A COMPARISON OF METABOLIC CHARACTERISTICS AMONG C57BL/6NTAC, C57BL/6J AND C57BL/6JBOM DIET INDUCED OBESE MICE WITH ENVIRONMENTAL CONDITIONING

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.

Modeling the Obesity Epidemic in RodentsModeling 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

The Diet-Induced Obese (DIO) ModelThe 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

The C57BL/6 Mouse As The DIO MouseThe 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

% o

f B

W G

ain

B6 M

ale

Balb M

ale

B6 Fem

ale

Balb F

emal

e0

20

40

60

80

Multiple C57BL/6 Strains ExistMultiple 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.

1948 to J

C57BL/6J1951 to N

C57BL/6N

1971 to J Bom

1991 to N Tac

1988 to J Bom TacC57BL/6 J Bom

C57BL/6 JBomTac

C57BL/6NTac

Nnt (-)

Experimental Design To Test Variations Among C57BL/6 Substrains As DIO Models

Environment

Conditioning Site

Bar Harbor, ME

Germantown, NY

Cranbury, NJ

Diet

Regular Diet

(Purina Pico

Rodent Chow 5053

(13.2 % Kcal)

Research Diets

RD12492 (60 % Fat

Kcal)

Genetic

C57BL/6J

C57BL/6NTac

C57BL/6JBom

Tac

Experimental GroupsExperimental Groups

Substrain Source Location of High Fat Diet Conditioning

Shipping Age Abbreviation

C57BL/6NTac Taconic Taconic, Germantown, NY

14 weeks NTac DIO

C57BL/6J The Jackson Laboratory The Jackson Laboratory, Bar Harbor, ME

14 weeks J DIO

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

Experimental Protocol For Metabolic CharacterizationExperimental Protocol For Metabolic Characterization

Age Time on Diet Week

14 8 -215 9 -1

T.Col & lipoprintAdiponectinFFATriglycerides

17 11 218 12 319 13 420 14 521 15 6

LeptinCorticosteronePancreatic InsulinClinical ChemHematology

22 16 7 Fed Bleed

restInsulin Tolerance Test

16 10 1 fasted bleed

single housing acclimationacclimation

10 Males

23 17 8 WAT weights

Food IntakeGlucose Tolerance+ Insulin

DEXA

All DIO mice started feeding on the high-fat diet at 6 weeks of age

Body WeightsBody Weights

15 16 17 18 19 20 21 22 2320

30

40

50NTac DIO

J DIO

Age in Weeks

Bo

dy

wei

gh

t (g

)

JBomTac DIO Cranbury

NTac DIO Cranbury

J DIO Cranbury

NTac Regular Diet

J Regular Diet

JBomTac Reg Diet

• 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)

Food Intake After 11 Weeks on a HFD (With Regular Diet Controls)Food Intake After 11 Weeks on a HFD (With Regular Diet Controls)

No differences between any of the groups in total food intake

Food Intake/24 hrs

0.0

2.5

5.0

7.5NTac DIOJ DIONTac DIO CranburyJ DIO CranburyJBomTac DIO CranburyNTac Reg DietJ Reg DietJBomTac Reg Diet

Gra

ms

DEXA Analysis of Body Composition After 13 Weeks of HFDDEXA Analysis of Body Composition After 13 Weeks of HFD

NTac DIO mice have higher adiposity than the J DIO mice from the respective location

% Fat

0

10

20

30

40

50***

per

cen

t

NTac DIOJ DIONTac DIO CranburyJ DIO CranburyJBomTac DIO CranburyNTac Reg DietJ Reg DietJBomTac Reg

Lean Mass

0

10

20

30

gra

ms

Oral Glucose Tolerance Test After 12 Weeks on a HFD-GlucoseOral Glucose Tolerance Test After 12 Weeks on a HFD-Glucose

0 15 30 45 60 75 90 105 1200

250

500

750NTac DIOJ DIO

Time (min)

Glu

cose

(m

g/d

L)

JBomTac DIO Cranbury

NTac DIO Cranbury

J DIO Cranbury

NTac Reg Diet

J Reg Diet

JBomTac Reg Diet

• 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.

Oral Glucose Tolerance Test After 12 Weeks on a HFD-InsulinOral Glucose Tolerance Test After 12 Weeks on a HFD-Insulin

0 15 30 45 60 75 90 105 1200

5

10NTac DIO

J DIO

Time (min)

Insu

lin (

ng

/ml)

*

NTac DIO Cranbury

J DIO Cranbury

JBomTac DIO Cranbury

*

NTac Reg Diet

J Reg Diet

JBomTac Reg Diet

• 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

Pancreatic Insulin ContentPancreatic Insulin Content

• 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

0

5

10

15NTac DIO

J DIO Cranbury

J DIO

NTac Reg DietJ Reg Diet

JBomTac DIO Cranbury

NTac DIO Cranbury

JBomTac Reg Diet g in

sulin

/mg

pro

tein

***

###

#

##

Insulin Tolerance Test After 15 Weeks on a HFD – Percent of BaselineInsulin Tolerance Test After 15 Weeks on a HFD – Percent of Baseline

0 30 60 90 1200

50

100

150 NTac DIO

J DIO

Time (min)

Pe

rce

nt NTac DIO Cranbury

JBomTac DIO Cranbury

J DIO Cranbury

NTac Regular Diet

J Regular Diet

JBomTac Regular Diet

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.

Fasted Chemistries – Lipids, Adiponectin (an Adipokine)Fasted Chemistries – Lipids, Adiponectin (an Adipokine)

Free Fatty Acids

0.0

0.5

1.0

1.5

FF

A (

mm

ol/l

)

NTac DIOJ DIONTac DIO CranburyJ DIO Cranbury

JBomTac DIO CranburyNTac Reg DietJ Reg DietJBomTac Reg Diet

***

Cholesterol

0

100

200

mg

/dl

**

• Free fatty acids were significantly higher in the NTac DIO than J DIO but the reverse was true for the corresponding regular diet groups.

• NTac DIO mice had higher cholesterol levels than J DIO mice.

• Adiponectin levels were lower in both NTac DIO groups compared to the corresponding J DIO group

Adiponectin

0

10000

20000

30000

ng

/ml

*** ***

Fed Chemistries – Obesity Related HormonesFed Chemistries – Obesity Related Hormones

Corticosterone

0

100

200

300

ng

/ml

Serum Leptin Levels

0

1000

50000

100000

150000

Un

it (p

g/m

l)

TAC DIOJAX DIOTAC DIO CranburyJAX DIO CranburyJ Bom Tac DIO CranburyTAC Reg DietJAX Reg DietJ Bom Tac Reg Diet

Serum leptin and corticosterone levels were elevated in all DIO models

SummarySummary

• 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.

Nicotinamide Nucleotide Transhydrogenase MutationNicotinamide Nucleotide Transhydrogenase Mutation

http://www.taconic.com/wmspage.cfm?parm1=760Origin: 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.

http://jaxmice.jax.org/strain/000664.htmlA 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.

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.

Closing RemarksClosing 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