Functional imaging of islet cells in situ

                 It is important to understand the function of pancreatic islet cells within a physiological context. Besides insulin secreting beta-cells, islets also contain glucagon secreting alpha-cells and somatostatin secreting delta-cells, among others. Interactions between these different cell types may be of great importance to physiological islet function. For this reason we have been working in Oxford to establish the functional imaging of single islet cells within intact pancreatic islets.

                 The focus presently has been on performing ratiometric calcium imaging to monitor the intracellular calcium responses of single cells within islets. We are using a Zeiss LSM510 Meta confocal imaging system to achieve this. Examples of this work are shown to the right. In the future this methodology may be extended to studies monitoring mitochondrial membrane potential and exocytotic function within the islets. We have the ability as well to perform multi-photon imaging, and have done some work using this.

The Project: This work is not simply about establishing a technique within the lab, but is indeed focused on examining an important physiological question: How is pancreatic alpha-cell activity regulated?

                 Alpha-cells secrete the important regulatory hormone glucagon, which acts in a manner opposite that of insulin to raise blood glucose when it is too low. Thus, glucagon secretion is stimulated by hypoglycaemia and is inhibited by hyperglycaemia. Although entry of calcium  into the alpha-cell through voltage-dependent channels is important from this, the mechanism by which glucose controls alpha-cell function is unknown. Several hypotheses exist: 1) the paracrine regulation of alpha-cell activity by substances released from beta-cells (such as GABA, zinc or insulin itself); 2) an effect on peripheral glucose-sensors to ultimately elicit the nervous control of glucagon release; 3) a metabolic effect of glucose on the alpha-cell by which intracellular calcium stores are regulated to affect a store-operated membrane potential response; and 4) a metabolic effect of glucose on alpha-cell Katp channels that mimics that which occurs in beta-cells (however because of the compliment of ion channels in the alpha-cells this suppresses electrical activity).

                 We are exploring hypothesis number 4 by monitoring the calcium response of single alpha-cells within the islet, and manipulating Katp channel activity under various conditions. These experiments should provide important information about the effects of glucose on alpha-cells within an in situ context– the intact islet.

Above: A mouse islet loaded with FuraRed and Fluo4 for ratiometric calcium measurements.

Below: Ratiometric calcium measurements from a rat islet demonstrate synchronized activity in beta-cells throughout the islet.

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The MacDonald Islet Biology Lab

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