Effects of diabetes on fructose 2, 6-P2, glucose 1, 6-P2 and 6-phosphofructo 2-kinase in rat liver

The levels of fructose 2,6-P2 and 6-phosphofructo 2-kinase have been found to be decreased in the liver of both ketotic and non-ketotic diabetic rats, a good correlation between fall of hepatic fructose 2,6-P2, ketonemia and glycemia being observed. The "total" 6-phosphofructo 2-kinase activity and the "active" (non-phosphorylated) from of the enzyme were decreased to a different extent, resulting in a fall of the "active"/"total" activity ratio. Hepatic levels of glucose 1,6-P2 were lowered only in ketotic diabetes. Insulin treatment normalized all the values studied. Insulin administration to control rats decreased the hepatic levels of fructose 2,6-P2 and did not affect glucose 1,6-P2 levels. It also decreased the "active" form of 6-phosphofructo 2-kinase, without significantly altering the "total" activity.     

Metabolism of glucose 1,6-P2. I. Enzymes involved in the synthesis of glucose 1,6-P2 in pig tissues

Pig tissues show four enzymatic activities of glucose 1,6-P2 synthesis: (A) 2 [glucose 1-P]----glucose 1,6-P2 + glucose; (B) glucose 1-P + ATP----glucose 1,6-P2 + ADP; (C) glucose 1-P + fructose 1,6-P2----glucose 1,6-P2 + fructose 6-P; (D) glucose 1-P + glycerate 1,3-P2----glucose 1,6-P2 + glycerate 3-P. Brain is the tissue with highest capability of glucose 1,6-P2 synthesis. With the exception of skeletal muscle, activity "D" represents the highest activity of glucose 1,6-P2 synthesis. In muscle, activity "B" is the major activity. The existence of a specific glucose 1,6-P2 synthase which catalyzes reaction "D" is confirmed. Two peaks of such an enzyme are isolated by ion-exchange chromatography. There is an enzyme which specifically catalyzes reaction "C", not previously described. There is a glucose 1-P kinase not identical to phosphofructokinase.     

Vanadate activates pentose phosphate pathway and glycolysis, and raises fructose 2,6-bisphosphate concentration in slices of lactating rat mammary gland.

In mammary gland slices from lactating rats, vanadate increased the rate of glucose oxidation via the pentose phosphate pathway by 36% and raised the glucose flux via glycolysis by 47%. Furthermore, vanadate increased the fructose 2,6-bisphosphate (Fru-2,6-P2) level by 33%. The effect of vanadate on glucose oxidation was compared to the effect of insulin. The present data indicate that 0.5mM vanadate has an effect on glucose utilization similar to that of insulin but does not reach the same level.     

Metabolism of glucose 1,6-P2--III. Partial purification and characterization of glucose 1,6-P2 synthase from pig skeletal muscle

1. Glycerate 1,3-P2-dependent glucose, 1,6-P2 synthase has been purified 2000-fold from pig skeletal muscle, with a yield of 75%. 2. The enzyme possesses fructose 1,6-P2-dependent glucose 1,6-P2 synthase and phosphoglucomutase activities, which represent 0.1 and 60% of the main activity, respectively. 3. Both glucose 1-P and glucose 6-P can act as acceptors of the phosphoryl group from glycerate 1,3-P2. 4. The Km values are 19 microM and 67 nM for glucose 1-P and glycerate 1,3-P2, respectively. 5. The enzyme is inhibited by glycerate 2,3-P2, fructose 1,6-P2, glycerate 3-P, phosphoenolpyruvate and lithium, the inhibition pattern varying with the compound.     

Glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels in different types of rat skeletal muscle

1. The concentration of glycogen, glucose 1,6-P2, fructose 2,6-P2 and the content of glycogen phosphorylase, phosphofructokinase, 6-phosphofructo 2-kinase and glucose 1,6-P2 phosphatase activity, have been determined in rat muscles which differ in their fiber composition: extensor digitorum longus, gastrocnemius, diaphragm and soleus. 2. Glucose 1,6-P2 concentration seems to be related to the glycolytic capacity of the muscle, while fructose 2,6-P2 concentration does not. 3. No significant relationship exists between the fiber type and the content in glucose 1,6-P2 phosphatase and 6-phosphofructo 2-kinase activities.     

Metabolism of glucose 1,6-P2--II. Glucose 1,6-P2 phosphatase in pig muscle

Most of the glucose 1,6-P2 phosphatase activity of pig skeletal muscle is present in the cytosolic fraction. Four peaks of glucose 1,6-P2 phosphatase activity are obtained when the cytosolic fraction from pig muscle is subjected to DE-cellulose chromatography. All the peaks hydrolyze other phosphocompounds in addition to glucose 1,6-P2. The glucose 1,6-P2 phosphatase activity of the main peak shows an optimal neutral pH. It is activated by divalent cations, Mg2+ being more effective than Mn2+. The addition of Ca2+ or EGTA does not affect the enzymatic activity. IMP does not possess any effect. It is concluded that this enzyme is different from the glucose 1,6-P2 phosphatases found in mouse brain cytosol and rat skeletal muscle.     

Effect of vanadate on the glucose-1,6-bisphosphate synthase and glucose-1,6-bisphosphatase activities of phosphoglucomutase

Phosphoglucomutase, in addition to catalyzing the interconversion of glucose 1-P and glucose 6-P, catalyzes both the synthesis of glucose 1,6-P2 from glucose monophosphate and either fructose 1,6-P2 or glycerate 1,3-P2, and the hydrolysis of glucose 1,6-P2. Vanadate inhibits the mutase activity, activates the synthase activities, and does not affect the phosphatase activity. These effects suggest that the "exchange" step postulated for the phosphoglucomutase pathway is specifically inhibited by vanadate.     

Activation of muscle phosphofructokinase by fructose 2,6-bisphosphate and fructose 1,6-bisphosphate is differently affected by other regulatory metabolites

Fructose-2,6-P2 and fructose-1,6-P2 are strong activators of muscle phosphofructokinase. They have been shown to be competitive in binding studies, and it is generally thought that they affect the physical and catalytic properties of the enzyme in the same manner. However, there are indications in published data that the effects of the two fructose bisphosphates on phosphofructokinase are not identical. To examine this possibility, the kinetics of activation of rat skeletal muscle phosphofructokinase by the two fructose bisphosphates were compared in the presence of other regulatory metabolites. Citrate greatly increased the K0.5 of the enzyme for fructose-2,6-P2, with little effect on the maximum activation. In contrast, citrate greatly decreased the maximum activation by fructose-1,6-P2, with only a small effect on the K0.5. Changes in the concentrations of the inhibitor ATP or the activator AMP similarly altered the K0.5 for fructose-2,6-P2, but altered the maximum activation by fructose-1,6-P2. Finally, when fructose-1,6-P2 was added in the presence of a given concentration of fructose-2,6-P2, phosphofructokinase activity was decreased if the activation by fructose-2,6-P2 alone was greater than the maximum activation by fructose-1,6-P2 alone. These results are consistent with competition of the two fructose bisphosphates for the same binding site, but indicate that the conformational changes produced by their binding are different.     

Increased hepatic fructose 2,6-bisphosphate after an oral glucose load does not affect gluconeogenesis

The generally accepted metabolic concept that fructose 2,6-bisphosphate (Fru-2,6-P2) inhibits gluconeogenesis by directly inhibiting fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P2 in intact animals, a novel NMR method was developed using [U-13C]glucose and 2H2O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats. Liver Fru-2,6-P2 increased approximately 10-fold shortly after the glucose load, reached a maximum at 60 min, and then dropped to base-line levels by 150 min. The gastric contribution to plasma glucose reached approximately 50% at 30 min after the glucose load and gradually decreased thereafter. Although the contribution of glycogen to plasma glucose was small, glucose formed from gluconeogenesis was substantial throughout the study period even when liver Fru-2,6-P2 was high. Liver glycogen repletion was also brisk throughout the study period, reaching approximately 30 micromol/g at 3 h. These data demonstrate that Fru-2,6-P2 does not inhibit gluconeogenesis significantly in vivo.     

Euglycemic hyperinsulinemia increases glucose 1,6-bisphosphate in human skeletal muscle

1. The effects of physiologic concentrations of insulin on the contents of glucose 1,6-bisphosphate (glucose 1,6-P2) and regulators of glucose 1,6-P2 synthase in intact human skeletal muscle have been investigated. 2. Insulin increased glucose 1,6-P2 from a basal value of 70 +/- 6 to 135 +/- 12 mumol/kg dry wt (P less than 0.001). 3. Activation of synthase could not be associated with changes in its inhibitors (fructose 1,6-P2, Pi, citrate) or its substrate glucose 6-P.     

Cardiac expression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase inhibits glycolysis, promotes hypertrophy, impairs myocyte function, and reduces insulin sensitivity

Glycolysis is important to cardiac metabolism and reduced glycolysis may contribute to diabetic cardiomyopathy. To understand its role independent of diabetes or hypoxic injury, we modulated glycolysis by cardiac-specific overexpression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (kd-PFK-2). PFK-2 controls the level of fructose 2,6-bisphosphate (Fru-2,6-P(2)), an important regulator of glycolysis. Transgenic mice had over 2-fold reduced levels of Fru-2,6-P(2). Heart weight/body weight ratio indicated mild hypertrophy. Sirius red staining for collagen was significantly increased. We observed a 2-fold elevation in glucose 6-phosphate and fructose 6-phosphate levels, whereas fructose 1,6-bisphosphate was reduced 2-fold. Pathways branching off of glycolysis above phosphofructokinase were activated as indicated by over 2-fold elevated UDP-N-acetylglucosamine and glycogen. The kd-PFK-2 transgene significantly inhibited glycolysis in perfused hearts. Insulin stimulation of metabolism and Akt phosphorylation were sharply reduced. In addition, contractility of isolated cardiomyocytes was impaired during basal and hypoxic incubations. The present study shows that cardiac overexpression of kinase-deficient PFK-2 reduces cardiac glycolysis that produced negative consequences to the heart including hypertrophy, fibrosis, and reduced cardiomyocyte function. In addition, metabolic and signaling responses to insulin were significantly decreased.     

Active hepatic glycogen synthesis from gluconeogenic precursors despite high tissue levels of fructose 2,6-bisphosphate

When fasted rats ate regular lab chow there was a lag time of about 2 h before the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2) in liver began to rise from its low basal level. By contrast, in animals refed on a sucrose-based diet hepatic [Fru-2,6-P2] increased 20-fold (to a value of approximately 12 nmol/g wet weight) during the first hour. These responses correlated with differences in the ability of the two diets to increase the circulating [insulin]/[glucagon] ratio and thus to elevate the ratio of 6-phosphofructo-2-kinase to fructose-2, 6-bisphosphatase. Liver glycogen was deposited briskly in both groups of rats. To assess its mechanism of synthesis (directly from glucose versus indirectly via the gluconeogenic pathway), animals eating the chow or sucrose diets received intravenous infusions of [14C]bicarbonate, [1-14C] fructose, and 3H2O. After isolation, the glycogen was subjected to positional isotopic analysis of its glucose residues. The results established that regardless of the diet the bulk of liver glycogen was gluconeogenic in origin. The fact that with sucrose feeding carbon flow through hepatic fructose-1,6-bisphosphatase remained active despite high levels of Fru-2,6-P2 (a potent inhibitor of this enzyme in vitro) presents a metabolic paradox. Conceivably, the suppressive effect of Fru-2, 6-P2 on hepatic fructose-1,6-bisphosphatase is overridden in vivo by some unknown factor or factors generated in response to sucrose feeding. Alternatively, metabolic zonation in liver might result in the coexistence of hepatocytes rich in Fru-2,6-P2 (high glycolytic, low gluconeogenic, low glycogenic capacitites) with cells depleted of Fru-2,6-P2 (low glycolytic, high gluconeogenic, high glycogenic capacities).     

Regulation of glycolysis during acclimation of scallops (Patinopecten yessoensis Jay) to anaerobiosis

Some glycolytic metabolites in the adductor muscle were measured after transfer of scallops from aerobic to anaerobic saltwater for 12 h. The level of octopine increased gradually during the initial 3 h incubation, and thereafter the level increased rapidly up to 12 h. The ATP level also did not show any significant change for the initial 3 h, and then decreased rapidly. The fructose 2,6-biphosphate (Fru 2,6-BP) level increased drastically during the initial 3 h incubation, but thereafter the level did not show any significant change up to 12 h. In the short-term effects of anaerobiosis for 90 min, the level of fructose 6-phosphate (Fru 6-P) increased just after transfer to anaerobiosis, and then its level decreased. In contrast, the fructose 1,6-biphosphate (Fru 1,6-BP) level increased greatly, at the time when both glucose 6-phosphate (Glc 6-P) and Fru 6-P decreased. The Fru 2,6-BP level did not any significant change during the initial 15 min incubation, but thereafter the level increased gradually up to 90 min. Scallop 6-phosphofructo 1-kinase (EC 2.7.1.11) (PFK1) was strongly activated by 1 microM Fru 2,6-BP when 0.2 mM Fru 6-P was used as a substrate, but the activity was not affected at 5 mM Fru 6-P. In view of these results, the regulation mechanism of glycolysis is discussed.     

Insulin-like effects of vanadate on glucokinase activity and fructose 2,6-bisphosphate levels in the liver of diabetic rats

Streptozotocin diabetic rats showed more than a 4-fold increase in blood glucose levels, whereas hepatic glycogen, fructose 2,6-bisphosphate concentration, and 6-phosphofructo-2-kinase activity were decreased. The "total" 6-phosphofructo-2-kinase and the "active" (nonphosphorylated) form of the enzyme were decreased to a different extent, resulting in a fall of the "active"/"total" activity ratio. Vanadate administration for a 2-week period restored the altered values in the diabetic rats without modifying significantly in the control animals any of the parameters studied. Glucokinase activity was essentially lacking in the diabetic animals, and vanadate treatment restored the activity to about 65% of its control value, a good correlation between the recovery of the enzyme and the blood glucose level being observed. These results show an insulin-like effect of vanadate in the whole animal and suggest that insulin and vanadate possess similar actions on hepatic intracellular events.     

Vanadate restores glucose 6-phosphate in diabetic rats: a mechanism to enhance glucose metabolism

Vanadate mimics the metabolic actions of insulin. In diabetic rodents, vanadate also sensitizes peripheral tissues to insulin. We have analyzed whether this latter effect is brought about by a mechanism other than the known insulinomimetic actions of vanadium in vitro. We report that the levels of glucose 6-phosphate (G-6-P) in adipose, liver, and muscle of streptozotocin-treated (STZ)-hyperglycemic rats are 77, 50, and 58% of those in healthy control rats, respectively. Normoglycemia was induced by vanadium or insulin therapy or by phlorizin. Vanadate fully restored G-6-P in all three insulin-responsive peripheral tissues. Insulin did not restore G-6-P in muscle, and phlorizin was ineffective in adipose and muscle. Incubation of diabetic adipose explants with glucose and vanadate in vitro increased lipogenic capacity three- to fourfold (half-maximally effective dose = 11 +/- 1 microM vanadate). Lipogenic capacity was elevated when a threshold level of approximately 7.5 +/- 0.3 nmol G-6-P/g tissue was reached. In summary, 1) chronic hyperglycemia largely reduces intracellular G-6-P in all three insulin-responsive tissues; 2) vanadate therapy restores this deficiency, but insulin therapy does not restore G-6-P in muscle tissue; 3) induction of normoglycemia per se (i.e., by phlorizin) restores G-6-P in liver only; and 4) glucose and vanadate together elevate G-6-P in adipose explants in vitro and significantly restore lipogenic capacity above the threshold of G-6-P level. We propose that hyperglycemia-associated decrease in peripheral G-6-P is a major factor responsible for peripheral resistance to insulin. The mechanism by which vanadate increases peripheral tissue capacity to metabolize glucose and to respond to the hormone involves elevation of this hexose phosphate metabolite and the cellular consequences of this elevated level of G-6-P.     

Phosphofructokinase 2 and glycolysis in HT29 human colon adenocarcinoma cell line. Regulation by insulin and phorbol esters.

Kinetic properties of phosphofructokinase 2 (PFK2) and regulation of glycolysis by phorbol 12-myristate 13-acetate (PMA) and insulin were investigated in highly glycolytic HT29 colon cancer cells. PFK2 was found to be inhibited by citrate and, to a lesser extent, by phosphoenolpyruvate and ADP, but to be insensitive to inhibition by sn-glycerol phosphate. From these kinetic data, PFK2 from HT29 cells appears different from the liver form, but resembles somewhat the heart isoenzyme. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels, glucose consumption and lactate production are increased in a dose-dependent manner in HT29 cells treated with PMA or insulin. The increase in Fru-2,6-P2 can be related to an increase in the Vmax. of PFK2, persisting after the enzyme has been precipitated with poly(ethylene glycol), without change in the Km for fructose 6-phosphate. The most striking effects of PMA and insulin on Fru-2,6-P2 production are observed after long-term treatment (24 h) and are abolished by actinomycin, cycloheximide and puromycin, suggesting that protein synthesis is involved. Furthermore, the effects of insulin and PMA on glucose consumption, lactate production, Fru-2,6-P2 levels and PFK2 activity are additive, and the effect of insulin on Fru-2,6-P2 production is not altered by pre-treatment of the cells with the phorbol ester. This suggests that these effects are exerted by separate mechanisms.     

The tissue distribution of fructose-2,6-p2 and fructose-6-P,2-kinase in rats and the effect of starvation diabetes and hypoglycemia on hepatic fructose-2,6-P2 and fructose-6-P,2-kinase

The tissue distribution of fructose-2,6-P₂ and fructose-6-P,2-kinase in rats was determined. The highest concentration of fructose-2,6-P₂ was found in liver, followed by brain, heart muscle, kidney, testis and skeletal muscle in decreasing order. Similar results were obtained with fructose-6-P,2-kinase activities in these tissues. Starvation, streptozotocin-induced diabetes or hypoglycemia lowers the fructose-2,6-P₂ levels and fructose-6-P,2-kinase activity in the liver.     

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.     

Regulation of fructose 2,6-bisphosphate concentration in white adipose tissue.

Injection of insulin to fed rats diminished the concentration of fructose 2,6-bisphosphate in white adipose tissue. Incubation of epididymal fat-pads or adipocytes with insulin stimulated lactate release and sugar detritiation and also decreased fructose 2,6-bisphosphate concentration. Such a decrease was, however, not observed in fat-pads from starved or alloxan-diabetic rats. Incubation of adipocytes from fed rats with various concentrations of glucose or fructose led to a dose-dependent rise in fructose 2,6-bisphosphate which correlated with lactate output and detritiation of 3-3H-labelled sugar. In adipocytes from fed rats, palmitate stimulated the detritiation of [3-3H]glucose without affecting lactate production and fructose 2,6-bisphosphate concentration. Incubation of epididymal fat-pads from fed rats in the presence of antimycin stimulated lactate output but decreased fructose 2,6-bisphosphate concentration. Changes in lipolytic rates brought about by noradrenaline, insulin, adenosine and corticotropin in adipocytes from fed rats were not related to changes in fructose 2,6-bisphosphate or to rates of lactate output. In fed rats, the activity of 6-phosphofructo-2-kinase was not changed after treatment of adipocytes with insulin, noradrenaline or adenosine. It is suggested that the decrease in fructose 2,6-bisphosphate concentration observed after insulin treatment can be explained by the increase in sn-glycerol 3-phosphate, an inhibitor of 6-phosphofructo-2-kinase.