- Email: [email protected]
Chronic administration of NaHS and L-Cysteine restores cardiovascular changes induced by high-fat diet in rats
Journal Pre-proof Chronic administration of NaHS and L-Cysteine restore cardiovascular changes induced by high-fat diet in rats Carolina B. Gomez, Saúl Huerta de la Cruz, Grecia J. Medina-Terol, Jesus H. Beltran-Ornelas, Araceli Sánchez-López, Diana L. Silva-Velasco, David Centurión PII:
S0014-2999(19)30659-4
DOI:
https://doi.org/10.1016/j.ejphar.2019.172707
Reference:
EJP 172707
To appear in:
European Journal of Pharmacology
Received Date: 22 August 2019 Revised Date:
25 September 2019
Accepted Date: 26 September 2019
Please cite this article as: Gomez, C.B., de la Cruz, Saú.Huerta., Medina-Terol, G.J., Beltran-Ornelas, J.H., Sánchez-López, A., Silva-Velasco, D.L., Centurión, D., Chronic administration of NaHS and L-Cysteine restore cardiovascular changes induced by high-fat diet in rats, European Journal of Pharmacology (2019), doi: https://doi.org/10.1016/j.ejphar.2019.172707. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
European Journal of Pharmacology
Chronic administration of NaHS and L-Cysteine restore cardiovascular changes induced by high-fat diet in rats
Carolina B. Gomez, Saúl Huerta de la Cruz, Grecia J. Medina-Terol, Jesus H. BeltranOrnelas, Araceli Sánchez-López, Diana L. Silva-Velasco and David Centurión.
Departamento de Farmacobiología, Cinvestav Unidad Coapa, Czda. de los Tenorios 235, Col. Granjas-Coapa, Del. Tlalpan, C.P. 14330, México D.F., México.
Correspondence: Dr. David Centurión at the above address Phone: (Int.)-(52)-(55)5483 2866 Fax: NA Email: [email protected] Web page: http://farmacobiologia.cinvestav.mx/Personal-Académico/Dr-D-CenturiónPacheco
Abstract Hydrogen sulfide plays an important role in the regulation of the cardiovascular system, insulin secretion, and glucose homeostasis. The aim of the present study was to examine the effects of chronic treatment with sodium hydrosulfide (NaHS), L-Cysteine (L-Cys) and DL-Propargylglycine (DL-PAG) on the changes induced by a high-fat diet (HFD) in zoometric and metabolic variables as well as cardiovascular changes such as hypertension and sympathetic hyperactivity. For this purpose, male Wistar rats were fed a normal fat diet (NFD) or HFD for 12 weeks. Next, the HFD rats were divided into 5 subgroups which received daily i.p. injections during 4 weeks of: (1) nothing (no injection, Control); (2) vehicle (PBS; 1 ml/kg); (3) NaHS (5.6 mg/kg); (4) L-Cys (300 mg/kg); or (5) DL-PAG (1 mg/kg). Then, an oral glucose tolerance test, hormone serum levels and blood pressure were determined. The cardiovascular responses to stimulation of the vasopressor sympathetic tone or intravenous administration of the agonists noradrenaline (α1/2-adrenoceptors), methoxamine (α1-adrenoceptors) and UK 14,304 (α2-adrenoceptors) were determined in pithed rats. Lastly, the heart, liver and adipose tissue were weighted. HFD significantly increased: (1) zoometric variables, which were decreased by NaHS and L-Cys; (2) metabolic variables, ameliorated by DLPAG; (3) haemodynamic variables, which were reversed by NaHS and L-Cys; and (4) the vasopressor responses induced by sympathetic stimulation, which were diminished by NaHS and L-Cys. In conclusion, chronic treatment with NaHS and L-Cys are effective in reducing adipose tissue and ameliorating the cardiovascular changes induced by obesity; meanwhile, DL-PAG ameliorates metabolic variables.
Keywords: Hydrogen sulfide; obesity; hypertension; cardiovascular.
1. Introduction Hydrogen sulfide (H2S) is a novel gasotransmitter that plays an important role in the regulation of the cardiovascular system. This gas produces complex cardiovascular responses including vasodilatation (Zhao et al., 2001), inhibition of the sympathetic nervous system (Centurion et al., 2018; Centurion et al., 2016), cardioprotection (Hu et al., 2008), and inhibition of renin activity during renovascular hypertension (Lu et al., 2010). Besides, it has been observed that biosynthesis and plasma levels of H2S are significantly reduced during obesity (Whiteman et al., 2010), diabetes mellitus (Jain et al., 2010) and systemic hypertension (Popov, 2013), which may suggest that treatment with H2S releasing drugs could have a beneficial effect in metabolic diseases. Indeed, in recent years, several studies have suggested that during obesity, the levels of H2Sproducing enzymes are reduced in brown and white adipose tissue (Katsouda et al., 2017). Moreover, in mouse C2C12 skeletal muscle myoblast line, expression of the enzymes involved in the production of H2S, cystathionine gamma-lyase (CSE), cystathionine-beta-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) were decreased in palmitic acid-induced insulin resistance (Chen et al., 2017). Therefore, H2S could be implicated in the regulation of insulin release (Beltowski et al., 2018; Chen et al., 2017; Kaneko et al., 2006; Untereiner and Wu, 2017), glucose homeostasis (Untereiner and Wu, 2017), insulin signalling pathway (Chen et al., 2017) and the regulation of incretins and adipokines (Beltowski et al., 2018). Although H2S seems to be beneficial during obesity, there is still a debate whether this rotten smell gas has deleterious or beneficial effects. Indeed, Geng et al. (2013) demonstrated differential effects in high-fat diet (HFD) mice as DL-Propargylglycine (DL-
PAG), an inhibitor of CSE, increased lipolysis and reduced insulin resistance; while GYY4137, a slow H2S donor, inhibited lipolysis and reduced insulin resistance (Geng et al., 2013). More recently, it has been suggested that H2S/CSE pathway promotes differentiation of adipocytes by sulfhydration of PPAR-γ (Peroxisome Proliferator Activated Receptors) (Yang et al., 2018). Despite the above findings, up to date, no study has analysed the effect of chronic administration of an H2S donor on the cardiovascular changes in systemic vasculature produced by obesity in rats. Thus, this study has examined the effects of chronic treatment with sodium hydrosulfide (NaHS), L-Cysteine (L-Cys) and DL-PAG over zoometric, metabolic and cardiovascular changes induced by HFD in rats. These data could shed further light on the controversial effects of H2S on obesity. 2. Materials and methods 2.1. Animals 7 weeks old 36 male Wistar rats (200-250 g) provided by our own laboratory animal facility were housed in plastic cages, maintained under standardized conditions (22 ± 2 °C, 50% humidity and 12/12 h light-dark cycle) and provided with food (either normal or high-fat diet) and water ad libitum. All animal procedures and the protocols of the present study were approved by the Institutional Ethics Committee (Cicual-Cinvestav) following the established regulations by the Mexican Official Norm for the Use and Welfare of Laboratory Animals (NOM-062-ZOO-1999), and following the Guide for the Care and Use of Laboratory Animals in the USA (Council, 2011). 2.2. Diet
The 36 animals were randomly divided into two main groups (Fig. 1A), which were fed with: (1) normal fat diet (NFD, n = 6), and (2) high-fat diet (HFD, n = 30). The NFD was the LabDiet® 5008 pellets and HFD was made with LabDiet® 5008 powder (550 g) mixed with Nutella® (150 g) and lard (300 g). The nutritional information of NFD and HFD is shown in Table 1. Each day, food was replaced with new pellets. Both diets were given during 16 weeks without interruptions. 2.3. Pharmacological treatments As shown in Fig. 1A, after 12 weeks of feeding, the HFD group was divided into five subgroups. Each subgroup received, respectively, intraperitoneal (i.p.) daily administration during 4 weeks of: (1) nothing (no injection, HFD Control); (2) vehicle (phosphate buffer saline, PBS; 1 ml/kg); (3) NaHS (5.6 mg/kg, inorganic H2S donor); (4) L-Cys (300 mg/kg, H2S-producing enzymes substrate); and (5) DL-PAG (1 mg/kg, CSE inhibitor). It should be mentioned that the NFD group did not receive any pharmacological treatment. 2.4. Oral glucose tolerance test and hormone assays After 12 weeks of feeding, both NFD and HFD control groups were fasted for 12 hours; then an oral glucose tolerance test (OGTT) and hormone assays were performed. For OGTT, blood glucose levels (tail vein) were measured at baseline and 5, 10, 15, 30, 60, 90 and 120 min after oral administration of glucose (1 g/kg) through glucometer (AccuCheck®, Roche of Mexico). At the same time, blood was collected for measurement of serum insulin by ELISA (Enzyme-linked Immunosorbent Assay) kit from ALPCO (Salem, NH, USA).
Furthermore, fasting plasma levels of leptin and total ghrelin were measured by ELISA kit from R&D Systems (Minneapolis, MN, USA) and Millipore (Burlington, MA, USA), respectively, as suggested by the provider. The procedures above mentioned were repeated after the chronic administration of vehicle, NaHS, L-Cys or DL-PAG, i.e., after 16 weeks with HFD (see Fig. 1B). 2.5. Determination of arterial blood pressure and heart rate in conscious animals Arterial blood pressure and heart rate were measured by a tail-cuff method using a LE 5001 automatic blood pressure recorder (Letical, PanLab, Barcelona, Spain). Animals were immobilized for a short period in acrylic cages and the tail was exposed to a heating lamp for 15 minutes. A cuff was placed in the tail, which was inflated to stop blood flow, as suggested by the provider. The tension released allowed to determinate systolic blood pressure (SBP) through the sensor; and calculate heart rate (HR), diastolic blood pressure (DBP) and mean blood pressure (MBP). The measurements were done at 12 and 16 weeks in all groups, representing the beginning and the end of the chronic treatment (see Fig. 1B). 2.6. Evaluation of cardiovascular function At the end of the pharmacological treatments, animals were anaesthetized with isoflurane (3%) and the trachea was cannulated. Animals were pithed by inserting a stainless-steel rod through the orbit and foramen magnum into the vertebral foramen (Centurión et al., 2009) aiming the destruction of the central nervous system. As animals were unconscious under these conditions, they were artificially ventilated with room air using a positive pressure pump (7025 rodent ventilator, Ugo Basile, Comerio, VA, Italy) at 80 strokes/min and a stroke volume of 20 ml/kg, as previously described (Kleinman and Radford, 1964). After a bilateral vagotomy, catheters were placed in: (1) right
femoral vein for drug administration and (2) the left carotid artery to record arterial blood pressure and heart rate with a pressure transducer (RX104A, Biopac Systems Inc., Goleta, CA, USA). Both parameters were recorded simultaneously using a data acquisition unit (MP150A-CE, Biopac Systems Inc., Goleta, CA). DBP was determined, as this is the blood pressure when the left ventricle is relaxed and thus could indirectly represent the systemic vascular resistance that regulates arterial blood pressure and blood flow within organs. The body of each pithed rat was maintained at 37 °C by a lamp and monitored with a rectal thermometer. 2.6.1 Experimental design in pithed rats After the procedure mentioned above, the 36 rats went under the same protocol. First, the vasopressor responses induced by selective preganglionic (T7-T9) stimulation of the vasopressor sympathetic outflow were determined. For this purpose, the stainless-steel rod was replaced by an enamelled bipolar electrode except for 1 cm length 9 cm from the tip. The uncovered segment was placed at the T7-T9 region of the spinal cord allowing the selective stimulation of the sympathetic nerves that supply the systemic vasculature (Gillespie and Muir, 1967). Before electrical stimulation, in order to avoid electrically-induced muscle twitching, animals received an intravenous (i.v.) bolus of gallamine (25 mg/kg). The preganglionic vasopressor sympathetic outflow was stimulated with an S88X square pulse stimulator (Grass Technologies, Warwick, RI, USA) by applying 10 s trains of monophasic, rectangular pulses (2 ms, 60 V), at increasing frequencies (0.03, 0.1, 0.3, 1 and 3 Hz). Once the stimuli-response curve was completed, the vasopressor responses induced by i.v. bolus injections of: (1) exogenous noradrenaline (0.03, 0.1, 0.3, 1 and 3 µg/kg; α1- and α2-adrenoceptor agonist, endogenous ligand); (2) methoxamine (1, 3, 10, 30 and 100 µg/kg; a selective α1-
adrenoceptor agonist); and (3) UK 14,304 (0.03, 0.1, 0.3, 1, 3, 10 and 30 µg/kg; a selective α2-adrenoceptor agonist) were evaluated. The electrical stimuli as well as the i.v. bolus injections of the compounds mentioned above were given using a sequential schedule of 0.5 log unit increments. The interval between the different stimulation frequencies or doses was determined depending on the duration of the resulting vasopressor response. Once this protocol was ended, artificial ventilation was turned off and the tissue collection was made by performing a thoracoabdominal incision. 2.7. Drugs In addition to the anaesthetic isoflurane (Fluriso™, Vet Ones, Boise, ID, USA), the compounds used in this study (obtained from the sources indicated) were: sodium hydrosulfide monohydrate (PubChem CID: 28015), L-Cysteine (PubChem CID: 5862), DL-Propargylglycine (PubChem CID: 95575), gallamine triethiodide (PubChem CID: 6172), (±)-noradrenaline bitartrate (PubChem CID: 168929), methoxamine hydrochloride (PubChem CID: 6081) and UK 14,304 (PubChem CID: 2435) (all from Sigma Chemical Co., St. Louis, MO, USA). Noradrenaline, methoxamine, UK 14,304 and gallamine were dissolved in physiological saline. NaHS, L-Cysteine, and DL-Propargylglycine were dissolved in phosphate buffer saline, pH 7.4 and 25 °C; new solutions were prepared every single day. 2.8. Statistical analysis All results in tables and figures are present as mean ± S.E.M. The differences in zoometric, metabolic and haemodynamic variables within groups of animals were evaluated by non-parametric Mann-Whitney U-test. The data obtained in the OGTT are presented as area under the curve (AUC). AUC was calculated by the trapezoidal rule.
The difference between the control group and vehicle in the AUC was evaluated by ttest, while differences between vehicle group and chronic treatments were compared by using one-way analysis of variance. The maximum changes in diastolic blood pressure to either electrical stimulation or α-adrenoceptors agonists as well as the changes in heart rate induced by noradrenaline were determined. The difference between the changes in DBP or heart rate within groups of animals was evaluated by a Tukey´s test, once the Two-Way Analysis of Variance reached statistical significance. Statistical significance was accepted at P<0.05. 3. Results 3.1. Zoometric and metabolic changes produced by high-fat diet and pharmacological treatments Table 2 shows the changes in zoometric variables induced by high-fat diet and the pharmacological treatments. High-fat diet (HFD control) produced a significant increase in body weight and weight of the liver as well as visceral and epididymal adipose tissue, but not in heart weight, when compared with normal fat diet group (NFD Control). Interestingly, body weight was significantly decreased by chronic treatment with NaHS and L-Cys compared to HFD control group, while DL-PAG treatment failed to change this variable. Besides, liver weight was significantly decreased by chronic treatment with NaHS, L-Cys, and DL-PAG when compared with vehicle and HFD control. Further, chronic treatment with vehicle (PBS), significantly diminished visceral adipose tissue compared versus HFD control. Nevertheless, chronic treatments with NaHS and L-Cys significantly diminished visceral adipose tissue when compared with vehicle. Moreover, epididymal adipose tissue was significantly decreased by treatment with NaHS;
conversely, this variable was significantly increased by DL-PAG when compared to vehicle. As shown in table 3, HFD: (1) significantly reduced fasting total ghrelin levels and Matsuda index; (2) significantly increased fasting leptin, insulin, glucose levels and HOMA index, when compared to NFD. It is important to highlight that these variables were measured after 12 weeks of diet in NFD and HFD control groups. After pharmacological treatments, NaHS and DL-PAG induced a partial reduction in fasting leptin levels when compared to vehicle. Chronic treatment with DL-PAG significantly decreased fasting blood glucose levels, HOMA index and increased Matsuda index compared to vehicle. On the other hand, chronic treatment with NaHS or L-Cys did not modify the above variables. Fig. 2 illustrates the effect of NFD, HFD (upper panel) and chronic treatments in HFD animals (lower panel) on blood glucose and serum insulin levels during the OGTT, calculated as area under the curve. Thus, 12 weeks HFD significantly increased blood glucose levels (Fig. 2A) and serum insulin levels (Fig. 2B) during OGTT compared to normal fat diet. Otherwise, the chronic treatments during four weeks with NaHS, L-Cys or DL-PAG did not modify blood glucose levels (Fig. 2C) or serum insulin levels (Fig. 2D) when compared to vehicle in HFD rats. 3.2. Haemodynamic changes induced by high-fat diet and pharmacological treatments Table 4 shows the effect of normal fat diet, high-fat diet and high-fat diet plus chronic treatments (vehicle, NaHS, L-Cys or DL-PAG) on several haemodynamic variables measured at 12 and 16 weeks in conscious animals. After 12 weeks of feeding with the high-fat diet, a significant increase in all the haemodynamic variables was observed in
HFD control when compared with NFD control group. This increase was maintained until 16 weeks of feeding with HFD. Focusing on the pharmacological treatments, the comparisons were performed within the values obtained before and after the chronic treatment. Interestingly, chronic treatment with NaHS significantly reduced all haemodynamic variables when compared to 12 weeks, i.e. before pharmacological treatment. Notably, haemodynamic values in HFD rats treated with NaHS were similar to those obtained from normal fat diet at 16 weeks. In addition, chronic treatment with L-Cys significantly diminished systolic and mean blood pressure, while DL-PAG significantly increased heart rate compared to the same treatment at 12 weeks. 3.3. Effect of high-fat diet on the cardiovascular responses induced by stimulation of the sympathetic nervous system and administration of α1/2-adrenoceptors agonists Fig. 3 shows the effect of HFD on the vasopressor responses induced by sympathetic stimulation and i.v. bolus injections of noradrenaline, methoxamine and UK 14,304. In normal fat diet rats, electrical stimulation of the sympathetic outflow or i.v. administration of noradrenaline, methoxamine and UK 14,304 produced frequency dependent or dosedependent increases in DBP. Interestingly, HFD significantly increased the vasopressor responses induced by electrical stimulation (1 and 3 Hz) and methoxamine (100 µg/kg) without significant changes on the vasopressor responses induced by noradrenaline or UK 14,304, when compared with NFD rats. 3.4. Effect of chronic administration of vehicle, NaHS, L-Cys, and DL-PAG on the cardiovascular responses induced by stimulation of the sympathetic nervous system and administration of α1/2-adrenoceptors agonists
The effect of daily i.p. injections during four weeks of vehicle, NaHS, L-Cys, and DLPAG on the cardiovascular responses induced by sympathetic stimulation and i.v. bolus injections of noradrenaline, methoxamine and UK 14,304 in HFD rats is shown in Figs. 4-7. As shown in Fig. 4, chronic administration of vehicle (PBS) did not alter the vasopressor responses induced by sympathetic stimulation or i.v. injections of methoxamine, noradrenaline or UK 14,304 when compared with those obtained in high-fat diet rats. Fig. 5 shows the effect of the chronic treatment with NaHS (5.6 mg/kg) in the vasopressor responses induced by stimulation of the sympathetic nervous system. Interestingly, chronic administration of NaHS: (1) significantly reduced vasopressor responses induced by electrical stimulation at the frequencies of 1 and 3 Hz; (2) significantly reduced the vasopressor responses induced by 0.3 µg/kg noradrenaline and 30 µg/kg methoxamine; and (3) failed to significantly modify the responses induced by UK 14,304, when compared to vehicle. Furthermore, Fig. 6 shows the effect of chronic treatment with L-Cys (300 mg/kg) on the vasopressor responses induced by stimulation of the sympathetic outflow or α1/2adrenoceptor agonists. L-Cys significantly diminished vasopressor responses induced by electrical stimulation at 1 and 3 Hz, and those induced by 30 µg/kg methoxamine when compared to vehicle. In marked contrast, L-Cys did not produce significant changes in the vasopressor responses induced by noradrenaline or UK 14,304. Fig. 7 shows the effect of chronic administration of DL-PAG (1 mg/kg) on the vasopressor responses induced by stimulation of sympathetic outflow or α1/2adrenoceptor agonists. DL-PAG significantly diminished the vasopressor response induced by 30 µg/kg of methoxamine compared to vehicle group. Further, this inhibitor
did not change the responses induced by electrical stimulation, or i.v. injections of noradrenaline and UK 14,304. Lastly, the tachycardic responses induced by i.v. injections of noradrenaline are presented in Fig. 8. Thus, high-fat diet induced an increase in tachycardic responses at 0.3-3 µg/kg of noradrenaline when compared to normal fat diet. Surprisingly, vehicle induced a significant decrease in heart rate at 1 µg/kg of noradrenaline compared to HFD control. Interestingly, chronic treatment with L-Cys (300 mg/kg) significantly decreased the tachycardic responses induced by 1 and 3 µg/kg of noradrenaline while chronic treatment with NaHS and DL-PAG failed to affect these responses. 4. Discussion 4.1. General H2S has gained extensive attention in exploring its potential therapeutic use for the treatment of cardiovascular diseases including ischemia-reperfusion injury (Elrod et al., 2007; Zhu et al., 2007), stroke (Qu et al., 2008) and hypertension (Meng et al., 2015; Yang et al., 2008). Nowadays, several lines of evidence suggest a link between H2S and metabolic diseases including insulin resistance, obesity and type 2 diabetes mellitus. In this respect, it has been recently studied the effects of HFD in H2S production and the expression of the synthesizing enzymes in several tissues (Beltowski et al., 2018). Indeed, HFD (35% fat) fed to male Wistar rats during 18 weeks produced a downregulation in CBS and CSE in liver (Bravo et al., 2011); meanwhile, in mice fed a 16% fat diet during 16 weeks diminished CSE as well as H2S synthesis in liver, kidney and lung (Peh et al., 2014). These results suggest downregulation of H2S-synthesizing enzymes and a reduction in H2S production in animals fed a HFD.
Our results demonstrate that: (1) HFD significantly increased the cardiovascular responses induced by sympathetic stimulation, haemodynamic, zoometric and metabolic variables; and (2) chronic administration of NaHS and L-Cys reversed the cardiovascular, haemodynamic and zoometric changes induced by HFD while DL-PAG ameliorated metabolic variables. 4.2. Zoometric and metabolic variables Our results demonstrate that 12 weeks HFD in rats developed obesity, hyperglycaemia, hyperleptinemia and whole-body insulin resistance (Table 3). Remarkably, we also demonstrate that chronic administration of NaHS for four weeks reduced body weight in HFD rats (Table 2). In this regard, Wu et al. (2016) demonstrated that chronic treatment with NaHS (50 µmol/kg) in mice fed HFD (45%) significantly reduced body weight and kidney triacylglycerol levels, which suggests an important role of H2S in lipid metabolism (Wu et al., 2016). This finding could explain weight and adipose tissue loss in our experimental model after chronic treatment with NaHS and L-Cys. It also could explain the loss of liver weight (Table 2). Admittedly, the role of H2S in the regulation of lipid metabolism is still controversial. In this context, several mechanisms supporting H2Sinduced lipolysis have been suggested: (1) increase in cAMP release and subsequent PKA activation (Beltowski and Jamroz-Wisniewska, 2016), (2) decrease in lipid synthesis by a down-regulation of fatty acid synthase with an increase in expenditure of lipids by an up-regulation in carnitine palmitoyltransferase-1 expression (Wu et al., 2015), (3) increase of tissue lipoprotein lipase activity and fatty acid mobilization from adipose tissue (Kawasaki et al., 2010) and (4) induce PPARα nuclear translocation and ATP binding cassette transporter A1 up-regulation leading to a decrease in lipids accumulation (Li et al., 2016). On the other hand, it has been demonstrated that (1) H2S
promotes lipid storage in adipocytes through PPARγ C139 site sulfhydration (Cai et al., 2016) and (2) H2S inhibition with DL-PAG increases PKA-perilipin1/HSL pathway stimulating the lipids catalytic reaction (Geng et al., 2013). Our results also showed that chronic treatment with NaHS and L-Cys for four weeks significantly diminished fasting leptin levels. In this respect, it has been suggested, in a model of Cdo1–/– (cysteine dioxygenase) mice, that elevated levels of cysteine promote cysteine metabolism by desulfhydration, which leads to a higher production of H2S. This increase in H2S levels could underlie the low leptin levels possibly by suppressing leptin expression (Niewiadomski et al., 2016). On the other hand, chronic treatment with DLPAG significantly diminished leptin levels. According to our findings, Geng et al. (2013), demonstrated that treatment with PAG as well as GYY4137 decreased fasting glucose and insulin levels as HOMA index by AMPK pathway (Geng et al., 2013). Hence, it could be argued that modulation of CSE/H2S system could lead to the same result. Also, it should be kept in mind that H2S is a gasotransmitter with dual effects in different organs and systems (Wang, 2002). A significant improvement in insulin sensitivity by H2S has been proposed in white adipocyte tissue from fructose-induced insulin resistance in rats (Feng et al., 2009; Geng et al., 2013) while an impairment of glucose utilization has been suggested in hepatocytes (Zhang et al., 2013). Under our experimental conditions, NaHS or L-Cys did not change HOMA-index or insulin serum levels (Table 3). Thus, the effect of H2S on insulin resistance may depend on the experimental conditions. 4.3. Haemodynamic variables Obesity is associated with endothelial dysfunction, sympathetic hyperactivity (Hall et al., 2010) and hypertension (Jiang et al., 2016). Under our experimental conditions, HFD-
produced hypertension after 12 weeks, which was maintained until 16 weeks (Table 4). Consistent with the above, diets containing high levels of saturated fatty acids elevated blood pressure and exacerbated spontaneous hypertension (Aguila and Mandarim-deLacerda, 2003). Indeed, our data demonstrated that chronic treatment with NaHS diminished heart rate, systolic, diastolic and mean blood pressure while L-Cys reduced systolic and mean blood pressure (see Table 4). According to our results, administration of H2S donors reduced blood pressure in different animal models such as spontaneously hypertensive rats (Zhao et al., 2008), Dahl salt-sensitive rats (Huang et al., 2015) and a nitric oxide synthase inhibition model (Zhong et al., 2003). Also, it has been shown that chronic administration of NaHS reduced oxidative stress and improved endothelial dysfunction in a streptozotocininduced experimental model of diabetes in mice (Ng et al., 2017). In this regard, hypertension was observed in CSE-/- knock-out mice and i.v. bolus injections of NaHS induced dose-dependent transitory decreases in systolic blood pressure of anaesthetized mice (Yang et al., 2008). Several studies have demonstrated that daily administration of N-acetylcysteine, a stable analogue of cysteine, diminished blood pressure in spontaneously hypertensive rats (Vasdev et al., 2009). Consistent with our results, two-week (Cabassia et al., 2001) or four-week (Girouard et al., 2003) oral treatment of N-acetylcysteine (4 g/kg of body weight per day in drinking water) reduced systolic blood pressure and mean blood pressure, respectively. A possible explanation of the antihypertensive effect of L-Cys has been proposed including an endothelium-dependent relaxation via a reduction in GSSG/GSH ratio (Cabassia et al., 2001). Nevertheless, if the effect of L-Cys is due to its antioxidant effect and/or an increase in H2S production remains to be determined.
As expected, conversely to NaHS, chronic treatment with DL-PAG increased heart rate (Table 4) probably due to inhibition of endogenous H2S production. In this regard, DLPAG significantly increased renal sympathetic nerve activity in anaesthetised rats (Guo et al., 2016). However, DL-PAG failed to modify blood pressure (see Table 4). To explain the lack of effect of DL-PAG in blood pressure, we should keep in mind that blood pressure is regulated by different mechanisms including local, hormonal, renal and neural mechanisms. 4.4. NaHS and L-Cys reversed the cardiovascular changes induced by high-fat diet During obesity, it has been suggested a depletion of H2S in small blood vessel (Candela et al., 2016) and a reduction in the production of H2S (Katsouda et al., 2017). In our model, we found that HFD increased the vasopressor responses induced by sympathetic stimulation (Fig. 3A). Interestingly, that increase was diminished by chronic treatment with NaHS and L-Cys (Fig. 5A and 6A). In this respect, we have previously demonstrated that acute administration of NaHS inhibited vasopressor (Centurion et al., 2016) and cardioaccelerator (Centurion et al., 2018) sympathetic outflow in a pithed rat model. Therefore, the reduction of blood pressure and heart rate by NaHS could be explained, at least in part, by the inhibition of sympathetic outflow. However, we cannot rule out the activation of other mechanisms activated by hydrogen sulfide such as Ssulfuration of KATP channels, blockade of L-type calcium channels, antioxidant effect or inhibition of renin angiotensin system. On the other hand, it has been demonstrated that in isolated aortic rings with periadventitial adipose tissue, the inhibition of CSE enzyme with DL-PAG (10 mM), enhanced phenylephrine-induced contractile response. However, under our experimental conditions, DL-PAG failed to produce a significant effect on the
cardiovascular responses (Fig. 7). In this respect, it is possible that the dose chosen could not have been enough to produce the effect expected (Fang et al., 2009).
5. Conclusion In conclusion, chronic treatment with NaHS and L-Cys was effective in reversing high-fat diet-induced cardiovascular changes. These effects could involve a reduction of the sympathetic tone. These results invite us to explore deeper into the mechanism underlying this long-term effect. Acknowledgments The authors acknowledge Conacyt Mexico for its partial financial support (Grant No. 252702). Conflict of interest None.
References Aguila, M.B., Mandarim-de-Lacerda, C.A., 2003. Heart and blood pressure adaptations in Wistar rats fed with different high-fat diets for 18 months. Nutrition 19, 347-352. Beltowski, J., Jamroz-Wisniewska, A., 2016. Hydrogen Sulfide in the Adipose TissuePhysiology, Pathology and a Target for Pharmacotherapy. Molecules 22. Beltowski, J., Wojcicka, G., Jamroz-Wisniewska, A., 2018. Hydrogen sulfide in the regulation of insulin secretion and insulin sensitivity: Implications for the pathogenesis and treatment of diabetes mellitus. Biochem. Pharmacol. 149, 6076. Bravo, E., Palleschi, S., Aspichueta, P., Buque, X., Rossi, B., Cano, A., Napolitano, M., Ochoa, B., Botham, K.M., 2011. High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomocysteinemia caused by down regulation of the transsulphuration pathway. Lipids Health Dis. 10, 60. Cabassia, A., Dumont, E.C., Girouard, H., Bouchard, J.-F., Le Jossec, M., Lamontagne, D., Besner, J.-G., de Champlain, J., 2001. Effects of chronic N-acetylcysteine treatment on the actions of peroxynitrite on aortic vascular reactivity in hypertensive rats. J. Hypertens. 19, 1233-1244. Cai, J., Shi, X., Wang, H., Fan, J., Feng, Y., Lin, X., Yang, J., Cui, Q., Tang, C., Xu, G., Geng, B., 2016. Cystathionine gamma lyase-hydrogen sulfide increases peroxisome proliferator-activated receptor gamma activity by sulfhydration at C139 site thereby promoting glucose uptake and lipid storage in adipocytes. Biochim. Biophys. Acta 1861, 419-429.
Candela, J., Velmurugan, G.V., White, C., 2016. Hydrogen sulfide depletion contributes to microvascular remodeling in obesity. Am J Physiol Heart Circ Physiol 310, H1071-1080. Centurión, D., Cobos-Puc, L.E., Ramírez-Rosas, M.B., Gómez-Díaz, B., SánchezLópez, A., Villalón, C.M., 2009. Pithed rat model for searching vasoactive drugs and studying the modulation of the sympathetic and non- adrenergic noncholinergic outflow, in: Rocha, L.L., Granados-Soto, V. (Eds.), Models of Neuropharmacology. Transworld Research Network, Kerala, India, pp. 91-97. Centurion, D., de la Cruz, S.H., Castillo-Santiago, S.V., Becerril-Chacon, M.E., TorresPerez, J.A., Sanchez-Lopez, A., 2018. NaHS prejunctionally inhibits the cardioaccelerator sympathetic outflow in pithed rats. Eur. J. Pharmacol. 823, 3540. Centurion, D., De la Cruz, S.H., Gutierrez-Lara, E.J., Beltran-Ornelas, J.H., SanchezLopez, A., 2016. Pharmacological evidence that NaHS inhibits the vasopressor responses induced by stimulation of the preganglionic sympathetic outflow in pithed rats. Eur. J. Pharmacol. 770, 40-45. Chen, X., Zhao, X., Lan, F., Zhou, T., Cai, H., Sun, H., Kong, W., Kong, W., 2017. Hydrogen Sulphide Treatment Increases Insulin Sensitivity and Improves Oxidant Metabolism through the CaMKKbeta-AMPK Pathway in PA-Induced IR C2C12 Cells. Sci. Rep. 7, 13248. Council, N.R., 2011. Guide for the Care and Use of Laboratory Animals: Eighth Edition. The National Academies Press, Washington, DC. Elrod, J.W., Calvert, J.W., Morrison, J., Doeller, J.E., Kraus, D.W., Tao, L., Jiao, X., Scalia, R., Kiss, L., Szabo, C., Kimura, H., Chow, C.-W., Lefer, D.J., 2007.
Hydrogen
sulfide
attenuates
myocardial
ischemia-reperfusion
injury
by
preservation of mitochondrial function. Proceedings of the National Academy of Sciences 104, 15560. Fang, L., Zhao, J., Chen, Y., Ma, T., Xu, G., Tang, C., Liu, X., Geng, B., 2009. Hydrogen sulfide derived from periadventitial adipose tissue is a vasodilator. J. Hypertens. 27, 2174-2185. Feng, X., Chen, Y., Zhao, J., Tang, C., Jiang, Z., Geng, B., 2009. Hydrogen sulfide from adipose tissue is a novel insulin resistance regulator. Biochem. Biophys. Res. Commun. 380, 153-159. Geng, B., Cai, B., Liao, F., Zheng, Y., Zeng, Q., Fan, X., Gong, Y., Yang, J., Cui, Q.H., Tang, C., Xu, G.H., 2013. Increase or decrease hydrogen sulfide exert opposite lipolysis, but reduce global insulin resistance in high fatty diet induced obese mice. PLoS One 8, e73892. Gillespie, J.S., Muir, T.C., 1967. A method of stimulating the complete sympathetic outflow from the spinal cord to blood vessels in the pithed rat. Br. J. Pharmacol. 30, 78-87. Girouard, H., Chulak, C., Wu, L., Lejossec, M., de Champlain, J., 2003. N-acetylcysteine improves nitric oxide and alpha-adrenergic pathways in mesenteric beds of spontaneously hypertensive rats. Am. J. Hypertens. 16, 577-584. Guo, Q., Wu, Y., Xue, H., Xiao, L., Jin, S., Wang, R., 2016. Perfusion of Isolated Carotid Sinus With Hydrogen Sulfide Attenuated the Renal Sympathetic Nerve Activity in Anesthetized Male Rats. Physiol. Res. 65, 413-423.
Hall, J.E., da Silva, A.A., do Carmo, J.M., Dubinion, J., Hamza, S., Munusamy, S., Smith, G., Stec, D.E., 2010. Obesity-induced hypertension: role of sympathetic nervous system, leptin, and melanocortins. J. Biol. Chem. 285, 17271-17276. Hu, Y., Chen, X., Pan, T.T., Neo, K.L., Lee, S.W., Khin, E.S., Moore, P.K., Bian, J.S., 2008. Cardioprotection induced by hydrogen sulfide preconditioning involves activation of ERK and PI3K/Akt pathways. Pflugers Arch 455, 607-616. Huang, P., Chen, S., Wang, Y., Liu, J., Yao, Q., Huang, Y., Li, H., Zhu, M., Wang, S., Li, L., Tang, C., Tao, Y., Yang, G., Du, J., Jin, H., 2015. Down-regulated CBS/H2S pathway is involved in high-salt-induced hypertension in Dahl rats. Nitric Oxide 46, 192-203. Jain, S.K., Bull, R., Rains, J.L., Bass, P.F., Levine, S.N., Reddy, S., McVie, R., Bocchini, J.A., 2010. Low levels of hydrogen sulfide in the blood of diabetes patients and streptozotocin-treated rats causes vascular inflammation? Antioxid Redox Signal 12, 1333-1337. Jiang, S.Z., Lu, W., Zong, X.F., Ruan, H.Y., Liu, Y., 2016. Obesity and hypertension. Exp. Ther. Med. 12, 2395-2399. Kaneko, Y., Kimura, Y., Kimura, H., Niki, I., 2006. L-Cysteine Inhibits Insulin Release From the Pancreatic -Cell: Possible Involvement of Metabolic Production of Hydrogen Sulfide, a Novel Gasotransmitter. Diabetes 55, 1391-1397. Katsouda, A., Szabo, C., Papapetropoulos, A., 2017. Reduced adipose tissue H2S in obesity. Pharmacol. Res. 128, 190-199. Kawasaki, M., Miura, Y., Funabiki, R., Yagasaki, K., 2010. Effects of Simultaneous Dietary Fish Oil Ingestion and Sulfur Amino Acid Supplementation on the Lipid
Metabolism in HepatomaBearing Rats with Hyperlipidemia. J Nutr Sci Vitaminol 56, 247-254. Kleinman, L.I., Radford, E.P., Jr., 1964. Ventilation standards for small mammals. J. Appl. Physiol. 19, 360-362. Li, D., Xiong, Q., Peng, J., Hu, B., Li, W., Zhu, Y., Shen, X., 2016. Hydrogen Sulfide UpRegulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARalpha. Int J Mol Sci 17. Lu, M., Liu, Y.H., Goh, H.S., Wang, J.J., Yong, Q.C., Wang, R., Bian, J.S., 2010. Hydrogen sulfide inhibits plasma renin activity. J Am Soc Nephrol 21, 993-1002. Meng, G., Ma, Y., Xie, L., Ferro, A., Ji, Y., 2015. Emerging role of hydrogen sulfide in hypertension and related cardiovascular diseases. Br. J. Pharmacol. 172, 55015511. Ng, H.H., Yildiz, G.S., Ku, J.M., Miller, A.A., Woodman, O.L., Hart, J.L., 2017. Chronic NaHS treatment decreases oxidative stress and improves endothelial function in diabetic mice. Diab. Vasc. Dis. Res. 14, 246-253. Niewiadomski, J., Zhou, J.Q., Roman, H.B., Liu, X., Hirschberger, L.L., Locasale, J.W., Stipanuk, M.H., 2016. Effects of a block in cysteine catabolism on energy balance and fat metabolism in mice. Ann. N. Y. Acad. Sci. 1363, 99-115. Peh, M.T., Anwar, A.B., Ng, D.S., Atan, M.S., Kumar, S.D., Moore, P.K., 2014. Effect of feeding a high fat diet on hydrogen sulfide (H2S) metabolism in the mouse. Nitric Oxide 41, 138-145. Popov, D., 2013. An outlook on vascular hydrogen sulphide effects, signalling, and therapeutic potential. Arch. Physiol. Biochem. 119, 189-194.
Qu, K., Lee, S.W., Bian, J.S., Low, C.M., Wong, P.T., 2008. Hydrogen sulfide: neurochemistry and neurobiology. Neurochem. Int. 52, 155-165. Untereiner, A., Wu, L., 2017. Hydrogen Sulfide and Glucose Homeostasis: A Tale of Sweet and the Stink. Antioxid. Redox Signal. Vasdev, S., Singal, P., Gill, V., 2009. The antihypertensive effect of cysteine. Int. J. Angiol. 1, 7-21. Wang, R., 2002. Two´s company, three´s a crowd-can H2S be the third endogenous gaseous transmitter. FASEB J. 16, 1792-1798. Whiteman, M., Gooding, K.M., Whatmore, J.L., Ball, C.I., Mawson, D., Skinner, K., Tooke, J.E., Shore, A.C., 2010. Adiposity is a major determinant of plasma levels of the novel vasodilator hydrogen sulphide. Diabetologia 53, 1722-1726. Wu, D., Gao, B., Li, M., Yao, L., Wang, S., Chen, M., Li, H., Ma, C., Ji, A., Li, Y., 2016. Hydrogen Sulfide Mitigates Kidney Injury in High Fat Diet-Induced Obese Mice. Oxid. Med. Cell Longev. 2, 715-718. Wu, D., Zheng, N., Qi, K., Cheng, H., Sun, Z., Gao, B., Zhang, Y., Pang, W., Huangfu, C., Ji, S., Xue, M., Ji, A., Li, Y., 2015. Exogenous hydrogen sulfide mitigates the fatty liver in obese mice through improving lipid metabolism and antioxidant potential. Med Gas Res 5, 1. Yang, G., Ju, Y., Fu, M., Zhang, Y., Pei, Y., Racine, M., Baath, S., Merritt, T.J.S., Wang, R., Wu, L., 2018. Cystathionine gamma-lyase/hydrogen sulfide system is essential for adipogenesis and fat mass accumulation in mice. Biochim. Biophys. Acta Mol. Cell. Biol. Lipids 1863, 165-176.
Yang, G., Wu, L., Jiang, B., Yang, W., Qi, J., Cao, K., Meng, Q., Mustafa, A.K., Mu, W., Zhang, S., Snyder, S.H., Wang, R., 2008. H2S as a physiologic vasorelaxant hypertension in mice with deletion of CSE. Science 322, 587-590. Zhang, L., Yang, G., Untereiner, A., Ju, Y., Wu, L., Wang, R., 2013. Hydrogen sulfide impairs glucose utilization and increases gluconeogenesis in hepatocytes. Endocrinology 154, 114-126. Zhao, W., Zhang, J., Lu, Y., Wang, R., 2001. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J. 20, 6008-6016. Zhao, X., Zhang, L., Zhang, C., Zeng, X., Yan, H., Jin, H., Tang, C., 2008. Regulatory effect of hydrogen sulfide on vascular collagen content in spontaneously hypertensive rats. Hypertens. Res. 31, 1619–1630. Zhong, G., Chen, F., Cheng, Y., Tang, C., Du, J., 2003. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase. J. Hypertens. 21, 1879-1885. Zhu, Y.Z., Wang, Z.J., Ho, P., Loke, Y.Y., Zhu, Y.C., Huang, S.H., Tan, C.S., Whiteman, M., Lu, J., Moore, P.K., 2007. Hydrogen sulfide and its possible roles in myocardial ischemia in experimental rats. J. Appl. Physiol. 102, 261-268.
Table 1. Composition of the normal and high-fat diet. 100 g NFD %
100 g HFD
grams
kcal
%
grams
kcal
Carbohydrates 50.3
50.3
201.2
26.37
36
144
Proteins
23.6
23.6
94.4
10.14
13.8
55.4
Lipids
6.7
6.7
60.3
63.5
38.5
346.8
Total kcal
355.9
546.1
Note: This information was obtained from the label of the products used. NFD, normal fat diet; HFD, high fat diet.
Table 2. Effect of: (1) normal fat diet (NFD control) and high-fat diet (HFD control) after 12 weeks of diet; or (2) daily i.p. injections for 4 weeks of vehicle (PBS, 1 ml/kg), NaHS (5.6 mg/kg), L-Cys (300 mg/kg) and DL-PAG (1 mg/kg) in HFD rats on several zoometric variables. NFD
HFD
Control
Control
HFD Vehicle
NaHS
L-Cys
DL-PAG
Zoometric Variables Body 492.7±17.1 568.3±24.2a 552.9±23.9 490.7±10.5b 490.3±27.9b 501.7±49.7 weight (g) Heart (g)
1.8±0.08
1.8±0.1
2±0.1
1.9±0.1
2±0.2
2.03±0.1
Liver (g)
17.8±1
20.1±1.5a
19.4±1.5
17.5±0.8b,c
17.2±0.8b,c
14.5±0.6b,c
VAT (g)
6.8±0.6
26.2±4.04a
17.03±2.1b
14.8±2.3b,c
10.2±1.5b,c
18.8±2.1b
EAT (g)
7.3±0.4
16.7±2.1a
15.02±1.6
11.9±1.1b
12.1±1.7
20±1.9c
Each value represents mean ± S.E.M. of 6 animals. a, P < 0.05 vs. NFD control; b, P < 0.05 vs. HFD control; c, P < 0.05 vs. vehicle. VAT, visceral adipose tissue; EAT, epididymal adipose tissue.
Table 3. Effect of: (1) normal fat diet (NFD control) and high-fat diet (HFD control) after 12 weeks of diet; or (2) daily i.p. injections for 4 weeks of vehicle (PBS, 1 ml/kg), NaHS (5.6 mg/kg), L-Cys (300 mg/kg) and DL-PAG (1 mg/kg) in HFD rats on several metabolic variables. HFD
NFD
HFD
Control
Control
Vehicle
NaHS
L-Cys
DL-PAG
1.04±0.1
1.07±0.06
0.94±0.09
0.92 ± 0.15
6969.1 ± 1197.2
4329.5 ± 679.3
0.94 ± 0.18
1.3 ± 0.3
0.8 ± 0.1
0.52 ± 0.3
117.7 ± 5.9
102.2 ± 4.5
115.5 ± 5.7
95.5 ± 5.5
Metabolic Variables b
Ghrelin (ng/ml)
2.8 ± 0.2
0.89 ± 0.1
Leptin (ng/ml)
687.8 ± 137.1
7969.9 ± 876.8
Insulin (ng/ml)
0.31 ± 0.03
0.9 ± 0.6
Glucose (mg/dl)
80.3 ± 4.5
111.2 ± 5.5
HOMA Index
1.9 ± 0.1
6.9 ± 2.1
b
7.8 ± 1.5
9.5 ± 2.9
11.215 ± 4.5
1.4 ± 0.3
d,f
Matsuda Index
5.1 ± 0.7
2.4 ± 0.7
a
1.6 ± 0.2
1.5 ± 0.3
1.5 ± 0.4
6.2 ± 1.2
c,f
b
b
b
d,e
4456.8 ± 632.9
c
4063 ± 803.6
d, e
e
Each value represents mean ± S.E.M. of 6 animals. a, P < 0.05 vs. NFD; b, P < 0.01 vs. NFD; c, P < 0.05 vs. HFD; d, P < 0.01 vs. HFD; e, P < 0.05 vs. vehicle; f, P < 0.01 vs. vehicle.
Table 4. Effect of: (1) normal fat diet (NFD control) and high-fat diet (HFD control); or (2) daily i.p. injections for four weeks of vehicle (PBS, 1 ml/kg), NaHS (5.6 mg/kg), L-Cys (300 mg/kg) and DL-PAG (1 mg/kg) in HFD rats on haemodynamic variables measured in conscious animals. 12 weeks
16 weeks
SBP
MBP
HR
DBP (mm
HR
SBP
(bpm)
(mm Hg)
(mm
(bpm)
(mm Hg) Hg)
Hg)
DBP
MBP
(mm
(mm
Hg)
Hg)
81±0.5
91±1
NFD 382±18
103±3
81±0.9
87±2
355±10
423±15
160±4
131±6
399±30
159±4
126±3
137±3
419±20
411±19
146±2
115±2
125±2
336±20
390±12
164±3
121±4
135±4
364±15
353±5
165±2
117±3
132±2
394±12
113±3
Control HFD a
a
140±5
a
416±10
a
164±6
a
130±4
a
141±5
117±3
b
132±2
a
Control HFD + 165±2
Vehicle HFD +
c
113±2
d
81±0.8
d
d
142±5
e
109±5
119±5
119±1
132±1
92±1
NaHS HFD + e
L-Cys HFD + f
159±3
DL-PAG
Each value represents mean ± S.E.M. of 6 animals. a, P < 0.01 vs. NFD; b, P < 0.05 vs. vehicle (12 weeks); c, P < 0.05 vs NaHS (12 weeks); d, P < 0.01 vs NaHS (12 weeks); e, P < 0.05 vs L-Cys (12 weeks); f, P < 0.05 vs DL-PAG (12 weeks). HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure.
Figure Legends Fig. 1. (A) Experimental protocol and (B) time course of the nutritional and pharmacological treatments. HFD, high-fat diet; NFD, normal fat diet; NaHS, sodium hydrosulfide; L-Cys, L-Cysteine; DL-PAG, DL-Propargylglycine; HR, heart rate; BP, blood pressure. Fig. 2. Area under the curve (AUC) of blood glucose and serum insulin levels during the oral glucose tolerance test of control and treatment groups. Each bar represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. NFD control. Fig. 3. Effect of normal fat diet (NFD control) or high-fat diet (HFD control) during 16 weeks on the vasopressor responses induced by: (A) sympathetic stimulation, (B) noradrenaline, (C) methoxamine and (D) UK 14,304. Each point represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. NFD control. Fig. 4. Vasopressor responses induced by: (A) sympathetic stimulation; or i.v. bolus injections of: (B) noradrenaline, (C) methoxamine or (D) UK 14,304 in HFD Control and HFD+Vehicle groups. Each point represents mean ± S.E.M. of 6 animals. Fig. 5. Effect of NaHS treatment (5.6 mg/kg) on: (A) Sympathetic stimulation or i.v. bolus injections of (B) noradrenaline, (C) methoxamine or (D) UK 14,304-induced vasopressor responses in high fat diet rats. Each point represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. vehicle; **, P < 0.01 vs. vehicle; ***, P < 0.001 vs. vehicle. Fig. 6. Effect of L-Cys (300 mg/kg) on the vasopressor responses induced by: (A) sympathetic stimulation; (B) noradrenaline; (C) methoxamine; and (D) UK 14,304 in high-fat diet (HFD) rats. Each point represents mean ± S.E.M. of 6 animals. ***, P < 0.001 vs. vehicle.
Fig. 7. Effect of daily i.p. injections for four weeks of DL-PAG (1 mg/kg) on the vasopressor responses induced by: (A) sympathetic stimulation; (B) noradrenaline; (C) methoxamine; and (D) UK 14,304 in high-fat diet (HFD) rats. Each point represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. vehicle. Fig. 8. Effect of: (A) normal fat diet (NFD), (B) high-fat diet (HFD) or daily i.p. injections for 4 weeks of (C) NaHS (5.6 mg/kg), (D) L-Cys (300 mg/kg) and (E) DL-PAG (1 mg/kg) in HFD rats on the tachycardic responses induced by i.v. bolus of noradrenaline. Each point represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. NFD control; **, P < 0.01 vs NFD control; #, P < 0.05 vs. HFD control; $$$, P < 0.001 vs. HFD+Vehicle.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
S0014-2999(19)30659-4
DOI:
https://doi.org/10.1016/j.ejphar.2019.172707
Reference:
EJP 172707
To appear in:
European Journal of Pharmacology
Received Date: 22 August 2019 Revised Date:
25 September 2019
Accepted Date: 26 September 2019
Please cite this article as: Gomez, C.B., de la Cruz, Saú.Huerta., Medina-Terol, G.J., Beltran-Ornelas, J.H., Sánchez-López, A., Silva-Velasco, D.L., Centurión, D., Chronic administration of NaHS and L-Cysteine restore cardiovascular changes induced by high-fat diet in rats, European Journal of Pharmacology (2019), doi: https://doi.org/10.1016/j.ejphar.2019.172707. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
European Journal of Pharmacology
Chronic administration of NaHS and L-Cysteine restore cardiovascular changes induced by high-fat diet in rats
Carolina B. Gomez, Saúl Huerta de la Cruz, Grecia J. Medina-Terol, Jesus H. BeltranOrnelas, Araceli Sánchez-López, Diana L. Silva-Velasco and David Centurión.
Departamento de Farmacobiología, Cinvestav Unidad Coapa, Czda. de los Tenorios 235, Col. Granjas-Coapa, Del. Tlalpan, C.P. 14330, México D.F., México.
Correspondence: Dr. David Centurión at the above address Phone: (Int.)-(52)-(55)5483 2866 Fax: NA Email: [email protected] Web page: http://farmacobiologia.cinvestav.mx/Personal-Académico/Dr-D-CenturiónPacheco
Abstract Hydrogen sulfide plays an important role in the regulation of the cardiovascular system, insulin secretion, and glucose homeostasis. The aim of the present study was to examine the effects of chronic treatment with sodium hydrosulfide (NaHS), L-Cysteine (L-Cys) and DL-Propargylglycine (DL-PAG) on the changes induced by a high-fat diet (HFD) in zoometric and metabolic variables as well as cardiovascular changes such as hypertension and sympathetic hyperactivity. For this purpose, male Wistar rats were fed a normal fat diet (NFD) or HFD for 12 weeks. Next, the HFD rats were divided into 5 subgroups which received daily i.p. injections during 4 weeks of: (1) nothing (no injection, Control); (2) vehicle (PBS; 1 ml/kg); (3) NaHS (5.6 mg/kg); (4) L-Cys (300 mg/kg); or (5) DL-PAG (1 mg/kg). Then, an oral glucose tolerance test, hormone serum levels and blood pressure were determined. The cardiovascular responses to stimulation of the vasopressor sympathetic tone or intravenous administration of the agonists noradrenaline (α1/2-adrenoceptors), methoxamine (α1-adrenoceptors) and UK 14,304 (α2-adrenoceptors) were determined in pithed rats. Lastly, the heart, liver and adipose tissue were weighted. HFD significantly increased: (1) zoometric variables, which were decreased by NaHS and L-Cys; (2) metabolic variables, ameliorated by DLPAG; (3) haemodynamic variables, which were reversed by NaHS and L-Cys; and (4) the vasopressor responses induced by sympathetic stimulation, which were diminished by NaHS and L-Cys. In conclusion, chronic treatment with NaHS and L-Cys are effective in reducing adipose tissue and ameliorating the cardiovascular changes induced by obesity; meanwhile, DL-PAG ameliorates metabolic variables.
Keywords: Hydrogen sulfide; obesity; hypertension; cardiovascular.
1. Introduction Hydrogen sulfide (H2S) is a novel gasotransmitter that plays an important role in the regulation of the cardiovascular system. This gas produces complex cardiovascular responses including vasodilatation (Zhao et al., 2001), inhibition of the sympathetic nervous system (Centurion et al., 2018; Centurion et al., 2016), cardioprotection (Hu et al., 2008), and inhibition of renin activity during renovascular hypertension (Lu et al., 2010). Besides, it has been observed that biosynthesis and plasma levels of H2S are significantly reduced during obesity (Whiteman et al., 2010), diabetes mellitus (Jain et al., 2010) and systemic hypertension (Popov, 2013), which may suggest that treatment with H2S releasing drugs could have a beneficial effect in metabolic diseases. Indeed, in recent years, several studies have suggested that during obesity, the levels of H2Sproducing enzymes are reduced in brown and white adipose tissue (Katsouda et al., 2017). Moreover, in mouse C2C12 skeletal muscle myoblast line, expression of the enzymes involved in the production of H2S, cystathionine gamma-lyase (CSE), cystathionine-beta-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) were decreased in palmitic acid-induced insulin resistance (Chen et al., 2017). Therefore, H2S could be implicated in the regulation of insulin release (Beltowski et al., 2018; Chen et al., 2017; Kaneko et al., 2006; Untereiner and Wu, 2017), glucose homeostasis (Untereiner and Wu, 2017), insulin signalling pathway (Chen et al., 2017) and the regulation of incretins and adipokines (Beltowski et al., 2018). Although H2S seems to be beneficial during obesity, there is still a debate whether this rotten smell gas has deleterious or beneficial effects. Indeed, Geng et al. (2013) demonstrated differential effects in high-fat diet (HFD) mice as DL-Propargylglycine (DL-
PAG), an inhibitor of CSE, increased lipolysis and reduced insulin resistance; while GYY4137, a slow H2S donor, inhibited lipolysis and reduced insulin resistance (Geng et al., 2013). More recently, it has been suggested that H2S/CSE pathway promotes differentiation of adipocytes by sulfhydration of PPAR-γ (Peroxisome Proliferator Activated Receptors) (Yang et al., 2018). Despite the above findings, up to date, no study has analysed the effect of chronic administration of an H2S donor on the cardiovascular changes in systemic vasculature produced by obesity in rats. Thus, this study has examined the effects of chronic treatment with sodium hydrosulfide (NaHS), L-Cysteine (L-Cys) and DL-PAG over zoometric, metabolic and cardiovascular changes induced by HFD in rats. These data could shed further light on the controversial effects of H2S on obesity. 2. Materials and methods 2.1. Animals 7 weeks old 36 male Wistar rats (200-250 g) provided by our own laboratory animal facility were housed in plastic cages, maintained under standardized conditions (22 ± 2 °C, 50% humidity and 12/12 h light-dark cycle) and provided with food (either normal or high-fat diet) and water ad libitum. All animal procedures and the protocols of the present study were approved by the Institutional Ethics Committee (Cicual-Cinvestav) following the established regulations by the Mexican Official Norm for the Use and Welfare of Laboratory Animals (NOM-062-ZOO-1999), and following the Guide for the Care and Use of Laboratory Animals in the USA (Council, 2011). 2.2. Diet
The 36 animals were randomly divided into two main groups (Fig. 1A), which were fed with: (1) normal fat diet (NFD, n = 6), and (2) high-fat diet (HFD, n = 30). The NFD was the LabDiet® 5008 pellets and HFD was made with LabDiet® 5008 powder (550 g) mixed with Nutella® (150 g) and lard (300 g). The nutritional information of NFD and HFD is shown in Table 1. Each day, food was replaced with new pellets. Both diets were given during 16 weeks without interruptions. 2.3. Pharmacological treatments As shown in Fig. 1A, after 12 weeks of feeding, the HFD group was divided into five subgroups. Each subgroup received, respectively, intraperitoneal (i.p.) daily administration during 4 weeks of: (1) nothing (no injection, HFD Control); (2) vehicle (phosphate buffer saline, PBS; 1 ml/kg); (3) NaHS (5.6 mg/kg, inorganic H2S donor); (4) L-Cys (300 mg/kg, H2S-producing enzymes substrate); and (5) DL-PAG (1 mg/kg, CSE inhibitor). It should be mentioned that the NFD group did not receive any pharmacological treatment. 2.4. Oral glucose tolerance test and hormone assays After 12 weeks of feeding, both NFD and HFD control groups were fasted for 12 hours; then an oral glucose tolerance test (OGTT) and hormone assays were performed. For OGTT, blood glucose levels (tail vein) were measured at baseline and 5, 10, 15, 30, 60, 90 and 120 min after oral administration of glucose (1 g/kg) through glucometer (AccuCheck®, Roche of Mexico). At the same time, blood was collected for measurement of serum insulin by ELISA (Enzyme-linked Immunosorbent Assay) kit from ALPCO (Salem, NH, USA).
Furthermore, fasting plasma levels of leptin and total ghrelin were measured by ELISA kit from R&D Systems (Minneapolis, MN, USA) and Millipore (Burlington, MA, USA), respectively, as suggested by the provider. The procedures above mentioned were repeated after the chronic administration of vehicle, NaHS, L-Cys or DL-PAG, i.e., after 16 weeks with HFD (see Fig. 1B). 2.5. Determination of arterial blood pressure and heart rate in conscious animals Arterial blood pressure and heart rate were measured by a tail-cuff method using a LE 5001 automatic blood pressure recorder (Letical, PanLab, Barcelona, Spain). Animals were immobilized for a short period in acrylic cages and the tail was exposed to a heating lamp for 15 minutes. A cuff was placed in the tail, which was inflated to stop blood flow, as suggested by the provider. The tension released allowed to determinate systolic blood pressure (SBP) through the sensor; and calculate heart rate (HR), diastolic blood pressure (DBP) and mean blood pressure (MBP). The measurements were done at 12 and 16 weeks in all groups, representing the beginning and the end of the chronic treatment (see Fig. 1B). 2.6. Evaluation of cardiovascular function At the end of the pharmacological treatments, animals were anaesthetized with isoflurane (3%) and the trachea was cannulated. Animals were pithed by inserting a stainless-steel rod through the orbit and foramen magnum into the vertebral foramen (Centurión et al., 2009) aiming the destruction of the central nervous system. As animals were unconscious under these conditions, they were artificially ventilated with room air using a positive pressure pump (7025 rodent ventilator, Ugo Basile, Comerio, VA, Italy) at 80 strokes/min and a stroke volume of 20 ml/kg, as previously described (Kleinman and Radford, 1964). After a bilateral vagotomy, catheters were placed in: (1) right
femoral vein for drug administration and (2) the left carotid artery to record arterial blood pressure and heart rate with a pressure transducer (RX104A, Biopac Systems Inc., Goleta, CA, USA). Both parameters were recorded simultaneously using a data acquisition unit (MP150A-CE, Biopac Systems Inc., Goleta, CA). DBP was determined, as this is the blood pressure when the left ventricle is relaxed and thus could indirectly represent the systemic vascular resistance that regulates arterial blood pressure and blood flow within organs. The body of each pithed rat was maintained at 37 °C by a lamp and monitored with a rectal thermometer. 2.6.1 Experimental design in pithed rats After the procedure mentioned above, the 36 rats went under the same protocol. First, the vasopressor responses induced by selective preganglionic (T7-T9) stimulation of the vasopressor sympathetic outflow were determined. For this purpose, the stainless-steel rod was replaced by an enamelled bipolar electrode except for 1 cm length 9 cm from the tip. The uncovered segment was placed at the T7-T9 region of the spinal cord allowing the selective stimulation of the sympathetic nerves that supply the systemic vasculature (Gillespie and Muir, 1967). Before electrical stimulation, in order to avoid electrically-induced muscle twitching, animals received an intravenous (i.v.) bolus of gallamine (25 mg/kg). The preganglionic vasopressor sympathetic outflow was stimulated with an S88X square pulse stimulator (Grass Technologies, Warwick, RI, USA) by applying 10 s trains of monophasic, rectangular pulses (2 ms, 60 V), at increasing frequencies (0.03, 0.1, 0.3, 1 and 3 Hz). Once the stimuli-response curve was completed, the vasopressor responses induced by i.v. bolus injections of: (1) exogenous noradrenaline (0.03, 0.1, 0.3, 1 and 3 µg/kg; α1- and α2-adrenoceptor agonist, endogenous ligand); (2) methoxamine (1, 3, 10, 30 and 100 µg/kg; a selective α1-
adrenoceptor agonist); and (3) UK 14,304 (0.03, 0.1, 0.3, 1, 3, 10 and 30 µg/kg; a selective α2-adrenoceptor agonist) were evaluated. The electrical stimuli as well as the i.v. bolus injections of the compounds mentioned above were given using a sequential schedule of 0.5 log unit increments. The interval between the different stimulation frequencies or doses was determined depending on the duration of the resulting vasopressor response. Once this protocol was ended, artificial ventilation was turned off and the tissue collection was made by performing a thoracoabdominal incision. 2.7. Drugs In addition to the anaesthetic isoflurane (Fluriso™, Vet Ones, Boise, ID, USA), the compounds used in this study (obtained from the sources indicated) were: sodium hydrosulfide monohydrate (PubChem CID: 28015), L-Cysteine (PubChem CID: 5862), DL-Propargylglycine (PubChem CID: 95575), gallamine triethiodide (PubChem CID: 6172), (±)-noradrenaline bitartrate (PubChem CID: 168929), methoxamine hydrochloride (PubChem CID: 6081) and UK 14,304 (PubChem CID: 2435) (all from Sigma Chemical Co., St. Louis, MO, USA). Noradrenaline, methoxamine, UK 14,304 and gallamine were dissolved in physiological saline. NaHS, L-Cysteine, and DL-Propargylglycine were dissolved in phosphate buffer saline, pH 7.4 and 25 °C; new solutions were prepared every single day. 2.8. Statistical analysis All results in tables and figures are present as mean ± S.E.M. The differences in zoometric, metabolic and haemodynamic variables within groups of animals were evaluated by non-parametric Mann-Whitney U-test. The data obtained in the OGTT are presented as area under the curve (AUC). AUC was calculated by the trapezoidal rule.
The difference between the control group and vehicle in the AUC was evaluated by ttest, while differences between vehicle group and chronic treatments were compared by using one-way analysis of variance. The maximum changes in diastolic blood pressure to either electrical stimulation or α-adrenoceptors agonists as well as the changes in heart rate induced by noradrenaline were determined. The difference between the changes in DBP or heart rate within groups of animals was evaluated by a Tukey´s test, once the Two-Way Analysis of Variance reached statistical significance. Statistical significance was accepted at P<0.05. 3. Results 3.1. Zoometric and metabolic changes produced by high-fat diet and pharmacological treatments Table 2 shows the changes in zoometric variables induced by high-fat diet and the pharmacological treatments. High-fat diet (HFD control) produced a significant increase in body weight and weight of the liver as well as visceral and epididymal adipose tissue, but not in heart weight, when compared with normal fat diet group (NFD Control). Interestingly, body weight was significantly decreased by chronic treatment with NaHS and L-Cys compared to HFD control group, while DL-PAG treatment failed to change this variable. Besides, liver weight was significantly decreased by chronic treatment with NaHS, L-Cys, and DL-PAG when compared with vehicle and HFD control. Further, chronic treatment with vehicle (PBS), significantly diminished visceral adipose tissue compared versus HFD control. Nevertheless, chronic treatments with NaHS and L-Cys significantly diminished visceral adipose tissue when compared with vehicle. Moreover, epididymal adipose tissue was significantly decreased by treatment with NaHS;
conversely, this variable was significantly increased by DL-PAG when compared to vehicle. As shown in table 3, HFD: (1) significantly reduced fasting total ghrelin levels and Matsuda index; (2) significantly increased fasting leptin, insulin, glucose levels and HOMA index, when compared to NFD. It is important to highlight that these variables were measured after 12 weeks of diet in NFD and HFD control groups. After pharmacological treatments, NaHS and DL-PAG induced a partial reduction in fasting leptin levels when compared to vehicle. Chronic treatment with DL-PAG significantly decreased fasting blood glucose levels, HOMA index and increased Matsuda index compared to vehicle. On the other hand, chronic treatment with NaHS or L-Cys did not modify the above variables. Fig. 2 illustrates the effect of NFD, HFD (upper panel) and chronic treatments in HFD animals (lower panel) on blood glucose and serum insulin levels during the OGTT, calculated as area under the curve. Thus, 12 weeks HFD significantly increased blood glucose levels (Fig. 2A) and serum insulin levels (Fig. 2B) during OGTT compared to normal fat diet. Otherwise, the chronic treatments during four weeks with NaHS, L-Cys or DL-PAG did not modify blood glucose levels (Fig. 2C) or serum insulin levels (Fig. 2D) when compared to vehicle in HFD rats. 3.2. Haemodynamic changes induced by high-fat diet and pharmacological treatments Table 4 shows the effect of normal fat diet, high-fat diet and high-fat diet plus chronic treatments (vehicle, NaHS, L-Cys or DL-PAG) on several haemodynamic variables measured at 12 and 16 weeks in conscious animals. After 12 weeks of feeding with the high-fat diet, a significant increase in all the haemodynamic variables was observed in
HFD control when compared with NFD control group. This increase was maintained until 16 weeks of feeding with HFD. Focusing on the pharmacological treatments, the comparisons were performed within the values obtained before and after the chronic treatment. Interestingly, chronic treatment with NaHS significantly reduced all haemodynamic variables when compared to 12 weeks, i.e. before pharmacological treatment. Notably, haemodynamic values in HFD rats treated with NaHS were similar to those obtained from normal fat diet at 16 weeks. In addition, chronic treatment with L-Cys significantly diminished systolic and mean blood pressure, while DL-PAG significantly increased heart rate compared to the same treatment at 12 weeks. 3.3. Effect of high-fat diet on the cardiovascular responses induced by stimulation of the sympathetic nervous system and administration of α1/2-adrenoceptors agonists Fig. 3 shows the effect of HFD on the vasopressor responses induced by sympathetic stimulation and i.v. bolus injections of noradrenaline, methoxamine and UK 14,304. In normal fat diet rats, electrical stimulation of the sympathetic outflow or i.v. administration of noradrenaline, methoxamine and UK 14,304 produced frequency dependent or dosedependent increases in DBP. Interestingly, HFD significantly increased the vasopressor responses induced by electrical stimulation (1 and 3 Hz) and methoxamine (100 µg/kg) without significant changes on the vasopressor responses induced by noradrenaline or UK 14,304, when compared with NFD rats. 3.4. Effect of chronic administration of vehicle, NaHS, L-Cys, and DL-PAG on the cardiovascular responses induced by stimulation of the sympathetic nervous system and administration of α1/2-adrenoceptors agonists
The effect of daily i.p. injections during four weeks of vehicle, NaHS, L-Cys, and DLPAG on the cardiovascular responses induced by sympathetic stimulation and i.v. bolus injections of noradrenaline, methoxamine and UK 14,304 in HFD rats is shown in Figs. 4-7. As shown in Fig. 4, chronic administration of vehicle (PBS) did not alter the vasopressor responses induced by sympathetic stimulation or i.v. injections of methoxamine, noradrenaline or UK 14,304 when compared with those obtained in high-fat diet rats. Fig. 5 shows the effect of the chronic treatment with NaHS (5.6 mg/kg) in the vasopressor responses induced by stimulation of the sympathetic nervous system. Interestingly, chronic administration of NaHS: (1) significantly reduced vasopressor responses induced by electrical stimulation at the frequencies of 1 and 3 Hz; (2) significantly reduced the vasopressor responses induced by 0.3 µg/kg noradrenaline and 30 µg/kg methoxamine; and (3) failed to significantly modify the responses induced by UK 14,304, when compared to vehicle. Furthermore, Fig. 6 shows the effect of chronic treatment with L-Cys (300 mg/kg) on the vasopressor responses induced by stimulation of the sympathetic outflow or α1/2adrenoceptor agonists. L-Cys significantly diminished vasopressor responses induced by electrical stimulation at 1 and 3 Hz, and those induced by 30 µg/kg methoxamine when compared to vehicle. In marked contrast, L-Cys did not produce significant changes in the vasopressor responses induced by noradrenaline or UK 14,304. Fig. 7 shows the effect of chronic administration of DL-PAG (1 mg/kg) on the vasopressor responses induced by stimulation of sympathetic outflow or α1/2adrenoceptor agonists. DL-PAG significantly diminished the vasopressor response induced by 30 µg/kg of methoxamine compared to vehicle group. Further, this inhibitor
did not change the responses induced by electrical stimulation, or i.v. injections of noradrenaline and UK 14,304. Lastly, the tachycardic responses induced by i.v. injections of noradrenaline are presented in Fig. 8. Thus, high-fat diet induced an increase in tachycardic responses at 0.3-3 µg/kg of noradrenaline when compared to normal fat diet. Surprisingly, vehicle induced a significant decrease in heart rate at 1 µg/kg of noradrenaline compared to HFD control. Interestingly, chronic treatment with L-Cys (300 mg/kg) significantly decreased the tachycardic responses induced by 1 and 3 µg/kg of noradrenaline while chronic treatment with NaHS and DL-PAG failed to affect these responses. 4. Discussion 4.1. General H2S has gained extensive attention in exploring its potential therapeutic use for the treatment of cardiovascular diseases including ischemia-reperfusion injury (Elrod et al., 2007; Zhu et al., 2007), stroke (Qu et al., 2008) and hypertension (Meng et al., 2015; Yang et al., 2008). Nowadays, several lines of evidence suggest a link between H2S and metabolic diseases including insulin resistance, obesity and type 2 diabetes mellitus. In this respect, it has been recently studied the effects of HFD in H2S production and the expression of the synthesizing enzymes in several tissues (Beltowski et al., 2018). Indeed, HFD (35% fat) fed to male Wistar rats during 18 weeks produced a downregulation in CBS and CSE in liver (Bravo et al., 2011); meanwhile, in mice fed a 16% fat diet during 16 weeks diminished CSE as well as H2S synthesis in liver, kidney and lung (Peh et al., 2014). These results suggest downregulation of H2S-synthesizing enzymes and a reduction in H2S production in animals fed a HFD.
Our results demonstrate that: (1) HFD significantly increased the cardiovascular responses induced by sympathetic stimulation, haemodynamic, zoometric and metabolic variables; and (2) chronic administration of NaHS and L-Cys reversed the cardiovascular, haemodynamic and zoometric changes induced by HFD while DL-PAG ameliorated metabolic variables. 4.2. Zoometric and metabolic variables Our results demonstrate that 12 weeks HFD in rats developed obesity, hyperglycaemia, hyperleptinemia and whole-body insulin resistance (Table 3). Remarkably, we also demonstrate that chronic administration of NaHS for four weeks reduced body weight in HFD rats (Table 2). In this regard, Wu et al. (2016) demonstrated that chronic treatment with NaHS (50 µmol/kg) in mice fed HFD (45%) significantly reduced body weight and kidney triacylglycerol levels, which suggests an important role of H2S in lipid metabolism (Wu et al., 2016). This finding could explain weight and adipose tissue loss in our experimental model after chronic treatment with NaHS and L-Cys. It also could explain the loss of liver weight (Table 2). Admittedly, the role of H2S in the regulation of lipid metabolism is still controversial. In this context, several mechanisms supporting H2Sinduced lipolysis have been suggested: (1) increase in cAMP release and subsequent PKA activation (Beltowski and Jamroz-Wisniewska, 2016), (2) decrease in lipid synthesis by a down-regulation of fatty acid synthase with an increase in expenditure of lipids by an up-regulation in carnitine palmitoyltransferase-1 expression (Wu et al., 2015), (3) increase of tissue lipoprotein lipase activity and fatty acid mobilization from adipose tissue (Kawasaki et al., 2010) and (4) induce PPARα nuclear translocation and ATP binding cassette transporter A1 up-regulation leading to a decrease in lipids accumulation (Li et al., 2016). On the other hand, it has been demonstrated that (1) H2S
promotes lipid storage in adipocytes through PPARγ C139 site sulfhydration (Cai et al., 2016) and (2) H2S inhibition with DL-PAG increases PKA-perilipin1/HSL pathway stimulating the lipids catalytic reaction (Geng et al., 2013). Our results also showed that chronic treatment with NaHS and L-Cys for four weeks significantly diminished fasting leptin levels. In this respect, it has been suggested, in a model of Cdo1–/– (cysteine dioxygenase) mice, that elevated levels of cysteine promote cysteine metabolism by desulfhydration, which leads to a higher production of H2S. This increase in H2S levels could underlie the low leptin levels possibly by suppressing leptin expression (Niewiadomski et al., 2016). On the other hand, chronic treatment with DLPAG significantly diminished leptin levels. According to our findings, Geng et al. (2013), demonstrated that treatment with PAG as well as GYY4137 decreased fasting glucose and insulin levels as HOMA index by AMPK pathway (Geng et al., 2013). Hence, it could be argued that modulation of CSE/H2S system could lead to the same result. Also, it should be kept in mind that H2S is a gasotransmitter with dual effects in different organs and systems (Wang, 2002). A significant improvement in insulin sensitivity by H2S has been proposed in white adipocyte tissue from fructose-induced insulin resistance in rats (Feng et al., 2009; Geng et al., 2013) while an impairment of glucose utilization has been suggested in hepatocytes (Zhang et al., 2013). Under our experimental conditions, NaHS or L-Cys did not change HOMA-index or insulin serum levels (Table 3). Thus, the effect of H2S on insulin resistance may depend on the experimental conditions. 4.3. Haemodynamic variables Obesity is associated with endothelial dysfunction, sympathetic hyperactivity (Hall et al., 2010) and hypertension (Jiang et al., 2016). Under our experimental conditions, HFD-
produced hypertension after 12 weeks, which was maintained until 16 weeks (Table 4). Consistent with the above, diets containing high levels of saturated fatty acids elevated blood pressure and exacerbated spontaneous hypertension (Aguila and Mandarim-deLacerda, 2003). Indeed, our data demonstrated that chronic treatment with NaHS diminished heart rate, systolic, diastolic and mean blood pressure while L-Cys reduced systolic and mean blood pressure (see Table 4). According to our results, administration of H2S donors reduced blood pressure in different animal models such as spontaneously hypertensive rats (Zhao et al., 2008), Dahl salt-sensitive rats (Huang et al., 2015) and a nitric oxide synthase inhibition model (Zhong et al., 2003). Also, it has been shown that chronic administration of NaHS reduced oxidative stress and improved endothelial dysfunction in a streptozotocininduced experimental model of diabetes in mice (Ng et al., 2017). In this regard, hypertension was observed in CSE-/- knock-out mice and i.v. bolus injections of NaHS induced dose-dependent transitory decreases in systolic blood pressure of anaesthetized mice (Yang et al., 2008). Several studies have demonstrated that daily administration of N-acetylcysteine, a stable analogue of cysteine, diminished blood pressure in spontaneously hypertensive rats (Vasdev et al., 2009). Consistent with our results, two-week (Cabassia et al., 2001) or four-week (Girouard et al., 2003) oral treatment of N-acetylcysteine (4 g/kg of body weight per day in drinking water) reduced systolic blood pressure and mean blood pressure, respectively. A possible explanation of the antihypertensive effect of L-Cys has been proposed including an endothelium-dependent relaxation via a reduction in GSSG/GSH ratio (Cabassia et al., 2001). Nevertheless, if the effect of L-Cys is due to its antioxidant effect and/or an increase in H2S production remains to be determined.
As expected, conversely to NaHS, chronic treatment with DL-PAG increased heart rate (Table 4) probably due to inhibition of endogenous H2S production. In this regard, DLPAG significantly increased renal sympathetic nerve activity in anaesthetised rats (Guo et al., 2016). However, DL-PAG failed to modify blood pressure (see Table 4). To explain the lack of effect of DL-PAG in blood pressure, we should keep in mind that blood pressure is regulated by different mechanisms including local, hormonal, renal and neural mechanisms. 4.4. NaHS and L-Cys reversed the cardiovascular changes induced by high-fat diet During obesity, it has been suggested a depletion of H2S in small blood vessel (Candela et al., 2016) and a reduction in the production of H2S (Katsouda et al., 2017). In our model, we found that HFD increased the vasopressor responses induced by sympathetic stimulation (Fig. 3A). Interestingly, that increase was diminished by chronic treatment with NaHS and L-Cys (Fig. 5A and 6A). In this respect, we have previously demonstrated that acute administration of NaHS inhibited vasopressor (Centurion et al., 2016) and cardioaccelerator (Centurion et al., 2018) sympathetic outflow in a pithed rat model. Therefore, the reduction of blood pressure and heart rate by NaHS could be explained, at least in part, by the inhibition of sympathetic outflow. However, we cannot rule out the activation of other mechanisms activated by hydrogen sulfide such as Ssulfuration of KATP channels, blockade of L-type calcium channels, antioxidant effect or inhibition of renin angiotensin system. On the other hand, it has been demonstrated that in isolated aortic rings with periadventitial adipose tissue, the inhibition of CSE enzyme with DL-PAG (10 mM), enhanced phenylephrine-induced contractile response. However, under our experimental conditions, DL-PAG failed to produce a significant effect on the
cardiovascular responses (Fig. 7). In this respect, it is possible that the dose chosen could not have been enough to produce the effect expected (Fang et al., 2009).
5. Conclusion In conclusion, chronic treatment with NaHS and L-Cys was effective in reversing high-fat diet-induced cardiovascular changes. These effects could involve a reduction of the sympathetic tone. These results invite us to explore deeper into the mechanism underlying this long-term effect. Acknowledgments The authors acknowledge Conacyt Mexico for its partial financial support (Grant No. 252702). Conflict of interest None.
References Aguila, M.B., Mandarim-de-Lacerda, C.A., 2003. Heart and blood pressure adaptations in Wistar rats fed with different high-fat diets for 18 months. Nutrition 19, 347-352. Beltowski, J., Jamroz-Wisniewska, A., 2016. Hydrogen Sulfide in the Adipose TissuePhysiology, Pathology and a Target for Pharmacotherapy. Molecules 22. Beltowski, J., Wojcicka, G., Jamroz-Wisniewska, A., 2018. Hydrogen sulfide in the regulation of insulin secretion and insulin sensitivity: Implications for the pathogenesis and treatment of diabetes mellitus. Biochem. Pharmacol. 149, 6076. Bravo, E., Palleschi, S., Aspichueta, P., Buque, X., Rossi, B., Cano, A., Napolitano, M., Ochoa, B., Botham, K.M., 2011. High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomocysteinemia caused by down regulation of the transsulphuration pathway. Lipids Health Dis. 10, 60. Cabassia, A., Dumont, E.C., Girouard, H., Bouchard, J.-F., Le Jossec, M., Lamontagne, D., Besner, J.-G., de Champlain, J., 2001. Effects of chronic N-acetylcysteine treatment on the actions of peroxynitrite on aortic vascular reactivity in hypertensive rats. J. Hypertens. 19, 1233-1244. Cai, J., Shi, X., Wang, H., Fan, J., Feng, Y., Lin, X., Yang, J., Cui, Q., Tang, C., Xu, G., Geng, B., 2016. Cystathionine gamma lyase-hydrogen sulfide increases peroxisome proliferator-activated receptor gamma activity by sulfhydration at C139 site thereby promoting glucose uptake and lipid storage in adipocytes. Biochim. Biophys. Acta 1861, 419-429.
Candela, J., Velmurugan, G.V., White, C., 2016. Hydrogen sulfide depletion contributes to microvascular remodeling in obesity. Am J Physiol Heart Circ Physiol 310, H1071-1080. Centurión, D., Cobos-Puc, L.E., Ramírez-Rosas, M.B., Gómez-Díaz, B., SánchezLópez, A., Villalón, C.M., 2009. Pithed rat model for searching vasoactive drugs and studying the modulation of the sympathetic and non- adrenergic noncholinergic outflow, in: Rocha, L.L., Granados-Soto, V. (Eds.), Models of Neuropharmacology. Transworld Research Network, Kerala, India, pp. 91-97. Centurion, D., de la Cruz, S.H., Castillo-Santiago, S.V., Becerril-Chacon, M.E., TorresPerez, J.A., Sanchez-Lopez, A., 2018. NaHS prejunctionally inhibits the cardioaccelerator sympathetic outflow in pithed rats. Eur. J. Pharmacol. 823, 3540. Centurion, D., De la Cruz, S.H., Gutierrez-Lara, E.J., Beltran-Ornelas, J.H., SanchezLopez, A., 2016. Pharmacological evidence that NaHS inhibits the vasopressor responses induced by stimulation of the preganglionic sympathetic outflow in pithed rats. Eur. J. Pharmacol. 770, 40-45. Chen, X., Zhao, X., Lan, F., Zhou, T., Cai, H., Sun, H., Kong, W., Kong, W., 2017. Hydrogen Sulphide Treatment Increases Insulin Sensitivity and Improves Oxidant Metabolism through the CaMKKbeta-AMPK Pathway in PA-Induced IR C2C12 Cells. Sci. Rep. 7, 13248. Council, N.R., 2011. Guide for the Care and Use of Laboratory Animals: Eighth Edition. The National Academies Press, Washington, DC. Elrod, J.W., Calvert, J.W., Morrison, J., Doeller, J.E., Kraus, D.W., Tao, L., Jiao, X., Scalia, R., Kiss, L., Szabo, C., Kimura, H., Chow, C.-W., Lefer, D.J., 2007.
Hydrogen
sulfide
attenuates
myocardial
ischemia-reperfusion
injury
by
preservation of mitochondrial function. Proceedings of the National Academy of Sciences 104, 15560. Fang, L., Zhao, J., Chen, Y., Ma, T., Xu, G., Tang, C., Liu, X., Geng, B., 2009. Hydrogen sulfide derived from periadventitial adipose tissue is a vasodilator. J. Hypertens. 27, 2174-2185. Feng, X., Chen, Y., Zhao, J., Tang, C., Jiang, Z., Geng, B., 2009. Hydrogen sulfide from adipose tissue is a novel insulin resistance regulator. Biochem. Biophys. Res. Commun. 380, 153-159. Geng, B., Cai, B., Liao, F., Zheng, Y., Zeng, Q., Fan, X., Gong, Y., Yang, J., Cui, Q.H., Tang, C., Xu, G.H., 2013. Increase or decrease hydrogen sulfide exert opposite lipolysis, but reduce global insulin resistance in high fatty diet induced obese mice. PLoS One 8, e73892. Gillespie, J.S., Muir, T.C., 1967. A method of stimulating the complete sympathetic outflow from the spinal cord to blood vessels in the pithed rat. Br. J. Pharmacol. 30, 78-87. Girouard, H., Chulak, C., Wu, L., Lejossec, M., de Champlain, J., 2003. N-acetylcysteine improves nitric oxide and alpha-adrenergic pathways in mesenteric beds of spontaneously hypertensive rats. Am. J. Hypertens. 16, 577-584. Guo, Q., Wu, Y., Xue, H., Xiao, L., Jin, S., Wang, R., 2016. Perfusion of Isolated Carotid Sinus With Hydrogen Sulfide Attenuated the Renal Sympathetic Nerve Activity in Anesthetized Male Rats. Physiol. Res. 65, 413-423.
Hall, J.E., da Silva, A.A., do Carmo, J.M., Dubinion, J., Hamza, S., Munusamy, S., Smith, G., Stec, D.E., 2010. Obesity-induced hypertension: role of sympathetic nervous system, leptin, and melanocortins. J. Biol. Chem. 285, 17271-17276. Hu, Y., Chen, X., Pan, T.T., Neo, K.L., Lee, S.W., Khin, E.S., Moore, P.K., Bian, J.S., 2008. Cardioprotection induced by hydrogen sulfide preconditioning involves activation of ERK and PI3K/Akt pathways. Pflugers Arch 455, 607-616. Huang, P., Chen, S., Wang, Y., Liu, J., Yao, Q., Huang, Y., Li, H., Zhu, M., Wang, S., Li, L., Tang, C., Tao, Y., Yang, G., Du, J., Jin, H., 2015. Down-regulated CBS/H2S pathway is involved in high-salt-induced hypertension in Dahl rats. Nitric Oxide 46, 192-203. Jain, S.K., Bull, R., Rains, J.L., Bass, P.F., Levine, S.N., Reddy, S., McVie, R., Bocchini, J.A., 2010. Low levels of hydrogen sulfide in the blood of diabetes patients and streptozotocin-treated rats causes vascular inflammation? Antioxid Redox Signal 12, 1333-1337. Jiang, S.Z., Lu, W., Zong, X.F., Ruan, H.Y., Liu, Y., 2016. Obesity and hypertension. Exp. Ther. Med. 12, 2395-2399. Kaneko, Y., Kimura, Y., Kimura, H., Niki, I., 2006. L-Cysteine Inhibits Insulin Release From the Pancreatic -Cell: Possible Involvement of Metabolic Production of Hydrogen Sulfide, a Novel Gasotransmitter. Diabetes 55, 1391-1397. Katsouda, A., Szabo, C., Papapetropoulos, A., 2017. Reduced adipose tissue H2S in obesity. Pharmacol. Res. 128, 190-199. Kawasaki, M., Miura, Y., Funabiki, R., Yagasaki, K., 2010. Effects of Simultaneous Dietary Fish Oil Ingestion and Sulfur Amino Acid Supplementation on the Lipid
Metabolism in HepatomaBearing Rats with Hyperlipidemia. J Nutr Sci Vitaminol 56, 247-254. Kleinman, L.I., Radford, E.P., Jr., 1964. Ventilation standards for small mammals. J. Appl. Physiol. 19, 360-362. Li, D., Xiong, Q., Peng, J., Hu, B., Li, W., Zhu, Y., Shen, X., 2016. Hydrogen Sulfide UpRegulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARalpha. Int J Mol Sci 17. Lu, M., Liu, Y.H., Goh, H.S., Wang, J.J., Yong, Q.C., Wang, R., Bian, J.S., 2010. Hydrogen sulfide inhibits plasma renin activity. J Am Soc Nephrol 21, 993-1002. Meng, G., Ma, Y., Xie, L., Ferro, A., Ji, Y., 2015. Emerging role of hydrogen sulfide in hypertension and related cardiovascular diseases. Br. J. Pharmacol. 172, 55015511. Ng, H.H., Yildiz, G.S., Ku, J.M., Miller, A.A., Woodman, O.L., Hart, J.L., 2017. Chronic NaHS treatment decreases oxidative stress and improves endothelial function in diabetic mice. Diab. Vasc. Dis. Res. 14, 246-253. Niewiadomski, J., Zhou, J.Q., Roman, H.B., Liu, X., Hirschberger, L.L., Locasale, J.W., Stipanuk, M.H., 2016. Effects of a block in cysteine catabolism on energy balance and fat metabolism in mice. Ann. N. Y. Acad. Sci. 1363, 99-115. Peh, M.T., Anwar, A.B., Ng, D.S., Atan, M.S., Kumar, S.D., Moore, P.K., 2014. Effect of feeding a high fat diet on hydrogen sulfide (H2S) metabolism in the mouse. Nitric Oxide 41, 138-145. Popov, D., 2013. An outlook on vascular hydrogen sulphide effects, signalling, and therapeutic potential. Arch. Physiol. Biochem. 119, 189-194.
Qu, K., Lee, S.W., Bian, J.S., Low, C.M., Wong, P.T., 2008. Hydrogen sulfide: neurochemistry and neurobiology. Neurochem. Int. 52, 155-165. Untereiner, A., Wu, L., 2017. Hydrogen Sulfide and Glucose Homeostasis: A Tale of Sweet and the Stink. Antioxid. Redox Signal. Vasdev, S., Singal, P., Gill, V., 2009. The antihypertensive effect of cysteine. Int. J. Angiol. 1, 7-21. Wang, R., 2002. Two´s company, three´s a crowd-can H2S be the third endogenous gaseous transmitter. FASEB J. 16, 1792-1798. Whiteman, M., Gooding, K.M., Whatmore, J.L., Ball, C.I., Mawson, D., Skinner, K., Tooke, J.E., Shore, A.C., 2010. Adiposity is a major determinant of plasma levels of the novel vasodilator hydrogen sulphide. Diabetologia 53, 1722-1726. Wu, D., Gao, B., Li, M., Yao, L., Wang, S., Chen, M., Li, H., Ma, C., Ji, A., Li, Y., 2016. Hydrogen Sulfide Mitigates Kidney Injury in High Fat Diet-Induced Obese Mice. Oxid. Med. Cell Longev. 2, 715-718. Wu, D., Zheng, N., Qi, K., Cheng, H., Sun, Z., Gao, B., Zhang, Y., Pang, W., Huangfu, C., Ji, S., Xue, M., Ji, A., Li, Y., 2015. Exogenous hydrogen sulfide mitigates the fatty liver in obese mice through improving lipid metabolism and antioxidant potential. Med Gas Res 5, 1. Yang, G., Ju, Y., Fu, M., Zhang, Y., Pei, Y., Racine, M., Baath, S., Merritt, T.J.S., Wang, R., Wu, L., 2018. Cystathionine gamma-lyase/hydrogen sulfide system is essential for adipogenesis and fat mass accumulation in mice. Biochim. Biophys. Acta Mol. Cell. Biol. Lipids 1863, 165-176.
Yang, G., Wu, L., Jiang, B., Yang, W., Qi, J., Cao, K., Meng, Q., Mustafa, A.K., Mu, W., Zhang, S., Snyder, S.H., Wang, R., 2008. H2S as a physiologic vasorelaxant hypertension in mice with deletion of CSE. Science 322, 587-590. Zhang, L., Yang, G., Untereiner, A., Ju, Y., Wu, L., Wang, R., 2013. Hydrogen sulfide impairs glucose utilization and increases gluconeogenesis in hepatocytes. Endocrinology 154, 114-126. Zhao, W., Zhang, J., Lu, Y., Wang, R., 2001. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J. 20, 6008-6016. Zhao, X., Zhang, L., Zhang, C., Zeng, X., Yan, H., Jin, H., Tang, C., 2008. Regulatory effect of hydrogen sulfide on vascular collagen content in spontaneously hypertensive rats. Hypertens. Res. 31, 1619–1630. Zhong, G., Chen, F., Cheng, Y., Tang, C., Du, J., 2003. The role of hydrogen sulfide generation in the pathogenesis of hypertension in rats induced by inhibition of nitric oxide synthase. J. Hypertens. 21, 1879-1885. Zhu, Y.Z., Wang, Z.J., Ho, P., Loke, Y.Y., Zhu, Y.C., Huang, S.H., Tan, C.S., Whiteman, M., Lu, J., Moore, P.K., 2007. Hydrogen sulfide and its possible roles in myocardial ischemia in experimental rats. J. Appl. Physiol. 102, 261-268.
Table 1. Composition of the normal and high-fat diet. 100 g NFD %
100 g HFD
grams
kcal
%
grams
kcal
Carbohydrates 50.3
50.3
201.2
26.37
36
144
Proteins
23.6
23.6
94.4
10.14
13.8
55.4
Lipids
6.7
6.7
60.3
63.5
38.5
346.8
Total kcal
355.9
546.1
Note: This information was obtained from the label of the products used. NFD, normal fat diet; HFD, high fat diet.
Table 2. Effect of: (1) normal fat diet (NFD control) and high-fat diet (HFD control) after 12 weeks of diet; or (2) daily i.p. injections for 4 weeks of vehicle (PBS, 1 ml/kg), NaHS (5.6 mg/kg), L-Cys (300 mg/kg) and DL-PAG (1 mg/kg) in HFD rats on several zoometric variables. NFD
HFD
Control
Control
HFD Vehicle
NaHS
L-Cys
DL-PAG
Zoometric Variables Body 492.7±17.1 568.3±24.2a 552.9±23.9 490.7±10.5b 490.3±27.9b 501.7±49.7 weight (g) Heart (g)
1.8±0.08
1.8±0.1
2±0.1
1.9±0.1
2±0.2
2.03±0.1
Liver (g)
17.8±1
20.1±1.5a
19.4±1.5
17.5±0.8b,c
17.2±0.8b,c
14.5±0.6b,c
VAT (g)
6.8±0.6
26.2±4.04a
17.03±2.1b
14.8±2.3b,c
10.2±1.5b,c
18.8±2.1b
EAT (g)
7.3±0.4
16.7±2.1a
15.02±1.6
11.9±1.1b
12.1±1.7
20±1.9c
Each value represents mean ± S.E.M. of 6 animals. a, P < 0.05 vs. NFD control; b, P < 0.05 vs. HFD control; c, P < 0.05 vs. vehicle. VAT, visceral adipose tissue; EAT, epididymal adipose tissue.
Table 3. Effect of: (1) normal fat diet (NFD control) and high-fat diet (HFD control) after 12 weeks of diet; or (2) daily i.p. injections for 4 weeks of vehicle (PBS, 1 ml/kg), NaHS (5.6 mg/kg), L-Cys (300 mg/kg) and DL-PAG (1 mg/kg) in HFD rats on several metabolic variables. HFD
NFD
HFD
Control
Control
Vehicle
NaHS
L-Cys
DL-PAG
1.04±0.1
1.07±0.06
0.94±0.09
0.92 ± 0.15
6969.1 ± 1197.2
4329.5 ± 679.3
0.94 ± 0.18
1.3 ± 0.3
0.8 ± 0.1
0.52 ± 0.3
117.7 ± 5.9
102.2 ± 4.5
115.5 ± 5.7
95.5 ± 5.5
Metabolic Variables b
Ghrelin (ng/ml)
2.8 ± 0.2
0.89 ± 0.1
Leptin (ng/ml)
687.8 ± 137.1
7969.9 ± 876.8
Insulin (ng/ml)
0.31 ± 0.03
0.9 ± 0.6
Glucose (mg/dl)
80.3 ± 4.5
111.2 ± 5.5
HOMA Index
1.9 ± 0.1
6.9 ± 2.1
b
7.8 ± 1.5
9.5 ± 2.9
11.215 ± 4.5
1.4 ± 0.3
d,f
Matsuda Index
5.1 ± 0.7
2.4 ± 0.7
a
1.6 ± 0.2
1.5 ± 0.3
1.5 ± 0.4
6.2 ± 1.2
c,f
b
b
b
d,e
4456.8 ± 632.9
c
4063 ± 803.6
d, e
e
Each value represents mean ± S.E.M. of 6 animals. a, P < 0.05 vs. NFD; b, P < 0.01 vs. NFD; c, P < 0.05 vs. HFD; d, P < 0.01 vs. HFD; e, P < 0.05 vs. vehicle; f, P < 0.01 vs. vehicle.
Table 4. Effect of: (1) normal fat diet (NFD control) and high-fat diet (HFD control); or (2) daily i.p. injections for four weeks of vehicle (PBS, 1 ml/kg), NaHS (5.6 mg/kg), L-Cys (300 mg/kg) and DL-PAG (1 mg/kg) in HFD rats on haemodynamic variables measured in conscious animals. 12 weeks
16 weeks
SBP
MBP
HR
DBP (mm
HR
SBP
(bpm)
(mm Hg)
(mm
(bpm)
(mm Hg) Hg)
Hg)
DBP
MBP
(mm
(mm
Hg)
Hg)
81±0.5
91±1
NFD 382±18
103±3
81±0.9
87±2
355±10
423±15
160±4
131±6
399±30
159±4
126±3
137±3
419±20
411±19
146±2
115±2
125±2
336±20
390±12
164±3
121±4
135±4
364±15
353±5
165±2
117±3
132±2
394±12
113±3
Control HFD a
a
140±5
a
416±10
a
164±6
a
130±4
a
141±5
117±3
b
132±2
a
Control HFD + 165±2
Vehicle HFD +
c
113±2
d
81±0.8
d
d
142±5
e
109±5
119±5
119±1
132±1
92±1
NaHS HFD + e
L-Cys HFD + f
159±3
DL-PAG
Each value represents mean ± S.E.M. of 6 animals. a, P < 0.01 vs. NFD; b, P < 0.05 vs. vehicle (12 weeks); c, P < 0.05 vs NaHS (12 weeks); d, P < 0.01 vs NaHS (12 weeks); e, P < 0.05 vs L-Cys (12 weeks); f, P < 0.05 vs DL-PAG (12 weeks). HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure.
Figure Legends Fig. 1. (A) Experimental protocol and (B) time course of the nutritional and pharmacological treatments. HFD, high-fat diet; NFD, normal fat diet; NaHS, sodium hydrosulfide; L-Cys, L-Cysteine; DL-PAG, DL-Propargylglycine; HR, heart rate; BP, blood pressure. Fig. 2. Area under the curve (AUC) of blood glucose and serum insulin levels during the oral glucose tolerance test of control and treatment groups. Each bar represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. NFD control. Fig. 3. Effect of normal fat diet (NFD control) or high-fat diet (HFD control) during 16 weeks on the vasopressor responses induced by: (A) sympathetic stimulation, (B) noradrenaline, (C) methoxamine and (D) UK 14,304. Each point represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. NFD control. Fig. 4. Vasopressor responses induced by: (A) sympathetic stimulation; or i.v. bolus injections of: (B) noradrenaline, (C) methoxamine or (D) UK 14,304 in HFD Control and HFD+Vehicle groups. Each point represents mean ± S.E.M. of 6 animals. Fig. 5. Effect of NaHS treatment (5.6 mg/kg) on: (A) Sympathetic stimulation or i.v. bolus injections of (B) noradrenaline, (C) methoxamine or (D) UK 14,304-induced vasopressor responses in high fat diet rats. Each point represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. vehicle; **, P < 0.01 vs. vehicle; ***, P < 0.001 vs. vehicle. Fig. 6. Effect of L-Cys (300 mg/kg) on the vasopressor responses induced by: (A) sympathetic stimulation; (B) noradrenaline; (C) methoxamine; and (D) UK 14,304 in high-fat diet (HFD) rats. Each point represents mean ± S.E.M. of 6 animals. ***, P < 0.001 vs. vehicle.
Fig. 7. Effect of daily i.p. injections for four weeks of DL-PAG (1 mg/kg) on the vasopressor responses induced by: (A) sympathetic stimulation; (B) noradrenaline; (C) methoxamine; and (D) UK 14,304 in high-fat diet (HFD) rats. Each point represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. vehicle. Fig. 8. Effect of: (A) normal fat diet (NFD), (B) high-fat diet (HFD) or daily i.p. injections for 4 weeks of (C) NaHS (5.6 mg/kg), (D) L-Cys (300 mg/kg) and (E) DL-PAG (1 mg/kg) in HFD rats on the tachycardic responses induced by i.v. bolus of noradrenaline. Each point represents mean ± S.E.M. of 6 animals. *, P < 0.05 vs. NFD control; **, P < 0.01 vs NFD control; #, P < 0.05 vs. HFD control; $$$, P < 0.001 vs. HFD+Vehicle.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8