1. A COMPARISON OF METABOLIC CHARACTERISTICS
AMONG C57BL/6NTAC, C57BL/6J AND
C57BL/6JBOM DIET INDUCED OBESE MICE WITH
ENVIRONMENTAL CONDITIONING
Michael D. Hayward, Ph.D.

2. Modeling the Obesity Epidemic in Rodents
• An obesity epidemic is believed to be
responsible for the increase in:
– Type 2 diabetes
– Atherosclerosis
– Hypertension
• Monogenic lines of obese rodents have
been known for many years
− ob/ob and db/db: leptin and leptin receptor
mutants
− Ay/a (yellow agouti): an ectopically
overexpressed melanocortin receptor antagonist
− MC-4 KO: induced melanocortin receptor KO
A diet-induced model of obesity is thought to be
more reflective of most cases of human obesity

3. The Diet-Induced Obese (DIO) Model
• DIO characteristics
– Obese-> increased adiposity
– Glucose intolerant
– Insulin resistant
– Mild hyperglycemia
• Diet-induced models of obesity in rodents are more relevant
to the development of type 2 diabetes
– A large percentage of type 2 diabetes appears to be polygenic
– Weight gain and insulin resistance are diet-related
– DIO models early stages of type 2 diabetes (not polydipsia,
polyuria, glucosuria or weight loss)
Consistent with many human characteristics of obesity and
pre-diabetes, dyslipidemia

4. The C57BL/6 Mouse As The DIO Mouse
• Not just environmentally caused, genetics are
involved in the generation of a DIO mouse
– Only some strains are sensitive to DIO (C57BL/6 is
sensitive, BALB/c is resistant)
– C57BL/6 is the strain of choice for generating DIO
mice Body Weight Gain after 9 Weeks of HFD
80
% of BW Gain
60
40
20
0
e
e
e
e
al
al
al
al
M
M
m
m
Fe
Fe
b
6
B
al
b
6
B
B
al
B

5. Multiple C57BL/6 Strains Exist
• Divergence of N substrain and J substrain occurred in 1951.
• Possible polymorphisms could contribute to differences in DIO
conditioning.
• One known polymorphism, a null mutation in the Nnt gene,
occurred in C57BL/6J between 1976 and 1984. The mutation
does not exist in many other C57BL/6 substrains.
Nnt (-)
C57BL/6J
1951 to N 1971 to J Bom
C57BL/6 J Bom
1948 to J
1988 to J Bom Tac
C57BL/6 JBomTac
C57BL/6N
1991 to N Tac
C57BL/6NTac

6. Experimental Design To Test Variations Among C57BL/6
Substrains As DIO Models
Genetic
Environment
Conditioning Site Diet
C57BL/
C57BL/ C57BL/
6JBom
6J 6NTac
Tac
Regular
Research
Diet (Purina
Bar Harbor, Germantown, Cranbury, Diets
Pico Rodent
ME NY NJ RD12492
Chow 5053
(60 % Fat
(13.2 %
Kcal)
Kcal)

7. Experimental Groups
Substrain Source Location of High Fat Shipping Age Abbreviation
Diet Conditioning
C57BL/6NTac Taconic Taconic, Germantown, 14 weeks NTac DIO
NY
C57BL/6J The Jackson Laboratory The Jackson 14 weeks J DIO
Laboratory, Bar Harbor,
ME
C57BL/6NTac Taconic Taconic Cranbury, NJ 4 weeks NTac DIO
Cranbury
C57BL/6JBom Taconic Taconic Cranbury, NJ 4 weeks JBomTac DIO
Cranbury
C57BL/6J The Jackson Laboratory Taconic Cranbury, NJ 4 weeks J DIO Cranbury
C57BL/6NTac Taconic None 14 weeks NTac Reg Diet
C57BL/6JBom Taconic None 14 weeks JBomTac Reg
Diet
C57BL/6J The Jackson Laboratory None 14 weeks J Reg Diet

8. Experimental Protocol For Metabolic
Characterization
Age Time on Diet Week 10 Males
14 8 -2 single housing acclimation
15 9 -1 acclimation
T.Col & lipoprint
Adiponectin
16 10 1 fasted bleed
FFA
Triglycerides
17 11 2 Food Intake
18 12 3 Glucose Tolerance+ Insulin
19 13 4 DEXA
20 14 5 rest
21 15 6 Insulin Tolerance Test All DIO mice
Leptin started feeding
22 16 7 Fed Bleed
Corticosterone on the high-fat
Pancreatic Insulin diet at 6 weeks of
23 17 8 WAT weights Clinical Chem age
Hematology

9. Body Weights
50
NTac DIO
Body weight (g)
J DIO
NTac DIO Cranbury
40
JBomTac DIO Cranbury
J DIO Cranbury
NTac Regular Diet
30
J Regular Diet
JBomTac Reg Diet
20
15 16 17 18 19 20 21 22 23
Age in Weeks
•NTac C57BL/6 DIO mice were heavier than J on the HFD, regardless of
conditioning location
•NTac C57BL/6 DIO mice were heavier than JBomTac DIO mice
•Mice conditioned in Cranbury were heavier than the corresponding
substrain of mice from the two commercial supply locations (Bar Harbor or
Germantown)

10. Food Intake After 11 Weeks on a HFD
(With Regular Diet Controls)
Food Intake/24 hrs
7.5
NTac DIO
J DIO
5.0
Grams
NTac DIO Cranbury
J DIO Cranbury
2.5 JBomTac DIO Cranbury
NTac Reg Diet
0.0 J Reg Diet
JBomTac Reg Diet
No differences between any of the groups
in total food intake

11. DEXA Analysis of Body Composition After
13 Weeks of HFD
% Fat Lean Mass
50 30
NTac DIO
40
*** J DIO
NTac DIO Cranbury
percent
20
grams
30 J DIO Cranbury
JBomTac DIO Cranbury
20 NTac Reg Diet 10
J Reg Diet
10
JBomTac Reg
0 0
NTac DIO mice have higher adiposity than
the J DIO mice from the respective location

12. Oral Glucose Tolerance Test After 12
Weeks on a HFD-Glucose
750 NTac DIO
J DIO
Glucose (mg/dL)
NTac DIO Cranbury
500
J DIO Cranbury
JBomTac DIO Cranbury
250 NTac Reg Diet
J Reg Diet
JBomTac Reg Diet
0
0 15 30 45 60 75 90 105 120
Time (min)
• NTac DIO and J DIO (from Bar Harbor or Germantown) did not
differ from each other
• J DIO from Cranbury were more impaired than NTac or JBomTac
Male mice were fasted for 16 hrs and were administered 2 g/kg of glucose po.

13. Oral Glucose Tolerance Test After 12
Weeks on a HFD-Insulin
10 *
Insulin (ng/ml) NTac DIO
J DIO
NTac DIO Cranbury
J DIO Cranbury
5 JBomTac DIO Cranbury
NTac Reg Diet
J Reg Diet
*
JBomTac Reg Diet
0
0 15 30 45 60 75 90 105 120
Time (min)
•Tac DIO mice were more hyperinsulinemic than other mice,
regardless of conditioning site
•Cranbury conditioned mice were generally more
hyperinsulinemic than the DIO mice from the respective
locations, consistent with the body weight differences

14. Pancreatic Insulin Content
g insulin/mg protein
15
NTac DIO
### J DIO
10 # NTac DIO Cranbury
## *** J DIO Cranbury
JBomTac DIO Cranbury
5
NTac Reg Diet
J Reg Diet
0 JBomTac Reg Diet
• Insulin content of NTac DIO was significantly higher than J DIO (from
Bar Harbor or Germantown)
• Both groups of NTac DIO mice had increased insulin content compared
to regular diet fed NTac
• The J DIO mice conditioned in Cranbury had increased insulin content
compared to regular diet fed J
• Pancreatic insulin content was consistent with insulin levels during
OGTT

15. Insulin Tolerance Test After 15 Weeks on
a HFD – Percent of Baseline
150
NTac DIO
J DIO
NTac DIO Cranbury
Percent
100
JBomTac DIO Cranbury
J DIO Cranbury
NTac Regular Diet
50
J Regular Diet
JBomTac Regular Diet
0
0 30 60 90 120
Time (min)
NTac and JBomTac DIOs show more insulin resistance than J DIO
mice, regardless of conditioning site.
The 21 weeks old male mice were fasted for 3 hours while acclimating to a procedure room, near the end of the light cycle.
The mice received 1.0 U/kg of normal insulin (Humulin R, Eli Lilly) by intraperitoneal injection.

16. Fasted Chemistries – Lipids, Adiponectin
(an Adipokine)
Free Fatty Acids Cholesterol
1.5 NTac DIO 200 **
FFA (mmol/l)
** J DIO
1.0 * NTac DIO Cranbury
J DIO Cranbury
mg/dl
JBomTac DIO Cranbury 100
0.5 NTac Reg Diet
J Reg Diet
JBomTac Reg Diet
0.0 0
Adiponectin
•Free fatty acids were significantly higher in the
*** NTac DIO than J DIO but the reverse was true for
30000 ***
the corresponding regular diet groups.
20000
ng/ml
• NTac DIO mice had higher cholesterol levels than
10000 J DIO mice.
0 •Adiponectin levels were lower in both NTac DIO
groups compared to the corresponding J DIO group

17. Fed Chemistries – Obesity Related
Hormones
Serum Leptin Levels Corticosterone
150000
TAC DIO
300
100000 JAX DIO
Unit (pg/ml)
TAC DIO Cranbury
50000 JAX DIO Cranbury
200
ng/ml
J Bom Tac DIO Cranbury
TAC Reg Diet
1000 JAX Reg Diet
J Bom Tac Reg Diet
100
0 0
Serum leptin and corticosterone levels were
elevated in all DIO models

18. Summary
• The B6NTac substrain was heavier and gained weight more
quickly
– An increase in adiposity was verified by DEXA scan
– Leptin and corticosterone levels were also correlated with the relative adiposity of the different
experimental groups.
• Glucose tolerance was affected most in the B6J substrain
• Hyperinsulinemia was highest in B6NTac substrain
– The pancreatic insulin content was consistent with this observation.
• B6NTac were more insulin resistant
– Adiponectin levels were consistent with the relative insulin sensitivity in the experimental groups.

19. Nicotinamide Nucleotide
Transhydrogenase Mutation
http://jaxmice.jax.org/strain/000664.html
A naturally occurring deletion in nicotinamide nucleotide transhydrogenase (Nnt) exons 7-11 occurred in
C57BL/6J sometime prior to 1984. This deletion results in the absence of the NNT protein, and is
associated with impaired glucose homeostasis control and reduced insulin secretion. This mutation is not
found in C57BL/6JEi, C57BL/6N, C57BL/6NJ, C57BL/6ByJ, C57BL/10J, C57L/J, or C58/J (Toye AA, et al,
Diabetologia, 2005). Since C57BL/6JEi separated from C57BL/6J in 1976, the Nnt deletion arose
sometime between 1976 and 1984.
http://www.taconic.com/wmspage.cfm?parm1=760
Origin: C57BL/6 litters were received in 1991 at F151 from the NIH Animal Genetic Resource. Cesarean
derived in 1991 at Taconic, a foundation colony is maintained in gnotobiotic isolators. Origin is as follows:
to NIH in 1951 from Jax at F32; to Jax in 1948 from Hall.
J Bom are WT at the NNT locus, but are also not as heavy as the
NTac, suggesting this gene has little to do with the body weight
differences.
While the NNT mutation has been hypothesized to affect insulin
secretion, the NTac mice had higher insulin secretion than the
JBom mice.

20. Closing Remarks
• Genetics vs environment in a DIO model
– Showed examples of contribution by both factors
– Influences of environment were evident (could be differences in cage
style, housing density, etc)
– Clear differences exist between the NTac, J and J Bom substrains
(polymorphism, like NNT mutation etc)
Regardless of environment, it appears that the C57BL/6N
substrain gains weight and develops insulin resistance sooner
than C57BL/6JBom and C57BL/6J
• Choosing the right mouse
– Focus of study (hyperglycemia, insulin resistance, rate of obesity, etc.)
– If genetically modified model, source of ES cells should be consistent
with substrain used for breeding