Therapeutic effects of ghrelin and growth hormone releasing peptide 6 on gastroparesis in streptozotocin-induced diabetic guinea pigs in vivo and in vitro

Ghrelin, a 28-amino-acid peptide synthesized in the endocrine cells of the gastric mucosa, was discovered in 1999 as the endogenous ligand for the growth hormone secretagogue receptor, now often referred to as the ghrelin receptor.^1,2 The most characteristic actions of ghrelin include the stimulation of growth hormone (GH) release,^1,3,4 the regulation of appetite and nutrient ingestion^5-7 and the improvement of digestive motility.^8-10 When injected into mice,^8,11 rats,^9,10 or dogs,¹²ghrelin accelerated the gastric emptying of a liquid or solid meal, with the site of action of ghrelin suggested to involve the enteric nervous system. In rats and mice, a gastroprokinetic-like activity of ghrelin was observed in vitro as an increase in neuronally-mediated contractions evoked by electrical field stimulation.^13,14Growth hormone releasing peptide 6 (GHRP-6) is a synthetic peptide that causes release of GH, similar to the effect of ghrelin; however, as yet its mechanisms are unknown.

Delayed gastrointestinal transit is a well-known complication of diabetes, and can lead to discomforting gastrointestinal symptoms, frequent vomiting, emaciation, and unpredictable excursion of blood glucose, which all impair the quality of life in diabetic patients.¹⁵ At its worst, gastroparesis can lead to intractable vomiting and an inability to feed, and carries a poor prognosis.^16,17 Present management of diabetic gastroparesis involves empirical use of prokinetic drugs such as domperidone, metoclopramide, cisapride¹⁶ and erythromycin.¹⁸ The effects of these drugs, however, are unpredictable. One possible explanation for this lack of sustained response to treatment is that gastroparesis may be originally associated with progressive autonomic neuropathy.^19,20

In the present study, we investigated the potential therapeutic significance of ghrelin and GHRP-6 in diabetic guinea pigs with gastric motility disorders.

Chemicals
Rat ghrelin, GHRP-6, and D-Lys³-GHRP-6 were obtained from Tocris Cookson (Bristol, UK). Atropine sulphate, phenol red and streptozotocin were obtained from Sigma (St Louis, MO, USA).

Animals
EWG/B guinea pigs of either sex (weighing 200–250 g) were obtained from the experimental Animal Center of the Shanghai Academia Sinica, China. All procedures were approved by the Medical Ethics Committee of Shanghai Jiaotong University. Guinea pigs were housed in stainless steel cages at a controlled temperature ((22±2)°C) and 60%–65% relative humidity with a normal 12:12 hour light/dark cycle. Six guinea pigs were randomly selected as normal controls, and the rest were fed with a high-fat diet. After exposure to the high-fat diet for 3 weeks, the guinea pigs were fasted overnight with free access to water and injected i.p. with streptozotocin (STZ; 280 mg/kg body weight) dissolved in citrate buffer (50 mg/ml); the dose of STZ-induced diabetes in guinea pigs as previously described.²¹ Seventy-two hours later, the fasting blood glucose levels of the guinea pigs were determined using the glucose oxidase method with a Glucose Analyzer (Shanghai Roche Company, China). Guinea pigs with a blood glucose level greater than 11.1 mmol/L were defined as diabetic guinea pigs. Diabetic guinea pigs continued to feed without control of blood glucose for six weeks, and the guinea pigs that were defined to be diabetic guinea pigs with gastroparesis, as confirmed by subsequent tests, were then used for further studies.

Gastric emptying in vivo
The diabetic guinea pigs were allowed to have free access to water 12 hours before the experiment. The diabetic guinea pigs were injected i.p. with either ghrelin (0, 10, 20, 50, or 100 μg/kg) or GHRP-6 (0, 10, 20, 50, or 100 μg/kg) in a random order. Modulation of the effects of the GHS-R agonists by pharmacological blockers was tested by i.p. administration of atropine (1 mg/kg) or D-Lys³-GHRP-6 (5 μmol/kg) at 15 minutes before administration of the GHS-R agonist (ghrelin 100 μg/kg or GHRP-6 100 μg/kg). Each drug treatment group consisted of 6 diabetic guinea pigs. An additional group of 6 normal guinea pigs were injected with saline (control group).

Immediately after the injection of the drug 2 ml of phenol red semi-liquid test meal (50 mg/100 ml in 0.9% NaCl with 1.5% methylcellulose) was administered intragastrically with an orogastric cannula. The guinea pigs were sacrificed 20 minutes later. The stomach was clamped with a string above the lower esophageal sphincter and a string beneath the pylorus to prevent leakage of phenol red. The stomach was cut just beneath the strings and was frozen at –70°C until measurement of gastric emptying. Gastric emptying was determined spectrophotometrically using a previously described method.^14,22The stomach of each guinea pig was cut just above the lower esophageal sphincter and the pyloric sphincter. Phenol red remained largely in the lumen of the stomach, although some was trapped in the mucus layer of the stomach, and a very small amount of phenol red was reabsorbed in the mucosa after 20 minutes.

Next, the stomach and its contents were placed in 15 ml of 0.1 mol/L NaOH and the stomach was minced for 30 seconds; these samples therefore contained the total amount of phenol red present in the stomach. The samples were further diluted to 30 ml with 0.1 mol/L NaOH, and left at room temperature for 1 hour. Five milliliters of the supernatant were then centrifuged at 800 g for 20 minutes. The absorbance was read at a wavelength of 546 nm with a spectrophotometer (Shanghai Yixian Company, China), and the phenol red content present in the stomach was calculated. The percentage of gastric emptying of the guinea pigs was calculated as (infusion–remaining)/(infusion) ×100%.

Contractility measurements in vitro
Diabetic guinea pigs were sacrificed by cervical dislocation, and the stomach was removed and rinsed with ice cold saline. Circular muscle strips, freed from mucosa (length 10–12 mm, width 2 mm) were cut from the fundus and suspended vertically in an organ bath filled with Krebs solution (120.9 mmol/L NaCl, 2.0 mmol/L NaH₂PO₄, 15.5 mmol/L NaHCO₃, 5.9 mmol/L KCl, 1.25 mmol/L CaCl₂, 1.2 mmol/L MgCl₂, and 11.5 mmol/L glucose) warmed at 37°C and gassed with 95% O₂/5% CO₂. One end of the strip was fixed to a hook on the bottom of the chamber while the other end was connected by a thread to an external isometric force transducer (Harvard Appartus, South Natick, USA) at the top. After 1 hour equilibration at optimal stretch (0.75 g), the reproducibility of the contractile response to carbachol (0.1 μmol/L) was assessed. Mechanical responses in the smooth muscle strips were measured using an isometric force transducer and stored on a computer for analysis using the SMUP-E biological signal processing system (Chengdu Equipment Factory, China). To investigate the modification of neuroeffector transmission by the GHS-R agonists the response was studied in the presence or absence of carbachol (0.1 μmol/L), which when used was added to the tissue bath 5 minutes before application of the GHS-R agonists. The effect of the GHS-R agonists on spontaneous or carbachol (0.1 μmol/L)-induced contractile activity in diabetic guinea pig fundic muscle strips was studied by measuring the mean contractile amplitude of the muscle strips. Each drug treatment group consisted of 6 diabetic guinea pigs.

Measurement of the growth hormone secretagogue receptor by reverse transcriptase polymerase chain reaction
Total RNA was prepared from diabetic guinea pig fundic muscle strips using the Trizol reagent (Invitrogen, CA, USA). Single-stranded cDNA was synthesized using an oligo (dT) anchor primer and Superscript^TM II RNase H^- reverse transcriptase (Gibco BRL, USA). The obtained cDNA served as a template for polymerase chain reaction, consisting of 35 cycles of amplification (95°C for 10 minutes, 94°C for 50 seconds, 60°C for 30 seconds, 72°C for 30 seconds), with a final elongation for 10 minutes at 72°C using 0.5 U of Taq DNA polymerase (Promega, Sweden) and 0.5 μmol/L primers (forward: 5′-CGACC- TGCTCTGCAAACTC-3′ and reverse: 5′-CACGCCCA- CCAGCACGAAGA-3′). PCR using intron-spanning mouse β-actin primers (forward: 5′-CCTGTATGCCT- CTGGTCGTA-3′ and reverse: 5′-CCATCTCCTGCTCG- AAGTCT-3′) demonstrated that cDNA was present and devoid of genomic DNA contamination. The expected sizes of GHS-R and β-actin fragments were 217 bp and 260 bp, respectively. All primers were selected from conserved regions identified by the alignment of published sequences for GHS-R mRNA in GenBank. PCR products were separated by electrophoresis on a 1.4% agarose gel, and the separated products were photographed.

Statistical analysis
Data are represented as means±standard error (SE). The effect of ghrelin and GHRP-6 on gastric emptying and muscle strips contractile amplitude was analyzed by one way analysis of variance (ANOVA) followed by Dunnett's test. P <0.05 was considered to be statistically significant.

Contractility in vivo
The gastric emptying rate of the diabetic guinea pigs was significantly reduced compared with that of the normal guinea pigs ((23.90±1.28)% vs (28.10±1.32)%, respectively, n=6, P <0.05; Figure 1A). In the diabetic guinea pigs, ghrelin significantly accelerated gastric emptying of the semi-liquid test meal at doses of 20, 50, and 100 μg/kg ((23.90±1.28)% control vs (28.20±2.03)%, (37.40±1.86)%, and (38.90±2.41)%, respectively, n=6, all P <0.05 vs saline injection; Figure 1B). Similarly, GHRP-6 increased gastric emptying dose-dependently with significant effects at 20, 50, and 100 μg/kg (n=6, all P <0.05 vs saline injection; Figure 1B).

The effect of ghrelin or GHRP-6 on diabetic guinea pig gastric emptying was characterized pharmacologically. Ghrelin (100 μg/kg) or GHRP-6 (100 μg/kg) was unable to reverse the inhibition of gastric emptying due to pretreatment with 1 mg/kg atropine ((25.60±2.78)% vs (38.90±2.41)% or (26.10±2.32)% vs (39.40±2.06)%, respectively, n=6, all P <0.05; Figure 2). Pretreatment of diabetic guinea pigs with D-Lys³-GHRP-6 (5 μmol/kg) also delayed the accelerated gastric emptying induced by ghrelin or GHRP-6 ((24.90±1.56)% vs (38.90±2.41)% or (27.20±1.86)% vs (39.40±2.06)%, respectively, n=6, all P <0.05; Figure 2).

Contractility in vitro
Fundic strips from the diabetic guinea pigs showed spontaneous contractile activity after 1 hour of equilibration. Ghrelin (0.01–10 μmol/L) or GHRP-6 (0.01–10 μmol/L) did not significantly change spontaneous contractile responses in the strips (Table 1). However, in the presence of carbachol (0.1 μmol/L), ghrelin increased the carbachol-induced contractile amplitude at 0.1, 1, and 10 μmol/L (n=6, P <0.05; Table 2). GHRP-6 also increased the carbachol-induced contractile amplitude at 0.1, 1, and 10 μmol/L (n=6, P <0.05; Table 2).

Expression of the ghrelin receptor in guinea pig fundic strips
The presence of GHS-R mRNA in the guinea pig fundic smooth muscle strips was verified by reverse transcriptase polymerase chain reaction (RT-PCR) with gene-specific primers. Analysis of the PCR products by electrophoresis revealed a band with the expected length of 217 bp (Figure 3).

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Figure 3. Expression of GHS-R mRNA in gastric fundic strips from diabetic guinea pigs. The band at 217 bp corresponds to the amplified GHS-R cDNA product with the expected length. The band at 260 bp corresponds to the amplified β-actin cDNA product with the expected length. GHS-R: growth hormone secretagogue receptor.

In the present study, ghrelin and the synthetic peptide GHRP-6 improved gastric emptying in diabetic guinea pigs with gastroparesis. This effect may be mediated through potentiation of the peripheral cholinergic pathways in the enteric nervous system.

Ghrelin, a recently discovered peptide hormone, is primarily produced by endocrine cells in the oxyntic mucosa of the stomach in rat and human.^1,23 Ghrelin has also been found in the small intestine, testis, pituitary gland, ovary, liver, pancreas, kidney, placenta and hypothalamus in both humans and rodents.^1,13 Ghrelin is a natural ligand for GHS-R, and its receptor is found all over the body including the bowel, pancreas, stomach, heart, lungs, and brain.^1,23,24 In addition to its effect on growth hormone secretion by activating GHS-R in the pituitary gland, ghrelin enhances appetite, increases food intake,²⁵ mediates energy balance, regulates glucose metabolism and insulin release,²⁶ stimulates gastric acid secretion²⁷ and promotes anxiety.²⁸

It is well known that many gastrointestinal peptides participate in the regulation of gastrointestinal functions. Ghrelin is one of these candidate gastrointestinal peptides because it is predominantly present in gastric endocrine cells and is secreted into the bloodstream. In fact, the potential for ghrelin and its synthetic peptide GHRP-6 as prokinetic agents has been shown previously both in vitro and in vivo.

Previous studies^29,30 on the effect of ghrelin on gastric motility have demonstrated the involvement of vagal and central ghrelin receptors. Thus, the effect of ghrelin on gastric emptying is blocked by atropine and vagotomy in rats and mice.^24,29 Peripheral ghrelin may stimulate fasted small intestinal motor activity through receptors on vagal afferents which activate neuropeptide Y-containing neurons in the brain, as suggested by experiments in rats.³⁰

In addition to the known vagal pathways, ghrelin and GHRP-6 accelerate gastric emptying and small intestinal transit by activating cholinergic excitatory pathways in the enteric neuron system.^12,14,31 Moreover, ghrelin has been shown to increase gastric emptying in patients with gastroparesis, and ghrelin or its analogues have been proposed as a new class of prokinetic agents for the treatment of gastroparesis.^32,33

In the present study, the gastric emptying rate in the diabetic guinea pigs was significantly reduced relative to the normal guinea pigs, while ghrelin and GHRP-6 accelerated gastric emptying of the diabetic guinea pigs with gastroparesis. In the presence of atropine which delayed gastric emptying, ghrelin and GHRP-6 (100 μg/kg) failed to accelerate gastric emptying. D-Lys³-GHRP-6 also delayed gastric emptying induced by GHS-R agonists. Gastric emptying is a complex process involving excitatory and inhibitory nerves, which may contribute to both acceleration and retardation of the emptying process.

Atropine, which blocks cholinergic nerves, delayed gastric emptying, probably by interfering with gastric accommodation and pyloric relaxation, as suggested by the inability of ghrelin to overcome the delay induced by atropine. The GHS-R antagonist, D-Lys³-GHRP-6, also blocked the effect of the GHS-R agonists, indicating that the effect of GHS-R agonists on gastric motility occurs through GHS-R, and is unlikely to involve cross interaction with other receptors. Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in fundic strips taken from the diabetic guinea pigs, also indicating that GHS-R agonists accelerate gastric emptying of semi-liquid through the activation of GHS-R receptors, possibly located on local cholinergic enteric nerves. Moreover, the presence of GHS-R mRNA in the strip preparations was confirmed by RT-PCR.

In conclusion, ghrelin and its synthetic peptide GHRP-6 increase gastric emptying in diabetic guinea pigs with gastroparesis, potentially via activation of peripheral cholinergic pathways in the enteric nervous system. Although further studies are needed to determine the underlying mechanisms, we propose that ghrelin or its analogues may represent a new class of prokinetic agents for the treatment of diabetic gastroparesis.

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