Peptides 62 (2014) 159–163

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Effect of endogenous galanin on glucose transporter 4 expression in cardiac muscle of type 2 diabetic rats Penghua Fang b,c , Mingyi Shi b,a , Lili Guo b , Biao He b , Qian Wang c , Mei Yu d , Ping Bo b,a,∗ , Zhenwen Zhang a,b,∗ a

Department of Endocrinology, Clinical Medical College, Yangzhou University, Yangzhou, China Research Institution of Combining Chinese Traditional and Western Medicine, Medical College, Yangzhou University, Yangzhou, China c Department of Physiology, Nanjing University of Chinese Medicine Hanlin College, Taizhou, China d Affiliated Taizhou Hospital, Nanjing University of Chinese Medicine, Taizhou, China b

a r t i c l e

i n f o

Article history: Received 18 November 2013 Received in revised form 1 October 2014 Accepted 1 October 2014 Available online 18 October 2014 Keywords: Galanin M35 GLUT4 Cardiac muscle

a b s t r a c t Although galanin has been shown to increase glucose transporter 4 (GLUT4) expression in skeletal muscle and adipocytes of rats, there is no literature available about the effect of galanin on GLUT4 expression in cardiac muscle of type 2 diabetic rats. In this study, we investigated the relationship between intracerebroventricular administration of M35, a galanin receptor antagonist, and GLUT4 expression in cardiac muscle of type 2 diabetic rats. The rats tested were divided into four groups: rats from healthy and type 2 diabetic drug groups were injected with 2 ␮M M35 for three weeks, while both control groups with 2 ␮l vehicle control. The euglycemic-hyperinsulinemic clamp test was conducted for an index of glucose infusion rates. The cardiac muscle was processed for determination of GLUT4 expression levels. The present study showed that the plasma insulin and retinol binding protein 4 (RBP4) levels were higher in both drug groups than controls respectively. Moreover, the results showed the inhibitive effect of central M35 treatment on glucose infusion rates in the euglycemic-hyperinsulinemic clamp test and GLUT4 expression levels in the cardiac muscle. These results demonstrate that endogenous galanin, acting through its central receptor, has an important attribute to increase GLUT4 expression, leading to enhance insulin sensitivity and glucose uptake in cardiac muscle of type 2 diabetic rats. Galanin and its fragment can play a significant role in regulation of glucose metabolic homeostasis in cardiac muscle and galanin is an important hormone relative to diabetic heart. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Type 2 diabetes mellitus is a worldwide rapidly increasing disease, which especially is accompanied by high morbidity from cardiovascular disease [32,33]. The diabetic heart is closely associated with insulin resistance that causes glucose metabolism disorders [26,29], which result in cardiac ischemia, myocardial infarction and cell death [13,23,28]. Although the precise mechanism, by which diabetes and insulin resistance lead to heart lesions, remains poorly understood, the reduced capacity to utilize glucose could make great contributions to heart lesions [13,28]. Thus, therapeutic interventions focusing on reducing insulin resistance and

∗ Corresponding author at: Research Institution of Combining Chinese Traditional and Western Medicine, Medical College, Yangzhou University, Yangzhou 225001, China. Tel.: +86 0514 87825993; fax: +86 0514 87341733. E-mail addresses: [email protected] (P. Bo), [email protected] (Z. Zhang). http://dx.doi.org/10.1016/j.peptides.2014.10.001 0196-9781/© 2014 Elsevier Inc. All rights reserved.

enhancing myocardial glucose metabolism may greatly improve prevention and treatment of diabetic heart. Glucose transporter 4 (GLUT4) is the most abundant glucose transporter isoform and primarily contributes to insulinstimulated glucose uptake in the cardiac muscle [3,27]. Similar to skeletal muscle, GLUT4 levels in the myocardium were reduced in type 2 diabetic animals and patients [5,6,11,24,28]. The reduction in the GLUT4 levels contributes significantly to elevated insulin resistance and myocardial dysfunction of cardiac muscle [3,27]. Therefore, restoration of GLUT4 levels is very important to enhance glucose metabolism and to assuage myocardial dysfunction in the diabetic heart. Galanin, a 29/30-amino-acid neuropeptide, was isolated in 1983 from porcine intestine by Tatemoto et al. [30]. Galanin distributes widely throughout the central and peripheral nervous system as well as other tissues [17]. The physiological effects of galanin occur through binding to one or more of the three identified receptor subtypes, GALR1-3. All of the subtype receptors are G-proteincoupled receptors and distribute in the hypothalamus, amygdala,

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hippocampus, thalamus, brainstem, spinal cord, dorsal root ganglia, heart, liver, lung, kidney and intestine [17]. Recently, there is increasing evidence focused on the important role of galanin as a critical factor to reduce insulin resistance and to benefit glucose uptake in insulin-independent diabetes [9]. Firstly, galanin may accelerate food intake, body weight and adiposity of subjects [10]. I.c.v. injection of galanin into paraventricular nucleus (PVN) significantly increased daily caloric intake, weight of fat depots, circulating non-esterified fatty acids content and lipoprotein lipase activity in adipose tissue, but reduced circulating glucose levels of animals [34]. Secondly, galanin gene knock out mice have impaired glucose disposal due to reduced insulin response and insulin-independent glucose elimination [2], whereas the homozygous galanin transgenic C57BL/6J mice of the obese phenotype show an increase in metabolic rates of lipid and carbohydrate [25]. Thirdly, galanin secretion in healthy volunteers and diabetic patients is positively correlative with the blood glucose level [7,19,20], which is strongly related to insulin sensitivity. And animals with metabolic disorder of galanin easily suffer from type 2 diabetes mellitus [18]. These results demonstrate that the plasma galanin contents are closely associated with blood glucose levels and insulin sensitivity in humans. Finally, our and other laboratories found that galanin can enhance insulin sensitivity via increasing GLUT4 expression and translocation to the plasma membrane in myocytes and adipocytes of type 2 diabetic animals [4,12,14,16,22,35]. Nevertheless, there are few reports about the effect of galanin on the glucose uptake in the myocardium by now. Accordingly, in this study we used intracerebroventricular (i.c.v.) administration of M35, a galanin receptor antagonist, to evaluate the putative relationship between endogenous galanin and GLUT4 expression levels in cardiac muscle of type 2 diabetic rats. 2. Materials and methods 2.1. Drugs and reagents M35 and streptozotocin were purchased from Sigma Inc. (Sigma, USA). Anti-GLUT4 and Anti-␤-actin were purchased from Santa Cruz Biotechnology Inc., USA. RIPA was purchased from Bioteke Corporation (Beijing, China). BCATM protein assay kit was purchased from Pierce Chemical Company (Pierce, Rockford, USA). Trizol reagent from Gibco Invitrogen Inc. (Gibco Invitrogen, USA). Rat retinol binding protein 4 (RBP4) ELISA kit was purchased from Uscn Life Science, Inc. (Uscn Life Sci., China). Insulin radioimmunoassay kit was from China Institute of Atomic Energy (Beijing, China). 2.2. Type 2 diabetic models Healthy male Wistar rats weighing 150 ± 10 g were employed in this study. Animals were kept in standard polypropylene cage and maintained under standard laboratory conditions of temperature 21 ± 2 ◦ C, relative humidity 50 ± 15%, 12 h light–dark cycles, diet (59% fat, 21% protein and 20% carbohydrate) and water ad libitum. All animals used were closely monitored to ensure that none experienced undue stress or discomfort. Eight weeks later, forty of rats were treated with an i.p. of streptozotocin (35 mg/kg) in 0.1 mM citrate buffer (pH = 4.5) under a fasting state. The tail blood of the rats was weekly taken to determine the blood glucose level with a Glucometer (HMD Biomedical, Taiwan) during the study. After another four weeks, animals with fasting blood glucose concentration over 11.1 mmol/L were taken as models of type 2 diabetes. The thirty two diabetic rats were randomly distributed into diabetic control group (DC, n = 16) and diabetic drug group with M35 (D-M35, n = 16). In addition, thirty two healthy rats were attributed

to healthy control group (HC, n = 16) and healthy drug group with M35 (H-M35, n = 16). All animal procedures used were performed in accordance to the Guiding Principles for Care and Use of Experimental Animals. The experiments were approved by the Animal Studies Committee of Yangzhou University. 2.3. Intracerebroventricular cannulation and injection The method for animal preparation and i.c.v. injection is similar to that described previously [35]. In brief, animals were anesthetized with 3% amobarbital sodium (50 mg/kg i.p.) and stereotaxically implanted with a guide cannula into the lateral ventricle: anterior-posterior (AP), −0.8 mm; L, 1.4 mm; and V, 3.3 mm. The cannula was cemented to four jeweler’s screws attached to the skull and closed with an obturator. Its location was judged by the flow of cerebrospinal fluid. All rats were allowed to recover from surgery for 7 days to minimize nonspecific stress responses. Rats from both drug groups were injected with 2 ␮M in 2 ␮l artificial cerebrospinal fluid (in mM: 133.3 NaCl, 3.4 KCl, 1.3 CaCl2 , 1.2 MgCl2 , 0.6 NaH2 PO4 , 32.0 NaHCO3 , and 3.4 glucose, pH 7.4 by 0.5 M hydrochloric acid) for three weeks, while rats from control group with 2 ␮l vehicle control. The injectors remained in place for an additional 3 min following the injections to assure complete drug delivery. 2.4. Blood sample handling and tissue collection On the next day after the last i.c.v. injection half of rats in every group (n = 8) were anesthetized i.p. by 3% amobarbital sodium (50 mg/kg) dissolved in physiological saline. All animals were used to collected 1 ml of artery blood and 2 g cardiac muscle. The blood was centrifuged at 3500 r.p.m. for 10 min to obtain the plasma. Lastly, the plasma and cardiac muscle were stored at −80 ◦ C. Once the above experiments were completed, each rat was humanely sacrificed under anesthesia, by infusion of amobarbital sodium and saturated potassium chloride. 2.5. Hyperinsulinemic euglycemic clamp tests In the hyperglycemic clamp tests, other half of rats in every group (n = 8) were anesthetized and catheterized in the right carotid artery and left jugular vein after fasted 12 h as previously described [35]. The animals were infused with insulin at a constant rate of 2 mU/kg min into the jugular vein until the end of the test. And 10% glucose was infused at variable rates as needed to clamp glucose levels at 5 ± 0.5 mmol/L. The glucose infusion rates were calculated corresponding to the last 6 samplings at the clamp level. Once above experiments were completed, each rat was euthanized by infusion of amobarbital sodium and saturated potassium chloride. Their brains were checked to confirm the correct implantation of the cannulas. 2.6. Blood RBP4 and insulin assay RBP4 was analyzed by an enzyme-linked immunosorbent assay (Uscn Life Sci., Inc. Wuhan, China). Insulin was analyzed by a radioimmunoassay. According to the manufacturer’s specification, all measurements were performed in duplicate, and the mean of the two measurements was considered. 2.7. RT-PCR analysis To determine the GLUT4 mRNA level, the total RNA from 1 g of the frozen cardiac muscle was isolated by Trizol according to the manufacturer’s instructions. The concentration of the RNA was calculated by spectrophotometric assays of

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260/280 nm, and the integrity of the RNA was assessed by running samples on a 1% formaldehyde agarose gel in TAE buffer (40 mmol/L tris–acetic acid, 1 mmol/L EDTA). cDNA was synthesized from 1 ␮g RNA using MMLV reverse transcriptase. Real-time quantitative PCR was performed for GLUT4 mRNA expression levels using real-time fluorescent detection in an Applied Biosystems 7500 (96 Wells) real-time PCR instrument (ABI 7500, USA). The oligonucleotide primers were as follows: GLUT4 forward 5 -ACAGGGCAAGGATGGTAGA3 , reverse 5 -TGGAGGGGAACAAGAAAGT-3 , ␤-actin 5 -GGCTGTGTTGTCCCTGTATG-3 , reverse 5 forward AATGTCACGCACGATTTCC-3 . Amplification condition was: an initial denaturation at 95 ◦ C for 10 min; 95 ◦ C for 15 s, 62 ◦ C for 60 s, 40 cycles. Results were analyzed with reference to ␤-actin, which was used as an endogenous housekeeping gene. 2.8. Western blot analysis Total membrane proteins of cardiac muscle were extracted using RIPA agents as described previously [21] and quantified with BCA protein assay kit. In brief, 1 g of the cardiac muscle was washed, minced and homogenized in cold TES buffer (20 mM Tris, 1 mM EDTA, 250 mM sucrose, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4, 4 ◦ C). The homogenates were centrifuged at 1200 × g for 10 min (4 ◦ C). The supernatants were centrifuged at 50,000 × g for 30 min (4 ◦ C). And the pellet of this centrifugation was resuspended in TES buffer. Western blot analyses were used to determine the GLUT4 levels in the crude membrane fraction of cardiac muscle. Briefly, 50 ␮g of samples were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) according to the method described before [14]. And then separated proteins were transferred to a polyvinylidene difluoride filter membranes. Membranes were blocked in Tris-buffered saline (pH = 7.5) containing 0.05% Tween-20 (1× TBST) and 5% skimmed milk for 2 h at room temperature and then probed overnight at 4 ◦ C with anti-GLUT4 antibody and anti-␤-actin antibody, respectively. Membranes were washed three times with 1× TBST for 10 min and then incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibody. Finally, immunoreactive bands were visualized by chemiluminescence and quantified by densitometry using a Quantity One analysis software (Bio-Rad). The relative of the GLUT4 concentration in membranes was calculated as the GLUT4/␤-actin × 100%. 2.9. Statistical analysis Data for each respective study were analyzed separately and presented as mean ± SEM. For all experiments, comparisons between the means of multiple groups were analyzed by 2-way ANOVA. Statistical significance was defined as P < 0.05. Statistical analysis of the data was performed using the SPSS statistical software for Windows (Version 10.0).

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Fig. 1. The change of plasma insulin concentration after the i.c.v. injection of M35 in type 2 diabetic rats (n = 8). The plasma insulin concentration was higher in healthy and diabetic groups with M35 compared with each control group respectively. The insulin concentration was higher in diabetic control (DC) compared with healthy control (HC). All data shown are the means ± SEM. *P < 0.05 and **P < 0.01 vs. DC; # P < 0.05 and ## P < 0.01 vs. HC. + P < 0.05 and ++ P < 0.01 vs. H-M35.

insulin and RBP4 contents were enhanced by 27.6% (5.18 ± 0.73 vs. 4.06 ± 0.55, P < 0.05) and 12.3% (3.02 ± 0.41 vs. 2.69 ± 0.63, P < 0.05) in the healthy drug group compared with the healthy control group respectively. Furthermore, the insulin and RBP4 contents were elevated by 24% (7.07 ± 1.18 vs. 5.7 ± 0.65, P < 0.05) and 29.3% (5.91 ± 0.59 vs. 4.57 ± 0.31, P < 0.05) in the diabetic drug group as compared with the diabetic controls respectively. These results suggest that activation of central galanin receptors may inhibit insulin and RBP4 secretion to ameliorate insulin resistance. 3.2. Hyperinsulinemic-euglycemic clamping During the clamp tests, the glucose infusion rates were markedly decreased by i.c.v. injection with M35 (P < 0.0001). Fig. 3 showed that the infusion rates were decreased by 15% (20.07 ± 2.07 vs. 23.61 ± 2.28, P < 0.05) and 23% (13.1 ± 1.22 vs. 17.01 ± 1.35, P < 0.05) in the H-M35 and D-M35 group compared with each control group respectively. In addition, the rate in the diabetic control group was attenuated by 27.9% (17.01 ± 1.35 vs. 23.61 ± 2.28, P < 0.01) compared with the healthy control group. 3.3. The expression of GLUT4 mRNA in membranes of cardiac muscle The central administration of M35 significantly inhibited the GLUT4 mRNA expression level in cardiac muscle of rats (P < 0.0001). As shown in Fig. 4, comparison of H-M35 and D-M35 groups with each control group displayed that the GLUT4 mRNA expression

3. Results 3.1. The changes of blood insulin and RBP4 levels To determine the effect of central M35 on insulin and RBP4 secretion, we contrasted the plasma insulin and RBP4 concentration in the both drug groups with the corresponding control groups. As shown in Figs. 1 and 2, the i.c.v. injection of M35 significantly elevated the insulin (P < 0.0001) and RBP4 (P < 0.0001) concentrations respectively. The insulin and RBP4 contents were enhanced by 40.4% (5.7 ± 0.65 vs. 4.06 ± 0.55, P < 0.01) and 69.9% (4.57 ± 0.31 vs. 2.69 ± 0.63, P < 0.01) in the diabetic control group compared with the healthy control group respectively. Besides, the

Fig. 2. The change of plasma RBP4 concentration after the i.c.v. injection of M35 in type 2 diabetic rats (n = 8). The plasma RBP4 concentration was higher in healthy and diabetic groups with M35 compared with each control group respectively. The RBP4 concentration was higher in diabetic control (DC) compared with healthy control (HC). All data shown are the means ± SEM. *P < 0.05 and **P < 0.01 vs. DC; # P < 0.05 and ## P < 0.01 vs. HC. + P < 0.05 and ++ P < 0.01 vs. H-M35.

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Fig. 3. The i.c.v. administration of M35 significantly reduced glucose infusing rates in hyperinsulinemic-euglycemic clamp tests. The glucose infusing rates were lower in H-M35 and D-M35 groups compared with each control group respectively (n = 8). The glucose infusing rate was lower in diabetic control (DC) compared with healthy control (HC). The data shown are the means ± SEM. *P < 0.05 and **P < 0.01 vs. DC; # P < 0.05 and ## P < 0.01 vs. HC. + P < 0.05 and ++ P < 0.01 vs. H-M35.

levels were reduced by 22.5% (9.45 ± 1.32 vs. 12.19 ± 2.02, P < 0.05) and 35% (3.8 ± 1.92 vs. 5.85 ± 0.81, P < 0.05) respectively. Besides, the GLUT4 mRNA expression level was significantly reduced by 52% (5.85 ± 0.81 vs. 12.19 ± 2.02, P < 0.01) in the diabetic control group compared with the healthy control group. 3.4. GLUT4 protein levels in membranes of cardiac muscle The effect of galanin on GLUT4 expression levels were also monitored by measuring the GLUT4 protein levels in the membrane fraction of the cardiac muscle of rats by immunoblotting. The GLUT4 protein levels in membranes of the cardiac muscle were significantly lower in both drug groups than each control group. Fig. 5 showed that the central administration of M35 significantly reduced GLUT4 protein levels in the membrane fraction of the cardiac muscle of rats (P < 0.0001). Comparison of H-M35 and D-M35 groups with each control group displayed that the GLUT4 protein levels in the membranes were reduced by 17.5% (28.76 ± 2.55 vs. 34.84.±3.89, P < 0.05) and 25% (15.8 ± 2.25 vs. 21.07 ± 2.44, P < 0.05) respectively. In addition, the GLUT4 protein level in membranes of diabetic control group was reduced by 39.5% (21.07 ± 2.44 vs. 34.84 ± 3.89, P < 0.01) compared with the healthy control group. 4. Discussion To date, a number of peptides, including RBP4, have been identified as important biomarkers of developing insulin resistance [8], as they all respond to glucose intake in a dose-dependent manner. The plasma high RBP4 may be taken as the marker of

Fig. 4. The i.c.v. injection of M35 significantly decreased GLUT4 mRNA expression levels in cardiac muscle of type 2 diabetic rats. The GLUT4 mRNA expression levels were lower in H-M35 and D-M35 groups compared with each control group respectively (n = 8). The GLUT4 mRNA expression level was lower in diabetic control (DC) compared with healthy control (HC). The data shown are the means ± SEM. *P < 0.05 and **P < 0.01 vs. DC; # P < 0.05 and ## P < 0.01 vs. HC. + P < 0.05 and ++ P < 0.01 vs. H-M35.

Fig. 5. The i.c.v. injection of M35 significantly decreased GLUT4 protein in cardiac muscle of type 2 diabetic rats. The central M35 treatment reduced GLUT4 immunoreactivity in cell membranes of cardiac muscle in both drug groups compared with each control group respectively (n = 8). The GLUT4 immunoreactivity in cell membranes of cardiac muscle was lower in diabetic control (DC) compared with healthy control (HC). The data shown are the means ± SEM. *P < 0.05 and **P < 0.01 vs. DC; # P < 0.05 and ## P < 0.01 vs. HC. + P < 0.05 and ++ P < 0.01 vs. H-M35.

deteriorating insulin resistance [8]. In this study, we found that the central administration of M35 elevated the plasma RBP4 and insulin levels of diabetic and healthy rats compared with each control group respectively. These results suggesting that central galanin may inhibit RBP4 and insulin secretion to benefit the insulin sensitivity. Furthermore, our study demonstrated that activated galanin in brain may elevate the insulin sensitivity and benefit glucose clearance in the hyperinsulinemic-euglycemic clamp test, which is an important method to directly assess insulin sensitivity of animals [14]. An elevation of the glucose infusion rate in the hyperinsulinemic-euglycemic clamp test indicates a development of insulin sensitivity. Our study demonstrated that the insulin sensitivity in both M35 treatment groups were lower compared with each control group in the clamp tests, suggesting that central treatment of M35 increased insulin resistance, i.e. activated galanin system in brain may elevate the insulin sensitivity and glucose clearance, which is beneficial to improve the function of the diabetic heart by reducing insulin resistance. There is considerable evidence indicating the impaired ability to utilize glucose in cardiac muscle of type 2 diabetes [13,23,28]. Maintenance of cardiac glucose metabolism is important for normal cardiac function. The cardiac muscle is quantitatively the very important target tissues for insulin-stimulated glucose disposal which predominantly depends on GLUT4 [3,15,27]. The GLUT4 expression level is lower in cardiac muscle of animals and patients with type 2 diabetes [5,6,11,27]. Besides, previous studies revealed that in the myocardium of insulin resistant patients with cardiac hypertrophy and ischemic injury the GLUT4 contents were decreased, which represents an obstacle in glucose uptake into the cardiomyocytes [5,6,24,31]. Therefore, it is important that the maintenance of GLUT4 protein concentration contributes to the myocardial energy supply [1]. Here our findings demonstrated that M35, combined with its central galanin receptor, reduced the GLUT4 mRNA expression levels of membranes of cardiac muscle. Moreover, GLUT4 protein contents in the membranes were lower in both drug groups compared with each control group. These results suggested that treatment of M35 inhibited GLUT4 expression, i.e. central endogenous galanin may enhance the GLUT4 expression in membranes of cardiac muscle, which results in benefiting energy metabolism of diabetic heart. Whereas, malfunction of this process can lead to insulin resistance and diabetic heart. The present study indicated that endogenous galanin enhanced GLUT4 expression levels via its central receptors in type 2 diabetic rats. However, the precise signal pathway of galanin induced GLUT4 expression in

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cardiac muscle is unclear. Further investigation for signal pathway is necessary to clarify the role of three galanin subtype receptors in regulation of GLUT4 expression in cardiac muscle of type 2 diabetic rats to unravel the correlation between energy metabolism of diabetic heart and the regulative function of galanin subtype receptors in central nervous system. In conclusion, these results suggest a beneficial effect of endogenous galanin, act as a metabolic regulator via its central receptors, on GLUT4 expression in cardiac muscle of type 2 diabetic rats. These findings contribute to our understanding of the diabetic cardiovascular complications.

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Conflict of interest The authors have no conflicts of interest to disclose. Acknowledgements This work was supported by the Grant of National Natural Scientific Fund of China to Ping Bo (81173392) and in part by the Grant of National Health and Family Planning Commission of China (W201309) to Zhenwen Zhang.

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