A single-islet microplate assay to measure mouse and human islet insulin secretion

Abstract

One complication to comparing β-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.

Decreased Consumption of Branched-Chain Amino Acids Improves Metabolic Health

“…We isolated pancreatic islets from mice on the Control, Low BCAA and Low AA diets, and examined ex vivo glucose stimulated insulin secretion as well as the metabolic and Ca 2+ oscillations that drive secretion (Merrins et al, 2016; Merrins et al, 2013; Truchan et al, 2015). Islets isolated from the mice fed Low BCAA and Low AA diets secreted significantly less insulin per islet than islets from the mice fed Control diet (Figure 3A), while the islet insulin content was also decreased by both diets, significantly in the case of the Low AA diet (Figure 3B).…”

Short‐term methionine deprivation improves metabolic health via sexually dimorphic, mTORCI‐independent mechanisms

“…Islets were isolated and an ex vivo glucose‐stimulated insulin secretion assay was performed (29, 31). Isolated islets were transferred to a 96‐well V‐bottom plate and incubated with RPMI 1640 medium with 10% fetal bovine serum, penicillin‐streptomycin, and 11.1 mM glucose for 48 h (32). In brief, after a 48 h incubation period, islets were treated with a low‐glucose (1.7 mM) Krebs‐Ringer bicarbonate buffer preincubation solution for 45 min, followed by a stimulatory high‐glucose (16.7 mM) Krebs‐Ringer bicarbonate buffer solution for 45 min, as well as by a stimulatory high‐glucose (16.7 mM), in combination with 10 nM of exendin‐4, a peptide agonist of the glucagon‐like peptide receptor, for 45 min.…”

Cholecystokinin expression in the β-cell leads to increased β-cell area in aged mice and protects from streptozotocin-induced diabetes and apoptosis

“…Single-islet insulin secretion assays were performed as described in Ref. 50. A total of 14 -30 isolated islets were individually placed in 96-well v-bottom plates and incubated for 24 h in regular islet media.…”