The Emi2 Protein of Saccharomyces cerevisiae is a Hexokinase Expressed under Glucose Limitation

Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg ²⁺ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.     

Hexokinase 2 Inhibition and Biological Effects of BNBZ and Its Derivatives: The Influence of the Number and Arrangement of Hydroxyl Groups

Hexokinase 2 (HK2), an enzyme of the sugar kinase family, plays a dual role in glucose metabolism and mediating cancer cell apoptosis, making it an attractive target for cancer therapy. While positive HK2 expression usually promotes cancer cells survival, silencing or inhibiting this enzyme has been found to improve the effectiveness of anti-cancer drugs and even result in cancer cell death. Previously, benitrobenrazide (BNBZ) was characterized as a potent HK2 inhibitor with good anti-cancer activity in mice, but the effect of its trihydroxy moiety (pyrogallol-like) on inhibitory activity and some cellular functions has not been fully understood. Therefore, the main goal of this study was to obtain the parent BNBZ (2a) and its three dihydroxy derivatives 2b–2d and to conduct additional physicochemical and biological investigations. The research hypothesis assumed that the HK2 inhibitory activity of the tested compounds depends on the number and location of hydroxyl groups in their chemical structure. Among many studies, the binding affinity to HK2 was determined and two human liver cancer cell lines, HepG2 and HUH7, were used and exposed to chemicals at various times: 24 h, 48 h and 72 h. The study showed that the modifications to the structures of the new BNBZ derivatives led to significant changes in their activities. It was also found that these compounds tend to aggregate and exhibit toxic effects. They were found to contribute to: (a) DNA damage, (b) increased ROS production, and (c) disruption of cell cycle progression. It was observed that, HepG2, occurred much more sensitive to the tested chemicals than the HUH7 cells; However, regardless of the used cell line it seems that the increase in the expression of HK2 in cancer cells compared to normal cells which have HK2 at a very low level, is a serious obstacle in anti-cancer therapy and efforts to find the effective inhibitors of this enzyme should be intensified.     

Isolation and characterization of a hexokinase mutant of Schwanniomyces castellii: Consequences on cell production in continuous culture

A mutant of Schwanniomyces castellii with reduced glucose phosphorylation and with practically no phosphorylation of fructose was investigated. Carbon catabolite represion of α-glucosidase and amylases was reduced. Repression of β-galactosidase was normal. We have compared in continuous culture this mutant strain with wild type and another previously described mutant. The relationship between the specific rate of glucose consumption (Qs) and residual glucose concentration (s) in an inverse mode, suggests that there may be two types of transport of glucose. Mutation at the phosphorylation level causes apparent modification of the kinetic parameters of glucose uptake rate. The consequence of mutation at the phosphorylation level on biomass production was discussed.     

Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol.     

Limonia fruit as a food supplement to regulate fluoride‐induced hyperglycaemia and hyperlipidaemia

BACKGROUND: Limonia fruit pulp is edible and used in a number of food preparations. This fruit is also used as a folk medicine to treat various ailments and reportedly possesses antihyperglycaemic and antihyperlipidaemic activities. The purpose of the present study was to examine the potential of Limonia acidissima L. (LA) fruit pulp in regulating the carbohydrate and lipid profiles in fluoride‐exposed rats. RESULTS: Exposure to fluoride (100 mg l^?1 NaF) resulted in significant increases in plasma and hepatic carbohydrate and lipid profiles. Administration of LA fruit powder (2.5, 5 and 10 g kg^?1) in the diet for 4 weeks resulted in significant decreases in plasma glucose and lipid profiles and hepatic glucose‐6‐phosphatase activity and significant increases in hepatic glycogen content and hexokinase activity and plasma high‐density lipoprotein cholesterol. Phytochemical analysis of the LA fruit pulp revealed the presence of fibres, phytosterols, saponins, polyphenols, flavonoids and ascorbic acid. CONCLUSION: Consumption of LA fruit pulp is beneficial in controlling hyperglycaemia and hyperlipidaemia in fluoride‐induced toxicity. Since fibres, phytosterols, saponins, polyphenols, flavonoids and total ascorbic acid are known to influence both carbohydrate and lipid metabolisms, the decline in carbohydrate and lipid levels in LA‐administered fluoride‐exposed rats could be attributed to the phytoconstituents of the fruit. Copyright © 2012 Society of Chemical Industry     

Effects of branched-chain amino acids on glucose uptake and lactose synthesis rates in bovine mammary epithelial cells and lactating mammary tissue slices

Even though supplementations of essential AA (EAA) are often related to increased lactose yields in dairy cows, underlying mechanisms connecting EAA availability to the mammary glands and lactose synthesis are poorly understood. The objective of this study was to examine the effects of branched-chain AA (BCAA) including Leu, Ile, and Val on (1) glucose transporter (GLUT1) abundance and glucose uptake, (2) the abundance of proteins regulating lactose synthesis pathway, and (3) fractional synthesis rates of lactose (FSR) using bovine mammary epithelial cells (BMEC) and mammary tissues slices (MTS). The BMEC (n = 4) were allocated randomly to regular Dulbecco's Modified Eagle Medium with Ham's F12 (DMEM/F12) medium (+EAA) or +EAA deficient (by 90%) in all EAA (?EAA), all BCAA (?BCAA), only Leu (?Leu), only Ile (?Ile) or only Val (?Val). Western immunoblotting analyses, depletion of glucose in media, and a proteomic analysis were performed to determine the abundance of GLUT1 in the cell membrane, net glucose uptake, and the abundance of enzymes involved in lactose synthesis pathway in BMEC, respectively. The MTS (n = 6) were allocated randomly to DMEM/F12 medium having all EAA and ¹³C-glucose at concentrations similar to plasma concentrations of cows (+EAA_p), and +EAA_p deprived of all BCAA (?BCAA_p) or only Leu (?Leu_p) for 3 h. The ¹³C enrichments of free glucose pool in MTS (E_Glu-free) and the enrichments of glucose incorporated into lactose in MTS and media [E_{Lactose-bound (T&M)}] were determined and used in calculating FSR. In BMEC, ?BCAA increased the fraction of total GLUT1 translocated to the cell membrane and the fraction that was potentially glycosylated compared with +EAA. Among individual BCAA, only ?Leu was associated with a 63% increase in GLUT1 translocated to the cell membrane and a 40% increase in glucose uptake of BMEC. The ?BCAA tended to be related to a 75% increase in the abundance of hexokinase in BMEC. Deprivation of Leu tended to increase glucose uptake of MTS but did not affect E_Glu-free, E_{Lactose-bound (T&M)}, or FSR relative to +EAA_p. On the other hand, ?BCAAp did not affect glucose uptake of MTS but was related to lower E_{Lactose-bound (T&M)}, or FSR relative to +EAA_p. Considering together, decreasing Leu supply to mammary tissues enhances GLUT1 and thus glucose uptake, which, however, does not affect lactose synthesis rates. Moreover, the deficiency of other BCAA, Ile, and Val alone or together with the deficiency of Leu seemed to decrease lactose synthesis rates without affecting glucose uptake. The data also emphasize the importance of addressing the effect of the supply of other nutrients to the mammary glands than the precursor supply in describing the synthesis of a milk component.     

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (GGS1FDP1BYP1CIF1GLC6TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of d-glucose and an equal concentration of radiolabelled l-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total d-glucose measured (intracellular+periplasmic/extracellular) and the total l-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mm-glucose to 0·5–2 mm in the wild-type strain, ±10 mm in a hxk1Δ hxk2Δ glk1Δ and 2–3 mm in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity. © 1998 John Wiley & Sons, Ltd.     

杏干制过程中己糖激酶和果糖激酶调控糖代谢的研究

以绿熟期杏果为材料,分析了热风干制和自然晒制过程中己糖激酶、果糖激酶、蔗糖相关酶活性及糖含量的变化规律,旨在明确干制过程中关键酶调控糖代谢的作用机制。结果表明,果糖、葡萄糖、蔗糖占可溶性总糖的比例在干制前分别为33.52%、47.25%、45.22%,干制后分别平均为9.70%、28.24%、54.16%,果糖、葡萄糖和蔗糖呈显著负相关,因此干制条件下杏果中蔗糖的积累主要来源于己糖的转化。干制期间杏果中分解类酶活性(NI、SSs、AI)降低,且与果糖、葡萄糖呈正相关,分解类酶的调控以NI和AI为主。蔗糖合成类酶(SPS、SSs)在蔗糖代谢中起到辅助调控作用且热风40~50℃温度处理能有效地提高其活性,使蔗糖含量及蔗糖占可溶性总糖的比例增大。果糖激酶和己糖激酶也在干制前期略有升高,高活性的果糖激酶和己糖激酶利于增加果实中己糖的消耗,为蔗糖的积累提供基础。     

Human pancreatic glucokinase (GlkB) complements the glucose signalling defect of Saccharomyces cerevisiae hxk2 mutants

Human pancreatic glucokinase (GlkB, hexokinase IV) has been expressed in Saccharomyces cerevisiae. The recombinant protein showed similar enzyme kinetics to those described for the original enzyme. When expressed in hxk2 yeast mutants, GlkB complemented both the glucose induction and the glucose repression defects present in the mutant. It was also functional in regulating the activity of the Snf1 kinase complex in response to glucose, participating in the regulation of the Reg1/Glc7 phosphatase complex, as its yeast counterpart.