Name: measuring the rate of lipolysis in ex vivo murine adipose tissue and primary preadipocytes differentiated in vitro
Uploaded: 2023-03-17T13:00:00
Description: Watch this Scientific Journal Video about Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro at JoVE.com

This work was supported by the US National Institutes of Health grant R01DK126944 to S.M.R.

The use of all animals was approved by the Institutional Animal Care and Use Committee (IACUC) at Weill Cornell Medical College of Cornell University.
1. Preparation of buffers and collection plates
<ol>
	<li>Make 5% bovine serum albumin (BSA) by dissolving 5 g of BSA in 100 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) without phenol red. Gently stir the BSA to dissolve (shaking is counterproductive). Once the BSA is fully dissolved, filter-sterilize the media wit...

Here, we provide a basic protocol for measuring the rate of lipolysis in adipocytes and ex vivo adipose tissue. To quantify lipolysis, it is important to measure lipolytic rate in the linear phase. We use a serial sampling technique, where a large fraction of media is collected and replaced with fresh media at regular intervals. This semiconservative method allows for the addition of fresh BSA with FFA buffering capacity and delays feedback inhibition, extending the duration of linear lipolysis. This experimenta...

Excess nutrients are stored in white adipose tissue in the form of triglycerides in the neutral lipid core of lipid droplets. Triglyceride stores are mobilized via lipolysis, a process by which the fatty acid side chains are sequentially cleaved by adipose tissue triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase (MGL), resulting in the release of free fatty acids (FFAs) and the glycerol backbone1,2. Lipolysis is activated by catecholamine signaling in the adipose tissue. Sympathetic nerve terminals locally release catecholamines, which bind to &#946;-adrenergic receptors on the adipocyte plasma membrane. Upon ligand binding, these G-protein coupled receptors (GPCRs) activate adenylyl cyclase via G&#945;s. Subsequent activation of protein kinase A (PKA) by cAMP results in the upregulation of both ATGL and HSL. The phosphorylation of perilipin-1 by PKA causes the dissociation of ABHD5 (also known as CGI-58), which binds and coactivates ATGL3. PKA directly phosphorylates HSL, promoting its translocation from the cytosol to the lipid droplet, where interaction with phosphorylated perilipin-1 further promotes its lipase activity4,5,6,7. The third lipase involved in lipolysis, MGL, does not appear to be regulated by catecholamine signaling8. Importantly, triglyceride synthesis in adipocytes is mediated by the glycerol lipid synthesis pathway, which does not involve the formation of monoglycerides as an intermediate; instead, glycerol-3-phosphate acyl transferases catalyze the formation of lysophosphatidic acid, which is combined with another fatty acyl-CoA to form phosphatidic acid, and then isomerized to diglycerides before the final synthesis of triglycerides (Figure 1)9,10,11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65106/65106fig01.jpg" /> 
Figure 1: Lipolysis and glycerol lipid synthesis pathways. Top: Lipolytic pathway; enzymes shown in red: adipose tissue triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and monoglyceride lipase (MGL). Bottom: glycerol lipid synthesis pathway; enzymes shown in green: diglyceride acyltransferase (DGAT), phosphatidic acid phosphatase (PAP), lysophosphatidic acid acyltransferase (LPAT, also known as LPAATs), and glycerol-3-phosphate acyltransferase (GPAT). Lipids: triglyceride (TG), diglyceride (DG), monoglyceride (MG), free fatty acid (FFA), fatty acyl-CoA (FA-CoA), lysophosphatidic acid (LPA), and phosphatidic acid (PA). Other metabolites: inorganic phosphate (Pi) and glycerol 3-phosphate (G3P). <a href="https://www.jove.com/files/ftp_upload/65106/65106fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Extracellular adenosine is another important regulator of lipolysis, working through Gs- and Gi-coupled GPCRs to impact adenyl cyclase activity. The predominant adenosine receptor in adipocytes, ADORA1, inhibits adenylyl cyclase, and thus lipolysis through the activation of Gi12. Expressed at lower levels, and primarily in brown adipocytes, ADORA2A activates lipolysis via Gs signaling13. ADORA1 impacts both basal lipolysis and the response to adrenergic agonists. The effect of adenosine on lipolysis can be controlled by adding adenosine deaminase to neutralize adenosine, as well as the ADORA1-specific agonist phenylisopropyladenosine14,15. Hormonal activation of Gq-coupled GPCRs can also affect lipolysis via the activation of phospholipase C and protein kinase C16,17,18,19. Inflammatory signals also impact lipolytic rates. TLR4 activation by LPS (and other endotoxins) increases the lipolytic rate by activating ERK, which phosphorylates perilipin-1 and HSL20. TNF-&#945; also activates lipolysis via ERK and NF-&#954;B activation, as well as transcriptional downregulation of the phosphodiesterase PDE-3B and CIDEC21,22,23. IL-6 has also been associated with increased adipocyte lipolysis, especially in mesenteric adipose tissue, whose FFA release impacts hepatic steatosis and gluconeogenesis24,25,26.
Lipolysis is suppressed during the fed state by insulin. AKT phosphorylates and activates PDE-3B to suppress cAMP signaling and prevent PKA activation27. Insulin also transcriptionally downregulates ATGL28. Obesity promotes catecholamine resistance through a variety of mechanisms, including the downregulation of &#946;-adrenergic receptors in adipocytes29,30,31,32,33. Adipocytes express all three &#946;-adrenergic receptors (&#946;-1, &#946;-2, and &#946;-3). While &#946;-1 and &#946;-2 adrenergic receptors are ubiquitously expressed, the &#946;-3 adrenergic receptor is predominately expressed in adipocytes in mice34,35. Adrb3 expression is induced by C/EBP&#945; during adipogenesis36. The &#946;-3 adrenergic receptor is highly expressed in mature adipocytes. The activation of &#946;-1 and &#946;-2 adrenergic receptors is self-limiting due to feedback inhibition by &#946;-arrestin37. Feedback inhibition of the &#946;-3 adrenergic receptor is mediated by other signaling pathways, which reduce Adrb3 expression33,38,39.
Numerous compounds can be used to activate adipocyte lipolysis. Catecholamines are major physiological activators of lipolysis. Norepinephrine (or noradrenaline) and epinephrine (or adrenaline) activate all three &#946;-adrenergic receptors40. Norepinephrine and epinephrine also effect lipolysis via activation of &#945;-adrenergic receptor signaling41. Commonly used &#946;-adrenergic receptor agonists include isoproterenol, which is a non-selective &#946;-adrenergic receptor agonist, and the &#946;-3 adrenergic receptor agonists CL-316,243 and mirabegron42. Given that adipocytes predominantly express the &#946;-3 adrenergic receptor, we use CL-316,243 as an example here. Its specificity for the &#946;-3 adrenergic receptor also makes it a relatively specific activator of adipocyte catecholamine signaling, that can also be safely used in vivo. Note that the commonly used concentration of 10 &#181;M CL-316,243 in cell culture is orders of magnitude higher than the ~0.1 &#181;M dose required to achieve a maximal response33. Forskolin bypasses the adrenergic receptor, directly activating adenylyl cyclase and downstream lipolytic signaling. There are many more activators, as well as suppressors of lipolysis. When selecting a compound to stimulate lipolysis, the receptor-specificity and downstream signaling pathways should be carefully considered within the experimental design.
The rate of lipolysis in white adipose tissue is an important metabolic factor impacting cold tolerance and nutrient availability during fasting or exercise43,44,45,46. The purpose of this protocol is to measure the rate of lipolysis in adipocytes and adipose tissue, which will facilitate the understanding of adipocyte metabolism and how it may impact the metabolic phenotype of various murine models. To quantify the lipolytic rate, we measure the appearance of lipolytic products in the media (i.e., FFAs and glycerol). The method relies on the release of lipolytic products from the adipocyte into the media. Since white adipocytes express low levels of glycerol kinase, glycerol reuptake rates are low47. Conversely, the production of FFAs and glycerol by metabolic pathways other than lipolysis should also be considered. Adipocytes appear to express a phosphatase with activity against glycerol-3 phosphate, enabling the production of glycerol from glycerol-3-phosphate derived from glucose48,49,50. Glycolysis is a source of glycerol-3-phosphate used for FFA re-esterification in white adipocytes. When glucose levels are limited, glyceroneogenesis requires other 3-carbon sources, such as lactate and pyruvate51. The channeling of FFAs released by lipolysis within the cell and their metabolic fate is poorly understood; FFAs released by lipolysis must be converted to fatty acyl-CoA, before being re-esterified or undergoing &#946;-oxidation. It appears that FFAs released by lipolysis likely exit the cell before being taken back up and converted to fatty acyl-CoA52,53,54,55,56,57,58,59,60,61,62. FFAs can be sequestered outside of the cell by albumin. Importantly, long-chain FFAs are known to feedback-inhibit lipolysis if they are not sequestered by albumin63,64,65,66,67. Thus, optimizing the FFA buffering capacity of the media during the lipolysis assay is critical. The procedure described here is similar to previously published methods to measure the lipolytic rate in adipocytes and ex vivo adipose tissue from mice and humans15,68,69,70,71. This protocol differs through the use of serial sampling; by performing serial sampling, we can internally validate that lipolysis is being measured in the linear phase and utilize multiple measurements to calculate the rate of lipolysis, thereby reducing measurement error to increase confidence in the final calculated value. The drawback of serial sampling is that the assay requires more time and reagents; however, the longer timeframe reduces impact of measurement error on the standard error of the estimates of the rate. Additionally, this protocol measures both FFA and glycerol release, and considers the ratio of FFA:glycerol release with the goal of achieving a 3:1 ratio, as would be expected from complete lipolysis and release of lipolytic products into the media72.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-Well tissue culture treated plate</td>
			<td>Corning Inc</td>
			<td>3527</td>
			<td>Must be tissue culture treated for adipocyte differntiation</td>
		</tr>
		<tr>
			<td>48-Well flat bottom plate with lid</td>
			<td>Corning Inc</td>
			<td>353078</td>
			<td>Can be tissue culture treated</td>
		</tr>
		<tr>
			<td>6-Well flat bottom plate with lid</td>
			<td>Corning Inc</td>
			<td>353046</td>
			<td>Can be tissue culture treated</td>
		</tr>
		<tr>
			<td>96-Well PCR Plate</td>
			<td>USA sceintific</td>
			<td>1402-9100</td>
			<td>Any conical 0.2 mL PCR plate will be convenient&#160;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td>FFA free BSA such as A8806, is also commonly used. The BSA should not have detectable FFA, also lot to lot variations in BSA can impact the observed rate of lipolysis</td>
		</tr>
		<tr>
			<td>CL-316,243</td>
			<td>Sigma Aldrich</td>
			<td>C5976</td>
			<td>CAS #: 138908-40-4 availaible from other suppliers</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>PHCBI</td>
			<td>MCO-170AICUVH</td>
			<td>CO2 should ideally be set to 10% for adipose tissue, however 5% CO2 will also work</td>
		</tr>
		<tr>
			<td>DMEM, low glucose, no phenol red</td>
			<td>Thermofischer</td>
			<td>11054020</td>
			<td>Any phenol red free media should work, DMEM/F12, RPMI, but should contain volatile buffering capacity, i.e. biocarbonate</td>
		</tr>
		<tr>
			<td>FFA-free Bovine serum albumin</td>
			<td>Equitech-Bio, Inc,&#160;</td>
			<td>BAH66</td>
			<td></td>
		</tr>
		<tr>
			<td>Free Glycerol Reagent</td>
			<td>Sigma Aldrich</td>
			<td>F6428</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Standard Solution</td>
			<td>Sigma Aldrich</td>
			<td>G7793&#160;</td>
			<td>This can also be made by diluting glycerol to the desired concentration</td>
		</tr>
		<tr>
			<td>HR Series NEFA Standard Solution</td>
			<td>Fujifilm</td>
			<td>276-76491</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Series NEFA-HR (2) Color Reagent A</td>
			<td>Fujifilm</td>
			<td>999-34691</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Series NEFA-HR (2) Color Reagent B</td>
			<td>Fujifilm</td>
			<td>991-34891</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Series NEFA-HR (2) Solvent A&#160;</td>
			<td>Fujifilm</td>
			<td>995-34791</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Series NEFA-HR (2) Solvent B&#160;</td>
			<td>Fujifilm</td>
			<td>993-35191</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological Incubator</td>
			<td>Fischer Scientific</td>
			<td>S28668</td>
			<td>Any incubator at 37C can be used</td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well Plates</td>
			<td>Thermo Scientific</td>
			<td>269620</td>
			<td>Any optically clear, flat bottom 96-well plate works</td>
		</tr>
		<tr>
			<td>Silicone Laboratory Benchtop Mat</td>
			<td>VWR</td>
			<td>76045-300</td>
			<td>Glass plate can also be used. Absorbant surfaces are not recommended</td>
		</tr>
		<tr>
			<td>Spectrophotometer/Microplate Reader</td>
			<td>Molecular devices</td>
			<td>SpectraMax i3x&#160;</td>
			<td>Any plate reader that can read at 540, 550 and 660 mm will work</td>
		</tr>
		<tr>
			<td>V Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>810531</td>
			<td></td>
		</tr>
		<tr>
			<td>WypAll X70 Wipers</td>
			<td>Kimberly-Clark</td>
			<td>41200</td>
			<td>Any high quality paper towel will work</td>
		</tr>
	</tbody>
</table>

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.

We measured the basal and stimulated lipolytic rate of in vitro differentiated adipocytes. Primary preadipocytes from inguinal white adipose tissue were differentiated into adipocytes by the treatment of confluent cells with 5 &#181;M dexamethasone, 0.5 mM IBMX, 1 &#181;g/mL insulin, and 1 &#181;M troglitazone for 4 days, followed by an additional 3 day treatment with 1 &#181;g/mL insulin. Cells were incubated in media without insulin for 24 h prior to the lipolysis assay. At time = 0h, the cells were washed onc...

Watch this Scientific Journal Video about Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro at JoVE.com

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.

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 ...

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