Direct Inhibition of the Pancreatic beta -Cell ATP-regulated Potassium Channel by alpha -Ketoisocaproate*

Robert Bränström, Suad Efendic', Per-Olof Berggren, and Olof LarssonDagger

From the Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, S-171 76 Stockholm, Sweden

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

The ATP-regulated potassium (KATP) channel plays an essential role in the control of insulin release from the pancreatic beta -cell. In the present study we have used the patch-clamp technique to study the direct effects of alpha -ketoisocaproate on the KATP channel in isolated patches and intact pancreatic beta -cells. In excised inside-out patches, the activity of the KATP channel was dose-dependently inhibited by alpha -ketoisocaproate, half-maximal concentration being approximately 8 mM. The blocking effect of alpha -ketoisocaproate was fully reversible. Stimulation of channel activity by the addition of ATP/ADP (ratio 1) did not counteract the inhibitory effect of alpha -ketoisocaproate. In the presence of the metabolic inhibitor sodium azide, alpha -ketoisocaproate was still able to inhibit single channel activity in excised patches and to block whole cell KATP currents in intact cells. No effect of alpha -ketoisocaproate could be obtained on either the large or the small conductance Ca2+-regulated K+ channel. Enzymatic treatment of the patches with trypsin prevented the inhibitory effect of alpha -ketoisocaproate. Based on these observations, it is unlikely that the blocking effect of alpha -ketoisocaproate is due to an unspecific effect on K+ channel pores. Leucine, the precursor of alpha -ketoisocaproate, did not affect KATP channel activity in excised patches. Our findings are compatible with the view that alpha -ketoisocaproate not only affects the beta -cell stimulus secretion coupling by generation of ATP but also by direct inhibition of the KATP channel.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

An increase in plasma glucose concentration is the major physiological stimulus for insulin release from the pancreatic beta -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, alpha -ketoisocaproate, is of particular interest since it is exclusively metabolized in mitochondria (3, 4). Accordingly, several studies have shown that alpha -ketoisocaproate stimulates insulin secretion (3, 4), initiates electrical activity (5), and inhibits the beta -cell KATP channel in intact cells monitored in the cell-attached configuration of the patch-clamp technique (6-8). The fact that metabolism of alpha -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 beta -cells to alpha -ketoisocaproate (4, 6). In the present study we have further investigated the effects of alpha -ketoisocaproate on the KATP channel and demonstrate that the substance directly inhibits the beta -cell KATP channel.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

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+. alpha -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 beta -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.
I<SUB>X</SUB>=<LIM><OP>∑</OP><LL>j<UP>=1</UP></LL><UL>N</UL></LIM> <FR><NU>(i<SUB>i</SUB>−i<SUB>B</SUB>)</NU><DE>N</DE></FR> (Eq. 1)
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.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

KATP Channel Activity Is Inhibited by alpha -Ketoisocaproate-- In the intact beta -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 alpha -ketoisocaproate. When exposing patches to 20 mM alpha -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 alpha -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 alpha -ketoisocaproate in inside-out patches.


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Fig. 1.   The effects of alpha -ketoisocaproate on KATP channel activity in inside-out patches from pancreatic beta -cells. A, typical recording of KATP currents in the presence of 20 mM alpha -ketoisocaproate. In the presence alpha -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 alpha -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 alpha -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 alpha -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 alpha -ketoisocaproate. No alteration in single-channel unitary conductance during exposure to alpha -ketoisocaproate could be observed under these conditions. This experimental protocol was repeated four times with similar results.

Since alpha -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 alpha -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 alpha -ketoisocaproate, no alteration in unitary conductance was observed (Fig. 1C).

In a series of experiments we exposed patches to four different concentrations of alpha -ketoisocaproate ranging from 2 to 20 mM. It is clear that alpha -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 alpha -ketoisocaproate (NPO), and the activity assessed in the standard intracellular solution before the addition of alpha -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 alpha -ketoisocaproate on KATP channel activity in inside-out patches. A, typical recording of KATP currents in the presence of different concentrations of alpha -ketoisocaproate. In the presence of 20 mM alpha -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 alpha -ketoisocaproate led to a decrease in channel activity from 2.06 to 0.98, and finally, exposing the patch to 5 mM alpha -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 alpha -ketoisocaproate. Ordinate, normalized channel activity (NPO/ NPO Control). Abscissa, concentration in mM of alpha -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 alpha -ketoisocaproate. In control solution, the distribution of channel open time could be described by a single exponential function with a time constant (tau ) of 37.6 ms. A total of 856 events were analyzed. In the presence of 20 mM alpha -ketoisocaproate, the distribution of open time could be fitted with a tau  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.

We further examined the effects of alpha -ketoisocaproate on kinetic properties of the KATP channel. In Fig. 2C we analyzed the open-time distribution in the presence of alpha -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 alpha -ketoisocaproate, with a mean open time of 29.7 ± 2.1 ms (n = 4; n.s.).

The Inhibitory Effects alpha -Ketoisocaproate on the KATP Channel Is Not Dependent on Metabolism-- To further verify that alpha -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 beta -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 alpha -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 alpha -ketoisocaproate, respectively, in the presence of NaN3.


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Fig. 3.   Effects of NaN3 on alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -ketoisocaproate (bottom trace). Glucose and alpha -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.

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 beta -cell. We therefore estimated the time from the addition of alpha -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 alpha -ketoisocaproate at low and high temperature. No significant change in the latency between the addition and appearance of action potential was observed using alpha -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 alpha -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, alpha -ketoisocaproate (bottom trace) still potently inhibited channel activity. Taken together, these data strongly suggest that alpha -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 alpha -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 alpha -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 alpha -ketoisocaproate in lacking a methyl group. Like alpha -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 alpha -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 (alpha -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.

Specific Effect on the KATP Channel-- In contrast to the potent inhibition of the KATP channel by alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -ketoisocaproate. D, compiled data of the effect of 20 mM alpha -ketoisocaproate from nine patches before (left) and after (right) trypsin. Each symbol represents a different patch. C denotes channel closed.

To further investigate whether the effect of alpha -ketoisocaproate is specific for the KATP channel, we studied the effects of alpha -ketoisocaproate on channel activity in trypsin-modified patches. Exposure of isolated membrane patches to 20 µg/ml trypsin for approx 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 alpha -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 alpha -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 alpha -ketoisocaproate, respectively. Based on these observations, it is highly unlikely that the effect of alpha -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, alpha -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 beta -cell (6-8, 21). In addition, blocking of the respiratory cycle reduces the insulinotrophic effect of alpha -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 alpha -ketoisocaproate directly and reversibly inhibits the KATP channel in the pancreatic beta -cell without involving metabolism of the substance. Since the resting potential in the intact beta -cell is mainly determined by the activity of the KATP channel (14), it is clear that alpha -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 alpha -ketoisocaproate as a tool to study the role of mitchondrial metabolism in intact beta -cells should be interpreted cautiously. To what extent direct effects of alpha -keto acids on KATP channel activity are involved in the beta -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 alpha -ketoisocaproate on the KATP channel versus effects resulting from metabolism of the substance. In this context it is interesting that alpha -ketoisocaproate stimulates insulin release more rapidly than glucose (4). The direct effect of alpha -ketoisocaproate on the KATP channel may explain this difference in kinetics in the stimulation of insulin release.

    FOOTNOTES

* This work was supported by the Swedish Medical Research Grants 04X-09891, 03X-09890, and 19X-00034 and grants from the Swedish Diabetes Association, the Nordic Insulin Foundation, Funds of the Karolinska Institute, Novo Nordisk Pharma AB, and the Swedish Society for Medical Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Rolf Luft Center for Diabetes Research, Dept. of Molecular Medicine, L1:02, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden. Tel.: 46-8-517-2744; Fax: 46-8-30 34 58; E-mail: olof.larsson{at}molmed.ki.sé.

1 The abbreviations used are: KATP, ATP-regulated K+ channel; alpha -ketoisocaproate, 4-methyl-2-oxopentanoate; nS, nanosiemens; N.S., not significant.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

  1. Berggren, P. O., and Larsson, O. (1994) Biochem. Soc. Trans. 22, 12-18[Medline] [Order article via Infotrieve]
  2. Cook, D. L., and Hales, C. N. (1984) Nature 311, 271-273[Medline] [Order article via Infotrieve]
  3. Panten, U., Christians, J., Kriegstein, E., von Poser, W., and Hasselblatt, A. (1974) Diabetologia 10, 149-154[Medline] [Order article via Infotrieve]
  4. Hutton, J. C., Sener, A., Herchuelz, A., Atwater, I., Kawazu, S., Boschero, A. C., Somers, G., Devis, G., and Malaisse, W. J. (1980) Endocrinology 106, 203-219[Abstract]
  5. Henquin, J. C., and Meissner, H. P. (1981) Am. J. Physiol. 240, E245-E252[Abstract/Free Full Text]
  6. Ashcroft, F. M., Ashcroft, S. J., and Harrison, D. E. (1987) J. Physiol. (Lond.) 385, 517-529[Abstract]
  7. Eddlestone, G. T., Ribalet, B., and Ciani, S. (1989) J. Membr. Biol. 109, 123-134[Medline] [Order article via Infotrieve]
  8. Ribalet, B., Eddlestone, G. T., and Ciani, S. (1988) J. Gen. Physiol. 92, 219-237[Abstract]
  9. Hellman, B. (1965) Ann. N. Y. Acad. Sci. 131, 541-558[Medline] [Order article via Infotrieve]
  10. Lacy, P. E., and Kostianovsky, M. (1967) Diabetes 16, 35-39[Medline] [Order article via Infotrieve]
  11. Lernmark, A. (1974) Diabetologia 10, 431-438[Medline] [Order article via Infotrieve]
  12. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pfuegers Arch. Eur. J. Physiol. 391, 85-100[Medline] [Order article via Infotrieve]
  13. Colquhoun, D., and Sigworth, F. J. (1983) in Single-Channel Recording (Sakmann, B., and Neher, E., eds), pp. 191-263, Plenum Press Corp., New York
  14. Ashcroft, F. M., and Rorsman, P. (1990) Biochem. Soc. Trans. 18, 109-111[Medline] [Order article via Infotrieve]
  15. Hopkins, W. F., Fatherazi, S., Peter-Riesch, B., Corkey, B. E., and Cook, D. L. (1992) J. Membr. Biol. 129, 287-295[Medline] [Order article via Infotrieve]
  16. Larsson, O., Ämmälä, C., Bokvist, K., Fredholm, B., and Rorsman, P. (1993) J. Physiol. (Lond.) 463, 349-365[Abstract]
  17. Kakei, M., Noma, A., and Shibasaki, T. (1985) J. Physiol. (Lond.) 363, 441-462[Abstract]
  18. Horie, M., Irisawa, H., and Noma, A. (1987) J. Physiol. (Lond.) 387, 251-272[Abstract]
  19. Arkhammar, P., Nilsson, T., Rorsman, P., and Berggren, P.-O. (1987) J. Biol. Chem. 262, 5448-5454[Abstract/Free Full Text]
  20. Freinkel, N., Younsi, C. E., and Dawson, R. M. C. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3403-3407[Abstract]
  21. Panten, U., Kriegstein, E., von, Poser, W., Schonborn, J., and Hasselblatt, A. (1972) FEBS Lett. 20, 225-228[CrossRef][Medline] [Order article via Infotrieve]
  22. Proks, P., and Ashcroft, F. M. (1993) Pfluegers Arch. Eur. J. Physiol. 424, 63-72[Medline] [Order article via Infotrieve]
  23. Bränström, R., Corkey, B. E., Berggren, P. O., and Larsson, O. (1997) J. Biol. Chem. 272, 17390-17394[Abstract/Free Full Text]
  24. Benyon, R. J., and Boyd, J. S. (1989) Proteolytic Enzymes: A Practical Approach, Oxford University Press, Oxford


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