A highly energetic process couples calcium influx through L-type calcium channels to insulin secretion in pancreatic beta-cells

Abstract

Calcium (Ca(2+)) influx is required for the sustained secretion of insulin and is accompanied by a large rate of energy usage. We hypothesize that the energy usage reflects a process [Ca(2+)/metabolic coupling process (CMCP)] that couples Ca(2+) to insulin secretion by pancreatic islets. The aim of the study was to test this hypothesis by testing the effect of inhibiting candidate Ca(2+)-sensitive proteins proposed to play a critical role in the CMCP. The effects of the inhibitors on oxygen consumption rate (OCR), a reflection of ATP usage, and insulin secretion rate (ISR) were compared with those seen when L-type Ca(2+) channels were blocked with nimodipine. We reasoned that if a downstream Ca(2+)-regulated site was responsible for the OCR associated with the CMCP, then its inhibition should mimic the effect of nimodipine. Consistent with previous findings, nimodipine decreased glucose-stimulated OCR by 36% and cytosolic Ca(2+) by 46% and completely suppressed ISR in rat pancreatic islets. Inhibitors of three calmodulin-sensitive proteins (myosin light-chain kinase, calcineurin, and Ca(2+)/calmodulin-dependent protein kinase II) did not meet the criteria. In contrast, KN-62 severed the connection between Ca(2+) influx, OCR, and ISR without interfering with Ca(2+) influx. In the presence of nimodipine or KN-62, potentiators of ISR, acetylcholine, GLP-1, and arginine had little effect on insulin secretion, suggesting that the CMCP is also essential for the amplification of ISR. In conclusion, a KN-62-sensitive process directly mediates the effects of Ca(2+) influx via L-type Ca(2+) channels on OCR and ISR, supporting the essential role of the CMCP in mediating ISR.

Fig. 1.

Conceptual design of study to test hypothesized model of the role of calcium (Ca²⁺)/metabolic coupling process (CMCP) in glucose-stimulated insulin secretion. A model by Henquin (18) proposed that influx of Ca²⁺ triggers the release of insulin. Into this framework, we define a CMCP that reflects the large energy (ATP) usage that occurs upon activation of both Ca²⁺ influx through L-type Ca²⁺ channels and metabolic rate (as depicted by the circular symbol denoting summation of the 2 input signals). The model reflects the observation that energy usage associated with amplification and exocytosis of secretory granules is small, and, like triggering of Ca²⁺ influx, activation of the CMCP is essential for insulin secretion to occur. The hypothesis was tested in this study by screening inhibitors of Ca²⁺-sensitive kinases for their ability to mimic the effects of nimodipine on insulin secretion rate (ISR) and oxygen consumption rate (OCR) without affecting Ca²⁺ influx. In addition, the absolute dependency of ISR on CMCP activation was tested by measuring the potentiation of ISR by glucagon-like peptide-1 (GLP-1), acetylcholine (Ach), and arginine (Arg) in the presence of nimodipine or 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) (see text for more detailed description of model and study design).

Fig. 2.

Effect of diazoxide, Bay K 8644, and nimodipine on ISR, OCR, NAD(P)H, and Ca²⁺. Islets were perifused in the presence of 3 mM glucose for 90 min. Glucose was increased to 20 mM at time 0, and diazoxide (50 μM) and Bay K 8644 (10 μM) were applied at 45 and 90 min, respectively. OCR and ISR data were obtained concomitantly from the same perifusions. Samples for ISR were collected every 5 min and measured for indicated time points. The cytosolic Ca²⁺ [represented as ratio of fluorescence (F340/F380)] and normalized NAD(P)H levels were imaged using an inverted fluorescence microscope and digital camera (see materials and methods).

Fig. 3.

Effect of various doses of KN-62 on OCR and ISR. Islets were perifused in the presence of 3 mM glucose for 90 min, and subsequently glucose was increased to 20 mM at time 0. At 45-min intervals, KN-62 was added sequentially at 0.1, 1, and 10 μM, and then to test whether its effects were irreversible, KN-62 was washed out of the perifusion chamber.

Fig. 4.

Effect of nimodipine and KN-62 on glucose-stimulated Ca²⁺ influx. Rates of Ca²⁺ uptake were measured as described in materials and methods in the presence of indicated agents. Results are the average of 3 separate experiments, where results from each individual experiment were normalized to the islet-associated radioactivity obtained at 20 mM glucose. Statistical analysis was performed using ANOVA with a post hoc Bonferonni test. Ca²⁺ influx in the presence 20 mM glucose was significantly different from Ca²⁺ influx in the presence of 3 mM glucose. The presence of nimodipine, but not KN-62, significantly decreased Ca²⁺ influx.

Fig. 5.

Effect of KN-62 and nimodipine on ISR, OCR, NAD(P)H, and Ca²⁺. Conditions were as described in the legend of Fig. 2, except that the protocol used involved applying KN-62 (10 μM) and nimodipine (5 μM) at 45 and 90 min, respectively (A), or nimodipine (5 μM) and KN-62 (10 μM) at 45 and 90 min, respectively (B).

Fig. 6.

Effect of KN-62 and nimodipine on Ca²⁺ (A) and ISR and OCR (B). A: islets were perifused in the presence of 3 mM glucose for 90 min. Glucose was increased to 20 mM at time 0, and at 45 min, nimodipine (10 nM), KN-62 (10 μM), or control solution was applied. Subsequently, at 90 min, 5 μM nimodipine was applied to the islets that had received the test agents. Cytosolic Ca²⁺ is plotted as the fractional change relative to stimulation by 20 mM glucose. Each curve represents the average of multiple perifusions (n = 3, control; n = 4, nimodipine; n = 5, KN-62). B: for measurements of OCR and ISR, conditions were as described in the legend of Fig. 2, except that the protocol used involved applying 10 nM and 5 μM nimodipine at 45 and 90 min, respectively.

Fig. 7.

Effect of KN-62 and nimodipine on ISR, OCR, and Ca²⁺ on unstimulated islets. A: conditions were as described in the legend of Fig. 2, except that the protocol used involved adding KN-62 (10 μM) to the inflow media 45 min prior to glucose being increased to 20 mM and nimodipine (5 μM) being added 45 min after glucose was increased. B: nimodipine (5 μM) was added in the presence of 3 mM glucose.

Fig. 8.

Effect of potentiators of glucose-stimulated insulin secretion in the presence and absence of nimodipine (A) or KN-62 (B) on ISR. ISR was measured on islets incubated in wells of 96-well plates containing the indicated concentrations of glucose, GLP-1 (100 nM), Arg (10 mM), Ach (10 μM), KCl (30 mM), and Bay K 8644 (10 μM) without (filled bars) or with 5 μM nimodipine (A, open bars) or 10 μM KN-62 (B, open bars). Statistical analysis was performed using ANOVA with a post hoc Bonferonni test, and differences were considered significant if P < 0.005. ISR for 3 mM glucose was significantly different from all ISRs in the presence of 20 mM glucose in the absence of inhibitors. ISRs in the presence of 20 mM glucose with inhibitors were statistically different from all ISRs in the absence of inhibitors except for Bay K 8644 + or − KN-62. In the absence of inhibitors, ISRs in the presence of GLP-1, Arg, Ach, KCl, and Bay K 8644 were statistically different from 20 mM glucose alone.

Fig. 9.

Effect of KN-62 and nimodipine on stimulation of ISR, OCR, and Ca²⁺ by Bay K 8644. Conditions were as described in the legend of Fig. 2, except that the protocol used involved applying KN-62 (10 μM) to glucose-stimulated islets, and subsequently, Bay K 8644 (10 μM) and nimodipine (5 μM) were applied at 45-min intervals.