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Modulation of glucagon-like peptide-1 release by berberine: In vivo and in
vitro studies
Yunli Yu, Li Liu, Xinting Wang, Xiang Liu, Xiaodong Liu *, Lin Xie, Guangji Wang
Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, PR China
1. Introduction
Glucagon-like peptide-1 (GLP-1) is a gut-derived hormone
secreted from intestinal L cells in response to nutrient ingestion. It
is produced by a tissue-specific post-translational process of its
precursor proglucagon peptide by the prohormone convertase 3
[1]. GLP-1 exerts important effects on regulating glucose metabo-
lism, stimulating glucose-dependent insulin secretion, promoting
beta-cell proliferation, as well as inhibiting glucagon release,
gastric emptying and food intake [2]. The physiological properties
which GLP-1 possesses make it a subject of intensive investigation
as a potential treatment of diabetes mellitus.
Berberine ([C
20
H
18
NO
4
]
+
), a major active constituent of
Rhizoma coptidis, has been successfully used for treating diabetes
[3]. It was reported that berberine may stimulate glucose uptake
[4], modulate lipids metabolism and scavenge free radical [5],
enhance insulin sensitivity and stimulate insulin secretion [6].
However, its hypoglycemic mechanism remains unclear because of
its poor absorption and low concentration in plasma. Recently, we
reported that complex of Rhizoma coptidis Huang-Lian-Jie-Du-
Tang [7] and its bioactive compound berberine [8] may increase
GLP-1(7–36) amide secretion in STZ-induced diabetic rats,
accompanied by increase of insulin levels, which indicated that
enhancement of GLP-1 release may contribute to beneficial effects
of berberine on diabetes mellitus.
The NCI-H716, an enteroendocrine cell line, was derived from
cells present in ascites fluid of a 33-year-old Caucasian male with
poorly differentiated adenocarcinoma of the colon after treatment
with 5-fluorouracil [9]. The cell line was described to display some
endocrine feature [10,11], including secretory granules and
chromogranin A. Several neurohormonal receptors such as
muscarinic receptors [12], leptin receptors [13] and G
a
s-coupled
receptor [14] were identified on the cell line. The NCI-H716 cell
Biochemical Pharmacology 79 (2010) 1000–1006
ARTICLE INFO
Article history:
Received 3 October 2009
Accepted 20 November 2009
Keywords:
Berberine
Glucagon-like peptide-1
Prohormone convertase 3
Proglucagon
Protein kinase C-dependent pathway
ABSTRACTS
Glucagon-like peptide (GLP)-1 is a potent glucose-dependent insulinotropic gut hormone released
from intestinal L cells. Our previous studies showed that berberine increased GLP-1 secretion in
streptozotocin-induced diabetic rats. The aim of this study was to investigate whether berberine
affected GLP-1 release in normal rats and in NCI-H716 cells. Proglucagon and prohormone convertase 3
genes regulating GLP-1 biosynthesis were analyzed by RT-PCR. Effects of pharmac ological inhibitors on
berberine-mediated GLP-1 release were studied. In vivo, 5-week treatment of be rberine enhanced GLP-
1 secretion induced by glucose load and promoted proglucagon mRNA expression as well as L cell
proliferation in intestine. In vitro, berberine concentration-dependently stimulated GLP-1 release in
NCI-H716 cells. Berberine also promoted both prohormone convertase 3 and proglucagon mRNA
expression. Chelerythrine (inhibitor of PKC) concentration-dependently suppressed berberine-
mediated GLP-1 secretion. Compound C (inhibitor of AMPK) also inhibited berberine-mediated
GLP-1 secretion. But only low concentrations of H89 (inhibitor of PKA) showed inhibitory effects on
berberine-mediated GLP-1 release. The present results demonstrated that berberine showed its
modulation on GLP-1 via promoting GLP-1 secretion and GLP-1 biosynthesis. Some signal pathways
including PKC-dependent pathway were involved in this process. Elucidation of mechanisms
controlling berberine-mediated GLP-1 secretion may facilitate the understanding of berberine’s
antidiabetic effects.
ß2009 Elsevier Inc. All rights reserved.
Abbreviations: GLP-1, glucagon-like peptide 1; PKC, protein kinase C; PKA, protein
kinase A; AMPK, AMP-activated protein kinase; KRB, Krebs–Ringer bicarbonate
buffer; FBG, fasted blood glucose; DPP-IV, dipeptidyl peptidase-IV; Compound C, 6-
[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine;
H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihy-
drochloride; ANOVA, analysis of variance.
* Corresponding author at: Key Laboratory of Drug Metabolism and Pharmaco-
kinetics, China Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing 210009, PR
China. Tel.: +86 25 83271006; fax: +86 25 85306750.
E-mail addresses: chrisyu1255@hotmail.com (Y. Yu), shmily9989@hotmail.com
(L. Liu), wxinting1986@yahoo.com.cn (X. Wang), liuxiang0715@yahoo.com.cn
(X. Liu), xdliu@cpu.edu.cn (X. Liu), jsxielin@sina.com.cn (L. Xie),
guangjiwang@yahoo.com.cn (G. Wang).
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
0006-2952/$ – see front matter ß2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2009.11.017
line highly expressed GLP-1 and was widely used as a unique
human model to study the regulation of GLP-1 secretion [15]. GLP-
1 secretion from NCI-H716 cells is regulated by multiple
extracellular secretagogues through some signal pathways [16].
The aim of this study was to investigate: (1) whether berberine
treatment also increased GLP-1 secretion in normal rat; (2)
berberine-mediated GLP-1 secretion in NCI-H716 cells; and (3)
some intracellular signaling pathways involved in regulation of
GLP-1 release induced by berberine. It is expected to obtain an
improved understanding of the mechanism regulating GLP-1
secretion by berberine using NCI-H716 cells, thereby providing an
alternative prospect to the use of berberine in the treatment of
diabetes mellitus.
2. Materials and methods
2.1. Materials
Berberine (purity: 98%) was purchased from Nanjing Qingze
Pharmaceutical Technology Ltd. CO. (Nanjing, China). Pentobarbi-
tal was bought from Sigma Chemical Co. (St. Louis, MO, USA). Both
DPP-IV inhibitor and GLP-1 active ELISA kit were purchased from
Linco Research (St. Charles, MI, USA). Primers for proglucagon
gene, PC 3 gene and DNA Taq polymerase used in RT-PCR were
provided by Xinlun Biotechnical Laboratory (Shanghai, China).
Rabbit anti-GLP-1 antibody was purchased from Phoenix Phar-
maceuticals (Mountain View, CA, USA). Primary antibody of insulin
was obtained from Maixin Biotechnical Company (Fuzhou, China).
H89 was purchased from Beyotime Institute of Biotechnology
(Shanghai, China). Chelerythrine and Compound C were purchased
from Sigma Chemical Co. (St. Louis, MO, USA). Bovine serum alume
(BSA) was obtained from Amresco Co. (Solon, OH, USA). Both RPMI
1640 and DMEM were purchased from Invitrogen Co. (Carlsbad,
CA, USA). Matrigel was bought from Becton Dickinson Biosciences
(Bedford, MA, USA). Fetal bovine serum (FBS) was bought from PAA
Laboratory (Chicago, IL, USA).
2.2. Animals
Male Sprague–Dawley rats (180–220 g), purchased from B&K
Universal Group Ltd. (Shanghai, China), were used in the study and
housed under controlled room of humidity (50
5%) and temper-
ature (23 18C) with a 12 h light/12 h darkness cycle. They were fed
commercial stock diet and water ad libitum. The studies were
approved by the Animal Ethics Committee of China Pharmaceutical
University.
2.3. Berberine treatment and sample collection
Rats were acclimated for 3 days before the experiment and
divided into three groups randomly. One group served as control
group, received only vehicle. The other two groups served as
berberine-treatedgroups, which orallyreceived low dose(60 mg/kg)
or high dose(120 mg/kg) of berberine once a day for 5 weeks. During
the treatment, fasted blood glucose (FBG) was monitored once a
week, both body weight and food intake was monitored once a day.
On day 35 of the treatment, rats were fasted for 12 h. Two hours
after last treatment, portal vein cannulation was performed
according to the method described previously [17] under sodium
pentobarbital (60 mg/kg, i.p.) anesthesia. Then, the rats orally
received 2.5 g/kg of glucose. Portal blood (300
m
L) was collected
in tube containing EDTA and DPP-IV inhibitor (10
m
l/ml) via the
cannula before and subsequently at 10, 20 and 30 min following
glucose load. Plasma samples were obtained and stored at 80 8Cfor
assessing GLP-1(7–36) amide and insulin. Then the rats were
immediately sacrificed, the pancreas, segments of distal ileum (4 cm
above the junction with the caecum) and proximal colon (4 cm of
intestine below the junction with the caecum) were obtained and
immersed in liquid nitrogen, and stored at 80 8C for peptide
analysis. Parts of tissues were used for immunohistochemistry and
RT-PCR analysis. GLP-1 and insulin measurement, immunohis-
tochemistry and RT-PCRwere performed as described previously [7].
2.4. Cell culture
Human NCI-H716 cells were obtained from the American Type
Culture Collection (Manassas, USA). Cells were grown in suspen-
sion at 37 8C, 5% CO
2
. The culture medium was RPMI 1640
supplemented with 10% FBS, 2 mM
L
-glutamine, 100 IU/ml
penicillin and 100
m
g/ml streptomycin. Endocrine differentiation
was induced by seeding cells in dishes coated with Matrigel, in
high-glucose DMEM, 10% FBS, 2 mM
L
-glutamine, 100 IU/ml
penicillin, and 100
m
g/ml streptomycin [15].
2.5. Effect of berberine on secretion of GLP-1 from NCI-H716 cells
Two days before the experiments, 1.5 10
6
cells were seeded in
12-well culture plates coated with Matrigel and containing high-
glucose DMEM, 10% FBS, 2 mM
L
-glutamine, 100 IU/ml penicillin
and 100
m
g/ml streptomycin. On the day of the experiment,
medium was replaced by Krebs–Ringer bicarbonate buffer (KRB)
buffer (128.8 mmol/l NaCl, 4.8 mmol/l KCl, 1.2 mmol/l KH
2
PO
4
,
1.2 mmol/l MgSO
4
, 2.5 mmol/l CaCl
2
, 5 mmol/l NaHCO
3
, and
10 mmol/l HEPES, pH 7.4) [18] containing 0.2% BSA and different
concentrations of berberine (0
m
M, 1
m
M, 10
m
M or 100
m
M).
Following incubating at 37 8C for 2 h, the supernatants were
collected with the addition of 50
m
g/ml phenylmethylsulfonyl
fluoride and stored at 80 8C for analysis. The cells were scraped
off and sonicated in a homogenization buffer (1 M HCl containing
5% formic acid, 1% trifluoroacetic acid and 1% NaCl). GLP-1(7–36)
amide in supernatant and cells was measured by a GLP-1 active
ELISA kit, respectively, according to the manufacturer’s protocol.
Protein content of the cells was determined using the Bradford
protein assay. The GLP-1 content was normalized for the total
protein of the cells. At the same time, the cell viability was
measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay [19]. No damage in cells was found
for all the tested agents within tested concentrations.
2.6. RT-PCR analysis of proglucagon mRNA and prohormone
convertase 3 mRNA
A total of 3 10
6
cells were seeded in 6-well culture plates
coated with Matrigel and incubated for 24 h. Medium was replaced
by serum-free medium containing 0.2% BSA without or with
100
m
M berberine. The cells were re-incubated for 24 h and were
washed with cold-PBS and stored at 80 8C for RT-PCR analysis.
Total RNA was isolated from each well with Trizol reagent
(Invitrogen Co., USA). RT-PCR was performed according to the
protocol provided with the TwoStep RT-PCR kit (BestBio, Shanghai,
China). The sequences of the forward and reverse primers were: 5
0
-
GTAATGCTGGTACAAGGCAG-3
0
and 5
0
-TTATAAAGTCCCTGGCGGCA-
3
0
for the proglucagon gene, 5
0
-CGCTGACCTGCACAATGACT-3
0
and
5
0
-CAGACAACCAGGTGCTGCAT-3
0
for prohormone convertase 3
gene, 5
0
-GCTGAGAACGGGAAGCTTGT-3
0
and 5
0
-TCTCCATGGTGGT-
GAAGACG-3
0
for internal control GAPDH gene. After denaturing at
94 8C for 5 min, the amplification was obtained by 33 cycles of 94 8C
for 30 s, 59 8C for 30 s and 72 8C for 30 s each. A final extension stepat
72 8C for 5 min was performed. 27 cycles were performed for the
amplification for GAPDH gene. PCR products were subjected to
electrophoresis on 2.5% agarose gel, and visualized by means of
ethidium bromide staining. Densitometric quantification was
Y. Yu et al. / Biochemical Pharmacology 79 (2010) 1000–1006
1001
recorded usingJeda image analysis system 3.3(Jiangsu Jeda Science-
Technology Co. Ltd., Nanjing, China).
2.7. Effect of pharmacological inhibitors on berberine-mediated GLP-1
release
Differentiated cells were starved in FBS-free DMEM containing
0.2% BSA for 1.5 h, then incubated with KRB containing 0.2% BSA
and 100
m
M berberine co-administrated with pharmacological
inhibitors chelerythrine (0.4
m
M, 2
m
M and 10
m
M), H89 (2
m
M,
10
m
M and 50
m
M) or Compound C (2
m
M, 10
m
M and 50
m
M) for
2 h. GLP-1(7–36) amide in supernatant and cells was measured as
described above. Effects of the pharmacological inhibitors (2
m
M
chelerythrine, 10
m
M H89 and 10
m
M Compound C) on GLP-1
secretion were also investigated.
2.8. Statistical analysis
Results were expressed as mean
standard error (S.E.M.).
Statistical differences among groups were evaluated by one-way of
analysis of variance (ANOVA). If analysis was significant, the
differences between groups were estimated using Student–New-
man–Keuls multiple comparison post hoc test. A pvalue of less than
0.05 indicated a significant difference.
3. Results
3.1. Effects of berberine treatment on body weight, food intake and
blood glucose concentration of normal rats
Five-week berberine treatment did not affect body weight (data
not shown), but may decrease FBG levels (Fig. 1A) and food intake
(Fig. 1B) of normal rats, significant decreases were found in rats
treated with high dose of berberine (120 mg/kg).
3.2. Effects of berberine treatment on insulin level
Plasma insulin levels were measured following glucose load
(Fig. 2A). It was found that glucose load may induce increase of
insulin level in plasma, the peak concentration occurred at 10 min.
High dose of berberine showed a trend to increase plasma insulin
level, but no statistical difference was found.
Fig. 1. Effects of berberine treatment on fasted blood glucose concentration (A) and food intake (B) during the 5 weeks of treatment. Rats received vehicle (^, CT) and
berberine at 60 mg/kg (&, BL) and 120 mg/kg (~, BH), respectively. Values are expressed as mean
S.E.M. (n= 5–7). *p<0.05, **p<0.01 vs. CT rats.
Fig. 2. Effects of 5-week berberine treatment on portal vein insulin level (A), pancreas insulin (B) and beta-cell mass (C) in normal rats. Rats received vehicle (^, CT) and
berberine at 60 mg/kg (&, BL) and 120 mg/kg (~, BH), respectively. Plasma samples were collected before and 10, 20 and 30 min following glucose load. Pancreas samples
were obtained at 30 min after glucose load. Insulin level was assessed by ELISA. Beta-cell proliferation was presented by beta-cell mass, detected by immunohistochemistry
technique. Data are means
S.E.M. (n= 4–5), **p<0.01 vs. CT rats.
Y. Yu et al. / Biochemical Pharmacology 79 (2010) 1000–1006
1002
The pancreas insulin levels were measured at 30 min after
glucose load. It was found that high dose of berberine significantly
increased pancreas insulin level, which induced a 2-fold increase
compared with control rats (Fig. 2B, p<0.01). However, low dose
of berberine treatment showed a trend to decrease pancreas
insulin level, although no significance was found.
The beta-cell volume density corresponds to the ratio of insulin
immunoreactivity area to pancreatic parenchymal area. The beta-
cell mass was calculated by multiplying the beta-cell volume
density by the weight of the pancreas [20]. The data from
immunohistochemistry showed a trend to increase beta-cell mass
in berberine-treated rats (Fig. 2C).
3.3. Effects of berberine treatment on GLP-1(7–36) amide in normal
rats
GLP-1 levels in portal plasma were investigated after glucose
load (Fig. 3A) and area under concentration–time curves from 0 to
30 min (AUC
30
) was estimated using linear trapezoidal rule. It was
found that glucose load may induce GLP-1 release. High dose of
berberine (120 mg/kg) may enhance GLP-1 release induced by
glucose load, peak level occurred at 20 min, accompanied by higher
values of AUC
30
(478.15
54.60 pMmin in rats treated with
120 mg/kg berberine vs. 320.40 27.08 pMmin in control rats,
p<0.05). However, low level of GLP-1 was observed in rats treated
with 60 mg/kg berberine, even lower than that of control rats.
GLP-1(7–36) amide in ileum and colon was measured at 30 min
after glucose load (Fig. 3B). Lower GLP-1 levels were found in both
ileum and colon of rats treated with berberine.
It was found that berberine treatment may increase
proglucagon mRNA expression in ileum in a dose-dependent
manner, 60 mg/kg and 120 mg/kg of berberine treatment may
induce 1.5-fold increase and 2.3-fold increase compared with
control rats, respectively (Fig. 3C). A trend of increase number of
GLP-1-positive L cells in intestine was found in berberine-
treated rats (Fig. 3D), and no morphologic changes in intestinal L
cells were observed between CT rats and berberine-treated rats
(Fig. 4A–F).
3.4. Effects of berberine on GLP-1 secretion in NCI-H716 cells
To determine the effectof berberineon GLP-1 secretion,NCI-H716
cellswere incubated withKRB in presenceof different concentrations
of berberine (0
m
M, 1
m
M, 10
m
M and 100
m
M) for 2 h. GLP-1 levels
in both medium (secreted) and cells were measured (Fig. 5). It was
found that berberine treatment may increase GLP-1 secretion
(medium) in a concentration-dependent manner (Fig. 5A), which
GLP-1 level increased from basal level 0.34
0.02 pM/mg protein to
0.51 0.03 pM/mg protein in presence of 100 mM berberi ne. Berberine
also increased cellular GLP-1 level, significant increases were found in
cells treated with 100 mM berberine (Fig. 5B, p<0.01).
3.5. Effects of berberine on proglucagon mRNA and prohormone
convertase 3 mRNA expression in NCI-H716 cells
NCI-H716 cells were treated with 100
m
M berberine for 24 h,
proglucagon mRNA and prohormone convertase 3 mRNA, which
controlled biosynthesis of GLP-1 were investigated using RT-PCR
analysis. Data from RT-PCR showed that berberine treatment may
increase both proglucagon mRNA expression and prohormone
convertase 3 mRNA expression, which induced 1.5-fold increase
for the two genes compared with cells without berberine
treatment (Fig. 6).
3.6. Effects of pharmacological inhibitors on berberine-mediated GLP-
1 release in NCI-H716 cell
To determine whether some cell signaling pathways were
involved in the regulation of berberine-mediated GLP-1 secretion,
NCI-H716 cells were incubated in presence of 100
m
M berberine
co-administrated with different pharmacological inhibitors. It was
found that chelerythrine inhibited berberine-mediated GLP-1
release in a dose-dependent manner, and presence of 10
m
M
chelerythrine resulted in 50% decrease of GLP-1 release (p<0.01,
Fig. 7A). Compound C also inhibited berberine-mediated GLP-1
release within the concentration range tested, but no concentra-
tion-dependent manner was found. Only low concentrations of
Fig. 3. Effects of 5-week berberine treatment on GLP-1(7–36) amide in normal rats. (A) Portal vein GLP-1 level, area under concentration–time curves from 0 to30min
(AUC
30
) was calculated, (B) GLP-1 content in intestinal tract of normal rats, (C) proglucagon mRNA expression in ileum, and (D) intestinal L cell proliferation. Rats received
vehicle (&, CT) and berberine at 60 mg/kg (&, BL) and 120 mg/kg (~, BH), respectively. Plasma samples were collected before and 10, 20 and 30 min following glucose load.
Intestinal samples were obtained at 30 min after glucose load. GLP-1(7–36) amide level was assessed by ELISA. Proglucagon mRNA expression was operated using RT-PCR and
L cell proliferation was estimated by immunohistochemistry. Data are means
S.E.M. (n= 4–5), *p<0.05, **p<0.01 vs. CT rats.
Y. Yu et al. / Biochemical Pharmacology 79 (2010) 1000–1006
1003
H89 (2
m
M and 10
m
M) may suppress berberine-mediated GLP-1
secretion. In contrast, high concentration (50
m
M) of H89 showed a
trend to enhance berberine-mediated GLP-1 secretion.
Effects of the pharmacological inhibitors themselves on GLP-1
release were investigated. The results demonstrated that similarly
to berberine, tested concentrations (2
m
M chelerythrine, 10
m
M
H89 or 10
m
M Compound C) of the pharmacological inhibitors
themselves may induce GLP-1 release, significant increase were
found in H89-treated cells and Compound C-treated cells.
However, co-administration of the same concentration of the
pharmacological inhibitors and berberine may significantly
suppress GLP-1 release (Fig. 7B).
4. Discussion
Berberine has been widely used as an antidiabetic agent, but its
mechanism of action is still obstacle because of poor absorption
and low concentration in plasma. Our previous studies showed
that berberine may increase GLP-1 secretion in experimental
diabetic rats [8], suggesting that berberine showed antidiabeitc
effect partly via promoting GLP-1 secretion. The present study was
focused on extending the previous findings and further providing
direct evidences using in vivo and in vitro experiments.
In vivo studies showed that berberine treatment may lower
fasted blood glucose, accompanied by increase of insulin levels in
plasma and pancreatic tissue, as well as beta-cell mass. 120 mg/kg
of berberine treatment may significantly increase GLP-1 release
induced by glucose load, accompanied by higher AUC
30
of GLP-1 in
portal plasma. Peripherally released GLP-1 enter brain areas and
participate in the regulation of anorexic response [21], which
indicated that suppression of food intake induced by berberine
may partly due to enhancement of GLP-1 release. It was well-
known that GLP-1 exerts important effects on regulating glucose
Fig. 4. The morphology of immunohistochemistry about intestinal L cell (final magnification 200). (A)–(C) are sections from ileum, CT, BL and BH rats respectively; (E)–(G)
are sections from colon, CT, BL and BH rats respectively. Rats received vehicle (CT) and berberine at 60 mg/kg (BL) and 120 mg/kg (BH) for 5 weeks, respectively. Intestinal
samples were obtained at 30 min after glucose load.
Fig. 5. Release of GLP-1(7–36) amide from NCI-H716 cells in response to 2-h incubation with medium alone, berberine (1
m
M, 10
m
M, and 100
m
M). (A) Secreted GLP-1 level,
(B) cellular GLP-1 level. GLP-1 concentration was measured by ELISA. Data are means
S.E.M. (n= 5), **p<0.01 vs. CT.
Fig. 6. Proglucagon gene and prohormone convertase 3 (PC3) gene expression in
NCI-H716 cells. Cells were treated with medium alone (CT) and berberine (100
m
M,
BBR) for 24 h. Proglucagon and PC3 mRNA levels were operated using RT-PCR. Data
are means
S.E.M. (n= 5), *p<0.05 vs. CT.
Y. Yu et al. / Biochemical Pharmacology 79 (2010) 1000–1006
1004
homeostasis via stimulating insulin secretion, beta-cell prolifera-
tion, inhibiting food intake [2]. All these results further supported
our previous findings that berberine showed its antidiabetic effects
partly via increasing GLP-1 release [8]. It was noticed that
berberine promoted proglucagon mRNA expression and L cell
proliferation in intestine of normal rats as well as diabetic rats [8].
It is known that proglucagon gene is precursor gene of GLP-1.
These findings further indicated the possibility of enhancement of
GLP-1 biosynthesis by berberine.
The present studies also showed that GLP-1 content in ileum
and colon of berberine-treated rats at 30 min following glucose
load was lower than that in control rats, although increase of
proglucagon mRNA expression and L cell proliferation was
showed. The mechanism resulting in this phenomenon was
unclear. These decreases may partly come from large amount of
GLP-1 release from tissue into blood, which was in agreement with
lower level of GLP-1 in portal plasma at 30 min after glucose load.
Similar phenomenon was found in previous result [8].
NCI-H716 cells were served as in vitro models of the intestinal L
cell to further investigate the effect of berberine on regulating GLP-
1 release. In vitro study demonstrated that berberine may
stimulate GLP-1 secretion from the NCI-H716 cells in a dose-
dependent manner, accompanied by increased both cellular GLP-1
level and total GLP-1 content, which indicated that berberine may
increase GLP-1 release as well as promote GLP-1 biosynthesis.
Effects of berberine on proglucagon gene and prohormone
convertase 3 gene in NCI-H716 cells, which regulate GLP-1
biosynthesis, were further investigated to verify the deduction.
Data from RT-PCR analysis showed that berberine treatment may
promote proglucagon mRNA expression, which supported in vivo
results. GLP-1 is derived from tissue-specific post-translational
processing of the proglucagon peptide by prohormone convertase
3 in L cells [1,22]. The present study also showed that berberine
treatment may up-regulate expression of prohormone convertase
3 mRNA. These findings indicated that up-regulation of the two
upstream genes may be one of the reasons that berberine
promoted GLP-1 biosynthesis.
Some reports showed that the PKC-dependent pathways were
involved in the regulation of GLP-1 secretion in NCI-H716 cells
[15,23]. Our present study showed that chelerythrine, inhibitor of
PKC-dependent pathway, suppressed berberine-mediated GLP-1
secretion in NCI-H716 cells in a concentration-dependent fashion.
No study to date reported on the direct interaction between
berberine and PKC pathway in GLP-1 secretion. A study using
cultured human liver cell model showed that berberine-induced
insulin receptor expression via activating the PKC-dependent
pathway [24], which indicated that berberine was connected with
PKC-dependent pathway, to some extend at least. These findings
revealed that activation of PKC-dependent pathway may be
involved in regulation of GLP-1 secretion mediated by berberine,
although a report showed inhibitory effect of berberine on the
activation of PKC
a
using glioma cells [25]. Chelerythrine itself
stimulated GLP-1 release, which indicated that PKC-mediated
regulation of GLP-1 secretion from the intestinal L cell is complex
process, multiple isoforms of PKC may be involved in. It was
reported that PKC
z
was required in oleic acid-induced GLP-1
secretion from GLUTag L cells and primary rat intestinal L cells
[23]. However, our results still provided a clue to the involvement
of PKC pathway in berberine-induced GLP-1 secretion.
cAMP-dependent GLP-1 secretion was observed in the human,
murine and rat L cell [8,26,27], but whether this was regulated
through a PKA-dependent pathway has not been identified [16].In
our study, the effect of H89 (PKA inhibitor) on berberine-mediated
GLP-1 secretion in NCI-H716 was ambiguous. Although a report
showed that 10
m
M H89 may suppress glucose-induced GLP-1
release in GLUTag cells [28], the present study gave a contrast
result that 10
m
M H89 itself enhanced GLP-1 release in NCI-H716.
We also found that low concentrations (2
m
Mor10
m
M) of H89
significantly inhibited the berberine-mediated GLP-1 secretion
from NCI-H716 cells. However, the high concentration (50
m
M) of
H89 showed enhanced berberine-mediated GLP-1 secretion. H89 is
marketed as a selective and potent inhibitor of PKA, but its mode of
specific inhibition of PKA is still unclear. There was reports that
H89 inhibited at least 8 other kinases [29].
Fig. 7. (A) Effects of berberine (100
m
M) with chelerythrine (0.4
m
M, 2
m
M, and 10
m
M), H89 (2
m
M, 10
m
M, and 50
m
M) and Compound C (2
m
M, 10
m
M, and 50
m
M) on GLP-
1 secretion in NCI-H716 cells. (B) Effects of chelerythrine (2
m
M), H89 (10
m
M) and Compound C (10
m
M) with or without berberine on GLP-1 secretion in NCI-H716 cells.
GLP-1 concentration is measured by ELISA. Data are means
S.E.M. (n= 5), *p<0.05, **p<0.01 berberine vs. CT.
#
p<0.05,
##
p<0.01 vs. BBR.
Y. Yu et al. / Biochemical Pharmacology 79 (2010) 1000–1006
1005
AMPK is a major intermediate in facilitating the beneficial
effects of berberine [30,31]. Several reports showed that AMPK lay
on upstream of p38 MAPK [4] and MAPK pathway is involved in
GLP-1 secretion in NCI-H716 cells [16]. We supposed that there
exist some correlations among berberine, AMPK and GLP-1. As
results in the present study showed that Compound C, inhibitor of
AMPK-dependent pathway, inhibited berberine-mediated GLP-1
secretion in NCI-H716 cells, no concentration-dependent response
was found. Similarly to two other pharmacological inhibitors
chelerythrine and H89, Compound C-induced GLP-1 release
suggested the effect of AMPK on GLP-1 secretion was also complex
process.
The main finding in the present study was that berberine
modulated GLP-1 release and biosynthesis in rats, which were
supported by data from NCI-H716 cells. Berberine enhanced GLP-1
biosynthesis partly via up-regulating both proglucagon and
prohormone convertase 3 gene expression; and PKC-dependent
pathways was involved in GLP-1 release induced by berberine
although roles of other signal pathways were not excluded.
Understanding the mechanisms controlling berberine-mediated
GLP-1 alteration allowed development of recognition of poor
absorbed berberine exerts good effects on hyperglycemia.
Acknowledgements
This work was supported by the National Science foundation of
China (No. 30873123) and the Postgraduate Novel foundation of
Simcere Pharmaceutical Group (2008).
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