Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7-37)

Abstract

Non-insulin-dependent diabetes mellitus (NIDDM, type 2 diabetes) is a disorder of glucose homeostasis characterized by hyperglycaemia, peripheral insulin resistance, impaired hepatic glucose metabolism, and diminished glucose-dependent secretion of insulin from pancreatic beta-cells. Glucagon-like-peptide-1(7-37) (GLP-1) is an intestinally derived hormone that may be useful for the treatment of NIDDM because it acts in vivo to increase the level of circulating insulin, and thus lower the concentration of blood glucose. This therapeutic effect may result from the ability of GLP-1 to compensate for a defect in the glucose signalling pathway that regulates insulin secretion from beta-cells. In support of this concept we report here that GLP-1 confers glucose sensitivity to glucose-resistant beta-cells, a phenomenon we term glucose competence. Induction of glucose competence by GLP-1 results from its synergistic interaction with glucose to inhibit metabolically regulated potassium channels that are also targeted for inhibition by sulphonylurea drugs commonly used in the treatment of NIDDM. Glucose competence allows membrane depolarization, the generation of action potentials, and Ca2+ influx, events that are known to trigger insulin secretion.

FIG. 1

Synergism of glucose and GLP-1 to depolarize β-cells. a, Trace 1: membrane potential recordings from a β-cell exhibiting weak sensitivity to 10 mM glucose repeatedly applied for 30 s as indicated. Trace 2: recordings from the same cell during application of 10 nM GLP-1 (left) or combined application of 10 mM glucose and 10 nM GLP-1 (right). Initial resting potential (V_m)−62 mV. b, trace 1: a cel1 that initially failed to respond to 10 mM glucose, but which responded to 10 mM glucose after pretreatment with 10 nM GLP-1 (V_m −66 mV). Trace 2: a cell that initially failed to respond to 10 nM GLP-1, but which responded to 10 nM GLP-1 after pretreatment with 10 mM glucose (V_m −60 mV). The initial insensitivity of β-cells to glucose or GLP-1 did not result from their failure to express functional l_KATP channels because application of 10 nM glyburide resulted in rapid membrane depolarization (42 ± 11 mV, ±s.e.m., n = 10 cells), as illustrated (b, traces 1,2). b, −5 pA of hyperpolarizing pipette current was applied to repolarize the membrane after application of 10 nM glyburide. Repolarization confirms the integrity of the membrane/pipette seal. c, Cumulative dose–response analysis summarizing the interaction of glucose and GLP-1 to depolarize β-cells. The action of GLP-1(7-37) but not GLP-1(8-37) exhibited dose-dependence under conditions in which the test solution also contained 10 mM glucose (left). In contrast, the action of GLP-1(7-37) but not 10 nM glyburide was abrogated when the test solution contained no added glucose (right). Statistical significance was determined by Student's t-test. Probability values (*P ⩽ 0.05, **P ⩽ 0.005) are expressed relative to control (10 mM glucose and no GLP-1). c, The number of cells tested was 50 for GLP-1(7-37) and 10 mM glucose, 25 for GLP-1(8-37) and 10 mM glucose, 20 for GLP-1(7-37) and 0 mM glucose, and 20 for glyburide and 0 mM glucose. The EC₅₀ value for GLP-1(7-37) was calculated as 1 nM by establishing a cumulative dose–response relationship in which the action of the peptide was assessed over a concentration range of 0.01–100 nM. The EC₅₀ was defined as the concentration of GLP-1(7-37) that depolarized 50% of the cells by ⩾20 mV when the cells were simultaneously challenged with 10 mM glucose. METHODS. Islets were isolated from pancreata of male rats (200–250 g) by collagenase digestion, suspended in culture medium (RPMI 1640 containing 11.1 mM glucose, 10% fetal bovine serum, 100 μg ml⁻¹ streptomycin, 100 U ml⁻¹ penicillin G), and maintained in culture 1–4 days. Single-cell suspensions were prepared by trypsin–EDTA digestion and mechanical dispersion of cultured islets, and cells were plated on Falcon tissue culture dishes coated with concanavalin-A for short-term (⩾16 h) culture. Under these conditions, ⩾90% of the 10–15 μm diameter cells secrete insulin, as determined by the reverse haemolytic plaque assay, and are therefore considered to be β-cells. The fact that ~80% of our cells responded to glyburide indicates that the primary rat cell cultures are indeed enriched with β-cells. For perforated-patch recordings the pipette solution contained 240 μg ml⁻¹ nystatin and (in mM): 10KCl, 10NaCl, 70K₂SO₄, 2MgCl₂, 10HEPES (pH 7.35, 295 mOsm). In Figs 1-3 the bath solution contained 138 NaCl, 5.6 KCl, 1.2 MgCl₂, 2.6 CaCl₂, 10 HEPES (pH 7.35, 300 mOsm), and was maintained at 32 °C by a peltier device mounted on the stage of an inverted microscope. All recordings were obtained from solitary cells not organized in clusters, and recordings were initiated within 30 min after transfer of the cells to the glucose-free solution. Pipettes were prepared from borosilicate glass capillary tubes and fire polished to tip resistances of 2–8 MΩ. Measurements were made using an EPC-9 amplifier (bandwidth 10 kHz) interfaced with the Instrutech Data Aquisition and Analysis system (Instrutech Corp., Mineola, NY). The signal was stored on videotape, low-pass filtered (0.5–3.0 kHz, 4-pole Bessel filter, −3 dB attenuation), digitized (100 Hz for recordings of the membrane potential, 5–10 kHz for unitary current recordings), and selected recordings were sampled. Following seal formation, perforation was achieved within 5–10 min, at which time values for the series resistance (R_s), slow capacitance compensation (C_s), and the resting membrane potential were 10–20 MΩ, 6–8 pF and −61 ± 5 mV (n = 50), respectively. No correction was made for liquid junction potentials. Test substances were applied by pressure ejection from ‘puffer’ pipettes. The kinetics of this delivery system were established by measuring the rates of onset (time constant, τ, 1.5 s) and offset (τ, 10 s) of 60 mM KCl-induced depolarizations. The change in membrane potential in response to glucose or GLP-1 was measured as the difference between the resting potential and the plateau potential. GLP-1 was obtained from Scios Nova.

FIG. 2

A frequency-of-response histogram (a) and scatter plot (b) analysis summarizing the interaction of glucose and GLP-1 to depolarize β-cells. When initially challenged with 10 mM glucose the majority of cells exhibited a ⩽15mV depolarizing response, as indicated by either the fully shaded histogram bars in a, or the position of the triangles relative to the x-axis in b (where one triangle equals one cell except for the n = 4 cells triangle). When subsequently challenged with a combined application of 10 mM glucose and 10 nM GLP-1, the distribution of the histogram plot was shifted to the right (a, cross-hatched bars) and a subpopulation of β-cells labelled set 3 was rendered glucose competent (b, as indicated by the position of the triangles relative to the y-axis). In contrast, cells that comprised set 2 exhibited a dose-dependent depolarizing response to glucose over a concentration range of ~7–20 mM glucose and were constitutively glucose competent. Each illustration depicts results obtained from the same 25 cells. Test substances were applied for 30 s at 10-min intervals, and only cells exhibiting a resting membrane potential of at least −55 mV were included in the data analysis. More prolonged (3–5 min) application of 10 or 20 mM glucose (without GLP-1) to glucose-insensitive β-cells that comprised sets 1 and 3 did not significantly increase the magnitude of their depolarizing response. Such glucose resistance may reflect metabolic dysfunctions resulting from disaggregation of the islets and the loss of cell-to-cell contacts, or alternatively the loss of paracrine/endocrine influences that regulate glucose responsiveness.

FIG. 3

Glucose and GLP-1 synergize to decrease the resting membrane conductance and inhibit the activity of single l_KATP channels, a, The membrane conductance was monitored by perforated-patch recording in the voltage clamp mode under conditions in which the bath solution contained no added glucose. Application for 30 s of either 10 mM glucose or 10 nM GLP-1 was without significant effect on the magnitude of evoked current responses to ±10 mV voltage steps from a holding potential of −80 mV, whereas the evoked currents were reversibly inhibited by simultaneous application of 10 mM glucose and 10 nM GLP-1. The conductance decreased 80% from 2.0 nS to 0.4 nS and no shift in the holding current was observed. Outward currents are indicated by upward deflections, b, Cell-attached patch recordings of unitary inward currents measured when the bath solution contained no added glucose (traces 1 and 4), 20 mM glucose (trace 2), 20 mM glucose and 10 nM GLP-1 (trace 3), or 10 nM glyburide (trace 5), each applied for 30 s. Inward currents are indicated by downward deflections from a closed level (C) to three superimposed levels of openings (0₁–₃). Filter 1 kHz, sample rate 5kHz. c, The l–V relationship for unitary currents recorded in the cell-attached patch configuration. The slope of the l–V relationship decreased at very negative pipette potentials (inward rectification), reversed direction when the pipette potential (V_p) was more negative than −70 mV, and the single channel conductance inferred from the slope of the linear portion of the l–V relationship indicated a value of 60 pS, as expected for l_KATP, d, Histogram analysis summarizing the actions of glucose, GLP-1, and glyburide, as illustrated in b, traces 1–5, to inhibit l_KATP. The effects of these test substances were assessed by determining the frequency of occurrence of 500-ms oscilloscope traces that exhibited no channel activity (blanks). Fifty traces were recorded before, during the peak effect, and after recovery from each test substance. METHODS, a, For measurements of the resting membrane conductance the command potential was shifted by ±10 mV for 1.5 s at a frequency of 0.1 Hz. The conductance was monitored while simultaneously compensating for the series resistance by 80%. Glucose and GLP-1 were applied at 4-min intervals to avoid priming effects that are observed at shorter intervals, b, For cell-attached-patch recordings the pipette solution contained (in mM): 140 KCI, 5CaCI₂, 5MgCI₂, 10 HEPES (pH adjusted to 7.35 with KOH, 305 mOsm) and V_p was +50 mV. Test substances were applied at 10-min intervals, and the current traces illustrated are representative of the peak effects observed. In b, trace 2, the relatively small effect of 20 mM glucose on channel activity was accompanied by a decreased unitary current amplitude, suggestive of a decreased driving force for K⁺, possibly due to a depolarizing action of glucose at the whole-cell level. The decreased unitary current amplitude observed in trace 2 may therefore suggest an action of glucose to depolarize β-cells, not simply by inhibiting l_KATP channels, but also by inducing an uncharacterized conductance change occurring in the membrane outside the patch.