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Increase of cardiac M2-muscarinic receptor gene expression in type-1 but not in type-2 diabetic rats
Neuroscience Letters 441 (2008) 201–204
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Increase of cardiac M2 -muscarinic receptor gene expression in type-1 but not in type-2 diabetic rats Liang-Ming Lee a , Cheng Kuei Chang b , Kai-Chun Cheng c , Dai-Huang Kou d , I-Min Liu d , Juei-Tang Cheng c,∗ a
Department of Urology, College of Medicine and Wan Fang Hospital, Taipei Medical University, Taipei City, Taiwan, ROC Department of Surgery, Mackay Memorial Hospital, and Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei City, Taiwan, ROC c Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan, ROC d Department of Pharmacy, Tajen University, Yen-Pou, Ping Tung Shien, Taiwan, ROC b
a r t i c l e
i n f o
Article history: Received 30 April 2008 Received in revised form 30 May 2008 Accepted 2 June 2008 Keywords: Cardiac M2 -muscarinic receptor Gene expression Hyperglycemia Insulin resistance
a b s t r a c t Changes of cardiac M2 -muscarinic receptor (M2 -mAChR) gene expression was investigated in type-1 like diabetic rats induced by intravenous injection of streptozotocin (STZ) and type-2 like diabetic rats induced by fed with fructose-rich chow. Systolic blood pressure (SBP) in STZ-diabetic rats was significantly lower than that in age-matched non-diabetic rats, while the SBP in type-2 like diabetic rats was higher than in non-diabetic rats. Also, the mRNA or protein level of cardiac M2 -mAChR in STZ-diabetic rats was markedly higher than non-diabetic rats, but it was not observed in type-2 like diabetic rats as compared to agematched non-diabetic rats. Arecaidine propargyl ester (APE), the agonist of M2 -mAChR, produced a marked reduction of heart rate in STZ-diabetic rats but made less influence on heart rate in fructose-fed rats or non-diabetic rats. The results suggest that cardiac M2 -mAChR gene expression is raised in type-1 like diabetic rats but not in type-2 like diabetic rats, this difference mainly due to hyperglycemia, for the production of hypotension in diabetic disorders. © 2008 Published by Elsevier Ireland Ltd.
Diabetes is a disease characterized by chronic hyperglycemia secondary to a reduction in the functional efficacy and/or a deficiency of insulin. In fact, patients with diabetes have a shorter life span and a lesser quality of life, mainly as a result of macrovascular and/or microvascular complications [16]. Although chronic complications are late occurring, alterations in autonomic nervous tone as an early development of these complications are focused in the course of diabetes [1]. Actually, the earliest detectable feature of cardiac neuropathy is defective parasympathetic control in diabetic patients [1]. The parasympathetic branch of the autonomic nervous system plays an important role in the regulation of cardiac function responding to an activation of muscarinic cholinergic receptors (mAChR) from the released neurotransmitter, acetylcholine [6]. The M2 receptor (M2 -mAChR) is believed to be the most common subtype of mAChR expressed in cardiac muscle; stimulation of M2 mAChR inhibits adenylyl cyclase activity to result in a decrease of heart rate and/or ventricular contractility [6]. Due to the difference between human and animal and/or the stage of diabetes, the
∗ Corresponding author. Tel.: +886 6 237 2706; fax: +886 6 238 6548. E-mail address: [email protected] (J.-T. Cheng). 0304-3940/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2008.06.003
response of blood pressure is varied; changes of blood pressure in diabetic complications seem not so simple. Chronic diabetes is widely linked to hypertension [4], but hypotension has been ubiquitously described in early stage of diabetes [11]. An impairment of cardiovascular function in streptozotocin (STZ)-diabetic rats has been mentioned within 5 days-to-3 months of induction [4]. In our previous study, hyperglycemia was observed to be responsible for an increase in cardiac M2 -mAChR gene expression of type-1 like diabetic rats [8,10]. Also, it has been documented that an increase in the number of atrial muscarinic receptors, evaluated by radioligand binding assay, was associated with the cardiac dysfunction in 30-day diabetic rats [3]. Otherwise, insulin resistance is mentioned as a major factor in the pathogenesis of cardiovascular disease [13]. Patients with insulin resistance suffer dyslipidemia, abdominal obesity, high blood pressure, and hyperinsulinemia in addition to a higher sympathetic activity [13]. However, role of insulin resistance or hyperinsulinemia in the regulation of cardiac M2 -mAChR gene expression is still not clear. It has been established that feeding rats a high-fructose diet induces insulin resistance and multiple metabolic syndromes similar to humans [9]. Actually, the duration of high fructose feeding and the lipid content and type of the diets lead to discrepancies in
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the degree of insulin resistance and in the metabolic parameters. In our previous study, an increase in plasma glucose associated with hyperinsulinemia definitively suggests the impaired insulin action in 6-week fructose-fed rats [9]. In addition, characteristic signs of diabetes in type-1 like diabetic rats, such as a decrease in body weight, and an increase in blood glucose levels, reach a peak at 4 weeks after STZ injection and approach a steady-state within 8 weeks of the onset of diabetes, while animals with diabetes for 12–16 weeks were considered as the later stage of diabetes [20]. Therefore, the present study employed the whole hearts of type-1 like diabetic rats induced by STZ injection for 8 weeks and insulin resistance rats induced by 8-week fructose chow feeding, to investigate the role of hyperglycemia or hyperinsulinemia in the changes of cardiac M2 -mAChR. Male Wistar rats aged 8 weeks were obtained from the Animal Center of National Cheng Kung University Medical College. They were maintained in a temperature-controlled room (25 ± 1 ◦ C) and kept on a 12:12 light–dark cycle (light on at 06:00 h). Food and water were available ad libitum. The rats were divided into two groups for experiment. One group of rats were received an intravenous injection of 60 mg/kg streptozotocin (STZ; Sigma–Aldrich, Inc., St. Louis, MO, U.S.A.). Rats with plasma glucose concentration of 400 mg/dl or greater in addition to polyuria and other diabetic disorders were considered as type-1 diabetic animals. All studies were carried out 8 weeks after the injection of STZ. Otherwise, two groups of age-matched rats were also used; one group was randomly assigned to receive the fructose-rich chow (containing 60% fructose, 5% fat, and 20% protein; Teklad, Madison, WI, U.S.A.) for 8 weeks to induce the insulin resistance. The remaining rats receiving standard chows (Purina Mills, LLC, St. Louis, MO, U.S.A.) for same period (8-week) were taken as the control group. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as the guidelines of the Animal Welfare Act. The plasma levels of glucose and insulin were determined using blood samples collected from tail vein of fasting animals. The levels of plasma glucose were estimated by an analyzer (Quik-Lab, Ames, Miles Inc., Elkhart, IN 46515, U.S.A.) using a commercial kit (Catalog #COD12503) from BioSystem (Costa Brava, Barcelona, Spain). The plasma insulin was determined by rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Catalog #EZRMI-13K) of LINCO Research, Inc. (St. Charles, MO, U.S.A.). Samples from each individual were analyzed in triplicate at the same time. The test compounds used in the present study did not affect the binding of peptide with antibodies. Data are expressed as the mean ± S.E.M. for the number (n) of animals in the group as indicated in tables and figures. Repeated measures analysis of variance (ANOVA) was used to analyze the changes in plasma glucose and other parameters. The Dunnett range post-hoc comparisons were used to determine the source of significant differences where appropriate. A P-value <0.05 was considered statistically significant. As shown in Table 1, 8 weeks after fructose feeding, the body weight of rats was significantly raised as compared with the normal chow-fed group. However, the body weight of diabetic rats induced by STZ for 8 weeks was markedly reduced. The fasting plasma glucose levels of STZ-diabetic rats were significantly higher than those of non-diabetic rats, but the plasma insulin level in STZ-diabetic rats was markedly lower than that in non-diabetic group (Table 1). Animals maintained on the 8week high-fructose diet exhibited higher plasma glucose than normal chow-fed non-diabetic group. However, the plasma glucose level in high-fructose fed rats was lower than that of STZ-diabetic group (Table 1). Besides, the plasma insulin level in high-fructose
Table 1 General conditions of each group
Duration of diabetes (weeks) Body weight (g) Plasma insulin (U/ml) Plasma glucose (mg/dl) SBP (mmHg) Heart rate (beats/min)
Non-diabetic rats
STZ-diabetic rats
Fructose-fed rats
0 252.6 ± 8.7 22.4 ± 5.2 92.7 ± 4.5 115.6 ± 2.8 348.4 ± 9.1
8 182.3 ± 7.5* 0.3 ± 0.2** 448.2 ± 9.3** 96.5 ± 3.2* 318.4 ± 8.6*
8 283.5 ± 5.4* 183.6 ± 6.5** 168.6 ± 3.7** 132.7 ± 3.5* 345.7 ± 7.8
Values (mean ± S.E.M.) were obtained for each group of 8 animals. * P < 0.05 compared to the values of non-diabetic rats. ** P < 0.01 compared to the values of non-diabetic rats.
fed rats was significantly higher than that in non-diabetic group (Table 1). In addition to hyperinsulinemia, insulin resistance is associated with fructose intake in animal models [9]. The in vivo evaluation of insulin sensitivity is widely used intravenous insulin challenge test [22]. Thus, we employed this insulin challenge test in both STZdiabetic rats and fructose-fed rats using 1.0 IU/kg of short-acting human insulin (Novo Nordisk A/S, Bagsvaerd, Denmark). Blood samples (0.2 ml) from the tail vein were drawn at 30 min following the challenge test to measure the change of plasma glucose. The difference in the response to exogenous insulin was compared in two groups. The plasma glucose level in STZ-diabetic rats treated with exogenous insulin (1.0 IU/kg) at 30 min later was markedly (P < 0.01) decreased from 443.3 ± 7.6 mg/dl to 310.4 ± 5.4 mg/dl, the plasma glucose lowering activity induced by this exogenous insulin treatment was about 30.2 ± 3.8% in type-1 diabetic rats. Indeed, role of endogenous insulin in STZ-diabetic rat is negligible and plasma glucose lowering response to exogenous insulin is believed as the direct action of insulin. The plasma glucose level in fructose-fed rats treated with exogenous insulin (1.0 IU/kg) at 30 min later was remained at 177.9 ± 6.3 mg/dl, which was near to that before insulin treatment (184.7 ± 5.8 mg/dl). Then, the plasma glucose lowering activity of exogenous insulin (1.0 IU/kg) in this type-2 diabetic rat was only 3.7 ± 1.2%. The ability of insulin to stimulate glucose disposal assessed by this method is markedly impaired in the rats received fructose-rich chow, indicating a decline of insulin sensitivity in peripheral tissues associated with insulin resistance. Thus, fructose-fed rats could be served as a reliable model of insulin resistance similar to type-2 diabetes. Insulin resistance is recognized as a major factor in metabolic syndrome accompanying hypertension [14]. Then, the difference in systolic blood pressure (SBP) was investigated in STZ-diabetic rats and fructose-fed rats. The blood pressure of the tail artery and heart rate was measured non-invasively with a photoelectric volume oscillometer (MK2000, Muromachi Kikai Co. Ltd., Tokyo, Japan) using the tail cuff device around the tail of rat. The measurements for SBP were recorded in quadruplicate for each rat and the average blood pressure was calculated. We observed that the SBP in STZ-diabetic rats was significantly lower than that in age-matched non-diabetic rats, while the SBP in fructose-fed rats was higher than that of non-diabetic rats (Table 1). It is possible that chronic exposure to high-level of insulin observed in type-2 diabetes may make the blood vessels less sensitive to insulin’s vasodilator effect [7]. Also, it has been documented that the muscarinic receptor-induced relaxation was not critical to the early increase of blood pressure in rat after 8-weeks fructose feeding, whereas the early dysfunction in alpha-adrenergic mechanism may initiate hypertension and alter vascular wall in insulin resistance [17]. Actually, an increase in the number of atrial muscarinic receptors has been observed in 30-day STZ-diabetic rats by radioligand binding assay [3]. Thus, we focus on the gene expression of M2 -mAChR on whole heart of experimental
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Fig. 1. The gene expression of cardiac M2 -mAChR in STZ-diabetic rats and fructose-fed rats. (A) Upper panel is the representative autoradiograph from Northern blot of mRNA level for M2 -mAChR or -actin in isolated hearts of diabetic rats. Similar results were obtained in other 5 replications. Quantification of mRNA levels using M2 -mAChR/-actin, expressed as mean with S.E.M. (n = 6 per group) in each column, is indicated in the lower panel. (B) Upper panel shows the Western blot of representative protein level for M2 -mAChR or actin in hearts isolated from diabetic rats. Quantification of protein levels using M2 -mAChR/actin, expressed as mean with S.E.M (n = 6 per group) in each column, is indicated in the lower panel. ** P < 0.01 represents significance compared to age-matched non-diabetic rats.
diabetes, instead of limiting to atria or ventricles, to distinguish the role of hyperglycemia or insulin resistance in the regulation of cardiac functions in diabetes. The M2 -mAChR protein expression in the heart tissue was measured by Western immunoblotting. The preparation of membrane fraction from whole heart was performed on ice. The isolated heart tissue was lysed in 10 ml of pH 7.4 Tris/EDTA buffer at 4 ◦ C and homogenized for 15 s. The membrane fraction was obtained by centrifugation at 20,000 g for 15 min. After homoge-
Fig. 2. Effects of M2 -muscarinic receptors agonist, APE, on the heart rates in Wistar rats (䊉), STZ-diabetic rats () and fructose-fed rats (). Rats were intravenously injected with APE at the indicated dose. The changes in heart rates were recorded at 5 min interval for 30 min. The results are expressed as the mean ± S.E.M. obtained from each group of 7 animals. * P < 0.05 and ** P < 0.01 for comparisons of heart rates between post-injection and pre-injection (0) in each group.
nization, the protein content was determined by BioRad protein dye binding assay (Bio-Rad Laboratories, Richmond, CA, U.S.A.). Protein samples (9 g) were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (10% acrylamide gel) using Bio-Rad Mini-Protein II system. The separated proteins were blotted onto nitrocellulose. After treatment with M2 -subtype specific antimAChR antibody (1:1000) (Affinity Bioreagents, Inc., CO, U.S.A.; Catalog #MA3-044), immunostaining was performed for peroxidase activity by incubation in Tris-buffer (10 mmol/l). The intensity of the blot incubated with goat polyclonal antibody (1:1000) to bind actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.; Catalog #sc-1616) was used to ensure that the amount of protein loaded into each lane of the gel was constant. Autoradiography was developed using an enhanced chemiluminescence development system (Amersham International, Buckinghamshire, U.K.). The resulting immunoblots were quantified by a scanning densitometer (Hoefer, San Francisco, CA, U.S.A.). Measurement of mRNA level for M2 -mAChR in heart tissue was performed by Northern blotting analysis. Total RNA was extracted from heart using UltraspecTM -II RNA extraction system (Bioteck, Houston, TX, U.S.A.) as indication of the manufacturer. RNA (20 g) was denatured and aliquots of total RNA were then size-fractionated in a 1.2% agarose/formaldehyde gel. The RNA was transferred to a Hybond-N membrane (Amersham International). M2 -mAChR mRNA levels were detected using prime-labeled full-length cDNA under stringent hybridization conditions [18]. Intensity of the mRNA blot was quantified by scanning densitometry (Hoefer, San Francisco, CA, U.S.A.). The response of -actin was used as internal standard. The mRNA level of cardiac M2 -mAChR in STZ-diabetic rats was markedly higher than in non-diabetic rats (Fig. 1A); also, changes in protein level of cardiac M2 -mAChR were associated with steadystate levels of mRNA encoding this receptor (Fig. 1B). An increase of cardiac M2 -mAChR gene expression was indeed observed
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during the early stage of type-1 like diabetes. However, no significant difference regarding the mRNA and protein levels of cardiac M2 -mAChR can be obtained between fructose-fed rats and nondiabetic rats (Fig. 1). It seems that hyperglycemia but not insulin resistance is associated with an elevation of M2 -mAChR gene expression accounting for the cardiac alteration in diabetes. The effect of arecaidine propargyl ester (APE), an agonist for M2 mAChR [19] on heart rate of diabetic rats was also investigated. In each group of rats, APE (Sigma–Aldrich, Inc.) was intravenously injected at the indicated dose and the changes in heart rates were recorded at 5 min interval for 30 min. As shown in Fig. 2, heart rate decreased in STZ-diabetic rats (10.6 ± 2.4%) by APE at 1.0 mg/kg. The reduction of heart rate was more pronounced to 20.6 ± 4.2% in STZ-diabetic rats responding to 2 mg/kg APE (Fig. 2). However, even at this high dose of 2 mg/kg, the reduction of heart rate induced by APE was only 5.8 ± 2.4% and 3.4 ± 2.1% in fructose-fed rats and non-diabetic rats, respectively (Fig. 2). It means that APE is more effective in type-1 diabetic rats with higher M2 -mAChR than in type-2 diabetic rats. Thus, hyperglycemia could be considered a key factor in cardiac alteration that is associated with an increase of cardiac M2 -mAChR gene expression leading to hypotension observed in 8 weeks type-1 diabetic rats. The mechanism for increase of M2 -mAChR gene expression by hyperglycemia is still unclear. Actually, muscarinic receptor populations regulated by the degree of effective neurotransmission has been documented; these receptor populations are decreased by chronic exposure to agonists or by inhibition of acetylcholinesterase [5], and they are increased by exposure to antagonists [21]. Also, it has been postulated that an axonopathyrelated neurotransmitter release defect leads to accumulation of nerve-ending neurotransmitters might result in an up-regulation of the postsynaptic M2 -mAChR in animal [18]. Clinically, heart disease is one of the major causes of death in diabetic patients, due in part to the accumulation of advanced glycation end products (AGE) resulting from chronic hyperglycemia [2]. It has been suggested that suitable glycemic control in patients with diabetes for 8 years does not lead to an effective reduction in AGE levels, illustrating the negative correlation between hyperglycemia and the advanced diabetic complications that occur in chronic diabetes [12]. It appears that hyperglycemia may elevate cardiac M2 -mAChR gene expression to account for the changes of cardiovascular function during the early stage of diabetes, whereas the metabolic effects induced by sustained hyperglycemia may lower cardiac M2 -mAChR to give advanced complications in the terminal stages of diabetes [15]. Thus, more studies are required for the understanding of detailed mechanism(s) in near future. In conclusion, both mRNA and protein levels of cardiac M2 mAChR are increased in type-1 but not in type-2 diabetic rats. Thus, hyperglycemia is important in the increase of cardiac M2 -mAChR that leads to hypotension observed at the early stage of diabetes. Acknowledgements We appreciate the kind assistance of Miss F.Y. Jou in blotting analysis. Thanks are also due to Professor S.S. Liu for generously supplying the plasmids containing the cDNA of -actin. The
present study was supported in part by a grant from the National Science Council (NSC 94-2320-B-006-089) of the Republic of China. References [1] A.J. Boulton, A.I. Vinik, J.C. Arezzo, V. Bril, E.L. Feldman, R. Freeman, R.A. Malik, R.E. Maser, J.M. Sosenko, D. Ziegler, Diabetic neuropathies: a statement by the American Diabetes Association, Diabetes Care 28 (2005) 956–962. [2] M.E. Cooper, Importance of advanced glycation end products in diabetesassociated cardiovascular and renal disease, Am. J. Hypertens. 17 (2004) 31S–38S. [3] P. Dall’Ago, V.O. Silva, K.L. De Angelis, M.C. Irigoyen, R. Fazan Jr., H.C. Salgado, Reflex control of arterial pressure and heart rate in short-term streptozotocin diabetic rats, Braz. J. Med. Biol. Res. 35 (2002) 843–849. [4] K. De Angelis, B.D. Schaan, C.Y. Maeda, P. Dall’Ago, R.B. Wichi, M.C. Irigoyen, Cardiovascular control in experimental diabetes, Braz. J. Med. Biol. Res. 35 (2002) 1091–1100. [5] H. Gazit, I. Silman, Y. Dudai, Administration of an organophosphate causes a decrease in muscarinic receptor levels in rat brain, Brain Res. 174 (1979) 351– 356. [6] R.D. Harvey, A.E. Belevych, Muscarinic regulation of cardiac ion channels, Br. J. Pharmacol. 139 (2003) 1074–1084. [7] T. Heise, K. Magnusson, L. Heinemann, P.T. Sawicki, Insulin resistance and the effect of insulin on blood pressure in essential hypertension, Hypertension 32 (1998) 243–248. [8] I.M. Liu, C. K. Chang, S.W. Juang, D.H. Kou, Y.C. Tong, K.C. Cheng, J.T. Cheng, Role of hyperglycaemia in the pathogenesis of hypotension observed in type-1 diabetic rats, Int. J. Exp. Path. (2008), in press. [9] T.P. Liu, S.W. Juang, J.T. Cheng, Y.C. Tong, The role of sorbitol pathway and treatment effect of aldose reductase inhibitor ONO2235 in the up-regulation of cardiac M2 -muscarinic receptors in streptozotocin-induced diabetic rats, Neurosci. Lett. 383 (2005) 131–135. [10] I.M. Liu, T.F. Tzeng, S.S. Liou, T.W. Lan, Myricetin, A naturally occurring flavonol, ameliorates insulin resistance induced by a high-fructose diet in rats, Life Sci. 81 (2007) 1479–1488. [11] C.Y. Maeda, T.G. Fernandes, H. Timm, M.C. Irigoyen, Autonomic dysfunction in short-term experimental diabetes, Hypertension 26 (1995) 1000–1004. [12] C.J. Mentink, B.K. Kilhovd, G.J. Rondas-Colbers, P.A. Torjesen, B.H. Wolffenbuttel, Time course of specific AGEs during optimised glycaemic control in type 2 diabetes, Neth. J. Med. 64 (2006) 10–16. [13] D. Moller, K. Kaufman, Metabolic syndrome: a clinical and molecular perspective, Ann. Rev. Med. 56 (2005) 45–62. ´ E. Nardi, S. Cottone, P. Cusimano, A. Palermo, F. Incalcaterra, M.E. [14] G. Mule, Giandalia, G. Cerasola, Impact of the metabolic syndrome on total arterial compliance in essential hypertension patients, J. Cardiometab. Syndr. 2 (2007) 84–90. [15] M.J. Sheetz, G.L. King, Molecular understanding of hyperglycemia’s adverse effects for diabetic complications, JAMA 288 (2002) 2579–2588. [16] B.E. Sobel, D.J. Schneider, Cardiovascular complications in diabetes mellitus, Curr. Opin. Pharmacol. 5 (2005) 143–148. [17] Y. Takagawa, M.E. Berger, M.L. Tuck, M.S. Golub, Impaired endothelial alpha2 adrenergic receptor-mediated vascular relaxation in the fructose-fed rat, Hypertens. Res. 25 (2002) 197–202. [18] Y.C. Tong, W.T. Chin, J.T. Cheng, Alterations in M2 -muscarinic receptor protein and mRNA in diabetic rat urinary bladders, Neurosci. Lett. 277 (1999) 173–176. [19] V. Tumiatti, J. Wehrle, C. Hildebrandt, U. Moser, G. Dannhardt, E. Mutschler, G. Lambrechtm, Muscarinic properties of compounds related to arecaidine propargyl ester, Arzneimittelforschung 50 (2000) 11– 15. [20] M. Wei, L. Ong, M.T. Smith, F.B. Ross, K. Schmid, A.J. Hoey, D. Burstow, L. Brown, The streptozotocin-diabetic rat as a model of the chronic complications of human diabetes, Heart Lung Circ. 12 (2003) 44–50. [21] B.C. Wise, M. Shoji, J.F. Kuo, Decrease or increase in cardiac muscarinic cholinergic receptor number in rats treated with methacholine or atropine, Biochem. Biophys. Res. Commun. 92 (1980) 1136–1142. [22] Y.C. Wu, J.H. Hsu, I.M. Liu, S.S. Liou, H.C. Su, J.T. Cheng, Increase of insulin sensitivity in diabetic rats received die-huang-wan, a herbal mixture used in Chinese traditional medicine, Acta Pharmacol. Sin 23 (2002) 1181–1187.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Increase of cardiac M2 -muscarinic receptor gene expression in type-1 but not in type-2 diabetic rats Liang-Ming Lee a , Cheng Kuei Chang b , Kai-Chun Cheng c , Dai-Huang Kou d , I-Min Liu d , Juei-Tang Cheng c,∗ a
Department of Urology, College of Medicine and Wan Fang Hospital, Taipei Medical University, Taipei City, Taiwan, ROC Department of Surgery, Mackay Memorial Hospital, and Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei City, Taiwan, ROC c Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan, ROC d Department of Pharmacy, Tajen University, Yen-Pou, Ping Tung Shien, Taiwan, ROC b
a r t i c l e
i n f o
Article history: Received 30 April 2008 Received in revised form 30 May 2008 Accepted 2 June 2008 Keywords: Cardiac M2 -muscarinic receptor Gene expression Hyperglycemia Insulin resistance
a b s t r a c t Changes of cardiac M2 -muscarinic receptor (M2 -mAChR) gene expression was investigated in type-1 like diabetic rats induced by intravenous injection of streptozotocin (STZ) and type-2 like diabetic rats induced by fed with fructose-rich chow. Systolic blood pressure (SBP) in STZ-diabetic rats was significantly lower than that in age-matched non-diabetic rats, while the SBP in type-2 like diabetic rats was higher than in non-diabetic rats. Also, the mRNA or protein level of cardiac M2 -mAChR in STZ-diabetic rats was markedly higher than non-diabetic rats, but it was not observed in type-2 like diabetic rats as compared to agematched non-diabetic rats. Arecaidine propargyl ester (APE), the agonist of M2 -mAChR, produced a marked reduction of heart rate in STZ-diabetic rats but made less influence on heart rate in fructose-fed rats or non-diabetic rats. The results suggest that cardiac M2 -mAChR gene expression is raised in type-1 like diabetic rats but not in type-2 like diabetic rats, this difference mainly due to hyperglycemia, for the production of hypotension in diabetic disorders. © 2008 Published by Elsevier Ireland Ltd.
Diabetes is a disease characterized by chronic hyperglycemia secondary to a reduction in the functional efficacy and/or a deficiency of insulin. In fact, patients with diabetes have a shorter life span and a lesser quality of life, mainly as a result of macrovascular and/or microvascular complications [16]. Although chronic complications are late occurring, alterations in autonomic nervous tone as an early development of these complications are focused in the course of diabetes [1]. Actually, the earliest detectable feature of cardiac neuropathy is defective parasympathetic control in diabetic patients [1]. The parasympathetic branch of the autonomic nervous system plays an important role in the regulation of cardiac function responding to an activation of muscarinic cholinergic receptors (mAChR) from the released neurotransmitter, acetylcholine [6]. The M2 receptor (M2 -mAChR) is believed to be the most common subtype of mAChR expressed in cardiac muscle; stimulation of M2 mAChR inhibits adenylyl cyclase activity to result in a decrease of heart rate and/or ventricular contractility [6]. Due to the difference between human and animal and/or the stage of diabetes, the
∗ Corresponding author. Tel.: +886 6 237 2706; fax: +886 6 238 6548. E-mail address: [email protected] (J.-T. Cheng). 0304-3940/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2008.06.003
response of blood pressure is varied; changes of blood pressure in diabetic complications seem not so simple. Chronic diabetes is widely linked to hypertension [4], but hypotension has been ubiquitously described in early stage of diabetes [11]. An impairment of cardiovascular function in streptozotocin (STZ)-diabetic rats has been mentioned within 5 days-to-3 months of induction [4]. In our previous study, hyperglycemia was observed to be responsible for an increase in cardiac M2 -mAChR gene expression of type-1 like diabetic rats [8,10]. Also, it has been documented that an increase in the number of atrial muscarinic receptors, evaluated by radioligand binding assay, was associated with the cardiac dysfunction in 30-day diabetic rats [3]. Otherwise, insulin resistance is mentioned as a major factor in the pathogenesis of cardiovascular disease [13]. Patients with insulin resistance suffer dyslipidemia, abdominal obesity, high blood pressure, and hyperinsulinemia in addition to a higher sympathetic activity [13]. However, role of insulin resistance or hyperinsulinemia in the regulation of cardiac M2 -mAChR gene expression is still not clear. It has been established that feeding rats a high-fructose diet induces insulin resistance and multiple metabolic syndromes similar to humans [9]. Actually, the duration of high fructose feeding and the lipid content and type of the diets lead to discrepancies in
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the degree of insulin resistance and in the metabolic parameters. In our previous study, an increase in plasma glucose associated with hyperinsulinemia definitively suggests the impaired insulin action in 6-week fructose-fed rats [9]. In addition, characteristic signs of diabetes in type-1 like diabetic rats, such as a decrease in body weight, and an increase in blood glucose levels, reach a peak at 4 weeks after STZ injection and approach a steady-state within 8 weeks of the onset of diabetes, while animals with diabetes for 12–16 weeks were considered as the later stage of diabetes [20]. Therefore, the present study employed the whole hearts of type-1 like diabetic rats induced by STZ injection for 8 weeks and insulin resistance rats induced by 8-week fructose chow feeding, to investigate the role of hyperglycemia or hyperinsulinemia in the changes of cardiac M2 -mAChR. Male Wistar rats aged 8 weeks were obtained from the Animal Center of National Cheng Kung University Medical College. They were maintained in a temperature-controlled room (25 ± 1 ◦ C) and kept on a 12:12 light–dark cycle (light on at 06:00 h). Food and water were available ad libitum. The rats were divided into two groups for experiment. One group of rats were received an intravenous injection of 60 mg/kg streptozotocin (STZ; Sigma–Aldrich, Inc., St. Louis, MO, U.S.A.). Rats with plasma glucose concentration of 400 mg/dl or greater in addition to polyuria and other diabetic disorders were considered as type-1 diabetic animals. All studies were carried out 8 weeks after the injection of STZ. Otherwise, two groups of age-matched rats were also used; one group was randomly assigned to receive the fructose-rich chow (containing 60% fructose, 5% fat, and 20% protein; Teklad, Madison, WI, U.S.A.) for 8 weeks to induce the insulin resistance. The remaining rats receiving standard chows (Purina Mills, LLC, St. Louis, MO, U.S.A.) for same period (8-week) were taken as the control group. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as the guidelines of the Animal Welfare Act. The plasma levels of glucose and insulin were determined using blood samples collected from tail vein of fasting animals. The levels of plasma glucose were estimated by an analyzer (Quik-Lab, Ames, Miles Inc., Elkhart, IN 46515, U.S.A.) using a commercial kit (Catalog #COD12503) from BioSystem (Costa Brava, Barcelona, Spain). The plasma insulin was determined by rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Catalog #EZRMI-13K) of LINCO Research, Inc. (St. Charles, MO, U.S.A.). Samples from each individual were analyzed in triplicate at the same time. The test compounds used in the present study did not affect the binding of peptide with antibodies. Data are expressed as the mean ± S.E.M. for the number (n) of animals in the group as indicated in tables and figures. Repeated measures analysis of variance (ANOVA) was used to analyze the changes in plasma glucose and other parameters. The Dunnett range post-hoc comparisons were used to determine the source of significant differences where appropriate. A P-value <0.05 was considered statistically significant. As shown in Table 1, 8 weeks after fructose feeding, the body weight of rats was significantly raised as compared with the normal chow-fed group. However, the body weight of diabetic rats induced by STZ for 8 weeks was markedly reduced. The fasting plasma glucose levels of STZ-diabetic rats were significantly higher than those of non-diabetic rats, but the plasma insulin level in STZ-diabetic rats was markedly lower than that in non-diabetic group (Table 1). Animals maintained on the 8week high-fructose diet exhibited higher plasma glucose than normal chow-fed non-diabetic group. However, the plasma glucose level in high-fructose fed rats was lower than that of STZ-diabetic group (Table 1). Besides, the plasma insulin level in high-fructose
Table 1 General conditions of each group
Duration of diabetes (weeks) Body weight (g) Plasma insulin (U/ml) Plasma glucose (mg/dl) SBP (mmHg) Heart rate (beats/min)
Non-diabetic rats
STZ-diabetic rats
Fructose-fed rats
0 252.6 ± 8.7 22.4 ± 5.2 92.7 ± 4.5 115.6 ± 2.8 348.4 ± 9.1
8 182.3 ± 7.5* 0.3 ± 0.2** 448.2 ± 9.3** 96.5 ± 3.2* 318.4 ± 8.6*
8 283.5 ± 5.4* 183.6 ± 6.5** 168.6 ± 3.7** 132.7 ± 3.5* 345.7 ± 7.8
Values (mean ± S.E.M.) were obtained for each group of 8 animals. * P < 0.05 compared to the values of non-diabetic rats. ** P < 0.01 compared to the values of non-diabetic rats.
fed rats was significantly higher than that in non-diabetic group (Table 1). In addition to hyperinsulinemia, insulin resistance is associated with fructose intake in animal models [9]. The in vivo evaluation of insulin sensitivity is widely used intravenous insulin challenge test [22]. Thus, we employed this insulin challenge test in both STZdiabetic rats and fructose-fed rats using 1.0 IU/kg of short-acting human insulin (Novo Nordisk A/S, Bagsvaerd, Denmark). Blood samples (0.2 ml) from the tail vein were drawn at 30 min following the challenge test to measure the change of plasma glucose. The difference in the response to exogenous insulin was compared in two groups. The plasma glucose level in STZ-diabetic rats treated with exogenous insulin (1.0 IU/kg) at 30 min later was markedly (P < 0.01) decreased from 443.3 ± 7.6 mg/dl to 310.4 ± 5.4 mg/dl, the plasma glucose lowering activity induced by this exogenous insulin treatment was about 30.2 ± 3.8% in type-1 diabetic rats. Indeed, role of endogenous insulin in STZ-diabetic rat is negligible and plasma glucose lowering response to exogenous insulin is believed as the direct action of insulin. The plasma glucose level in fructose-fed rats treated with exogenous insulin (1.0 IU/kg) at 30 min later was remained at 177.9 ± 6.3 mg/dl, which was near to that before insulin treatment (184.7 ± 5.8 mg/dl). Then, the plasma glucose lowering activity of exogenous insulin (1.0 IU/kg) in this type-2 diabetic rat was only 3.7 ± 1.2%. The ability of insulin to stimulate glucose disposal assessed by this method is markedly impaired in the rats received fructose-rich chow, indicating a decline of insulin sensitivity in peripheral tissues associated with insulin resistance. Thus, fructose-fed rats could be served as a reliable model of insulin resistance similar to type-2 diabetes. Insulin resistance is recognized as a major factor in metabolic syndrome accompanying hypertension [14]. Then, the difference in systolic blood pressure (SBP) was investigated in STZ-diabetic rats and fructose-fed rats. The blood pressure of the tail artery and heart rate was measured non-invasively with a photoelectric volume oscillometer (MK2000, Muromachi Kikai Co. Ltd., Tokyo, Japan) using the tail cuff device around the tail of rat. The measurements for SBP were recorded in quadruplicate for each rat and the average blood pressure was calculated. We observed that the SBP in STZ-diabetic rats was significantly lower than that in age-matched non-diabetic rats, while the SBP in fructose-fed rats was higher than that of non-diabetic rats (Table 1). It is possible that chronic exposure to high-level of insulin observed in type-2 diabetes may make the blood vessels less sensitive to insulin’s vasodilator effect [7]. Also, it has been documented that the muscarinic receptor-induced relaxation was not critical to the early increase of blood pressure in rat after 8-weeks fructose feeding, whereas the early dysfunction in alpha-adrenergic mechanism may initiate hypertension and alter vascular wall in insulin resistance [17]. Actually, an increase in the number of atrial muscarinic receptors has been observed in 30-day STZ-diabetic rats by radioligand binding assay [3]. Thus, we focus on the gene expression of M2 -mAChR on whole heart of experimental
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Fig. 1. The gene expression of cardiac M2 -mAChR in STZ-diabetic rats and fructose-fed rats. (A) Upper panel is the representative autoradiograph from Northern blot of mRNA level for M2 -mAChR or -actin in isolated hearts of diabetic rats. Similar results were obtained in other 5 replications. Quantification of mRNA levels using M2 -mAChR/-actin, expressed as mean with S.E.M. (n = 6 per group) in each column, is indicated in the lower panel. (B) Upper panel shows the Western blot of representative protein level for M2 -mAChR or actin in hearts isolated from diabetic rats. Quantification of protein levels using M2 -mAChR/actin, expressed as mean with S.E.M (n = 6 per group) in each column, is indicated in the lower panel. ** P < 0.01 represents significance compared to age-matched non-diabetic rats.
diabetes, instead of limiting to atria or ventricles, to distinguish the role of hyperglycemia or insulin resistance in the regulation of cardiac functions in diabetes. The M2 -mAChR protein expression in the heart tissue was measured by Western immunoblotting. The preparation of membrane fraction from whole heart was performed on ice. The isolated heart tissue was lysed in 10 ml of pH 7.4 Tris/EDTA buffer at 4 ◦ C and homogenized for 15 s. The membrane fraction was obtained by centrifugation at 20,000 g for 15 min. After homoge-
Fig. 2. Effects of M2 -muscarinic receptors agonist, APE, on the heart rates in Wistar rats (䊉), STZ-diabetic rats () and fructose-fed rats (). Rats were intravenously injected with APE at the indicated dose. The changes in heart rates were recorded at 5 min interval for 30 min. The results are expressed as the mean ± S.E.M. obtained from each group of 7 animals. * P < 0.05 and ** P < 0.01 for comparisons of heart rates between post-injection and pre-injection (0) in each group.
nization, the protein content was determined by BioRad protein dye binding assay (Bio-Rad Laboratories, Richmond, CA, U.S.A.). Protein samples (9 g) were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (10% acrylamide gel) using Bio-Rad Mini-Protein II system. The separated proteins were blotted onto nitrocellulose. After treatment with M2 -subtype specific antimAChR antibody (1:1000) (Affinity Bioreagents, Inc., CO, U.S.A.; Catalog #MA3-044), immunostaining was performed for peroxidase activity by incubation in Tris-buffer (10 mmol/l). The intensity of the blot incubated with goat polyclonal antibody (1:1000) to bind actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.; Catalog #sc-1616) was used to ensure that the amount of protein loaded into each lane of the gel was constant. Autoradiography was developed using an enhanced chemiluminescence development system (Amersham International, Buckinghamshire, U.K.). The resulting immunoblots were quantified by a scanning densitometer (Hoefer, San Francisco, CA, U.S.A.). Measurement of mRNA level for M2 -mAChR in heart tissue was performed by Northern blotting analysis. Total RNA was extracted from heart using UltraspecTM -II RNA extraction system (Bioteck, Houston, TX, U.S.A.) as indication of the manufacturer. RNA (20 g) was denatured and aliquots of total RNA were then size-fractionated in a 1.2% agarose/formaldehyde gel. The RNA was transferred to a Hybond-N membrane (Amersham International). M2 -mAChR mRNA levels were detected using prime-labeled full-length cDNA under stringent hybridization conditions [18]. Intensity of the mRNA blot was quantified by scanning densitometry (Hoefer, San Francisco, CA, U.S.A.). The response of -actin was used as internal standard. The mRNA level of cardiac M2 -mAChR in STZ-diabetic rats was markedly higher than in non-diabetic rats (Fig. 1A); also, changes in protein level of cardiac M2 -mAChR were associated with steadystate levels of mRNA encoding this receptor (Fig. 1B). An increase of cardiac M2 -mAChR gene expression was indeed observed
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during the early stage of type-1 like diabetes. However, no significant difference regarding the mRNA and protein levels of cardiac M2 -mAChR can be obtained between fructose-fed rats and nondiabetic rats (Fig. 1). It seems that hyperglycemia but not insulin resistance is associated with an elevation of M2 -mAChR gene expression accounting for the cardiac alteration in diabetes. The effect of arecaidine propargyl ester (APE), an agonist for M2 mAChR [19] on heart rate of diabetic rats was also investigated. In each group of rats, APE (Sigma–Aldrich, Inc.) was intravenously injected at the indicated dose and the changes in heart rates were recorded at 5 min interval for 30 min. As shown in Fig. 2, heart rate decreased in STZ-diabetic rats (10.6 ± 2.4%) by APE at 1.0 mg/kg. The reduction of heart rate was more pronounced to 20.6 ± 4.2% in STZ-diabetic rats responding to 2 mg/kg APE (Fig. 2). However, even at this high dose of 2 mg/kg, the reduction of heart rate induced by APE was only 5.8 ± 2.4% and 3.4 ± 2.1% in fructose-fed rats and non-diabetic rats, respectively (Fig. 2). It means that APE is more effective in type-1 diabetic rats with higher M2 -mAChR than in type-2 diabetic rats. Thus, hyperglycemia could be considered a key factor in cardiac alteration that is associated with an increase of cardiac M2 -mAChR gene expression leading to hypotension observed in 8 weeks type-1 diabetic rats. The mechanism for increase of M2 -mAChR gene expression by hyperglycemia is still unclear. Actually, muscarinic receptor populations regulated by the degree of effective neurotransmission has been documented; these receptor populations are decreased by chronic exposure to agonists or by inhibition of acetylcholinesterase [5], and they are increased by exposure to antagonists [21]. Also, it has been postulated that an axonopathyrelated neurotransmitter release defect leads to accumulation of nerve-ending neurotransmitters might result in an up-regulation of the postsynaptic M2 -mAChR in animal [18]. Clinically, heart disease is one of the major causes of death in diabetic patients, due in part to the accumulation of advanced glycation end products (AGE) resulting from chronic hyperglycemia [2]. It has been suggested that suitable glycemic control in patients with diabetes for 8 years does not lead to an effective reduction in AGE levels, illustrating the negative correlation between hyperglycemia and the advanced diabetic complications that occur in chronic diabetes [12]. It appears that hyperglycemia may elevate cardiac M2 -mAChR gene expression to account for the changes of cardiovascular function during the early stage of diabetes, whereas the metabolic effects induced by sustained hyperglycemia may lower cardiac M2 -mAChR to give advanced complications in the terminal stages of diabetes [15]. Thus, more studies are required for the understanding of detailed mechanism(s) in near future. In conclusion, both mRNA and protein levels of cardiac M2 mAChR are increased in type-1 but not in type-2 diabetic rats. Thus, hyperglycemia is important in the increase of cardiac M2 -mAChR that leads to hypotension observed at the early stage of diabetes. Acknowledgements We appreciate the kind assistance of Miss F.Y. Jou in blotting analysis. Thanks are also due to Professor S.S. Liu for generously supplying the plasmids containing the cDNA of -actin. The
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