Static Glucose-stimulated Insulin Secretion (GSIS) Protocol - Human Islets v2 (protocols.io.wy4ffyw)

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2′- O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2′- O-Me-cAMP-AM was associated with depolarization and an increase of [Ca2+]i that reflected both Ca2+ influx and intracellular Ca2+ mobilization in islet β-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2′- O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca2+-dependent exocytosis.

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Glucose Responsiveness of β-Cells Depends on Fatty Acids

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/a-0884-2919 ◽

2019 ◽

Vol 128 (10) ◽

pp. 644-653

Author(s):

Felicia Gerst ◽

Christine Singer ◽

Katja Noack ◽

Dunia Graf ◽

Gabriele Kaiser ◽

...

Keyword(s):

Fatty Acids ◽

Insulin Secretion ◽

Cell Function ◽

Human Islets ◽

In Vitro Tests ◽

High Concentration ◽

Β Cell Function ◽

Glucose Stimulated Insulin Secretion ◽

Glucose Responsiveness

AbstractGlucose-stimulated insulin secretion (GSIS) is the gold standard for β-cell function. Both experimental and clinical diabetology, i. e., preceding transplantation of isolated human islets, depend on functional testing. However, multiple factors influence GSIS rendering the comparison of different in vitro tests of glucose responsiveness difficult. This study examined the influence of bovine serum albumin (BSA)-coupled fatty acids on GSIS. Isolated islet preparations of human donors and of 12-months old mice displayed impaired GSIS in the presence of 0.5% FFA-free BSA compared to 0.5% BSA (fraction V, not deprived from fatty acids). In aged INS-1E cells, i. e. at a high passage number, GSIS became highly sensitive to FFA-free BSA. Readdition of 30 µM palmitate or 30 µM oleate to FFA-free BSA did not rescue GSIS, while the addition of 100 µM palmitate and the raise of extracellular Ca2+from 1.3 to 2.6 mM improved glucose responsiveness. A high concentration of palmitate (600 µM), which fully activates FFA1, largely restored insulin secretion. The FFA1-agonist TUG-469 also increased insulin secretion but to a lesser extent than palmitate. Glucose- and TUG-induced Ca2+oscillations were impaired in glucose-unresponsive, i. e., aged INS-1E cells. These results suggest that fatty acid deprivation (FFA-free BSA) impairs GSIS mainly through an effect on Ca2+sensitivity.

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The Effect of Recovery Warm-up Time Following Cold Storage on the Dynamic Glucose-stimulated Insulin Secretion of Isolated Human Islets

Cell Transplantation ◽

10.1177/0963689720908278 ◽

2020 ◽

Vol 29 ◽

pp. 096368972090827

Author(s):

Oscar Alcazar ◽

Alejandro Alvarez ◽

Camillo Ricordi ◽

Elina Linetsky ◽

Peter Buchwald

Keyword(s):

Insulin Secretion ◽

Cold Storage ◽

Islet Cell ◽

Cell Function ◽

Human Islets ◽

Second Phase ◽

Islet Cell Transplantation ◽

Warm Up ◽

Recovery Times ◽

Glucose Stimulated Insulin Secretion

Standardized islet characterization assays that can provide results in a timely manner are essential for successful islet cell transplantation. A critical component of islet cell quality is β-cell function, and perifusion-based assessments of dynamic glucose-stimulated insulin secretion (GSIS) are the most informative method to assess this, as they provide the most complex in vitro evaluation of GSIS. However, protocols used vary considerably among centers and investigators as they often use different low- and high-glucose concentrations, exposure-times, flow-rates, oxygen concentrations, islet numbers, analytical methods, measurement units, and instruments, which result in different readouts and make comparisons across platforms difficult. Additionally, the conditions of islet storage and shipment prior to assessment may also affect islet function. Establishing improved standardized protocols for perifusion GSIS assays should be an integral part of the ongoing effort to increase the rigor of human islet studies. Here, we performed detailed evaluation of GSIS of human islets using a fully automated multichannel perifusion instrument following various warm-up recovery times after cold storage that corresponds to current shipping conditions (8°C). We found that recovery times shorter than 18 h (overnight) resulted in impaired insulin secretion. While the effects were relatively moderate on second-phase insulin secretion, first-phase peaks were restored only following 18-h incubation. Hence, the biphasic profile of dynamic GSIS was considerably affected when islets were not allowed to recover for a sufficient time after being maintained in cold. Accordingly, while cold storage might improve islet cell survival during shipment and prolong the length of culture, functional assessments should be performed only after allowing for at least overnight recovery at physiological temperatures.

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Resveratrol Potentiates Glucose-stimulated Insulin Secretion in INS-1E β-Cells and Human Islets through a SIRT1-dependent Mechanism

Journal of Biological Chemistry ◽

10.1074/jbc.m110.176842 ◽

2010 ◽

Vol 286 (8) ◽

pp. 6049-6060 ◽

Cited By ~ 104

Author(s):

Laurène Vetterli ◽

Thierry Brun ◽

Laurianne Giovannoni ◽

Domenico Bosco ◽

Pierre Maechler

Keyword(s):

Insulin Secretion ◽

Human Islets ◽

Β Cells ◽

Glucose Stimulated Insulin Secretion ◽

Dependent Mechanism

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Static Glucose-stimulated Insulin Secretion (GSIS) Protocol - Human Islets v3 (protocols.io.wztff6n)

protocols.io ◽

10.17504/protocols.io.wztff6n ◽

2019 ◽

Author(s):

Aliya Spigelman ◽

Jocelyn Manning ◽

Patrick MacDonald

Keyword(s):

Insulin Secretion ◽

Human Islets ◽

Glucose Stimulated Insulin Secretion

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1,25-Dihydroxyvitamin D3 enhances glucose-stimulated insulin secretion in mouse and human islets: a role for transcriptional regulation of voltage-gated calcium channels by the vitamin D receptor

The Journal of Steroid Biochemistry and Molecular Biology ◽

10.1016/j.jsbmb.2018.07.004 ◽

2019 ◽

Vol 185 ◽

pp. 17-26 ◽

Cited By ~ 6

Author(s):

Lilja Kjalarsdottir ◽

Sarah A. Tersey ◽

Mridula Vishwanath ◽

Jen-Chieh Chuang ◽

Bruce A. Posner ◽

...

Keyword(s):

Vitamin D ◽

Transcriptional Regulation ◽

Insulin Secretion ◽

Calcium Channels ◽

Vitamin D Receptor ◽

Human Islets ◽

Dihydroxyvitamin D3 ◽

Voltage Gated ◽

Voltage Gated Calcium Channels ◽

Glucose Stimulated Insulin Secretion

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Perilipin Is Present in Islets of Langerhans and Protects against Lipotoxicity When Overexpressed in the β-Cell Line INS-1

Endocrinology ◽

10.1210/en.2008-0913 ◽

2009 ◽

Vol 150 (7) ◽

pp. 3049-3057 ◽

Cited By ~ 20

Author(s):

Jörgen Borg ◽

Cecilia Klint ◽

Nils Wierup ◽

Kristoffer Ström ◽

Sara Larsson ◽

...

Keyword(s):

Insulin Secretion ◽

Cell Line ◽

Islets Of Langerhans ◽

Prolonged Exposure ◽

Human Islets ◽

Lipid Storage ◽

Β Cells ◽

Β Cell ◽

Glucose Stimulated Insulin Secretion ◽

In Cells

Lipids have been shown to play a dual role in pancreatic β-cells: a lipid-derived signal appears to be necessary for glucose-stimulated insulin secretion, whereas lipid accumulation causes impaired insulin secretion and apoptosis. The ability of the protein perilipin to regulate lipolysis prompted an investigation of the presence of perilipin in the islets of Langerhans. In this study evidence is presented for perilipin expression in rat, mouse, and human islets of Langerhans as well as the rat clonal β-cell line INS-1. In rat and mouse islets, perilipin was verified to be present in β-cells. To examine whether the development of lipotoxicity could be prevented by manipulating the conditions for lipid storage in the β-cell, INS-1 cells with adenoviral-mediated overexpression of perilipin were exposed to lipotoxic conditions for 72 h. In cells exposed to palmitate, perilipin overexpression caused increased accumulation of triacylglycerols and decreased lipolysis compared with control cells. Whereas glucose-stimulated insulin secretion was retained after palmitate exposure in cells overexpressing perilipin, it was completely abolished in control β-cells. Thus, overexpression of perilipin appears to confer protection against the development of β-cell dysfunction after prolonged exposure to palmitate by promoting lipid storage and limiting lipolysis.

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Dopamine-Mediated Autocrine Inhibitory Circuit Regulating Human Insulin Secretion in Vitro

Molecular Endocrinology ◽

10.1210/me.2012-1101 ◽

2012 ◽

Vol 26 (10) ◽

pp. 1757-1772 ◽

Cited By ~ 43

Author(s):

Norman Simpson ◽

Antonella Maffei ◽

Matthew Freeby ◽

Steven Burroughs ◽

Zachary Freyberg ◽

...

Keyword(s):

Insulin Secretion ◽

Secretory Granules ◽

Human Islets ◽

Negative Regulator ◽

Β Cells ◽

Regulatory Circuit ◽

Glucose Stimulated Insulin Secretion ◽

Insulin Secretion In Vitro

Abstract We describe a negative feedback autocrine regulatory circuit for glucose-stimulated insulin secretion in purified human islets in vitro. Using chronoamperometry and in vitro glucose-stimulated insulin secretion measurements, evidence is provided that dopamine (DA), which is loaded into insulin-containing secretory granules by vesicular monoamine transporter type 2 in human β-cells, is released in response to glucose stimulation. DA then acts as a negative regulator of insulin secretion via its action on D2R, which are also expressed on β-cells. We found that antagonism of receptors participating in islet DA signaling generally drive increased glucose-stimulated insulin secretion. These in vitro observations may represent correlates of the in vivo metabolic changes associated with the use of atypical antipsychotics, such as increased adiposity.

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