Transcript Dieting with Dinitrophenol - National Center for Case Study

A Diet to Die For: An Exploration
of Oxidative Phosphorylation
by
Terry Platt, Department of Biology,
University of Rochester
Eric Ribbens, Department of Biological
Sciences, Western Illinois University
1
The “Energy Burner”
“Cheryl! Come here!” Charles called to his twin sister
excitedly.
“What?” asked Cheryl.
“You know how my JV wrestling coach wants me to get
down to the 145-pound weight category? Well, it’s here!”
“What’s here?”
“The solution! DNP!”
2
Just a Few Little Pills
Cheryl picked up the invoice. “Seventy-one bucks for 11
tablets? What is musle-man.com anyhow?”
Charles nodded. “It’s not cheap, but it’s been
scientifically proven to work. Here are the instructions.
Take one pill the first day, two tablets each of the next
four days…”
“So this really works? What does it do?”
3
The Plan…
“I don’t know, and I don’t care. But it should help me
get rid of these eight pounds in only a week. It’s fast,
and it’s proven to work.”
Cheryl was intrigued. “Maybe this could help me trim
back too. I could sure use it in a few crucial places…
Hey, does your coach know about this? Maybe it could
help the whole team!”
“Heck, no!” Charles exclaimed, “It’s my secret for now;
besides, he wouldn’t care – he just wants us to win,
and this is my first college match!”
4
…Goes Awry
Two days later, Cheryl met her brother crossing campus
after dinner and learned that he had taken an extra two
pills in the afternoon, since he thought he wasn’t losing
weight fast enough to make his cutoff next weekend.
“You know, you don’t look so good – you seem kind of
flushed, and you’re breathing pretty fast – have you been
running?”
“Actually,” Charles confided, “I haven’t, and I’m a little
scared because my body feels like its racing, even though
I’m not…”
5
You Can Lose More Than Weight
“I feel kind of nauseous, too, and weak in the knees,”
continued Charles, “and I’m sweating like a horse – look
how damp my shirt is.”
“We better get you to Student Health,” urged Cheryl. “The
sooner the better.”
“Well, okay, but don’t tell my coach about this, or he might
not let me compete on Saturday!”
Fortunately, Student Health saw him quickly. They
immediately called an ambulance for emergency treatment,
as they recognized that there was more than Charles’
weight at stake.
6
Two Days Later …
A scared Cheryl and her parents listened nervously to the
emergency room doctor.
“You said he was taking DNP, 2,4-dinitrophenol? Well,
you’ve all had a very serious scare, but it looks like he’s
going to be okay. We’ll keep him here for a few days, just
to be certain.”
Cheryl sniffed. “He’ll be mad to miss his match, and I don’t
understand! It was working so well. And it was for sale
over the internet! What happened?”
Dr. Adams frowned.
7
A Diet You May Die For
“Medically, DNP is one of those drugs where the
‘therapeutic dose,’ which is the amount that will
produce desired results in half of those who take it, is
only a little lower than the ‘lethal dose’ (LD50), where
half of those who take it die. For drugs to be medically
approved, the LD50 must be much higher than the
therapeutic dose.”
“Oh my gosh,” Cheryl said to her parents in a whisper.
“Those two extra little pills could have killed my
brother!”
8
Charles is Alive and Stable
While Charles was recovering, Cheryl found a paper “Dying to be
thin: A dinitrophenol related fatality.”* The authors note that 2,4
dinitrophenol:
– “was originally used as an explosive and later introduced in the 1930’s to
stimulate metabolism and promote weight loss. Concerns…led to DNP
being banned as a dietary aid in 1938.”
– A 22-yr-old male patient arrived at the ER 16 hr after his last dose of DNP
with a temperature of 102 degrees Fahrenheit and sweating profusely.
– He became agitated and delirious, was mechanically cooled and given IV
drug treatment to counter the DNP, but within another hour his heart
slowed, stopped, and, despite resuscitative efforts, he died.
– “Advertisements claim DNP safe at the dose our patient ingested. It is
widely available and with the potential to cause severe toxicity is an
understudied public health concern.”
*McFee et al. (2004) Vet. Hum. Toxicol. 46: 251-254.
9
Why Does DNP Do This?
• Cheryl read some more. In an article titled “Weight loss
and 2,4-dinitrophenol poisoning,”*
– a 27-yr-old female admitted to the ER “complaining of fatigue,
nausea, and excessive sweating” had begun taking new diet tablets
the week before, and had “doubled the recommended dose for faster
results.” Despite heroic efforts to save her, she died 7 hours after
admission.
– the authors stated that “DNP causes a hyper-metabolic state by
uncoupling oxidative phosphorylation. Energy is released in the
mitochondria as heat… Toxic doses will result in uncontrolled
thermogenesis leading to hyperthermia and systemic responses to
elevated body temperature.”
*Tewari et al. (2009) Brit. J. Anaesthesia 102: 566-567.
10
What is Oxidative Phosphorylation?
• Cheryl had only a vague recollection of this
process, recalling that it had something to do
with metabolic breakdown of energy rich
compounds, electrons, phosphorylation, and
the role of ATP.
• What do you remember, and what do you
think?
11
CQ#1: Choose the description below that
best completes the statement “oxidative
phosphorylation is the process in
mitochondria by which”:
A. Electrons reduce O2 to H2O and this causes
ATP to be made.
B. Glycolysis produces more ATP than is
needed to activate its pathway.
C. Oxygen is used to cause phosphorylation of
biological molecules.
D. Synthesis of ATP is dependent on the
passage of electrons through the electron
transport chain.
12
Here’s a Quick Review
“Ox Phos, as it’s often called, is carried out in your
mitochondria…,” began Dr. Adams.
Cheryl interrupted, “Oh, yeah, I remember - they’re ‘the
powerhouses of the cell’!”
“...where much of your cellular energy, in the form of ATP, is
made. The majority of electrons derived from oxidation
[remember OILRIG?] of nutrients (sugars, fats, proteins) in
your body end up passing through the electron transport
chain (ETC) that is embedded in the mitochondrial inner
membrane. They combine in the end with molecular oxygen
to produce water. But that’s only the first half, the
‘oxidative’ part, of the story.”
13
What About Phosphorylation?
“This is the part I have trouble with,” said Cheryl,
“because what I don’t get is how converting oxygen into
water helps you make ATP.”
Dr. Adams replied, “Well, you’re in good company. For
two decades or more, most scientists studying this
process also didn’t get it.
“So let’s just look for a moment at the relationship
between the ETC and the activity of the enzyme, ATP
synthase, that makes ATP in mitochondria by
“phosphorylating” ADP with inorganic phosphate
(usually termed Pi). That’s the second half.”
14
Mitochondrial Experiments
• Scientists have isolated mitochondria from the cells they
normally occupy, and they retain many of their functions,
including the ability to carry out “Ox Phos.”
• All they require is some substrate that could be oxidized
(succinate works well), ADP + Pi (as precursors), and
appropriate buffers and salts.
• ETC activity can be detected by the consumption of oxygen
(using an oxygen electrode).
• ATP synthesis can be measured by the appearance of
labeled ATP from radioactive ADP precursor.
15
Some Questions
“Let’s see if you have the basics, Cheryl,” Dr. Adams
proposed.
“First, I’m sure you know that cyanide is a nasty
poison – that’s because it blocks Component IV
(cytochrome oxidase) of the ETC.”
“What do you think will happen if you add cyanide to
a test tube with mitochondria that are actively
carrying out Ox Phos?”
16
CQ#2: You incubate isolated intact mitochondria in a
buffered solution containing succinate (an oxidizable
substrate) and ADP plus Pi. Upon adding cyanide (an
inhibitor of Complex IV, cyto-chrome oxidase), you
examine the effect on oxygen consumption and the
production of ATP. What do you predict?
A.
B.
C.
D.
Oxygen will not be consumed, and no ATP will be
produced.
Oxygen will be consumed, but no ATP will be
produced.
Oxygen will not be consumed, but ATP will be
produced.
Oxygen will be consumed, and ATP will be
produced.
17
Cyanide
added
Succinate
added
ADP + Pi
added
ATP synthesized
Oxygen consumed
The answer is A.
Cyanide inhibits Complex
IV, known as cytochrome
oxidase, preventing the
transfer of electrons to
molecular oxygen.
Time
Conclusion: Cyanide prevents oxygen consumption, and
because ATP production is also prevented, ATP synthesis
must require electron transport.
18
Another Experiment
“Now, in a similar situation, instead of using
cyanide, you add an inhibitor of ATP synthase,
such as the antibiotic oligomycin.
Think carefully, because the answer to this may
be a little tricky…”
19
CQ#3: You incubate isolated intact mitochondria in a
buffered solution with succinate (an oxidizable
substrate) and ADP plus Pi. Upon adding oligomycin,
an antibiotic inhibitor of ATP synthase, you examine the
effect on oxygen consumption and the production of
ATP. What do you predict?
A.
B.
C.
D.
Oxygen will not be consumed, and no ATP will be
produced.
Oxygen will be consumed, but no ATP will be
produced.
Oxygen will not be consumed, but ATP will be
produced.
Oxygen will be consumed, and ATP will be produced.
20
Oligomycin
added
Succinate
added
ADP + Pi
added
ATP synthesized
Blocking ATP
synthase also
prevents the
consumption of
oxygen. How
can this be?
Oxygen consumed
The answer is A.
Time
Conclusion: Not only does ATP synthesis require electron
transport, but electron transport requires ATP synthesis.
21
The Concept of “Coupling”
Dr. Adams continued, “The obvious part is that because
ATP synthase is blocked, no ATP can be synthesized.
The challenging question is: how does this prevent the
consumption of oxygen, since none of the components
of the Electron Transport Chain have been directly
affected?
Scientists have decided that this unexpected result
should be called ‘coupling’ between electron transport
and ATP synthesis.”
22
How Does Coupling Work?
• For many years, this phenomenon was
enigmatic; it wasn’t until a British scientist
named Peter Mitchell came up with his
unusual “Chemiosmotic Hypothesis” that
things began to make sense.
• In parallel, it was becoming clearer how the
enzyme ATP synthase worked – as a molecular
motor!
23
Motors in Cells?
Dr. Adams smiled. “Elegant enzyme experiments have
shown that this enzyme is a true molecular motor, if you
can believe that – driven by the flow of protons through a
channel, which
causes physical
rotation of a…
…but let’s look at
a picture!”
ATP synthase:
24
What About the Protons?
• “That’s pretty neat, but where do the protons come from to
make it work?” Cheryl wanted to know.
• “Ah, you’ve hit on the key question that helps understand
coupling, and in the process reveals how DNP works! But, one
step at a time.
• As examination of the ETC progressed, data indicated that
protons were being pumped out of the mitochondria in concert
with the passage of electrons down the ETC.
• Many scientists argued that this proton pumping was only a
tangential byproduct of electron transport, and unrelated to its
main function.
• Peter Mitchell, however, suspected a direct involvement, and
many of his experiments began to support it.”
25
Three Linked Events
Remember what you know about the inner membrane of
mitochondria?
1. Electron transport occurs laterally between complexes
embedded in the lipid bilayer, ending with the reduction
of molecular oxygen to water.
2. In concert with this electron transport, protons are
pumped out of the mitochondria into the intermembrane space by Complexes I, III and IV, creating a
higher pH (less acidic) within the matrix.
3. ATP synthase (Complex V) is a molecular motor also in
the lipid bilayer, driven by proton passage through a
channel that lets them back in again.
26
It Starts to Make Sense
Dr. Adams continued: “With the realization
that ATP synthase required the passage of
protons, driven by a proton gradient (which
Mitchell called his ‘Proton Motive Force’),
where they could flow ‘downhill,’ the puzzle
became resolved….
Before that, it was believed that….well, never
mind,” explained Dr. Adams. “Let’s look at a
picture you’ve seen before.”
27
Peter Mitchell’s Chemiosmotic
Hypothesis
Protons are pumped out (by the ETC) and flow back in
(via ATP synthase) to make ATP.
28
What Happens with DNP?
“With this in mind, remember when you read
that DNP was an ‘uncoupler’ of Ox Phos? What
would that mean in terms of the ETC and ATP
synthesis?” asked Dr. Adams.
Cheryl thought, and came up with an idea.
What idea do you have for this question?
29
CQ#4: You incubate isolated intact mitochondria in a
buffered solution with succinate (an oxidizable
substrate) and ADP plus Pi. You add oligomycin (the
ATP synthase inhibitor), then the compound DNP (2,4dinitrophenol), and examine the effect on oxygen
consumption and the production of ATP. What do you
predict? Hint: What happened when people took this as
a weight-loss drug?
A.
B.
C.
D.
Oxygen will not be consumed, and no ATP will be
produced.
Oxygen will be consumed, but no ATP will be
produced.
Oxygen will not be consumed, but ATP will be
produced.
Oxygen will be consumed, and ATP will be
produced.
30
Oligomycin
added
Succinate
added
ADP + Pi
added
DNP
added
ATP synthesized
Oxygen consumed
The answer is B: DNP acts as an uncoupler!
Time
Recall: Electron transport requires ATP synthesis except in the presence of DNP, where oxygen continues
to be consumed, though no ATP is being made. The
mitochondria are said to be uncoupled. How is this?
31
What Does DNP Actually Do?
“Here’s the structure of DNP, with a ring that looks sort of
like benzene – do you think it would be more soluble in
water, or in a hydrophobic solvent?”
32
Phyllic or Phobic?
Cheryl responded, “Hydrophobic – like greasy stuff,
right? I’ve watched my dad use benzene to get the
grease off his hands when he’s been working on the
car, and I know that it doesn’t mix with water.”
“Yes, good thinking.” Dr. Adams circled the –OH group
on his diagram. “But, unlike benzene, you can see
that DNP is a ‘weak acid’ due to its –OH group, which
just means that some of the time it can shed its
proton, becoming –O- + H+. This is unlike HCl, which
is a strong acid, and thus completely dissociates in
water. So here’s an application to think about…”
33
CQ#5: You have created some artificial membrane
vesicles (spherical lipid-bilayer enclosed droplets)
that have a higher pH inside than the aqueous
solution outside. To sample 1 of these vesicles you
add a little HCl, and to the other (samplle 2) you add
some DNP (with equivalent acidity) and then measure
the internal pH of the vesicles. What do you predict?
A. Internal pH will be lowered for both 1 and 2.
B. Internal pH will be unchanged for both 1 and 2.
C. Internal pH will be unchanged for 1 but lowered for 2.
D. Internal pH will be lowered for 1 but unchanged for 2.
34
“The correct answer is C,” Dr. Adams lectured.
“And here’s why: when protonated, that is, carrying its H+
proton as you saw in the figure, DNP will be not only
uncharged, but very hydrophobic. It is hence readily
soluble in lipid bilayers such as those enclosing these
vesicles (and those of the mitochondrial inner membrane).
Thus DNP can readily carry protons across the membrane
from a higher concentration (lower pH) outside, and
release them to the lower concentration (higher pH)
inside. This equilibrates the pH inside and outside.
HCl cannot do this, because it is not hydrophobic, and its
charged ion species (H+ and Cl-) cannot cross lipid bilayer
structures.”
35
What Happens With a Leak?
“That is really cool,” Cheryl blurted out, “and if we think of
mitochondria as just sort of glorified vesicles, then DNP would
make their inside pH the same as the outside pH – the
difference would be gone! But wouldn’t that be a big problem,
because now the Electron Transport Chain would have to keep
working like crazy with its proton pumping just to keep up? Sort
of like a bicycle tire with a leak, when you’re trying to fill it?”
“Exactly right!” Dr. Adams exclaimed, “And what do you think
that ‘working like crazy’ might need a lot of, and generate a lot
of…?”
36
What Happens to Wasted Energy?
“A lot of oxygen!” Cheryl said, ”Because to pump all
those protons, oxygen has to be there to accept the
electrons being transported along at the same time.”
“And heat!” cried Dr. Adams. “Does this fit with the
effects you know that DNP has on people?”
Cheryl thought. “Yes: rapid breathing because they need
oxygen, sweating from the increase in body
temperature due to the heat generated, and fatigue
because ATP stops being made, even though lots of
calories are being burned.”
37
To Lose Weight, You May
Lose Your Life…
• Dr. Adams summarized, “So you now realize why DNP is so
dangerous. Although it will cause weight loss, it’s within a
very narrow dosage window, and small physiological
differences between individuals may shift its effects into
the lethal range. The ‘If one pill is good, two pills must be
better’ approach has a deadly flaw with this drug. Your
brother is actually a very lucky young man.
• But Cheryl, did you know that some organisms co-opt this
concept and use it in a well-controlled fashion?”
38
A Controlled Energy Burn!
• Human babies, bears, and some rodents have
“brown fat” - specialized fat cells, much richer than
normal in mitochondria (which is why they are
brown).
• The inner membranes of these mitochondria have a
protein called thermogenin.
• It provides a proton channel from outside to inside,
dissipating the proton gradient without making ATP,
thus causing the ETC to run faster, creating...heat!
39
For Warm Babies and Bears
H+
H+
H+
H+
H+
X
Heat
H+
Thermogenin
(uncoupling
protein)
H+
• Heat generation by “nonshivering thermo-genesis”
keeps human babies (who
have a high surface to
volume ratio) warm when
they need to be.
• Thus organisms have
evolved a mechanism to
control what DNP does in
an uncontrolled way, and
to exploit it in a helpful
way.
H+
40
Epilogue – A Look Back in Time
In the 1950’s and 60’s, the mechanism for how energy derived
from the transport of electrons was connected to the
generation of ATP was regarded as mysterious.
• Many eminent scientists believed that there had to be a
transient high energy phosphoryl chemical intermediate, which
was nicknamed X~P.
• This intermediate was predicted to capture the energy released
during electron transport, and then transfer it to ADP, forming
ATP – a “Chemical Coupling” model.
• When Peter Mitchell proposed the “Chemiosmotic Hypothesis,”
it was highly controversial.
41
Alternative Possibilities
• The Chemiosmotic Hypothesis seemed to fit some
observations that could not be accounted for by
Chemical Coupling, including:
– The higher pH inside vs. outside (lower Ph, thus more
protons) of the mitochondrial matrix.
– The known pumping of protons outward, regarded by
many as being peripheral to oxidative phosphorylation
• Nonetheless, Mitchell had many serious and vocal
critics who seemed to resent any challenge to the
Chemical Coupling model.
42
Hypothesis Testing
• The Chemical Coupling model predicted that ATP
synthesis could not occur in the absence of electron
transport, because X~P was only formed by the action
of the ETC.
• The Chemiosmotic Hypothesis predicted that the
driving force for ATP synthesis comes from the proton
gradient (or “Proton Motive Force” as Mitchell termed
it) directly.
• Eventually, experiments like the following one were
performed. What do you think will happen?
43
CQ#6. You isolate intact mitochondria and equilibrate them in a
buffered solution at pH 9, containing 0.1 M KCl and ADP plus Pi,
but without succinate. You collect them by centrifugation, and
quickly resuspend them in a new buffer at pH 7, without KCl , but
with valinomycin (a K+ ionophore). Note: the K+ rushing out will
create a huge positive charge differential. What do you predict
will be the result on oxygen consumption and the production of
ATP?
A. Oxygen will not be consumed, and no ATP will be
produced.
B. Oxygen will be consumed, but no ATP will be produced.
C. Oxygen will not be consumed, but ATP will be produced.
D. Oxygen will be consumed, and ATP will be produced.
44
Start with equilibrated mitochondria
[K+] = [Cl-] = 0.1 M
H+ = 10-9 M
H+ = 10-9 M
[K+] = [Cl-] = 0.1 M
H+
Intermembrane
space
• Mitochondria are placed in
pH 9 buffer and 0.1 M KCl,
and allowed to reach
equilibrium.
The proton (10-9 M) and the
potassium (0.1 M)
concentrations are thus
identical in the intermembrane space and the
matrix.
These mitochondria are
now shifted….
H+ = 10-9 M
45
Can an Artificial Proton Gradient Drive
ATP Synthesis?
...into pH 7 buffer, no KCl,
with valinomycin present
Intermembrane
space H+
H+
H+
H+
H+ = 10-7 M
Cl-
Cl-
ClH+
ClCl-
K+
H+ = 10-9 M
K+
[K+] < [Cl-]
valinomycin
Cl-
Now, DpH = 2, and K+
flows out, giving a charge
imbalance (negative
inside) that helps pull in
protons.
The answer is C. ATP is
synthesized, in the
complete absence of any
electron transport or
oxygen consumption.
46
What Did This Experiment Prove
• ATP synthesis correlated with the formation of an
electrochemical gradient of protons, and
• In the complete absence of any electron transport!
• This result supported the Chemiosmotic Hypothesis
developed by Peter Mitchell, and ruled out those that
require ATP synthesis to be solely dependent on electron
transport.
• Many scientific opponents vigorously challenged it as
being heretical if not impossible, and it took them another
decade or more to be convinced by Mitchell’s proposal.
47
Behind Mitchell’s Novel Proposal
Peter Mitchell’s insights into DNP’s function as an uncoupler
led him, in part, to set aside traditional thinking and to
propose his controversial alternative, the “Chemiosmotic
Hypothesis.”
Throughout his studies, he kept an open mind, and was always
trying to test between alternative hypotheses rather than
trying to “prove” a favorite one. This was in striking
contrast to many of those who either did not understand or
could not accept his ideas.
His ideas revolutionized the way in which we think about
oxidative phosphorylation, photosynthesis, and energy
transduction in living cells.
48
A Brilliant but Modest Researcher
In 1978, Peter Mitchell flew to Stockholm to accept his
Nobel Prize and offered a gracious comment to the
scientific community in his acceptance speech:
"I find most remarkable and admirable...the
altruism and generosity with which former
opponents of the chemiosmotic hypothesis have
not only come to accept it, but have actively
promoted it to the status of a theory."
49
Image Credits
Except where noted below, figures appearing in the presentation were created or adapted by
author Terry Platt.
Slide 24
Description: ATP synthase molecular machine.
Source: Image from the Materials Research Science and Engineering Center on Structured
Interfaces at the University of Wisconsin-Madison with funding from the National Science
Foundation under award numbers DMR-1121288, DMR-0520527 and DMR-0079983. Any
opinions, findings, and conclusions or recommendations expressed in this report are those of
the authors and do not necessarily reflect the views of the Foundation.“
Clearance: Used with permission.
Slide 28
Description: Schematic diagram of the mitochondrial electron transport chain.
Source: Wikimedia Commons, by user Fvasconcellos,
http://commons.wikimedia.org/wiki/File:Mitochondrial_electron_transport_chain%E2%80%
94Etc4.svg
Clearance: Released into the public domain by the image author.
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