Delphinidin reduces glucose uptake in mice jejunal tissue and human intestinal cells lines through FFA1/GPR40

International Journal of Molecular Sciences Article Delphinidin Reduces Glucose Uptake in Mice Jejunal Tissue and Human Intestinal Cells Lines through FFA1/GPR40 Jorge Hidalgo 1, Stefanie Teuber 1, Francisco J. Morera 1, Camila Ojeda 1, Carlos A. Flores 2, María A. Hidalgo 1, Lucía Núñez 3,4, Carlos Villalobos 3,4 and Rafael A. Burgos 1,* 1Institute of Pharmacology and Morphophysiology, Universidad Austral de Chile, 5110566 Valdivia, Chile; [email protected] (J.H.); [email protected] (S.T.); fjmor[email protected] (F.J.M.); [email protected] (C.O.); [email protected] (M.A.H.) 2Centro de Estudios Científicos (CECs), Avenida Arturo Prat 514, 511046 Valdivia, Chile; [email protected] 3Instituto de Biología y Genética Molecular (IBGM), Consejo Superior de Investigaciones Científicas (CSIC), 47003 Valladolid, Spain; [email protected] (L.N.); [email protected] (C.V.) 4Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid, 47003 Valladolid, Spain *Correspondence: [email protected]; Tel.: +56-63-229-3015 Academic Editors: Nuno Mateus and Iva Fernandes Received: 21 February 2017; Accepted: 27 March 2017; Published: 5 April 2017 Abstract: Anthocyanins are pigments with antihyperglycemic properties, and they are potential candidates for developing functional foods for the therapy or prevention of Diabetes mellitus type 2 (DM2). The mechanism of these beneficial effects of anthocyanins are, however, hard to explain, given their very low bioavailability due to poor intestinal absorption. We propose that free fatty acid receptor 1 (FFA1, also named GPR40), is involved in an inhibitory effect of the anthocyanidin delphinidin over intestinal glucose absorption. We show the direct effects of delphinidin on the intestine using jejunum samples from RF/J mice, and the human intestinal cell lines HT-29, Caco-2, and NCM460. By the use of specific pharmacological antagonists, we determined that delphinidin inhibits glucose absorption in both mouse jejunum and a human enterocytic cell line in a FFA1-dependent manner. Delphinidin also affects the function of sodium-glucose cotransporter 1 (SGLT1). Intracellular signaling after FFA1 activation involved cAMP increase and cytosolic Ca 2+ oscillations originated from intracellular Ca 2+ stores and were followed by store-operated Ca 2+ entry. Taken together, our results suggest a new GPR-40 mediated local mechanism of action for delphinidin over intestinal cells that may in part explain its antidiabetic effect. These findings are promising for the search for new prevention and pharmacological treatment strategies for DM2 management. Keywords: delphinidin; anthocyanins; SGLT1; glucose; GPR40; FFA1 1. Introduction Intestinal glucose uptake control is a pharmacological target used for the control of hyperglycaemia [ 1 ]. Metformin, an effective drug used as a first-line treatment in Diabetes mellitus type 2 (DM2), is accumulated in the mucosa of the intestine increasing glucose turnover and contributing to its antihyperglycaemic effect [ 2 ]. In isolated rat jejunal loops, metformin inhibited the glucose-induced short-circuit current, thus reducing the activity of sodium-glucose transporter 1 (SGLT1), and simultaneously increasing the recruitment of glucose transporter 2 (GLUT2) to the apical membrane of the rat jejunum [ 3 ]. Since metformin increases intestinal glucose uptake and also lactate production [ 4 , 5 ], this phenomenon can cause intolerance to the treatment [ 5 ] and contribute to the development of lactic acidosis [1]. Int. J. Mol. Sci. 2017,18, 750; doi:10.3390/ijms18040750 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017,18, 750 2 of 18 Diet is considered a useful non-pharmacological strategy for the control of glycaemia [ 1 ]. Recently, considerable attention has been focused on dietary constituents that may be beneficial for the prevention and treatment of diabetes. Diet consumption of polyphenols such as anthocyanins has been associated with a lower risk of DM2 [6–8]. Anthocyanins, which belong to the flavonoids group, are red or purple plant pigments, present in the form of glycosides in berries. Beyond their anti-oxidant properties, it has been documented that anthocyanins have anti-diabetic properties [9]. Dietary bilberry extract reduces the blood glucose level and enhances insulin sensitivity in type 2 diabetic mice, modulating GLUT4 in white adipose tissue and skeletal muscle, glucose output, and lipid metabolism via AMP-activated protein kinase activation [9]. Anthocyanins such as cyanidin-3-glucoside and delphinidin-3-glucoside showed a high-level capability to stimulate insulin secretion from rodent pancreatic β cells in the presence of 4 and 10 mmol/L glucose concentrations [ 10 ]. A formulation of anthocyanins or delphinidin 3-sambubioside-5-glucoside decreased fasting blood glucose levels in obese C57BL/6J mice, while in vitro it decreased glucose production in rat liver cells and increased glucose uptake in L6 myotubes [ 11 ]. Anthocyanins can be directly absorbed from the gastrointestinal tract, however they show first-pass metabolism (toward anthocyanidins, their aglycone form) by the gut microflora and hence have poor systemic availability [12,13] . In fact, anthocyanins and the sugar-free form, anthocyanidins, are actively transported out of intestinal tissues and endothelia, limiting their bioavailability in plasma [ 14 ]. These data suggest that another unknown mechanism might be involved in the control of glycaemia by these pigments. Recently, it has been described that delphinidin can directly induce the release of glucagon-like peptide-1 from enteroendocrine L-cells, and therefore, it could stimulate glucose-dependent insulin secretion in pancreatic β -cells via free fatty acid receptor 1 (FFA1) [ 15 ]. In spite of this, patients with DM2 treated with a standardised bilberry extract (36% (w/w) anthocyanins) had reduced postprandial glycaemia, but showed no significant increase of the GLP-1 level in plasma [ 16 ]. Delphidin increases cytosolic free calcium, by releasing Ca 2+ from intracellular stores and by increasing Ca 2+ entry in endothelial [ 17 ] and T cells [ 18 ]. It has been widely demonstrated that the intracellular calcium concentration is involved in glucose transport [19]. However, by using L-type Ca2+ channel blockers, an increase in the transport of hexose in rabbit jejunal tissue has been demonstrated, suggesting a more complex role of calcium [ 20 , 21 ]. FFA1 agonist can activate calcium flux via the phospholipase C and L-type Ca 2+ channel [ 22 ], however, the direct effect of these drugs on glucose transport has not yet been assessed. In this paper, we show evidence that delphinidin can reduce glucose uptake in the intestine via stimulation of FFA1, by mechanisms other than incretin release. 2. Results 2.1. Delphinidin Inhibits Glucose Absorption in Mouse Jejunal In order to initially evaluate a possible inhibitory effect of delphinidine on intestinal glucose transport, we decided to study its effect on the electrogenic sodium-coupled glucose transport. Experiments with mouse intestinal preparations in the Ussing chamber (Figure 1A,B) showed that treatment of the jejunal mucosa with 100 µ M delphinidin is able to reduce the short-circuit current induced by the addition of 10 mM glucose to the mucosal side. The differences were statistically significant. Since the electrophysiological study carried out only allows for the evaluation of the function of the SGLT1 co-transporter, we decided to complement it by performing uptake assays with a radiolabeled glucose analogue in everted sacs of mouse jejunum sections. The results showed that treatment with 100 µ M delphinidin significantly inhibited the incorporation of 3-O-methyl-glucose [ 3 H] (3-OMG) in the mouse intestine (Figure 1C), an effect that is similar to that of phlorizin 1 mM, a pharmacological inhibitor of the electrogenic transport of glucose, which was used as a positive control. Int. J. Mol. Sci. 2017,18, 750 3 of 18 Int. J. Mol. Sci. 2017, 18, 750 3 of 17 pharmacological inhibitor of the electrogenic transport of glucose, which was used as a positive control. Figure 1. Delphinidin inhibits intestinal glucose absorption. (A) Sample tracing showing changes in short-circuit current during an experiment using 10 mM glucose and 100 μm delphinidin; (B) Summary of Ussing experiments in Rockefeller mice (RF/J); (C) Effect of DMSO 0.2% (control), delphinidin 100 μM, or phlorizin on 3-O-methyl-glucose-3H absorption. A glucose uptake is shown as counts per minute per gram of tissue (CPM/g). Bars represent mean ± SEM of at least six different animals. * p < 0.05 compared to glucose; ** p < 0.01 *** p < 0.001 compared to the control. In order to determine whether this inhibition of glucose uptake was mediated by the activation of FFA1, we repeated the experiment but added a pre-incubation step of 15 min with pharmacological antagonists of this receptor or vehicle (Figure 2A,B). In these trials, 100 μM delphinidin and the synthetic FFA1 agonist, TAK 875, inhibited the incorporation of glucose significantly (Figure 2A). Tissues pretreated with any of the FFA1 receptor antagonists, DC260126 (Figure 2A), or GW1100 (Figure 2B), interfered with the inhibition of the intestinal glucose uptake produced by delphinidin. In order to study the molecular mechanisms of signaling related to the inhibitory effect of delphinidin Glucose 0 2 4 6 -14 -12 -10 -8 Time (min) Isc (μA cm-2) Delphinidin A Glucose 10mM M μ Gluc. + Delphinidin 100 -5 -4 -3 -2 -1 0 ΔIsc (μA cm-2) B Control M μ Delphinidin 100 Phlorizin 1mM 0 2000 4000 6000 8000 *** ** 3-OMG uptake (CPM/g) C * Figure 1. Delphinidin inhibits intestinal glucose absorption. ( A ) Sample tracing showing changes in short-circuit current during an experiment using 10 mM glucose and 100 µ m delphinidin; ( B ) Summary of Ussing experiments in Rockefeller mice (RF/J); ( C ) Effect of DMSO 0.2% (control), delphinidin 100 µ M, or phlorizin on 3-O-methyl-glucose-3H absorption. A glucose uptake is shown as counts per minute per gram of tissue (CPM/g). Bars represent mean ± SEM of at least six different animals. *p< 0.05 compared to glucose; ** p< 0.01 *** p< 0.001 compared to the control. In order to determine whether this inhibition of glucose uptake was mediated by the activation of FFA1, we repeated the experiment but added a pre-incubation step of 15 min with pharmacological antagonists of this receptor or vehicle (Figure 2A,B). In these trials, 100 µ M delphinidin and the synthetic FFA1 agonist, TAK 875, inhibited the incorporation of glucose significantly (Figure 2A). Tissues pretreated with any of the FFA1 receptor antagonists, DC260126 (Figure 2A), or GW1100 (Figure 2B), interfered with the inhibition of the intestinal glucose uptake produced by delphinidin. In order to study the molecular mechanisms of signaling related to the inhibitory effect of delphinidin on the uptake of glucose in the intestine, we decided to interfere with the activation of the PI3K/Akt kinase pathway through the pharmacological inhibitor of PI3K, LY294002 (Figure 2C), and to Int. J. Mol. Sci. 2017,18, 750 4 of 18 interfere with the mobilization of intracellular calcium by the use of the intracellular calcium chelator BAPTA-AM (Figure 2D). In tissues pretreated with 50 µ M BAPTA-AM, delphinidin showed no significant inhibition on glucose uptake, whereas in tissues pretreated with the vehicle (0.2% DMSO) or 10 µM LY294002, delphinidin still showed a significant inhibitory effect on glucose uptake. Int. J. Mol. Sci. 2017, 18, 750 4 of 17 on the uptake of glucose in the intestine, we decided to interfere with the activation of the PI3K/Akt kinase pathway through the pharmacological inhibitor of PI3K, LY294002 (Figure 2C), and to interfere with the mobilization of intracellular calcium by the use of the intracellular calcium chelator BAPTA-AM (Figure 2D). In tissues pretreated with 50 μM BAPTA-AM, delphinidin showed no significant inhibition on glucose uptake, whereas in tissues pretreated with the vehicle (0.2% DMSO) or 10 μM LY294002, delphinidin still showed a significant inhibitory effect on glucose uptake. Figure 2. Inhibition of intestinal glucose uptake produced by delphinidin depends on FFA1 and intracellular calcium. Uptake of 3-O-methyl-glucose [3H] is expressed in counts per minute per gram of tissue (CPM/g). The effects of FFA1 antagonist DC260126 (A) and GW1100 (B) on delphinidin are depicted. The effect of FFA1 agonist TAK875 on 3-OMG uptake, is shown (A). The effect of LY294002, a PI3K Inhibitor, (C) or BAPTA-AM (D) are shown. Additionally Phlorizin, a SGT1 inhibitor, was used. The bars represent the mean ± standard error of the mean of at least six animals. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control. Delph. = 100 μM delphinidin. B A Control M μ Delphinidin 100 M + Delph. μ GW1100 10 Phlorizin 1mM 0 2000 4000 6000 8000 * ** *** 3-OMG uptake (CPM/g) Control M μ Delphinidin 100 M + Delph. μDC260126 10 M μ TAK875 10 0 500 1000 1500 2000 ** ** * 3-OMG uptake (CPM/g) DC Control Mμ Delphinidin 100 M + Delph. μ LY294002 10 0 2000 4000 6000 ** 3-OMG uptake (CPM/g) Control M μ Delphinidin 100 M + Delph. μ BAPTA 50 0 1000 2000 3000 * 3-OMG uptake (CPM/g) Figure 2. Inhibition of intestinal glucose uptake produced by delphinidin depends on FFA1 and intracellular calcium. Uptake of 3-O-methyl-glucose [ 3 H] is expressed in counts per minute per gram of tissue (CPM/g). The effects of FFA1 antagonist DC260126 ( A ) and GW1100 ( B ) on delphinidin are depicted. The effect of FFA1 agonist TAK875 on 3-OMG uptake, is shown ( A ). The effect of LY294002, a PI3K Inhibitor, ( C ) or BAPTA-AM ( D ) are shown. Additionally Phlorizin, a SGT1 inhibitor, was used. The bars represent the mean ± standard error of the mean of at least six animals. * p< 0.05, ** p< 0.01, *** p< 0.001, compared with the control. Delph. = 100 µM delphinidin. 2.2. Presence of FFA1 in Caco-2 and HT-29 Having observed the inhibitory effect of delphinidin on the intestinal absorption of glucose, and its dependence of calcium and the activation of FFA1, we decided to study in more detail the signaling involved using a cellular model. The presence of the FFA1 receptor was detected in the Int. J. Mol. Sci. 2017,18, 750 5 of 18 HT-29 enterocyte-like human cell line by Western blot (Figure 3A) and by immunofluorescence using a confocal microscope (Figure 3B), and was confirmed by immunofluorescence measured by flow cytometry (Figure 3C) and qRT-PCR (Figure 3D). Int. J. Mol. Sci. 2017, 18, 750 5 of 17 2.2. Presence of FFA1 in Caco-2 and HT-29 Having observed the inhibitory effect of delphinidin on the intestinal absorption of glucose, and its dependence of calcium and the activation of FFA1, we decided to study in more detail the signaling involved using a cellular model. The presence of the FFA1 receptor was detected in the HT29 enterocyte-like human cell line by Western blot (Figure 3A) and by immunofluorescence using a confocal microscope (Figure 3B), and was confirmed by immunofluorescence measured by flow cytometry (Figure 3C) and qRT-PCR (Figure 3D). Figure 3. HT-29 and Caco-2 cells express FFA1. (A) Immunoblot of FFA1 in HT-29 and Caco-2 cells. A single band near 28 kDa was observed in at least three different assays. The β-actin loading control is shown. Immunofluorescence microscopy of FFA1 in HT-29 Cells and Caco-2 Cells. (B) Monoclonal anti-FFA1 antibody was used as the primary antibody, with an Alexa-488-bound fluorescent secondary antibody. Nuclei were stained using Hoechst 33342. Scale bar corresponds to 10 μm; (C) Flow cytometry of FFA1 in HT-29 cells. Monoclonal anti-FFA1 rabbit antibody and secondary Alexa-488 bound antibody were used. Results are shown as counts in the Y axis, and Figure 3. HT-29 and Caco-2 cells express FFA1. ( A ) Immunoblot of FFA1 in HT-29 and Caco-2 cells. A single band near 28 kDa was observed in at least three different assays. The β -actin loading control is shown. Immunofluorescence microscopy of FFA1 in HT-29 Cells and Caco-2 Cells. ( B ) Monoclonal anti-FFA1 antibody was used as the primary antibody, with an Alexa-488-bound fluorescent secondary antibody. Nuclei were stained using Hoechst 33342. Scale bar corresponds to 10 µ m; ( C ) Flow cytometry of FFA1 in HT-29 cells. Monoclonal anti-FFA1 rabbit antibody and secondary Alexa-488 bound antibody were used. Results are shown as counts in the Y axis, and fluorescence intensity in the X axis. (Representative experiment from two were carried out); ( D ) qRT-PCR of FFA1 in HT-29 cells. The amplicon for FFA1 is shown in red. Int. J. Mol. Sci. 2017,18, 750 6 of 18 2.3. Delphinidin Induces Intracellular Calcium Release in HT-29 Cells via FFA1 Using the HT-29 enterocytic line, intracellular calcium measurements in cell populations were carried out with the FURA-2AM fluorescent probe (Figure 4). It was observed that delphinidin was able to induce an increase in intracellular calcium concentrations in a dose-dependent manner (Figure 4B). Upon pre-incubation with the FFA1 antagonist GW1100 (10 µM) for 15 min, the intracellular calcium signal induced by 50 µM delphinidin was diminished (Figure 4C,D) Int. J. Mol. Sci. 2017, 18, 750 6 of 17 fluorescence intensity in the X axis. (Representative experiment from two were carried out); (D) qRTPCR of FFA1 in HT-29 cells. The amplicon for FFA1 is shown in red. 2.3. Delphinidin Induces Intracellular Calcium Release in HT-29 Cells via FFA1 Using the HT-29 enterocytic line, intracellular calcium measurements in cell populations were carried out with the FURA-2AM fluorescent probe (Figure 4). It was observed that delphinidin was able to induce an increase in intracellular calcium concentrations in a dose-dependent manner (Figure 4B). Upon pre-incubation with the FFA1 antagonist GW1100 (10 μM) for 15 min, the intracellular calcium signal induced by 50 μM delphinidin was diminished (Figure 4C,D) Figure 4. Delphinidin increases intracellular Ca2+ concentration in HT-29 Fura-2 AM loaded cells. The changes in cytosolic calcium concentration were evaluated by spectrofluorimetric assays; (A) The representative fluorescence record of cytosolic calcium changes induced by delphinidin is depicted; (B) Mean data from dose-response experiments in HT-29 cells; (C) Intracellular calcium increases induced by delphinidin were inhibited with 10 μM GW1100 (FFA1 antagonist); (D) Comparison of the peak observed calcium response with and without FFA1 antagonist treatment. Bars represent mean ± SEM of 3–5 different experiments. * p < 0.05. 2.4. Delphinidin Induces Intracellular Calcium Oscilations in HT-29 Cells in a Ca2+ Store-Dependent Manner In order to examine the intracellular calcium signals induced by delphinidin in more detail, we decided to perform measurements at the single cell level (Figure 5). At this resolution, it was observed that the intracellular calcium signals produced by delphinidin were actually in the form of oscillations (Figure 5A), that usually started within a few minutes of treatment and which were recorded at concentrations of delphinidin that were even 20 times lower than those effective at inhibiting the Figure 4. Delphinidin increases intracellular Ca 2+ concentration in HT-29 Fura-2 AM loaded cells. The changes in cytosolic calcium concentration were evaluated by spectrofluorimetric assays; ( A ) The representative fluorescence record of cytosolic calcium changes induced by delphinidin is depicted; ( B ) Mean data from dose-response experiments in HT-29 cells; ( C ) Intracellular calcium increases induced by delphinidin were inhibited with 10 µ M GW1100 (FFA1 antagonist); ( D ) Comparison of the peak observed calcium response with and without FFA1 antagonist treatment. Bars represent mean ±SEM of 3–5 different experiments. * p< 0.05. 2.4. Delphinidin Induces Intracellular Calcium Oscilations in HT-29 Cells in a Ca 2+ Store-Dependent Manner In order to examine the intracellular calcium signals induced by delphinidin in more detail, we decided to perform measurements at the single cell level (Figure 5). At this resolution, it was observed that the intracellular calcium signals produced by delphinidin were actually in the form of oscillations (Figure 5A), that usually started within a few minutes of treatment and which were recorded at concentrations of delphinidin that were even 20 times lower than those effective at inhibiting the transport of glucose analogues in the jejunum from mice (Figure 5C). On the other hand, 50 µM Delphinidin-3,5-glucoside did not induce the effect observed with delphinidin. Int. J. Mol. Sci. 2017,18, 750 7 of 18 Int. J. Mol. Sci. 2017, 18, 750 7 of 17 transport of glucose analogues in the jejunum from mice (Figure 5C). On the other hand, 50 μM Delphinidin-3,5-glucoside did not induce the effect observed with delphinidin. Figure 5. Delphinidin induces intracellular calcium oscillations in HT-29 cells. (A) Delphinidininduced intracellular calcium oscillations at the single-cell level. Each colored line represents calcium recordings of individual cells in the same microscopic field. The horizontal bar depicts the duration of delphinidin perfusion; (B) Representative tracing of the mean calcium (± SEM) response of 16 cells treated for 6 min with either Delphinidin-3,5-glucoside 50 μM (red) or Delphinidin 50 μM (black); (C) Area-under-the-curve (AUC) data from dose-response experiments on HT-29 cells. Bars represent mean ± SEM of 16–20 cells. *** p < 0.001. The characteristic oscillations observed after stimulation with delphinidin could also be reproduced in an extracellular calcium-free medium (Figure 6A), resulting in a smaller average magnitude. It was also observed that the subsequent addition of calcium to the extracellular medium caused a rapid increase in its cytosolic concentration (Figure 6A,B). This calcium influx likely corresponds to the store-operated calcium entry (SOCE) commonly triggered by the emptying of intracellular calcium stores. After the observation that calcium oscillations can be generated in the absence of extracellular calcium, we decided to confirm if intracellular stores are involved in intracellular calcium oscillations by using 30 μM cyclopiazonic acid, a specific inhibitor of the sarcoplasmic and endoplasmic reticulum Ca 2+ -ATPase (SERCA) of intracellular deposits, that depletes Ca 2+ stores passively. In this condition, the stimulation with delphinidin was not able to generate the oscillations of intracellular calcium (Figure 6C,D). Figure 5. Delphinidin induces intracellular calcium oscillations in HT-29 cells. ( A ) Delphinidin-induced intracellular calcium oscillations at the single-cell level. Each colored line represents calcium recordings of individual cells in the same microscopic field. The horizontal bar depicts the duration of delphinidin perfusion; ( B ) Representative tracing of the mean calcium ( ± SEM) response of 16 cells treated for 6 min with either Delphinidin-3,5-glucoside 50 µ M ( red ) or Delphinidin 50 µ M ( black ); ( C ) Area-under-the-curve (AUC) data from dose-response experiments on HT-29 cells. Bars represent mean ±SEM of 16–20 cells. *** p< 0.001. The characteristic oscillations observed after stimulation with delphinidin could also be reproduced in an extracellular calcium-free medium (Figure 6A), resulting in a smaller average magnitude. It was also observed that the subsequent addition of calcium to the extracellular medium caused a rapid increase in its cytosolic concentration (Figure 6A,B). This calcium influx likely corresponds to the store-operated calcium entry (SOCE) commonly triggered by the emptying of intracellular calcium stores. After the observation that calcium oscillations can be generated in the absence of extracellular calcium, we decided to confirm if intracellular stores are involved in intracellular calcium oscillations by using 30 µ M cyclopiazonic acid, a specific inhibitor of the sarcoplasmic and endoplasmic reticulum Ca 2+ -ATPase (SERCA) of intracellular deposits, that depletes Ca 2+ stores passively. In this condition, the stimulation with delphinidin was not able to generate the oscillations of intracellular calcium (Figure 6C,D). Int. J. Mol. Sci. 2017,18, 750 8 of 18 Int. J. Mol. Sci. 2017, 18, 750 8 of 17 Figure 6. Delphinidin-induces Ca2+ store-dependent calcium oscillations and triggers store-operated Ca2+ entry in HT-29 Fura-2 AM loaded cells. (A) Cells in Ca2+-free medium were perfused with 5 μM delphinidin. Each colored line represents the Ca2+ concentrations of a single cell. The horizontal bar depicts the duration of perfusion. Before the end of the experiment, extracellular Ca2+ containing medium was perfused; (B) Mean ± SEM recording from all cells in a representative experiment; (C) Cells in HEPES/Ca2+ buffer were pre-treated with 30 μM cyclopiazonic acid (SERCA inhibitor) until the stabilization of Ca2+ readouts and then the cells were stimulated with delphinidin 5 μM for 7 min. Each colored line represents a recording from individual cells. The horizontal bar depicts the duration of perfusion; (D) Mean ± SEM data from all cells in a representative experiment. Data are mean ± SEM of 18 cells. 2.5. Delphinidin Induces Intracellular Calcium Oscilations via FFA1 in Colon Normal Cells Although HT-29 cells are used as a model for human enterocytes, they may behave differently from normal human intestinal mucosa cells [23] as they are derived from adenocarcinoma. With this in mind, we decided to test the same experiment using normal human colon mucosa cells NCM460. Intracellular calcium oscillations, induced by delphinidin but not Delphinidin-3,5-glucoside, also occurred in these cells, this time at minimum concentrations up to 200 times lower than those that had an effect on the uptake of glucose analogues in the mouse jejunum (Figure 7A,B). DelphinidinAB CD 02468101214 0.2 0.4 0.6 0.8 1.0 1.2 Ca2+ 1mM No calcium Ratio F340/F380 Time (min) Delphinidin 5μM 0 2 4 6 8 101214 0.2 0.4 0.6 0.8 1.0 1.2 Ratio F340/F380 Time (min) Delphinidin 5μM Ca2+ 1mM No calcium 0 2 4 6 8 10 12 14 16 18 20 22 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CPA 30μM Ratio F340/F380 Time (min) Delphinidin 5μM 0 2 4 6 8 10 12 14 16 18 20 22 0.2 0.4 0.6 0.8 1.0 1.2 CPA 30μM Ratio F340/F380 Time (min) Delphinidin 5μM Figure 6. Delphinidin-induces Ca 2+ store-dependent calcium oscillations and triggers store-operated Ca 2+ entry in HT-29 Fura-2 AM loaded cells. ( A ) Cells in Ca 2+ -free medium were perfused with 5 µ M delphinidin. Each colored line represents the Ca 2+ concentrations of a single cell. The horizontal bar depicts the duration of perfusion. Before the end of the experiment, extracellular Ca 2+ containing medium was perfused; ( B ) Mean ± SEM recording from all cells in a representative experiment; ( C ) Cells in HEPES/Ca 2+ buffer were pre-treated with 30 µ M cyclopiazonic acid (SERCA inhibitor) until the stabilization of Ca 2+ readouts and then the cells were stimulated with delphinidin 5 µ M for 7 min. Each colored line represents a recording from individual cells. The horizontal bar depicts the duration of perfusion; ( D ) Mean ± SEM data from all cells in a representative experiment. Data are mean ±SEM of 18 cells. 2.5. Delphinidin Induces Intracellular Calcium Oscilations via FFA1 in Colon Normal Cells Although HT-29 cells are used as a model for human enterocytes, they may behave differently from normal human intestinal mucosa cells [ 23 ] as they are derived from adenocarcinoma. With this in mind, we decided to test the same experiment using normal human colon mucosa cells NCM460. Intracellular calcium oscillations, induced by delphinidin but not Delphinidin-3,5-glucoside, also occurred in these cells, this time at minimum concentrations up to 200 times lower than those that had an effect on the uptake of glucose analogues in the mouse jejunum (Figure 7A,B). Delphinidin-induced Int. J. Mol. Sci. 2017,18, 750 9 of 18 calcium oscillations could also be seen in calcium-free extracellular conditions, and adding calcium before the end of measurements once again prompted a fast calcium influx (Figure 7C,D). Int. J. Mol. Sci. 2017, 18, 750 9 of 17 induced calcium oscillations could also be seen in calcium-free extracellular conditions, and adding calcium before the end of measurements once again prompted a fast calcium influx (Figure 7C,D). Figure 7. Delphinidin induces intracellular calcium oscillations and store-operated calcium entry in NCM460 Fura-2 AM loaded cells. (A) Representative recording (mean ± SEM) corresponding to 19 cells treated with either Delphinidin-3,5-glucoside (black triangles) or delphinidin (empty circles). The vertical symbols represent SEM; (B) Area-under-the-curve (AUC) data from dose-response experiments on NCM460 cells; (C) NCM460 cells in calcium-free medium were perfused with delphinidin. Each colored line represents calcium concentrations of a single cell. The horizontal bar depicts the duration of perfusion; (D) Mean data ± SEM from all cells in a representative experiment from at least 20 cells. *** p < 0.001. To determine if intracellular store-dependent calcium oscillations produced by delphinidin were secondary to the activation of FFA1, calcium measurements were performed on cells exposed to 5 μM delphinidin after being pretreated with the FFA1 antagonist, GW1100 (Figure 8). A significantly lower response to delphinidin was noted in NCM460 cells pretreated with 10 μM GW1100 versus the vehicle (Figure 8B). 2.6. Delphinidin Induces Intracellular Calcium Release, cAMP, and Glucose Uptake via FFA1 To assess whether the effects of delphinidin observed on glucose transport in mouse jejunum would also be replicated in a human enterocytic cell line, assays were performed for the uptake of radiolabeled glucose analogs into Caco-2 cells. These cells were selected for glucose uptake assays due to their tendency to spontaneously differentiate into a polarized monolayer of mature enterocytes [23], emulating an epithelial barrier. In cells stimulated with delphinidin at a concentration of 50 μM for 15 min, glucose uptake was significantly reduced compared to the vehicle, Figure 7. Delphinidin induces intracellular calcium oscillations and store-operated calcium entry in NCM460 Fura-2 AM loaded cells. ( A ) Representative recording (mean ± SEM) corresponding to 19 cells treated with either Delphinidin-3,5-glucoside (black triangles) or delphinidin (empty circles). The vertical symbols represent SEM; ( B ) Area-under-the-curve (AUC) data from dose-response experiments on NCM460 cells; ( C ) NCM460 cells in calcium-free medium were perfused with delphinidin. Each colored line represents calcium concentrations of a single cell. The horizontal bar depicts the duration of perfusion; ( D ) Mean data ± SEM from all cells in a representative experiment from at least 20 cells. *** p< 0.001. To determine if intracellular store-dependent calcium oscillations produced by delphinidin were secondary to the activation of FFA1, calcium measurements were performed on cells exposed to 5 µ M delphinidin after being pretreated with the FFA1 antagonist, GW1100 (Figure 8). A significantly lower response to delphinidin was noted in NCM460 cells pretreated with 10 µ M GW1100 versus the vehicle (Figure 8B). 2.6. Delphinidin Induces Intracellular Calcium Release, cAMP, and Glucose Uptake via FFA1 To assess whether the effects of delphinidin observed on glucose transport in mouse jejunum would also be replicated in a human enterocytic cell line, assays were performed for the uptake of radiolabeled glucose analogs into Caco-2 cells. These cells were selected for glucose uptake assays due to their tendency to spontaneously differentiate into a polarized monolayer of mature enterocytes [ 23 ], emulating an epithelial barrier. 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