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Central nitric oxide inhibition modifies metabolic adjustments induced by exercise in rats
Neuroscience Letters 410 (2006) 152–156
Central nitric oxide inhibition modifies metabolic adjustments induced by exercise in rats Ana Cristina R. Lacerda, Umeko Marubayashi, Cl´audio H. Balthazar, Laura H.R. Leite, Cˆandido C. Coimbra ∗ Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil Received 8 June 2006; received in revised form 25 August 2006; accepted 26 September 2006
Abstract The influence of the central nervous system on metabolic function is of interest in situations deviating from basal states, such as during exercise. Our previous study in rats demonstrated that central nitric oxide (NO) blockade increases metabolic rate, reducing mechanical efficiency during exercise. To assess the role of brain nitric oxide in the plasma glucose, lactate and free fatty acids (FFAs) concentrations of rats submitted to an incremental exercise protocol on a treadmill until fatigue, 1.43 mol (2 l) of Nω -nitro-l-arginine methyl ester (l-NAME, n = 6), a NO synthase inhibitor, or 2 l of 0.15 M NaCl (SAL, n = 6) was injected into the lateral cerebral ventricle (icv) of male Wistar rats immediately before exercise (starting at 10 m/min, with increments of 1 m/min every 3 min until fatigue, 10% inclination). Blood samples were collected through a chronic jugular catheter at rest and during exercise until fatigue. During exercise, the l-NAME-treated animals had the following metabolic response compared to controls: (1) an increased hyperglycemic response during the first 60% of time to fatigue; (2) higher plasma lactate levels; and (3) a significant transitory increase in plasma free fatty acids during the dynamic phase of exercise that returned to basal levels earlier than controls during the steady state phase of exercise. In addition l-NAME-treated rats fatigued earlier than controls. The data indicate that the inhibition of the brain nitrergic system induced by icv l-NAME treatment disrupted the accuracy of the neural mechanism that regulates plasma glucose and free fatty acids mobilization during exercise in rats. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Exercise; Glucose; Lactate; Free fatty acids; Fatigue
The central regulation of hepatic glucose production, catecholamine outflow from the adrenal medulla, and the mobilization of free fatty acids (FFAs) from the adipose tissue during exercise have been shown to be mediated by direct sympathetic innervation [7,14]. In such cases, during exercise, the sympathetic drive acts redundantly together with some hormones to increase FFAs mobilization, to stimulate liver glyconeogenesis and glycogenolysis, as well as to inhibit peripheral glucose uptake, thus conserving blood glucose, and inducing FFAs utilization as energetic fuel [4]. In our previous study, intracerebroventricular (icv) infusion of Nω -nitro-l-arginine methyl este [l-NAME—a nitric oxide
∗ Corresponding author at: Departamento de Fisiologia e Biof´ısica, Instituto de Ciˆencias Biol´ogicas/UFMG, Av. Antˆonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil. Tel.: +55 31 34992936; fax: +55 31 34992924. E-mail address: [email protected] (C.C. Coimbra).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.09.067
synthase (NOS) blocker] induced a significant increase in metabolic rate and a decrease in mechanical efficiency, reducing running performance in rats [12]. However, we do not know whether fuel sources were also altered with the higher metabolic rate as a result of l-NAME-treated rats. Matsumura et al. [16] demonstrated that intracerebroventricular infusion of l-NAME increases plasma epinephrine and norepinephrine concentrations at rest, inducing hyperglycemia. They attributed these findings mainly to the enhanced sympathetic outflow. However, it is not known whether this central inhibitory effect on sympathetic outflow by brain NO would affect plasma glucose, lactate, and FFAs levels during exercise in the same extension as it did in metabolic rate [12]. Therefore, the influence of the central nervous system on metabolic adjustments during exercise is of particular interest. Exercise represents a physiological state in which metabolic and hormonal adaptations are required to increase the supply of glucose and FFAs levels to the working muscle and to maintain an adequate flow of glucose to the
A.C.R. Lacerda et al. / Neuroscience Letters 410 (2006) 152–156
brain [6]. It has been well established that the sympathoadrenal system is activated during exercise, resulting in increased plasma concentrations of epinephrine and norepinephrine [7]. However, there is no evidence about the relative role of central nitric oxide (NO) on metabolic adjustments during exercise. So, we hypothesized that brain NO inhibition might be induce a much more increase on sympathetic outflow to the liver, adipose tissue, and adrenal medulla during exercise. As a result, we could observe a hyperglycemic and lactic response and an augmented free fatty acids mobilization, despite of the metabolic demands of the exercising muscles. The aim of this study was to assess the effects of the central administration of the NOS inhibitor l-NAME on plasma glucose, lactate and FFAs concentrations of untrained rats submitted to progressive exercise until fatigue. Male Wistar rats (200–280 g) were individually housed under 14/10 h, light–dark cycles and had free access to water and rat chow. Following anesthesia achieved using 2,2,2tribromoethanol (300 mg/kg body weight i.p.), the rats were fixed to a stereotaxic apparatus (David Kopf Instruments, M900, Tujunga, CA, USA) and a guide cannula (22 G) was implanted into the right lateral cerebral ventricle using a previously described technique [15,20]. A chronic jugular vein catheter was also implanted into these animals as described previously [14]. All animals were allowed to recover for at least 1 week before being submitted to the experiments. The rats were familiarized with exercise on the motor-driven treadmill by running at 10% inclination for 5 min per day for the 5 consecutive days prior to the experiments. In the first and second day of familiarization, they exercised at a speed of 10 m min−1 , while in the subsequent days of familiarization they exercised at a speed of 11, 13 and 15 m min−1 consecutively. The purpose of this preliminary exercise was to show the animals in which direction to run. All experiments were approved by the Ethics Committee for the Care and Use of Laboratory Animals at the Federal University of Minas Gerais and were carried out in accordance with the regulations described in the Committee’s Guiding Principles Manual (protocol 012/05). All the proceedings were carried out between 13:00 and 17:00 h at a room temperature of 21 ± 2 ◦ C. On the day of the experiment, the animals were allowed to rest for 1 h in the rodent treadmill chamber before being submitted to the test. A needle (30 G) protruding 0.3 mm from the tip of the guide cannula was introduced into the right lateral cerebral ventricle by connecting it to a Hamilton syringe. Immediately prior to exercise, 2.0 L of 0.15 M NaCl (n = 6) or 2.0 L of l-NAME (1.43 mol, n = 6) was injected into the right lateral ventricle. The dose of brain lNAME was based on the results of our previous experiments that showed that the response of reduction in workload was clearly l-NAME dose-dependent [11]. Rats were randomly assigned to groups receiving either saline or l-NAME solution. Immediately after the injections, the animals were submitted to progressive running exercise until reaching fatigue. Progressive work was performed on a motor-driven treadmill (Columbus Instruments, OH, USA, Modular Treadmill) at a constant slope of 10◦ . The rats started running at 10 m min−1 and treadmill speed was increased by 1 m min−1 every 3 min until
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Fig. 1. Effect of icv injection of l-NAME (1.43 mol/2 L) or 0.15 M NaCl (2 L, SAL) on workload until fatigue. n = 6 in each group. * Significantly different from the control group (p < 0.05).
fatigue. Fatigue was defined as the point at which the animals were no longer able to keep pace with the treadmill [25,26]. Time to fatigue (minutes) and workload (kgm) were considered indexes of running performance. During the experimental situation, blood samples (0.3 mL) were collected at rest, using a jugular vein catheter [8], before l-NAME or SAL intracerebroventricular infusion, at 3, 6, 9, 12, 15, and 21 min following initiation of exercise and at fatigue point. The blood volume collected in each sample was replaced by normal donated blood to avoid reduction in the blood volume of the animal. The blood samples were centrifuged and the plasma separated. The plasma samples were then frozen (−20 ◦ C) until biochemical analyses were carried out using the enzymatic method of glucose and lactate oxidase (Glucose Analyser YSI 300 Plus), and the spectrophotometric method of FFAs (WACO kit). Workload (W; kgm) was calculated as W = body weight (kg) × TTF × treadmill speed (m min−1 ) × sin θ (treadmill inclination) [1,2,15], where TTF is time to fatigue (minutes). The data are reported as mean + S.E.M. Two experimental groups and six sequential times on treadmill defined a split plot design with six replications. Target response mean between groups were compared at each treadmill speed. Also, within each group, mean at each treadmill speed was compared to rest levels solely. All comparisons were made using Student’s t-test (α ≤ 0.05). Intracerebroventricular injection of l-NAME in untrained normal rats resulted in a marked decrease in workload (lNAME: 6.0 ± 0.6 kgm versus SAL: 7.8 ± 0.6 kgm, p < 0.01) (Fig. 1) and time to fatigue (l-NAME: 19.6 ± 2.5 min versus SAL: 23.3 ± 1.9 min, p < 0.01) compared to SAL-treated rats. As seen in Fig. 2A and D, exercise induced a rise in plasma glucose level in control animals, which was only evident as early as 12 m/min (9 min of exercise) (2A) and 80% of maximal work (2D), and continued to rise until fatigue. l-NAME-treated rats exhibited a rise in plasma glucose concentration throughout the experiment, however showing an increased hyperglycemic response compared to control animals during the first 60% of maximal work.
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Fig. 2. Effect of icv injection of l-NAME (1.43 mol/2 L) or 0.15 M NaCl (2 L, SAL) on increase in plasma glucose (A and D; basal level—l-NAME: 5.4 ± 0.2 mM vs. SAL:5.8 ± 0.2 mM), lactate (B and E; basal level—l-NAME: 1.9 ± 0.3 mM vs. 1.8 ± 0.1 mM), and free fatty acid concentrations (C and F; basal level—l-NAME: 0.46 ± 0.05 mM vs. SAL: 0.66 ± 0.10 mM). On the left panel, the values represent the animals that were still exercising. The number in the brackets represents the rats that were not fatigued. Values are expressed as mean ± S.E.M. n = 6 in each group. * p < 0.05 compared with saline-treated group. + p < 0.05 compared with corresponding basal value.
Plasma lactate level increased in both groups during the progressive exercise until fatigue. However, much higher values were recorded in l-NAME-treated rats during all the experimental protocol compared to SAL-treated animals (Fig. 2B and E). The analysis under the lactate curve showed that plasma lactate accumulation during exercise in l-NAME-treated rats was ∼25% higher than control (p ≤ 0.02). Plasma FFAs levels in the control animals remained stable throughout the experiment. On the other hand, there
was a marked increase in plasma FFAs concentration in l-NAME-treated rats that was evident as early as 3 min (10 m/min) and started higher than control during the first 20% of maximal work. Furthermore, at this speed and at this stage in the exercise, the plasma FFAs levels of l-NAMEtreated animals were ∼15% higher than those of the controls (p < 0.01). Thereafter, plasma FFAs level returned to basal values and remained stable throughout the experiment (Fig. 2C–F).
A.C.R. Lacerda et al. / Neuroscience Letters 410 (2006) 152–156
The results of the present study show that inhibition of the nitric oxide pathway in the CNS by icv injection of l-NAME shifts the balance of substrate utilization during exercise. lNAME treatment increased plasma glucose concentration and glycolytic flux as showed by the huge increase in plasma lactate accumulation. Furthermore, the inhibition of the brain nitrergic system promoted high levels of plasma free fatty acids in the initial 20% of maximal work. This data suggested an increased peripheral sympathetic outflow in the liver and adipose tissue. To the best of our knowledge, this is the first study describing the role of brain nitric oxide in the neural control of plasma glucose and lactate levels, and free fatty acids mobilization from adipose tissue during exercise. It has generally been assumed that the regulation of energy metabolism during exercise is a very sensitive feedback mechanism that depends on the metabolic demands of the exercising muscles and the ambient glucose and free fatty acids concentrations in plasma [6]. The experiment reported here with saline-injected rats is in agreement with this assumption. In these animals, the blood concentrations of glucose and FFAs remained unchanged during exercise, since the feedback regulation mechanism accurately matched the hepatic glucose production and FFAs mobilization from adipose tissue to the peripheral uptake of these metabolites. Our results with l-NAME injection into the cerebral ventricle suggests that this agent disrupted the feedback adjustments during exercise, since the match between energy production and utilization was not as accurate as observed in saline animals. In l-NAME-treated rats both plasma glucose and FFAs concentrations increased during exercise. The metabolic profile observed after l-NAME injection suggests a defective feedback mechanism adjustment in these animals. Therefore, because of this increase in blood glucose and in free fatty acids, the reduction in running performance in l-NAME-treated rats may not be related to availability of an energy substrate. Interestingly, the high lactate concentration observed in lNAME-treated rats was even higher than the lactate threshold described in the literature for running rats (3.0–4.0 mM for rats) at 80% of VO2max [13,19]. Such a high concentration indicates a new regulation point of the renovation rate of the plasma lactate. This shift in lactate during exercise is in agreement with the previous observations of an increased sympathoadrenal activity in l-NAME-treated rats [12]. The findings of various studies involving icv administration of NOS blockers [17,18,24] or administration within specific sites in the central nervous system (CNS) [18,21], are all in general agreement with the view that the central nitric oxide system is inhibitory to overall sympathetic outflow. Matsumura et al. [16] demonstrated that intracerebroventricular infusion of l-NAME increases epinephrine and norepinephrine plasmatic concentrations at rest, inducing hyperglycemia. They attributed these findings mainly to the enhanced sympathetic outflow. Our results are in agreement with the general idea that central NO plays a role in reducing sympathetic tonus [17,18,21]. As central regulation of hepatic glucose production, catecholamine outflow from the adrenal medulla, and the mobilization of free fatty acids from the adipose tissue during exercise are mediated by direct
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sympathetic innervation together with some hormones [4,7,14], we believe that the hyperglycemia, the increased plasma lactate concentration, and the high level of free fatty acids in the initial 20% of time to fatigue induced by l-NAME treatment might be related to a much more accentuated increase on sympathetic outflow to the liver, adipose tissue, and adrenal medulla. The exact location and precise pathways involved in the nitrergic mediation of metabolic homeostasis during exercise still require clarification. However, hypothalamic regions expressing NOS, such as the preoptic area or paraventricular nucleus, are possible sites. It has been established that the POA/AH is an integrative region for the maintenance of metabolic, vasomotor and thermal homeostasis [3,5,9,10,22,23]. However, further research is necessary to identify the exact location of nitrergic mediation involved in normal metabolic homeostasis during exercise. In summary, the results of this study indicate that central NO plays an important role in metabolic adjustment during exercise and that central blockage of nitric oxide synthase by icv injection of l-NAME in rats induces increased glycolytic flux as shown by a higher lactate accumulation in plasma, and high levels of plasma free fatty acids in the initial stages of exercise. Acknowledgements The authors are grateful to Dr. Ivan Barbosa Machado Sampaio for help with the statistical analysis. The technical assistance of Mr. Andr´e Luis Pimenta de Faria is also acknowledged. This study was supported by grants from CNPq, CAPES, and FAPEMIG. References [1] G.A. Brooks, C.M. Donovan, T.P. White, Estimation of anaerobic energy production and efficiency in rats during exercise, J. Appl. Physiol. 56 (1984) 520–525. [2] G.A. Brooks, T.P. White, Determination of metabolic and heart rate responses of rats to treadmill exercise, J. Appl. Physiol. 45 (1978) 1009–1015. [3] C.C. Coimbra, R.H. Migliorin, Cold induced free fatty acid mobilization is impaired in rats with lesions in preoptic area, Neurosci. Lett. 88 (1988) 1–5. [4] R.H. Coker, M. Kjaer, Glucoregulation during exercise: the role of the neuroendocrine system, Sports Med. 35 (2005) 575–583. [5] M.L. Ferreira, U. Marubayashi, C.C. Coimbra, The medial preoptic area modulates the increase in plasma glucose and free fatty acid mobilization induced by cold exposure, Brain Res. Bull. 49 (1999) 189–193. [6] H. Galbo, Exercise physiology: humoral function, Sport Sci. Rev. 1 (1992) 65–93. [7] H. Galbo, E.A. Richter, N.J. Christensen, J.J. Holst, Sympathetic control of metabolic and hormonal responses to exercise in rats, Acta Physiol. Scand. 102 (1978) 441–449. [8] P.G. Harms, S.R.A. Ojeda, A rapid and simple procedure for chronic cannulation of rat jugular vein, J. Appl. Physiol. 36 (1974) 391–392. [9] H. Hasegawa, T. Ishiwata, T. Saito, T. Yazawa, Y. Aiahara, R. Meeusen, Inhibition of the preoptic area and anterior hypothalamus by tetrodotoxin alters thermoregulatory functions in exercising rats, J. Appl. Physiol. 98 (2005) 1458–1462. [10] T. Ishiwata, H. Hasegawa, T. Yazawa, M. Otokawa, Y. Aihara, Functional role of the preoptic area and anterior hypothalamus in thermoregulation in freely moving rats, Neurosci. Lett. 325 (2002) 167–170.
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[11] A.C.R. Lacerda, U. Marubayashi, C.H. Balthazar, C.C. Coimbra, Nitric oxide pathway is an important modulator of heat loss in rats during exercise, Brain Res. Bull. 67 (2005) 110–116. [12] A.C.R. Lacerda, U. Marubayashi, C.H. Balthazar, C.C. Coimbra, Evidence that brain nitric oxide inhibition increases metabolic cost of exercise, reducing running performance in rats, Neurosci. Lett. 393 (2006) 260–263. [13] J. Langfort, R. Zarzaczny, W. Pilis, H. Kaciuba-Uscilko, K. Nazar, S. Porta, Effect of sustained hyperadrenalinemia on exercise performance and lactate threshold in rats, Comp. Biochem. Physiol. A Physiol. 114A (1996) 51–55. [14] N.R.V. Lima, C.C. Coimbra, U. Marubayashi, Effect of intracerebroventricular injection of atropine on metabolic responses during exercise in untrained rats, Physiol. Behav. 64 (1998) 69–74. [15] N.R.V. Lima, W. Pereira, A.M. Reis, C.C. Coimbra, U. Marubayashi, Prolactin release during exercise in normal and adrenodemedullated untrained rats submitted to central cholinergic blockade with atropine, Horm. Behav. 40 (2001) 526–532. [16] K. Matsumura, I. Abe, T. Tsuchihashi, M. Fujishima, Central nitric oxide attenuates the baroreceptor reflex in conscious rabbits, Am. J. Physiol. 274 (1998) 1142–1149. [17] M. Monda, S. Amaro, A. Sullo, B. De Luca, Nitric oxide reduces body temperature and sympathetic input to brown adipose tissue during PGE1hyperthermia, Brain Res. Bull. 38 (1995) 489–493. [18] K.P. Patel, Y. Li, Y. Hirooka, Role of nitric oxide in central sympathetic outflow, Exp. Biol. Med. 226 (2001) 814–824.
[19] W. Pilis, R. Zarzeczny, J. Langfort, H. Kaciuba-Uscilko, K. Nazar, J. Wojtyna, Anaerobic threshold in rats, Comp. Biochem. Physiol. Comp. Physiol. 102A (1993) 285–289. [20] A.G. Rodrigues, N.R.V. Lima, C.C. Coimbra, U. Marabayashi, Intracerebroventricular physostigmine facilitates heat loss mechanisms in running rats, J. Appl. Physiol. 97 (2004) 333–338. [21] W.A. Saad, I.F.M.S. Guarda, L.A.A. Camargo, G. Garcia, L.I. Gutierrez, W.A. Saad, S. Sim˜oes, R. Guarda, Lateral hypothalamus lesions influences water and salt intake, and sodium and urine excretion, and arterial blood pressure induced by l-NAME and FK 409 injections into median preoptic nucleus in conscious rats, Life Sci. 75 (2004) 685–697. [22] G.L. Santos, J.V.P. Leite, C.C. Coimbra, Metabolic adjustments induced by exposure to elevated ambient temperature is impaired in rats bearing lesions in the preoptic area, Braz. J. Med. Biol. Res. 23 (1990) 831–834. [23] G.L. Santos, J.V.P. Leite, C.C. Coimbra, Metabolic adjustment during adaptation to high ambient temperature in preoptic-lesioned rats, Braz. J. Med. Biol. Res. 24 (1991) 1169–1172. [24] E. Simon, Nitric oxide as a peripheral and central mediator in temperature regulation, Amino Acids 14 (1998) 87–93. [25] D.D. Soares, N.R.V. Lima, C.C. Coimbra, U. Marubayashi, Evidence that tryptophan reduces mechanical efficiency and running performance in rats, Pharmacol. Biochem. Behav. 74 (2003) 357–362. [26] D.D. Soares, N.R.V. Lima, C.C. Coimbra, U. Marubayashi, Intracerebroventricular tryptophan increases heating and heat storage rate in exercising rats, Pharmacol. Biochem. Behav. 78 (2004) 255–261.
Central nitric oxide inhibition modifies metabolic adjustments induced by exercise in rats Ana Cristina R. Lacerda, Umeko Marubayashi, Cl´audio H. Balthazar, Laura H.R. Leite, Cˆandido C. Coimbra ∗ Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil Received 8 June 2006; received in revised form 25 August 2006; accepted 26 September 2006
Abstract The influence of the central nervous system on metabolic function is of interest in situations deviating from basal states, such as during exercise. Our previous study in rats demonstrated that central nitric oxide (NO) blockade increases metabolic rate, reducing mechanical efficiency during exercise. To assess the role of brain nitric oxide in the plasma glucose, lactate and free fatty acids (FFAs) concentrations of rats submitted to an incremental exercise protocol on a treadmill until fatigue, 1.43 mol (2 l) of Nω -nitro-l-arginine methyl ester (l-NAME, n = 6), a NO synthase inhibitor, or 2 l of 0.15 M NaCl (SAL, n = 6) was injected into the lateral cerebral ventricle (icv) of male Wistar rats immediately before exercise (starting at 10 m/min, with increments of 1 m/min every 3 min until fatigue, 10% inclination). Blood samples were collected through a chronic jugular catheter at rest and during exercise until fatigue. During exercise, the l-NAME-treated animals had the following metabolic response compared to controls: (1) an increased hyperglycemic response during the first 60% of time to fatigue; (2) higher plasma lactate levels; and (3) a significant transitory increase in plasma free fatty acids during the dynamic phase of exercise that returned to basal levels earlier than controls during the steady state phase of exercise. In addition l-NAME-treated rats fatigued earlier than controls. The data indicate that the inhibition of the brain nitrergic system induced by icv l-NAME treatment disrupted the accuracy of the neural mechanism that regulates plasma glucose and free fatty acids mobilization during exercise in rats. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Exercise; Glucose; Lactate; Free fatty acids; Fatigue
The central regulation of hepatic glucose production, catecholamine outflow from the adrenal medulla, and the mobilization of free fatty acids (FFAs) from the adipose tissue during exercise have been shown to be mediated by direct sympathetic innervation [7,14]. In such cases, during exercise, the sympathetic drive acts redundantly together with some hormones to increase FFAs mobilization, to stimulate liver glyconeogenesis and glycogenolysis, as well as to inhibit peripheral glucose uptake, thus conserving blood glucose, and inducing FFAs utilization as energetic fuel [4]. In our previous study, intracerebroventricular (icv) infusion of Nω -nitro-l-arginine methyl este [l-NAME—a nitric oxide
∗ Corresponding author at: Departamento de Fisiologia e Biof´ısica, Instituto de Ciˆencias Biol´ogicas/UFMG, Av. Antˆonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil. Tel.: +55 31 34992936; fax: +55 31 34992924. E-mail address: [email protected] (C.C. Coimbra).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.09.067
synthase (NOS) blocker] induced a significant increase in metabolic rate and a decrease in mechanical efficiency, reducing running performance in rats [12]. However, we do not know whether fuel sources were also altered with the higher metabolic rate as a result of l-NAME-treated rats. Matsumura et al. [16] demonstrated that intracerebroventricular infusion of l-NAME increases plasma epinephrine and norepinephrine concentrations at rest, inducing hyperglycemia. They attributed these findings mainly to the enhanced sympathetic outflow. However, it is not known whether this central inhibitory effect on sympathetic outflow by brain NO would affect plasma glucose, lactate, and FFAs levels during exercise in the same extension as it did in metabolic rate [12]. Therefore, the influence of the central nervous system on metabolic adjustments during exercise is of particular interest. Exercise represents a physiological state in which metabolic and hormonal adaptations are required to increase the supply of glucose and FFAs levels to the working muscle and to maintain an adequate flow of glucose to the
A.C.R. Lacerda et al. / Neuroscience Letters 410 (2006) 152–156
brain [6]. It has been well established that the sympathoadrenal system is activated during exercise, resulting in increased plasma concentrations of epinephrine and norepinephrine [7]. However, there is no evidence about the relative role of central nitric oxide (NO) on metabolic adjustments during exercise. So, we hypothesized that brain NO inhibition might be induce a much more increase on sympathetic outflow to the liver, adipose tissue, and adrenal medulla during exercise. As a result, we could observe a hyperglycemic and lactic response and an augmented free fatty acids mobilization, despite of the metabolic demands of the exercising muscles. The aim of this study was to assess the effects of the central administration of the NOS inhibitor l-NAME on plasma glucose, lactate and FFAs concentrations of untrained rats submitted to progressive exercise until fatigue. Male Wistar rats (200–280 g) were individually housed under 14/10 h, light–dark cycles and had free access to water and rat chow. Following anesthesia achieved using 2,2,2tribromoethanol (300 mg/kg body weight i.p.), the rats were fixed to a stereotaxic apparatus (David Kopf Instruments, M900, Tujunga, CA, USA) and a guide cannula (22 G) was implanted into the right lateral cerebral ventricle using a previously described technique [15,20]. A chronic jugular vein catheter was also implanted into these animals as described previously [14]. All animals were allowed to recover for at least 1 week before being submitted to the experiments. The rats were familiarized with exercise on the motor-driven treadmill by running at 10% inclination for 5 min per day for the 5 consecutive days prior to the experiments. In the first and second day of familiarization, they exercised at a speed of 10 m min−1 , while in the subsequent days of familiarization they exercised at a speed of 11, 13 and 15 m min−1 consecutively. The purpose of this preliminary exercise was to show the animals in which direction to run. All experiments were approved by the Ethics Committee for the Care and Use of Laboratory Animals at the Federal University of Minas Gerais and were carried out in accordance with the regulations described in the Committee’s Guiding Principles Manual (protocol 012/05). All the proceedings were carried out between 13:00 and 17:00 h at a room temperature of 21 ± 2 ◦ C. On the day of the experiment, the animals were allowed to rest for 1 h in the rodent treadmill chamber before being submitted to the test. A needle (30 G) protruding 0.3 mm from the tip of the guide cannula was introduced into the right lateral cerebral ventricle by connecting it to a Hamilton syringe. Immediately prior to exercise, 2.0 L of 0.15 M NaCl (n = 6) or 2.0 L of l-NAME (1.43 mol, n = 6) was injected into the right lateral ventricle. The dose of brain lNAME was based on the results of our previous experiments that showed that the response of reduction in workload was clearly l-NAME dose-dependent [11]. Rats were randomly assigned to groups receiving either saline or l-NAME solution. Immediately after the injections, the animals were submitted to progressive running exercise until reaching fatigue. Progressive work was performed on a motor-driven treadmill (Columbus Instruments, OH, USA, Modular Treadmill) at a constant slope of 10◦ . The rats started running at 10 m min−1 and treadmill speed was increased by 1 m min−1 every 3 min until
153
Fig. 1. Effect of icv injection of l-NAME (1.43 mol/2 L) or 0.15 M NaCl (2 L, SAL) on workload until fatigue. n = 6 in each group. * Significantly different from the control group (p < 0.05).
fatigue. Fatigue was defined as the point at which the animals were no longer able to keep pace with the treadmill [25,26]. Time to fatigue (minutes) and workload (kgm) were considered indexes of running performance. During the experimental situation, blood samples (0.3 mL) were collected at rest, using a jugular vein catheter [8], before l-NAME or SAL intracerebroventricular infusion, at 3, 6, 9, 12, 15, and 21 min following initiation of exercise and at fatigue point. The blood volume collected in each sample was replaced by normal donated blood to avoid reduction in the blood volume of the animal. The blood samples were centrifuged and the plasma separated. The plasma samples were then frozen (−20 ◦ C) until biochemical analyses were carried out using the enzymatic method of glucose and lactate oxidase (Glucose Analyser YSI 300 Plus), and the spectrophotometric method of FFAs (WACO kit). Workload (W; kgm) was calculated as W = body weight (kg) × TTF × treadmill speed (m min−1 ) × sin θ (treadmill inclination) [1,2,15], where TTF is time to fatigue (minutes). The data are reported as mean + S.E.M. Two experimental groups and six sequential times on treadmill defined a split plot design with six replications. Target response mean between groups were compared at each treadmill speed. Also, within each group, mean at each treadmill speed was compared to rest levels solely. All comparisons were made using Student’s t-test (α ≤ 0.05). Intracerebroventricular injection of l-NAME in untrained normal rats resulted in a marked decrease in workload (lNAME: 6.0 ± 0.6 kgm versus SAL: 7.8 ± 0.6 kgm, p < 0.01) (Fig. 1) and time to fatigue (l-NAME: 19.6 ± 2.5 min versus SAL: 23.3 ± 1.9 min, p < 0.01) compared to SAL-treated rats. As seen in Fig. 2A and D, exercise induced a rise in plasma glucose level in control animals, which was only evident as early as 12 m/min (9 min of exercise) (2A) and 80% of maximal work (2D), and continued to rise until fatigue. l-NAME-treated rats exhibited a rise in plasma glucose concentration throughout the experiment, however showing an increased hyperglycemic response compared to control animals during the first 60% of maximal work.
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Fig. 2. Effect of icv injection of l-NAME (1.43 mol/2 L) or 0.15 M NaCl (2 L, SAL) on increase in plasma glucose (A and D; basal level—l-NAME: 5.4 ± 0.2 mM vs. SAL:5.8 ± 0.2 mM), lactate (B and E; basal level—l-NAME: 1.9 ± 0.3 mM vs. 1.8 ± 0.1 mM), and free fatty acid concentrations (C and F; basal level—l-NAME: 0.46 ± 0.05 mM vs. SAL: 0.66 ± 0.10 mM). On the left panel, the values represent the animals that were still exercising. The number in the brackets represents the rats that were not fatigued. Values are expressed as mean ± S.E.M. n = 6 in each group. * p < 0.05 compared with saline-treated group. + p < 0.05 compared with corresponding basal value.
Plasma lactate level increased in both groups during the progressive exercise until fatigue. However, much higher values were recorded in l-NAME-treated rats during all the experimental protocol compared to SAL-treated animals (Fig. 2B and E). The analysis under the lactate curve showed that plasma lactate accumulation during exercise in l-NAME-treated rats was ∼25% higher than control (p ≤ 0.02). Plasma FFAs levels in the control animals remained stable throughout the experiment. On the other hand, there
was a marked increase in plasma FFAs concentration in l-NAME-treated rats that was evident as early as 3 min (10 m/min) and started higher than control during the first 20% of maximal work. Furthermore, at this speed and at this stage in the exercise, the plasma FFAs levels of l-NAMEtreated animals were ∼15% higher than those of the controls (p < 0.01). Thereafter, plasma FFAs level returned to basal values and remained stable throughout the experiment (Fig. 2C–F).
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The results of the present study show that inhibition of the nitric oxide pathway in the CNS by icv injection of l-NAME shifts the balance of substrate utilization during exercise. lNAME treatment increased plasma glucose concentration and glycolytic flux as showed by the huge increase in plasma lactate accumulation. Furthermore, the inhibition of the brain nitrergic system promoted high levels of plasma free fatty acids in the initial 20% of maximal work. This data suggested an increased peripheral sympathetic outflow in the liver and adipose tissue. To the best of our knowledge, this is the first study describing the role of brain nitric oxide in the neural control of plasma glucose and lactate levels, and free fatty acids mobilization from adipose tissue during exercise. It has generally been assumed that the regulation of energy metabolism during exercise is a very sensitive feedback mechanism that depends on the metabolic demands of the exercising muscles and the ambient glucose and free fatty acids concentrations in plasma [6]. The experiment reported here with saline-injected rats is in agreement with this assumption. In these animals, the blood concentrations of glucose and FFAs remained unchanged during exercise, since the feedback regulation mechanism accurately matched the hepatic glucose production and FFAs mobilization from adipose tissue to the peripheral uptake of these metabolites. Our results with l-NAME injection into the cerebral ventricle suggests that this agent disrupted the feedback adjustments during exercise, since the match between energy production and utilization was not as accurate as observed in saline animals. In l-NAME-treated rats both plasma glucose and FFAs concentrations increased during exercise. The metabolic profile observed after l-NAME injection suggests a defective feedback mechanism adjustment in these animals. Therefore, because of this increase in blood glucose and in free fatty acids, the reduction in running performance in l-NAME-treated rats may not be related to availability of an energy substrate. Interestingly, the high lactate concentration observed in lNAME-treated rats was even higher than the lactate threshold described in the literature for running rats (3.0–4.0 mM for rats) at 80% of VO2max [13,19]. Such a high concentration indicates a new regulation point of the renovation rate of the plasma lactate. This shift in lactate during exercise is in agreement with the previous observations of an increased sympathoadrenal activity in l-NAME-treated rats [12]. The findings of various studies involving icv administration of NOS blockers [17,18,24] or administration within specific sites in the central nervous system (CNS) [18,21], are all in general agreement with the view that the central nitric oxide system is inhibitory to overall sympathetic outflow. Matsumura et al. [16] demonstrated that intracerebroventricular infusion of l-NAME increases epinephrine and norepinephrine plasmatic concentrations at rest, inducing hyperglycemia. They attributed these findings mainly to the enhanced sympathetic outflow. Our results are in agreement with the general idea that central NO plays a role in reducing sympathetic tonus [17,18,21]. As central regulation of hepatic glucose production, catecholamine outflow from the adrenal medulla, and the mobilization of free fatty acids from the adipose tissue during exercise are mediated by direct
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sympathetic innervation together with some hormones [4,7,14], we believe that the hyperglycemia, the increased plasma lactate concentration, and the high level of free fatty acids in the initial 20% of time to fatigue induced by l-NAME treatment might be related to a much more accentuated increase on sympathetic outflow to the liver, adipose tissue, and adrenal medulla. The exact location and precise pathways involved in the nitrergic mediation of metabolic homeostasis during exercise still require clarification. However, hypothalamic regions expressing NOS, such as the preoptic area or paraventricular nucleus, are possible sites. It has been established that the POA/AH is an integrative region for the maintenance of metabolic, vasomotor and thermal homeostasis [3,5,9,10,22,23]. However, further research is necessary to identify the exact location of nitrergic mediation involved in normal metabolic homeostasis during exercise. In summary, the results of this study indicate that central NO plays an important role in metabolic adjustment during exercise and that central blockage of nitric oxide synthase by icv injection of l-NAME in rats induces increased glycolytic flux as shown by a higher lactate accumulation in plasma, and high levels of plasma free fatty acids in the initial stages of exercise. Acknowledgements The authors are grateful to Dr. Ivan Barbosa Machado Sampaio for help with the statistical analysis. The technical assistance of Mr. Andr´e Luis Pimenta de Faria is also acknowledged. This study was supported by grants from CNPq, CAPES, and FAPEMIG. References [1] G.A. Brooks, C.M. Donovan, T.P. White, Estimation of anaerobic energy production and efficiency in rats during exercise, J. Appl. Physiol. 56 (1984) 520–525. [2] G.A. Brooks, T.P. White, Determination of metabolic and heart rate responses of rats to treadmill exercise, J. Appl. Physiol. 45 (1978) 1009–1015. [3] C.C. Coimbra, R.H. Migliorin, Cold induced free fatty acid mobilization is impaired in rats with lesions in preoptic area, Neurosci. Lett. 88 (1988) 1–5. [4] R.H. Coker, M. Kjaer, Glucoregulation during exercise: the role of the neuroendocrine system, Sports Med. 35 (2005) 575–583. [5] M.L. Ferreira, U. Marubayashi, C.C. Coimbra, The medial preoptic area modulates the increase in plasma glucose and free fatty acid mobilization induced by cold exposure, Brain Res. Bull. 49 (1999) 189–193. [6] H. Galbo, Exercise physiology: humoral function, Sport Sci. Rev. 1 (1992) 65–93. [7] H. Galbo, E.A. Richter, N.J. Christensen, J.J. Holst, Sympathetic control of metabolic and hormonal responses to exercise in rats, Acta Physiol. Scand. 102 (1978) 441–449. [8] P.G. Harms, S.R.A. Ojeda, A rapid and simple procedure for chronic cannulation of rat jugular vein, J. Appl. Physiol. 36 (1974) 391–392. [9] H. Hasegawa, T. Ishiwata, T. Saito, T. Yazawa, Y. Aiahara, R. Meeusen, Inhibition of the preoptic area and anterior hypothalamus by tetrodotoxin alters thermoregulatory functions in exercising rats, J. Appl. Physiol. 98 (2005) 1458–1462. [10] T. Ishiwata, H. Hasegawa, T. Yazawa, M. Otokawa, Y. Aihara, Functional role of the preoptic area and anterior hypothalamus in thermoregulation in freely moving rats, Neurosci. Lett. 325 (2002) 167–170.
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