Aberrant Intracellular pH Regulation Limiting Glyceraldehyde-3-Phosphate Dehydrogenase Activity in the Glucose-Sensitive Yeast tps1Δ Mutant.

Whereas the yeast Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH4Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant.IMPORTANCE Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast tps1Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.

Metabolic control analysis of hepatic glycogen synthesis in vivo.

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Optimization of synthesis of carbohydrates and 1-phenyl-3-methyl-5-pyrazolone (PMP) by response surface methodology (RSM) for improved carbohydrate detection.

Reducing sugars can react with 1-phenyl-3-methyl-5-pyrazolone (PMP) to form sugar-PMP derivatives, which can be detected by HPLC-UV or HPLC-DAD due to their high UV absorbance at 248 nm. Six different sugars were synthesized with PMP with aid of response surface methodology (RSM), by which the parameters of the synthesis were designed within temperature ranged between 60 °C and 90 °C, and time from 60 to 180 min, respectively. Consequently, optimal conditions of the glucose (Glu)-, glucosamine (GluN)-, galactose (Gal)-, glucuronic acid (GluA), galacturonic acid (GalA) and glucose-6-phosphate (G6P-PMP) reactions were determined at 71 °C for 129 min, 73 °C for 96 min, 70 °C for 117 min, 75 °C for 151 min, 76 °C for 144 min, and 70 °C for 154 min, respectively. Experiments demonstrated that unique functional groups and delicate differences of carbohydrates' inner pH environment could significantly influence the sugar-PMP reactions. However, sugar stereoisomers did not have remarkable impacts on the reactions.

Simplest Chemosensor Array for Phosphorylated Saccharides.

We believe that "the simpler we are, the more complete we become" is a key concept of chemical sensing systems. In this work, a "turn-on" fluorescence chemosensor array relying on only two self-assembled molecular chemosensors with ability of both qualitative and quantitative detection of phosphorylated saccharides has been developed. The easy-to-prepare chemosensor array was fabricated by in situ mixing of off-the-shelf reagents (esculetin, 4-methylesculetin, and 3-nitrophenylboronic acid). The fluorescence-based saccharide sensing system was carried out using indicator displacement assay accompanied by photoinduced electron transfer (PeT) under various pH conditions. The simultaneous recognition of 14 types of saccharides including glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) was achieved with a successful classification rate of 100%. We also succeeded in the quantitative analysis of a mixture of glucose (Glc), as an original substrate, G6P and F6P, as enzymatic products in pseudoglycolysis pathway. Finally, levels of Glc and F6P in human induced pluripotent stem (hiPS) cells were indirectly monitored by using our proposed chemosensor array. Glc and F6P in supernatants of hiPS cells were classified by linear discriminant analysis as a pattern recognition model and the observed clusters represent the activity of hiPS cells. The results show the high accuracy of the proposed chemosensor array in detection of phosphorylated and similarly modified saccharides.

Online Liquid Chromatography-Sheath-Flow Surface Enhanced Raman Detection of Phosphorylated Carbohydrates.

Online detection and quantification of three phosphorylated carbohydrate molecules: glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate was achieved by coupling sheath-flow surface enhanced Raman spectroscopy (SERS) to liquid chromatography. The presence of an alkanethiol (hexanethiol) self-assembled monolayer adsorbed to a silver SERS-active substrate helps retain and concentrate the analytes of interest at the SERS substrate to improve the detection sensitivity significantly. Mixtures of 2 µM of phosphorylated carbohydrates in pure water as well as in cell culture media were successfully separated by HPLC, with identification using the sheath-flow SERS detector. The quantification of each analyte was achieved using partial least-squares (PLS) regression analysis and acetonitrile in the mobile phases as an internal standard. These results illustrate the utility of sheath-flow SERS for molecular specific detection in complex biological samples appropriate for metabolomics and other applications.

'Last In-First Out': seasonal variations of non-structural carbohydrates, glucose-6-phosphate and ATP in tubers of two Arum species.

Knowledge on the metabolism of polysaccharide reserves in wild species is still scarce. In natural sites we collected tubers of Arum italicum Mill. and A. maculatum L. - two geophytes with different apparent phenological timing, ecology and chorology - during five stages of the annual cycle in order to understand patterns of reserve accumulation and degradation. Both the entire tuber and its proximal and distal to shoot portion were utilised. Pools of non-structural carbohydrates (glucose, sucrose and starch), glucose-6-phosphate and ATP were analysed as important markers of carbohydrate metabolism. In both species, starch and glucose content of the whole tuber significantly increased from sprouting to the maturation/senescence stages, whereas sucrose showed an opposite trend; ATP and glucose-6-phosphate were almost stable and dropped only at the end of the annual cycle. Considering the two different portions of the tuber, both ATP and glucose-6-phosphate concentrations were higher in proximity to the shoot in all seasonal stages, except the flowering stage. Our findings suggest that seasonal carbon partitioning in the underground organ is driven by phenology and occurs independently of seasonal climate conditions. Moreover, our results show that starch degradation, sustained by elevated ATP and glucose-6-phosphate pools, starts in the peripheral, proximal-to-shoot portion of the tuber, consuming starch accumulated in the previous season, as a 'Last In-First Out' mechanism of carbohydrate storage.

Rewiring the Glucose Transportation and Central Metabolic Pathways for Overproduction of N-Acetylglucosamine in Bacillus subtilis.

N-acetylglucosamine (GlcNAc) is an important amino sugar extensively used in the healthcare field. In a previous study, the recombinant Bacillus subtilis strain BSGN6-PxylA -glmS-pP43NMK-GNA1 (BN0-GNA1) had been constructed for microbial production of GlcNAc by pathway design and modular optimization. Here, the production of GlcNAc is further improved by rewiring both the glucose transportation and central metabolic pathways. First, the phosphotransferase system (PTS) is blocked by deletion of three genes, yyzE (encoding the PTS system transporter subunit IIA YyzE), ypqE (encoding the PTS system transporter subunit IIA YpqE), and ptsG (encoding the PTS system glucose-specific EIICBA component), resulting in 47.6% increase in the GlcNAc titer (from 6.5 ± 0.25 to 9.6 ± 0.16 g L-1 ) in shake flasks. Then, reinforcement of the expression of the glcP and glcK genes and optimization of glucose facilitator proteins are performed to promote glucose import and phosphorylation. Next, the competitive pathways for GlcNAc synthesis, namely glycolysis, peptidoglycan synthesis pathway, pentose phosphate pathway, and tricarboxylic acid cycle, are repressed by initiation codon-optimization strategies, and the GlcNAc titer in shake flasks is improved from 10.8 ± 0.25 to 13.2 ± 0.31 g L-1 . Finally, the GlcNAc titer is further increased to 42.1 ± 1.1 g L-1 in a 3-L fed-batch bioreactor, which is 1.72-fold that of the original strain, BN0-GNA1. This study shows considerably enhanced GlcNAc production, and the metabolic engineering strategy described here will be useful for engineering other prokaryotic microorganisms for the production of GlcNAc and related molecules.

Application of capillary enzyme micro-reactor in enzyme activity and inhibitors studies of glucose-6-phosphate dehydrogenase.

In this study, we present an on-line measurement of enzyme activity and inhibition of Glucose-6-phosphate dehydrogenase (G6PDH) enzyme using capillary electrophoresis based immobilized enzyme micro-reactor (CE-based IMER). The IMER was prepared using a two-step protocol based on electrostatic assembly. The micro-reactor exhibited good stability and reproducibility for on-line assay of G6PDH enzyme. Both the activity as well as the inhibition of the G6PDH enzyme by six inhibitors, including three metals (Cu(2+), Pb(2+), Cd(2+)), vancomycin, urea and KMnO4, were investigated using on-line assay of the CE-based IMERs. The enzyme activity and inhibition kinetic constants were measured using the IMERs which were found to be consistent with those using traditional off-line enzyme assays. The kinetic mechanism of each inhibitor was also determined. The present study demonstrates the feasibility of using CE-based IMERs for rapid and efficient on-line assay of G6PDH, an important enzyme in the pentosephosphate pathway of human metabolism.

Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae stopped by maintaining the pH of the medium in a pH-stat at pH 8.0 or 9.0. Studying its main physiological capacities and comparing cells after incubation at pH 6.0 vs. 8.0 or 9.0, we found that (a) fermentation was moderately decreased by high pH and respiration was similar and sensitive to the addition of an uncoupler, (b) ATP and glucose-6-phosphate levels upon glucose addition increased to similar levels and (c) proton pumping and K(+) transport were also not affected; all this indicating that energy mechanisms were preserved. Growth inhibition at high pH was also not due to a significant lower amino acid transport by the cells or incorporation into proteins. The cell cycle stopped at pH 9.0, probably due to an arrest as a result of adjustments needed by the cells to contend with the changes under these conditions, and microarray experiments showed some relevant changes to this response.

Identification of sensory attributes, instrumental and chemical measurements important for consumer acceptability of grilled lamb Longissimus lumborum.

In this study, important eating quality attributes that influence consumer liking for grilled lamb loin have been identified using preference mapping techniques. The eating quality attributes identified as driving the consumer liking of lamb loin steaks were "tenderness", "sweet flavour", "meaty aftertaste", "roast lamb flavour" and "roast lamb aftertaste". In contrast, the texture attribute "rubbery" and the flavour attributes "bitter flavour" and "bitter aftertaste" had a negative influence on consumer perceptions. Associations were observed between eating quality and a number of instrumental and chemical measurements. Warner Bratzler Shear Force showed an association with "rubbery" texture and a negative association with "tenderness" and consumer liking scores. The compounds, glucose, glucose-6-phosphate, inosine, inosine monophosphate and adenosine monophosphate were associated with the attributes, "sweet flavour","meaty aftertaste", "roast lamb flavour", "roast lamb aftertaste" and with consumer scores for liking of lamb which is probably caused by the role some of these compounds play as precursors of flavour and as taste compounds.

Milk metabolites as indicators of mammary gland functions and milk quality.

The assumption, that metabolites derived from the activity of the mammary gland epithelial cells reflect changes in milk secretion and its coagulation properties, was tested in dairy cows. The experiment included cows with uninfected udders and cows with one of the glands infected by different bacteria specie. Analysis were carried at the cow level (including all four glands), or at the gland level. High and significant correlations among the concentrations of lactose, glucose, glucose-6-posphate, milk related respiratory index (the ratio between the concentrations of citrate/lactate+malate in milk) and milk-derived glycolytic index (the ratio between glucose-6-phosphate and glucose in milk) and milk clotting parameters were found. The physiological basis for these relations and their ability to predict the deterioration in milk quality in subclinically infected glands and in glands previously clinically infected with Escherichia coli are discussed.

Signal amplification of glucosamine-6-phosphate based on ribozyme glmS.

Ribozyme glmS based isothermal amplification assay is developed for the colorimetric detection of glucosamine-6-phosphate (GlcN6P). Upon binding to the metabolite target GlcN6P, self-cleavage of glmS ribozyme is initiated to release RNA fragment that can trigger the cascade signal amplification to release large amount of G-quadruplex DNAzymes as reporter for colorimetric detection. Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. This assay not only offers a convenient detection of GlcN6P with high specificity and sensitivity, but also provides a platform for high-throughput screening of antibiotics based on glmS riboswitches.

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.

Untargeted metabolomic profiling in saliva of smokers and nonsmokers by a validated GC-TOF-MS method.

A GC-TOF-MS method was developed and validated for a metabolic fingerprinting in saliva of smokers and nonsmokers. We validated the method by spiking 37 different metabolites and 6 internal standards to saliva between 0.1 µM and 2 mM. Intraday coefficients of variation (CVs) (accuracies) were on average, 11.9% (85.8%), 8.2% (88.9%), and 10.0% (106.7%) for the spiked levels 25, 50, and 200 µM, respectively (N = 5). Interday CVs (accuracies) were 12.4% (97%), 18.8% (95.5%), and 17.2% (105.9%) for the respective levels of 25, 50, and 200 µM (N = 5). The method was applied to saliva of smokers and nonsmokers, obtained from a 24 h diet-controlled clinical study, in order to identify biomarkers of endogenous origin, which could be linked to smoking related diseases. Automated peak picking, integration, and statistical analysis were conducted by the software tools MZmine, Metaboanalyst, and PSPP. We could identify 13 significantly altered metabolites in smokers (p < 0.05) by matching them against MS libraries and authentic standard compounds. Most of the identified metabolites, including tyramine, adenosine, and glucose-6-phosphate, could be linked to smoking-related perturbations and may be associated with established detrimental effects of smoking.

Glycogen is the primary source of glucose during the lag phase of E. coli proliferation.

In the studies of Escherichia coli (E. coli), metabolomics analyses have mainly been performed using steady state culture. However, to analyze the dynamic changes in cellular metabolism, we performed a profiling of concentration of metabolites by using batch culture. As a first step, we focused on glucose uptake and the behavior of the first metabolite, G6P (glucose-6-phosphate). A computational formula was derived to express the glucose uptake rate by a single cell from two kinds of experimental data, extracellular glucose concentration and cell growth, being simulated by Cell Illustrator. In addition, average concentration of G6P has been measured by CE-MS. The existence of another carbon source was suggested from the computational result. After careful comparison between cell growth, G6P concentration, and the computationally obtained curve of glucose uptake rate, we predicted the consumption of glycogen in lag phase and its accumulation as an energy source in an E. coli cell for the next proliferation. We confirmed our prediction experimentally. This behavior indicates the importance of glycogen participation in the lag phase for the growth of E. coli. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.

Unsupervised analysis of small animal dynamic Cerenkov luminescence imaging.

Clustering analysis (CA) and principal component analysis (PCA) were applied to dynamic Cerenkov luminescence images (dCLI). In order to investigate the performances of the proposed approaches, two distinct dynamic data sets obtained by injecting mice with (32)P-ATP and (18)F-FDG were acquired using the IVIS 200 optical imager. The k-means clustering algorithm has been applied to dCLI and was implemented using interactive data language 8.1. We show that cluster analysis allows us to obtain good agreement between the clustered and the corresponding emission regions like the bladder, the liver, and the tumor. We also show a good correspondence between the time activity curves of the different regions obtained by using CA and manual region of interest analysis on dCLIT and PCA images. We conclude that CA provides an automatic unsupervised method for the analysis of preclinical dynamic Cerenkov luminescence image data.

Metabolomics of supragingival plaque and oral bacteria.

Dental caries is initiated by demineralization of the tooth surface through acid production by sugar metabolism of supragingival plaque microflora. To elucidate the sugar metabolic system, we used CE-MS to perform metabolomics of the central carbon metabolism, the EMP pathway, the pentose-phosphate pathway, and the TCA cycle in supra- gingival plaque and representative oral bacteria, Streptococcus and Actinomyces. Supragingival plaque contained all the targeted metabolites in the central carbon metabolism, except erythrose 4-phosphate in the pentose-phosphate pathway. After glucose rinse, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and pyruvate in the EMP pathway and 6-phosphogluconate, ribulose 5-phosphate, and sedoheptulose 7-phosphate in the pentose-phosphate pathway, and acetyl CoA were increased. Meanwhile, 3-phosphoglycerate and phosphoenolpyruvate in the EMP pathway and succinate, fumarate, and malate in the TCA cycle were decreased. These pathways and changes in metabolites observed in supragingival plaque were similar to the integration of metabolite profiles in Streptococcus and Actinomyces.

In vivo imaging of schistosomes to assess disease burden using positron emission tomography (PET).

BACKGROUND: Schistosomes are chronic intravascular helminth parasites of humans causing a heavy burden of disease worldwide. Diagnosis of schistosomiasis currently requires the detection of schistosome eggs in the feces and urine of infected individuals. This method unreliably measures disease burden due to poor sensitivity and wide variances in egg shedding. In vivo imaging of schistosome parasites could potentially better assess disease burden, improve management of schistosomiasis, facilitate vaccine development, and enhance study of the parasite's biology. Schistosoma mansoni (S. mansoni) have a high metabolic demand for glucose. In this work we investigated whether the parasite burden in mice could be assessed by positron emission tomography (PET) imaging with 2-deoxy-2[(18)F]fluoro-D-glucose (FDG). METHODOLOGY/PRINCIPAL FINDINGS: Live adult S. mansoni worms FDG uptake in vitro increased with the number of worms. Athymic nude mice infected with S. mansoni 5-6 weeks earlier were used in the imaging studies. Fluorescence molecular tomography (FMT) imaging with Prosense 680 was first performed. Accumulation of the imaging probe in the lower abdomen correlated with the number of worms in mice with low infection burden. The total FDG uptake in the common portal vein and/or regions of elevated FDG uptake in the liver linearly correlated to the number of worms recovered from infected animals (R(2) =0.58, P<0.001, n = 40). FDG uptake showed a stronger correlation with the worm burden in mice with more than 50 worms (R(2) = 0.85, P<0.001, n = 17). Cryomicrotome imaging confirmed that most of the worms in a mouse with a high infection burden were in the portal vein, but not in a mouse with a low infection burden. FDG uptake in recovered worms measured by well counting closely correlated with worm number (R(2) = 0.85, P<0.001, n = 21). Infected mice showed a 32% average decrease in total FDG uptake after three days of praziquantel treatment (P = 0.12). The total FDG uptake in untreated mice increased on average by 36% over the same period (P = 0.052). CONCLUSION: FDG PET may be useful to non-invasively quantify the worm burden in schistosomiasis-infected animals. Future investigations aiming at minimizing non-specific FDG uptake and to improve the recovery of signal from worms located in the lower abdomen will include the development of more specific radiotracers.

An enzymatic fluorimetric assay to quantitate 2-deoxyglucose and 2-deoxyglucose-6-phosphate for in vitro and in vivo use.

Previously, we developed a microplate assay to quantitate 2-deoxyglucose (2DG) and 2-deoxyglucose-6-phosphate in samples for in vitro and in vivo use. In this assay system, four different reaction mixtures were used, and the difference in the reactivity of the two types of glucose-6-phosphate dehydrogenase (G6PDH) variants was used. Because G6PDH from tolura yeast was no longer available, we modified our assay system for the use of G6PDH from Leuconostoc. Using this improved assay system, concentrations of glucose, 2DG, glucose-6-phosphate, and 2-deoxyglucose-6-phosphate were easily measured. This assay may be useful for measuring uptake of 2DG without the use of radioisotopes.

Effects of fasting prior to slaughter on pH development and energy metabolism post-mortem in M. longissimus dorsi of pigs.

The aim of this study was to investigate the effect of pre-slaughter fasting time, sex and feeding regime on the development of energy metabolism and pH in M. longissimusdorsi (LD) post-mortem in pigs. Two hundred and seventy pigs of the commercial Norwegian crossbreed Noroc (LYLD) were used involving two sexes (gilts and castrates), two feeding regimes (restricted and ad libitum) and four different fasting treatments: (F4) 4h fasting, (F175) 17.5h fasting on the farm, (FO175) 17.5h fasting overnight at the abattoir, and (FO265) 26.5h fasting overnight at the abattoir. Additionally the pigs experienced two different abattoir lairage times as fasting treatment F4 and F175 had a lairage time of 1.5h, while fasting treatment FO175 and FO265 had a lairage time of 23.0 h. A short fasting time of 4 h led to a delayed degradation of glycogen, slow decline in pH and a lower ultimate pH(45 h) post-mortem (pHu) in the LD compared with a fasting time of 26.5 h which resulted in a rapid breakdown of glycogen and pH decline early post-mortem and a high pHu. Proglycogen was degraded in favour of macroglycogen under anaerobic conditions post-mortem. Feeding the animals in the morning before delivery if slaughtered the same day, results in low pH reduction rate and a low pHu compared with pigs fasted overnight either on farm or at the abattoir. Aiming a higher pHu in LD it should be recommended not to feed the pigs in the morning at the day of slaughter.