In a previous study, the administration of a grape seed procyanidin extract (GSPE) in female Wistar rats improved insulin resistance, reduced insulin production, and modulated apoptosis biomarkers in the pancreas. Considering that pharmacokinetic and pharmacodynamic parameters in females are different from these parameters in males, the aim of the present study was to evaluate the effects of GSPE on male Wistar cafeteria-induced obese rats. The results have confirmed that the cafeteria model is a robust model mimicking a prediabetic state, as these rats display insulin resistance, increased insulin synthesis and secretion, and increased apoptosis in the pancreas. In addition, GSPE treatment (25 mg/kg of GSPE for 21 days) in male rats improves insulin resistance and counteracts the cafeteria-induced effects on insulin synthesis. However, the administration of the extract enhances the cafeteria-induced increase in Bax protein levels, suggesting increased apoptosis. This result contradicts previous results from cafeteria-fed female rats, in which GSPE seemed to counteract the increased apoptosis induced by the cafeteria diet.
Procyanidins are the second most abundant natural phenolic after lignin, and they are widely distributed in fruits, berries, beans, nuts, cocoa, and wine [
Obesity has become a worldwide problem, leading to an explosion of obesity-related health issues [
Several animal models have been used to study obesity, including both genetic and diet-induced obesity models. However, the cafeteria diet is a more robust model to reproduce the diet in Western society [
The pharmacokinetics and pharmacodynamics in females are different from the same parameters in males because of the female’s unique anatomy and physiology [
The procyanidin extract was derived from grape seed and contained the following: catechin (58
Wistar male rats weighting between 250–330 g were purchased from Charles River Laboratories (Barcelona, Spain) and housed in animal quarters at 22°C with a 12 h light/dark cycle. After 1 week in quarantine, the animals were divided in two groups, a diet-control group (7 animals) fed a standard diet (Panlab A03) and a cafeteria group (21 animals) fed a cafeteria diet (bacon, biscuits with pâté, biscuits with cheese, muffins, carrots, and milk with sugar) in addition to standard chow and water. Every day at 9 AM, food was withdrawn, and it was replaced at 6 PM. Obesity was induced in the animals on the cafeteria diet for 52 days. Afterwards, the diet-control group and 7 animals from the cafeteria-fed group were sacrificed, as a reference for the state of the animals before the beginning of treatment. The rest of the cafeteria-fed rats were divided in two subgroups (7 animals/group). These two groups were the (i) cafeteria group: rats treated with a vehicle (gum arabic 5% w/v) and the (ii) GSPE-treated group: rats treated with 25 mg of GSPE/kg of body weight (bw) per day. The treatment was administrated every evening for 21 days before the replacement of the food. Three days before the end of the treatment and after 8 h of fasting, blood was collected from the tails of the rats to measure glucose and insulin levels. At the end of the treatment regimen and after 3 h of fasting, the animals were anesthetised using sodium pentobarbital (50 mg/kg of bw, Sigma-Aldrich, St. Louis, MO) and sacrificed by abdominal aorta exsanguination. The pancreas was isolated from all of the animals, frozen immediately in liquid nitrogen, and stored at −80°C until analysis. All of the procedures were approved by the Experimental Animals Ethics Committee of the Universitat Rovira i Virgili.
Insulin plasma levels were assayed using an ELISA method following the manufacturer’s instructions (Mercodia, Uppsala, Sweden). Glucose plasma levels were determined using an enzymatic colorimetric kit (QCA, Amposta, Spain).
The HOMA-IR and HOMA-
Triglycerides (TAG) and nonesterified fatty acids (NEFAs) from the pancreas were extracted by homogenising the tissue with PBS containing 0.1% triton X-100 (Sigma-Aldrich, St. Louis, MO), and their concentrations were determined using enzymatic colorimetric kits (QCA, Amposta, Spain for TAG and Wako chemicals GmbH, Neuss, Germany for NEFAs).
The pancreas was homogenised with six volumes of PBS containing 50 mM EDTA at pH 7.4 and centrifuged at 3000 g for 5 min at 4°C. Reactive oxygen species (ROS) in the supernatants were quantified using 20
Total RNA from the pancreas was extracted using the RNeasy Mini Kit (Qiagen, Barcelona, Spain). cDNA was generated with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain) and was subjected to quantitative Real-Time PCR amplification using the TaqMan Master Mix (Applied Biosystems, Madrid, Spain). Specific TaqMan probes (Applied Biosystems, Madrid, Spain) were used for each gene: Rn99999125_m1 for
The protein levels of Bax and Bcl-2 were quantified by Western Blot as previously described [
The results are expressed as the mean ± SEM. Effects were assessed using Student’s
We first examined the effects of the cafeteria diet on pancreatic insulin production after 52 days of diet administration. Insulin and glucose plasma levels were quantified at day 49, and the cafeteria diet-fed rats showed significantly higher plasma insulin levels and no changes in glucose levels (Table
Effects of the cafeteria diet and GSPE treatment on plasmatic glucose and insulin levels, HOMA-IR and HOMA-
Standard diet | Cafeteria diet | Cafeteria + vehicle | Cafeteria + GSPE | |
---|---|---|---|---|
Glucose (mM) | 4.13 ± 0.2 | 4.49 ± 0.3 | 4.76 ± 0.2 | 4.88 ± 0.2 |
Insulin (ng/mL) | 1.23 ± 0.1 | 2.47 ± 0.2*** | 2.78 ± 0.2 | 1.80 ± 0.3‡ |
Insulin/Glucose | 6.02 ± 0.6 | 12.40 ± 1.6** | 13.25 ± 0.7 | 8.39 ± 1.2‡ |
HOMA-IR | 4.97 ± 0.5 | 10.75 ± 1.0*** | 14.66 ± 2.8 | 8.37 ± 1.2† |
HOMA- |
609.45 ± 93.4 | 1508.98 ± 388.9# | 1084.1 ± 13.7 | 750.58 ± 121.2‡ |
Effects of cafeteria diet on gene expression in the pancreas. *
Standard diet | Cafeteria diet | |
---|---|---|
|
1.22 ± 0.3 | 5.37 ± 1.4* |
|
1.16 ± 0.3 | 3.71 ± 0.8* |
|
1.15 ± 0.3 | 0.28 ± 0.1* |
|
1.08 ± 0.2 | 1.64 ± 0.4 |
|
1.27 ± 0.5 | 0.24 ± 0.1# |
|
1.31 ± 0.4 | 1.12 ± 0.3 |
After induction of obesity via the cafeteria diet, rats were treated with 25 mg/kg of bw GSPE for 21 days, concomitantly with cafeteria diet administration. The animals treated with the procyanidin extract had lower insulinemia and decreased HOMA-IR and HOMA-
Effects of GSPE treatment of cafeteria-fed rats on gene expression in the pancreas. **
Cafeteria + vehicle | Cafeteria + GSPE | |
---|---|---|
|
1.16 ± 0.3 | 0.55 ± 0.1# |
|
1.04 ± 0.2 | 0.2 ± 0.1** |
|
1.12 ± 0.2 | 0.96 ± 0.3 |
|
1.13 ± 0.3 | 1.18 ± 0.2 |
|
1.67 ± 0.4 | 0.95 ± 0.4 |
|
1.15 ± 0.3 | 1.80 ± 0.3 |
To examine the effects of the cafeteria diet and GSPE on apoptosis and proliferation in the pancreas, several markers were analysed at the gene and protein level.
The cafeteria-fed rats showed a decrease in the antiapoptotic marker Bcl-2 at both the gene (Table
Protein expression of the apoptosis markers Bcl-2 and Bax and the calculated ratio of Bcl-2/Bax in: (a) standard-diet-fed rats and cafeteria-diet-fed rats and (b) in GSPE-treated rats and vehicle-treated rats assessed by Western Blot. Data are shown as the mean ± SEM. *
The administration of GSPE had no effect on Bcl-2 and Bax at gene expression compared to the cafeteria diet (Table
Finally, we also analysed Cyclin D2, a proliferation marker, but no changes were observed in the cafeteria-fed animals or in the GSPE-treated rats (Table
Pancreas malfunction is in part due to the accrual of TAG in its cells. To measure it, we examined the TAG content in this tissue and found that TAG triplicated its levels in the pancreas of cafeteria-fed rats compared to the standard-diet-fed rats (
TAG content in the pancreas of cafeteria-fed rats treated with GSPE or vehicle, expressed as
In contrast, the content of NEFAs was not modified neither in the cafeteria group compared to the standard-diet-fed rats (
ROS content in the pancreas was also analysed, and no significant differences were observed in either the cafeteria-fed rats or in the GSPE-treated animals (Figure
ROS content in the pancreas, expressed as fluorescence arbitrary units/(mg/mL) of protein and normalised to the respective control group. Data are shown as the mean ± SEM.
This study was designed to examine the effects of the cafeteria diet on insulin production in male Wistar rats by evaluating pancreas functionality, apoptosis, and proliferation. We have also evaluated the effects of procyanidins on these processes, since procyanidins were shown to have positive effects on glucose metabolism under conditions of slightly disrupted homeostasis [
We had previously shown that 17 weeks of a cafeteria diet led to insulin resistance, high plasma insulin levels, and increased insulin synthesis and secretion in female Wistar rats [
Increased deposits of fat are associated with obesity and lead to an increase in free fatty acids (FFAs). The induction of apoptosis in
The results from the apoptosis markers are in accordance with the data obtained in the previous study which evaluated the cafeteria diet in Wistar female rats [
Taken together, the data suggests that the effects of the cafeteria diet on insulin synthesis, secretion, and apoptosis are not influenced by gender or treatment duration.
Once the effects of the cafeteria have been established, we have analyzed the effects of a GSPE treatment on the cafeteria-fed animals. After 52 days of cafeteria diet administration, male Wistar rats have been treated with 25 mg/kg of GSPE for 21 days concomitant with the cafeteria diet. The GSPE treatment has decreased insulin production, plasma insulin levels, and pancreatic insulin Cpe gene expression compared to the cafeteria-vehicle-fed rats. Moreover, the decreased insulin production could be at least in part explained through GSPE’s lipid-lowering effect, since the triglyceride content has also been reduced in the pancreas of GSPE-treated rats. Previously, we showed that 25 mg of GSPE/kg of bw administered to female Wistar rats for 30 days resulted in decreased insulin production and reduced triglyceride content in the pancreas, likely via decreased fatty acid synthesis and increased
GSPE has counteracted the effects of the cafeteria diet reducing the accumulation of triglycerides in the pancreas but has not counteracted the cafeteria-diet effects on the apoptosis markers. Instead, GSPE-treated rats have shown an increase in Bax protein levels and a decreased ratio of Bcl-2/Bax, suggesting an enhancement in the apoptosis. Therefore, the lipid-lowering effects of GSPE do not involve a reduction in apoptosis in the pancreas of cafeteria-fed male rats. Actually,
The apoptosis marker results conflict with those in the previous study of female Wistar rats, which showed that 25 and 50 mg/kg of GSPE seemed to counteract the deleterious effects of the cafeteria diet by inhibiting the down-regulation of Bcl-2 protein expression after 10 and 30 days of treatment [
In conclusion, the present study has confirmed that the cafeteria model is a suitable reproduction of the prediabetic state. This model induces an insulin resistance state, shows increased insulin synthesis and secretion, and exhibits increased apoptosis in the pancreas. Moreover, GSPE treatment in male rats treated with 25 mg/kg of GSPE for 21 days improves the insulin resistance state and counteracts the cafeteria-induced effects on insulin synthesis. However, procyanidins enhance the elevated levels of Bax, a pro-apoptotic protein observed in the cafeteria-fed rats, potentially suggesting an increase in apoptosis. This result indicates that the effects of GSPE on apoptosis markers are dose, time, and/or gender dependent.
The authors declare no conflict of interests.
The authors thank the members of the Nutrigenomics group with whom they have collaborated to care for and sample the animals. They also thank Pol Andres for assistance in analysing gene and protein expressions. This study was supported by Grants AGL2011-23879 and AGL2008-00387/ALI from the Ministerio de Educación y Ciencia (MEC) of the Spanish Government. Fellowships have been given to L. Cedó from the Generalitat de Catalunya; A. Castell-Auví from the Ministerio de Educación of Spanish Government; and V. Pallarès from de Universitat Rovira i Virgili.