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 arbejde blev understøttet af AHA-prisen nr. 15BGIA23250030, NIH&#39;s National Institute of General Medical Sciences under prisen nummer 8P20 GM103432-12 og University of Wyoming Fakultetstilskud i Aid to BT.

Alle protokoller følger retningslinjerne for dyresundhed ved University of Wyoming. 1. Husdyr og fodring BEMÆRK: Voksen af ​​voksne vildtype mus (C57BL / 6) (12 til 24 uger i alderen) blev opdrættet i forsøgsdyrfaciliteten i henhold til godkendte protokoller for institutionelle dyresundheds- og brugskomitéer (IACUC). <ol><li> Start fra uge 6, husmus i grupper på fire i separate bure og tilfældigt tildele dem til fodergrupper af NCD eller HFD (± 0,01...

<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. P., Mornex, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+steady+state+and+nonsteady-state+glycerol+kinetics+in+humans+using+deuterium-labeled+tracer.">Determination of steady state and nonsteady-state glycerol kinetics in humans using deuterium-labeled tracer.</a> J Lipid Res. 28 (4), 414-422 (1987).</li><li>Gilker, C. D., Pesola, G. R., Matthews, D. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skinny+on+fat:+lipolysis+and+fatty+acid+utilization+in+adipocytes.">The skinny on fat: lipolysis and fatty acid utilization in adipocytes.</a> Trends Endocrinol Metab. 20 (9), 424-428 (2009).</li><li>Jaworski, K., Sarkadi-Nagy, E., Duncan, R. E., Ahmadian, M., Sul, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+triglyceride+metabolism.+IV.+Hormonal+regulation+of+lipolysis+in+adipose+tissue.">Regulation of triglyceride metabolism. IV. Hormonal regulation of lipolysis in adipose tissue.</a> Am J Physiol Gastrointest Liver Physiol. 293 (1), G1-G4 (2007).</li><li>Jeppesen, J., Kiens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+limitations+to+fatty+acid+oxidation+during+exercise.">Regulation and limitations to fatty acid oxidation during exercise.</a> J Physiol. 590 (5), 1059-1068 (2012).</li><li>Nakamura, M. T., Yudell, B. E., Loor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+energy+metabolism+by+long-chain+fatty+acids.">Regulation of energy metabolism by long-chain fatty acids.</a> Prog Lipid Res. 53, 124-144 (2014).</li><li>Murray, A. J., Panagia, M., Hauton, D., Gibbons, G. F., Clarke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+free+fatty+acids+and+peroxisome+proliferator-activated+receptor+alpha+in+the+control+of+myocardial+uncoupling+protein+levels.">Plasma free fatty acids and peroxisome proliferator-activated receptor alpha in the control of myocardial uncoupling protein levels.</a> Diabetes. 54 (12), 3496-3502 (2005).</li><li>Barbera, M. J., 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=Peroxisome+proliferator-activated+receptor+alpha+activates+transcription+of+the+brown+fat+uncoupling+protein-1+gene.+A+link+between+regulation+of+the+thermogenic+and+lipid+oxidation+pathways+in+the+brown+fat+cell.">Peroxisome proliferator-activated receptor alpha activates transcription of the brown fat uncoupling protein-1 gene. 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Fordelingen af ​​TGL i glycerol og fedtsyrer katalyseres af ATGL 9 under basal lipolyse og orkestreret ved en række proteiner, herunder aktivering af adenylylcyclase / PKA-afhængig vej under stimuleret lipolyse 21 , 22 , 23 . Forøgelse af lipolyse øger plasmaniveauerne af fedtsyrer til transport og energiforbrug 24 . Fedtsyrer optages af mitochondrier som acetyl CoA, som b...

Fedtvæv opbevarer energi som fedt 2 og fedtsyreoxidation kræves til termogenese 3 , 4 . Fedtsyrer indtaget gennem kostvaner pakkes sammen med apoproteiner i chylomicroner og leveres til forskellige væv i kroppen via blodcirkulation. Selvom de fleste celler i kroppen opbevarer en energibesparelse, opbevarer fedtvæv overskydende energi som fedt 5 , 6 . Lipolyse i fedtvæv reguleres af komplekse processer, og molekylære detaljer af lipolyse forbliver stadig vage 7 . Lipolyse er en proces, hvorved triglycerider (TGL), der opbevares i fedtvæv, hydrolyseres til dannelse af glycerol og fedtsyrer (FA) af enzymet adipose triglycerid lipase (ATGL) 8 . Ændringer i basal og stimuleret lipolyse er et karakteristisk træk ved fedme. BAsal lipolyse reguleres af ATGL-aktivering 9 , som omdanner TGL til diacylglycerol (DAG), der efterfølgende hydrolyseres til monoacylglycerol (MAG). Aktivering af hormonfølsom lipase (HSL) via adenylylcyclase aktiverer stimulering af proteinkinase A (PKA) med cyklisk adenosinmonophosphat (cAMP) og forårsager lipolyse. Måling af lipolyse, basal og stimuleret, er derfor vigtig for at analysere aktiviteten af ​​proteiner involveret i denne proces. Desuden kan unraveling den molekylære regulering af lipolyse være gavnlig for at udvikle nye terapeutiske strategier mod fedme 10 . Da molekyler, som stimulerer lipolyse og fedtsyreoxidation, er potentielle kandidater til faldende fedtstoffer, der er lagret i depoter, er det vigtigt at anvende et robust assay for reproducerbarhed. Tidligere offentliggjorte data tyder på, at aktivering af TRPV1 protein udtrykt i hvidt fedtvæv med CAP forbedret basalOg FSK (adenylylcyklaseaktivator) -stimuleret lipolyse i indinale fedtpuder 11 . Tidligere forskning tyder også på, at langsigtet aktivering af TRPV1 ved CAP aktiverer PKA 12 . Da aktiveringen af ​​PKA stimulerer lipolyse 13 , 14 , der måler både basal og PKA-afhængig stimuleret lipolyse i indinale fedtpuder isoleret fra NCD eller HFD (± CAP) -fedmus efter 32 ugers fodring af de respektive diæt, vil valideringen af ​​TRPV1&#39;s rolle Aktivering i lipolyse. Denne artikel beskriver en effektiv metode til bestemmelse af basal og stimuleret lipolyse. Skønt andre metoder, der anvender radioaktive isotoper af glycerol og kedelig højtryksvæskekromatografi eller gaschromatografi / massespektrometri til målinger 15 , 16 er tilgængelige, giver denne metode en mere direkte, enkel og omkostningseffektivTeknik til bestemmelse af lipolyse i fedtvæv.

<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 proces, hvorved lipidet opbevaret som triglycerider i fedtvæv hydrolyseres til glycerol og fedtsyrer. Denne artikel beskriver metoden til måling af basal og forskolin (FSK) -stimuleret lipolyse i de indinale fedtpuder, der er isoleret fra vildtype-mus, der er fodret med enten normal chow diet (NCD), fedtfattig kost (HFD) eller en fedtfattig diæt indeholdende 0,01 % Af capsaicin (CAP; transient receptorpotential vanilloid subfamily 1 (TRPV1) agonist) i 32 uger. Fremgangsmåden beskrevet her for at udføre ex vivo lipolyse er vedtaget fra Schweiger et al. 1 Vi præsenterer en detaljeret protokol til måling af glycerolniveauer ved UV-synlig (UV / VIS) spektrofotometri. Den her beskrevne metode kan bruges til at isolere fedtpuder til lipolyse med succes for at opnå konsistente resultater. Protokollen beskrevet for induinal fedt puder kan let udvides til at måle lipolyse i andre væv.

For at evaluere effekten af ​​CAP på basal og stimuleret lipolyse, målte denne forskning lipolyse i inguinal adipose fedt puder isoleret fra NCD eller HFD (± CAP) -fed vildtype mus. De repræsentative resultater for den basale og FSK-stimulerede lipolyse til de indinale fedtpuder er angivet i tabel I. Basal- og FSK-stimuleret glycerolfrigivelse i nærvær af Triacsin C, som hæmmer acylcoA-syntetase og forhindrer regenerering af TGL. Som vist i <strong class="xfig...

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 protokol beskriver metoden til bestemmelse af basal og forskolinstimuleret lipolyse i fedtpuder, der opnås fra normal chow diet (NCD) eller fedtsyre (HFD) ± capsaicin fodret vildtype-mus. Som et indeks for lipolyse blev glycerolfrigivelse målt fra inguinal adipose fatpads.

Måling af basal og forskolin-stimuleret lipolyse i fedtpads

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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