Glucose-Dependent Promotion of Insulin Release From Mouse Pancreatic Islets by the Insulin-Mimetic Compound L-783,281

RESEARCH DESIGN AND METHODS

Materials.

Reagents of analytical grade and deionized water were used. Collagenase, HEPES, and bovine serum albumin (fraction V) were obtained from Boehringer Mannheim GmbH (Mannheim, Germany). Tetramethylbenzidine and insulin-peroxidase came from Sigma (St. Louis, MO). The rat insulin standard was from Novo Nordisk (Bagsvaerd, Denmark). Immunoglobulin G–certified microtiter plates were purchased from Nunc (Roskilde, Denmark). The mouse insulin antibodies were raised in guinea pigs in our laboratory. The insulin analog L-783,281 was obtained from Merck.

Preparation and culture of islets.

Pancreatic islets were collagenase isolated from C57Bl/6j mice (B&K, Sollentuna, Sweden). The islets were cultured for 1 to 4 days in 11 mmol/l glucose in RPMI culture medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 μg/ml strepotomycin, and 30 μg/ml gentamicin. The experiments were approved by the local animal ethics committee.

Perifusion of islets.

The kinetics of insulin release were studied essentially as described previously (12). A single islet was placed in a 10-μl chamber (thermostat at 37°C) and perifused at a constant flow rate with medium containing 3 mmol/l glucose. The experiments were performed in medium supplemented with 1 mg/ml albumin and containing (in mmol/l): NaCl 125, KCl 5.9, MgCl₂ 1.2, CaCl₂ 1.3, and HEPES 25, titrated to pH 7.4 with NaOH. The flow rate was kept constant throughout each experiment with the aid of a peristaltic pump placed before the islet. Flow variation of 150 to 200 μl/min was allowed between experiments. After 60 to 75 min of introductory perifusion, the perifusate was collected in 20-s fractions directly into microtiter plates.

Insulin measurements.

Insulin was assayed by a competitive enzyme-linked immunosorbent assay (ELISA) with the insulin antibody immobilized directly onto the solid phase. Amounts of insulin down to 100 amol were obtained from linear standard curves in semilogarithmic plots. The insulin-mimetic compound did not interfere with the insulin assay in the concentrations used in the experiments (data not shown).

Data analysis.

Frequency determination of insulin pulses was done by Fourier transformation using the Igor software (Wave Metrics Inc, Lake Oswego, OR). Determination of significant insulin pulses was based on the signal-to-noise ratio as described previously (13).

Statistical analysis.

Results are presented as means ± SE. Differences in rates of secretion and frequencies were evaluated with Students’ two-tailed t test for paired observations.

RESULTS

Insulin release from individual islets perifused in the presence of 3 mmol/l glucose was 10.5 ± 1.4 pg/min and pulsatile (0.35 ± 0.03 oscillations/min) (Fig. 1). When 10 μmol/l L-783,281 was added to the perifusion medium, there was no change in the rate of secretion, but the pulse frequency decreased significantly (P < 0.02) to 0.22 ± 0.04 oscillations/min. Islets were also perifused in the presence of 11 mmol/l glucose (Fig. 2). Upon introduction of the higher glucose concentration, an initial accentuated peak of insulin was followed by insulin pulses with declining amplitudes (not shown). After 30 min, L-783,281 was added to the perifusion medium containing 11 mmol/l glucose. The addition of the compound increased insulin release from 15.6 ± 4.8 to 64.3 ± 16.9 pg/min. The effect of L-783,281 on the pulse frequency was heterogeneous, and no statistical change could be established.

DISCUSSION

In the present study, we show that the insulin-mimetic compound L-783,281 increases the rate of glucose-stimulated but not basal insulin release from the individual islet of Langerhans measured by an immunological method. At basal but not stimulatory glucose concentration, the compound clearly modulated the frequency of the pulsatile insulin release. L-783,281 was used to obtain β-cell IR activation without applying insulin, which would not have been distinguished from secreted insulin by the assay. The insulin mimetic, which was recently described (14), is a nonpeptidyl metabolite derived from a fungal extract that activates the IR tyrosine kinase, stimulates insulin gene expression, and decreases blood glucose levels in murine models of type 2 diabetes (14,15). In a previous study, insulin-stimulated exocytosis in β-cells measured by amperometry was similarly stimulated at basal and stimulatory glucose concentrations (11). Whether the conflicting results with regard to the glucose-dependency of insulin stimulation on insulin release in the present study depend on the usage of L-783,281 rather than insulin remains to be elucidated.

The mechanisms involved in insulin-induced insulin release have been proposed to involve regulation of the intracellular Ca²⁺ homeostasis via SERCA3 (SERCA, sarco/endoplasmic reticulum calcium ATPase) (15,16). This concept was supported by findings of insulin-induced cytoplasmic Ca²⁺ mobilization from the endoplasmic reticulum (17). The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) plays a pivotal role in the regulation of glucose-induced insulin release (18). Oscillations in [Ca²⁺]_c are closely followed by pulses of insulin release (19). The [Ca²⁺]_c oscillations, which appear at stimulatory glucose concentrations, have been found to depend on paracrine factors promoting mobilization of Ca²⁺ from intracellular stores (20). The L-783,281–induced change in pulse frequency was observed at basal glucose, when [Ca²⁺]_c is nonoscillatory. If β-cell IR activation at basal glucose also induces changes in [Ca²⁺]_c, these changes may be important for the observed alteration in frequency. Insulin resistance in type 2 diabetes may be associated with impaired handling of Ca²⁺ in the endoplasmic reticulum (15,16). Such impaired handling may contribute to the deranged plasma insulin pattern observed in such individuals (21). In healthy subjects, blood insulin levels fluctuate regularly (22).

The importance of the expression of the β-cell IR was also apparent when the receptor was selectively knocked out in the β-cell in the so-called βIRKO mouse, which displays a secretory response to glucose similar to that of type 2 diabetes (23). In contrast to other autocrine feedback systems, the β-cells seem to signal via positive feedback. This seemingly dangerous self-promoting secretion of the hypoglycemic hormone may be explained by the present observation of glucose dependency of the β-cell IR–mediated effect.