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The energy-metabolism oscillation in aerobic chemostat cultures of yeast is a periodic change of the respiro-fermentative and respiratory phase. In the respiro-fermentative phase, the NADH level was kept high and respiration was suppressed, and glucose was anabolized into trehalose and glycogen at a rate comparable to that of catabolism. On the transition to the respiratory phase, cAMP levels increased triggering the breakdown of storage carbohydrates and the increased influx of glucose into the glycolytic pathway activated production of glycerol and ethanol consuming NADH. The resulting increase in the NAD+/NADH ratio stimulated respiration in combination with a decrease in the level of ATP, which was consumed mainly in the formation of biomass accompanying budding, and the accumulated ethanol and glycerol were gradually degraded by respiration via NAD+-dependent oxidation to acetate and the respiratory phase ceased after the recovery of NADH and ATP levels. However, the mRNA levels of both synthetic and degradative enzymes of storage carbohydrates were increased around the early respiro-fermentative phase, when storage carbohydrates are being synthesized, suggesting that the synthetic enzymes were expressed directly as active forms while the degradative enzymes were activated late by cAMP. In summary, the energy-metabolism oscillation is basically regulated by a feedback loop of oxido-reductive reactions of energy metabolism mediated by metabolites like NADH and ATP, and is modulated by metabolism of storage carbohydrates in combination of post-translational and transcriptional regulation of the related enzymes. A potential mechanism of energy-metabolism oscillation is proposed.
Biological rhythms are considered to be ubiquitous in eukaryotic and prokaryotic organisms and are synchronized so the organism can adapt to environmental changes. Circadian rhythm is the most common of the biological rhythms and the molecular clock mechanism has been studied extensively. However, no circadian rhythm has been discovered in yeast and instead two kinds of ultradian rhythms of energy metabolism have been reported. One is a KCN-induced oscillation of the glycolytic pathway and the other is an energy-metabolism oscillation (EMO) found in aerobic chemostat cultures. The KCN-induced oscillations were evoked after addition of glucose by inhibiting mitochondrial respiration with cyanide  and show a periodicity of 1–2 min as monitored by measuring the level of NAD(P)H. The glycolytic pathway has been physically proven to be a sustained oscillator by Prigogine and co-workers [2,3]. Theoretically, the glycolytic pathway oscillates under the primary control of phosphofructokinase 1 (Pfk1p) which is activated auto-catalytically by its own product ADP leading to a nonlinear accumulation of NADH in combination with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). NADH acts as the feed-forward activator of a glycolytic pathway facilitating the fermentation and ATP produced by fermentation acts as a feedback inhibitor by inhibiting the kinase reaction of the enzymes hexokinase and Pfk1p. Experimentally, the ATP level oscillated with an inversed phase to that of the NADH level, supporting the theory . Although the NADH oscillation ceases within 30 min in batch cultures, it lasts as long as several days in chemostat cultures  proving that the glycolytic pathway is a sustained oscillator comprising a dissipative structure.
The other ultradian oscillation called EMO is usually monitored by measuring the dissolved oxygen (DO) level and shows a periodicity of approximately 4 h [6–9], while a similar EMO with a short-period (about 40 min) has been studied [10,11]. EMO arises spontaneously under glucose- and nitrogen-limited conditions dependent on a high cell-density (∼ 5 × 108 cells·mL−1)  and is sustained synchronization of the cell division cycle and a periodic change in the factors involved in energy metabolism such as CO2 production, O2 uptake, glucose and ethanol concentrations, and amounts of storage carbohydrates [6–9]. EMO is considered to be a periodic change between respiratory and respiro-fermentative phases in which oxygen demands are relatively high and low, respectively. We previously reported that the NADH level oscillated in the same phase as the DO level  being high in the respiro-fermentative phase and low in the respiratory phase. However, we found later that the ATP level, which is supposed to act as a feedback inhibitor, was low in the respiratory phase , suggesting that EMO oscillates with a distinct, if similar, mechanism from the KCN-induced oscillation of the glycolytic pathway. On the other hand, in the short-period EMO, Lloyd et al.  found that the NADH level oscillated in the same phase as the DO level and Klevecz et al.  reported that the mRNA levels of most genes show peaks during the two distinct time windows within the reductive phase, and only 10% of genes are transcribed in the oxidative phase suggesting that gene expression is influenced by redox states of cells. They also reported that the oscillation become synchronized by the rhythmic secretion of metabolites like acetaldehyde and hydrogen sulfide . Therefore, it seems likely that a periodic change of intracellular redox potentials and/or levels of some metabolites underlie the control of EMO. However, as even the central pathway of the energy metabolism contains many oxidoreductase reactions and many intermediates, the mechanism by which EMO oscillates remains largely unknown.
We isolated the gene GTS1 as a candidate rhythm-related gene from a yeast cDNA library using oligonucleotides that encoded an Ala-Gln repeat in Gts1p [17,18] and, during the course of studying the role of GTS1, we found that EMO is coupled with a fluctuation in the trehalose level and that the trehalose level began to increase in the early respiro-fermentative phase and decrease in the late respiratory phase after the elevation of the cAMP level [13,19]. Furthermore, we found that the transcription of TPS1 encoding trehalose-6-phosphate synthase 1 (Tps1p), a regulatory enzyme in trehalose synthesis, was periodically regulated in EMO peaking in the late respiratory phase [13,20] and that EMO was destabilized in a transformant in which the synthesis of trehalose was inhibited. So, considering that Tps1p has been reported to regulate glucose influx for glycolysis [21,22], we suggested that the metabolism of storage carbohydrates like trehalose is also involved in the stabilization of EMO . Recently, Jules et al.  reported that yeast cells in a batch culture on trehalose generate transient short-period oscillations of respiration and cell division and that intracellular degradation of glycogen and trehalose plays an important role for the generation of EMO. However, the mechanism by which the regulation of trehalose and glycogen metabolism stabilize EMO remains to be elucidated.
In this report, to investigate the mechanism of EMO, we determined profiles of the levels of metabolic variables and found that EMO is the periodic change between reductive and oxidative states with respect to the NAD+/NADH ratio, thereby forming a feedback loop of dehydrogenase reactions. Furthermore, we found that the synthesis and breakdown of storage carbohydrates play an important role in regulation of EMO.
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In this communication, we studied on the mechanism of EMO in yeast and showed that the oscillation of the energy metabolism is basically regulated by the periodic change between reductive and oxidative states of NADH, thereby forming a feedback loop of dehydrogenase reactions in the energy metabolism. NADH produced from the upper part of the glycolytic pathway (from glucose phosphorylation to pyruvate production) activates ethanol production in the lower part (from pyruvate decarboxylation to ethanol production) and then the resulting increase of NAD+ activates oxidation of ethanol and mitochondrial respiration. We further showed that metabolism of trehalose and glycogen plays an important role in the regulation of EMO. The synthesis of them attenuates the glucose flux for glycolysis in the early respiro-fermentative phase and the breakdown induces the facilitation of respiration on the shift to the respiratory phase.
The potential mechanism of EMO is suggested as follows (Fig. 7). First, in the respiro-fermentative phase shown in Figure 7A, glucose is catabolized mainly by respiration and anabolized into storage carbohydrates at similar flux rates when calculated from OUR (Fig. 1A) and the storage carbohydrate-related glucose flux rate (Fig. 3B). Although this phase has been called ‘respiro-fermentative’, ethanol production is suppressed (Fig. 2A) so that reoxidation of NADH decreases. It has been proposed that a metabolic function of trehalose synthesis is to restrict the flux of glucose for glycolysis by inhibiting the early steps of the glycolytic pathway. The glycolytic pathway is designed based on the ‘turbo’ principle, as mutants having a defect in TPS1 cannot grow on glucose-accumulating glucose-phosphates until depletion of ATP, its own product, occurs . Thus, it is likely that the attenuation of the early steps of glycolysis is caused, at least in part, by the flux of glucose into storage carbohydrates suppressing the overflow of pyruvate to the ethanol production which may lead to premature shift to the respiratory phase. In addition, the synthesis of trehalose is involved in the recovery of inorganic phosphate, which is required for the GAPDH reaction to produce NADH , via trehalose-6-phosphate phosphatase. The synthesis of glycogen and trehalose is facilitated by expression of synthetic genes encoding regulatory enzymes like Tps1p  and Gsy2p in this phase (Fig. 5B). Our result showed that the degradative enzymes of storage carbohydrates were also expressed in this phase, but were thought to remain inactive until the transition to the respiratory phase when they are activated by phosphorylation . Although the production of trehalose increased earlier than that of glycogen in the early respiro-fermentative phase, glycogen was three times more abundant than trehalose, suggesting that glycogen is a main carbohydrate reservoir in cells during EMO (Fig. 3).
Figure 7. Summary of energy metabolism during EMO. Schematic presentation of the metabolism of glucose in the respiro-fermentative (A), respiratory (C) and transitional phases (B,D). Numbers indicate the approximate flux rate of glucose expressed in fmol·min−1·cell−1. Enzymes involved are indicated by the genes encoding them: TDHs, GAPDHs; GDP1/2, Glycerol-3-phosphate dehydrogenases (DH); PYK1, pyruvate kinase; ADHs, alcohol DHs; GCY1, glycerol DH; ALDs, aldehyde DHs; PFK2, phosphofructokinase 2; TPS1, trehalose-6-phosphate synthase 1; GSY2, glycogen synthase 2; NTH1/2, neutral trehalases and GPH1, glycogen phosphorylase 1. Pi indicates inorganic phosphate. T-shaped arrows indicate inhibitory action.
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Secondly, during the transition to the respiratory phase shown in Figure 7B, ethanol as well as glycerol were rapidly accumulated consuming NADH as a reductive force for related dehydrogenases; ethanol was produced through the reduction of acetaldehyde by NADH-dependent alcohol dehydrogenases (encoded by the ADH gene family). The total amount of glycerol was much less than that of ethanol (< 1/20) although the intracellular concentration of glycerol was higher than that of ethanol at the peaks. The result suggested that the role of glycerol as a carbohydrate reservoir is considered to be small but rather glycerol, known as a major osmolyte in cells, may increase the cellular ‘turgor pressure’ which is proposed to facilitate bud formation . Alternatively, as either hyper- or hypo-osmotic pressure induces calcium pulse responses in cells [31,32], glycerol production may be involved in the signal-transfer pathway considering that calcium is known to act in coupling with the Ras-cAMP pathway . The fact that glycerol production started earlier than that of ethanol in the late respiro-fermentative phase (Fig. 2), when the bud formation and cAMP production started (Fig. 3), supported these speculations. To start the acceleration of ethanol and glycerol production, the glucose influx into the glycolytic pathway has to be facilitated. We suggested that glucose was supplied from the storage carbohydrates mediated by cAMP which increased prior to this phase (Fig. 3A). The cAMP-dependent PKA was activated leading to the degradation of trehalose and glycogen by activating neutral trehalases (encoded by NTH1/2) and glycogen phosphorylase (GPH1). In contrast, the Ras/PKA signaling pathway has been reported to inactivate glycogen synthase  and the mutation of RAS2 in which the cAMP level decreased induced hyperaccumulation of glycogen . Furthermore, the activity of purified glycogen synthase was inhibited after phosphorylation by PKA in vitro. Thus, it is probable that the degradative and synthetic enzymes for glycogen were inversely regulated by PKA in this phase. Furthermore, a few lines of evidence have been reported which support the acceleration of the flux rate of the glycolytic pathway by cAMP in this phase. (1) In addition to the conventional proposal that Pfk1p is activated by its own product ADP in an autocatalytic fashion [2,3], phosphofructokinase 2 (Pfk2p encoded by PFK2) which produces fructose-2,6-bisphosphate (F-2,6-BP), a potent activator of Pfk1p, is exclusively dependent on PKA [37,38]. (2) The pyruvate kinase is reportedly activated by PKA in the presence of the activator fructose-1,6-bisphosphate (FBP) , increasing production of pyruvate. In this respect, Wittmann et al.  reported recently that FBP oscillated peaking at the early respiratory phase out of phase of 2- and 3-phosphoglycerate, and phosphoenolpyruvate, suggesting that FBP activates pyruvate kinase leading to progressively increasing glycolytic flux. These results suggested that the released glucose flowed into the glycolytic pathway leading to ethanol production which was facilitated by PKA, in combination with NADH which is not only an feed-forward activator for the ethanol production in glycolysis, but also an inhibitor of PDC suppressing the flux of puruvate into the citric acid cycle.
Thirdly, in the early respiratory phase (Fig. 7C), NADH was mostly oxidized to NAD+ as a result of the ethanol and glycerol production and the resulting increase in the NAD+/NADH ratio activated mitochodrial respiration in combination with the increased flux of glucose from storage carbohydrates for glycolysis. Ethanol and glycerol produced in the early respiratory phase are oxidized to acetate via acetaldehyde using NAD+ as an oxidative cofactor of glycerol-3-phosphate dehydrogenase (GPD1/2), alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDs), and acetate is further oxidized in mitochondria via acetyl-CoA . Alternatively, glycerol may be oxidized by mitochondria using FAD not to influence the NAD+/NADH ratio in the cytoplasm via the glycerol-3-phosphate shuttle or external NADH dehydrogenase on mitochondria . As a result, OUR increased more than twofold that in the respiro-fermentative phase, suggesting that the internal environment of cells became oxidative. There have been reports that GAPDH (encoded by the TDH gene family), mildly oxidized by either H2O2 or nitric oxide, exhibits acyl phosphatase activity with which GAPDH directly produces 3-phosphoglycerate without ATP production, and thus facilitates glycolysis [42–44]. Therefore, it is possible that glycolysis in the respiratory phase is facilitated by this modification due to reactive oxygen species in addition to the cAMP action described above. The doubling of OUR in this phase means that ATP production was also doubled. However, the fact that the ATP level decreased while the AMP and ADP levels increased in the respiratory phase  suggested that ATP was consumed at a higher rate than it was produced. The decrease in the ATP level is required together with the increase in the NAD+ level to stimulate the activation of mitochondrial respiration via PDC. We found that ATP is mainly used for the biomass formation for budding (Fig. 3). The predominant formation of biomass in the respiratory phase of EMO is in good agreement with a previous result .
Finally (Fig. 7D), by the end of the respiratory phase, both ATP  and NADH levels (Fig. 1B) recover because the biomass formation and acetate oxidation were completed, and act as feedback inhibitors to suppress respiration in mitochondria. In addition, the trehalose and glycogen synthesis started attenuating the glucose influx into glycolysis, which promotes the shift to the respiro-fermentative phase.
It should be noted that EMO is a dissipative structure that spontaneously operates in chemostat cultures, obeying the second law of thermodynamics, which states that spontaneously occurring reactions increase entropy (energy specifying the amount of randomness or disorder) in the universe. Dissipative structures are defined as sustained oscillators operating far from equilibrium of energy according to the second law of thermodynamics [2,3]. In other words, they are oscillators that spontaneously operate by dissipating energy so that they self-organize vivid structures (or systems). The dissipation of energy means a series of reactions consisting of uptake of high-ordered (energy-rich) materials from the environment and subsequent decomposition of these materials into low-ordered materials releasing free energy and entropy, which are used for work and are excreted into the environment, respectively. Living organisms are complex examples of dissipative structures. EMO in yeast is a sustained oscillator that operates spontaneously along with the decomposition of glucose into carbon dioxide and water, synchronizing various metabolic pathways in an oscillatory fashion, including the cell-replication cycle, metabolism of storage carbohydrates and glycerol, gene expression, and so on, all of which require ATP, a product of EMO.
We have previously reported that in gts1Δ, in which expression of TPS1 is attenuated, EMO is destabilized and disappears , and it is indicated herein that GSY2-deleted transformants are unable to generate EMO (Fig. 6), suggesting that the normal synthesis of storage carbohydrates is required for stabilization of EMO. So far, it seems likely that trehalose synthesis functions as a primary source of inorganic phosphate, while glycogen is a primary source of glucose upon the transfer to the respiratory phase. However, whether the roles of glycogen and trehalose in the stabilization of EMO are distinct from one another remains to be clarified. In addition, although we suggested that cAMP/PKA is deeply involved in the control of EMO by regulating glycogen synthesis and various enzyme activities, it is likely that many other kinases are also involved. For example, PAS and Pho85p kinases inhibit glycogen synthase via phosphorylation and promote mRNA translation or the cell cycle . Furthermore, it was recently reported that the TOR (target of rapamycin) and cAMP/PKA signaling pathways coordinately stimulate the transcription of ribosomal proteins and rRNA  which accounts for over 60% of all transcription. Studies on the involvement of these kinases in EMO should be conducted in the future.
In this communication, we for the first time presented a potential mechanism of EMO by systemically determining profiles of the levels of metabolic variables. It should be mentioned, however, that there have been several studies on the metabolism of some intermediates in EMO [8,40,47]. There are no significant differences between previous results and ours: for example, ethanol and acetate were produced and degraded in a sequential manner in the respiratory phase [8,47]; the cAMP level increased at the shift to the respiratory phase activating trehalases , and trehalose and glycogen were metabolized similarly peaking in the late respiro-fermentative phase . On the other hand, there are some significant differences between the metabolism of EMO and the short-period EMO [10,11]. For example, the ethanol level did not oscillate significantly while the acetaldehyde level oscillated with a high amplitude and the trehalose level did not significantly oscillate . So, possibly, there are some differences between the mechanisms of short- and long-period EMO.
Very recently, Tu et al.  reported the results of microarray analysis of gene expression in a long-period EMO using a diploid yeast strain CEN.PK. They showed that over half of genes were expressed periodically during EMO and that genes encoding proteins having a common function exhibit similar temporal expression patterns. Roughly, the genes involved in the protein synthesis and cell division are expressed in the respiratory (named the OX phase in their report) and early respiro-fermentative phases (R/B phase), respectively, and those involved in nonrespiratory modes of metabolism and protein degradation are in the late respiro-fermentative phase (R/C phase). Thus, it is likely that about half of genes were expressed in the phase when their encoded proteins are required to function. Furthermore, their supplemental data indicated that the genes encoding the synthetic and degradative enzymes of storage carbohydrates are all expressed in the late respiro-fermentative. Although the phase of expression of the genes seems earlier than that shown in this report, their result also indicated that post-transcriptional regulation is required for the enzymes to function properly. The problem whether and how the gene expression and post-transcriptional regulation are influenced by the redox change of EMO remains to be elucidated.