Name: assessing whole-body lipid-handling capacity in mice
Uploaded: 2020-11-24T14:00:00
Description: Watch this Scientific Journal Video about Assessing Whole-Body Lipid-Handling Capacity in Mice at JoVE.com

This work is supported by the National Institutes of Health (NIH), grant R00-DK114498, and the United States Department of Agriculture (USDA), grant CRIS: 3092-51000-062 to Y. Z.

Animals are housed in standardized conditions following animal-care and experimental protocols approved by the Institutional Animal Care and Use Committee of the Baylor College of Medicine (BCM). Animals are fed a standard or special diet, water ad libitum, and kept with a 12-hour day/night cycle.
1. Measuring of fasting serum lipids
<ol>
	<li>Transfer mice to a new cage after 5 PM and fast with free access to water, overnight (with around 16 hours of fasting before the experiment). The over...

<ol>
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The three assays described function robustly in the lab, with a few critical considerations. Overnight fasting is required for determining fasting serum lipid levels and oral intralipid tolerance test. For oral intralipid tolerance test, it is critical to spin the blood at room temperature to minimize the formation of a fat layer, especially at the 1- and 2-hour time points; it is important not to discard this fat layer if it forms. Make sure to transfer the supernatant with the lipid layer, and pipet gently to mix them ...

Carbohydrates and lipids are two major substrates for energy metabolism. Aberrant lipid metabolism results in many human diseases, including type II diabetes, cardiovascular diseases, fatty liver diseases, and cancers. Dietary lipids, mainly triglycerides, are absorbed through the intestine into the lymphatic system and enter the venous circulation in chylomicrons near the heart1. Lipids are carried by lipoprotein particles in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase at peripheral organs such as muscle and adipose tissue2. The remaining cholesterol-rich remnant particles are cleared by the liver3. Mice have been widely used in laboratories as a research model to study lipid metabolism. With comprehensive genetic toolsets available and a relatively short breeding cycle, they are a powerful model for studying how lipids are absorbed, synthesized, and metabolized.
Due to the complexity of lipid metabolism, sophisticated lipidomics studies or isotopic tracer studies are usually used to quantify collections of lipid species or lipid-related metabolic fluxes and fates4,5. This creates a massive challenge for researchers without specialized equipment or expertise. In this paper, we present three assays that can serve as initial tests before technically challenging techniques are used. They are non-terminal procedures for the mice, and thus very useful for identifying potential differences in lipid-handling capacity and narrowing down the processes affected.
First, measuring fasting serum lipid molecules can help one ascertain a mouse&#8217;s overall lipid profile. Mice should be fasted, because many lipid species rise after meals, and the extent of the increase is strongly affected by the composition of the diet. Many lipid molecules, including total cholesterol, triglyceride, and non-esterified fatty acid (NEFA), can be measured using a commercial kit and a plate reader that can read absorbance.
Second, an oral intralipid tolerance test evaluates lipid-handling capability as a net effect of absorption and metabolism. An orally administered intralipid&#160;causes a spike in circulating triglyceride levels (1&#8211;2 hours), after which the serum triglyceride levels&#160;return to basal levels (4&#8211;6&#160;hours). This assay offers information about how well a mouse can handle the exogenous lipids. Heart, liver, and brown adipose tissue are active consumers of triglycerides, whereas white adipose tissue stores it as an energy reserve. Changes in these functions will lead to differences in the test results.
Lastly, promoting lipolysis to mobilize stored lipids is considered a possible strategy for weight loss. The &#946;3-adrenergic receptor signaling pathway in the adipose tissue plays an important role in adipocyte lipolysis, and human genetics have identified a loss-of-function polymorphism Trp64Arg in &#946;3-adrenergic receptor&#160;correlated with obesity6. CL 316,243, a specific and potent &#946;3-adrenergic receptor agonist, stimulates adipose tissue lipolysis and the release of glycerol. Evaluation of a mouse&#8217;s response to CL 316,243 can provide valuable information on the development, improvement, and understanding of the efficacy of the compound.
Collectively, these tests can be used as an initial screen for changes in the lipid metabolic state of mice. They are chosen for the accessibility of the instruments and reagents. With the results derived from these assays, researchers can form an overall picture of the metabolic fitness of their animals and decide on more sophisticated and targeted approaches.

<table>
	<tbody>
		<tr>
			<td>20% Intralipid</td>
			<td>Sigma Aldrich</td>
			<td>I141</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes 1ml</td>
			<td>Shaotong</td>
			<td>B07F1KRMYN</td>
			<td></td>
		</tr>
		<tr>
			<td>CL 316,243 Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C5976</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Feeding Needles (18 Gauge)</td>
			<td>Kent Scientific</td>
			<td>FNC-18-2-2</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</td>
			<td>G7793</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>999-34691</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>991-34891</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>995-34791</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>993-35191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vedco</td>
			<td>50989-161-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix Plus Chemistry Reference Kit</td>
			<td>Verichem</td>
			<td>9500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-222-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Microhematrocrit Capillary Tube, Not Heparanized</td>
			<td>Fisher Scientific</td>
			<td>22-362-574</td>
			<td></td>
		</tr>
		<tr>
			<td>NEFA STANDARD SOLUTION</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>276-76491</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Triglycerides Reagent</td>
			<td>Fisher Scientific</td>
			<td>TR22421</td>
			<td></td>
		</tr>
		<tr>
			<td>Total Cholesterol Reagents</td>
			<td>Thermo Scientifi</td>
			<td>TR13421</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Henry Schein</td>
			<td>11695-4022-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We show with three excerpts that each assay offers valuable information about the mice&#39;s lipid metabolism. For C57BL/6J male mice, challenged by eight weeks of high-fat diet (HFD) feeding&#160; starting at eight weeks of age, total cholesterol levels were significantly elevated, while serum triglyyceride and NEFA were not (Table 1), suggesting that triglyceride and NEFA in the blood are not predominantly regulated by a dietary fat challenge. In the second cohort of mice, C57BL/6J and C57BL6/N&#160;su...

Watch this Scientific Journal Video about Assessing Whole-Body Lipid-Handling Capacity in Mice at JoVE.com

Assessing Whole-Body Lipid-Handling Capacity 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 ...

Assessing lipid metabolism is a cornerstone of evaluating metabolic function and it is considered essential for in vivo metabolism studies. Due to the complexity of lipid metabolism, sophisticated lipidomic studies or isotopic tracer studies are usually used, creating a massive challenge for researchers without specialized equipment or expertise. This protocol demonstrates easy and accessible assays for assessing lipid metabolism in mice.Demonstrating the procedure will be Mingyang Huang, a research assistant from the Zhu laboratory. Begin by transferring mice to a new cage and fasting them overnight with free access to water per 16 hours before the experiment, which ensures complete emptying of the mice's gastrointestinal tracts. On the next morning, make a superficial incision in the tail vein of the mouse.And draw 25 microliters of blood from the incision into a glass capillary. Quickly blow the blood into a microcentrifuge tube. Stop the bleeding using septic powders, then refill the feed in the cage and make sure the mice show no signs of extreme stress.Perform the blood withdrawal on all mice. Allow the blood to clot by leaving it undisturbed at room temperature for one hour, then spin the clotted blood samples at 2000 x g at four degrees Celsius for 10 minutes in a refrigerated benchtop microcentrifuge. Collect the supernatant for lipid metabolite analysis.After 5:00 PM, weigh the mice to calculate the intralipid volume that will be given to them on the next day. Then, transfer them into a new cage and fast them overnight. On the next morning, mark the tales of the mice housed in one cage to help identify them in the subsequent bleeding steps.Make a nick in the tail vein and drop 15 microliters of blood from the incision into a glass capillary, and quickly blow the blood into a microcentrifuge tube for T 0 serum. Gavage mice with 20%intralipid using an 18 gauge gavage needle at a ratio of 15 microliters per gram of body weight using the pre-fasting body weight. Draw blood at 1, 2, 3, 4, 5, and 6 hours.Spin the blood samples at 2000 x g for 10 minutes in a microcentrifuge. Transfer the supernatant, including the floating fat layer to a PCR tube for storage. The supernatant can be stored at minus 20 degrees Celsius for several weeks until analysis.After the last blood collection, stop the bleeding using septic powders, refill the feed in the cage and make sure the mice show no signs of extreme stress. Load two microliters of triglyceride standard and collected supernatants into a 96-well plate. Add 200 microliters of triglyceride reagent and let the plate incubate for five minutes at 37 degrees Celsius for color development.Measure the absorbance at 500 nanometers with a reference wavelength of 660 nanometers in a plate reader and calculate the sample's concentration. In the morning, weigh the mice to calculate the amount of diluted CL 316, 243 compound solution needed for the experiment. Transfer the mice into a new cage with free access to water and fast them for four hours.Make enough 1X CL compound solution from the 50X stock that was prepared by dissolving five milligrams of CL 316, 243 powder in one milliliter sterile saline or PBS. Use sterile saline or PBS for the control treatment group. Filter through a Millipore 0.22 micron PES membrane filter unit to sterilize the solution.Mark the tales of the mice housed in the same cage for easy identification during the bleeding step. Make a nick in the tail vein and draw 15 microliters of blood from the incision into a glass capillary, then quickly flow the blood into a microcentrifuge tube for the T 0 sample. Inject diluted CL compound solution intraperitoneally at a volume of 10 microliters per gram of body weight.Use a maximum of five mice for each 60 minute experiment or 10 mice for a two person team. Draw blood at 5, 15, 30, and 60 minutes after the injection. After the last blood collection, stop the bleeding using septic powders, refill the feed in the cage, and make sure the mice show no signs of extreme stress.Spin the blood samples at 2000 x g at four degrees Celsius for five minutes in a refrigerated microcentrifuge, then transfer the supernatant to a PCR tube for storage. The supernatant can be stored at minus 20 degrees Celsius for several weeks until analysis. Load one microliter of 2X serially diluted glycerol standards and collected supernatants into a 96-well plate.Add 100 microliters of free glycerol reagent and let the plate incubate for five minutes at 37 degrees Celsius for the color to develop. Measure the absorbance at 540 nanometers using a laboratory plate reader and calculate the concentration of each sample. When C57BL6/J male mice were challenged by eight weeks of a high-fat diet, total cholesterol levels were significantly elevated.Meanwhile, serum triglyceride and NEFA were not, suggesting that triglyceride and NEFA in the blood are not predominantly influenced by chronic high-fat diet challenge. In the second cohort of mice, substrains of C57BL6 were fed the high-fat diet for eight weeks starting at eight weeks of age. Their serum triglyceride levels were compared after an oral intralipid challenge.The 6/J substrain had a significantly higher peak compared to the 6/N substrain, indicating an enhanced absorption or a much slower triglyceride clearance. For eight week old C57BL6/J mice fed on normal chow, a single CL 316, 243 treatment led to a significant increase in serum glycerol. However, daily intraperitoneal pretreatment of mice with CL compound for one week led to a blunted reaction to the CL compound treatment, suggesting a quick development of resistance to the compound.Simplicity is both the power and weakness of this protocol. It does not impose a significant technical hurdle on experimenters, but the results need careful interpretation. Measurement of a specific lipid molecule by mass spectrometry or more comprehensive lipidomics should be performed for followup studies.An isotope tracing technique is usually used to understand the flux of lipid metabolism.

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