Metabolism
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How is NADH from glycolysis normally re-oxidised?
We have seen in the exercise on Breathless after sprinting that under anaerobic conditions the NADH that is formed in glycolysis is re-oxidised at the expense of pyruvate being reduced to lactate. However, we have also seen in the exercise on Life-threatening acidosis in an alcoholic - and in a hunger striker given intravenous glucose that under aerobic conditions pyruvate is oxidised to acetyl CoA, and then undergoes complete oxidation to carbon dioxide and water.
If you incubate isolated mitochondria in an oxygen electrode with NADH you do not observe any consumption of oxygen, regardless of how much ADP is added.
(See the exercise on Overheating after overdosing on E - and slimming by taking dinitrophenol for more information on the oxygen electrode).
What conclusion can you draw from this observation?
This suggests that NADH cannot cross the mitochondrial membrane.
If NADH does cannot cross the mitochondrial membrane, it cannot be re-oxidised via the electron transport chain. This means that we are left with the problem of how NADH produced in the cytosol can be re-oxidised.
In skeletal muscle lactate dehydrogenase acts to reduce pyruvate to lactate, in order to re-oxidise NADH. However, the form of lactate dehydrogenase in heart muscle acts preferentially in the opposite direction, oxidising lactate to pyruvate. Heart takes up lactate from the bloodstream and uses it as a metabolic fuel.
In the following experiments, isolated cardiomyocytes (heart muscle cells) were used to study the metabolism of lactate under various conditions.
Fluoropyruvate
is a potent inhibitor of pyruvate dehydrogenase. While it might also be expected
to inhibit lactate dehydrogenase acting in the direction of reduction of pyruvate
to lactate, it would not be expected to inhibit the oxidation of lactate to
pyruvate, especially in cardiomyocytes, whose lactate dehydrogenase has a very
much higher affinity for lactate than for pyruvate.
Cardiomyocytes were incubated with 100 mmol /L lactate and 1 mmol /L fluoropyruvate in an oxygen electrode. There was consumption of oxygen that was dependent on the addition of ADP, and a P:O ratio of ~2.5 was observed.
After 30 minutes the reaction mixture was pipetted out into a vial and frozen in liquid nitrogen. The cells were disrupted by repeated cycles of thawing and freezing, then centrifuged to remove mitochondria.
There was a significant accumulation of pyruvate in the cytosol.
What conclusions can you draw from these results?
These results suggest that lactate has been oxidised to pyruvate, with the reduction of NAD to NADH, and that the resultant NADH has been re-oxidised in the mitochondria. However, we know that NADH does not cross the mitochondrial membrane.
In further studies with cardiomyocytes incubated with lactate and fluoropyruvate, a variety of inhibitors of electron transport or oxidative phosphorylation were used. The following results were obtained:
final saturation with
oxygen |
pyruvate |
|
| control | 20% |
5 mmol / L |
| + potassium cyanide | 98% |
0 |
| + rotenone | 98% |
0 |
| + antimycin A | 98% |
0 |
| + oligomycin | 95% |
0 |
What conclusions can you draw from these results?
Can you name the site of action of each of the inhibitors used?
These results confirm the suggestion that the NADH is being re-oxidised in the mitochondria, since inhibition of cytochrome oxidase (by cyanide), inhibition of complex I (by rotenone or antimycin A) and inhibition of ATP synthase (by oligomycin) all lead to inhibition of oxygen consumption and pyruvate formation. If there were any other way in which the NADH formed by lactate dehydrogenase could be re-oxidised then inhibition of the electron transport chain or ATP synthase would not inhibit pyruvate formation under these conditions.
In the next series of experiments the cells were incubated with [14C]lactate, with and without the addition of fluoropyruvate, and the cytosol preparation was subjected to high pressure liquid chromatography linked to a scintillation counter to permit determination of radioactivity in metabolites. Apart from lactate, four compounds were found to be labelled: aspartate, oxaloacetate and malate.
The results were as follows (figures show dpm in the sample):
incubation with fluoropyruvate |
incubation without fluoropyruvate |
|
| pyruvate | 9170 ± 120 |
3890 ± 130 |
| oxaloacetate | 0 |
4905 ± 98 |
| malate | 0 |
5105 ± 105 |
| aspartate | 0 |
5059 ± 97 |
What conclusions can you draw from these results?
The results in the presence of fluoropyruvate confirm what we have already seen, that lactate can be oxidised to pyruvate, which accumulates in the cytosol if it cannot undergo reaction with pyruvate dehydrogenase in the mitochondria.
The
results in the absence of fluoropyruvate are more interesting. They show that
much of the pyruvate has now entered the mitochondria, and a number of compounds
in the cytosol have become labelled. This means that they must have been formed
in the mitochondria from the acetyl CoA produced by pyruvate dehydrogenase.
(You will see in later exercises that malate and oxaloacetate are intermediates
in the citric acid cycle, and would be expected to become labelled if [14C]pyruvate
is provided.
What is the chemical relationship between oxaloacetate and malate?
Oxaloacetate
is a product of oxidation of malate - the enzyme malate dehydrogenase catalyses
the following reaction shown on the right.
What is the relationship between oxaloacetate and aspartate?
Oxaloacetate
is the keto-acid corresponding to aspartate.
Aspartate transaminase catalyses the reaction shown on the right.
In a further series of experiments, isolated mitochondria were incubated in an oxygen electrode with pyruvate, oxaloacetate, malate or aspartate, and the following results were obtained:
| substrate | pyruvate |
oxaloacetate |
malate |
aspartate |
| final % saturation with oxygen | 20 |
98 |
19 |
21 |
What conclusions can you draw from these results?
We already know from the exercise on Overheating after overdosing on E - and slimming by taking dinitrophenol that malate can enter mitochondria and be oxidised, linked to the electron transport chain and phosphorylation of ADP to ATP, and previous results in this exercise have shown that pyruvate also enter the mitochondria. These results conform that, and also show that aspartate can cross the mitochondrial membrane. However, oxaloacetate cannot cross the mitochondrial membrane - there is no consumption of oxygen when the mitochondria are provided with oxaloacetate
In
the next series of experiments isolated heart muscle cells were incubated with
lactate in the oxygen electrode, with and without the addition of methylene
aspartate, a specific inhibitor of aspartate transaminase.
The following results were obtained:
final saturation with
oxygen |
pyruvate |
|
| control | 20% |
5 mmol / L |
| + methylene aspartate | 98% |
0 |
What conclusions can you draw from these results?
Transamination of aspartate / oxaloacetate seems to be essential for the oxidation of lactate, and therefore presumably for the transfer into the mitochondria of the reducing equivalents on the NADH formed by oxidation of lactate.
From the information you have deduced to date, can you propose a pathway for transfer of reducing equivalents from cytosolic NADH into the mitochondrion?
We know
that oxaloacetate can be reduced to malate in the cytosol, and that malate enters
the mitochondria.
We also know that aspartate can cross the mitochondrial membrane, while oxaloacetate cannot, and that transamination of aspartate / oxaloacetate is essential for transfer of reducing equivalents into the mitochondria.
Therefore it seems likely that the sequence of events is as shown on the right. This pathway is known as the malate-aspartate shuttle, because it involves exchange of mitochondrial aspartate for cytosolic malate:
Aspartate from the mitochondria crosses to the cytosol.
Aspartate is transaminated (at the expense of ketoglutarate) to yield oxaloacetate
Oxaloacetate is reduced to malate by cytosolic NADH
Malate enters the mitochondrion and is oxidised to oxaloacetate, yielding NADH inside the mitochondrion
Oxaloacetate is transaminated to aspartate, which leaves the mitochondrion.
Overall there is transport into the mitochondrion of reducing equivalents from cytosolic NADH to yield NADH inside the mitochondrion.
It is also known, although not shown in the diagram above, that aspartate can only leave the mitochondria in exchange for glutamate entering, so there is a continuing supply of glutamate in the mitochondria to act as the amino donor to oxaloacetate. In turn, the ketoglutarate formed by transamination of glutamate leaves the mitochondria in exchange for malate entering.