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Quantitative monitoring of insulin secretion from single islets of Langerhans in parallel on a microfluidic chip.

ABSTRACT: Quantification of insulin release from pancreatic islets of Langerhans is of interest for diabetes research. Typical insulin secretion experiments are performed using offline techniques that are expensive, slow, have low-throughput, and require multiple islets. We have developed a microfluidic device for high-throughput, automated, and online monitoring of insulin secretion from individual islets in parallel. This chip consists of 15 channel networks each capable of superfusing a single islet and mixing superfusate from each islet online with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for a competitive immunoassay. The resulting continuous reaction streams are periodically injected onto parallel electrophoresis channels where the mixtures are separated. The resulting traces are used to quantify relative insulin released from islets. Serial immunoassays were performed at 10 s intervals on all 15 channels, corresponding to 5400 immunoassays per hour, to create temporally resolved insulin release profiles that captured single islet secretion dynamics. The chip was used to demonstrate that free fatty acid induced lipotoxicity in islets eliminates pulsatile insulin secretion.

SUBMITTER: Dishinger JF

PROVIDER: S-EPMC2679996 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Quantitative monitoring of insulin secretion from single islets of Langerhans in parallel on a microfluidic chip.

Dishinger John F JF Reid Kendra R KR Kennedy Robert T RT

Analytical chemistry 20090401 8

Quantification of insulin release from pancreatic islets of Langerhans is of interest for diabetes research. Typical insulin secretion experiments are performed using offline techniques that are expensive, slow, have low-throughput, and require multiple islets. We have developed a microfluidic device for high-throughput, automated, and online monitoring of insulin secretion from individual islets in parallel. This chip consists of 15 channel networks each capable of superfusing a single islet an ...[more]

PMID: 19364142

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Project description:Effects on insulin release, cyclic AMP content and protein phosphorylation of agents modifying cyclic AMP levels have been tested in intact rat islets of Langerhans. Insulin release induced by glucose was potentiated by dibutyryl cyclic AMP, glucagon, cholera toxin and 3-isobutyl-1-methylxanthine (IBMX); the calmodulin antagonist trifluoperazine reversed these potentiatory effects. Inhibition by trifluoperazine of IBMX-potentiated release was, however, confined to concentrations of IBMX below 50 microM; higher concentrations, up to 1 mM, were resistant to inhibition by trifluoperazine. IBMX-potentiated insulin release was also inhibited by 2-deoxyadenosine, an inhibitor of adenylate cyclase. In the absence of glucose, IBMX at concentrations up to 1 mM did not stimulate insulin release and in the presence of 3.3 mM-glucose IBMX was effective only at a concentration of 1 mM; under the latter conditions trifluoperazine again did not inhibit insulin secretion. The maximum effect on insulin release was achieved with 25 microM-IBMX. Islet [cyclic AMP] was increased by IBMX, with the maximum rise occurring with 100 microM-IBMX. The increase in [cyclic AMP] elicited by IBMX was more rapid than that induced by cholera toxin. Trifluoperazine did not significantly affect islet cyclic AMP levels under any of the conditions tested. When islets were incubated with [32P]Pi, radioactivity was incorporated into islet ATP predominantly in the gamma-position. The rate of equilibration of label was dependent on medium Pi and glucose concentration and at optimal concentrations of these 100% equilibration of internal [32P]ATP with external [32P]Pi required a period of 3h. Radioactivity was incorporated into islet protein and, in response to an increase in islet [cyclic AMP], the major effect was on a protein of Mr 15 000 on sodium dodecyl sulphate/polyacrylamide gels. The extent of phosphorylation of the Mr-15 000 protein was correlated with the level of cyclic AMP: phosphorylation in response to IBMX was inhibited by 2-deoxyadenosine but not by trifluoperazine. Fractionation of islets suggested that the Mr-15 000 protein was of nuclear origin: the protein co-migrated with histone H3 on acetic acid/urea/Triton gels. In the islet cytosol a number of proteins were phosphorylated in response to elevation of islet [cyclic AMP]: the major species had Mr values of 18 000, 25 000, 34 000, 38 000 and 48 000. Culture of islets with IBMX increased the rate of [3H]-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)

Dataset Information

Quantitative monitoring of insulin secretion from single islets of Langerhans in parallel on a microfluidic chip.

Publications

Quantitative monitoring of insulin secretion from single islets of Langerhans in parallel on a microfluidic chip.

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