The glucagon-like peptide 1 receptor is essential for postprandial lipoprotein synthesis and secretion in hamsters and mice

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1 DOI.7/s-9-- ARTICLE The glucgon-like peptide receptor is essentil for postprndil lipoprotein synthesis nd secretion in hmsters nd mice J. Hsieh & C. Longuet & C. L. Bker & B. Qin & L. M. Federico & D. J. Drucker & K. Adeli Received: September 9 / Accepted: October 9 # Springer-Verlg 9 Abstrct Aims/hypothesis Glucgon-like peptide (GLP-) receptor (GLP-R) gonists nd dipeptidyl peptidse- (DPP-) inhibitors ttenute postprndil lipemi through mechnisms tht remin uncler. As dyslipidemi is contributing risk fctor for crdiovsculr disese in type dibetes, we exmined the mechnisms linking phrmcologicl nd physiologicl regultion of GLP- ction to control of postprndil lipid metbolism. J. Hsieh nd C. Longuet re joint first uthors; D. J. Drucker nd K. Adeli re joint senior co-uthors. Electronic supplementry mteril The online version of this rticle (doi:.7/s-9--) contins supplementry mteril, which is vilble to uthorised users. J. Hsieh : K. Adeli Deprtment of Biochemistry, University of Toronto, Toronto, ON, Cnd J. Hsieh : C. L. Bker : L. M. Federico : K. Adeli Moleculr Structure nd Function, Reserch Institute, The Hospitl for Sick Children, University of Toronto, Toronto, ON, Cnd C. Longuet : D. J. Drucker Deprtment of Medicine, Smuel Lunenfeld Reserch Institute, Mount Sini Hospitl, University of Toronto, Toronto, ON, Cnd D. J. Drucker () Mt Sini Hospitl SLRI, University Ave TCP-, Toronto, ON, Cnd MG X e-mil: d.drucker@utoronto.c B. Qin Beltsville Humn Nutrition Reserch Centre, Agriculturl Reserch Service, US Deprtment of Agriculture, Beltsville, MD, USA Methods Postprndil lipid synthesis nd secretion were ssessed in norml nd fructose-fed hmsters nd in wildtype mice tht were treted with or without sitgliptin. Apolipoprotein B- (ApoB-) synthesis nd secretion were lso exmined in primry enterocyte cultures. The importnce of exogenous vs endogenous GLP-R signlling for regultion of intestinl lipoprotein synthesis nd secretion ws ssessed in mice nd hmsters treted with the GLP-R gonist exendin-, the GLP-R ntgonist exendin(9-9) nd in Glpr +/+ vs Glpr / mice. Results Sitgliptin decresed fsting plsm tricylglycerol, predominntly in the VLDL frction, s well s postprndil tricylglycerol-rich lipoprotein (TRL)-tricylglycerol, TRLcholesterol nd TRL-ApoB- in hmsters nd mice. GLP- R ctivtion with exendin- lone lso decresed plsm nd TRL-ApoB- in hmsters nd mice, nd reduced secretion of ApoB- in hmster enterocyte cultures. Conversely, blockde of endogenous GLP-R signlling by the ntgonist exendin(9-9) or genetic elimintion of GLP- R signlling in Glpr / mice enhnced TRL-ApoB- secretion in vivo. Co-dministrtion of exendin(9-9) lso bolished the hypolipidemic effect of sitgliptin. Conclusions/interprettion Potentition of endogenous incretin ction vi DPP- inhibition or phrmcologicl ugmenttion of GLP-R signlling reduces intestinl secretion of tricylglycerol, cholesterol nd ApoB-. Moreover, endogenous GLP-R signlling is essentil for the control of intestinl lipoprotein biosynthesis nd secretion. Keywords Dipeptidyl peptidse-. Incretins. Lipids. Tricylglycerol Abbrevitions ApoB- Apolipoprotein B- DPP Dipeptidyl peptidse-.7/s-9-- Proof only-not for redistribution

2 FPLC Fst protein liquid chromtogrphy GIP Gstric inhibitory peptide GLP- Glucgon-like peptide GLP-R GLP- receptor TRL Tricylglycerol-rich lipoprotein Introduction The growing incidence of type dibetes is mjor problem [] nd my be ssocited with vriety of lipid bnormlities tht pose crdiovsculr disese risk fctors, including hypertricylglycerolemi, incresed levels of smll dense LDL nd low levels of HDL []. At the top of this cscde re intestinlly derived chylomicrons contining tricylglycerol-rich polipoprotein B- (ApoB-), which re secreted following ft ingestion nd peripherlly ctbolised to generte remnnt prticles []. Insulin resistnce is positively correlted with remnnt-like prticle cholesterol [] nd remnnt-like prticle tricylglycerol is gretly incresed in some dibetic popultions []. Thus, it is of interest to identify therpeutic strtegies tht cn limit postprndil tricylglycerol-rich lipoprotein (TRL) secretion. The ingestion of nutrients, including ft, provokes the secretion of gut-derived hormones, including two incretins: gstric inhibitory peptide (GIP) from duodenl K cells [] nd glucgon-like peptide- (GLP-) from ilel enteroendocrine L cells [7]. These peptides stimulte insulin secretion in glucose-dependent mnner nd preserve pncretic bet cell function nd mss in preclinicl studies, s reviewed []. A number of extr-pncretic effects hve lso been demonstrted following exogenous dministrtion of these two hormones. GLP- slows gstric emptying nd induces norectic effects [9], while GIP hs nbolic effects on dipose tissue [] nd regultes dipokine secretion []. However, GLP- nd GIP re rpidly inctivted by dipeptidyl peptidse- (DPP-)-medited clevge, thus limiting their durtion of ction. In type dibetes, the incretin response is blunted, due lrgely to reduced incretin ction []. Therefore, there is considerble interest in enhncing incretin ction for the tretment of type dibetes mellitus. DPP- inhibitors protect endogenous GLP- nd GIP from N-terminl degrdtion thereby prolonging their bioctivity. DPP- inhibition lowers fsting nd postprndil glycemi in ptients with type dibetes mellitus [, ]; hence there is considerble interest in understnding the effects of incretins nd DPP- inhibitors on plsm lipid profiles []. Acute dministrtion of GIP reduces circulting chylomicrons, probbly by promoting tricylglycerol ctbolism by dipose tissue [, 7]; GLP- lso ttenutes postprndil tricylglycerol secretion [, 9], lthough the exct underlying mechnisms re not cler. In the present study, we used wild-type mice nd hmster model of postprndil dyslipidemi [] to study the role of incretin ction in controlling intestinl lipid nd lipoprotein metbolism. The fructose-fed hmster exhibits intestinl overproduction of TRL [], long with berrnt insulin signlling in the bsorptive enterocyte []. Mice nd hmsters were treted with sitgliptin or exendin- to potentite incretin ction with or without exendin(9-9) to ssess the importnce of exogenous GLP- receptor (GLP- R) ctivtion vs endogenous bsl GLP-R signlling for regultion of intestinl lipoprotein metbolism. Methods Animls Mle Syrin golden hmsters (Mesocricetus urtus) weighing to g were purchsed from Chrles River (Montrel, QC, Cnd) nd housed individully with room lighting set for h light drk cycle nd free ccess provided to food nd wter. Animls were cclimtised for week prior to being given either stndrd chow diet or fructose-enriched pelleted hmster diet contining % fructose nd % csein (Dyets, Bethlehem, PA, USA) for dys to induce insulin resistnce []. Hmsters were rndomised to receive either sitgliptin phosphte monohydrte (Merck, Whitehouse Sttion, NJ, USA) ( mg/kg) or wter by orl gvge, ech given dily. Chow-fed hmsters were given sitgliptin for weeks while fructose-fed hmsters received sitgliptin for weeks. Blood ws collected in the morning for mbient (fed) mesurements nd fter h fst by retro-orbitl bleeding. The hmsters were then killed for the ex vivo protocol or underwent the in vivo protocol. All niml protocols were pproved by the Animl Ethics Committee of the Hospitl for Sick Children, University of Toronto. Wild-type C7BL/ J mice (Jckson Lbortory, Br Hrbor, ME, USA), Glpr / nd ge nd sex-mtched Glpr +/+ littermte controls fed norml chow were studied t to weeks of ge nd mintined under h light drk cycle. Tretments in mice used single dose of sitgliptin ( mg/kg body weight) or exendin- ( nmol/kg) (dministrtion, see figure legends). Assessment of intestinl lipoprotein production by in vivo Triton-WR9 infusion Hmsters were nesthetised with isoflurne dministered through vporiser. A cnnul ws inserted into the right jugulr vein, exteriorised t the bck of the neck, filled with heprinised sline ( IU/ml) nd seled. The hmsters were llowed to recover nd fter h fst, conscious hmsters were given μl olive oil lod vi orl gvge, followed min lter by vehicle.7/s-9-- Proof only-not for redistribution

3 (sline), exendin- ( nmol/kg) (Bchem, Torrnce, CA, USA), exendin(9-9) ( nmol/kg) (Bchem) or both, dministered vi intrperitonel injection, nd by seprte bolus of Triton-WR9 tht ws diluted % (wt/vol.) (. g/kg) nd injected into the jugulr vein to inhibit lipoprotein ctbolism nd uptke [, ]. Blood ( μl) ws collected from the jugulr cnnul into lithium heprincoted tubes (BD Biosciences, Frnklin Lkes, NJ, USA) t bseline nd t, nd min. The orl gvge, peptide dministrtion, Triton infusion nd blood collection were ll performed on conscious nimls in the bsence of nesthetics. The jejunum ws excised under isoflurne nesthesi. Mice fsted for or h were given μl olive oil orlly. At min fter gvge, blood smple ws collected vi the til vein (time ). Acute sitgliptin dministrtion ws given orlly to conscious mice min prior to olive oil gvge. At min fter ft lod, Triton WR-9 (. g/kg body weight of % [wt/vol.] solution prepred in PBS) ws injected vi the til vein with or without exendin- ( nmol/kg). Blood smples were collected vi til bleed without nesthetics for plsm tricylglycerol nd cholesterol ssy, nd ApoB- immunoblotting. At 9 min fter Triton WR-9 injection, mice were killed nd crdic puncture ws performed for plsm tricylglycerol nd cholesterol ssy, ApoB- western blot nlysis nd TRL purifiction s described below. Isoltion of tricylglycerol-rich lipoproteins To isolte the TRL frction of the plsm, blood smples were first centrifuged for min t C nd, g to seprte the plsm lyer. Plsm ( μl) ws overlyed with ml potssium bromide solution (density. g/ml) in ml ultrcentrifuge tube nd centrifuged for 7 min t, g nd t C using rotor (SW Ti; Beckmn Coulter, Mississug, ON, Cnd). The TRL frction (Svedberg flottion rte [Sf] >) ws collected s the top μl of the tube content. Chemiluminescent immunoblotting ApoB- immunoblotting ws performed on TRL frctions nd diluted plsm (:) by SDS-PAGE nlysis s previously described []. Membrnes were then incubted in electrochemiluminescence detection regents (GE Helthcre, Pisctwy, NJ, USA) nd exposed to Hyperfilm (Denville Scientific, Metuchen, NJ, USA). Quntittive nlysis ws performed using n AlphImger imging densitometer (Alph Innotech, Sn Lendro, CA, USA). Ex vivo metbolic lbelling of intct primry enterocytes Primry enterocytes were isolted from hmster intestinl tissue s described []. Resuspended villi were incubted with mmol/l glucose nd pmol/l exendin-. Hmster enterocytes were preincubted in methioninefree MEM t 7 C for min nd pulsed with. to. MBq/ml of [ S]methionine for min. The cells were then chsed with unlbelled mmol/l methionineenriched DMEM. Immunoprecipittion, SDS-PAGE nd fluorogrphy ApoB- immunoprecipittion nd SDS-PAGE were performed s described previously []. The ApoB- bnds were excised nd quntittive nlysis ws performed using liquid scintilltion counter. No mjor differences were noticed in the trichlorocetic cid protein precipittion counts between the control nd experimentl groups. Medi counts were normlised to initil cellulr trichlorocetic cid counts. Rdioctivity incorportion into ApoB- ws lso visulised with the Storm Phosphor Imger (Moleculr Dynmics, Sunnyvle, CA, USA). Plsm mesurements Plsm tricylglycerol nd cholesterol were determined by n enzymtic-bsed colorimetric ssy (Rndox, Crumlin, UK). Fst protein liquid chromtogrphy of plsm lipoproteins Hmster plsm ( μl) ws filtered through. μmol/l micro-spin polysulphone filter (Alltech; Mndel Scientific, Lchine, QC, Cnd) to remove mcroprticles. Plsm ws then subjected to gel filtrtion through HR / GL Superose column (Phrmci, Uppsl, Sweden) with solution of mmol/l Tris, mmol/l NCl, mmol/l CCl,μmol/l DTPA nd.% NN (wt/vol.), ph 7. pumped t flow rte of. ml/min. Sttisticl nlysis All results re presented s men±sem. Sttisticl comprisons were performed using Student s t test when compring two groups nd two-wy ANOVA with the Bonferroni post hoc test s indicted in the text nd figure legends. Results A DPP- inhibitor ttenutes dyslipidemi in fructose-fed hmsters As incresed incretin ction cn ttenute postprndil plsm lipid ccumultion [, 9, ], we exmined the consequences of DPP- inhibition for ccumultion of tricylglycerol nd cholesterol in hmsters fed high-fructose diet (%), which promotes dyslipidemi nd mild insulin resistnce []. A high-fructose diet for dys prior to strt of sitgliptin tretment produced significnt increse in plsm tricylglycerol nd cholesterol (p=. nd p=., respectively) (Fig. b, c), without chnges in blood glucose or body weight (Tble ).7/s-9-- Proof only-not for redistribution

4 b c Sitgliptin mg/kg or vehicle Chow feeding Sitgliptin mg/kg or vehicle % Fructose-feeding or Time (weeks) Plsm tricylglycerol (mmol/l) d e f Sitgliptin mg/kg or vehicle % Fructose-feeding Time (weeks) Tricylglycerol (mmol/l) Plsm totl cholesterol (mmol/l) Bseline Endpoint Bseline Endpoint Cholesterol (mmol/l) VLDL LDL HDL VLDL LDL HDL Lipoprotein Lipoprotein Fig. Chnges in plsm lipids following chronic dministrtion of sitgliptin. Blood smples were drwn from hmsters fed either regulr chow or high-fructose diet supplemented with vehicle or sitgliptin for to weeks. b Plsm tricylglycerol nd cholesterol (c) levels were determined prior to nd fter ech dosing period. White brs, chow vehicle; htched brs, chow sitgliptin; blck brs, fructose vehicle; crossed brs, fructose sitgliptin. d Lipoproteins were seprted from plsm of hmsters fed high-fructose diet supplemented with vehicle or sitgliptin s shown. Seprtion ws by FPLC frctiontion into VLDL/chylomicron remnnts, LDL nd HDL. e Levels of lipoprotein tricylglycerol nd cholesterol (f) were determined. Blck brs, vehicle; crossed brs, sitgliptin. n=9 for plsm lipid vribles nlysed by pired t test (b, c); n= for FPLC vribles nlysed by Student s t test (e, f); p<. fructose-fed sitgliptin vs fructose-fed vehicle; p<. fructose-fed vehicle vs chow-fed vehicle or fructose-fed sitgliptin vs chow-fed sitgliptin nd no differences in postprndil glucose excursions (dt not shown). After weeks of sitgliptin tretment, the fructose-induced increse of plsm tricylglycerol, but not tht of plsm cholesterol ws significntly ttenuted (p<.) (Fig. b, c). Fst protein liquid chromtogrphy (FPLC) ws performed to seprte the vrious densities of lipid prticles. In fructose-fed hmsters, levels of VLDL- nd LDLtricylglycerol nd -cholesterol were significntly higher thn in control nimls (dt not shown). Sitgliptin significntly reduced levels of VLDL-tricylglycerol by threefold (Fig. e). LDL- nd HDL-tricylglycerol levels were slightly but not significntly decresed by sitgliptin tretment (Fig. e). Wheres totl plsm cholesterol ws not ltered by sitgliptin, LDL-cholesterol ws reduced nd significnt decrese in VLDL- nd HDL-cholesterol (Fig. f, d) ws observed in sitgliptin-treted vs control nimls (p<.). A DPP- inhibitor decreses intestinl production of TRLtricylglycerol nd TRL-cholesterol To understnd how sitgliptin modultes plsm lipoprotein levels, we ssessed intestinl lipoprotein production nd secretion fter tretment of chow fed hmsters with sitgliptin for weeks. Sitgliptin slightly reduced TRL cholesterol (Fig. b) nd ApoB- (Fig. d) in TRL frction fter n cute ft lod, while the ccumultion of TRL-tricylglycerol ws unffected (Fig. c). In contrst, sitgliptin significntly reduced the mount of tricylglycerol in TRL frction 9 min fter the ft lod (Fig. g) in fructose-fed hmsters nd lowered TRL-cholesterol (Fig. f) nd ApoB- (Fig. h) compred with vehicle-treted high-fructose fed nimls. Slope clcultions for TRL-cholesterol nd -tricylglycerol secretion in fructose-fed hmsters (cholesterol: 7.±. vs.±.7 mmol l min, sitgliptin vs control; tricylglycerol.7±. vs.±.7 mmol l min, sitgliptin vs control) suggested Tble Body mss nd blood glucose in chow-fed nd fructose-fed hmsters chroniclly treted with sitgliptin Vlues re men±se, n= per group None of the vlues were sttisticlly significnt s determined by two-wy ANOVA Chrcteristics Chow-fed Fructose-fed Vehicle Sitgliptin Vehicle Sitgliptin Body weight (g) Bseline.±. 9.±..7±.9.±. Endpoint.±. 9.±.7 9.±. 9.±. Fsting glucose (mmol/l) Bseline.7±..±..±..±. Endpoint.7±..±..±..±..7/s-9-- Proof only-not for redistribution

5 Sitgliptin mg/kg or vehicle e b c d TRL-cholesterol (mmol/l) TRL-tricylglycerol (mmol/l) f g h TRL-cholesterol (mmol/l).... Chow feeding Sitgliptin mg/kg or vehicle % Fructose-feeding. 9 then Time (weeks) then Time (weeks) TRL-tricylglycerol (mmol/l) TRL-ApoB- mss (rbitrry units 7 ) TRL-ApoB- mss (rbitrry units 7 ) Fig. Chnges in TRL lipid mss following chronic sitgliptin dministrtion. Hmsters were fed regulr chow (b d) or(e) highfructose diet (f h) nd dministered either vehicle or sitgliptin for to weeks. Hmsters were then ft-loded, dministered Triton WR- 9 nd blood ws drwn t,,, 9 nd min (, e). Plsm ws spun to isolte TRL frction nd levels of cholesterol, tricylglycerol nd ApoB- were determined. Grphs show TRL levels of (b) cholesterol, (c) tricylglycerol nd (d) ApoB- in chowfed hmsters given vehicle (white circles) or sitgliptin (blck circles). f Cholesterol, (g) tricylglycerol nd (h) ApoB- content of TRL frctions from fructose-fed hmsters given vehicle (white squres) or sitgliptin (blck squres). n= for ech group; p<. s nlysed by two-wy ANOVA with the Bonferroni post hoc test tht sitgliptin retrds intestinl postprndil cholesterol nd tricylglycerol secretion. To determine whether the effect of sitgliptin on plsm levels of cholesterol nd tricylglycerol ws restricted to hmsters, chylomicron production ws ssessed in chowfed mice. Sitgliptin significntly decresed cholesterol in plsm (Fig. b) nd in the TRL-enriched frction (Tble ) fter n cute ft lod; furthermore, plsm tricylglycerol (Fig. c) nd TRL-tricylglycerol (Tble ) were significntly reduced 9 min fter triton injection in sitgliptintreted mice. The ccumultion of ApoB- in plsm ws lso decresed by sitgliptin (Fig. d). Phrmcologicl ctivtion of GLP-R mimics the effects of sitgliptin on intestinl lipid bsorption As GLP- nd GIP regulte plsm lipid levels nd re DPP- substrtes, we mesured intestinl lipid bsorption in mice fter cute dministrtion of the DPP--resistnt peptides, D-Al -GIP nd exendin-. As shown in Fig., D-Al -GIP significntly incresed levels of plsm tricylglycerol nd ApoB- fter n cute ft lod (Fig. b, c). GIP, therefore, could not hve been responsible for the ctions of sitgliptin on intestinl lipid bsorption. In contrst, the GLP-R gonist exendin- reduced plsm nd TRL-tricylglycerol nd ApoB- ccumultion (Fig. e, f, Tble ), but no effect ws noted on plsm nd TRL-cholesterol (Fig. c, Tble ). Hence, ctivtion of GLP-R but not GIP receptor signlling mimics the ctions of sitgliptin on intestinl lipid bsorption. Insulin nd glucgon re involved in the regultion of lipemi nd ctivtion of GLP-R signlling hs been shown to stimulte insulin secretion nd inhibit glucgon secretion. Accordingly, we mesured plsm insulin nd.7/s-9-- Proof only-not for redistribution

6 Sitgliptin mg/kg or vehicle 9 Plsm cholesterol (mmol/l) b c d 7 ApoB- mss (rbitrry units 7 ) Fig. Chnges in postprndil lipid secretion following single cute dministrtion of sitgliptin in mice. Wild-type mice fsted for h were given mg/kg of sitgliptin (blck circles) prepred in wter by orl gvge or wter only (white circles). At min fter sitgliptin tretment, mice were dministered μl olive oil nd injected intrvenously min lter with Triton WR9 (. g/kg ). Blood smples were collected prior to i.v. injection (time ), s well s t, nd 9 min fter injection for mesurement of plsm (b) cholesterol, (c) tricylglycerol nd (d) ApoB- s described. n= per group; p<., p<. s determined by two-wy ANOVA with the Bonferroni post hoc test Tble TRL frction lipid msses determined by colorimetric ssys nd TRL-ApoB- mss determined by immunoblotting in mouse studies Tretment group TRL-tricylglycerol (mmol/l) TRL-cholesterol (mmol/l) TRL-ApoB- mss (% control) A Control.±..±..±. Sitgliptin.±..7±.7 7.±. B Control.±..±..±. Insulin.9±.7 n.d. n.d. D-Al -GIP.7±..9±. 7.±.9 Exendin-.±..9±. 9.7±. C Control.±. n.d..±. Exendin-.±. n.d. 7.±7. D Wild-type.7±. n.d..±. Glpr /.±. n.d..±. Vlues re shown s men±sem Tretment groups: A: Mice were given sitgliptin min prior to ft lod. For methods nd plsm levels of tricylglycerol, cholesterol nd ApoB-, see Fig. B: Mice were given insulin, D-Al -GIP or exendin- min fter ft lod. For plsm levels of tricylglycerol, cholesterol nd ApoB-, see Fig. e nd ESM Fig. b C: Mice were given exendin- h fter ft lod. For plsm levels of tricylglycerol nd ApoB-, see Fig. f, g D: Glpr / mice nd littermte controls were given n orl ft lod to monitor intestinl lipid bsorption. For tricylglycerol nd ApoB- plsm levels, see Fig. c, d p<. vs control of respective group s determined by Student s t test n.d., not determined.7/s-9-- Proof only-not for redistribution

7 D-Al GIP, Exendin- or PBS 9 Plsm cholesterol (mmol//l) Exendin- or PBS 9 d e f ApoB- (% of bsl) ApoB- (% bseline) g h i b c ApoB- (% of bsl) 7 Fig. Phrmcologicl ctivtion of GLP-R mimics the effect of sitgliptin on postprndil lipid excursion. Wild-type mice fsted for h were gvged with olive oil t ( f) or min (g i) nd injected (t min) with triton WR-9 (. g/kg body weight) nd with (b, c) D-Al -GIP ( nmol/kg, smll blck squres) or (d f) exendin- ( nmol/kg, lrge blck squres). White symbols, control. Blood smples were collected prior to i.v. injection (time ), s well s t, nd 9 min fter injection. (b, e, h) Tricylglycerol, (d) cholesterol nd (c, f, i) ApoB- secretion were mesured. n= per group; p<. s determined by two-wy ANOVA with the Bonferroni post hoc test glucgon levels fter n orl ft lod, with or without sitgliptin or exendin- tretment. Both sitgliptin nd exendin- significntly incresed plsm insulin levels compred with controls (Electronic supplementry mteril [ESM] Fig. ). However, this trnsient difference in plsm insulin levels is unlikely to ccount for the effect of sitgliptin or exendin- on intestinl lipid bsorption or for the difference in mgnitude of the effect of those two tretments, s plsm insulin levels were no longer significntly different between the groups min fter the ft lod (ESM Fig. ). More importntly, exogenous insulin dministrtion hd no effect on tricylglycerol ccumultion in mice (ESM Fig. b). Furthermore, cute dministrtion of sitgliptin or exendin- to fsted mice in ssocition with subsequent orl ft lod hd no significnt effect on plsm glucgon levels (ESM Fig. c). As exendin- is rpid nd potent inhibitor of gstric emptying, which in turn might contribute to decresed intestinl lipid bsorption, we monitored intestinl lipid bsorption when exendin- ws dministered h fter the orl ft lod to fcilitte entry of olive oil into the mouse smll bowel. Under these conditions, exendin- still significntly decresed tricylglycerol nd ApoB- ccumultion in plsm (Fig. h, i) nd TRL frction (Tble ). Endogenous GLP-R signlling is required for control of postprndil lipemi We next investigted the role of GLP- in the regultion of intestinl lipid bsorption in mice nd hmsters fter n cute ft lod. Exendin- decresed tricylglycerol nd cholesterol in VLDL/chylomicron remnnt-sized lipoproteins in chow-fed hmsters not given bolus of Triton WR-9 (Fig. b) nd significntly reduced levels of ApoB- in the TRL frction 9 min fter ft lod (Fig. c). The GLP-R ntgonist, exendin(9-9), blocked the exendin--medited reduction of TRL-ApoB- nd, dministered lone, ugmented levels of TRL-ApoB- min fter ft lod (Fig. c), indicting tht endogenous bsl GLP-R signlling modultes postprndil lipemi. Consistent with dt obtined using the GLP-R ntgonist exendin(9-9) in hmsters, tricylglycerol ccumultion in plsm (Fig. e) nd TRL frction (Tble ) ws significntly enhnced in Glpr / mice compred with Glpr +/+ littermte controls, despite the fct tht Glpr / mice hve gstric emptying rte similr to Glpr +/+ littermte controls [7]. There ws lso significntly greter.7/s-9-- Proof only-not for redistribution

8 Exendin- nd/or Exendin(9-9) or PBS 9 d 9 g Exendin(9-9) or PBS Sitgliptin mg/kg or vehicle 9 b Lipids (mmol/l) e h Plsm cholesterol (mmol/l) Frction VLDL LDL HDL c TRL-ApoB- mss (% bseline) f ApoB- (% of bsl) i 9 Fig. Role of GLP- in postprndil lipemi nd sitgliptin ction. c Acute effect of the GLP-R gonist exendin- nd ntgonist exendin(9-9) on ApoB- metbolism in chow-fed hmsters. (b) Representtive FPLC profile of plsm from chow-fed hmsters injected intrperitonelly with exendin- without intrvenous Triton WR-9. (c) Hmsters were chllenged with ft lod prior to Triton WR-9 dministrtion. TRL ApoB- mss ws mesured by immunoblotting. Dt re normlised to bseline vlues for ech tretment. n= per group; p<. for exendin(9-9) or exendin(9-9)+exendin- vs control; p<. exendin- vs control s determined by two-wy ANOVA with the Bonferroni post hoc test. d f Glpr / mice (blck squres) or littermte control mice (white dimonds) fsted for h were given orlly μl of olive oil. At min fter gvge, mice were injected i.v. with Triton WR9 (. g/kg). Blood smples were TRL ApoB- mss in GLP-R-deficient mice (Tble ). Therefore, the modultory ctions of bsl GLP-R signlling on intestinl lipid bsorption re not due to regultion of gstric emptying nd implicte the endogenous GLP-R in the control of intestinl lipid bsorption. Intct GLP-R signlling is required for the hypolipidemic ction of sitgliptin To determine whether the ctions of sitgliptin on postprndil lipid secretion required GLP-R signlling, the GLP-R ntgonist exendin(9-9) ws codministered with sitgliptin min before the ft lod. Sitgliptin ttenuted postprndil tricylglycerol excursions in mice (Fig. i), but did not significntly ffect plsm cholesterol levels (Fig. h). Furthermore, the GLP-R ntgonist exendin(9-9) eliminted the hypolipidemic ctions of sitgliptin on plsm tricylglycerol levels (Fig. i). collected prior to i.v. injection (time ), s well s t, nd 9 min fter injection. Grphs show tricylglycerol (e) nd ApoB- (f) mesurement s described. n= per group; p<. s determined by two-wy ANOVA with the Bonferroni post hoc test. g i Mice were fsted for h nd dministered sitgliptin ( mg/kg; blck circles) or vehicle (white circles) by orl gvge. GLP-R signlling ws blocked by co-dministering the GLP-R ntgonist exendin9-9 (blck dimonds) or PBS (white squres) by intrperitonel injection. After min ( min), mice were chllenged with n orl ft lod nd given Triton WR-9 by intrvenous injection t min. (h) Cholesterol nd (i) tricylglycerol secretion ws mesured s described. n= per group; p<., p<. sitgliptin vs control or sitgliptin+exendin (9-9) s determined by two-wy ANOVA with the Bonferroni post hoc test Cellulr S-lbelled ApoB- (normlised to TCA count) b Medi S-lbelled ApoB- (cpm normlised to TCA count) 9 Fig. Direct effects of exendin- on intestinl ApoB- metbolism ex vivo. Enterocytes from chow-fed hmsters were nlysed. () Cellulr nd secreted (b) levels of ApoB- were determined in primry enterocytes metboliclly lbelled with [ S]methionine in medium contining pmol/l exendin-. Direct tretment with exendin- significntly decresed secretion in newly synthesised ApoB-. White circles, control; blck circles, exendin-. n= per group; p<., p<. s determined by two-wy ANOVA with the Bonferroni post hoc test.7/s-9-- Proof only-not for redistribution

9 Exendin- directly reduces enterocyte ApoB- secretion We next evluted the effect of exendin- tretment on enterocytes isolted from chow-fed hmsters. Pulse chse studies in primry enterocytes showed tht cellulr ApoB- ws not chnged in exendin--treted hmsters (Fig. ). In contrst, exendin- decresed the mount of secreted ApoB- from enterocytes of chow-fed hmsters (Fig. b) (p<. t min, p<. t 9 min). Hence GLP-R ctivtion directly regultes enterocyte ApoB- secretion ex vivo. Discussion Dipeptidyl peptidse- inhibitors exert their ctions in prt vi ugmenttion of GLP- ction, which leds to reduction of glucgon, increses in insulin nd reduced glycemi in humn prticipnts [, 9]. Moreover, GLP- dministrtion reduced postprndil circulting lipid levels in humn prticipnts [], lthough the underlying mechnisms remin uncertin. In the current study, we found tht sitgliptin decresed tricylglycerol ccumultion, specificlly in the VLDL frction, in hmsters nd mice. Sitgliptin tretment lso decresed ApoB- ccumultion in plsm, specificlly in the intestinlly produced TRL frction. Tretment with DPP- inhibitors cn significntly decrese postprndil tricylglycerol, cholesterol nd ApoB- levels in humns [, ]; however, DPP- inhibition hs miniml effects on fsting lipid levels []. Our dt indicte tht prominent spect of sitgliptin s effects on plsm tricylglycerol levels is the decrese in levels of VLDL. This suggests tht the mjor effect of ugmenting GLP- ction is the reduction in number nd/or size of lrge TRL prticles. Our observtions demonstrte tht sitgliptin nd exendin- exert qulittively similr effects on postprndil lipid profiles, consistent with the notion tht incresed GLP- ction is likely to be responsible for the reduced circulting lipid levels nd reduced intestinlly derived TRL observed following sitgliptin dministrtion. Nevertheless, exendin- more potently reduced postprndil tricylglycerol synthesis nd secretion compred with sitgliptin, consistent with the reltively greter GLP-R ctivtion chieved with exendin- reltive to sitgliptin. Moreover, inhibition of DPP- ctivity is lso ssocited with potentition of GIP ction, which might be expected to prtilly ttenute the hypolipidemic effects medited through the GLP-R. Although incresed levels of insulin my lso reduce plsm tricylglycerol levels nd ApoB- secretion from the intestine [], dministrtion of exogenous insulin did not ffect intestinl lipid bsorption (ESM Fig. b), ruling out insulin s meditor of sitgliptin s or exendin- s effects on postprndil lipemi. We lso present dt clrifying the puttive role(s) of GIP or glucgon in the ctions of sitgliptin on intestinl lipid secretion. Although glucgon hs been reported to inhibit lipid secretion from heptocytes [], circulting levels of glucgon were not reduced following sitgliptin dministrtion in our current experiments. Furthermore D-Al -GIP did not reduce, but ctully promoted postprndil tricylglycerol nd ApoB- secretion. These experiments, tken together with dt using exendin(9-9) nd Glpr / mice implicte GLP- s the predominnt meditor of sitgliptin ction on intestinl ApoB- secretion. Our dt lso demonstrte overlpping effects of sitgliptin nd exendin- tretment on plsm nd TRL lipoprotein levels. Sitgliptin decresed cholesterol, tricylglycerol nd ApoB- levels in the TRL frction of fructose-fed hmsters. Similrly, exendin- decresed ApoB- secretion in chow-fed hmsters nd in freshly isolted primry enterocyte cultures. These observtions suggest for the first time tht GLP- directly regultes lipoprotein ssembly nd/or secretory mchinery in the enterocyte. However, dditionl work is required to understnd the moleculr mechnism underlying the suppressive effect of GLP- on ApoB--TRL production. An importnt spect of our studies is the demonstrtion tht reduction or elimintion of GLP-R signlling results in detectble chnges in postprndil lipoprotein profiles. Specificlly, dministrtion of exendin(9-9) lone resulted in incresed levels of TRL ApoB- in hmsters, wheres levels of ApoB- nd tricylglycerol mss were incresed in Glpr / compred with Glpr +/+ mice. Hence, these findings estblish n essentil role for bsl levels of GLP-R signlling in the control of intestinl lipoprotein synthesis/secretion in vivo. In summry, our dt support n importnt role for the GLP- R signlling system in regulting intestinl lipid nd lipoprotein metbolism. Augmenttion of GLP-R signlling lowers postprndil circulting levels of tricylglycerol, cholesterolrich TRL nd chylomicrons contining ApoB-. The current study suggests tht enhnced GLP- ction, chieved vi DPP- inhibition or use of GLP-R gonists, my contribute to control of postprndil lipid excursion through control of intestinl lipoprotein synthesis nd secretion. Acknowledgements This work ws supported in prt by CIHR operting grnts MOP-9 (to K. Adeli) nd MOP-7 (to D. J. Drucker), nd by the Cnd Reserch Chirs Progrm (D. J. Drucker). Dulity of interest D. J. Drucker hs served s consultnt to Merck Inc. The remining uthors declre tht there is no dulity of interest ssocited with this mnuscript. References. Zimmet P, Alberti KG, Shw J () Globl nd societl implictions of the dibetes epidemic. Nture :7 77.7/s-9-- Proof only-not for redistribution

10 . Ginsberg HN () Insulin resistnce nd crdiovsculr disese. J Clin Invest :. Mero N, Syvnne M, Tskinen MR (99) Postprndil lipid metbolism in dibetes. Atherosclerosis (Suppl ):S S. Ohnishi H, Sitoh S, Tkgi S et l () Reltionship between insulin-resistnce nd remnnt-like prticle cholesterol. Atherosclerosis :7 7. Schefer EJ, McNmr JR, Shh PK et l () Elevted remnnt-like prticle cholesterol nd triglyceride levels in dibetic men nd women in the Frminghm Offspring Study. Dibetes Cre : Lu WJ, Yng Q, Sun W et l () Using the lymph fistul rt model to study the potentition of GIP secretion by the ingestion of ft nd glucose. Am J Physiol Gstrointest Liver Physiol 9: G G 7. Ikoubov R, Izzo A, Yeung A, Whiteside CI, Brubker PL (7) Protein kinse Czet is required for oleic cid-induced secretion of glucgon-like peptide- by intestinl endocrine L cells. Endocrinology :9 9. Drucker DJ () The biology of incretin hormones. Cell Metb : 9. Bggio LL, Hung Q, Brown TJ, Drucker DJ () Oxyntomodulin nd glucgon-like peptide- differentilly regulte murine food intke nd energy expenditure. Gstroenterology 7:. Miywki K, Ymd Y, Bn N et l () Inhibition of gstric inhibitory polypeptide signling prevents obesity. Nt Med :7 7. Hnsoti T, Mid A, Flock G et l (7) Extrpncretic incretin receptors modulte glucose homeostsis, body weight, nd energy expenditure. J Clin Invest 7:. Meier JJ, Nuck MA () Incretins nd the development of type dibetes. Curr Dib Rep :9. Aschner P, Kipnes MS, Lunceford JK et l () Effect of the dipeptidyl peptidse- inhibitor sitgliptin s monotherpy on glycemic control in ptients with type dibetes. Dibetes Cre 9: 7. Rz I, Hnefeld M, Xu L et l () Efficcy nd sfety of the dipeptidyl peptidse- inhibitor sitgliptin s monotherpy in ptients with type dibetes mellitus. Dibetologi 9: 7. Amori RE, Lu J, Pitts AG (7) Efficcy nd sfety of incretin therpy in type dibetes: systemtic review nd met-nlysis. JAMA 9:9. Wsd T, McCorkle K, Hrris V et l (9) Effect of gstric inhibitory polypeptide on plsm levels of chylomicron triglycerides in dogs. J Clin Invest : 7 7. Kim SJ, Nin C, McIntosh CH (7) Activtion of lipoprotein lipse by glucose-dependent insulinotropic polypeptide in dipocytes. A role for protein kinse B, LKB, nd AMP-ctivted protein kinse cscde. J Biol Chem :7 7. Meier JJ, Gethmnn A, Gotze O et l () Glucgon-like peptide bolishes the postprndil rise in triglyceride concentrtions nd lowers levels of non-esterified ftty cids in humns. Dibetologi 9: 9. Qin X, Shen H, Liu M et l () GLP- reduces intestinl lymph flow, triglyceride bsorption, nd polipoprotein production in rts. Am J Physiol Gstrointest Liver Physiol :G9 G99. Tghibiglou C, Rshid-Kolver F, Vn Iderstine SC et l () Heptic very low density lipoprotein-apob overproduction is ssocited with ttenuted heptic insulin signling nd overexpression of protein-tyrosine phosphtse B in fructose-fed hmster model of insulin resistnce. J Biol Chem 77:79. Hidri M, Leung N, Mhbub F et l () Fsting nd postprndil overproduction of intestinlly derived lipoproteins in n niml model of insulin resistnce. Evidence tht chronic fructose feeding in the hmster is ccompnied by enhnced intestinl de novo lipogenesis nd ApoB-contining lipoprotein overproduction. J Biol Chem 77:. Federico LM, Nples M, Tylor D, Adeli K () Intestinl insulin resistnce nd berrnt production of polipoprotein B lipoproteins in n niml model of insulin resistnce nd metbolic dyslipidemi: evidence for ctivtion of protein tyrosine phosphtse-b, extrcellulr signl-relted kinse, nd sterol regultory element-binding protein-c in the fructose-fed hmster intestine. Dibetes :. Tghibiglou C, Crpentier A, Vn Iderstine SC et l () Mechnisms of heptic very low density lipoprotein overproduction in insulin resistnce. Evidence for enhnced lipoprotein ssembly, reduced intrcellulr ApoB degrdtion, nd incresed microsoml triglyceride trnsfer protein in fructose-fed hmster model. J Biol Chem 7:. Tggrt C, Gibney J, Owens D et l (997) The role of dietry cholesterol in the regultion of postprndil polipoprotein B levels in dibetes. Dibet Med :. Zoltowsk M, Ziv E, Delvin E et l () Cellulr spects of intestinl lipoprotein ssembly in Psmmomys obesus: model of insulin resistnce nd type dibetes. Dibetes :9. Ebert R, Nuck M, Creutzfeldt W (99) Effect of exogenous or endogenous gstric inhibitory polypeptide (GIP) on plsm triglyceride responses in rts. Horm Metb Res :7 7. Bggio LL, Hung Q, Brown TJ, Drucker DJ () A recombinnt humn glucgon-like peptide (GLP)--lbumin protein (lbugon) mimics peptidergic ctivtion of GLP- receptordependent pthwys coupled with stiety, gstrointestinl motility, nd glucose homeostsis. Dibetes :9. Bergmn AJ, Stevens C, Zhou Y et l () Phrmcokinetic nd phrmcodynmic properties of multiple orl doses of sitgliptin, dipeptidyl peptidse-iv inhibitor: double-blind, rndomized, plcebo-controlled study in helthy mle volunteers. Clin Ther : 7 9. Nuck MA, Meininger G, Sheng D, Terrnell L, Stein PP (7) Efficcy nd sfety of the dipeptidyl peptidse- inhibitor, sitgliptin, compred with the sulfonylure, glipizide, in ptients with type dibetes indequtely controlled on metformin lone: rndomized, double-blind, non-inferiority tril. Dibetes Obes Metb 9:9. Mtikinen N, Mnttri S, Schweizer A et l () Vildgliptin therpy reduces postprndil intestinl triglyceride-rich lipoprotein prticles in ptients with type dibetes. Dibetologi 9:9 7. Boschmnn M, Engeli S, Dobberstein K et l (9) Dipeptidylpeptidse-IV inhibition ugments postprndil lipid mobiliztion nd oxidtion in type dibetic ptients. J Clin Endocrinol Metb 9:. Levy E, Sinnett D, Thibult L et l (99) Insulin modultion of newly synthesized polipoproteins B- nd B- in humn fetl intestine: gene expression nd mrna editing re not involved. FEBS Lett 9:. Longuet C, Sinclir EM, Mid A et l () The glucgon receptor is required for the dptive metbolic response to fsting. Cell Metb :9 7.7/s-9-- Proof only-not for redistribution

11 Electronic supplementry mteril Plsm insulin (pmol/l) d Plsm glucgon (pg/ml) 7 min min min min Control Sitgliptin Exendin- Control Sitgliptin Exendin- b e Plsm GLP- (pmol/l) Control Insulin min min Control Sitgliptin f Plsm exendin- (pmol/l) c Insulin or PBS 9 min min ESM Fig., d f Mice fsted for h were dministered single dose of sitgliptin t mg/kg body weight (, d, e) or exendin-, nmol/kg body weight, (, d, f) nd chllenged min lter with n orl ft lod. Blood ws collected by crdic puncture t nd min fter the olive oil gvge ( nd min fter initil sitgliptin dose). Insulin (Mouse Insulin Ultrsensitive Elis; Alpco Dignostic, Slem, NH, USA) (), glucgon (Mouse Endocrine Lincoplex; Millipore, Billeric, MA, USA) (d), totl GLP- (Mouse Totl GLP- Elis; Alpco Dignostic) (e) nd exendin- (Exendin- EIA kit; Phoenix Phrmceuticls, Burlingme, CA, USA) (f) were mesured t min nd min) fter olive oil gvge. n= per group; p<. control vs sitgliptin; p<. control vs exendin- s determined by two-wy ANOVA. c Mice were chllenged with n orl ft lod nd then injected with insulin (. mu/kg body weight) or PBS min lter nd concomitntly with intrvenous Triton WR-9 (. mg/kg body weight) to mesure tricylglycerol secretion (b). n= per group; p>. s determined by two-wy ANOVA