650

Chlorogenic acid differentially affects postprandial

glucose and glucose-dependent insulinotropic
polypeptide response in rats
Jasmine M. Tunnicliffe, Lindsay K. Eller, Raylene A. Reimer, Dustin S. Hittel, and
Jane Shearer
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 05/19/15

Abstract: Regular coffee consumption significantly lowers the risk of type 2 diabetes (T2D). Coffee contains thousands of
compounds; however, the specific component(s) responsible for this reduced risk is unknown. Chlorogenic acids (CGA)
found in brewed coffee inhibit intestinal glucose uptake in vitro. The objective of this study was to elucidate the mecha-
nisms by which CGA acts to mediate blood glucose response in vivo. Conscious, unrestrained, male Sprague–Dawley rats
were chronically catheterized and gavage-fed a standardized meal (59% carbohydrate, 25% fat, 12% protein), administered
with or without CGA (120 mg·kg–1), in a randomized crossover design separated by a 3-day washout period. Acetamino-
phen was co-administered to assess the effects of CGA on gastric emptying. The incretins glucagon-like peptide-1 (GLP-1)
and glucose-dependent insulinotropic peptide (GIP) were measured. GLP-1 response in the presence of glucose and CGA
was further examined, using the human colon cell line NCI-H716. Total area under the curve (AUC) for blood glucose was
significantly attenuated in rats fed CGA (p < 0.05). Despite this, no differences in plasma insulin or nonesterified fatty acids
were observed, and gastric emptying was not altered. Plasma GIP response was blunted in rats fed CGA, with a lower peak
concentration and AUC up to 180 min postprandially (p < 0.05). There were no changes in GLP-1 secretion in either the in
vivo or in vitro study. In conclusion, CGA treatment resulted in beneficial effects on blood glucose response, with altera-
For personal use only.

tions seen in GIP concentrations. Given the widespread consumption and availability of coffee, CGA may be a viable pre-
vention tool for T2D.
Key words: Coffee, chlorogenic acid, blood glucose, type 2 diabetes, GIP, GLP-1, incretins.
Résumé : La consommation régulière de café diminue significativement le risque de diabète de type 2 (T2D). On ne connaît
pas le constituant spécifique responsable de la diminution du risque parmi les milliers de constituants contenus dans le café.
L’acide chlorogénique (CGA) présent dans le café infusé inhibe la captation intestinale du glucose in vitro. Cette étude se
propose d’élucider le mécanisme par lequel le CGA intervient dans la réponse du glucose sanguin in vivo. On insère de fa-
çon chronique un cathéter à des rats Sprague–Dawley conscients et sans restriction puis on leur donne par gavage un repas
standard (59 % de sucres, 25 % de gras, 12 % de protéines), avec ou sans CGA (120 mg·kg–1), en se conformant à un devis
aléatoire contrebalancé; entre les traitements, on compte 3 jours d’élimination. En même temps, on administre de l’acétami-
nophène afin d’évaluer les effets du CGA sur la vidange gastrique. On évalue les concentrations des incrétines GLP-1 (pep-
tide-1 semblable au glucagon) et GIP (peptide insulinotrope dépendant du glucose). On étudie plus à fond la réponse du
GLP-1 en présence du glucose et de CGA par l’analyse de la lignée cellulaire NCI-H716. Chez les rats ayant reçu du CGA,
on observe une diminution significative de la surface totale sous la courbe (AUC) de glucose (p < 0,05). Toutefois, on n’ob-
serve aucune différence des concentrations plasmatiques d’insuline et d’acides gras libres; la vidange gastrique n’est pas mo-
difiée. La réponse plasmatique du GIP est émoussée chez les rats ayant reçu du CGA; jusqu’à 180 min après le repas, on
observe une concentration de pointe plus faible et une plus petite AUC (p < 0,05). On n’observe aucune variation de la sé-
crétion de GLP-1 dans les conditions in vivo et in vitro de l’étude. En conclusion, le traitement au CGA procure des effets
bénéfiques sur la réponse sanguine du glucose et sur les modifications des concentrations de GIP. Du fait de la consomma-
tion généralisée et de la disponibilité du café, le CGA apparaît comme un outil de prévention viable du T2D.
Mots‐clés : café, acide chlorogénique, glucose sanguin, diabète de type 2, GIP, GLP-1, incrétines.
[Traduit par la Rédaction]

Received 29 April 2011. Accepted 6 June 2011. Published at www.nrcresearchpress.com/apnm on 6 October 2011.
J.M. Tunnicliffe. Faculty of Kinesiology, University of Calgary, Calgary, AB T2N 1N4, Canada.
L.K. Eller. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB T2N 1N4,
Canada.
R.A. Reimer, D.S. Hittel, and J. Shearer. Faculty of Kinesiology, University of Calgary, Calgary, AB T2N 1N4, Canada; Department of
Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada.
Corresponding author: Jasmine Tunnicliffe (e-mail: jmtunnic@ucalgary.ca).

Appl. Physiol. Nutr. Metab. 36: 650–659 (2011) doi:10.1139/H11-072 Published by NRC Research Press
 Tunnicliffe et al.
Introduction
Type 2 diabetes (T2D) is rapidly rising in prevalence.
Characterized by either inadequate insulin production or the
inability to utilize insulin produced, T2D results in elevated
blood glucose levels (Reaven 1993). Epidemiological studies
show decaffeinated and caffeinated coffee consumption to be
correlated to reduced risk of T2D in a dose-dependent man-
ner (Higdon and Frei 2006; Salazar-Martinez et al. 2004;
Soriguer et al. 2004; Yamaji et al. 2004). A systematic re-
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 05/19/15
view by van Dam and Hu (2005) found relative risk reduc-
tions of 0.65 ± 0.12 (95% confidence interval) with ≥6 cups
per day. Despite convincing epidemiological evidence, the
specific coffee component(s) and physiological mechanisms
responsible for these anti-diabetic effects are unclear. Be-
cause caffeine, ingested either alone or as a part of coffee,
acutely impairs insulin sensitivity, a component of coffee
other than caffeine is likely responsible (Battram et al.
2006; Graham et al. 2001; Greer et al. 2001; van Dijk et al.
2009).
Chlorogenic acids (CGA) are the main antioxidants in
brewed coffee, and are found in concentrations similar to caf-
feine (Clifford 1999). Upon ingestion, small amounts of CGA
are absorbed intact from the stomach and small intestine,
with the remainder undergoing hydrolysis and subsequent ab-
sorption in the colon (Lafay et al. 2006a, 2006b; Olthof et al.
For personal use only.
2003; Renouf et al. 2010). CGA have a prolonged presence
in the intestine, with only 43% of orally administered CGA
being recovered intact from the small intestine of rats 1 h
after ingestion (Azuma et al. 2000). Consumption of glucose
together with coffee (Battram et al. 2006; Johnston et al.
2003; Shearer et al. 2007), CGA (Bassoli et al. 2008), or
CGA-enriched coffee (Thom 2007) reduces the rise in blood
glucose concentration, potentially explaining the anti-diabetic
effects of coffee. Of interest in this study, the timing and
macronutrient composition of a meal may also play a role in
the protective effects of coffee and CGA. Sartorelli and col-
leagues (2010) recently found the greatest benefits of coffee
on T2D when individuals consumed coffee at lunch, often
the largest meal of the day.
Although the exact physiological mechanisms by which
coffee and CGA lower blood glucose are not known, studies
have shown that these compounds reduce intestinal glucose
absorption and inhibit hepatic glucose output in vitro (Rodri-
guez de Sotillo and Hadley 2002; Welsch et al. 1989). There
is also evidence to suggest that CGA may alter the release of
gastrointestinal hormones that enhance insulin secretion
(Baggio and Drucker 2007; Elrick et al. 1964). Glucose-de-
pendent insulinotropic peptide (GIP) and glucagon-like pep-
tide-1 (GLP-1) 1, known as incretins, are responsible for
50%–70% of insulin response after a meal, and act in a syn-
ergistic manner (Baggio and Drucker 2007; Nauck et al.
1993). GIP is secreted by K-cells dispersed throughout the
intestine, but proximally concentrated in the jejunum and re-
leased in response to the absorption of fat, glucose, and pro-
tein (Cataland et al. 1974; Deacon 2005; Elliott et al. 1993;
Falko et al. 1975). GLP-1 is secreted from distally concen-
trated intestinal L-cells in response to the presence of carbo-
hydrates, fatty acids, essential amino acids, or fiber (Baggio
and Drucker 2007; Holst 2007). It would be expected that
the inhibition of glucose absorption in the intestine would
651
lower the rate of GIP secretion while increasing GLP-1, ow-
ing to sustained glucose presence in the intestine. Johnston
and colleagues (2003) have shown both caffeinated and non-
caffeinated coffee ingestion to reduce GIP, and decaffeinated
coffee to enhance GLP-1 response following a 25 g oral glu-
cose tolerance test in human subjects. Greenberg et al. (2009)
also found GIP to be lower after decaffeinated coffee con-
sumption. Coffee consumed prior to nutrient intake may not
produce the same effect, as neither decaffeinated coffee nor
CGA consumed 30 min before an oral glucose tolerance test
altered incretin response (Olthof et al. 2011). As such, differ-
ential incretin response has been proposed as a mechanism
by which coffee consumption lowers glucose response, and
thus T2D risk (McCarty 2005).
To gain insight into the protective effects of CGA on glu-
cose control, the objective of this study was to determine the
effects of CGA consumed with a mixed meal. Novel findings
show CGA to have no effect on the rate of gastric emptying,
but to profoundly influence GIP release. These results are in-
dependent from those of plasma insulin and fatty acid levels,
which did not change with CGA administration. Additionally,
the ability of CGA to inhibit glucose absorption, and thereby
alter GLP-1 response in vitro, was further explored using the
human colon cell line NCI-H716. GLP-1 was not found to be
affected by CGA, either in vivo or in vitro.
Materials and methods
Animals
Procedures were approved by the University of Calgary
Animal Care and Use Committee (protocol No. BI 2008-42),
and abide by the Canadian Association for Laboratory Ani-
mal Science guidelines for animal experimentation. Male
Sprague–Dawley rats, weighing 260–300 g, were housed in-
dividually in a temperature-controlled room with a 12 h light /
12 h dark cycle. Rats were given ad libitum access to drink-
ing water, and were maintained on a standard diet (5001 Lab-
oratory Rodent Diet, Purina, Richmond, Ind., USA). Energy
density of the rat chow (5001 Laboratory Rodent Diet) was
234.0 g·kg–1 of energy as protein, 45.0 g·kg–1 as fat, and
499.0 g·kg–1 as carbohydrate. Experiments were performed
on chronically catheterized, unrestrained, conscious rats. This
model allows blood to be collected with minimal stress on
the animal, as stress responses can alter insulin, glucose, and
free fatty acid concentrations (Cyr et al. 2007; Remage-
Healey and Romero 2001; Sapolsky et al. 2000).
Surgery
Rats underwent surgery to implant a catheter into the right
carotid artery, as described elsewhere (Halseth et al. 1998).
Briefly, the surgical procedure involved anesthetizing the rats
with isoflurane, making a midline incision, and isolating the
left common carotid artery. The artery was catheterised with
PE50 tubing under sterile conditions. The inserted catheter
was flushed with heparinized saline (20 U heparin·mL–1),
then exteriorized and secured at the back of the neck, where
it was stopped with a stainless steel plug. Rats were allowed
to recover for 4–5 days prior to the experiments, and weight
gain was monitored daily. Only animals who regained weight
to presurgery levels were used in the experiments.
Published by NRC Research Press
 652 Appl. Physiol. Nutr. Metab. Vol. 36, 2011

Table 1. Oral gavage composition. Ku, Osaka, Japan). Acetaminophen levels were determined
using Acetaminophen-SL Assay (Diagnostic Chemicals Lim-
Ingredient g·100 g–1 % kcal
ited, Charlottetown, P.E.I., Canada).
Corn starch 32.41 29.40
Sucrose 29.79 30.03 Cell model
Protein (casein) 13.58 12.32
The NCI-H716 cell line is an enteroendocrine L-cell line
Lard 4.85 11.00
derived from a poorly differentiated human colon adenocarci-
Soybean oil 6.79 15.40
noma; it has been shown to secrete GLP-1 in a regulated
Dyetrose 4.85 0.00
manner (Park et al. 1987). Methods were adapted from Re-
AIN-93M Mineral Mix 3.39 0.72
imer and colleagues (2001). Briefly, identical cell culture
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 05/19/15

AIN-93-VX Vitamin Mix 0.97 0.95

conditions and GLP-1 secretion study techniques were uti-
L-cystine 0.17 0.18
lized.
Choline bitartrate 0.19 0.00
Acetaminophen 3.01 0.00
Cell culture
Total 100.00 100.00
Cells were grown in suspension in RPMI 1640 medium
Note: On each experiment day, oral gavage dose was cal- (Life Technologies, Carlsbad, Calif., USA), supplemented
culated (4 g·kg–1 BW) for the meal tolerance test. On the
randomly assigned treatment day, 120 mg·kg–1 BW chloro-
with 10% fetal bovine serum (Life Technologies), 2 mmol·L–1
genic acid (CGA) was added to the gavage mixture. L-glutamine (Life Technologies), 100 IU·mL–1 penicillin
(Life Technologies), and 100 µg·mL streptomycin (Life
Experimental protocol Technologies). Media mixture was added every 3–4 days
Experiments were performed in a crossover design, with until the desired cell density was reached, then every
animals randomly assigned to initial placebo or CGA treat- 7 days for proliferation maintenance.
ment. Experiments were separated by a 3-day washout pe-
riod. Rats were fasted overnight (12 h) with ad libitum Secretion study and analysis
access to water. The arterial catheter was connected to addi- Two days prior to the experiments, 1 × 106 cells were
tional PE50 tubing to allow for blood collection, and was seeded into 12-well plates coated with Matrigel (Becton Dick-
For personal use only.

flushed with heparinized saline (10 U heparin·mL–1) between
blood draws to prevent clotting. Baseline blood was collected
∼5 min before oral gavage. On each experiment day, rats
were weighed and a meal dose (4 g·kg–1 body weight (BW))
was calculated for the meal tolerance test. The oral gavage
consisted of 59% carbohydrate, 25% fat, and 12% protein
(Table 1), with a dry food (1 g)/water ratio of 1.5 mL. Acet-
aminophen (124 mg·kg–1 BW) was added to the mixture to
track the rate of gastric emptying, as it is minimally absorbed
in the stomach and rapidly absorbed in the small intestine,
and does not require the removal of the stomach for analysis
(Hatanaka et al. 1994; Porsgaard et al. 2003). Blood was col-
lected at 15, 30, 45, 60, 90, 120, and 180 min after meal in-
gestion. For animals receiving CGA, the meal contained
120 mg·kg–1 BW CGA (Sigma–Aldrich Canada, Oakville,
Ont., Canada).
Plasma measurements
At each time point, blood glucose was measured with a
whole blood glucose monitor (OneTouch Ultra 2, Milpitas,
Calif., USA). Additional samples were collected in a chilled
EDTA-coated tube, containing 1 µL Diprotin A, a dipeptidyl
peptidase-IV inhibitor to prevent GLP-1 and GIP degradation
(Balkan et al. 1999). Immediately after collection, plasma
was separated by centrifugation and stored at –80 °C until
analysis. Analysis of insulin and total GIP was carried out
using the Rat Gut Hormone Panel LINCOplex Kit (Millipore
Corporation, Billerica, Mass., USA). Samples of plasma
(12.5 mL) were analyzed using antibody-immobilized beads
specific for these 2 hormones. Analysis of hormone concen-
tration was determined using Luminex100 (Luminex Corpora-
tion Inc., Austin, Tex.). Active GLP-1 was quantified using
the Glucagon-Like Peptide-1 (Active) ELISA kit (Millipore
Corporation). Nonesterified free fatty acids (NEFA) were
measured using HR Series NEFA-HR (2) kit (Wako, Chuo-
inson, Franklin Lakes, N.J., USA) to induce endocrine differ-
entiation for GLP-1 secretion (de Bruïne et al. 1993). Media
was simultaneously changed to low-glucose Dulbecco’s
modified Eagle’s medium (Life Technologies), supplemented
with 10% fetal bovine serum, 2 mmol·L–1 L-glutamine, 100
IU·mL–1 penicillin, and 100 µg·mL–1 streptomycin, as de-
scribed above. Cells were washed, and the differentiation
media was replaced with Krebs–Ringer bicarbonate buffer,
containing 0.2% bovine serum albumin. CGA was added to
the Krebs–Ringer bicarbonate buffer solution, and the pH
was adjusted to 7.2 with NaOH. Cells were incubated in
six 12-well plates, with treatments in duplicate, of control
(no test agents, no glucose), positive control (2% meat hy-
drosylate), and either 100 mmol·L–1 5-CGA with various
concentrations of glucose or 5% glucose with various con-
centrations of 5-CGA. After the 2 h incubation period,
supernatants and cells were collected separately. Superna-
tants were removed, and 50 mg·mL–1 anti-protease phenyl-
methylsulfonyl fluoride (Sigma), with 2 µL·mL–1 DPP-IV
inhibitor Diprotin A (Calbiochem, Gibbstown, N.J.), was
added to the collection to prevent GLP-1 degradation. Cells
were scraped with lysis buffer, containing 50 mg·mL–1
phenylmethylsulfonyl fluoride and 2 µL·mL–1 Diprotin A.
Both cells and supernatants were stored at –80 °C until
analysis. GLP-1 was measured using the GLP-1 (7–36) Ac-
tive RIA kit (Linco Research, St. Charles, Mo., USA) assay
after 10-fold dilution of both cells and supernatants.
Statistical analysis
In each of the CGA and no-CGA treatments, differences
between baseline body mass and blood glucose, GIP, GLP-1,
insulin, NEFA, and acetaminophen were calculated by sub-
tracting baseline values from the intervention values (Sigma-
Stat for Windows, version 3.5, San Jose, Calif., USA). Total
area under the curve (AUC) was calculated using the trape-

Published by NRC Research Press

 Tunnicliffe et al.
Table 2. Baseline characteristics of rats fed placebo or CGA.
Characteristic Placebo CGA
Body weight, g 266.80±4.90 264.90±4.70
Fasting NEFA, mmol·L–1 0.65±0.04 0.59±0.04
Fasting glucose, mmol·L–1 5.46±0.24 5.18±0.19
Fasting insulin, pg·mL–1 347.50±47.30 364.40±73.40
Fasting GIP, pg·mL–1 51.70±8.30 60.00±4.10
Fasting GLP-1, pmol·L–1 6.06±1.02 5.64±0.93
Note: Measurements were made following a 12 h overnight fast. All
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 05/19/15
blood samples were obtained from the arterial blood catheter. Data repre-
sent means ± SE, n = 12. There were no significant differences. GIP, glu-
cose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide-1;
NEFA, nonesterified free fatty acids.
zoidal method. Comparison of results for individual time
points and AUC were done using paired t tests. Differences
between treatments in the NCI-H716 cell line were deter-
mined with 1-way analysis of variance. Differences with p <
0.05 were considered significant. Data are expressed as
means ± SE.
Results
In vivo rat feeding study
Baseline measurements
For personal use only.
There were no significant differences in baseline blood
glucose, plasma insulin, GIP, GLP-1, or NEFA between the
CGA and placebo groups, or between the values for each of
the experiment days (Table 2).
Blood glucose
Blood glucose rapidly increased from fasting levels with
both placebo and CGA treatments following gavage, and con-
centrations were significantly higher at 15 min in both groups
(p < 0.001) (Fig. 1). However, blood glucose concentration
in rats was significantly lower at 60 min (p = 0.024) in the
CGA group than in the placebo group. Blood glucose levels
appeared to be attenuated over the course of the experiment
for rats given CGA treatment. AUC was significantly lower
in the CGA group than in the placebo group (p = 0.021) (Ta-
Table 3). Blood glucose did not return to baseline in either
CGA- or placebo-treated rats 180 min after meal gavage
(p < 0.001).
Incretins
Plasma GIP increased above baseline following gavage in
both groups, with a maximum response occurring at 60 min
in placebo-treated rats, compared with 45 min in CGA-
treated rats (Fig. 2A). Ingesting CGA in a mixed-meal gav-
age resulted in significantly reduced GIP levels at 30 min
(p = 0.036) and 60 min (p = 0.006). There was a signifi-
cantly lower GIP AUC (p = 0.029) in the CGA group over
180 min than in the placebo group (Table 3). Plasma GIP
concentrations returned to baseline after 180 min in both
groups. GLP-1 concentrations decreased shortly after meal
ingestion in both groups, then increased again between 45
and 90 min (Fig. 2C). Despite attenuated secretion of GIP
with CGA, there were no statistically significant differences
in GLP-1 between groups at any time point, or in the AUC
over 180 min (Table 3).
653
Fig. 1. Blood glucose in response to mixed-meal gavage with or
without chlorogenic acid (CGA). Results represent the mean ± SE,
n = 12. *, Significant difference between placebo and CGA at
60 min (p = 0.024).
10 CGA
Placebo
9
Blood glucose (mmol·L –1)
8 *
7
6
5
0
0 30 60 90 120 150 180
Time (min)
Insulin and NEFA
There was a rapid rise in plasma insulin in both CGA- and
placebo-fed rats following mixed-meal administration
(Fig. 2B). Insulin concentration peaked at 15 min in both
treatment groups, and dropped steadily thereafter. Although
the rise in blood glucose was attenuated by CGA, no corre-
sponding declines in plasma insulin were observed between
groups at any time point or for the AUC over 180 min (Ta-
ble 3). Although blood glucose concentrations did not return
to baseline after 180 min, plasma insulin reached a plateau
45 min after meal ingestion. NEFA concentration did not
change after oral gavage in either treatment group (Fig. 2D).
In the fasted state, NEFA concentrations are typically ele-
vated, as glucose stores are depleted. Postprandially, endoge-
nous fatty acids decrease as circulating blood glucose
increases. Despite changes in glucose, the blood levels of
NEFA we saw were not affected by CGA treatment (Table 3).
Gastric emptying
Gastric emptying was assessed with the administration and
subsequent appearance in the blood of acetaminophen.
Plasma levels of acetaminophen peaked 15–30 min after the
meal gavage, and steadily decreased thereafter in both the
CGA and placebo treatment groups (Fig. 3). Although sev-
eral factors can affect gastric emptying, including GLP-1 se-
cretion, no significant differences in the concentration of
plasma acetaminophen were observed over the 180 min of
study (p = 0.667). The lack of changes in gastric emptying
between the groups suggests that any differences observed in
the blood measures of interest can be attributed to the ab-
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 654 Appl. Physiol. Nutr. Metab. Vol. 36, 2011

Table 3. Total area under the curve (AUC) for blood glucose, plasma insulin, NEFA, GIP, GLP-1, and
acetaminophen in rats fed placebo or CGA in a mixed meal.

Placebo AUC CGA AUC p

Blood glucose, mmol·L–1 × 3 h 1360.5±25.9 1255.6±28.0 0.021
Plasma insulin, pg·mL–1 × 3 h 91 298.0±10 308.0 94 932.0±15 004.0 >0.050
Plasma NEFA, mmol·L–1 × 3 h 125.2±11.3 125.3±16.2 >0.050
Plasma GIP, pg·mL–1 × 3 h 30 510.0±3683.0 20 678.0±2694.0 0.029
Plasma GLP-1, pmol·L–1 × 3 h 1015.0±79.1 948.8±119.3 >0.050
Plasma acetaminophen, mg·mL–1 × 3 h 3400.5±429.1 3173.3±541.8 0.667
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 05/19/15

Note: Data represent mean ± SE, n = 12. Differences are considered statistically significant if p < 0.05.

Fig. 2. Plasma glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), insulin, and nonesterified free fatty acid
(NEFA) in rats fed placebo or CGA with a mixed meal. (A) Plasma GIP response; (B) plasma insulin response; (C) plasma GLP-1 response;
(D) plasma NEFA response. Results represent the mean ± SE, n = 12. *, Difference between placebo and CGA at 30 min (p = 0.036) and
60 min (p = 0.006).
400 1400 B
A
CGA CGA
*
* 1200 Placebo
Placebo
300

Plasma Insulin (pg·mL–1 )

Plasma GIP (pg·mL–1 )

1000

800
200
For personal use only.

600

400
100

200

0 0
0 30 60 90 120 150 180 0 30 60 90 120 150 180
Time (min) Time (min)

8 1.0
C D

7
Plasma NEFA (mmol·L–1)

0.8
6
Plasma GLP-1 (pmol·L–1 )

5
0.6

3 0.4

CGA
2 CGA Placebo
0.2
Placebo
1

0 0
0 30 60 90 120 150 180 0 30 60 90 120 150 180
Time (min) Time (min)

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 Tunnicliffe et al.
Fig. 3. Blood acetaminophen as a measure of gastric emptying in
response to mixed-meal gavage with or without CGA. Results repre-
sent the mean ± SE, n = 12. No significant differences between
treatments were found.
60
50
Plasma Acetaminophen (µg·mL –1)
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 05/19/15
40
30
20
10
0 lowered the glycemic index of the meal administered. A low-
0 30 60 90 120
Time (min)
For personal use only.
sence or presence of CGA, rather than to alterations in nu-
trient arrival to the gut.
NCI-H716 in vitro study
GLP-1 secretion
This experiment indirectly explored the ability of CGA to
inhibit glucose uptake by measuring GLP-1 secretion without
the confounding effect of altered GIP levels. Meat hydrosy-
late was used as a positive control, as it has previously been
shown to promote GLP-1 secretion (Reimer et al. 2001). Our
results corroborate this finding; meat hydrosylate signifi-
cantly increased GLP-1 secretion, compared with control
(p < 0.05). CGA had no effect on GLP-1 secretion, regard-
less of the concentration tested or the amount of glucose
present (Figs. 4A and B).
Discussion
Although the protective effect of coffee on the develop-
ment of T2D is well established, the mechanism of action
has not been clarified. This study examined the role of
CGA, the main polyphenol found in coffee, in altering glu-
cose concentration and incretin response. Employing a con-
scious animal model, we show that oral CGA (120 mg·kg–1)
intake in combination with a mixed meal has a negligible im-
pact on the rate of gastric emptying, attenuates postprandial
blood glucose levels with no corresponding decrease in insu-
lin response, and alters GIP but not GLP-1 secretion in rats.
Additionally, we found no alteration in GLP-1 release in
NCI-H716 colon cells incubated with CGA with or without
glucose.
To understand the mechanisms by which CGA mediates
blood glucose response, gastric emptying was evaluated. The
possibility that CGA delays the rate of gastric emptying has
655
not been considered in previous studies. In addition, data on
polyphenolic intake and gastric emptying are scarce, with one
study finding that ferulic acid, another coffee-related com-
pound, increases the rate of gastric emptying and gastrointes-
tinal transit time (Badary et al. 2006). Acetaminophen
administration allows for indirect measure of gastric empty-
CGA
ing, as it is not absorbed in the stomach but is rapidly ab-
Placebo sorbed in the small intestine (Hatanaka et al. 1994;
Porsgaard et al. 2003). Thus, novel findings from this study
show that CGA does not alter the rate of gastric emptying
when consumed with a meal.
Despite no differences in gastric emptying, we found that
acute CGA consumption lowers the postprandial rise in blood
glucose concentration, compared with placebo treatment.
Although blood glucose can be affected by several factors,
including glucose production in the liver and muscle uptake,
absorption of intact CGA is very low, with effects on muscle
and (or) liver from an acute dose likely to be insignificant. In
this experiment, the results are most likely due to a reduction
in the rate of glucose absorption from a standardized meal,
which was composed of rapidly digestible carbohydrates.
Thus, our results show that CGA may have effectively
150 180 glycemic diet is one of the recommendations to prevent or
treat T2D, as it helps prevent chronic hyperglycemia (Brand-
Miller et al. 2003; Neff 2003). It is possible that the chronic
intake of CGA lowers glucose absorption when consumed
with or close to a meal, reducing the risk of T2D. To exam-
ine the mechanism by which CGA administration lowers
blood glucose response, the incretin hormones GIP and
GLP-1 were assessed. Previous work on the impact of coffee
on these hormones in humans has shown variable results
(Greenberg et al. 2009; Johnston et al. 2003; Olthof et al.
2011). The lack of consistent findings may stem from indi-
vidual variability, a factor that was minimized with the use
of an isolated coffee component in an animal model in the
our study.
We observed a large reduction in GIP secretion with CGA
administration. Conversely, no differences were observed for
GLP-1. The reduction in GIP may be one factor that ex-
plains, in part, the attenuated blood glucose rise observed in
our study with CGA administration. Johnston et al. (2003)
also noted a decrease in GIP and blood glucose following de-
caffeinated coffee consumption by human subjects, compared
with controls, following an oral glucose tolerance test. How-
ever, a subsequent study administering decaffeinated coffee
failed to observe differential changes in glucose and insulin
levels, compared with placebo, despite a lower total GIP re-
sponse (Greenberg et al. 2009). Additionally, the results of
this study show no effect of GIP on NEFA levels following
a meal. It has been speculated that GIP plays a role in post-
prandial regulation of triglycerides via adipocytes; however,
no direct effects of GIP on NEFA concentration have been
found in humans (Asmar et al. 2010).
Results of incretin analysis demonstrated an expected de-
crease in GIP secretion, whereas no increase in GLP-1 secre-
tion with CGA administration was observed. GLP-1
production and secretion is chiefly located in distal segments
of the gut, as opposed to the more proximal production of
GIP (Baggio and Drucker 2007). Although reduced glucose
absorption in the proximal gut could result in increased glu-
Published by NRC Research Press
 656
Fig. 4. (A) Cells incubated with 10–4 mol·L–1 CGA and varying concentrations of glucose for 2 h. (B) Cells incubated with 5% glucose and
varying concentrations of CGA for 2 h. Values shown are means ± SE, expressed as a percentage of control (0 mol·L–1 CGA, 0% glucose).
MH, meat hydrosylate. *, p < 0.05 vs. other treatment groups.
400 A *
GLP-1 secretion (% control)
300
200
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 05/19/15
100
0
Control
800 B
GLP-1 secretion (% control)
700
600
500
400
300
200
For personal use only.
100
0
Control
cose presence further down in the lumen, nearly all sugars
are absorbed in the upper small intestine (Wright et al.
2003). An increase in glucose presence in the duodenum and
jejunum may, therefore, not cause a corresponding increase
in GLP-1. Additionally, GIP stimulates GLP-1 secretion (Ro-
berge and Brubaker 1993); therefore, a reduction in GIP se-
cretion following CGA treatment could counteract any
potential increase in GLP-1. To further explore the role of
GLP-1 with CGA, a cell line was employed. NCI-H716 cells
incubated with CGA showed no alteration in GLP-1 secre-
tion, even among different glucose concentrations. This is in
contrast to previous work, which showed that the addition of
3% and 10% glucose resulted in an approximately 1.5- and
2.7-fold increase, respectively, in GLP-1 secretion, compared
with controls (Jang et al. 2007). The results indicate that, at
the concentrations studied, intact CGA may be unable to alter
glucose uptake in GLP-1-secreting cells. Although Welsch et
al. (1989) found a significant decrease in glucose uptake with
10–3 mol·L–1 5-CGA, they used rat membrane vesicles, which
contain absorptive and enteroendocrine cells. Thus, it may be
that CGA affects glucose uptake in absorptive cells, rather
than the GLP-1-secreting L-cells, although we did not see
changes in GLP-1 secretion in our in vivo study.
Unexpectedly, we could not attribute differences in rat
blood glucose with CGA treatment to changes in plasma in-
sulin. No differences in insulin were observed between CGA
and placebo treatment, suggesting that CGA does not directly
alter insulin secretion. This result substantiates previous work
Appl. Physiol. Nutr. Metab. Vol. 36, 2011
MH 0% glucose 2% glucose 5% glucose 10% glucose
CGA (10 –4 mol·L–1)
*
MH 0 mol·L–1 10–5 mol·L–1 10–4 mol·L–1 10–3 mol·L–1
CGA CGA CGA CGA
5% glucose
in humans, which showed no differences in insulin AUC
after caffeinated or decaffeinated coffee consumption, despite
differences in GIP levels (Johnston et al. 2003). In contrast,
the recent finding that decaffeinated coffee intake was posi-
tively associated with acute insulin response was not sup-
ported by our results; however, that was a cross-sectional
study in which neither volume nor timing of coffee ingestion
was recorded (Loopstra-Masters et al. 2011). Insulin is se-
creted by pancreatic b-cells in response to elevated blood
glucose; incretins are thought to be responsible for 50%–70%
of the insulin response following a meal (Baggio and
Drucker 2007; Nauck et al. 1993). The relative contributions
of GIP and GLP-1 to the stimulation of insulin secretion are
debated, although it is clear that they each contribute (Elahi
et al. 1994; Fehmann et al. 1989; Siegel et al. 1992). GIP cir-
culates in higher concentrations than GLP-1, yet GLP-1 ap-
pears to be a more potent stimulator (Nauck et al. 1993;
Vilsbøll et al. 2001). In our study, GLP-1 secretions were
not altered with CGA treatment; however, GIP was signifi-
cantly reduced.
The in vivo study employed a conscious, unrestrained ani-
mal model for blood collection. This allowed for more phys-
iologically relevant data, as stress is known to alter insulin,
glucose, and free fatty acid concentrations (Cyr et al. 2007;
Remage-Healey and Romero 2001; Sapolsky et al. 2000).
Additionally, we used a mixed-meal gavage, as opposed to
an oral glucose tolerance test, as humans typically eat meals
composed of several food groups, not single nutrients, such
Published by NRC Research Press
 Tunnicliffe et al.
as glucose. Although coffee consumption may not always be
accompanied by a meal, CGA remains in the small intestine
for up to 6 h after administration (Azuma et al. 2000). A
dose comparable to that expected from usual daily coffee
consumption in humans is 500–1000 mg·day–1, or 7–
20 mg·kg–1·day–1 (Clifford 1999). However, studies looking
at CGA absorption and bioavailability in rats have used
much higher doses, from ∼25 to 285 mg·kg–1·day–1 (Azuma
et al. 2000; Dupas et al. 2006; Gonthier et al. 2003). For this
project, a median dose of 120 mg·kg–1 was used to ensure
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 05/19/15
that enough of the compound was present to have an acute
effect on glucose absorption. Despite our positive findings,
showing that a major constituent of coffee protects against
hyperglycemia, there is evidence that caffeine, caffeinated
coffee, and decaffeinated coffee impair whole-body insulin
sensitivity (Graham et al. 2001; Greenberg et al. 2009). Ad-
ditionally, results from chronic intake may differ from those
found acutely, and timing of intake may be a confounder
(Johnston et al 2003; Loopstra-Masters et al. 2011; Olthof et
al. 2011). These contradictions highlight the need for further
study in this area.
In conclusion, CGA may have a protective effect on ele-
vated blood glucose and reduce GIP secretion by slowing
the rate of glucose appearance from the intestine into the cir-
culation. Indeed, CGA may reduce the glycemic index of
foods, especially if ingested prior to or with a meal. Since
For personal use only.
coffee is the main source of CGA, this compound may ex-
plain the risk reduction for T2D seen in coffee drinkers.
Acknowledgements
The authors gratefully acknowledge the technical assis-
tance of Lin Su, Olivia T. Brusselers, and Megan C. Hallam.
Funding: J.S. holds salary support awards from the Alberta
Heritage Foundation for Medical Research, the Heart and
Stroke Foundation of Canada, and the Canadian Diabetes As-
sociation. Author disclosures: The authors declare no con-
flicts of interest. Author contributions: J.M.T. and J.S.
designed the research and wrote the paper. L.K.E. assisted
with the surgeries and biochemical analysis. R.A.R. assisted
with the incretin analysis and edited the manuscript. D.S.H.
provided technical support and manuscript editing. J.S. had
primary responsibility for the final content. All authors read
and approved the final manuscript.
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