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 mitochondria as acetyl CoA, which is used to produce energy.
The degree and extent of lipolysis can efficiently be measured by glycerol release from adipose tissues (Table 1 and Figure 1). Hydrolysis of TGL produces fatty acids and glycerol. Previous research showed that dietary supplementation of CAP significantly enhanced the basal as well as FSK-stimulated glycerol release from inguinal and brown fat pads11,17. The increased production of fatty acids from enhanced lipolysis could serve as feed to produce heat and thermogenesis since CAP also significantly increased the expression of mitochondrial UCP-1 in the inguinal fat pads. Consistent to this notion, previous research suggests that CAP enhanced the expression of peroxisome proliferator activated receptor alpha (PPAR&#945;) in inguinal fat pads, which plays a critical role in the mitochondrial fatty acid oxidation and in the transcriptional upregulation of UCP-125,26,27. Consequently, CAP also increased metabolic activity, heat production and energy expenditure in HFD-fed mice28,29. Further, HFD feeding suppressed stimulated lipolysis while CAP increased basal and FSK-stimulated PKA-dependent lipolysis as well as enhanced insulin sensitivity30. Therefore, as described by the model in Figure 2, it is reasonable to interpret that CAP increases basal and stimulated lipolysis without causing insulin resistance as the excess fat is burned to produce heat by mitochondrial UCP-1 upregulation.
This article describes the method of measuring glycerol release from white adipose tissues. Although several methods using stable isotopes of glycerol are available, they require either high performance liquid chromatography or gas chromatography/mass spectrometry for measurements15,16. Also, glycerol radio-tracer measurements are associated with non-specificities31. Alternate methods include determining mRNA and protein levels of lipases and regulatory proteins involved in lipolysis. Colorimetric methods are also available to determine the concentration of long chain fatty acids in circulation. The method described here is very efficient in analyzing glycerol release from adipose tissues as it can be done using a plate reader capable of fluorescence and absorbance measurement. Also, the simplified analytical method for glycerol release described in this article provides improved stability and precision32.
Standard operating procedures should be practiced as this method involves surgical techniques to isolate fat pads. This protocol recommends the use of fat free BSA. This may cause air bubble formation, which will interfere with absorbance reading. Therefore, care should be taken while handling samples to prevent air bubbles. Precaution should be taken not to shake or invert samples during BSA treatment, unless such a step is recommended in the protocol. Also, the fat extraction step from inguinal fat is critical since the presence of fat will interfere with inguinal adipose tissue protein determination. Additionally, during the preparation of glycerol standards, it is important to mix the contents by inversion and not shake the vial.
The method described here can be used to successfully isolate inguinal fat pads for several assays including lipolysis measurements. The lipolysis protocol described for inguinal fat pads can readily be extended to the measurement of lipolysis in other tissues. The methods are optimized for all kinds of fat pads, as well as preadipocytes.
In summary, this article describes the method for measuring basal and FSK-stimulated glycerol release in inguinal adipose fat pads isolated from NCD or HFD (&#177; CAP)-fed mice. The presented data suggest that TRPV1 activation by CAP increased the basal and FSK-stimulated lipolysis and prevented the accumulation of lipids in the adipose tissues. These data demonstrate a critical role of TRPV1 protein in the regulation of lipolysis in white adipose tissue.

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><tbody><tr><td>Capsaicin</td><td>Sigma, USA</td><td>M2028</td><td>TRPV1 agonist</td></tr><tr><td>Forskolin</td><td>Sigma, USA</td><td>F6886</td><td>Adenylyl cyclase activator</td></tr><tr><td>DMEM</td><td>GE healthcare and life sciences, UT, USA</td><td>SH30081.01</td><td></td></tr><tr><td>High fat diet</td><td>Research diets, New Brunswick, USA</td><td>D12492</td><td>Abbreviated as HFD</td></tr><tr><td>Tris</td><td>Amresco, USA</td><td>O497</td><td></td></tr><tr><td>Sodium chloride</td><td>Thermofisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma, USA</td><td>D6750</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma, USA</td><td>D9163</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>Sigma, USA</td><td>S6508</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma, USA</td><td>P8340</td><td></td></tr><tr><td>Free Glycerol reagent</td><td>Sigma USA</td><td>F6428</td><td></td></tr><tr><td>DMSO</td><td>Sigma, USA</td><td>D8779</td><td></td></tr><tr><td>Triacsin C</td><td>Sigma, USA</td><td>T4540</td><td>Acyl CoA transferase inhibitor</td></tr><tr><td>Bovine serum albumin</td><td>Sigma, USA</td><td>A7030</td><td></td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>31998-8</td><td></td></tr><tr><td>Methanol</td><td>Thermofisher Scientific, USA</td><td>A412-1</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Amresco, USA</td><td>O583</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma, USA</td><td>L3371</td><td></td></tr><tr><td>Bicinchoninic acid reagent</td><td>Sigma, USA</td><td>BCA1-1KT</td><td></td></tr><tr><td>UV-VIS Spectrophotometer</td><td>Pharmacia Biotech, NJ, USA</td><td>Ultrospec 2000</td><td></td></tr><tr><td>Normal chow diet</td><td>Labdiet.com</td><td>500I</td><td>abreviated as NCD</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Laboratory, CT, USA</td><td>Stock number000664</td><td>wild type mice</td></tr><tr><td>Parafilm</td><td>Heathrow Scientific, USA</td><td>HS 234526A</td><td></td></tr><tr><td>Glycerol standard</td><td>Sigma, USA</td><td>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 Figure 1, HFD suppressed the FSK-stimulated lipolysis, and CAP increased both basal and FSK-stimulated lipolysis. Triacsin C was used to inhibit the regeneration of TGL from glycerol and fatty acids11. All data are expressed as means &#177; S.E.M. Comparisons between groups are analyzed using one-way ANOVA and post hoc analyses were performed using Student&#39;s t test. Sample sizes are set to determine whether the mean value of an outcome variable in one group differed significantly from that in another group. A p-value &#60; 0.05 is considered as statistically significant.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55625/55625fig1.jpg" /> 
Figure 1: CAP Increases Basal and FSK-stimulated Glycerol Release in Inguinal Fat Pads. Bar graphs represent the mean &#177; S.E.M. of basal and FSK (10 &#181;M)-stimulated glycerol release (nmol/mg protein/h) in the inguinal fat pads isolated from NCD or HFD (&#177; CAP)-fed wild type mice. **Represents statistical significance for p-value &#60;0.05 for n = 6. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55625/55625fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55625/55625fig2.jpg" /> 
Figure 2: Hydrolysis of Triglycerides (TGL) Results in the Generation of Free Fatty Acids (FFA) and Glycerol. FFA undergoes mitochondrial &#946;-oxidation. This, along with the upregulation of uncoupling protein 1 (UCP-1) stimulates thermogenesis. Glycerol release is measured by the protocol described in the article. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55625/55625fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lipolysis 
			(nmoles glycerol /mg of protein/h)</td>
			<td>NCD 
			(n = 6)</td>
			<td>HFD 
			(n = 6)</td>
			<td>HFD + CAP 
			(n = 6)</td>
		</tr>
		<tr>
			<td>Basal</td>
			<td>16.22 &#177; 1.28</td>
			<td>17.16 &#177; 1.48</td>
			<td>52.07 &#177; 2.66</td>
		</tr>
		<tr>
			<td>FSK Stimulated 
			(with Triacsin C)</td>
			<td>54.88 &#177; 3.27</td>
			<td>41.23 &#177; 4.43</td>
			<td>72.04 &#177; 4.16</td>
		</tr>
	</tbody>
</table>
Table 1: Effect of CAP on Basal and FSK Stimulated Lipolysis in Inguinal Fat Pads. Mean basal and FSK-stimulated glycerol release &#177; S.E.M measured in inguinal fat adipose tissue obtained from NCD, HFD or HFD + CAP-fed wild type mice.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.6K Views.  University of Wyoming.  Dit protocol beschrijft de methode voor het bepalen van basale en forskolin gestimuleerde lipolyse in inguineuze vetkussens die worden verkregen uit normale Chow Diet (NCD) of met hoge vet dieet (HFD) ± capsaicine gevoed wild type muizen. Als een index voor lipolyse werd de glycerol-afgifte gemeten van ingewanden van adipose vetpads. 

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

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1&#945; protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of ...

Developmental Biology

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

Behavior

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Medicine

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Immunology and Infection

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Biology

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium (2H)-Labeling Approach

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat mass or distribution are regulated by changes in the synthesis and breakdown (i.e., turnover) of adipose cells and triacylglycerols. Evidence suggests that the manner and magnitude of subcutaneous adipose tissue expansion (i.e., hypertrophy vs. hyperplasia) and turnover can influence metabolic health, as adipogenesis has been implicated in the pathogenesis of obesity and related diseases. Despite the potential role of adipose turnover in human health, there is a lack of knowledge about the in vivo kinetics of adipose cells. This is due, in part, to the slow turnover rate of the cells in adipose tissue and the practical complexity of directly labeling their metabolic precursors in vivo. Herein, we describe methods to measure in vivo adipose kinetics and turnover rates in humans through the consumption of deuterium (2H)-labeled water. The incorporation of 2H into the deoxyribonucleotide moieties of DNA in pre-adipocytes and adipocytes provides an accurate measure of cell formation and death (adipose turnover). Overall, this is an innovative approach to measuring in vivo adipose kinetics and represents a substantive departure from other in vitro assessments.

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium 2H-Labeling Approach

Presented here is a protocol to measure in vivo adipose tissue kinetics in humans using the deuterium (2H)-labeling method.

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat ...

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

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