Endothelial IRE1α promotes thrombospondin-1 mRNA decay and supports metabolic stress adaptation of pancreatic islets

Domain: biology
Published: 2026-01-09
Editorial outcome: major_revision

Abstract
Vascularendothelialcells(ECs)playpivotalrolesinmaintainingmetabolic
tissuehomeostasis,andECdysfunctionisassociatedwithobesityandmeta-
bolicdisorders.ThemammalianERstresssensorIRE1αkinase/RNaseresponds	ometaboliccues,butitremainsunclearwhetherendothelialIRE1αisimpli-
catedincontrollingsystemicmetabolism.Hereweshowthatgeneticdeple-
tionofIRE1αinECsleadstomaladaptationofpancreaticisletsunderobesity-
associatedmetabolicstress.Wefindthatinhigh-fatdiet-fedmalemice,lossof
IRE1αinECshasnosignificantimpactuponadiposity,butunexpectedly
resultsinglucoseintolerancewithimpairedinsulinsecretion,accompaniedby
bluntedintra-isletangiogenesisandcompensatoryisletgrowth.Mechan-
istically,IRE1αRNasedecaysthemRNAencodingtheendogenousanti-
angiogenicfactorthrombospondin-1(THBS1/TSP1)inisletECs.Thesefindings
thusuncoveracriticalroleoftheendothelialIRE1αsuppressionofTHBS1in
governingthevascularsupportthatenablesthefunctionaladaptationofislets
tometabolicstress.

Introduction
Vascular endothelial cells (ECs) form a highly plastic and dynamic vascularized metabolic organs like adipose tissue, ECs are being
monolayerthatinterfacescirculatingbloodandtissues,andtheycan recognizedascriticalgatekeepersandactiveregulatorsinthesystemic
readilyshiftfromdormanttoangiogenicstatesduringtissuerevas- controlofmetabolismviaadjustingtissue-specifictransportofnutri-
cularizationaccordingtophysiologicdemands1.ECsplaypivotalroles entssuchasglucoseandfattyacidsaswellashormoneslikeinsulin6–8.
intissuehomeostasisviagoverningoxygenandnutrientsupply,hor- Constantlyexposedtovariouscirculatingfactorsandmetabolites,ECs
monetransport,immunecelltrafficking,andwastedisposalbetween can be viewed as energy/nutrient sensors and are susceptible to
bloodandperfusedorgans.Tomeetspecificlocalneedsandfunctions, metabolic perturbations and pathogenicstimuli, whichmay leadto
ECs exhibit heterogeneous structures and phenotypes in different disruption of their angiocrine interactions with other cell types.
organs/tissues2,3.Recentsingle-celltranscriptomicsstudieshavealso Indeed, emerging evidence indicates that EC dysfunction is closely
revealedremarkableheterogeneityandmolecularsignaturesoftissue- associated with metabolic dysregulation that underlies obesity and
specificECs,reflectingtheirlocalfunctionalcharacteristicsunderthe relatedmetabolicconditionssuchastype2diabetes,cardiovascular
influence of specific tissue microenvironments4,5. In highly diseasesandsteatohepatitis9,10.Forinstance,angiogenesisisacritical
contributorinsupportingadiposetissueexpansionremodelingandits (epiWATandscWAT)andbrownadiposetissue(BAT)(Supplementary
metabolic function in response to environmental changes such as Fig.1c).H&Estainingrevealednoovertchangesinliverfataccumu-
nutrient excess, while impaired angiogenic capacity and decreased lation or adipocyte enlargement inscWAT/BAT of HFD-fed Ern1EC-KO
vascularity are thought to promote adipose inflammation and dys- mice relative to their Ern1fl/fl counterparts (Supplementary Fig. 1d).
function in obesity8,11–13. Moreover, multiple mechanisms have been Interestingly,whileexhibitingslightlymoreincreasesinserumtrigly-
proposedtounderliethepathologicalECdysfunctioninvariousdis- ceride (TG) and total cholesterol(TC) levels,Ern1EC-KO mice had sig-
ease settings, including hypoxia, oxidative stress, and endoplasmic nificantly higher elevations in their hepatic TG and TC content
reticulum(ER)stressaswell8,14,15.

ERstressarisesfromanoverloadofunfolded/misfoldedproteinsat following HFD feeding (Supplementary Fig. 1e–h). This implies an
theERandisconnectedtothepathogenicprogressionofmanymeta- impactofendothelialIRE1αdeficiencyuponhepaticlipidmetabolism.
bolic and inflammatory diseases16. ER stress triggers the activation of Moreimportantly,glucosetolerancetest(GTT)showedthatHFD-fed
threecanonicalbranchesoftheunfoldedproteinresponse(UPR),which maleErn1EC-KOmicehadsignificantlyreducedabilitytocontrolblood
aremediatedbythreeER-localizedtransmembranesignaltransducers, glucoselevelsrelativetotheirErn1fl/fllittermates(Fig.1f).Unexpect-
inositol-requiringenzyme1(IRE1),PKR-likeERkinase(PERK),andacti- edly, insulin tolerance test (ITT) revealed no differences in insulin-
vatingtranscriptionfactor6(ATF6)16,17.IRE1isthemostconservedER induced lowering of blood glucose levels (Fig. 1g), suggesting that
stress sensor with a cytoplasmic segment possessing dual protein IRE1αdeficiencyinECsdidnotaffecttheinsulinsensitivityinErn1EC-KO
kinase/endoribonuclease (RNase) activities18,19. Upon activation of its micefollowingHFDfeeding.Consistently,weobservedsimilarlevels
RNaseactivitythroughautophosphorylationandoligomerization,IRE1 of insulin-stimulated Akt phosphorylation in the livers, scWAT or
notonlycatalyzestheunconventionalsplicingofthemRNAencoding musclefromNC-orHFD-fedErn1EC-KOandErn1fl/flmice(Supplementary
X-boxbindingprotein(XBP1)togenerateanactivetranscriptionfactor Fig.2a,b).Thisthenledustotestiftheyhaddifferentinsulinpro-
XBP1s20,21,butalsodegradesselectmRNAsthroughaprocesstermed duction capacities in the face of obesity. Indeed, we detected sig-
“regulated IRE1-dependent decay” (RIDD)22,23. Two isoforms of IRE1, nificantly blunted hyperinsulinemia with comparable serum glucose
IRE1αandIRE1β,arepresentinmammals,withIRE1αfoundtobeubi- levels infasted HFD-fed Ern1EC-KO micerelative to their Ern1fl/fl coun-
quitously expressed across different tissues and cells19. Many studies terparts(Fig.1h).Inaccordance,HFD-fedErn1EC-KOmicealsoexhibited
haveshownthatmammalianIRE1αservesasamulti-functionalmeta- markedreductionsintheirseruminsulinlevelsfollowingglucosesti-
bolicsensorthatregulatesmanyaspectsofenergymetabolismindif- mulation, despite significantly higher glucose levels in the blood
ferent metabolic organs24, including thermogenic fat remodeling and (Fig.1i).Bycontrast,femaleErn1EC-KOandErn1fl/flmiceshowedsimilarly
adiposeinflammationthroughitscelltype-dependentRNaseactionsin lowerdegreeofbodyweightgainthantheirmalecounterpartsupon
adipocytesandmacrophages,respectively25,26.Moreover,ERstresshas HFDfeedingfor12weekswithcomparableglucoseintoleranceandno
alsobeenimplicatedinregulatingangiogenesis27,andinparticular,the overt changes in serum insulin levels (Supplementary Fig. 3a–d),
IRE1α-XBP1pathwayinECshasbeendocumentedtocross-talkwiththe reflecting the phenotypic sexual dimorphism in their sensitivity to
vascularendothelialgrowthfactor(VEGF)receptorsignalingandreg- HFD-inducedobesity,includingpancreaticisletadaptationasreported
ulate endothelial proliferation and angiogenesis in the state of in documented studies31. In addition, without tamoxifen treatment,
ischemia28.Itremainslargelyunknown,however,whetherIRE1αinECs maleErn1fl/fl;VE-cad-CreERT2miceandtheirErn1fl/fl littermateshad no
can respond to distinctlocal stress signals from tissue microenviron- phenotypicdifferencesintheirmetabolicparametersfollowingHFD
ments and exert tissue-specific regulatory actions upon metabolic feeding,includingglucosetolerance,fastedseruminsulinlevels,and
homeostasis.

Inthisstudy,wegeneratedamousemodelinwhichIRE1αwas pancreaticisletβcellmass(SupplementaryFig.4a–g).Thisexcludes
specificallydepletedinECsinadultsandinvestigatedwhetherendo- thepossibilitythatthe metabolicchangesinHFD-fedErn1EC-KOmice
thelial IRE1α could regulate systemic metabolism under nutritional resultedmerelyfromCreexpressionintheabsenceofIRE1αdepletion.
stress,presumablyimpactingthemetabolicfunctionofadiposetissue Together,theseresultsdemonstrateacriticalroleofendothelialIRE1α
via influencing the angiogenic capacity. Surprisingly, we found an in the control of glucose homeostasis under obesity-associated
unanticipatedroleofendothelialIRE1αinspecificallyregulatingthe metabolic stress in male mice, predominately through influencing
adaptiveresponseofpancreaticisletstoovernutrition-inducedmeta- thecapacityofinsulinproductioninpancreaticislets.

Results
EndothelialIRE1αabrogationleadstoimpairedglucosetoler-
ancewithlowerinsulinproductionupondiet-inducedobesityin
malemice
ToinvestigatethemetabolicfunctionofIRE1αinECsunderthestress
ofexcessnutrition,weintercrossedfloxedIRE1α(Ern1fl/fl)mice29with
the VE-cadherin-CreERT2 line30 to generate a mouse model with
tamoxifen (Tam)-induced EC-specific IRE1α knockout (Ern1EC-KO) in
adults (Fig. 1a),and challenged them to high-fat diet (HFD) feeding
(Fig. 1b). Immunoblot analysis revealed apparent ablation of IRE1α
proteininCD31-positiveECs(CD31+)butnotinCD31-negativecells
(CD31-) isolated from the livers, lungs and heart of Ern1EC-KO mice
relativetothatfromErn1fl/flmice,indicatingefficientendothelialIRE1α
depletion(Fig.1c).Whenmaintainedonanormalchow(NC)dietor
following chronic HFDfeeding,male Ern1EC-KO mice and their Ern1fl/fl
littermatesshowednosignificantdifferencesintheirbodyweightgain
(Fig.1d),dailyfoodintake(SupplementaryFig.1a),oxygenconsump-
tion (Supplementary Fig. 1b), body fat mass (Fig. 1e), or the tissue
weight of liver, epididymal and subcutaneous white adipose tissue
(epiWATandscWAT)andbrownadiposetissue(BAT)(Supplementary
Fig.1c).H&Estainingrevealednoovertchangesinliverfataccumu-
lation or adipocyte enlargement inscWAT/BAT of HFD-fed Ern1EC-KO
mice relative to their Ern1fl/fl counterparts (Supplementary Fig. 1d).
Interestingly,whileexhibitingslightlymoreincreasesinserumtrigly-
ceride (TG) and total cholesterol(TC) levels,Ern1EC-KO mice had sig-
nificantly higher elevations in their hepatic TG and TC content
following HFD feeding (Supplementary Fig. 1e–h). This implies an
impactofendothelialIRE1αdeficiencyuponhepaticlipidmetabolism.
Moreimportantly,glucosetolerancetest(GTT)showedthatHFD-fed
maleErn1EC-KOmicehadsignificantlyreducedabilitytocontrolblood
glucoselevelsrelativetotheirErn1fl/fllittermates(Fig.1f).Unexpect-
edly, insulin tolerance test (ITT) revealed no differences in insulin-
induced lowering of blood glucose levels (Fig. 1g), suggesting that
IRE1αdeficiencyinECsdidnotaffecttheinsulinsensitivityinErn1EC-KO
micefollowingHFDfeeding.Consistently,weobservedsimilarlevels
of insulin-stimulated Akt phosphorylation in the livers, scWAT or
musclefromNC-orHFD-fedErn1EC-KOandErn1fl/flmice(Supplementary
Fig.2a,b).Thisthenledustotestiftheyhaddifferentinsulinpro-
duction capacities in the face of obesity. Indeed, we detected sig-
nificantly blunted hyperinsulinemia with comparable serum glucose
levels infasted HFD-fed Ern1EC-KO micerelative to their Ern1fl/fl coun-
terparts(Fig.1h).Inaccordance,HFD-fedErn1EC-KOmicealsoexhibited
markedreductionsintheirseruminsulinlevelsfollowingglucosesti-
mulation, despite significantly higher glucose levels in the blood
(Fig.1i).Bycontrast,femaleErn1EC-KOandErn1fl/flmiceshowedsimilarly
lowerdegreeofbodyweightgainthantheirmalecounterpartsupon
HFDfeedingfor12weekswithcomparableglucoseintoleranceandno
overt changes in serum insulin levels (Supplementary Fig. 3a–d),
reflecting the phenotypic sexual dimorphism in their sensitivity to
HFD-inducedobesity,includingpancreaticisletadaptationasreported
in documented studies31. In addition, without tamoxifen treatment,
maleErn1fl/fl;VE-cad-CreERT2miceandtheirErn1fl/fl littermateshad no
phenotypicdifferencesintheirmetabolicparametersfollowingHFD
feeding,includingglucosetolerance,fastedseruminsulinlevels,and
pancreaticisletβcellmass(SupplementaryFig.4a–g).Thisexcludes
thepossibilitythatthe metabolicchangesinHFD-fedErn1EC-KOmice
resultedmerelyfromCreexpressionintheabsenceofIRE1αdepletion.
Together,theseresultsdemonstrateacriticalroleofendothelialIRE1α
in the control of glucose homeostasis under obesity-associated
metabolic stress in male mice, predominately through influencing
thecapacityofinsulinproductioninpancreaticislets.

IRE1αablationinECsdampensthecompensatorygrowthof
pancreaticisletsandβcellsinmalemice
Because obesity-associated insulin resistance triggers physiological
compensatorygrowth ofpancreaticislets with robustlyincreased β
cell mass32,we then examined whether endothelial IRE1αdeficiency
affectedtheadaptivechangesofpancreaticisletsinhandlingobesity-
associatedmetabolicstress.Immunoblotanalysisfurtherrevealedthe
efficientdepletionofIRE1αproteininCD31+ECsfrompancreas,pre-
sumablyincludingintra-isletECs,ofErn1EC-KOmicerelativetothatfrom
Ern1fl/fl mice (Fig. 2a). Histochemical and morphometric analyses
showednoapparentalterationsinthepancreaticarchitectureorthe
structural organization of islets in NC- or HFD-fed male Ern1fl/fl and
Ern1EC-KOmice(Fig.2b).However,markedincreasesinthenumber(by
~56.3%), averaged size of islets (by ~42.8%) and β cell mass were
observedinHFD-fedErn1fl/flmicerelativetotheirNC-fedcounterparts,
allofwhich were substantiallybluntedinErn1EC-KOmice(Fig.2c,d),
includingthenumberofsmallaswellaslargeislets(Fig.2e).Next,we
askedifisletcellproliferationorcelldeathisinvolved.Whole-mount
Ki67immunostainingofislets33showedthatHFD-fedErn1EC-KOmice,
relativetotheirHFD-fedErn1fl/fllittermates,hadsignificantreductions
inKi67-positive(Ki67+)cellsfromIns+βcellseitherintotalisletsorin
islets of varying sizes (Fig. 2f, g). Moreover, TUNEL analyses also
revealedsignificantlyhighernumberofTUNEL-positive(TUNEL+)
nucleifromβcells(Ins+)inHFD-fedErn1EC-KOislets(Fig.2h,i).Thus,
these data suggest that loss of endothelial IRE1α leads to defective
compensatorygrowthofisletsinmalemice,whichisinparallelwith
decreasedβcellproliferationandincreasedβcelldeathundermeta-
bolicstressconditions.

IRE1αdeficiencyinECscausesisletdysfunctionunderdietary
obesityinmalemice
ToexamineifIRE1αdepletioninECshadanimpactuponthecellular
composition of islets, we performed immunostaining analysis of
insulin and glucagon in pancreatic sections (Fig. 3a). We observed
significantlyelevatedareasofβcells(Ins+)andslightlyincreasedareas
ofαcells (Gcg+)inHFD-fed versus NC-fed Ern1fl/fl islets,whichwere
significantly and appreciably, respectively, lowered in HFD-fed
Ern1EC-KO islets (Fig. 3b, c). However, no significant changes in the
areapercentageofβcells(Ins+)orαcells(Gcg+)wereseeninNC-or
HFD-fedErn1EC-KOversusErn1fl/flislets(Fig.3d,e).Wethenwonderedif
endothelial IRE1α deficiency affected the secretory functionof pan-
creatic islets. Whereas isolated primary islets from HFD-fed Ern1fl/fl
miceshowedhighercapacityofglucose-stimulatedinsulinsecretion
than their NC-fed counterparts, islets from HFD-fed Ern1EC-KO mice
exhibitednearlyatotallossofsuchaugmentation(Fig.3f),whileno
alterationsintheinsulincontentweredetectedintheseislets(Fig.3g).
Notably,quantitativeRT-PCRprofilingshowedappreciabledecreases
inHFDfeeding-inducedupregulationofinsulin1(Ins1)andinsulin2
(Ins 2) mRNA expression in Ern1EC-KO islets (Supplementary Fig. 5a),
while no significant changes were seen in the expression of critical
transcription regulators34 involved in insulin biogenesis or β cell
development and regeneration, including pancreas/duodenum
homeobox protein 1 (Pdx1), Neurogenin 3 (Ngn3) or musculoapo-
neurotic fibrosarcoma oncogene family protein A (Mafa) (Supple-
mentaryFig.5b).Interestingly,suggestinganisletangiogenicresponse
suchasVEGFsignaling35isinvolved,wedetectedasignificantupre-
gulationofFlk1(fetalliverkinase1,i.e.,murineVEGFR2),butnotFlt1
(FMS-like tyrosine kinase 1, i.e., murine VEGFR1) or Vegfa mRNA
expression in HFD-fed Ern1fl/fl islets in comparison to their NC-fed
counterparts,whichwasabrogatedinErn1EC-KOmice(Supplementary
Fig.5c).Furthermore,wecharacterizedinsulinsecretiondynamicsvia
islet perifusion assays and observed significant reductions in both
phaseIandphaseIIsecretionfollowingglucosestimulationaswellas
uponKClstimulationinHFD-fedErn1EC-KOislets(Fig.3h,i).Inlinewith
their defective secretory capacity, fluorescent imaging analysis also
revealed decreased calcium mobilization in HFD-fed Ern1EC-KO islets
following high glucosestimulation (Fig.3j,k). Collectively,these ex
vivo data demonstrate that IRE1α ablation in ECs could impair the
enhancement of the secretory function of islets, and particularly β
cells,inthefaceofobesity-inducedmetabolicstress.

IRE1αdepletioninECslimitsHFDfeeding-inducedincreaseof
intra-isletvasculardensityinmalemice
Mature pancreatic islets are known to be highly vascularized and
possess a dense capillary network, ~5 times denser than the sur-
roundingexocrinearea,whichnotonlysupportstheefficientsecre-
tionofislethormoneslikeinsulinintothecirculation,butalsoactsto
regulate β cell mass and function36–38. Therefore, we went on to
investigateifIRE1αdeficiencyinECsdisruptedtheadaptivecompen-
sationofisletsthroughaffectingintra-isletvascularremodelingsuch
asangiogenesisduringmetabolicstress.Immunostaininganalysisof
CD31 as well as CD3439 in pancreatic sections showed a significant
increase(by~23.9%)ofcapillarydensityinisletsfromHFD-fedmale
Ern1fl/flmicerelativetothatfromNC-fedgroup,whichwasmarkedly
diminishedinErn1EC-KOanimals(Fig.4a,b,SupplementaryFig.6a,b).
QuantitativeRT-PCRanalysisalsorevealedbluntedupregulationofthe
expressionofPecam1(encodingplateletandendothelialcelladhesion
molecule1,i.e.,CD31)inErn1EC-KOisletsfollowingHFDfeedingrelative
to that in Ern1fl/fl islets (Fig. 4c). We then performed whole-mount
immunofluorescentstaining33ofCD31+ECsinisolatedisletsformore
thoroughvisualization oftotalintra-isletvascular network(Fig.4d).
Consistently, a significant increase of intra-islet CD31+ ECs was
observedinErn1fl/flmicefollowingHFDfeeding,whichwascompletely
diminishedinErn1EC-KOislets(Fig.4e).Thisisalsoinparallelwithsig-
nificantlybluntedHFDfeeding-elicitedincreaseofIns+βcellsinislets
from Ern1EC-KO mice as compared to Ern1fl/fl control mice (Fig. 4e).
Moreover,significantreductionswereobservedinCD31+Ki67+double-
positivesignalsfromErn1EC-KOrelativetoErn1fl/flisletsafterHFDfeeding
(Fig.4f,g),whereasnoTUNELsignalsweredetectedwithinCD31+ECs
in these islets (Fig. 4h). By contrast, we observed no significant
alterationsintheabundanceofCD31+orCD34+ECsinliversectionsof
NC-orHFD-fedErn1EC-KOandErn1fl/flmice(SupplementaryFig.6c,d).
NordidwedetectsignificantchangesofPecam1mRNAabundancein
Ern1EC-KOliversrelativetothatinErn1fl/fllivers(SupplementaryFig.6e).
Whole-mountimmunostainingofCD31inepWATorscWAT(Supple-
mentaryFig.6f,g)aswellasIHCstainingofCD34inscWAT(Supple-
mentaryFig.6h)alsorevealednosignificantchangesintheirvascular
densityinNC-orHFD-fedErn1EC-KOandErn1fl/flmice.Inaddition,real-
timebloodflowmeasurements40exhibitednoapparentdifferencesin
thebloodperfusionwithinpancreasoringuinalWATofNC-orHFD-
fed male Ern1EC-KO miceand their Ern1fl/fl littermates (Supplementary
Fig.7a–d).Althoughitremainsunknownifthebloodperfusionwithin
the islets was affected, these results suggest that IRE1α in ECs can
respond to the unique microenvironmental cues of metabolically
stressed islets, promoting intra-islet endothelial proliferation/angio-
genesisforadaptivevascularremodelingtoenableislet’scompensa-
torygrowthandaugmentedsecretorycapacity.

EndothelialIRE1αdepletionsubstantiallyimpactsislettran-
scriptomewithenhancedThbs1expressioninECs
TogainfurtherinsightintohowIRE1αpromotesintra-isletangiogen-
esisundermetabolicstress,weperformedbulkRNA-seqanalysisof
primary islets isolated from Ern1fl/fl and Ern1EC-KO mice following 16-
weeks of HFD feeding. Notably, rMATS analysis used for assessing
alternative splicing events revealed overt exon 2 deletion in Ern1
mRNAfromErn1EC-KOislets(SupplementaryFig.8a),confirmingeffec-
tiveIRE1αknockoutinisletECs.Comparisonofgeneexpressionpro-
files between Ern1fl/fl and Ern1EC-KO islets showed 573 genes whose
expressionwasinfluencedbyIRE1αabrogationinECs.Geneontology
(GO)biologicalprocess(BP)analysisrevealedthatIRE1αablationin
ECs affected a variety of regulatory processes in islets, including
intercellularinteractions,nutrientandhypoxiaresponses,inflamma-
tion, insulin secretion, as well as EC proliferation/migration and ER
stress (Fig. 5a). Notably, among enriched genes related to insulin
secretion (Supplementary Fig. 8b) are downregulated expression in
Ern1EC-KO islets of Cartpt (Cocaine- and amphetamine-regulated tran-
scriptprotein)andGpr119(Gprotein-coupledreceptor119),twocru-
cial regulators of islet or β cell secretion function41–43 whose
upregulationinresponsetoHFDfeedingwassignificantlydampened
in islets by endothelial IRE1α deficiency (Supplementary Fig. 8c).
Importantly, among top-ranked genes whose expression was most
robustlyupregulatedordownregulatedasaresultofIRE1αablation
(Fig.5b,c)andfromtheenrichedgenesimplicatedinregulationofEC
proliferation(Fig.5d),weidentifiedThbs1codingforThrombospondin
1(TSP1),awell-establishedendogenousantiangiogenicfactorthatnot
onlyinhibitsECproliferationanditsproangiogenicresponses,butalso
exertsmultipleregulatoryeffectsuponglucoseandlipidmetabolism
throughaffectingthefunctionofadiposetissueaswellaspancreatic
islets44–46. Then we confirmed by quantitative RT-PCR that Thbs1
expression was indeed significantly enhanced in HFD-fed Ern1EC-KO
islets(Fig.5e),whichwasinparallelwithhighercontentofTSP1protein
inisletswithendothelialIRE1αdeficiency(Fig.5f).Bycontrast,while
weobservedincreasedserumTSP1levelsinHFD-fedmice,wedidnot
detect significant changes resulting from endothelialIRE1α ablation
(SupplementaryFig.9a,b).ThisindicatesthatECsaremostlikelynot
themajorproductionsourceforcirculatingTSP1levels.
SinceTSP1hasbeenshowntobeexpressedintheendotheliumof
islets47,48,wefurtheranalyzedthesingle-cellRNA-seqdata(accession
No.GSE203376)fromprimarypancreaticisletsinnormalchow-versus
HFD-fedmice49andisolatedtheECclusterbydimensionalityreduction
alongwithothercelltypes(SupplementaryFig.10a,b).Remarkably,we
alsoobservedthattheexpressionofThbs1,butnotKlf6(Krüppel-like
factor6)orSncg(Synucleingamma),wasspecificallyrestrictedtoECs
alongwiththeECmarkerCdh5andanangiogenictranscriptionfactor
Sox18(SRY-relatedHMG-box18)(SupplementaryFig.10c).Moreover,
up-regulatedThbs1expressionwasseeninHFD-fedintra-isletECsas
comparedtothatinnormalchow-fedcontrolgroup(Supplementary
Fig.10d).Inaddition,GOanalysisofHFDfeeding-regulatedgenesin
intra-islet ECs, using the Findmarkers function with |log FC|>0.25,
revealedanenrichmentofpathwaysrelatedtoresponsetounfolded
protein or protein folding as well as angiogenesis (Supplementary
Fig.10e).Therefore,itismostlikelythattheobservedupregulationof
Thbs1 in HFD-fed Ern1EC-KO islets occursspecificallyinisletECs,sug-
gestingtheexistenceofauniqueIRE1α-TSP1axisinECsthatregulates
intra-isletangiogenesisandthecompensatoryresponseofisletsunder
metabolicstress.

IRE1αsuppressestheexpressionofThbs1topromoteendothe-
lialcellproliferation
Next, we tested if IRE1α regulates the proliferation of ECs through
alteringTSP1expressionusingthemurineislet-derivedMS1endothe-
lialcellline50,whichwasCD31-positivebyimmunofluorescentstaining
(Supplementary Fig. 11a), as well as primary human umbilical vein
endothelialcells(HUVECs).Giventhatglucoseanditsmetabolismare
theprimaryfactordrivingcompensatoryβ-cellproliferationintheface
of insulin resistance32,51 and that high glucose was documented to
inhibitangiogenesisinisolatedhumanpancreaticisletswithincreased
TSP1expressionlevels52,wefirstexaminedifhighglucoseregulates
Thbs1expressioninMS1cellsandHUVECs.Indeed,highglucosesti-
mulationresultedinsignificantelevationsinThbs1/THBS1mRNAand
TSP1proteinexpressionlevelsalongwithmarkedlyhighersecretionof
TSP1proteinfrombothMS1cellsandHUVECs,andlentiviralshRNA
knockdown of the expression of IRE1α significantly augmented this
glucoseupregulationofTSP1production(Fig.6a–f).Moreover,CCK-8
and cell-count assays showed that IRE1α knockdown led to sig-
nificantly reduced growth and viability of MS1 cells following high
glucosestimulation(Fig.6g),whereaslentiviralshRNAknockdownof
TSP1expressioninthefaceofIREαknockdown(Fig.6h)effectively
reversedthisgrowthdefectofMS1cellsresultingfromIREαdeficiency
(Fig.6i).ThesedatasuggestthatIREαmayacttosustainendothelial
cell proliferationthrough suppressing Thbs1expression toblockits
autocrineantiangiogenicactivity.
Then, we tested the direct effect of TSP1 protein upon EC pro-
liferationandinterrogatedthepotentialmechanism(s)thatmediatesits
antiangiogenicactions.CCK-8assayshowedthattreatmentfor48hours
with recombinant human TSP1 protein significantly reduced the pro-
liferationofMS1cells(SupplementaryFig.11b)buthadnoimpactupon
Min6βcells(SupplementaryFig.11c).Asithasbeendocumentedthat
CD36, CD47 or SIRPα (signal-regulatory protein alpha) may mediate
TSP1’ssuppressiveeffectsuponendothelialandvascularfunctions53–55,
we analyzed the islet single-cell RNA-seq data (accession No.
GSE203376) and found that Cd36 was poorly expressed in islet cells,
while Cd47 but not Sirp was most abundantly expressed in islet ECs
(Supplementary Fig. 11d). Quantitative RT-PCR analyses revealed that
IRE1αdeficiencydidnotaffecttheexpressionofCd47orSirpinMS1cells
(SupplementaryFig.11e).GiventhereportedroleoftheTSP1-CD47axis
in age-related EC deterioration in angiogenesis56, we asked if CD47
mediatesTSP1suppressionofECproliferation.Indeed,weobservedthat
theCD47neutralizingantibodyreversed,atleastpartially,theinhibitory
effect of TSP1 protein upon MS1 cell viability from the CCK-8 assay
(Fig. 6j). In addition, because TSP1 has been shown to impede eNOS
activationinarterialendothelium57,wewonderedifIRE1αregulationof
TSP1mightalsohavesuchaneffectinisletECs.Notably,weobserved
decreasedlevelsofeNOSproteinaswellasitsphosphorylationactiva-
tion in HFD-fed Ern1EC-KO islets relative to their Ern1fl/fl counterparts
(SupplementaryFig.12a,b),butdetectednosignificantchangesineNOS
protein or phosphorylation levels in TSP1-treated MS1 cells with or
withoutinsulinstimulation(SupplementaryFig.12c,d).Thus,whilethe
TSP1-CD47axismaylargelyaccountfortheimpairedintra-isletangio-
genesisresultingfromendothelialIRE1αablation,defectiveeNOSacti-
vationisalsolikelytoserveasacontributingfactor.

IRE1αRNasepromotesThbs1mRNAdecayinendothelialcells
TodeterminehowIRE1αnegativelyregulatesTSP1expressioninECs,
wefirstaskedifitsRNaseactivitywasinvolved.TreatmentofMS1cells
with4μ8C,achemicalinhibitorofIRE1αRNaseactivity58,59,potently
blocked Xbp1 mRNA splicing and significantly augmented glucose-
inducedupregulationofThbs1mRNAabundance(Fig.7a).InIRE1α-
knockoutHEK-293Tcells,IRE1αoverexpressionresultedinareduction
of TSP1 protein level, which could be blocked by 4μ8C treatment,
whereasXBP1s-Flagoverexpressiondidnothaveasuppressiveeffect
(Fig.7b).Furthermore,overexpressionofthewild-type(WT)human
IRE1α,butnotitsRNase-deficientmutantIRE1α-K907A60,resultedin
robustXbp1mRNAsplicingwithsignificantreductionsinthemRNA
abundanceofTHBS1aswellasBLOC1S1,atypicalRIDDsubstrateof
IRE1α (Fig. 7c). Consistently, overexpression of IRE1α-WT but not
IRE1α-K907Aledtoalowerlevelofco-expressedTSP1protein(Fig.7d).
TodeterminehowIRE1αnegativelyregulatesTSP1expressioninECs,
wefirstaskedifitsRNaseactivitywasinvolved.TreatmentofMS1cells
with4μ8C,achemicalinhibitorofIRE1αRNaseactivity58,59,potently
blocked Xbp1 mRNA splicing and significantly augmented glucose-
inducedupregulationofThbs1mRNAabundance(Fig.7a).InIRE1α-
knockoutHEK-293Tcells,IRE1αoverexpressionresultedinareduction
of TSP1 protein level, which could be blocked by 4μ8C treatment,
whereasXBP1s-Flagoverexpressiondidnothaveasuppressiveeffect
(Fig.7b).Furthermore,overexpressionofthewild-type(WT)human
IRE1α,butnotitsRNase-deficientmutantIRE1α-K907A60,resultedin
robustXbp1mRNAsplicingwithsignificantreductionsinthemRNA
abundanceofTHBS1aswellasBLOC1S1,atypicalRIDDsubstrateof
IRE1α (Fig. 7c). Consistently, overexpression of IRE1α-WT but not
IRE1α-K907Aledtoalowerlevelofco-expressedTSP1protein(Fig.7d).
ToaffirmthisIRE1αRNase-dependentsuppressionofTSP1expression,
we established an experimental ER stress condition by treating
MS1 cells with the chemical ER stressor tunicamycin (Tm), which
inducedtheactivationofIRE1αRNaseactivityasindicatedbyefficient
Xbp1 mRNA splicing along with significant decreases in the mRNA
abundance of both Bloc1s1 and Thbs1 (Supplementary Fig. 13a).
Moreover, Tm-induced Xbp1 mRNA splicing and downregulation of
Bloc1s1andThbs1mRNAexpressionweresigificantlyattenuatedby
lentiviralshRNAknockdownofIRE1αexpressionorbytreatmentwith
4μ8C (Supplementary Fig. 13a,b). In addition, we performed mRNA
stabilityanalysisandobservedsustainedstabilityofThbs1mRNAalong
withBloc1s1mRNAasaresultofIRE1αdeficiencyor4μ8Cinhibitionof
IRE1α RNase following actinomycin D treatment of ER stressed
MS1 cells (Supplementary Fig. 13c). These results demonstrate that
IRE1α RNase controls Thbs1 mRNA stability, likely through its RIDD
activity. Furthermore, we identified the presence of a conserved
hairpinstructurecontainingaputativeIRE1αcleavagesitefortypical
RIDD substrates23 within both human and mouse Thbs1 mRNAs
(Fig.7e).UsinginvitrosynthesizedRNAtranscriptscorrespondingto
nucleotide(nt)1-1770and1771-3510ofthecodingregionofhuman
THBS1mRNA,wewereabletodetecttheircleavagesbyrecombinant
human IRE1α protein, which could be effectively blocked by 4μ8C.
Interestingly, more efficient cleavages were seen for the nucleotide
1771-3510substrateofTHBS1RNAcontainingapredictablestem-loop
hairpinthanthenucleotide1-1770substratewithoutsuchaRIDDsig-
naturestructure(Fig.7f),suggestingthatthenucleotide1-1770sub-
stratemayrepresentaninvivoexampleofRNAsundergoingIRE1α-
dependentcleavagethroughtheso-called“RIDDlackingendomotif”
(RIDDLE)61.Tofurtherconfirmthatthenucleotide1771-3510substrate
isabonafadeRIDDsubstrateofIRE1α,wesynthesizedamutantRNA
molecule (THBS1Mut) that harbors a G-to-C mutation at the putative
IRE1αcleavagesitewithinthehairpinstructure(Fig.7g).Whereaswe
detected a clear IRE1α-mediated cleavage of the wild-type THBS1WT
RNAsubstrate,whichagaincouldbeinhibitedby4μ8C,amuchfainter
cleavage was observed for the THBS1Mut RNA substrate (Fig. 7g).
Together,thesedatasuggestthatIRE1αemploysitsRNaseactivityto
promoteThbs1mRNAdecayviaRIDD(LE)inECs.

TSP1abrogationinECscorrectspancreaticisletdysfunction
resultingfromendothelialIRE1αdeficiencyinmalemice
Finally,wegeneratedtamoxifen-inducibleErn1EC-KO;Thbs1EC-KOdouble-
knockoutmicetoverifythemetabolicimportanceinvivoofIRE1α-
directedsuppressionofTSP1inECsduringdietaryobesity.Immuno-
blotanalysisofECsfromthelivers,lungsandheartsshowedefficient
depletion of both IRE1α and TSP1 following tamoxifen treatment
(Supplementary Fig. 14a). In comparison to HFD-fed Ern1fl/fl;Thbs1fl/fl
controlmice,HFD-fedErn1EC-KO;Thbs1EC-KOmiceexhibitedsimilarbody
weightgain(Fig.8a)anddailyfoodintake(Fig.8b),slightlyimproved
glucose tolerance (Fig. 8c) and insulin-responsive glycemic control
(Fig. 8d). Moreover, no significant changes were detected in their
serum insulin or blood glucose levels following glucose injection
(Fig.8e).ThesephenotypesareinsharpcontrasttothoseofHFD-fed
Ern1EC-KOmicethatdisplayedimpairedglucosetoleranceandinsulin
secretioncapacityrelativetotheirErn1fl/fllittermates(Fig.1).Further-
more,unlikeinHFD-fedErn1EC-KOmice,weobservednoreductionsin
theintra-isletvasculardensity(Fig.8f),isletPecam1mRNAabundance
(Fig.8g),isletsizesornumbers(Fig.8h,i),orislet’sglucose-stimulated
insulin secretion capacity (Fig. 8j, k) in HFD-fed Ern1EC-KO;Thbs1EC-KO
mice relative to their Ern1fl/fl;Thbs1fl/fl counterparts. Comparable vas-
culardensitywasalsoobservedinscWATofHFD-fedErn1EC-KO;Thbs1EC-
KOmiceandErn1fl/fl;Thbs1fl/fllittermates(SupplementaryFig.14b).These
data demonstrate that endothelial ablation of TSP1 normalized the
pancreaticisletdysfunctioncausedbyIRE1αdeficiencyinECsduring
dietaryobesity.ThissuggeststhatTSP1inisletECsserves,inlargepart,
as a key effector molecule mediating IRE1α’s regulatory actions in
maintainingtheendothelialsupportfortheadaptivecompensationof
isletfunctionunderobesity-associatedmetabolicstress.

Methods
Animals
AllanimalstudieswereconductedinaccordancewiththeInstitutional
Guidelines for the humane treatmentofanimals, with experimental
protocols(No.WDSKY0201701−2_18120D)approvedbytheCommit-
teeonEthicsintheCareandUseofLaboratoryAnimals,CollegeofLife
Sciences,WuhanUniversity.Ern1fl/flmice,inwhichthe121-nucleotide
exon 2 of the Ern1 allele was flanked by loxP, were generated at
ShanghaiResearchCenterforModelOrganismsandhadbeenback-
crossedintothegeneticbackgroundofC57BL/6J29.Tocreateadult
Ern1EC-KOmicewithEC-specificIRE1αknockout,wefirstintercrossed
Ern1fl/fl mice with VE-cadherin-Cre-ERT2 mice expressing the Cre
recombinaseunderthecontroloftheVE-cadheringenepromoter30to
generateErn1fl/fl;VE-cadherin-Cre-ERT2mice.Ern1EC-KOmiceweresubse-
quently produced after daily intraperitoneal injections of 2mg
tamoxifen(dissolvedina10:1sunflowerseedoil/ethanolmixtureat
10mg/mL)for5consecutivedaysattheageof4weeks,withErn1fl/fl
micetreatedlikewiseasthecontrolgroup.Thbs1fl/flmice(StrainNo.
T013681) were obtained from GemPharmatech (Nanjing, China).
Ern1fl/fl;Thbs1fl/fl;VE-cadherin-Cre-ERT2miceweregeneratedasthesec-
ond filial generation from Ern1fl/fl;VE-cadherin-Cre-ERT2 and Thbs1fl/fl
mice. Ern1EC-KO;Thbs1EC-KO double-knockout mice were subsequently
producedaftertamoxifeninjections.Animalswerehousedinlabora-
torycagesat22±3°Cwithahumidityof35±5%undera12-hdark/light
cycle(lightsonat7:00a.m.).Fordiet-inducedobesity,age-matched
maleandfemalemiceofdifferentgenotypeswererandomlyallocated
togroups(2micepercage)andfedahigh-fatdiet(HFD,60%kcalfat;
ResearchDiets)for16weeks,startingat8weeksofage.Foranimal
euthanasia,mice weresacrificed bycervical dislocation under anes-
thesia via i.p. injection of 2,2,2-Tribromoethanol (250mg/kg body
weightformice,Sigma-Aldrich,T48402).

Metabolicphenotyping
Whole-bodyfatmasswasmeasuredby1H-nuclearmagneticresonance
(NMR) spectroscopy (Minispec LF50, BRUKER). To analyze energy
expenditure,single-housedmicewereacclimatedfor24htoanOxy-
MaxComprehensiveLaboratoryAnimalMonitoringSystem(CLAMS,
Columbus Instruments) before oxygen consumption and physical
activity were monitored for 24h. Oxygen consumption (VO ) was
2
calculatedafternormalizationtotheleanmass.Forglucosetolerance
test(GTT),micewerefastedfor16hbeforeintraperitonealinjectionof
glucose at 1.0g/kg body weight for NC-fed mice or 1.5g/kg body
weightforHFD-fedmice.Forinsulintolerancetest(ITT),micewere
fastedfor4handtheninjectedwithinsulin(Roche)at0.75U/kgor
1.00U/kgbodyweightforNC-orHFD-fedmice,respectively.Blood
glucosewasdeterminedwithaglucometer(FreeStyle)beforeinjection
(0min)orat15,30,60or90minafterinjection.Forglucosestimu-
latedinsulinsecretion(GSIS)analysisinvivo,micewerefastedfor16h
beforeintraperitonealinjectionofglucoseasinGTTanalysis.Blood
wascollectedfromtailveinforinsulinmeasurement,andserumlevels
ofinsulinweremeasuredbytheinsulinELISAkit(Millipore,EZRMI-
13K)accordingtothemanufacturers’instructions.Formeasurement
ofserumTSP1levels,tail-veinbloodwascollectedandallowedtostand
atroomtemperaturefor45min,followedbycentrifugationtwicefor
10minat1000×g.ThesupernatantwasusedtodetermineserumTSP1
levelswiththeELISAkit(JONLNBIO,JL30686)accordingtotheman-
ufacturer’sinstructions.

Isolationofmouseliver,lung,heartandpancreas
endothelialcells
For isolation of lung, and heart ECs, tissues were harvested from
mice,mincedandthendigestedwith1.5mg/mLcollagenasetypeI
(Sigma-Aldrich, C0130; added in RPMI 1640 with 5%FBS, 25mM
HEPES and 1% Pen/Strep) at 37°C for 45min. Tissues were then
resuspendedinRed-Blood-Celllysisbufferfor5minandterminated
with double volume of DMEM, followed by filtering through a
100-µmnylonmesh.CellpelletswereresuspendedinPBSwith5%
FBS and incubated with the fluorochrome-conjugated antibody
FITC-anti-CD31 (BD, 553372). ECs were then sorted by Dynabeads
(ThermoFisherScientific,11035)beforebeingcollectedforWestern
immunoblotanalysis.
ForisolationofliverECs,micewereanesthetizedandliverswere
perfused with Kreb’s buffer (7mg/mL NaCl, 2mg/mL NaHCO and
3
3.6mg/mL glucose) containing 0.5mg/mL collagenase I (Yeasen,
40507ES) successively through the inferior vena cava. Livers were
dissectedandtransferredintotubesthrougha40-μmcellmesh,and
thenthevolumewasadjustedto50mLwithRPMI1640.Hepatocytes
wereremovedbythreecyclesoflow-speedcentrifugationat60xgfor
2min,andthesupernatantswerethencentrifugedat400xgfor10min
to separate hepatic nonparenchymal cells. Pelleted cells were re-
suspendedin3.3mLofRPMI1640,andthengentlylayeredonthetop
of6mLof25%Percoll(Sigma-Aldrich,17-0891-02)over5mLof50%
Percoll. After gradient centrifugation at 900xg for 20min at room
temperaturewiththebrakesetto0,cellsattheinterlayerbetweenthe
25%and 50% Percoll solution were transferred and re-suspended in
50mLofRPMI1640.Followingincubationwithanti-CD31,ECswere
similarlysortedbyDynabeadsandcollectedforsubsequentWestern
immunoblotanalysis.
ForisolationofpancreasECs,micewereanesthetizedandwhole-
body perfused through heart with 50mL Kreb’s buffer containing
0.5mg/mL collagenase II (Yeasen, 40508ES60), 0.5mg/mL col-
lagenase IV (Yeasen, 40510ES60), 0.5mg/mL dispase II (Yeasen,
40104ES60) and 0.2mg/mL trypsin inhibitor (Beyotime, SG2031).
Engorged pancreas was harvested and minced, and exocrine and
endocrinecellswereremovedbytwocyclesoflow-speedcentrifuga-
tionat60xgfor2min.Supernatantswerethencentrifugedat400xg
for 10min. Pellets wereresuspended and incubated with anti-CD31,
and ECs were sorted by Dynabeads and collected for subsequent
Westernimmunoblotanalysis.

Histology,immunohistochemistryandβcellmassanalysis
Pancreas,liver,brownandwhiteadiposetissueswereisolatedfrom
miceandfixedin10%neutralformalinforatleast48hat4°C.Tissue
werethendesiccatedandembeddedinparaffinbeforebeingsliced
into4-µmthicksectionsforhematoxylinandeosin(H&E)(Servicebio)
stainingorimmunohistochemistry(IHC).ForIHCanalysis,pancreas,
liver and adiposetissue sections were mounted onadhesion slides,
deparaffinizedandrehydratedusingastandardprotocol(2xXylene,2
x100%Ethanol,95%v/vEthanol,70%v/vEthanol,50%v/vEthanol,and
water;eachfor5min).Antigenretrievalwasdonebyboilingtheslides
intheCitratebuffer(10mMsodiumcitrate,0.05%v/vTween20,pH
6.0)inamicrowaveovenfor10min.Slideswerethencooleddownto
roomtemperaturefor20min,rinsedwithPBSandthenplacedina
humiditychamberforblockingandpermeabilizationintheblocking
buffer(2%v/vgoatserum,1%w/vBSA,0.1%v/vTritonX-100,0.05%v/v
Tween 20 in PBS) for 1.5h at room temperature. Slides were then
incubatedovernightat4°Cwithanti-Insulin(ABclonal,A19066;dilu-
ted1:400),anti-CD31(RDsystems,AF3628-SP;diluted1:200)oranti-
CD34(ABclonal,A19015;diluted1:200)primaryantibodyinPBSsup-
plemented with 1% BSA and 0.05% Tween-20 with IgG staining as
negativecontrol.SlideswerewashedthreetimeswithPBSandthen
incubatedwithsecondaryantibodies(ABclonal,AS014orAS029)ata
1:500dilutionfor1hatroomtemperature.Afterwashingthreetimes
with PBS, slides were stained using a 3,3’-diaminobenzidine (DAB)
substratekit(Abcam,ab64238)followingthemanufacturer’sinstruc-
tions.AfterrinsingwithPBStostopthechromogenicreaction,slides
were counterstained with hematoxylin QS (Vector Laboratories) for
2minbeforeanalysisbymicroscopy(LeciaAperioVERSA8).
Forwhole-mountimmunofluorescencestaining,freshlyisolated
islets,scWATorepiWATtissuelumpswerefixedin4%PFAat4°Cfor
1h,andpermeabilizedwithPBSTbuffer(0.1%TritonX-100inPBS)for
30min.IsletswereblockedinPBSTbufferwith10%FBSfor2h,and
WATtissueswereblockedinMABTbuffer(11.6g/LMaleicacid,8.7g/L
NaCl,0.1%Tween20)with10%FBSfor2h.Tissueswerethenincubated
with anti-Insulin (Abcam, ab10983), anti-CD31 (BD Pharmingen,
553372), anti-Perilipin-1 (CST, 3470), or anti-Ki67 (BD, 612472; CST,
9127)primaryantibodyintheblockingbufferat4°Cfor24h,followed
bywashing3timeswithPBSTbufferbeforeincubationwiththesec-
ondaryantibody(ThermoFisherScientific,A-11012)ata1:200dilution
at4°Cfor24h.TissuesweremountedwithAntifadeReagent(Vector
laboratories,H-1200),andimaginganalysiswasdoneusingLeicaTCS
SP8ConfocalMicroscopy.
Theisletnumberandsizeineachpancreassectionalongwithβ
cellmassineachmousewerequantifiedfromatotalof15–18pan-
creassectionsanalyzedbyIHCpermousegroup(3sections/mouse,
atleast90-μmapart;5-6mice/group).Theaverageisletsizeineach
sectionwascalculatedbydividingtheareaofinsulin-positive(Ins+)
regionbyisletnumber.Forβcellmassanalysis,totalIns+areaand
totalpancreasareaforeachsectionweremeasured,andβcellmass
wasthendeterminedthroughmultiplyingthepancreasweightwith
the ratio of Ins+ area/pancreas area. The percentage of CD31+ or
CD34+areaineachisletwascalculatedbydividingCD31+orCD34+
areabyisletarea.AllIHCquantificationwasdoneusingtheCase-
ViewerSoftware(Servicebio).
The glucagon-positive(Gcg+) α and insulin-positive(Ins+) β cell
areaswerequantifiedfromimmunofluorescentstainingofpancreas
sections,andtheareapercentageofαandβcellsperisletforeach
mousewascalculatedbydividingtheGcg+orIns+areabyisletarea(12-
20isletspersection,1section/mouse,6-9mice/group).Thenumberof
CD31 and Ki67 double-positive (CD31+/Ki67+) cells, insulin and Ki67
double-positive (Ins+/Ki67+) cells, and insulin and TUNEL double-
positive(Ins+/TUNEL+)cellsineachisletwasalsoquantified,allusing
theImageScopeSoftware(Leicav12.3.3.5048)andImageJSoftware.

Animalsbloodperfusionanalysis
Micewereanaesthetizedwithsevofluranewhiletheirbodytempera-
turewaskeptat37°C.Thebloodflowinthepancreasandinguinal
white adiposetissue wasmeasured using moorFLPI-2 Laser Speckle
ContrastImager(MoorInstruments,Axminster,Devon,UK)following
themanufacturer’sinstructions.Coloredimageswereacquiredfrom
1500framesduringthe1-minutecapturetime.

TUNELAssayandCellViabilityAnalysis
Celldeath in islets wasanalyzed using the DeadEndTM Fluorometric
TUNEL System (Promega, G3250) according to the manufacturer’s
instructions. TUNEL signals were visualized by fluorescence
microscopy.
Murineislet-derivedMS1endothelialcellswerefromATCC(CRL-
2279)andculturedinDMEMwith5%FBS.MS1cellswereculturedin
DMEMwith16mMglucoseandnoFBSforcellproliferationassays.
CellsweretreatedwithrecombinanthumanTSP1protein(ABclonal,
RP01197)in the absenceorpresenceofanti-CD47 neutralizing anti-
body(BioXCell-InVivoMAb,BE0283;CloneMIAP410)orIgGcontrol
(Bio X Cell-InVivoMAb, BE0083; Clone MOPC-21). MIN6 cells were
kindly provided by Dr. Peter Arvan from University of Michigan
Medical School and were cultured in RPMI 1640 with 10% FBS and
0.05mMβ-mercaptoethanol.MIN6cellsmaintainedinRPMI1640with
2000mg/LglucoseandnoFBSweretreatedwithTSP1proteinpriorto
viability analysis. Cell viability was assessed by the Enhanced Cell
Counting Kit-8(CCK8)accordingtothemanufacturer’s instructions
(BeyotimeBiotechnology,C0041).

Primaryisletisolationandinsulinsecretionanalysis
MicewereeuthanizedandinjectedwithHanks-balancedsaltsolution
containingCollagenaseV(Sigma-Aldrich,C9263)intotheircommon
bile duct. Distended pancreas was then isolated and digested for
20min at 37°C. For GSIS experiments, pancreases from 3 mice per
groupwerepooledandfiltered,followedbygentlecentrifugationin
Histopaque®-1119 (Sigma-Aldrich, 11191). Islets were then isolated by
handpickunderadissectingmicroscopeandcollectedinRPMImed-
iumcontaining10%fetalbovineserum,50μg/mLstreptomycin,and
50U/mLpenicillin.Isletswereculturedovernightat37°Cinahumi-
dified,5%CO atmospherebeforefurtherexperiments.ForGSISana-
2
lysis,isletspooledfrom3miceanddividedindesiredreplicateswere
incubated for 30min in HEPES-balanced Krebs-Ringer bicarbonate
(KRBH) buffer (135mM NaCl, 3.6mM KCl, 0.5mM MgSO ·7H O,
4 2
0.5mMNaH PO ,2mMNaHCO ,10mMHEPES,1.5mMCaCl and0.1%
2 4 3 2
BSA)withoutglucose.Afterincubationfor1hinKRBHcontaininglow
glucose(2.8mM),isletswerethenstimulatedinKRBHcontaininghigh
glucose(16.8mM)for1h.Supernatantswerecollected,andisletswere
homogenized inlysis bufferforproteinextractionat4°C.Secreted
insulinandisletinsulincontentweremeasuredbyELISA(Millipore,
EZRMI-13K)andnormalizedtothetotalisletproteincontent.

Isletperifusionandcalciumimaginganalyses
Primary islets were isolated from mice and cultured overnight
beforeperifusionexperiments.Foreachassay,perifusionchambers
wereloadedwith150IEQislets,and isletswerefirstperifusedfor
30min with KRBH buffer containing 2.8mM glucose, followed by
perifusion at <1mL/min flow rate with KRBH buffer containing
2.8mMglucose,16.8mMglucose,2.8mMglucoseand30mMKCl
for 25, 30, 20 and 20min, respectively. Fractions were collected
everyminute,andisletswererecollectedfromthechamberafterthe
assayfortotalproteincontentmeasurementusingtheBCAprotein
assay.Insulinsecretionateachtimepointwasdeterminedusingthe
HTRF insulin assay kit (Cisbio, 62INSPEC) according to the manu-
facturer’sinstructions.Foranalyzingthebiphasicinsulinsecretion,
phaseIandphaseIIofinsulinsecretionwerebasedontheplateau
formationofthetwophasesandquantifiedastheareaundercurve
(AUC).Insulinlevelswerenormalizedtotheaveragebaselineinsulin
secretion at low glucose (2.8mM) and presented as fold insulin
secretion.
Forcalciumimaginganalysis,isolatedprimaryisletswerecul-
tured overnight and washed twice with KRBH buffer containing
2mM glucose, followed by loading for 45min with KRBH buffer
containing 5.6µM Fluo-4 AM (Thermo Fisher Scientific, F14201),
0.4‰PluronicF127(Sigma-Aldrich,P2443),and2mMglucose.Islets
werethenwashedtwicewithKRBHbuffercontaining2mMglucose,
seeded on 35-mm glass-bottom dish (MatTek), and cultured for
another15min.Isletswereimagedinahumidified37°Ccellculture
chamberwith5%CO thatwasmountedonaZeissLSM800inverted
2
confocal microscope. Continuous images were acquired with 10-s
intervals,startingat2minafterglucosesupplementationtoreach
a final concentration of 16.8mM until fluorescent plateaus
were reached. Fluorescent intensity dynamics were analyzed
usingImageJ.

Isolationofprimaryhumanumbilicalveinendothelialcells
(HUVECs)
HumanumbilicalcordswereobtainedfromWuhanMaternalandChild
HealthHospitalofHubeiprovince,TongjiMedicalCollege,Huazhong
UniversityofScienceandTechnologywithethicalapprovalfromthe
Clinical Trial Ethics Committee and written consent from patients.
HUVECswereisolatedfromumbilicalcordveinwithPBScontaining
1mg/mLCollagenase1(Yeasen,40507ES)andculturedinVascuLife
EnGS-MvEndothelialMedium(LifelineCellTechnology,LL-0004)in
T-25flaskswhichwerecoatedwith1%gelatinfor30min.Cellswere
cultured at 37°C with 5% CO overnight and maintained in fresh
2
mediumbeforefurtheranalysis.

Geneexpressionanalysis
TotalRNAfromtissueorculturedcellswasextractedandpurifiedwith
TRIzol reagent (Sigma-Aldrich, 93289). cDNA was synthesized by
M-MLVreversetranscriptase(ThermoFisherScientific,M1682).Rela-
tiveabundanceofmRNAwasdeterminedbyqPCRusingSYBRGreen
(Abclonal, RK21203) on StepOne Real-Time PCR Systems (Applied
Biosystems,4376357),withvaluesnormalizedtoActborRplp0asthe
internalcontrolusingtheΔΔ-Ctmethod.Oligonucleotideprimerpairs
used are listed in Supplementary Table 1 and all oligonucleotides
purchasedfromSangonBiotech.

Virusconstructionandproduction
ForknockdownoftheexpressionofIRE1αorTSP1,lentivirusparticles
wereproducedbyco-transfectionofHEK-293Tcellswith5μgpsPAX2
and 5μg pMD2.G packaging plasmid together with 10μg pLKO.1-
shScramble-Puro, pLKO.1-shScramble-Hygro, pLKO.1-shErn1-Puro,
pLKO.1-shERN1-Puro or pLKO.1-shThbs1-Hygro plasmid. Cell culture
medium containing lentivirus particles was harvested at 48h post
transfectionandusedtoincubateovernightwithMS1cellsorHUVECs
pluspolybrene(5μg/mL),followedbyculturingincompletemedium
containingpuromycin(1μg/mL)orhygromycin(100μg/mL)for48h.
ThetargetsequencesforshRNAsagainstIRE1αandTSP1arelistedin
SupplementaryTable2.

Expressionconstructsandtransienttransfection
IRE1αknockoutHEK-293TcellsweregeneratedbyCRISPR/Cas9with
Lenti-CRISPR-v2plasmid,with5’-GTTGTTGTTTGTGTCAACGC-3’used
as the guide sequence. The IRE1αWT and IRE1αK907A constructs were
subclonedfromplasmidspreviouslydescribed63withpLVX-IRES-Neo
backbone.TheexpressionconstructsforTHBS1andXBP1sweregen-
eratedbycloningtherespectivesegmentsofcodingsequencepro-
ducedbyPCRintothepLVX-IRES-Myc-NeoorpCMV-tag4Abackbone.
Forproteinoverexpression,transienttransfectionexperimentswere
performedusingLipofectamine2000(Invitrogen,11668019)accord-
ingtothemanufacturer’sinstructions.

Westernimmunoblotanalysis
TissuesamplesorcellswerelysedinRIPAbuffer(1%TritonX-100,1%
NP-40, 150mM NaCl, 0.5 % sodium deoxycholate, 0.1 % SDS, and
50mM Tris-HCL, PH7.4) containing complete protease inhibitor
cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). The
supernatantofcelllysateswastransferredintonewmicrotubesand
mixedwithonevolumeofMPSB(62.5mMTris-HCl,15%SDS,8MUrea,
10%glyceroland100mMDTT,pH=6.8)and20%volumeof6xloading
buffer(300mMTris-HCl,12%SDS,0.6%bromophenolblue,60%gly-
cerol and 600mM DTT, pH=7.4) after 1,2000xg centrifugation for
10min.Themixturewasthenincubatedfor30minat37°C.Proteins
wereseparatedbySDS-PAGE,transferredtonitrocellulosemembrane,
andprobedwiththedesiredantibody.Proteinlevelswerequantified
with NIH ImageJ software. Antibodies were diluted in the blocking
solutionandusedasfollowsandarelistedinSupplementaryTable4:
anti-IRE1α,1:1000(CST,3294);anti-XBP1,1:1000(ABcam,ab220783);
anti-Flag, 1:2000 (ABclonal, AE005); anti-Myc, 1:2000 (ABclonal,
AE010);anti-β-Actin,1:50000(ABclonal,AC026);anti-phospho-IRE1α-
S724, 1:1000 (ABclonal, AP1146); anti-Akt, 1:2000 (CST, 9272); anti-
phospho-Akt-S473, 1:2000 (CST, 4060); anti-TSP1, 1:1000 (CST,
37879); anti-eNOS, 1:1000 (CST, 32027); anti-phospho-eNOS-S1177,
1:1000(CST,9570)andanti-α-Tubulin,1:5000(Sigma-Aldrich,T6199).

RNA-seqanalysis
TotalRNAwasextracted from isolated islets ofHFD-fed Ern1fl/fl and
Ern1EC-KO mice and then subjected to commercial RNA-seq analysis
(Beijing Genomics Institute). Three independent islet samples per
group were analyzed. Paired-end,150-ntreads wereobtained from
eachsequencinglane.ThesequencingdatawerefilteredwithSOAP-
nuke(v1.5.2)79by(1)removingreadscontainingsequencingadapters;
(2)removingreadswhoselow-qualitybaseratio(basequalitylessthan
orequalto5)ismorethan20%;(3)removingreadswhoseunknown
base(‘N’base)ratioismorethan5%.Cleanreadswereobtainedand
storedintheFASTQformat,andweremappedtothemousegenome,
UCSCmm10(~94.03%alignment),usingtheHISAT2(v2.0.4)80.The
Bowtie2(v2.2.5)81wasappliedtoalignthecleanreadstothereference
codinggeneset,andtheexpressionleveloftargetgenewascalculated
bytheRSEM(v1.2.12)82.Differentialexpressionanalysiswasperformed
using the DESeq2(v 1.4.5)83. Differential expression P values are
obtainedfromDESeq2two-sidedWaldtestsofthenullhypothesisthat
log fold-change=0foreachgene,withBenjamini-HochbergFDRcor-
2
rectionacrossallgenes.Thecriteriaforconsideringagenebeingdif-
ferentiallyregulatedwerebasedonalog (fold-change)greaterthan
2
0.5 (either direction) and a significant P value<0.01 versus control
analyzed by two-sided hypergeometric (Fisher’s exact) tests with
Benjamini-HochbergFDRcorrection.GOandpathwaygroupingand
enrichmentanalysiswereperformedbytheclusterProfiler(v3.16.1)84.
Fordetectionofgeneticknockoutevents,Ern1mRNAofisletsfrom
HFD-fedErn1fl/flandErn1EC-KOmice(n=3pergroup)wasanalyzedby
rMATS(v4.1.0)85.Exon2inclusionlevelswereshownasIncLevel.Islet
RNA-seq data have been deposited in the NCBI Gene Expression
OmnibuswiththeaccessionnumberGSE263586.
ForscRNA-seqanalysis,cellswithlessthan200detectablegenes
or greater than 20% mitochondrial genes (calculated with the Per-
centageFeatureSetfunctionintheSeuratpackage86)wereexcluded
fromfurtheranalysis.DatawerenormalizedusingtheNormalizeData
functionintheSeuratpackagetoidentifygeneswithhighvariability.
“Anchors”betweendatasetswereidentifiedanddatafromdifferent
samples were integrated using the Find Integration Anchors and
IntegrateDataintheSeuratpackage,respectively.Datawerescaled,
andprincipalcomponentanalysis(PCA)wasperformedbasedonthe
top2000high-variabilitygenes.Top7principalswereusedfordis-
tributedstochasticneighborembedding(t-SNE)construction.Feature
plotsandviolinplotsweregeneratedbyfunctionsinSeuratpackage.

ActinomycinDPulse-chaseAnalysis
The stability of mRNAs of interest in MS1 cells was determined by
addition of 5μg/mL actinomycin D (Act D) (MedChemExpress, HY-
17559)togetherwith10μg/mLtunicamycin(Tm)aftercellswerepre-
treated with DMSOor10μM4μ8C.Total RNAwas extracted atthe
indicatedtimeforqRT-PCRanalysis.TheamountofthetargetmRNA
at each time point was determined relative to Rplp0 mRNA as the
internalcontrol,andplottedrelativetoitsvalueattime0thatwas
consideredas1.

RNAdecayanalysis
Oligonucleotide primers containing a minimal T7 RNA polymerase
promoter(5’-TAATACGACTCACTATAGG-3’)wereusedforPCRfrom
expressionplasmidDNAtemplatestogenerateDNAfragmentscon-
tainingT7promotersequenceupstreamofthe1-1770or1771-3510nt
segmentofthecodingsequenceofhumanTHBS1,orthefullcoding
regionofhumanXBP1.Invitrotranscriptionreactionswereperformed
using the T7high-yield RNA synthesis kit (TransGen Biotech, JT101)
followingthemanufacturer’srecommendations.ThetranscribedRNAs
were treated for 20min with DNase and purified by EasyPure RNA
Purification Kit (TransGen Biotech, ER701). For RNA decay analysis,
~2μg transcribedRNAor ~0.1nmole synthetic RNAsubstrates were
incubated for 1h with 1μg recombinant hIRE1α protein plus 10μM
4μ8C or DMSO in the RNA cleavage buffer (40mM HEPES pH 7.0,
10mMMg(OAc) ,50mMKOAc,5mMDDT).RNAsampleswerethen
2
analyzedby3%Agarosegelor20%TBE-UreaPAGEgelelectrophoresis,
followedbyvisualizationusingYeaRedNucleicAcidGelStain(Yeasen,
10202ES76).TheoligonucleotideprimersforPCRproductionofTHBS1
and XBP1 templates for in vitro transcription are listed in Supple-
mentaryTable3.

Statisticalanalysis
Alldataarepresentedasthemean±SEM.Statisticalanalysiswasper-
formedwithunpairedtwo-tailedStudent’st-testortwo-wayanalysisof
variance (ANOVA) with Fisher’s LSD test using GraphPad Prism 7.0.
P<0.05wasconsideredtobestatisticallysignificant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the present manuscript the authors test the idea that IRE1α alters the vascular network of islets to impeach their function through intersecting antiangiogenic TSP1. The hypothesis is tested using a range of approaches foremost being mutant mice that lack IRE1α in vascular endothelial cells uniformly and pathway suppression with siRNA methods. Young mice lacking IRE1α were fed a diet high in fat and metabolic readouts characterized. The text is further supported by helpful supplemental figures with control data. Overall, the composition is clear and easy to follow. A few minor usage issues were encountered but not enough to detract from the reading of the text. The data support the conclusions mostly, although certain aspects lack signaling direction and specificity and these points are explained in detail below.\nStrategic/novelty: The hypothesis is new, as are the findings. However, a link between IRE1α and TSP1 was noted (J Cell Mol Med. 2022 Oct;26(20):5303-5314). Further, links between human islets and TSP1 and the dominant TSP1 receptor CD47 was published (Am J Physiol Endocrinol Metab. 2023 Apr 1;324(4): E347-E357). Also, a link between TSP1-CD47 and glucose homeostasis was made in mice on a HFD (Cells. 2020 Jul 15;9(7):1695). These points are emphasized not to lessen the present work. Still these references should be covered in the Introduction and/or Discussion section of the manuscript to give a balanced presentation of the existing literature in relation to the new findings.\nBalance in presentation: Not to belabor the ideas noted above, but here is an example of limited and erroneous use of citations by the authors. They cite the following 2 papers when introducing their data on TSP1 – (1) Molecular basis for the regulation of angiogenesis by thrombospondin-1 and -2. Cold Spring Harb Perspect Med 2, a006627 (2012) from Jack Lawler at Harvard. The issue here is the year of publication 2012 so it misses many more recent findings. The second citation, Thrombospondin 1 in Metabolic Diseases. Front Endocrinol (Lausanne) 12, 638536 (2021), was from folks in Pennsylvania that have done only a little work in this area. More importantly, one should always cite primary papers and in this case some that show how TSP1 limits ECs, vascular pathways, blood flow, self-renewal (see effects on cMyc), and metabolism. See Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13141-6 and others by this same group.\nMethods: Please provide detailed information on animal usage: ACUC protocol #, anesthesia and euthanasia methods, balancing and mitigation of the cage effect, randomization of animals into treatment groups (was a software program used for this and if yes please state). Explain how cohort size was arrived at. Please provide details on the methods used to blind the researchers to treatment groups, etc. Justify using young mice and if only one sex was used justify why both were not studied.\nData: Counting CD31+ cells is a common metric employed to characterize vascularization. Yet, it gives no information concerning real-time tissue perfusion. This is a valid point as TSP1 seems to limit perfusion of various vascular beds separate from inhibiting cell and specifically EC proliferation and migration. Some analysis of organ (i.e., pancreas) and organoid (i.e., islet) blood flow should be made in the mice from the various treatment groups.\nThe paper is light on details on the signaling mechanism(s) at play. We do not the source of the TSP1 in the islets that is putatively the cause of issues and a target for IRE1α. A change in mRNA suggests a cell type may be the source but not more. The literature finds many stressed cell types from many species and people make TSP1. However, some data already pointed out in mice lacking the TSP1 signal versus circulating cell lacking TSP1 found the effect resided in the parenchyma and not in the circulating cells.\nWe do not know the receptors that secreated TSP1 acts through in the islets to negatively impact glucose homeostasis. One place to begin would be to look at WT and IRE1α null EC receptor patterns for primary TSP1 receptors such as CD36, CD47, beta integrins, and SIRPα. TSP1 apparently has several domains that can bind to one of these receptors and adversely alter redox pathways and metabolism. After the authors define the cell surface landscape, they should treat cells with TSP1 ± TSP1 receptor blockers and then characterize proliferation, cell death, etc.\nThe authors might also consider testing possible direct effects of IRE1α on vascular nitric oxide. See Mol Cells. 2018 Nov 30;41(11):964-970 and Aging (Albany NY). 2023 Dec 13;15(23):13608-13627.\nWhy was there had only a partial loss in IRE1α protein in the lung EC of the mutant mice. All westerns need molecular marker dye lane. I find just placing the expected MW adjacent to a blot leaves out key information that as the reader I wish to consider for myself. Included the original full blots too.\nMany blots of TSP1 find it at about 150 kD. The blot in Fig. 5F has it assigned a MW of 170. Please comment on this. BTW, TSP1 is up in the plasma of people with T1 and T2 diabetes. The Discussion section should point this out and cite the several papers that showed this.\nThis being the case, I am wondering if more detail in plasma TSP1 analysis can be given. Care is needed to not activate platelets as they were found decades ago to hold lots of pre-made TSP1. And the lack of impact of HF diet on plasma TSP1 is curious. Would a longer disease timeline alter this?\nThe TSP1 translation blocking morpholino gave only a partial suppression. Was a dose and time interval study done to optimize the conditions?\nAdditional information: A table summarizing all antibodies, manufacture details, dilutions, and validation of specificity information should be included.\nFigures: These were nicely done, and one can get the point fast. And the legends were well done with n numbers and stat test used included for each.", "reviewer_2": "The main finding of this manuscript is that genetic depletion of IRE1 alpha in endothelial cells leads to glucose intolerance, blunted intra-islet angiogenesis, compensatory islet growth and enhanced endothelial cells thbs1 expression in high-fat diet-fed mice. Further, the author report that genetic deletion of thbs1 in endothelial cells corrected the defects in glucose homeostasis observed in HFD-fed mice with genetic deletion of IRE1alpha in endothelial cells. The authors conclude that their findings “uncover a critical role of the endothelial IRE1alpha suppression of THBS1 in governing the vascular support that enables the functional adaptation of islets during metabolic stress”. However, substantial issues have been identified that limit the interpretation of the data.\nComments:\n- The genetic mouse models need further validation, including confirmation that gene expression and protein levels of IRE1alpha and THBS1 are significantly reduced in other tissues than lung, especially in islets and in tissues involved in glucose homeostasis, but also confirmation that their levels are not altered in other cell types than endothelial cells.\n- For the in vivo studies, the authors must show data from VE-cadherin-Cre-ERT2 mice without tamoxifen injection to confirm that data are not related to the CRE expression only.\n- Based on the data shown on both Figure 1 and figure 8, the authors cannot conclude that there is glucose intolerance and deficient insulin production. Indeed, the authors report insulin data without taking in account and correcting for glucose data at the same time. The authors can absolutely not conclude that fasting insulin and glucose-stimulated insulin secretion are decreased without reporting the glucose data, since insulin is dependent on glucose levels. Further, on both figure 1 and figure 8, glucose tolerance must be calculated as iAUC during the OGTT.\n- The authors must report data in female and/or at least justify why they did only report data in male mice\n- Information about the methods and experiments is lacking throughout the manuscript. For example, there is no information regarding how blood was drawn for insulin measurement (Fig 1) or how islets were processed for insulin content measurement (Fig 2). For histo and immunohistochemistry, the authors must have used IgG isotype as negative controls for staining. The n are very low for several experiments; only representative images of one experiment are shown (e.g. Fig 1b, Fig S1,..) and some figures show only replicate of one experiment (e.g. GSIS and perifusion data on Fig3).\n- Beta-cell and alpha cell area and mass rather than % of alpha and beta-cell must be correctly calculated and reported to allow the authors to conclude there are changes in islet morphology.", "reviewer_3": "In this report, the authors demonstrate that genetic deletion of the inositol-requiring enzyme 1 (IRE1α) specifically in the endothelial cells of the adult mice, results in the poor response of the vascular cells to the metabolic stress induced by enforced obesity secondary to chronic high fat diet (HFD) intake. These abnormalities in IRE1α deficient endothelial cells were manifested in subtle glucose intolerance with impaired insulin secretion. The mechanism of this metabolic dysregulation was mediated through upregulation of the mRNA encoding presumed anti-angiogenic agent thrombospondin-1 (THBS1/TSP1). IRE1α RNase15 mediated mRNA decay of THBS1 presumably modulate the overall expression of this factor in SV40 large T antigen immortalized MS1 mouse endothelial cells. Knock down of THBS1 in endothelial cells restored the islet vascular defects arising from endothelial cell-IRE1α deficiency in response to HDF induced obesity.\nComments:\nThe mechanism by which neo-angiogenesis might play a role in the regulation of obesity induced metabolic dysfunction is not known. Demonstration that IRE1α deficiency through upregulation of THBS1 might interfere with angiogenic activity in the islets thereby contributing to metabolic derangements afforded by chronic HDF induced obesity could have therapeutic implications.\nHowever, the data presented are not that convincing and many of the conclusions are not strongly supported by the data provided.\n1) The majority of the changes in glucose levels and insulin intolerance in the IRE1α deficient mice are only marginal, which are corrected with the THBS1 knock down. Indeed, the pathophysiological consequences of those metabolic changes are not readily apparent. Are there any pathological changes in the cardiovascular function, liver metabolism or kidney function in the IRE1α deficient mice ?\n2) It is unclear whether the subtle differences in hyperglycemia and insulin intolerance are primarily due to pancreatic dysfunction or increase in the adipose tissue vascular mass changes secondary to the chronic HFD induced obesity? Is there a correlation with the increase vascular mass index with the metabolic changes potentially in the adipose tissue of the IRE1α or THBS1 deficient mice.\n3) The authors claim that there could be a defect in the islet angiogenesis in the IRE1α or THBS1 deficient mice. However, whether there are changes in the vessel density and as to how these changes lead to metabolic abnormalities observed is not well characterized. In figure 4 the authors use CD31 (PECAM) as the marker for endothelial cell staining. However, CD31 staining is not sufficient to specifically stain endothelial cells as CD31 is also expressed on hematopoietic cells including inflammatory monocytes and macrophages. Other vascular specific markers such as VE-cadherin or CD34 might be considered.\n4) The use of mouse MS1 SV40 large T antigen immortalized endothelial cells could be fraught with artifacts because metabolically these cells employ oxidative phosphorylation, but not glycolysis, secondary to oncogenic transformation possibly obscuring the effects I IRE1α and XBP1 mediated stress responses. Use of more primary isolated endothelial cells, preferably from the metabolic organs might be more revealing.\n5) In IRE1α deficient mice there are also changes in the expression and RNA decay of other factors. Then, is THBS1 dysregulation the main factor that contributes to the pathogenesis of metabolic dysfunction in the IRE1α mice? Why the authors elected to focus on THBS1 and not the other factors.\n6) THBS1 has many different pleiotropic effects on regulating blood vessel integrity. Besides potential anti-angiogenic effect, THSP1 could also modulate homeostatic functions of blood vessels not only in the pancreas but also in adipose or liver vasculature. Then, to what extent the metabolic changes in IRE1α or THSP1 deficient mice, could be attributed to its anti-angiogenic activity?"}, "author_response": "Point-by-point response to the reviewers’ comments:\nReviewer #1:\nIn the present manuscript the authors test the idea that IRE1α alters the vascular network\nof islets to impeach their function through intersecting antiangiogenic TSP1. The\nhypothesis is tested using a range of approaches foremost being mutant mice that lack\nIRE1α in vascular endothelial cells uniformly and pathway suppression with siRNA\nmethods. Young mice lacking IRE1α were fed a diet high in fat and metabolic readouts\ncharacterized. The text is further supported by helpful supplemental figures with control\ndata. Overall, the composition is clear and easy to follow. A few minor usage issues\nwere encountered but not enough to detract from the reading of the text. The data\nsupport the conclusions mostly, although certain aspects lack signaling direction and\nspecificity and these points are explained in detail below.\nResponse:\nMany thanks for the Reviewer’s constructive and positive comments.\n1) Strategic/novelty: The hypothesis is new, as are the findings. However, a link\nbetween IRE1α and TSP1 was noted (J Cell Mol Med. 2022 Oct;26(20):5303-5314).\nFurther, links between human islets and TSP1 and the dominant TSP1 receptor CD47\nwas published (Am J Physiol Endocrinol Metab. 2023 Apr 1;324(4): E347-E357). Also,\na link between TSP1-CD47 and glucose homeostasis was made in mice on a HFD (Cells.\nStill these references should be covered in the Introduction and/or Discussion section\nof the manuscript to give a balanced presentation of the existing literature in relation to\nthe new findings.\nBalance in presentation: Not to belabor the ideas noted above, but here is an example\nof limited and erroneous use of citations by the authors. They cite the following 2 papers\nwhen introducing their data on TSP1 – (1) Molecular basis for the regulation of\nangiogenesis by thrombospondin-1 and -2. Cold Spring Harb Perspect Med 2, a006627\n(2012) from Jack Lawler at Harvard. The issue here is the year of publication 2012 so\nit misses many more recent findings. The second citation, Thrombospondin 1 in\nMetabolic Diseases. Front Endocrinol (Lausanne) 12, 638536 (2021), was from folks\nin Pennsylvania that have done only a little work in this area. More importantly, one\nshould always cite primary papers and in this case some that show how TSP1 limits\nECs, vascular pathways, blood flow, self-renewal (see effects on cMyc), and\nmetabolism. See Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13141-6 and others\nby this same group.\nResponse:\nWe sincerely appreciate the Reviewer’s important point that we should have more\ncarefully cited relevant primary papers on TSP1. In our revised manuscript, we included\nseveral original papers (Ref #46, 48, 53-57) when introducing our data on TSP1,\nits\nlinks to human islets, as well its signaling mechanisms, as suggested by the Reviewer.\nBecause we could not find in this paper (J Cell Mol Med. 2022 Oct;26(20):5303-5314)\nany data showing a clear link between IRE1α and TSP1, we chose not to cite it in order\nto avoid confusion to the readers.\n2) Methods: Please provide detailed information on animal usage: ACUC protocol #,\nanesthesia and euthanasia methods, balancing and mitigation of the cage effect,\nrandomization of animals into treatment groups (was a software program used for this\nand if yes please state). Explain how cohort size was arrived at. Please provide details\non the methods used to blind the researchers to treatment groups, etc. Justify using\nyoung mice and if only one sex was used justify why both were not studied.\nResponse:\nFollowing the Reviewer’s suggestions, we provided detailed information on animal\nexperiments, including ACUC protocol #, anesthesia and euthanasia methods, and\nmanual randomization of animals into treatment groups without using a software\nprogram. Mice were maintained at 2 animals per cage without environmental changes,\nthus reducing possible cage effects. Samples sizes were determined based on previous\nexperience, literature standards, and pilot study data. In our studies, no blinding was\nemployed as the experimental design required the investigators to know the genotyping\ninformation on each animal. However, based on the randomization of animal groups,\nwe could keep ourselves completely unbiased when collecting and analyzing our data\neven without blinding performance. Young male mice have been routinely used for\ndiet-induced obesity studies, because females or aged male mice are known to be\nmetabolically insensitive to high-fat feeding. Still, we performed dietary obesity\nanalysis for female Ern1EC-KO and Ern1fl/fl mice, which indeed showed lower sensitivity\nto HFD-induced obesity (data were presented in Supplemental Fig. S3a-d of the revised\nmanuscript). This is in accordance with results from previously documented studies (e.g.\nPMID: 33308894).\n3) Data: Counting CD31+ cells is a common metric employed to characterize\nvascularization. Yet, it gives no information concerning real-time tissue perfusion. This\nis a valid point as TSP1 seems to limit perfusion of various vascular beds separate from\ninhibiting cell and specifically EC proliferation and migration. Some analysis of organ\n(i.e., pancreas) and organoid (i.e., islet) blood flow should be made in the mice from\nthe various treatment groups.\nResponse:\nWe followed the suggestions from the Reviewer and measured the blood perfusion in\npancreas and white adipose tissue of NC- and HFD-fed mice using MoorFLPI-2 blood\nflow imager. We did not observe apparent changes of blood flow in these tissues\nexamined (data were presented in Supplemental Fig. S7), suggesting that endothelial\nIRE1α ablation likely had no obvious impact upon the blood flow rate. So far, we have\nnot been able to find reliable methodology that can be readily utilized to precisely\nmeasure the blood flow within the islets, and we wish not to speculate too much on this\npoint.\n4) The paper is light on details on the signaling mechanism(s) at play. We do not the\nsource of the TSP1 in the islets that is putatively the cause of issues and a target for\nIRE1α. A change in mRNA suggests a cell type may be the source but not more. The\nliterature finds many stressed cell types from many species and people make TSP1.\nHowever, some data already pointed out in mice lacking the TSP1 signal versus\ncirculating cell lacking TSP1 found the effect resided in the parenchyma and not in the\ncirculating cells. We do not know the receptors that secreted TSP1 acts through in the\nislets to negatively impact glucose homeostasis. One place to begin would be to look at\nWT and IRE1α null EC receptor patterns for primary TSP1 receptors such as CD36,\nCD47, beta integrins, and SIRPα. TSP1 apparently has several domains that can bind\nto one of these receptors and adversely alter redox pathways and metabolism. After the\nauthors define the cell surface landscape, they should treat cells with TSP1 ± TSP1\nreceptor blockers and then characterize proliferation, cell death, etc.\nThe authors might also consider testing possible direct effects of IRE1α on vascular\nnitric oxide. See Mol Cells. 2018 Nov 30;41(11):964-970 and Aging (Albany NY).\nResponse:\nFollowing the suggestions from the Reviewer, we further explored the signaling\nmechanisms at play. Based on the scRNA-seq analysis of islets (data were presented in\nSupplemental Fig. S11), we focused on the TSP1-CD47 axis in ECs and examined the\neffect of blocking CD47 with a neutralizing antibody in MS1 cells upon cell\nproliferation (data were presented in Fig. 6j). In addition, we also examined the impact\nof IRE1α ablation upon islet eNOS as well as that of TSP1 upon eNOS in MS1 cells\n(data were presented in Supplemental Fig. S12). While we could not detect changes in\neNOS in TSP1-treated MS1 cells, we detected decreased eNOS expression in islets with\nIRE1α deficiency in ECs. This again supports the multi-tasked nature of IRE1α function,\nand as a matter of fact, we have been studying, as an independent research project,\nthis\ninteresting point in more detail with respect to its mechanistic function in blood pressure\ncontrol.\n5) Why was there had only a partial loss in IRE1α protein in the lung EC of the mutant\nmice. All westerns need molecular marker dye lane. I find just placing the expected\nMW adjacent to a blot leaves out key information that as the reader I wish to consider\nfor myself. Included the original full blots too. Many blots of TSP1 find it at about 150\nkD. The blot in Fig. 5F has it assigned a MW of 170. Please comment on this. BTW,\nTSP1 is up in the plasma of people with T1 and T2 diabetes. The Discussion section\nshould point this out and cite the several papers that showed this. This being the case, I\nam wondering if more detail in plasma TSP1 analysis can be given. Care is needed to\nnot activate platelets as they were found decades ago to hold lots of pre-made TSP1.\nAnd the lack of impact of HF diet on plasma TSP1 is curious. Would a longer disease\ntimeline alter this?\nResponse:\nUsually the EC fraction from the lung would contain cells of other types due to non-\nspecific binding to the beads used, from which IRE1α protein can still be detected by\nWestern immunoblot analysis. To confirm the efficiency of IRE1α depletion, we\noptimized the isolation procedure and analyzed ECs isolated from the liver, lung and\nheart, and our results more clearly showed efficient ablation of IRE1α in Ern1EC-KO ECs\n(data were presented in Fig. 1c). Due to the limited space for figures containing all the\nimmunoblots, we chose to show one MW marker near the protein of interest without\nincluding the MW marker dye lane. We will provide all the original full blots in the\nSupplemental Materials and the Reporting Summary as requested by the journal if\naccepted for publication. The immunoblot of TSP1 in the previous Fig. 5F showed a\nMW position below 170, which appeared closer to 170 because of shorter running time\nof the PAGE. For the association of TSP1 with human diabetes, we added a recent\nreview (PMID: 34106789) on the topic in Discussion to avoid citing too many reference\npapers. For the impact of HFD feeding upon circulating TSP1, our data also showed\nincreased serum TSP1 levels in the obese group (presented in Supplemental Fig S9),\nwhich is in line with reported results. We used the standard protocol for serum TSP1\nprotein measurement as described in the Methods section. While it is not easy to assess\nwhether platelets may have produced a major proportion of circulating TSP1,\napparently no significant contributions could be seen from EC-derived TSP1 protein in\nthe context of endothelial IRE1α depletion.\n6) The TSP1 translation blocking morpholino gave only a partial suppression. Was a\ndose and time interval study done to optimize the conditions? Additional information:\nA table summarizing all antibodies, manufacture details, dilutions, and validation of\nspecificity information should be included.\nResponse: We actually utilized lentiviral shRNA to knock down the expression of TSP1\nin MS1 cells (Fig 6h). Usually a 36-48-hour infection would do for the knockdown\nexperiment, and an incomplete suppression of the target gene is not surprising. We\nfollowed the Reviewer’s suggestions and included a summary table for all the desired\ninformation (Supplemental Table S4).\nReviewer #2 (Remarks to the Author):\nThe main finding of this manuscript is that genetic depletion of IRE1 alpha in\nendothelial cells leads to glucose intolerance, blunted intra-islet angiogenesis,\ncompensatory islet growth and enhanced endothelial cells thbs1 expression in high-fat\ndiet-fed mice. Further, the author report that genetic deletion of thbs1 in endothelial\ncells corrected the defects in glucose homeostasis observed in HFD-fed mice with\ngenetic deletion of IRE1alpha in endothelial cells. The authors conclude that their\nfindings “uncover a critical role of the endothelial IRE1alpha suppression of THBS1 in\ngoverning the vascular support that enables the functional adaptation of islets during\nmetabolic stress”. However, substantial issues have been identified that limit the\ninterpretation of the data.\nResponse:\nWe sincerely appreciate the Reviewer’s constructive and critical comments.\nComments\n1) The genetic mouse models need further validation, including confirmation that gene\nexpression and protein levels of IRE1alpha and THBS1 are significantly reduced in\nother tissues than lung, especially in islets and in tissues involved in glucose\nhomeostasis, but also confirmation that their levels are not altered in other cell types\nthan endothelial cells.\nResponse:\nWe followed the suggestions from the Reviewer and further validated the genetic\nmouse model. We analyzed additional tissues involved in glucose homeostasis,\nincluding the liver and heart. We confirmed the efficient depletion of IRE1α and\nTHBS1 in CD31+ endothelial cells in Ern1EC-KO and Ern1EC-KO;Thbs1EC-KO mice,\nusing CD31- cells as the negative control. These data were presented in Fig.1c and\nSupplemental Fig. S14a. Owing to the technical difficulty in isolating islet ECs,\nwhich is generally known to the relevant research field, we analyzed the RNA-seq\ndata and confirmed the efficient deletion of the exon 2 of Ern1 gene in Ern1EC-KO\nmice (Supplemental Fig. S8a).\n2) For the in vivo studies, the authors must show data from VE-cadherin-Cre-ERT2\nmice without tamoxifen injection to confirm that data are not related to the CRE\nexpression only.\nFollowing the suggestions from the Reviewer, we performed metabolic phenotyping\nanalyses of Ern1fl/fl and Ern1fl/fl;VE-cad-CreERT2 mice without tamoxifen treatment\nfollowing HFD feeding for 12 weeks. We confirmed that the observed changes in islet\nadaptation of HFD-fed Ern1EC-KO mice are not related to the Cre expression only. These\ndata were presented in Supplemental Fig. S4.\n3) Based on the data shown on both Figure 1 and figure 8, the authors cannot conclude\nthat there is glucose intolerance and deficient insulin production. Indeed, the authors\nreport insulin data without taking in account and correcting for glucose data at the same\ntime. The authors can absolutely not conclude that fasting insulin and glucose-\nstimulated insulin secretion are decreased without reporting the glucose data, since\ninsulin is dependent on glucose levels. Further, on both figure 1 and figure 8, glucose\ntolerance must be calculated as iAUC during the OGTT.\nResponse:\nWe totally understand the Reviewer’s concerns and presented the metabolic data more\nclearly. We included the iAUC for the GTT results in both Fig 1f and Fig 8c, which\nshowed significant impairment of glucose tolerance in HFD-fed Ern1EC-KO but not in\nHFD-fed Ern1EC-KO;Thbs1EC-KO mice relative to their control littermates. We also\nincluded the data on serum glucose levels of HFD-fed Ern1EC-KO mice after a 4-hour\nfast when showing their serum insulin levels (Fig. 1h), which suggests the reduced\ninsulin levels did not result from lower glucose levels. For glucose-stimulated insulin\nsecretion in vivo, we observed marked reductions in insulin levels from HFD-fed\nErn1EC-KO mice (Fig. 1i) following glucose stimulation during the GTT (Fig. 1f),\nindicating significantly impaired insulin production even in the face of higher blood\nglucose levels in HFD-fed Ern1EC-KO mice.\n4) The authors must report data in female and/or at least justify why they did only report\ndata in male mice.\nResponse:\nWe followed the suggestions from the Reviewer and performed dietary obesity analysis\nfor female Ern1EC-KO and Ern1fl/fl mice, which showed lower sensitivity to HFD-\ninduced obesity (data were presented in Supplemental Fig. S3 of the revised\nmanuscript). This is in accordance with results from previously documented studies (e.g.\nPMID: 33308894). This is why male mice are usually used in the field for metabolic\nstudies of dietary obesity.\n5) Information about the methods and experiments is lacking throughout the manuscript.\nFor example, there is no information regarding how blood was drawn for insulin\nmeasurement (Fig 1) or how islets were processed for insulin content measurement (Fig\n2). For histo and immunohistochemistry, the authors must have used IgG isotype as\nnegative controls for staining. The n are very low for several experiments; only\nrepresentative images of one experiment are shown (e.g. Fig 1b, Fig S1,..) and some\nfigures show only replicate of one experiment (e.g. GSIS and perifusion data on Fig3).\nResponse:\nWe appreciate these important points from the Reviewer and included the information\nabout the methods and experiments in the Methods section, including how blood was\ndrawn for insulin measurement and how islets were processed for insulin content\nmeasurement. For immunohistochemistry staining analysis of Insulin, CD31 and CD34\nfrom pancreas sections, we used IgG as the negative control (Supplemental Fig. S6a).\nFor key metabolic phenotypes, we combined the n from repeated independent cohorts\nfor reaching a higher n number. Usually we presented representative images from at\nleast two independent experiments. We removed the flow-cytometry data in Fig 1b of\nthe previous manuscript as we employed a better method for isolating tissue ECs as\ndescribed in the Methods section. For islet perifusion data, we showed results from\npooled islets of HFD-fed mice (n=3 mice for each group, 2 independent animal cohorts).\nFor islet GSIS assay, we presented data from pooled islets in one animal cohort study\n(Fig 3f), and similar data from another independent study, which is difficult to combine\nwhen presenting the actual insulin valuse, is shown below for your information.\nPrimary islets of HFD-fed Ern1EC-KO mice showed decreased glucose stimulated\ninsulin secretion.\n6) Beta-cell and alpha cell area and mass rather than % of alpha and beta-cell must be\ncorrectly calculated and reported to allow the authors to conclude there are changes in\nislet morphology.\nResponse:\nFollowing the Reviewer’s suggestions, we calculated β cell and α cell areas in islets of\nthe pancreas sections (presented in Fig. 3b and Fig. 3c), in addition to their proportions\nin islets of Ern1EC-KO mice after HFD feeding. We also determined β cell mass in\nprimary islets from these models in Fig 4e.\nReviewer #3 (Remarks to the Author):\nIn this report, the authors demonstrate that genetic deletion of the inositol-requiring\nenzyme 1 (IRE1α) specifically in the endothelial cells of the adult mice, results in the\npoor response of the vascular cells to the metabolic stress induced by enforced obesity\nsecondary to chronic high fat diet (HFD) intake. These abnormalities in IRE1α deficient\nendothelial cells were manifested in subtle glucose intolerance with impaired insulin\nsecretion. The mechanism of this metabolic dysregulation was mediated through\nupregulation of the mRNA encoding presumed anti-angiogenic agent thrombospondin-\n1 (THBS1/TSP1). IRE1α RNase15 mediated mRNA decay of THBS1 presumably\nmodulate the overall expression of this factor in SV40 large T antigen immortalized\nMS1 mouse endothelial cells. Knock down of THBS1 in endothelial cells restored the\nislet vascular defects arising from endothelial cell-IRE1α deficiency in response to HDF\ninduced obesity.\nComments:\nThe mechanism by which neo-angiogenesis might play a role in the regulation of\nobesity induced metabolic dysfunction is not known. Demonstration that IRE1α\ndeficiency through upregulation of THBS1 might interfere with angiogenic activity in\nthe islets thereby contributing to metabolic derangements afforded by chronic HDF\ninduced obesity could have therapeutic implications.\nHowever, the data presented are not that convincing and many of the conclusions are\nnot strongly supported by the data provided.\nResponse:\nWe really appreciate the Reviewer’s constructive and critical comments.\n1) The majority of the changes in glucose levels and insulin intolerance in the IRE1α\ndeficient mice are only marginal, which are corrected with the THBS1 knock down.\nIndeed, the pathophysiological consequences of those metabolic changes are not readily\napparent. Are there any pathological changes in the cardiovascular function, liver\nmetabolism or kidney function in the IRE1α deficient mice ?\nResponse:\nWe do agree with Reviewer in that endothelial IRE1α depletion did not lead to very\ndramatic changes in glucose metabolism and insulin intolerance, but it did result in\nimpaired islet adaptation following HFD feeding. While we are very interested in\nconducting more thorough studies such as the effects on their cardiovascular function,\nliver metabolism and kidney function in this animal model, we did not observe apparent\npathological changes in their heart or kidney function. Surely enough, we will look into\nopportunities for collaborating with colleagues in relevant fields to investigate these\nimportant questions using appropriate stress conditions in the future. In the current\ndietary obesity model, we conducted more experiments to examine lipid metabolism in\nthe liver, and we observed elevated levels of hepatic TAG and cholesterol in HFD-fed\nErn1EC-KO mice (data presented in Supplemental Fig. S1g and S1h), despite no apparent\nhistological changes in the liver via H/E staining analysis. Because this study focused\non glucose metabolism under obesity-induced metabolic stress, we would like to further\ninvestigate in more mechanistic detail the role of endothelial IRE1α in hepatic lipid\nmetabolism following NASH-diet feeding or liver injuries. In this case, we will actively\nseek more sufficient research funding support for these highly interesting studies in the\nfuture.\n2) It is unclear whether the subtle differences in hyperglycemia and insulin intolerance\nare primarily due to pancreatic dysfunction or increase in the adipose tissue vascular\nmass changes secondary to the chronic HFD induced obesity? Is there a correlation with\nthe increase vascular mass index with the metabolic changes potentially in the adipose\ntissue of the IRE1α or THBS1 deficient mice.\nResponse:\nThis is a very insightful point that, in fact, led us to establish initially the DIO model,\nas we anticipated a likely impact of endothelial IRE1α depletion upon adipose tissue\nfunction in obesity. Unexpectedly but intriguingly, we found that the intra-islet ECs are\nmost sensitive to the loss of IRE1α during dietary obesity. We did not observe\nsignificant alterations in the vascular density in adipose tissue of HFD-fed Ern1EC-KO\nmice (Supplemental Fig. S6e-g) or HFD-fed Ern1EC-KO;Thbs1EC-KO mice (Supplemental\nFig. S14b). This may reflect the distinct regulatory actions of endothelial IRE1α in\ndifferent tissues.\n3) The authors claim that there could be a defect in the islet angiogenesis in the IRE1α\nor THBS1 deficient mice. However, whether there are changes in the vessel density and\nas to how these changes lead to metabolic abnormalities observed is not well\ncharacterized. In figure 4 the authors use CD31 (PECAM) as the marker for endothelial\ncell staining. However, CD31 staining is not sufficient to specifically stain endothelial\ncells as CD31 is also expressed on hematopoietic cells including inflammatory\nmonocytes and macrophages. Other vascular specific markers such as VE-cadherin or\nCD34 might be considered.\nResponse:\nFollowing the Reviewer’s suggestions, we more carefully characterized the vascular\ndensity in pancreatic islets, liver and adipose tissue of NC- and HFD-fed Ern1EC-KO\nmice using CD31 as well as CD34 as the EC staining marker (Supplemental Fig. S6).\nConsistently, we detected significantly reduced abundance of intra-islet ECs but not in\nthe liver or adipose tissue of HFD-fed Ern1EC-KO mice, suggesting defective angiogenic\ncapacity in the islets under metabolic stress.\n4) The use of mouse MS1 SV40 large T antigen immortalized endothelial cells could\nbe fraught with artifacts because metabolically these cells employ oxidative\nphosphorylation, but not glycolysis, secondary to oncogenic transformation possibly\nobscuring the effects I IRE1α and XBP1 mediated stress responses. Use of more\nprimary isolated endothelial cells, preferably from the metabolic organs might be more\nrevealing.\nResponse:\nWe appreciate this important point from the Reviewer. We followed the suggestions\nand used primary HUVECs for dissecting the effect of IRE1α knockdown upon TSP1\nexpression (Supplemental Fig 6b, 6d and 6f). Notably, similar results were obtained as\nin MS1 cells.\n5) In IRE1α deficient mice there are also changes in the expression and RNA decay of\nother factors. Then, is THBS1 dysregulation the main factor that contributes to the\npathogenesis of metabolic dysfunction in the IRE1α mice? Why the authors elected to\nfocus on THBS1 and not the other factors.\nResponse:\nWe identified THBS1 as one of the top-ranked downregulated target genes as a result\nof IRE1α ablation (Fig. 5). Moreover, based on the scRNA-seq analysis of HFD-fed\nislets (accession No. GSE203376), we found that its expression is specifically restricted\nto ECs along with the EC marker Cdh5 (Fig S10). Given TSP1 as a well-established\nendogenous antiangiogenic factor that not only inhibits EC proliferation but also exerts\nmultiple regulatory effects upon glucose and lipid metabolism through affecting the\nadipose tissue as well as pancreatic islets, we chose to focus on this molecule as a\npotential downstream effector of IRE1α. Indeed, depletion of TSP1 in the context of\nIRE1α ablation completely corrected the metabolic defects during dietary obesity (Fig\n8), supporting that THBS1 dysregulation is the main factor contributing to the\nmetabolic dysfunction of islets in EC-specific IRE1α-deficient mice during dietary\nobesity.\n6) THBS1 has many different pleiotropic effects on regulating blood vessel integrity.\nBesides potential anti-angiogenic effect, THSP1 could also modulate homeostatic\nfunctions of blood vessels not only in the pancreas but also in adipose or liver\nvasculature. Then, to what extent the metabolic changes in IRE1α or THSP1 deficient\nmice, could be attributed to its anti-angiogenic activity?\nResponse:\nWe agree with the Reviewer in that IRE1α or TSP1 in ECs may exert additional\nregulatory effects upon the endocrine cells in the islets, e.g. through intercellular\ninteractions that remain to be elucidated, in addition to their anti-angiogenic activity.\nNonetheless, our results together from the animal models and cell-based assays indicate\nthat the effect of TSP1’s anti-angiogenic activity in islets most likely serves as one\nmajor contributor to the metabolic changes arising from endothelial IRE1α deficiency,\nas we observed no overt changes in vascular density in the liver or adipose tissue of\nHFD-fed Ern1EC-KO mice. We discussed this important point in the Discussion section."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors presented a revised manuscript with more text, and new methods, data, and references. They are thanked for the extra effort. As well, the response to my comments was nicely done.\nA few things came up on reading the new draft.\nSeveral papers from the Kentucky group should be mentioned that deal with the TSP1 effect in HFD - PLoS One.\n2011;6(10): e26656/ Am J Physiol Renal Physiol. 2013 Sep 15;305(6): F871-80/Sci Rep. 2023 Nov 18;13(1):20193.\nThe authors isolate and look at ECs from other organs and this was a nice addition. Could the implications for the paper\nextend to peripheral and coronary vascular disease?\nPlease indicate the clone, manufacturer, concentration, etc. of the CD47 antibody used in the cell viability study. The CD47\nare reported to behave as TSP1 agonists and antagonists. If the Ab worked in the cultured cells, would it work in the mice +/-\nIRE1α? This would link the soluble TSP1 to the cell surface receptor/target.\nPlease confirm the identity of the murine islet EC line as they seem to be first described in the 1990s. These data could be\nplaced in a supplement.", "reviewer_2": "I appreciate the authors' efforts in addressing some of my initial concerns. However, significant scientific limitations remain,\nparticularly regarding the methodology and data interpretation.\n1. Regarding the validation of the genetic mouse model, I appreciate the authors' additional validation efforts, including the\nanalysis of other tissues involved in glucose homeostasis. However, the provided evidence remains insufficient. The RNA-seq\ndata alone does not adequately confirm efficient gene deletion in endothelial cells within islets. Additionally, the Western\nblot presented are only a representative image, with n=2, which is inadequate for proper validation. A more robust\nquantification, such as qPCR or Western blot from multiple biological replicates, is necessary to strengthen the conclusions.\n2. Regarding the Cre Control mice, I appreciate the authors' efforts in addressing this concern and providing additional data.\nHowever, it would have been valuable to include insulin measurements in these control mice to fully assess the potential\neffects of Cre expression. The absence of such data limits the ability to completely rule out confounding factors.\n3. I remain unconvinced about the authors' conclusions regarding glucose intolerance and insulin secretion in their mouse\nmodels. A major issue is that glucose tolerance data (Fig. 1f, Fig. 8c) is reported at week 20, whereas insulin levels are\nshown separately at week 24 (Fig. 1i, Fig. 8e). The authors never present glucose and insulin data together during glucose\ntolerance tests, which is inappropriate and prevents a proper interpretation of β-cell function, as I previously mentioned.\nFurthermore, they report an increase in fasting insulin levels without any change in fasting glucose levels (Fig. 1h). How do\nthe authors explain this finding if there is no alteration in insulin sensitivity? This raises concerns about the interpretation of\ntheir metabolic phenotyping and suggests a need for additional analyses to clarify the mechanisms at play.\n4. I appreciate that the authors have now included data from female mice. However, this aspect must at least be properly\ndiscussed in the manuscript and not only reported in the results section. Additionally, all sections of the manuscript should\nbe revised to clearly state that the reported findings apply only to male mice. This is essential for transparency and aligns\nwith Nature Communications’ guidelines on sex as a biological variable.\n5. I still have major methodological Issues and concerns regarding Data Presentation:\n- There are major methodological flaws in the way β-cell and α-cell areas and mass are calculated. The authors do not report\ncell areas relative to total islet area, which is the correct approach. Since they claim to observe differences in islet size, this\nalone could explain the observed changes in β-cell area. Similarly, β-cell mass should always be normalized to total\npancreatic mass, which was not done.\n- Another inconsistency lies in the choice of methodology: in some cases, the authors report results from isolated islets rather\nthan pancreatic sections, without providing any justification or methodological details. This raises concerns about data\ninterpretation and reproducibility. Additionally, as the authors themselves acknowledge, studying endothelial cells within\nislets is technically challenging. Given that the majority of endothelial cells die within a few hours of islet isolation and\nculture, how can the authors reliably assess β-cell markers in isolated islets rather than in pancreatic sections? This\nmethodological choice is problematic and further weakens their conclusions.\n- Regarding histological analyses, the authors present data for each islet analyzed within a single mouse, but with a limited\nnumber of mice per group. This is an inappropriate method for statistical analysis, as each mouse should be considered an\nindependent biological replicate (n=1), rather than pooling all islets from the same mouse as individual data points. A\nstriking example is Fig. 4b, where the authors plot 80–100 analyzed islets per mouse in a statistical test based on only n=4\nper group, which is methodologically incorrect.\n- Additionally, in Fig. 3f–k, islets were pooled from three mice per group and then divided into three replicates for GSIS\nanalysis. Pooling islets from different mice and then artificially generating replicates significantly reduces biological\nvariability, making the results appear more statistically favorable. This approach is not methodologically rigorous and once\nagain weakens the interpretation of the findings.\n- More broadly, the sample sizes (n=3–4 per group) remain too low across multiple experiments to support robust\nconclusions. Increasing n and using appropriate statistical approaches are necessary to strengthen the validity of the\nfindings.", "reviewer_3": "The authors have performed additional experiments and provided clarifications for the concerns raised int he previous\nreview.\nThey now provide a more detailed analyses of:\n1) Vascular density in the islets by performing staining with CD31 and CD34. However, CD34 might not be expressed on all\nof the capillary endothelial cells. Staining with CDH5 (VEcadherin) could have been more revealing.\n2) They now use human umbilical vein endothelial cells in addition to immortalized endothelial cell cell lines.\n3)) They provide explanation that insulin intolerance in the IREa vascular knock out mice is not due off targeted effect of the\nthrombospondin on the adipose or muscle or liver tissue."}, "author_response": "Point-by-point response to the reviewers’ comments:\nReviewer #1:\n1) The authors presented a revised manuscript with more text, and new methods, data,\nand references. They are thanked for the extra effort. As well, the response to my\ncomments was nicely done. A few things came up on reading the new draft. Several\npapers from the Kentucky group should be mentioned that deal with the TSP1 effect\nin HFD - PLoS One. 2011;6(10): e26656/ Am J Physiol Renal Physiol. 2013 Sep\n15;305(6): F871-80/Sci Rep. 2023 Nov 18;13(1):20193.\nResponse:\nWe wish to thank the Reviewer’s positive feedback. We have now included one paper\n(Ref. 73) from the Kentucky group that dealt with the TSP1 effect in HFD-fed mice\n(PLoS One. 2011;6(10): e26656), which we somehow forgot to cite in the prior version\nof the manuscript. We chose not to cite the other two papers from the same group (Am\nJ Physiol Renal Physiol. 2013 Sep 15;305(6): F871-80 and Sci Rep. 2023 Nov\n18;13(1):20193), because neither appears to have relevance to our mechanistic studies\non the metabolic functions of endothelial TSP1 in the islets.\n2) The authors isolate and look at ECs from other organs and this was a nice addition.\nCould the implications for the paper extend to peripheral and coronary vascular\ndisease?\nResponse: We appreciate the Reviewer’s enthusiasm on potential functions of IRE1α\nin ECs from other organs. Honestly, we are also very interested in conducting more\nthorough studies using this genetic mouse model to investigate the IRE1α-TSP1 axis\nin the context of peripheral and coronary vascular disease. While we have got some\npreliminary results from vascular injury and liver regeneration models, we will have\nto seek more sufficient research funding support as well as opportunities for\ncollaborating with colleagues in relevant fields in order to carry out these highly\ninteresting studies in the future.\n3) Please indicate the clone, manufacturer, concentration, etc. of the CD47 antibody\nused in the cell viability study. The CD47 are reported to behave as TSP1 agonists\nand antagonists. If the Ab worked in the cultured cells, would it work in the mice +/-\nIRE1α? This would link the soluble TSP1 to the cell surface receptor/target.\nResponse:\nWe apologize for forgetting to include the clone, manufacturer and concentration of the\nCD47 antibody used in the cell viability study in the previous manuscript, which we\nhave provided now in the Methods and figure legend (Fig 6j).\nWe also appreciate the Reviewer’s insightful thought. It should be a great idea to test\nthe effect of blocking the TSP1-CD47 signaling within the islets using the anti-CD47\nantibody (which worked in the cultured cells) in mice with or without IRE1α in ECs.\nHowever, we anticipate that an extraordinarily large amount of antibody would be\nneeded for such animal treatment experiments, which can be well beyond our financial\ncapability. Moreover, given the multiple functions of CD47 via its effects upon\nmacrophages, stem cells, etc, it can also be very difficult to detect its metabolic impact\nsolely resulting from blocking the TSP1-CD47 pathway within the islets. We hope the\nReviewer will agree with us in that our current study should come to focus on the\nmechanistic action of the IRE1α-TSP1 pathway, instead of the TSP1-Cd47 signaling,\nwithin the islets in our animal model.\n4) Please confirm the identity of the murine islet EC line as they seem to be first\ndescribed in the 1990s. These data could be placed in a supplement.\nResponse:\nWe analyzed the MS1 cells through immunofluorescent staining of the CD31 marker\n(Suppl. Fig 11a) and confirmed its identity as the murine islet EC line.\nReviewer #2:\nI appreciate the authors' efforts in addressing some of my initial concerns. However,\nsignificant scientific limitations remain, particularly regarding the methodology and\ndata interpretation.\nResponse:\nWe sincerely appreciate the Reviewer’s constructive feedback and critical comments\nregarding the methodology and data interpretation.\n1) Regarding the validation of the genetic mouse model, I appreciate the authors'\nadditional validation efforts, including the analysis of other tissues involved in\nglucose homeostasis. However, the provided evidence remains insufficient. The\nRNA-seq data alone does not adequately confirm efficient gene deletion in endothelial\ncells within islets. Additionally, the Western blot presented are only a representative\nimage, with n=2, which is inadequate for proper validation. A more robust\nquantification, such as qPCR or Western blot from multiple biological replicates, is\nnecessary to strengthen the conclusions.\nResponse:\nWe followed the suggestions from the Reviewer and further validated the genetic\nmouse model. We performed additional Western immunoblot analyses of IRE1α\nprotein using isolated primary ECs, including ECs from pancreas, and quantified the\nresults from multiple mice. These data were presented in Fig 1c and Fig 2a, which\nconfirmed the efficient IRE1α ablation in ECs of Ern1EC-KO mice.\n2) Regarding the Cre Control mice, I appreciate the authors' efforts in addressing this\nconcern and providing additional data. However, it would have been valuable to\ninclude insulin measurements in these control mice to fully assess the potential\neffects of Cre expression. The absence of such data limits the ability to completely rule out\nconfounding factors.\nFollowing the suggestions from the Reviewer, we measured serum insulin levels and\ncalculated β-cell mass in Ern1fl/fl and Ern1fl/fl;VE-cad-CreERT2 mice without tamoxifen\ntreatment following HFD feeding for 12 weeks. These results were shown in Suppl. Fig.\n4e-4g, indicating that no difference could be detected in their pancreatic islet\nphenotypes.\n3) I remain unconvinced about the authors' conclusions regarding glucose intolerance\nand insulin secretion in their mouse models. A major issue is that glucose tolerance\ndata (Fig. 1f, Fig. 8c) is reported at week 20, whereas insulin levels are shown\nseparately at week 24 (Fig. 1i, Fig. 8e). The authors never present glucose and insulin\ndata together during glucose tolerance tests, which is inappropriate and prevents a\nproper interpretation of β-cell function, as I previously mentioned.\nResponse:\nWe now fully understand the Reviewer’s concerns. We included the suggested\nmetabolic data from additional measurements for the relevant animal experiments. We\ndetermined serum glucose levels in HFD-fed animals, i.e. Ern1fl/fl and Ern1EC-KO mice\nas well as Ern1fl/fl;Thbs1fl/fl and Ern1EC-KO; Thbs1 EC-KO mice, simultaneously following\nglucose stimulation during GSIS in vivo. These results were now added to Fig. 1i and\nFig. 8e of our revised manuscript, which consistently indicate altered insulin secretion\ncapacity of β-cells as a result of endothelial IRE1α ablation.\n4) Furthermore, they report an increase in fasting insulin levels without any change in\nfasting glucose levels (Fig. 1h). How do the authors explain this finding if there is no\nalteration in insulin sensitivity? This raises concerns about the interpretation of their\nmetabolic phenotyping and suggests a need for additional analyses to clarify the\nmechanisms at play.\nResponse:\nWe appreciate this thoughtful point from the Reviewer. Under the fasted state, the\nserum insulin levels reflect the basal secretion ability of the pancreatic islets, whereas\nblood glucose levels are largely controlled by hepatic glucose production. Because no\novert changes of insulin resistance were observed between HFD-fed Ern1fl/fl and\nErn1EC-KO mice, including insulin signaling in the liver (Suppl. Fig. 2b), it is not\nsurprising that they showed similar blood glucose levels. Otherwise, significantly\nhigher glucose levels should have been observed in HFD-fed Ern1EC-KO mice. We\ndetected significantly decreased insulin levels in fasted HFD-fed Ern1EC-KO mice\nrelative to their Ern1fl/fl controls, which suggests a decreased basal insulin secretion\ncapacity. This fully supports our proposed mechanisms at play under HFD feeding-\ninduced metabolic stress.\n5) I appreciate that the authors have now included data from female mice. However,\nthis aspect must at least be properly discussed in the manuscript and not only reported\nin the results section. Additionally, all sections of the manuscript should be revised to\nclearly state that the reported findings apply only to male mice. This is essential for\ntransparency and aligns with Nature Communications’ guidelines on sex as a biological\nvariable.\nResponse:\nWe discussed the sexual dimorphism in the sensitivity to HFD-induced obesity model\nwhen comparing male and female mice (lines 123-128), which has been reported in\nmany dietary obesity model studies using mice in the genetic background of\nC57BL/6J. We also indicated in all sections of the manuscript that the reported results\nwere from experiments using male animals, including the subtitles as well as the\nNature Portfolio Reporting Summary.\n6) I still have major methodological Issues and concerns regarding Data Presentation:\na) There are major methodological flaws in the way β-cell and α-cell areas and mass\nare calculated. The authors do not report cell areas relative to total islet area, which is\nthe correct approach. Since they claim to observe differences in islet size, this alone\ncould explain the observed changes in β-cell area. Similarly, β-cell mass should\nalways be normalized to total pancreatic mass, which was not done.\nResponse:\nWe followed the Reviewer’s advice and analyzed β-cell and α-cell areas relative to total\nislet areas (Fig 3b-3e), and also calculated β-cell mass by normalizing to total pancreatic\nmass (Fig 2d).\nb) Another inconsistency lies in the choice of methodology: in some cases, the authors\nreport results from isolated islets rather than pancreatic sections, without providing\nany justification or methodological details. This raises concerns about data\ninterpretation and reproducibility. Additionally, as the authors themselves\nacknowledge, studying endothelial cells within islets is technically challenging. Given\nthat the majority of endothelial cells die within a few hours of islet isolation and\nculture, how can the authors reliably assess β-cell markers in isolated islets rather than\nin pancreatic sections? This methodological choice is problematic and further\nweakens their conclusions.\nResponse:\nWe understand the Reviewer’s concerns. In fact, we used both pancreatic sections\n(Fig 2b-2e, Fig 3a-3e and Fig 4a-4b) and isolated islets (Fig 2f-2i and Fig 4d-4h) to\nanalyze β-cells and CD31+ ECs. We used isolated islets as an ex vivo model to\nfurther confirm our observations from pancreatic sections, and we obtained similar\nresults that consistently support and strengthen our conclusions. In particular, we used\na whole-mount IF staining protocol for isolated islets that were immediately fixed\nprior to staining to avoid cell death. We provided detailed description of these\nexperimental procedures in our manuscript. While we acknowledged that it is very\ndifficult to isolate primary islet ECs for molecular analysis, we were able to assess\nboth β-cell and EC markers from these isolated islets.\nc) Regarding histological analyses, the authors present data for each islet analyzed\nwithin a single mouse, but with a limited number of mice per group. This is an\ninappropriate method for statistical analysis, as each mouse should be considered an\nindependent biological replicate (n=1), rather than pooling all islets from the same\nmouse as individual data points. A striking example is Fig. 4b, where the authors\nplot 80–100 analyzed islets per mouse in a statistical test based on only n=4\nper group, which is methodologically incorrect.\nResponse:\nWe appreciate the Reviewer’s comments on this issue. Following the suggestions\nfrom the Reviewer, we included more data from analysis of the histograms of\nadditional mice, and determined β-cell and α-cell area as well as CD31+ EC area in\nthe islets from 6 or 9 mice per group (n=6 or 9 for Fig 3b and 3c; n=6 for Fig 4b).\nd) Additionally, in Fig. 3f–k, islets were pooled from three mice per group and then\ndivided into three replicates for GSIS analysis. Pooling islets from different mice and\nthen artificially generating replicates significantly reduces biological variability,\nmaking the results appear more statistically favorable. This approach is not\nmethodologically rigorous and once again weakens the interpretation of the findings.\nResponse:\nWe understand the Reviewer’s concerns regarding the utilization of islets pooled from\nmultiple mice. We did not describe previously the islet data correctly in the legend of\nFig 4f, which caused some misunderstanding. For higher islet recovery efficiency to\nperform GSIS analysis, islets were obtained using pooled pancreases from 3 mice per\ngroup by centrifugation before hand-pick isolation, a procedure often employed in the\nislet research field. Moreover, we believe using pooled islets for each group of mice\nmay actually reduce the variations arising from the genetic background of each\nindividual animal within the group. These ex vivo results from pooled islets may more\naccurately reflect the effects of genetic manipulations upon the function of the islets\nper se. In addition, we showed the data that represent 2 independent experiments\nusing pooled islets from n=6 mice. For more clarity, we described the islet isolation\nand analysis more clearly in the Methods and figure legend of the revised manuscript.\ne) More broadly, the sample sizes (n=3–4 per group) remain too low across multiple\nexperiments to support robust conclusions. Increasing n and using appropriate\nstatistical approaches are necessary to strengthen the validity of the findings.\nResponse:\nWe appreciate the Reviewer’s comments. We included additional data from analyses\nof more mice for each group in most of our animal studies, particularly for the\nmetabolic phenotyping results (at least n=6 per group) and critical islet analyses from\npancreas sections (at least n=5 per group). We used statistical analysis methods based\non our previous experiences from various animal model studies.\nReviewer #3:\nThe authors have addressed my remaining minor comments.\nResponse:\nMany thanks!"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors provided a revised text and letter of explanation. The requested clarification concerning a CD47 antibody employed in the paper was made to the text. The authors are thanked again for continuing to improve the final draft.", "reviewer_4": "The authors have adequately address our previous concerns."}, "author_response": "Point-by-point response to the reviewers’ comments:\nReviewer #1:\n1) The team provided a further revised manuscript with more information and data. A\nresponse letter covered details on the changes made and some that were not doable.\nOverall, I feel this was a good effort on their part. The feedback was satisfactory. A\nsmall point on the CD47 antibody (Bio X Cell-InVivoMAb, BE0283) employed by\nthe authors. I do not recognize the terminology. The more common nomenclature\nis MIAP410 which I suggest using along with the company product information.\nThis is important. Also, the team should be aware that MIAP401 was published as\na regulator of macrophage inflammation; see PMID: 34591795 and PMID:\n31337788. Thus, it is not clear if it actually blocks TSP1 binding to CD47.\nResponse:\nWe really appreciate the Reviewer’s positive comments on our revision. As for the\nCD47 antibody, MIAP410 is actually the clone # for the commercial CD47 antibody\n(Bio X Cell-InVivoMAb, BE0283) that we used as recommended previously by the\nReviewer. Now we have included this clone # of the CD47 antibody in the Methods of\nthe revised manuscript. We also agree with the Reviewer in that it is currently not clear\nif the antibody can directly block TSP1 binding to CD47.\n2) The team did say that running a test on removing the TSP1-CD47 signal from mice\nmight be financially prohibitive, and such a study might be confounded by changes\non immune cells that also show CD47. For these reasons they opted to not test this.\nSomething thing for the team to think about: 1) There are less expensive means to\nlower the signal such a using an oligo that blocks translation of the CD47 and/\nor TSP1 RNA. Such a molecule is relatively cheap and could be given every 7-10 days\nvia i.p. injection.\nResponse:\nWe also appreciate this thoughtful point from the Reviewer. We believe that it would\nbe very difficult to deliver anti-sense oligonucleotides targeting CD47 or TSP1 to the\nislets, and i.p. injection may generate similar systemic effects via affecting many other\ntissues. Our current study focused on the mechanistic action of the IRE1α-TSP1\npathway within the islets in our animal models of obesity. However, we would very\nmuch like to seek more research funding support as well as opportunities for\ncollaborating with colleagues in relevant fields to further investigate the role of IRE1α\nin regulating the TSP1-CD47 axis in other disease models, such as muscle atrophy,\nusing these approaches suggested by the Reviewer in the future.\nReviewer #3:\nThe authors have addressed my remaining minor comments.\nResponse:\nMany thanks!\nReviewer #4:\nSummary: The authors demonstrate the necessity of the Endoplasmic Reticulum (ER)\nstress sensor IRE1 in endothelial cells (EC) for adaptation of pancreatic islets to\ncontinuous high fat diet (HFD) conditions. This was achieved using CreER mouse lines\nthat allowed for inducible deletion of IRE1 from ECs. Under HFD conditions, male KO\nmice exhibited worse control of blood glucose levels and reduced insulin levels in\nresponse to feeding, a reduction in islet number and area, and decreased\nislet vascularization compared to control mice. Transcriptomic analysis revealed an increase\nin Thrombospondin 1 (Thbs1) transcript, an anti-angiogenic factor, in islets of KO mice\nislets. Thbs1 transcript was further confirmed to be suppressed by IRE1 via splicing\nthrough IRE1a’s ribonuclease activity. Knockout of Thbs1 in combination with IRE1\nin ECs restored phenotypes seen in control mice, confirming the role of Tbhs1 in\nsuppressing pancreatic islet adaptation. This study elucidates the role that IRE1 activity\nin ECs plays in pancreatic islet adaptation in response to metabolic burden through its\nregulation of Thbs1. This work will provide valuable insight into the broad role that ER\nstress sensors in ECs play in adapting to stress conditions such as continuous HFD, and\nthe role of ECs in sensing and adapting to metabolic burden in the pancreatic islet\nmicroenvironment. This is fascinating and well-done work.\nResponse:\nWe sincerely thank the Reviewer for the positive feedback and comments.\n1) The authors show that IRE1α in ECs is necessary for adaptation of pancreatic islets\nto metabolic stress but do comment in depth on what the source of ER stress in islet\nECs during HFD feeding is. What is causing ER proteins to misfold in islet ECs\nunder HFD? The authors could consider revising the manuscript to include a\ndiscussion of the mechanism by which ER stress is induced in islet ECs to begin\nwith.\nResponse:\nWe appreciate this thoughtful point from the Reviewer. It has been well established that\nthe ER is highly sensitive to metabolic perturbations, and ER stress ensues upon nutrient\noverloading that causes changes of ER protein folding task or its remodeling. In\npancreatic islet β-cells, IRE1α has been shown to respond to glucose stimulation and\nregulate insulin biosynthesis, while promoting the compensatory islet growth in the\nstate of obesity. Presumably, islet ECs can also go through metabolic ER stress upon\nexposure to increased levels of glucose, insulin and fatty acids in the state of HFD-\ninduced obesity and insulin resistance. We briefly mentioned these points in the\nDiscussion instead of the Introduction, in order to maintain the flow of our manuscript.\n2) In the introduction, the authors refer to IRE1 initially (line 103-110) and then start\nreferring to IRE1α in line 111 without explanation for the change. For clarity, the\nauthors could clarify that IRE1α is specifically the mammalian form of IRE1 when\nthe change in naming occurs.\nResponse:\nThis is an important point for clarification. We indicated that IRE1α is one of the two\nisoforms in mammals and found to be ubiquitously expressed across different tissues.\n3) The authors used western blot analysis to determine if IRE1α is knocked out in EC\ncells following induced Cre recombination in a cell-type specific manner (Figure\n1C, Figure 2A). However, quantification of IRE1α protein levels is only provided\nfor CD31+ cells and not CD31- cells. Evidence of selective knockout requires both\nproof of decreased IRE1α levels in ECs as well as lack of knockout in other cell\nlines. IRE1α protein levels could be quantified for CD31- cells to show that IRE1α\nlevels are not decreasing. Additionally, the β-actin loading control is not visible in\nheart tissue for CD31- cells (Figure 1a, Supp Figure 14a). These blots could be\nrepeated using either a stain-free gel to quantify protein loading or with\nimmunoblotting of another housekeeping gene. This is necessary to make any\nquantitative or qualitative conclusions regarding protein expression in CD31- heart\ntissue.\nResponse:\nFollowing the suggestions from the Reviewer, we quantified the IRE1α protein levels\nin both CD31+ and CD31- cells, now shown in Figure 1c and Figure 2a. We also used\nα-tubulin instead of β-actin as a loading control in the heart in Figure 1c and Supp\nFigure 14a.\n4) Histochemical and morphometric analysis were used by the authors to make\nconclusions about pancreatic architecture and structural organization of islets\n(Figure 2b, 8h). However, the authors only use insulin (β-cell) staining to identify\nchanges in structural organization of islets. It would be helpful to stain for other cell\ntypes (α-cells (glucagon), δ-cells (somatostatin)) to more conclusively show that\nthere are not changes in the composition and structural organization of islets.\nResponse:\nWe appreciate the Reviewer’s point. For histochemical and morphometric analysis of\nislets from pancreatic sections, insulin has been routinely used because β-cells are the\nmost abundant cell type and can more accurately reflect changes in the structural\norganization of islets during metabolic stress. In addition, we performed\ninsulin/glucagon co-staining in pancreas sections from both NC- and HFD-fed mice in\nFigure 3a-e to further examine the structural organization of islets. Due to their low\nabundance in the islets, δ-cells have been rarely used for this purpose via somatostatin\nstaining.\n5) In Figures 3B and 3C, “pixel” is spelled incorrectly on the x-axis (spelled as “piexl”).\nPlease fix this axis labeling.\nResponse:\nWe fixed this typo in Figures 3b, 3c and Figure 4e now. Many thanks!\n6) The authors use immunostaining to show that there are no significant alterations in\nCD31+ cells in the liver (Supplementary Fig 6c and 6d). Why not perform qPCR\non these tissue slices as well to detect CD31? It would be more convincing as there\nis quite a bit of variability that can occur with immunostaining.\nResponse:\nWe followed the Reviewer’s suggestion and performed qRT-PCR analysis of Pecam1\nmRNA encoding CD31 in NC- and HFD-fed livers, which is presented in\nSupplementary Fig 6e.\n7) Bulk RNA sequencing was used by the authors to look at transcript level changes\nin primary islets. It is difficult to make much out of this data because of the variety\nof cell types that exist in islets. β-cells and ECs could have been sorted out with\nFACS prior to bulk RNA sequencing to get more useful information out of this data\nset.\nResponse:\nWe appreciate the Reviewer’s thoughtful point. RNA-seq analysis using primary islets\nmay reveal gene expression programs reflecting the functional changes of islets, but\nfurther isolating islet ECs can be very challenging for obtaining sufficient amount of\nECs. Moreover, we think isolated ECs through flow-cytometry sorting may hardly\nreflect their cellular state within the islets and thus unlikely provide meaningful\ntranscriptomic results. However, we will seek more funding support for conducting\nscRNA-seq studies of islets to address these potentially important questions in the\nfuture.\n8) The authors use rMATs analysis from bulk RNA sequencing data to show ~40%\nexon 2 deletion in IRE1α. Does this percentage make sense based on the percentage\nof EC cells in an islet? Is it possible that Cre could be expressed in or transferred to\nother islet cells types?\nResponse:\nWe fully understand the Reviewer’s thoughtful point. We intended to show the effective\ndepletion of exon 2 in the KO islets based on rMATs analysis from bulk RNA\nsequencing data, which is hardly an accurate quantification of deletion efficiency in this\ncase. Therefore, we chose to remove the description of ~40% exon 2 deletion in the text\nto avoid misunderstanding. Nonetheless, this just directly confirmed the effective exon\n2 depletion in the islet ECs, although we cannot exclude the possibility of higher\nexpression of Ern1 in islet ECs as a result of its exon 2 deletion. This is also supported\nby Western immunoblot analysis showing depletion of IRE1α protein in CD31+ but not\nin CD31- cells of the pancreas (Figure 2a), indicating no Cre expression in or its\ntransferring to other islet cells types.\n9) Based on the bulk RNA sequencing of islets, is there evidence of ER stress in islets\nor EC cells in HFD-diet control or KO mice compared to samples from NC-diet\nmice? Were transcripts of ER stress markers that are not exclusively dependent on\nIRE1a activity (e.g. Bip, CHOP) upregulated?\nResponse:\nWe appreciate the Reviewer’s interesting point. We did not perform bulk RNA\nsequencing of NC-fed islets because we observed no metabolic alterations in NC-fed\nErn1EC-KO mice. However, from scRNA-seq analysis (accession No. GSE203376), we\nshowed GO analysis of HFD feeding-regulated genes in intra-islet ECs, revealing an\nenrichment of pathways related to unfolded protein or protein folding, such as increased\nexpression of the Hsp (Heat shock proteins, Hspa1a and Hspa1b) genes and Atf3\n(Activating transcription factor 3) gene, presented in Supplemental Figure 10e. It is not\nsurprising that no ER stress markers such as Bip and CHOP could be identified, given\nthe poor depth of sequencing for intra-islet ECs as reported in many islet studies.\n10) Please clarify whether the RIDD(LE) cleavage target is the 1-1770 substrate or the\n1771-3510 substrate (lines 432-436). This sentence is confusing as the authors\nmention the cleavage of both substrates before making a claim that they have\nidentified an example of a RIDD(LE) cleavage target.\nResponse:\nWe appreciate the Reviewer’s point and modified the text for better clarity, indicating\nthat the 1-1770 substrate is a RIDDLE target while the 1771-3510 substrate is a bona\nfade hairpin-containing RIDD substrate of IRE1α.\n11) The authors state that there is no apparent cleavage of the THBS1 mutant RNA\nsubstrate (Figure 7g, Lines 441-442). There does appear to be a faint cleavage band\nin the gel, so this conclusion should be tempered.\nResponse:\nWe agree and modified our description, stating that “a much fainter cleavage was\nobserved for the THBS1Mut RNA substrate”.\n12) The authors do not mention which post-hoc statistical test they use when concluding\nstatistical significance between groups following ANOVA. Please include this to\nyour methods section.\nResponse:\nThanks for noting this. We included this information in Methods, i.e. using two-way\nanalysis of variance (ANOVA) with Fisher’s LSD test.\n13) The authors may wish to clarify whether additional groups are statistically different\nthroughout the manuscript. For example, is there a significant reduction in islet\nnumber in the control diet fed mice in Fig 2C? Additionally, the authors may wish\nto highlight whether statistical tests were not carried out or whether significance\nwas not found in other cases where they compare between NC and HFD groups\nand/or do not note significance.\nResponse:\nWe followed these suggestions and indicated all the significant differences found\nbetween groups in all of our results. No significant difference was found otherwise,\nwith or without indicating that in the figures.\n14) In Supplementary Figure 10, the authors misspell “alpha” (spelled as “alfa”) in the\nlabeling for figures a and c. Please correct the spelling.\nResponse:\nWe corrected the misspelled “alpha” in Supplemental Figures 10a and 10c and\nSupplemental Figure 11d. Many thanks!"}]

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