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 overnight fasting ensures complete emptying of the mice&#8217;s gastrointestinal tracts. 
	NOTE: Mice eat their feces during fasting, so food withdrawal cannot ensure they are adequately fasted.</li>
	<li>The next morning, grasp the mouse by the tail and place it on a surface. Pull back gently on the tail of the mouse to place the mouse in the restrainer. Place the nose restraint to retrain the movement of the mouse while still allowing the mouse to breathe. Tighten the knob of the nose restraint. Monitor chest movements to make sure the animal is breathing normally and minimize the time a mouse spends in restrainers.</li>
	<li>Make a superficial incision (nick) in the tail vein of the free-moving mouse, and draw 25 &#181;L of blood from the incision into a glass capillary (filling about 1/3 of the capillary) without restraining the mouse. Quickly blow the blood into a microcentrifuge tube.</li>
	<li>Stop the bleeding using styptic powders, refill the feed in the cage, and make sure the mice show no signs of&#160;stress.</li>
	<li>Complete the blood withdrawal for all the mice.</li>
	<li>Allow the blood to clot by leaving it undisturbed at room temperature for 1 hour. Spin the clotted blood samples at 2,000 x g at 4 &#176;C for 10 minutes in a refrigerated benchtop microcentrifuge, and transfer supernatant (serum) for analysis. 
	NOTE: Serum can be stored at &#8211;20 &#176;C for several weeks until analysis. For long-term storage, keep the serum at &#8211;70&#176;C.</li>
	<li>Analyze each lipid metabolite using the manufacturer&#8217;s provided protocol.</li>
</ol>
2. Oral Intralipid Tolerance Test
<ol>
	<li>After 5 PM, weigh the mice for the calculation of the intralipid volume to be given to them the next day. Then, transfer the mice into a new cage and fast them overnight (16 hours).</li>
	<li>The next morning, mark tails of the mice housed in one cage to help identify them in the subsequent bleeding steps.</li>
	<li>Make a nick in the tail vein and draw 15 &#181;L of blood from the incision into a glass capillary (filling about 1/5 of the capillary), and quickly blow the blood into a microcentrifuge tube for T = 0 serum. 
	NOTE: There is no need to stop the bleeding during the assay unless the mice show excess bleeding.</li>
	<li>Gavage mice 20% intralipid using an 18G gavage needle at a ratio of 15 &#181;l per gram of bodyweight, using the pre-fasting bodyweight. Stack each mouse by 1 minute. 
	NOTE: Weigh the animal and determine the appropriate gavage needle size. Generally, an 18 gauge gavage needle is appropriate for mice &#62;25 g, and a 20-22 gauge gavage needle would be more appropriate for smaller animals. Scruff the mouse, grasping the skin over the shoulders to hold the animal&#8217;s head in place. Place the gavage needle in the mouth and then gently advance along the upper palate, until&#160;the esophagus is reached. The tube should pass without resistance. Once proper depth is reached, the Intralipid can be slowly administered. Gently remove the needle following the same angle during needle insertion after administrating the Intralipid. Return the animal to the cage and monitor for signs of labored breathing or other distress. 
	NOTE: Researchers who are inexperienced with oral gavage or tail bleeding techniques can stack each mouse by 2 minutes or even longer.</li>
	<li>Draw blood at T = 1, 2, 3, 4, 5, and 6 hours: Draw 15 &#181;L of blood (1/5 capillary) per mouse through tail bleeding, and quickly blow the blood into a microcentrifuge tube.</li>
	<li>Spin the blood samples at 2,000 x g at room temperature 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 &#8211;20 &#176;C for several weeks until analysis. 
	NOTE: The supernatant should be plasma. If some samples have already clotted by the time of centrifugation, it does not affect triglyceride measurement.</li>
	<li>After the last blood collection, stop the bleeding using styptic powders, refill the feed in the cage, and make sure the mice show no signs of extreme stress.</li>
	<li>Load 2 &#181;L of triglyceride standard and collected supernatants into a 96-well plate.</li>
	<li>Add 200 &#181;L of triglyceride reagent and let the plate incubate for 5 minutes at 37 &#176;C for color development.</li>
	<li>Measure the absorbance at 500 nm with a reference wavelength of 660 nm in a laboratory plate reader, and calculate the sample&#8217;s concentration.</li>
</ol>
3. &#946;3 Adrenergic Receptor Agonist CL 316,243 Stimulated Lipolysis Assay
<ol>
	<li>Prepare CL 316,243 as a stock solution of 5 mg/mL (50x) in sterile&#160;saline, and store at &#8211;20&#176;C until use.</li>
	<li>In the morning, weigh the mice to calculate the amount of diluted CL 316,243 solution needed for the experiment. The mouse will receive 10 &#181;L per gram of bodyweight of diluted CL 316,243, for a final dose of 1 mg/kg bodyweight.</li>
	<li>Transfer the mice into a new cage with free access to water, and fast them for 4 hours.</li>
	<li>Make enough 1x CL 316,243 solution from 50x stock using saline. The final concentration of 1x CL 316,243 solution is 0.1 mg/mL. Use saline&#160;for the control treatment group.</li>
	<li>Mark the tails of the mice housed in the same cage for easy identification during the bleeding steps.</li>
	<li>Make a nick in the tail vein, and draw 15 &#181;L of blood from the incision into a glass capillary (filling about 1/5 of the capillary), and quickly blow the blood into a microcentrifuge tube for T = 0 sample. 
	NOTE: There is no need to stop the bleeding during the assay unless the mice show excess bleeding.</li>
	<li>Inject diluted CL 316,243 solution (or control if included in the experiment) intraperitoneally at a volume of 10 &#181;L/g bodyweight. Stack each mouse by 1 minute. Use a maximum of 5 mice for each 60-minute experiment, or 10 mice for a two-person team.</li>
	<li>Draw blood at T = 5, 15, 30, 60 minutes: draw 15 &#181;L of blood (1/5 capillary) per mouse through tail bleeding.</li>
	<li>After the last blood collection, stop the bleeding using styptic powders, refill the feed in the cage, and make sure the mice show no signs of extreme stress.</li>
	<li>Spin blood samples at 2,000 x g at 4 &#176;C for 5&#160;minutes in a refrigerated microcentrifuge. Transfer the supernatant to a PCR tube for storage. The supernatant can be stored at &#8211;20&#176;C for several weeks until analysis.</li>
	<li>Load 1 &#181;L of 2x serially diluted glycerol standards (0.156, 0.312, 0.625, 1.25, and 2.5 mg/ml Trioleine-equivalent concentrations) and collected supernatants into a 96-well plate. Add 100 &#181;L of free glycerol reagent, and let the plate incubate for 5 minutes at 37 &#176;C for the color to develop.</li>
	<li>Measure the absorbance at 540 nm using a laboratory plate reader, and calculate the sample&#8217;s concentration.</li>
</ol>

<ol><li>Dixon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+chylomicron+uptake+into+lacteals%5BTitle%5D)+AND+%22Annals+of+the+New+York+Academy+of+Sciences%22%5BJournal%5D)">Mechanisms of chylomicron uptake into lacteals.</a> Annals of the New York Academy of Sciences. 1207, Suppl 1 52-57 (2010).</li>
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<li>Burla, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MS-based+lipidomics+of+human+blood+plasma%3A+a+community-initiated+position+paper+to+develop+accepted+guidelines%5BTitle%5D)+AND+%22Journal+of+Lipid+Research%22%5BJournal%5D)">MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines.</a> Journal of Lipid Research. 59 (10), 2001-2017 (2018).</li>
<li>Umpleby, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hormone+measurement+guidelines%3A+Tracing+lipid+metabolism%3A+the+value+of+stable+isotopes%5BTitle%5D)+AND+%22Journal+of+Endocrinology%22%5BJournal%5D)">Hormone measurement guidelines: Tracing lipid metabolism: the value of stable isotopes.</a> Journal of Endocrinology. 226 (3), 1-10 (2015).</li>
<li>Mitchell, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+paired+sibling+analysis+of+the+beta-3+adrenergic+receptor+and+obesity+in+Mexican+Americans%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">A paired sibling analysis of the beta-3 adrenergic receptor and obesity in Mexican Americans.</a> Journal of Clinical Investigation. 101 (3), 584-587 (1998).</li>
<li>Mahoney, L. B., Denny, C. A., Seyfried, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caloric+restriction+in+C57BL%2F6J+mice+mimics+therapeutic+fasting+in+humans%5BTitle%5D)+AND+%22Lipids+in+Health+and+Disease%22%5BJournal%5D)">Caloric restriction in C57BL/6J mice mimics therapeutic fasting in humans.</a> Lipids in Health and Disease. 5, 13(2006).</li>
<li>Hayek, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+fat+increases+high+density+lipoprotein+%28HDL%29+levels+both+by+increasing+the+transport+rates+and+decreasing+the+fractional+catabolic+rates+of+HDL+cholesterol+ester+and+apolipoprotein+%28Apo%29+A-I.+Presentation+of+a+new+animal+model+and+mechanistic+studies+in+human+Apo+A-I+transgenic+and+control+mice%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Dietary fat increases high density lipoprotein (HDL) levels both by increasing the transport rates and decreasing the fractional catabolic rates of HDL cholesterol ester and apolipoprotein (Apo) A-I. Presentation of a new animal model and mechanistic studies in human Apo A-I transgenic and control mice.</a> Journal of Clinical Investigation. 91 (4), 1665-1671 (1993).</li>
<li>Hogarth, C. A., Roy, A., Ebert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+evidence+for+the+absence+of+a+functional+cholesteryl+ester+transfer+protein+gene+in+mice+and+rats%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+-+Part+B%3A+Biochemistry+%26+Molecular+Biology%22%5BJournal%5D)">Genomic evidence for the absence of a functional cholesteryl ester transfer protein gene in mice and rats.</a> Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology. 135 (2), 219-229 (2003).</li>
<li>Tall, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functions+of+cholesterol+ester+transfer+protein+and+relationship+to+coronary+artery+disease+risk%5BTitle%5D)+AND+%22Journal+of+Clinical+Lipidology%22%5BJournal%5D)">Functions of cholesterol ester transfer protein and relationship to coronary artery disease risk.</a> Journal of Clinical Lipidology. 4 (5), 389-393 (2010).</li>
<li>Singh, A. K., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Triglyceride+and+cardiovascular+risk%3A+A+critical+appraisal%5BTitle%5D)+AND+%22Indian+Journal+of+Endocrinology+and+Metabolism%22%5BJournal%5D)">Triglyceride and cardiovascular risk: A critical appraisal.</a> Indian Journal of Endocrinology and Metabolism. 20 (4), 418-428 (2016).</li>
<li>Miller, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Triglycerides+and+cardiovascular+disease%3A+a+scientific+statement+from+the+American+Heart+Association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association.</a> Circulation. 123 (20), 2292-2333 (2011).</li>
<li>Dron, J. S., Hegele, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+Hypertriglyceridemia%5BTitle%5D)+AND+%22Frontiers+in+Endocrinology+%28Lausanne%29%22%5BJournal%5D)">Genetics of Hypertriglyceridemia.</a> Frontiers in Endocrinology (Lausanne). 11, 455(2020).</li>
<li>Dole, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+relation+between+non-esterified+fatty+acids+in+plasma+and+the+metabolism+of+glucose%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">A relation between non-esterified fatty acids in plasma and the metabolism of glucose.</a> Journal of Clinical Investigation. 35 (2), 150-154 (1956).</li>
<li>Bartelt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brown+adipose+tissue+activity+controls+triglyceride+clearance%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Brown adipose tissue activity controls triglyceride clearance.</a> Nature Medicine. 17 (2), 200-205 (2011).</li>
<li>de Souza, C. J., Burkey, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beta+3-adrenoceptor+agonists+as+anti-diabetic+and+anti-obesity+drugs+in+humans%5BTitle%5D)+AND+%22Current+Pharmaceutical+Design%22%5BJournal%5D)">Beta 3-adrenoceptor agonists as anti-diabetic and anti-obesity drugs in humans.</a> Current Pharmaceutical Design. 7 (14), 1433-1449 (2001).</li>
<li>Braun, K., Oeckl, J., Westermeier, J., Li, Y., Klingenspor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-adrenergic+control+of+lipolysis+and+thermogenesis+in+adipose+tissues%5BTitle%5D)+AND+%22Journal+of+Experimental+Biology%22%5BJournal%5D)">Non-adrenergic control of lipolysis and thermogenesis in adipose tissues.</a> Journal of Experimental Biology. 221, Pt Suppl 1 (2018).</li>
</ol>

The authors have nothing to disclose.

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>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></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...

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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 ...

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Medicine

3.9K Views.  Baylor College of Medicine. This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Video: Assessing Whole-Body Lipid-Handling Capacity in Mice

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Biochemistry

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biology

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for atherosclerosis research. They are used to study the impact of a various genetic factors and different cell types on atherosclerotic lesion formation and as well as test the development of new therapies. Isolation, excision of the whole aorta, and quantification of Oil Red O-stained atherosclerotic lesions are basic morphometric methods used to evaluate atherosclerotic burden. The goal of this protocol is to describe an optimized, step-by-step surgical method to dissect, perfuse-fix, isolate, stain, image and analyze atherosclerotic lesions in mouse aortas with Oil Red O. Because atherosclerotic lesions can form anywhere in the entire aortic tree, this whole aorta Oil Red O staining method has the advantage of evaluating lipid-laden plaques in the entire aorta and all branches in a single mouse. In addition to Oil Red O staining, fresh isolated whole aortas can be used for variety of in vitro and in vivo experiments and cell isolations.

This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Investigating HFpEF Pathophysiology in a Hyperlipidemia&#45;Induced Mouse Model

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

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 ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Summary

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Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

A Murine Model of Hyperlipidemia-Induced Heart Failure with Preserved Ejection Fraction

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

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