Haowei Song

Matrix metalloproteinase-9 degrades amyloid-β fibrils in vitro and compact plaques in situ

The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-β fibrils (fAβ), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Aβ (sAβ) could degrade fAβ as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAβ and that this ability is not shared by other sAβ-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAβ was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAβ breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAβ were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Aβ that are cleaved during its degradation. Only MMP-9 digests contained fragments (Aβ1-20 and Aβ1-30) from fAβ1-42 substrate; the corresponding cleavage sites are thought to be important for β-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAβ and may contribute to ongoing clearance of plaques from amyloid-laden brains.

Matrix Metalloproteinases Expressed by Astrocytes Mediate Extracellular Amyloid-β Peptide Catabolism

It has been postulated that the development of amyloid plaques in Alzheimers disease (AD) may result from an imbalance between the generation and clearance of the amyloid-β peptide (Aβ). Although familial AD appears to be caused by Aβ overproduction, sporadic AD (the most prevalent form) may result from impairment in clearance. Recent evidence suggests that several proteases may contribute to the degradation of Aβ. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of Aβ degradation. In this study, we examined the possibility that matrix metalloproteinases (MMPs), proteases known to be expressed and secreted by astrocytes, could play a role in extracellular Aβ degradation. We found that astrocytes surrounding amyloid plaques showed enhanced expression of MMP-2 and MMP-9 in aged amyloid precursor protein (APP)/presenilin 1 mice. Moreover, astrocyte-conditioned medium (ACM) degraded Aβ, lowering levels and producing several fragments after incubation with synthetic human Aβ1–40 and Aβ1–42. This activity was attenuated with specific inhibitors of MMP-2 and -9, as well as in ACM derived from mmp-2 or -9 knock-out (KO) mice. In vivo, significant increases in the steady-state levels of Aβ were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls. Furthermore, pharmacological inhibition of the MMPs with N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM 6001) increased brain interstitial fluid Aβ levels and elimination of half-life in APPsw mice. These results suggest that MMP-2 and -9 may contribute to extracellular brain Aβ clearance by promoting Aβ catabolism.

Insulin Secretory Responses and Phospholipid Composition of Pancreatic Islets from Mice That Do Not Express Group VIA Phospholipase A2 and Effects of Metabolic Stress on Glucose Homeostasis

Studies involving pharmacologic or molecular biologic manipulation of Group VIA phospholipase A2 (iPLA2β) activity in pancreatic islets and insulinoma cells suggest that iPLA2β participates in insulin secretion. It has also been suggested that iPLA2β is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA2β-null mice by homologous recombination and have reported that they exhibit reduced male fertility and defective motility of spermatozoa. Here we report that pancreatic islets from iPLA2β-null mice have impaired insulin secretory responses to d-glucose and forskolin. Electrospray ionization mass spectrometric analyses indicate that the abundance of arachidonate-containing PC species of islets, brain, and other tissues from iPLA2β-null mice is virtually identical to that of wild-type mice, and no iPLA2β mRNA was observed in any tissue from iPLA2β-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA2β-null and wild-type mice are essentially identical under normal circumstances, but iPLA2β-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the β-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA2β-null mice than in wild-type mice, but PLA2β-null mice have greater responsiveness to exogenous insulin than do wild-type mice fed a high fat diet. These and previous findings thus indicate that iPLA2β-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages.

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.

Algorithms for automatic processing of data from mass spectrometric analyses of lipids

Lipidomics comprises large-scale studies of the structures, quantities, and functions of lipid molecular species. Recently developed mass spectrometric methods for lipid analyses, especially electrospray ionization (ESI) tandem mass spectrometry, permit identification and quantitation of an enormous variety of distinct lipid molecular species from small amounts of biological samples but generate a huge amount of experimental data within a brief interval. Processing such data sets so that comprehensible information is derived from them requires bioinformatics tools, and algorithms developed for proteomics and genomics have provided some strategies that can be directly adapted to lipidomics. The structural diversity and complexity of lipids, however, also requires the development and application of new algorithms and software tools that are specifically directed at processing data from lipid analyses. Several such tools are reviewed here, including LipidQA. This program employs searches of a fragment ion database constructed from acquired and theoretical spectra of a wide variety of lipid molecular species, and raw mass spectrometric data can be processed by the program to achieve identification and quantification of many distinct lipids in mixtures. Other approaches that are reviewed here include LIMSA (Lipid Mass Spectrum Analysis), SECD (Spectrum Extraction from Chromatographic Data), MPIS (Multiple Precursor Ion Scanning), FIDS (Fragment Ion Database Searching), LipidInspector, Lipid Profiler, FAAT (Fatty Acid Analysis Tool), and LIPID Arrays. Internet resources for lipid analyses are also summarized.

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.

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

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)

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.

Mice deficient in Group VIB phospholipase A2 (iPLA2γ) exhibit relative resistance to obesity and metabolic abnormalities induced by a Western diet

Phospholipases A(2) (PLA(2)) play important roles in metabolic processes, and the Group VI PLA(2) family is comprised of intracellular enzymes that do not require Ca(2+) for catalysis. Mice deficient in Group VIA PLA(2) (iPLA(2)beta) develop more severe glucose intolerance than wild-type (WT) mice in response to dietary stress. Group VIB PLA(2) (iPLA(2)gamma) is a related enzyme distributed in membranous organelles, including mitochondria, and iPLA(2)gamma knockout (KO) mice exhibit altered mitochondrial morphology and function. We have compared metabolic responses of iPLA(2)gamma-KO and WT mice fed a Western diet (WD) with a high fat content. We find that KO mice are resistant to WD-induced increases in body weight and adiposity and in blood levels of cholesterol, glucose, and insulin, even though WT and KO mice exhibit similar food consumption and dietary fat digestion and absorption. KO mice are also relatively resistant to WD-induced insulin resistance, glucose intolerance, and altered patterns of fat vs. carbohydrate fuel utilization. KO skeletal muscle exhibits impaired mitochondrial beta-oxidation of fatty acids, as reflected by accumulation of larger amounts of long-chain acylcarnitine (LCAC) species in KO muscle and liver compared with WT in response to WD feeding. This is associated with increased urinary excretion of LCAC and much reduced deposition of triacylglycerols in liver by WD-fed KO compared with WT mice. The iPLA(2)gamma-deficient genotype thus results in a phenotype characterized by impaired mitochondrial oxidation of fatty acids and relative resistance to the metabolic abnormalities induced by WD.

Group VIA PLA2 (iPLA2β) Is Activated Upstream of p38 Mitogen-activated Protein Kinase (MAPK) in Pancreatic Islet β-Cell Signaling

Background: Group VIA Phospholipase A2 (iPLA2β) is activated in β-cell signaling. Results: β-Cell p38 MAPK is activated by an iPLA2β-dependent mechanism and participates in insulin secretion and apoptosis. Conclusion: p38 MAPK is a downstream effector of iPLA2β activation in β-cells. Significance: Understanding how insulin secretion is impaired and β-cell apoptosis occurs could suggest therapeutic strategies for type 2 diabetes mellitus. Group VIA phospholipase A2 (iPLA2β) in pancreatic islet β-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 iPLA2β activity and amplified by iPLA2β overexpression. While exploring signaling events that occur downstream of iPLA2β activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA2β expression level, and that it is stimulated by the iPLA2β reaction product arachidonic acid. The insulin secretagogue d-glucose also stimulates β-cell p38 MAPK phosphorylation, and this is prevented by the iPLA2β inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA2β 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 β-cell MAPK kinase isoforms and p38 MAPK, and bromoenol lactone prevents both events. Others have reported that iPLA2β 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 β-cell p38 MAPK phosphorylation. Thapsigargin-induced β-cell apoptosis and ceramide generation are also prevented by the p38 MAPK inhibitor PD169316. These observations indicate that p38 MAPK is activated downstream of iPLA2β in β-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA2β activation, and that p38 MAPK is involved in the β-cell functional responses of insulin secretion and apoptosis in which iPLA2β participates.