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In brain, muscle, and pancreatic islets, depolarization induces an increase in respiration, which is dependent on
calcium influx. The goal of this study was to assess the
quantitative significance of this effect in islets relative to
glucose-stimulated ATP turnover, to examine the molecular
mechanism mediating the changes, and to investigate the
functional implications with respect to insulin secretion.
Glucose (320 mmol/l) increased steady-state levels of cytochrome c reduction (32 66%) in isolated rat islets, reflecting
an increased production of NADH, and oxygen consumption
rate (OCR) by 0.32 nmol/min/100 islets. Glucose-stimulated
OCR was inhibited 30% by inhibitors of calcium influx (diazoxide or nimodipine), whereas a protein synthesis inhibitor
(emetine) decreased it by only 24%. None of the inhibitors
affected cytochrome c reduction, suggesting that calciums
effect on steady-state OCR is mediated by changes in ATP
usage rather than the rate of NADH generation. 3-isobutyl-1methylxanthine increased insulin secretion but had little
effect on OCR, indicating that the processes of movement and
exocytosis of secretory granules do not significantly contribute to ATP turnover. At 20 mmol/l glucose, a blocker of
sarcoendoplasmic reticulum calcium ATPase (SERCA) had
little effect on OCR despite a large increase in cytosolic
calcium, further supporting the notion that influx of calcium,
not bulk cytosolic calcium, is associated with the increase in
ATP turnover. The glucose dose response of calcium influx
dependent OCR showed a remarkable correlation with insulin secretion, suggesting that the process mediating the effect
of calcium on ATP turnover has a role in the amplification
pathway of insulin secretion. Diabetes 55:3509 3519, 2006
Perifusion system. A perifusion system was used that allows for simultaneous measurement of OCR and cytochrome c reduction and collection of
outflow fractions for subsequent measurement of ISR. Handpicked rat islets
(n 750) were loaded into the chamber with Cytodex beads (Amersham
Biosciences, Piscataway, NJ) and sandwiched between two layers of Cytopore
beads (Amersham Biosciences), as previously described in detail (14,37,38).
Flow rate was set to 80 l/min for all perifusion experiments; chamber
volume was 400 l. Data were corrected for delays in the flow system by
referencing the insulin measurements to glucose measured in the outflow
fractions, and the OCR and cytochrome c reduction data were referenced to
the response to antimycin A, which was given at the end of each flow culture
experiment, as previously described (14,29,30). However, data were not
corrected for dispersion of the signals, and, consequently, the temporal
resolution of the kinetic responses is limited to 4 5 min.
Measurement of OCR. OCR was calculated as the flow rate times the
difference between inflow and outflow oxygen tension, which was measured
by detecting the phosphorescence lifetime of an oxygen-sensitive dye that was
painted on the inside of the perifusion chamber (38). Unlike the previous
report (38), phosphorescent lifetimes were monitored using either a PMOD
5000 Frequency Domain Phosphorometer (Oxygen Enterprises, Philadelphia,
PA) or an MFPF-100 multifrequency phase fluorometer lifetime measurement
system made by TauTheta Instruments (Boulder, CO) where the end of the
excitation light guide (one-eighth inch; Edmund Industrial Optics, Barrington,
NJ, or a 2-mm fiberoptic patch cord; TauTheta part no. SFO-026) illuminated
by a 405-nm light emitting diode was just touching the outside of the glass
opposite where the dye was painted and the detecting light guide situated at
a 90 angle. For the Oxygen Enterprise System, a 48-kHz, 16-bit Sigma-Delta
digitizer was used to average the phosphorescence signal (700 nm) over 10 ms
per scan. Ten scans were performed at each measurement point, and the
signal was averaged over a 100-ms interval; lifetime was calculated as
described (39) for each interval, with measurements being repeated every
30 s. The MFPF100 measured the luminescent lifetime of phosphorescence
using an avalanche photodiode where the light emitting diode was sinusoidally modulated at 5 kHz and phase shift measured over an interval of 0.5 s
and repeated every 30 s.
Measurement of cytochrome c reduction. Absorption due to cytochrome c
was measured by light transmission at 550 nm through the bed of islets/
Cytodex beads, as described previously (14).
Imaging and quantification of cytosolic calcium. Cytosolic calcium was
measured as reflected by fluorescence detection as described by Hille and
colleagues (40), except that fura-2AM was used instead of indo-1. Briefly, islets
were loaded with dye by incubating them in 4 mol/l fura-2AM (Invitrogen,
Carlsbad, CA) for 20 min at 37C. Subsequently, the islets were pipetted into
a temperature-controlled perifusion dish (Bioptechs, Butler, PA) next to a
semicircle-shaped section of 650 micron-OD glass capillary tubing that maintained the position of the islets. The perifusion dish was mounted onto the
stage of a Nikon Eclipse TE-2000-U inverted microscope. Fluorescent emission was detected at 510 nm by a Pixelfly camera (PCO Imaging, Kelheim,
Germany) during alternating excitation at either 340 or 380 nm.
Protein synthesis. The amount of L-[35S]-methionine incorporated into the
TCA-precipitable fraction was determined as follows. Islets were preincubated for 60 min in Krebs-Ringer bicarbonate solution containing 20 mmol/l
glucose and subsequently for 60 min in the presence of [35S]-methionine (4.1
Ci), either with or without 5 mol/l nimodipine or in the presence of varying
amounts of emetine. Thereafter, 300 l of 25% TCA was added. Samples were
vortexed for 10 s and then centrifuged for 3 min at 3,270g. The supernatant
was removed, 400 l of 5% TCA was added, and the samples were again
vortexed and centrifuged. After aspirating the supernatant, 50 l of 0.5% SDS
was added and samples placed on a rocker at 4C until the pellet dissolved
(2 h). The amount of cell-associated radioactivity was determined as
previously described (41).
Insulin measurements. Insulin was measured by enzyme-linked immunosorbent assay per the manufacturers instruction (ALPCO, Windham, NH).
Protocols and data analysis. The basic protocol for the perifusion experiments entailed a 90-min baseline period at 3 mmol/l glucose, followed by
stimulation with 20 mmol/l glucose (45 min) and 45 min in the presence of an
effector of the process of interest. In some experiments, a second agent that
reversed the effect of the inhibitor was added to the perifusate during an
additional 40-min period. To assess the effects of agents, steady-state values of
OCR and cytochrome c reduction were calculated by averaging a portion of
each kinetic curve obtained before and after the agent came in contact with
the islets in the chamber. Typically, this portion was the last 15 min before the
next change in perifusate composition, except where noted. Thus, steadystate averages were calculated at 3 mmol/l glucose from 15 to 0 min, at 20
mmol/l glucose from 30 to 45 min, for agent 1 from 75 to 90 min, and for agent
2 from 115 to 130 min. ISR, which does not actually reach a steady state, was
DIABETES, VOL. 55, DECEMBER 2006
FIG. 2. Control studies: concomitant measurement of OCR, cytochrome c reduction, and ISR in the absence of effectors. Islets were perifused with
buffer containing 3 mmol/l glucose for 90 min. Glucose was increased to 20 mmol/l, and parameters were measured continuously for a time
spanning both experimental protocols used in subsequent experiments (average of six perifusions).
also averaged for each perifusion composition, except time windows of 25
to 0, 20 45, 6590, and 105130 min were used.
The following equations were then used to calculate the steady-state
changes induced by agent 1 relative to glucose-stimulated OCR, cytochrome c
reduction, and ISR.
OCRagent 1 OCR20 mM glc
OCRagent 1
100
100
OCRglc
OCR20 mM glc OCR3 mM glc
(1)
(2)
100
ISRagent 1
ISRagent 1 ISR20 mM glc
100
ISR20 mM glc
ISR20 mM glc
(3)
When a second agent was tested, the steady-state changes were calculated as
follows:
100
OCRagent 2 OCRagent 1
OCRagent 2
100
OCR glc
OSR20 mM glc OCR3 mM glc
ISRagent 2
ISRagent 2ISRagent 1
100
ISR20 mM glc
ISR20 mM glc
(4)
(5)
(6)
For OCR and cytochrome c reduction data, unpaired t tests were used to
compare steady-state changes in response to an agent to control studies at
identical time points where the agents were not present. For the ISR data, due
to the variability in responses that were typically seen, the requirement for
normally distributed data was not met, and an unpaired Mann-Whitney U test
DIABETES, VOL. 55, DECEMBER 2006
was used. Calculations for both tests were carried out using Kaleidagraph
(Synergy Software, Reading, PA).
RESULTS
TABLE 1
Contribution of calcium and insulin secretion to glucose-stimulated OCR, cytochrome c reduction, and ISR
Target process
Agent
Calcium influx
Nimodipine (n 7)
Diazoxide (n 9)
Glibenclamide (n 9)*
A23187 (n 5)
Insulin secretion
Clonidine (n 4)
IBMX (n 4)
Calcium uptake by ER
CPA (n 8)
Protein synthesis
Emetine (n 6)
Control (agent 1)
None (n 6)
Control (agent 2)
None (n 6)
OCR
(% change from
Eqs. 1 and 4)
Cytochrome c reduction
(% change from
Eqs. 2 and 5)
ISR
(% change from
Eqs. 3 and 6)
1.9 3.0
1.7 1.9
28.1 13.3
0.9 3.5
1.5 2.0
2.1 16.9
Data are average SE or average SE (P value). P values were generated either from an unpaired t test (OCR and cytochrome c data) or
Mann-Whitney U test (ISR data), relative to the control experiments. Steady-state calculations were made on data from each experiment (the
composites of which are shown in Figs. 27) using Eqs. 1 6. Glybenclamide was tested following diazoxide (Fig. 3B), and IBMX was tested
following clonidine (Fig. 4). For A23187, since the effect on OCR was transient, OCRagent1 was calculated as the average from 53 to 60 min
(Fig. 5). ER, endoplasmic reticulum.
FIG. 3. Effect of inhibition of calcium influx on OCR, cytochrome c reduction, and ISR. Forty-five minutes after islets were stimulated with 20
mmol/l glucose, calcium influx was inhibited either by direct inhibition of L-type calcium channels (5 mol/l nimodipine; A) or by opening the KATP
channels (50 mol/l diazoxide; B) (average of seven [A] or nine [B] perifusions). In the latter case, glybenclamide (1 mol/l) was subsequently
added to the inflow buffer.
3513
FIG. 4. Effect of increasing influx of calcium via an ionophore, in the presence of an L-type calcium channel blocker. Islets were perifused in the
presence of 3 mmol/l glucose and 5 mol/l nimodipine for 90 min; at t 0, glucose concentration was raised to 20 mmol/l for 45 min; subsequently,
10 mol/l A23198 was added. OCR (top), ISR (middle), and cytochrome c reduction (data not shown) were measured concomitantly (average of
five perifusions). Bottom: Detection of fluorescence from a calcium-sensitive dye, using a similar temporal protocol except that each time period
lasted 10 min as indicated (average of responses by three islets).
FIG. 5. Inhibition of protein synthesis by emetine decreased glucose-stimulated OCR. Forty-five minutes after islets were stimulated with 20
mmol/l glucose, protein synthesis was inhibited with 10 mol/l emetine (average of six perifusions).
FIG. 6. Effect of an 2-adrenergic agonist and a phosphodiesterase inhibitor on OCR, cytochrome c reduction, and ISR. Forty-five minutes after
stimulating islets with glucose, ISR was inhibited by 1 mol/l clonidine and subsequently stimulated with 0.25 mmol/l IBMX (average of four
perifusions).
FIG. 7. Effect of inhibition of SERCA. Using the flow culture system, islets were perifused in the presence of 3 mmol/l glucose for 90 min; at t
0, glucose was raised to 20 mmol/l for 45 min; subsequently, 50 mol/l CPA was added. OCR (top), ISR (middle), and cytochrome c reduction (data
not shown) were measured concomitantly (average of four perifusions). Bottom: Detection of fluorescence from a calcium-sensitive dye, using
a similar temporal protocol except that each time period lasted 20 min (average of responses by three islets).
FIG. 8. Glucose dependency of OCR, ISR, and calcium influx dependent OCR. Using the protocol in Fig. 3A, steady-state values of glucosestimulated OCR (OCRglc) and (ISRglc) and the decrement in OCR in
response to blocking calcium influx by nimodipine (OCRnim) were
calculated (as described in RESULTS) from perifusions done at 3 (n 5),
8 (n 5), 12 (n 7), or 20 mmol/l (n 8) glucose. Data were
normalized by dividing by the value of the parameter obtained at 20
mmol/l glucose.
This study was funded by grants from the National Institutes of Health (DK17047, DK063986, and P30 DK17047S1).
Special thanks to Drs. Duk-Su Koh and Bertil Hille for
help on the calcium studies. Rats used in this study were
kindly provided by Dr. ke Lernmark.
DIABETES, VOL. 55, DECEMBER 2006
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