Glucagon-Like Peptide-1 Increases Mitochondrial Biogenesis and Function in INS-1 Rat Insulinoma Cells

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1 Brief Report Endocrinol Met 215;3: pissn X eissn Glucgon-Like Peptide-1 Increses Mitochondril Biogenesis nd Function in INS-1 Rt Insulinom Cells Mi Yeon Kng*, Te Jung Oh, Young Min Cho Deprtment of Internl Medicine, Seoul Ntionl University College of Medicine, Seoul, Kore Glucgon-like peptide-1 (GLP-1) is gut-derived incretin hormone tht increses glucose-stimulted insulin secretion in pncretic β-cells. Since mitochondril function is crucil to insulin secretion, we hypothesized tht GLP-1 my increse mitochondril iogenesis in pncretic β-cells. We treted INS-1 rt insulinom cells with GLP-1 or exendin-4 for 48 hours nd mesured mitochondril mss nd function. Both GLP-1 nd exendin-4 incresed mitochondril mss y pproximtely 2%. The mitochondri/cytosol rtio ws incresed from 7.6±3.12% to 1.53±2.7% y exendin-4. In ddition, GLP-1 incresed the mitochondril memrne potentil nd oxygen consumption. Prolifertor-ctivted receptor-gmm coctivtor 1α expression ws incresed pproximtely 2-fold y GLP-1 tretment. In conclusion, the present study presents evidence for new mechnism of ction y which GLP-1 improves pncretic β-cell function vi enhnced mitochondril mss nd performnce. Keywords: Glucgon-like peptide-1; Mitochondri; Dietes mellitus; Bet cell; Insulin INTRODUCTION Mitochondril dysfunction cuses oth insulin resistnce nd β-cell dysfunction, leding to glucose intolernce nd dietes [1,2]. In skeletl muscle, decresed mitochondril ftty cid oxidtion results in incresed mounts of cytosolic long-chin cyl CoA nd dicylglycerol, which leds to incresed serine phosphoryltion of insulin receptor sustrte-1, therey eliciting insulin resistnce [3]. In pncretic β-cells, decresed ATP genertion due to impired mitochondril oxidtive phosphoryltion is linked to impired insulin secretion, vi mechnism tht reduces closure of the ATP-sensitive potssium chnnel [4]. In this regrd, mesures to improve mitochondril function, either in the skeletl muscle or in the pncretic β-cell, should e effective for treting dietes. Glucgon-like peptide-1 (GLP-1) is gut-derived incretin hormone tht stimultes insulin secretion nd suppresses glucgon secretion, inhiits gstric emptying, nd reduces ppetite nd food intke [5,6]. Becuse of its efficcy in lowering lood glucose y stimulting β-cell insulin secretion, GLP-1 nlogues nd incretin enhncers (i.e., dipeptidyl peptidse-4 inhiitors) re widely used in the clinic to tret dietes [7]. Interestingly, it ws reported tht GLP-1 stimultes ATP synthesis in pncretic MIN6 β-cells [8], suggesting new mechnism for improving β-cell function using GLP-1 therpy. In this study, we exmined the effect of GLP-1 nd its nlogue Received: 14 July 214, Revised: 27 August 214, Accepted: 29 August 214 Corresponding uthor: Young Min Cho Deprtment of Internl Medicine, Seoul Ntionl University College of Medicine, 11 Dehk-ro, Jongno-gu, Seoul , Kore Tel: , Fx: , E-mil: ymchomd@snu.c.kr *Current ffilition: Deprtment of Internl Medicine, St. Crollo Hospitl, 221 Sunkwng-ro, Suncheon, Kore Copyright 215 Koren Endocrine Society This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution Non-Commercil License ( licenses/y-nc/3./) which permits unrestricted non-commercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited

2 GLP-1 nd Mitochondril Biogenesis on mitochondril iogenesis in pncretic β-cells. METHODS INS-1 cell culture nd glucose-stimulted insulin secretion INS-1 rt insulinom cells (pssges 28 to 34) were grown in monolyer culture in RPMI-164 supplemented with 1% fetl ovine serum, 1 mm 4-(2-hydroxyethyl)-1-piperzineethnesulfonic cid, 2 mm L-glutmine, 1 mm sodium pyruvte, 5 μm β-mercptoethnol, 1 U/mL penicillin, nd 1 μg/ml streptomycin, t 37 C in humidified tmosphere of 5% CO2 nd 95% ir. For glucose-stimulted insulin secretion, INS-1 cells were seeded in 24-well pltes nd treted with, 1, or 2 nm GLP-1 (Sigm-Aldrich, St. Louis, MO, USA) for 48 hours. Cells were then wshed twice with Kre s ringer icronte uffer (KRBB), incuted in KRBB t 37 C for 1 hour, nd exposed to 5 or 1 mm glucose for 1 hour. The culture superntnt ws collected nd stored t 2 C until ssyed for insulin concentrtion using enzyme-linked immunosorent ssy (ELISA, Linco Reserch, St. Chrles, MO, USA). Mesurement of mitochondril mss nd memrne potentil INS-1 cells (1 1 5 ) were seeded in six-well pltes nd incuted with GLP-1 (1 to 4 nm) or exendin-4 (1 to 2 nm, Sigm-Aldrich) for 48 hours. Mitochondril mss ws mesured y 1-n-nonyl-cridine ornge stining (NAO, Invitrogen, Crlsd, CA, USA), nd the mitochondril memrne potentil ws mesured using tetrmethylrhodmine ethyl ester perchlorte (TMRE, Invitrogen) with FACSCliur flow cytometer (Becton Dickinson, Frnklin Lkes, NJ, USA), following the mnufcturers protocols. The strength of NAO or TMRE stining ws expressed s the men fluorescence intensity (MFI). Trnsmission electron microscopy INS-1 cells (5 1 5 ) were seeded in 6-mm pltes nd incuted with exendin-4 for 48 hours. Cells were collected nd processed for electron microscopy using stndrd methods. Ten rndom pictures were tken t mgnifiction of 5,, using n H-71 trnsmission electron microscope (Hitchi, Tokyo, Jpn). The volume density of mitochondri ws estimted using pointcounting method in linded fshion y two seprte exminers. For ech set of 1 pictures, the verge volume density ws clculted, nd the men of 1 vlues ws used to estimte the volume density for ech individul cell. Mesurement of oxygen consumption Oxygen consumption ws mesured using high-resolution respirometer (Oxygrph-2k, Orooros Instruments, Innsruck, Austri) ccording to the mnufcturer s instructions. A suspension of INS-1 cells t cells/ml in 2-mL volume of culture medium (RPMI-164) ws mesured in the respirometer t 37 C. Reverse trnscriptse polymerse chin rection Totl RNA from cells ws prepred using n RNesy Mini kit (Qigen, Vlenci, CA, USA). cdna ws otined from 1 μg RNA using rndom hexmers nd vin myelolstosis virus reverse trnscriptse (Invitrogen). Smples were mplified in Gene Amp polymerse chin rection (PCR) system 96 (Perkin-Elmer/Cetus, Norwlk, CT, USA). Digitl imges of the PCR products seprted on 1% grose gels were nlyzed using Gel Doc 2 (BioRd Inc., Richmond, CA, USA). Sttisticl nlysis Dt re presented s mens±sd. Nonprmetric methods, including Kruskl-Wllis tests, Wilcoxon signed-rnk tests, nd Mnn-Whitney tests, were used to evlute sttisticl significnce. Vlues for P<.5 were considered significnt. RESULTS Effects of GLP-1 nd its nlogue on insulin secretion nd mitochondril iogenesis in INS-1 cells GLP-1 tretment of INS-1 cells for 48 hours enhnced glucosestimulted insulin secretion (Fig. 1A) nd mitochondril mss (Fig. 1B, C). GLP-1 t 1, 2, nd 4 nm incresed NAO stining intensity to ±11.54, ±15.47, nd ±12.89 MFI, respectively (n=1) (Fig. 1C). A similr result ws otined using nother mitochondril dye (MitoTrcker Green, dt not shown). When mitochondril density ws mesured using point-counting method on trnsmission electron microscopy imges, the mitochondri/cytosol re rtio ws significntly incresed y 1 nm exendin-4, from 7.6±3.12% to 1.53± 2.7% (Fig. 1D, E). The expression of prolifertor-ctivted receptor-gmm coctivtor 1 α (PGC1α), key regultor of mitochondril iogenesis, ws incresed drmticlly fter 1 hour of GLP-1 tretment; fter 4 hours, expression decresed grdully ut remined ove control levels for up to 48 hours of tretment Copyright 215 Koren Endocrine Society 217

3 Kng MY, et l. Insulin concentrtions in medi (ng/ml) GLP-1 conc. (nm) Glucose conc mm 2 mm A Counts GLP-1 (1 nm) FL1-Height B NAO intensity (MFI) GLP-1 concentrtion (nm) C P< GLP-1 2 nm C 1 hr 4 hr 8 hr 24 hr 48 hr 1 5 PGC1α GAPDH Exendin-4 (1 nm) D E F Mitochondri/cytosol re rtio Fig. 1. Effects of glucgon-like peptide-1 (GLP-1) or exendin-4 on insulin secretion (A, n=6), mitochondril mss (C, n=1), mitochondril density (E, n=1), nd prolifertor-ctivted receptor-gmm coctivtor 1 α (PGC1α) expression (F) in INS-1 cells. (B) A representtive fluorescence ctivted cell sorting nlysis for 1-n-nonyl-cridine ornge stining (NAO) intensity, s summrized in (C). (D) Representtive trnsmission electron microscopy imges used to estimte the mitochondri/cytosol re rtios shown in concentrtion (conc). MFI, men fluorescence intensity; GAPDH, glycerldehyde 3-phosphte dehydrogense. P<.5; P<.1 compred with the control TMRE intenslty (MFI) Oxygen consumption rte (μmol/sec/15 cells) GLP-1 concentrtion (nm) Exendin-4 (1 nm) A GLP-1 (1 nm) B Fig. 2. Glucgon-like peptide-1 (GLP-1) increses mitochondri memrne potentil (A, n=6) nd cellulr oxygen consumption rte (B, n=4). TMRE, tetrmethylrhodmine ethyl ester perchlorte; MFI, men fluorescence intensity. P<.5; P<.1 compred with control Copyright 215 Koren Endocrine Society

4 GLP-1 nd Mitochondril Biogenesis (Fig. 1F). Effects of GLP-1 nd exendin-4 on mitochondril function in INS-1 cells The intensity of TMRE stining, which is n indictor of the strength of the mitochondril memrne potentil, ws incresed in INS-1 cells treted with GLP-1 or exendin-4 for 48 hours (Fig. 2A). In line with this finding, the oxygen consumption rte of cells treted with GLP-1 for 48 hours exhiited significnt increse reltive to controls (56.3±2.8 μmol/sec/1 5 cells vs. 42.±1.6 μmol/sec/1 5 cells, P<.5) (Fig. 2B). DISCUSSION Becuse mitochondril oxidtive phosphoryltion is crucil to glucose-stimulted insulin secretion [4], the mechnisms mediting the effects of GLP-1 on mitochondri in pncretic β-cells deserve further study. One possile mechnism is the moiliztion of clcium into the mitochondril mtrix fter GLP-1 tretment, which hs een reported to enhnce mitochondril function in pncretic MIN6 β-cells, possily through the ctivtion of severl Kres cycle dehydrogenses [8] nd lso in vsculr smooth muscle cells [9]. Interestingly, in heptocytes, nonpeptide frgments of GLP-1 (i.e., GLP-1 [28-36] mide) were shown to enter the cytoplsm rpidly nd trget mitochondri in GLP-1 receptor-independent mnner, nd to e ssocited with decresed gluconeogenesis nd oxidtive stress [1]. However, in the present study, exendin-4 lso exhiited stimultory effect on mitochondril mss nd function. Therefore, signling through the GLP-1 receptor ppers to e sufficient to increse oth mitochondril mss nd function in pncretic β-cells. We report here novel effect of GLP-1 on mitochondril iogenesis nd function. In INS-1 cells, GLP-1 nd exendin-4 tretment triggered n increse in mitochondril mss, mitochondril density, mitochondril memrne potentil, nd oxygen consumption. These increses were ccompnied y upregultion of PGC1α, key regultor of mitochondril iogenesis [11]. However, further studies re needed to chrcterize the time-course of GLP-1-stimulted iogenesis, s it reltes to tht of PGC1α expression. In conclusion, the present study provides evidence for new mechnism of ction of GLP-1 in pncretic β-cells, linking mitochondril iogenesis nd function to glucose-stimulted insulin secretion. CONFLICTS OF INTEREST Y.M.C. hs received lecture or consulttion fees from MSD, Lilly, Novrtis, Astr-Zenec, nd Boehringer-Ingelheim. ACKNOWLEDGMENTS We thnk Dr. Hyung Jin Choi for the helpful discussions nd Ms. So Hee Kim for her technicl ssistnce. This study ws supported in prt y the Seoul Ntionl University Hospitl Reserch Fund nd y KES Reserch Grnt Awrd. REFERENCES 1. Cho YM, Prk KS, Lee HK. Genetic fctors relted to mitochondril function nd risk of dietes mellitus. Dietes Res Clin Prct 27;77 Suppl 1:S Kwk SH, Prk KS, Lee KU, Lee HK. Mitochondril metolism nd dietes. J Dietes Investig 21;1: Lowell BB, Shulmn GI. Mitochondril dysfunction nd type 2 dietes. Science 25;37: Mechler P, Wollheim CB. Mitochondril function in norml nd dietic et-cells. Nture 21;414: Cho YM, Fujit Y, Kieffer TJ. Glucgon-like peptide-1: glucose homeostsis nd eyond. Annu Rev Physiol 214;76: Cho YM, Merchnt CE, Kieffer TJ. Trgeting the glucgon receptor fmily for dietes nd oesity therpy. Phrmcol Ther 212;135: Cho YM, Widemn RD, Kieffer TJ. Clinicl ppliction of glucgon-like peptide 1 receptor gonists for the tretment of type 2 dietes mellitus. Endocrinol Met (Seoul) 213;28: Tsuoi T, d Silv Xvier G, Holz GG, Jouville LS, Thoms AP, Rutter GA. Glucgon-like peptide-1 moilizes intrcellulr C2+ nd stimultes mitochondril ATP synthesis in pncretic MIN6 et-cells. Biochem J 23;369(Pt 2): Morles PE, Torres G, Sotomyor-Flores C, Pen-Oyrzun D, River-Mejis P, Predes F, Chiong M. GLP-1 promotes mitochondril metolism in vsculr smooth muscle cells y enhncing endoplsmic reticulum-mitochondri coupling. Biochem Biophys Res Commun 214;446: Toms E, Stnojevic V, Hener JF. GLP-1-derived nonpeptide GLP-1(28-36)mide trgets to mitochondri nd suppresses glucose production nd oxidtive stress in iso- Copyright 215 Koren Endocrine Society 219

5 Kng MY, et l. lted mouse heptocytes. Regul Pept 211;167: Puigserver P, Spiegelmn BM. Peroxisome prolifertor-ctivted receptor-gmm coctivtor 1 lph (PGC-1 lph): trnscriptionl coctivtor nd metolic regultor. Endocr Rev 23;24: Copyright 215 Koren Endocrine Society