Chapter 3 – Cellular Energy Metabolism
1. Mitochondrial oxidation of diet-derived carbon-based fuels
Metabolic fuel is imported into cells in one of three main forms: lipid, carbohydrate
and amino acids. In all cases, the carbon backbone of these molecules is eventually
imported into mitochondria for oxidation if required to maintain ATP synthesis. The
exception to this rule occurs in the complete absence of oxygen, where metabolic fuels
are broken down to lactate (in mammals; other endproducts are possible in nonmammals) via anaerobic glycolysis. In most animal cells, this is not sustainable over the
long term, and upon return to normoxia any accumulated lactate is oxidized. Oxidation
of metabolic fuel in mitochondria is directly coupled to the creation of a proton motive
force (PMF) across the mitochondrial inner membrane, and indirectly coupled to the
synthesis of ATP from ADP by the ATP synthase (also called the FoF1ATPase). The
term ‘oxidative phosphorylation’ (OxPhos) refers to the process in its entirety. It is
important to understand how OxPhos is organized.
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The fundamental unit of the bioenergetic organelle is the mitochondrion (plural =
mitochondria). However, it should be noted that most recent data demonstrate that
mitochondria normally exist as a reticulum, with tubule-like branches that extend
throughout the cell. Furthermore, this reticulum is dynamic. It appears to be continually
altering its shape, with individual tubules ‘pinching off’ from the network and then
rejoining elsewhere. Nonetheless, it remains common for people to refer to a
‘mitochondrion’, implying a discrete ovoid entity (the first good electron microscopy
images implied this kind of structure), and I will use this terminology here.
Mitochondria are enveloped by two distinct membranes: the inner membrane and
the outer membrane. For the purpose of the following discussion of bioenergetics, we
will basically ignore the outer membrane, which is quite freely permeable to molecules
with molecular weights <1500 Daltons and therefore does not contribute directly to
OxPhos The inner membrane is actually organized into two regions, though the regions
are fully continuous. The first region is referred to as the ‘inner boundary membrane’,
which encapsulates the interior of the organelles. The second region, ‘cristae’, refers to
tubular processes which penetrate into the interior of the mitochondrial matrix and
emerge out the other side. It is within the cristae that the machinery of OxPhos is
localized.
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The interior of the mitochondrion defined by the inner boundary membrane is the
‘matrix’. It is in the mitochondrial matrix that the enzymes of the tricarboxylic acid
(TCA) cycle reside. Molecules to be oxidized within mitochondria are transformed into
one of the TCA cycle intermediates so that they can enter the cycle. (It’s called a cycle
because it continually regenerate its intermediates) The enzymes of the TCA cycle
catalyze the reduction of the so-called ‘reducing equivalents’, NADH and FADH2 from
NAD+ and FAD+, respectively. It is these reducing equivalents that will donate the high
energy electrons to the ‘electron transfer chain’, thus driving the process of OxPhos.
There are five respiratory complexes: Complex I accepts electrons from NADH
and passes them, via Coenzyme Q, to complex III; Complex II accepts electrons from
FADH2 and passes them, via Coenzyme Q, to complex III; Complex III accepts electrons
from Coenzyme Q, transferring them via cytochrome C to Complex IV. This latter
enzyme transfers the electrons to oxygen, in the process reducing oxygen to water (thus
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oxygen is the ‘electron sink’). This is illustrated in movie form at this website:
http://vcell.ndsu.nodak.edu/animations/etc/first.htm
In complexes I, III and IV the redox reactions undergone during electron transfer
are linked to the extrusion (pumping) of protons out of the mitochondrial matrix. This
thus establishes the PMF. The PMF provides the energy to drive ATP synthesis from
ADP and Pi. The ATP synthase (complex V) is a huge, multi-protein-subunit rotary
motor that spans the crystal membrane (there are many of them per unit mitochondrion).
As protons flow down their concentration gradient and back into the matrix, they propel
the ATP synthase, thus driving the synthesis of ATP. Peter Mitchell received the Nobel
Prize in Chemistry in 1978 for outlining the above in his ‘chemiosmotic hypothesis, first
presented in 1961.
In 1997, John Walker and Paul Boyer shared a Nobel prize for their work in
elucidating the structure of the ATP synthase using X-ray crystallography. With this
information, we now understand the structure of this large mult-protein reverse pump to
look something like this:
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More recently, Masasuke Yoshida has demonstrated that the ATP synthase
functions as a rotary motor. As H+ pass through the core of the enzyme from
intermembrane space to matrix, the membrane-bound Fo portion rotates and this is linked
to the phosphorylation of ADP. Yoshida and colleagues created a movie showing their
experiment directly showing the rotary behaviour of the synthase. View a movie of their
experiment in action at: http://www.k2.phys.waseda.ac.jp/F1movies/F1Prop.htm
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ATP is used up at approximately the same rate as its synthesis, so that there is no
‘storage’ of ATP within the cell; nor does the intracellular [ATP] change markedly in
most animal cells, even when huge changes in ATP turnover may occur, as during the
rest to work transition in skeletal muscle. Thus, this is a dynamical system. Also, in
most cells, there is no way to ‘store’ oxygen (but see chapter 4). It must therefore be
continually supplied at the same rate that it is used up. The rate at which it must be
supplied to a cell is entirely dependent upon the rate at which that cell is cleaving ATP to
ADP. This rate of ATP usage is a reflection of the work the cell is performing. This is
referred to as ‘ATP turnover’. ATP turnover is sustained by oxidative phosphorylation,
and therefore the rate of oxygen consumption during this process is an indication of the
rate of cellular work.
The efficiency of OxPhos
An interesting observation that has been made in all eukaryotic cells in which it
has been studied is that PMF formation (via respiration) is not perfectly coupled to ATP
synthesis. One might expect that all of the protons pumped out of the mitochondrial
matrix would re-enter via the ATP synthase; however, a substantial proportion of these
protons is able to re-enter the matrix via other pathway(s).
In mitochondria isolated from any eukaryotic cell, the PMF established by
respiration-linked proton translocation from the matrix is gradually dissipated unless
there is an ongoing oxidation of metabolic fuel. Thus, even when the ATP synthase is
not working, either in the absence of ADP, or in the presence of its inhibitor oligomycin
(neither of these represents a physiologically normal situation in a live cell),
mitochondria continue to consume oxygen and pump protons. This observation was
made long ago in isolated mitochondria, but was assumed to be due to damage that
occurred during isolation of the organelle. However, subsequent measurements in intact
cells, and even in whole tissues, have indicated that it is a real property of mitochondria
in their normal working environment.
Measurements made in cells and working tissues indicate that the proportion of
oxygen consumption dedicated to simply maintaining the PMF in the face of this proton
leak is about 20% of SMR, a substantial amount! The original observations were made in
mammals, and so it was assumed that this was related to the strategy of endothermy,
allowing heat production that could be harnessed to maintain body temperature above
ambient. However, a similar 20% of cellular MR is consumed by proton leak in the cells
of ectotherms, including reptiles. Therefore, it must have some other purpose.
The realization that cellular oxidative metabolism has this seemingly wasteful
design has actually occurred fairly recently (in the past 10-15 years), and it remains
unknown why this would be the case. 20% of SMR is a lot of energy to be expended on a
process unless it is either an unavoidable design feature or has a specific function.
Interestingly, mitochondrial membranes are more permeable to protons than other
cellular membranes that are not involved in OxPhos, suggesting that it could be avoided.
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Perhaps then, there is a very good reason for the existence of the proton leak in
mitochondria from apparently all animal cells. A number of theories have been put forth.
Two leading theories are: (1) perhaps the uncoupling works similarly to a clutch in a car,
allowing an idling speed where there is some energy consumption but the motor is not
turned off completely. It may be that with this design, it is possible to get up and running
again quickly; (2) at high PMF, mitochondria tend to produce free radicals (reactive
oxygen species; see chapter 4) and the proton leak might be a mechanism to prevent PMF
from becoming too high if [ADP] or Pi becomes temporarily diminished.
Research into the physiological significance of proton leak has benefited from the
identification of specific proteins that catalyze OxPhos uncoupling. The first uncoupling
protein (originally thermogenin; now UCP1; see Chapter 5) was discovered and
described in brown adipose tissue, a specialized form of adipose found in small
mammals. It is not normally present in any other cell types. In 1997, several
homologues to UCP1 (UCP2, UCP3, and others) were discovered and found to be
expressed in a variety of cell types in mammals. It was initially thought that these might
provide the explanation for proton leak; however, although they are widely expressed
they are not ubiquitous, and even mitochondria that have no UCPs nonetheless have
proton leak. Thus, both the mechanism and physiological function of the mitochondrial
proton leak remain incompletely understood, though it has now been established as one
of the major contributors to cellular MR.
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Cellular Energy Utilization and the Molecular Origin of
Standard Metabolic Rate in Mammals
Animals may be composed of enormous numbers of cells. For example, it takes
approximately 300 trillion cells to make an adult human. These cells may bear virtually
no physical resemblance to each other: a neuron and a liver cell appear to have very little
in common. However, they do have the same basic metabolic design in that many of the
energy-consuming reactions are driven by ATP, and oxidative phosphorylation
continually synthesizes ATP to replenish it as it is used up. Oxidative phosphorylation
can be considered as two distinct but coupled processes: (1) oxidation of molecules
derived from the diet (or mitochondrial respiration); (2) ATP synthesis using the PMF.
While ATP can also be made anaerobically (anaerobic glycolysis), this pathway
can sustain the ATP demand for only a short time. Over the long haul, mammalian
metabolism is aerobic. That is, all of the reactions of energy metabolism eventually end
up supplying electrons to oxidative phosphorylation, with the concomitant conversion of
oxygen to water. Because of this, we can measure metabolic rate as the rate at which the
body consumes oxygen.
Thus, whole body metabolic rate is the culmination of all individual cellular
metabolic rates. We can examine whole body metabolic rate as the sum of its constituent
parts. An initial observation is that different tissues and organs make different
contributions to the metabolic rate of the body under standard conditions (see glossary for
definition of standard metabolic rate; SMR). For example, in humans the liver, heart,
kidney and brain are responsible for 55% of oxygen consumption (i.e. metabolic rate)
under standard conditions, despite combining for only 4.9% of overall body mass.
Clearly, the remaining 95% of the human body by mass must be considerably less active
under standard conditions. This remaining proportion includes the skeleton, adipose
tissue and inactive skeletal muscle, all of which have very low metabolic rates under
SMR conditions. However, skeletal muscle can become the major oxygen-consuming
tissue under working conditions.
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As the human SMR was partitioned into contributions made by individual tissues
and organs, so can the metabolic rate of a cell be partitioned. When removed from the
body (isolated) and placed into a bathing medium, an individual cell continues to have a
measurable metabolic rate. This can be measured readily using an oxygen electrode. The
oxygen electrode, contained within a sealed chamber, records the rate at which isolated
animal cells (also in the chamber) use up oxygen. Now, if individual activities that the
cell engages in are inhibited, one can estimate the contribution they make to overall
cellular metabolism. Before examining these contributions, we must remind ourselves of
the way cellular energy metabolism works.
The cell is organized into discrete compartments separated by membranes. With
respect to cellular energy metabolism, the important compartments are the cytosol
(contained within the cell membrane but not including the interior of organelles) and the
mitochondria (enclosed within a double membrane structure). The individual metabolic
reactions can then be considered with respect to their location:
Cytosol: glycolysis – one molecule of glucose split into to molecules of pyruvate
Mitochondria: pyruvate conversion to acetyl-CoA
lipid conversion to acetyl-CoA
amino acid conversion to acetyl-CoA or other TCA cycle intermediates
TCA cycle - acetyl-CoA derived from above substrates enters it; NADH
and FADH2 (reducing equivalents) are produced from NAD+ and FAD+
respiratory chain – NADH & FADH2 from TCA cycle provide electrons to
complexes I or II, respectively. As electrons passed from one respiratory
complex to another respiratory complexes pump protons from matrix
ATP synthase – proton motive force drives synthesis of ATP from ADP
As you can see, the majority of energy metabolism relies on the mitochondria. For this
reason, mitochondria are often referred to as the ‘powerhouses’ of the cell. This is true,
but keep in mind that these organelles do not produce energy. Rather, they ‘transduce’
energy, i.e. convert it from one form to another. To do this, they require oxygen, as the
energy transduction is dependent on the presence of an electron sink. Actually, there are
other possibilities: some deep sea hydrothermal vent animals utilize hydrogen sulphide
as an electron sink instead of oxygen.
Approximately 90% of cellular oxygen usage is mitochondrial. But, there are
some other reactions that consume oxygen: egs. NADPH oxidases, cytochrome P450
oxidases, xanthine oxidase, monoamine oxidase (see Chapter4). Also, not all of the
oxygen consumed by mitochondria to build the PMF (∆Ψm in the figure above) is
coupled to the synthesis of ATP (approximately 20% of oxygen consumption associated
with proton pumping is uncoupled from ATP synthesis because some of the proton leak;
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see above). Thus, only 80% of the 90%, or approximately 72% of total body oxygen
consumption, is linked to ATP synthesis.
The major consumers of ATP in the cell (SMR)
Remember that the vast majority of work done by a cell depends upon proteins. If
we consider the case of muscle (particularly skeletal or cardiac), the shortening of
individual cells occurs via interactions between myosin and actin that consume ATP.
Also, the continual cycling of Ca2+ out of and into the sarcoplasmic reticulum (SR)
requires the activity of the Ca2+ ATPase to pump Ca2+ into the SR. And, the activity of
the Na+/K+ ATPase is required to maintain Na+ and K+ gradients across the sarcopasmic
membrane. There are many other ATP-consuming reactions as well, but studies in which
individual activities are inhibited suggest that a few individual proteins or processes are
responsible for the bulk of ATP consumption in many types of cells.
Na+/K+ ATPase: 5 to 70% of cellular metablic rate
All cells (not just excitable ones) maintain Na+ and K+ gradients between the
cytosol and extracellular fluid. One purpose of this gradient is to drive the import and
export of specific molecules across the membrane (facilitated transport), often against the
concentration gradient of that molecule (secondary active transport). For example,
glucose import into many cells relies on this. Thus, the plasma membrane contains many
co-transporters that will move molecules across the membrane in concert with Na+ and/or
K+ movements (symport or antiport). The gradient itself is maintained by the Na+/K+
ATPase at the expense of ATP hydrolysis. As the metabolic activity of a cell increases,
so does the rate of material movement across the membrane (in either direction).
Therefore, simultaneously, the rate of Na+/K+ ATPase activity increases. The gradient
never collapses; rather, these increases and decreases in activity of membrane
transporters and Na+/K+ ATPase necessarily occur in parallel.
The contribution this one molecular machine (the Na+/K+ ATPase) makes to the
energy budget of a cell (and by extrapolation of the body) can be estimated by inhibiting
it and measuring the change in metabolic rate. Alternatively, it can be measured using
radiolabelled K+ or Rb+ (the latter is recognized as K+ by the Na+/K+ ATPase).
Experiments where this has been done indicate that the pump makes a substantial
contribution to cellular metabolic rate, though the magnitude differs between tissues. In
kidney and brain, Na+/K+ ATPase activity accounts for 50-60% of total cellular oxygen
consumption! In other tissues, it is less, however. By experimentally measuring the
contribution of Na+/K+ ATPase to the metabolic rates of individual cell types and then
multiplying that number by the amount of that cell type present in the body, one can
calculate that approximately 20% of whole body SMR is used to drive the Na+/K+
ATPase. Therefore, fully one-fifth of the oxygen you consume at rest goes simply to
drive this one type of molecular machine.
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Ca2+ ATPase: 5 to 30% of cellular metabolic rate
Ca2+ is an important intracellular messenger in the cytosol of most cells, and
therefore its concentration is strictly regulated. Ca2+ is stored within specific organelles,
particularly the endoplasmic reticulum (or sarcoplasmic reticulum (SR) in muscle) and
mitochondria. The role of the Ca2+ ATPase in muscle provides an excellent example of
its function. The cycles of contraction/relaxation that occur in working skeletal muscle
are dependant upon very rapid increases and decreases in [Ca2+] in the cytosol, and this is
accomplished by storing lots of Ca2+ in the SR and using a gated Ca2+ channel to release
it, and the Ca2+ ATPase to pump it rapidly back in. The contribution this one activity
makes to skeletal muscle metabolism is estimated to be 6% for muscles at rest, but up to
60% for rapidly contracting muscle. Similarly, it is <1% of MR of resting heart muscle,
but 18-36% of MR of contracting heart (of course only the latter condition is
physiologically relevant). It is substantially lower in other cell types. It is estimated that
the Ca2+ ATPase accounts for about 5% of SMR. During exercise or work, this percent
will increase as skeletal muscle makes a progressively greater contribution to whole body
metabolic rate.
Actinomyosin ATPase: 5-6% of SMR
The actinomyosin ATPase is abundant only in striated muscle, i.e.
cardiomyocytes and skeletal muscle cells. However, both types are active even under
SMR conditions. The heart beats at a reduced rate, the diaphragm contracts regularly,
and skeletal muscle isometric contractions maintain posture. It is estimated that
actinomyosin ATPase activity to sustain these activities might contribute 5-6% to oxygen
consumption.
Protein synthesis: 3 to 74% of cellular metabolic rate
They don’t make bodies like they used to. Actually they more or less do, but
they’re just not built to last. Any given cell in a human body is, at any time, using
typically 10-20% of the genes present in its genome. That’s because all cells in a body
have the same genetic material (usually; certain episodes of CSI notwithstanding), but
they have very different functions. In any cell, the process of gene transcription is neverending. There is an ongoing need to synthesize new proteins because the old ones just
keep breaking down! Protein half-lives vary greatly, from several minutes to many
weeks, but all of them eventually wear out and need to be replaced. Making new proteins
is actually quite an expensive undertaking: genetic material must be unwrapped and
exposed, then unwound, transcribed, exported to ribosomes, translated, packaged and
possibly further modified. The cost of protein synthesis as a proportion of cellular
metabolic rate is estimated by either directly measuring incorporation of radiolabelled
amino acids or by inhibiting protein synthesis and measuring the resultant change in
metabolic rate. These measurements, made many times by many laboratories in many
different tissues, can be summarized by the statement that protein synthesis appears to
account for ~20% of whole body SMR. It makes a particularly large contribution to
cellular metabolic in tissues that turn over rapidly. For example, in the epithelium of the
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gastrointestinal tract cells replicate constantly at a relatively fast rate, because they are
constantly being lost and ‘digested’. Therefore, there is an ongoing need to replace lost
cells with new ones, and this means synthesizing all of the proteins necessary to make the
new cells. For this reason, protein synthesis is the largest single contributor to metabolic
rate in intestinal epithelial cells. It makes a much smaller contribution in cell types that
do not replicate at all, such as neurons in the brain.
Other cellular activities
Too many other cellular processes to enumerate here are also continually active
and, together, consume the remainder of ATP produced within the cell. These include
various substrate cycles, turnover of RNA and DNA, reversible phosphorylation of
proteins, gluconeogenesis and urea synthesis.
The analysis of oxygen consumption thus can be summarized as in the figure
below. The vast majority (90%) of oxygen consumed in the body is consumed in
mitochondria (respiration). All of this mitochondrial oxygen consumption is used to
pump protons out of the mitochondrial matrix, creating a gradient to drive ATP synthesis.
However, because 20% of the protons pumped out simply leak back in, only about 80%
is directly coupled to ATP synthesis as protons driving the ATP synthase. Therefore,
about 72% of oxygen consumption is coupled to ATP synthesis. ATP is always
synthesized at rates equal to the overall rate of its usage (to do otherwise is lethal). The
contributions of individual protein or pathway activities can be quantified, producing the
rightmost bar in the figure below. The two most significant molecular sinks of ATP are
protein synthesis and the Na+/K+ ATPase.
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Size and cellular metabolism
One of the more interesting trends in biology is that between size and metabolic
rate. As an animal grows larger, it adds living tissue that requires maintenance and
therefore a constant supply of oxidizable fuel and oxygen. However, there is not a 1:1
relationship between added tissue mass and the fuel required to sustain that mass. Rather,
as the body grows larger, the metabolism required to sustain a unit of tissues actually
declines. In other words, the metabolic rate per gram body mass declines as body mass
increases.
The graph above (from Brody, 1945; Bioenergetics and Growth) shows two
trends. The first is simply that larger animals have a greater BMR. The more interesting
trend however, from the perspective of this course, is that BMR/kg correlates negatively
with body mass. What is the mechanistic basis underlying this trend?
Firstly, some of the relationship can be explained by the fact that the relative size
of many highly metabolically active internal organs decreases with increasing body mass.
The graph below (from Stahl, 1965; Science) shows the relationship in mammals
between body mass and % of body mass that is kidney. The important thing to note is
that a 10 times increase in body mass can be sustained by an approximately 8 times
increase in kidney mass. In other words, it takes less kidney to maintain whole body
BMR as the body becomes larger. Similar trends are observed for other organs, including
brain and liver.
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This is interesting, but quantitatively it is insufficient to completely explain the
allometric scaling relationship. Also, it wouldn’t be particularly relevant to a cell
physiology course to end the story here. It turns out that, in addition to being
proportionally smaller, the organs of larger animals consume less oxygen per unit mass in
the basal state. This was originally demonstrated by Krebs (1956; ref), and has more
recently been confirmed by Paul Else and Tony Hulbert (ref), as shown in the figure
below.
liver
kidney cortex
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The graph shows that, for slices of kidney and liver tissue the metabolic rate per
unit mass decreases as the mass of the animal from which they were taken increases.
Thus, in both instances, and in other tissues as well, the mass specific metabolic rates of
tissue slices (and therefore of the organs from which they are taken) are negatively
correlated with body mass.
This same relationship holds in individual cells isolated from a specific organ.
Porter and Brand showed the negative correlation between the basal metabolic rates of
individual hepatocytes (liver cells) and species body mass.
Thus, it seems that some of the allometry of BMR can be explained by the fact
that individual cells and the tissues they comprise work more slowly in larger animals.
The energy consumed per unit time per cell actually decreases as an animal grows larger.
The question next becomes: How?
It is not because the hepatocytes of the larger animals are themselves larger. As
the graph below shows, hepatocytes from the largest animals have almost exactly the
same volume and mass as those from the smallest animals. In fact, from this observation
we can extrapolate that organs (and animals) get bigger by adding additional cells, rather
than by increasing the size of these cells. This is shown below for hepatocytes isolated
from a wide range of mammals.
Hepatocyte volume
(ul/mg dry mass)
10
1
1
15
100
10000
Body weight (g)
1000000
So, what makes the cells of smaller animals work so much faster than those from
larger animals? Recall that the basic machinery of the cell is made from proteins. Thus,
it is possible that cells might become faster (i.e. higher metabolic rate) by adding more
protein. While this could be true, empirical evidence indicates that it is not. The graph
below shows that total protein per unit tissue mass is quite constant for various tissues
when examined over a range of animal masses. Indeed, if anything it increases as animal
mass does.
250
liver
200
150
kidney
100
50
0
1
10
100
1000
10000
100000
1000000
However, if we confine our analysis to specifically those proteins that make
substantial contributions to cellular metabolic rate, interesting trends do begin to emerge.
The major ATP producers in animal cells are the mitochondria, which we can think of as
ATP-producing machines. If we look specifically at mitochondria and ask if there are
more of them in the faster cells of small mammals, the answer appears to be yes. The
volume of an individual hepatocyte that is occupied by mitochondria is greater in a
mouse than in a horse, by a factor of approximately two (Fig from Porter and Brand).
The negative correlation (log scale) of these parameters is significant.
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ATP synthesis takes place via oxidative phosphorylation, which is organized
across the mitochondrial inner membrane. Generally, any increase in the rate of
oxidative phosphorylation includes a concomitant increase in the overall amount (surface
area) of this inner membrane. If one measures the surface area of the inner membrane
and compares it to body weight, it is also seen to correlate negatively with animal body
mass. This is shown below for liver, kidney and brain in a figure from Else and Hulbert
(1985; American Journal of Physiology). This observation makes sense because we
know that the cellular metabolic rate, and therefore the rate of oxidative phosphorylation,
declines with increasing size. Thus, we have identified mitochondrial abundance and the
magnitude of the scapholding for oxidative phosphorylation (inner membrane) as
important underlying differences between the cells of smaller and larger animals.
However, this is also only a partial explanation. The function of mitochondria is
to continually rephosphorylate ADP. Cellular [ATP] remains remarkably invariant over
quite large ranges of work rate, species and cell types. Thus, if the cells of smaller
animals were simply making all this ATP without a parallel increase in the rate of its use,
all of the available ADP would rapidly be used up and a lot of ATP would accumulate.
We know that this represents a pathological state. Therefore, the ATP-consuming
reactions must be changing in concert with changes in ATP synthesis. One of the major
ATP-consuming enzymes identified above was the Na+/K+ ATPase of the cell membrane,
responsible for maintaining the transmembrane gradients of Na+ and K+ that are essential
for molecular trafficking across the membrane. Experimentally, the activity of the
Na+/K+ ATPase in animal cells and tissues can be measured indirectly as the rate at which
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cells accumulate a radioactive K+ analogue. Couture and Hulbert showed that the rate of
K+ uptake thus estimated scaled negatively with body mass for either liver or kidney
tissue slices, as shown in the figure below.
liver
kidney
Thus, cells of larger animals also have lower Na+K+ ATPase activities. It is likely
that much of the metabolic machinery is similarly reduced in the cells of larger animals,
although in most cases we do not yet have data to demonstrate this.
However, one of the major consumers of oxygen (non-productively) is the proton
leak across the mitochondrial inner membrane. We know that this accounts for about
20% of mitochondrial oxygen consumption, and therefore any substantial change in the
rate of proton leak could make a substantial contribution to these allometric relationships.
It is therefore important to note that the rate of proton leak (at a constant membrane
potential) is greater in hepatocytes of smaller mammals, as shown in the graph below
from Porter and Brand. Thus, a significant portion of the greater metabolic rate of mouse
hepatocytes relative to horse hepatocytes occurs due to a greater leak of protons across
the mitochondrial inner membrane. So, scaling of both ATP-consuming, ATP-producing,
and energetically non-productive reactions contribute to the allometric relationship
between cellular metabolic rate and body mass in animals.
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One question that remains to be answered is: what causes the metabolic rates of
cells to scale this way? Is it an intrinsic property of cells of small species to adopt faster
metabolic rates? One way this question can be addressed is to measure the metabolic
rates of cells in laboratory culture. Cells can be isolated from mammals of vastly different
mass, maintained in culture, and their metabolic rates then measured. This approach was
used by Brown and Stuart, who found that metabolic rate scaling was absent in cells
propagated for many generations in culture, as shown in the figure below.
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In culture, cells from all species are grown in the presence of blood serum derived
from fetal calves. Thus, they are all receiving the same environmental cues with respect
to growth rate. Similarly, all cells are grown in the same kind of media, with constant
levels of glucose and other metabolic fuels. Therefore, while this result was obtained for
just one cell type, a fibroblast, it suggests that the higher metabolic rate of mouse cells
might not be an intrinsic property, but rather originate from differences in the
environment within a mouse versus within a horse. These differences might include
parameters such as thyroid hormone and/or growth hormone (both of which affect
metabolic rate).
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