Glucose-mediated control of ghrelin release from primary cultures of gastric mucosal cells

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DOI: 10.1152/ajpendo.00041.2012 · Source: PubMed
Abstract
The peptide hormone ghrelin is released from a distinct group of gastrointestinal cells in response to caloric restriction, whereas its levels fall after eating. The mechanisms by which ghrelin secretion is regulated remain largely unknown. Here, we have used primary cultures of mouse gastric mucosal cells to investigate ghrelin secretion, with an emphasis on the role of glucose. Ghrelin secretion from these cells upon exposure to different d-glucose concentrations, the glucose antimetabolite 2-deoxy-d-glucose, and other potential secretagogues was assessed. The expression profile of proteins involved in glucose transport, metabolism, and utilization within highly enriched pools of mouse ghrelin cells and within cultured ghrelinoma cells was also determined. Ghrelin release negatively correlated with d-glucose concentration. Insulin blocked ghrelin release, but only in a low d-glucose environment. 2-Deoxy-d-glucose prevented the inhibitory effect of high d-glucose exposure on ghrelin release. mRNAs encoding several facilitative glucose transporters, hexokinases, the ATP-sensitive potassium channel subunit Kir6.2, and sulfonylurea type 1 receptor were expressed highly within ghrelin cells, although neither tolbutamide nor diazoxide exerted direct effects on ghrelin secretion. These findings suggest that direct exposure of ghrelin cells to low ambient d-glucose stimulates ghrelin release, whereas high d-glucose and glucose metabolism within ghrelin cells block ghrelin release. Also, low d-glucose sensitizes ghrelin cells to insulin. Various glucose transporters, channels, and enzymes that mediate glucose responsiveness in other cell types may contribute to the ghrelin cell machinery involved in regulating ghrelin secretion under these different glucose environments, although their exact roles in ghrelin release remain uncertain.
Glucose-mediated control of ghrelin release from primary cultures
of gastric mucosal cells
Ichiro Sakata,
1,2
* Won-Mee Park,
1
* Angela K. Walker,
1
Paul K. Piper,
3
Jen-Chieh Chuang,
1
Sherri Osborne-Lawrence,
1
and Jeffrey M. Zigman
1,3,4
1
Division of Hypothalamic Research, Department of Internal Medicine, University of Texas Southwestern Medical Center,
Dallas, Texas;
2
Graduate School of Science and Engineering, Division of Life Sciences, Saitama University, Saitama, Japan;
3
Division of Endocrinology and Metabolism, Department of Internal Medicine; and
4
Department of Psychiatry, University of
Texas Southwestern Medical Center, Dallas, Texas
Submitted 23 January 2012; accepted in final form 12 March 2012
Sakata I, Park WM, Walker AK, Piper PK, Chuang JC,
Osborne-Lawrence S, Zigman JM. Glucose-mediated control of
ghrelin release from primary cultures of gastric mucosal cells. Am J
Physiol Endocrinol Metab 302: E1300 –E1310, 2012. First published
March 13, 2012; doi:10.1152/ajpendo.00041.2012.—The peptide hor-
mone ghrelin is released from a distinct group of gastrointestinal cells
in response to caloric restriction, whereas its levels fall after eating.
The mechanisms by which ghrelin secretion is regulated remain
largely unknown. Here, we have used primary cultures of mouse
gastric mucosal cells to investigate ghrelin secretion, with an empha-
sis on the role of glucose. Ghrelin secretion from these cells upon
exposure to different
D-glucose concentrations, the glucose antime-
tabolite 2-deoxy-
D-glucose, and other potential secretagogues was
assessed. The expression profile of proteins involved in glucose
transport, metabolism, and utilization within highly enriched pools of
mouse ghrelin cells and within cultured ghrelinoma cells was also
determined. Ghrelin release negatively correlated with
D-glucose
concentration. Insulin blocked ghrelin release, but only in a low
D-glucose environment. 2-Deoxy-D-glucose prevented the inhibitory
effect of high
D-glucose exposure on ghrelin release. mRNAs encod-
ing several facilitative glucose transporters, hexokinases, the ATP-
sensitive potassium channel subunit Kir6.2, and sulfonylurea type 1
receptor were expressed highly within ghrelin cells, although neither
tolbutamide nor diazoxide exerted direct effects on ghrelin secretion.
These findings suggest that direct exposure of ghrelin cells to low
ambient
D-glucose stimulates ghrelin release, whereas high D-glucose
and glucose metabolism within ghrelin cells block ghrelin release.
Also, low
D-glucose sensitizes ghrelin cells to insulin. Various glucose
transporters, channels, and enzymes that mediate glucose responsive-
ness in other cell types may contribute to the ghrelin cell machinery
involved in regulating ghrelin secretion under these different glucose
environments, although their exact roles in ghrelin release remain
uncertain.
secretion
THE PEPTIDE HORMONE GHRELIN is the endogenous ligand of the
growth hormone secretagogue receptor (GHSR) and is named
for its ability to stimulate growth hormone release (32, 59).
Ghrelin also regulates gastrointestinal motility, chronic stress-
induced mood-related behaviors, and alcohol-seeking behav-
iors, among many other actions (1, 13, 15, 18, 31, 32, 34, 37).
Perhaps best studied are ghrelin’s actions in signaling and
responding to states of energy insufficiency. Regarding its role
in signaling energy-insufficient states, ghrelin levels are known
to rise prior to set meals, following food deprivation, and after
weight loss linked to exercise, cachexia, and anorexia nervosa
(9, 10, 33, 39, 42, 45, 55, 57, 60). Several lines of evidence
suggest that the rise in plasma ghrelin upon caloric restriction
is likely related, at least in part, to binding of norepinephrine
released from the sympathetic nervous system to
1
-adrenergic
receptors embedded in the plasma membranes of ghrelin cells
(14, 28, 41, 63). Regarding ghrelin’s role in responding to
energy-insufficient states, infusions of ghrelin or GHSR ago-
nists increase body weight via proorexigenic actions and/or
decreases in energy expenditure (1, 43, 53, 59, 61). Ghrelin
shifts fuel preference away from metabolic utilization of fat as
an energy source and increases the expression of fat storage-
promoting enzymes (51, 56, 59). Also, ghrelin plays an oblig-
atory role in mediating various hedonic components of eating
(4). Of note, ghrelin’s orexigenic actions seemingly help re-
verse the rises in ghrelin induced originally by energy insuffi-
ciency, as evidenced by a lowering of its circulating levels
following a meal (10, 11). In lean humans, studies with
isocaloric drinks have demonstrated that ingested proteins are
most effective in lowering ghrelin, whereas ingested lipids are
least effective; carbohydrates result in the largest initial drop
and also a subsequent rebound above preprandial levels (20). It
can be postulated that ghrelin secretion might be regulated by
nutrients acting directly at the level of the ghrelin cell.
Here, we have focused on the mediation of ghrelin secretion
by the interaction of D-glucose directly with the ghrelin cell.
This was achieved by establishing a model in which pools of
dispersed mouse gastric mucosal cells, containing both ghrelin
cells and nonghrelin cells, were grown in primary culture. The
isolation and culture protocol offered by this in vitro system
disrupts the usual gastric mucosal cell-cell interactions and
thus facilitates the identification of direct ghrelin secretagogues
and conditions linked to modulation of ghrelin secretion.
MATERIALS AND METHODS
Animals. Male C57Bl6/J mice aged 8 –12 wk fed a standard chow
diet (Teklad Global 16% Protein Rodent Diet no. 2016; Harland
Laboratory, Madison, WI) were used for harvesting cells. Animal
studies were approved by the Institutional Animal Care and Use
Committees at University of Texas Southwestern Medical Center and
Saitama University.
Isolation of gastric mucosal cells. The following isolation protocol
was reported previously, albeit in less detail (48, 50). Following
terminal anesthetization of mice using chloral hydrate (700 mg/kg
body wt ip), the stomachs were exposed, and surgical suture was used
* These authors contributed equally to this work.
Address for reprint requests and other correspondence: J. M. Zigman, UT
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-
9077 (e-mail: jeffrey.zigman@utsouthwestern.edu).
Am J Physiol Endocrinol Metab 302: E1300–E1310, 2012.
First published March 13, 2012; doi:10.1152/ajpendo.00041.2012.
0193-1849/12 Copyright
©
2012 the American Physiological Society http://www.ajpendo.orgE1300
to tie off the lumen of the stomach proximally from the esophagus and
distally from the duodenum. The stomachs were quickly excised such
that the sutures remained attached to the stomachs and were placed in
cold PBS (Gibco, Gaithersburg, MD). A small, 5-mm-long incision
was placed in the nonglandular forestomach, and the stomach was
emptied of the bulk of any ingested food matter by gently pushing the
food out through the incision using a blunt forceps. The stomach was
turned inside out by pushing the distal part of the stomach through the
incision in the forestomach. Next, a blunt 20-gauge metal needle
attached to a syringe was advanced into the inside-out stomach
through the forestomach incision, and a piece of suture was used to
tighten the incisional opening against the needle shaft. Cold DMEM
with no glucose (Gibco) was injected into the stomach to inflate it,
after which the needle was removed and the incisional opening
completely tied off using the previously placed suture. The procedure
from time of anesthetization to inflation of the stomach took 15–20
min, and each animal was done sequentially. The inflated, inside-out
stomachs were placed temporarily in DMEM with no glucose on ice
while awaiting the isolation of all the specimens. In our hands, the
remaining steps of the isolation protocol described below were de-
layed another 20 min while these specimens were transported to the
next location.
Next, the stomachs were removed from the cold DMEM and gently
and rapidly brushed with a Kimwipe to remove any residual food
particles. Our preparations for the ghrelin secretion studies (see
below) were done using three stomachs, and all of them were placed
into a single 50-ml conical tube containing 5 ml of digestion solution
[2.5 mg of Dispase II (Roche Diagnostics, Indianapolis, IN) per 1 ml
of PBS] per stomach [our preparations for fluorescent-activated cell
sorting (FACS) separation were done using 3–5 stomachs, with the
4th or 4th and 5th stomachs being placed in a second 50-ml conical
tube containing another 5 ml of digestion solution per stomach]. The
tube was then incubated in a 37°C water bath for 90 min. The mucosal
layer of each stomach was isolated by securing the forestomach with
a forceps such that the stomach was held against the inside lip of a
100-ml glass beaker containing 10 ml of room-temperature DMEM-
F-12 (which happened to contain 17.49 mM
D-glucose; Mediatech,
Manassas, VA) supplemented with 10% (vol/vol) FBS (Atlanta Bio-
logicals, Lawrenceville, GA) and 100 U/ml penicillin plus 100 g/ml
streptomycin sulfate (Gibco), and then a polyethylene transfer pipet
(13-711-7M; Fisher Scientific, Pittsburgh, PA) was used to squirt
some of the media over the sides of the stomach; while squirting, the
transfer pipet was also scraped against the stomach as a further means
to mechanically release the mucosal cells into the media. The cell
suspension for each stomach was transferred to a 15-ml conical tube
(1 tube for the cell suspension from each stomach), after which the
tubes were centrifuged at 1,200 rpm for 3 min. For each tube, the
supernatant was removed with a polyethylene transfer pipet, and
the remaining cellular pellet was resuspended with another transfer
pipet in 2.5 ml of 0.25% trypsin-EDTA (Gibco), followed by incu-
bation in a 37°C water bath for 5 min. To inactivate the trypsin, 10 ml
of DMEM-F-12 supplemented with 10% FBS was added, after which
the cells were again resuspended and then collected by filtering
through a 100-m nylon mesh (Cell Strainer, 352360; BD Falcon,
Bedford, MA). The filtered cells were centrifuged at 1,200 rpm for 3
min, the supernatants were removed, and then the cellular pellets from
all three stomachs were resuspended in a total of 5 ml of DMEM-F-12 supp-
lemented with 10% FBS, 100 U/ml penicillin plus 100 g/ml strep-
tomycin, and 50 M octanoate-BSA [note that the octanoate-BSA
conjugate was prepared as a 10-mM stock solution by dissolving
16.62 mg of sodium octanoate (Sigma, St. Louis, MO) in 6.67 ml of
0.9% NaCl (Sigma) and adding 3.33 ml of fatty acid-free BSA
(Sigma)]. The number and concentration of isolated cells were deter-
mined using a hemocytometer. On average, we were able to isolate
2 10
6
cells from each stomach. The same medium was used to
dilute the cells to a final concentration of 1 10
5
cells/ml.
Ghrelin secretion studies. Studies were run on isolated gastric
mucosal cells from a total of three C57BL6/J mice at a time since
greater variation was observed when cells were isolated from more than
three mice at a time, suggesting that extended isolation time impairs
cell recovery and function. The cells were placed into the wells of
poly-
L-lysine-coated 24-well plates (1 10
5
cells/well in 1 ml of
medium) and incubated in humidified 95% air and 5% CO
2
at 37°C.
Upon initial plating, most of the mixed population of cells was not in
contact with one another, with the exception of the occasional dou-
blets. For the Fig. 1 studies, after a 16- to 18-h incubation, the medium
was aspirated from the cells and replaced with serum-free DMEM
containing 100 U/ml penicillin, 100 g/ml streptomycin sulfate, 50
M octanoate-BSA, and 5 mM glucose with or without one of the
following reagents: epinephrine (10 M; Sigma), norepinephrine (10
M; Sigma), insulin (100 nM; Sigma), somatostatin (100 nM; Phoe-
nix Pharmaceuticals, Belmont, CA), secretin (100 nM; Phoenix Phar-
maceuticals), endothelin-1 (100 nM; Peptides International, Louis-
ville, KY), or PMA (20 M; Tocris Bioscience, Minneapolis, MN).
The cells were exposed to these potential secretagogues for6hin
humidified 95% air and 5% CO
2
at 37°C. For the Fig. 2
and Fig. 3
studies, which examined the effects of D-glucose, the plated cells were
treated similarly after the 16- to 18-h incubation for6hinserum-free
DMEM containing 100 U/ml penicillin, 100 g/ml streptomycin
sulfate, 50 M octanoate-BSA, and the stated concentrations of
D-glucose, L-glucose (Sigma), norepinephrine, insulin, 2-deoxy-D-
glucose (Sigma), tolbutamide (Sigma), and diazoxide (Sigma). Stock
solutions of tolbutamide and diazoxide were prepared in DMSO,
resulting in DMSO concentrations within the media of 0.05 (vol/vol)
and 0.025% (vol/vol), respectively; 0.05% (vol/vol) DMSO was also
included in the appropriate control wells for Fig. 3, D and E.
Fig. 1. Secretion of ghrelin upon exposure of primary
cultures of dispersed mouse gastric mucosal cells to pep-
tide hormones and other compounds. Levels of acyl ghrelin
(A) and desacyl ghrelin (B) released into the culture me-
dium (DMEM; 100 U/ml penicillin, 100 g/ml streptomy-
cin sulfate, 50 M octanoate-BSA, and 5 mM glucose)
upon 6-h incubation of dispersed mouse gastric mucosal
cells in medium alone (control) or medium containing
epinephrine (10 M), norepinephrine (10 M), insulin
(100 nM), somatostatin (100 nM), secretin (100 nM),
endothelin-1 (100 nM), and PMA (20 M). Each bar
represents the ghrelin level in the medium relative to that
observed under the control condition (A: 462.6 9.2
pg/ml; B: 1,995.4 45.7 pg/ml) and is shown as the
mean SE for 3 wells containing 1 10
5
cells/well in 500
l of medium. ***P 0.001, level of statistical signifi-
cance.
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Following the 6-h incubations, the medium was immediately collected
and centrifuged at 3,000 rpm for 5 min. To stabilize acyl ghrelin, the
supernatants had 1 N HCl added to achieve a final HCl concentration
of 0.1 N and were stored at 80°C.
Of note, it is customary when preparing plasma for subsequent
assay of acyl ghrelin to add a protease inhibitor such as PHMB
(p-hydroxymercuribenzoate) prior to the HCl stabilization step as
another step to stabilize the acyl group (26, 35, 58). To test the
requirement for the protease inhibitor with the current cell culture-
based system, pilot secretion studies were performed as described
above in serum-free DMEM medium (containing penicillin, strepto-
mycin, and octanoate-BSA) plus 1, 5, or 10 mM
D-glucose (as
described above) with or without 0.05% DMSO. Each condition was
run four times in each of two separate trials. After the usual 6-h
incubation, a stock solution of PHMB was added to the supernatants
from one-half of the wells (to achieve a final concentration of 1 mM)
prior to the HCl stabilization step. PHMB had no statistically signif-
icant effect on assayed acyl ghrelin levels, and thus for all subsequent
trials, PHMB was not used.
Measurement of ghrelin levels. Ghrelin levels were determined
using rat acylated ghrelin (no. 10006307) and rat unacylated ghrelin
(no. 10008953) ELISA kits (Cayman Chemical, Ann Arbor, MI).
Absorbance data were collected via a BioTek spectrophotometer,
using the KC Junior program (BioTek Instruments, Winooski, VT).
Isolation of ghrelin cell-enriched and non-ghrelin cell-enriched
pools of gastric mucosal cells using ghrelin-humanized Renilla reni-
formis green fluorescent protein transgenic mice. Gastric mucosal
cells were isolated from ghrelin-humanized Renilla reniformis green
fluorescent protein (hrGFP) reporter mice (hrGFP10) (48), as de-
scribed above. After being washed in PBS, cells were resuspended in
FACS buffer (Hanks’ balanced salt solution containing 3% fetal
bovine serum, 0.5 mM EDTA, 0.1% BSA, 10 U/ml DNase I, and 20
mg/ml glucose). The cells were sorted with a DakoCytomation MoFlo
(Dako, Carpinteria, CA) at the University of Texas Southwestern
Flow Cytometry Multi-User Core Facility on the basis of size,
complexity, and intensity of GFP fluorescence (at 530 nm) and
fluorescence at 585 nm. Three independent preparations (3–5 mice
were used for each independent preparation) were included in the
subsequent analyses.
RNA extraction and quantitative real-time polymerase chain
reaction. The hrGFP-positive pools and the hrGFP-negative pools
were adjusted to contain a matched number of cells (6,500, 10,000,
and 12,500 cells for preparations 1, 2, and 3, respectively). The cells
in each pool were collected by centrifugation at 4°C at 3,000 rpm for
10 min. Total RNA was extracted from these cellular pellets using a
standard guanidium thiocyanate-phenol-chloroform extraction proto-
col after the addition of RNA STAT-60 (Tel-Test, Friendswood, TX).
Total RNA was also extracted from two different mouse ghrelinoma
cell lines, SG-1 and PG-1 (63), using RNA STAT-60. Concentration
and relative purity of the RNA were determined using a Nanodrop
1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA),
and it was stored at 80°C until use. Complementary DNA was
synthesized for each individual sample using Superscript III reverse
transcriptase (Life Technologies, Grand Island, NY). Quantitative
PCR was performed using the iTaq SYBR Supermix (Bio-Rad Lab-
oratories, Hercules, CA) and an ABI 7300 Real-Time PCR System
(Applied Biosystems, Foster City, CA). Initial template denaturation
(3 min at 95°C) was performed, followed by 40 cycles of denaturation
(15 s at 95°C), annealing, and extension (45 s at 60°C). Reactions
were evaluated by the comparative threshold cycle (C
T
) method, using
cyclophilin as the invariant control gene. Such was done separately for
each of the three different hrGFP-positive pools, each of the three
different hrGFP-negative pools, and each of the preparations of SG-1
and PG-1 cells. Previously, we have reported comparing the C
T
values
of several genes amplified from FACS-separated gastric mucosal cells
with the C
T
values of a separate housekeeping gene, 36B4, and
observed results similar to those determined with cyclophilin (50).
Sequences of primers are listed in Table 1. Primers used (Table 1)
were as published previously [ghrelin, cyclophilin, and ghrelin O-
acyltransferase (48, 50)], newly designed [somatostatin receptor
(SSTR)2, SSTR5, secretin receptor, GLUT2, hexokinase 1, hexoki-
nase 2, and hexokinase 3], or gifts from Joyce Repa (University of
Texas Southwestern Medical Center). The newly designed primers
were designed using Primer Express Software (Life Technologies).
These primer pairs were further tested for specificity to the gene of
interest by analysis using the Basic Local Alignment Search Tool and
Primer-BLAST (National Center for Biotechnology Information).
Only those primer pairs for which neither of the two primers bound
with 100% specificity to the gene of interest, did not bind to another
gene of interest, and/or did not amplify another random DNA frag-
ment 1 kb in size were used. The efficiencies of the primers were
validated by verifying a slope of approximately 3.3 when the logs of
the cDNA concentration at different serial dilutions were compared
with the C
T
(cDNA dilutions ranged from 50 to 0.016 ng of cDNA).
The primers were designed to amplify regions of cDNA that in the
corresponding genomic DNA span introns to further ensure the
amplification of cDNA derived from mRNA rather than residual
genomic DNA.
Statistical analysis. Data are expressed as means SE. GraphPad
Prism 5 software (GraphPad Software, San Diego, CA) was used to
perform all statistical analyses. One-way anaylsis of variance
(ANOVA) followed by Dunnett’s post hoc test was used to assess the
effects on desacyl ghrelin and/or acyl ghrelin secretion of various
reagents (all in a single
D-glucose environment; Fig. 1), D-glucose
concentration (Fig. 2), norepinephrine, insulin, and 2-deoxy-
D-glucose
for each of three different
D-glucose concentrations (Fig. 3A), and
Fig. 2. Effect of glucose on secretion of ghrelin from
primary cultures of dispersed mouse gastric mucosal cells.
Levels of acyl ghrelin (A) and desacyl ghrelin (B) released
into the culture medium upon 6-h incubation of dispersed
mouse gastric mucosal cells in medium (DMEM; 100
U/ml penicillin, 100 g/ml streptomycin sulfate, and 50
M octanoate-BSA) containing 1, 5, and 10 mM D-glu-
cose. Each bar represents the ghrelin level in the medium
relative to that observed upon incubation in 5 mM D-glu-
cose (A: 789.1 48.1 pg/ml; B: 1,919.8 83.3 pg/ml) and
is shown as the mean SE for 4 wells containing 1 10
5
cells/well in 500 l of medium. **P 0.01 and ***P
0.001, level of statistical significance.
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D-glucose concentration L-glucose (Fig. 3B) or DMSO, tolbut-
amide, and diazoxide for each of three different D-glucose concentra-
tions (Fig. 3C). Two-way ANOVA was used to assess the effects of
D-glucose concentration and tolbutamide (and the interaction between
them) on norepinephrine-stimulated acyl ghrelin levels (Fig. 3D) and
on insulin-influenced acyl ghrelin levels (Fig. 3E). For the quantitative
real-time polymerase chain reaction (qPCR) studies, we determined
the mean SE for each of the tissue types and then used a one-way
ANOVA followed by Dunnett’s post hoc test to determine statistical
significance. P 0.05 was considered statistically significant.
RESULTS
Validation of dispersed mouse gastric mucosal cell pri-
mary culture model. In the current study, we examined
ghrelin secretion from preparations of dispersed mouse
gastric mucosal cells grown for a brief time in primary
culture. Here, cells comprising the gastric mucosa of adult
mice were enzymatically and mechanically separated from
the stomach, dispersed such that they were no longer in
contact with one another, and then studied in primary
culture. Approximately 0.3 to 1% of this mixed population
of gastric mucosal cells is thought to be ghrelin cells (48).
A similar isolation protocol and primary cell culture system
was described previously for rat stomach cells, although in
that system, the isolated cells were first exposed to Percoll
centrifugation to enrich for ghrelin cells prior to plating
(49). Previously, we also used an identical isolation protocol to
prepare gastric mucosal cells from ghrelin-hrGFP (humanized
Renilla reniformis green fluorescent protein) transgenic reporter
mice for FACS to generate highly enriched pools of ghrelin cells
(see more below) (48). Of note, despite several manipulations to
the protocol, the survivability of the hrGFP-positive gastric mu-
cosal cells was shown to drop significantly following their enrich-
Fig. 3. Impact of ambient D-glucose concen-
tration on the ghrelin secretagogic effects of
various reagents. Levels of acyl ghrelin re-
leased into the culture medium upon 6-h
incubation of dispersed mouse gastric muco-
sal cells in medium (DMEM; 100 U/ml pen-
icillin, 100 g/ml streptomycin sulfate, and
50 M octanoate-BSA) containing various
concentrations of D-glucose 10 M nor-
epinephrine (NE), 100 nM insulin (Ins), or
10 mM 2-deoxy-D-glucose (2DG) (A), 10
mM L-glucose (B), 0.05% (vol/vol) DMSO,
500 M tolbutamide (Tolb), or 100 M
diazoxide (DZX) (C), 10 MNE 500 M
Tolb (D), and 100 nM Ins 500 M Tolb
(E). In A, the patterned bars in each set
represent the ghrelin levels in the medium
relative to that observed upon incubation in
1 (636.5 37.0 pg/ml), 5 (444.7 34.8
pg/ml), or 10 mM D-glucose (348.9 10.1
pg/ml) alone (open bars) and are shown as
means SE for 2 different cell preparations
each containing 3 wells. In B, each bar
represents the ghrelin level in the medium
relative to that observed upon incubation in
1mMD-glucose (853.8 6.9 pg/ml) and is
shown as the mean SE for 4 wells. In C,
the patterned bars in each set represent the
ghrelin levels in the medium relative to that
observed upon incubation in 1 (624.2 95.7
pg/ml), 5 (376.3 51.7 pg/ml), or 10 mM
D-glucose (313.7 35.2 pg/ml) alone (open
bars) and are shown as means SE for 2
different cell preparations each containing
2–3 wells. In D and E, each bar represents
the ghrelin level in the medium relative to
that observed upon incubation in 1 mM D-
glucose 10 M NE (884.4 63.7 pg/ml)
and1mMD-glucose 100 nM Ins (378.0
86.0 pg/ml), respectively, and is shown as
the mean SE for 2 different cell prepara-
tions each containing 2–3 wells. Wells for all
these studies contained 1 10
5
cells/well in
500 l of medium. Asterisks in A denote the
level of statistical significance relative to the
open bar of the set, and asterisks in B denote
the significant effect of the condition noted.
*P 0.05, **P 0.01, and ***P 0.001.
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ment by FACS analysis, thus precluding studies of these enriched
pools in primary culture.
To confirm the survivability and functionality of the ghrelin
cells within the nonsorted, dispersed gastric mucosal cell
primary culture model used here, we first treated the cells with
two adrenergic agonists, epinephrine and norepinephrine. Pre-
vious studies have consistently demonstrated a ghrelin secre-
tory action for epinephrine and norepinephrine. For instance, in
vivo studies in rats and mice have shown induction of ghrelin
secretion upon stimulation of the sympathetic nervous system
(41, 63). Both of these compounds also potently stimulate
ghrelin release when infused via microdialysis probes into the
gastric mucosa of rats (14) and when added to the culture
media of immortalized ghrelinoma cell lines (28, 63). These
data, along with the finding of high levels of
1
-adrenergic
receptors on ghrelin cells isolated from ghrelin-hrGFP reporter
mice, suggest that these catecholaminergic agents may act
directly on ghrelin cells to stimulate ghrelin release (63). Here,
both epinephrine and norepinephrine (10 M) potently stimu-
lated ghrelin release from the dispersed gastric mucosal cell
primary culture system (Fig. 1), thus confirming this system as
a valid model with which to investigate ghrelin secretion.
Regulation of ghrelin secretion by other peptide hormones
and compounds. We also investigated the effects of the peptide
hormones insulin, somatostatin, secretin, and endothelin-1 on
ghrelin release using the dispersed gastric mucosal cell primary
culture model. These hormones were studied given previous
work suggesting that they play a role in ghrelin secretion (see
below). When cells were incubated in the presence of 5 mM
D-glucose, none of the mean acyl ghrelin levels that accumu-
lated in the culture media in response to 100 nM insulin
(413.1 29.3 pg/ml), 100 nM somatostatin (390.3 24.7
pg/ml), 100 nM secretin (414.8 17.3 pg/ml), or 100 nM
endothelin-1 (487.4 53.8 pg/ml) was statistically different
from that released under basal conditions (462.6 9.2 pg/ml)
when assessed using 1-way ANOVA (Fig. 1A). Nor did any of
these hormones affect levels of desacyl ghrelin that accumu-
lated in the culture media (Fig. 1B).
Effects of the phorbol ester PMA were also examined. This
compound directly alters the activity of key modulators down-
stream of G protein-coupled and other plasma membrane-
bound receptor signaling cascades, which in other systems
affect secretory activity. PMA and other phorbol esters bind to
the C1b regulatory domain of members of the conventional
protein kinase C family, including PKC, PKC, and PKC,
and activate those PKC isozymes, thus bypassing their normal
activation by binding of diacylglycerol generated by phospho-
lipase C (7). Phorbol esters also bind members of the novel
PKC and RasGRP families. Here, 20 M PMA potently
increased both acyl ghrelin and desacyl ghrelin in the culture
medium (Fig. 1).
mRNA expression profiles of ghrelin cells. Since the dis-
persed gastric mucosal cell primary culture model consists
primarily of nonghrelin cells in addition to ghrelin cells, we
next sought to determine whether highly purified populations
of ghrelin cells express the receptors and molecular targets for
the peptide hormones and compound tested. We prepared
highly enriched pools of gastric ghrelin cells by taking advan-
tage of the hrGFP reporter present in a previously described
line of ghrelin hrGFP transgenic mice (hrGFP10 line) (48).
qPCR was performed on mRNAs isolated from both the
hrGFP-positive (ghrelin cell-enriched) and the hrGFP-negative
(non-ghrelin cell-enriched) pools, and the average levels of the
mRNA species of interest relative to that of the housekeeping
gene cyclophilin were determined. Previously, this method has
been used to confirm expression within ghrelin cells of pro-
hormone convertases 1/3 and 2 (48), ghrelin O-acyltransferase
(50), and
1
-adrenergic receptor (63). Similar analyses were
done using mouse ghrelinoma cell lines SG-1 and PG-1 estab-
lished previously from mice bearing ghrelinomas induced by a
tissue-specific SV40 T-antigen transgene (63).
Table 1. Quantitative real-time PCR primer sequences
Mouse Gene Sequences of Primer Sets
Ghrelin 5=-GTCCTCACCACCAAGACCAT-3=
5=-TGGCTTCTTGGATTCCTTTC-3=
GOAT 5=-TCCACAGCCTGGCTCTTTAAAC-3=
5=-GCCGCGTGGAGGAGAGA-3=
Insulin receptor 5=-CGAGTGCCCGTCTGGCTATA-3=
5=-GGCAGGGTCCCAGACATG-3=
SSTR1 5=-GCTACGCCAAGATGAAGACCG-3=
5=-GCTCATCAGCAATAGCCAGGTTT-3=
SSTR2 5=-ACCCCAGCCCTGAAAGG-3=
5=-CGCAGCTGTTGGCATAGGT-3=
SSTR3 5=-CTACGGCTTCCTCTCCTACCG-3=
5=-CGACGTGATGGTCTTAGCAGGA-3=
SSTR4 5=-ACTGGAGGTGCTGAGGAAGA-3=
5=-TCTTGGTGAAAGGGACTTGC-3=
SSTR5 5=-TGGCTGACGTGTTGTTTATGTTG-3=
5=-ACCAGGCGGCACAAGAAG-3=
Secretin receptor 5=-TACTCTTCCCCGCAGATGAC-3=
5=-AGATAGAGGCCCTCCACCAG-3=
Endothelin receptor A 5=-GCGGATCGCCCTTAGTGA-3=
5=-GATGACAACCAAGCAGAAGACAGT-3=
Endothelin receptor B 5=-CTGCGAAATGCTCAGGAAGAA-3=
5=-CGAGGACCAGGCAGAAGACT-3=
PKC 5=-GAAACTCACAGACTTCAACTT-3=
5=-ATCTTGATGGCGTACAGTTC-3=
PKC 5=-AATGGCAACAGGGACCGGAT-3=
5=-TACCCTTCCGCTCTGAGAGC-3=
PKC 5=-TGGCCGATGCTGACAACTGC-3=
5=-TGGGGGATGGAGAAGGGATG-3=
GLUT1 5=-CGTCGTTGGCATCCTTATTG-3=
5=-GAGGCCACAAGTCTGCATTG-3=
GLUT2 5=-TCCAACCACACTCAGGGGTG-3=
5=-AAGCTGAGGCCAGCAATCTG-3=
GLUT3 5=-TGTTGGACTCTTTGTCAACCG-3=
5=-GGCCAGCAAGTTGACTAGAAGC-3=
GLUT4 5=-CCTTTCTCATTGGCATCATTTC-3=
5=-CACGGCCAAGACATTGTTG-3=
GLUT5 5=-GGGCCGTCAATGTGTTCAT-3=
5=-CCGACGAGGAGTAGGAATCG-3=
Glucokinase 5=-CCGTGATCCGGGAAGAGAA-3=
5=-GGGAAACCTGACAGGGATGAG-3=
Hexokinase 1 5=-CGAGCCTAGATTGCGGAATC-3=
5=-AAATTCCTCTCTCCTTTTTACAGCAT-3=
Hexokinase 2 5=-GTCTCGGATATTGAAGACGATAAGGA-3=
5=-GCGCGTGGACACAATCTG-3=
Hexokinase 3 5=-CTGACAGTGTCTGTGGGAGTAGATG-3=
5=-GGCTAGCTTCCGGACTGTTG-3=
Kir6.2 5=-GCAGAGCCCAGGTACCGTACT-3=
5=-CGTTGCAGTTGCCTTTCTTG-3=
SUR1 5=-TCTCGCCTTTTTCCGAATG-3=
5=-TGTCGATGCCATCAATGATA-3=
Cyclophilin 5=-TGGAGAGCACCAAGACAGACA-3=
5=-TGCCGGAGTCGACAATGAT-3=
GOAT, ghehlin O-acyltransferase; SSTR, somatostatin receptor; GLUT,
glucose transporter; SUR1, sulfonylurea type 1 receptor.
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To confirm adequacy of the FACS separation, we first
assessed levels of ghrelin and ghrelin O-acyltransferase within
each pool. Messenger RNAs for ghrelin and ghrelin O-acyl-
transferase were significantly higher within the hrGFP-positive
pools, as expected (Table 2).
Insulin receptor mRNA was observed in both the hrGFP-
negative pools and the ghrelin cell-enriched pools (with higher
levels observed in the latter) as well as in both ghrelinoma cell
lines. Expression of five different SSTRs was also examined.
Marked elevations in SSTR1 and SSTR3 mRNA were ob-
served in the hrGFP-positive pools compared with the hrGFP-
negative pools. SSTR2 and SSTR5 were increased slightly
within the hrGFP-positive pools, whereas SSTR4 was not
detected in either FACS-separated pool. All of these SSTRs
were observed in the ghrelinoma cell lines. Secretin receptor
mRNA was observed in both FACS-separated pools but was
barely detectable in the ghrelinoma cell lines. mRNAs for
entothelin receptors A and B were barely detected or not
detected in the FACS-separated cells and the ghrelinoma cells.
mRNAs encoding PKC and PKC were found in both FACS-
separated pools and both ghrelinoma cell lines, although PKC
levels in the ghrelinoma cell lines were much lower than in the
hrGFP-positive pools; PKC mRNA was not detected in any
sample.
Effects of glucose on ghrelin release. We next investigated
the effects of exposing the primary cultures to different con-
centrations of D-glucose. Compared with levels of acyl ghrelin
released into the media in an environment of 5 mM D-glucose,
which is equivalent to a normal blood glucose level (90 mg/dl),
increased D-glucose (10 mM, which is equivalent to 180 mg/dl)
lowered the amount of acyl ghrelin released by 16% (Fig. 2A).
On the other hand, when ambient D-glucose levels were low-
ered to 1 mM (the equivalent of 18 mg/dl), acyl ghrelin release
was enhanced, resulting in a 31% increase in acyl ghrelin
levels in the media (Fig. 2A). Similar effects of raising and
lowering D-glucose were noted for desacyl ghrelin (Fig. 2B).
These changes were statistically significant.
The effect of glucose on ghrelin release was explored further
by incubating the cells with different concentrations of D-glu-
cose together with norepinephrine, insulin, 2-deoxy-D-glucose,
or L-glucose (Fig. 3, A and B). Norepinephrine (10 M) was
effective at stimulating ghrelin secretion not only in the pres-
ence of 5 mM D-glucose, as had been observed in Fig. 1, but
also in the presence of lower (1 mM) and higher (10 mM)
D-glucose (Fig. 3A). The D-glucose-dependent pattern of ghre-
lin release induced by norepinephrine seemed to mirror that
observed in the absence of norepinephrine only at a higher
mean level (Fig. 3A). Also, as had been observed in Fig. 1,
insulin (100 nM) had no statistically significant effect on
ghrelin release in the condition of 5 mM
D-glucose; insulin was
similarly ineffective at altering ghrelin release in the presence
of 10 mM
D-glucose (Fig. 3 A). However, when the cells were
Table 2. Relative levels of mRNAs in FACS-separated gastric mucosal cells from hrGFP mice and in 2 ghrelinoma cell lines
mRNA
FACS-Separated Gastric Mucosal Cells Ghrelinoma Cell Lines
Non-ghrelin cell-enriched (hrGFP-negative) pools Ghrelin cell-enriched (hrGFP-positive) pools SG-1 PG-1
Ghrelin 1.0 (28.9 0.6)# 45,529 2,635* 533 34.6 1,086 180
GOAT 1.0 (33.7.0.2) 691 216* 140 16.6 99.4 6.0
Insulin receptor 1.0 (29.5 0.3) 4.50 1.71 4.1 0.7 8.5 2.2*
SSTR1 1.0 (33.1 0.2) 32.1 11.8* 4.0 0.5 2.2 1.3
SSTR2 1.0 (30.7 0.5) 2.8 0.9 1.3 0.2 2.9 1.2
SSTR3 1.0 (34.0 0.9) 155.5 31.8* 3.8 0.9 26.1 0.5
SSTR4 Not detected† Not detected† (29.42 0.34)‡ (29.42 0.17)‡
SSTR5 1.0 (33.4 0.4) 3.5 0.7* 0.8 0.4 0.2 0.0
Secretin receptor 1.0 (29.0 0.6) 1.4 0.6* 0.004 0.001* 0.007 0.003*
Endothelin receptor A 1.0 (33.5 0.4) 2.9 0.3* 0.4 0.1 0.2 0.07
Endothelin receptor B Not detected† Not detected† Not detected† (33.5 0.2)‡
PKC 1.0 (33.7 0.2) 1.4 0.4 2.2 0.2 4.3 1.8*
PKC 1.0 (26.3 0.7) 1.0 0.3 Not detected† 0.070 0.003
PKC Not detected† Not detected† Not detected† Not detected†
GLUT1 1.0 (26.6 0.5) 1.6 0.3 1.4 0.1 3.1 0.3*
GLUT2 Not detected† Not detected† Not detected† Not detected†
GLUT3 1.0 (33.2 0.4) Not detected† 59.6 6.9* 221 11*
GLUT4 1.0 (32.8 0.3) 13.2 1.2 87.8 17.6* 246 13*
GLUT5 1.0 (31.0 0.6) 74.3 11.0* 120 22.0* 199 17*
Glucokinase 1.0 (31.2 0.4) 75.6 15.5* 8.4 2.0 32.2 2.7
Hexokinase 1 1.0 (27.3 0.5) 3.5 0.0 13.9 1.4* 8.5 0.8*
Hexokinase 2 1.0 (28.5 0.7) 1.2 0.1 3.1 0.3* 1.5 0.6
Hexokinase 3 Not detected† Not detected† (29.2 0.6)‡ (32.9 0.3)‡
Kir6.2 1.0 (31.6 0.7) 106 4* 129 25* 144 10*
SUR1 1.0 (31.9 0.5) 104 24 187 33* 326 31*
Values are means SE. FACS, fluorescent-activated cell sorting; hrGFP, humanized Renilla reniformis green fluorescemt protein. Ghrelin cell-enriched
(hrGFP-positive) and non-ghrelin cell-enriched (hrGFP-negative) gastric mucosal cells were FACS separated, and mRNAs from these pools and from SG-1 and
PG-1 ghrelinoma cell lines were extracted and quantified by quantitative real-time PCR, as described in MATERIALS AND METHODS. Cyclophilin mRNA was used
as an invariant control. Each value represents the amount of mRNA relative to that in the hrGFP-negative pool, which is arbitrarily defined at 1.0 and is shown
as the means SE of 3 different preparations. Each determination was done in duplicate. #Values in parentheses denote the mean SE of threshold cycle values.
*Level significantly different from non-ghrelin cell-enriched pools (P 0.05), as determined by 1-way ANOVA followed by Dunnett’s post hoc analysis.
†Threshold cycle value 35. ‡In instances when mRNA was not detected in the hrGFP-negative pools but was detected in 1 or more of the other cell types
(thereby precluding a relative comparison with the levels observed in the hrGFP-negative pools), the threshold cycle values for the other cell types are listed in
parentheses.
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exposed to 1 mM D-glucose, insulin did restrict ghrelin release
in a statistically significant manner (Fig. 3A). The glucoprivic
D-glucose analog 2-deoxy-D-glucose, which is taken up into
cells where it subsequently blocks D-glucose metabolism, was
also tested. The addition of 2-deoxy-D-glucose (10 mM) to
culture medium containing 10 mM D-glucose blocked the
expected decrease in ghrelin release otherwise observed upon
ambient D-glucose concentration being raised (Fig. 3A). Com-
pared with incubations without 2-deoxy-D-glucose, the addi-
tion of 2-deoxy-D-glucose raised ghrelin secretion observed
under conditions of 10 mM D-glucose and 5 mM D-glucose,
whereas no statistically significant difference in ghrelin secre-
tion was observed upon the addition of 2-deoxy-D-glucose to
culture medium with 1 mM D-glucose (Fig. 3A).
The importance of D-glucose metabolism within the cells as
a mechanism for D-glucose-induced inhibition of ghrelin re-
lease was suggested further upon incubation of the primary
cultures with L-glucose, which is the diastereoisomer of D-glu-
cose and does not serve as a substrate for glycolysis (Fig. 3B).
In contrast to the reduction in ghrelin release observed upon
incubation of the cells in 11 mM D-glucose compared with 1
mM D-glucose, the addition of 10 mM L-glucose had no effect
on ghrelin release (Fig. 3B).
Mechanistic aspects of glucose-mediated changes to ghrelin
release. The enhanced ghrelin release observed in the primary
culture system upon 2-deoxy-D-glucose-induced glucopriva-
tion, which impersonates a state of low ambient
D-glucose,
supports the assertions that ghrelin secretion is inversely pro-
portional to the ambient
D-glucose concentrations to which
ghrelin cells are directly exposed and that metabolism of
D-glucose is essential to decrease ghrelin release. Thus, we next
investigated the expression of various glucose metabolism-
related enzymes and channels that help other cell types sense
and respond to changes in D-glucose concentration, using a
qPCR-based strategy.
Messenger RNAs encoding several members of the glucose
transporter (GLUT) family of facilitative glucose transporters
were localized to ghrelin cell-enriched (hrGFP-positive) gastric
mucosal cell pools (Table 2). These included GLUT1, GLUT4,
and GLUT5. GLUT1 expression was nearly as high in the
hrGFP-negative pools as in the hrGFP-positive pools. GLUT2
was not detected within either pool, whereas GLUT3 seemed
restricted to the non-ghrelin cell-enriched pools. Nonetheless,
GLUT3 was observed at fairly high levels in both ghrelinoma
cell lines. GLUT4 was higher in both ghrelinoma cell lines, and
GLUT5 was higher within the hrGFP-positive pools and the
ghrelinoma cell lines.
Messenger RNA encoding glucokinase, which is known to
catalyze the rate-limiting step in glucose metabolism in pan-
creatic -cells, was also highly enriched in the hrGFP-positive
pool, as were mRNAs encoding the inwardly rectifying potas-
sium channel Kir6.2 and the high-affinity sulfonylurea type 1
receptor (SUR1; Table 2). Expression of hexokinase 1 and
hexokinase 2, which, similar to glucokinase, also phosphory-
late glucose, was modest within both FACS-separated pools,
whereas hexokinase 3 was not detected. Glucokinase, hexoki-
nase 1, hexokinase 2, Kir6.2, and SUR1 also were expressed in
ghrelinoma cells, as was a low level of hexokinase 3.
A complex of Kir6.2 and SUR1 comprises the well-charac-
terized pancreatic -cell ATP-sensitive potassium (K
ATP
)
channel (27). Within the pancreatic -cell, this Kir6.2-SUR1
complex plays a key role in insulin secretion, whereby closure
of K
ATP
channels and the ensuing depolarization of -cells is
the major pathway by which
D-glucose-induced insulin secre-
tion, as well as sulfonyurea-induced insulin secretion, occurs
(8). The opening of K
ATP
channels is the mechanism by which
the drug diazoxide works to inhibit insulin secretion in cases of
congenital hyperinsulinism (25, 30). Because of the presence
of both Kir6.2 and SUR1 at such high levels within the ghrelin
cell-enriched pools and the ghrelinoma cell lines, we examined
the effects of both the sulfonylurea tolbutamide and diazoxide
on ghrelin release from the primary cell cultures (Fig. 3C).
Once again, acyl ghrelin release was inversely proportional to
the amount of
D-glucose in the media (1 vs. 5 vs. 10 mM
D-glucose). However, concentrations of tolbutamide (500 M)
and diazoxide (100 M) shown in other in vitro studies to
influence insulin release from pancreatic -cells and/or islets
(25, 40) neither enhanced nor inhibited ghrelin secretion at
D-glucose concentrations simulating hypoglycemic, normogly-
cemic, or hyperglycemic conditions (Fig. 3C). Nor did tolbu-
tamide significantly alter the effects of norepinephrine or
insulin on ghrelin release (Fig. 3, D and E).
DISCUSSION
Here, we used primary cultures of dispersed mouse gastric
mucosal cells to investigate ghrelin secretion. This model was
validated by the demonstration of ghrelin secretion by epineph-
rine and norepinephrine, which were shown previously to have
the same effect in vitro, using ghrelinoma cell lines, as well as
in vivo (14, 28, 41, 63). Neither insulin, somatostatin, secretin,
nor endothelin-1 had a statistically significant effect on ghrelin
release from the dispersed cells upon incubation in the pres-
ence of 5 mM
D-glucose. Similarly to the catecholamines used,
the phorbol ester PMA, which is best characterized as an
activator of several members of the protein kinase C family,
markedly enhanced ghrelin secretion. Messenger RNAs encod-
ing the receptors and direct molecular targets of these peptide
hormones and compounds were found at varying levels within
highly enriched pools of ghrelin cells and within two different
mouse ghrelinoma cell lines at levels ranging from very high
(relative to non-ghrelin cell-enriched pools of gastric mucosal
cells) to barely detectable. Compared with that observed in an
environment of 5 mM
D-glucose, which simulates a normogly-
cemic state, ghrelin release from the cultured cells was inhib-
ited by high
D-glucose (10 mM) and stimulated by low D-glu-
cose (1 mM). Metabolism of
D-glucose within the cells seemed
to be essential for its inhibitory effect on ghrelin release since
the reductions in ghrelin levels achieved by incubation in
media with both 5 and 10 mM
D-glucose (compared with the
higher ghrelin release observed in 1 mM
D-glucose) were
blocked by the glucoprivic agent 2-deoxy-D-glucose. Corrob-
orating this latter point,
L-glucose had no effect on ghrelin
release. However, an inhibitory effect of insulin on ghrelin
release was observed, such as that which occurred only in
conditions of low
D-glucose (1 mM). Messenger RNAs encod-
ing several channels and enzymes responsible for mediating
the effects of glucose on secretion in other cell types, including
hexokinase 1, hexokinase 2, glucose transporters GLUT1,
GLUT4, and GLUT5, glucokinase, and both components of the
pancreatic -cell K
ATP
channel (Kir6.2 and SUR1), were all
expressed highly within ghrelin cells, with the latter five genes
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also being highly enriched within ghrelin cells compared with
other gastric mucosal cell types. Despite the presence of the
K
ATP
channel within ghrelin cells, neither the sulfonylurea
agent tolbutamide (a K
ATP
channel blocker) nor diazoxide (a
K
ATP
channel activator) exerted any apparent direct effect on
ghrelin secretion.
There are several noteworthy discussion points and impli-
cations for these results. The first relates to the use of the
dispersed gastric mucosal cell system to study ghrelin secre-
tion. This model provides for gastric mucosal cells that have
been physically separated from their neighbors and helps to
isolate any observed effects on ghrelin secretion to direct
effects on ghrelin cells. Caveats of the system include possible
indirect paracrine effects of substances released into the culture
medium by the other nonghrelin cells that comprise the ma-
jority of cells in culture. Possible untoward effects of the
enzymatic and mechanical dispersion protocols might also
influence the observed results. It is assumed that disruptive
mechanical changes to the cells are magnified with FACS
analysis, precluding FACS-separated ghrelin cells from ghre-
lin-hrGFP mice from being studied in primary culture, al-
though mRNA expression data from highly enriched, FACS-
separated pools of ghrelin cells does help provide perspective
into the secretion data obtained using the primary culture
system. As used here, the dispersed gastric mucosal cell system
was able to confirm findings of catecholamine-induced ghrelin
secretion observed in immortalized mouse ghrelinoma cell
lines and using various other modalities (28, 63). Although the
mouse ghrelinoma cell lines are predicted to be key tools to
studying ghrelin secretion in the future, the variations in
mRNA expression described here (for instance, undetectable
GLUT3 in ghrelin cell-enriched pools vs. markedly elevated
GLUT3 within SG-1 and PG-1 cells) expose some potential
limitations of the ghrelinoma cell lines as models of normal
ghrelin cell function. Similar types of differences result in
altered glucose sensitivity within immortalized pancreatic
-cell lines, thereby somewhat limiting their utility as an exact
replica of wild-type -cells (12).
The ability of
D-glucose to regulate ghrelin secretion at the
level of the ghrelin cell was not necessarily unexpected, espe-
cially when viewed from the perspective of known prandial-
related changes in ghrelin levels (10, 11). Perhaps more tanta-
lizing would be to speculate on the relevance of this finding to
ghrelin’s important role as a regulator of blood glucose. Sev-
eral studies have now demonstrated clearly that ghrelin in-
creases blood glucose levels, likely via multiple mechanisms.
Ghrelin administration to rodents dose-dependently increases
fasting blood glucose, lowers insulin levels, and attenuates
insulin responses during glucose tolerance testing (15, 16).
Similar ghrelin-mediated effects on blood glucose and/or in-
sulin release have been demonstrated in isolated rodent islets
and pancreata, in ghrelin-overexpressing mice, and in humans
(3, 6, 15, 16, 47). Ghrelin also directly stimulates glucagon
secretion from pancreatic -cells (5). Conversely, GHSR de-
letion lowers blood glucose, enhances insulin sensitivity, and
lowers plasma glucagon (5, 36, 46, 64). Ghrelin deletion also
improves glucose tolerance, whereas simultaneous deletion of
both leptin and ghrelin improves the insulin-resistant pheno-
type characteristic of leptin deficiency (16, 17, 54). Strikingly,
mice lacking ghrelin O-acyltransferase show a progressive
decline in fasting blood glucose to the point of near death after
only 1 wk of 60% calorie restriction (62). Administration of
either acyl ghrelin or growth hormone to ghrelin O-acyltrans-
ferase-deficient mice normalizes blood glucose and prevents
death under these conditions, thus suggesting an essential role
for ghrelin in maintaining a minimum blood glucose level to
allow the survival of severely calorie-restricted mice (62). The
findings here demonstrating enhanced ghrelin secretion upon
exposure of ghrelin cells to low ambient D-glucose (or simu-
lation of a hypoglycemic state by exposure of ghrelin cells to
2-deoxy-D-glucose) and decreased ghrelin secretion upon ex-
posure of ghrelin cells to high ambient D-glucose, together with
published work describing ghrelin-induced elevations in blood
glucose, suggest that ghrelin participates in blood glucose
homeostatic pathways in an analogous manner to its role in
body weight homeostasis. As mentioned previously, ghrelin
levels rise in energy-insufficient states to stimulate food intake
and fat storage, after which levels again fall. The findings here
support a model by which ghrelin also both signals hypogly-
cemic states (its plasma levels rise) and responds to hypogly-
cemic states (by reducing insulin release and insulin sensitivity
and by enhancing growth hormone and glucagon secretion) and
then also responds appropriately upon reversal of the hypogly-
cemia (its plasma levels fall) (Fig. 4).
We also investigated the ability of insulin to directly interact
with ghrelin cells to regulate ghrelin secretion. Such an action
would be relevant not only to ghrelin’s actions in signaling and
responding to hypoglycemia but also its actions on eating and
body weight. Previously, it has been proposed that meal-related
changes in insulin might contribute to meal-related fluxes in
circulating ghrelin (20). However, the results of previous
studies examining the ability of insulin to directly interact with
ghrelin cells to affect ghrelin release have been inconsistent.
Our own group did not observe any statistically significant
decrease in ghrelin release from SG-1 or PG-1 mouse ghreli-
noma cell lines (63), whereas another stomach ghrelinoma cell
line (MGN3-1) generated using a similar transgenic strategy
did reduce its release of ghrelin in a statistically significant
manner and also reduced ghrelin and ghrelin O-acyltransferase
mRNA levels upon exposure to insulin (28, 29). Here, reduc-
tion in ghrelin release by insulin appeared dependent on the
ambient D-glucose concentration. As such, whereas insulin was
ineffective at reducing ghrelin release in a statistically signif-
icant manner in the presence of either 5 or 10 mM
D-glucose,
Fig. 4. Ghrelin’s roles in blood glucose control. The findings here support a
model by which ghrelin both signals and responds to hypoglycemic states.
Ghrelin secretion is stimulated when low blood glucose levels are sensed by
ghrelin cells. Following ghrelin secretion, blood glucose is raised, at least in
part, as a result of ghrelin’s ability to reduce insulin release and insulin
sensitivity and enhance growth hormone secretion and glucagon secretion. The
raised blood glucose then feeds back upon the ghrelin cells, leading to a
reduction in ghrelin secretion.
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it did reduce ghrelin release from the dispersed gastric mucosal
cell primary cultures in conditions of 1 mM D-glucose. Insu-
lin’s dependency on glucose concentration may explain our
previous findings in the SG-1 and PG-1 cell lines, which were
performed in culture medium with 5.56 mM D-glucose (63),
although it wouldn’t explain the findings in MGN3-1 cells,
which were performed at 25 mM D-glucose (28, 29). The
presence of insulin receptor mRNAs within highly enriched
populations of ghrelin cells as well as within the SG-1, PG-1,
and MGN3-1 ghrelinoma cells (28, 29) does support the idea
that insulin can act directly on ghrelin cells to modulate ghrelin
cell physiology.
Indeed, previous in vivo studies in humans also support the
notion that both insulin and D-glucose can regulate ghrelin
release (19, 20, 38). In one of these studies, infusion of insulin
with purposeful maintenance of normoglycemia (90 mg/dl),
achieved by infusion of dextrose, led to a rapid fall in acyl
ghrelin levels, suggesting that insulin suppresses ghrelin re-
lease independently of the degree of glycemia (19). Subsequent
hypoglycemia (50 mg/dl), achieved by maintaining the insulin
infusion rate but lowering the dextrose infusion rate, had no
significant effect on ghrelin levels, suggesting that hypoglyce-
mia does not reverse the fall in ghrelin induced by hyperinsu-
linemia (19). Induction of hyperglycemia (160 mg/dl),
achieved by maintaining the insulin infusion rate but raising
the dextrose infusion rate, resulted in a further decline in
plasma ghrelin, suggesting that high blood glucose also sup-
presses ghrelin release independently of insulin (19). Obvi-
ously, there are some differences in D-glucose-related sensitiv-
ity to insulin in this human study (19) and the current in vitro
culture system that will need to be explored further.
Of note and as mentioned above, ingestion by healthy lean
human volunteers of drinks composed predominantly of car-
bohydrates had been shown to have a biphasic effect on plasma
ghrelin, first lowering its levels below baseline and subse-
quently raising its levels above baseline (20). The late over-
shoot of plasma ghrelin observed in that study coincided with
blood glucose levels that were decreased below baseline and
insulin levels that were only slightly elevated above baseline.
This observation prompted the authors of that study to hypoth-
esize that the late postcarbohydrate overshoot in plasma ghrelin
may have resulted from lowered intracellular glucose metabo-
lism associated with blood glucose levels that had decreased
below baseline (20). Our findings here upon exposure of the
primary cultures to low D-glucose or to the antimetabolite
2-deoxy-
D-glucose support such a hypothesis. Also of interest
as it relates to ghrelin’s proposed roles in responding to states
of both energy insufficiency and hypoglycemia, food intake in
rats induced by 2-deoxy-D-glucose glucoprivation has been
shown to be blunted by pretreatment with anti-ghrelin antibod-
ies (52).
The mechanisms by which low D-glucose and high D-glu-
cose states directly regulate ghrelin release have admittedly not
been fully determined by the studies performed here. What
does seem apparent, though, by the findings with 2-deoxy-D-
glucose and L-glucose is that metabolism of D-glucose within
ghrelin cells does contribute to an inhibition of ghrelin release.
Entry of D-glucose into the ghrelin cell may occur via transport
by one or more members of the facilitative glucose transporter
family identified within ghrelin cells, including GLUT1,
GLUT4, and GLUT5. Once within the ghrelin cell, metabolism
might begin via interaction with glucokinase or one of the
hexokinases, which were also identified within ghrelin cells.
Glucokinase plays a key role in glucose metabolism within the
pancreatic -cell, where it serves as an important glucose
sensor, and within other cell types. Further downstream, me-
tabolites of
D-glucose might change the activity of the Kir6.2/
SUR1 K
ATP
channel, the components of which also were
identified within ghrelin cells, although exposure of the pri-
mary gastric mucosal cell pools to the sulfonylurea tolbutamide
or to diazoxide, which are expected to close or open the K
ATP
channel, respectively, had no effect on ghrelin release. How-
ever, although a mechanism by which glucose transport fol-
lowed by glucose metabolism, K
ATP
channel closure, cellular
depolarization, and an ensuing stimulatory effect on secretion
seems intuitive in a system such as the pancreatic -cell (in
which a high glucose state is associated with insulin secretion),
such an equivalent pathway within ghrelin cells does not seem
obvious (since ghrelin secretion is expected in low glucose
states). In this regard, ghrelin cells seem more akin to pancre-
atic -cells, in which glucagon secretion occurs in the setting
of low glucose. Similarly to both pancreatic -cells and ghrelin
cells, pancreatic -cells express Kir6.2/SUR1 complexes, and
they also contain K
ATP
conductance (2, 22, 44). Of note,
whereas tolbutamide increases insulin release, its application to
pancreatic -cells has been shown in many (but not all) studies
to suppress glucagon release (21–23). It has been proposed that
the opposite effects of
D-glucose and tolbutamide on insulin
secretion by pancreatic -cells and on glucagon secretion by
pancreatic -cells are the consequence of the two different cell
types being equipped with different sets of voltage-dependent
ion channels that lead to different electrophysiological charac-
teristics (22). A similar phenomenon might occur within gas-
tric ghrelin cells. Other similar features between ghrelin cells
and pancreatic -cells include lack of GLUT2 expression,
whereas pancreatic -cells contain high levels of GLUT2 (24).
Differential expression of GLUT2, and more broadly, members
of the facilitative glucose transport family, has been associated
with differences in glucose transport kinetics (24) and may
contribute to the differential cellular responses to
D-glucose.
More work will be required to more clearly define the mech-
anism by which D-glucose interacts with glucose transporters,
glycolytic enzymes, and ion channels within the ghrelin cell to
regulate ghrelin release.
We also investigated the effects of somatostatin, secretin,
and endothelin-1 on ghrelin release based on published reports
of such activity. For instance, gastric submucosal microinfu-
sion of both secretin and endothelin-1 stimulated ghrelin se-
cretion in rats, whereas somatostatin inhibited ghrelin release
(14). Using that same technique, neither insulin nor
D-glucose
affected ghrelin release (14). MGN3-1 mouse ghrelinoma cells
demonstrated somatostatin-induced suppression of ghrelin se-
cretion and expression of SSTR2, SSTR3, SSTR5, and SSTR5
(but not SSTR1) but a lack of an effect of secretin on ghrelin
release (28, 29). Here, we failed to demonstrate a statistically
significant effect of somatostatin and secretin on ghrelin re-
lease. This is despite the finding of receptors for those peptide
hormones in ghrelin cell-enriched pools of gastric mucosal
cells, which would otherwise suggest the ability of somatosta-
tin and secretin to affect ghrelin cell physiology in some way.
It is possible that the sensitivity to ambient
D-glucose concen-
tration, which is observed here for norepinephrine and insulin,
E1308 CONTROL OF GHRELIN RELEASE BY GLUCOSE
AJP-Endocrinol Metab doi:10.1152/ajpendo.00041.2012 www.ajpendo.org
may also help to explain a lack of responsiveness to soma-
tostatin and secretin. It also is possible that the effects of these
agents on ghrelin secretion might be apparent only upon
simultaneous exposure to other hormones. Endothelin-1 had no
effect on ghrelin release here, which might coincide with the
barely detectable or lack of detectable levels of its receptors
within ghrelin cells.
In conclusion, we have used a dispersed gastric mucosal cell
primary culture system and qPCR on highly enriched pools of
ghrelin cells to investigate ghrelin secretion. In particular, the
enhanced ghrelin release observed in low D-glucose conditions
and the reduced ghrelin release observed in high D-glucose
conditions, when viewed together with ghrelin’s roles in the
setting of severe caloric restriction to normalize and prevent
life-threatening falls in blood glucose (62), help solidify an
important role for ghrelin in blood glucose homeostasis.
ACKNOWLEDGMENTS
We acknowledge Chelsea Migura for maintenance of the animal colony and
Joyce Repa (University of Texas Southwestern Medical Center) for the
generous gift of qPCR primers and for the critical read-through of the
manuscript.
GRANTS
These studies were made possible through the support of an International
Research Alliance grant with the Novo Nordisk Foundation Center for Basic
Metabolic Research (to J. M. Zigman), National Institutes of Health Grants
1-K08-DK-068069, 1-R01-DA-024680, and 1-R01-MH-085298 (to J. M. Zig-
man), and an Endocrine Fellows Foundation Development Grant in Diabetes,
Obesity, and Fat Cell Biology (to P. K. Piper).
DISCLOSURES
The authors have no potential conflicts of interest, financial or otherwise.
AUTHOR CONTRIBUTIONS
I.S. and J.M.Z. did the conception and design of the research; I.S., W.-M.P.,
A.K.W., P.K.P., J.-C.C., S.O.-L., and J.M.Z. performed the experiments; I.S.,
W.-M.P., A.K.W., P.K.P., J.-C.C., and J.M.Z. analyzed the data; I.S., W.-M.P.,
A.K.W., P.K.P., J.-C.C., and J.M.Z. interpreted the results of the experiments;
I.S., W.-M.P., A.K.W., and J.M.Z. prepared the figures; I.S., W.-M.P., and
J.M.Z. drafted the manuscript; I.S., W.-M.P., A.K.W., and J.M.Z. edited and
revised the manuscript; J.M.Z. approved the final version of the manuscript.
REFERENCES
1. Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, Makino
S, Fujimiya M, Niijima A, Fujino MA, Kasuga M. Ghrelin is an
appetite-stimulatory signal from stomach with structural resemblance to
motilin. Gastroenterology 120: 337–345, 2001.
2. Bokvist K, Olsen HL, Hoy M, Gotfredsen CF, Holmes WF, Buschard
K, Rorsman P, Gromada J. Characterisation of sulphonylurea and
ATP-regulated K
channels in rat pancreatic A-cells. Pflugers Arch 438:
428 –436, 1999.
3. Broglio F, Arvat E, Benso A, Gottero C, Muccioli G, Papotti M, van
der Lely AJ, Deghenghi R, Ghigo E. Ghrelin, a natural GH secretagogue
produced by the stomach, induces hyperglycemia and reduces insulin
secretion in humans. J Clin Endocrinol Metab 86: 5083–5086, 2001.
4. Chuang JC, Perello M, Sakata I, Osborne-Lawrence S, Savitt JM,
Lutter M, Zigman JM. Ghrelin mediates stress-induced food-reward
behavior in mice. J Clin Invest 121: 2684 –2692, 2011.
5. Chuang JC, Sakata I, Kohno D, Perello M, Osborne-Lawrence S,
Repa JJ, Zigman JM. Ghrelin directly stimulates glucagon secretion
from pancreatic alpha-cells. Mol Endocrinol 25: 1600 –1611, 2011.
6. Colombo M, Gregersen S, Xiao J, Hermansen K. Effects of ghrelin and
other neuropeptides (CART, MCH, orexin A and B, and GLP-1) on the
release of insulin from isolated rat islets. Pancreas 27: 161–166, 2003.
7. Colon-Gonzalez F, Kazanietz MG. C1 domains exposed: from diacyl-
glycerol binding to protein-protein interactions. Biochim Biophys Acta
1761: 827–837, 2006.
8. Cook DL, Hales CN, Satin LS. Glucose suppresses ATP-inhibited
K-channels in pancreatic beta-cells. Adv Exp Med Biol 211: 63–67, 1986.
9. Cummings DE, Foster KE. Ghrelin-leptin tango in body-weight regula-
tion. Gastroenterology 124: 1532–1535, 2003.
10. Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE,
Weigle DS. A preprandial rise in plasma ghrelin levels suggests a role in
meal initiation in humans. Diabetes 50: 1714 –1719, 2001.
11. Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger
EP, Purnell JQ. Plasma ghrelin levels after diet-induced weight loss or
gastric bypass surgery. N Engl J Med 346: 1623–1630, 2002.
12. D’Ambra R, Surana M, Efrat S, Starr RG, Fleischer N. Regulation of
insulin secretion from beta-cell lines derived from transgenic mice insuli-
nomas resembles that of normal beta-cells. Endocrinology 126: 2815–
2822, 1990.
13. Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsu-
kura S. Ghrelin acts in the central nervous system to stimulate gastric acid
secretion. Biochem Biophys Res Commun 280: 904 –907, 2001.
14. de la Cour CD, Norlen P, Hakanson R. Secretion of ghrelin from rat
stomach ghrelin cells in response to local microinfusion of candidate
messenger compounds: a microdialysis study. Regul Pept 143: 118–126,
2007.
15. Dezaki K, Hosoda H, Kakei M, Hashiguchi S, Watanabe M, Kangawa
K, Yada T. Endogenous ghrelin in pancreatic islets restricts insulin
release by attenuating Ca
2
signaling in beta-cells: implication in the
glycemic control in rodents. Diabetes 53: 3142–3151, 2004.
16. Dezaki K, Kakei M, Yada T. Ghrelin uses Galphai2 and activates
voltage-dependent K
channels to attenuate glucose-induced Ca
2
signal
-
ing and insulin release in islet beta-cells: novel signal transduction of
ghrelin. Diabetes 56: 2319 –2327, 2007.
17. Dezaki K, Sone H, Koizumi M, Nakata M, Kakei M, Nagai H, Hosoda
H, Kangawa K, Yada T. Blockade of pancreatic islet-derived ghrelin
enhances insulin secretion to prevent high-fat diet-induced glucose intol-
erance. Diabetes 55: 3486 –3493, 2006.
18. Diano S, Farr SA, Benoit SC, McNay EC, da Silva I, Horvath B,
Gaskin FS, Nonaka N, Jaeger LB, Banks WA, Morley JE, Pinto S,
Sherwin RS, Xu L, Yamada KA, Sleeman MW, Tschop MH, Horvath
TL. Ghrelin controls hippocampal spine synapse density and memory
performance. Nat Neurosci 9: 381–388, 2006.
19. Flanagan DE, Evans ML, Monsod TP, Rife F, Heptulla RA, Tambo-
rlane WV, Sherwin RS. The influence of insulin on circulating ghrelin.
Am J Physiol Endocrinol Metab 284: E313–E316, 2003.
20. Foster-Schubert KE, Overduin J, Prudom CE, Liu J, Callahan HS,
Gaylinn BD, Thorner MO, Cummings DE. Acyl and total ghrelin are
suppressed strongly by ingested proteins, weakly by lipids, and biphasi-
cally by carbohydrates. J Clin Endocrinol Metab 93: 1971–1979, 2008.
21. Franklin I, Gromada J, Gjinovci A, Theander S, Wollheim CB.
Beta-cell secretory products activate alpha-cell ATP-dependent potassium
channels to inhibit glucagon release. Diabetes 54: 1808 –1815, 2005.
22. Göpel SO, Kanno T, Barg S, Weng XG, Gromada J, Rorsman P.
Regulation of glucagon release in mouse -cells by KATP channels and
inactivation of TTX-sensitive Na
channels. J Physiol 528: 509 –520,
2000.
23. Gromada J, Ma X, Hoy M, Bokvist K, Salehi A, Berggren PO,
Rorsman P. ATP-sensitive K
channel-dependent regulation of glucagon
release and electrical activity by glucose in wild-type and SUR1
/
mouse
alpha-cells. Diabetes 53, Suppl 3: S181–S189, 2004.
24. Heimberg H, De Vos A, Pipeleers D, Thorens B, Schuit F. Differences
in glucose transporter gene expression between rat pancreatic alpha- and
beta-cells are correlated to differences in glucose transport but not in
glucose utilization. J Biol Chem 270: 8971–8975, 1995.
25. Henquin JC, Nenquin M, Sempoux C, Guiot Y, Bellanne-Chantelot C,
Otonkoski T, de Lonlay P, Nihoul-Fekete C, Rahier J. In vitro insulin
secretion by pancreatic tissue from infants with diazoxide-resistant con-
genital hyperinsulinism deviates from model predictions. J Clin Invest
121: 3932–3942, 2011.
26. Hosoda H, Doi K, Nagaya N, Okumura H, Nakagawa E, Enomoto M,
Ono F, Kangawa K. Optimum collection and storage conditions for
ghrelin measurements: octanoyl modification of ghrelin is rapidly hydro-
lyzed to desacyl ghrelin in blood samples. Clin Chem 50: 1077–1080,
2004.
27. Inagaki N, Gonoi T, Clement JP 4th, Namba N, Inazawa J, Gonzalez
G, Aguilar-Bryan L, Seino S, Bryan J. Reconstitution of IKATP: an
inward rectifier subunit plus the sulfonylurea receptor. Science 270:
1166 –1170, 1995.
E1309CONTROL OF GHRELIN RELEASE BY GLUCOSE
AJP-Endocrinol Metab doi:10.1152/ajpendo.00041.2012 www.ajpendo.org
28. Iwakura H, Ariyasu H, Hosoda H, Yamada G, Hosoda K, Nakao K,
Kangawa K, Akamizu T. Oxytocin and dopamine stimulate ghrelin
secretion by the ghrelin-producing cell line, MGN3–1 in vitro. Endocri-
nology 152: 2619 –2625, 2011.
29. Iwakura H, Li Y, Ariyasu H, Hosoda H, Kanamoto N, Bando M,
Yamada G, Hosoda K, Nakao K, Kangawa K, Akamizu T. Establish-
ment of a novel ghrelin-producing cell line. Endocrinology 151: 2940
2945, 2010.
30. James C, Kapoor RR, Ismail D, Hussain K. The genetic basis of
congenital hyperinsulinism. J Med Genet 46: 289 –299, 2009.
31. Jerlhag E, Egecioglu E, Landgren S, Salome N, Heilig M, Moechars D,
Datta R, Perrissoud D, Dickson SL, Engel JA. Requirement of central
ghrelin signaling for alcohol reward. Proc Natl Acad Sci USA 106:
11318 –11323, 2009.
32. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K.
Ghrelin is a growth-hormone-releasing acylated peptide from stomach.
Nature 402: 656 –660, 1999.
33. Kojima S, Nakahara T, Nagai N, Muranaga T, Tanaka M, Yasuhara
D, Masuda A, Date Y, Ueno H, Nakazato M, Naruo T. Altered ghrelin
and peptide YY responses to meals in bulimia nervosa. Clin Endocrinol
(Oxf) 62: 74 –78, 2005.
34. Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB. Ghre-
lin—a hormone with multiple functions. Front Neuroendocrinol 25: 27–
68, 2004.
35. Liu J, Prudom CE, Nass R, Pezzoli SS, Oliveri MC, Johnson ML,
Veldhuis P, Gordon DA, Howard AD, Witcher DR, Geysen HM,
Gaylinn BD, Thorner MO. Novel ghrelin assays provide evidence for
independent regulation of ghrelin acylation and secretion in healthy young
men. J Clin Endocrinol Metab 93: 1980 –1987, 2008.
36. Longo KA, Charoenthongtrakul S, Giuliana DJ, Govek EK, McDon-
agh T, Qi Y, DiStefano PS, Geddes BJ. Improved insulin sensitivity and
metabolic flexibility in ghrelin receptor knockout mice. Regul Pept 150:
55–61, 2008.
37. Lutter M, Sakata I, Osborne-Lawrence S, Rovinsky SA, Anderson
JG, Jung S, Birnbaum S, Yanagisawa M, Elmquist JK, Nestler EJ,
Zigman JM. The orexigenic hormone ghrelin defends against depressive
symptoms of chronic stress. Nat Neurosci 11: 752–753, 2008.
38. McCowen KC, Maykel JA, Bistrian BR, Ling PR. Circulating ghrelin
concentrations are lowered by intravenous glucose or hyperinsulinemic
euglycemic conditions in rodents. J Endocrinol 175: R7–R11, 2002.
39. Misra M, Miller KK, Kuo K, Griffin K, Stewart V, Hunter E, Herzog
DB, Klibanski A. Secretory dynamics of ghrelin in adolescent girls with
anorexia nervosa and healthy adolescents. Am J Physiol Endocrinol Metab
289: E347–E356, 2005.
40. Mourad NI, Nenquin M, Henquin JC. Metabolic amplifying pathway
increases both phases of insulin secretion independently of -cell actin
microfilaments. Am J Physiol Cell Physiol 299: C389 –C398, 2010.
41. Mundinger TO, Cummings DE, Taborsky GJ Jr. Direct stimulation of
ghrelin secretion by sympathetic nerves. Endocrinology 147: 2893–2901,
2006.
42. Nagaya N, Uematsu M, Kojima M, Date Y, Nakazato M, Okumura H,
Hosoda H, Shimizu W, Yamagishi M, Oya H, Koh H, Yutani C,
Kangawa K. Elevated circulating level of ghrelin in cachexia associated
with chronic heart failure: relationships between ghrelin and anabolic/
catabolic factors. Circulation 104: 2034 –2038, 2001.
43. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa
K, Matsukura S. A role for ghrelin in the central regulation of feeding.
Nature 409: 194 –198, 2001.
44. Nielsen LB, Ploug KB, Swift P, Ørskov C, Jansen-Olesen I, Chiarelli
F, Holst JJ, Hougaard P, Pörksen S, Holl R, de Beaufort C, Gam-
meltoft S, Rorsman P, Mortensen HB, Hansen L; Hvidøre Study
Group. Co-localisation of the Kir6.2/SUR1 channel complex with gluca-
gon-like peptide-1 and glucose-dependent insulinotrophic polypeptide
expression in human ileal cells and implications for glycaemic control in
new onset type 1 diabetes. Eur J Endocrinol 156: 663–671, 2007.
45. Otto B, Cuntz U, Fruehauf E, Wawarta R, Folwaczny C, Riepl RL,
Heiman ML, Lehnert P, Fichter M, Tschöp M. Weight gain decreases
elevated plasma ghrelin concentrations of patients with anorexia nervosa.
Eur J Endocrinol 145: 669 –673, 2001.
46. Qi Y, Longo KA, Giuliana DJ, Gagne S, McDonagh T, Govek E,
Nolan A, Zou C, Morgan K, Hixon J, Saunders JO, Distefano PS,
Geddes BJ. Characterization of the insulin sensitivity of ghrelin receptor
KO mice using glycemic clamps. BMC Physiol 11: 1, 2011.
47. Reed JA, Benoit SC, Pfluger PT, Tschöp MH, D’Alessio DA, Seeley
RJ. Mice with chronically increased circulating ghrelin develop age-
related glucose intolerance. Am J Physiol Endocrinol Metab 294: E752–
E760, 2008.
48. Sakata I, Nakano Y, Osborne-Lawrence S, Rovinsky SA, Lee CE,
Perello M, Anderson JG, Coppari R, Xiao G, Lowell BB, Elmquist JK,
Zigman JM. Characterization of a novel ghrelin cell reporter mouse.
Regul Pept 155: 91–98, 2009.
49. Sakata I, Tanaka T, Yamazaki M, Tanizaki T, Zheng Z, Sakai T.
Gastric estrogen directly induces ghrelin expression and production in the
rat stomach. J Endocrinol 190: 749 –757, 2006.
50. Sakata I, Yang J, Lee CE, Osborne-Lawrence S, Rovinsky SA,
Elmquist JK, Zigman JM. Colocalization of ghrelin O-acyltransferase
and ghrelin in gastric mucosal cells. Am J Physiol Endocrinol Metab 297:
E134 –E141, 2009.
51. Shimbara T, Mondal MS, Kawagoe T, Toshinai K, Koda S, Yamagu-
chi H, Date Y, Nakazato M. Central administration of ghrelin preferen-
tially enhances fat ingestion. Neurosci Lett 369: 75–79, 2004.
52. Solomon A, De Fanti BA, Martinez JA. Peripheral ghrelin participates in
the glucostatic signaling mediated by the ventromedial and lateral hypo-
thalamus neurons. Peptides 27: 1607–1615, 2006.
53. Strassburg S, Anker SD, Castaneda TR, Burget L, Perez-Tilve D,
Pfluger PT, Nogueiras R, Halem H, Dong JZ, Culler MD, Datta R,
Tschöp MH. Long-term effects of ghrelin and ghrelin receptor agonists on
energy balance in rats. Am J Physiol Endocrinol Metab 295: E78 –E84,
2008.
54. Sun Y, Asnicar M, Saha PK, Chan L, Smith RG. Ablation of ghrelin
improves the diabetic but not obese phenotype of ob/ob mice. Cell Metab
3: 379 –386, 2006.
55. Tanaka M, Naruo T, Muranaga T, Yasuhara D, Shiiya T, Nakazato
M, Matsukura S, Nozoe S. Increased fasting plasma ghrelin levels in
patients with bulimia nervosa. Eur J Endocrinol 146: R1–R3, 2002.
56. Theander-Carrillo C, Wiedmer P, Cettour-Rose P, Nogueiras R,
Perez-Tilve D, Pfluger P, Castaneda TR, Muzzin P, Schürmann A,
Szanto I, Tschöp MH, Rohner-Jeanrenaud F. Ghrelin action in the
brain controls adipocyte metabolism. J Clin Invest 116: 1983–1993, 2006.
57. Tolle V, Kadem M, Bluet-Pajot MT, Frere D, Foulon C, Bossu C,
Dardennes R, Mounier C, Zizzari P, Lang F, Epelbaum J, Estour B.
Balance in ghrelin and leptin plasma levels in anorexia nervosa patients
and constitutionally thin women. J Clin Endocrinol Metab 88: 109 –116,
2003.
58. Trivedi A, Babic S, Chanoine JP. Pitfalls in the determination of human
acylated ghrelin plasma concentrations using a double antibody enzyme
immunometric assay. Clin Biochem 45: 178 –180, 2012.
59. Tschöp M, Smiley DL, Heiman ML. Ghrelin induces adiposity in
rodents. Nature 407: 908 –913, 2000.
60. Wisse BE, Frayo RS, Schwartz MW, Cummings DE. Reversal of
cancer anorexia by blockade of central melanocortin receptors in rats.
Endocrinology 142: 3292–3301, 2001.
61. Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA,
Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR. Ghrelin
causes hyperphagia and obesity in rats. Diabetes 50: 2540 –2547, 2001.
62. Zhao TJ, Liang G, Li RL, Xie X, Sleeman MW, Murphy AJ, Valen-
zuela DM, Yancopoulos GD, Goldstein JL, Brown MS. Ghrelin O-
acyltransferase (GOAT) is essential for growth hormone-mediated sur-
vival of calorie-restricted mice. Proc Natl Acad Sci USA 107: 7467–7472,
2010.
63. Zhao TJ, Sakata I, Li RL, Liang G, Richardson JA, Brown MS,
Goldstein JL, Zigman JM. Ghrelin secretion stimulated by {beta}1-
adrenergic receptors in cultured ghrelinoma cells and in fasted mice. Proc
Natl Acad Sci USA 107: 15868 –15873, 2010.
64. Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE,
Jones JE, Deysher AE, Waxman AR, White RD, Williams TD, Lachey
JL, Seeley RJ, Lowell BB, Elmquist JK. Mice lacking ghrelin receptors
resist the development of diet-induced obesity. J Clin Invest 115: 3564
3572, 2005.
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AJP-Endocrinol Metab doi:10.1152/ajpendo.00041.2012 www.ajpendo.org
    • "Increased blood glucose (Flanagan et al. 2003; Shiiya et al. 2002; Sim et al. 2014) and insulin concentrations (Broglio et al. 2004; Cummings & Overduin, 2007; Iwakura et al. 2015) are associated with decreased total ghrelin levels. High concentrations of glucose directly suppressed ghrelin secretion potentially through GLUT1, 4, 5 receptors and glucokinase that are abundant on ghrelin producing cells in mice (Sakata et al. 2012). The inhibitory effect of insulin is also at least partially direct, as ghrelin producing cells express insulin receptors (Iwakura et al. 2010) and insulin directly impeded ghrelin secretion in rat stomach cells (Gagnon & Anini, 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: The physiological control of appetite regulation involves circulating hormones with orexigenic (appetite-stimulating) and anorexigenic (appetite-inhibiting) properties that induce alterations in energy intake via perceptions of hunger and satiety. As the effectiveness of exercise to induce weight loss is a controversial topic, there is considerable interest in the effect of exercise on the appetite-regulating hormones such as acylated ghrelin, peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and pancreatic polypeptide (PP). Research to date suggests short-term appetite regulation following a single exercise session is likely affected by decreases in acylated ghrelin and increases in PYY, GLP-1, and PP. Further, this exercise-induced response may be intensity-dependent. In an effort to guide future research, it is important to consider how exercise alters the circulating concentrations of these appetite-regulating hormones. Potential mechanisms include blood redistribution, sympathetic nervous system activity, gastrointestinal motility, cytokine release, free fatty acid concentrations, lactate production, and changes in plasma glucose and insulin concentrations. This review of relevant research suggests blood redistribution during exercise may be important for suppressing ghrelin, while other mechanisms involving cytokine release, changes in plasma glucose and insulin concentrations, SNS activity, and muscle metabolism likely mediate changes in the anorexigenic signals PYY and GLP-1. Overall, changes in appetite-regulating hormones following acute exercise appear to be intensity-dependent, with increasing intensity leading to a greater suppression of orexigenic signals and greater stimulation of anorexigenic signals. However, there is less research on how exercise-induced responses in appetite-regulating hormones differ between sexes or different age groups. A better understanding of how exercise intensity and workload affect appetite across the sexes and life stages will be a powerful tool in developing more successful strategies for managing weight.
    Full-text · Article · Dec 2015
    • "Insulin, glucagon, oxytocin, somatostatin , dopamine, glucose, and long-chain fatty acids all have been shown to regulate ghrelin secretion through their direct interaction with ghrelin cells [253,288,307e310]. In addition, all of these models, as well as related in vivo studies, have been used to confirm that the catecholamines norepinephrine and epinephrine act as direct ghrelin secretagogues [253,287,288,299,307] . These data are supported by high levels of b1-adrenergic receptor expression in ghrelin cells enriched from the stomach of ghrelin-green fluorescent protein reporter mice as well as in the SG-1 and PG-1 ghrelin cell lines [253]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. Scope of review: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. Major conclusions: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.
    Full-text · Article · Mar 2015
    • "Furthermore, the glucoprivic agent 2-deoxy-D-glucose prevents the inhibitory effect of high glucose exposure on ghrelin release, suggesting a requirement for glucose entry into the ghrelin cell and subsequent metabolism for its inhibitory effects on ghrelin secretion [71]. This is further supported by the expression by ghrelin cells of mRNAs encoding several channels and enzymes responsible for mediating the effects glucose responsiveness and metabolism in other cell types, including several facilitative glucose transporters (GLUT1, GLUT4, and GLUT5), hexokinases (including glucokinase), and both components of the pancreatic b-cell KATP channel (the ATP-sensitive potassium channel subunit Kir6.2 and the sulfonylurea type 1 receptor SUR1) [71]. In evaluating the observed lower ghrelin levels in DIO, one might expect known stimulators of ghrelin secretion e such as norepinephrine e to no longer be potent and/or inhibitors of ghrelin secretion e such as glucose e to be more potent. "
    [Show abstract] [Hide abstract] ABSTRACT: The current study examined potential mechanisms for altered circulating ghrelin levels observed in diet-induced obesity (DIO) and following weight loss resulting from Roux-en-Y gastric bypass (RYGB). We hypothesized that circulating ghrelin levels were altered in obesity and after weight loss through changes in ghrelin cell responsiveness to physiological cues. We confirmed lower ghrelin levels in DIO mice and demonstrated elevated ghrelin levels in mice 6 weeks post-RYGB. In both DIO and RYGB settings, these changes in ghrelin levels were associated with altered ghrelin cell responsiveness to two key physiological modulators of ghrelin secretion – glucose and norepinephrine. In DIO mice, increases in ghrelin cell density within both the stomach and duodenum and in somatostatin-immunoreactive D cell density in the duodenum were observed. Our findings provide new insights into the regulation of ghrelin secretion and its relation to circulating ghrelin within the contexts of obesity and weight loss.
    Full-text · Article · Oct 2014
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