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Adaptive upregulation of gastric and hypothalamic ghrelin receptors and increased plasma ghrelin in a model of cancer chemotherapy-induced dyspepsia
Regulatory Peptides 148 (2008) 33–38
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Adaptive upregulation of gastric and hypothalamic ghrelin receptors and increased plasma ghrelin in a model of cancer chemotherapy-induced dyspepsia N.M. Malik a, G.B.T. Moore b, R. Kaur b, Y.-L. Liu a, S.L. Wood b, R.W. Morrow b, G.J. Sanger b, P.L.R. Andrews a,⁎ a b
Division of Basic Medical Sciences, St. George's, University of London, London, SWl7 ORE, UK Neurology and Gastrointestinal-CEDD, GlaxoSmithKline, Harlow, Essex CMl9 5AW, UK
A R T I C L E
I N F O
Article history: Received 20 July 2007 Received in revised form 4 January 2008 Accepted 14 March 2008 Available online 25 March 2008 Keywords: Ghrelin GHRL Ghrelin receptor GHSR Cisplatin Dexamethasone Dyspepsia
A B S T R A C T Chemotherapy treatment can lead to delayed gastric emptying, early satiety, anorexia, nausea and vomiting, described collectively as the cancer-associated dyspepsia syndrome (CADS). Administration of ghrelin (GHRL), an endogenous orexigenic peptide known to stimulate gastric motility, has been shown to reduce the symptoms of CADS induced in relevant animal models with the potent chemotherapeutic agent, cisplatin. We examined the effects in the rat of cisplatin (6 mg/kg i.p.) treatment on the expression of GHRL and ghrelin receptor (GHSR) mRNAs in the hypothalamus and the stomach at a time-point (2 days) when the effects of cisplatin are pronounced. In addition, plasma levels of GHRL (acylated and total including des-acyl GHRL) were measured and the effect on these levels of treatment with the synthetic glucocorticoid dexamethasone (2 mg/kg s.c. bd.) was investigated. Cisplatin increased GHSR mRNA expression in the stomach (67%) and hypothalamus (52%) but not GHRL mRNA expression and increased the percentage of acylated GHRL (7.03 ± 1.35% vs. 11.38 ± 2.40%) in the plasma. Dexamethasone reduced the plasma level of acylated GHRL and the percentage of acylated GHRL to values below those in animals treated with saline alone (7.03 ± 1.35% vs. 2.60 ± 0.49%). Our findings support the hypothesis that an adaptive upregulation of the ghrelin receptor may occur during cancer chemotherapy-associated dyspepsia. This may have a role in defensive responses to toxic challenges to the gut. In addition, our results provide preliminary evidence for glucocorticoid modulation of plasma ghrelin levels. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Ghrelin (GHRL) is an endogenous orexigenic, anti-emetic and gastrointestinal (GI) prokinetic peptide secreted mainly from the stomach but also from elsewhere, to act on ghrelin receptors (GHSR) within the hypothalamus (particularly the arcuate nucleus) and on gastrointestinal tract vagal, enteric (e.g. [1–5]) and spinal [6] nerve pathways. Patients with cancer being treated with cytotoxic drugs such as cisplatin, may experience a number of undesirable symptoms including emesis, dyspepsia, and anorexia [7]. Administration of exogenous ghrelin has been shown to have the potential to reduce each of these symptoms in relevant animal models treated with cisplatin as an exemplar cytotoxic agent: emesis in the ferret [5]; anorexia in the rat and mouse [8]; delayed gastric emptying in the mouse [8]. In addition ghrelin has been shown to have gastroprotective effects in a rodent model of ischaemia reperfusion injury [9]. Studies in cancer patients with impaired appetites have shown that ghrelin administration can increase energy intake and meal appreciation of a buffet meal [10]. Together, these results reveal the
⁎ Corresponding author. Fax: +44 208 725 2993. E-mail address: [email protected] (P.L.R. Andrews). 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2008.03.005
therapeutic potential of ghrelin receptor agonists but in addition, suggest that if released in response to cytotoxic drugs, ghrelin could act as an endogenous agent attempting to ameliorate the undesirable effects of cytotoxic drugs on emesis, food intake and gastric function. To investigate this hypothesis we have examined the effects in the rat of cisplatin treatment on the expression of GHR and ghrelin-R mRNA in the hypothalamus and the stomach, measured 2 days after treatment and at the time when food intake is at a nadir, delayed gastric emptying is established and pica (argued to be indicative of nausea and/or vomiting in rats which lack a vomiting reflex [11]) is present. Expression levels were also measured 7 days after cisplatin when previous studies have shown that the above effects have subsided [11,12]. In addition, plasma levels of ghrelin (acylated and total including des-acyl ghrelin) were measured and the effects on these levels of treatment with the synthetic glucocorticoid dexamethasone, were investigated. The latter has been shown in related studies to reduce cisplatin-induced pica, delayed gastric emptying, reduced locomotor activity and food intake [13] measured over 2 days after cisplatin administration. Our findings support the hypothesis that an adaptive upregulation of the function of ghrelin may occur during cancer chemotherapy-associated dyspepsia. In addition, the results provide preliminary evidence for glucocorticoid modulation of ghrelin levels in the plasma.
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2. Methods 2.1. Animals Male Wistar rats (350 g to 400 g.) were obtained from A. Tuck & Son Ltd (Essex, UK) and housed in a temperature and humidity controlled room with 12 h light: 12 h dark cycle (lights on at 07.00 am). The rats were caged individually in clear plastic cages with food (Bantin and Kingman, Hull, UK) and water provided ad libitum. Body weight, food and water were monitored daily and the overall health of the animals assessed twice daily against pre-set humane end points for the entire experimental period. All experiments were performed under the UK Animals (Scientific Procedures) Act 1986. 2.2. Drug administration 2.2.1. Study 1 Following a 3 day habituation period in the cages, the rats were given an intra-peritoneal injection (i.p.) of either sterile saline (154 mM NaCl, dose volume: 0.4 ml/100 g) or cisplatin (6 mg/kg). The dose of cisplatin used in the current experiments is comparable to that in previous studies, e.g. Refs. [12,14]. The cisplatin injection was prepared at a concentration of 1.5 mg/ml by dissolving 7.5 mg cisplatinum (II) diammine dichloride (Sigma, Poole, UK) in 5 ml sterile saline (154 mM NaCl) with 90 s of sonication. Animals given either cisplatin or saline alone were culled either 2 or 7 days following administration and tissues removed for the molecular studies described in Section 2.3 below. 2.2.2. Study 2 An additional study was performed on groups of animals which received either cisplatin (6 mg/kg i.p) or saline (i.p.) as described above on day 1 and in addition received either dexamethasone (2 mg/ kg) or saline subcutaneously (s.c.) 1 h before either the cisplatin (6 mg/ kg i.p.) or saline (i.p.). This dose is twice that used in the rat by Rudd et al. [14] but in their study the dose of cisplatin used was half that in the present study (3 mg/kg vs 6 mg/kg). In addition, Rudd et al. [14] used the i.p. route for dexamethasone as opposed to the s.c route in the present study which was considered to be more appropriate for minimising peritoneal irritation when 4 injections were required. At
17.00h, depending upon the group, animals received either a further injection of saline (s.c.) or dexamethasone (2 mg/kg s.c.). On day 2 these animals were given either saline (s.c.) or dexamethasone (2 mg/ kg s.c.) at 09.00h and 17.00h. Animals were culled at 10.00h on day 3 (i.e. 48 h after the start of the study) without either any further dexamethasone or saline. 2.3. Measurement of expression levels of mRNA At either 2 or 7 days after either cisplatin or saline treatment, animals in study 1 were killed between 10.00 and 11.00h by a rising concentration of CO2 and cervical dislocation (Schedule 1 method) and placed on a bed of dry ice for tissue removal. Hypothalami were taken and snap-frozen in liquid nitrogen prior to storage at −70 °C. The stomach was removed from the same animals, opened, contents removed and weighed (see below) and the tissue washed in chilled sterile saline to remove adherent particulate matter. The stomach was then divided into proximal (the non-glandular region with stratified squamous epithelium externally identifiable as the pale region with a relatively thin wall) and distal (glandular region consisting of the “proper” gastric region and the pyloric gland region but excluding the pyloric sphincter and recognisable by the relatively thickened mucosa [15]) by cutting along the demarcating mucosal ridge and then snapfrozen in liquid nitrogen prior to storage at −70 °C. Total RNA was isolated from the hypothalamus using the RNeasy® Mini kit (Qiagen, Crawley, UK) according to the manufacturer's instructions and from the stomach samples using TRIzol® reagent (Invitrogen, Paisley, UK) as previously described [17]. Relative quantification of mRNA transcripts was performed using the RT-PCR-based 5′ nuclease assay with TaqMan® probes (TaqMan assay, reviewed in [16]). Reverse transcriptase (RT) and negative control RT-minus (NoRT) reactions containing random 9-mers and DNase-treated total RNA (0.21 μg for the hypothalamus samples and 1 μg for the stomach region samples) were performed with no template controls (NoRNA) as before [18]. TaqMan assay oligonucleotide primers (Proligo France SAS, Paris, France) and probes (Applied Biosystems, Warrington, UK) were designed and TaqMan analysis performed as previously described [17] (see Table 1 for oligonucleotide sequences). For each transcript measurement, equal amounts of cDNA were added to each reaction (equivalent to 10 or 20 ng total RNA for the mRNAs and 100 pg for 18S
Table 1 Oligonucleotide sequences of gene-specific TaqMan assay primers and probes Rat RNA (RGD gene symbol)
Accession number (amplicon position in parentheses)
Primer or probe
Primer or probe sequence
Ghrl
AB029433 (151–276)
Ghsr
AB001982 (751–880)
Actb
AF122902.1 (131–216)
Hprt
M63983 (590–683)
Ppia
M19533 (296–401)
Rnr1
M11188 (12–107)
Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe
5′-TCCAAGAAGCCACCAGCTAAAC-3′ 5′-AACATCGAAGGGAGCATTGAAC-3′ FAM-5′-CTTCTGCTTGTCCTCTGTCCTCTGGGTG-3′-TAMRA 5′-CTCCGGGACCAGAACCACA-3′ 5′-CCAGAGAGCCAGGCTCGAA-3′ FAM-5′-CAAACACCACCACAGCAAGCATCTTCACT-3′-TAMRA 5′-ACCCTAAGGCCAACCGTGAA-3′ 5′-CACAGCCTGGATGGCTACGT-3′ FAM-5′-CCCAGATCATGTTTGAGACCTTCAACACCC-3′-TAMRA 5′-GGTGAAAAGGACCTCTCGAAGTG-3′ 5′-ATAGTCAAGGGCATATCCAACAACA-3′ FAM-5′-CCAGACTTTGTTGGATTTGAAATTCCAGACAA-3′-TAMRA 5′-ATGAGAACTTCATCCTGAAGCATACA-3′ 5′-TCAGTCTTGGCAGTGCAGATAAA-3′ FAM-5′-CCTGGCATCTTGTCCATGGCAAATG-3′-TAMRA 5′-ACCTGGTTGATCCTGCCAGTAG-3′ 5′-AGCCATTCGCAGTTTCACTGTAC-3′ FAM-5′- TCAAAGATTAAGCCATGCATGTCTAAGTACGCAC-3fs-TAMRA
FAM: fluorogenic probe reporter dye 6-carboxyfluorescein; TAMRA: fluorogenic probe quencher dye 6-carboxytetramethylrhodamine; RGD: rat genome database (http://rgd.mcw. edu/); Ghrl: ghrelin; Ghsr: ghrelin receptor or growth hormone secretagogue receptor; Actb: β-actin; Hprt: hypoxanthine phosphoribosyltransferase; Ppia: peptidylprolyl isomerase A (cyclophilin); Rnr1: 18S rRNA.
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Table 2 Cisplatin treatment increased ghrelin receptor, but not ghrelin mRNA expression (A) without affecting the mRNA expression levels of reference housekeeping genes (B) A. Tissue
Distal stomach
Proximal stomach
Distal stomach
Proximal stomach
Hypothalamus
RNA
Ghrl
Ghrl
Ghsr
Ghsr
Ghsr
Saline-treated, RAU Cisplatin-treated, RAU Fold-change
24.26 (19.72–29.85), n = 8 29.70 (24.88–35.46), n = 8 1.22 (0.96–1.57)
0.12 (0.03–0.45), n = 8 0.12 (0.02–0.89), n = 7 0.96 (0.12–8.03)
0.037 (0.028–0.050), n = 8 0.062 (0.041–0.093), n = 8 1.67 (1.06–2.63)*
0.009 (0.004–0.020), n = 7 0.010 (0.006–0.018), n = 4 1.18 (0.42–3.31)
5.37 (3.96–7.28), n = 7 8.16 (5.69–11.70), n = 6 1.52 (1.01–2.29)*
B. Tissue
Distal stomach
Distal stomach
Proximal stomach
Proximal stomach
Hypothalamus
Hypothalamus
RNA
Rnr1 (18S rRNA)
Ppia
Rnr1 (18S rRNA)
Actb
Ppia
Hprt
Saline-treated, RAU Cisplatin-treated, RAU Fold-change
537.54 (447.68–645.44), n=8 528.36 (461.13–605.39), n=8 0.98 (0.80–1.21)
29.35 (24.62–34.98), n=8 35.44 (29.03–43.27), n=8 1.21 (0.95–1.54)
39.96 (31.60–50.53), n=8 54.10 (40.30–72.61), n=7 1.35 (0.97–1.89)
2.96 (2.23–3.92), n=8 2.70 (2.00–3.64), n=7 0.91 (0.63–1.32)
7.13 (6.22–8.18), n = 7
4.78 (4.14–5.52), n=7 5.60 (4.79–6.54), n=6 1.17 (0.97–1.41)
7.80 (6.28–9.68), n=6 1.09 (0.88–1.36)
Adult male Wistar rats treated with either saline (i.p.) or cisplatin (6 mg/kg i.p.) and tissues collected 2 days later for RNA. TaqMan real-time RT-PCR data in relative arbitrary units (RAU) expressed as geometric mean with 95% confidence limits and as ratio to geometric mean of saline controls with 95% confidence limits; *p b 0.05; n = 4–8.
rRNA). Reaction conditions were as follows: 50 °C for 2 min, 95 °C for 10 min then 45–50 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative standard curves were constructed using a 3-fold serial dilution of rat pituitary, corpus-antrum, forestomach or hypothalamus total cDNA (equivalent to 150 to 0.008 or 0.0025 ng total RNA for mRNA measurements, and 900 to 0.046 or 0.015 pg total RNA for 18S rRNA), the choice of cDNA being dependent on the mRNA being studied. 2.4. Analysis of acylated and total, including des-acylated ghrelin concentrations Blood samples were collected by cardiac puncture (animals killed by a Schedule 1 procedure see above) from the animals in study 2 and delivered into tubes containing potassium-EDTA on ice. Plasma was separated by centrifugation, frozen and stored at −20 °C until analysis. Plasma levels of acylated and des-acyl ghrelin were determined by enzyme-linked immunosorbent assay using commercially available kits (ELISA; active ghrelin and desacyl ghrelin ELISA kit, LINCO Research Inc, USA) according to the manufacturer's instructions. The standard curve was reconstituted in stripped rat plasma. In brief, plasma from the rats were mixed with activated charcoal at a ratio of 0.2 g/ml and incubated at room temperature for 1 h with occasional shaking, followed by centrifugation at 3000 ×g for 30 min at 4 °C. Stripped plasma was aliquoted and stored in −20 °C until use. Prior to measurement, plasma samples were spun at 16000 ×g for 5 min to remove aggregates. For the ELISA of active ghrelin, plasma was acidified with 0.05 M HCL and 0.01 M phenylmethylsulfonyl fluoride. The detection limit for the assay was 2.5 fmol/ml. 2.5. Measurement of body weight, food and water intake, kaolin consumption and gastric contents Food and water intake was measured by weighing the food hopper or water bottle on each day of the experiment between 10.00h and 11.00h and body weight was measured at the same time. Spilled food present in the cage was subtracted from the above measurement. Pica was assessed by measurement of kaolin consumption as previously described [11]. Post-mortem gastric contents were removed from the stomach and the wet weight measured. 2.6. Statistical analysis Results of the mRNA studies are presented as the ratio to the geometric mean of the saline-treated controls at each time point with
95% confidence intervals for the ratio. The transcript data were analyzed using one-way analysis of variance (ANOVA) after a log10transformation (performed in Microsoft® Excel 97; Microsoft Corporation, Redmond, WA, USA with an add-in toolkit for the analysis of real-time PCR data (PRISM Training and Consultancy Ltd., Cambridge, UK). Differences were considered significant when P b 0.05. Results from the remainder of the study are presented as mean ± S.E.M (n = number of animals). Statistical analysis of plasma levels of ghrelin were undertaken using ANOVA followed by the Tukey honest significant difference test allowing for multiple comparisons. Other analyses used either paired or unpaired (non parametric) sample ttests as appropriate. Differences were considered significant when p b 0.05. 3. Results 3.1. The effect of cisplatin on the expression of ghrelin and the ghrelin receptor Ghrelin and ghrelin receptor mRNA were detected in both the proximal and distal regions of the stomach in animals treated with saline. Two days after treatment with cisplatin, there were no changes in the levels of ghrelin mRNA expression, but the levels of ghrelin receptor mRNA were increased in the distal region of the stomach (67% increase; p b 0.05, n = 8; Table 2). No changes in either ghrelin or ghrelin receptor expression were found in either region of the stomach 7 days after cisplatin treatment (data not shown). In the hypothalamus ghrelin receptor mRNA expression increased by 52% (p b 0.05, n = 6–7; Table 2) 2 days after cisplatin administration but no change was found 7 days after cisplatin administration (data not shown). 3.2. The effect of cisplatin and dexamethasone on blood plasma levels of ghrelin In animals treated with saline alone, both acylated and desacylated ghrelin were present in the plasma with the des-acylated form being present at concentrations approximately 10-times higher than for the acylated ghrelin (respectively 25.8 ± 5.7 and 313 ± 46 fmol/ ml of acylated and des-acylated ghrelin; Fig. 1). Two days after cisplatin treatment, the mean concentration of acylated ghrelin increased to 45.3 ± 8.5 fmol/ml, but this increase was not statistically significant (Fig. 1A). Likewise although the plasma concentration of the des-acylated form of ghrelin increased to 412 ± 55 fmol/ml this
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treated and saline-treated animals to a comparable level which was below that for the control animals treated with saline alone (Fig. 1C). 3.3. Effect of cisplatin and dexamethasone on body weight, food and water intake, kaolin consumption and gastric contents
Fig. 1. The effect of saline, cisplatin (6mg/kg, i.p.) and dexamethsasone (2 mg/kg s.c.) on the plasma concentration of acylated (A) and des-acylated (B) ghrelin and on the percentage of acylated to total ghrelin (C). Values are mean ± S.E.M. (n = 7–8); ⁎⁎ p b 0.01, ⁎⁎⁎p b 0.001 compared to corresponding control or to cisplatin saline as appropriate.
increase was not statistically significant (Fig. 1B). Following treatment with dexamethasone alone, the mean plasma concentrations of acylated ghrelin tended to be lower than those found in the salinetreated animals (respectively 11.6 ± 2.4 and 25.8 ± 5.7 fmol/ml; Fig. 1A), although this difference was not statistically significant (Fig. 1A). Treatment with dexamethasone did not significantly change the concentrations of the des-acylated form of ghrelin (Fig. 1B). In rats treated with both cisplatin and dexamethasone, there was a significant reduction in the blood concentrations of acylated ghrelin, compared with the cisplatin-treated rats (p = 0.003; Fig. 1A). Interestingly, in these animals, the concentration of des-acylated ghrelin also tended to be larger than in the cisplatin-treated animals, although again, any change was not statistically significant. Calculation of the percentage of acylated to total ghrelin in individual animals revealed that this increased in response to cisplatin although not significantly, however dexamethasone treatment significantly (p b 0.001) reduced the percentage in both the cisplatin-
In the study measuring plasma ghrelin levels, total food intake over 2 days decreased significantly in animals treated with cisplatin–saline in comparison to those treated with saline alone (cisplatin–saline 30.1 ± 4.1 g. n = 7 vs. saline–saline 49.0 ± 3.0 g. n = 8, p b 0.01). Although there was a reduction in food intake on both days it was only statistically significant on day 2 (day 1 saline–saline 23.4 ± 1.74 g vs. day 1 cisplatin– saline19.43 ± 1.99 g, ns; day 2 saline–saline 25.63 ± 1.47 g vs. day 2 cisplatin–saline 10.71 ± 2.24 g, p b 0.0001). This reduced food intake in the cisplatin-treated animals was unaffected by dexamethasone administration to cisplatin-treated animals (total intake over 2 days cisplatin–saline 30.1 ± 4.1 g. n = 7 vs. cisplatin–dexamethasone 32.0 ± 3.6 g. n = 8; day1 cisplatin–saline 19.43 ± 1.99 g vs. day 1 cisplatin– dexamethasone 20.63 ± 2.06 g; day 2 cisplatin–saline 10.71 ± 2.24 g vs day 2 cisplatin–dexamethasone 11.38 ± 2.08 g) and although dexamethasone alone did significantly reduce food intake when compared to animals treated with saline alone the effect was only significant on day 2 of treatment (day 1 saline 23.38 ± 1.74 g vs. day 1 saline–dexamethasone 24.50 ± 1.18 g; day 2 saline–saline 25.63. ± 1.47 g vs day 2 saline– dexamethasone 14.13 ± 1.04 g, p b 0.0001). Water consumption was not significantly affected on either day by any of the treatments (data not shown). Kaolin consumption over the 48 h of the study was significantly increased in animals treated with cisplatin (saline–saline 0.22 ± 0.05 g vs. cisplatin–saline 3.36 ± 0.90 g, p b 0.01). In animals treated with dexamethasone kaolin consumption was not significantly different from that in animals treated with saline (saline–dexamethasone 0.97 ± 0.67 g vs. saline–saline 0.22 ± 0.05 g) but dexamethasone did reduce kaolin consumption in cisplatin-treated rats (cisplatin–saline 3.36 ± 0.90 g vs. cisplatin–dexamethasone 1.36 ± 0.41 g) but this effect was not statistically significant. Animals treated with cisplatin had a significant reduction in body weight over 2 days (374 ± 8.5 g. vs. 357 ± 6.5 g. n = 7, p b 0.005) as did animals treated with both cisplatin and dexamethasone (364 ± 8.6 g vs. 324 ±8.2 g n = 8, p b 0.0001) but the weight loss in the latter group was significantly greater (p b 0.001) than the former. Saline and dexamethasone treatment resulted in a significant weight loss (364 ± 10 g vs. 323 ± 10 g, n = 8, p b 0.0005) which was not significantly different from that recorded in the animals treated with both dexamethasone and cisplatin. Cisplatin treatment led to an increase in the wet weight of gastric contents measured 2 days later (saline–saline 3.00 ± 0.43 g, n = 8 vs. saline–cisplatin 8.32 ± 1.90 g, n = 7, p b 0.01). Treatment with dexamethasone alone was without significant effect on the weight of gastric contents in comparison to saline-treated animals (saline– saline 3.00 ± 0.43 g, n = 8, vs. saline–dexamethasone 3.1 ± 0.43 g, n = 8). Dexamethasone treatment in animals given cisplatin resulted in a reduction in the weight of gastric contents but this failed to reach statistical significance (saline–cisplatin 8.32 ± 1.90 g, n = 7, vs. cisplatin– dexamethasone 4.9 ± 0.7 g., n = 8). In the molecular biology study the cisplatin treatment resulted in reductions in body weight and food intake and an increase in the weight of gastric contents measured 48 h after administration (data not shown) with values not significantly different from those in animals treated in a similar way in the plasma ghrelin study reported above. In animals studied 7 days after cisplatin administration food intake and body weight although still significantly reduced were higher than at 2 days after cisplatin. Water intake was significantly (p b 0.01) elevated compared to pre-cisplatin administration and at 2 days after cisplatin. In contrast to animals treated with cisplatin and culled at 2 days those culled at 7 days had significantly lower wet weight of gastric content compared to animals treated with saline (saline 2.72 ± 0.5 g. vs. cisplatin 0.50 ± 0.23 g, p = 0.01.).
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3.4. Relationship between plasma acylated and total ghrelin and the reduction in body weight Examination of the percentage body weight reduction over the 2 days of the study in the three groups which lost weight (cisplatin, cisplatin + dexamethasone, saline+ dexamethasone) revealed a linear relationship (r2 = 0.61 p b 0.0001) with the percentage of acylated to total ghrelin; higher weight loss being associated with lower percentages of acylated to total ghrelin. The points clustered into two groups and an arbitrary split was made at a percentage of acylated to total ghrelin of 10% and further analysis undertaken. A mean weight loss of −3.3 ± 0.9% (n = 5) occurred in animals with a percentage acylated to total ghrelin of 15.0 ± 0.9% in contrast to a mean weight loss of −10.1 ± 0.41% (n = 18) in animals with a percentage acylated to total ghrelin of 2.5 ± 0.35%. These weight reductions and acylated ghrelin percentages are significantly different from each other (p b 0.001) and are also different (p b 0.001) from the percentage of 7.0 ± 1.3% and weight gain of + 1.4 ± 0.6% the saline + saline group. 4. Discussion The different effects of ghrelin on gastric functions (e.g. appetite, emesis, motility; see Introduction) and the ability of exogenouslyadministered ghrelin to ameliorate cancer chemotherapy-associated dyspepsia in rodent models of this condition [8] prompted the present study. In short, we investigated the possibility that secretion of ghrelin might be a component of the body's defensive strategy to protect the stomach from injury or other dyspepsia-inducing stimuli and thereby attempt to ameliorate the anorectic effects of the injury. The results have shown multiple-adaptive changes in the ghrelin system in response to treatment with cisplatin, including upregulation in ghrelin receptor mRNA expression in the stomach and hypothalamus and an increase in the plasma level of acylated but not des-acylated ghrelin. In this study the plasma and molecular measurements were made 48 h after administration of cisplatin, when the effects of cisplatin on the various parameters are established; future studies should examine the changes whilst the effects of cisplatin are developing. It is notable that no increase in ghrelin mRNA was observed in either the hypothalamus or the stomach, in marked contrast to the increases in plasma levels of ghrelin. This apparent discrepancy merits further study. One possibility is that any change in the level of ghrelin mRNA was transitory and could not be detected 48 h after administration of cisplatin. Another possibility is that the plasma changes reflect an acute effect of cisplatin on ghrelin secretion rather than an effect of cisplatin treatment on de novo synthesis of ghrelin. Irrespective of the explanation, the combination of responses observed supports our hypothesis that the ghrelin system is involved in defensive responses to toxic challenges to the gut. The expected functional effect of this combination of changes would be to stimulate food intake via effects of ghrelin on vagal afferents and the hypothalamus [18,19] and enhance gastric emptying by an action on the brain, the vagus and the enteric neurones [4,20]. Whilst a substantial reduction in food intake and retention of gastric contents occurs in the cisplatin-treated animals it is likely that this would be greater in the absence of these adaptive changes but testing of this requires studies using selective ghrelin receptor antagonists and possibly, the use of cytotoxic agents less injurious than cisplatin. Thus, it should be noted that cisplatin produces a marked reduction in food intake (beginning on day 1 and greater magnitude on day 2 after cisplatin) and delay in gastric emptying (beginning on day 1 and continuing on day 2 after cisplatin; see [12] for references) and hence a protective role of endogenous ghrelin may be more apparent in a milder model of dyspepsia in which ghrelin receptor agonists could also be investigated. Our findings find some support in the observations of Shimizu et al. [21] who found an increase in plasma ghrelin concentrations in patients experiencing a reduction of food intake 8 and 21 days after
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the start of anti-cancer chemotherapy; in those patients where food intake was not changed, there was no change in the plasma concentrations of ghrelin. Similarly, in a pilot study in a different, less severe form of dyspepsia (functional dyspepsia), an increase in plasma acylated ghrelin levels correlated with a subjective measure of symptoms [22]. A novel finding in the present study is the effect of the glucocorticoid receptor agonist dexamethasone on the plasma levels of ghrelin. Dexamethasone reduced the plasma level of acylated ghrelin and percentage of acylated to total ghrelin to levels below those in animals treated with saline alone. This observation indicates that dexamethasone either preferentially affects the transcription, translation or post-translational processes [23] leading to the synthesis of acylated ghrelin, or affects the mechanism by which acylated ghrelin is secreted from the X/A cells in the stomach [24]. A genomic site of action of dexamethasone is supported by the presence of a binding half-site for glucocorticoid response elements in a region upstream from the start codon of the 5′ flanking region of the ghrelin gene [25]. The mechanism by which dexamethasone influences ghrelin requires investigation using molecular techniques to differentiate between a direct effect of dexamethasone such as described above and an indirect effect on pro-inflammatory mediators affected by cisplatin. Preliminary studies have revealed an upregulation of gastric iNOS and TNFα mRNA by cisplatin (unpublished observations and [26]). In cisplatin-treated animals dexamethasone also reduced the level of acylated ghrelin and the percentage of acylated to total ghrelin to values below those in animals treated with saline alone and identical to the values in animals treated with saline and dexaemethasone. This indicates that dexamethasone treatment not only influences the basal secretion of acylated ghrelin but also prevents the adaptive increase in levels stimulated by cisplatin. In addition, irrespective of other factors an elevation in ghrelin would be expected when food intake is reduced but this was not seen in the dexamethasone treated animals despite their relatively low intake. This may explain why the reduction in food intake is similar in the saline–cisplatin, cisplatin–dexamethasone and saline–dexamethasone treated animals. Although this should be a potent stimulus to ghrelin secretion [19,27] the plasma levels of ghrelin differ between the groups and in addition the animals in the two dexamethasone treated groups lost similar amounts of weight and in both cases this was significantly greater than in the saline–cisplatin group. This anorectic effect could be secondary to an effect of the reduced secretion of acylated ghrelin on energy balance [28,29], the secretion of growth hormone [24] as well as to the effects of dexamethasone on growth hormone and secretion (see [30,31] for refs). Consequently, this study provides preliminary in vivo evidence that glucocorticoid modulation of ghrelin synthesis and secretion may be a significant control mechanism, an observation supported by an in vitro study showing glucocorticoid downregulation of human ghrelin gene expression at the transcriptional level [32]. As the present study used a relatively high single dose of dexamethasone further investigation is needed to examine the dose response relationship of this effect. In conclusion the results indicate that administration of the cytotoxic anti-cancer agent cisplatin tends to increase plasma acylated ghrelin concentrations and the percentage contribution of acylated to total ghrelin levels, accompanied by a modest increase in ghrelin receptor mRNA levels in both the distal stomach and the hypothalamus. It is argued that these changes represent a defensive response to ameliorate the damaging effects of cisplatin (possibly mediated by substances such as iNOS and TNFα-see above) and perhaps other noxious stimuli, on gastrointestinal function and energy balance. Conversely, we have identified a novel, potent suppressive effect on plasma levels of ghrelin of a synthetic glucocorticoid, often used clinically to treat the consequences of cytotoxic drug treatment and the molecular mechanisms underlying this effect require investigation.
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References [1] Date Y, Murakami N, Toshinai K, Matsukura S, Niijima A, Matsuo H, Kangawa K, Nakazato M. The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology 2002;123:1120–8. [2] Dass NB, Munonyara M, Bassil AK, Hervieu GJ, Osbourne S, Corcoran S, Morgan M, Sanger GJ. Growth hormone secretagogue receptors in rat and human gastrointestinal tract and the effects of ghrelin. Neuroscience 2003;120:443–53. [3] Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams JR, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van der Ploeg LH, Howard AD, MacNeil DJ, Qian S. Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology 2004;145:2607–12. [4] Peeters TL. Ghrelin: a new player in the control of gastrointestinal functions. Gut 2005;54:1638–49. [5] Rudd JA, Ngan MP, Wai MK, King AG, Witherington J, Andrews PL, Sanger GJ. Antiemetic activity of ghrelin in ferrets exposed to the cytotoxic anti-cancer agent cisplatin. Neurosci Lett 2006;392:79–83. [6] Shimizu Y, Chang EC, Shafton AD, Ferens DM, Sanger GJ, Witherington J, Furness JB. Evidence that stimulation of ghrelin receptors in the spinal cord initiates propulsive activity in the colon of the rat. J Physiol 2006;576:329–38. [7] Nelson K, Walsh D, Sheehan F. Cancer and chemotherapy-related upper gastrointestinal symptoms: the role of abnormal gastric motor function and its evaluation in cancer patients. Support Care Cancer 2002;10:455–61. [8] Liu YL, Malik NM, Sanger GJ, Andrews PL. Ghrelin alleviates cancer chemotherapyassociated dyspepsia in rodents. Cancer Chemother Pharmacol 2006;58:326–33. [9] Brzozowski T, Konturek PC, Sliwowski Z, Pajdo R, Drozdowicz D, Kwiecien S, Burnat G, Konturek SJ, Pawlik WW. Prostaglandin/cyclooxygenase pathway in ghrelininduced gastroprotection against ischemia-reperfusion injury. J Pharmacol Exp Ther 2006;319:477–87. [10] Neary NM, Small CJ, Wren AM, Lee JL, Druce MR, Palmieri C, Frost GS, Ghatei MA, Coombes RC, Bloom SR. Ghrelin increases energy intake in cancer patients with impaired appetite: acute, randomized, placebo-controlled trial. J Clin Endocrinol Metab 2004;89:2832–6. [11] Liu YL, Malik N, Sanger GJ, Friedman MI, Andrews PL. Pica—a model of nausea? Species differences in response to cisplatin. Physiol Behav 2005;85:271–7. [12] Malik NM, Liu YL, Cole N, Sanger GJ, Andrews PL. Differential effects of dexamethasone, ondansetron and a tachykinin NK1 receptor antagonist (GR205171) on cisplatin-induced changes in behaviour, food intake, pica and gastric function in rats. Eur J Pharmacol 2007;555:164–73. [13] Malik NM, Moore GB, Smith G, Liu YL, Sanger GJ, Andrews PL. Behavioural and hypothalamic molecular effects of the anti-cancer agent cisplatin in the rat: a model of chemotherapy-related malaise? Pharmacol Biochem Behav 2006;83:9–20. [14] Rudd JA, Yamamoto K, Yamatodani A, Takeda N. Differential action of ondansetron and dexamethasone to modify cisplatin-induced acute and delayed kaolin consumption (“pica”) in rats. Eur J Pharmacol 2002;454:47–52. [15] Stevens CE, Hume ID. Comparative physiology of the vertebrate digestive system. Cambridge, UK: Cambridge University Press; 1998. 400 pp.
[16] Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000;25:169–93. [17] Moore GB, Himms-Hagen J, Harper ME, Clapham JC. Overexpression of UCP-3 in skeletal muscle of mice results in increased expression of mitochondrial thioesterase mRNA. Biochem Biophys Res Commun 2001;283:785–90. [18] 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 2001;120:337–45. [19] De Ambrogi M, Volpe S, Tamanini C. Ghrelin: central and peripheral effects of a novel peptydil hormone. Med Sci Monit 2003;9(9):RA217–24. [20] Bassil AK, Dass NB, Sanger GJ. The prokinetic-like activity of ghrelin in rat isolated stomach is mediated via cholinergic and tachykininergic motor neurones. Eur J Pharmacol 2006;544:146–52. [21] Shimizu Y, Nagaya N, Isobe T, Imazu M, Okumura H, Hosoda H, Kojima M, Kangawa K, Kohno N. Increased plasma ghrelin level in lung cancer cachexia. Clin Cancer Res 2003;9:774–8. [22] Shinomiya T, Fukunaga M, Akamizu T, Irako T, Yokode M, Kangawa K, Nakai Y, Nakai Y. Plasma acylated ghrelin levels correlate with subjective symptoms of functional dyspepsia in female patients. Scand J Gastroenterol 2005;40:648–53. [23] Gualillo O, Lago F, Casanueva FF, Dieguez C. One ancestor, several peptides posttranslational modifications of preproghrelin generate several peptides with antithetical effects. Mol Cell Endocrinol 2006;256:1–8. [24] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growthhormone-releasing acylated peptide from stomach. Nature 1999;402:656–60. [25] Kishimoto M, Okimura Y, Nakata H, Kudo T, Iguchi G, Takahashi Y, Kaji H, Chihara K. Cloning and characterization of the 5(′)-flanking region of the human ghrelin gene. Biochem Biophys Res Commun 2003;305:186–92. [26] Gale D, Blakemore SJ, Cook, Kidwai S, Moore I, Moore GBT, Moore SE, Sanger GJ, Holbrook J, Liu Y-L, Malik N, Andrews PLR. Modulation of gene expression in the glandular and non-glandular regions of the rat stomach after treatment with the anti-cancer drug cisplatin. Gastroenterology 2005;128(A-545):T1762. [27] Kim MS, Yoon CY, Park KH, Shin CS, Park KS, Kim SY, Cho BY, Lee HK. Changes in ghrelin and ghrelin receptor expression according to feeding status. NeuroReport 2003;14:1317–20. [28] 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 2001;409:194–8. [29] Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407:908–13. [30] Miller TL, Mayo KE. Glucocorticoids regulate pituitary growth hormone-releasing hormone receptor messenger ribonucleic acid expression. Endocrinology 1997;138: 2458–65. [31] Luque RM, Kineman RD, Park S, Peng XD, Gracia-Navarro F, Castano JP, Malagon MM. Homologous and heterologous regulation of pituitary receptors for ghrelin and growth hormone-releasing hormone. Endocrinology 2004;145:3182–9. [32] Kaji H, Kishimoto M, Kirimura T, Iguchi G, Murata M, Yoshioka S, Iida K, Okimura Y, Yoshimoto Y, Chihara K. Hormonal regulation of the human ghrelin receptor gene transcription. Biochem Biophys Res Commun 2001;284:660–6.
Contents lists available at ScienceDirect
Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Adaptive upregulation of gastric and hypothalamic ghrelin receptors and increased plasma ghrelin in a model of cancer chemotherapy-induced dyspepsia N.M. Malik a, G.B.T. Moore b, R. Kaur b, Y.-L. Liu a, S.L. Wood b, R.W. Morrow b, G.J. Sanger b, P.L.R. Andrews a,⁎ a b
Division of Basic Medical Sciences, St. George's, University of London, London, SWl7 ORE, UK Neurology and Gastrointestinal-CEDD, GlaxoSmithKline, Harlow, Essex CMl9 5AW, UK
A R T I C L E
I N F O
Article history: Received 20 July 2007 Received in revised form 4 January 2008 Accepted 14 March 2008 Available online 25 March 2008 Keywords: Ghrelin GHRL Ghrelin receptor GHSR Cisplatin Dexamethasone Dyspepsia
A B S T R A C T Chemotherapy treatment can lead to delayed gastric emptying, early satiety, anorexia, nausea and vomiting, described collectively as the cancer-associated dyspepsia syndrome (CADS). Administration of ghrelin (GHRL), an endogenous orexigenic peptide known to stimulate gastric motility, has been shown to reduce the symptoms of CADS induced in relevant animal models with the potent chemotherapeutic agent, cisplatin. We examined the effects in the rat of cisplatin (6 mg/kg i.p.) treatment on the expression of GHRL and ghrelin receptor (GHSR) mRNAs in the hypothalamus and the stomach at a time-point (2 days) when the effects of cisplatin are pronounced. In addition, plasma levels of GHRL (acylated and total including des-acyl GHRL) were measured and the effect on these levels of treatment with the synthetic glucocorticoid dexamethasone (2 mg/kg s.c. bd.) was investigated. Cisplatin increased GHSR mRNA expression in the stomach (67%) and hypothalamus (52%) but not GHRL mRNA expression and increased the percentage of acylated GHRL (7.03 ± 1.35% vs. 11.38 ± 2.40%) in the plasma. Dexamethasone reduced the plasma level of acylated GHRL and the percentage of acylated GHRL to values below those in animals treated with saline alone (7.03 ± 1.35% vs. 2.60 ± 0.49%). Our findings support the hypothesis that an adaptive upregulation of the ghrelin receptor may occur during cancer chemotherapy-associated dyspepsia. This may have a role in defensive responses to toxic challenges to the gut. In addition, our results provide preliminary evidence for glucocorticoid modulation of plasma ghrelin levels. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Ghrelin (GHRL) is an endogenous orexigenic, anti-emetic and gastrointestinal (GI) prokinetic peptide secreted mainly from the stomach but also from elsewhere, to act on ghrelin receptors (GHSR) within the hypothalamus (particularly the arcuate nucleus) and on gastrointestinal tract vagal, enteric (e.g. [1–5]) and spinal [6] nerve pathways. Patients with cancer being treated with cytotoxic drugs such as cisplatin, may experience a number of undesirable symptoms including emesis, dyspepsia, and anorexia [7]. Administration of exogenous ghrelin has been shown to have the potential to reduce each of these symptoms in relevant animal models treated with cisplatin as an exemplar cytotoxic agent: emesis in the ferret [5]; anorexia in the rat and mouse [8]; delayed gastric emptying in the mouse [8]. In addition ghrelin has been shown to have gastroprotective effects in a rodent model of ischaemia reperfusion injury [9]. Studies in cancer patients with impaired appetites have shown that ghrelin administration can increase energy intake and meal appreciation of a buffet meal [10]. Together, these results reveal the
⁎ Corresponding author. Fax: +44 208 725 2993. E-mail address: [email protected] (P.L.R. Andrews). 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2008.03.005
therapeutic potential of ghrelin receptor agonists but in addition, suggest that if released in response to cytotoxic drugs, ghrelin could act as an endogenous agent attempting to ameliorate the undesirable effects of cytotoxic drugs on emesis, food intake and gastric function. To investigate this hypothesis we have examined the effects in the rat of cisplatin treatment on the expression of GHR and ghrelin-R mRNA in the hypothalamus and the stomach, measured 2 days after treatment and at the time when food intake is at a nadir, delayed gastric emptying is established and pica (argued to be indicative of nausea and/or vomiting in rats which lack a vomiting reflex [11]) is present. Expression levels were also measured 7 days after cisplatin when previous studies have shown that the above effects have subsided [11,12]. In addition, plasma levels of ghrelin (acylated and total including des-acyl ghrelin) were measured and the effects on these levels of treatment with the synthetic glucocorticoid dexamethasone, were investigated. The latter has been shown in related studies to reduce cisplatin-induced pica, delayed gastric emptying, reduced locomotor activity and food intake [13] measured over 2 days after cisplatin administration. Our findings support the hypothesis that an adaptive upregulation of the function of ghrelin may occur during cancer chemotherapy-associated dyspepsia. In addition, the results provide preliminary evidence for glucocorticoid modulation of ghrelin levels in the plasma.
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2. Methods 2.1. Animals Male Wistar rats (350 g to 400 g.) were obtained from A. Tuck & Son Ltd (Essex, UK) and housed in a temperature and humidity controlled room with 12 h light: 12 h dark cycle (lights on at 07.00 am). The rats were caged individually in clear plastic cages with food (Bantin and Kingman, Hull, UK) and water provided ad libitum. Body weight, food and water were monitored daily and the overall health of the animals assessed twice daily against pre-set humane end points for the entire experimental period. All experiments were performed under the UK Animals (Scientific Procedures) Act 1986. 2.2. Drug administration 2.2.1. Study 1 Following a 3 day habituation period in the cages, the rats were given an intra-peritoneal injection (i.p.) of either sterile saline (154 mM NaCl, dose volume: 0.4 ml/100 g) or cisplatin (6 mg/kg). The dose of cisplatin used in the current experiments is comparable to that in previous studies, e.g. Refs. [12,14]. The cisplatin injection was prepared at a concentration of 1.5 mg/ml by dissolving 7.5 mg cisplatinum (II) diammine dichloride (Sigma, Poole, UK) in 5 ml sterile saline (154 mM NaCl) with 90 s of sonication. Animals given either cisplatin or saline alone were culled either 2 or 7 days following administration and tissues removed for the molecular studies described in Section 2.3 below. 2.2.2. Study 2 An additional study was performed on groups of animals which received either cisplatin (6 mg/kg i.p) or saline (i.p.) as described above on day 1 and in addition received either dexamethasone (2 mg/ kg) or saline subcutaneously (s.c.) 1 h before either the cisplatin (6 mg/ kg i.p.) or saline (i.p.). This dose is twice that used in the rat by Rudd et al. [14] but in their study the dose of cisplatin used was half that in the present study (3 mg/kg vs 6 mg/kg). In addition, Rudd et al. [14] used the i.p. route for dexamethasone as opposed to the s.c route in the present study which was considered to be more appropriate for minimising peritoneal irritation when 4 injections were required. At
17.00h, depending upon the group, animals received either a further injection of saline (s.c.) or dexamethasone (2 mg/kg s.c.). On day 2 these animals were given either saline (s.c.) or dexamethasone (2 mg/ kg s.c.) at 09.00h and 17.00h. Animals were culled at 10.00h on day 3 (i.e. 48 h after the start of the study) without either any further dexamethasone or saline. 2.3. Measurement of expression levels of mRNA At either 2 or 7 days after either cisplatin or saline treatment, animals in study 1 were killed between 10.00 and 11.00h by a rising concentration of CO2 and cervical dislocation (Schedule 1 method) and placed on a bed of dry ice for tissue removal. Hypothalami were taken and snap-frozen in liquid nitrogen prior to storage at −70 °C. The stomach was removed from the same animals, opened, contents removed and weighed (see below) and the tissue washed in chilled sterile saline to remove adherent particulate matter. The stomach was then divided into proximal (the non-glandular region with stratified squamous epithelium externally identifiable as the pale region with a relatively thin wall) and distal (glandular region consisting of the “proper” gastric region and the pyloric gland region but excluding the pyloric sphincter and recognisable by the relatively thickened mucosa [15]) by cutting along the demarcating mucosal ridge and then snapfrozen in liquid nitrogen prior to storage at −70 °C. Total RNA was isolated from the hypothalamus using the RNeasy® Mini kit (Qiagen, Crawley, UK) according to the manufacturer's instructions and from the stomach samples using TRIzol® reagent (Invitrogen, Paisley, UK) as previously described [17]. Relative quantification of mRNA transcripts was performed using the RT-PCR-based 5′ nuclease assay with TaqMan® probes (TaqMan assay, reviewed in [16]). Reverse transcriptase (RT) and negative control RT-minus (NoRT) reactions containing random 9-mers and DNase-treated total RNA (0.21 μg for the hypothalamus samples and 1 μg for the stomach region samples) were performed with no template controls (NoRNA) as before [18]. TaqMan assay oligonucleotide primers (Proligo France SAS, Paris, France) and probes (Applied Biosystems, Warrington, UK) were designed and TaqMan analysis performed as previously described [17] (see Table 1 for oligonucleotide sequences). For each transcript measurement, equal amounts of cDNA were added to each reaction (equivalent to 10 or 20 ng total RNA for the mRNAs and 100 pg for 18S
Table 1 Oligonucleotide sequences of gene-specific TaqMan assay primers and probes Rat RNA (RGD gene symbol)
Accession number (amplicon position in parentheses)
Primer or probe
Primer or probe sequence
Ghrl
AB029433 (151–276)
Ghsr
AB001982 (751–880)
Actb
AF122902.1 (131–216)
Hprt
M63983 (590–683)
Ppia
M19533 (296–401)
Rnr1
M11188 (12–107)
Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe Forward (sense) Reverse (antisense) Fluorogenic probe
5′-TCCAAGAAGCCACCAGCTAAAC-3′ 5′-AACATCGAAGGGAGCATTGAAC-3′ FAM-5′-CTTCTGCTTGTCCTCTGTCCTCTGGGTG-3′-TAMRA 5′-CTCCGGGACCAGAACCACA-3′ 5′-CCAGAGAGCCAGGCTCGAA-3′ FAM-5′-CAAACACCACCACAGCAAGCATCTTCACT-3′-TAMRA 5′-ACCCTAAGGCCAACCGTGAA-3′ 5′-CACAGCCTGGATGGCTACGT-3′ FAM-5′-CCCAGATCATGTTTGAGACCTTCAACACCC-3′-TAMRA 5′-GGTGAAAAGGACCTCTCGAAGTG-3′ 5′-ATAGTCAAGGGCATATCCAACAACA-3′ FAM-5′-CCAGACTTTGTTGGATTTGAAATTCCAGACAA-3′-TAMRA 5′-ATGAGAACTTCATCCTGAAGCATACA-3′ 5′-TCAGTCTTGGCAGTGCAGATAAA-3′ FAM-5′-CCTGGCATCTTGTCCATGGCAAATG-3′-TAMRA 5′-ACCTGGTTGATCCTGCCAGTAG-3′ 5′-AGCCATTCGCAGTTTCACTGTAC-3′ FAM-5′- TCAAAGATTAAGCCATGCATGTCTAAGTACGCAC-3fs-TAMRA
FAM: fluorogenic probe reporter dye 6-carboxyfluorescein; TAMRA: fluorogenic probe quencher dye 6-carboxytetramethylrhodamine; RGD: rat genome database (http://rgd.mcw. edu/); Ghrl: ghrelin; Ghsr: ghrelin receptor or growth hormone secretagogue receptor; Actb: β-actin; Hprt: hypoxanthine phosphoribosyltransferase; Ppia: peptidylprolyl isomerase A (cyclophilin); Rnr1: 18S rRNA.
N.M. Malik et al. / Regulatory Peptides 148 (2008) 33–38
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Table 2 Cisplatin treatment increased ghrelin receptor, but not ghrelin mRNA expression (A) without affecting the mRNA expression levels of reference housekeeping genes (B) A. Tissue
Distal stomach
Proximal stomach
Distal stomach
Proximal stomach
Hypothalamus
RNA
Ghrl
Ghrl
Ghsr
Ghsr
Ghsr
Saline-treated, RAU Cisplatin-treated, RAU Fold-change
24.26 (19.72–29.85), n = 8 29.70 (24.88–35.46), n = 8 1.22 (0.96–1.57)
0.12 (0.03–0.45), n = 8 0.12 (0.02–0.89), n = 7 0.96 (0.12–8.03)
0.037 (0.028–0.050), n = 8 0.062 (0.041–0.093), n = 8 1.67 (1.06–2.63)*
0.009 (0.004–0.020), n = 7 0.010 (0.006–0.018), n = 4 1.18 (0.42–3.31)
5.37 (3.96–7.28), n = 7 8.16 (5.69–11.70), n = 6 1.52 (1.01–2.29)*
B. Tissue
Distal stomach
Distal stomach
Proximal stomach
Proximal stomach
Hypothalamus
Hypothalamus
RNA
Rnr1 (18S rRNA)
Ppia
Rnr1 (18S rRNA)
Actb
Ppia
Hprt
Saline-treated, RAU Cisplatin-treated, RAU Fold-change
537.54 (447.68–645.44), n=8 528.36 (461.13–605.39), n=8 0.98 (0.80–1.21)
29.35 (24.62–34.98), n=8 35.44 (29.03–43.27), n=8 1.21 (0.95–1.54)
39.96 (31.60–50.53), n=8 54.10 (40.30–72.61), n=7 1.35 (0.97–1.89)
2.96 (2.23–3.92), n=8 2.70 (2.00–3.64), n=7 0.91 (0.63–1.32)
7.13 (6.22–8.18), n = 7
4.78 (4.14–5.52), n=7 5.60 (4.79–6.54), n=6 1.17 (0.97–1.41)
7.80 (6.28–9.68), n=6 1.09 (0.88–1.36)
Adult male Wistar rats treated with either saline (i.p.) or cisplatin (6 mg/kg i.p.) and tissues collected 2 days later for RNA. TaqMan real-time RT-PCR data in relative arbitrary units (RAU) expressed as geometric mean with 95% confidence limits and as ratio to geometric mean of saline controls with 95% confidence limits; *p b 0.05; n = 4–8.
rRNA). Reaction conditions were as follows: 50 °C for 2 min, 95 °C for 10 min then 45–50 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative standard curves were constructed using a 3-fold serial dilution of rat pituitary, corpus-antrum, forestomach or hypothalamus total cDNA (equivalent to 150 to 0.008 or 0.0025 ng total RNA for mRNA measurements, and 900 to 0.046 or 0.015 pg total RNA for 18S rRNA), the choice of cDNA being dependent on the mRNA being studied. 2.4. Analysis of acylated and total, including des-acylated ghrelin concentrations Blood samples were collected by cardiac puncture (animals killed by a Schedule 1 procedure see above) from the animals in study 2 and delivered into tubes containing potassium-EDTA on ice. Plasma was separated by centrifugation, frozen and stored at −20 °C until analysis. Plasma levels of acylated and des-acyl ghrelin were determined by enzyme-linked immunosorbent assay using commercially available kits (ELISA; active ghrelin and desacyl ghrelin ELISA kit, LINCO Research Inc, USA) according to the manufacturer's instructions. The standard curve was reconstituted in stripped rat plasma. In brief, plasma from the rats were mixed with activated charcoal at a ratio of 0.2 g/ml and incubated at room temperature for 1 h with occasional shaking, followed by centrifugation at 3000 ×g for 30 min at 4 °C. Stripped plasma was aliquoted and stored in −20 °C until use. Prior to measurement, plasma samples were spun at 16000 ×g for 5 min to remove aggregates. For the ELISA of active ghrelin, plasma was acidified with 0.05 M HCL and 0.01 M phenylmethylsulfonyl fluoride. The detection limit for the assay was 2.5 fmol/ml. 2.5. Measurement of body weight, food and water intake, kaolin consumption and gastric contents Food and water intake was measured by weighing the food hopper or water bottle on each day of the experiment between 10.00h and 11.00h and body weight was measured at the same time. Spilled food present in the cage was subtracted from the above measurement. Pica was assessed by measurement of kaolin consumption as previously described [11]. Post-mortem gastric contents were removed from the stomach and the wet weight measured. 2.6. Statistical analysis Results of the mRNA studies are presented as the ratio to the geometric mean of the saline-treated controls at each time point with
95% confidence intervals for the ratio. The transcript data were analyzed using one-way analysis of variance (ANOVA) after a log10transformation (performed in Microsoft® Excel 97; Microsoft Corporation, Redmond, WA, USA with an add-in toolkit for the analysis of real-time PCR data (PRISM Training and Consultancy Ltd., Cambridge, UK). Differences were considered significant when P b 0.05. Results from the remainder of the study are presented as mean ± S.E.M (n = number of animals). Statistical analysis of plasma levels of ghrelin were undertaken using ANOVA followed by the Tukey honest significant difference test allowing for multiple comparisons. Other analyses used either paired or unpaired (non parametric) sample ttests as appropriate. Differences were considered significant when p b 0.05. 3. Results 3.1. The effect of cisplatin on the expression of ghrelin and the ghrelin receptor Ghrelin and ghrelin receptor mRNA were detected in both the proximal and distal regions of the stomach in animals treated with saline. Two days after treatment with cisplatin, there were no changes in the levels of ghrelin mRNA expression, but the levels of ghrelin receptor mRNA were increased in the distal region of the stomach (67% increase; p b 0.05, n = 8; Table 2). No changes in either ghrelin or ghrelin receptor expression were found in either region of the stomach 7 days after cisplatin treatment (data not shown). In the hypothalamus ghrelin receptor mRNA expression increased by 52% (p b 0.05, n = 6–7; Table 2) 2 days after cisplatin administration but no change was found 7 days after cisplatin administration (data not shown). 3.2. The effect of cisplatin and dexamethasone on blood plasma levels of ghrelin In animals treated with saline alone, both acylated and desacylated ghrelin were present in the plasma with the des-acylated form being present at concentrations approximately 10-times higher than for the acylated ghrelin (respectively 25.8 ± 5.7 and 313 ± 46 fmol/ ml of acylated and des-acylated ghrelin; Fig. 1). Two days after cisplatin treatment, the mean concentration of acylated ghrelin increased to 45.3 ± 8.5 fmol/ml, but this increase was not statistically significant (Fig. 1A). Likewise although the plasma concentration of the des-acylated form of ghrelin increased to 412 ± 55 fmol/ml this
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treated and saline-treated animals to a comparable level which was below that for the control animals treated with saline alone (Fig. 1C). 3.3. Effect of cisplatin and dexamethasone on body weight, food and water intake, kaolin consumption and gastric contents
Fig. 1. The effect of saline, cisplatin (6mg/kg, i.p.) and dexamethsasone (2 mg/kg s.c.) on the plasma concentration of acylated (A) and des-acylated (B) ghrelin and on the percentage of acylated to total ghrelin (C). Values are mean ± S.E.M. (n = 7–8); ⁎⁎ p b 0.01, ⁎⁎⁎p b 0.001 compared to corresponding control or to cisplatin saline as appropriate.
increase was not statistically significant (Fig. 1B). Following treatment with dexamethasone alone, the mean plasma concentrations of acylated ghrelin tended to be lower than those found in the salinetreated animals (respectively 11.6 ± 2.4 and 25.8 ± 5.7 fmol/ml; Fig. 1A), although this difference was not statistically significant (Fig. 1A). Treatment with dexamethasone did not significantly change the concentrations of the des-acylated form of ghrelin (Fig. 1B). In rats treated with both cisplatin and dexamethasone, there was a significant reduction in the blood concentrations of acylated ghrelin, compared with the cisplatin-treated rats (p = 0.003; Fig. 1A). Interestingly, in these animals, the concentration of des-acylated ghrelin also tended to be larger than in the cisplatin-treated animals, although again, any change was not statistically significant. Calculation of the percentage of acylated to total ghrelin in individual animals revealed that this increased in response to cisplatin although not significantly, however dexamethasone treatment significantly (p b 0.001) reduced the percentage in both the cisplatin-
In the study measuring plasma ghrelin levels, total food intake over 2 days decreased significantly in animals treated with cisplatin–saline in comparison to those treated with saline alone (cisplatin–saline 30.1 ± 4.1 g. n = 7 vs. saline–saline 49.0 ± 3.0 g. n = 8, p b 0.01). Although there was a reduction in food intake on both days it was only statistically significant on day 2 (day 1 saline–saline 23.4 ± 1.74 g vs. day 1 cisplatin– saline19.43 ± 1.99 g, ns; day 2 saline–saline 25.63 ± 1.47 g vs. day 2 cisplatin–saline 10.71 ± 2.24 g, p b 0.0001). This reduced food intake in the cisplatin-treated animals was unaffected by dexamethasone administration to cisplatin-treated animals (total intake over 2 days cisplatin–saline 30.1 ± 4.1 g. n = 7 vs. cisplatin–dexamethasone 32.0 ± 3.6 g. n = 8; day1 cisplatin–saline 19.43 ± 1.99 g vs. day 1 cisplatin– dexamethasone 20.63 ± 2.06 g; day 2 cisplatin–saline 10.71 ± 2.24 g vs day 2 cisplatin–dexamethasone 11.38 ± 2.08 g) and although dexamethasone alone did significantly reduce food intake when compared to animals treated with saline alone the effect was only significant on day 2 of treatment (day 1 saline 23.38 ± 1.74 g vs. day 1 saline–dexamethasone 24.50 ± 1.18 g; day 2 saline–saline 25.63. ± 1.47 g vs day 2 saline– dexamethasone 14.13 ± 1.04 g, p b 0.0001). Water consumption was not significantly affected on either day by any of the treatments (data not shown). Kaolin consumption over the 48 h of the study was significantly increased in animals treated with cisplatin (saline–saline 0.22 ± 0.05 g vs. cisplatin–saline 3.36 ± 0.90 g, p b 0.01). In animals treated with dexamethasone kaolin consumption was not significantly different from that in animals treated with saline (saline–dexamethasone 0.97 ± 0.67 g vs. saline–saline 0.22 ± 0.05 g) but dexamethasone did reduce kaolin consumption in cisplatin-treated rats (cisplatin–saline 3.36 ± 0.90 g vs. cisplatin–dexamethasone 1.36 ± 0.41 g) but this effect was not statistically significant. Animals treated with cisplatin had a significant reduction in body weight over 2 days (374 ± 8.5 g. vs. 357 ± 6.5 g. n = 7, p b 0.005) as did animals treated with both cisplatin and dexamethasone (364 ± 8.6 g vs. 324 ±8.2 g n = 8, p b 0.0001) but the weight loss in the latter group was significantly greater (p b 0.001) than the former. Saline and dexamethasone treatment resulted in a significant weight loss (364 ± 10 g vs. 323 ± 10 g, n = 8, p b 0.0005) which was not significantly different from that recorded in the animals treated with both dexamethasone and cisplatin. Cisplatin treatment led to an increase in the wet weight of gastric contents measured 2 days later (saline–saline 3.00 ± 0.43 g, n = 8 vs. saline–cisplatin 8.32 ± 1.90 g, n = 7, p b 0.01). Treatment with dexamethasone alone was without significant effect on the weight of gastric contents in comparison to saline-treated animals (saline– saline 3.00 ± 0.43 g, n = 8, vs. saline–dexamethasone 3.1 ± 0.43 g, n = 8). Dexamethasone treatment in animals given cisplatin resulted in a reduction in the weight of gastric contents but this failed to reach statistical significance (saline–cisplatin 8.32 ± 1.90 g, n = 7, vs. cisplatin– dexamethasone 4.9 ± 0.7 g., n = 8). In the molecular biology study the cisplatin treatment resulted in reductions in body weight and food intake and an increase in the weight of gastric contents measured 48 h after administration (data not shown) with values not significantly different from those in animals treated in a similar way in the plasma ghrelin study reported above. In animals studied 7 days after cisplatin administration food intake and body weight although still significantly reduced were higher than at 2 days after cisplatin. Water intake was significantly (p b 0.01) elevated compared to pre-cisplatin administration and at 2 days after cisplatin. In contrast to animals treated with cisplatin and culled at 2 days those culled at 7 days had significantly lower wet weight of gastric content compared to animals treated with saline (saline 2.72 ± 0.5 g. vs. cisplatin 0.50 ± 0.23 g, p = 0.01.).
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3.4. Relationship between plasma acylated and total ghrelin and the reduction in body weight Examination of the percentage body weight reduction over the 2 days of the study in the three groups which lost weight (cisplatin, cisplatin + dexamethasone, saline+ dexamethasone) revealed a linear relationship (r2 = 0.61 p b 0.0001) with the percentage of acylated to total ghrelin; higher weight loss being associated with lower percentages of acylated to total ghrelin. The points clustered into two groups and an arbitrary split was made at a percentage of acylated to total ghrelin of 10% and further analysis undertaken. A mean weight loss of −3.3 ± 0.9% (n = 5) occurred in animals with a percentage acylated to total ghrelin of 15.0 ± 0.9% in contrast to a mean weight loss of −10.1 ± 0.41% (n = 18) in animals with a percentage acylated to total ghrelin of 2.5 ± 0.35%. These weight reductions and acylated ghrelin percentages are significantly different from each other (p b 0.001) and are also different (p b 0.001) from the percentage of 7.0 ± 1.3% and weight gain of + 1.4 ± 0.6% the saline + saline group. 4. Discussion The different effects of ghrelin on gastric functions (e.g. appetite, emesis, motility; see Introduction) and the ability of exogenouslyadministered ghrelin to ameliorate cancer chemotherapy-associated dyspepsia in rodent models of this condition [8] prompted the present study. In short, we investigated the possibility that secretion of ghrelin might be a component of the body's defensive strategy to protect the stomach from injury or other dyspepsia-inducing stimuli and thereby attempt to ameliorate the anorectic effects of the injury. The results have shown multiple-adaptive changes in the ghrelin system in response to treatment with cisplatin, including upregulation in ghrelin receptor mRNA expression in the stomach and hypothalamus and an increase in the plasma level of acylated but not des-acylated ghrelin. In this study the plasma and molecular measurements were made 48 h after administration of cisplatin, when the effects of cisplatin on the various parameters are established; future studies should examine the changes whilst the effects of cisplatin are developing. It is notable that no increase in ghrelin mRNA was observed in either the hypothalamus or the stomach, in marked contrast to the increases in plasma levels of ghrelin. This apparent discrepancy merits further study. One possibility is that any change in the level of ghrelin mRNA was transitory and could not be detected 48 h after administration of cisplatin. Another possibility is that the plasma changes reflect an acute effect of cisplatin on ghrelin secretion rather than an effect of cisplatin treatment on de novo synthesis of ghrelin. Irrespective of the explanation, the combination of responses observed supports our hypothesis that the ghrelin system is involved in defensive responses to toxic challenges to the gut. The expected functional effect of this combination of changes would be to stimulate food intake via effects of ghrelin on vagal afferents and the hypothalamus [18,19] and enhance gastric emptying by an action on the brain, the vagus and the enteric neurones [4,20]. Whilst a substantial reduction in food intake and retention of gastric contents occurs in the cisplatin-treated animals it is likely that this would be greater in the absence of these adaptive changes but testing of this requires studies using selective ghrelin receptor antagonists and possibly, the use of cytotoxic agents less injurious than cisplatin. Thus, it should be noted that cisplatin produces a marked reduction in food intake (beginning on day 1 and greater magnitude on day 2 after cisplatin) and delay in gastric emptying (beginning on day 1 and continuing on day 2 after cisplatin; see [12] for references) and hence a protective role of endogenous ghrelin may be more apparent in a milder model of dyspepsia in which ghrelin receptor agonists could also be investigated. Our findings find some support in the observations of Shimizu et al. [21] who found an increase in plasma ghrelin concentrations in patients experiencing a reduction of food intake 8 and 21 days after
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the start of anti-cancer chemotherapy; in those patients where food intake was not changed, there was no change in the plasma concentrations of ghrelin. Similarly, in a pilot study in a different, less severe form of dyspepsia (functional dyspepsia), an increase in plasma acylated ghrelin levels correlated with a subjective measure of symptoms [22]. A novel finding in the present study is the effect of the glucocorticoid receptor agonist dexamethasone on the plasma levels of ghrelin. Dexamethasone reduced the plasma level of acylated ghrelin and percentage of acylated to total ghrelin to levels below those in animals treated with saline alone. This observation indicates that dexamethasone either preferentially affects the transcription, translation or post-translational processes [23] leading to the synthesis of acylated ghrelin, or affects the mechanism by which acylated ghrelin is secreted from the X/A cells in the stomach [24]. A genomic site of action of dexamethasone is supported by the presence of a binding half-site for glucocorticoid response elements in a region upstream from the start codon of the 5′ flanking region of the ghrelin gene [25]. The mechanism by which dexamethasone influences ghrelin requires investigation using molecular techniques to differentiate between a direct effect of dexamethasone such as described above and an indirect effect on pro-inflammatory mediators affected by cisplatin. Preliminary studies have revealed an upregulation of gastric iNOS and TNFα mRNA by cisplatin (unpublished observations and [26]). In cisplatin-treated animals dexamethasone also reduced the level of acylated ghrelin and the percentage of acylated to total ghrelin to values below those in animals treated with saline alone and identical to the values in animals treated with saline and dexaemethasone. This indicates that dexamethasone treatment not only influences the basal secretion of acylated ghrelin but also prevents the adaptive increase in levels stimulated by cisplatin. In addition, irrespective of other factors an elevation in ghrelin would be expected when food intake is reduced but this was not seen in the dexamethasone treated animals despite their relatively low intake. This may explain why the reduction in food intake is similar in the saline–cisplatin, cisplatin–dexamethasone and saline–dexamethasone treated animals. Although this should be a potent stimulus to ghrelin secretion [19,27] the plasma levels of ghrelin differ between the groups and in addition the animals in the two dexamethasone treated groups lost similar amounts of weight and in both cases this was significantly greater than in the saline–cisplatin group. This anorectic effect could be secondary to an effect of the reduced secretion of acylated ghrelin on energy balance [28,29], the secretion of growth hormone [24] as well as to the effects of dexamethasone on growth hormone and secretion (see [30,31] for refs). Consequently, this study provides preliminary in vivo evidence that glucocorticoid modulation of ghrelin synthesis and secretion may be a significant control mechanism, an observation supported by an in vitro study showing glucocorticoid downregulation of human ghrelin gene expression at the transcriptional level [32]. As the present study used a relatively high single dose of dexamethasone further investigation is needed to examine the dose response relationship of this effect. In conclusion the results indicate that administration of the cytotoxic anti-cancer agent cisplatin tends to increase plasma acylated ghrelin concentrations and the percentage contribution of acylated to total ghrelin levels, accompanied by a modest increase in ghrelin receptor mRNA levels in both the distal stomach and the hypothalamus. It is argued that these changes represent a defensive response to ameliorate the damaging effects of cisplatin (possibly mediated by substances such as iNOS and TNFα-see above) and perhaps other noxious stimuli, on gastrointestinal function and energy balance. Conversely, we have identified a novel, potent suppressive effect on plasma levels of ghrelin of a synthetic glucocorticoid, often used clinically to treat the consequences of cytotoxic drug treatment and the molecular mechanisms underlying this effect require investigation.
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