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Important concepts from colloidal physical chemistry such as coacervation, phase transitions, emergent properties and ionic association, are currently emerging in the lexicon of cellular biology, prompted mostly by recent experimental observations of liquid phase coexistence in the cell cytosol. Nevertheless, from an historical point of view, the application of these concepts in cell biology is not new. They were key concepts into the so-called protoplasmic doctrine, an alternative (and largely forgotten) approach to cell physiology. The most complete theory originating from this line of thinking was the Association-Induction Hypothesis (AIH), introduced by Gilbert N. Ling in 1962. The AIH, which envisions living cells as complex dynamical colloidal systems, provides ample theory and experimental evidence to call into question the now dominant view of living cells as fluid-filled vesicles. This review attempts to present and discuss the usefulness of the AIH to understand a series of experimental observations from our laboratory from living suspensions of the yeast Saccharomyces cerevisiae exhibiting glycolytic oscillations. Particularly, the AIH helped us integrate, in a mechanistic sense, the basis of a strong temporal coupling observed between ATP and a series of cellular properties such as intracellular water dipolar relaxation, intracellular K + concentration, among many others, where the colloidal physical chemistry of the cell interior plays a fundamental role., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

My graduate and postdoctoral training in metabolism and enzymology eventually led me to study the short- and long-term regulation of glucose and lipid metabolism. In the early phase of my career, my trainees and I identified, purified, and characterized a variety of phosphofructokinase enzymes from mammalian tissues. These studies led us to discover fructose 2,6-P 2 , the most potent activator of phosphofructokinase and glycolysis. The discovery of fructose 2,6-P 2 led to the identification and characterization of the tissue-specific bifunctional enzyme 6-phosphofructo-2-kinase:fructose 2,6-bisphosphatase. We discovered a glucose signaling mechanism by which the liver maintains glucose homeostasis by regulating the activities of this bifunctional enzyme. With a rise in glucose, a signaling metabolite, xylulose 5-phosphate, triggers rapid activation of a specific protein phosphatase (PP2ABδC), which dephosphorylates the bifunctional enzyme, thereby increasing fructose 2,6-P 2 levels and upregulating glycolysis. These endeavors paved the way for us to initiate the later phase of my career in which we discovered a new transcription factor termed the carbohydrate response element binding protein (ChREBP). Now ChREBP is recognized as the masterregulator controlling conversion of excess carbohydrates to storage of fat in the liver. ChREBP functions as a central metabolic coordinator that responds to nutrients independently of insulin. The ChREBP transcription factor facilitates metabolic adaptation to excess glucose, leading to obesity and its associated diseases.

The goal of this laboratory exercise is to give upper-level undergraduate students an introduction to sterile technique in mammalian cell culture and metabolism. The experiment can be completed within a 3-h lab period and can be performed either in conjunction with other biochemistry/metabolism experiments or used as a stand-alone experiment. In this experiment, students are tasked with relating the acidification of cell culture medium to metabolism in order to elucidate the mechanism of action for a compound. Students can relate their experimental results to topics covered on glycolysis and oxidative phosphorylation in upper-level biochemistry classes as well as gain valuable experience relating metabolism to drug discovery., (© 2020 International Union of Biochemistry and Molecular Biology.)

Zacharias Dische's discovery of feedback inhibition in metabolism was one of the most important in the history of biochemistry. However, his paper was written and published under very difficult circumstances in wartime and passed almost completely unnoticed. It is almost never cited, and the discovery itself is usually attributed to later work of others. Here I provide a discussion of Dische's work, a translation of his paper into English, and a transcription of the original French version, which is almost unobtainable anywhere., (Copyright © 2020. Published by Elsevier B.V.)

In the center stage of this third and last part of the author's treatise about the life and the oeuvre of the German humanist, biochemist, and Nobel Prize Winner Otto MEYERHOF (1884-1951) is the first half of his life. As a young man, in the age of 16 and after a severe renal disease, Otto MEYERHOF spent 1900/1901 five months in Egypt, where he, led by his cousin Max MEYERHOF, Egyptologist and Ophthalmologist, studied the country and its people as well as its architecture and history. During his medical courses he became acquainted with the philosopher Leonard NELSON, who stimulated him to study the Kantian-Friesian philosophy, about which he published several papers. Simultaneously MEYERHOF became seriously interested in GOETHE'S method of natural investigation. About this topic he gave two famous lectures, 1909 in Berlin and 1949 in his American exile in New York. From 1903 MEYERHOF attended medical courses and conducted advanced training courses for workers at the University of Berlin. 1907 in the Heidelberg philosophical circle MEYERHOF became acquainted with Otto WARBURG. 1909 MEYERHOF earned a doctorate in medicine with the thesis "Contributions to a psychological theory of mental disorders". In the years 1911/12 a deep change occurred in MEYERHOF'S scientific career, in the course of which he turned to active experimental research in natural science. However, MEYERHOF remained true to philosophy and his friendship to Leonard NELSON. When Otto MEYERHOF in 1911 obtained a Badian scholarship for experimental research work in the Zoological Station in Naples, a tight collaboration with Otto WARBURG in sea urchin egg biology began. In 1912 MEYERHOF published a famous paper on "The energetics of cellular processes", which became the guideline for his prospective scientific work. In the same year he habilitated at the Medical School of the Kiel University with an enzymological study, and he joined the Physiological Institute of Kiel as unpaid private lecturer. Because of a severe chronic renal disease, which burdened himself from childhood, he became exempted from military service. In the years after 1917 he published several papers on fermentation, glycolysis, and respiration of animal cells and yeast and started after 1918 an extensive experimental project on "Muscle Metabolism and Mechanical Work". In this study he brought together different aspects of muscle metabolism and muscle activity: aerobiosis and anaerobiosis, muscular work, muscular exhaustion, and muscular recovery with glycogen degradation, glycogen synthesis as well as lactic acid formation and lactic acid utilization with muscular oxygen uptake. With this comprehensive experimental approach MEYERHOF in only few years built up a grandiose work about the correlations between muscle metabolism and muscular work. For this brilliant research Otto MEYERHOF and his British colleague Sir Archibald Vivian HILL received the Nobel Prize 1922 for Physiology or Medicine. The two investigators received the honor for their discoveries in the coordination of muscular performance with chemical, physical and thermodynamic processes, MEYERHOF "for his discovery of the fixed relationship between oxygen consumption and lactic acid metabolism in muscle" and HILL "for his research into the quantitative relations between heat production and muscular work". As explicated in the two preceding papers of the author Otto MEYERHOF and his first and longest collaborator Karl LOHMANN from 1925 till 1938 clarified chemically most of the intermediates and enzymatic reactions of the glycolytic pathway, also named Embden-Meyerhof-Pamas-pathway. Because of the antijewish pogrome in Germany MEYERHOF escaped 1938 from Heidelberg and accepted a French offer to continue his research in Paris. But after the German troops occupied France MEYERHOF again had to flee. He, his wife and their youngest son Walter breached through France, Spain to Portugal. From Lisbon he arrived by ship USA.

Enolase (EC 4.2.1.11) is a cytosolic metalloenzyme responsible for the conversion of 2-phosphoglycerate into phosphoenolpyruvate, the second to last step in glycolysis. In mammals, enolase is encoded by three homologous genes. These gene products not only possess distinct biochemical and immunological properties but also show different tissue distribution. Besides its glycolytic function, α-enolase plays a variety of roles in pathophysiological settings including oncogenesis, tumor progression, ischemia, and bacterial infection. The expression levels of α-enolase have been attributed diagnostic and prognostic value in a number of tumors. Furthermore, neuron-specific α-enolase is released into the cerebrospinal fluid as well as in the systemic circulation upon traumatic brain injury and ischemic episodes. Thus, the measurement of the enzymatic activity of enolase is relevant for diverse fields of investigation, including oncometabolism. Here, we described simple and rapid protocols to measure the activity of enolase in lysates from mammalian cells and tissues., (© 2014 Elsevier Inc. All rights reserved.)

ATP is commonly considered as the main energy unit of the cell and participates in a variety of cellular processes. Thus, intracellular ATP concentrations rapidly vary in response to a wide variety of stimuli, including nutrients, hormones, cytotoxic agents, and hypoxia. Such alterations not necessarily affect cytosolic and mitochondrial ATP to similar extents. From an oncological perspective, this is particularly relevant in the course of tumor progression as well as in the response of cancer cells to therapy. In normal cells, mitochondrial oxidative phosphorylation (OXPHOS) is the predominant source of ATP. Conversely, many cancer cells exhibit an increased flux through glycolysis irrespective of oxygen tension. Assessing the relative contribution of glycolysis and OXPHOS to intracellular ATP production is fundamental not only for obtaining further insights into the peculiarities and complexities of oncometabolism but also for developing therapeutic and diagnostic tools. Several techniques have been developed to measure intracellular ATP levels including enzymatic methods based on hexokinase, glucose-6-phosphate dehydrogenase, and firefly luciferase. Here, we summarize conventional methods for measuring intracellular ATP levels and we provide a detailed protocol based on cytosol- and mitochondrion-targeted variants of firefly luciferase to determine the relative contribution of glycolysis and OXPHOS to ATP synthesis., (© 2014 Elsevier Inc. All rights reserved.)

Evidence accumulating over the past decade has linked alterations in bioenergetic metabolism to the pathogenesis of several diseases, including inflammatory conditions and cancer. However, the mutual relationship between the effector functions and the metabolism of immune cells has begun to emerge only recently. Similar to malignant cells, both innate and adaptive immune cells undergo a metabolic reprogramming that is required for effector functions, de facto underlying the elicitation of a robust immune response. These changes allow immune cells not only to rapidly respond to pathogens or (pre)malignant cells but also to adapt to changing microenvironmental conditions. Targeting the metabolic alterations of malignant cells has been the subject of an intense wave of investigation, resulting in the identification of promising therapeutic strategies. Since the inflammatory milieu and the tumor microenvironment are similar, the metabolism of immune cells and its regulation has recently come under renewed interest as a target for immunotherapy. Here, we describe different tools and techniques to study the bioenergetic metabolism of cultured cells, using immune cells as a model. Our methodological approach relies on an extracellular flux analyzer, an instrument that enables the real-time measurement of the two central pathways used by living cells to generate adenosine triphosphate: glycolysis and oxidative phosphorylation. This instrument and similar technological innovations have transformed the study of cellular metabolism, unveiling its profound impact on various immunologic and oncological disorders., (© 2014 Published by Elsevier Inc.)

An increased flux through glycolysis supports the proliferation of cancer cells by providing additional energy in the form of ATP as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Thus, glycolysis and other metabolic pathways that control cell proliferation may represent valuable targets for therapeutic interventions and diagnostic procedures. In this context, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect shifts in glucose catabolism, while determining the activity of rate-limiting glycolytic enzymes can provide insights into points of metabolic regulation. Moreover, metabolomic studies can be used to generate large, integrated datasets to track changes in carbon flux through glycolysis and its collateral anabolic pathways. As discussed here, these approaches can reveal and quantify the metabolic alterations that underlie malignant cell proliferation., (© 2014 Elsevier Inc. All rights reserved.)

Background: Given the complex mechanisms underlying biochemical processes systems biology researchers tend to build ever increasing computational models. However, dealing with complex systems entails a variety of problems, e.g. difficult intuitive understanding, variety of time scales or non-identifiable parameters. Therefore, methods are needed that, at least semi-automatically, help to elucidate how the complexity of a model can be reduced such that important behavior is maintained and the predictive capacity of the model is increased. The results should be easily accessible and interpretable. In the best case such methods may also provide insight into fundamental biochemical mechanisms., Results: We have developed a strategy based on the Computational Singular Perturbation (CSP) method which can be used to perform a "biochemically-driven" model reduction of even large and complex kinetic ODE systems. We provide an implementation of the original CSP algorithm in COPASI (a COmplex PAthway SImulator) and applied the strategy to two example models of different degree of complexity - a simple one-enzyme system and a full-scale model of yeast glycolysis., Conclusion: The results show the usefulness of the method for model simplification purposes as well as for analyzing fundamental biochemical mechanisms. COPASI is freely available at http://www.copasi.org.

The origins of oxidative phosphorylation, initially known as aerobic phosphorylation, grew out of three research areas of muscle metabolism, creatine phosphorylation, aerobic metabolism of lactic acid in muscle, and studies on the nature and role of adenosine triphosphate (ATP). Much of this work centred round the laboratory of Otto Meyerhof, and most of those contributing to the study of aerobic phosphorylation were influenced by that laboratory: particularly Lipmann and also Ochoa. The work of Engelhardt on ATP levels in blood also appears to have been influenced by the studies of Meyerhof's laboratory. However, with the work of Kalckar, influenced by Lipmann, biochemists began to realise the potential importance of the process. This was confirmed and extended by Belitzer and Ochoa and the theoretical contribution of Lipmann. Thus it is not easy to identify a single point that marked the initiation of studies in this field. The early work was based on the use of tissue homogenates and cells but ultimately this approach limited research possibilities. The development of techniques based on mitochondria opened up new possibilities and brought this first phase of the study of oxidative phorphorylation to a close., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

An integral part of any systems biology approach is the modelling and simulation of the respective system under investigation. However, the values of many parameters of the system have often not been determined or are not identifiable due to technical experimental difficulties or other constraints. Sensitivity analysis is often employed to quantify the importance of each of the model's parameters in the behaviour of the system. This approach can also be useful in identifying those parts of the system that are most sensitive with the potential of becoming drug targets. A problem of the commonly used methods of sensitivity analysis is that they constitute local methods meaning that they depend directly on the exact parameter space, which in turn is not known exactly. One way to circumvent this problem is to carry out sensitivity analysis over a wide range of values for all parameters, but this is handicapped by expensive computations when the systems are high dimensional. Another approach is to employ global sensitivity analysis, which in this context is mostly based on random sampling methods. In this paper we present an efficient approach that involves using numerical optimizing methods that search a wide region of parameter space for a given model to determine the maximum and minimum values of its metabolic control coefficients. A relevant example for drug development is presented to demonstrate the strategy using the software COPASI.

Conversion of glucose to pyruvate via reactions homologous to the non-phosphorylated Entner-Doudoroff (non-P ED) pathway could be achieved in the presence of two amino acid catalysts, cysteine and histidine: cystine oxidizes glucose to gluconic acid by the reaction homologous to glucose dehydrogenase and histidine changes gluconic acid to 2-keto-3-deoxy gluconic acid, then to pyruvate by the reaction homologous to gluconic acid dehydratase and 2-keto-3-deoxy gluconate aldolase, respectively. Pyruvate can be converted to acetyl CoA by the reaction with CoA, TPP and FAD in the presence of cysteine and histidine, which resembles pyruvate dehydrogenase reaction. It was found that gluconic acid dehydration alone is non-specific, in contrast to other reactions. The non-P ED pathway is used by some extreme thermophiles in bacteria and archaea, usually thought as the oldest among the contemporary organisms. This study suggests the possible contribution of amino acid to the origin of the glycolytic pathway.

We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

Genome data as well as biochemical studies have indicated that--as a peculiarity within hyperthermophilic Archaea--Thermoproteus tenax uses three different pathways for glucose metabolism, a variant of the reversible EMP (Embden-Meyerhof-Parnas) pathway and two different modifications of the ED (Entner-Doudoroff) pathway, a non-phosphorylative and a semi-phosphorylative version. An overview of the three different pathways is presented and the physiological function of the variants is discussed.

Functional properties of biochemical networks depend on both the network structure and the kinetic parameters. Extensive data on metabolic network topologies have been collected in databases, but much less information is available about the kinetic constants or metabolite concentrations. Depending on the values of these parameters, metabolic fluxes and control coefficients may vary within a wide range. Nevertheless, some of the parameters may have little influence on the observables of interest. We address the question whether, despite uncertainty about kinetic parameters, probabilistic statements can be made about dynamic network features. To this end, we perform a variability analysis of the parameters: assuming that the parameters follow statistical distributions, we compute the resulting distributions of the network properties like metabolic fluxes, concentrations, or control coefficients by Monte Carlo simulation. In this manner, we study systematically the possible distributions arising from typical topologies of biochemical networks such as linear chains, branched networks, and signaling and gene expression cascades. This analysis reveals how much information about dynamic behavior can be drawn from structural knowledge.

It has hitherto not been possible to analyze the control of oscillatory dynamic cellular processes in other than qualitative ways. The control coefficients, used in metabolic control analyses of steady states, cannot be applied directly to dynamic systems. We here illustrate a way out of this limitation that uses Fourier transforms to convert the time domain into the stationary frequency domain, and then analyses the control of limit cycle oscillations. In addition to the already known summation theorems for frequency and amplitude, we reveal summation theorems that apply to the control of average value, waveform, and phase differences of the oscillations. The approach is made fully operational in an analysis of yeast glycolytic oscillations. It follows an experimental approach, sampling from the model output and using discrete Fourier transforms of this data set. It quantifies the control of various aspects of the oscillations by the external glucose concentration and by various internal molecular processes. We show that the control of various oscillatory properties is distributed over the system enzymes in ways that differ among those properties. The models that are described in this paper can be accessed on http://jjj.biochem.sun.ac.za.

Today different database systems for molecular structures (genes and proteins) and metabolic pathways are available. All these systems are characterized by the static data representation. For progress in biotechnology the dynamic representation of this data is important. The metabolism can be characterized as a complex biochemical network. Different models for the quantitative simulation of biochemical networks are discussed, but no useful formalization is available. This paper shows that the theory of Petrinets is useful for the quantitative modeling of biochemical networks.

In prior work we demonstrated the implementation of logic gates, sequential computers (universal Turing machines), and parallel computers by means of the kinetics of chemical reaction mechanisms. In the present article we develop this subject further by first investigating the computational properties of several enzymatic (single and multiple) reaction mechanisms: we show their steady states are analogous to either Boolean or fuzzy logic gates. Nearly perfect digital function is obtained only in the regime in which the enzymes are saturated with their substrates. With these enzymatic gates, we construct combinational chemical networks that execute a given truth-table. The dynamic range of a network's output is strongly affected by "input/output matching" conditions among the internal gate elements. We find a simple mechanism, similar to the interconversion of fructose-6-phosphate between its two bisphosphate forms (fructose-1,6-bisphosphate and fructose-2,6-bisphosphate), that functions analogously to an AND gate. When the simple model is supplanted with one in which the enzyme rate laws are derived from experimental data, the steady state of the mechanism functions as an asymmetric fuzzy aggregation operator with properties akin to a fuzzy AND gate. The qualitative behavior of the mechanism does not change when situated within a large model of glycolysis/gluconeogenesis and the TCA cycle. The mechanism, in this case, switches the pathway's mode from glycolysis to gluconeogenesis in response to chemical signals of low blood glucose (cAMP) and abundant fuel for the TCA cycle (acetyl coenzyme A).

The discovery in 1861 by Louis Pasteur that more yeast is formed aerobically than anaerobically per gram of glucose was the first clue to the difference in efficiency of glycolysis and oxidative phosphorylation. During the first half of the 20th century the pathway of glycolysis was untraveled. Individual enzymes and cofactors were isolated and characterized. A reconstituted system of all enzymes and cofactors catalyzed steady-state glycolysis, provided an appropriate ATPase was added. The need for an ATPase, clearly demonstrated in 1945 by Otto Meyerhof, remains an important aspect of glycolysis that has been sorely neglected by textbooks. The coupling of oxidation and phosphorylation and the formation of the high-energy intermediate 1,3-diphosphoglycerate, discovered by Otto Warburg, are the key reactions of glycolysis. A high-energy intermediate formed during this process was identified as a thiolester. Early concepts of the mechanism of oxidative phosphorylation based on this model led to some frustrating and confusing years of search for high-energy intermediates. Important contributions from the laboratories of Boyer, Cohn, Chance, Green, Lardy, and Lehninger elucidated the properties of the mitochondrial process. Then Peter Mitchell proposed in 1961, 100 years after the publication by Pasteur, that the "high-energy intermediate" is an electrochemical proton gradient generated by the electron transport chain and utilized by a proton turbine (the mitochondrial ATPase complex) to generate ATP. This concept is now widely accepted. Several problems remain to be solved. How are the protons translocated during electron transport? How many protons per site? What is the mechanism of ATP generation during proton flux via the mitochondrial ATPase?

Understanding metabolic energy transformation began with the realization of an 'intrusion' of phosphate into the mechanism of alcoholic fermentation. The discovery of an analogous participation of phosphate in muscle glycolysis connected the metabolic generation of energy-rich phosphate bonds fed into a common transmitter, adenosine triphosphate (ATP), with the production of mechanical energy through the finding that the phosphoryl group of creatine phosphate transferred to ATP could supply the energy for muscle contraction. In this way, a functional applicability of the energy of the phosphate bond was first shown. This observation was soon followed by the recognition that the phosphoanhydride bond of ATP provided the driving force in biosynthetic reactions; in this type of bond, metabolic energy apparently collects before it is transmitted for functional and biosynthetic use. The storage of energy in ATP was first detected in anaerobic energy-yielding reactions but soon was also found in respiratory and photosynthetic energy production. However, the mechanism by which energy derived from metabolites was converted into phosphate-bond energy in the latter processes appeared to differ from that of anaerobic energy transmission. Whereas phosphorylated compounds mediate the latter in homogeneous solutions, aerobic phosphorylation and photophosphorylation in prokaryotes seem to require special submembranous structures; and in eukaryotes, energy conversion is a function of special organelles, the mitochondria and chloroplasts. The evolutionary aspects of the transition from prokaryotes to eukaryotes are of considerable interest. In conclusion, the relevance of an apparent prokaryotic origin of the energy-transforming organelles in the eukaryotes will be commented on.

1. The dissimilation of a number of externally added hexose phosphates and 5'-nucleotides by the perfused rat heart is described, and non-specific esterase and 5'-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of (14)CO(2) from [U-(14)C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-(14)C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-(14)C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.

An allosteric model of an open monosubstrate enzyme reaction is analyzed for the case where the enzyme, containing two protomers, is activated by the product. It is shown that this system can lead to instabilities beyond which a new state organized in time or in space (dissipative structure) can be reached. The conditions for both types of instabilities are presented and the occurrence of a temporal structure, consisting of a limit cycle behavior, is determined numerically as a function of the important parameters involved in the system. Sustained oscillations in the product and substrate concentrations are shown to occur for acceptable values of the allosteric and kinetic constants; moreover, they seem to be favored by substrate activation. The model is applied to phosphofructokinase, which is the enzyme chiefly responsible for glycolytic oscillations and which presents the same pattern of regulation as the allosteric enzyme appearing in the model. A qualitative and quantitative agreement is obtained with the experimental observations concerning glycolytic self-oscillations.

Abstract Background The role of ELAVL1 in the progression of various tumors has been demonstrated. Our research aims to investigate how ELAVL1 controls the glycolytic process in nasopharyngeal carcinoma cells through the HMGB3/β-catenin pathway. Methods The expression of ELAVL1 was detected in clinical tumor samples and nasopharyngeal carcinoma cell lines. A subcutaneous tumor model was established in nude mice to investigate the role of ELAVL1 in tumor progression. The relationship between HMGB3 and ELAVL1 was validated by RNA pull down and RIP assays. TOPFlash/FOPFlash reporter assay was used to detect β-catenin activity. Assay kits were utilized to measure glucose consumption, lactate production, and G6PD activity in nasopharyngeal carcinoma cells. Western blot was conducted to detect the expression of glycolysis-related proteins. The glycolytic capacity was analyzed through extracellular acidification rate (ECAR). Results In both clinical samples and nasopharyngeal carcinoma cell lines, the expression levels of ELAVL1 mRNA and protein were found to be upregulated. Knockdown of ELAVL1 significantly inhibited the in vivo proliferation of nasopharyngeal carcinoma and suppressed the glycolytic capacity of nasopharyngeal carcinoma cells. ELAVL1 interacts with HMGB3, leading to an increase in the stability of HMGB3 mRNA. Overexpression of HMGB3 elevated the reduced β-catenin activity caused by sh-ELAVL1 and reversed the inhibitory effect of sh-ELAVL1 on cellular glycolytic capacity. Treatment with β-catenin inhibitor (FH535) effectively suppressed the promotion of glycolytic capacity induced by HMGB3 overexpression. Conclusions ELAVL1 promotes glycolysis in nasopharyngeal carcinoma cells by interacting with HMGB3 to stabilize HMGB3 mRNA, thereby activating β-catenin pathway. Therefore, targeting the ELAVL1-HMGB3-β-catenin axis has the potential to be a novel approach for treating nasopharyngeal carcinoma.

Abstract Objective Renal ischemia/reperfusion injury (IRI) is a major cause of acute kidney injury (AKI), which is associated with high incidence and mortality. AST-120 is an oral carbonaceous adsorbent that can alleviate kidney damage. This study aimed to explore the effects of AST-120 on renal IRI and the molecular mechanism. Methods A renal IRI mouse model was established and administrated AST-120, and differentially expressed genes were screened using RNA sequencing. Renal function and pathology were analyzed in mice. Hypoxia/reoxygenation (H/R) cell model was generated, and glycolysis was evaluated by detecting lactate levels and Seahorse analysis. Histone lactylation was analyzed by western blotting, and its relationship with hexokinase 2 (HK2) was assessed using chromatin immunoprecipitation. Results The results showed that HK2 expression was increased after IRI, and AST-120 decreased HK2 expression. Knockout of HK2 attenuated renal IRI and inhibits glycolysis. AST-120 inhibited renal IRI in the presence of HK2 rather than HK2 absence. In proximal tubular cells, knockdown of HK2 suppressed glycolysis and H3K18 lactylation caused by H/R. H3K18 lactylation was enriched in HK2 promoter and upregulated HK2 levels. Rescue experiments revealed that lactate reversed IRI that suppressed by HK2 knockdown. Conclusions In conclusion, AST-120 alleviates renal IRI via suppressing HK2-mediated glycolysis, which suppresses H3K18 lactylation and further reduces HK2 levels. This study proposes a novel mechanism by which AST-120 alleviates IRI.

Abstract Background One of major challenges in breast tumor therapy is the existence of breast cancer stem cells (BCSCs). BCSCs are a small subpopulation of tumor cells that exhibit characteristics of stem cells. BCSCs are responsible for progression, recurrence, chemoresistance and metastasis of breast cancer. Ca2+ signalling plays an important role in diverse processes in cancer development. However, the role of Ca2+ signalling in BCSCs is still poorly understood. Methods A highly effective 3D soft fibrin gel system was used to enrich BCSC-like cells from ER+ breast cancer lines MCF7 and MDA-MB-415. We then investigated the role of two Ca2+-permeable ion channels Orai1 and Orai3 in the growth and stemness of BCSC-like cells in vitro, and tumorigenicity in female NOD/SCID mice in vivo. Results Orai1 RNA silencing and pharmacological inhibition reduced the growth of BCSC-like cells in tumor spheroids, decreased the expression levels of BCSC markers, and reduced the growth of tumor xenografts in NOD/SCID mice. Orai3 RNA silencing also had similar inhibitory effect on the growth and stemness of BCSC-like cells in vitro, and tumor xenograft growth in vivo. Mechanistically, Orai1 and SPCA2 mediate store-operated Ca2+ entry. Knockdown of Orai1 or SPCA2 inhibited glycolysis pathway, whereas knockdown of Orai3 or STIM1 had no effect on glycolysis. Conclusion We found that Orai1 interacts with SPCA2 to mediate store-independent Ca2+ entry, subsequently promoting the growth and tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1 mediate store-operated Ca2+ entry, promoting the growth and tumorigenicity of BCSC-like cells via a glycolysis-independent pathway. Together, our study uncovered a well-orchestrated mechanism through which two Ca2+ entry pathways act through distinct signalling axes to finely control the growth and tumorigenicity of BCSCs.

Abstract Background Mesenchymal stem cells (MSCs) are one of the most widely studied adult stem cells, while MSC replicative senescence occurs with serial expansion in vitro. We determined whether miR-34a can regulate MSC senescence by directly targeting glycolytic key enzymes to influence glycolysis. Methods Detected the effects of miR-34a on MSC senescence and glycolytic metabolism through gene manipulation. Bioinformatics prediction and luciferase reporter assay were applied to confirm that HK1 is a direct target of miR-34a. The underlying regulatory mechanism of miR-34a targeting HK1 in MSC senescence was further explored by a cellular function recovery experiment. Results In the current study, we revealed that miR-34a over-expression exacerbated senescence-associated characteristics and impaired glycolytic metabolism. Then we identified hexokinase1 (HK1) as a direct target gene of miR-34a. And HK1 replenishment reversed MSC senescence and reinforced glycolysis. In addition, miR-34a-mediated MSC senescence and lower glycolytic levels were evidently rescued following the co-treatment with HK1 over-expression. Conclusion The miR-34a-HK1 signal axis can alleviate MSC senescence via enhancing glycolytic metabolism, which possibly provides a novel mechanism for MSC senescence and opens up new possibilities for delaying and suppressing the occurrence and development of aging and age-related diseases.

Abstract Background Ginsenoside Rh2 (G-Rh2), a steroidal compound extracted from roots of ginseng, has been extensively studied in tumor therapy. However, its specific regulatory mechanism in non-small cell lung cancer (NSCLC) is not well understood. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in various malignant tumors. We investigated the impact of G-Rh2 on the malignant progression of NSCLC and how it regulated PDK4 to influence tumor aerobic glycolysis and mitochondrial function. Method We examined the inhibitory effect of G-Rh2 on NSCLC through I proliferation assay, migration assay and flow cytometry in vitro. Subsequently, we verified the ability of G-Rh2 to inhibit tumor growth and metastasis by constructing subcutaneous tumor and metastasis models in nude mice. Proteomics analysis was conducted to analyze the action pathways of G-Rh2. Additionally, we assessed glycolysis and mitochondrial function using seahorse, PET-CT, Western blot, and RT-qPCR. Result Treatment with G-Rh2 significantly inhibited tumor proliferation and migration ability both in vitro and in vivo. Furthermore, G-Rh2 inhibited the tumor’s aerobic glycolytic capacity, including glucose uptake and lactate production, through the HIF1-α/PDK4 pathway. Overexpression of PDK4 demonstrated that G-Rh2 targeted the inhibition of PDK4 expression, thereby restoring mitochondrial function, promoting reactive oxygen species (ROS) accumulation, and inducing apoptosis. When combined with sodium dichloroacetate, a PDK inhibitor, it complemented the inhibitory capacity of PDKs, acting synergistically as a detoxifier. Conclusion G-Rh2 could target and down-regulate the expression of HIF-1α, resulting in decreased expression of glycolytic enzymes and inhibition of aerobic glycolysis in tumors. Additionally, by directly targeting mitochondrial PDK, it elevated mitochondrial oxidative phosphorylation and enhanced ROS accumulation, thereby promoting tumor cells to undergo normal apoptotic processes.

PURPOSE Hypoxic training is beneficial for improving endurance performance. Hypoxia-inducible factor (HIF) is a key transcription factor that regulates the hypoxic-responsive pathway. Stabilization of HIF by inactivating prolyl hydroxylase domain protein 2 (PHD2) under low oxygen tension, triggers angiogenesis, erythropoiesis, and glycolysis. Glycolytic capacity is essential to perform anaerobic exercise. This study aims to verify whether the upregulation of glycolytic metabolism under the hypoxic-responsive pathway enhances exercise capacity. METHODS We employed tamoxifen-inducible conditional PHD2 knock-out (KO) mice that exhibited stabilized HIF-alpha. To evaluate anaerobic exercise capacity, a rodent treadmill exercise test was performed. RNA sequencing (RNAseq) analysis was conducted using C2C12 myoblasts after PHD2 siRNA transfection. RESULTS PHD2KO mice exhibited increased red blood cell counts, hemoglobin, and hematocrit values, along with enhanced maximal exercise performance compared to littermate wild-type (WT) mice. PHD2 deficiency promoted maximal exercise capacity and resulted in lower blood lactate levels. RNAseq analysis revealed that PHD2-deficient myoblasts increased the expression of genes related to glycolysis, gluconeogenesis, and muscle contraction. CONCLUSIONS These findings suggest that PHD2 deficiency-induced activation of the hypoxic-responsive pathway enhances maximal exercise capacity.

Abstract Background Fluvoxamine is one of the selective serotonin reuptake inhibitors (SSRIs) that are regarded as the first-line drugs to manage mental disorders. It has been also recognized with the potential to treat inflammatory diseases and viral infection. However, the effect of fluvoxamine on autoimmune diseases, particularly type 1 diabetes (T1D) and the related cellular and molecular mechanisms, are yet to be addressed. Method Herein in this report, we treated NOD mice with fluvoxamine for 2 weeks starting from 10-week of age to dissect the impact of fluvoxamine on the prevention of type 1 diabetes. We compared the differences of immune cells between 12-week-old control and fluvoxamine-treated mice by flow cytometry analysis. To study the mechanism involved, we extensively examined the characteristics of CD4+ T cells with fluvoxamine stimulation using RNA-seq analysis, real-time PCR, Western blot, and seahorse assay. Furthermore, we investigated the relevance of our data to human autoimmune diabetes. Result Fluvoxamine not only delayed T1D onset, but also decreased T1D incidence. Moreover, fluvoxamine-treated NOD mice showed significantly attenuated insulitis coupled with well-preserved β cell function, and decreased Th1 and Th17 cells in the peripheral blood, pancreatic lymph nodes (PLNs), and spleen. Mechanistic studies revealed that fluvoxamine downregulated glycolytic process by inhibiting phosphatidylinositol 3-kinase (PI3K)-AKT signaling, by which it restrained effector T (Teff) cell differentiation and production of proinflammatory cytokines. Conclusion Collectively, our study supports that fluvoxamine could be a viable therapeutic drug against autoimmunity in T1D setting.

Abstract Background Neovascular eye disease is characterized by pathological neovascularization, with clinical manifestations such as intraocular exudation, bleeding, and scar formation, ultimately leading to blindness in millions of individuals worldwide. Pathologic ocular angiogenesis often occurs in common fundus diseases including proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), and retinopathy of prematurity (ROP). Anti-vascular endothelial growth factor (VEGF) targets the core pathology of ocular angiogenesis. Main body In recent years, therapies targeting metabolism to prevent angiogenesis have also rapidly developed, offering assistance to patients with a poor prognosis while receiving anti-VEGF therapy and reducing the side effects associated with long-term VEGF usage. Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key enzyme in targeted metabolism, has been shown to have great potential, with antiangiogenic effects and multiple protective effects in the treatment of neovascular eye disease. In this review, we summarize the mechanisms of common types of neovascular eye diseases; discuss the protective effect and potential mechanism of targeting PFKFB3, including the related inhibitors of PFKFB3; and look forward to the future exploration directions and therapeutic prospects of PFKFB3 in neovascular eye disease. Conclusion Neovascular eye disease, the most common and severely debilitating retinal disease, is largely incurable, necessitating the exploration of new treatment methods. PFKFB3 has been shown to possess various potential protective mechanisms in treating neovascular eye disease. With the development of several drugs targeting PFKFB3 and their gradual entry into clinical research, targeting PFKFB3-mediated glycolysis has emerged as a promising therapeutic approach for the future of neovascular eye disease.

Ovarian cancer is a common, highly lethal tumor. Herein, we reported that S-phase kinase-associated protein 2 (Skp2) is essential for the growth and aerobic glycolysis of ovarian cancer cells. Skp2 was upregulated in ovarian cancer tissues and associated with poor clinical outcomes. Using a customized natural product library screening, we found that xanthohumol inhibited aerobic glycolysis and cell viability of ovarian cancer cells. Xanthohumol facilitated the interaction between E3 ligase Cdh1 and Skp2 and promoted the Ub-K48-linked polyubiquitination of Skp2 and degradation. Cdh1 depletion reversed xanthohumol-induced Skp2 downregulation, enhancing HK2 expression and glycolysis in ovarian cancer cells. Finally, a xenograft tumor model was employed to examine the antitumor efficacy of xanthohumol in vivo. Collectively, we discovered that xanthohumol promotes the binding between Skp2 and Cdh1 to suppress the Skp2/AKT/HK2 signal pathway and exhibits potential antitumor activity for ovarian cancer cells. [ABSTRACT FROM AUTHOR]

The anaerobic capacity (AC[La]+EPOCfast) method is of interest as it can differentiate between the contributions of phosphocreatine (PCr) and glycolytic energy, assess the anaerobic metabolism during a single effort, and evaluate various exercise modalities. However, no previous studies have investigated whether the superiority of the PCr and glycolytic systems can be assessed in athletes with high anaerobic capacity. This study aimed to compare the AC[La]+EPOCfast method in seven sprinters (SP) and seven middle-distance runners (MD). The participants underwent a graded exercise test and constant-load exercise at 115% ˙VO2max until exhaustion using a bicycle ergometer. AC[La]+EPOCfast was calculated as the sum of oxygen equivalents from the phosphagen (EPCr) and glycolytic (ELa) pathways. This study reveals that there was no significant difference in AC[La]+EPOCfast or maximal accumulated oxygen deficit (MAOD) between the two groups. Although EPCr was not significantly different between groups, ELa was significantly higher in the SP group than in the MD group. In conclusion, the AC[La]+EPOCfast method can be used to evaluate high and low anaerobic capacities. However, further studies on athletes with high aerobic capacity are required. [ABSTRACT FROM AUTHOR]

Abstract Background Hair follicle stem cells (HFSCs) typically remain quiescent and are activated only during the transition from telogen to anagen to ensure that the hair follicle enters a new cycle. The metabolic behavior of stem cells in tissues is regulated by macroautophagy/autophagy, and changes in HFSC metabolism directly affect their activation and maintenance. However, the role of autophagy in the regulation of HFSC metabolism and function remains unclear. Methods Back skin samples were obtained from mice at different hair follicle cycle stages, and immunofluorescence staining was used to monitor autophagy in HFSCs. Mouse and human hair follicles were treated with rapamycin (Rapa, an autophagy activator) or 3-methyladenine (3-MA, an autophagy inhibitor). The effects of autophagy on the hair follicle cycle and HFSC were investigated by imaging, cell proliferation staining, and HFSC-specific marker staining. The influence and mechanism of autophagy on HFSC metabolism were explored using RNA sequencing, real-time polymerase chain reaction, immunohistochemical staining, and detection of lactate and glucose concentrations. Finally, the influence of autophagy-induced glycolysis on HFSC and the hair follicle cycle was verified by stem cell characteristics and in vivo functional experiments. Results Autophagy in HFSC was highest during the transition from telogen to anagen. Inhibiting autophagy with 3-MA led to early entry into catagen and prolonged telogen, whereas Rapa promoted autophagy and hair growth. Autophagy activated HFSC by increasing the expression and activity of HFSC lactate dehydrogenase (Ldha), thereby transforming HFSC metabolism into glycolysis. Inhibition of Ldha expression counteracted the effects of autophagy. Conclusions Autophagy activated HFSC by promoting the transition from HFSC metabolism to glycolysis, ultimately initiating the hair follicle cycle and promoting hair growth. Graphical Abstract

Background: The influence of homeostatically regulated physiological processes, including cardiorespiratory fitness (VO2max), on the response to physical stressors such as acclimatisation and marching, remains understudied. We aimed to investigate the effects of summer and winter acclimatisation and marching on cortisol levels and blood lactate, to gain insight into the role of these physiological processes in the stress response. Methods: Two groups of young Europeans, classified as poor (PCF; n=9) and good physical condition (GCF; n=21), based on a VO2MAX threshold of 40 mL O2/ kg/min, underwent 2-h March (6-7 km/h) in winter (5˚C) and summer (32˚C). Commercial tests, UniCel DxI Access Cortisol assay and EKF Biosen Clinic/GP assay were used for cortisol and lactate blood measurements (morning samples and those taken immediately after marches), respectively. Results: Basal cortisol levels were significantly higher at 5°C than at 32°C (PCF group: P=0.0079; cortisol dropped after the March at 5°C in both groups, but increased at 32°C only in PCF. Basal lactate levels were higher at 32°C only in the GCF group (compared to PCF, P=0.0014) and post-marching (compared to basal values, P=0.0002). Conclusion: Cold exposure elicits a more significant stress response, based on higher basal cortisol levels, in individuals with worse physical fitness. Anaerobe metabolism prevails in participants with better physical fitness exposed to high ambient temperature based on increased basal and post-marching lactate levels. The study is significant for individual training/performance optimisation as it indicates the association between physiological stress responses and individual physical fitness levels.

Abstract An intrinsic link between metabolism and function in immune cells, and in particular macrophages, has been well established recently. However, the molecular mechanisms controlling the metabolic switch in these sentinel cells for their integral roles in host defense, inflammation, homeostasis, and pathogenesis remain largely unknown. Here, we identify the master transcription factor NF-κB RelA as a vital cell-intrinsic checkpoint restricting aerobic glycolysis to favor mitochondrial oxidative phosphorylation (OXPHOS) and “M2” activation (alternative anti-inflammatory and pro-tumorigenic activation, in contrast to classical pro-inflammatory and anti-tumor M1 activation) of macrophages under oncogenic stress. RelA specific knockdown or genetic deletion in macrophages causes metabolism to shift away from OXPHOS toward glycolysis, resulting in drastically decreased oxygen consumption but significantly increased lactate and ATP production. The metabolic change in RelA deficient cells is associated with the decrease in the expressions of the OXPHOS gene SCO2 as well as the M2 marker and function genes arginase-1 and VEGF. These data suggest that RelA induces SCO2 expression to enhance OXPHOS and restrict glycolysis in macrophages for their pro-tumorigenic activation.

Abstract Background Medial vascular calcification is commonly identified in chronic kidney disease (CKD) patients and seriously affects the health and life quality of patients. This study aimed to investigate the effects of protein arginine methyltransferase 3 (PRMT3) on vascular calcification induced by CKD. Methods A mice model of CKD was established with a two-step diet containing high levels of calcium and phosphorus. Vascular smooth muscle cells (VSMCs) were subjected to β-glycerophosphate (β-GP) treatment to induce the osteogenic differentiation as an in vitro CKD model. Results PRMT3 was upregulated in VSMCs of medial artery of CKD mice and β-GP-induced VSMCs. The inhibitor of PRMT3 (SGC707) alleviated the vascular calcification and inhibited the glycolysis of CKD mice. Knockdown of PRMT3 alleviated the β-GP-induced osteogenic transfomation of VSMCs by the repression of glycolysis. Next, PRMT3 interacted with hypoxia-induced factor 1α (HIF-1α), and the knockdown of PRMT3 downregulated the protein expression of HIF-1α by weakening its methylation. Gain of HIF-1α reversed the PRMT3 depletion-induced suppression of osteogenic differentiation and glycolysis of VSMCs. Conclusion The inhibitory role of PRMT3 depletion was at least mediated by the regulation of glycolysis upon repressing the methylation of HIF-1α.