4.2 The effects of a green rooibos extract on the reproductive function of obesity-induced insulin
4.2.1 Biometric data before and after initiation of GRT treatment
Water and food intake as well as the change in body weight and glucose levels were monitored during the 16-week treatment period before onset of GRT treatment (from 0 to 10 weeks) and during the treatment phase (from week 11–16). Additionally, visceral fat weight in relation to the total body weight (adiposity index) were determined in four groups (LC, LC+GRT, OB and OB+GRT).
4.2.1.1 Food intake
Figure 4.1 illustrates the mean food intake in both lean (LC) and obese (OB) animal groups at 10 weeks before the start of GRT treatment. The average food intake was significantly higher in the OB
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compared to the LC group (18.72 ± 0.19 g/rat/day vs. 13.90 ± 0.08 g/rat/day, p<0.001). Treatment with GRT was started at 11 weeks of the diet period. Both groups (LC and OB) were further divided in two subgroups: LC+GRT and OB+GTR. Food intake was further monitored daily until 16 weeks.
According to a two-way ANOVA, diet significantly increased food intake between untreated OB and LC (16.49 ± 0.59 g/rat/day vs 13.52 ± 0.14 g/rat/day, p<0.001) as well as treated OB+GRT and LC group (17.88 ± 0.76 g/rat/day vs 13.52 ± 0.14 g/rat/day, p<0.001). The OB+GRT group showed a significant higher food intake when compared to LC+GRT group (17.88 ± 0.76 g/rat/day vs 13.99 ± 0.77 g/rat/day, p<0.001) (Figure 4.2). However, GRT treatment did not show any effect on food intake in the LC+GRT or OB+GRT animals when compared either to the LC or OB animals.
LC OB
0 5 10 15 20
***
Treatment groups Mean food intake (g/rat/day)
Figure 4.1: Mean food intake of the OB and LC animals over 10 weeks
Data are presented as mean ± SEM. ***p<0.001 according to Student’s t-test, LC: lean control, OB: obese group. N=14 per group.
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LC
LC+GRT
OB
OB +G
RT 0
5 10 15
20 ***
***
***
Treatment groups Mean food intake (g/rat/day )
Figure 4.2: Mean food intake of the treatment groups from 11 to 16 weeks
Data are presented as mean ± SEM. ***p<0.001 according to 2-way ANOVA, LC: lean control, LC+GRT:
lean group treated with GRT, OB: obese group, OB+GRT: obese animals treated with GRT. N=7 per group.
4.2.1.2 Water intake
Before onset of treatment, the OB group consumed less water than the LC group (16.07 ± 0.44 mL/rat/day vs 22.02 ± 0.42 mL/rat/day, p<0.001) (Figure 4.3). Similarly, after GRT treatment, the average water intake in the OB group was less compared to that of the LC group (16.20 ± 1.59 mL/rat/day vs 20.73 ± 0.50 mL/rat/day, p<0.001) (Figure 4.4). There was no significant difference between LC+GRT and the LC group and between OB+GRT and the OB group. The OB+GRT group showed a significant decrease in water intake when compared to the LC+GRT group (15.68 ± 1.00 mL/rat/day vs 20.47 ± 1.04 mL/rat/day, p<0.001) as well as compared to the LC group (15.68 ± 1.00 mL/rat/day vs 20.73 ± 0.50 mL/rat/day, p<0.001) (Figure 4.4). According to the two-way ANOVA, diet, and not GRT treatment, influence water consumption.
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Figure 4.3: Mean water intake of lean and obese animals over 10 weeks
Data are presented as mean ± SEM. ***p<0.0001 according to Student’s t-test, LC: lean control, OB: obese group. N=14 per group.
Figure 4.4: Mean water intake from week 11 to week 16 in treatment groups
Data are presented as mean ± SEM. ***p<0.001 according to a two-way ANOVA, LC: lean control, LC+GRT:
lean group treated with GRT, OB: obese group, OB+GRT: obese animals treated with GRT. N=7 per group.
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4.2.1.3 Body weights
Initially, animal body weights (BWs) were measured (120.00 ± 10.00g) and grouped into different treatment groups. The changes in BW were monitored before and after GRT treatment. In week 10, the OB group showed a trend of increase in the mean BW at baseline (before the onset of GRT treatment) when compared to the LC group (299.00 ± 5.51 g vs 285.40 ± 4.44 g, p>0.05) (Figure 4.5).
In week 16, the OB animals presented a significant higher BW when compared to the LC animals (378.12 ± 11.69 g vs 346.30 ± 7.05 g, p<0.05) (Figure 4.6). The OB+GRT group had a significant lower BW compared to the OB group (340.00 ± 9.01 g vs 378.12 ± 11.69 g). Additionally, the LC+GRT group did not present with changes in BW compared to the LC group as well as compared to OB+GRT groups (Figure 4.6).
LC OB
0 100 200 300 400
Treatment groups Mean body weight (g)
Figure 4.5: Mean body weight of lean and obese group in week 10
Data are presented as mean ± SEM. p>0.05 according to Student’s t-test, LC: lean control group, OB: obese group. N=14 per group.
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LC
LC +GRT
OB
OB +G
RT 0
100 200 300 400
500 *
*
Treatment groups Mean body weight (g)
Figure 4.6: Mean body weight per experimental group at the time of sacrifice
Data are presented as mean ± SEM. * p<0.05 according to a one-way ANOVA, LC: lean control, LC+GRT:
lean group treated with GRT, OB: obese group, OB+GRT: obese animals treated with GRT. N=7 per group.
4.2.1.4 Intra-peritoneal fat weight
Intra-peritoneal (IP) fat weight was expressed as a ratio of the BW and presented as an adiposity index (IP/BW*100) expressed in %. The adiposity index was significantly higher in the OB group compared to the LC group (3.60 ± 0.10% vs 1.96± 0.32%, p<0.001). The OB+GRT group showed a significantly lower adiposity index compared to the OB group (2.06 ± 0.07% vs 3.60 ± 0.10%, p<0.001) and there was no difference between OB+GRT and LC groups. The ingestion of GRT in the LC+GRT group did not show any significant difference in the adiposity index compared to the LC as well as compared to OB+GRT groups (Figure 4.7).
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LC
LC +
GRT OB
OB+G RT 0
1 2 3
4 ***
***
Treatment groups Adiposity index (%)
Figure 4.7: Adiposity index in the experimental groups at the time of sacrifice
Data are presented as mean ± SEM. ***p<0.001 according to a one-way ANOVA, LC: lean control, LC+GRT:
lean group treated with GRT, OB: obese group, OB+GRT: obese animals treated with GRT. N=7 per group.
4.2.1.5 Insulin sensitivity and glucose levels
4.2.1.5.1 OGTT results at baseline level in week 10
After oral administration of the 50% sucrose solution, the monitoring of glucose levels for 2 hours OB animals showed higher glucose levels compared to the LC animals at different time points respectively: 0 min (5.99 ± 0.24 mmol/L vs 4.87 ± 0.21 mmol/L, p<0.05), 10 min (7.24 ± 0.12 mmol/L vs 6.23 ± 0.21 mmol/L, p<0.05), 15 min (7.95 ± 0.16 mmol/L vs 6.31 ± 0.40 mmol/L, p<0.01) and 30 min 7.64 ± 0.11 mmol/L vs 6.73 ± 0.27 mmol/L, p<0.001) (Figure 4.8). Furthermore, according to the area under the curve (AUC), the OB group showed significantly increased blood glucose levels compared to the LC group (768.00 ± 17.45 arbitrary units vs 690.80 ± 13.45 arbitrary units, p<0.05) (Figure 4.9).
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0 3 5 10 15 20 25 30 45 60 90 120
4 5 6 7
8 *
LC OB
**
*
***
Time (minutes) Glucose levels (mmol / L )
Figure 4.8: Glucose levels in two experimental animal groups (LC and OB) at 10th week on diet Data are presented as mean ± SEM. *p<0.05 vs OB according to a two-way ANOVA, LC: lean control group, OB: obese group. N=7 per group.
LC OB
0 200 400 600 800 1000
**
Treatment groups AUC (Arbitrary Units)
Figure 4.9: AUC representing the effect of diet on glucose tolerance of LC and OB at week 10 Data are presented as mean ± SEM. *p<0.05 according to Student’s t-test, LC: lean control group, OB: obese group. N=14 per group.
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4.2.1.5.2 OGTT results after GRT treatment
In week 15 (i.e. after 5 weeks of GRT treatment), the blood glucose levels in the different treatment groups were changed at different time checking points after gavaging animals with 50% sucrose solution. The OB group presented with significantly higher glucose levels in the plasma when compared to the LC group at 10 min, 15 min, 20 min, 25 min, 30 min and 60 min time points respectively (7.77 ± 0.27 mmol/L vs 6.37 ± 0.12 mmol/L, p<0.001), (8.17 ± 0.21 mmol/L vs 6.91 ± 0.25 mmol/L, p<0.01), (8.17 ± 0.24 mmol/L vs 6.87 ± 0.18 mmol/L, p<0.001),8.00 + 0.30 mmol/L vs 6.67+ 0.17 mmol/L, p<0.001), (7.74 ± 0.21 mmol/L vs 6.48 ± 0.25, p<0.01) and (6.97 + 0.33 mmol/L vs 5.89 ± 0.14 mmol/L, p<0.01), (Figure 10). According to the calculation of AUC, the OB presented higher blood glucose levels compared to the LC group (820.70 ± 21.49 arbitrary units vs 714.00 ± 12.14 arbitrary units, p<0.05) (Figure 4.11).
GRT treatment in the LC group (LC+GRT) and in the OB group (OB+GRT) did not show any significant effect on glucose levels when compared to the untreated group (LC and OB). However, it was demonstrated that the two treated groups (OB+GRT and LC+GRT) were significantly different at 0 min (6.19 ± 0.20 mmol/L, vs 5.06 ± 0.06 mmol/L, p<0.01),5 min (7.13 ± 0.25 mmol/L vs 5.83
± 0.19 mmol/L, p<0.001), 10 min (7.77 ± 0.25 mmol/L vs 6.40 ± 0.21 mmol/L, p<0.001), 15 min (8.20 ± 0.27 mmol/L vs 6.36 ± 0.12 mmol/L, p<0.001), 20 min (8.10 ± 0.30 mmol/L vs 6.39 ± 0.13 mmol/L, p<0.001), 25 min (8.04 ± 0.28 mmol/L vs 6.23 ± 0.18 mmol/L, p<0.001), 30 min (7.81 ± 0.33 mmol/L vs 6.24 ± 0.19 mmol/L, p<0.001), 45 min (7.29 ± 0.27 mmol/L vs 5.54 ± 0.21, p<0.001), 60 min (7.09 ± 0.35 mmol/L vs 5.44 ± 0.01 mmol/L, p<0.001) and 90 min (6.34 ± 0.39 mmol/L vs 5.09 ± 0.16 mmol/L, p<0.01) (Figure 4.10). These significant differences were also observed when calculating the AUC where the OB+GRT group showed a significant increase of AUC compared to the LC+GRT (839.60 ± 32.64 arbitrary units vs 667.70 ± 14.21 arbitrary units, p<0.001) as well compared to the LC group (839.60 ± 32.64 arbitrary units vs 714.00 ± 12.14 arbitrary units, p<0.01) (Figure 4.11).
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0 3 5 10 15 20 25 30 45 60 90 120
4 5 6 7 8 9
**
$$
$ $$$ $$
$$
$$$
$$
LC LC+GRT OB OB+GRT
*** ******
**
Time (minutes)
Glucose levels mmol/L
**
Figure 4.10: Glucose levels after treating animals with GRT
Data are presented as mean ± SEM. *p<0.05, **p<0.01 OB vs LC group, $p<0.05, $$p<0.01, $$$p<0.005 OB+GRT vs LC+GRT according to a two-way ANOVA. LC: lean control, LC+GRT: lean group treated with GRT, OB: obese group, OB+GRT: obese group treated with GRT. N=7 per group.
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LC
LC+GR
T OB
OB +G
RT 0
200 400 600 800
1000 ***
*
**
Treatment groups AUC (Arbitrary Units)
Figure 4.11: AUC representation of the effect of diet in glucose tolerance of LC and OB
*p<0.05, **p<0.01 according to ANOVA, LC: lean control, LC+GRT: lean group treated with GRT, OB:
obese group, OB+GRT: obese animals treated with GRT. N=7 per group.
4.2.1.5.3 Non-fasting blood glucose levels
On the day of sacrifice, an increase in the non-fasting blood glucose levels was observed in the OB group compared to the LC group (7.5 ± 0.2 mmol/ L vs 6.7 ± 0.1 mmol/L, p<0.05). GRT treatment in OB (OB+GRT) resulted in a significant decrease in glucose levels when compared to the untreated OB group (6.8 ± 0.1 mmol/L vs 7.5 ± 0.2 mmol/ L, p<0.05). There was no difference between LC and OB+GRT groups. No significant difference in glucose levels was also observed between LC and LC+GRT as well as between LC+ GRT and OB+GRT groups (Figure 4.12).
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LC
LC +GRT
OB
OB +GRT 0
2