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.
