Chronic exposure to
-hydroxybutyrate inhibits glucose-induced insulin release from pancreatic islets by decreasing NADH contents
Mihoko Takehiro,1
Shimpei Fujimoto,1
Makiko Shimodahira,1
Dai Shimono,1
Eri Mukai,1
Koichiro Nabe,1
Razvan Gheorghe Radu,1
Rieko Kominato,1
Yo Aramaki,1
Yutaka Seino,1,2 and
Yuichiro Yamada1
1Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University, Kyoto; and 2Kansai-Denryoku Hospital, Osaka, Japan
Submitted 5 April 2004
; accepted in final form 9 October 2004
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ABSTRACT
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To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM D-
-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+]i) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+]i efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.
islet; reduced nicotinamide adenine dinucleotide; adenosine 5'-triphosphate
KETONE BODIES ARE PRODUCED IN THE LIVER and are used peripherally as an energy source when glucose is not readily available. Ketosis is seen in various physiological conditions such as fasting, prolonged exercise, and high-fat diet. Hyperketonemia is also caused by absolute or relative insulin deficiency in type 1 diabetic patients or type 2 diabetic patients, respectively (18).
The importance of insulin in control of ketone body production in the liver is well established (18). The function of these substrates in regulating insulin output from pancreatic
-cells is less well understood. In short-term exposure of <60 min, ketone bodies such as D-
-hydroxybutyrate (HB) are oxidized by islet cells and enhance insulin secretion in the presence of stimulatory levels of glucose (4, 10, 15, 28, 29, 31). However, little is known of the effect of prolonged exposure to ketone bodies on pancreatic
-cells and insulin secretion. Zhou et al. (47) have reported that 48-h exposure to HB reduces high glucose-induced insulin secretion from human pancreatic islets without affecting pyruvate dehydrogenase (PDH) activity or PDH kinase activity, although the mechanism of the inhibitory effect is unclear.
The mechanism of glucose-stimulated insulin release from pancreatic
-cells is well documented. Glucose entry into the
-cells accelerates glycolysis and glucose oxidation to increase the ATP content and ATP-to-ADP ratio, which closes the ATP-sensitive K+ (KATP) channels. The decrease in K+ conductance depolarizes the membrane and opens the voltage-dependent Ca2+ channels (VDCC). Increased Ca2+ influx through VDCCs increases the intracellular Ca2+ concentration ([Ca2+]i) to a level that triggers exocytosis of the insulin granules (13). It has been shown that glucose can also promote insulin release KATP channel independently by increasing Ca2+ efficacy in stimulation-secretion coupling, which may be due at least in part to the direct effect of increased ATP derived from glucose metabolism on exocytosis (1, 13). The effect of chronic exposure to ketone bodies in this mechanism is not known, however.
In the present study, insulin release, intracellular Ca2+ and metabolism, and the efficacy of Ca2+ in the exocytotic system were investigated in HB-exposed islets.
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MATERIALS AND METHODS
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Materials.
NADH, ADP, 2-amino-2-methyl-1-propanol, oxaloacetate, RPMI 1640 medium, alcohol dehydrogenase, aspartate transaminase, malate dehydrogenase, GDP
S, diadenosine pentaphosphate (DAPP), antimycin A, L-
-hydroxybutyrate, lithium acetoacetate, aminooxyacetate, and potassium aspartate were obtained from Sigma Chemicals (St. Louis, MO). Fura PE-3 AM was obtained from Calbiochem (La Jolla, CA). Succinate and HB were purchased from Aldrich (Steimheim, Germany). D-Hydroxybutyrate dehydrogenase was obtained from Toyobo (Osaka, Japan). 3H2O, [5-3H]glucose, and [U-14C]glucose were obtained from Amersham (Buckinghamshire, UK). All other reagents are of analytic grade and were obtained from Nacalai Tesque (Kyoto, Japan). Sodium acetoacetate was prepared and incubated for over 16 h at 20°C, from ethyl acetoacetate and NaOH (each 1 M), followed by three successive washes with diethyl ether to remove residual ethyl acetoacetate and ethanol formed by hydrolysis as previously described (29). The solution was then flushed with N2 for 60 min and stored at 80°C.
Acetoacetate determination.
Acetoacetate was determined as previously described (12). Briefly, the reaction mixture containing 50 µl of sample, 120 mM triethanolamine (pH 7.0), 375 µM NADH, 1 unit/ml D-hydroxybutyrate dehydrogenase, and 1.25 mg/ml oxamic acid in a total volume of 400 µl was incubated for 15 min at 37°C. The reaction was stopped by heating the mixture at 100°C for 2 min. The concentration was determined by measuring the decrease in absorbance at 340 nm using lithium acetoacetate as standard.
Animals.
Male Wistar rats were obtained from Shimizu (Kyoto, Japan). The animals were fed standard lab chow ad libitum and allowed free access to water in an air-conditioned room with a 12:12-h light-dark cycle until used in the experiments. All experiments were carried out with rats aged 812 wk. The animals were maintained and used in accordance with the Guidelines for Animal Experiments of Kyoto University.
Islet isolation and culture.
Islets of Langerhans were isolated from Wistar rats by collagenase digestion as described previously (6). Islets were cultured for 15 h in RPMI 1640 medium (containing 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 8.3 mM glucose) with or without
-hydroxybutyrate and acetoacetate at 37°C in humidified air containing 5% CO2.
Measurement of insulin release from intact islets.
Insulin release from intact islets was monitored by use of static incubation and perifusion conditions, as described previously (6), with Krebs-Ringer bicarbonate buffer (KRBB) supplemented with 0.2% BSA and 10 mM HEPES adjusted to pH 7.4 (KRBB medium). For static incubation experiments, cultured islets were preincubated at 37°C for 30 min with 2.8 mM glucose. Groups of five islets were then batch incubated for 30 min in 0.7 ml of KRBB medium with 2.8 and 16.7 mM glucose. At the end of the incubation period, islets were pelleted by centrifugation (15,000 g, 180 s), and aliquots of the buffer were sampled.
For perifusion experiments, groups of 20 cultured islets were placed in each of the parallel chambers (400 µl each) of a perifusion apparatus and perifused with KRBB medium at a rate of 0.7 ml/min at 37°C. The medium was continuously gassed with 95% O2-5% CO2. Islets were perifused for 30 min to establish a stable insulin secretory rate at the basal level of glucose, and the glucose concentration was then raised to 16.7 mM.
The amount of immunoreactive insulin was determined by RIA, using rat insulin as standard (6). Insulin contents and DNA contents of islets were measured as previously described (8).
Measurement of [Ca2+]i and reduced pyridine nucleotide fluorescence.
For [Ca2+]i measurement, cultured islets were loaded with fura PE-3 as previously described (7). The islets were immediately placed in a heat-controlled chamber on the stage of an inverted microscope kept at 36 ± 1°C, superfused with KRBB medium containing 2.8 mM glucose for 30 min, and subsequently exposed to the medium containing a high concentration of glucose. These islets were excited successively at 340 and 380 nm, and the fluorescence emitted at 510 nm was captured by a charge-coupled device (CCD) camera (Micro Max 5-MHz System; Roper Industries, Trenton, NJ). The 340-nm (F340) and 380-nm (F380) fluorescence signals were detected every 20 s, and the ratios (F340/F380) were calculated. In vitro calibration was performed as previously described (7). For reduced pyridine nucleotide [NAD(P)H] measurement, the islets without dye were excited successively at 360 nm, and the fluorescence emitted at 470 nm was captured every 20 s (9) by the CCD camera described above. Changes in NAD(P)H fluorescence signal were expressed as percent control values by dividing the signal at a given time by the average signal at 2.8 mM glucose during the last 1 min before stimulation.
Images of [Ca2+]i and NAD(P)H were analyzed with the Meta Fluor image analyzing system (Universal Imaging, West Chester, PA).
Measurement of insulin release from permeabilized islets.
Insulin release from permeabilized islets was determined as previously described (6). After preincubation with 2.8 mM glucose for 30 min, cultured islets were washed twice in cold potassium aspartate buffer (KA buffer) containing 140 mM potassium aspartate, 7 mM MgSO4, 2.5 mM EGTA, 30 mM HEPES, and 0.5% BSA (pH 7.0), with CaCl2 added to a Ca2+ concentration of 30 nM. The islets were permeabilized by high-voltage discharge in KA buffer and washed once with the same buffer. Groups of five electrically permeabilized islets were batch incubated for 30 min at 37°C in 0.4 ml of KA buffer with various concentrations of Ca2+ and ATP. At the end of the incubation period, permeabilized islets were pelleted by centrifugation (15,000 g, 180 s), and aliquots of the buffer were sampled for immunoreactive insulin determination.
Measurement of adenine nucleotide contents.
After groups of cultured intact islets were preincubated at 2.8 mM glucose for 30 min, groups of 15 islets were batch incubated in 0.5 ml of KRBB medium containing 2.8 or 16.7 mM glucose at 37°C for 30 min. Incubation was stopped by the addition of 0.1 ml of 2 M HClO4. The tubes were immediately mixed with vortex and sonicated in ice-cold water for 3 min. They were then centrifuged (3,000 g, 3 min), and a fraction (0.4 ml) of the supernatant was mixed with 100 µl of 2 M HEPES and 100 µl of 1 M Na2CO3. ATP and ADP were assayed by a luminometric method as previously described (8). For measurement of the sum of ATP + ADP, ADP was first converted into ATP by adding 210 µl of solution containing 20 mM HEPES (pH 7.75), 3 mM MgCl2, 1.5 mM phosphoenolpyruvate, and 2.2 U/ml pyruvate kinase to 70 µl of extracts, with incubation at 37°C for 30 min. The ATP concentration in solutions was measured by adding 200 µl of luciferin-luciferase solution (Turner Designs, Sunnyvale, CA) to a fraction of sample (100 µl) in a bioluminometer (Luminometer model 20e, Turner Designs). For measurement of ATP, the same procedure was performed except that the incubation step was done without pyruvate kinase. The ADP concentration was calculated as the difference between the value of ATP + ADP and ATP from the same sample. To draw a standard curve and to ascertain that the conversion of ADP into ATP was complete, blanks and ADP and ATP standards were run through the entire procedure, including the extraction steps.
Measurement of NADH contents.
NADH contents were assayed with an enzymatic cycling system for NADH amplification (17, 45). After preincubation at 2.8 mM glucose, batches of 20 cultured islets were incubated in 50 µl of KRBB medium containing 2.8 or 16.7 mM glucose. After incubation at 37°C for 30 min, 20 µl of solution containing 40 mM NaOH and 0.5 mM cysteine were added to each tube. The tubes were immediately mixed with vortex and sonicated in the ice-cold water for 3 min. They were then centrifuged (3,000 g, 3 min), and a fraction (30 µl) of the supernatant was incubated for 20 min at 60°C to destroy any remaining NAD. For the first step in NADH amplification, the reaction mixture containing 30 µl of extract, 100 mM HEPES-NaOH buffer (pH 7.5), 1 mM dithiothreitol, 0.2 mg/ml BSA, 280 mM ethanol, 2 mM oxaloacetate, 16 U/ml alcohol dehydrogenase, and 4 U/ml malate dehydrogenase in a total volume of 100 µl was incubated for 30 min at 30°C. The reaction was stopped by heating the mixture at 100°C for 2 min. In the second step, malate formed in the first step was assayed by incubating 100 µl of sample solution in 50 mM 2-amino-2-methyl-1-propanol-HCl buffer (pH 9.9) containing 0.18 mM NAD, 0.1 mg/ml BSA, 9 mM glutamate, 0.8 unit/ml malate dehydrogenase, and 1 U/ml aspartate transaminase in a total volume of 600 µl for 30 min at 30°C. The NADH formed was measured by fluorometry (Shimazu RF-5000, Kyoto, Japan) at a 340-nm excitation and a 450-nm emission. To draw a standard curve, blanks and NADH standards were run through the entire procedure.
Measurement of mitochondrial ATP production.
Mitochondrial suspension from cultured islets was prepared by repeated centrifugation, as previously described (14, 16, 19). First, isolated islets were homogenized in solution A consisting of (in mM) 50 HEPES, 100 KCl, 1.8 ATP, 1 EGTA, and 2 MgCl2 and 0.5 mg/ml BSA (electrophoretically homogeneous; pH 7.00 at 37°C with KOH). After precipitation of cell debris and nuclei by centrifugation, the supernatant was centrifuged more rapidly (10,000 g) to obtain a pellet containing the mitochondrial fraction. The precipitation diluted by 200 µl of solution A was centrifuged again and rinsed three times in solution consisting of (in mM) 20 HEPES, 3 KH2PO4, 1 EGTA, 12 sodium gluconate, 0.3 MgCl2, 148 potassium gluconate, and 4 carnitine and 0.5 mg/ml BSA (electrophoretically homogeneous), adjusted to pH 7.10 with KOH (solution B). The mitochondrial fraction in 500 µl of solution B was kept on ice until use. To measure ATP production by oxidative phosphorylation, the reaction was started by adding 5 µl of mitochondrial suspension to 495 µl of prewarmed solution B (37°C) supplemented with the mitochondrial substrates, 50 µM ADP, and 1 µM DAPP adjusted to pH 7.10. DAPP is a specific inhibitor of adenylate kinase used to measure ATP production by oxidative phosphorylation exclusively. To normalize the mass of the intact mitochondria obtained, ATP production by adenylate kinase, one of the mitochondrial intermembrane kinases, was measured in the presence of ADP but without mitochondrial substrates or DAPP in parallel incubations (1921). Reaction was stopped by addition of 0.5 µM antimycin A. The samples were cooled to room temperature, and ATP concentration in the solutions was measured by adding luciferin-luciferase solution to each sample with a bioluminometer. ATP production was determined as the ratio of ATP production by oxidative phosphorylation to that by adenylate kinase. To draw a standard curve, blank and ATP standards were added to parallel samples containing the complete incubation mixture except the mitochondrial suspension.
Measurement of glucose utilization and oxidation.
Glucose utilization and oxidation were measured, using the previously described method (3) with a slight modification. Cultured islets were preincubated in KRBB medium with 2.8 mM glucose at 37°C for 30 min. For utilization, batches of 30 islets for each condition were incubated at 37°C for 90 min in 150 µl of medium containing 1.5 µCi of [5-3H]glucose. Aliquots of the incubation medium (100 µl) and 20 µl of 1 M HCl were transferred to small tubes and placed into a glass vial containing 0.5 ml of H2O. The capped vials were incubated overnight at 34°C to vaporize 3H2O from the solution. The inner tube was then lifted out, and the disintegrations per minute of water-melting 3H2O in the vial were counted. In a parallel incubation, the recovery ratio of 3H2O was measured with 3H2O. After subtraction of blank disintegrations per minute from sample disintegrations per minute, glucose utilization was calculated with the disintegrations per minute, specific radioactivity of [5-3H]glucose, and recovery ratio of 3H2O. For oxidation, all procedures were the same as for utilization except the use of [U-14C]glucose (0.5 µCi/tube) and 0.5 ml of hydroxide of hyamine 10-X (Packard, Meriden, CT) in place of [5-3H]glucose and 0.5 ml of H2O, respectively. After subtraction of blank disintegrations per minute from sample disintegrations per minute, glucose oxidation was calculated using the disintegrations per minute and specific radioactivity of [U-14C]glucose.
Statistical analysis.
Results are expressed as means ± SE. Statistical significance was evaluated by unpaired Students t-test. P < 0.05 was considered significant.
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RESULTS
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Effect of chronic exposure to
-hydroxybutyrate on glucose-induced insulin release from intact islets.
Addition of 10 mM HB to RPMI 1640 medium did not alter the H+ concentration measured by the H+ electrode (data not shown). Exposure to 1 mM HB for 15 h did not alter 16.7 mM glucose-induced and basal insulin release. However, exposure to 2 mM HB reduced 16.7 mM glucose-induced insulin release but enhanced basal insulin release. No further inhibitory effect on high glucose-induced insulin release and no further enhancement of basal insulin release at 5 mM HB were observed (Table 1, top). Insulin release in the presence of 200 µM diazoxide, 16.7 mM glucose, and 30 mM K+ from HB-cultured islets was also reduced by chronic exposure to 5 mM HB for 15 h (HB 1.22 ± 0.03 vs. control 2.15 ± 0.09 ng·islet1·30 min1, n = 5, P < 0.01). Exposure to 5 mM L-
-hydroxybutyrate for 15 h did not alter 16.7 mM glucose-induced and basal insulin release (Table 1, bottom). The DNA contents and insulin contents of islets cultured with 5 mM HB for 15 h were similar to controls (DNA contents: HB 26.6 ± 1.9 vs. control 27.1 ± 1.8 ng/islet, n = 30, not significant; insulin contents: HB 34.3 ± 1.9 vs. control 30.6 ± 1.4 ng/islet, n = 30, not significant).
In perifusion experiments, insulin release from HB-cultured islets in the presence of 2.8 mM glucose for 5 min before high-glucose exposure was increased compared with control islets (Fig. 1A, inset; mean value from 5 to 0 min: HB 0.21 ± 0.01 vs. control 0.16 ± 0.01 ng·20 islets1·min1, n = 6, P < 0.01). Insulin release began to elevate
1 min after exposure to high glucose in both groups of islets (Fig. 1A). Insulin release from HB-cultured islets 110 min after 16.7 mM glucose exposure was similar to that from control islets. Insulin release from HB-cultured islets for the second and third 10-min period after high-glucose exposure was less than from control islets (Fig. 1B; mean value from 20 to 30 min: HB 1.93 ± 0.14 vs. control 2.66 ± 0.07 ng·20 islets1·min1, n = 6, P < 0.01).

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Fig. 1. Effect of D- -hydroxybutyrate (HB) on biphasic 16.7 mM glucose-induced insulin release from intact islets. Islets were cultured with or without 5 mM HB for 15 h. After 2 groups of islets were perifused for 30 min (30 to 0 min) with 2.8 mM glucose (G2.8), they were stimulated with 16.7 mM glucose (G16.7; 030 min). Values represent means ± SE of 6 determinations in the same experiments. Experiments using the same protocol were repeated 3 times to ascertain reproducibility. A: time course of high glucose-induced biphasic insulin release from control and HB-cultured islets. All values in HB-treated islets after 13 min, except those at 16 min, were significantly less than the corresponding control values (at 13 min, P < 0.05; at 20, 25, and 30 min, P < 0.01). Inset: time course of basal insulin release for 5 min (5 to 0 min) in the same experiment. All values in HB-treated islets from 5 to 0 min were significantly greater than the corresponding control values (at 5 and 0 min, P < 0.05; at 2 and 0 min, P < 0.01). B: average values of insulin release from control and HB-cultured islets during the time indicated. Values are calculated from the data in A. *P < 0.05 vs. control; **P < 0.01 vs. control.
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Effect of chronic exposure to acetoacetate on glucose-induced insulin release from intact islets.
Because acetoacetate (AcA) is spontaneously decarboxylated to acetone, a decline in the concentration of AcA in the medium may occur during the culture period. As expected, decreases in the concentrations of AcA were observed in the medium; however, >65% AcA remained in the medium at the end of the 15-h culture period (Table 2). Exposure to AcA (initial concentration 210 mM) for 15 h did not affect 16.7 mM glucose-induced insulin release. However, chronic exposure to high concentrations of AcA (initial concentration 10 mM) augmented basal insulin release (Table 2).
[Ca2+]i elevation in intact islets induced by a high concentration of glucose.
Figure 2A shows [Ca2+]i elevation induced by a high concentration of glucose in control and HB-cultured islets. In the presence of 2.8 mM glucose for 5 min before exposure to high glucose, [Ca2+]i of HB-cultured islets was higher than in control islets (average from 5 to 0 min: HB 134.8 ± 10.4, n = 17, vs. control 86.7 ± 7.6 nM, n = 13, P < 0.01). However, [Ca2+]i of HB-cultured islets at 110 min and for the second and third 10-min period after exposure to high glucose was similar to that of controls (Fig. 2B).

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Fig. 2. Intracellular Ca2+ concentration ([Ca2+]i) elevation induced by 16.7 mM glucose in control and HB-cultured intact islets. Islets were cultured with or without 5 mM HB for 15 h. After preincubation with 2.8 mM glucose for 30 min, the islets were stimulated with 16.7 mM glucose at time 0. A: time course of [Ca2+]i in control and HB-cultured islets. Values represent means ± SE of 13 (control) or 17 (HB) determinations from several experiments. B: average values of [Ca2+]i from control and HB-cultured islets during the time indicated. Values are calculated from the data in A. *P < 0.01 vs. control.
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Insulin release from electrically permeabilized islets.
In the presence of 5 mM ATP, raising the Ca2+ concentration from 30 nM to 3 µM elicited a concentration-dependent increase in insulin release from both electrically permeabilized control and HB-cultured islets. In the presence of 5 mM ATP, insulin release at 30 nM, 100 nM, 1 µM, and 3 µM Ca2+ was greater in HB-cultured islets than in control islets (Fig. 3A). Augmentation of insulin release at 30 nM Ca2+ in HB-cultured islets also was found in the absence of ATP, indicating an effect of the ATP-independent and Ca2+-independent component of insulin release (6) (Fig. 3B). When the increment of insulin release in the presence of 30 nM Ca2+, which is due to chronic exposure to HB, was subtracted from the insulin release from HB-cultured islets to evaluate Ca2+-dependent components of insulin release (7), the values at 100 nM, 1 µM, and 3 µM were found to be similar to the values from control islets (Fig. 3A). Insulin release without ATP at 30 nM Ca2+ from control islets was not affected by 500 µM GDP
S, a stable GDP analog, although that from HB-cultured islets was significantly suppressed by the analog (Fig. 3B).
Time course of NAD(P)H fluorescence in intact islets.
NAD(P)H fluorescence began to elevate immediately after exposure to high glucose in both groups of islets and reached plateau within 5 min after exposure (Fig. 4). Attenuation of NAD(P)H fluorescence was observed from 2 min in HB-cultured islets (at 2 min: HB 1.16 ± 0.01, n = 15, vs. control 1.38 ± 0.03 arbitrary units, n = 12, P < 0.01). The average of fluorescence during the first, second, and third 10-min period was lower in HB-cultured islets than in control islets (from 0 to 10 min: HB 1.23 ± 0.01, n = 15, vs. control 1.50 ± 0.03, n = 12, P < 0.01; from 10 to 20 min: HB 1.32 ± 0.02, n = 15, vs. control 1.68 ± 0.04, n = 12, P < 0.01; from 20 to 30 min: HB 1.31 ± 0.03, n = 15, vs. control 1.68 ± 0.04 arbitrary units, n = 12, P < 0.01).

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Fig. 4. Time course of NAD(P)H elevation induced by 16.7 mM glucose in control and HB-cultured islets. Islets were cultured with or without 5 mM HB for 15 h. After perifusion with 2.8 mM glucose for 30 min, they were exposed to 16.7 mM glucose at time 0 for 30 min. NAD(P)H signals were measured at 20-s intervals from 4 to 30 min. Values represent means ± SE of 12 (control) and 15 (HB) determinations from several experiments.
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Reduced pyridine nucleotide contents and adenine nucleotide contents.
NADH contents in the presence of 16.7 mM were greater than in the presence of 2.8 mM glucose in both control and HB-cultured islets. NADH contents of HB-cultured islets in the presence of 2.8 mM glucose and in the presence of 16.7 mM glucose were more and less than control islets, respectively (Table 3, top). The ATP contents and the ATP-to-ADP ratio in the presence of 16.7 mM were greater than in the presence of 2.8 mM glucose in both control and HB-cultured islets. The ATP contents and ATP-to-ADP ratio of HB-cultured islets in the presence of 16.7 mM glucose were less than in control islets (Table 3, bottom).
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Table 3. Reduced pyridine nucleotide and adenine nucleotides contents in the presence of basal and stimulated levels of glucose in control and HB-cultured islets
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ATP production by mitochondria from islets.
ATP production by mitochondria from control and HB-cultured islets in the presence of various substrates and inhibitors is shown in Table 4. ATP production by mitochondria from HB-cultured islets in the presence of succinate was decreased compared with that from control islets. Antimycin A, a complex III inhibitor in the respiratory chain, inhibited ATP production dramatically in the presence of succinate in both control and HB-cultured islets. Rotenone, a complex I inhibitor in the respiratory chain, also inhibited ATP production in the presence of succinate in both control and HB-cultured islets. However, in the presence of succinate and rotenone, no significant difference in ATP production was observed in control and HB-cultured islets. Mitochondrial ATP production of HB-cultured islets was similar to that of control islets in the presence of tetramethyl-p-phenyldiamine and ascorbate.
Glucose utilization and glucose oxidation.
Glucose utilization and glucose oxidation in the presence of 16.7 mM glucose were greater than in the presence of 2.8 mM glucose in both control and HB-cultured islets. Glucose utilization and glucose oxidation of HB-cultured islets in the presence of 2.8 mM glucose were greater than in control islets. However, glucose utilization and glucose oxidation of HB-cultured islets in the presence of 16.7 mM glucose were not different from those of control islets (Table 5).
Insulin release in the presence of aminooxyacetate.
Glucose (16.7 mM)-induced insulin release from HB-cultured islets was reduced compared with that from control islets (HB 2.22 ± 0.08 vs. control 2.89 ± 0.16 ng·islet1·30 min1, n = 7, P < 0.01). Aminooxyacetate (AOA), an aspartate aminotransferase inhibitor, reduced 16.7 mM glucose-induced insulin release from both control and HB-cultured islets. However, in the presence of AOA, 16.7 mM glucose-induced insulin release from HB-cultured islets was not different from that of control islets (HB 1.98 ± 0.06 vs. control 1.91 ± 0.07 ng·islet1·30 min1, n = 7, not significant; Fig. 5).

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Fig. 5. Glucose (16.7 mM)-induced insulin release from control and HB-cultured islets in the presence of aminooxyacetate (AOA). Islets were cultured with or without 5 mM HB for 15 h. After cultured intact islets were preincubated with 2.8 mM glucose for 30 min, they were incubated with 16.7 mM glucose with (+) or without 5 mM AOA for 30 min. Values represent means ± SE of 7 determinations. *P < 0.01 vs. control without AOA; **P < 0.05 vs. HB-cultured islets without AOA.
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DISCUSSION
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We find that chronic exposure to HB reduces high glucose-induced insulin secretion and augments basal insulin secretion from pancreatic islets. Insulin secretion is reduced by diminished ATP, which is at least partly due to lower ATP production resulting from the reduced supply of NADH to the respiratory chain. Basal insulin secretion is increased due to augmentation of the Ca2+-independent and ATP-independent component of insulin release, in which a GTP-sensitive site may play a role.
In the present study, chronic exposure to >2 mM HB affected insulin secretion, this concentration of HB augmenting glucose-induced insulin acutely in vitro (4, 10, 15, 28, 29, 31) and being frequently observed in serum in various pathophysiological states (18). Acute exposure to the L-isomer was ineffective on insulin release, presumably due to no capacity to oxidize the isomer in islets (4, 28). To exclude nonspecific effects of HB, the chronic effect of the L-isomer was also examined. Because the L-isomer did not affect insulin release, the effect of the D-isomer is presumably linked to its metabolism. Moreover, chronic exposure to AcA, another ketone body, did not affect glucose-induced insulin release. HB is oxidized to AcA by D-
-hydroxybutyrate dehydrogenase, a mitochondrial enzyme, also present in islets (28), producing NADH in mitochondria. Accordingly, alteration of mitochondrial redox potential during culture may be involved in reduced glucose-induced insulin release from HB-cultured islets. Interestingly, acute exposure to exogenous HB or AcA increases or decreases [6-14C]glucose oxidation (28), respectively, which is strongly affected by anaplerosis (41). Anaplerosis plays an important role in determination of the cytosolic level of metabolic factors, including malonyl-CoA (5), NADPH (24), and nonessential amino acids (41) in pancreatic
-cells. The chronic effects of these factors should be taken into account when considering reduced glucose-induced insulin release by HB.
In pancreatic
-cells, intracellular Ca2+ and ATP are the most important regulators of insulin secretion, although there may be other independent metabolic signals (2, 2527, 34). Ca2+ and ATP directly affect the exocytotic system and enhance insulin release synergistically in experiments using single
-cells (37, 43) and permeabilized islets (7). In the present study, we have compared [Ca2+]i, intracellular ATP, and [Ca2+]i efficacy in the exocytotic system at clamped concentrations of ATP in control and HB-exposed islets.
Chronic exposure to HB decreases insulin release by 16.7 mM glucose during a short period around the peak in the first phase and mostly during the second phase of biphasic secretion, 10 min after exposure. [Ca2+]i in the presence of 16.7 mM glucose in HB-cultured islets was similar to that in control islets during the second phase. Moreover, insulin release in the presence of diazoxide, high glucose, and a depolarizing concentration of K+, in which [Ca2+]i is clamped independently of glucose metabolism (10), is also reduced by chronic exposure to HB. Apparently, [Ca2+]i does not play a role in the lower response to high glucose in HB-cultured islets. In addition, Ca2+ efficacy at clamped concentration of ATP in the exocytotic system was similar in the two groups of islets. On the other hand, NAD(P)H fluorescence was less in the HB-cultured islets in the presence of high glucose. Moreover, both ATP contents and ATP-to-ADP ratio of HB-cultured islets 30 min after exposure to high glucose were reduced compared with control islets. Decreased intracellular ATP reduces Ca2+ efficacy in the exocytotic system (7, 37, 43). Therefore, the lower ATP level due to the reduced redox state may play a major role in the attenuation of insulin secretion from HB-cultured islets in response to high glucose.
NAD(P)H autofluorescence dominantly reflects the redox state of mitochondria (32) and is widely used as an index of the intracellular redox state in intact islets (9, 33, 36). However, the method cannot discriminate between NADH and NADPH signals. Therefore, NADH, the supply of which accelerates mitochondrial oxidative phosphorylation, was determined using the enzymatic method. NADH contents of HB-cultured islets were lower in the presence of high glucose, partly accounting for the decreased ATP level in these islets.
To determine whether oxidative phosphorylation in the mitochondrial respiratory chain is suppressed in HB-cultured islets, mitochondrial ATP production was examined in the presence of various substrates and inhibitors. In the presence of succinate, which directly renders electrons to complex II (39), ATP production in HB-cultured islets is less than in controls. The fact that ATP production in both groups of islets in the presence of succinate is partially inhibited by rotenone, a complex I inhibitor (39), indicates that increased NADH derived from succinate also renders electrons to complex I and that electrons from complex I, in addition to electrons from complex II, augment oxidative phosphorylation in the presence of succinate. Tetramethyl-p-phenyldiamine with ascorbate renders electrons to cytochrome C, which transfers electrons to complex IV (39). The fact that ATP production in the presence of succinate and rotenone and in the presence of tetramethyl-p-phenyldiamine and ascorbate is similar in the two groups of islets indicates that the respiratory chain downstream of complex II is not affected by chronic exposure to HB. Accordingly, the decrease in ATP production in the presence of succinate may be due to decreased NADH content in the matrix of mitochondria derived from succinate.
NADH in the matrix of mitochondria is produced in a reaction catalyzed by isocitrate dehydrogenase,
-ketoglutarate dehydrogenase, and malate dehydrogenase in the TCA cycle, and by PDH (23, 38). Malate dehydrogenase also participates in the malate/aspartate shuttle, which carries NADH produced in cytosol to the matrix of mitochondria (22, 40). PDH activity is not affected by chronic exposure to HB in human islets (47). The fact that glucose utilization and glucose oxidation were not affected indicates that isocitrate dehydrogenase,
-ketoglutarate dehydrogenase, and PDH, which catalyze reactions accompanying production of NADH and CO2, were not affected by chronic exposure to HB. Malate dehydrogenase catalyzes reactions that produce NADH without production of CO2 that are involved in both the TCA cycle and malate/aspartate shuttle. AOA (5 mM) reduced glucose-induced insulin release from rat islets without affecting glucose oxidation, because AOA does not affect the TCA cycle and inhibits the malate/aspartate shuttle by blocking transamination reactions (30). Glucose-induced insulin release in the presence of AOA was not reduced by chronic exposure to HB, suggesting that chronic exposure to HB affects the malate/aspartate shuttle.
With the use of electrically permeabilized islets incubated at low Ca2+ without ATP, it was revealed that augmentation of basal insulin release from HB-cultured islets does not necessarily require elevation of [Ca2+]i, although [Ca2+]i was higher in HB-cultured islets in the presence of basal levels of glucose than in control islets. This augmentation is not due to nonspecific leakage of insulin and to slow washout of insulin, since it was suppressed by the GDP analog, which is also compatible with the fact that GTP augments insulin release in the exocytotic process directly and Ca2+ independently (35).
Postprandial insulin release is inhibited in vivo in the starved state (11, 42, 46) and in type 2 diabetic patients with ketosis (44). Although the present results suggest suppressed insulin release in such pathophysiological conditions, these results may well not be found in vivo, as elevation of basal insulin secretion is not observed in the fasting state.
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GRANTS
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This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grants for Leading Project for Biosimulation from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Health and Labor Sciences Research Grants for Research on Human Genome, Tissue Engineering Food Biotechnology, from the Ministry of Health, Labor and Welfare of Japan; Health and Labor Sciences Research Grants for Comprehensive Research on Aging and Health from the Ministry of Health, Labor and Welfare of Japan; and Establishment of International Center of Excellence (COE) for Integration of Transplantation Therapy and Regenerative Medicine (COE program of the Ministry of Education, Culture, Sports, Science and Technology of Japan).
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ACKNOWLEDGMENTS
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We thank S. Akagi for technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. Fujimoto, Dept. of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto Univ., 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: fujimoto{at}metab.kuhp.kyoto-u.ac.jp)
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.
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REFERENCES
|
---|
- Aizawa T, Komatsu M, Asanuma N, Sato Y, and Sharp GWG. Glucose action "beyond ionic events" in the pancreatic
cell. Trends Pharmacol Sci 19: 496499, 1998.[CrossRef][ISI][Medline]
- Antinozzi PA, Segall L, Prentki M, McGarry JD, and Newgard CB. Molecular or pharmacologic perturbation of the link between glucose and lipid metabolism is without effect on glucose-stimulated insulin secretion. A re-evaluation of the long-chain acyl-CoA hypothesis. J Biol Chem 273: 1614616154, 1998.[Abstract/Free Full Text]
- Ashcroft SJ, Weerasinghe LC, Bassett JM, and Randle PJ. The pentose cycle and insulin release in mouse pancreatic islets. Biochem J 126: 525532, 1972.[ISI][Medline]
- Biden TJ and Taylor KW. Effects of ketone bodies on insulin release and islet-cell metabolism in the rat. Biochem J 212: 371377, 1983.[ISI][Medline]
- Farfari S, Schulz V, Corkey B, and Prentki M. Glucose-regulated anaplerosis and cataplerosis in pancreatic
-cells: possible implication of a pyruvate/citrate shuttle in insulin secretion. Diabetes 49: 718726, 2000.[Abstract]
- Fujimoto S, Ishida H, Kato S, Okamoto Y, Tsuji K, Mizuno N, Ueda S, Mukai E, and Seino Y. The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in
-cells. Endocrinology 139: 11331140, 1998.[Abstract/Free Full Text]
- Fujimoto S, Mukai E, Hamamoto Y, Takeda T, Takehiro M, Yamada Y, and Seino Y. Prior exposure to high glucose augments depolarization-induced insulin release by mitigating the decline of ATP level in rat islets. Endocrinology 143: 213221, 2002.[Abstract/Free Full Text]
- Fujimoto S, Tsuura Y, Ishida H, Tsuji K, Mukai E, Kajikawa M, Hamamoto Y, Takeda T, Yamada Y, and Seino Y. Augmentation of basal insulin release from rat islets by preexposure to a high concentration of glucose. Am J Physiol Endocrinol Metab 279: E927E940, 2000.[Abstract/Free Full Text]
- Gilon P and Henquin JC. Influence of membrane potential changes on cytoplasmic Ca2+ concentration in an electrically excitable cell, the insulin-secreting pancreatic B-cell. J Biol Chem 267: 2071320720, 1992.[Abstract/Free Full Text]
- Goberna R, Tamarit J Jr, Osorio J, Fussganger R, Tamarit J, and Pfeiffer EF. Action of B-hydroxy butyrate, acetoacetate and palmitate on the insulin release in the perfused isolated rat pancreas. Horm Metab Res 6: 256260, 1974.[ISI][Medline]
- Grey NJ, Goldring S, and Kipnis DM. The effect of fasting, diet, and actinomycin D on insulin secretion in the rat. J Clin Invest 49: 881889, 1970.[ISI][Medline]
- Harano Y, Ohtsuki M, Ida M, Kojima H, Harada M, Okanishi T, Kashiwagi A, Ochi Y, Uno S, and Shigeta Y. Direct automated assay method for serum or urine levels of ketone bodies. Clin Chim Acta 151: 177183, 1985.[CrossRef][ISI][Medline]
- Henquin JC. Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes 49: 17511760, 2000.[Abstract]
- Idahl LA and Lembert N. Glycerol 3-phosphate-induced ATP production in intact mitochondria from pancreatic B-cells. Biochem J 312: 287292, 1995.[ISI][Medline]
- Ikeda T, Yoshida T, Ito Y, Murakami I, Mokuda O, Tominaga M, and Mashiba H. Effect of
-hydroxybutyrate and acetoacetate on insulin and glucagon secretion from perfused rat pancreas. Arch Biochem Biophys 257: 140143, 1987.[CrossRef][ISI][Medline]
- Kajikawa M, Fujimoto S, Tsuura Y, Mukai E, Takeda T, Hamamoto Y, Takehiro M, Fujita J, Yamada Y, and Seino Y. Ouabain suppresses glucose-induced mitochondrial ATP production and insulin release by generating reactive oxygen species in pancreatic islets. Diabetes 51: 25222529, 2002.[Abstract/Free Full Text]
- Kato T, Berger SJ, Carter JA, and Lowry OH. An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases. Anal Biochem 53: 8697, 1973.[ISI][Medline]
- Laffel L. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes Metab Res Rev 15: 412426, 1999.[CrossRef][ISI][Medline]
- Lembert N and Idahl LA.
-Ketoisocaproate is not a true substrate for ATP production by pancreatic
-cell mitochondria. Diabetes 47: 339344, 1998.[Abstract]
- Lembert N, Idahl LA, and Ammon HP. K-ATP channel independent effects of pinacidil on ATP production in isolated cardiomyocyte or pancreatic
-cell mitochondria. Biochem Pharmacol 65: 18351841, 2003.[ISI][Medline]
- Lembert N, Joos HC, Idahl LA, Ammon HP, and Wahl MA. Methyl pyruvate initiates membrane depolarization and insulin release by metabolic factors other than ATP. Biochem J 354: 345350, 2001.[CrossRef][ISI][Medline]
- MacDonald MJ. Evidence for the malate aspartate shuttle in pancreatic islets. Arch Biochem Biophys 213: 643649, 1982.[ISI][Medline]
- MacDonald MJ. Differences between mouse and rat pancreatic islets: succinate responsiveness, malic enzyme, and anaplerosis. Am J Physiol Endocrinol Metab 283: E302E310, 2002.[Abstract/Free Full Text]
- MacDonald MJ. The export of metabolites from mitochondria and anaplerosis in insulin secretion. Biochim Biophys Acta 1619: 7788, 2003.[ISI][Medline]
- MacDonald MJ and Fahien LA. Glutamate is not a messenger in insulin secretion. J Biol Chem 275: 3402534027, 2000.[Abstract/Free Full Text]
- Maechler P, Kennedy ED, Pozzan T, and Wollheim CB. Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic
-cells. EMBO J 16: 38333841, 1997.[Abstract/Free Full Text]
- Maechler P and Wollheim CB. Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis. Nature 402: 685689, 1999.[CrossRef][ISI][Medline]
- Malaisse WJ, Lebrun P, Rasschaert J, Blachier F, Yilmaz T, and Sener A. Ketone bodies and islet function: 86Rb handling and metabolic data. Am J Physiol Endocrinol Metab 259: E123E130, 1990.[Abstract/Free Full Text]
- Malaisse WJ, Lebrun P, Yaylali B, Camara J, Valverde I, and Sener A. Ketone bodies and islet function: 45Ca handling, insulin synthesis, and release. Am J Physiol Endocrinol Metab 259: E117E122, 1990.[Abstract/Free Full Text]
- Malaisse WJ, Malaisse-Lagae F, and Sener A. The stimulus-secretion coupling of glucose-induced insulin release: effect of aminooxyacetate upon nutrient-stimulated insulin secretion. Endocrinology 111: 392397, 1982.[Abstract]
- Metzger P, Franken P, and Balasse EO. Permissive role of glucose on the insulinotropic effect of ketone bodies in vivo. Horm Metab Res 5: 313315, 1973.[ISI][Medline]
- Patterson GH, Knobel SM, Arkhammar P, Thastrup O, and Piston DW. Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet
cells. Proc Natl Acad Sci USA 97: 52035207, 2000.[Abstract/Free Full Text]
- Pralong WF, Spat A, and Wollheim CB. Dynamic pacing of cell metabolism by intracellular Ca2+ transients. J Biol Chem 269: 2731027314, 1994.[Abstract/Free Full Text]
- Prentki M, Tornheim K, and Corkey BE. Signal transduction mechanisms in nutrient-induced insulin secretion. Diabetologia 40, Suppl 2: S32S41, 1997.[CrossRef]
- Proks P, Eliasson L, Ammala C, Rorsman P, and Ashcroft FM. Ca2+- and GTP-dependent exocytosis in mouse pancreatic
-cells involves both common and distinct steps. J Physiol 496: 255264, 1996.[Abstract]
- Rocheleau JV, Head WS, Nicholson WE, Powers AC, and Piston DW. Pancreatic islet
-cells transiently metabolize pyruvate. J Biol Chem 277: 3091430920, 2002.[Abstract/Free Full Text]
- Rorsman P. The pancreatic
-cell as a fuel sensor: an electrophysiologists viewpoint. Diabetologia 40: 487495, 1997.[CrossRef][ISI][Medline]
- Salway JG. Biosynthesis of ATP I: ATP, the molecule that powers metabolism. In: Metabolism at a Glance (2nd ed.). Malden, MA: Blackwell Science, 1999, p. 1213.
- Salway JG. Biosynthesis of ATP II: mitochondrial respiratory chain. In: Metabolism at a Glance (2nd ed.). Malden, MA: Blackwell Science, 1999, p. 1415.
- Salway JG. The oxidation of cytosolic NADH:the malate/aspartate shuttle and the glycerol phosphate shuttle. In: Metabolism at a Glance (2nd ed.). Malden, MA: Blackwell Science, 1999, p. 1617.
- Schuit F, De Vos A, Farfari S, Moens K, Pipeleers D, Brun T, and Prentki M. Metabolic fate of glucose in purified islet cells. Glucose-regulated anaplerosis in beta cells. J Biol Chem 272: 1857218579, 1997.[Abstract/Free Full Text]
- Solomon SS, Ensinck JW, and Williams RH. Effect of starvation on plasma immunoreactive insulin and non-suppressible insulin-like activity in normal and obese humans. Metabolism 17: 528534, 1968.[CrossRef][ISI][Medline]
- Takahashi N, Kadowaki T, Yazaki Y, Ellis-Davies GC, Miyashita Y, and Kasai H. Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells. Proc Natl Acad Sci USA 96: 760765, 1999.[Abstract/Free Full Text]
- Tanaka K, Moriya T, Kanamori A, and Yajima Y. Analysis and a long-term follow up of ketosis-onset Japanese NIDDM patients. Diabetes Res Clin Pract 44: 137146, 1999.[CrossRef][ISI][Medline]
- Taniguchi S, Okinaka M, Tanigawa K, and Miwa I. Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets. J Biochem (Tokyo) 127: 289295, 2000.[Abstract]
- Vance JE, Buchanan KD, and Williams RH. Effect of starvation and refeeding on serum immunoreactive glucagon and insulin levels. J Lab Clin Med 72: 290297, 1968.[ISI][Medline]
- Zhou YP and Grill V. Long term exposure to fatty acids and ketones inhibits B-cell functions in human pancreatic islets of Langerhans. J Clin Endocrinol Metab 80: 15841590, 1995.[Abstract]
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