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Phenolic Compounds from Fermented Berry Beverages Modulated Gene and Protein Expression to Increase Insulin Secretion from Pancreatic #-cells in vitro Michelle H. Johnson, and Elvira Gonzalez de Mejia J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 11 Mar 2016 Downloaded from http://pubs.acs.org on March 11, 2016

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Journal of Agricultural and Food Chemistry

Phenolic Compounds from Fermented Berry Beverages Modulated Gene and Protein Expression to Increase Insulin Secretion from Pancreatic β-cells in vitro Michelle H. Johnson1, Elvira Gonzalez de Mejia1,2* 1

Division of Nutritional Sciences, 2Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL

*Correspondence: Elvira González de Mejía, Phone: (217) 244-3196; Fax: (217) 265-0925; Email: [email protected]

Running title: In vitro incretin effect of ANC and PAC from fermented berry beverage.

1 ACS Paragon Plus Environment


1

ABSTRACT: Berries are a rich source of bioactive phenolic compounds that are able to bind

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and inhibit the enzyme dipeptidyl peptidase-IV (DPP-IV), a current target for type-2 diabetes

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therapy. The objectives were to determine the role of berry phenolic compounds to modulate

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incretin-cleaving DPP-IV and its substrate glucagon-like peptide-1 (GLP-1), insulin secretion

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from pancreatic β-cells, and genes and proteins involved in the insulin secretion pathway using

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cell culture. Anthocyanins (ANC) from 50%blueberry- 50%blackberry (Blu-Bla) and 100%

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blackberry (Bla) fermented beverages at 50 µM cyanidin-3-glucoside equivalents increased (p
0.05) by treatments of sitagliptin at 0.5 µM, resveratrol at 5 µM, or

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by VitaBlue®, ANC 50%Blu-Bla, ANC 100%Bla, PAC 50%Blu-Bla, and PAC 100%Bla at 50

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µM in cyanidin-3-glucoside or epicatechin equivalents for ANC or PAC, respectively.

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Dual-Layered Simulated Absorption. Phenolic compounds were applied to the apical side of

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human epithelial Caco-2 cells grown in a monolayer inserts above iNS-1E pancreatic β-cells to


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determine the effect of transported ANC or PAC compounds or their metabolites on insulin

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secretion (Supporting Figure 1). This dual-cell system is a combination of both human and rat

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cells as there is currently no appropriate human β-cell line that is physiologically relevant16;

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however, they were not grown as a co-culture adjacent to each other to avoid any cross-species

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concerns. Hanging cell culture inserts contained a polystyrene membrane with a 0.4 µM pore

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that allowed transport of phenolic compounds19. Caco-2 cells were seeded on the apical side of

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24-well cell culture inserts at a density of 5*104 cells /cm2 alone, with volume of culture media at

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0.4 mL. On day 18, pancreatic iNS-1E cells were seeded onto 24-well plates at a density of

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1*105 cells /cm2, with 1 mL of culture medium. Only those cells that successfully formed a

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monolayer, as determined by Transepithelial Electrical Resistance (TEER) measurement to

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insure the integrity of the monolayer, were used for the experiment; TEER measurement with

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Millicel-ERS volt-ohm-meter (Millipore, Billerica, MA) was taken daily and before and after the

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experiment to insure the integrity of the monolayer in each well. After 20 days, Caco-2 cells

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reached confluence (average TEER for all wells was 300.9 ± 4.8 Ω*cm2); insuring that only

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compounds that passed through the Caco-2 cells would be able to affect the pancreatic β-cells

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growing on the inserts below. Treatments (sitagliptin, VitaBlue®, ANC 50%Blu-Bla and

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100%Bla, or PAC 50%Blu-Bla and 100%Bla) were added to the apical side for 24 h, and cells

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were layered by transferring the inserts into the 24-well plates containing the iNS-1E cells; Caco-

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2 cell permeability was assayed after exposure to the compounds and after incubation, so the

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selective permeability of the Caco-2 monolayer was not lost; TEER for all wells was 300.0 ±

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23.5 after the layering of the inserts and addition of the samples. After 24 h, the media from both

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the apical and basolateral side and both cell lysates were collected, centrifuged at 4 °C for 10 min

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at 1000 g to remove cell debris, and stored for analysis of insulin or for GLP-1 as follows. The



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terms apical and basolateral were used to describe the upper and lower sides of the Caco-2 cells,

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respectively, and were used to indicate the physiological system they are meant to model. The

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apical side models the intestinal fluid containing bioactive compounds after consumption, while

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the basolateral side models the compounds that are absorbed and could impact other organs in

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the body, such as the pancreas.

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Evaluation of Insulin Secretion after Glucose Stimulation (GSIS). Measurement of insulin

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release from INS-1E cells was performed according to Asfari et al.20, with modifications. INS-

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1E cells were plated 8 x 104 cells/well in 24-well plates and incubated for 3 days at 37 ºC. On

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the day of the experiment, the media was replaced by RPMI 1640 without glucose supplemented

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with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% HEPES, 1% sodium pyruvate, and

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50 µM β-mercaptoethanol and incubated for 2 h as starvation. The cells were then gently washed

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3 times with modified Krebs-Ringer-bicarbonate-HEPES buffer (KRBH), containing 135 mM

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NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 10 mM HEPES, 2 mM NaHCO3, and

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1.5 mM CaCl2. Cells were incubated in 0-glucose modified KRBH or treatments dissolved in the

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0-glucose modified KRBH buffer, and incubated for 30 min. The KRBH was replaced by KRBH

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with 0.1% bovine serum albumin (BSA) containing 0, 2.5, or 20 mM glucose for glucose

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stimulation, and incubated for another 30 min. The 0-glucose KRBH treated wells were used as

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the negative control.

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For the dual-cell system, INS-1E cells were plated (8 x 104 cells/well in 24-well plates) on

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day 18 and incubated for 3 days at 37 ºC, in a humidified 95% air, 5% CO2 atmosphere.

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Following the 24 h layered-cell treatment, INS-1E media was changed to 0-glucose RPMI media

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for 2 h before experiment (as starvation) before stimulation with 0 or 20 mM glucose modified-

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KRBH.


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In both direct and dual-layered experiments, the medium was collected and cleared by

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centrifugation at 15,000 g for 1 min, and stored at -20 ºC until assayed by Insulin ELISA

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(Abcam, Cambridge, MA). To determine insulin content, each standard (recombinant human

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insulin, 0-300 µIU/mL) or sample was loaded into the supplied wells and allowed to adhere

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overnight at 4 °C with gentle shaking. After, the solution was discarded and the wells were

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washed of non-adherent material. Then, a biotinylated insulin detection antibody was added to

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each well and incubated for 1 h at room temperature with gentle shaking, followed by additional

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washing. Next, HRP-Streptavidin solution was added to each well, incubated for 45 min at room

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temp, and wells washed again. Finally, the provided substrate reagent was added to each well,

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incubated for 30 min at room temperature in the dark with gentle shaking until the stop solution

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was added and the absorbance read at 450 nm on an ELx808™ Microplate Reader (Biotek

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Instruments, Winooski, VT). Insulin content was calculated using the standard curve y = 0.0014x

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- 0.0068, R² = 0.986.

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Dipeptidyl Peptidase-IV Inhibition. Activity of DPP-IV was measured using DPP-IV Glo™

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Protease Assay (Promega, Madison, WI) following manufacturer’s instructions. Cell lysates

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collected from Caco-2 cells, plated at 2*105 cells/well in 6-well plates following direct treatment

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with samples for 4, 8, 24, and 48 h were collected, normalized for protein content, and diluted to

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a final concentration of 10 ng of protein per well. The provided Glo-substrate was added, and

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the content of the wells was gently mixed using an Synergy2 multi-well plate reader (Biotek,

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Winooski, VT) at medium intensity for 4 s. DPP-IV cleavage of the provided Gly-Pro-amino

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methyl coumarin (AMC) substrate generates a luminescent signal by luciferase reaction, with the

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amount of DPP-IV enzyme available to bind Gly-Pro-AMC proportional to relative light units

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(RLU) produced. This signal in RLU was measured after 30 min, and treatments were compared



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to wells containing cell lysates treated with media only as control of no DPP-IV enzyme

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inhibition.

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Adipokine Expression. The effect on adipokine expression were carried out on non-glucose

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stimulated INS-1E cells without using the dual-cell system in order to determine the direct action

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of the compounds tested. Pancreatic iNS-1E cells plated at 2*105 cells/well in 6-well plates were

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treated with 50 µM ANC 50%Blu-Bla or ANC 100%Bla, sitagliptin (0.5 µM), or media only for

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24 h. Cells were washed twice with DPBS before collecting cell lysates using a lysis buffer

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containing 1% Igepal CA-630, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% SDS, 2 mM

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EDTA, 10 µg/mL Aprotinin, 10 µg/mL Leupeptin, and 10 µg/mL Pepstatin. Cell lysates were

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diluted to 200 µg protein, mixed with a cocktail of biotinylated detection antibodies, and

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incubated overnight with the Proteome Profiler Rat Adipokine Array nitrocellulose membranes

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(R&D Systems, Minneapolis, MN) containing capture and control antibodies spotted in

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duplicate. The membrane was then washed to remove unbound material before application of

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streptavidin-HRP and chemiluminescent detection reagents. The chemiluminescent signal

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produced was captured using a GelLogic 4000 Pro Imaging System (Carestream Health, Inc.,

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Rochester, NY) and analyzed for pixel intensity.

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Gene Expression. Gene expression analyses were carried out on non-glucose stimulated INS-1E

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cells without the dual-cell system in order to determine the direct action of the compounds tested.

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iNS-1E cells growing at 1*105 cells/well in a 6-well plate were treated with 50 µM

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ANC50%Blu-Bla and ANC100%Bla. After treatment for 24 h, RNA was collected using an

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RNAeasy Kit (Qiagen, Germantown, MD), quantified, and checked for quality. cDNA was

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synthesized using a Qiagen RT2 First Strand synthesis kit. Quantitative RT-PCR was performed

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using a 7900HT Fast Real-Time PCR System Cycler (Applied Biosystems, Foster City, CA) and


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a targeted array (PARN-030ZA RT2 Profiler PCR Array, Qiagen, Germantown, MD) that

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profiles the expression of 84 insulin-responsive genes. Thermocycling conditions were: 1 cycle

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at 95 °C for 10 min and then 40 cycles at 95 °C for 15 s followed by 60 °C for 1 min before a

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dissociation stage. The relative mRNA expression levels of each gene were calculated using the

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2-∆∆Ct method in reference to β2 microglobulin, present in all nucleated cells.

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Active Glucagon-Like Peptide 1 Measurement. The active form of GLP-1 was quantified from

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both the apical and basolateral media using an ELISA (EGLP-35K, EMD Millipore, Billerica,

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MA). Per manufacturer’s instructions, the active GLP-1 present in media was captured in a 96-

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well plate by a monoclonal antibody that binds the N-terminal region of the active GLP-1

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molecule. The wells were washed to remove unbound materials, and anti-GLP-1 alkaline

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phosphatase detection conjugate was added to bind the immobilized GLP-1. Next, the unbound

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conjugate was washed, and bound detection conjugate was quantified by addition of methyl

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umbelliferyl phosphate, which forms a fluorescent signal proportional to the concentration of

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active GLP-1 in the unknown sample. Fluorescence was read using a FL600 Microplate

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Fluorescence Reader (Bio-Tek, Winooski, VT) with excitation/emission wavelength of 360/460

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nm and quantified using GLP-1 standards ranging from 2-100 pM with equation of y = 0.44x -

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2.52, R² = 0.95.

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Western Blot Analysis. To measure the effect of phenolic compounds on DPP-IV expression

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and resulting active GLP-1, equal amounts of Caco-2 or iNS-1E cell lysate protein (20 µg) were

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loaded in each well of 4-20% gradient SDS-polyacrylamide gels. After electrophoresis, proteins

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were transferred to polyvinylidene difluoride membranes and blocked with 3% non-fat dry milk

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in 0.1% TBST for 1 h at 4 °C. After blocking, the membranes were washed with 0.1% TBST (5

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times, 5 min each) and incubated with primary antibodies (1:500) overnight at 4 °C. The



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membranes were washed again and incubated with anti- IgG horseradish peroxidase conjugate

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secondary antibodies (1:2500) for 2 h at room temperature. After incubation and repeated

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washing, the membranes were prepared for detection using a 1:1 mixture of chemiluminescent

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reagents A (luminol solution) and B (peroxide solution) (GE Healthcare Biosciences, Pittsburgh,

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PA). The membrane pictures were taken on a GelLogic 4000 Pro Imaging System (Carestream

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Health, Inc., Rochester, NY). Relative expression of DPP-IV and GLP-1R were normalized to

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GAPDH.

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Statistical Analysis. Means of at least three replicates of independent duplicates were generated

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and adjusted with Tukey’s post hoc comparisons using Statistical Analysis System Software,

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version 9.3 (Cary, NC). Significant differences were reported at p-values < 0.05.

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RESULTS

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Anthocyanins present in beverages were predominantly delphinidin derivatives.

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Comparing the predominant anthocyanins (% of total ANC present) identified in 50% blueberry-

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50% blackberry (50%Blu-Bla) to those from the 100% blackberry (100%Bla) beverage, both had

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delphinidin-3-arabinoside as the most predominant ANC (70.8 and 80.3%, respectively). The

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50%Blu-Bla ANC contained a higher percentage of malvidin-3-arabinoside (7.9%) in

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comparison to 100%Bla beverage (0.5%), petunidin-3-glucoside (3.3 vs 0.6%), delphinidin-3-

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glucoside (2.5 vs 0.6%), petunidin-3-arabinoside (1.7 vs 0.6%) and malvidin-3-glucoside (1.6 vs

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0.4%). The ANC extracted from 100%Bla contained higher percentages of cyanidin derivatives

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with more identified cyanidin-3-arabinoside than 50%Blu-Bla (7.5% vs 0.6%), cyanidin-3-

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galactoside (1.5 vs none identified) and cyanidin-6-acetyl-3-glucoside (2.8 vs 0.4%). A summary

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of the predominant anthocyanins (% of total present) can be seen in Supporting Table 1.


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Glucose-stimulated insulin secretion was increased by anthocyanin treatments when

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directly applied to iNS-1E cells but was not different from media-only control. Insulin

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secretory response of iNS-1E cells following glucose stimulation and pre-treatment by

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sitagliptin, VitaBlue®, or ANC and PAC from 50%Blu-Bla or 100%Bla was measured to

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determine their ability to act as insulin secretagogues in cell culture. Direct treatment of the

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phenolic compounds tested did not reduce the cellular viability or the proliferation of iNS-1E

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pancreatic β-cells. Cell viability was 104.2 ± 5.8%, 104.1 ± 7.7%, 108.8 ± 5.5%, 87.8 ± 3.6%,

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100.4 ± 8.8%, 100.0 ± 14.0%, and 100.4 ± 1.7% for sitagliptin, resveratrol, VitaBlue®,

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ANC50%Blu-Bla, PAC50%Blu-Bla, ANC100%Bla and PAC100%Bla, respectively (p < 0.05).

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When iNS-1E cells were not stimulated with glucose (0 mM), there was an increase of insulin

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secretion seen by all treatments except PAC 50% and ANC 100% (Figure 1); this increase was

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significantly higher than media-only treated cells (106.9 ± 4.3 µIU/mL) for sitagliptin (289.3 ±

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18.9 µIU/mL), VitaBlue® (254.6 ± 41.4 µIU/mL), ANC50%Blu-Bla (232.7 ± 30.0 µIU/mL),

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and PAC100%Bla (272.7 ± 36.7 µIU/mL). Sitagliptin enhanced the secretion of insulin without

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any glucose or GLP-1 present. Glucose-stimulation at 2.5 mM did not alter insulin secretion

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from iNS-1E cells among treatments (Figure 1A). As expected18,19, stimulating the β-cells with

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20 mM glucose induced insulin secretion in media-only treated cells (301.0 ± 33.3 µIU/mL).

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Within treatment, the ability to increase insulin secretion in response to glucose was most

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increased by the ANC 50%Blu-Bla (452.7 ± 105.1µIU/mL) and ANC 100%Bla (376.0 ± 29.6

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µIU/mL). The glucose-stimulated (with 20 mM glucose) insulin secretion from iNS-1E cells was

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increased only following 30 min pretreatment by ANC 50%Blu-Bla compared to media-only

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treated cells. There were also statistical increases in insulin following treatment of ANC



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50%Blu-Bla and ANC 100%Bla when unstimulated with no glucose (0 mM) present compared

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to the media-only unstimulated control.

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Dipeptidyl Peptidase-IV Expression and Activity in Caco-2 Cells Treated with Blueberry

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Extracts for 24 h were Reduced. Due to limited bioavailability, the ability of anthocyanins to

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affect glucose homeostasis in the body may be mediated by the effects of the compounds or their

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metabolites within the gastrointestinal tract. The greatest reduction of DPP-IV activity was seen

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in Caco-2 cells after treatment for 24 h by 23.6% ± 2.6 for Res and 10.7% ± 1.5 for VitaBlue®,

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respectively, compared to other timepoints that were measured (4, 8, and 48 h, data not shown),

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therefore the 24 h timepoint was used for all following experiments. As such, Caco-2 cells were

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treated with media only, sitagliptin, resveratrol, a commercial berry extract (VitaBlue®), and

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anthocyanin and proanthocyanidin extracts from a fermented berry beverage, and the activity and

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amount of DPP-IV protein expression was measured. Exposure during dual-layering with

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treatments for 24 h reduced DPP-IV protein expression by VitaBlue® (69.5%), ANC 50%Blu-

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Bla (48.7%) and PAC 50%Blu-Bla (54.0%) in Caco-2 cells (Figure 1B). As expected, there was

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no reduction seen by the DPP-IV inhibitor sitagliptin on expression of DPP-IV as the mechanism

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of inhibition is competitive binding within the active site rather than an inhibition of the

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enzyme’s expression; the IC50 of DPP-IV for sitagliptin was reported as 0.06 ± 0.03 µM21. There

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was no reduction in the expression of DPP-IV by resveratrol or 100%Bla extracts.

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Proteins of iNS-1E Cells Related to Insulin Growth Factor Binding were Increased with

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Anthocyanins from 50% Blueberry-50% Blackberry Treatment

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Following treatment with anthocyanins from 50%Blu-Bla or anthocyanins from 100%Bla,

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the relative expression of adipokines in iNS-1E proteins was measured to determine the impact

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of anthocyanins on proteins in pancreatic β-cells (Figure 2 and Supporting Figure 2).


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Circulating adipokine proteins DPP-IV, TNFα, IL-6, and IGFBP-1 were not affected by either

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anthocyanin treatment compared to the control (data not shown). As these are secreted proteins,

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potential effects may not have been detected due to screening of the whole cell lysate rather than

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secreted proteins. The proteins with highest detected pixel intensity that were significantly

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increased by treatment with ANC 50%Blu-Bla in comparison to the control were insulin-like

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growth factor binding proteins 3 (IGFBP-3, fold-change 1.58 ± 0.01), insulin-like growth factor

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2 (IGF-II, fold-change 1.40 ± 0.19), vascular endothelial growth factor (VEGF, fold-change 1.53

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± 0.06), and insulin-like growth factor binding proteins 2 (IGFBP-2, fold-change 1.26 ± 0.01)

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(Figure 2A).

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Other adipokine proteins that were less highly expressed (lower pixel intensity) were also

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modulated by treatment with anthocyanins (Figure 2B), with the trend being an increase in

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protein expression by ANC 50%Blu-Bla and a slight decrease by ANC 100%Bla treatment

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relative to the control. Adipokines that were affected differentially between ANC from either

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50%Blu-Bla or 100%Bla control include leukemia inhibitory factor (LIF), IGFBP-5 and -6,

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fibroblast growth factor 21 (FGF-21), monocyte chemoattractant protein-1 (MCP-1), and

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Lipocalin-2.

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Genes Affected in Specific Pathways Related to Insulin Secretion were Up-Regulated

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Following Anthocyanin Treatment. Due to the ability of ANC from 50%Blu-Bla or 100%Bla

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to act as insulin secretagogues with no glucose stimulation, the expression of genes involved in

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insulin secretion in iNS-1E cells was measured without any glucose present and compared to the

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media-only treated cells. Genes linked to insulin secretion that were most up-regulated by either

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anthocyanin treatment are shown in Table 1, while Table 2 indicates the genes that were most

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down-regulated. Overall, ANC from 50%Blu-Bla were able to up-regulate 28 genes in the array



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(fold-change >1.5), while ANC from 100%Bla up-regulated 14 genes. Of genes most up-

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regulated, there was an effect on insulin, insulin receptors and insulin binding including Ins1 and

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Ins2, as well as Dok3, Igfbp1, and Igf1r. Dok3 (docking protein 3) plays a key role in insulin

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binding and Ras protein signal transduction, and was up-regulated by 8.5 and 6.5-fold for ANC

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50%Blu-Bla and 100%Bla, respectively. There was an increase of Glucagon (Gcg) by 3.0 and

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2.0-fold compared to the control for ANC 50%Blu-Bla and 100%Bla, respectively. Igf1r

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(insulin-like growth factor 1 receptor) was increased by 2.3 ± 0.6 for ANC 50%Blu-Bla and by

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1.6 ± 0.3 fold for ANC 100%Bla. Igfbp1 (insulin-like growth factor binding protein 1) is a

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modulator of insulin growth factor (IGF) bioavailability and was also up-regulated by both ANC

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treatments. Other genes that were up-regulated by both ANC treatments included Rras (related

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RAS viral, r-ras, oncogene homolog), Akt2 (v-akt murine thymoma viral oncogene homolog 2)

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that is related to phosphatidylinositol 3-kinase (PI3-K) mediated signaling, Hras (Harvey rat

332

sarcoma virus oncogene), and Frs2 (fibroblast growth factor receptor substrate 2). The gene for

333

ins2 (insulin 2) and Irs2 were up-regulated only by ANC 50%Blu-Bla, however this ANC

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50%Blu-Bla treatment did not strongly down-regulate many genes studied.

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Table 2 indicates the genes that were most down-regulated by either anthocyanin treatment.

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Overall, ANC from 50%Blu-Bla were able to down-regulate with fold-change < -1.5 only 7

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genes, while ANC from 100%Bla down-regulated the expression of 16 genes in the array. G6pc

338

(glucose-6-phosphatase, catalytic subunit) was down-regulated by both anthocyanin treatments

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by -1.7 for both ANC 50%Blu-Bla and ANC 100%Bla. Pparg (peroxisome proliferator-activated

340

receptor gamma) and Ins1 (insulin 1) were down-regulated only by ANC50%Blu-Bla (-2.11 ±

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0.14 and -1.77 ± 0.13, respectively). Genes most strongly down-regulated by the ANC 100%Bla

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treatment were Gpd1, Insl3, Pklr, Shc1, Ptpn1, and Fbp1.


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Utilizing IPA software that takes into consideration all of the genes that were affected in the

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array, Figure 3 illustrates the primary predicted downstream pathways including glucose

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metabolism affected by ANC from 50%Blu-Bla (A) or from 100%Bla (C) and cell viability

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affected by ANC from 50%Blu-Bla (B) or from 100%Bla (D). This analysis indicated that both

347

ANC treatments would lead to an improvement of glucose metabolism and cell vitality and

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identifies the primary ways that ANC may act upon pancreatic β-cells to lead to an improvement

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in insulin secretion.

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Glucose-Stimulated Insulin Secretion Following Treatment in the Simulated Absorption

351

Model of Caco-2 Cells on Inserts over iNS-1E cells. To simulate absorption, Caco-2 cells that

352

had formed a confluent monolayer on cell culture inserts were treated with compounds that could

353

cross into the basolateral side if transported through the epithelial cells. After treatment for 24 h,

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iNS-1E cells growing on the wells below were evaluated for response to glucose stimulation. As

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seen with the direct treatment, all groups increased the amount of insulin secreted without any

356

glucose stimulation compared to the media-only, except ANC 100%Bla (Figure 4). When cells

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were stimulated with 20 mM glucose, insulin secretion was increased by VitaBlue® (291.1 ± 7.6

358

µIU/mL), ANC 50%Blu-Bla (274.4 ± 37.9 µIU/mL) and ANC 100%Bla (300.8 ± 5.1 µIU/mL)

359

compared to media-only treated cells. However, following layering of the cells, the effect to

360

increase insulin secretion seen by ANC (Figure 1A) was maintained only by treatment with

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ANC from 100% Blackberry.

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GLP-1 and GLP-1R were Increased following 100% Blackberry Proanthocyanidin

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Treatment, but this Increase did not Correlate Directly with Insulin Secretory Results. To

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measure the effect of inhibiting DPP-IV in order to increase biologically active GLP-1 in the

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media that could stimulate insulin release from pancreatic β-cells, GLP-1 was measured in both



Page 18 of 43

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the apical and basolateral sides of the dual-layered system. Total GLP-1 was not measured as this

367

would include the inactive metabolites of GLP-1 after cleavage by DPP-IV, and thus these other

368

forms of GLP-1 would not lead to stimulation of insulin secretion. Total active GLP-1 in the

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supernatant of Caco-2 (apical side) and of iNS-1E cells (basolateral side) was increased

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following only the PAC 100%Bla treatment (Figure 5A). Active GLP-1 of the apical side only

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was increased from media-only treated cells following treatment with sitagliptin (39.1 ± 13.3

372

pM) that was mirrored by VitaBlue® (53.1 ± 13.1 pM), PAC 50%Blu-Bla (51.4 ± 12.9 pM), and

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PAC 100%Bla (50.8 ± 8.7 pM), There was an increased expression of GLP-1R in iNS-1E cells

374

following the treatment for 24 h by PAC100%Bla (relative intensity of 3.0 ± 1.9); however, other

375

treatments did not differ from the media-only treated cells (Figure 5B). This increase in the

376

GLP-1R for PAC 100%Bla correlates with the increase seen in the total active GLP-1 in the

377

media of the dual-cell system, however, these results do not correlate with the modulation of

378

insulin secretion seen. Taken together, a proposed mechanism of action that compounds from

379

ANC may have within pancreatic β-cells is presented in Figure 6.

380

DISCUSSION

381

The aim of the study was to assess the insulin secretory response in iNS-1E pancreatic beta-

382

cells to follow the lines of research into incretin-based therapies; the approach was to set a

383

predetermined amount of glucose stimulation based on postprandial levels and the results were

384

compared to the media-only cells. This study demonstrated the role of phenolic compounds

385

(such as ANC and PAC) from fermented berry beverages to modulate incretin-cleaving hormone

386

DPP-IV and its substrate GLP-1 to increase insulin secretion from pancreatic β-cells. Bioactive

387

compounds (ANC and PAC) from fermented berry beverages reduced incretin-cleaving hormone

388

DPP-IV protein activity and protein expression in Caco-2 cells, and ANC increased insulin


Page 19 of 43


389

secretion of iNS-1E pancreatic β-cells both directly and following a dual-cell layered model of

390

simulated absorption.

391

The finding that the ANC and PAC treatments are also able to stimulate insulin secretion even

392

without the presence of any glucose can be attributed to the glycosidic side chains of these

393

compounds allowing them to act as glucose mimetics22. Previous studies have indicated that

394

procyanidins, part of the proanthocyanidin class of flavonoids, may play a role to modify insulin

395

synthesis and secretion leading to decreased insulin production23, while proanthocyanidins in

396

this study were seen to increase insulin secretion without glucose stimulation. This difference

397

could be due to the degree of polymerization of compounds from grapes in the previous study

398

rather than those present in blackberries used in this study4. While much higher concentrations

399

than what could be achieved in plasma for these compounds were used, the concentration of 50

400

µM was chosen based on efficacy in previous studies4, 22. Further, the proposed cell culture

401

system is a simulated absorption and the cell culture concentration of 50 µM of ANC in C3G

402

equivalents translates to feasible consumption of 3 mg of ANC by a human, assuming their

403

intestinal fluid volume is 150 mL24 and that 1% of this would reach their β-cells. Previous

404

characterization of the extracts indicated that delphinidin was the primary ANC present4 and

405

therefore it is likely that the primary metabolites protocatechuic acid andphloroglucinaldehyde in

406

the cell media could be the compounds exerting the biological activities observed25-26.

407

That the increase in insulin secretion by treatments compared to media-only with no glucose

408

stimulation was not also observed when stimulated with 20 mM glucose is an interesting finding,

409

and suggests the mechanism to increase insulin secretion may not be directly mediated by

410

glucose-stimulation. Previously, anthocyanins present in fruits were shown to increase the

411

insulin secretion following 4 and 10 mM glucose in pancreatic β-cells in vitro, with the highest



Page 20 of 43

412

increase due to cyanidin-3-glucoside and cyanidin-3-galactoside22. The primary anthocyanin

413

present in the ANC100%Bla was identified as delphidin-3-arabinoside4, which has three

414

hydroxyl groups in its B-ring, while the ANC50%Blu-Bla contained higher amounts of cyanidin

415

derivatives. The difference in ability of these anthocyanins to act as insulin secretagogues may

416

be attributed to the difference in the concentration of different anthocyanins present and their

417

structure27. Major metabolites of ANC that are produced due to metabolic transformation by the

418

gut microbiota have been identified as protocatechuic acid (PCA), along with other phenolic

419

acids such as vanillic acid, syringic acid, caffeic acid, and ferulic acid that can be identified in

420

plasma following ANC consumption28,29. Proanthocyanidins may be partially metabolized by

421

intestinal microbiota to phenolic acids and other derivatives. Most observed benefits from

422

consumption of ANC are likely due to the metabolites such as PCA which are present in

423

circulation at much higher concentrations30,31. As ANC are prone to transformation in the

424

intestine, it is likely that the primary degradation products such as PCA, phloroglucinaldehyde

425

and their conjugates25,26 were the responsible compounds for the effects seen, rather than the

426

parent anthocyanin compounds. In the simulated absorption model, the polymerized compounds

427

were no longer able to act as insulin secretagogues, or may have been cleaved into their smaller

428

components such as procyanidins that have previously been shown to decrease insulin

429

secretion23. While this model will not address the metabolism of the extracts before they reach

430

the part of the gut that is represented by the Caco-2 cells nor the effect that gut microbiota or the

431

role of the liver in metabolism of the phenolic compounds present, it is an attempt to go a step

432

beyond direct treatment of the extracts to the cell line that is currently a limiting factor to

433

translation of in vitro results to in vivo effects. Both the direct and dual-layered simulated


Page 21 of 43


434

absorption models support the idea that at 24 h, none of the parent ANC remains, and the effects

435

seen can be due to degradation products or conjugates.

436

Resveratrol was evaluated as a control of a purified stilbenoid, which may be present in the

437

PAC fractions and was previously seen to be the most potent inhibitor of DPP-IV activity using

438

computational modeling12. In this study we found no reduction in the expression of DPP-IV by

439

resveratrol indicating that previously seen inhibition may act through competitive inhibition

440

rather than reduction in protein expression. As VitaBlue® (extract standardized to 20%

441

anthocyanins) is an ANC-enriched extract from blueberry that contains both ANC and PAC, and

442

since no reduction was seen by the 100%Bla extracts, the effect to reduce DPP-IV activity and

443

expression appears to be due to compounds present only in blueberries. Previous studies have

444

indicated that phenolic compounds, especially anthocyanins, are strong inhibitors of the enzyme

445

DPP-IV12. Grape seed procyanidins have been found previously to inhibit Caco-2 expressed

446

DPP-IV activity by about 20%, with longer treatment leading to inhibition not seen by higher

447

concentrations for 24 h32, which agrees with the results seen in our study. While a reduction in

448

the protein expression would lead to a reduction in the cleavage of GLP-1, inhibition through

449

competitive binding of the active site rather than a reduction of protein expression would allow

450

the Caco-2 cells expressing DPP-IV to maintain their normal function, however there is limited

451

data regarding intestinal DPP-IV activity. Further, considering the exposure times and

452

temperatures of these cell culture conditions, it is likely that the parent anthocyanins are not the

453

responsible compounds behind the effects observed, especially considering recent publications

454

indicating rapid degradation of anthocyanins into phenolic acids within the gastrointestinal

455

tract26.



456

Page 22 of 43

It has been previously proposed that cytokines secreted from adipose have an effect on the

457

function of the pancreas to secrete insulin, and that their secretion from adipocytes may be

458

modulated by anthocyanin treatment33, and it is also known that the pancreas itself can secrete

459

these compounds34. IGFBP-2 binds to IGF-I and IGF-II, and these proteins are involved in

460

insulin-signaling pathway and in the cellular response to various stimuli. Since ANC 50%Blu-

461

Bla increased the pixel intensity of these proteins more than ANC 100%Bla, anthocyanins from

462

blueberries may contribute more than those from blackberries to the increased expression of

463

these proteins. An increase of VEGF by anthocyanins is supported by previous research that

464

purified C3G protected against endothelial dysfunction in mice34. Although adiponectin was not

465

measured in the present study, the increase in VEGF may be linked to increased expression of

466

adiponectin; recent studies investigating the role of anthocyanins to affect adipocytes have

467

indicated that the expression of adiponectin can be increased by anthocyanins35. We have also

468

demonstrated a comparable trend when using ANC blends similar to those used in this study36.

469

The role of VEGF in inhibiting apoptosis indicates a potential mechanism by which

470

anthocyanins may protect β-cells from loss of mass of the pancreas.

471

LIF is a secreted cytokine with growth factor activity, related to the role of IGFBP-5, which

472

plays a key role in growth factor binding as well as signal transduction and cellular response to

473

cAMP37. IGFBP-6 also is involved in cell growth and has a higher affinity for IGF-II than IGF-I,

474

which also agrees with the observed increased expression of IGF-II over IGF-I. Another

475

adipokine that is a member of the growth factor family, FGF-21, is involved in regulation of

476

glucose import beyond its activities related to cell and tissue growth and may lead to increases in

477

proliferation or mass of β-cells as well as increases in insulin transcription38. Lipocalin-2 is

478

involved in apoptosis and inflammatory response, indicating that the reduced expression of this


Page 23 of 43


479

adipokine by the ANC from 100%Bla would be more protective against inflammatory-mediated

480

cell death than ANC from 50%Blu-Bla. Finally, MCP-1 is involved in cellular calcium ion

481

homeostasis, response to ATP, and VEFG signaling, indicating the compounds present in

482

ANC50%Blu-Bla may be more responsive to intracellular signaling than ANC100%Bla.

483

It is important to note that the adipokines that have been reported to have the most significant

484

deleterious effects on the function and proliferation and eventual failure of β-cells, including

485

TNFα and DPP-IV38, were not increased in our study. These results also agree with our results

486

from the western blot in that there was no change by the ANC treatments to affect the expression

487

of DPP-IV. Overall, an increase in these cytokines produced by the β-cell may have a protective

488

role in defense against infection and eventual loss of pancreatic function; however, the

489

physiological relevance of these results may be impacted by the presence of circulating

490

adipokines from adipose, and should be considered as only direct effects on protein expression

491

within the pancreatic β-cell.

492

Modulation of genes related to insulin secretion as well as protein expression may help to

493

understand the mechanisms through which phenolic compounds could affect GSIS. In this study,

494

ANC50%Blu-Bla and ANC100%Bla were used based on initial insulin secretion analysis which

495

indicated the greatest effects by these two treatments. Glucagon (Gcg) induces glucose

496

production, regulates carbohydrate and protein metabolism, and also induces transcription of

497

GLP-1, an important inducer of insulin secretion following food intake. Igf1r is the receptor for

498

Igf-1 which is involved in the induction of cell cycle progression and survival, and therefore the

499

induction of this gene may allow for an increase in survival of pancreatic β-cells in response to

500

excess glucose. The ins2 gene encodes pre-pro-insulin, which is processed to form the insulin

501

hormone involved in glucose and lipid metabolism, while Irs2 encodes the insulin receptor 23 ACS Paragon Plus Environment


Page 24 of 43

502

substrate necessary for insulin signaling to occur. Up-regulation of these genes related to insulin

503

secretion and an increase in the gene that induces GLP-1 may be the mechanism through which

504

anthocyanins increased insulin secretion following glucose stimulation. The presence of malvidin

505

or cyanidin structures in blueberry may be able to preferentially affect certain genes such as Ins2

506

and Irs2, however, there is a need for future studies to address the elucidation of which ANC

507

degradation products and phenolic compounds are present in media able to reach the target cells.

508

Previous studies have indicated the importance of the GLP-1 receptor to regulate endogenous

509

glucose production39 and to enhance insulin secretion40, therefore the relative amount of the

510

GLP-1R protein in the pancreatic β-cells following the dual-cell experiment was measured in

511

addition to GLP-1. Kato et al.41 indicated that delphinidin, also the major anthocyanin in the

512

ANC from 100% blackberry, increased the secretion of GLP-1 from intestinal cells due to the

513

structural differences of the three hydroxyl groups. In the present study, we expanded on the role

514

of anthocyanins to have a potential to contribute to the prevention and treatment of diabetes

515

through a DPP-IV-inhibitory mechanism, leading to prolonged action of GLP-1. The similarity

516

of some treatment effects to the control sitagliptin, which acts to inhibit DPP-IV through

517

competitive inhibition, suggests these compounds were acting through a DPP-IV inhibitory

518

mechanism to increase GLP-1. Further, as VitaBlue® contains a mixture of ANC and PAC, and

519

acted similarly to the PAC-only treatments rather than the ANC treatments, it seems the more

520

complex polyphenolic compounds as present in VitaBlue® or PAC had an effect to increase

521

GLP-1 on the apical side. This is also supported by a previous study demonstrating that

522

procyanidins could increase GLP-1 levels in plasma of mice42.

523 524

Genes down-regulated by both ANC treatments included those involved in gluconeogenesis such as the G6pc enzyme which catalyzes the conversion of D-glucose 6-phosphate and water to


Page 25 of 43


525

D-glucose and phosphate. Additionally, Gpd1 (glycerol-3-phosphate dehydrogenase 1, soluble)

526

is involved in carbohydrate and lipid metabolism and the glycerol-phosphate shuttle, Insl3

527

(insulin-like 3) is a ligand for the LGR8 relaxin receptor that leads to stimulation of adenylate

528

cyclase and increase of cAMP, and Pklr (pyruvate kinase, liver and RBC) catalyzes the

529

conversion of ATP and pyruvate to ADP and phosphoenolpyruvate in glycolysis. Down-

530

regulation of these genes following treatment by ANC 100%Bla could be due to the primary

531

anthocyanin present, delphinidin-3-arabinoside4, rather than cyanidin or malvidin derivatives

532

contributed by fermented blueberries. These three down-regulated genes are all involved in

533

energy metabolism, and thus increasing ATP through downregulation of Pklr, for example,

534

would allow for the glucose-stimulated increase of insulin secretion resulting from the increased

535

ATP to ADP ratio to occur. Both Slc2a1, a transporter for glucose and other hexoses that may be

536

involved in response to osmotic and metabolic stress, and Jun, a transcription factor that acts as a

537

proto-oncogene and may be involved in activated TLR4 signaling, were down-regulated,

538

indicating that ANC from blackberry may affect pancreatic β-cell response to the extracellular

539

state of the body. Pparγ is a ligand-activated transcription factor that mediates expression of

540

genes involved in lipid metabolism. Other studies have indicated that cyanidin is an agonistic

541

ligand of PPARα43, and therefore down-regulation of this gene could be due to binding ligand

542

similarity by cyanidin present in 50%Blu-Bla. Other studies have found that cyanidin-3-

543

glucoside up-regulated PPARɣ activity and mRNA levels in adipocytes44, 45; however, in this

544

study we found a down regulation of the gene for this protein. This difference could be due to

545

diverse roles or regulation of PPARɣ by various cell types in response to anthocyanins. Lastly,

546

Ptpn1 (protein tyrosine phosphatase, non-receptor type 1) has phosphotyrosine phosphatase

547

activity and is a negative regulator of insulin-signaling, and is also a current target of type-2



Page 26 of 43

548

diabetes therapy. Other flavonoids from licorice or botanical roots have been identified as natural

549

inhibitors of PTPN146, 47, however there is a need for more studies with flavonoids from

550

fermented berries. The modulation of these genes to indicate a predicted downstream effect

551

involving glucose metabolism and cell viability may be only seen phenotypically with a longer

552

treatment of the cells, potentially explaining why there was no promotion of GSIS after 24 h. The

553

suggested effects from the IPA analysis, however, are important to direct future projects

554

investigating molecular mechanisms of anthocyanins to modulate glucose metabolism.

555

Our results indicated that the ability of phenolic compounds to act as insulin secretagogues in

556

cell culture is possibly linked to enhanced insulin receptor signaling and up-regulation of

557

proteins IGFBP-2 and -3, IGF-II, and VEGF. Additionally, the ANC treatments up-regulated

558

genes of insulin-receptor associated proteins and GLP-1, and down-regulated PTP1B in iNS-1E

559

cells. Therefore, ANC and PAC from fermented berry beverages may be acting through a

560

mechanism beyond DPP-IV inhibition to increase incretin-mediated insulin secretion in

561

pancreatic β-cells. These effects, however, are likely mediated by degradation products of parent

562

ANC and should be further evaluated in vivo, and in a more complete physiological model

563

system.

564 565

ABBREVIATIONS: ANC, anthocyanin; 100%Bla, 100% blackberry fermented beverage;

566

50%Blu-Bla, 50%blueberry-blackberry fermented beverage; DPP-IV, dipeptidyl peptidase IV;

567

EMEM Eagle’s Minimum Essential Medium; GLP-1, glucagon-like peptide-1; GSIS, glucose

568

stimulated insulin secretion; KRBH, Krebs-Ringer-bicarbonate-HEPES buffer; PAC,

569

proanthocyanidins; PCA, protocatechuic acid; RLU, relative light units, Res, resveratrol; Sitag,

570

sitagliptin; TEER, transepithelial electrical resistance.


Page 27 of 43


571

ACKNOWLEDGMENTS

572

Thanks to Mr. Diego Luna for help with figures related to gene expression.

573 574

Supporting Information Description:

575 576

Supporting Figure 1. Diagram of the dual-cell simulated absorption system. Caco-2 cells were

577

seeded on the apical side of 24-well cell culture inserts at a density of 5*104 cells /cm2 alone,

578

with volume of culture media at 0.4 mL. Only those cells that successfully formed a monolayer,

579

as determined by TEER measurement to insure the integrity of the monolayer were used for the

580

experiment. On day 18, pancreatic iNS-1E cells were seeded onto 24-well plates at a density of

581

1*105 cells /cm2, with 1 mL of culture medium. After 20 days, Caco-2 cells reached confluence

582

and treatments (sitagliptin, VitaBlue®, ANC 50%Blu-Bla and 100%Bla, or PAC 50%Blu-Bla

583

and 100%Bla) were added to the apical side for 24 h before cells were layered by transferring the

584

inserts into the 24-well plates containing the iNS-1E cells. Diagram adapted from Castell-Auvi et

585

al.19.

586 587 588

Supporting Figure 2. Blots that were generated and the location of each protein detected.

589

Supporting Table 1. Anthocyanins present in each ANC blend at >1% of total present as

590

measured by the area under the curve.

591 592 593 594 595 27 ACS Paragon Plus Environment


Page 28 of 43

596

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37. Rat Genes, Rat Genome Database, Medical College of Wisconsin, Milwaukee, Wisconsin. URL (http://rgd.mcw.edu/) (April 2014). 38. Dunmore, S.J.; Brown, J.E.P. The role of adipokines in β-cell failure of type-2 diabetes. J. Endocrinol. 2013, 216(1), T37-45.

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Ca2+/calmodulin-dependent kinase II pathway. PLoS One. 2015, 10(5):e0126157).

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Figure Captions

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Figure 1. A) Glucose-stimulated insulin secretion from iNS-1E cells following 30 min

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pretreatment by anthocyanin (ANC) or proanthocyanidins (PAC) from a 50% blueberry-50%

739

blackberry blend or from 100% blackberry (50%Blu-Bla or 100%Bla, respectively, sitagliptin

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(Sitag), or VitaBlue®. Values are means ± SEM, n=4-8. A # indicates significant difference (p

744

0.05). B) Relative DPP-IV expression in Caco-2 cells following treatments for 24 h. Values are

745

means ± SEM. Means with different letters are significantly different (p < 0.05).

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Figure 2. Rat adipokine array of thirty proteins detected in iNS-1E after 24 h treatment by

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anthocyanins from a 50% blueberry-50% blackberry blend or from 100% blackberry (ANC

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50%Blu-Bla or ANC 100%Bla, respectively. A) The pixel intensity of the most expressed

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proteins indicated in membranes and B) pixel intensity of proteins less highly expressed sorted

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by largest to smallest pixel intensity based on the ANC 50%Blu-Bla treatment. Different letters

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in A) indicate significant differences for each protein (p < 0.05); while in B) * indicates

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significant difference from the media-only control for each protein and # indicates significant

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differences between ANC treatment and the media-only control.

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Figure 3. Gene expression results as interpreted using Ingenuity® Pathway Analysis at FDR

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Phenolic Compounds from Fermented Berry ... - ACS Publications

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