The Role of Trehalose and Glycogen in the Survival of Aging Saccharomyces cerevisiae Cells

The role of the storage carbohydrates trehalose and glycogen in the survival of aging Saccharomyces cerevisiae cells was studied. Culture aging for one week did not reduce cell viability. During this period, the cells accumulated the storage carbohydrates and showed increased activity of the glycolytic enzymes hexokinase and phosphofructokinase. However, further aging led to a drastic drop in cell viability and to a decrease in the cellular content of trehalose and glycogen and in the activity of hexokinase and phosphofructokinase. The possible reasons for these changes are discussed.     

Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in Saccharomyces cerevisiae

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO₂ production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.     

Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation

Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae. Alterations in trehalose synthesis affect carbon source-dependent growth, accumulation of glycogen and sporulation. Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins. In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components. The tps1-H223Y allele (glc6-1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild-type TPS1 because, unlike the wild-type enzyme, TPS activity in tps1-H223Y cells is not inhibited by phosphate. Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1. Furthermore, high-copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait. These results suggest that the trehalose-6-phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.     

Role of reserve carbohydrates in the growth dynamics of Saccharomyces cerevisiae

The purpose of this study was to explore the role of glycogen and trehalose in the ability of Saccharomyces cerevisiae to respond to a sudden rise of the carbon flux. To this end, aerobic glucose-limited continuous cultures were challenged with a sudden increase of the dilution rate from 0.05 to 0.15 h(-1). Under this condition, a rapid mobilization of glycogen and trehalose was observed which coincided with a transient burst of budding and a decrease of cell biomass. Experiments carried out with mutants defective in storage carbohydrates indicated a predominant role of glycogen in the adaptation to this perturbation. However, the real importance of trehalose in this response was veiled by the unexpected phenotypes harboured by the tps1 mutant, chosen for its inability to synthesize trehalose. First, the biomass yield of this mutant was 25% lower than that of the isogenic wild-type strain at dilution rate of 0.05 h(-1), and this difference was annulled when cultures were run at a higher dilution rate of 0.15 h(-1). Second, the tps1 mutant was more effective to sustain the dilution rate shift-up, apparently because it had a faster glycolytic rate and an apparent higher capacity to consume glucose with oxidative phosphorylation than the wild type. Consequently, a tps1gsy1gsy2 mutant was able to adapt to the dilution rate shift-up after a long delay, likely because the detrimental effects from the absence of glycogen was compensated for by the tps1 mutation. Third, a glg1Deltaglg2Delta strain, defective in glycogen synthesis because of the lack of the glycogen initiation protein, recovered glycogen accumulation upon further deletion of TPS1. This recovery, however, required glycogen synthase. Finally, we demonstrated that the rapid breakdown of reserve carbohydrates triggered by the shift-up is merely due to changes in the concentrations of hexose-6-phosphate and UDPglucose, which are the main metabolic effectors of the rate-limiting enzymes of glycogen and trehalose pathways.     

松针瘿蚊越冬幼虫体内酶活性的时序变化

昆虫的越冬耐寒过程与糖酵解、磷酸己糖途径和抗冻保护性物质合成等一些中间代谢有关的酶有关。该文对松针瘿蚊Thecodiplosis japonensis老熟幼虫1998/1999越冬期间体内上述代谢酶活性的变化进行了研究。越冬期间体内糖原磷酸化酶活性明显地增加,糖酵解有关的酶（己糖激酶、乳酸脱氢酶和醛缩酶）活性较低,以保证更多的碳源（糖原）转化成海藻糖。越冬期间,体内葡萄糖-6-磷酸脱氢酶活性增高所产生的还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH),可为细胞在亚低温状态下发挥正常功能以及体内抗冻保护性物质的合成提供还原动力,同时通过调节体内海藻糖酶活性来维持越冬期间较高含量的海藻糖和移除春季体内累积的过多的海藻糖。     

D2O effects on association behavior and binding properties of phospho- and dephospho-cytoplasmic malic dehydrogenase

Changes in the concentration of three major carbohydrates, e.g., glycogen, trehalose, and cellulose, were determined during differentiation of Dictyostelium discoideum in a stage study. These three carbohydrates consituted 50–63% of the total carbohydrates. Total carbohydrate content per cell aliquot did not change between the aggregation and sorocarp stages of differentiation. The isolation, purification, and characterization of cellulose is described. Cellulose consisted of an alkali-insoluble (alpha) and an alkali-soluble (beta) fraction. Total cellulose accumulated from very low amounts in late pseudoplasmodium cells to about 35% of carbohydrates in mature sorocarps at a rate of 0.07 μmole glucose equiv/min/ml packed cell volume. Purified alkali-insoluble cellulose constituted about 19% of total carbohydrates in mature sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Trehalose constituted 10–11% of the carbohydrates in sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Glycogen, comparing several methods of determination, was rapidly degraded between the culmination and sorocarp stages of differentiation at an average rate of 0.05 μmole glucose equiv/min/ml packed cell volume. The major portion of glycogen was soluble in TCA and constituted 35% of total carbohydrates in aggregated cells and about 11% in mature sorocarps. A minor fraction of glycogen, about 15% of total carbohydrates in aggregated cells, was solubilized by KOH from a TCA precipitate. A mild acidic treatment of solubilized cell constituents increased the glycogen content by 55%, as judged by an enzymatic assay.     

A Highlights from MBoC Selection: Trehalose Is a Key Determinant of the Quiescent Metabolic State That Fuels Cell Cycle Progression upon Return to Growth

When conditions are unfavorable, virtually all living cells have the capability of entering a resting state termed quiescence or G0. Many aspects of the quiescence program as well as the mechanisms governing the entry and exit from quiescence remain poorly understood. Previous studies using the budding yeast Saccharomyces cerevisiae have shown that upon entry into stationary phase, a quiescent cell population emerges that is heavier in density than nonquiescent cells. Here, we show that total intracellular trehalose and glycogen content exhibits substantial correlation with the density of individual cells both in stationary phase batch cultures and during continuous growth. During prolonged quiescence, trehalose stores are often maintained in favor over glycogen, perhaps to fulfill its numerous stress-protectant functions. Immediately upon exit from quiescence, cells preferentially metabolize trehalose over other fuel sources. Moreover, cells lacking trehalose initiate growth more slowly and frequently exhibit poor survivability. Together, our results support the view that trehalose, which is more stable than other carbohydrates, provides an enduring source of energy that helps drive cell cycle progression upon return to growth.     

Comparative analysis of glycogen and trehalose accumulation in methylotrophic and nonmethylotrophic yeasts

Trehalose and glycogen accumulate in certain yeast species when they are exposed to unfavorable growth conditions. Accumulations of these reserve carbohydrates in yeasts provide resistance to stress conditions. The results of this study indicate that certain Pichia species do not accumulate high levels of glycogen and trehalose under normal growth conditions. However, depending on the Pichia species, both saccharides accumulate at high levels when the Pichia cells are exposed to unfavorable or stress-inducing growth conditions. Growth on glycerol or methanol mostly led to trehalose accumulation in Pichia species tested in this study. It was shown that the metabolic pathways for glycogen and trehalose biosynthesis are present in Pichia species. However, it appears that the biosynthesis of trehalose and glycogen may be regulated in different manners in Pichia species than in the yeast S. cerevisiae.     

Metabolites of the Free-Living Amoeba Phreatamoeba balamuthi Analyzed by 13C- and 31P-NMR Spectroscopy: Occurrence of Phosphoinositol Diphosphates

ABSTRACT. Phreatamoeba balamuthi is a free-living heterotrophic amoeba that lacks mitochondria. Metabolites of axenically-grown cells were characterized by natural-abundance ¹³C-NMR and ³¹P-NMR spectroscopy on acellular perchloric acid extracts. The amoebae were found to contain glycogen and trehalose as storage carbohydrates, together with putrescine and several amino acids, most prominently proline; we propose that proline and trehalose may serve in osmoregulation. Glycerophosphocholine and glycerophosphoethanolamine were present with their phosphomonoester derivatives, phosphocholine and phosphoethanolamine. Along with inorganic phosphate, inorganic pyrophosphate, nucleoside diphosphates, nucleoside triphosphates and NAD, P. balamuthi amoebae also contained unusual phosphoinositol diphosphates in large quantities (0.5 μmol/g wet cells).     

Destabilization of energy-metabolism oscillation in the absence of trehalose synthesis in the chemostat culture of yeast

Energy-metabolism oscillation (EMO) in yeast is basically regulated by a feedback-loop of redox reactions and modulated by the metabolism of storage carbohydrates like glycogen and trehalose. We found that EMO of the transformant tps1Delta deleted of TPS1 encoding trehalose-6-phosphate synthase fluctuated unsteadily with a short wavelength in the absence of trehalose synthesis, while EMO was gradually destabilized with the wavelength increasing as storage in a frozen state was prolonged. During EMO, whereas the fluctuations in levels of the oxygen uptake rate, NAD(P)H and cAMP were attenuated, the glycerol level fluctuated with high amplitude and the levels of glycogen and ethanol fluctuated with similar amplitudes to those in the wild type. Thus, EMO barely operated in tps1Delta depending on the increase of glycerol synthesis as a source of inorganic phosphate in place of trehalose synthesis and fairly conserved fluctuation in the level of ethanol as a synchronizing agent.     

Relationships between mutations affecting protein kinase and accumulation of energy reserves in Saccharomyces cerevisiae

Abstract Independently discovered mutations which alter cyclic-AMP dependent protein kinase activity in Saccharomyces cerevisiae are analysed in relation to trehalose and glycogen storage. The defective trehalose and glycogen accumulation in strains which bear the glc1 mutation results from abnormal activation of trehalase by a protein kinase which has partially lost its cAMP dependence. Cells bearing the bcy1 mutation produce an altered protein kinase due to extremely low levels of the cAMP-binding protein. This altered kinase activates trehalase, resulting in low trehalose contents in these cells. In cell-free extracts of control strains (S288C and 7Q-2D), which produce normal levels of glycogen and trehalose, the enzyme trehalase is mainly found in an inactive, cryptic form. Each of the haploid strains containing one of the mutant genes (glc1, glc4-1 and bcy1) is defective in both trehalose and glycogen accumulation and exhibits low activation ratios of trehalase by protein kinase. Genetic complementation experiments clearly establish that the bcy1 mutation involves a different gene to that altered by the glc1 mutation, since the resulting diploid behaved normally. Strain AM9-10D, previously classified as wild-type (normal for bcy1 ), is defective in the accumulation of trehalose and glycogen and exhibits almost all trehalose in the active form.     

Investigations of the influence of carbon starvation on the carbohydrate storage compounds (trehalose, glycogen), viability, adenylate pool, and adenylate energy charge in Cellulomonas sp. (DSM20108)

During conditions of energy and carbon excess Cellulomonas sp. accumulates intracellularly two different carbohydrate storage products in different relative concentrations: trehalose and glycogen. During carbon starvation these compounds are degraded at different rates and are therefore characterized metabolically by different half-life periods (glycogen 1.6 h, trehalose 34 h). Other parameters which bear some relation to viability during conditions of stress are compared with these half-life periods. The half-life period of the adenylate energy charge EC_A (52 h) is similar to the trehalose half-life period, and it is concluded that it is trehalose which is essential for long-term survival while glycogen is used in the very early stages of carbon starvation to produce energy for metabolism under these conditions. Evidence is presented that two mechanisms are active for the stabilization of the intracellular adenylate energy charge: specific excretion and adenylate degradation.     

Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.     

Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation.

The amounts of glycogen and trehalose have been measured in cells of a prototrophic diploid yeast strain subjected to a variety of nutrient limitations. Both glycogen and trehalose were accumulated in cells deprived specifically of nirogen, sulfur, or phosphorus, suggesting that reserve carbohydrate accumulation is a general response to nutrient limitation. The patterns of accumulation and utilization of glycogen and trehalose were not identical under these conditions, suggesting that the two carbohydrates may play distinct physiological roles. Glycogen and trehalose were also accumulated by cells undergoing carbon and energy limitation, both during diauxic growth in a relatively poor medium and during the approach to stationary phase in a rich medium. Growth in the rich medium was shown to be carbon or energy limited or both, although the interaction between carbon source limitation and oxygen limitation was complex. In both media, the pattern of glycogen accumulation and utilization was compatible with its serving as a source of energy both during respiratory adaptation and during a subsequent starvation. In contrast, the pattern of trehalose accumulation and utilization seemed compatible only with the latter role. In cultures that were depleting their supplies of exogenous glucose, the accumulation of glycogen began at glucose concentrations well above those sufficient to suppress glycogen accumulation in cultures growing with a constant concentration of exogenous glucose. The mechanism of this effect is not clear, but may involve a response to the rapid rate of change in the glucose concentration.     

Glucokinase overexpression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats.

Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats. Glucokinase overexpression using adenovirus restored glucose metabolism in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose 6-phosphate levels increased. Moreover, glycogen deposition was greatly enhanced due to the activation of glycogen synthase. Pyruvate kinase was also activated, and fructose-2,6-bisphosphate concentration and lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but glucokinase overexpression improved their glucose utilization and storage.     

Behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel and performing alcoholic fermentation

Summary The behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel was studied during their continuous function in an ethanol-producing reactor. Polymerization destroys 40% to 80% of the cells, depending on their physiological state. A three day adaptation phase is required before ethanol production stabilizes and this phase corresponds to an increase in cell concentration in the gels and to protein synthesis. The amounts of DNA, glucan, glycogen and trehalose are different in entrapped and free cells. Microscopic observation shows that 75% to 85% of the cells lose their integrity and that the remainder appear to multiply normally. Within a gel particle, both viability and fermentation activity are heterogeneous. A high percentage of cells have low viability and low fermentation activity. A proportion of cells remains capable of forming colonies and these cells have higher fermentation activity and are preferentially localized at the surface of gel particles.     

The Drosophila NR4A nuclear receptor DHR38 regulates carbohydrate metabolism and glycogen storage

Animals balance nutrient storage and mobilization to maintain metabolic homeostasis, a process that is disrupted in metabolic diseases like obesity and diabetes. Here, we show that DHR38, the single fly ortholog of the mammalian nuclear receptor 4A family of nuclear receptors, regulates glycogen storage during the larval stages of Drosophila melanogaster. DHR38 is expressed and active in the gut and body wall of larvae, and its expression levels change in response to nutritional status. DHR38 null mutants have normal levels of glucose, trehalose (the major circulating form of sugar), and triacylglycerol but display reduced levels of glycogen in the body wall muscles, which constitute the primary storage site for carbohydrates. Microarray analysis reveals that many metabolic genes are mis-regulated in DHR38 mutants. These include phosphoglucomutase, which is required for glycogen synthesis, and the two genes that encode the digestive enzyme amylase, accounting for the reduced amylase enzyme activity seen in DHR38 mutant larvae. These studies demonstrate that a critical role of nuclear receptor 4A receptors in carbohydrate metabolism has been conserved through evolution and that nutritional regulation of DHR38 expression maintains the proper uptake and storage of glycogen during the growing larval stage of development.     

Factors affecting accumulation and degradation of curdlan, trehalose and glycogen in cultures of Cellulomonas flavigena strain KU (ATCC 53703)

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.     

Effect of glucose starvation on germ-tube production byCandida albicans

By incubating starved and unstarved yeast cells in synthetic media with a pH of 4.5 or 6.7 at 37°C the effect of a 3 hours' glucose starvation on germ-tube production byCandida albicans was evaluated. In addition the endocellular content of total carbohydrates, glycogen, trehalose and proteins after and before the starvation were dosed. The most interesting result was the overcoming of the pH-regulated dimorphism, thanks to the starvation treatment. Infact the starved cultures produced germtubes indifferently in neutral or acid media, whereas the filamentation of the unstarved cultures was more copious in pH 6.7 medium. The endocellular content of trehalose and protein was unchanged, whereas total carbohydrates and glycogen showed a shortage after the 3 hours' glucose starvation. The possible involvements of these metabolic changes in the regulation of dimorphic transition are discussed.