Increased glycolysis as protective adaptation of energy depleted,degenerating human hibernating myocardium

In the current study on human hibernating myocardium (HHM), we tested the hypothesis that increased glycolysis might exert a positive effect during a supply-demand balance situation by augmentation of myocardial energy formation. In 14 patients HHM was preoperatively detected by clinical methods and validated by the recovery of contractile function three months following revascularization. During open-heart surgery, transmural biopsies were removed from the hibernating areas and analyzed using biochemical and morphologic methods. Metabolite contents were normalized for the degree of fibrosis (control: 9.8 ± 0.5%, HHM 28.1 ± 3.0%; p < 0.05), providing values for cardiomyocytes only. In energy depleted HHM, severe intracellular degeneration, glycogen accumulation and myocyte loss were found. Elevated lactate levels (2.22 ± 0.26 vs. 25.38 ± 3.53 mol/wet wt, p < 0.001) were indicative of an increased anaerobic glycolytic flux. In conclusion the presence of abundant intracellular glycogen and an increased anaerobic glycolysis in HHM is indicative of a protective adaptation of this myocardium, which might balance energy deficit and may limit structural damage.     

A self-perpetuating vicious cycle of tissue damage in human hibernating myocardium

Recently, we proposed the hypothesis that a vicious cycle exists in human hibernating myocardium (HM) between the progression of myocyte degeneration and the development of fibrosis [1]. We now investigated the pathomechanism of this cycle in more detail and established a correlation between the severity of the morphological changes and the degree of postoperative functional recovery of HM.HM was diagnosed by dobutamine echocardiography, thallium-201 scintigraphy and radionuclide ventriculography. Functional recovery was present at 3 months after coronary bypass surgery but remained unchanged at 15 months. Forty patients were subdivided into 2 groups: A with complete and B with incomplete recovery. Biopsies taken during surgery and studied by electron microscopy, immunocytochemistry, rt-PCR, and morphometry revealed myocyte degeneration and inflammatory and fibrinogenic changes in a widened interstitial space. We report here for the first time an upregulation of TGF-₁ evident by a 5-fold increase of fibroblasts and macrophages exhibiting a TGF-₁ content 3-fold larger than in control, and a > 3-fold increase in TGF-₁ mRNA by rt-PCR. The number of angiotensin converting enzyme (ACE) containing structures was increased (n/mm²: control - 11.4, A - 17.6, B - 19.2, control vs. A and B, p < 0.05). Fibrosis was more severe in group B than A or control (%: C - 10.1; A - 21.2; B - 40.6; p < 0.05). Capillary density was significantly reduced (n/mm²: C - 1152; A - 782; B - 579, p < 0.05) and intercapillary distance was widened (m: C - 29.5, A - 36.1, B - 43.3, p < 0.05). The number of CD 3 (n/mm²: C - 5.0; A - 9.6; B - 9.4, ns) and CD 68 positive cells (n/mm²: C - 37.2; A - 80.7; B - 55.0, C vs. A p < 0.05) was elevated in HM as compared to control indicating an inflammatory reaction. Cut-off points for functional recovery are fibrosis > 32%, capillary density < 660/mm² and intercapillary distance > 39.0 m.In HM a self-perpetuating vicious cycle of tissue alterations leads to progressive replacement fibrosis and continuous intracellular degeneration which should be interrupted by early revascularization.     

Metabolic flux analysis in biotechnology processes

Metabolic flux analysis (MFA) has become a fundamental tool of metabolic engineering to elucidate the metabolic state of the cell and has been applied to various biotechnological processes. In recent years, considerable technical advances have been made. Developments of analytical instruments allow us to determine ¹³C labeling distribution of intracellular metabolites with high accuracy and sensitivity. Moreover, kinetic information of intracellular label distribution during isotopic instationary enables us to calculate metabolic fluxes with shortened experimental time and decreased amount of labeled substrate. The ¹³C MFA may be one of the most promising approaches for the target estimation to improve strain performances and production processes.     

Light-inducible flux control of triosephosphate isomerase on glycolysis in Escherichia coli

An engineering tool for controlling flux distribution on metabolic pathways to an appropriate state is highly desirable in bioproduction. An optogenetic switch, which regulates gene expression by light illumination is an attractive on/off switchable system, and is a promising way for flux control with an external stimulus. We demonstrated a light-inducible flux control between glycolysis and the methylglyoxal (MGO) pathway in Escherichia coli using a CcaS/CcaR system. CcaR is phosphorylated by green light and is dephosphorylated by red light. Phosphorylated CcaR induces gene expression under the cpcG2 promoter. The tpiA gene was expressed under the cpcG2 promoter in a genomic tpiA deletion strain. The strain was then cultured with glucose minimum medium under green or red light. We found that tpiA messenger RNA level under green light was four times higher than that under red light. The repression of tpiA expression led to a decrease in glycolytic flux, resulting in slower growth under red light (0.25 hr ⁻¹) when compared to green light (0.37 hr ⁻¹). The maximum extracellular MGO concentration under red light (0.2 mM) was higher than that under green light (0.05 mM). These phenotypes confirm that the MGO pathway flux was enhanced under red light.     

Metabolic flux analysis in mammalian cell culture

Mammalian cell culture metabolism is characterized by glucoglutaminolysis, that is, high glucose and glutamine uptake combined with a high rate of lactate and non-essential amino acid secretion. Stress associated with acid neutralization and ammonia accumulation necessitates complex feeding schemes and limits cell densities achieved in fed-batch culture. Conventional and constraint-based metabolic flux analysis has been successfully used to study the metabolic phenotype of mammalian cells in culture, while ¹³C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale ¹³C metabolic flux analysis, which is required to improve confidence in the network models and their predictions, remains a major challenge. Advances in both modeling and analytical techniques are bringing this challenge within sight.     

Schemes of flux control in a model of Saccharomyces cerevisiae glycolysis.

We used parameter scanning to emulate changes to the limiting rate for steps in a fitted model of glucose-derepressed yeast glycolysis. Three flux-control regimes were observed, two of which were under the dominant control of hexose transport, in accordance with various experimental studies and other model predictions. A third control regime in which phosphofructokinase exerted dominant glycolytic flux control was also found, but it appeared to be physiologically unreachable by this model, and all realistically obtainable flux control regimes featured hexose transport as a step involving high flux control.     

Metabolic flux control analysis of branch points: an improved approach to obtain flux control coefficients from large perturbation data

An overview of published approaches for the metabolic flux control analysis of branch points revealed that often not all fundamental constraints on the flux control coefficients have been taken into account. This has led to contradictory statements in literature on the minimum number of large perturbation experiments required to estimate the complete set of flux control coefficients C(J) for a metabolic branch point. An improved calculation procedure, based on approximate Lin-log reaction kinetics, is proposed, providing explicit analytical solutions of steady state fluxes and metabolite concentrations as a function of large changes in enzyme levels. The obtained solutions allow direct calculation of elasticity ratios from experimental data and subsequently all C(J)-values from the unique relation between elasticity ratio's and flux control coefficients. This procedure ensures that the obtained C(J)-values satisfy all fundamental constraints. From these it follows that for a three enzyme branch point only one characterised or two uncharacterised large flux perturbations are sufficient to obtain all C(J)- values. The improved calculation procedure is illustrated with four experimental cases.     

Metabolic flux analysis of postburn hepatic hypermetabolism

The hepatic response to severe injury is characterized by a marked upregulation of glucose, fatty acid, and amino acid turnover, which, if persistent, predisposes the patient to progressive organ dysfunction. To study the effect of injury on liver intermediary metabolism, metabolic flux analysis was applied to isolated perfused livers of burned and sham-burned rats. Intracellular fluxes were calculated using metabolite measurements and a stoichiometric balance model. Significant flux increases were found for multiple pathways, including mitochondrial electron transport, the TCA and urea cycles, gluconeogenesis, and pentose phosphate pathway (PPP). The burn-induced increase in gluconeogenesis did not significantly increase glucose output. Instead, glucose-6-phosphate was diverted into the PPP. These changes were paralleled by increases in glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activities. Given that G6PDH and GR are the most significant NADPH producers and consumers in the liver, respectively, and that GR is responsible for recycling the free radical scavenger glutathione, these data are consistent with the notion that hepatic metabolic changes are in part due to the induction of liver antioxidant defenses.     

Metabolic flux analysis of carbon balance in Lactobacillus strains

Metabolic flux analyses were performed based on the carbon balance of six different Lactobacillus strains used in this study. Results confirmed that L. delbrueckii, L. plantarum ATCC 21028, L. plantarum NCIMB 8826 ΔldhL1, L. plantarum NCIMB 8826 ΔldhL1‐pCU‐PxylAB, and L. plantarum NCIMB 8826 ΔldhL1‐pLEM415‐xylAB metabolized glucose via EMP: whereas, L. brevis metabolized glucose via PK pathway. Xylose was metabolized through the PK pathway in L. brevis, L. plantarum NCIMB 8826 ΔldhL1‐pCU‐PxylAB and L. plantarum NCIMB 8826 ΔldhL1‐pLEM415‐xylAB. Operation of both EMP and PK pathways was found in L. brevis, L. plantarum NCIMB 8826 ΔldhL1‐pCU‐PxylAB, and L. plantarum NCIMB 8826 ΔldhL1‐pLEM415‐xylAB when glucose plus xylose were used as carbon source. The information of detailed carbon flow may help the strain and biomass selection in a designed process of lactic acid biosynthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1397–1403, 2016     

Ecological control analysis: being(s) in control of mass flux and metabolite concentrations in anaerobic degradation processes

Identification of the functional groups of microorganisms that are predominantly in control of fluxes through, and concentrations in, microbial networks would benefit microbial ecology and environmental biotechnology: the properties of those controlling microorganisms could be studied or monitored specifically or their activity could be modulated in attempts to manipulate the behaviour of such networks. Herein we present ecological control analysis (ECA) as a versatile mathematical framework that allows for the quantification of the control of each functional group in a microbial network on its process rates and concentrations of intermediates. In contrast to current views, we show that rates of flow of matter are not always limited by a single functional group; rather flux control can be distributed over several groups. Also, control over intermediate concentrations is always shared. Because of indirect interactions, through other functional groups, the concentration of an intermediate can also be controlled by functional groups not producing or consuming it. Ecological control analysis is illustrated by a case study on the anaerobic degradation of organic matter, using experimental data obtained from the literature. During anaerobic degradation, fermenting microorganisms interact with terminal electron-accepting microorganisms (e.g. halorespirers, methanogens). The analysis indicates that flux control mainly resides with fermenting microorganisms, but can shift to the terminal electron-accepting microorganisms under less favourable redox conditions. Paradoxically, halorespiring microorganisms do not control the rate of perchloroethylene and trichloroethylene degradation even though they catalyse those processes themselves.     

Long-term preservation of myocardial energetic in chronic hibernating myocardium

We previously reported that the myocardial energetic state, as defined by the ratio of phosphocreatine to ATP (PCr/ATP), was preserved at baseline (BL) in a swine model of chronic myocardial ischemia with mild reduction of myocardial blood flow (MBF) 10 wk after the placement of an external constrictor on the left anterior descending coronary artery. It remains to be seen whether this stable energetic state is maintained at a longer-term follow-up. Hibernating myocardium (HB) was created in minipigs (n = 7) by the placement of an external constrictor (1.25 mm internal diameter) on the left anterior descending coronary artery. Function was assessed with MRI at regular intervals until 6 mo. At 6 mo, myocardial energetic in the HB was assessed by (31)P-magnetic resonance spectrometry and myocardial oxygenation was examined from the deoxymyoglobin signal using (1)H-magnetic resonance spectrometry during BL, coronary vasodilation with adenosine, and high cardiac workload with dopamine and dobutamine (DpDb). MBF was measured with radiolabeled microspheres. At BL, systolic thickening fraction was significantly lower in the HB compared with remote region (34.4 ± 9.4 vs. 50.1 ± 10.7, P = 0.006). This was associated with a decreased MBF in the HB compared with the remote region (0.73 ± 0.08 vs. 0.97 ± 0.07 ml · min(-1) · g, P = 0.03). The HB PCr/ATP at BL was normal. DpDb resulted in a significant increase in rate pressure product, which caused a twofold increase in MBF in the HB and a threefold increase in the remote region. The systolic thickening fraction increased with DpDb, which was significantly higher in the remote region than HB (P < 0.05). The high cardiac workload was associated with a significant reduction in the HB PCr/ATP (P < 0.02), but this response was similar to normal myocardium. Thus HB has stable BL myocardial energetic despite the reduction MBF and regional left ventricular function. More importantly, HB has a reduced contractile reserve but has a similar energetic response to high cardiac workload like normal myocardium.     

Metabolic flux analysis and metabolic engineering of microorganisms

Recent advances in metabolic flux analysis including genome-scale constraints-based flux analysis and its applications in metabolic engineering are reviewed. Various computational aspects of constraints-based flux analysis including genome-scale stoichiometric models, additional constraints used for the improved accuracy, and several algorithms for identifying the target genes to be manipulated are described. Also, some of the successful applications of metabolic flux analysis in metabolic engineering are reviewed. Finally, we discuss the limitations that need to be overcome to make the results of genome-scale flux analysis more realistically represent the real cell metabolism.     

Metabolic flux analysis in plants: coping with complexity

Theory and experience in metabolic engineering both show that metabolism operates at the network level. In plants, this complexity is compounded by a high degree of compartmentation and the synthesis of a very wide array of secondary metabolic products. A further challenge to understanding and predicting plant metabolic function is posed by our ignorance about the structure of metabolic networks even in well-studied systems. Metabolic flux analysis (MFA) provides tools to measure and model the functioning of metabolism, and is making significant contributions to coping with their complexity.
This review gives an overview of different MFA approaches, the measurements required to implement them and the information they yield. The application of MFA methods to plant systems is then illustrated by several examples from the recent literature. Next, the challenges that plant metabolism poses for MFA are discussed together with ways that these can be addressed. Lastly, new developments in MFA are described that can be expected to improve the range and reliability of plant MFA in the coming years.     

Development of bifunctional biosensors for sensing and dynamic control of glycolysis flux in metabolic engineering

Glycolysis is the primary metabolic pathway in all living organisms. Maintaining the balance of glycolysis flux and biosynthetic pathways is the crucial matter involved in the microbial cell factory. Few regulation systems can address the issue of metabolic flux imbalance in glycolysis. Here, we designed and constructed a bifunctional glycolysis flux biosensor that can dynamically regulate glycolysis flux for overproduction of desired biochemicals. A series of positive-and negative-response biosensors were created and modified for varied thresholds and dynamic ranges. These engineered glycolysis flux biosensors were verified to be able to characterize in vivo fructose-1,6-diphosphate concentration. Subsequently, the biosensors were applied for fine-tuning glycolysis flux to effectively balance the biosynthesis of two chemicals: mevalonate and N-acetylglucosamine. A glycolysis flux-dynamically controlled Escherichia coli strain achieved a 111.3 g/L mevalonate titer in a 1L fermenter.     

Metabolic reconstruction and flux analysis of industrial Pichia yeasts

Pichia yeasts have been recognized as important microbial cell factories in the biotechnological industry. Notably, the Pichia pastoris and Pichia stipitis species have attracted much research interest due to their unique cellular physiology and metabolic capability: P. pastoris has the ability to utilize methanol for cell growth and recombinant protein production, while P. stipitis is capable of assimilating xylose to produce ethanol under oxygen-limited conditions. To harness these characteristics for biotechnological applications, it is highly required to characterize their metabolic behavior. Recently, following the genome sequencing of these two Pichia species, genome-scale metabolic networks have been reconstructed to model the yeasts’ metabolism from a systems perspective. To date, there are three genome-scale models available for each of P. pastoris and P. stipitis. In this mini-review, we provide an overview of the models, discuss certain limitations of previous studies, and propose potential future works that can be conducted to better understand and engineer Pichia yeasts for industrial applications.     

Metabolic flux network and analysis of fermentative hydrogen production

Fermentative hydrogen production (FHP) has received a great R & D interest in recent decades, as it offers a potential means of producing H₂ from a variety of renewable resources, even wastewater via a low energy continuous process. Various extracellular metabolites including ethanol, acetate, butyrate and lactate can be produced during the fermentation, building a complex metabolic network of the FHP. Except for the recognition of its complexity, the metabolic flux network has not been well understood. Studies on biochemical reactions and metabolic flux network associated with the FHP in anaerobic fermentation system have only been drawn attention in recent years. This review summarizes the biochemical reactions taking place in the metabolic network of FHP. We discuss how the key operation factors influence metabolism in the FHP process. Recently developed and applied technologies for metabolic flux analysis have been described. Future studies on the metabolic network to enhance fermentative hydrogen production by strict anaerobes are recommended. It is expected that this review can provide useful information in terms of fundamental knowledge and update technology for scientists and research engineers in the field of biological hydrogen production.     

Metabolic flux analysis of cultured hepatocytes exposed to plasma

Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.     

Metabolic flux analysis in Streptomyces coelicolor under various nutrient limitations

Metabolic flux analysis was applied to Streptomyces coelicolor continuous culture data obtained under nitrogen, phosphate, sulfate, and potassium limitations. The metabolic reaction network involved more than 200 reactions describing the major pathways as well as the secondary metabolism for the production of actinorhodin and excretion of certain metabolites. Linear programming was used for the optimization of specific growth rates and energy requirements. Two types of specific growth rates, stoichiometric and theoretical, were defined, maximized, and compared in order to investigate the microbial potential. Potassium limitation led to the largest and nitrogen limitation to the smallest difference between the stoichiometric and theoretical specific growth rates. Although the value of the maximum theoretical specific growth rate was close to that of the experimental specific growth rate with potassium limitation, this difference was the largest in the case of nitrogen limitation. Energy requirements during different nutrient limitations were also investigated. The model indicated that although the highest actinorhodin production rate was with nitrogen limitation, this was accompanied with the undesired excretion of certain metabolites.