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Nestlé Research Center (R.A.R., C.D., S.G., K.M.), P.O. Box 44, Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland; and Pharmacology Group (V.N.-M., U.T.R.), School of Pharmacy, University of Lausanne, 1015 Lausanne, Switzerland
Address all correspondence and requests for reprints to: Dr. Katherine Macé, Nestlé Research Center, P.O. Box 44, Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland. E-mail: catherine.mace{at}rdls.nestle.com
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Introduction |
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Increasing insulin secretion is a key goal in the treatment of type 2 diabetes. Stimulation of the endogenous release of GLP-1 is an attractive alternative to parenteral administration of the peptide. For this purpose, a number of cellular models of animal origin have been used to study the regulation of GLP-1 secretion, such as isolated canine L cells (5), fetal rat intestinal cell cultures (6), and the murine enteroendocrine cell lines STC-1 (7) and GLUTag (8). These cellular models have provided useful information regarding the signaling pathways that regulate proglucagon gene expression and GLP-1 secretion. The activation of PKA was reported to induce both proglucagon gene expression and GLP-1 secretion, whereas PKC activation increased only GLP-1 secretion (2, 5, 8). These animal cellular models do not necessarily exhibit the same intracellular regulating mechanisms that are active in human L cells. Indeed, glucose-dependent insulinotropic polypeptide, a potent stimulator of GLP-1 secretion in rodent models, is ineffective on human L cells in vivo (8, 9, 10). Mixed meals as well as oral administration of carbohydrates, lipids, and amino acids have been shown to stimulate GLP-1 secretion in humans (11, 12, 13). The mechanisms through which such regulation occurs in humans have been poorly characterized, probably because of the difficulty in isolating a truly homogeneous L cell population. The NCI-H716 cell line, derived from a poorly differentiated adenocarcinoma of human cecum (14), has been described to display some endocrine features, in particular the formation of secretory granules and chromogranin A expression (15). Furthermore, this cell line expresses several neurohormonal receptors, including receptors for gastrin, serotonin, and somatostatin (16). The purpose of this study was to evaluate whether the NCI-H716 cell line represents a valuable human cellular model for studying the regulation of GLP-1 expression and secretion.
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Materials and Methods |
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Cell line and culture conditions
Human NCI-H716 cells were obtained from the American Type Culture Collection (Manassas, VA). The NCI-H716 cell line was
initiated from ascites fluid of a 33-yr-old Caucasian male with poorly
differentiated adenocarcinoma of the colon (14). This is a
hypotriploid human cell line with a modal chromosome number of 61
occurring in 28% of cells. Commonly occurring colorectal tumor markers
such as CA 19-9, TAG-72, and CEA were not expressed by these cells
(14). For proliferation maintenance, the cells were grown
in suspension in RPMI 1640 supplemented with 10% FBS, 2 mM
L-glutamine, 100 IU/ml penicillin, and 100 µg/ml
streptomycin. Cell adhesion and endocrine differentiation were
initiated by growing cells in dishes coated with Matrigel (Becton Dickinson and Co., Bedford, MA) in high-glucose DMEM, 10% FBS,
2 mM L-glutamine, 100 IU/ml penicillin, and 100
µg/ml streptomycin as described previously (15).
Secretion studies
Two days before the experiments, 1 x
106 cells were seeded in 12-well culture plates
coated with Matrigel. On the day of the experiment, supernatants were
replaced by KRB containing 0.2% (wt/vol) BSA with or without test
agents. The solutions were adjusted to pH 7.2. Cells were incubated for
2 h at 37 C with the different effectors or with DMSO or BSA (when
fatty acids were used as effectors) alone as a control. The 2-h cell
incubation with the different effectors did not affect cell viability
(data not shown). Supernatants were collected with the addition of 50
µg/ml phenylmethylsulfonyl fluoride and frozen at -80 C for
subsequent analysis. Cells were scraped off and sonicated in a
homogenization buffer [1 N HCl containing 5% (vol/vol)
HCOOH, 1% (vol/vol) trifluoroacetic acid, and 1% (wt/vol) NaCl]. To
normalize GLP-1 content, cell homogenate proteins were
measured using the Bio-Rad Laboratories, Inc. (Munich,
Germany) protein assay. Peptides were extracted from the cell media and
homogenates using an alcohol extraction method as described by the
supplier of the GLP-1 (7-36) Total RIA Kit (Linco Research, Inc., St. Charles, MO). This kit, used to determine
concentrations of GLP-1, measures GLP-1
(7-36) and GLP-1 (9-36) with less than 0.4%
cross-reactivity with GLP-1 (7-37). The
ED50 of the assay was 72 pM. The
intraassay coefficient of variance was 2.3%. Biologically active
GLP-1 (7-36) was measured as described by the supplier of
the GLP-1 (Active) RIA Kit (Linco Research, Inc.). This kit reacts less than 0.1% with GLP-1
(9-36).
RNA isolation and RT-PCR
A total of 2 x 106 cells were seeded
in six-well culture plates coated with Matrigel and grown as described
above. After 24 h, fresh medium without serum but containing 0.2%
BSA and the test agents were added. After 18 h, cells were washed
with HBSS and stored at -80 C until RNA extraction using the RNeasy
Total RNA Purification System (QIAGEN AG, Basel,
Switzerland).
RT was performed with an input of 1 µg of total RNA using the first strand cDNA synthesis kit for RT-PCR (avian myeloblastosis virus; Roche Biomedical, Basel, Switzerland) with oligo d(T)15 as primer. Primers used for the amplification of cDNAs of interest were synthesized by Mycrosynth (Windisch, Switzerland). The sequences of the forward and reverse primers were (respectively): 5'-GTAATGCTGGTACAAGGCAG-3' and 5'-TTATAAAGTCCCTGGCGGCA-3' for the proglucagon gene, 5'-TATCGCAGAGAACGGATGGC-3' and 5'-TTGGAAACGCCAAGC-3' for the cholecystokinin gene, 5'-GCTGACTGATACACTCCAAG-3' and 5'-TCCCAGTCTGCTGCATAGAA-3' for the c-fos gene, 5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-TCTAGACGGCAGGTCAGGTCCACC-3' for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and 5'-GTTGCTATCCAGGCTGTG-3' and 5'-CATAGTCCGCCTAGAAGC-3' for the actin gene. PCR involved heating for two cycles to 98 C for 1 min, 60 C for 2 min, and 72 C for 2 min and then cycling 28 times through a 1-min denaturation step at 94 C, a 1-min annealing step at 60 C, and a 2-min extension step at 72 C in a DNA thermal cycler apparatus (Bioconcept, Allschwil, Switzerland). The 30 cycles used for the detection of the proglucagon transcripts correspond to the linear portion of the amplification curve (data not shown). GAPDH or actin primers were included in the reaction as internal controls. PCR products (10 µl) were separated on a 2% agarose gel and visualized by ethidium bromide staining. Quantification of the PCR products was performed using the densitometric NIH Imager Program.
Northern blotting
Northern blotting was performed as previously described
(17) with minor modifications. Actin was used as the
control gene to correct for any total RNA loading differences. A 472-bp
PCR-generated fragment of the proglucagon gene and a 739-bp
PCR-generated fragment of the actin gene were labeled by nick
translation with 
-32P (Amersham Pharmacia Biotech, Little Chalfont, UK). A single
proglucagon transcript of 1.6 kb and a single ß-actin transcript of
1.8 kb were detected.
Immunofluorescence
Cells were plated in four-well glass chamber slides at 0.2
x 106 cells/well and incubated for 2 days at 37
C. Immunofluorescence was then performed as previously described
(18), except that cells were incubated overnight at 4 C
with the rabbit antihuman GLP-1 antibody diluted 1:100
(Affiniti Research Products Ltd., Exeter, UK), which, according to the
supplier, reacts with the mid- to C-terminal domain of
GLP-1 (1-19) and recognizes GLP-1 (1-37),
(1-36amide), and (7-36amide).
Cytosolic calcium measurement
Cytosolic free calcium concentration
([Ca2+]c) was measured by
Fura-2 fluorescence. NCI-H716 cells grown on Matrigel-coated glass
coverslips were washed three times with PSS buffer and incubated in the
dark for 45 min at room temperature with Fura-2/AM (5 µM)
in PSS buffer containing 0.01% Pluronic F-127. Cells were washed six
times with PSS, and the coverslip was analyzed in a
thermo-regulated chamber (37 C) on a Nikon (Tokyo,
Japan) Diaphot inverted epifluorescence microscope with a PhoCal
cell fluorescence analyzer (Life Science Resources, Cambridge, UK). The
cells were illuminated with alternating light of 340 and 380 nm from a
rotating filter wheel (6.25 hertz). Emission was monitored at 510 nm,
and data were analyzed using the PhoCal software. Calibrations were
performed by treating the cells with CaCl2 (6
mM) and ionomycin (10 µM) to obtain the
maximal signal, followed by the addition of EGTA (20 mM) to
obtain the minimal signal. Background fluorescence, obtained by
quenching the Ca2+ signal with
MnCl2 (10 mM), was subtracted from
the signals. Results are given as fluorescence intensity ratio (340:380
nm) and as [Ca2+]c
calculated as described (19).
Calculations and statistics
Data in the figures are presented as means ±
SEM and represents at least three experiments measured in
duplicate. Differences between treatments were determined using the
one-way ANOVA model. Statistical significance is defined as
P 
0.05.
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Results |
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shows that GLP-1 staining
was uniformly distributed in all cells. At a higher magnification
(x400), the immunofluorescent staining was visible as discrete dots
scattered in the cytoplasm, indicating that intracellular
GLP-1 storage was restricted to secretory vesicles (data
not shown).
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, both palmitic and oleic acid
stimulated GLP-1 secretion, with a maximal effect at 0.8
mM (1.8 ± 0.2-fold and 1.6 ± 0.1-fold increase,
respectively, compared with the control value). MH also induced a
concentration-dependent increase in GLP-1 release, with a
plateau at 1% MH corresponding to a 5.0 ± 0.9-fold stimulation
(Fig. 2B).
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). GRP at 0.7 µM
increased 2.2 ± 0.2-fold GLP-1 secretion, whereas
GRP at 0.07 µM did not show significant effects (Fig. 3A).
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, forskolin (50 µM) and PMA (1
µM) induced 1.7 ± 0.2-fold and 4.3 ± 0.6-fold
increases of GLP-1 secretion, respectively. Simultaneous
activation of both PKA- and PKC-dependent pathways with 50
µM forskolin and 1 µM PMA resulted in a
4.6 ± 0.8-fold increase (Fig. 3B).
Calcium participates in the mechanism of GLP-1
secretion in NCI-H716 cells
To investigate whether cytosolic calcium is involved in
GLP-1 secretion in NCI-H716 cells, the amount of
GLP-1 released in the medium of cells treated with the
calcium ionophore ionomycin was measured. As shown in Fig. 3B, a 2-h
ionomycin treatment induced a concentration-dependent increase in
GLP-1 secretion, with a plateau at 2 µM
corresponding to a 4.5 ± 0.2-fold stimulation. As expected,
ionomycin was also able to increase
[Ca2+]c in NCI-H716 cells
(Fig. 4A). Both ionomycin-induced
GLP-1 secretion and
[Ca2+]c were completely
abolished when cells were incubated with EGTA (Fig. 4A and data not
shown). Interestingly, the addition of 0.7 µM GRP on
NCI-H716 cells led to an immediate increase in
[Ca2+]c, followed by a
sustained plateau (Fig. 4B).
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). These results obtained by RT-PCR
(Fig. 5A) were confirmed by Northern blot analysis (Fig. 5B). Using
this technique, no change in proglucagon mRNA was detected except a
small yet significant decrease after 18 h of incubation with
forskolin/IBMX and 2% MH. A time course performed at 6, 12, 18, 24,
and 48 h confirmed that no change occurred in the levels of
proglucagon mRNA with the same test compounds (data not shown). On the
contrary, the expression of the early response gene c-fos
was 1.8- or 2.6-fold induced when forskolin/IBMX (10
µM each) or PMA (1 µM),
respectively, was applied to the cells for 1 h (Fig. 6). These data confirmed that the PKA and
the PKC pathways are functional in these cells. Carbachol, palmitic
acid, and oleic acid were also tested for their ability to modulate
proglucagon mRNA expression in parallel to GLP-1
secretion, but none of them modified proglucagon mRNA after 18 h
of incubation (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A uniform population of differentiated NCI-H716 cells showed a high GLP-1 content (2.2 ± 0.45 pmol/106 cells) and an increased secretory capacity, with a basal release of 4.2 ± 0.9% of the total GLP-1 cell content during a 2-h incubation period. For comparison, the mouse enteroendocrine STC-1 cell line has been described to contain 1.2 ± 0.2 pmol GLP-1/106 cells with a secretion rate of 1.4 ± 0.3% during the same interval (7). Contrary to the secretin tumor cell line STC-1 (25) and the mouse L cell line GLUTag (26), the NCI-H716 cell line was unable to express the cholecystokinin gene as tested by RT-PCR (data not shown). This discrepancy could be attributable to the colonic origin of the NCI-H716 cells, cholecystokinin being localized in endocrine cells of the duodenal and jejunal mucosa but not in the terminal ileum and colon (27).
In agreement with studies of intestinal L cells of animal origin, the release of GLP-1 by NCI-H716 cells was increased by protein hydrolysates (20). However, although exposure of the ileum to peptone stimulated GLP-1 secretion in the isolated vascularly perfused rat intestine (20, 28), ileal perfusion of peptone had no effect or only weak effects on plasma GLP-1 in humans (29).
Both palmitic acid and oleic acid stimulated GLP-1 secretion in NCI-H716 cells, whereas only unsaturated fatty acids were effective in the rat enteroendocrine cell line and in fetal rat intestinal cells (8, 30). In healthy subjects, olive oil induced higher concentrations of GLP-1 and glucose-dependent insulinotropic polypeptide than butter (13). Further work will be needed to assess the structure-activity relationship of fatty acids with regard to the stimulation of GLP-1 secretion in NCI-H716 cells.
Neural stimulation of GLP-1 also constitutes an important part in the control of GLP-1 secretion. In particular, cholinergic activation is considered an efficient signal in humans, because atropine has been shown to decrease GLP-1 release after an oral load (31). In NCI-H716 cells, carbachol, a cholinergic agonist, elicited a small but significant increase in GLP-1 secretion. This effect is consistent with observations made in rodent cellular models (24, 32). GRP is a neuropeptide expressed in the central nervous system and peripheral organs, which functions both as gut hormone and neuromediator. It stimulates GLP-1 release in humans, rats, and dogs and represents another important part of the vagal signal, apart from the adrenergic and cholinergic pathways (33). Placement of fat into the duodenum of rats has been reported to induce the release of GLP-1 from the distal intestine through a GRP-dependent mechanism (34). Furthermore, when applied at 0.1 µM, GRP stimulated GLP-1 secretion in the isolated vascularly perfused rat ileum (27). Here, 0.7 µM GRP significantly enhanced GLP-1 secretion in NCI-H716 cells, although to a lesser extent than in an animal organ model (27). The relatively high effective dose of GRP allowing stimulation of GLP-1 secretion in NCI-H716 cells could be attributable to the limited GRP receptor gene expression observed with RT-PCR in these cells compared with colon cancer CaCo-2 cells (data not shown). Interestingly, GRP-enhanced GLP-1 secretion was correlated with an increase of cytosolic calcium levels in NCI-H716 cells. Similarly, Ca2+ ionophore strongly stimulated GLP-1 secretion in these cells, as previously reported in fetal rat intestinal cell cultures (32). The stimulation of the calcium signaling pathway by GRP has been described previously for the control of chromogranin A and neurotensin secretion in pancreatic cells transfected with a human GRP receptor (35). In pancreatic and chromaffin cells, calcium entry through specific channels is responsible for the exocytosis of secretory granules (36, 37). Therefore, it is tempting to speculate that a similar mechanism occurs in enteroendocrine cells. Nevertheless, further investigation is required to determine whether a calcium-dependent exocytosis mechanism exists in these cells.
As previously described in rodent cellular models (8), the
PKA- and PKC-dependent pathways were involved in the regulation of
GLP-1 secretion in the human NCI-H716 cell line. However,
proglucagon mRNA levels did not change in the NCI-H716 cells when
incubated with PKA activators, which are known to up-regulate
proglucagon gene expression in animal cell lines (8).
Genomic alterations arising in human colorectal tumor cell lines could
be responsible for the lack of transcriptional response observed in the
NCI-H716 cells. Nevertheless, the expression of c-fos, an
early response gene, was up-regulated after NCI-H716 cells were
incubated with forskolin or PMA, indicating effective PKA and PKC
pathways (38). Interestingly, promoter sequence
differences between the rodent and human proglucagon gene, which could
lead to potential differences in the mechanisms used for
tissue-specific regulation, have been recently described
(39). Nucleotide changes were observed between the two
promoters at the binding sites of transcription factors, including
cAMP-responsive element-binding protein, pax6, HNF-3, and HNF-3ß
(39). Modifications in the interaction of one or more of
these factors with the human proximal promoter could explain the
differences in proglucagon gene regulation observed between rodent and
human cells. This hypothesis requires further investigation.
In conclusion, this study indicates that differentiated NCI-H716 cells, which express high levels of GLP-1, represent a useful cellular model for the study of the regulation of GLP-1 secretion in humans. With this model, we demonstrated that GRP stimulates GLP-1 secretion, probably through an increase of cytosolic calcium. Although the pathways regulating GLP-1 secretion in these human enteroendocrine cells appear to be similar to those previously described in rodent cells, the potential interspecies divergence in the regulation of proglucagon gene expression underscores the importance of further studying GLP-1 regulation in human models.
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Acknowledgments |
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Footnotes |
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1 Current address: Faculties of Kinesiology and Medicine, University
of Calgary, 2500 University Drive NW, Calgary, Alberta, T2N1N4
Canada. 
Abbreviations: [Ca2+]c, Cytosolic free calcium concentration; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLP-1, glucagon-like peptide-1; GRP, gastrin-releasing peptide; IBMX, 3-isobutyl-1-methylxanthine; KRB, Krebs-Ringer bicarbonate buffer; MH, meat hydrolysate; PMA, phorbol 12-myristate 13-acetate; PSS, physiological salt solution.
Received February 6, 2001.
Accepted for publication June 7, 2001.
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
) (for both fatty acids, the fatty acid-to-BSA ratio
was 5:1) (A) and MH (•) (B). Secretion into the medium is normalized
to the total cellular content and expressed as a percentage of the
control value. Results represent means ± SEM of three
experiments. *, P 
