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Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens Page 1
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
• In concert with the passage of electrons along the are uncouplers of oxidative phosphorylation, though
electron transport chain, protons are pumped this was not known at the time (Parascandola, 1974).
outwards, resulting in establishment of an
electrochemical gradient. In-Class PowerPoint Presentation
• ATP synthesis within mitochondria is normally Slide 1: Title slide.
“coupled” to electron transport, in that inhibiting Slides 2–6: Charles, a college freshman wrestler,
either process blocks the other one. purchases DNP (2,4-dinitrophenol) on the Internet in
• Energy for ATP synthesis is directly derived from an order to lose weight, but things take a frightening turn
electrochemical proton gradient. when he displays alarming symptoms and has to be
• Situations that “uncouple” oxidative phosphorylation rushed to the Emergency Room.
do so by dissipating or eliminating the proton gradient, Slides 7–10: Charles survives. His twin sister Cheryl
so that electron transport continues unabated but asks the attending physician a series of questions that
ATP synthesis stops. covers the major concepts of the case. Students learn
• Biological “uncoupling” by the protein thermogenin about the medical and historical aspects of DNP
is used by organisms to keep warm. use. This includes reference to two deaths described
in the literature, one due to a mere doubling of the
Misconceptions recommended dose.
• Diet products are safe and have no side effects. Slide 11: Poses the central question of “What is
• Any product you obtain through the internet or public oxidative phosphorylation?” which students should be
media sources cannot be harmful. familiar with from their Case Handout/assignment, and
• The electron transport chain is directly connected to where it fits in with cellular respiration overall. This is
ATP synthesis. an opportunity to remind students that this differs from
• The connection between electron transport and ATP ATP made during glycolysis, for example, which is called
synthesis is mysterious. “substrate-level” phosphorylation.
• The transfer of electrons is obligatory for ATP Slide 12: Clicker Question 1 (CQ#1) is a review
synthesis. question, and will determine whether students have a
• Oxidative phosphorylation “uncouplers” allow ATP basic understanding of what this central process actually is.
synthesis without electron transport. Slides 13–16: Review of oxidative phosphorylation
and how it is measured in vitro, setting the stage for
CLASSROOM MANAGEMENT a question about what happens when you block the
This case was designed to be used with personal response process. A reminder here about “OILRIG” as an
systems (“clickers”) in large classes of 100 or more acronym—“oxidation is loss, reduction is gain” (of
students in an introductory biology course. It is designed electrons), and that the electrons are donated to the
for one 75-minute class period, or may be expanded Electron Transport Chain (ETC) by the soluble electron
slightly and spread over two sequential 50-minute classes. carriers NADH and FADH2 (derived from the vitamins
Some advance reading and thinking by the students is niacin and riboflavin).
required. Student-student discussion in class may be Slide 17: CQ#2 tests whether students understand
beneficial for some of the clicker questions or other from their reading that blocking the ETC with cyanide
concepts. (an inhibitor of Complex IV, cytochrome oxidase) will
also prevent the synthesis of ATP.
Teaching the Case Slides 18–19: These slides answer the question
Before the class period in which the case is presented, (yes, ATP synthesis is also blocked) and pose a parallel
students read the chapter in their text corresponding to question about inhibition with oligomycin, which
oxidative phosphorylation (which must not be confused inhibits ATP synthase.
with photophosphorylation). They should also read the Slides 20–21: CQ#3 explores whether students
Case Handout (included at the end of these teaching understand the meaning of “coupling”– that not only
notes) and think about the questions posed in it. This does inhibiting electron transport prevent the synthesis
account is based on factual observations from that time of ATP, but inhibiting ATP synthesis knocks out the
in history—both the dyes and the munitions ingredients transport of electrons.
Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens Page 2
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
Note to Instructors: It may be useful to have each provided a mechanism to do so (either through ATP
student provide an individual clicker answer first; synthase or via DNP transport).
then, after you look at the distribution of answers, Slides 35–38: Provide the analogy of keeping a bicycle
have students discuss the question with neighboring tire inflated when it has a slow leak (continued pumping
students to provide “collective” answers, and see whether is necessary). The ETC tries to compensate by pumping
understanding has improved. This technique may be protons ever faster, which requires faster electron
used on subsequent questions as well. transport. In turn, this consumes a lot of oxygen (the
Slides 22–23: Show that blocking ATP synthase ultimate repository for those electrons), while failing to
also prevents the ETC from continuing functionally, restore the gradient (because of the DNP-caused “leak”).
and sets the stage for a structural glimpse of the ATP This eliminates the means by which protons could drive
synthase as a physical molecular motor. It is driven by ATP synthase and generates instead a significant amount
the passage of protons through a channel as favored by of heat. Another analogy is revving a car engine without
their concentration and charge gradient (higher and the clutch being engaged: you burn a lot of gasoline
more positive outside the mitochondria), which turns a (consuming oxygen) and make a lot of heat from the
motor that can thereby synthesize ATP. engine, but don’t go anywhere. As an “uncoupler” of
Slides 24–25: Show the motor, driven by “proton oxidative phosphorylation, DNP is very toxic.
flux”—their origin from those that were pumped Slides 39–40: Some animals have evolved a biological
outwards by the ETC, thereby creating a substantial strategy for harnessing this process through the use of an
electrochemical potential. uncoupling protein called thermogenin. It is a controlled
Slides 26–28: Link the proton excess outside to the biological “uncoupler,” which functions, for example, to
action of ETC and to the ability of the mitochondria keep human infants warm; at present, how its activity is
to make ATP, culminating in a visual schematic of Peter cellularly regulated to prevent runaway uncoupling like
Mitchell’s Chemiosmotic Hypothesis. DNP is not understood.
Slide 29: Introduces DNP, which the students have Note to Instructors: The presentation could end
read about in their pre-class assignment in the context here for many courses. For those where additional
of weight loss and establishing the Food and Drug time is available, the remaining slides offer a historical
Administration, and asks what effect it might have on glimpse at the origins of Peter Mitchell’s Chemiosmotic
“coupling.” Hypothesis (Mitchell, 1961). It flew in the face of
Slide 30: CQ#4 invites students to predict what will conventional thinking at the time, yet application of the
happen when DNP is added to the “blocked” situation principles of “strong inference” (Platt, 1964)—testing
outlined in CQ#3 previously. The answer is surprising— the predictions of alternative hypotheses—ultimately
oxygen is now consumed at a high rate, although no vindicated the model, for which Mitchell received the
ATP is made. This is called “uncoupling,” and puts DNP Nobel Prize in 1978.
in the category of other chemical “uncouplers” (like the Slides 41–43: These slides pose the favored
coal-tar dyes mentioned in the Case Handout), though Chemical Coupling model of the time as a contrast to
fails to explain why it has this effect. An extra credit the Chemiosmotic Hypothesis, and set the stage for an
exercise might be to look at the structures of the two experimental test that can distinguish between them.
mentioned dyes, dinitronaphthol and dinitrocresol. Few current textbooks address this controversy (Scott
Slides 31–33: These provide a visual depiction of Freeman’s Biological Science, 3rd ed., 2008 is an exception),
uncoupling, and delve into the chemical properties of which raged strongly for well over a decade, yet it is an
DNP to determine how it might work. excellent demonstration of the importance of adhering
Slide 34: CQ#5 poses a hypothetical situation to to scientific method to achieve scientific understanding.
see whether students can predict that DNP (but not Slides 44–47: CQ#6 has a complex question as its
HCl) will allow the “leakage” of protons across a lipid “stem,” but is essentially asking whether ATP synthesis
bilayer to equilibrate a pH difference. This is relevant to can be driven solely by a chemiosmotic gradient; note
the high pH observed inside the mitochondrial matrix, that no oxidizable substrate is present. After the shift
compared to outside, and thus why protons would be to the new buffer, valinomycin-mediated export of K+
energetically favored to move from outside to inside if makes the outside positive charge high, and the pH shift
makes the outside proton concentration high (by 100-
Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens Page 3
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens Page 4
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
•
Copyright held by the National Center for Case
Study Teaching in Science, University at Buffalo, State
University of New York. Originally published May 21,
2012. Please see our usage guidelines, which outline our
policy concerning permissible reproduction of this work.
Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens Page 5
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
CASE HANDOUT
for
“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
“By the end of the two and a half decades between 1940 and 1965, the field of oxidative phosphorylation
was littered with the smouldering conceptual remains of numerous exploded energy-rich chemical
intermediates; the remarkable uncoupling action of 2,4-dinitrophenate [dinitrophenol] … remained
obscure; and the process of hypothesis-building, needed to keep faith with the chemical-coupling notion,
reached such fantastic proportions as to be hardly intelligible to those outside the field.”
—Peter Mitchell (1978 Nobel address)
Prologue: Coal-tar dyes were used in the late 19th century to color food; one favorite in Europe was Martius yellow
(dinitronaphthol), used in pastries and macaroni where the color implied the foods were rich in eggs. A related dye
with similar properties was Victoria Yellow (dinitocresol). Some deaths occurred due to the use of these dyes, which
called attention to their potential danger in food products. This led French and German scientists to study toxicity
of these coal-tar dyes with dogs: in moderate doses, the symptoms included increased respiration and rise in body
temperature (to 44° C in one case) before death ensued. Because of their toxicity, these two dyes were banned in
Europe for use in coloring food by 1900, and within a few years were similarly banned in the United States, although
seven other coal-tar dyes could legally continue to be used in food coloration.
During and after World War I, munitions factories in France commonly made explosives from 40% 2,4-dinitrophenol
(DNP) and 60% picric acid (trinitrophenol) rather than the trinitro-toluene (TNT) manufactured in Britain and
the U.S. Overweight workers in these factories were observed to lose weight, felt fatigued, and sometimes displayed
excessive sweating and elevated body temperatures. Various studies between 1910 and 1920 confirmed in animal
experiments the previously observed toxicity of dinitrophenol, without being able to identify the mechanism of its
action. One interesting possibility, that increased heat production was due to “a stimulation of cellular oxidation,”
became favored—a remarkable insight, considering that ATP itself was not discovered until 1929!
In the U.S., Stanford scientists learned of the early French studies and repeated many of those experiments with
similar outcomes. This appeared to present a genuine method for “free” weight loss, and by 1933 physicians had
carried out clinical studies, establishing dosage levels that were able to hold the metabolic rate of obese patients at a
level 40% higher than normal with no adverse effects. This resulted in weight loss without dieting, which was great
news for patent medicine promoters, who seized upon this finding with glee. They began marketing “miracle cures” for
obesity, and some physicians started to prescribe DNP as a weight loss remedy. By 1935, more than 100,000 people
were estimated to have been “treated” with DNP. Unfortunately, deaths attributable to DNP had begun to be reported.
Concern spread after a 1933 Newsweek headline blared “Diet and Die with Excess Alpha Dinitrophenol,” which told
“of a physician who had been ‘literally cooked to death’ when he took an overdose of the drug in an attempt to lose
weight rapidly” (Parascandola, 1974).
The amount of dinitrophenol that causes harmful effects varies among individuals, because the amount necessary to
realize moderate weight loss (therapeutic dose) and the amount that produces death (lethal dose) are separated by less
than a factor of ten. Given the propensity of patients to believe that “if one pill is good, two pills must be twice as
good,” it is easy to see how DNP was able to claim so many lives in so short a time. Common signs and symptoms in-
clude increased basal metabolic rate (the rate that you use energy at rest), rapid breathing, increased sweating, a feeling
Case Handout for “A Diet to Die For” by Platt and Ribbens Page 1
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
of warmth (even in chilly or cold conditions), and weight loss. Your heart rate, breathing rate, and body temperature
rise. Cataracts, skin rashes, and decreases in white blood cell counts are also associated with DNP ingestion.
At that time, the government had no control over “cosmetic” medications if no medical claims were made on the
label—and in any case “obesity” was not considered a disease. To a large extent, the deaths caused by misuse of DNP
forced the adoption of a new stricter “Food, Drug and Cosmetic Act” by Congress in 1938. Shortly thereafter, DNP
was banned as a weight loss drug by a newly formed governmental agency: the U.S. Food and Drug Administration
(FDA). This was one of its first official actions, and in fact the abuse of this “drug” may have been instrumental
in Congressional chartering of the FDA, an institution that we now take for granted. The current mandate of the
FDA has become to monitor for safety and efficacy “all food except for meat and poultry; all prescription and non-
prescription drugs; all blood products, vaccines, and tissues for transplantation; all medical equipment and all devices
that emit radiation, including microwave ovens; all animal drugs and feed, and even all cosmetics.” Yet even today,
DNP is available over the Internet.
It took another two to three decades to confirm that 2,4-dinitrophenol acted as an uncoupling agent in mitochondria
both in vitro and in vivo, though the mechanism of its action remained obscure until the late 1960’s. Due to the
pioneering efforts of Dr. Peter Mitchell, supported by those of his scientific colleagues open-minded enough to
consider his “Chemiosmotic Hypothesis” as a serious alternative to the “Chemical Coupling Hypothesis” (previously
assumed by many to be correct), we now understand how DNP works. In the class period(s) ahead you will explore
the mechanism of DNP action to illuminate the currently accepted cellular model for coupling between the electron
transport chain and ATP synthesis in the process called “oxidative phosphorylation.”
Background reading for class: The text chapter covering oxidative phosphorylation.
Ask yourself: 1. What is meant by “coupling” of oxidative phosphorylation?
2. What is meant by “uncoupling” of oxidative phosphorylation?
3. Under what kinds of conditions will these effects occur?
Case Handout for “A Diet to Die For” by Platt and Ribbens Page 2