Este trabajo fue apoyado por el Premio de la AHA N º 15BGIA23250030, el Instituto Nacional de Ciencias Médicas Generales de los NIH bajo el número de adjudicación 8P20 GM103432-12 y la Universidad de Wyoming Beca de la Facultad de Ayuda a BT.

Todos los protocolos siguen las directrices de cuidado de animales de la Universidad de Wyoming. 1. Vivienda y Alimentación Animal NOTA: Los ratones machos adultos de tipo salvaje (C57BL / 6) (de 12 a 24 semanas de edad) fueron criados en el centro para animales de investigación de acuerdo con los protocolos aprobados por el Comité de Uso y Cuidado de Animales Institucionales (IACUC). <ol><li> A partir de la semana 6 de edad, casa ratones en grupos de cuatro en jaulas separadas y asignarlos al azar en grupos de alimentación de NCD o HFD (± 0,01% CAP) hasta la semana 38 de edad. NOTA: CAP es un agonista de la proteína del canal TRPV1 expresada en tejidos adiposos 11 , 17 . Mezcle la tapa con HFD en una licuadora dentro de la campana y transfiera la mezcla mezclada a una bandeja que contenga pequeñas particiones de 1 en 2 . Mantenga la bandeja dentro de un congelador de -20 ° C. Retire la bandeja que contiene la dieta HFD + CAP del congelador después de 24 h y guárdela en un recipiente a -20 &amp;# 176; C congelador hasta su uso. </li><li> (22,8 ± 2,0 ° C, 45 - 50% de humedad) con un ciclo de 12/12 de luz / oscuridad con acceso a la dieta designada y agua ad libitum . </li><li> Al final de 38 semanas, se disecan los tejidos adiposos inguinales y se usan para experimentos de lipólisis (secciones 2-7). </li></ol> 2. Preparación de ratones para los experimentos <ol><li> Anestesiar los ratones inyectando ketamina y xilazina (10 mg / kg y 80 mg / kg de peso corporal, respectivamente). Inyectar 0,01 ml de la mezcla / 10 g de peso corporal de ratón. </li><li> Confirme la anestesia profunda con una pinza firme del dedo del pie. Si hay un reflejo del pedal, vuelva a probar el ratón después de al menos 30 s. </li><li> Use un ungüento para ojos para prevenir la sequedad de los ojos durante la anestesia. No deje los ratones desatendidos durante ninguno de los procedimientos. </li><li> Euthanize ratones mediante la inyección de una alta dosis de ketamina y mezcla de xilazina inyección (10 mg / kg y 80 mg / kg de peso corporal, respectivamente) mixturE (0,01 ml / 10 g de peso corporal) seguido de dislocación cervical. NOTA: Este método de eutanasia es aprobado por el IACUC de la Universidad de Wyoming. </li></ol> 3. Aislamiento de las almohadillas de grasa adiposa inguinal <ol><li> Coloque el ratón (alimentado con NCD o HFD (± CAP) durante 32 semanas) acostado a su izquierda para el procedimiento. NOTA: Colocando el ratón en el lado izquierdo, la extremidad anterior izquierda y el miembro posterior izquierdo estarán en la plataforma de disección, mientras que la extremidad anterior derecha y el miembro posterior derecho estarán orientados hacia fuera de la plataforma. </li><li> Esterilizar la superficie de la piel con una almohadilla de gasa de 2 pulgadas 2 empapada en aproximadamente 2,5 ml de etanol al 70%. Haga un corte lateral de 2 - 3 mm a través de la piel usando un bisturí para revelar la capa grasa subyacente. </li><li> Haga un corte de 1 cm (dependiendo del tamaño del ratón) a través de la piel usando un bisturí justo debajo de la caja torácica a través de la superficie dorsal que une las dos incisiones laterales. </li><li> Despegue la solapa de la piel arrastrándola cuidadosamenteUsando fórceps estériles y dejar la almohadilla subcutánea intacta por no cortar las almohadillas de grasa. Esta es la almohadilla de grasa que yace bajo la piel. </li><li> Disecar la almohadilla de grasa cuidadosamente desde el músculo subyacente y la fascia con un par de tijeras. Tire de la almohadilla de grasa como se corta del músculo subyacente. NOTA: El peso y el tamaño de las almohadillas de grasa depende del tipo de ratones (NCD o HFD (± CAP) -fed). </li><li> Utilice pinzas para transferirlo a una placa de Petri que contenga solución salina tamponada con fosfato (PBS) hasta los experimentos de lipólisis, a temperatura ambiente (~ 15 min). </li><li> Aislar las almohadillas de grasa del lado derecho del ratón como se describe en los pasos 3.2-3.6. </li></ol> 4. Liberación basal de glicerol de almohadillas de grasa inguinal <ol><li> Use tijeras afiladas para cortar alrededor de 20 mg de tejido graso en 5 a 8 piezas. </li><li> Incubar las piezas cortadas de almohadillas de grasa inguinal en 200 μl de medio de incubación (Medio de Eagel Modificado de Dulbecco (DMEM) que contiene ácido graso al 2%Libre de albúmina de suero bovino (BSA)) a 37ºC, 5% de CO 2 y 95% de humedad durante 60 min. <ol><li> Recoger el medio de incubación y congelar a -80 ° C para el ensayo de lipólisis. NOTA: Las piezas cortadas se usan para determinar la concentración de proteína después de la extracción de lípidos. </li><li> Transferir las piezas de grasa cortada con la ayuda de pinzas en 1 ml de solución de extracción (cloroformo: metanol (2: 1, v / v) y 1% de ácido acético glacial) e incubar durante 60 min a 37 ° C bajo agitación vigorosa a 100ºC Rpm. Deseche la solución de extracción de grasa. NOTA: Este paso extraerá la grasa de las piezas cortadas. Deseche la solución de extracción de grasa ya que interferirá con la determinación de proteínas. </li><li> Transferir el tejido (de la etapa 4.2.2) con una pinza en un tubo estéril de microcentrífuga que contenga 500 μl de solución de lisis (NaOH 0,3 N que contiene dodecilsulfato de sodio al 0,1%) y se incuba durante la noche (12 h) a 55ºC bajo agitación vigorosa A 100 rpm. </li></ol></li><li> Determine la concentración de proteína del tejido (paso 4.2.3) usando el reactivo de ácido bicinconínico (BCA) y BSA como estándar 18 . </li><li> Descongelar el medio congelado (paso 4.2.1) sobre hielo y determinar el contenido de glicerol del medio usando un reactivo de glicerol libre como se describe en las secciones de protocolo 6 y 7 6 . <ol><li> Extrapolar la concentración de glicerol en muestras de la curva estándar trazada usando los estándares de glicerol 19 y expresar la lipólisis como nanomoles de glicerol por mg de proteína por h 20 . </li></ol></li></ol> 5. Lipolisis estimulada por FSK <ol><li> Preincuba aproximadamente 20 mg de almohadilla de grasa inguinal (5 a 8 piezas cortadas) obtenida de un ratón salvaje de NCD o HFD (± CAP) en 200 μL de DMEM que contiene BSA sin ácido graso al 2%, FSK 10 μM y Triacsina C 5 μM en 37 ° C en 5% de CO2 y 95% de humedad durante 60 min. <li> Transferir las piezas de tejido con pinzas a un medio idéntico e incubar durante otros 60 min a 37 ° C, 5% de CO 2 y 95% de humedad. Recoger el medio de incubación y almacenar a -80 ° C hasta el ensayo de lipólisis. NOTA: La estimulación de la lipólisis causa una liberación rápida de ácidos grasos y glicerol dentro de los 15 minutos, que es lineal durante la primera hora, y las mesetas a partir de entonces. Dado que la tasa de lipólisis estimulada es más alta y estable entre la primera y segunda hora de estimulación FSK, este protocolo midió la lipólisis en el estado estimulado después del segundo período de incubación como se describió anteriormente [ 1] . Por lo tanto, ambos pasos experimentales (5.1 y 5.1.1) son necesarios. </li></ol></li><li> Para extraer la grasa de las almohadillas de grasa inguinal, transferir las piezas cortadas de las almohadillas de grasa inguinal (obtenidas de ratones alimentados con NCD o HFD (± CAP) del paso 5.1 con la ayuda de pinzas en 1 ml de solución de extracción (cloroformo: metanol VV) y ácido acético glacial al 1%) e incubar durante 60 minutos a 37 ° C bajo agitación vigorosa a 100 rpm. Deseche la grasa extraída. </li><li> Transferir el tejido (de la etapa 5.2) utilizando pinzas en un tubo de microcentrífuga estéril que contiene 500 μl de solución de lisis (NaOH 0,3 N que contiene SDS al 0,1%) e incubar durante la noche (12 h) a 55ºC bajo agitación vigorosa a 100 rpm. </li><li> Determine la concentración de proteína y el contenido de glicerol del tejido, tal como se describe en los pasos 4.3 y 4.4, respectivamente. </li></ol> 6. Preparación del reactivo libre de glicerol <ol><li> Reconstituya el reactivo de glicerina 19 en 40 ml de agua desionizada en un vial de vidrio de color ámbar, coloque un tapón en el vial y mezcle bien invirtiendo 10 veces. No mezcle agitando. </li><li> Guarde el vial a 4 ° C dentro de un refrigerador protegido de la luz cubriendo el vial completamente con papel de aluminio. </li><li> Proceder a la preparación del estándar de glicerol (sección 7). </Li </ol> 7. Preparación de la Norma de Glicerol y Determinación del Contenido de Glicerol <ol><li> Hacer una reserva de 1 mM diluyendo 35 μL del stock suministrado con 65 μl de agua desionizada. NOTA: El stock de glicerol suministrado por el fabricante es 2,8 mM de glicerol. </li><li> Tome cinco cubetas de metacrilato desechable de 1 cm de longitud. Etiquetar las cubetas usando un marcador como 0, 1,25, 2,5, 5 y 10 nmol estándar. </li><li> Añadir 0, 1,25, 2,5, 5 y 10 μl de 1 mM de glicerol estándar a las respectivas cubetas marcadas. Completar el volumen de cada cubeta a 10 μl con agua desionizada. </li><li> Hacer una dilución de 1 a 10 de todas las muestras (del paso 4.4 o 5.1.1) añadiendo 10 μl de la muestra a 90 μl de agua desionizada en tubos de centrifugación pre-etiquetados de 500 μl. Añadir 10 μl de las muestras diluidas en las cubetas marcadas con el respectivo número de identificación de la muestra. </li><li> Encienda el espectrofotómetro UV-VIS y ajuste la ondaLongitud hasta 540 nm. </li><li> Calentar el reactivo de glicerina libre a temperatura ambiente manteniendo el vial (paso 6.2) a temperatura ambiente durante 15 min. </li><li> Establecer una serie de cubetas marcadas como blanco &quot;0&quot; nmol estándar (paso 7.3), normas (paso 7.3) y muestras (etapa 7.4). </li><li> Añadir 10 μl de cada uno de los estándares y de la muestra en la respectiva cubeta marcada. Utilice &quot;0&quot; nmol estándar como blanco. </li><li> Añadir 0,8 ml de reactivo de glicerol libre en cada cubeta, conteniendo los estándares de glicerol usando una pipeta de 1 ml. </li><li> Cubra la cubeta con una película de parafina de plástico cuadrada de 1,5 cm y mezcle el contenido invirtiendo la cubeta durante 3 veces y déjela a temperatura ambiente durante 10 minutos. </li><li> Colocar la cubeta en el espectrofotómetro UV-VIS y registrar la absorbancia (540 nm de longitud de onda). </li><li> Trazar la curva estándar utilizando las concentraciones de las normas (nmol) en el eje X y la absorbancia registrada en el eje Y. </li><li> Calcular la concentración de glYcerol en las muestras (nmol) por extrapolación usando la curva estándar trazado en la etapa 7.12. </li><li> Multiplicar la concentración de muestras (nmol / cuvette) con el factor de dilución 10 (véase el paso 7.4) y 20 (volumen total) </li><li> Dividir la concentración de glicerol en cada muestra (paso 7.13) por el mg de proteína calculado por el método BCA. NOTA: Dado que las muestras de almohadillas de grasa inguinal se incuban durante 60 min, representan los resultados como nmol de glicerol / mg de proteína / h. </li></ol>

<ol>
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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>

Los autores no tienen nada que revelar.

El proceso de degradación de TGL en glicerol y ácidos grasos es catalizada por ATGL 9 durante la lipólisis basal y orquestada por una serie de proteínas, incluyendo la activación de la adenilil ciclasa / PKA dependiente vía durante la lipolisis estimulada [ 21 , 22 , 23] . Aumento de la lipólisis aumenta los niveles plasmáticos de ácidos grasos para el transporte y el uso de energía [ <sup clas...

Los tejidos adiposos almacenan la energía como grasa 2 y la oxidación de ácidos grasos es necesaria para la termogénesis 3 , 4 . Los ácidos grasos ingeridos a través de las dietas se empaquetan junto con apoproteínas en quilomicrones y entregado a diferentes tejidos en el cuerpo a través de la circulación sanguínea. Aunque la mayoría de las células del cuerpo almacenan una reserva de energía, el tejido adiposo almacena el exceso de energía como grasa 5 , 6 . La lipólisis en el tejido adiposo está regulada por procesos complejos y los detalles moleculares de la lipólisis siguen siendo vagos 7 . La lipólisis es un proceso por el cual los triglicéridos (TGL) almacenados en el tejido adiposo son hidrolizados para producir glicerol y ácidos grasos (FA) por la enzima grasa triglicérida adiposa (ATGL) 8 . Alteraciones en la lipolisis basal y estimulada es una característica de la obesidad. El bAsal está regulada por la activación 9 de ATGL, que convierte TGL en diacilglicerol (DAG), que posteriormente se hidroliza en monoacilglicerol (MAG). La activación de la lipasa sensible a las hormonas (HSL) a través de la adenilil ciclasa activa la estimulación de la proteína quinasa A (PKA) dependiente del adenosina monofosfato cíclico (cAMP) y provoca la lipólisis. La medición de la lipólisis, basal y estimulada, es, por lo tanto, importante para analizar la actividad de las proteínas implicadas en este proceso. También, desentrañar la regulación molecular de la lipólisis puede ser beneficioso para desarrollar nuevas estrategias terapéuticas contra la obesidad [ 10] . Dado que las moléculas que estimulan la lipólisis y la oxidación de ácidos grasos son potenciales candidatos para disminuir las grasas almacenadas en los depósitos, es importante emplear un ensayo robusto para la reproducibilidad. Los datos publicados anteriormente sugieren que la activación de la proteína TRPV1 expresada en tejido adiposo blanco por CAP mejorada basalY FSK (activador de adenilil ciclasa) lipólisis estimulada en almohadillas de grasa inguinal [ 11] . La investigación anterior también sugiere que la activación a largo plazo de TRPV1 por PAC activa PKA [ 12] . Puesto que la activación de la PKA estimula la lipólisis 13 , 14 , la medición de la lipólisis estimulada basal y dependiente de PKA en almohadillas de grasa inguinal aisladas de ratones alimentados con NCD o HFD (± CAP) después de 32 semanas de alimentación de las dietas respectivas validará el papel de TRPV1 Activación en la lipólisis. Este artículo describe un método eficiente para determinar la lipolisis basal y estimulada. Aunque existen otros métodos que emplean isótopos radiactivos de glicerol y una cromatografía líquida de alto rendimiento tediosa o cromatografía de gases / espectrometría de masas para las mediciones 15 , 16 , este método ofrece una forma más directa, simple y rentableTécnica para determinar la lipólisis en tejidos adiposos.

<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

La lipólisis es un proceso mediante el cual el lípido almacenado como triglicéridos en los tejidos adiposos se hidroliza en glicerol y ácidos grasos. Este artículo describe el método para la medición de la lipolisis estimulada basal y forskolin (FSK) en las almohadillas de grasa inguinal aisladas de ratones de tipo salvaje alimentados con dieta normal de chow (NCD), dieta con alto contenido de grasa (HFD) % De capsaicina (CAP, transitorio receptor potencial vanilloid subfamilia 1 (TRPV1) agonista) durante 32 semanas. El método descrito aquí para realizar la lipólisis ex vivo se adopta a partir de Schweiger et al. 1 Presentamos un protocolo detallado para medir los niveles de glicerol mediante espectrofotometría UV-Visible (UV / VIS). El método descrito aquí puede utilizarse para aislar con éxito almohadillas de grasa inguinal para mediciones de lipólisis para obtener resultados consistentes. El protocolo descrito para almohadillas de grasa inguinal se puede extender fácilmente para medir la lipólisis en otros tejidos.

Para evaluar el efecto de la CAP sobre la lipólisis basal y estimulada, esta investigación midió la lipólisis en almohadillas de grasa adiposa inguinal aisladas de ratones de tipo salvaje alimentados con NCD o HFD (± CAP). Los resultados representativos de la lipolisis basal y estimulada por FSK para las almohadillas de grasa inguinal se dan en la Tabla I. La liberación de glicerol basal y estimulada por FSK en presencia de Triacsina C, que inhibe acil coA sintetas...

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

Este protocolo describe el método para determinar la lipólisis basal y estimulada por forskolina en almohadillas de grasa inguinal obtenidas de dieta normal de chow (NCD) o dieta con alto contenido de grasa (HFD) ± capsaicina alimentada con ratones de tipo salvaje. Como índice para la lipólisis, la liberación de glicerol se midió a partir de almohadillas de grasa adiposa inguinal.

Medición de la lipólisis estimulada por Basal y Forskolin en las almohadillas de grasa adiposa inguinal

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 vistas.  University of Wyoming.  Este protocolo describe el método para determinar la lipólisis basal y estimulada por forskolina en almohadillas de grasa inguinal obtenidas de dieta normal de chow (NCD) o dieta con alto contenido de grasa (HFD) ± capsaicina alimentada con ratones de tipo salvaje. Como índice para la lipólisis, la liberación de glicerol se midió a partir de almohadillas de grasa adiposa inguinal. 

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.

Preparación de hidrolizado de queratina de plumas de pollo y su aplicación en cosméticos

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

Investigación

Bioquímica

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.

Aislamiento y medidas respiratoria de las mitocondrias de Arabidopsis thaliana

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.

Medida simultánea de superóxido de Hidrógeno peróxido y la producción de NADH deshidrogenasas mitocondriales Flavin-que contiene

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.

Colección de tejido adiposo ratón y procesamiento para el análisis de ARN

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.

Medición de consumo de oxígeno mitocondrial en las fibras Permeabilized de Drosophila utilizando cantidades mínimas de tejido

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.

In Situ Medición y correlación de la densidad celular y emisión de luz de bacterias bioluminiscentes

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.

Medición de cortisol en Koala (Phascolarctos cinereus) Piel

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.

Medición directa del compromiso objetivo de KDM1A mediante inmunoensayos basados en chemoprobe

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.

Interacciones con y la permeabilización de membrana de las mitocondrias cerebrales por Amiloide Fibrils

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.

Extracción rápida y rentable de ARN de tejido pancreático de rata

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