Role of Rab3 in peripheral tissue insulin resistance

Lead Research Organisation: University of Bath
Department Name: Biology and Biochemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Chen YC (2017) Feeding influences adipose tissue responses to exercise in overweight men. in American journal of physiology. Endocrinology and metabolism

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Edinburgh RM (2020) Lipid Metabolism Links Nutrient-Exercise Timing to Insulin Sensitivity in Men Classified as Overweight or Obese. in The Journal of clinical endocrinology and metabolism

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Hengist A (2019) Fructose and metabolic health: governed by hepatic glycogen status? in The Journal of physiology

 
Description Endosomal sorting complexes in insulin-stimulated GLUT4 recycling
Amount £14,089 (GBP)
Funding ID 19/0005983 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 08/2020
 
Title Dataset for "Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization" 
Description This dataset contains the SAXS data obtained at i22 (Diamond Light Source) and ID02 beamline (ESRF). Other data such as ELISA and Circular Dichroism are also included. All data is organised by figures presented in the research paper. Majority of data is stored in excel or csv format with processing done in MatLab for graphical presentation. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL https://researchdata.bath.ac.uk/id/eprint/771
 
Title Dataset for "Lipid metabolism links nutrient-exercise timing to insulin sensitivity in overweight men" 
Description The aim of the present work was to assess the acute and chronic effects of manipulating nutrient-exercise timing on lipid metabolism, skeletal muscle adaptation, and oral glucose insulin sensitivity in overweight and obese men. This project comprised two experiments. We first assessed the acute metabolic and mRNA responses to manipulating nutrient-exercise timing (Acute Study), followed by a 6-week randomized controlled trial to assess the longer-term adaptations in response to nutrient-exercise timing (Training Study). We showed that in overweight/obese, but otherwise healthy men (mean±SD for age: 30 ± 10 years for acute study, 35 ± 9 years for training study and BMI: 30.2±3.5 kg/m-2 for acute study, 30.9±4.5 kg/m-2 for training study) a single exercise bout before versus after nutrient provision increased lipid utilization at the whole-body level, but also in both type I (p<0.01) and type II muscle fibers (p=0.02). We then used a 6-week intervention to show sustained, 2-fold increases in lipid utilization with exercise training before versus after nutrient provision (p<0.01). An oral glucose-derived estimate of peripheral insulin sensitivity (OGIS index) increased when training was performed before versus after nutrient provision (25±38 vs -21±32 mL/min/m-2; p=0.01) and this was associated with increased lipid utilization during exercise (r=0.50, p=0.02). Regular exercise prior to nutrient provision augmented remodelling of skeletal muscle phospholipids and muscle expression of the glucose transport protein GLUT4 (p<0.05). These responses were observed despite similar changes in body mass, waist-to-hip ratio, and oxidative capacity. Therefore: 1) experiments investigating exercise training and metabolic health need to control for nutrient-exercise timing; 2) exercise performed before versus after nutrient intake may exert beneficial effects on lipid utilization and oral glucose insulin sensitivity. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
 
Title Dataset for "Thermal resilience of ensilicated lysozyme via calorimetric and in vivo analysis" 
Description This dataset contains a variety of data formats used to characterise the thermal resilience of ensilicated lysozyme. All raw data formats have been converted to *.csv or similar Excel format, which is editable. MATLAB is used to generate all graphs and files have been included in each folder where applicable. Specifically, data were generated using analytical methods such a differential scanning calorimetry, circular dichroism spectroscopy, enzyme-linked immunosorbent assay (ELISA) and thermogravimetric analysis. Data is sorted based on the figure order described in the article, including supplementary figures. Each folder contains a brief explanation on the contents. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL https://researchdata.bath.ac.uk/id/eprint/895
 
Title Dataset for 'Pre-Exercise Breakfast Ingestion versus Extended Overnight Fasting Increases Postprandial Glucose Flux after Exercise in Healthy Men: Pre-exercise feeding and postprandial glucose flux' 
Description The aim of this study was to characterise postprandial glucose flux after exercise in the fed versus overnight fasted-state and to investigate potential underlying mechanisms. In a randomized order, twelve men underwent breakfast-rest (BR; 3 h semi-recumbent), breakfast-exercise (BE; 2 h semi-recumbent before 60-min of cycling (50% peak power output) and overnight fasted-exercise (FE; as per BE omitting breakfast) trials. An oral glucose tolerance test (OGTT) was completed post-exercise (post-rest on BR). Dual stable isotope tracers ([U-13C] glucose ingestion and [6,6-2H2] glucose infusion) and muscle biopsies were combined to assess postprandial plasma glucose kinetics and intramuscular signaling, respectively. Plasma intestinal fatty acid binding (I-FABP) concentrations were determined as a marker of intestinal damage. The results from this study showed that consuming breakfast before exercise increases post-exercise postprandial plasma glucose disposal, which is offset (primarily) by increased appearance rates of orally-ingested glucose. Therefore, metabolic responses to fed-state exercise cannot be readily inferred from studies conducted in a fasted state. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
 
Title Dataset for 'Skipping breakfast before exercise creates a more negative 24-hour energy balance: A randomized controlled trial in healthy physically active young men' 
Description The aim of this study was to assess the effect of pre-exercise breakfast omission versus consumption on 24-h energy balance. The data set contains data about 12 healthy men who completed preliminary testing followed by three trials in a randomized order (separated >1 week): a breakfast of oats and milk (431 kcal; 65 g CHO, 11 g FAT, 19 g PRO) followed by rest (BR); breakfast before exercise (BE; 60 min cycling at 50% peak power output); and overnight-fasting before exercise (FE). These results from the study suggested that, neither exercise energy expenditure nor the restricted energy intake via breakfast omission were completely compensated for post-exercise. Therefore, in healthy men, pre-exercise breakfast omission creates a more negative daily energy balance and could be a useful strategy to induce a short-term energy deficit. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
 
Title Feeding influences adipose tissue responses to exercise in overweight men 
Description Dataset for the following study: Feeding profoundly affects metabolic responses to exercise in various tissues but the effect of feeding status on human adipose tissue responses to exercise has never been studied. Ten healthy overweight men aged 26 ± 5 years (mean ± SD) with a waist circumference of 105 ± 10 cm walked at 60% of maximum oxygen uptake under either FASTED or FED conditions in a randomised, counterbalanced design. Feeding comprised 648 ± 115 kcal 2 h before exercise. Blood samples were collected at regular intervals to examine changes in metabolic parameters and adipokine concentrations. Adipose tissue samples were obtained at baseline and one hour post-exercise to examine changes in adipose tissue mRNA expression and secretion of selected adipokines ex-vivo. Adipose tissue mRNA expression of PDK4, ATGL, HSL, FAT/CD36, GLUT4 and IRS2 in response to exercise were lower in FED compared to FASTED conditions (all p = 0.05). Post-exercise adipose IRS2 protein was affected by feeding (p = 0.05), but Akt2, AMPK, IRS1, GLUT4, PDK4 and HSL protein levels were not different. Feeding status did not impact serum and ex-vivo adipose secretion of IL-6, leptin or adiponectin in response to exercise. This is the first study to show that feeding prior to acute exercise affects post-exercise adipose tissue gene expression and we propose that feeding is likely to blunt long-term adipose tissue adaptation to regular exercise. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
 
Description Imprinted genes defining a novel mammalian growth regulatory axis MR/S008233/1 
Organisation University of Bath
Department Department of Biology and Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution I am co-investigator on this MRC project grant. My contribution is to help with the development of the adipose tissue cell models and with the investigation of the signalling pathways involved in the Grb10 signalling during development.
Collaborator Contribution I am co-investigator on this MRC project grant. My contribution is to help with the development of the adipose tissue cell models and with the investigation of the signalling pathways involved in the Grb10 signalling during development.
Impact No output yet
Start Year 2019
 
Description Rab3 interactome 
Organisation University of Sydney
Country Australia 
Sector Academic/University 
PI Contribution This collaboration is part of the awarded project. We are in the process of generating samples that will be then sent to Prof David James's lab in Australia for proteomic analysis.
Collaborator Contribution The team lead by Prof David James in Australia will be performing proteomic analysis on samples generated by us to elucidate the Rab3 interacting partners in adipose cells and mapping the Rab3 interactive.
Impact We are currently generating the samples for analysis. Therefore there is no output generated yet.
Start Year 2017
 
Description Facebook page to promote our human intervention study 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We are recruiting participant for our human intervention study as part of the research grant. To be able to reach more members of the general public and to engage our current study participants we have created an Facebook page where people can contact us and follow the progress of the study.
Year(s) Of Engagement Activity 2018,2019
URL https://www.facebook.com/mrcrabstudy/
 
Description Research conference organisation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Myself, Dylan Thompson and Javier Gonzalez together with Kei Sakamoto (Nestle Research Institute) were the organisers of a Biochemical Society Focused meeting entitles "Insulin and exercise signalling for glucose homeostasis and metabolic health". This meeting was held at the University of Bath, from the 6th till the 8th of September 2017. 45 participants took part, from UK, Germany, Switzerland, Sweden, Danemark and the USA. At the meeting Prof Geoff Holman delivered the Sir Philip Randle award lecture 2017 from the Biochemical Society. The conference was a great success with a lot of positive feedback from colleagues and post-graduate students. They all appreciated the wide variety of the research programme and the interdisciplinarity of the talks. In the aftermath of the event some of our colleagues expressed interest in transforming this event in a regular event with a 2 year reoccurrence.
Year(s) Of Engagement Activity 2017
URL https://www.biochemistry.org/Events/tabid/379/Filter/64/MeetingNo/SA202/view/Conference/Default.aspx