1 Institute of Internal Medicine, Endocrinology, and Metabolism and S. Signorelli Diabetes Center, Ospedale Garibaldi, and 2 Section of Biochemistry and Molecular Biology, Department of Chemistry, Faculty of Medicine, University of Catania, 95123 Catania, Italy
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ABSTRACT |
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Exposure of rat pancreatic islets to 20 mM leucine for 24 h reduced insulin release in response to glucose (16.7 and 22.2 mM). Insulin release was normal when the same islets were stimulated with leucine (40 mM) or glyburide (1 µM). To investigate the mechanisms responsible for the different effect of these secretagogues, we studied several steps of glucose-induced insulin secretion. Glucose utilization and oxidation rates in leucine-precultured islets were similar to those of control islets. Also, the ATP-sensitive K+ channel-independent pathway of glucose-stimulated insulin release, studied in the presence of 30 mM K+ and 250 µM diazoxide, was normal. In contrast, the ATP-to-ADP ratio after stimulation with 22.2 mM glucose was reduced in leucine-exposed islets with respect to control islets. The decrease of the ATP-to-ADP ratio was due to an increase of ADP levels. In conclusion, prolonged exposure of pancreatic islets to high leucine levels selectively impairs glusoce-induced insulin release. This secretory abnormality is associated with (and might be due to) a reduced ATP-to-ADP ratio. The abnormal plasma amino acid levels often present in obesity and diabetes may, therefore, affect pancreatic islet insulin secretion in these patients.
adenosine triphosphate; adenosine diphosphate; -cell
desensitization; amino acids; adenine nucleotides; adenosine
triphosphate potassium channel-independent pathway
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INTRODUCTION |
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TYPE 2 DIABETES is characterized by inadequate pancreatic -cell
insulin release in response to glucose and by impaired insulin action
(5, 6, 16, 19, 20, 31). In these patients, the altered
insulin secretory pattern depends, at least in part, on the negative
influence of chronic high glucose and/or free fatty acid (FFA) plasma
concentrations (gluco- or lipotoxicity). These metabolites are believed
to affect pancreatic
-cells function by chronic stimulation (by
either glucose or FFA or both) and consequent "desensitization" to
glucose (2, 11, 23, 26, 28, 30, 33, 37).
In contrast to the effects of glucose and FFA, less studied are the effects of amino acids, the third class of nutrient insulin secretagogues. Increased amino acid levels (and in particular leucine, isoleucine, and valine in the concentration range of 0.1-0.3 mM) have been described in both type 1 and type 2 diabetes and in obesity (12, 13, 38) as a consequence of impaired insulin effect on amino acid uptake and metabolism. Also, in animal models of diabetes, insulin action deficiency increased amino acid plasma concentrations up to 1.5 mM (4, 35).
The aim of this study was to investigate whether chronic (24 h) exposure of cultured rat pancreatic islets to an elevated leucine concentration (20 mM) might affect insulin secretion stimulated by glucose or other secretagogues. Leucine at 20 mM was used, because this is a concentration that gives a maximal insulin stimulation, similar to that obtained with 16.7 mM glucose in vitro (22).
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MATERIALS AND METHODS |
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Materials. Crude collagenase was obtained from Boehringer Mannheim (Mannheim, Germany). Culture medium CMRL-1066, heat-inactivated fetal calf serum, glutamine, and gentamycin were obtained from GIBCO (Glasgow, UK). D-[5-3H]glucose and D-[U-14C]glucose were from Amersham (Amersham, Buckinghamshire, UK). Leucine, diazoxide, glyburide, ATP, ADP, phosphoenolpyruvate, and pyruvate kinase were from Sigma (St. Louis, MO). All other chemicals were of analytical grade.
Islet preparation and culture conditions. Pancreatic islets were isolated by the collagenase method from fed male Wistar rats (200-250 g) injected intraperitoneally with 0.2 ml of a 0.2% pilocarpine solution 2 h before being killed by decapitation. Purified islets were cultured overnight at 5.5 mM glucose in CMRL-1066 medium containing 10% fetal calf serum, 2 mM L-glutamine, and gentamycin at 37°C in a 95% air-5% CO2 atmosphere and then at 5.5 mM glucose with or without 20 mM leucine for 24 h.
Insulin secretion. Islets cultured with or without leucine were washed twice in Krebs-Ringer HEPES buffer (containing in mM: 136 NaCl, 5.4 KCl, 2.5 CaCl2, 0.8 MgSO4, 0.3 Na2HPO4, 0.4 KH2PO4, and 10 HEPES and 0.25% BSA, pH 7.35).
In the experiments with 30 mM KCl (high K+ medium), NaCl was reduced to 111.4 mM. Batches of five purified islets were then incubated in 1 ml of buffer with appropriate concentration of glucose and test substances (30-min incubation at 37°C). Insulin in the medium was then measured by radioimmunoassay. Results are expressed as insulin released in the medium (pg · isletGlucose utilization. Islet utilization of glucose was determined by measuring the formation of 3H20 from D-[5-3H]glucose, as previously described (27). Groups of 15 islets were incubated in 40 µl of Krebs-Ringer bicarbonate buffer (KRBB) (in mM: 118 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3) supplemented with 10 mM HEPES (pH 7.4) containing 2 µCi D-[5-3H]glucose at 2.8 or 16.7 mM glucose. The incubation was carried out in 1.5-ml Eppendorf tubes inside an airtight, sealed 20-ml glass scintillation vial that contained 500 µl of distilled water. After 2 h at 37°C, the reaction was stopped by adding 0.5 M HCl (100 µl, injected through the rubber seal). Scintillation vials were then incubated overnight at 50°C, and water radioactivity was measured. Under these conditions, the recovery from known amount of 3H2O was fairly constant, ranging from 50 to 60%.
Glucose oxidation. Glucose oxidation was determined by measuring the formation of 14CO2 from D-[U-14C]glucose (29). Groups of 15 islets were incubated in a 1.5-ml Eppendorf tube containing 100 µl of KRBB containing 3 µCi D-[U-14C]glucose (specific activity: 302 mCi/mM) plus nonradioactive glucose to a final concentration of either 2.8 or 16.7 mM. The tubes, suspended in standard 20-ml glass scintillation vials, were gassed with O2-CO2 (95:5) and capped airtight. The vials were then shaken continuously at 37°C for 120 min. The islets' metabolism was stopped by injection of 100 µl of 0.05 mM antimycin A (dissolved in 70% ethanol) into the tube. This was immediately followed by an injection of 250 µl of hyamine hydroxide (New England Nuclear, Boston, MA) into the vials. 14CO2 was liberated from the incubation medium by subsequent injection into the tubes of 100 µl of 0.4 mM Na2HPO4 solution adjusted to pH 6.0. After 2 h at room temperature (to allow the liberated 14CO2 to be trapped by hyamine hydroxide), the cup was removed, 8 ml of a scintillation fluid were added to each flask, and the radioactivity was measured in a liquid scintillation counter.
Measurements of adenine nucleotides in incubated islets.
Adenine nucleotides were measured according to Detimary et al.
(9). After the incubation with 2.8 or 22.2 mM glucose, an aliquot of medium was taken for insulin assay while the tubes remained
at 37°C. The islets were incubated for another 5 min. The incubation
was stopped by the addition of 0.125 ml of tricarboxylic acid (TCA) to
a final concentration of 5%. The tubes were then vortexed, left on ice
for 5 min, and centrifuged in a microfuge (Beckman). A fraction (0.4 ml) of the supernatant was mixed with 1.5 ml of diethyl ether, and the
ether phase containing TCA was discarded. This procedure was repeated
three times to ensure complete elimination of TCA. The extracts were
then diluted with 0.4 ml of a buffer containing 20 mM HEPES, 3 mM
MgCl2, and KOH as required to adjust pH to 7.75 (assay
buffer). The diluted extracts were frozen at 70°C until the day of
the assay, which started with an appropriate further dilution. ATP and
ADP were assayed in triplicate by a luminometric method
(18). To measure total ATP + ADP, ADP was first
converted into ATP by mixing 100 µl of the diluted extract with 300 µl of assay buffer supplemented with 1.5 mM
phosphoenolpyruvate and 2.3 U/ml pyruvate kinase with
incubation at room temperature for 15 min. Samples with known
concentrations of ADP, without ATP, were run in parallel to check that
the transformation was complete. ATP was measured by the addition of
100 µl of an ATP-monitoring reagent containing luciferase and
luciferin (Sigma). The emitted light was measured in a luminometer
(Turner TD-20/20). To measure only ATP, the same previously described
procedure was followed, except that, in the first incubation step,
pyruvate kinase was lacking. ADP levels were then calculated by
subtracting ATP from the total ATP + ADP. Blanks and ATP standards
were run through the entire procedure, including the extraction steps.
Statistical analysis. The statistical significance of differences between means was assessed by Student's t-test for unpaired data when only two groups of data were compared or by an analysis of variance (ANOVA) followed by Newman-Keul's test for comparison of dose-response curve.
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RESULTS |
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Insulin release.
A 24-h incubation with increasing leucine concentrations resulted in a
dose-dependent decrease of glucose-induced insulin release. The effect
was significant at 20 mM leucine (915.2 ± 149.9 vs. 1,719.7 ± 156.0 pg · islet1 · 30 min
1, means ± SE, n = 6, P < 0.01) that was then used for the further experiments (Fig. 1). In islets cultured
for 24 h with 20 mM leucine, basal insulin release (in the absence
of glucose) was higher than in control islets (110 ± 13.3 vs.
46 ± 12.1 pg · islet
1 · 30 min
1, n = 5, P < 0.01).
The insulin response to increasing glucose concentrations was
statistically different in the two groups of islets (P < 0.0001 by the ANOVA test). In particular, in islets preexposed to
leucine, the secretory response to 16.7 and 22.2 mM glucose was reduced
compared with control islets (711.9 ± 90 vs. 1,413.6 ± 209.8 pg · islet
1 · 30 min
1
at 16.7 mM and 737.6 ± 164.3 vs. 1,640.7 ± 158.7 pg · islet
1 · 30 min
1 at
22.2 mM glucose, means ± SE, n = 5, P < 0.01 and P < 0.001, respectively)
(Fig. 2). The sensitivity to glucose,
calculated as the glucose concentration at which the 50% of maximal
effect (EC50) was observed, was similar in the two groups
of islets (10.5 ± 0.9 vs. 8.3 ± 1.2 mM glucose in control
and leucine-exposed islets, respectively, n = 5).
Because in islets preexposed to leucine intracellular insulin content
was reduced (47.1 ± 5 vs. 75.6 ± 9 ng/islet in control
islets, n = 5, P < 0.05), we
investigated whether the impaired insulin release was related to the
lower intracellular insulin content. Accordingly, in islets preexposed to leucine, we tested the secretory effects of glyburide or leucine, two secretagogues that stimulate insulin secretion with mechanisms different from the mechanism of glucose. No difference between islets
preexposed to leucine or control islets was observed in the secretory
response to glyburide 1 µM (843.3 ± 44.2 vs. 926 ± 176.5 pg · islet
1 · 30 min
1 in
control and leucine-exposed islets, respectively, n = 4) or to leucine (40 mM) itself (775.2 ± 153.6 vs. 713.2 ± 123.0 pg · islet
1 · 30 min
1, n = 4) (Fig.
3).
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Glucose utilization and oxidation.
To investigate whether the reduced secretory response to glucose was
due to changes in glucose metabolism, we measured glucose utilization
and oxidation. Preexposure to leucine significantly increased basal
glucose utilization, measured at 2.8 mM glucose (59.3 ± 3.9 vs.
43.4 ± 1.9 pmol · islet1 · 120 min
1 in control islets, P < 0.05).
Glucose utilization at 16.7 mM was not different in the two groups of
islets. Glucose oxidation was unaffected by islet preexposure to
leucine at both 2.8 and 16.7 mM glucose (Fig.
4).
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ATP-sensitive K+ channel-independent
pathway.
According to recent data (1, 3, 15), the regulation of
insulin secretion by glucose involves both ATP-sensitive K+
(K1 · 30 min
1, n = 4) (Fig.
5).
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Relationship between insulin release and adenine nucleotide levels.
Because -cell intracellular adenine nucleotides, and in particular
the ATP-to-ADP ratio, is believed to play a critical role in
glucose-induced insulin release (10), we measured both
adenine nucleotide content and insulin secretion in the two groups of islets in the presence of basal (2.8 mM) or stimulating (22.2) glucose
concentrations (Table 1). In control
islets, in response to glucose concentration increase, ATP levels
increased, ADP levels decreased, and as a consequence, the ATP-to-ADP
ratio clearly increased. In contrast, in leucine-preexposed islets,
both ATP and ADP levels increased in response to glucose stimulation,
and therefore, the ATP-to-ADP ratio was unchanged and significantly lower than in control islets (Table 1). In leucine-exposed islets, these changes in the ATP-to-ADP ratio were associated with a reduced insulin release in response to glucose (Table 1).
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DISCUSSION |
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Our data indicate that the chronic exposure of rat pancreatic
islets to high (20 mM) leucine levels selectively impairs
glucose-induced insulin release. Insulin secretion induced by other
secretagogues like glyburide or leucine itself is unaffected, despite
the reduced insulin content of leucine-preexposed islets. We found that
the mechanism of the selective secretory impairment of
glucose-stimulated secretion in islets exposed to high leucine
concentrations is unrelated to the K
The cause of increased ADP levels in response to the glucose stimulation in leucine-exposed islets is unknown. An increase of ATP hydrolysis (with subsequent ADP formation) or a decrease of ATP formation from ADP (with subsequent ADP accumulation) are among the various possibilities. Exposure to high leucine levels did not affect the response of pancreatic islets to leucine itself or to the sulfonylurea glyburide. These observations suggest that the secretory pathway distal to the ATP-sensitive K+ channels is normal in leucine-preexposed islets and indicate that the reduced intracellular insulin content of leucine-preexposed islets is not a limiting factor for a quantitatively normal insulin release after appropriate stimulation.
Leucine enters the pancreatic -cell and is metabolized in the
mitochondria. The mechanism by which it affects insulin release is
still under debate. It has been reported that it may increase ATP
levels by its own catabolism (21, 22, 25) and
allosterically activate glutamate dehydrogenase (17, 36).
The effect on nucleotide levels is controversial. Under our
experimental conditions, we did not observe an increase of ATP levels
in response to acute leucine stimulation. Moreover, in a recent study
of Ronner et al. (32), the addition of amino acids did not
affect the concentration of ATP and free ADP, yet amino acids greatly
stimulated insulin release. The authors proposed that amino acids may
regulate insulin release both by an unknown amino acid sensor and by
the K
-cell.
In conclusion, we found that a prolonged exposure to high leucine
levels selectively impairs glucose-induced insulin release from
isolated rat pancreatic islets. This secretory alteration is associated
with (and might be due to) a reduced ATP-to-ADP ratio. Because elevated
amino acid levels, including leucine, have been described in both type
1 and type 2 diabetes, this mechanism might contribute to the -cell
secretory abnormalities observed in diabetic patients in poor metabolic control.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Anello, Division of Endocrinology, Ospedale Garibaldi, Piazza S. Maria di Gesú, 95123 Catania, Italy (E-mail: segmeint{at}mbox.unict.it).
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.
Received 14 February 2001; accepted in final form 13 July 2001.
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