Name: measurement of basal and forskolin-stimulated lipolysis in inguinal adipose fat pads
Uploaded: 2017-07-21T12:30:00
Description: Watch this Scientific Journal Video about Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads at JoVE.com

Dette arbeidet ble støttet av AHA-prisen nr. 15BGIA23250030, NIHs nasjonale institutt for generell medisinsk vitenskap, under prisnummer 8P20 GM103432-12 og fakultetstilskudd for Universitetet i Wyoming i hjelp til BT.

Alle protokoller følger retningslinjene for dyrepleie av University of Wyoming. 1. Husdyr og fôring MERK: Voksne mannlige villtype mus (C57BL / 6) (alder 12 til 24 uker) ble oppdrettet i forskningsdyrfasiliteten i henhold til godkjente protokoller for Institutt for dyrepleie og brukskomité (IACUC). <ol><li> Fra og med uke 6 går husmus i grupper på fire i separate bur og tildeler dem tilfeldig til fôringsgrupper av NCD eller HFD (± 0,01% CAP) til uke 38 i...

<ol>
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W., Obin, M. S., Greenberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+triglyceride+lipase+regulates+basal+lipolysis+and+lipid+droplet+size+in+adipocytes.">Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes.</a> J Cell Biochem. 105 (6), 1430-1436 (2008).</li><li>Kolditz, C. I., Langin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+lipolysis.">Adipose tissue lipolysis.</a> Curr Opin Clin Nutr Metab Care. 13 (4), 377-381 (2010).</li><li>Baskaran, P., Krishnan, V., Ren, J., Thyagarajan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsaicin+induces+browning+of+white+adipose+tissue+and+counters+obesity+by+activating+TRPV1+channel-dependent+mechanisms.">Capsaicin induces browning of white adipose tissue and counters obesity by activating TRPV1 channel-dependent mechanisms.</a> Br J Pharmacol. 173 (15), 2369-2389 (2016).</li><li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRPV1+by+dietary+capsaicin+improves+endothelium-dependent+vasorelaxation+and+prevents+hypertension.">Activation of TRPV1 by dietary capsaicin improves endothelium-dependent vasorelaxation and prevents hypertension.</a> Cell Metab. 12 (2), 130-141 (2010).</li><li>Ding, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+lipolysis+response+to+adipose+afferent+reflex+involved+in+impaired+activation+of+adrenoceptor-cAMP-PKA-hormone+sensitive+lipase+pathway+in+obesity.">Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity.</a> Sci Rep. 6, 34374 (2016).</li><li>Ohyama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combination+of+exercise+and+capsinoid+supplementation+additively+suppresses+diet-induced+obesity+by+increasing+energy+expenditure+in+mice.">A combination of exercise and capsinoid supplementation additively suppresses diet-induced obesity by increasing energy expenditure in mice.</a> Am J Physiol Endocrinol Metab. 308 (4), E315-E323 (2015).</li><li>Beylot, M., Martin, C., Beaufrere, B., Riou, J. 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Fordelingen av TGL i glycerol og fettsyrer katalyseres av ATGL 9 under basal lipolyse og orkestrert av en rekke proteiner, inkludert aktivering av adenylylcyklase / PKA-avhengig vei under stimulert lipolyse 21 , 22 , 23 . Forbedring av lipolyse øker plasmanivåene av fettsyrer for transport og energibruk 24 . Fettsyrer tas opp av mitokondrier som acetyl CoA, som brukes til å pro...

Fettvev lagrer energi som fett 2 og fettsyreoksydasjon er nødvendig for termogenese 3 , 4 . Fettsyrer inntatt gjennom dietter pakkes sammen med apoproteiner i chylomikroner og leveres til forskjellige vev i kroppen via blodsirkulasjon. Selv om de fleste celler i kroppen lagrer et reserve av energi, beholder fettvev overflødig energi som fett 5 , 6 . Lipolyse i fettvev reguleres av komplekse prosesser, og molekylære detaljer av lipolyse forblir fortsatt vage 7 . Lipolyse er en prosess hvor triglyserider (TGL) lagret i fettvev hydrolyseres for å produsere glyserol og fettsyrer (FA) av enzymet adipose triglycerid lipase (ATGL) 8 . Endringer i basal og stimulert lipolyse er et karakteristisk trekk ved fedme. BAsal lipolyse er regulert av ATGL-aktivering 9 , som omdanner TGL til diacylglycerol (DAG), som deretter hydrolyseres til monoacylglycerol (MAG). Aktivering av hormonfølsom lipase (HSL) via adenylylcyklase aktiverer stimulering av cyklisk adenosinmonofosfat (cAMP) avhengig av proteinkinase A (PKA) og forårsaker lipolyse. Måling av lipolyse, basal og stimulert, er derfor viktig for å analysere aktiviteten til proteiner involvert i denne prosessen. Også unraveling molekylær regulering av lipolyse kan være gunstig for å utvikle nye terapeutiske strategier mot fedme 10 . Siden molekyler som stimulerer lipolyse og fettsyreoksidasjon er potensielle kandidater for å redusere fett lagret i depoter, er det viktig å anvende et robust assay for reproduserbarhet. Tidligere publiserte data antyder at aktivering av TRPV1 protein uttrykt i hvitt fettvev ved CAP forbedret basalOg FSK (adenylyl-syklaseaktivator) -stimulert lipolyse i inngangsfettputer 11 . Tidligere forskning tyder også på at langsiktig aktivering av TRPV1 ved CAP aktiverer PKA 12 . Siden aktiveringen av PKA stimulerer lipolyse 13 , 14 , som måler både basal og PKA-avhengig stimulert lipolyse i inguinal fettputer isolert fra NCD eller HFD (± CAP) -fedmus etter 32 ukers føding av de respektive dietter, vil valideringen av rollen som TRPV1 Aktivering i lipolyse. Denne artikkelen beskriver en effektiv metode for å bestemme basal og stimulert lipolyse. Selv om andre metoder som benytter radioaktive isotoper av glycerol og kjedelig høyytelsesvæskekromatografi eller gasskromatografi / massespektrometri for målinger 15 , 16 er tilgjengelige, gir denne metoden en mer direkte, enkel og kostnadseffektivTeknikk for å bestemme lipolyse i fettvev.

<table border="0" cellpadding="0" cellspacing="0" width="694">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Capsaicin</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">M2028</td>
			<td style="width:189px;">TRPV1 agonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forskolin</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">F6886</td>
			<td>Adenylyl cyclase activator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM</td>
			<td style="width:171px;">GE healthcare and life sciences, UT, USA</td>
			<td style="width:119px;">SH30081.01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High fat diet</td>
			<td style="width:171px;">Research diets, New Brunswick, USA</td>
			<td style="width:119px;">D12492</td>
			<td>Abbreviated as HFD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:171px;">Amresco, USA</td>
			<td style="width:119px;">O497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:171px;">Thermofisher Scientific</td>
			<td style="width:119px;">BP358-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium deoxycholate</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">D6750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">D9163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium orthovanadate</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">S6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Free Glycerol reagent</td>
			<td style="width:171px;">Sigma USA</td>
			<td style="width:119px;">F6428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">D8779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triacsin C</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">T4540</td>
			<td>Acyl CoA transferase inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine serum albumin</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:119px;">31998-8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methanol</td>
			<td style="width:171px;">Thermofisher Scientific, USA</td>
			<td style="width:119px;">A412-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hydroxide</td>
			<td style="width:171px;">Amresco, USA</td>
			<td style="width:119px;">O583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium dodecyl sulfate</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">L3371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bicinchoninic acid reagent</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">BCA1-1KT</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UV-VIS Spectrophotometer</td>
			<td style="width:171px;">Pharmacia Biotech, NJ, USA</td>
			<td style="width:119px;">Ultrospec 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal chow diet</td>
			<td style="width:171px;">Labdiet.com</td>
			<td style="width:119px;">500I</td>
			<td>abreviated as NCD</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57BL/6 mice</td>
			<td style="width:171px;">Jackson Laboratory, CT, USA</td>
			<td style="width:119px;">Stock number000664</td>
			<td>wild type mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:171px;">Heathrow Scientific, USA</td>
			<td style="width:119px;">HS 234526A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol standard</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">G7793</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of basal and forskolin-stimulated lipolysis in inguinal adipose fat pads

Lipolyse er en prosess hvorved lipidet lagret som triglyserider i fettvev hydrolyseres til glycerol og fettsyrer. Denne artikkelen beskriver metoden for måling av basal og forskolin (FSK) -stimulert lipolyse i inguinal fettputer isolert fra villtype mus som er matet enten normal chow diett (NCD), høyt fett diett (HFD) eller en høy fett diett som inneholder 0,01 % Av capsaicin (CAP; transient reseptor potensiell vanilloid subfamily 1 (TRPV1) agonist) i 32 uker. Fremgangsmåten beskrevet her for å utføre ex vivo lipolyse er vedtatt fra Schweiger et al. 1 Vi presenterer en detaljert protokoll for måling av glycerol nivåer ved UV-synlig (UV / VIS) spektrofotometri. Metoden beskrevet her kan brukes til å isolere fettpudder for lipolyse for å oppnå konsistente resultater. Protokollen beskrevet for inguinal fettputer kan lett bli utvidet til å måle lipolyse i andre vev.

For å evaluere effekten av CAP på basal og stimulert lipolyse, ble denne undersøkelsen målt lipolyse i inngangsvektfettputer isolert fra NCD eller HFD (± CAP) -fed-vildtype-mus. De representative resultatene for den basale og FSK-stimulerte lipolyse for inguinal fettputer er gitt i tabell I. Basal- og FSK-stimulert glyserolfrigivelse i nærvær av Triacsin C, som hemmer acylcoA-syntetase og forhindrer regenerering av TGL. Som vist i figur 1</str...

Watch this Scientific Journal Video about Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads at JoVE.com

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

Denne protokollen beskriver metoden for å bestemme basal og forskolinstimulert lipolyse i inguinal fettputer oppnådd fra normal chow diett (NCD) eller høyt fett diett (HFD) ± capsaicin matet villtype mus. Som en indeks for lipolyse ble glyserolfrigivelsen målt fra inguinal adipose fettputer.

Måling av basal og forskolinstimulert lipolyse i fettputer

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes ...

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