Diet Research Data：Effects of Long-Term Quick Fat Feeding on Male Wistar Hannover Rats

 

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１.Objective

The purpose of this study was to collect basic data through a long-term feeding study (50 weeks) of a high-fat diet in rats and to gather data on obesity and diabetic lesions (hyperglycemia and complications) expected to develop due to long-term feeding of a high-fat diet.

 

２.Materials and Methods

（1）Experimental Site

The experiment was conducted in the rat and mouse breeding room (conventional) of the Central Research Institute of Nippon Compound Feed Co., Ltd. (currently Feed One Co., Ltd.).

（2）Diet

• CE-2: Crude fat content 5.9%, Fat kcal% 15.2%, Soluble non-nitrogenous substance content 49.0%, NFE kcal% 56.1%
• Quick Fat (QF): Crude fat content 14.6%, Fat kcal% 32.4%, Soluble non-nitrogenous substance content 44.0%, NFE kcal% 43.4%

（3）Animals

Male BrlHan:WIST@Jcl (GALAS) rats (Wistar Hannover rats) were used as subjects.

（4）Housing

• Temperature and humidity: Temperature = 21-25°C, Humidity = 40-60%
• Lighting: 12-hour light-dark cycle (lights on 9:00-21:00)
• Cages: Two rats per polypropylene rat cage (345×403×177mm)
• Diet: Ad libitum
• Drinking water: Ad libitum

（5）Experimental Methods

Rats were introduced at 4 weeks of age and acclimatized for 1 week (fed CE-2). After acclimatization, rats were divided into groups so that there were no differences in body weight and blood glucose levels between groups. Feeding of the experimental diet started from 5 weeks of age. Six rats in each group were fed for 25 weeks (until 30 weeks of age), and 8 rats were fed for 50 weeks (until 55 weeks of age). Biopsy was performed under isoflurane anesthesia after a 15-hour fast, and the weights of various organs were measured and blood was analyzed. Blood glucose levels were measured using a Glucotest PROR (Sanwa Kagaku Kenkyusho Co., Ltd.). Student's T-test was used for statistical analysis.

 

３.Results

The results are described below.

 

1.Body Weight

Changes in Body Weight

Body weight was measured weekly. The points and vertical lines in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each age, and * indicates a significant difference (p < 0.05).

 

2.Food Intake

Trends in Food Intake

Food intake represents the average daily intake per animal at each age. The points and vertical lines in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each age, and * indicates a significant difference (p < 0.05).

 

3.Casual Blood Glucose Levels

Trends in Casual Blood Glucose Levels

Blood samples were collected from the tail vein every 5 weeks at 1 pm, 4 hours after lights on. Whole blood was measured using a whole blood glucose meter (Glucotest PROR / Sanwa Kagaku Kenkyusho Co., Ltd.). The vertical bars and lines in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each age, and * indicates a significant difference (p < 0.05).

 

4.Fasting Blood Glucose Levels

Trends in Fasting Blood Glucose Levels

Measurements were taken at 5, 15, 25, 40, and 55 weeks (3, 10, 20, 35, and 50 weeks after starting the experimental diet). After a 15-hour fast (18:00-09:00), blood was collected from the tail vein, and whole blood was measured using a whole blood glucose meter (Glucotest PROR / Sanwa Kagaku Kenkyusho Co., Ltd.). The vertical bars and lines in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each age, and * indicates a significant difference (p < 0.05).

 

5.Oral Glucose Tolerance Test

Results of Oral Glucose Tolerance Test at Each Age

The test was conducted at 5, 15, 25, 40, and 55 weeks of age (3, 10, 20, 35, and 50 weeks after starting the experimental diet). After a 15-hour fast (18:00-09:00), a glucose solution (2g/kg body weight) was orally administered via gastric gavage, and blood was collected from the tail vein before administration (0 min) and 30, 60, and 120 min after administration. Whole blood was measured using a whole blood glucose meter (Glucotest PROR / Sanwa Kagaku Kenkyusho Co., Ltd.). The points and vertical lines in the figure represent the mean ± standard error. Statistical analysis was performed between groups at each time point, and * indicates a significant difference (p < 0.05).

 

 

Plasma Insulin Concentrations During Oral Glucose Tolerance Test at Each Age

経Blood was collected simultaneously during the blood glucose measurement during the oral glucose tolerance test, and plasma obtained by centrifugation was used for insulin measurement. An ELISA Insulin kit (Morinaga Seikagaku Kenkyusho Co., Ltd.) was used for insulin measurement. The figure shows the mean ± standard error. Statistical analysis was performed between groups at each time point, and * indicates a significant difference (p < 0.05).

 

 

6.Major Organ Weights at Biopsy

Major Organ Weights at Each Age

Biopsy was performed at 30 and 55 weeks of age. After a 15-hour fast, the abdomen was opened under isoflurane anesthesia, and each organ was removed and weighed. The vertical bars and lines in the figure represent the mean ± standard error. Statistical analysis was performed between groups, and * indicates a significant difference (p < 0.05).

 

 

Weights of Different Adipose Tissues at Each Age

Biopsy was performed at 30 and 55 weeks of age. After a 15-hour fast, the abdomen was opened under isoflurane anesthesia, and the weight of each adipose tissue was measured. The vertical bars and lines in the figure represent the mean ± standard error. Statistical analysis was performed between groups, and * indicates a significant difference (p < 0.05).

 

 

7.Blood Analysis at Biopsy

Changes in Serum Lipid-Related Substances at Each Age

Biopsy was performed at 30 and 55 weeks of age. After a 15-hour fast, the abdomen was opened under isoflurane anesthesia, blood was collected from the posterior vena cava, and serum total cholesterol (T-Cho), HDL-cholesterol (HDL-Cho), triglycerides (TG), and free fatty acids (NEFA) levels were measured. The vertical bars and lines in the figure represent the mean ± standard error. Statistical analysis was performed between groups, and * indicates a significant difference (p < 0.05).

 

8.Adiponectin and Leptin Concentrations at Biopsy

Plasma Leptin Concentrations at Each Age

The figure shows the mean ± standard error. * indicates a significant difference (p < 0.05). Blood was collected from the posterior vena cava at biopsy, and plasma obtained by centrifugation was used for leptin measurement. A Leptin/Rat ELISA kit (Morinaga Seikagaku Kenkyusho Co., Ltd.) was used for leptin measurement.

 

Serum Adiponectin Concentrations at Each Age

The figure shows the mean ± standard error. Blood was collected from the posterior vena cava at biopsy, and serum was used for adiponectin measurement. A mouse/rat adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd.) was used for adiponectin measurement.

 

9.Liver and Adipose Tissue Observation

Tissue Photographs at 55 Weeks of Age (Liver, Adipose Tissue; HE staining, ×300)

Upper left: Liver of the CE-2 group, Upper right: Liver of the QF group, Lower left: Adipose tissue of the CE-2 group, Lower right: Adipose tissue of the QF group. In the liver of the QF group, more severe fat degeneration was observed compared to the CE-2 group. In adipose tissue, relatively well-shaped adipocytes were observed in the CE-2 group, while in the QF group, many cells had irregular shapes and a mixture of large and small cells.

 

Evaluation of Liver and Adipose Tissue

Scoring was performed numerically based on the "frequency" and "severity" of findings: 0: no evidence, 0.5: rare slight, 1: slight = 1, 1.5: slight ~ moderate, 2.0: moderate, 2.5: moderate ~ severe, 3.0: severe.

 

 

Results of Tissue Evaluation

Note: Fat degeneration refers to findings of fat accumulation accompanied by degeneration and disintegration of hepatocytes. On the other hand, fat droplet degeneration refers to findings of deposition of large and small fat droplets in the cytoplasm.