이 작품은 보너스 번호 8P20 GM103432-12하에 NIH 일반 의학 연구소의 AHA Award No. 15BGIA23250030 및 BT 보조원 인 와이오밍 대학 (University of Wyoming Faculty Grant)에 의해 지원되었습니다.

모든 프로토콜은 와이오밍 대학교의 동물 관리 지침을 따릅니다. 1. 동물 주거 및 수유 참고 : 동물계 야생 동물 (C57BL / 6) (12 ~ 24 주령)은 기관 동물 관리 및 사용위원회 (IACUC) 승인 프로토콜에 따라 연구 동물 시설에서 자란다. <ol><li> 6 주부터 시작하여 4 마리의 그룹으로 된 쥐를 별도의 우리에 넣고 38 주까지 NCD 또는 HFD (+ 0.01 % CAP)의 수유 그룹에 무작위로 할당합니다. 참고 : CAP는 지방 조직 11 , 17 에서 발현되는 TRPV1 채널 단백질의 작용제입니다. 후드 내부의 믹서기에서 HFD와 CAP를 섞은 다음 혼합 된 혼합물을 작은 1 / 2 파티션이있는 트레이로 옮깁니다. 트레이를 -20 ° C의 냉동고 안에 보관하십시오. 24 시간 후 냉동실에서 HFD + CAP식이가 들어있는 트레이를 꺼내어 -20 &amp;사용하기 전까지 # 176; C 냉동고. </li><li> 지정된식이 요법과 물을 자유롭게 섭취 할 수있는 12 / 12-light / dark cycle의 기후 조절 환경 (22.8 ± 2.0 ° C, 45 - 50 % 습도)의 하우스 마우스. </li><li> 38 주가 끝날 때 사타구니 지방 조직을 해부하고 지방 분해 실험에 사용합니다 (2-7 절). </li></ol> 2. 실험용 마우스 준비 <ol><li> ketamine과 xylazine 혼합물 (각각 10 ㎎ / ㎏ 및 80 ㎎ / kg 체중)을 주사하여 쥐를 마취시킨다. 혼합물 0.01 mL / 마우스 10 g을 주입하십시오. </li><li> 단단한 발가락으로 깊은 마취를 확인하십시오. 페달 반사가 있으면 적어도 30 초 후에 마우스를 다시 테스트하십시오. </li><li> 마취 중 눈의 건조를 방지하기 위해 수의사 용 연고를 사용하십시오. 어떤 과정에서도 마우스를 방치하지 마십시오. </li><li> 케타민 및 자일 라진 혼합 주사 (각각 10 mg / kg 및 80 mg / kg 체중)의 높은 용량을 주입하여 마우스를 안락사하십시오.e (0.01 ml / 10 g 체중)로 자궁 경부 탈구를 시행 하였다. 참고 :이 안락사 방법은 와이오밍 대학교 IACUC의 승인을 받았습니다. </li></ol> 3. 사타구니 지방 지방 패드의 격리 <ol><li> 절차를 위해 왼쪽에 놓인 마우스 (NCD 또는 HFD (± CAP)를 32 주 동안 먹임)를 놓습니다. 참고 : 마우스를 왼쪽에 놓으면 왼쪽 forelimb와 왼쪽 hindlimb가 해부 플랫폼에 있고 오른쪽 forelimb와 오른쪽 hindlimb는 플랫폼에서 멀어집니다. </li><li> 약 2.5 mL의 70 % 에탄올에 담근 2 인치 2 거즈 패드로 피부 표면을 소독하십시오. 밑에있는 지방층을 드러내 기 위해 메스를 사용하여 피부를 2 - 3 mm 측면 절단하십시오. </li><li> 두 측면 incisions을 합치면 등면에 걸쳐 갈비뼈 케이지 바로 아래 메스를 사용하여 피부를 통해 1cm 잘라 내기 (마우스의 크기에 따라) 확인하십시오. </li><li> 피부 뚜껑을 조심스럽게 떼어서 껍질을 벗기십시오.살균 포셉을 사용하고 지방 패드를 절단하지 않고 그대로 피하 패드를 남겨주세요. 이것은 피부 아래에있는 뚱뚱한 패드입니다. </li><li> 가위를 사용하여 아래 근육과 근막에서 조심스럽게 지방 패드를 해부하십시오. 그것이 기본 근육에서 잘라 지방 패드를 당겨. 참고 : 뚱뚱한 패드의 무게와 크기는 쥐의 유형에 따라 다릅니다 (NCD 또는 HFD (± CAP) -fed). </li><li> 지방 제거 실험까지 실온 (~ 15 분)까지 인산 완충 식염수 (PBS)가 들어있는 페트리 접시에 핀셋을 사용하여 옮깁니다. </li><li> 3.2-3.6 단계에서 설명한대로 마우스의 오른쪽에서 지방 패드를 분리합니다. </li></ol> 4. 사타구니 지방으로부터의 기초 글리세롤 방출 <ol><li> 날카로운 가위로 약 20mg의 지방 조직을 5-8 개로 자릅니다. </li><li> 배양 배지 (2 % 지방산을 포함하는 Dulbecco의 수정 Eagel의 매체 (DMEM)의 200 μL에 사타구니 지방 패드의 절단 조각을 품어37 ℃, 5 % CO 2 및 95 % 습도에서 60 분 동안 무균 소 혈청 알부민 (BSA)으로 처리 하였다. <ol><li> 부화 배지를 수집하고 lipolysis 분석을 위해 -80 ° C에서 동결. 참고 : 잘라 조각은 지질 추출 후 단백질 농도의 결정에 사용됩니다. </li><li> 추출 용액 (클로로포름 : 메탄올 (2 : 1, v / v) 및 1 % 빙초산) 1 mL에 핀셋을 사용하여 잘라 낸 지방 조각을 옮기고 37 ℃에서 60 분 동안 항온 배양 한 후 100 ℃에서 격렬한 동요 rpm. 지방 추출 용액을 버린다. 참고 :이 단계는 절단 조각에서 지방을 추출합니다. 지방 추출 용액은 단백질 결정을 방해하므로 폐기하십시오. </li><li> 500 μL의 용해 용액 (0.1 % 나트륨 도데 실 설페이트가 포함 된 0.3 N NaOH; SDS)이 들어있는 살균 된 미세 튜브에 핀셋을 사용하여 (4.2.2 단계에서) 조직을 옮기고 55 ° C에서 밤새도록 배양합니다 (12 시간). 100 rpm에서. </li></ol></li><li> 조직 (단계 4.2.3) 표준으로서 18 bicinchoninic 산 (BCA) 시약 및 BSA를 사용하여 단백질 농도를 결정한다. </li><li> 얼음 냉동 매질 (단계 4.2)에 녹여 프로토콜 부 (6, 7) (6)에 기재된 바와 같이 자유 글리세롤 시약을 사용하여 매체의 글리세롤 함량을 결정한다. <ol><li> 글리세롤 표준 19을 사용하여 플로팅 된 표준 곡선에서 샘플의 글리세롤 농도를 외삽하고 20 분당 단백질 당 나노 몰 글리세롤로 지방 분해를 나타냅니다. </li></ol></li></ol> 5. FSK- 자극 된 지방 분해 <ol><li> 2 % 지방산이없는 BSA, 10 μM FSK 및 5 μM Triacsin C가 함유 된 200 μL DMEM에 NCD 또는 HFD (± CAP)가 투여 된 야생형 마우스에서 얻은 사타구니 지방 패드 (5 ~ 8 조각) 약 20 mg을 사전 인큐베이션 37 ℃ 5 % CO 2 및 95 % 습도에서 60 분. </strong&gt; <ol><li> 37 ° C, 5 % CO 2 및 95 % 습도에서 추가 60 분 동안 동일한 배지 및 부화로 핀셋을 사용하여 조직 조각을 전송합니다. lipolysis 분석 때까지 incubation 매체와 상점을 -80 ° C에서 수집하십시오. 참고 : 지방 분해의 자극은 지방산과 글리세롤의 빠른 방출을 15 분 이내에 일으키며, 처음 1 시간 동안 선형이며 이후 평지가됩니다. 자극 지질 분해 속도는 FSK 자극의 제 1 및 제 2 시간 사이에서 더 안정하기 때문에 이전에 한 바와 같이, 자극 된 상태에서이 프로토콜 측정 지질 분해 제 잠복기 다음. 따라서 실험 단계 (5.1 및 5.1.1)가 모두 필요합니다. </li></ol></li><li> 사타구니 지방 패드에서 지방을 추출하려면 추출 용액 (클로로포름 : 메탄올 (2) 1 mL)에 핀셋을 사용하여 단계 5.1의 사타구니 지방 패드 (NCD 또는 HFD (± CAP) : 1. v /v)와 1 % glacial acetic acid)에 넣고 100 rpm으로 격렬하게 흔들어 주면서 37 ℃에서 60 분간 배양한다. 추출 된 지방을 버립니다. </li><li> 100 μl에서 격렬하게 흔들어 55 ° C에서 하룻밤 (12 H) 품어과 용해 솔루션 (0.1 N SDS 함유) 용해 액 500 μL를 포함하는 멸균 microfuge 튜브에 핀셋을 사용하여 (단계 5.2에서) 조직을 전송합니다. </li><li> 단계 4.3 및 4.4에 각각 설명 된대로 조직의 단백질 농도와 글리세롤 함량을 결정합니다. </li></ol> 6. 유리 글리세롤 시약의 제조 <ol><li> 앰버 색 유리 병에 40mL의 탈 이온수에 글리세롤 시약 19 를 넣고 바이알에 마개를 놓고 10 번 뒤집어서 잘 섞는다. 흔들어서 섞지 마십시오. </li><li> 유리 병을 알루미늄 호일로 완전히 덮어서 빛으로부터 보호되는 냉장고 안에 4 ℃에서 보관하십시오. </li><li> 글리세롤 표준 준비 (섹션 7). </li&gt; </ol> 7. 글리세롤 표준의 제조 및 글리세롤 함량의 결정 <ol><li> 공급 된 물질 35 μL를 탈 이온수 65 μL로 희석하여 1 mM의 원료를 만드십시오. 참고 : 제조사가 공급 한 글리세롤 표준 원액은 2.8 mM 글리세롤입니다. </li><li> 1cm 길이의 일회용 메타 크릴 레이트 큐벳 5 개를 가져갑니다. 0, 1.25, 2.5, 5 및 10 nmol 표준으로 마커를 사용하여 큐벳에 라벨을 붙이십시오. </li><li> 각각의 표시된 cuvettes에 1mM 글리세롤 표준의 0, 1.25, 2.5, 5 및 10μL를 추가합니다. 탈 이온수로 각 큐벳의 부피를 10 μL로 만듭니다. </li><li> 신선한 500 - μL prelabel 원심 튜브에 탈 이온수 90 μL에 샘플 10 μL를 추가하여 모든 시료 (단계 4.4 또는 5.1.1에서)를 1에서 10 희석합니다. 묽게 한 견본의 10μL를 각각 견본 ID 수로 레테르를 붙인 cuvettes로 추가하십시오. </li><li> UV-VIS 분광 광도계를 켜고 물결을 설정합니다.길이는 540nm입니다. </li><li> 바이알 (단계 6.2)을 실온에서 15 분간 유지하여 유리 글리세롤 시약을 실온으로 가온시킨다. </li><li> 일련의 표지 된 큐벳을 빈 &quot;0&quot;nmol 표준 (7.3 단계), 표준 (7.3 단계) 및 시료 (7.4 단계)로 설정하십시오. </li><li> 표준 및 시료 각각 10 μL를 해당 레이블이 표시된 큐벳에 넣으십시오. &quot;0&quot;nmol 표준을 공란으로 사용하십시오. </li><li> 1 ML 피펫을 사용하여 글리세롤 표준을 포함하는 각 큐벳에 무료 글리세롤 시약 0.8 ML을 추가합니다. </li><li> 큐벳을 1.5 cm 정사각형 플라스틱 파라핀 필름으로 덮고 큐벳을 3 회 뒤집어 내용물을 혼합하고 실온에서 10 분간 정치합니다. </li><li> UV-VIS 분광 광도계에 큐벳을 놓고 흡광도 (540 nm 파장)를 기록합니다. </li><li> X 축의 기준 농도 (nmol)와 Y 축의 기록 흡광도를 사용하여 표준 곡선을 플롯합니다. </li><li> gl 농도를 계산하십시오.(7.12)에서 플롯 한 표준 곡선을 사용하여 외삽 법에 의해 샘플 (nmol)에서 결정 하였다. </li><li> 희석 배수 10 (단계 7.4 참조) 및 20 (총량)으로 시료 농도 (nmol / cuvette)를 곱합니다. </li><li> BCA 방법으로 계산 된 단백질 mg에 의해 각 시료 (단계 7.13)의 글리세롤 농도를 나눕니다. 참고 : 사타구니 지방 패드 샘플은 60 분 incubated 때문에, 글리세롤 / mg 단백질 / h의 nmol로 결과를 나타냅니다. </li></ol>

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저자는 공개 할 것이 없습니다.

글리세롤과 지방산으로의 TGL 분해 과정은 기저 지방 분해 동안 ATGL 9에 의해 촉매되고 자극 된 지방 분해 동안 아데 닐릴 사이 클라 제 / PKA 의존 경로의 활성화를 포함하는 단백질 어레이에 의해 조율된다 21 , 22 , 23 . 지방 분해의 향상은 교통 및 에너지 사용량 24 지방산의 혈장 수준을 증가?...

지방 조직은 에너지를 지방 2 로 저장하고 지방산 산화는 열 생성 3 , 4에 필요합니다. 다이어트를 통해 섭취 된 지방산은 아포 단백질과 함께 키로 마이크론으로 포장되어 혈액 순환을 통해 신체의 다른 조직으로 전달됩니다. 신체의 대부분의 세포가 에너지를 저장하지만, 지방 조직은 과도한 에너지를 지방으로 저장합니다 5 , 6 . 지방 조직에서 지방 분해는 복잡한 프로세스에 의해 규제되고 지방 분해의 분자 세부 사항은 아직 7 모호한 남아있다. 지방 분해는 지방 조직에 저장된 트리글리 세라이드 (TGL)를 가수 분해하여 효소 지방 트리글리세리드 리파아제 (ATGL) 8에 의해 글리세롤 및 지방산 (FA)을 생산하는 과정입니다. 기저 및 자극 된 지방 분해의 변형은 비만의 특징입니다. The basal lipolysis는 TGL을 diacylglycerol (DAG)로 전환시키는 ATGL 활성화 9에 의해 조절되며,이어서 DAG가 monoacyl glycerol (MAG)로 가수 분해됩니다. adenylyl cyclase 를 통한 호르몬 민감성 lipase (HSL)의 활성화는 cyclic adenosine monophosphate (cAMP) 의존성 protein kinase A (PKA) 자극을 활성화시키고 지방 분해를 일으킨다. 따라서, 지방 분해, 기초 및 자극의 측정은이 과정에 관여하는 단백질의 활성을 분석하는 데 중요합니다. 또한, lipolysis의 분자 조절을 풀어 비만 10 새로운 치료 전략을 개발하는 것이 유리할 수 있습니다. 지방 분해와 지방산 산화를 자극하는 분자는 저장소에 저장된 지방을 감소시키는 잠재적 인 후보이므로, 재현성에 대한 견고한 분석을 이용하는 것이 중요합니다. 이전에 발표 된 데이터는 CAP에 의해 백색 지방 조직에서 발현 된 TRPV1 단백질의 활성화가 기저부 강화및 사타구니 지방 패드에 FSK (adenylyl cyclase 활성제) 자극 lipolysis 11 . 이전의 연구는 또한 CAP에 의한 TRPV1의 장기간 활성화가 PKA 12를 활성화 시킨다는 것을 제안한다. PKA의 활성화가 lipolysis 13 , 14를 자극하기 때문에 NBC 또는 HFD (± CAP)가 투여 된 사료에서 32 주 후에 각 사료를 먹인 사타구니 지방 패드에서 기초 및 PKA 의존성 지방 분해를 측정 한 결과 TRPV1 지방 분해의 활성화. 이 기사는 기저 및 자극 지방 분해를 결정하는 효율적인 방법을 설명합니다. 글리세롤 지루한 고성능 액체 크로마토 그래피 측정 또는 (15, 16)를 사용할 수있는 용 기체 크로마토 그래피 / 질량 분석기의 방사성 동위 원소를 이용하는 다른 방법이 있지만,이 방법은 더 직접적 간단하고 비용 효율적인 제공지방 조직에서의 지방 분해를 측정하는 기술.

<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

지방 분해는 지방 조직에 트리글리 세라이드로 저장된 지질을 글리세롤과 지방산으로 가수 분해하는 과정입니다. 이 기사에서는 정상적인식이 요법 (NCD), 고지방식이 요법 (HFD) 또는 0.01을 포함하는 고지식식이 요법을받은 야생형 마우스에서 격리 된 사타구니 지방 패드에서 basal and forskolin (FSK) - 자극 된 지방 분해 측정 방법을 설명합니다 capsaicin (CAP; 일시적인 수용체 잠재 성 vanilloid subfamily 1 (TRPV1) agonist)을 32 주간 투여 하였다. 체외 지방 분해를 수행하기 위해 여기에 기술 된 방법은 Schweiger et al. 1 UV-Visible (UV / Vis) 분광 광도계로 글리세롤 농도를 측정하기위한 상세한 프로토콜을 제시합니다. 여기에 설명 된 방법은 일관된 결과를 얻기 위해 지질 분해 측정을 위해 사타구니 지방 패드를 성공적으로 분리하는 데 사용할 수 있습니다. 사타구니 지방 패드에 설명 된 프로토콜을 다른 조직에서 지방 분해를 측정하기 위해 쉽게 확장 할 수 있습니다.

기초 및 자극 lipolysis에 대한 CAP의 효과를 평가하기 위해,이 연구는 NCD 또는 HFD (± CAP)가 투여 된 야생형 마우스로부터 격리 된 사타구니 지방 지방 패드에서 지방 분해를 측정했습니다. 사타구니 지방 패드에 대한 기초 및 FSK 자극 lipolysis에 대한 대표 결과는 표 1 에 나와 있습니다. 아실 CoA 신테 타제를 억제하고 TGL의 재생을 방지하는 Triacsin C의 ?...

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

이 프로토콜은 일반식이 다이어트 (NCD) 또는 고지 방식 (HFD) ± 캡사이신 야생 동물 먹이에서 얻은 사타구니 지방 패드에서 기초 및 forskolin - 자극 lipolysis를 결정하는 방법을 설명합니다. 지방 분해 지수는 사타구니 지방 지방 패드에서 글리세롤 방출을 측정했습니다.

사타구니 지방 지방 패드에서 기저 및 포스 콜린 자극 된 지방 분해의 측정

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 조회수.  University of Wyoming.  이 프로토콜은 일반식이 다이어트 (NCD) 또는 고지 방식 (HFD) ± 캡사이신 야생 동물 먹이에서 얻은 사타구니 지방 패드에서 기초 및 forskolin - 자극 lipolysis를 결정하는 방법을 설명합니다. 지방 분해 지수는 사타구니 지방 지방 패드에서 글리세롤 방출을 측정했습니다. 

비디오: 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.

각 질의 닭 깃털 및 화장품에의 응용에서의 준비

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

연구

생화학

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.

절연 및 호흡 측정의 미토 콘 드리 아 애기 thaliana 에서

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.

초과/수소 과산화 수소와 NADH 생산 미토 콘 드리 아 Dehydrogenases 플 라빈을 포함 하 여 동시 측정

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.

마우스 지방 조직 수집 및 RNA 분석에 대 한 처리

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.

조직의 최소 금액을 사용 하 여 초파리의 Permeabilized 섬유에 미토 콘 드리 아 산소 소비량의 측정

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.

제자리에서 측정 및 셀 밀도의 상관 관계 및 발광 박테리아의 발광

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.

코알라의 코티솔 측정(파스콜아크토시네레우스)모피

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.

화학 프로브 기반 면역 분석제사용 KDM1A 표적 결합의 직접 측정

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.

아밀로이드 피브릴스뇌 미토콘드리아의 상호 작용 및 막 관과화

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.

쥐 췌장 조직의 신속하고 비용 효율적인 RNA 추출

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