A comparison of the effects of diabetes induced with either alloxan or streptozotocin and of starvation on the activities in rat liver of the key enzymes of gluconeogenesis

1. Measurements of the activities in rat liver of the four key enzymes involved in gluconeogenesis, i.e. pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), have been carried out, all four enzymes being measured in the same liver sample. Changes in activities resulting from starvation and diabetes have been studied. Changes in concentration (activity/unit wet weight of tissue) were compared with changes in the hepatic cellular content (activity/unit of DNA). 2. Each enzyme was found to increase in concentration during starvation for up to 3 days, but only glucose 6-phosphatase and phosphoenolpyruvate carboxykinase showed a significant rise in content. Fructose 1,6-diphosphatase appeared to decrease in content somewhat during the early stages of starvation. 3. There was a marked increase in the concentration of all four enzymes in non-starved rats made diabetic with alloxan or streptozotocin, for the most part similar responses being found for the two diabetogenic agents. On starvation, however, the enzyme contents in the diabetic animals tended to fall, often with streptozotocin-treated animals to values no greater than for the normal overnight-starved rat. Deprivation of food during the period after induction of diabetes with streptozotocin lessened the rise in enzyme activity. 4. The results are compared with other published values and factors such as substrate and activator concentrations likely to influence activity in vivo are considered. 5. Lack of correlation of change in fructose 1,6-diphosphatase with the other enzymes questions whether it should be included in any postulation of control of gluconeogenic enzymes by a single gene unit.

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The development of gluconeogenesis in rat liver. Effects of glucagon and ether

Biochemical Journal ◽

10.1042/bj1200385 ◽

1970 ◽

Vol 120 (2) ◽

pp. 385-392 ◽

Cited By ~ 22

Author(s):

Helen Philippidis ◽

F. J. Ballard

Keyword(s):

Rat Liver ◽

Redox State ◽

Phosphoenolpyruvate Carboxykinase ◽

Fold Increase ◽

Similar Increase ◽

Ether Anaesthesia ◽

Foetal Liver ◽

Foetal Rat ◽

Newborn Animals

1. Administration of glucagon to foetal rats produced a 10–15-fold increase in hepatic phosphoenolpyruvate carboxykinase activity together with a similar increase in the overall pathway of pyruvate conversion into glycogen in liver slices. 2. Glucagon was without effect on gluconeogenesis in vivo, which remained at approx. 0.1% of the incorporation as measured in newborn animals. 3. The apparent discrepancy between these results was due to the ether anaesthesia that was required for experimentation in vivo. Under conditions when minimal ether was used, the rates of labelling of glycogen from [3-14C]pyruvate in vivo were increased 10–20-fold and there was an additional stimulus by glucagon. 4. Ether anaesthesia produced a more reduced redox state of the foetal liver cytosol and lowered the ATP/ADP concentration ratio. 5. It is proposed that these effects are significant in the limitation of gluconeogenesis in the foetal rat liver, so that only with high phosphoenolpyruvate carboxykinase activity, high ATP concentration and a relatively oxidized cytosol redox state will a functional gluconeogenic pathway be present.

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Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase

Biochemical Journal ◽

10.1042/bj1770833 ◽

1979 ◽

Vol 177 (3) ◽

pp. 833-846 ◽

Cited By ~ 31

Author(s):

M C Scrutton ◽

I Beis

Keyword(s):

Rat Liver ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Specific Activity ◽

Competitive Inhibitor ◽

Product Inhibition ◽

Detectable Effect ◽

Formyltetrahydrofolate Dehydrogenase ◽

Hydrolysis Of

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7–0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

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Differential effects of tryptophan on glucose synthesis in rats and guinea pigs

Biochemical Journal ◽

10.1042/bj1760817 ◽

1978 ◽

Vol 176 (3) ◽

pp. 817-825 ◽

Cited By ~ 30

Author(s):

S A Smith ◽

K R F Elliott ◽

C I Pogson

Keyword(s):

Rat Liver ◽

Phosphoenolpyruvate Carboxykinase ◽

Glucose Production ◽

Rat Hepatocytes ◽

Liver Cells ◽

Isolated Rat Hepatocytes ◽

Rat Liver Cells ◽

Glucose Output ◽

Isolated Rat

1. Tryptophan inhibition of gluconeogenesis in isolated rat liver cells is characterized by a 20 min lag period before linear rates of glucose output are attained. 2. Half-maximal inhibition of gluconeogenesis in isolated rat hepatocytes is produced by approx. 0.1 mM-tryptophan. 3. Tryptophan inhibits gluconeogenesis from all substrates giving rise to oxaloacetate, but stimulates glycerol-fuelled glucose production. 4. Gluconeogenesis in guinea-pig hepatocytes is insensitive to tryptophan. 5. Changes in metabolite concentrations in rat liver cells are consistent with a locus of inhibition at the step catalysed by phosphoenolpyruvate carboxykinase. 6. Inhibition of gluconeogenesis persists in cells from rats pretreated with tryptophan in vivo. 7. Tryptophan has no effect on urea production from alanine, but decreases [1-14C]palmitate oxidation to 14CO2 and is associated with an increased [hydroxybutyrate]/[acetoacetate] ratio. 8. These results are discussed with reference to the control of gluconeogenesis in various species.

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A physiological role of Mn2+ in the regulation of cytosolic phosphoenolpyruvate carboxykinase from rat liver is unlikely

Biochemical Journal ◽

10.1042/bj2920365 ◽

1993 ◽

Vol 292 (2) ◽

pp. 365-370 ◽

Cited By ~ 7

Author(s):

S Maggini ◽

F B Stoecklin-Tschan ◽

S Mörikofer-Zwez ◽

P Walter

Keyword(s):

Rat Liver ◽

Phosphoenolpyruvate Carboxykinase ◽

Physiological Role ◽

Chelating Agent ◽

Molecular Forms ◽

Experimental Conditions ◽

Free System ◽

Inactivation Curve ◽

Gluconeogenic Enzyme

A cytosolic cell-free system prepared from rat liver was used to study the effect of bivalent cations on the activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Steady-state concentrations of oxaloacetate in the range 5-50 microM were generated from increasing concentrations of malate+fumarate (10:1); 2 mM ITP and 3 mM Mg2+ were added as cofactors. Micromolar concentrations of Mn2+, Fe2+ and, to a lesser extent, of Zn2+ and Co2+ were shown to stimulate PEPCK activity. Vmax. (mumol/min per g of liver) increased from 0.67 to 1.68 on addition of 5 microM Fe2+ and to 2.34 with 2 microM Mn2+, whereas no significant effect on the Km for oxaloacetate was observed. The apparent K(a) values (total) were 0.62 microM for Mn2+, 1.48 microM for Zn2+, 1.92 microM for Co2+ and 3.37 microM for Fe2+, being 2-8-fold lower than the corresponding published values. Variations of the free Mn2+ concentration were obtained (a) by increasing the Mn2+ concentration (i.e. activation curve) and (b) by simultaneous addition of Mn2+ and increasing concentrations of the chelating agent EGTA (i.e. inactivation curve). Different results were obtained for the activation and inactivation curves. The inactivation curve showed that PEPCK activity was almost unaffected by variations of the free Mn2+ concentration over the range 0.05-0.15 microM. Under comparable experimental conditions, rat liver arginase (another Mn(2+)-dependent enzyme) was completely inactivated. From kinetic evidence, the existence of two distinct molecular forms of cytosolic rat liver PEPCK with different Mn2+ affinities is postulated. Considering the high affinity of PEPCK for Mn2+ and its relative insensitivity to changes in the free Mn2+ concentration, it seems rather unlikely that changes in the free cation concentration play a major role in regulating PEPCK activity in vivo.

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