Experiments with isolated liver cells - the citric acid cycle and warming up post-operative patients

They were using freshly prepared muscle tissue, which will have contained a considerable amount of glycogen; this is what is providing the substrate for glycolysis and onward oxidation.

These experiments show a considerably greater increase in oxygen consumption than can be accounted for by the total oxidation of the citrate that was added to the incubation. Complete oxidation of 3 mmol of citrate would consume only 300 µL of oxygen, while over 90 minutes there was additional consumption of 806 µL of oxygen, more than two and a half times more than would be accounted for by the citrate.

Citrate obviously has a catalytic effect, increasing the rate at which other substrates can be oxidised. It was this observation, together with previous studies showing a similar catalytic effect of fumarate, oxaloacetate or succinate, which led Krebs and Johnson to propose a cyclic pathway in which a two carbon compound was added to oxaloacetate to form citrate, then oxidised to yield oxaloacetate again.

At this time the identity of the two carbon compound was unknown, but was assumed to be an acetate derivative. We now know that it is acetyl CoA, which is the end product of both aerobic glycolysis (in cells that have mitochondria) and also beta-oxidation of fatty acids.

Click here for a printable version of the full citric acid cycle

Experiment 1

Isolated hepatocytes were incubated for 40 min in a phosphate / bicarbonate / carbon dioxide buffer system, with [U-14C]palmitate (i.e., the C16 fatty acid, palmitate, labelled with 14C in all 16 carbon atoms), at a specific radioactivity of 103 dpm /mmol, with and without the addition of 60 mmol/L malonate and / or oxaloacetate.

One set of incubations was set up to act as an unincubated control, in which the perchloric acid was added at the beginning of the incubation.

After collection of the carbon dioxide for measurement of radioactivity, the denatured incubation mixture was extracted with a chloroform:methanol mixture to separate unmetabolised fatty acid (palmitate, in the organic phase) from water-soluble metabolites (in the aqueous layer). The radioactivity in both phases was determined.

This experiment with isolated hepatocytes incubated with [14C]palmitate shows that there is a negligible amount of radioactivity in water-soluble compounds under normal conditions, with most of the radioactivity being recovered in carbon dioxide.

Adding oxaloacetate increases the rate at which palmitate is oxidised to carbon dioxide - this is the same catalytic effect as observed by Krebs and Johnson - a small increase in the amount of one of the carrier molecules in the cycle increases the rate at which acetyl CoA arising from the beta-oxidation of palmitate can be oxidised.

The addition of malonate, as an inhibitor of succinate dehydrogenase, leads to more or less complete inhibition of the oxidation of palmitate, as would be expected from inhibition of the cycle. However, adding oxaloacetate plus malonate permits considerable metabolism of palmitate, but with negligible formation of radioactive carbon dioxide. The cycle is blocked by the inhibition of succinate dehydrogenase and succinate accumulates, with net consumption of the added oxaloacetate.

Although 2 mol of carbon dioxide are formed for each mol of acetyl CoA + oxaloacetate forming citrate and being oxidised to fumarate, the two carbon atoms that are lost as carbon dioxide are not the two that were added from acetyl CoA. Check this for yourself by labelling the two carbon atoms from acetyl CoA added to oxaloacetate to form citrate on the printable version of the cycle.

The intermediates of the citric acid cycle all become labelled during incubation with [14C]acetyl CoA, and radioactive carbon dioxide will only be released during the second and subsequent turns of the cycle.

succinate

You have seen that citrate behaves asymmetrically, in that the two carbon atoms added from acetyl CoA are not the same two as are released as carbon dioxide in the first turn of the cycle (when succinate dehydrogenase is inhibited by malonate). However, citrate is a symmetrical molecule. Carbons 1 and 2 are equivalent to carbons 5 and 6.

Citrate must be passed directly from the active site of citrate synthase onto the active site of aconitase, without entering into free solution. If it were free in solution then it would behave symmetrically, and on average half the carbon dioxide released in the first turn of the cycle would be labelled.

This metabolic chanelling of citrate directly from the active site of one enzyme onto the active site of the next in the pathway provides an important metabolic control. Citrate is also required as the precursor for fatty acid synthesis, but cannot be removed from the mitochondria unless the citric acid cycle is acting fast enough to provide the ATP that the cell requires. When the cycle is acting at a fast enough rate then citrate is formed in excess of the need for ATP synthesis and can be released from citrate synthase into free solution, and can then be exported to the cytosol for fatty acid synthesis.

Experiment 2

Isolated hepatocytes were incubated with lactate, glutamate and / or palmitate as substrates. At the end of the experiment lactate, palmitate, glutamate and glucose (after acid hydrolysis of glycogen) were determined. The results are shown as the change during the incubation, compared with similar incubations which were stopped with perchloric acid at the beginning of the experiment.

Incubation (1) shows that lactate can not only undergo complete oxidation to carbon dioxide and water, but can also be a substrate for fatty acid synthesis (the increase in palmitate) and gluconeogenesis (the increase in glucose). You have already seen in the exercises on carbohydrate metabolism that pyruvate (which is formed from lactate in the liver) can either be decarboxylated and oxidised to acetyl CoA, or be carboxylated to oxaloacetate, which is the substrate for gluconeogenesis.

Incubation (2) shows that palmitate can undergo complete oxidation to carbon dioxide and water, but cannot be a substrate for gluconeogenesis. This should be obvious. When palmitate is being metabolised, 2 carbons are added to oxaloacetate to yield citrate, but two carbons are lost in each turn of the citric acid cycle, so there is no increase in the amount of oxaloacetate in the cell. This means that oxaloacetate cannot be withdrawn for gluconeogenesis, which is ATP expensive, since otherwise the citric acid cycle would slow down, reducing the amount of ATP available.

In the diagram of the citric acid cycle above (and in the printable version) the reaction of succinyl CoA synthetase is shown as being linked to the phosphorylation of GDP to GTP. This is so in liver and other tissues that can catalyse gluconeogenesis, but in other tissues (e.g. muscle, brain and adipose tissue) the reaction is linked to phosphorylation of ADP to ATP. The role of the liver isoenzyme of succinyl CoA synthetase is to provide the GTP that is needed for the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate in glycolysis.

Incubation (3) shows that when both lactate and palmitate are present in the incubation all of the lactate that is consumed is used for gluconeogenesis - 2.4 µmol of lactate(a 3-carbon compound) yields 1.2 µmol of glucose (a 6-carbon compound). At the same time, more palmitate is consumed than when palmitate was the sole substrate.

The additional consumption of palmitate is to provide the ATP and GTP needed for gluconeogenesis from lactate.

Incubation (4) shows that glutamate is metabolised to yield glucose (as well as complete oxidation to carbon dioxide). Glutamate can be deaminated (or transaminated) to yield 2-oxoglutarate, which increases the total amount of citric acid cycle intermediates in the cell, and so leads to a net increase in oxaloacetate, which can then be used for gluconeogenesis.

Experiment 3

Isolated hepatocytes were incubated with lactate labelled with 14C in carbon-2 ([14C-2]lactate) or palmitate labelled with 14C in all carbon atoms ([U-14C]palmitate); in each case the specific radioactivity of the labelled substrate in the incubation medium was 103 dpm /mmol. In a further series of incubations with [U-14C]palmitate, non-radioactive glutamate was also added.

1) Label from [14C]lactate appears as carbon dioxide and glucose. This is as you would expect from the results of experiment 2; lactate can undergo both complete oxidation and also provide a substrate for gluconeogenesis.

2) Label from palmitate with no other additions only appears as carbon dioxide. There is no incorporation of label from palmitate into glucose. Again this is as you would expect from the results of experiment 2; fatty acids can never be a substrate for gluconeogenesis because when palmitate is being metabolised, 2 carbons are added to oxaloacetate to yield citrate, but two carbons are lost in each turn of the citric acid cycle, so there is no increase in the amount of oxaloacetate in the cell.

3) When glutamate is added, label from [14C]palmitate appears in glucose. There is no formation of glucose from palmitate, but there is formation of glucose from glutamate, via 2-oxoglutarate and oxaloacetate. The whole of the cellular pool of oxaloacetate has been labelled by the [14C]palmitate, and therefore some of the oxaloacetate which is used for gluconeogenesis contains atoms of 14C from palmitate.

Warming up post-operative patients

More than half of all patients undergoing surgery with general anaesthesia experience some degree of hypothermia, defined as a core body temperature below 35ºC. This can lead to cardiovascular and respiratory problems, impaired blood coagulation, and increased recovery time. Anaesthesia impairs central thermoregulation and lowers the threshold for vasoconstriction, so redistributing blood (and hence heat) to the periphery. At the same time, fluids are infused at room temperature rather than body temperature, and the muscle relaxant drugs used in anaesthesia inhibit shivering.

The blue line in the graph on the right shows this fall in core body temperature in control subjects undergoing coronary artery bypass surgery. The red line shows the thermic effect of infusing a mixture of amino acids - not only do the patients' body temperatures not fall, they actually increase as a result of the amino acid infusion.

[Moriyama T, Tsuneyoshi I, Omae T, et al. The effect of amino-acid infusion during off-pump coronary arterial bypass surgery on thermogenic and hormonal regulation. J Anesth. 2008;22(4):354-60]

Many amino acids yield citric acid cycle intermediates as a result of metabolism of their carbon skeletons:

• 2-Oxoglutarate comes from arginine, glutamine, glutamate, histidine and proline
• Succinyl CoA comes from isoleucine, methionine and valine
• Fumarate comes from aspartate, asparagine, phenylalanine and tyrosine
• Oxaloacetate comes from aspartate and asparagine

The citric acid cycle intermediates formed from these amino acids lead to a considerable increase in the amount of oxaloacetate in the cell - well above the amount that is required for maximum activity of the cycle for ATP formation. There are two possible fates for this surplus oxaloacetate:

1) It may be used for gluconeogenesis. This is ATP (and GTP) expensive, and there will be an increased rate of metabolism to provide the ATP and GTP needed. The net effect of formation and utilisation of more ATP is heat production, and hence an increase in body temperature.

2) If the oxaloacetate is not used for gluconeogenesis it must undergo complete oxidation. For this to occur it must be carboxylated to phosphoenolpyruvate at the expense of GTP and then dephosphorylated to pyruvate and oxidised to acetyl CoA - again this is a thermogenic reaction sequence. In addition, the metabolism of ammonium arising from transamination and deamination of the amino acids is a thermogenic process.

Just for amusement, this photograph of Sir Hans Krebs on his (motor) cycle was published in The Biochemist in 2010 with the caption "The Krebs' cycle".

Key points from this exercise:

• Addition of citrate leads to a considerably greater increase in oxygen consumption than is accounted for by the amount of citrate added, because it has a catalytic effect, increasing the rate at which what we now know is a cyclic pathway can operate. Addition of any of the other intermediates of the cycle has a similar catalytic effect.
• When radioactive palmitate is provided as the substrate, it undergoes beta-oxidation to radioactive acetyl CoA, followed by complete oxidation in the citric acid cycle to yield radioactive carbon dioxide.
• However, if malonate is added as an inhibitor of succinate dehydrogenase,and oxaloacetate is provided to permit continued uptake of acetyl CoA into citrate, there is no formation of radioactive carbon dioxide, and radioactive succinate, a water-soluble compound, accumulates. This is because the two carbon atoms that are lost from citrate in the first turn of the cycle are not the two that have been added from acetyl CoA.
• Citrate is a symmetrical molecule, but in the cycle it behaves asymmetrically. This means that it must be passed directly from citrate synthase onto aconitase, without going into free solution. This metabolic chanelling provides regulation of the metabolism of citrate. When more citrate is being formed than is required for cycle activity and ATP formation, it can leave citrate synthase and go into free solution (because aconitase is saturated) and can be exported from the mitochondrion for fatty acid synthesis in the cytosol.
• Lactate and glutamate can undergo complete oxidation in the citric acid cycle, or can be used for synthesis of fatty acids and glucose. Palmitate, which yields acetyl CoA, cannot be used for gluconeogenesis (because the reaction of pyruvate dehydrogenase is irreversible, and for each two carbon atoms added as acetyl CoA, two are lost in the cycle, so there is no net increase in the amount of oxaloacetate in the cell. If lactate and palmitate are provided, all of the lactate is used for gluconeogenesis, and there is greater oxidation of palmitate than when only palmitate is provided, since there is a need to produce the ATP needed for gluconeogenesis from lactate.
• When non-radioactive glutamate and radioactive palmitate are added, there is radioactive labelling of the glucose formed from glutamate, because all of the intracellular pool of oxaloacetate has become labelled from the radioactive palmitate. Palmitate (or any other fatty acid or ketone body) is not, and cannot be, a substrate for gluconeogenesis.
• When amino acids that yield citric acid cycle intermediates are metabolised they lead to an increase in oxaloacetate, but are not completely oxidised unless the oxaloacetate first undergoes carboxylation to phosphoenolpyruvate, then dephosphorylation to pyruvate and oxidation to acetyl CoA. This involves synthesis and utilisation of ATP, and hence is a thermogenic process, leading to an increase in body temperature. Similarly, if the oxaloacetate is used for gluconeogenesis, there must be increased formation of ATP, which is used in gluconeogenesis, so again this is thermogenic (see also the exercise breathless after sprinting).