ME TAB O L IS M CL I N ICA L A N D EX P ER IM EN T AL 6 3 ( 2 0 14 ) 14 9 9 –1 50 2
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
Brief Report
Acute growth hormone administration increases myoglobin expression and Glut4 translocation in rat cardiac muscle cells Rogério Antônio Laurato Sertié a,⁎, Andréa Laurato Sertié b , Gisele Giannocco c , Leonice Lourenço Poyares d , Maria Tereza Nunes d a
Laboratório de Fisiologia do Tecido Adiposo, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil Centro de Pesquisa Experimental, Hospital Israelita Albert Einstein, São Paulo, SP, Brazil c Faculdade de Medicina do ABC, Santo André, SP, Brazil d Laboratório de Regulação Hormonal e Expressão Gênica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil b
A R T I C LE I N FO Article history:
AB S T R A C T Objective. Oxygen (O2) and glucose are important energy sources for the heart. This study
Received 26 May 2014
sought to investigate the effects of acute growth hormone (GH) administration on the
Accepted 21 August 2014
expression of myoglobin (Mb) and Glut4 glucose transporter, two important limiting factors for O2 and glucose utilization for energy production, in cardiac muscle cells of treated rats.
Keywords:
Methods. Male Wistar rats were sacrificed at 30, 45, 90 and 120 min after a single dose of
Acute growth hormone
intraperitoneal (ip) rat GH (1.5 mg/kg) or vehicle administration, and total RNA and protein
Cardiovascular function
(from whole cell or subcellular fractions) were extracted from cardiomyocytes (left
Myoglobin
ventricles) of these animals.
Glucose transporter Glut4
Results. Acute GH injection led to a significant increase in both Mb mRNA and protein levels, and stimulated Glut4 protein translocation to the plasma membrane of cardiac cells. Conclusions. These results suggest that GH exerts some of its effects on cardiomyocytes shortly after the first administration inducing the expression of proteins potentially involved in cardiac performance. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
The heart is a target organ for the growth hormone (GH). There is ample evidence that the GH/insulin-like growth factor 1 (IGF-1) axis exert major effects on the cardiovascular system [1]. Animal and human studies have shown that both short- and long-term GH administrations improve myocardial performance in heart failure [2–5]. Moreover, even acute GH
injection has also been demonstrated to exert beneficial effects on cardiovascular functions in patients with cardiac insufficiency [6,7]. It has been suggested that the GH/IGF-1 axis regulates cardiac growth, stimulates myocardial contractility and influences the vascular system [1]. However, the exact cellular and molecular basis of GH actions on the heart, mainly it acute actions, are still poorly explored and have not been fully elucidated.
Abbreviations: GH, growth hormone; Mb, myoglobin; Glut4, glucose transporter 4; O2, oxygen; IGF-1, insulin-like growth factor 1; M, microsomal fraction; PM, plasma membrane fraction. ⁎ Corresponding author. Tel.: +55 11 30917248. E-mail address: [email protected] (R.A.L. Sertié). http://dx.doi.org/10.1016/j.metabol.2014.08.012 0026-0495/© 2014 Elsevier Inc. All rights reserved.
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ME TAB O L IS M CL I N ICA L A N D EX PE R IM EN T AL 6 3 ( 2 0 14 ) 14 9 9 –15 0 2
The heart's pumping efficiency depends, among other factors, on a constant supply of oxygen (O2) and nutrients, of which glucose is an important source of energy for the heart [8]. Therefore, we hypothesized that the acute GH administration improves cardiac performance through, at least in part, rapid stimulation of O2 and glucose utilization as substrates for energy metabolism by the heart. To begin to test this hypothesis, in the present study we aimed to verify the effects of acute GH injection on myoglobin (Mb) and glucose transporter Glut4 gene and protein expression, two proteins highly expressed in cardiac muscle which are important limiting factors for O2 and glucose utilization, in cardiomyocytes of treated rats.
2.
Methods
2.1.
Animals and treatments
All procedures were approved by Institute of Biomedical Sciences – University of São Paulo (ICB-USP) Animal Ethics Committees. Male Wistar rats (45 days old, 200 g body weight) from the ICB-USP Animal Resources Center were maintained on ad libitum chow and tap water. Rooms were kept at a constant temperature (23 ± 1 °C) under 12 h light/12 h dark lighting conditions (lights on at 0700 h). Rats were acutely treated with rat GH (National Hormone & Pituitary Program-NHPP-NIDDK, USA) (1.5 mg/kg b.w. ip) or saline (0.9% NaCl) and were sacrificed by decapitation at 30, 45, 90 and 120 min after the treatment (n = 5 per group/per time). The hearts were removed and the left ventricle was separated for mRNA and protein extraction.
2.2.
Procedures
2.2.1.
Northern blotting
Total RNA from left ventricles was isolated using acid guanidinium thiocyanate–phenol–chloroform extraction method. Ten micrograms of total RNA was used for the analyses. Blotted membranes were hybridized with 32P-labeled rat Mb or Glut4 cDNA probes for 16 h at 42 °C. Membranes were washed under high stringency conditions and subjected to autoradiography. All blots were stripped and re-hybridized with a 32Plabeled RNA probe specific for 18S ribosomal RNA. Band intensities were quantified by phosphor imaging using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Mb and Glut4 mRNA levels were normalized to 18S rRNA and presented as percentage of saline control. Values represent mean ± SEM from 5 rats per group for each time point.
2.2.2.
Western blotting
Left ventricle samples of treated animals were homogenized in a buffer containing 10 mmol/L TRIS, 1 mmol/L EDTA and 250 mmol/L sucrose. Plasma membrane (PM) and light microsomes (M) fractions were obtained by differential centrifugation as described previously [9]. Equal amounts of proteins (30 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes by Western blotting. Membranes were then blocked and incubated with the following primary antibodies: anti-Glut4 (1/1000, Chemicon, Temecula, CA) and anti-myoglobin (1/1000; Sigma). The blots were subsequently incubated with 2 μCi of 125I-protein A (30 μCi/μg) (Amersham
pharmacia) in blocking buffer at room temperature for 3 h and then washed again for 2 h. Band intensities were detected as described above. Mb and Glut4 protein levels were presented as percentage of saline control. Data represent mean ± SEM from 5 rats per group for each time point.
3.
Statistics
All statistical analyses were carried out using GraphPad Prism version 4 for Windows. The effects of treatment on expression of genes or proteins were determined using one-way ANOVA test followed by Bonferroni's test for multiple comparisons. Significant differences were accepted when p < 0.05.
4.
Results
We first investigated the acute effects (30, 45, 90 and 120 min) of a single-dose of exogenous rat GH administration (1.5 mg/ kg b.w. ip) on Mb gene and protein expression in solubilized cardiac muscle cells (left ventricle) extracts of treated rats. We observed a significant increase in Mb mRNA levels at 90 min after hormone injection, followed by a significant increase in Mb protein levels at 120 min after injection when compared to the control group (saline-treated animals) (Fig. 1). These results suggest that Mb is a GH target gene and its expression is stimulated shortly after GH injection. We next examined Glut4 mRNA levels and Glut4 protein translocation in cardiac muscle (left ventricle) of rats after acute administration of GH. While no significant difference in Glut4 mRNA levels was observed, Glut4 protein content in microsomal (M) fraction was significantly reduced at 30 and 45 min after GH administration followed by a significant increase in plasma membrane (PM) fraction at 120 min after GH administration when compared to control (saline only) (Fig. 2). These results suggest that GH promotes Glut4 translocation to the plasma membrane of cardiac muscle cells soon after its first administration.
5.
Discussion
Here, we showed that acute GH administration stimulates Mb mRNA and protein levels in cardiac muscle cells of treated rats. Previous reports have shown that GH controls gene expression in target tissues such as adipose tissue [10], liver [11] and skeletal muscle [12]. However, to our knowledge, this is the first study describing the effects of acute GH treatment on Mb levels in the heart. Mb stores O2 in the muscle cell and transports it from the sarcolemma to the mitochondria, favoring oxidative phosphorylation in response to muscle energy demands [13]. Therefore, it is possible that the rapid increase in O2 availability to cardiomyocytes may be a contributing factor to improve heart performance after acute GH injection. Even if further experiments are required to confirm this hypothesis, our results are encouraging and should stimulate more research along similar lines. Our current results also suggest that whereas acute GH administration has no effects on Glut4 mRNA expression, it stimulates cardiac muscle cells to translocate Glut4 protein to
ME TAB O L IS M CL I N ICA L A N D EX P ER IM EN T AL 6 3 ( 2 0 14 ) 14 9 9 –1 50 2
1501
Fig. 1 – Effects of acute GH administration on Mb expression in cardiac muscle cells (left ventricles) of rats. (A) Representative northern blotting analysis of Mb mRNA levels. Band intensities were densitometrically evaluated and the data were normalized to 18S rRNA levels and are expressed as the mean percentage ± SEM relative to the saline-treated control; n = 5 rats per group per time point. It was observed that after 90 min of GH administration there was a significant increase in Mb expression compared to control (*p < 0.05). (B) Representative immunoblot analysis of Mb protein levels. Band intensities were evaluated by densitometric analysis and the data are expressed as the mean percentage ± SEM of saline control; n = 5 rats per group per time point. It was observed that at 120 min after GH injection there was a significant increase in Mb protein expression compared to control (⁎p < 0.05).
their surfaces. Glut4 is the most abundant and the only insulinregulated glucose transporter and its translocation to the plasma membrane represents an important mechanism by which the rate of glucose entry into cardiomyocytes is regulated. Previous reports suggest that while prolonged GH treatment may exert anti-insulin effects and impair glucose uptake and metabolism [14,15], short-term GH administration mimics the effects of insulin as it decreases blood glucose levels, increases Glut4 translocation in ovary cells that overexpress GH receptor and Glut4 [16], and stimulates glucose uptake
and lipogenesis in adipocytes [11,17]. Our current results are in accordance with these studies and show that acute GH injection may also cause insulin-like effects in cardiac muscle cells. The insulin-like effects of GH have been attributed to the fact that GH and insulin act through common intracellular signal transduction pathways, the MAPK and the PI3K/AKT pathways, which control Glut4 translocation and glucose uptake by the cells [15,16]. It is well established that cardiac metabolism can be altered in several cardiovascular disorders and optimizing cardiac
Fig. 2 – Effects of acute GH administration on Glut4 expression/translocation in cardiac muscle cells (left ventricles) of rats. (A) Representative northern blotting analysis of Glut4 mRNA levels. Band intensities were densitometrically evaluated and the data were normalized to 18S rRNA and are presented as the mean percentage ± SEM relative to the saline-treated control; n = 5 rats per group per time point. No significant differences were observed when compared to the control group. (B) Representative immunoblot analysis of Glut4 protein levels in sub-cellular fractions of cardiomyocytes. Band intensities were evaluated by densitometric analysis and Glut4 protein levels are presented as the mean percentage ± SEM relative to the control group (saline only); n = 5 rats per group per time point. Significant decrease of Glut4 protein was observed in the microsomal (M) fraction after 30 and 45 min of GH administration, followed by a significant increase in the amount of Glut4 in the plasma membrane (PM) fraction at 120 min compared to control (*p < 0.05).
1502
ME TAB O L IS M CL I N ICA L A N D EX PE R IM EN T AL 6 3 ( 2 0 14 ) 14 9 9 –15 0 2
energy metabolism has been considered an attractive approach to treating such disorders [18,19]. The single dose of GH used in the present study was based on previous studies showing the cardiobeneficial effects of short-term GH administration (1– 3 mg/kg/day × 1–4 weeks) in rats with experimental myocardial infarction [5]. Although our study design did not include direct monitoring of changes in cardiac function following acute GH injection, the results obtained here suggest that some of the effects of GH on cardiomyocytes initiate shortly after its injection stimulating the expression of proteins potentially involved in energy metabolism and cardiac performance.
6.
Conclusion
In conclusion, our data suggest that GH stimulates the expression of Mb and the translocation of Glut4 to the plasma membrane of adult rat cardiomyocytes shortly after being injected. Further studies exploring acute GH use in cardiac failure therapy should be persuaded.
Author contributions RALS: design and conduct of the study, data analysis and manuscript preparation. ALS: data interpretation and manuscript writing. GG and LLP: assisted with experiment execution. MTN: coordination of research.
Funding This work was supported by grants from CNPq (National Counsel of Technological and Scientific Development), FAPESP (São Paulo Research Foundation), and CAPES (Higher Education Co-Ordination Agency).
Acknowledgments We thank the Institute of Biomedical Sciences for its vital support of this project. We thank Dr Ubiratan Fabre Machado and Dr Maristela Mitiko Okamoto for their assistance in carrying out the study of glucose transporters.
Conflict of interest The authors declare no conflict interest.
REFERENCES
[1] Castellano G, Affuso F, Conza PD, Fazio S. The GH/IGF-1 axis and heart failure. Curr Cardiol Rev 2009;5(3):203–15.
[2] Abdu TA, Neary R, Elhadd TA, Akber M, Clayton RN. Coronary risk in growth hormone deficient hypopituitary adults: increased predicted risk is due largely to lipid profile abnormalities. Clin Endocrinol (Oxf) 2001;55(2):209–16. [3] Colao A, Marzullo P, Di Somma C, Lombardi G. Growth hormone and the heart. Clin Endocrinol (Oxf) 2001;54(2): 137–54. [4] Le Corvoisier P, Hittinger L, Chanson P, Montagne O, Macquin-Mavier I, Maison P. Cardiac effects of growth hormone treatment in chronic heart failure: a meta-analysis. J Clin Endocrinol Metab 2007;92(1):180–5. [5] Marleau S, Mulumba M, Lamontagne D, Ong H. Cardiac and peripheral actions of growth hormone and its releasing peptides: relevance for the treatment of cardiomyopathies. Cardiovasc Res 2006;69(1):26–35 [Epub 2005 Oct 10. Review]. [6] Volterrani M, Desenzani P, Lorusso R, d'Aloia A, Manelli F, Giustina A. Haemodynamic effects of intravenous growth hormone in congestive heart failure. Lancet 1997;12:1067–8. [7] Giustina A, Volterrani M, Manelli F, Desenzani P, Poiesi C, Lorusso R, et al. Endocrine predictors of acute hemodynamic effects of growth hormone in congestive heart failure. Am Heart J 1999;137(6):1035–43. [8] Lopaschuk GD. Targets for modulation of fatty acid oxidation in the heart. Curr Opin Investig Drugs 2004;5:290–4. [9] Yonemitsu S, Nishimura H, Shintani M, Inoue R, Yamamoto Y, Nakao K. Troglitazone induces GLTU4 translocation in L6 myotubes. Diabetes 2001;50:1093–101. [10] Houseknecht KL, Portocarrero CP, Ji S, Lemenager R, Spurlock ME. Growth hormone regulates leptin gene expression in bovine adipose tissue: correlation with adipose IGF-1 expression. J Endocrinol 2000;164(1):51–7. [11] Tanner JW, Leingang KA, Mueckler MM, Glenn KC. Cellular mechanism of the insulin-like effect of growth hormone in adipocytes. Rapid translocation of the HepG2-type and adipocyte/muscle glucose transporters. Biochem J 1992;15 (282):99–106. [12] Clasen BF, Krusenstjerna-Hafstrøm T, Vendelbo MH, Thorsen K, Escande C, Møller N, et al. Gene expression in skeletal muscle after an acute intravenous GH bolus in human subjects: identification of a mechanism regulating ANGPTL4. J Lipid Res 2013;54(7):1988–97. [13] Giordano FJ. Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest 2005;115(3):500–8. [14] Christiansen JS, Jørgensen JO, Pedersen SA, Müller J, Jørgensen J, Møller J, et al. GH-replacement therapy in adults. Horm Res 1991;36(Suppl 1):66–72. [15] Miquet JG, Giani JF, Martinez CS, Muñoz MC, González L, Sotelo AI, et al. Prolonged exposure to GH impairs insulin signaling in the heart. J Mol Endocrinol 2011;47(2):167–77. [16] Yokota I, Hayashi H, Matsuda J, Saijo T, Naito E, Ito M, et al. Effect of growth hormone on the translocation of GLUT4 and its relation to insulin-like and anti-insulin action. Biochim Biophys Acta 1998;1404(3):451–6. [17] Yakar S, Liu JL, LeRoith D. The growth hormone/insulin-like growth factor-I system: implications for organ growth and development. Pediatr Nephrol 2000;14(7):544–9. [18] Nagoshi T, Yoshimura M, Rosano GM, Lopaschuk GD, Mochizuki S. Optimization of cardiac metabolism in heart failure. Curr Pharm Des 2011;17(35):3846–53 [Review]. [19] Wang KC, Lim CH, McMillen IC, Duffield JA, Brooks DA, Morrison JL. Metabolism. Alteration of cardiac glucose metabolism in association to low birth weight: experimental evidence in lambs with left ventricular hypertrophy. Metabolism 2013;62(11):1662–72.
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
Brief Report
Acute growth hormone administration increases myoglobin expression and Glut4 translocation in rat cardiac muscle cells Rogério Antônio Laurato Sertié a,⁎, Andréa Laurato Sertié b , Gisele Giannocco c , Leonice Lourenço Poyares d , Maria Tereza Nunes d a
Laboratório de Fisiologia do Tecido Adiposo, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil Centro de Pesquisa Experimental, Hospital Israelita Albert Einstein, São Paulo, SP, Brazil c Faculdade de Medicina do ABC, Santo André, SP, Brazil d Laboratório de Regulação Hormonal e Expressão Gênica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil b
A R T I C LE I N FO Article history:
AB S T R A C T Objective. Oxygen (O2) and glucose are important energy sources for the heart. This study
Received 26 May 2014
sought to investigate the effects of acute growth hormone (GH) administration on the
Accepted 21 August 2014
expression of myoglobin (Mb) and Glut4 glucose transporter, two important limiting factors for O2 and glucose utilization for energy production, in cardiac muscle cells of treated rats.
Keywords:
Methods. Male Wistar rats were sacrificed at 30, 45, 90 and 120 min after a single dose of
Acute growth hormone
intraperitoneal (ip) rat GH (1.5 mg/kg) or vehicle administration, and total RNA and protein
Cardiovascular function
(from whole cell or subcellular fractions) were extracted from cardiomyocytes (left
Myoglobin
ventricles) of these animals.
Glucose transporter Glut4
Results. Acute GH injection led to a significant increase in both Mb mRNA and protein levels, and stimulated Glut4 protein translocation to the plasma membrane of cardiac cells. Conclusions. These results suggest that GH exerts some of its effects on cardiomyocytes shortly after the first administration inducing the expression of proteins potentially involved in cardiac performance. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
The heart is a target organ for the growth hormone (GH). There is ample evidence that the GH/insulin-like growth factor 1 (IGF-1) axis exert major effects on the cardiovascular system [1]. Animal and human studies have shown that both short- and long-term GH administrations improve myocardial performance in heart failure [2–5]. Moreover, even acute GH
injection has also been demonstrated to exert beneficial effects on cardiovascular functions in patients with cardiac insufficiency [6,7]. It has been suggested that the GH/IGF-1 axis regulates cardiac growth, stimulates myocardial contractility and influences the vascular system [1]. However, the exact cellular and molecular basis of GH actions on the heart, mainly it acute actions, are still poorly explored and have not been fully elucidated.
Abbreviations: GH, growth hormone; Mb, myoglobin; Glut4, glucose transporter 4; O2, oxygen; IGF-1, insulin-like growth factor 1; M, microsomal fraction; PM, plasma membrane fraction. ⁎ Corresponding author. Tel.: +55 11 30917248. E-mail address: [email protected] (R.A.L. Sertié). http://dx.doi.org/10.1016/j.metabol.2014.08.012 0026-0495/© 2014 Elsevier Inc. All rights reserved.
1500
ME TAB O L IS M CL I N ICA L A N D EX PE R IM EN T AL 6 3 ( 2 0 14 ) 14 9 9 –15 0 2
The heart's pumping efficiency depends, among other factors, on a constant supply of oxygen (O2) and nutrients, of which glucose is an important source of energy for the heart [8]. Therefore, we hypothesized that the acute GH administration improves cardiac performance through, at least in part, rapid stimulation of O2 and glucose utilization as substrates for energy metabolism by the heart. To begin to test this hypothesis, in the present study we aimed to verify the effects of acute GH injection on myoglobin (Mb) and glucose transporter Glut4 gene and protein expression, two proteins highly expressed in cardiac muscle which are important limiting factors for O2 and glucose utilization, in cardiomyocytes of treated rats.
2.
Methods
2.1.
Animals and treatments
All procedures were approved by Institute of Biomedical Sciences – University of São Paulo (ICB-USP) Animal Ethics Committees. Male Wistar rats (45 days old, 200 g body weight) from the ICB-USP Animal Resources Center were maintained on ad libitum chow and tap water. Rooms were kept at a constant temperature (23 ± 1 °C) under 12 h light/12 h dark lighting conditions (lights on at 0700 h). Rats were acutely treated with rat GH (National Hormone & Pituitary Program-NHPP-NIDDK, USA) (1.5 mg/kg b.w. ip) or saline (0.9% NaCl) and were sacrificed by decapitation at 30, 45, 90 and 120 min after the treatment (n = 5 per group/per time). The hearts were removed and the left ventricle was separated for mRNA and protein extraction.
2.2.
Procedures
2.2.1.
Northern blotting
Total RNA from left ventricles was isolated using acid guanidinium thiocyanate–phenol–chloroform extraction method. Ten micrograms of total RNA was used for the analyses. Blotted membranes were hybridized with 32P-labeled rat Mb or Glut4 cDNA probes for 16 h at 42 °C. Membranes were washed under high stringency conditions and subjected to autoradiography. All blots were stripped and re-hybridized with a 32Plabeled RNA probe specific for 18S ribosomal RNA. Band intensities were quantified by phosphor imaging using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Mb and Glut4 mRNA levels were normalized to 18S rRNA and presented as percentage of saline control. Values represent mean ± SEM from 5 rats per group for each time point.
2.2.2.
Western blotting
Left ventricle samples of treated animals were homogenized in a buffer containing 10 mmol/L TRIS, 1 mmol/L EDTA and 250 mmol/L sucrose. Plasma membrane (PM) and light microsomes (M) fractions were obtained by differential centrifugation as described previously [9]. Equal amounts of proteins (30 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes by Western blotting. Membranes were then blocked and incubated with the following primary antibodies: anti-Glut4 (1/1000, Chemicon, Temecula, CA) and anti-myoglobin (1/1000; Sigma). The blots were subsequently incubated with 2 μCi of 125I-protein A (30 μCi/μg) (Amersham
pharmacia) in blocking buffer at room temperature for 3 h and then washed again for 2 h. Band intensities were detected as described above. Mb and Glut4 protein levels were presented as percentage of saline control. Data represent mean ± SEM from 5 rats per group for each time point.
3.
Statistics
All statistical analyses were carried out using GraphPad Prism version 4 for Windows. The effects of treatment on expression of genes or proteins were determined using one-way ANOVA test followed by Bonferroni's test for multiple comparisons. Significant differences were accepted when p < 0.05.
4.
Results
We first investigated the acute effects (30, 45, 90 and 120 min) of a single-dose of exogenous rat GH administration (1.5 mg/ kg b.w. ip) on Mb gene and protein expression in solubilized cardiac muscle cells (left ventricle) extracts of treated rats. We observed a significant increase in Mb mRNA levels at 90 min after hormone injection, followed by a significant increase in Mb protein levels at 120 min after injection when compared to the control group (saline-treated animals) (Fig. 1). These results suggest that Mb is a GH target gene and its expression is stimulated shortly after GH injection. We next examined Glut4 mRNA levels and Glut4 protein translocation in cardiac muscle (left ventricle) of rats after acute administration of GH. While no significant difference in Glut4 mRNA levels was observed, Glut4 protein content in microsomal (M) fraction was significantly reduced at 30 and 45 min after GH administration followed by a significant increase in plasma membrane (PM) fraction at 120 min after GH administration when compared to control (saline only) (Fig. 2). These results suggest that GH promotes Glut4 translocation to the plasma membrane of cardiac muscle cells soon after its first administration.
5.
Discussion
Here, we showed that acute GH administration stimulates Mb mRNA and protein levels in cardiac muscle cells of treated rats. Previous reports have shown that GH controls gene expression in target tissues such as adipose tissue [10], liver [11] and skeletal muscle [12]. However, to our knowledge, this is the first study describing the effects of acute GH treatment on Mb levels in the heart. Mb stores O2 in the muscle cell and transports it from the sarcolemma to the mitochondria, favoring oxidative phosphorylation in response to muscle energy demands [13]. Therefore, it is possible that the rapid increase in O2 availability to cardiomyocytes may be a contributing factor to improve heart performance after acute GH injection. Even if further experiments are required to confirm this hypothesis, our results are encouraging and should stimulate more research along similar lines. Our current results also suggest that whereas acute GH administration has no effects on Glut4 mRNA expression, it stimulates cardiac muscle cells to translocate Glut4 protein to
ME TAB O L IS M CL I N ICA L A N D EX P ER IM EN T AL 6 3 ( 2 0 14 ) 14 9 9 –1 50 2
1501
Fig. 1 – Effects of acute GH administration on Mb expression in cardiac muscle cells (left ventricles) of rats. (A) Representative northern blotting analysis of Mb mRNA levels. Band intensities were densitometrically evaluated and the data were normalized to 18S rRNA levels and are expressed as the mean percentage ± SEM relative to the saline-treated control; n = 5 rats per group per time point. It was observed that after 90 min of GH administration there was a significant increase in Mb expression compared to control (*p < 0.05). (B) Representative immunoblot analysis of Mb protein levels. Band intensities were evaluated by densitometric analysis and the data are expressed as the mean percentage ± SEM of saline control; n = 5 rats per group per time point. It was observed that at 120 min after GH injection there was a significant increase in Mb protein expression compared to control (⁎p < 0.05).
their surfaces. Glut4 is the most abundant and the only insulinregulated glucose transporter and its translocation to the plasma membrane represents an important mechanism by which the rate of glucose entry into cardiomyocytes is regulated. Previous reports suggest that while prolonged GH treatment may exert anti-insulin effects and impair glucose uptake and metabolism [14,15], short-term GH administration mimics the effects of insulin as it decreases blood glucose levels, increases Glut4 translocation in ovary cells that overexpress GH receptor and Glut4 [16], and stimulates glucose uptake
and lipogenesis in adipocytes [11,17]. Our current results are in accordance with these studies and show that acute GH injection may also cause insulin-like effects in cardiac muscle cells. The insulin-like effects of GH have been attributed to the fact that GH and insulin act through common intracellular signal transduction pathways, the MAPK and the PI3K/AKT pathways, which control Glut4 translocation and glucose uptake by the cells [15,16]. It is well established that cardiac metabolism can be altered in several cardiovascular disorders and optimizing cardiac
Fig. 2 – Effects of acute GH administration on Glut4 expression/translocation in cardiac muscle cells (left ventricles) of rats. (A) Representative northern blotting analysis of Glut4 mRNA levels. Band intensities were densitometrically evaluated and the data were normalized to 18S rRNA and are presented as the mean percentage ± SEM relative to the saline-treated control; n = 5 rats per group per time point. No significant differences were observed when compared to the control group. (B) Representative immunoblot analysis of Glut4 protein levels in sub-cellular fractions of cardiomyocytes. Band intensities were evaluated by densitometric analysis and Glut4 protein levels are presented as the mean percentage ± SEM relative to the control group (saline only); n = 5 rats per group per time point. Significant decrease of Glut4 protein was observed in the microsomal (M) fraction after 30 and 45 min of GH administration, followed by a significant increase in the amount of Glut4 in the plasma membrane (PM) fraction at 120 min compared to control (*p < 0.05).
1502
ME TAB O L IS M CL I N ICA L A N D EX PE R IM EN T AL 6 3 ( 2 0 14 ) 14 9 9 –15 0 2
energy metabolism has been considered an attractive approach to treating such disorders [18,19]. The single dose of GH used in the present study was based on previous studies showing the cardiobeneficial effects of short-term GH administration (1– 3 mg/kg/day × 1–4 weeks) in rats with experimental myocardial infarction [5]. Although our study design did not include direct monitoring of changes in cardiac function following acute GH injection, the results obtained here suggest that some of the effects of GH on cardiomyocytes initiate shortly after its injection stimulating the expression of proteins potentially involved in energy metabolism and cardiac performance.
6.
Conclusion
In conclusion, our data suggest that GH stimulates the expression of Mb and the translocation of Glut4 to the plasma membrane of adult rat cardiomyocytes shortly after being injected. Further studies exploring acute GH use in cardiac failure therapy should be persuaded.
Author contributions RALS: design and conduct of the study, data analysis and manuscript preparation. ALS: data interpretation and manuscript writing. GG and LLP: assisted with experiment execution. MTN: coordination of research.
Funding This work was supported by grants from CNPq (National Counsel of Technological and Scientific Development), FAPESP (São Paulo Research Foundation), and CAPES (Higher Education Co-Ordination Agency).
Acknowledgments We thank the Institute of Biomedical Sciences for its vital support of this project. We thank Dr Ubiratan Fabre Machado and Dr Maristela Mitiko Okamoto for their assistance in carrying out the study of glucose transporters.
Conflict of interest The authors declare no conflict interest.
REFERENCES
[1] Castellano G, Affuso F, Conza PD, Fazio S. The GH/IGF-1 axis and heart failure. Curr Cardiol Rev 2009;5(3):203–15.
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