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A grape seed extract increases active glucagon-like peptide-1 levels after an oral glucose load in rats† Cite this: Food Funct., 2014, 5, 2357
Noemi Gonza´lez-Abu´ın,a Neus Mart´ınez-Micaelo,a Maria Margalef,a Mayte Blay,a ab a a Anna Arola-Arnal,a Begon ˜ a Muguerza, Anna Arde´vol* and Montserrat Pinent We have previously reported that procyanidins, a class of flavonoids, improve glycemia and exert an incretin-like effect, which was linked to their proven inhibitory effect on the dipeptidyl-peptidase 4 (DPP4) activity. However, their actual effect on incretin levels has not been reported yet. Therefore, in the present study we have evaluated whether a grape seed extract enriched in procyanidins (GSPE) modulates plasma incretin levels and attempted to determine the mechanisms involved. An acute GSPE treatment in healthy Wistar female rats prior to an oral glucose load induced an increase in plasma active glucagon-like peptide-1 (GLP-1), which was accompanied by an increase in the plasma insulin/glucose ratio and a simultaneous decrease in glucose levels. In agreement with our previous studies, the intestinal DPP4 activity was inhibited by the GSPE treatment. We have also assayed in vitro whether this inhibition occurs in inner intestinal tissues close to GLP-1-producing cells, such as the endothelium of the capillaries. We have found that the main compounds absorbed by intestinal CaCo-2 cells after an acute treatment with GSPE are catechin, epicatechin, B2 dimer and gallic acid, and that they inhibit the Received 20th May 2014 Accepted 16th July 2014
DPP4 activity in endothelial HUVEC cells in an additive way. Moreover, an increase in plasma total GLP-1 levels was found, suggesting an increase in GLP-1 secretion. In conclusion, our results show that GSPE
DOI: 10.1039/c4fo00447g
improves glycemia through its action on GLP-1 secretion and on the inhibition of the inner intestinal
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DPP4 activity, leading to an increase in active GLP-1 levels, which, in turn, may affect the insulin release.
Introduction The high prevalence of type 2 diabetes mellitus highlights the need to identify new effective therapies,1 including the use of natural products that could help avoid the development of this pathology. One approach could be to improve the ability of some foods to increase the life-time of glucagon-like peptide-1 (GLP-1), an incretin hormone produced by the Lcells in the distal ileum and colon that is secreted aer oral consumption of nutrients, mainly glucose. This hormone has a wide range of biological activities, including increased insulin biosynthesis and secretion, inhibition of glucagon secretion, enhancement of b cell mass proliferation and neogenesis, reduction in food intake, and enhancement of satiety (reviewed in ref. 2 and 3). Certain natural compounds have been reported to promote GLP-1 secretion. Chlorogenic acid, a major phenolic compound in coffee, was shown to increase GLP-1 production and secretion by L-cells and improve glycemia in mice aer
Departament de Bioqu´ımica i Biotecnologia, Universitat Rovira i Virgili, Tarragona, Spain. E-mail: [email protected]; Fax: +34-977-558232; Tel: +34-977-559566
a
b
Centre Tecnol` ogic de Nutrici´o i Salut (CTNS), TECNIO, CEICS, Reus 43204, Spain
† Electronic supplementary 10.1039/c4fo00447g
information
(ESI)
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available.
See
DOI:
oral glucose load.4,5 Similarly, an Ilex paraguariensis leaf extract, which is rich in the phenolic compound 3,5-O-dicaffeoyl-D-quinic acid, was shown to improve glycemia and insulinemia in mice fed a high-fat diet (HFD), a result that could be related to higher active plasma GLP-1 levels.6,7 Berberine, a major active constituent of Rhizoma coptidis, was also reported to improve glycemia, insulinemia and GLP-1 levels aer oral glucose challenge.8 Resveratrol, a polyphenolic compound presented in such plants as red grapes, exhibits anti-hyperglycemic effect, which has been attributed to increase in GLP-1 levels.9 Berries, a rich source of polyphenols, have also been shown to enhance GLP-1 levels and improve insulin concentrations aer sucrose consumption.10 Altogether, it appears that some phenolic compounds have the ability to enhance glucose-stimulated GLP-1, though the exact mechanisms by which these natural compounds exert such effects have not been elucidated yet. The GLP-1 half-life is less than two minutes due to its rapid cleavage by the enzyme dipeptidyl-peptidase 4 (DPP4).11 In fact, synthetic DPP4 inhibitors, such as vildagliptin or sitagliptin, are being currently used for the treatment of type 2 diabetes mellitus because of their capacity to improve glycemic control by avoiding rapid incretin cleavage.12,13 We have previously reported that a grape seed extract enriched in procyanidins (GSPE) inhibited the intestinal DPP4 activity and increased the plasma
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insulin/glucose ratio in rats in response to orally administered glucose, suggesting an incretinomimetic effect,14 which could help explain the antihyperglycemic effect of GSPE.15–17 Therefore, the aim of the present study was to investigate if a grape seed extract enriched in procyanidins modulates glucosestimulated GLP-1 levels, and to elucidate the mechanisms by which this effect is produced.
Results GSPE enhances glucose-induced active GLP-1 levels To investigate if GSPE modulates incretin levels in vivo, active GLP-1 and total GIP levels in plasma were analyzed aer an oral glucose load (2 g of glucose per kg of bw). As shown in Fig. 1a, the active GLP-1 levels are higher in rats treated with GSPE for 1 hour than in the control group. In contrast, Fig. 1b shows decreased total GIP levels aer GSPE treatment. The effect of these changes on glycaemia and insulinemia were also analyzed, and we have found that they were improved by the GSPE treatment. There was a reduction in plasma glucose levels in the rats treated with GSPE compared to controls (Fig. 1c), concomitantly with a higher insulin/glucose ratio in
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the GSPE-treated rats compared to the control group, as shown in Fig. 1d. The GSPE effects are similar to those found in the positive control group treated with Vildagliptin, a DPP4 inhibitor (Fig. 1). The increase in active GLP-1 levels was dependent on the presence of glucose, since it was only found in animals under OGTT treatment, but not in fasted GSPE-treated animals (Control: 6.1 0.5 pM; GSPE: 5.8 0.3 pM). In fact, GSPE did not modify glycemia (Dglucose: 2.26 0.34 and 1.57 0.29, for control and GSPE, respectively) or insulinemia (insulin/ glucose ratio: 0.23 0.04 and 0.22 0.05, for control and GSPE, respectively) in these animals.
GSPE increases GLP-1 levels by inhibiting the intestinal DPP4 activity To analyze whether an acute dose of GSPE exerts the hypoglycemic effect through its previously described effects as a DPP4 inhibitor, we have assayed the DPP4 activity in plasma and the intestine. The plasma DPP4 activity was not modied aer 1 hour of GSPE treatment (Fig. 2a), whereas GSPE decreased the
Fig. 1 GSPE effect on plasma active GLP-1, total GIP, glucose and insulin levels. An oral load of 2 g glucose per kg of bw was administered in rats treated with 1 g GSPE per kg of bw or 1 mg vildagliptin per kg of bw for 40 minutes (E). Effect of the oral glucose load on the plasma active GLP-1 levels (A), total GIP levels (B), glucose levels (C), and insulin concentration per plasma glucose (D) after 20 minutes. The data are displayed as the mean SEM. a and b, statistically significant differences with P < 0.05.
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also found. Other forms present in GSPE (shown in Table 1 of ESI†), such as epicatechin gallate and procyanidin trimer, and other products of intestinal absorption, such as glucuronide forms, were also detected in the basolateral medium but their levels were too low to be quantied. We have used the basolateral media to treat HUVEC cells for 1 hour and, as shown in Fig. 3, the endothelial DPP4 activity was inhibited by approximately 32% (Fig. 3a). No signicant changes were observed in DPP4 gene expression (results not shown). To assess whether DPP4 is directly inhibited by the pure absorbed molecules found in the basolateral medium, the in vitro DPP4 activity was assayed in the presence of these molecules. DPP4 isolated from HUVEC cells was incubated for 1 hour with 100 mg L1 of each pure molecule, at a concentration similar to that found in the basolateral medium. Catechin, procyanidin B2, and gallic acid signicantly inhibited the DPP4 activity, showing between 4 and 8% inhibition (Fig. 3b). The sum of the inhibitions obtained by each of the four compounds was 21.19% 5.74. This sum was not signicantly different from the inhibition achieved by a mixture of all the compounds (26%, Fig. 3b), and it was also not different from the inhibition produced by the basolateral medium on the indirect co-cultures (P > 0.05). GSPE increases GLP-1 levels by increasing its release
Fig. 2 Effect of GSPE on DPP4. Effects on the plasma DPP4 activity (A) and intestinal DPP4 activity (B), and DPP4 gene expression (C) in rats treated with 1 g of GSPE per kg of bw or 1 mg vildagliptin per kg of bw for 1 hour concomitant with an oral glucose load. The data are displayed as the mean SEM. *Significant differences vs. control group with P < 0.05.
intestinal DPP4 activity by approximately 28%, as shown in Fig. 2b. Vildagliptin also reduced the intestinal DPP4 activity by approximately 37%, which is a newly described effect; and inhibited the plasma DPP4 activity, causing up to 29% inhibition, as expected. Both the GSPE and Vildagliptin treatments have signicantly up-regulated the intestinal DPP4 gene expression (Fig. 2c). Because in vivo measurements are obtained using the whole intestine, we have used an in vitro approach to show that GSPE inhibits DPP4 located in the capillaries, as GLP-1 is secreted into intestinal capillaries. First, HUVEC endothelial cells were directly treated with 25 mg of GSPE per L, a non-toxic dose, and an inhibition on the DPP4 activity of 39% 4 (P ¼ 0.026) was observed. Nevertheless, GSPE is absorbed and metabolized by the intestine before reaching the endothelium of the capillaries; in order to mimic it, CaCo-2 cells were grown in Millicell hanging cell culture inserts and treated, on their apical side, with 250 mg of GSPE per L for 1 hour. By chromatographic analysis (HPLC-MS/MS) we have identied the avanol components of the media. As shown in Table 1, the basolateral medium contained catechin, epicatechin, gallic acid, and procyanidin dimer. Sulfate forms of catechin and epicatechin were
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To analyze whether an acute dose of GSPE also acts by increasing GLP-1 release, total GLP-1 levels in plasma were measured aer 1 h of GSPE treatment. As shown in Fig. 4, total GLP-1 levels are higher aer the GSPE treatment both in fasted (Fig. 4a) and glucose-induced groups (Fig. 4b). As expected, the commercial DPP4 inhibitor Vildagliptin did not change total GLP-1 levels (Fig. 4b).
Discussion Although the antihyperglycemic effect of GSPE has been previously reported18 and its incretinomimetic effect has been suggested,14 its actual effect on incretin levels have not been described yet. The current study shows that an extract from grape seeds enriched in procyanidins increases the glucoseinduced active GLP-1 levels in plasma. This effect could be explained by two mechanisms: the inhibition of endothelial DPP4 activity and the increase in GLP-1 release. Previously, a few studies reported the effect of polyphenols on GLP-1 levels.8–10 Resveratrol, a polyphenolic compound present in red grapes, was shown to increase plasma GLP-1 levels in HFD-induced diabetic mice, which was linked to an amelioration of the glucose prole and an increase in the insulin level in response to OGTT.9 However, the exact mechanisms responsible for such effects were not further elucidated. In our case, we have also found an amelioration of the insulin/ glucose plasma prole as a possible consequence of increased active GLP-1. Although our current experiment is an acute treatment, GSPE has been shown to decrease the HOMA-IR index in cafeteria-fed rats;15,19 thus, the chronic effects on incretin levels deserve further study.
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Table 1
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Flavonols and their metabolites in the basolateral media collected from CaCo-2 inserts, after treating apically with 250 mg of GSPE per La
Compound
Total amount (mM)
Total amount (mg L1)
Catechin Epicatechin Procyanidin dimer(3) Gallic acid
0.21 0.06 0.28 0.06 0.12 0.03 0.29 0.02
60.09 16.54 79.86 18.39 67.05 15.26 49.68 2.70
Metabolite Catechin-sulfate(1) Epicatechin-sulfate(2) Methyl-epicatechin-o-sulfate(2)
0.12 0.01 0.16 0.01 0.21 0.02
44.38 3.77 58.87 4.63 78.48 8.11
a Compounds not quantied: epicatechin gallate(4), dimer gallate(3), trimer(3), EGCG, procyanidin dimer gallate(3); metabolites not quantied: epicatechin-glucuronide(2), methyl-catechin-o-sulfate(1), methyl-catechin-glucuronide(1), methyl-epicatechincatechin-glucuronide(1), (2) (2) (2) glucuronide , 3-o-methyl-epicatechin , 4-o-methyl-epicatechin ; metabolite not detected: epicatechin gallate glucuronide(2).
Effect of absorbed molecules and pure compounds in GSPE on endothelial DPP4 activity. CaCo-2 cells were apically treated with 250 mg of GSPE per L for 1 hour, the basolateral medium was collected and used to incubate HUVEC cells for 1 hour (n ¼ 4) (A). DPP4 extracted from HUVEC cells incubated with 100 mg L1 catechin (Cat), epicatechin (EC), procyanidin B2 (B2) or gallic acid (GA), and a mixture of 100 mg L1 each (Mix) for 1 hour (n ¼ 4) (B). The data are displayed as the mean SEM. *Significant differences with P < 0.05. Fig. 3
Regarding the mechanism leading to the GLP-1 increase, we have previously shown that GSPE inhibited DPP4.14 Indeed, inhibition of the DPP4 enzyme is an emerging strategy to improve glycemia, and several DPP4 inhibitors are currently being used in the pharmacological treatment of type 2 diabetes.20–22 Previous14 and current experiments report no in vivo effects of GSPE on the plasma DPP4 activity. However, we have found inhibition of the intestinal DPP4 activity due to acute (1 hour) GSPE treatment concomitant with an oral glucose load. These results are in agreement with our previous results that also show reduced intestinal DPP4 activity due to acute and chronic treatments in healthy and cafeteria-fed rats.14
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GSPE on total GLP-1 in plasma. Plasma total GLP-1 levels in fasted rats that received a dose of 1 g of GSPE per kg of bw for 1 h (A), and in rats that received a dose of 1 g of GSPE per kg of bw or 1 mg of vildagliptin per kg of bw and, after 40 minutes of treatment, were administered with an acute load of 2 g of glucose per kg of bw for 20 minutes (B). The data are displayed as the mean SEM. *, a and b, statistically significant differences with P # 0.05. Fig. 4
Commercial DPP4 inhibitors, such as the positive control vildagliptin,23 have been commonly reported to exert their incretinomimetic effect via inhibition of the plasma DPP4 activity, thereby delaying the GLP-1 cleavage in plasma (reviewed in ref. 22). However, it was also shown in healthy humans that the increase in plasma GLP-1 levels is not proportional to plasma DPP4 inhibition, suggesting that routes other than plasma DPP4 inhibition are involved.24 Moreover, Hansen et al.25 have reported that half of the newly secreted GLP-1 is N-terminally degraded prior to reaching systemic circulation, suggesting cleavage soon aer its secretion by the DPP4 present in the endothelium of the capillaries adjacent to the GLP-1-secreting cells. A recent study has reported that low
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active doses of sitagliptin, another DPP4 inhibitor, cannot exert the same effect on glucose homeostasis when administered intravenously versus when orally administered, which was attributed to the selective inhibition of intestinal DPP4 activity caused by the oral administration of sitagliptin.26 In addition, the present study shows that vildagliptin can also inhibit the intestinal DPP4 activity, an effect that has not been reported to date. All these ndings support the relevance of the DPP4 enzyme located in tissues close to site of GLP-1 production. Regarding the effects on the other incretin hormone GIP, which is also a target for the DPP4 enzyme, GSPE was found to decrease its total levels aer the oral glucose load, a similar effect to that found for vildagliptin. These results are in concordance with previously reported effects of the commercial DPP4 inhibitor sitagliptin, which was also found to decrease plasma total GIP levels with the concomitant increase in active GLP-1.27 Thus, we hypothesize that the inhibition of inner intestinal DPP4 by the absorbed/metabolized forms of GSPE could be, at least in part, responsible for the increased active GLP-1 plasma levels. In our experiments, DPP4 activity was determined using lysates of the whole intestine; thus, the GSPE-reduced DPP4 activity could also be due to direct inhibition of the enzyme present on the brush border membrane of the absorptive cells. Therefore, we have used an in vitro approach to test our hypothesis: we have analyzed whether the molecules contained in GSPE and/or their metabolites absorbed by intestinal cells (Caco-2 cells cultured on hanging inserts to simulate the intestinal barrier) are able to decrease the activity of DPP4 present in the endothelium of the capillaries (HUVEC cells). To this end, we have used indirect co-cultures due to the low viability of HUVEC cells cultured directly under the Caco-2 monolayer (results not shown). Our results show that the collected basolateral medium of the Caco-2 cells treated with GSPE signicantly decreased the activity of endothelial DPP4, supporting the hypothesis that GSPE might act on the DPP4 present in the endothelium of the capillaries, potentially decreasing GLP-1 cleavage and leading to an increase in active GLP-1 levels. Our in vitro experiments also have led us to identify the molecules responsible for the DPP4 inhibitory effect, which is of importance since previous studies showed that the effect on enzyme modulation depends on the type of the phenolic compound: quercetin and a avonoid-rich extract from Pilea microphylla were also previously shown to inhibit DPP4,28 but other avonoids, such as curcumin29 and avone-8acetic acid30 did not. Thus, catechin, epicatechin, procyanidin dimer and gallic acid, the pure molecules absorbed by the Caco2 monolayer, were tested in vitro, and it was found that the additive effect of these molecules equaled the effect of the basolateral medium. Therefore, we describe for the rst time the inhibition of DPP4 by pure compounds: catechin, gallic acid, and B2 dimer, molecules that can be absorbed in the intestine.31 The decrease in DPP4 activity induced by GSPE could be due to the direct effect of these molecules, since we did not nd reduced DPP4 gene expression either in vivo or in vitro, despite our nding that GSPE modulates DPP4 gene expression.14
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In addition to inhibition of the GLP-1-inactivating enzyme, the increase in active GLP-1 plasma levels could be also due to higher GLP-1 secretion, as suggested for other avonoids.9,10,32 Thus, we have measured total GLP-1 levels, and found that GSPE increased GLP-1 levels in glucose-loaded animals, pointing out the increased secretion. Besides, GSPE induced higher active GLP-1 levels than Vildagliptin, despite Vildagliptin has a broader DPP4 inhibitory capacity, reinforcing that GSPE might also increase active GLP-1 levels by inducing higher GLP-1 secretion. In addition, we have found that such increased secretion of GLP-1 is dependent on the presence of glucose, since in the fasted condition we did not nd an increase in active GLP-1 forms. But, surprisingly, we did also nd higher total GLP-1 levels in fasted animals. Active GLP-1 was measured by using the GLP-1 (active) ELISA kit which is specic for GLP1(7-36), mainly secreted from the intestine aer glucose stimulation.33 In contrast, under fasting conditions, pancreas is the main production site for GLP-1-like molecules, mainly the GLP1(1-36)amide form which is not sensitive to DPP4 and whose activity is still unknown.33 Therefore we hypothesize that, under fasting conditions, GSPE activates the secretion of GLP-1-like molecules from pancreas, which has been clearly identied as an important target for procyanidins;34,35 while, under glucose stimulation, GSPE enhances GLP-1-like intestinal secretions induced by glucose.
Experimental Reagents The grape seed extract enriched in procyanidins (GSPE) was obtained from Les D´ eriv´ es R´ esiniques et Terp´ eniques (Dax, France). According to the manufacturer, the extract contains essentially monomeric (21.3%), dimeric (17.4%), trimeric (16.3%), tetrameric (13.3%) and oligomeric (5–13 U; 31.7%) procyanidins. The small molecules (up to trimers) present in GSPE were characterized, in more detail, by reverse-phase chromatographic analyses by our research group.36 Cell culture reagents were obtained from BioWhittaker (Veviers, Belgium) and EmbryoMax. 0.1% Gelatin Solution was purchased from Millipore (Madrid, Spain). For the HPLC-MS/MS analysis, pure molecules ((+)-catechin, ()-epicatechin, gallic acid, epigallocatechin gallate and procyanidin B2) and the internal standard (IS) pyrocatechol were purchased from Fluka/Sigma-Aldrich (Madrid, Spain). Acetone (HPLC grade), methanol (HPLC grade) and phosphoric acid were purchased from Sigma-Aldrich (Madrid, Spain). Ultrapure water was obtained using a Milli-Q Advantage A10 system from Millipore (Madrid, Spain), and glacial acetic acid was purchased from Panreac (Barcelona, Spain). Animal experimental procedures Female Wistar rats weighing between 200 and 225 g were purchased from Charles River Laboratories (Barcelona, Spain), housed in animal quarters at 22 C with a 12 h light/12 h dark cycle and maintained for 1 week in quarantine. The animals had ad l´ıbitum food access and the food was withdrawn at 8 p.m. Food Funct., 2014, 5, 2357–2364 | 2361
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on the day before sacrice. In both experiments, the animals were anesthetized with 50 mg of pentobarbital per kg of body weight (bw) and sacriced by bleeding through the aorta artery. The blood was collected, and animal tissues were immediately frozen in liquid nitrogen and stored at 80 C until further analysis. All the procedures were approved by the Experimental Animal Ethics Committee of the Universitat Rovira i Virgili according to Law 214/1997 of the Catalonian Department of Environment and Housing (published in BOE (DOGC number 2450)). Experiment 1. At 8 a.m. on the day of the experiment, the rats were divided into three groups (6 animals per group): (i) control group, rats treated with the vehicle (tap water), (ii) GSPE group, rats treated with 1 g of GSPE per kg of body weight (bw), and (iii) positive control group, rats treated with 1 mg of Vildagliptin per kg of bw (Axon Medchem, Groningen, The Netherlands). The treatment was administered acutely by oral gavage. Tail blood samples were collected before treatment administration. Aer 40 minutes of GSPE/Vildagliptin gavage, an oral glucose load (2 g of glucose per kg of bw) was administered; 20 minutes later, the animals were sacriced. A schematic diagram of the animal experiment is shown in Fig. 1. A portion of the collected blood was treated with commercial DPP4 inhibitor (Millipore, Madrid, Spain) and serine protease inhibitor, Pefabloc SC, (Roche, Barcelona, Spain). Experiment 2. At 8 a.m. on the day of the experiment, the rats were divided into two groups (6 animals per group): (i) control group, rats treated with the vehicle (tap water), and (ii) GSPE group, rats treated with 1 g of GSPE per kg of bw. The treatment was administered acutely by oral gavage. Tail blood samples were collected before treatment administration, and, aer 1 h of GSPE gavage, the animals were sacriced.
Plasma measurements Plasma glucose concentrations were assayed using an enzymatic colorimetric kit (GOD-PAP method from QCA, Tarragona, Spain). The plasma insulin and active GLP-1 concentrations were determined using Rat Insulin ELISA/Ultrasensitive Rat Insulin ELISA (Mercodia, Uppsala, Sweden) and GLP-1 (active) ELISA (EGLP-35K) (Millipore, Madrid, Spain) kits, respectively, following the manufacturer's instructions. Total GIP and GLP-1 plasma levels were measured using Rat/Mouse GIP (total) (EZRMGIP-55K) and GLP-1 total ELISA (EZGLP1T-36K) (Millipore, Madrid, Spain) kits, respectively, following the manufacturer's instructions.
Cell lines and indirect co-culture Caco-2 cells were obtained from the American Type Culture Collection (ATCC) (LGC Standards S.L.U., Barcelona, Spain) and cultured in DMEM supplemented as previously described.37 HUVEC cells were obtained from Cascade Biologics™ (Carlsbad, CA, USA), cultured in 0.1% gelatin-coated asks and maintained in Endothelial Growth Medium-2 (EGM-2).
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Caco-2 cells were seeded onto Millicell hanging cell culture inserts (Millipore) for 6-well plates at a cell density of 1.3 104 cells per cm2. The volume of the culture medium was 1 mL on the apical side and 2 mL on the basolateral side. The transepithelial electrical resistance (TEER) of Caco-2 cells was monitored daily by using the Millicell-ERS System (Millipore); when a conuent monolayer was formed (21 days of culture), the cells were treated with 250 mg GSPE per L on their apical side. The basolateral media was collected aer 1 hour of GSPE treatment and stored at 20 C. HUVEC cells were seeded in 6-well plates coated with 0.1% gelatin at a cell density of 8–10.5 104 cells per cm2. When cells reached 80–90% of conuence, they were treated with the collected basolateral media for 1 hour or with 25 mg of GSPE per L for 2 hours. Measurement of DPP4 activity DPP4 was extracted from rat intestine and HUVEC cells as previously described (ref. 14 and 38, respectively). Briey, the intestines were homogenized with lysis buffer (PBS containing 100 KIU mL1 aprotinin and 1% Triton X-100), centrifuged at 1000 g at 4 C for 10 minutes to eliminate the cellular debris, and centrifuged twice at 20 000 g at 4 C for 10 minutes. The HUVEC cells were washed twice with 1 PBS, harvested in 100 mM Tris–HCl, and centrifuged. The supernatant was collected; the pellet was dispersed in the same buffer supplemented with 2% Triton X-100 and, aer centrifugation, the supernatant was added to the rst supernatant and stored at 80 C until further analysis. The DPP4 activity in the cell and intestinal lysates and rat plasma, was measured by following the manufacturer's instructions provided for the DPP4 Drug Discovery Kit-AK499 (Enzo Life Sciences International, Inc.), with a few modications based on the volume of the DPP4-containing sample, and adjusted to the nal volume with Tris–HCl buffer. The protein concentration in all samples was determined as previously reported39 and used to normalize the DPP4 activity values. DPP4 gene expression Total RNA from HUVEC cells was extracted using an RNAeasy Kit (Qiagen, Hilden, Germany) and the total RNA from the intestine was extracted using TRIzol reagent following the manufacturer's instructions. The cDNA was generated using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain). Quantitative PCR amplication and detection for DPP4 was performed using the following TaqMan assay-on-demand probes (Applied Biosystems, Madrid, Spain): Rn00562910_m1 and Hs00175210_m1 for DPP4 for rat and HUVEC cells, respectively. The results were normalized to bactin Rn00667869_m1 for rat and cyclophilin Hs99999904_m1 (Ppia) for HUVEC cells. The relative mRNA expression levels were calculated using the DDCt method. Chromatographic analysis of procyanidins and their metabolites Standards preparation. A 200 mg L1 stock standard mixture in methanol of (+)-catechin, ()-epicatechin, gallic acid and
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epigallocatechin gallate and 100 mg L1 of procyanidin B2 were prepared weekly and stored in a dark ask at 20 C. This stock standard solution was diluted daily to the desired concentration with an acetone : water : acetic acid (70 : 29.5 : 0.5, v/v/v) solution and stored under the same conditions until the chromatographic analysis. Micro-solid phase extraction. Prior to the chromatographic analysis, cell medium samples were pre-treated by using off-line m-SPE using OASIS® HLB m-Elution Plates 30 mm (Waters, Barcelona, Spain) following the methodology for biological samples as previously described.40 Instrumental conditions. The eluted solution was directly analyzed using a 1200 LC Series coupled to a 6410 QqQ-MS/MS (Agilent Technologies, Palo Alto, USA). The chromatographic separation of both apical and basolateral media was performed on a Zorbax C18 column (150 mm 2.1 mm i.d., 3.5 mm particle size, Agilent Technologies). The procyanidins and their metabolites were analyzed by ESI as the ionization technique and in negative mode, as previously described.41 Direct inhibition of DPP4 activity by pure compounds in vitro DPP4 was isolated from untreated HUVEC cells as described above and incubated with 100 mg L1 of catechin, epicatechin, procyanidin B2 or gallic acid, either alone or together, for 1 hour. Then, the DPP4 activity was determined as described above. Data analyses The results are expressed as mean SEM. The effects were assessed by ANOVA or Student's t-test. All calculations were performed using SPSS soware version 17.0 (SPSS, Chicago, USA).
Conclusions This study indicates that acute treatment with GSPE, concomitant to a glucose load, favours lower glycemia caused by the higher active GLP-1 levels observed in the GSPE-treated group. The higher active GLP-1 levels might be due to increased enteric glucose-induced secretion plus the additive effect of the absorbed avanol forms inhibiting the intestinal DPP4.
Conflict of interest The authors declare that they have no conict of interest.
Acknowledgements We would like to acknowledge the technical support of Niurka Llopiz and Naroa Mendizuri from the Universitat Rovira i Virgili. Thanks are conveyed to members of the Nutrigenomics group collaborated for caring and sampling of the animals. This study was supported by a grant (AGL2011-23879) from the Spanish government. Noem´ı Gonz´ alez-Abu´ın is the recipient of an FPI fellowship from the Spanish Ministry of Science and Innovation (MICINN), and Neus Mart´ınez-Micaelo and Maria This journal is © The Royal Society of Chemistry 2014
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Margalef are recipients of fellowships from the Universitat Rovira i Virgili. N. G.-A. has analyzed data and wrote the manuscript; N. M.-M. and M. M. contributed to research data; MT. B., A. A.-A. and B. M. contributed to discussion; A. A. and M. P. contributed to the experimental design and discussion, and reviewed the manuscript.
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31 A. Serra, A. Maci` a, M.-P. Romero, J. Valls, C. Blad´ e, L. Arola and M.-J. Motilva, Br. J. Nutr., 2010, 103, 944–952. 32 Y. Yamashita, M. Okabe, M. Natsume and H. Ashida, Biosci., Biotechnol., Biochem., 2013, 77, 888–891. 33 J. van Del, L. Uttenthal, O. Hermida, T. Fontela and M. Ghiglione, Regul. Pept., 1997, 70, 191–198. 34 A. Castell-Auv´ı, L. Ced´ o, V. Pallar` es, M. T. Blay, M. Pinent, M. J. Motilva, S. Garcia-Vallv´ e, G. Pujadas, P. Maechler and A. Ard´ evol, J. Nutr. Biochem., 2012, 23, 1565–1572. 35 L. Cedo, A. Castell-Auvi, V. Pallares, C. U. Mohien, I. Baiges, M. Blay, A. Ardevol and M. Pinent, Food Chem., 2012, 135, 1948–1956. 36 M. Qui˜ nones, L. Guerrero, M. Suarez, Z. Pons, A. Aleixandre, L. Arola and B. Muguerza, Food Res. Int., 2013, 51, 587–595. 37 A. Alvaro, R. Rosales, L. Masana and J.-C. Vallv´ e, J. Nutr. Biochem., 2010, 21, 518–525. 38 A. P. Silva, C. Cavadas, B. Ba¨ısse-Agushi, O. Spertini, H. R. Brunner and E. Grouzmann, Regul. Pept., 2003, 116, 71–79. 39 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254. 40 M.-P. Mart´ı, A. Pantale´ on, A. Rozek, A. Soler, J. Valls, A. Maci` a, M.-P. Romero and M.-J. Motilva, J. Sep. Sci., 2010, 33, 2841–2853. 41 A. Serra, A. Maci` a, M.-P. Romero, M.-J. Salvad´ o, M. Bustos, J. Fern´ andez-Larrea and M.-J. Motilva, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2009, 877, 1169–1176.
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A grape seed extract increases active glucagon-like peptide-1 levels after an oral glucose load in rats† Cite this: Food Funct., 2014, 5, 2357
Noemi Gonza´lez-Abu´ın,a Neus Mart´ınez-Micaelo,a Maria Margalef,a Mayte Blay,a ab a a Anna Arola-Arnal,a Begon ˜ a Muguerza, Anna Arde´vol* and Montserrat Pinent We have previously reported that procyanidins, a class of flavonoids, improve glycemia and exert an incretin-like effect, which was linked to their proven inhibitory effect on the dipeptidyl-peptidase 4 (DPP4) activity. However, their actual effect on incretin levels has not been reported yet. Therefore, in the present study we have evaluated whether a grape seed extract enriched in procyanidins (GSPE) modulates plasma incretin levels and attempted to determine the mechanisms involved. An acute GSPE treatment in healthy Wistar female rats prior to an oral glucose load induced an increase in plasma active glucagon-like peptide-1 (GLP-1), which was accompanied by an increase in the plasma insulin/glucose ratio and a simultaneous decrease in glucose levels. In agreement with our previous studies, the intestinal DPP4 activity was inhibited by the GSPE treatment. We have also assayed in vitro whether this inhibition occurs in inner intestinal tissues close to GLP-1-producing cells, such as the endothelium of the capillaries. We have found that the main compounds absorbed by intestinal CaCo-2 cells after an acute treatment with GSPE are catechin, epicatechin, B2 dimer and gallic acid, and that they inhibit the Received 20th May 2014 Accepted 16th July 2014
DPP4 activity in endothelial HUVEC cells in an additive way. Moreover, an increase in plasma total GLP-1 levels was found, suggesting an increase in GLP-1 secretion. In conclusion, our results show that GSPE
DOI: 10.1039/c4fo00447g
improves glycemia through its action on GLP-1 secretion and on the inhibition of the inner intestinal
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DPP4 activity, leading to an increase in active GLP-1 levels, which, in turn, may affect the insulin release.
Introduction The high prevalence of type 2 diabetes mellitus highlights the need to identify new effective therapies,1 including the use of natural products that could help avoid the development of this pathology. One approach could be to improve the ability of some foods to increase the life-time of glucagon-like peptide-1 (GLP-1), an incretin hormone produced by the Lcells in the distal ileum and colon that is secreted aer oral consumption of nutrients, mainly glucose. This hormone has a wide range of biological activities, including increased insulin biosynthesis and secretion, inhibition of glucagon secretion, enhancement of b cell mass proliferation and neogenesis, reduction in food intake, and enhancement of satiety (reviewed in ref. 2 and 3). Certain natural compounds have been reported to promote GLP-1 secretion. Chlorogenic acid, a major phenolic compound in coffee, was shown to increase GLP-1 production and secretion by L-cells and improve glycemia in mice aer
Departament de Bioqu´ımica i Biotecnologia, Universitat Rovira i Virgili, Tarragona, Spain. E-mail: [email protected]; Fax: +34-977-558232; Tel: +34-977-559566
a
b
Centre Tecnol` ogic de Nutrici´o i Salut (CTNS), TECNIO, CEICS, Reus 43204, Spain
† Electronic supplementary 10.1039/c4fo00447g
information
(ESI)
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available.
See
DOI:
oral glucose load.4,5 Similarly, an Ilex paraguariensis leaf extract, which is rich in the phenolic compound 3,5-O-dicaffeoyl-D-quinic acid, was shown to improve glycemia and insulinemia in mice fed a high-fat diet (HFD), a result that could be related to higher active plasma GLP-1 levels.6,7 Berberine, a major active constituent of Rhizoma coptidis, was also reported to improve glycemia, insulinemia and GLP-1 levels aer oral glucose challenge.8 Resveratrol, a polyphenolic compound presented in such plants as red grapes, exhibits anti-hyperglycemic effect, which has been attributed to increase in GLP-1 levels.9 Berries, a rich source of polyphenols, have also been shown to enhance GLP-1 levels and improve insulin concentrations aer sucrose consumption.10 Altogether, it appears that some phenolic compounds have the ability to enhance glucose-stimulated GLP-1, though the exact mechanisms by which these natural compounds exert such effects have not been elucidated yet. The GLP-1 half-life is less than two minutes due to its rapid cleavage by the enzyme dipeptidyl-peptidase 4 (DPP4).11 In fact, synthetic DPP4 inhibitors, such as vildagliptin or sitagliptin, are being currently used for the treatment of type 2 diabetes mellitus because of their capacity to improve glycemic control by avoiding rapid incretin cleavage.12,13 We have previously reported that a grape seed extract enriched in procyanidins (GSPE) inhibited the intestinal DPP4 activity and increased the plasma
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insulin/glucose ratio in rats in response to orally administered glucose, suggesting an incretinomimetic effect,14 which could help explain the antihyperglycemic effect of GSPE.15–17 Therefore, the aim of the present study was to investigate if a grape seed extract enriched in procyanidins modulates glucosestimulated GLP-1 levels, and to elucidate the mechanisms by which this effect is produced.
Results GSPE enhances glucose-induced active GLP-1 levels To investigate if GSPE modulates incretin levels in vivo, active GLP-1 and total GIP levels in plasma were analyzed aer an oral glucose load (2 g of glucose per kg of bw). As shown in Fig. 1a, the active GLP-1 levels are higher in rats treated with GSPE for 1 hour than in the control group. In contrast, Fig. 1b shows decreased total GIP levels aer GSPE treatment. The effect of these changes on glycaemia and insulinemia were also analyzed, and we have found that they were improved by the GSPE treatment. There was a reduction in plasma glucose levels in the rats treated with GSPE compared to controls (Fig. 1c), concomitantly with a higher insulin/glucose ratio in
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the GSPE-treated rats compared to the control group, as shown in Fig. 1d. The GSPE effects are similar to those found in the positive control group treated with Vildagliptin, a DPP4 inhibitor (Fig. 1). The increase in active GLP-1 levels was dependent on the presence of glucose, since it was only found in animals under OGTT treatment, but not in fasted GSPE-treated animals (Control: 6.1 0.5 pM; GSPE: 5.8 0.3 pM). In fact, GSPE did not modify glycemia (Dglucose: 2.26 0.34 and 1.57 0.29, for control and GSPE, respectively) or insulinemia (insulin/ glucose ratio: 0.23 0.04 and 0.22 0.05, for control and GSPE, respectively) in these animals.
GSPE increases GLP-1 levels by inhibiting the intestinal DPP4 activity To analyze whether an acute dose of GSPE exerts the hypoglycemic effect through its previously described effects as a DPP4 inhibitor, we have assayed the DPP4 activity in plasma and the intestine. The plasma DPP4 activity was not modied aer 1 hour of GSPE treatment (Fig. 2a), whereas GSPE decreased the
Fig. 1 GSPE effect on plasma active GLP-1, total GIP, glucose and insulin levels. An oral load of 2 g glucose per kg of bw was administered in rats treated with 1 g GSPE per kg of bw or 1 mg vildagliptin per kg of bw for 40 minutes (E). Effect of the oral glucose load on the plasma active GLP-1 levels (A), total GIP levels (B), glucose levels (C), and insulin concentration per plasma glucose (D) after 20 minutes. The data are displayed as the mean SEM. a and b, statistically significant differences with P < 0.05.
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also found. Other forms present in GSPE (shown in Table 1 of ESI†), such as epicatechin gallate and procyanidin trimer, and other products of intestinal absorption, such as glucuronide forms, were also detected in the basolateral medium but their levels were too low to be quantied. We have used the basolateral media to treat HUVEC cells for 1 hour and, as shown in Fig. 3, the endothelial DPP4 activity was inhibited by approximately 32% (Fig. 3a). No signicant changes were observed in DPP4 gene expression (results not shown). To assess whether DPP4 is directly inhibited by the pure absorbed molecules found in the basolateral medium, the in vitro DPP4 activity was assayed in the presence of these molecules. DPP4 isolated from HUVEC cells was incubated for 1 hour with 100 mg L1 of each pure molecule, at a concentration similar to that found in the basolateral medium. Catechin, procyanidin B2, and gallic acid signicantly inhibited the DPP4 activity, showing between 4 and 8% inhibition (Fig. 3b). The sum of the inhibitions obtained by each of the four compounds was 21.19% 5.74. This sum was not signicantly different from the inhibition achieved by a mixture of all the compounds (26%, Fig. 3b), and it was also not different from the inhibition produced by the basolateral medium on the indirect co-cultures (P > 0.05). GSPE increases GLP-1 levels by increasing its release
Fig. 2 Effect of GSPE on DPP4. Effects on the plasma DPP4 activity (A) and intestinal DPP4 activity (B), and DPP4 gene expression (C) in rats treated with 1 g of GSPE per kg of bw or 1 mg vildagliptin per kg of bw for 1 hour concomitant with an oral glucose load. The data are displayed as the mean SEM. *Significant differences vs. control group with P < 0.05.
intestinal DPP4 activity by approximately 28%, as shown in Fig. 2b. Vildagliptin also reduced the intestinal DPP4 activity by approximately 37%, which is a newly described effect; and inhibited the plasma DPP4 activity, causing up to 29% inhibition, as expected. Both the GSPE and Vildagliptin treatments have signicantly up-regulated the intestinal DPP4 gene expression (Fig. 2c). Because in vivo measurements are obtained using the whole intestine, we have used an in vitro approach to show that GSPE inhibits DPP4 located in the capillaries, as GLP-1 is secreted into intestinal capillaries. First, HUVEC endothelial cells were directly treated with 25 mg of GSPE per L, a non-toxic dose, and an inhibition on the DPP4 activity of 39% 4 (P ¼ 0.026) was observed. Nevertheless, GSPE is absorbed and metabolized by the intestine before reaching the endothelium of the capillaries; in order to mimic it, CaCo-2 cells were grown in Millicell hanging cell culture inserts and treated, on their apical side, with 250 mg of GSPE per L for 1 hour. By chromatographic analysis (HPLC-MS/MS) we have identied the avanol components of the media. As shown in Table 1, the basolateral medium contained catechin, epicatechin, gallic acid, and procyanidin dimer. Sulfate forms of catechin and epicatechin were
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To analyze whether an acute dose of GSPE also acts by increasing GLP-1 release, total GLP-1 levels in plasma were measured aer 1 h of GSPE treatment. As shown in Fig. 4, total GLP-1 levels are higher aer the GSPE treatment both in fasted (Fig. 4a) and glucose-induced groups (Fig. 4b). As expected, the commercial DPP4 inhibitor Vildagliptin did not change total GLP-1 levels (Fig. 4b).
Discussion Although the antihyperglycemic effect of GSPE has been previously reported18 and its incretinomimetic effect has been suggested,14 its actual effect on incretin levels have not been described yet. The current study shows that an extract from grape seeds enriched in procyanidins increases the glucoseinduced active GLP-1 levels in plasma. This effect could be explained by two mechanisms: the inhibition of endothelial DPP4 activity and the increase in GLP-1 release. Previously, a few studies reported the effect of polyphenols on GLP-1 levels.8–10 Resveratrol, a polyphenolic compound present in red grapes, was shown to increase plasma GLP-1 levels in HFD-induced diabetic mice, which was linked to an amelioration of the glucose prole and an increase in the insulin level in response to OGTT.9 However, the exact mechanisms responsible for such effects were not further elucidated. In our case, we have also found an amelioration of the insulin/ glucose plasma prole as a possible consequence of increased active GLP-1. Although our current experiment is an acute treatment, GSPE has been shown to decrease the HOMA-IR index in cafeteria-fed rats;15,19 thus, the chronic effects on incretin levels deserve further study.
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Table 1
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Flavonols and their metabolites in the basolateral media collected from CaCo-2 inserts, after treating apically with 250 mg of GSPE per La
Compound
Total amount (mM)
Total amount (mg L1)
Catechin Epicatechin Procyanidin dimer(3) Gallic acid
0.21 0.06 0.28 0.06 0.12 0.03 0.29 0.02
60.09 16.54 79.86 18.39 67.05 15.26 49.68 2.70
Metabolite Catechin-sulfate(1) Epicatechin-sulfate(2) Methyl-epicatechin-o-sulfate(2)
0.12 0.01 0.16 0.01 0.21 0.02
44.38 3.77 58.87 4.63 78.48 8.11
a Compounds not quantied: epicatechin gallate(4), dimer gallate(3), trimer(3), EGCG, procyanidin dimer gallate(3); metabolites not quantied: epicatechin-glucuronide(2), methyl-catechin-o-sulfate(1), methyl-catechin-glucuronide(1), methyl-epicatechincatechin-glucuronide(1), (2) (2) (2) glucuronide , 3-o-methyl-epicatechin , 4-o-methyl-epicatechin ; metabolite not detected: epicatechin gallate glucuronide(2).
Effect of absorbed molecules and pure compounds in GSPE on endothelial DPP4 activity. CaCo-2 cells were apically treated with 250 mg of GSPE per L for 1 hour, the basolateral medium was collected and used to incubate HUVEC cells for 1 hour (n ¼ 4) (A). DPP4 extracted from HUVEC cells incubated with 100 mg L1 catechin (Cat), epicatechin (EC), procyanidin B2 (B2) or gallic acid (GA), and a mixture of 100 mg L1 each (Mix) for 1 hour (n ¼ 4) (B). The data are displayed as the mean SEM. *Significant differences with P < 0.05. Fig. 3
Regarding the mechanism leading to the GLP-1 increase, we have previously shown that GSPE inhibited DPP4.14 Indeed, inhibition of the DPP4 enzyme is an emerging strategy to improve glycemia, and several DPP4 inhibitors are currently being used in the pharmacological treatment of type 2 diabetes.20–22 Previous14 and current experiments report no in vivo effects of GSPE on the plasma DPP4 activity. However, we have found inhibition of the intestinal DPP4 activity due to acute (1 hour) GSPE treatment concomitant with an oral glucose load. These results are in agreement with our previous results that also show reduced intestinal DPP4 activity due to acute and chronic treatments in healthy and cafeteria-fed rats.14
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GSPE on total GLP-1 in plasma. Plasma total GLP-1 levels in fasted rats that received a dose of 1 g of GSPE per kg of bw for 1 h (A), and in rats that received a dose of 1 g of GSPE per kg of bw or 1 mg of vildagliptin per kg of bw and, after 40 minutes of treatment, were administered with an acute load of 2 g of glucose per kg of bw for 20 minutes (B). The data are displayed as the mean SEM. *, a and b, statistically significant differences with P # 0.05. Fig. 4
Commercial DPP4 inhibitors, such as the positive control vildagliptin,23 have been commonly reported to exert their incretinomimetic effect via inhibition of the plasma DPP4 activity, thereby delaying the GLP-1 cleavage in plasma (reviewed in ref. 22). However, it was also shown in healthy humans that the increase in plasma GLP-1 levels is not proportional to plasma DPP4 inhibition, suggesting that routes other than plasma DPP4 inhibition are involved.24 Moreover, Hansen et al.25 have reported that half of the newly secreted GLP-1 is N-terminally degraded prior to reaching systemic circulation, suggesting cleavage soon aer its secretion by the DPP4 present in the endothelium of the capillaries adjacent to the GLP-1-secreting cells. A recent study has reported that low
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active doses of sitagliptin, another DPP4 inhibitor, cannot exert the same effect on glucose homeostasis when administered intravenously versus when orally administered, which was attributed to the selective inhibition of intestinal DPP4 activity caused by the oral administration of sitagliptin.26 In addition, the present study shows that vildagliptin can also inhibit the intestinal DPP4 activity, an effect that has not been reported to date. All these ndings support the relevance of the DPP4 enzyme located in tissues close to site of GLP-1 production. Regarding the effects on the other incretin hormone GIP, which is also a target for the DPP4 enzyme, GSPE was found to decrease its total levels aer the oral glucose load, a similar effect to that found for vildagliptin. These results are in concordance with previously reported effects of the commercial DPP4 inhibitor sitagliptin, which was also found to decrease plasma total GIP levels with the concomitant increase in active GLP-1.27 Thus, we hypothesize that the inhibition of inner intestinal DPP4 by the absorbed/metabolized forms of GSPE could be, at least in part, responsible for the increased active GLP-1 plasma levels. In our experiments, DPP4 activity was determined using lysates of the whole intestine; thus, the GSPE-reduced DPP4 activity could also be due to direct inhibition of the enzyme present on the brush border membrane of the absorptive cells. Therefore, we have used an in vitro approach to test our hypothesis: we have analyzed whether the molecules contained in GSPE and/or their metabolites absorbed by intestinal cells (Caco-2 cells cultured on hanging inserts to simulate the intestinal barrier) are able to decrease the activity of DPP4 present in the endothelium of the capillaries (HUVEC cells). To this end, we have used indirect co-cultures due to the low viability of HUVEC cells cultured directly under the Caco-2 monolayer (results not shown). Our results show that the collected basolateral medium of the Caco-2 cells treated with GSPE signicantly decreased the activity of endothelial DPP4, supporting the hypothesis that GSPE might act on the DPP4 present in the endothelium of the capillaries, potentially decreasing GLP-1 cleavage and leading to an increase in active GLP-1 levels. Our in vitro experiments also have led us to identify the molecules responsible for the DPP4 inhibitory effect, which is of importance since previous studies showed that the effect on enzyme modulation depends on the type of the phenolic compound: quercetin and a avonoid-rich extract from Pilea microphylla were also previously shown to inhibit DPP4,28 but other avonoids, such as curcumin29 and avone-8acetic acid30 did not. Thus, catechin, epicatechin, procyanidin dimer and gallic acid, the pure molecules absorbed by the Caco2 monolayer, were tested in vitro, and it was found that the additive effect of these molecules equaled the effect of the basolateral medium. Therefore, we describe for the rst time the inhibition of DPP4 by pure compounds: catechin, gallic acid, and B2 dimer, molecules that can be absorbed in the intestine.31 The decrease in DPP4 activity induced by GSPE could be due to the direct effect of these molecules, since we did not nd reduced DPP4 gene expression either in vivo or in vitro, despite our nding that GSPE modulates DPP4 gene expression.14
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In addition to inhibition of the GLP-1-inactivating enzyme, the increase in active GLP-1 plasma levels could be also due to higher GLP-1 secretion, as suggested for other avonoids.9,10,32 Thus, we have measured total GLP-1 levels, and found that GSPE increased GLP-1 levels in glucose-loaded animals, pointing out the increased secretion. Besides, GSPE induced higher active GLP-1 levels than Vildagliptin, despite Vildagliptin has a broader DPP4 inhibitory capacity, reinforcing that GSPE might also increase active GLP-1 levels by inducing higher GLP-1 secretion. In addition, we have found that such increased secretion of GLP-1 is dependent on the presence of glucose, since in the fasted condition we did not nd an increase in active GLP-1 forms. But, surprisingly, we did also nd higher total GLP-1 levels in fasted animals. Active GLP-1 was measured by using the GLP-1 (active) ELISA kit which is specic for GLP1(7-36), mainly secreted from the intestine aer glucose stimulation.33 In contrast, under fasting conditions, pancreas is the main production site for GLP-1-like molecules, mainly the GLP1(1-36)amide form which is not sensitive to DPP4 and whose activity is still unknown.33 Therefore we hypothesize that, under fasting conditions, GSPE activates the secretion of GLP-1-like molecules from pancreas, which has been clearly identied as an important target for procyanidins;34,35 while, under glucose stimulation, GSPE enhances GLP-1-like intestinal secretions induced by glucose.
Experimental Reagents The grape seed extract enriched in procyanidins (GSPE) was obtained from Les D´ eriv´ es R´ esiniques et Terp´ eniques (Dax, France). According to the manufacturer, the extract contains essentially monomeric (21.3%), dimeric (17.4%), trimeric (16.3%), tetrameric (13.3%) and oligomeric (5–13 U; 31.7%) procyanidins. The small molecules (up to trimers) present in GSPE were characterized, in more detail, by reverse-phase chromatographic analyses by our research group.36 Cell culture reagents were obtained from BioWhittaker (Veviers, Belgium) and EmbryoMax. 0.1% Gelatin Solution was purchased from Millipore (Madrid, Spain). For the HPLC-MS/MS analysis, pure molecules ((+)-catechin, ()-epicatechin, gallic acid, epigallocatechin gallate and procyanidin B2) and the internal standard (IS) pyrocatechol were purchased from Fluka/Sigma-Aldrich (Madrid, Spain). Acetone (HPLC grade), methanol (HPLC grade) and phosphoric acid were purchased from Sigma-Aldrich (Madrid, Spain). Ultrapure water was obtained using a Milli-Q Advantage A10 system from Millipore (Madrid, Spain), and glacial acetic acid was purchased from Panreac (Barcelona, Spain). Animal experimental procedures Female Wistar rats weighing between 200 and 225 g were purchased from Charles River Laboratories (Barcelona, Spain), housed in animal quarters at 22 C with a 12 h light/12 h dark cycle and maintained for 1 week in quarantine. The animals had ad l´ıbitum food access and the food was withdrawn at 8 p.m. Food Funct., 2014, 5, 2357–2364 | 2361
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on the day before sacrice. In both experiments, the animals were anesthetized with 50 mg of pentobarbital per kg of body weight (bw) and sacriced by bleeding through the aorta artery. The blood was collected, and animal tissues were immediately frozen in liquid nitrogen and stored at 80 C until further analysis. All the procedures were approved by the Experimental Animal Ethics Committee of the Universitat Rovira i Virgili according to Law 214/1997 of the Catalonian Department of Environment and Housing (published in BOE (DOGC number 2450)). Experiment 1. At 8 a.m. on the day of the experiment, the rats were divided into three groups (6 animals per group): (i) control group, rats treated with the vehicle (tap water), (ii) GSPE group, rats treated with 1 g of GSPE per kg of body weight (bw), and (iii) positive control group, rats treated with 1 mg of Vildagliptin per kg of bw (Axon Medchem, Groningen, The Netherlands). The treatment was administered acutely by oral gavage. Tail blood samples were collected before treatment administration. Aer 40 minutes of GSPE/Vildagliptin gavage, an oral glucose load (2 g of glucose per kg of bw) was administered; 20 minutes later, the animals were sacriced. A schematic diagram of the animal experiment is shown in Fig. 1. A portion of the collected blood was treated with commercial DPP4 inhibitor (Millipore, Madrid, Spain) and serine protease inhibitor, Pefabloc SC, (Roche, Barcelona, Spain). Experiment 2. At 8 a.m. on the day of the experiment, the rats were divided into two groups (6 animals per group): (i) control group, rats treated with the vehicle (tap water), and (ii) GSPE group, rats treated with 1 g of GSPE per kg of bw. The treatment was administered acutely by oral gavage. Tail blood samples were collected before treatment administration, and, aer 1 h of GSPE gavage, the animals were sacriced.
Plasma measurements Plasma glucose concentrations were assayed using an enzymatic colorimetric kit (GOD-PAP method from QCA, Tarragona, Spain). The plasma insulin and active GLP-1 concentrations were determined using Rat Insulin ELISA/Ultrasensitive Rat Insulin ELISA (Mercodia, Uppsala, Sweden) and GLP-1 (active) ELISA (EGLP-35K) (Millipore, Madrid, Spain) kits, respectively, following the manufacturer's instructions. Total GIP and GLP-1 plasma levels were measured using Rat/Mouse GIP (total) (EZRMGIP-55K) and GLP-1 total ELISA (EZGLP1T-36K) (Millipore, Madrid, Spain) kits, respectively, following the manufacturer's instructions.
Cell lines and indirect co-culture Caco-2 cells were obtained from the American Type Culture Collection (ATCC) (LGC Standards S.L.U., Barcelona, Spain) and cultured in DMEM supplemented as previously described.37 HUVEC cells were obtained from Cascade Biologics™ (Carlsbad, CA, USA), cultured in 0.1% gelatin-coated asks and maintained in Endothelial Growth Medium-2 (EGM-2).
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Caco-2 cells were seeded onto Millicell hanging cell culture inserts (Millipore) for 6-well plates at a cell density of 1.3 104 cells per cm2. The volume of the culture medium was 1 mL on the apical side and 2 mL on the basolateral side. The transepithelial electrical resistance (TEER) of Caco-2 cells was monitored daily by using the Millicell-ERS System (Millipore); when a conuent monolayer was formed (21 days of culture), the cells were treated with 250 mg GSPE per L on their apical side. The basolateral media was collected aer 1 hour of GSPE treatment and stored at 20 C. HUVEC cells were seeded in 6-well plates coated with 0.1% gelatin at a cell density of 8–10.5 104 cells per cm2. When cells reached 80–90% of conuence, they were treated with the collected basolateral media for 1 hour or with 25 mg of GSPE per L for 2 hours. Measurement of DPP4 activity DPP4 was extracted from rat intestine and HUVEC cells as previously described (ref. 14 and 38, respectively). Briey, the intestines were homogenized with lysis buffer (PBS containing 100 KIU mL1 aprotinin and 1% Triton X-100), centrifuged at 1000 g at 4 C for 10 minutes to eliminate the cellular debris, and centrifuged twice at 20 000 g at 4 C for 10 minutes. The HUVEC cells were washed twice with 1 PBS, harvested in 100 mM Tris–HCl, and centrifuged. The supernatant was collected; the pellet was dispersed in the same buffer supplemented with 2% Triton X-100 and, aer centrifugation, the supernatant was added to the rst supernatant and stored at 80 C until further analysis. The DPP4 activity in the cell and intestinal lysates and rat plasma, was measured by following the manufacturer's instructions provided for the DPP4 Drug Discovery Kit-AK499 (Enzo Life Sciences International, Inc.), with a few modications based on the volume of the DPP4-containing sample, and adjusted to the nal volume with Tris–HCl buffer. The protein concentration in all samples was determined as previously reported39 and used to normalize the DPP4 activity values. DPP4 gene expression Total RNA from HUVEC cells was extracted using an RNAeasy Kit (Qiagen, Hilden, Germany) and the total RNA from the intestine was extracted using TRIzol reagent following the manufacturer's instructions. The cDNA was generated using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain). Quantitative PCR amplication and detection for DPP4 was performed using the following TaqMan assay-on-demand probes (Applied Biosystems, Madrid, Spain): Rn00562910_m1 and Hs00175210_m1 for DPP4 for rat and HUVEC cells, respectively. The results were normalized to bactin Rn00667869_m1 for rat and cyclophilin Hs99999904_m1 (Ppia) for HUVEC cells. The relative mRNA expression levels were calculated using the DDCt method. Chromatographic analysis of procyanidins and their metabolites Standards preparation. A 200 mg L1 stock standard mixture in methanol of (+)-catechin, ()-epicatechin, gallic acid and
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epigallocatechin gallate and 100 mg L1 of procyanidin B2 were prepared weekly and stored in a dark ask at 20 C. This stock standard solution was diluted daily to the desired concentration with an acetone : water : acetic acid (70 : 29.5 : 0.5, v/v/v) solution and stored under the same conditions until the chromatographic analysis. Micro-solid phase extraction. Prior to the chromatographic analysis, cell medium samples were pre-treated by using off-line m-SPE using OASIS® HLB m-Elution Plates 30 mm (Waters, Barcelona, Spain) following the methodology for biological samples as previously described.40 Instrumental conditions. The eluted solution was directly analyzed using a 1200 LC Series coupled to a 6410 QqQ-MS/MS (Agilent Technologies, Palo Alto, USA). The chromatographic separation of both apical and basolateral media was performed on a Zorbax C18 column (150 mm 2.1 mm i.d., 3.5 mm particle size, Agilent Technologies). The procyanidins and their metabolites were analyzed by ESI as the ionization technique and in negative mode, as previously described.41 Direct inhibition of DPP4 activity by pure compounds in vitro DPP4 was isolated from untreated HUVEC cells as described above and incubated with 100 mg L1 of catechin, epicatechin, procyanidin B2 or gallic acid, either alone or together, for 1 hour. Then, the DPP4 activity was determined as described above. Data analyses The results are expressed as mean SEM. The effects were assessed by ANOVA or Student's t-test. All calculations were performed using SPSS soware version 17.0 (SPSS, Chicago, USA).
Conclusions This study indicates that acute treatment with GSPE, concomitant to a glucose load, favours lower glycemia caused by the higher active GLP-1 levels observed in the GSPE-treated group. The higher active GLP-1 levels might be due to increased enteric glucose-induced secretion plus the additive effect of the absorbed avanol forms inhibiting the intestinal DPP4.
Conflict of interest The authors declare that they have no conict of interest.
Acknowledgements We would like to acknowledge the technical support of Niurka Llopiz and Naroa Mendizuri from the Universitat Rovira i Virgili. Thanks are conveyed to members of the Nutrigenomics group collaborated for caring and sampling of the animals. This study was supported by a grant (AGL2011-23879) from the Spanish government. Noem´ı Gonz´ alez-Abu´ın is the recipient of an FPI fellowship from the Spanish Ministry of Science and Innovation (MICINN), and Neus Mart´ınez-Micaelo and Maria This journal is © The Royal Society of Chemistry 2014
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Margalef are recipients of fellowships from the Universitat Rovira i Virgili. N. G.-A. has analyzed data and wrote the manuscript; N. M.-M. and M. M. contributed to research data; MT. B., A. A.-A. and B. M. contributed to discussion; A. A. and M. P. contributed to the experimental design and discussion, and reviewed the manuscript.
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