1. The interaction of Nrf2 and Glyoxalase I in response to lipid
loading in Hepatocytes
FARYA MUBARIK
URN 6176280
B.Sc. Nutrition (B400)
Supervisor: Dr. J.B. Moore
Faculty of Health and Medical Science
University of Surrey
Submitted May 2015

2. 1
Title
The interaction of Nrf2 and Glyoxalase I in response to lipid loading in Hepatocytes.
Author: Farya Mubarik, Ciaran Fisher and J. Bernadette Moore
Author’s affiliation
Department of Nutritional Sciences, School of Biosciences and Medicine, Faculty of Health
and Medical Sciences the University of Surrey, United Kingdom.
Corresponding Author
J. Bernadette Moore, Senior Lecturer in Molecular Biology, Department of Nutritional
Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences the
University of Surrey, United Kingdom. E-mail j.b.moore@surrey.ac.uk
Contribution
JBM developed the concept. FM was responsible for literature review, conducting the
experiment, acquisition of data, statistical analysis and interpretation of data as well as write-
up of the paper. CPF supervised the experimental procedures. JBM provided inputs to the
structure and writing of the manuscript. Supervisor and author have read and approved the
final draft.
Word Count
3586 words
Figures and Tables
7 Figures
Conflict of Interest
There are no conflicts of interest to declare.

3. 2
Abstract
Background: The Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) serves a master
regulator of antioxidant response-driven gene transcription such as glyoxalase I. The aims of
this study were to determine the optimal dose and duration of MG132 treatment for
proteasomal inhibition of Nrf2 and to examine cell viability in response to oleic acid (OA)
and palmitic acid (PA) in HepG2 cells. Methods: HepG2 cells were exposed to 100, 200,
300, 400, 500 µM of PA and OA for 24 hrs, separately. Cell viability was assessed by Alamar
Blue assay. Nrf2 accumulation was determined by exposing HepG2 cells to 0, 2.5, 5, 10, 20
µM of MG132 for 0, 2, 4, 6, 8 hrs. Whole cell lysate was then assessed for the accumulation
of Nrf2 by immunoblotting. Results: We found a dose- and time-dependent increase in
accumulation of Nrf2 in response to MG132 treatment. The optimal expression of Nrf2 was
identified at 5μM of MG132 after 2 hrs of incubation. Both fatty acids were found to have no
significant effect on cell viability. Conclusion: In conclusion, duration and concentration of
inhibition of 26S proteasome leads to an accumulation of Nrf2. Interestingly, double bands
were detected suggestive of posttranslational modification, which may act as a signal for Nrf2
translocation and/or degradation. Nrf2 is an inducer of glyoxalase I gene. In vitro study
suggests that lipid accumulation down regulates glyoxalase I protein levels. Therefore,
combination of lipid accumulation and proteasomal may result in dual signal for glyoxalase I
expression.
Words: 247
Key Words
Nrf2 accumulation, Glyoxalase, MG132, Oleate, Palmitate
Abbreviations
Antioxidant Response element, ARE; MG, Methylglyoxal; ROS, Reactive Oxygen Species;
Nuclear factor-erythroid 2 p45 subunit-related factor 2, Nrf2; Kelch-like ECH-associated
protein 1, Keap 1; Ubiquitin Proteasome System, UPS; DMSO, Dimethyl Sulfoxide; PBS,
Phosphate-buffered Saline

4. 3
Introduction
Diabetes is characterized by uncontrolled hyperglycaemia as a result of defect in
insulin signalling (1). Hyperglycaemia leads to the formation of advanced glycated end
products (AGEs). These are a diverse group of highly oxidant compounds which are
produced by the non-enzymatic glycation of protein and lipids (2,3). AGEs have known to
generate reactive oxygen species (ROS) which pose a threat to genomic integrity (4-6).
Accumulation of AGEs is also associated with ageing, retinal and liver complications (3,7-9).
Methylglyoxal (MG) is one of the most reactive AGE precursors (8), found elevated in
hyperglycaemic conditions and diabetes. High MG levels are associated with later diabetic
complications (10). These induce an irreversible production of MG-derived AGEs.
Consequently, AGEs causes glycation of proteins, which disrupts the normal function by
changes in molecular conformations, altering the enzymatic activity and interfering with the
receptor functioning (11).
MG is a dicarbonyl product of glucose autoxidation and lipid peroxidation (12). MG
promotes synthesis of ROS which can cause oxidative stress in the body leading to further
significant levels of oxidative damage (13). In healthy individuals, MG is detoxified by
glyoxalase system present in the cytosol of all the cells. However, diabetes and other chronic
diseases are associated with the down regulation of glyoxalase I, which results in
accumulation of MG and as a consequence glycation of circulating proteins and lipids (See
Fig. 1) (14-17). The glyoxalase system is an efficient enzymatic detoxification scheme which
protects against the formation of MG-derived AGEs damages, by metabolising MG into D-
lactate in two steps (18,19). The glyoxalase I gene provides protection against oxidative
damage by expressing a specific cytoprotective detoxifying enzyme (20-22). Recent research
shows that there are three potential antioxidant response elements (ARE) found in the
promoter region of glyoxalase I gene (20). AREs are the genomic sequences which are
responsible for encoding detoxifying and anti-oxidant enzymes (23). These AREs are
activated in response to stress via a specific transcription factor called Nrf2 (nuclear factor-
erythroid 2 p45 subunit-related factor 2). Nrf2 is from “cap n collar” family of Nuclear
Erythroid Factor (24,25). It co-ordinates the increased expression of genes associated with
protection against oxidative damage.
In vitro studies have shown that the expression of the glyoxalase I gene is largely
controlled by the transcription factor Nrf2 (20). Nrf2 mediates an anti-stress response and as a

5. 4
result expression of glyoxalase I is initiated to protect against MG dicarbonyl stress. In the
basal state, Nrf2 is found complexed with Keap 1 (Kelch-like ECH-associated protein 1) in
the cytosol, which regulates it through ubiquitin and proteasomal mediated turnover. Nrf2
(110 kDa) has been described as “a master regulator of cellular defence mechanism” and is
responsible for eliminating carcinogens and toxins (26). Under homeostatic conditions, Nrf2
is constantly synthesised and degraded in the cells to maintain cell-cycle progression.
However, in response to cellular stress it dissociates with Keap 1 and translocates into the
nucleus to bind with the small Maf protein on AREs target genes, which includes: NADPH
quinone oxidoreductase 1, glutathione transferase isoforms, and glutamate cysteine ligase
(See Fig. 2) (25,27). Nrf2 also plays a role in lipid metabolism in the liver; higher levels of
Nrf2 are associated with reduced expression of enzymes involved in fatty acids synthesis
(28).
In eukaryotic cells, many proteins are degraded via ubiquitin proteasome system
(UPS) (29,30). It is a highly regulated and controlled system localised in the cytoplasm of the
cells. The UPS degrades misfolded, malfunctioned proteins and proteins which exceed
cellular requirements, in a step-by-step manner. In addition to its housekeeping role, current
literature suggests that an imbalance of UPS contributes towards the pathogenesis of several
diseases such as cancer and Parkinson’s disease (31-33). The proteasome may be inhibited in
vitro or in vivo, by proteasomal inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal
(MG132). MG132 inhibits proteasomal degradation of Nrf2 and it also induces the nuclear
translocation of Nrf2 in human vascular endothelial cells (34,35).
Recent research by the Moore laboratory has identified modulation of glyoxalase I
levels in response to hepatic lipid accumulation (36,37). In addition to the down regulation
of glyoxalase I in mice fed high fat diet, in vitro investigation demonstrated that oleic acid-
induced lipid accumulation decreased the glyoxalase I protein levels (See Fig. 3 A and B).
This appeared to be mediated through posttranslational modification of glyoxalase I, first by
acetylation and then by ubiquitination; upon proteasomal inhibition (MG132) glyoxalase I
levels increase and are found to be polyubiquitinated (See Fig. 3 C).
Another research has demonstrated that Eicosapentaenoic acid and Docosahexaenoic
acid can induce the Nrf2 antioxidant defence mechanism (38). Therefore, lipid accumulation
may have a dual effect on the expression of glyoxalase I gene, driving the acetylation and
turnover of glyoxalase I at the same time as Nrf2 induces mRNA expression of the

6. 5
glyoxalase gene. This multi-induction pathway therefore calls into question whether MG132-
induced Nrf2 leads to a concomitant up regulation of glyoxalase I. To explore this research
question, we examined the optimal dose and duration of MG132 treatment for proteasomal
inhibition and Nrf2 accumulation; first optimising antibody detection of Nrf2 and examining
cell viability in response to Oleic acid and Palmitic acid, prior to using a co-treatment
approach to examine time and dose effects of MG132 on Nrf2 levels in immortalised human
hepatocytes (HepG2 cells).
Methodology
Cell Culturing
Human hepatocellular carcinoma (HepG2) cells were purchased from European Collection of
Cell Culture (ECACC; Salisbury, UK). Cells were grown in low glucose (5.5 mM)
Dulbecco’s modified Eagle’s medium (Lonza; Slough, UK), with 1g/L glucose, 10% FBS
(Fisher Scientific, Loughborough, UK), 100 µM non-essential amino acids (NEAA), 2mM L-
glutamine, 10,000 U/ml penicillin and 10,000 U/ml streptomycin (all Lonza; Slough, UK).
An automated cell counter (Bio-Rad, UK) was used to analyse the cell count. All of the
treatments were performed at approximately 70% confluence, between passages 19 and 27.
Fatty Acid Treatment and Alamar Blue Assay
Cells were seeded in a 96-well microtiter plate in five technical replicates 72 hrs before fatty
acid treatment, at a seeding density of 30,000 cells/cm2
, with maintenance media described
above. Cells were incubated at 37o
C and 5% CO2, in humidified HERA cell incubator
(Buckinghamshire, UK).
Oleic acid (OA; Sigma-Aldrich, Gillingham, UK) and Palmitic acid (PA; Sigma-Aldrich,
Gillingham, UK) dissolved in dimethyly sulfoxide (DMSO; Sigma-Aldrich, Gillingham, UK)
were complexed with Fatty Acid Free Bovine Serum Albumin (FAF-BSA; Sigma-Aldrich,
Gillingham, UK) for 1hr at 37 o
C with mixing every 10 min, in two separate centrifuges
tubes. These were sterile filtered prior to the complexing. Two sets of different concentration
treatments of 100, 200, 300, 400, 500 µM of 200µL were prepared for a 24 hrs treatment
period. DMSO in FAF-BSA was used for vehicle.
In order to measure cell viability, cells were incubated with 20µL Alamar Blue (Sigma-
Aldrich, Gillingham, UK) for 2 hrs at 22hrs of fatty acid treatment. Prior to the Alamar Blue

7. 6
assay cells were rinsed with PBS. After 2 hrs incubation, fluorescence was measures in an
automated plate reader (FLUOstar, Omega; Ortenberg, Germany) in arbitrary fluorescence
units following excitation at a wavelength of 590 nm.
MG132 Treatment
Cells were seeded in T25 plates 72 hrs before fatty acid treatment, at a seeding density of
30,000 cells/cm2
. MG132 (Calbiochem (Merck), Darmstadt, Germany) in DMSO solvent
(Sigma-Aldrich, Gillingham, UK) was dissolved with serum free media to prepare a set of
different concentration treatments of 0, 2.5, 5, 10, 20 µM of 3ml each. Prior to the treatment,
cells were washed twice with PBS. Cells were then incubated with the treatment for 0, 2, 4, 6
and 8 hrs for all (5) sets of treatment concentrations.
Protein Extraction
A 20x stock solution of Protease Inhibitor (PI) was prepared prior to the lysis by mixing one
EDTA free-complete ultra-tablets (Roche; Welwyn, UK) in 500 µL of distilled deionized
water. After exposure to MG132, cells were washed with 5 ml phosphate-buffered saline
(PBS) twice and lysed with 500ul of radioimmunoprecipitation buffer (Sigma-Aldrich;
Gillingham, UK.) containing 1x PI for 5 min on ice. The whole cell lysate was collected in a
centrifuge tube and centrifuged at 8000g for 10 min at 4o
C. Supernatant was collected and
stored at -80 o
C. Total protein concentration was measured by using a BCA Protein Assay Kit
(Thermo Fisher Scientific, USA) according to manufacturer’s instructions. Standards and
lysed samples were added in a 96-well microtiter plate in duplicates. Plate was incubated at
37o
C for 30 min in an automated plate reader (FLUOstar, Omega; Ortenberg, Germany).
After incubation absorbance was measured at 562 nm wave length on the same plate reader.
Immunoblotting
Immunoblot was performed using whole cell lysates. First, protein samples were prepared by
adding 2x reducing Lamelli buffer (Sigma-Aldrich; Gillingham UK), than denatured by
heating at 60 o
C for 10 min. Equal amounts of denatured protein (10 μg) were subjected to
12% sodium dodecyl sulphate-polyacrylamide (SDS) gels at room temperature. Protein
extract was separated on the gel based on their molecular weight at 150 V voltages, in 1x
Tris-Glycine-SDS buffer. Separated proteins were then transferred on to polyvinylidene
diflouride (PVDF) membranes from the gels at 300 mA of current, for 2 hrs in 1x transfer

8. 7
buffer. Prior to the transfer, PVDF membranes were activated in methanol for 5 min. After
the transfer, membranes were than blocked by 0.1% of Marvel milk in 1x Tris-buffered
saline, 0.1% tween-20 (TBS-T) overnight. Blocking time varied for two membrane, 19 hrs
and 45 min and 13 hrs and 30 min. Membranes were than probed by primary antibodies,
rabbit anti-Nrf2 antibody [Catalogue # (EP1808Y) (ab62352)]; (1:1000 dilution, Abcam,
Cambridge, UK), mouse anti-alpha Tubulin [Catalogue # (TU-01) (ab7750)]; (1:5000
dilution, Abcam; Cambridge, UK). After 3hrs of incubation at room temperature, membranes
were washed three times with 1x TBS-T for 5 min. Membranes were incubated with
appropriate fluorescent dye-labelled secondary antibodies for 1 hr at room temperature.
Secondary antibodies were donkey anti-rabbit and donkey anti-mouse antibodies (1:10000
dilution, Odyssey; Cambridge, UK). Fluorescent bands were visualised by Infrared detector
(LICOR Odyssey CLx, UK). The results were analysed using Image Studio Lit version 4.0
software.
Anti Nrf2 Optimisation Experiment
Prior to above experiments anti Nrf2 antibody was optimised using HepG2 cells seeded at a
density of 30,000 cells/cm2
, with maintenance media described above, 72 hrs before the
experiment. HepG2 cells were incubated with 10 µM of MG132 for 4 hrs, followed by
protein extraction and immunoblotting as mentioned above. 10 and 20 µg of protein samples
were subjected to 12 % SDS gels. Total seven PVDF membranes were probed by primary
antibody on different days, rabbit anti-Nrf2 antibody [Catalogue # (EP1808Y) (ab62352)];
(Abcam; Cambridge, UK) at 1:2000 vs 1:4000 and 1:1000 and 1:2000 dilutions and rabbit,
anti-Nrf2 antibody [Catalogue # (C-20): sc-722)]; (Santa Cruz Biotechnology; Texas, USA)
at 1:500 and 1:200 vs 1:500 dilutions. Cells were taken from passages 2-19 at different. Rest
of the protocol was same as mentioned above.
Data Analysis
For quantitative data analysis, cell viability in response to fatty acid treatment relative to the
untreated cells (vehicle), was expressed as a percentage (%), calculated in Microsoft Excel
2007. The ratio of the protein of interest (Nrf2) and the Loading control (alpha Tubulin) was
also calculated in Microsoft Excel 2007. The ratios were normalized by dividing the Nrf2
protein levels with average alpha Tubulin level. All graphs were made using Graph Pad
Prism 6.

9. 8
Results
Effects of fatty acids on cell viability
In order to study the effects of lipid loading on cell viability, HepG2 cells were exposed to
100, 200, 300, 400, 500 µM of PA and OA for 24 hrs, separately. Alamar Blue assay was
performed to assess cell viability in response OA and PA. Both fatty acids found to have no
significant effect on cell viability (See Fig. 4 A and B). Cells were taken from the same
passage and seeded into different wells (n=5 Technical replicates).
Antibody Optimization
With a view of using anti-Nrf2 antibody for assessing Nrf2 accumulation, antibody
optimization experiments were performed prior to the determination of optimal dose and
duration of MG132 treatment. Untreated samples and samples treated with 10µM of MG132
for 4 hrs, resulted in accumulation of Nrf2 protein (See Fig. 5). Strong Nrf2 bands were
detected with Santa Cruz anti-Nrf2 (1:500 dilution) at 60 kDa in untreated samples. However,
very light or no bands were detected in repeated experiments with 1:500 and 1:200 dilution in
MG132 treated and untreated samples at 60 kDa.
Similarly, very light Nrf2 protein bands were detected with Abcam anti-Nrf2 antibody in the
region of 110 kDa, with 1:2000, 1:4000 dilution in MG132 treated and untreated samples
(See Fig. 5). However, 1:1000 dilution resulted in prominent protein band at 110kDa.
Proteasomal inhibition causes accumulation of Nrf2 protein in a dose-dependent
manner
In order to study the dose and time-dependent effect of MG132 on Nrf2 accumulation, a time
course experiment was performed. HepG2 cells were exposed to 0, 2.5, 5, 10, 20 µM
concentrations of MG132 for 0, 2, 4, 6 and 8 hrs. Proteasome inhibitor MG132 was found to
increase the accumulation of Nrf2 in a time and dose-dependent manner (See Fig. 6 A and B).
Nrf2 protein bands were detected in the region of approximately 110 kDa. This was closer to
the observed band size of 100 kDa, indicated by manufacturer. Treatment with 5μM of
MG132 for 2hrs of duration caused an optimal build-up of Nrf2 protein (Nrf2/alpha Tubulin
ratio= 16.26) (See Fig 6. C). Loading control alpha Tubulin showed an equal expression and
even loading of the samples (Mean ± SD = 3248 ± 496).
Both blots showed double bands in the region of 110 kDa in response to higher dose and
longer duration of proteasomal inhibitor (MG132) treatments (20 μM after 2hrs; 2.5, 5, 10, 20

10. 9
μM after 4hrs 6hrs and 8hrs) suggestive of posttranslational modification of Nrf2. An
increase in the intensity of band with MG132 treatment concentration and duration was
observed (See Fig. 6 D).
Discussion
Mechanism of inhibition
This study found that duration and concentration of proteasomal inhibition by MG132 leads
to an accumulation of Nrf2 proteins in a time and dose-dependent manner, in human
hepatocyte (HepG2 cells). MG132 is a peptide aldehyde which acts as a substrate (Nrf2)
analogue. It tightly binds to the catalytic sites of the 26 S proteasome and effectively blocking
its activity, which results in a build-up of proteins (Nrf2) (39,40). In addition to the
accumulation of Nrf2, the presence and increasing intensity of double bands in response to
longer duration and higher dose effect of MG132 is an indicative of an increase in molecular
weight of Nrf2 owing to posttranslational modification. These modifications usually involve
addition of covalent group such as phosphorylation at Serine 40 and tyrosine residue (41-43).
This may lead to a change in the weight of Nrf2 proteins (phosphorylated) and shift of band.
Role of phosphorylation in activation of Nrf2
Previous laboratory research by Pi et al (2007) suggests that, Nrf2 is activated by
phosphorylation, in response to increased oxidative stress (44). The two identified
phosphorylated form of Nrf2 were detected at 98 kDa and 118 kDa in response to stress
inducers in HaCat cell (human keratinocyte cell line) (44). However, only Nrf2 at 98 kDa has
transcriptional activity, whereas Nrf2 at 118kDa had higher susceptibility of degradation. It
has also been indicated that phosphorylation takes place in two steps and Casein Kinase-2
(CK2) enzyme is critical to this process (Nrf2---Nrf2 98--- Nrf2 118) (44). Bryan et al
(2013) suggests that phosphorylation is one of the regulatory mechanisms for Nrf2 (43).
Phosphorylation at Serine 40 residue disrupts the association between Keap 1 andNrf2, which
promotes the Nrf2 translocation into the nucleus. Once in the nucleus after the transcriptional
activity, phosphorylation at tyrosine-568 residue causes the exclusion and degradation of
Nrf2 (43).
Our results also showed double bands which could also be potentially due to phosphorylation
of Nrf2 as a result of oxidative stress. Longer duration and higher concentration of
proteasomal inhibition can lead to oxidative stress and results in higher molecular weight and

11. 10
stronger top band (47,48). Therefore, in order to see the whole picture of Nrf2 regulatory
mechanism and optimum Nrf2 activity, nuclear and cytoplasmic extracts must be analysed
separately for phosphorylation after MG132 treatment.
Dual Signal for Glyoxalase 1
Previous in vitro experiments demonstrated that fatty acids (EPA and DHA) can induce Nrf2
antioxidant defence mechanism (38). Lipid accumulation decreases cell viability and causes
an imbalance between anti-oxidant/oxidant mechanisms resulting in lipid peroxidation and a
build-up of free radicals (45). However, our results demonstrated no significant effect on cell
viability on one sample (n=1; 5 Technical replicates). Therefore, further replication is needed
to examine cell viability.
In addition to the effect of lipid accumulation on Nrf2, earlier in vitro research has confirmed
that fatty acid treatment of Hepg2 cells for 24 hrs increased the intracellular lipid
accumulation and down regulated glyoxalase I protein levels as compared to the controls
(36,37). In response to lipid loading of oleic acid, glyoxalase I was first hyperacetylated,
ubiquinated and degraded, leading to an increase in reactive methylglyoxal (36,37). MG132
was used to inhibit 26S proteasome and subsequently accumulation of glyoxalase I protein
levels. However, our findings suggest that MG132 disrupts the normal cell signalling
mechanism. It act as an accumulator of Nrf2, which could potentially lead to an increased
intracellular levels of this transcription factor, where it may act as signal for manufacturing of
glyoxalase I (See Fig. 7) (35).
Toxicity of MG132
MG132 is toxic to the cells after prolonged treatment at higher concentrations (34,46). The
toxicity is induced via several pathways. The inhibition of proteasome leads to an
accumulation of Nrf2 as well as other misfolded and malfunctioned proteins (47). This causes
an increase in oxidative stress and contribute towards the generation of free radicals (47,48).
In order to eliminate the stress, degradation pathways such as lysosome and autophagy are
activated as a survival mechanism. In addition to degradation, laboratory experiments have
shown an up-regulation of genes involved in oxidative stress defence and an increased
expression of neuro-protective heat shock proteins to reverse the effects of MG132 mediated
damage (49). However, if the damage is severe than programmed cell death (apoptosis) is
induced. Therefore, it would be useful to evaluate the cell viability in response to MG132
treatment, in future.

12. 11
In addition to the toxicity of Mg132, in vitro experiments have shown that Mg132 is very
likely to be metabolised by CYP3A enzyme (a member of cytochrome P450 Phase I
enzyme). These enzymes make MG132 ineffective after 18 hrs of treatment in primary
human hepatocytes (34). Thus, CYP3A inhibition could be considered for an effective
proteosomal inhibition by MG132. However, HepG2 cell line have low levels of Phase I
enzymes (50,51).
Strength, Limitations and Future Directions
HepG2 Cell line Model
HepG2 is a human carcinoma cell line, derived from the liver tissue of a 15 year old male
Caucasian (52). Owing to their well differentiated growth, these are frequently used in a
variety of preclinical settings such as hepatotoxicity, liver metabolism etc. (53).
Immunofluorescence analysis of Human Protein Atlas (a proteomic database) has detected
high levels of Nrf2 expression in HepG2 cell line, with an antibody score of >2500 units (54).
This makes it a suitable model for Nrf2 investigating studies.
In comparison with primary human hepatocytes, HepG2 has significantly lower expression
and activity levels of certain protein including Phase I enzymes, which are involved in drug
metabolism (50,51). Therefore, this makes it less appropriate for predicting human
metabolism studies. Primary human hepatocytes have higher genotypic sensitive against
promutagens, as compared to HepG2 cells (50). However, availability of human liver
material remains limited. It is also challenging to maintain human hepatocytes as these lose
their metabolic activity sooner (50). Research has identified HepG2 as a suitable model for
toxicological studies owing to higher expression of certain enzyme, organelles and especially
mitochondrial DNA (55,56) . However, results must be interpreted cautiously.
Methodological limitations of present research
Western blotting was employed to assess the protein expression. It is a widely used technique
in molecular biology to identify specific proteins. However, it is only a semi quantitative
method to assess the protein accumulation. Methods such as ELISA or mass
spectrophotometry methods could be used for absolute quantification.
Another limitation of this study was the sample size. Due to time constrains the results were
based upon only one sample of cells. In order to obtain a concrete conclusion the experiments
must be repeated with fewer variations (e.g. incubation duration.) and additional replications.

13. 12
Nrf2 is maintained at equilibrium inside the cell by constant synthesis and degradation cycle
(27). However, further research could analyse Nrf2 mRNA levels in response to proteasomal
inhibition to assess the Nrf2 induction at mRNA level. In addition, assessment of correlation
between activity and expression of Nrf2 protein could be examined. Similarly, expression and
activity level of glyoxalase I should be determined from the same sample. Furthermore,
investigation of expression and activity of glyoxalase I and Nrf2 in fatty acid treated cells
upon optimal proteasomal inhibition would be an appropriate approach to gain an overall
view.
Conclusion
In conclusion, we have demonstrated that the duration and concentration of proteasomal
inhibition leads to an accumulation of Nrf2. It has also appeared that Nrf2 may have been
phosphorylated in response to proteasomal inhibition. Nrf2 induces the expression of
glyoxalase I and a combination of lipid accumulation and MG132 treatment likely results in.
dual signal. However, these findings remain inconclusive as further replication is required for
concrete results. Therefore, experiments must be repeated in combination with other
suggested experiments including assessment of expression and activity levels of glyoxalase I
and Nrf2 in response to lipid accumulation and proteasomal inhibition.
Acknowledgments
With thanks to Moore laboratory research team for their guidance throughout the project.

14. 13
Appendix
Figure 1: Intracellular formation and removal of methylglyoxal. During normal
metabolism, methylglyoxal is metabolism by the glyoxalase system into lactate and provides
protection against oxidative damage. In case of increased glucose supply, increased
methylglyoxal contributes towards the production of advanced glycated end products (AGE)
(adapted from Kiefer, et al, 2013) (57)

15. 14
Figure 2: Schematic overview of Keap 1- Nrf2 pathway. Under homeostatic conditions,
Nrf2 is complexed with Keap 1 in the cytosol, which regulates its activity. Nrf2 is constantly
synthesised and degraded via ubiquitination-proteosomal pathway. Under stress, Nrf2
translocates into the nucleus, where it co-binds with the small Maf protein on anti-response
element (ARE) target gene, and mediates the anti-stress response gene expression.

16. 15
Figure 3: Glyoxalase I regulation in response to lipid loading. A: Intracellular lipid
accumulation in response to 24 hr incubation of HepG2 cell with oleic acid (OA) and
palmitic acid (PA). B: Immunoblotting and relative expression of Glyoxalase I in response to
oleic acid and palmitic acid treatment of HepG2 cells. C: Immunoprecipitant of oleic acid
treated cells. Cells were also treated with the proteasome inhibitor MG132; then
immunoblotted with rat Alpha-Glyoxalase I and mouse Alpha-Ubiquitin (36,37).

17. 16
Figure 4: Effect of 24hrs fatty acids treatment on HepG2 cell viability. Relative cell
viability measured by Alamar Blue assay in response to A: Oleic Acid. And B: Palmitic
Acid. (n=1; 5 Technical replicates).

18. 17
Figure 5: Anti-Nrf2 antibody Optimisation. HepG2 cells were either treated with10M of
MG132 or untreated (V). Extracted protein samples were run on 12%SDS gels and subjected
to immunoblot analysis using indicated commercial antibodies with different dilutions of
anti-nrf2 antibody from Santa Cruz biotechnology and Abcam.

19. 18
A
B
Figure 6: A and B: Dose and time-dependent effects of MG132 on accumulation of Nrf2
in HepG2 cells. HepG2 cells were treated with vehicle (DMSO, 0.1%) or MG132 (0, 2.5, 5,
10, 20 µM) for 0, 2, 4, 6, 8 hrs. (10 µg of protein/lane).C: Quantification of Nrf2 bands.
The intensity of bands was quantified using Image Studio Lit version 4.0 software. Results
were normalised with alpha Tubulin levels and expressed as relative ratio of Nrf2 protein
levels. Immunoblotting was performed with same sets of lysed protein samples on two
different days (n=1). D: Quantification of post translationally modified of Nrf2 bands
after proteasomal inhibitor (MG132) treatments 20 μM after 2hrs; 2.5, 5, 10, 20 μM after 4hrs
6hrs and 8hrs (n=1).

20. 19
Figure 7: Schematic overview of interaction between Nrf2 pathway and lipid loading.
Fatty acid and proteasome inhibitor (MG132) disrupts the Keap1 1-nrf2 pathway, causing the
accumulation of Nrf2 in response, which may than translocate into the nucleus, leading to an
increased expression of Glyoxalase I.

21. 20
References
(1) American Diabetes Association. Diagnosis and classification of diabetes mellitus.
Diabetes Care 2010 Jan;33 Suppl 1:S62-9.
(2) Basta G, Schmidt AM, De Caterina R. Advanced glycation end products and vascular
inflammation: implications for accelerated atherosclerosis in diabetes. Cardiovasc Res 2004
The Oxford University Press;63(4):582-592.
(3) Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced Glycation End Products:
Sparking the Development of Diabetic Vascular Injury. Circulation 2006 August
08;114(6):597-605.
(4) Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role
in inflammatory disease and progression to cancer. Biochem J 1996;313:17-29.
(5) Schmidt AM, Hori O, Brett J, Yan SD, Wautier JL, Stern D. Cellular receptors for
advanced glycation end products. Implications for induction of oxidant stress and cellular
dysfunction in the pathogenesis of vascular lesions. Arterioscler Thromb 1994
Oct;14(10):1521-1528.
(6) Schumacker PT. Reactive oxygen species in cancer cells: live by the sword, die by the
sword. Cancer cell 2006;10(3):175-176.
(7) Singh R, Barden A, Mori T, Beilin L. Advanced glycation end-products: a review.
Diabetologia 2001 February 2001;44(2):129-146.
(8) Wang X, Desai K, JES THORN CLAUSEN, Wu L. Increased methylglyoxal and
advanced glycation end products in kidney from spontaneously hypertensive rats. Kidney Int
2004 Dec 2004;66(6):2315-21.
(9) Šebeková K, Kupčová V, Schinzel R, Heidland A. Markedly elevated levels of plasma
advanced glycation end products in patients with liver cirrhosis – amelioration by liver
transplantation. J Hepatol 2002 1;36(1):66-71.

22. 21
(10) Kilhovd BK, Giardino I, Torjesen PA, Birkeland KI, Berg TJ, Thornalley PJ, et al.
Increased serum levels of the specific AGE-compound methylglyoxal-derived
hydroimidazolone in patients with type 2 diabetes. Metab Clin Exp 2003 2;52(2):163-167.
(11) Singh VP, Bali A, Singh N, Jaggi AS. Advanced glycation end products and diabetic
complications. The Korean Journal of Physiology & Pharmacology 2014;18(1):1-14.
(12) Cantero A, Portero-Otín M, Ayala V, Auge N, Sanson M, Elbaz M, et al. Methylglyoxal
induces advanced glycation end product (AGEs) formation and dysfunction of PDGF
receptor-ß: implications for diabetic atherosclerosis. The FASEB Journal 2007 October
01;21(12):3096-3106.
(13) Desai K, Chang T, Wang H, Banigesh A, Dhar A, Liu J, et al. Oxidative stress and
aging: Is methylglyoxal the hidden enemy? 2001;3(88):273-84.
(14) Rabbani N, Thornalley PJ. Glyoxalase in diabetes, obesity and related disorders.
2011;22(3):309-317.
(15) Miyata T, De Strihou, Charles Van Ypersele, Imasawa T, Yoshino A, Ueda Y, Ogura H,
et al. Glyoxalase I deficiency is associated with an unusual level of advanced glycation end
products in a hemodialysis patient. Kidney Int 2001;60(6):2351-2359.
(16) Kuhla B, Boeck K, Schmidt A, Ogunlade V, Arendt T, Münch G, et al. Age-and stage-
dependent glyoxalase I expression and its activity in normal and Alzheimer's disease brains.
Neurobiol Aging 2007;28(1):29-41.
(17) Kalousová M, Germanová A, Jáchymová M, Mestek O, Tesav̌ V, Zima T. A419C
(E111A) polymorphism of the glyoxalase I gene and vascular complications in chronic
hemodialysis patients. Ann N Y Acad Sci 2008;1126(1):268-271.
(18) Thornalley PJ. Advances in glyoxalase research. Glyoxalase expression in malignancy,
anti-proliferative effects of methylglyoxal, glyoxalase I inhibitor diesters and S-d-
lactoylglutathione, and methylglyoxal-modified protein binding and endocytosis by the
advanced glycation endproduct receptor. Crit Rev Oncol 1995 8;20(1–2):99-128.

23. 22
(19) Rabbani N, Thornalley PJ. The Critical Role of Methylglyoxal and Glyoxalase 1 in
Diabetic Nephropathy. Diabetes 2014 January 01;63(1):50-52.
(20) Xue M, Rabbani N, Momiji H, Imbasi P, Anwar MM, Kitteringham N, et al.
Transcriptional control of glyoxalase 1 by Nrf2 provides a stress-responsive defence against
dicarbonyl glycation. 2012;443(1):213-222.
(21) Thornalley PJ. Glyoxalase I--structure, function and a critical role in the enzymatic
defence against glycation. 2003;31:1343-8.
(22) Reddy SP. The Antioxidant Response Element and Oxidative Stress Modifiers in
Airway Diseases. 2008;8(5):376-383.
(23) Wang X, Tomso DJ, Chorley BN, Cho H, Cheung VG, Kleeberger SR, et al.
Identification of polymorphic antioxidant response elements in the human genome. Human
Molecular Genetics 2007 May 15;16(10):1188-1200.
(24) Jaiswal AK. Regulation of genes encoding NAD(P)H:quinone oxidoreductases. Free
Radical Biology and Medicine 2000 8;29(3–4):254-262.
(25) Hardwick RN, Fisher CD, Canet MJ, Lake AD, Cherrington NJ. Diversity in
Antioxidant Response Enzymes in Progressive Stages of Human Nonalcoholic Fatty Liver
Disease. Drug Metabolism and Disposition 2010 December 01;38(12):2293-2301.
(26) Lau A, Tian W, Whitman SA, Zhang DD. The predicted molecular weight of Nrf2: it is
what it is not. Antioxidants & redox signaling 2013;18(1):91-93.
(27) Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1?Nrf2
pathway in stress response and cancer evolution. Genes to Cells 2011;16(2):123-140.
(28) Chambel SS, Santos-Gonçalves A, Duarte TL. Incomplete reference<br />The Dual
Role of Nrf2 in Nonalcoholic Fatty Liver Disease: Regulation of Antioxidant Defenses and
Hepatic Lipid Metabolism. The Dual Role of Nrf2 in Nonalcoholic Fatty Liver Disease:
Regulation of Antioxidant Defenses and Hepatic Lipid Metabolism 2015.
(29) Voges D, Zwickl P, Baumeister W. The 26S proteasome: a molecular machine designed
for controlled proteolysis. Annu Rev Biochem 1999;68(1):1015-1068.

24. 23
(30) Heinemeyer W, Ramos P, Dohmen R. Ubiquitin-proteasome system. Cellular and
Molecular Life Sciences CMLS 2004;61(13):1562-1578.
(31) Naujokat C, Hoffmann S. Role and function of the 26S proteasome in proliferation and
apoptosis. Laboratory investigation 2002;82(8):965-980.
(32) Frankland-Searby S, Bhaumik SR. The 26S proteasome complex: an attractive target for
cancer therapy. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 2012;1825(1):64-
76.
(33) Crawford LJ, Walker B, Irvine AE. Proteasome inhibitors in cancer therapy. Journal of
cell communication and signaling 2011;5(2):101-110.
(34) Lee C, Kumar V, Riley RI, Morgan ET. Metabolism and Action of Proteasome
Inhibitors in Primary Human Hepatocytes. Drug Metabolism and Disposition 2010 December
01;38(12):2166-2172.
(35) Sahni SK, Rydkina E, Sahni A. The proteasome inhibitor MG132 induces nuclear
translocation of erythroid transcription factor Nrf2 and cyclooxygenase-2 expression in
human vascular endothelial cells. Thromb Res 2008;122(6):820-825.
(36) Hepatic Glyoxalase 1 is Downregulated and Hyperacetylated in Pediatric Non-alcoholic
Fatty Liver Disease. HEPATOLOGY: WILEY-BLACKWELL 111 RIVER ST, HOBOKEN
07030-5774, NJ USA; 2013.
(37) Moore JB, Weeks ME, Fisher CP, Fitzpatrick E, Dhawan A, Oviedo-Orta, E. & Spanos,
C. Proteomic identification of novel biomarkers of paediatric non-alcoholic fatty liver
disease. . 2013(56(S2): 66.).
(38) Gao L, Wang J, Sekhar KR, Yin H, Yared NF, Schneider SN, et al. Novel n-3 Fatty Acid
Oxidation Products Activate Nrf2 by Destabilizing the Association between Keap1 and
Cullin3. Journal of Biological Chemistry 2007 January 26;282(4):2529-2537.
(39) Guo N, Peng Z. MG132, a proteasome inhibitor, induces apoptosis in tumor cells. Asia‐
Pacific Journal of Clinical Oncology 2013;9(1):6-11.

25. 24
(40) Lee DH, Goldberg AL. Proteasome inhibitors: valuable new tools for cell biologists.
Trends Cell Biol 1998;8(10):397-403.
(41) Purdom-Dickinson SE, Sheveleva EV, Sun H, Chen QM. Translational control of nrf2
protein in activation of antioxidant response by oxidants. Mol Pharmacol 2007
Oct;72(4):1074-1081.
(42) Bloom DA, Jaiswal AK. Phosphorylation of Nrf2 at Ser40 by protein kinase C in
response to antioxidants leads to the release of Nrf2 from INrf2, but is not required for Nrf2
stabilization/accumulation in the nucleus and transcriptional activation of antioxidant
response element-mediated NAD(P)H:quinone oxidoreductase-1 gene expression. J Biol
Chem 2003 Nov 7;278(45):44675-44682.
(43) Bryan HK, Olayanju A, Goldring CE, Park BK. The Nrf2 cell defence pathway: Keap1-
dependent and-independent mechanisms of regulation. Biochem Pharmacol 2013;85(6):705-
717.
(44) Pi J, Bai Y, Reece JM, Williams J, Liu D, Freeman ML, et al. Molecular mechanism of
human Nrf2 activation and degradation: role of sequential phosphorylation by protein kinase
CK2. Free Radical Biology and Medicine 2007;42(12):1797-1806.
(45) Yao HR, Liu J, Plumeri D, Cao YB, He T, Lin L, et al. Lipotoxicity in HepG2 cells
triggered by free fatty acids. Am J Transl Res 2011 May 15;3(3):284-291.
(46) Zanotto-Filho A, Braganhol E, Battastini AMO, Moreira JCF. Proteasome inhibitor
MG132 induces selective apoptosis in glioblastoma cells through inhibition of PI3K/Akt and
NFkappaB pathways, mitochondrial dysfunction, and activation of p38-JNK1/2 signaling.
Invest New Drugs 2012;30(6):2252-2262.
(47) Han YH, Park WH. MG132 as a proteasome inhibitor induces cell growth inhibition and
cell death in A549 lung cancer cells via influencing reactive oxygen species and GSH level.
Hum Exp Toxicol 2010 Jul;29(7):607-614.
(48) Legesse-Miller A, Raitman I, Haley EM, Liao A, Sun LL, Wang DJ, et al. Quiescent
fibroblasts are protected from proteasome inhibition-mediated toxicity. Mol Biol Cell 2012
Sep;23(18):3566-3581.

26. 25
(49) Yew EHJ, Cheung NS, Choy MS, Qi RZ, Lee AY, Peng ZF, et al. Proteasome inhibition
by lactacystin in primary neuronal cells induces both potentially neuroprotective and pro‐
apoptotic transcriptional responses: a microarray analysis. J Neurochem 2005;94(4):943-956.
(50) Wilkening S, Stahl F, Bader A. Comparison of primary human hepatocytes and
hepatoma cell line Hepg2 with regard to their biotransformation properties. Drug Metab
Dispos 2003 Aug;31(8):1035-1042.
(51) Westerink WM, Schoonen WG. Cytochrome P450 enzyme levels in HepG2 cells and
cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicology in
vitro 2007;21(8):1581-1591.
(52) Public Health England. General Cell Collection: Hep G2. 2015; Available at:
https://www.phe-
culturecollections.org.uk/products/celllines/generalcell/detail.jsp?refId=85011430&collection
=ecacc_gc. Accessed 03/19, 2015.
(53) Knasmüller S, Mersch-Sundermann V, Kevekordes S, Darroudi F, Huber W, Hoelzl C,
et al. Use of human-derived liver cell lines for the detection of environmental and dietary
genotoxicants; current state of knowledge. Toxicology 2004;198(1):315-328.
(54) The Human Protein Atlas. NFE2L2. 2015; Available at:
http://www.proteinatlas.org/ENSG00000116044-NFE2L2/cell/HPA002990. Accessed 03/15,
2015.
(55) Pinti M, Troiano L, Nasi M, Ferraresi R, Dobrucki J, Cossarizza A. Hepatoma HepG2
cells as a model for in vitro studies on mitochondrial toxicity of antiviral drugs: which
correlation with the patient? J Biol Regul Homeost Agents 2003 Apr-Jun;17(2):166-171.
(56) Guo L, Dial S, Shi L, Branham W, Liu J, Fang JL, et al. Similarities and differences in
the expression of drug-metabolizing enzymes between human hepatic cell lines and primary
human hepatocytes. Drug Metab Dispos 2011 Mar;39(3):528-538.
(57) Kiefer AS, Fleming T, Eckert GJ, Poindexter BB, Nawroth PP, Yoder MC.
Methylglyoxal concentrations differ in standard and washed neonatal packed red blood cells.
Pediatr Res 2013;75(3):409-414.

27. 26