Molecular Typing of Salmonella gallinarum by Random Amplification of

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					               Studies on Production of High Fat Diet Induced Obesity C57BL/6NCrjBgi Mice
                 Chungtaek Han*, Sanghoon Lee, Seungbin Lim, Hyunseok Kong, Soohyeon Kim, Sangkyun Lee and Jaejin Jang
                                                                                      HaeEun Biomedical Research Institute, Orient Bio, Inc.
                                                                          143-1 Sangdaewon-Dong, Jungwon-Gu, Seongnam-Si, Gyeonggi-Do 462-120, Korea



                                                           Abstract                                                                                                                                                                                          Results and Discussion
  To save researcher’s animal room space, time and expense, we developed a standard protocol for production of diet                                                1. Body Weight Changes
 induced obesity C57BL/6NCrjBgi mice. This model should better reflect the physiology of the majority of human                                                        The mean body weight of D-45 Group was significantly increased compared to D-10 Group as early as 1 week after
 obesity than mouse genetic models of obesity with defects in leptin or leptin signaling. In this study, we have                                                      initiation of diet. At the final week of diet, the average weight of D-45 Group is 44.20±2.42 g. It shows
 monitored general clinical signs, weekly body weight changes, weekly food intake, organ weights and glucose                                                          approximately 14.69 g more than 29.51±2.19 g in D-10 Group (Figure 1). D-45 Group mice have 2 times more
 tolerance on male C57BL/6NCrjBgi mice fed high (D12451, Rodent Diet with 45 kcal% fat, Research Diets Inc., New                                                      average weight than D-10 Group in overall experimental period (Figure 2).
 Brunswick, NJ; D-45 Group) and low (D12450B, Rodent Diet with 10 kcal% fat; D-10 Group) fat diets for 16 weeks.                                                  50                                                                                                                                                                                                                                   4.
                                                                                                                                                                                                                                                                                                                                                                                                        0                                     D -45
                                                                                                                                                                                     D -45                                                                                                *** ***                                                                                                                      **                     D -10
                                                                                                                                                                                                                                                                                    ***
                                                                                                                                                                                     D -10                                                                 ***         ***
                                                                                                                                                                                                                                                     ***                                                                                                                                                                           *** ***
 The results are summarized as followings.                                                                                                                        40                                                                           ***                                               20
                                                                                                                                                                                                                                                                                               44.
                                                                                                                                                                                                                                                                                                                                                                                                        0
                                                                                                                                                                                                                                                                                                                                                                                                       3.                                                                  ***
                                                                                                                                                                                                                                       ***




                                                                                                                                                                                                                                                                                                                                                                                (g /m o use/w eek)
                                                                                                                                                                                                                                                                                                                                                                                                                                                            ** ***




                                                                                                                                                                                                                                                                                                                                                                                                   n
  1) The mean body weight of D-45 Group had significantly increased compared to D-10 Group as early as 1 week




                                                                                                                                              B o d y W ei ht )




                                                                                                                                                                                                                                                                                                                                                                               B o d y w ei ht g ai
                                                                                                                                                                                                                                 ***                                                                                                                                                                                                                                                             ***




                                                                                                                                                          g (g
                                                                                                                                                                                                                          ***                                                                                                                                                                                                                   **                               ***
                                                                                                                                                                                                                                                                                                                                                                                                                                                      **
                                                                                                                                                                                                                   ***                                                                                                                                                                                                                                                                 ***              ***
     after initiation of diet.                                                                                                                                    30                                         ***                                                                                                                                                                                                                                                                                              **           **




                                                                                                                                                                                                                                                                                                                                                                                           g
                                                                                                                                                                                                      ***                                                                                                                                                                                               0
                                                                                                                                                                                                                                                                                                                                                                                                       2.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 **
                                                                                                                                                                                                ***
  2) Food intake was not significantly different between two groups, but caloric intake and food efficiency ratio                                                                       ***
                                                                                                                                                                                                                                                                                                 51
                                                                                                                                                                                                                                                                                               29.

     were significantly increased in D-45 Group.                                                                                                                  20
                                                                                                                                                                          56
                                                                                                                                                                        16.
                                                                                                                                                                                ***
                                                                                                                                                                                                                                                                                                                                                                                                        0
                                                                                                                                                                                                                                                                                                                                                                                                       1.
                                                                                                                                                                                                                                                                                                                 D-10                                            D-45
  3) Absolute organ weights of spleen, liver, pancreas and kidneys in D-45 Group were significantly increased                                                             65
                                                                                                                                                                        15.
     compared to D-10 Group.                                                                                                                                      10                                                                                                                                                                                                                                    0
                                                                                                                                                                                                                                                                                                                                                                                                       0.
                                                                                                                                                                         4       5       6      7     8       9    10     11     12    13      14    15     16         17           18    19     20                                                                                                                      5          6     7      8    9     10 11 12 13 14 15 16 17 18 19 20
  4) The significantly increased fat deposit in epididymal and perirenal area was found in D-45 Group.                                                                                                                   W e e ks o f d ie t                                                                                                                                                                                                                          W e e ks o f d ie t
  5) The D-45 Group developed glucose intolerance compared to D-10 Group.
                                                                                                                                                                  . Figure 1. Body weight changes of C57BL/6NCrjBgi mice                                                                                                                                                                . Figure 2. Body weight gain of C57BL/6NCrjBgi mice
                                                                                                                                                                              fed high and low fat diets for 16 weeks                                                                                                                                                                               fed high and low fat diets for 16 weeks
  In summary, the first characterization data of C57BL/6NCrjBgi mice fed high fat diet for 16 weeks is very similar to                                                                       Values represent means±S.D. (n=50)                                                                                                                                                                                                      Values represent means±S.D. (n=50)
 the most frequently used C57BL/6J diet induced obesity (DIO) mouse model of Jackson Laboratory, USA. Therefore                                                                              ***:Significantly different from control (p<0.001)                                                                                                                                                                                      **,***:Significantly different from control (p<0.01 and
                                                                                                                                                                                                                                                                                                                                                                                                                                     p<0.001, respectively)
 the results obtained in this study were considered to be a useful reference data in obesity and type II diabetes research
 using DIO mouse model.                                                                                                                                            2. Food Intake, Caloric Intake and Food efficiency
                                                                                                                                                                      Weekly food intake of D-45 Group was slightly higher than those of D-10 Group during the overall experimental
 Key words: C57BL/6NCrjBgi mice, DIO model, obesity, glucose tolerance                                                                                                period with no statistically significant difference. There was no food intake changes according to the age (Figure
                                                                                                                                                                      3A). Caloric intake of D-45 Group was significantly increased compare to D-10 Group (Figure 3B). Food
                                                                                                                                                                      efficiency ratio was tend to decrease with increase of age in both groups and D-45 Group show 2 times more
                                                        Introduction                                                                                                  higher than D-10 Group (Figure 3C).
                                                                                                                                                                                                                                                                           B 16.
                                                                                                                                                                                                                                                                               0
                                                                                                                                                                                                                                                                                                                                                                                                                         C 1.
                                                                                                                                                                                                                                                                                                                                                                                                                            5                 D -45
                                                                                                                                                  0
                                                                                                                                               A 3.                                                                                                                                                   D -45
                                                                                                                                                                                     D -45
                                                                                                                                                                                                                                                                                                      D -10                                                                                                                           *** *** D -10




                                                                                                                                                                                                                                                                                                                                                                                                            Food ef i i ncy r to
                                                                                                                                                                                     D -10                                                                                                                                                                                                                                                    ***




                                                                                                                                                                                                                                                                                                                                                                                                                             a i
  The increasing prevalence of overweight and obesity is an important public health problem contributing to significant                                           2.
                                                                                                                                                                   8
                                                                                                                                                                                                                                                                                                                                                                                                                                    2
                                                                                                                                                                                                                                                                                                                                                                                                                                   1.
                                                                                                                                                                                                                                                                                      0
                                                                                                                                                                                                                                                                                    14.




                                                                                                                                                                                                                                                                 (kcal/mouse/day)
                                                                                                                                                                                                                                                                   Caloric intake
 excess of the associated disease and mortality. People who are obese or overweight have a higher risk of disease                                                                                                                                                                                                                                                *** *** *** *** ***                                                                                  *** *** ***
                                                                                                                                       (g/mouse/day)




                                                                                                                                                                                                                                                                                                                 ***                                                                                                                                                                             ***
                                                                                                                                        Food intake




                                                                                                                                                                                                                                                                                           ***                                                                                                                                      9
                                                                                                                                                                                                                                                                                                                                                                                                                                   0.
                                                                                                                                                                   6
                                                                                                                                                                  2.                                                                                                                             *** ***   ***             *** *** *** *** ***                                                                                                            ***                       *** ***
 including coronary heart disease, diabetes, hypercholesterolemia, hypertension, degenerative joint disease and




                                                                                                                                                                                                                                                                                                                                                                                                                    (% )
                                                                                                                                                                                                                                                                                                                       ***




                                                                                                                                                                                                                                                                                                                                                                                                                   fc e
                                                                                                                                                                                                                                                                                                                                                                                                                                                                ***                                    *** ***
                                                                                                                                                                                                                                                                                      0
                                                                                                                                                                                                                                                                                    12.                                                                                                                                                                                                                          *** ***
 obstructive sleep apnoea. Life expectancy is reduced by obesity, mainly through the effect of increased weight on                                                2.
                                                                                                                                                                   4                                                                                                                                                                                                                                                                6
                                                                                                                                                                                                                                                                                                                                                                                                                                   0.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           ***
 related conditions.                                                                                                                                                                                                                                                                  0
                                                                                                                                                                                                                                                                                    10.                                                                                                                                             3
                                                                                                                                                                                                                                                                                                                                                                                                                                   0.
                                                                                                                                                                   2
                                                                                                                                                                  2.


  The diet induced obesity (DIO) model has been largely used in obesity and type II diabetes research, however the                                                2.
                                                                                                                                                                   0                                                                                                                8.
                                                                                                                                                                                                                                                                                     0                                                                                                                                              0
                                                                                                                                                                                                                                                                                                                                                                                                                                   0.
                                                                                                                                                                          5 6         7 8 9 10 11 12 13 14 15 16 17 18 19 20                                                               5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20                                                                                                     5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
 time necessary for the animals to become obese differs greatly between diets. Recently, The Jackson Laboratory
                                                                                                                                                                                                          W e e ks o f d ie t                                                                                            W eeks of diet                                                                                                                                  W eeks of diet
 completed characterizing the growth curves, blood glucose levels, and percent body fat of C57BL/6J males placed on
 standard DIO regimens.                                                                                                                                                   Figure 3. Food intake(A), caloric intake(B) and food efficiency ratio(C) of C57BL/6NCrjBgi mice fed high and low fat diets for 16 weeks
                                                                                                                                                                                                Values represent means±S.D.(n=50); ***: Significantly different from control (p<0.001)

 The DIO model better reflect the human obesity than genetic models of obesity, as there are only a few patients
 reported in the literature in whom mutations of leptin or leptin receptor genes account for the obesity. Obese humans                                                 3. Organ Weights
 present high levels of leptin correlated to fat mass, indicating that the vast majority of human obesity cannot be                                                       The absolute organ weight of spleen, liver, pancreas, thymus and kidneys of D-45 Group was significantly increased
 attributed to defects in leptin or leptin receptors.                                                                                                                     compared to D-10 Group, however organ weight of brain, heart, lung, adrenal glands, testes and eididymides was
                                                                                                                                                                          not significantly different between two groups. In the relative organ weight, the weight of D-45 Group was
                                                            Purpose                                                                                                       significantly decreased compared to D-10 Group (Table 3).
                                                                                                                                                                             Table 3. Absolute and relative organ weights of C57BL/6NCrjBgi mice fed high and low fat diets for 16 weeks
  The purpose of this study was first to establish and characterize a model that mimics the physiological changes                                                                                                                                                                        Absolute weight(㎎)                                                                                                                         Relative weight(% of terminal BW)
 occurring in obesity by using a same source of high fat diet as that found in the Jackson Laboratory’s standard DIO                                                           Groups
                                                                                                                                                                                                                                                                 D-10                                                             D-45                                                                                                  D-10                                                           D-45
 regimens. Secondly, we would like to provide scientists with this new information regarding DIO services by Orient
                                                                                                                                                                               Terminal body weight (g)                                                28.31±1.63                                                         40.95±2.60***                                                                                    28.31±1.63                                                   40.95±2.60***
 Bio, Inc.
                                                                                                                                                                               Brain                                                                   395.93±6.58                                                        393.73±21.85                                                                                         1.40±0.08                                                    0.96±0.08***
                                                                                                                                                                               Spleen                                                                  67.67±5.31                                                          74.93±7.96**                                                                                        0.24±0.02                                                    0.18±0.01***

                                          Materials and Methods                                                                                                                Heart                                                                 134.80±10.68                                                         141.67±12.16                                                                                         0.48±0.03                                                    0.35±0.03***
                                                                                                                                                                               Lung                                                                  198.67±10.27                                                         200.33±12.41                                                                                         0.70±0.05                                                    0.49±0.03***
 1. Animals and Group Assignment                                                                                                                                               Liver                                                                 1088.33±73.09                                                     1257.60±115.82***                                                                                       3.86±0.35                                                    3.07±0.20***
    Male C57BL/6NCrjBgi mice (3 weeks, Orient Bio, Ltd., Korea) were randomly assigned 2 groups. Each group                                                                    Pancreas                                                                115.80±6.89                                                      140.93±11.53***                                                                                        0.41±0.03                                                    0.35±0.03***
    consist of 50 mice and fed high fat D12451-Rodent Diet with 45 kcal% fat (Research Diets, New Brunswick, NJ)
                                                                                                                                                                               Thymus                                                                  59.87±4.82                                                         73.80±8.23***                                                                                        0.21±0.02                                                    0.03±0.05***
    and low fat D12450B-Rodent Diet with 10 kcal% fat (Research Diets, New Brunswick, NJ).
                                                                                                                                                                               Kidneys                                                               309.07±18.29                                                       346.00±17.06***                                                                                        1.09±0.09                                                    0.85±0.06***
 2. The Origin of C57BL/6NCrjBgi Mice                                                                                                                                          Adrenal glands                                                              5.13±0.83                                                         4.87±0.72                                                                                 0.018±0.003                                                     0.011±0.002***
    CC Little (1921, mating of female 57 with male 52 from Miss Abby Lathrop's stock) → The Jackson Lab. (1948)                                                                Testes                                                                166.80±12.78                                                         171.80±10.06                                                                                         0.59±0.05                                                    0.42±0.03***
    → NIH (1951, F32) → Charles River USA (1974, F78) → Charles River Japan (1976, F101) → Orient Bio (2001,
                                                                                                                                                                               Epididymides                                                            65.53±4.09                                                           66.40±2.95                                                                                 0.161±0.013                                                     0.113±0.013***
    F118)
                                                                                                                                                                               Values represent means±S.D.(n=20); **, ***: Significantly different from control (p<0.01 and p<0.001, respectively)


  Table 1. Group assignment                                                                                                                                            4. Body Fat Deposit
       Groups                   Diet ID                   Diet source       %kcal fat          No. of animals                                                             Absolute and relative epididymal and perirenal adipose depot weights of D-45 Group were significantly increased
                                                                                                                                                                          compared to D-10 Group (Table 4 and Figure 4).
        D-10                    D12450B                  Research Diets        10                     50
                                                                                                                                                                             Table 4. Absolute and relative adipose depot weights of C57BL/6NCrjBgi mice fed high and low fat diets for 16 weeks
        D-45                    D12451                   Research Diets        45                     50
                                                                                                                                                                                                                                                                                               Absolute weight(g)                                                                                                                       Relative weight(% of terminal BW)
                                                                                                                                                                               Groups
   Table 2. The Composition of high and low fat diets                                                                                                                                                                                                                               D-10                                               D-45                                                                                              D-10                                                           D-45

         Experimental groups                           D-10                           D-45                                                                                     Terminal body weight(g)                                                           28.31±1.63                                                    40.95±2.60***                                                                                       28.31±1.63                                               40.95±2.60***
               Product                                D12450B                        D12451                                                                                    Sum of fat pads                                                                      1.28±0.20                                                   3.43±0.30***                                                                                        4.52±0.53                                                8.37±0.66***
                                               gm%               Kcal%       gm%              Kcal%
                                                                                                                                                                               Epididymal fat pad                                                                   0.91±0.14                                                   2.34±0.27***                                                                                        3.21±0.38                                                5.71±0.66***
                Protein                        19.2               20          24               20
             Carbohydrate                      67.3               70          41               35                                                                              Perirenal fat pad                                                                    0.37±0.06                                                   1.09±0.12***                                                                                        1.32±0.18                                                2.66±0.25***
                  Fat                           4.3               10          24               45
                                                                                                                                                                               Values represent means±S.D.(n=20); ***: Significantly different from control (p<0.001); Epididymal and perirenal depot weights were each the sum of left and right depots
                 Total                                            100                          100
               Kcal/gm                         3.85                          4.73
                                                                                                                                                                                                                                Epididymal fat pad
              Ingredients                      gm                 kcal        gm              kcal
                Casein                         200                800         200              800
               L-Cystine                         3                 12          3               12                                                                                                                                                                                                                                                                500                                                                                             ***
             Corn Starch                       315                1260       72.8              291                                                                                                                                                                                                                                                                                         D -45(n=10)
                                                                                                                                                                                                                                                                                                                                                                                           D -10(n=10)                                  ***
            Maltodextrin 10                     35                140         100              400          D12450B(10 kcal% fat)                                                                                                                                                                                                                                400                                                                                                                    ***
                Sucrose                        350                1400       172.8             691
                                                                                                                                                                                                                                                                                                                                          Blood Glucose(mg/dL)




               Cellulose                        50                 0           50               0                                                                                                                                                                                                                                                                300
                                                                                                                                                                                                                                        D-10                                                          D-45                                                                                                                                                                                                          ***
             Soybean Oil                        25                225          25              225                                                                           D-45
                 Lard                           20                180        177.5            1598                                                                                                                              perirenal fat pad                                                                                                                200
         Mineral Mix S10026                     10                 0           10               0                                                                                                                                                                                                                                                                                     ***

         Dicalcium Phosphate                    13                 0           13               0                                                                                                                                                                                                                                                                100
          Calcium Carbonate                     5.5                0          5.5               0
           Potassium Citrate                   16.5                0         16.5               0                                                                                                                                                                                                                                                                   0
         Vitamin Mix V10001                     10                 40          10              40                                                                                                                                                                                                                                                                                        0                                              30                       60                         90                      120

           Choline Bitartrate                    2                 0           2                0                                                                                                                                                                                                                                                                                                                                                           m
                                                                                                                                                                                                                                                                                                                                                                                                                                                          Ti e(m in)

         FD&C Yellow Dye#5                     0.05                0                                                                                                                                                                                                                                                                    Figure 5. Effect of glucose tolerance test (75mg/kg, ip) in C57BL/6NCrjBgi
                                                                                                                D12451(45 kcal% fat)                                                                                                            D-10                                                   D-45
          FD&C Red Dye#40                                                    0.05               0                                                                            D-10                                                                                                                                                                 mice fed high and low fat diet for 16 weeks
                 Total                        1055.05             4057      858.15            4057                                                                     .Figure 4. Representative photos of visceral fat accumulation in                                                                                                                                   Values represent means±S.D.(n=10); ***: Significantly different from control (p<0.001)
                                                                                                                                                                                  C57BL/6NCrjBgi mice fed high and low fat diets for 16 weeks

3. Observations                                                                                                                                                        5. Glucose Tolerance
   During the study, general clinical signs, weekly body weight changes and weekly food intake were evaluated. Organ                                                      In glucose tolerance test of D-10 Group, baseline glucose level was 86.57 mg/dL. After 30 minutes of glucose
   weights and adipose pads were weighed at necropsy. After 16 weeks on diet treatment (20 weeks age), 16 hours                                                           injection, glucose level was increased dramatically and reached 303.15 mg/dL and then slowly decreased. After
   fasted glucose tolerance test was conducted.                                                                                                                           120 minutes of glucose injection, the level showed 136.81 mg/dL. In glucose tolerance test of D-45 Group,
                                                                                                                                                                          baseline glucose level was 136.70 mg/dL and glucose level was increased dramatically at 30 and 60 minutes after
4. Statistical Analysis                                                                                                                                                   glucose injection, the level was reached 375.00 and 428.00 mg/dL, respectively and then slowly decreased. After
   The data were presented as means ±SD and analyzed for statistical significance with ANOVA for repeated measures                                                        120 minutes of glucose injection, the level showed 230.20 mg/dL (Figure 5). The D-45 Group developed glucose
   and Student’s t-test; all differences were considered significant when p<0.05, p<0.01, and p<0.001 (SAS, 1987).                                                        intolerance compared to D-10 Group.