Name: assessing whole-body lipid-handling capacity in mice
Uploaded: 2020-11-24T14:00:00
Description: Watch this Scientific Journal Video about Assessing Whole-Body Lipid-Handling Capacity in Mice at JoVE.com

Dette arbejde er støttet af National Institutes of Health (NIH), tilskud R00-DK114498, og United States Department of Agriculture (USDA), tilskud CRIS: 3092-51000-062 til Y. Z.

Dyr er anbragt under standardiserede forhold efter dyrepleje og forsøgsprotokoller godkendt af den institutionelle Animal Care and Use Committee af Baylor College of Medicine (BCM). Dyr fodres med en standard eller speciel kost, vand ad libitum og holdes med en 12-timers dag / nat cyklus.
1. Måling af faste serum lipider
<ol><li>Overfør mus til et nyt bur efter kl. 17 og hurtigt med fri adgang til vand natten over (med ca. 16 timers faste før eksperimentet). Den overnattende faste sikrer...

<ol>
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De tre beskrevne analyser fungerer robust i laboratoriet, med et par kritiske overvejelser. Overnight faste er nødvendig for bestemmelse faste serum lipid niveauer og oral intralipid tolerance test. For oral intralipid tolerance test, er det afgørende at dreje blodet ved stuetemperatur for at minimere dannelsen af et fedtlag, især på 1- og 2-timers tid punkter; det er vigtigt ikke at kassere dette fedtlag, hvis det dannes. Sørg for at overføre supernatanten med lipidlaget, og pipet forsigtigt for at blande dem samm...

Kulhydrater og lipider er to store substrater for energimetabolisme. Afvigende lipid metabolisme resulterer i mange menneskelige sygdomme, herunder type II diabetes, hjerte-kar-sygdomme, fedtlever sygdomme, og kræft. Kosten lipider, hovedsagelig triglycerider, absorberes gennem tarmen ind i lymfesystemet og kommer ind i venøscirkulationen i chylomicrons nær hjertet1. Lipider bæres af lipoproteinpartikler i blodbanen, hvor fedtsyremoietierne frigøres ved virkningen af lipoprotein lipase ved perifere organer som muskel- og fedtvæv2. De resterende kolesterolrige restpartikler ryddes af leveren3. Mus har været meget udbredt i laboratorier som en forskningsmodel til at studere lipid metabolisme. Med omfattende genetiske værktøjssæt til rådighed og en relativt kort avlscyklus er de en stærk model til at studere, hvordan lipider absorberes, syntetiseres og metaboliseres.
På grund af kompleksiteten af lipid metabolisme, sofistikerede lipidomics undersøgelser eller isotop tracer undersøgelser bruges normalt til at kvantificere samlinger af lipid arter eller lipid-relaterede metaboliske fluxes og skæbner4,5. Dette skaber en massiv udfordring for forskere uden specialiseret udstyr eller ekspertise. I dette papir, præsenterer vi tre analyser, der kan tjene som indledende tests, før teknisk udfordrende teknikker anvendes. De er ikke-terminale procedurer for musene, og dermed meget nyttige til at identificere potentielle forskelle i lipid-håndtering kapacitet og indsnævring af de berørte processer.
For det første kan måling af sende serum lipidmolekyler hjælpe en med at fastslå en mus samlede lipidprofil. Mus skal fastes, fordi mange lipidarter stiger efter måltider, og omfanget af stigningen påvirkes stærkt af kostens sammensætning. Mange lipidmolekyler, herunder total kolesterol, triglycerid og ikke-esterificeret fedtsyre (NEFA), kan måles ved hjælp af et kommercielt kit og en pladelæser, der kan læse absorbans.
For det andet, en oral intralipid tolerance test vurderer lipid-håndtering kapacitet som en nettoeffekt af absorption og stofskifte. En mundtligt administreret intralipid forårsager en stigning i cirkulerende triglyceridniveauer (1-2 timer), hvorefter serum triglyceridniveauet vender tilbage til basalniveauer (4-6 timer). Denne analyse giver oplysninger om, hvor godt en mus kan håndtere de udefrakommende lipider. Hjerte, lever, og brun fedtvæv er aktive forbrugere af triglycerider, mens hvidt fedtvæv gemmer det som en energireserve. Ændringer i disse funktioner vil føre til forskelle i testresultaterne.
Endelig, fremme lipolyse at mobilisere lagrede lipider betragtes som en mulig strategi for vægttab. Den β3-adrenergic receptor signalering vej i fedtvæv spiller en vigtig rolle i adipocyt lipolyse, og human genetik har identificeret et tab-of-funktion polymorfi Trp64Arg i β3-adrenergic receptor korreleret med fedme6. CL 316,243, en specifik og potent β3-adrenergic receptor agonist, stimulerer fedtvæv lipolyse og frigivelse af glycerol. Evaluering af en mus svar på CL 316,243 kan give værdifulde oplysninger om udvikling, forbedring og forståelse af effekten af forbindelsen.
Kollektivt, disse tests kan bruges som en indledende skærm for ændringer i lipid metaboliske tilstand af mus. De vælges til tilgængeligheden af instrumenter og reagenser. Med resultaterne stammer fra disse analyser, forskere kan danne et samlet billede af metaboliske egnethed af deres dyr og træffe beslutning om mere sofistikerede og målrettede tilgange.

<table>
	<tbody>
		<tr>
			<td>20% Intralipid</td>
			<td>Sigma Aldrich</td>
			<td>I141</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes 1ml</td>
			<td>Shaotong</td>
			<td>B07F1KRMYN</td>
			<td></td>
		</tr>
		<tr>
			<td>CL 316,243 Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C5976</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Feeding Needles (18 Gauge)</td>
			<td>Kent Scientific</td>
			<td>FNC-18-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Free Glycerol Reagent</td>
			<td>Sigma Aldrich</td>
			<td>F6428</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Standard Solution</td>
			<td>Sigma</td>
			<td>G7793</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>999-34691</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>991-34891</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>995-34791</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>993-35191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vedco</td>
			<td>50989-161-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix Plus Chemistry Reference Kit</td>
			<td>Verichem</td>
			<td>9500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-222-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Microhematrocrit Capillary Tube, Not Heparanized</td>
			<td>Fisher Scientific</td>
			<td>22-362-574</td>
			<td></td>
		</tr>
		<tr>
			<td>NEFA STANDARD SOLUTION</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>276-76491</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Triglycerides Reagent</td>
			<td>Fisher Scientific</td>
			<td>TR22421</td>
			<td></td>
		</tr>
		<tr>
			<td>Total Cholesterol Reagents</td>
			<td>Thermo Scientifi</td>
			<td>TR13421</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Henry Schein</td>
			<td>11695-4022-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing whole-body lipid-handling capacity in mice

Vurdering lipid metabolisme er en hjørnesten i evalueringen metabolisk funktion, og det anses for afgørende for in vivo metabolisme undersøgelser. Lipider er en klasse af mange forskellige molekyler med mange veje involveret i deres syntese og stofskifte. Et udgangspunkt for evaluering lipid hæmostase for ernæring og fedme forskning er nødvendig. Dette papir beskriver tre nemme og tilgængelige metoder, der kræver lidt ekspertise eller praksis at mestre, og som kan tilpasses af de fleste laboratorier til at screene for lipid-metabolisme abnormiteter i mus. Disse metoder er (1) måling flere faste serum lipid molekyler ved hjælp af kommercielle kits (2) assaying for kosten lipid-håndtering kapacitet gennem en oral intralipid tolerance test, og (3) evaluere respons på en farmaceutisk forbindelse, CL 316,243, i mus. Sammen vil disse metoder give et højt niveau overblik over lipid håndtering kapacitet i mus.

Vi viser med tre uddrag, at hver analyse giver værdifulde oplysninger om musenes lipid metabolisme. For C57BL/6J hanmus, udfordret af otte ugers fedtrig kost (HFD) fodring starter ved otte ugers alder, samlede kolesterolniveauer var signifikant forhøjet, mens serum triglyyceid og NEFA ikke var (tabel 1), tyder på, at triglycerid og NEFA i blodet ikke overvejende er reguleret af en kosten fedt udfordring. I den anden kohorte af mus blev C57BL/6J og C57BL6/N undertræne af C57BL6 fodret HFD i otte uger,...

Watch this Scientific Journal Video about Assessing Whole-Body Lipid-Handling Capacity in Mice at JoVE.com

Assessing Whole-Body Lipid-Handling Capacity in Mice

Dette papir giver tre nemme og tilgængelige analyser til vurdering af lipid metabolisme i mus.

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