Bu çalışma, AHA Ödülü No. 15BGIA23250030, NIH Ulusal Tıp Bilimleri Enstitüsü, 8P20 GM103432-12 ödülü ve Wyoming Üniversitesi Teknik Destek Bursu adı altında BT tarafından desteklenmiştir.

Tüm protokoller, Wyoming Üniversitesi&#39;ndeki hayvan bakım kılavuzlarına uymaktadır. 1. Hayvan Barınağı ve Beslenme NOT: Yetişkin vahşi tipli fareler (C57BL / 6) (12-24 haftalık yaşlar), Kurumsal Hayvan Bakımı ve Kullanım Komitesi (IACUC) onaylı protokollere göre araştırma hayvan tesislerinde yetiştirilmiştir. <ol><li> Yaşın 6. haftasından itibaren ev fareleri dört grup halinde ayrı kafeste gruplandı ve rastgele onları 38 yaşına kadar NBH veya HFD (±% 0.01 CAP) beslenme gruplarına atadılar. NOT: CAP, yağ dokuları 11 , 17&#39;de ifade edilen TRPV1 kanal proteininin bir agonistidir. Kaput içinde bir karıştırıcıda HFD ile CAP karıştırın ve küçük 1 2 bölümleri ihtiva eden bir tepsiye harmanlanmış karışımın aktarın. Tepsiyi -20 ° C dondurucuda saklayın. HFD + CAP diyetini içeren tepsiyi 24 saat sonra dondurucudan çıkarın ve -20 ° C&#39;de bir kapta saklayın.# 176; C dondurucu kullanana kadar. </li><li> Evde beslenen fareler, iklimle kontrol edilen bir ortamda (22.8 ± 2.0 ° C,% 45-50 nem) 12/12 ışık / karanlık döngüsü ile belirlenmiş diyet ve suya serbestçe erişebilirler. </li><li> 38 haftanın sonunda, inguinal yağ dokularını inceleyin ve lipoliz deneyleri için kullanın (bölümler 2-7). </li></ol> 2. Deneyler için Fare Hazırlama <ol><li> Ketamin ve ksilazin karışımı (sırasıyla 10 mg / kg ve 80 mg / kg vücut ağırlığı) enjekte ederek farelere anestezi uygulayın. 0.01 mL karışım / 10 g vücut ağırlığı fare enjekte edin. </li><li> Sıkı bir anestezi sıkı bir ayak parmağı çimdikle teyit edin. Pedal refleksi varsa fareyi en az 30 saniye sonra tekrar test edin. </li><li> Anestezi sırasında gözlerin kuruluğunu önlemek için veteriner merhemi kullanın. İşlemlerden herhangi birisi sırasında fareleri gözetimsiz bırakmayın. </li><li> Yüksek dozda ketamin ve xylazine karışım enjeksiyonu (sırasıyla 10 mg / kg ve 80 mg / kg vücut ağırlığı) karışımını enjekte ederek fareleri euthanize edinE (0.01 ml / 10 g vücut ağırlığı) ve ardından servikal dislokasyon. NOT: Bu ötenazi yöntemi Wyoming Üniversitesinin IACUC tarafından onaylanmıştır. </li></ol> 3. Inguinal Adipose Fat Pad&#39;lerin İzolasyonu <ol><li> Prosedür için solunda kalan fareyi (NCD veya HFD (± CAP) ile 32 hafta boyunca beslenir) yerleştirin. NOT: Fareyi sol tarafa yerleştirerek sağ ön ayak ve sağ arka platform platformdan uzaklaşırken sol ön ayak ve sol arka bacak diseksiyon platformunda olacaktır. </li><li> 2.5 inç% 70 etanol batırılmış 2 inç 2 gazlı bezle cilt yüzeyini sterilize edin. Altta yağ tabakası ortaya çıkarmak için bir neşter kullanarak deriden 2 - 3 mm&#39;lik bir yanal kesim yapın. </li><li> Dorsal yüzeyin iki yanal kesisini birleştiren kaburga kafesinin hemen altında bir neşter kullanarak cilt boyunca 1 cm&#39;lik bir kesik yapın (fare boyutuna bağlı olarak). </li><li> Deriyi sürükleyerek deriyi soyun.Steril forseps kullanarak ve yağ pedleri keserek cilt altı pedini sağlam bırakın. Bu, cildin altında yatan yağ yastığı. </li><li> Bir çift makas kullanarak yağ bandını alttaki kas ve fasyadan dikkatlice ayırın. Yağ pedini, alttaki kası kesildiği için çekin. NOT: Yağ yastıklarının ağırlığı ve boyutu farelerin türüne (NCD veya HFD (± CAP) -fed) bağlıdır. </li><li> Oda sıcaklığında (~ 15 dakika) lipoliz deneyi olana kadar fosfat tamponlu salin (PBS) içeren bir Petri kabına aktarmak için cımbız kullanın. </li><li> Adımlar 3.2-3.6&#39;da açıklandığı gibi yağ yastıklarını farenin sağ tarafından ayırın. </li></ol> 4. İnguinal Yağ Keçelerinden Bazal Gliserol Salınımı <ol><li> 5 ila 8 parçaya yaklaşık 20 mg yağ dokusu kesmek için keskin makas kullanın. </li><li> Inüinal yağ yastıklarının kesme parçalarını 200 uL inkübasyon ortamında (% 2 yağ asidi içeren Dulbecco Modifiye Eagel&#39;s Medium (DMEM) kuluçkalayın37 ° C &#39;de içermeyen sığır serum albümini (BSA)),% 5 CO2 ve 60 dakika için% 95 nem. <ol><li> İnkübasyon ortamı toplayın ve lipoliz testi için -80 ° C&#39;de dondurun. NOT: Kesilen parçalar, lipit ekstraksiyonu sonrası protein konsantrasyonunun belirlenmesi için kullanılır. </li><li> Kesilen yağ parçalarını cımbız yardımıyla 1 mL ekstraksiyon çözeltisine (kloroform: metanol (2: 1, v / v) ve% 1 buzlu asetik asit) aktarın ve 100 ° C&#39;de çalkalarken 37 ° C&#39;de 60 dakika inkübe edin rpm. Yağ ekstraksiyon çözeltisini atın. NOT: Bu adım kesilen parçalardan yağ çıkaracaktır. Yağ ekstraksiyon çözeltisini, protein belirlenmesini etkileyeceğinden atın. </li><li> Dokuyu (adım 4.2.2&#39;den), cımbızı kullanarak, 500 μL lizis çözeltisi (% 0.1 sodyum dodesil sülfat içeren 0.3 N NaOH; SDS) içeren steril bir mikrofuge tüpüne aktarın ve gece boyunca (12 saat) 55 ° C&#39;de kuvvetli çalkantı altında inkübe edin 100 rpm&#39;de. </li></ol></li><li> Doku (aşama 4.2.3) standart 18 olarak bisinkoninik asit (BCA) reaktif ve BSA kullanılarak protein konsantrasyonunu belirleyin. </li><li> Donmuş ortamı buz üzerinde çözün (adım 4.2.1) ve protokol bölümleri 6 ve 7&#39;de açıklandığı gibi serbest bir gliserol reaktifi kullanarak ortamın gliserol içeriğini belirleyin 6 . <ol><li> Gliserol standartları 19 ile çizilen standart eğriden alınan örneklerdeki gliserol konsantrasyonunu hesaplayın ve lipoliz, saat 20 başına mg protein başına nanomole gliserol olarak ifade edin. </li></ol></li></ol> 5. FSK ile uyarılan lipoliz <ol><li> % 2 yağ asidi içermeyen BSA, 10 uM FSK ve 5 uM Triacsin C içeren 200 μL DMEM&#39;de NCD veya HFD (± CAP) ile beslenmiş yabani tip fareden elde edilen yaklaşık 20 mg kasık yağ yastığını (5 ila 8 kesilmiş parça) önceden inkübe edin. 60 dakika boyunca 37 ° C,% 5 CO2 ve% 95 nem. </strong&gt; <ol><li> 37 ° C,% 5 CO2 ve% 95 nem ile bir 60 dakika daha aynı ortama cımbız kullanarak doku parçaları aktarın ve inkübe edilir. Kuluçka ortamı toplayın ve lipoliz testine kadar -80 ° C&#39;de saklayın. NOT: Lipoliz stimülasyonu, ilk saatlerde doğrusal olan 15 dakika içinde yağ asitleri ve gliserol&#39;ün hızlı bir şekilde serbest bırakılmasına ve bundan sonraki yaylalara neden olur. Uyarılmış lipoliz oranı FSK Uyarımın birinci ve ikinci saat arasında daha yüksek ve stabil olduğu daha önce tarif edildiği gibi 1, uyarılmış halde bu protokol ölçülen lipoliz ikinci kuluçka dönemi geçirmektedir. Bu nedenle, hem deneysel adımlar (5.1 ve 5.1.1) gereklidir. </li></ol></li><li> Kasıktaki yağ yastıklarından yağ almak için, cımbız yardımıyla 5.1 adımdan 1 mL ekstraksiyon solüsyonu (kloroform: metanol (2 mi) ile kasık yağ pedleri kesitleri (NCD veya HFD (± CAP) -fed farelerden) : 1 v /V) ve% 1 buzlu asetik asit) ve 100 rpm&#39;de şiddetli çalkalama altında 37 ° C&#39;de 60 dakika inkübe edin. Çıkarılan yağları atın. </li><li> Dokuyu (adım 5.2&#39;den) püskürtücüler kullanarak, 500 μL liziz çözeltisi (% 0.1 SDS içeren 0,3 N NaOH) içeren steril bir mikrofaj tüpüne aktarın ve 100 rpm&#39;de şiddetli çalkalama altında 55 ° C&#39;de gece boyunca (12 saat) inkübe edin. </li><li> Sırasıyla 4.3 ve 4.4 adımlarında açıklandığı gibi doku protein konsantrasyonunu ve gliserol içeriğini belirleyin. </li></ol> 6. Serbest Gliserol Reaktifinin Hazırlanması <ol><li> Gliserol reaktifi 19&#39;u , amber renkli bir cam şişede 40 mL&#39;lik deiyonize su içinde yeniden bir araya getirin, şişeye bir tıpa yerleştirin ve 10 kez ters çevirerek iyice karıştırın. Çalkalamak suretiyle karıştırmayın. </li><li> Şişeyi, alüminyum folyo ile tamamen kaplayarak, ışığa karşı korunan bir buzdolabının içine 4 ° C&#39;de saklayın. </li><li> Gliserol standardının hazırlanmasına geçin (bölüm 7). </li&gt; </ol> 7. Gliserol Standardının Hazırlanması ve Gliserol İçeriğinin Belirlenmesi <ol><li> Sağlanan stok 35 mcL deiyonize su 65 mcL seyreltilerek 1 mM stok olun. NOT: Üreticiden temin edilen gliserol standart stok 2.8 mM gliserol&#39;dür. </li><li> 1 cm uzunluğunda yol uzunluğunda tek kullanımlık metakrilat küvetler alın. Bir işaretleyiciyi 0, 1.25, 2.5, 5 ve 10 nmol standart olarak kullanarak küvetler etiketleyin. </li><li> Etiketli küvetler için 0, 1.25, 2.5, 5 ve 10 uL 1 mM gliserol standardı ekleyin. Deiyonize su ile her küvette 10 μL&#39;ye kadar hacim oluşturun. </li><li> Taze 500-μL önceden etiketlenmiş santrifüj tüplerine 90 μL deiyonize suya 10 uL örnek ekleyerek tüm örneklerin (adım 4.4 veya 5.1.1&#39;den) 1 ila 10 seyreltmesini yapın. 10 uL seyreltilmiş numuneleri ilgili numune tanımlama numarasıyla etiketlenmiş küvete ekleyin. </li><li> UV-VIS spektrofotometresini açın ve dalgayı ayarlayınUzunluğunu 540 nm&#39;ye çıkarır. </li><li> Şişeyi (adım 6.2) oda sıcaklığında 15 dakika tutarak serbest gliserol reaktifini oda sıcaklığına ısıtın. </li><li> Boş &quot;0&quot; nmol standardı (adım 7.3), standartlar (adım 7.3) ve örnekler (adım 7.4) olarak bir dizi etiketlenmiş küvet ayarlayın. </li><li> Standardın ve numunenin her birinden 10 uL, ilgili etiketli küvete ekleyin. &quot;0&quot; nmol standardını boş olarak kullanın. </li><li> Gliserol standartlarını 1 mL&#39;lik bir pipet kullanarak içeren, her bir küvete 0,8 mL serbest gliserol reaktif ekleyin. </li><li> Küveti 1.5 cm kare plastik parafin film ile örtün ve küveti 3 kez ters çevirerek karıştırın ve oda sıcaklığında 10 dakika bekletin. </li><li> Küveti UV-VIS spektrofotometresine yerleştirin ve absorbansı (dalga boyu 540 nm) kaydedin. </li><li> Standart eğrileri, X ekseni standartlarının (nmol) konsantrasyonlarını ve kaydedilen absorbansı Y ekseni ile çizin. </li><li> Gl konsantrasyonunu hesaplaÖrnek 7.12&#39;de çizilen standart eğri kullanılarak ekstrapolasyon ile örneklerde (nmol) yserol. </li><li> Seyreltme faktörü 10 (adım 7.4&#39;e bakın) ve 20 (toplam hacim) ile numunelerin konsantrasyonunu (nmol / küvet) çarpın </li><li> Her numunedeki (adım 7,13) gliserol konsantrasyonunu BCA metodu ile hesaplanan protein mg oranında bölün. NOT: Kasık yağ yastığı numuneleri 60 dakika kuluçka yapılacağından, sonuçlar gluterol / mg protein / saat olarak nmol olarak gösterilir. </li></ol>

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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. A link between regulation of the thermogenic and lipid oxidation pathways in the brown fat cell.</a> J Biol Chem. 276 (2), 1486-1493 (2001).</li><li>Leung, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsaicin+as+an+anti-obesity+drug.">Capsaicin as an anti-obesity drug.</a> Prog Drug Res. 68, 171-179 (2014).</li><li>Hursel, R., Westerterp-Plantenga, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermogenic+ingredients+and+body+weight+regulation.">Thermogenic ingredients and body weight regulation.</a> Int J Obes (Lond). 34 (4), 659-669 (2010).</li><li>Kang, J. H., 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=Dietary+capsaicin+reduces+obesity-induced+insulin+resistance+and+hepatic+steatosis+in+obese+mice+fed+a+high-fat+diet.">Dietary capsaicin reduces obesity-induced insulin resistance and hepatic steatosis in obese mice fed a high-fat diet.</a> Obesity (Silver Spring). 18 (4), 780-787 (2010).</li><li>Winkler, B., Steele, R., Altszuler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+glycerol+uptake+to+plasma+glycerol+concentration+in+the+normal+dog.">Relationship of glycerol uptake to plasma glycerol concentration in the normal dog.</a> Am J Physiol. 216 (1), 191-196 (1969).</li><li>Dugan, C. E., Cawthorn, W. P., MacDougald, O. A., Kennedy, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+microfluidic+enzyme+assays+for+simultaneous+detection+of+lipolysis+products+from+adipocytes.">Multiplexed microfluidic enzyme assays for simultaneous detection of lipolysis products from adipocytes.</a> Anal Bioanal Chem. 406 (20), 4851-4859 (2014).</li></ol>

Yazarların açıklayacak bir şeyi yok.

TGL&#39;nin gliserol ve yağ asitleri haline dönüşümü, bazal lipoliz sırasında ATGL 9 ile katalize edilir ve uyarılan lipoliz 21 , 22 , 23 sırasında adenil siklaz / PKA&#39;ya bağımlı yolağın aktivasyonu da dahil olmak üzere bir dizi protein ile düzenlenir. Lipolizin arttırılması, ulaşım ve enerji kullanımı için yağ asitlerinin plazma seviyelerini arttırır 24...

Yağ dokuları yağ 2 olarak enerji depolarlar ve termojenez 3 , 4 için yağ asidi oksidasyonu gerekir. Diyetle yutulan yağ asitleri apoproteinler ile birlikte şilomikronlara paketlenir ve kan dolaşımıyla vücuttaki farklı dokulara aktarılır. Vücuttaki çoğu hücrenin bir enerji rezervi depolamasına rağmen, yağ dokusu yağ olarak fazla enerji depolar 5 , 6 . Yağ dokusundaki lipoliz kompleks proseslerle düzenlenir ve lipolizin moleküler detayları halen belirsiz kalmaktadır 7 . Lipoliz, yağ dokusunda depolanan trigliseridlerin (TGL), yağlı trigliserid lipaz (ATGL) 8 enzimi ile gliserol ve yağ asitleri (FA) üretmek üzere hidrolizlenmesiyle oluşan bir işlemdir. Bazal ve uyarılmış lipolizdeki değişiklikler obezitenin karakteristik bir özelliğidir. BAsal lipoliz, TGL&#39;yi diasilgliserol (DAG) haline getiren, daha sonra monoasil gliserole (MAG) hidrolize edilen ATGL aktivasyonu 9 ile düzenlenir. Adenillyla siklaz yoluyla hormona duyarlı lipazın (HSL) aktivasyonu siklik adenozin monofosfat (cAMP) bağımlı protein kinaz A (PKA) uyarılmasını aktive eder ve lipolize neden olur. Bu nedenle, bazal ve uyarılmış lipoliz ölçümü, bu prosese dahil olan proteinlerin aktivitesini analiz etmek için önemlidir. Ayrıca, lipoliz moleküler düzenleme çözülüyor obezite 10 karşı yeni tedavi stratejilerinin geliştirilmesi faydalı olabilir. Lipoliz ve yağ asidi oksidasyonunu uyaran moleküller, depolarda depolanan yağları azaltmak için potansiyel aday olduğundan, tekrarlanabilirlik için sağlam bir test kullanmak önemlidir. Daha önce yayınlanmış veriler, beyaz yağ dokusunda ifade edilen TRPV1 proteininin CAP ile güçlendirilmiş bazalVe kasık yağ pedlerinde FSK (adenillyla siklaz aktivatörü) uyarılmış lipoliz 11 . Önceki araştırmalar ayrıca, TRPV1&#39;in CAP tarafından uzun süreli aktive edilmesinin PKA 12&#39;yi aktive ettiğini ileri sürmektedir. PKA&#39;nın aktivasyonu, lipozizi uyardığından , 13 , 14 , ilgili diyetlerin beslenmesinden 32 hafta sonra NCD veya HFD (± CAP) ile beslenen farelerden izole edilmiş kasık yağ pedlerinde bazal ve PKA&#39;ya bağlı uyarılmış lipolizin ölçülmesiyle TRPV1&#39;in rolü doğrulanacaktır Lipoliz aktivasyonu. Bu makale, bazal ve uyarılan lipolizin belirlenmesinde etkili bir yöntem anlatmaktadır. Gliserolün radyoaktif izotoplarını kullanan diğer yöntemler ve ölçümler 15 , 16 için sıkıcı yüksek performanslı sıvı kromatografisi veya gaz kromatografisi / kütle spektrometresi mevcut olsa da, bu yöntem daha doğrudan, basit ve uygun maliyetli bir yöntem sunmaktadırYağ dokusundaki lipolizi belirleme tekniği.

<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

Lipoliz, yağ dokularında trigliserit olarak depolanan lipitin, gliserol ve yağ asitlerine hidrolize edilmesiyle ilgili bir işlemdir. Bu makale, normal yemeklik diyet (NCD), yüksek yağlı diyet (HFD) ya da 0.01&#39;i içeren yüksek yağlı bir diyet ile beslenen vahşi tipli farelerden izole edilen kasıktaki yağ yastıklarında bazal ve forskolin (FSK) uyarılmış lipolizin ölçülmesine yönelik yöntemi açıklamaktadır % Kapsaisin (CAP; geçici reseptör potansiyeli vaniloid altfamilı 1 (TRPV1) agonisti) 32 hafta boyunca. Ex vivo lipoliz gerçekleştirmek için burada tarif edilen yöntem Schweiger ve ark. 1 UV-Görünür (UV / VIS) spektrofotometri ile gliserol düzeylerini ölçmek için ayrıntılı bir protokol sunmaktayız. Burada açıklanan yöntem, tutarlı sonuçlar elde etmek için kasık yağ pedlerinin lipoliz ölçümleri için başarıyla izole edilmesi için kullanılabilir. Kasık yağ pedleri için tanımlanan protokol, diğer dokulardaki lipolizin ölçülmesi için kolaylıkla genişletilebilir.

CAP&#39;nin bazal ve uyarılmış lipoliz üzerindeki etkisini değerlendirmek için, bu araştırma, NCD veya HFD (± CAP) -fed vahşi tipli farelerden izole edilmiş inguinal yağlı yastıklardaki lipolizi ölçtü. Kasık yağ pedleri için bazal ve FSK ile uyarılan lipoliz için temsili sonuçlar Tablo I&#39;de verilmektedir. Asil coA sentetazı inhibe eden ve TGL&#39;nin yenilenmesini önleyen Triacsin C&#39;nin varlığında bazal ve FSK ile uyarılmış gliserol...

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

Bu protokol, normal yem diyetinden (NCD) veya yüksek yağlı diyetten (HFD) ± kapsaisin ile beslenen vahşi tipli farelerde elde edilen kasık yağ tabletlerinde bazal ve forkolinin uyardığı lipoliz belirleme metodunu açıklamaktadır. Lipoliz için bir indeks olarak, inguinal yağlı yağ pedlerinden gliserol salınımı ölçülmüştür.

İnguinal Yağ Yastıklarında Basal ve Forskolin ile uyarılmış Lipoliz Ölçümleri

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

measurement-basal-forskolin-stimulated-lipolysis-inguinal-adipose-fat

Research

JoVE Journal

Biochemistry

9.5K Görüntüleme.  University of Wyoming.  Bu protokol, normal yem diyetinden (NCD) veya yüksek yağlı diyetten (HFD) ± kapsaisin ile beslenen vahşi tipli farelerde elde edilen kasık yağ tabletlerinde bazal ve forkolinin uyardığı lipoliz belirleme metodunu açıklamaktadır. Lipoliz için bir indeks olarak, inguinal yağlı yağ pedlerinden gliserol salınımı ölçülmüştür. 

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

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hidrolizat tavuk tüyleri ve kozmetik kendi uygulamasında hazırlanması

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Biyokimya

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Yalıtım ve solunum ölçümleri mitokondri Arabidopsis thaliana üzerinden

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

Süperoksit/hidrojen peroksit ve NADH üretim mitokondriyal Dehydrogenases Flavin içeren tarafından eş zamanlı ölçüm

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

Fare yağ dokusu toplama ve işleme RNA analizi için

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

Doku çok az miktarda kullanarak Drosophila Permeabilized liflerinin mitokondrial oksijen harcama ölçme noktalar

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

Situ Ölçüm ve korelasyon hücre yoğunluğu ve kollarındaki bakterilerin ışık emisyon

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

Koalak'da Kortizol Ölçümü (Phascolarctos cinereus) Kürk

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the interpretation of the biological activity of any compound in drug development or in basic research projects. In epigenetics, target engagement is most often assessed by the analysis of proxy markers instead of measuring the union of the compound to the target. Downstream biological readouts that have been analyzed include the histone mark modulation or gene expression changes. KDM1A is a lysine demethylase that removes methyl groups from mono- and dimethylated H3K4, a modification associated with the silencing of gene expression. Modulation of the proxy markers is dependent on the cell type and function of the genetic make-up of the cells investigated, which can make interpretation and cross-case comparison quite difficult. To circumvent these problems, a versatile protocol is presented to assess the dose effects and dynamics of the direct KDM1A target engagement. The assay described makes use of a KDM1A chemoprobe to capture and quantify uninhibited enzyme, can be broadly applied to cells or tissue samples without the need for genetic modification, has an excellent window of detection, and can be used both for basic research and analysis of clinical samples.

Here, we present a protocol to measure KDM1A target engagement in a human or animal cell, tissue or blood samples treated with KDM1A inhibitors. The protocol employs chemoprobe tagging of the free KDM1A enzyme and direct quantification of the target occupation using chemoprobe-based immunoassays and can be used in preclinical and clinical studies.

Chemoprobe tabanlı bağışıklık sistemini kullanarak KDM1A hedef etkileşimi doğrudan ölçümü

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

Amiloid Fibrils ile Beyin Mitokondrisi ile Etkileşimler ve Membran Permeabilizasyonu

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Rat Pankreas Dokusunun Hızlı ve Uygun Maliyetli RNA Ekstraksiyonu

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...