Aerobic Metabolism and the Role of Insulin in Regulation Flashcards
LO #1: Summarize the effects of insulin on glucose and fat metabolism in the well-fed state
In the well-fed state, dietary carbohydrate provides the vast majority of the energy needs of the
body when at rest. Fatty acids make a small contribution primarily to meet energy needs of the
heart. The brain relies solely on glucose for its energy source. Most energy in the body is
produced by complete oxidation of acetyl CoA leading to ATP production. Notable exceptions
are red blood cells (erythrocyte), which lack mitochondria and, therefore, the ability to carry out
oxidative metabolism, and anaerobic muscle.
Dietary carbohydrates and fats are oxidized to CO2 and H2O in peripheral tissues to drive
synthetic reactions and sustain cell function. After a typical meal, a significant portion of dietary
glucose bypasses the liver. The pancreas responds to elevated blood glucose by releasing
insulin from the β-cells (Figure 1). The large influx of dietary glucose does not create a severe
hyperglycemia because insulin maintains blood glucose concentration within a normal range.
This maintenance of glucose balance is defined as blood glucose homeostasis. Insulin
accelerates the storage and metabolism of glucose in a variety of tissues especially liver,
muscle and adipose (Figure 1). Surplus fuel is converted to glycogen and fat under the positive
influence of insulin. Thus, insulin lowers blood glucose through its anabolic effects on glucose
uptake by muscle and adipose tissue, and on glycolysis and glycogenesis in liver and muscle.
Insulin is essential for the proper metabolism of all carbohydrates. Just about everything that
happens to glucose, amino acids, and fat in the well-fed (absorptive) state depends upon a high
ratio of insulin to glucagon (a hormone signal of low blood glucose)
LO #2: Describe the consequence and mechanism of vitamin deficiencies on the enzymatic
activities (E1, E2, E3) of the pyruvate dehydrogenase complex
Acetyl coenzyme A (acetyl CoA), the key product of the reaction, is mostly oxidized in the citric
acid cycle, but may also be used for the synthesis of fatty acids (lipogenesis). Thus PDH
provides an important link between glycolysis and the citric acid cycle. PDH is a multi-enzyme
complex of three activities (Figure 6). E1 requires thiamine diphosphate (TDP) as a prosthetic
group (covalently attached to E1) to remove the CO2 from pyruvate. E2 transfers the acetate
product from E1 onto Coenzyme A, a co-substrate (coenzyme), that binds to E2. An oxidized
lipoic acid prosthetic group is used in the E2 reaction process. E3 uses the FAD prosthetic group
to restore lipoic acid to its original state. Then NAD+, a second co-substrate coenzyme),
regenerates the FAD to its original oxidized state.
E1: Thiamine
E2: Pantothenic Acid
E3: Riboflavin, Niacin
LO #3: Discuss the regulation of pyruvate dehydrogenase by acetyl-CoA, NADH/NAD+, ADP, and
insulin
Regulation of the PDH complex includes both allosteric control and covalent modification
(phosphorylation) (Figure 7). The simplest regulation is feedback inhibition by excess amounts
of acetyl CoA and NADH, respectively. Acetyl CoA, which inhibits E2, accumulates under
conditions where oxidation of fatty acids produces large amounts of acetyl CoA, such as in liver
during food deprivation, in muscle when oxidizing fats for energy, or when the citric acid cycle
oxidizes acetyl CoA slowly because the cycle is inhibited. NADH, which allosterically inhibits E3,
accumulates when it is oxidized to NAD+ more slowly than its rate of formation.
Covalent modification involves adding a phosphate group to the E1 component. E1 either is
“active” (non-phosphorylated form; upper Figure 7) or “inactive” (phosphorylated form; lower
Figure 7). PDH kinase phosphorylates E1 with ATP providing the phosphate. PDH kinase is
activated allosterically by excess amounts of the PDH reaction products (i.e., acetyl CoA and
NADH). This is logical because the appearance of excess products requires that their
production be decreased. These products inactivate the PDH complex either directly by
allosteric feedback inhibition or indirectly by activating PDH kinase. PDH kinase when activated
phosphorylates, and thereby inactivates, the complex. These effects of acetyl CoA and NADH
on PDH kinase are counteracted by a high concentration of NAD+ resulting in a high ratio of
NAD+/NADH that allosterically inhibits PDH kinase. ADP, the signal of low energy levels in the mitochondrion, also inactivates PDH kinase. Inhibition of PDH kinase maintains E1 in its dephosphorylated active state to continue to provide acetyl CoA to boost energy levels.
LO #4: Identify the citric acid cycle reactions that are regulated by NADH and ADP
[1] Citrate synthase initiates the cycle. It catalyzes the condensation of oxaloacetate with the
acetate carbons derived from acetyl CoA that is produced from PDH, β-oxidation of fatty acids,
or oxidation of ketone bodies.
[2] Isocitrate dehydrogenase, the first of four oxidation-reduction reactions in the cycle and
requires NAD+ as a coenzyme. CO2 is released, the first of two carbons lost in the cycle. This
step also is the first site of NADH production for ATP formation via oxidative phosphorylation.
The reaction is stimulated by ADP as a signal of low energy in the mitochondrion. The reaction
is inhibited by NADH to ensure that when NADH accumulates more is not produced.
STEP 1 RELEVANT
[3] α-Ketoglutarate dehydrogenase is the second oxidation-reduction reaction and also requires
NAD+ as a coenzyme. A second CO2 is produced along with a second NADH that yields additional ATP. The reaction is inhibited by NADH using the same logic as in the preceding
reaction. STEP 1 RELEVANT
[4] Succinyl CoA synthetase catalyzes a substrate level phosphorylation reaction.
The GTP product transfers its high-energy phosphate to form ATP.
[5] Succinate dehydrogenase is the only enzyme of the cycle attached to the inner membrane of
the mitochondria. It is an oxidation-reduction reaction that uses FAD rather than NAD+. The
FADH2 product is oxidized via complex II of the electron transport chain. Unlike NADH, FADH2
generates just 1.5 ATP via oxidative phosphorylation.
[6] Malate dehydrogenase is the final oxidation-reduction reaction with NAD+ as the electron
acceptor. This is the final reaction of the cycle and regenerates the oxaloacetate needed to
restart the cycle.
LO #5: Discuss the role of the respiratory chain in ATP formation, link this relationship to the
development of pH and charge gradients across the inner mitochondrial membrane and
discuss the significance of respiratory control
The free energy released from the redox reactions in complexes I, III and IV pumps protons
from the mitochondrial matrix into the intermembrane space (Figure 12). The outer
mitochondrial membrane is freely permeable to protons allowing the pH to equilibrate between
the intermembrane space and the cytoplasm. The impermeability of the inner membrane prevents protons from diffusing back into the matrix. Consequently, a pH gradient forms across
the inner membrane. The pH in the matrix space is more basic (lower proton concentration)
than in the intermembrane space and the cytoplasm so that both compartments are more acidic.
Part of the energy from pumping out protons is conserved by forming this pH gradient. Influx of
protons is harnessed for synthesis of ATP. Because protons carry a positive charge, their
unequal distribution across the inner membrane also creates a charge gradient.
Respiratory control depends on the availability of ADP. When the concentration of ADP
increases in the mitochondrial matrix, ADP opens the proton channel. As protons move through
the channel driven by the pH gradient, respiration increases to compensate for the decline in the
pH gradient. In this way, oxygen consumption (respiration) is controlled. When the matrix
concentration of ADP is low, ATP synthesis ceases allowing the pH gradient to build up.
Consequently, oxygen use diminishes. Then as ATP needs rise respiration again accelerates in
response to ADP. These events correlate with the change in lung respiration during exercise.
LO #6: Explain the roles of the malate-aspartate and glycerol phosphate NADH shuttles during
aerobic metabolism
Under aerobic conditions, NADH produced by glycolysis must be oxidized by the mitochondria.
This event regenerates NAD+ for glycolysis and produces additional energy by feeding electrons
to the respiratory chain. Because no translocase exists to move NADH directly into the matrix,
mitochondrial oxidation of cytoplasmic NADH occurs indirectly. In muscle, brain and other
tissues, but not liver, this oxidation is accomplished by the malate-aspartate shuttle (Figure
14). The key points to know for this shuttle are that i) malate is the electron carrier, ii) the shuttle
generates an additional 2.5 ATP from glycolysis by malate dehydrogenase in the citric acid
cycle producing NADH and iii) the glutamate-aspartate transporter makes the shuttle
unidirectional.
Liver uses the glycerol phosphate shuttle in the same way that muscle and other tissues use the
malate-aspartate shuttle (Figure 15). The key points to know are that i) glycerol-3-phosphate is
the electron carrier, ii) the shuttle generates an additional 1.5 ATP from glycolysis by
mitochondrial glycerol-3-phosphate dehydrogenase producing FADH2.
LO #7: Discuss the actions of uncouplers, oligomycin and complex I inhibitors in relation to the rate
of respiration, synthesis of ATP, flux through the citric acid cycle and PDH, and the
formation of lactate.
Important concepts to recall are that decreased respiration
inhibits the citric acid cycle and PDH because of increased NADH and that when NADH
accumulates, a disproportionate amount of the pyruvate, which can no longer be oxidized in the
mitochondria, is reduced to lactate potentially causing lactic acidosis.
Uncouplers are hydrophobic molecules that bind protons. Their
hydrophobicity allows them to artificially carry protons across the inner membrane into the
mitochondrial matrix. This action collapses the pH gradient by equilibrating protons across the
membrane. Thus these events provide a constant recycling of H+, so that no net gradient is formed.
Without a pH gradient, synthesis of ATP ceases. Even though ATP is no longer synthesized, the
electron transport chain operates at a high rate of respiration because protons are pumped out rapidly
attempting to restore the pH gradient. Energy is released as heat and the body temperature rises. With
uncoupling and no ATP synthesis, reduction of oxygen to water via the respiratory chain is no longer
linked to ATP synthesis so that respiratory control is lost. Because respiration remains rapid, the citric
acid cycle and pyruvate dehydrogenase reaction continue at a rapid rate as NADH is maximally
oxidized. Excessive oxidation of NADH restricts the formation of lactate in uncoupled cells since
NADH is needed to reduce pyruvate to lactate. Naturally occurring uncoupler proteins play important
roles in physiology of obesity and thermogenesis.
Effect of oligomycin (Figure 17c): Oligomycin is an antibiotic that binds to the Fo component
preventing proton flow to the F1-ATPase. The rate of respiration is decreased because the pH
gradient reaches a maximum with blockage of the proton channel. Proton pumping ceases because
there is insufficient energy to make the gradient larger. Respiration decreases even in the presence
of large amounts of ADP and phosphate. This blockage causes ATP synthesis to decline and ADP
to increase. Despite the increase of mitochondrial ADP, the citric acid cycle and pyruvate
dehydrogenase will be inhibited by the marked increase in NADH due to the decline of the rate of
the respiratory chain. The rise in NADH will ultimately lead to reduction of pyruvate to lactate.
Lactate concentrations will be particularly large because pyruvate dehydrogenase is inhibited both
by NADH and by acetyl CoA that cannot be oxidized by the citric acid cycle.
Inhibitors of electron transport (Figure 17d): Rotenone, a rodent poison, directly inhibits electron
transport at Complex I. The metabolic changes with this inhibitor match those in a patient with a
defect in complex I. Although inhibition of complex I blocks electron flow from NADH, small amounts
of ATP are still synthesized from FADH2-linked substrates. Respiration will be reduced but not
completely inhibited. Because oxidation of NADH is prevented, flux through the citric acid cycle and
pyruvate dehydrogenase will be inhibited. Lactate formation will be enhanced. Antimycin A inhibits
complex III. Cyanide, carbon monoxide, and azide inhibit complex IV.
