Control of insulin secretion by cytochrome C and calcium signaling in islets with impaired metabolism

Abstract

The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal.

Keywords: Calcium; Calcium Signaling; Cytochrome c; Cytochrome c Translocation; Insulin Secretion; Mitochondrial Metabolism; Oxygen Consumption; Pancreatic Islet.

FIGURE 1.

KIC- and glucose-stimulated ISR by islets cultured in RPMI 1640 medium containing either 11 mm (A) or 3 mm (B) glucose for 40 h. Islets were perifused in the presence of 3 mm glucose for 90 min. Subsequently, 10 mm KIC was added and removed, and 20 mm glucose and nimodipine were added to and removed from the inflow at the times shown on the graph. Fractions were collected and insulin was assayed to determine the ISR (average ± S.E., n = 4). Statistical analysis was carried out by comparing steady-state values (determined by averaging data obtained in the final 15 min of each experimental condition) before and after each change in medium composition using a paired t test (*, p < 0.05; **, p < 0.01).

FIGURE 2.

KIC- and glucose-stimulated OCR by islets cultured in RPMI 1640 medium containing either 11 mm or 3 mm glucose for 40 h. Islets were perifused in the presence of 3 mm glucose for 90 min. Subsequently, 10 mm KIC was added and removed, and 20 mm glucose and nimodipine were added to and removed from the inflow at the times shown on the graph. *, p < 0.05; **, p <0.01. Statistics were also calculated comparing islets cultured at 3 and 11 mm glucose. Difference in response to KIC is nonsignificant; responses to 20 mm glucose and nimodipine were different for the two culture conditions (p < 0.05).

FIGURE 3.

KIC- and glucose-stimulated cytosolic Ca²⁺ or ⁴⁵Ca²⁺ influx by islets cultured in RPMI 1640 medium containing either 11 mm or 3 mm glucose for 40 h. A and B, islets were perifused in the presence of 3 mm glucose for 90 min. Subsequently, 10 mm KIC was added and removed, and 20 mm glucose and nimodipine were added to and removed from the inflow at the times shown on the graph. Cytosolic Ca²⁺ was imaged fluorescently in real time and displayed as the change in relative fluorescence relative to the steady-state value obtained at 3 mm glucose (average ± S.E., n = 3). *, p < 0.05; **, p < 0.01. C, Ca²⁺ influx was measured on islets in 24-well plates in the presence of the indicated compounds. Analysis of variance with a Bonferroni's post hoc test was carried out on all conditions; no differences were seen between the islets cultured at 3 mm glucose versus those cultured at 11 mm glucose.

FIGURE 4.

Effect of L-type Ca²⁺ channel flux on glucose-stimulated cytosolic Ca²⁺ (A) and ISR (B) by islets cultured in RPMI 1640 medium containing 3 mm glucose for 40 h. Islets were perifused in the presence of 3 mm glucose for 90 min. Subsequently, glucose was raised to 20 mm glucose followed by 10 μm BayK8644 and by 5 μm nimodipine for the Ca²⁺ measurements. Cytosolic Ca²⁺ was imaged fluorescently in real time and displayed as the change in relative fluorescence relative to the steady-state value obtained at 3 mm glucose (average ± S.E., n = 3). *, p < 0.05; **, p < 0.01.

FIGURE 5.

KIC- and glucose-stimulated reduced cytochrome c by islets cultured in RPMI 1640 medium containing either 11 mm or 3 mm glucose for 40 h. Islets were perifused in the presence of 3 mm glucose for 90 min. Subsequently, 10 mm KIC was added and removed, and 20 mm glucose and nimodipine were added to and removed from the inflow at the times shown on the graph. Data are displayed as the change in cytochrome c reduction relative to a blank and maximal signal (in the presence of antimycin A and KCN) (average ± S.E., n = 4). Asterisks denote significance of the change relative to the preceding condition as described in the legend for Fig. 1. *, p < 0.05; **, p <0.01. Statistics were also calculated comparing islets cultured at 3 and 11 mm glucose. Difference in response to KIC is nonsignificant; responses to 20 mm glucose and nimodipine were different for the two culture conditions (p < 0.05).

FIGURE 6.

Effect of a GKa on cytochrome c reduction, OCR, ISR, and cytosolic Ca²⁺, in islets with impaired glucose-stimulated ISR. A–C, islets cultured for 40 h in the presence of 3 mm glucose were perifused in the presence of 3 mm glucose for 90 min. Subsequently, 10 mm KIC was added and removed, and 20 mm glucose and the GKa were added to the inflow at the times shown on the graph. Cytochrome c reduction, OCR, and ISR were measured concomitantly using the flow culture system and expressed as average ± S.E., n = 4. OCR data are displayed as the change in OCR relative to the steady-state value obtained at 3 mm glucose. Steady-state values of OCR at 3 mm glucose were 0.11 ± 0.03 nmol/min/100 islets (n = 4). D, effect of GKa on Ca²⁺-sensitive OCR by islets cultured at 3 mm glucose for 40 h. Islets were perifused in the presence of 3 mm glucose for 90 min. Subsequently, 20 mm glucose, a GKa, and a Ca²⁺ channel blocker (nimodipine) were added sequentially to the inflow at the times shown on the graph. OCR and Ca²⁺ were expressed as average ± S.E., n = 4. OCR data are displayed as the change in OCR relative to the steady-state value obtained at 3 mm glucose (0.19 ± 0.04 nmol/min/100 islets (n = 4)). E, comparison of Ca²⁺-sensitive OCR by islets cultured for 40 h in 11 mm glucose or 3 mm glucose. Large black symbols from Fig. 3, OCR response to nimodipine from in islets cultured at 3 mm glucose; small black symbols from panel D), islets cultured at 3 mm glucose and exposed to the GKa; gray symbols from Fig. 2), islets cultured at 11 mm glucose. Data were normalized by subtracting the steady-state value for the final 5 min prior to the exposure to nimodipine. F, effect of GKa on Ca²⁺ by islets cultured at 3 mm glucose for 40 h. Islets were perifused in the presence of 3 mm glucose for 90 min. Subsequently, 20 mm glucose, the GKa, and an L-type Ca²⁺ channel blocker (nimodipine) were added sequentially to the inflow at the times shown on the graph. Ca²⁺ was expressed as average ± S.E., n = 4 (*, p < 0.05; **, p < 0.01).

FIGURE 7.

Effect of glucose, a GKa, and staurosporine on appearance of cytochrome c in cytosol. Extracted cytosolic protein from islets following permeabilization was analyzed by Western blot for the presence of cytochrome c, and the density of the cytochrome c bands was quantified. The blot was subsequently reprobed for actin. A, rat islets (500 per condition) were incubated in the presence of the indicated agents (n = 4). Extracts equivalent to 200 islets were loaded on to the gel. Due to the variability in quantifying Western bands, data were normalized to the band densities generated in response to 3 mm glucose; the ratio of the bands was 1.04 ± 0.28 for islets cultured at 3 mm versus 11 mm glucose, which was not statistically different from 1. *, p < 0.05; **, p < 0.01. B, mouse islets harvested from either Bax/Bak DKO or control mice were incubated in the presence of 20 mm glucose (500 islets) as in A or cultured overnight in the presence of 3 μm staurosporine (60 islets) (n = 3). Extracts equivalent to 30 islets were loaded on to the gel. Note that the actin bands for staurosporine-treated islet extracts were not visible. Statistics were carried out comparing the effects of agents as compared with their normalized controls using paired t tests where * denotes p < 0.05. NS indicates that difference in response to 20 mm glucose is nonsignificant.

FIGURE 8.

Effect of fasting on ISR (A), OCR (B), calcium (C), and cytochrome c signaling (D). Islets were harvested from rats that were either fed or fasted for 40 h prior to isolation. Perifusions of the islets were initiated within 1 h of isolation using the same protocol used for islets cultured for 40 h. Data are displayed as the average ± S.E., where OCR, cytochrome c, and ISR have n = 4–7, and Ca²⁺ data are n = 3–4 (*, p < 0.05; **, p < 0.01).