Predominant role of active versus facilitative glucose transport for glucagon-like peptide-1 secretion

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1 Dietologi (22) :2 2 DOI.7/s ARTICLE Predominnt role of ctive versus fcilittive glucose trnsport for glucgon-like peptide- secretion H. E. Prker & A. Adrienssens & G. Rogers & P. Richrds & H. Koepsell & F. Reimnn & F. M. Grile Received: 7 Ferury 22 /Accepted: 2 April 22 /Pulished online: 2 My 22 # The Author(s) 22. This rticle is pulished with open ccess t Springerlink.com Astrct Aims/hypothesis Severl glucose-sensing pthwys hve een implicted in glucose-triggered secretion of glucgonlike peptide- (GLP-) from intestinl L cells. One involves glucose metolism nd closure of ATP-sensitive K chnnels, nd nother exploits the electrogenic nture of N - coupled glucose trnsporters (SGLTs). This study imed to elucidte the role of these distinct mechnisms in glucosestimulted GLP- secretion. Methods uptke into L cells (either GLUTg cells or cells in primry cultures, using new trnsgenic mouse model comining proglucgon promoter-driven Cre recominse with ROSA2tdRFP reporter) ws monitored with the FLII 2 Pglu-7μδ glucose sensor. Effects of phrmcologicl nd genetic interference with SGLT or fcilittive glucose trnsport (GLUT) on intrcellulr glucose ccumultion nd metolism (mesured y NAD(P)H utofluorescence), cytosolic C 2 (monitored with Fur2) nd GLP- secretion (ssyed y ELISA) were ssessed. Results L cell glucose uptke ws dominted y GLUTmedited trnsport, eing olished y phloretin ut not Electronic supplementry mteril The online version of this rticle (doi:.7/s ) contins peer-reviewed ut unedited supplementry mteril, which is ville to uthorised users. H. E. Prker : A. Adrienssens : G. Rogers : P. Richrds : F. Reimnn () : F. M. Grile () Cmridge Institute for Medicl Reserch, Wellcome Trust/MRC Building, Addenrooke s Hospitl, Box 39, Hills Rod, Cmridge CB2 XY, UK e-mil: fmg23@cm.c.uk e-mil: fr222@cm.c.uk H. Koepsell Institute of Antomy nd Cell Biology, University of Würzurg, Würzurg, Germny phloridzin. NAD(P)H utofluorescence ws glucose dependent nd enhnced y glucokinse ctivtor. In GLUTg cells, ut not primry L cells, phloretin prtilly impired glucose-dependent secretion, nd suppressed n mplifying effect of glucose under depolrising high K conditions. The key importnce of SGLT in GLUTg nd primry cells ws evident from the impirment of secretion y phloridzin or Sglt knockdown nd filure of glucose to trigger cytosolic C 2 elevtion in primry L cells from Sglt knockout mice. Conclusions/interprettion SGLT cts s the luminl glucose sensor in L cells, ut intrcellulr glucose concentrtions re lrgely determined y GLUT ctivity. Although L cell glucose metolism depends prtilly on glucokinse ctivity, this plys only minor role in glucose-stimulted GLP- secretion. Keywords Glucgon-like peptide- (GLP-). Glucokinse. K ATP chnnel. L cells. SGLT Arevitions BAC Bcteril rtificil chromosome CFP Cyn fluorescent protein CMV Cytomeglovirus FRET Förster resonnce energy trnsfer GIP -dependent insulinotropic polypeptide GKA Glucokinse ctivtor GLP- Glucgon-like peptide- K ATP ATP-sensitive potssium chnnel αmg α-methyl-d-glucopyrnoside NMDG N-Methyl-D-glucmine RFP Red fluorescent protein SGLT Sodium-dependent glucose trnsporter SI Smll intestine tdrfp Tndem red fluorescent protein YFP Yellow fluorescent protein

2 2 Dietologi (22) :2 2 Introduction Glucgon-like peptide- (GLP-) is n incretin hormone secreted from intestinl L cells, locted throughout the gut epithelium prticulrly in the ileum nd colon []. GLP- ugments insulin secretion in glucose-dependent mnner, nd, together with glucose-dependent insulinotropic polypeptide (GIP), is responsile for up to 7% of the insulin response to food intke in helthy individuls [2, 3]. As GLP- dditionlly inhiits glucgon secretion, slows gstric emptying nd enhnces stiety, it is n ttrctive trget for phrmceuticl interventions. Recent therpies tht increse GLP- ctivity, y the use of degrdtion-resistnt mimetics or inhiition of GLP- clevge y dipeptidyl peptidse IV, improve glycemi in ptients with type 2 dietes, demonstrting the success of trgeting the GLP- xis. Current interest in studying pthwys underlying GLP- relese could led to new therpeutic strtegies for incresing endogenous GLP- secretion. GLP- relese is triggered y ingestion of crohydrtes, fts nd protein, nd is elieved to reflect, t lest in prt, the direct sensing of luminl nutrients vi the picl processes of L cells. The relese of GLP- nd GIP fter glucose ingestion ccounts for the incretin effect, chrcterised y enhnced insulin relese triggered y orl compred with intrvenous glucose [, ]. This difference cn most esily e explined y luminl glucose sensor, shielded from vritions in the plsm glucose concentrtion. The identity of this sensor for detecting luminl sugrs hs een much deted ut not fully elucidted. Initil studies using the GLUTg cell line suggested tht L cells my use the clssicl glucose-sensing mchinery used y the pncretic et cell, involving glucose phosphoryltion y glucokinse, enhnced glycolytic nd mitochondril metolism, nd closure of ATP-sensitive potssium (K ATP ) chnnels []. More recent studies confirmed tht the K ATP chnnel suunits, Kir.2 nd SUR, nd Glucokinse re expressed t high levels in purified mouse L cells nd tht the proteins re detectle y immunostining in humn Lcells[7 9]. Electrophysiologicl nd secretion studies hve demonstrted tht K ATP chnnels re functionl in murine L cells nd tht sulfonylures cn stimulte GLP- secretion from primry colonic cultures [7]. An element of glucose sensing y L cells is, however, clerly independent of metolism, s GLP- secretion is lso stimulted y non-metolisle sugrs such s methylα-glucopyrnoside (αmg) nd 3-O-methylglucose in whole nimls [], intestinl preprtions [], GLUTg cells nd primry intestinl cultures [7, 2].Erlyinvivostudies showed tht luminl sugr stimultion of GLP- secretion is N dependent nd tht the specificity of the response prlleled the sugr specificity of N -coupled glucose trnsport [, 3]. This led to the identifiction of distinct glucose-sensing pthwy in L cells, resulting from the ctivity of N -coupled glucose trnsporters (SGLTs) [2]. These concomitntly crry N ions for ech glucose molecule trnsported, therey generting smll depolrising currents sufficient to trigger electricl ctivity nd C 2 entry nd consequent incresed GLP- secretion. As the reltive importnce of SGLTs, intrcellulr glucose levels nd metolism for glucose-dependent GLP- secretion is uncler, we imed to further investigte the roles of these mechnisms in determining L cell glucose fluxes nd GLP- relese. Methods Animl models Animl procedures were pproved y the locl ethics committee nd conformed with UK Home Office regultions. Sglt / mice [] on C7BL/ ckground were crossed with GLU-Venus trnsgenic mice [7]. Sglt / nd Sglt / littermtes received glucose/glctose-reduced diet (Altromin, Lge, Germny). Lelling of intestinl L cells with red fluorescent protein (RFP) ws chieved y crossing Ros2tdRFP reporter mice []with mice expressingcre recominse under the control of the proglucgon promoter (GLU-Cre2 mice). GLU-Cre2 mice were creted using construct sed on the cteril rtificil chromosome (BAC) RP23-33C7 (Children s Hospitl Oklnd Reserch Institute, Oklnd, CA, USA) in which the sequence etween the proglucgon strt codon in exon 2 nd the stop codon in exon ws replced y icre using Red/ET recomintion technology (Generidges, Heidelerg, Germny) (see electronic supplementry mteril [ESM Methods/Tle ] for more detils). Tissue culture Intestines from 3- to -month-old mice were collected, nd the epithelil cells cultured s descried previously [7]. The upper ( top third) smll intestine (SI) comprised cm length distl to the stomch, nd the colon ws tken distl to the ileocolic junction. Aliquots were plted on to 2-well pltes or 3 mm glss-ottomed dishes (MtTek, Ashlnd, MA, USA) coted with Mtrigel (BD Biosciences, Oxford, UK) for 2 8 h nd incuted t 37 C in % CO 2. GLUTg cells were cultured s descried previously [, 7]. Intrcellulr glucose mesurements The FLII 2 Pglu- 7μδ Förster resonnce energy trnsfer (FRET) glucose sensor [8] ws cloned into pshuttle-cmv (Qiogene, Crlsd, CA, USA) for genertion of denoviruses [9]. GLUTg cells were trnsfected with pcdna3. contining the glucose sensor under cytomeglovirus (CMV) promoter control (Addgene, Cmridge, MA, USA), using Lipofectmine 2 (Invitrogen, Pisley, UK). Cells were then seeded

3 Dietologi (22) : on to Mtrigel-coted glss-ottomed dishes nd imged 2 8 h lter. Before imging, GLUTg cells were incuted in sline uffer (see elow) for min t room temperture. Twody-old primry colonic cultures from GLU-Cre- tdrfp mice were trnsduced with denovirus encoding the FLII 2 Pglu-7μδ glucose sensor nd imged 72 h lter. Before ech experiment, colonic cultures were incuted in forskolin ( μmol/l) nd 3-isoutyl--methylxnthine ( μmol/l) for 3 min t 37 C. L cells were identified y their RFP fluorescence nd chrcteristic morphology. FRET imging ws performed using n inverted fluorescence microscope (Nikon Eclipse TE2-S or Olympus IX7) with oil immersion ojective. The FRET proe ws excited every s t 3/ nm using 7 W xenon rc lmp nd monochromtor (Cirn Reserch, Fvershm, UK) controlled y MetFluor softwre (Moleculr Devices, Wokinghm, UK). Emission ws recorded with CCD cmer (QuntEM Photometrics, Tucson, AZ, USA or Orc-ER, Hmmtsu Photonics, Welwyn Grden City, UK) ehind n Optosplit II imge splitter (Cirn Reserch) equipped with cyn fluorescent protein (CFP) nd yellow fluorescent protein (YFP) emission filter sets. Fluorescence ws recorded from individul cells, ckground corrected nd expressed s the rtio YFP/CFP. For nlysis, dt were verged over 2 s, nd pek responses normlised y dividing y the pretretment seline. Immunohistochemistry Tissues were fixed with % (w/v) prformldehyde for 2 h, cryoprotected in 3% sucrose nd emedded in Optiml Cutting Temperture compound (OCT; CellPth, Newton, UK). Tissue sections (8 μm) were permeilised with.% Tween, locked with 3% BSA for h, nd incuted with :3 diluted glucgon ntiody (ctlogue no. sc-39, Snt Cruz Biotechnology, Snt Cruz, CA, USA) or : diluted Glut2 ntiody (ctlogue no. sc-78; Snt Cruz) overnight t room temperture. Tissues were then incuted for h t room temperture with Alex 88-conjugted got nti-rit ntiody or donkey nti-got Alex Fluor 33 (:3 dilution; Invitrogen) nd Hoechst stin. Tissue smples stined with secondry ntiody lone served s controls. Imges were cptured using either n inverted fluorescence (Olympus IX7) or confocl (Zeiss LSM) microscope nd processed using Volocity nd MetFluor softwre. Assy of glucokinse ctivity Glucokinse ctivity ws determined using n dpted pyridine nucleotide-coupled ssy [2]. Briefly, 3 7 GLUTg cells were homogenised in uffer contining (in mmol/l) 2 Hepes, KCl, 2 dithiothreitol nd EDTA (ph 7.), nd the resultnt superntnt frction ws mixed with ssy uffer contining (in mmol/l) Hepes, MgCl 2, ATP,.% BSA, KCl, dithiothreitol, β-nad,. -thio-d-glucose -phosphte, 3-O-methyl-N-cetylglucosmine (Axxor, Exeter, UK), 2. IU/ml glucose--phosphte dehydrogense from Leuconostoc mesenteroides nd glucose ( mmol/l) in 9-well plte. NADH fluorescence (excittion 32 nm, emission nm) ws mesured every min for 2 h t 3 C using Fluoroskn Ascent plte reder (Thermo Scientific, Bsingstoke, UK). NAD(P)H imging in GLUTg cells NAD(P)H levels were imged in GLUTg cells cultured for 2 8 h on Mtrigelcoted glss-ottomed dishes. Cells were wshed with nd incuted in sline uffer for min, mounted on n Olympus IX7 microscope with oil-immersion ojective, nd imged using n Orc-ER CCD cmer nd Metfluor softwre. Cells were perfused with sline uffer (plus glucose s indicted) t room temperture. Autofluorescence t 3/ nm excittion, /8 nm emission, ws mesured every s, ckground corrected, nd verged over s periods. Men fluorescence under test conditions ws normlised y dividing y the men of selines mesured efore ddition, nd fter wshout, of the test regent. Anlysis of primry L cells y flow cytometry Single-cell digests were fixed with % prformldehyde in PBS for 3 min t room temperture nd locked with PBS/% got serum t C overnight. Cells were permeilised with.% vol./vol. Triton X- in PBS/% got serum for 3 min t room temperture, nd then incuted with or without primry ntiody detecting proglucgon (source s ove; :2 dilution) in PBS/% got serum t room temperture for 3 h. Cells were rinsed three times in PBS/ % got serum nd incuted for h with secondry ntiody (Alex Fluor 88; Invitrogen, Eugene, OR, USA; ctlogue no. A-3 t :3 dilution). After three wshes with PBS, cells were nlysed using BD LSRFortess nlyser (BD Biosciences, Sn Jose, CA, USA) equipped with 88 nm nd nm lsers for excittion of Alex Fluor 88 nd RFP, respectively. Dt were nlysed using FlowJo 7. softwre (Tree Str, Ashlnd OR, USA). For NAD(P)H mesurements from primry L cells, SI tissue from GLU-Venus mice ws digested to single cells [7] nd nlysed on the sme dy. Cells were wshed nd resuspended in sline uffer contining.% BSA nd vrious glucose concentrtions ( 3 mmol/l) min efore nlysis. From ech condition, events were ssessed using n LSRFortess nlyser nd FlowJo 7. softwre. Venus-positive L cells were selected on the sis of their chrcteristic fluorescence when excited t 88 nm, nd utofluorescence ws ssessed y excittion t 3 nm (emission nm). The geometric men of the 3 nm fluorescence intensity distriution from ech cell popultion ws clculted nd normlised to tht of the zero glucose control mesured on the sme dy.

4 28 Dietologi (22) :2 2 Smll interfering RNA (sirna) knockdown GLUTg cells were trnsfected with nmol/l scrmled or Sglt sirna (Qigen, Crwley, UK) for 2 h using Lipofectmine 2. Cells were reseeded into 2-well pltes nd used 2 h lter. Knockdown efficiency ws ssessed using Tqmn ssys for Slc (sglt) nd Act (β-ctin) (Applied Biosystems, Pisley, UK) [7, 2]. GLP- secretion Secretion studies on GLUTg nd primry intestinl cultures were performed 2 3 h fter plting in Mtrigel-coted 2-well pltes. Cultures were incuted with test regents in sline uffer contining.% ftty cid-free BSA for 2 h t 37 C. Cell lystes were then collected from primry cultures s descried previously [7]. Superntnt frctions nd lystes were ssyed using either GLP--ctive ELISA kit (Millipore, Wtford, UK) or totl GLP- ssy (MesoScle Discovery, Githersurg, MD, USA). For primry cells, GLP- secretion ws expressed s frction of the totl hormone content per well, normlised to sl secretion mesured in prllel. For GLUTg cells, superntnt frction concentrtions were normlised to sl levels in prllel control wells. Clcium imging Experiments were performed on - to 8- dy-old cultures from Sglt / mice nd wild-type littermtes, crossed into the GLU-Venus ckground. Cells were loded in 7 μmol/l fur2-am (Invitrogen, UK) nd.% pluronic F27, nd incuted in sline uffer contining mmol/l glucose nd 3 μmol/l eserine, for 3 min. Experiments were performed using the Olympus IX7 imging system descried ove. Fur2 ws excited t 3 nd 38 nm (emission /8 nm), nd Venus t 7 nm (emission 3/ nm). Fur2 fluorescence mesurements were tken every 2 s, ckground corrected, nd expressed s the 3/ 38 nm rtio. Men fluorescence rtios were determined over 2 s, nd responses expressed s the mximum rtio chieved during stimultion divided y the men of the rtios mesured efore nd fter wshout. Cells were included in the nlysis if they responded to the positive controls, mmol/l glutmine nd 3 mmol/l KCl. Solutions Sline uffer contined (mmol/l). KCl, 38 NCl,.2 NHCO 3,.2 NH 2 PO, 2. CCl 2,.2 MgCl 2 nd HEPES (ph 7., NOH). In some experiments, KCl ws incresed with equivlent reduction in NCl, in others N ws replced y N-methyl-D-glucmine (NMDG). Where possile, solutions were prepred s, stock. Glucokinse ctivtor (GKA) [22] (AstrZenec, Mcclesfield, UK) ws dissolved in DMSO. Chemicls were supplied y Sigm Aldrich (Poole, UK) unless otherwise stted. Dt nlysis Dt re presented s mens±sem. Significnce ws ssessed using Student s t test (Microsoft Excel) or y one- or two-wy ANOVA followed y Bonferroni or Dunnett s post hoc tests (Grph Pd Prism Softwre, Sn Diego, CA, USA). p<. ws considered significnt. Results uptke pthwys in L cells Intrcellulr glucose levels were monitored in GLUTg cells trnsiently producing the FLII 2 Pglu-7μδ glucose FRET proe [8]. Initil experiments confirmed the specificity of the sensor, s glucose ( or mmol/l), ut not glutmine ( mmol/l) or the glucose nlogue αmg ( mmol/l), elicited significnt increse in the YFP/CFP rtio (Fig.,). To determine whether glucose entry into L cells is primrily medited y N -coupled (SGLT) or fcilittive (GLUT) glucose trnsport, we exmined the effects of extrcellulr N replcement nd phrmcologicl inhiitors of oth clsses of trnsporter (Fig. c e). The response to mmol/l glucose ws impired in the presence of or μmol/l phloridzin, ut ppered incresed rther thn decresed in the sence of N. Although the incresed glucose signl following N sustitution could reflect prdoxicl incresed net glucose influx, it might lso e cused y reduced rte of glucose metolism or n ltered intrcellulr ionic composition/ph ffecting the properties of the glucose sensor. A roust inhiition of glucose uptke ws oserved in the presence of either phloretin ( or μmol/l) or cytochlsin B ( μmol/l), oth of which trget the GLUT fmily. As we previously reported n -fold higher expression of Sglt mrna in primry L cells compred with GLUTg cells [7], we imed to perform similr experiments in L cells in primry culture. To enle the use of YFP/CFP-sed FRET sensors, we developed mouse model in which L cells re identifile y their red fluorescence. Trnsgenic mice were generted in which Cre-recominse expression is driven y the proglucgon promoter (GLU-Cre2). Crossing these with Ros2tdRFP reporter mice [] resulted in red fluorescence in glucgon-positive pncretic lph cells (Fig. 2) nd GLP--contining L cells (Fig. 2), s expected. The efficiency of L cell trgeting ws quntified y FACS nlysis of colonic epithelil cell suspensions from GLU-Cre2 tdrfp mice, reveling tht >7% of proglucgon-positive cells hd undergone Cre-medited recomintion (Fig. 2c e). We detected frction of cells (<3%) with red fluorescence not stining for proglucgon, corresponding to smll numer of red fluorescent cells in primry colonic cultures not showing the typicl morphology of L cells. These were excluded morphologiclly from experiments on mixed epithelil primry cultures trnsduced with newly mde denovirus expressing FLII 2 Pglu-7μδ. In L cells in primry culture, mmol/l glucose elicited n verge.3-fold increse in the YFP/CFP rtio, which ws

5 Dietologi (22) : YFP/CFP (mmol/l). min Reltive YFP/CFP rtio mmol/l: 2. αmggln c Reltive YFP/CFP rtio N - d e. 39 YFP/CFP 2 8 Phlt Phlz Cyt B min Reltive YFP/CFP rtio μmol/l: Phlz Phlt CytB Fig. uptke into GLUTg cells. () Representtive trce showing the rw FRET YFP/CFP rtio monitored in single GLUTg cell producing FLII 2 Pglu-7μδ. (,,. mmol/l) ws pplied s indicted y the horizontl rs. () Men normlised YFP/ CFP rtio, recorded s in (). (., or mmol/l, pplied in rndom order), αmg ( mmol/l) nd glutmine (Gln, mmol/l) were pplied s indicted. Error rs represent SEM from n cells s indicted ove the rs from two to four seprte experiments. (c) Men normlised YFP/CFP rtio, recorded s in (), elicited y mmol/l glucose pplied inslineuffer(n ) or fter N sustitution y NMDG (N )inn cells s indicted ove the olished y phloretin ( μmol/l) ut unffected y phloridzin ( μmol/l) (Fig. 2f,g). The glucose trnsporter GLUT2 ws detected in the solterl memrne of enterocytes nd L cells (Fig. 2h). -stimulted chnges in L cell metolism To investigte whether elevtion of intrcellulr glucose trnsltes into metolic chnges within GLUTg cells, we monitored the utofluorescence signl ttriuted to NAD(P)H y reltime imging (Fig. 3). dose-dependently incresed NAD(P)H utofluorescence, eliciting mximl.8-fold increse t mmol/l (Fig. 3). To monitor NAD(P)H in primry L cells, we developed FACS nlysis-sed technique for use with cutely dispersed SI epithelil cells. In the Venus-lelled L cell supopultion, glucose dose-dependently shifted the histogrm of NAD(P)H utofluorescence rightwrds (Fig. 3 c,d). Role of glucokinse in L cells To exmine the role of the gene encoding glucokinse (Gck), which is expressed in rs. (d) Representtive trce, recorded s in (). ( mmol/l), phloretin (Phlt, μmol/l), phlorizdin (Phlz, μmol/l) or cytochlsin B (Cyt B, μmol/l) ws pplied s indicted. (e) Men normlised YFP/CFP responses, recorded s in (d), to glucose ( mmol/l), phloridzin (Phlz,, μmol/l), phloretin (Phlt,, μmol/l) or cytochlsin (CytB, μmol/l) in n2 29 cells s indicted ove the rs from five to seven seprte experiments. Sttisticl significnce ws tested y ANOVA followed y Student s t test to compre responses to seline (p<., p<., p<.) or y Dunnett s test to compre responses to glucose ( p<.) GLUTg nd L cells t mrna levels comprle to those found in pncretic et cells [7], we mesured enzyme ctivity in GLUTg cell extrcts [2]. Consistent with functionl glucokinse production, the enzymtic rte incresed with incresing glucose concentrtions in the mmol/l rnge, t which low-ffinity hexokinses would e sturted, nd significnt (p<.) further increse ws oserved when glucokinse ctivtor, GKA (3 μmol/l), ws dded to the cell extrct (Fig. ). Addition of GKA to 3 mmol/l glucose lso significntly incresed the NAD(P)H utofluorescence in GLUTg cells (Fig. ). In secretion experiments, GKA cused smll ut significnt increse in glucosestimulted GLP- relese from GLUTg cells (p<. y two-wy ANOVA, Fig. c). However, under the conditions tested, we were unle to detect n effect of GKA on GLP- secretion from primry intestinl cultures (Fig. d). Effect of intrcellulr glucose on GLP- secretion To evlute whether glucose-dependent GLP- relese requires ctivtion of n intrcellulr trget, such s signl generted

6 2 Dietologi (22) :2 2 c d e Frequency (AU) f YFP/CFP YFP/CFP h Log green fluorescence (GLP) Per cent Cre cells lso GLP A min Phloridzin Phloretin min from glucose metolism, we exmined whether GLUT inhiition, which lrgely olished intrcellulr glucose trnsients, ffected GLP- secretion. In primry intestinl cultures, the secretory response to mmol/l glucose ws not significntly impired y phloretin ( or μmol/l), wheres the higher dose olished secretion from GLUTg cells (Fig.,). In contrst, in oth model systems, glucose-triggered secretion ws significntly reduced when SGLTs were inhiited y phloridzin, with more profound effects evident in the primry cultures (Fig.,). Consistent with dominnt role of electrogenic N -coupled glucose uptke in primry L cells, no further glucose-dependent 8 2 g Reltive YFP/CFP rtio.3.2 Per cent GLP A cells lso Cre μmol/l: Phlz Phlt R Fig. 2 Mechnisms of glucose uptke in primry L cells. (,) Coloclistion of direct tdrfp fluorescence (red) with glucgon immunofluorescence (green) in () pncretic nd () colonic tissue slices from GLU-Cre2 tdrfp mouse. Blue represents Hoechst fluorescence. (c) FACS nlysis performed on colonic digests from GLU-Cre2 tdrfp mice stined with n ntiody ginst proglucgon nd green fluorescent secondry ntiody. Frequency histogrms represent the green fluorescence of ll tdrfp-positive cells (drk grey shding) or ll strongly green fluorescent cells (tdrfp-positive nd -negtive cells; light grey shding). The non-shded distriution represents the green fluorescence of tdrfp-positive cells when primry proglucgon ntiody ws omitted. Frequencies re given in ritrry units (AU). (d,e) FACS-determined percentges of tdrfp-producing cells stining for proglucgon (d), nd of proglucgon-positive cells tht contin tdrfp from three independent experiments. (f) Intrcellulr glucose, monitored s YFP/CFP rtio, in individul primry L cells identified y tdrfp fluorescence. ( mmol/l), phloridzin ( μmol/l) or phloretin ( μmol/l) ws pplied s indicted. (g) Men normlised FRET responses, recorded s in (f) from n 2 cells (s indicted ove the rs) from more thn five seprte experiments. p<., p<. vs seline y single-fctor t tests. Sttisticl comprison etween glucose nd other conditions ws ssessed y ANOVA nd Dunnett s test; p<.. (h) Immunofluorescent loclistion of GLUT2 in duodenl slice. Left, phse-contrst imge of villus, with superimposed Venus fluorescence (green) identifying n L cell; middle, GLUT2 immunofluorescence (red); right, overly of Venus fluorescence nd GLUT2 immunofluorescence increse in GLP- secretion ws oserved in primry cultures depolrised y KCl (Fig. c). -dependent mplifiction of secretion ws, however, seen under these conditions in GLUTg cells (Fig. d), which ws sensitive to phloretin ut not phloridzin (Fig. e). Role of SGLT in glucose-triggered GLP- secretion To further confirm the role of SGLT in glucose-stimulted GLP- secretion, we crossed GLU-Venus mice with the recently descried Sglt knockout mouse model []. ( mmol/l) triggered rise in cytosolic C 2 in identified L cells in colonic cultures from Sglt / ut not Sglt / mice (Fig.,). Glutmine, in contrst, stimulted similr C 2 responses in L cells from oth wild-type nd knockout tissue cultures. This is consistent with the previous oservtion tht Sglt knockout selectively olishes glucose-stimulted GLP- secretion []. As the impired C 2 response nd GLP- secretion of L cells in this SGLT-deficient model my still rise from defect in glucose uptke into neighouring cells rther thn the enteroendocrine cells themselves, we lso knocked down Sglt in GLUTg cells using sirna, which decresed mrna expression y % (Fig. c). Sglt knockdown lrgely olished the effects of oth glucose ( mmol/l) nd αmg ( mmol/l) on GLP- relese (Fig. d). Discussion The mechnism underlying glucose sensing y L cells is topic of recent dete, with sweet tste receptors (TsR2/3)

7 Dietologi (22) : AU c Frequency (AU). 8 2 min Log fluorescence (NAD(P)H) [23, 2], SGLT [2] nd K ATP chnnels [] ech suggested to ply role. We demonstrte here tht genetic or phrmcologicl interference with SGLT olishes glucose-triggered C 2 responses nd GLP- secretion from L cells in primry culture. This is consistent with the inhiition of GLP- secretion y phloridzin from perfused intestinl preprtions [] nd the more recent oservtion of impired glucose-triggered GLP- secretion in Sglt knockout mice []. The ltter oservtions re incomptile with the concept of n piclly locted glucose receptor on the surfce of the L cell, s inhiition of glucose sorption would, if nything, tend to increse exposure of such receptor to luminl glucose. Although they could e explined y solterlly expressed receptor which is exposed to elevted glucose concentrtions fter SGLT-dependent sorption through enterocytes, the current findings, in which oth sides of the cells re exposed to glucose, rgue ginst ny mjor role of n extrcellulr receptor. This is consistent with our previous oservtion tht rtificil sweeteners, t concentrtions tht sturte TsR2/3 receptors, did not trigger GLP- Reltive NAD(P)H utofluorescence d Reltive NAD(P)H utofluorescence (mmol/l). 3 (mmol/l) Fig. 3 -dependent chnges in L cell metolism. () Representtive trce representing NAD(P)/NAD(P)H utofluorescence (ritrry units, AU) from GLUTg cell upon glucose ppliction (.,,, 2 mmol/l). () Men normlised utofluorescence, recorded s in (). n8 cells from five seprte experiments. (c) Smoothed frequency histogrms showing the utofluorescence intensity of primry L cells in smll intestinl digests in the sence (white-shded) nd presence (drk grey-shded) of mmol/l glucose. For ech condition, million events were counted, of which.% were Venus positive. (d) Normlised men utofluorescence intensities of primry L cells, nlysed s in (c), from four to five mice nlysed independently s indicted ove the rs. p<., p<., p<. vs seline, y Student s single-smple t test Velocity (AU) c Reltive GLP- secretion (mmol/l) Reltive NAD(P)H utofluorescence GKA relese from primry intestinl cultures [7]. A metolic sensing mechnism downstrem of SGLT-medited glucose uptke could lso e envisioned. Monitoring intrcellulr glucose levels, however, llowed us to dissocite the stimultory ction of N -coupled glucose uptke from possile downstrem metolic effects, s SGLT inhiition hd only minor effects on intrcellulr glucose levels, wheres GLUT inhiitors lrgely olished glucose uptke in L cells ut did not significntly impir GLP- secretion. The finding tht (mmol/l) d Reltive GLP- secretion GKA Fig. Role of glucokinse in GLUTg nd primry L cells. () Glucokinse ctivity in GLUTg extrcts vs glucose concentrtion in the sence (lck squres, solid line) nd presence (white circles, dotted line) of GKA (3 μmol/l). Velocity represents the increse in fluorescence over time in ritrry units (AU). Dt were fitted using hyperol plot with the eqution yv mx [glucose]/(s. [glucose]), giving vlues of 3 nd mmol/l for S., the sustrte concentrtion t which hlf the mximl velocity (V mx ) is reched, in the sence nd presence of GKA, nd V mx under oth conditions of.7 AU (n). () Men normlised NAD(P)H utofluorescence responses in GLUTg cells fter ddition of glucose (3 mmol/l) nd GKA (3 μmol/l). n cells from three seprte experiments. Sttisticl comprison to utofluorescence in the sence of glucose (p<.) nd etween the sence/presence of GKA ( p<.) were ssessed y Student s t test. (c) GLP- secretion from GLUTg cells in response to vrious concentrtions of glucose, with GKA (3 μmol/l, lck rs) or without GKA (white rs). Secretion ws normlised to prllel seline mesurements nd the numer of wells is shown ove the rs. Sttisticl comprisons were ssessed y two-wy ANOVA (p.7 for GKA vs no GKA, nd p<. for effect of glucose concentrtions, with no significnt interction), followed y post hoc Bonferroni s test; p<. vs respective controls in the sence or presence of GKA. (d) GLP- secretion from primry upper SI cultures in response to mmol/l glucose with or without GKA (3 μmol/l). Secretion ws clculted reltive to GLP- content, normlised to sl secretion mesured in prllel, with the numer of wells given ove the rs. Significnce ws ssessed y one-smple t test; p<. vs control

8 22 Dietologi (22) :2 2 GLP secretion (% of glucose response) Phlz Phlt GLP secretion (% of glucose response) Phlz 3 Phlt c d e Reltive GLP- secretion KCl (mmol/l) Diz (mmol/l) Gluc (mmol/l) Reltive GLP- secretion Reltive GLP- secretion Phlt Phlz Fig. Phrmcologicl modultion of glucose-stimulted GLP- secretion. GLP- secretion from () GLUTg cells nd () primry upper SI cultures, in glucose ( mmol/l), phloretin (Phlt, or μmol/l) nd phloridzin (Phlz, or μmol/l), s indicted. Incrementl responses to glucose re shown for ech condition, normlised to the incrementl glucose response in the sence of inhiitor (%). Error rs represent SEM nd the numer of wells is given ove the rs. Significnce ws ssessed y one-wy ANOVA nd Dunnett s test vs glucose only; p<., p<.. (c) GLP- secretion from primry upper SI cultures in 3 or 7 mmol/l extrcellulr K nd 3 μmol/l dizoxide (Diz) in the sence (white rs) or presence (lck rs) of glucose ( or mmol/l) s indicted. Error rs represent SEM nd the numer of wells is given ove the rs. Secretion ws clculted reltive to GLP- content nd normlised to sl secretion mesured in prllel. (d,e) GLP- secretion from GLU- Tg cells in 3 mmol/l extrcellulr K nd 3 μmol/l dizoxide nd (d) vrious glucose concentrtions or (e) mmol/l glucose with or without phloretin ( μmol/l, Phlt) or phloridzin ( μmol/l, Phlz) s indicted. Secretion ws normlised to seline secretion mesured in prllel. Error rs represent SEM nd the numer of wells is given ove the rs. Significnce ws determined y one-wy ANOVA followed y one-smple t tests nd Dunnett s test vs glucose in the sence of inhiitor; p<., p<., p<. Sglt knockdown in GLUTg cells impired oth glucosend αmg-triggered secretion rgues for mechnism intrinsic to the enteroendocrine cells, rther thn involving coupling through neighouring enterocytes. It lso demonstrtes dominnt role of SGLT over SGLT3, which is lso produced in GLUTg nd primry L cells [7]. We thus conclude tht the electrogenic uptke itself vi piclly loclised SGLT [7] is the mjor glucose-sensing mechnism in L cells. To enle the use in primry tissues of FRET-sed sensors employing YFP nd CFP, we generted new BAC trnsgenic mouse model expressing the gene encoding Cre recominse under control of the proglucgon promoter, trgeting enteroendocrine L cells s well s pncretic lph cells unlike the shorter promoter constructs used previously to drive Cre expression in lph cells [2]. Quntifiction y FACS nlysis reveled tht, lthough the mjority of proglucgon-stined cells in the colon lso contined the Cre reporter in GLU-Cre2 tdrfp mice, 3% did not. This suggests tht smll ut significnt numer of L cells my escpe Cre recomintion, nd should e tken into ccount when GLU-Cre2 mice re used in future conditionl gene knockout experiments. Cre-medited ctivtion of RFP production ws lso evident in some cells tht did not stin for proglucgon, consistent with the oservtion tht smll proportion of the red fluorescent cells in primry colonic cultures did not exhiit morphology typicl of L cells. These cells were redily identifile nd could e voided in singlecell imging experiments, ut re likely to reflect trnsient trnsgene ctivtion in different cell popultion during development. Similr findings in the other founder GLU-Cre strins (see ESM Tle 2) suggest tht this is not merely n rtefct of the prticulr trnsgene integrtion site.

9 Dietologi (22) : Sglt / Sglt -/- c Sglt/β-ctin mrna Con Sglt. 3/38 Glutmine min Glutmine. 3/38 min d Reltive GLP- secretion.. Con Previous nlysis detected Glut, Glut2 nd Glut expression in primry murine L cells, with Glut2 evident in L cells from the SI, nd Glut in those from the colon [7]. GLUTg cells notly lck Glut2 [2], ut express Glut3,s determined y Affymetrix microrrys (dt not shown). The role of GLUTs in incretin hormone secretion is uncler, s their phrmcologicl inhiition hd no significnt effect on glucose-stimulted GLP- secretion in primry cultures, lthough mice lcking GLUT2 showed reduced plsm Reltive 3/38 fluorescence Glutmine αmg Fig. Effect of genetic interference with SGLT ction. () C 2 concentrtions in Venus-positive L cells in colonic cultures, monitored s the fur2 3/38 nm fluorescence rtio. Trces from wild-type nd n Sglt / mouse re shown fter ddition of glucose ( mmol/l) nd glutmine ( mmol/l). The verticl r represents chnge in the fluorescence rtio of.. () Men normlised C 2 responses in L cells recorded s in () from colonic cultures of control (white rs) nd Sglt / (lck rs) mice. Error rs represent SEM nd the numer of cells is given ove the rs. p<., p<. vs seline nd p<. etween genotypes ssessed using Student s t test. (c) Sglt expression in GLUTg cells trnsfected with Sglt or scrmled (Con) sirna, s determined using quntittive RT-PCR, nd normlised to β-ctin. mrna from four experiments were nlysed for ech r. Dt re presented s geometric men nd upper SEM clculted from the log (se 2) dt. Significnce ws nlysed y Student s t test on the non-trnsformed ΔC t dt; p<.. (d) GLP- secretion from GLUTg cells trnsfected with scrmled (white rs) or Sglt sirna (lck rs). Cells were incuted in the sence of dditions (Con) or in the presence of glucose ( mmol/l) or αmg ( mmol/l). Secretion ws normlised to seline mesured in prllel on the sme dy. Error rs represent SEM, nd the numer of wells tested for ech concentrtion is indicted ove the r. Significnce ws clculted y two-wy ANOVA nd Bonferroni test; p<., p<. vs seline; p<., p<. etween knock-down nd control GLP- concentrtions following orl glucose, nd lower intestinl GLP- content [2]. Wheres SGLT is piclly locted on L cells [], GLUT2 ppered loclised to the solterl surfce of L cells nd enterocytes, suggesting tht intrcellulr glucose concentrtions would reflect solterl rther thn luminl glucose levels. Wheres GLP- secretion is predominntly stimulted y orl rther thn systemic glucose delivery, GLP- relese from the perfused pig intestine ws found to e influenced lso y vsculr glucose levels [27], possily through ltertion of the intrcellulr glucose concentrtion in L cells. Whether intrcellulr glucose metolism plys ny role in determining GLP- secretion remins uncertin. In GLU- Tg ut not primry L cells, we oserved strong inhiition of secretion when glucose uptke ws completely locked nd n mplifying ction of glucose under depolrising conditions. The lnce etween the metolic nd electrogenic effects of glucose is thus slightly different etween the cell line nd primry culture, with more dominnt role for SGLT-sed glucose sensing in the ltter. NAD(P)H utofluorescence mesurements suggest tht GLUTg nd primry L cells increse their metolic rte in response to extrcellulr glucose elevtion, consistent with our previous oservtion tht ATP concentrtions in GLUTg cells re elevted upon exposure to mmol/l glucose [28]. NAD(P)H chnges occurring t glucose concentrtions ove mmol/l would e consistent with the recruitment of Glucokinse, which is known to e expressed in enteroendocrine cells [7, 9, 29 3]. Glucokinse ctivity ws demonstrle in GLUTg cell extrcts, nd ws responsive to the glucokinse ctivtor, GKA. The oserved S. vlue ( 3 mmol/l) in the sence of GKA is lower thn the expected vlue of -8 mmol/ l[2], possily reflecting incomplete inhiition of hexokinses I III or dditionl regultion of enzyme ctivity y unknown fctors in GLUTg cell extrcts. GKA significntly ffected NAD(P)H utofluorescence t 3 mmol/l glucose in GLUTg cells, demonstrting tht glucokinse exhiits t lest some control over the metolic flux in L cells, ut hd only smll effect on glucose-stimulted GLP- secretion from GLUTg cells nd no effect on secretion from primry cultures. The present study demonstrtes tht metolism plys t est minor role in glucose-stimulted GLP- secretion in primry cultures, consistent with the finding tht nonmetolisle glucose nlogues such s αmg re effective stimuli of GLP- relese in vivo nd in vitro [,,, 32]. Although phloretin olished glucose ccumultion in GLUTg nd primry L cells, its effect on glucosestimulted GLP- secretion ws restricted to the cell line, suggesting tht glucose metolism does not enhnce secretion in the context of predominnt SGLT-medited stimulus. Future work should ddress whether the glucokinse/ K ATP chnnel mchinery exerts longer-term effects on

10 2 Dietologi (22) :2 2 L cells or enles modultion of GLP- secretion y neurohormonl or lterntive nutritionl stimuli. Acknowledgements We would like to thnk M. Bowen nd M. Grtin (Cmridge Institute for Medicl Reserch [CIMR]) nd A. Petrunkin nd V. Romshov (CIMR) for help with microscopy nd flow cytometry, respectively. D. Drucker (Smuel Lunenfeld Reserch Institute, Toronto, Cnd) kindly provided the GLUTg cell line. R. Sprengel (Mx Plnck Institute for Medicl Reserch, Heidelerg, Germny) nd H.J. Fehling (University of Ulm, Ulm, Germny) kindly provided plsmid contining icre sequence nd the Ros2- tdrfp reporter mice, respectively. The glucokinse ctivtor, GKA, ws gift from AstrZenec. Funding This work ws funded y Wellcome Trust grnts to F.M. Grile nd F. Reimnn (WT8837 nd WT82) nd y the Deutsche Forschungsgemeinschft to H. Koepsell (Grnt SFB 87/ C). GLP- ssys nd mouse husndry were supported y the MRC Centre for Oesity nd Relted Metolic Diseses (Cmridge). Dulity of interest AstrZenec ws not involved in the reserch descried in this mnuscript eyond the gift of GKA, ut pproved the mnuscript prior to sumission. The uthors declre tht there is no other dulity of interest ssocited with this mnuscript. Contriution sttement HEP, AA, GR nd PR designed nd performed experiments, HK generted nd provided the Sglt knockout mice nd contriuted to design nd interprettion of experiments involving these mice. HEP, FR nd FMG designed the study nd drfted the mnuscript. All uthors contriuted to criticl revision of the mnuscript nd pproved the finl version. Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution License which permits ny use, distriution, nd reproduction in ny medium, provided the originl uthor(s) nd the source re credited. References. Eissele R, Goke R, Willemer S et l (992) Glucgon-like peptide- cells in the gstrointestinl trct nd pncres of rt, pig nd mn. 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