A COMPARISON OF METABOLIC CHARACTERISTICSAMONG C57BL/6NTAC, C57BL/6J ANDC57BL/6JBOM DIET INDUCED OBESE MICE WITHENVIRONMENTAL CONDITIONING Michael D. Hayward, Ph.D.
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 KOA diet-induced model of obesity is thought to bemore reflective of most cases of human obesity
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
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
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 Bom1948 to J 1988 to J Bom Tac C57BL/6 JBomTac C57BL/6N 1991 to N Tac C57BL/6NTac
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)
Experimental GroupsSubstrain Source Location of High Fat Shipping Age Abbreviation Diet ConditioningC57BL/6NTac Taconic Taconic, Germantown, 14 weeks NTac DIO NYC57BL/6J The Jackson Laboratory The Jackson 14 weeks J DIO Laboratory, Bar Harbor, MEC57BL/6NTac Taconic Taconic Cranbury, NJ 4 weeks NTac DIO CranburyC57BL/6JBom Taconic Taconic Cranbury, NJ 4 weeks JBomTac DIO CranburyC57BL/6J The Jackson Laboratory Taconic Cranbury, NJ 4 weeks J DIO CranburyC57BL/6NTac Taconic None 14 weeks NTac Reg DietC57BL/6JBom Taconic None 14 weeks JBomTac Reg DietC57BL/6J The Jackson Laboratory None 14 weeks J Reg Diet
Experimental Protocol For MetabolicCharacterization 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
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 ofconditioning location•NTac C57BL/6 DIO mice were heavier than JBomTac DIO mice•Mice conditioned in Cranbury were heavier than the correspondingsubstrain of mice from the two commercial supply locations (Bar Harbor orGermantown)
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 DietNo differences between any of the groupsin total food intake
DEXA Analysis of Body Composition After13 Weeks of HFD % Fat Lean Mass 50 30 NTac DIO 40 *** J DIO NTac DIO Cranburypercent 20 grams 30 J DIO Cranbury JBomTac DIO Cranbury 20 NTac Reg Diet 10 J Reg Diet 10 JBomTac Reg 0 0NTac DIO mice have higher adiposity thanthe J DIO mice from the respective location
Oral Glucose Tolerance Test After 12Weeks 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 JBomTacMale mice were fasted for 16 hrs and were administered 2 g/kg of glucose po.
Oral Glucose Tolerance Test After 12Weeks 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 morehyperinsulinemic than the DIO mice from the respectivelocations, consistent with the body weight differences
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
Insulin Tolerance Test After 15 Weeks ona 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 DIOmice, 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) 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
Fed Chemistries – Obesity Related Hormones Serum Leptin Levels Corticosterone 150000 TAC DIO 300 100000 JAX DIOUnit (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
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.
Nicotinamide NucleotideTranshydrogenase Mutationhttp://jaxmice.jax.org/strain/000664.htmlA naturally occurring deletion in nicotinamide nucleotide transhydrogenase (Nnt) exons 7-11 occurred inC57BL/6J sometime prior to 1984. This deletion results in the absence of the NNT protein, and isassociated with impaired glucose homeostasis control and reduced insulin secretion. This mutation is notfound 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 arosesometime between 1976 and 1984.http://www.taconic.com/wmspage.cfm?parm1=760Origin: C57BL/6 litters were received in 1991 at F151 from the NIH Animal Genetic Resource. Cesareanderived 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 theNTac, suggesting this gene has little to do with the body weightdifferences.While the NNT mutation has been hypothesized to affect insulinsecretion, the NTac mice had higher insulin secretion than theJBom mice.
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