PAGE DE GARDE DES RAPPORTS DE STAGE
Stage d’exécution 
Stage élève – ingénieur 
Stage année de césure :
12 mois 
1er semestre
Stage projet de fin d’études
2ème semestre

Date du stage
du 6/01/2014
au 31/07/2014
Année
2013/2014
DE MONTGOLFIER Oriane
Spécialité : CGP
CONFIDENTIALITE du RAPPORT :
 OUI
Development of xanthohumol derivatives as mild mitochondrial
uncouplers for treatment of metabolic syndrome
Développement de dérivés de xanthohumol en tant qu’agents découplants
de la chaîne respiratoire mitochondriale pour le traitement du syndrome
metabolique
Plant cell, 2008 [2]
Linus Pauling Institute / Oregon State University
Corvallis, OR. USA
Dr. Jan Frederik Stevens
Principal Investigator, Linus Pauling Institute
Associate Professor of Medicinal Chemistry, College of Pharmacy
Co-Director, OSU Biomolecular Mass Spectrometry Facility
PFE at Linus Pauling Institute – Master thesis
1
Acknowledgements
I would like to thank first Dr Stevens for giving me the opportunity to work on this
challenging project in his lab. I really enjoyed being able to combine for the same project
both organic synthesis and biochemistry/cell culture experience that I got the chance to
learn the techniques in the lab, thanks to Dr Miranda.
I thank all those I worked with, as a team, who helped me, gave me some advice and were
patient with all my questions: Fred Stevens, Jaewoo Choi, Val Miranda, Gerd Bobe, Yu Zhen,
Ralph Reed and LeeCole Legette. I thank also Jackilyn Toftner for her work on the DXN
synthesis, Elizabeth Axton and Eunice Lee for sharing the lab and some coffee breaks. Thanks
also to the personnel of the LPI for its availability and kindness typical of Oregon.
Finally I thank my family for their support and especially my uncle and my aunt who made
me want to study Sciences, Chemistry and Biochemistry.
PFE at Linus Pauling Institute – Master thesis
1
Contents
Abbreviations.......................................................................................................................................... 3
Table of schemes .................................................................................................................................... 4
Table of figures ....................................................................................................................................... 4
Introduction ............................................................................................................................................ 5
I.
Development of the synthesis of dihydroxanthohumol (DXN) .................................................... 6

Experimental procedures ........................................................................................................... 8

Results and discussion ................................................................................................................ 9
II. The effects of dihydroxanthohumol (DXN) as a mild mitochondrial uncoupler in comparison
with xanthohumol (XN) and tetrahydroxanthohumol (TXN) .............................................................. 10

Experimental procedures .......................................................................................................... 12

Results ....................................................................................................................................... 15
MTT assay ...................................................................................................................................... 15
Optimization of Seahorse assay conditions (cell density, oligomycin and FCCP concentrations) 15
Comparison of the uncoupling effects of XN, DXN, TXN ............................................................... 16
Dose-response effect of DXN ........................................................................................................ 18
JC1 Assay ....................................................................................................................................... 20

Discussion.................................................................................................................................. 22
Conclusion ............................................................................................................................................. 25
Bibliography .......................................................................................................................................... 26
Appendices............................................................................................................................................ 29
Appendix 1: Hydrogenation mechanism by using the Wilkinson catalyst ........................................ 29
Appendix 2: NMR Spectra of DXN ..................................................................................................... 30
Appendix 3: Mass Spectra of DXN (Q1, Product ion, and proposed fragmentation pattern)........... 37
Appendix 4: HPLC chromatogram to identify a mixture of DXN, XN and TXN .................................. 39
Appendix 5: Effect of XN, DXN and TXN on cell viability (MTT assay) ............................................... 40
Appendix 6: Cell density and FCCP concentration optimization experiment (Seahorse assay)........ 41
Appendix 7: Oligomycin concentration optimization experiment (Seahorse assay) ........................ 42
Appendix 8: Tables presenting the results (OCR and ECAR) of the statistical analysis ..................... 43
Summary ............................................................................................................................................... 49
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Abbreviations
8-PN
8-prenylnaringenin
ADP
adenosine diphosphate
ATP
adenosine triphosphate
Δψm
transmembrane (electrical) potential
DNP
dinitrophenol
DXN
dihydroxanthohumol
ETC
electron transfer chain
FAD
flavin adenine dinucleotide, fully oxidized form
FADH2
flavin adenine dinucleotide, reduced form
FBS
fetal bovine serum
FCCP
carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone
NAD+
nicotinamide adenine dinucleotide, oxidized form
NADH
nicotinamide adenine dinucleotide, reduced form
OCR
oxygen consumption rate
P/O
ATP/Oxygen ratio
ROS
reactive oxygen species
TXN
tetrahydroxanthohumol
UCP
uncoupling protein
XN
xanthohumol
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Table of schemes
Scheme 1: Metabolic conversion of XN into desmethylxanthohumol (DMX) and 6- and 8prenylnaringenin (6-PN and 8-PN). XN is spontaneously converted into isoxanthohumol (IX) by
intramolecular Michael addition. 8-PN is formed from DMX or from IX by CYP-mediated
demethylation (from [21]). ..................................................................................................................... 7
Scheme 2: Synthesis of DXN from XN ..................................................................................................... 8
Table of figures
Figure 1: Common structure of flavonoids, chalcones and the xanthohumol ........................................ 6
Figure 2: Wilkinson catalyst..................................................................................................................... 9
Figure 3: Electron transport chain (http://www.dbriers.com) ............................................................. 10
Figure 4 : Representative OCR profile of the Seahorse assay. 80% of the total OCR is mitochondrial
OCR. In this example, 51% of the total OCR is non ATP linked OCR, i.e., non mitochondrial OCR and
proton leak OCR. That value is determined by injection of oligomycin (by inhibiting ATP synthase).
49% of the total OCR is thus coupled to ATP synthesis. The injection of an uncoupler increased the
OCR until reaching a maximal OCR [1]. ................................................................................................. 11
Figure 5: A to F: Comparison of XN, DXN and TXN as mild mitochondrial uncouplers. DXN and TXN
cause...................................................................................................................................................... 17
Figure 6 A to F: DXN causes mitochondrial uncoupling in cells. C2C12 cells were sequentially treated
with oligomycin (1 μM) and the indicated concentration of DXN (uncoupler) (A, C and E). Changes in
OCR and ECAR after injection of test compounds were plotted as bars (B, D and F) (mean ± SEM, n =
3).* Indicates p < 0.05 by ANOVA procedure in PROC MIXED. ............................................................. 19
Figure 7: XN, DXN and TXN are able to depolarize the mitochondrial transmembrane potential
measured as a decrease in the ratio aggregate/monomers. * indicates p < 0.05. .............................. 21
Figure 8 : Representative ECAR profile of the Seahorse assay. The maximal lactate acidification is
determined by injection of oligomycin (inhibition of ATP synthase). The ECAR is then decreased by
injection of an uncoupler such as DXN or TXN at 5 μM. ....................................................................... 24
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Introduction
This report describes the development of xanthohumol derivatives as mild mitochondrial
uncouplers for treatment of metabolic syndrome. The metabolic syndrome [3] comprises
multiple risk factors for cardiovascular disease. It is clinically diagnosed by one or more of
the following conditions: atherogenic dyslipidemia, elevated blood pressure, insulin
resistance and/or glucose intolerance leading both to diabetes, proinflammatory state,
prothrombotic state and abdominal obesity. Obesity [4] results from an energy imbalance
where fuel intake is in excess compared to energy expenditure. Among the
pharmacotherapeutic strategies to treat obesity, targeting mitochondrial uncoupling
appears to be one of them, as mitochondria are the cellular source of energy. Xanthohumol
(XN; 3'-[3,3-dimethyl allyl]-2',4',4-trihydroxy-6'-methoxychalcone), a major prenylated
flavonoid from hops used for making beer, has shown anti-obesity effects by inducing a
reduction in body weight gain in the Zucker rat model of obesity and metabolic syndrome.
XN can increase energy expenditure by mild mitochondrial uncoupling [5]. However, some
concerns have been expressed about the use of XN in dietary supplements [6], as 8prenylnaringenin (8-PN), one of its metabolites [7-9], is the most potent phytoestrogen
known to date and since it can act as a weak agonist of estrogen receptors in the body [1012]. 8-PN is generated from XN via a cyclic intramolecular rearrangement of the α,βunsaturated ketone. Reduction of this critical alkene will prevent the formation of the
estrogenic metabolite 8-PN. Synthesis of a XN analogue that displays no estrogenic activity
would be an attractive therapeutic option to treat the metabolic syndrome. This project is
aimed at finding a way to synthesize DXN from XN and then studying its biological effects as
a mild mitochondrial uncoupler in comparison with XN and tetrahydroxanthohumol (TXN). I
have worked as part of the team of Dr. Stevens’ laboratory at the Linus Pauling Institute
(Oregon State University, Corvallis, OR, USA). I developed a method for the synthesis of DXN
and supervised an undergraduate student to optimize the synthesis yield. I have learned the
cell culture techniques necessary to design and execute diverse biochemical experiments in
order to investigate the effects as mitochondrial uncouplers, potency and toxicity of DXN,
TXN and XN using mouse skeletal muscle myoblasts (C2C12 cells).
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I.
Development of the synthesis of dihydroxanthohumol (DXN)
Xanthohumol (XN; 3'-[3,3-dimethyl allyl]-2',4',4-trihydroxy-6'-methoxychalcone) is a major
prenylated chalcone and it is also the main prenylflavonoid found in hops. Flavonoids are a
large family of polyphenolic compounds synthesized by plants and chalcones are made of an
aromatic ketone and an enone (see figure 1). XN consists thus of two aromatic rings
substituted with hydroxyls and one methoxyl group, one α-β unsaturated double bond
(enone) and one isoprenyl unit (3,3-dimethylallyl).
Figure 1: Common structure of flavonoids, chalcones and the xanthohumol
XN was first isolated from hops in 1913 [13]. Several chalcones relatives to XN and XN
derivatives have then been extracted, identified and studied [10,14-17]. XN has a wide
spectrum of biological activities and has shown beneficial effect on human health [14] by
preventing cancer and having some anti-obesity effects [18]. Found mainly in beer, its
natural amounts are too small to have a significant effect through a dietary consumption. XN
could thus be taken in the form of dietary supplements.
The cyclic intramolecular rearrangement of the α,β-unsaturated ketone and the enzymatic
O-demethylation lead to 8-prenylnaringenin, one of the main metabolites of XN [612,19,20](see figure 2). Some concerns have been expressed about the use of XN in dietary
complements [6,19]. Reduction of the α,β alkene will prevent the formation of 8prenylnaringenin and its effects as a weak agonist of estrogen receptors in the body.
Synthesis of a XN analogue (DXN) that displays no estrogenic activity could be a new
therapeutic option to treat metabolic syndrome.
PFE at Linus Pauling Institute
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Scheme 1: Metabolic conversion of XN into desmethylxanthohumol (DMX) and 6- and 8prenylnaringenin (6-PN and 8-PN). XN is spontaneously converted into isoxanthohumol (IX) by
intramolecular Michael addition. 8-PN is formed from DMX or from IX by CYP-mediated
demethylation (from [21]).
The hydrogenation of both double bonds of XN leads to tetrahydroxanthohumol, one of the
xanthohumol derivatives; however, our interest was focused on the selective reduction in αβ unsaturated carbonyl position in xanthohumol. Besides, the chemoselective reduction of
α-β unsaturated carbonyl compounds can form three principal products: the 1,4-conjugate
reduction product (DXN), the product of the reduction of carbonyl function (1,2-reduction)
rather than the unsaturated carbon-carbon bond (obtaining of an α,β-unsaturated alcohol),
and the over-reduced product to generate the saturated alcohol.
Dihydroxanthohumol (4,2′,4′-Trihydroxy-6′-methoxy-3-prenyl-α,β-dihydrochalcone; α,βdihydroxanthohumol, see scheme 2) synthesis is first mentioned in 1960 [22] but the
procedure is not detailed. It was then isolated as a chalcone from hops in 1999 [17]. In 2003,
DXN was obtained as a result of a microbial transformation of xanthohumol by the fungus
Fusarium tricinctum [23]. In the present paper, we report our results relating to the catalytic
transfer hydrogenation of XN by using two different experimental procedures. Catalytic
transfer hydrogenation is known to be used for organic synthesis without the use of special
equipment or molecular hydrogen gas. Different methods have been developed using
selenium, indium, nickel and a complex palladium catalyst. Hydride-based systems have also
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been studied; the variation of the chemoselectivity of the reduction depends on the type of
hydride agent, the solvent, the substrate and the specific conditions used.
Scheme 2: Synthesis of DXN from XN
 Experimental procedures
XN, isolated from hops, was a gift from Dr. Martin Biendl (from Hopsteiner, Inc., New York).
The ionic liquid and Wilkinson catalysts were purchased from TCI America, Pd catalyst from
Alfa aesar, the sodium borohydride from JT Baker, the ammonium formate from Sigma.
The chemoselectivity of the catalytic transfer of hydrogen depends on the type of hydride
agent, the solvent, the substrate and the specific conditions used. We first tried a palladiumcatalyzed reduction system in combination with sodium borohydride and acetic acid as
hydrogen donor in toluene and dichloromethane [24]. Selectivity strongly depends on the
solvent used: non-polar solvents such as toluene favoring the 1,4-conjugate reduction of
the olefinic bond. However, xanthohumol is nearly insoluble in toluene so that this reaction
condition was not desirable. We chose dichloromethane as a suitable solvent for the
synthesis, even if xanthohumol was not completely dissolved in dichloromethane. Next, we
explored rhodium-catalyzed hydrogenation of α,β-unsaturated ketones by using an ionic
liquid [25]. The use of the Wilkinson catalyst (RhCl(PPh3)3; figure 2) selectively achieves 1,4conjugate reduction of the olefinic bond product over the 1,2-reduction of the carbonyl
product (see mechanism of the catalyzed hydrogenation in appendix 1). Besides, the double
bond of interest is more reactive than the prenyl group because the phenol proximity makes
the reactivity site planar and thus easy to attack by the Rhodium catalyst. The ionic liquid,
[bmim][BF4], was used as solvent and ammonium formate as the source of hydrogen. The
[bmim][BF4] as an ionic solvent and ammonium formate as hydrogen source were used.
PFE at Linus Pauling Institute
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Ionic liquids have the advantages to have a good solvent power for many organic chemical
reactions, a high miscibility, a high thermal stability, low vapor pressure and other
interesting physical properties.
Figure 2: Wilkinson catalyst
 Results and discussion
With the palladium-catalyzed hydrogenation method, we observed the formation of an
undesired cyclic product by intramolecular rearrangement of XN under acidic condition.
Therefore, we did not further pursue this method. Furthermore, the use of toluene and
dichloromethane resulted in low yields due to the poor solubility of XN in these solvents.
We successfully obtained DXN by catalytic hydrogenation using the Wilkinson catalyst
method by stirring the reaction mixture at 90oC for 3h. Various combinations of organic
solvent or organic solvent with ionic liquid were investigated. The best results (conversion
and yield) were obtained by using 10% mol of RhCl(PPh3)3, ammonium formate (4 eq), and
[bmim][BF4] ionic liquid (6.3 eq, 8.5 mmol), which showed a 89% conversion of XN into DXN
by HPLC (see conditions in Appendix 4). The reaction mixture was extracted with ethyl
acetate and washed with water and the organic layer was dried with anhydrous sodium
sulfate (Na2SO4) and evaporated under reduced pressure. The crude product was purified by
flash chromatography on a silica gel column eluted with ethyl acetate: hexane (1:1.7, v/v) as
eluent. DXN was obtained as a pale yellow solid for a yield of 44%. The compound was
characterized by NMR and MS-MS analysis (see appendices 2 and 3). A quantity of 4 g of
DXN was successfully synthesized and purified in order to be used for future animal studies.
An Oregon State University undergraduate chemistry student further optimized the method
under my guidance, and together we succeeded to increase the isolated yield to 62% by
extracting the reaction mixture with diethyl ether.
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II.
The effects of dihydroxanthohumol (DXN) as a mild
mitochondrial uncoupler in comparison with xanthohumol
(XN) and tetrahydroxanthohumol (TXN)
The mitochondrion, an organelle that is unique by its inner and outer membrane structure,
is involved in several essential cellular tasks such as continuous cellular energy supply in the
form of adenosine triphosphate (ATP) and cell life cycle (growth, death [26]). ATP is
produced both by oxidation of carbohydrates, lipids and proteins through the citric acid
cycle and by beta oxidation of fatty acids. Most of the reduced compounds, NADH and
FADH2, products of the citric acid cycle, are reoxidized with production of energy in the form
of ATP by the enzymes of the electron transport chain (ETC; figure 3) in the inner membrane
of the mitochondria. During the respiratory chain, NADH (or FADH2) is oxidized to NAD+ in
complex I (or FAD in complex II), the electrons relayed are ultimately transferred to O2 to
produce H2O. At the same time, protons are pumped to the intermembrane space (through
complexes I, III and IV) creating thus a proton motive force p. It has two components: Δψm,
the transmembrane (electrical) potential, and ΔpHm, the chemical potential gradient,
between the intermembrane space and the mitochondrial matrix. This proton motive force
provides the driving force for flowing back protons into the matrix through the ATP synthase
(complex V) thereby producing ATP by phosphorylation of adenosine diphosphate (ADP).
ATP synthesis in the mitochondria is therefore coupled to the oxidation of NADH and FADH2
leading to the transfer of electrons through the ETC.
Figure 3: Electron transport chain (http://www.dbriers.com)
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Previous studies [27,28] have associated a high coupling efficiency and a high proton motive
force to fat deposition into tissues and generation of reactive oxygen species (ROS).
Similarly, a low coupling efficiency leads to a decrease in both fat stores and production of
mitochondrial oxygen species. The ATP/Oxygen (P/O) ratio measures the efficiency of
oxidative phosphorylation and is a convenient means to examine mitochondrial coupling
[29]. The P/O ratio is the number of molecules of ATP synthesized per each pair of electrons
traversing the ETC. In theory, 3 ATP molecules are formed by oxidation of one molecule of
NADH. Beginning with NADH, 10 H+ are pumped across the inner mitochondrial membrane
and would result in the formation of three molecules of ATP, yielding a P/O value of ~3.
Experimentally, the P/O ratio is measured by using an oxygen electrode. The measurement
of the oxygen consumption rate (OCR) represents the uptake of oxygen by mitochondria to
reduce it to water. The OCR is thus an indicator of the mitochondrial respiration. The actual
yield of ATP synthesized by protons pumped is lower than the theoretical amount (P/O = 2.5
for NADH [29]). That can be explained by the fact that some energy created by the proton
motive force is used for purposes other than oxidative phosphorylation, lowering thus the
P/O ratio. These processes involve adenine-nucleotide exchange by adenine nucleotide
translocase, pyruvate and phosphate transport and natural uncoupling proteins (UCP 1, 2, 3)
[27].
OCR % baseline
Mitochondrial uncouplers are chemicals able to dissipate the proton gradient: they carry
protons into the matrix bypassing the proton channel of ATP synthase and therefore they
prevent the production of ATP, resulting in a decrease in the coupling efficiency and in the
value of the P/O ratio. These uncoupling agents uncouple the ETC from ADP phosphorylation
since they still permit electron transfer along the respiratory chain to O 2 to occur even in the
absence of ATP synthesis. The oxygen consumption rate is also increased (see figure 4). The
“proton leak” across the membrane created by uncouplers decreases the proton motive
force and generates heat instead of ATP.
Oligomycin
120
110
100
90
80
70
60
50
40
30
20
10
0
Uncoupler
ATP linked
OCR
total OCR
0
uncoupler
linked OCR
non ATP
linked OCR
10 20 30 40 50 60 70 80 90 100
Time (min)
Figure 4 : Representative OCR profile of the Seahorse assay. 80% of the total OCR is mitochondrial
OCR. In this example, 51% of the total OCR is non ATP linked OCR, i.e., non mitochondrial OCR and
proton leak OCR. That value is determined by injection of oligomycin (by inhibiting ATP synthase).
49% of the total OCR is thus coupled to ATP synthesis. The injection of an uncoupler increased the
OCR until reaching a maximal OCR [1].
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The hydrophobic character of the uncouplers makes them soluble in the mitochondrial
membrane. They can be weak acids able to collapse the proton gradient by binding a proton
on the acidic side of the membrane (in the outer membrane space), diffusing through the
membrane and releasing the proton in the alkaline matrix. Uncoupling proteins (UCP 1, 2, 3)
play a major role in mitochondrial function by controlling both heat and ROS species [28,30].
The collapse of the mitochondrial membrane potential leads to a rapid consumption of
oxygen and energy (in form of heat) since the ETC is again working, and thus, without ATP
production. ROS are primarily produced in mitochondria: excess electrons are transferred to
oxygen which is converted to ROS such as superoxide and hydrogen peroxide. The rate of
production of ROS is higher when the proton gradient is also higher [30]. Therefore, reducing
the electrochemical transmembrane potential by the action of UCP proteins works against
the formation of ROS species. UCP-1 is found exclusively in brown adipose tissue and is
responsible to regulate adaptive thermogenesis. UCP-2 is more widely expressed and UCP-3
is expressed in skeletal muscle. Increase of UCP activity in skeletal muscle has been
associated with protection against obesity [27,28,30]. Mitochondrial uncoupling can be
realized by transgenic mice overexpressing UCPs [30] or by compounds (such as transretinoic acid and trans-retinal [31]) that induce UCP expression. In the past, therapy with
uncoupling agents has been used for treatment of obesity. In the 1930’s, the compound
dinitrophenol (DNP) was efficient at causing weight loss in humans by decreasing coupling
efficiency and increasing energy expenditure [27]. DNP has, however, a narrow therapeutic
window (potential overdose risk) and dangerous side effects that make this compound not
safe as a therapeutic. It is thus still challenging to find an efficient and safe mild
mitochondrial uncoupler in order to treat obesity and obesity-related disorders.
Xanthohumol has previously [5,18] induced a reduction in body weight gain in the Zucker rat
model of obesity and metabolic syndrome. Xanthohumol treatment has been shown [5] to
decrease the generation of ROS by inducing an adaptative stress response. Xanthohumol is a
potent pro-estrogenic agent [7-12] and therefore it is important to inhibit or prevent the
formation of the estrogenic metabolite of xanthohumol, i.e., 8-prenylnaringenin (8-PN). In
part one, I have described the synthesis of the dihdroxanthohumol, which cannot be
metabolically converted into 8-PN. This study investigates the biological effects of DXN as a
potential mitochondrial uncoupler in comparison with xanthohumol and
tetrahydroxanthohumol using mouse skeletal muscle myoblasts (C2C12 cells).
 Experimental procedures
Chemicals
Xanthohumol, isolated from hops, and tetrahydroxanthohumol were gifts from Dr. Martin
Biendl and Dr. Robert Smith, respectively (both from Hopsteiner, Inc., New York).
Dihydroxanthohumol was synthesized according to the previously described procedure (part
1).
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Oligomycin
and
the
synthetic
uncoupler,
FCCP
(carbonyl
cyanide-4(trifluoromethoxy)phenylhydrazone), were purchased from Seahorse Bioscience (North
America, USA). MTT was obtained from Sigma (Missouri, USA).
JC-1 staining agent and Cell-Based Assay Buffer tablets were purchased as an assay kit from
the company Cayman Chemical (Ann Arbor, USA).
Cell culture and treatments
Mouse C2C12 skeletal muscle myoblasts (purchased from ATCC, Manassas, VA, USA) were
first seeded in 75 cm2 flasks using a culture medium consisting of DMEM (Life Technologies,
Grand Island, NY, USA), 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA,
USA), high glucose, L-glutamine, phenol red, 1 mM sodium pyruvate and 100 units/mL
penicillin, 100 g/mL streptomycin. The C2C12 cells were incubated at 37°C in a humidified
atmosphere of 5% CO2 until they were confluent. Then they were obtained by trypsinization
and seeded at a specific density in the plates of choice depending on the experiment.
MTT assay
For cell viability experiments (MTT assay), C2C12 cells were plated in 96-well plates at a
density of 4,000 cells per well in 200 μL of growth medium (identical to the culture medium).
After 48h of incubation, the medium was removed and the cells were treated with fresh
solutions of various concentrations of XN, DXN or TXN in phenol red-free growth medium.
The final concentrations of XN, DXN, TXN were 1, 2, 5, 8, 10, 25, 50 μM, all in quadruplicate
wells, in the 96-well plates. The medium used consists of phenol red free DMEM (Life
Technologies, Grand Island, NY, USA), 10 % FBS, 1 % glutamine, 1 mM of sodium pyruvate
and 100 units/mL penicillin, 100 μg/mL streptomycin. The plates were then incubated for
24h or 1h. After removing the medium, the cells were treated with 0.5 mg/mL solution of
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in phenol red-free growth
medium and incubated for two hours. After removing the medium and adding a solution of
acidified isopropanol to each well, the absorbance of each well was measured at 570 nm by
using a spectrometer (SpectraMax 190). Cell viability of drug-treated cells is displayed as a
percentage of control cells i.e. cells not treated.
Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) measurements
OCR and ECAR measurements were performed with a Seahorse XF 24 Analyzer (Seahorse
Bioscience). Cells were plated 24 hours prior to measurements at a density of 40,000 cells
per well within growth medium in a 24-well plate. The cells were then allowed to adhere for
24 hours (37°C, 5 % CO2). Prior to the assay, the cells were washed with freshly prepared
running media consisting of Seahorse XF Assay medium (Seahorse Bioscience, North
America, USA), 10 mM glucose and 1 mM sodium pyruvate. The pH of the medium was
adjusted to 7.4 with 1 M NaOH. The cells were then equilibrated for one hour at 37°C
without CO2. The compounds were injected during the assay; OCR and ECAR were measured
at 9 minutes measurement intervals. The solutions of oligomycin, FCCP, XN, DXN and TXN
were freshly prepared in running medium. The final concentrations in the wells were: 1 μM
for oligomycin, 5 μM for XN, DXN and TXN and 2, 5, 8, 25 μM for the dose response
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experiments with DXN. Ethanol was added to the control wells not to exceed a final
concentration of 0.1%. All the compounds were tested in quadruplicate on the 24-well plate.
XN is highly non-polar and it is our experience [5] that FBS should be present in order to
dissolve XN. Thus, XN, DXN and TXN were first dissolved in ethanol and the resulting
solutions were diluted 100-fold in running medium containing 1% of FBS. After a further 10fold dilution into the media in wells, only 0.1% of FBS was present during OCR
measurements. This amount appeared to dissolve up to 25 μM of DXN at 37°C and was well
below the 1-2% limit recommended by Seahorse Bioscience. The concentration of
oligomycin and the cell density used were selected based on preliminary experiments.
Measurement of mitochrondrial transmembrane potential changes using the JC-1 assay
Cells were plated at a density of 80,000 cells per well in black 96-well plates and incubated
for 24 hours at 37°C (5% CO2). They were then treated with fresh solutions of various
concentrations of XN, DXN or TXN in growth medium. The final concentrations of XN, DXN,
TXN were 1, 2, 5, 8, 25, 50 μM, all in quadruplicate wells, in the 96-well plates. After one
hour of exposure to the compounds at 37°C (5% CO2), the cells were treated with 10 μL of a
JC-1 staining solution (freshly prepared by diluting 11 fold 100 μL of the JC-1 agent furnished
from the assay kit). After 15 to 30 minutes of incubation (37°C, 5% CO2), the cells were
washed three times with the Cell-Based Assay Buffer (prepared by dissolving three buffer
tablets of the kit in 300 mL of MilliQ water). The plates were then analyzed by a fluorescent
plate reader (SpectraMax Gemini XS) at 560-595 nm and 485-535 nm.
Statistical analysis
The cellular experiments (MTT assay and JC-1 assay) were analyzed according to ANOVA
procedure, Dunnett. A p value < 0.05 was considered significant.
Seahorse data were analyzed in SAS 9.2 (SAS Institute Inc., Cary, NC). To show a consistent
baseline, OCR values (in pmol/min/mg protein) and ECAR values (in mpH/min/mg protein)
for each value were divided prior to statistical analysis by the average of the four pre
oligomycin treatment values (0, 8, 17, and 25 min) for the corresponding well and are shown
as % pre oligomycin values. The original scale for OCR and for ECAR values was utilized for
the OCR/ECAR ratio (in pMol/mpH). To account for repeated measures within wells over
time, we utilized a repeated-measures-in-time design using ANOVA procedures in PROC
MIXED. Fixed effects in the model were treatment (control, 2 μM DXN, 5 μM XN, 5 μM DXN,
and 5 μM TXN for the compound study and control, 2, 5, 8, and 25 μM DXN for the DXN
dosage study), time (0, 8, 17, 25, 34, 43, 52, 60, 69, 78, 86, and 95 min), and their
interaction. The random effect was the experiment (5 experiments for the compound study
and 3 experiments for the DXN dosage study). To account for repeated measures within
wells over time, a first order homogeneous variance-covariance matrix [AR(1)] was fitted for
each well. Using the ESTIMATE statement, a priori contrasts were constructed by comparing
the changes in compound treated cells from the last two time points prior to compound
treatment (average 52 and 60 min) to the changes in control cells during the same time
period. This was done for each compound and dosage for 69, 78, 86, and 95 min separately
and for all four time points combined. Results are shown in the text and the graphs as least
squares means and their corresponding SEM (standard error of mean). A P-value of 0.05 was
considered statistically significant and is shown as an asterisk in the graphs.
PFE at Linus Pauling Institute
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The bar graphs showing the xanthohumol-induced change in OCR, ECAR, and OCR/ECAR,
graphed as % change to control, are calculated as follows: first, we calculated, using the
trapezoidal rule, the area under the curve pre oligomycin treatment (0 to 25 min), post
oligomycin treatment (25 to 60 min, and post compound treatment (60 to 95 min). Next we
calculated the ATP-associated OCR fraction {[(post oligomycin OCR/pre oligomycin OCR) -1] x
100}, the glycolysis reserve fraction {[(post oligomycin ECAR/pre oligomycin ECAR) -1] x 100},
and the compound-induced changes in OCR, ECAR, and OCR/ECAR {[(post xanthohumol
OCR/ECAR, or OCR/ECARCompound/post oligomycin OCR/ECAR, or OCR/ECARCompound)-1] x 100}.
The calculated values were used for the statistical analysis in PROC MIXED. The fixed effect
in the model was treatment (as described previously) and the random effect was experiment
(as described previously). Using the ESTIMATE statement, a priori contrasts were
constructed by comparing the compound induced changes to the changes in control cells
during the same time period. Those least squared means estimates and SEM, displayed as %
change to control, are shown in the text and graphs. For the calculation of the area 21, only
the two last values between 52 and 60 min were considered. (Control, 2 μM DXN, 5 μM XN,
5 μM DXN, and 5 μM TXN for the compound study and control, 2, 5, 8, and 25 μM DXN for
the DXN dosage study fitted to the same statistical model as described in the previous
paragraph with the exception that time and its interaction was not included in the model. A
P-value of 0.05 was considered statistically significant and is shown as star in the graphs.
 Results
MTT assay
Two MTT assays were conducted (see appendix 5): after one hour and 24 hours of exposure.
After one hour of exposure, XN, DXN and TXN did not significantly affect cell viability up to
50 M. After 24 hours of exposure, cell viability was decreased at 8 M of DXN, 25 M of
TXN and 50 M of XN.
Optimization of Seahorse assay conditions (cell density, oligomycin and FCCP concentrations)
As advised by Seahorce Bioscience, running a cell titration assay with FCCP at different
concentrations prior to any Seahorse experiment is an important step to determine proper
cell density for the cell line used. Then, oligomycin was tested at different concentrations at
the proper cell density in order to determine the optimal working concentration. These
optimization and titration assays offered us starting conditions in our investigation of the
uncoupling effects on the mitochondria (see appendices 6 and 7).
In the cell titration assay, cells were plated at 10,000, 20,000 and 40,000 cells/wells and
FCCP was injected at 0.3 M or 0.5 M. The maximal OCR was obtained at a density of
40,000 cells/well at a concentration of 0.5 μM of FCCP. The values of OCR obtained were
between 205 and 234 picomole/min for the control baseline. An increase was observed
after injection of FCCP: 517 to 498 picomole/min and 605 to 518 picomole/min for 0.3 M of
FCCP and for 0.5 M of FCCP, respectively. The values of OCR obtained at this density were
in the range of the typical values obtained for C2C12 muscle mouse cells so the density of
40,000 cells/wells was chosen for the following experiments. After injection of FCCP,
especially at 0.5 M, the cells increased their OCR, meaning that they behaved as expected
in the presence of a mitochondrial uncoupler. DXN was also injected at 2 M at 20,000
PFE at Linus Pauling Institute
15
cells/well to have an idea of its potential effects on OCR. We then observed an increase of
OCR of 204% of the baseline (see appendix appendix 6).
In the oligomycin optimization assay, oligomycin was injected at 0 M, 0.5 M 1 M and 2
M followed by an injection of 0.5 M of FCCP. After injection of oligomycin, the resulting
decrease in OCR was the most significant at a concentration of 1 M. This concentration was
thus the optimized working concentration for the next experiments.
Comparison of the uncoupling effects of XN, DXN, TXN
80% of the oxygen consumed by the cells is due to mitochondrial respiration [32]. The ATP
synthase is the major pathway whereby protons reenter the mitochondrial matrix and it is
also coupled to the respiration chain. Therefore we measured cellular OCR after injection of
the ATP synthase inhibitor, oligomycin, to investigate whether the addition of either XN,
DXN or TXN could stimulate the OCR, when ATP synthesis is inhibited. We used a diluted
solution of ethanol in the running medium (0.01 % final concentration in wells) as a vehicle
control in order to measure the significance of the impacts of the compounds on the OCR. At
5 M concentration, we observed that DXN and TXN were able to significantly increase the
OCR in the presence of oligomycin: by 28.7 ± 6.9 % for DXN and by 56.8 ± 6.9 % for TXN,
respectively, compared to the baseline and for an average of five independent experiments
(see figure 5 A and B). A concentration of 5 M appeared to be sufficient for both DXN and
TXN to have a significant effect on the OCR. TXN was found to be most potent as a
mitochondrial uncoupler among our three compounds. XN did not demonstrate a significant
increase in the OCR at 5 M in contrast to a previous study investigating its mitochondrial
uncoupling effects [5]. The effects of DXN at 2 M were similar to the effects of XN at 5 M
(on average, 11.3 ± 6.9 % and 8.9 ± 7.0 % for XN at 5 M and DXN at 2 M, respectively).
After injection of oligomycin in each well, the ECAR increased until reaching a plateau with
the value 160.1 ± 5.1 % (see figure 5 C and D). The injection of 5 M of XN, DXN and TXN and
2 M of DXN resulted in a significant decrease in ECAR of 4.7 ± 2.0 %, 17.8 ± 2.0 %, 27.5 ± 2.0
%, 5.9 ± 2.0 % respectively, for an average of five independent experiments.
PFE at Linus Pauling Institute
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O lig o m y c in
120
110
100
90
80
70
60
50
40
30
20
10
0
Com pound
*
*
*
**
*
*
*
TXN 5 M
DXN 5 M
XN 5 M
DXN 2 M
C o n tro l
0
10
20
30
40
50
60
70
80
90 100
 O C R ( % C h a n g e t o C o n t r o l)
O C R ( % P r e O lig o m y c in )
A
B
*
60
50
40
30
20
10
0
DXN
T IM E (m in )
O lig o m y c in
180
(2  M )
Com pound
160
140
*
*
120
*
*
100
*
*
*
*
*
*
80
60
40
20
0
0
10
20
30
40
50
60
70
80
90 100
*
Com pound
TXN
(5  M )
*
-15
*
-25
*
-35
DXN
(2 M)
 O C R /E C A R ( % C h a n g e t o C o n t r o l)
O C R /E C A R ( p M o l/m p H )
O lig o m y c in
13
12
11
10
9
8
7
6
5
4
3
2
1
0
DXN
(5  M )
-5
T IM E (m in )
E
XN
(5  M )
D
ECAR (% Change to Control)
E C A R ( % P r e O lig o m y c in )
C
*
XN
(5 M)
DXN
(5 M)
TXN
(5 M)
F
*
100
*
*
*
0
20
40
60
**
80
**
*
100
T IM E (m in )
80
60
*
40
20
0
DXN
(2  M )
XN
DXN
TXN
(5  M )
(5  M )
(5  M )
Figure 5: A to F: Comparison of XN, DXN and TXN as mild mitochondrial uncouplers. DXN and TXN cause
mitochondrial uncoupling in cells. C2C12 cells were sequentially treated with oligomycin (1 μM) and 5 μM of the test
compounds (A, C and E). Changes in OCR and ECAR after injection of test compounds were plotted as bars (B, D and
F) (mean ± SEM, n = 5).* Indicates p < 0.05 by ANOVA procedure in PROC MIXED.
PFE at Linus Pauling Institute
17
Dose-response effect of DXN
Because we were first primarily interested in DXN, we tested different concentrations (2, 5,
8, 25 µM) of this compound on the same plate. We observed a dose-effect relationship (see
figure 6 A and B): the greater the dose, the greater the OCR was increased in the presence of
oligomycin, i.e., by 6 ± 6 %, 30.8 ± 6.0 %, 55.1 ± 6.0 % at a concentration of 2 M, 5 M and 8
M, respectively, for an average of three independent experiments. The concentrations of 5
μM and 8 μM thus showed a significant OCR increase. At higher dose (25 µM), the cells
exhibited an initial increase in the OCR that was immediately followed by a sharp decrease.
After injection of oligomycin into each well, the ECAR increased until reaching a plateau with
the value 151.1 ± 3.4 % (see figure 6 C and D). The injection of 2 M, 5 M, 8 M and 25 M
of DXN resulted in a significant decrease in ECAR of 8.0 ± 2.4 %, 25.2 ± 2.3 %, 31.6 ± 2.3, 49.8
± 2.3 %, respectively.
PFE at Linus Pauling Institute
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O lig o m y c in
120
110
100
90
80
70
60
50
40
30
20
10
0
Com pound
*
*
*
*
*
*
*
*
*
*
*
D XN 25  M
DXN 8 M
DXN 5 M
DXN 2 M
C o n tro l
0
10
20
30
40
50
60
70
80
90 100
 O C R ( % C h a n g e t o C o n t r o l)
O C R ( % P r e O lig o m y c in )
A
B
70
*
60
*
50
*
40
30
20
10
0
2
T IM E (m in )
O lig o m y c in
180
Com pound
160
*
*
140
120
**
100
80
*
*
*
*
*
*
*
*
*
*
*
60
40
20
0
0
10
20
30
40
50
60
70
80
90 100
-5
-15
*
-25
*
-35
*
-45
*
-55
2
Com pound
*
*
**
*
*
**
**
0
10
20
30
40
50
60
5
8
25
DXN (M)
70
*
80
*
90 100
T IM E (m in )
 O C R /E C A R ( % C h a n g e t o C o n t r o l)
O C R /E C A R ( p M o l/m p H )
O lig o m y c in
13
12
11
10
9
8
7
6
5
4
3
2
1
0
25
D
T IM E (m in )
E
8
D X N ( M )
ECAR (% Change to Control)
E C A R ( % P r e O lig o m y c in )
C
5
F
*
140
120
*
100
80
*
60
40
20
0
2
5
8
25
D X N ( M )
Figure 6 A to F: DXN causes mitochondrial uncoupling in cells. C2C12 cells were sequentially treated with
oligomycin (1 μM) and the indicated concentration of DXN (uncoupler) (A, C and E). Changes in OCR and ECAR
after injection of test compounds were plotted as bars (B, D and F) (mean ± SEM, n = 3).* Indicates p < 0.05 by
ANOVA procedure in PROC MIXED.
PFE at Linus Pauling Institute
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JC1 Assay
To determine whether XN, DXN and TXN were acting as protonophores, i.e., being able to
transport protons across the mitochondrial inner membrane instead of transport via the
proton channel of the ATP synthase, we measured the mitochondrial membrane potential
variations. Mitochondria are especially responsible of key events in healthy and apoptotic
cells. While the proton motive force (p) drives ATP synthesis, the electrical component,
Δψm, is primarily responsible for the generation of ROS and NADH reduction. Alteration of
the mitochondrial activity results in a depolarization or loss of mitochondrial transmembrane
potential [26,33]. Δψm is thus an important parameter of mitochondrial function and can be
used as an indicator of cell health. Qualitative variations, like a loss of Δψm, can be studied
by evaluating the changes in fluorescence intensity of cells stained with cationic dyes such as
rhodamine-123 or the new cytofluorometric lipophilic cationic dye, 5,6-dichloro-2-[3-(5,6dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propen-1-yl]-1,3-diethyl-1Hbenzimidazolium, monoiodide (JC-1). JC-1 can selectively enter into mitochondria and
reversibly change color from green to red as the membrane potential increases. In healthy
cells with high mitochondrial transmembrane potential, JC-1 spontaneously forms
complexes known as J-aggregates with intense red fluorescence with excitation and
emission at 560 nm and 595 nm, respectively. In apoptotic or unhealthy cells with low
potential difference (or in our case of a dissipation of the proton gradient), JC-1 remains in
the monomeric form, which shows only intense green fluorescence with excitation and
emission at 485 nm and 535 nm, respectively. The ratio of fluorescence intensity of Jaggregates to fluorescence intensity of J-monomers can be used as an indicator of cell heath
or mitochondrial state. After treatment with various concentrations of the compounds and
spectrometric measurements, the ratios of aggregates to monomers (expressed as percent
of control) were obtained (see figure 7). A decrease in the values of the ratios compared to
the control is indicative of an alteration in the potential across the mitochondrial membrane.
The ratios changed significantly from the control values for XN, DXN, and TXN at 5 M, 8 M,
25 M and 50 M. The higher the dose, the greater the loss of transmembrane potential. In
order to confirm the directional dye behavior, we treated a group with different
concentrations (0.5 M and 2.5 M) of the protonophore FCCP, which was able to collapse
the charge gradient at 2.5 M with a ratio value of aggregates to monomers of 33.0 ± 5.6 %.
PFE at Linus Pauling Institute
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Ratio aggreagate/monomers (% control)
120
110
100
90
80
70
60
50
40
30
20
10
0
DXN
TXN
XN
* **
*
* *
*
*
**
0.5
2.5
1
2
FCCP
5
8
25
*
**
50
M
Test compounds
Figure 7: XN, DXN and TXN are able to depolarize the mitochondrial transmembrane potential measured as a
decrease in the ratio aggregate/monomers. * indicates p < 0.05.
PFE at Linus Pauling Institute
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 Discussion
For our experiments, we used C2C12 skeletal muscle cells because of their high
bioenergetics activity. Previous studies [5,34] have shown that dysregulation of fatty acids
oxidation and glucose metabolism are associated with reduced mitochondrial oxidative
activity in muscle cells. Therefore, investigating the effects of uncouplers on OCR in C2C12
muscle cells was a model of cellular energy generation and expenditure.
XN [5], DXN and TXN (this thesis) were demonstrated to act as mitochondrial uncouplers on
C2C12 cells by their ability to increase significantly the OCR after selective inhibition of the
ATP synthase by oligomycin. As investigated in a previous study [5], the α,β unsaturated
ketone functionality of xanthohumol is thus not required for acting as a mitochondrial
uncoupler. The results of the two main experiments (DXN dose response and comparison of
XN, DXN and TXN) are consistent with each other: on one side, DXN effect on the OCR
increased with the dose injected and on the other side TXN was more potent than DXN. DXN
was more potent than XN.
80% of the oxygen consumed by the cells is due to mitochondrial [32]. After inhibition of the
respiratory chain by oligomycin, the 51.4 ± 6.3 % residual OCR (value for the DXN dosage
experiment) corresponded to the non-coupled to ATP synthesis (proton leak and cytosolic
respiration, see figure 4). 49 % of the OCR was thus coupled to oxidative phosphorylation.
These values agree with other studies [32,35] stating that 54% of the total OCR is coupled to
ATP synthesis.
The doses tested were low enough to get a mild mitochondrial effect in order to reduce the
proton motive force and to expend energy without showing toxicity. A cell viability
experiment (MTT assay) was conducted prior to the Seahorse experiments to verify that the
concentrations used (5 M for all compounds and 2 to 25 M for DXN dose-response) were
not toxic to the cells after one hour of test compound exposure. However, at higher dose (25
µM), the cells exhibited an initial increase in the OCR that was rapidly followed by a sharp
decrease. This finding can be explained as a response to a toxic insult. In addition, a previous
study [5] showed that at higher concentrations, XN and TXN inhibit OCR. In fact, at higher
concentrations, the cells rapidly increased their OCR but they were not able to maintain the
OCR due to toxicity because the mitochondrial integrity is essential for the cell viability.
However, 25 M was not toxic to cells in the MTT assay. MTT assay is based on the ability of
the cells to reduce the MTT complex exclusively via mitochondrial succinic dehydrogenases.
However, MTT conversion has been shown [36] to be a complex process dependent on
multiple factors (critical role of cell membrane function for example). If DXN, TXN and XN act
on the cell membrane, they will thus have effects on MTT uptake and excretion. The MTT
assay has been also shown to be less sensitive to alteration of ETC activity than some
respirometry analysis.
XN, DXN and TXN have shown to act as mitochondrial uncoupers able to depolarize the
mitochondrial transmembrane membrane. The mechanism of action of the uncouplers can
be a direct effect (by acting as protonophores dissipating the proton gradient) or an indirect
PFE at Linus Pauling Institute
22
effect by stimulating UCP expression. The influence of XN and its derivatives on UCP activity
could be investigated by using UCP-knockout and UCP-overexpressing mice [30,31]. The
induction of UCP-expression would require time (interactions with translation proteins
processes). However, in the Seahorse assays, the uncoupling effect is immediately observed
after injection of the uncoupling compounds so the effect of XN derivatives must be direct
and not UCP-dependent.
Using mitochondrial uncouplers as therapeutics can be an issue because of possible toxicity
at higher doses and side effects such as plasma membrane depolarization at even low doses.
FCCP has been found to depolarize the plasma membrane from concentrations [26,37]
above 2.5 M. Therefore, it would be important to verify that for DXN and TXN. Besides, the
integrity of the mitochondrial function has to be controlled: it is important to verify that the
uncouplers do not affect ADP phosphorylation and cell membrane and mitochondrial
enzymes activity. That can be conducted by mitochondrial toxicity assay (ToxGlo™ Promega),
measuring mitochondrial enzymes activity [38] and also by adding mitochondrial complexes
inhibitors. The pharmacokinetics of DXN versus TXN has to be studied in order to determine
their bioavailability in comparison with XN [21,39].
In the Seahorse experiments conducted, the injection of oligomycin induced a decrease in
the OCR and an increase in the ECAR. The cells were not able anymore to supply ATP via
oxidative phosphorylation so they shifted to glycolysis, an anaerobic process, to provide
energy [4]. Glycolysis generates pyruvate that can be converted to lactate. In animals,
lactate is converted back to pyruvate that can normally enter the citric acid cycle to be
oxidized and generate energy and electrons flowing through the respiration chain. In the
case of an excess of lactate, when it cannot be converted and enter the citric acid cycle that
does not work, the environment is acidified (the pH decreases) because of the presence of
lactic acid, and the ECAR increases. After injection of the uncoupler DXN, the respiration
chain is uncoupled and is working versus the oxidative phosphorylation, the OCR increases
and some heat is produced by dissipating the proton gradient (see figure 8). Pyruvate is able
to enter the citric acid cycle so less of this substrate is converted to lactate resulting in a light
decrease in the ECAR compared to prior injection of DXN. ATP is still produced though, since
ECAR is not equal to zero, allowing the cells to live. The cells are continuously trying to
restore the energetic equilibrium. In reality and in therapies, the uncoupler is added without
prior injection of ATP synthase inhibitor. There is thus still some production of ATP through
oxidative phosphorylation (but less than usual) and there is a continuous competition
between the flow of protons through ATP synthase forming ATP and the dissipation of the
proton motive force via the uncoupler generating heat. An increase in ECAR is expected
since the cells will shift to glycolysis to supply the cells in the amount of energy needed, as
seen in figure D (appendix 6) with injection of 2 μM of DXN. The experiment need to be
repeated and to be conducted also for TXN.
PFE at Linus Pauling Institute
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ECAR % baseline
Oligomycin
180
160
Uncoupler
140
120
100
80
basal lactate
acidification
60
unc. linked
decrease in
lactate
production
maximal
lactate
acidification
40
20
0
0
10 20 30 40 50 60 70 80 90 100
Time (min)
Figure 8 : Representative ECAR profile of the Seahorse assay. The maximal lactate acidification is
determined by injection of oligomycin (inhibition of ATP synthase). The ECAR is then decreased by
injection of an uncoupler such as DXN or TXN at 5 μM.
PFE at Linus Pauling Institute
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Conclusion
The mitochondrial uncoupling effect of xanthohumol can be increased by synthetic
conversion into dihydroxanthohumol and tetrahydroxanthohumol. We confirmed that
xanthohumol and its two derivatives are able to dissipate the proton gradient, possibly by
acting as protonophores, thereby increasing cellular oxygen consumption without ATP
production. The significance of these findings is that xanthohumol and its synthetic
derivatives increase energy expenditure and may be used therapeutically to treat obesity
and metabolic syndrome. DXN has the advantage over XN in that it lacks pro-estrogenic
effects and reactivity towards proteins. DXN has the advantage over TXN in that its
mitochondrial uncoupling effects are milder and may therefore be less toxic. The cell culture
findings described in this thesis set the stage for pre-clinical studies in animals.
PFE at Linus Pauling Institute
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Bibliography
1. Dott W, Mistry P, Wright J, Cain K, Herbert KE: Modulation of mitochondrial bioenergetics in a
skeletal muscle cell line model of mitochondrial toxicity. Redox Biol 2014, 2:224-233.
2. Nagel J, Culley LK, Lu Y, Liu E, Matthews PD, Stevens JF, Page JE: EST analysis of hop glandular
trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of
xanthohumol. Plant Cell 2008, 20:186-200.
3. Grundy SM, Brewer HB, Cleeman JI, Smith SC, Lenfant C, Association AH, National Heart Ln, and
Blood Institute: Definition of metabolic syndrome: Report of the National Heart, Lung, and
Blood Institute/American Heart Association conference on scientific issues related to
definition. Circulation 2004, 109:433-438.
4. Rogge MM: The role of impaired mitochondrial lipid oxidation in obesity. Biol Res Nurs 2009,
10:356-373.
5. Kirkwood JS, Legette LL, Miranda CL, Jiang Y, Stevens JF: A metabolomics-driven elucidation of the
anti-obesity mechanisms of xanthohumol. J Biol Chem 2013, 288:19000-19013.
6. Bolca S, Possemiers S, Maervoet V, Huybrechts I, Heyerick A, Vervarcke S, Depypere H, De
Keukeleire D, Bracke M, De Henauw S, et al.: Microbial and dietary factors associated with
the 8-prenylnaringenin producer phenotype: a dietary intervention trial with fifty healthy
post-menopausal Caucasian women. Br J Nutr 2007, 98:950-959.
7. Yilmazer M, Stevens JF, Deinzer ML, Buhler DR: In vitro biotransformation of xanthohumol, a
flavonoid from hops (Humulus lupulus), by rat liver microsomes. Drug Metab Dispos 2001,
29:223-231.
8. Yilmazer M, Stevens JF, Buhler DR: In vitro glucuronidation of xanthohumol, a flavonoid in hop
and beer, by rat and human liver microsomes. FEBS Lett 2001, 491:252-256.
9. Jirásko R, Holcapek M, Vrublová E, Ulrichová J, Simánek V: Identification of new phase II
metabolites of xanthohumol in rat in vivo biotransformation of hop extracts using highperformance liquid chromatography electrospray ionization tandem mass spectrometry. J
Chromatogr A 2010, 1217:4100-4108.
10. Guo J, Nikolic D, Chadwick LR, Pauli GF, van Breemen RB: Identification of human hepatic
cytochrome P450 enzymes involved in the metabolism of 8-prenylnaringenin and
isoxanthohumol from hops (Humulus lupulus L.). Drug Metab Dispos 2006, 34:1152-1159.
11. Possemiers S, Heyerick A, Robbens V, De Keukeleire D, Verstraete W: Activation of proestrogens
from hops (Humulus lupulus L.) by intestinal microbiota; conversion of isoxanthohumol
into 8-prenylnaringenin. J Agric Food Chem 2005, 53:6281-6288.
12. Possemiers S, Bolca S, Grootaert C, Heyerick A, Decroos K, Dhooge W, De Keukeleire D, Rabot S,
Verstraete W, Van de Wiele T: The prenylflavonoid isoxanthohumol from hops (Humulus
lupulus L.) is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the
human intestine. J Nutr 2006, 136:1862-1867.
13. Power FB, Tutin F, Rogerson H: The Constituents of Hops. J. Chem. Soc., Trans. 1913, 103:12671292.
14. Stevens JF, Page JE: Xanthohumol and related prenylflavonoids from hops and beer: to your
good health! Phytochemistry 2004, 65:1317-1330.
15. Stevens JF, Ivancic M, Hsu VL, Deinzer ML: Prenylflavonoids from Humulus lupulus.
Phytochemistry 1997, 44:1575-1585.
16. Stevens JF, Taylor AW, Nickerson GB, Ivancic M, Henning J, Haunold A, Deinzer ML:
Prenylflavonoid variation in Humulus lupulus: distribution and taxonomic significance of
xanthogalenol and 4'-O-methylxanthohumol. Phytochemistry 2000, 53:759-775.
17. Etteldorf N, Etteldorf N, Becker H: New chalcones from hop Humulus lupulus. Z. Naturforsch., C
J. Biosci. 1999, 54:610-612.
PFE at Linus Pauling Institute
26
18. Legette LL, Luna AY, Reed RL, Miranda CL, Bobe G, Proteau RR, Stevens JF: Xanthohumol lowers
body weight and fasting plasma glucose in obese male Zucker fa/fa rats. Phytochemistry
2013, 91:236-241.
19. Milligan SR, Kalita JC, Pocock V, Van De Kauter V, Stevens JF, Deinzer ML, Rong H, De Keukeleire
D: The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.)
flavonoids. J Clin Endocrinol Metab 2000, 85:4912-4915.
20. Possemiers S, Verstraete W: Oestrogenicity of prenylflavonoids from hops: activation of prooestrogens by intestinal bacteria. Environ Microbiol Rep 2009, 1:100-109.
21. Legette L, Ma L, Reed RL, Miranda CL, Christensen JM, Rodriguez-Proteau R, Stevens JF:
Pharmacokinetics of xanthohumol and metabolites in rats after oral and intravenous
administration. Mol Nutr Food Res 2012, 56:466-474.
22. Anteunis M: An experimental contribution to the study of the isomerization of α,β and β,γunsaturated ketones. Verhandel. Koninkl. Vlaam. Acad. Wetenschap., Belg., Kl. Wetenschap.
1960, 22:1-86.
23. Tronina T, Bartmańska A, Filip-Psurska B, Wietrzyk J, Popłoński J, Huszcza E: Fungal metabolites
of xanthohumol with potent antiproliferative activity on human cancer cell lines in vitro.
Bioorg Med Chem 2013, 21:2001-2006.
24. Russo AT, Amezcua KL, Huynh VA, Rousslang ZM, Cordes DB: A simple borohydride-based
method for selective 1,4-conjugate reduction of α,β-unsaturated carbonyl compounds.
Tetrahedron Lett. 2011, 52:6823-6826.
25. Baan Z, Finta Z, Keglevich G, Hermecz I: Unexpected chemoselectivity in the rhodium-catalyzed
transfer hydrogenation of α,β-unsaturated ketones in ionic liquids. Green Chem. 2009,
11:1937-1940.
26. Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA: Mitochondrial membrane potential
probes and the proton gradient: a practical usage guide. Biotechniques 2011, 50:98-115.
27. Harper ME, Green K, Brand MD: The efficiency of cellular energy transduction and its
implications for obesity. Annu Rev Nutr 2008, 28:13-33.
28. Kim JA, Wei Y, Sowers JR: Role of mitochondrial dysfunction in insulin resistance. Circ Res 2008,
102:401-414.
29. Hinkle PC: P/O ratios of mitochondrial oxidative phosphorylation. Biochim Biophys Acta 2005,
1706:1-11.
30. Li B, Nolte LA, Ju JS, Han DH, Coleman T, Holloszy JO, Semenkovich CF: Skeletal muscle
respiratory uncoupling prevents diet-induced obesity and insulin resistance in mice. Nat
Med 2000, 6:1115-1120.
31. Echtay KS, Esteves TC, Pakay JL, Jekabsons MB, Lambert AJ, Portero-Otín M, Pamplona R, VidalPuig AJ, Wang S, Roebuck SJ, et al.: A signalling role for 4-hydroxy-2-nonenal in regulation of
mitochondrial uncoupling. EMBO J 2003, 22:4103-4110.
32. Brand MD, Chien LF, Ainscow EK, Rolfe DF, Porter RK: The causes and functions of mitochondrial
proton leak. Biochim Biophys Acta 1994, 1187:132-139.
33. Nicholls DG, Ward MW: Mitochondrial membrane potential and neuronal glutamate
excitotoxicity: mortality and millivolts. Trends Neurosci 2000, 23:166-174.
34. Mihalik SJ, Goodpaster BH, Kelley DE, Chace DH, Vockley J, Toledo FG, DeLany JP: Increased levels
of plasma acylcarnitines in obesity and type 2 diabetes and identification of a marker of
glucolipotoxicity. Obesity (Silver Spring) 2010, 18:1695-1700.
35. Rolfe DF, Brand MD: Contribution of mitochondrial proton leak to skeletal muscle respiration
and to standard metabolic rate. Am J Physiol 1996, 271:C1380-1389.
36. Hynes J, Hill R, Papkovsky DB: The use of a fluorescence-based oxygen uptake assay in the
analysis of cytotoxicity. Toxicol In Vitro 2006, 20:785-792.
37. Kenwood BM, Weaver JL, Bajwa A, Poon IK, Byrne FL, Murrow BA, Calderone JA, Huang L,
Divakaruni AS, Tomsig JL, et al.: Identification of a novel mitochondrial uncoupler that does
not depolarize the plasma membrane. Mol Metab 2014, 3:114-123.
PFE at Linus Pauling Institute
27
38. Boudina S, Sena S, O'Neill BT, Tathireddy P, Young ME, Abel ED: Reduced mitochondrial oxidative
capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity.
Circulation 2005, 112:2686-2695.
39. Legette L, Karnpracha C, Reed RL, Choi J, Bobe G, Christensen JM, Rodriguez-Proteau R, Purnell
JQ, Stevens JF: Human pharmacokinetics of xanthohumol, an antihyperglycemic flavonoid
from hops. Mol Nutr Food Res 2014, 58:248-255.
PFE at Linus Pauling Institute
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Appendices
Appendix 1: Hydrogenation mechanism by using the Wilkinson catalyst
The Wilkinson catalyst dissociates first to some extent in the solvent (Solv) used ; a solvent molecule
fills the vacant site. Steps: (1) addition, (2) alkene addition, (3) migratory insertion, (4) reductive
elimination of the alkane, regeneration of the catalyst
Halpern, Chem. Com. 1973, 629; J. Mol. Cat. 1976, 2, 65; Inorg. Chim. Acta. 1981, 50, 11
PFE at Linus Pauling Institute
29
Appendix 2: NMR Spectra of DXN
Figure A: DXN molecule annotated. Figures B to F are, respectively, 1H(700 MHz), 13C(175MHz), COSY,
HSQC, HMBC
A
PFE at Linus Pauling Institute
30
B
p
u
c,e b,f
t
h,q
n
r
PFE at Linus Pauling Institute – Master thesis
g
32
C
c+e b+f
m
o
s
l
i
k
PFE at Linus Pauling Institute
a
d
r
j
n
p
33
h
u
g qt
b,f
D
q g
h
r
c,e
PFE at Linus Pauling Institute
34
E
ut
p
c,e
b,f
n
h,q
r
t
q
u
g
h
p
n
j
l
b,f
r
PFE at Linus Pauling Institute
35
g
c,e
s
d
F
p
n
j
l
o
m
k
PFE at Linus Pauling Institute
Hn ↔ Co
Hn ↔ Cl
Hn ↔ Cj
Hp ↔ Co
36
PFE at Linus Pauling Institute
37
Appendix 3: Mass Spectra of DXN (Q1, Product ion, and proposed fragmentation pattern)
PFE at Linus Pauling Institute – Master thesis
37
+Q1: 3.183 min from Sample 1 (TuneSampleID) of MT20140129134902.wiff (Heated Nebulizer)
Max. 3.1e5 cps.
357.0
3.1e5
3.0e5
2.8e5
2.6e5
2.4e5
2.2e5
In te n s ity , c p s
2.0e5
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
419.3
8.0e4
6.0e4
261.4
4.0e4
270.9
266.1
2.0e4
255.3
260
270
280
362.8
300.7
284.4
286.8
297.0
290
345.1
309.3
323.6
299.0
300
387.6
327.0
295.1
279.2
267.3
263.4
0.0
250
340.2
323.0
285.0
310
320
331.2
359.4
338.3
345.9 352.7
330
340
m/z, Da
350
378.9
369.3
364.8
373.5
360
383.9
413.4
395.9 403.2
375.6 381.8 393.7
370
380
390
400
408.9
410
+MS2 (357.00): 0.653 to 0.921 min from Sample 1 (TuneSampleID) of MT20140129135533.wiff (Heated Nebulizer)
420
Max. 4.6e6 cps.
301.1
4.6e6
4.4e6
4.2e6
4.0e6
195.1
3.8e6
3.6e6
3.4e6
3.2e6
3.0e6
In te n s ity , c p s
2.8e6
2.6e6
2.4e6
2.2e6
283.1
2.0e6
1.8e6
1.6e6
1.4e6
107.2
1.2e6
1.0e6
207.1
8.0e5
6.0e5
153.1
4.0e5
137.2
2.0e5
0.0
177.1
149.2
80
100
120
140
251.2
181.2
139.1
160
180
200
220
m/z, Da
240
357.2
269.1
229.1
260
280
300
320
340
360
Figure A: Q1
PFE at Linus Pauling Institute
38
Figure B: Product ion
Figure C: proposed fragmentation pattern of DXN
PFE at Linus Pauling Institute
39
Appendix 4: HPLC chromatogram to characterize a mixture of DXN, XN and TXN
AU
0.10
XN
13.308
DXN
9.666
10.661
0.15
TXN
0.05
0.00
5.00
10.00
15.00
20.00
Minutes
4 μl injection of solutions of 0.07 mg/ml of DXN, XN and TXN (each of them) in methanol into a 2 X 150 mm Phenomenex
Luna 5 micron PFP column. The sample was eluted with 0.5 ml/min, 46 % acetonitrile in 0.1% trifluoroacetic acid in water,
220 nm chromatogram, Waters 600 HPLC system. Note: Isocratic runs do not have retention times as consistent as
gradient runs and therefore for further mixture reactions analysis, peaks can be identified using their UV spectra (210 to
500 nm). Method developed by Dr. Ralph Reed in the lab.
PFE at Linus Pauling Institute – Master thesis
39
Appendix 5: Effect of XN, DXN and TXN on cell viability (MTT assay)
Cell viability (% of control)
A
XN
150
DXN
125
100
*
*
75
TXN
*
50
**
25
0
1
2
5
8
10
25
50
Test compound (M)
Cell viability (% of control)
B
XN
DXN
TXN
100
*
50
0
1
2
5
8
10
25
50
Test compound (M)
Figures A and B: After one hour of exposure (B), XN, DXN and TXN did not significantly affect cell
viability up to 50 M. After 24 hours of exposure (A), cell viability was decreased at 8 M of DXN, 25 M
of TXN and 50 M of XN. Cell viability of drug-treated cells is displayed as a percentage of control cells
i.e. cells not treated.
* Indicates p < 0.05
PFE at Linus Pauling Institute – Master thesis
40
Appendix 6: Cell density and FCCP concentration optimization experiment (Seahorse assay)
B
Compound
275
250
225
200
175
150
125
100
75
50
25
0
FCCP  M
FCCP 3 M
control
15
10
5
0
0
10
20
30
40
50
60
70
0
TIME (min)
C
275
250
225
200
175
150
125
100
75
50
25
0
10
20
50
60
70
50
60
70
50
60
70
20
10
0
0
10
20
30
40
50
60
70
0
10
20
TIME (min)
E
Compound
600
550
500
450
400
350
300
250
200
150
100
50
0
30
40
TIME (min)
50
ECAR (mpH/min)
OCR (pmol/min)
40
Compound
30
DXN 2 M
30
TIME (min)
D
Compound
ECAR (mpH/min)
OCR (pmol/min)
Compound
20
ECAR (mpH/min)
OCR (pmol/min/min)
A
F
Compound
40
30
20
10
0
0
10
20
30
40
50
60
70
0
10
20
TIME (min)
30
40
TIME (min)
Figures A to F: cell titration assay. C2C12 cells were perturbed by injection of uncoupler (0.3 μM
or 0.5 μM of FCCP or 2 μM DXN) at different cells densities: 10,000 (A & B), 20,000 (C & D),
40,000 (E & F) cells/well.
PFE at Linus Pauling Institute – Master Thesis
41
Appendix 7: Oligomycin concentration optimization experiment (Seahorse assay)
OCR (% Pre Oligomycin)
A
Oligomycin Compound
275
250
225
200
175
150
125
100
75
50
25
0
0
OL 0 M + FCCP
OL M + FCCP
OL 0 M + FCCP
OL  M + FCCP
OL  M + DXN
10 20 30 40 50 60 70 80 90 100
TIME (min)
ECAR (% Pre Oligomycin)
B
Oligomycin
200
Compound
175
150
125
100
75
OL
OL
OL
OL
OL
50
25
0.5 mM + FCCP
1 mM + FCCP
2 mM + FCCP
1 mM + DXN
0 mM + FCCP
0
0
10 20 30 40 50 60 70 80 90 100
TIME (min)
Figure A and B: Optimization of oligomycin concentration. C2C12 cells were sequentiallly treated with
oligomycin at the concentration indicated and 0.5 μM of FCCP.
PFE at Linus Pauling Institute – Master Thesis
42
Appendix 8: Tables presenting the results (OCR and ECAR) of the statistical analysis
Tables A to F: Effects of XN, DXN and TXN as mitochondrial uncouplers in cells. C2C12 cells were sequently treated with oligomycin (1 μM) and 5 μM of the
test compounds (A to C, average of 5 experiments) or the indicated concentration of DXN (D to F, average of 3 experiments). All the values are expressed in
percent of control (except for the time points of the OCR/ECAR, expressed in picomol/mpH). *Indicates p < 0.005 by ANOVA procedure in PROC MIXED.
A
OCR
Time
0
8
17
25
34
43
52
60
69
78
86
95
Area
12
23
21
Control
Avg
SEM
99,5
3,69
99,7
3,69
99,15
3,69
101,6
3,69
38,55
3,69
35,05
3,69
34,6
3,69
37,9
3,69
36,95
3,69
37,45
3,69
38,8
3,69
38,05
3,69
-52,53
38,72
XN5
Avg
100,95
99,65
99,4
100,1
45,75
44,2
44,7
47,15
48,85
51,05
51,75
53,2
SEM
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
DXN5
Avg
99,45
100,5
99,4
100,55
42
37,8
37,55
39,3
48,25
53,05
59,85
64,3
69-95
SEM
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
2,24
2,53
11,26
43 to 60
6,87
28,67
6,87
TXN5
Avg
99,4
100,35
100,55
99,85
41,5
36,6
36,28
37,15
50,95
66,75
81
90,7
25 to 60
56,79
SEM
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
3,69
DXN2
Avg
99,43
100,06
100,01
102,27
41,53
38,43
38,32
39,48
39,85
43,8
46,64
48,95
6,87
8,88
PFE at Linus Pauling Institute – Master thesis
P-value
SEM XN5 vs. C DXN5 vs. C TXN5 vs. C DXN2 vs. C
3,75
3,75
3,75
3,75
3,75
3,75
3,75
3,75
3,75
0,43
0,001
<0.0001
0,93
3,75
0,27
0,0002
<0.0001
0,31
3,75
0,42
<0.0001
<0.0001
0,21
3,75
0,22
<0.0001
<0.0001
0,07
0,25
<0.0001
<0.0001
0,18
6,97
0,1
<0.0001
43
<0.0001
0,21
B
ECAR
Time
0
8
17
25
34
43
52
60
69
78
86
95
Area
12
23
21
Control
Avg
SEM
99,2
4,93
98,8
4,93
100,5
4,93
101,55
4,93
126,35
4,93
154,75
4,93
155,1
4,93
159,35
4,93
164,75
4,93
165,5
4,93
165,8
4,93
163,85
4,93
45,07
160,05
XN5
Avg
104,4
96,15
99,3
100
128,5
161,8
162,55
163,1
168,55
163,55
156,9
154,45
SEM
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
DXN5
Avg
99,25
99,6
100
101,15
134
160,35
160,85
164,2
150,25
142,75
137,85
134,25
69-95
1,99
-17,77
5,36
5,41
-4,68
43 to 60
SEM
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
TXN5
Avg
98,65
100,55
100,75
100,05
138
152,3
158,25
161,8
135,5
120,85
118,6
115,65
25 to 60
1,99
-27,45
PFE at Linus Pauling Institute – Master Thesis
SEM
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
4,93
DXN2
Avg
95,2
100,51
100,78
101,46
132,99
161,93
163,99
165,56
165,56
164,46
162,2
161,35
1,99
-5,93
P-value
SEM XN5 vs. C DXN5 vs. C TXN5 vs. C DXN2 vs. C
4,99
4,99
4,99
4,99
4,99
4,99
4,99
4,99
4,99
0,68 <0.0001
<0.0001
0,12
4,99
0,15 <0.0001
<0.0001
0,1
4,99
0,01 <0.0001
<0.0001
0,05
4,99
0,01 <0.0001
<0.0001
0,1
0,03
<0.0001
<0.0001
0,04
2,02
44
0,02 <0.0001
<0.0001
0,004
C
Ratio
Time
0
8
17
25
34
43
52
60
69
78
86
95
Area
12
23
Control
Avg
SEM
9,47
0,77
9,41
0,77
9,23
0,77
9,21
0,77
2,93
0,77
2,08
0,77
2,02
0,77
2,14
0,77
2,02
0,77
2,04
0,77
2,18
0,77
2,19
0,77
-63,99
XN5
Avg
10,58
11,2
10,67
10,74
3,68
2,77
2,81
2,95
2,93
3,15
3,4
3,53
SEM
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
DXN5
Avg
10,83
10,84
10,75
10,63
3,3
2,47
2,44
2,47
3,3
3,78
4,47
4,97
69-95
1,56
13,41
10,75
41,3
SEM
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
TXN5
Avg
10,82
10,53
10,68
10,52
3,3
2,53
2,3
2,32
3,51
4,9
6,12
7,36
25 to 60
10,75
92,08
PFE at Linus Pauling Institute – Master Thesis
SEM
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
0,77
10,75
DXN2
Avg
9,99
9,34
9,38
9,41
2,93
2,09
2,06
2,09
2,14
2,35
2,55
2,7
11,94
P-value
SEM XN5 vs. C DXN5 vs. C TXN5 vs. C DXN2 vs. C
0,78
0,78
0,78
0,78
0,78
0,78
0,78
0,78
0,78
0,79
0,03
0,003
0,76
0,78
0,56
0,01 <0.0001
0,56
0,78
0,51
0,003 <0.0001
0,56
0,78
0,44
0,0006 <0.0001
0,47
0,49
0,001 <0.0001
0,51
10,89
45
0,22
0,0002 <0.0001
0,28
D
OCR
Time
0
8
17
25
34
43
52
60
69
78
86
95
Control
Avg
107,33
106,08
98,25
88,83
47,67
47,25
49,5
51,83
50,25
53,33
54
55,17
SEM
5,4
5,4
5,4
5,4
5,4
5,4
5,4
5,4
5,4
5,4
5,4
5,4
DXN2
DXN5
DXN8
DXN25
P-value
Avg
SEM
Avg
SEM
Avg
SEM
Avg
SEM DXN2 vs. C DXN5 vs. C DXN8 vs. C DXN25 vs. C
107,66
5,4 110,58
5,4 115,25
5,4 106,5
5,4
101,47
5,4 106,5
5,4 104,17
5,4 104,33
5,4
100,93
5,4 95,92
5,4 94,42
5,4 98,83
5,4
92,84
5,4 86,92
5,4 86,42
5,4 89,75
5,4
48,47
5,4 49,25
5,4 48,75
5,4
49,5
5,4
47,93
5,4 50,92
5,4 48,83
5,4 49,58
5,4
51,02
5,4 52,75
5,4 52,58
5,4 52,92
5,4
51,57
5,4 54,25
5,4 55,17
5,4 55,42
5,4
55,2
5,4 60,75
5,4 66,13
5,4 111,17
5,4
0,32
0,04
0,002
<0.0001
55,57
5,4 70,67
5,4
82
5,4 89,75
5,4
0,76
0,005
<0.0001
<0.0001
59,2
5,4
81
5,4 100,33
5,4 58,33
5,4
0,43
<0.0001
<0.0001
0,88
61,93
5,4 92,17
5,4 115,25
5,4
45
5,4
0,31
<0.0001
<0.0001
0,02
69-95
0,35
<0.0001
<0.0001
<0.0001
Area
12
23
21
-44,9
4,56
51,37
5,51
6,33 43 to 60
6,08
30,78
25 to 60
5,95 55,09
PFE at Linus Pauling Institute – Master Thesis
5,95
40,62
5,95
46
0,37
<0.0001
<0.0001
<0.0001
E
ECAR
Time
0
8
17
25
34
43
52
60
69
78
86
95
Control
Avg
93,25
93,5
102,75
110,5
121,83
148,58
151,42
150,42
159,17
158
158,92
158,17
SEM
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
DXN2
Avg
93,69
95,72
101,87
108,14
127,24
151,78
153,51
153,1
151,96
152,6
146,78
144,87
SEM
4,32
4,32
4,32
4,32
4,32
4,32
4,32
4,32
4,32
4,32
4,32
4,32
DXN5
Avg
94,92
95,08
101,25
108,83
134,67
148,75
147
149,25
125,83
122,92
117,5
114,33
69-95
SEM
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
DXN8
Avg
97,67
91,25
103,75
107,5
133,67
147,08
146,83
150
117,5
111,92
107,75
103,08
SEM
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
4,17
DXN25
P-value
Avg
SEM DXN2 vs. C DXN5 vs. C DXN8 vs. C
91,58
4,17
97,17
4,17
101,58
4,17
109,67
4,17
134,58
4,17
146,25
4,17
152,92
4,17
152,58
4,17
96,42
4,17
0,05 <0.0001
<0.0001
81,42
4,17
0,18 <0.0001
<0.0001
75,08
4,17
0,02 <0.0001
<0.0001
71,67
4,17
0,02 <0.0001
<0.0001
0,02 <0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
2,31
-49,76
<0.0001
DXN25 vs. C
Area
12
23
21
40,79
151,16
1,5
-7,99
3,36 43 to 60
2,36
-25,23
25 to 60
2,31 -31,61
PFE at Linus Pauling Institute – Master Thesis
2,31
47
0,001 <0.0001
<0.0001
F
Ratio
Time
0
8
17
25
34
43
52
60
69
78
86
95
Control
Avg
10,01
9,87
8,32
7,18
3,37
2,71
2,78
2,91
2,72
2,89
2,88
2,99
SEM
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
DXN2
Avg
10,1
9,68
8,94
7,89
3,39
2,81
2,92
2,94
3,17
3,21
3,55
3,75
SEM
0,56
0,56
0,56
0,56
0,56
0,56
0,56
0,56
0,56
0,56
0,56
0,56
DXN5
Avg
10,38
9,99
8,7
7,63
3,18
2,96
3,08
3,11
4,34
5,08
6,14
7,11
69-95
SEM
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
10,59
60,17
10,35
DXN8
Avg
9,55
9,32
7,63
6,83
2,78
2,58
2,75
2,83
4,53
5,83
7,48
8,88
SEM
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
0,54
DXN25
P-value
Avg
SEM DXN2 vs. C DXN5 vs. C DXN8 vs. C
8,97
0,54
8,35
0,54
7,61
0,54
6,34
0,54
2,83
0,54
2,53
0,54
2,65
0,54
2,76
0,54
8,88
0,54
0,48
0,007
0,0003
8,39
0,54
0,72
0,003 <0.0001
5,84
0,54
0,43 <0.0001
<0.0001
4,79
0,54
0,4 <0.0001
<0.0001
0,42 <0.0001
<0.0001
DXN25 vs. C
<0.0001
<0.0001
<0.0001
0,01
<0.0001
Area
12
23
-56,47
2,06
10,51
PFE at Linus Pauling Institute – Master Thesis
100,31
10,35
130,81
10,35
48
0,33 <0.0001
<0.0001
<0.0001
4ème PAGE DE COUVERTURE DES RAPPORTS DE STAGE
Stage d’exécution 
Stage élève – ingénieur 
Stage année de césure :
12 mois 
1er semestre
Stage projet de fin d’études
2ème semestre

Date du stage
du 6/01/2014
au 31/07/2014
Année
2013/2014
DE MONTGOLFIER Oriane
Spécialité : CGP
Summary
The metabolic syndrome comprises multiple risk factors for cardiovascular disease including
abdominal obesity. Obesity results from an energy imbalance where fuel intake is in excess
compared to energy expenditure. Among the pharmacotherapeutic strategies to treat
obesity, targeting mitochondrial uncoupling appears to be one of them, as mitochondria are
the cellular source of energy. Xanthohumol (XN; 3'-[3,3-dimethyl allyl]-2',4',4-trihydroxy-6'methoxychalcone), a major prenylated hops flavonoid, has shown anti-obesity effects by
inducing a reduction in body weight gain in the Zucker rat model of obesity and metabolic
syndrome. XN can increase energy expenditure by mild mitochondrial uncoupling. However,
some concerns have been expressed about the use of XN in dietary supplements, as 8prenylnaringenin (8-PN), one of its metabolites, can act as a weak agonist of estrogen
receptors in the body. Synthesis of a XN analogue (dihydroxanthohumol, DXN) that displays
no estrogenic activity would be an attractive therapeutic option to treat the metabolic
syndrome. This project is aimed at finding a way to synthesize DXN from XN and then
determining its biological effects as a mild mitochondrial uncoupler in comparison with XN
and tetrahydroxanthohumol (TXN), a second XN derivative. The synthesis of DXN has been
successfully achieved with 89% of conversion via a Wilkinson catalyzed hydrogenation
(chemoselective reduction of the α,β-unsaturated ketone of XN). Then the biological activity
of DXN, XN and TXN was investigated. Oxygen Consumption Rate (OCR) and Extra Cellular
Acidification rate (ECAR) measurements were conducted by using the Seahorse assay. The
results obtained have shown that the mitochondrial uncoupling effect of XN can be
increased by synthetic conversion into its derivatives that are able to dissipate the proton
motive force thereby increasing cellular oxygen consumption without ATP production
leading to energy expenditure. XN derivatives act as mild mitochondrial uncouplers at 5 uM.
They have shown to be able to act as protonophores by depolarizing the transmembrane
mitochondrial potential in the JC-1 assay. DXN has the advantage over TXN in that its
mitochondrial uncoupling effects are milder and may therefore be less toxic. The cell culture
findings described in this thesis set the stage for pre-clinical studies in animals. This project
will also be published in a scientific journal.
PFE at Linus Pauling Institute – Master Thesis
49