This work was supported by the AHA Award No. 15BGIA23250030, the National Institute of General Medical Sciences of the NIH under award number 8P20 GM103432-12 and the University of Wyoming Faculty Grant in Aid to BT.

All protocols follow the animal care guidelines of the University of Wyoming.
1. Animal Housing and Feeding
NOTE: Adult male wild type mice (C57BL/6) (age 12 to 24 weeks) were bred in the research animal facility as per the Institutional Animal Care and Use Committee (IACUC) approved protocols.
<ol>
	<li>Starting from week 6 of age, house mice in groups of four in separate cages and randomly assign them into feeding groups of NCD or HFD (&#177; 0.01% CAP) until week 38 of age. 
	NOTE: CAP is an agonist of TRPV1 channel protein expressed in adipose tissues11,17. Mix CAP with HFD in a blender inside the hood and transfer the blended mixture to a tray containing small 1 in2 partitions. Keep the tray inside a -20 &#176;C freezer. Remove the tray containing the HFD + CAP diet from the freezer after 24 h and store in a container at -20 &#176;C freezer until use.</li>
	<li>House mice in a climate-controlled environment (22.8 &#177; 2.0 &#176;C, 45 - 50% humidity) with a 12/12-light/dark cycle with access to designated diet and water ad libitum.</li>
	<li>At the end of 38 weeks, dissect inguinal adipose tissues and use for lipolysis experiments (sections 2-7).</li>
</ol>
2. Preparing Mice for the Experiments
<ol>
	<li>Anesthetize mice by injecting ketamine and xylazine mixture (10 mg/kg and 80 mg/kg body weight, respectively). Inject 0.01 mL of the mixture/10 g body weight of mouse.</li>
	<li>Confirm deep anesthesia by a firm toe pinch. If there is a pedal reflex, test the mouse again after at least 30 s.</li>
	<li>Use vet eye ointment to prevent dryness of eyes during anesthesia. Do not leave mice unattended during any of the procedures.</li>
	<li>Euthanize mice by injecting a high dose of ketamine and xylazine mixture injection (10 mg/kg and 80 mg/kg body weight, respectively) mixture (0.01 ml/10 g body weight) followed by cervical dislocation. 
	NOTE: This method of euthanasia is approved by the IACUC of the University of Wyoming.</li>
</ol>
3. Isolation of Inguinal Adipose Fat Pads
<ol>
	<li>Place the mouse (fed with NCD or HFD (&#177; CAP) for 32 weeks) lying on its left for the procedure. 
	NOTE: By placing the mouse on the left side, the left forelimb and left hindlimb will be on the dissection platform, while the right forelimb and right hindlimb will be facing away from the platform.</li>
	<li>Sterilize the skin surface with a 2 inch2 gauze pad soaked in about 2.5 mL of 70% ethanol. Make a 2 - 3 mm lateral cut through the skin using a scalpel to reveal the underlying fatty layer.</li>
	<li>Make a 1 cm cut (depending on the size of the mouse) through the skin using a scalpel just below the rib cage across the dorsal surface joining the two lateral incisions.</li>
	<li>Peel the skin flap by dragging it carefully using sterile forceps and leave the subcutaneous pad intact by not cutting the fat pads. This is the fat pad lying under the skin.</li>
	<li>Dissect the fat pad carefully from the underlying muscle and fascia using a pair of scissors. Pull the fat pad as it is cut from the underlying muscle. 
	NOTE: The weight and size of the fat pads depends on the type of mice (NCD or HFD (&#177; CAP)-fed).</li>
	<li>Use tweezers to transfer it to a Petri dish containing phosphate buffered saline (PBS) until the lipolysis experiments, at room temperature (~15 min).</li>
	<li>Isolate the fat pads from the right side of the mouse as described in steps 3.2-3.6.</li>
</ol>
4. Basal Glycerol Release from Inguinal Fat Pads
<ol>
	<li>Use sharp scissors to cut about 20 mg of fat tissue into 5 to 8 pieces.</li>
	<li>Incubate the cut pieces of inguinal fat pads in 200 &#181;L of incubation medium (Dulbecco&#39;s Modified Eagel&#39;s Medium (DMEM) containing 2% fatty acid free bovine serum albumin (BSA)) at 37 &#176;C, 5% CO2 and 95% humidity for 60 min.
	<ol>
		<li>Collect the incubation medium and freeze at -80 &#176;C for lipolysis assay. 
		NOTE: The cut pieces are used for determination of protein concentration after lipid extraction.</li>
		<li>Transfer the cut fat pieces with the help of tweezers into 1 mL of extraction solution (chloroform:methanol (2:1, v/v) and 1% glacial acetic acid) and incubate for 60 min at 37 &#176;C under vigorous shaking at 100 rpm. Discard the fat extraction solution. 
		NOTE: This step will extract fat from the cut pieces. Discard the fat extraction solution as it will interfere with the protein determination.</li>
		<li>Transfer the tissue (from step 4.2.2) using tweezers into a sterile microfuge tube containing 500 &#181;L of lysis solution (0.3 N NaOH containing 0.1% sodium dodecyl sulfate; SDS) and incubate overnight (12 h) at 55 &#176;C under vigorous shaking at 100 rpm.</li>
	</ol>
	</li>
	<li>Determine the protein concentration of tissue (step 4.2.3) using the bicinchoninic acid (BCA) reagent and BSA as standard18.</li>
	<li>Thaw the frozen medium (step 4.2.1) on ice and determine the glycerol content of the medium using a free glycerol reagent as described in protocol sections 6 and 76.
	<ol>
		<li>Extrapolate the concentration of glycerol in samples from the standard curve plotted using glycerol standards19 and express lipolysis as nanomoles glycerol per mg protein per h20.</li>
	</ol>
	</li>
</ol>
5. FSK-stimulated lipolysis
<ol>
	<li>Preincubate about 20 mg of inguinal fat pad (5 to 8 cut pieces) obtained from NCD or HFD (&#177; CAP)-fed wild type mouse in 200 &#181;L DMEM containing 2% fatty acid free BSA, 10 &#181;M FSK and 5 &#181;M Triacsin C at 37 &#176;C in 5% CO2 and 95% humidity for 60 min.
	<ol>
		<li>Transfer the tissue pieces using tweezers to an identical medium and incubate for a further 60 min at 37 &#176;C, 5% CO2 and 95% humidity. Collect the incubation medium and store at -80 &#176;C until the lipolysis assay. 
		NOTE: Stimulation of lipolysis causes a rapid release of fatty acids and glycerol within 15 min, which is linear during the first hour, and plateaus thereafter. Since the rate of stimulated lipolysis is higher and stable between the first and second hour of FSK stimulation, this protocol measured lipolysis in the stimulated state following the second incubation period as described previously1. So, both the experimental steps (5.1 and 5.1.1) are necessary.</li>
	</ol>
	</li>
	<li>To extract fat from inguinal fat pads, transfer the cut pieces of inguinal fat pads (obtained from NCD or HFD (&#177; CAP)-fed mice) from step 5.1 with the help of tweezers into 1 mL of extraction solution (chloroform:methanol (2:1. v/v) and 1% glacial acetic acid) and incubate for 60 min at 37 &#176;C under vigorous shaking at 100 rpm. Discard the extracted fat.</li>
	<li>Transfer the tissue (from step 5.2) using tweezers into a sterile microfuge tube containing 500 &#181;L of lysis solution (0.3 N NaOH containing 0.1% SDS) and incubate overnight (12 h) at 55 &#176;C under vigorous shaking at 100 rpm.</li>
	<li>Determine the protein concentration and glycerol content of tissue as described in steps 4.3 and 4.4, respectively.</li>
</ol>
6. Preparation of Free Glycerol Reagent
<ol>
	<li>Reconstitute the glycerol reagent19 in 40 mL of deionized water in an amber colored glass vial, place a stopper on the vial and mix well by inverting 10 times. Do not mix by shaking.</li>
	<li>Store the vial at 4 &#176;C inside a refrigerator protected from light by covering the vial completely with aluminum foil.</li>
	<li>Proceed to the preparation of glycerol standard (section 7).</li>
</ol>
7. Preparation of Glycerol Standard and Determination of Glycerol Content
<ol>
	<li>Make a stock of 1 mM by diluting 35 &#181;L of the supplied stock with 65 &#181;L of deionized water. 
	NOTE: The manufacturer supplied glycerol standard stock is 2.8 mM glycerol.</li>
	<li>Take five 1-cm path length disposable methacrylate cuvettes. Label the cuvettes using a marker as 0, 1.25, 2.5, 5 and 10 nmol standard.</li>
	<li>Add 0, 1.25, 2.5, 5 and 10 &#181;L of 1 mM glycerol standard to the respective labeled cuvettes. Make up the volume of each cuvette to 10 &#181;L with deionized water.</li>
	<li>Make 1 to 10 dilution of all samples (from step 4.4 or 5.1.1) by adding 10 &#181;L of the sample to 90 &#181;L of deionized water into fresh 500-&#181;L prelabeled centrifuge tubes. Add 10 &#181;L of the diluted samples into the cuvettes labeled with the respective sample identification number.</li>
	<li>Switch on the UV-VIS spectrophotometer and set the wavelength to 540 nm.</li>
	<li>Warm the free glycerol reagent to room temperature by keeping the vial (step 6.2) at room temperature for 15 min.</li>
	<li>Set up a series of labelled cuvettes as blank &#34;0&#34; nmol standard (step 7.3), standards (step 7.3) and samples (step 7.4).</li>
	<li>Add 10 &#181;L of each of the standard and sample into the respective labeled cuvette. Use &#34;0&#34; nmol standard as blank.</li>
	<li>Add 0.8 mL of free glycerol reagent into each cuvette, containing the glycerol standards using a 1 mL pipette.</li>
	<li>Cover the cuvette with a 1.5 cm square plastic paraffin film and mix the contents by inverting the cuvette for 3 times and set aside at room temperature for 10 min.</li>
	<li>Place the cuvette in the UV-VIS spectrophotometer and record the absorbance (540 nm wavelength).</li>
	<li>Plot the standard curve using the concentrations of the standards (nmol) in X-axis and the recorded absorbance in the Y-axis.</li>
	<li>Calculate the concentration of glycerol in the samples (nmol) by extrapolation using the standard curve plotted in step 7.12.</li>
	<li>Multiply the concentration of samples (nmol/cuvette) with the dilution factor 10 (refer to step 7.4) and 20 (total volume)</li>
	<li>Divide the concentration of glycerol in each sample (step 7.13) by the mg of protein calculated by the BCA method. 
	NOTE: Since the inguinal fat pad samples are incubated for 60 min, represent the results as nmol of glycerol/mg protein/h.</li>
</ol>

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The authors have nothing to disclose.

The breakdown process of TGL into glycerol and fatty acids is catalyzed by ATGL9 during basal lipolysis and orchestrated by an array of proteins including the activation of adenylyl cyclase/PKA-dependent pathway during stimulated lipolysis21,22,23. Enhancement of lipolysis increases the plasma levels of fatty acids for transportation and energy use24. Fatty acids are taken up by mi...

Adipose tissues store energy as fat2 and fatty acid oxidation is required for thermogenesis3,4. Fatty acids ingested through diets are packaged along with apoproteins into chylomicrons and delivered to different tissues in the body via blood circulation. Although most cells in the body store a reserve of energy, adipose tissue stores excess energy as fat5,6. Lipolysis in adipose tissue is regulated by complex processes and the molecular details of lipolysis still remain vague7.
Lipolysis is a process by which triglycerides (TGL) stored in adipose tissue are hydrolyzed to produce glycerol and fatty acids (FA) by the enzyme adipose triglyceride lipase (ATGL)8. Alterations in basal and stimulated lipolysis is a characteristic feature of obesity. The basal lipolysis is regulated by ATGL activation9, which converts TGL to diacylglycerol (DAG), that is subsequently hydrolyzed to monoacyl glycerol (MAG). Activation of hormone sensitive lipase (HSL) via adenylyl cyclase activates cyclic adenosine monophosphate (cAMP) dependent protein kinase A (PKA) stimulation and causes lipolysis. Measurement of lipolysis, basal and stimulated, is, therefore, important to analyze the activity of proteins involved in this process. Also, unraveling the molecular regulation of lipolysis may be beneficial to develop novel therapeutic strategies against obesity10. Since, molecules that stimulate lipolysis and fatty acid oxidation are potential candidates for decreasing fats stored in depots, it is important to employ a robust assay for reproducibility.
Previously published data suggest that activation of TRPV1 protein expressed in white adipose tissue by CAP enhanced basal and FSK (adenylyl cyclase activator)-stimulated lipolysis in inguinal fat pads11. Previous research also suggests that long-term activation of TRPV1 by CAP activates PKA12. Since the activation of PKA stimulates lipolysis13,14, measuring both basal and PKA-dependent stimulated lipolysis in inguinal fat pads isolated from NCD or HFD (&#177; CAP)-fed mice after 32 weeks of feeding the respective diets will validate the role of TRPV1 activation in lipolysis.
This article describes an efficient method of determining basal and stimulated lipolysis. Although other methods that employ radioactive isotopes of glycerol and tedious high performance liquid chromatography or gas chromatography/mass spectrometry for measurements15,16 are available, this method offers a more direct, simple and cost effective technique to determine lipolysis in adipose tissues.

<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

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes the method for the measurement of basal and forskolin (FSK)-stimulated lipolysis in the inguinal fat pads isolated from wild type mice fed either normal chow diet (NCD), high fat diet (HFD) or a high fat diet containing 0.01% of capsaicin (CAP; transient receptor potential vanilloid subfamily 1 (TRPV1) agonist) for 32 weeks. The method described here for performing ex vivo lipolysis is adopted from Schweiger et al.1 We present a detailed protocol for measuring glycerol levels by UV-Visible (UV/VIS) spectrophotometry. The method described here can be used to successfully isolate inguinal fat pads for lipolysis measurements to obtain consistent results. The protocol described for inguinal fat pads can readily be extended to measure lipolysis in other tissues.

To evaluate the effect of CAP on basal and stimulated lipolysis, this research measured lipolysis in inguinal adipose fat pads isolated from NCD or HFD (&#177; CAP)-fed wild type mice. The representative results for the basal and FSK-stimulated lipolysis for the inguinal fat pads are given in Table I. Basal and FSK-stimulated glycerol release in the presence of Triacsin C, which inhibits acyl coA synthetase and prevents the regeneration of TGL. As shown in <strong class="...

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

This protocol describes the method of determining basal and forskolin-stimulated lipolysis in inguinal fat pads obtained from normal chow diet (NCD) or high fat diet (HFD) &plusmn; capsaicin fed wild type mice. As an index for lipolysis, glycerol release was measured from inguinal adipose fat pads.

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

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Research

JoVE Journal

Biochemistry

9.5K Views.  University of Wyoming. This protocol describes the method of determining basal and forskolin-stimulated lipolysis in inguinal fat pads obtained from normal chow diet (NCD) or high fat diet (HFD) ± capsaicin fed wild type mice. As an index for lipolysis, glycerol release was measured from inguinal adipose fat pads.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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