Biology Reference
In-Depth Information
9.1.3.3.2
Methods
1.
Fast mice to eliminate TAG in blood from a previous meal. This time period may
be different depending on the diet the mouse is consuming. Generally, 4 h of
fasting at the beginning of the light cycle is appropriate unless a high fat diet is
being consumed. Before starting the blood collection, it is important to weigh the
mouse to estimate maximum blood collection volumes (less than10% of their
bodyweight) for a survival bleed.
2.
Collect blood via submandibular bleed (vasculature behind the jaw bone) using
a lancet. Collect
50
m
l of blood into a microcentrifuge tube with 0.5
m
lof
0.5 M EDTA. Place the tube on ice. Alternatively, blood collection may be done
via tail bleed using a capillary tube, if desired.
3.
For postprandial triglyceridemic response—gavage the mouse with oil, the
amount of oil depends on the question being addressed. To mimic a high fat meal
in a mouse, 200
l of oil is appropriate. Start the timer and collect blood at
1, 2, 3, and 4 h post oral oil gavage. Keep blood samples on ice until further
processing and analysis.
4.
For TAG secretion rate—inject the mouse via IP with Tyloxapol solution at a
dose of 500 mg/kg, wait 30 min, and gavage the mouse with oil. To mimic a high
fat meal in a mouse, 200
m
l of oil is appropriate. Start the timer and collect blood
at 2 and 4 h post oral oil gavage.
5.
After collecting all time points, centrifuge the blood at 4
C at 3000
m
g
. Analyze
plasma for TAG concentration immediately.
6.
For postprandial triglyceridemic response—use 10
l plasma (in duplicate) for
measuring TAG by Wako L-Type TG M Kit (Microtiter Procedure).
7.
For TAG secretion rate—the plasma after spinning will likely be opaque with a
white/pinkish tint. Dilute the plasma collected at 2 h by 10 and the plasma
collected at 4 h by 20 in PBS. Use 10
m
l (in duplicate) diluted plasma for
determination of TAG concentration by Wako L-Type TG M Kit (Microtiter
Procedure). If samples do not fall within the standard curve, dilute as appropriate.
m
9.1.3.3.3
Results
Plot the results from the TAG concentrations over time (
Fig. 9.4
). For the postpran-
dial triglyceridemic response, if the mice were gavaged with 200
l of oil, TAG
levels will peak at either 1 or 2 h and a decrease to baseline by 4 h. The response
will vary depending on the volume of oil gavaged. For the TAG secretion rate,
the points at 0, 2, and 4 h should result in a linear fit. This line can be used to calculate
a slope.
m
9.2
DISCUSSION
A mechanistic understanding of dietary fat absorption is essential for determining
factors important in regulating energy balance and blood lipid concentrations—
contributors to obesity, cardiovascular disease, and diabetes. This chapter highlights