Project
protocol
—
Contents
Workflow
and
sampling
Equipment
Reagents,
supplies,
and
solutions
Procedures
Data
References
Workflow
and
sampling
Workflow
Age
(wk)
|
Category
|
Procedure
performed
|
Data
collected
|
1-20 |
Growth
curve
|
Weekly
bw
measurements
|
Weekly
body
weight
(bw)
|
10
|
Blood
chemistry |
Glucose
assay,
glucose
tolerance
|
Serum
glucose
|
20
|
Blood
chemistry |
Glucose
assay,
glucose
tolerance
|
Serum
glucose
|
20-30
|
Body
composition
and
organ
metrics,
blood
chemistry |
Necropsy
and
organ
weights,
glucose
and
lipid
assays,
hormone
assay
|
Serum
glucose,
cholesterol,
triglycerides,
HDL,
insulin,
and
leptin;
heart,
fat
pad,
spleen,
liver,
and
kidney
weights
|
Equipment
Top
loading
balance:
for
measurement
of
body
weights
Blood
glucose
meter
Glucometer
Dex
(Bayer)
Reagents,
supplies,
solutions
Composition
of
low-
and
high-fat
diet
(Harlan
Teklad
catalog
#TD88137)
and
the
low-fat
diet
(Research
Diets
catalog
#D12284):
Components
|
Food
type |
(1
Kg)
High
fat
diet
(g) |
(1
Kg)
Low
fat
diet
(g)
|
| Casein
|
Protein
|
195 |
197
|
| Sugar
|
Carbohydrate |
341
|
307
|
| Corn
starch
|
Carbohydrate
|
150
|
313
|
| Cellulose
|
Fiber
|
50
|
30
|
| Corn
oil
|
Vegetable
fat |
- |
58
|
| Hydrogenated
coconut
oil |
Vegetable
fat
|
- |
7 |
| Anhydrous
milk
fat
|
Milk
fat |
210
|
- |
| Cholesterol
|
- |
1.5
|
- |
| Energy
from
fat
|
- |
42%
|
15%
|
| Kilojoules
per
gram
|
- |
18.95
|
16.99
|
Table
1.
Composition
of
high-
and
low-fat
diets
as
given
in
Cheverud
et.
al
in
Diabetes,
volume
53,
December
2004,
page
3329.
10%
glucose
solution
HemoCue
glucose
system
(HemoCue
AB,
Angelholm,
Sweden)
HemoCue
Beta-glucose
analyzer
(HemoCue)
vs.
Yellow
Springs
Instrument
(YSI
2300
STAT;
YSI)
glucose
oxidase
analyzer
Sodium
pentobarbital
anesthetic
solution
Procedures
I.
Weekly
body
weight
measurements
a.
At
3-wk
of
age,
while
the
mice
are
being
weaned,
the
weanlings
are
randomly
assigned
to
low-
or
high-fat
diet.
b.
While
maintained
in
their
respective
dietary
regime,
the
mice
are
also
weighed
weekly
until
20-wk
of
age.
Parental
strains:
Large
(LG/J)
white
mouse
on
the
left
and
Small
(SM/J)
agouti
mouse
on
the
right
(photo
from
The
Cheverud
Lab).
II.
Blood
glucose
measurement
and
glucose
tolerance
test
a.
At
10-
and
20-wk
of
age,
the
mice
are
fasted
for
4
hrs
before
blood
is
drawn
to
measure
serum
glucose.
b.
After
obtaining
fasting
glucose
levels,
the
mice
are
then
given
intraperitoneal
injection
of
0.01
mL
of
10%
glucose
solution
per
gram
body
weight.
c.
Blood
samples
are
taken
at
15,
30,
60,
and
120
min
for
glucose
tolerance
test.
d.
A
blood
glucose
meter
is
used
to
measure
glucose
levels
in
a
drop
of
blood
sample
according
manufacturer's
instruction.
III.
Necropsy,
body
composition,
blood
chemistry,
and
hormone
measurements
a.
Between
20-30-wk
of
age,
the
mice
are
again
fasted
for
4
hrs
and
prepared
for
necropsy.
b.
For
restraint,
mice
are
given
i.p.
injection
of
sodium
pentobarbital
before
obtaining
blood
via
cardiac
puncture,
which
is
a
terminal
procedure.
c.
Tail
lengths
are
measured
as
an
added
indicator
of
growth
(skeletal).
d.
The
thoracic
and
abdominal
cavities
are
opened
for
the
dissection
and
subsequent
weighing
of
the
heart,
spleen,
liver,
and
kidneys.
e.
The
different
fat
pads
are
dissected
and
weighed
(see
Figure)
Note:
To
facilitate
the
dissection
of
the
gonadal
fat
pads,
the
entire
uterus
and
ovaries
or
testes
can
be
removed
from
their
attachments
to
the
pelvic
floor.
Likewise,
the
entire
intestinal
loop
can
also
be
removed
from
its
attachment
to
the
root
of
the
mesentery,
and
then
straightened
and
unwound
for
the
dissection
of
the
mesenteric
fat
pad.
In
contrast,
both
the
inguinal
and
the
perirenal
fat
pads
may
be
dissected
in
situ.
f.
Collected
blood
is
centrifuged
for
the
separation
of
plasma,
and
its
storage-
to
be
analyzed
at
a
later
date
for
blood
lipids
(free
fatty
acid,
cholesterol,
HDL,
and
triglycerides)
and
hormones
(insulin
and
leptin).
Data
collected
by
investigator
Weekly
body
weight
(from
1-wk
to
20-wk
of
age),
10
and
20-wk
serum
glucose
plus
glucose
tolerance
and
glucose
clearance
(AUC),
20-30wk
plasma
lipid
(cholesterol,
HDL,
triglycerides,
and
free
fatty
acid)
levels,
and
hormone
(insulin
and
leptin)
levels,
followed
by
necropsy
body
weight,
organ
(liver,
spleen,
kidney,
heart)
weights,
fat
pads
(gonadal,
inguinal,
mesenteric,
and
perirenal)
weights.
MPD
calculated
measurements:
fat
pad
weight
as
%
of
body
weight.
Definitions
&
formulas
AUC:
Area
under
the
curve
(graph
of
blood
glucose
levels
following
i.p.
injection
of
10%
glucose
challenge,
typically
obtained
at
15,
30,
60,
and
120
min),
which
is
indicative
of
a
mouse's
ability
to
clear
glucose.
Mice
with
low
AUC
reflect
a
greater
efficiency
in
clearing
glucose,
as
well
as
healthier
insulin
mobilization
and
sensitivity.
Glucose
clearance:
log10[baseline
glucose
(mg/dL)
÷
glucose
(mg/dL)
2
hrs
post-injection]
Total
adiposity
index
=
[total
weight
of
isolated
fat
pads
(g)
÷
necropsy
body
weight
(g)]
Percent
adiposity
=
[weight
of
isolated
fat
pad
(g)
÷
necropsy
body
weight
(g)]
x
100
growth
rate
=
log10[bw
÷
age]
(g/wk)
pre-weaning
=
from
birth
to
three
weeks
of
age.
post-weaning
=
from
3
wks
of
age
to
10
wks
of
age.
adult
=
10
wks
old
and
beyond.
Test age:
various