1521-0103/354/3/279–289$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.115.225508
J Pharmacol Exp Ther 354:279–289, September 2015
Intestinal Sodium Glucose Cotransporter 1 Inhibition
Enhances Glucagon-Like Peptide-1 Secretion in
Normal and Diabetic Rodents
Takahiro Oguma, Keiko Nakayama, Chiaki Kuriyama, Yasuaki Matsushita, Kumiko Yoshida,
Kumiko Hikida, Naoyuki Obokata, Minoru Tsuda-Tsukimoto, Akira Saito, Kenji Arakawa,
Kiichiro Ueta, and Masaharu Shiotani
Research Division, Mitsubishi Tanabe Pharma Corporation, Toda-shi, Saitama, Japan
Received April 27, 2015; accepted June 22, 2015
Introduction
Long-term hyperglycemia leads to toxic effects in various
tissues and organs (Rossetti et al., 1990; Leahy et al., 1992;
Nathan, 1993), and therapeutic strategies to treat patients
with type 2 diabetes mellitus (T2DM) are currently focused on
controlling blood glucose levels (Blonde, 2010; Bennett et al.,
2012; Inzucchi et al., 2012; Qaseem et al., 2012). Many classes
of oral antihyperglycemic drugs (OADs) have been developed,
but it is still difficult to maintain long-term glycemic control in
most patients with T2DM because of the limited efficacy and
safety issues of drugs (Kahn et al., 2006; Blonde, 2010; Qaseem
et al., 2012). Recently, multiple functions of incretins, such as
glucagon-like peptide-1 (GLP-1), have been investigated. GLP-1
is secreted from enteroendocrine L cells in the distal part of
the small intestine in response to dietary stimulation (Orskov
All authors are employees of Mitsubishi Tanabe Pharma Corporation and
have not received financial support from any other institution.
T.O. and K.N. contributed equally to this work.
dx.doi.org/10.1124/jpet.115.225508.
increase in combination treatment with sitagliptin, a dipeptidyl
peptidase-4 (DPP4) inhibitor, in normoglycemic mice and rats.
CGMI, the most potent SGLT1 inhibitor among them, enhanced
glucose-induced, but not fat-induced, plasma aGLP-1 increase at a
lower dose compared with canagliflozin. Both CGMI and canagliflozin delayed intestinal glucose absorption after oral administration
in normoglycemic rats. The combined treatment of canagliflozin
and a DPP4 inhibitor increased plasma aGLP-1 levels and improved glucose tolerance compared with single treatment in
both 8- and 13-week-old Zucker diabetic fatty rats. These results
suggest that transient inhibition of intestinal SGLT1 promotes GLP-1
secretion by delaying glucose absorption and that concomitant
inhibition of intestinal SGLT1 and DPP4 is a novel therapeutic
option for glycemic control in type 2 diabetes mellitus.
et al., 1993) and exerts antidiabetic effects, such as enhancing
glucose-dependent insulin secretion and inhibiting gastric
emptying, food intake, and glucagon secretion (Drucker, 2003).
Dipeptidyl peptidase-4 (DPP4) inhibitors, a novel class of OADs,
delay enzymatic degradation and inactivation of GLP-1, thereby
increasing insulin release and decreasing glucagon secretion
in a glucose-dependent manner (Drucker, 2003; Fukuda-Tsuru
et al., 2012).
Sodium glucose cotransporter (SGLT) 2 is predominantly
located on the apical side of renal proximal tubules and plays
a critical role in glucose reabsorption from glomerular
filtrates (Kanai et al., 1994a; Hediger et al., 1995; You et al.,
1995; Abdul-Ghani et al., 2013). SGLT2 inhibitors, the newest
class of OADs, lower plasma glucose levels independent of
insulin action by inhibiting renal glucose reabsorption (Oku
et al., 1999, 2000; Arakawa et al., 2001; Ueta et al., 2005). On
the other hand, SGLT1 is highly expressed on the brushborder membrane of villus enterocytes in the proximal part of
the small intestine and is responsible for dietary glucose
absorption (Takata et al., 1992; Freeman et al., 1993; Kanai
et al., 1994b, 1995). Glucose stimulates GLP-1 secretion from
ABBREVIATIONS: aGLP-1, active glucagon-like peptide-1; AMG, a-methyl-D-glucopyranoside; AUC, area under the curve; CGMI, 3-(4cyclopropylphenylmethyl)-1-(b-D-glucopyranosyl)-4-methylindole; DPP4, dipeptidyl peptidase-4; ELISA, enzyme-linked immunosorbent assay; GI,
gastrointestinal; GLP-1, glucagon-like peptide-1; GLUT, glucose transporter; OAD, oral antihyperglycemic drug; OGTT, oral glucose tolerance test;
SD, Sprague-Dawley; SGLT, sodium glucose cotransporter; T2DM, type 2 diabetes mellitus; ZDF, Zucker diabetic fatty.
279
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ABSTRACT
The sodium glucose cotransporter (SGLT) 1 plays a major role
in glucose absorption and incretin hormone release in the
gastrointestinal tract; however, the impact of SGLT1 inhibition
on plasma glucagon-like peptide-1 (GLP-1) levels in vivo is
controversial. We analyzed the effects of SGLT1 inhibitors on
GLP-1 secretion in normoglycemic and hyperglycemic rodents
using phloridzin, CGMI [3-(4-cyclopropylphenylmethyl)-1-(b-Dglucopyranosyl)-4-methylindole], and canagliflozin. These compounds are SGLT2 inhibitors with moderate SGLT1 inhibitory
activity, and their IC50 values against rat SGLT1 and mouse
SGLT1 were 609 and 760 nM for phloridzin, 39.4 and 41.5 nM for
CGMI, and 555 and 613 nM for canagliflozin, respectively. Oral
administration of these inhibitors markedly enhanced and
prolonged the glucose-induced plasma active GLP-1 (aGLP-1)
280
Oguma et al.
Materials and Methods
Reagent and Chemicals
Canagliflozin, CGMI, sitagliptin, and voglibose were synthesized at
the Medicinal Chemistry Laboratory at Mitsubishi Tanabe Pharma
Corporation (Toda-shi, Saitama, Japan). Phloridzin was purchased
from Sigma-Aldrich (St. Louis, MO). All other chemicals were purchased from commercial sources and were of reagent or tissue-culture
grade.
Cell-Based Assays
SGLTs Inhibition Assay. Expression plasmids containing human SGLTs, rat SGLTs, and mouse SGLTs were stably transfected
into Chinese hamster ovary–K1 cells. Cells were seeded into 24-well
plates at a density of 4 105 cells/well and cultured for 2 days. The
cells were washed with assay buffer (50 mM HEPES, 20 mM Tris
base, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 137 mM NaCl at pH
7.4) and were preincubated with the compound solutions for
10 minutes. Transport reaction was initiated by adding 0.3 or 0.5 mM
AMG (a-methyl-D-glucopyranoside) (Sigma-Aldrich) in the presence
of [14C]AMG (PerkinElmer, Waltham, MA) and was incubated for
120 minutes at 37°C. Radioactive counts in cells were assessed using
a liquid scintillation counter (PerkinElmer). Protein concentration
was determined using the Coomassie Plus protein assay kit (Pierce,
Rockford, IL).
Facilitated Glucose Transporter Inhibition Assay. Glucose
transporter (GLUT) 1 activity was assessed in L6 myoblast cells
(Health Science Research Resources Bank, Osaka, Japan) (Mitsumoto
et al., 1991). Cells were seeded into 24-well plates at a density of
3 105 cells/well and cultured for 24 hours. Prior to the transport
experiment, the cells were washed with Krebs-Ringer-phosphateHEPES buffer (150 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.25 mM
CaCl2, 10 mM HEPES, and 2.9 mM Na2HPO4 at pH 7.4) and were
preincubated with the compound solution for 5 minutes. Transport
reaction was initiated by adding 2-deoxyglucose (Tokyo Chemical
Industry, Tokyo, Japan) and [3H]2-deoxyglucose (American Radiolabeled Chemicals, St. Louis, MO) and was incubated for 30 minutes
at room temperature. Incorporated radioactivity was determined
using a liquid scintillation counter (PerkinElmer). Protein concentration
was determined using the Coomassie Plus protein assay kit (Pierce).
DPP4 Inhibition Assay. Inhibitory activities of test compounds
against rat DPP4 were measured using serum collected from 7-weekold male Sprague-Dawley (SD) rats (Charles River Japan, Yokohama,
Japan). Rat serum and Gly-Pro 4-methylcoumaryl-7-amide were mixed
with the assay buffer (phosphate-buffered saline containing 0.003%
Brij-35 solution) to initiate the enzyme reaction, as described previously
(Fukuda-Tsuru et al., 2012). The fluorescence intensity of Gly-Pro
4-methylcoumaryl-7-amide was measured using a microplate reader
(Molecular Devices, Sunnyvale, CA) after 1-hour incubation at 37°C.
In Vivo Studies
Animals and Test Compound Administration. All animal
experimental procedures were approved by the Institutional Animal
Care and Use Committee of Mitsubishi Tanabe Pharma Corporation
and met the Japanese Experimental Animal Research Association
standards, as defined in the Guidelines for Animal Experiments. Male
C57BL/6J mice were purchased from CLEA Japan (Kanagawa, Japan).
Male SD rats and Zucker diabetic fatty (ZDF) rats were purchased from
Charles River Japan. During an acclimatization period of at least 1 week,
the animals were housed in a temperature- and humidity-controlled
environment with 12-hour light/dark cycles. They were provided water ad
libitum and a standard commercial diet. Test compounds for oral gavage
were prepared in 0.5% carboxymethylcellulose containing 0.2% Tween
80. The vehicle, instead of the drug, was administered in the control
groups.
Oral Glucose Tolerance Test in Mice. After overnight fasting,
9-week-old C57BL/6J mice were simultaneously treated with test
compounds and glucose solution at 2 g/kg body weight. Blood was
collected from the abdominal aorta into chilled tubes containing EDTA
(Dojindo Laboratories, Kumamoto, Japan) and a DPP4 inhibitor
(Millipore, Billerica, MA) while under anesthesia with isoflurane (Escain;
Mylan, Tokyo, Japan) before and at 5, 30, 60, 120, and 180 minutes after
administration. After centrifugation, plasma was stored at 280°C until
the measurement of plasma aGLP-1.
Oral Glucose Tolerance Test and Fat Tolerance Test in
Rats. After overnight fasting, test compounds and glucose solution
(2 g/kg p.o.) were simultaneously administered to 7- to 8-week-old SD
rats. Blood was collected from the tail vein before and at 10, 30, 60,
120, and 180 minutes after administration. Oral glucose tolerance
tests (OGTTs) in 8- or 13-week-old ZDF rats were performed using the
same protocol. In the fat tolerance test, 0.3 mg/kg of CGMI and water
or a fat emulsion, 2 g/kg Intralipos (Otsuka Pharmaceutical Factory,
Tokushima, Japan), instead of glucose solution were orally administered. Blood was collected into chilled tubes containing EDTA and
a DPP4 inhibitor, and after centrifugation, plasma was stored at 280°C
until the measurement of plasma glucose, insulin, and aGLP-1.
Sucrose Loading Test in Normal Rats. Overnight-fasted
7-week-old SD rats were orally treated with CGMI and sucrose solution (2.5 g/kg) simultaneously. Blood was collected from the tail vein at
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the gastrointestinal (GI) tract, and there is an increasing body
of evidence that SGLT1 is a sensor linking glucose to incretin
release (Moriya et al., 2009; Gorboulev et al., 2012). However,
the observation that the SGLT1 inhibitor increased portal
GLP-1 levels after intestinal glucose infusion contradicts this
hypothesis (Shibazaki et al., 2012). The relative contributions of SGLT1 in glucose-induced GLP-1 secretion remain
controversial.
Canagliflozin, a first-in-class SGLT2 inhibitor with potent
antihyperglycemic activity (Nomura et al., 2010; Liang et al.,
2012; Cefalu et al., 2013; Kuriyama et al., 2014), has modest
SGLT1 inhibitory potency compared with other highly selective SGLT2 inhibitors (Grempler et al., 2012). A recent clinical
study of canagliflozin found delayed intestinal glucose absorption accompanied by increased plasma GLP-1 levels in
healthy subjects (Polidori et al., 2013). Furthermore, the combined treatment with canagliflozin and teneligliptin, a DPP4
inhibitor, increased plasma active GLP-1 (aGLP-1) levels in
diabetic rats (Oguma et al., 2015). Because plasma GLP-1 levels
were not potentiated in SGLT2-deficient mice after a meal
challenge (Powell et al., 2013), it has been suggested that GLP-1
elevation by canagliflozin is mediated by a mechanism other
than the inhibition of SGLT2. However, the contribution of
SGLT1 inhibition to increased plasma GLP-1 levels has not been
directly determined in either animals or humans.
In the present study, we hypothesized that pharmacological
inhibition of intestinal SGLT1 increases GLP-1 secretion and,
when combined with DPP4 inhibition, the increase in plasma
aGLP-1 has a therapeutic potential for T2DM. To address
this hypothesis, we used three SGLT2 inhibitors: phloridzin,
CGMI [3-(4-cyclopropylphenylmethyl)-1-(b-D-glucopyranosyl)4-methylindole], and canagliflozin. A previous report showed
that phloridzin reduced glucose-induced GLP-1 release (Moriya
et al., 2009). CGMI is reported to have more potent inhibitory
activity against SGLT1 than canagliflozin (Nomura et al.,
2014). We explored the effects of these compounds on plasma
GLP-1 levels and intestinal carbohydrate absorption and
evaluated the combined effect of canagliflozin and sitagliptin,
a DPP4 inhibitor, in normal and diabetic rodents.
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Intestinal SGLT1 Inhibition Enhances GLP-1 Secretion In Vivo
Statistical Analysis
Data in all figures were presented as the arithmetic means 6 S.E.M.
for each group, and IC50 values in Tables 1 and 2 were expressed as the
geometric mean of two to three experiments (Fleming et al., 1972;
Stórustovu and Ebert, 2006; Kuriyama et al., 2014). The area under
the curve (AUC) and incremental AUC (DAUC, defined as the AUC
occurring above the baseline value) were calculated by the trapezoidal
rule. Statistical differences between vehicle and single treatment
groups or between single and combination treatment groups were
TABLE 1
Inhibitory effects of SGLT inhibitors on SGLT1 and SGLT2
All values are expressed as the geometric mean of two to three experiments.
IC50
Human
Rat
SGLT1
SGLT2
SGLT1
241
22.1
663
23.8
1.39
4.2
609
39.4
555
Mouse
SGLT2
SGLT1
SGLT2
760
41.5
613
65.6
6.31
5.6
nmol/l
Phloridzin
CGMI
Canagliflozina
a
Data from Kuriyama et al. (2014).
49.1
2.73
3.7
TABLE 2
Inhibitory effects of SGLT inhibitors and sitagliptin on rat GLUT and
DPP4
All values are expressed as the geometric mean of two to three experiments.
IC50
Rat GLUT
Rat DPP4
nmol/l
.10,000
.10,000
.10,000a
N.D.
Phloridzin
CGMI
Canagliflozin
Sitagliptin
.10,000
.10,000
.10,000
12.5
N.D., not determined.
a
Data from Kuriyama et al. (2014).
determined by one- or two-way analysis of variance followed by
Student’s t test (Hwee et al., 2015; Wang et al., 2015), unless otherwise
indicated in figure legends. Statistical analyses were performed using
a SAS-based system (SAS Institute, Cary, NC) or Prism software
(GraphPad Software, San Diego, CA). Differences assessed by each test
were considered statistically significant at P , 0.05.
Results
SGLT1 Inhibition and Selectivity. Tables 1 and 2
summarize the effect of the compounds used in this study on
the activities of SGLTs, GLUTs, and DPP4. Similar to
phloridzin, CGMI and canagliflozin inhibited human, rat,
and mouse SGLT1 (Table 1). These compounds also inhibited
SGLT2 activity, and CGMI was the most potent SGLT1
inhibitor among the three compounds. These SGLT inhibitors
did not inhibit GLUT1 and DPP4 activity, and sitagliptin
potently inhibited DPP4 activity (Table 2).
Effects of Phloridzin and Canagliflozin in Combination with Sitagliptin on Plasma aGLP-1 Levels in
Normal Mice and Rats. To determine whether oral treatment of phloridzin and canagliflozin affects GLP-1 secretion,
we analyzed the time course of plasma GLP-1 levels during
OGTT. For this purpose, the in vivo effects of these inhibitors
were examined in combination with sitagliptin to prevent
aGLP-1 degradation. In normoglycemic mice and rats, plasma
aGLP-1 levels were not significantly changed after oral glucose
loading. In mice treated with 10 mg/kg sitagliptin, the plasma
aGLP-1 level was increased at 5 minutes after glucose loading (Fig. 1A). In contrast, in mice treated with 500 mg/kg
phloridzin, the plasma aGLP-1 level was unchanged at
5 minutes after glucose loading, but increased at 30 minutes
and thereafter. Moreover, combined treatment of phloridzin and
sitagliptin increased and prolonged plasma aGLP-1 elevation
compared with either monotherapy at 30 minutes and later
following glucose loading. Similar results were obtained in rats
(Fig. 1, B and C).
The combined treatment of canagliflozin (0.3–30 mg/kg)
and sitagliptin (10 mg/kg) also increased plasma aGLP-1
levels in a dose-dependent manner in normoglycemic mice
and rats (Fig. 2). Monotherapy with canagliflozin increased
plasma total GLP-1 at 30 minutes after glucose loading in
normoglycemic rats (Fig. 3A), and canagliflozin did not affect
plasma DPP4 activity in combination with or without
sitagliptin at 30 minutes after the oral administration of
compounds (Fig. 3B). These data suggest that canagliflozin
does not prevent inactivation but enhances GLP-1 secretion.
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0, 30, 60, 120, and 180 minutes after administration for plasma glucose
monitoring. In another set of experiments, the rats were anesthetized
with pentobarbital sodium (Tokyo Chemical Industry) (50 mg/kg i.p.) at
1 hour after administration, and the small intestine was removed and
divided into three parts: upper small intestine (lower segment 20 cm
from the pylorus), middle small intestine (segment between upper and
lower intestine), and lower small intestine (upper segment 20 cm from
the ileocecal junction). The contents of these intestinal segments were
collected with 5 ml of ice-cold saline, and the volume of the contents was
measured. Carbohydrates in collected samples were hydrolyzed with
H2SO4 by boiling and neutralized with NaOH. Glucose contents were
then measured. For calculating sucrose dosage, the sucrose solution
was hydrolyzed, and the glucose concentration was determined in the
same way.
For evaluating the effects of canagliflozin and voglibose, the rats
were anesthetized 1 or 6 hours after administration and GI tracts
were removed and divided into five parts: upper small intestine,
middle small intestine, lower small intestine, cecum, and large
intestine. Other procedures were performed in the same way as the
experiment with CGMI. The intraluminal contents of canagliflozin in
a portion of the fluid collected from the upper small intestine were
determined by liquid chromatography–tandem mass spectrometry,
API 4000 (AB SCIEX, Framingham, MA) equipped with a Cadenza
CD C-18 column, 2.0-mm i.d. 50 mm, 3 mm (Imtakt Corporation,
Kyoto, Japan), after deproteinization with acetonitrile followed by
dilution with 0.1 mol/l ammonium acetate.
Determination of Metabolic Parameters. Glucose concentrations in plasma and GI contents were determined using a glucose
CII-test Wako kit (Wako Pure Chemical Industries, Osaka, Japan).
Insulin concentrations were measured using an ultrasensitive rat
insulin enzyme-linked immunosorbent assay (ELISA) kit (Morinaga
Institute of Biologic Science, Yokohama, Japan). Plasma active GLP-1
was determined by an ELISA kit (Millipore or Epitope Diagnostics,
San Diego, CA) after solid phase extraction with an Oasis HLB
mElution plate (Waters, Milford, MA) (Franz and Li, 2012; Kinoshita
and Kondo, 2015). Plasma total GLP-1 was determined by an ELISA
kit (Meso Scale Discovery, Rockville, MD). The baseline values are
defined as plasma GLP-1 concentrations at 0 hours in the vehicletreated group, and plasma GLP-1 relative levels were normalized and
expressed as the ratio to the baseline in the each figure. aGLP-1
baseline values ranged from 0.10 to 1.17 pmol/l in all experiments,
and the total GLP-1 baseline value was 0.62 pmol/l.
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Oguma et al.
Effects of CGMI and Canagliflozin in Combination
with Sitagliptin on Plasma aGLP-1 Levels in Normal
Rats. Subsequently, we analyzed the involvement of SGLT1
in GLP-1 secretion in detail using CGMI and canagliflozin,
which are potent and weak SGLT1 inhibitors, respectively. In
sitagliptin-treated rats, glucose loading increased plasma
aGLP-1 concentrations in a manner dependent on the dose of
CGMI (0.1–1 mg/kg) (Fig. 4A). The efficacy of 0.3 mg/kg CGMI
in increasing plasma aGLP-1 levels was almost equivalent to
that of 10 mg/kg canagliflozin (Fig. 4B). This dose ratio of
CGMI and canagliflozin was consistent with the inhibitory
activities of these compounds against SGLT1.
To further investigate whether intestinal SGLT1 inhibition
contributes to elevated plasma aGLP-1, we compared the
stimulant for GLP-1 secretion, glucose as SGLT1 substrate
and water or fat as a non-SGLT1 substrate. Oral water
loading did not increase plasma aGLP-1 levels in rats
treated with sitagliptin alone and with combination CGMI
Fig. 2. Effects of canagliflozin combined with sitagliptin on plasma GLP-1 levels during OGTT in C57BL/6J mice and SD rats. (A) Time course of plasma
aGLP-1 relative levels with sitagliptin (10 mg/kg) and canagliflozin (10 mg/kg) treatment in mice. (B and C) Time course and AUC0–2 hours of plasma
aGLP-1 relative levels with sitagliptin (10 mg/kg) and canagliflozin (0.3–30 mg/kg) treatment in rats. Canagliflozin, sitagliptin, and glucose solution (2 g/kg)
were simultaneously administered to mice and rats at time 0 by oral gavage. Blood was collected from the abdominal vein in mice or the tail vein in rats.
Data are presented as the mean 6 S.E.M. (N = 4–6). **P , 0.01 for sitagliptin compared with vehicle; ||P , 0.01 for canagliflozin compared with vehicle;
##
P , 0.01 for sitagliptin + canagliflozin compared with sitagliptin; $P , 0.05 and $$P , 0.01 for sitagliptin + canagliflozin compared with canagliflozin
group; &&P , 0.01 versus sitagliptin group.
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Fig. 1. Effects of phloridzin combined with sitagliptin on plasma aGLP-1 levels during OGTT in C57BL/6J mice and SD rats. (A) Time course of plasma
aGLP-1 relative levels with sitagliptin (10 mg/kg) and phloridzin (500 mg/kg) treatment in mice. (B and C) Time course and AUC0–3 hours of plasma
aGLP-1 relative levels with sitagliptin (10 mg/kg) and phloridzin (300 mg/kg) treatment in rats. Phloridzin, sitagliptin, and glucose solution (2 g/kg) were
simultaneously administered to 8-week-old C57BL/6J mice or 7-week-old SD rats at time 0 by oral gavage. Blood was collected from the abdominal vein
in mice and the tail vein in rats. Data are presented as the mean 6 S.E.M. (N = 5–6). *P , 0.05 and **P , 0.01 for sitagliptin compared with vehicle;
|
P , 0.05 and ||P , 0.01 for phloridzin compared with vehicle; #P , 0.05 and ##P , 0.01 for sitagliptin + phloridzin compared with sitagliptin; $P , 0.05
and $$P , 0.01 for sitagliptin + phloridzin compared with phloridzin group.
Intestinal SGLT1 Inhibition Enhances GLP-1 Secretion In Vivo
283
and sitagliptin treatment (Fig. 5, A and B). Although oral fat
loading also increased plasma aGLP-1 levels in sitagliptintreated rats, the plasma aGLP-1 increase induced by fat
was not enhanced by combined treatment with CGMI and
sitagliptin in contrast to that induced by glucose (Fig. 5, C
and D).
Fig. 4. Effects of CGMI combined with sitagliptin on plasma aGLP-1 levels during OGTT in SD rats. (A and B) Time course and AUC0–3 hours of plasma
aGLP-1 relative levels. CGMI (0.1–1 mg/kg), canagliflozin (10 mg/kg), sitagliptin (10 mg/kg), and glucose solution (2 g/kg) were administered to SD rats
at time 0 by oral gavage, and blood was collected from the tail vein. Data are presented as the mean 6 S.E.M. (N = 6). *P , 0.05 versus vehicle group;
##
P , 0.01 versus sitagliptin group; and $P , 0.05 and $$P , 0.01 versus sitagliptin group by one-way analysis of variance with Dunnett’s post hoc test.
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Fig. 3. Effects of canagliflozin on plasma total GLP-1 levels and DPP4 activity during OGTT in SD rats. (A) Plasma total GLP-1 relative levels at 0 and
30 minutes after glucose administration in canagliflozin (0.3–30 mg/kg) treated rats. (B) Plasma DPP4 activity at 0 and 30 minutes after glucose
administration in rats. Canagliflozin, sitagliptin, and glucose solution (2 g/kg) were simultaneously administered to SD rats at time 0 by oral gavage. Blood
was collected from the tail vein in rats. Data are presented as the mean 6 S.E.M. (N = 6). ++P , 0.01 versus vehicle group by one-way analysis of variance
with Dunnett’s post hoc test. In plasma DPP4 activity, there were no significant differences between the canagliflozin (1–10 mg/kg) versus vehicle group and
between the sitagliptin + canagliflozin (1–10 mg/kg) versus sitagliptin group by one-way analysis of variance with Dunnett’s post hoc test, respectively.
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Fig. 5. Effects of CGMI combined with sitagliptin on plasma aGLP-1 levels during oral glucose or fat tolerance test in SD rats. (A and B) Time course and
AUC0–3 hours of plasma aGLP-1 relative levels after glucose or water loading. (C and D) Time course and AUC0–3 hours of plasma aGLP-1 relative levels
after glucose or fat loading. CGMI (0.3 mg/kg), sitagliptin (10 mg/kg), and glucose solution (2 g/kg) were administered to SD rats at time 0 by oral gavage,
and blood was collected from the tail vein. Data are presented as the mean 6 S.E.M. (N = 6). **P , 0.01 versus vehicle-treated glucose-loading group;
##
P , 0.01 versus sitagliptin-treated glucose-loading group; $$P , 0.01 versus vehicle-treated fat-loading group.
Intestinal SGLT1 Inhibition Enhances GLP-1 Secretion In Vivo
would be 35.4 and 6.2 mmol/l at 1 and 6 hours, respectively,
after administration in the upper small intestine. Intraluminal canagliflozin concentration was estimated to be more
than 10 times higher than the IC50 (555 nmol/l) for rat SGLT1
inhibition. Therefore, it is suggested that canagliflozin and
CGMI transiently inhibited and delayed intestinal carbohydrate absorption in contrast to the sustained inhibition
induced by an a-glucosidase inhibitor.
Effect of Canagliflozin in Combination with Sitagliptin on Plasma aGLP-1 Levels during OGTT in ZDF
Rats. ZDF rats rapidly progress from normoglycemia to
frank diabetes within 12 weeks of age and are ultimately
characterized by extensive insulin resistance, hyperinsulinemia, hyperglycemia, and hyperlipidemia similar to obese
patients with T2DM (Szocs et al., 2008; Watanabe et al.,
2015). In 8-week-old ZDF rats, in which the fasting blood
glucose concentration is not increased, the plasma aGLP-1
level was increased in canagliflozin- and sitagliptin-treated
groups and was further augmented in the combined treatment group (Fig. 8, E and F). Inhibition of DPP4 by sitagliptin
increased insulin levels and reduced plasma glucose levels
induced by oral glucose (Fig. 8, A–D). Additional canagliflozin
treatment further reduced the plasma glucose excursion in
OGTT. In 13-week-old ZDF rats, in which the fasting blood
glucose level is extensively increased, sitagliptin increased
both plasma aGLP-1 and insulin levels and reduced plasma
glucose levels after glucose loading (Fig. 9). Additional treatment of canagliflozin in combination with sitagliptin treatment further increased the aGLP-1 level and extensively
reduced the increase of plasma glucose excursion. The effect of
Fig. 6. Effects of CGMI on carbohydrate
absorption in the small intestine and
plasma glucose levels in sucrose-loaded
SD rats. (A) Carbohydrate content in
upper, middle, lower, and total segment
of small intestine after sucrose loading.
(B and C) Time course and AUC0–3 hours
of plasma glucose levels after sucrose
loading. CGMI (0.3 mg/kg) and sucrose
solution (2.5 g/kg) were simultaneously
administered to SD rats by oral gavage.
Intestinal contents were collected from
the small intestine divided into three
parts. Data are presented as the mean 6
S.E.M. (N = 6). **P , 0.01 versus vehicle
group.
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Effect of CGMI, Canagliflozin, and Voglibose on
Intestinal Carbohydrate Absorption in Rats. As only
glucose-induced aGLP-1 increase was enhanced by the combination of CGMI and a DPP4 inhibitor, we then analyzed the
effects of CGMI, canagliflozin, and voglibose on intestinal
carbohydrate absorption in a sucrose loading test. Voglibose,
an a-glucosidase inhibitor, was used as a positive control to
validate the experiment of intestinal carbohydrate contents
(Bischoff, 1994; Goke et al., 1994). Fasted SD rats were orally
treated with 0.3 mg/kg CGMI, 10 mg/kg canagliflozin, or
0.1 mg/kg voglibose and sucrose (2.5 g/kg) simultaneously.
CGMI significantly increased the residual carbohydrate
contents in the middle and lower parts of the small intestine
at 1 hour after sucrose administration (Fig. 6A). Although
peak plasma glucose concentrations following sucrose loading
were suppressed, the AUC0–3 hours of plasma glucose was not
affected by CGMI treatment (Fig. 6, B and C).
Similar to CGMI, both canagliflozin and voglibose significantly increased residual carbohydrate contents in the upper
and middle parts of the small intestine at 1 hour after sucrose
loading (Fig. 7A). Voglibose increased cecal carbohydrate
contents at 6 hours after sucrose loading, indicating sustained
inhibition of carbohydrate absorption (Fig. 7B). Canagliflozin,
however, did not increase residual carbohydrate contents at
6 hours after sucrose loading. The intraluminal amount of
canagliflozin in the upper small intestine at 1 and 6 hours
after administration was 93.86 6 64.81 and 16.11 6 11.87
nmol, respectively. Considering that the water volume in the
rat whole small intestine is 11.1 g/kg body weight (McConnell
et al., 2008), the intraluminal concentration of canagliflozin
285
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Oguma et al.
combination treatment on plasma aGLP-1 and glucose levels
was greater than that of the canagliflozin single treatment.
Discussion
Although SGLT1 was the first molecule identified within
the SGLT family and its role of dietary glucose absorption has
been studied well (Kanai et al., 1994b, 1995), the role of
SGLT1 on incretin secretion in vivo is still controversial. It
has been demonstrated that SGLT1 is crucial for incretin
secretion because phloridzin blocked the increase in portal
aGLP-1 levels immediately after glucose administration into
the small intestine (Moriya et al., 2009). Furthermore, in
Sglt12/2 mice, plasma GLP-1 levels at 5 minutes after oral
glucose challenge are reduced and glucose fails to stimulate
incretin secretion from intestinal cultures in vitro (Gorboulev
et al., 2012). In these previous studies, however, GLP-1 levels
were only measured in an early phase at 5 minutes after
glucose loading, but not in delayed phases at 30 minutes and
later. In the present study, phloridzin did not suppress
plasma aGLP-1 levels at 5 minutes, but rather enhanced them
at 30 minutes and later after glucose loading in normoglycemic
mice, especially in combination with sitagliptin. Similarly,
CGMI and canagliflozin, an SGLT2 inhibitor with a moderate
inhibitory activity against SGLT1, enhanced GLP-1 secretion
and increased plasma aGLP-1 levels in combination with
sitagliptin in glucose-loaded SD rats without affecting basal
plasma aGLP-1 levels. Because plasma GLP-1 levels were not
potentiated in SGLT2-deficient mice after a meal challenge
(Powell et al., 2013), plasma aGLP-1 increase by phloridzin,
CGMI, and canagliflozin would be attributed to SGLT1
inhibition.
CGMI, a potent SGLT1 inhibitor, increased glucoseinduced, but not fat-induced, plasma aGLP-1 increase at
a lower dose compared with canagliflozin. Moreover, the
residual carbohydrate contents after oral sucrose ingestion
were increased in the middle and lower part of small intestine
and the peak plasma glucose concentration was delayed in
animals orally treated with CGMI. Total absorption of
carbohydrates was not inhibited, as the AUC of plasma glucose levels following oral sucrose challenge was not reduced
by CGMI. Therefore, the results indicate that intestinal
SGLT1 inhibition suppresses carbohydrate absorption in the
upper segment of the GI tract and increases delivery to the
lower segment. Because glucose is a representative stimulant
of GLP-1 secretion, it is likely that nonabsorbed glucose
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 7. Effects of canagliflozin on carbohydrate absorption in the GI tract in
sucrose-loaded SD rats. (A and B) Carbohydrate content in the upper small intestine, middle small intestine, lower
small intestine, cecum, large intestine,
and total segment of intestine 1 hour
(A) or 6 hours (B) after sucrose loading.
Canagliflozin (10 mg/kg) or voglibose
(0.1 mg/kg) and sucrose solution (2.5 g/kg)
were simultaneously administered to SD
rats by oral gavage. Intestinal contents
were collected from the GI tract divided
into five parts. Data are presented as the
mean 6 S.E.M. (N = 4–6). *P , 0.05 and
**P , 0.01 versus vehicle group.
Intestinal SGLT1 Inhibition Enhances GLP-1 Secretion In Vivo
287
delivered into the lower segment facilitates the release of
GLP-1 from L cells, which are abundantly located in the lower
part of the small intestine.
CGMI and canagliflozin transiently inhibited carbohydrate
absorption in the upper small intestine after oral administration. Although SGLT1 is abundantly present in the
intestine and plays a critical role in intestinal glucose
absorption (Gorboulev et al., 2012), SGLT2 is rarely expressed
in the intestine (Chen et al., 2010). Therefore, it is suggested
that the effects of these compounds are mediated mainly by
inhibition of SGLT1. A previous study (Kuriyama et al., 2014)
has shown that the plasma Cmax level of canagliflozin
following oral administration is below the IC50 value for
SGLT1; therefore, it is unlikely that canagliflozin inhibits
intestinal SGLT1 as a systemic effect. Although it is difficult
to determine the precise concentration of canagliflozin in the
upper small intestine from the remaining intraluminal
content, the estimated concentration markedly exceeded the
IC50 value for SGLT1. The concentrations of CGMI and
canagliflozin were transiently high enough to inhibit SGLT1
and delay the absorption of glucose in the upper small
intestine after oral administration. It is suggested that
transient inhibition of SGLT1 in the small intestine delayed
the absorption of glucose and the nonabsorbed glucose
delivered to the lower small intestine augmented GLP-1
secretion in the L cells.
Sustained inhibition of carbohydrate absorption in the
small intestine is accompanied by severe intestinal symptoms, as shown in the phenotype of SGLT1 deficiency
(Gorboulev et al., 2012) and adverse effects of a-glucosidase
inhibitors (Goke et al., 1994; Martin and Montgomery, 1996;
Scott and Spencer, 2000). Systemic inhibition of SGLT1
suppresses glucose absorption in the entire GI tract, and
nonabsorbed glucose delivered into the cecum and colon
facilitates microbial fermentation and production of short
chain fatty acids, which in turn activate GLP-1 secretion from
L cells (Gorboulev et al., 2012; Powell et al., 2013). In contrast
to the sustained inhibition of carbohydrate absorption in the
entire digestive tract by agents, such as the a-glucosidase
inhibitor, transient inhibition of SGLT1 only delayed glucose
absorption and enhanced GLP-1 secretion without increasing residual carbohydrate contents in the cecum and
colon. This suggests that transient and moderate SGLT1
inhibition leads to GLP-1 release without adverse effects in
digestive systems.
Because SGLT1 is abundantly expressed in the duodenum
in patients with T2DM (Freitas et al., 2008; Gerich, 2010) and
aGLP-1 increases nutrient-induced insulin release (Nauck
et al., 1997; Gromada et al., 1998), we determined the
potential of concomitant inhibition of SGLT1 and DPP4
in hyperglycemic animals. In ZDF rats, an animal model
exhibiting progressive diabetes similar to human T2DM,
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Fig. 8. Effects of canagliflozin combined with sitagliptin on glucose, insulin, and aGLP-1 levels during OGTT in 8-week-old ZDF rats. (A and B) Time
course and DAUC0–3 hours of plasma glucose levels. (C and D) Time course and DAUC0–3 hours of plasma insulin levels. (E and F) Time course and
DAUC0–3 hours of plasma aGLP-1 relative levels. Canagliflozin (10 mg/kg), sitagliptin (10 mg/kg), and glucose solution (2 g/kg) were administered to
8-week-old ZDF rats at time 0 by oral gavage, and blood was collected from the tail vein. Data are presented as the mean 6 S.E.M. (N = 6). *P , 0.05 and
**P , 0.01 versus vehicle group; ||P , 0.01 versus sitagliptin group; $$P , 0.01 versus canagliflozin group.
288
Oguma et al.
insulin resistance develops at 7 weeks of age and plasma
glucose levels increase by 13 weeks of age (Szocs et al., 2008;
Watanabe et al., 2015). Plasma aGLP-1 levels in ZDF rats
were higher after combination treatment with canagliflozin
and sitagliptin compared with monotherapy of either drug at
both 8 and 13 weeks of age. In sitagliptin-treated ZDF rats at
both early and middle stages of diabetes, canagliflozin
treatment enhanced the increase in plasma aGLP-1 levels
and further reduced plasma glucose excursion during OGTT.
These antihyperglycemic effects are likely to be mediated by
the enhancement of GLP-1 secretion and the delay of glucose
absorption because of intestinal SGLT1 inhibition in addition
to the suppression of renal glucose reabsorption by SGLT2
inhibition. Although glucose response in OGTT was markedly
suppressed, the plasma insulin response in the combined
treatment group was not significantly different from that in
the sitagliptin-treated group. It is likely that increased
plasma aGLP-1 enhances glucose-induced insulin release
in OGTT in spite of suppressed glucose levels. These results
suggest that concomitant inhibition of SGLT1 and DPP4
increases plasma aGLP-1 levels and improves glucose tolerance in hyperglycemia.
In summary, the present study demonstrates that transient
and moderate inhibition of SGLT1 in the proximal part of the
intestine increases the release of GLP-1 through increasing
glucose concentrations in the distal part of the small intestine.
Concomitant inhibition of SGLT1 and DPP4 is a novel
therapeutic option for the improvement of glucose tolerance
and hyperglycemia in T2DM.
Acknowledgments
Canagliflozin was developed by Mitsubishi Tanabe Pharma Corporation
in collaboration with Janssen Research & Development, LLC.
Authorship Contributions
Participated in research design: Oguma, Nakayama, Kuriyama,
Matsushita, Hikida, Obokata, Tsuda-Tsukimoto, Arakawa, Ueta,
Shiotani.
Conducted experiments: Oguma, Nakayama, Kuriyama, Matsushita,
Yoshida, Hikida, Obokata, Tsuda-Tsukimoto, Ueta, Shiotani.
Performed data analysis: Oguma, Nakayama, Hikida.
Wrote or contributed to the writing of the manuscript: Oguma,
Saito, Ueta.
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