Studies with GIP/Ins cells indicate secretion by gut K cells
is KATP channel independent
Song Yan
Wang1,
Maggie M.-Y.
Chi2,
Lin
Li1,
Kelle H.
Moley2, and
Burton M.
Wice1
Division of Metabolism, Departments of 1 Internal
Medicine and 2 Obstetrics and Gynecology, Washington
University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
K cells
are a subpopulation of enteroendocrine cells that secrete
glucose-dependent insulinotropic polypeptide (GIP), a hormone that
promotes glucose homeostasis and obesity. Therefore, it is important to
understand how GIP secretion is regulated. GIP-producing (GIP/Ins) cell
lines secreted hormones in response to many GIP secretagogues except
glucose. In contrast, glyceraldehyde and methyl pyruvate stimulated
hormone release. Measurements of intracellular glucose 6-phosphate,
fructose 1,6-bisphosphate, and pyruvate levels, as well as glycolytic
flux, in glucose-stimulated GIP/Ins cells indicated that glycolysis was
not impaired. Analogous results were obtained using glucose-responsive
MIN6 insulinoma cells. Citrate levels increased similarly in
glucose-treated MIN6 and GIP/Ins cells. Thus pyruvate entered the
tricarboxylic acid cycle. Glucose and methyl pyruvate stimulated 1.4- and 1.6-fold increases, respectively, in the ATP-to-ADP ratio in
GIP/Ins cells. Glyceraldehyde profoundly reduced, rather than
increased, ATP/ADP. Thus nutrient-regulated secretion is independent of
the ATP-dependent potassium (KATP) channel. Antibody
staining of mouse intestine demonstrated that enteroendocrine cells
producing GIP, glucagon-like peptide-1, CCK, or somatostatin do not
express detectable levels of inwardly rectifying potassium (Kir) 6.1 or
Kir 6.2, indicating that release of these hormones in vivo may also be
KATP channel independent. Conversely, nearly all cells
expressing chromogranin A or substance P and ~50% of the cells
expressing secretin or serotonin exhibited Kir 6.2 staining. Compounds
that activate calcium mobilization were potent secretagogues for
GIP/Ins cells. Secretion was only partially inhibited by verapamil,
suggesting that calcium mobilization from intracellular and
extracellular sources, independent from KATP channels,
regulates secretion from some, but not all, subpopulations of
enteroendocrine cells.
K cells; glucose-dependent insulinotropic polypeptide; glucagon-like peptide-1; hormone secretion; inwardly rectifying
potassium channel; ATP-dependent potassium channels; insulin
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INTRODUCTION |
ENTEROENDOCRINE
(EE) CELLS are a complex population of diffusely distributed
hormone-producing intestinal epithelial cells that play important roles
in regulating and integrating many aspects of gastrointestinal and
whole animal physiology (1, 32, 48, 52, 57, 66). Although
they represent <1% of the intestinal epithelial cells, EE cells as a
whole represent the largest endocrine organ in the body. There are at
least 16 different subpopulations of EE cells based on the major
product(s) produced and secreted by individual cells (1, 48,
57). The specific product(s) is dependent on a cell's position
along the crypt to villus and stomach to colonic axes of the gut
(49, 57).
Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like
peptide-1 (GLP-1) are EE cell-derived "incretin" hormones that are
released in the circulation immediately after ingestion of a meal and
potentiate insulin secretion after binding to receptors on islet
-cells (32). GIP is produced and secreted by gut K cells located in the stomach and proximal small intestine, whereas GLP-1 is a product of gut L cells located in the distal small intestine
(13, 52, 65, 66, 73). Mice lacking GIP (34) or GLP-1 (53) receptors exhibit impaired first-phase
glucose-stimulated insulin release or glucose intolerance,
respectively. In addition to effects on glucose homeostasis, GIP
promotes obesity in mice fed a high-fat diet (33). Thus it
is important to understand the molecular mechanisms that regulate GIP
and GLP-1 secretion.
Incretin-producing EE cells respond to changes in the concentrations of
lumenal nutrients but are refractive to changes in the levels of
nutrients in the blood (11, 13, 52). Glucose (5, 62,
65), protein hydrolysates (70), specific amino acids (64), and fat (12) are the major
nutrients that stimulate GIP release. Gastrin-releasing peptide (GRP),
a hormone produced and secreted by enteric neurons, also stimulates GIP
release (46). Conversely, somatostatin (SST) inhibits
secretion from gut K cells (32, 66). GLP-1 secretion is
also under nutritional (11, 44), hormonal (4,
45), and neuronal (47) control (10, 18). Interestingly, GIP stimulates GLP-1 secretion from gut L
cells (4, 45).
Because GIP is secreted by gut K cells with a temporal pattern and in
response to similar nutrients as insulin secretion by islet
-cells,
it has been proposed that engineering gut K cells to produce insulin is
a potential gene therapy to treat diabetes (7, 39). To
begin to test this hypothesis, novel GIP-producing cell lines were
established and engineered to express the human insulin gene (GIP/Ins
cells). Like K cells in vivo, GIP/Ins cells secreted both insulin and
GIP in response to the GIP secretagogues arginine, bombesin, and
protein hydrolysates (39). However, glucose failed to
stimulate hormone release from the cells even though they express
glucokinase, the major glucose-sensing enzyme used by islet
-cells
and hepatocytes. This observation is consistent with published results
that demonstrated glucose-stimulated GIP release was dependent on sugar
uptake by enterocytes (62) and suggests that gut K cells
may not directly sense glucose in the lumen of the gut (see Ref.
39 for detailed discussion). In contrast to glucose,
glyceraldehyde and methyl pyruvate were good secretagogues for GIP/Ins
cells, indicating that these cells can sense changes in the
intracellular concentrations of specific metabolic intermediates.
We have begun to study the regulation of gut K cell physiology by using
the well-characterized islet
-cell as a model (17, 21).
In islet
-cells, glucose metabolism results in an increase in the
intracellular ATP-to-ADP ratio. This, in turn, inhibits the
ATP-dependent potassium (KATP) channel, resulting in cell depolarization, influx of calcium, and finally exocytosis of insulin from secretory granules. Methyl pyruvate is thought to stimulate insulin release from
-cells by increasing the mitochondrial ATP levels (20, 31). However, the situation concerning
glyceraldehyde-stimulated insulin release is more complicated. After
phosphorylation to glyceraldehyde-3-phosphate by triose kinase,
glyceraldehyde can be metabolized via glycolysis and the tricarboxylic
acid cycle to ultimately increase the intracellular ATP-to-ADP ratio.
If this pathway is active in K cells, lack of glucose-stimulated insulin secretion by GIP/Ins cells could result from an inability to
metabolize glucose in the early steps of glycolysis. However, it has
also been proposed that glyceraldehyde, in the presence of
Pi, can be directly phosphorylated by glyceraldehyde
phosphate dehydrogenase to generate the nonmetabolizable
intermediate glyceraldehyde-1-phosphate (24). This
reaction would increase the intracellular NADH-to-NAD ratio and,
consequently, mitochondrial ATP synthesis. Taken together, these
observations raised the possibility that glucose cannot be metabolized
to glyceraldehyde-3-phosphate by GIP/Ins cells and methyl pyruvate, and
glyceraldehyde stimulated hormone release by increasing the
intracellular ratio of ATP to ADP independently of early steps in
glycolysis. However, RT-PCR analyses of mRNAs prepared from 10 independently derived GIP-producing cell lines indicated that these
cells express only very low levels of inward rectifying potassium
channel 6.1 (Kir 6.1), inward rectifying potassium channel 6.2 (Kir
6.2), sulfonylurea receptor 1 (SUR 1), sulfonylurea receptor 2A (SUR
2A), and sulfonylurea receptor 2B (SUR 2B), suggesting that
nutrient-stimulated hormone secretion by gut K cells may be independent
of KATP channels altogether (39). This
hypothesis is also supported by the observation that sulfonylurea
compounds and potassium channel opening (KCO) drugs had relatively
little effect on secretion by GIP/Ins cells (39). Interestingly, islet
-cells exhibit both KATP
channel-dependent and -independent mechanisms of secretion
(50). To begin to sort out biochemical and molecular
mechanisms that regulate secretion from K cells, the intracellular
concentrations of specific glycolytic and citric acid cycle
intermediates were determined in GIP/Ins cells cultured in the absence
of glucose vs. 25 mM glucose, glyceraldehyde, or methyl pyruvate. As a
control, similar measurements were performed using insulin-secreting
MIN6 cells, a well-characterized, glucose-responsive,
-cell line
(35, 67). Results of these analyses indicated that glucose
is rapidly metabolized by the GIP/Ins cells and secretion is
independent of the intracellular ATP-to-ADP ratio. These results are
consistent with the RT-PCR data indicating that KATP
subunit channels are expressed at very low levels in GIP/Ins cells.
Double-label immunohistochemical techniques were then used to
demonstrate that gut K cells in vivo, as well as gut endocrine cells
that secrete GLP-1, SST, and CCK, do not express either Kir 6.1 or Kir
6.2. Therefore, depolarization by these gut endocrine cells is
fundamentally different from that by islet
-cells. However, several
specific subpopulations of EE cells do express Kir 6.2, revealing an
unexpected level of complexity concerning mechanisms of hormone
secretion by different subtypes of EE cells.
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MATERIALS AND METHODS |
Cells and culture conditions.
All cells were cultured in an atmosphere of 5% CO2-95%
air and 100% humidity. MIN6 cells (passage 25-40) were cultured
in DMEM containing 15% FCS, as previously described (39,
67), and GIP/Ins cells were cultured in DMEM containing 10% FCS
(39).
Hormone secretion assays.
Insulin or GIP secretion was measured as previously described
(39). Briefly, GIP/Ins or MIN6 cells were plated in
12-well tissue culture dishes. When nearly confluent, cells were washed two times with PBS (containing calcium and magnesium) and then preincubated for 60 min at 37°C in insulin secretion assay buffer (glucose-free KRBH-Alb; see Ref. 51). Cells were then
refed secretion buffer containing the indicated secretagogue. Later (90 min), buffers were collected, centrifuged to remove detached cells, and
then assayed for human insulin or GIP production by RIA.
Measurement of intracellular metabolite levels.
Metabolite levels were determined in cells cultured under the same
conditions that were used to measure hormone secretion (39) except cells were plated in 60-mm dishes
(51). Thirty minutes after addition of assay buffer
containing the indicated secretagogue, cells were washed two times with
PBS (0°C; <5 s/wash) and then lysed in 700 µl of 0.05 N sodium
hydroxide (0°C). DNA was sheared by passing lysates through a
QIAshredder (Qiagen, Valencia, CA). To prepare alkali-treated extracts,
a 240-µl aliquot of the cell lysate was heated at 80°C for 20 min
and then neutralized by addition of 120 µl of a solution containing
0.05 N HCl plus 0.1 M Tris base. Acid-treated extracts were prepared by
adding 60 µl of 0.3 N HCl to a 240-µl aliquot of untreated cell
lysate, heating for 20 min at 80°C, and then neutralizing with 60 µl of 0.2 M Tris base. Neutralized extracts were stored at
80°C.
All metabolite values were normalized to the amount of protein in the
original cell lysate. Alkali-treated extracts were used for the
determination of ATP, ADP, pyruvate, fructose 1,6-bisphosphate (F-1,6-P2), and citrate. Glucose 6-phosphate
(G-6-P) and glutamate were measured on acid-treated
extracts. Metabolites were assayed in a direct, or linked, coupled
enzymatic reaction that either oxidized or reduced NAD(P)(H), as
described previously (8, 22, 41, 42, 69). Pyridine
nucleotides generated in the enzymatic reaction were measured using a
Farrand A4 Fluorometer containing a 7-60 excitation filter and
5-57 plus 3-73 emission filters.
Measurement of lactic acid production.
GIP/Ins cells were plated in six-well tissue culture dishes and treated
as described for insulin secretion assays (39). After the
90-min incubation with or without secretagogues, secretion buffers were
collected and centrifuged to remove detached cells. Lactic acid in the
buffer was then determined using lactate dehydrogenase (Sigma Chemical,
St. Louis, MO). All values are normalized to the amount of cell protein
in each dish.
Antibody staining.
The entire mouse small intestine was removed en bloc immediately after
the animal was killed and then flushed with PBS followed by freshly
prepared 4% paraformaldehyde in PBS. The intestine was then cut
longitudinally along its entire length. After fixation for a total of
1 h at room temperature, the intestine was stored in 70% alcohol
(at least overnight) and then rolled up from the duodenum to distal
ileum. The resulting "Swiss roll" (14, 15) was cut in
half with a razor blade along the duodenal to ileal axis and then
impregnated with 2% agar in 5% phosphate-buffered formalin. The Swiss
rolls were then embedded in paraffin, sectioned, and stained as
described below. Other tissues were fixed and embedded in paraffin
without the use of agar.
Tissue sections were deparaffinized in xylene and rehydrated in graded
alcohols followed by water and then PBS. After antigen retrieval using
1 mM EDTA (38), sections were washed with PBS and then
blocked using BACKGROUNDSNIPER (Biocare Medical, Walnut Creek, CA). Sections were then incubated for 60 min at room temperature with the appropriate primary antibodies [diluted to the indicated concentration in Da Vinci Green Primary Antibody Diluent (Biocare Medical)]. After three washes with PBS, sections were incubated for 45 min at room temperature with the indicated secondary antibodies diluted
in Da Vinci Green Primary Antibody Diluent. Sections were then washed,
and nuclei were stained with bis-benzimide and mounted in PBS-glycerol
as described (68).
The following antibodies were used to stain the tissue sections
reported here: goat anti-Kir 6.1 (catalog no. sc-11225; 2 µg/ml;
Santa Cruz Biotechnology, Santa Cruz, CA); goat anti-Kir 6.2 (catalog
no. sc-11228; 2 µg/ml; Santa Cruz Biotechnology); rabbit anti-GIP
(catalog no. IHC-7154, 1 µg/ml; Peninsula Laboratories, Torrance,
CA); rabbit anti-GLP-1 (catalog no. IHC-7123, 1 µg/ml; Peninsula
Laboratories); rabbit anti-SST-14 (catalog no. IHC-8001, 1 µg/ml;
Peninsula Laboratories); rabbit anti-substance P (SP; IHC-7451, 1 µg/ml; Peninsula Laboratories); rabbit anti-CCK (catalog no.
IHC-61014, 1 µg/ml; Peninsula Laboratories); rabbit anti-CGA (catalog
no. 20085, 1:1,000 dilution; DiaSorin, Stillwater, MN); rabbit
anti-secretin and rabbit anti-serotonin were kindly provided by Dr.
Jeffrey Gordon of our university and used at 1 µg/ml. All fluorochrome-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were raised in donkeys and preadsorbed to reduce cross-reactivity with IgG from other species. Secondary antibodies were
used at a 1:1,000 dilution.
Quantifying Kir 6.2 and gut hormone double positive cells.
Single sections of paraffin-embedded mouse small intestine were
incubated with goat anti-Kir 6.2 plus rabbit anti-GIP, GLP-1, secretin,
serotonin, SST-14, SP, CCK, or chromogranin A (CGA; see above). Bound
primary antibodies were detected using Cy3-conjugated donkey anti-sheep
IgG (red) plus FITC-conjugated donkey anti-rabbit IgG (green) secondary
antibodies. Individual cells expressing a specific gut hormone (green)
were identified using a fluorescent microscope. The filters were then
changed to determine whether Kir 6.2 (red) was expressed by that same
cell. Typically, a total of 300 green cells was analyzed for
coexpression of Kir 6.2.
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RESULTS |
Glycolysis is not impaired in GIP/Ins cells.
Methyl pyruvate (data not shown) and glyceraldehyde, but not glucose,
stimulate hormone release from GIP/Ins cells (Fig. 1 and also see Ref.
39). Glyceraldehyde enters
glycolysis downstream of phosphofructokinase 1. This raised the
possibility that GIP/Ins cells cannot metabolize glucose through early
steps of glycolysis. To address this issue, the intracellular
concentrations of several glycolytic intermediates were determined in
cells cultured under the same conditions used for the hormone secretion
assays. Cells were incubated for 1 h in insulin secretion assay
buffer (no glucose) to deplete intracellular metabolites and then refed
buffer with or without 25 mM glucose. Later (30 min), cells were
harvested, extracted, and then assayed for the indicated metabolite. As
a control, similar measurements were performed using glucose-responsive MIN6 insulinoma cells. As shown in Tables
1 and 2, addition of
glucose caused a 56-fold increase in
G-6-P levels in GIP/Ins cells, whereas MIN6 cells exhibited
a 7-fold increase in the level of this metabolite. That
G-6-P levels increased eightfold more in GIP/Ins cells
compared with MIN6 cells indicated that glucose phosphorylation was not
limiting and suggested that phosphofructokinase 1, the next regulated
step in glycolysis, could possibly be inactive in GIP/Ins cells.
However, addition of glucose caused 143- and 38-fold increases in
F-1,6-P2 levels in GIP/Ins and MIN6 cells, respectively. Thus phosphofructokinase 1 activity is not limiting in
GIP/Ins cells. Finally, the levels of pyruvate, the end product of
glycolysis, were determined. Addition of glucose resulted in 2.4- and
2-fold increases in pyruvate levels in GIP/Ins and MIN6 cells,
respectively. Therefore, glucose-stimulated increases in the levels of
glycolytic intermediates were greater in GIP/Ins vs. MIN6 cells even
though glucose stimulates hormone release from MIN6, but not GIP/Ins,
cells.

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Fig. 1.
Insulin secretion by glucose (Glc)-dependent
insulinotropic polypeptide (GIP)/Ins cells. The indicated clone of
GIP/Ins cells was plated in 12-well tissue culture-treated dishes and
cultured for 3-4 days until ~80% confluent. At this time, cells
were washed and preincubated at 37°C in insulin secretion assay
buffer for 60 min. Assay buffer was then replaced with 1 ml of fresh
buffer containing the indicated secretagogue and again incubated at
37°C. Later (90 min), buffers were collected, and detached cells were
removed by centrifugation. Supernatants were then assayed for
immunoreactive human insulin by RIA. Glc, 25 mM glucose; IBMX, 1.5 mM
IBMX; PMA, 10 6 M phorbol 12-myristate 13-acetate; meGlut,
5 mM L-glutamic acid dimethyl ester; Glyc, 25 mM
glyceraldehyde. *P < 0.05 and +P < 0.005. Results from 1 experiment are shown (n = 4 for
each condition). Essentially identical results were obtained in 2
(GIP/Ins clone 10) or 1 (clone 12) additional
experiments.
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Table 1.
Intracellular metabolite levels in GIP/Ins clone 10 and MIN6 cells
stimulated for 30 min with 25 mM glucose, glyceraldehyde, or methyl
pyruvate
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Although the preceding results strongly suggest that glycolysis is not
impaired in GIP/Ins cells, it is possible that metabolite levels do not
reflect the actual flux of carbon through glycolysis. To address this
issue, GIP/Ins cells were incubated for 90 min in insulin secretion
assay buffer either with or without 25 mM glucose. The amount of lactic
acid in the secretion buffer was then determined. As shown in Fig.
2, GIP/Ins clones 10 and
12 incubated with glucose produced 45 and 75 µg
lactate · mg
protein
1 · 90 min
1,
respectively. Next, lactate secretion was measured in GIP/Ins cells
treated with glyceraldehyde. It is important to note that glyceraldehyde can be metabolized to lactate via glycolysis or to
glyceraldehyde 1-phosphate plus NADH by glyceraldehyde phosphate dehydrogenase. If this latter reaction is operative, the increased NADH
levels would drive production of lactate from pyruvate. Thus lactate
production from glyceraldehyde-treated cells represents the maximal
flux through the downstream reactions of glycolysis. As shown in Fig.
2, metabolism of glyceraldehyde or glucose generated similar amounts of
lactate, indicating that flux through glycolysis is not preventing
glucose-stimulated hormone release from GIP/Ins cells.

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Fig. 2.
Glycolysis is not impaired in GIP/Ins cells. The
indicated clone of GIP/Ins cells was treated as described in Fig. 1,
except cells were plated in 6-well dishes. After 90 min of incubation
and removal of detached cells, the amount of lactic acid in the
supernatants was determined using lactate dehydrogenase. Values were
then normalized to the amount of protein in each well.
+P < 0.005. Results from 1 experiment are shown
(n = 4 for each condition). Essentially identical
results were obtained in 2 (GIP/Ins clone 10) or 1 (clone 12) additional experiments.
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Secretion by GIP/Ins cells is independent of the intracellular
ATP-to-ADP ratio.
The previous results indicate that lack of glycolysis cannot account
for the inability of glucose to stimulate hormone release from GIP/Ins
cells. Pyruvate generated via glycolysis appears to enter the
tricarboxylic acid cycle, since the intracellular concentration of
citrate and the glucose-stimulated increase in citrate levels are
similar in GIP/Ins and MIN6 cells (Tables 1 and 2). Because the
ATP-to-ADP ratio plays a critical role in regulating KATP
channel activity and cell depolarization in islet
-cells, this ratio
was determined on GIP/Ins and MIN6 cells treated with glucose,
glyceraldehyde, or methyl pyruvate. The addition of glucose or methyl
pyruvate to GIP/Ins cells resulted in similar increases in the
ATP-to-ADP ratio (1.4- vs. 1.6-fold increases, respectively),
suggesting that secretion was independent of ATP-to-ADP ratios.
Surprisingly, glyceraldehyde caused a dramatic decrease in the
intracellular ATP-to-ADP ratio (just the opposite of what would be
expected if this fuel secretagogue stimulated secretion via
KATP channels). In MIN6 cells, methyl pyruvate caused a
greater increase in the ATP-to-ADP ratio than did glucose (2.6- vs.
1.8-fold), yet glucose was a better secretagogue than methyl pyruvate
(Fig. 3). These observations suggest that
methyl pyruvate stimulated secretion from
-cells by
KATP-dependent and -independent mechanisms. However,
glyceraldehyde had little effect on the ATP-to-ADP ratio in MIN6 cells,
indicating that, as was observed with GIP/Ins cells, glyceraldehyde
stimulates hormone secretion independently from effects on the
intracellular ATP-to-ADP ratio. Interestingly, addition of
glyceraldehyde to MIN6 or GIP/Ins cells caused a nearly fourfold
increase in the intracellular levels of pyruvate, whereas glucose
increased pyruvate levels only approximately twofold for both cell
types.

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Fig. 3.
Insulin secretion in glucose-responsive MIN6 cells. MIN6
cells were plated in 12-well dishes and assayed for rat insulin
secretion, as described in Fig. 1 (n = 4 for each
condition). Other add, other addition; mePyr, 25 mM methyl pyruvate;
Glut, 5 mM glutamate; Gln, 5 mM glutamine. *P < 0.05 and +P < 0.005. #Amount of insulin secreted by all 4 samples was >10 ng/ml, the upper limits of the RIA.
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Gut K cells in vivo do not express Kir 6.1 or Kir 6.2.
The preceding results suggest that secretion by gut K cells is
KATP channel independent. This result would be consistent
with our published observation that GIP-producing cell lines express very low levels of Kir 6.1, Kir 6.2, SUR 1, SUR 2A, and SUR 2B. To
address the issue of whether the GIP/Ins cells reflect what occurs in
vivo, double-label immunohistochemical studies were performed to
determine whether gut K cells express KATP channels. These
channels are expressed in a variety of cell types, including islet
-, vascular smooth muscle, and heart cells, and are composed of four
subunits of a regulatory protein SUR and four subunits of a Kir isoform
(74). Kir 6.2 is the major Kir subunit expressed in islet
-cells. Thus sections of paraffin-embedded mouse pancreas were
stained with goat anti-Kir 6.2 plus guinea pig anti-insulin antibodies
(Fig. 4). As expected, Kir 6.2 was
expressed in insulin-containing islet
-cells. Next, sections of
paraffin-embedded mouse small intestine were stained with goat anti-Kir
6.2 plus rabbit anti-GIP antibodies. As shown in Fig.
5, the Kir 6.2 antibodies recognized an
antigen present in a rare population of cells scattered throughout the
intestinal epithelium (red). Preincubation of this antibody with the
peptide against which it was raised abolished all staining (data not
shown). This pattern of staining is consistent with Kir 6.2 being
expressed in EE cells. However, when the same section was examined for
expression of GIP (green), it was apparent that Kir 6.2 and GIP
expression was discordant. A quantitative analysis revealed that only
2.6% of the gut K cells expressed Kir 6.2 (Table 3). Next, sections of mouse small
intestine were examined for expression of Kir 6.1 plus GIP. As shown in
Fig. 6, only very weak, diffuse
cytoplasmic staining of the intestinal epithelium was observed with the
antibodies directed against Kir 6.1. Importantly, the antibodies did
not stain the plasma membrane where KATP channels associated with cell depolarization would be localized. No specific GIP
and Kir 6.1 colocalization was observed (Fig. 6C). This
observation is consistent with our previous results that showed that
none of 10 independently derived GIP-producing cell lines contained detectable levels of Kir 6.1 transcripts (39). Conversely,
paraffin-embedded sections of mouse heart stained strongly with the
same Kir 6.1 antibodies (Fig. 6A), and this staining was
abolished if the antibodies were preincubated with the peptide used to
immunize the rabbits (data not shown).

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Fig. 4.
Inwardly rectifying potassium (Kir) channel 6.2 is expressed in
pancreatic islet -cells. An adult mouse pancreas was fixed in 4%
paraformaldehyde, embedded in paraffin, and then sectioned. A single
section was deparaffinized, rehydrated using graded alcohols, blocked,
and then incubated for 1 h at room temperature with guinea pig
anti-insulin antibodies plus goat anti-peptide antibodies specific for
Kir 6.2. The section was then washed and incubated with FITC- and
Cy3-conjugated donkey anti-guinea pig plus anti-goat antibodies. Nuclei
were counterstained with bis-benzimide. Sections were examined under a
fluorescent microscope. A-C were photographed to
visualize only insulin (green), Kir 6.2 (red), or nuclei (blue).
D is a merged image of A-C. Note that Kir
6.2 is expressed in insulin-positive islet -cells (open arrow points
to a representative cell) and insulin-negative islet cells (filled
arrow).
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Fig. 5.
Gut K cells in vivo do not express Kir 6.2. A single section of
adult mouse small intestine was treated in the same fashion as the
pancreas described in Fig. 4 and then incubated for 1 h at room
temperature with rabbit anti-GIP antibodies plus goat anti-peptide
antibodies specific for Kir 6.2. The section was then washed and
incubated with FITC- and Cy3-conjugated donkey anti-rabbit and goat
antibodies. Nuclei were counterstained with bis-benzimide. Sections
were examined under a fluorescent microscope. A-C were
photographed to visualize only GIP (green), Kir 6.2 (red), or nuclei
(blue). D is a merged image of A-C.
Intestinal villi are pointing to the top in A-D. Arrowheads
point to cells that expressed GIP but not Kir 6.2; white arrows point
to cells that expressed Kir 6.2 but not GIP; the black arrow with white
outline points to a rare (<3%) cell that coexpressed GIP and Kir 6.2. The faint staining of cells located within the lamina propria
represents autofluorescence observed even in the absence of primary
antibodies. All specific staining was abolished if the primary
antibodies were omitted (data not shown). Preincubation of the Kir 6.2 antibody with the peptide against which it was raised abolished Kir 6.2 straining (data not shown).
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Fig. 6.
Kir 6.1 expression in murine heart and small intestine.
Heart (A) and small intestine (B and
C) were treated as described in Fig. 4. Single sections were
then stained with antibodies to Kir 6.1 (red) and/or GIP (green).
Nuclei were visualized by staining with bis-benzimide (blue).
Preincubation of the Kir 6.1 antibodies with the peptide used to
generate the antibodies abolished all staining of Kir 6.1 (data not
shown). Exposures in A and B are identical so
that the relative levels of Kir 6.1 protein expression in the two
different tissues could be compared. C is the same as
B except GIP staining (green areas indicated by arrowheads)
was included.
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Complex pattern of Kir 6.2 expression in other EE cell populations.
The pattern of Kir 6.2 expression in the intestine suggested that this
KATP subunit is expressed in subpopulations of EE cells. Therefore, additional double-label immunohistochemical studies were
performed using goat anti-Kir 6.2 antibodies plus rabbit antibodies
directed against a host of individual EE cell products. Several
selected examples of the staining patterns are shown in Fig.
7, and a quantitative presentation of all
of the results is presented in Table 3. Like GIP, the incretin hormone
GLP-1 is secreted immediately after ingestion of a meal. We were unable to identify a single GLP-1-positive L cell that was also positive for
Kir 6.2 (Table 3). Similarly, 100 and 99.3% of the cells that stained
positive for CCK and SST, respectively, did not express Kir 6.2 (data
not shown). In stark contrast, 98% of the cells that expressed CGA
also expressed Kir 6.2 (Fig. 7, A-D). In the intestinal
epithelium, SP is expressed predominantly in the crypts, and 95% of
these cells also expressed Kir 6.2. Of the few SP-positive cells that
were located on the villi, ~50% stained positive for Kir 6.2. Both
secretin (Fig. 7, E-H)- and serotonin-producing EE cells
exhibited an intermediate phenotype with 57 and 36% of each
population, respectively, also expressing Kir 6.2.

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Fig. 7.
Kir 6.2 expression in chromogranin A (CGA)- and secretin-producing
enteroendocrine (EE) cells. Samples of mouse small intestine were
stained as described in Fig. 4 except sections were incubated with goat
anti-Kir 6.2 antibodies (red) plus rabbit anti-CGA (green;
A-D) or rabbit anti-secretin (green;
E-H). Black arrows with white outlines point to cells
that coexpress Kir 6.2 plus CGA (D) or secretin
(H). In H, arrowhead points to secretin-producing
cells that do not coexpress Kir 6.2, and solid white arrows point to
cells that express Kir 6.2 but not secretin.
|
|
Secretion from GIP/Ins cells is both dependent and independent of
L-type calcium channels.
The preceding results suggest that gut K cells do not express plasma
membrane-associated KATP channels and that secretion may be
completely independent of the cytoplasmic ATP-to-ADP ratio. Because
intracellular calcium is required for exocytosis, experiments were
conducted to determine whether L-type calcium channels participate in
hormone secretion by GIP/Ins cells. It has been reported that protein
kinase C (PKC) can activate L-type calcium channels (16, 54, 56,
61, 63, 72). Thus the effects of phorbol 12-myristate 13-acetate
(PMA) on both GIP and insulin secretion by GIP/Ins cells were examined.
As shown in Figs. 1 and 8, addition of
10
6 M PMA to GIP/Ins clones 10 and
12 resulted in a fivefold increase in insulin release from
GIP/Ins cells. This increase is similar to that observed after addition
of meat hydrolysate, one of the most potent secretagogues that we have
identified to date for GIP/Ins cells (39). Next, we
examined the effects of verapamil, an inhibitor of L-type calcium
channels, on PMA-stimulated insulin release from GIP/Ins cells. As
shown in Fig. 8, increasing concentrations of verapamil inhibited
insulin secretion from GIP/Ins cells. However, even 0.3 mM verapamil
inhibited secretion by only ~50%. In contrast, 0.03 mM verapamil
almost completely inhibited KCl-stimulated insulin release from the
GIP/Ins cells. As with PMA, verapamil only partially prevented
bombesin-stimulated insulin release (data not shown). Similar results
were obtained when GIP release was determined (Fig.
9), indicating that these results are not
an artifact of measuring insulin, rather than GIP release, from GIP/Ins
cells. These results indicate that, depending on the secretagogue,
calcium mobilization can occur via verapamil-sensitive and -insensitive calcium channels or from intracellular stores.

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Fig. 8.
Verapamil partially inhibits PMA-stimulated insulin
release from GIP/Ins cells. GIP/Ins clone 12 cells were
treated as described in Fig. 1 except the indicated concentration of
verapamil (or vehicle alone) was added 30 min before addition of either
30 mM KCl or 10 6 M PMA. Verapamil was added again along
with the indicated secretagogue. Similar results were obtained in at
least 2 additional experiments using a single concentration of
verapamil (0.1 mM). *P 0.03 and **P 0.003.
|
|

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|
Fig. 9.
GIP and insulin are released simultaneously from GIP/Ins
cells. GIP release was measured in GIP/Ins clone 12 cells as
described in Figs. 1 and 8 except aprotinin was included in the
secretion buffer, as previously described (39). Bom,
10 6 M bombesin; Ver, 0.1 mM verapamil; KCl, 30 mM
potassium chloride. Note that, like its effect on insulin release from
GIP/Ins cells, verapamil completely inhibited KCl-stimulated GIP
release, whereas this inhibitor of L-type calcium channels only
partially inhibited PMA-stimulated GIP release. *P < 0.001.
|
|
Ryanodine receptors (RyRs) mobilize calcium from intracellular stores
and are present in
-cells (19). RyRs can be activated by 1.5 mM IBMX, caffeine, or theophylline. Consistent with this, addition of IBMX to MIN6 cells resulted in a fivefold increase in
insulin release (Fig. 3). In contrast, addition of IBMX, either alone
or along with glucose, had no effect on insulin release by GIP/Ins
clone 10 or clone 12 cells (Fig. 1).
F-1,6-P2 activates RyRs, and, as shown in Tables
1 and 2, the intracellular concentration of this glycolytic
intermediate increased 143-fold in GIP/Ins cells treated with 25 mM
glucose. Taken together, these results strongly suggest that RyRs do
not play a major role in hormone release from GIP/Ins cells.
Although somewhat controversial, intracellular glutamate has been
proposed to represent an important secretagogue for
-cells (25, 26, 71). However, glutamate levels were increased
similarly in GIP/Ins and MIN6 cells after addition of glucose and
actually decreased after addition of glyceraldehyde or methyl pyruvate (Tables 1 and 2). Methyl glutamate, a cell-permeable form of glutamate,
failed to stimulate insulin release from GIP/Ins cells (Fig. 1) and had
very little effect on MIN6 cells (Fig. 3). Furthermore, addition of
glucose plus glutamine, which profoundly increases intracellular
glutamate levels (25), had no effect on secretion by MIN6
(Fig. 3) or GIP/Ins (data not shown) cells. Extracellular glutamate is
a secretagogue for islet
-cells and increased insulin release from
MIN6 cells >15-fold. In contrast, addition of glutamate with or
without glucose failed to stimulate insulin release from GIP/Ins cells
(data not shown). These results argue against intracellular or
extracellular glutamate representing a secretagogue in K cells.
 |
DISCUSSION |
Although both islet
- and gut K cells secrete hormones in
response to similar nutrients, peptide release by gut K cells appears to be KATP channel independent. This conclusion is based on
several convergent observations. First, cytoplasmic ATP/ADP regulates plasma membrane KATP channel activity. Addition of either
glucose or methyl pyruvate to GIP/Ins cells resulted in similar
increases in the intracellular ATP-to-ADP ratio, but only methyl
pyruvate stimulated hormone release. It should also be noted that
metabolism of methyl pyruvate generates ATP in the mitochondria
(31). Thus the measured ATP-to-ADP ratio represents the
maximal ratio present in the cell cytoplasm, and the ATP-to-ADP ratio
in GIP/Ins cells treated with glucose is probably greater than that in
methyl pyruvate-treated cells. Second, the ATP-to-ADP ratio is nearly
three times higher in GIP/Ins cells treated with 25 mM glucose vs.
glyceraldehyde. However, glyceraldehyde, but not glucose, stimulated
hormone release from GIP/Ins cells. Third, RT-PCR analysis of RNA
samples prepared from 10 independently derived GIP-producing cell lines
demonstrated that these cells express much lower levels of Kir 6.2 and
SUR 1 than MIN6 insulinoma cells (39). Furthermore, none
of the 10 cell lines expressed detectable levels of Kir 6.1, SUR 2A, or
SUR 2B transcripts (39). Thus GIP-producing cell lines
express, at best, very low levels of KATP channel subunits.
Fourth, differential sensitivities to SURs and KCOs have been used to
demonstrate that islet
-cells, heart, and vascular smooth muscle
cells express KATP channels composed of different
combinations of subunits (74). KATP channels
in pancreas, heart, and vascular smooth muscle are all inhibited by
glyburide within a 5- to 20-fold range of concentrations. In contrast,
GIP/Ins cells are 30,000-fold less sensitive to glyburide than
-cells (39). KATP channels in pancreas are
more sensitive to diazoxide than are the channels present in heart and
vascular smooth muscle cells, yet GIP/Ins cells are at least 330-fold
less sensitive to this KCO drug than
-cells (39). Thus
either the GIP/Ins cells do not express KATP channels or
the channels are incredibly distinct from those expressed by other
tissues. It is possible that low levels of KATP channels
play a role in maintaining GIP/Ins cells in a hyperpolarized state
rather than in allowing cells to depolarize. However, diazoxide had no
effect on basal GIP or insulin secretion from GIP/Ins cells
(unpublished observation). This result would argue against this
possibility. Fifth, if the GIP-producing cell lines reflect what occurs
in vivo, one would predict that K cells do not express KATP
channels. Double-label antibody staining clearly demonstrated that K
cells in vivo do not express detectable levels of Kir 6.1 or Kir 6.2. Our antibodies against SUR 1 and 2 did not stain either small intestine
or positive control tissues. Thus it is unknown whether these proteins
are expressed in gut K cells in vivo. However, because functional KATP channels require both SUR and Kir subunits and neither
Kir 6.1 nor Kir 6.2 are present in gut K cells, KATP
channels may not be expressed in gut K cells. In a previous report
(39), we demonstrated that, unlike gut K cells in vivo,
GIP/Ins cells do not secrete hormones in response to glucose, and it
was hypothesized that this major GIP "secretagogue" does not act
directly on gut K cells. Rather, glucose uptake and metabolism by
adjacent enterocytes is required for glucose-stimulated GIP release by
K cells (see Ref. 39 for detailed discussion). This
hypothesis is consistent with the results presented in this paper,
since glucose is rapidly metabolized by GIP/Ins cells even though it
does not stimulate hormone release. Furthermore, K cells in vivo do not
express detectable levels of Kir 6.1 or Kir 6.2, and hormone release
from GIP-producing cell lines is independent from the increased
ATP-to-ADP ratio that is generated via a high rate of glucose
metabolism. EE cells that produce GLP-1, CCK, and SST also release
hormones in response to nutrients. It is interesting to note that very
few individual cells that expressed any of these hormones also
expressed detectable levels of Kir 6.2. Therefore, nutrient sensing by
four different subpopulations of EE vs. islet
-cells appears to
occur via distinct mechanisms (see below). However, GIP/Ins cells
secreted GIP and insulin after addition of 30 mM KCl, suggesting that
ATP-independent potassium channels play a role in depolarizing gut K
cells. In support of this hypothesis, it has been reported that parent
STC-1 cells express at least two types of ATP-independent potassium channels (58).
Although not expressed in gut K cells, Kir 6.2 was present in specific
subpopulations of EE cells. Most notably, nearly all of the EE cells
that expressed CGA or SP also expressed Kir 6.2. This suggests that
secretion of CGA and SP immunoreactive hormones and GIP is regulated by
distinct mechanisms. More surprising was the observation that only 43 or 64% of secretin- or serotonin-producing cells, respectively,
coexpressed Kir 6.2. This raises the possibility that secretion of
these hormones is regulated by ATP/ADP-sensitive and
ATP/ADP-insensitive mechanisms. Alternatively, SP- and
secretin-producing EE cells that also express Kir 6.2 may contain
combinations of hormones that are distinct from those that are Kir 6.2 negative. In either case, Kir 6.2 expression can be used to further
define specific subpopulations of gut endocrine cells.
On the basis of results using a Simian virus (SV) 40 T
antigen-transformed cell line, it has been suggested that glucose
stimulates secretion of GLP-1 from gut L cells via closure of
KATP channels (40). However, comparison of
results in Figs. 1, 2, and 6 of that paper reveal that there is no
correlation between the effects of glucose or tolbutamide on the firing
of action potentials and GLP-1 secretion. Furthermore, tolbutamide
stimulated secretion only ~2-fold, whereas glucose plus IBMX and
forskolin stimulated secretion ~15-fold. Thus, at best,
KATP channels play a minor role in regulating secretion by
gut L cells. This conclusion is consistent with our in vivo
immunohistochemical studies indicating that gut L cells, like gut K
cells, do not express detectable Kir 6.1 or Kir 6.2. Patch-clamp
studies have also demonstrated the presence of KATP
channels in parent STC-1 cells (2, 28, 30, 58). Thus it
has been proposed that they play a role in regulating CCK secretion
(2, 27, 28). However, the results presented in this paper
suggest that KATP channels play, at best, a very minor role
in regulating CCK secretion, since CCK-producing cells in vivo do not
express detectable levels of Kir 6.1 or Kir 6.2. STC-1 is a
heterogeneous hormone-producing cell line, and the previous studies
most likely measured KATP channels in CGA-, secretin-, SP-,
or serotonin-expressing cells, since these cells express Kir 6.2 in
vivo. STC-1 cells exhibit at least three types of potassium channel
activities (58) and also express L-type calcium channels.
Bombesin, sodium oleate, phenylalanine, barium chloride, and PMA are
all potent CCK secretagogues for STC-1 cells (6, 29, 30, 58,
59), whereas glucose is a very weak stimulant (27).
Inhibition of L-type calcium channel activity prevented
secretagogue-stimulated CCK release. Thus, as with gut K cells,
secretagogues probably stimulate CCK release by their ability to
mobilize calcium independently of activation of KATP channels. Because SST-producing cells do not express Kir 6.1 or Kir 6.2 in vivo, it seems likely that EE cell secretion of this hormone is also
KATP channel independent.
The results of these studies raise the question as to what controls
calcium mobilization and thus hormone secretion from not only gut K
cells but also the other nutrient-responsive EE cell populations.
Evidence presented in this paper supports the notion that the RyRs are
not involved in calcium mobilization by GIP-producing cells.
Verapamil-sensitive L-type calcium channels play a role in regulating
secretion by GIP/Ins cells since KCl-stimulated hormone release was
completely blocked by verapamil. However, verapamil-insensitive
mechanisms also play an important role in regulating hormone release
from GIP/Ins cells, since this drug inhibited PMA- and
bombesin-stimulated insulin and GIP release by only ~50%. That
verapamil inhibited meat hydrolysate-stimulated hormone release from
GIP/Ins cells (39) suggests that protein hydrolysates may
be directly depolarizing GIP/Ins cells, as was observed after addition
of KCl. Bombesin binds to the GRP receptor and activates phospholipase
C, resulting in increases in inositol phosphate, diacylglycerol, and
intracellular calcium levels (60). Thus bombesin
could potentially mobilize calcium via activation of PKC as well as
from intracellular stores via inositol 1,4,5-trisphosphate receptors
(IP3Rs). IP3Rs are expressed in secretory granules of neuroendocrine
cells and are thought to facilitate secretion by controlling calcium
release from the granules (3). There are at least three
different IP3Rs in mammalian cells, and studies are currently underway
to determine whether any of these isoforms are expressed in gut K cells
in vivo.
Results presented in this and our previous study (39) have
provided unexpected insights into not only gut K cell physiology but
also the regulation of secretion by other EE cell populations. Insulin
and GIP are released from islet
- and gut K cells, respectively, in
response to similar nutrients immediately after ingestion of a meal
(5, 52, 64, 66). This is not surprising, since GIP
potentiates glucose-stimulated insulin release from
-cells. Thus GIP
actually lies upstream of insulin in terms of regulating glucose
homeostasis. EE and islet
-cells are both derived from the primitive
gut endoderm and express many of the same transcription factors and
processing enzymes (23, 36, 37, 55). In fact, transgenes
expressed using the rat insulin promoter (RIP) are frequently expressed
in EE cells and in islet
-cells. For example, the STC-1 EE cell
line, from which the GIP/Ins cells were derived, was isolated from a
murine intestinal carcinoma that arose in a double-transgenic mouse
generated by crossing RIP/SV40 T antigen and RIP/Polyoma small T
antigen mice (43). The intimate relationship between EE
and islet
-cells is further illustrated by the fact that, in some
lower invertebrates that lack islets but contain a brain-gut axis,
insulin is produced and secreted by gut endocrine cells
(9). Thus it is quite surprising that gut K cells do not
appear to express KATP channels and seem to sense glucose in a distinct fashion from islet
-cells. Similarly, why don't other
nutrient-responsive gut endocrine cell populations that produce and
secrete GLP-1, SST, and CCK express KATP channels? On the
other hand, why do EE cells that express CGA immunoreactive peptides
express Kir 6.2 as do islet
-cells? Although these are philosophical
questions, it is clear that GIP and insulin secretion from gut K cells
and islet
-cells, respectively, occurs via very distinct mechanisms.
Because of the roles for GIP in maintaining blood glucose homeostasis
and promoting obesity, it is important to understand the molecular
mechanisms that regulate hormone production and secretion by gut K cells.
 |
ACKNOWLEDGEMENTS |
We thank Drs. David Kipnis, Paul Schlesinger, and Mitsuyoshi Saito
for helpful discussions.
 |
FOOTNOTES |
This work was supported in part by a Career Development Award from the
American Diabetes Association (B. M. Wice) and National Institute
of Diabetes and Digestive and Kidney Diseases Grants 5 P60 DK-20579 and
DK-52574 (Digestive Diseases Research Core Center of Washington University).
Address for reprint requests and other correspondence:
B. M. Wice, Division of Metabolism, Dept. of Internal
Medicine, Washington Univ. School of Medicine, Campus Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail:
bwice{at}im.wustl.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 30, 2002;10.1152/ajpendo.00398.2002
Received 1 November 2002; accepted in final form 29 December 2002.
 |
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