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• W2018541716 abstract "In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the “Crabtree effect.” We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into “Crabtree-positive,” where the Crabtree effect occurs, and “Crabtree-negative,” where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect. In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the “Crabtree effect.” We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into “Crabtree-positive,” where the Crabtree effect occurs, and “Crabtree-negative,” where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect. In aerobic organisms, glycolysis and oxidative phosphorylation are coordinated to fulfill the cell energy demand. In some conditions, such as glucose addition to the cells, one can observe an increase in glycolytic flux, whereas respiration is inhibited. This has been observed in tumoral cells (1.Crabtree H.G. Biochem. J. 1929; 22: 1289-1298Crossref Google Scholar), nontumoral proliferating cells (2.Greiner E.F. Guppy M. Brand K. J. Biol. Chem. 1994; 269: 31484-31490Abstract Full Text PDF PubMed Google Scholar), some bacteria (3.Mustea I. Muresian T. Cancer. 1967; 20: 1499-1501Crossref PubMed Scopus (12) Google Scholar), and some yeast species (4.Van Urk H. Voll W.S. Scheffers W.A. Van Dijken J.P. Appl. Environ. Microbiol. 1990; 56: 281-287Crossref PubMed Google Scholar). In all of these cases, glucose induces a transition to a mostly fermentative metabolism. This phenomenon has been named the “Crabtree effect,” after its discoverer (1.Crabtree H.G. Biochem. J. 1929; 22: 1289-1298Crossref Google Scholar). The physiological events that could clearly explain the occurrence of the Crabtree effect are currently unknown, although many hypotheses have been laid (4.Van Urk H. Voll W.S. Scheffers W.A. Van Dijken J.P. Appl. Environ. Microbiol. 1990; 56: 281-287Crossref PubMed Google Scholar, 5.Gatt S. Racker E. J. Biol. Chem. 1959; 234: 1015-1023Abstract Full Text PDF PubMed Google Scholar, 6.Chapman C. Bartley W. Biochem. J. 1969; 111: 609-613Crossref PubMed Scopus (15) Google Scholar, 7.Rodriguez-Enriquez S. Juarez O. Rodriguez-Zavala J.S. Moreno-Sanchez R. Eur. J. Biochem. 2001; 268: 2512-2519Crossref PubMed Scopus (106) Google Scholar). It has been proposed, for instance, that it could originate from a competition between mitochondria and glycolytic enzymes for free ADP and inorganic phosphate (5.Gatt S. Racker E. J. Biol. Chem. 1959; 234: 1015-1023Abstract Full Text PDF PubMed Google Scholar, 8.Sanchez N.S. Calahorra M. González-Hernandez J.C. Peña A. Yeast. 2006; 23: 361-374Crossref PubMed Scopus (29) Google Scholar). Indeed, the respiration of isolated mitochondria is decreased in the presence of ADP-consuming systems, such as reconstituted glycolysis or the phosphocreatine/creatine kinase system (5.Gatt S. Racker E. J. Biol. Chem. 1959; 234: 1015-1023Abstract Full Text PDF PubMed Google Scholar). Nevertheless, after glucose addition, ADP levels remain constant or even increase in yeast (6.Chapman C. Bartley W. Biochem. J. 1969; 111: 609-613Crossref PubMed Scopus (15) Google Scholar, 9.Beauvoit B. Rigoulet M. Bunoust O. Raffard G. Canioni P. Guerin B. Eur. J. Biochem. 1993; 214: 163-172Crossref PubMed Scopus (45) Google Scholar) and hepatoma cells (7.Rodriguez-Enriquez S. Juarez O. Rodriguez-Zavala J.S. Moreno-Sanchez R. Eur. J. Biochem. 2001; 268: 2512-2519Crossref PubMed Scopus (106) Google Scholar). Furthermore, in both models, there is a transient decrease in cytoplasmic Pi levels (7.Rodriguez-Enriquez S. Juarez O. Rodriguez-Zavala J.S. Moreno-Sanchez R. Eur. J. Biochem. 2001; 268: 2512-2519Crossref PubMed Scopus (106) Google Scholar, 10.den Hollander J.A. Ugurbil K. Brown T.R. Shulman R.G. Biochemistry. 1981; 20: 5871-5880Crossref PubMed Scopus (176) Google Scholar), pointing to a possible role of Pi or phosphate potential (ΔGp) in this process. It has been proposed that one of the short term events leading to the Crabtree effect is an overflow through pyruvate decarboxylase, since it has been observed that in Crabtree-positive yeast strains, its activity increases after a glucose pulse (4.Van Urk H. Voll W.S. Scheffers W.A. Van Dijken J.P. Appl. Environ. Microbiol. 1990; 56: 281-287Crossref PubMed Google Scholar). Nonetheless, pyruvate decarboxylase seems to be an important bypass of pyruvate dehydrogenase during oxidative metabolism (11.Boubekeur S. Bunoust O. Camougrand N. Castroviejo M. Rigoulet M. Guerin B. J. Biol. Chem. 1999; 274: 21044-21048Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). It was also proposed that changes in mitochondrial outer membrane permeability could be critical for the regulation of the Crabtree effect (12.Zizi M. Forte M. Blachly-Dyson E. Colombini M. J. Biol. Chem. 1994; 269: 1614-1616Abstract Full Text PDF PubMed Google Scholar, 13.Lee A.C. Zizi M. Colombini M. J. Biol. Chem. 1994; 269: 30974-30980Abstract Full Text PDF PubMed Google Scholar). From results obtained with reconstituted systems (12.Zizi M. Forte M. Blachly-Dyson E. Colombini M. J. Biol. Chem. 1994; 269: 1614-1616Abstract Full Text PDF PubMed Google Scholar) and with mitochondria isolated from potato tubers (13.Lee A.C. Zizi M. Colombini M. J. Biol. Chem. 1994; 269: 30974-30980Abstract Full Text PDF PubMed Google Scholar), it was suggested that cytosolic NADH produced by glycolysis could close the voltage-dependent anionic channel and consequently limit the passage of molecules such as ADP toward the intermembrane space. In permeabilized yeast cells, it has been shown that NADH is not involved in the voltage-dependent anionic channel closure and that in situ produced NADH is channeled through voltage-dependent anionic channel to the intermembrane space, where the external NADH dehydrogenases are located (14.Averet N. Aguilaniu H. Bunoust O. Gustafsson L. Rigoulet M. J. Bioenerg. Biomembr. 2002; 34: 499-506Crossref PubMed Scopus (22) Google Scholar). Another possible effector involved in the Crabtree effect is Ca2+ (15.Evtodienko Y.V. Teplova V.V. Duszyński J. Bogucka K. Wojtczak L. Cell Calcium. 1994; 15: 439-446Crossref PubMed Scopus (22) Google Scholar). In Ehrlich ascites tumors and in Zajdela hepatoma cells, it has been observed that there is a glucose-induced increase in cytoplasmic calcium levels along with an enhanced mitochondrial uptake of this cation. Inside the mitochondria, Ca2+ would inhibit ATP synthase by enhancing the interaction with IF1, its inhibitory subunit (16.Wojtczak L. Teplova V.V. Bogucka K. Czyz A. Makowska A. Wieckowski M.R. Duszynski J. Evtodienko Y.V. Eur. J. Biochem. 1999; 263: 495-501Crossref PubMed Scopus (41) Google Scholar). However, is not clear whether this Ca2+ accumulation is a common event in all Crabtree-positive cells, since in AS-D30 hepatoma cells, calcium levels are not modified after glucose addition (7.Rodriguez-Enriquez S. Juarez O. Rodriguez-Zavala J.S. Moreno-Sanchez R. Eur. J. Biochem. 2001; 268: 2512-2519Crossref PubMed Scopus (106) Google Scholar). Thus, to date, no clear experimental results have allowed determination of the early event leading to the Crabtree effect. With regard to this Crabtree effect, yeast species are either negative (e.g. Candida utilis) or positive (e.g. Saccharomyces cerevisiae). Yeast thus constitutes a good experimental model to study this effect. Under its alternative designation of glucose repression, the Crabtree effect has indeed been thoroughly studied in S. cerevisiae, in which short term and long term events have been defined. Regarding the latter class of events, when S. cerevisiae grows using high glucose concentrations as carbon source, it represses oxidative metabolism by down-regulating the synthesis of mitochondrial respiratory chain components and by inhibiting enzymatic activities of the Krebs and glyoxylate cycles. At the same time, the expression of glycolytic enzymes is enhanced (reviewed in Ref. 17.Gancedo J.M. Microbiol. Mol. Biol. Rev. 1998; 62: 334-361Crossref PubMed Google Scholar). Whereas long term effects have been thoroughly studied and are very well understood, the origin of the short term events is ill defined. It has been proposed that during glucose repression, metabolic intermediates could have a regulatory role in both short and long term events, functioning as “metabolic messengers” (18.Thevelein J.M. Yeast. 1994; 10: 1753-1790Crossref PubMed Scopus (314) Google Scholar). For instance, in mutants that accumulate different glycolysis metabolites, the transcription of glycolytic genes increases, pointing to a relationship between internal metabolite levels and enzyme expression (19.Muller S. Boles E. May M. Zimmermann F.K. J. Bacteriol. 1995; 177: 4517-4519Crossref PubMed Google Scholar) (e.g. glucose phosphorylation to glucose 6-phosphate (G6P) 4The abbreviations used are: G6P, glucose 6-phosphate; T6P, trehalose 6-phosphate; F6P, fructose 6-phosphate; JO2, respiratory flux; F16bP, fructose 1,6-bisphosphate. seems to be important for signaling processes induced by glucose (20.Rolland F. Wanke V. Cauwenberg L. Ma P. Boles E. Vanoni M. de Winde J.H. Thevelein J.M. Winderickx J. FEMS Yeast Res. 2001; 1: 33-45PubMed Google Scholar, 21.Colombo S. Ronchetti D. Thevelein J.M. Winderickx J. Martegani E. J. Biol. Chem. 2004; 279: 46715-46722Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 22.Tisi R. Baldassa S. Belotti F. Martegani E. FEBS Lett. 2002; 520: 133-138Crossref PubMed Scopus (56) Google Scholar); also, trehalose 6-phosphate (T6P) might regulate the glycolytic flux by modulating hexokinase activity (23.Blazquez M.A. Lagunas R. Gancedo C. Gancedo J.M. FEBS Lett. 1993; 329: 51-54Crossref PubMed Scopus (271) Google Scholar)). Therefore, a yeast mutant that lacks trehalose 6-phosphate synthase (Tps1p) overaccumulates glycolysis hexose phosphates in response to glucose (24.Thevelein J.M. Hohmann S. Trends Biochem. Sci. 1995; 20: 3-10Abstract Full Text PDF PubMed Scopus (370) Google Scholar). However, it has not been clearly demonstrated whether metabolic intermediates could function as messengers in long or short term regulatory events. In S. cerevisiae, cytoplasmic levels of glycolysis hexose phosphates increase after glucose addition to the culture medium (25.Diderich J.A. Raamsdonk L.M. Kuiper A. Kruckeberg A.L. Berden J.A. Teixeira de Mattos M.J. van Dam K. FEMS Yeast Res. 2002; 2: 165-172PubMed Google Scholar, 26.Ernandes J.R. De Meirsman C. Rolland F. Winderickx J. de Winde J. Brandao R.L. Thevelein J.M. Yeast. 1998; 14: 255-269Crossref PubMed Scopus (38) Google Scholar). Given the possible role of glycolysis intermediates as metabolic messengers, we investigated whether some of these could contribute to the short term Crabtree effect (i.e. function as signaling molecules during glucose-induced repression of oxidative metabolism). In order to study such a process, we took advantage of the existence of Crabtree-positive (S. cerevisiae) and Crabtree-negative (C. utilis) yeast strains. In both kinds of isolated mitochondria, we found that low, physiological concentrations of G6P and fructose 6-phosphate (F6P) stimulated the respiratory flux (JO2), and this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself inhibited mitochondrial respiration only in mitochondria isolated from S. cerevisiae. We also observed that in the yeast mutant Δtps1, which accumulates F16bP in response to glucose addition, the Crabtree effect is enhanced as compared with the parental strain. Moreover, F16bP-mediated inhibition of respiratory flux was also observed in isolated rat liver mitochondria. Based on these results and taking into account that F16bP accumulates in the cell cytoplasm under certain conditions, we propose that F16bP has an effector role in the repression of oxidative metabolism observed in the course of the Crabtree effect. Yeast Strains and Growth Conditions—S. cerevisiae (Yeast Foam, an industrial strain) and C. utilis (laboratory strain) were used for mitochondria preparation. Cultures were obtained by growing cells in YPL medium (1% yeast extract, 0.1% potassium phosphate, 0.12% ammonium sulfate, supplemented with 2% lactate as carbon source, pH 5.5). Yeast cells were harvested in midlog growth phase for spheroplasts and mitochondria preparation. The laboratory strains W303 1-A (wild type: Mat a, ade 2-1, trp1-1, leu 2-3/112, his 3-11-15, ura 3-1, can 1-100, GAL, SUC2) and YSH 648 (Mat a, ade 2-1, trp1-1, leu 2-3/112, his 3-11-15, ura 3-1, can 1-100, GAL, SUC, tps1::TRP1) (27.Noubhani A. Bunoust O. Rigoulet M. Thevelein J.M. Eur. J. Biochem. 2000; 267: 4566-4576Crossref PubMed Scopus (31) Google Scholar) were grown in YPGal medium (1% yeast extract, 0.1% potassium phosphate, 0.12% ammonium sulfate, pH 5.5, supplemented with 2% galactose as carbon source) and collected at 1 unit of optical density. Spheroplasts and Mitochondria Preparation—Spheroplasts were obtained according to Avéret et al. (28.Averet N. Fitton V. Bunoust O. Rigoulet M. Guerin B. Mol. Cell. Biochem. 1998; 184: 67-79Crossref PubMed Google Scholar) and were resuspended in buffer A (1 m sorbitol, 1.7 mm NaCl, 0.5 mm EGTA, 10 mm KCl, 1 mm potassium phosphate, 10 mm Tris-HCl, 4 mm iodoacetate, and 1% bovine serum albumin, pH 6.8). Yeast mitochondria were isolated from spheroplasts as described elsewhere (29.Guérin B. Labbe P. Somlo M. Methods Enzymol. 1979; 55: 149-159Crossref PubMed Scopus (193) Google Scholar), and they were suspended in buffer B (0.6 m mannitol, 5 mm MES, 10 mm KCl, 1 mm potassium phosphate, pH 6.8). Rat liver mitochondria were obtained according to Saavedra-Molina et al. (30.Saavedra-Molina A. Uribe S. Devlin T.M. Biochem. Biophys. Res. Commun. 1990; 167: 148-153Crossref PubMed Scopus (31) Google Scholar) from male Wistar rats weighing 180–200 g and suspended in buffer C (250 mm sucrose, 10 mm Tris-HCl, 1 mm EGTA, pH 7.2). Protein determination was done using the biuret method with BSA as a standard. Respiration Assay—The rate of oxygen consumption was measured in a thermostatically controlled chamber at 28 °C equipped with a Clark electrode connected to a recorder. Spheroplasts (1 mg of protein/ml) or mitochondria (0.3 mg of protein/ml) were suspended in buffer A or B, respectively. NADH (1 mm for mitochondria or 10 mm for spheroplasts) or 100 mm ethanol was used as respiratory substrate. For rat liver mitochondria, 5 mm glutamate/malate was used. In order to achieve proper permeabilization, spheroplasts were incubated for 10 min with nystatin (20 μg/ml) at 28 °C before each experiment. For cytochrome c oxidase-mediated respiration, mitochondria (0.3 mg of protein/ml) were incubated in the presence of antimycin (2.5 μg/mg of protein), 2.5 mm ascorbate, and 100 μmN,N,N′,N′-tetramethyl-p-phenylenediamine. To induce state 3 respiration, ADP was added at the concentration indicated in the figure legends. To determine cellular respiration, galactose-grown cells (wild type W303 1-A and Δtps1) or lactate-grown cells (Yeast Foam and C. utilis) collected in midlog phase were placed in an oxygraph. Determination of Mitochondrial Complex III (Ubiquinol:Cytochrome c Oxidoreductase) Activity—Mitochondria (0.3 mg of protein/ml) were incubated in buffer B in the presence of 1 mm KCN, 2 mm ferricyanide, and 100 mm ethanol as respiratory substrate. Since isolated S. cerevisiae mitochondria do not have a complex I (31.Ohnishi T. Kawaguchi K. Hagihara B. J. Biol. Chem. 1966; 241: 1797-1806Abstract Full Text PDF PubMed Google Scholar) but rather internal and external NADH dehydrogenases that donate their electrons to the quinone pool, the ferricyanide reduction rate in these conditions is representative of the ubiquinol:cytochrome c oxidoreductase activity. Absorbance changes were followed at 436 nm in a Safas spectrophotometer (Monaco). The rate of ferricyanide reduction was calculated from the slope of absorbance change as a function of time. A molar extinction coefficient (ϵ) of 0.21 mm–1 cm–1 was used. Measurement of Mitochondrial Transmembranal Electrical Potential Difference (Δψ) by Fluorescent Probe Distribution—ΔΨ was estimated from fluorescent quenching of the lipophilic cationic dye rhodamine 123 (32.Emaus R.K. Grunwald R. Lemasters J.J. Biochim. Biophys. Acta. 1986; 850: 436-448Crossref PubMed Scopus (732) Google Scholar). Isolated mitochondria (0.3 mg/ml) were incubated in the mitochondrial buffer supplemented with rhodamine 123 (Sigma) (0.5 μg/ml) in the presence of 100 mm EtOH as respiratory substrate. The rhodamine fluorescence signal at each steady state was recorded with a Kontron SFM 25 fluorimeter at 28 °C. The excitation wavelength was 485 nm, and fluorescence emission was continuously collected at 525 nm. Metabolite Separation and Quantification by HPIC (High Performance Ionic Chromatography)—High Performance Ionic Chromatography (HPIC) was carried out on a DX 500 chromatography work station (Dionex, Sunnyvale, CA) equipped with GP50 gradient pump and ED50 electrochemical and UV detectors. System management and data acquisition were controlled through Peaknet 4.3 software (Dionex). Separation and quantification of sugar phosphates was carried out on a CarboPac PA10 column (250 × 4 mm) equipped with a Dionex PA1 guard column according to Ref. 33.Loret M.O. Pedersen L. François J. Yeast. 2007; 24: 47-60Crossref PubMed Scopus (57) Google Scholar. In yeast cells, the addition of large amounts of glucose results in a metabolic shift toward fermentation and in the accumulation of glycolysis hexose phosphates (25.Diderich J.A. Raamsdonk L.M. Kuiper A. Kruckeberg A.L. Berden J.A. Teixeira de Mattos M.J. van Dam K. FEMS Yeast Res. 2002; 2: 165-172PubMed Google Scholar, 26.Ernandes J.R. De Meirsman C. Rolland F. Winderickx J. de Winde J. Brandao R.L. Thevelein J.M. Yeast. 1998; 14: 255-269Crossref PubMed Scopus (38) Google Scholar). Since accumulation of hexose phosphates occurs concomitantly with an inhibition of respiration, we tested the possibility of a direct role of these intermediates in the regulation of mitochondrial oxidative phosphorylation. Each one of the three glycolysis hexose phosphates (G6P, F6P, and F16bP) was tested. Fig. 1 shows the effect of G6P, F6P, and F16bP on nonphosphorylating respiration. The respiration was stimulated in a concentration-dependent manner in the presence of G6P and F6P. Although the stimulation mediated by G6P was considerable, it should be stressed that cytosolic concentrations from 1 to 6 mm have been reported; thus, higher concentrations of G6P are not physiological, and only the stimulation induced by up to 5 mm G6P is physiologically meaningful. In the presence of F6P, this increase was less important, and for physiological concentrations (less than 1 mm), there was no effect. On the other hand, F16bP, at physiological concentrations (2–10 mm) (25.Diderich J.A. Raamsdonk L.M. Kuiper A. Kruckeberg A.L. Berden J.A. Teixeira de Mattos M.J. van Dam K. FEMS Yeast Res. 2002; 2: 165-172PubMed Google Scholar, 26.Ernandes J.R. De Meirsman C. Rolland F. Winderickx J. de Winde J. Brandao R.L. Thevelein J.M. Yeast. 1998; 14: 255-269Crossref PubMed Scopus (38) Google Scholar, 34.Gonzalez M.I. Stucka R. Blazquez M.A. Feldmann H. Gancedo C. Yeast. 1992; 8: 183-192Crossref PubMed Scopus (85) Google Scholar) induced an inhibition of the respiratory rate that reached 25%. This effect is not dependent on the respiratory substrate, since the same effect was also observed when using ethanol as substrate instead of NADH (data not shown). Under phosphorylation conditions, G6P and F6P had no significant effect on the respiratory rate (Fig. 2), whereas the inhibition in the presence of F16bP was still observed (20%). This indicates that the F16bP-mediated inhibition is present regardless of the respiratory state.FIGURE 2Effect of glycolysis-derived hexose phosphates on the respiratory flux of isolated yeast mitochondria under phosphorylating conditions. Reaction mixture and respiratory substrate were the same as in Fig. 1 except that 1 mm ADP was added. Respiratory rates were measured after the addition of various concentrations of glucose 6-phosphate (▪), fructose 6-phosphate (○), and fructose 1,6-bisphosphate (▴), as indicated. Results are expressed as mean values ± S.D. (n = 3). natO, nanoatoms of oxygen.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to determine whether the stimulation mediated by G6P and F6P was due to uncoupling, the mitochondrial transmembrane electrical potential (ΔΨm) was assessed in the presence of each glycolysis hexose phosphate. Up to 20 mm, the addition of either G6P or F6P did not change the transmembrane potential value (Table 1). Furthermore, the ΔΨm did not vary up to 8 mm F16bP and slightly (10 mV) decreased upon the addition of 20 mm F16bP (Table 1). Based on these results, the hypothesis of a possible uncoupling effect induced by G6P and F6P was ruled out. The F16bP-mediated decrease of the respiratory rate in either state, plus the null to slight decrease in ΔΨm strongly suggested that at least one of the mitochondrial respiratory complexes is inhibited.TABLE 1Mitochondrial transmembranal electrical potential difference in the presence of hexose phosphatesΔψ0 mm2 mm4 mm6 mm8 mm10 mm20 mmmVG6P178 ± 4178 ± 3175 ± 5177 ± 4174 ± 5175 ± 4173 ± 3F6P178 ± 4177 ± 5178 ± 5180 ± 4180 ± 3180 ± 5178 ± 3F16bP178 ± 4177 ± 5175 ± 3175 ± 4176 ± 7171 ± 6168 ± 3 Open table in a new tab During the fermentative shift, every hexose phosphate increases in the cytosol. Thus, the mitochondria are in the presence of all of these hexoses at the same time. Therefore, we tested whether the G6P-induced stimulation of the respiratory rate could be inhibited by F16bP. G6P (3 mm)-induced stimulation was reverted by 1.5 mm F16bP (Fig. 3). Moreover, the F16bP (7 mm)-induced inhibition was present (i.e. 25%; 154 nanoatoms of oxygen/min/mg of protein versus 204 nanoatoms of oxygen/min/mg of protein). This indicates that under physiological conditions, the effect of glycolysis hexose phosphates on oxidative phosphorylation is that of F16bP (i.e. an inhibition of the respiratory rate). As mentioned above and in view of the results observed for flux and ΔΨm in the presence of each hexose phosphate (see above), we identified the respiratory chain complex(es) whose activity is affected by these hexose phosphates. Complex IV (cytochrome c oxidase) catalyzes an irreversible step of the respiratory chain and has been identified as an important step for controlling the respiratory fluxes both in phosphorylating and nonphosphorylating conditions (35.Mazat J.P. Jean-Bart E. Rigoulet M. Guerin B. Biochim. Biophys. Acta. 1986; 849: 7-15Crossref Scopus (69) Google Scholar, 36.Rigoulet M. Guerin B. Denis M. Eur. J. Biochem. 1987; 168: 275-279Crossref PubMed Scopus (38) Google Scholar). Complex IV activity is strongly inhibited by F16bP (Fig. 4). This inhibition, which is similar regardless of the respiratory state, is important at low concentrations and reaches ∼30% of inhibition for 5 mm F16bP. Furthermore, G6P and F6P had no effects on cytochrome c oxidase activity (data not shown). We then assessed complex III (ubiquinol:cytochrome c oxidoreductase) activity in the presence of these glycolysis intermediates. Both hexose monophosphates stimulated the basal activity of complex III (Fig. 5). This activation was similar for both hexose monophosphates, reaching a maximal stimulation at ∼150% of the basal value. However, this maximal stimulation was obtained for nonphysiological concentrations of these intermediates, and only a slight stimulation was observed in the presence of physiological (3 mm G6P and 1 mm F6P) concentrations of hexose monophosphates. An F16bP-induced inhibition was also observed on this respiratory complex. Furthermore, hexose phosphate-mediated effects shown in Fig. 5 were sensitive to antimycin A (data not shown), indicating that the ferricyanide reduction rate assessed here was indeed mediated by complex III. Since hexose monophosphates and F16bP have, respectively, stimulatory and inhibitory effects over complex III activity (Fig. 5), we decided to determine whether these antagonistic effects could be observed on the functionally isolated complex III. Indeed, G6P-mediated stimulation of complex III could be reverted by F16bP, and basal activity was almost restored (Fig. 6). To this point, we have used mitochondria isolated from S. cerevisiae as an experimental model. However, to extrapolate our results to the in vivo situation, it was necessary to study mitochondria in a more physiological context. One approach that has been used to undertake bioenergetic studies in situ is the use of permeabilized cells (28.Averet N. Fitton V. Bunoust O. Rigoulet M. Guerin B. Mol. Cell. Biochem. 1998; 184: 67-79Crossref PubMed Google Scholar, 37.Fontaine E.M. Keriel C. Lantuejoul S. Rigoulet M. Leverve X.M. Saks V. Biochem. Biophys. Res. Commun. 1995; 213: 138-146Crossref PubMed Scopus (35) Google Scholar). Nystatin-permeabilized spheroplasts have been successfully employed to study yeast energetic metabolism (28.Averet N. Fitton V. Bunoust O. Rigoulet M. Guerin B. Mol. Cell. Biochem. 1998; 184: 67-79Crossref PubMed Google Scholar, 38.Dejean L. Beauvoit B. Alonso A.P. Bunoust O. Guérin B. Rigoulet M. Biochim. Biophys. Acta. 2002; 1554: 159-169Crossref PubMed Scopus (32) Google Scholar, 39.Chevtzoff C. Vallortigara J. Avéret N. Rigoulet M. Devin A. Biochim. Biophys. Acta. 2005; 1706: 117-125Crossref PubMed Scopus (51) Google Scholar). To determine the effect of F16bP on the mitochondrial respiratory rate in situ, we evaluated nystatin-permeabilized spheroplast respiration in the presence of this hexose. To avoid F16bP metabolism, we omitted the cofactors required for adequate function of the glycolytic enzymes and added iodoacetate in respiration buffer. Fig. 7 shows that in nystatin-permeabilized spheroplasts, F16bP inhibited the nonphosphorylating respiratory rates by 50%. The range of concentrations used to obtain the maximum inhibition was similar to that employed on isolated mitochondria: 6–10 mm for state 4 and 2–4 mm for state 3 (data not shown). Of note, these concentrations are within the physiological range (25.Diderich J.A. Raamsdonk L.M. Kuiper A. Kruckeberg A.L. Berden J.A. Teixeira de Mattos M.J. van Dam K. FEMS Yeast Res. 2002; 2: 165-172PubMed Google Scholar, 26.Ernandes J.R. De Meirsman C. Rolland F. Winderickx J. de Winde J. Brandao R.L. Thevelein J.M. Yeast. 1998; 14: 255-269Crossref PubMed Scopus (38) Google Scholar) (see Table 3). The additions of G6P and F6P were also tested. However, an exceedingly high concentration was required to observe a stimulation of respiratory flux (∼120 mm) (data not shown), indicating that there might be a constraint for metabolite diffusion toward mitochondria or the intermembrane space. A similar situation was observed in permeabilized cells for NADH in yeast (28.Averet N. Fitton V. Bunoust O. Rigoulet M. Guerin B. Mol. Cell. Biochem. 1998; 184: 67-79Crossref PubMed Google Scholar) and in hepatocytes (37.Fontaine E.M. Keriel C. Lantuejoul S. Rigoulet M. Leverve X.M. Saks V. Biochem. Biophys. Res. Commun. 1995; 213: 138-146Cro" @default.
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• W2018541716 title "Mitochondrial Oxidative Phosphorylation Is Regulated by Fructose 1,6-Bisphosphate" @default.
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