Secretome profiling of primary human skeletal muscle cells

The skeletal muscle is a metabolically active tissue that secretes various proteins. These so-called myokines have been proposed to affect muscle physiology and to exert systemic effects on other tissues and organs. Yet, changes in the secretory profile may participate in the pathophysiology of metabolic diseases. The present study aimed at characterizing the secretome of differentiated primary human skeletal muscle cells (hSkMC) derived from healthy, adult donors combining three different mass spectrometry based non-targeted approaches as well as one antibody based method. This led to the identification of 548 non-redundant proteins in conditioned media from hSkmc. For 501 proteins, significant mRNA expression could be demonstrated. Applying stringent consecutive filtering using SignalP, SecretomeP and ER_retention signal databases, 305 proteins were assigned as potential myokines of which 12 proteins containing a secretory signal peptide were not previously described. This comprehensive profiling study of the human skeletal muscle secretome expands our knowledge of the composition of the human myokinome and may contribute to our understanding of the role of myokines in multiple biological processes. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Large-scale production of selected type A trichothecenes: the use of HT-2 toxin and T-2 triol as precursors for the synthesis of <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-T-2 and <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-HT-2 toxin

The type A trichothecenes T-2 and HT-2 toxins are toxic secondary metabolites produced by fungi of the Fusarium genus. Their occurrence in cereals, especially in oats, implies health risks for the consumer. Therefore, it is an important task to develop selective and sensitive methods for the analysis of T-2 and HT-2 toxins, and to undertake further studies on their stability and toxicity. Although most toxins are commercially available, their high prices are the limiting factor on the realization of these experiments. Thus, we developed a method for large-scale production of T-2 and HT-2 toxin as well as T-2 triol and T-2 tetraol. T-2 toxin was obtained in gram quantities by biosynthetic production with cultures of F. sporotrichioides. As HT-2 toxin was only formed as a by-product, and T-2 triol and T-2 tetraol were not generated, these compounds were produced by alkaline hydrolysis of T-2 toxin. Separation and isolation of crude toxins was achieved by fast centrifugal partition chromatography (FCPC), which is an efficient tool for the large-scale purification of natural products. Using this fast and yield effective technique, several hundred milligrams of HT-2 toxin, T-2 triol, and T-2 tetraol were obtained. Subsequent, HT-2 toxin and T-2 triol were used for the large-scale synthesis of isotope-labeled T-2 and HT-2 toxin, respectively. Using these standards, an isotope dilution-(ID)-HPLC-MS/MS method for the quantification of T-2 and HT-2 toxin in different matrices was developed.     

Sphingomyelinase dependent apoptosis following treatment of pancreatic beta-cells with amyloid peptides Aß1-42 or IAPP

Amyloid peptides interfere with survival of pancreatic beta-cells. In some cells apoptosis is paralleled by ceramide-dependent alterations of ion channel activity. The purpose of the present study was to elucidate the dependence of amyloid peptides Aß_1-42 and islet amyloid polypeptide (IAPP)-induced cell death on ceramide formation and ion channel activity in murine pancreatic islet cells. As disclosed by TUNEL (terminal dUTP nick-end labelling) and cleaved caspase 3 staining, apoptotic cell death was induced by Aß_1-42, IAPP and exogenously added C2-ceramide in islet cells from wild type mice. In islet cells from acid sphingomyelinase-deficient mice (ASMKO) Aß_1-42 and IAPP but not exogenously added N-acetyl-d-sphingosine (C2-ceramide, 20 μM) failed to stimulate apoptosis. Immunofluorescent staining revealed a stimulatory effect of Aß_1-42 on ceramide formation. According to patch clamp experiments, administration of Aß_1-42 and IAPP significantly decreased outwardly rectifying whole cell currents in wild type but not in ASMKO islet cells. C2-ceramide but not inactive di-ceramide (20 μM) mimicked the inhibitory effect on Kv channel current. In conclusion, amyloid peptides induce apoptosis of pancreatic islet cells at least in part through activation of acid sphingomyelinase resulting in production of ceramide and subsequent inhibition of ion channel activity.     

Insulin sensitivity is reflected by characteristic metabolic fingerprints--a Fourier transform mass spectrometric non-targeted metabolomics approach

Background

A decline in body insulin sensitivity in apparently healthy individuals indicates a high risk to develop type 2 diabetes. Investigating the metabolic fingerprints of individuals with different whole body insulin sensitivity according to the formula of Matsuda, et al. (ISI_Matsuda) by a non-targeted metabolomics approach we aimed a) to figure out an unsuspicious and altered metabolic pattern, b) to estimate a threshold related to these changes based on the ISI, and c) to identify the metabolic pathways responsible for the discrimination of the two patterns.

Methodology and Principal Findings

By applying infusion ion cyclotron resonance Fourier transform mass spectrometry, we analyzed plasma of 46 non-diabetic subjects exhibiting high to low insulin sensitivities. The orthogonal partial least square model revealed a cluster of 28 individuals with alterations in their metabolic fingerprints associated with a decline in insulin sensitivity. This group could be separated from 18 subjects with an unsuspicious metabolite pattern. The orthogonal signal correction score scatter plot suggests a threshold of an ISI_Matsuda of 15 for the discrimination of these two groups. Of note, a potential subgroup represented by eight individuals (ISI_Matsuda value between 8.5 and 15) was identified in different models. This subgroup may indicate a metabolic transition state, since it is already located within the cluster of individuals with declined insulin sensitivity but the metabolic fingerprints still show some similarities with unaffected individuals (ISI >15). Moreover, the highest number of metabolite intensity differences between unsuspicious and altered metabolic fingerprints was detected in lipid metabolic pathways (arachidonic acid metabolism, metabolism of essential fatty acids and biosynthesis of unsaturated fatty acids), steroid hormone biosyntheses and bile acid metabolism, based on data evaluation using the metabolic annotation interface MassTRIX.

Conclusions

Our results suggest that altered metabolite patterns that reflect changes in insulin sensitivity respectively the ISI_Matsuda are dominated by lipid-related pathways. Furthermore, a metabolic transition state reflected by heterogeneous metabolite fingerprints may precede severe alterations of metabolism. Our findings offer future prospects for novel insights in the pathogenesis of the pre-diabetic phase.     

Medium Chain Acylcarnitines Dominate the Metabolite Pattern in Humans under Moderate Intensity Exercise and Support Lipid Oxidation

Background

Exercise is an extreme physiological challenge for skeletal muscle energy metabolism and has notable health benefits. We aimed to identify and characterize metabolites, which are components of the regulatory network mediating the beneficial metabolic adaptation to exercise.

Methodology and Principal Findings

First, we investigated plasma from healthy human subjects who completed two independent running studies under moderate, predominantly aerobic conditions. Samples obtained prior to and immediately after running and then 3 and 24 h into the recovery phase were analyzed by a non-targeted (NT-) metabolomics approach applying liquid chromatography-qTOF-mass spectrometry. Under these conditions medium and long chain acylcarnitines were found to be the most discriminant plasma biomarkers of moderately intense exercise. Immediately after a 60 min (at 93% V_IAT) or a 120 min run (at 70% V_IAT) a pronounced, transient increase dominated by octanoyl-, decanoyl-, and dodecanoyl-carnitine was observed. The release of acylcarnitines as intermediates of partial β-oxidation was verified in skeletal muscle cell culture experiments by probing ¹³C-palmitate metabolism. Further investigations in primary human myotubes and mouse muscle tissue revealed that octanoyl-, decanoyl-, and dodecanoyl-carnitine were able to support the oxidation of palmitate, proving more effective than L-carnitine.

Conclusions

Medium chain acylcarnitines were identified and characterized by a functional metabolomics approach as the dominating biomarkers during a moderately intense exercise bout possessing the power to support fat oxidation. This physiological production and efflux of acylcarnitines might exert beneficial biological functions in muscle tissue.     

Engineered Cystine Knot Miniproteins as Potent Inhibitors of Human Mast Cell Tryptase β

Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase β, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase β tetramer with an overall equilibrium dissociation constant K_i of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase β described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors.     

qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL

Background  

Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST.     

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.