Diet Research Data：Effects of High Fat Diet 32 Feeding on Male C57BL/6J and KK-Ay Mice

 

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１.Objective

This study aimed to compare and examine the effects of a high-fat diet (High Fat Diet 32, or HFD32) on normal mice and obese type 2 diabetes model mice.

 

２.Materials and Methods

（1）Experimental Site

The experiment was conducted in the mouse breeding facility (conventional) of Nippon Compound Feed Manufacturing Co., Ltd. (currently Feed One Co., Ltd.) Central Research Laboratory.

（2）Diets

• CE-2: Crude fat content 4.6%, Fat kcal% 11.9%, soluble non-nitrogenous substance content 51.4%, NFE kcal% 59.3%
• High Fat Diet 32 (HFD32): Crude fat content 32.0%, Fat kcal% 56.7%, soluble non-nitrogenous substance content 29.4%, NFE kcal% 23.2%

（3）Animals

Five-week-old male KK-Ay/Ta Jcl (obese type 2 diabetes model) and C57BL/6JJcl (normal) mice were used. There were 6 mice per group.

（4）Housing Conditions

• Temperature and humidity: Temperature = 24-27°C, Humidity = 50-70%
• Lighting: 12-hour light/12-hour dark cycle (lights on 9:00 AM - 9:00 PM)
• Cages: Individually housed in polycarbonate cages with sterilized wood chip bedding
• Diet: Ad libitum
• Drinking water: Ad libitum tap water

（5）Experimental Procedure

Five-week-old KK-Ay and C57BL/6J mice were introduced and acclimatized for 1 week. Mice were then randomly divided into two groups (CE-2 group and HFD32 group) based on body weight and fasting blood glucose levels. The experimental diets were fed from 5 weeks of age to 12 weeks of age. After a 15-hour fast, mice were sacrificed. Blood samples were collected for analysis of serum lipid-related substances (triglycerides, non-esterified fatty acids, total cholesterol), blood glucose, and insulin levels. Statistical analysis was performed using Student's t-test.

 

３.Results

The results are described below.

 

1.Body Weight

Changes in body weight in each strain

Data points and vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and an asterisk (*) indicates a significant difference (p < 0.05).

2.Food and Water Intake

Changes in food intake in each strain

Data points and vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and an asterisk (*) indicates a significant difference (p < 0.05).

Changes in water intake in each strain

Data points and vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and an asterisk (*) indicates a significant difference (p < 0.05).

 

3.Fasting Blood Glucose Levels

Changes in fasting blood glucose levels in each strain

Blood was collected from the tail vein every 2 weeks at 1 PM. Whole blood was measured using a whole blood glucose meter (GlucoTest PROR, Sanwa Kagaku Kenkyusho Co., Ltd.). Vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and an asterisk (*) indicates a significant difference (p < 0.05).

 

4.Oral Glucose Tolerance Test

Oral Glucose Tolerance Test Results in C57BL/6J Mice at 8 and 12 Weeks of Age - 1

The oral glucose tolerance test was conducted at 8 and 12 weeks of age. After an overnight fast (20 hours), mice were orally administered a glucose solution (2 g/kg body weight) using a gavage needle. Blood was collected from the tail vein before administration (0 min) and 30, 60, and 120 min after administration. The obtained blood was stored as plasma at -40°C. Blood glucose levels were measured using the Glucose CⅡ-test Wako (Wako Pure Chemical Industries, Ltd.). Data points and vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and an asterisk (*) indicates a significant difference (p < 0.05).

Oral Glucose Tolerance Test Results in C57BL/6J Mice at 8 and 12 Weeks of Age - 2

【Blood glucose sum and area under the curve (AUC)】

Blood glucose sum was calculated by summing the blood glucose levels at each time point during the oral glucose tolerance test. The area under the glucose curve was calculated using the following formula:

Area under the glucose curve (mg/dl·h) = {(a + 2b + 3c + 2d) × 1/4} - 2a
(a, b, c, and d represent blood glucose levels at 0, 30, 60, and 120 minutes, respectively)

Vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups, and an asterisk (*) indicates a significant difference (p < 0.05).

Oral Glucose Tolerance Test Results in KK-Ay Mice at 8 and 12 Weeks of Age - 1

The oral glucose tolerance test was conducted at 8 and 12 weeks of age. After an overnight fast (20 hours), mice were orally administered a glucose solution (2 g/kg body weight) using a gavage needle. Blood was collected from the tail vein before administration (0 min) and 30, 60, and 120 min after administration. The obtained blood was stored as plasma at -40°C. Blood glucose levels were measured using the Glucose CⅡ-test Wako (Wako Pure Chemical Industries, Ltd.). Data points and vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and an asterisk (*) indicates a significant difference (p < 0.05).

Oral Glucose Tolerance Test Results in KK-Ay Mice at 8 and 12 Weeks of Age - 2

【Blood glucose sum and area under the curve (AUC)】

Blood glucose sum was calculated by summing the blood glucose levels at each time point during the oral glucose tolerance test. The area under the glucose curve was calculated using the following formula:

Area under the glucose curve (mg/dl·h) = {(a + 2b + 3c + 2d) × 1/4} - 2a
(a, b, c, and d represent blood glucose levels at 0, 30, 60, and 120 minutes, respectively)

Vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups, and an asterisk (*) indicates a significant difference (p < 0.05).

5.Insulin Tolerance Test

Insulin Tolerance Test Results in C57BL/6J mice at 8 and 12 Weeks of Age

An insulin solution (0.75 IU/kg body weight) was administered intraperitoneally, and blood was collected from the tail vein before administration (0 min) and 20, 40, 60, 80, and 120 min after administration. Whole blood was measured using a whole blood glucose meter (GlucoTest PROR, Sanwa Kagaku Kenkyusho Co., Ltd.). Data points and vertical bars in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and an asterisk (*) indicates a significant difference (p < 0.05).

Insulin Tolerance Test Results in KK-Ay mice at 8 and 12 Weeks of Age

An insulin solution (0.75 IU/kg body weight) was administered intraperitoneally, and blood was collected from the tail vein before administration (0 min) and 20, 40, 60, 80, and 120 min after administration. Whole blood was measured using a whole blood glucose meter (GlucoTest PROR, Sanwa Kagaku Kenkyusho Co., Ltd.). Data points and vertical bars in the graph represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and an asterisk (*) indicates a significant difference (p < 0.05).

6.Blood Analysis at Biopsy

Serum and Plasma Analysis at Biopsy

Values in the table represent the mean ± standard error. Blood was collected at Biopsy (12 weeks of age). After a 20-hour fast, animals were anesthetized with ether, and blood was collected from the posterior vena cava and stored as plasma and serum at -40°C. Total cholesterol, triglycerides, non-esterified fatty acids, and glucose were measured using commercial assay kits (Cholesterol E-test Wako, Triglyceride E-test Wako, NEFA test Wako, and Glucose CⅡ-test Wako, respectively). Insulin was measured using an insulin assay kit (Morigana Seikagaku Kenkyusho Co., Ltd.). Statistical analysis was performed between groups, and an asterisk (*) indicates a significant difference (p < 0.05).

 

7.Weights of Major Organs at Biopsy

Organ Weights

Values in the table represent the mean ± standard error. Statistical analysis was performed between groups, and an asterisk (*) indicates a significant difference (p < 0.05).

Weights of major organs and regional adipose tissue at Biopsy