Concentrations Of Metabolic Intermediates Research Articles

A preliminary study on a novel bioaugmentation technique enhancing lactic acid production by mixed cultures fermentation

The paper is a preliminary study on the selection of lactic acid producing microorganisms from a mixed microbial population via bioaugmentation. The bioaugmentation technique is based on pH sudden variations occurring in sequential batch steps of a dark fermentation process applied to simple substrates. Different conditions are tested and compared. The structure of microbial communities and concentrations of metabolic intermediates are analyzed to study the possible substrate conversion routes. Obtained results indicate that the initial mixed culture produced a lactic acid percentage of 5% in terms of CODLA/CODPRODUCTS. In the most favourable conditions, the selected culture produced a lactic acid percentage of 59%. The analysis of the composition of microbial communities before and after the bioaugmentation processes, indicates that lactic acid production mainly results from the population change to bacteria belonging to the genus Bacillus. Indeed, the relative abundance of Bacilli increased from 0.67%, to 8.40% during the bioaugmentation cycle.

Drug Target Selection for Trypanosoma cruzi Metabolism by Metabolic Control Analysis and Kinetic Modeling.

In the search for therapeutic targets in the intermediary metabolism of trypanosomatids the gene essentiality criterion as determined by using knock-out and knock-down genetic strategies is commonly applied. As most of the evaluated enzymes/transporters have turned out to be essential for parasite survival, additional criteria and approaches are clearly required for suitable drug target prioritization. The fundamentals of Metabolic Control Analysis (MCA; an approach in the study of control and regulation of metabolism) and kinetic modeling of metabolic pathways (a bottom-up systems biology approach) allow quantification of the degree of control that each enzyme exerts on the pathway flux (flux control coefficient) and metabolic intermediate concentrations (concentration control coefficient). MCA studies have demonstrated that metabolic pathways usually have two or three enzymes with the highest control of flux; their inhibition has more negative effects on the pathway function than inhibition of enzymes exerting low flux control. Therefore, the enzymes with the highest pathway control are the most convenient targets for therapeutic intervention. In this review, the fundamentals of MCA as well as experimental strategies to determine the flux control coefficients and metabolic modeling are analyzed. MCA and kinetic modeling have been applied to trypanothione metabolism in Trypanosoma cruzi and the model predictions subsequently validated in vivo. The results showed that three out of ten enzyme reactions analyzed in the T. cruzi anti-oxidant metabolism were the most controlling enzymes. Hence, MCA and metabolic modeling allow a further step in target prioritization for drug development against trypanosomatids and other parasites.

Short-Term Temporal Metabolic Behavior in Halophilic Cyanobacterium Synechococcus sp. Strain PCC 7002 after Salt Shock.

In response to salt stress, cyanobacteria increases the gene expression of Na+/H+ antiporter and K+ uptake system proteins and subsequently accumulate compatible solutes. However, alterations in the concentrations of metabolic intermediates functionally related to the early stage of the salt stress response have not been investigated. The halophilic cyanobacterium Synechococcus sp. PCC 7002 was subjected to salt shock with 0.5 and 1 M NaCl, then we performed metabolomics analysis by capillary electrophoresis/mass spectrometry (CE/MS) and gas chromatography/mass spectrometry (GC/MS) after cultivation for 1, 3, 10, and 24 h. Gene expression profiling using a microarray after 1 h of salt shock was also conducted. We observed suppression of the Calvin cycle and activation of glycolysis at both NaCl concentrations. However, there were several differences in the metabolic changes after salt shock following exposure to 0.5 M and 1 M NaCl: (i): the main compatible solute, glucosylglycerol, accumulated quickly at 0.5 M NaCl after 1 h but increased gradually for 10 h at 1 M NaCl; (ii) the oxidative pentose phosphate pathway and the tricarboxylic acid cycle were activated at 0.5 M NaCl; and (iii) the multi-functional compound spermidine greatly accumulated at 1 M NaCl. Our results show that Synechococcus sp. PCC 7002 acclimated to different levels of salt through a salt stress response involving the activation of different metabolic pathways.

Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism.

Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery. The following protocol is a relatively simple method that is not only sensitive for small concentrations of metabolic intermediates but can also be used in vivo or in vitro to determine the integrity of various metabolic pathways, such as flux changes within specific metabolite pools. We used this protocol to determine the role of phosphoenolpyruvate carboxykinase 1 (Pck1) gene in mouse macrophage cells to determine the percent contribution from a precursor of 13C labeled glucose into specific CAC metabolite pools.

Quantifying the efficiency of Saccharomyces cerevisiae translocation tags.

Compartmentalization of metabolic pathways into organelles of the yeast Saccharomyces cerevisiae has been used to improve chemical production. Pathway compartmentalization aids chemical production by bringing enzymes into close proximity to one another, placing enzymes near key starting metabolites or essential co-factors, increasing the effective concentration of metabolic intermediates, and providing a more suitable chemical environment for enzymatic activity. Although several translocation tags have been used to localize enzymes to different yeast organelles, their translocation efficiencies have not been quantified. Here, we systematically quantify the translocation efficiencies of 10 commonly used S. cerevisiae tags by localizing green fluorescent protein (GFP) into three yeast organelles: the mitochondrion (4 tags), the vacuole (3 tags), and the peroxisome (3 tags). Further, we investigate whether plasmid copy number or mRNA levels vary with tag translocation efficiency. Quantification of the efficiencies of S. cerevisiae translocation tags provides an important resource for bioengineering practitioners when choosing a tag to compartmentalize their desired protein. Finally, these efficiencies can be used to determine the percentage of enzyme compartmentalization and, thus, help better quantify effects of compartmentalization on metabolic pathway efficiency.

1H NMR Metabolomics Reveals Association of High Expression of Inositol 1, 4, 5 Trisphosphate Receptor and Metabolites in Breast Cancer Patients

1H NMR is used to detect alterations in metabolites and their linkage to metabolic processes in a number of pathological conditions including breast cancer. Inositol 1, 4, 5 trisphosphate (IP3R) receptor is an intracellular calcium channel known to regulate metabolism and cellular bioenergetics. Its expression is up regulated in a number of cancers. However, its linkage to metabolism in disease conditions has not been evaluated. This study was designed to determine the association if any, of these metabolites with altered expression of IP3R in breast cancer. We used 1H NMR to identify metabolites in the serum of breast cancer patients (n = 27) and performed Real-time Polymerase Chain Reaction analysis for quantifying the expression of IP3R type 3 and type 2 in tissues from breast cancer patients (n = 40). Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) clearly distinguished patients with high/low IP3R expression from healthy subjects. The present study revealed high expression of IP3R type 2 and type 3 in human breast tumor tissue compared to adjacent non-tumorous tissue. Moreover, patients with ≥ 2-fold increase in IP3R (high IP3R group) had significantly higher concentration of metabolic intermediates compared to those with < 2-fold increase in IP3R (low IP3R group). We observed an increase in lipoprotein content and the levels of metabolites like lactate, lysine and alanine and a decrease in the levels of pyruvate and glucose in serum of high IP3R group patients when compared to those in healthy subjects. Receiver operating characteristic (ROC) curve analysis was performed to show the clinical utility of metabolites. In addition to the human studies, functional relevance of IP3Rs in causing metabolic disruption was observed in MCF-7 and MDA MB-231 cells. Results from our studies bring forth the importance of metabolic (or metabolomics) profiling of serum by 1H NMR in conjunction with tissue expression studies for characterizing breast cancer patients. The results from this study provide new insights into relationship of breast cancer metabolites with IP3R.

Kinetic folding design of aptazyme-regulated expression devices as riboswitches for metabolic engineering.

Recent developments in the fields of synthetic biology and metabolic engineering have opened the doors for the microbial production of biofuels and other valuable organic compounds. There remain, however, significant metabolic hurdles to the production of these compounds in cost-effective quantities. This is due, in part, to mismatches between the metabolic engineer's desire for high yields and the microbe's desire to survive. Many valuable compounds, or the intermediates necessary for their biosynthesis, prove deleterious at the desired production concentrations. One potential solution to these toxicity-related issues is the implementation of nonnative dynamic genetic control mechanisms that sense excessively high concentrations of metabolic intermediates and respond accordingly to alleviate their impact. One potential class of dynamic regulator is the riboswitch: cis-acting RNA elements that regulate the expression of downstream genes based on the presence of an effector molecule. Here, we present combined methods for constructing aptazyme-regulated expression devices (aREDs) through computational cotranscriptional kinetic folding design and experimental validation. These approaches can be used to engineer aREDs within novel genetic contexts for the predictable, dynamic regulation of gene expression in vivo.

Long-chain acylcarnitine content determines the pattern of energy metabolism in cardiac mitochondria

In the heart, a nutritional state (fed or fasted) is characterized by a unique energy metabolism pattern determined by the availability of substrates. Increased availability of acylcarnitines has been associated with decreased glucose utilization; however, the effects of long-chain acylcarnitines on glucose metabolism have not been previously studied. We tested how changes in long-chain acylcarnitine content regulate the metabolism of glucose and long-chain fatty acids in cardiac mitochondria in fed and fasted states. We examined the concentrations of metabolic intermediates in plasma and cardiac tissues under fed and fasted states. The effects of substrate availability and their competition for energy production at the mitochondrial level were studied in isolated rat cardiac mitochondria. The availability of long-chain acylcarnitines in plasma reflected their content in cardiac tissue in the fed and fasted states, and acylcarnitine content in the heart was fivefold higher in fasted state compared to the fed state. In substrate competition experiments, pyruvate and fatty acid metabolites effectively competed for the energy production pathway; however, only the physiological content of acylcarnitine significantly reduced pyruvate and lactate oxidation in mitochondria. The increased availability of long-chain acylcarnitine significantly reduced glucose utilization in isolated rat heart model and in vivo. Our results demonstrate that changes in long-chain acylcarnitine contents could orchestrate the interplay between the metabolism of pyruvate-lactate and long-chain fatty acids, and thus determine the pattern of energy metabolism in cardiac mitochondria.

Metabolite Changes Signal Genetic Regulatory Mechanisms for Robust Cell Behavior

ABSTRACTExploiting mechanisms of utilizing the sugar d-galactose in Escherichia coli as a model system, we explored the consequences of accumulation of critical intermediates of the d-galactose metabolic pathways by monitoring cell growth, metabolites, and transcript profiles. These studies revealed both metabolic network changes far from the d-galactose pathway and changes in the global gene regulatory network. The concentration change of a critical intermediate disturbs the equilibrium state, generating a ripple effect through several metabolic pathways that ends up signaling up- or downregulation of specific sets of genes in a programmed manner to cope with the imbalance. Such long-range effects on metabolites and genetic regulatory mechanisms not only may be a common feature in bacteria but very likely operate during cellular development and differentiation in higher organisms as well as in disease cells, like cancer cells.

Explicit consideration of topological and parameter uncertainty gives new insights into a well‐established model of glycolysis

Previous models of glycolysis in the sleeping sickness parasite Trypanosoma brucei assumed that the core part of glycolysis in this unicellular parasite is tightly compartimentalized within an organelle, the glycosome, which had previously been shown to contain most of the glycolytic enzymes. The glycosomes were assumed to be largely impermeable, and exchange of metabolites between the cytosol and the glycosome was assumed to be regulated by specific transporters in the glycosomal membrane. This tight compartmentalization was considered to be essential for parasite viability. Recently, size-specific metabolite pores were discovered in the membrane of glycosomes. These channels are proposed to allow smaller metabolites to diffuse across the membrane but not larger ones. In light of this new finding, we re-analyzed the model taking into account uncertainty about the topology of the metabolic system in T. brucei, as well as uncertainty about the values of all parameters of individual enzymatic reactions. Our analysis shows that these newly-discovered nonspecific pores are not necessarily incompatible with our current knowledge of the glycosomal metabolic system, provided that the known cytosolic activities of the glycosomal enzymes play an important role in the regulation of glycolytic fluxes and the concentration of metabolic intermediates of the pathway.

Microwave irradiation decreases ATP, increases free [Mg2+], and alters in vivo intracellular reactions in rat brain

Rapid inactivation of metabolism is essential for accurately determining the concentrations of metabolic intermediates in the in vivo state. We compared a broad spectrum of energetic intermediate metabolites and neurotransmitters in brains obtained by microwave irradiation to those obtained by freeze blowing, the most rapid method of extracting and freezing rat brain. The concentrations of many intermediates, cytosolic free NAD(P)(+) /NAD(P)H ratios, as well as neurotransmitters were not affected by the microwave procedure. However, the brain concentrations of ATP were about 30% lower, whereas those of ADP, AMP, and GDP were higher in the microwave-irradiated compared with the freeze-blown brains. In addition, the hydrolysis of approximately 1 μmol/g of ATP, a major in vivo Mg(2+) -binding site, was related to approximately five-fold increase in free [Mg(2+) ] (0.53 ± 0.07 mM in freeze blown vs. 2.91 mM ± 0.48 mM in microwaved brains), as determined from the ratio [citrate]/[isocitrate]. Consequently, many intracellular properties, such as the phosphorylation potential and the ∆G' of ATP hydrolysis were significantly altered in microwaved tissue. The determinations of some glycolytic and TCA cycle metabolites, the phosphorylation potential, and the ∆G' of ATP hydrolysis do not represent the in vivo state when using microwave-fixed brain tissue.

Structure and Function of Assemblages of Bacteria and Archaea in Model Anaerobic Aquifer Columns: Can Functional Instability Be Practically Beneficial?

Biodegradable organic carbon is often added to aquifers to stimulate microbial reduction of oxidized contaminants. This carbon also stimulates fermenters, which generate important metabolites that can fuel contaminant reduction and may enhance dissolution of hydrophobic compounds. Therefore, understanding how different methods of carbon addition affect the fermentative community will enable design of more effective remediation strategies. Our research objective was to evaluate the microbial communities that developed in model aquifer columns in response to pulsed or continuous molasses input. Results indicated that the continuously fed column produced relatively low concentrations of metabolic intermediates and had a greater proportion of Bacteria and methanogens, as measured by quantitative polymerase chain reaction, near the column inlet. In contrast, the pulsed-fed column generated periodic high concentrations of metabolic intermediates, with Bacteria and methanogens distributed throughout the length of the column. The community structures of Bacteria and Archaea, measured via automated ribosomal intergenic spacer analysis, in the pulsed-fed column were significantly different from those in the control column (not fed). The microbial community composition of the continuously fed column, however, became increasingly similar to the control column along the column length. These results demonstrate that a strategy of pulsed carbon addition leads to activity that is associated with functional instability, in terms of the production of periodic pulses of fermentation products and changing carbon concentration, and may be advantageous for remediation by producing large quantities of beneficial intermediates and resulting in more homogenously distributed biomass.

I-1. Changes of metabolic intermediate concentrations and enzymatic activities in muscle and hepatopancreas of red sea bream Pagrus major and mackerel Scomber australasicus in response to fishery stresses

I-1. Changes of metabolic intermediate concentrations and enzymatic activities in muscle and hepatopancreas of red sea bream Pagrus major and mackerel Scomber australasicus in response to fishery stresses

Effect of Caloric Restriction with and without Exercise on Metabolic Intermediates in Nonobese Men and Women

The objective of the study was to evaluate whether serum concentrations of metabolic intermediates are related to adiposity and insulin sensitivity (Si) in overweight healthy subjects and compare changes in metabolic intermediates with similar weight loss achieved by diet only or diet plus exercise. This was a randomized controlled trial. The cross-sectional study included 46 (aged 36.8 ± 1.0 yr) overweight (body mass index 27.8 ± 0.7 kg/m(2)) subjects enrolled in a 6-month study of calorie restriction. To determine the effect of diet only or diet plus exercise on metabolic intermediates, 35 subjects were randomized to control (energy intake at 100% of energy requirements); CR (25% calorie restriction), or CR+EX: (12.5% CR plus 12.5% increase in energy expenditure by exercise). Serum concentrations of eight fatty acids, 15 amino acids, and 45 acylcarnitines (ACs) measured by targeted mass spectrometry. In overweight subjects, the concentrations of C2 AC and long-chain ACs were positively associated with percent fat (R(2) = 0.75, P = 0.0001) and Si (R(2) = 0.12, P = 0.05). The percent fat (R(2) = 0.77, P < 0.0001), abdominal visceral fat (R(2) = 0.64, P < 0.0001), and intrahepatic fat (R(2) = 0.30, P = 0.0002) were positively associated with fatty acid concentrations. There was a significant increase in an AC factor (comprised of C2 and several medium chain ACs) in the CR group (P = 0.01). In nonobese subjects, fasted serum ACs are associated with Si and fat mass. Despite similar weight loss, serum ACs increase with CR alone but not CR+EX. A greater improvement in Si with weight loss during CR+EX interventions may be related to improved coupling of β-oxidation and tricarboxylic acid cycle flux induced by exercise.

Exercise-Induced Changes in Metabolic Intermediates, Hormones, and Inflammatory Markers Associated With Improvements in Insulin Sensitivity

OBJECTIVETo understand relationships between exercise training-mediated improvements in insulin sensitivity (SI) and changes in circulating concentrations of metabolic intermediates, hormones, and inflammatory mediators.RESEARCH DESIGN AND METHODSTargeted mass spectrometry and enzyme-linked immunosorbent assays were used to quantify metabolic intermediates, hormones, and inflammatory markers at baseline, after 6 months of exercise training, and 2 weeks after exercise training cessation (n = 53). A principal components analysis (PCA) strategy was used to relate changes in these intermediates to changes in SI.RESULTSPCA reduced the number of intermediates from 90 to 24 factors composed of biologically related components. With exercise training, improvements in SI were associated with reductions in by-products of fatty acid oxidation and increases in glycine and proline (P < 0.05, R2 = 0.59); these relationships were retained 15 days after cessation of exercise training (P < 0.05, R2 = 0.34).CONCLUSIONSThese observations support prior observations in animal models that exercise training promotes more efficient mitochondrial β-oxidation and challenges current hypotheses regarding exercise training and glycine metabolism.

In silico modeling and metabolome analysis of long-stored erythrocytes to improve blood storage methods

There is currently no effective method for preventing ATP and 2,3-bisphosphoglycerate (2,3-BPG) depletion during long-term erythrocyte storage in the cold, although these metabolites are strongly associated with cell viability and oxygen delivery after transfusion. Metabolite reduction is caused by whole metabolic networks in the cell, which are regulated by various physical or chemical factors. Mathematical modeling is a powerful tool for integrating such complex and dynamic systems. Here, we developed a mathematical model to predict metabolism in erythrocytes preserved with a mannitol-adenine-phosphate solution (MAP) at 4 °C, by modifying a published model of large-scale erythrocyte metabolism. Our model successfully reproduced the reported decreases in ATP and 2,3-BPG during storage. Analysis of our model identified several enzymatic reactions and factors related to ATP and 2,3-BPG depletions, which may serve as possible targets for improving blood storage methods. We also performed metabolome analysis of laboratory-made MAP-stored erythrocytes using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which provided a comprehensive view of the metabolism dynamics. Alterations in the metabolic intermediate concentrations after long storage were qualitatively predicted by the model. Finally, through further systematic analysis, we also discuss the usability of our model.

Enhancing Production of Adapted Bacteria To Degrade Chlorinated Aromatics

The study's aim was to enhance the production of adapted bacteria for degrading chlorobenzene (CB) and o-dichlorobenzene (DCB). Batch cultures fed by pulses of substrate (SPB, substrate pulse batch process) were successfully performed in a 12-L bioreactor: 240 mg L-1 h-1 of CB/DCB mixture was eliminated, and 50 mg L-1 h-1 of dry weight was produced. Studying the substrate removal dynamics, the concentration of metabolic intermediates and chlorides, and the cellular integrity allowed the correct proceeding of bacterial cultivation. The substrate loading rate was optimized to obtain the highest biomass productivity with the lowest toxicity and inhibiting effects. The time interval between each pulse was decreased gradually so that a continuous substrate feeding was finally attained. In the continuous feeding technique, both cell productivity and CB/DCB elimination capacity were twice the values obtained with the SPB. Results come up to industrial prospects and present the continuous feeding technique as an attractive adaptation of SPB to degrade chlorinated solvents.

Metabolic design principles: Chemical and physical determinants of cell chemistry

SummaryBiochemical defense can be viewed, not only as mechanisms that deal with toxicmolecules and with the consequences of damage to macromolecules but, also, to a setof design principles that ensures that the occurrence of unwanted outcomes isminimized, or even eliminated. Such design principles include those responsible forthe conservation of solvent capacity, the maintenance of constant osmotic pressure,and the avoidance of unwanted reactions and of unnecessary increases in body mass.Unwanted reactions are minimized by means of very low concentrations ofmetabolic intermediates as well as by the avoidance of highly reactive intermediates,such as aldehydes. This may have favored the selection of glucose as the principalmonosaccharide in animals as it has the lowest proportion of straight-chain structure(and, therefore, of free aldehyde groups) of all the aldohexoses. Glycogen may havebeen selected as the major storage form of carbohydrate because (i) its a-1,4-glycosidic linkages ensure that it has no free aldehyde groups, and (ii) its polymericnature exerts almost no osmotic pressure. The conservation of solvent capacity isfacilitated by the use of activated intermediates that drive reactions at very lowconcentrations of intermediates. The avoidance of unnecessary increases in mass isfacilitated by the utilization of fat, rather than carbohydrate, as our principal caloricstore.AcknowledgmentsThe author’s work is supported by grants from the Canadian Institutes for HealthResearch and by the Canadian Diabetes Association. JTB is a Senior Investigator ofthe Canadian Institutes for Health Research.References

Response of enzyme activities and metabolic intermediate concentrations to a long burst of exercise and following resting in muscle and the hepatopancreas of carp

ABSTRACT: The responses of enzyme activities and metabolic intermediate concentrations to a long burst of exercise and following resting were examined in muscle and the hepatopancreas of carp Cyprinus carpio. A 15 min burst of exercise made the fish so exhausted that they could not swim any more. In muscle, the exercise decreased glycogen content significantly and increased lactate content significantly, resulting in a lowered pH. Furthermore, the burst of exercise decreased phosphofructokinase (PFK) activity significantly, although it changed adenosine-5′-monophosphate, adenosine-5′-diphosphate, adenosine-5′-triphosphate, fructose-6-phosphate and citrate concentrations within ranges that could activate PFK. In the hepatopancreas, the exercise increased glucose-6-phosphatase and fructose-1,6-bis-phosphatase activities, and glucose and lactate concentrations in the serum, and it decreased glycogen content. Even at 3 h resting after the burst of exercise, the fish had not completely restored many parameters. These results suggest that although the fish tried to enhance in vivo muscular glycolysis through the activation of PFK by changes in metabolic intermediate concentrations, glycolysis seemed to decrease markedly through the inhibition of PFK as a result of the lowered pH. However, the hepatopancreas made a contribution to muscular exercise through glucose supplementation by enhanced gluconeogenesis and glycogenolysis, indicating the presence of an interdependence of carbohydrate metabolism between muscle and the hepatopancreas in the fish.

Response of enzyme activities and metabolic intermediate concentrations to epinephrine administration in hepatopancreas and muscle of carp

SUMMARY: An experiment was carried out to investigate the effect of an intraperitoneal glucagon administration (60 μg/kg) on enzyme activities and metabolic intermediate concentrations in the hepatopancreas and muscle of common carp Cyprinus carpio. In the hepatopancreas, glycogen phosphorylase a activity together with cyclic adenosine-5′-monophosphate (AMP) concentration were significantly increased, and glycogen content was significantly decreased, at 1 h after the glucagon administration. Additionally, glucose-6-phosphatase and fructose-1,6-bisphosphatase activities as well as plasma glucose and free amino acid concentrations were increased after its administration. In the muscle, glucose and glycogen contents increased after the administration. Furthermore, the hormone administration also increased phosphofructokinase activity together with fructose-6-phosphate, AMP, and adenosine-5′-diphosphate concentrations and decreased adenosine-5′-triphosphate concentration. From 3 h after the administration, many parameter concentrations in both tissues showed a tendency to recover to the initial levels. These results suggest that glucagon administration enhanced glycogenolysis and gluconeogenesis in the hepatopancreas, and as a consequence, glucose was released into the bloodstream. The blood glucose seems to be metabolized through enhanced glycolysis and glycogenesis in the muscle.