Insulin Stimulates Membrane Conductance in a Liver Cell Line

Abstract

Activation of insulin receptors stimulates a rapid increase in the ion permeability of liver cells. To evaluate whether this response involves insertion of ion channels, plasma membrane turnover was measured in a model liver cell line using the fluorescent membrane marker FM1-43. Under basal conditions, the rate of constitutive membrane turnover was ϳ2%min ؊1 , and balanced exocytosis and endocytosis maintained the total cell membrane area constant. Exposure to insulin stimulated a transient increase in membrane turnover of up to 10-fold above constitutive rates. The response was concentration-dependent (0.001-10 M). Insulin also caused a parallel increase in membrane conductance as measured by whole-cell patch clamp recording due to opening of Cl ؊ -and K ؉ -selective ion channels. The insulin-stimulated membrane turnover did not appear to involve the constitutive recycling compartments, suggesting that a distinct pool of vesicles may be involved. The effects of insulin on membrane turnover and membrane conductance were inhibited by blockers of phosphoinositide 3-kinase LY294002 and wortmannin or by disrupting microtubule assembly with nocodazole. Taken together, these findings indicate that insulin stimulates recruitment of new membranes through phosphoinositide 3-kinase-dependent mechanisms. Thus, regulated insertion of a separate population of ion channel-containing vesicles may represent one mechanism for mediating the changes in membrane conductance that are essential for the cellular response to insulin.Liver cells exhibit rapid regulation of solute transport across the plasma membrane in response to changing physiological demands. Changes in the concentration of solutes in the extracellular space are followed by increased uptake of glucose, amino acids, and bile acids as well as an increase in the activity of specific enzymes that regulate glucose metabolism and bile formation. While many of the individual transport proteins involved have been molecularly defined, little is known regarding the cellular strategies for regulating the number, type, and activity of transporters in the plasma membrane under defined physiological conditions. In other cells, insertion or retrieval of transport proteins through vesicular exocytosis or endocytosis regulates the protein composition of the plasma membrane (1). In adipocytes, for example, insulin-stimulated glucose uptake is mediated by insertion of glucose transporter GLUT4-containing vesicles, leading to an increase in total transport capacity (2). These effects appear to be mediated in part by phosphoinositide 3-kinase (PI 3-kinase).1 This kinase phosphorylates phosphatidylinositols at the D3 position, and the resulting lipid-signaling molecules have been implicated in recruitment or activation of proteins essential for vesicular transport (3-6). In liver, insulin receptor binding leads to an increase in ion channel activity, and the resulting increases in membrane conductance appear to be an early and essential step necessary for subsequent changes in transport ...

Volume-sensitive Tyrosine Kinases Regulate Liver Cell Volume through Effects on Vesicular Trafficking and Membrane Na+ Permeability

“…6). Under basal conditions in the presence of FM1-43, fluorescence intensity increases at a rate of 2.03 Ϯ 0.07%/min as a result of constitutive exocytosis and continuous exposure of new vesicular membranes to FM1-43 in the bath (11). Exposure of cells to hypertonic media increased the rate of exocytosis to 2.84 Ϯ 0.21% min Ϫ1 (n ϭ 5, p Ͻ 0.01).…”

“…Measurement of Membrane Turnover-The rate of exocytosis was assessed by real-time imaging using a fluorescent probe FM1-43 (Molecular Probes, OR) as described (11). FM1-43 has two specific properties that permit its use in this capacity.…”

“…replace ϳ25% of the plasma membrane within minutes (11). However, the insulin response differs in that exocytosis is sensitive to colchicine and displays different kinetics, with a rapid burst of exocytosis followed after 2-3 min by a return to constitutive levels (11).…”

“…Notably, volumesensitive tyrosine kinases have been shown to modulate the trafficking of epidermal growth factor receptors in liver cells (12); both insulin receptor and p60 c-src activities are closely associated with PI 3-kinase, which is known to regulate exoand endocytosis (11,33). To assess the possibility that membrane trafficking events may contribute to the NSC response, two complementary approaches were utilized.…”

“…To assess the possibility that membrane trafficking events may contribute to the NSC response, two complementary approaches were utilized. First, using patch clamp techniques, NSC currents were measured during hypertonic exposure in the absence versus presence of wortmannin (50 nM), a PI 3-kinase inhibitor known to inhibit exocytosis in HTC cells (11,34) (Fig. 5).…”