Central Administration of Resistin into the Paraventricular Nucleus (PVN) Produces Significant Cardiovascular Responses

Introduction: Resistin, a complex multimeric structure which is secreted by adipose tissue and circulates in the blood, acts on the hypothalamus to increase sympathetic nerve activity, inhibit appetite and is associated with obesity, insulin resistance and cardiovascular disorders. In this study, we survey the cardiovascular effects of direct injection of resistin into specific cell group of the paraventricular nucleus (PVN) that is known as one of the centers which control the baseline of arterial pressure and heart rate. Methods: Adult male rats were anesthetized with urethane (1.4g/kg intraperitoneally). Arterial pressure (AP) and heart rate (HR) were monitored before and after treatment. Resistin (1, 3 and 5μg/rat), norepinephrine (2.5 nM), muscimol (250ng/rat) and saline as control (vehicle solution, 1μl) were injected into the PVN parvocellular neurons. Results: The results showed that resistin (3 and 5μg/rat) caused a significant increase in AP, HR and high QRS compared to control group and prior to its injection. Injection of norepinephrine into the PVN evoked a significant increase in AP, HR and QRS amplitude, whereas injection of muscimol significantly decreased these parameters. In the control group, saline injection into the PVN had no significant effect on these parameters. Conclusion: It can be concluded that the PVN can be one of the important central areas for actions of resistin which had obvious effects on HR and AP. These results provide a base for future studies to explore the role of resistin in cardiovascular responses in conditions like metabolic syndrome and hypertension. D ow nl oa de d fr om p pj . p hy ph a. ir at 7 :2 0 + 03 30 o n S at ur da y Ja nu ar y 25 th 2 02 0 217 | Physiol Pharmacol 21 (2017) 216-224 Akbari et al. mammary gland (Steppan et al., 2001). Resistin expression is regulated in a nutritional-, ageand gender-specific manner (Morash et al., 2004) and is influenced by other factors including tumor necrosis factor-κ (Fasshauer et al., 2001a, b), β-adrenergic agonists (Fasshauer et al., 2001a) and testosterone (Ling et al., 2001). Metabolic syndrome refers to a set of conditions that include hypertension, insulin resistance, obesity and increased levels of blood lipids. Type II diabetes is characterized by insulin resistance in certain peripheral tissues and is associated with obesity (Steppan et al., 2001). Type II diabetes and obesity are major risk factors for the development of cardiovascular complications, especially hypertension and premature death (Frankel et al., 2009; Steppan et al., 2001). Studies in the last decade have shown that increase of resistin is involved in the development of diabetes, insulin resistance, obesity, cardiovascular disorders, hypertension and heart failure (Frankel et al., 2009; Steppan et al., 2001). Resistin is also involved in insulin signaling in 3T3-L1 adipocytes and expression of suppressor of cytokine signaling-3 either 3T3-L1 adipocytes or murine adipose tissues (Steppan et al., 2005). Moreover, some studies have shown that resistin, like leptin, exists in cerebrospinal fluid (Kos et al., 2007) and is widely expressed (mRNA and protein) in the pituitary (Morash et al., 2004) and hypothalamus nuclei including arcuate nucleus and the ventromedial nucleus (Tovar et al., 2005) which plays a central role in control of food intake and energy homeostasis. The hypothalamus has various nuclei with different functions, such as the paraventricular nucleus (PVN) which is involved in control of food intake, energy homeostasis and cardiovascular activity. The PVN can be one of the most important brain nuclei which controls the physiological response to energetic challenges (Hill, 2012), environmental stress and cardiovascular diseases (Benarroch, 2005). The PVN contains two general populations of neurons, the magnocellular neurons project to the median eminence and the posterior pituitary, and parvocellular elements that project to the anterior pituitary, hindbrain, spinal cord and other areas of the brain (Benarroch, 2005). Recent studies showed that the PVN contains autonomic neurons which are directly associated with cardiac sympathetic nerve by sympathetic preganglionic neurons (Dampney, 1994; Ranson et al., 1998). In addition, the PVN, as a forebrain structure, contains sympathetic pre-motor neurons (Dampney, 1994). There is increasing evidence that the PVN is implicated in disorders such as congestive heart failure (CHF), hypertension and metabolic syndrome (Benarroch, 2005). Hence, the PVN may be one of the major central areas for actions of resistin, like leptin (Shih et al., 2003). On the other hand, it was reported that there is a relationship between plasma concentration of resistin and metabolic syndrome, CHF and hypertension (Bhalla et al., 2010; Takeishi et al., 2007). However, the relationship between central resistin and cardiovascular responses has not been examined to date. Thus, the aim of the present study was to survey the effect of direct injection of resistin into the PVN on arterial blood pressure (AP) and heart rate (HR) in adult male rat. Materials and methods General procedures All investigations conducted in this study are in accordance with the ‘Guiding Principles for the Care and Use of Research Animals’ approved by Shiraz University. Adult male Sprague-Dawley rats (280-320 g, age 9-10 weeks), colony-bred in the Animal House Center, Shiraz, Iran, were housed in the animal room under controlled lighting (12 h light: 12 h darkness) and temperature (20 ± 2°C) conditions and had free access to pelleted food (formulated and made by Javaneh Khorasan Company, Iran) and tap water. To study the effect of injection of resistin into the PVN parvocellular neurons on AP and HR, rats were randomly allocated in six equal groups (n=6) as follows: Group 1: injection of 1μl saline as vehicle (as control group); all of the procedures were exactly the same in all groups; however, instead of saline other drugs were injected into the PVN in the other group. All injections were performed in the same volume (1μl). Group 2: injection of resistin (1μg/rat, Phoenix Pharmaceuticals Inc., Karlsruhe, Germany); group 3: injection of resistin (3μg/rat); group 4: injection of resistin (5μg/rat); group 5: injection of norepinephrine (2.5nM, a α1 receptor agonist; Sigma) and group 6: injection of muscimol (250ng/rat, a GABAA receptor agonist; Sigma). All injections were made unilaterally into the PVN parvocellular neurons. D ow nl oa de d fr om p pj . p hy ph a. ir at 7 :2 0 + 03 30 o n S at ur da y Ja nu ar y 25 th 2 02 0 Resistin and cardiovascular responses Physiol Pharmacol 21 (2017) 216-224 | 218 Experimental procedures and cardiovascular response All animals were anesthetized with urethane (1.4 g/kg intraperitoneally). The paw pinch reflex was used to assess the depth of anesthesia. Continuous cardiovascular response measurements were made directly using polyethylene catheters (PE-50) inserted into the left femoral artery. Polyethylene catheter was filled with heparinized saline, and was connected to a pressure transducer (Grass Instrument Company, USA). Heart rate and arterial pressure were continuously recorded by both a Grass polygraph (Model 7D Polygraph, Grass Instrument Co., USA) and a computer program written in this laboratory and by a digital electrocardiography (ECG) recorder (SuzukenKenz, ECG 110, Suzuken Co., Japan). Body temperature was monitored by rectal temperature and maintained in the range 37-38°C. The animals were placed in prone position in a stereotaxic apparatus (Stoelting Co., USA) and a small hole was drilled through the parietal bone over the PVN. Stereotaxic position for injection of drugs into the PVN was selected from the Rat Brain Atlas of Paxinos and Watson (Paxinos and Watson, 2005). Coordination of PVN was as follows: 7.44 mm rostral to the interaural line, right+0.5-0.7mm from the medial suture and 7.8 mm deep from the bregma. Following insertion of catheter in femoral artery and stereotaxic here was a waiting period of 20-25 minutes before measuring cardiovascular variables. Arterial pressure and heart rate were then recorded for an additional 15 to 30 minutes before injections, and for a period of 45 minutes following injection of resistin, vehicle and other drugs in the PVN parvocellular neurons.