Eur J Nutr DOI 10.1007/s00394-016-1192-2
ORIGINAL CONTRIBUTION
Beneficial effects of a red wine polyphenol extract on high‑fat diet‑induced metabolic syndrome in rats Nathalie Auberval1 · Stéphanie Dal1 · Elisa Maillard1 · William Bietiger1 · Claude Peronet1 · Michel Pinget1,2,3 · Valérie Schini‑Kerth2 · Séverine Sigrist1
Received: 25 August 2015 / Accepted: 14 February 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract Purpose Individuals with metabolic syndrome (MS) show several metabolic abnormalities including insulin resistance, dyslipidaemia, and oxidative stress (OS). Diet is one of the factors influencing the development of MS, and current nutritional advice emphasises the benefits of fruit and vegetable consumption. Here, we assessed the effects of naturally occurring antioxidants, red wine polyphenols (RWPs), on MS and OS. Methods Wistar rats (n = 20) weighing 200–220 g received a high-fat diet (HFD) for 2 months before they were divided into two groups that received either HFD only or HFD plus 50 mg/kg RWPs in their drinking water for an additional 2 months. A control group (n = 10) received a normal diet (ND) for 4 months. Results Rats receiving HFD increased body weight over 20 % throughout the duration of the study. They also showed increased blood levels of C-peptide, glucose, lipid peroxides, and oxidised proteins. In addition, the HFD increased OS in hepatic, pancreatic, and vascular tissues, as well as induced pancreatic islet cell hyperplasia and hepatic steatosis. Addition of RWPs to the HFD attenuated these * Séverine Sigrist s.sigrist@ceed‑diabete.org 1
UMR DIATHEC, EA 7294, Centre Européen d’Etude du Diabète, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Bld René Leriche, 67200 Strasbourg, France
2
UMR 7175 CNRS, Pharmacologie et Physico‑Chimie, Faculté de Pharmacie, Université de Strasbourg, 67400 Illkirch, France
3
Departement d’Endocrinologie, Diabète, Maladies Métaboliques, Pôle NUDE, Hôpitaux Universitaires de Strasbourg, (HUS), 67000 Strasbourg, France
effects on plasma and tissue OS and on islet cell hyperplasia. However, RWPs had no effect on blood glucose levels or hepatic steatosis. Conclusions RWPs showed an antioxidant mechanism of action against MS. This result will inform future animal studies exploring the metabolic effects of RWPs in more detail. In addition, these findings support the use of antioxidants as adjunctive nutritional treatments for patients with diabetes. Keywords Metabolic syndrome · Oxidative stress · Highfat-diet rats · Red wine polyphenols
Introduction Metabolic syndrome (MS), a condition that encompasses multiple factors such as an elevated blood glucose level (associated with diabetes or a prediabetic state), high cholesterol, high blood pressure, and abdominal obesity [1, 2], affects nearly a quarter of the world’s adult population [3]. It increases the risk of developing type 2 diabetes by fivefold [4]; this is significant, considering that of the 356 million people worldwide who live with diabetes, 90 % have type 2 diabetes. Environmental factors, particularly food intake, are one of primary causes of type 2 diabetes. Indeed, consumption of a diet that is high in energy, protein, fat, and sugars, but low in fruits and vegetables, increases the risk of becoming overweight or obese [5]. In turn, obesity increases the risk of developing type 2 diabetes [6], which is characterised by insulin resistance, and leads to chronic hyperglycaemia and hyperinsulinism. Both of these metabolic alterations are associated with hyperlipidaemia, which causes oxidative stress (OS) [7–9]. OS results from an imbalance between
13
pro-oxidant and antioxidant species in favour of oxidised entities [10]. It promotes the development of MS and type 2 diabetes by disrupting insulin secretion and increasing insulin resistance. In turn, insulin resistance induces hyperinsulinism, which generates OS by stimulating hydrogen peroxide secretion in adipocytes [11] and superoxides in endothelial cells [12], causing endothelial dysfunction and vascular complications. Meanwhile, insulin can inhibit fatty acid oxidation and catalase activity [13], while an unbalanced diet (nutritional failure, nutritional deficiency, or an excessively rich diet) can induce OS by compromising antioxidant defence systems. Insulin resistance in adipocytes and myocytes is responsible for some complications, such as the accumulation of lipids and triglycerides in organs and tissues not normally used for lipid storage; these include the cardiovascular system [14], pancreas, and liver [15]. Insulin resistanceinduced endothelial dysfunction is caused by blood vessel exposure to high levels of lipids and glucose [16]. These high levels also enhance hepatic fatty acid synthesis and strongly inhibit their β-oxidation, leading to fat accumulation, which causes non-alcoholic fatty liver disease [17, 18] and steatosis [19]. Furthermore, insulin resistance induces the storage of hepatic glucose in the form of glycogen [20]. In the pancreas, it causes islet hyperplasia and increases insulin secretion [21]. Epidemiological studies have shown that regular consumption of polyphenol-rich foods such as fruits and vegetables, and beverages such as red wine, is associated with a reduced risk of cardiovascular disease [22]. Intake of red wine polyphenols (RWPs) has also been shown to prevent angiotensin II-induced hypertension and endothelial dysfunction by inhibiting the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent formation of superoxide anions [23] and by preventing age-induced endothelial dysfunction [24, 25], age-related impairment of skeletal muscle mitochondrial function [26], and metabolic disorders [27]. In fact, a red wine extract (ProvinolsTM) was shown to improve glucose metabolism in Zucker fatty rats by reducing plasma glucose and fructosamine levels [27]. Based on this information related to antioxidants and diabetic pathology, we wanted to assess the effect of RWP supplementation in an established animal model of MS. Previously, Auberval et al. [28] demonstrated that a highfat diet (HFD) containing 21.4 % fat and 50.0 % carbohydrate induced MS (and subsequently type 2 diabetes) in healthy young rats. MS was assessed by monitoring obesity, insulin resistance, OS, and widespread tissue changes. The present study fed rats an HFD for 2 months to induce MS and then continued administering the HFD or the HFD+RWPs for 2 months. The effects of these treatments on metabolic parameters and on the function and OS levels of the pancreas, liver, and mesenteric artery were assessed
13
Eur J Nutr
and compared to those of a group of rats fed a normal diet (ND) for 4 months.
Methods This study was performed in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH publication No. 85–23, revised 1996). All efforts were made to minimise animal suffering and to reduce the number of animals. Animals and diets Fifteen male Wistar rats (DEPRE, Saint Doulchard, France) weighing 200–220 g upon arrival were housed in a climatecontrolled room (22 ± 2 °C and 60 % relative humidity) with a 12-h light/dark cycle. The rats had ad libitum access to food and water. The animals were randomly divided into three equal groups, with at least five animals per group. The ND group received a standard diet (A04, SAFE, Augy, France) for 4 months; this had an Atwater fuel energy of 2.8 kcal/g and 60 % carbohydrate, 3.1 % crude fat, 16.1 % crude protein, 3.9 % crude fibres, and 5.1 % ash for 4 months. The HFD group received WESTERN RD (SDS; Special Diets Services, Saint Gratien, France) for 2 months to induce MS and for a further 2 months to develop diabetes; this diet had an Atwater fuel energy of 4.6 kcal/g and comprised 50 % crude carbohydrate, 21.4 % crude fat, 17.5 % crude protein, 3.5 crude fibre, and 4.1 % ash [28]. The HFD+RWPs group was fed the HFD for 2 months, followed by concomitant administration of HFD and 50 mg/kg/day RWPs in the drinking water for an additional 2 months. All rats were weighed once a week throughout the study period. The RWP extract used in this study was generously provided by Dr. M. Moutounet (National Institute of Agronomic Research, Montpellier, France). This extract was prepared from French red wine (Corbières A.O.C.) as previously described [29] and analysed by Dr. P.-L. Teissedre (Département d’Œnologie, Bordeaux, France). Briefly, phenolic compounds were adsorbed on a preparative column. The alcohol was then desorbed, and the alcoholic eluent was gently evaporated prior to lyophilisation of the concentrated residue, which was finely sprayed to obtain the RWP extract as a dry powder. One litre of red wine produced 2.9 g of extract, which contained 471 mg/g of total phenolic compounds, expressed as gallic acid. Phenolic levels in the RWP extract were measured by high-performance liquid chromatography. The extract contained 8.6 mg/g catechin, 8.7 mg/g epicatechin, dimers (B1 6.9 mg/g; B2 8.0 mg/g; B3 20.7 mg/g; and B4 0.7 mg/g), anthocyanins (malvidin-3-glucoside, 11.7 mg/g; peonidin-3-glucoside,
Eur J Nutr
0.66 mg/g; and cyanidin-3-glucoside, 0.06 mg/g), and phenolic acids (gallic acid, 5.0 mg/g; caffeic acid, 2.5 mg/g; and caftaric acid, 12.5 mg/g). The RWPs were dissolved in absolute ethanol at a final concentration of 3.3 %, and this vehicle was administered to the ND and HFD groups.
Auberval et al. [28]. The assay signal was read at 450 nm. Samples were analysed in duplicate, and the results were expressed in mg glycogen/mg liver [31].
Sample preparation
Paraformaldehyde-fixed liver and pancreas tissues were embedded in paraffin blocks and cut into 4-μm sections. Liver sections were stained with haematoxylin and eosin, according to standard procedures, in order to define the degree of steatosis [32]. The grade of steatosis was defined using low-to-medium power microscopy to evaluate parenchymal involvement: a score of 0 indicated 66 % involvement. Insulin immunohistochemical staining was performed on the pancreatic sections using a mouse anti-insulin primary antibody (1/1000; Sigma), followed by a goat biotinylated secondary antibody (1/200; Sigma). The sections were exposed to ExtrAvidin peroxidase solution (1/20; Sigma), and the signal was developed using a solution containing 3-amino-9-ethylcarbazole. After counterstaining the sections with Harris’ haematoxylin, coverslips were mounted using Aquatex® (Merck, Darmstadt, Germany), an aqueous mounting medium. For determination of the islet surface area, at least three sections were examined per rat. Islet surface area was measured using Nikon NIS Elements Br software (Nikon, Tokyo, Japan), and the data distribution was represented by box and whiskers plots showing the median, the first and third quartiles, and the range.
At the end of the 4-month study period, each rat was anaesthetised with a mixture of xylazine (5 mg/kg) and ketamine (40 mg/kg), and blood was drawn from the abdominal aorta into tubes containing ethylenediaminetetraacetic acid. Plasma samples were then stored at −80 °C. All rats were killed at this stage. A piece of fresh liver was weighed and placed in a tube containing sodium acetate buffer for glycogen determination. The rest of the liver and the pancreas were either placed in 4 % paraformaldehyde for subsequent paraffin embedding, sectioning and staining, or embedded in Tissue-Tek® O.C.T. compound (Electron Microscopy Sciences, Hatfield, PA, USA), directly frozen in liquid nitrogen and stored at −80 °C prior to determination of OS. A piece of mesenteric artery was also sampled and embedded in Tissue-Tek® O.C.T. compound. Metabolic analyses Plasma glucose and C-peptide levels were measured with a glucose RTU® kit from bioMérieux (Marcy-l’Étoile, France) and Rat C-peptide enzyme-linked immunosorbent assay (ELISA kit) from Mercodia (Uppsala, Sweden), respectively, according to the manufacturers’ protocols. C-peptide was measured, rather than insulin, to evaluate insulinaemia because it is more stable in blood and is not affected by haemolysis. Results were expressed in g/L for plasma glucose and pmol/L for plasma C-peptide. Plasma triglyceride levels were measured with a Triglycerides Quantification Kit (BioVision Research Products, Mountain View, CA, USA) in accordance with the manufacturer’s protocol, and the results were expressed in mmol/L. Lipid peroxides were determined using the thiobarbituric acid reactive substances method, as described by Auberval et al. [28]. Results were expressed in µmol/L and were normalised to the total protein level in the sample, which was determined using the Bradford method [30]. Carbonylated proteins were measured using an OxiSelectTM ELISA (Cell Biolabs, Inc., San Diego, CA, USA), according to the manufacturer’s instruction. The assay signal was measured by spectrophotometry at 450 nm, and the data were expressed as nmol/mg of total protein, which was determined by Bradford assay. The hepatic glycogen content was determined using 100 mg fresh liver, according to the protocol described by
Histological analyses
Determination of tissue OS The fluorescent dye, dihydroethidine (DHE; SigmaAldrich), was used to evaluate in situ formation of reactive oxygen species (ROS) as previously described by DalRos et al. [25]. Non-fixed pancreas, liver and mesenteric artery tissues were cut into 10-µm sections, treated with DHE (2.5 µM) and incubated in a light-protected humidified chamber at 37 °C for 30 min. The level of ROS was determined using a microscope to quantify whole-tissue fluorescence (NIS-Elements BR; Nikon) in five arbitrarily selected fields. The mean value for each section was calculated, and the results of the ND, HFD, and HFD + RWPs groups were compared. Statistical analyses Statistical tests were conducted using STATISTICA® version 10, Statsoft. The significance of differences between the three study groups (ND, HFD, and HFD + RWPs) was evaluated using analysis of variance with Tukey’s honestly
13
Eur J Nutr
significant difference test, after validating the data normality; five animals were analysed in each group for metabolic studies and three animals were analysed per group for the histological studies. Data were reported as the mean ± standard error of the mean (SEM) for all parameters, except for islet surface area, which was reported as the median and interquartile range. The significant influence of differences between the study groups was represented in the figures and tables using an asterisk (*). Differences with a p value of
ORIGINAL CONTRIBUTION
Beneficial effects of a red wine polyphenol extract on high‑fat diet‑induced metabolic syndrome in rats Nathalie Auberval1 · Stéphanie Dal1 · Elisa Maillard1 · William Bietiger1 · Claude Peronet1 · Michel Pinget1,2,3 · Valérie Schini‑Kerth2 · Séverine Sigrist1
Received: 25 August 2015 / Accepted: 14 February 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract Purpose Individuals with metabolic syndrome (MS) show several metabolic abnormalities including insulin resistance, dyslipidaemia, and oxidative stress (OS). Diet is one of the factors influencing the development of MS, and current nutritional advice emphasises the benefits of fruit and vegetable consumption. Here, we assessed the effects of naturally occurring antioxidants, red wine polyphenols (RWPs), on MS and OS. Methods Wistar rats (n = 20) weighing 200–220 g received a high-fat diet (HFD) for 2 months before they were divided into two groups that received either HFD only or HFD plus 50 mg/kg RWPs in their drinking water for an additional 2 months. A control group (n = 10) received a normal diet (ND) for 4 months. Results Rats receiving HFD increased body weight over 20 % throughout the duration of the study. They also showed increased blood levels of C-peptide, glucose, lipid peroxides, and oxidised proteins. In addition, the HFD increased OS in hepatic, pancreatic, and vascular tissues, as well as induced pancreatic islet cell hyperplasia and hepatic steatosis. Addition of RWPs to the HFD attenuated these * Séverine Sigrist s.sigrist@ceed‑diabete.org 1
UMR DIATHEC, EA 7294, Centre Européen d’Etude du Diabète, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Bld René Leriche, 67200 Strasbourg, France
2
UMR 7175 CNRS, Pharmacologie et Physico‑Chimie, Faculté de Pharmacie, Université de Strasbourg, 67400 Illkirch, France
3
Departement d’Endocrinologie, Diabète, Maladies Métaboliques, Pôle NUDE, Hôpitaux Universitaires de Strasbourg, (HUS), 67000 Strasbourg, France
effects on plasma and tissue OS and on islet cell hyperplasia. However, RWPs had no effect on blood glucose levels or hepatic steatosis. Conclusions RWPs showed an antioxidant mechanism of action against MS. This result will inform future animal studies exploring the metabolic effects of RWPs in more detail. In addition, these findings support the use of antioxidants as adjunctive nutritional treatments for patients with diabetes. Keywords Metabolic syndrome · Oxidative stress · Highfat-diet rats · Red wine polyphenols
Introduction Metabolic syndrome (MS), a condition that encompasses multiple factors such as an elevated blood glucose level (associated with diabetes or a prediabetic state), high cholesterol, high blood pressure, and abdominal obesity [1, 2], affects nearly a quarter of the world’s adult population [3]. It increases the risk of developing type 2 diabetes by fivefold [4]; this is significant, considering that of the 356 million people worldwide who live with diabetes, 90 % have type 2 diabetes. Environmental factors, particularly food intake, are one of primary causes of type 2 diabetes. Indeed, consumption of a diet that is high in energy, protein, fat, and sugars, but low in fruits and vegetables, increases the risk of becoming overweight or obese [5]. In turn, obesity increases the risk of developing type 2 diabetes [6], which is characterised by insulin resistance, and leads to chronic hyperglycaemia and hyperinsulinism. Both of these metabolic alterations are associated with hyperlipidaemia, which causes oxidative stress (OS) [7–9]. OS results from an imbalance between
13
pro-oxidant and antioxidant species in favour of oxidised entities [10]. It promotes the development of MS and type 2 diabetes by disrupting insulin secretion and increasing insulin resistance. In turn, insulin resistance induces hyperinsulinism, which generates OS by stimulating hydrogen peroxide secretion in adipocytes [11] and superoxides in endothelial cells [12], causing endothelial dysfunction and vascular complications. Meanwhile, insulin can inhibit fatty acid oxidation and catalase activity [13], while an unbalanced diet (nutritional failure, nutritional deficiency, or an excessively rich diet) can induce OS by compromising antioxidant defence systems. Insulin resistance in adipocytes and myocytes is responsible for some complications, such as the accumulation of lipids and triglycerides in organs and tissues not normally used for lipid storage; these include the cardiovascular system [14], pancreas, and liver [15]. Insulin resistanceinduced endothelial dysfunction is caused by blood vessel exposure to high levels of lipids and glucose [16]. These high levels also enhance hepatic fatty acid synthesis and strongly inhibit their β-oxidation, leading to fat accumulation, which causes non-alcoholic fatty liver disease [17, 18] and steatosis [19]. Furthermore, insulin resistance induces the storage of hepatic glucose in the form of glycogen [20]. In the pancreas, it causes islet hyperplasia and increases insulin secretion [21]. Epidemiological studies have shown that regular consumption of polyphenol-rich foods such as fruits and vegetables, and beverages such as red wine, is associated with a reduced risk of cardiovascular disease [22]. Intake of red wine polyphenols (RWPs) has also been shown to prevent angiotensin II-induced hypertension and endothelial dysfunction by inhibiting the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent formation of superoxide anions [23] and by preventing age-induced endothelial dysfunction [24, 25], age-related impairment of skeletal muscle mitochondrial function [26], and metabolic disorders [27]. In fact, a red wine extract (ProvinolsTM) was shown to improve glucose metabolism in Zucker fatty rats by reducing plasma glucose and fructosamine levels [27]. Based on this information related to antioxidants and diabetic pathology, we wanted to assess the effect of RWP supplementation in an established animal model of MS. Previously, Auberval et al. [28] demonstrated that a highfat diet (HFD) containing 21.4 % fat and 50.0 % carbohydrate induced MS (and subsequently type 2 diabetes) in healthy young rats. MS was assessed by monitoring obesity, insulin resistance, OS, and widespread tissue changes. The present study fed rats an HFD for 2 months to induce MS and then continued administering the HFD or the HFD+RWPs for 2 months. The effects of these treatments on metabolic parameters and on the function and OS levels of the pancreas, liver, and mesenteric artery were assessed
13
Eur J Nutr
and compared to those of a group of rats fed a normal diet (ND) for 4 months.
Methods This study was performed in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH publication No. 85–23, revised 1996). All efforts were made to minimise animal suffering and to reduce the number of animals. Animals and diets Fifteen male Wistar rats (DEPRE, Saint Doulchard, France) weighing 200–220 g upon arrival were housed in a climatecontrolled room (22 ± 2 °C and 60 % relative humidity) with a 12-h light/dark cycle. The rats had ad libitum access to food and water. The animals were randomly divided into three equal groups, with at least five animals per group. The ND group received a standard diet (A04, SAFE, Augy, France) for 4 months; this had an Atwater fuel energy of 2.8 kcal/g and 60 % carbohydrate, 3.1 % crude fat, 16.1 % crude protein, 3.9 % crude fibres, and 5.1 % ash for 4 months. The HFD group received WESTERN RD (SDS; Special Diets Services, Saint Gratien, France) for 2 months to induce MS and for a further 2 months to develop diabetes; this diet had an Atwater fuel energy of 4.6 kcal/g and comprised 50 % crude carbohydrate, 21.4 % crude fat, 17.5 % crude protein, 3.5 crude fibre, and 4.1 % ash [28]. The HFD+RWPs group was fed the HFD for 2 months, followed by concomitant administration of HFD and 50 mg/kg/day RWPs in the drinking water for an additional 2 months. All rats were weighed once a week throughout the study period. The RWP extract used in this study was generously provided by Dr. M. Moutounet (National Institute of Agronomic Research, Montpellier, France). This extract was prepared from French red wine (Corbières A.O.C.) as previously described [29] and analysed by Dr. P.-L. Teissedre (Département d’Œnologie, Bordeaux, France). Briefly, phenolic compounds were adsorbed on a preparative column. The alcohol was then desorbed, and the alcoholic eluent was gently evaporated prior to lyophilisation of the concentrated residue, which was finely sprayed to obtain the RWP extract as a dry powder. One litre of red wine produced 2.9 g of extract, which contained 471 mg/g of total phenolic compounds, expressed as gallic acid. Phenolic levels in the RWP extract were measured by high-performance liquid chromatography. The extract contained 8.6 mg/g catechin, 8.7 mg/g epicatechin, dimers (B1 6.9 mg/g; B2 8.0 mg/g; B3 20.7 mg/g; and B4 0.7 mg/g), anthocyanins (malvidin-3-glucoside, 11.7 mg/g; peonidin-3-glucoside,
Eur J Nutr
0.66 mg/g; and cyanidin-3-glucoside, 0.06 mg/g), and phenolic acids (gallic acid, 5.0 mg/g; caffeic acid, 2.5 mg/g; and caftaric acid, 12.5 mg/g). The RWPs were dissolved in absolute ethanol at a final concentration of 3.3 %, and this vehicle was administered to the ND and HFD groups.
Auberval et al. [28]. The assay signal was read at 450 nm. Samples were analysed in duplicate, and the results were expressed in mg glycogen/mg liver [31].
Sample preparation
Paraformaldehyde-fixed liver and pancreas tissues were embedded in paraffin blocks and cut into 4-μm sections. Liver sections were stained with haematoxylin and eosin, according to standard procedures, in order to define the degree of steatosis [32]. The grade of steatosis was defined using low-to-medium power microscopy to evaluate parenchymal involvement: a score of 0 indicated 66 % involvement. Insulin immunohistochemical staining was performed on the pancreatic sections using a mouse anti-insulin primary antibody (1/1000; Sigma), followed by a goat biotinylated secondary antibody (1/200; Sigma). The sections were exposed to ExtrAvidin peroxidase solution (1/20; Sigma), and the signal was developed using a solution containing 3-amino-9-ethylcarbazole. After counterstaining the sections with Harris’ haematoxylin, coverslips were mounted using Aquatex® (Merck, Darmstadt, Germany), an aqueous mounting medium. For determination of the islet surface area, at least three sections were examined per rat. Islet surface area was measured using Nikon NIS Elements Br software (Nikon, Tokyo, Japan), and the data distribution was represented by box and whiskers plots showing the median, the first and third quartiles, and the range.
At the end of the 4-month study period, each rat was anaesthetised with a mixture of xylazine (5 mg/kg) and ketamine (40 mg/kg), and blood was drawn from the abdominal aorta into tubes containing ethylenediaminetetraacetic acid. Plasma samples were then stored at −80 °C. All rats were killed at this stage. A piece of fresh liver was weighed and placed in a tube containing sodium acetate buffer for glycogen determination. The rest of the liver and the pancreas were either placed in 4 % paraformaldehyde for subsequent paraffin embedding, sectioning and staining, or embedded in Tissue-Tek® O.C.T. compound (Electron Microscopy Sciences, Hatfield, PA, USA), directly frozen in liquid nitrogen and stored at −80 °C prior to determination of OS. A piece of mesenteric artery was also sampled and embedded in Tissue-Tek® O.C.T. compound. Metabolic analyses Plasma glucose and C-peptide levels were measured with a glucose RTU® kit from bioMérieux (Marcy-l’Étoile, France) and Rat C-peptide enzyme-linked immunosorbent assay (ELISA kit) from Mercodia (Uppsala, Sweden), respectively, according to the manufacturers’ protocols. C-peptide was measured, rather than insulin, to evaluate insulinaemia because it is more stable in blood and is not affected by haemolysis. Results were expressed in g/L for plasma glucose and pmol/L for plasma C-peptide. Plasma triglyceride levels were measured with a Triglycerides Quantification Kit (BioVision Research Products, Mountain View, CA, USA) in accordance with the manufacturer’s protocol, and the results were expressed in mmol/L. Lipid peroxides were determined using the thiobarbituric acid reactive substances method, as described by Auberval et al. [28]. Results were expressed in µmol/L and were normalised to the total protein level in the sample, which was determined using the Bradford method [30]. Carbonylated proteins were measured using an OxiSelectTM ELISA (Cell Biolabs, Inc., San Diego, CA, USA), according to the manufacturer’s instruction. The assay signal was measured by spectrophotometry at 450 nm, and the data were expressed as nmol/mg of total protein, which was determined by Bradford assay. The hepatic glycogen content was determined using 100 mg fresh liver, according to the protocol described by
Histological analyses
Determination of tissue OS The fluorescent dye, dihydroethidine (DHE; SigmaAldrich), was used to evaluate in situ formation of reactive oxygen species (ROS) as previously described by DalRos et al. [25]. Non-fixed pancreas, liver and mesenteric artery tissues were cut into 10-µm sections, treated with DHE (2.5 µM) and incubated in a light-protected humidified chamber at 37 °C for 30 min. The level of ROS was determined using a microscope to quantify whole-tissue fluorescence (NIS-Elements BR; Nikon) in five arbitrarily selected fields. The mean value for each section was calculated, and the results of the ND, HFD, and HFD + RWPs groups were compared. Statistical analyses Statistical tests were conducted using STATISTICA® version 10, Statsoft. The significance of differences between the three study groups (ND, HFD, and HFD + RWPs) was evaluated using analysis of variance with Tukey’s honestly
13
Eur J Nutr
significant difference test, after validating the data normality; five animals were analysed in each group for metabolic studies and three animals were analysed per group for the histological studies. Data were reported as the mean ± standard error of the mean (SEM) for all parameters, except for islet surface area, which was reported as the median and interquartile range. The significant influence of differences between the study groups was represented in the figures and tables using an asterisk (*). Differences with a p value of
