J Physiol Biochem (2014) 70:417–423 DOI 10.1007/s13105-014-0319-2
ORIGINAL PAPER
Exercise prevents leptin-induced increase in blood pressure in Sprague–Dawley rats K. Farhana & I. Effendi & Brinnell Caszo & Nuraliza Abdul Satar & HJ Singh
Received: 1 October 2013 / Accepted: 29 January 2014 / Published online: 8 April 2014 # University of Navarra 2014
Abstract Although leptin has been shown to increase blood pressure (BP), it is however unclear if this increase can be prevented by exercise. This study therefore investigated the effect of leptin treatment with concurrent exercise on blood pressure (BP), sodium output, and endothelin-1 (ET-1) levels in normotensive rats. Male Sprague–Dawley rats weighing 250–270 g were divided into four groups consisting of a control group (n=6), leptin-treated (n=8), non-leptin-treated exercise group (n=8), and a leptin-treated exercise group (n=8). Leptin was given subcutaneously daily for 14 days (60 μg/kg/day). Animals were exercised on a treadmill for 30 min at a speed of 0.5 m/s and at 5° incline four times per week. Measurement of systolic blood pressure (SBP) and collection of urine samples for estimation of sodium and creatinine was done once a week. Serum samples were collected at the end of the experiment for determination of sodium, creatinine and ET-1. At day 14, mean SBP and serum ET-1 level in the leptin-treated group was significantly higher than that in
K. Farhana : I. Effendi (*) : N. A. Satar Faculty of Medicine, Universiti Teknologi Mara, Sg.Buloh, Selangor, Malaysia e-mail: [email protected] B. Caszo Faculty of Medicine, University Pertahanan Nasional Malaysia, Kem Sungai Besi, 57000 Kuala Lumpur, Malaysia H. Singh Faculty of Medicine, & Institute of Medical Molecular Biology, Universiti Teknologi Mara, Sg. Buloh, Selangor, Malaysia
the control group whereas mean SBP and serum ET-1 level was significantly lower in the leptin-treated exercise group than those in leptin-treated and control groups. Creatinine clearance, urinary sodium excretion, and urine output were not different between the four groups. Regular treadmill exercise prevents leptininduced increases in SBP in rats, which might in part result from increased urinary sodium excretion and preventing the leptin-induced increases in serum ET-1 concentration. Keywords Blood pressure . Leptin . Urinary sodium excretion . Endothelin-1 . Exercise
Introduction Obesity is associated with increased risk of cardiovascular disease, and the prevalence of hypertension is higher in obese and overweight populations [30, 31]. The precise reason for this is not clear, but recent studies have shown that adipose tissue secretes numerous metabolically active mediators including leptin, adiponectin, visfatin, resistin, and chemerin [25, 28]. Among these, leptin has emerged as an important hormone that might provide a link between obesity and hypertension [42]. Results from studies on the effects of leptin on blood pressure however remain somewhat equivocal. Animal studies in vivo and in vitro seem to indicate that leptin has a blood pressure-lowering effect [16, 17, 40]. On the other hand, higher plasma leptin levels have been associated with hypertension in both men and women [2, 14, 42]. Long-term leptin
418
administration to normal nonpregnant [37] or pregnant rats [20] has been shown to increase blood pressure. The precise reason for the differences is unclear, but it seems to suggest that leptin might have a dual effect depending on the duration of exposure. Numerous mechanisms have been proposed to explain the leptin-induced increase in blood pressure. Some of these include (a) increase in sympathetic nerve activity, particularly in the kidneys and the adrenal gland possibly involving the caudal nucleus tractus solitarii; (b) impairment of baroreflex sensitivity that might contribute permissively to increases in arterial blood pressure; (c) altered endothelial function and vasomotor tone; and (d) changes in salt and water handling by the kidney where leptin has been shown to alter renal Na+-K+-ATPase activity [3, 4, 19, 27, 29]. The effects of leptin on renal sodium handling, however, seem to depend on the duration of leptin exposure. Short-term leptin administration increases sodium excretion but chronic leptin treatment, on the other hand, seems to impair sodium excretion [7, 27, 41]. Interestingly, the natriuretic effect of leptin is somewhat diminished in obese and hypertensive rats where chronic hyperleptinemia is often also present [6, 39, 41]. The reason for this is not known, but it might be inferred that this diminished response alters salt and water balance consequently resulting in sodium retention, increased intravascular volume and cardiac output, which, together with high sympathetic activity, could lead to elevation of blood pressure. In the management of hypertension, both pharmacological and nonpharmacological approaches are often used. The latter involves regular physical exercise. Regular physical activity is an important means of fighting overweight and obesity [12], and physical activity is often recommended as a nonpharmacological way of managing hypertension as it also reduces the risk of coronary artery disease in hypertensive individuals [1, 23]. The impact of exercise on leptin-induced increase in blood pressure in non-obese individuals has not been investigated before. We hypothesize that regular physical exercise reduces the impact of leptin on blood pressure, which might involve changes in renal salt and water handling and serum endothelin-1 (ET-1) levels. The main aim of this study, therefore, was to determine the effect of exercise on leptininduced increases in blood pressure, serum ET-1 levels, and urinary sodium excretion in normal Sprague–Dawley rats.
K. Farhana et al.
Methods Animals and experimental procedure Male Sprague–Dawley rats, aged 8–10 weeks and weighing 250–270 g were divided into four groups, namely, control (n=6), leptin-treated non-exercised (n=8), exercise only (n=8), and leptin-treated exercised (n=8) groups. All animals were housed individually in metabolic cages with a 12:12 light/dark cycle and had excess ad libitum to food and water. Leptin (BioVision Inc, Milpitas, CA, USA) was given subcutaneously once daily at a dose of 60 μg/kg in 0.1 ml normal saline for 14 days. Non-leptin-treated control and the exercised group received 0.1 ml normal saline subcutaneously once daily for 14 days. Body weight was measured on days 0, 7, and 14. All aspects of animal care and experimentation were approved by the Institute’s Animal Care and Users Committee, Faculty of Medicine, Universiti Teknologi MARA. Exercise procedure Rats were exercised four times per week on a small animal treadmill (Columbus Instruments, Columbus, OH, USA) for 30 min each time at a speed of 0.5 m/s and at a 5° incline. All exercise was performed at the same time of the day. A three-track treadmill was used, and three animals were exercised each time. The observer was present at all times during the exercise to ensure the safety of the animals during exercise. Blood pressure measurements Blood pressure was measured on days 0, 7, and 14, using tail-cuff plethysmography (Kent Scientific, Torrington, CT, USA). All blood pressure measurements were made in conscious rats. To ensure accuracy and reproducibility of measurements, animals were acclimatized to restraint and tail-cuff inflation a couple of days before the commencement of the actual measurement and then for about 15 min of familiarization prior to each measurement. All blood pressure recordings were made at the same time of the day. The restrainers used during the measurement of blood pressure were made of transparent acrylic and fitted with darkened nose cones. After the placement of the animals in the restrainers, an appropriately sized blood pressure cuff was placed on the tail and they were then placed on a thermostat-
Exercise prevents leptin-induced increase in blood pressure
controlled platform (Kent Scientific) at 37 ○C. Body temperature was measured frequently by infrared thermometer to ensure the animals remained sufficiently warm (Thermoworks, China). The ambient room temperature was maintained at 22 °C. According to the recommendations of the manufacturer, a set of 25 measurements of blood pressure was considered as a single cycle. Cycles were repeated if necessary, up to a maximum of two cycles. All animals went through 10-trial measurements of blood pressure before data was collected for analysis. The occlusion cuff maximum pressure was set at 250 mmHg. A computer algorithm provided in the software of the instrument selected the data points based on the following parameters: minimum tail blood volume, duration between systolic and diastolic blood pressure measurements, and the overall shape of the pressure trace, and determined the acceptable readings. Sample collection Twenty-four hour urine samples were collected on days 0, 7, and 14 using metabolic cages. On day 14, after the completion of blood pressure measurements and collection of urine samples, the rats were euthanized by decapitation and blood was collected into EDTAcontaining tubes and centrifuged for 15 min at 3,000 rpm (1,000×g) in a refrigerated centrifuge. Serum and urine concentrations of creatinine and sodium were then estimated (Hitachi, Japan). The levels of ET-1 in serum were measured using ELISA (ET-1 Assay kit; IBL Japan). Statistical analyses Statistical analysis was performed using two-way ANOVA (SPSS 16.0) followed by post hoc analysis (Bonferroni and Tukey). A p value of
ORIGINAL PAPER
Exercise prevents leptin-induced increase in blood pressure in Sprague–Dawley rats K. Farhana & I. Effendi & Brinnell Caszo & Nuraliza Abdul Satar & HJ Singh
Received: 1 October 2013 / Accepted: 29 January 2014 / Published online: 8 April 2014 # University of Navarra 2014
Abstract Although leptin has been shown to increase blood pressure (BP), it is however unclear if this increase can be prevented by exercise. This study therefore investigated the effect of leptin treatment with concurrent exercise on blood pressure (BP), sodium output, and endothelin-1 (ET-1) levels in normotensive rats. Male Sprague–Dawley rats weighing 250–270 g were divided into four groups consisting of a control group (n=6), leptin-treated (n=8), non-leptin-treated exercise group (n=8), and a leptin-treated exercise group (n=8). Leptin was given subcutaneously daily for 14 days (60 μg/kg/day). Animals were exercised on a treadmill for 30 min at a speed of 0.5 m/s and at 5° incline four times per week. Measurement of systolic blood pressure (SBP) and collection of urine samples for estimation of sodium and creatinine was done once a week. Serum samples were collected at the end of the experiment for determination of sodium, creatinine and ET-1. At day 14, mean SBP and serum ET-1 level in the leptin-treated group was significantly higher than that in
K. Farhana : I. Effendi (*) : N. A. Satar Faculty of Medicine, Universiti Teknologi Mara, Sg.Buloh, Selangor, Malaysia e-mail: [email protected] B. Caszo Faculty of Medicine, University Pertahanan Nasional Malaysia, Kem Sungai Besi, 57000 Kuala Lumpur, Malaysia H. Singh Faculty of Medicine, & Institute of Medical Molecular Biology, Universiti Teknologi Mara, Sg. Buloh, Selangor, Malaysia
the control group whereas mean SBP and serum ET-1 level was significantly lower in the leptin-treated exercise group than those in leptin-treated and control groups. Creatinine clearance, urinary sodium excretion, and urine output were not different between the four groups. Regular treadmill exercise prevents leptininduced increases in SBP in rats, which might in part result from increased urinary sodium excretion and preventing the leptin-induced increases in serum ET-1 concentration. Keywords Blood pressure . Leptin . Urinary sodium excretion . Endothelin-1 . Exercise
Introduction Obesity is associated with increased risk of cardiovascular disease, and the prevalence of hypertension is higher in obese and overweight populations [30, 31]. The precise reason for this is not clear, but recent studies have shown that adipose tissue secretes numerous metabolically active mediators including leptin, adiponectin, visfatin, resistin, and chemerin [25, 28]. Among these, leptin has emerged as an important hormone that might provide a link between obesity and hypertension [42]. Results from studies on the effects of leptin on blood pressure however remain somewhat equivocal. Animal studies in vivo and in vitro seem to indicate that leptin has a blood pressure-lowering effect [16, 17, 40]. On the other hand, higher plasma leptin levels have been associated with hypertension in both men and women [2, 14, 42]. Long-term leptin
418
administration to normal nonpregnant [37] or pregnant rats [20] has been shown to increase blood pressure. The precise reason for the differences is unclear, but it seems to suggest that leptin might have a dual effect depending on the duration of exposure. Numerous mechanisms have been proposed to explain the leptin-induced increase in blood pressure. Some of these include (a) increase in sympathetic nerve activity, particularly in the kidneys and the adrenal gland possibly involving the caudal nucleus tractus solitarii; (b) impairment of baroreflex sensitivity that might contribute permissively to increases in arterial blood pressure; (c) altered endothelial function and vasomotor tone; and (d) changes in salt and water handling by the kidney where leptin has been shown to alter renal Na+-K+-ATPase activity [3, 4, 19, 27, 29]. The effects of leptin on renal sodium handling, however, seem to depend on the duration of leptin exposure. Short-term leptin administration increases sodium excretion but chronic leptin treatment, on the other hand, seems to impair sodium excretion [7, 27, 41]. Interestingly, the natriuretic effect of leptin is somewhat diminished in obese and hypertensive rats where chronic hyperleptinemia is often also present [6, 39, 41]. The reason for this is not known, but it might be inferred that this diminished response alters salt and water balance consequently resulting in sodium retention, increased intravascular volume and cardiac output, which, together with high sympathetic activity, could lead to elevation of blood pressure. In the management of hypertension, both pharmacological and nonpharmacological approaches are often used. The latter involves regular physical exercise. Regular physical activity is an important means of fighting overweight and obesity [12], and physical activity is often recommended as a nonpharmacological way of managing hypertension as it also reduces the risk of coronary artery disease in hypertensive individuals [1, 23]. The impact of exercise on leptin-induced increase in blood pressure in non-obese individuals has not been investigated before. We hypothesize that regular physical exercise reduces the impact of leptin on blood pressure, which might involve changes in renal salt and water handling and serum endothelin-1 (ET-1) levels. The main aim of this study, therefore, was to determine the effect of exercise on leptininduced increases in blood pressure, serum ET-1 levels, and urinary sodium excretion in normal Sprague–Dawley rats.
K. Farhana et al.
Methods Animals and experimental procedure Male Sprague–Dawley rats, aged 8–10 weeks and weighing 250–270 g were divided into four groups, namely, control (n=6), leptin-treated non-exercised (n=8), exercise only (n=8), and leptin-treated exercised (n=8) groups. All animals were housed individually in metabolic cages with a 12:12 light/dark cycle and had excess ad libitum to food and water. Leptin (BioVision Inc, Milpitas, CA, USA) was given subcutaneously once daily at a dose of 60 μg/kg in 0.1 ml normal saline for 14 days. Non-leptin-treated control and the exercised group received 0.1 ml normal saline subcutaneously once daily for 14 days. Body weight was measured on days 0, 7, and 14. All aspects of animal care and experimentation were approved by the Institute’s Animal Care and Users Committee, Faculty of Medicine, Universiti Teknologi MARA. Exercise procedure Rats were exercised four times per week on a small animal treadmill (Columbus Instruments, Columbus, OH, USA) for 30 min each time at a speed of 0.5 m/s and at a 5° incline. All exercise was performed at the same time of the day. A three-track treadmill was used, and three animals were exercised each time. The observer was present at all times during the exercise to ensure the safety of the animals during exercise. Blood pressure measurements Blood pressure was measured on days 0, 7, and 14, using tail-cuff plethysmography (Kent Scientific, Torrington, CT, USA). All blood pressure measurements were made in conscious rats. To ensure accuracy and reproducibility of measurements, animals were acclimatized to restraint and tail-cuff inflation a couple of days before the commencement of the actual measurement and then for about 15 min of familiarization prior to each measurement. All blood pressure recordings were made at the same time of the day. The restrainers used during the measurement of blood pressure were made of transparent acrylic and fitted with darkened nose cones. After the placement of the animals in the restrainers, an appropriately sized blood pressure cuff was placed on the tail and they were then placed on a thermostat-
Exercise prevents leptin-induced increase in blood pressure
controlled platform (Kent Scientific) at 37 ○C. Body temperature was measured frequently by infrared thermometer to ensure the animals remained sufficiently warm (Thermoworks, China). The ambient room temperature was maintained at 22 °C. According to the recommendations of the manufacturer, a set of 25 measurements of blood pressure was considered as a single cycle. Cycles were repeated if necessary, up to a maximum of two cycles. All animals went through 10-trial measurements of blood pressure before data was collected for analysis. The occlusion cuff maximum pressure was set at 250 mmHg. A computer algorithm provided in the software of the instrument selected the data points based on the following parameters: minimum tail blood volume, duration between systolic and diastolic blood pressure measurements, and the overall shape of the pressure trace, and determined the acceptable readings. Sample collection Twenty-four hour urine samples were collected on days 0, 7, and 14 using metabolic cages. On day 14, after the completion of blood pressure measurements and collection of urine samples, the rats were euthanized by decapitation and blood was collected into EDTAcontaining tubes and centrifuged for 15 min at 3,000 rpm (1,000×g) in a refrigerated centrifuge. Serum and urine concentrations of creatinine and sodium were then estimated (Hitachi, Japan). The levels of ET-1 in serum were measured using ELISA (ET-1 Assay kit; IBL Japan). Statistical analyses Statistical analysis was performed using two-way ANOVA (SPSS 16.0) followed by post hoc analysis (Bonferroni and Tukey). A p value of
