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

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.

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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

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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

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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

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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 37oC 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 oC 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

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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 4oC. Supernatant was collected and

stored at -80 oC. 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

37oC 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 oC 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

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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.

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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

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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

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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.

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.

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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.

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)

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.

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).

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).

17

Figure 5: Anti-Nrf2 antibody Optimisation. HepG2 cells were either treated with10M 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.

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).

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.

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.

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.

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.

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.

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.

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.

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