Three-week-old male, Sprague-Dawley rats were obtained from CLEA (Tokyo, Japan). Rats were housed individually in stainless steel wire-bottom cages in a room maintained at 22 ± 2 °C and 55 ± 5% relative humidity with a 12 h cycle of light and dark. Rats were given free access to tap water throughout the experiment. They were fed a commercial diet (CE-2; CLEA) for 1 wk prior to the experiment. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Josai University.

The rats were randomly divided into six groups of 6 rats each. Each group was fed either the high-fat high-sucrose diet (HFS; lard 50%kcal, sucrose 25%kcal) based on the modified version of the AIN-93G [¹⁰] as a control (Ctrl group) or the HFS containing 0.25% (VL group), 1.25% (L group), 2.50% (M group), 5.00% (H group) or 12.30% (VH group) fenugreek seeds powder (Gaban Co., Tokyo, Japan) for 12 wks (Table 1). Almost the same energy intake was maintained for all groups by adjusting the dietary intake, and the food consumption in all groups except for the VH group was approximately equivalent to the ad libitum consumption under these conditions. After 12 wks, the rats were individually transferred into the metabolic cage, and urine and feces were separately collected for 1 day. At the end of the experiment, the rats were killed with pentobarbital sodium (100 mg/kg, ip). Blood samples were collected from the abdominal aorta, and the plasma was separated after centrifugation (2,000 × g for 20 min at 4°C) and stored at -30°C until analyzed. After the collection of the blood sample, the liver, interscapular brown adipose tissue (IBAT) and epididymal white adipose tissue (EWAT) were immediately excised, weighed and stored at -30°C for further analyses.

At the 10th wk, the rats were deprived of food for 12 h. Then they were given oral glucose (2 g/kg body weight) for the oral glucose tolerance test (OGTT) and an intraperitoneal injection of human regular insulin (0.75 U/kg body weight) for the intraperitoneal insulin tolerance test (IPITT). Blood glucose concentrations were measured by Ascensia™ DexterZII (Bayer Medical, Tokyo, Japan) using tail blood samples at 0, 30, 60 and 120 min after glucose administration and at 0, 15, 30, 45, 60 and 120 min after insulin administration. Plasma insulin concentrations were measured using a commercial kit (Rat Insulin ELISA kit, Shibayagi Co., Gunma, Japan) using separated tail plasma samples at 0, 30, 60 and 120 min after glucose administration.

Plasma triglyceride, total cholesterol, AST and ALT were measured by colorimetric slides using the Fuji Dri-Chem 3500 (Fujifilm Corp., Tokyo, Japan).

The hepatic and fecal lipids were extracted in accordance with the method of Bligh and Dyer [¹¹] and the method of Folch [¹²], respectively. Each extract was solubilized by Triton-X100 (Wako Pure Chemical Industries, Osaka, Japan), and the lipids concentrations were determined enzymatically by means of commercial kits (Triglycerides E-Test Wako, Cholesterol E-Test Wako, Total Bile Acid Test Wako; Wako Pure Chemical Industries).

All data are expressed as the mean ± SEM. Statistical analyses were carried out using Statistical Package for Social Sciences (SPSS12.0J for Windows; SPSS Japan, Tokyo, Japan). In the three-group comparison, the effects of treatment were analyzed using one-way analysis of variance (ANOVA), and the differences among means were tested by means of the Tukey's honestly significant difference (HSD) test. In the two-group comparison, the effects of treatment were analyzed through a T-test. In OGTT and IPITT, the effects of treatment were analyzed by means of repeated ANOVA. Differences were considered significant at P < 0.05.

The body weight gain, the EWAT weight and the IBAT weight decreased significantly in the VH group compared to the other groups. No significant difference was found in the liver weight among the groups (Table 2).

No significant difference was found in AST, ALT levels or AST/ALT in the liver among the groups (Table 2). Thus fenugreek intake did not show any liver injury.

The fasting blood glucose levels decreased significantly in the M and VH groups compared to the Ctrl group, whereas no significant difference was found in the fasting plasma insulin levels or insulin resistance index, HOMA-IR (homeostasis model assessment as an index of insulin resistance), among groups (Table 3).

In OGTT, the blood glucose levels at 120 min in the VH group were significantly lower than those in the Ctrl group (Figure 1A), and the plasma insulin levels at 120 min in the VH group were significantly lower than those in other groups (Figure 1B). There was no significant difference of the area under the curve (AUC) of glucose among the groups (Figure 1C), but the VH group showed significant lower AUC of insulin compared to the Ctrl group (Figure 1D).

In IPITT, the blood glucose levels at 60 and 120 min in the VH group were significantly lower than those of the other groups (Figure 2A). AUC of the blood glucose in the VH group was also significantly lower than those of the other groups (Figure 2B).

Although the plasma triglyceride and total cholesterol levels increased slightly in some groups, no dose dependent effect was found (Table 2). However, the hepatic triglyceride and total cholesterol levels decreased significantly in the M and VH groups compared to the Ctrl group (Figure 3). In feces, the triglyceride levels in the VH group increased remarkably compared to the levels the in the other groups. The total cholesterol levels increased significantly in the L group, the H group and the VH group compared to the Ctrl group. Fenugreek elevated the total bile acid levels in feces in a dose-dependent manner (Figure 4).

Ctrl: Control group, VL: Very low dose of fenugreek group (0.25%), L: Low dose of fenugreek group (1.25%), M: Middle dose of fenugreek group (2.5%), H: High dose of fenugreek group (5.0%), VH: Very high dose of fenugreek group (12.3%). ¹Based on the AIN-93G and modified. ^2,3,4Purchased from Oriental Yeast Co., Tokyo, Japan. ³Vitamin mixture was based on the AIN-93 formation, and added with tartaric acid choline. ⁴Mineral mixture was based on the AIN-93G formation. ⁵Purchased from Wako Pure Chemical Industries, Osaka, Japan. ⁶Purchased from Gaban Co., Tokyo, Japan.

Ctrl: Control group, VL: Very low dose of fenugreek group (0.25%), L: Low dose of fenugreek group (1.25%), M: Middle dose of fenugreek group (2.5%), H: High dose of fenugreek group (5.0%), VH: Very high dose of fenugreek group (12.3%). ¹EER (mg/kJ) = Body weight gain (mg)/Total food intake (kJ). ²EWAT: Epididymal white adipose tissue. ³IBAT: Interscapular brown adipose tissue. ⁴TG: Triglyceride. ⁵TC: Total cholesterol. ⁶The relative values for Normal rats' data; AST/ALT = AST (U/L)/ALT (U/L)/Normal rats' data. Values are expressed as means ± SEM for 6 rats. Means without a common superscript letter are significantly different, P < 0.05 (ANOVA). Asterisks indicate a difference from Ctrl: P < 0.05 (t-test).

Ctrl: Control group, VL: Very low dose of fenugreek group (0.25%), L: Low dose of fenugreek group (1.25%), M: Middle dose of fenugreek group (2.5%), H: High dose of fenugreek group (5.0%), VH: Very high dose of fenugreek group (12.3%). ¹Relative values of (fasting blood glucose × fasting blood insulin) compared to the mean value of the Ctrl group. Values are expressed as means ± SEM for 6 rats. Means without a common superscript letter are significantly different, P < 0.05 (ANOVA).