The effects of apelin treatment on a rat model of type 2 diabetes

Advances in Medical Sciences 60 (2015) 94–100

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Original Research Article

The effects of apelin treatment on a rat model of type 2 diabetes Raziye Akcılar a,*, Sebahat Turgut b, Vildan Caner c, Aydın Akcılar d, Ceylan Ayada a, ¨ zcan b Levent Elmas c, T. Olgun O a

University of Dumlupınar, Faculty of Medicine, Department of Physiology, Ku¨tahya, Turkey University of Pamukkale, Faculty of Medicine, Department of Physiology, Denizli, Turkey c University of Pamukkale, Faculty of Medicine, Department of Medical Biology, Denizli, Turkey d University of Dumlupınar, Faculty of Medicine, Ku¨tahya, Turkey b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 July 2014 Accepted 25 November 2014 Available online 6 December 2014

Purpose: Apelin is an adipokine that plays a role in the regulation of many biological functions in mammals including the neuroendocrine, cardiovascular, immune systems, glucose homeostasis and obesity. It can act via autocrine, paracrine, endocrine, and exocrine signaling. We aimed to identify the role of apelin pathophysiology of diabetes. Material/methods: 37 male Wistar Albino rats aged 8–10 weeks were divided in four experimental groups as: control group (C) control + apelin group (C + A), diabetic group (D) diabetic + apelin group (D + A). Apelin and apelin receptor mRNA gene expressions in heart and aorta tissue were determined by real-time polymerase chain reaction. The plasma levels of insulin and plasma apelin were determined by ELISA. Results: Plasma levels of insulin, glucose, blood pressure levels were significantly lower in D + A group. There was no statistically significant difference for level of apelin between diabetic groups. On the other hand, differences for apelin and APJ mRNA expression in heart and vascular tissue were found significant between groups. Conclusions: Apelin can be used as a therapeutic agent in the treatment of type II diabetes in the future. ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Keywords: Apelin Apelin receptor (APJ) Type II diabetes Alloxan Glucose metabolism Blood pressure

1. Introduction Type 2 diabetes mellitus (T2DM) is a metabolic disorder which is characterized by hyperglycemia in the context of insulin resistance and relative lack of insulin [1]. Insulin resistance, which is the inability of cells to respond adequately to normal levels of insulin, occurs primarily within the muscles, liver, and fat tissue [2]. In the liver, insulin normally suppresses glucose release. However, in the setting of insulin resistance, the liver inappropriately releases glucose into the blood [3]. T2DM prevalence and incidence is rapidly increasing worldwide. T2DM is a chronic disease leading to macro- and microvascular complications, which results in severe illness and premature death, with elevated personal and economic costs [4]. Apelin is a newly identified adipokine which derives from a 77 amino acids precursor. It has several active forms including apelin-12, apelin-13, apelin-17, apelin-19 and apelin-36. Among them, apelin-13 has the highest abundancy and activity [5]. Apelin * Corresponding author at: University of Dumlupınar, Faculty of Medicine, Department of Physiology, Ku¨tahya, Turkey. Tel.: +90 0507 953 94 74; fax: +90 274 265 22 85. E-mail address: [email protected] (R. Akcılar).

exerts its function by binding and activating the angiotensin receptor related G protein-coupled receptor, APJ [6]. Apelin and APJ are widely expressed in various tissues, including adipose, brain, lung and kidney [7]. Increasing evidence suggests apelin is involved in the regulation of multiple physiological functions, including food intake, blood pressure and glucose utilization [8,9]. Apelin signaling may have an important role in the physiopathology of diseases such as hypertension, heart failure, cardiovascular disease, type 2 diabetes, and obesity, although their effects and functions are still unclear. The physiological effects of apelin on diabetes are not fully known. Apelin and APJ have not been studied in high fat diet alloxan induced type 2 diabetes model before. Therefore, this study aimed to investigate the possible alterations in blood pressure, plasma insulin, blood glucose level and in the renin–angiotensin system in response to apelin in type II diabetic and healthy rats. 2. Materials and methods 2.1. Animals and experimental conditions Thirty-seven Wistar Albino 8–10-week-old male rats (180–300 g) were obtained from the Experimental Research Unit of the University

http://dx.doi.org/10.1016/j.advms.2014.11.001 1896-1126/ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

R. Akcılar et al. / Advances in Medical Sciences 60 (2015) 94–100

of Pamukkale (Denizli, Turkey). They were maintained on a 12/12 h light–dark cycle under controlled temperature and humidity. The animals were fed standard rat chow and given water ad libitum. All protocols used in this study were approved by the Pamukkale University Ethics Committee on animal research (2010/026).

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were recorded, and ‘‘stock concentrations’’ of each sample were calculated. Complementary DNAs (cDNA) were synthesized by QuantiTect Reverse Transcription Kit (Qiagen). Reverse transcription was carried out 42 8C for 15 min followed by incubation at 95 8C for 3 min. cDNAs were stored at 20 8C until used in the realtime polymerase chain reaction (RT-PCR).

2.2. Experimental design The male animals were selected randomly and divided into four experimental groups: Control group (C) (n = 10) received a single dose of saline given intraperitoneally (i.p.) for 17 days. (II) Control + apelin group (C + A) (n = 10) was treated with pyroglutamylated apelin-13 (200 mg kg1 day1 i.p.) for 17 days [10]. (III) Diabetic group (D) (n = 9) was administered with high fat emulsion (10 mL/kg) for 10 days and injected 120 mg/kg alloxan (Sigma–Aldrich, St. Louis, MO, USA) i.p. at day 11 and day 12, respectively [11,12]. At day 17, the blood glucose levels were determined. The animals with blood glucose levels greater than 300 mg/dL were accepted for inclusion as diabetic. (IV) Diabetic + apelin group (D + A) (n = 8) was given i.p. pyroglutamylated apelin-13 (200 mg kg1 day1) to diabetic animals for 17 days [10]. 2.3. Blood glucose and blood pressure measurement Blood glucose and blood pressure levels were measured before starting the study and at the end of the experiment. Blood samples were collected from the tail of each animals after 12 h fasting. The tail was embedded in alcohol and about 1 mm of its end was cut and a drop of blood was used for the blood glucose test with the help of a glucometer (Plusmed Co., Izmir, Turkey). Systolic blood pressures were measured from rats as described previously [13]. 2.4. Blood samples and measurements At the end of the experimental period, all the animals were anesthetized with ketamin/xylazine HCl (75 mg/kg/10 mg/kg intraperitonealy). Blood samples were collected in heparinized tubes. After centrifugation, plasma samples were stored at 80 8C until analysis. The plasma ACE2, angiotension II, angiotensinogen, endothelin-1, apelin and insulin concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) method using an ultra sensitive rat ELISA kit (Diagnostic Product Corporation, Los Angeles, CA, USA) in a multiplate ELISA reader (das, Digital and Analog Systems, Vimercate, MI, Italy). Homeostatic model assessment (HOMA-IR) score was calculated using fasting plasma insulin and fasting blood glucose concentrations measured at the end of the experimental period according to the following formula: HOMA-IR [(Fasting plasma insulin in U/L  fasting blood glucose in mmol/L)/22.5] [14,15].

2.6. Quantitative real-time reverse transcriptase-polymerase chain reaction Relative quantitative analysis of target gene (APJ and apelin) and an internal reference gene (b-actin) was done using the RTPCR system (Light-Cycler 480, Roche, Berlin, Germany). Primers and probes ‘‘Universal Probe Library (UPL) (Roche)’’ were designed for target gene. ‘‘Mouse b-actin Single Assay’’ (Assay ID: 500152, Roche) was used including both primers and original probes for the reference gene (Table 1). Final reaction volume for the analysis of apelin and APJ gene expression was performed in 20 mL volume; 0.5 mL from each primer (final concentration: 0.5 mM), 0.2 mL probe (final concentration: 0.2 mM), 10 mL of LightCycler probes Master Mix (Roche), 4 mL cDNA sample, and 4.8 mL PCR-grade water. The cycling conditions were 95 8C for 10 min, followed by 45 cycles at 95 8C for 10 s, 60 8C for 30 s, and 72 8C for 1 s. At the end of the cycles, a cooling step at 40 8C for 30 s was performed for each reaction. All runs included one negative-templated control consisting of PCR-grade water instead of cDNA. The RT-PCR phases of the samples were completed with optimized protocols, and relative quantitative expression levels of samples were determined. Both target gene and reference gene expression levels were performed with LightCycler Relative Quantitative software (version 3). 2.7. Statistical analysis Statistical analysis was done with SPSS (Statistical Package for Social Sciences, Chicago, IL, USA) 10.0 pocket program. The results were expressed as means  standard error (SE). ‘‘Kruskal–Wallis variance analysis’’ and ‘‘Mann–Whitney U test’’ were used for statistics, with p values 0.05 accepted as statistically significant.

3. Results 3.1. Blood pressure, blood glucose, percentage of body weight and heart weights Significant differences were observed in the levels of percentage of body weight [(final body weight/initial body weight)  100] among the groups, p = 0.000. The level of percentage of body weight in the D, D + A groups were observed to be significantly lower than in C and C + A (p = 0.000, p = 0.001, p = 0.001 and 0.003) (Table 2).

2.5. Isolation of total RNA and synthesis of complementary DNA Heart and aorta tissues of each rat were carefully cleaned of fat and connective tissue and were excised and weighed. The atrium was removed from the heart, and the right and the left ventricles were separated and weighed. Heart and left ventricle weights were normalized to body weight (left ventricle weight/body weight ratio). Total RNA was isolated from fresh heart and aorta tissues using RNeasy kits (RNeasy Mini kit, Cat No. 74104, Qiagen, Germany) according to the manufacturer’s instruction. The quantity and quality of RNA sample were determined with the spectrophotometer method (Biophotometer, Eppendorf, Hamburg, Germany). ‘‘Concentration, A260, A280, A260/280’’ values of the samples

Table 1 Primers and probes used in analysis of expression APJ, apelin and b-actin mRNA (50 !30 ). APJ Primers UPL No. Apelin Primers UPL No.

50 -CACCAAGACCCAGTGCTACAT-30 (forward) 50 -AGGCCCACTCTGAGTTTGAA-30 (reverse) Probe No.: #49 (04 688 104 001) 50 -CTATGTTGACTGGGCCCTTG-30 (forward) 50 -CCACTCTCCTTCCTTTCTTCG-30 (reverse) Probe No.: #58 (04 688 554 001)

b-Actin Single assay

ID: 500 152 (Cat. REF. 05 532 957 001)

R. Akcılar et al. / Advances in Medical Sciences 60 (2015) 94–100

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Table 2 The levels of blood pressure, blood glucose, % body weight and heart weights in control (C), control + apelin (C + A), diabetes mellitus (D), diabetes mellitus + apelin (D + A) groups (mean  SE). Groups

C (n = 10)

C + A (n = 10)

D (n = 9)

D + A (n = 8)

p

% BW (g) HW/BW (mg/g) LVW/BW (mg/g) BP (mm-Hg) Blood glucose (mg/dL)

28.8  1.45a 2.70  0.05ab 2.15  0.84a 122.4  1.40a 114.1  6.96a

27.2  2.04b 3.41  0.20a 1.28  0.09b 115.9  2.16ab 117.3  2.22b

15.1  1.87ab 2.90  0.39 1.01  0.54ab 123.4  0.60bc 436.7  33.7abc

11.9  3.58ab 3.36  0.11b 1.49  0.07 119.3  1.87c 332.0  13.3abc

0.000 0.003 0.002 0.026 0.000

p shows the differences between all groups (Kruskal–Wallis test). In each line, the difference between the means with same letters is significant, p  0.05 (Mann–Whitney U test). % BW: % body weight [(final body weight/initial body weight)  100]; HW/BW: heart weight/body weight; LVW/BW: left ventricular weight/body weight; BP: blood pressure.

C and C + A (p = 0.000 and p = 0.013). High levels of endothelin-1 were found in the C + A (p = 0.028) compared with the C group (Table 3). There were significant differences in plasma apelin levels of rats in all the groups, p = 0.05. Plasma apelin levels were significantly higher in the C + A, D and D + A groups than in C group (p = 0.043, p = 0.024 and p = 0.05) (Table 3). There were significant differences in plasma insulin levels of rats in the all groups (p = 0.019). The low levels of insulin were found in the D + A group compared with the C and D (p = 0.006 and p = 0.015) (Table 3). There were significant differences in HOMA-IR levels of rats in the all groups (p = 0.001). The high levels of HOMA-IR were found in the D group compared with the C, C + A and D + A groups (p = 0.000, p = 0.000 and p = 0.006) (Table 3).

There were significant differences in heart weight/body weight (HW/BW) rate and left ventricular weight (LVW)/BW levels among the groups, p = 0.003 and p = 0.002. The levels of HW/BW were found significantly higher in C + A, and D + A groups than in C, which were p = 0.000. In addition, LVW/BW levels were significantly lower in the D than in C, C + A, and D + A groups (p = 0.001, p = 0.03, and p = 0.000, respectively) (Table 2). At the end of the experiment, blood pressures were observed significantly different among all the groups (p = 0.026). Blood pressures were significantly decreased in the C + A group compared with the C group and in the D + A group compared with the D group (p = 0.04 and p = 0.01) In addition, A higher level of blood pressure was found in the DM group than in the C + A group (p = 0.008) (Table 2). There were significant differences in blood glucose levels of rats in the all groups (p = 0.000). In addition, blood glucose levels were significantly higher in the D, D + A, than in C and C + A groups (p = 0.000). The low levels of blood glucose in the D + A were found compared with the DM (p = 0.05) (Table 2).

3.3. Levels of apelin and APJ gene mRNA expression in the heart and vascular tissue Apelin and APJ gene mRNA expression levels were determined as the relative according to b-actin gene mRNA levels in the cardiac and vascular tissue samples of the groups in this study. The differences in the apelin gene mRNA expression levels in the heart tissue among C (0.05  0.006), C + A (0.04  0.008), D (0.28  0.11) and D + A (0.84  0.26) groups were significant (p = 0.016). In addition, the levels of apelin gene mRNA expression in the D and D + A groups were significantly higher than in C and C + A groups (p  0.05) (Fig. 1). No significant differences were observed in the apelin gene mRNA expression levels in the vascular tissue among C (0.02  0.004), C + A (0.02  0.005), D (0.13  0.05) and D + A (0.16  0.07) groups, p = 0.307 (Fig. 2). In addition, the high levels of apelin gene mRNA expression in the vascular tissue were not significant in the D and D + A group compared with the C and C + A (p = 0.167, p = 0.373, p = 0.146 and p = 0.287, respectively) (Fig. 2). There were significant differences in APJ gene mRNA expression levels in the heart tissue among C (0.09  0.005), C + A (0.06  0.01), D (0.13  0.07) and D + A (0.03  0.01) groups (p = 0.038) (Fig. 3). High levels of APJ gene mRNA expression were not significant in the D

3.2. Levels of plasma ACE2, angiotensinogen, angiotensin-II, endothelin-1, apelin, and insulin The differences in the plasma ACE2 among the all groups were significant (p = 0.015). The level of plasma ACE2 in the D group was observed to be significantly higher than in C and C + A groups (p = 0.02 and p = 0.001) (Table 3). No significant differences were observed in the plasma angiotensin II levels among the all groups, p = 0.749 (Table 3). There were significant differences in plasma angiotensinogen levels among the all groups, p = 0.001. In addition, plasma angiotensinogen levels were significantly lower in the C + A, D and D + A groups than in C group (p = 0.008, p = 0.001 and p = 0.003 respectively). The levels of plasma angiotensinogen in the C + A group compared with the D + A group were found significantly higher (p = 0.01) (Table 3). There were significant differences in plasma endothelin-1 levels of rats in all the groups, p = 0.003. In addition, plasma endothelin-1 levels were significantly higher in the D group than in

Table 3 The levels of plasma ACE2, angiotensin-II, angiotensinogen, endothelin-1, apelin, and insulin in control (C), control + apelin (C + A), diabetes mellitus (DM), diabetes mellitus + apelin (DM + A) groups (mean  SE). Groups ACE2 (IU/mL) Angiotensin-II (ng/mL) Angiotensinogen (ng/mL) Endothelin-1 (ng/mL) Apelin (pg/mL) Insulin (ng/mL) HOMA-IR

C (n = 10)

C + A (n = 10) a

32.1  10.6 3.62  0.67 485.4  60.6a 3.13  1.07ab 0.16  0.02abc 0.60  0.16a 2.81  0.54a

b

27.1  4.61 4.81  1.04 287.8  29.06ab 8.05  1.30ac 0.26  0.04a 0.52  0.12 3.22  0.76b

D (n = 9) ab

77.2  13.3 5.49  1.20 205.7  48.5a 18.2  2.98bc 0.23  0.02b 0.89  0.16b 21.03  4.52abc

p shows the differences between all groups (Kruskal–Wallis test). In each line, the difference between the means with same letters is significant, p  0.05 (Mann–Whitney U test).

D + A (n = 8)

p

48.3  12.5 6.07  2.49 103.1  55.4ab 13.3  6.02 0.20  0.01c 0.25  0.04ab 4.46  0.88c

0.015 0.200 0.000 0.002 0.036 0.029 0.001

R. Akcılar et al. / Advances in Medical Sciences 60 (2015) 94–100

Fig. 1. Apelin mRNA expression in heart tissues of control (C), control + apelin (C + A), diabetes mellitus (D), diabetes mellitus + apelin (D + A) groups. Data are expressed as means  standard error (SE). Statistically significant differences are indicated: *yp  0.05. * shows significance between D and D + A with the C group (Mann–Whitney U test). y shows significance between D and D + A with the C + A group (Mann–Whitney U test).

group compared with the other groups. The APJ gene mRNA expression levels in the C + A and D + A groups were significantly lower than in the C group, which were p = 0.034 and p = 0.007, respectively (Fig. 3). There were no significant differences in APJ gene mRNA expression levels in the vascular tissue among the C (0.05  0.01), C + A (0.06  0.01), D (0.18  0.08) and D + A (0.13  0.12) groups (p = 0.038) (Fig. 4). In addition, the high levels of APJ gene mRNA expression in the vascular tissue were not significant in the D and D + A group compared with the C and C + A (p > 0.05) (Fig. 4). 4. Discussion The significant increase was observed in the proportion of blood glucose, plasma ACE2, endothelin-1 and HOMA-IR scores while percentage of body weight is decreased in alloxan-treated type

Fig. 2. Apelin mRNA expression in vascular tissues of control (C), control + apelin (C + A), diabetes mellitus (D), diabetes mellitus + apelin (D + A) groups. Data are expressed as means  standard error (SE).

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Fig. 3. APJ mRNA expression in heart tissues of control (C), control + apelin (C + A), diabetes mellitus (D), diabetes mellitus + apelin (D + A) groups. Data are expressed as means  standard error (SE). Statistically significant differences are indicated: *p  0.05. * shows significance between C + A and D + A with the C group (Mann– Whitney U test).

2 diabetic rats. These results can be considered as an indicator of forming of type 2 diabetes in rats. In this study, an increase was observed in plasma ACE2 level in the D group compared with the C and C + A groups. In the study performed by Bindom [16], it was shown that ACE2 over expression may ameliorate glucose homeostasis in diabetic mice and prevent the development of pancreatic b-cell dysfunction. Ye et al. [17] demonstrated that ACE2 protein expression increased in renal cortical tubules from the young db/db mice with early diabetes. In addition increased renal ACE2 expression may be protective for the kidneys in the early phases of diabetes [17]. There were not significant increases in the plasma level of ANG II and insulin in the D group compared to the C and C + A groups. Recent studies have shown that ANG II signaling causes not only insulin resistance but also lowers nitric oxide (NO) production via

Fig. 4. APJ mRNA expression in vascular tissues of control (C), control + apelin (C + A), diabetes mellitus (D), diabetes mellitus + apelin (D + A) groups. Data are expressed as means  standard error (SE).

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R. Akcılar et al. / Advances in Medical Sciences 60 (2015) 94–100

activation of NADPH oxidase and ROS production [18] and angiotensin II is known to inhibit the insulin-signaling pathway [19]. In this study, increasing ANG II levels may have prevented the increased insulin in the D group. In this study, an increase was observed in plasma endothelin-1 (ET-1) in the D groups compared with C and C + A groups. Recent experimental reports have demonstrated that increased circulating levels of ET-1, a potent vasoconstrictor peptide, was found in patients with diabetes [20]. One study showed that cell proliferation and ET-1 production increased in type 2 diabetes [21,22]. A study by Mastumoto et al. showed that the expression of both ET-1 and the receptors increased in the streptozotocin-induced diabetes model [23]. Raised ET-1 levels were determined in diabetic patients with retinopathy, diabetic hypertensive patients and patients with microalbuminuria [24]. All these studies show that ET-1 and the receptors play important roles in diabetes-induced complications. In humans, several studies have evaluated the relationship between the serum concentration of apelin and diabetes, although results are still controversial. Li et al. [25] reported that the apelin levels of 2-h post glucose load were found to be significantly increased compared with the fasting apelin levels in normal, impaired glucose tolerance and type 2 diabetic subjects. Fasting and post-load plasma apelin concentrations were higher in subjects with type 2 diabetes and impaired glucose tolerance than in those with normal glucose tolerance [25]. Soriguer et al. [26] showed that apelin levels in morbidly obese patients were significantly higher than in controls only when the obese patients were diabetic. Boucher et al. [27] have demonstrated that increased plasma apelin levels were reported in obesity in association with hyperinsulinemia. In contrast, two reports demonstrated that plasma apelin concentrations were lower in newly diagnosed type 2 diabetes patients than in controls [28,29]. A longitudinal study is needed to clarify whether the difference in plasma apelin precedes the onset of type 2 diabetes. In our study, plasma apelin levels and apelin–APJ mRNA expression in the heart and vascular tissue were increased in the D group compared with the C group. Dray et al. [30] showed that apelin plasma levels were significantly increased in diabetic patients. Apelin and APJ expression were increased in adipose tissue in the insulin-resistant high-fat (HF)-fed mice, compared with control but not in db/db mice. In addition, in skeletal muscle, apelin expression was similar in control and HF-fed mice and decreased in db/db mice. APJ expression was decreased in both HF-fed and db/ db mice [30]. A study has shown that both mRNA and protein levels of apelin receptor were diminished in the aortas from adult spontaneously diabetic db/db mice. Diminished expression of APJ receptor observed in the current study may be, responsible for reduced phosphorylations of Ser473–Akt and Ser1177–eNOS in the aortas from db/db mice [31]. In this study, there was no difference in blood glucose levels in the C + A group compared with the C group. But blood pressure has decreased despite the high level of ET-1 in the C + A [32,33]. In addition, the blood pressure was observed to be not changed despite the high levels of plasma ACE2, ANG II and ET-1 in D group. The reason for the unchanged of blood pressure might be increasing levels of plasma apelin and apelin–APJ mRNA expression in the heart and vascular tissue. Bagi et al. [34], have shown that mice with type 2 diabetes mellitus have elevated systolic blood pressures and increased peripheral vascular resistance [34]. In another study, it was shown that blood pressure did not change in diabetic rats [35]. However, the nature of the mechanisms has not yet been fully elucidated. There has not been any study in the literature between apelin and type 2 diabetes mellitus in blood pressure regulation to compare with the results of this study. Several other studies have demonstrated that apelin

have brought contradictory results in blood pressure regulation. Kagiyama et al. [36] reported that intracerebroventricular injection of apelin-13 increased blood pressure and heart rate while Reaux et al. [37] were not able to observe significant blood pressure changes. Nagano et al. [38] indicated that apelin treatment in mice affects blood pressure in cases where blood pressure that is relatively low under normal conditions becomes elevated under the pathological conditions [38]. In addition, in the study performed by Akcılar et al. [32], the authors found that intraperitoneal administration of apelin reduces blood pressure in DOCA-salt hypertensive rats. In this study and previous studies on blood pressure, the dose and type of administration of apelin differ leading to the differences in the results. In this study, there were significant decreases blood glucose and blood pressure in the D + A group compared with the D group. The low levels of plasma ACE2, angiotensinogen and ET-1 were not statistically significant and ANG II levels did not change in the D + A group. According to these findings, although reduction in these parameters may contribute to the fall of blood glucose and blood pressure in the animals of the D + A group, it cannot be considered as the only contributing factor. These results suggest that apelin can be effective with other pathways in regulating blood glucose and blood pressure. However, apelin could not lower the blood glucose and blood pressure of the animals in the D + A group as well as in the C and C + A groups to the completely normative level. The reason for the reduction of blood glucose and blood pressure might be different ways in D + A group. In this study, the low level of plasma insulin was statistically significant in the D + A group compared with the C and D group. Recently, Dray et al. [39] demonstrated that chronic apelin treatment not only improved insulin sensitivity but also increased insulin-stimulated glucose uptake in soleus muscle [39]. Apelin has been known to inhibit pancreatic insulin secretion [40]. In another study, it was shown that apelin treatment also decreased body adiposity and serum levels of insulin and triglycerides in obese mice fed a high-fat diet without influencing food intake [41]. In a study by Attane´ et al. [42], it was shown that chronic apelin treatment prevented hyperinsulinemia and reduced hyperglycemia in high-fat diet mice [42]. In this study, there were significant increases plasma apelin in the C + A and D + A group compared with the C group. But, there were no significant differences in plasma apelin levels of D + A group compared with the D group. Boucher et al. [27] demonstrated that apelin synthesis is stimulated by insulin. The reason for the unchanged of plasma apelin levels might arise from decreasing the levels of insulin in D + A group. In addition, while apelin mRNA expression increased in the heart tissue of the D + A group compared with the C + A group, a decrease was found in APJ mRNA expression. Apelin and APJ mRNA expression was significantly increased in vascular tissue of D + A group. Another reason for the decrease in blood glucose and blood pressure was increased apelin mRNA expression in cardiac and vascular tissue of rats in this group, leading to increased levels of NO. In this study, NO levels were not considered. In a study by Reaux-Le et al. [43], it was shown that apelin had diuretic effect, and accordingly, caused by the loss of water [43]. Therefore, decreasing blood volume caused a decrease in blood pressure due to diuretic effect of apelin in the D + A group. In this study, the low level of HOMA-IR was statistically significant in the D + A group compared with the D group. Correlations between apelin and insulin resistance, a majo¨r characteristic of obesity and type 2 diabetes, have been demonstrated by several authors. Erdem et al. [28] demonstrated a negative correlation with HOMA-IR in newly diagnosed type 2 diabetes mellitus. Tasci et al. [44] have reported a mild to moderate negative correlation between apelin and HOMA-IR in

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patients with elevated low-density lipoprotein cholesterol (LDL-C). In contrast, Li et al. [25] described a positive correlation with HOMA-IR in patients with impaired glucose tolerance and type 2 diabetic subjects. Hosoya et al. [45] have shown that plasma apelin levels increased markedly in insulin resistance and hyperinsulinemia. In view of these associations, Castan-Laurell et al. [46] suggest that apelin may act as an insulin sensitizing agent and may be a potential target for diabetes treatment, that is, given its potent activity in energy metabolism and ability to improve insulin sensitivity [46]. In this study, high level of plasma ET-1 was statistically significant in the C + A group compared with the C group. However, it seems there is less plasma ET-1 in D + A group than that in D group. The reason for reduction in ET-1 might arise from decreasing the levels of insulin in D + A group. There were increases plasma insulin in the C + A compared with the D + A group. A study by Sliwin´ska-Mosson´ et al. [47] indicated that high insulin concentrations induce the secretion of ET-1 by the endothelial cells of the pancreas [47] In addition, Potenza et al. [21] demonstrated that insulin acutely stimulates ET-1 secretion through MAPK-dependent signaling pathways while up-regulating ET-1 gene expression through PI3K-dependent inactivation of GSK3b in cultured endothelial cells [21]. Therefore, decreasing insulin levels caused a decrease in ET-1 levels in the D + A group compared with the C + A group. Angiotensinogen was observed significantly decreased in the C + A, D and D + A groups compared with the C group. In a study by Gabriely et al. [48] it was shown that there was no change in angiotensinogen gene expression during hyperinsulinemia in the obese rats, which suggests that obesity may be associated with resistance to the effect of insulin to suppress angiotensinogen gene expression. In addition, physiological hyperinsulinemia induced a significant decrease in fat and liver angiotensinogen gene expression in the lean animals [48]. There has not been any study in the literature between apelin and angiotensinogen to compare with the results of this study. The reason for the reduction of angiotensinogen might be two different ways in D and apelin application groups: it may be due to excessive weight loss in D rats. Apelin may lead to inhibiting the production of angiotensinogen directly or increasing ET-1 can cause a decrease in levels of angiotensinogen by reducing the renin secretion. Previous studies have shown that increasing the ET-1 level reduces renin secretion from the kidney [49,50]. In this study, renin levels in rats were not considered. In this study, a significant increase in the proportion of HW/BW in the C + A and D + A groups are seen when compared with the C group. There has not been any study in the literature between the proportion of HW/BW and apelin to compare with the results of this study. The reason for the increase in the proportion of HW/BW may occur from positive inotropic effects of apelin. Chandrasekaran et al. [51] have demonstrated that apelin is a peripheral vasodilator, powerful inotrope [51]. It was observed that percentage of BW was significantly lower in the D and D + A groups compared with the C and C + A groups. The role of apelin and its receptor in the central control of food intake remains a matter of debate. In a study by O’Shea et al. [52], it was demonstrated that intracerebroventricular (icv) apelin-12 injection has been shown to inhibit nocturnal food intake in rats [52]. Similarly, Sunter et al. [53] reported that icv apelin-13 reduced food intake in both fasted and fed rats. More recently, Clarke et al. [54] showed that the icv administration of apelin-13 decreased food and water intake in diet-induced obese (DIO) rats on the control diet. However, another study reported that the icv infusion of apelin-13 for 10 days significantly increased food intake on days 3–7 of infusion in C57BL/6 mice [55], whereas Taheri et al. [56] found that the icv injection of apelin-13 had no significant

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effect on food intake. These observations suggest that apelin may play a role in the central control of food intake. In this study and previous studies on this subject, the dose and type of administration of apelin differ leading to the differences in the results. As a result, in high fat diet alloxan induced type 2 diabetic rats, plasma apelin levels and apelin–APJ mRNA expression levels are increased. These findings suggest that apelin could have a strong anti-type 2 diabetic activity and also ameliorate other diabetesrelated complications. Apelin may act as an insulin sensitizing agent and may be a potential target for diabetes treatment, that is, given its potent activity in energy metabolism and ability to improve insulin sensitivity. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Financial disclosure This study was supported by Pamukkale University Research Fund (Project No.: 2011 SBE001). References [1] Kumar V, Fausto N, Abbas AK, Cotran RS, Robbins SL. Robbins and Cotran pathologic basis of disease. 7th ed. Philadelphia, PA: Saunders; 2005. p. 1194– 5, ISBN 0-7216-0187-1. [2] Diabetes mellitus a guide to patient care. Philadelphia: Lippincott Williams & Wilkins; 2007. 15, ISBN 978-1-58255-732-8. [3] Williams textbook of endocrinology. 12th ed. Philadelphia: Elsevier/Saunders; 2011. p. 1371–435, ISBN 978-1-4377-0324-5. [4] Stumvoll M, Goldstein BJ, van Haeften TW. Type 2 diabetes: principles of pathogenesis and therapy. Lancet 2005;365:1333–46. [5] Ladeiras-Lopes R, Ferreira-Martins J, Leite-Moreira AF. The apelinergic system: the role played in human physiology and pathology and potential therapeutic applications. Arq Bras Cardiol 2008;90:343–9. [6] Habata Y, Fujii R, Hosoya M, Fukusumi S, Kawamata Y, Hinuma S, et al. Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum. Biochim Biophys Acta 1999;1452:25–35. [7] Medhurst AD, Jennings CA, Robbins MJ, Davis RP, Ellis C, Winborn KY, et al. Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin. J Neurochem 2003;84:1162–72. [8] Reaux A, De Mota N, Skultetyova I, Lenkei Z, El Messari S, Gallatz K, et al. Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain. J Neurochem 2001;77:1085–96. [9] Reaux A, Gallatz K, Palkovits M, Llorens-Cortes C. Distribution of apelin synthesizing neurons in the adult rat brain. Neuroscience 2002;113:653–62. [10] Falcao-Pires I, Goncalves N, Henriques-Coelho T, Moreira-Goncalves D, Roncon Albuquerque R, Leite-Moreira AF. Apelin decreases myocardial injury and improves right ventricular function in monocrotaline-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 2009;296:2007–14. [11] Ai J, Wang N, Du J, Yang M, Liu P, Du Z, et al. Establishment of type 2 diabetic animal model in Wistar rats. Chin Pharmacol Bull 2004;20:1309–12. [12] Ai J, Wang N, Yang M, Du ZM, Zhang YC, Yang BF. Development of Wistar rat model of insulin resistance. World J Gastroenterol 2005;11:3675–9. [13] Erken HA, Erken G, Genc¸ O. Blood pressure measurement in freely moving rats by the tail cuff method. Clin Exp Hypertens 2013;35:1–5. http://dx.doi.org/ 10.3109/10641963.2012.685534. [14] Ibrahim MA, Islam MS. Anti-diabetic effects of the acetone fraction of Senna singueana stem bark in a type 2 diabetes rat model. J Ethnopharmacol 2014;153(2):392–9. [15] Roma´n CL, Flores LE, Maiztegui B, Raschia MA, Del Zotto H, Gagliardino JJ. Islet NADPH oxidase activity modulates b-cell mass and endocrine function in rats with fructose-induced oxidative stress. Biochim Biophys Acta 2014;1840(12): 3475–82. http://dx.doi.org/10.1016/j.bbagen.2014.09.011. [16] Bindom SM, Hans CP, Xia H, Boulares AH, Lazartigues E. Angiotensin I-converting enzyme type 2 (ACE2) gene therapy improves glycemic control in diabetic mice. Diabetes 2010;59(10):2540–8. [17] Ye M, Wysocki J, Naaz P, Salabat MR, LaPointe MS, Batlle D. Increased ACE 2 and decreased ACE protein in renal tubules from diabetic mice: a renoprotective combination? Hypertension 2004;43:1120–5. [18] Huisamen B, Peˆrel SJ, Friedrich SO, Salie R, Strijdom H, Lochner A. ANG II type I receptor antagonism improved nitric oxide production and enhanced eNOS and PKB/Akt expression in hearts from a rat model of insulin resistance. Mol Cell Biochem 2011;349(12):21–31. [19] Rakugi H, Ogihara T. Role of angiotensin in the metabolic syndrome and cardiovascular complications. Nihon Rinsho 2002;60(10):1898–903.

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