by LC/MS/MS scanning, and the levels in iPLA(2)γ-KD cells exceeded those in control cells. iPLA(2)γ-KD INS-1 cells also exhibited higher levels of apoptosis than control cells when incubated with STZ or with IL-1ß and IFN-γ. These findings suggest that iPLA(2)γ promotes ß-cell proliferation and that its expression is increased during inflammation or oxidative stress as a mechanism to mitigate membrane injury that may enhance ß-cell survival.

Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARγ activation to decrease diet-induced obesity.

De novo lipogenesis in adipocytes, especially with high fat feeding, is poorly understood. We demonstrate that an adipocyte lipogenic pathway encompassing fatty acid synthase (FAS) and PexRAP (peroxisomal reductase activating PPARγ) modulates endogenous PPARγ activation and adiposity. Mice lacking FAS in adult adipose tissue manifested increased energy expenditure, increased brown fat-like adipocytes in subcutaneous adipose tissue, and resistance to diet-induced obesity. FAS knockdown in embryonic fibroblasts decreased PPARγ transcriptional activity and adipogenesis. FAS-dependent alkyl ether phosphatidylcholine species were associated with PPARγ and treatment of 3T3-L1 cells with one such ether lipid increased PPARγ transcriptional activity. PexRAP, a protein required for alkyl ether lipid synthesis, was associated with peroxisomes and induced during adipogenesis. PexRAP knockdown in cells decreased PPARγ transcriptional activity and adipogenesis. PexRAP knockdown in mice decreased expression of PPARγ-dependent genes and reduced diet-induced adiposity. These findings suggest that inhibiting PexRAP or related lipogenic enzymes could treat obesity and diabetes.

Group VIA PLA2 (iPLA2ß) is activated upstream of p38 mitogen-activated protein kinase (MAPK) in pancreatic islet ß-cell signaling.

Group VIA phospholipase A(2) (iPLA(2)ß) in pancreatic islet ß-cells participates in glucose-stimulated insulin secretion and sarco(endo)plasmic reticulum ATPase (SERCA) inhibitor-induced apoptosis, and both are attenuated by pharmacologic or genetic reductions in iPLA(2)ß activity and amplified by iPLA(2)ß overexpression. While exploring signaling events that occur downstream of iPLA(2)ß activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA(2)ß expression level, and that it is stimulated by the iPLA(2)ß reaction product arachidonic acid. The insulin secretagogue D-glucose also stimulates ß-cell p38 MAPK phosphorylation, and this is prevented by the iPLA(2)ß inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA(2)ß in INS-1 cells and in mouse islets, and the p38 MAPK inhibitor PD169316 prevents both responses. The SERCA inhibitor thapsigargin also stimulates phosphorylation of both ß-cell MAPK kinase isoforms and p38 MAPK, and bromoenol lactone prevents both events. Others have reported that iPLA(2)ß products activate Rho family G-proteins that promote MAPK kinase activation via a mechanism inhibited by Clostridium difficile toxin B, which we find to inhibit thapsigargin-induced ß-cell p38 MAPK phosphorylation. Thapsigargin-induced ß-cell apoptosis and ceramide generation are also prevented by the p38 MAPK inhibitor PD169316. These observations indicate that p38 MAPK is activated downstream of iPLA(2)ß in ß-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA(2)ß activation, and that p38 MAPK is involved in the ß-cell functional responses of insulin secretion and apoptosis in which iPLA(2)ß participates.

Oxidative modifications of the C-terminal domain of tropoelastin prevent cell binding.

Tropoelastin (TE), the soluble monomer of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, and smooth muscle cells (SMCs). The C-terminal domain of TE interacts with cell receptors, and these interactions play critical roles in elastic fiber assembly. We recently found that oxidation of TE prevents elastic fiber assembly. Here, we examined the effects of oxidation of TE on cell interactions. We found that SMCs bind to TE through heparan sulfate (HS), whereas fetal lung fibroblasts (WI-38 cells) bind through integrin α(v)ß(3) and HS. In addition, we found that oxidation of TE by peroxynitrite (ONOO(-)) prevented binding of SMCs and WI-38 cells and other elastogenic cells, human dermal fibroblasts and fetal bovine chondrocytes. Because the C-terminal domain of TE has binding sites for both HS and integrin, we examined the effects of oxidation of a synthetic peptide derived from the C-terminal 25 amino acids of TE (CT-25) on cell binding. The CT-25 peptide contains the only two Cys residues in TE juxtaposed to a cluster of positively charged residues (RKRK) that are important for cell binding. ONOO(-) treatment of the CT-25 peptide prevented cell binding, whereas reduction of the CT-25 peptide had no effect. Mass spectrometric and circular dichroism spectroscopic analyses showed that ONOO(-) treatment modified both Cys residues in the CT-25 peptide to sulfonic acid derivatives, without altering the secondary structure. These data suggest that the mechanism by which ONOO(-) prevents cell binding to TE is by introducing negatively charged sulfonic acid residues near the positively charged cluster.

Effects of endoplasmic reticulum stress on group VIA phospholipase A2 in beta cells include tyrosine phosphorylation and increased association with calnexin.

The Group VIA phospholipase A(2) (iPLA(2)ß) hydrolyzes glycerophospholipids at the sn-2-position to yield a free fatty acid and a 2-lysophospholipid, and iPLA(2)ß has been reported to participate in apoptosis, phospholipid remodeling, insulin secretion, transcriptional regulation, and other processes. Induction of endoplasmic reticulum (ER) stress in ß-cells and vascular myocytes with SERCA inhibitors activates iPLA(2)ß, resulting in hydrolysis of arachidonic acid from membrane phospholipids, by a mechanism that is not well understood. Regulatory proteins interact with iPLA(2)ß, including the Ca(2+)/calmodulin-dependent protein kinase IIß, and we have characterized the iPLA(2)ß interactome further using affinity capture and LC/electrospray ionization/MS/MS. An iPLA(2)ß-FLAG fusion protein was expressed in an INS-1 insulinoma cell line and then adsorbed to an anti-FLAG matrix after cell lysis. iPLA(2)ß and any associated proteins were then displaced with FLAG peptide and analyzed by SDS-PAGE. Gel sections were digested with trypsin, and the resultant peptide mixtures were analyzed by LC/MS/MS with database searching. This identified 37 proteins that associate with iPLA(2)ß, and nearly half of them reside in ER or mitochondria. They include the ER chaperone calnexin, whose association with iPLA(2)ß increases upon induction of ER stress. Phosphorylation of iPLA(2)ß at Tyr(616) also occurs upon induction of ER stress, and the phosphoprotein associates with calnexin. The activity of iPLA(2)ß in vitro increases upon co-incubation with calnexin, and overexpression of calnexin in INS-1 cells results in augmentation of ER stress-induced, iPLA(2)ß-catalyzed hydrolysis of arachidonic acid from membrane phospholipids, reflecting the functional significance of the interaction. Similar results were obtained with mouse pancreatic islets.