Role of Rab3 in peripheral tissue insulin resistance

Diabetes is a chronic metabolic disease affecting 415 million people worldwide and 3.3 million people in the UK. Ninety percent of the people suffering from diabetes have type 2 diabetes. Type 2 diabetes is characterised by the inability of muscle and fat tissues to respond to physiological levels of insulin (peripheral tissue insulin resistance), and to restore the normal levels of sugars in the bloodstream. The development of peripheral tissue insulin resistance and type 2 diabetes is strongly linked to lifestyle and to obesity, although the underlying mechanisms are incompletely resolved.
In healthy individual when insulin combines with its receptor on target tissues (muscle and fat tissues) this initiates a cascade of linked reactions that ultimately result in the fusion of membrane vesicles containing the glucose transporter protein (called GLUT4) with the surface membrane of cells. This latter process increases the availability of glucose transporter molecules and thereby increases glucose transport into the cell.
When fat cells are under stress, as for example when they need to store large quantities of nutrients in overweight people, they secrete small molecules, called cytokines, which can trigger inflammatory responses in the surrounding tissues. Such a cytokine is TNFa. Secretion of TNFa from fat cells has been linked to the development of a low-grade chronic inflammation in overweight subjects and to the development of insulin resistance. TNFa also has a direct effect on the fat cells themselves. It induces a cascade of events within cells, which alters the ability of the cells to respond to insulin and to increase the numbers of glucose transporters GLUT4 at the cell surface. In our recently published study, we reported our discovery that a small protein called Rab3 is important for the targeting of GLUT4 to the surface membrane of cells. More recently, we also discovered that in adipose cells TNFa treatment induces a very marked decrease in the number of Rab3 proteins in the cells.
Therefore, in the current proposal, we aim to investigate the role played by Rab3 in the development of peripheral tissue insulin resistance. To achieve this aim we will use cellular models of adipose cells to investigate the molecular interactions between Rab3 and other proteins in the cell that act as molecular links between insulin action at its receptor and the GLUT4 transporter. We will investigate how these interactions are affected by treatment with TNFa or other molecules known to induce the state of insulin resistance. We will make use of unique tools that were recently developed in our laboratory to monitor the activity of Rab3 and the movement of GLUT4 to the cell surface. These experiments will allow us to understand the mechanisms of action of Rab3 in the context of the development of the insulin-resistant state. We will also investigate in humans, undergoing a diet and exercise intervention programme, whether Rab3 is affected in a manner that reveals underlying mechanisms involved in the control of modulating the insulin sensitivity in adipose tissues and skeletal muscles. The outcome from this project will have important implications for our understanding of the mechanisms of the development of peripheral tissue insulin resistance and for the development of new, targeted therapies for treatment of insulin resistant subjects and people with type 2 diabetes.

The aim of our study is to investigate the role of Rab3 isoforms and their interacting partners in the development of the peripheral tissue insulin resistance. Our previous work has identified that isoforms of the small GTPase Rab3 and their effector Noc2 (RPH3AL) are activated by insulin and that they play a role the final stages of insulin-stimulated GLUT4 translocation to the plasma membrane in adipocytes. Our preliminary data demonstrate that treatment of adipocytes with TNFa, a pro-inflammatory cytokine known to induce insulin resistance, decreases the levels of Rab3 and Noc2 in cells and this effect is reversed by treatment with the PPARg agonist rosiglitazone. In addition, analysis of human adipocytes from obese insulin resistant subjects showed that the levels of Rab3 isoforms are reduced compared to weight-matched insulin-sensitive subjects. These novel findings led us to hypothesise that alterations in the activation and abundance of Rab3 isoforms and their interacting partners, play a key role in mediating pro-inflammatory cytokine induced insulin resistance by disrupting the final stages of GLUT4 translocation in adipocytes. We propose to study how TNFa alters Rab3 isoforms expression at transcriptional and post-translational level. We will compare TNFa action on Rab3 with other treatments known to induce insulin resistance, such as chronic insulin treatment, or high palmitate treatment. We will study the molecular interactions between the Rab3 guanidine exchange factor DENN/MADD and Rab3 and their reported association with the TNFa receptor. Using proteomic, biochemical and cell biology approaches, we will determine how insulin regulates these interactions and how TNFa alters their function. To understand the role played by Rab3 in whole body insulin sensitivity, we will carry out a human intervention study to determine whether restoring glucose tolerance in overweight and insulin resistant subjects affects Rab3 isoforms' expression and regulation.

Economic impact
Beneficiaries: Pharmaceutical and Food industry (improved products and sales); NHS (improved therapies and interventions)
There is a constant demand from the pharmaceutical industry for novel drug targets for treatment of insulin resistance and Type 2 diabetes. Our work on Rab3 will define new pathways which could be used as new drug targets. We have extensive collaborations with UK based pharmaceutical and food companies including Glaxo-SmithKline, AstraZeneca and Unilever that have helped translate our research into practice. One example outcome has been the testing of the potency of an insulin sensitizer that was developed into a highly successful anti-diabetes drug Rosiglitazone (annual drug sale in 2006 peaked at approximately $2.5bn). Information obtained in this collaboration was used to mechanistically validate the drug. Our work was supported by AstraZeneca by two consecutive CASE studentships. We also have a long-standing collaborations with Unilever, which have sponsored two CASE studentships to explore how different diets and exercise regimes will affect the development of obesity and insulin resistance. With these continuing links, we will be able to translate new output on the proposed project into drug screening programs with an expected impact on new drug sales. In addition, our findings will facilitate the understanding of the pathways underlying the development of insulin resistance and this will be of interest for the food industry as they can develop food or food supplements designed to interact with these pathways. Diabetes treatment has economic consequences to the NHS (below).
Social and human health impact
Beneficiaries: General public; NHS; Charities; Policy Makers
Diabetes is a major global health problem with more than 415 million people worldwide currently living with this condition. 3.3 million people in the UK are now diagnosed with diabetes and it is estimated that 1 million more people may have pre-diabetes. Diabetes can lead to complications including kidney failure, coronary heart disease, stroke, blindness and foot amputation. It is estimated that Diabetes treatment (mainly type 2) accounts for 5% of all NHS expenditure. A detailed understanding of the underlying biological system is key for developing effective diagnostic tools, causal intervention strategies and in supporting public health advice (through government departments and charities). Providing a full pathway from insulin receptor to glucose transporter will impact on strategies for the control of glycaemia, energy homeostasis and bodyweight. Our inter-disciplinary approach provides the opportunity to translate directly our mechanistic findings into whole body human physiological functions and to identify important players in these processes. Therefore there is potential to change behaviours and improve the clinical interventions with associated improvements in health outcomes.
The targeting of this fundamental research area will reduce animal use by generating more targeted drug discovery. It will also provide a focused and directed range of parameters to be monitored in human subjects undergoing clinical drug trials.
Skills and training impact
Beneficiaries: Next generation research students; Pharmaceutical companies; Health coaching.
The proposed inter-disciplinary research will involve postgraduate students and research technicians who will benefit from training in the novel methods and approaches used by our team. The combination of whole-body human physiology and cellular and biochemical expertise will provide a unique all-round training programme for our post-graduate students and technicians and equip them with a wide range of skills. People trained in this way will be able to pass on skills to a range of employment sectors but specifically sectors concerned with treatments for obesity and type 2 diabetes, and with those in human health related areas such as exercise and diet coaching.