Leucine Culture Reveals That ATP Synthase Functions as a Fuel Sensor in Pancreatic β-Cells
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m405309200
ISSN1083-351X
AutoresJichun Yang, Ryan Wong, Xujing Wang, Jacob A. Moibi, Martin J. Hessner, Scott R. Greene, Jianmei Wu, Siam Sukumvanich, Bryan A. Wolf, Zhiyong Gao,
Tópico(s)Adenosine and Purinergic Signaling
ResumoOur goal was to investigate whether leucine culture affects β-cell glucose sensing. One-day culture of rat islets with 10 mm leucine had no effect on glucose-induced insulin secretion. One-week leucine culture decreased the threshold for glucose-induced insulin secretion and increased maximal insulin secretion at 30 mm glucose. Glucose-induced cytosolic free Ca2+ was increased at 1 week but not at 1 day of leucine culture. Without glucose, ATP content was not different with or without leucine culture for 1 week. With 20 mm glucose, ATP content was higher by 1.5-fold in islets cultured for 1 week with leucine than those without leucine. Microarray experiments indicated that culture of RINm5F cells with leucine increased expression of ATP synthase β subunit 3.2-fold, which was confirmed by real time reverse transcription-PCR analysis (3.0- ± 0.4-fold) in rat islets at 1 week but not after 1 day with leucine culture. Down-regulation of ATP synthase β subunit by siRNA decreased INS1 cell ATP content and insulin secretion with 20 mm glucose. Overexpression of ATP synthase β subunit in INS1 cell increased insulin secretion in the presence of 5 and 20 mm glucose. In conclusion, one-week leucine culture of rat islets up-regulated ATP synthase and increased ATP content, which resulted in elevated [Ca2+] levels and more insulin exocytosis by glucose. Depletion of ATP synthase β subunit with siRNA produced opposite effects. These data reveal the fuel-sensing role of mitochondrial ATP synthase in the control of ATP production from glucose and the control of glucose-induced insulin secretion. Our goal was to investigate whether leucine culture affects β-cell glucose sensing. One-day culture of rat islets with 10 mm leucine had no effect on glucose-induced insulin secretion. One-week leucine culture decreased the threshold for glucose-induced insulin secretion and increased maximal insulin secretion at 30 mm glucose. Glucose-induced cytosolic free Ca2+ was increased at 1 week but not at 1 day of leucine culture. Without glucose, ATP content was not different with or without leucine culture for 1 week. With 20 mm glucose, ATP content was higher by 1.5-fold in islets cultured for 1 week with leucine than those without leucine. Microarray experiments indicated that culture of RINm5F cells with leucine increased expression of ATP synthase β subunit 3.2-fold, which was confirmed by real time reverse transcription-PCR analysis (3.0- ± 0.4-fold) in rat islets at 1 week but not after 1 day with leucine culture. Down-regulation of ATP synthase β subunit by siRNA decreased INS1 cell ATP content and insulin secretion with 20 mm glucose. Overexpression of ATP synthase β subunit in INS1 cell increased insulin secretion in the presence of 5 and 20 mm glucose. In conclusion, one-week leucine culture of rat islets up-regulated ATP synthase and increased ATP content, which resulted in elevated [Ca2+] levels and more insulin exocytosis by glucose. Depletion of ATP synthase β subunit with siRNA produced opposite effects. These data reveal the fuel-sensing role of mitochondrial ATP synthase in the control of ATP production from glucose and the control of glucose-induced insulin secretion. Glucose is the main secretagogue of insulin secretion from pancreatic β-cells. The mechanisms of glucose-induced insulin secretion have been studied extensively. Through glycolysis and oxidation, glucose increases pancreatic β-cell ATP/ADP ratio, which closes ATP-sensitive potassium (KATP) channels and depolarizes the cell membrane. This results in an influx of extracellular Ca2+ and increase of free cytosolic [Ca2+] that stimulates exocytosis of insulin granules (1Ashcroft F.M. Rorsman P. Prog. Biophys. Mol. 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In pancreatic β-cells, most of the intracellular ATP comes from the oxidation of glucose-derived pyruvate and oxidation of NADH in the mitochondria via the electron transport chain. Damage or inhibition of ATP synthesis results in β-cell dysfunction and impairs glucose-stimulated insulin secretion (8Brownlee M. J. Clin. Investig. 2003; 112: 1788-1790Crossref PubMed Scopus (187) Google Scholar, 9Krauss S. Zhang C.Y. Scorrano L. Dalgaard L.T. St. Pierre J. Grey S.T. Lowell B.B. J. Clin. Investig. 2003; 112: 1831-1842Crossref PubMed Scopus (334) Google Scholar). Some recent publications indicate that superoxide produced by hyperglycemia activates uncoupling-protein-2 (UCP2) and destroys the proton gradient between inner and outer mitochondrial membranes. This negatively affects the activity of ATP synthase, decreases ATP production, and impairs glucose-stimulated insulin secretion of pancreatic β-cells resulting in diabetes (8Brownlee M. J. Clin. 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As the rate-limiting enzyme of glucose metabolism, it is believed that glucokinase sets a strict control on glucose metabolism in pancreatic β-cells. However, overexpression of glucokinase or hexokinase I fails to increase the maximal insulin output induced by glucose, although it decreases the threshold for glucose-induced insulin secretion in pancreatic β-cells (10Antinozzi P.A. Ishihara H. Newgard C.B. Wollheim C.B. J. Biol. Chem. 2002; 277: 11746-11755Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 20Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (119) Google Scholar, 21Becker T.C. Noel R.J. Johnson J.H. Lynch R.M. Hirose H. Tokuyama Y. Bell G.I. Newgard C.B. J. Biol. Chem. 1996; 271: 390-394Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 22Grimsby J. Sarabu R. Corbett W.L. Haynes N.E. Bizzarro F.T. Coffey J.W. Guertin K.R. Hilliard D.W. Kester R.F. Mahaney P.E. Marcus L. Qi L. Spence C.L. Tengi J. Magnuson M.A. Chu C.A. Dvorozniak M.T. Matschinsky F.M. Grippo J.F. Science. 2003; 301: 370-373Crossref PubMed Scopus (443) Google Scholar). Thus, glucokinase may not be the only rate-limiting step in glucose-induced insulin secretion of pancreatic β-cells. Other factor(s) may also be rate-limiting and contribute to the tight control of glucose-induced insulin secretion. As the key enzyme catalyzing the conversion of ADP and Pi to ATP in the electron transport chain, ATP synthase (complex V) may play an important role in ATP synthesis and hence glucose-induced insulin secretion. Thus, the aim of this study was to investigate the role of ATP synthase in glucose-induced insulin secretion by attempting to change its expression level with leucine culture and siRNA. Isolation and Culture of Rat Islets—Islets were isolated from 4–6 male Sprague-Dawley rats (220–300 g) by collagenase P digestion and Ficoll gradient centrifugation as described in detail previously (14Gao Z.Y. Li G. Najafi H. Wolf B.A. Matschinsky F.M. Diabetes. 1999; 48: 1535-1542Crossref PubMed Scopus (93) Google Scholar). Before culturing, the islets were washed at least three times with leucine-free RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 μg/ml streptomycin, and 100 IU/ml penicillin in one sterile conical tube. Batches of 50–100 islets were cultured in 10 ml of RPMI 1640 with or without 10 mm leucine at 37 °C in 5% CO2, 95% air in a 6-cm dish for 1 day or 1 week. Medium was changed every three days. Insulin Secretion of Perifused Rat Islets—Fresh or cultured rat islets were washed once with 10 ml of pre-warmed KRB-G0 (0 mm glucose, 115 mm NaCl, 24 mm NaHCO3, 5 mm KCl, 1 mm MgCl2, 2.5 mm CaCl2, 25 mm Hepes (pH 7.4), and 0.1% bovine serum albumin) and perifused with KRB-G0 at 37 °C at a flow rate of 1 ml/min as described in detail previously (14Gao Z.Y. Li G. Najafi H. Wolf B.A. Matschinsky F.M. Diabetes. 1999; 48: 1535-1542Crossref PubMed Scopus (93) Google Scholar). In each experiment, 30 islets of similar sizes were hand picked under microscopy and put in the perifusion chamber. The perifusion protocol was as follows: pre-perifusion in G0 for 60 min, followed by 10 min with G0, 20 min with G2, 20 min with G5, 20 min with G10, 20 min with G30, and 20 min with 30 mm KCl. G0, G2, G5, G10, and G30 represent KRB containing 0, 2, 5, 10 and 30 mm glucose, respectively. Insulin secretion was measured by radioimmunoassay at the University of Pennsylvania Diabetes Center. Free Cytosolic [Ca2+] Measurement—Islets were loaded in 3 ml of KRB-G0 with 2.0 μm fura-2 AM and 0.2 mg/ml pluronic F-127 at 37 °C in 5% CO2, 95% air for 30 min. Islets were placed in the center of a perifusion chamber and fixed onto the tip of a glass micropipet by slight suction. They were perifused with KRB at a flow rate of 1 ml/min at 37 °C. The method for measuring [Ca2+] of rat islets was described in detail previously (14Gao Z.Y. Li G. Najafi H. Wolf B.A. Matschinsky F.M. Diabetes. 1999; 48: 1535-1542Crossref PubMed Scopus (93) Google Scholar). The perifusion protocol was as follows: 5 min with G0, G2, G5, G10, G30, and 30 mm KCl, respectively. Two to three islets of each condition were analyzed for each islet preparation. Data were collected from at least four islet preparations, and each preparation used four different rats. When calculating the increase of free cytosolic [Ca2+] in each experiment, the average concentration of cytosolic [Ca2+] between 0–5 min under KRB-G0 was set as baseline. For a given condition, the absolute value of cytosolic [Ca2+] was calculated as the mean concentration during the entire 5-min period of stimulation, whereas the net increase of [Ca2+] was obtained by subtracting the baseline value from the absolute value. Insulin Secretion and ATP Content Measurement in Incubated Islets—Islets were washed once with 10 ml of prewarmed KRB-G0 and then incubated in 10 ml of KRB-G0 at 37 °C in 5% CO2, 95% air for 30 min. Islets were then divided into batches of five islets and transferred into tubes containing 1 ml of KRB with 0, 5, or 20 mm glucose. Islets were incubated at 37 °C in 5% CO2, 95% air for 1 h in a shaking water bath. After incubation, 950 μl of supernatant was removed for insulin radioimmunoassay. Lysis buffer (450 μl) for ATP extraction (Roche Applied Science) was added into each tube containing islets. Islets were sonicated on ice for 40 pulses at 60% of maximal output. Sonicated samples were then frozen and thawed twice with dry ice to ensure complete lysis of cells. Islet lysate insulin content was assayed by radioimmunoassay. Islet lysate ATP content was measured using the ATP Bioluminescence Assay Kit HS II (Roche Applied Science). Fifty-μl samples were measured in triplicate. Data were collected from at least 15 batches of islets in four independent experiments. Quantitative Real Time RT-PCR assay—After treatments, islets were transferred to a 1.5-ml sterile tube and washed once with 1 ml of sterile diethyl pyrocarbonate-treated water. Total RNA was extracted from the islets using the RNeasy mini kit (Qiagen, Inc.), and agarose gel electrophoresis was used to analyze the quality of total RNA. Real-time RT-PCR was performed with One-step RT-PCR Master Mix reagents kit (TaqMan®). The cycling conditions for RT-PCR were as follows: 48 °C for 30 min and 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. Quantitative values were obtained as threshold PCR cycle number (Ct) when the increase in fluorescent signal of PCR product showed exponential amplification. Target gene mRNA level was normalized to that of β-actin in the same sample. In brief, the relative expression level of the target gene compared with that of β-actin was calculated as 2–ΔCt, where ΔCt = Cttarget gene – Ctβ-actin. The ratio of relative expression of the target gene in leucine-treated islets to that of untreated islets was then calculated as 2–ΔΔCt, where ΔΔCt = ΔCttreated islet – ΔCtnon-treated islet (23Fitzhugh D.J. Naik S. Caughman S.W. Hwang S.T. J. Immunol. 2000; 165: 6677-6681Crossref PubMed Scopus (103) Google Scholar, 24Lin X. Irwin D. Kanazawa S. Huang L. Romeo J. Yen T.S. Peterlin B.M. J. Virol. 2003; 77: 8227-8236Crossref PubMed Scopus (79) Google Scholar, 25Kutlu B. Darville M.I. Cardozo A.K. Eizirik D.L. Diabetes. 2003; 52: 348-355Crossref PubMed Scopus (76) Google Scholar). Each sample was measured in duplicate for each experiment. Down-regulation of ATP Synthase by siRNA Cassette—Four siRNA cassettes targeting rat ATP synthase β subunit were designed and synthesized as PCR product (Genescript, Inc.). The target sequence of cassette A, which is under the control of human U6 promoter, is 5′-AGATGAGTGTTGAACAGGAAA-3′, and that of cassette B, which is under the control of human H1 promoter, is 5′-ATGGCACTGAAGGCTTGGTTA-3′. The target sequence of cassette C is 5′-TGGTGGTGATTCTTTCCTGCA-3′ under the control of U6 promoter and that of cassette D is 5′-CACCTGCTAAGATCTGCTGG-3′ under control of H1 promoter. The day before transfection, the INS1 cells were seeded in 24-well plates with antibiotic-free RPMI 1640 (10% fetal bovine serum) so that the cells could grow more quickly in culture and reach 80% confluence the next day. For transfection of each well, 0.15 or 0.30 μgof siRNA cassette DNA was diluted into 25 μl of Opti-MEM I (serum- and antibiotic-free) and mixed gently. Then, 4 μl of Plus™ Reagent (Invitrogen) was added and mixed gently. The mixture was incubated at room temperature for 15 min. At the same time, 1 μl of Lipofectamine™ 2000 (Invitrogen) was added to 25 μl of Opti-MEM I and mixed gently before the mixture was incubated at room temperature for 15 min. After incubation, the two mixtures were combined to generate DNA-Lipofectamine 2000 complex and incubated at room temperature for 15 min. During the incubation, the culture medium in 24-well plates was replaced with 200 μl of Opti-MEM I. The DNA-Lipofectamine 2000 complex was added into the well, and the plate was rocked gently back and forth. Cells were then incubated at 37 °C in 5% CO2-95% air for 3 h before 1 ml of complete RPMI 1640 was added into each well. The cells were cultured for 2 days. The cells were washed twice with 1 ml of KRB-G0 before they were preincubated in 1 ml of KRB-G0 for 1 h. They were then incubated in 1 ml of KRB with 0 mm or 20 mm glucose for 1 h. All KRB was transferred into a 1.5-ml tube and centrifuged at 15,000 rpm at 4 °C for 10 min. The supernatant was collected for insulin measurement by radioimmunoassay. Lysis buffer (200 μl/well) (Roche Applied Science) was added, and the cells were scraped on ice. The cell lysate was transferred into a 1.5-ml tube and centrifuged at 4 °C at 15,000 rpm for 15 min. The ATP content in the supernatant was assayed as above, and the data was normalized to its protein content. For real time RT-PCR, 48 h after transfection the cells were washed twice with 1 ml of ice-cold phosphate-buffered saline followed by extraction of total RNA. RT-PCR was performed as described above. Cloning of ATP Synthase β Subunit from INS1 Cells—Two specific primers, 5′-GAC GGG CCC AA ATG TTG AGT CTT GTG GGG CG T GTG-3′ containing an ApaI site and 5′-GAC GCG GCC GCA TCA CGA CCC ATG CTC CTC TGC CAG-3′ containing a NotI site, were synthesized. The full-length ATP synthase β subunit cDNA (GenBank™ accession number P19044) of 1.6 kb was amplified using rat INS1 cell mRNA as a template and cloned into pShuttle vector with the restriction enzymes ApaI and NotI. Overexpression of ATP Synthase β Subunit in INS1 Cells—Forty-eight h after transfection, cells were washed once with cold phosphate-buffered saline. Total RNA was extracted from the cells, and real time RT-PCR was performed as above. For insulin secretion, INS1 cells were seeded in a 24-well plate and transfected with pShuttle-ATP-synthase-β subunit. 48 h after transfection, the cells were washed twice with 1 ml of prewarmed KRB and preincubated in 1 ml of KRB-G0 for 1 h. Cells were then incubated in 1 ml of KRB containing 0, 5, or 20 mm glucose for 1 h, respectively. The cells transfected with empty pShuttle vector were used as control. Culturing Islets with Leucine Increases Glucose-induced Insulin Secretion—To investigate whether leucine can affect glucose-induced insulin secretion, rat islets were cultured in complete RPMI 1640 (11 mm glucose, 10% fetal bovine serum) with or without 10 mm leucine for 1 day or 1 week. The time course of perifusion is shown in Fig. 1, and the averaged insulin secretion during each condition is presented in Fig. 2. After a 1-day culture, both untreated (Leu0) and leucine-treated (Leu10) islets had similar basal insulin secretion in the absence of glucose (19.5 ± 4.7 nanounits/islet·min versus 13.7 ± 2.2 nanounits/islet·min, p > 0.05). With 2 mm glucose, insulin secretion of untreated and leucine-treated islets was 21.6 ± 4.1 nanounits/islet·min versus 22.2 ± 8.3 nanounits/islet· min (p > 0.05). With 5 mm glucose, insulin secretion of untreated and leucine-treated islets was 44.4 ± 26.4 nanounits/islet·min versus 42.6 ± 17.1 nanounits/islet·min (p > 0.05). With 10 mm glucose, insulin secretion of untreated and leucine-treated islets was 62.9 ± 20.1 nanounits/islet·min versus 87.9 ± 38.3 nanounits/islet·min (p > 0.05). With 30 mm glucose, insulin secretion of untreated and leucine-treated islets was 75.8 ± 8.8 nanounits/islet·min versus 115.4 ± 45.8 nanounits/islet·min (p > 0.05). When stimulated with 30 mm KCl, insulin secretion of untreated islets was 83.8 ± 20.3 nanounits/islet·min, which was not different from that of leucine-treated islets (129.6 ± 35.4 nanounits/islet·min, p > 0.05) (Fig. 2A). Thus, leucine treatment for 1 day failed to enhance glucose-induced insulin secretion in rat islets under all glucose concentrations tested and 30 mm KCl (Fig. 1A).Fig. 2Effects of leucine culture on average insulin secretion in perifused islets. The effects of 1-day (A) and 1-week treatment (B) with leucine on average insulin secretion are presented. G0, G2, G5, G10, and G30 represent 0, 2, 5, 10, and 30 mm glucose in KRB, respectively. The data are mean ± S.E. of six independent experiments from 12 different rats. There was no significant difference among three groups of islets at 1 day. #, p < 0.05 comparing treated and untreated islets. *, p < 0.05 comparing treated and fresh islets.View Large Image Figure ViewerDownload Hi-res image Download (PPT) After a 1-week culture, both untreated and leucine-treated islets had similar basal insulin secretion without glucose (36.8 ± 19.1 nanounits/islet·min versus 45.9 ± 17.1 nanounits/islet·min, p > 0.05) (Figs. 1 and 2). Even with 2 mm glucose, the average insulin secretion of leucine-treated islets (91.0 ± 18.7 nanounits/islet·min) was higher than that of untreated islets (29.5 ± 6.5 nanounits/islet·min, p < 0.05). With 5 mm glucose, the average insulin secretion was even higher (139.2 ± 24.3 nanounits/islet·min), whereas that of untreated islets was not different from its baseline (47.8 ± 12.9 nanounits/islet·min p > 0.05, Figs. 1B and 2B). With 10 mm glucose, insulin secretion of the treated islets (383.4 ± 127.6 nanounits/islet·min) was 4.3-fold higher than that of untreated islets (90.3 ± 9.5 nanounits/islet·min). With 30 mm glucose, insulin secretion of the treated islets (470.0 ± 137.5 nanounits/islet·min, p < 0.05) was 3.1-fold higher than that of untreated islets (154.3 ± 25.5 nanounits/islet·min, p < 0.05). With 30 mm KCl, insulin secretion of treated islets (468.4 ± 98.3 nanounits/islet·min) was 2.2-fold higher than that of untreated islets (214.1 ± 26.5 nanounits/islet·min, p < 0.05) (Fig. 1B). The glucose-induced insulin secretion in rat islets cultured without leucine was similar to that of fresh islets (Figs. 1 and 2). Thus, treatment of rat islets with 10 mm leucine decreased the threshold for glucose-induced insulin secretion and increased the maximal insulin output significantly in a time-dependent manner. Culturing Islets with Leucine Increases Glucose-induced Rise of Free Cytosolic [Ca2+]—Free cytosolic [Ca2+] plays a vital role in exocytosis of insulin granules in pancreatic β-cells. The effects of leucine treatment on glucose-induced changes of islet-free cytosolic [Ca2+] were also assayed. The basal [Ca2+] level without glucose of 1-day-cultured islets (68 ± 7 nm) was the same as that of fresh islets (76 ± 5 nm, p > 0.05). The KCl-induced [Ca2+] change was smaller in islets cultured for 1 day without (126 ± 22 nm) or with (125 ± 10 nm) leucine than the change in fresh islets (204 ± 36 nm, p < 0.05, Fig. 3A). However, there was no significant difference between the two culture conditions with all glucose concentrations tested or with 30 mm KCl (Figs. 3A and 4A). Both 1-day-treated and untreated islets had a smaller Ca2+ response and a smaller insulin secretion peak response to KCl than did fresh islets (Figs. 1A and 3A). Therefore, there was no significant difference in the glucose-induced change of free cytosolic [Ca2+] between 1-day-treated islets and untreated islets.Fig. 4Effects of leucine culture on average changes in free cytosolic [Ca2+] in rat islets. In each experiment, the average concentration of [Ca2+] from 0–5 min without glucose was set as baseline. The net change in [Ca2+] equals the average concentration of [Ca2+] for 5 min of stimulation minus the baseline. The open bars represents the control group without leucine, and the filled bars represents the leucine-treated group. A shows the islets cultured with 10 mm leucine for 1 day. B shows the islets cultured with 10 mm leucine for 1 week. The results are mean ± S.E. of at least eight islets in four independent experiments from four different rats. *, p < 0.05; **, p < 0.01 when compared with the untreated control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Basal [Ca2+] without glucose was the same in islets cultured for 1 week with (72 ± 4 nm) or without (68 ± 6 nm) 10 mm leucine (p > 0.05). With 2 mm glucose, free cytosolic [Ca2+] of treated islets began to increase by 3 ± 1 nm (p < 0.05) compared with basal level, whereas that of untreated islets was the same as baseline (68 ± 6nm) (Fig. 4B). With 5 mm glucose, free cytosolic [Ca2+] of treated islets increased by 16 ± 4 nm (p < 0.05) when compared with baseline, whereas that of untreated islets was still the same as baseline (Fig. 4B). Glucose at 10 mm increased free cytosolic [Ca2+]by33 ± 6nm in treated islets but only 3 ± 1 nm in untreated islets. Glucose at 30 mm increased free cytosolic [Ca2+]by47 ± 6nm (p < 0.05) in treated islets but only by 9 ± 1 nm in untreated islets. Treated islets also had a significantly larger increase of [Ca2+] (80 ± 7 nm) than did untreated islets (33 ± 2 nm) when stimulated with 30 mm KCl compared with baseline (Figs. 3B and 4B). Therefore, there was a significant elevation of glucose-induced free cytosolic [Ca2+] in 1-week-treated islets compared with untreated islets (Figs. 3, B and C, and 4B). The dose-response curve of glucose-induced cytosolic Ca2+ rise was shifted to the left by leucine treatment. Effect of Leucine Culture on Islet ATP Content and Insulin Secretion—In islets cultured for 1 day without leucine, ATP content was 2.2 ± 0.2 pmol/islet without glucose and 3.2 ± 0.6 pmol/islet with 5 mm glucose or 3.1 ± 0.3 pmol/islet with 20 mm glucose (p > 0.05). In 1-day leucine-treated islets, ATP was 1.8 ± 0.3 pmol/islet without glucose and 2.8 ± 0.5 pmol/islet (p > 0.05) at 5 mm glucose or 2.9 ± 0.4 (p > 0.05) pmol/islet at 20 mm glucose (Fig. 5A). We did not detect significant difference in basal insulin secretion without glucose in the same islets cultured for 1 day with leucine (337 ± 81 nanounits/islet·h) or without leucine (243 ± 55 nanounits/islet·h). Glucose (5 mm)-induced insulin secretion was the same in islets cultured with leucine (488 ± 196 nanounits/islet·h) or without leucine (380 ± 79 nanounits/islet·h). Insulin secretion tended to be higher in the presence of 20 mm glucose, and there was no difference between the two culture conditions (1045 ± 425 nanounits/islet·h versus 1123 ± 290 nanounits/islet·h) (Fig. 5B). In islets cultured for 1 week without leucine, ATP content was increased by 1.6-fold at 5 mm glucose (2.6 ± 0.3 pmol/islet, p < 0.01) and by 1.6-fold at 20 mm glucose (2.5 ± 0.1 pmol/islet, p < 0.01) when compared with ATP content without glucose (1.6 ± 0.2 pmol/islet). These data are consistent with those published by other research groups (4Detimary P. Jonas J.C. Henquin J.C. J. Clin. Investig. 1995; 96: 1738-1745Crossref PubMed Scopus (109) Google Scholar, 5Detimary P. Van den Berghe G. Henquin J.C. J. Biol. Chem. 1996; 271: 20559-20565Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 7Detimary P. Dejonghe S. Ling Z. Pipeleers D. Schuit F. Henquin J.C. J. Biol. Chem. 1998; 273: 33905-33908Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In 1-week leucine-treated islets, compared with G0 (1.5 ± 0.2 pmol/islet), ATP content was increased by 2.5-fold at 5 mm glucose (3.8 ± 0.3 pmol/islet, p < 0.01) and 2.5-fold at 20 mm glucose (3.7 ± 0.4 pmol/islet, p < 0.01), both of which were significantly higher than the ATP content of untreated islets (p < 0.01). In 1-week leucine-treated islets, basal insulin secretion without glucose (368 ± 68 nanounits/islet·h) was the same as that of untreated islets (302 ± 37 nanounits/islet·h, p > 0.05). Glucose at 5 mm induced 3.1-fold more insulin secretion in islets cultured for 1 week treated with leucine (2160 ± 251 nanounits/islet·h) than without leucine (694 ± 160 nanounits/islet·h, p < 0.05). Glucose at 20 mm induced a 1.9-fold increase in insulin secretion in islets cultured for 1 week treated with leucine (4060 ± 710 nanounits/islet·h) than without leucine (2180 ± 406 nanounits/islet·h, p < 0.05, Fig. 6B). Insulin content was significantly lower in islets cultured with leucine for 1 week and then incubated with 5 mm glucose (8.7 ± 1.5 microunits/islet) compared with control (18.6 ± 3.3 microunits/islet, p < 0.05). Sim
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