Investigating Adipocyte-Derived Extracellular Vesicles in Obesity using Mouse Models

Abstract

Overvægt og fedme er et hastigt voksende samfundsproblem, hvor dysfunktionelle fedtceller (adipocytter) er forbundet med en ubalance i udskillelsen af fedtvævshormoner, øget inflammation og metabolisk dysfunktion. Det bidrager til udviklingen af en lang række sygdomme, heriblandt type 2-diabetes and hjertekarsygdomme. Ud over de velkendte fedtvævshormoner kan adipocytter også kommunikere med andre væv via små partikler kaldet ekstracellulære vesikler, der indeholder nukleinsyrer og proteiner. Adipocyt-afledte vesikler menes at spille en vigtig rolle i udviklingen af fedmerelaterede sygdomme. Under overvægt øges mængden af vesikler i blodet, og indholdet af adipocytafledte vesikler er blevet linket til insulinresistens og systemisk inflammation. Samtidig producerer cellernes mitokondrier også flere reaktive iltstoffer (ROS), hvilket kan stimulere vesikelfrigivelse gennem endnu ukendte mekanismer. Det er dog teknisk udfordrende at undersøge vesikler fra en bestemt celletype, da det i blodet er svært at spore vesikler tilbage til deres oprindelige celletype. Desuden stammer langt størstedelen af vesikler i blodet fra blodceller, mens kun en lille andel stammer fra andre væv, såsom fedtvævet. Dette understreger behovet for nye, følsomme metoder til at spore celletype-specifikke vesikler, såsom adipocyt-afledte vesikler, i en levende organisme. I mit ph.d.-projekt har jeg evalueret følsomheden af to musemodeller – CD9-EGFP og CD63-NanoLuc – til at spore adipocyt-afledte vesikler i levende mus. Derudover anvendte vi en CD9-EGFP-vesikel reporter cellelinje til at undersøge den underliggende mekanisme, hvorved ROS stimulerer vesikelfrigivelse. De to studier beskrives i detaljer nedenfor:Studie I: I dette studie testede vi to musemodellers evne til at spore adipocyt-afledte-vesikler med henblik på at studere deres distribution ved begyndende overvægt. Den første model, CD9-EGFP, baserede sig på et grønt fluorescerende protein til at mærke adipocyt-afledte vesikler. Denne model viste sig dog ikke at være følsom nok til at detektere adipocyt-afledte vesikler i blodet, hverken ved brug af AAV virusteknologi eller transgen avling. For at øge følsomheden udviklede vi CD63-NanoLuc modellen, hvor vesikler spores via et luminescerende NanoLuc protein og et HA-mærke. Vi inkluderede også en kontrolmodel (sec-NanoLuc), som repræsenterer frit adipocyt-udskilt protein. I cellekulturer validerede vi, at CD63-NanoLuc og sec-NanoLuc specifikt reporterede henholdsvist vesikler og frit udskilt protein. I mus var CD63- NanoLuc modellen følsom nok til at spore adipocyt-afledte vesikler i blodet ved brug af AAV-teknologi. Derudover observerede vi højere NanoLuc-niveauer i forskellige fedtvæv hos CD63-NanoLuc mus, mens sec-NanoLuc mus havde højere niveauer i plasma og urin, hvilket indikerede forskellige distributionsmønstre. På trods af detektering af NanoLuc og HA-mærke i nyrerne og urin, så var CD63-NanoLuc proteinet kun fundet i adipocyt-afledte vesikler i plasma, hvilket indikerede, at intakte adipocyt-afledte vesikler ikke filtreres af nyrerne. Vi anvendte CD63-NanoLuc og sec-NanoLuc musene til at undersøgte effekten af to ugers fedtrig kost, hvilket havde minimal påvirkning på kropsvægten og fedtvævsspecifikke markører. Kun CD63-NanoLuc mus viste signifikant forhøjede NanoLuc niveauer i brunt fedtvæv, visceralt hvidt fedtvæv, lunger, nyrer, og urin. Dette indikerede, at adipocyt-afledte vesiklers frigivelse er påvirket tidligt i udviklingen af overvægt. Vi konkluderede, at en NanoLuc-baseret model muliggør sporing af celletype-specifikke vesikler i levende mus. Derudover fremhævede vi vigtigheden af at inkludere en kontrol for udskilt protein for at kunne adskille vesiklers effekt fra proteiners i forbindelse med at studere deres rolle under sygdom.Studie II: I dette studie undersøgte vi de molekylære mekanismer, hvorved mitokondrie-afledt ROS stimulerer frigivelsen af vesikler. Tidligere observerede vi, at ROS-induceret vesikelfrigivelse, stimuleret af Dichloroacetat (DCA), var forbundet med øget vakuolær fluorescens af vores vesikelsporingsprotein CD9-EGFP. Dette indikerede en mulig sammenhæng mellem vesikelfrigivelse og pH hos CD9-EGFP-positive vakuoler. Den vakuolære pH reguleres blandt af vakuolære proton pumper (v-ATPaser), hvis aktivitet kan påvirkes af ROS-sensitive enzymer såsom calcineurin og nSMase2. Vi testede derfor hypotesen, at mitokondrie-afledt ROS fremmer vesikelfrigivelse via hæmning af calcineurin, aktivering af neutral sphingomyelinase 2 (nSMase2) og hæmning af v-ATPase. Hæmning af nSMase2 med GW4869 havde ingen effekt på den basale vesikelfrigivelse, men formåede at blokere DCA-induceret vesikelfrigivelse, dog uden at påvirke den vakuolære CD9-EGFP-fluorescens. Da andre har vist, at calcineurin negativt regulerer nSMase2’s aktivitet, undersøgte vi calcineurins rolle i DCAinduceret vesikelfrigivelse. Hæmning af calcineurin med Cyclosporin A (CsA) efterlignende effekten af DCA med øget vesikelfrigivelse og kraftigere vakuolær CD9-EGFP-fluorescens. Denne CsA-medieret effekt blev hverken yderligere forstærket af DCA eller hæmmet af antioxidanten 4-Hydroxy-TEMPO. Dette tyder på, at ROS virker opstrøms for calcineurin. Aktivering af v-ATPase med EN6 påvirkede ikke den basale vesikelfrigivelse, men blokerede DCA-induceret vesikelfrigivelse dog uden at ændre den vakuolære CD9-EGFP-fluorescens. Selvom den vakuolære pH påvirkede DCA-induceret vesikelfrigivelse, tyder vores resultater på, at mitokondrie-afledt ROS fremmer vesikelfrigivelse via calcineurin- og nSMase2-afhængige mekanismer.Obesity is characterized by impaired adipose tissue function and adysregulated hormone secretion, which is linked to systemicinflammation and metabolic dysfunction, increasing the risk of comorbidities like type 2 diabetes and cardiovascular disease. Beyondclassical adipokines, adipocytes also participate in interorgancommunication via extracellular vesicles (EVs) containing nucleic acidsand proteins. Adipocyte-derived EVs may play a significant role in thepathogenesis of obesity. In obesity, circulating EV levels are increasedand adipose tissue-derived EV cargo has been linked to insulinresistance and inflammation. Obesity is also associated with increasedmitochondrial production of reactive oxygen species (ROS) known tostimulate EV secretion, yet through unknown mechanisms. Oncereleased, EVs enter the extracellular space as a heterogeneouspopulation originating from various cell types. Additionally, the majority ofEVs in the circulation derive from blood cells with less than 1% comingfrom other sources including adipose tissue. This makes it challenging tostudy adipocyte-derived EV dynamics, which is crucial for understandingtheir physiological and pathophysiological roles. Therefore, novelmethodologies are needed to monitor adipocyte-derived EVs with highsensitivity in vivo.During my PhD, I have evaluated the sensitivity of two EV reportersystems – The CD9-EGFP and CD63-NanoLuc system – to monitoradipocyte-derived EVs secretion in vivo. Additionally, we used a CD9-EGFP reporter cell line to investigate the underlying mechanism of ROSinduced EV secretion. These two studies are described in greater detailbelow:Study I: We evaluated the sensitivity of two EV reporter systems tomonitor adipocyte-derived EV abundance and biodistribution in vivo,focusing on mice on chow diet and high-fat diet (HFD).We first evaluated the CD9-EGFP reporter system. To induce adipocytespecific CD9-EGFP expression, we compared adeno-associated viral(AAV) mediated and transgene adiponectin (Adipoq)-driven Creexpression. AAV-mediated delivery of Cre recombinase driven by a short human adiponectin (hAdipoq) promoter induced off-targets effects inother non-adipose tissues, while crossbreeding with Adipoq-Cre micerestricted CD9-EGFP expression to adipose tissues. However, CD9-EGFP remained undetectable in plasma, indicating low reportersensitivity. To enhance the reporter sensitivity, we designed a Credependent CD63-NanoLuc reporter with human CD63 fused toNanoluciferase (NanoLuc) and hemagglutinin (HA). We included acontrol to monitor cellular protein secretion (sec-NanoLuc). In vitro, weconfirmed that CD63-NanoLuc localized to EVs, while sec-NanoLuc wasfreely secreted. In vivo, AAV-mediated delivery to Adipoq-Cre miceinduced adipose tissue-specific Cre activation enabling differentiationbetween EV donors and recipients. CD63-NanoLuc mice had the highestluciferase levels in adipose tissues, while sec-NanoLuc mice had thehighest levels in plasma and urine, reflecting distinct biodistributionpatterns of EVs and proteins. HA-tag and luciferase activity weredetected in kidneys and urine, but CD63-NanoLuc was only EVassociated in plasma, indicating that intact adipocyte-derived EVs are notfiltered by the kidneys. We used CD63-NanoLuc and sec-NanoLuc mice to study how two weeksof HFD affects EV and secreted protein abundance and biodistribution.Despite minimal changes in body weight and adipogenic markers, onlyHFD-fed CD63-NanoLuc mice showed significantly increased luciferaselevels, including in interscapular brown adipose tissue (iBAT), epididymalwhite adipose tissue (eWAT), lungs, kidneys and urine, indicating that EVdynamics are affected in the early stages of obesity development. Inconclusion, we introduced a sensitive NanoLuc-based reporter systemallowing monitoring of cell type-specific EVs in vivo. Additionally, wehighlighted the value of including a secreted protein control to distinguishEV-specific contributions and demonstrated that the reporter systemscan be employed to study EV and secreted protein dynamics in healthand disease.Study II: We explored the molecular mechanism by which mitochondrialROS stimulate EV secretion. We previously demonstrated that increasedEV secretion by ROS – stimulated by Dichloroacetate (DCA) – wasassociated with enhanced vacuolar fluorescence of our EV reporter CD9-EGFP, indicating a link between EV secretion and the pH of CD9-EGFPpositive vacuoles. The vacuolar pH can be regulated by vacuolar H+-ATPases (v-ATPases), which may be regulated by ROS-sensitive enzymes including calcineurin and neutral sphingomyelinase 2(nSMase2). Thus, we hypothesized that mitochondrial ROS promote EVsecretion via calcineurin inhibition, nSMase2 activation, and v-ATPaseinhibition. nSMase2 inhibition by GW4869 had no effect on basal EVsecretion, but blocked DCA-induced EV secretion without affecting thevacuolar CD9-EGFP fluorescence. Since calcineurin negativelyregulates nSMase2 activity, we tested calcineurin’s role in DCA-inducedEV secretion. Calcineurin inhibition by Cyclosporine A (CsA) significantlyenhanced EV secretion and enhanced vacuolar CD9-EGFPfluorescence, mimicking DCA effects. These CsA-mediated effects werenot further enhanced by DCA or reversed by the ROS scavenger 4-Hydroxy-TEMPO, suggesting that ROS may act upstream of calcineurin.V-ATPase activation by EN6 had no effect on basal EV secretion, butblocked DCA-induced EV secretion, yet without altering the vacuolarCD9-EGFP fluorescence. In conclusion, while the vacuolar pH did affectEV secretion our findings indicate that mitochondrial ROS promote EVsecretion via. calcineurin- and nSMase2 dependent mechanisms