Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis.

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.

Lipoprotein-based drug delivery.

Lipoproteins (LPs) are circulating heterogeneous nanoparticles produced by the liver and intestines. LPs play a major role in the transport of dietary and endogenous lipids to target cells through cell membrane receptors or cell surface-bound lipoprotein lipase. The stability, biocompatibility, and selective transport of LPs make them promising delivery vehicles for various therapeutic and imaging agents. This review discusses isolation, manufacturing, and drug loading techniques used for LP-based drug delivery, as well as recent applications for diagnosis and treatment of cancer, atherosclerosis, and other life-threatening diseases.

Lipoprotein Processing and Sorting in Helicobacter pylori.

Our current understanding of lipoprotein synthesis and localization in Gram-negative bacteria is based primarily on studies of Escherichia coli Newly synthesized E. coli prolipoproteins undergo posttranslational modifications catalyzed by three essential enzymes (Lgt, LspA, and Lnt). The mature lipoproteins are then sorted to the inner or outer membrane via the Lol system (LolABCDE). Recent studies suggested that this paradigm may not be universally applicable among different classes of proteobacteria. In this study, we conducted a systematic analysis of lipoprotein processing and sorting in Helicobacter pylori, a member of the Epsilonproteobacteria that colonizes the human stomach. We show that H. pylorilgt, lspA, and lnt homologs can complement conditionally lethal E. coli mutant strains in which expression of these genes is conditionally regulated. Mutagenesis studies and analyses of conditionally lethal H. pylori mutant strains indicate that lgt and lspA are essential for H. pylori growth but lnt is dispensable. H. pylorilolA and the single lolC (or lolE) homolog are also essential genes. We then explored the role of lipoproteins in H. pylori Cag type IV secretion system (Cag T4SS) activity. Comparative analysis of the putative VirB7 homolog CagT in wild-type and lnt mutant H. pylori strains indicates that CagT undergoes amino-terminal modifications consistent with lipidation, and we show that CagT lipidation is essential for CagT stability and Cag T4SS function. This work demonstrates that lipoprotein synthesis and localization in H. pylori diverge from the canonical pathways and that lipidation of a T4SS component is necessary for H. pylori Cag T4SS activity.IMPORTANCE Bacterial lipoproteins have diverse roles in multiple aspects of bacterial physiology, antimicrobial resistance, and pathogenesis. Dedicated pathways direct the posttranslational lipidation and localization of lipoproteins, but there is considerable variation in these pathways among the proteobacteria. In this study, we characterized the proteins responsible for lipoprotein synthesis and localization in Helicobacter pylori, a member of the Epsilonproteobacteria that contributes to stomach cancer pathogenesis. We also provide evidence suggesting that lipidation of CagT, a component of the H. pylori Cag T4SS, is required for delivery of the H. pylori CagA oncoprotein into human gastric cells. Overall, these results constitute the first systematic analysis of H. pylori lipoprotein production and localization pathways and reveal how these processes in H. pylori differ from corresponding pathways in model proteobacteria.

Bacterial lyso-form lipoproteins are synthesized via an intramolecular acyl chain migration.

All bacterial lipoproteins share a variably acylated N-terminal cysteine residue. Gram-negative bacterial lipoproteins are triacylated with a thioether-linked diacylglycerol moiety and an N-acyl chain. The latter is transferred from a membrane phospholipid donor to the α-amino terminus by the enzyme lipoprotein N-acyltransferase (Lnt), using an active-site cysteine thioester covalent intermediate. Many Gram-positive Firmicutes also have N-acylated lipoproteins, but the enzymes catalyzing N-acylation remain uncharacterized. The integral membrane protein Lit (lipoprotein intramolecular transacylase) from the opportunistic nosocomial pathogen Enterococcus faecalis synthesizes a specific lysoform lipoprotein (N-acyl S-monoacylglycerol) chemotype by an unknown mechanism that helps this bacterium evade immune recognition by the Toll-like receptor 2 family complex. Here, we used a deuterium-labeled lipoprotein substrate with reconstituted Lit to investigate intramolecular acyl chain transfer. We observed that Lit transfers the sn-2 ester-linked lipid from the diacylglycerol moiety to the α-amino terminus without forming a covalent thioester intermediate. Utilizing Mut-Seq to analyze an alanine scan library of Lit alleles, we identified two stretches of functionally important amino acid residues containing two conserved histidines. Topology maps based on reporter fusion assays and cysteine accessibility placed both histidines in the extracellular half of the cytoplasmic membrane. We propose a general acid base-promoted catalytic mechanism, invoking direct nucleophilic attack by the substrate α-amino group on the sn-2 ester to form a cyclic tetrahedral intermediate that then collapses to produce lyso-lipoprotein. Lit is a unique example of an intramolecular transacylase differentiated from that catalyzed by Lnt, and provides insight into the heterogeneity of bacterial lipoprotein biosynthetic systems.

Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants.

Mycoplasma agalactiae exhibits antigenic variation by switching the expression of multiple surface lipoproteins called Vpmas. Although implicated to have a significant influence on the pathogenicity, their exact role in pathogen-host interactions has not been investigated so far. Initial attachment to host cells is regarded as one of the most important steps for colonization but this pathogen lacks the typical mycoplasma attachment organelle. The aim of this study was to determine the role of Vpmas in adhesion of M. agalactiae to host cells. 'Phase-Locked' Mutants (PLMs) steadily expressing single well-characterized Vpma lipoproteins served as ideal tools to evaluate the role of each of the six Vpmas in cytadhesion, which was otherwise not possible due to the high-frequency switching of Vpmas in the wildtype strain PG2. Using in vitro adhesion assays with HeLa and sheep mammary epithelial (MECs) and stromal (MSCs) cells, we could demonstrate differences in the adhesion capabilities of each of the six PLMs compared to the wildtype strain. The PLMV mutant expressing VpmaV exhibited the highest adhesion rate, whereas PLMU, which expresses VpmaU showed the lowest adhesion values explaining the reduced in vivo fitness of PLMU in sheep during experimental intramammary and conjunctival infections. Furthermore, adhesion inhibition assays using Vpma-specific polyclonal antisera were performed to confirm the role of Vpmas in M. agalactiae cytadhesion. This led to a significant decrease (p<0.05) in the adhesion percentage of each PLM. Immunofluorescence staining of TX-114 phase proteins extracted from each PLM showed binding of the respective Vpma to HeLa cells and MECs proving the direct role of Vpmas in cytadhesion. Furthermore, as adhesion is a prerequisite for cell invasion, the ability of the six PLMs to invade HeLa cells was also evaluated using the gentamicin protection assay. The results showed a strong correlation between the adhesion rates and invasion frequencies of the individual PLMs. This is the first report that describes a novel function of Vpma proteins in cell adhesion and invasion. Besides the variability of these proteins causing surface antigenic variation, the newly identified phenotypes are likely to play critical roles in the pathogenicity potential of this ruminant pathogen.

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein's invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.

Relationships between the expression of hepatocyte nuclear factors and factors essential for lipoprotein production in a human mesenchymal stem cell line, UE7T-13.

To clarify the mechanisms regulating lipoprotein production by hepatocyte nuclear factors (HNFs), we generated four kinds of transfectants in human bone marrow mesenchymal stem cells: UE7T-13, stably expressing FOXA2 (also known as HNF3ß), HNF4α, HNF1α or co-expressing HNF4α, and HNF1α (HNF4α/HNF1α). In HNF4α/HNF1α transfectants, cellular contents of triglycerides (TG) and cholesterol were markedly higher than in UE7T-13 cells and comparable to those in human hepatoma HepG2 cells. However, TG and cholesterol, which are secreted from cells as components of lipoproteins, were hardly detected in the medium for any of the transfectants. ApoB100 and MTP, which are essential for the formation and secretion of lipoproteins, were undetectable and detected at low levels, respectively, in HNF4α/HNF1α transfectants. We suggest that enforced co-expression of HNF4α and HNF1α is effective for cellular lipid accumulation, while additional factors are probably required for lipoprotein formation and secretion.

Mechanism of lipid-lowering action of the dipeptidyl peptidase-4 inhibitor, anagliptin, in low-density lipoprotein receptor-deficient mice.

AIMS/INTRODUCTION: Dipeptidyl peptidase-4 inhibitors are used for treatment of patients with type 2 diabetes. In addition to glycemic control, these agents showed beneficial effects on lipid metabolism in clinical trials. However, the mechanism underlying the lipid-lowering effect of dipeptidyl peptidase-4 inhibitors remains unclear. Here, we investigated the lipid-lowering efficacy of anagliptin in a hyperlipidemic animal model, and examined the mechanism of action. MATERIALS AND METHODS: Male low-density lipoprotein receptor-deficient mice were administered 0.3% anagliptin in their diet. Plasma lipid levels were assayed and lipoprotein profile was analyzed using high-performance liquid chromatography. Hepatic gene expression was examined by deoxyribonucleic acid microarray and quantitative polymerase chain reaction analyses. Sterol regulatory element-binding protein transactivation assay was carried out in vitro. RESULTS: Anagliptin treatment significantly decreased the plasma total cholesterol (14% reduction, P < 0.01) and triglyceride levels (27% reduction, P < 0.01). Both low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol were also decreased significantly by anagliptin treatment. Sterol regulatory element-binding protein-2 messenger ribonucleic acid expression level was significantly decreased at night in anagliptin-treated mice (15% reduction, P < 0.05). Anagliptin significantly suppressed sterol regulatory element-binding protein activity in HepG2 cells (21% decrease, P < 0.001). CONCLUSIONS: The results presented here showed that the dipeptidyl peptidase-4 inhibitor, anagliptin, exhibited a lipid-lowering effect in a hyperlipidemic animal model, and suggested that the downregulation of hepatic lipid synthesis was involved in the effect. Anagliptin might have beneficial effects on lipid metabolism in addition to a glucose-lowering effect.

Analysis of the genomic sequences and metabolites of Serratia surfactantfaciens sp. nov. YD25T that simultaneously produces prodigiosin and serrawettin W2.

BACKGROUND: Gram-negative bacteria of the genus Serratia are potential producers of many useful secondary metabolites, such as prodigiosin and serrawettins, which have potential applications in environmental bioremediation or in the pharmaceutical industry. Several Serratia strains produce prodigiosin and serrawettin W1 as the main bioactive compounds, and the biosynthetic pathways are co-regulated by quorum sensing (QS). In contrast, the Serratia strain, which can simultaneously produce prodigiosin and serrawettin W2, has not been reported. This study focused on analyzing the genomic sequence of Serratia sp. strain YD25T isolated from rhizosphere soil under continuously planted burley tobacco collected from Yongding, Fujian province, China, which is unique in producing both prodigiosin and serrawettin W2. RESULTS: A hybrid polyketide synthases (PKS)-non-ribosomal peptide synthetases (NRPS) gene cluster putatively involved in biosynthesis of antimicrobial serrawettin W2 was identified in the genome of YD25T, and its biosynthesis pathway was proposed. We found potent antimicrobial activity of serrawettin W2 purified from YD25T against various pathogenic bacteria and fungi as well as antitumor activity against Hela cells. Subsequently, comparative genomic analyses were performed among a total of 133 Serratia species. The prodigiosin biosynthesis gene cluster in YD25T belongs to the type I pig cluster, which is the main form of pig-encoding genes existing in most of the pigmented Serratia species. In addition, a complete autoinducer-2 (AI-2) system (including luxS, lsrBACDEF, lsrGK, and lsrR) as a conserved bacterial operator is found in the genome of Serratia sp. strain YD25T. Phylogenetic analysis based on concatenated Lsr and LuxS proteins revealed that YD25T formed an independent branch and was clearly distant from the strains that solely produce either prodigiosin or serrawettin W2. The Fe (III) ion reduction assay confirmed that strain YD25T could produce an AI-2 signal molecule. Phylogenetic analysis using the genomic sequence of YD25T combined with phylogenetic and phenotypic analyses support this strain as a member of a novel and previously uncharacterized Serratia species. CONCLUSION: Genomic sequence and metabolite analysis of Serratia surfactantfaciens YD25T indicate that this strain can be further explored for the production of useful metabolites. Unveiling the genomic sequence of S. surfactantfaciens YD25T benefits the usage of this unique strain as a model system for studying the biosynthesis regulation of both prodigiosin and serrawettin W2 by the QS system.

Global and hepatocyte-specific ablation of Bmal1 induces hyperlipidaemia and enhances atherosclerosis.

Circadian rhythms controlled by clock genes affect plasma lipids. Here we show that global ablation of Bmal1 in Apoe-/- and Ldlr-/- mice and its liver-specific ablation in Apoe-/- (L-Bmal1-/-Apoe-/-) mice increases, whereas overexpression of BMAL1 in L-Bmal1-/-Apoe-/- and Apoe-/-mice decreases hyperlipidaemia and atherosclerosis. Bmal1 deficiency augments hepatic lipoprotein secretion and diminishes cholesterol excretion to the bile. Further, Bmal1 deficiency reduces expression of Shp and Gata4. Reductions in Shp increase Mtp expression and lipoprotein production, whereas reductions in Gata4 diminish Abcg5/Abcg8 expression and biliary cholesterol excretion. Forced SHP expression normalizes lipoprotein secretion with no effect on biliary cholesterol excretion, while forced GATA4 expression increases cholesterol excretion to the bile and reduces plasma lipids in L-Bmal1-/-Apoe-/- and Apoe-/- mice. Thus, our data indicate that Bmal1 modulates lipoprotein production and biliary cholesterol excretion by regulating the expression of Mtp and Abcg5/Abcg8 via Shp and Gata4.

The roles of bile acids and sphingosine-1-phosphate signaling in the hepatobiliary diseases.

Based on research carried out over the last decade, it has become increasingly evident that bile acids act not only as detergents, but also as important signaling molecules that exert various biological effects via activation of specific nuclear receptors and cell signaling pathways. Bile acids also regulate the expression of numerous genes encoding enzymes and proteins involved in the synthesis and metabolism of bile acids, glucose, fatty acids, and lipoproteins, as well as energy metabolism. Receptors activated by bile acids include, farnesoid X receptor α, pregnane X receptor, vitamin D receptor, and G protein-coupled receptors, TGR5, muscarinic receptor 2, and sphingosine-1-phosphate receptor (S1PR)2. The ligand of S1PR2, sphingosine-1-phosphate (S1P), is a bioactive lipid mediator that regulates various physiological and pathophysiological cellular processes. We have recently reported that conjugated bile acids, via S1PR2, activate and upregulate nuclear sphingosine kinase 2, increase nuclear S1P, and induce genes encoding enzymes and transporters involved in lipid and sterol metabolism in the liver. Here, we discuss the role of bile acids and S1P signaling in the regulation of hepatic lipid metabolism and in hepatobiliary diseases.

Organochloride pesticides induced hepatic ABCG5/G8 expression and lipogenesis in Chinese patients with gallstone disease.

BACKGROUND: Organochlorine pesticides (OCPs) are one kind of persistent organic pollutants. Although they are reported to be associated with metabolic disorders, the underlying mechanism is unclear. We explored the association of OCPs with gallstone disease and its influence on hepatic lipid metabolism. MATERIALS AND METHODS: OCPs levels in omentum adipose tissues from patients with and without gallstone disease between 2008 and 2011 were measured by GC-MS. Differences of gene expression involved in hepatic lipid metabolism and hepatic lipids content were compared in liver biopsies between groups with high and low level of OCPs. Using HepG2 cell lines, the influence on hepatic lipid metabolism by individual OCP was evaluated in vitro. RESULTS: In all patients who were from non-occupational population, there were high levels of ß-hexachlorocyclohexane (ß-HCH) and p',p'-dichloroethylene (p',p'-DDE) accumulated in adipose tissues. Both ß-HCH and p', p'-DDE levels were significantly higher in adipose tissues from patients with gallstone disease (294.3± 313.5 and 2222± 2279 ng/g of lipid) than gallstone-free controls (282.7± 449.0 and 2025±2664 ng/g of lipid, P< 0.01) and they were strongly related with gallstone disease (P for trend = 0.0004 and 0.0138). Furthermore, higher OCPs in adipose tissue led to increase in the expression of hepatic cholesterol transporters ABCG5 and G8 (+34% and +27%, P< 0.01) and higher cholesterol saturation index in gallbladder bile, and induced hepatic fatty acids synthesis, which was further confirmed in HepG2 cells. CONCLUSIONS: OCPs might enhance hepatic secretion of cholesterol into bile via ABCG5/G8 which promoting gallstone disease as well as lipogenesis.

Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor.

Mycobacterium tuberculosis executes numerous defense strategies for the successful establishment of infection under a diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface-localized lipoprotein, LprI, which is exclusively present in M. tuberculosis complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin (HbN)) and both are synchronously up-regulated in M. tuberculosis during macrophage infection. Recombinant LprI, expressed in Escherichia coli, exhibited strong binding (Kd ≤ 2 nm) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein-labeled Micrococcus lysodeikticus from lysozyme-mediated hydrolysis. Expression of the lprI gene in Mycobacterium smegmatis (8-10-fold) protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis and survival during intracellular infection of peritoneal and monocyte-derived macrophages, known to secrete lysozyme, and in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of the glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2-3-fold in secondary cell lines in the presence of lysozyme in comparison with isogenic cells expressing these genes individually. Thus, specific advantage against macrophage-generated lysozyme, conferred by the combination of LprI-HbN during invasion of M. tuberculosis, may have vital implications on the pathogenesis of tuberculosis.

Immunostimulation by phospholipopeptide biosurfactant from Staphylococcus hominis in Oreochromis mossambicus.

The immunostimulatory effect of phospholipopeptide biosurfactant from Staphylococcus hominis (GenBank Accession No: KJ564272) was assessed with Oreochromis mossambicus. The non-specific (serum lysozyme activity, serum antiprotease activity, serum peroxidase activity and serum bactericidal activity), specific (bacterial agglutination assay) immune responses and disease resistance activity against Aeromonas hydrophila were examined. Fish were intraperitonially injected with water soluble secondary metabolite (biosurfactant) of S. hominis at a dose of 2 mg, 20 mg and 200 mg kg(-1) body weight. Commercial surfactant surfactin (sigma) at 20 mg kg(-1) was used as standard and saline as negative control. All the doses of water soluble biosurfactant tested, significantly enhanced the specific, nonspecific immunity and disease resistance from the day of post administration of phospholipopeptide biosurfactant till the tail of the experimental period. These results clearly indicated that the secondary metabolite isolated from S. hominis stimulates the immunity of finfish thereby could enhance aquaculture production.

The expression of genes involved in jejunal lipogenesis and lipoprotein synthesis is altered in morbidly obese subjects with insulin resistance.

The dyslipidemia associated with type 2 diabetes mellitus (T2DM) is an important risk factor for atherosclerotic cardiovascular disease. However, until now little attention has been paid to the role that the intestine might have. The aim of this research was to determine the relation between insulin resistance and intestinal de novo lipogenesis/lipoprotein synthesis in morbidly obese subjects and to study the effect of insulin on these processes. Jejunal mRNA expression of the different genes involved in the intestinal de novo lipogenesis/lipoprotein synthesis was analyzed in three groups of morbidly obese subjects: Group 1 with low insulin resistance (MO-low-IR), group 2 with high insulin resistance (MO-high-IR), and group 3 with T2DM and treatment with metformin (MO-metf-T2DM). In addition, intestinal epithelial cells (IECs) from MO-low-IR were incubated with different doses of insulin/glucose. In Group 2 (MO-high-IR), the jejunal mRNA expression levels of apo A-IV, ATP-citrate lyase (ACLY), pyruvate dehydrogenase (lipoamide) beta (PDHB), and sterol regulatory element-binding protein-1c (SREBP-1c) were significantly higher and acetyl-CoA carboxylase alpha (ACC1) and fatty-acid synthase lower than in Group 1 (MO-low-IR). In Group 3 (MO-metf-T2DM), only the ACLY and PDHB mRNA expressions were significantly higher than in Group 1 (MO-low-IR). The mRNA expression of most of the genes studied was significantly linked to insulin and glucose levels. The incubation of IEC with different doses of insulin and glucose produced a higher expression of diacylglycerol acyltransferase 2, microsomal triglyceride transfer protein, apo A-IV, SREBP-1c, and ACC1 when both, glucose and insulin, were at a high concentration. However, with only high insulin levels, there were higher apo A-IV, PDHB and SREBP-1c expressions, and a lower ACLY expression. In conclusion, the jejunum of MO-high-IR has a decreased mRNA expression of genes involved in de novo fatty-acid synthesis and an increase of genes involved in acetyl-CoA and lipoprotein synthesis. This effect is attenuated by metformin. In addition, the expression of most of the genes studied was found to be regulated by insulin.

Outer membrane lipoprotein biogenesis: Lol is not the end.

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.

LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2.

High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR) through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages (BMDMs) cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1), but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2) was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2-/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients.

LRP5 and plasma cholesterol levels modulate the canonical Wnt pathway in peripheral blood leukocytes.

Inflammation is triggered after invasion or injury to restore homeostasis. Although the activation of Wnt/ß-catenin signaling is one of the first molecular responses to cellular damage, its role in inflammation is still unclear. It was our hypothesis that the low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and the canonical Wnt signaling pathway are modulators of inflammatory mechanisms. Wild-type (WT) and LRP5(-/-) mice were fed a hypercholesterolemic (HC) diet to trigger dislipidemia and chronic inflammation. Diets were supplemented with plant sterol esters (PSEs) to induce LDL cholesterol lowering and the reduction of inflammation. HC WT mice showed increased serum cholesterol levels that correlated with increased Lrp5 and Wnt/ß-catenin gene expression while in the HC LRP5(-/-) mice Wnt/ß-catenin pathway was shut down. Functionally, HC induced pro-inflammatory gene expression in LRP5(-/-) mice, suggesting an inhibitory role of the Wnt pathway in inflammation. Dietary PSE administration downregulated serum cholesterol levels in WT and LRP5(-/-) mice. Furthermore, in WT mice PSE increased anti-inflammatory genes expression and inhibited Wnt/ß-catenin activation. Hepatic gene expression of Vldlr, Lrp2 and Lrp6 was increased after HC feeding in WT mice but not in LRP5(-/-) mice, suggesting a role for these receptors in the clearance of plasmatic lipoproteins. Finally, an antiatherogenic role for LRP5 was demonstrated as HC LRP5(-/-) mice developed larger aortic atherosclerotic lesions than WT mice. Our results show an anti-inflammatory, pro-survival role for LRP5 and the Wnt signaling pathway in peripheral blood leukocytes.

Glucose metabolism in pigs expressing human genes under an insulin promoter.

BACKGROUND: Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown. METHODS: On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon. RESULTS: This preliminary study did not show definite evidence of ß-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed. CONCLUSIONS: Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models.