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Biologia

Kvantitativ bestemmelse af de novo fedtsyresyntese i brunt fedtvæv under anvendelse af deuteriumoxid

Published: May 12, 2023
doi:
10.3791/64219
, , ,
1UCD Conway Institute and UCD Institute of Food and Health, School of Agriculture and Food Science,University College Dublin, 2Division of Endocrinology,Cincinnati Children’s Hospital Medical Center, Cincinnati, 3Division of Developmental Biology,Cincinnati Children’s Hospital Medical Center, Cincinnati, 4Department of Pediatrics,University of Cincinnati College of Medicine

Summary

Her præsenterer vi en billig kvantitativ metode, der anvender deuteriumoxid og gaskromatografi massespektrometri (GCMS) til analyse af total fedtsyre de novo lipogenese i brunt fedtvæv in vivo.

Abstract

Fedtsyresyntese er en kompleks og meget energikrævende metabolisk vej med vigtige funktionelle roller i styringen af helkropsmetabolisk homeostase og andre fysiologiske og patologiske processer. I modsætning til andre vigtige metaboliske veje, såsom glukosebortskaffelse, vurderes fedtsyresyntese ikke rutinemæssigt funktionelt, hvilket fører til ufuldstændige fortolkninger af metabolisk status. Derudover mangler der offentligt tilgængelige detaljerede protokoller, der er egnede til nytilkomne på området. Her beskriver vi en billig kvantitativ metode, der anvender deuteriumoxid og gaskromatografi massespektrometri (GCMS) til analyse af total fedtsyre de novo-syntese i brunt fedtvæv in vivo. Denne metode måler syntesen af produkterne af fedtsyresyntase uafhængigt af en kulstofkilde, og den er potentielt nyttig for stort set ethvert væv, i enhver musemodel og under enhver ekstern forstyrrelse. Der gives nærmere oplysninger om prøveforberedelsen til GCMS og downstream-beregninger. Vi fokuserer på analysen af brunt fedt på grund af dets høje niveauer af de novo fedtsyresyntese og kritiske roller i opretholdelsen af metabolisk homeostase.

Introduction

Fedme og tilknyttede metaboliske sygdomme er en pandemi, der bringer nuværende og fremtidige generationeri fare 1,2. Almindeligvis forenklet som følge af ubalancen mellem energiindtag og udgifter påvirker den metaboliske dysregulering forbundet med fedme et stort antal metaboliske veje, der styres af miljømæssige og endogene faktorer3. Imidlertid testes kun få veje rutinemæssigt i dyremodeller for metabolisk dysregulering.

Som et eksempel måles glukosebortskaffelse rutinemæssigt ved glukose- og insulintolerancetest, sandsynligvis på grund af enkelheden ved at bruge bærbare glukosemonitorer4. Helkropsglukose- og lipidoxidationsrelative hastigheder estimeres også rutinemæssigt baseret på respirationsudvekslingsforholdet fra indirekte kalorimetriassays 5,6. Imidlertid vurderes størstedelen af alle andre aspekter af stofskiftet ikke rutinemæssigt funktionelt. Dette fører til ufuldstændige fortolkninger af metabolisk status og savnede terapeutiske muligheder. En af de vigtigste sådanne veje er de novo lipogenese.

De novo lipogenese (DNL) er den proces, hvorved nye fedtsyrer genereres fra prækursorer. Glukose anses for at være den vigtigste forløber, der bidrager til DNL7 i hele kroppen, men andre forløbere, såsom acetat, fruktose, lactat og forgrenede aminosyrer, har vist sig at være relevante kulstofkilder på en rumlig og tilstandsafhængig måde 8,9,10,11,12. DNL er en vigtig bidragyder til metabolisk homeostase og er afgørende for normal udvikling13. Derudover har ændringer i DNL været forbundet med kræft 14,15 og metabolisk 16,17,18 og hjerte-kar-sygdomme 19,20.

DNL-vejen består af de centrale enzymatiske komponenter ATP-citratlyase (ACLY), acetyl-CoA-carboxylase (ACC1/2) og fedtsyresyntase (FAS), der primært producerer palmitat, en 16-carbonmættet fedtsyre. Imidlertid kan ulige kæde og forgrenede fedtsyrer også produceres ved lavere hastigheder9. Elongasser og desaturaser modificerer yderligere disse fedtsyrer og skaber en bred vifte af fedtsyrer, der er nyttige til en række funktioner (f.eks. Langsigtet energilagring og manipulation af membranfluiditet).

Ekspressionen af DNL enzymatiske maskiner styres af et kort antal transkriptionsfaktorer. De mest velbeskrevne til dato omfatter sterol regulatorisk element bindende protein (SREBP) familie, kulhydrat respons element bindende protein (ChREBP), og lever X-receptor (LXR) 21,22,23,24,25,26. På trods af en tilsyneladende overlapning i deres funktioner er individuelle regler baseret på celletypedominans og fysiologiske eller patologiske tilstande blevet rapporteret21,22,27,28.

Bemærkelsesværdigt nok er en række hæmmere for udvalgte trin i DNL-vejen blevet godkendt til klinisk brug eller er i de prækliniske eller kliniske udviklingsstadier for en række sygdomme, herunder fedme, ikke-alkoholisk fedtleversygdom / ikke-alkoholisk steatohepatitis (NAFLD / NASH) og hjerte-kar-sygdom29. Disse bestræbelser fremhæver DNL’s relevans for sundhed og sygdom.

I de senere år er anvendelsen af metoder til kvantitativ vurdering af de novo fedtsyresyntese steget30. Den mest almindelige metode til vurdering af dette er brugen af tungt mærket vand (D2O), hvor det tungt mærkede brint inkorporeres i acylkæder under syntese både direkte og indirekte via deuteriumudveksling med hydrogenerne fra DNL-substraterne NAPDH, acetyl-CoA og malonyl-CoA. Selvom denne tilgang vinder popularitet, mangler der offentligt tilgængelige detaljerede protokoller, der passer til nyankomne på området. Her skitserer vi en metode til kvantitativ vurdering af de novo-syntesen af produkter af FAS ved hjælp af D2O og gaskromatografi massespektrometri (GCMS) med beregninger, der tidligere er udviklet af Lee et al.31. Denne metode måler de novo fedtsyresyntese uafhængigt af en kulstofkilde, og den er potentielt nyttig for stort set ethvert væv, i enhver musemodel og under enhver ekstern forstyrrelse. Her fokuserer vi på analysen af brunt fedtvæv (BAT) på grund af dets høje niveauer af DNL og kritiske roller i opretholdelsen af metabolisk homeostase.

Protocol

Alle forsøg blev godkendt af Institutional Animal Care and Use Committee på Cincinnati Children’s Hospital Medical Center. 1. Fremstilling af D2O BEMÆRK: For at undgå eksperimentel variation skal du forberede tilstrækkelig opløsning/drikkevand til alle mus i løbet af eksperimentet. Til intraperitoneal injektion: generer 0,9% w / v saltvand D 2 O ved at opløse 9 g NaCl pr. Liter D 2 O.Filtrer gennem et ikke-pyr…

Representative Results

Baseret på D 2 O-doseringen beskrevet i trin 1 finder vi typisk, at kropsvand beriges i området2,5% til 6%, og at et baselineniveau for deuteriumberigelse i kropsvand hurtigt opnås på 1 time og opretholdes i undersøgelsens varighed via 8% beriget drikkevand (figur 1). Kontinuerlig steady state kropsvandberigelse er en antagelse af de beregninger, der anvendes i trin 6, og derfor anbefaler vi eksperimentel validering af kinetikken for kropsvandsberigelse i nye eksperi…

Discussion

Forståelse af balancen og interaktionen mellem komplekse metaboliske veje er et uundværligt skridt i retning af at forstå det biologiske grundlag for metaboliske relaterede sygdomme. Her viser vi en ikke-invasiv og billig metode til at bestemme ændringer i de novo fedtsyresyntese. Denne metode er tilpasset fra tidligere offentliggjorte metoder, der udviklede beregninger til estimering af de novo-synteseflux fra fedtsyredeuteriumberigelse31 og til anvendelse af deuterium-aceto…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Vi takker Sanchez-Gurmaches og Wallace laboratoriets medlemmer for værdifulde diskussioner. Dette arbejde blev støttet af tilskud fra American Heart Association (18CDA34080527 til JSG og 19POST34380545 til RM), NIH (R21OD031907 til JSG), en CCHMC Trustee Award, en CCHMC Center for Pediatric Genomics Award og en CCHMC Center for Mendelian Genomics & Therapeutics Award. Dette arbejde blev delvist støttet af NIH P30 DK078392 fra Digestive Diseases Research Core Center i Cincinnati. Indholdet er udelukkende forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter fra National Institutes of Health. RT og MW blev støttet af et UCD Ad Astra Fellowship.

Materials

4 mL Glass Vials Fisher Scientific 14-955-334
0.2 µm filter Olympus Plastic 25-244
26G needeled syringes BD 309597
Acetone Merck 34850
Acetonitrile Merck 900667
Blue GC screw cap with septa Agilent 5190-1599
Centrifuge Eppendorf 5424R
Chloroform Sigma 366927
Deuterium oxide Sigma 151882
Di-tert-butyl-4-methylphenol (BHT)
Select FAME Column		 Merck B1378
Di-tert-butyl-4-methylphenol (BHT)
Select FAME Column		 Agilent CP7419
EDTA tube Sarstedt 411395105
Ethanol Merck 51976
Hexadecenoic-d31 Acid Larodan 71-1631
Hexane Merck 34859
Methanol Merck 34860
Microcentrifuge tube Olympus Plastic 24-282
Mouse environmental chamber Caron Caron 7001-33
Potasium Chloride Fisher Bioreagents BP366-500
Potasium Phosphate MP Biomedicals 194727
SafeLock microcentrifuge tubes Eppendorf 30120086
Screw top amber GC vial Agilent 5182-0716
Sodium Chloride Fisher Bioreagents BP358-212
Sodium Hydroxide Merck S5881
Sodium Phosphate, dibasic Fisher Bioreagents BP332-500
Sodium Sulfate Merck 239313
Sulfuric Acid Merck 258105
Vial insert Agilent 5183-2088
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Tags

Fatty Acid SynthesisDeuterium OxideNon-invasiveIsotope-labeled GlucoseMetabolic StatusChloroform-methanol ExtractionFatty Acid MethylationGC-MS Analysis
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Citazione di questo articolo
Turner, R., Mukherjee, R., Wallace, M., Sanchez-Gurmaches, J. Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide. J. Vis. Exp. (195), e64219, doi:10.3791/64219 (2023).
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