Peptide Production and Secretion in Glutag, NCI-H716, and STC-1 Cells: a Comparison to Native L-Cells

56:3

r e kuhre and others Peptide production in GLP1- 56:3 201–211 Research producing cell lines

Peptide production and secretion in GLUTag, NCI-H716, and STC-1 cells: a comparison to native L-cells

Rune Ehrenreich Kuhre1, Nicolai Jacob Wewer Albrechtsen1, Carolyn Fiona Deacon1, Emilie Balk-Møller1, Jens Frederik Rehfeld2, Frank Reimann3, Fiona Mary Gribble3 and Jens Juul Holst1 Correspondence 1Department of Biomedical Sciences and NNF Center for Basic Metabolic Research, the Panum Institute, should be addressed University of Copenhagen, Copenhagen, Denmark to J J Holst 2Department of Clinical Biochemistry Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Email 3Cambridge Institute for Medical Research and MRC Metabolic Diseases Unit, University of Cambridge, [email protected] Cambridge, United Kingdom

Abstract

GLUTag, NCI-H716, and STC-1 are cell lines that are widely used to study mechanisms Key Words underlying secretion of glucagon-like peptide-1 (GLP-1), but the extent to which they ff GLP-1-producing cell resemble native L-cells is unknown. We used validated immunoassays for 14 different lines ff peptide production hormones to analyze peptide content (lysis samples; n = 9 from different passage numbers) or peptide secretion in response to buffer (baseline), and after stimulation ff L-cells with 50 mM KCl or 10 mM glucose + 10 µM forskolin/3-isobutyl-1-methylxanthine (n = 6 also different passage numbers). All cell lines produced and processed proglucagon into GLP-1, GLP-2, glicentin, and oxyntomodulin in a pattern (prohormone convertase (PC)1/3

Journal of Molecular Endocrinology dependent) similar to that described for human gut. All three cell lines showed basal secretion of GLP-1 and GLP-2, which increased after stimulation. In contrast to freshly isolated murine L-cells, all cell lines also expressed PC2 and secreted large amounts of pancreatic glucagon. Neurotensin and somatostatin storage was low and secretion was not consistently increased by stimulation. STC-1 cells released more glucose-dependent insulinotropic polypeptide than GLP-1 at baseline (P < 0.01) and KCl elevated its secretion (P < 0.05). Peptide YY, which normally co-localizes with GLP-1 in distal L-cells, was not detected in any of the cell lines. GLUTag and STC-1 cells also expressed vasoactive intestinal peptide, but none expressed pancreatic polypeptide or insulin. GLUTag contained and secreted large amounts of CCK, while NCI-H716 did not store this peptide and STC-1 contained low amounts. Our results show that hormone production in cell line Journal of Molecular Endocrinology models of the L-cell has limited similarity to the natural L-cells. (2016) 56, 201–211

Introduction

Enteroendocrine cells are derived from a common The gut hormone, glucagon-like peptide-1 (GLP-1), is a pluripotent stem cell that also gives rise to enterocytes, key hormone in the regulation of blood glucose and goblet cells, and Paneth cells (Roth et al. 1992), but there satiety in humans. It is produced by enteroendocrine are pronounced differences with respect to the expression L-cells located in the gut epithelium, and is particularly pattern of the different gut hormones along the intestine. known for its incretin actions , that is, the enhancement

http://jme.endocrinology-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-15-0293 Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 06:38:32PM via free access

10.1530/JME-15-0293 Journal of Molecular Endocrinology secretion of a panel of intestinal and pancreatic hormones. resemble naturalL-cellsbyexamining theirexpressionand to explorewhatextent these ‘L-cellmodels’actually the lines areimmortal,whichalso distinguishesthemfrom produced bynativeL-cells.Furthermore,allofthesecells process, andsecretehormonesthatnormallyarenot depending ontheirrespectivedifferentiationstate,express, from carcinomas, we hypothesized that they may, contain somatostatin(SST)(Egerodet al.2012). et al. 2003,Svendsen et al. 2015).None,however, seemsto (PYY) (Egerod whereas distalL-cellstypicallyalsoproducepeptideYY L-cells may also contain CCK and neurotensin (NT), hormones not derived from proglucagon. Thus, proximal L-cells exist,inwhichGLP-1isco-localizedwithother groups haveprovidedevidencethatsomepopulationsof 1994).Recently,(Holst 2010,et al. however, several to generateglucagonandthemajorproglucagonfragment but heretheprecursorispredominantlyprocessedbyPC2 peptide. Thepancreaticα-cellsalsoproduceproglucagon, into oxyntomodulin(OX)andglicentin-relatedpancreatic glicentin (Holst2007).Glicentinmaybeprocessedfurther in theformationofGLP-1,togetherwithGLP-2and processing oftheglucagonprecursor, proglucagon,results 1991, Leeet al.1992,Rindi1990). colon (Bruïne ascites fluidfromahumanwithadenocarcinoma ofthe respectively, whileNCI-H716cellswerederivedfrom tumors of the large bowel and small intestine of mice, from carcinomas; GLUTag andSTC-1cellsoriginatedfrom characterized inanydetail.Allthreecelllinesarederived of resemblancetonaturalL-cells,butthishasnotbeen with thesecelllinescruciallydependsontheirdegree unveiled. Naturally, thephysiologicalrelevanceofstudies mechanisms leading to the secretion of GLP-1 began to be and NCI-H716,inthe1990sthatspecificmolecular three GLP-1-producingcelllines,namelyGLUTag, STC-1, secretion. Therefore,itwasnotuntilthedevelopmentof underlying molecularmechanismscouplingstimulusto vs indirecteffectsontheL-cell,orfordissecting sense, theyarenotalwaysidealfordistinguishingdirect useful forstudyingphysiologicalprocessesinthebroad GLP-1 haveutilizedanimalmodelsandwhiletheseare II diabetes.Manystudiesinvestigatingthesecretionof and therefore,GLP-1-baseddrugsareusedtotreattype limit postprandialbloodglucoselevels(Holst2007), to of meal-stimulatedinsulinsecretion.Thisserves DOI: 10.1530/JME-15-0293 http://jme.endocrinology-journals.org Research As GLUTag, NCI-H716,andSTC-1cellsall arederived In theL-cell,prohormoneconvertase(PC)1/3 natural L-cells.We, therefore,undertookthisstudy . 1994,Grantet al. 1992,Drucker et al et al. t al. 2012,Mortensen al.2012,Habibet et r

© 2016SocietyforEndocrinology kuhre andothers Printed inGreatBritain KCl, 4.2 stimulants). Thebufferconsistedof138 10 previously (Kuhre thorough mechanicaldisruptionbyscraping,asdescribed ice-cold lysisbufferfor5 assayed non-extracted. stored at processed forhormonequantificationwhilekeptonice or to freshEppendorftubesandsampleswereeitherdirectly cells ordebris.Theresultingsupernatantsweretransferred centrifuged (1500 to whichwasadded50 incubated for2 study. Onthefollowingday, cells(~80%confluent)were 24 (Cat. No.354234;BDBiosciences)18 – plates precoatedwithmatrigelbasementmembrane serum, 2.5%(vol/vol)FBS. Sigma-Aldrich) supplementedwith15%(vol/vol)horse for STC-1:highglucose(4.5 with 10%(vol/vol)FBSand1%p/s;iii) RPMI 1640(Cat.No.R8750,Sigma-Aldrich)supplemented Life Technologies); ii)forNCI-H716:lowglucose(2.0 (p/s), andGlutaMAX(200 Aldrich), 0.05 sodium deoxycholatemonohydrate (Cat.No. D5670, 1% IgepalCA-630(Cat.No. I8896, Sigma-Aldrich),0.12 Sigma-Aldrich) (pH 0.1% (wt/vol)fattyacid-freeBSA(Cat.No.A-603-10G, 1.2 at 37°C,5%CO protocols (Kuhre Cells wereculturedfollowingpreviouslydescribed Cell work Materials andmethods penicillin (10,000 with 10%(vol/vol)fetalbovineserum(FBS),1% DMEM (CatNo.6046,Sigma-Aldrich)supplemented 2001), andcomprisedi)forGLUTag: lowglucose(1 Katsuma from severalgroups(Drucker Cell mediumcompositionwasbasedonpublications (Cat. No.C6731,Sigma-Aldrich)forNCI-H716cells. and STC-1cells,oranti-attachmentcoatedT25flasks Thermo FisherScientific)forGLUTagNo. EW-01930-54, and transferredtofreshnucleon-coatedT75flasks(Cat. GmbH, Darmstadt,Germany), 0.15 producing celllines Peptide productioninGLP1- Published byBioscientifica Ltd. µM forskolin/3-isobutyl-1-methylxanthine(FSK/IBMX, mM MgCl Lysis sampleswereobtainedbyincubatingthecellswith For secretionstudies,cellswereplatedonto24-well mM NaHCO −20°C until analysis. In either case, samples were et al. 2005,Reimann & Gribble2002,Reimeret al. 2 M Tris– , and10 h withbuffer(baselinesecretion)or 2 2014a,b).Inbrief,cellswerekept et al. 85% confluent,thensplit until80 – t al.2014a et g, 4°C,5 U/mL)/streptomycin (10,000 = 3 7). Supernatantswereobtainedand , 1.2 HCl (Cat. No. A1087, AppliChem A1087,AppliChem HCl (Cat.No. min while kept on ice, followed by min whilekeptonice,followedby Downloaded fromBioscientifica.com at09/29/202106:38:32PM mM KClor10 mM HEPESsupplementedwith mM; Cat.No.35050061,Gibco, mM NaH min) toremoveanyfloating g/L) DMEM(CatNo.6429, 2009, 1994,Eikiet al. et al. ). Lysis bufferconsistedof: M NaCl,andone 2 PO 56 mM NaCl,4.5 : 4 mM glucoseand 3 , 2.5 h beforethe mM CaCl µg/mL) Sigma- 202 tablet tablet mM g/L) g/L) M via freeaccess 2 , Journal of Molecular Endocrinology supplementary datagivenattheendofthisarticle). on supplementary Tableassays isprovidedinSupplementary 1(seesection epitopes, sensitivity, specificity, andlinearrangeforthe regarding assay type (RIA or ELISA), antibody codes, Information the sensitivepartofstandard curves. assay bufferssothatallpositivemeasurementswerewithin available ELISA. Samples were diluted in the respective RIA, in-housesandwichELISA,orvalidatedcommercially samples weremeasuredusingwell-characterizedin-house instructions. Hormonecontentsoflysisandsecretion BCA ProteinAssayKitaccordingtothemanufacturer’s Protein contentinlysissampleswasmeasuredusing Biochemical measurements fractions fromanyonthesecontrolruns(datanotshown). with buffer;noimmunoreactive moieties were detected in internal controls.Betweenruns,thecolumnwaswashed In allruns, concentrations were analyzedasdescribedpreviously. albumin and sodium calibrators (Holst 1983). Peptide as thedifferencebetweenelutionvolumesof volume for the substance in question,V using volume of the column. The column was precalibrated corresponding to approximately 1/50 of the fractionation effluents werecollectedautomaticallyinfractions same sodiumphosphatebufferasabove.Gelfiltration column (Pharmacia),equilibrated,andelutedwiththe by gelfiltrationonaSephadexG-50SF-packedK16-100 (4°C, 4 Pooled extractedcelllysissamples(n Gel filtrationanalysis Protein AssayKit(Cat.No. 71285-3,Millipore). Merck). Total proteincontentwasquantifiedbyaBCA with 0.1%(wt/vol)humanserumalbumin(Cat.No.12666, reconstituted insodiumphosphatebuffersupplemented compressed airovernight.Thepurifiedsampleswere trifluoroacetic acidanddriedunderagentlestreamof Waters, Milford,MA,USA),elutedwith70%ethanol purified usingSep-PakC18cartridges(Cat.No. WAT036810, Samples werestoredat− 05056489001, F. Hoffmann-LaRoche,Basel,Switzerland). EDTA-free proteaseinhibitorcocktail/50 volume, andV calculated as: K OX, andglicentin.Coefficientsofdistributionwere proglucagon-derived peptides: glucagon, GLP-1, GLP-2, http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0293 Research 125 min, 4500 I-labeled albumin(V 22 Na and i is the available inner volume determined istheavailableinnervolumedetermined d g = (V ) and the supernatants were fractionated ) andthesupernatantswerefractionated e − 125 V I-labeled albumin were added as I-labeled albuminwereaddedas 0 80° ) /V C, afterwhich i , where V 0 ), 22 r

e Na (V

9) were centrifuged 9) werecentrifuged andothers 0 i ), and unlabeled ), andunlabeled is the exclusion istheexclusion e is the elution is the elution Printed inGreatBritain mL (Cat. No. mL (Cat.No. peptides were peptides were +

0.1% 0.1%

(x-36 utilizing aC-terminalantibodythatbindsamidated was measuredusinganin-houseRIA(antibody89390) al. 2015a).Total GLP-1 directed) (Wewer Albrechtsenet and Mab26.1asadetectingantibody(NH2-terminally F5 ascatchingantibody(COOH-terminallydirected) ELISA involving two monoclonalantibodies: GLP-1 was measuredusinganin-housetwo-sitesandwich Proglucagon-derived gut peptides displaying nocross-reactivity with gastrin(Rehfeld1998). and tyrosyl-O-sulfatedCCK-58,-33,-22,-8)while which measures all bioactive CCK forms (i.e. amidated was measuredusinganin-houseRIA(antibody92128), Gut peptidesnotderivedfromproglucagon (Wewer Albrechtsenet al.2015b). with equalaffinityforOXandglicentin(antibody645) was measuredusingaC-terminallydirectedin-houseRIA prevent anyDPP-4-mediateddegradation.Glicentin/OX added tothesamples(finalconcentration0.01 valine pyrrolidide(agenerousgiftfromNovoNordisk)was et al. 2000).For measurements of intact GLP-1 and GLP-2, antibody thattargetsN-terminallyGLP-2(Hartmann an in-houseRIA(antibody92160),whichemploys (Orskov murine/porcine PYY standards and Bubendorf, Switzerland) that bindsmurinePYY, with using aside-viewingantibody (Cat.No.T-4093, Bachem, 2015). ForGLUTag andSTC-1,PYY thus measuringtotalNT(antibody 3D97)(Kuhre NT wasmeasuredusinganin-houseN-terminalRIA, amide, GLP-2(1–33),orglucagon(Deacon (3–42) orthestructurallyrelatedpeptides:GLP-1(7/936) of intactGIP(1–42),andcross-reacts< (antibody 98171), which reacts with the N-terminal part Intact, bioactiveGIPwasmeasuredusingin-houseRIA truncated metaboliteGIP(3–42)(Krarup&Holst1984 ). which reactsfullywithintactGIPandtheN-terminally using aC-terminallydirectedantiserum(code:80867-5), 125 peptides (Stadil&Rehfeld1973).GIP equimolar potencywithoutcross-reactivitywithCCK gastrins (gastrin-71,-34,-17,and-14)inplasmawith amidated) of gastrin-17andbindsallbioactive(i.e. (antibody 2604),whichisdirectedagainsttheC-terminus in CgA.Gastrinwasmeasuredusinganin-house RIA et al. 1999)targetingtheN-terminusofsequence340-348 assayed byin-houseRIA(antibody95058)(BørglumJensen as standard(Rehfeld1998).ChromograninA(CgA)was producing celllines Peptide productioninGLP1- Published byBioscientifica Ltd. I-labeled sulfatedCCK-8wasusedastracerand amide t al.1994).GLP-2(intact)wasmeasuredusing et ) but not glycine (x-37) extended GLP-1 isoforms Downloaded fromBioscientifica.com at09/29/202106:38:32PM 125 56 total total I-labeled porcine : 3 wasmeasured 0.1% withGIP wasmeasured GLP-1 (intact) t al.2000). et mM) to 203 t al. et CCK via freeaccess

Journal of Molecular Endocrinology 1 into 1– intestine asdescribedpreviously. Tissues werechopped at sitesinproximalanddistal smallintestineandlarge collected, whereastissuefor L-cell isolationwascollected 10– tissue from the upper small intestine (incorporating cold Leibovitz-15(L-15)medium.ForK-cellpopulation dislocationandthegutwascollected intoice- cervical In bothcases,miceaged2– ( K-cell populationswereisolatedfromYFP-Venus mice positive andL-cell-negativeepithelialcellpopulations. promoter (Reimann protein YFP-Venus under control of the proglucagon GLU-Venus transgenicmice,expressingthefluorescent Microarray (Holst &Bersani1991). 28 possess themidsequence,includinggut-derivedSST1 – 1758-5), which reacts with all molecular forms of SST that in-house RIAemployingaside-viewingantibody(code: 347-07) PP(Dirksen bind human(antibody7198)ormurineHYB measured usingin-houseRIAsemployingantibodiesthat Orskov respectively) as described previously (Brand human (NCI-H716)insulin(antibody2006-3 and 2004-3, RIAs, reactingwitheithermurine(GLUTag andSTC-1)or (Orskov the C-terminalofpancreaticglucagon(antibody4305) in-house RIAemployinganantibodydirectedagainst For gelfiltrationdata, glucagonwasmeasured usingan or truncatedforms(Wewer Albrechtsenetal.2014)). glucagon andneitherC-norN-terminallyelongated glucagon (thusonlymeasuringfullyprocessedpancreatic Sweden), targetingboththeN-andC-terminalof sandwich ELISA(Cat.No.10-1271-01,Mercodia, Uppsala, glucagon wasmeasuredusingacommercially available 2015). ForNCI-H716,PYY 240,PerkinElmer)(Svendsen NEX No. (Cat. PYY Pancreatic peptides Muckadell 1977). previously described(Fahrenkrug&Schaffalitzkyde polypeptide (VIP)wasmeasuredusingin-houseRIA human PYY (Torang RIA (code:MAB8500),reactingwiththemidpartof Parker DOI: 10.1530/JME-15-0293 http://jme.endocrinology-journals.org Research mg/mL collagenase XI in calcium-free Hanks balanced collagenaseXIincalcium-freeHanks balanced mg/mL 15 cm of gut, immediately distal to the pylorus was cm of gut, immediately distal to the pylorus was 2 et al. 1991).Pancreaticpolypeptide(PP)(total)was t al.2009)directedbythepreproGIP-promoter. et et al. 1991 ). Insulinwasmeasuredusingin-house

mm piece and digested twice for 30 for mm piece and digested twice 2008),wereusedtoobtainL-cell- et al. 2013).SSTwasmeasuredusing et al. t al.2015). Vasoactive intestinal et For content analysis (Fig. total wasquantifiedbyin-house 6

r months were killed by months werekilledby

min with t al. et 2),

presented asmean ± Cell contentandsecretiondata(Figs. Data analysis previously (Habib -2 expressionwasdeterminedbymicroarrayasdescribed mice withglucagon-drivenVenus expression.PC1/3and nonfluorescent isletcellsinsinglecellpreparationsfrom side scatter characteristics distinguishing them from other and were isolatedonthebasisoftheirsize,usingforward promoter, respectively(Adriaenssens was controlledbytheproglucagonandsomatostatin from mice in which the expression of fluorescent proteins Venus positiveα β 2008).Theα 2009,Reimannet al. 2012,Parkeret al. et al. dominated byenterocytes)asdescribedpreviously(Habib >95% pureVenus-positive or-negativecells(presumably High Wycombe, UK)wasusedtoseparatepopulationsof MoFlo cell sorter (488 in L-15supplementedwith10%(vol/vol)FBS.Cytomation centrifuged at300 filtered through70 salt solutionat37° fractions (expressedasK peptide concentrations(pM)ineachofthecollected (Fig. PC, prohormoneconvertase. from pancreaticmouseα-andβ-cellsGLUTag andSTC-1cells. collected fromidenticalanatomical sites asK-andL-cellswereisolated intestine, andcolonicL-cells(LC+),reference tissue(K–,LPandLC) mouse K-cells(K+)andproximalL-cells (LP+)collectedfromthesmall β-cells. ExpressionlevelsofPC1/3and PC2areshownforFACS-sorted murine K-andL-cells,nonepancreaticα- Microarray expressionofPC1/3andPC2inGLUTag andSTC-cells,isolated Figure 1 producing celllines Peptide productioninGLP1- -, andδ Published byBioscientifica Ltd. Expression by microarray 1000 1500 2000 2500 5000 7000 9000 500 1) asmeans,andgelfiltrationdata(Fig. 0 -cells wereisolatedasdescribedpreviously. Inbrief, K+ PC1/3 PC2

K- -cells andeYFP-positiveδ Prohormone convertaseexpression et al. 2012,Reimann2008). g C (Sigma-Aldrich). Cell suspensions were C (Sigma-Aldrich).Cellsuspensionswere μ LP+ for5 M nylon strainers (BD Biosciences) and M nylonstrainers(BDBiosciences)and s . e nm excitation;Beckman Coulter, . min, and pellets were resuspended min, andpelletswereresuspended

Downloaded fromBioscientifica.com at09/29/202106:38:32PM LP- m d values).Forcellcontentdata, . , microarrayexpressiondata LC+

LC-

GLUTag et al. 2015).Theβ 56 -cells were isolated -cells wereisolated : 3

STC-1 2 and 3)were

α 204 4) as -cells -cells β via freeaccess -, -, Journal of Molecular Endocrinology GraphPad Prism5andedited inAdobeIllustrator. considered tobesignificant. Graphswereconstructedin stimuli values against baseline. A statistical significancewasassessed bypairedt-test,testing (2–10 against therespectiveassay’s lowerlimitofdetection t-test, for each peptide testing the measured concentrations contents, statistical significance was tested by one-sample concentration (nonnormalized)values(pM)forcell in confluence.Secretiondatawereexpressedasabsolute concentration (mg/L)toadjustforintragroupvariation peptide concentrations(pM)werenormalizedtoprotein (total); SST, somatostatin(total);VIP, vasoactiveintestinal polypeptide. peptide-2; NT, neurotensin(total);PP, pancreaticpolypeptide; PYY, PYY oxyntomodulin; GLP-1,glucagon-likepeptide-1;GLP-2, chromogranin A;GIP(total),gastricinhibitorypeptide;GLI/OX,glicentin/ ***P ** P <0.01, *P <0.05, significance betweenbaselineandstimulantareindicatedwithstars; cells. Dataareshownasmean ± Gut andpancreatichormonecontentsinGLUTag, NCI-H716,andSTC-1 Figure 2 http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0293 Research C B A Pcg. derivedgutpeptides Peptide content

pM, depending on the assay). For secretion, Peptide content (pmol/mg total protein) Peptide content (pmol/mg total protein) (pmol/mg total protein) 10 20 30 0 1 2 3 0 1 2 3 4 5 GLP-1 GLP-10 GLP-1

GLI/OX GLI/OX GLI/OX **** ** **** **** **** GLP-2 GLP-2 **** GLP-2 **** **** ** CCK CCK CCK **** *** .0, **** P <0.0001. < 0.001,

CGA CGA CGA Gastrin Gastrin **** Gastrin ** s NCI-H716 Other gutpeptides . GLUTa * e . STC-1 m GIP GIP GIP **** . relativetobaseline.Levelof **** NT NT NT **** ** PYY PYY PYY r g

Glucagon Glucagon Glucagon e

Insulin Insulin Insulin ** andothers Printed inGreatBritain Pancreatic peptides

PP PP PP

SST **** SST SST **** **** < 0.05 was

baseline. The secretion of NT and SST was not altered by significantly enhancedthe secretioncomparedwith CgA wassecretedinlow concentrations,butKCl baseline vsKCl;P both stimulantswerecomparedwithbaseline(P concentrations and showed near significant increases after FSK/IBMX) (Fig. both stimulants(50 high concentrationsandtheirsecretionwasenhancedby GLUTag cellssecretedGLP-1,GLP-2, and glucagon in and STC-1cells Secretory repertoire ofGLUTag, NCI-H716, were PYYnegative. relatively highconcentrationsofVIP. Allthreecelllines but significantconcentrationsofCCK,gastrin,NT, and In addition,STC-1cellscontainedcomparativelylow, significantly higherthanthelevelsofGLP-1(P <0.001). and similarlevelsofintactGIP(datanotshown),both highlevelsoftotalGIP(Fig. STC-1 cellscontainedvery NCI-H716 cellsalsocontainedlowlevelsofCgAandNT. PP. GLUTag cellscontainedhighlevelsofCCKandNT. also storedglucagonandSST, butneitherinsulinnor in theothercells,glicentindominated.Allthreecelllines were glicentinandOXpresentincomparableamounts; in a1:1ratio in allcell lines, but only in NCI-H716 cells GLP-2, and glicentin/OX. GLP-1 and GLP-2 were present contained thegut-derivedproglucagonproductsGLP-1, relative amountsofeachpeptide.Allthreecelllines respect to the peptides they contained, as well as the Overall, thethreecelllinesvariedconsiderablywith Hormone contentofGLUTag, NCI-H716,andSTC-1cells PC1/3 andPC2,asexpected. β-cellsexpressedboth of PC2butnoPC1/3,whileprimary Primary both expressedPC2toabouthalfthelevelofPC1/3. GLUTag andSTC-1cellsexpressedPC1/3but,inaddition, not detected in K-orL-cells or inreference gut tissue. from sameanatomicalsites(Fig. intestine, respectivelybutnotinreferencetissuecollected murineL-cellsfromthesmallintestineandlarge primary murineK-cellsandby PC1/3 wasexpressedbyprimary GLUTag andSTC-1cells,α-β-cells Expression ofPC2andPC1/3inprimaryK-L-cells, Results producing celllines Peptide productioninGLP1- Published byBioscientifica Ltd. -cells from the mouse expressed very highlevels α-cells fromthemouseexpressedvery =0.086, baseline vs glucagon +FSK/IBMX). 3 mM KCland10 A). CCK was secreted in high A). CCKwassecretedinhigh Downloaded fromBioscientifica.com at09/29/202106:38:32PM 1). PC2expressionwas mM glucagon +10 mM 56 : 3 0.054, =0.054, 205 µ M via freeaccess 2) Journal of Molecular Endocrinology peptide YY; SST, somatostatin. methylxanthine; NT, neurotensin;PP, pancreaticpolypeptide; PYY, glucagon-like peptide-2;glu,glucagon;IBMX,3-isobutyl-1- GLI/OX, glicentin/oxyntomodulin;GLP-1,glucagon-likepeptide-1;GLP-2, CgA, chromograninA;FSK,forskolin;GIP, gastricinhibitorypeptide; ***P <0.001, **P <0.01, stimulant areindicatedwithstars;*P <0.05, shown asmean ± Secretory repertoireofGLUTag, NCI-H716,andSTC-1cells.Dataare Figure 3 and onlybyglucagon secretion ofSSTwassignificantly elevatedbystimulation large amountsofNT, glucagon,andSST, butonly the stimulants (Fig. and GLP-2theirsecretionwasenhancedbyboth response tothestimuli.NCI-H716cellssecretedGLP-1 the stimuli.GIPwasnotsecretedinbasalstateor in both stimuli.GIPsecretionwas notdetectedinanyofthe low concentrations andits secretion was enhanced by DOI: 10.1530/JME-15-0293 http://jme.endocrinology-journals.org C B A Research

Concentration (pM) Buffer (baseline) 100 200 300 400 500 Concentration (pM) Concentration (pM) 100 200 300 400 500 100 200 300 400 500 0 0 GLP-1 GLP-1 GLP-1 0 ** * **

s GLP-2 *

. GLP-2 GLP-2 e . m * * . Levelofsignificancebetweenbaselineand * 3 *

CCK *

B). NCI-H716alsosecretedrelatively CCK CCK

CgA CgA CgA 50 mMKCl ** + * FSK/IBMX. CgAwassecreted in Gastrin Gastrin Gastrin NCI-H716 GLUTag STC-1

GIP GIP GIP * * r

e NT NT NT 10 mMglu+10µMFSK/IBMX

SST SST SST ** * n =6. and ≈0.66,respectively)(Holst1983). glucagon secretion (P elevation ofthesecretionGLP-1comparedwithbasal but the stimulants onlycaused a borderlinesignificant treatment groups. STC-1 cells released GLP-1andGLP-2, glucagon (K material waselutedat the positionofunlabeledpancreatic for allthethreecelllines,majorityofimmunoreactive Gel filtrationpatternofextractableglucagonshowedthat, Processing ofproglucagon-derived patterns peptides secreted PYY. by oneorbothofthestimulants.Nonecelllines the basalstate,andforeach,secretionwasenhanced of SSTandCgAwerealsosecretedbytheSTC-1cellat baseline. Highconcentrationsofglucagonandlowlevels but neitherstimuluselevateditssecretioncomparedwith secreted inconcentrationssimilartoGLP-1andGLP-2, its secretionwaselevatedbybothstimulants.CCK both GIPassays)thanGLP-1atbaseline(P STC-1 cellsactuallysecretedmoreGIP(measuredusing positive controlreached significance (Fig. (K synthetic unlabeledGLP-1andGLP-2(Ørskov at elution positions close to that previously described for the majorityofimmunoreactivematerialwasalsofound the NCI-H716 cells (Holst 1983). For GLP-1 and GLP-2, immunoreactive moietieswerealsofound,particularlyin distal smallintestine,respectively (Pedersen in CCKandPYYexpression (mRNA)inproximaland intestinal L-cellsalsoresulted indramaticreductions al.2006).Supportingthis,cell-specific ablationof et Mortensen L-cell populationthatco-expressPYY(Habib CCK and/orGIPtosomedegree,andamoredistal peptides derivedfromPC1/3processing,oftenco-express intestinal populationthat,inadditiontotheproglucagon subpopulations of L-cellsseemtoexist:aproximalsmall expression analysisonFACS-sorted mouseL-cells,two studies of L-cells from human, porcine, and rat tissues and According todataobtainedfromimmunohistochemical Discussion K cell linesatelutionpositionsclosetopreviouslyreported peaks forglicentinandOXwereidentifiedallthree producing celllines Peptide productioninGLP1- d Published byBioscientifica Ltd. dGLP-1 valuesforunlabeledsyntheticglicentinandOX(≈0.35

≈ 0.55 and K + FSK/IBMX), while the GLP-2 response to the . 2015,Theodorakis . 2003,Svendsen et al et al

= d

0.054, baselinevsKCl;P ≈ 0.85),althoughsmallamountsoflarger dGLP-2 Downloaded fromBioscientifica.com at09/29/202106:38:32PM

≈ 0.50 (Buhl 56 .3 bsln vs baseline =0.13, 1988). Single et al. : 3 3 C). However, .1, and <0.01), . 2013). et al 2012, et al. et al. 1991) 206 via freeaccess Journal of Molecular Endocrinology were definedasV graphs. Foreachcellline,nineextracted cell-lysissampleswerepooled. immunoreactivity (pM)isplottedagainst coefficient ofdistribution (K Processing patternsofproglucagon-derived gutpeptides.Gelfiltration-basedcharacterizationofthe processing patternofproglucagon.Eluted Figure 4 http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0293 Research D C Glucagon B A

Hormoneconcentration (pM) GL Hormoneconcentration (pM) Hormoneconcentration (pM) GL

GL Hormoneconcentration(pM) 100 200 300 400 500 100 150 200 250 100 150 10 15 50 0 50 andV 0 0 5 0 0 I/ 0.00 0.0 0.00 0.0 P-2 P-1 OX V V V V 0 0 0 0 .1 GL i Distribution c 0.2 0.20 0.2 , respectively(indicatedongraphs). RepresentativeK Distribution coef Distribution coef Distribution coeff .2 .3 GLI UT 0.40 0.40 0.4 0.4 GL GL oeffic ag .5 P- P- Glucago 2 fi fi 0.6 0.60 0.6 icient (K cient (K 1 cient (K .6 ient (K OX r

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© 2016SocietyforEndocrinology 0.81 kuhre 0.8 0.80 0.8 n d d d ) ) ) d ) .9 andothers 1.0 1.0 1.0 V V V V .0 i i i i Printed inGreatBritain G E F H Hormoneconcentration (pM) Hormone concentration (pM) Hormoneconcentration (pM) Hormone concentration (pM) 100 200 300 400 200 400 600 100 150 20 40 60 80 50 0 0 0 0 0.00 0.00 0.0 0.00 V V V V 0 0 0 0 0.2 NCI-H716 Distribution coeffi Distribution coef Distribution coef Distribution coef .2 .2 .2 d ). Elutionfractioncontainingthecalibrators GLI 0.40 0.40 0.40 0.40 GL producing celllines Peptide productioninGLP1- GL Published byBioscientifica Ltd. P- d valuesforeachofthemeasuredpeptides areindicatedontopof P- fi 2 fi fi cient (K cient (K Glucag cient (K cient (K .6 .6 .6 .6 1 OX 0.81 0.81 0.81 0.81 on d d d d ) ) ) ) V V V V .0 .0 .0 .0 i i i i J K L Hormone concentration (pM) Hormone concentration (pM) Hormoneconcentration(pM) Hormone concentration (pM) I 10 20 30 40 50 10 10 20 30 10 15 20 0 0 2 4 6 8 0 0 5 0.00 0.00 0.00 0.00 V V V V Downloaded fromBioscientifica.com at09/29/202106:38:32PM 0 0 0 0 Distribution coef Distribution coeffi Distribution coef Distribution coef .2 .2 .2 .2 STC-1 GLI 22 0.40 0.40 0.40 0.40 Na and GL GL P- P- fi fi fi Glucag 2 cient (K cient (K cient (K cient (K .6 .6 .6 .6 56 1 125 OX : 3 I-labeled albumin on 0. 0. 0. 0. d d d d ) ) ) ) 81 81 81 81 V V V .0 V .0 .0 .0 i i i i 207 via freeaccess Journal of Molecular Endocrinology assays forPYYmeasurement in this study, weconsiderit lower limit of detection sensitivity (< highlevels,combinedwiththe study werepresentatvery However, giventhattheotherpeptides measuredinour level ofdetectionasreported bytheassaymanufacturers. measured concentrations that eitherare below oratthelower et al. 2013),butthestudybyGeraedts and secretionfromSTC-1cells(Geraedts A fewstudieshavereportedmeasurablePYYstorage in the lysatesorsecretionsamplesfromanyofcelllines. often co-localizes with GLP-1 in distal L-cells, in either ( in cellsexpressingCCK,GIP, GLP-1/2,NT, andPYY substance P, whileayetlaterlineagecommitmentresults then resultsincellsproducingghrelin/motilinand results in expression of SST. A later lineage commitment result fromanearlylineagecommitmentthatsolely express SST(Egerod unexpected, sincenaturalL-cellsarenotthoughtto incubation mediumofallthreecelllineswasentirely that arenot‘classical’L-cellproducts. three celllinesproduceandsecreteseveralotherpeptides that despiteproducingGLP-1andGLP-2asexpected,all 10 gutand4pancreaticpeptides.Themajorfindingis repertoireofthethreecelllinesbyassayingfor secretory sporadically beeninvestigatedpreviously. the peptide profiles of thevarious celllineshave only patterns reflectthoseofnaturalL-cells.Nevertheless, depends on whether the peptide contents and secretory value ofresultsgeneratedwiththesecelllinesnaturally characteristic ofnativeL-cells.Furthermore,thetranslational the polarization(apicalvsbasolateralsurfaces),whichis their limitations,inparticularwithrespecttolackof Reimann calcium andpatch-clampstudies(Reimann&Gribble2002, live they aresuitableforcellbiologicalstudies,instance, has been established. Such cell lines are useful because L-cells because nomethodforcultureofpureprimary mostly becausetheyareeasytomaintainincultureand frequently usedasmodelstostudyL-cellphysiology, than previouslythought. Therefore, it appearsthatL-cellsare more plurihormonal expression analysis inadditionto GIP (Parker Similarly, insortedmurineK-cells,PYYwasdetectedby Egerod DOI: 10.1530/JME-15-0293 http://jme.endocrinology-journals.org Research Unexpectedly, wedidnotdetectanyPYY, which The detectionofSSTinboththeextractsand We, therefore,characterizedthepeptidecontentand The GLUTag, NCI-H716,andSTC-1celllinesare monitoring of the intracellular concentration of free et al. 2012). . 2004).However, thesecelllinesalsohave et al t al.2012),asSST-producing cells et r

hormones doesnotnecessarily correlatetosecretory studies wereincluded,since cellularstorageofpeptide 1998, Sidhuet al.2000). Hand produce andsecreteCCK(Cordier-Bussat the abilityofGLUTag andinparticular STC-1cells,to the presenceofGLP-2inGLUTag and STC-1 cells,and Our results alsoconfirmdatafrom other studiesregarding 1994), insulin was not detected in any of the cell lines. previous study in GLUTag and STC-1 cells (Drucker ( any elongatedortruncatedsplicevariantsofglucagon) glucagon, anddetectsneitherglicentinnorOX(nor employed ishighlyspecificforfullyprocessedpancreatic contained inthemiddlepartofglicentin),butassay in theglucagonassay(theentiresequenceis The finding wasnotdue tocross-reactivityofglicentin pancreatic thatGLUTagthe observation andSTC-1cells,likethe glucagon in relatively high amounts,consistentwith GLUTag cells(Habib our previouslyreportedfailuretodetectGIPmRNAin GIP inGLUTag andNCI-H716cells,inagreementwith did notdetectsignificantamountsofstoredorsecreted epithelial cultures(Parker sucralose failedtostimulateGIPsecretionfrommixed sweetener sucralose(Margolskee reported fromGLUTag cellsinresponsetotheartificial . 2002).GIPsecretionhasalsobeen 1995, Ramshuret al glucose responsiveness(Cheung of stimuli,althoughthecelllinesdifferedintheir GIP- lines engineeredtoexpressinsulinundercontrolofthe neutralized (Kieffer enhanced whenco-secretedsomatostatinwasimmune glucose-dependent GIP-secretion,whichwasfurther enriched subclone(STC-6-14)wasestablished,exhibiting stained for GIP (Rindi ~ has been demonstrated previously, although only shown forintactGIP).ExpressionofGIPinSTC-1cells both N-andC-terminallydirectedassay(dataonly with equivalentconcentrationsbeingmeasuredusing in thehighestamountsfromSTC-1cellswasGIP, line mayshowvariabilitywithrespecttoPYYexpression. unlikely thatourresultsareerroneous.Instead,thiscell producing celllines Peptide productioninGLP1- t al.2014).Inaccordancewitha Wewer Albrechtsenet 7% ofthecellsintumorsgaverisetocellline Published byBioscientifica Ltd. A majorstrengthofthis study isthatsecretion All threecelllinesalsocontainedandsecreted Surprisingly, the peptide that was stored and released promoter secretedinsulininresponsetoanumber . . 2013,Nemoz-Gaillard et al . 2010,Handet al et al α -cell (butunlikenaturalL-cells),expressPC2. . 1995).OtherSTC-1-derivedcell et al et al. 2012). . 1990). Subsequently, a GIP- et al Downloaded fromBioscientifica.com at09/29/202106:38:32PM . 2009).Inthisstudy, we et al . . 2000,Kiefferet al et al t al.2007),although et 56 : 3 t al.1997, et 208 . et al via freeaccess Journal of Molecular Endocrinology paracrine directly fromthe perfused organs, where hormone secretion can be assessed models includinginvivostudies,orstudiesusingisolated caution and lines investigatedhereshouldbeinterpretedwithsome unsuccessful. In attempts tocultureisolated(mouse)L-cellshavebeen L-cells inresponseto to studythesecretory al . 2003).Itwouldbeofimmensescientificinterest et activating humanandratproglucagonpromoters(Cao seem toberegulatedbythesamemechanismsasthose the expressionofproglucagoninthiscelllinedoesnot stage of Supporting thatNCI-H716cellsrepresentanaberrant cells seemtoresembleneitherproximalnordistalL-cells. nor PYY, but most restricted rather than suggest thattheycouldbeusedasamodelforproximal express andsecretehighlevelsofCCKbutnotPYY, which suitable forGLP-1secretionstudies.GLUTag cellsalso concentrations. Therefore,thiscelllinemaybeless whereas GIPwasbothexpressedandsecretedinhigh secretion wasonlyweaklyelevatedbythestimulants, STC-1 cellsresembledL-cellstheleast,sinceGLP-1 of theexpressedpeptides.Ofthreecelllinestested, while NCI-H716 cells contain and secrete larger amounts different peptidesthanthe(human)NCI-H716cells, cells (derivedfrommice)containandsecretemore lines alsostoredandsecretedglucagon. 1991),althoughunlikenativeL-cells,allthreecellet al. ( glicentin, andOXasthatdescribedforhumanpig of theproglucagonprecursorintoGLP-1andGLP-2, lines havethesamePC1/3-dependentprocessingpattern In summary, ouranalysisindicatesthatallthreecell processing patternoftheproglucagon-derivedpeptides. strength ofthisstudyisthatwealsoanalyzedthe secreted differedonlytwo-tothree-fold.Another for NCI-H716cells),whereastheamountsactually in theamountsofpeptidesstored(6– Emphasizing this, the cell lines differed considerably be recruitedtotherapidlyreleasablevesiclepool. vesicles,whichthenhavetopeptide intosecretory sensing mechanismsandintracellularsortingofthe bymolecularactivation oftheexocytoticmachinery output, aspeptidesecretion,forinstance,dependson Buhl http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0293 Research Comparing thethreecelllines,GLUTag andSTC-1 et al. 1988,Holst1983,Orskovet al . 1987, Ørskov differentiation, anotherstudyconcludedthat relationships, and storing andsecretingSST. Therefore,NCI-716 distal nativeL-cells.NCI-H716showedthe preferably validatedinmore physiological peptide conclusion, resultsobtainedwiththecell relevant organ,whilevasculature, different stimuli,butunfortunately, pattern, expressingneitherCCK products fromisolatednative innervations arepreserved. innervations r

Cheung AT, Dayanandan B, Lewis JT, Korbutt GS, Rajotte RV,Cheung AT, Lewis JT, Korbutt GS, Dayanandan B, 2003Aberrant &Drucker DJ Irwin DM Choi C, Flock G, Cao X, Buhl T, Thim L, Kofod H, Orskov C, Harling H & Holst JJ 1988Naturally &Holst JJ Harling H Orskov C, Kofod H, Buhl T, Thim L, 9 June2015. Sessions oftheAmericanDiabetesAssociation,Boston,MA,5 – Some ofthefindingsthisstudywerepresentedat75thScientific Prior presentation E B M, FR,MG,JandHapprovedfinalversionofthemanuscript. FMG,JR,andHeditedrevised manuscript.REK,CFD, F R, REK preparedfiguresanddraftedthemanuscript.NJWA,CFD,EBM, R E K, N J W A, E B M, J F R, F R, and F M G produced and analyzed data. R EK,NJWA,andHwereresponsibleforideadesignofthestudy. Author contribution and theMRC(grantMRC_MC_UU_12012). supported bytheWellcome Trust (WT106262/Z/14/Z andWT106263/Z/14/Z) other aspectsofthisstudy. Work intheReimann/Gribble laboratorieswas body hadnoinfluenceonthedesign,conduction,interpretation,or for BasicMetabolicResearch(Novo Nordisk Foundation).Thefunding This studywassupportedbyagranttoJHfromtheNovoNordiskCentre Funding perceived asprejudicingtheimpartialityofresearchreported. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest JME-15-0293 This islinkedtotheonlineversionofpaperathttp://dx.doi.org/10.1530/ Supplementary data Adriaenssens A, Lam BY, Billing L, Skeffington K, Sewing S, Reimann F & Reimann F Sewing S, Skeffington K, Lam BY, Billing L, Adriaenssens A, References ne A, Dinjens WM, Pijls MJ, Linden EM, Rousch MM, Moerkerk P, Rousch MM, Linden EM, Pijls MJ, Dinjens WM, Bruïne A, Kristensen JS Warberg J, Svendsen I, Knigge U, Jorgensen PN, Brand CL, ofsequence- 1999Library T, &Rehfeld JF Børglum Jensen Hilsted L producing celllines Peptide productioninGLP1- Published byBioscientifica Ltd. 290 1959–1962. dependent insulinreleasefromgenetically engineeredKcells.Science 2000Glucose- Wolfe MM &Kieffer TJ Boylan MO, Bryer-Ash M, en.2002-0049) NCI-H716 cellline.Endocrinology regulation ofhumanintestinalproglucagongeneexpressioninthe 8621–8624. ofBiologicalChemistry human smallintestine.Journal occurring productsofproglucagon111–160intheporcine and 311–320. epithelium. mechanisms regulatingsomatostatin-producingD-Cellsinthegastric 2015Atranscriptome-ledexplorationofmolecular Gribble F endocrine differentiationincolorectalcancer. Virchows Archiv 1992NCI-H716cellsasamodelfor &Bosnian F Goeij APM ofPhysiology fed andfastedrats.AmericanJournal 1995Roleofglucagoninmaintenanceeuglycemia & Holst JJ Chemistry specific radioimmunoassaysforhumanchromograninA.Clinical 1301) 45 549–560. Endocrinology (doi:10.1126/science.290.5498.1959) 156 3924–3936. Downloaded fromBioscientifica.com at09/29/202106:38:32PM 144 2025–2033. (doi:10.1210/en.2015- 56 : 3 269 E469–477. (doi:10.1210/ 263

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