Abstract
Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment.
Highlights
Insulin is an anabolic hormone that regulates metabolism and energy homeostasis
Specific detection of the stable EuK-XL665 fluorescence energy transfer (FRET) signal was significantly enhanced by the addition of a time delay that filtered out interference from transient assay buffer and protein autofluorescence (Fig 1C)
We used this detection method to measure insulin released from cells or islets treated in the following manner: (1) insulin-secreting cells or islets were stimulated with secretagogue, followed immediately by (2) the transfer of the insulin-containing supernatant to a separate plate where the donor and acceptor-coupled insulin antibodies were added, incubated and measured directly without the need for additional wash steps (Fig 1D)
Summary
Insulin is an anabolic hormone that regulates metabolism and energy homeostasis. The release of insulin by pancreatic beta cells in response to increases in extracellular glucose promotes glucose uptake in insulin-sensitive tissues [1]. Disruption of the regulation of insulin secretion leads to profound global metabolic effects that can result in diabetes mellitus and tissue damage [2]. Many aspects of insulin release, including the mechanisms regulating glucose-stimulated insulin secretion (GSIS), require further study. Several antipsychotic drugs, which target DA D2-like receptors including D2 (D2R) and D3 (D3R) receptors, significantly dysregulate insulin secretion [3,4]. A rapid, cost-effective and scalable assay for quantitating insulin levels would facilitate further studies of GSIS and drug-induced
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