Clinical and experimental studies have shown that obesity

Transcription

1 Leptin Acts in the Forebrain to Differentially Influence Baroreflex Control of Lumbar, Renal, and Splanchnic Sympathetic Nerve Activity and Heart Rate Baoxin Li, Zhigang Shi, Priscila A. Cassaglia, Virginia L. Brooks Abstract Although leptin is known to increase sympathetic nerve activity (SNA), we tested the hypothesis that leptin also enhances baroreflex control of SNA and heart rate (HR). Using α-chloralose anesthetized male rats, mean arterial pressure (MAP), HR, lumbar SNA (LSNA), splanchnic SNA (SSNA), and renal SNA (RSNA) were recorded before and for 2 hours after lateral cerebroventricular leptin or artificial cerebrospinal fluid administration. Baroreflex function was assessed using a 4-parameter sigmoidal fit of HR and SNA responses to slow ramp (3 5 minutes) changes in MAP, induced by intravenous infusion of nitroprusside and phenylephrine. Leptin (3 μg) increased (P<0.05) basal LSNA, SSNA, RSNA, HR, and MAP, and the LSNA, SSNA, RSNA, and HR baroreflex maxima. Leptin also increased gain of baroreflex control of LSNA and RSNA, but not of SSNA or HR. The elevations in HR were eliminated by pretreatment with methscopalamine, to block parasympathetic nerve activity; however, after cardiac sympathetic blockade with atenolol, leptin still increased basal HR and MAP and the HR baroreflex maximum and minimum. Leptin (1.5 μg) also increased LSNA and enhanced LSNA baroreflex gain and maximum, but did not alter MAP, HR, or the HR baroreflex. Lateral cerebroventricular artificial cerebrospinal fluid had no effects. Finally, to test whether leptin acts in the brain stem, leptin (3 μg) was infused into the 4th ventricle; however, no significant changes were observed. In conclusion, leptin acts in the forebrain to differentially influence baroreflex control of LSNA, RSNA, SSNA, and HR, with the latter action mediated via suppression of parasympathetic nerve activity. (Hypertension. 2013;61: ) Online Data Supplement Key Words: lumbar sympathetic nerve activity male rats methscopolamine parasympathetic renal sympathetic nerve activity splanchnic sympathetic nerve activity Clinical and experimental studies have shown that obesity is associated with hypertension, attributable at least in part to increases in sympathetic nerve activity (SNA). 1,2 Considerable effort has been directed toward identifying the underlying mechanisms. As recently reviewed, 2,3 1 potential candidate is leptin, a 16-kDa peptide hormone produced predominantly by white adipocytes. Acute leptin administration increases SNA to multiple organs, including brown adipose tissue, the hindlimb, adrenal gland, and kidney, 3 and chronic leptin infusion produces hypertension. 2,3 In addition, leptindeficient obese mice (ob/ob mice) fail to exhibit hypertension, despite massive obesity. 2,3 Another deleterious consequence of obesity is impaired sensitivity of the baroreceptor reflex. 4 6 Decreased baroreflex function has been identified as a risk factor for subsequent adverse cardiovascular events in humans with type 2 diabetes mellitus. 7 However, the mechanism is unknown. Although leptin has been considered, 8 current information is conflicting. For example, monogenic forms of rodent obesity attributable to loss of leptin or to mutations of the leptin receptor exhibit decreased baroreflex gain (BRG), 9 12 suggesting that leptin enhances baroreflex function. Conversely, systemic administration of leptin was reported to be ineffective, 13 and microinjection of leptin into the nucleus tractus solitarius (NTS) suppressed baroreflex control of heart rate (HR). 14 Therefore, 1 aim of the present experiments was to further investigate whether leptin influences baroreflex function. Given the well-established sympathoexcitatory effects of leptin, we hypothesized that leptin would also enhance the SNA responses to decreases in arterial pressure (ie, BRG). A key feature of these experiments is that we compared the effects of leptin on baroreflex control of lumbar SNA (LSNA), renal SNA (RSNA), as well as splanchnic SNA (SSNA). In addition, to test whether leptin influences baroreflex control of HR via changes in the activity of the sympathetic nervous system or the parasympathetic nervous system, experiments were performed to determine whether the effects of leptin on the HR baroreflex were eliminated by systemic pretreatment Received October 27, 2012; first decision January 11, 2013; revision accepted January 22, From the Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR (B.L., Z.S., P.A.C., V.L.B.); and Department of Endocrinology, Baoding First Central Hospital, Baoding, People s Republic of China (B.L.). The online-only Data Supplement is available with this article at /-/DC1. Correspondence to Virginia L. Brooks, Department of Physiology and Pharmacology, Oregon Health & Science University, 1381 SW Sam Jackson Park Rd L334, Portland, OR brooksv@ohsu.edu 2013 American Heart Association, Inc. Hypertension is available at DOI: /HYPERTENSIONAHA

2 Li et al Leptin and Baroreflex Control of SNA and HR 813 with drugs that blocked cholinergic (methscopolamine) or β-adrenergic (atenolol) receptors. Leptin has been shown to increase SNA after selective microinjection into several hypothalamic nuclei and into the NTS. 14,20 Therefore, to activate these multiple sites concurrently, leptin was infused via a lateral cerebral ventricle (LV). In addition, to begin to identify the sites at which leptin acts to modify baroreflex function, we compared the effects of leptin infusion via the LV, which will activate receptors in both the hypothalamus and NTS, versus the fourth ventricle (4 V), which targets brain stem leptin receptors. Methods Animals Forty male ( g) Sprague Dawley rats (Charles River Laboratories, Inc) were used for these experiments. All of the rats were acclimated for 1 week before experimentation in a room with a 12-hour:12-hour light/dark cycle, with food (LabDiet 5001, Richmond, IN) and water provided ad libitum. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional (Oregon Health & Science University) Animal Care and Use Committee. Surgery Under isoflurane anesthesia, a tracheal tube, femoral arterial and venous catheters, and a recording electrode around the lumbar, splanchnic, or renal sympathetic nerve were implanted (see the online-only Data Supplement for details). After preparing the rat for intracerebroventricular (icv) infusions, a loading dose of α-chloralose (50 mg kg 1 ; Sigma) was administered intravenously over 30 minutes, while isoflurane was slowly withdrawn, and this was followed by a continuous infusion of α-chloralose (25 mg kg 1 h 1 ) for the duration of the experiment. Throughout the experiment, artificial ventilation with 100% oxygen was maintained, and respiratory rate and tidal volume were adjusted to maintain expired CO 2 at 3.5% to 4.5%. Anesthetic depth was regularly confirmed by the lack of a pressor or withdrawal response to a foot or tail pinch. After completion of surgery and the α-chloralose loading dose, rats were allowed to stabilize for 60 minutes before experimentation. Baroreflex Function Pulsatile and mean arterial pressures (MAPs), HR, and SNA were continuously recorded, and baroreflex function was assessed as previously described 21 (see the online-only Data Supplement for details). Briefly, complete baroreflex curves were constructed by fitting a sigmoidal curve to the changes in HR and SNA induced by slow changes in arterial pressure after intravenous infusion of nitroprusside and phenylephrine. Absolute values of BRG and the mean±sem of other sigmoidal parameters (baroreflex maxima, minima, and midpoint) are depicted in the figures. Intracerebroventricular Infusions With a flat skull and using bregma and the dorsal surface of the dura as zero, single-barreled glass pipettes drawn to a small tip were used for both LV and 4 V infusions. Coordinates for positioning the LV cannulae were as follows (mm from bregma): 1.0 caudal, 1.4 lateral, and 4.2 dorsal. A pipette was placed into the 4 V using the following coordinates (mm): 2.0 caudal to the interaural line, on the midline, and 7.3 ventral to the skull surface. Leptin (R&D Systems) was dissolved in artificial cerebrospinal fluid (CSF) containing (in mmol/l): 128 NaCl, 2.6 KCl, 1.3 CaCl 2, 0.9 MgCl 2, 20 NaHCO 3, 1.3 Na 2 HPO 4, and 2.0 dextrose, ph 7.4. Correct pipette placement was confirmed at the end of the experiment by infusing 100 nl of 2.5% Alcian blue in 0.5 mmol/l per liter of sodium acetate via the same pipette, removing the brain, and verifying the presence of dye in the cerebroventricles. Experimental Protocols After stabilization, basal baroreflex function was established by producing 2 baroreflex curves, 30 minutes apart, with similar gains; the final control curve was used for data analysis. Protocol 1 After reestablishment of basal values, LSNA and HR BRG were measured 1 and 2 hours after LV injection of a dose of leptin that has been shown to inhibit food intake when administered in conscious rats 22,23 (3 μg in 3 μl, followed by 5 μg/h; n=5), a lower leptin dose (1.5 μg in 1.5 μl, followed by 5 μg/h; n=5) or the artificial CSF vehicle (0.6 μl/min; n=5). In separate groups of rats, SSNA (n=6) or RSNA (n=5) and HR BRG were measured after LV injection of the higher leptin dose. The HR and MAP results from the 3 groups of rats receiving the higher leptin dose were combined (n=16). Protocol 2 After establishing basal baroreflex function, either methscopolamine (1 mg/kg; n=4) or atenolol (2 mg/kgh; n=5) was administered intravenously, at doses previously documented to block cardiac parasympathetic and sympathetic contributions, respectively. 24 Thirty minutes later, baroreflex curves were generated to assess the effects of the blockade alone. Then, leptin was administered icv (3 μg followed by 5 μg/h), and baroreflex function was reassessed after 1 and 2 hours. Protocol 3 After control measurements, LSNA and HR BRG were measured 1 and 2 hours after leptin (3 μg, followed by 5 μg/h; n=5) administration into the 4 V. Statistical Analysis In each protocol, basal data were taken as the average of the 30 second period before each BRG measurement. All data are presented as means±sem. Between-group differences were evaluated using 1- or 2-way ANOVA for repeated measures and the post hoc Newman Keuls test. P values <0.05 were considered statistically significant. Results LV Leptin Administration Increases Gain of Baroreflex Control of LSNA and RSNA, but Not of SSNA or HR LV leptin (3 μg) administration increased basal LSNA and RSNA after 1 and 2 hours; the lower dose of leptin (1.5 μg) increased LSNA only at 2 hours (Figures 1 and 2). In contrast, basal SSNA was not significantly elevated until 2 hours after 3 μg leptin (Figure 2). MAP and HR also increased 2 hours after leptin (3 μg) injection; however, 1.5 μg leptin did not significantly alter MAP or HR (Table 1). Basal values were not significantly changed during icv infusion of artificial CSF (Figure 1; Table 1). Baroreflex control of LSNA, SSNA, RSNA, and HR were differentially altered by icv leptin. Leptin increased LSNA (3 and 1.5 μg doses) and RSNA BRG (Figures 3 and 4); the LSNA and RSNA reflex maximum and range were also elevated (Figures 3 and 4 and Tables S1 and S2 in the online-only Data Supplement; see Figure S1 for representative experiment). On the other hand, whereas the SSNA maximum was elevated 1 and 2 hours after leptin, BRG was not significantly increased (Figure 4; Table S3). Similarly, the higher dose of leptin increased the HR reflex maximum and BP 50 after 2 hours (Table S4), without significantly altering HR BRG (Figure 5). Leptin increased the baroreflex minimum of LSNA and HR (Figures 3 and 5; Tables S1 and S4), but not of RSNA and SSNA (Figure 4 and Tables S2 and S3).

3 814 Hypertension April 2013 Table 1. Effect of LV Leptin and acsf on Basal MAP and HR Figure 1. Effects of lateral cerebroventricular (LV) leptin or artificial cerebrospinal fluid (acsf) on baseline lumbar sympathetic nerve activity (LSNA). LV leptin (3 μg, n=5; top) increased basal LSNA at 1 and 2 hours, whereas the lower dose (1.5 μg, n=5) increased basal LSNA only at 2 hours (middle). acsf (n=5) had no effects on LSNA. *P<0.05 vs CON (control); P<0.05 vs acsf at the same time. Methscopolamine Blocks the Effect of Leptin to Increase HR and Alter Baroreflex Control of HR Methscopolamine increased basal HR, without altering MAP; maximum and minimum baroreflex HR were increased, and HR BRG was reduced (Table 2; Figure 6). More importantly, after methscopolamine, leptin failed to alter basal MAP, HR, or influence baroreflex control of HR (Figure 6; Table S5). To Figure 2. Effects of lateral cerebroventricular (LV) leptin on baseline renal sympathetic nerve activity (RSNA) and splanchnic SNA (SSNA). LV leptin (3 μg) increased basal RSNA (n=5, top) at 1 and 2 hours, whereas SSNA (n=6; bottom) was increased only at 2 hours. *P<0.05 vs CON (control). Variable Control Leptin 1 h Leptin 2 h 3 μg leptin (n=16) MAP, mm Hg 103±4 106±4 118±5* HR, bpm 328±14 357±16 388±19* 1.5 μg leptin (n=5) MAP, mm Hg 105±4 102±2 99±2 HR, bpm 345±22 342±16 355±19 acsf (n=5) MAP, mm Hg 102±7 98±7 98±14 HR, bpm 342±9 355±9 360±5 acsf indicates artificial cerebrospinal fluid; HR, heart rate; LV, lateral cerebral ventricle; and MAP, mean arterial pressure. *P<0.05 vs control; P<0.05 vs acsf, same time. The MAP and HR results were combined from the separate sets of rats used for studies of lumbar sympathetic nerve activity (SNA), splanchnic SNA, and renal SNA. These data were not different between groups (2-way repeated measures ANOVA, insignificant group and interaction). determine whether the failure of leptin to increase HR was a result of a maximal elevation after methscopolamine, the β-agonist isoproterenol (1 μg/kg) was injected intravenously after methscopolamine administration and 2 hours of leptin infusion. Notably, isoproterenol increased HR from 409±19 to 495±23 bpm, a value higher than the baroreflex HR maximum (446±2 bpm; P<0.05; n=3). Atenolol reduced basal MAP and HR and decreased the baroreflex HR maximum and the HR range (Figure 6; Table 2; Table S5). After atenolol, 2 hours of leptin infusion increased basal MAP and HR and elevated the baroreflex HR maximum and minimum (Figure 6; Table S5). Interestingly, HR gain was reduced 2 hours after initiating the leptin infusion (Figure 6) in atenolol-treated rats. 4 V Leptin Does Not Alter Basal or Baroreflex Control of LSNA, HR, and MAP In sharp contrast to the potent effects of leptin administered via the LV, basal MAP, HR, and LSNA, as well as BRG and baroreflex parameters, were not altered during 4 V leptin infusion (Figure 7; Table S6). Discussion The major goal of this study was to test the hypothesis that leptin acts centrally to enhance baroreflex control of SNA and HR and, if so, to begin to investigate the mechanisms. The important new findings are as follows: (1) leptin increases SSNA (as well as LSNA, RSNA, HR, and MAP, as previously reported 19 ); (2) central leptin exerts differential effects on baroreflex control of LSNA, RSNA, SSNA, and HR; (3) the ability of leptin to increase HR and its baroreflex regulation is eliminated by pretreatment with methscopolamine, to block the parasympathetic nervous system, but not by pretreatment with atenolol, to block cardiac sympathetic effects; and (4) these actions of leptin are not observed when leptin is infused via the 4 V. Collectively, these data indicate that central leptin acts in the forebrain to influence baroreflex control of SNA and HR, with the latter action mediated via suppression of cardiac parasympathetic tone.

4 Li et al Leptin and Baroreflex Control of SNA and HR 815 Figure 4. Effects of leptin on baroreflex control of renal sympathetic nerve activity (RSNA) and splanchnic SNA (SSNA). Leptin (lateral cerebroventricular; 3 μg) increased the RSNA (n=5; top) baroreflex maximum and gain (inset) after 1 and 2 hours. Leptin also increased the SSNA (n=6; bottom) baroreflex maximum at 1 and 2 hours, and the BP50 at 2 hours (n=6; *P<0.05); however, the SSNA baroreflex gain and minimum were unchanged (top; inset). *P<0.05 vs CON (control). Figure 3. Effects of lateral cerebroventricular (LV) leptin or artificial cerebrospinal fluid (acsf) on baroreflex control of lumbar sympathetic nerve activity (LSNA). Both doses of LV leptin (1.5 and 3 μg) increased baroreflex gain (insets), as well as the LSNA baroreflex maximum. In contrast, acsf had no effects on the baroreflex. *P<0.05 vs CON (control); P<0.05 vs acsf at the same time. Previous studies have demonstrated that intravenous or central leptin administration increases LSNA, RSNA, HR, and MAP 19,25,26 ; our results confirm this work. A new finding, however, is that leptin also acts centrally to increase SSNA. Dunbar et al 26 reported that icv leptin decreases splanchnic blood flow, which contributes to its pressor effect. The increase in SSNA that we observed suggests that the sympathetic nervous system likely mediates this action. Plasma leptin levels increase after a meal. 27 Therefore, the central action of leptin to increase SSNA may help to maintain arterial pressure at a time when the splanchnic circulation is dilating to accept and distribute absorbed nutrients. In addition, in parallel to the actions of insulin, 28 meal-induced increases in leptin may contribute to enhanced baroreflex control of LSNA. Monogenetic impairment of the actions of leptin, attributable either to loss of leptin or mutations of the leptin receptor, depresses the baroreflex, 9 12 suggesting that leptin may support or enhance baroreflex function. However, these rodent models exhibit obesity, and diet-induced obesity also decreases baroreflex control of HR 4,5,24 and, in 1 human study, SNA, 29 confounding this suggestion. The present study demonstrates that exogenous leptin administration differentially influences baroreflex function. Increases in BRG were observed for LSNA and RSNA, but not for SSNA or HR. However, leptin increased the baroreflex maximum and range of LSNA, RSNA, SSNA, and HR, as well as the baroreflex minimum of LSNA and HR. Previously, intravenous leptin was shown to increase the range and maximum of baroreflex control of RSNA in mice, without enhancing gain. 13 Different species or routes of leptin administration may explain these divergent results. The mechanism by which leptin differentially alters baroreflex control of various autonomic efferents was not identified, but may arise from actions of leptin at different brain sites, or within a site on distinct neurons. In support of this supposition, previous studies have demonstrated that leptin acts differentially to increase SNA at several hypothalamic sites containing leptin receptors. 16,18,19 We found that leptin increases the baroreflex minimum for LSNA and HR (but not for SSNA or RSNA; see also Reference 13); therefore, leptin can increase LSNA and HR even when baroreflex suppression is maximal at high pressures, suggesting that the neurocircuitry that mediates the increases in LSNA and HR is at least in part independent of, or distal to, brain baroreflex pathways. However, the finding that leptin also increases LSNA BRG suggests an additional interaction with baroreflex pathways. Thus, the brain neurons that initiate the effects of leptin on baroreflex control of different sympathetic nerves may also project to multiple sites in the brain stem and spinal cord. Obesity mutes leptin s effect to increase SNA to brown adipose tissue and LSNA, yet leptin-induced increases in RSNA are preserved or enhanced. 30,31 Because leptin increases gain of baroreflex control of LSNA, brain resistance to leptin could explain in part the decrease in muscle SNA BRG reported in obese humans compared with after weight loss. 29 On the

5 816 Hypertension April 2013 Figure 5. Effects of lateral cerebroventricular leptin or artificial cerebrospinal fluid (acsf) on baroreflex control of heart rate (HR). Although neither the lower dose of leptin (n=5) nor acsf (n=5) influenced the HR baroreflex, the higher dose of leptin (top) increased the HR baroreflex maximum and minimum (n=16) without altering gain. *P<0.05 vs CON (control). P<0.05 vs acsf at the same time. other hand, because leptin does not influence SSNA BRG, its loss does not likely contribute to the decreased SSNA gain observed in Zucker rats. 10 Indeed, Schreihofer et al 10 came to the same conclusion, because they found that SSNA BRG is not impaired in juvenile Zucker rats, but only in older Zucker rats that exhibit considerable obesity. Although the ability of leptin to increase HR has been noted before, 14,18,32 the mechanism has not been directly Table 2. Effects of Methscopolamine and Atenolol on MAP and HR, Before and After Intracerebroventricular Leptin (3 μg) Variable Control Methscopolamine Leptin 1 h Leptin 2 h MAP, 118±5 118±4 117±8 109±7 mm Hg HR, bpm 355±13 403±4* 409±8* 411±7* Control Atenelol Leptin 1 h Leptin 2 h MAP, 113±5 92±2* 102±5* 106±5 mm Hg HR, bpm 351±13 318±8* 326±6* 348±13 HR indicates heart rate; and MAP, mean arterial pressure. *P<0.05 vs control. P<0.05 vs atenolol. Figure 6. Effects of leptin on the heart rate (HR) baroreflex were blocked by methscopolamine (METH), but not atenolol (ATEN). Top, Methscopolamine (1 mg/kg, iv; n=4; solid gray line and circles) elevated the HR baroreflex curve, increased the HR maximum and minimum (P<0.05), but reduced the HR gain (inset; *P<0.05). However, after methscopolamine, leptin did not affect the baroreflex control of HR (dashed and dotted lines). Bottom, Atenolol (2 mg/kg h, iv; n=5; solid gray line and circles) depressed the HR baroreflex curve and reduced the HR maximum (P<0.05). After atenolol, leptin increased the HR maximum and minimum and reduced the HR gain (inset; P<0.05). *P<0.05 vs CON (control); P<0.05 vs atenolol. investigated. Nevertheless, previous work has provided indirect evidence that leptin may suppress parasympathetic control of the heart. First, Arnold et al 14 reported that NTS microinjection of leptin decreased spontaneous baroreflex sensitivity and the reflex bradycardia after bolus intravenous injections of phenylephrine, each of which reflect primarily cardiovagal activity. Second, correlations between leptin and indices of cardiac parasympathetic activity have been noted in humans. 33 Using a pharmacological approach, we directly tested this hypothesis. Administration of methscopolamine to block the parasympathetic nervous system completely prevented the ability of leptin to elevate HR and right-shift the baroreflex curve. This was not attributable to a HR ceiling effect, because after methscopolamine and leptin treatment, intravenous injection of the β-adrenergic agonist, isopreteronol, to mimic cardiac sympathoexcitation, increased HR even higher. Interestingly, methscopolamine also prevented the pressor response to LV leptin. Methscopolamine does not cross the blood brain barrier, but it can exhibit some cholinergic nicotinic blocking activity. 34 Therefore, 1 explanation for this finding is that methscolpolamine inhibited ganglionic transmission. In support, we have found that intravenous methscopolamine decreases SNA and arterial pressure when administered after leptin infusion (Li et al, unpublished observations). On

6 Li et al Leptin and Baroreflex Control of SNA and HR 817 Figure 7. Effects of 4 V leptin (3 μg) on basal lumbar sympathetic nerve activity (LSNA) and baroreflex control of LSNA and heart rate (HR). No significant changes in basal LSNA or in baroreflex control of LSNA and HR were observed after 4 V leptin (3 μg) infusion. the other hand, in rats in which cardiac sympathetic activity was blocked with atenolol, leptin increased HR, MAP, and right-shifted the baroreflex curve. Collectively, these data suggest that leptin increases HR at any given MAP by suppressing parasympathetic control of the heart. One of the hallmarks of obesity in humans and experimental animals is suppression of cardiac parasympathetic activity 4,24,35 in association with elevated leptin levels. Therefore, we speculate that leptin may contribute to impaired vagal activity during obesity. Interestingly, after atenolol, leptin infusion also markedly decreased BRG. Thus, leptin may also suppress the response of the parasympathetic nervous system to changes in arterial pressure, at least when cardiac sympathetic activity is also reduced. In humans, obesity can reduce sympathetic activity to the heart 1 ; therefore, leptin may also contribute to the impaired cardiac baroreflex sensitivity that is observed. Leptin has been shown to increase SNA and HR after microinjection into several hypothalamic nuclei (arcuate nucleus, dorsal medial hypothalamus, lateral hypothalamus, and ventromedial hypothalamus) 18,19,36 as well as the NTS. 14,20 However, as we previously found with insulin, 37 leptin infusion into the 4 V, to restrict its actions to the hindbrain, was ineffective. The variance of our study with previous work may be explained by a failure of sufficient leptin to reach its active sites in the NTS, because at least in 1 report, 20 larger doses were used. Regardless of the explanation, our studies show that leptin can act in the forebrain to increase SSNA and influence baroreflex control of LSNA, RSNA, SSNA, and HR. As discussed, in view of the variable effects on baroreflex control of SNA and HR that were observed, >1 site may be involved. Although the results of this study clearly demonstrate a differential effect of leptin on baroreflex function, there are several limitations that must be considered. First, the experiments were performed in anesthetized rats. Although we acknowledge that it will be important to repeat these experiments in conscious animals, it is noteworthy that α-chloralose was chosen as the anesthetic, because baroreflex function is relatively preserved. Indeed, the values of LSNA BRG measured in the present study are essentially identical to values obtained in conscious rats. 38,39 Second, the concentrations of leptin achieved in the CSF were certainly supraphysiological. However, high doses are required to establish a significant diffusion gradient, because as a large peptide only limited amounts of leptin penetrate brain from the CSF and only very slowly. 40 The fact that the stimulatory effects of leptin on LSNA in the present study were related more to the initial bolus dose than to the total infused amount supports this contention. Importantly, in rats, intravenous infusion of doses that increase plasma leptin within the physiological-pathophysiological range increase SNA similarly to the present study, 25 strongly suggesting that such levels of plasma leptin would also enhance baroreflex control. Third, the short-term effects of leptin that we observed may underestimate the potency of long-term elevations. In support, in agreement with previous work, 16,25 we found that the SNA responses to LV leptin are slowly developing and may not have peaked within our 2-hour study period. Moreover, chronic intracerebroventricular infusion of significantly lower doses of leptin are sufficient to elevate arterial pressure and suppress food intake. 41 Perspectives In both humans and animals, elevations in plasma leptin occur soon after beginning a high-fat diet and accruing excess adiposity, in association with activation of the sympathetic nervous system Thus, the hypothesis that increased leptin contributes to obesity-induced sympathoexcitation has received considerable attention. 2,3 Another hallmark of obesity is depressed basal parasympathetic nerve activity and impaired HR baroreflex function, which also rapidly develops. 4 6,24,35,44 The present results suggest that leptin may also contribute to these cardiovascular consequences. On the other hand, in obese humans, baroreflex control of muscle SNA has been reported to be depressed 29,45 or preserved. 46,47 Therefore, if leptin is involved in SNA baroreflex impairment, then the factor(s) that reduce brain sensitivity to leptin s action to enhance BRG must vary among individuals. Future experiments are required to identify the factors that modify sensitivity of the brain to leptin

7 818 Hypertension April 2013 and to determine whether and how the impact of these factors varies among obese individuals. Sources of Funding This study was supported in part by a National Institutes of Health grant HL and a Grant-in-Aid from the American Heart Association (12GRNT ). None. Disclosures References 1. Esler M, Straznicky N, Eikelis N, Masuo K, Lambert G, Lambert E. Mechanisms of sympathetic activation in obesity-related hypertension. Hypertension. 2006;48: Hall JE, da Silva AA, do Carmo JM, Dubinion J, Hamza S, Munusamy S, Smith G, Stec DE. Obesity-induced hypertension: role of sympathetic nervous system, leptin, and melanocortins. J Biol Chem. 2010;285: Rahmouni K. Leptin-induced sympathetic nerve activation: signaling mechanisms and cardiovascular consequences in obesity. Curr Hypertens Rev. 2010;6: Beske SD, Alvarez GE, Ballard TP, Davy KP. Reduced cardiovagal baroreflex gain in visceral obesity: implications for the metabolic syndrome. Am J Physiol Heart Circ Physiol. 2002;282:H630 H Straznicky NE, Lambert GW, Lambert EA. Neuroadrenergic dysfunction in obesity: an overview of the effects of weight loss. Curr Opin Lipidol. 2010;21: Arone LJ, Mackintosh R, Rosenbaum M, Leibel RL, Hirsch J. Autonomic nervous system activity in weight gain and weight loss. Am J Physiol. 1995;269(1 pt 2):R222 R Okada N, Takahashi N, Yufu K, Murozono Y, Wakisaka O, Shinohara T, Anan F, Nakagawa M, Hara M, Saikawa T, Yoshimatsu H. Baroreflex sensitivity predicts cardiovascular events in patients with type 2 diabetes mellitus without structural heart disease. Circ J. 2010;74: Grassi G. Leptin, sympathetic nervous system, and baroreflex function. Curr Hypertens Rep. 2004;6: Goncalves AC, Tank J, Diedrich A, Hilzendeger A, Plehm R, Bader M, Luft FC, Jordan J, Gross V. Diabetic hypertensive leptin receptordeficient db/db mice develop cardioregulatory autonomic dysfunction. Hypertension. 2009;53: Schreihofer AM, Mandel DA, Mobley SC, Stepp DW. Impairment of sympathetic baroreceptor reflexes in obese Zucker rats. Am J Physiol Heart Circ Physiol. 2007;293:H2543 H Davis G. Baroreflex and somato-reflex control of blood pressure, heart rate and renal sympathetic nerve activity in the obese Zucker rat. Exp Physiol. 2011;96: Hilzendeger AM, Goncalves AC, Plehm R, Diedrich A, Gross V, Pesquero JB, Bader M. Autonomic dysregulation in ob/ob mice is improved by inhibition of angiotensin-converting enzyme. J Mol Med. 2010;88: Hausberg M, Morgan DA, Chapleau MA, Sivitz WI, Mark AL, Haynes WG. Differential modulation of leptin-induced sympathoexcitation by baroreflex activation. J Hypertens. 2002;20: Arnold AC, Shaltout HA, Gallagher PE, Diz DI. Leptin impairs cardiovagal baroreflex function at the level of the solitary tract nucleus. Hypertension. 2009;54: Harlan SM, Morgan DA, Agassandian K, Guo DF, Cassell MD, Sigmund CD, Mark AL, Rahmouni K. Ablation of the leptin receptor in the hypothalamic arcuate nucleus abrogates leptin-induced sympathetic activation. Circ Res. 2011;108: Rahmouni K, Morgan DA. Hypothalamic arcuate nucleus mediates the sympathetic and arterial pressure responses to leptin. Hypertension. 2007;49: Satoh N, Ogawa Y, Katsuura G, Numata Y, Tsuji T, Hayase M, Ebihara K, Masuzaki H, Hosoda K, Yoshimasa Y, Nakao K. Sympathetic activation of leptin via the ventromedial hypothalamus: leptin-induced increase in catecholamine secretion. Diabetes. 1999;48: Marsh AJ, Fontes MA, Killinger S, Pawlak DB, Polson JW, Dampney RA. Cardiovascular responses evoked by leptin acting on neurons in the ventromedial and dorsomedial hypothalamus. Hypertension. 2003;42: Montanaro MS, Allen AM, Oldfield BJ. Structural and functional evidence supporting a role for leptin in central neural pathways influencing blood pressure in rats. Exp Physiol. 2005;90: Mark AL, Agassandian K, Morgan DA, Liu X, Cassell MD, Rahmouni K. Leptin signaling in the nucleus tractus solitarii increases sympathetic nerve activity to the kidney. Hypertension. 2009;53: Cassaglia PA, Hermes SM, Aicher SA, Brooks VL. Insulin acts in the arcuate nucleus to increase lumbar sympathetic nerve activity and baroreflex function in rats. J Physiol (Lond). 2011;589(pt 7): Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Invest. 1996;98: Lee JH, Cha MJ, Yoo SB, Moon YW, Noh SJ, Jahng JW. Leptin blocks the fasting-induced increase of perk1/2 in the paraventricular nucleus of rats. Regul Pept. 2010;162: McCully BH, Brooks VL, Andresen MC. Diet-induced obesity severely impairs myelinated aortic baroreceptor reflex responses. Am J Physiol Heart Circ Physiol. 2012;302:H2083 H Haynes WG, Morgan DA, Walsh SA, Mark AL, Sivitz WI. Receptormediated regional sympathetic nerve activation by leptin. J Clin Invest. 1997;100: Dunbar JC, Hu Y, Lu H. Intracerebroventricular leptin increases lumbar and renal sympathetic nerve activity and blood pressure in normal rats. Diabetes. 1997;46: Lee MJ, Fried SK. Integration of hormonal and nutrient signals that regulate leptin synthesis and secretion. Am J Physiol Endocrinol Metab. 2009;296:E1230 E Young CN, Deo SH, Chaudhary K, Thyfault JP, Fadel PJ. Insulin enhances the gain of arterial baroreflex control of muscle sympathetic nerve activity in humans. J Physiol (Lond). 2010;588(pt 18): Grassi G, Seravalle G, Colombo M, Bolla G, Cattaneo BM, Cavagnini F, Mancia G. Body weight reduction, sympathetic nerve traffic, and arterial baroreflex in obese normotensive humans. Circulation. 1998;97: Rahmouni K, Morgan DA, Morgan GM, Mark AL, Haynes WG. Role of selective leptin resistance in diet-induced obesity hypertension. Diabetes. 2005;54: Prior LJ, Eikelis N, Armitage JA, Davern PJ, Burke SL, Montani JP, Barzel B, Head GA. Exposure to a high-fat diet alters leptin sensitivity and elevates renal sympathetic nerve activity and arterial pressure in rabbits. Hypertension. 2010;55: Ren J. Leptin and hyperleptinemia from friend to foe for cardiovascular function. J Endocrinol. 2004;181: Piestrzeniewicz K, Łuczak K, Lelonek M, Wranicz JK, Goch JH. Obesity and heart rate variability in men with myocardial infarction. Cardiol J. 2008;15: Brown JH, Laiken N. Muscarinic receptor agonists and antagonists. In: Brunton LL, Chabner BA, Knollmann BC, eds. Goodman & Gilman s Pharmacological Basis of Therapeutics. New York: McGraw-Hill; 2011: Van Vliet BN, Hall JE, Mizelle HL, Montani JP, Smith MJ Jr. Reduced parasympathetic control of heart rate in obese dogs. Am J Physiol. 1995;269(2 pt 2):H629 H Enriori PJ, Sinnayah P, Simonds SE, Garcia Rudaz C, Cowley MA. Leptin action in the dorsomedial hypothalamus increases sympathetic tone to brown adipose tissue in spite of systemic leptin resistance. J Neurosci. 2011;31: Pricher MP, Freeman KL, Brooks VL. Insulin in the brain increases gain of baroreflex control of heart rate and lumbar sympathetic nerve activity. Hypertension. 2008;51: Xu L, Brooks VL. Sodium intake, angiotensin II receptor blockade, and baroreflex function in conscious rats. Hypertension. 1997;29(1 pt 2): Xu L, Collister JP, Osborn JW, Brooks VL. Endogenous angiotensin II supports lumbar sympathetic activity in conscious, sodium deprived rats: role of area postrema. Am J Physiol. 1998;275:R46 R Maness LM, Kastin AJ, Farrell CL, Banks WA. Fate of leptin after intracerebroventricular injection into the mouse brain. Endocrinology. 1998;139: Dubinion JH, da Silva AA, Hall JE. Chronic blood pressure and appetite responses to central leptin infusion in rats fed a high fat diet. J Hypertens. 2011;29:

8 Li et al Leptin and Baroreflex Control of SNA and HR Gentile CL, Orr JS, Davy BM, Davy KP. Modest weight gain is associated with sympathetic neural activation in nonobese humans. Am J Physiol Regul Integr Comp Physiol. 2007;292:R1834 R Muntzel MS, Al-Naimi OA, Barclay A, Ajasin D. Cafeteria diet increases fat mass and chronically elevates lumbar sympathetic nerve activity in rats. Hypertension. 2012;60: Armitage JA, Burke SL, Prior LJ, Barzel B, Eikelis N, Lim K, Head GA. Rapid onset of renal sympathetic nerve activation in rabbits fed a high-fat diet. Hypertension. 2012;60: Grassi G, Dell Oro R, Facchini A, Quarti Trevano F, Bolla GB, Mancia G. Effect of central and peripheral body fat distribution on sympathetic and baroreflex function in obese normotensives. J Hypertens. 2004;22: Alvarez GE, Ballard TP, Beske SD, Davy KP. Subcutaneous obesity is not associated with sympathetic neural activation. Am J Physiol Heart Circ Physiol. 2004;287:H414 H Alvarez GE, Beske SD, Ballard TP, Davy KP. Sympathetic neural activation in visceral obesity. Circulation. 2002;106: Novelty and Significance What Is New? Leptin acts in the brain to increase splanchnic sympathetic nerve activity (SSNA). Leptin increases lumbar SNA and renal SNA baroreflex gain (but not gain of baroreflex control of SSNA or heart rate) and increases lumbar SNA, SSNA, renal SNA, and heart rate baroreflex maxima and range. Leptin increases heart rate via suppression of parasympathetic nerve activity. Leptin exerts these effects via an action in the forebrain. What Is Relevant? Obesity increases leptin. Therefore, leptin may contribute to the suppression of basal and baroreflex-mediated changes in parasympathetic nerve activity commonly observed in obese individuals and identified as a risk factor for adverse cardiovascular events. After a meal, like insulin, increases in leptin may contribute to enhanced lumbar SNA baroreflex gain and may counteract splanchnic vasodilation via increases in SSNA. Summary Leptin acts in the forebrain to differentially influence baroreflex control of lumbar SNA, SSNA, renal SNA, and heart rate, with the latter action mediated via suppression of parasympathetic nerve activity.