Rune E. Kuhre, Charlotte R. Frost, Berit Svendsen, and Jens J. Holst

Molecular Mechanisms ofGlucose-Stimulated GLP-1Secretion From Perfused RatSmall IntestineDiabetes 2015;64:370–382 | DOI: 10.2337/db14-0807

Glucose is an important stimulus for glucagon-likepeptide 1 (GLP-1) secretion, but the mechanisms of secre-tion have not been investigated in integrated physiologicalmodels. We studied glucose-stimulated GLP-1 secretionfrom isolated perfused rat small intestine. Luminal glucose(5% and 20% w/v) stimulated the secretion dose depen-dently, but vascular glucose was without significant effectat 5, 10, 15, and 25 mmol/L. GLP-1 stimulation by luminalglucose (20%) secretion was blocked by the voltage-gatedCa channel inhibitor, nifedipine, or by hyperpolarization withdiazoxide. Luminal administration (20%) of the nonmetabo-lizable sodium-glucose transporter 1 (SGLT1) substrate,methyl-a-D-glucopyranoside (a-MGP), stimulated release,whereas the SGLT1 inhibitor phloridzin (luminally) abolishedresponses to a-MGP and glucose. Furthermore, in theabsence of luminal NaCl, luminal glucose (20%) did notstimulate a response. Luminal glucose-stimulated GLP-1secretion was also sensitive to luminal GLUT2 inhibition(phloretin), but in contrast to SGLT1 inhibition, phloretindid not eliminate the response, and luminal glucose (20%)stimulated larger GLP-1 responses than luminal a-MGP inmatched concentrations. Glucose transported by GLUT2may act after metabolization, closing KATP channels sim-ilar to sulfonylureas, which also stimulated secretion. Ourdata indicate that SGLT1 activity is the driving force forglucose-stimulated GLP-1 secretion and that KATP-channelclosure is required to stimulate a full-blown glucose-induced response.

Glucagon-like peptide 1 (GLP-1) is an incretin hormonewith many effects that can be exploited for the treatment

of type 2 diabetes and obesity (1). As an incretin, GLP-1potentiates glucose-induced insulin secretion, and it haslong been known that intake of macronutrients (triglycer-ides, proteins, and carbohydrates) stimulates GLP-1 secre-tion. Among these, the effects of glucose on the incretinsystem have been studied in the greatest detail, and glu-cose appears to be one of the most effective incretinstimuli in humans and rodents (2–4). On the basis of stud-ies of the GLP-1–expressing cell lines, GLUTag, NCI-H716,and STC-1 (5–7), primary L cells and cultures of dispersedintestinal cells (8), several molecular mechanisms havebeen suggested to link glucose exposure to GLP-1 secretion.These include 1) sweet taste receptors, which are expressedin GLUTag cells and in primary murine L cells (8,9);2) electrogenic uptake through the sodium-glucose trans-porter 1 (SGLT1), which couples uptake of one glucosemolecule and two Na ions and causes cell depolarization(10,11); and 3) by electroneutral GLUT2-mediated glucoseuptake with KATP-channel closure secondary to intracellu-lar glucose metabolism causing cell depolarization by pre-venting leakage of K ions. Because intestinal epithelialcells in culture have lost their polarity and all relevantparacrine and neural influences, whether these findingstranslate to intact physiological systems is uncertain;however, the mechanisms of secretion appear to be acti-vated in the gut before glucose has reached the systemiccirculation because glucose stimulates GLP-1 secretionfrom isolated canine ileal loops (12). For instance, it isuncertain whether glucose stimulates GLP-1 secretion byapically or basolaterally activated mechanisms and it is

Novo Nordisk Foundation Center for Basic Metabolic Research and Department ofBiomedical Sciences, the Panum Institute, University of Copenhagen, Copenhagen,Denmark

Corresponding author: Jens J. Holst, jjholst@sund.ku.dk.

Received 21 May 2014 and accepted 15 August 2014.

© 2015 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, andthe work is not altered.

See accompanying article, p. 338.

370 Diabetes Volume 64, February 2015

METABOLISM

equally uncertain to what extent the enteric nervous sys-tem, which is known to have a synergistic role on GLP-1secretion, may affect the suggested mechanisms of secre-tion outlined above.

GLUT2 is predominantly located on the basolateralside of the enterocyte but may be recruited to the apicalsurface upon the presence of glucose in the lumen(13,14). Theoretically, glucose could therefore stimulateGLP-1 secretion by basolateral GLUT2-facilitated glucoseuptake rather than or in addition to apical SGLT1- and/orGLUT2-mediated uptake. In contrast, SGLT1 has not beenlocalized to the basolateral side of enterocytes or L cells,making this scenario unlikely for SGLT1–mediated glucose-stimulated GLP-1 release. The aim of the current studywas, therefore, to investigate the mechanisms of glucose-stimulated GLP-1 secretion in a more physiological setting,which allows discrimination between apical and basolateralactivated mechanisms. To achieve this, we studied theeffects of glucose on the isolated perfused rat small intes-tine. The advantage of this preparation is that cell polarityis retained, nerve and blood vessel connections are intact,and the local cell environment (including contact withneighboring cells) is undisturbed, meaning that data fromthis model are more likely to be transferrable to intactphysiological systems. We examined the effects of vascular(intra-arterial) glucose and luminal glucose stimulation andthe importance of intracellular glucose metabolism, KATP-channel closure, voltage-sensitive Na (NaV) and voltage-gated(V-gated) Ca channels, sweet taste receptors, facilita-tive (GLUT2-mediated) glucose uptake, and electrogenic(SGLT1)-mediated glucose uptake for GLP-1 secretion.

RESEARCH DESIGN AND METHODS

Animals and Perfusion ProtocolStudies were conducted with permission from the DanishAnimal Experiments Inspectorate (2013-15-2934-00833)and the local ethics committee (EMED, P-12-099 and P-13-240) in accordance with the guidelines of Danishlegislation governing animal experimentation (1987) andthe National Institutes of Health (publication number 85-23). Male Wistar rats (;250 g) were obtained fromTaconic (Ejby, Denmark) and housed two per cage, withad libitum access to standard chow and water, and kepton a 12:12-h light-dark cycle.

Nonfasted rats were anesthetized by subcutaneousinjection with Hypnorm/midazolam (0.079 mg fentanylcitrate + 2.5 mg fluanisone + 1.25 mg midazolam) andplaced on a 37°C heated table. The abdominal cavity wasopened, and the entire large intestine and approximatelytwo-thirds of the small intestine were carefully removed,leaving the most proximal part of the small intestine(;32 cm) in situ. The intestinal length retained variedlittle (coefficient of variation = 9.70%) between experi-ments. A plastic tube was placed in the lumen, the in-testinal contents were carefully removed by flushingwith isotonic (0.9%; 0.31 Osmol/L) NaCl (room tempera-ture), and the intestine was luminally perfused with

a steady flow of NaCl (0.250 mL/min). A catheter wasinserted into the cranial/superior mesenteric artery, andthe intestine was vascularly perfused (7.5 mL/min) withperfusion buffer gassed with 95% O2 and 5% CO2 (Krebs-Ringer bicarbonate buffer femented with 0.1% BSA, 5% dex-tran T-70 [Pharmacosmos, Holbaek, Denmark], 3.5 mmol/Lglucose, 10 mmol/L 3-isobutyl-1-methylxanthine, and 5mmol/L pyruvate, fumarate, and glutamate; pH 7.4). Theperfusion medium was collected through a metal catheterplaced in the hepatic portal vein, and the rat was killed byperforation of the diaphragm as soon as proper flow wasapparent. The perfusion buffer was warmed (37°C) by pass-ing it through a Uniper UP-100 perfusion system (HugoSachs; Harvard Apparatus, March-Hugstetten, Germany),and perfusion pressure was measured continuously.

StimulantsThe luminal stimuli were glucose (5% or 20% [w/v]); 0.36and 1.42 Osmol/L, respectively), mannitol (20% [w/v];1.41 Osmol/L), or the nonmetabolizable sugar, methyl-a-D-glucopyranoside (a-MGP; 20% [w/v]; 1.34 Osmol/L),perfused in presence or absence of the SGLT1 inhibitorphloridzin (10 mmol/L) or the GLUT2 inhibitor phloretin(1 mmol/L) applied to the lumen. Moreover, the lumenwas stimulated with the artificial sweeteners sucraloseand acesulfame K, which are 800 and 260 times sweeterthan glucose, respectively, meaning that the applied con-centrations of 0.25% (w/v) sucralose and 0.78% (w/v) ace-sulfame K were each equivalent to a sweetness 10 timesgreater than 20% (w/v) glucose. For all luminal stimulationexperiments except one (luminal glucose with and withoutNaCl), stimulants were diluted in isotonic saline (0.9% w/v)applied at an initial rate of 2.5 mL/min for the first 2 minto replace the saline solution in the lumen and then at0.250 mL/min throughout the rest of the stimulation pe-riod. Immediately after each stimulation, the lumen wasflushed with a similar bolus of 0.9% NaCl (2.5 mL/min) for2 min, followed by infusion at a flow rate of 0.250 mL/minbetween stimulations.

In the luminal glucose with and without the NaClexperiment, the gut was perfused as outlined above, butimmediately after the first glucose stimulation and untilthe end of the second glucose stimulation, isotonic salinewas replaced with NaCl-deprived Milli-Q water balancedwith iso-osmolar KCl (0.11% [w/v]). In other experiments,the gut was vascularly (arterially) perfused with KCl (50mmol/L), the KATP-closing sulfonylureas tolbutamide (500mmol/L) or gliclazide (500 mmol/L), the KATP-channelopener diazoxide (500 mmol/L), the NaV channel blockerlidocaine (100 mmol/L), the NaV activator veratridine(10 mmol/L), or the V-gated Ca channel blocker nifedipine(10 mmol/L). Additional experiments assessed GLP-1 re-sponses when glucose was administered intra-arterially atphysiological (5 mmol/L and 10 mmol/L) and pathophys-iological (15 mmol/L and 25 mmol/L) concentrations.Bombesin (10 nmol/L; BBS), a known GLP-1 secre-tagogue, was included in all experiments as a positive

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control. All drugs were obtained from Sigma-Aldrich(Brøndby, Denmark). Drugs applied to the luminal sideof the gut were diluted in 0.9% NaCl, and stimulants forthe vascular side were diluted in perfusion buffer. Toaid solubility, diazoxide, gliclazide, lidocaine, nifedipine,phloretin, and tolbutamide were first dissolved in DMSOand then diluted further in perfusion medium, with DMSOconcentrations never exceeding a final concentration of0.1%, which did not cause GLP-1 secretion in controlexperiments (n = 6; data not shown).

After an equilibration period of 30 min, effluentsamples were collected each minute, immediately chilledon ice, and transferred to 220°C within 30 min. In ran-domly chosen experiments, PO2, PCO2, lactate concentra-tions, and pH were measured with a Radiometer ABL 700blood gas analyzer (Radiometer Copenhagen, Copenha-gen, Denmark) in perfusate samples collected from thearterial and venous sides of the preparation at the startand end of the experiment, respectively. The intestine wasexcised and stored in formaldehyde (4°C) for later histo-logical examination (embedded in paraffin and stainedwith hematoxylin and eosin, as described previously[15]). The perfused preparations showed regular peristal-tic activity (recorded as changes in perfusion pressure),which increased with respect to frequency and amplitudein response to stimulation with BBS, suggesting preservedenteric nervous function.

Biochemical MeasurementsVenous effluent glucose concentration was measured bythe glucose-oxidase method using the hand-held Accu-Chek Compact Plus meter (Roche, Mannheim, Germany).Effluent GLP-1 concentrations were measured usinga total GLP-1 radioimmunoassay (measuring intact pep-tide + the primary metabolite, GLP-1 [9–36amide]) usingantiserum code no. 89390, recognizing the amidated C-terminus of the molecule, as described previously (16).Because the gut was perfused at a constant rate, concen-trations parallel output (= secretion). The choice of tar-geting the amidated (x-36amide) rather than the glycineextended (x-37) isoform was based on a recent studyshowing that GLP-1 is predominantly amidated in rats(17). The experimental detection limit was 1 pmol/L forthe 89390 assay, with an intra-assay coefficient of varia-tion of 6%.

Statistical AnalysisData are expressed as means 6 1 SEM. Responses wereassessed by comparing averaged concentrations during thestimulation period with mean basal levels from a period ofsimilar duration: immediately before the stimulation (5–8consecutive 1-min observations) and at the end of thefollowing equilibrium period (5–8 observations). The re-sponse period was defined as the period from the startof stimulant application until GLP-1 concentrations wereback to baseline levels. Statistical significance was assessedby paired t test; P , 0.05 was considered significant.

RESULTS

Luminal Glucose Stimulates GLP-1 Secretion Fromthe Perfused Rat Small Intestine in a Dose-and Absorption-Dependent MannerLuminal stimulation of the intestine with 5% (w/v)glucose caused a small, but significant, elevation inglucose and GLP-1 (total) concentrations in the venouseffluent compared with basal levels (glucose: from 3.53 60.1 to 4.42 6 0.3 mmol/L, GLP-1: from 14.9 6 0.5 to20.2 6 0.6 pmol/L, P , 0.0001, n = 6; Fig. 1A–C), withgreater responses being elicited by 20% (w/v) glucose(glucose: from 4.46 6 0.1 to 7.87 6 0.4 mmol/L, GLP-1: from 19.6 6 0.9 to 39.5 6 2.6 pmol/L, P , 0.0001, n =6; Fig. 1E–G). A positive linear relationship was foundbetween venous glucose and GLP-1 concentrations forboth treatments (R2 = 0.68 for 5% [w/v] glucose, R2 =0.87 for 20% [w/v] glucose, P , 0.001; n = 6; Fig. 1Dand H). Control experiments demonstrated similarly en-hanced GLP-1 responses to two consecutive stimulationperiods with luminal glucose (20% [w/v]) compared withbasal levels (stimulation 1: from 22.7 6 1.2 to 42.3 6 2.3pmol/L, P , 0.0001; stimulation 2: from 27.1 6 0.8 to43.2 6 2.5 pmol/L, P , 0.0001; P . 0.05 for stimulation1 vs. stimulation 2, n = 6; Fig. 2A and B). The responseswere not related to osmolality, because luminally admin-istered mannitol (20% [w/v]) did not stimulate secretion(from 14.6 6 0.5 to 14.8 6 0.4, P = 0.73, n = 6; Fig. 2Eand F). All experiments included administration of BBS asa positive control, in each case resulting in a robust GLP-1response (Figs. 1–7).

L-Cell Depolarization Causes GLP-1 Secretion byActivation of V-Gated Ca ChannelsIn the initial experiments, we assessed whether celldepolarization causes GLP-1 secretion from the perfusedrat intestine. Vascular administration of KCl increasedGLP-1 secretion (from 8.60 6 0.4 to 47.8 6 13 pmol/L,P, 0.05, n = 6), but in the presence of the V-gated (V-type)Ca channel inhibitor nifedipine, the response was mark-edly reduced (from 7.83 6 0.5 to 15.3 6 1.4 pmol/L, P ,0.01; Fig. 3C and D) and significantly smaller than with-out nifedipine (P , 0.05). Control studies showed thatthe secretory responses to two consecutive KCl stimula-tions resulted in responses that both were significantlydifferent from their respective baselines (stimulation 1:from 12.7 6 1.7 to 42.2 6 12.3 pmol/L, stimulation 2:from 6.29 6 3.7 to 22.2 6 4.1 pmol/L, both P , 0.05)but were not significantly different from each other (P =0.08; Fig. 3A and B), although the second response tendedto be smaller. Moreover, BBS-induced secretion wasunaffected by nifedipine (BBS: from 13.9 6 1.0 to65.6 6 10 pmol/L, BBS + nifedipine: from 12.7 6 0.3to 49.9 6 8.9 pmol/L, P , 0.05, n = 6; Fig. 3E and F).

Glucose Stimulates GLP-1 Secretion by NaV-Independent Activation of V-Gated Ca ChannelsThe effect of luminal glucose (20% [w/v]) was also lost inthe presence of nifedipine (glucose: from 9.19 6 0.6 to

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25.1 6 0.6 pmol/L, P , 0.01; glucose + nifedipine: from7.90 6 0.6 to 10.6 6 1.9 pmol/L, P . 0.05, n = 6; Fig. 2Cand D). The addition of the hyperpolarizing agent diazoxidealso abolished the GLP-1 response to luminal glucose (20%[w/v] glucose: from 12.0 6 0.6 to 31.8 6 1.9 pmol/L, P ,0.0001; glucose + diazoxide: from 16.9 6 1.5 to 14.6 6 0.5,P . 0.05, n = 6; Fig. 3G and H).

We next investigated the importance of NaV channelactivation for GLP-1 secretion by stimulating the gutwith luminal glucose (20% [w/v]) in the presence orabsence of lidocaine, an analgesic that blocks NaV chan-nels (18). Again, luminal glucose (20% [w/v]) stimu-lated GLP-1 secretion (from 14.8 6 1.2 to 26.7 6 2.7pmol/L, P , 0.0001), but the response was fully main-tained in the presence of lidocaine (from 15.0 6 1.5 to27.1 6 2.0 pmol/L, P , 0.0001, n = 6; Fig. 4A and B).Control experiments confirmed that this was not dueto inadequate NaV-channel blockade, because channelactivation by veratridine (19) caused GLP-1 secretion(veratridine: from 16.8 6 0.5 to 33.5 6 7.3, P , 0.01,n = 6), but not in the presence of lidocaine (veratridine +lidocaine: from 18.3 6 1.1 to 22.9 6 0.6, P . 0.05, n = 6;Fig. 4C and D). In other experiments, veratridine-stimulated GLP-1 secretion (from 10.4 6 0.4 to 22.3 64.2 pmol/L, P , 0.05) was abolished by costimulationwith nifedipine (from 11.6 6 0.6 to 13.9 6 1.1 pmol/L,

P . 0.05, n = 6; Fig. 4E and F), indicating that theresponse to Na+ channel activation also involved theV-gated Ca channels.

Glucose Stimulates GLP-1 Secretion by SGLT1 UptakeWe next investigated the role of Na-coupled glucoseuptake through SGLT1 for GLP-1 secretion. Luminalglucose 20% (w/v) stimulated a GLP-1 response (glucose:from 12.5 6 0.3 to 21.1 6 2.0 pmol/L, P , 0.01) but notin the absence of luminal NaCl (glucose 2 NaCl: 12.5 60.3 to 12.7 6 0.4, P . 0.05, n = 6; Fig. 5A and B). Inother experiments, 20% (w/v) luminal glucose resulted ina robust GLP-1 response (glucose: from 17.3 6 0.4 to37.8 6 2.1 pmol/L, P , 0.0001), but this was completelylost in the presence of the SGLT1 inhibitor, phloridzin(glucose + phloridzin: from 20.1 6 1.0 to 20.9 6 0.9pmol/L, P . 0.05, n = 6; Fig. 5C and D). Perfusion ofthe gut with the nonmetabolizable SGLT1 substrate,a-MGP, also significantly increased GLP-1 secretion(from 15.7 6 0.5 to 29.4 6 0.7 pmol/L, P , 0.0001,n = 6), which could be completely blocked by the admin-istration of phloridzin (from 17.5 6 0.7 to 17.7 6 0.4pmol/L, P . 0.05, n = 6; Fig. 5E and F). When applied inthe same experiment, luminal glucose and luminal a-MGP(both 20% [w/v]) both stimulated GLP-1 secretion (glu-cose: from 14.6 6 1.1 to 30.6 6 2.9, a-MGP: 16.1 6 0.5

Figure 1—Glucose stimulates GLP-1 secretion from the perfused rat intestine by a dose- and absorption-dependent manner. Data areshown as means 6 1 SEM. GLP-1 total concentrations (pmol/L) (black line) and venous glucose concentrations (mmol/L) (blue line)are shown in response to 5% (w/v) luminal glucose (A) or 20% (w/v) luminal glucose (E). Averaged GLP-1 total concentrations (pmol/L) are shownat baseline and in response to 5% (w/v) glucose (B) or 20% (w/v) glucose (F). Averaged venous glucose concentrations (pmol/L) are shown atbaseline and in response to 5% (w/v) glucose (C) or 20% (w/v) glucose (G). D and H: Correlation analysis of venous GLP-1 (total) concentration(pmol/L) and venous glucose concentrations (mmol/L). BBS was included at the end of the experiments as a positive control. Statisticalsignificance between stimulant and baseline levels was tested by paired t test. ****P < 0.0001 (n = 6).

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to 22.0 6 0.8, both P , 0.001, n = 6), but the response toglucose was significantly higher than the response toa-MGP (P , 0.01; Fig. 6G and H).

Glucose Stimulates GLP-1 Secretion by GLUT2-Mediated Uptake and KATP-Channel ClosureWe next investigated the effect of GLUT2 inhibition onglucose-stimulated GLP-1 secretion. Phloretin, a widelyused GLUT2-specific inhibitor (20,21), significantly re-duced, but did not eliminate, glucose-stimulated GLP-1 se-cretion (glucose: from 13.9 6 0.6 to 30.9 6 2.8 pmol/L,glucose + phloretin: from 12.4 6 1.3 to 17.2 6 3.4 pmol/L,P , 0.001 for both stimulations compared with respectivebaselines, P , 0.0001 between responses, n = 6; Fig. 6Aand B), whereas the secretory response to a-MGP was notsensitive to phloretin (a-MGP: from 14.1 6 0.3 to 29.4 62.4 pmol/L, a-MGP + phloretin: from 14.76 0.4 to 25.161.0 pmol/L, P , 0.0001 between responses and respectivebaselines, P . 0.05 between responses; Fig. 5G and H).

To investigate the consequences of KATP-channel clo-sure, we stimulated the gut with the two sulfonylureadrugs: gliclazide and tolbutamide. Both drugs significantly

elevated GLP-1 levels compared with basal levels (tolbu-tamide: from 14.7 6 0.2 to 28.6 6 5.2 pmol/L, gliclazide:from 15.2 6 0.3 to 24.6 6 2.4 pmol/L, P , 0.05, n = 8;Fig. 6C and D). Furthermore, glucose-stimulated GLP-1secretion was lost by blockage of ATP synthesis with2,4-dinitrophenol (glucose: from 7.49 6 0.7 to 23.5 63.0 pmol/L, P , 0.0001, glucose + 2,4-dinitrophenol:from 9.24 6 1.3 to 6.61 6 0.4 pmol/L, P . 0.05, n =6; Fig. 6E and F).

Neither Vascular Glucose nor Sweet Taste Receptor–Activating Compounds Are Major GLP-1 StimuliIntravascularly administered glucose within the physio-logical range (5 and 10 mmol/L) had no effect on GLP-1secretion (P . 0.05), but a minor increase was observedat the 15 mmol/L concentration (from 21.7 6 0.5 to25.2 6 0.6 pmol/L, P , 0.001, n = 6; Fig. 7A and B).However, 25 mmol/L vascular glucose concentrationswere ineffective in a separate experiment (P . 0.05,n = 6; data not shown). Some reports have indicatedthat sweet taste receptor activation causes GLP-1 secretion.In our hands, however, neither sucralose (0.25% w/v)

Figure 2—Luminal glucose-stimulated GLP-1 secretion involves V-gated Ca channel activation. GLP-1 total concentrations and averagedGLP-1 total concentrations at baseline and stimulation are shown as means 6 1 SEM in response to two subsequent stimulations with20% (w/v) luminal glucose (A and B), 20% (w/v) luminal glucose with and without vascular nifedipine (10 mmol/L) (C and D), and 20% (w/v)mannitol (osmolarity control) (E and F ). BBS was included at the end of all experiments as a positive control. ****P < 0.0001 betweenstimulant and baseline, ####P < 0.0001 between stimulations (n = 6).

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nor acesulfame K (0.78% w/v) increased GLP-1 secretion(sucralose: from 16.9 6 0.4 to 16.5 6 0.4 pmol/L,acesulfame K: from 17.9 6 0.5 to 16.7 6 0.3 pmol/L,P . 0.05, n = 6) when administered luminally (Fig. 7Cand D).

DISCUSSION

It has long been known that oral glucose intake stimulatesGLP-1 secretion in humans, but detailed analysis of themechanisms involved, including discrimination betweenapically and basolaterally activated mechanisms is, of

Figure 3—GLP-1 is secreted in response to L-cell depolarization and V-gated Ca channel activation and by depolarization-independentactivation of phospholipase C pathways, but hyperpolarization abolishes glucose-induced GLP-1 secretion. GLP-1 total concentrationsand averaged GLP-1 total concentrations at baseline and stimulation are shown as means 6 1 SEM in response to two subsequentvascular KCl (50 mmol/L) stimulations (A and B), vascular KCl (50 mmol/L) with and without vascular nifedipine (10 mmol/L) (C and D),vascular BBS with and without vascular nifedipine (10 mmol/L) (E and F ), and 20% (w/v) luminal glucose with and without vascular diazoxide(500 mmol/L) (G and H). BBS was included at the end of all experiments as a positive control. Statistical significance between stimulant andbaseline or between stimulants was tested by paired t test. *P < 0.05, **P < 0.01, ****P < 0.0001 between stimulant and baseline; #P <0.05, ####P < 0.0001 between stimulations (n = 6).

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course, not possible in vivo. Significant insight into themolecular mechanisms of GLP-1 secretion has beengained from studies of GLP-1–secreting cell lines and,more recently, through studies of primary L cells andprimary murine gut epithelial cell cultures. According tothe classical model of intestinal glucose absorption, glu-cose crosses the apical membrane of the enterocyte viaSGLT1 and exits across the basolateral membranethrough GLUT2. More recent evidence indicates, however,that the presence of glucose in the lumen recruits GLUT2to the apical side of the enterocyte within minutes, andduring assimilation of a meal, this component of absorp-tion may be several times greater than the active SGLT1–mediated absorption, as reviewed (13,14). Because GLUTagand primary murine L cells express both SGLT1 andGLUT2 transporters (8), glucose may, therefore, stimu-late GLP-1 secretion by a basolateral uptake secondary tointestinal glucose absorption (by neighboring entero-cytes) rather than by apical mechanisms of the L cells

themselves, activated by luminal glucose. We, therefore,investigated if vascular glucose stimulated GLP-1 secre-tion and the role of apical SGLT1 and GLUT2 activity forluminal glucose-stimulated GLP-1 secretion. Vascular glu-cose was without effect in physiological concentrationsand did not consistently stimulate secretion at supraphys-iological doses, whereas luminal glucose robustly anddose-dependently stimulated secretion.

Glucose absorption has been shown to be more impor-tant for GLP-1 secretion than the mere presence ofglucose in the lumen (22). We therefore investigated theeffect of luminal NaCl depletion (substituting Na with K).In absence of NaCl, the stimulatory effect of luminalglucose was completely lost, suggesting that the mecha-nisms of secretion depend on Na-coupled uptake. A sim-ilar observation was made by another group studyingperfused rat ileum (23). Indeed, glucose uptake bySGLT1 was both necessary and sufficient to cause glucose-stimulated GLP-1 secretion because a-MGP and glucose

Figure 4—NaV activation stimulates GLP-1 secretion by V-gated Ca channel activation. GLP-1 total concentrations and averaged GLP-1total concentrations at baseline and stimulation are shown as means 6 1 SEM in response to 20% (w/v) luminal glucose with and withoutvascular lidocaine (100 mmol/L) (A and B), vascular veratridine (10 mmol/L) with and without vascular lidocaine (100 mmol/L) (C and D), andvascular veratridine (10 mmol/L) with and without vascular nifedipine (10 mmol/L) (E and F ). BBS was included at the end of all experimentsas a positive control. *P < 0.05, ****P < 0.0001 between stimulant and baseline (n = 6).

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both caused GLP-1 secretion, whereas blockade of the trans-porter (with phloridzin) completely abolished the responseto both secretagogues. Consistent with this, studies fromother groups on GLUTag cells and primary mouse intestinecultures have shown that a-MGP and glucose both stimu-late phloridzin-sensitive GLP-1 secretion and that a-MGPalso stimulates GLP-1 secretion after oral administration inmice, whereas glucose did not stimulate GLP-1 secretion inSGLT12/2 mice (8,11,24–27).

However, in our model (Fig. 8), glucose-stimulatedGLP-1 secretion was also sensitive to apical GLUT2 block-age, but in contrast to SGLT1 inhibition, phloretin didnot eliminate the response to luminal glucose, althoughthe applied dose was sufficient to inhibit GLUT2 activityin other models (21,27). In support of this, glucose-stimulated GLP-1 secretion to an oral gavage has beenfound reduced, but not abolished, in GLUT22/2 knockoutmice compared with wild-type littermates, and the same

Figure 5—SGLT1 activity is both sufficient and necessary for glucose to induce GLP-1 secretion. GLP-1 total concentrations andaveraged GLP-1 total concentrations at baseline and stimulation are shown as means 6 1 SEM in response to 20% (w/v) luminalglucose with and without NaCl (A and B), 20% (w/v) luminal glucose with and without luminal phloridzin (10 mmol/L) (C and D),a-MGP with and without luminal phloridzin (10 mmol/L) (E and F ), and 20% (w/v) luminal a-MGP with and without luminal phloretin(1 mmol/L) (G and H). BBS was included at the end of all experiments as positive control. **P < 0.01, ****P < 0.0001 between stimulantand baseline; ##P < 0.01, ####P < 0.0001 between stimulations (n = 6).

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pattern has been observed in studies on GLUTag cells(27,28). Our results therefore are in good agreementwith work performed on isolated/single cells, but onlyour approach allows analysis of the differential effectscaused by apical or basolateral GLUT2 inhibition. Al-though SGLT1-mediated transport involves cotransportof two Na ions for each glucose molecule, glucose uptake

via GLUT2 is electroneutral. In this case, secretion mightdepend on glucose metabolism and formation of ATP,with actions on the KATP channels, similar to glucose-activation of pancreatic b-cells. Indeed, expression analy-sis in primary murine L cells (and GLP-1–secreting celllines) indicated that L cells do express the KATP-channelsubunits SUR1 and Kir6.2 and that tolbutamide also

Figure 6—Glucose stimulates GLP-1 secretion by GLUT2-mediated uptake, causing closure of KATP channels, and KATP-channel closure isrequired for glucose to stimulate a full-blown response. GLP-1 total concentrations and averaged GLP-1 total concentrations at baselineand stimulation are shown as means 6 1 SEM in response to 20% (w/v) luminal glucose with and without luminal phloretin (1 mmol/L) (Aand B), vascular tolbutamide or vascular gliclazide (both 500 mmol/L) (C and D), 20% (w/v) luminal glucose with and without vascular2,4-dinitrophenol (E and F ), and luminal glucose and luminal a-MGP (both 20% [w/v]) (G and H). BBS was included at the end of allexperiments as a positive control. *P < 0.05, ***P < 0.001, ****P < 0.0001 between stimulant and baseline; ##P < 0.01, ####P < 0.0001between stimulations (n = 6, A, B, E, and F; n = 8, C and D).

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stimulates GLP-1 secretion from primary dispersed mu-rine cultures and GLUTag cells (8,29). In contrast, inhumans, sulfonylureas do not seem to influence GLP-1secretion (27), although SUR1 was found colocalizedwith GLP-1 in human L cells (30). Like the b-cells, humanL cells also express the rate-limiting glucose sensor, glu-cokinase, but glucose-stimulated GLP-1 secretion is notreduced in individuals with glucokinase mutations(MODY 2) (31–33), although these individuals requireabnormally high glucose concentrations to generate ade-quate metabolism-dependent insulin secretion.

We therefore investigated the effects of high doses ofsulfonylureas on the perfused gut. Tolbutamide andgliclazide both stimulated GLP-1 secretion, supportingthat KATP-channel closure does stimulate GLP-1 secretionfrom the murine L cell in a physiological setting. Althoughnot investigated in present study, the discrepancy be-tween our findings and the lack of GLP-1 response tosulfonylureas in humans could be related to the level ofKATP-channel expression in L cells. Thus, the KATP-channelsubunits Kir6.2 and SUR1 are expressed to a greater de-gree in isolated murine b-cells compared with isolatedmurine L cells and GLUTag and STC-1 cells (8). It is, there-fore, possible that the therapeutic doses of sulfonylureasused to control blood sugar levels in type 2 diabeticpatients may not be high enough to have demonstrableeffects on the L cell in vivo. Furthermore, the lack of effectof glucokinase mutations may simply reflect a predominantrole of SGLT1 in GLP-1 secretion after oral glucose intake.However, at high concentrations, KATP-channel closure,

subsequent to L-cell glucose uptake, may function to fur-ther enhance SGLT1-driven secretion. Indeed, we foundthat when the rat gut was luminally stimulated withsimilarly high concentrations (20% [w/v]) of a-MGP andglucose, respectively, in the same experiments, glucosestimulated GLP-1 secretion to a larger extent thana-MGP.

Studies of GLUTag and primary L cells have shown thatL cells are electrically excitable, firing action potentials inresponse to glucose (8,11). Propagation of the actionpotentials across the membranes of the L cells wouldthen result in depolarization and influx of Ca throughV-gated Ca channels. Action potentials cannot easily berecorded from L cells in our preparation, but we used lido-caine to examine the consequences of blocking the NaVchannels responsible for the action potentials. This, how-ever, did not affect luminal glucose-induced GLP-1 se-cretion, although the applied dose has been shownto powerfully inhibit channel activity in other studies(18,34). This is consistent with reported findings onGLUTag cells (35). Furthermore, we used veratridine toinvestigate the secretory responses to NaV-channel activa-tion. Veratridine powerfully stimulated GLP-1 secretion,and this response could be blocked by the same doses oflidocaine, suggesting that they were indeed sufficient to in-hibit channel activity. A probable consequence of Na entryafter NaV-channel activation and entry via SGLT1 would bedepolarization, activation of V-gated Ca channels, and se-cretion. This concept was supported by the observation thatnifedipine abolished veratridine-induced GLP-1 secretion.

Figure 7—Vascular glucose or sweet taste receptor activation does not cause GLP-1 release. GLP-1 total concentrations and averagedGLP-1 total concentrations at baseline and stimulation are shown as means6 1 SEM in response to vascular glucose at 5, 10, and 15 mmol/L(A and B) and to 0.25% (w/v) luminal sucralose and 0.78% (w/v) luminal acesulfame K (C and D). BBS was included at the end of allexperiments as a positive control. ***P < 0.001 (n = 6).

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To directly investigate the influence of depolarizationfor GLP-1 secretion in our preparation, we added 50mmol/L KCl to the perfusion medium. This caused clearGLP-1 secretion, which could be abolished by nifedipine,indicating that cell depolarization causes GLP-1 secretionby V-gated Ca-channel activation and uptake of Ca fromthe extracellular space. In further support of this concept,nifedipine and diazoxide (a KATP-channel opener, causingefflux of K and hyperpolarization of the cell) both abol-ished glucose-stimulated GLP-1 secretion. In contrast, ni-fedipine did not affect the secretory responses to thephospholipase C activator, BBS, which stimulates GLP-1secretion by mobilization of Ca from intracellular storesrather than by depolarization. Thus, in the isolated per-fused rat intestine, glucose-stimulated GLP-1 release doesnot depend on firing of action potentials, generated byactivation of NaV channels, but does require cell mem-brane depolarization and activation of L-type Ca channelswith subsequent entry of extracellular Ca2+. Diazoxidealso has inhibitory effects on insulin secretion from thepancreatic b-cell, and because of this effect was previouslyused to treat hypoglycemia. From our data it may, how-ever, be speculated diazoxide may also have inhibitedGLP-1 secretion in the human subjects receiving this treat-ment, but to our knowledge, no data are available on this.

GLUTag cells and primary murine L cells have beenshown to express the sweet taste receptor subunits T1R2and T1R3 as well as the signaling molecule gustducin.Combined with reports demonstrating that artificialsweeteners cause GLP-1 secretion in GLUTag cells andmice (9,36), this has led to the suggestion that the L cellmay “taste” sugars and secrete GLP-1 in response (37). Inour hands, however, the artificial sweeteners sucraloseand acesulfame K (at doses corresponding to 10 timesthe sweetness of a 20% [w/v] glucose solution) did notenhance GLP-1 secretion, and our study, therefore, doesnot support the view that glucose (or other sugars) shouldstimulate GLP-1 secretion by activation of sweet taste recep-tors, in agreement with evidence from other studies by severalindependent groups on primary murine intestinal cultures inrodents or humans (8,24,38,39). It might be argued that thesweet taste receptors might generate afferent vagal signalsthat eventually lead to a reflex-generated GLP-1 response in-volving the central nervous system. In our preparation, thiswould not be possible. However, there is little evidence thatGLP-1 secretion is vagally regulated (40), and, as mentioned,the artificial sweeteners are ineffective in humans.

Taken together, our data show that glucose most likelystimulates GLP-1 secretion from the perfused rat smallintestine by SGLT1-mediated uptake from the lumen,

Figure 8—Suggested model for glucose-stimulated GLP-1 release. Glucose stimulates GLP-1 release by luminal uptake by 1) electrogenicSGLT1, which couples the uptake of one glucose molecule to two Na ions and causes cell membrane depolarization per se, and by 2) KATP-channel closure secondary to uptake through SGLT1 and GLUT2, causing cell membrane depolarization by intracellular metabolism to ATPand closure of ATP-sensitive K channels (KATP). Both pathways stimulate secretion by activation of voltage-gated Ca channels (V-type),whereas further enhancement of depolarization by NaV activation does not seem to be essential, causing uptake of extracellular Ca and Ca-induced mobilization of Ca from intracellular stores, collectively causing activation of the exocytotic machinery and GLP-1 secretion. CICR,Ca-induced Ca release.

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causing GLP-1 secretion by activation of V-gated Cachannels through coupled Na uptake and depolarization.However, glucose may also stimulate GLP-1 secretion viaelectroneutral GLUT2-mediated uptake with subsequentmetabolism, ATP formation, and KATP-channel closure.This hypothesis is supported by the elimination of theGLP-1 response after administration of the oxidativephosphorylation blocker 2,4-dinitrophenol. However, be-cause GLUT2 inhibition, in contrast to SGLT1 inhibition,did not completely abolish glucose-stimulated GLP-1 se-cretion, KATP-channel closure may function to enhanceSGLT1-driven GLP-1 secretion when glucose concentrationsare high. Our data therefore indicate that SGLT1-mediateduptake is the major activator of glucose-stimulated GLP-1secretion and that closure of KATP channels is required tostimulate a full-blown response to glucose.

Acknowledgments. The authors thank Carolyn F. Deacon (Novo NordiskFoundation Center for Basic Metabolic Research, Department of Biomedical Sci-ences, the Panum Institute, University of Copenhagen) for editing the manuscript,Nicolai Jacob Wever Albrechtsen (also at Novo Nordisk Foundation Center forBasic Metabolic Research) for many fruitful discussions, and Musa Büyükuslu(Parenkymkirurgisk afsnit, Sydvestjysk Sygehus, Denmark) for making Fig. 8.Funding. The study was supported by grants from the Novo Nordisk Centerfor Basic Metabolic Research (Novo Nordisk Foundation, Denmark) and theEuropean Union’s Seventh Framework Programme for Research, TechnologicalDevelopment, and Demonstration Activities (Grant No. 266408).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. R.E.K. and J.J.H. contributed to the conceptionand design of the study and interpreted results. R.E.K. performed experiments,analyzed data, prepared figures, and drafted the manuscript. R.E.K., C.R.F., B.S.,and J.J.H. approved final version of the manuscript. C.R.F. and B.S. developedthe study model. J.J.H. supervised experiments and edited and revised themanuscript. J.J.H. is the guarantor of this work and, as such, had full accessto all the data in the study and takes responsibility for the integrity of the data enthe accuracy of the data analysis.Prior Presentation. The main findings of this study were presented at the74th Scientific Sessions of the American Diabetes Association, San Francisco,CA, 13–17 June 2014.

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