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Updated: Dec 14, 2025
A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Published on: June 25, 2014
Fresh and cultured mouse islets differ in their response to nutrient stimulation
Mai Morsi1, Torben Schulze1, Eike Früh1
1Institute of Pharmacology, Toxicology and Clinical Pharmacy, Technische Universität Braunschweig, Braunschweig, Germany.
Insights
Freshly isolated mouse islets show more variable insulin secretion kinetics in response to nutrients compared to cultured islets. This difference may stem from how cultured islets utilize endogenous metabolites.
Area of Science:
- Endocrinology
- Cell Biology
- Metabolism
Background:
- Insulin secretion is crucial for glucose homeostasis.
- Understanding stimulus-secretion coupling in pancreatic islets is key to diabetes research.
- Islet culture conditions can alter cellular function and responsiveness.
Purpose of the Study:
- To compare nutrient-induced insulin secretion kinetics between fresh and cultured mouse islets.
- To investigate differences in stimulus-secretion coupling parameters.
- To explore the impact of culture conditions on islet function.
Main Methods:
- Perifusion of freshly isolated and 22-h-cultured NMRI mouse islets.
- Stimulation with glucose, alpha-ketoisocaproic acid, tolbutamide, and KCl.
- Measurement of insulin secretion rates.
- Assessment of cytosolic Ca2+ concentration and ATP/ADP ratio.
- Monitoring of mitochondrial reducing equivalents (NAD(P)H/FAD fluorescence).
Main Results:
- Cultured islets exhibited a typical biphasic insulin secretion pattern, while fresh islets showed a continuously ascending rate with glucose.
- Both fresh and cultured islets responded similarly to non-nutrient secretagogues (tolbutamide, KCl).
- Cultured islets displayed a larger glucose-induced rise in cytosolic Ca2+ and ATP/ADP ratio, with altered mitochondrial activity.
- Modifying culture media resulted in variable insulin release but consistently predominant first-phase kinetics.
Conclusions:
- Nutrient-induced insulin secretion kinetics are more variable in fresh mouse islets than in cultured islets.
- The uniform kinetics in cultured islets might be due to altered endogenous metabolite utilization.
- These findings highlight the impact of culture on islet function and responsiveness.
Abstract:
Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be typical for mouse islets. This was also the response of cultured islets to the nutrient secretagogue alpha-ketoisocaproic acid, whereas the secretion of fresh islets increased similarly fast but remained strongly elevated. The responses of fresh and cultured islets to purely depolarizing stimuli (tolbutamide or KCl), however, were closely similar. Signs of apoptosis and necrosis were rare in both preparations. In cultured islets, the glucose-induced rise of the cytosolic Ca2+ concentration started from a lower value and was larger as was the increase of the ATP/ADP ratio. The prestimulatory level of mitochondrial reducing equivalents, expressed as the NAD(P)H/FAD fluorescence ratio, was lower in cultured islets, but increased more strongly than in fresh islets. When culture conditions were modified by replacing RPMI with Krebs-Ringer medium and FCS with BSA, the amount of released insulin varied widely, but the kinetics always showed a predominant first phase. In conclusion, the secretion kinetics of fresh mouse islets is more responsive to variations of nutrient stimulation than cultured islets. The more uniform kinetics of the latter may be caused by a different use of endogenous metabolites.
