Development of a Coculture System to Evaluate the Bioactivity of Plant Extracts on Pancreatic β-Cells

Abstract

Natural plant extracts are candidates for the development of new functional foods. Most of them are usually complex mixtures of molecules of uncertain bioavailability that are often partially metabolized before they finally reach the target cells in vivo. In vitro studies of the bioactivity of these extracts suggest that their direct application to some cell cultures might be a long way from becoming a reality. To overcome this limitation, we seeded Caco-2 cells onto culture inserts and after 21 days, cocultured these with INS-1E on the base of the well. After 24 hours of coculture, TEER (transepithelium electrical resistance) measurements indicated no changes in the permeability of the Caco-2 barrier. We also found no changes in either the ability of Caco-2 cells to metabolize the flavan-3-ol component of a grape-seed procyanidin-rich extract, or in the flavanols' ability to pass through the barrier. However, the expression of the Caco-2 SGLT-1 gene increased due to the coculture. GSIS (glucose stimulated insulin secretion) was maintained in the INS-1E cells with higher levels of insulin secretion despite the fact that the insulin gene expression was unmodified by the cocultivation. Furthermore, we found that in some of the assays requiring several medium changes there was a tendency to lose β-cells. Neutral red assay showed that seeded cells should only be cocultured for a short time to obtain a higher consistency. In conclusion, four hours coculture with Caco-2 cells and INS-1E is a suitable method for checking the bioactivity of natural plant extracts of unknown bioavailability on β-cells.

Key words

coculture - pancreatic β‐cells - Caco‐2 cells - insulin secretion - plant extracts - bioavailability

References

• 1 Aggett P J, Antoine J M, Asp N G, Bellisle F, Contor L, Cummings J H, Howlett J, Müller D J, Persin C, Pijls L T, Rechkemmer G, Tuijtelaars S, Verhagen H. PASSCLAIM: Consensus on criteria.  Eur J Nutr. 2005;  44 5-30
• 2 European Union . Council directive 86/609/EEC of 24 November 1986 on the approximation of lows, regulations and administrative provisions of the member states regarding the protection of animals used for experimental and other scientific purposes.  OJL. 1986;  358 1-28
• 3 Manach C, Williamson G, Morand C, Scalbert A, Remesy C. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies.  Am J Clin Nutr. 2005;  81 230S-242S
• 4 Hackman R M, Polagruto J A, Zhu Q Y, Sun B, Fuji H, Keen C L. Flavanols: digestion, absorption and bioactivity.  Phytochem Rev. 2008;  7 195-208
• 5 Pinent M, Castell A, Baiges I, Montagut G, Arola L, Ardévol A. Bioactivity of flavonoids on insulin-secreting cell.  Compr Rev Food Sci Food Saf. 2008;  7 299-308
• 6 Kroon P A, Clifford M N, Crozier A, Day A J, Donovan J L, Manach C, Williamson G. How should we assess the effects of exposure to dietary polyphenols in vitro?.  Am J Clin Nutr. 2004;  80 15-21
• 7 Kim M, Ryu G, Kang J, Sim S, Min D, Rhie D, Yoon S, Hahn S, Jeong I, Hong K, Kim M, Jo Y. Inhibitory effects of epicatechin on interleukin-1β-induced inducible nitric oxide synthase expression in RINm5F cells and rat pancreatic islets by down-regulation of NF-κB activation.  Biochem Pharmacol. 2004;  68 1775-1785
• 8 Mackenzie G G, Carrasquedo F, Delfino J M, Keen C L, Fraga C G, Oteiza P I. Epicatechin, catechin, and dimeric procyanidins inhibit PMA-induced NF-{kappa}B activation at multiple steps in jurkat T cells.  FASEB J. 2004;  18 167-169
• 9 Vaidyanathan J B, Walle T. Cellular uptake and efflux of the tea flavonoid (−)epicatechin-3-gallate in the human intestinal cell line Caco-2.  J Pharmacol Exp Ther. 2003;  307 745-752
• 10 Baba S, Osakabe N, Natsume M, Terao J. Absorption and urinary excretion of procyanidin B2 [epicatechin-(4[beta]-8)-epicatechin] in rats.  Free Radic Biol Med. 2002;  33 142-148
• 11 Waltner-Law M E, Wang X L, Law B K, Hall R K, Nawano M, Granner D K. Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production.  J Biol Chem. 2002;  277 34933-34940
• 12 Bose M, Lambert J D, Ju J, Reuhl K R, Shapses S A, Yang C S. The major green tea polyphenol, (−)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice.  J Nutr. 2008;  138 1677-1683
• 13 Chan K Y, Zhang L, Zuo Z. Intestinal efflux transport kinetics of green tea catechins in Caco-2 monolayer model.  J Pharm Pharmacol. 2007;  59 395-400
• 14 Serra A, Macià A, Romero M, Salvadó M, Bustos M, Fernández-Larrea J, Motilva M. Determination of procyanidins and their metabolites in plasma samples by improved liquid chromatography–tandem mass spectrometry.  J Chromatogr B. 2009;  877 1169-1176
• 15 Garcia-Ramirez B, Fernandez-Larrea J, Salvado M J, Ardevol A, Arola L, Blade C. Tetramethylated dimeric procyanidins are detected in rat plasma and liver early after oral administration of synthetic oligomeric procyanidins.  J Agric Food Chem. 2006;  54 2543-2551
• 16 Kris-Etherton P M, Hu F B, Ros E, Sabate J. The role of tree nuts and peanuts in the prevention of coronary heart disease: multiple potential mechanisms.  J Nutr. 2008;  138 1746S-1751S
• 17 Sambuy Y, De Angelis I, Ranaldi G, Scarino M L, Stammati A, Zucco F. The caco-2 cell line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell functional characteristics.  Cell Biol Toxicol. 2005;  21 1-26
• 18 Tsukura Y. Effects of capsaicin on cellular damage and monolayer permeability in human intestinal Caco-2 cells.  Biol Pharm Bull. 2007;  30 1982-1986
• 19 Yang X. Establishment of Caco-2 cell monolayer model and standard operation procedure for assessing intestinal absorption of chemical components of traditional chinese medicine.  Zhong Xi Yi Jie He Xue Bao. 2007;  5 634-641
• 20 Yamashita S, Furubayashi T, Kataoka M, Sakane T, Sezaki H, Tokuda H. Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells.  Eur J Pharm Sci. 2000;  10 195-204
• 21 Hyung Choi S, Nishikawa M, Sakoda A, Sakai Y. Feasibility of a simple double-layered coculture system incorporating metabolic processes of the intestine and liver tissue: application to the analysis of benzo[a]pyrene toxicity.  Toxicol In Vitro. 2004;  18 393-402
• 22 Miyazaki J, Araki K, Yamato E, Ikegami H, Asano T, Shibasaki Y, Oka Y, Yamamura K. Establishment of a pancreatic b cell line that retains glucose inducible insulin secretion: special reference to expression of glucose transporter isoforms.  Endocrinology. 1990;  127 126-132
• 23 Merglen A, Theander S, Rubi B, Chaffard G, Wollheim C B, Maechler P. Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells.  Endocrinology. 2004;  145 667-678
• 24 Borenfreund E, Puerner J A. Toxicity determined in vitro by morphological alternations and neutral red absorption.  Toxicol Lett. 1985;  24 119-123
• 25 Suganami T, Nishida J, Ogawa Y. A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor {alpha}.  Arterioscler Thromb Vasc Biol. 2005;  25 2062-2068
• 26 Nollevaux G, Devillé C, El Moualij B, Zorzi W, Deloyer P, Schneider Y, Peulen O, Dandrifosse G. Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21).  BMC Cell Biol. 2006;  7 20
• 27 Neville H, McClenaghan P, Flatt R. Engineering cultured insulin-secreting pancreatic B-cell lines.  J Mol Med. 1999;  77 235-243
• 28 Markowska M, Oberle R, Juzwin S, Hsu C, Gryszkiewicz M, Streeter A J. Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis.  J Pharmacol Toxicol Methods. 2001;  46 51-55
• 29 Konsoula R, Barile F A. Correlation of in vitro cytotoxicity with paracellular permeability in mortal rat intestinal cells.  J Pharmacol Toxicol Methods. 2007;  55 176-183
• 30 Deprez S, Mila I, Huneau J F, Tome D, Scalbert A. Transport of proanthocyanidin dimer, trimer, and polymer across monolayers of human intestinal epithelial Caco-2 cells.  Antioxid Redox Signal. 2001;  3 957-967
• 31 Walgren R A, Lin J, Kinne R K, Walle T. Cellular uptake of dietary flavonoid quercetin 4′-beta-glucoside by sodium-dependent glucose transporter SGLT1.  J Pharmacol Exp Ther. 2000;  294 837-843
• 32 Galijatovic A, Otake Y, Walle U K, Walle T. Extensive metabolism of the flavonoid chrysin by human Caco-2 and hep G2 cells.  Xenobiotica. 1999;  29 1241-1256
• 33 Galijatovic A, Otake Y, Walle U K, Walle T. Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in caco-2 cells – potential role in carcinogen bioinactivation.  Pharm Res. 2001;  18 374-379
• 34 Tsang C, Auger C, Mullen W, Bornet A, Rouanet J, Crozier A, Teissedre P. The absorption, metabolism and excretion of flavan-3-ols and procyanidins following the ingestion of a grape seed extract by rats.  Br J Nutr. 2005;  94 170