Pivotal role of the Keap1-Nrf2 pathway in the pathogenesis and prevention of non-alcoholic steatohepatitis induced cirrhosis

Lead Research Organisation: University of Dundee
Department Name: Cancer Research

Abstract

Non-alcoholic steatohepatitis (NASH) is a common disease that involves fat accumulation in liver cells and inflammation of the liver. Its incidence is increasing rapidly because it is prevalent in individuals with diabetes and those who are obese; it has been estimated that approximately 2.0% of the adult population in the UK may have NASH. This trend is alarming, and has huge cost implications for the National Health Service, because NASH can give rise to liver fibrosis, cirrhosis and cancer, none of which are treatable. It is thought that NASH arises in certain individuals because their livers do not cope with metabolic changes produced by diet, diabetes or the environment. In each of these cases, it is envisaged that oxidative stress represents a common consequence of the physiological and environmental insults that produce liver disease. The activity of a protein known as Nrf2 is emerging as the most important determinant of susceptibility to NASH for three reasons: (i) it keeps fat production and storage in the liver low; (ii) it helps maintain high levels of anti-inflammatory proteins and (iii) it is a vital component in maintaining high levels of natural anti-oxidant agents in the liver.

We hypothesize that Nrf2 normally functions to minimise the likelihood of NASH arising, and that by increasing its activity using drugs will protect 'at risk' individuals of developing NASH and the disease progressing further. To date, experiments that have implicated Nrf2 in determining susceptibility to NASH or prevention of the disease have used a specialized, non-standard diet (deficient in certain key molecules), which is not relevant to the human disease. Unfortunately, no standard animal model exists that recapitulates the entire spectrum of liver pathology (i.e., from steatosis, to NASH, to fibrosis, to cirrhosis and to hepatoma), which we can use to test the role of Nrf2 in this disease. In a pilot study, we have found that feeding a 'Western' high fat (HF) diet to mice for 24 weeks produced mild NASH, whereas feeding the same diet to mice totally deficient of Nrf2 for the same period produced full-blown NASH with fibrosis and cirrhosis. We now wish to confirm these preliminary findings by studying a larger number of animals on the HF diet, and by examining the time-course of the disease using biomarkers (i.e. monitoring levels of certain disease-associated molecules in the blood).

In order to prove whether Nrf2 is important in the development of NASH and its progression to fibrosis and cirrhosis, we first require at least one reliable animal model that reflects the human disease. To this end, we will generate two models, which we anticipate will develop liver disease at different rates: firstly, we will feed normal mice a 'Western' high fat (HF) diet; secondly, we will feed normal mice a 'Western' high fat + fructose (i.e. high levels of sugar; HFF) diet. A detailed time course of the development of fatty liver, NASH, fibrosis and cirrhosis in mice fed these two diets will be established using clinical chemistry, histology and biochemical measurements of pathological changes associated with development of the disease. Once the rate of progression of the disease has been established in the normal mice placed on the 'Western' HF and HFF diets, we will examine the contribution that Nrf2 makes to protection against NASH by testing whether removal of this protein results in an accelerated appearance of NASH, fibrosis and cirrhosis when these mice are fed the same diets. Lastly, we will examine whether activation of Nrf2, either by genetic means or by treatment with a certain type of drug, inhibits the appearance of NASH, fibrosis, or cirrhosis when the animals are fed the same 'Western' HF and HFF diets.

Technical Summary

Part 1. We will perform a longditudinal study of C57BL/6 Nrf2+/+ mice in which the development of disease will be monitored by measuring plasma markers of steatosis, insulin sensitivity, inflammation, oxidative stress and fibrosis. Upon sacrifice of the animals, we will perform various histological examinations (H&E, Van Glieson, reticulin, Oil Red O and TUNEL staining), and measure changes in expression of key hepatic genes associated with lipid metabolism, inflammation, redox status and fibrosis. Furthermore, in the first part of the project we will also test whether administration of a 'Western' high fat/high fructose (HFF) diet increases the rate at which NASH, fibrosis and cirrhosis develop in C57BL/6 Nrf2+/+ mice, and examine whether both the HF and HFF diets provide appropriate models for human liver disease. The rate at which NASH developes and progresses in wild-type mice fed the HFF diet will be evaluated using the same measurements employed for mice on the HF diet, and compared with the latter.

Part 2. We will use the same criteria set out above to test the prediction that loss of Nrf2 results in a rapid acceleration in the rate at which NASH, fibrosis and cirrhosis develop in C57BL/6 Nrf2-/- mice placed on the HF and HFF diets.

Part 3. We will test the hypothesis that either genetic or pharmacological upregulation of Nrf2 provides protection against NASH, fibrosis and cirrhosis in mice placed on the HF or HFF diets: genetic upregulation of Nrf2 will be studied in C57BL/6 Keap1-floxed (i.e., Keap1loxP/loxP) mice in which Keap1, an E3 ubiquitin ligase substrate adaptor protein that represses Nrf2, is substantially downregulated; pharmacological upregulation of Nrf2 will be achieved using the potent chemopreventive agent TBE31, which is a strong inhibitor of Keap1. As one control, we will generate Keap1-floxed::Nrf2-/- and place them on the HF and HFF diets. As a second control, we will treat Nrf2-/- mice placed on the HF and HFF diets with TBE31.

Planned Impact

This project seeks to determine whether NF-E2-related factor 2 (Nrf2) normally functions to decrease the likelihood of NASH, fibrosis and cirrhosis arising through exposure to a 'Western' diet, namely one that is high in fat and in sugar. Nrf2 is a transcription factor that is critical to the processes through which cells in the body respond to stress and inflammation. The studies described in this application will demonstrate whether in the absence of Nrf2 transgenic mice show accelerated NASH and cirrhosis, and conversely whether NASH and cirrhosis can be prevented through genetic or pharmacological activation of Nrf2.

It is estimated that 20% of the adult UK population have fatty liver as a consequence of the marked increase in obesity and type II diabetes mellitus. Of these, at least 10% (i.e., 2% of the Nation's adult population) are thought to have NASH, a significant percentage of whom will progress on to develop liver fibrosis and cirrhosis and ultimately end-stage liver disease. More alarmingly, the rising prevalence of obesity and type 2 diabetes in childhood in the UK means that NASH is increasingly seen in young adults. This will substantially shorten life quality, life expectancy, and the contribution that affected individuals make to society. At present, there is no effective treatment for fibrosis and cirrhosis other than liver transplantation, and there are clearly only a finite number of suitable donor livers for this purpose. The potential cost implications to the NHS of a rapidly rising incidence of NASH is truly grave.

In this context, it is clear that alternative strategies to prevent NASH are urgently required and would be of great benefit to society. The preliminary studies performed in our laboratory provide strong support for a role for Nrf2 in the prevention of NASH in an animal model of obesity. The current project aims to build on this exciting data and will directly test the hypothesis that Nrf2 markedly influences susceptibility to NASH, and also that up-regulation of Nrf2 inhibits the onset of NASH and its progression to fibrosis and cirrhosis. Most excitingly, a variety of xenobiotics, including the drug dimethyl fumarate (BG-12) and the food preservative butylated hydroxyanisole (BHA, E320), are known to up-regulate Nrf2. Assuming our hypothesis is correct, a clear opportunity then exists to intervene pharmacologically to activate Nrf2 and stimulate expression of its target genes that collectively inhibit hepatic lipid accumulation, inflammation, oxidative stress and apoptosis.

Within our research collaboration grouping, John Dillon and Rory McCrimmon are best placed to translate the findings from our mouse experiments to the clinic because they run Hepatology and diabetes outpatient clinics in Tayside, and through the Dundee Clinical Research Centre, have access to a new state-of-the-art facility for conducting human trails. We envisage that our initial clinical studies will focus on dimethyl fumarate, which is already being used in phase III trials to treat multiple sclerosis, or the food preservative BHA. This means a drug is, almost immediately, available for use in a human clinical trial to prove the therapeutic concept of up-regulation of Nrf2, leading to rapid translation of our experimental findings into human disease.

In summary, the work described herein may result in outcomes that:
(i) Offer novel pharmacological-based treatments for NASH, which can be fast-tracked into small clinical trials, with the strong possibility that better drugs may be rapidly developed in association with Pharmaceutical companies.
(ii) Enhance the quality of life (and may extend life) for a substantial proportion of the adult (young and old) population.
(iii) Improve the effectiveness of NHS care and treatment and reduce NHS costs associated with liver disease treatment and management.
 
Title Antibodies relevant to the Keap1-Nrf2-ARE pathway 
Description We have immunopurified antibodies against transcription factor Nrf2 that are very specific [relative to commercially available ones]. These have to be regenerated from antiserum stocks on a regular basis. We also have nice antibodies against a number of important Nrf2-target genes. 
Type Of Material Antibody 
Provided To Others? Yes  
Impact I would estimate that more than 20 publications have utilized our reagents. 
 
Title Transgenic mouse lines 
Description We have Nrf2-/-, Keap1-floxed and Nrf1-floxed mouse lines. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2006 
Provided To Others? Yes  
Impact At least 50 publications have arisen from the transgenic mice mentioned above. 
 
Description Biochemical Society Focused Meeting- The Keap1/Nrf2 signalling pathway in health and disease 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Along with Dr Maria O'Connell (University of East Anglia), I organised a "Focused Meeting" that was held through the Biochemical Society of Great Britain that was held in Robinson College Cambridge on 6-8th January. We had around 120 participants from around the world (Europe, Japan and USA), that varied from graduate students through to senior professors. The presentations by invited speakers were published as an edition of the Biochemical Society Transactions. We also had media coverage.
Year(s) Of Engagement Activity 2015
 
Description British Toxicology Society Annual Congress 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I was invited to participate at the Annual congress of the British Toxicology Society that was held in the Hilton Newcastle Gateshead between 16th and 18th April 2018, at which I gave a talk on "Nrf2 and the antioxidant response".
Year(s) Of Engagement Activity 2018
 
Description British Toxicology Society, Autumn Meeting 2010 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact to develop collaborations and encourage student interest

lively open discussions
Year(s) Of Engagement Activity 2010
 
Description Colloquium called NRF2 and Cancer held in The Gateway, University of St Andrews, Scotland, UK on 27th February to 2nd March 2018 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I gave a 45-minute introductory lecture entitled 'Historical background to the Nrf2 in cancer field' and later an additional 30-minute talk entitled 'Reversal of insulin resistance and non-alcoholic steatohepatitis by pharmacological activation of Nrf2'. The colloquium was a closed meeting about the role of Nrf2 in cancer that was held to discuss recent unpublished results and promote collaboration between the leading international researchers in the Nrf2 field. The meeting also included approximately ten postgraduate students and postdoctoral assistants from the University of St Andrews and the University of Dundee who are currently engaged on Nrf2-related projects. The participants were from around the world and included: Dr Tom Kensler (USA), Dr Hozumi Motohashi (Japan), Dr Gina DeNicola (USA), Dr Donna Zhang (USA), Dr Antonio Cuadrado (Spain), Dr Christine Chio (USA), Dr Karen Liby (USA), Dr Albena Dinkova-Kostova (UK), Dr Masayuki Yamamoto (Japan), Dr Martin Bergo (Spain), Dr Michael Sporn (USA), Dr Geoff Wells (UK), and Dr David Harrison (UK).
Year(s) Of Engagement Activity 2018
 
Description International Society for the Study of Xenobiotics, 13th European meeting, 2015 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Along with Professor C. Roland Wolf and Professor Anne Daley I helped organise the 13th European International Society for the Study of Xenobiotics (ISSX) 2015 meeting that was held at the University of Strathclyde, Glasgow, on 22nd-25th June 2015. This international meeting was concerned primarily with the study of drugs and their metabolism and disposition as applied to the pharmaceutical industry. It was attended by graduate students and international experts in the area, totaling around 350. My specific responsibility was to organise and chair a session entitled 'Advances in pathways of chemically-induced oxidative stress and DNA damage'. After the meeting I was approached by AstraZeneca for information about Nrf2 and oxidative stress. The meeting helped foster collaboration between my lab and several others including that of Roberto Sitia (Italy) and Ken Tew (USA).
Year(s) Of Engagement Activity 2015
 
Description International Society for the study of Xenobiotics, 11th conference, held in Busan, South Korea on 13th-16th June 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I gave a 40 minute talk entitled "Regulation of Nrf2 by redox and other cellular signalling pathways, and its influence on xenobiotic and lipid metabolism" to the ISSX meeting
Year(s) Of Engagement Activity 2016
 
Description Keynote lecture to Scottish Biomedical Postdoctoral Researcher Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I gave the Scientific keynote address at the 4th Scottish Biomedical Postdoctoral Researcher Conference on the 1st June 2018, which was entitled "Multifaceted roles of the transcription factor Nrf2 in controlling insulin resistance-associated comorbid disease" that was attended by postdoctoral researchers from the Scottish universities including the Universities of Edinburgh, Glasgow, St Andrews, Aberdeen and Strathclyde.
Year(s) Of Engagement Activity 2018
 
Description Keynote talk to the University of South Carolina (at Columbia) Colon Cancer Center Annual Retreat, held 5th January, 2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact I gave the keynote lecture entitled "Molecular regulation of transcription factor Nrf2 and its therapeutic manipulation to treat lung cancer and liver disease" to the Cancer Center
Year(s) Of Engagement Activity 2017
 
Description Seminar given to the Divisions of Pharmacology and Cancer at the University of South Carolina at Charleston, USA, on 9th January 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact I gave a 50-minute lunchtime seminar to the Division of Pharmacology that was entitled "Molecular regulation of transcription factor Nrf2 and its therapeutic manipulation to treat lung cancer and liver disease"
Year(s) Of Engagement Activity 2017
 
Description The Environmental Response IV 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Environmental Response IV meeting was held in Tohoku University, Sendai, Japan on 28th Februray to 2nd March 2014. It was attended by the major researchers in the Nrf2 field from across the world. Along with Thomas Kensler (University of Pittsburgh, USA), Steven Kleeberger (NIEHS, USA) and Donna Zhang (University of Arizona, USA), I was on the organising committee. The meeting included presentations about the basic biology of Nrf2 and also its translational significance for human health. It was well attended.

to encourage collaborations, discussions and students to visit the lab
Year(s) Of Engagement Activity 2014
 
Description Transcription factor NRF2: new opportunities for pharmaceutical innovations in chronic diseases 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I attended a conference entitled "Transcription factor NRF2: new opportunities for pharmaceutical innovations in chronic diseases" that was held at the Universidad Autonoma, Madrid between 11th and 13th April 2018, at which I gave a talk on Nrf2 and liver disease. The purpose of the meeting was to bring together academic researchers and researchers in the pharmaceutical and biotech industries involved in developing new agents that activate NRF2 with a view of accelerating clinical trials.
Year(s) Of Engagement Activity 2018
 
Description Workshop called Better Cancer Therapy from Redox Biology, held in The Banbury Center, Cold Spring Harbor Laboratory, NY, USA, on 10th-13th April 2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I gave a 25-minute talk at the workshop entitled "The mechanisms of repression of transcription factor Nrf2 and its crosstalk with lipid metabolism". This was a closed meeting and its intention was to provide a better understanding of the roles of antioxidants and reactive oxygen species in tumourigenesis and how researchers/clinicians might exploit them to improve therapy for cancer patients. The participants were international and included: Dr Jim Watson, Dr Dave Tuveson, Dr Arne Holmgren, Dr Christine Winterbourn, Dr Jakob Ursula, Dr Nick Tonks, Dr Navdeep Chandel, Dr Paul Schumacker, Dr Karen Vousden, Dr Tak Mak, Dr Edward Schmidt, Dr Martin Bergo, Dr Nissim Hay, Dr Michael Espey, Dr Christine Chio, Dr Masayuki Yamamoto, Dr Thales Papagiannokopolus, Dr Gina DeNicola, Dr Donna Zhang, Dr Tobias Dick, Dr Vsevolo Belousov, Dr Kevin Brindle, Dr Christopher Chang, Dr Yi Yang, Dr Michael Murphy, Dr Tom Miller, Dr David Boothman, Dr Garry Buettner, Dr Douglas Spitz, Dr Sean Morrison and Dr Elizabeth Parkinson.
Year(s) Of Engagement Activity 2017