General Overview of Intermediary Metabolism

Just about anything you eat is metabolized to carbon dioxide & water with the concomitant synthesis of ATP to provide the chemical energy necessary to build and repair cellular components or to sustain muscle contraction.

ATP used up by muscle contraction can be regenerated through 4 processes: 1-MK 2-CPK

3-Glycolysis 4-Oxidative Phosphorylation

An Exercise-Centric View of Metabolism

The Myokinase reaction: ADP + ADP → ATP + AMP

is impossible to sustain at maximal rates because AMP cannot beregenerated to ADP and ATP; it can, however, be used to generatefumarate 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]

Minerals(functional) components of enzymes, molecular bindingfactors, catalysts . . .

Vitaminsco-factors, antioxidants, gene regulators . . .

Amino AcidsNRG, amino acid sequence produces structure of enzymes,

DNA, RNA, NAD+, FAD+ . . .

Fatty AcidsNRG, part of structure of some enzymes, Phospholipids . . .

CarbohydratesNRG, component of DNA, RNA, ATP, NAD+, FAD+ . . .

Just for fun we should not view ATP generation for energy as the only metabolism happening:

A Nutritionist’s View of Metabolism (& Cell Function)

Enzymes perform chemical reactions of metabolism- requires (protein) synthesis of enzymes (duh!)- many enzymes contain minerals as part of their structure- many enzymes require co-factors which contain vitamins as part of their structure.

Protein synthesis- requires DNA, RNA, mRNA, GTP . . .- synthesis enzymes require Cr+?, Mg++, Zn+ to function

Synthesis of DNA, RNA, ATP, GTP . . .- folic acid, B12, glucose, aa, PO4 (structure)- enzymes for synthesis require Zn+,Cr+?, Mg++, to function

Getting back to the ATP thing, these are some of the major metabolic pathways for

resting and exercise metabolism:

Glycolysis: produces pyruvate for acetyl CoA production in mitochondria, produces NADH (electrons) for ETC in mitochondria, anaerobic production of ATP

MK & CPK: anaerobic production of ATP

TCA: accepts acetyl-CoA for citrate synthesis, production of NADH (electrons) for ETC, “anaerobic” production of GTP

β-oxidation: produces acetyl CoA for TCA

Transamination: produces pyruvate, or acetyl CoA, or TCA intermediates

ETC: electrons from TCA cycle & glycolysis are “joined” to oxygen to make water & the production of ATP

Pentose Phosphate Pathway: production of ribose and NADPH for nucleotide and other synthesis processes

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 is broken down to pyruvate in the cytosol.

In order to prevent glucose from building up inside the cell and producing a huge osmotic (and gradient) problem it is immediately phosphorylated by the enzyme hexokinase (using up one ATP molecule). The resulting glucose 6-phosphate can either be stored as glycogen or progress through the glycolytic pathway.

Step 3, catalyzed by the enzyme Phosphofructokinase (PFK) is the rate-limiting step in glycolysis. It converts fructose 6-P to fructose 1,6 bisP.

After several more steps two pyruvates & 2 NADH + H+ are made with 4 ATP being produced; a net gain of 2 ATP for this pathway.

The 2 pyruvates and 2 NADH + H+ are picked up by the mitochondria.

©C. Murray Ardies, 2008

Inside the mitochondria, the pyruvate is converted to 2 acetyl CoA, 2 CO2 and 2 (more) NADH + H+ by the enzyme PDH.

The acetyl CoA condenses with oxaloacetate (OAA) to synthesize citrate and leave behind the CoA.

Through a series of enzyme reactions, the citrate is converted back into OAA with the concomitant production of 1 GTP, 1 FADH2, 3 NADH + H+, and 2 CO2.

The NADH and FADH are transferred to the ETC while most of the CO2 diffuses to the blood (and lungs) for disposal. The GTP can be used for protein synthesis or converted to ATP for use as “NRG”. ©C. Murray Ardies, 2008

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 activities can exceed the maximal velocities of mitochondrial enzymes by about 40x.

Regulation of glucose levels in the blood is very important

Normal Fasting (Serum) ~ 70 – 100 mg/dl Elevated = Diabetes Low = Hypoglycemia

Insulin from pancreas stimulates uptake of glucose into muscle cells by activating the GLUT4 transporter. When serum levels of glucose decrease too much, then the pancreas releases glucagon to stimulate the liver and kidney to break down glycogen to glucose and release glucose into the blood (glycogenolysis). Adrenals will release cortisol if levels stay too low to enhance breakdown of protein in muscles to release amino acids so the liver can pick up the amino acids and make them into glucose (gluconeogenesis).

Epinephrine stimulates the triglyceride lipase to cleave fatty acids off of the glycerol; making lots of fatty acids available for NRG.

Fatty acids are transported into the mitochondria by a carnitine transporter which is in the membrane. (Note that ascorbic acid is required for carnitine synthesis.)

Once inside the mitochondria the fatty acids are oxidized into two-carbon fragments (acetate) which are utilized by the TCA cycle in the form of Acetyl-CoA.

The central role of

glycolysis and TCA cycle in intermediary metabolism is illustrated by all the different compounds which originate from these pathways and which can be metabolized

through these pathways

Glycolysis + PDH, β-Oxidation & some transamination reactions produce acetyl CoA (different transamination reactions produce TCA cycle intermediates)

The GTP produced can be used for protein synthesis while the NADH+H+/FADH2 donate their electrons to the electron transport chain

In the simplest terms, NADH passes its electrons to complex I while FADH gives them to Co-Q.

The electrons are passed alomg the ETC to complex IV where they are “joined” to oxygen.

At the same time, hydrogens are transferred from the matrix to the inter- membrane space, creating a proton gradient.

When the protons move through the ATP synthase to return to the matrix, their movement through the synthase powers the regeneration of ATP from ADP + PO4

Note from the preceding 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 (and other mitochondrial uncoupling proteins) which are 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 much less than the proton cost-based ~ 2.5 ATP & ~ 1.5 ATP; a yield that will diminish at higher temperature and higher calcium conditions (think exercise here).

Theoretical efficiency of ATP production from 1 molecule of glucose:

Glycolysis produces 2 pyruvate molecules and 2 NADH + H+ (enters the mitochondria as FADH2), PDH produces 1 NADH + H+ and 1 acetyl CoA for each pyruvate molecule, and TCA cycle produces 3 NADH + H+ and 1 FADH2 for each pyruvate molecule for a total of 9 NADH + H+ (x 2.5) to produce 22.5 ATP, 3 FADH2 (x 1.5) to produce 4.5 ATP, and a net gain of 2 ATP in glycolysis and ~1.5 ATP (GTP) in TCA cycle for a grand total of ~ 30 ATP for each glucose molecule oxidized to 6 CO2 and 12 H2O.

(It costs H+ to transport GTP (ATP) out of the mitochondria which makes the yield less than the 2 GTP actually produced)

6 CO2 produced for 6 O2 (RER: 6/6 = 1) used and ~ 30 ATP produced gives 30/6 = ~ 5 ATP/C and 30/6 = ~ 5 ATP/O2 used

Theoretical efficiency of ATP production from 1 molecule of palmitate, a 16 carbon saturated fatty acid.

7 turns of β-oxidation produces 7 NADH + H+ and 7 FADH2 and 8 acetyl CoA.

Each acetyl CoA produces 3 NADH + H+, 1 FADH2, and 1 GTP for a total production of 31 NADH + H+ (x 2.5) to produce 77.5 ATP, 15 FADH2 (x 1.5) to produce 22.5 ATP and 8 GTP to produce a net of 6 ATP for a grand total of 106 ATP for each palmitate molecule metabolized to CO2 and H2O.

16 CO2 produced for 23 oxygen used (RER: 16/23 = 0.695) and 106 ATP produced gives 106/16 = 6.625 ATP/C and 106/23 = 4.6 ATP/O2 used.

In comparing fat to glucose as a substrate it is clear that glucose gives a better ATP yield per oxygen used: ~ 5 for glucose vs. ~ 4.6 for palmitate indicating that glucose is a better substrate to use when efficiencies of ATP synthesis through the ETC are increasingly diminished as metabolic intensity increases (think exercise).

On the other hand, fat gives a better ATP yield per carbon stored: ~ 6.625 for palmitate vs. ~ 5 for glucose. Thus fat makes for a better storage form of substrate; especially when you consider that glycogen is hydrophilic: 1:1 (or greater) water:glycogen.

Summary Of Substrate Use:

ATP Yield / ATP Yield/ Water O2 Used CO2 Content

(stored)

Glucose/ ~ 5/ ~ 5 ~5:1Glycogen ~ 5.2 ~ 5.2

Palmitate ~ 4.6 ~ 6.625 0

Of course, these are the theoretical maximums of total ATP yield: ~ 106 ATP for each palmitate molecule metabolized to 16 CO2 and 46 H2O & ~ 30 ATP for each glucose molecule oxidized to 6 CO2 and 12 H2O.

It doesn’t really happen that way in vivo…

If TCA intermediates leak out, they must be replaced or the cycle shuts down: w/o oxaloacetate to condense with Acetyl CoA, citrate simply cannot be made and the cycle is done for… and if they leak out… they obviously can’t be oxidized as a source of electrons – another reason for less than 2.5 ATP / NADH…

Notice that heat increases the permeability of the mitochondria membrane to H+. That means that efficiency of ATP synthesis MUST be less than 2.5 ATP / NADH and 1.5 / FADH… even at rest!

The membranes are in fact, permeable to several compounds produced in the mitochondria, including some produced in the TCA cycle (especially citrate, oxaloacetate & succinyl Co-A).

Regulation of PDH, TCA cycle, and ETC are exceedingly important; if they worked fast all of the time then the electron carriers NAD and FAD would be full of electrons (FADH2 and NADH + H+) and they can easily autooxidize to produce superoxide anions.

These radicals react to produce peroxide:

O2- + O2

- H2O2 + O2

Peroxide and superoxide can then react to produce the dreaded hydroxyl radical:

O2- + H2O2

.OH + -OH + O2

Note that as molecular oxygen picks up electrons and reacts with other oxygen radicals (ROS) the end result is the production of water and hydroxyl radicals.

In the presence of free iron, rates of ROS chemistry are greatly accelerated! Highly relevant if ROS-mediated iron release from aconitase occurs…

Recall the oxygen radical chemistry from the previous slide which produces the hydroxyl radical:

O2- + O2

- H2O2 + O2

O2- + H2O2 .OH + -OH + O2

In order to protect ourselves from the consequences of these reactions we have antioxidant enzymes which help avoid the problem:

SuperoxideDismutase

O2- + O2

- H2O2 + O2

Catalase

H2O2 + H2O2 2 H2O + O2

The enzymes SOD and CAT reduce oxygen radicals to water and oxygen, preventing the build-up of the hydroxyl radicals and reducing ROS damage – an important consideration for exercise (recall IOM recommendations) since exercise can greatly enhance the production of ROS.

Manganese or copper/zinc

iron

Other means exist to reduce ROS-mediated damage such as ingesting sufficient amounts of the antioxidant: vitamin C (ascorbic acid)Notice that ascorbate can beregenerated byglutathione

Vitamin E also acts as an antioxidant; although it specifically acts on lipid radicals, not water-soluble ones like ascorbic acid.

Notice, however, that it can be regenerated by ascorbic acid/glutathione (and also directly by glutathione).

Because a high rate of metabolism during exercise is apotent ROSgenerator, regular exerciseenhances the need for antioxidants.

Note that GDH produces ammonia (toxic) while ALT produces alanine (from pyruvate).

Both PC and ALT may have important implications for high-intensity exercise:

Pyruvate can be made into useful non-lactate products and the alanine diffuses out of the cell very easily, lactate does not!

Recall that Ile & Val are metabolized mainly in muscle and also can be made into Succinyl Co-A; normal aa metabolic pathways that also can be considered anapleurotic…

So… if TCA intermediates leak out, how do we regenerate the OAA? … Through what are called: Anapleurotic reactions: The major anapleurotic reactions are catalyzed by the enzymes: pyruvate carboxylase; glutamate dehydrogenase and alanine aminotransferase.

Overview of the major anapleurotic reactions…

PDH activity is enhanced by NAD+, Co-A, Ca++, and insulin and inhibited by Acetyl CoA, NADH + H+, and ATP

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 while the ATP synthase is activated by Ca++ and ETC as well.

-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.

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 out-competing 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

heat and radical formation at higher 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.

An interesting issue….recall the major anapleurotic reactions…

…what might happen with a specific nutritional deficit … such as say…ascorbic acid???

Ascorbic acid a necessary co-factor carnitine synthesis as well as an antioxidant

Because aconitase and Co-Q are especially sensitive to oxygen-radical attack, ascorbic acid deficiency should lead to a decrease in β-oxidation of fatty acids, a decrease in α-KG production from citrate, a decrease in FADH coupling to Co-Q, and an increase it citrate leaking

The major compensatory mechanism(s) is simply to accelerate the velocity of the glycolytic and anapleurotic enzymes to make up for the deficits…