From the Rolf Luft Center for Diabetes Research, Department of
Molecular Medicine, Karolinska Institute,
S-171 76 Stockholm, Sweden
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INTRODUCTION |
An increase in plasma glucose concentration is the major
physiological stimulus for insulin release from the pancreatic
-cell. Glucose metabolism leads to membrane depolarization and
initiation of a characteristic pattern in electrical activity
concomitant with fluctuations in cytosolic-free
Ca2+-concentration (1). Depolarization results from an
increase in the ATP/ADP ratio, thereby inducing a closure of the
ATP-dependent K+ (KATP)
channel (2).1An increase in
mitochondrial metabolism plays an important role in the response to
glucose and several other fuel secretagogues. Among other fuel
secretagogues, the deamination product of the amino acid
L-leucine,
-ketoisocaproate, is of particular interest since it is exclusively metabolized in mitochondria (3, 4). Accordingly, several studies have shown that
-ketoisocaproate stimulates insulin secretion (3, 4), initiates electrical activity (5),
and inhibits the
-cell KATP channel in intact cells monitored in the cell-attached configuration of the patch-clamp technique (6-8). The fact that metabolism of
-ketoisocaproate is
confined to the mitochondria indicates that a product in Krebs cycle is
involved in modulation of channel activity. The most likely candidate
is ATP, which has been shown to be elevated after exposure of
-cells
to
-ketoisocaproate (4, 6). In the present study we have further
investigated the effects of
-ketoisocaproate on the
KATP channel and demonstrate that the substance
directly inhibits the
-cell KATP channel.
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EXPERIMENTAL PROCEDURES |
Preparation--
Adult obese mice (gene ob/ob) of
both sexes were obtained from a local colony (9). The mice were starved
for 24 h and then killed by decapitation. Pancreatic islets were
isolated by a collagenase technique (10), and a cell suspension was
prepared and washed essentially as described previously (11). The cells
were resuspended in RPMI 1640 culture medium (Flow Laboratories,
Scotland, UK) containing 11 mM glucose supplemented with
10% fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml
streptomycin, and 60 µg/ml gentamycin. Collagenase was obtained from
Boehringer Mannheim. The cell suspension was seeded into Petri dishes
(Corning Glass, Corning, NY) and incubated at 37 °C in 5%
CO2 for 1-3 days.
Solutions--
The bath solution (i.e. the
"intracellular" solution) consisted of 125 mM KCl, 1 mM MgCl2, 10 mM EGTA, 30 mM KOH, and 5 mM HEPES-KOH (pH 7.15) unless
otherwise indicated. In the experiments using the inside-out
configuration, the pipettes were filled with standard extracellular
solution containing 138 mM NaCl, 5.6 mM KCl,
1.2 mM MgCl2, 2.6 mM CaCl2,
and 5 mM HEPES-NaOH (pH 7.40). In the perforated patch
experiments, the pipette solution contained 10 mM KCl, 76 mM K2SO4, 10 mM NaCl, 1 mM MgCl2, and 10 mM NaOH (pH 7.15),
and 200 µg of amphotericin B/ml (dissolved in Me2SO). The
final concentration of Me2SO was less than 0.1%. ATP and
ADP (both supplied by Sigma) were added to the intracellular solution as indicated. When nucleotides were added as their Na+ salt
(ADP), Mg2+ was added to maintain an excess of
Mg2+.
-Ketoisocaproate-Na+ salt was obtained
from two different suppliers (Sigma and Fluka Chemie AG, Neu-Ulm,
Switzerland), and 2-oxopentanoate was obtained from Aldrich.
Trypsin-EDTA was purchased from Life Technologies, Inc.
Electrophysiology--
KATP channel
activity and membrane potential were recorded using the patch-clamp
technique (12). Pipettes were coated with Sylgard resin (Dow Corning,
Kanagawa, Japan) near their tips to reduce capacitance transients and,
finally, were fire-polished. Currents were recorded using an Axopatch
200 patch-clamp amplifier (Axon Instruments, Inc., Foster City, CA).
Experiments were stored on magnetic tape using a video cassette
recorder (JVC, Tokyo, Japan) and a digital data recorder (VR-10B,
Instrutech Corp., Elmont, NY). The recorded signal was stored with an
upper cut-off frequency of 2 kHz. Patches were excised into a
nucleotide-free solution, and 0.1 mM ATP was first added to
test for channel inhibition. With the solutions used, ion currents were
outward, and channel records were displayed according to the
convention; upward deflections denote outward currents. All experiments
were performed at room temperature (20-24 °C), and channel activity
was measured at 0 mV unless otherwise indicated. Whole cell
KATP currents and
-cell membrane potential
were recorded using the perforated patch configuration of the
patch-clamp technique.
For analysis of single channel kinetics, records were low-pass filtered
at 0.2 kHz using an 8-pole Bessel filter (Frequency Devices, Haverhill,
MA) digitized at 0.8 kHz using a TL-1 DMA interface (Axon Instruments)
and stored in a computer. Open time kinetics were determined using
in-house software by digitizing segments of the current records
(~60-s long) and forming histograms of base-line and open-level data
points. Analysis of the distribution of KATP
channel open times was restricted to segments containing no more than
three active channels. Events were identified using a 50% amplitude
criterion. The kinetic constants were derived by approximation of the
data to exponential functions by the method of maximum likelihood (13).
Channel activity (NPO) was calculated as the mean
current (IX) divided by the single channel current
amplitude. The mean current (IX) was calculated
according to Equation 1.
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(Eq. 1)
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where N is the number of samples,
ij is the current observed in sample j, and
iB is the value of a user-defined base line. Results
are expressed as mean ± S.E., and statistical significance was
analyzed using paired or unpaired Student's t tests.
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RESULTS AND DISCUSSION |
KATP Channel Activity Is Inhibited by
-Ketoisocaproate--
In the intact
-cell, the activity of the
KATP channel is the main regulator of resting
membrane potential, and the intracellular ATP/ADP ratio is considered
to constitute the primary determinator of channel activity (14). It is
obvious that substances that are capable of modulating channel activity
may also influence resting membrane potential. Fig.
1A shows a typical trace
exposing an excised patch to 20 mM
-ketoisocaproate.
When exposing patches to 20 mM
-ketoisocaproate, channel
activity (NPO) decreased to 31 ± 5%
(n = 6; p < 0.001) compared with what
was found before the addition of the substance. In Fig. 1B,
we exposed isolated patches to an ATP/ADP ratio of 1, which efficiently
stimulates KATP channel activity (15). Upon the
addition of the nucleotides, mean current increased with 247 ± 74% (n = 3) and induced a typical kinetic pattern with
openings of long duration (16). The addition of
-ketoisocaproate (20 mM) to the patch in the continued presence of nucleotides
reduced channel activity to 26 ± 7% (n = 4;
p < 0.05). These data clearly show that the
KATP channel can be directly modulated by
-ketoisocaproate in inside-out patches.

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Fig. 1.
The effects of -ketoisocaproate on
KATP channel activity in inside-out patches
from pancreatic -cells. A, typical recording of
KATP currents in the presence of 20 mM -ketoisocaproate. In the presence
-ketoisocaproate, NPO decreased approximately
4-fold, from 2.42 to 0.55. Returning to standard bath solution, channel
activity increased rapidly to 1.42. B, in the presence of
100 µM ATP and 100 µM ADP (ratio 1),
KATP channel activity displayed a kinetic
pattern characterized by openings of long duration. The addition of
-ketoisocaproate, in the presence of nucleotides, significantly
decreased channel current. NPO was reduced from
0.96, during exposure to nucleotides alone, to 0.29 after the inclusion
of 20 mM -ketoisocaproate in the perifusion buffer. The
trace is a representative recording out of five with similar results.
C, NaCl, 20 mM, was added to a modified bath
solution where the K+ concentration was reduced to 125 mM. NaCl was subsequently exchanged for 20 mM
-ketoisocaproate, Na+ salt, maintaining a constant
concentration of Na+. Channel activity during control was
4.07, which decreased to 0.43 in the presence of -ketoisocaproate.
No alteration in single-channel unitary conductance during exposure to
-ketoisocaproate could be observed under these conditions. This
experimental protocol was repeated four times with similar
results.
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Since
-ketoisocaproate is a Na+ salt, the decrease in
single channel unitary conductance from 19.1 ± 0. 3 picosiemens
to 14.3 ± 0.4 picosiemens (n = 7;
p < 0.01) after the addition of this fuel secretagogue
could be explained by the presence of Na+ alone. In the
millimolar range, Na+ is known to interact with channel
conductance (17, 18). Thus, the obtained decreases in channel
conductance by the various concentrations of the
-ketoisocaproate-Na+ salt were identical to those
reported earlier by Na+ (17). When correcting for the
Na+ concentration during and before exposure of the patches
to
-ketoisocaproate, no alteration in unitary conductance was
observed (Fig. 1C).
In a series of experiments we exposed patches to four different
concentrations of
-ketoisocaproate ranging from 2 to 20 mM. It is clear that
-ketoisocaproate
dose-dependently decreased KATP
channel activity (Fig. 2A).
Compiled data of the concentration-inhibition relation is shown in Fig.
2B. Values are expressed as the ratio of channel activity
obtained in the presence of
-ketoisocaproate (NPO), and the activity assessed in the standard
intracellular solution before the addition of
-ketoisocaproate
(NPO Control). The mean value points were
fitted to the Hill equation. Estimates of the concentration causing a
50% reduction in channel activity was found to be 8.1 mM,
with a Hill coefficient of 2.3, which suggests that the block involves
the binding of more than one molecule to the
KATP channel complex.

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Fig. 2.
Dose-response and kinetic effects of
-ketoisocaproate on KATP channel activity in
inside-out patches. A, typical recording of
KATP currents in the presence of different
concentrations of -ketoisocaproate. In the presence of 20 mM -ketoisocaproate, NPO decreased
approximately 4-fold (to 0.57), compared with before the addition of
the substance (2.01). Exposing the same patch to 10 mM
-ketoisocaproate led to a decrease in channel activity from 2.06 to
0.98, and finally, exposing the patch to 5 mM
-ketoisocaproate resulted in a NPO of 1.08, compared with 1.78 before the addition of the substance. B,
compiled data on the concentration-inhibition relationship of
KATP channel activity after inclusion of
different concentrations of -ketoisocaproate. Ordinate,
normalized channel activity (NPO/
NPO Control). Abscissa,
concentration in mM of -ketoisocaproate. The data were
fitted to the Hill equation. Results are presented as mean values
±S.E.M. for 4-6 observations. C, frequency
versus lifetime histograms of channel openings under control
condition and in the presence of -ketoisocaproate. In control
solution, the distribution of channel open time could be described by a
single exponential function with a time constant ( ) of 37.6 ms. A
total of 856 events were analyzed. In the presence of 20 mM
-ketoisocaproate, the distribution of open time could be fitted with
a of 33.1 ms (978 events). The insets show typical
channel activity on an expanded time scale. Arrowheads
indicate the current level when the channel is closed. A sample
frequency of 0.8 kHz was used, and the bin width was set to 25 ms.
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We further examined the effects of
-ketoisocaproate on kinetic
properties of the KATP channel. In Fig.
2C we analyzed the open-time distribution in the presence of
-ketoisocaproate using patches containing no more than three
simultaneously active channels. Insets show examples of
channel openings under the respective experimental conditions on an
expanded time scale. The mean duration of openings under control
conditions was 34.1 ± 1.1 ms (n = 5), which is
similar to what has been reported earlier for the
KATP channel (16). The same type of
channel activity was observed in the presence of
-ketoisocaproate,
with a mean open time of 29.7 ± 2.1 ms (n = 4;
n.s.).
The Inhibitory Effects
-Ketoisocaproate on the KATP
Channel Is Not Dependent on Metabolism--
To further verify that
-ketoisocaproate has the ability to directly inhibit the
KATP channel without involving metabolism of the
substance, we investigated KATP channel activity
in intact cells in the presence of the metabolic inhibitor sodium azide (NaN3) using the perforated patch configuration of the
patch-clamp technique. It is well established that the input
conductance in the unstimulated
-cell is dominated by the
KATP channel and this conductance is virtually
completely inhibited by the sulfonylurea compound tolbutamide (14). To
measure input conductance, we used a voltage-clamp protocol with ±5-mV
excursions from a holding potential of
70 mV. As shown in Fig.
3A, the addition of 1 mM NaN3 in the presence of 3 mM
glucose increased input conductance 7-fold (698 ± 78%;
p < 0.05; n = 6). However, the
addition of
-ketoisocaproate in the continued presence of
NaN3 decreased input conductance to 50 ± 3%
(n = 5; p < 0.01), whereas no
significant change was observed when increasing the glucose
concentration to 15 mM (106 ± 6%; n = 3; n.s.). Fig. 3B shows compiled data of a
series of measurements of input conductance after the addition of
glucose and
-ketoisocaproate, respectively, in the presence of
NaN3.

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Fig. 3.
Effects of NaN3 on
-ketoisocaproate-induced inhibition of KATP
channel activity in intact cells (A-C) using the
perforated-patch configuration and in excised inside-out patches
(D). A, cells were voltage-clamped
(V-C) at a holding potential of 70 mV, and voltage
excursions of +/ 5mV (200 ms) were performed (top left). In
the presence of 1 mM NaN3, input conductance
increased more than 5-fold, from 0.9 nS to 5.2 nS (bottom
left). The addition of 15 mM glucose in the presence
of NaN3 did not affect the KATP
currents. In the presence of NaN3, input conductance was
estimated to 3.0 nS and 3.1 nS before and after the addition of
glucose, respectively (top right). Bottom right
shows currents in the presence and absence of -ketoisocaproate in
the continued presence of sodium azide. The addition of the keto acid
decreased the conductance from 5.3 nS to 2.5 nS. B, compiled
data on the effects on input conductance after the addition of 15 mM glucose and 20 mM -ketoisocaproate in the
presence of 1 mM NaN3. The recordings were made
at 34 °C, and the effect of glucose was estimated after 120 s,
which should be sufficient time for the fuel to act. C,
effects of temperature on membrane potential when stimulated with 20 mM -ketoisocaproate (top panels). At low
temperature (26 °C; left), the addition of the keto acid
caused the cell to depolarize and display continues action potentials.
The time from the addition of -ketoisocaproate to the appearance of
action potentials was estimated to 14.5 s. The time was not
significantly affected by increasing the temperature to 34 °C (13.2 s; right). Lower panels, at 26 °C, the time
span from the addition of 15 mM glucose to the appearance
of action potentials was 55.1 s (left). Elevating the
temperature to 34 °C resulted in a shortening of the time to
31.5 s (right). D, 1 mM
NaN3 did not affect KATP channel
currents in inside-out patches (top trace);
NPO before azide was estimated to 3.72 and after addition of azide to 3.45. The small decrease in channel
activity could well be accounted for by spontaneous channel run down.
In the presence of NaN3, channel activity was calculated to
6.17, which decreased to 1.99 after the addition of 20 mM
-ketoisocaproate (bottom trace). Glucose and
-ketoisocaproate were added using a puffer system in A,
B, and C, allowing a momentaneous application and
precise estimation of time from the addition of a substance to membrane
depolarization. ** p < 0.01, compared with
NaN3 alone.
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We have previously shown that glucose-induced membrane depolarization
and increase in cytosolic Ca2+ concentration are slower at
room temperature compared with 37 °C (19), most likely reflecting a
lower metabolic rate in the
-cell. We therefore estimated the time
from the addition of
-ketoisocaproate or 15 mM glucose
to the appearance of action potentials at high (34 °C) and low
(26 °C) temperature. Top traces in Fig. 3C show recordings of membrane potential after the addition of 20 mM
-ketoisocaproate at low and high temperature. No
significant change in the latency between the addition and appearance
of action potential was observed using
-ketoisocaproate, 11.2 ± 0.9 s versus 9.8 ± 1.6 s at low and high
temperature, respectively (n = 4; n.s.). In
contrast, when adding 15 mM glucose (bottom
traces), a significant delay was observed when lowering the
temperature to 26 °C, 37.7 ± 6.7 s at 34 °C 86.2 ± 20.5 s at 26 °C (n = 6; p < 0.05). It should be pointed out that it has been reported that the
onset of insulin secretion is more rapid after stimulation with
-ketoisocaproate compared with stimulation with glucose (4).
Finally, we studied the effect of NaN3 in inside-out
patches. As seen in Fig. 3D, channel activity remained
unchanged after the addition of NaN3 to the bath. In the
presence of NaN3,
-ketoisocaproate (bottom trace) still potently inhibited channel activity. Taken together, these data strongly suggest that
-ketoisocaproate can block
KATP channel activity independent of metabolism
of the keto acid.
Effects on KATP Channel Activity by
L-Leucine and 2-Oxopentanoate--
Several studies have
described insulinotrophic effects of L-leucine (3, 20, 21).
Because of the blocking effect of
-ketoisocaproate on the
KATP channel, we also investigated whether the
precursor amino acid, L-leucine, had direct effects on the channel in excised patches. As shown in Fig.
4A, the addition of 20 mM L-leucine to an isolated patch neither
effected single KATP channel activity nor
channel mean open time (30.4 ± 2.8 ms; n = 4;
n.s.). Subsequent inclusion of 20 mM
-ketoisocaproate in the same patch significantly inhibited channel
activity. L-Arginine, which has a close structural
resemblance to L-leucine, was also without effect on
KATP channel activity (data not shown).
Oxopentanoate, which is derived from pentanoic acid, differs from
-ketoisocaproate in lacking a methyl group. Like
-ketoisocaproate, 2-oxopentanoate potently inhibited
KATP channel currents (Fig. 4B).

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Fig. 4.
. Effects on KATP
channel activity by L-leucine and 2-oxopentanoate.
A, inclusion of L-leucine (20 mM) in
the intracellular solution did not affect KATP
channel activity. NPO was estimated to 2.39 under
control conditions and 2.31 in the presence of the amino acid, whereas
the addition of 20 mM -ketoisocaproate to the same patch
reduced channel activity to 0.57. B, the addition of
2-oxopentanoate reduced channel activity from 7.05 to 0.89. The
structures of L-leucine, 4-metyl-2-oxopentanoic acid
( -ketoisocaproate), and 2-oxopentanoate are shown in C.
The vertical calibration bar represents 5 pA in A
and 10 pA in B. The above experiments were repeated at least
four times with similar results.
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Specific Effect on the KATP Channel--
In contrast
to the potent inhibition of the KATP channel by
-ketoisocaproate, no effect could be seen on the large conductance Ca2+-regulated K+ (KBK) channel
(14). As seen in Fig. 5A,
exposing inside-out patches to 20 mM
-ketoisocaproate,
no effect on the mean current of KBK channel could be
monitored. In a series of experiments, KBK channel activity
(NPO) was assessed to 0.61 ± 0.09 during
control solution, compared with 0.60 ± 0.10 in the presence of
-ketoisocaproate (n = 3; n.s.). The
activity of the small Ca2+-regulated K+
conductance channel (KSK) (14) was also unaffected after
inclusion of 20 mM
-ketoisocaproate in the bath
solution (data not shown).

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Fig. 5.
. Specific effect on the
KATP channel. A, to record the
Ca2+-activated large conductance KBK channel,
EGTA was omitted and 100 µM CaCl2 was added
to the bath solution. Currents were recorded at a membrane potential of
0 mV. Typical recording showing that the open probability of
KBK channel was unchanged during exposure to 20 mM -ketoisocaproate. This experimental protocol was
repeated three times with identical results. B, amplitude
histograms from the trace in A in the absence and presence
of 20 mM -ketoisocaproate. C, the top trace
shows a typical recording of single KATP channel
currents after an approximately 5-min exposure of the patch to 20 µg/ml trypsin. Channel activity was unchanged after exposure of the
patch to 20 mM -ketoisocaproate. D, compiled
data of the effect of 20 mM -ketoisocaproate from nine
patches before (left) and after (right) trypsin.
Each symbol represents a different patch. C denotes channel
closed.
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To further investigate whether the effect of
-ketoisocaproate is
specific for the KATP channel, we studied the
effects of
-ketoisocaproate on channel activity in trypsin-modified
patches. Exposure of isolated membrane patches to 20 µg/ml trypsin
for
5 min has been reported to alter KATP
channel activity with a specific pattern. Thus, trypsin treatment takes
away the activating effect of MgADP and the inhibitory effect of
sulfonylureas on KATP channel activity, whereas
the inhibitory effect of ATP remains, although with slightly decreased
efficiency (22, 23). Evidently this action of trypsin treatment results
from its specific proteolytic effects on the
KATP channel complex (22), trypsin having a
primary affinity for arginine and lysine residues (24). After trypsin modification, inclusion of
-ketoisocaproate (20 mM) in
the bath solution was unable to affect KATP
channel activity (Fig. 5C). This experimental protocol was
repeated four times, and in all four patches the inhibitory effect of
-ketoisocaproate on KATP channel activity was
lost subsequent to trypsin treatment (Fig. 5D). Channel
activity (NPO) was 3.1 ± 0.5 and
3.0 ± 0.5 (n = 4; n.s.) before and
after the addition of
-ketoisocaproate, respectively. Based on these
observations, it is highly unlikely that the effect of
-ketoisocaproate is due to a nonspecific block of any K+
channel pore but rather involves a specific interaction with the
KATP channel protein complex.
Concluding Remarks--
It has been known for the last decade that
the deamination product of L-leucine,
-ketoisocaproate,
is a potent stimulator of insulin secretion (21). A number of studies
have demonstrated that this compound, like glucose, causes inhibition
of the KATP channel in the pancreatic
-cell
(6-8, 21). In addition, blocking of the respiratory cycle reduces the
insulinotrophic effect of
-ketoisocaproate (4). These observations
have suggested a role for oxidative phosphorylation-generated
messengers to promote closure of the KATP
channel. In contrast to these findings, the results presented in this
study clearly show that
-ketoisocaproate directly and reversibly
inhibits the KATP channel in the pancreatic
-cell without involving metabolism of the substance. Since the resting potential in the intact
-cell is mainly determined by the
activity of the KATP channel (14), it is clear
that
-ketoisocaproate will also influence membrane potential and
thereby insulin secretion by direct inhibition of the channel. Thus, in
view of our findings, the use of
-ketoisocaproate as a tool to study
the role of mitchondrial metabolism in intact
-cells should be
interpreted cautiously. To what extent direct effects of
-keto acids
on KATP channel activity are involved in the
-cell stimulus-secretion coupling is at present difficult to assess.
In our view, the concentrations required to affect the activity of the
KATP channel, as found in the present study, are
not likely to be reached during physiological conditions.
The present design does not allow us to evaluate relative contribution
of the direct effect of
-ketoisocaproate on the
KATP channel versus effects resulting
from metabolism of the substance. In this context it is interesting
that
-ketoisocaproate stimulates insulin release more rapidly than
glucose (4). The direct effect of
-ketoisocaproate on the
KATP channel may explain this difference in
kinetics in the stimulation of insulin release.