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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, Chapel Hill, NC 27599, USA
Figure 1. Metformin
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 diabetics1-3. 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 administration4,
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 membrane5. Under normal physiology,
GLUT2 is known to translocate to the apical membrane of intestinal
epithelia in response to a meal6. A previous study suggests an
increase in GLUT2 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
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% CO2 and 90% relative humidity.
Cells were seeded at 120,000 cells/cm2 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
GLUT1-3. All mRNA levels were normalized to 18s rRNA.
2-deoxy-D-Glucose (2DG) Uptake. Caco-2 cell monolayers were preincubated
for 30 min with transport buffer (Hank’s Balanced Salt Solution with 10 mM
HEPES) with or without 5 mM metformin. To initiate uptake, 2DG solution
(10mM) 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 NaOH/0.1% SDS. 2DG
BL
was quantified by liquid scintillation
Image courtesy of Beverly Knight
spectrometry.
2-deoxy-D-Glucose (2DG) Transport. Caco-2 cell monolayers were
preincubated for 30 min with transport buffer with or without 10 mM metformin.
To initiate transport, 2DG solution (100mM) was added to the AP compartment.
At the designated timepoints, 10µM samples were collected from the BL
compartment, and 2DG was quantified by liquid scintillation spectrometry.
30.0
25.0
20.0
15.0
10.0
5.0
0.0
12.5mM Glu
25mM Glu
50mM Glu
Concentration-dependent transport of 2DG suggests transporter
saturation.
Conclusions
 Trends favoring decreased AP glucose uptake may be
reflective of the inhibitory effect of metformin on energydependent transport of 2DG concentrations not high
enough to recruit GLUT 2 transporters.
 Lack of statistical significance may be due to experimental
conditions of high 2DG concentration that saturated
transport.
6000
5000
4000
3000
 Further studies are needed to confirm the effects of
metformin using lower concentrations of 2DG.
2000
1000
0
AP uptake
AP+Met
BL uptake
BL+Met
Metformin (10mM) treatment was associated with a trend towards
decreased AP uptake of 2DG and its increased BL uptake.
Absorptive Flux (µmol/min)
Figure 4. AP to BL Transport of 2DG in the
Presence and Absence of Metformin
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.
Figure 5. Concentration-dependent AP to BL
Transport of 2DG
 Trends towards increased BL 2DG 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 2DG in the
Presence and Absence of Metformin
Average pmol/min/mg
Metformin (Figure 1) is an anti-diabetic drug widely used to treat
type 2 diabetes mellitus.
Methods
Papp (cm/sec)
Purpose
120
100
80
60
40
20
0
5 mM glucose
Metformin
10 mM
Metformin
(10mM) + 5
glucose
(10mM) + 10
mM glucose
mM glucose
Conditions in BL compartment
A trend towards enhanced 2DG transport was observed with metformin
(10mM) treatment, with 100 mM 2DG solution in the AP compartment.
Acknowledgements
Funding for the project was provided by NIH. Sponsor
award # 5-RO1-DK088097-01-02.
References
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SE, Schumann WC, Petersen KF, Landau BR, and Shulman GI (2000).
Mechanism by Which Metformin Reduces Glucose Production in Type 2 Diabetes
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) 5aminoimidazole-4-carboxamide riboside (AICAR) enhances GLUT2-dependent
jejunal glucose transport: a possible role for AMPK. Biochem J 385:485-491.
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intestinal sugar absorption. Diabetes 54:3056-3062.