Insulin is known to regulate pancreatic β-cell function through the activation of cell surface insulin receptors, phosphorylation of insulin receptor substrate (IRS)-1 and -2, and activation of phosphatidylinositol (PI) 3-kinase. However, an acute effect of insulin in modulating β-cell electrical activity and its underlying ionic currents has not been reported. Using the perforated patch clamp technique, we found that insulin (1–600 nmol/l) but not IGF-1 (100 nmol/l) reversibly hyperpolarized single mouse β-cells and inhibited their electrical activity. The dose-response relationship for insulin yielded a maximal change (mean ± SE) in membrane potential of −13.6 ± 2.0 mV (P < 0.001) and a 50% effective dose of 25.9 ± 0.1 nmol/l (n = 63). Exposing patched β-cells within intact islets to 200 nmol/l insulin produced similar results, hyperpolarizing islets from −47.7 ± 3.3 to −65.6 ± 3.7 mV (P < 0.0001, n = 11). In single cells, insulin-induced hyperpolarization was associated with a threefold increase in whole-cell conductance from 0.6 ± 0.1 to 1.7 ± 0.2 nS (P < 0.001, n = 10) and a shift in the current reversal potential from −25.7 ± 2.5 to −63.7 ± 1.0 mV (P < 0.001 vs. control, n = 9; calculated K+ equilibrium potential = −90 mV). The effects of insulin were reversed by tolbutamide, which decreased cell conductance to 0.5 ± 0.1 nS and shifted the current reversal potential to −25.2 ± 2.3 mV. Insulin-induced β-cell hyperpolarization was sufficient to abolish intracellular calcium concentration ([Ca2+]i) oscillations measured in pancreatic islets exposed to 10 mmol/l glucose. The application of 100 nmol/l wortmannin to inactivate PI 3-kinase, a key enzyme in insulin signaling, was found to reverse the effects of 100 nmol/l insulin. In cell-attached patches, single ATP-sensitive K+ (KATP) channels were activated by bath-applied insulin and subsequently inhibited by wortmannin. Our data thus demonstrate that insulin activates the KATP channels of single mouse pancreatic β-cells and islets, resulting in membrane hyperpolarization, an inhibition of electrical activity, and the abolition of [Ca2+]i oscillations. We thus propose that locally released insulin might serve as a negative feedback signal within the islet under physiological conditions.
Address correspondence and reprint requests to Leslie S. Satin, Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Box 980524, Richmond, VA 23298-0524. E-mail:[email protected]
Received for publication 10 May 2001 and accepted in revised form 2 August 2001. Posted on the World Wide Web at http://www.diabetes.org/diabetes_rapids on 6 September 2001.
[Ca2+]i, intracellular calcium concentration; ED50, 50% effective dose; ER, endoplasmic reticulum; IRS, insulin receptor substrate; KATP, ATP-sensitive K+ channel; NPo, mean channel activity; PI, phosphatidylinositol; R, fluorescence ratio.