Transcript Slide 1

Basic Concepts of Regulation of
Intermediary Metabolism
An Exercise-Centric View of Metabolism
ATP used up by muscle contraction can be regenerated through 4 processes:
1-MK
2-CPK
3-Glycolysis
4-Oxidative Phosphorylation
The Myokinase reaction:
ADP + ADP → ATP + AMP
is impossible to sustain at maximal rates because AMP cannot be
regenerated to ADP and ATP; it can, however, be used to generate
fumarate through the purine nucleotide cycle (costs NRG):
AMP + H2O → IMP + NH4+
IMP + Aspartate + GTP → AMP + Fumarate + GDP + Pi
The Creatine Phosphokinase reaction:
ADP + CP ↔ ATP + C
is impossible to sustain at maximum activity because of relatively
limited supplies of CP in the cell and CP can’t be regenerated very
quickly if the vast majority of ATP is being used up for the
contractile demands. It is however, more active than MK because it
has a lower Km: ~0.02 mM vs. ~0.120mM; [ADP] rest ~ .08, Ex
~0.15) therefore CPK is always active – indicating an important role
in shuttling regulating ATP/ADP/Pi while MK is active only at higher
[ADP]
Glycolysis (+ PDH), β-Oxidation, & several transamination
reactions lead to the production of acetyl CoA which transfers
the acetate group to oxaloacetate in the mitochondria for
further breakdown to CO2 and H2O.
Glycolysis is the metabolic pathway through which glucose 6phosphate is “broken down” to pyruvate (or lactate) in the
cytosol.
Note that there are other metabolic pathways for “glucose”
metabolism: storage as glycogen, glycerol synthesis for
Triglyceride synthesis, pentose-phosphate pathway for
synthesis of a variety of other compounds (including ribose and
NADPH).
Remember These
Pathways!
REGULATION OF GLYCOLYSIS
The rate limiting enzyme of glycolysis: PFK, is the slowest one of the
pathway. It is regulated predominantly by ATP, citrate, AMP, H+ and
fructose 2,6 Bis-P; with the 2,6 BP being the major regulator in liver and a
relatively minor regulator in muscle.
ATP, H+, and citrate greatly inhibit it, promoting storage of glucose as
glycogen whenever ATP levels are high. When ATP levels decrease (slightly)
as a result of extreme rates of ATP use, such as with maximum muscle
contraction, the inhibition of PFK is attenuated and rates of pyruvate and
ATP production increase.
When AMP levels increase from increasing MK activity due to increasing
production of ADP during exercise rates of glycolysis are greatly speeded up.
Maximally stimulated rates of glycolytic enzyme activity can exceed the
maximal capacity of mitochondrial enzymes by about 40x.
Regulation of TCA & ETC
PDH activity allosterically regulated by: NAD+, Co-A, Ca++, and insulin (not
really), enhancing activity, and by Acetyl CoA, NADH + H+, and ATP,
inhibiting avtivity
Citrate synthase is inhibited by ATP
α-ketoglutarate dehydrogenase activity is inhibited by NADH + H+ and
Succinyl CoA and both α-ketoglutarate dehydrogenase and isocitrate
dehydrogenase are activated by Ca++
Cytochrome oxidase activity is enhanced by ADP and thyroid hormone (T2),
inhibited by ATP, while the ATP synthase and the ETC is activated by Ca++.
-note that just about all of the regulated enzymes in mitochondria can be
activated by Ca++ (they are actually more sensitive to calcium than the other
allosteric regulators) ensuring increased ATP supply immediately as it is
needed while there are multiple inhibitors to prevent unnecessary electron
transfer (kinase-decrease/phosphatase-increase).
Recall from the ETC slide that some of the hydrogens that make it to the
intermembrane space can leak out when the membrane gets too hot and some
leak back into the matrix through the mitochondrial permeability transition
which is activated by calcium coming in through the calcium uniporter.
Also note that electron carriers can autooxidize directly to oxygen, creating
oxygen radicals (Co-Q is the major site of autooxidation) with as much as 5% of
resting oxygen use due to this phenomenon.
All of these processes represent a significant amount of electron transfer to
oxygen without concomitant ATP synthesis and all become much more active
when exercising hard, creating interesting complications when trying to
interpret oxygen consumption and its association with athletic performance.
When accounting for ATP synthesis on the basis of the actual proton cost, you
will get ~ 2.5 ATP for each NADH + H+ and ~ 1.5 ATP for each FADH2.
Because of hydrogen leaks & oxygen-radical chemistry, actual yields of ATP
from the electron donors are actually less than the proton cost-based ~ 2.5 ATP
& ~ 1.5 ATP; a yield that will diminish at high temperature and high calcium
conditions (think exercise here).
Recall that H+ and citrate inhibit glycolysis and that it can only be maximally stimulated a lot by
AMP (citrate leaks out of the mitochondria when you make lots of it!). This ensures that rates of
glycolysis will more or less match rates of oxidative phosphorylation at all rates of ATP demand,
at least until rates of glycolysis speed up due to increasing muscle contraction.
We must remember that both mitochondria and LDH enzymes co-exist in cells and therefor they
“compete” for the pyruvate that is produced by glycolysis. Because the muscle form of LDH
maintains [lactate] >> {pyruvate] some of the pyruvate produced through glycolysis will always
be converted to lactate while some is picked up by the mitochondria. Losing lactate from the cell
would be awkward because then it wouldn’t be made into pyruvate by LDH so it is transported
out of the cell only at relatively high [lactate]. Thus lactate diffuses out of muscle cells at high
rates of lactate production (i.e. during exercise) and is an indicator that the LDH is outcompeting the mitochondria for pyruvate - a situation that can change when more mitochondria
are synthesized.
Oxygen consumption will continue to increase at higher workloads (up to the your maximum
ability to remove electrons from “food”) while increasing inefficiencies in oxidative
phosphorylation occur at increasing temperature-, ROS-, and calcium-loads.
Thus your maximum capacity to produce ATP through oxidative (aerobic) pathways; “aerobic
max” (to make ATP), is reached at a workload somewhat similar to that producing the
appearance of lactate in the blood (lactate threshold, OBLA). This approximates to that point
where increasing inefficiencies in coupling O2 consumption to ATP synthesis (due to increasing
work-loads) match the increasing rate of flow of electrons from substrates to O2 through the
metabolic pathways to produce no net gain in “aerobically” produced ATP.
… and another reminder . . .
Abbreviated Summary of Regulation
When you put all this regulation of metabolism stuff together you can see why the relative
use of carbohydrates and lipids changes so dramatically as the intensity of exercise
changes . . .
To make it easier, you think about metabolic pathways rather then individual enzymes
and focus only on a couple of the molecules that regulate the pathways (ATP, ADP, AMP –
ok, that was a few):
There are 6 basic “pathways” 4 of which can regenerate ATP and 3 of which supply
Acetyl CoA to the TCA cycle of the Oxidative Phosphorylation (Ox) pathway and/or
electrons for the Electron Transport Chain of the Ox pathway:
Pathway
Abbr.
Products
Regulators
Glycolytic Pathway
Oxidative Phosphorylation
TCA
ETC
Myokinase
Creatinephosphokinase
β-oxidation
Transamination
Gly
Ox
(ATP + Acetyl CoA + e-)
(ATP/H2O)
(e-, ie.: NADH/FADH)
(ATP/H2O)
(ATP)
(ATP)
(Acetyl CoA + e-)
(Acetyl CoA)
ATP↓
ATP ↓
MK
CPK
β-Ox
Tam
ADP ↑
ADP ↑
“ADP” ↑
“ADP” ↑
A table of substrate use based on “Regulators” follows:
AMP↑ Ca++ ↑
ADP ↑ Ca++ ↑
Ca++ ↑
Ca++ ↑
Notice that at rest FFA provide the
majority of the cells energy
requirements . . . and insulin can
greatly increase uptake of glucose
into the cell . . .
Because the energy needs of the cells have
not changed all that much after a meal,
most of the additional uptake of glucose is
handled in the storage pathways while
some is metabolized at the expense of FFA
Muscle contraction can activate glucose
transport independent of insulin and
activate TG breakdown independent of
epinephrine, greatly increasing glucose
uptake and FFA availability . . .
As a result of muscle contraction, all
metabolic pathways of ATP generation
increase while those of storage and
synthesis decrease . . . Because the
glycolysis pathway increases so much
faster than the others, glycogen stores in
the muscle will decrease very quickly
compared to TG stores . . .