Anthocyanins from purple corn activate free fatty acid-receptor 1 and glucokinase enhancing in vitro insulin secretion and hepatic glucose uptake.

The objective of this study was to evaluate the ability of anthocyanins (ANC) present in purple corn to enhance insulin secretion and hepatic glucose uptake in pancreatic cells and hepatocytes, through activation of the free fatty acid receptor-1 (FFAR1) and glucokinase (GK), respectively. Using a dual-layer cell culture with Caco-2 cells, INS-1E or HepG2 cells were treated with an anthocyanin-rich extract from the pericarp of purple corn (PCW), as well as pure ANC cyanidin-3-O-glucoside (C3G), peonidin-3-O-glucoside, pelargonidin-3-O-glucoside. Delphinidin-3-O-glucoside (D3G) was used for comparative purposes. Semipurified C3G (C3G-P) and condensed forms (CF-P) isolated from PCW were also used. At 100 μM, the pure ANC enhanced glucose-stimulated insulin secretion (GSIS) in INS-1E cells ranging from 18% to 40% (p<0.05) compared to untreated cells. PCW increased GSIS by 51%. D3G was the most effective anthocyanin activating FFAR1 (EC50: 196.6 μM). PCW had activating potential on FFAR1 (EC50: 77 μg/mL). PCW, as well as C3G and D3G increased the expression of FFAR1, PLC, and phosphorylation of PKD, related to the FFAR1-dependent insulin secretory pathway. The treatment with 100 μM of P3G and C3G increased (p<0.05) glucose uptake in HepG2 cells by 19% and 31%. PCW increased the glucose uptake in HepG2 cells by 48%. It was determined that CF-P was the most effective for activating GK (EC50: 39.9 μM) and the PCW extracts had an efficacy of EC50: 44 μg/mL. The ANC in purple corn also reduced AMPK phosphorylation and PEPCK expression in HepG2 cells, known to be related to reduction in gluconeogenesis. It is demonstrated for the first time that dietary ANC can enhance the activity of novel biomarkers FFAR1 and GK and potentially ameliorate type-2 diabetes comorbidities

Identification and Comparison of Peptides from Chickpea Protein Hydrolysates Using Either Bromelain or Gastrointestinal Enzymes and Their Relationship with Markers of Type 2 Diabetes and Bitterness

The chickpea (Cicer arietinum L.) is one of the most important pulses worldwide. The objective was to identify, compare and evaluate peptides from chickpea hydrolysates produced by two enzymatic treatments. The antidiabetic potential and bitterness of the peptides and induction of bitter receptors were identified in silico. Proteins were isolated from the Kabuli variety. Peptides were produced from the proteins using a simulated digestive system (pepsin/pancreatin, 1:50 Enzyme/Protein, E/P), and these peptides were compared with those produced via bromelain hydrolysis (1:50 E/P). The protein profiles, sequences and characteristics of the peptides were evaluated. The biochemical inhibition and molecular docking of dipeptidyl peptidase-IV (DPP-IV), &alpha;-amylase and &alpha;-glucosidase were also studied. The molecular docking identified peptides from enzymatic hydrolysis as inhibitors of DPP-IV. The high hydrophobicity of the peptides indicated the potential for bitterness. There was no correlation between peptide length and DPP-IV binding. Peptides sequenced from the pepsin/pancreatin hydrolysates, PHPATSGGGL and YVDGSGTPLT, had greater affinity for the DPP-IV catalytic site than the peptides from the bromelain hydrolysates. These results are in agreement with their biochemical inhibition, when considering the inhibition of sitagliptin (54.3 &micro;g/mL) as a standard. The bitter receptors hTAS2R38, hTAS2R5, hTAS2R7 and hTAS2R14 were stimulated by most sequences, which could be beneficial in the treatment of type 2 diabetes. Chickpea hydrolysates could be utilized as functional ingredients to be included in the diet for the prevention of diabetes

Black bean peptides inhibit glucose uptake in Caco-2 adenocarcinoma cells by blocking the expression and translocation pathway of glucose transporters

The objective was to evaluate the effect of black bean protein fraction (PFRA), and its derived peptides on glucose uptake, SGLT1 and GLUT2 expression and translocation on Caco-2 cells. The effect of treatments was evaluated on glucose uptake, protein expression and localization and gene expression on Caco-2 cells. PFRA (10 mg/mL) lowered glucose uptake from 27.4% after 30 min to 33.9% after 180 min of treatment compared to untreated control (p < 0.05). All treatments lowered GLUT2 expression after 30 min of treatment compared to untreated control (31.4 to 48.6%, p < 0.05). Similarly, after 24 h of treatment, GLUT2 was decreased in all treatments (23.5% to 48.9%) (p < 0.05). SGLT1 protein expression decreased 18.3% for LSVSVL (100 μM) to 45.1% for PFRA (10 mg/mL) after 24 h. Immunofluorescence microscopy showed a decrease in expression and membrane translocation of GLUT2 and SGLT1 for all treatments compared to untreated control (p < 0.05). Relative gene expression of SLC2A2 (GLUT2) and SLC5A1 (SGLT1) was downregulated significantly up to two-fold change compared to the untreated control after 24 h treatment. Black bean protein fractions are an inexpensive, functional ingredient with significant biological potential to reduce glucose uptake and could be used as an adjuvant in the treatment of colorectal cancer. Keywords: Black bean protein fraction, Colorectal cancer, Glucose uptake, GLUT2, SGLT

Effect of the anthocyanins from purple corn on insulin secretion with and without a FFAR1 inhibitor in pancreatic cells.

<p>A) Effect of the free fatty acid receptor-1 (FFAR1) antagonist GW-1100 in the insulin secreting activity of pure anthocyanins ANC and ANC-rich extracts from purple corn in iNS-1E pancreatic β-cells compared to positive control C+ (glucose). B) Insulin secreting activity of pure ANC and ANC-rich extracts from purple corn in glucose-starved pancreatic β-cells compared to negative control C- (starving conditions). The cells were treated with different concentrations of pure ANC (1 μM– 100 μM) cyanidin-3-<i>O</i>-glucoside (C3G), delphinidin-3-<i>O</i>-glucoside (D3G), pelargonidin-3-<i>O</i>-glucoside (Pr3G), peonidin-3-<i>O</i>-glucoside (P3G), the semipurified C3G from purple corn pericarp (C3GP), catechin-(4,8)-cyanidin-3,5-diglucoside condensed form (CF), the purple corn pericarp water extract (PCW) (0.125 mg/mL– 1.0 mg/mL), and a FFAR1 agonist TAK-875 (5 μM– 20 μM) in the presence or absence of 10 μM GW-1100. Results are expressed as the mean ± SD of three independent experiments. Bars with different letters means statistical difference (p < 0.05) as determined by Tukey’s test.</p

Characterization and isolation of anthocyanins present in the purple corn pericarp water extract.

<p>A) Quantification of anthocyanins present in PCW. The results are expressed as the mean ± SD of three independent determinations. B) Representative chromatogram of purple corn pericarp water extract (PCW) at 520 nm showing the different anthocyanins (ANC) identified as follows: 1 –condensed form of catechin-(4,8)-cyanidin-3,5-diglucoside; 2 –cyanidin-3-<i>O</i>-glucoside; 3—pelargonidin-3-<i>O</i>-glucoside; 4 –peonidin-3-<i>O</i>-glucoside; 5 –cyanidin-3-(6’-malonylglucoside); 6—pelargonidin-3-(6’-malonylglucoside); 7—peonidin-3-(6’-malonylglucoside). HPLC chromatogram of C) the semi-purified condensed form and D) semi-purified cyanidin-3-<i>O</i>-glucoside, isolated from PCW. Representative MS² spectra of E) the semi-purified condensed form, and F) the semi-purified cyanidin-3-<i>O</i>-glucoside, showing the fragmentation pattern and structures of the compounds isolated from PCW.</p

Summary of the effects of the anthocyanins from purple corn on the free fatty acid receptor-1 (FFAR1)-dependent effects on pancreatic β-cells, and in glucose metabolism of hepatic cells.

<p>The anthocyanins from purple corn activated FFAR1 in iNS-1E pancreatic cells and increased the glucose-dependent insulin secretion. In HepG2 cells, the anthocyanins activated glucokinase activity and promoted the phosphorylation of AMPK. Markers with an up- or down-arrow indicate modulation in response to the ANC of purple corn.</p

Fractionation of the purple corn pericarp water extract.

<p>A-J) Chromatograms from HPLC identification of the different fractions obtained from the purple corn pericarp water extract (PCW). K) Preparative phase liquid chromatography output at 280 nm showing the different fractions collected. The anthocyanins (ANC) were identified as follows: 1 –condensed form of catechin-(4,8)-cyanidin-3,5-diglucoside; 2 –cyanidin-3-<i>O</i>-glucoside; 3—pelargonidin-3-<i>O</i>-glucoside; 4 –peonidin-3-<i>O</i>-glucoside; 5 –cyanidin-3-(6’-malonylglucoside); 6—pelargonidin-3-(6’-malonylglucoside); 7—peonidin-3-(6’-malonylglucoside). The chromatograms shown in this figure were used exclusively for ANC identification purposes.</p

Effect of anthocyanins from purple corn on glucose uptake and GK-activating potential in HepG2 cells.

<p>A) Effect of pure anthocyanins (ANC) and ANC-rich extracts from purple corn in glucose uptake in HepG2 cells. The cells were treated with 0.4 mg/mL of the ANC-rich extract from purple corn pericarp water extract (PCW), 50 μM of pure ANC cyanidin-3-<i>O</i>-glucoside (C3G), delphinidin-3-<i>O</i>-glucoside (D3G), pelargonidin-3-<i>O</i>-glucoside (Pr3G), peonidin-3-<i>O</i>-glucoside (P3G), the semipurified C3G from purple corn pericarp (C3GP) and catechin-(4,8)-cyanidin-3,5-diglucoside condensed form (CF). Results are expressed as the mean ± SD of three independent experiments. Bars with different letters means statistical difference (p < 0.05) as determined by Tukey’s test. B) GK activation by ANC from purple corn as determined using a biochemical assay. A non-linear regression was used to determine the half-maximal effective concentration (EC₅₀) based on the method by Sebaugh [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200449#pone.0200449.ref061" target="_blank">61</a>]. The cells were treated with a glucokinase agonist Ro-28-1675 (1 μM– 100 μM), different concentrations of pure ANC (1 μM– 300 μM), and PCW (0.125 mg/mL– 0.5 mg/mL). The results are expressed as units of glucokinase activity per milligram of protein in the cell lysates (Units/mg protein) versus the log concentration of the samples.</p

Effect of pure anthocyanins and anthocyanins-rich extracts from purple corn in glucose-stimulated insulin secretion in iNS-1E pancreatic β-cells.

<p>Modulation of insulin secretion in iNS-1E cells as a response to A) cyanidin-3-<i>O</i>-glucoside (C3G), peonidin-3-<i>O</i>-glucoside (P3G), pelargonidin-3-<i>O</i>-glucoside (Pr3G), delphinidin-3-<i>O</i>-glucoside (D3G); and B) the semipurified C3G from purple corn pericarp (C3GP) catechin-(4,8)-cyanidin-3,5-diglucoside condensed form (CF), purple corn pericarp water extract (PCW). The treatments with pure ANC were from 1 μM to 100 μM, and the anthocyanin-rich extracts from 0.125 mg/mL to 1.0 mg/mL, the free fatty acid receptor-1 (FFAR1) agonist TAK-875 was tested from 5 μM to 20 μM. The results are expressed as the mean ± SD of three independent experiments. A dotted line representing 100% was added for reference. Bars with a star means statistical difference (p < 0.05) compared to untreated cells as determined by Dunnet’s test.</p

Effect of anthocyanins from purple corn on proteins related to glucose metabolism in HepG2 cells.

<p>A) Effect of the pure anthocyanins (ANC) and ANC-rich extracts from purple corn on HepG2 hepatic cells on expression of proteins related to glucose metabolism. A representative image of the immunoblots is shown. Results are expressed as the mean ± SD of three independent experiments. Bars with different letters means statistical difference (p < 0.05) as determined by Tukey’s test. B) Confocal laser scanning microscopy depicting two-dimensional immunofluorescence localization of nuclei (blue) and GK (green). The quantification determined by the intensity (AU) over area (μm²) of free fatty acid receptor-1 (FFAR1) is shown. Data represents the mean ± SE of four independent fields of view from two independent cellular replicates. Means with different letters at the same time point are significantly different (p < 0.05) according to Tukey’s test. The cells were treated with 100 μM of pure ANC cyanidin-3-<i>O</i>-glucoside (C3G), delphinidin-3-<i>O</i>-glucoside (D3G), semipurified C3G from purple corn pericarp (C3GP), catechin-(4,8)-cyanidin-3,5-diglucoside condensed form (CF), and 0.5 mg/mL of the ANC-rich extracts from purple corn pericarp water extract (PCW) in comparison to just insulin (1 μM).</p