METFORMIN MODULATES GLUCOSE UPTAKE AND TRANSPORT IN CACO2

METFORMIN MODULATES GLUCOSE UPTAKE AND TRANSPORT IN CACO-2 CELL MONOLAYERS Deanna Wung, Ravindra V. Alluri, Chester L. Costales, Bryan Mackowiak, Purav Bhatt, Ruth S. Everett, and Dhiren R. Thakker Division of Pharmacotherapy and Experimental Therapeutics, Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy The University of North Carolina at Chapel Hill, NC 27599, USA Methods Metformin (Figure 1) is an anti-diabetic drug widely used to treat type 2 diabetes mellitus. Caco-2 Cell Culture. Caco-2 cells were cultured at 37ºC in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 1% antibiotic-antimycotic in 5% CO 2 and 90% relative humidity. Cells were seeded at 120, 000 cells/cm 2 on 12 -well Transwell™ plates and cultured for 21 -28 days prior to transport experiments. Real-Time (RT) PCR. Total RNA was isolated from Caco-2 cells and reverse transcribed using a Superscript® III First-strand synthesis kit (Invitrogen). Human small intestinal total RNA was purchased from Zyagen. RT-PCR was conducted using Taqman assays (Applied Biosystems) to determine relative expression of GLUT 1 -3. All m. RNA levels were normalized to 18 s r. RNA. 2 -deoxy-D-Glucose (2 DG) Uptake. Caco-2 cell monolayers were preincubated for 30 min with transport buffer (Hank’s Balanced Salt Solution with 10 m. M HEPES) with or without 5 m. M metformin. To initiate uptake, 2 DG solution (10 m. M) was added to the donor Schematic of Caco-2 Transwell System compartment. After 10 minutes, cells were washed with ice cold transport buffer three times and then lysed AP with 0. 1 N Na. OH/0. 1% SDS. 2 DG BL was quantified by liquid scintillation Image courtesy of Beverly Knight spectrometry. 2 -deoxy-D-Glucose (2 DG) Transport. Caco-2 cell monolayers were preincubated for 30 min with transport buffer with or without 10 m. M metformin. To initiate transport, 2 DG solution (100 m. M) was added to the AP compartment. At the designated timepoints, 10µM samples were collected from the BL compartment, and 2 DG was quantified by liquid scintillation spectrometry. Studies show that in vivo, metformin decreases hepatic glucose production and intestinal glucose transport, and increases intestinal glucose utilization, all of which contribute to blood glucose-lowering effects of metformin in diabetics 1 -3. Increased intestinal glucose utilization is thought to contribute to metformin-induced intestinal lactic acidosis. Studies show that the highest metformin accumulation occurs in the intestine following oral administration 4, which raises the question whether high metformin intestinal concentrations modulate cell function. Since intestinal glucose absorption occurs from the apical (AP) and basolateral (BL) membranes, it warrants investigating whether high metformin concentrations affect intestinal glucose absorption and glucose transporter (GLUT) translocation to membranes of intestinal cells. Little is known about how metformin regulates GLUT transporters, although some evidence shows that metformin promotes their translocation to the AP membrane 5. Under normal physiology, GLUT 2 is known to translocate to the apical membrane of intestinal epithelia in response to a meal 6. A previous study suggests an increase in GLUT 2 translocation to the AP membrane of mouse jejunal tissue following metformin treatment due to the activation of the cellular energy sensor, AMP-activated protein kinase (Figure 2)5. Figure 2. Intestinal Glucose Absorption via Glucose Transporters 25. 0 20. 0 15. 0 10. 0 5. 0 0. 0 12. 5 m. M Glu 25 m. M Glu 50 m. M Glu Concentration-dependent transport of 2 DG suggests transporter saturation. Conclusions v Trends favoring decreased AP glucose uptake may be reflective of the inhibitory effect of metformin on energydependent transport of 2 DG concentrations not high enough to recruit GLUT 2 transporters. v Lack of statistical significance may be due to experimental conditions of high 2 DG concentration that saturated transport. 6000 5000 4000 3000 v Further studies are needed to confirm the effects of metformin using lower concentrations of 2 DG. 2000 1000 0 AP uptake AP+Met BL uptake BL+Met Absorptive Flux (µmol/min) Metformin (10 m. M) treatment was associated with a trend towards decreased AP uptake of 2 DG and its increased BL uptake. This study aims to elucidate the effect of metformin on glucose uptake and transport via GLUT transporter translocation to AP and BL membranes in Caco-2 cell monolayers, a wellestablished model of human intestinal epithelia. 30. 0 v Trends towards increased BL 2 DG uptake and increased AP to BL overall transport may be indicative of a mechanism to increase glucose utilization. Figure 3. AP and BL Uptake of 2 DG in the Presence and Absence of Metformin Average pmol/min/mg Figure 1. Metformin Figure 5. Concentration-dependent AP to BL Transport of 2 DG Papp (cm/sec) Purpose Figure 4. AP to BL Transport of 2 DG in the Presence and Absence of Metformin 120 100 80 60 40 20 0 5 m. M glucose Metformin 10 m. M glucose Metformin (10 m. M) + 5 (10 m. M) + 10 m. M glucose Conditions in BL compartment A trend towards enhanced 2 DG transport was observed with metformin (10 m. M) treatment, with 100 m. M 2 DG solution in the AP compartment. Acknowledgements Funding for the project was provided by NIH. Sponsor award # 5 -RO 1 -DK 088097 -01 -02. References 1. Hundal RS, Krssak M, Dufour S, Laurent D, Lebon V, Chandramouli V, Inzucchi SE, Schumann WC, Petersen KF, Landau BR, and Shulman GI (2000). Mechanism by Which Metformin Reduces Glucose Production in Type 2 Diabetes 49: 2063– 2069 2. Ikeda T, Iwata K, Murakami H (2000). Inhibitory effect of metformin on intestinal glucose absorption in the perfused rat intestine Gastrointestinal and Renal Pharmacology, Vol 59 (7), pp 887– 890 3. Bailey CJ, Mynett KJ, Page T (1994). Importance of the intestine as a site of metformin-stimulated glucose utilization Br J Pharmacol, 112, pp. 671– 675 4. Wilcock C, Bailey CJ (1994) Accumulation of metformin by tissues of the normal and diabetic mouse. Xenobiotica 24(1): 49 -57. 5. Walker J, Jijon HB, Diaz H, Salehi P, Churchill T, and Madsen KL (2005) 5 aminoimidazole-4 -carboxamide riboside (AICAR) enhances GLUT 2 -dependent jejunal glucose transport: a possible role for AMPK. Biochem J 385: 485 -491. 6. Kellett GL and Brot-Laroche E (2005) Apical GLUT 2: a major pathway of intestinal sugar absorption. Diabetes 54: 3056 -3062.