Effect of a high fructose diet on the activities of enzymes implicated in the conversion of fructose to glucose by the liver and the intestinal mucosa of the rat

The activity of enzymes implicated in the metabolic pathway of fructose to glucose conversion was shown in rat liver and intestine. In rats on normal diet, the specific activity of glucose-6-phosphatase, fructokinase, fructose-1,6-diphosphatase and triokinase was low in the intestine confirming that sugar conversion is not operative in this organ. In rats on a fructose diet, all the specific enzymatic activities tested were increased except for the hepatic triokinase and triose phosphate isomerase and for the intestinal triose phosphate isomerase. The intestine acquires the possibility to transform fructose to glucose by modifying the activities of enzymes implicated in the same metabolic pathway as that intervening in the liver.     

Carbohydrate metabolism in sheep when adding carboxyline to their diet

The content of glycogen and glucose, as well as aldolase, phosphofructokinase, phosphoglucomutase, glucose-6-phosphatase and fructose-1,6-diphosphatase activities in liver tissue and the same activities in skeletal muscle of sheep were determined under the influence of prolonged addition of carboxyline separately and in combination with methionine, diammonium phosphate and potassium iodine to their diet. It is established that under the influence of carboxyline the glycogen content as well as aldolase and fructose-1,6-diphosphatase activities rise significantly in the liver of the tested animals. In the skeletal muscle only aldolase activity increases.     

Dietary and hormonal regulation of some enzyme activities associated with gluconeogenesis in rabbit liver.

1. Starvation increases the activity of cytosolic P-enolpyruvate carboxkinase in rabbit liver some 4-5 fold but does not alter the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase or glucose-6-phosphatase.2. Alloxan-induced diabetes increases the activities of cytosolic P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase approx. 6-, 2- and 2-fold, respectively. Again the activity of mitochondrial P-enolpyruvate carboxykinase is not altered. 3. Administration of mannoheptulose rapidly increases blood glucose levels and also causes a significant increase in cytosolic P-enolpyruvate carboyxkinase activity within 4 h. The activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase are not affected. 4. Administration of hydrocortisone also increases blood glucose levels and the activities of cytosolic P-enolpyruvate carboxykinase and glucose-6-phosphatase are significantly increased within 12h. Again, mitochondrial P-enolpyruvate carboxykinase and fructose-1,6-diphosphatase activities remain unaffected. 5. The observations that (A) the activity of cytosolic P-enolpyruvate carboxykinase responds to more situations conducive to gluconeogenesis than do the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase, and (B) cytosolic P-enolpyruvate carboxykinase activity is rapidly adaptive under appropriate circumstances, suggests that this particular enzyme's activity plays an important role in the regulation of gluconeogenesis in rabbits.     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Radiation dose response of some gluconeogenic enzyme activities in rats

Negative correlation was found between the activity of liver glucose-6-phosphatase (EC 3.1.3.9) and the increasing radiation dose 24 h after continuous irradiation of rats. A dose response of increased fructose-1,6-diphosphatase (EC 3.1.3.11) was not confirmed.     

Undifferentiated patterns of key carbohydrate-metabolizing enzymes in injured livers. I. Acute carbon-tetrachloride intoxication of rat.

In acute CCL4 intoxication of rats significantly increased activities of hepatic low-Km hexokinases, glucose-6-phosphate dehydrogenase, phosphofructokinase, aldolase A and pyruvate kinase M2 with concurrently decreased activities of glucokinase, glucose-6-phosphatase, fructose-1,6-diphosphatase, aldolase B and pyruvate kinase L were observed. The resulting enzyme pattern was apparently different from that in dietary induction. Principal component analysis revealed that the degree of enzyme deviation in the injured liver was much greater than that in the regenerating liver after partial hepatectomy and was closer to that in fetal liver or hepatoma tissue.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: distinction of the 1-monodeutero-isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy.

The 1H NMR spectrum obtained with the alpha- and beta-anomers of D-[1-2H]fructose 6-phosphate generated from D-glucose 6-phosphate sequentially exposed in D2O to phosphoglucoisomerase, phosphofructokinase and fructose-1,6-diphosphatase differed from that recorded when the deuterated ketohexose phosphate was produced from D-mannose 6-phosphate sequentially exposed in D2O to phosphomannoisomerase, phosphofructokinase and fructose-1,6-diphosphatase. The identification of the 2 isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy provides a new tool to assess the relative extent of interconversion of hexose phosphates in the reactions catalyzed by phosphoglucoisomerase and phosphomannoisomerase, respectively.     

Biochemical compartmentation of fish tissues. Distribution of gluconeogenic enzymes in the brain

1. Specific glucose-6-phosphatase and fructose-1,6-diphosphatase activity were found to be biochemically compartmentalized in four parts of the brain in nine nutritionally important fishes. 2. Glucose-6-phosphatase and fructose-1,6-diphosphatase activity were highest in the cerebrum and lowest in the cerebellum. 3. Piscivorous fishes had the highest gluconeogenic enzyme content, followed by catfishes and major carps. 4. After the liver and muscles, the various parts of the brain play an important role in carbohydrate metabolism. 5. A direct relationship between the stage of evolution and elevation of gluconeogenic enzyme levels was observed. 6. It is evident from the results and the discussion that evolution modifies the biochemical organization of fishes in general and of their brain in particular.     

Glutamine synthetase and fructose-1, 6-diphosphatase activity in the putamen of control and Huntington's disease brain post mortem

There is a linear negative correlation between the activities of glutamine synthetase and fructose-1, 6-diphosphatase in normal Human putamen autopsy samples, and also in the Huntington's disease putamen. However, glutamine synthetase activity is reduced in choreic brain samples, while fructose-1, 6-diphosphatase activity is normal. The ratio of fructose-1, 6-diphosphatase to glutamine synthetase is therefore increased in Huntington's disease. The products of the two reactions, glutamine and fructose-6-phosphate, are the starting substrates for glycolipid and glycoprotein biosynthesis, via the glutamine:fructose-6-phosphate aminotransferase catalysed formation of glucoseamine-6-phosphate. The alternative metabolic route of fructose-6-phosphate leads to glycogen. The availability of glutamine, and the activity of glutamine synthetase may control fructose-6-phosphate metabolism, and the increased ratio of fructose-1,6-diphosphatase to glutamine synthetase in Huntington's disease may explain the accumulation of glycogen, and the reduction in ganglioside levels reported in this state.     

The value of enzyme histochemical techniques in the classification of fibre types of human skeletal muscle

Summary Classification of human skeletal muscle into type I and type II fibres is frequently based on their weak or strong staining with the myosin adenosine triphosphatase reaction. In order to evaluate the reliability of this screening technique a combined histochemical and biochemical study was performed on normal and diseased skeletal muscle of human subjects. In the present investigation activities of enzymes which play a role in the aerobic and anaerobic pathways and which can characterize fibre type, were examined in human muscle specimens with disease of the neuromuscular system.Special attention is given to the maximal activities of phosphofructokinase and fructose-1,6-diphosphatase, the rate limiting enzymes for the regulation of the glycolysis and gluconeogenesis respectively. Moreover the activities of enzymes of the pentose phosphate pathway are determined.A most important feature of the biochemical findings is that the constancy of activity ratios of the examined enzymes, as is found apparently normal human skeletal muscle, was frequently not present in diseased human skeletal muscle. From these results and from the histochemical results it can be concluded that for fibre classification in diseased human skeletal muscle the histochemical demonstration of myosin ATPase activity exclusively is not sufficient, but that it is necessary to apply other enzyme histochemical techniques too.Moreover it was found that in diseased human skeletal muscles the activity of the NADPH regenerating enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was strongly increased. A third observation was the relative decrease of the activity of the examined aerobic enzymes in affected muscle fibres of neurogenic muscle diseases.     

Fructose-1, 6-diphosphatase and acid hexose phosphatase of Escherichia coli

Fraenkel, D. G. (Albert Einstein College of Medicine, New York, N.Y.), and B. L. Horecker. Fructose-1,6-diphosphatase and acid hexose phosphatase of Escherichia coli. J. Bacteriol. 90:837-842. 1965.-The conversion of fructose-1,6-diphosphate to fructose-6-phosphate (fructose-1,6-diphosphatase activity) is essential for growth of Escherichia coli on glycerol, acetate, or succinate, but is unnecessary for growth on hexoses or pentoses. It has sometimes been assumed that fructose-1,6-diphosphatase activity is due to a nonspecific acid hexose phosphatase. We have now obtained a number of one-step mutants which have lost the ability to grow on glycerol, succinate, or acetate, but which grow normally on hexoses; these mutants are deficient in a fructose-1,6-diphosphatase which can be assayed spectrophotometrically in the presence of Mg(++) and low concentrations of substrate. These mutants still possess the nonspecific acid hexose phosphatase, which does not require Mg(++) and is active only at much higher concentrations of fructose-1,6-diphosphate. Evidence is presented to support the hypothesis that the newly described activity is the physiological fructose-1,6-diphosphatase. The acid hexose phosphatase is a different enzyme whose function remains unknown.     

Do non-equilibrium reactions in the glucose-to-triose phosphate pathway exist in the liver?

The circadian changes in the contents of intermediates of the initial reactions of the glycolytic pathway in pigeon liver were studied. the concentrations of glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate and triose phosphates were found to change synchronously, being maximal at the dark time and minimal during the light daytime. The glycogen content in the liver decreased steadily between 12.00 and 09.00. The diurnal variations in the concentrations of metabolite pairs (glucose and glucose-6-phosphate, glucose-6-phosphate and fructose-6-phosphate, fructose-6-phosphate and fructose-1.6-diphosphate, fructose-1.6-diphosphate and triose phosphates) appeared to correlate significantly. The results obtained suggest that in the liver at least there are no limiting i. e. physiologically non-equilibrium reactions in the carbohydrate metabolic pathway from glucose to triose phosphates.     

Phosphoglucomutase: its role in the response of pancreatic islets to glucose epimers and anomers

Rat pancreatic islets display phosphoglucomutase activity. The velocity of glucose-1-phosphate conversion to glucose-6-phosphate is increased in a dose-related fashion by glucose-1,6-bisphosphate. The islet homogenate, like purified muscle phosphoglucomutase, also catalyzes the synthesis of glucose-1,6-bisphosphate from glucose-6-phosphate and fructose-1,6-bisphosphate. The rate of the latter reaction is about 10,000 times lower than that of glucose-1-phosphate conversion to glucose-6-phosphate in the presence of glucose-1,6-bisphosphate. D-glucose and D-mannose, but not D-galactose nor D-fructose, markedly increase the islet content in glucose-1,6-bisphosphate. Such a content is twice higher in islets exposed for 5 minutes to alpha-D-glucose than in islets exposed to beta-D-glucose. The process of glucose-1,6-bisphosphate synthesis, as catalyzed by the alpha-stereospecific phosphoglucomutase, may play a role in the metabolic and, hence, secretory responses of the islets to glucose epimers and anomers.     

Development of gluconeogenic enzymes in fetal sheep liver and kidney

In the sheep, the system of enzymes necessary for conversion of nonhexose substrates to glucose becomes active during late fetal life. Glucose-6-phosphatase and fructose-1,6-diphosphatase, two of the four key gluconeogenic enzymes, appear in significant amounts between 100 and 120 days gestation. Phosphoenolpyruvate carboxykinase activity is comparable to mature animals as early as 45 days gestation. Two aminotransferases, necessary to allow amino acid access to the gluconeogenic pathway, likewise have substantial activity as early as 45 days gestation. Hence, the surge of glucose-6-phosphatase and fructose-1,6-diphosphatase at 100-120 days gestation makes possible the endogenous production of new glucose by fetal sheep at a time when the amount of glucose transferred from the maternal circulation is less than the total aerobic substrate utilized by the fetus. Both renal cortex and liver have similar developmental patterns for the gluconeogenic enzymes, although renal cortex generally shows greater activity than liver. This observation holds true for tissue from both fetal and mature animals.     

DDT toxicity: variations in tissue non-specific phosphomonoesterases and gluconeogenic enzymes in three teleosts

Three fish species were exposed to a sublethal dose (0.35 mg/l) of DDT continuously for a period of 50 days and the effect of hepatic and renal acid and alkaline phosphatases, glucose-6-phosphatase and fructose-1,6-diphosphatase activities was observed at 15, 30 and 45 days. Exposure to DDT at 15 days led to the fall and increase thereafter (at 30 and 45 days) in the activities of acid phosphatase, glucose-6-phosphatase and fructose-1,6-diphosphatase in hepatic tissue, where as alkaline phosphatase in liver registered an increase at 15, 30 and 45 days DDT exposure. In renal tissue the trend of 4 phosphatases was same as that of alkaline phosphatase in the liver. The changes in these 4 phosphatases were more pronounced in C. punctatus than in G. batrachus and L. rohita.     

Activity of enzymes related to carbohydrate metabolism in the HT 29 colon adenocarcinoma cell line and tumor

Activity of several enzymes of the glycogen and carbohydrate metabolism is studied in HT 29 colon adenocarcinoma cell line and in HT 29 tumors developed in nude mice, by reference to the normal human colon mucosa. Activity of glycogen synthase, glycogen phosphorylase, pyruvate kinase, fructose-1,6-diphosphatase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase is found to be increased in both the cultured cells and the tumors. It indicates that the biochemical strategy of malignant cells, due to the neoplastic transformation process, involves specific changes in the carbohydrate metabolism of tumor as well as in vitro growing correspondent cell line.     

Enzyme activities associated with maize kernel amyloplasts

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.     

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.     

Phosphohydrolase activity of the isolated, brush-border membrane of Hymenolepis diminuta (Cestoda) following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

Following electrophoresis of isolated, brush-border membranes of Hymenolepis diminuta on SDS-polyacrylamide gels, three distinct areas of alpha-naphthyl phosphate (NP) hydrolysis were detected; these corresponded to proteins with molecular weights of 106,800, 172,700, and greater than 340,000 Daltons. Hydrolysis of NP was inhibited by adenosine triphosphate, adenosine;5'-monophosphate, p-nitrophenyl-phosphate, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, molybdate, ethylenediaminetetraacetate (EDTA), and ethyleneglycol-bis-(beta-amino-ethyl)-N,N'-tetraacetate (EGTA), but not by fluoride. Inhibition of NP hydrolysis by EDTA was relieved in the presence of Mg++ or Ca++. Heating the isolated, brush-border membrane in the presence of SDS for 5 min at 95 C destroyed all enzymatic activity. These characteristics indicated that the enzyme(s) responsible for NP hydrolysis (following separation of membrane proteins by SDS-polyacrylamide gel electrophoresis) were the same enzymes responsible for the phosphohydrolase activity associated with intact and solubilized, brush-border membrane preparations of H. diminuta.     

Some observations on mitochondrial-bound hexokinase and creatine kinase of the heart

A large part of the hexokinase activity of the rat brain 20,000g supernatant became mitochondrial bound when incubated with rat heart mitochondria which had been pretreated with glucose-6-phosphate. This binding was dependent on small-molecular compounds (as yet unidentified) of the brain supernatant. Divalent cations, spermine, and pentalysine strongly stimulated the binding of brain supernatant hexokinase to heart mitochondria. Inorganic phosphate, alpha-glycerophosphate, and fructose-1,6-diphosphate showed some stimulatory effect. No effect was observed with insulin or glucose. Mitochondria isolated from hearts of fasted rats had less specific hexokinase activity than mitochondria from fasted and then carbohydrate refed rats. This dietary treatment had no significant effect on the total heart hexokinase activity. Oligomycin did not inhibit the formation of creatine phosphate or glucose-6-phosphate by isolated rabbit heart mitochondria incubated in the presence of phosphoenolpyruvate and pyruvate kinase. However, the presence of creatine inhibited the formation of glucose-6-phosphate when the ATP/ADP ratio was low, indicating that creatine kinase has a greater access to ATP/ADP translocation than has hexokinase.