Effect of cAMP of glycolysis and glycogenolysis in the liver and adrenals of white rats

The rates of glycolysis and glycogenolysis an the rate of lactate formation from glucoso-6-phosphate (G-6-Ph) in the liver were reduced during stress (starvation). On the contrary, these activities in the adrenals were increased. The rates of lactate formation from fructose diphosphate remained unchanged in both organs. The results obtained attest to the inhibition in the liver and activation in the adrenals of phosphorylase, hexokinase and phosphofructokinase. The degree of hexokinase inhibition in the liver depended on the presence of cAMP, ATP and MgCl2 in the incubation medium and was a consequence of enzymatic phosphorylation. Unlike 2', 3'-AMP, the inhibitory effect of CAMP was highly specific. The protein inhibitor of protein kinase completely reversed the inhibitory effect of cAMP on hexokinase. In the adrenals, cAMP slightly increased the rates of glycolysis and lactate formation from G-6-Ph because of allosteric effects of cAMP. The activation rather than inhibition of glycolysis in the adrenals during stress is probably caused by the absence in this tissue of cAMP-dependent protein kinase which phosphorylates hexokinase.     

Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats

We examined the effects of high-fat diet (HFD) and exercise training on insulin-stimulated whole body glucose fluxes and several key steps of glucose metabolism in skeletal muscle. Rats were maintained for 3 wk on either low-fat (LFD) or high-fat diet with or without exercise training (swimming for 3 h per day). After the 3-wk diet/exercise treatments, animals underwent hyperinsulinemic euglycemic clamp experiments for measurements of insulin-stimulated whole body glucose fluxes. In addition, muscle samples were taken at the end of the clamps for measurements of glucose 6-phosphate (G-6-P) and GLUT-4 protein contents, hexokinase, and glycogen synthase (GS) activities. Insulin-stimulated glucose uptake was decreased by HFD and increased by exercise training (P < 0.01 for both). The opposite effects of HFD and exercise training on insulin-stimulated glucose uptake were associated with similar increases in muscle G-6-P levels (P < 0.05 for both). However, the increase in G-6-P level was accompanied by decreased GS activity without changes in GLUT-4 protein content and hexokinase activities in the HFD group. In contrast, the increase in G-6-P level in the exercise-trained group was accompanied by increased GLUT-4 protein content and hexokinase II (cytosolic) and GS activities. These results suggest that HFD and exercise training affect insulin sensitivity by acting predominantly on different steps of intracellular glucose metabolism. High-fat feeding appears to induce insulin resistance by affecting predominantly steps distal to G-6-P (e.g., glycolysis and glycogen synthesis). Exercise training affected multiple steps of glucose metabolism both proximal and distal to G-6-P. However, increased muscle G-6-P levels in the face of increased glucose metabolic fluxes suggest that the effect of exercise training is quantitatively more prominent on the steps proximal to G-6-P (i.e., glucose transport and phosphorylation).     

Action of cytochalasin A, a sulfhydryl-reactive agent, on sugar metabolism and membrane-bound adenosine triphosphatase of yeast.

Cytochalasin A at 10-20 mug/ml inhibits growth and sugar uptake by Saccharomyces strain 1016. The effects of cytochalasin A in intact cells were completely prevented when 1 mM cysteine or dithiothreitol was added along with cytochalasin A, but were not eliminated by thiols added after inhibition had occurred. Purified yeast hexokinase, glucose-6-P dehydrogenase, phosphofructokinase and aldolase were not sensitive to cytochalasin A (20 mug/ml). Glyceraldehyde-3-P dehydrogenase was strongly inhibited by cytochalasin A (5 mug/ml); activity was promptly restored by thiols. Anaerobic glycolysis was inhibited by cytochalasin A or by iodoacetate; unlike iodoacetate, cytochalasin A did not cause accumulation of sugar phosphates. In contrast, cytochalasin A, but not iodoacetate, inhibited isolated membrane-bound ATPases. Cytochalasin A is a sulfhydryl-reactive agent and has membrane-related effects (adenosine triphosphatase) which may well be the basis of its interference with energy-dependent uptake of solutes.     

Carbohydrate metabolism in human skeletal muscle during exercise is not regulated by G-1,6-P2

Glucose 1,6-bisphosphate (G-1,6-P2) is a potent activator of phosphofructokinase (PFK) and an inhibitor of hexokinase in vitro. It has been suggested that increases in G-1,6-P2 are a main means by which PFK can achieve significant catalytic function in vivo despite falling pH and that increases in G-1,6-P2 will inhibit hexokinase in vivo. The purpose of the present study was to determine whether contraction-induced changes in flux through PFK and hexokinase are associated with changes in G-1,6-P2 in skeletal muscle. Ten men performed bicycle exercise for 10 min at 40 and 75% of maximal O2 uptake (VO2max) and to fatigue [4.8 +/- 0.6 (SE) min] at 100% VO2max. Biopsies were obtained from the quadriceps femoris muscle at rest and after each work load and analyzed for G-1,6-P2. G-1,6-P2 averaged 111 +/- 13 mumol/kg dry wt at rest and 121 +/- 16, 123 +/- 15, and 123 +/- 11 mumol/kg dry wt after the low-, moderate-, and high-intensity exercise bouts, respectively (P less than 0.05 for all means vs. rest). Flux through PFK was estimated to increase exponentially as the exercise intensity increased and muscle pH decreased at the higher work loads, whereas flux through hexokinase was estimated to increase during exercise at 40 and 75% VO2max but decrease sharply at 100% VO2max. These data demonstrate that flux through neither PFK nor hexokinase is mediated by changes in G-1,6-P2 in human skeletal muscle during short-term dynamic exercise.     

Cardiac phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase increases glycolysis, hypertrophy, and myocyte resistance to hypoxia

During ischemia and heart failure, there is an increase in cardiac glycolysis. To understand if this is beneficial or detrimental to the heart, we chronically elevated glycolysis by cardiac-specific overexpression of phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) in transgenic mice. PFK-2 controls the level of fructose-2,6-bisphosphate (Fru-2,6-P2), an important regulator of phosphofructokinase and glycolysis. Transgenic mice had over a threefold elevation in levels of Fru-2,6-P2. Cardiac metabolites upstream of phosphofructokinase were significantly reduced, as would be expected by the activation of phosphofructokinase. In perfused hearts, the transgene caused a significant increase in glycolysis that was less sensitive to inhibition by palmitate. Conversely, oxidation of palmitate was reduced by close to 50%. The elevation in glycolysis made isolated cardiomyocytes highly resistant to contractile inhibition by hypoxia, but in vivo the transgene had no effect on ischemia-reperfusion injury. Transgenic hearts exhibited pathology: the heart weight-to-body weight ratio was increased 17%, cardiomyocyte length was greater, and cardiac fibrosis was increased. However, the transgene did not change insulin sensitivity. These results show that the elevation in glycolysis provides acute benefits against hypoxia, but the chronic increase in glycolysis or reduction in fatty acid oxidation interferes with normal cardiac metabolism, which may be detrimental to the heart.     

Effects of insulin and work on fructose 2,6-bisphosphate content and phosphofructokinase activity in perfused rat hearts

The effects of insulin and increased cardiac work on glycolytic rate, metabolite content, and fructose 2,6-bisphosphate (Fru-2,6-P2) content were studied in isolated perfused rat hearts. Steady-state rates of glycolysis increased 5-fold with the addition of insulin to the perfusate or by increasing cardiac pressure-volume work and correlated well in most conditions with changes in substrate concentration (Fru-6-P) and with concentration of the activator, Fru-2,6-P2. There was no correlation with changes in other well known regulators including citrate, ATP, AMP, Pi, or cytosolic phosphorylation potential. Using phosphofructokinase purified from hearts perfused under identical conditions, allosteric kinetic experiments were performed using the metabolite and effector concentrations determined from in vivo experiments. Reaction rates for phosphofructokinase calculated in vitro agreed well with the glycolytic rates measured in vivo and correlated with changes in Fru-6-P but not with other effectors. However, higher Fru-2,6-P2 levels were more effective in maintaining phosphofructokinase activity at high ATP and citrate levels. Kinetic experiments did not indicate a covalent modification of phosphofructokinase. These data indicate that control of cardiac phosphofructokinase and glycolysis may be accomplished by changes in the availability of substrate, Fru-6-P, and activator, Fru-2,6-P2, rather than by citrate, adenine nucleotides, or cytosolic phosphorylation potential as previously suggested.     

Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase; the role of glucose 1,6-bisphosphate.

ATP and citrate, the well known inhibitors of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), were found to inhibit the activities of the multiple forms of phosphoglucomutase (alpha-D-glucose 1,6-bisphosphate: alpha-D-glucose 1-phosphate phosphotransferase, EC 2.7.5.1) from rat muscle and adipose tissue. This inhibition could be reversed by an increase in the glucose 1,6-bisphosphate (Glc-1,6-P2) concentration. Other known activators (deinhibitors) of phosphofructokinase, viz. cyclic AMP, AMP, ADP or Pi, had no direct deinhibitory action on the ATP or citrate inhibited multiple phosphoglucomutases. Cyclic AMP and AMP, could however lead indirectly to deinhibition of the phosphoglucomutases, by activating phosphofructokinase which catalyzes the ATP-dependent phosphorylation of glucose 1-phosphate to form Glc-1,6-P2, the la-ter then released the multiple phosphoglucomutases from ATP or citrate inhibition. The Glc-1,6-P2 was also found to exert a selective inhibitory effect on hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type II, the predominant form in skeletal muscle. This selective inhibition by Glc-1,6-P2 was demonstrated on the multiple hexokinases which were resolved by cellogel electrophoresis or isolated by chromatography on DEAE-cellulose. Based on the in vitro studies it is suggested that during periods of highly active epinephrine-induced glycogenolysis in muscle, the Glc-1,6-P2, produced by the cyclic AMP-stimulated reaction of phosphofructokinase with glucose 1-phosphate, will release the phosphoglucomutases from ATP or citrate inhibition, and will depress the activity of muscle type II hexokinase.     

On the mechanism of glycolysis stimulation by mitochondria from Guérin epithelioma

The experiments were performed to determine the factor(s) responsible for the stimulatory effect on glycolysis in the cytosol (post-microsomal supernatant) of mitochondria isolated from Guérin epithelioma. It was found that epithelioma mitochondria contain bound hexokinase which constitutes about 50% of the total cellular hexokinase activity. The solubilized and partially purified enzyme, when added to the cytosol, stimulated glycolysis. The stimulatory effect of mitochondria on glycolysis was associated with the decrease of adenylate energy charge which was caused by an apparently very fast production of ADP in the hexokinase reaction. A large part of ATP hydrolyzed in this process was converted to IMP and NH3, which can additionally stimulate glycolysis through its stimulatory effect on phosphofructokinase. It is therefore suggested that the stimulatory effect of epithelioma mitochondria on glycolysis can be explained by production of ADP by the hexokinase associated with these mitochondria.     

Glycolytic enzymes in the premolt field crab Paratelphusa hydrodromus (Milne-Edwards) (Crustacea)

The quantitative assay of hexokinase (HK), phosphorylase, phosphofructokinase (PFK), glucose 6-phosphate dehydrogenase (G-6-PDH), glycerol 3-phosphate dehydrogenase (G-3 PDH) and lactate dehydrogenase (LDH) revealed that coxal muscles compared to hepatopancreas contained higher activities of all the enzymes investigated. It appears that the coxal muscles of the premolt field crab has carbohydrate-based fuel economy. The hepatopancreas is a rich source of lipid and very poor source of glycogen. The activity of G-6-PDH is moderately high in the hepatopancreas. It seems that in this lipogenic tissue conversion of G-6-P to triose phosphate occurs predominately via pentose-phosphate pathway thus generating NADPH for lipogenesis. The relative G-3PDH ad LDH activities in hepatopancreas and coxal muscles led us to believe that the reconversion of NAD from NADH in hepatopancreas nd muscle flexor is effected by glycerol 3-phosphate shuttle, whereas in muscle extensor it is achieved by both G-3PDH and LDH activities.     

Metabolite Concentrations and Phase Relations in d-Fructose Induced Glycolytic Oscillations

The frequency of glycolytic oscillations in yeast cells is only slightly modified by the consumption rate of either glucose or fructose, but it is reduced to 1 /2 to 2/3 if the ketohexose is the only substrate. Phase relations between NADH, adenine nucleotides and sugar phosphates (G-6-P, F-6-P, FDP, DAP) are independent of the hexose fermented. With fructose as a substrate the amplitudes of adenylates and hexose phosphates are distinctly smaller than with glucose. The maximum of G-6-P is higher with glucose and the minimum concentration of FDP is higher with fructose. In the transition to anaerobiosis FDP, adenosine 5′-diphosphate and adenosine 5′-phosphate are extremely high, whereas G-6-P, F-6-P and ATP concentrations are lower if fructose is the substrate fermented. The results are indicative for different control characteristics of the phosphofructokinase step, but on their basis it cannot be distinguished between a direct interaction of fructose (or one of its derivatives) on the phosphofructokinase kinetics or the existence of a bypass for fructose which might be able to withdraw a part of the substrate from the control point at the phosphofructokinase.     

The enzyme activity of carbohydrate metabolism in the liver of swine during the transition from prenatal to postnatal development

Studies have ben been made on the activity of hexokinase, glucokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, as well as NADP-dependent dehydrogenases (malate and citrate) in the liver of foetuses and newborn piglets in relation to their age, fasting and reaction to injection of adaptive hormones (insulin and cortisol). It was shown that postpartum adaptation of carbohydrate metabolism in porcine liver is associated with activation of the glycolysis and with the increase in the activity of NADPH-generating dehydrogenases. In fasting newborn piglets the rate of carbohydrate catabolism increases. The effects of the investigated factors are different in the liver of 1-day piglets (sensitive to fasting) and 5-day animals (less sensitive). In is suggested that low ability of newborn piglets to maintain physiological level of glucose in the blood is associated with active glycolysis in the liver and ineffectiveness of the hormone-substrate mechanisms which control tissue glycaemia.     

Glucose metabolism and template synthesis in the mitotic cycle of human diploid fibroblasts

The correlation between the rates of protein and nucleic acid synthesis and the activity of the key enzymes of glycolysis (hexokinase, phosphofructokinase) and pentose phosphate cycle (glucose-6-phosphate dehydrogenase) in the mitotic cycle of human diploid fibroblasts synchronized by double thymidine block was studied. It was found that the removal of the thymidine block is followed by short-term (presumably, non-specific) simultaneous stimulation of matrix syntheses, as well as by glycolytic and pentose phosphate cycle enzyme syntheses. By the beginning of the S-phase, all the processes appear to be inhibited, followed by gradual activation of glycolysis and pentose phosphate cycle reactions. The implementation of the cell cycle is concomitant with stepwise transitions of protein and hexokinase synthesis rates and ATP content to one of the following levels--basal, intermediate or maximal. Changes in the activity of glucose-6-phosphate dehydrogenase in the course of the cell cycle appear as oscillations, those in phosphofructokinase as alternative states. At stage M, the oscillatory processes are temporarily quenched, whereas the ATP content occupies an intermediate level. In contrast with diploid fibroblasts, in transformed T9 cells the enzyme activity is much higher, and the fluctuations in activity throughout the cell cycle are less noticeable. Presumably, in transformed cells the enzyme activity is at the maximum level and is not prone to effector regulation.     

Glycolysis in human erythrocytes containing elevated concentrations of 2, 3-P2-glycerate.

In studies on the mechanism of the inhibitory effect of 2, 3-diphosphoglycerate on glycolysis in human erythrocytes, the following results were obtained: 1) Glucose consumption and lactate production are reduced by 70 and 40 per cent relative to normal erythrocytes in red blood cells containing five times the normal amount of 2, 3, -P2-glycerate ("high-diphosphoglycerate" cells) at an extracellular pH of 7.4. The marked dependency of glycolysis on the extracellular pH observed in normal erythrocytes is almost completely lost in the "high-diphosphoglycerate" cells. 2) About 50 per cent of the inhibition of glycolysis in "high-diphosphoglycerate" cells can be accounted for by the 2, 3-P2-glycerate-induced decrease of the red-cell pH. This fall of the red-cell pH which occurs as a conswquence of the Donnan effect of the non-pentrating 2, 3-P2-glycerate anion leads to a reduction of the glycolytic rate due to the properties of the enzyme phosphofructokinase. 3) The remaining part of the inhibitory effect must be attributed to an inhibition by 2, 3-P2-glycerate of glycolytic enzymes. From measurements of glycolytic rates and of the concentrations of glycolytic intermediates in the absence and presence of methylene blue it is concluded that the hexokinase reaction is inhibited by an elevation of 2, 3-P2-glycerate concentration in "high-diphosphoglycerate" cells suggests that also the enzyme pyruvate kinase is inhibited by 2, 3-P2-glycerate. 4) The dependencies of net-change of 2, 3-P2-glycerate concentration on the red-cell pH are identical in normal and "high-diphosphoglycerate" cells indicating that the balance between formation and decomposition of 2, 3-P2-glycerate is the same in erythrocytes with normal and very high compositions of 2, 3-P2-glycerate.     

Selective tonic inhibition of G-6-Pase catalytic subunit, but not G-6-P transporter, gene expression by insulin in vivo

The regulation of glucose-6-phosphatase (G-6-Pase) catalytic subunit and glucose 6-phosphate (G-6-P) transporter gene expression by insulin in conscious dogs in vivo and in tissue culture cells in situ were compared. In pancreatic-clamped, euglycemic conscious dogs, a 5-h period of hypoinsulinemia led to a marked increase in hepatic G-6-Pase catalytic subunit mRNA; however, G-6-P transporter mRNA was unchanged. In contrast, a 5-h period of hyperinsulinemia resulted in a suppression of both G-6-Pase catalytic subunit and G-6-P transporter gene expression. Similarly, insulin suppressed G-6-Pase catalytic subunit and G-6-P transporter gene expression in H4IIE hepatoma cells. However, the magnitude of the insulin effect was much greater on G-6-Pase catalytic subunit gene expression and was manifested more rapidly. Furthermore, cAMP stimulated G-6-Pase catalytic subunit expression in H4IIE cells and in primary hepatocytes but had no effect on G-6-P transporter expression. These results suggest that the relative control strengths of the G-6-Pase catalytic subunit and G-6-P transporter in the G-6-Pase reaction are likely to vary depending on the in vivo environment.     

Sequence of insulin effects on cytoskeletal and cytosolic phosphofructokinase, mitochondrial hexokinase, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels, and the antagonistic action of calmodulin inhibitors, in diaphragm muscle.

1. Time-curves of insulin effects on energy-producing systems in different cellular compartments of rat diaphragm muscle have revealed: (a) a rapid (within minutes) and transient stimulatory effect of insulin on cytoskeletal phosphofructokinase and aldolase and mitochondrial hexokinase. (b) A slower and consistent stimulatory effect on glucose 1,6-bisphosphate level, with concomitant gradual activation of cytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin. (c) Lactate concentration correlated with the stimulation of cytoskeletal and cytosolic glycolysis. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source of ATP for the rapid actions of insulin, whereas cytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.     

Increase in glucose 1,6-bisphosphate levels, activation of phosphofructokinase and phosphoglucomutase, and inhibition of glucose 1,6-bisphosphatase in muscle induced by trifluoperazine

Injection of trifluoperazine (TFP) to rats induced a significant rise in the level of glucose 1,6-bisphosphate (Glc-1,6-P2) in muscle. This increase in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a marked activation of both enzymes, when assayed in the absence of exogenous Glc-1,6-P2 under conditions in which these enzymes are sensitive to regulation by endogenous Glc-1,6-P2. Glucose-1,6-bisphosphatase (the enzyme that degrades Glc-1,6-P2) was markedly inhibited following the injection of TFP, which may account for the rise in the Glc-1,6-P2 level. Previous results from this laboratory have revealed that muscle damage or weakness is characterized by a decrease in Glc-1,6-P2 levels, leading to a marked reduction in the activities of phosphoglucomutase and phosphofructokinase (the rate-limiting enzyme in glycolysis). The present results suggest that TFP treatment may have a beneficial effect on the depressed glycolysis in muscle weakness or damage.     

Activities of glycolytic enzymes in rapidly proliferating and differentiated C6 glioma cells

Comparisons of glycolytic enzymes between rapidly proliferating and Bt2 cAMP-induced differentiated C6 glioma cells have been made. Rapidly proliferating cells had higher concentrations of glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-bisphosphate compared to morphologically differentiated cells. Under maximally activating conditions, the specific activity and Vmax of hexokinase and phosphofructokinase enzymes were reduced by approximately 3- and 28-fold, respectively, in differentiated cells, without any change in Km values. These results suggest that hexokinase and phosphofructokinase occupy special control positions and the rate of glycolysis is correlated with cellular proliferation of C6 glioma cells.     

Regulation of glycolysis and level of the Crassulacean acid metabolism

Glycolysis shows different patterns of operation and different control steps, depending on whether the level of Crassulacean acid metabolism (CAM) is low or high in the leaves of Kalanchoe blossfeldiana v.Poelln., when subjected to appropriate photoperiodic treatments: at a low level of CAM operation all the enzymes of glycolysis and phosphoenol pyruvate (PEP) carboxylase present a 12 h rhythm of capacity, resulting from the superposition of two 24h rhythms out of phase; phosphofructokinase appears to be the main regulation step; attainment of high CAM level involves (1) an increase in the peak of capacity occurring during the night of all the glycolytic enzymes, thus achieving an over-all 24h rhythm, in strict allometric coherence with the increase in PEP carboxylase capacity, (2) the establishment of different phase relationships between the rhythms of enzyme capacity, and (3) the control of three enzymic steps (phosphofructokinase, the group 3-P-glyceraldehyde dehydrogenase — 3-P-glycerate kinase, and PEP carboxylase). Results show that the hypothesis of allosteric regulation of phosphofructokinase (by PEP) and PEP carboxylase (by malate and glucose-6-P) cannot provide a complete explanation for the temporal organization of glycolysis and that changes in the phase relationships between the rhythms of enzyme capacity along the pathway and a strict correlation between the level of PEP carboxylase capacity and the levels of capacity of the glycolytic enzymes are important components of the regulation of glycolysis in relation to CAM.Abbreviations CAM crassulacean acid metabolism - F-6-P fructose-6-phosphate - F-bi-P fructose-1,6 biphosphate - G-3-PDH 3-phosphoglyceraldehyde dehydrogenase (NAD), EC 1.2.1.12 - G-6-P glucose-6-phosphate - GSH reduced glutathion - GDH glycerolphosphate dehydrogenase, EC 1.1.1.8 - PEP phosphoenol pyruvate - PEPC PEP carboxylase, EC 4.1.1.31 - PFK phosphofructokinase, EC 2.7.1.11 - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PGM phosphoglycerate phosphomutase, EC 5.4.2.1 - T.P. triose phosphates - TPI triose phosphate isomerase, EC 5.3.1.1     

Control of glycolysis by orthophosphate and adenosine triphosphate in soluble extracts of germinating pea seeds

Summary Control of aerobic glycolysis by adenosine triphosphate and orthophosphate has been studied in cell-free extracts of germinating pea seeds. Orthophosphate accelerates glycolysis under all conditions studied. At high concentrations of magnesium ion ATP accelerates glycolysis, whereas at lower magnesium concentrations ATP severely inhibits glycolysis. The inhibitory effect of ATP is markedly relieved by orthophosphate. Metabolite analyses suggest an important regulatory role of phosphofructokinase and show that low ratios of F-6-P: FDP accompany the appearance of a high rate of glycolysis, and vice versa. Thus, ATP raises the F-6-P: FDP ratio at low magnesium levels, while P_i lowers this ratio. At high Mg²⁺ (where ATP accelerates glycolysis), ATP causes a low F-6-P: FDP ratio to appear. At low Mg²⁺ concentration, orthophosphate accelerates glycolysis by activation of phosphofructokinase; at high magnesium concentration, the chief effect of orthophosphate is its long-known role in facilitating the oxidation of triose phosphate.