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Originally published In Press as doi:10.1074/jbc.M411358200 on February 8, 2005

J. Biol. Chem., Vol. 280, Issue 15, 15247-15256, April 15, 2005
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Analysis of Rat Insulin II Promoter-Ghrelin Transgenic Mice and Rat Glucagon Promoter-Ghrelin Transgenic Mice*

Hiroshi Iwakura{ddagger}, Kiminori Hosoda{ddagger}§, Choel Son{ddagger}, Junji Fujikura{ddagger}, Tsutomu Tomita{ddagger}, Michio Noguchi{ddagger}, Hiroyuki Ariyasu{ddagger}, Kazuhiko Takaya{ddagger}||, Hiroaki Masuzaki{ddagger}, Yoshihiro Ogawa{ddagger}, Tatsuya Hayashi{ddagger}, Gen Inoue{ddagger}, Takashi Akamizu||, Hiroshi Hosoda||**, Masayasu Kojima{ddagger}{ddagger}, Hiroshi Itoh{ddagger}, Shinya Toyokuni¶, Kenji Kangawa||**, and Kazuwa Nakao{ddagger}

From the {ddagger}Department of Medicine and Clinical Science, Endocrinology and Metabolism and Department of Pathology and Biology of Diseases, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, the ||Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, the {ddagger}{ddagger}Department of Molecular Genetics, Institute of Life Science, Kurume University, Fukuoka 839-0861, and the **Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan

Received for publication, October 5, 2004 , and in revised form, February 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The pancreatic tissue ghrelin concentration measured by C-terminal radioimmunoassay (RIA) and plasma desacyl ghrelin concentration of RIP-G Tg were about 1000 and 3.4 times higher than those of nontransgenic littermates, respectively. The pancreatic tissue n-octanoylated ghrelin concentration measured by N-terminal RIA and plasma n-octanoylated ghrelin concentration of RIP-G Tg were not distinguishable from those of nontransgenic littermates. RIP-G Tg showed suppression of glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion, pancreatic insulin mRNA and peptide levels, {beta} cell mass, islet architecture, and GLUT2 and PDX-1 immunoreactivity in RIP-G Tg pancreas were not significantly different from those of nontransgenic littermates. Islet batch incubation study did not show suppression of insulin secretion of RIP-G Tg in vitro. The insulin tolerance test showed lower tendency of blood glucose levels in RIP-G Tg. Taking lower tendency of triglyceride level of RIP-G Tg into consideration, these results may indicate that the suppression of insulin secretion is likely due to the effect of desacyl ghrelin on insulin sensitivity. RGP-G Tg, in which the pancreatic tissue ghrelin concentration measured by C-RIA was about 50 times higher than that of nontransgenic littermates, showed no significant changes in insulin secretion, glucose metabolism, islet mass, and islet architecture. The present study raises the possibility that desacyl ghrelin may have influence on glucose metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ghrelin is a 28-amino acid peptide with unique modification of acylation, which is essential for its biological action (1). Ghrelin was originally identified in rat stomach as an endogenous ligand for an orphan receptor, which has been so far called growth hormone secretagogue receptor (GHS-R)1 (1). Ghrelin expression is detected in the stomach, intestine, hypothalamus, pituitary gland, kidney, placenta, and testis (2–6). Ghrelin is involved in a wide variety of the functions, including the regulation of growth hormone release, food intake, gastric acid secretion, gastric motility, blood pressure, and cardiac output (7–19).

Recently Date et al. (20) reported that ghrelin is present in {alpha} cells of normal human and rat pancreatic islets. Volante et al. (21) described ghrelin-expression in {beta} cells of human islet. Wierup et al. and Prado et al. reported that ghrelin-expressing cells are a new islet cell type distinct from {alpha}, {beta}, {delta}, and PP cells in human, rat, and mouse islets (22–24). Although there was no apparent change of plasma insulin levels in ghrelin null mouse (25, 26), which may indicate that ghrelin is not a direct regulator of insulin secretion in the physiological condition, there have been several reports of the effect of pharmacological dose of ghrelin on insulin secretion. Broglio et al., Egido et al., and Reimer et al. have reported that ghrelin has an inhibitory effect on insulin secretion (27–30). Adeghate et al., Date et al., and Lee et al. have reported that ghrelin stimulates insulin secretion (20, 31, 32). Salehi et al. have reported ghrelin has both inhibitory and stimulatory effects depending on its concentration (33). Therefore, there is still a lot of controversy about the localization of ghrelin in the pancreas and the effects of ghrelin on the insulin secretion. As for the effects of desacyl ghrelin on insulin secretion, Broglio et al. (34) have reported that acute desacyl ghrelin administration has no effect on insulin secretion in human but that it counteracts the inhibitory effect of n-octanoylated ghrelin on insulin secretion when co-administrated with n-octanoylated ghrelin (35).

Here we developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic mice (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The purpose of this study was to clarify the effect of transgenic overexpression of ghrelin cDNA in pancreatic islets.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generating RIP- and RGP-ghrelin Transgenic Mice—Mouse stomach cDNA library was constructed from 1 µg of mouse stomach poly(A)+ RNA with a cDNA synthesis kit (Amersham Biosciences). Mouse ghrelin cDNA was isolated from this library, using rat ghrelin cDNA as a probe. A fusion gene comprising RIP and mouse ghrelin cDNA coding sequences was designed. The purified fragment (10 µg/ml) was micro-injected into the pronucleus of fertilized C57/B6J mice (SLC, Shizuoka, Japan) eggs. The viable eggs were transferred into the oviducts of pseudopregnant female ICR mice (SLC) using standard techniques. Transgenic founder mice were identified by Southern blot analysis of tail DNAs using the mouse ghrelin cDNA fragment as a probe. RGP-G Tg was generated similarly. Transgenic mice were used as heterozygotes. Animals were maintained on standard rat food (CE-2, 352 kcal/100 g, Japan CLEA, Tokyo, Japan) on a 12-h light/12-h dark cycle. All experimental procedures were approved by the Kyoto University Graduate School of Medicine Committee on Animal Research.

Immunohistochemistry—Formalin-fixed, paraffin-embedded tissue sections were immunostained using the avidin-biotin peroxidase complex method (Vectastain "ABC" Elite kit, Vector Laboratories, Burlingame, CA) as described previously (36). Serial sections were used, and the thickness of each section was 5 µm. Sections were incubated with anti-C-terminal ghrelin [13–28] (1:1000 at final dilution), anti-N-terminal ghrelin [1–11] (1:2000) (1), which recognizes the n-octanoylated portion of ghrelin, anti-glucagon (1:500), anti-insulin (1:500), anti-somatostatin (1:500), anti-pancreatic polypeptide (PP, 1:500, DAKO, Glostrup, Denmark), anti-PDX-1 (1:2000, kindly provided by Christopher V. E. Wright) (37), and anti-GLUT2 (1:200, kindly provided by Bernard Thorens) (38) antisera. Quantification of {beta} cell area was performed in insulin-stained sections by using Axio Vision (Carl Zeiss, Hallbergmoos, Germany) and Scion Image (Scion Corp., Frederick, MD). Ten sections (200-µm interval) for each mouse (n = 5) were analyzed. The percentage of {beta} cell area in the pancreas was determined by dividing the area of all insulin-positive cells in one section by the total area of the section.

Measurements of Plasma and Tissue Ghrelin Concentrations— Plasma was sampled from 10-week-old RIP-G Tg and their nontransgenic littermates under ad libitum feeding states considering the promoter activity. From RGP-G Tg and their littermates, it was sampled after overnight fast. Blood was withdrawn from the retroorbital vein or the proximal end of the portal vein under ether anesthesia, immediately transferred to chilled siliconized glass tubes containing Na2EDTA (1 mg/ml) and aprotinin (1000 KIU/ml, Ohkura Pharmaceutical, Kyoto, Japan), and centrifuged at 4 °C. Hydrogen chloride was added to the samples at a final concentration of 0.1 N immediately after separation of plasma. Plasma was immediately frozen and stored at -80 °C until assay. Plasma ghrelin concentration was determined by desacyl ghrelin enzyme-linked immunosorbent assay kit and active-ghrelin enzyme-linked immunosorbent assay kit that recognizes n-octanoylated ghrelin (Mitsubishi Kagaku Iatron, Tokyo, Japan).

As for measurement of tissue ghrelin concentration, pancreata or stomachs were taken from the 8-week-old male mice. The rumen was removed from the stomach. Samples were diced and boiled for 5 min in the 10-fold v/w of water. Acetic acid was added to each solution so that the final concentration was adjusted to 1 M, and the tissues were homogenized. The supernatants were obtained after centrifugation. Tissue ghrelin concentration was determined by radioimmunoassay (RIA) using anti-ghrelin [13–28] antiserum (C-RIA) and anti-ghrelin [1–11] antiserum (N-RIA) as described previously (39).

Measurements of Body Weight and Food Consumption—Mice were housed individually and were allowed free access to standard rat chow. Body weights of mice were measured weekly. Daily food intake was measured by weighing the pellets between 9:00 and 10:00 a.m.

Measurements of % Body Fat and Visceral/Subcutaneous Fat Mass Ratio—Forty-week-old mice were anesthetized with pentobarbital. Percent body fat and visceral/subcutaneous fat mass ratio of mice were measured by Latheta LTC-100 (ALOKA, Tokyo, Japan).

Glucose and Insulin Tolerance Tests—For the glucose tolerance test, after overnight fast, the mice were injected with 1.5 g/kg glucose intraperitoneally. For the insulin tolerance test, after a 4-h fast, mice were injected with 2.0 milliunits/g human regular insulin (Novolin R; Novo Nordisk, Bagsvaerd, Denmark) intraperitoneally. Blood was sampled from the tail vein before and 15, 30, 60, 90, and 120 min after the injection. Blood glucose levels were determined by glucose oxidase method using Glutest sensor (Sanwa Kagaku, Kyoto, Japan).

Insulin Release—After overnight fast, the mice were injected with 3.0 g/kg glucose or 0.25 g/kg L-arginine intraperitoneally. Plasma was sampled from the tail vein before and 2, 5, 15, 30, and 60 min after the injection using heparin coated tubes. The measurement of insulin concentration was carried out by enzyme-linked immunosorbent assay using ultra-sensitive rat insulin kit (Morinaga, Yokohama, Japan).

Pancreatic Insulin Concentration—As for measurement of pancreatic insulin concentration, pancreata were obtained from the mice under the ether anesthesia and homogenized in acid-ethanol. The supernatants were used for assay after centrifugation.

Batch Incubation of Islet—Under the pentobarbital anesthesia, Type IV collagenase (Worthington, Lakewood, NJ) dissolved in Hanks' balanced salt solution (1.5 mg/ml) was injected into mouse pancreatic duct. Pancreas was removed and incubated at 37 °C for 14 min. After washing out collagenase by Hanks' balanced salt solution, islets were collected by Ficoll gradient and manually picked up so that the sizes of the islets were equal. Islets were incubated at 37 °C in RPMI1640 containing 10% fetal calf serum for 2 h and then in Krebs-Ringer bicarbonate buffer containing 3.3 mM glucose and 0.2% bovine serum albumin for 30 min. Five islets were incubated at 37 °C in 500 µl of Krebs-Ringer bicarbonate buffer containing 0.2% bovine serum albumin and 3.3 or 8.7 or 16.7 mM glucose for 1 h. After centrifugation, the supernatants were collected. Insulin concentrations in supernatants were determined by rat insulin kit (Morinaga, Yokohama, Japan).

Northern Blot Analysis and Real-time Quantitative RT-PCR—Total RNA was extracted from pancreata using RNeasy mini kit (Qiagen K.K., Tokyo, Japan). Filters containing 5 µg of total RNA were prepared. Northern blot analyses were performed as described previously (36) using the mouse insulin II cDNA and human {beta}-actin cDNA (Clontech, Palo Alto, CA) as probes. To confirm that approximately equal amounts of total RNA were assayed in Northern blot hybridization analysis, the density of 18 S rRNA in the gel and signal of {beta}-actin in each lane was used. The hybridization signal intensity was quantitated using an image analyzer BAS-2500 (Fuji Photo Film, Tokyo, Japan). Reverse transcription (RT) was performed with random hexamer and Super-Script II reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed with ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The following primers and TaqMan probes were used: mouse GHS-R (sense, 5'-CACCAACCTCTACCTATCCAGCAT-3'; antisense, 5'-CTGACAAACTGGAAGAGTTTGCA-3'; TaqMan probe, 5'-TCCGATCTGCTCATCTTCCTGTGCATG-3'); mouse ghrelin (sense, 5'-GCATGCTCTGGATGGACATG-3'; antisense, 5'-TGGTGGCTTCTTGGATTCCT-3'; TaqMan probe, 5'-AGCCCAGAGCACCAGAAAGCCCA-3').

Lipid Measurements—Blood was collected from the retroorbital vein of 35-week-old RIP-G Tg and their nontransgenic littermates. After separation of serum, total cholesterol, triglyceride, free fatty acid, and HDL-cholesterol levels in serum were determined by Cholesterol E-test Wako, Triglyceride E-test Wako, NEFA C-test Wako, and HDL-cholesterol E-test Wako (Wako Pure Chemical Industries, Osaka, Japan).

Statistical Analysis—All values were expressed as means ± S.E. Statistical significance of difference in mean values was assessed by repeated measures analysis of variance or Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution of Ghrelin in Normal Mouse Pancreas—We first examined which cell type of islet cells expresses ghrelin in mouse by immunohistochemistry using anti-C-terminal ghrelin antiserum. In the most of the islets no ghrelin-like immunoreactivity was detected. C-terminal ghrelin-like immunoreactivity was observed in the periphery of minor proportion of islets of wild type mice (Fig. 1A). Most of the ghrelin-positive cells were also glucagon-positive by serial section analysis (Fig. 1B), whereas most of the glucagon-positive cells were not ghrelin-positive.



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  		FIG. 1.
A, C-terminal ghrelin-like immunoreactivity in adult mouse islet. The staining was observed in the peripheral region of the islet. B, glucagon-like immunoreactivity in serial section.

 
Generation of RIP- and RGP-ghrelin Transgenic Mice—A fusion gene comprising RIP and mouse ghrelin cDNA coding sequences was designed so that ghrelin expression might be targeted to the pancreatic {beta} cells (Fig. 2A). The ghrelin mRNA level of RIP-G Tg in pancreas determined by quantitative RT-PCR was about 215 times higher than that of nontransgenic littermates (215.3 ± 40.6 versus 1.0 ± 0.025 arbitrary units, n = 5, p < 0.01). There was also an increase in ghrelin mRNA levels in brain of RIP-G Tg (242.6 ± 17.6 versus 89.1 ± 27.3 arbitrary unit, n = 5, p < 0.01). To confirm the expression of ghrelin transgene in pancreatic {beta} cells, we performed an immunohistochemical analysis using anti-C-terminal ghrelin antiserum. C-terminal ghrelin-like immunoreactivity was observed in the nearly whole area of the islets of the RIP-G Tg (Fig. 2C), whereas it was only seen in the periphery of the islets of their nontransgenic littermates (Fig. 1A). Immunohistochemical analysis using anti-N-terminal ghrelin antiserum showed the same staining pattern (Fig. 2D), indicating that n-octanoylated ghrelin may be produced in {beta} cells of this transgenic mouse. We also stained the brain section of RIP-G Tg. No ghrelin-like immunoreactivity was detected either with anti-C-terminal or anti-N-terminal ghrelin antisera (data not shown). The pancreatic tissue ghrelin concentration of RIP-G Tg measured by C-RIA was about 1000 times higher than that of their nontransgenic littermates (1024 ± 108.9 fmol/mg versus 1.2 ± 0.1 fmol/mg, n = 5, p < 0.01). This concentration was about one third of the nontransgenic stomach concentration (3558.1 ± 51.0 fmol/mg, n = 5). The pancreatic tissue ghrelin concentration of RIP-G Tg measured by N-RIA tended to be also higher than that of their nontransgenic littermates (0.054 ± 0.017 fmol/mg versus 0.038 ± 0.006fmol/mg, n = 5, NS; not significant), but it did not reach statistical significance. Plasma desacyl ghrelin concentration of RIP-G Tg was about 3.4 times higher than that of nontransgenic littermates under the ad libitum feeding states (2805.5 ± 236.4 versus 825.9 ± 244.4 fmol/ml, n = 5, p < 0.01, Fig. 2G). We also measured desacyl ghrelin levels in portal vein of the mice. In the nontransgenic mice, the portal desacyl ghrelin level was significantly higher than that in retroorbital vein (1108.0 ± 257.3 fmol/ml versus 825.9 ± 244.4 fmol/ml, n = 5, p < 0.05, Fig. 2G). The desacyl ghrelin concentration collected from portal vein of RIP-G Tg at the same time was much higher than that of nontransgenic littermates (3671.8 ± 328.6 versus 1108.0 ± 257.3 fmol/ml, n = 5, p < 0.01, Fig. 2G). The step-up of desacyl ghrelin concentration from retroorbital vein to portal vein of RIP-G Tg was significantly higher than that of nontransgenic littermates (866.3 ± 182.2 fmol/ml versus 262.9 ± 59.8 fmol/ml, p < 0.01, Fig. 2H). Plasma n-octanoylated ghrelin levels in retroorbital and portal vein of RIP-G Tg tended to be higher than those of their nontransgenic littermates (retroorbital: 78.5 ± 13.4 versus 66.1 ± 7.1 fmol/ml, n = 5, NS; portal: 104.6 ± 15.3 versus 71.4 ± 9.0 fmol/ml, n = 5, NS), but it did not reach statistical significance.



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  		FIG. 2.
A, structure of RIP-ghrelin transgene. B, structure of RGP-ghrelin transgene. C and D, pancreatic islet of RIP-ghrelin transgenic mouse stained with anti-C-terminal ghrelin (C) and anti-N-terminal ghrelin antisera (D). E and F, pancreatic islet of RGP-ghrelin transgenic mouse stained with anti-C-terminal ghrelin (E) and anti-N-terminal ghrelin antisera (F). G, plasma ghrelin levels collected from retroorbital and portal veins in RIP-G Tg. *, p < 0.05 compared with retroorbital vein. #, p < 0.01 compared with nontransgenic littermates. H, the step-up of ghrelin concentration from retroorbital vein to portal vein in RIP-G Tg. #, p < 0.01 compared with their nontransgenic littermates.

 
We also generated RGP-G Tg, in which ghrelin expression is targeted to the pancreatic {alpha} cells (Fig. 2B). The ghrelin mRNA level Tg in pancreas of RGP-G determined by quantitative RT-PCR was about 16 times higher than that of nontransgenic littermates (16.3 ± 1.7 versus 1.0 ± 0.24 arbitrary unit, n = 5, p < 0.01). The ghrelin mRNA level in duodenum of RGP Tg was not statistically different from that of nontransgenic littermates (520.1 ± 111.1 versus 379.1 ± 37.6 arbitrary unit, n = 5, NS). The ghrelin mRNA level in brain of RGP Tg was not distinguishable from that of nontransgenic littermates (72.0 ± 6.4 versus 71.8 ± 7.8 arbitrary unit, n = 5, NS). Immunohistochemical analysis showed ghrelin-like immunoreactivity in the periphery of the pancreatic islet of RGP-ghrelin transgenic mouse by both anti-C-terminal ghrelin and anti-N-terminal ghrelin antisera (Fig. 2, E and F). The pancreatic tissue ghrelin concentrations of RGP-G Tg measured by C-RIA were about 50 times higher than those of their nontransgenic littermates (48.9 ± 2.5 fmol/mg versus 1.2 ± 0.1 fmol/mg, n = 5, p < 0.01). The pancreatic tissue ghrelin concentration of RGP-G Tg measured by N-RIA tended to be higher than that of their nontransgenic littermates (0.076 ± 0.019 fmol/mg versus 0.038 ± 0.006 fmol/mg, n = 5, NS), but it did not reach statistical significance. The plasma desacyl ghrelin concentrations in retroorbital vein were not elevated in RGP-G Tg after over night fasting compared with nontransgenic littermates (661.6 ± 38.0 versus 1024.7 ± 27.1 fmol/ml, n = 5). The portal desacyl ghrelin concentrations of RGP-G Tg were also indistinguishable from those of their nontransgenic littermate (1320.6 ± 164.7 versus 1442.9 ± 361.5 fmol/ml, n = 5, NS). Plasma n-octanoylated ghrelin levels in retroorbital and portal vein of RGP-G Tg were indistinguishable from those of their nontransgenic littermates (retroorbital: 98.3 ± 18.7 versus 133.5 ± 25.3 fmol/ml, n = 5, NS; portal: 154.3 ± 20.7 versus 198.9 ± 34.9 fmol/ml, n = 5, NS). Body Weight, Food Consumption, and Percent Body Fat— There was no significant difference in body weight and food intake between RIP-G Tg and their nontransgenic littermates (Fig. 3). Percent body fat and visceral/subcutaneous ratio of RIP-G Tg were not different from those of nontransgenic littermates (Fig. 2, C and D). No significant changes were observed in RGP-G Tg, either (data not shown).



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  		FIG. 3.
A, body weight of RIP-G Tg (Tg) and their nontransgenic littermates (non). B, food intake of RIP-GTg (Tg) and their nontransgenic littermates (non). Percent body fat (C) and visceral/subcutaneous fat ratio (D) of RIP-GTg and their nontransgenic littermates (non).

 
Glucose Metabolism and Insulin Secretion—Although no significant differences in blood glucose levels were noted between RIP-G Tg and their nontransgenic littermates on the fasting state and intraperitoneal glucose tolerance tests (Fig. 4, A and C), plasma insulin levels 2 and 30 min after the glucose injection were significantly decreased in RIP-G Tg compared with those in their nontransgenic littermates (Fig. 4D). Suppression of insulin secretion was not observed in RIP-G Tg on intraperitoneal injection of arginine (Fig. 4G). Blood glucose level of RIP-G Tg in the insulin tolerance test tended to be lower than those of their nontransgenic littermates, but it did not reach statistical significance (Fig. 4H).



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  		FIG. 4.
A and B, blood glucose levels after overnight fast in RIP-G Tg (A) and RGP-G Tg (B) (Tg) and their nontransgenic littermates (non). C and E, intraperitoneal (IP) glucose tolerance test (1.5 g/kg) in RIP-G Tg (C) and RGP-G Tg (E) (Tg) and their nontransgenic littermates (non). D and F, plasma insulin concentration after intraperitoneal glucose (3g/kg) injection in RIP-G Tg (D) and RGP-G Tg (F) (Tg) and their nontransgenic littermates (non). G, plasma insulin concentration after intraperitoneal arginine (0.25g/kg) injection in RIP-G Tg (Tg) and their nontransgenic littermates (non). H, insulin (2.0 units/kg) tolerance test in RIP-G Tg (Tg) and their nontransgenic littermates (non). Values are represented as mean ± S.E. *, p < 0.05 compared with nontransgenic littermates.

 
No significant differences in blood glucose or insulin levels were observed between RGP-G Tg and their nontransgenic littermates on the fasting state, ad libitum feeding, or intraperitoneal glucose or arginine injection (Fig. 4, B, E, and F, and data not shown). Blood glucose levels on insulin tolerance test showed no differences between RGP-ghrelin and their nontransgenic littermates (data not shown).

Islet Architecture and {beta} Cell Mass—We studied the tissue sections of RIP-G Tg to explore the effect of ghrelin on the islet architecture and {beta} cell mass. There were no obvious abnormalities in the intra islet cytoarchitecture and cell number of insulin, glucagon, somatostatin, and PP cells in the islets of the RIP-G Tg (Fig. 5A–D). The intensity of staining of these four islet hormones in the islets of the RIP-G Tg was not apparently different from those of nontransgenic littermates. The ratio of the {beta} cell area to whole pancreas was not changed significantly (Fig. 5I). We also studied the tissue sections of RGP-G Tg and found no significant differences (Fig. 5, E–H, and J).



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  		FIG. 5.
Islet morphology and {beta} cell area in RIP-G Tg (A–D) and RGP-G Tg (E–H). The sections were stained with anti-insulin (A and E), anti-glucagon (B and F), anti-somatostatin (C and G), and anti-PP antiserum (D and H). I and J, the ratio of {beta} cell area to that of whole section in RIP-G Tg (I) and RGP-G Tg (J). non, nontransgenic littermates; Tg, RIP-G Tg; NS, not significant.

 
Expression of Insulin mRNA and Insulin Content—Because RIP-G Tg showed suppression of insulin secretion, we examined pancreatic mRNA expression and peptide content of insulin in RIP-G Tg and their nontransgenic littermates by Northern blot analysis and RIA. The insulin mRNA in RIP-G Tg did not differ from those of their nontransgenic littermates (Fig. 6, A and B). No significant differences of insulin contents were observed between RIP-G Tg and their nontransgenic littermates (Fig. 6C).



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  		FIG. 6.
mRNA level and peptide content of insulin in RIP-G Tg (Tg) and their nontransgenic littermates (non) pancreas. A, representative blot of Northern blot analysis of insulin; B, insulin mRNA levels; C, insulin peptide contents. NS, not significant.

 
PDX-1 and GLUT2 Immunoreactivity—We examined the immunoreactivity of PDX-1 and GLUT2 in RIP-G Tg. The staining intensities of PDX-1 and GLUT2 in the RIP-G Tg (Fig. 7, A and C) were not apparently different from those in the nontransgenic littermates (Fig. 7, B and D).



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  		FIG. 7.
A and B, immunoreactivity of Glut-2 in the islet of RIP-G Tg (A) and nontransgenic littermates (B). C and D, immunoreactivity of PDX-1 in the islet of RIP-G Tg (C) and nontransgenic littermates (D).

 
Expression of GHS-R mRNA—To rule out possible downregulation of GHS-R due to chronic exposure to high level ghrelin, we measured the expression level of GHS-R mRNA in pancreas and pituitary by real-time quantitative RT-PCR. There were no significant differences in GHS-R mRNA levels between RIP-G Tg and their nontransgenic littermates either in pancreas (Fig. 8A) or in pituitary (Fig. 8B).



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  		FIG. 8.
mRNA level of GHS-R determined by quantitative RT-PCR in pancreas (A) and pituitary (B) of RIP-G Tg (Tg) and their nontransgenic littermates (non). NS, not significant.

 
Batch Incubation of Islets—The insulin secretion from isolated islet of RIP-G Tg by batch incubation was indistinguishable from that of nontransgenic littermates, in 3.3 or 8.7 or 16.7 mM glucose conditions (Fig. 9).



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  		FIG. 9.
Batch incubation study of isolated islets of RIP-G Tg (Tg) and their nontransgenic littermates (non).

 
Lipid Metabolism—Plasma total cholesterol level of RIP-G Tg tended to be lower than those of nontransgenic littermates, but it did not reach statistical significance (total cholesterol: 85.4 ± 6.9 versus 79.4 ± 7.5 mg/dl, n = 6, NS). The plasma triglyceride level of RIP-G Tg tended to be lower than that of nontransgenic littermates, but it did not reach statistical significance (154.5 ± 11.0 versus 136.9 ± 10.3 mg/dl, n = 6, NS). Free fatty acid level and HDL-cholesterol level of RIP-G Tg were not significantly different from those of nontransgenic littermates (free fatty acid; 0.44 ± 0.05 versus 0.48 ± 0.07 mEq/liter, n = 6, NS, HDL-cholesterol; 46.1 ± 2.3 versus 44.9 ± 3.4 mg/dl, n = 6, NS).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In wild-type mice, no ghrelin-like immunoreactivity was detected in most of the islets. C-terminal ghrelin-like immunoreactivity was observed in the periphery of minor proportion of islets of wild type mice, which is consistent with a previous report (24). By the serial section analysis, most of the ghrelin-producing cells also showed glucagon-like immunoreactivity. These findings indicate that ghrelin was expressed in minor proportion of mouse pancreatic {alpha} cells. Expression of ghrelin was not detected in pancreatic {beta} cells of wild type mice.

In the present study we developed RIP-G Tg, in which pancreatic ghrelin concentration measured by C-RIA was ~1000 times higher than that of nontransgenic littermates. By immunohistochemistry using anti-C-terminal ghrelin [13–28] antiserum we detected C-terminal ghrelin-like immunoreactivity in almost the whole area of islets. Therefore, because ghrelin was not detected in {beta} cells of control mice by immunohistochemistry, ghrelin transgene driven by RIP was considered to be expressed in {beta} cells.

We also found about 3 times higher expression level of ghrelin mRNA in the brain of RIP-G Tg compared with that of nontransgenic littermates, which could not be detected by immunohistochemistry. Although a small amount of ghrelin has been reported to be expressed in brain, which can be detected by immunohistochemistry only after colchicine treatment (1), there have been controversies as to whether this small amount of ghrelin in the brain has a biological role. Because the food intake of RIP-G Tg was not different from that of nontransgenic littermates, the ghrelin produced by transgene in the brain seems not to show bioactive effect of n-octanoylated ghrelin.

By immunohistochemistry using anti-ghrelin [1–11] antiserum that recognizes the n-octanoylated portion of ghrelin, ghrelin-like immunoreactivity was also demonstrated in nearly whole area of islets of RIP-G Tg, indicating the production of n-octanoylated ghrelin in {beta} cells. This finding indicates that the mechanism of acylation may exist not only in pancreatic {alpha} cells but also in {beta} cells. This is reasonable, because {alpha} and {beta} cells are pancreatic endocrine cells derived from common precursor cells (40). Because the N-RIA/C-RIA ratio of the pancreatic tissue ghrelin concentration of RIP-G Tg was much lower than that of the stomach (0.0053% versus 11.67%, p < 0.01), the ability of acylation in {beta} cell might be lower than that of in ghrelinproducing cell in the stomach (X/A-like cell). It is possible that exocrine pancreatic enzymes might interfere with the results, although these were inactivated by boiling before extraction. The other possibility is that because of the formalin fixation of ghrelin in the tissue section the epitope recognized by immunohistochemistry using anti-ghrelin [1–11] antiserum might not be exactly the same as that recognized by N-RIA or enzymelinked immunosorbent assay. Because the amount of n-octanoylated ghrelin was so little that it could not be detected by RIA if any, we considered that the phenotype of these transgenic mice is due to the effect of desacyl ghrelin. Desacyl ghrelin has been shown not to activate GHS-R (39). There have been several reports saying that desacyl ghrelin has biological activities, such as promoting adipogenesis (41), inhibition of cell proliferation, inhibition of apoptosis (42), and counteracting the effect of n-octanoylated ghrelin (35).

We showed here that the ghrelin level in portal vein is significantly higher than that in retroorbital vein in wild type mouse. Ghrelin has been reported to be mainly synthesized in stomach and intestine. The step-up of plasma ghrelin level in gastric vein has been reported previously (43), but there has been no report showing the step-up of plasma ghrelin level in portal vein as compared with that in systemic circulation. The present study is the first report of the step-up of plasma ghrelin levels in portal vein. Moreover, the step-up of desacyl ghrelin in RIP-G Tg was much higher than that in control littermates, indicating overproduction of desacyl ghrelin by transgene in the pancreas.

The body weight, percent body fat, and food consumption of RIP-G Tg were not significantly different from those of nontransgenic littermates. Recently, we and Asakawa et al. have reported the studies of {beta}-actin promoter ghrelin transgenic mouse (44, 45), in which plasma desacyl ghrelin levels were 30 and 50 times higher than those of their nontransgenic littermates. These transgenic mice were reported to show small phenotype, although some discrepancy of interpretation regarding on etiology exists. Asakawa et al. reported that the triglyceride level of {beta}-actin promoter ghrelin transgenic mouse was lower, but that cholesterol level and free fatty acid level were not changed compared with their nontransgenic littermates. The triglyceride levels of our RIP-G Tg only showed lower tendency compared with that of nontransgenic littermates. The lack of small phenotype and milder phenotype of lipid metabolism in RIP-G Tg may result from the fact that plasma desacyl ghrelin level of RIP-G Tg was only 3.4 times higher than those of nontransgenic littermates.

The tissue sections of the pancreas of these transgenic mice showed no apparent disarrangement in the islet architecture and in {beta} cell mass. There have been several reports on the transgenic mice overexpressing humoral factors in the {beta} cells, such as parathyroid hormone-related peptide, hepatocyte growth factor, and insulin-like growth factor-I (46–49). Some of these transgenic mice showed islet hypertrophy or disarrangement of the endocrine cells in the islet (46–49). Our observation showed that desacyl ghrelin might have no apparent effects on the islet architecture and {beta} cell mass.

In the present study plasma insulin levels after the 3.0 g/kg glucose injection were significantly lower in RIP-G Tg than those in nontransgenic littermates, although there was no significant difference in plasma insulin levels between RIP-G Tg and nontransgenic littermates on the fasting state. To rule out the decreased production of insulin caused by exogenous insulin promoter, we measured insulin mRNA level and content in the pancreata of our transgenic mice. The insulin mRNA level and content from the transgenic mice were not significantly different from those from nontransgenic littermates. Therefore, the insulin production might not be disturbed in these mice either in transcriptional or translational levels. The immunoreactivity of PDX-1, which is the master regulator of the pancreas development and essential for insulin transcription, in RIP-G Tg {beta} cell was not different from that in {beta} cells of nontransgenic littermates. These results suggest that the suppression of glucose-stimulated insulin secretion in RIP-G Tg might not be due to the transcriptional dysregulation of insulin caused by injection of exogenous insulin promoter.

RIP-G Tg did not show decreased-insulin secretion in response to arginine. Arginine is known to stimulate insulin secretion by the mechanisms that are different from those used by glucose, although the detail remains controversial (50, 51). However, it seems certain that arginine somehow evoked Ca2+ influx into the {beta} cell, and that leads to the exocytosis of insulincontaining vesicles (52, 53). So at least, the decreased insulin secretion in RIP-G Tg might not be due to disorders in exocytosis process. Egido (28) reported that ghrelin inhibits insulin secretion from rat pancreas in response to arginine in vitro, however, there has been no report on the effect of desacyl ghrelin on arginine-induced insulin secretion.

The immunoreactivity of GLUT2, glucose transporter in the pancreatic {beta} cell, in RIP-G Tg {beta} cells, was indistinguishable from that in the {beta} cells of nontransgenic littermates. Although immunohistochemistry is not so suitable for quantitative analysis, at least no apparent decreased expression or disposition of GLUT2 in RIP-G Tg {beta} cell exists. Chronic exposure to the high level of desacyl ghrelin may not influence on GLUT2 expression.

We performed a batch incubation study of RIP-G Tg islet. The insulin secretion from isolated islets of RIP-G Tg was indistinguishable from that of nontransgenic littermates. This finding indicates that insulin secretion was not affected by overexpression of ghrelin transgene in vitro but was affected in vivo. The different observations in vitro and in vivo may be explained by dilution of ghrelin produced by transgene with the incubation buffer. Alternatively, suppression of insulin secretion of RIP-G Tg was not due to the effect of desacyl ghrelin on insulin secretion from {beta} cell but on insulin sensitivity. Recently Gauna et al. (55) reported that co-administration of desacyl ghrelin and active ghrelin improves insulin sensitivity in humans (54) and that desacyl ghrelin suppress glucose output from liver. Although an insulin tolerance test did not show a statistically significant difference in blood glucose levels between RIP-G Tg and their nontransgenic littermates, there was a tendency for lower blood glucose levels of RIP-G Tg. Moreover, plasma triglyceride levels of RIP-G Tg showed lower tendency. Taken together, these results may indicate that desacyl ghrelin may improve insulin sensitivity of RIP-G Tg. The suppression of insulin secretion of RIP-G Tg is likely due to the effect of desacyl ghrelin on insulin sensitivity.

To explore if chronic exposure to high level desacyl ghrelin may influence the expression level of GHS-R, we investigated the mRNA level of GHS-R in the pancreas and pituitary of RIP-G Tg. No significant differences were found in GHS-R mRNA levels in pancreas or in pituitary between RIP-G Tg and their nontransgenic littermates. These findings indicate that chronic exposure to high level desacyl ghrelin might not influence the GHS-R mRNA expression level.

We also developed RGP-G Tg. The pancreatic tissue ghrelin concentrations determined by C-RIA of RGP-G Tg were about 50 times higher than those of their nontransgenic littermates, indicating that ghrelin was overexpressed in RGP-G Tg. However, there was no obvious phenotype regarding insulin secretion and pancreatic morphology. Considering the observation that portal ghrelin levels were not elevated in RGP-G Tg compared with those in their nontransgenic littermates, the amount of secreted ghrelin from {alpha} cell may not outstrip the amount from stomach.

In summary we developed RIP-G Tg, in which pancreatic desacyl ghrelin content was ~1,000 times higher than that in control littermates. We detected n-octanoylated ghrelin-like immunoreactivity in pancreatic {beta} cells by immunohistochemistry, indicating that the mechanism of acylation may exist not only in pancreatic {alpha} cells but also in {beta} cells. The glucose-stimulated insulin secretion of RIP-G Tg was decreased. There were no abnormalities with the arginine-induced insulin secretion, pancreatic histology, pancreatic insulin mRNA levels, and insulin content in the RIP-G Tg. The absence of insulin suppression in the islet batch incubation study, lower tendency of blood glucose levels in insulin tolerance test, and lower tendency of plasma triglyceride level may indicate that the suppression of insulin secretion of RIP-G Tg is likely due to the effect of desacyl ghrelin on insulin sensitivity. Although we also developed RGP-G Tg with a 50-fold increase of pancreatic desacyl ghrelin content, we did not find obvious phenotype regarding insulin secretion and pancreatic morphology. The present study raises the possibility that desacyl ghrelin may have an influence on glucose metabolism.


   FOOTNOTES
 
* This work was supported by research grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the Japanese Ministry of Health, Labor and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 81-75-751-3172; Fax: 81-75-771-9452; E-mail: kh{at}kuhp.kyoto-u.ac.jp.

1 The abbreviations used are: GHS-R, growth hormone secretagogue receptor; RIP-G Tg, rat insulin II promoter-ghrelin transgenic; RGP-G Tg, rat glucagon promoter-ghrelin transgenic mice; RIA, radioimmunoassay; C-RIA, anti-ghrelin [13–28] antiserum; N-RIA, anti-ghrelin [1–11] antiserum; RT, reverse transcription; HDL, high density lipoprotein; PP, pancreatic polypeptide. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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Acylated and Unacylated Ghrelin Promote Proliferation and Inhibit Apoptosis of Pancreatic {beta}-Cells and Human Islets: Involvement of 3',5'-Cyclic Adenosine Monophosphate/Protein Kinase A, Extracellular Signal-Regulated Kinase 1/2, and Phosphatidyl Inositol 3-Kinase/Akt Signaling
Endocrinology, February 1, 2007; 148(2): 512 - 529.
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 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
W. Wei, X. Qi, J. Reed, J. Ceci, H.-Q. Wang, G. Wang, E. W. Englander, and G. H. Greeley Jr.
Effect of chronic hyperghrelinemia on ingestive action of ghrelin
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2006; 290(3): R803 - R808.
[Abstract] [Full Text] [PDF]

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 Proc. Natl. Acad. Sci. USAHome page
A. Doi, T. Shono, M. Nishi, H. Furuta, H. Sasaki, and K. Nanjo
IA-2beta, but not IA-2, is induced by ghrelin and inhibits glucose-stimulated insulin secretion
PNAS, January 24, 2006; 103(4): 885 - 890.
[Abstract] [Full Text] [PDF]

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