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.
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.
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.
Organisations
People |
ORCID iD |
| Joanna Poulton (Principal Investigator) | |
| Karl Morten (Co-Investigator) |