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Biochemistry

Arteriovenøs metabolomics til måling af in vivo metabolitudveksling i brunt fedtvæv

Published: October 06, 2023
doi:
10.3791/66012
, , , , , , ,
1Department of Biological Sciences,Sungkyunkwan University (SKKU), 2Department of Biochemistry, College of Natural Sciences,Chungnam National University, 3Department of Biotechnology, College of Life Sciences and Biotechnology,Korea University

Summary

I denne protokol skitseres metoder, der er relevante for BAT-optimeret arteriovenøs metabolomics ved anvendelse af GC-MS i en musemodel. Disse metoder giver mulighed for erhvervelse af værdifuld indsigt i BAT-medieret metabolitudveksling på organismeniveau.

Abstract

Brunt fedtvæv (BAT) spiller en afgørende rolle i reguleringen af metabolisk homeostase gennem en unik energiforbrugsproces kendt som ikke-rystende termogenese. For at opnå dette bruger BAT en varieret menu af cirkulerende næringsstoffer til at understøtte dets høje metaboliske efterspørgsel. Derudover udskiller BAT metabolitafledte bioaktive faktorer, der kan tjene som enten metaboliske brændstoffer eller signalmolekyler, hvilket letter BAT-medieret intravævs- og/eller intervævskommunikation. Dette tyder på, at BAT aktivt deltager i systemisk metabolitudveksling, en interessant funktion, der begynder at blive udforsket. Her introducerer vi en protokol til in vivo museniveau optimeret BAT arteriovenøs metabolomics. Protokollen fokuserer på relevante metoder til termogeniske stimuleringer og en arteriovenøs blodprøvetagningsteknik ved hjælp af Sulzers vene, som selektivt dræner interscapulært BAT-afledt venøst blod og systemisk arterielt blod. Dernæst demonstreres en gaskromatografibaseret metabolomics-protokol, der anvender disse blodprøver. Anvendelsen af denne teknik bør udvide forståelsen af BAT-reguleret metabolitudveksling på organniveau ved at måle BAT’s nettooptagelse og frigivelse af metabolitter.

Introduction

Brunt fedtvæv (BAT) besidder en unik energiforbrugsegenskab kendt som ikke-rystende termogenese (NST), som involverer både mitokondriel afkoblingsprotein 1 (UCP1)-afhængige og UCP1-uafhængige mekanismer 1,2,3,4,5. Disse karakteristiske egenskaber implicerer BAT i reguleringen af systemisk metabolisme og patogenesen af metaboliske sygdomme, herunder fedme, type 2-diabetes, hjerte-kar-sygdomme og kræftkakeksi 6,7,8. Nylige retrospektive undersøgelser har vist en omvendt sammenhæng mellem BAT-masse og / eller dens metaboliske aktivitet med fedme, hyperglykæmi og kardiometabolisk sundhed hos mennesker 9,10,11.

For nylig er BAT blevet foreslået som et metabolisk dræn, der er ansvarligt for at opretholde NST, da det kræver betydelige mængder cirkulerende næringsstoffer som termogenisk brændstof 6,7. Desuden kan BAT generere og frigive bioaktive faktorer, kaldet brune adipokiner eller BATokines, der fungerer som endokrine og/eller parakrin signaler, hvilket indikerer dets aktive involvering i metabolisk homeostase på systemniveau 12,13,14,15. Derfor bør forståelsen af BAT’s næringsstofmetabolisme øge vores forståelse af dens patofysiologiske betydning hos mennesker ud over dens konventionelle rolle som et termoregulerende organ.

Metabolomiske undersøgelser, der anvender stabile isotopsporere, i kombination med klassiske undersøgelser af næringsstofoptagelse ved hjælp af ikke-metaboliserbare radiosporstoffer, har signifikant forbedret vores forståelse af, hvilke næringsstoffer der fortrinsvis optages af BAT, og hvordan de udnyttes 16,17,18,19,20,21,22,23,24,25,26,27. For eksempel har radioaktive sporstofundersøgelser vist, at koldaktiveret BAT optager glukose, lipoproteinbundne fedtsyrer og forgrenede aminosyrer 16,17,18,19,20,21,22,23,27. Nylig isotopsporing kombineret med metabolomiske undersøgelser har gjort det muligt for os at måle den metaboliske skæbne og flux af disse næringsstoffer i væv og dyrkede celler 24,25,26,28,29,30. Disse analyser fokuserer dog primært på den individuelle udnyttelse af næringsstoffer, hvilket efterlader os med begrænset viden om BAT’s systemniveau roller i organmetabolitudveksling. Spørgsmål vedrørende den specifikke serie af cirkulerende næringsstoffer, der forbruges af BAT, og deres kvantitative bidrag i form af kulstof og kvælstof er fortsat uklare. Derudover er udforskningen af, om BAT kan generere og frigive metabolitafledte BATokiner (f.eks. lipokiner) ved hjælp af næringsstoffer, lige begyndt 12,13,14,15,31,32.

Arteriovenøs blodanalyse er en klassisk fysiologisk tilgang, der bruges til at vurdere den specifikke optagelse eller frigivelse af cirkulerende molekyler i organer / væv. Denne teknik er tidligere blevet anvendt på interscapular BAT hos rotter til måling af ilt og flere metabolitter, hvorved BAT blev etableret som det vigtigste sted for adaptiv termogenese med dets kataboliske potentiale 33,34,35,36,37. For nylig blev en arteriovenøs undersøgelse med rotteinterscapular BAT kombineret med en trans-omics tilgang, hvilket førte til identifikation af uopdagede BATokiner frigivet af termogent stimuleret BAT38.

Nylige fremskridt inden for højfølsom gaskromatografi- og væskekromatografi-massespektrometri (GC-MS og LC-MS)-baserede metabolomics har genoplivet interessen for arteriovenøse undersøgelser til kvantitativ analyse af organspecifik metabolitudveksling 39,40,41. Disse teknikker, med deres høje opløsningsevne og massenøjagtighed, muliggør omfattende analyse af en bred vifte af metabolitter ved hjælp af små prøvemængder.

I overensstemmelse med disse fremskridt tilpassede en nylig undersøgelse med succes arteriovenøs metabolomics til undersøgelse af BAT på museniveau, hvilket muliggjorde kvantitativ analyse af metabolitudvekslingsaktiviteter i BAT under forskellige betingelser42. Denne artikel præsenterer en BAT-målrettet arteriovenøs metabolomics-protokol, der bruger GC-MS i en C57BL/6J-musemodel.

Protocol

Alle eksperimenter blev udført med godkendelse af Sungkyunkwan University Institutional Animal Care and Use Committee (IACUC). Mus blev anbragt i et IACUC-godkendt dyreanlæg beliggende i et renrum indstillet til 22 °C og 45% fugtighed efter en daglig 12 timers lys/mørk cyklus. De blev opbevaret i ventilerede stativer og havde adgang til en standard chow-diæt ad libitum (bestående af 60% kulhydrat, 16% protein og 3% fedt). Strøelse og redematerialer blev skiftet på ugentlig basis. Til denne undersøgelse blev der …

Representative Results

Figur 1 illustrerer det eksperimentelle skema for BAT-optimeret AV-metabolomics. Som nævnt i protokolafsnittet gennemgår mus temperaturakklimatisering ved hjælp af gnaverinkubatorer eller modtager farmakologisk administration såsom β-adrenerge receptoragonister for at opnå differentielt stimuleret brunt fedtvæv. Derefter bedøves mus, og blodprøver indsamles til metabolomisk analyse (figur 1A). Til blodprøvetagning opsamles venøst blod, der specifikt d…

Discussion

Et kritisk skridt i forståelsen af BAT’s metaboliske potentiale i hele kroppens energibalance er at definere, hvilke næringsstoffer det indtager, hvordan de metabolisk behandles, og hvilke metabolitter der frigives i kredsløbet. Denne protokol introducerer en specialiseret arteriovenøs prøveudtagningsteknik, der giver adgang til venøs vaskulatur af interscapular BAT og systemisk arteriel vaskulatur i C57BL/6J-mus, som for nylig blev udviklet og valideret af Park et al42. Nedenfor er nøglepu…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker alle medlemmer af Choi og Jung laboratorierne for metodologisk diskussion. Vi takker C. Jang og D. Guertin for råd og feedback. Vi takker M.S. Choi for den kritiske læsning af manuskriptet. Dette arbejde blev finansieret af NRF-2022R1C1C1012034 til S.M.J.; NRF-2022R1C1C1007023 til D.W.C; NRF-2022R1A4A3024551 til S.M.J. og D.W.C. Dette arbejde blev støttet af Chungnam National University for W.T.K. Figur 1 og figur 2 blev oprettet ved hjælp af BioRender (http://biorender.com/).

Materials

0.5-20 µL Filter Tips Axygen AX.TF-20-R-S
1 mL Syringe with attached needle – 26 G 5/8" BD Biosciences 309597
Agilent 5977B GC/MSD (mass selective detector) Agilent G7077B
Agilent 7693A Autosampler Agilent G4513A
Agilent 8890 GC System Agilent G3542A
Agilent J&W GC column (Capilary column) HP-5MS UI Agilent 19091S-433UI
Agilent MassHunter Workstation software_MS Quantitative analysis(Quant-My-way) Agilent G3335-90240
C57BL/6J mouse DBL C57BL/6JBomTac
CentriVap -50 °C Cold Trap (with Stainless steel Lid) LABCONCO  7811041
DL-Norvaline Sigma-Aldrich N7502-25G
Eppendorf centrifuge 5430R Eppendorf 5428000210
Eppendorf Safe-Lock Tubes 1.5 mL Eppendorf 30120086
Glass insert 250 μL  Agilent 5181-1270
Methanol (LC-MS grade) Sigma-Aldrich Q34966-1L
Methoxyamine hydrochloride Sigma-Aldrich 226904-5G
Microvette 200 Serum, 200 µL, cap red, flat base Sarstedt 20.1290.100
MTBSTFA Sigma-Aldrich 394882-100ML
Pyridine(anhydrous, 99.8%) Sigma-Aldrich 270970-100ML
Refrigerated CentriVap Complete Vaccum Concentrators LABCONCO  7310041
Rodent diet SAFE SAFE R+40-10
Rodent incubator Power scientific RIT33SD
Ultra-Fine Pen Needles – 29 G 1/2" BD Biosciences 328203
Vial Cap 9 mm Agilent 5190-9067
Vial, ambr scrw wrtn 2 mL Agilent 5190-9063
Vial, ambr scrw wrtn 2 mL+A2:C40 Axygen PCR-02-C
DOWNLOAD MATERIALS LIST

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Keywords: Arteriovenous MetabolomicsMetabolite ExchangeEnergy ExpenditureThermogenesisMouse ModelGas Chromatography-based Metabolomics
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Lee, S., Lim, G., Kim, S., Kim, H., Roh, Y. J., Kim, W., Choi, D. W., Jung, S. M. Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue. J. Vis. Exp. (200), e66012, doi:10.3791/66012 (2023).
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