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Quercetin-3- O -glucoside Improves Glucose Tolerance in Rats and Decreases Intestinal Sugar Uptake in Caco-2 Cells

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Abstract

Flavonoid-rich foods intake has been associated with lower risk of non-communicable chronic diseases. Quercetin is the most abundant flavonoid in nature (fruits, vegetables, leaves and grains) as well as the most consumed flavonol. This study aims to investigate the potential effects of its conjugated form quercetin-3-O-glucoside (or isoquercetin) on glucose metabolism in rats and Caco-2 cells. To analyse the effect of quercetin-3-O-glucoside on postprandial hyperglycemia, an oral glucose tolerance test (OGTT) was conducted in Wistar rats. Additionally, Caco-2 cells were used to determine the effect of quercetin- 3-O-glucoside (30 to 60 μM) on mRNA expression of genes involved in glucose uptake by RT-PCR. Thereby, in vivo studies demonstrated that quercetin-3-Oglucoside decreased blood glucose levels evaluated by OGTT in rats. Furthermore, in the presence of Na+, quercetin-3-O-glucoside inhibited methylglucoside (MG) uptake in enterocytes and both sodium dependent glucose transporter-1 (SGLT1)- and glucose transporter-2 (GLUT2)-mediated glucose uptake were downregulated in Caco-2 cells incubated with quercetin-3-O-glucoside. In summary, our results show that quercetin-3-O-glucoside improves postprandial glycemic control in rats and reduces sugar uptake in Caco-2 cells, possible by decreasing the expression of glucose transporters (SGLT1 and GLUT2) according to the results obtained through RT-PCR.
Quercetin-3-O-glucoside Improves Glucose Tolerance in Rats and
Decreases Intestinal Sugar Uptake in Caco-2 Cells
Denys Torres-Villarreala, Alberto Camachob,c, Fermín I. Milagrod, Rocío Ortiz-Lopeze and
Ana Laura de la Garzaa,f,*
aUniversidad Autonoma de Nuevo Leon, Facultad de Salud Pública y Nutrición, Monterrey, Nuevo León, México
bUniversidad Autonoma de Nuevo Leon, Facultad de Medicina, Monterrey, Nuevo León, México
cUniversidad Autonoma de Nuevo Leon, Unidad de Neurociencias, Centro de Investigación y Desarrollo en
Ciencias de la Salud, Monterrey, Nuevo León, México
dDepartment of Nutrition, Food Science and Physiology, Centre for Nutrition Research, University of Navarra,
Pamplona, Spain
eEscuela de Medicina, Tecnológico de Monterrey, Monterrey, Nuevo León, México
fUniversidad Autonoma de Nuevo Leon, Unidad de Nutrición, Centro de Investigación y Desarrollo en Ciencias
de la Salud, Monterrey, Nuevo León, México
ana.dlgarzah@uanl.mx
Received: August 22nd, 2017; Accepted: September 11th, 2017
Flavonoid-rich foods intake has been associated with lower risk of non-communicable chronic diseases. Quercetin is the most abundant flavonoid in nature
(fruits, vegetables, leaves and grains) as well as the most consumed flavonol. This study aims to investigate the potential effects of its conjugated form
quercetin-3-O-glucoside (or isoquercetin) on glucose metabolism in rats and Caco-2 cells. To analyse the effect of quercetin-3-O-glucoside on postprandial
hyperglycemia, an oral glucose tolerance test (OGTT) was conducted in Wistar rats. Additionally, Caco-2 cells were used to determine the effect of quercetin-
3-O-glucoside (30 to 60 µM) on mRNA expression of genes involved in glucose uptake by RT-PCR. Thereby, in vivo studies demonstrated that quercetin-3-O-
glucoside decreased blood glucose levels evaluated by OGTT in rats. Furthermore, in the presence of Na+, quercetin-3-O-glucoside inhibited methylglucoside
(MG) uptake in enterocytes and both sodium dependent glucose transporter-1 (SGLT1)- and glucose transporter-2 (GLUT2)-mediated glucose uptake were
downregulated in Caco-2 cells incubated with quercetin-3-O-glucoside. In summary, our results show that quercetin-3-O-glucoside improves postprandial
glycemic control in rats and reduces sugar uptake in Caco-2 cells, possible by decreasing the expression of glucose transporters (SGLT1 and GLUT2)
according to the results obtained through RT-PCR.
Keywords: Diabetes, GLUT2, Glycosidic flavonoid, Hyperglycemia, SGLT1.
Type 2 diabetes mellitus (DM2) is a metabolic disorder
characterized by long periods of elevated glucose in blood
(hyperglycemia), which leads to insulin resistance in peripheral
tissues and/or impairments in insulin secretion from pancreatic β-
cells [1]. Insulin resistance is defined as the inability of cells to
respond adequately to normal levels of plasma insulin [1].
Therefore, high concentrations of postprandial glucose levels in
serum have been associated with an increased risk of developing
diabetes [2]. Thus, control of postprandial glycemia has been
proposed as a possible strategy to prevent the development of DM2
and other complications associated with obesity, such as
cardiovascular disease [3]. A therapeutic strategy for reducing
postprandial hyperglycemia is the partial inhibition or delay of
glucose absorption in the gut by inhibiting glucose transporters [2].
Intestinal glucose absorption occurs in two phases: the first one
involves the absorption through the enterocytes apical membrane by
the sodium-dependent glucose transporter-1 (SGLT1), which
transports glucose against a concentration gradient depending on the
period of digestion and the amount of carbohydrates ingested; and,
the second one, where glucose is transported mainly by the glucose
transporter-2 (GLUT2), located on the enterocytes basolateral
surface and, transported by facilitated diffusion from the enterocyte
into the interstitial space [4,5].
Experimental data from the last decades has proposed the use of
phenolic compounds and other plant-derived molecules for the
treatment of diabetes and other related disorders [6]. Theoretically,
these compounds offer safer alternatives than pharmaceutical
products for glycemia regulation [6, 7].
Flavonoids are compounds which their molecular structure is
composed by one or more phenolic rings, widely known by their
biological and pharmacological effects and have been used as
dietary supplements for various metabolic diseases [8, 9].
Epidemiological studies suggest that the incidence of chronic
diseases, such as obesity and diabetes, are inversely correlated with
the consumption of fruit and vegetables rich in flavonoids [9].
Quercetin is the most abundant flavonoid in nature as well as the
most frequently consumed [10]. Previous studies have shown that
some flavonoids reduce intestinal glucose absorption due to its
inhibition uptake through SGLT1 and GLUT2 transporters [11, 12].
However, the acute inhibitory effects of other related flavonoids,
such as quercetin-3-O-glucoside, on dietary glucose uptake have not
been thoroughly investigated. Thus, the aim of the current study is
to determine the effect of quercetin-3-O-glucoside on glycemic
control in rats and to analyse its action on the modulation of the
intestinal glucose uptake including the key genes SGLT1 and
GLUT2, using Caco-2 human cells.
NPC Natural Product Communications 2017
Vol. 12
No. 11
1709 - 1712
1710 Natural Product Communications Vol. 12 (11) 2017 Torres-Villarreal et al.
Effect of quercetin-3-O-glucoside on postprandial
hyperglycemia in rats. An oral glucose tolerance test (OGTT)
was used to analyse the effect of quercetin-3-O-glucoside on
postprandial hyperglycemia in Wistar rats. Oral administration of
quercetin-3-O-glucoside caused a significant reduction of blood
glucose levels at 30 and 60 minutes (p < 0.01 and p < 0.05,
respectively) when compared to the glucose group (Figure 1).
However, significant differences were not found when the area
under the curve (AUC) was analysed between groups (Figure 2).
Figure 1: Effect of quercetin-3-O-glucoside on postprandial hyperglycemia after oral
glucose administration in Wistar rats. Results are expressed as the mean + SD.
Statistical analysis was performed using ANOVA test and Dunnett’s test was used to
analyze differences in the mean of quercetin group with glucose group (n = 5/group) *
p < 0.05; ** p < 0.01.
Figure 2: Area under the curve. Effect of quercetin-3-O-glucoside on postprandial
hyperglycemia after oral glucose administration of glucose in Wistar rats. Results are
expressed as the mean + SD. Statistical analysis was performed using ANOVA test and
Dunnett’s test was used to analyze differences in the mean of quercetin group with
glucose group (n = 5/group).
Thus, although no differences were found in AUC between glucose
and quercetin groups, the OGTT showed that, thirty and sixty
minutes after glucose administration (2 g/kg bw) quercetin-3-O-
glucoside induced a decrease in glucose levels, suggesting an
antihyperglycemic effect. OGTT is a method that can be used to
measure glucose homeostasis in an in vivo model [13]. Vitor et al,.
identified that P.tridentatum, a plant used in Portuguese herbal
products, contains isoquercetin [14]. Likewise, Paulo et al.,
demonstrated that oral isoquercetin administration (100 mg/kg)
isolated from P.tridentatum, decreased blood glucose levels thirty
minutes after the glucose load [15].
Furthermore, previous studies have shown that the
antihyperglycemic effect of flavonoids may be due to the inhibition
of digestive enzymes but also because of glucose transporters
inhibition found in the intestine [16]. In this context, Caco-2 cells
have features that mimic enterocytes in the intestine and are
commonly used as a model to study the intestinal absorption of
nutrients [17]. Thus, an in vitro study with Caco-2 cells using
phlorizin, as a positive control, was conducted.
Quercetin-3-O-glucoside inhibits methylglucoside (MG) uptake
and modulates the expression of glucose transporters in Caco-2-
cells. As shown in Figure 3, quercetin-3-O-glucoside inhibited MG
uptake in Caco-2 cells in a dose-dependent manner, being
statistically significant in the cells treated with 60 µM of quercetin-
3-O-glucoside (56%). This result suggests an inhibition of SGLT1-
mediated glucose uptake by quercetin-3-O-glucoside.
Figure 3: Inhibitory effect of quercetin-3-O-glucoside on the uptake of MG in Caco-2
cells. Results are expressed as the mean + SD. Statistical analysis was performed using
ANOVA test and Dunnett’s test to analyze the differences in the mean of each group
with control group (n = 6/group). ** p < 0.01.
To investigate the molecular mechanism associated to the
antihyperglycemic effect of quercetin-3-O-glucoside, glucose
transporters SGLT1 and GLUT2 mRNA expression was measured
by RT-PCR. Our results show that GLUT2 expression levels were
statistically downregulated in the three groups of cells treated with
quercetin-3-O-glucoside (p < 0.01). On the other hand, regarding
SGLT1 gene, statistical significance was only observed in the cells
treated with 60 µM of quercetin-3-O-glucoside (p < 0.01) (Figure
4).
Figure 4: Effect of quercetin-3-O-glucoside on mRNA expression of SGLT1 and
GLUT2 in Caco-2 cells. Results are expressed as fold changes compared to
housekeeping (18S rRNA gene), and shown as mean + SD. Statistical analysis was
performed using ANOVA test and Dunnett’s test to analyze the differences in the
mean of each group with control group (normalized to 1), n=6; ** p < 0.01.
Thereby, these results suggest that quercetin-3-O-glucoside might
contribute to the inhibition of glucose uptake by decreasing the
mRNA levels of glucose transporters in Caco-2 cells. In this
context, several studies, have confirmed that some phenolic
compounds are able to interact with glucose transporters to limit
sugar uptake [18]. However, the affinity of the flavonoids for
SGLT1 glucose transporter depends on the nature and position of
the chemical groups in the phenyl ring and the monosaccharide
molecule [17]. In this sense, quercetin absorption depends on its
chemical structure and the absorption of some glycosidic forms of
quercetin, such as quercetin-3-O-glucoside, involves a
deglycosylation process in the small intestine [19]. This process
occurs to remove the sugar molecule attached and the main
Antihyperglycemic effect of isoquercetin Natural Product Communications Vol. 12 (11) 2017 1711
mechanism for quercetin deglycosylation involves hydrolysis by the
lactase phlorizin hydrolase (LPH) in the brush border of the small
intestine epithelial cells [20]. In this context, quercetin-3-O-
glucoside may impair glucose intestinal uptake by competing with
glucose for binding to the SGLT1-glucose transporter [21]. These
results are supported by different investigations suggesting that
glucose absorption may be significantly reduced by 10-100 µM
quercetin-3-O-glucoside administration in part by its interaction
with the glucose transporter GLUT2 [19,22].
In summary, our results provide evidence that dietary quercetin-3-
O-glucoside may act as an important modulator of intestinal glucose
absorption and might be useful for improving glucose tolerance
after a meal. This enables to propose quercetin-3-O-glucoside as a
potential therapeutic alternative for diabetes treatment based on its
antihyperglycemic properties.
Experimental
Experimental animals: Fifteen Wistar rats (250-300 g) were
randomly assigned into three groups (n=5) and were kept in an
isolated room at a constant temperature between 21 and 23°C,
controlled humidity (50 + 10%) and subjected to cycles of 12 hours:
12 h artificial light/darkness. Rats were fasted for 12 h with free
access to water and orally administered by gastric intubation (5
ml/kg bw) as follows: Control group: water; Glucose group: 2 g
glucose/kg bw in a 30% w/v solution; Quercetin group: 2 g
glucose/kg bw in a 30% w/v solution and 100 mg/kg bw of
quercetin-3-O-glucoside. Fasting glucose was measured before oral
gavage (0’) and after the corresponding oral administration of each
group at different time lapses (30’, 60’, 90’, 120’, 180’) from a
blood drop extracted from tail vein puncture using a glucometer and
blood glucose test strips (Accu-Chek Performa, Eypro S.A. de C.V).
Areas under the curve (AUC) were calculated according to the
following formula [23]:
AUC 0-180min = 30 × [G30 + G60 + G90 + G120 + (G0 + (G180 x 2) / 2]
Cell culture: Human colon adenocarcinoma cell line Caco-2 (HTB-
37) was obtained from the ATCC (American Type Culture
Collection). Cells were cultured in a moist environment of 95% air:
5% CO2 at a temperature of 37°C in a Dulbecco's Modified Eagle's
Medium (DMEM, Gibco Invitrogen, Paisley, UK) supplemented
with 10% fetal bovine serum (FBS), 1% of non-essential amino
acids, 1% penicilin (1000 U/mL), 1% streptomycin (100 µg/mL)
and 1% amphotericin (250 U/mL). Once cells reached an 80%
confluence, they were dissociated with 0.05% trypsin and
subcultured in a flask at a density of 2.5 × 104 cells/cm2.
Methylglucoside (MG) uptake in human Caco-2 cells: To measure
glucose uptake, Caco-2 cells were seeded at 6 × 104 cells cm
-2
density in 24-well culture plates. Culture medium was replaced
every 2 days and the experiments were performed 17−21 days post
seeding. The effect of quercetin-3-O-glucoside on the MG uptake
was evaluated at different concentrations (30, 45 and 60 µM). To
perform this, Caco-2 cells were pre-incubated 2 hours in glucose-
free DMEM. After washing with PBS, 0.5 mL of buffer containing
0.1 mM α-MG with traces of 14C MG (0.2 μCi/mL) was added to
the cells. Substrate uptake was measured in the presence and in the
absence of quercetin-3-O-glucoside (30, 45 and 60 µM) after 15
minutes incubation. MG uptake was stopped using ice-cold free-
substrate buffer followed by aspiration. The cells were washed
twice again with ice cold buffer and finally solubilized in 500 μL of
1% Triton X-100 in 0.1 N NaOH. Samples (100 μL) were taken to
measure radioactivity with a liquid scintillation [17]. MG uptake
values were corrected for protein concentration quantified by the
Bradford method [24].
RNA extraction, reverse transcription and quantitative real time
polymerase chain reaction (RT-PCR) analysis: Total RNA was
extracted from Caco-2 cells using RNeasy Mini Kit according to
manufacturer’s instructions (Qiagen, Germantown, Md., USA).
RNA concentration and quality were measured with a Nanodrop
Spectrophotometer 1000 (Thermo Scientific, Delaware, USA).
Then, RNA samples (2 µg) were reverse-transcribed to cDNA using
the Moloney Murine Leukemia Virus Reverse Transcriptase (M-
MLV RT) (Invitrogen) following the supplier’s specifications. RT-
PCR was performed according to the manufacturer´s instructions
using ABI PRISM® 7000 Sequence Detection System. The
following pre-designed TaqMan® Assays-on-Demand by Applied
Biosystems (Texas, USA) were used: SGLT1 (SLC5A1),
Hs01573790_m1; GLUT2 (SLC2A2), Hs01096908_m1. The
reference gene used to normalize the results was 18s (18S rRNA,
probe referenced as Hs99999901_s1). All samples were analyzed in
triplicate. The relative expression level of each gene was calculated
by the 2−∆∆CT method.
Statistical analysis: All the results are expressed as mean ±
standard deviation (SD) of the mean. The statistical analyses were
performed using SPSS Statistics 15.0 software (SPSS Inc., Chicago,
IL). Statistical significance of differences among the groups was
evaluated using ANOVA test followed by Dunnett’s post hoc test.
P-value < 0.05 was set as statistically significant.
Acknowledgments The authors wish to thank PRODEP
(Programa para el Desarrollo Profesional Docente) for the financial
support. Also the authors wish to acknowledge “CONACYT
(Consejo Nacional de Ciencia y Tecnología México)” for the
grant awarded to Denys Torres-Villarreal.
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