BIOMEDICAL REPORTS 15: 73, 2021 Coffea arabica bean extract inhibits glucose transport and disaccharidase activity in Caco‑2 cells ATCHARAPORN ONTAWONG1, ACHARAPORN DUANGJAI1 and CHUTIMA SRIMAROENG2 1Division of Physiology, School of Medical Sciences, University of Phayao, Muang Phayao, Phayao 56000; 2Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Nong Khai 52000, Thailand Received February 3, 2021; Accepted June 14, 2021 DOI: 10.3892/br.2021.1449 Abstract. The major constituents of Coffea arabica (coffee), or sucrase‑isomaltase cleaves the α‑glucoside bonds of including caffeine, chlorogenic acid and caffeic acid, exhibit disaccharides, including sucrose, to monosaccharides. The antihyperglycemic properties in in vitro and in vivo models. absorption of dietary monosaccharides by the intestine may However, whether Coffea arabica bean extract (CBE) regu‑ be one of the risk factors associated with diabetes mellitus (1). lates glucose uptake activity and the underlying mechanisms Glucose derived from starch or sucrose is taken up by the involved remain unclear. The aim of the present study was epithelial cells through the brush border membrane (BBM), to examine the effects of CBE on glucose absorption and predominantly by a sodium‑dependent glucose co‑transporter identify the mechanisms involved using an in vitro model. (SGLT1) (2,3). A previous study has revealed that glucose The uptake of a fluorescent glucose analog into Caco‑2 transporter 2 (GLUT2) is expressed at the BBM of enterocytes colorectal adenocarcinoma cells was determined. The expres‑ during the digestive phase and may affect circulatory glucose sion levels of sodium glucose co‑transporter 1 (SGLT1) and concentration (2). Subsequently, glucose effluxes across the baso‑ glucose transporter 2 (GLUT2) were evaluated. In addition, lateral membrane (BLM) to the blood circulation via GLUT2. glycoside hydrolase enzyme activity was investigated. It Therefore, any factor that influences the activity of SGLT1 and was observed that CBE inhibited disaccharidase enzyme GLUT2 also alters the absorption and metabolism of glucose. activity. Furthermore, CBE exerted an inhibitory effect on Previous studies have reported that intestinal SGLT1 mRNA intestinal glucose absorption by downregulating SGLT1‑ and expression is increased in streptozotocin‑induced diabetic rats GLUT2‑mediated 5' AMP‑activated protein kinase phosphory‑ and mice (4,5). Additionally, Otsuka Long‑Evans Tokushima lation and suppressing hepatocyte nuclear factor 1α expression. Fatty rats exhibiting increased intestinal SGLT1 mRNA expres‑ These data suggest that CBE may attenuate glucose absorption sion combined with impaired glucose tolerance ultimately and may have potentially beneficial antihyperglycemic effects developed insulin resistance and hyperinsulinemia (6). Similarly, in the body; however, the mechanisms underlying the effects patients with type 2 diabetes mellitus also exhibit elevated intes‑ of CBE must be elucidated through further investigation. tinal glucose uptake as a result of increased SGLT1 mRNA and protein expression (7). Furthermore, GLUT2 protein expression Introduction is increased at the BBM of enterocytes in streptozotocin‑induced diabetic rats (8) and in response to elevated glucose levels in the Type 2 diabetes is a disease that occurs when blood glucose levels intestinal lumen (9). Therefore, intestinal SGLT1 and GLUT2 are high, which is referred to as hyperglycemia. The severity of have a significant impact on the progression of diabetes mellitus. hyperglycemia is determined by an imbalance between glucose Coffea arabica is one of the most widely consumed bever‑ entering and leaving the circulation. Carbohydrates from the ages. In traditional Chinese medicine, the green Coffea bean diet are hydrolyzed by digestive enzymes. α‑Glucosidase is classified as a blood circulation warmer, liver qi (Chinese concept of energy) and menstrual regulation, stimulant, heart orifice opening, detoxification and tonification herb (a traditional Chinese medicine therapeutic concept that nourishes and replen‑ ishes the qi and blood of weakened individuals) (10). At present, Correspondence to: Dr Atcharaporn Ontawong, Division of Physiology, School of Medical Sciences, University of Phayao, the Coffea species C. arabica (Arabica) and C. canephora 19 Moo 2 Mae‑Ka, Muang Phayao, Phayao 56000, Thailand (Robusta) are economically important in the global coffee market. E‑mail: [email protected] The major constituents of Coffea include caffeine, chlorogenic acid, diterpenes and trigonelline (11). Moderate intake of Coffea Abbreviations: CBE, Coffea arabica bean extract; GLUT2, has been linked to a reduced risk of chronic diseases, including glucose transporter 2; SGLT1, sodium glucose co‑transporter 1 type 2 diabetes, Parkinson's disease and liver disease (12‑14). As mentioned above, there are several studies on the antidiabetic Key words: CBE, 5' AMP‑activated protein kinase, glucose uptake, potential of roast Coffea. However, to the best of our knowledge, GLUT2, hepatocyte nuclear factor 1α, SGLT1 the effects of Coffea arabica bean extract (CBE) on glucose absorption and the related mechanisms remain unknown. The 2 ONTAWONG et al: COFFEE BEAN EXTRACT INHIBITS GLUCOSE TRANSPORT aim of the present study was to examine the effects of CBE on Glucose transport in human intestinal Caco‑2 cells. glucose absorption in human Caco‑2 colorectal adenocarcinoma Differentiated Caco‑2 cells were washed three times with PBS cells. Additionally, the molecular mechanisms involved in (pH 7.4) and incubated with 2 mM 2‑NBDG in the presence glucose transport in an in vitro model were determined. or absence of 0.1‑1,000 µg/ml CBE, a molar ratio of four major active compounds of chlorogenic acid (29.62 µg/ml), caffeine Materials and methods (5.11 µg/ml), caffeic acid (3.16 µg/ml), ferulic acid (1.54 µg/ml), phlorizin (0.5 mM) and phloretin (1 mM) for 4 h. The medium Chemicals. DMEM/F12, CelLytic MT mammalian tissue was removed and the cells were washed three times with ice‑cold lysis/extraction reagents, phloretin, chlorogenic acid, PBS. The fluorescence intensity was measured using a Synergy™ caffeine, caffeic acid and ferulic acid were purchased from HT microplate reader (BioTek Instruments, Inc.) at excitation Sigma‑Aldrich; Merck KGaA. FBS was purchased from Gibco; and emission wavelengths of 485 and 535 nm, respectively. Thermo Fisher Scientific, Inc. Polyclonal rabbit anti‑SGLT1 (1:250; cat. no. 07‑1417), GLUT2 (1:250; cat. no. 07‑1402‑I), Determination of cell viability. The viability of Caco‑2 cells 5' AMP‑activated protein kinase (AMPK; 1:250; cat. no. 07‑1417) after exposure to CBE and other test compounds was determined and phospho‑AMPK (1:250; cat. no. 07‑363) were purchased using an MTT assay. Caco‑2 cells were seeded at a density of from Merck KGaA. Monoclonal mouse alkaline phosphatase 5x104 cells/well in 96‑well plates and cultured for 18‑21 days (ALP; 1:500; cat. no. NB110‑3638) antibodies were obtained at 37˚C. On the day of the experiment, culture medium with from Novus Biologicals, LLC. Monoclonal mouse anti‑β‑actin or without test compounds was added and the cells were incu‑ (1:500; cat. no. ab8224) was purchased from Abcam. bated for 4 h at 37˚C. After exposure, the cells were washed 2‑(N‑(7‑nitrobenz‑2‑oxa‑1,3‑diazol‑4‑yl)amino)‑2‑deoxyglucose with PBS, culture medium containing MTT reagent was added (2‑NBDG) was obtained from Thermo Fisher Scientific, Inc. and the cells were incubated for the next 4 h at 37˚C. At the Phlorizin was purchased from Tocris Bioscience. All other chem‑ end of the experiment, the MTT solution was aspirated and the icals with high purity were obtained from commercial sources. cells were washed once with ice‑cold PBS. DMSO was added to each well, and cells were incubated for a further 30 min at CBE extract preparation, purification and qualification. Coffea 37˚C. The absorbance of dissolved formazan was measured at arabica beans were kindly provided by Hillkoff, and a voucher a wavelength of 570 nm using an M965 AccuReader microplate specimen (no. NU003806) was deposited at the herbarium reader (Metertech, Inc.). The sample detected at a wavelength of the Faculty of Biology, Naresuan University, Phitsanulok, of 680 nm was also used as a reference. Thailand. Dried Coffea arabica beans were weighed and blended thoroughly, followed by 100˚C water infusion for Inhibition of disaccharidase activity in Caco‑2 cells. The dose 30 min. Subsequently, the liquid extract was filtered through of CBE used in this experiment was inferred from the glucose filter paper (Whatman plc; GE Healthcare Life Sciences) transport experiment, which revealed that CBE at a dose of three times. The filtrate was evaporated using a lyophilizer 100 µg/ml reduced glucose transport to a similar extent to (Labconco). The chemical constituents of CBE, caffeine, that observed with CBE at a dose of 1,000 µg/ml. Therefore, chlorogenic acid, caffeic acid and ferulic acid (Sigma‑Aldrich; CBE at a dose of 100 µg/ml was selected for further experi‑ Merck KGaA), were used as references for validation and ments. Inhibition of sucrase enzyme activity was determined quantitation by high‑performance liquid chromatography as previously described, with some modifications (17). Briefly, (HPLC) with diode array detection on an Agilent 1200 series differentiated Caco‑2 cells were rinsed three times with Hank's chromatograph (LabX) at the Science and Technology Service balanced salt solution (HBSS) (Sigma‑Aldrich; Merck KGaA). Centre, Faculty of Science, Chiang Mai University (Chiang The cells were subsequently incubated with HBSS containing Mai, Thailand) according to the ISO/IEC 17025 method 28 mM sucrose, which is an α‑glucosidase substrate, in the pres‑ (International Organization for Standardization, 2005) (15). ence or absence of 100 µg/ml CBE, a molar ratio of four major The flow rate was 0.5 ml/min. The mobile phase was a binary active compounds of CBE at 100 µg/ml of chlorogenic acid solvent system consisting of 2% acetic acid dissolved in HPLC (29.62 µg/ml), caffeine (5.11 µg/ml), caffeic acid (3.16 µg/ml), water (solvent A) and absolute methanol (solvent B).