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A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Published on: June 25, 2014
Effects of muscarinic receptor type 3 knockout on mouse islet secretory responses
Walter S Zawalich1, Kathleen C Zawalich, Gregory J Tesz
1Yale University School of Nursing, 100 Church Street South, New Haven, CT 06536-0740, USA. Walter.Zawalich@Yale.edu
Abstract:
The impact of muscarinic type 3 receptor knockout (M3KO) on the cholinergic regulation of insulin secretion and phospholipase C (PLC) activation was determined. Islets isolated from control, wild-type mice or heterozygotes responded with comparable insulin secretory responses to 15 mM glucose. This response was markedly amplified by the inclusion of 10 microM carbachol. While 15 mM glucose-induced release remained similar to wild-type and heterozygote responses in M3KO mice, the stimulatory impact of carbachol was abolished. Stimulation with 15 mM glucose plus 50 microM carbachol increased fractional efflux rates of myo-[2-3H]inositol from control wild-type and heterozygote islets but not from M3KO islets. Fed plasma insulin levels of M3KO mice were reduced 68% when compared to values obtained from combined wild-type and heterozygote animals. These studies support the conclusion that the M3 receptor in islets is coupled to PLC activation and insulin secretion and that cholinergic stimulation of the islets may play an important role in the regulation of plasma insulin levels.
Insights
Muscarinic type 3 receptors are crucial for cholinergic regulation of insulin secretion. Knockout mice lacking these receptors showed abolished carbachol-stimulated insulin release and reduced plasma insulin levels.
Area of Science:
- Endocrinology
- Molecular Pharmacology
Background:
- Cholinergic signaling regulates insulin secretion.
- Muscarinic receptors mediate these effects, but the specific subtype involved in pancreatic islets is not fully elucidated.
Purpose of the Study:
- To investigate the role of the muscarinic type 3 receptor (M3R) in cholinergic regulation of insulin secretion and phospholipase C (PLC) activation in pancreatic islets.
Main Methods:
- Utilized M3 receptor knockout (M3KO) mice and control wild-type/heterozygote littermates.
- Assessed insulin secretion in response to glucose and carbachol stimulation.
- Measured myo-[2-3H]inositol efflux to determine PLC activation.
- Quantified fed plasma insulin levels.
Main Results:
- M3KO islets exhibited abolished carbachol-stimulated insulin secretion compared to controls.
- Glucose-stimulated insulin release was unaffected by M3R absence.
- Carbachol failed to stimulate myo-[2-3H]inositol efflux in M3KO islets, indicating impaired PLC activation.
- M3KO mice displayed significantly reduced fed plasma insulin levels (68% lower).
Conclusions:
- The M3 receptor is essential for cholinergic stimulation of insulin secretion and PLC activation in pancreatic islets.
- Cholinergic signaling via M3 receptors plays a significant role in regulating plasma insulin levels.
