Page 1 of 37 Diabetes

Glutamine-elicited secretion of glucagon-like peptide 1 (GLP-1) is governed by

an activated glutamate dehydrogenase

Lotta E. Andersson1, Liliya Shcherbina2, Mahmoud Al-Majdoub1, Neelanjan Vishnu1,

Claudia Balderas Arroyo4, Jonathan Aste Carrara4, Claes B. Wollheim3, Malin Fex1,
Hindrik Mulder1, Nils Wierup2, and Peter Spégel1,4*

1
Unit of Molecular Metabolism, Department of Clinical Sciences, Lund University
Diabetes Centre, CRC, Skåne University Hospital, 205 02 Malmö, Sweden
2
Neuroendocrine Cell Biology, Department of Clinical Sciences, Lund University
Diabetes Centre, CRC, Skåne University Hospital, 205 02 Malmö, Sweden
3
Lund University Diabetes Centre, CRC, Skåne University Hospital, 205 02 Malmö,
Sweden and Department of Cell Physiology and Metabolism, University Medical
Centre, 1 rue Michel-Servet, Geneva 4, Switzerland
4
Department of Chemistry, Centre for Analysis and Synthesis, Lund University, SE-
221 00, Lund Sweden

Running head: GDH and GLP-1 secretion

Word count: 3783

Number of figures: 8

*
Address correspondence to: Peter Spégel, MSc, PhD, Department of Chemistry,
Centre for Analysis and Synthesis, Lund University, SE-221 00 Lund, Sweden.
Phone: +46 (0)40 391009. E-mail: peter.spegel@chem.lu.se

1
Diabetes Publish Ahead of Print, published online December 11, 2017
 Diabetes Page 2 of 37

ABSTRACT

Glucagon-like peptide-1 (GLP-1), secreted from intestinal L-cells, glucose-

dependently stimulates insulin secretion from beta-cells. This glucose-dependence

prevents hypoglycemia, rendering GLP-1 analogues a useful and safe treatment

modality in type-2 diabetes. While the amino acid glutamine is a potent elicitor of

GLP-1 secretion, the responsible mechanism remains unclear.

We investigated how GLP-1 secretion is metabolically coupled in L-cells (GLUTag)

and in vivo in mice, using the insulin-secreting cell-line INS-1 832/13 as reference. A

membrane-permeable glutamate analogue (dimethylglutamate; DMG), acting down-

stream of electrogenic transporters, elicited similar alterations in metabolism as

glutamine in both cell-lines. Both DMG and glutamine alone elicited GLP-1 secretion

in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH)

was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological

inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG.

In conclusion, our results suggest that non-electrogenic nutrient uptake and

metabolism play an important role in L-cell stimulus secretion coupling. Metabolism

of glutamine and related analogues by GDH in the L-cell may explain why GLP-1

secretion, but not that of insulin, is activated by these secretagogues in vivo.

Key words: Stimulus-secretion coupling, metabolite profiling, metabolomics,

mitochondrial metabolism

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Glucagon-like peptide-1 (GLP-1), secreted from intestinal L-cells, potentiates insulin

secretion from the pancreatic beta-cells (1). Importantly, this potentiation is glucose-

dependent, i.e., GLP-1 stimulates insulin secretion when blood glucose levels are

elevated (2). Hence, GLP-1 is unlikely to induce severe hypoglycemia, a dreaded

complication of insulin treatment. Moreover, GLP-1 delays gastric emptying leading

to reduced hunger and food intake (1). These features of GLP-1 underlie the

increasing use of GLP-1 analogues in treatment of type-2 diabetes.

Currently, therapeutic actions of GLP-1 in vivo are mediated by administration of

GLP-1 analogues or dipeptidyl peptidase 4 (DPP-4) inhibitors. However, DPP-4 has

important physiological roles other than degradation of GLP-1 (3), and indiscriminate

inhibitors may affect functions also of other biological processes, resulting in a

potentially wide range of physiological effects. For instance, DPP-4 has been shown

to suppress tumor growth and inhibit malignancies (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 beta-cell, which gradually deteriorates during

progression of the disease (6). In fact, studies have revealed that the GLP-1

secretagogue glutamine potentiates GLP-1 secretion in healthy as well as in subjects

with type-2 diabetes (5). Notably, glutamine does not elicit secretion of insulin from

the pancreatic beta-cell (7). Levels of glutamine are tightly regulated by glutaminase,

glutamine synthetase and glutamate dehydrogenase (GDH). The amino acid functions

both as a modulator of ammonia levels and a substrate for energy production (8). An

increased understanding of stimulus-secretion coupling in the L-cell could enable

nutritional or pharmacological enhancement of GLP-1 secretion.

L-cells are difficult to isolate from the intestine due to their relatively low abundance.

Moreover, survival of L-cells in purified cultures is poor (9). Beta-cell stimulus-

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secretion coupling, on the other hand, has been extensively studied in isolated

pancreatic islets, sorted beta-cells and multiple clonal cell lines (10). Only few

immortalized L-cell lines are available, with the GLUTag cell-line being recognized

as a useful and relevant model of the primary L-cell (11).

Studies of stimulus-secretion coupling in the beta-cell have revealed two major

pathways underlying secretion of insulin. In both of these, increased blood glucose

levels result in an increase in glucose uptake and phosphorylation followed by

glycolysis and complete oxidation of the sugar in mitochondria. 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 exocytosis of

insulin granules (12). In addition to this triggering pathway, an amplifying pathway

has been shown to account for a large proportion of secreted insulin. Multiple

metabolites have been implicated in the amplifying pathway, but a functional role for

most of them has been questioned due to conflicting results from later studies (13;

14). Studies on stimulus-secretion coupling in the L-cell have revealed similar

mechanisms as in the pancreatic beta-cell (15). However, in contrast to the beta-cell,

which expresses Glut1/2, the L-cells also express electrogenic sugar and amino acid

transporters (16). Hence, Na+-coupled nutrient uptake has been suggested to account

for a large proportion of GLP-1 secretion (15). Moreover, the L-cell has also been

shown to respond to stimulation with peptones (17).

In the present study, we examined stimulus secretion-coupling in GLUTag cells, and

used the widely studied beta-cell model, INS-1 832/13, as reference. Results from

these studies were validated in vivo in mice.

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Page 5 of 37 Diabetes

RESEARCH DESIGN AND METHODS

Cell culture. GLUTag cells were cultured in Dulbecco’s modified Eagle’s medium

containing 5.6 mmol/L glucose and supplemented with 10% fetal bovine serum at

37°C in a humidified atmosphere containing 95% air and 5% CO2. INS-1 832/13 cells

were cultured in RPMI-1640 containing either 5.6 or 11.1 mmol/L glucose and

supplemented with 10% fetal bovine serum, 10 mmol/L HEPES, 2 mmol/L glutamine,

1 mmol/L sodium pyruvate and 50 µmol/L 2-mercaptoethanol at 37°C in a humidified

atmosphere containing 95% air and 5% CO2. Cells were seeded in 24-well tissue

culture plates 24 or 48 hours prior to experiments.

Hormone secretion. GLUTag and INS-1 832/13 cells were incubated as previously

described in detail (15; 18). Briefly, GLUTag cells were washed twice with 0.5 mL of

PBS and then incubated in 0.5 mL HEPES-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%, w/w), and various concentrations of glucose, glutamine, dimethyl

glutamate (DMG), and leucine for 2 hours. INS-1 832/13 cells were washed with 0.5

mL PBS and pre-incubated in 0.5 mL HBSS, with 2.8 mmol/L glucose for 2 hours.

Then, cells were incubated in 0.5 mL HBSS containing the same secretagogues as

used for the GLUTag-cells for 1 hour. An epigallocatechin (EGCG) stock solution

was prepared by dissolving EGCG (4 mmol/L) in water containing 1 mmol/L ascorbic

acid. EGCG and ascorbic acid were then added both to pre-incubation and incubation

media at 20 µmol/L and 0.5 mmol/L, respectively (19). Secreted GLP-1 and insulin

were determined in the supernatant after centrifugation using an ELISA to active

GLP-1 (7-36) (EMD Millipore, Billerica, MA) or human insulin (Mercodia,

Stockholm, Sweden), respectively, according to manufacturer’s instructions.

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 Diabetes Page 6 of 37

Secretion of hormones was normalized to protein levels, determined by the

bicinchoninic acid (BCA) assay.

Metabolite profiling. Cells from the hormone secretion assays were swiftly washed

with 1 mL ice-cold PBS prior to quenching of metabolism by adding 300 µL

methanol at -80°C. Metabolites were extracted and derivatized as previously

described (20; 21). The derivatized metabolites were analyzed on an Agilent 6890N

gas chromatograph (Agilent Technologies, Atlanta, GA) equipped with an Agilent

7683B auto-sampler (Agilent Technologies) and coupled to a LECO Pegasus III

TOFMS electron impact time-of-flight mass spectrometer (LECO Corp., St. Joseph,

MI) as previously described (22), and on a 5973 inert GC/MS system (Agilent

Technologies) in single ion monitoring mode.

Respiration. Oxygen consumption rate (OCR) was measured by the XF24

Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) as previously

described (23). Cells were pre-incubated for 1 hour at basal glucose levels (0 mmol/L

for GLUTag cells, 2.8 mmol/L for INS-1 832/13 cells) at 37°C in air after which

respiration was measured in the absence of glucose, followed by addition of either

leucine or glutamine. Oligomycin, carbonyl cyanide-p-trifluoromethoxy-

phenylhydrazone (FCCP) and rotenone were injected as described previously (23).

Nucleotide end point measurements. Basal levels of ATP were measured in

GLUTag and INS-1 832/13 cells cultured in 24-well plates and incubated in glucose

free HBSS (GLUTag) or HBSS supplemented with 2.8 mmol/L glucose (INS-1

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Page 7 of 37 Diabetes

832/13). Subsequently, the incubation buffer was removed, cells were washed in PBS

and lysed by addition of 100 µL lysis buffer. Finally, cells were snap-frozen on dry-

ice/ethanol and levels of ATP determined using a luciferase-based luminescence

assay (BioThema, Handen, Sweden).

Nucleotides were also analyzed by HPLC with UV-detection at 254 nm using an

XBridge Amide column (4.6 mm x 150mm, 3.5 μm). Briefly, cells were

deproteinized by adding 1.2 mol/L HClO4, followed by centrifugation and

removal of lipids from the supernatant by extraction with CHCl3. Samples were

neutralized by addition of 2 mol/L K2CO3, filtered and diluted 4-fold in

acetonitrile. Nucleotides were eluted using a linear gradient composed of A:

acetonitrile and B: 2 mmol/L KH2PO4 starting at 75% A and ending at 62% A in

10 min.

Single live cell ATP/ADP-ratio measurements. Single cell ATP/ADP ratio

measurements were carried out using the pericam-based genetically encoded ATP

biosensor Perceval HR (24). Cells were seeded onto poly-D-lysine coated 8-well

chambered cover glasses (Lab-Tek, Thermo Scientific) at a density of 70,000

cells/cm2. 24 hours after seeding, cells at ~50% confluency were transfected with 1 µg

of plasmid encoding Perceval HR (Addgene ID:49083). On the day of imaging, 48 h

after transfection, cells were pre-incubated at 37°C in 400 µL buffer P (135 mM

NaCl, 3.6 mM KCl, 1.5 mM CaCl2, 0.5 mM MgSO4, 0.5 mM Na2HPO4, 10 mM

HEPES, 5 mM NaHCO3, pH 7.4) containing 0 or 2.8 mM glucose for GLUTag and

INS-1 832/13 cells, respectively. After 1.5 hours of incubation, cells were imaged

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 Diabetes Page 8 of 37

with 490 nm excitation and 535 nm emission filter settings on a Zeiss LSM510

inverted confocal fluorescence microscope.

RNA isolation, quantitative real-time PCR and glutamate dehydrogenase (GDH)

activity. Total RNA was extracted from GLUTag and INS-1 832/13 cells using the

RNAeasy RNA purification kit (Qiagen, Hilden, Germany) according to

manufacturer’s protocol. RNA concentrations were determined using a NanoDrop

Spectrophotometer (Thermo Scientific, Waltham, MA). Equal quantities of total RNA

were reversely transcribed using RevertAid First-Strand cDNA synthesis kit

(Fermentas, Vilnius, Lithuania) in reactions containing 500 ng of total RNA.

Quantitative real-time PCR (q-PCR) was performed using the TaqMan gene

expression assay (glutamate dehydrogenase (GDH) isoform GLUD1, regulator of

GDH activity sirtuin-4 (SIRT4) and mitochondrial transcription factor A (TFAM)),

using a 7900HT Fast Real-Time System (Applied Biosystems, Foster City, CA). The

qPCR was carried out as previously described (25). Gene expression was quantified

by the comparative Ct method, in which the amount of target is expressed as 2-∆∆Ct

using hypoxanthine-guanine phosphoribosyl transferase (HPRT1) as reference gene.

GDH activity was measured as previously described in detail (26).

Colorectal infusion in mice. Fasted (2 hours) female C57BL/6 mice (n=24; Janvier

Labs, Rennes, France) were anesthetized with midazolam (0.4 mg/mouse;

Dormicum®; Hoffmann-La Roche, Basel, Switzerland) and 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 temperature and divided into

three groups. Retro orbital blood samples were taken at time zero with a Luer

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Page 9 of 37 Diabetes

capillary glass pipette rinsed in EDTA. Thereafter, mice were infused colorectally

(27) with 1 mL of either physiological NaCl (9 mg/L) (control), 10 mmol/L glutamine

or 10 mmol/L DMG in physiological NaCl. The concentration of secretagogues in our

study are in the same range as postprandial intestinal concentrations of the amino acid

(28). EGCG was infused at 1 mmol/L in 0.5 mmol/L ascorbic acid diluted in

physiological NaCl. The infusion rate was 1 mL/minute. After 10 minutes an

additional blood sample was taken. Blood samples from both time points were

centrifuged and plasma was separated from blood cells. Plasma samples were

immediately assayed for total GLP-1 (EMD Millipore, Merck Millipore, Billerica,

MA).

Statistical analysis. All data are presented as means ± S.E.M. for the indicated

number of experiments. Metabolite data were log2-transformed prior to assessment of

differences between groups by the paired Student’s t-test or ANOVA with

Bonferroni’s test post hoc when more than two groups were compared. Orthogonal

projection to latent structures discriminant analysis (OPLS-DA) was performed in

SIMCA 13.0 (Umetrics, Umeå, Sweden) on mean-centered and unit variance scaled

data.

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 Diabetes Page 10 of 37

RESULTS

Glucose-stimulated hormone secretion from INS-1 832/13 and GLUTag cells is

associated with increased mitochondrial metabolism. Glucose elicited secretion of

GLP-1 and insulin from GLUTag and INS-1 832/13 cells, respectively (Figure 1). To

further examine to which extent metabolism of the hexose is involved in this process

we profiled changes in metabolism elicited by glucose using gas chromatography /

mass spectrometry. To this end, we generated data on levels of amino acids, fatty

acids, glycolytic-, pentose phosphate pathway-, and tricarboxylic acid (TCA)-cycle

intermediates. Data were analyzed by OPLS-DA; one model was calculated for each

of the cell types with the glucose level as discriminating variable. Data were then

visualized in a shared and unique structures (SUS)-like plot (Figure 2A), derived from

the loadings, scaled as correlations, of the OPLS-DA models (29). The significance of

the loadings, obtained by jack-knifing, highlighted metabolites, the levels of which

were uniquely increased by glucose in GLUTag or INS-1 832/13 cells, as well as

changes in metabolite levels common to both cell-types. Clearly, glucose elicited

similar changes in metabolism in GLUTag and INS-1 832/13 cells. Thus, levels of

glycolytic- and TCA-cycle-intermediates increased in both cell-types (Figure 2B and

C). Glutamate levels increased 5-fold (p<0.001) and 1.3-fold (p<0.05) in GLUTag

and INS-1 832/13 cells, respectively. Aspartate levels decreased in both cell types;

0.6-fold (p<0.001) in GLUTag cells and 0.5-fold (p<0.001) in INS-1 832/13 cells.

Glycerol-3-phosphate levels increased after glucose stimulation only in the INS-1

832/13 cells (1.5-fold, p<0.001). Levels of glutamine were unaltered in both cell

types.

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Glutamine stimulates GLP-1 secretion from GLUTag cells in absence of the

glutamate dehydrogenase activator leucine. After having established that

mitochondrial metabolism is activated by glucose in both GLUTag and INS-1 832/13

cells, we investigated whether the cell lines responded similarly also to glutamine.

Glutamine alone was found to potently stimulate GLP-1 secretion from GLUTag

cells, whereas it was ineffective in stimulating insulin secretion from INS-1 832/13

cells (Figure 3A). Activation of GDH with leucine did not affect glutamine-induced

GLP-1 secretion, but permitted insulin secretion in response to glutamine. Leucine

alone did neither induce GLP-1 secretion from GLUTag cells, nor insulin secretion

from INS-1 832/13. These differences between cells was not caused by the differing

culturing conditions used, as INS-1 832/13 cells cultured for 48 h at 5.6 mM glucose

showed a similar pattern of insulin secretion as those cultured at 11.1 mM glucose

(Figure 3B)

Metabolite profiling revealed that addition of glutamine alone increased levels of

TCA-cycle intermediates in GLUTag cells. Notably, levels of these intermediates

were unaffected by leucine (Figure 3C). In INS-1 832/13 cells, on the other hand,

glutamine was largely ineffective in stimulating mitochondrial metabolism in the

absence of leucine (Figure 3D). Hence, these results suggest that GDH is active in the

GLUTag cells even in the absence of the allosteric activator leucine, which is required

for glutamine-activated TCA metabolism and hormone secretion in beta-cells.

Metabolism and hormone secretion in GLUTag and INS-1 832/13 cells

stimulated with a membrane-permeable glutamate analogue mirror those

elicited by glutamine. To further investigate whether glutamine-elicited hormone

secretion has a metabolic component, we stimulated GLUTag and INS-1 832/13 cells

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 Diabetes Page 12 of 37

with the membrane-permeable glutamate analogue DMG, which bypasses Na+-

coupled plasma membrane transporters. Similar to glutamine, DMG induced GLP-1

secretion from GLUTag cells independent of leucine, whereas leucine was required

for DMG to provoke insulin secretion from INS-1 832/13 cells (Figure 4A).

The metabolic response elicited by either glutamine or DMG in the GLUTag cells

was similar with regards to GLP-1 secretion and the increase in levels of TCA-cycle

intermediates, with the exception of aspartate (Figure 4B and Supplemental Figure

1,); the level of this metabolite increased to a greater extent when stimulated with

glutamine (2.4-fold by glutamine vs 1.7-fold by DMG, p<0.001). In INS-1 832/13

cells, on the other hand, glutamine and DMG failed to raise levels of TCA-cycle

intermediates in the absence of the allosteric GDH activator leucine (Figure 4C and

Supplemental Figure 1).

Glutamate dehydrogenase activity does not differ between lysed GLUTag and

INS-1 832/13 cells. Thus far, our results indicate that GDH activity differs between

GLUTag and INS-1 832/13 cells. To examine this further, we assessed GDH activity

in lysed cells. To reflect the conditions used in our previous experiments, we used

glutamine as substrate, which via deamination by glutaminase produces glutamate and

ammonia. Then, we determined NADH generated by the GDH-catalyzed oxidation of

glutamate to α-ketoglutarate. However, these analyses showed no difference in

activity between GLUTag and INS-1 832/13 cells, estimated as the glutamine elicited

AUC (Figure 5A).

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Glutamate dehydrogenase is activated in intact GLUTag cells. Next, we aimed to

determine enzyme activity in intact cells. As a measure of GDH activity linked to

TCA-cycle metabolism and oxidative phosphorylation, we monitored the oxygen

consumption rate (OCR) after stimulation with glutamine followed by leucine, or vice

versa. In GLUTag cells, leucine was ineffective in stimulating OCR, whereas

glutamine potently increased OCR, which did not increase further upon subsequent

addition of leucine (Figure 5B). In contrast, both leucine and glutamine were required

to elicit a robust increase in OCR in INS-1 832/13 cells (Figure 5C). These results

further support that GDH exists in an enhanced activity state in GLUTag cells.

Regulation of glutamate dehydrogenase activity. GDH activity is regulated both

allosterically, by e.g., amino acids and nucleotides, and covalently by ADP

ribosylation, catalyzed by SIRT4 (7; 30). Unexpectedly, the GLUD1/SIRT4

expression ratio was 55% (p<0.05) lower in GLUTag compared to INS-1 832/13 cells

(Figure 6A). Moreover, basal levels of ATP, reflecting the energy status of the cell,

were 2.9-fold (p<0.05) higher in GLUTag cells (Figure 6B). We also determined

cytosolic ATP/ADP-ratio using Perceval HR (Figure 6C). The maximal response

(Figure 6D) and AUC of the ATP/ADP-ratio (Figure 6E) in response to glutamine

stimulation was higher in GLUTag cells, as compared to INS-1 832/13. Addition of

leucine did not impact the ATP/ADP-ratio in either of the cell-types. Levels of ADP,

AMP, GMP, GDP and GTP did not differ between cell-types. Of note, basal levels of

leucine were 7.4-fold (p<0.01) higher in GLUTag cells than in INS-1 832/13 cells

(Figure 6F). Given leucine's role as allosteric activator of GDH, its higher basal level

may explain the increased GDH activity in the GLUTag cells.

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 Diabetes Page 14 of 37

Inhibition of glutamate dehydrogenase reduces secretion of GLP-1 in response to

glutamine and dimethylglutamate. To further examine the role of GDH in

glutamine-elicited GLP-1 secretion, we inhibited the enzyme using EGCG. Secretion

of GLP-1 in response to both glutamine and DMG was significantly reduced in the

presence of EGCG (Figure 7A). Notably, Gln was still effective in eliciting GLP-1

secretion in presence of the inhibitor (p<0.001), whereas DMG was not. Reduced

secretion of GLP-1 in response to the secretagogues in presence of EGCG was

paralleled by an almost complete abolishment of the glutamine- and DMG-elicited

increases in levels of TCA-cycle intermediates (Figure 7B-C).

Colorectal infusion of dimethylglutamate in mice elicits GLP-1 secretion. To gain

physiological support for our findings, we examined whether the membrane

permeable glutamate analogue DMG could affect GLP-1 secretion in vivo in mice.

Colorectal infusion of either glutamine or DMG yielded a 2.1-fold (p<0.05) and a 2.0-

fold (p<0.05), respectively, stronger stimulation of GLP-1 secretion as compared to

the control (Figure 7D). Inhibition of GDH by EGCG abrogated secretion of GLP-1

in response to DMG (Figure 7E). A schematic depiction of differences in stimulus-

secretion coupling between the L- and beta-cell is shown in Figure 8.

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DISCUSSION

Metabolic coupling in nutrient-stimulated insulin secretion from the pancreatic beta-

cell has been comprehensively studied. These studies have highlighted mitochondria

as key in the generation of signals that trigger and amplify secretion of the hormone.

The L-cell, on the other hand, is less well characterized, but studies have revealed

mechanisms similar to those in the beta-cell (15), as well as mechanisms that may be

unique to the L-cell (16). As opposed to the beta-cell, secretion of GLP-1 from the L-

cell has also been shown to be governed by sodium-coupled nutrient uptake, implying

an action essentially independent of intracellular metabolism of the secretagogue (15).

In the present study, we investigated the metabolic component of stimulus-secretion

coupling in the L-cell. To facilitate these analyses, we compared the L-cell model

GLUTag with the well established beta-cell model INS-1 832/13. Conditions were

selected from the literature (15; 18), and hence differ between GLUTag and INS-1

832/13 cells, but allow comparison with previous studies in these cells. Secretion of

insulin in response to glucose, glutamine and leucine was not altered when INS-1

832/13 cells were pre-cultured for 48 h at the lower glucose levels that were used for

the GLUTag cells. It must, however, be acknowledged that metabolism in these

immortalized cell-lines may not exactly mirror metabolism in vivo.

We found that exposure of GLUTag cells or L-cells in vivo to nutrients and stimuli

acting upstream and downstream of electrogenic transporters yielded qualitatively

similar results. Hence, our data support previous studies that have indicated a

metabolic component, involving ATP-production and KATP-channel closure,

analogous to that observed in the beta-cell, in L-cell glutamine- (31), glucose- (9; 15),

and fructose-sensing (15). It needs to be taken into account that glutamine has also

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 Diabetes Page 16 of 37

been suggested to act via a cAMP-dependent non-electrogenic sensing mechanism

(32).

It is possible that different sensing mechanisms may govern GLP-1 secretion from

different parts of the gastrointestinal tract. GLUTag cells are derived from colonic

tumors, and the in vivo administration of glutamine and DMG mainly targeted the

more distal segments of the gastrointestinal tract. Oral glucose tolerance tests

conducted in sodium-glucose linked transporter (SGLT1) knock out mice have

revealed a blunted early secretion of GLP-1 (33), but increased intestinal levels of

glucose to associate with an exaggerated late secretion of the hormone (34). Hence,

our data support that non-electrogenic nutrient uptake and sensing may play a more

important role in the more distal sections of the gastrointestinal tract.

Our results also provide evidence that glutamine-elicited GLP-1 secretion requires an

active GDH. When the enzyme was inhibited, both mitochondrial metabolism and

GLP-1 secretion became insensitive to DMG. However, whereas GLP-1 secretion

was significantly reduced in response to glutamine, the amino acid was still effective

in eliciting secretion of the hormone. This was paralleled by an almost abolished

response in mitochondrial metabolism, suggesting that glutamine may act via

additional mechanisms. This contrasts with the beta-cell, in which GDH activation by,

e.g. leucine, is necessary for glutamine-stimulated insulin secretion (19; 35-39). As a

matter of fact, mutations resulting in escape from nucleotide inhibition and

constitutive activation of GDH cause hyperinsulinemic hypoglycemia (7). Moreover,

GDH activity is also regulated by SIRT4 that inhibits GDH activity via ADP-

ribosylation (30). Hence, silencing of SIRT4 in beta-cells results in glutamine-

stimulated insulin secretion (40; 41). We could not find any differences in SIRT4

mRNA levels between GLUTag and INS-1 832/13 cells. Rather the relative levels of

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SIRT4 to GLUD1 were higher in the former, as was the overall energy state, reflected

by total cellular ATP levels. Instead, metabolite profiling revealed higher basal levels

of leucine in the GLUTag cells. It is possible that this elevation of basal leucine levels

serves to keep GDH in an active state in GLUTag cells, thereby promoting secretion

of GLP-1.

The ATP/ADP-ratio changed only slowly upon stimulation of GLUTag cells with

glutamine and did not increase significantly upon addition of leucine to INS-1 832/13

cells. Such slow responses are consistently observed and are likely due to

consumption of ATP by Ca2+-ATPases (42), and in this case also leucine-elicited

energy utilizing protein synthesis (43).

Whereas low GDH activity is a prerequisite for normal beta-cell function, the

opposite holds true for astrocytes (44). Astrocytes rely on GDH activity to enable

glutamate clearance and energy production required for glutamate uptake (45). In

humans, but not rodents, these cells express the GTP-insensitive GDH isoform

GLUD2, enabling these pivotal functions to operate also at a high energy state.

Notably, the neurotransmitter glutamate has been shown to be secreted from GLUTag

cells (46), as well as alpha- (47; 48), and beta-cells (49). Indeed, enteroendocrine cells

and cells of the nervous system share multiple features, which previously were

misinterpreted as a result of both cell lineages being derived from the neural crest

(50).

In conclusion, our data suggest that non-electrogenic nutrient uptake and metabolism

play an equally important role in nutrient sensing in colonic L-cells as they do in the

pancreatic beta-cells. An activated state of GDH is essential in the L-cells, whereas

activity of this enzyme needs to be inactivated in the beta-cell in the absence of

glucose. Our results support that glutamine and related amino acid analogues may

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offer a means of increasing GLP-1 levels without affecting insulin secretion directly

in vivo.

Acknowledgements: We thank Dr.. Daniel J. Drucker, Mount Sinai Hospital,

Toronto, Canada for permitting us to use the GLUTag cells.

Author contributions: LEA, LS, MAM, NV and MF designed and performed

experiments, analyzed data, interpreted results and edited the manuscript. CBA and

JAC designed and performed experiments and edited the manuscript. NW, HM and

CW provided intellectual guidance and co-wrote the paper. PS conceived and directed

the project, analyzed data, interpreted results, and wrote the first draft of the paper

together with LEA. PS is the guarantor of this work and, as such, had full access to all

the data in the study and takes responsibility for the integrity of the data and the

accuracy of the data analysis.

Duality of interests: The authors have no conflicts of interest to declare.

Funding: This work was supported by grants from Swedish Research Council, Novo

Nordisk-, Swedish Diabetes-, Crafoord-, Lars Hierta’s Minne-, Fredrik och Ingrid

Thuring’s-, O.E. och Edla Johansson’s Vetenskapliga-, Åke Wibergs-, Direktör

Albert Påhlssons-, Magnus Bergvalls-, Inga and John Hain's-, and Hjelt Foundations,

and the Royal Physiographic Society. Equipment grant from Knut and Alice

Wallenberg’s Foundations is acknowledged.

18
Page 19 of 37 Diabetes

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23. Malmgren S, Nicholls DG, Taneera J, Bacos K, Koeck T, Tamaddon A, Wibom R,
Groop L, Ling C, Mulder H, Sharoyko VV: Tight coupling between glucose and
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25. Krus U, Kotova O, Spegel P, Hallgard E, Sharoyko VV, Vedin A, Moritz T,
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27. Adrian TE, Gariballa S, Parekh KA, Thomas SA, Saadi H, Al Kaabi J, Nagelkerke
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Gottfries J, Moritz T, Trygg J: Visualization of GC/TOF-MS-based metabolomics
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Valenzuela DM, Yancopoulos GD, Karow M, Blander G, Wolberger C, Prolla TA,
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Page 21 of 37 Diabetes

31. Reimann F, Williams L, da Silva Xavier G, Rutter GA, Gribble FM: Glutamine
potently stimulates glucagon-like peptide-1 secretion from GLUTag cells.
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32. Tolhurst G, Zheng Y, Parker HE, Habib AM, Reimann F, Gribble FM: Glutamine
Triggers and Potentiates Glucagon-Like Peptide-1 Secretion by Raising Cytosolic
Ca2+ and cAMP. Endocrinology 2011;152:405-413
33. Gorboulev V, Schurmann A, Vallon V, Kipp H, Jaschke A, Klessen D, Friedrich
A, Scherneck S, Rieg T, Cunard R, Veyhl-Wichmann M, Srinivasan A, Balen D,
Breljak D, Rexhepaj R, Parker HE, Gribble FM, Reimann F, Lang F, Wiese S,
Sabolic I, Sendtner M, Koepsell H: Na(+)-D-glucose cotransporter SGLT1 is
pivotal for intestinal glucose absorption and glucose-dependent incretin
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34. Powell DR, DaCosta CM, Gay J, Ding ZM, Smith M, Greer J, Doree D, Jeter-Jones
S, Mseeh F, Rodriguez LA, Harris A, Buhring L, Platt KA, Vogel P, Brommage R,
Shadoan MK, Sands AT, Zambrowicz B: Improved glycemic control in mice
lacking Sglt1 and Sglt2. Am J Physiol Endocrinol Metab 2013;304:E117-130
35. Sener A, Somers G, Devis G, Malaisse WJ: The stimulus-secretion coupling of
amino acid-induced insulin release. Biosynthetic and secretory responses of rat
pancreatic islet to L-leucine and L-glutamine. Diabetologia 1981;21:135-142
36. Sener A, Malaisse WJ: L-leucine and a nonmetabolized analogue activate
pancreatic islet glutamate dehydrogenase. Nature 1980;288:187-189
37. Fahien LA, MacDonald MJ, Kmiotek EH, Mertz RJ, Fahien CM: Regulation of
insulin release by factors that also modify glutamate dehydrogenase. J Biol Chem
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38. Stanley CA: Hyperinsulinism/hyperammonemia syndrome: insights into the
regulatory role of glutamate dehydrogenase in ammonia metabolism. Molecular
Genetics and Metabolism 2004;81:S45-S51
39. Liu YJ, Cheng H, Drought H, MacDonald MJ, Sharp GW, Straub SG: Activation
of the KATP channel-independent signaling pathway by the nonhydrolyzable
analog of leucine, BCH. Am J Physiol Endocrinol Metab 2003;285:E380-389
40. Argmann C, Auwerx J: Insulin secretion: SIRT4 gets in on the act. Cell
2006;126:837-839
41. Maechler P, Carobbio S, Rubi B: In beta-cells, mitochondria integrate and
generate metabolic signals controlling insulin secretion. Int J Biochem Cell Biol
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42. Wiederkehr A, Park KS, Dupont O, Demaurex N, Pozzan T, Cline GW,
Wollheim CB: Matrix alkalinization: a novel mitochondrial signal for sustained
pancreatic beta-cell activation. EMBO J 2009;28:417-428
43. Yang J, Chi Y, Burkhardt BR, Guan Y, Wolf BA: Leucine metabolism in
regulation of insulin secretion from pancreatic beta cells. Nutr Rev 2010;68:270-
279
44. Schousboe A, Bak LK, Waagepetersen HS: Astrocytic Control of Biosynthesis
and Turnover of the Neurotransmitters Glutamate and GABA. Front Endocrinol
(Lausanne) 2013;4:102
45. Karaca M, Frigerio F, Migrenne S, Martin-Levilain J, Skytt DM, Pajecka K,
Martin-Del-Rio R, Gruetter R, Tamarit-Rodriguez J, Waagepetersen HS, Magnan C,
Maechler P: GDH-Dependent Glutamate Oxidation in the Brain Dictates
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46. Uehara S, Jung SK, Morimoto R, Arioka S, Miyaji T, Juge N, Hiasa M, Shimizu K,
Ishimura A, Otsuka M, Yamamoto A, Maechler P, Moriyama Y: Vesicular storage
and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal
intestinal L cells. J Neurochem 2006;96:550-560
47. Moriyama Y, Hayashi M: Glutamate-mediated signaling in the islets of
Langerhans: a thread entangled. Trends Pharmacol Sci 2003;24:511-517
48. Hayashi M, Yamada H, Uehara S, Morimoto R, Muroyama A, Yatsushiro S,
Takeda J, Yamamoto A, Moriyama Y: Secretory granule-mediated co-secretion of
L-glutamate and glucagon triggers glutamatergic signal transmission in islets of
Langerhans. J Biol Chem 2003;278:1966-1974
49. Feldmann N, del Rio RM, Gjinovci A, Tamarit-Rodriguez J, Wollheim CB,
Wiederkehr A: Reduction of plasma membrane glutamate transport potentiates
insulin but not glucagon secretion in pancreatic islet cells. Mol Cell Endocrinol
2011;338:46-57
50. Andrew A, Kramer B, Rawdon BB: The origin of gut and pancreatic
neuroendocrine (APUD) cells--the last word? J Pathol 1998;186:117-118

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Page 23 of 37 Diabetes

FIGURE LEGENDS

FIGURE 1 Glucose elicits hormone secretion from GLUTag and INS-1 832/13

cells. GLP-1 and insulin secretion from GLUTag and INS-1 832/13 cells,

respectively, at 0 mmol/L (white bars), 2.8 mmol/L (black bars), 10 mmol/L (grey

bars) and 16.7 mmol/L glucose (checkered bars). Data are expressed as means ±

S.E.M. for n=5. Differences between groups were assessed by ANOVA followed by

Bonferroni’s test post hoc, *p<0.05, **p<0.01, ***p<0.001.

FIGURE 2 Glucose elicits similar changes in metabolism in GLUTag and INS-

1 832/13 cells. (A) SUS-like plot derived from 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 and top decrease

and increase, respectively after glucose stimulation of INS-1 832/13 cells. Hence,

glucose elicits increased levels of metabolites found in the top right section and

decreased levels of those found in the lower left corner in both cell types (shared).

Metabolites in the central right and left section increase and decrease, respectively,

uniquely in GLUTag cells. Metabolites in the central top and lower sections increase

and decrease, respectively, uniquely in INS-1 832/13 cells. The shared and unique

fields are indicated by + for increase, - for decrease and 0 for unaltered for

GLUTag/INS-1 832/13. Only significantly altered metabolites are named. (B) Plots of

relative levels of metabolites in central glucose metabolism in GLUTag cells at basal

(white bars) and stimulatory glucose (black bars). (C) Plots of relative levels of

23
 Diabetes Page 24 of 37

metabolites in INS-1 832/13 cells at basal (white bars) and stimulatory glucose (black

bars) levels. Data are expressed as means ± S.E.M. for n=4. Differences between

groups were assessed by the paired Student's t-test on log transformed data, *p<0.05,

**p<0.01, ***p<0.001. AKGA, alpha-ketoglutarate; DiHAP, dihydroxy

acetonephosphate; Cit, citrate; Creat, creatinine; Fruct, fructose; FruP, fructose 1- and

6-phosphate; Fum, fumarate; Glu6P, glucose 6-phosphate; Gly2P, glycerol 2-

phosphate; Gly3P, glycerol 3-phosphate; GlyA3P, 3-phosphoglycerate; IsoCit,

isocitrate; Lac, lactate; Mal, malate; pGlu, pyroglutamate; Pyr, pyridine; Succ,

succinate; Udi, unknown disacharide.

FIGURE 3 Glutamine stimulates mitochondrial metabolism and GLP-1

secretion from GLUTag cells in the absence of leucine, whereas leucine is

required for glutamine-elicited mitochondrial metabolism and insulin secretion

from INS-1 832/13 cells. (A) GLP-1 and insulin secretion 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; white bars), 10 mmol/L leucine (black bars), 10 mmol/L glutamine

(grey 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 mM glucose for 48

h as in those cultured at 11.1 mM glucose. (C) Plots of relative levels of metabolites

involved in mitochondrial metabolism in GLUTag cells 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 each of glutamine and leucine (grey 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

24
Page 25 of 37 Diabetes

glutamine (black bars) or the combination of 10 mmol/L each of glutamine and

leucine (grey bars). Data are expressed as means ± S.E.M. for (A) n=5 (GLUTag),

n=14 (INS-1 832/13) and (B-D) n=4. Differences between groups were assessed by

ANOVA followed by Bonferroni's test post hoc on log transformed data, *p<0.05,

**p<0.01, ***p<0.001. Abbreviations as in Figure 1.

FIGURE 4 Dimethyl glutamate-elicited metabolism and hormone secretion

mirrors that of glutamine in GLUTag and INS-1 832/13 cells. (A) GLP-1 and

insulin secretion 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; white bars), 10 mmol/L

leucine (black bars), 10 mmol/L dimethyl glutamate (DMG, grey bars), and the

combination of 10 mmol/L leucine and 10 mmol/L DMG (checkered bars). (B) Plots

of relative levels of metabolites involved in mitochondrial metabolism in GLUTag

cells at basal (0 mmol/L glucose, white bars) and after stimulation with 10 mmol/L

DMG (black bars) or the combination of 10 mmol/L each of DMG and leucine (grey

bars). (C) 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 DMG (black bars) or the combination of 10 mmol/L each of DMG

and leucine (grey bars). Data are expressed as means ± S.E.M. for 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 between groups were assessed by ANOVA followed by

Bonferroni’s test post hoc, *p<0.05, **p<0.01, ***p<0.001.

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 Diabetes Page 26 of 37

FIGURE 5 Glutamate dehydrogenase activity does not differ between lysed

GLUTag and INS-1 832/13 cells but is activated in intact GLUTag cells. (A)

Glutamate dehydrogenase activity in GLUTag and INS-1 832/13 cells monitored in

cell extracts as NADH fluorescence. Glutamine (1mM) and purified GDH was added

as indicated by the arrows (B) Oxygen consumption rate (OCR) in GLUTag cells

after stimulation with glutamine (10 mmol/L) and leucine (10 mmol/L). Leucine was

added either before (black symbols) or after (grey symbols) glutamine. The

combination of leucine and glutamine does not increase OCR above the level

observed for glutamine alone. (C) OCR in response to glutamine and leucine in INS-1

832/13 cells. The combination of glutamine and leucine potently stimulates OCR.

Data are expressed as means ± S.E.M. for n=4. Differences between groups and the

basal condition were assessed by the paired Student's t-test, *p<0.05, **p<0.01,

***p<0.001.

FIGURE 6 Levels of factors regulating glutamate dehydrogenase activity

differ between INS-1 832/13 and GLUTag cells. (A) mRNA levels of Sirt4 and

Glud1, normalized to TFAM in GLUTag (white bars) and INS-1 832/13 (black bars)

cells. (B) Basal ATP levels, 0 mmol/L glucose for GLUTag (white bars) and 2.8

mmol/L glucose for INS-1 832/13 (black bars). (C) Cytosolic ATP/ADP-ratio

assessed by Perceval HR. The maximum response (D), and AUC (E) after glutamine

(10 mM) stimulation was higher in GLUTag compared to INS-1 832/13 cells. (F)

Basal levels of leucine in GLUTag (white bars) and INS-1 832/13 cells (black bars).

Data are expressed as means ± S.E.M. for n=3 (A,B,F) and n=184 and n=212 single

cells for GLUTag and INS-1 832/13, respectively, in (C-E). Differences between

26
Page 27 of 37 Diabetes

groups were assessed by ANOVA followed by Bonferroni’s test post hoc (A) and the

unpaired Student´s t-test (B-F), *p<0.05, ***p<0.001.

FIGURE 7 GDH is essential in glutamine- and dimethylglutamate-elicited

GLP-1 secretion. (A) Inhibition of glutamate dehydrogenase (GDH) in GLUTag

cells with epigallocatechin (EGCG, 20 µmol/L) abolishes secretion of GLP-1 in

response to glutamine (Gln, 10 mmol/L) and dimethylglutamate (DMG, 10 mmol/L).

Inhibition of GDH with EGCG is associated with a blunted response in levels of

TCA-cycle intermediates in response to (B) glutamine and (C) dimethylglutamate.

(D) Colorectal infusion of glutamine (Gln, 10 mmol/L) and dimethylglutamate

(DMG; 10 mmol/L,) elicits 2-fold higher GLP-1 secretion as compared to control

(Ctrl, 0.9% NaCl) 10 min post infusion (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 are expressed as means ± S.E.M. for n=4 in (A-C) and n=9 (number of mice;

control), n=8 (glutamine) and n=7 (DMG) in (D), and n=8 in (E). Differences

between groups were assessed by the Student's t-test (E) or ANOVA followed by

Bonferroni’s test post hoc. $p<0.05, $$p<0.01, $$$p<0.001, versus control, and

*p<0.05, **p<0.01, ***p<0.001, versus indicated condition.

FIGURE 8 Glutamine and dimethylglutamate metabolism in L-cells and beta-

cells. In the L-cell, glutamine elicits increased TCA-cycle metabolism, mitochondrial

respiration and GLP-1 secretion. In the beta-cell, on the other hand, 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

27
 Diabetes Page 28 of 37

ADP. Thus, glutamine stimulated GLP-1 secretion from the L-cell is governed by an

active GDH, whereas this enzyme is inactive in the beta-cell. Dimethylglutamate

(DMG), which bypasses sodium-coupled transporters, and glutamine yield

qualitatively similar responses in both cell types. Hence, non-electrogenic nutrient

uptake and metabolism plays an important role in stimulus-secretion coupling in both

the L-cell and the beta-cell.

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Page 29 of 37 Diabetes

Figure  1  

500 600
**
* ***
*
400 ***

Insulin secretion
GLP-1 secretion
(pmol/mg/hr)

400

(µU/mg/hr)
300

200
200
100

0 0
GLUTag INS-1 832/13
0 mM glucose 10 mM glucose
2.8 mM glucose 16.7 mM glucose
 Diabetes Page 30 of 37

Figure 2

GlyA3P AKGA +/+  

(A) FruP
IsoCit Cit
INS-1 832/13 low vs high glucose.p(corr)[1]

0/+   Mal
Fum Glu6P
DiHAP Ala
Gly3P
0,6 Gly2P Succ Udi Glu pGlu

Pyr Ser
0,2 Fruct
-­‐/0   +/0  

-0,2

Lac
-0,6 Pro
0/-­‐  
Asp Creat
-­‐/-­‐  
-1
-1 -0,8 -0,6 -0,4 -0,2 0 0,2 0,4 0,6 0,8
GLUTag low vs high glucose.p(corr)[1]

(B)
0 mM glucose 10 mM glucose
14 5 16 7
***
*** *** 14 *** ***
12 6
4 *** ***
Fold to 0 mM glucose

12
10 *** 5
3 10 ***
8 4
*** ** 8
6 2 3
6
4 *** 2
4
1 *** ***
2 2 * 1

0 0 0 0

(C)
2.8 mM glucose 16.7 mM glucose
100 4 25 3
***
Fold to 2.8 mM glucose

80 *** 20 ***
*** 3
2
60 15
** ***
2 *
40 10 ***
*** *** 1
1 ***
20 5
*
0 0 0 0
Page 31 of 37 Diabetes

Figure 3

(A) (B)
1000 1000 1200
** ***
**
800 800
1000

Insulin secretion
Insulin secretion
GLP-1 secretion

800 ***
(pmol/mg/hr)

(µU/mg/hr)
(µU/mg/hr)
600 600
***
600
400 400
400
200 200 ***
200
0 0
0
GLUTag INS-1 832/13

Gln+Leu
Gln

Leu
Ctrl

Glucose
Basal glucose 10 mM glutamine
10 mM leucine 10 mM glutamine + 10 mM leucine

(C)
0 mM glucose 10 mM glutamine 10 mM glutamine + 10 mM leucine
20 45
18 *** 40 ***
***
16 35
Fold to 0 mM glucose

14 ***
30 ***
12 ***
25
10
20
8 ***
*** 15
6 ***
***
*** *** 10
4 **
**
* 5 ***
2 ***
0 0
Cit IsoC AKGA Succ Fum Mal Asp Glu Gln

(D)
2.8 mM glucose 10 mM glutamine 10 mM glutamine + 10 mM leucine
12 35
*** ***
30
Fold to 2.8 mM glucose

10 ***
25
8
20
6
15
*** *** ***
4 *** *** 10 *** ***
** ***
**
2 5

0 0
Cit IsoC AKGA Succ Fum Mal Asp Glu Gln
 Diabetes Page 32 of 37

Figure 4
(A)
800 4000
**
**
600 *** 3000

Insulin secretion
GLP-1 secretion
(pmol/mg/hr)

(µU/mg/hr)
400 2000

200 1000

0 0
GLUTag INS-1 832/13
Basal glucose 10 mM DMG
10 mM leucine 10 mM DMG + 10 mM leucine

(B) (C)
0 mM glucose 10 mM DMG 2.8 mM glucose 10 mM DMG
10 mM DMG + 10 mM leucine 10 mM DMG + 10 mM leucine
18 10
****** ***
16 9
Fold to 2.8 mM glucose
Fold to 0 mM glucose

14 8
*** 7
12
*** *** 6 ***
10 ***
5
8 *** ***
*** 4 *** ***
6 ***
*** ** *** 3 ***
4 * *** *** ***
2
* *
2 * 1
0 0
Page 33 of 37 Diabetes
 Diabetes Page 34 of 37

Figure 6
(A) (B)

8 180
Gene expression ratio to Tfam

Glutag
6 INS-1 832/13

ATP levels (mmol/mg)

4 135
2 *
0.05 90
0.04
0.03 *
45
0.02
0.01
0.00 0
Sirt4 Glud1

(C) (D) (E) (F)

GLUTag
10 mM Leu INS-1 832/13
Ctrl
4 mM FCCP
2.0 0.6 3¥ 10 02
D max perceval HR 520 emission

21
Perceval HR emission 520 nm

Oligomycin

Log2 (Basal leucine levels)

AUC Perceval HR (a.u)

10 mM Gln

1.5 0.4 2¥ 10 02

18
*** ***
***
1.0 0.2 1¥ 10 02

GLUTag

INS-1 832/13
0.5 0.0 0 15
0 200 400 600 800 1000
Time [s]
Page 35 of 37 Diabetes

Figure 7
(A) (B)
-EGCG +EGCG
1200 200
** *
Ctrl
GLP-1 (pmol//L/mg protein)

$$$
1000 $ Gln

Metabolite levels (AU)

150
$$
* Gln+EGCG
800

600 100
$$
**
NS
400
50 $$
**
200
$$
**
0 0
Ctrl Gln DMG Fum Mal IsoCit Cit
(C) (D)
80
$$ ** Ctrl
0 min
20 *
DMG
Metabolite levels (AU)

60 DMG+EGCG 10 min

$
* 16 *
GLP-1 (pmol/L)

40

$
* 12
20
8
0

Fum Mal IsoCit Cit 4

(E)
0
Ctrl Gln DMG
1.5
**
GLP-1 secretion (fold to 0 min)

1.0

0.5

0.0
DMG DMG+EGCG
 Diabetes Page 36 of 37

Figure 8

GLUTag INS-1 832/13

Na+ Na+
Glucose DMG Glutamine Glucose DMG Glutamine
SGLT1/2 GLUT1/2

Glucose Glucose

Glutamine Glutamine
Glycolysis Glycolysis
DMG DMG

Pyruvate Pyruvate
Glutamate Glutamate

GDH X GDH
TCA TCA α-KG
α-KG
cycle cycle

GTP ADP GTP ADP

ATP Leu ATP Leu
SIRT4 SIRT4
OxPhos OxPhos

Mitochondrion Mitochondrion

GLP-1 Insulin
Page 37 of 37 Diabetes

Supplemental  Figure  S1  

(A) 0/+   +/+  

Orn Mal
pGlu Glu Fum
1 Put
Ile GlyA3P IsoCit Pro AKGA
Gly Cit Ala Asp
DMG.p(corr)[1]

0,5 Thr Val

GABA
FruP
InosP
0 -­‐/0   +/0  

Creat
-0,5
0/-­‐  
-­‐/-­‐  
-1
-1 -0,8 -0,6 -0,4 -0,2 0 0,2 0,4 0,6 0,8

Gln.p(corr)[1]
(B)

+/+  
0/+  
pGlu Glu
1 Ala Asp
Pro GlyA3P
DMG.p(corr)[1]

0,5
GABA

0 Orn
-­‐/0   IsoCit +/0  
Ser
Creat
-0,5 Thr

-­‐/-­‐   0/-­‐  
-1
-1 -0,5 0 0,5 1

Gln.p(corr)[1]

Dimethyl  glutamate-­‐elicited  metabolism  mirrors  the  impact  of  glutamine  in  GLUTag  and  INS-­‐1  
832/13  cells.  SUS-­‐like  plots  derived  from  OPLS-­‐DA  models  discrimina>ng  between  GLUTag  (A)  
or  INS-­‐1  832/13  (B)  cells  s>mulated  with  glutamine  or  DMG,  compared  to  basal  glucose  (0  
mmol/L  for  GLUTag  and  2.8  mmol/L  for  INS-­‐1  832/13).  The  plot  is  interpreted  as  described  in  
Figure  2A.