Role of mitochondrial calcium transport in the regulation of insulin secretion

Insulin secretion is essential for the normal regulation of blood glucose levels and becomes defective in all forms of diabetes mellitus, a disease affecting ~5 % of the population of westernised societies. Pancreatic beta cells are the body's sole source of circulating insulin and, in healthy individuals, respond to elevated glucose levels with the enhanced metabolism of the sugar. This leads to a cascade of biochemical events culminating in the influx into the cell of Ca2+ and the fusion of insulin-containing granules at the cell surface.
Mitochondria play a particularly important role in beta cell glucose recognition and enable these cells to breakdown the sugar almost completely to CO2 and H2O. This efficient "oxidative" metabolism, which increases steeply as glucose concentrations rise, helps the cells to synthesise large quantities of ATP. The resulting shift in the ratio of ATP to its precursor ADP leads to the closure of ATP-sensitive K* (KATP) channels on the plasma membrane. Voltage-sensitive Ca2+ channels then permit Ca2+ influx to trigger the release ("exocytosis") of insulin.
Increases in intracellular Ca2+ also prompt the enhanced consumption of ATP to fuel processes including ion pumping out of the cell, secretory granule movement, and so on. Findings over the past few decades have indicated that, in order to meet this demand, mitochondria contain a group of three enzymes involved in the breakdown of glucose-derived carbon in the "citrate cycle", which are strongly regulated by Ca2+ ions. The Ca2+ sensitive dehydrogenases, as well as a regulatory subunit of the ATP synthase, thus offer the potential to permit mitochondrial ATP output to match ATP demand by the rest of the cell.
Whilst providing an attractive hypothesis, for which substantial correlative evidence exists, the above model has proved difficult to test formally due to the absence of molecular or pharmacological tools with which to intervene to block (or enhance) mitochondrial Ca2+ uptake. This situation has changed in the last ~12 months with the discovery by our collaborators (Rosario Rizzuto and colleagues), and others, of the identity of the mitochondrial Ca2+ uniporter, as MCU. Other components, including a Ca2+ binding subunit MICU1, and a Na+-Ca2+ exchange protein, NCLX, were also identified in 2010.
In order to test both the involvement of these components, and the role of mitochondrial Ca2+ uptake in triggering or sustaining insulin secretion, we have established a combined system for recording both the electrical activity, and Ca2+ concentrations within defined subcellular domains - notably the mitochondria and the cytosol - of primary pancreatic islet beta cells. Further combining this technique with the use of "short hairpin RNAs" (shRNAs), delivered using lentiviral vectors, we propose here firstly to explore the effects on mitochondrial transport, metabolism, and on glucose- (and other stimulus-) regulated insulin secretion of depleting beta cells of each of these components individually.
We shall next use a new mouse model in which shRNAs can be delivered in vivo and with high selectivity to the pancreatic beta cell, to determine the impact of interfering with mitochondrial Ca2+ uptake in the beta cell on insulin secretion and hence whole body glucose homeostasis. Our findings will be substantiated by achieving a more complete "knockout" of one or more of the transporter genes in these cells using more conventional genetic "recombination" approaches in mice.
Subject to progress, we shall also explore the changes in the expression of the mitochondrial Ca2+ transporter genes in the context of various rodent models of T2 diabetes, and in human islets from healthy donors and T2 diabetes patients. The insights gained will thus provide a deeper understanding of a fundamental aspect of beta cell biology, and may provide findings which can be translated into new treatments for diabetes.

Glucose stimulates the release of insulin through mechanisms involving the activation of intracellular metabolism, increases in cytosolic ATP/ADP ratio, plasma membrane depolarisation, and Ca2+ influx. Mitochondria contain at least three intramitochondrial dehydrogenases which are strongly regulated by Ca2+ ions and it has been proposed that the accumulation of Ca2+ into these organelles serves to further stimulate ATP synthesis in response to glucose. The development of recombinant probes for Ca2+, which can be selectively targeted to specific subcellular domains including the mitochondrial matrix, has allowed correlative evidence to be provided in support of this model. However, a robust test of this hypothesis, involving interventions which specifically block Ca2+ accumulation within mitochondria, has been missing.

Here, we propose to take advantage of recent exciting advances in the identification of the molecular players involved in mitochondrial Ca2+ transport to achieve this goal. Specifically, we shall silence the expression of the putative mitochondrial uniporter components MCU (encoded by ccdc109a and-b) and MlCU1, as well as the efflux-mediator NCLX. We shall then use single beta cells in which both the membrane potential is controlled by perforated patch, whilst mitochondrial and cytosolic Ca2+ are monitored with a recombinant targeted probe (mitochondrial Pericam) or Fura Red, respectively. The newly-developed ATP sensor "Perceval" will next be used in the same system to monitor the impact on [ATP/ADP]cyt. Finally, we shall use a newly-developed mouse model, or CreLoxP-mediated deletion of flox'd alleles, to test the impact of silencing these genes on insulin secretion and glucose homeostasis in vivo.

The research proposed here is likely to benefit both the general population, in terms of improvements in healthcare, as well as the UK Pharmaceutical industry.

1. The general population of the UK. Type 2 diabetes affects ~3 m UK subjects and ~30 m Europeans (mean prevalence 8.4%; http://www.euphix.org/object_document/o4858n27165.html). These values are predicted to grow further in an "epidemic" driven by increasingly sedentary lifestyles and obesity. The complications of the disease include stroke, retinopathy, neuropathy, renal failure, cardiovascular disease and now cancer. The increased prevalence of this disease was recently predicted to contribute to a lowered overall life expectancy in the UK (http://www.independent.co.uk/life-style/health-and-wellbeing/health-news/diabetes-may-cause-first-fall-in-life-expectancy-for-200-years-966914.html) for the first time in 200 years. Treatment of diabetes is estimated to cost ~£8000 per year per patient, or £ 24 billion in total: diabetic patients are 3.5 times more likely to be admitted for hospital treatment than the rest of the population (http://www.physorg.com/news151077389.html). These direct economic costs together account for 7-13 % of health care costs in most developed societies (IDF Diabetes Atlas, 2003) and are further aggravated by increased absenteeism and decreased individual productivity (ADA: Diabetic Care 31, 596, 2008).

Pancreatic beta cell loss or dysfunction are cardinal elements of diabetes mellitus, and strategies to rejuvenate or replace these cells, as explored here, are likely to be key to the development of new therapeutic approaches for this disease and its complications.
In particular this work will address roadblocks in diabetes research as identified recently by the European Commission's Support Action "DIAMAP: A Road Map for Diabetes in Europe" (http://www.diamap.eu/; 7th Sep, 2010) including the development of: "novel therapies based on beta cell mass and function" and "a lack of appropriate models that mimic the human condition".

2. The UK Pharmaceutical Industry. The global market for anti-diabetes drugs is estimated to be worth ~$30 billion. Following its joint Workshop with the Association of British Pharmaceutical Industries (ABPI) in March 2011, the MRC concluded that pancreatic beta cell biology should become a Strategic Priority, alongside Stratified Medicine.
New drug targets and leads are desperately needed for the Pharmaceutical industry to produce novel approaches to diabetes treatment. By addressing highly promising new targets to regulate mitochondrial metabolism and thus the efficiency of glucose-stimulated insulin secretion, the proposed study will enhance feeds of new Intellectual Property to this sector. As a member of the trans-European diabetes research network "IMIDIA" (http://www.imidia.org/) the PI already interacts with several UK and Europe-based companies (eg Astra Zeneca, Sanofi Aventis, Boehringer-Ingelheim, Novo Nordisk and Novartis), and has established a collaboration with Pfizer (US) to initiate high throughput screens for small molecule regulators of genes involved in the control of b cell mass and function. Similarly, with Cenix, an SME based in Dresden, he has begun a collaboration to use genome-wide RNAis screens to identify endogenous regulators of such genes.
The Co-PI directly involved in the project will enhance his professional skills with training in basic biomedical research and thus develop his skill set for application in both the academic and commercial sectors.