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Plasma ghrelin and obestatin levels are increased in spontaneously hypertensive rats
Peptides 31 (2010) 297–300
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Plasma ghrelin and obestatin levels are increased in spontaneously hypertensive rats Zhao-Feng Li a,b,1, Zhi-Fu Guo a,1, Jiang Cao a, Jian-Qiang Hu a, Xian-Xian Zhao a, Rong-Liang Xu a, Xin-Miao Huang a, Yong-Wen Qin a,*, Xing Zheng a,* a b
Department of Cardiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China Department of Cardiology, No. 88 Hospital of PLA, Tai’an City, Shandong Province 271000, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 September 2009 Received in revised form 10 November 2009 Accepted 16 November 2009 Available online 26 November 2009
Obestatin, encoded by the same gene as ghrelin, was first described as a physiological opponent of ghrelin. We investigated fasting plasma ghrelin and obestatin levels in spontaneously hypertensive rats and Wistar-Kyoto rats. We found that ghrelin levels, obestatin levels and the ratio of ghrelin to obestatin were significantly higher in spontaneously hypertensive rats than Wistar-Kyoto rats. Systolic blood pressure and diastolic blood pressure were positively correlated; however, heart period and baroreflex sensitivity were negatively correlated with ghrelin levels. Systolic blood pressure was positively correlated, whereas baroreflex sensitivity was negatively correlated with obestatin levels. In addition, systolic blood pressure was a significantly independent variable of ghrelin levels, obestatin levels, and the ghrelin to obestatin ratio in a multiple regression analysis. Our data suggests that there is a disturbance of ghrelin and obestatin in the circulation of spontaneously hypertensive rats and the ghrelin/obestatin system might play a role in blood pressure regulation. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Ghrelin Obestatin Blood pressure Spontaneously hypertensive rats
1. Introduction Ghrelin was first identified from rat stomachs, and subsequent studies have shown that ghrelin has a multiplicity of physiological functions such as stimulating appetite, initiating food intake, controlling gastric motility and modulating energy metabolism [5,14]. In addition, there are some researches demonstrating that ghrelin is intimately correlated with blood pressure. Firstly, Nagaya et al. found that the intravenous injection of ghrelin could decrease peripheral artery resistance, leading to a drop in blood pressure without an increase in heart rate (HR) in healthy volunteers [10]. Secondly, Matsumura et al. also found that the intravenous injection of ghrelin could elicit dose-related decreases in arterial pressure and HR, and intracerebroventricular injection of ghrelin could decrease arterial pressure, HR, and renal sympathetic nerve activity in conscious rabbits [9]. Finally, Makino et al. once found that fasting plasma ghrelin concentration was significantly higher in patients with pregnancy-induced hypertension (PIH) than normal pregnant women [8]. These results suggest that ghrelin may play an important role in blood pressure regulation.
In 2005, Zhang et al. first reported that they found a ghrelinassociated peptide, encoded by the same gene as ghrelin, and named it obestatin [16]. An interesting finding was that obestatin, though derived from the same peptide precursor as ghrelin, suppressed food intake, inhibited jejunal contraction, decreased body weight gain, and antagonized the actions of ghrelin when both peptides were co-administered [16]. In our department, Guo et al. showed that obese individuals would display a disturbance of ghrelin and obestatin levels and suggested that this might play a role in the pathophysiological progress of the disease [4]. Recently, Anderwald-Stadler et al. found that the fasting plasma obestatin level was negatively correlated with SBP in insulin-resistant humans, suggesting a possible role of obestatin in the regulation of blood pressure [2]. To the best of our knowledge, plasma obestatin profiles in relation to ghrelin in hypertension have not been studied. Therefore, the aim of this study was to investigate the fasting plasma ghrelin and obestatin levels and their relationship with blood pressure, HR and the baroreflex function in SHR, the animal model of human essential hypertension [11]. 2. Materials and methods
* Corresponding authors at: Department of Cardiology, Changhai Hospital, Second Military Medical University, Changhai Road 168, Shanghai 200433, China. Tel.: +86 21 8187 3202; fax: +86 21 5522 5366. E-mail addresses: [email protected] (Y.-W. Qin), [email protected] (X. Zheng). 1 The first two authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.11.018
2.1. Animals Rats (Slac Laboratory Animal Co. Ltd., Shanghai, China), 14 male SHR (age: 12–13 weeks; body weight: 140–250 g) and 12 male WKY (age: 12–13 weeks; body weight: 200–260 g) were housed in
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groups in a temperature-controlled room (23–25 8C) under a 12-h light/12-h dark cycle (8:00 am to 8:00 pm light, 8:00 pm to 8:00 am dark) and maintained on pelleted chow with free access to water. Animal protocols were in compliance with the institutional guidelines. 2.2. Blood pressure and heart period measurements SBP, DBP and HP of rats were continuously recorded using a previously described technique [15]. Briefly, two days before the blood pressure recording, the rats were anesthetized with a combination of ketamine (40 mg/kg, intraperitonea) and diazepam (6 mg/kg, intraperitonea). Two polyethylene catheters (a PE10 (ID 0.3 mm, OD 0.58 mm; Biotrol, France) fused to a PE-50 (ID 0.58 mm, OD 0.96 mm; Biotrol, France)) filled with heparinized 0.9% NaCl (50 U/ml) were inserted into the lower abdominal aorta via the left femoral artery and the left femoral vein for blood pressure recording and intravenous phenylephrine injection, respectively. The two catheters were tunneled subcutaneously under the skin of the back to exit between the scapulae and were plugged with a short piece of stainless steel wire. The rats were then allowed to recover from anesthesia for two days in individual cylindrical cages containing food and water. The two catheters were flushed twice daily with a solution of heparinized NaCl (0.9%). The recording of arterial pressure and the intravenous injection of phenylephrine were performed in unrestrained rats after two days of recovery. The venous catheter was connected to a syringe for saline or phenylephrine injections. The arterial catheter was connected to a pressure transducer through a rotating swivel allowing the rats to move freely in the cage. After 2 h of stabilization, blood pressure signals and HP were recorded on an automatic blood pressure recording and analysis system by a microcomputer. SBP, DBP, MAP and HP values were determined online. A continuity of 1-h recordings was performed immediately after a 2-h period of stabilization. The mean values of these parameters were calculated. 2.3. Determination of baroreflex sensitivity At the end of the continuity of 1-h recordings, the baroreflex sensitivity (BRS) of the rats was conducted in the previously mentioned blood pressure recording condition. BRS was measured in the conscious rat using the previously described method [3,7]. Briefly, a bolus injection of phenylephrine was used to induce blood pressure elevation. The dose of phenylephrine (5–10 mg/kg) was adjusted to raise SBP approximately 30 10 mmHg. There exists a delay (approximately 1 s) between the elevation of blood pressure (stimulus) and the prolongation of heart rate (response) for arterial baroreflex. In rats, the heart rate is approximately 5 or 6 beats per second. So, HP was plotted against SBP for linear regression analysis for 2–8 shifts (calculated by computer); the slope with the largest correlation coefficient (r) of HP/SBP was expressed as BRS (ms/ mmHg). The mean of the two measurements with the proper dosage was taken as the final result. At the end of the determination of BRS, the rats were killed. 2.4. Blood sampling and hormone assay Before the blood pressure recording, blood was drawn from the aortic catheter after a 12-h overnight fast. Blood samples for measurement of ghrelin, obestatin, and insulin were immediately transferred to chilled polypropylene tubes containing EDTA–2Na (1 mg/ml) and aprotinin (500 U/ml), centrifuged at 4 8C, 1600 g for 15 min, and then plasma samples were stored at 80 8C until assayed.
Plasma ghrelin and obestatin levels were measured using commercial RIA kits (Phoenix Pharmaceuticals, Belmont, CA, USA) according to the manufacturer’s instructions. Sensitivity of the obestatin assay was 170.2 pg/ml and sensitivity of the ghrelin assay was 27.7 pg/ml. The intra-assay CV was less than 5%, and the inter-assay CV was less than 10%. Plasma insulin levels were measured using an ELISA kit (Phoenix Biotech Co., Ltd., Beijing, China). The lower and upper detection limits were 5 mIU/ml and 160 mIU/ml, respectively. The intra-assay CV was less than 10%, and the inter-assay CV was less than 15%. 2.5. Statistical analysis Results are expressed as the mean SE. Differences between groups were assessed with the two-side unpaired Student’s t-test. Correlations between ghrelin, obestatin, ghrelin to obestatin ratio and SBP, DBP, MAP, HP, BRS, insulin, and body weight were examined by bivariate correlations (Pearson’s correlation). Multiple regression analysis was further used to assess the relationships between ghrelin, obestatin, ghrelin to obestatin ratio and SBP, DBP, MAP, HP, BRS, insulin, and body weight. A value of P < 0.05 was considered statistically significant. All of the analyses were performed using SPSS for Windows (Version 10.0; SPSS Inc., Chicago, IL). 3. Results 3.1. Animals (Table 1) Body weight is lower in SHR group compared with WKY group (P < 0.01). SBP, DBP and MAP were significantly higher in SHR group compared with WKY group (all P < 0.01). However, HP was significantly lower in SHR group compared with WKY group (P < 0.05). BRS was significantly lower in SHR group compared with WKY group (P < 0.01). Insulin concentration was significantly lower in SHR group compared with WKY group (P < 0.01). 3.2. Differences in ghrelin, obestatin, ghrelin to obestatin ratio (Fig. 1) Both ghrelin and obestatin plasma levels were significantly higher in SHR group compared with WKY group (649.4 55.3 pg/ ml vs 106.3 8.4 pg/ml, P < 0.01; 44.6 2.3 pg/ml vs 38.6 1.5 pg/ ml, P < 0.05 respectively) (Fig. 1A and B). Moreover, the ratios of ghrelin to obestatin in SHR group were significantly higher compared with WKY group (15.1 1.5 vs 2.8 0.2, P < 0.01) (Fig. 1C). 3.3. Correlations of ghrelin, obestatin, and ghrelin to obestatin ratio with various parameters HP, BRS, body weight and insulin levels were negatively correlated with ghrelin levels and the ratio of ghrelin to obestatin (Table 2). SBP, DBP and MAP were positively correlated with Table 1 Characterization of the rats in the study.
Number Age (w) Body weight (g) SBP (mmHg) DBP (mmHg) MAP (mmHg) HP (ms) BRS (ms/mmHg) Insulin (mIU/ml) Data are means SE. * P < 0.01 vs WKY. # P < 0.05 vs WKY.
WKY
SHR
12 12–13 220.5 7.9 115.8 3.3 77.4 2.4 96.6 2.4 171.8 4.4 0.74 0.009 26.6 2.4
14 12–13 175.1 9.4* 145.4 2.0* 91.8 1.6* 115.2 1.5* 153.6 6.8# 0.50 0.02* 11.3 0.8*
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were further assessed in multiple regression analysis. In a multiple regression model including body weight, insulin, SBP, DBP, MAP, HP and BRS, body weight and SBP were independent variables of ghrelin level (standardized coefficient = 0.377; P = 0.004; standardized coefficient = 0.615; P < 0.0005, respectively); and SBP was an independent determinant of the fasting obestatin level (standardized coefficient = 0.460; P = 0.018); SBP and body weight were significantly independent determinants of the ghrelin to obestatin ratio (standardized coefficient = 0.567; P = 0.01; standardized coefficient = 0.332; P = 0.037, respectively). None of these parameters showed any significant correlation with ghrelin, obestatin, and the ratio of ghrelin to obestatin in SHR group or WKY group when considered alone. 4. Discussion This study showed that fasting plasma ghrelin and obestatin levels, and the ratio of ghrelin to obestatin were significantly increased in SHR group compared with WKY group and that SBP was positively correlated with ghrelin, obestatin and ghrelin to obestatin ratio in rats. Makino et al. [8] first reported that fasting plasma ghrelin levels were significantly increased in PIH patients. Based on the fact that ghrelin could decrease blood pressure [9,10], they postulated that a compensatory increase of ghrelin occurred in PIH [8]. In this study, we found a similar phenomenon that SHR had a higher fasting plasma ghrelin level. SBP and DBP were both positively correlated with ghrelin. SBP was also a significantly independent variable of ghrelin levels in multiple regression model analysis. However, Po¨ykko¨ et al. found that fasting ghrelin levels were low in patients with hypertension and were negatively associated with SBP and DBP [12]. Recently, Akamizu et al. also found that fasting plasma acylated ghrelin levels were negatively correlated with SBP in healthy elderly females [1]. Based on the small amount of available data at present, the differences may be due to the different cohorts studied or different pathophysiologic mechanisms between the different types of hypertension such as PIH and essential hypertension. The difference of species between humans and rats may be another reason. Nevertheless, the available data suggests there is a disturbance of circulating ghrelin in hypertension and that ghrelin might play a role in the regulation of blood pressure. As we know, obestatin and ghrelin are encoded by the same gene and derived from the same peptide precursor. It was not surprising that ghrelin levels were positively correlated with obestatin levels and fasting plasma obestatin levels were significantly higher in SHR group compared with WKY group. Moreover, the ratio of ghrelin to obestatin was significantly increased in SHR, which may suggest there was more of a profound disturbance of ghrelin compared with obestatin. In addition, in this study we also found that HP and BRS were negatively correlated with ghrelin and the ratio of ghrelin to obestatin. All this suggests that there are disturbances of both ghrelin and obestatin in the
Fig. 1. Fasting plasma ghrelin levels (A), obestatin levels (B), ghrelin to obestatin ratio (C) in SHR and WKY. **P < 0.01 compared with WKY, *P < 0.05 compared with WKY.
ghrelin levels and the ratio of ghrelin to obestatin (Table 2). SBP and MAP were positively correlated with obestatin levels. However, BRS was negatively correlated with obestatin levels (Table 2). The ghrelin levels were also positively correlated with the obestatin levels (Table 2). Based on the simple correlation coefficients, the relationship between ghrelin, obestatin, ghrelin to obestatin ratio and body weight, insulin, blood pressure, and BRS
Table 2 Correlations between ghrelin, obestatin, ghrelin to obestatin ratio and related parameters. Parameters
Ghrelin r
Weight SBP DBP MAP HP Insulin BRS Ghrelin Obestatin
Obestatin P
0.704 0.816 0.635 0.738 0.546 0.689 0.791 1.000 0.393
<0.0005 <0.0005 <0.0005 <0.0005 0.004 <0.0005 <0.0005 – 0.047
r
Ghrelin to obestatin ratio P
0.160 0.460 0.358 0.413 0.213 0.253 0.453 0.393 1.000
0.435 0.018 0.072 0.036 0.295 0.213 0.020 0.047 –
r
P 0.633 0.743 0.600 0.691 0.429 0.658 0.717 0.937 0.108
0.001 <0.0005 0.001 <0.0005 0.029 <0.0005 <0.0005 <0.0005 0.599
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plasma of SHR and that the ghrelin/obestatin system might play a role in the regulation of blood pressure. To our knowledge, this is the first study reporting that there is a disturbance of obestatin in the circulation of SHR. Recently, Ren et al. reported that the plasma concentration of obestatin was significantly lower in the pregnant women with PIH than non-pregnant women but significantly higher than normal pregnant women [13]. They also found that the plasma obestatin concentrations of the normal pregnant women and the pregnant women with PIH were positively correlated with the mean arterial blood pressure [13]. Anderwald-Stadler et al. found that the fasting plasma obestatin level was negatively correlated with SBP in insulin-resistant humans [2]. All this suggests that obestatin might play a role in the regulation of blood pressure in humans or animals. However, our earlier study showed that bolus intravenous injection of obestatin did not change the blood pressure level in SHR [6]. From these results, we could not determine the role of obestatin in the regulation of blood pressure. Since the results concerning the relationship between obestatin and hypertension are controversial, more detailed studies of circulating obestatin and ghrelin profiles in human or different types of hypertension models to confirm or refute our initial results are eagerly anticipated.
[2]
[3]
[4]
[5]
[6]
[7] [8]
[9] [10]
[11]
Acknowledgements
[12]
Funding: This study was supported by a grant from the National Nature Science Foundation of China (no.: 30700380). Disclosure of interest: The authors have nothing to disclose.
[13] [14] [15]
References [1] Akamizu T, Murayama T, Teramukai S, MuIUra K, Bando I, Irako T, et al. Plasma ghrelin levels in healthy elderly volunteers: the levels of acylated ghrelin in elderly females correlate positively with serum IGF-I levels and bowel move-
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ment frequency and negatively with systolic blood pressure. J Endocrinol 2006;188:333–44. Anderwald-Stadler M, Krebs M, Promintzer M, Mandl M, Bischof MG, Nowotny P, et al. Plasma obestatin is lower at fasting and not suppressed by insulin in insulin-resistant humans. Am J Physiol Endocrinol Metab 2007;293:E1393–8. Fu YJ, Shu H, Miao CY, Wang MW, Su DF. Restoration of baroreflex function by ketanserin is not blood pressure dependent in conscious freely moving rats. J Hypertens 2004;22:1165–72. Guo ZF, Zheng X, Qin YW, Hu JQ, Chen SP, Zhang Z. Circulating preprandial ghrelin to obestatin ratio is increased in human obesity. J Clin Endocrinol Metab 2007;92:1875–80. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kagawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402: 656–60. Li ZF, Song SW, Qin YW, Zhang JL, Zhao XX, Zhang BL, et al. Bolus intravenous injection of obestatin does not change blood pressure level in spontaneously hypertensive rat. Peptides 2009;30:1928–30. Liu AJ, Ma XJ, Shen FM, Liu JG, Chen H, Su DF. Arterial baroreflex: a novel target for preventing stroke in rat hypertension. Stroke 2007;38:1916–23. Makino Y, Hosoda H, Shibata K, Makino I, Kojima M, Kangawa K, et al. Alteration of plasma ghrelin levels associated with the blood pressure in pregnancy. Hypertension 2002;39:781–4. Matsumura K, Tsuchihashi T, Fujii K, Abe I, Iida M. Central ghrelin modulates sympathetic activity in conscious rabbits. Hypertension 2002;40:694–9. Nagaya N, Kojima M, Uematsu M, Yamagishi M, Hosoda H, Oya H, et al. Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol Regul Integr Comp Physiol 2001;280:R1483–7. Norman Jr RA, Coleman TG, Dent AC. Continuous monitoring of arterial pressure indicates sinoaortic denervated rats are not hypertensive. Hypertension 1981;3:119–25. Po¨ykko¨ SM, Kellokoski E, Ho¨rkko¨ S, Kauma H, Kesa¨niemi YA, Ukkola O. Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes 2003;52:2546–53. Ren AJ, He Q, Shi JS, Guo ZF, Zheng X, Lin L, et al. Association of obestatin with blood pressure in the third trimesters of pregnancy. Peptides 2009;30:1742–5. Soares JB, Leite-Moreira AF. Ghrelin, des-acyl ghrelin and obestatin: three pieces of the same puzzle. Peptides 2008;29:1255–70. Xie HH, Miao CY, Liu JG, Su DF. Effects of long-term treatment with candesartan on organ damages in sinoaortic denervated rats. J Cardiovasc Pharmacol 2003;41:325–31. Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, Klein C, et al. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005;310:996–9.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Plasma ghrelin and obestatin levels are increased in spontaneously hypertensive rats Zhao-Feng Li a,b,1, Zhi-Fu Guo a,1, Jiang Cao a, Jian-Qiang Hu a, Xian-Xian Zhao a, Rong-Liang Xu a, Xin-Miao Huang a, Yong-Wen Qin a,*, Xing Zheng a,* a b
Department of Cardiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China Department of Cardiology, No. 88 Hospital of PLA, Tai’an City, Shandong Province 271000, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 September 2009 Received in revised form 10 November 2009 Accepted 16 November 2009 Available online 26 November 2009
Obestatin, encoded by the same gene as ghrelin, was first described as a physiological opponent of ghrelin. We investigated fasting plasma ghrelin and obestatin levels in spontaneously hypertensive rats and Wistar-Kyoto rats. We found that ghrelin levels, obestatin levels and the ratio of ghrelin to obestatin were significantly higher in spontaneously hypertensive rats than Wistar-Kyoto rats. Systolic blood pressure and diastolic blood pressure were positively correlated; however, heart period and baroreflex sensitivity were negatively correlated with ghrelin levels. Systolic blood pressure was positively correlated, whereas baroreflex sensitivity was negatively correlated with obestatin levels. In addition, systolic blood pressure was a significantly independent variable of ghrelin levels, obestatin levels, and the ghrelin to obestatin ratio in a multiple regression analysis. Our data suggests that there is a disturbance of ghrelin and obestatin in the circulation of spontaneously hypertensive rats and the ghrelin/obestatin system might play a role in blood pressure regulation. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Ghrelin Obestatin Blood pressure Spontaneously hypertensive rats
1. Introduction Ghrelin was first identified from rat stomachs, and subsequent studies have shown that ghrelin has a multiplicity of physiological functions such as stimulating appetite, initiating food intake, controlling gastric motility and modulating energy metabolism [5,14]. In addition, there are some researches demonstrating that ghrelin is intimately correlated with blood pressure. Firstly, Nagaya et al. found that the intravenous injection of ghrelin could decrease peripheral artery resistance, leading to a drop in blood pressure without an increase in heart rate (HR) in healthy volunteers [10]. Secondly, Matsumura et al. also found that the intravenous injection of ghrelin could elicit dose-related decreases in arterial pressure and HR, and intracerebroventricular injection of ghrelin could decrease arterial pressure, HR, and renal sympathetic nerve activity in conscious rabbits [9]. Finally, Makino et al. once found that fasting plasma ghrelin concentration was significantly higher in patients with pregnancy-induced hypertension (PIH) than normal pregnant women [8]. These results suggest that ghrelin may play an important role in blood pressure regulation.
In 2005, Zhang et al. first reported that they found a ghrelinassociated peptide, encoded by the same gene as ghrelin, and named it obestatin [16]. An interesting finding was that obestatin, though derived from the same peptide precursor as ghrelin, suppressed food intake, inhibited jejunal contraction, decreased body weight gain, and antagonized the actions of ghrelin when both peptides were co-administered [16]. In our department, Guo et al. showed that obese individuals would display a disturbance of ghrelin and obestatin levels and suggested that this might play a role in the pathophysiological progress of the disease [4]. Recently, Anderwald-Stadler et al. found that the fasting plasma obestatin level was negatively correlated with SBP in insulin-resistant humans, suggesting a possible role of obestatin in the regulation of blood pressure [2]. To the best of our knowledge, plasma obestatin profiles in relation to ghrelin in hypertension have not been studied. Therefore, the aim of this study was to investigate the fasting plasma ghrelin and obestatin levels and their relationship with blood pressure, HR and the baroreflex function in SHR, the animal model of human essential hypertension [11]. 2. Materials and methods
* Corresponding authors at: Department of Cardiology, Changhai Hospital, Second Military Medical University, Changhai Road 168, Shanghai 200433, China. Tel.: +86 21 8187 3202; fax: +86 21 5522 5366. E-mail addresses: [email protected] (Y.-W. Qin), [email protected] (X. Zheng). 1 The first two authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.11.018
2.1. Animals Rats (Slac Laboratory Animal Co. Ltd., Shanghai, China), 14 male SHR (age: 12–13 weeks; body weight: 140–250 g) and 12 male WKY (age: 12–13 weeks; body weight: 200–260 g) were housed in
298
Z.-F. Li et al. / Peptides 31 (2010) 297–300
groups in a temperature-controlled room (23–25 8C) under a 12-h light/12-h dark cycle (8:00 am to 8:00 pm light, 8:00 pm to 8:00 am dark) and maintained on pelleted chow with free access to water. Animal protocols were in compliance with the institutional guidelines. 2.2. Blood pressure and heart period measurements SBP, DBP and HP of rats were continuously recorded using a previously described technique [15]. Briefly, two days before the blood pressure recording, the rats were anesthetized with a combination of ketamine (40 mg/kg, intraperitonea) and diazepam (6 mg/kg, intraperitonea). Two polyethylene catheters (a PE10 (ID 0.3 mm, OD 0.58 mm; Biotrol, France) fused to a PE-50 (ID 0.58 mm, OD 0.96 mm; Biotrol, France)) filled with heparinized 0.9% NaCl (50 U/ml) were inserted into the lower abdominal aorta via the left femoral artery and the left femoral vein for blood pressure recording and intravenous phenylephrine injection, respectively. The two catheters were tunneled subcutaneously under the skin of the back to exit between the scapulae and were plugged with a short piece of stainless steel wire. The rats were then allowed to recover from anesthesia for two days in individual cylindrical cages containing food and water. The two catheters were flushed twice daily with a solution of heparinized NaCl (0.9%). The recording of arterial pressure and the intravenous injection of phenylephrine were performed in unrestrained rats after two days of recovery. The venous catheter was connected to a syringe for saline or phenylephrine injections. The arterial catheter was connected to a pressure transducer through a rotating swivel allowing the rats to move freely in the cage. After 2 h of stabilization, blood pressure signals and HP were recorded on an automatic blood pressure recording and analysis system by a microcomputer. SBP, DBP, MAP and HP values were determined online. A continuity of 1-h recordings was performed immediately after a 2-h period of stabilization. The mean values of these parameters were calculated. 2.3. Determination of baroreflex sensitivity At the end of the continuity of 1-h recordings, the baroreflex sensitivity (BRS) of the rats was conducted in the previously mentioned blood pressure recording condition. BRS was measured in the conscious rat using the previously described method [3,7]. Briefly, a bolus injection of phenylephrine was used to induce blood pressure elevation. The dose of phenylephrine (5–10 mg/kg) was adjusted to raise SBP approximately 30 10 mmHg. There exists a delay (approximately 1 s) between the elevation of blood pressure (stimulus) and the prolongation of heart rate (response) for arterial baroreflex. In rats, the heart rate is approximately 5 or 6 beats per second. So, HP was plotted against SBP for linear regression analysis for 2–8 shifts (calculated by computer); the slope with the largest correlation coefficient (r) of HP/SBP was expressed as BRS (ms/ mmHg). The mean of the two measurements with the proper dosage was taken as the final result. At the end of the determination of BRS, the rats were killed. 2.4. Blood sampling and hormone assay Before the blood pressure recording, blood was drawn from the aortic catheter after a 12-h overnight fast. Blood samples for measurement of ghrelin, obestatin, and insulin were immediately transferred to chilled polypropylene tubes containing EDTA–2Na (1 mg/ml) and aprotinin (500 U/ml), centrifuged at 4 8C, 1600 g for 15 min, and then plasma samples were stored at 80 8C until assayed.
Plasma ghrelin and obestatin levels were measured using commercial RIA kits (Phoenix Pharmaceuticals, Belmont, CA, USA) according to the manufacturer’s instructions. Sensitivity of the obestatin assay was 170.2 pg/ml and sensitivity of the ghrelin assay was 27.7 pg/ml. The intra-assay CV was less than 5%, and the inter-assay CV was less than 10%. Plasma insulin levels were measured using an ELISA kit (Phoenix Biotech Co., Ltd., Beijing, China). The lower and upper detection limits were 5 mIU/ml and 160 mIU/ml, respectively. The intra-assay CV was less than 10%, and the inter-assay CV was less than 15%. 2.5. Statistical analysis Results are expressed as the mean SE. Differences between groups were assessed with the two-side unpaired Student’s t-test. Correlations between ghrelin, obestatin, ghrelin to obestatin ratio and SBP, DBP, MAP, HP, BRS, insulin, and body weight were examined by bivariate correlations (Pearson’s correlation). Multiple regression analysis was further used to assess the relationships between ghrelin, obestatin, ghrelin to obestatin ratio and SBP, DBP, MAP, HP, BRS, insulin, and body weight. A value of P < 0.05 was considered statistically significant. All of the analyses were performed using SPSS for Windows (Version 10.0; SPSS Inc., Chicago, IL). 3. Results 3.1. Animals (Table 1) Body weight is lower in SHR group compared with WKY group (P < 0.01). SBP, DBP and MAP were significantly higher in SHR group compared with WKY group (all P < 0.01). However, HP was significantly lower in SHR group compared with WKY group (P < 0.05). BRS was significantly lower in SHR group compared with WKY group (P < 0.01). Insulin concentration was significantly lower in SHR group compared with WKY group (P < 0.01). 3.2. Differences in ghrelin, obestatin, ghrelin to obestatin ratio (Fig. 1) Both ghrelin and obestatin plasma levels were significantly higher in SHR group compared with WKY group (649.4 55.3 pg/ ml vs 106.3 8.4 pg/ml, P < 0.01; 44.6 2.3 pg/ml vs 38.6 1.5 pg/ ml, P < 0.05 respectively) (Fig. 1A and B). Moreover, the ratios of ghrelin to obestatin in SHR group were significantly higher compared with WKY group (15.1 1.5 vs 2.8 0.2, P < 0.01) (Fig. 1C). 3.3. Correlations of ghrelin, obestatin, and ghrelin to obestatin ratio with various parameters HP, BRS, body weight and insulin levels were negatively correlated with ghrelin levels and the ratio of ghrelin to obestatin (Table 2). SBP, DBP and MAP were positively correlated with Table 1 Characterization of the rats in the study.
Number Age (w) Body weight (g) SBP (mmHg) DBP (mmHg) MAP (mmHg) HP (ms) BRS (ms/mmHg) Insulin (mIU/ml) Data are means SE. * P < 0.01 vs WKY. # P < 0.05 vs WKY.
WKY
SHR
12 12–13 220.5 7.9 115.8 3.3 77.4 2.4 96.6 2.4 171.8 4.4 0.74 0.009 26.6 2.4
14 12–13 175.1 9.4* 145.4 2.0* 91.8 1.6* 115.2 1.5* 153.6 6.8# 0.50 0.02* 11.3 0.8*
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were further assessed in multiple regression analysis. In a multiple regression model including body weight, insulin, SBP, DBP, MAP, HP and BRS, body weight and SBP were independent variables of ghrelin level (standardized coefficient = 0.377; P = 0.004; standardized coefficient = 0.615; P < 0.0005, respectively); and SBP was an independent determinant of the fasting obestatin level (standardized coefficient = 0.460; P = 0.018); SBP and body weight were significantly independent determinants of the ghrelin to obestatin ratio (standardized coefficient = 0.567; P = 0.01; standardized coefficient = 0.332; P = 0.037, respectively). None of these parameters showed any significant correlation with ghrelin, obestatin, and the ratio of ghrelin to obestatin in SHR group or WKY group when considered alone. 4. Discussion This study showed that fasting plasma ghrelin and obestatin levels, and the ratio of ghrelin to obestatin were significantly increased in SHR group compared with WKY group and that SBP was positively correlated with ghrelin, obestatin and ghrelin to obestatin ratio in rats. Makino et al. [8] first reported that fasting plasma ghrelin levels were significantly increased in PIH patients. Based on the fact that ghrelin could decrease blood pressure [9,10], they postulated that a compensatory increase of ghrelin occurred in PIH [8]. In this study, we found a similar phenomenon that SHR had a higher fasting plasma ghrelin level. SBP and DBP were both positively correlated with ghrelin. SBP was also a significantly independent variable of ghrelin levels in multiple regression model analysis. However, Po¨ykko¨ et al. found that fasting ghrelin levels were low in patients with hypertension and were negatively associated with SBP and DBP [12]. Recently, Akamizu et al. also found that fasting plasma acylated ghrelin levels were negatively correlated with SBP in healthy elderly females [1]. Based on the small amount of available data at present, the differences may be due to the different cohorts studied or different pathophysiologic mechanisms between the different types of hypertension such as PIH and essential hypertension. The difference of species between humans and rats may be another reason. Nevertheless, the available data suggests there is a disturbance of circulating ghrelin in hypertension and that ghrelin might play a role in the regulation of blood pressure. As we know, obestatin and ghrelin are encoded by the same gene and derived from the same peptide precursor. It was not surprising that ghrelin levels were positively correlated with obestatin levels and fasting plasma obestatin levels were significantly higher in SHR group compared with WKY group. Moreover, the ratio of ghrelin to obestatin was significantly increased in SHR, which may suggest there was more of a profound disturbance of ghrelin compared with obestatin. In addition, in this study we also found that HP and BRS were negatively correlated with ghrelin and the ratio of ghrelin to obestatin. All this suggests that there are disturbances of both ghrelin and obestatin in the
Fig. 1. Fasting plasma ghrelin levels (A), obestatin levels (B), ghrelin to obestatin ratio (C) in SHR and WKY. **P < 0.01 compared with WKY, *P < 0.05 compared with WKY.
ghrelin levels and the ratio of ghrelin to obestatin (Table 2). SBP and MAP were positively correlated with obestatin levels. However, BRS was negatively correlated with obestatin levels (Table 2). The ghrelin levels were also positively correlated with the obestatin levels (Table 2). Based on the simple correlation coefficients, the relationship between ghrelin, obestatin, ghrelin to obestatin ratio and body weight, insulin, blood pressure, and BRS
Table 2 Correlations between ghrelin, obestatin, ghrelin to obestatin ratio and related parameters. Parameters
Ghrelin r
Weight SBP DBP MAP HP Insulin BRS Ghrelin Obestatin
Obestatin P
0.704 0.816 0.635 0.738 0.546 0.689 0.791 1.000 0.393
<0.0005 <0.0005 <0.0005 <0.0005 0.004 <0.0005 <0.0005 – 0.047
r
Ghrelin to obestatin ratio P
0.160 0.460 0.358 0.413 0.213 0.253 0.453 0.393 1.000
0.435 0.018 0.072 0.036 0.295 0.213 0.020 0.047 –
r
P 0.633 0.743 0.600 0.691 0.429 0.658 0.717 0.937 0.108
0.001 <0.0005 0.001 <0.0005 0.029 <0.0005 <0.0005 <0.0005 0.599
300
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plasma of SHR and that the ghrelin/obestatin system might play a role in the regulation of blood pressure. To our knowledge, this is the first study reporting that there is a disturbance of obestatin in the circulation of SHR. Recently, Ren et al. reported that the plasma concentration of obestatin was significantly lower in the pregnant women with PIH than non-pregnant women but significantly higher than normal pregnant women [13]. They also found that the plasma obestatin concentrations of the normal pregnant women and the pregnant women with PIH were positively correlated with the mean arterial blood pressure [13]. Anderwald-Stadler et al. found that the fasting plasma obestatin level was negatively correlated with SBP in insulin-resistant humans [2]. All this suggests that obestatin might play a role in the regulation of blood pressure in humans or animals. However, our earlier study showed that bolus intravenous injection of obestatin did not change the blood pressure level in SHR [6]. From these results, we could not determine the role of obestatin in the regulation of blood pressure. Since the results concerning the relationship between obestatin and hypertension are controversial, more detailed studies of circulating obestatin and ghrelin profiles in human or different types of hypertension models to confirm or refute our initial results are eagerly anticipated.
[2]
[3]
[4]
[5]
[6]
[7] [8]
[9] [10]
[11]
Acknowledgements
[12]
Funding: This study was supported by a grant from the National Nature Science Foundation of China (no.: 30700380). Disclosure of interest: The authors have nothing to disclose.
[13] [14] [15]
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