ARTICLE. Keywords Lipotoxicity. MicroRNA. Pancreatic beta cells. Stearic acid

Transcription

1 Dietologi (6) 59:7 57 DOI.7/s5639 ARTICLE Elevted irulting steri id leds to mjor lipotoxi effet on mouse pnreti et ells in hyperlipidemi vi mir35pmedited PERK/p53dependent pthwy Huimin Lu & Liuyi Ho & Songto Li & Song Lin & Lin Lv & Yng Chen & Hongli Cui & Tinqi Zi & Xi Chu & Lixin N & Chngho Sun, Reeived: 8 August 5 /Aepted: 5 Jnury 6 /Pulished online: Mrh 6 # SpringerVerlg Berlin Heidelerg 6 Astrt Aims/hypothesis Serum steri id (C8:) is elevted in individuls with hyperlipidemi nd type dietes. However, the lipotoxiity indued y inresed steri id in et ells hs not een well desried. This study imed to exmine the dverse effets of steri id on et ells nd the potentil mehnisms through whih these re medited. Methods Three groups of C57BL/6 mie were fed norml diet or highsteriid/highplmitiid diet for weeks, respetively. The mirorna (mir) profiles of islets were determined y mirorry sreening. Islet injury ws deteted with ostining using the TUNEL ssy nd insulin lelling. A lentivirl vetor expressing ntimirna35p oligonuleotide (AMO35p) ws injeted into mie vi n intrdutl pnreti route. Results In oth mouse islets nd ultured rt insulinom I ells, steri id exhiited stronger lipotoxi role thn other ftty ids, owing to repression of B ell CLL/ lymphom (BCL) nd BCLlike (BCLW) y steri id stimultion of mir35p. The steriidindued lipotoxiity nd redution in insulin seretion were llevited y AMO35p. Further investigtions in I ells reveled tht p53 ws involved in steriidindued elevtion of mir35p, owing in prt to tivtion of protein kinselike endoplsmi retiulum kinse (PERK). Conversely, silening PERK llevited steriidindued p53, mir3 5p nd lipotoxiity. Conlusions/interprettion These findings provide new insight for understnding the moleulr mehnisms underlying not only the deleterious impt of steriidindued lipotoxiity ut lso poptosis in et ells nd progression to type dietes. Keywords Lipotoxiity. MiroRNA. Pnreti et ells. Steri id Huimin Lu, Liuyi Ho nd Songto Li ontriuted eqully to this work Eletroni supplementry mteril The online version of this rtile (doi:.7/s5639) ontins peerreviewed ut unedited supplementry mteril, whih is ville to uthorised users. Lixin N nlixin3@63.om Chngho Sun hnghosun@63.om Deprtment of Nutrition nd Food Hygiene, Puli Helth College, Hrin Medil University, 57 Bojin Rod, Nngng Distrit, Hrin, Hei Longjing Provine 58, People s Repuli of Chin Reserh Institute of Food, Nutrition nd Helth, SinoRussin Medil Reserh Center, Hrin Medil University, Hrin, People s Repuli of Chin Arevitions AMO35p AntimiRNA35p oligonuleotide ATF6 Ativting trnsription ftor 6 BCL B ell CLL/lymphom BCLW BCLlike CHOP CCAAT/enhner inding protein homologous protein ER Endoplsmi retiulum GSIS Gluoseindued insulin seretion Highplmitiid diet Highsteriid diet IRE Inositolrequiringenzyme LentiAMO35p Lentivirl vetor expressing ntimirna35p oligonuleotide mirna/mir MiroRNA

2 8 Dietologi (6) 59:7 57 MTT PERK qpcr SFA Introdution 3(,5Dimethylthizolyl),5 diphenylhtetrzolium romide Protein kinselike endoplsmi retiulum kinse Quntittive PCR Sturted ftty ids Lipotoxiity refers to the ellulr dysfuntion used y n elevted level of NEFA. Aumulting evidene indites tht lipotoxiity ontriutes to et ell injury nd plys ritil role in the development of type dietes [ 3]. NEFA is hemilly lssified into sturted nd unsturted ftty ids. It is widely epted tht lipotoxiity is mostly indued y longhin sturted ftty ids (SFA), suh s plmiti (C6:) nd steri id (C8:), wheres unsturted ftty ids re, in generl, less toxi, nd my even e protetive [ 6]. There is growing evidene tht hroni onsumption of diet rih in sturted fts inreses irulting SFA levels nd the risk of type dietes [7, 8]. It hs een oserved tht NEFA profiles in individuls with oesity or hyperlipidemi re sustntilly ltered ompred with helthy individuls; suh hnges re regrded s n importnt risk ftor for type dietes[7, 9]. Plmiti id is widely employed s representtive ftty id to investigte the reltionship etween SFA nd type dietes euse it ounts for the highest perentge of dietry NEFA for the humn ody. However, whether other ltertions in NEFA profiles hve detrimentl role in et ell destrution nd type dietes is unknown. Our previous study reveled tht the profiles of oth fsting nd postprndil serum NEFA were signifintly ltered in ptients with hyperlipidemi [7]. Most strikingly, we oserved tht only the proportion of steri id in the serum inresed drmtilly in the postprndil stte, whih implies tht ptients with hyperlipidemi hve onsiderly higher level of serum steri id most of the time. In niml models of insulin resistne, the rtio etween plmiti nd steri ids ws deresed from 3: in the norml group to : in mie fed highft diet [7]. Reserh in vitro hs lerly shown tht inresing steri id stimultes severe lipotoxiity in heptoytes nd skeletl musle ells [5,, ]. Added to our previous oservtion tht n inresed proportion of steri id in the serum is losely relted to insulin resistne [7], we proposed tht rising the proportion of steri id in the lood mkes the gretest ontriution to et ell lipotoxiity reltive to other NEFA, nd plys dominnt role in type dietes progression. Moreover, we sought to explore the moleulr mehnisms underlying the destrutive effet of steri id on et ells. Although the moleulr mehnisms of steriidindued lipotoxiity re not fully understood, it is evident tht enhned endoplsmi retiulum (ER) stress is entrl ontriutor to lipotoxiity resulting from trnsriptionl reprogrmming [], tivtion of Jun Nterminl kinse [3], CCAAT/enhner inding protein homologous protein (CHOP) [], p53 nd mitohondril poptosis pthwys [5, 6]. Importntly, mirornas (mirnas), whih re involved in posttrnsriptionl regultion under ER stress, hve emerged s key regultors of heptoyte nd rdi musle lipotoxiity [7, 8]. In islet et ells, the involvement of mirna in steriidmedited lipotoxiity is still unler. In this study, we hypothesised tht inresing steri id indues more severe et ell lipotoxiity thn other NEFA nd its underlying mehnism is relted to mirna (mir) 35p. Methods Mterils Stok solutions of steri nd plmiti ids (Sigm, St Louis, MO, U) were prepred s desried previously [9]. The oligonuleotides nd lentivirus re desried in the eletroni supplementry mteril (ESM) Methods. Animl experiments All experimentl proedures followed guidelines for the re nd use of nimls estlished y Hrin Medil University nd pproved y the Animl Experimenttion Ethis Committee of Hrin Medil University. Sevenweekold mle C57BL/6 mie were purhsed from Vitl River Lortories (Beijing, Chin) nd ompletely rndomised to reeive norml diet, highsteriid diet () or highplmitiid diet () (ESM Tle ). A lentivirl vetor expressing ntimirna 35p oligonuleotide (AMO35p) (lentiamo35p) ( 8 TU/ml) ws slowly injeted into mie vi the pnreti dut ( μl) fter feeding for weeks, s desried previously []. Further mesurements were performed t weeks postlentivirl infetion. See ESM Methods. Experimenters were lind to group ssignment nd outome ssessment. Serum NEFA profile nd gluose nd lipid mesurement Blood smples were olleted for further detetion. See ESM Methods. mirna mirorry nlysis Oeioteh (Shnghi, Chin) performed the mirna mirorry work, proessing smples from mouse islets (n = per group). See ESM Methods. Costining using TUNEL ssy nd insulinlelled islets Islet injury ws deteted with the In Situ Cell Deth Detetion Kit (TUNEL Fluoresene Kit, Rohe, Indinpolis, IN,

3 Dietologi (6) 59: U). Islets were lelled with ntiinsulin ntiody (Cell Signling Tehnology, Minnepolis, MN, U), nd viewed y onfol mirosopy. See ESM Methods. Pnreti insulin ontent The pnres ws extrted using the id ethnol method s desried previously [], nd the insulin level ws deteted. Intrvenous gluose tolerne tests IVGTTs were performed on overnightfsted mie. For the mesurement of insulin nd gluose levels, lood smples were olleted t,, 5,,, 3 nd 6 min fter gluose dministrtion (.75 g/kg) to the til vein. Bet ell mss nd histohemil nlysis The pnreti tissues were fixed, emedded in prffin nd stined with ntiinsulin nd ntiglugon ntiodies. See ESM Methods. Insulin grnules nlysis Eletron mirosopy ws employed to nlyse the ontent of insulin grnules in fixed pnreti tissues s desried previously []. Cell ulture Rt insulinom ell line I ells were ultured nd treted with 6 μmol/l steri or other ftty ids for h. Mouse pnreti islets were isolted s desried previously [3]. See ESM Methods. Experimenters were lind to group ssignment nd outome ssessment. Cell viility mesurements Cell viility ws determined y ssessment of ltte dehydrogense (LDH) relese, propidium iodide (PI) stining, 3(,5dimethylthizolyl),5diphenylHtetrzolium romide (MTT) ssy nd Hoehst stining. See ESM Methods. Trnsfetion proedures I ells were trnsfeted with oligonuleotides or reominnt plsmid using Lipofetmine (Invitrogen), s desried previously [], nd used in detetion ssys 8 h fter trnsfetion. Luiferse tivity ssy The luiferse tivity ws deteted using luiferse tivity ssy. See ESM Methods. Quntittive PCR The mrna nd mirna levels were determined y quntittive PCR (qpcr). See ESM Methods. The primer sequenes re shown in ESM Tle. Western lot The western lot proedure hs een desried previously [5]. See ESM Methods. Insulin mesurements Insulin levels in mouse serum nd ell ulture medi were determined y mouse/rt insulin ELI kit (Lino Reserh, St Chrles, MO, U). To ssess gluoseindued insulin seretion (GSIS), ells were inuted in seretion uffer (see ESM Methods) for n dditionl 6 min with.8 or mmol/l gluose. Sttistis Vlues re expressed s men ± SD. Multiple groups were nlysed with onewy ANOVA followed y Student Newmn Keuls test. Twogrouponly omprisons were rried out y t test. A twosided p vlue <.5 ws onsidered sttistilly signifint. Repetedmesures ANOVA ws used to nlyse the gluose nd insulin levels during IVGTT. Results Steri id indues more severe lipotoxiity thn plmiti id As shown in ESM Fig.,, only steri nd plmiti ids hd n oviously lipotoxi effet on tested ells. The dose response urves for plmiti nd steri ids re shown in ESM Fig.. A mouse model with different steri/plmiti id rtios ws susequently estlished to mimi nd investigte the lipotoxiity of inresed proportions of steri id in hyperlipidemi. The model ws suessfully estlished, s evidened y signifint elevtion of irulting steri nd plmiti id levels in the nd groups, respetively, ompred with ontrols (ESM Tle 3). Importntly, the plmiti id:steri id rtio ws lower thn ontrols in the group (.7: vs.7:, respetively) ut higher in the group (6.:) (ESM Fig. ). Body weight, totl holesterol (TC), triylglyerol (TG), nd LDLholesterol (LDL C) were mrkedly higher in oth nd mie, wheres HDLholesterol (HDLC) ws signifintly lower thn in ontrol mie (ESM Tle ). TUNEL results showed tht pnreses from mie ontined more TUNELpositive islets thn pnreses from mie, nd et ell mss ws lower in the group (Fig. ). Susequently, the levels of leved PARP nd leved spse3 ws inresed in pnreti islets (Fig. ). Further, the impired gluose tolerne nd seondphse insulin seretion in response to gluose were signifintly enhned in the group ompred with the group (Fig., d). Also, immunohistohemil nlysis of pnreti tissue showed tht the islets of mie omprised more glugonpositive ells thn oth norml mie nd the group (Fig. e). These dt suggest tht feeding is more detrimentl to islet ells thn feeding. We next inuted I ells in ulture medi with different rtios of plmiti/steri id for h nd mesured ytotoxiity with Hoehst 333 stining (Fig. f) nd MTT ssy (Fig. g). The results showed tht lipotoxiity ws enhned in the presene of higher proportion of steri id, nd levels of leved PARP nd leved spse3 proteins were elevted ordingly (Fig. h). Additionlly, GSIS in steriidtreted I superntnt frtions ws drmtilly lower thn in plmitiidtreted ells (Fig. i).

4 5 Dietologi (6) 59:7 57 Insulin TUNEL PARP Cleved PARP Cspse 3 Cleved spse 3 αtuulin d e Insulin Glugon f PA PA/=: PA/=: PA/=: PA/=: PA/=: g i h PARP Cleved PARP Cspse 3 Cleved spse 3 PA Ct rl /S PA PA A= /S : PA A= /S : PA A= /S : A PA = /S : A= : αtuulin Fig. Comprison of the lipotoxi effet of steri id () nd plmiti id (PA) on islets nd I ells. TUNEL stining (sle r, μm) (), western lot nlysis of leved PARP nd leved spse 3 (), lood gluose () nd insulin (d) levels during IVGTT nd immunohistohemil nlysis of pnres setions (sle r, μm) (e), from ontrol, nd mie; n = 6 per group. In () islets re red nd TUNELpositive regions re green. In (, d) irles, ontrol; squres, ; inverted tringles,. p <.5 nd p <. vs ontrol; p <.5 nd p <. vs. Lipotoxi effet of different PA: molr rtios (totl onentrtion, 6 μmol/l) on I ells mesured y Hoehst stining (sle r, μm) (f), MTT ssy (g), immunolots of leved PARP/leved spse 3 (h) nd GSIS (i). p <.5 vs ontrol; p <.5 vs PA. The test ws performed five times., ontrol Steri id inreses mir35p levels in oth mouse islets nd I ells In islets, mirnas were differentilly expressed (ten up nd 3 downregulted.fold hnge nd p <.5), wheres in islets, 77 mirnas were highly dysregulted (3 up nd 6 downregulted.fold hnge nd p <.5), ompred with norml mie (ESM Tle 5). A het mp of nd islet mirna expression rtios (log sle) is shown in ESM Fig. 3. Furthermore, we sreened out six upregulted nd 3 downregulted mirnas tht responded to n, ompred with oth ontrol nd groups (. fold hnge nd p <.5, Fig. ). Among these, mir35p expression inresed the

5 Dietologi (6) 59: Fig. Differentil expression of mirnas in islets nd I ells. () Het mps nd hierrhil lustering of expression rtios (log log sle) of mirnas in mouse islets ompred with oth nd ontrol mie. High reltive expression is shown in red nd low reltive expression in green. n = per group. (, ) qpcr verifies upregultion of mir35p in mie () nd I ells(). p <.5nd p <. vs ontrols; p <.5 vs or plmiti id; n = 6 per group; eh in vitro test ws performed five times., ontrol; PA, plmiti id;, steri id most, 6.35fold in nd.57fold in mie, respetively. Quntittive PCR verified tht mir35p levels inresed y 9% in nd 97% in islets, respetively, ompred with norml islets (Fig. ). In I ells exposed to different rtios of plmiti/steri ids, mir35p expression ws mrkedly upregulted ompred with ontrols in ells exposed to higher proportion of steri id, (Fig. ). mir35p ontriutes to steriidindued lipotoxiity in islets nd I ells Trnsfetion of AMO 35p into steriidtreted I ells effiiently redued intrellulr mir35p levels (ESM Fig. ), inhiited steriidindued ell deth (Fig. 3, ), nd reversed steriidinresed leved PARP nd leved spse 3 levels(fig. 3). Aordingly, steriidderesed GSIS ws pprently restored fter mir35p inhiition (Fig. 3d). However, trnsfeting only mir35p into I ells lso indued ell deth nd redution in GSIS, whih were reversed y AMO35p (ESM Fig. 5 d). In vivo, the lenti AMO35p ws suessfully delivered into the pnres s evidened y the strong green fluoresene (ESM Fig. 6), with endogenous mir35p expression lmost ompletely silened (up to 95%) in islets (ESM Fig. 6). indued islet dmge in mie ws roustly llevited y lentiamo 35p injetion, onsistent with the deresed numer of TUNELpositive islets nd inresed et ell mss (Fig. 3e), deresed levels of leved PARP nd leved spse3 proteins (Fig. 3f) nd improved gluose tolerne (Fig. 3g). Importntly, the firstphse insulin relese ws... S3 S S S C C3 C C P3 P P P mmumir85p mmumir33p mmumir563p mmumir5p mmumir35p mmumir665p mmumir893 mmumir75p mmumir385p mmumir3575p mmumir639 mmumir95 mmumir6653p mmumir6975p mmumir79 mmumir89 mmumir89 mmumir675p mmumir935p mir35p/u6 (reltive expression vs ) mir35p/u6 (reltive expression vs ) signifintly enhned nd seondphse insulin seretion ws deresed fter lentiamo35p injetion (Fig. 3h), whih indites tht inhiition of mir35p ould enhne the pity to inrese GSIS. Also, the numer of lph ells in the lentiamo35p group ws signifintly redued nd the norml ell distriution pttern ws restored (Fig. 3i). Additionlly, steriidmedited redutions in pnreti insulin ontent (ESM Fig. 6) nd insulin grnules (ESM Fig. 6d) were pprently reversed. The ody weight nd serum lipid level were not ltered (ESM Tle 6). mir35p speifilly represses BCL nd BCLW expression Expression of B ell CLL/lymphom (BCL) nd BCLlike (BCLW) ws redued in oth islets nd steriidtreted I ells (Fig., ). AMO35p tretment, either in vitro or in vivo, mrkedly reversed steriidmedited downregultion of BCL nd BCLW proteins, (Fig., d). In I ells overexpressing mir35p, levels of BCL s well s BCLW were deresed, ut were effiiently resued y ddition of AMO35p (Fig. e). Furthermore, luiferse ssy verified tht overexpression of mir35p inhiited luiferse tivity in humn emryoni kidney 93 (HEK93) ells trnsfeted with plsmid rrying the 3 UTR of either the Bl or the Bll gene (Fig. f). p53 is involved in steri idindued elevtion of mir 35p The mount of p53 protein inresed in islets nd steriidtreted I ells (ESM Fig. 7). Steriidindued lipotoxiity ws rogted fter silening p53 (lso known s Trp53), ompnied y 75% redution in steriidinresed mir35p (Fig. 5 ). Menwhile, 3 6 PA PA/=: PA/=: PA/=: PA/=: PA/=:

6 5 Dietologi (6) 59:7 57 f PARP Cleved PARP Cspse 3 Cleved spse 3 αtuulin h Plsm insulin (pmol/l) PARP Cleved PARP Cspse 3 Cleved spse 3 αtuulin AMO35p MT AMO35p e lentisr Insulin TUNEL lentisr AMO lentisr lentiamo35p, Time (min) lentisr (fold vs ) AUC insulin min (pmol/l min) MT AMO (fold vs ) AMO35p MT AMO35p 3 8, 6,,, Apoptoti ells (%) 3 AMO35p MT AMO35p 3 lenti AMO35p Cleved PARP Cell survivl rte (%) 8 AMO35p MT AMO35p Cleved PARP Cleved spse 3 % TUNELpositive ells/islet re Cleved spse 3 lentisr lentisr lentiamo35p lentisr lentisr lentiamo35p lentisr lentisr lentiamo35p Insulin seretion (ng [mg protein] h ) Gluose (mmol/l) AMO35p MT AMO35p lentisr lentisr lentiamo35p Blood gluose (mmol/l) , 6,, AUC insulin 6 min (pmol/l min), g lentisr lentisr lentiamo35p d Bet ell mss (mg) 3 lentisr lentisr lentiamo35p Time (min) i Insulin Glugon lentisr AUC gluose 6 min (mmol/l min) lentisr, 8 6 lentisr lentisr lentiamo35p lenti AMO35p

7 Dietologi (6) 59: Fig. 3 Inhiition of mir35p improves steriidindued lipotoxiity in islets nd I ells. Hoehst stining (sle r, μm) (), MTT ssy (), immunolotting of leved PARP/ leved spse 3 () nd GSIS(d) showing the effet of AMO35p on poptosis in steri idtreted I ells. p <.5 vs ontrol; p <.5 vs ; eh test ws performed five times. TUNEL ssy (e), western lot for leved PARP leved spse 3 (f), lood gluose (g) nd insulin (h) level during IVGTT, nd immunohistohemil nlysis of pnreti setions (sle r, μm) (i), showing the effet of lenti AMO35p on islets. In (e) islets re red nd TUNELpositive regions re green (sle r, μm). Cirles, ontrol lentivirl vetor expressing srmled AMO oligonuleotide (lentisr); squres lentisr; inverted tringles, lentiamo35p. p <.5 nd p <. vs ontrol lentisr; p <.5 nd p <. vs lentisr. n = 6 per group. AMO, ntimirna oligonuleotide;, ontrol; MT, mutnt; PA, plmiti id;, steri id steriideliited upregultion of leved PARP nd leved spse3, s well s the downregultion of BCL nd BCLW protein expression, ws resued y p53 knokdown (Fig. 5d). PERK ontriutes to steriidindued upregultion of mir35p nd lipotoxiity Our in vitro results showed tht the trnsmemrne sensors of ER stress, inluding inositolrequiringenzyme (IRE), tivting trnsription ftor 6 (ATF6) nd PERK, re strongly upregulted y steri id tretment. Similrly, ER stress mrker proteins, suh s CHOP nd gluoseregulted protein 78 kd (GRP78), were lso enhned (ESM Fig. 7). Genetilly silening Perk (lso known s Eifk3) y smll interfering RNA (sirna) in I ells signifintly loked the steriidindued inrese in p53 expression (Fig. 6), mir35p level (Fig. 6) nd lipotoxiity (Fig. 6, d). Aordingly, steriidinresed expression of leved PARP nd leved spse 3, s well s suppressed BCL nd BCLW, returned to sl levels fter inhiition of Perk expression (Fig. 6e). However, gene silening of neither Ire nor Atf6 inhiited the steriidindued hnges deteted ove (ESM Fig. 8). Further, the phosphorylted level of eukryoti trnsltion initition ftor (eif)α ws signifintly enhned fter steri id exposure (ESM Fig. 9). The inhiitory effiieny of sirnas trgeting p53, Ire, Atf6 nd Perk re shown in ESM Fig.. Disussion In this study, we provide evidene tht, reltive to other NEFA, steri id is the mjor ontriutor to lipotoxiity in et ells, implying tht n inresed onentrtion of serum steri id plys fundmentl role in the development of et ell dysfuntion nd type dietes. Our mehnisti studies reveled tht mir35p upregultion modultes steriidindued lipotoxiity y inhiiting the prodution of BCL nd BCLW proteins. Additionl studies hve lso (fold vs ) AMO35p MT AMO35p BCL BCL BCL mir35p AMO35p MT AMO35p BCLW BCL BCLW αtuulin AMO35p MT AMO35p (fold vs ) e BCLW AMO35p MT AMO35p (fold vs ) BCL BCLW αtuulin BCL BCLW αtuulin BCL PA BCLW Bl pmirrb NC mir35p AMO35p BCLW PA demonstrted tht the ERstressrelted sensor PERK ontriutes to steriidindued upregultion of mir35p in p53dependent signlling pthwy. Although ltertions in the NEFA profiles of people with hyperlipidemi hve een widely investigted, the NEFA tht is most diretly ssoited with et ell lipotoxiity remins unler. Therefore, we first seleted NEFA with BCL BCLW αtuulin (fold vs ) d BCL BCLW αtuulin (fold vs ) f Reltive luiferse tivity lentisr BCL lentisr lentisr lentiamo35p PA lentisr lentiamo35p BCLW lentisr lentisr lentiamo35p Bll Fig. Repression of Bl nd Bll genes y mir35p. Downregultion of BCL/BCLW in islets () nd steri idtreted I ells (). Expression of BCL/BCLW y AMO35p in treted I ells () nd islets (d). (e) Effet of mir35p on BCL/BCLW expression. (f) Effet of mir35p on the 3 UTR of Bl nd Bll determined y luiferse tivity ssy. p <.5 vs ontrol, p <.5 for the indited omprison. n = 6 mie per group; eh in vitro test ws performed five times. AMO, ntimirna oligonuleotide;, ontrol; MT, mutnt; PA, plmiti id; pmirrb, pmir RB REPORT;, steri id

8 5 Dietologi (6) 59:7 57 Fig. 5 Involvement of p53 in steriidindued stimultion of mir35p in I ells. Effet of p53 sirna on mir3 5p levels (), ell viility (), poptosis (sle r, μm) (), nd leved PARP/spse 3/BCL/BCLW protein expression (d) with/without steri id. p <.5 vs ontrol; p <.5 vs sirnanegtive ontrol; the test ws performed five times., ontrol; NC, negtive ontrol;, steri id d mir35p/u6 (reltive expression vs ) 6 sirnanc sirnap53 PARP Cleved PARP Cspse 3 Cleved spse 3 BCL BCLW αtuulin sirnanc sirnap53 sirnanc sirnanc sirnap53 sirnap53 (fold vs ) Cell survivl rte (%) sirnanc sirnap53 Cleved PARP sirnanc sirnap53 Apoptoti ells (%) sirnanc sirnap53 Cleved spse BCL BCLW sttistilly disrepnt levels etween helthy nd hyperlipidemi individuls to test their ytotoxiity to pnreti et ells. We oserved tht only steri nd plmiti ids signifintly lowered ell viility. Inuting ells in medium ontining different rtios of plmiti id:steri id drmtilly enhned ytotoxiity, ut insulin seretion ws redued s the proportion of steri id ws inresed. The onentrtions of plmiti nd steri ids used in this study were seleted to void exessive ell deth sed on previous reports nd our previous experiments [7, 6, 7], though this does not hppen in humn or niml pnreti et ells in vivo [6, 7]. The onentrtions of plmiti nd steri ids employed in ultured ells were, in ft, lower thn in ptients with hyperlipidemi, ut indued more severe dmge thn in the in vivo system, whih ould e explined y the existene of protetive ftors (i.e. unsturted ftty ids [ 6]). Additionlly, we oserved tht eiospentenoi id (EPA; C:5), potentil protetive ftor, ws redued in oth nd mie, nd this might lso ontriute to highftdietindued lipotoxiity. Unlike the in vitro system, it is unrelisti to estlish niml models with only steri or plmitiidonstruted lipid profiles in lood. However, wellepted pproh to ompre different iologil funtion mong the different types of NEFA ws hieved y regulting the lood NEFA rtio using vrious oils s ft soure in the highft diet [8, 9]. In this study, models of hyperlipidemi with high irulting levels of either steri or plmiti id were estlished using lrd or plm oil s ft soure, onsistent with previous studies [8 3]. After weeks of feeding, the fsting serum levels of steri nd plmiti ids inresed in nd mie, respetively. The plmiti id:steri id rtio ws.7: nd 6.: in nd mie, respetively. Susequently, the mie showed signifint impirment of gluose tolerne nd GSIS ompred with mie, with n inresed numer of poptoti et ells leding to derese in et ell mss. Our results lerly show tht, in vivo, indues more dmge in islets thn, onsistent with the in vitro results. No differene ws oserved in the totl SFA ontent etween the nd groups, exluding the possiility tht the different extent of lipotoxiity ws used y something other thn differenes in lipid onentrtions. Additionlly, these results re lso onsistent with our previous findings [7, 3], inditing tht steri id ppers to e more importnt risk ftor for type dietes thn other NEFA. Therefore, gret ttention should e pid to elevted serum steri id, s it

9 Dietologi (6) 59: Fig. 6 Involvement of PERK in steri idindued stimultion of mir35p vi p53 in I ells. Effet of Perk sirna on p53 protein expression (), mir 35p levels (), ell survivl (), poptosis (sle r, μm) (d), nd leved PARP/leved spse 3/BCL/BCLW protein expression (e) with/without. p <.5 vs ontrol; p <.5vs sirnanegtive ontrol; the test ws performed five times., ontrol; NC, negtive ontrol;, steri id p53 βatin d sirnanc sirnanc sirnaperk sirnaperk e PARP Cleved PARP Cspse 3 Cleved spse 3 BCL BCLW βatin sirnanc sirnaperk ppers to hve greter negtive impt on et ell funtion. The mehnisti pthwys responsile for indued lipotoxiity in pnreti et ells re not fully understood. Reent evidene hs provided new lues tht mirnas re involved in the regultion of et ell filure nd type dietes [3 3]. In our study, we seleted 9 mirnas tht re differentilly expressed in islets of mie reltive to nd norml mie, sed on genehip mirorry nlysis. Among these mirnas, mir35p displyed the lrgest fold inrese, whih ws in line with previous oservtions tht high level of mir35p ws present in the islets of dieti d/d mie nd pnreti islets exposed to plmiti id [3]. To demonstrte the potentil role of mir35p in medited lipotoxiity in et ells, endogenous mir35p ws rogted y AMO35p in oth mie nd I ells. LentiAMO35p injetion meliorted indued islet poptosis in vivo. Aordingly, impired gluose tolerne nd GSIS in mie were lrgely restored. The derese in et ell mss seen in mie ws lso reversed. Insulin seretion nd et ell mss re two importnt ftors for regulting gluose homeostsis through welllned et ell ompenstion in the erly stge of type dietes, ut they grdully worsen [35, 36]. Our results indite tht mir 35p inhiition in islets ould proly improve the et ell dysfuntion nd further slow the progression of type dietes. In vitro, AMO35p relieved the ytotoxi effet of steri id nd reversed the redution in insulin seretion. Conversely, overexpression of mir35p signifintly enhned ytotoxiity nd redued GSIS. These results strongly suggest tht mir35p plys ruil role in elevting lipotoxiity indued y serum steri id. However, the steriidmedited derese in GSIS ws not ompletely reversed y mir35p inhiition, whih implies tht nonmir35pmedited mehnism operted in GSIS redution. We lso oserved tht mir35p levels were higher

10 56 Dietologi (6) 59:7 57 in islets nd steriidtreted I ells thn in islets nd the plmiti id group, respetively, whih suggests tht the greter dverse effet of steri over plmiti id on et ells, oth in vivo nd in vitro, is proly ttriutle to differentil regultion of mir35p. Previous studies hve indited tht mir35p is losely ssoited with poptosis through the BCL fmily in ner ells, tuulr ells nd rdiomyoytes [37 39]. The BCL fmily inludes oth ntipoptosis memers (BCL, BCL like [BCLXL] nd BCLW) nd propoptosis memers (BCLssoited X protein [BAX], BCLntgonist/killer [BAK] nd BCLssoited gonist of ell deth [BAD]) [, ]. In our study, we onfirmed tht BCL nd BCL W re diret trgets of mir35p using luiferse tivity ssys. However, how steri id regultes mir35p levels is still unler. Aumulting evidene supports p53 s lssi trnsription ftor regulting mir35p. To further investigte the involvement of p53 in mir35p upregultion fter longterm inution of I ells with steri id, we silened p53 nd then investigted whether mir35p levels were reversed, with n ssoited redution in lipotoxiity, whih would suggest tht the mehnism underlying indued stimultion of mir35p uses p53dependent pthwy. Enhned ER stress, ssoited with et ell lipotoxiity, trnsdues poptoti signls in the erly stge of type dietes []. Three mjor signlling pthwys seprtely initited y ER trnsmemrne sensor proteins, inluding IRE, PERK nd ATF6, re involved in ER stress [3]. We susequently knoked down Ire, Perk nd Atf6 with sirnas in steriidtreted I ells, nd found tht inhiition of Ire nd Atf6 filed to inhiit steriidindued stimultion of mir35p. However, trnsfetion of sirna for Perk ttenuted lipotoxiity nd mir35p levels on steri id tretment. These findings demonstrte tht mir35p might e promoted y PERK in onditions of ER stress. We lso disovered tht PERK exhiited n inhiitory effet on p53 protein expression, suggesting tht PERK p53 mir35p xis is mehnistilly involved in steriidindued lipotoxiity in et ells. Additionlly, the inflmmtory response ws the ommon retion during prolonged exposure to NEFA, whih in turn results in the ggrvtion of lipotoxiity. Chroni exposure to inflmmtory onditions leds to et ell dysfuntion nd poptosis [3,, 5]. A reent investigtion hs reveled tht the level of mir3 inresed onomitntly in ytokinetreted MIN6 ells nd humn pnreti islets, nd ontriuted to et ell dysfuntion [3]. Therefore, we proposed tht the indued inrese in mir35p nd further pnreti injury proly represented n importnt inflmmtory response pthwy. This needs to e lrified in future studies. In onlusion, we provide evidene tht elevted levels of irulting steri id indue severe lipotoxiity in pnreti et ells. It ppers tht upregultion of mir35p nd the ssoited inhiition of BCL/BCLW ply ritil role in mediting steri id ytotoxiity. Our results lso suggest regultory role for PERK in mir35p expression nd tht p53 is meditor of PERKindued mir35p upregultion. Colletively, these findings llow us to propose novel signlling pthwy linking steri id to lipotoxiity in et ells: steri id PERK p53mir35p BCL/BCLW. These findings provide novel insight into steriidindued lipotoxiity nd progression of type dietes, wherey lowering mir35p might e n effetive strtegy for improving lipotoxiity in et ells. Aknowledgements We thnk B. X. Wu (Deprtment of Nutrition nd Food Hygiene, Puli Helth College, Hrin Medil University) for helping with serum gluose nd lipid mesurement nd Q. N. Qu nd Y. Y. Liu (Deprtment of Nutrition nd Food Hygiene, Puli Helth College, Hrin Medil University) for help with niml feeding. Funding This work ws supported y the Stte Key Progrm of Ntionl Nturl Siene of Chin (grnt numer 839), nd the Ntionl Nturl Siene Foundtion of Chin (grnt numers 887, 888 nd 89). 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