To accomplish these goals we will: A) establish the stimulus-secretion coupling in human pancreatic islets from non-diabetic organ donors to obtain proof of concepts derived from rodent studies; these data will also establish the gold standard for efforts to differentiate embryonic stem cells into mature islet cells. B) Combine islet studies with genetic and metabolic studies in human

A) Our knowledge about the stimulus-secretion coupling in human islet cells is fragmentary. Therefore, we will analyse normal human donor islets for:

• pancreatic hormone release (insulin, glucagon and somatostatin) by in-house radioimmunoassay
• cytoplasmic Ca2+ homeostasis (by standard microfluorimetry or spatially resolved confocal recordings)
• islet histology and ultrastructure (to determine β cell mass and intracellular granule distribution, possibly keys to understand the secretion defects)
• electrophysiology(glucose regulation of the KATP channels, properties of voltage-gated Ca2+ currents and exocytosis)

Finally, islets from T2D individuals (compared with age- and BMI-matched donors) will help to pinpoint the site of defect in diabetes.

Certain biological pathways in the β cell are of such obvious biological importance that they can easily be identified as potential targets for malfunction in T2D. Such proteins include the ATP-sensitive K+ channel (KATP channel) voltage-gated Ca2+ channels, β cell redox system and microRNAs. Their association with diabetes will be carefully evaluated in genetic studies. These pathways, and others identified in genetic studies (below), will be manipulated by viral gene transfer and RNA interference techniques in primary human islet cells and later in cell models differentiated from human embryonic stem cells.

B) Identification of the (presumably many) genes responsible for suppressed insulin secretion in T2D is ongoing in the GK rat, commonly regarded as the best animal model of human T2D. We will use ‘congenic’ rat strains that carry parts of the major diabetes susceptibility locus in the GK rat on a non-diabetic background to address the cellular and molecular defects imposed by the encoded variation in insulin secretion using the state-of-the art methods outlined above.

The human diabetes genomics database will be applied in parallel: We anticipate that we will identify novel pathways of importance for disturbed β-cell function in the families with early- onset diabetes. This is because many of the SNPs may locate to genes with unknown function or with no information on how they contribute to the pathogenesis of diabetes. We shall explore their function combining viral gene transfer and RNA interference as outlined above.

The final answer to the question of how a certain SNP influences human physiology requires metabolic studies in carriers and non-carriers of the variants. Our metabolic research unit provides excellent opportunity to quantify effects on insulin secretion and action.

Given the concentration of competence in islet biology, diabetes genomics and clinical metabolism at the centre, we are in a unique position to dissect the central role of β-cell dysfunction in diabetes.