MODULATES 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, Chapel Hill, NC 27599, USA

Purpose Methods Figure 5. Concentration-dependent AP to BL Metformin (Figure 1) is an anti-diabetic drug widely used to treat Caco-2 Cell Culture. Caco-2 cells were cultured at 37ºC in Eagle's minimum Transport of 2DG type 2 diabetes mellitus. essential medium supplemented with 10% fetal bovine serum, 1% nonessential 30.0 amino acids, and 1% antibiotic-antimycotic in 5% CO2 and 90% relative humidity. Figure 1. Metformin 2 25.0 Cells were seeded at 120,000 cells/cm 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 20.0 transcribed using a Superscript® III First-strand synthesis kit (Invitrogen). Human 15.0

small intestinal total RNA was purchased from Zyagen. RT-PCR was conducted (cm/sec) using Taqman assays (Applied Biosystems) to determine relative expression of app app 10.0 GLUT1-3. All mRNA levels were normalized to 18s rRNA. P Studies show that in vivo, metformin decreases hepatic glucose 2-deoxy-D-Glucose (2DG) Uptake. Caco-2 cell monolayers were preincubated 5.0 production and intestinal glucose transport, and increases intestinal for 30 min with transport buffer (Hank’s Balanced Salt Solution with 10 mM glucose utilization, all of which contribute to blood glucose-lowering HEPES) with or without 5 mM metformin. To initiate uptake, 2DG solution 0.0 effects of metformin in diabetics1-3. Increased intestinal glucose (10mM) was added to the donor Schematic of Caco-2 Transwell System 12.5mM Glu 25mM Glu 50mM Glu utilization is thought to contribute to metformin-induced intestinal compartment. After 10 minutes, cells were washed with ice cold transport lactic acidosis. Studies show that the highest metformin Concentration-dependent transport of 2DG suggests transporter buffer three times and then lysed AP accumulation occurs in the intestine following oral administration4, saturation. with 0.1 N NaOH/0.1% SDS. 2DG BL which raises the question whether high metformin intestinal was quantified by liquid scintillation Image courtesy of Beverly Knight concentrations modulate cell function. Since intestinal glucose spectrometry. absorption occurs from the apical (AP) and basolateral (BL) 2-deoxy-D-Glucose (2DG) Transport. Caco-2 cell monolayers were Conclusions membranes, it warrants investigating whether high metformin preincubated for 30 min with transport buffer with or without 10 mM metformin.  Trends favoring decreased AP glucose uptake may be concentrations affect intestinal glucose absorption and glucose To initiate transport, 2DG solution (100mM) was added to the AP compartment. reflective of the inhibitory effect of metformin on energy- transporter (GLUT) translocation to membranes of intestinal cells. At the designated timepoints, 10µM samples were collected from the BL dependent transport of 2DG concentrations not high compartment, and 2DG was quantified by liquid scintillation spectrometry. enough to recruit GLUT 2 transporters. Little is known about how metformin regulates GLUT transporters, although some evidence shows that metformin promotes their  Trends towards increased BL 2DG uptake and increased translocation to the AP membrane5. Under normal physiology, AP to BL overall transport may be indicative of a GLUT2 is known to translocate to the apical membrane of intestinal Figure 3. AP and BL Uptake of 2DG in the mechanism to increase glucose utilization. epithelia in response to a meal6. A previous study suggests an Presence and Absence of Metformin

increase in GLUT2 translocation to the AP membrane of mouse

6000  Lack of statistical significance may be due to experimental jejunal tissue following metformin treatment due to the activation of conditions of high 2DG concentration that saturated the cellular energy sensor, AMP-activated protein kinase (Figure 5000 transport. 2)5. 4000 /min/mg Figure 2. Intestinal Glucose Absorption via 3000  Further studies are needed to confirm the effects of Glucose Transporters pmol 2000 metformin using lower concentrations of 2DG. 1000

Average 0 Acknowledgements AP uptake AP+Met BL uptake BL+Met

Funding for the project was provided by NIH. Sponsor Metformin (10mM) treatment was associated with a trend towards decreased AP uptake of 2DG and its increased BL uptake. award # 5-RO1-DK088097-01-02.

Figure 4. AP to BL Transport of 2DG in the References Presence and Absence of Metformin

120 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). 100

/min) Mechanism by Which Metformin Reduces Glucose Production in Type 2 Diabetes 80 Diabetes 49:2063–2069 mol 60 2. Ikeda T, Iwata K, Murakami H (2000). Inhibitory effect of metformin on intestinal (µ 40 glucose absorption in the perfused rat intestine Gastrointestinal and Renal Pharmacology, Vol 59 (7), pp 887–890 20 3. Bailey CJ, Mynett KJ, Page T (1994). Importance of the intestine as a site of 0 metformin-stimulated glucose utilization Br J Pharmacol, 112, pp. 671–675 5 mM glucose Metformin 10 mM Metformin 4. Wilcock C, Bailey CJ (1994) Accumulation of metformin by tissues of the normal This study aims to elucidate the effect of metformin on glucose and diabetic mouse. Xenobiotica 24(1):49-57. Absorptive Flux Flux Absorptive (10mM) + 5 glucose (10mM) + 10 mM glucose mM glucose 5. Walker J, Jijon HB, Diaz H, Salehi P, Churchill T, and Madsen KL (2005) 5- uptake and transport via GLUT transporter translocation to AP aminoimidazole-4-carboxamide riboside (AICAR) enhances GLUT2-dependent and BL membranes in Caco-2 cell monolayers, a well- Conditions in BL compartment jejunal glucose transport: a possible role for AMPK. Biochem J 385:485-491. established model of human intestinal epithelia. A trend towards enhanced 2DG transport was observed with metformin 6. Kellett GL and Brot-Laroche E (2005) Apical GLUT2: a major pathway of intestinal sugar absorption. Diabetes 54:3056-3062. (10mM) treatment, with 100 mM 2DG solution in the AP compartment.