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INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS |
Department of Surgery (H.-M.L., G.W., E.W.E., G.H.G.), University of Texas Medical Branch, Galveston, Texas 77555; and Department of Molecular Genetics (M.K.), Institute of Life Science, Karume-University, Karume, Fukuoka 839-0861, Japan
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Abstract |
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Introduction |
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The evidence to date indicates that ghrelin is an important endocrine peptide that links the gastrointestinal system and brain in the regulation of food intake and energy expenditure. Therefore, in the present article, the enteric distribution of ghrelin, ontogeny of stomach ghrelin gene expression, and influence of dietary and endocrine manipulations and vagotomy on stomach ghrelin levels were investigated. In some cases, plasma ghrelin concentrations were measured. We also examined the effects of iv administration of ghrelin on gastrin and insulin secretion in vivo in rats.
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Methods and Materials |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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Chemicals and peptides
All chemicals were obtained from Sigma (St. Louis,
MO). Synthetic peptides were purchased from Bachem
(Torrance, CA), with the exception of ghrelin. Synthetic ghrelin was
purchased from Bachem or supplied by M. Kojima.
Animal experiments
Gastrointestinal (GI) expression of ghrelin. Ghrelin
expression (i.e. mRNA levels) and peptide concentrations in
various regions of the GI tract were examined by Northern blotting
analysis and RIA of ghrelin peptide levels using total cellular RNA and
tissue extracts, respectively. Ad libitum (ad lib)-fed adult
male rats were killed and the mucosal epithelial layer of the stomach
fundus, stomach antrum, duodenum, jejunum, and colon scraped and
extracted for either total cellular RNA or ghrelin peptide. Previous
reports indicated by immunohistochemistry that ghrelin is expressed
only in the mucosal epithelium; we have confirmed that the muscle layer
of the GI tract does not produce ghrelin (data not shown). Ghrelin
expression was also analyzed in extracts of the rat pancreas, liver,
and kidney.
Ontogeny of stomach ghrelin gene expression. Findings in Exp 1 show that ghrelin is expressed primarily in the stomach fundus; therefore, the stomach fundus was chosen for the developmental study. Rat fetuses were harvested from timed-pregnant Sprague Dawley rats. Rat fetuses of both sexes were collected at 21 d gestation and the entire stomachs extirpated, taking care not to include pancreas. Litters were born at approximately 22 d gestation and were kept with their mothers until 21 d post partum. After birth, ad libitum-fed (i.e., nursing) male and female Sprague Dawley rat pups were sampled at frequent intervals. At 21 d gestation and at 1, 5, 7, 10, 12, 16, 18, and 22 d old, the entire stomach (full thickness, without the rumen) was extracted for total RNA and ghrelin peptide. For older animals the mucosal layer of only the stomach fundus was harvested. For fetal and some of the early postnatal samples, the stomach fundus specimens from three to four littermates were pooled to constitute one sample. Plasma was collected from nursing pups at 5, 12, 13, 17, and 21 d of age and 30 d of age from weaned pups for measurement of ghrelin levels. With the earlier ages, plasma from littermates was pooled to extract 1.2-ml plasma specimens. All tissue samples were removed quickly after animals were killed and were immediately homogenized in either an RNA or ghrelin peptide extraction solution for measurement of ghrelin expression and peptide levels, respectively. Samples were stored at -80 C until assays or Northern hybridizations were done.
Influence of dietary manipulations and a fasting-refeeding regimen on stomach ghrelin. Adult male Sprague Dawley rats were fed ad lib either a commercial, AIN-76A biscuit diet (Bio-Serve, Frenchtown, NJ) (composition: approximate percent of calories: fat 12%; protein 20%; carbohydrate 65%); a high-fat diet (fat 48%, beef tallow; protein 16%; carbohydrate 34%) or a low-protein diet (fat 12%; protein 5%, casein; carbohydrate 83%) for 30 d. Two percent of the fat calories were derived from safflower oil in the beef tallow diet to supply essential fatty acids. Animals were killed in the ad lib-fed condition. At time of sacrifice, AIN-76A-fed rats, high-fat diet rats, and low-protein diet rats weighed (mean ± SEM) 358 ± 8 g, 367 ± 5 g, and 282 ± 6 g, respectively. For the fasting-refeeding experiment, rats were fasted for 72 h; a portion of the fasted rats was then refed ad lib for 24 h before being killed. All rats had unrestricted access to water. The stomach fundal mucosa was harvested and extracted for total cellular RNA and ghrelin peptide. Plasma was collected for measurement ghrelin levels by RIA.
Influence of endocrine manipulations and vagotomy on stomach ghrelin expression and peptide levels and plasma ghrelin concentrations. Adult male Sprague Dawley rats were given synthetic rat gastrin-17 (150 µg/kg body weight (BW), 3 times a day for 4 d, sc), T4 (50 µg/100 g BW·d for 4 d, ip), GH (400 µg/kg BW, 3 times a day for 3 d, sc), leptin (240 µg/kg BW, 2 times a day for 4 d, sc), or insulin (40 U/kg, 2 times a day for 3 d, sc). The influence of thyroidectomy and truncal vagotomy on stomach ghrelin and plasma ghrelin levels was also examined. Surgeries were done as previously described (8, 9), and rats were killed approximately 14 d after thyroidectomy or vagotomy. Highly purified rat GH and recombinant mouse leptin were used (supplied by A. F. Parlow, National Hormone and Pituitary Program-NIDDK). GH, leptin, insulin, and gastrin were prepared in 0.154 M saline containing 0.1% BSA. T4 was prepared in a mixture of methanol-ammonium hydroxide (2 ml/0.4 ml) and appropriate dilutions made in 0.154 M saline.
Effects of ghrelin on insulin and gastrin secretion. Ad
lib-fed male Sprague Dawley rats (
115 g) were given synthetic rat
ghrelin (25 nmol) or saline-containing 0.1%BSA iv into the jugular
vein under ether anesthesia. This experiment was done at 0900–1130 h.
Serum was then collected at various times after iv ghrelin or vehicle.
Basal serum specimens were also collected from ad lib-fed rats. Serum
gastrin and insulin levels were measured with radioimmunoassays as
described previously (10). The sensitivity and
ID50 (50% inhibition of maximal binding) for the
gastrin and insulin assays are 6 and 20 pg/tube, and 4 and 40 pg/tube,
respectively. The gastrin antiserum does not recognize
cholecystokinin.
RNA purification and Northern blotting analysis. Tissues were homogenized immediately in 4 M guanidinium isothiocyanate containing 25 mM sodium citrate, pH 7.0, 0.5% sodium N-lauroylsarcosine, and 0.1 M ß-mercaptoethanol. Extracts were frozen at -80 C until purification by ultracentrifugation over a cesium chloride cushion (2 ml, 5.7 M) as described previously (11). RNA samples were separated on a 1% agarose gel (30 µg/lane) in a 20 mM 3-[N-morpholino]propanesulfonic acid running buffer system (11, 12) and then transferred to a nylon membrane and subjected to Northern hybridization. 32P-Labeled riboprobes prepared from Strip-EZ RNA kits (catalog no. 1366, Ambion, Inc., Austin, TX) were used for Northern hybridizations. Complementary RNA for rat ghrelin was supplied by M. Kojima (1). The 18S was used to normalize for variations in RNA loading and transfer. Expression levels of ghrelin or the 18S genes were quantitated by phosphoimaging.
Ghrelin RIA; extraction of tissue and plasma ghrelin
A double-antibody RIA procedure was used to measure tissue and
plasma ghrelin levels (1). The ghrelin antiserum was
generated in rabbits against a synthetic C-terminal fragment (residues
13–28 with an added N-terminal tyrosine) of rat ghrelin. The ghrelin
antiserum does not recognize other enteric peptides. The sensitivity
and ID50 are 0.01 and 0.2 ng/tube. The intra- and
interassay coefficients of variation are 13% and 20%, respectively.
Ghrelin peptide was extracted from rat tissues by homogenizing tissues
in approximately 10 volumes of 1 M acetic acid containing
20 mM HCl. Homogenates were then boiled for 20 min. The
supernatants were lyophilized and resuspended in assay buffer for the
ghrelin RIA. Extraction efficiency of tissue ghrelin is 75–80%. Rat
plasma (1.2 ml/rat) was extracted by use of C18 Sep-Paks
(Waters, Milford, MA). Plasma for ghrelin RIA was
collected into tubes prepared with EDTA (1 mg/ml blood) and a protease
inhibitor (Trasylol, 70 µg/ml blood). For ghrelin
extraction, 1.2 ml plasma is mixed with an equal volume of 0.9% NaCl
and the pH adjusted to 6.8–6.9 with 12 µl 1 N HCl/1 ml
plasma. Samples were mixed well and added to C18 Sep-Paks prepared with
two 4-ml chloroform washes, followed by two 3-ml methanol washes, two
3-ml washes of acetonitrile (ACN) containing 0.1% trifluoroacetic acid
(TFA) and two 3-ml 0.9% NaCl washes. Disposable glass syringes
are used to add the washes and syringes are changed after the methanol
wash. Plasma specimens are added after the saline wash, followed by two
3-ml saline washes and two 3-ml washes of 5% ACN containing 0.1% TFA.
Ghrelin is eluted off the Sep-Paks with 4 ml 60% ACN containing 0.1%
TFA into tubes containing 10 µl 0.1% Triton. ACN is then evaporated
from the samples and the samples lyophilized. A buffer of 480 µl RIA
is added to lyophilized samples and samples assayed at 100–200
µl/sample in duplicate in the ghrelin RIA. Extraction efficiency for
plasma ghrelin is more than 70%.
Statistics
Results are shown as means ± SE. Data were
analyzed by a one-way or two-way ANOVA followed by the Newman-Keuls
test where pertinent. Differences with a value of P <
0.05 were considered significant.
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Results |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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). The most abundant expression and
peptide concentrations were measured in mucosal extracts of the stomach
fundus and the lowest ghrelin expression and peptide levels in mucosal
extracts of the colon. Ghrelin mRNA and peptide levels of the stomach
fundus were approximately 10-fold greater than those measured in the
stomach-antrum. Ghrelin expression was not detected in extracts of
liver, kidney, pancreas, and the muscle layer of the stomach
(data not shown). A single approximately 0.7-kb transcript was found as
described earlier (1, 3).
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). Ghrelin mRNA was first detectable at
1 d of age and ghrelin peptide was first detectable at 7 d of
age. From 1 and 7 d of age, respectively, stomach ghrelin mRNA and
peptide levels increased progressively as a function of age through the
second and third weeks of life. Thereafter, ghrelin mRNA and peptide
levels attained a plateau through the end of the nursing period with
values that were approximately 10- to 20-fold and 3- to 4-fold higher
than the average ghrelin mRNA and peptide levels, respectively, early
after birth. Changes in stomach ghrelin mRNA levels and peptide
concentrations were parallel during ontogeny. In 5-d-old pups, plasma
ghrelin was undetectable; however, plasma levels of ghrelin increased
progressively during the late postnatal period (Fig. 2).
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). Stomach ghrelin peptide stores were
unchanged in fasted rats, compared with control ad lib-fed rats.
Refeeding of 72 h-fasted rats restored stomach ghrelin mRNA levels to
control expression levels. Refeeding of fasted rats increased stomach
ghrelin peptide stores significantly higher than control levels.
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).
In contrast, stomach ghrelin expression was significantly higher in
rats fed a low-protein diet, compared with AIN-76A-fed rats. Stomach
concentrations of ghrelin peptide were unchanged by either high-fat or
low-protein diets. Plasma ghrelin levels were significantly lower in
rats fed the high-fat diet, whereas plasma ghrelin levels were
significantly higher in rats fed the low-protein diet, compared with
rats fed the control diet. Plasma ghrelin levels in rats fed the
low-protein diet were approximately 2-fold greater when compared with
control rats.
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and 2. Thyroidectomy increased stomach mRNA
levels significantly (controls, 0.36 ± 0.05 vs.
thyroidectomized, 0.8 ± 0.08) but did not affect stomach ghrelin
peptide levels (data not shown). GH treatment decreased stomach ghrelin
mRNA levels significantly (controls, 1.4 ± 0.09 vs. GH
treatment, 0.5 ± 0.06). Vagotomy, insulin, and gastrin treatments
increased plasma ghrelin levels significantly. Vagotomy increased
plasma ghrelin levels approximately 3-fold (controls, 1418 ± 307
vs. vagotomy, 4128 ± 526 pg/ml). Gastrin and insulin
treatments increased plasma ghrelin levels approximately 2-fold
(controls, 1423 ± 328 vs. gastrin treated, 2836
± 349 pg/ml; controls, 2629 ± 521 vs. insulin
treated, 4678 ± 384 pg/ml). Thyroidectomy,
T4, and leptin treatments did not influence
plasma ghrelin levels.
Intravenous ghrelin stimulates insulin and gastrin secretion
Intravenous administration of ghrelin increased serum gastrin and
insulin levels significantly (Table 3).
Serum gastrin and insulin levels were increased significantly at 15 and
60 min after iv ghrelin, compared with control-basal levels and to
those levels of rats given iv vehicle. Serum levels of gastrin and
insulin did not change significantly 5 min after iv ghrelin (data not
shown).
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Discussion |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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In this study, we examined the effects of various endocrine
manipulations on stomach ghrelin expression and peptide levels. Because
ghrelin stimulates GH, insulin, and gastrin secretion, food intake and
body weight gain (1, 5, 6), we expected stomach ghrelin
expression and secretion to be influenced by metabolic factors. Our
results show that stomach ghrelin mRNA and peptide levels are
unaffected by leptin, T4, and insulin treatments.
An earlier study (5) showed that exogenous leptin
decreases stomach ghrelin expression significantly in mice. The
differences between the two studies might be explained by dosages of
leptin and species differences. The dose of leptin used in the mouse
study (57 µg/30 g mouse) was approximately 2-fold higher than that
used in our study (250 µg/225 g rat). We also showed that
thyroidectomy treatment increases stomach ghrelin expression
slightly but significantly, whereas vagotomy lowered stomach ghrelin
expression. Intriguingly, GH treatment lowered stomach ghrelin
expression levels, suggesting that a neuroendocrine feedback loop
exists between stomach ghrelin and pituitary GH. Further studies are
needed to explore whether GH modulates stomach ghrelin peptide stores
and secretion.
Truncal vagotomy increases plasma ghrelin levels indicating that the vagus nerve exerts a tonic inhibitory influence over ghrelin secretion. This finding agrees with the increase in ghrelin secretion observed during a fasted condition, a time when vagal (i.e. parasympathetic) activity is at a nadir. Because one of the documented, biological activities of ghrelin is to stimulate food intake (4, 5, 6, 7), ghrelin secretion is expected to increase during fasting. It can be pointed out that the increased secretion of stomach ghrelin with fasting is unique when contrasted to a majority of gut hormones whose secretion increases with food intake and decreases with fasting. The finding that ghrelin secretion decreases with food intake implies that either a nutrient, a gut or pancreatic hormone released by an ingested nutrient, or both modulate ghrelin secretion. Our present findings indicate that insulin is not involved in the postcibal decline in circulating ghrelin levels because insulin administration increases ghrelin secretion. This finding also bolsters a role for endogenous ghrelin in regulation of food intake. The finding that stomach ghrelin homeostasis (i.e. mRNA, peptide levels) and secretion are responsive to dietary manipulations, caloric restriction, and refeeding is expected because stomach ghrelin cells are exposed to the luminal contents.
Data given in this article show that gastrin clearly stimulates ghrelin secretion. These findings suggest that the reduction in ghrelin secretion in fasted rats with refeeding is not because of food-induced release of gastrin. Another gut peptide may inhibit ghrelin secretion.
The increased secretion of ghrelin with exogenous gastrin and insulin treatments, in view of the increased secretion of ghrelin with fasting, indicates that regulation of ghrelin secretion is complex and that its secretion is stimulated by seemingly contradictory signals. Gastrin and insulin secretion is reduced with fasting (10, 15).
The finding that truncal vagotomy disinhibits ghrelin secretion implies that its secretion is influenced by central neural mechanisms originating in the dorsal vagal complex of the medulla oblongata. The dorsal vagal complex has been shown to modulate gastrointestinal and pancreatic activity (16, 17, 18).
It is difficult to reconcile the elevation in plasma ghrelin levels during acute nutrient restriction (i.e. fasting and protein deprivation) in view of the GH-releasing action of ghrelin. It is conceivable that the GH-releasing action of ghrelin is repressed in the hypothalamus or pituitary, whereas its orexigenic actions are preserved during periods of caloric or nutritional restriction. Multiple ghrelin receptors may exert these various effects in the brain and pituitary. A more plausible explanation is that the protein deprivation causes increased ghrelin secretion to counter protein depletion by stimulating appetite and GH secretion that, in turn, stimulates protein synthesis.
Ghrelin secretion increases with a low-protein diet and decreases with a high-fat diet. Stomach ghrelin expression parallels ghrelin secretion. The decreased ghrelin secretion accompanying increased dietary fat may be owing to an inhibition of its secretion by luminal fat itself or by another enteric hormone or a metabolic signal that is sensitive or dependent on dietary fat. Fat-sensitive signals include PYY, glucagon-like peptide-1, neurotensin, and cholecystokinin from the intestine; leptin and resistin from adipocytes; and FFA from dietary fat (19, 20, 21). The reduction in plasma ghrelin levels with the high-fat diet agrees with a recent article showing decreased circulating ghrelin levels in obese humans (22). The authors of the human study suggest that the reduced plasma ghrelin levels reflect an adaptation to the excessive caloric intake in obese subjects. They also suggest that elevated plasma leptin and insulin levels in obese subjects underline the lower ghrelin secretion. In the present study, we show that exogenous leptin does not affect ghrelin secretion and that exogenous insulin stimulates ghrelin secretion. In the low-protein and high-fat diet rat experiments, the changes in ghrelin secretion may be triggered actually by the altered percentages of dietary carbohydrates. In the low-protein and the high-fat diets, carbohydrate percentages are increased and decreased, respectively. An earlier report indicates that dietary carbohydrates are a primary regulator of GH secretion (23). The hypothesis that dietary carbohydrates modulate ghrelin secretion is under study.
This study shows, for the first time, that ghrelin stimulates gastrin and insulin secretion. Circulating gastrin and insulin levels are increased significantly within 15 min after iv ghrelin administration. Although ghrelin receptors are present in the stomach and pancreas (24, 25, 26), the delayed elevations in serum gastrin and insulin levels at 15 min and again at 60 min suggests that ghrelin may act indirectly to stimulate gastrin and insulin secretion. The ghrelin-induced secretion of gastrin and insulin is not owing to an acute intake of food. Food intake was not stimulated in this experiment. Ghrelin also stimulates gastric acid secretion in rats (27). Therefore, the stimulatory action of ghrelin on acid secretion may be mediated partly by ghrelin-induced release of stomach gastrin.
Together, our findings when considered with the earlier reports from other laboratories, indicate that ghrelin is an important stomach hormone that links enteric nutrition with gastrin and insulin secretion, central neural regulation of GH secretion, growth, and food intake.
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Footnotes |
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This work was supported by grants from the National Institutes of Health (RO1 DK-15241, PO1 DK-35608).
Abbreviations: ACN, Acetonitrile; ad lib, ad libitum; BW, body weight; GHS-R, GH secretagogue receptor; GI, gastrointestinal; PYY, peptide YY; TFA, trifluoroacetic acid.
Received June 7, 2001.
Accepted for publication September 27, 2001.
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Y. Nishi, H. Hiejima, H. Mifune, T. Sato, K. Kangawa, and M. Kojima Developmental Changes in the Pattern of Ghrelin's Acyl Modification and the Levels of Acyl-Modified Ghrelins in Murine Stomach Endocrinology, June 1, 2005; 146(6): 2709 - 2715. [Abstract] [Full Text] [PDF]
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T. Sato, Y. Fukue, H. Teranishi, Y. Yoshida, and M. Kojima Molecular Forms of Hypothalamic Ghrelin and Its Regulation by Fasting and 2-Deoxy-D-Glucose Administration Endocrinology, June 1, 2005; 146(6): 2510 - 2516. [Abstract] [Full Text] [PDF]
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