A shift in quadriceps muscle metabolic profile toward decreased oxidative metabolism and increased glycolysis is a consistent finding in chronic obstructive pulmonary disease (COPD). Chronic ...inflammation has been proposed as a trigger of this pathological metabolic adaptation. Indeed, the proinflammatory cytokine TNF-α impairs muscle oxidative metabolism through activation of the nuclear factor-κB (NF-κB) pathway. Putative effects on muscle glycolysis, however, are unclear. We hypothesized that TNF-α-induced NF-κB signaling stimulates muscle glycolytic metabolism through activation of the glycolytic regulator hypoxia-inducible factor-1α (HIF-1α). Wild-type C2C12 and C2C12-IκBα-SR (blocked NF-κB signaling) myotubes were stimulated with TNF-α, and its effects on glycolytic metabolism and involvement of the HIF pathway herein were investigated. As proof of principle, expression of HIF signaling constituents was investigated in quadriceps muscle biopsies of a previously well-characterized cohort of clinically stable patients with severe COPD and healthy matched controls. TNF-α increased myotube glucose uptake and lactate production and enhanced the activity and expression levels of multiple effectors of muscle glycolytic metabolism in a NF-κB-dependent manner. In addition, TNF-α activated HIF signaling, which required classical NF-κB activation. Moreover, the knockdown of HIF-1α largely attenuated TNF-α-induced increases in glycolytic metabolism. Accordingly, the mRNA levels of HIF-1α and the HIF-1α target gene, vascular endothelial growth factor (VEGF), were increased in muscle biopsies of COPD patients compared with controls, which was most pronounced in the patients with high levels of muscle TNF-α. In conclusion, these data show that TNF-α-induced classical NF-κB activation enhances muscle glycolytic metabolism in a HIF-1α-dependent manner.
The filamentous fungus
Chrysosporium lucknowense (C1) is a rich source of cell wall degrading enzymes. In the present paper four arabinose releasing enzymes from C1 were characterized, among them one ...endoarabinanase, two arabinofuranosidases and one exoarabinanase. Combinations of these enzymes released up to 80% of the arabinose present in sugar beet arabinan to fermentable monosugars. Besides the main product arabinobiose, unknown arabinose oligomers are produced from highly branched arabinan when endoarabinanase was combined with exoarabinanase and/or arabinofuranosidase. All described arabinose releasing enzymes are temperature stable up to 50
°C and have a broad pH stability. This makes C1 arabinohydrolases suitable for many biotechnical applications, like co-fermentation bioethanol production.
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•T. longibrachiatum preparation was fractionated and supplemented to an enzyme mixture.•Synergistic potential was found by supplementation of acetyl xylan esterase 1 ...(Axe1).•Deacetylation efficiency by Axe1 is demonstrated and compared to AxeA (A. niger).•Acetic acid release by Axe1 was up to six times higher on model oligosaccharides.•MS demonstrated efficient deacetylation of O-acetyl-(4-O-methylglucurono)xylan.
Supplementation of a Trichoderma longibrachiatum preparation to an industrial Aspergillus niger/Talaromyces emersonii enzyme mixture demonstrated synergy for the saccharification of corn silage water-unextractable solids (WUS). Sub-fractions of the crude T. longibrachiatum preparation obtained after chromatography were analyzed regarding their hydrolytic activity. An acetyl xylan esterase 1 Axe1, carbohydrate esterase (CE) family 5-enriched sub-fraction closely mimicked the hydrolytic gain as obtained by supplementation of the complete, crude enzyme mixture (increase of 50%, 62% and 29% for Xyl, Ara and Glc, respectively). The acetic acid released from model polysaccharides (WUS) and oligosaccharides neutral (AcXOS) and acidic (AcUXOS) xylo-oligosaccharides by Axe1 was two and up to six times higher compared to the acetic acid released by acetyl xylan esterase A (AxeA, CE 1). Characterization of Axe1 treated AcXOS and AcUXOS revealed deacetylation of oligosaccharides that were not deacetylated by AxeA or the A. niger/T. emersonii preparation.
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•A-type RS-3 crystals are more resistant to digestion than B-type RS-3 crystals.•The higher the Mw of RS-3 preparations, the higher the resistance to digestion.•A low RS-3 ...polydispersity leads to lower digestibility.•A-type RS-3 crystals that escape digestion do not change in Mw (distribution).•Some B-type RS-3 crystals that escape digestion change in Mw (distribution).
Resistant starch type 3 (RS-3) holds great potential as a prebiotic by supporting gut microbiota following intestinal digestion. However the factors influencing the digestibility of RS-3 are largely unknown. This research aims to reveal how crystal type and molecular weight (distribution) of RS-3 influence its resistance. Narrow and polydisperse α-glucans of degree of polymerization (DP) 14–76, either obtained by enzymatic synthesis or debranching amylopectins from different sources, were crystallized in 12 different A- or B-type crystals and in vitro digested. Crystal type had the largest influence on resistance to digestion (A >>> B), followed by molecular weight (Mw) (high DP >> low DP) and Mw distribution (narrow disperse > polydisperse). B-type crystals escaping digestion changed in Mw and Mw distribution compared to that in the original B-type crystals, whereas A-type crystals were unchanged. This indicates that pancreatic α-amylase binds and acts differently to A- or B-type RS-3 crystals.
•PG/PL digestion with HILIC–MS/ELSD is a powerful approach for pectin analysis.•Novel GalA oligomeric structures released by PG/PL digestion are identified using LC–MS.•Descriptive parameters enable ...us to distinguish between substitution patterns in SBPs.•The degree of hydrolysis by PG/PL provides information on SBP distribution patterns.
Enzymatic fingerprinting was applied to sugar beet pectins (SBPs) modified by either plant or fungal pectin methyl esterases and alkali catalyzed de-esterification to reveal the ester distributions over the pectin backbone. A simultaneous pectin lyase (PL) treatment to the commonly used endo-polygalacturonase (endo-PG) degradation showed to be effective in degrading both high and low methylesterified and/or acetylated homogalaturonan regions of SBP simultaneously. Using LC-HILIC–MS/ELSD, we studied in detail all the diagnostic oligomers present, enabling us to discriminate between differently prepared sugar beet pectins having various levels of methylesterification and acetylation. Furthermore, distinction between commercially extracted and de-esterified sugar beet pectin having different patterns of substitution was achieved by using novel descriptive pectin parameters. In addition to DBabs approach for nonmethylesterified sequences degradable by endo-PG, the “degree of hydrolysis” (DHPG) representing all partially saturated methylesterified and/or acetylated galacturonic acid (GalA) moieties was introduced as a new parameter. Consequently, the description DHPL has been introduced to quantify all esterified unsaturated GalA oligomers.
Chronic obstructive pulmonary disease (COPD) is a multiorgan systemic disease. The systemic features are skeletal muscle weakness and cachexia, the latter being associated with systemic inflammation. ...The exact mechanisms underlying skeletal muscle dysfunction in COPD remain obscure. Recent evidence suggests involvement of the peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivator (PGC)-1alpha in regulation of skeletal muscle morphology and metabolism, and mitochondrial transcription factor A (TFAM) has been implicated in the process of mitochondrial biogenesis. The aim of the present exploratory study was, therefore, to compare these factors in the skeletal muscle of nine healthy control subjects and 14 COPD patients stratified by cachexia. PPAR-gamma, PPAR-delta and TFAM were measured at the mRNA and protein level by real-time quantitative PCR and Western blotting, respectively. PPAR-alpha and PGC-1alpha were meansured at the mRNA level. PPAR-delta and TFAM protein content, as well as PGC-1alpha mRNA levels, were decreased in the skeletal muscle of COPD patients compared with healthy controls. The cachectic COPD subgroup was further characterised by decreased PPAR-alpha mRNA expression and decreased TFAM protein and mRNA levels compared with noncachectic COPD patients. In addition, PPAR-alpha mRNA levels in skeletal muscle correlated negatively with inflammatory markers in plasma. Therefore, a disturbed expression of these regulatory factors may well underlie the disturbed skeletal muscle functioning in chronic obstructive pulmonary disease.
Aberrant skeletal muscle mitochondrial oxidative metabolism is a debilitating feature of chronic diseases such as chronic obstructive pulmonary disease, type 2 diabetes and chronic heart failure. ...Evidence in non-muscle cells suggests that glycogen synthase kinase-3β (GSK-3β) represses mitochondrial biogenesis and inhibits PPAR-γ co-activator 1 (PGC-1), a master regulator of cellular oxidative metabolism. The role of GSK-3β in the regulation of skeletal muscle oxidative metabolism is unknown.
We hypothesized that inactivation of GSK-3β stimulates muscle oxidative metabolism by activating PGC-1 signaling and explored if GSK-3β inactivation could protect against physical inactivity-induced alterations in skeletal muscle oxidative metabolism.
GSK-3β was modulated genetically and pharmacologically in C2C12 myotubes in vitro and in skeletal muscle in vivo. Wild-type and muscle-specific GSK-3β knock-out (KO) mice were subjected to hind limb suspension for 14days. Key constituents of oxidative metabolism and PGC-1 signaling were investigated.
In vitro, knock-down of GSK-3β increased mitochondrial DNA copy number, protein and mRNA abundance of oxidative phosphorylation (OXPHOS) complexes and activity of oxidative metabolic enzymes but also enhanced protein and mRNA abundance of key PGC-1 signaling constituents. Similarly, pharmacological inhibition of GSK-3β increased transcript and protein abundance of key constituents and regulators of mitochondrial energy metabolism. Furthermore, GSK-3β KO animals were protected against unloading-induced decrements in expression levels of these constituents.
Inactivation of GSK-3β up-regulates skeletal muscle mitochondrial metabolism and increases expression levels of PGC-1 signaling constituents. In vivo, GSK-3β KO protects against inactivity-induced reductions in muscle metabolic gene expression.
•Inactivation of GSK-3β increases mitochondrial metabolism in C2C12 muscle cells.•Inactivation of GSK-3β activates PGC-1 signaling in cultured C2C12 myotubes.•GSK-3β inactivation increased metabolic gene expression via PGC-1α in myotubes.•GSK-3β KO protects against HLS-induced loss of muscle oxidative gene expression.
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•The chain length distribution of several IMMP substructures is revealed.•Efficient inclusion of α-(1→6) linkages in IMMP amylopectin is amylose dependent.•Increased GTFB modification ...leads to longer α-(1→6) linked glycosidic chains.
In this study, we present an enzymatic fingerprinting method for the characterization of isomalto/malto-polysaccharides (IMMPs). IMMPs are produced by the modification of starch with the 4,6-α-glucanotransferase (GTFB) enzyme and consist of α-(1→4), α-(1→6) and α-(1→4,6) linked glucoses. Enzymes were used separately, simultaneously or in successive order to specifically degrade and/or reveal IMMP substructures. The enzymatic digests were subsequently analysed with HPSEC and HPAEC to reveal the chain length distribution (CLD) of different IMMP substructures. The presence of amylose in the substrate resulted in the formation of linear α-(1→6) linked glycosidic chains (13.5 kDa) in the former amylopectin fraction. The length of these chains indicates that GTFB transferase activity on amylopectin is more likely to elongate single amylopectin chains than to provide an even distribution. Enzymatic fingerprinting also revealed that the GTFB enzyme is capable of introducing large (20 kDa) linear α-(1→6) linked glycosidic chains in the α-glucan substrate.
Pectin, predominantly present within plant cell walls, is a dietary fiber that potentially induces distinct health effects depending on its molecular structure. Such structure-dependent health ...effects of pectin-derived galacturonic acid oligosaccharides (GalA-OS) are yet largely unknown. This study describes the influence of methyl-esterification and ∆4,5-unsaturation of GalA-OS through defined sets of GalA-OS made from pectin using defined pectinases, on the fermentability by individual fecal inocula. The metabolite production, OS utilization, quantity and size, methyl-esterification and saturation of remaining GalA-OS were monitored during the fermentation of GalA-OS. Fermentation of all GalA-OS predominantly induced the production of acetate, butyrate and propionate. Metabolization of unsaturated GalA-OS (uGalA-OS) significantly increased butyrate formation compared to saturated GalA-OS (satGalA-OS), while satGalA-OS significantly increased propionate formation. Absence of methyl-esters within GalA-OS improved substrate metabolization during the first 18 h of fermentation (99 %) compared to their esterified analogues (51 %). Furthermore, HPAEC and HILIC-LC-MS revealed accumulation of specific methyl-esterified GalA-OS, confirming that methyl-esterification delays fermentation. Fermentation of structurally distinct GalA-OS results in donor specific microbiota composition with uGalA-OS specifically stimulating the butyrate-producer Clostridium Butyricum. This study concludes that GalA-OS fermentation induces highly structure-dependent changes in the gut microbiota, further expanding their potential use as prebiotics.
► Pectolytic enzymes almost fully degrade sugar beet pectins. ► The HILIC column enables a good separation of most oligomers present in the digest. ► HILIC-ELSD-MS allows a complete identification ...and quantification of all oligomers. ► The precise location of acetyl groups within an oligomer can be elucidated.
The combined action of endo-polygalacturonase (endo-PGII), pectin lyase (PL), pectin methyl esterase (fungal PME) and RG-I degrading enzymes enabled the extended degradation of methylesterified and acetylated sugar beet pectins (SBPs). The released oligomers were separated, identified and quantified using hydrophilic interaction liquid chromatography (HILIC) with online electrospray ionization ion trap mass spectrometry (ESI-IT-MSn) and evaporative light scattering detection (ELSD). By MSn, the structures of galacturonic acid (GalA) oligomers having an acetyl group in the O-2 and/or O-3 positions eluting from the HILIC column were elucidated. The presence of methylesterified and/or acetylated galacturonic acid units within an oligomer reduced the interaction with the HILIC column significantly compared to the unsubstituted GalA oligomers. The HILIC column enables a good separation of most oligomers present in the digest. The use of ELSD to quantify oligogalacturonides was validated using pure GalA standards and the signal was found to be independent of the chemical structure of the oligomer being detected. The combination of chromatographic and enzymatic strategies enables to distinguish SBPs having different methylesters and acetyl group distribution.
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