Accurate quantification of beta-cell fraction in islet preparations

Measuring Glucose Stimulated Insulin Secretion (GSIS) ex-vivo in mouse and cadaveric human islets is the gold standard for assaying beta-cell function. Insulin secreted is usually normalized to protein or insulin content. Such normalization might be inadequate specifically in cases where protein or insulin content is changed. An alternative is to measure insulin secretion on a per-islet basis or normalize to DNA. The caveat with this method is that islets contain other cell types beside beta-cells and that both islet cell composition and function can be affected by conditions such as prolonged hyperglycemia. We reasoned that a more appropriate method would be to normalize insulin content or secreted insulin to beta-cell DNA. To this aim we developed a sensitive and specific Taqman-ddPCR assay using bisulfite converted islet DNA as a template to quantify beta-cell DNA in islets. The detection of beta-cell DNA is based on the presence of differentially methylated CpG sites in the mouse Ins2 and human INS genes. These sites are specifically unmethylated in beta cells while almost fully methylated in non-beta-cells.

We show that this method is quantitative, specific and sensitive enough to measure beta-cell DNA in mouse and human islets. We are currently calibrating this procedure to assess the fraction of alpha-cells in human islets.

Our ultimate goal is to use this strategy to quantify the main cell-type fractions (beta, alpha and delta) in islets. Such a resolution will be useful to monitor the effects of islet cell composition on beta-cell function.