Glutamine-Elicited Secretion of Glucagon-Like Peptide 1Is Governed by an Activated Glutamate DehydrogenaseLotta E. Andersson,1 Liliya Shcherbina,2 Mahmoud Al-Majdoub,1 Neelanjan Vishnu,1

Claudia Balderas Arroyo,3 Jonathan Aste Carrara,3 Claes B. Wollheim,4,5 Malin Fex,1 Hindrik Mulder,1

Nils Wierup,2 and Peter Spégel1,3

Diabetes 2018;67:372–384 | https://doi.org/10.2337/db16-1441

Glucagon-like peptide 1 (GLP-1), secreted from intestinalL cells, glucose dependently stimulates insulin secretionfrom b-cells. This glucose dependence prevents hypogly-cemia, rendering GLP-1 analogs a useful and safe treat-ment modality in type 2 diabetes. Although the amino acidglutamine is a potent elicitor of GLP-1 secretion, the re-sponsible mechanism remains unclear. We investigatedhow GLP-1 secretion is metabolically coupled in L cells(GLUTag) and in vivo inmice using the insulin-secreting cellline INS-1 832/13 as reference. A membrane-permeableglutamate analog (dimethylglutamate [DMG]), actingdownstream of electrogenic transporters, elicited similaralterations in metabolism as glutamine in both cell lines.Both DMG and glutamine alone elicited GLP-1 secretionin GLUTag cells and in vivo, whereas activation of gluta-mate dehydrogenase (GDH) was required to stimulate in-sulin secretion from INS-1 832/13 cells. Pharmacologicalinhibition in vivo of GDH blocked secretion of GLP-1 inresponse to DMG. In conclusion, our results suggest thatnonelectrogenic nutrient uptake and metabolism play animportant role in L cell stimulus-secretion coupling. Me-tabolism of glutamine and related analogs by GDH in theL cell may explain why GLP-1 secretion, but not that ofinsulin, is activated by these secretagogues in vivo.

Glucagon-like peptide 1 (GLP-1), secreted from intestinalL cells, potentiates insulin secretion from the pancreatic

b-cells (1). Importantly, this potentiation is glucose depen-dent (i.e., GLP-1 stimulates insulin secretion when bloodglucose levels are elevated) (2). Hence, GLP-1 is unlikelyto induce severe hypoglycemia, a dreaded complication ofinsulin treatment. Moreover, GLP-1 delays gastric empty-ing, leading to reduced hunger and food intake (1). Thesefeatures of GLP-1 underlie the increasing use of GLP-1analogs in treatment of type 2 diabetes.

Currently, therapeutic actions of GLP-1 in vivo aremediated by administration of GLP-1 analogs or dipeptidylpeptidase 4 (DPP-4) inhibitors. However, DPP-4 has impor-tant physiological roles other than degradation of GLP-1 (3),and indiscriminate inhibitors may also affect functions ofother biological processes, resulting in a potentially widerange of physiological effects. For instance, DPP-4 hasbeen shown to suppress tumor growth and inhibit malig-nancies (4); its inhibition may thus increase cancer risk.L cell function is relatively well preserved in type 2 diabetes(5), as opposed to that of the pancreatic b-cell, which grad-ually deteriorates during progression of the disease (6). Infact, studies have revealed that the GLP-1 secretagogueglutamine potentiates GLP-1 secretion in healthy subjectsas well as in those with type 2 diabetes (5). Notably, gluta-mine does not elicit secretion of insulin from the pancreaticb-cell (7). Levels of glutamine are tightly regulated by glu-taminase, glutamine synthetase, and glutamate dehydroge-nase (GDH). The amino acid functions as both a modulator

1Unit of Molecular Metabolism, Department of Clinical Sciences, Lund UniversityDiabetes Centre, Clinical Research Centre, Skåne University Hospital, Malmö,Sweden2Neuroendocrine Cell Biology, Department of Clinical Sciences, Lund UniversityDiabetes Centre, Clinical Research Centre, Skåne University Hospital, Malmö,Sweden3Centre for Analysis and Synthesis, Department of Chemistry, Lund University,Lund, Sweden4Lund University Diabetes Centre, Clinical Research Centre, Skåne UniversityHospital, Malmö, Sweden5Department of Cell Physiology and Metabolism, University Medical Centre, Ge-neva, Switzerland

Corresponding author: Peter Spégel, peter.spegel@chem.lu.se.

Received 23 November 2016 and accepted 7 December 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1441/-/DC1.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

372 Diabetes Volume 67, March 2018

METABOLISM

of ammonia levels and a substrate for energy production(8). An increased understanding of stimulus-secretion cou-pling in the L cell could enable nutritional or pharmacologicalenhancement of GLP-1 secretion.

L cells are difficult to isolate from the intestine due totheir relatively low abundance. Moreover, survival of L cellsin purified cultures is poor (9). b-Cell stimulus-secretioncoupling, in contrast, has been extensively studied in iso-lated pancreatic islets, sorted b-cells, and multiple clonalcell lines (10). Only few immortalized L cell lines are avail-able, with the GLUTag cell line being recognized as a usefuland relevant model of the primary L cell (11).

Studies of stimulus-secretion coupling in the b-cell haverevealed two major pathways underlying secretion of insu-lin. In both of these, increased blood glucose levels result inan increase in glucose uptake and phosphorylation followedby glycolysis and complete oxidation of the sugar in mitochon-dria. The ATP generated thereby acts on the ATP-sensitive K+

channel, leading to its closure, membrane depolarization,opening of voltage-gated Ca2+ channels, and finally exocy-tosis of insulin granules (12). In addition to this triggeringpathway, an amplifying pathway has been shown to accountfor a large proportion of secreted insulin. Multiple metab-olites have been implicated in the amplifying pathway, buta functional role for most of them has been questioned dueto conflicting results from later studies (12,13). Studies onstimulus-secretion coupling in the L cell have revealed sim-ilar mechanisms as in the pancreatic b-cell (14). However,in contrast to the b-cell, which expresses Glut1/2, theL cells also express electrogenic sugar and amino acid trans-porters (15). Hence, Na+-coupled nutrient uptake has beensuggested to account for a large proportion of GLP-1 secre-tion (14). Moreover, the L cell has also been shown to re-spond to stimulation with peptones (16).

In the current study, we examined stimulus-secretioncoupling in GLUTag cells and used the widely studied b-cellmodel INS-1 832/13 as a reference. Results from thesestudies were validated in vivo in mice.

RESEARCH DESIGN AND METHODS

Cell CultureGLUTag cells were cultured in DMEM containing 5.6 mmol/Lglucose and supplemented with 10% FBS at 37°C in a humid-ified atmosphere containing 95% air and 5% CO2. INS-1832/13 cells were cultured in RPMI 1640 containing either5.6 or 11.1 mmol/L glucose and supplemented with 10%FBS, 10 mmol/L HEPES, 2 mmol/L glutamine, 1 mmol/Lsodium pyruvate, and 50 mmol/L 2-mercaptoethanol at37°C in a humidified atmosphere containing 95% air and5% CO2. Cells were seeded in 24-well tissue-culture platesfor 24 or 48 h prior to experiments.

Hormone SecretionGLUTag and INS-1 832/13 cells were incubated as pre-viously described in detail (14,17). Briefly, GLUTag cellswere washed twice with 0.5 mL PBS and then incubatedin 0.5 mL Hanks’ balanced salt solution (HBSS) containing

a DPP-4 inhibitor (0.1 mmol/L diprotin A; Sigma-Aldrich,St. Louis, MO), fatty acid–free BSA (0.2%, weight forweight), and various concentrations of glucose, glutamine,dimethylglutamate (DMG), and leucine for 2 h. INS-1832/13 cells were washed with 0.5 mL PBS and preincu-bated in 0.5 mL HBSS with 2.8 mmol/L glucose for 2 h.Then, cells were incubated in 0.5 mL HBSS containing thesame secretagogues as used for the GLUTag-cells for 1 h. Anepigallocatechin (EGCG) stock solution was prepared bydissolving EGCG (4 mmol/L) in water containing 1 mmol/Lascorbic acid. EGCG and ascorbic acid were then added toboth preincubation and incubation media at 20 mmol/L and0.5 mmol/L, respectively (18). Secreted GLP-1 and insulinwere determined in the supernatant after centrifugationusing an ELISA to active GLP-1 (7–36) (EMD Millipore,Billerica, MA) or human insulin (Mercodia, Stockholm, Swe-den), respectively, according to manufacturer’s instructions.Secretion of hormones was normalized to protein levels,determined by the bicinchoninic acid assay.

Metabolite ProfilingCells from the hormone secretion assays were swiftlywashed with 1 mL ice-cold PBS prior to quenching ofmetabolism by adding 300 mL methanol at 280°C. Metab-olites were extracted and derivatized as previously described(19,20). The derivatized metabolites were analyzed on anAgilent 6890N gas chromatograph (Agilent Technologies,Atlanta, GA) equipped with an Agilent 7683B autosampler(Agilent Technologies) and coupled to a LECO Pegasus IIITOFMS electron impact time-of-flight mass spectrometer(LECO Corp., St. Joseph, MI), as previously described(21), and on a 5973 inert GC/MS system (Agilent Technol-ogies) in single ion-monitoring mode.

RespirationOxygen consumption rate (OCR) was measured by the XF24Extracellular Flux Analyzer (Seahorse Bioscience, NorthBillerica, MA) as previously described (22). Cells were pre-incubated for 1 h at basal glucose levels (0 mmol/L forGLUTag cells and 2.8 mmol/L for INS-1 832/13 cells) at37°C in air, after which respiration was measured in theabsence of glucose, followed by addition of either leucine orglutamine. Oligomycin, carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone, and rotenone were injected as describedpreviously (22).

Nucleotide End Point MeasurementsGLUTag and INS-1 832/13 cells were cultured in 24-wellplates and incubated in glucose-free HBSS (GLUTag) orHBSS supplemented with 2.8 mmol/L glucose (INS-1 832/13).Subsequently, the incubation buffer was removed, and cellswere washed in PBS and lysed by addition of 100 mL lysisbuffer. Finally, cells were snap-frozen on dry ice/ethanoland levels of ATP determined using a luciferase-based lumi-nescence assay (BioThema, Handen, Sweden).

Nucleotides were also analyzed by high-performanceliquid chromatography with ultraviolet detection at 254 nmusing an XBridge Amide column (4.6 3 150 mm, 3.5 mm).

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Briefly, cells were deproteinized by adding 1.2 mol/L HClO4,followed by centrifugation and removal of lipids from thesupernatant by extraction with CHCl3. Samples were neu-tralized by addition of 2 mol/L K2CO3, filtered, and dilutedfourfold in acetonitrile. Nucleotides were eluted using a lin-ear gradient composed of A: acetonitrile and B: 2 mmol/LKH2PO4 starting at 75% A and ending at 62% A in 10 min.

Single Live-Cell ATP/ADP Ratio MeasurementsSingle-cell ATP/ADP ratio measurements were carried outusing the pericam-based genetically encoded ATP biosensorPerceval HR (23). Cells were seeded onto poly-D-lysine–coatedeight-well chambered cover glasses (Lab-Tek; Thermo Scien-tific, Waltham, MA) at a density of 70,000 cells/cm2. At24 h after seeding, cells at ;50% confluency were trans-fected with 1 mg plasmid encoding Perceval HR (AddgeneID: 49083). On the day of imaging, 48 h after transfec-tion, cells were preincubated at 37°C in 400 mL bufferP (135 mmol/L NaCl, 3.6 mmol/L KCl, 1.5 mmol/L CaCl2,0.5 mmol/L MgSO4, 0.5 mmol/L Na2HPO4, 10 mmol/LHEPES, and 5 mmol/L NaHCO3, pH 7.4) containing 0 or2.8 mmol/L glucose for GLUTag and INS-1 832/13 cells,respectively. After 1.5 h of incubation, cells were imagedwith 490-nm excitation and 535-nm emission filter settingson a Zeiss LSM510 inverted confocal fluorescence micro-scope (Carl Zeiss, Oberkochen, Germany).

RNA Isolation, Quantitative Real-time PCR, andGDH ActivityTotal RNA was extracted from GLUTag and INS-1 832/13cells using the RNAeasy RNA purification kit (Qiagen,Hilden, Germany) according to the manufacturer’s protocol.RNA concentrations were determined using a NanoDropSpectrophotometer (Thermo Scientific). Equal quantitiesof total RNA were reversely transcribed using the RevertAidFirst-Strand cDNA synthesis kit (Fermentas, Vilnius, Lith-uania) in reactions containing 500 ng total RNA. Quantita-tive real-time PCR was performed using the TaqMan geneexpression assay (GDH isoform GLUD1, regulator of GDHactivity sirtuin-4 [SIRT4], and mitochondrial transcriptionfactor A) using a 7900HT Fast Real-Time System (AppliedBiosystems, Foster City, CA). The quantitative real-timePCR was carried out as previously described (24). Gene ex-pression was quantified by the comparative threshold cyclemethod, in which the amount of target is expressed as22ΔΔCt using hypoxanthine guanine phosphoribosyl trans-ferase as reference gene. GDH activity was measured aspreviously described in detail (25).

Colorectal Infusion in MiceFasted (2 h) female C57BL/6 mice (n = 24; Janvier Labora-tories, Rennes, France) were anesthetized with midazolam(0.4 mg/mouse; Dormicum; Hoffmann-La Roche, Basel,Switzerland), fluanisone (0.9 mg/mouse), and fentanyl(0.02 mg/mouse; Hypnorm; Janssen, Beerse, Belgium).Mice were placed on a heating pad to maintain body tem-perature and divided into three groups. Retro-orbital bloodsamples were taken at time zero with a Luer capillary glass

pipette rinsed in EDTA. Thereafter, mice were infused col-orectally (26) with 1 mL physiological NaCl (9 mg/L; con-trol), 10 mmol/L glutamine, or 10 mmol/L DMG in physiologicalNaCl. The concentration of secretagogues in our studyare in the same range as postprandial intestinal concen-trations of the amino acid (27). EGCG was infused at1 mmol/L in 0.5 mmol/L ascorbic acid diluted in physiolog-ical NaCl. The infusion rate was 1 mL/min. After 10 min, anadditional blood sample was taken. Blood samples fromboth time points were centrifuged, and plasma was sepa-rated from blood cells. Plasma samples were immediatelyassayed for total GLP-1 (EMD Millipore; Merck Millipore,Billerica, MA).

Statistical AnalysisAll data are presented as means 6 SEM for the indi-cated number of experiments. Metabolite data were log2-transformed prior to assessment of differences betweengroups by the paired Student t test or ANOVA with Bon-ferroni test post hoc when more than two groups werecompared. Orthogonal projection to latent structures dis-criminant analysis (OPLS-DA) was performed in SIMCA 13.0(Umetrics, Umeå, Sweden) on mean-centered and unit var-iance scaled data.

RESULTS

Glucose-Stimulated Hormone Secretion From INS-1832/13 and GLUTag Cells Is Associated With IncreasedMitochondrial MetabolismGlucose elicited secretion of GLP-1 and insulin fromGLUTag and INS-1 832/13 cells, respectively (Fig. 1). Tofurther examine to which extent metabolism of the hexoseis involved in this process, we profiled changes in metabolismelicited by glucose using gas chromatography/mass spectrom-etry. To this end, we generated data on levels of amino acids,fatty acids, and glycolytic, pentose phosphate pathway, andtricarboxylic acid (TCA) cycle intermediates. Data were

Figure 1—Glucose elicits hormone secretion from GLUTag and INS-1832/13 cells. GLP-1 and insulin secretion from GLUTag and INS-1832/13 cells, respectively, at 0 mmol/L (white bars), 2.8 mmol/L (blackbars), 10 mmol/L (gray bars), and 16.7 mmol/L glucose (checkeredbars). Data are expressed as means 6 SEM for n = 5. Differencesbetween groups were assessed by ANOVA followed by Bonferronitest post hoc. *P , 0.05; **P , 0.01; ***P , 0.001.

374 GDH and GLP-1 Secretion Diabetes Volume 67, March 2018

Figure 2—Glucose elicits similar changes in metabolism in GLUTag and INS-1 832/13 cells. A: Shared and unique structures–like plot derivedfrom the loadings of the predictive component, scaled as correlations (p(corr)[1]), of OPLS-DA models discriminating between cells at basal(0 mmol/L for GLUTag and 2.8 mmol/L for INS-1 832/13 cells) and stimulatory (10 mmol/L for GLUTag and 16.7 mmol/L for INS-1 832/13 cells)glucose levels. Metabolites to the left and right decrease and increase, respectively, in GLUTag cells, whereas metabolites on the bottom andtop decrease and increase, respectively, after glucose stimulation of INS-1 832/13 cells. Hence, glucose elicits increased levels of metabolitesfound in the top right section and decreased levels of those found in the lower left corner in both cell types (shared). Metabolites in the centralright and left section increase and decrease, respectively, uniquely in GLUTag cells. Metabolites in the central top and lower sections increaseand decrease, respectively, uniquely in INS-1 832/13 cells. The shared and unique fields are indicated by + for increase, 2 for decrease, and0 for unaltered for GLUTag/INS-1 832/13. Only significantly altered metabolites are named. B: Plots of relative levels of metabolites in centralglucose metabolism in GLUTag cells at basal (white bars) and stimulatory glucose (black bars). C: Plots of relative levels of metabolites in INS-1832/13 cells at basal (white bars) and stimulatory glucose (black bars) levels. Data are expressed as means 6 SEM for n = 4. Differencesbetween groups were assessed by the paired Student t test on log-transformed data. *P , 0.05; **P , 0.01; ***P , 0.001. AKGA,a-ketoglutarate; Cit, citrate; Creat, creatinine; DiHAP, dihydroxyacetone phosphate; Fruct, fructose; FruP, fructose 1- and 6-phosphate; Fum,fumarate; Glu6P, glucose 6-phosphate; GlyA3P, 3-phosphoglycerate; Gly2P, glycerol 2-phosphate; Gly3P, glycerol 3-phosphate; IsoCit (IsoC),isocitrate; Lac, lactate; Mal, malate; pGlu, pyroglutamate; Pyr, pyridine; Succ, succinate; Udi, unknown disaccharide.

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analyzed by OPLS-DA; one model was calculated for each ofthe cell types, with the glucose level as discriminating variable.Data were then visualized in a shared and unique structures–like plot (Fig. 2A), derived from the loadings, scaled as corre-lations, of the OPLS-DA models (28). The significance of theloadings, obtained by jackknifing, highlighted metabo-lites, the levels of which were uniquely increased by glucosein GLUTag or INS-1 832/13 cells, as well as changes in me-tabolite levels common to both cell types. Clearly, glucoseelicited similar changes in metabolism in GLUTag and INS-1832/13 cells. Thus, levels of glycolytic and TCA cycle intermedi-ates increased in both cell types (Fig. 2B and C). Glutamatelevels increased 5- (P , 0.001) and 1.3-fold (P , 0.05) inGLUTag and INS-1 832/13 cells, respectively. Aspartatelevels decreased in both cell types: 0.6-fold (P , 0.001) inGLUTag cells and 0.5-fold (P, 0.001) in INS-1 832/13 cells.Glycerol-3-phosphate levels increased after glucose stimula-tion only in the INS-1 832/13 cells (1.5-fold; P , 0.001).Levels of glutamine were unaltered in both cell types.

Glutamine Stimulates GLP-1 Secretion From GLUTagCells in Absence of the GDH Activator LeucineAfter having established that mitochondrial metabolism isactivated by glucose in both GLUTag and INS-1 832/13 cells,we investigated whether the cell lines responded similarlyalso to glutamine. Glutamine alone was found to potentlystimulate GLP-1 secretion from GLUTag cells, whereas it wasineffective in stimulating insulin secretion from INS-1 832/13 cells (Fig. 3A). Activation of GDH with leucine did notaffect glutamine-induced GLP-1 secretion, but permittedinsulin secretion in response to glutamine. Leucine alonedid not induce either GLP-1 secretion from GLUTag cells orinsulin secretion from INS-1 832/13. These differencesbetween cells were not caused by the differing culturingconditions used, as INS-1 832/13 cells cultured for 48 h at5.6 mmol/L glucose showed a similar pattern of insulin secre-tion as those cultured at 11.1 mmol/L glucose (Fig. 3B).

Metabolite profiling revealed that addition of glutaminealone increased levels of TCA cycle intermediates in GLUTagcells. Notably, levels of these intermediates were unaffectedby leucine (Fig. 3C). In INS-1 832/13 cells, in contrast,glutamine was largely ineffective in stimulating mitochon-drial metabolism in the absence of leucine (Fig. 3D). Hence,these results suggest that GDH is active in the GLUTag cellseven in the absence of the allosteric activator leucine, whichis required for glutamine-activated TCA metabolism andhormone secretion in b-cells.

Metabolism and Hormone Secretion in GLUTagand INS-1 832/13 Cells Stimulated Witha Membrane-Permeable Glutamate AnalogMirror Those Elicited by GlutamineTo further investigate whether glutamine-elicited hormonesecretion has a metabolic component, we stimulated GLUTagand INS-1 832/13 cells with the membrane-permeableglutamate analog DMG, which bypasses Na+-coupled plasmamembrane transporters. Similar to glutamine, DMG induced

GLP-1 secretion from GLUTag cells independent of leucine,whereas leucine was required for DMG to provoke insulinsecretion from INS-1 832/13 cells (Fig. 4A).

The metabolic response elicited by either glutamine orDMG in the GLUTag cells was similar with regard to GLP-1secretion and the increase in levels of TCA cycle inter-mediates, with the exception of aspartate (Fig. 4B and Sup-plementary Fig. 1); the level of this metabolite increased toa greater extent when stimulated with glutamine (2.4-fold byglutamine vs. 1.7-fold by DMG; P, 0.001). In INS-1 832/13cells, in contrast, glutamine and DMG failed to raise levelsof TCA cycle intermediates in the absence of the allostericGDH activator leucine (Fig. 4C and Supplementary Fig. 1).

GDH Activity Does Not Differ Between Lysed GLUTagand INS-1 832/13 CellsThus far, our results indicate that GDH activity differsbetween GLUTag and INS-1 832/13 cells. To examine thisfurther, we assessed GDH activity in lysed cells. To reflectthe conditions used in our previous experiments, we usedglutamine as substrate, which, via deamination by gluta-minase, produces glutamate and ammonia. Then we de-termined NADH generated by the GDH-catalyzed oxidationof glutamate to a-ketoglutarate. However, these analysesshowed no difference in activity between GLUTag and INS-1832/13 cells, estimated as the glutamine-elicited area underthe curve (AUC) (Fig. 5A).

GDH Is Activated in Intact GLUTag CellsNext, we aimed to determine enzyme activity in intact cells.As a measure of GDH activity linked to TCA cycle metabolismand oxidative phosphorylation, we monitored the OCR afterstimulation with glutamine followed by leucine or vice versa.In GLUTag cells, leucine was ineffective in stimulating OCR,whereas glutamine potently increased OCR, which did notincrease further upon subsequent addition of leucine (Fig.5B). In contrast, both leucine and glutamine were requiredto elicit a robust increase in OCR in INS-1 832/13 cells (Fig.5C). These results further support that GDH exists in anenhanced activity state in GLUTag cells.

Regulation of GDH ActivityGDH activity is regulated both allosterically by, for exam-ple, amino acids and nucleotides and covalently by ADPribosylation, catalyzed by SIRT4 (7,29). Unexpectedly, theGLUD1/SIRT4 expression ratio was 55% (P , 0.05) lowerin GLUTag compared with INS-1 832/13 cells (Fig. 6A).Moreover, basal levels of ATP, reflecting the energy statusof the cell, were 2.9-fold (P , 0.05) higher in GLUTag cells(Fig. 6B). We also determined cytosolic ATP/ADP ratio usingPerceval HR (Fig. 6C). The maximal response (Fig. 6D) andAUC of the ATP/ADP ratio (Fig. 6E) in response to glutaminestimulation were higher in GLUTag cells as compared withINS-1 832/13. Addition of leucine did not affect the ATP/ADPratio in either of the cell types. Levels of ADP, AMP, guanosinemonophosphate, guanosine diphosphate, and guanosine tri-phosphate did not differ between cell types. Of note, basallevels of leucine were 7.4-fold (P , 0.01) higher in GLUTag

376 GDH and GLP-1 Secretion Diabetes Volume 67, March 2018

Figure 3—Glutamine stimulates mitochondrial metabolism and GLP-1 secretion from GLUTag cells in the absence of leucine, whereas leucine isrequired for glutamine-elicited mitochondrial metabolism and insulin secretion from INS-1 832/13 cells. A: GLP-1 and insulin secretion fromGLUTag and INS-1 832/13 cells, respectively, at basal glucose (0 mmol/L for GLUTag and 2.8 mmol/L for INS-1 832/13; white bars), 10 mmol/Lleucine (black bars), 10 mmol/L glutamine (gray bars), and the combination of 10 mmol/L glutamine and 10 mmol/L leucine (checkered bars). B:Secretion of insulin in response to glucose, glutamine, glutamine + leucine, and leucine was similar in cells cultured at 5.6 mmol/L glucose for48 h and in those cultured at 11.1 mmol/L glucose. C: Plots of relative levels of metabolites involved in mitochondrial metabolism in GLUTagcells at basal (0 mmol/L glucose; white bars) and after stimulation with 10 mmol/L glutamine (black bars) or the combination of 10 mmol/L eachof glutamine and leucine (gray bars). D: Plots of relative levels of metabolites involved in mitochondrial metabolism in INS-1 832/13 cells at basal(2.8 mmol/L glucose; white bars) and after stimulation with 10 mmol/L glutamine (black bars) or the combination of 10 mmol/L each of glutamineand leucine (gray bars). Data are expressed as means 6 SEM for n = 5 (GLUTag) (A), n = 14 (INS-1 832/13) (A), and n = 4 (B–D). Differencesbetween groups were assessed by ANOVA followed by Bonferroni test post hoc on log-transformed data. *P , 0.05; **P , 0.01; ***P , 0.001.AKGA, a-ketoglutarate; Cit, citrate; Ctrl, control; Fum, fumarate; IsoC, isocitrate; Mal, malate; Succ, succinate.

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cells than in INS-1 832/13 cells (Fig. 6F). Given leucine’srole as allosteric activator of GDH, its higher basal level mayexplain the increased GDH activity in the GLUTag cells.

Inhibition of GDH Reduces Secretion of GLP-1in Response to Glutamine and DMGTo further examine the role of GDH in glutamine-elicitedGLP-1 secretion, we inhibited the enzyme using EGCG.Secretion of GLP-1 in response to both glutamine and DMGwas significantly reduced in the presence of EGCG (Fig. 7A).Notably, glutamine was still effective in eliciting GLP-1 se-cretion in presence of the inhibitor (P , 0.001), whereasDMG was not. Reduced secretion of GLP-1 in response to thesecretagogues in presence of EGCG was paralleled by an almostcomplete abolishment of the glutamine- and DMG-elicitedincreases in levels of TCA cycle intermediates (Fig. 7B and C).

Colorectal Infusion of DMG in Mice ElicitsGLP-1 SecretionTo gain physiological support for our findings, we examinedwhether the membrane-permeable glutamate analog DMGcould affect GLP-1 secretion in vivo in mice. Colorectal infusion

of either glutamine or DMG yielded a 2.1-fold (P , 0.05) anda 2.0-fold (P , 0.05), respectively, stronger stimulation ofGLP-1 secretion as compared with the control (Fig. 7D).Inhibition of GDH by EGCG abrogated secretion of GLP-1in response to DMG (Fig. 7E). A schematic depiction ofdifferences in stimulus-secretion coupling between the L celland b-cell is shown in Fig. 8.

DISCUSSION

Metabolic coupling in nutrient-stimulated insulin secretionfrom the pancreatic b-cell has been comprehensively stud-ied. These studies have highlighted mitochondria as key inthe generation of signals that trigger and amplify secretionof the hormone. The L cell, in contrast, is less well charac-terized, but studies have revealed mechanisms similar tothose in the b-cell (14), as well as mechanisms that maybe unique to the L cell (15). As opposed to the b-cell,secretion of GLP-1 from the L cell has also been shown tobe governed by sodium-coupled nutrient uptake, implying anaction essentially independent of intracellular metabolism ofthe secretagogue (14).

Figure 4—DMG-elicited metabolism and hormone secretion mirrors that of glutamine in GLUTag and INS-1 832/13 cells. A: GLP-1 and insulinsecretion from GLUTag and INS-1 832/13 cells, respectively, at basal glucose (0 mmol/L for GLUTag and 2.8 mmol/L for INS-1 832/13; whitebars), 10 mmol/L leucine (black bars), 10 mmol/L DMG (gray bars), and the combination of 10 mmol/L leucine and 10 mmol/L DMG (checkeredbars). B: Plots of relative levels of metabolites involved in mitochondrial metabolism in GLUTag cells at basal (0 mmol/L glucose, white bars) andafter stimulation with 10 mmol/L DMG (black bars) or the combination of 10 mmol/L each of DMG and leucine (gray bars). C: Plots of relativelevels of metabolites involved in mitochondrial metabolism in INS-1 832/13 cells at basal (2.8 mmol/L glucose; white bars) and after stimulationwith 10 mmol/L DMG (black bars) or the combination of 10 mmol/L each of DMG and leucine (gray bars). Data are expressed as means6 SEMfor n = 5 and n = 6 for GLUTag in A and B, respectively, and n = 14 and n = 6 for INS-1 832/13 in A and C, respectively. Differences betweengroups were assessed by ANOVA followed by Bonferroni test post hoc. *P, 0.05; **P, 0.01; ***P, 0.001. AKGA, a-ketoglutarate; Cit, citrate;Fum, fumarate; IsoC, isocitrate; Mal, malate; Succ, succinate.

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In the current study, we investigated the metaboliccomponent of stimulus-secretion coupling in the L cell. Tofacilitate these analyses, we compared the L cell modelGLUTag with the well-established b-cell model INS-1 832/13.Conditions were selected from the literature (14,17) andhence differ between GLUTag and INS-1 832/13 cells, butallow comparison with previous studies in these cells. Se-cretion of insulin in response to glucose, glutamine, andleucine was not altered when INS-1 832/13 cells were pre-cultured for 48 h at the lower glucose levels that were usedfor the GLUTag cells. It must, however, be acknowledgedthat metabolism in these immortalized cell lines may notexactly mirror metabolism in vivo.

We found that exposure of GLUTag cells or L cells in vivoto nutrients and stimuli acting upstream and downstreamof electrogenic transporters yielded qualitatively similarresults. Hence, our data support previous studies thathave indicated a metabolic component, involving ATPproduction and KATP channel closure, analogous to that

observed in the b-cell, in L cell glutamine (30), glucose(9,14), and fructose sensing (14). It needs to be taken intoaccount that glutamine has also been suggested to act viaa cAMP-dependent nonelectrogenic sensing mechanism (31).

It is possible that different sensing mechanisms mightgovern GLP-1 secretion from different parts of the gastro-intestinal tract. GLUTag cells are derived from colonictumors, and the in vivo administration of glutamine andDMG mainly targeted the more distal segments of the gas-trointestinal tract. Oral glucose tolerance tests conductedin sodium-glucose linked transporter 1 knockout mice haverevealed a blunted early secretion of GLP-1 (32) but in-creased intestinal levels of glucose to associate with an ex-aggerated late secretion of the hormone (33). Hence, ourdata support that nonelectrogenic nutrient uptake and sens-ing may play a more important role in the more distal sec-tions of the gastrointestinal tract.

Our results also provide evidence that glutamine-elicitedGLP-1 secretion requires an active GDH. When the enzyme

Figure 5—GDH activity does not differ between lysed GLUTag and INS-1 832/13 cells but is activated in intact GLUTag cells. A: GDH activity inGLUTag and INS-1 832/13 cells monitored in cell extracts as NADH fluorescence. Glutamine (1 mmol) and purified GDH were added asindicated by the arrows. B: OCR in GLUTag cells after stimulation with glutamine (10 mmol/L) and leucine (10 mmol/L). Leucine was addedeither before (black symbols) or after (gray symbols) glutamine. The combination of leucine and glutamine does not increase OCR above the levelobserved for glutamine alone. C: OCR in response to glutamine and leucine in INS-1 832/13 cells. The combination of glutamine and leucinepotently stimulates OCR. Data are expressed as means6 SEM for n = 4. Differences between groups and the basal condition were assessed bythe paired Student t test. *P , 0.05; **P , 0.01; ***P , 0.001. AU, arbitrary units; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone;OM, oligomycin.

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was inhibited, both mitochondrial metabolism and GLP-1secretion became insensitive to DMG. However, whereasGLP-1 secretion was significantly reduced in response toglutamine, the amino acid was still effective in elicitingsecretion of the hormone. This was paralleled by an almostabolished response in mitochondrial metabolism, sug-gesting that glutamine may act via additional mecha-nisms. This contrasts with the b-cell, in which GDHactivation by, for example, leucine is necessary for glutamine-stimulated insulin secretion (18,34–38). In fact, muta-tions resulting in escape from nucleotide inhibition andconstitutive activation of GDH cause hyperinsulinemichypoglycemia (7). Moreover, GDH activity is also regu-lated by SIRT4 that inhibits GDH activity via ADP ribosy-lation (29). Hence, silencing of SIRT4 in b-cells resultsin glutamine-stimulated insulin secretion (39,40). Wecould not find any differences in SIRT4 mRNA levels be-tween GLUTag and INS-1 832/13 cells. Rather the relativelevels of SIRT4 to GLUD1 were higher in the former, as wasthe overall energy state, reflected by total cellular ATP lev-els. Instead, metabolite profiling revealed higher basal levels

of leucine in the GLUTag cells. It is possible that this ele-vation of basal leucine levels serves to keep GDH in anactive state in GLUTag cells, thereby promoting secretionof GLP-1.

The ATP/ADP ratio changed only slowly upon stimula-tion of GLUTag cells with glutamine and did not increasesignificantly upon addition of leucine to INS-1 832/13 cells.Such slow responses are consistently observed and likelydue to consumption of ATP by Ca2+-ATPases (41) and, inthis case, also leucine-elicited energy using protein synthesis(42).

Whereas low GDH activity is a prerequisite for normalb-cell function, the opposite holds true for astrocytes (43).Astrocytes rely on GDH activity to enable glutamateclearance and energy production required for glutamateuptake (44). In humans, but not rodents, these cells expressthe GTP-insensitive GDH isoform GLUD2, enabling thesepivotal functions to also operate at a high-energy state.Notably, the neurotransmitter glutamate has been shownto be secreted from GLUTag cells (45), as well as a- (46,47)and b-cells (48). Indeed, enteroendocrine cells and cells of

Figure 6—Levels of factors regulating GDH activity differ between INS-1 832/13 and GLUTag cells. A: mRNA levels of Sirt4 and Glud1,normalized to mitochondrial transcription factor A (Tfam) in GLUTag (white bars) and INS-1 832/13 (black bars) cells. B: Basal ATP levels,0 mmol/L glucose for GLUTag (white bar) and 2.8 mmol/L glucose for INS-1 832/13 (black bar). C: Cytosolic ATP/ADP ratio assessed byPerceval HR. The maximum response (D) and AUC (E) after glutamine (10 mmol/L) stimulation was higher in GLUTag compared with INS-1832/13 cells. F: Basal levels of leucine in GLUTag (white bar) and INS-1 832/13 cells (black bar). Data are expressed as means 6 SEM for n =3 (A, B, and F) and n = 184 and n = 212 single cells for GLUTag and INS-1 832/13, respectively, in C–E. Differences between groups wereassessed by ANOVA followed by Bonferroni test post hoc (A) and the unpaired Student t test (B–F). *P, 0.05; ***P, 0.001. a.u., arbitrary units;Ctrl, control; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.

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Figure 7—GDH is essential in glutamine- and DMG-elicited GLP-1 secretion. A: Inhibition of GDH in GLUTag cells with EGCG (20 mmol/L)abolishes secretion of GLP-1 in response to glutamine (Gln; 10 mmol/L) and DMG (10 mmol/L). Inhibition of GDH with EGCG is associated witha blunted response in levels of TCA cycle intermediates in response to glutamine (B) and DMG (C). D: Colorectal infusion of glutamine(Gln; 10 mmol/L) and DMG (10 mmol/L) elicits twofold higher GLP-1 secretion as compared with control (Ctrl; 0.9% NaCl) 10 min postinfusion(t = 0, white bars; t = 10 min, black bars). E: In vivo inhibition of GDH by EGCG abolishes secretion of GLP-1 in response to DMG. Data areexpressed as means6 SEM for n = 4 in A–C; n = 9 (number of mice; control), n = 8 (glutamine), and n = 7 (DMG) in D; and n = 8 in E. Differencesbetween groups were assessed by the Student t test (E) or ANOVA followed by Bonferroni test post hoc. $P, 0.05; $$P, 0.01; $$$P, 0.001vs. control. *P , 0.05; **P , 0.01 vs. indicated condition. AU, arbitrary units; Cit, citrate; Fum, fumarate; IsoCit, isocitrate; Mal, malate.

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the nervous system share multiple features, which previ-ously were misinterpreted because of both cell lineages be-ing derived from the neural crest (49).

In conclusion, our data suggest that nonelectrogenicnutrient uptake and metabolism play an equally importantrole in nutrient sensing in colonic L cells as they do in thepancreatic b-cells. An activated state of GDH is essential inthe L cells, whereas activity of this enzyme needs to beinactivated in the b-cell in the absence of glucose. Ourresults support that glutamine and related amino acid ana-logs may offer a means of increasing GLP-1 levels withoutaffecting insulin secretion directly in vivo.

Acknowledgments. The authors thank Dr. Daniel J. Drucker, MountSinai Hospital, Toronto, Ontario, Canada, for permitting use of the GLUTagcells.

Funding. This work was supported by grants from Vetenskapsrådet (SwedishResearch Council), Novo Nordisk Foundation, Swedish Diabetes Foundation,Crafoordska Stiftelsen, Stiftelsen Lars Hiertas Minne, Fredrik och Ingrid ThuringsStiftelse, O.E. och Edla Johanssons Vetenskapliga Stiftelse, Åke Wibergs Stiftelse,Direktör Albert Påhlssons Stiftelse, Magnus Bergvalls Stiftelse, Inga and John HainFoundation, Hjelt Foundation, and Kungliga Fysiografiska Sällskapet i Lund (RoyalPhysiographic Society of Lund). An equipment grant from Knut och Alice WallenbergsStiftelse was also received.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. L.E.A., L.S., M.A.-M., N.V., and M.F. designed andperformed experiments, analyzed data, interpreted results, and edited the manuscript.C.B.A. and J.A.C. designed and performed experiments and edited the manuscript.C.B.W., H.M., and N.W. provided intellectual guidance and cowrote the paper. P.S.conceived and directed the project, analyzed data, interpreted results, andwrote the first draft of the paper together with L.E.A. P.S. is the guarantor ofthis work and, as such, had full access to all of the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

Figure 8—Glutamine and DMG metabolism in L cells and b-cells. In the L cell, glutamine elicits increased TCA cycle metabolism, mitochondrialrespiration, and GLP-1 secretion. In the b-cell, in contrast, glutamine and DMG are incapable of eliciting increased TCA cycle metabolism,respiration, and secretion of insulin in absence of an allosteric activator of GDH, such as leucine or ADP. Thus, glutamine-stimulated GLP-1secretion from the L cell is governed by an active GDH, whereas this enzyme is inactive in the b-cell. DMG, which bypasses sodium-coupledtransporters, and glutamine yield qualitatively similar responses in both cell types. Hence, nonelectrogenic nutrient uptake and metabo-lism plays an important role in stimulus-secretion coupling in both the L cell and b-cell. a-KG, a-ketoglutaric acid; OxPhos, oxidative phos-phorylation.

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References1. Drucker DJ. The biology of incretin hormones. Cell Metab 2006;3:153–1652. Göke R, Wagner B, Fehmann HC, Göke B. Glucose-dependency of the insulinstimulatory effect of glucagon-like peptide-1 (7-36) amide on the rat pancreas.Res Exp Med (Berl) 1993;193:97–1033. Barnett A. DPP-4 inhibitors and their potential role in the management of type 2diabetes. Int J Clin Pract 2006;60:1454–14704. Pro B, Dang NH. CD26/dipeptidyl peptidase IV and its role in cancer. HistolHistopathol 2004;19:1345–13515. Greenfield JR, Farooqi IS, Keogh JM, et al. Oral glutamine increases circulatingglucagon-like peptide 1, glucagon, and insulin concentrations in lean, obese, andtype 2 diabetic subjects. Am J Clin Nutr 2009;89:106–1136. Weir GC, Bonner-Weir S. Five stages of evolving beta-cell dysfunction duringprogression to diabetes. Diabetes 2004;53(Suppl. 3):S16–S217. Li M, Li C, Allen A, Stanley CA, Smith TJ. The structure and allosteric regulationof mammalian glutamate dehydrogenase. Arch Biochem Biophys 2012;519:69–808. Spanaki C, Plaitakis A. The role of glutamate dehydrogenase in mammalianammonia metabolism. Neurotox Res 2012;21:117–1279. Reimann F, Habib AM, Tolhurst G, Parker HE, Rogers GJ, Gribble FM. Glucosesensing in L cells: a primary cell study. Cell Metab 2008;8:532–53910. Prentki M, Matschinsky FM, Madiraju SR. Metabolic signaling in fuel-inducedinsulin secretion. Cell Metab 2013;18:162–18511. Brubaker PL, Schloos J, Drucker DJ. Regulation of glucagon-like peptide-1synthesis and secretion in the GLUTag enteroendocrine cell line. Endocrinology1998;139:4108–411412. Henquin JC. Regulation of insulin secretion: a matter of phase control andamplitude modulation. Diabetologia 2009;52:739–75113. Maechler P. Mitochondria as the conductor of metabolic signals for insulinexocytosis in pancreatic beta-cells. Cell Mol Life Sci 2002;59:1803–181814. Gribble FM, Williams L, Simpson AK, Reimann F. A novel glucose-sensingmechanism contributing to glucagon-like peptide-1 secretion from the GLUTag cellline. Diabetes 2003;52:1147–115415. Powell DR, Smith M, Greer J, et al. LX4211 increases serum glucagon-likepeptide 1 and peptide YY levels by reducing sodium/glucose cotransporter 1(SGLT1)-mediated absorption of intestinal glucose. J Pharmacol Exp Ther 2013;345:250–25916. Cordier-Bussat M, Bernard C, Levenez F, et al. Peptones stimulate both thesecretion of the incretin hormone glucagon-like peptide 1 and the transcription of theproglucagon gene. Diabetes 1998;47:1038–104517. Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, Newgard CB.Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependentand -independent glucose-stimulated insulin secretion. Diabetes 2000;49:424–43018. Pournourmohammadi S, Grimaldi M, Stridh MH, et al. Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenasein muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabeticstate? Int J Biochem Cell Biol 2017;88:220–22519. Spégel P, Sharoyko VV, Goehring I, et al. Time-resolved metabolomics analysisof b-cells implicates the pentose phosphate pathway in the control of insulin release.Biochem J 2013;450:595–60520. Gullberg J, Jonsson P, Nordström A, Sjöström M, Moritz T. Design of experi-ments: an efficient strategy to identify factors influencing extraction and derivatizationof Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry. Anal Biochem 2004;331:283–29521. Danielsson AP, Moritz T, Mulder H, Spégel P. Development and optimization ofa metabolomic method for analysis of adherent cell cultures. Anal Biochem 2010;404:30–3922. Malmgren S, Nicholls DG, Taneera J, et al. Tight coupling between glucose andmitochondrial metabolism in clonal beta-cells is required for robust insulin secretion.J Biol Chem 2009;284:32395–3240423. Tantama M, Martínez-François JR, Mongeon R, Yellen G. Imaging energy statusin live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio. NatCommun 2013;4:2550

24. Krus U, Kotova O, Spégel P, et al. Pyruvate dehydrogenase kinase 1 controlsmitochondrial metabolism and insulin secretion in INS-1 832/13 clonal beta-cells.Biochem J 2010;429:205–21325. Carobbio S, Ishihara H, Fernandez-Pascual S, Bartley C, Martin-Del-Rio R,Maechler P. Insulin secretion profiles are modified by overexpression of glutamatedehydrogenase in pancreatic islets. Diabetologia 2004;47:266–27626. Adrian TE, Gariballa S, Parekh KA, et al. Rectal taurocholate increases L cell andinsulin secretion, and decreases blood glucose and food intake in obese type 2 di-abetic volunteers. Diabetologia 2012;55:2343–234727. Adibi SA, Mercer DW. Protein digestion in human intestine as reflected in lu-minal, mucosal, and plasma amino acid concentrations after meals. J Clin Invest1973;52:1586–159428. Wiklund S, Johansson E, Sjöström L, et al. Visualization of GC/TOF-MS-basedmetabolomics data for identification of biochemically interesting compounds usingOPLS class models. Anal Chem 2008;80:115–12229. Haigis MC, Mostoslavsky R, Haigis KM, et al. SIRT4 inhibits glutamate de-hydrogenase and opposes the effects of calorie restriction in pancreatic betacells. Cell 2006;126:941–95430. Reimann F, Williams L, da Silva Xavier G, Rutter GA, Gribble FM. Glutaminepotently stimulates glucagon-like peptide-1 secretion from GLUTag cells. Di-abetologia 2004;47:1592–160131. Tolhurst G, Zheng Y, Parker HE, Habib AM, Reimann F, Gribble FM. Glutaminetriggers and potentiates glucagon-like peptide-1 secretion by raising cytosolic Ca2+and cAMP. Endocrinology 2011;152:405–41332. Gorboulev V, Schürmann A, Vallon V, et al. Na(+)-D-glucose cotransporterSGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretinsecretion. Diabetes 2012;61:187–19633. Powell DR, DaCosta CM, Gay J, et al. Improved glycemic control in mice lackingSglt1 and Sglt2. Am J Physiol Endocrinol Metab 2013;304:E117–E13034. Sener A, Somers G, Devis G, Malaisse WJ. The stimulus-secretion couplingof amino acid-induced insulin release. Biosynthetic and secretory responses of ratpancreatic islet to L-leucine and L-glutamine. Diabetologia 1981;21:135–14235. Sener A, Malaisse WJ. L-leucine and a nonmetabolized analogue activatepancreatic islet glutamate dehydrogenase. Nature 1980;288:187–18936. Fahien LA, MacDonald MJ, Kmiotek EH, Mertz RJ, Fahien CM. Regulation ofinsulin release by factors that also modify glutamate dehydrogenase. J Biol Chem1988;263:13610–1361437. Stanley CA. Hyperinsulinism/hyperammonemia syndrome: insights into theregulatory role of glutamate dehydrogenase in ammonia metabolism. Mol GenetMetab 2004;81(Suppl. 1):S45–S5138. Liu YJ, Cheng H, Drought H, MacDonald MJ, Sharp GW, Straub SG. Activationof the KATP channel-independent signaling pathway by the nonhydrolyzableanalog of leucine, BCH. Am J Physiol Endocrinol Metab 2003;285:E380–E38939. Argmann C, Auwerx J. Insulin secretion: SIRT4 gets in on the act. Cell 2006;126:837–83940. Maechler P, Carobbio S, Rubi B. In beta-cells, mitochondria integrate andgenerate metabolic signals controlling insulin secretion. Int J Biochem Cell Biol 2006;38:696–70941. Wiederkehr A, Park KS, Dupont O, et al. Matrix alkalinization: a novel mito-chondrial signal for sustained pancreatic beta-cell activation. EMBO J 2009;28:417–42842. Yang J, Chi Y, Burkhardt BR, Guan Y, Wolf BA. Leucine metabolism in regu-lation of insulin secretion from pancreatic beta cells. Nutr Rev 2010;68:270–27943. Schousboe A, Bak LK, Waagepetersen HS. Astrocytic control of biosynthesis andturnover of the neurotransmitters glutamate and GABA. Front Endocrinol (Lausanne)2013;4:102

44. Karaca M, Frigerio F, Migrenne S, et al. GDH-dependent glutamate oxidationin the brain dictates peripheral energy substrate distribution. Cell Reports 2015;13:365–375

45. Uehara S, Jung SK, Morimoto R, et al. Vesicular storage and secretion ofL-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells. JNeurochem 2006;96:550–560

diabetes.diabetesjournals.org Andersson and Associates 383

46. Moriyama Y, Hayashi M. Glutamate-mediated signaling in the isletsof Langerhans: a thread entangled. Trends Pharmacol Sci 2003;24:511–51747. Hayashi M, Yamada H, Uehara S, et al. Secretory granule-mediatedco-secretion of L-glutamate and glucagon triggers glutamatergic signaltransmission in islets of Langerhans. J Biol Chem 2003;278:1966–1974

48. Feldmann N, del Rio RM, Gjinovci A, Tamarit-Rodriguez J, Wollheim CB,Wiederkehr A. Reduction of plasma membrane glutamate transport potentiates in-sulin but not glucagon secretion in pancreatic islet cells. Mol Cell Endocrinol 2011;338:46–5749. Andrew A, Kramer B, Rawdon BB. The origin of gut and pancreatic neuroen-docrine (APUD) cells–the last word? J Pathol 1998;186:117–118

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