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Summary

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

Abstract

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.

Introduction

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 fates^4,5. 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’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 causes a spike in circulating triglyceride levels (1–2 hours), after which the serum triglyceride levels return to basal levels (4–6 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 β3-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 β3-adrenergic receptor correlated with obesity⁶. CL 316,243, a specific and potent β3-adrenergic receptor agonist, stimulates adipose tissue lipolysis and the release of glycerol. Evaluation of a mouse’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.

Protocol

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

1. 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’s gastrointestinal tracts.
   NOTE: Mice eat their feces during fasting, so food withdrawal cannot ensure they are adequately fasted.
2. 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.
3. Make a superficial incision (nick) in the tail vein of the free-moving mouse, and draw 25 µL 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.
4. Stop the bleeding using styptic powders, refill the feed in the cage, and make sure the mice show no signs of stress.
5. Complete the blood withdrawal for all the mice.
6. 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 °C for 10 minutes in a refrigerated benchtop microcentrifuge, and transfer supernatant (serum) for analysis.
   NOTE: Serum can be stored at –20 °C for several weeks until analysis. For long-term storage, keep the serum at –70°C.
7. Analyze each lipid metabolite using the manufacturer’s provided protocol.

2. Oral Intralipid Tolerance Test

1. 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).
2. The next morning, mark tails of the mice housed in one cage to help identify them in the subsequent bleeding steps.
3. Make a nick in the tail vein and draw 15 µL 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.
4. Gavage mice 20% intralipid using an 18G gavage needle at a ratio of 15 µl 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 >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’s head in place. Place the gavage needle in the mouth and then gently advance along the upper palate, until 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.
5. Draw blood at T = 1, 2, 3, 4, 5, and 6 hours: Draw 15 µL of blood (1/5 capillary) per mouse through tail bleeding, and quickly blow the blood into a microcentrifuge tube.
6. 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 –20 °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.
7. 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.
8. Load 2 µL of triglyceride standard and collected supernatants into a 96-well plate.
9. Add 200 µL of triglyceride reagent and let the plate incubate for 5 minutes at 37 °C for color development.
10. Measure the absorbance at 500 nm with a reference wavelength of 660 nm in a laboratory plate reader, and calculate the sample’s concentration.

3. β3 Adrenergic Receptor Agonist CL 316,243 Stimulated Lipolysis Assay

1. Prepare CL 316,243 as a stock solution of 5 mg/mL (50x) in sterile saline, and store at –20°C until use.
2. 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 µL per gram of bodyweight of diluted CL 316,243, for a final dose of 1 mg/kg bodyweight.
3. Transfer the mice into a new cage with free access to water, and fast them for 4 hours.
4. 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 for the control treatment group.
5. Mark the tails of the mice housed in the same cage for easy identification during the bleeding steps.
6. Make a nick in the tail vein, and draw 15 µL 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.
7. Inject diluted CL 316,243 solution (or control if included in the experiment) intraperitoneally at a volume of 10 µL/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.
8. Draw blood at T = 5, 15, 30, 60 minutes: draw 15 µL of blood (1/5 capillary) per mouse through tail bleeding.
9. 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.
10. Spin blood samples at 2,000 x g at 4 °C for 5 minutes in a refrigerated microcentrifuge. Transfer the supernatant to a PCR tube for storage. The supernatant can be stored at –20°C for several weeks until analysis.
11. Load 1 µL 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 µL of free glycerol reagent, and let the plate incubate for 5 minutes at 37 °C for the color to develop.
12. Measure the absorbance at 540 nm using a laboratory plate reader, and calculate the sample’s concentration.

Representative Results

We show with three excerpts that each assay offers valuable information about the mice's lipid metabolism. For C57BL/6J male mice, challenged by eight weeks of high-fat diet (HFD) feeding  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 substrains of C57BL6 were fed the HFD for eight weeks, starting at eight weeks of age. Their serum triglyceride levels were compared after an oral intralipid challenge. The results demonstrated a striking difference between 6N and 6J substrains, with 6J having a significantly highter peak in serum triglyceride levels after intralipid administration, indicating an enhanced absorption or a much slower triglyceride clearance (Figure 1). Lastly, for eight-week-old male C57BL/6J mice fed on normal chow (NC), a single CL 316,243 treatment does (1 mg/kg bodyweight) led to a significant increase in serum glycerol. However, daily intraperitoneal pretreatment of mice with 1 mg/kg bodyweight CL 316,243 for one week led to a blunted reaction to CL 316,243, suggesting the development of resistance to CL 3116,263 in those mice (Figure 2).

Serum Parameters	NC	HFD	P Value
Cholesterol (mg/dL)	132.7±10.3	202.3±8.4	0.0002
Triglyceride (mg/dL)	91.7±9.1	79.3±4.5	0.26
Non esterified fatty acids (mmol/L)	1.47±0.12	1.48±0.08	0.73

Table 1: Fasting lipid species in mice fed on normal chow (NC) or high-fat diet (HFD) for eight weeks. Data are expressed as mean values ± SEM. N = 6 for NC group, n = 12 for HFD group. P-value was determined using two-tailed Student’s t-test.

Figure 1: An example of results demonstrating the difference in serum triglyceride of C57BL/6 substrains after an oral intralipid challenge. Data are expressed as mean values ± SEM. N = 5 for each group. P-value was determined using two-tailed Student’s t-test at each time point. * p < .05, ** p < .01. Please click here to view a larger version of this figure.

Figure 2: An example of results demonstrating the development of resistance to CL 316,243 treatment in C57BL/6J mice after one week of daily CL 316,243 treatment. Data are expressed as mean values ± SEM. N = 5 for each group. P-value was determined using two-tailed Student’s t-test at each time point. ** p < .01. Please click here to view a larger version of this figure.

Discussion

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 together for triglyceride determination.

Interpretation of the fasting serum lipid levels
Fasting has been shown to lower total cholesterol levels in mice⁷, whereas chronically consuming high fat content diets increases total cholesterol levels⁸. There are two main types of cholesterol: high-density lipoprotein -cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). HDL-C is regarded as the “good” lipid in humans. It carries cholesterol and transports it to the liver to be flushed out of the system. LDL-Cs make up most of the cholesterol in the human serum, and they can build up in arteries, leading to major artery diseases. However, mice lack an important enzyme, cholesteryl ester transfer protein (CETP)⁹, that mediates the exchange of triglycerides for esterified cholesterol between HDL and apoB-lipoproteins¹⁰. This gives mice a completely different lipoprotein particle profile, with HDL being the main species. As a result, a change in total serum cholesterol levels primarily reflects changes in HDL-C levels.

In both mice and humans, high serum triglyceride levels can increase low-grade inflammation and may impair cardiac function^11,12. However, HFD does not increase serum triglyceride levels. Genetic factors may play a dominant role in serum triglyceride levels over metabolic conditions¹³. NEFA in the blood can be avidly absorbed and utilized by many organs, suppressed by insulin and feeding, and increased by epinephrine¹⁴. HFD feeding does not change serum NEFA level, suggesting the hormonal cue dominates the regulation of serum NEFA levels.

Interpreting oral intralipid clearance test
An orally administered intralipid is absorbed by the intestinal epithelial cells and carried in lipoprotein particles in the bloodstream, where it is liberated and used by peripheral organs. Changes in lipoprotein lipase activity, peripheral-tissue triglyceride uptake, and oxidation will affect the dynamics of serum triglyceride levels. For example, brown and beige adipocytes avidly oxidize fatty acids for heat production. Cold exposure significantly increases brown and beige adipocyte activity, accelerating plasma clearance of triglycerides¹⁵. The oral intralipid tolerance clearance test was crucial for evaluating the effects of cold exposure on triglyceride metabolism, as demonstrated in the paper¹⁵.

Evaluation of compounds targeting adipose tissue lipolysis
Activation of lipolysis is conveyed by the sympathetic nervous system, endocrine factors, and various metabolites. Many compounds have been put into development by pharmaceutical companies to promote adipose tissue lipolysis^16,17. Assessing their efficacy in pre-clinical animal models such as mice is critical for facilitating the development process. Here we use a β3- adrenergic receptor agonist, CL 316,243, as an example to illustrate how we can assess how a mouse responds to the compound and whether the mouse displays different levels of sensitivity to the compound in different metabolic states. As seen in the exemplary results, repeated use of CL 316,243 caused desensitization to the treatment in the mice. We used CL 316,243 to illustrate how we could assess a mouse’s response to acute treatment; more importantly, this concept and design can be easily applied to other molecules targeting adipose tissue lipid metabolism.

Limitations
A few selected lipid species offer limited information about lipid metabolism in mice. Due to the small amount of serum available from tail bleeding, this protocol measures only total cholesterol and does not distinguish HDL-C and LDL-C, as those assays require significant amounts of blood. Because mice are unique in the way they lack the CETP, total cholesterol is a good approximation, and more HDL-C in mice does not indicate a healthy lipid profile, so the additional information obtained by distinguishing the cholesterol in different lipoprotein particles is limited.

Serum lipid levels, including triglyceride levels, are usually a net effect of absorption and excursion by many organs acting in a very dynamic way. Interpreting the results usually requires an experimental setup with only one variable. As shown in the exemplary result, no specific conclusion regarding the lipid absorption or excursion can be made between C57BL/6J and C57BL/6N substrains of C57BL/6 mice. However, in the cited cold exposure study¹⁵, prior knowledge and sometimes assumptions can be used to exclude contributions from other variables, and authors were able to pin down to a specific tissue and discovered that brown adipose tissue contributed to the enhanced triglyceride clearance.

Lastly, metabolism is a dynamic process. The change of one metabolite in a lipid metabolic pathway provides only a snapshot of the overall state. To understand the flow, a more sophisticated flux study using isotope-tracing techniques is required.

In summary, the simplicity is both the power and weakness of this protocol. The three assays presented here are not designed for the study of specific lipid metabolism pathways, but rather to provide an initial screening or a starting point for evaluating lipid metabolism in general nutrition and obesity research.

Materials

20% Intralipid	Sigma Aldrich	I141
BD Slip Tip Sterile Syringes 1ml	Shaotong	B07F1KRMYN
CL 316,243 Hydrate	Sigma-Aldrich	C5976
Curved Feeding Needles (18 Gauge)	Kent Scientific	FNC-18-2-2
Free Glycerol Reagent	Sigma Aldrich	F6428
Glycerol Standard Solution	Sigma	G7793
HR SERIES NEFA-HR(2)COLOR REAGENT A	Fujifilm Wako Diagnostics	999-34691
HR SERIES NEFA-HR(2)COLOR REAGENT B	Fujifilm Wako Diagnostics	991-34891
HR SERIES NEFA-HR(2)SOLVENT A	Fujifilm Wako Diagnostics	995-34791
HR SERIES NEFA-HR(2)SOLVENT B	Fujifilm Wako Diagnostics	993-35191
Ketamine	Vedco	50989-161-06
Matrix Plus Chemistry Reference Kit	Verichem	9500
Micro Centrifuge Tubes	Fisher Scientific	14-222-168
Microhematrocrit Capillary Tube, Not Heparanized	Fisher Scientific	22-362-574
NEFA STANDARD SOLUTION	Fujifilm Wako Diagnostics	276-76491
Phosphate Buffered Saline	Boston Bioproducts	BM-220
Thermo Scientific Triglycerides Reagent	Fisher Scientific	TR22421
Total Cholesterol Reagents	Thermo Scientifi	TR13421
Xylazine	Henry Schein	11695-4022-1