Body weight
The mean body weights of the five experimental groups were similar at the start of the experiment. After the treatment of 56 days, the body weights of all experimental groups were significantly decreased compared to the control group (Figure 1). NT-treated groups and the d-fenfluramine (d-FF) treated group lost weight during the first 48 hours of treatment. After this time, the weight gain of the d-fenfluramine (d-FF) group paralleled that of the control group. By contrast, the NT-treated groups continued to gain weight at a slower rate than the control group. It should also be noted that the rats that were pair fed to the high dose NT group (NT-H) gained weight at the same rate as the NT-H group (Figure 2). On day 56 of the treatment, the rats administered low dose NT (NT-L) and high dose NT (NT-H) had gained 24.6% and 33.2% less weight than the control group respectively. The rats administered d-fenfluramine (d-FF) gained 12.3% less weight than the control group. After termination of treatment at 56 days, there was no increase in the rate of weight gain of the NT treated group (NT-H-R) over the next 14 days and their weight difference to the control group (Control-R) was maintained (Figure 1).
Food intake
The food intake of each group over the experimental period is illustrated in Figures 2 and 3. The rats in the control group consumed significantly more total food than the rats in any of the treatment groups over the 56-day period. The rats administered d-fenfluramine (d-FF) consumed less total food over the 56 day period than the control group. On the other hand, they consumed significantly more food than those in other groups, namely NT-H, NT-L and Pr-fed. The pair-fed rats ate slightly less than the NT-H group during the experiment, presumably due to spillage of the diet.
Feed Efficiency
In this study, feed efficiency is expressed as weight gain (g) divided by weight of food consumed (Figure 4). The feed efficiency of the d-FF group, which was still higher than those of the NT-H, NT-L and Pr-fed groups, decreased significantly compared to the control group. The feed efficiencies of the NT-L and NT-H groups were 15.6% and 22.5% lower than that of the control group respectively. The feed efficiency of the d-FF group was 7.8% lower than that of the control group. Body weight gain and food intake were closely correlated in individual experimental groups (R2 and P values for each group respectively: Control 0.719, P < 0.05; NT-H 0.907, P < 0.01; NT-L 0.796, P < 0.05; Pr-fed 0.694, P < 0.05; d-FF 0.798, P < 0.05) and in the combined data (Figure 5).
Organ weights
There were no significant differences in the weights of the kidney, spleen, liver, and gastrocnemius muscle among the groups after the 56-day experimental period (data not shown). The hearts of the rats in the control group were significantly larger than those in all other groups. However, when the weight of the heart was taken as a percentage of the body weight of the respective rat, there were no significant differences between the groups.
Parametrial and retroperitoneal white fat pads were significantly smaller in the NT-H group. They were also smaller in the NT-L and the Pr-fed groups but were not significantly different from either the NT-H group or the control group (Figure 6). There were no differences between the sizes of these adipose depots between the d-FF group and the control group. In the rats allowed to recover for two weeks after the end of treatment, the adipose depots remained smaller than those in the control group despite the fact that fat deposition increased significantly during the 2-week recovery period. The size of the inter-scapular brown adipose tissue depot was significantly reduced in all experimental groups compared to the control group (Figure 6).
Body composition
The percentage of total body fat was significantly reduced in the NT-H group and a clear dose-related reduction with NT treatment was observed (Figure 7), while body fat was not significantly altered in any other experimental groups. The percentage of body water, as expected, increased slightly in the NT groups. There were no significant effects of any treatment on body protein or ash (i.e. mineral content). Changes in body composition during the treatment period could not be calculated because no rats were sacrificed for analysis at time zero. However, from percentage compositions and final body weights, we calculated the differences in the body composition between the treatment groups and control group at the end of the experiment. Virtually all the differences in body weight between the NT-H group and control group can be attributed to the difference in body fat between these two groups (Figure 8).
Serum hormones
Serum leptin was reduced significantly in all the treatment groups compared to the control group (Figure 9). The differences in leptin values continued during the 14-day recovery period despite the withdrawal of NT, which might be due to a modest weight gain during that period. Serum leptin levels were correlated with percentage body fat for each individual experimental group (R2 and P values for each group: Control 0.887, P < 0.001; Control-R 0.627, P < 0.05; NT-H 0.855, P < 0.01; NT-H-R 0.99, P < 0.001; NT-L 0.933, P < 0.001; Pr-fed 0.875, P < 0.001; d-FF 0.769, P < 0.01) and the combined data (R2 = 0.708, P < 0.001) (Figure 10).
Serum metabolites
Serum prepared from trunk blood obtained at time of sacrifice (day 56) was used for clinical chemistry assays. The analyses included quantification of glucose, triglycerides, cholesterol, alkaline phosphatase, alanine aminotransferase, amylase, aspartate aminotransferase, chloride, cholesterol, creatinine phosphokinase, γ-glutamyl transpeptidase, glucose, hemoglobin, hematocrit, mean cell volume, platelets, potassium, sodium, triglycerides and white blood cells. One-way ANOVA plus Scheffes' Comparison of Means for each variable indicated that there were no significant differences between any of the serum analysis variables from the treatment groups and control group, nor among the treatment groups.
Safety and toxicity
A small degree of alopecia during the first week of treatment was observed around the chins of the rats in the NT-H group. The alopecia disappeared within the next seven days after the animal technician was advised to be more scrupulous with the use of the intubation tube.
Rats in the NT treatment groups had yellowish urine which stained fur around the genitalia. Urine was tested negative for bilirubin. Rats in both NT-H and NT-L groups had loose stools for the first few days. By the end of the first week, the stools of the rats in the NT-L group were well formed and their consistency appeared normal, whereas the consistency of the stools of the rats in the NT-H group still appeared softer than those in other groups. Soft stools were observed among the rats in the NT-H group from time to time during the course of study. Moreover, the stools of the rats in both the NT-H and NT-L groups appeared reddish throughout the duration of the experiment. A test for hemoglobin was negative. The rats in the NT-H group liked to rub and groom the fur around their mouths after intubation, which was not observed in other groups. During the recovery period when the NT treatment was withdrawn, the color of the urine and stools returned to normal and the rats stopped grooming around the mouth. No other differences in activity/lethargy, fur condition, skin condition, eye condition or other physical characteristics were observed.