Cell-Free Culture Supernatant of Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 Reduces the Pathogenicity of NetB-Positive Clostridium perfringens in a Chicken Intestinal Epithelial Cell Line.

The worldwide reduction in the use of antibiotics in animal feed is fueling the need for alternatives for the prevention and control of poultry intestinal diseases such as necrotic enteritis (NE), which is caused by Clostridium perfringens. This is the first report on the use of an intestinal epithelial chicken cell line (CHIC-8E11) to study the pathogenic traits of C. perfringens and to investigate the mode of action of cell-free supernatants (CFS) from probiotic Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 in reducing the pathogenicity of C. perfringens. The cell adhesion, permeability and cytotoxicity were assessed under challenge with four C. perfringens strains isolated from broiler NE episodes of differing geographical origin (CP1-UK; CP10-Sweden; 25037-CP01 and CP22-USA). All the C. perfringens strains could adhere to the CHIC-8E11 cells, with varying affinity (0.05-0.48% adhesion across the strains). The CFS from one out of two strains (CP22) increased the cell permeability (+4.5-fold vs. the control, p < 0.01), as measured by the fluorescein isothiocyanate-dextran (FD4) content, with NetB toxin implicated in this effect. The CFS from all the strains was cytotoxic against the CHIC-8E11 cells in a dose- and strain-dependent manner (cytotoxicity 23-62% across the strains when dosed at 50 µL/mL, as assessed by the MTT cell viability assay). Pre-treatment of the cells with CFS from B. animalis subsp. lactis AG02 but not L. acidophilus AG01 reduced the cell adhesion of three out of four C. perfringens strains (by 77-85% vs. the control, p < 0.001) and reduced the negative effect of two NetB-positive strains on the cell permeability. The CFS of both probiotics alleviated the cytotoxicity of all the C. perfringens strains, which was dependent on the dose. The results confirm the suitability of the CHIC-8E11 cell line for the study of host-pathogen cell interactions in the context of NE caused by C. perfringens and reveal a beneficial mode of action of B. animalis subsp. lactis AG02 in reducing C. perfringens cell adhesion and, together with L. acidophilus AG01, in reducing C. perfringens cytotoxicity.

Unravelling Effects of Rosemary (Rosmarinus officinalis L.) Extract on Hepatic Fat Accumulation and Plasma Lipid Profile in Rats Fed a High-Fat Western-Style Diet.

The objective of the study was to investigate the preventive effect on obesity-related conditions of rosemary (Rosmarinus officinalis L.) extract (RE) in young, healthy rats fed a high-fat Western-style diet to complement the existing knowledge gap concerning the anti-obesity effects of RE in vivo. Sprague Dawley rats (71.3 ± 0.46 g) were fed a high-fat Western-style diet (WD) or WD containing either 1 g/kg feed or 4 g/kg feed RE for six weeks. A group fed standard chow served as a negative control. The treatments did not affect body weight; however, the liver fat percentage was reduced in rats fed RE, and NMR analyses of liver tissue indicated that total cholesterol and triglycerides in the liver were reduced. In plasma, HDL cholesterol was increased while triglycerides were decreased. Rats fed high RE had significantly increased fasting plasma concentrations of Glucagon-like peptide-1 (GLP-1). Proteomics analyses of liver tissue showed that RE increased enzymes involved in fatty acid oxidation, possibly associated with the higher fasting GLP-1 levels, which may explain the improvement of the overall lipid profile and hepatic fat accumulation. Furthermore, high levels of succinic acid in the cecal content of RE-treated animals suggested a modulation of the microbiota composition. In conclusion, our results suggest that RE may alleviate the effects of consuming a high-fat diet through increased GLP-1 secretion and changes in microbiota composition.

Identification and Characterization of a Novel Species of Genus Akkermansia with Metabolic Health Effects in a Diet-Induced Obesity Mouse Model.

Akkermansia muciniphila is a well-known bacterium with the ability to degrade mucin. This metabolic capability is believed to play an important role in the colonization of this bacterium in the gut. In this study, we report the identification and characterization of a novel Akkermansia sp. DSM 33459 isolated from human feces of a healthy donor. Phylogenetic analysis based on the genome-wide average nucleotide identity indicated that the Akkermansia sp. DSM 33459 has only 87.5% similarity with the type strain A. muciniphila ATCC BAA-835. Akkermansia sp. DSM 33459 showed significant differences in its fatty acid profile and carbon utilization as compared to the type strain. The Akkermansia sp. DSM 33459 strain was tested in a preclinical obesity model to determine its effect on metabolic markers. Akkermansia sp. DSM 33459 showed significant improvement in body weight, total fat weight, and resistin and insulin levels. Interestingly, these effects were more pronounced with the live form as compared to a pasteurized form of the strain. The strain showed production of agmatine, suggesting a potential novel mechanism for supporting metabolic and cognitive health. Based on its phenotypic features and phylogenetic position, it is proposed that this isolate represents a novel species in the genus Akkermansia and a promising therapeutic candidate for the management of metabolic diseases.

Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo.

There is increasing evidence that multiple proteins involved in key regulatory processes in mitochondria are phosphorylated in mammalian tissues. Insulin regulates glucose metabolism by phosphorylation-dependent signaling and has been shown to stimulate ATP synthesis in human skeletal muscle. Here, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO(2) phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included the majority of novel sites. Phosphorylation sites detected more often or exclusively in insulin-stimulated samples include multiple sites in mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid metabolism, as well as several components of the newly defined mitochondrial inner membrane organizing system (MINOS). In conclusion, the present study demonstrates that insulin increases the phosphorylation of several mitochondrial proteins in human skeletal muscle in vivo and provides a first step in the understanding of how insulin potentially regulates mitochondrial processes by phosphorylation-dependent mechanisms.

Tissue specific phosphorylation of mitochondrial proteins isolated from rat liver, heart muscle, and skeletal muscle.

Phosphorylation of mitochondrial proteins in a variety of biological processes is increasingly being recognized and may contribute to the differences in function and energy demands observed in mitochondria from different tissues such as liver, heart, and skeletal muscle. Here, we used a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS on isolated mitochondria to investigate the tissue-specific mitochondrial phosphoproteomes of rat liver, heart, and skeletal muscle. In total, we identified 899 phosphorylation sites in 354 different mitochondrial proteins including 479 potential novel sites. Most phosphorylation sites were detected in liver mitochondria (594), followed by heart (448) and skeletal muscle (336), and more phosphorylation sites were exclusively identified in liver mitochondria than in heart and skeletal muscle. Bioinformatics analysis pointed out enrichment for phosphoproteins involved in amino acid and fatty acid metabolism in liver mitochondria, whereas heart and skeletal muscle were enriched for phosphoproteins involved in energy metabolism, in particular, tricarboxylic acid cycle and oxidative phosphorylation. Multiple tissue-specific phosphorylation sites were identified in tissue-specific enzymes such as those encoded by HMGCS2, BDH1, PCK2, CPS1, and OTC in liver mitochondria, and CKMT2 and CPT1B in heart and skeletal muscle. Kinase prediction showed an important role for PKA and PKC in all tissues but also for proline-directed kinases in liver mitochondria. In conclusion, we provide a comprehensive map of mitochondrial phosphorylation sites, which covers approximately one-third of the mitochondrial proteome and can be targeted for the investigation of tissue-specific regulation of mitochondrial biological processes.

Vesicular signalling and immune modulation as hedonic fingerprints: proteomic profiling in the chronic mild stress depression model.

Extensive preclinical research has focused at unravelling the underlying molecular mechanisms leading to depression and recovery. In this study, we investigated the quantitative changes in protein abundance in the ventral hippocampal granular cell layer. We compared different phenotypes from the chronic mild stress (CMS) model of depression using chronic administration with two selective serotonin reuptake inhibitors (SSRIs), escitalopram and sertraline. We isolated granular cells using Laser-Capture Microdissection (LCM) and we identified their regulated proteins using two-dimensional (2D) differential gel electrophoresis (DIGE) and tandem mass spectrometry (MS/MS). The majority of the proteins we identified were enzymes involved in different metabolic activities. Additional proteins were functionally classified as vesicular proteins and immune system proteins. Rab GDP dissociation inhibitor alpha (GDIA) and syntaxin-binding protein 1 (STXB1) were potential markers for stress reactivity. Dynamin 1 (DYN1), glutathione S-transferase omega-1 (GSTO1) and peroxiredoxin (PRDX6) were associated with treatment response. In addition, an imbalance between different post-translationally modified versions of DYN1 and GSTO1 potentially accounted for SSRI treatment refraction. In the present study, we searched for new markers of stress reactivity and treatment response as well as any underlying molecular mechanisms correlating to the development of anhedonia and antidepressant therapy refraction. Our results pointed towards an essential role of post-translational modifications in both vesicular and immune protein systems.

Identification of a potential biomarker panel for the intake of the common dietary trans fat elaidic acid (trans∆9-C18:1).

Trans fatty acid intake has been correlated to an unfavorable plasma lipoprotein profile and an increased cardiovascular disease risk. The present study aimed to identify a plasma protein biomarker panel related to human intake of elaidic acid. The human liver cell line HepG2-SF was used as a model system, and the cells were maintained for seven days in serum-free medium containing 100 µM elaidic acid (trans∆9-C18:1), oleic acid (cis∆9-C18:1) or stearic acid (C18:0). The secretomes were analyzed by stable isotope labeling of amino acids in cell culture (SILAC), difference in gel electrophoresis (DIGE) and gene expression microarray analysis. Twelve proteins were found to be differentially regulated based on SILAC data (>1.3 fold change, P-value<0.05), 13 proteins were found to be differentially regulated based on DIGE analysis (>1.3 fold change, P-value<0.05), and 17 mRNA transcripts encoding extracellular proteins were determined to be affected (>1.3 fold change, P-value<0.01) following the addition of elaidic acid compared to oleic acid or stearic acid. The results revealed that 37 proteins were regulated specifically in response to elaidic acid exposure, and nine of these proteins were confirmed to be regulated in this manner by using selected reaction monitoring mass spectrometry.

Candidate hippocampal biomarkers of susceptibility and resilience to stress in a rat model of depression.

Susceptibility to stress plays a crucial role in the development of psychiatric disorders such as unipolar depression and post-traumatic stress disorder. In the present study the chronic mild stress rat model of depression was used to reveal stress-susceptible and stress-resilient rats. Large-scale proteomics was used to map hippocampal protein alterations in different stress states. Membrane proteins were successfully captured by two-phase separation and peptide based proteomics. Using iTRAQ labeling coupled with mass spectrometry, more than 2000 proteins were quantified and 73 proteins were found to be differentially expressed. Stress susceptibility was associated with increased expression of a sodium-channel protein (SCN9A) currently investigated as a potential antidepressant target. Differential protein profiling also indicated stress susceptibility to be associated with deficits in synaptic vesicle release involving SNCA, SYN-1, and AP-3. Our results indicate that increased oxidative phosphorylation (COX5A, NDUFB7, NDUFS8, COX5B, and UQCRB) within the hippocampal CA regions is part of a stress-protection mechanism.

Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase.

In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin-pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction may influence the TSG-6/heavy Chain 2-mediated transfer of heavy chains observed during inflammation.

Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods.

Skeletal muscle is a key tissue site of insulin resistance in type 2 diabetes. Human myotubes are primary skeletal muscle cells displaying both morphological and biochemical characteristics of mature skeletal muscle and the diabetic phenotype is conserved in myotubes derived from subjects with type 2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery strategy using isobaric peptide tags for relative and absolute quantification (iTRAQ) and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring to identify, quantify, and validate changes in protein abundance among human myotubes obtained from nondiabetic lean, nondiabetic obese, and type 2 diabetic subjects, respectively. Using an optimized protein precipitation protocol, a total of 2832 unique proteins were identified and quantified using the iTRAQ strategy. Despite a clear diabetic phenotype in diabetic myotubes, the majority of the proteins identified in this study did not exhibit significant abundance changes across the patient groups. Proteins from all major pathways known to be important in type 2 diabetic subjects were well-characterized in this study. This included pathways like the trichloroacetic acid (TCA) cycle, lipid oxidation, oxidative phosphorylation, the glycolytic pathway, and glycogen metabolism from which all but two enzymes were found in the present study. None of these enzymes were found to be regulated at the level of protein expression or degradation supporting the hypothesis that these pathways are regulated at the level of post-translational modification. Twelve proteins were, however, differentially expressed among the three different groups. Thirty-six proteins were chosen for further analysis and validation using selected reaction monitoring based on the regulation identified in the iTRAQ discovery study. The abundance of adenosine deaminase was considerably down-regulated in diabetic myotubes and as the protein binds propyl dipeptidase (DPP-IV), we speculate whether the reduced binding of adenosine deaminase to DPP-IV may contribute to the diabetic phenotype in vivo by leading to a higher level of free DPP-IV to bind and inactivate the anti-diabetic hormones, glucagon-like peptide-1 and glucose-dependent insulintropic polypeptide.

Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes.

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.