In order for cells to live and perform their programmed functions, they must produce energy. This cellular energy is governed by ATP (adenosine triphosphate ) molecules. ATP is synthesized inside the cell during cellular respiration. Hydrolysis (breakdown of ATP under the action of water) of ATP releases energy stored in its phosphate bonds. The standard energy of ATP hydrolysis under physiological conditions is tightly regulated in all cells between − 53 and − 60 kJ/ mol. Energy from ATP is used to gate ion channels and maintain cell membrane potential.
There are several sources of ATP synthesis that can be used to maintain membrane potentials. The mitochondria produce most of the energy in normal mammalian cells. In cells with functional mitochondria, ATP is derived mostly from oxidative phosphorylation (OxPhos) where approximately 89% of the total cellular energy is produced (about 32/ 36 total ATP molecules during the complete oxidation of glucose). This value can differ among different cells depending on which shuttle systems are used in the transport of cytoplasmic reducing equivalents (nicotinamide adenine dinucleotide (reduced form), NADH) from the cytoplasm to the mitochondria. Through OxPhos, mitochondria produce the bulk of intracellular ATP, and hence are considered the cell's “power plants.” In addition, mitochondria regulate Ca2 + homeostasis and modulate several other metabolic circuitries including the Krebs cycle, the urea cycle, gluconeogenesis, ketogenesis, heme biosynthesis, fatty acid β-oxidation, steroidogenesis, metabolism of certain amino acids, and the formation of iron/ sulfur clusters. .....
Besides OxPhos, approximately 11% (4/ 36 total ATP molecules) of the total cellular energy is produced through substrate-level phosphorylation.
In oxidative phosphorylation, ATP is produced through electron transfer reactions in the presence of oxygen, using a gradient of protons across the inner mitochondrial membrane. Substrate level phosphorylation on the other hand, generates ATP through addition of phosphates to ADP from the substrate or an intermediate reaction product. There are two main pathways in mammalian cells for substrate level phosphorylation:
a) glycolysis in the cyptoplasm of the cell where glucose (substrate) is broken down generating net 2 ATP molecules
b) succinyl-CoA synthetase reaction in the TCA (tricarboxylic acid cycle or Kreb's cycle) cycle which takes place in the mitochondrial matrix and generates 2 ATP molecules.
Unlike OxPhos, substrate level phosphorylation does not require oxygen.
The number of ATP molecules produced from TCA cycle, substrate-level phosphorylation would need to increase if OxPhos were insufficient to maintain energy homeostasis. This would be similar to the increase in the number of ATP molecules produced through glycolysis when OxPhos is reduced. Nonoxidative energy production through amino acid fermentation and substrate-level phosphorylation has been documented in developing mammalian embryos, in diving animals, and in heart and kidney tissue under hypoxia.
An important point to consider is that the standard energy of ATP hydrolysis under physiological conditions is tightly regulated in all cells as mentioned earlier. Energy production in heart and liver which contain many mitochondria is largely through OxPhos while the red blood cells (erythrocytes) which do not have a nucleus or mitochondria produces energy through glycolysis. The membrane potentials for these cells are
heart : -86mV
red blood cells: -6mV
Despite the differences in resting membrane potentials as well as mechanism of energy production among various cells in the body, the free energy of ATP hydrolysis is maintained around -56 KJ/mol. This indicates that the balance of energy consumption and production (energy homeostasis) is independent of the energy source and the total amount of ATP produced.
. Any disturbance in this energy homeostasis affects cell function and cell viability. Normal cells have the capacity to balance the energy use and production with respiration and substrate level phosphorylation thus maintaining the free energy of ATP hydrolysis. However, energy dysregulation is the characteristic of cancer cells. Energy regulation in cancer cells
If OxPhos becomes compromised in a normal cell through some injury, then substrate level phosphorylation must increase to maintain the stable free energy of ATP hydrolysis and cell viability. The cell could reduce energy expenditure to balance the reduced energy production. The cell could also die through apoptosis (programmed cell death) or necrosis (stress induced uncontrolled cell death).
It is important to recognize that prolonged reliance on substrate-level phosphorylation for energy production in previously normally respiring cells produces genome instability, disorder, and increased proliferation, that is, the hallmarks of cancer. Entropy refers to the degree of disorder in systems and is the foundation of the second law of thermodynamics . Szent-Gyorgyi described cancer as a state of increased entropy, where randomness and disorder predominate . Protracted OxPhos insufficiency coupled with persistent compensatory fermentation increases entropy. Cells that do not increase fermentation energy to compensate for insufficient OxPhos simply die off and never become neoplastic. Adaptation to fermentation allows a cell to bypass mitochondrial-induced senescence. Cancer arises in those cells that bypass mitochondrial-induced senescence (akin to programmed cell death).
Warburg was the first to describe in detail the dependence of cancer cells on glucose and glycolysis in order to maintain viability following irreversible respiratory damage. He considered respiration and fermentation as the sole producers of energy within cells, and energy alone as the central issue of tumorigenesis. “We need to know no more of respiration and fermentation here than that they are energy-producing reactions and that they synthesize the energy-rich adenosine triphosphate, through which the energy of respiration and fermentation is then made available for life” [Warburg] . Warburg considered fermentation as the formation of lactate from glucose in the absence of oxygen. This type of energy is also produced in mammalian embryos and in our muscles during strenuous exercise.
A dependence on glucose with lactate production in the presence of oxygen later became known as the Warburg effect, which is essentially aerobic glucose fermentation or the continued production of lactic acid in the presence of O2. Why would cancer cells continue to ferment glucose in the presence of O2? Warburg attributed the aerobic fermentation in tumor cells to respiratory damage or respiratory insufficiency. Tumor cells grown in the presence of O2 behave as if they were abnormal facultative anaerobes in continuing to ferment in the presence of O2.
Here it may be worthwhile to understand the difference between respiration and fermentation. From a cellular perspective, fermentation is evolutionarily older than respiration. Fermentation is linked to relatively uncontrolled cell proliferation and less differentiation. Respiration in the presence of oxygen on the other hand is a characteristic of more complex, differentiated structures. So in the presence of injury to the mitochondria which disrupts respiration, cells which do not die but revert to fermentation as their main source of energy can eventually transform into malignant cancer cells.
There is considerable experimental evidence of mitochondrial injury in cancer cells. Mitochondria in tumors differ in number, size and shape from regular mitochondria. There is experimental evidence to show that OxPhos capability is closely linked to the structural integrity of healthy mitochondria; conversely when OxPhos capability is impaired, the morphology becomes abnormal. It has been reported that greater the degree of mitochondrial morphological abnormality, greater is the degree of malignancy of the tumour. Disputes about Warburg's Theory
One insufficiently rigorous criticism of Warburg's theory comes from the observation that cancer cells do use O2. This is then taken as an indication of normal respiration in cancer cells. However, there is experimental evidence to show that is not true.
Indeed, O2 consumption increases with increased malignancy in some tumor cells. Does this mean that respiration is normal or increased in such cells? Not necessarily. Warburg has attributed this phenomenon to defects in the coupling of respiration to ATP production . In other words, some cancer cells produce CO2 and consume O2, but produce insufficient energy through respiration.
Defects in the inner mitochondrial membrane of tumor cells dissipate the proton motive gradient, thus uncoupling the linkage between electron transport and ATP production through OxPhos.
When such uncoupling occurs between the electron transport mechanism (where oxygen is utilized as in OxPhos ) and usable energy production ( ATP synthesis as in OxPhos), there is often a production of heat. More aggressive tumours are known to be "hot". The author's hypothesis ties the heat produced in aggressive tumours to the mechanism of mitochondrial uncoupling. Role of amino acid fermentation
One of the criticisms of Warburg's hypothesis was that not all cancer cells showed elevated lactic acid production through glycolysis. The author provides evidence which supplied the missing link in Warburg's hypothesis.
Although many tumor cells have active TCA cycles and might appear to respire, in that they consume oxygen and produce CO2 and ATP in the mitochondria, I will present data showing that this is pseudo respiration in some cases. In other words, pseudo respiration has all the characteristics of respiration, but does not involve ATP synthesis through OxPhos. I propose that this apparent respiratory energy is derived from amino acid fermentation. Just as tumor cells ferment glucose in the presence of O2, some tumor cells also ferment glutamine and possibly other amino acids in the presence of elevated glucose and O2. Glucose and glutamine interact synergistically to drive tumor cell fermentation. Fermentation is the bioenergetic signature of tumor cells.
Mitochondrial amino acid fermentation is known to maintain metabolic homeostasis under hypoxia in several species of diving animals. Mitochondrial amino acid fermentation can also maintain metabolic homeostasis in the heart and kidney under low glucose and low O2 conditions. The possibility that tumor cells might also obtain energy through amino acid fermentation has not been considered previously as an alternative energy source to OxPhos. Although Warburg considered respiration and glucose fermentation as the sole producers of energy within cells, amino acid fermentation in the mitochondria can also produce energy through substrate-level phosphorylation.
We were the first group to report that Krebs cycle substrate-level phosphorylation might compensate for insufficient respiration in metastatic cancer cells . On the basis of preliminary studies, I suggest that energy through glutamine fermentation could compensate for insufficient or suppressed respiration in those tumor cells that can use glutamine for energy. While it is well known that glucose can be fermented, less is known about amino acid fermentation. Lactate is the by-product of glucose fermentation, whereas succinate, alanine, and aspartate are by-products of glutamine or amino acid fermentation under hypoxia (insufficient oxygen availability). ...........
The expression of lactate in the presence of O2 is abnormal and would indicate that the cells are fermenting. The degree of fermentation (lactate production) is positively correlated with the degree of malignant growth . Also, the less is the respiration, the greater is the fermentation.
....If the cells consume oxygen, it is unlikely that succinate would accumulate. Under high glucose, amino acid fermentation can occur whether or not succinate accumulates. Hence, it is important to account for the multiple variables required to assure that cells are actually using OxPhos alone or are using some combination of OxPhos and mitochondrial substrate-level phosphorylation to maintain their viability.
Using a bioluminescent-based in vitro ATP assay, we found that ATP production and cell viability were similar in the metastatic cells grown in media containing either glutamine alone or glucose alone. Shelton also showed that lactate production was significantly lower in the metastatic cells grown in glutamine than in the cells grown in glucose, indicating that these cells produce little lactate from glutamine alone. ...However, ATP synthesis and lactate production were significantly greater in the VM-M3 tumor cells grown in glucose and glutamine than that for the tumor cells grown in either glutamine alone or glucose alone . These findings show that glucose and glutamine work synergistically to enhance ATP synthesis, lactate production, and growth..... The synergy we found in the VM-M3 tumor cells was due to glutamine, as neither aspartate nor alanine (alternative nitrogen sources) could replace glutamine for the effect.
In other words, mitochondria are capable of metabolizing glutamine in these tumor cells. The question arose as to whether these cells were using the glutamine to produce energy through OxPhos or through mitochondrial fermentation. The role for glutamine in energy production would be in addition to the known role of glutamine in replenishing TCA cycle metabolites (anapleurosis).
We showed that tumor cell viability and ATP production were robust in either anoxia or cyanide as long as both glucose and glutamine were present in the media. Since anoxia (95% N2, 5% CO2) or cyanide (an inhibitor of complex IV respiration) inhibits OxPhos, the robust synergy seen for glucose and glutamine is unlikely due to significant energy from OxPhos.
We propose that the glucose/ glutamine energy synergy observed in our metastatic mouse cells arises from linked fermentation redox couples in the cytoplasm and mitochondria that synthesize ATP largely through nonoxidative substrate-level phosphorylations.