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• W2076270562 abstract "Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1-/-) and control mouse islets with d-[U-13C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and γ-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1-/- islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-β-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1-/- and glyburide-treated SUR1+/+ islets. Glucose oxidation and pyruvate cycling was increased in SUR1-/- islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1-/- islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1-/- islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation. Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1-/-) and control mouse islets with d-[U-13C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and γ-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1-/- islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-β-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1-/- and glyburide-treated SUR1+/+ islets. Glucose oxidation and pyruvate cycling was increased in SUR1-/- islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1-/- islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1-/- islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation. The pancreatic beta cells function as the predominant sensors and regulators of glucose, amino acid, and fatty acid levels of the mammalian organism, including man, by adjusting the minute to minute rate of insulin secretion such that these fuels are maintained at physiologically optimal blood concentrations under all nutritional conditions including feeding and fasting. This process of fuel-sensing and stimulation of insulin secretion requires that the various stimuli are transported into the beta cells and are metabolized to generate coupling factors that trigger and sustain the secretion of the hormone from large stores of insulin granules (1Matschinsky F.M. Magnuson M.A. Zelent D. Jetton T.L. Doliba N. Han Y. Taub R. Grimsby J. Diabetes. 2006; 55: 1-12Crossref PubMed Scopus (231) Google Scholar, 2Newgard C.B. Matschinsky F.M. Jefferson J. Cherrington A Handbook of Physiology. 2. Oxford University Press, London2001: 125-151Google Scholar, 3Henquin J.C. Diabetes. 2000; 49: 1751-1760Crossref PubMed Scopus (930) Google Scholar, 4Malaisse W.J. Diabetes Metab. Rev. 1986; 2: 243-259Crossref PubMed Scopus (21) Google Scholar). The diverse specific pathways that allow access to metabolism for glucose, amino acids, and fatty acids converge to a complex network of intermediary metabolism represented by the citric acid cycle, a considerable variety of metabolite and cofactor shuttles including the GABA 2The abbreviations used are: GABA, γ-aminobutyric acid; BCH, d,l-β-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid; GAD, glutamate decarboxylase; GC-MS, gas chromatography-mass spectrometry; GS, glutamine synthetase; ME, malic enzyme; MSO, methionine sulfoximine; SUR1, sulfonylurea receptor 1; VGB, vigabatrin; HPLC, high performance liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AAM, amino acid mixture. 2The abbreviations used are: GABA, γ-aminobutyric acid; BCH, d,l-β-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid; GAD, glutamate decarboxylase; GC-MS, gas chromatography-mass spectrometry; GS, glutamine synthetase; ME, malic enzyme; MSO, methionine sulfoximine; SUR1, sulfonylurea receptor 1; VGB, vigabatrin; HPLC, high performance liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AAM, amino acid mixture. shunt, and the processes of electron transport and oxidative phosphorylation, to mention just a few outstanding features of the biochemical maze of the beta cell.In the present study we have used uniformly labeled d-[13C]glucose and GC-MS methods to explore the role of intermediary metabolism of pancreatic islets isolated from normal and sulfonylurea receptor 1 (SUR1) knock-out (SUR1-/-) mice. SUR1-/- islets were chosen for comparison to control islets because they have profound changes of glucose and amino acid responsiveness, showing markedly reduced glucose but strikingly enhanced amino acid-induced insulin release (5Doliba N.M. Qin W. Vatamaniuk M.Z. Li C. Zelent D. Najafi H. Buettger C.W. Collins H.W. Carr R.D. Magnuson M.A. Matschinsky F.M. Am. J. Physiol. Endocrinol. Metab. 2004; 286: 834-843Crossref PubMed Scopus (51) Google Scholar, 6Li C. Buettger C. Kwagh J. Matter A. Daikhin Y. Nissim I.B. Collins H.W. Yudkoff M. Stanley C.A. Matschinsky F.M. J. Biol. Chem. 2004; 279: 13393-13401Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 7Doliba N.M. Qin W. Vatamaniuk M.Z. Buettger C.W. Collins H.W. Magnuson M.A. Kaestner K.H. Wilson D.F. Carr R.D. Matschinsky F.M. Am. J. Physiol. Endocrinol. Metab. 2006; 291: 525-535Crossref PubMed Scopus (24) Google Scholar). We used the conversion or incorporation of 13C carbon into CO2 and various amino acids, respectively, as readout for the integration of metabolic pathways. This approach allowed us to examine for the first time in isolated mouse islets and in one comprehensive study the concentration dependences of glucose oxidation and glutaminolysis, the operation of the so called “pyruvate cycling,” the nature of the glucose induced aspartate decrease (herein referred to as “aspartate switch”), the regulatory role of glutamate decarboxylase (GAD) in the operation of the beta cell GABA shunt, and glucose stimulation of glutamine synthesis. These studies provide new insights into the complex role of the metabolic network in fuel-stimulated authentic pancreatic islet cells as contrasted with studies using tumor-derived beta cell lines (8Simpson N.E. Khokhlova N. Oca-Cossio J.A. Constantinidis I. Diabetologia. 2006; 49: 1338-1348Crossref PubMed Scopus (16) Google Scholar, 9Lu D. Mulder H. Zhao P. Burgess S.C. Jensen M.V. Kamzolova S. Newgard C.B. Sherry A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2708-2713Crossref PubMed Scopus (219) Google Scholar).MATERIALS AND METHODSPancreatic Islet Source and Preparation—SUR1 knock-out mice were obtained from Dr. Mark A. Magnuson. The knockout procedure and genotyping were described by Shiota et al. (10Shiota C. Larsson O. Shelton K.D. Shiota M. Efanov A.M. Hoy M. Lindner J. Kooptiwut S. Juntti-Berggren L. Gromada J. Berggren P.O. Magnuson M.A. J. Biol. Chem. 2002; 277: 37176-37183Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Both SUR1-/- mice and control (SUR1+/+) mice (B6D2F1) were maintained on a 12-h light/dark cycle and were fed a standard rodent chow diet. Islet isolation and culture were described previously (11Li C. Najafi H. Daikhin Y. Nissim I.B. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In brief, islets were isolated by collagenase digestion and cultured for 3–4 days in RPMI 1640 medium containing 10 mm glucose.[U-14C]Glutamine Oxidation—[U-14C]glutamine (PerkinElmer Life Sciences) oxidation in the presence or absence of 10 mm d,l-β-2-amino-2-norbornane-carboxylic acid (BCH) was examined in cultured SUR1-/- and control islets. Separate experiments were performed in control islets cultured with 0.3 μm glyburide for 3 days and then using the same concentration of glyburide throughout the experiment to mimic SUR1-/- conditions. The experimental procedures were described previously (12Li C. Allen A. Kwagh J. Doliba N.M. Qin W. Najafi H. Collins H.W. Matschinsky F.M. Stanley C.A. Smith T.J. J. Biol. Chem. 2006; 281: 10214-10221Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar).Studies with [U-13C]Glucose—Batches of 1000 cultured islets from SUR1-/- and SUR1+/+ animals were preincubated with 95% O2, 5% CO2-equilibrated Krebs-Ringer bicarbonate buffer (115 mmol/liter NaCl, 24 mmol/liter NaHCO3, 5 mmol/liter KCl, 1 mmol/liter MgCl2, 2.5 mmol/liter CaCl2, 10 mm HEPES, pH 7.4) with 0.25% bovine serum albumin for 60 min ± 1 mm methionine sulfoximine (MSO), a glutamine synthetase (GS) inhibitor, or ±1.55 mm vigabatrin (VGB), a GABA transaminase inhibitor. The purpose of the preincubation was to reduce the possibility of influences of the complex culture medium. The islets were then incubated 120 min with a 4.0 mm physiological mixture of amino acids plus 300 μm NH4Cl as control or with a further addition of 5 or 25 mm [U-13C]glucose (Cambridge Isotope Laboratories, Inc., Andover, MA). In the high glucose condition the medium was further modified by adding 1 mm MSO or 1.55 mm VGB in accordance with the pretreatment protocol. The physiological mixture of the 4.0 mm 19 amino acids had the following composition: 0.44 mm alanine, 0.19 mm arginine, 0.04 mm aspartate, 0.09 mm citrulline, 0.12 mm glutamate, 0.5 mm glutamine, 0.30 mm glycine, 0.08 mm histidine, 0.09 mm isoleucine, 0.16 mm leucine, 0.37 mm lysine, 0.05 mm methionine, 0.07 mm ornithine, 0.08 mm phenylalanine, 0.35 mm proline, 0.57 mm serine, 0.27 mm threonine, 0.07 mm tryptophan, and 0.20 mm valine.After 120 min of incubation, the medium was sampled and stored to be assayed for insulin and glucagon by radioimmunoassay. Incubations were performed in tightly sealed tubes that had been gassed with 5% CO2 and 95% O2. After 120 min of incubation, the air was sampled by suction using a well sealed syringe, and 13CO2 enrichment was then measured by an isotope ratio mass spectrometer. The islets were then quickly washed twice with ice-cold glucose-free Hanks' buffer, and the cellular amino acids were extracted with ice-cold 6% perchloric acid. Assays of amino acids were performed as described previously (11Li C. Najafi H. Daikhin Y. Nissim I.B. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The 13C enrichment of amino acids was determined by GC-MS. ATP content and ATP/ADP ratio were measured using the method as previously published (13Li C. Matter A. Kelly A. Petty T.J. Najafi H. MacMullen C. Daikhin Y. Nissim I. Lazarow A. Kwagh J. Collins H.W. Hsu B.Y. Nissim I. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2006; 281: 15064-15072Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).A set of separate experiments was performed similarly but using 15N-containing instead of unlabeled NH4Cl (Cambridge Isotope Laboratories) to trace 15N fluxes in islets. These latter conditions included 300 μm 15NH4Cl with 4 mm amino acids alone or with additional 25 mm glucose or with 25 mm glucose plus 1 mm MSO. 15N amino acids were measured by GC-MS as described in previous reports (11Li C. Najafi H. Daikhin Y. Nissim I.B. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 13Li C. Matter A. Kelly A. Petty T.J. Najafi H. MacMullen C. Daikhin Y. Nissim I. Lazarow A. Kwagh J. Collins H.W. Hsu B.Y. Nissim I. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2006; 281: 15064-15072Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).GC-MS Methodology and Determination of 13C-Labeled Metabolites—GC-MS measurements of 13C isotopic enrichment were performed on either a Hewlett Packard 5970 Mass Selective Detector (MSD) and/or 5971 MSD coupled with a 5890 HP-GC, GC-MS Agilent system (6890 GC-5973 MSD) or Hewlett-Packard (HP-5970 MSD) using electron impact ionization with an ionizing voltage of -70 eV and an electron multiplier set to 2000 V.For measurement of the 13C enrichment in amino acids, samples were prepared as previously described (14Nissim I. Daikhin Y. Nissim I. Luhovyy B. Horyn O. Wehrli S.L. Yudkoff M. J. Biol. Chem. 2006; 281: 8486-8496Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 15Nissim I. Horyn O. Daikhin Y. Nissim I. Luhovyy B. Phillips P.C. Yudkoff M. Cancer Res. 2006; 66: 7824-7831Crossref PubMed Scopus (70) Google Scholar). Briefly, an aliquot of islet perchloric acid extract was purified by passage on an AG-1 (Cl-, 100–200 mesh; 0.5 × 2.5 cm) or AG-50 (H+, 100–200 mesh) column and then converted into the t-butyldimethylsilyl derivatives. Isotopic enrichment in glutamate isotopomers was monitored using ions at m/z 432, 433, 434, 435, 436, and 437 for M+1, M+2, M+3, M+4, and M+5 (containing 1–5 13C-enriched atoms), respectively. Isotopic enrichment in aspartate isotopomers was monitored using ions at m/z 418, 419, 420, 421, and 422 for M+1, M+2, M+3, and M+ 4 (containing 1–4 13C-enriched atoms), respectively. Isotopic enrichment in alanine was monitored using ions at m/z 232, 233, 234, and 235 for M+1, M+2, and M+3 (containing 1–3 13C-enriched atoms), respectively, and 13C enrichment in GABA was monitored using ions at m/z 274, 275, 276, 277, and 278 for M+1, M+2, M+3, and M+ 4 (containing 1–4 13C-enriched atoms), respectively.The production of 13CO2 was monitored as follows. After 120 min of incubation, air samples were taken from the sealed incubation tube. The latter was transferred into autosampler tubes for analysis. Isotopic enrichment in 13CO2 was determined by an isotope ratio mass spectrometer (Thermoquest Finnigan Delta Plus) using the m/z 44/45 ratio (14Nissim I. Daikhin Y. Nissim I. Luhovyy B. Horyn O. Wehrli S.L. Yudkoff M. J. Biol. Chem. 2006; 281: 8486-8496Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 15Nissim I. Horyn O. Daikhin Y. Nissim I. Luhovyy B. Phillips P.C. Yudkoff M. Cancer Res. 2006; 66: 7824-7831Crossref PubMed Scopus (70) Google Scholar).Materials—Chemicals were usually from Sigma except when stated otherwise.Calculations and Statistical Analyses—13C enrichment is expressed by molar percent enrichment, which is the mol fraction percent of analyte containing 13C atoms above natural abundance (14Nissim I. Daikhin Y. Nissim I. Luhovyy B. Horyn O. Wehrli S.L. Yudkoff M. J. Biol. Chem. 2006; 281: 8486-8496Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 15Nissim I. Horyn O. Daikhin Y. Nissim I. Luhovyy B. Phillips P.C. Yudkoff M. Cancer Res. 2006; 66: 7824-7831Crossref PubMed Scopus (70) Google Scholar). The production of 13C-labeled mass isotopomer was calculated by the product of (molar percent enrichment)/100 times concentration (nmol/1000 islets) and is expressed as nmol of 13C metabolite/1000 islets. All the data are presented as the mean ± S.E. Student's t tests were done when two groups were compared. Analysis of variances (one way analysis of variance) was used followed by the Bonferroni test when multiple groups were compared. Differences were considered significant when p < 0.05.Western Blot Analysis—Monoclonal rabbit anti-mouse GS, rabbit, anti-GAD (against GAD 65 and 67), and mouse anti-GAPDH antibodies (all from Sigma) were used as the primary antibodies, and donkey anti-rabbit IgG horseradish peroxidase conjugate (Amersham Biosciences) and goat anti-mouse horseradish peroxidase conjugate (Santa Cruz) were used as the secondary antibodies. A total of 20 μg of islets protein was used for Western blotting. Islets protein was extracted from cultured SUR1-/-, control islets, or control islets incubated with 0.3 μm glyburide for 24 h in Krebs-Ringer bicarbonate buffer supplemented with 0.25% bovine serum albumin.mRNA Analysis—Total RNA was isolated from islets using the Trizol (Invitrogen) method, including SUR1-/- islets, control islets, SUR1-/- islets incubated with 20 μm nimodipine for 4 and 24 h, and control islets incubated with 0.3 μm glyburide for 24 h. For the reverse transcription reaction, 40 μl of PCR reagent efficiently converts 1.0 μg of total RNA to cDNA (Invitrogen). The sequences of primers were as indicated: for GAD forward, CAC AAA CTC AGC GGC ATA GA; for GAD reverse, ATC TGG TTG CAT CCT TGG AG; for GAPDH forward, AAC TTT GGC ATT GTG GAA GG; for GAPDH reverse, GGA TGC AGG GAT GAT GTT CT. PCR products were 128 bp for GAD and 132 bp for GAPDH. Quantitative real-time PCR (Applied Biosystems SYBR Green Master Mix kit) was carried out in low reaction volumes in 384-well plates using 7.5 μl of 2× Syber green master mix, 0.2 μl of primer (10 μm), 1 μl of cDNA, and water added to bring the final volume of 15 μl. Thermal cycle conditions were 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Experiments were performed with the 7900HT sequence detection system (Applied Biosystems). Islets mRNA levels were calculated using serial-diluted GAPDH as the standard and internal controls. mRNA expression of malic enzyme isoforms 1, 2, and 3 in islets, brain, and liver was detected by reverse transcription-PCR and real-time PCR. The sequences of primers were designed to exclude the detection of genomic DNA as indicated: malic enzyme 1 (ME1) forward, GGG ATT GCT CAC TTG GTT GT; ME1 reverse, GTT CAT GGG CAA ACA CCT CT; ME2 forward, AGG AGA AGC TGC ACT TGG AA; ME2 reverse, GCA CCT GCC ACT CCA ATT AT; ME3 forward, TCT TCT ACC GGG TGT TGA CC; ME3 reverse, TAG CAG GAC AGG AAG GCA CT. The generated PCR products were 151 bp (ME1), 249 bp (ME2), and 326 bp (ME3). The PCR products were confirmed by sequencing.RESULTSProfiles of Major Amino Acids in SUR1-/- and Control Islets as Influenced by Glucose, MSO, and VGB—Earlier studies had indicated that incubation of cultured mouse pancreatic islets for 2 h in Krebs-Ringer bicarbonate buffer solution that lacked amino acids resulted in a substantial reduction of the intracellular amino acid pool by 1/2 to 2/3 (6Li C. Buettger C. Kwagh J. Matter A. Daikhin Y. Nissim I.B. Collins H.W. Yudkoff M. Stanley C.A. Matschinsky F.M. J. Biol. Chem. 2004; 279: 13393-13401Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 13Li C. Matter A. Kelly A. Petty T.J. Najafi H. MacMullen C. Daikhin Y. Nissim I. Lazarow A. Kwagh J. Collins H.W. Hsu B.Y. Nissim I. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2006; 281: 15064-15072Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This loss, amounting to as much as 10–15 mm intracellular amino acids, was not preventable by 5 or 25 mm glucose and was explained by a net efflux of amino acids due to the large downhill concentration gradient associated with nonphysiological incubation conditions. The present studies of intermediary metabolism of SUR1-/- islets using [U-13C]glucose were, therefore, performed in the presence of 3.5 mm concentrations of a physiological mixture of 18 amino acids plus 0.5 mm glutamine to approach physiological conditions. The medium also contained 300 μm ammonia to facilitate glutamine synthesis. Contents of the nine predominant amino acids that were reliably quantified in pancreatic islets of SUR1-/- and control mice after incubation for 2 h with 4 mm amino acids present but with varying glucose and drug additions as dictated by the aims of the study are shown in Table 1. The total amino acid contents under the various experimental conditions were not different statistically, ranging from 30 to 42 nmol/1000 islets. This extrapolates to an ∼15–21 mm pool size. Three amino acids (glycine, isoleucine, and leucine) were virtually unchanged by the SUR1 knock-out or the incubation conditions. The other six were, however, markedly influenced by the experimental manipulations. Three changes are particularly striking; 1) the substantial drop of aspartate levels to as low as ⅓ that of base line when glucose concentrations are increased, both in control and SUR1-/- islets, 2) the striking depletion of the GABA content by about 75% in SUR1-/- islets as compared with the controls whatever the conditions, and 3) the relatively high levels of alanine, glutamate, glutamine, and serine in SUR1-/- as compared with the controls in many conditions studied here such that the combined pool of these 4 amino acids was on average 52% higher in SUR1-/- islets than controls (i.e. 19 ± 1.7 versus 13 ± 1.1 nmol/1000 islets in glucose-free conditions). It is noteworthy that GABA was undetectable by HPLC in the incubation medium, which suggested the low intracellular GABA was not due to increased release. The careful comparison of the results of GABA and glutamate measurements in SUR1-/- islets with those in controls shows a classical “crossover” at the GABA decarboxylase step as illustrated in Fig. 1. The ratios of GABA/glutamate of SUR1-/- islets are at least 10-fold lower than those in the controls. The results strongly suggested that flux through the GABA shunt is greatly reduced in pancreatic islets of SUR1-/- mice. It is worth noting here that MSO, an inhibitor of glutamine synthesis, had relatively little impact on the amino acid profile of isolated islets incubated in the presence of glucose (realizing the fact, however, that glutamine was not measurable because MSO has the same chromatographic retention time as glutamine) and that VGB, an inhibitor of GABA transaminase, caused GABA to pile up as expected in the control and also in the SUR1-/- islets. Because of the marked inhibition of the GABA shunt, the possibility that glutamine and glutamate catabolism might also be impaired was tested by studying glutaminolysis. It was found that BCH-stimulated glutaminolysis was greatly enhanced in SUR1-/- islets, a phenomenon that was reproduced in model studies with glyburide-treated control islets (Fig. 2). The observation showed that the capacity of glutamine and glutamate oxidation is not reduced by the near complete block of the GABA shunt. In fact, both SUR1-/- and glyburide-treated control islets had increased BCH-stimulated glutaminolysis by 2- and 3-fold, respectively. The comprehensive analysis of the amino acid profiles of pancreatic islets as presented above is critical for the interpretation of tracer studies that use [U-13C]glucose because amino acids represent quantitatively a major sink for [13C]carbon.TABLE 1Concentrations of intracellular amino acid (nmol/1000 islets) G, glucose. NA, not detectable. HPLC measurements are interfered by MSO.G 0aNote that in all 3 G 0 and half of the G 25 and G 25/MSO experiments we used 300 μm 15NH4Cl instead of NH4Cl that was present in all the other experiments. Amino acids recorded in italics were not influenced by the experimental manipulations n = 3G 5 n = 3G 25aNote that in all 3 G 0 and half of the G 25 and G 25/MSO experiments we used 300 μm 15NH4Cl instead of NH4Cl that was present in all the other experiments. Amino acids recorded in italics were not influenced by the experimental manipulations n = 6G 25/MSOaNote that in all 3 G 0 and half of the G 25 and G 25/MSO experiments we used 300 μm 15NH4Cl instead of NH4Cl that was present in all the other experiments. Amino acids recorded in italics were not influenced by the experimental manipulations n = 6G 25/VGB n = 3SUR1–/–SUR1+/+SUR1–/–SUR1+/+SUR1–/–SUR1+/+SUR1–/–SUR1+/+SUR1–/–SUR1+/+Alanine2.7 ± 0.22.6 ± 0.33.5 ± 0.3bp < 0.05 vs. G 02.9 ± 0.34.6 ± 0.7bp < 0.05 vs. G 02.9 ± 0.24.1 ± 0.33.0 ± 0.24.1 ± 0.62.6 ± 0.1Aspartate13.9 ± 0.414.1 ± 1.07.8 ± 0.4cp < 0.01 vs. G 08.3 ± 0.6bp < 0.05 vs. G 05.1 ± 0.3cp < 0.01 vs. G 0,dp < 0.05 vs. SUR1+/+6.0 ± 0.3bp < 0.05 vs. G 07.0 ± 0.3ep < 0.01 vs. G 258.1 ± 0.5ep < 0.01 vs. G 254.8 ± 0.5ep < 0.01 vs. G 254.9 ± 0.2GABA0.3 ± 0.0fp < 0.01 vs. SUR1+/+1.8 ± 0.10.5 ± 0.0bp < 0.05 vs. G 0,fp < 0.01 vs. SUR1+/+1.8 ± 0.10.3 ± 0.0fp < 0.01 vs. SUR1+/+1.4 ± 0.1bp < 0.05 vs. G 00.3 ± 0.1fp < 0.01 vs. SUR1+/+1.4 ± 0.11.3 ± 0.2fp < 0.01 vs. SUR1+/+,gp < 0.05 vs. G 255.1 ± 0.5gp < 0.05 vs. G 25Glutamate11.5 ± 1.1dp < 0.05 vs. SUR1+/+7.3 ± 0.611.5 ± 1.08.3 ± 0.911.6 ± 1.77.7 ± 0.513.9 ± 1.111.2 ± 1.0ep < 0.01 vs. G 2511.1 ± 1.77.1 ± 0.3Glutamine1.0 ± 0.20.8 ± 0.12.2 ± 0.1bp < 0.05 vs. G 0,dp < 0.05 vs. SUR1+/+1.3 ± 0.0bp < 0.05 vs. G 02.6 ± 0.3cp < 0.01 vs. G 0,dp < 0.05 vs. SUR1+/+1.3 ± 0.1cp < 0.01 vs. G 0NANA2.8 ± 0.31.3 ± 0.1Glycine7.7 ± 0.59.4 ± 1.28.1 ± 0.312.0 ± 1.17.4 ± 0.87.3 ± 0.76.7 ± 0.37.1 ± 0.97.5 ± 0.77.9 ± 0.1Isoleucine0.5 ± 0.00.5 ± 0.00.4 ± 0.10.8 ± 0.30.5 ± 0.00.4 ± 0.00.4 ± 0.10.5 ± 0.00.5 ± 0.10.5 ± 0.0Leucine0.4 ± 0.00.3 ± 0.00.3 ± 0.00.3 ± 0.10.3 ± 0.10.2 ± 0.00.3 ± 0.00.2 ± 0.00.3 ± 0.00.2 ± 0.0Serine3.9 ± 0.3fp < 0.01 vs. SUR1+/+1.8 ± 0.23.5 ± 0.32.2 ± 0.34.3 ± 0.7dp < 0.05 vs. SUR1+/+2.0 ± 0.23.8 ± 0.3dp < 0.05 vs. SUR1+/+2.0 ± 0.23.9 ± 0.6dp < 0.05 vs. SUR1+/+2.0 ± 0.1Sum42 ± 339 ± 338 ± 238 ± 337 ± 430 ± 236 ± 234 ± 336 ± 431 ± 1a Note that in all 3 G 0 and half of the G 25 and G 25/MSO experiments we used 300 μm 15NH4Cl instead of NH4Cl that was present in all the other experiments. Amino acids recorded in italics were not influenced by the experimental manipulationsb p < 0.05 vs. G 0c p < 0.01 vs. G 0d p < 0.05 vs. SUR1+/+e p < 0.01 vs. G 25f p < 0.01 vs. SUR1+/+g p < 0.05 vs. G 25 Open table in a new tab FIGURE 2Increased capacity for glutaminolysis of SUR1-/- islets. [U-14C]Glutamine oxidation was measured in SUR1+/+ and SUR1-/- islets and SUR1+/+ islets that had been cultured with 0. 3 μm glyburide for 3 days and also exposed to the drug throughout the experiment. For SUR1+/+ islets: red open squares with red dashed line, without BCH; red solid squares with red line, with 10 mm BCH. For glyburide-treated: green open diamonds with dashed green line, without BCH; green solid diamonds with green line, with 10 mm BCH. SUR1-/- islets: blue open circles with blue dashed line, without BCH; blue solid circles with blue line, with 10 mm BCH. Statistical significance: compared with SUR1+/+ islets with BCH: *, p < 0.05, compared with SUR1-/- islets with BCH; #, p < 0.05. (n = 4–5) (the inset highlights the physiological glutamine range from 0.3 to 2 mm).View Large Image Figure ViewerDownload Hi-res image Download (PPT)General Description of Pancreatic 13C Labeling Profiles of Amino Acid Isotopomers Using [U-13C]Glucose as Substrate—As a measure of pathway fluxes, we used [U-13C]glucose in an attempt to follow carbon flux into carbon dioxide and five prominent amino acids (alanine, aspartate, GABA, glutamate, and glutamine) of SUR1-/- and control pancreatic islets. To meet the sensitivity requirements of the GC-MS analysis of these islet constituents, we needed 1000 islets for each condition incubated in 1 ml of Krebs-Ringer bicarbonate buffer for 2 h to achieve significant 13C labeling of amino acids. The results of limited ATP and ADP level determinations and of insulin and glucagon release rates demonstrated that the tissues were functionally well maintained (Figs. 3 and 4). The ATP levels (of 6–9 pmol/islet) and the ATP/ADP ratios (of 3–5) were within the expected ranges. To obtain more detailed quantitative information on the energy potential of islet cells during fuel stimulation would have required comprehensive measurements including ATP, ADP, AMP, inorganic phosphorus, phosphocreatine, and creatine to extrapolate to the effective free levels of these molecules, which was not possible in the present study. The insulin release data are consistent with published results which demonstrate functional preservation of islets under the present experimental conditions (for further details on the hormone release data, see below). The [13C]carbon dioxide enrichment data (APE) show that SUR1-/- islets incubated at basal 5 mm glucose oxidize glucose twice as effectively as controls (Fig. 3A) and that the relative enhancement of glucose oxidation by increasing the substrate level to 25 mm is far less pronounced than in controls (i.e. the rate barely doubles in SUR1-/- islets but increases 4-fold in con" @default.
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• W2076270562 date "2008-06-01" @default.
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• W2076270562 title "Elimination of KATP Channels in Mouse Islets Results in Elevated [U-13C]Glucose Metabolism, Glutaminolysis, and Pyruvate Cycling but a Decreased γ-Aminobutyric Acid Shunt" @default.
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• W2076270562 doi "https://doi.org/10.1074/jbc.m709235200" @default.
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