The mammalian pyruvate dehydrogenase complex: structure and regulation.

Mitochondrial and cytosolic contents of adenine nucleotides and phosphate were measured in perfused rat livers empolying a technique of fractionation of freeze-fixated tissue in non-aqueous solvents. From the subcellular contents the mitochondrial and cytosolic concentrations of ATP. ADP, AMP and phosphate and the phosphorylation potentials of the subcellular ATP systems were calculated. An attempt was made to elucidate the relationship between mitochnodrial and cytosolic adenine nucleoide systmes and the dependency on the metabolic state of the liver.



The following results were obtaines.



1
Under all mtabolic conditions studied the mitochnodrial ATP/ADP ratios were considerably lower than the cytosolic ratios (mitochondria: 0.1–0.7); cytosol: 2–11).

2
The ATP/ADP ratios calculated from overall tissue contents refflect mainly the cytosolic rations.

3
An inverse relationship was found between mitochondrial and cytosilic ATP/ADP ratios, i.e. when the mitchondrial ratios tended to increase, the cytosolic ratios decrease and vice versa.

4
The phosphorylation potentials calculated from the subcellular concertratopms were higher in the cytosol that in the mitochondria. The potential difference varied between 11 and 3 kJ/mol in livers from fed and starved rats, respectively.

5
In the presence of mitochondrial inhibitors, i.e. amytal, dinitrophenol and carboxyatractyloside the potential difference between the subcellular ATP systems decreased predominantly due to an increase in the mitocnodrial ATP/ADP ratios.

6
A correlation between mitochondrial ATP/ADP ratios and the respiratory rates was not obsrved, but the subcellular ratios appeared to correlate with the rate of glycolysi. When the rate of lactate + pyruvate production was increased, the cytosolic ATP/ ADP ratios were increased, too, wheres the mitochondrial ratios tended to decrease.

7
The adenine nucleotides in the cytosol appear to be in near equilibrium catalysed by the adenylate kinase. In the mitochondria, the AMP concentration is much lowe than to be expected under equilibrium conditions.





These results were discussed with respect to rate control of processes involved in ATP generation, i.e. Oxidative phosphorylation, adenine nucleotide translocation and glycolysis.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1978.tb12387.x

Subcellular Metabolite Concentrations

1
The digitonin fractionation procedure [Zuurendonk, P. F. and Tager, J. M. (1974) Biochim. Biophys. Acta, 333, 393–399] was used to determine the concentrations of adenine nucleotides and of inorganic phosphate in the mitochondrial and cytosolic compartments of hepatocytes isolated from the livers of starved rats.

2
Using glycerol 3-phosphate as a cytosolic marker, it was concluded that leakage of cytosolic metabolites was complete after 15 s exposure of the hepatocytes to 2 mM digitonin. The recovery of the adenine nucleotides after digitonin fractionation was measured. The recovery of ATP was about 100% and that of ADP and AMP 88–109%. Radioactively labelled ATP or ADP were added to the fractionation medium in order to test whether redistribution or metabolism of adenine nucleotides occurs during the fraction procedure. No significant redistribution of adenine nucleotides occurs during the fractionation. Although slight metabolic activity appears to occur in the cytosolic fraction this leads to a change of only 20% in ADP and no change in ATP. It is concluded that the digitonin fractionation procedure can be used to obtain an accurate estimation of the content of adenine nucleotides in the cytosolic and mitochondrial fractions of isolated rat-liver cells.

3
In isolated hepatocytes from fasted rats, 60% of total adenine nucleotides is found in the cytosolic fraction.

4
When isolated hepatocytes from fasted rats are incubated with alanine, the ATP/ADP ratio is 8.76 in the cytosolic fraction and 1.77 in the mitochondrial fraction. On addition of oleate there is no change in cytosolic ATP/ADP.

5
In hepatocytes incubated with alanine or alanine + oleate, the concentration of inorganic phosphate is 5-times higher in the mitochondrial fraction than in the cytosol.

6
It can be calculated that in isolated hepatocytes incubated with alanine the phosphorylation state ([ATP]/[ADP]x[Pi]) in the mitochondrial fraction is 105 M−1 (ΔGc= 12.0 kJ/mol) (2.87 kcal/mol) and that in the cytosolic fraction 2630 M−1 (ΔGc= 20.3 kJ/mol) (4.85 kcal/mol). The addition of oleate did not alter the value of the cytosolic phosphorylation state whereas that in the mitochondria was increased by about 20%.

7
The concentrations of the complexed and uncomplexed forms of the adenine nucleotides and inorganic phosphate in the cytosol were calculated assuming [K+]free= 100 mM, [Mg2+]free= 0.4 mM, pH = 7.0 and I= 0.25 M. The cytosolic concentration of uncomplexed ATP was calculated to be 170 μM, and that of uncomplexed ADP 115 μM. The latter value is about 10-times greater than the Km for ADP that has been reported for the adenine nucleotide translocator at low temperatures.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1978.tb12182.x

Intramitochondrial and Extramitochondrial Concentrations of Adenine Nucleotides and Inorganic Phosphate in Isolated Hepatocytes from Fasted Rats

Publisher Summary This chapter describes the hormonal control of hepatic gluconeogenesis, which is the process whereby lactate, pyruvate, glycerol, and certain amino acids are converted into glucose and glycogen. The liver is the major site of gluconeogenesis although the kidney becomes important during prolonged starvation. The most important function of gluconeogenesis is the maintenance of blood glucose levels during times when food intake is restricted and/or glycogen stores are depleted. Hormonal control of gluconeogenesis can be divided for convenience into three categories. The first involves the regulation of substrate supply. All gluconeogenic substrates reach the liver in subsaturating concentration. Thus, the regulation of substrate release into the blood from the extrahepatic tissues will directly affect hepatic glucose formation. The regulation of the level of cyclic AMP (cAMP) could also explain the inhibitory effect of insulin on gluconeogenesis. There is also evidence that insulin can suppress gluconeogenesis by a mechanism that is independent of changes in cAMP.

Hormonal control of hepatic gluconeogenesis.

1. The interconversion of pyruvate dehydrogenase between its inactive phosphorylated and active dephosphorylated forms was studied in skeletal muscle. 2. Exercise, induced by electrical stimulation of the sciatic nerve (5/s), increased the measured activity of (active) pyruvate dehydrogenase threefold in intact anaesthetized rated within 2 min. No further increase was seen after 15 min of stimulation. 3. In the perfused rat hindquarter, (active) pyruvate dehydrogenase activity was decreased by 50% in muscle of starved and diabetic rats. Exercise produced a twofold increase in its activity in all groups; however, the relative differences between fed, starved and diabetic groups persisted. 4. Perfusion of muslce with acetoacetate (2 mM) decreased (active) pyruvate dehydrogenase activity by 50% at rest but not during exercise. 5. Whole-tissue concentrations of pyruvate and citrate, inhibitors of (active) pyruvate dehydrogenase kinase and (inactive) pyruvate dehydrogenase phosphate phosphatase respectively, were not altered by excerise. A decrease in the ATP/ADP ratio was observed, but did not appear to be sufficient to account for the increase in (active) pyruvate dehydrogenase activity. 6. The results suggest that interconversion of the phosphorylated and dephosphorylated forms of pyruvate dehydrogenase plays a major role in the regulation of pyruvate oxidation by eomparison of enzyme activity with measurements of lactate oxidation in the perfused hindquarter [see the preceding paper, Berger et al. (1976)] suggest that pyruvate oxidation is also modulated by the concentrations of substrates, cofactors and inhibitors of (active) pyruvate dehydrogenase activity.

Glucose metabolism in perfused skeletal muscle. Pyruvate dehydrogenase activity in starvation, diabetes and exercise.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2875%2980811-8

E.A. Siess

Papers

Regulation of pyruvate dehydrogenase interconversion in isolated hepatocytes by the mitochondrial ATP/ADP ratio.