http://ajpendo.physiology.org/content/early/2004/09/21/ajpendo.00346.2004.full.pdf

Articles in PresS. Am J Physiol Endocrinol Metab (September 21, 2004). doi:10.1152/ajpendo.00346.2004
Bombesin and nutrients independently and additively regulate hormone release from
GIP/Ins cells
Lin Li and Burton M. Wice
From the Department of Internal Medicine, Division of Endocrinology, Diabetes & Metabolism,
Washington University School of Medicine, Saint Louis, Missouri 63110
Running title: Regulation of secretion from enteroendocrine cells
Address correspondence to:
Burton M. Wice, Ph.D.
Washington University School of Medicine
Department of Internal Medicine
Division of Endocrinology, Diabetes & Metabolism
Campus Box 8127
660 South Euclid Avenue
Saint Louis, Missouri 63110
Phone: 314-747-0423
Fax: 314-747-2692
Email: [email protected]
Copyright © 2004 by the American Physiological Society.
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Abstract:
Glucose-dependent insulinotropic polypeptide (GIP) regulates glucose homeostasis and high fat
diet-induced obesity and insulin resistance. Therefore, it is important to elucidate the
mechanisms that regulate GIP release. GIP is produced by K cells- a specific sub-type of small
intestinal enteroendocrine (EE) cell. Bombesin-like peptides produced by enteric neurons and
lumenal nutrients stimulate GIP release in vivo. We previously showed that PMA, bombesin,
meat hydrolysate, glyceraldehyde, and methyl-pyruvate increase hormone release from a GIPproducing EE cell line (GIP/Ins cells). Here we demonstrate that bombesin and nutrients
additively stimulate hormone release from GIP/Ins cells. In various cell systems, bombesin and
PMA regulate cell physiology by activating protein kinase D (PKD) signaling in a PKCdependent fashion whereas nutrients regulate cell physiology by inhibiting AMP-activated
protein kinase (AMPK) signaling. Western blot analyses of GIP/Ins cells using antibodies
specific for activated and/or phosphorylated forms of PKD, AMPK, and 1 substrate for each
kinase, revealed that bombesin and PMA, but not nutrients, activated PKC, but not PKD.
Conversely, nutrients, but not bombesin or PMA, inhibited AMPK activity. Pharmacological
studies showed that inhibition of PKC blocked bombesin and PMA stimulated hormone release
but activation of AMPK failed to suppress nutrient-stimulated hormone secretion. Forced
expression of constitutively active versus dominant negative PKDs or AMPKs failed to perturb
bombesin or nutrient-stimulated hormone release. Thus, in GIP/Ins cells, PKC regulates
bombesin-stimulated hormone release whereas nutrients may control hormone release by
regulating the activity of AMPK-related kinases, rather than AMPK itself. These results strongly
suggest that K cells in vivo independently respond to neuronal versus nutritional stimuli via 2
distinct signaling pathways.
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Keywords: K cells, PKC, PKD, AMPK, AMPK-related kinase, enteric neurons, nutrient sensing
4
Introduction:
EE cells are hormone producing intestinal epithelial cells. Although these singly
dispersed cells comprise less than 1% of the intestinal epithelium, as a whole they represent the
largest endocrine organ in the body. There are at least 16 different sub-populations of EE cells
based upon the major product(s) synthesized and secreted by individual cells (1). These
hormones play important roles in regulating gastrointestinal secretion, motility, and blood flow
and also regulate whole animal physiology (1,35,43,48,50,59). For example, glucagon-like
peptides-1 and -2 are important growth and trophic factors for islet β-cells and the intestine,
respectively (13). GIP, ghrelin, CCK, and peptide tyrosine tyrosine regulate food intake and/or
adiposity (3,5,40,49,57). GIP and glucagon like peptide-1 potentiate glucose-stimulated insulin
release and thus, play important roles in maintaining blood glucose homeostasis (18).
Surprisingly, little is known about the molecular mechanisms that regulate hormone release from
different sub-types of EE cells. Because GIP promotes both obesity and glucose-stimulated
insulin release, we have been particularly interested in understanding the molecular mechanisms
that regulate release of this hormone.
GIP is secreted in response to nutrients present in the lumen of the gut but not to those
circulating in the blood (12,18,48). GIP release is also regulated by molecules produced by
enteric neurons [e.g. bombesin-like peptides (29)], other enteroendocrine cells [e.g. somatostatin
inhibits GIP release (59)], and possibly by enterocytes (42,54,60). Therefore, K cells integrate
input from numerous sources in order to release appropriate amounts of GIP. Using GIP/Ins
cells (42), we have begun to study the regulation of K cell physiology using the wellcharacterized islet β-cell as a model.
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In β-cells, glucose metabolism increases the intracellular ATP/ADP ratio, which in turn,
inhibits KATP channels. This causes cell depolarization, influx of calcium via voltage-dependent
calcium channels, and finally exocytosis of insulin from secretory granules. Islet β-cells also
exhibit KATP channel-independent mechanisms of secretion which involve mobilization of
calcium from endoplasmic reticulum (ER) derived stores (44). Release of ER calcium stores can
be regulated by ryanodine receptors (27) and/or inositol 1,4,5-trisphosphate receptors (IP3Rs)
(7,19,55,56). Surprisingly, hormone release from EE cells that produce GIP, glucagon-like
peptide-1, CCK, or somatostatin, but not chromogranin A or serotonin appears to be mostly
independent from KATP channels and IP3Rs (42,61). EE cells that produce secretin or Substance
P express a heterogeneous phenotype with respect to expression of KATP channels and IP3Rs.
Hormone release from GIP/Ins cells is not regulated by ryanodine receptors (unpublished
observation). Therefore, different sub-types of EE cells exhibit unexpected complexity,
heterogeneity, and novelty concerning the molecules that regulate hormone release.
Bombesin, PMA, protein hydrolysates, glyceraldehyde, and methyl-pyruvate are
secretagogues for GIP/Ins cells (42). However, it is unknown which signaling pathways are
activated by these secretagogues. PKD is a serine/threonine protein kinase regulated by
diacylglycerol signaling (58). Bombesin and phorbol esters stimulate proliferation of 3T3 cells
by activating PKD via a PKC-dependent, GF-1 inhibitable signaling pathway (67,68). Thus,
bombesin and PMA could potentially stimulate hormone release from GIP/Ins cells by activating
PKC/PKD signaling. In many systems, AMPK activity is inhibited by nutrients which
coordinately turns on ATP consuming pathways and inhibits ATP generating pathways
(20,21,45). Hydrolyzed proteins, glyceraldehyde, and methyl-pyruvate are nutritionally rich
compounds. Thus, these secretagogues could potentially regulate hormone release from GIP/Ins
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cells by inhibiting AMPK activity. Results presented in this paper indicate that in GIP/Ins cells,
PKC signaling regulates hormone release stimulated by PMA and bombesin, but not nutrients.
Conversely, nutrients, but not PMA or bombesin, regulate AMPK signaling but AMPK-related
kinases rather than AMPK itself may regulate nutrient-stimulated hormone release.
Furthermore, signaling via bombesin and nutrients is independent and additive, which would
allow K cells to independently respond to neural versus nutritional stimuli.
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Experimental Procedures:
Cells and culture conditions: GIP/Ins Clone 10 cells are GIP-producing EE cells that
were engineered to express the human insulin gene. These cells secrete both insulin and GIP in
response to secretagogues (42,60). Thus, insulin release is a surrogate marker for GIP secretion
in these cells. Cells were cultured in an atmosphere of 5% CO2/95% air and 100% humidity in
DMEM containing 10% FBS as previously described (42).
Insulin secretion: Insulin secretion was measured essentially as described (47). Briefly,
cells were plated at ~105 cells/well in 12-well plates. When~80% confluent, cells were washed
twice with secretion buffer [Krebs-Ringer Bicarbonate buffer containing HEPES plus 0.1% BSA
(KRBH-Alb)] (47) and then pre-incubated at 37 oC in secretion buffer. One hour later, buffer
was replaced with fresh KRBH-Alb containing the appropriate secretagogue(s). When more
than 1 secretagogue was added to cells, both compounds were added at the same time and not
sequentially. Sixty minutes later, assay media were collected, centrifuged to remove any
detached cells, and assayed for human insulin by RIA. Bombesin (Calbiochem; San Diego, CA)
was added to the cells from a 100X stock solution prepared in KRBH-Alb. Meat hydrolysate
(Sigma; St. Louis, MO) was prepared as a 20% (W/V) stock solution in PBS. Methyl-pyruvate,
glyceraldehyde (Sigma), and 5-amino-4-imidazolecarboxamide riboside [(AICAR) Calbiochem]
were prepared as 100X stock solutions in water. GF-1 (Sigma) was prepared as 100X stock in
DMSO. GF-1 and AICAR were included during the pre-incubation and also added along with
the indicated secretagogue. All values are the average of quadruplicate samples.
Western blot analysis: Cells were treated exactly as described for insulin secretion
assays. Following collection of the KRBH-Alb, cells were washed twice with ice cold PBS and
lysed directly in 1X SDS-PAGE gel loading buffer. Cell proteins were then separated by SDS-
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PAGE and transferred to nitrocellulose membranes. Membranes were stained with Ponceau S to
confirm that samples were equally loaded and transferred. The names and catalogue numbers for
antibodies obtained from Cell Signaling Technology, Inc. (Beverly, MA) are: PKD/PKCµ
Antibody, #2052; Phospho-PKD/PKCµ (Ser744/748) Antibody, #2054; Phospho-PKD/PKCµ
(Ser916) Antibody, #2051; AMPKα Antibody, #2532; and Phospho-AMPKα(Thr172) antibody,
#2535. The names and catalogue numbers for antibodies obtained from Upstate USA, Inc
(Charlottesville, VA) are: Anti-Acetyl CoA Carboxylase, #07-439 and Anti-phospho-Acetyl
CoA Carboxylase (Ser79) #07-303. Antibodies to Kidins 220 phosphorylated at Serine 919
(#K1725-01) were obtained from United States Biological (Swampscott, MA). Western blots
were performed according to protocols provided with each antibody.
Transient transfection and human growth hormone (hGH) secretion assays: These assays
are similar to those described in which MIN6 insulinoma cells were co-transfected with a human
growth hormone (hGH) reporter construct plus a cDNA that encodes a putative regulatory
protein (11,31). The transiently produced hGH is stored by the endocrine cells and is released
along with insulin following stimulation with appropriate secretagogues. Like MIN6, GIP/Ins
cells store the transiently produced hGH and release it with appropriate GIP secretagogues.
Thus, by transfecting cDNAs that encode hGH along with cDNAs that encode constitutively
active or dominant negative AMPKs or PKDs, we can determine the effects of these protein
kinases on secretagogue-stimulated hormone release from GIP/Ins cells. GIP/Ins cells were
plated at 105 cells per well in 12-well dishes. The next day, quadruplicate wells of cells were
transfected with the indicated cDNAs (see below) using the LT-1 transfection reagent (Panvera,
Madison, WI) according to the manufacturer’s protocol. The following day, cells were washed
twice with fresh media, refed, incubated for an additional 24-hours, and then assayed for hGH
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release using the insulin secretion assay protocol. hGH levels were determined using a high
sensitivity ELISA (Abazyme, Needham, MA). Cells in each well were transfected with 0.25µg
of the pTKGH hGH reporter construct (Nichols Diagnostic, Inc, San Clemente, CA) plus 0.25µg
of the indicated control or test plasmid(s). The constitutively active α1-AMPK (CA-α1-AMPK),
dominant negative α1-AMPK (DN-α1-AMPK), and dominant negative α2-AMPK (DN-α2AMPK) constructs in pCDNA3 were generously provided by Dr. David Carling [(52,63)
Imperial College School of Medicine, University of London, London]. Constitutively active
(∆PH) and kinase dead (K612W; dominant negative) PKD1 constructs in pEGFP-N1 were
generously provided by Dr. Angelika Hausser [(23,28) Institute for Cell Biology and
Immunology, University of Stuttgart, Stuttgart, Germany].
Isolation of pools of stably transfected GIP/Ins cells: GIP/Ins cells were plated in 100mm dishes. The next day, cells were transfected with 1µg of pIRES/hygro (BD Biosciences
Clontech Palo Alto, CA) plus 9µg of pCDNA3, constitutively active AMPK, or dominant
negative α1 plus α2AMPKs (4.5µg of each). Forty-eight hours later, cells were re-fed complete
media containing hygromycin (400µg/ml). The next day, cells were trypsinized and re-plated at
different densities. Cells plated at low densities were allowed to grow as individual colonies,
which allowed estimation of the number and size of clones from cells transfected with each set of
plasmids. Cells re-plated at higher dilutions were trypsinized and maintained as pools until
selection was complete (4-weeks). Hygromycin treatment killed 100% of non-transfected cells
using this protocol.
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Results:
PMA and bombesin, but not nutrients, activate PKC signaling in GIP/Ins cells.
Bombesin and bombesin-like peptides stimulate hormone release from gut endocrine cells in the
absence of additional agonists (29,32,38). In many cell systems, bombesin and PMA activate
PKD signaling via a PKC-dependent, GF-1 inhibitable signaling pathway. Phosphorylation of
PKD at Ser744/748 or Ser916 profoundly increases PKD activity (26,67,68). Therefore, we
determined whether secretagogues increased phosphorylation of these residues in GIP/Ins cells.
Cells were pre-incubated for 60-minutes in the absence of secretagogues, treated for the
indicated time with secretagogues, and then analyzed by Western blots using antibodies against
total or specifically phosphorylated forms of PKD. As shown in Fig 1, bombesin profoundly
increased phosphorylation of PKD on Ser744/748. Maximal stimulation was observed within 15
minutes and increased phosphorylation was maintained for at least 90 minutes (Fig 1).
Throughout this period, total PKD levels or PKD phosphorylated at Ser916 remained relatively
constant. Addition of PMA to GIP/Ins cells resulted in similar patterns of PKD expression and
phosphorylation in a dose-dependent fashion (not shown). Meat hydrolysate is a nutrient-rich
mixture of peptides and amino acids that would be present in the lumen of the gut following
ingestion of a meal. Stimulation of GIP/Ins cells with meat hydrolysate did not alter total PKD
levels or PKD phosphorylation at Ser744/748 or Ser916. GIP/Ins cells do not secrete hormones
in response to glucose (42,60). However, glyceraldehyde and methyl-pyruvate are nutrients that
by-pass glycolysis and stimulate hormone release from these cells (42,60). As with meat
hydrolysate, glyceraldehyde and methyl-pyruvate had no affect on PKD phosphorylation (Fig 2).
Therefore, PKD is phosphorylated in response to PMA and bombesin, but not nutrients.
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PKC regulates bombesin, but not nutrient-, stimulated hormone release. GF-1 is a highly
selective inhibitor of phorbol ester activated PKCs but has no direct effect on PKD activity (58).
Addition of GF-1 to 3T3 cells prevents bombesin-stimulated PKD activation (58). As shown in
figure 2, GF-1 completely inhibited bombesin-stimulated PKD phosphorylation at Ser744/748
but had no effect on phosphorylation at Ser916. GF-1 had no apparent affect on PKD
phosphorylation in cells treated with meat hydrolysate. GF-1 completely prevented bombesinand PMA- stimulated hormone release but had no affect on hormone release from cells treated
with meat hydrolysate (Figure 3 and not shown). Therefore, PKC increases PKD
phosphorylation and also stimulates bombesin- or PMA-, but not nutrient-, stimulated hormone
release.
Kidins220 is selectively expressed in the brain and in neuroendocrine cells and is
phosphorylated on serine919 by activated PKD (24). Thus, Western blots were probed with
antibodies specific for Kidins220 phosphorylated on serine 919. Compared to unstimulated cells
at every time point examined, addition of bombesin, meat hydrolysate, glyceraldehyde, or
methyl-pyruvate did not alter phosphorylation of Kidins220 at Ser919 (Figure 1 and not shown)
suggesting that although PKD phosphorylation is increased, PKD activity may not regulate
hormone release from GIP/Ins cells. To directly test this hypothesis, a transient hGH secretion
assay was utilized to determine whether constitutively active and dominant negative (kinase
dead) PKDs (in pEGPF-N1) perturb secretagogue-stimulated hormone release from GIP/Ins cells
(See Experimental Procedures). Deletion of the pleckstrin homology domain of PKD (PKDdeltaPH) has been shown to produce a constitutively active form of PKD (23,25,28).
Conversely, transient over expression of a K612W kinase dead PKD (PKD-KD) has been shown
to suppress PKD mediated NF-κB-dependent gene expression (28). Empty pEGPF-N1 and
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pCDNA3 vectors served as controls. As shown in figure 4A, GIP/Ins cells transiently expressing
the hGH plus control vectors secrete hGH. As noted for insulin release from non-transfected
cells(figure 3), hGH secretion was increased following addition of bombesin. Importantly, the
kinase dead PKD did not suppress bombesin-stimulated hGH release and the constitutively
active PKD did not increase basal hGH release to levels observed following addition of
bombesin. Thus, PKD activity does not regulate hormone release from GIP/Ins cells. However,
since basal hGH release was similarly increased in cells expressing either PKD construct, it is
possible that the transiently expressed PKDs can interact with and sequester proteins that inhibit
hormone release from GIP/Ins cells. Taken together, these results indicate that bombesin- and
PMA-stimulated hormone release from GIP/Ins cells is PKC-dependent and does not require
PKD activity.
Nutrients, but not bombesin or PMA, inhibit AMPK activity in GIP/Ins cells. Next, we
investigated whether nutrients regulate AMPK, rather than PKC, activity in GIP/Ins cells.
AMPK activity is profoundly increased by phosphorylation at Thr172 (20,45). Typically,
AMPK phosphorylation and activity are high under nutrient poor conditions. GIP/Ins cells were
treated as described in the previous section and then analyzed for total AMPK or AMPK
phosphorylated at Thr172. In the absence of secretagogues, AMPK is highly phosphorylated at
Thr172 (figure 1). Addition of meat hydrolysate caused a rapid and profound decrease in AMPK
phosphorylation that was maintained for at least 90 minutes. Glyceraldehyde and methylpyruvate also inhibited phosphorylation of AMPK at Thr172 (figure 2). Conversely, treatment
of GIP/Ins cells with bombesin (figures 1 and 2) or PMA (not shown) did not reduce AMPK
phosphorylation.
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Activated AMPK phosphorylates ACC at Ser79 (2,11). As shown in figure 1, addition of
meat hydrolysate to GIP/Ins cells caused a rapid and profound decrease in phosphorylation of
ACC at Ser79 that paralleled the decrease in AMPK phosphorylation at Thr172. Similar
decreases were noted in AMPK and ACC phosphorylation when GIP/Ins cells were treated with
glyceraldehyde or methyl-pyruvate (figure 2). Conversely, treatment of cells with bombesin
(figures 1 and 2) or PMA (not shown) had no affect on the phosphorylation states of AMPK or
ACC. Therefore, nutritional secretagogues, but not bombesin or PMA, rapidly inhibit AMPK
activity and signaling in GIP/Ins cells.
AMPK does not regulate nutrient-stimulated hormone release from GIP/Ins cells.
AICAR is a cell permeable analogue of adenosine that can activate AMPK in vivo (53). AICAR
(0.1 to 3 mM) did not inhibit meat hydrolysate-stimulated hormone release from GIP/Ins cells
(not shown). However, addition of 1 mM AICAR reversed much of the meat hydrolysatestimulated decrease in AMPK phosphorylation (Figure 2) but did not prevent the meat
hydrolysate-regulated decrease in ACC phosphorylation (Figure 2). These observations suggest
that AMPK may not be the major regulator of ACC phosphorylation. Two sets of experiments
were conducted to directly determine whether AMPK regulates nutrient stimulated hormone
release from GIP/Ins cells. First, cells were transiently transfected with the hGH reporter
construct along with cDNA vectors that encode a constitutively active-AMPK versus dominant
negative α1 plus α2-AMPKs as described for PKD. Both dominant negative forms of AMPK
were included since inhibition of one AMPK isoform can increase expression of the other (36).
Control cells were transfected with empty pCDNA3 or EGFP vectors. Similar to insulin release
from non-transfected cells, GIP/Ins cells transfected with the hGH reporter plus either control
vector secreted increased amounts of hGH in response to bombesin and meat hydrolysate (Figure
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4B). However, the response to meat hydrolysate was somewhat blunted when compared to that
of non-transfected cells (Figure 3). Basal hGH release was increased to a similar extent in cells
transfected with the dominant negative or constitutively active AMPKs. Furthermore,
expression of the constitutively active AMPK failed to inhibit meat hydrolysate-stimulated hGH
release. As expected, expression of the AMPK plasmids had no effect on bombesin-stimulated
hGH release. Next, separate dishes of GIP/Ins cells were transfected with an empty pCDNA
vector or with constitutively active or α1 plus α2 dominant negative AMPK plasmids. Pools of
stably transfected cells were then isolated and assayed for insulin release as described in figure 3.
Each pool was composed of approximately 1500 similarly sized clones suggesting that AMPK
activity did not alter cell growth or viability (not shown). Addition of meat hydrolysate or
glyceraldehyde increased insulin release from the each of the 3 different pools of transfected
cells 3-fold and 4-fold, respectively (not shown). Therefore, nutrients, but not PMA or
bombesin, inhibit phosphorylation of AMPK in GIP/Ins cells. However, AMPK does not
regulate hormone release.
Bombesin and nutrient signaling pathways independently and additively regulate
hormone release from GIP/Ins cells. The previous results indicate that bombesin and nutrients
regulate different signaling pathways. Nutrient and bombesin signaling pathways could
converge at a common, downstream point or else represent parallel, independent signaling
pathways. To distinguish between these 2 possibilities, GIP/Ins cells were treated with varying
concentrations of bombesin and/or meat hydrolysate. As shown in Fig 5, hormone release was
maximally stimulated by 10-9 M bombesin alone (3-fold) or 0.3% meat hydrolysate alone (2.5fold). Addition of 10-9 M bombesin plus 0.3% meat hydrolysate resulted in a 6-fold increase in
15
hormone release. Therefore, bombesin- and nutrient-regulated signaling pathways are working
in an independent and additive manner to stimulate hormone release from GIP/Ins cells.
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Discussion:
Hormones produced by EE cells regulate a variety of important gastrointestinal and
whole animal physiologic events. Therefore, it is critical for EE cells to be able to tightly control
production and release of specific EE cell products. To accomplish this, each specific sub-type
of EE cell must be able to sense and respond not only to changes in specific lumenal contents,
but also to neurotransmitters released from adjacent neurons as well as to other regulatory
molecules produced by additional intestinal and non-intestinal cell types (35). Previous studies
from this laboratory have shown that different sub-types of EE cells utilize remarkably distinct
and heterogeneous sets of proteins to regulate hormone release (42,60,61).
In many cells, bombesin and PMA activate PKD signaling in a PKC-dependent, GF-1
inhibitable manner (9,34,64,67,68). Bombesin and PMA, but not nutrients, increased
phosphorylation of PKD on Ser744/748, but not on Ser916. Since GF-1 inhibited bombesin and
PMA-stimulated phosphorylation of PKD at Ser744/748, these 2 secretagogues activate PKC in
GIP/Ins cells. Site directed mutagenesis of positions 744/748 of PKD have demonstrated that
phosphorylation of these 2 residues alone is sufficient to regulate PKD activity (26,65,66).
However, addition of bombesin or PMA did not increase phosphorylation of Kidins 220, the only
known in vivo substrate for activated PKD and over expression of constitutively active or
dominant negative PKDs did not perturb secretagogue-stimulated hormone release. Since GF-1
inhibited phosphorylation of PKD and also prevented bombesin- and PMA-stimulated hormone
release, the effects bombesin and PMA are mediated by PKC and are mostly independent of
PKD activity. There have been 2 other reports that examined the potential role for PKD in
regulating hormone release from gut endocrine cells. In agreement with our results, Moore et al
presented evidence that PKC, but not PKD, regulates hormone release from purified human
17
antral gastrin-producing EE cells (38). The authors also showed that gastrin-producing EE cells
express at least 6 different isoforms of PKC. Thus, it will be important to determine which PKC
isoforms are expressed by GIP/Ins cells and to evaluate the physiologic role for each isozyme.
Conversely, Li et al concluded that PKD plays a central role in regulating neurotensin release
from BON cells (32). However, in these latter studies, expression of a constitutively active PKD
in BON cells increased basal neurotensin release only about 2-fold whereas PMA increased
neurotensin release about 10-fold. In addition, BON cells transfected with the gastrin releasing
peptide receptor exhibit a 10-fold increase in neurotensin release following addition of
bombesin. However, following transfection with a PKD siRNA, these same cells exhibited only
a 2-fold reduction in bombesin-stimulated neurotensin release even though PKD expression and
activation appeared to be completely blocked. Thus, PKC may also be much more important
than PKD for regulating neurotensin release.
Phosphorylation of AMPK at Thr172 greatly increases its enzyme activity (20). Addition
of meat hydrolysate, glyceraldehyde, or methyl-pyruvate, but not PMA or bombesin, to GIP/Ins
cells resulted in decreased phosphorylation of AMPK at Thr172. AMPK signaling also appeared
to be inhibited since these same nutrients suppressed phosphorylation of ACC. However, forced
expression of constitutively active versus dominant negative AMPKs did not perturb
secretagogue-stimulated hormone release from GIP/Ins cells. These results suggest that AMPK
signaling does not regulate hormone release but may be important for regulating other aspects of
K cell physiology. In contrast to our results, forced expression of a dominant negative AMPK in
MIN6 insulinoma cells led to unregulated insulin release whereas forced expression of a
constitutively active AMPK blocked glucose-stimulated insulin release (11). These results
18
further illustrate the remarkable differences in the proteins that regulate hormone release from
islet β cells versus GIP-producing EE cells.
It was initially unclear why AICAR, which can activate AMPK in other systems, failed to
inhibit insulin release from GIP/Ins cells. One possible explanation is that GIP/Ins cells do not
efficiently metabolize AICAR. Alternatively, there is some evidence that in islet β cells, AICAR
can be phosphorylated to ZMP, which can then be phosphorylated further to ZTP, an ATP
mimetic. This, in turn, could stimulate depolarization and subsequent hormone release from βcells by inhibiting KATP channel activity without inhibiting AMPK activity (45). However, KATP
channels do not regulate hormone release from GIP/Ins cells (42,60). Therefore, it is possible
that additional intermediates derived from metabolism of AICAR could inhibit, rather than
activate, AMPK. A much more intriguing and unifying explanation for our results is that
nutrients control hormone release from GIP/Ins cells by regulating activity of AMPK-related
kinases, rather than AMPK itself. Twelve human AMPK-related kinases have recently been
identified and partially characterized (33). Like AMPK, eleven of these AMPK-related kinases
are phosphorylated in their T-loops by LKB1, an AMPK kinase. Phosphorylation at these sites
increased enzyme activity >50-fold. In contrast to AMPK, the activity of LKB1 or the AMPKrelated kinases is not increased by AICAR. Although there were marked differences in the
relative rates, all of the AMPK-related kinases also phosphorylated each of 3 peptides that are
substrates for AMPK. Therefore, our results suggest that AMPK-related kinases regulate
hormone release from GIP/Ins cells since: 1) AICAR activated AMPK but did not inhibit
phosphorylation of ACC, a substrate for AMPK; 2) AICAR did not inhibit nutrient-stimulated
hormone release, and 3) forced expression of AMPKs did not perturb hormone release from
GIP/Ins cells.
19
It was predicted that PMA and bombesin versus nutrients would control hormone release
by regulating different signaling pathways. However, it was unexpected to see that there was no
apparent cross talk between the PKC and AMPK/AMPK-related kinase signaling pathways and
that hormone release regulated by these 2 pathways was additive. Since bombesin-like peptide
are produced by enteric neurons, these results imply that K cells independently sense and
respond to nutritional and neuronal stimuli.
As already discussed, K cells do not utilize many of the same regulatory proteins to
control hormone release that are used by other endocrine, excitatory, and some EE cells
(42,60,61). However, evidence presented in this paper indicates that GIP secretagogues regulate
2 conserved signaling pathways in GIP/Ins cells. This implies that steps leading to the activation
of PKC and/or inactivation of AMPK/AMPK-related kinases may represent critical steps in
regulating K cell physiology and GIP release in vivo. This also implies that PKC and
APMK/AMPK-related kinase signaling can regulate hormone release from other sub-types of EE
cells and that cell-specific differences in the regulatory cascades that activate or inactivate these
kinases control release of other hormones. For example, cell-specific molecules that regulate
bombesin receptor signaling could potentially confer differential responses to bombesin-like
peptides in distinct sub-types of EE cells. With respect to nutrients, GIP is released in response
to a different set of amino acids from those that stimulate gastrin and CCK release (59).
Therefore, amino acid transporters present on one type of EE cell but not on another could
potentially initiate cell-specific cascades that inhibit AMPK/AMPK-related kinase signaling and
thus, stimulate release of hormones from only a specific type of EE cell. However, it is also
possible that different sub-types of EE cells express distinct AMPK-related kinases and each of
these kinases can be regulated by metabolites derived from different nutrients.
20
There is a large body of biochemical, physiologic, and genetic evidence that indicates
GIP promotes obesity and insulin resistance (4,6,8,10,14-17,22,30,37,39,41,46,51,62).
Furthermore, mice lacking GIP receptors do not exhibit any serious detrimental phenotypes (37).
Therefore, inhibition of GIPR signaling is a potential strategy to ameliorate obesity and insulin
resistance. This could be accomplished by development of GIPR antagonists. An alternative
and complementary approach would be to inhibit GIP release from K cells. One advantage of
this latter approach is that potential drugs could be delivered orally to K cells, which could
minimize adverse side effects due to systemic drug delivery. A second advantage is that
different sub-types of EE cells utilize distinct proteins to control hormone release (42) suggesting
drugs that inhibit GIP release, but do not adversely affect other cell populations, could be
rationally developed (60,61). Therefore, as a first step in developing potential anti-obesity drugs,
it is critical to elucidate the mechanisms that regulate GIP release from K cells so that K cellspecific components of those pathways can be defined. Studies are underway to determine the
roles of PKC, PKD, AMPK, and AMPK-related kinase signaling pathways in regulating
hormone release from K cells in vivo, as well as from other sub-types of EE cells, and to identify
cell-specific components of these pathways.
21
Acknowledgments: The authors wish to thank Drs. David Kipnis and Ernesto Bernal for
helpful discussions. This work was partly supported by a Career Development Award from the
American Diabetes Association (BMW) and the Washington University Diabetes and Research
Training Center (NIH 5 P60 DK20579).
22
References:
1. Aiken KD, Kisslinger JA, and Roth KA. Immunohistochemical Studies Indicate
Multiple Enteroendocrine Cell Differentiation Pathways in the Mouse Proximal
Small Intestine. Developmental Dynamics 201: 636-70, 1994.
2. Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D,
and Small CJ. AMP-activated protein kinase plays a role in the control of food
intake. J Biol Chem 279: 12005-12008, 2004.
3. Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, Makino S, Fujimiya
M, Niijima A, Fujino MA, and Kasuga M. Ghrelin is an appetite-stimulatory
signal from stomach with structural resemblance to motilin. Gastroenterology
120: 337-345, 2001.
4. Baba AS, Harper JM, and Buttery PJ. Effects of gastric inhibitory polypeptide,
somatostatin and epidermal growth factor on lipogenesis in ovine adipose
explants. Comp Biochem Physiol B Biochem Mol Biol 127: 173-182, 2000.
5. Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM,
Brynes AE, Low MJ, Ghatei MA, Cone RD, and Bloom SR. Gut hormone
PYY(3-36) physiologically inhibits food intake. Nature 418: 650-654, 2002.
6. Beck B, Max JP, and Villaume C. Effects of somatostatin on the insulin- and gastric
inhibitory polypeptide-stimulated fatty acid esterification in rat adipose tissue. Int
J Obes 12: 41-47, 1988.
7. Blondel O, Bell GI, and Seino S. Inositol 1,4,5-trisphosphate receptors, secretory
granules and secretion in endocrine and neuroendocrine cells. Trends Neurosci
18: 157-161, 1995.
8. Chini EN, Chini CC, Kato I, Takasawa S, and Okamoto H. CD38 is the major
enzyme responsible for synthesis of nicotinic acid-adenine dinucleotide phosphate
in mammalian tissues. Biochem J 362: 125-130, 2002.
9. Chiu T and Rozengurt E. PKD in intestinal epithelial cells: rapid activation by phorbol
esters, LPA, and angiotensin through PKC. Am J Physiol Cell Physiol 280: C929C942, 2001.
10. Creutzfeldt W, Ebert R, Willms B, Frerichs H, and Brown JC. Gastric inhibitory
polypeptide (GIP) and insulin in obesity: increased response to stimulation and
defective feedback control of serum levels. Diabetologia 14: 15-24, 1978.
11. Da S, X, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Role for AMPactivated protein kinase in glucose-stimulated insulin secretion and preproinsulin
gene expression. Biochem J 371: 761-774, 2003.
12. Drucker DJ. Glucagon-Like Peptides. Diabetes 47: 159-169, 1998.
23
13. Drucker DJ. Glucagon-like peptides: regulators of cell proliferation, differentiation, and
apoptosis. Mol Endocrinol 17: 161-171, 2003.
14. Ebert R, Frerichs H, and Creutzfeldt W. Impaired feedback control of fat induced
gastric inhibitory polypeptide (GIP) secretion by insulin in obesity and glucose
intolerance. Eur J Clin Invest 9: 129-135, 1979.
15. Ebert R, Nauck M, and Creutzfeldt W. Effect of exogenous or endogenous gastric
inhibitory polypeptide (GIP) on plasma triglyceride responses in rats. Horm
Metab Res 23: 517-521, 1991.
16. Eckel RH, Fujimoto WY, and Brunzell JD. Gastric inhibitory polypeptide enhanced
lipoprotein lipase activity in cultured preadipocytes. Diabetes 28: 1141-1142,
1979.
17. Elahi D, Andersen DK, Muller DC, Tobin JD, Brown JC, and Andres R. The enteric
enhancement of glucose-stimulated insulin release. The role of GIP in aging,
obesity, and non-insulin-dependent diabetes mellitus. Diabetes 33: 950-957,
1984.
18. Fehmann H, Goke R, and Goke B. Cell and Molecular Biology of the Incretin
Hormones Glucagon-Like Peptide-1 and Glucose-Dependent Insulin Releasing
Polypeptide. Endocrine Reviews 16: 390-410, 1995.
19. Hagar RE and Ehrlich BE. Regulation of the type III InsP3 receptor and its role in beta
cell function. Cell Mol Life Sci 57: 1938-1949, 2000.
20. Hardie DG, Carling D, and Carlson M. The AMP-activated/SNF1 protein kinase
subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 67: 821855, 1998.
21. Hardie DG, Scott JW, Pan DA, and Hudson ER. Management of cellular energy by
the AMP-activated protein kinase system. FEBS Lett 546: 113-120, 2003.
22. Hauner H, Glatting G, Kaminska D, and Pfeiffer EF. Effects of gastric inhibitory
polypeptide on glucose and lipid metabolism of isolated rat adipocytes. Ann Nutr
Metab 32: 282-288, 1988.
23. Hausser A, Link G, Bamberg L, Burzlaff A, Lutz S, Pfizenmaier K, and Johannes
FJ. Structural requirements for localization and activation of protein kinase C mu
(PKC mu) at the Golgi compartment. J Cell Biol 156: 65-74, 2002.
24. Iglesias T, Cabrera-Poch N, Mitchell MP, Naven TJ, Rozengurt E, and Schiavo G.
Identification and cloning of Kidins220, a novel neuronal substrate of protein
kinase D. J Biol Chem 275: 40048-40056, 2000.
25. Iglesias T and Rozengurt E. Protein kinase D activation by mutations within its
pleckstrin homology domain. J Biol Chem 273: 410-416, 1998.
24
26. Iglesias T, Waldron RT, and Rozengurt E. Identification of in vivo phosphorylation
sites required for protein kinase D activation. J Biol Chem 273: 27662-27667,
1998.
27. Islam MS. The ryanodine receptor calcium channel of beta-cells: molecular regulation
and physiological significance. Diabetes 51: 1299-1309, 2002.
28. Johannes FJ, Horn J, Link G, Haas E, Siemienski K, Wajant H, and Pfizenmaier K.
Protein kinase Cmu downregulation of tumor-necrosis-factor-induced apoptosis
correlates with enhanced expression of nuclear-factor-kappaB-dependent
protective genes. Eur J Biochem 257: 47-54, 1998.
29. Kieffer TJ, Buchan AM, Barker H, Brown JC, and Pederson RA. Release of gastric
inhibitory polypeptide from cultured canine endocrine cells. Am J Physiol 267:
E489-E496, 1994.
30. Knapper JM, Puddicombe SM, Morgan LM, Fletcher JM, and Marks V. Glucose
dependent insulinotropic polypeptide and glucagon-like peptide-1(7-36)amide:
effects on lipoprotein lipase activity. Biochem Soc Trans 21: 135S, 1993.
31. Kotake K, Ozaki N, Mizuta M, Sekiya S, Inagaki N, and Seino S. Noc2, a putative
zinc finger protein involved in exocytosis in endocrine cells. J Biol Chem 272:
29407-29410, 1997.
32. Li J, O'Connor KL, Hellmich MR, Greeley GHJ, Townsend CMJ, and Evers BM.
The Role of Protein Kinase D in Neurotensin Secretion Mediated by Protein
Kinase C-{alpha}/-{delta} and Rho/Rho Kinase. J Biol Chem 279: 28466-28474,
2004.
33. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA,
Udd L, Makela TP, Hardie DG, and Alessi DR. LKB1 is a master kinase that
activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J
23: 833-843, 2004.
34. Matthews SA, Rozengurt E, and Cantrell D. Protein kinase D. A selective target for
antigen receptors and a downstream target for protein kinase C in lymphocytes. J
Exp Med 191: 2075-2082, 2000.
35. Miller, L.J. Gastrointestinal Hormones and Receptors. In: Textbook of Gastroenterology,
edited by T. Yamada, D.H. Alpers, L. Laine, C. Owyang, and D.W. Powell.
Philadelphia: Lippincott Williams & Wilkens, 1999, p. 35-66.
36. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F,
Ferre P, Birnbaum MJ, Stuck BJ, and Kahn BB. AMP-kinase regulates food
intake by responding to hormonal and nutrient signals in the hypothalamus.
Nature 428: 569-574, 2004.
25
37. Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H, Fujimoto S, Oku
A, Tsuda K, Toyokuni S, Hiai H, Mizunoya W, Fushiki T, Holst JJ, Makino
M, Tashita A, Kobara Y, Tsubamoto Y, Jinnouchi T, Jomori T, and Seino Y.
Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat Med
8: 738-742, 2002.
38. Moore ED, Ring M, Scriven DR, Smith VC, Meloche RM, and Buchan AM. The role
of protein kinase C isozymes in bombesin-stimulated gastrin release from human
antral gastrin cells. J Biol Chem 274: 22493-22501, 1999.
39. Morgan LM, Hampton SM, Tredger JA, Cramb R, and Marks V. Modifications of
gastric inhibitory polypeptide (GIP) secretion in man by a high-fat diet. Br J Nutr
59: 373-380, 1988.
40. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, and
Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 409:
194-198, 2001.
41. Oben J, Morgan L, Fletcher J, and Marks V. Effect of the entero-pancreatic
hormones, gastric inhibitory polypeptide and glucagon-like polypeptide-1(7-36)
amide, on fatty acid synthesis in explants of rat adipose tissue. J Endocrinol 130:
267-272, 1991.
42. Ramshur EB, Rull TR, and Wice BM. Novel insulin/GIP-producing cell lines provide
unexpected insights into gut K-cell function in vivo. Journal of Cellular
Physiology 192: 339-350, 2002.
43. Roth KA and Gordon JI. Spatial differentiation of the intestinal epithelium: Analysis of
enteroendocrine cells containing immunoreactive serotonin, secretin, and
substance P in normal and transgenic mice. Proceedings of the National Academy
of Sciences 87: 6408-6412, 1990.
44. Rutter GA. Nutrient-secretion coupling in the pancreatic islet beta-cell: recent advances.
Mol Aspects Med 22: 247-284, 2001.
45. Rutter GA, Da S, X, and Leclerc I. Roles of 5'-AMP-activated protein kinase (AMPK)
in mammalian glucose homoeostasis. Biochem J 375: 1-16, 2003.
46. Salera M, Giacomoni P, Pironi L, Cornia G, Capelli M, Marini A, Benfenati F,
Miglioli M, and Barbara L. Gastric inhibitory polypeptide release after oral
glucose: relationship to glucose intolerance, diabetes mellitus, and obesity. J Clin
Endocrinol Metab 55: 329-336, 1982.
47. Sandler, S. and D.L. Eizirik. Culture of human pancreatic islet cells. In: Methods in
Molecular Medicine: Human Cell Culture Protocols, edited by G.E. Jones.
Totowa, New Jersey: Humana Press, Inc., 1994, p. 391-407.
26
48. Schmier, W.E. Gastrointestinal Hormones. In: Textbook of Gastroenterology, edited by
T. Yamada. Philadelphia: JB Lippincott Company, 1995, p. 25-71.
49. Schwartz MW, Woods SC, Porte DJ, Seeley RJ, and Baskin DG. Central nervous
system control of food intake. Nature 404: 661-671, 2000.
50. Sjolund K, Sanden G, Hakanson R, and Sundler F. Endocrine cells in human
intestine: an immunocytochemical study. Gastroenterology 85: 1120-1130,
1983.
51. Starich GH, Bar RS, and Mazzaferri EL. GIP increases insulin receptor affinity and
cellular sensitivity in adipocytes. Am J Physiol 249: E603-E607, 1985.
52. Stein SC, Woods A, Jones NA, Davison MD, and Carling D. The regulation of AMPactivated protein kinase by phosphorylation. Biochem J 345 Pt 3: 437-443, 2000.
53. Sullivan JE, Brocklehurst KJ, Marley AE, Carey F, Carling D, and Beri RK.
Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a
cell-permeable activator of AMP-activated protein kinase. FEBS Lett 353: 33-36,
1994.
54. Sykes S, Morgan LM, English J, and Marks V. Evidence for preferential stimulation
of gastric inhibitory polypeptide secretion in the rat by actively transported
carbohydrates and their analogues. Journal of Endocrinology 85: 201-207, 1980.
55. Taylor CW, Genazzani AA, and Morris SA. Expression of inositol trisphosphate
receptors. Cell Calcium 26: 237-251, 1999.
56. Thrower EC, Hagar RE, and Ehrlich BE. Regulation of Ins(1,4,5)P3 receptor isoforms
by endogenous modulators. Trends Pharmacol Sci 22: 580-586, 2001.
57. Tschop M, Smiley DL, and Heiman ML. Ghrelin induces adiposity in rodents. Nature
407: 908-913, 2000.
58. Waldron RT, Iglesias T, and Rozengurt E. Phosphorylation-dependent protein kinase
D activation. Electrophoresis 20: 382-390, 1999.
59. Walsh, J.H. Gastrointestinal Hormones. In: Physiology of the Gastrointestinal Tract,
edited by L.R. Johnson. New York: Raven Press, 1994, p. 1-128.
60. Wang SY, Chi MM, Li L, Moley KH, and Wice BM. Studies with GIP/Ins Cells
Indicate Secretion by Gut K-Cells is KATP Channel-Independent. American
Journal of Physiology- Endocrinology 284: E988-E1000, 2003.
61. Wang SY, Liu J, Li L, and Wice BM. Individual sub-types of enteroendocrine cells in
the mouse small intestine exhibit unique patterns of inositol 1,4,5-trisphosphate
receptor expression. Journal of Histochemistry and Cytochemistry 52: 53-63,
2004.
27
62. Wasada T, McCorkle K, Harris V, Kawai K, Howard B, and Unger RH. Effect of
gastric inhibitory polypeptide on plasma levels of chylomicron triglycerides in
dogs. J Clin Invest 68: 1106-1107, 1981.
63. Woods A, Azzout-Marniche D, Foretz M, Stein SC, Lemarchand P, Ferre P,
Foufelle F, and Carling D. Characterization of the role of AMP-activated protein
kinase in the regulation of glucose-activated gene expression using constitutively
active and dominant negative forms of the kinase. Mol Cell Biol 20: 6704-6711,
2000.
64. Yuan J, Bae D, Cantrell D, Nel AE, and Rozengurt E. Protein kinase D is a
downstream target of protein kinase Ctheta. Biochem Biophys Res Commun 291:
444-452, 2002.
65. Yuan J, Slice L, Walsh JH, and Rozengurt E. Activation of protein kinase D by
signaling through the alpha subunit of the heterotrimeric G protein G(q). J Biol
Chem 275: 2157-2164, 2000.
66. Yuan J, Slice LW, and Rozengurt E. Activation of protein kinase D by signaling
through Rho and the alpha subunit of the heterotrimeric G protein G13. J Biol
Chem 276: 38619-38627, 2001.
67. Zhukova E, Sinnett-Smith J, and Rozengurt E. Protein kinase D potentiates DNA
synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol
esters in Swiss 3T3 cells. J Biol Chem 276: 40298-40305, 2001.
68. Zugaza JL, Waldron RT, Sinnett-Smith J, and Rozengurt E. Bombesin, vasopressin,
endothelin, bradykinin, and platelet-derived growth factor rapidly activate protein
kinase D through a protein kinase C-dependent signal transduction pathway. J
Biol Chem 272: 23952-23960, 1997.
28
Figure Legends:
Figure 1. Bombesin and meat hydrolysate regulate PKC and AMPK signaling, respectively,
in GIP/Ins cells. Cells were cultured in standard growth media until 80% confluent and preincubated for 60-minutes in KRBH-Alb without secretagogues. Fresh KRBH-Alb containing no
secretagogue (None), 10-7 M bombesin (Bomb), or 1% meat hydrolysate (MH) was then added to
cells. Cells were harvested for Western blots at the indicated time following addition of
secretagogues. From top to bottom panels, Western blots were probed with antibodies specific
for PKD phosphorylated at Ser744/748, PKD phosphorylated at Ser916, total PKD, Kidins 220
phosphorylated at Serine 919, AMPK phosphorylated at Thr172, total AMPK, and ACC
phosphorylated at Ser79. Bombesin, but not meat hydrolysate, increases PKC activity since
PKD phosphorylation is increased at Ser744/748. Conversely, meat hydrolysate, but not
bombesin, decreases AMPK signaling since this nutrient reduces AMPK-P and ACC-P levels.
Figure 2. PKC regulates PKD phosphorylation in GIP/Ins cells. Cells were treated as
described in figure 1 and harvested just before (0 minutes) or 60 minutes after addition of
secretagogues. Cells were analyzed by Western blots as described in Fig 1. When added, GF-1
(3.5 µM) and AICAR (1 mM) were present during the pre-incubation and the treatment with
secretagogues. Note that a) GF-1 inhibits bombesin-stimulated phosphorylation of PKD; b)
AICAR, which activates AMPK in many systems, partially activated AMPK in GIP/Ins cells
treated with meat hydrolysate; and c) meat hydrolysate, glyceraldehyde (Glycerald) and methylpyruvate (me-Pyr), but not bombesin, inhibited AMPK activation and signaling.
29
Figure 3. Inhibition of PKC prevents bombesin-, but not meat hydrolysate-stimulated
hormone release from GIP/Ins cells. Cells were treated with or without GF-1 plus bombesin
(Panel A) or meat hydrolysate (Panel B) as described in figures 1 and 2. Secretion buffer was
collected after 60 minutes and assayed for human insulin. Note that GF-1 inhibits bombesinstimulated insulin release and has no affect on meat hydrolysate-stimulated insulin release. GF-1
also completely inhibited PMA-stimulated hormone release (not shown).
Figure 4. PKD and AMPK do not regulate hormone release from GIP/Ins cells. GIP/Ins
cells were transiently co-transfected with an hGH reporter construct plus the indicated control,
PKD, or AMPK expression vector(s). Cells were then treated with no secretagogue (“None”),
with bombesin (Bomb) or meat hydrolysate (MH) and assayed for hGH released into the
medium. Abbreviations are: pCDNA3, pCDNA3 vector with no insert; EGFP, pEGFP-N1
vector with no insert; PKD-delta PH, constitutively active PKD in pEGFP-N1; PKD-KD, kinase
dead PKD in pEGFP-N1; CA-AMPK, constitutively active AMPK in pCDNA3; DN-AMPK,
equal amounts of dominant negative α1 plus α2 AMPKs in pCDNA3. Each condition was
tested in quadruplicate and results are means +/- SEM. Similar results were obtained in a second
independent experiment. The pCDNA3 and pEGFP controls for panels A and B are the same
samples. Note that cells expressing the constitutively active versus kinase dead or dominant
negative constructs respond similarly to all test conditions indicating that PKD or AMPK activity
does not regulate hormone release from GIP/Ins cells.
Figure 5. Bombesin and nutrients independently and additively regulate hormone release
from GIP/Ins cells. Secretion assays were conducted with GIP/Ins cells treated with the
30
indicated concentrations of bombesin and meat hydrolysate. Both secretagogues were added to
cells at the same time. Note that insulin release is additive in cells treated with maximal
concentrations of bombesin plus a maximal concentration of meat hydrolysate.