Reduced cytochrome C is an essential regulator of sustained insulin secretion by pancreatic islets

Abstract

Influx of calcium is an essential but insufficient signal in sustained nutrient-stimulated insulin secretion, and increased metabolic rate of the beta cell is also required. The aim of the study was to test the hypothesis that the reduced state of cytochrome c is a metabolic co-factor necessary for insulin secretion, over and above its participation in the ATP-generating function of electron transport/oxidative phosphorylation. We found that nutrient stimulation of insulin secretion by isolated rat islets was strongly correlated with reduced cytochrome c, and agents that acutely and specifically reduced cytochrome c led to increased insulin secretion, even in the face of decreased oxygen consumption and calcium influx. In contrast, neither sites 1 nor 4 of the electron transport chain were both necessary and essential for the stimulation of insulin secretion to occur. Importantly, stimulation of islets with glucose, α-ketoisocaproate, or glyceraldehyde resulted in the appearance of cytochrome c in the cytosol, suggesting a pathway for the regulation of exocytotic machinery by reduction of cytochrome c. The data suggest that the metabolic factor essential for sustained calcium-stimulated insulin secretion to occur is linked to reduction and translocation of cytochrome c.

FIGURE 1.

Hypothesized role of reduced cytochrome c in mediating glucose-stimulated insulin secretion. We previously defined a highly energetic, Ca²⁺-sensitive process that couples the influx of Ca²⁺ to the release of insulin secretion and denoted it as the calcium-metabolic coupling process or CMCP (24). We hypothesize that the CMCP is dually controlled by Ca²⁺ influx through L-type Ca²⁺ channels and a factor related to reduced cytochrome c. The model predicts that Ca²⁺-sensitive OCR and ISR are only activated when both calcium influx is stimulated and cytochrome c is reduced.

FIGURE 2.

Effect of increasing Ca²⁺ influx at 20 mm (A) and 3 mm (B) glucose on cytochrome c reduction, OCR, Ca²⁺, and ISR. Islets were perifused in the presence of 3 mm glucose for 90 min. Subsequently, 10 μm BayK 8644 was added to the in-flow after (A) or before (B) glucose (glc) concentration was raised to 20 mm for 45 min. Third from the top, detection of cytosolic Ca²⁺ by fluorescence imaging (measured in separate experiments). Top, second from top and bottom, OCR, cytochrome c reduction, and ISR were measured concomitantly using the flow culture system. Data are displayed as the change in signal relative to the steady-state value obtained at 3 mm glucose (determined by averaging data obtained in the final 15 min prior to the increase in glucose). Steady-state values of cytochrome c, OCR, and ISR at 3 mm glucose were 28 ± 2.7% (n = 7), 0.31 ± 0.057 nmol/min/100 islets (n = 8), and 0.12 ± 0.038 ng/min/100 islets (n = 10), respectively. 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.

FIGURE 3.

Effect of nutrient secretogogues (KIC and glyceraldehyde) and a blocker of l-type Ca²⁺ channels on NAD(P)H, cytochrome c reduction, OCR, cytosolic Ca²⁺, and ISR. Islets were perifused in the presence of 3 mm glucose (glc) for 90 min; at time = 0, 10 mm KIC (A) or 10 mm glyceraldehyde (B) was added for 45 min; subsequently, L-type Ca²⁺ channel activity was inhibited by nimodipine (5 μm) for 45 min. Top and fourth panel from top, detection of NAD(P)H and Ca²⁺ by fluorescence imaging. Second and third panel from the top and bottom panels: cytochrome c reduction, OCR, and ISR were measured concomitantly using the flow culture system. Data are displayed and analyzed as described in the legend of Fig. 2. For KIC studies, steady-state values of NAD(P)H, cytochrome c reduction, OCR, and ISR at 3 mm glucose were 8.6 ± 2.1 (n = 3), 23 ± 3.9 (n = 5), 0.35 ± 0.056 nmol/min/100 islets (n = 6), and 0.31 ± 0.061 ng/min/100 islets (n = 6), respectively. For the glyceraldehyde experiments, steady-state values of NAD(P)H, cytochrome c reduction, OCR, and ISR at 3 mm glucose were 17 ± 2.7 (n = 5), 22 ± 4.6 (n = 6), 0.39 ± 0.064 nmol/min/100 islets (n = 5), and 0.19 ± 0.095 ng/min/100 islets (n = 8), respectively.

FIGURE 4.

Effect of stimulation of the ETC at the level of cytochrome c oxidase and subsequent blockade of l-type Ca²⁺ channels. A, cytochrome c reduction, cytochrome c oxidase reduction, OCR, and ISR were measured in response to exposure to TMPD/ascorbate (0.1/5 mm) applied for 90 min at 3 mm glucose (glc), followed by the addition of 5 mm nimodipine. Because TMPD/ascorbate interfered with the oxygen-sensitive dye used in the flow experiments, for these OCR measurements, a Seahorse XF 24 was used. For reference, the OCR responses to an increase in glucose and subsequently nimodipine are shown (n = 6). Steady-state values of cytochrome c reduction and ISR at 3 mm glucose were 21 ± 10% (n = 4) and 0.17 ± 0.017 ng/min/100 islets (n = 4), respectively; cytochrome c oxidase was set to 0 at 3 mm glucose due to the difficulty of measuring a difference between this value and that in the presence of the antimycin A that is used for calibration. Due to non-uniform distribution of islets around the oxygen-sensitive dye in the well, well-to-well variation in the OCR data were normalized by dividing it by that obtained at 3 mm glucose. 70 islets were used for each well. 13 wells are included for average (filled circles). B, Ca²⁺ influx was measured in response to activation by 20 mm glucose, TMPD/ascorbate (0.1 mm/5 mm), 10 mm KIC, and 10 mm glyceraldehyde, in the absence or presence of nimodipine (5 mm) using radioactive ⁴⁵Ca²⁺ and normalized with the 3 mm glucose (glc) effect in each experiment. Statistical significance compared with control or 20 mm glucose or TMPD, is indicated by either a single asterisk for p < 0.05 or a double asterisk for p < 0.01. C, cytochrome c reduction, cytochrome c oxidase reduction, OCR, and ISR were measured as described for A, except that TMPD/ascorbate was added after an increase of glucose as indicated. Steady-state values of cytochrome c reduction and ISR at 3 mm glucose were 18.4 ± 3.4% (n = 6) and 0.23 ± 0.02 ng/min/100 islets (n = 5), respectively.

FIGURE 5.

Effect of KCN-induced increase in the reductive state of cytochrome c on ISR and OCR (A) and Ca²⁺ influx (B). A, islets were perifused in the presence of 3 mm glucose (glc) for 90 min; at time = 0, glucose was raised to 20 mm. After 45 min, islets were exposed to incrementally increasing levels of either KCN (1, 3 or 10 μm) or antimycin A (0.6, 1.8, or 6 ng/ml). Cytochrome c reduction, OCR, and ISR were measured concomitantly using the flow culture system. Steady-state values of cytochrome c reduction, OCR, and ISR at 3 mm glucose with KCN were 22 ± 3.2% (n = 6), 0.37 ± 0.035 nmol/min/100 islets (n = 6), and 0.25 ± 0.054 ng/min/100 islets (n = 6), respectively. Steady-state values of cytochrome c reduction, OCR, and ISR at 3 mm glucose with antimycin A were 25 ± 6.5% (n = 3), 0.42 ± 0.095 nmol/min/100 islets (n = 3), and 0.31 ± 0.036 ng/min/100 islets (n = 3), respectively. Steady-state values of cytochrome c reduction, OCR, and ISR at 3 mm glucose alone were 44.8 ± 3.8% (n = 3), 0.23 ± 0.03 nmol/min/100 islets (n = 6), and 0.12 ± 0.04 ng/min/100 islets (n = 6), respectively. As inclusion of two-sided error bars on individual data points made it difficult to distinguish the individual curves, we have shown just one-sided error bars. Inset, statistical comparison of effects of KCN relative to antimycin A at the lowest concentrations on cytochrome c and ISR. B, Ca²⁺ influx was measured in response to activation by 20 mm glucose, in the absence or presence of either 1 μm KCN or 0.6 ng/ml of antimycin A by using radioactive ⁴⁵Ca²⁺ and normalized with the 3 mm glucose effect in each experiment. If statistically significant compared with 20 mm glucose then *, p < 0.05; **, p < 0.01.

FIGURE 6.

Effect of K⁺ (30 mm) on cytochrome c reduction, OCR, and ISR and Ca²⁺ influx, in the presence of 50 μm diazoxide and 20 mm glucose. A, islets were perifused in the presence of 3 mm glucose (glc) and 50 μm diazoxide for 90 min. Diazoxide was present for the duration of the protocol. At time = 0, glucose was raised to 20 mm for 45 min; subsequently, K⁺ was raised from 5 to 30 mm (at the same time Na⁺ was lowered by 25 mm). Cytochrome c reduction, OCR, and ISR were measured concomitantly using the flow culture system. Data are displayed and analyzed as described in the legend of Fig. 2. Steady-state values of cytochrome c reduction, OCR, and ISR at 3 mm glucose were 25 ± 4.2% (n = 6), 0.45 ± 0.080 nmol/min/100 islets (n = 8), and 0.12 ± 0.045 ng/min/100 islets (n = 5), respectively. Normalized ⁴⁵Ca²⁺ influx (B) and static ISR (C) were measured in response to activation by 20 mm glucose, or 30 mm KCl, in the absence or presence of diazoxide (50 μm). Statistical significance compared with 20 mm glucose or 20 mm glucose plus diazoxide were denoted with: *, p < 0.05, and **, p < 0.01. D, islets were perifused as described in A except that K⁺ was raised from 5 to 30 mm prior to switching to 20 mm glucose. Steady-state values of cytochrome c reduction, OCR, and ISR at 3 mm glucose were 26.6 ± 2.1% (n = 6), 0.20 ± 0.02 nmol/min/100 islets (n = 6), and 0.03 ± 0.02 ng/min/100 islets (n = 4), respectively.

FIGURE 7.

Effect of nutrient secretagogues on appearance of cytochrome c in cytosol. Islets (100 per condition) were incubated in the presence of 3 mm glucose in KRB for 4 h and 45 min, before adding the indicated agents and incubating further for 15 min. Islets were then lysed and 30 mg of cytosolic protein were loaded into SDS gels as described under “Experimental Procedures.” Cytochrome c and cytochrome c oxidase subunit IV were detected using antibodies supplied in the kit and densitometry was used to quantify the intensity of the bands. Statistics were carried out comparing the effects of nutrient additions relative to control conditions, or the effect of nimodipine relative to 20 mm glucose, using paired t tests where, * denotes p < 0.05, and ** denotes p < 0.01. No cytochrome c oxidase bands were seen in the cytosolic fractions validating the fractionation process (data not shown).

FIGURE 8.

Summary of steady-state levels of reduced cytochrome c versus ISR under conditions where Ca²⁺ influx is elevated. Legend for conditions: 1, BayK 8644/3 mm Glc (Fig. 2B); 2, 3 mm Glc, 0.1 mm TMPD, 5 mm ascorbate (Fig. 4A); 3, 30 mm K⁺, 3 mm Glc (Fig. 6D); 4, 10 mm glyceraldehyde, 3 mm Glc (Fig. 3B); 5, 10 mm KIC, 3 mm Glc (Fig. 3A); 6, 20 mm Glc (Fig. 2A); 7, 30 mm K⁺, 20 mm Glc/diazoxide (Fig. 6A). Note that these are absolute values not changes as are shown in the graphs.