The importance of mitochondrial oxidative stress on beta cells in type II diabetes

Lead Research Organisation: University of Oxford
Department Name: Obstetrics and Gynaecology

Abstract

We aim to study natural mechanisms that protect people from diabetes in the beta cells that make insulin. This will help design treatments that may prevent people from developing diabetes.
Mitochondria are small parts of cells that generate energy. They also generate damaging by-products called free radicals, that are inactivated by an enzyme called superoxide dismutase (SOD). Mitochondria have their own DNA, mtDNA, a blueprint for proteins needed to make energy. Variations in mtDNA make diabetes more likely by reducing the energy supplied to the beta cells. We will use molecular techniques to lower SOD levels in cultured beta cells and determine whether this increases levels of the damaging free radicals. We will determine whether these changes decrease insulin production, as this would make diabetes likely.
Once beta cells are damaged they cannot readily be regenerated and this causes insulin deficiency. Our results may suggest novel treatments with drugs that counteract free radicals to prevent failure of the beta cells. Finding a treatment to prevent this damage would change how we treat patients whose mtDNA predisposes them to diabetes. This includes 140,000 diabetics in the UK and ~95% of people in Polynesia, where diabetes is very common.

Technical Summary

Beta-cell dysfunction is an important component of type 2 diabetes (T2D) that is increasingly implicated early in pathogenesis. Age associated mitochondrial oxidative stress has been implicated in the development of a number of condtions including T2D.

Our research objective is to investigate the effect of mitochondrial oxidative stress on cellular pathways associated with insulin secretion (B-cells). We will investigate the effects of mitochondrial reactive oxygen species (ROS), which may be increased as the result of respiratory chain dysfunction and/or impaired antioxidant defence systems, on mitochondrial function and insulin secretion in beta cells. We will test the hypothesis that mitochondrial ROS damage mitochondrial protein and mtDNA in B-cells and hence contribute to the pathogenesis of type II diabetes.

We have recently shown that 1) mtDNA mutations may be associated with increased ROS production and respiratory chain dysfunction and 2) high ROS levels in the mitochondrial superoxide dismutase (SOD2) knockout mouse are associated with major increases in the phosphorylation state of proteins associated with disease pathology (Morten unpublished). Hence, oxidative stress may impair both mitochondrial function and cell signaling in the B-cell, with a major impact on both insulin release.

In this proposal we will:-
1)Increase levels of ROS in immortalised mouse beta cells by targeting SOD2 with RNA interference (shRNA technology) to decrease cellular antioxidant defences. We will confirm specificity at the level of RNA and protein expression, and use ROS sensitive dyes to demonstrate increased ROS production .
2)Investigate the relationship between mitochondrial ROS and mtDNA damage in cultured mouse beta-cells and ES cells with mildly reduced Sod2 expression.
3)Use real time PCR, and fluorescence imaging to assess the effects of ROS on mtDNA copy number.
4)Determine the effect of elevated beta-cell ROS on mitochondrial function and mitochondrial protein turnover using a combination of sensitive assays which KM has developed for ROS and respiratory complexes enzymology, 2 D protein turnover analysis and sucrose gradient fractionation.
5)Determine the effect of mitochondrial ROS on B-cell insulin secretion and assess interactions with fatty acid loading. Total and secreted insulin will be measured by radioimmunoassay. ATP and calcium levels will be measured by fluroimetry and fluroscent techniques.

These studies will provide the groundwork to generate a mouse model of mitochondrial oxidative stress with which to investigate type II diabetes. Understanding the effect of ROS on insulin secretion will suggest novel therapies for preventing beta cell failure, such as antioxidant therapy, which can be tested in this model.

Publications

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Ashley N (2009) Mitochondrial DNA is a direct target of anti-cancer anthracycline drugs. in Biochemical and biophysical research communications

 
Description Cabinet office review
Geographic Reach National 
Policy Influence Type Participation in a national consultation
Impact Review of confidentiality of Records (K Lidbetter, Grade 6 Cabinet office)
 
Description HFEA Review of the effectiveness and safety of methods to avoid mitochondria disease
Geographic Reach National 
Policy Influence Type Citation in other policy documents
Impact Invited to give evidence at Human Fertilisation and Embryology Authority (HFEA) review, which attracted international speakers (US and Netherlands). I was cited in the policy document arising from this.
URL http://www.hfea.gov.uk/docs/2011-04-18_Mitochondria_review_-_final_report.PDF
 
Description I am a member of the Royal Society stem cell consultation group
Geographic Reach Europe 
Policy Influence Type Participation in advisory committee
Impact This consultation group submits evidence to governmental committees.; Royal Society report 2008
 
Description Invited to give 2 talks to HFEA committees
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact I suggested that HFEA should allow the Newcastle Mitochondrial research group working on nuclear transfer as a treatment for mitochondrial diseases should now be permitted to perform experiments on healthy single cell embryos rather than just abnormal ones.
 
Description Submissions to the HFEA open consultation on new techniques to prevent transmission of serious mitochondrial disease
Geographic Reach National 
Policy Influence Type Citation in other policy documents
Impact Our collaborative paper (with Drs Joerg Burgstaller and Iain Johnston) on mitochondrial DNA segregation suggests that an additional restriction on the practice of mitochondrial donation for preventing mtDNA disease. We made a presentation to the review, suggesting that there should be an acceptably close mitochondrial genetic distance between donors of permitted embryos and of permitted eggs. When two mtDNA haplotypes are present in a cell (mtDNA heteroplasmy), it is usually assumed that segregation (the proliferation of one haplotype over another) is negligible and best explained by random drift. An intensively studied mouse model showing tissue-specific mtDNA segregation is currently seen as an exception to this assumption. However, existing studies of mtDNA dynamics have focused on laboratory mouse strains with very limited genetic diversity, thus ignoring the fact that genetic differences between haplotypes may lead to proliferative differences. We have data showing that in the mouse, segregation between two mtDNA haplotypes is the rule rather than the exception and strongly depends on the genetic distance. Segregation is much less when mitochondrial haplotypes are closely related than when they are diverse. The dataset on which this is based is substantially larger than in any previous such studies. Hence, the mtDNA of the permitted embryo might increase to disease-causing levels in later generations. We cannot exclude the possibility that there might be segregation to significant levels during development in foetuses developing from embryos generated by nuclear transfer. The risk would be substantially reduced by matching the mtDNA haplotype of donors. These data were quoted in the HFEA report on the consultation.
URL http://www.hfea.gov.uk/docs/Third_Mitochondrial_replacement_scientific_review.pdf
 
Description Academy of Finland (JP and KM are collaborating with a paeditric neurologist (JU who is the PI)/Finish Paediatric association
Amount £120,000 (GBP)
Organisation Finnish Medical Association 
Sector Learned Society
Country Finland
Start  
 
Description BRC Clinical Fellowship
Amount £60,000 (GBP)
Organisation Oxford University Hospitals NHS Foundation Trust 
Department NIHR Oxford Biomedical Research Centre
Sector Public
Country United Kingdom
Start 10/2010 
End 09/2011
 
Description John Fell Small Award scheme
Amount £7,000 (GBP)
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start  
 
Description Medical Research Fund University of Oxford
Amount £10,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start  
 
Description Project grant
Amount £350,000 (GBP)
Funding ID MR/J010448/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 12/2014
 
Description Wellcome Trust project grant
Amount £251,826 (GBP)
Funding ID 0948685/Z/10/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2012 
End 03/2015
 
Description Williams Fund (Nanotherapeutics and cancer)
Amount £90,000 (GBP)
Organisation Oxford University Hospitals NHS Foundation Trust 
Department Williams Fund
Sector Hospitals
Country United Kingdom
Start 09/2010 
End 09/2013
 
Description newlife
Amount £15,000 (GBP)
Organisation Newlife 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2012
 
Title Collection of DNA from 30 human islets 
Description In collaboration with Dr Paul Johnson & Dr Neil Waker at OCDEM we have been collecting human islets from individuals donating their islets for research. This mainly consists of control individuals but also includes several diabetic samples 
Type Of Material Biological samples 
Year Produced 2008 
Provided To Others? Yes  
Impact We have shown a link between reduced levels of mtDNA and high BMI. 
 
Title Developing ImageStream to quantify mitophagy 
Description Developing ImageStream (fluorescence mitoscopy combined with cytometry in a single platfrom) to quantify mitophagy 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Successful grant application (Wellcome) and oral presentation at Biochemical Society meeting 
 
Title Human liver stem cell line 
Description Human liver stem cell line obtained from Reneuron. Transformed with a tamoxifen controlled C-myc allows large quantiies of the cells to be produced before they are differentiated. 
Type Of Material Cell line 
Year Produced 2012 
Provided To Others? No  
Impact This line will hopefully provide us with a better system for looking at hepatocyte ischaemia reperfusion injury 
 
Title Mouse model of mild mito dysfunction & nutient deprivation 
Description In our mouse model of mild mitochondrial dysfunction and nutritional deprivation, we found that early exposure to the anti-AIDS drug AZT interacts with low protein diet to impair foetal development. This combination appeared to impair the supply of insulin and hence glucose homeostasis in the mouse, perhaps as a result of impaired mitochondrial function 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact Maternal nutritional deprivation combined with AIDS therapy may influence both birth weight and onset of diabetes by effects on mitochondria. This has potential implications for management not only of type 2 diabetes but also for the AIDS pandemic 
 
Title Picogreen staining of mtDNA 
Description We developed a new method for studying the organisation of mtDNA in living cells using florescence microscopy. We can use this method diagnostically as a rapid screening method of patient-derived cultured cells. This is particularly useful posthumously, when a fibroblast cell line may be the only material available. Further work provided insights into the mode of action of and resistance to a major class of anti-cancer drugs (anthracylcines such as doxorubicin and daunorubicin). This may be very important for clinical oncology 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact We have shown that a major class of anti-cancer drugs (anthracylcines such as doxorubicin and daunorubicin) can induce gross re-organisation of mtDNA packaging into nucleoids. These changes may contribute to the long term mtDNA damage and toxicity associated with the clinical use of these drugs 
 
Title Purchase of oxygen control plate reader from BMG labtech 
Description The reader allows us to set up experiments where we change oxygen conditions i.e modelling ischaemia reperfsuion injury. The experiements are run in real time and we are developing assays to monitor mitochondrial function and reactive oxygen species generation through out the experiment. 
Type Of Material Improvements to research infrastructure 
Year Produced 2009 
Provided To Others? Yes  
Impact We can measure oxygen consumption in a 96 plate format. Our current work looks at oxygen consumption from whole islets, INS-1 (insulinoma) cells and a whole range of cell lines. By scalling down the process further we hope to look at mitochondria isolated from small part areas of the brain 
 
Title RedMIT mefs 
Description Cell line with DS red targeted to mitochondria for studying mitophagy 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact Biochemical society meeting oral presentation 
 
Title RedMIT/GFP-LC3 mouse 
Description Mice with fluorescent organelles for investigating mitophagy 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact Liver stem cells with fluorescent organelles 
 
Description Collaboration with Dr K Simon 
Organisation University of Oxford
Department Nuffield Department of Clinical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Research activity
Collaborator Contribution Access to ImageStream technology and Expertise Wellcome Trust Funding
Impact Submitted publications Abstracts accepted for Biochemical Society meeting, Mitochondrial Dynamics meeting, EUROMIT
Start Year 2009
 
Description Collaboration with Kings College London 
Organisation King's College London
Department School of Medicine KCL
Country United Kingdom 
Sector Academic/University 
PI Contribution Our current work focusses on mitochondrial turnover which will inform the mechanism of the associations Dr Toby Andrew is studying.
Collaborator Contribution We plan to discuss the analysis of Toby's data in respect of OPA1 and PARP and write a collaborative paper
Impact We plan to write a collaborative paper
Start Year 2009
 
Description Collaboration with MRC Epidemiology Unit 
Organisation University of Cambridge
Department MRC Epidemiology Unit
Country United Kingdom 
Sector Multiple 
PI Contribution I conceived this project and made the initial findings. Dr Holt then mapped a novel origin of mtDNA replication precisely to the location of the variant. I helped Dr Sims set up the screening technique by providing the positive controls of the sequence variants and advising on the genetic diversity and its interpretation.
Collaborator Contribution We collaborated with a data review and meta-analysis confirming my finding that a common mtDNA variant is a risk factor for type 2 diabetes. We are now screening a large case-control study to determine whether we can replicate our finding that a mtDNA variant that I characterised is significantly enriched in diabetics compared to matched controls. If confirmed my finding will link 2 large MRC groups located on the Addenbrookes campus in investigating type 2 diabetes. This is because the variant that I identified maps precisely to a novel origin of replication (see collaboration with Dr Ian Holt, Mitochondrial Biology Unit).
Impact We have had the meta-analysis paper in draft for 2 years, and are now carrying out the laboratory side of the study (large case-control study of type 2 diabetics).
 
Description Collaboration with Mitochondrial Biology Unit 
Organisation Medical Research Council (MRC)
Department MRC Mitochondrial Biology Unit
Country United Kingdom 
Sector Public 
PI Contribution I provide him with ideas and patient materials.
Collaborator Contribution Ian has provided reagents advice and expertise for many years
Impact We co-chaired an international workshop (163rd ENMC International Workshop: Nucleoid and nucleotide biology in syndromes of mitochondrial DNA depletion myopathy. 12-14 December 2008, Naarden, The Netherlands) resulting in 2 publications. Cross-ferlisation of ideas result in a PLOS-genetics paper in 2010 and a Nucleic Acids Research paper in 2009.
 
Description Investigation of patients with mitochondrial disease 
Organisation University of Oulu
Department Department of Paediatric Neurology
Country Finland 
Sector Academic/University 
PI Contribution JP collected the original cohort of patients, conceived the first proposal which was funded by The Sigrid Juselius Foundation, Finland (Co-applicant on Senior Research Fellowship for Dr J Uusimaa) 50,000 Eur and The Paediatric Foundation, Finlandand 20,000 Eur. JP and KM both co-authored the current application (Academy of Finland) on which both are collaborators.
Collaborator Contribution JP flew to Oulu for collaborative meeting which enabled discussions with several other groups
Impact Paper submitted to Epilepsia Prospective study of POLG1 mutations presenting in children with intractable epilepsy-prevalence and clinical features J Uusimaa, V Gowda, A McShane, C Smith, J Evans, A Shrier, M Narasimhan, A O'Rourke, Y Rajabally, F Cowan, C Fratter, J Poulton Paper in draft for submission to J Med Genet Benign Cytochrome Oxidase Deficiency or Reversible Infantile Respiratory Chain Deficiency: Evidence for genetic heterogeneity Johanna Uusimaa, Heinz Jungbluth, G Crisponi, Lucy Feng, Massimo Zeviani, Imelda Hughes, Eileen Treacy, Jacqueline Birks, Caroline Sewry, Francesco Muntoni, and Joanna Poulton
Start Year 2008
 
Description Nanomedicine collaboration with the Engineering and Materials Sciences 
Organisation University of Oxford
Department Department of Engineering Science
Country United Kingdom 
Sector Academic/University 
PI Contribution Karl Morten has set up and established a series of collaborations with the Engineering(Drs Alex Lubansky, Helen Townley & Professor Richard Darton) and Material Sciences (Professor Peter Dobson)
Collaborator Contribution Allowed us to establish a multidisciplinary collaboration
Impact This project has recieved funding from several sources: 1)SEEDA funded research grant to fund a SEEDA Professorial fellow to work with Dr Karl Morten and Prof's Phil Legrani and Pete Dobson on the microfluidic separation of biological Nanoparticles (£150k). 2)University of Oxford John Fell Fund, Pump priming grant. Microfluidic Separation of Biological Nanoparticles. Dr Karl Morten with Professor Ligrani (£90k). 3)University of Oxford Seed fund. Pump priming grant. Microfluidic Separation of Biological Nanoparticles. Dr Karl Morten with Prof Ligrani (£35k). 4)Camila Samuel Fund (Sir Ronald Grierson). Start up grant 6 months. Mitochondrial therapeutics, and specific vascular delivery using nanotechnology for novel cancer treatments. Dr Karl Morten with Miss E Rapa (£27k). 5) Investigation of aptamer drug delivery strategies for Neuroblastoma. Funded by Dr Chris Mitchells charitable funds Oct 2009- Sept 2010. 6) Separation of aptamer coupled Nanoparticles. Msc research project (Miss Alicja Bulsiewicz & Dr Alex Lubansky/ Professor Richard Darton, Dept of Engineering Science Nov 2009- Oct-2011).
Start Year 2007
 
Description Professor Adrian Harris 
Organisation Cancer Research UK
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution KJM does metabolic analysis
Collaborator Contribution New project ongoing
Impact Multidisciplinary (findings will be applied to clinical samples) Collaborative grant application KJM
Start Year 2010
 
Description Invited speaker, Murdoch Institute of Medical Research, Melbourne Australia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited speaker, Murdoch Institute of Medical Research, Melbourne Australia
Year(s) Of Engagement Activity 2017
 
Description BPNA 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary talk

Increased patient referrals
Year(s) Of Engagement Activity 2007,2012
 
Description CGS 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact JP Invited 30 minute talk

Increased (i) queries regarding patients and advances (including the diagnostic approach we set up as a result of findings in my MRC CEG) and (ii) referrals to our NCG funded diagnostic service
Year(s) Of Engagement Activity 2008,2011
 
Description CLIMB Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact JP invited to give an illustrated talk to families of mitochondrial patients for 40 minutes

Personal feedback from grateful families and commendation by chief exec of CLIMB as well as chairperson. Report to appear in organisation literature.
Year(s) Of Engagement Activity 2008
 
Description ENMC 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Chairing International workshop, co-chaired with IJ Holt (MRC Cambridge). Wrote meeting reports (professional and lay) that appear on web, 2 publications arose

My report of the meeting has appeared in Neuromuscular Disorders and a collaborative paper that was generated in BBA
Year(s) Of Engagement Activity 2008
 
Description Gordon conference 
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 2 poster presentations

Collaborations
Year(s) Of Engagement Activity 2006
 
Description Nottingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited seminar speaker, Paediatric dept, Nottingham

Collaboration
Year(s) Of Engagement Activity 2009
 
Description Seminar on the role of Mitochondrial ROS in disease to Oxford Hypoxia group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Seminar to the Oxford goups has resulted in a series of discussions about future collaborations in the areas of Heart failure and novel treatments for Cancer.

Collaborations
Year(s) Of Engagement Activity 2008
 
Description Seminar to the Oxford Islet transplantation unit on mitochondrial function and beta cell failure 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Seminar/discussion led to a series of collaborations with Mr Paul Johnson and Prof Patrik Rorsmann. This includes (i) new approaches to isolate islets for transplantation (collaboration with Engineering), (ii) investigating the role of the mtDNA 16189 diabetes susceptibility gene on islet function. This is a collaboration with a Swedish Group ( Dr Erik Renstrom), we have recently obtained ehtics to allow this collaboration to start

We are currently looking for funding for the islet separation work.
Year(s) Of Engagement Activity 2008
 
Description St Marys 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Talk in symposium at St Marys

Increased (i) queries regarding patients and advances (including the diagnostic approach we set up as a result of findings in my MRC CEG) and (ii) referrals to our NCG funded diagnostic service (iii)Collaboration discussions
Year(s) Of Engagement Activity 2008
 
Description Sydney 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited to give 60 min seminar to clinical scientists

Collaborative grant application to Wellcome Trust
Year(s) Of Engagement Activity 2008