PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Hypertension. Author manuscript; available in PMC 2010 November 1.
Published in final edited form as:
PMCID: PMC2789663
NIHMSID: NIHMS153811

LEPTIN IMPAIRS CARDIOVAGAL BAROREFLEX FUNCTION AT THE LEVEL OF THE SOLITARY TRACT NUCLEUS

Abstract

Circulating leptin is elevated in some forms of obesity-related hypertension, associated with impaired baroreflex function. Leptin receptors are present on vagal afferent fibers and neurons within the solitary tract nucleus, providing an anatomical distribution consistent with baroreflex modulation. While solitary tract nucleus microinjection of 144 fmol/60 nL leptin had no significant effect on baroreflex sensitivity for control of heart rate in urethane/chloralose anesthetized Sprague-Dawley rats, 500 fmol leptin impaired baroreflex sensitivity for bradycardia in response to increases in pressure (1.15 ± 0.04 versus 0.52 ± 0.12 msec/mm Hg; p < 0.01). Transgenic ASrAOGEN rats with low brain angiotensinogen have an upregulation of leptin receptor and p85 alpha mRNA in dorsal medulla relative to Sprague-Dawley rats. Consistent with these observations, the response to leptin was enhanced in ASrAOGEN rats, since both the 144 fmol (1.46 ± 0.08 versus 0.75 ± 0.10 msec/mm Hg; p < 0.001) and 500 fmol (1.36 ± 0.32 versus 0.44 ± 0.06 msec/mm Hg; p < 0.05) leptin microinjections impaired baroreflex sensitivity. At these doses, leptin microinjection had no effect on resting pressure, heart rate or the tachycardic response to decreases in pressure in Sprague-Dawley or ASrAOGEN rats. Thus, exogenous leptin at sites within the solitary tract nucleus impairs the baroreflex sensitivity for bradycardia induced by increases in arterial pressure, consistent with a permissive role in mediating increases in arterial pressure. Baroreflex inhibition was enhanced in animals with evidence of increased leptin receptor and relevant signaling pathway mRNA.

Keywords: leptin, solitary tract nucleus, transgenic rats, angiotensin, baroreflex

Introduction

Leptin is secreted by adipose cells in direct proportion to adiposity1 and can cross the blood brain barrier to activate hypothalamic pathways involved in satiety and energy expenditure2. Leptin actions at key hypothalamic nuclei also mediate cardiovascular responses including increases in sympathetic nervous system activity (SNA) and arterial pressure (AP),3 likely involving descending pathways to brainstem nuclei involved in direct control of AP and reflex modulation of autonomic function such as the solitary tract nucleus (NTS).4 The active long form of the leptin receptor, Ob-Rb, has been localized to the nodose ganglion, on vagal afferent fibers and on cells within brainstem areas such as the NTS in normotensive rats5,6 implicating leptin in direct actions on baroreflex sensitivity (BRS) for control of heart rate (HR). While the BRS is often impaired in conditions with elevated circulating leptin levels,7 a direct link between hyperleptinemia and brain sites mediating the effects on BRS is lacking.

Transgenic rats with low brain angiotensinogen (Aogen) resulting from glial over-expression of an antisense oligonucleotide to Aogen (ASrAOGEN) exhibit a 90% reduction in brain Aogen8 and decreased hypothalamic tissue levels of angiotensin (Ang) I with a similar trend for Ang II.9 ASrAOGEN rats have plasma leptin and insulin levels comparable to control Sprague-Dawley (SD) rats at 15-weeks of age,10 but show enhanced sensitivity to both hormones as detected with a glucose challenge11. The sensitivity to leptin for cardiovascular actions in these rats is currently unknown.

We assessed the effect of acute, site-specific NTS microinjection of leptin on baroreflex function and indices of autonomic balance, as well as resting AP and HR in SD in comparison with ASrAOGEN rats which might be expected to show enhanced sensitivity to leptin. The present study provides direct evidence that administration of exogenous leptin impairs BRS for control of HR in response to increases in AP within the NTS of SD rats as well as alters autonomic balance. In addition, ASrAOGEN rats with down-regulation of the endogenous brain renin-Ang system (RAS) exhibit increased sensitivity to exogenous leptin microinjection, consistent with an upregulation of leptin receptors and signaling pathways in this brain region.

Methods

The institutional animal care and use committee approved all procedures. For a detailed Methods section please see http://hyper.ahajournals.org.

Animals

Experiments were performed in 3 to 5 month-old male Hannover SD and transgenic (ASrAogen)680 rats obtained from the Hypertension and Vascular Research colony at the Wake Forest University School of Medicine.

Surgical Procedures and Hemodynamic Measures

As previously reported,12,13 rats were anesthetized with combination urethane-chloralose (750 mg and 35 mg per kg, respectively) via intraperitoneal injections with supplemental intravenous (IV) doses given as needed. Animals were instrumented with femoral artery and vein catheters and placed in a stereotaxic frame with the head tilted downward (45°) for surgical exposure of the dorsal medulla oblongata. Pulsatile AP and mean AP (MAP) were monitored, recorded and digitized using a Data Acquisition System (BIOPAC System Inc.; Acknowledge software Version 3.8.1; Santa Barbara, CA) and HR was determined from the AP wave. After obtaining stable measures of MAP and HR, baseline responses to BRS were established by bolus IV randomized injection of 3 doses (2, 5 and 10 μg/kg in 0.9% NaCl) of phenylephrine (PE) or sodium nitroprusside (NP), to determine the BRS for increases or decreases in AP, respectively. Assessment of BRS by bolus injections is more sensitive for detection of alterations in the bradycardic BRS relative to infusion determinations.14 The BRS for bradycardia and tachycardia was determined for each animal as the slope of the relationship between changes in MAP and the pulse interval generated from the 3 doses of PE and NP, independently.12,13 Reflex testing was completed within 30 min of leptin microinjection. Maximum transient changes in MAP and HR in response to NTS microinjection of leptin were measured and BRS testing was repeated at 10 min after the leptin microinjection, with each animal serving as its own control. Indices of sympathovagal function were also analyzed using Nevrokard software (Nevrokard SA-BRS; Medistar, Ljubljana, Slovenia).15 Consistent with the duration of recordings used in previous human and rodent studies,15-18 spontaneous BRS was determined from a minimum of 5 min of AP recordings obtained within 10 min of leptin injection, prior to the evoked baroreflex testing. Spontaneous BRS was calculated in the time [Sequence (Seq) Up, Seq Down and Seq All] and frequency domains [Low Frequency (LF) and High Frequency (HF) alpha indices]. Time domain analysis was used to assess changes in HR variability (HRV) measured as the standard deviation of the beat-to-beat interval. Blood pressure variability (BPV) was measured in the time domain as the standard deviation of the MAP.

NTS Microinjections

Rat recombinant leptin [Sigma; 144 or 500 fmol (0.002 and 0.008 μg, respectively) in a 60 nL volume of 15 mM HCl and 7.5 mM NaOH diluted to pH 7.4 in artificial cerebrospinal fluid (CSF)] or vehicle (60 nL) was microinjected bilaterally via pressure into the NTS [0.4 mm rostral, 0.4 mm lateral to the calamus scriptorius (caudal tip of the area postrema) and 0.4 mm below the dorsal surface] using a glass micropipette connected to a syringe as previously reported.12,13 The doses and volume of leptin were comparable to previous NTS microinjection studies in which the peptides effectively altered BRS.12,19,20 NTS microinjection of the vehicle solution had no significant effect on MAP, HR or BRS for control of HR in SD or ASrAOGEN rats (Table S1 and Figure S1, http://hyper.ahajournals.org), similar to previous studies in our laboratory.12,13 The vehicle had no effect on spontaneous BRS or BPV in SD and ASrAOGEN rats. However, HRV increased after injection of vehicle (1.81 ± 0.29 versus 2.32 ± 0.17 msec after vehicle; p < 0.05) in SD rats, with no effect of vehicle on HRV in ASrAOGEN rats. At the end of experiments, brains were removed, frozen and sectioned (30 μM) for localization of microinjection sites (Figure S2, http://hyper.ahajournals.org). Only data from injections within the medial NTS at rostro-caudal level −13.3 to −14.0 mm caudal to bregma were used in the analysis.

Quantification of leptin receptor and p85 alpha mRNA

Leptin receptor and phosphoinositide-3 kinase (PI3K) p85 alpha mRNA were measured in dorsal medullary tissue from separate groups of naive 15-week old SD (n = 9) and ASrAOGEN (n = 7) rats. Brains were removed and placed on dry ice for excision of 2 mm3 dorsal medullary sections. The sections were obtained from 1 mm in front of to 1 mm behind the usual placement of the pipette, corresponding to the expected injectate spread20 and including portions of area postrema, dorsal motor nucleus and nucleus gracilis. Isolation of RNA from excised tissue was assessed for concentration and stability. Total RNA (1 μg) was reverse transcribed using AMV reverse transcriptase in a 20 μL reaction mixture containing deoxyribonucleotides, random hexamers, and Rnase inhibitor in reverse transcriptase buffer as previously described.12,13 For real-time PCR, 2 μL of resultant cDNA was added to TaqMan Universal PCR Master Mix with the appropriate gene-specific primer/probe set for leptin receptor and p85 alpha (Applied Biosystems) and amplification was performed. All reactions were performed in triplicate. 18S ribosomal RNA served as the internal control. Results were quantified as Ct values, where Ct is the threshold cycle of PCR at which amplified product is first detected, and was defined as relative gene expression (ratio of target/control).

Analysis of Data

Values are presented as mean ± standard error of the mean. A 2-way ANOVA was utilized to compare data between ASrAOGEN and SD strains. Comparisons of changes in BRS and indices of sympathovagal function in response to leptin or vehicle were assessed using a one-sample paired t-test. Changes in MAP and HR over time and time-course experiments were analyzed by repeated-measures ANOVA with post-hoc Student-Newman-Keuls multiple comparisons. mRNA quantification was analyzed by an unpaired t-test between strains. The criterion for statistical significance was p < 0.05. Tests were performed using Prism 4.0 and InStat 3 (GraphPad Software, San Diego, CA).

Results

Effect of NTS microinjection of leptin on BRS for control of HR in SD rats

In SD rats, NTS microinjection of 144 fmol leptin impaired BRS for control of HR measured as the bradycardic response to increases in AP produced by PE by 22%, an effect that did not reach significance (Figure 1A, D). In contrast, the 500 fmol leptin dose significantly impaired BRS by 63% in SD rats (Figure 1B, D) indicative of a dose-dependent response for leptin actions on BRS within the NTS. There were no differences in baseline BRS values or PE-induced increases in AP (Figure S3) among SD rats receiving various doses of leptin. Time-course experiments showed that the BRS was impaired at 10 and 60 min with partial recovery at 120 min after NTS microinjection of the 500 fmol leptin dose in SD rats (Figure 1C). BRS for control of HR measured as the tachycardic response to decreases in AP produced by NP was not altered by NTS microinjection of 500 fmol leptin (Figure S4, http://hyper.ahajournals.org).

Figure 1
Effect of NTS leptin microinjection on BRS for control of HR evoked by PE in SD rats

Effect of NTS microinjection of leptin on BRS for control of HR in ASrAOGEN rats

The bradycardic BRS was significantly higher in anesthetized ASrAOGEN relative to SD rats at baseline (p < 0.01), with no differences in the PE-induced increases in AP between strains (Figure S3). NTS microinjection of both 144 and 500 fmol leptin doses impaired the bradycardic BRS in ASrAOGEN rats corresponding to a 50% and 68% reduction, respectively (Figure 2A, B, D). The 144 fmol leptin group shows data from younger (n = 3) and older (n = 5; 18 to 21 months) ASrAOGEN rats as there were no differences in BRS values at baseline or in response to leptin in younger (1.56 ± 0.15 msec/mm Hg baseline versus 0.81 ± 0.19 msec/mm Hg after leptin; p < 0.05) and older (1.40 ± 0.09 msec/mm Hg baseline versus 0.72 ± 0.12 msec/mm Hg after leptin injection; p < 0.01) rats. The 500 fmol leptin dose was only tested in younger ASrAOGEN rats. Similar to SD rats, there were no differences in baseline BRS or PE-induced increases in AP (Figure S3) among ASrAOGEN rats receiving varying leptin doses. In ASrAOGEN rats, the BRS remained suppressed at 10, 60 and 120 min after the leptin injection with no evidence of recovery over this time period (Figure 2C). There was no significant difference in baseline values of the tachycardic BRS in response to decreases in AP between anesthetized ASrAOGEN and SD rats. Similar to SD rats, NTS microinjection of 500 fmol leptin had no effect on the tachycardic BRS in ASrAOGEN rats (Figure S4).

Figure 2
Effect of NTS leptin microinjection on BRS for control of HR evoked by PE in ASrAOGEN rats

Leptin influence on indices of sympathovagal function

There were no differences in baseline indices of spontaneous BRS, HRV or BPV within groups of SD or ASrAOGEN rats. Indices of spontaneous BRS were also similar at baseline between anesthetized ASrAOGEN and SD rats (Table 1). However, baseline values of HRV, a measure of cardiac vagal tone, and BPV were significantly higher in ASrAOGEN relative to SD rats (Table 2; p < 0.001 and p < 0.05, respectively). Similar to the evoked baroreflex measurements, 144 fmol leptin had no significant effect on spontaneous BRS indices, HRV or BPV in SD rats. In contrast, the 144 fmol leptin dose significantly reduced spontaneous BRS (Seq All; p < 0.01) as well as HRV in ASrAOGEN rats. Specifically, vagal indices of the BRS were reduced (Seq Up and HFα) with no effect on sympathetic indices of the spontaneous BRS (Seq Down and LFα) in these animals. The 500 fmol leptin reduced vagal spontaneous BRS indices (Seq Up and HFα) and HRV in both SD and ASrAOGEN rats. In addition, 500 fmol leptin reduced the LFα sympathetic index in ASrAOGEN rats only (Table 1). While 500 fmol leptin significantly increased BPV in SD, there was no effect at either dose in ASrAOGEN rats on this parameter (Table 2).

Table 1
Leptin Influence on Indices of Spontaneous BRS
Table 2
Leptin Modulation of Heart Rate Variability and Blood Pressure Variability

MAP and HR responses to NTS microinjection of leptin

There were no significant differences in MAP or HR within groups of SD or ASrAOGEN rats. However, as previously reported,12 the pooled baseline MAP was significantly higher in anesthetized ASrAOGEN relative to SD rats (112 ± 5 mm Hg versus 85 ± 3 mm Hg, respectively; p < 0.001; n = 12 - 15 per group). The pooled baseline HR was also significantly higher in ASrAOGEN rats (345 ± 14 bpm versus 286 ± 9 bpm; p < 0.01). There was no significant effect of acute NTS microinjection of either 144 or 500 fmol leptin on resting MAP in SD or ASrAOGEN rats (Table S1, http://hyper.ahajournals.org). While leptin injection had no effect on resting HR in SD rats, HR was modestly reduced following 144 fmol leptin with no effect of the 500 fmol leptin in ASrAOGEN animals. Values of MAP and HR were not different from baseline at the time of reflex testing, at 10 minutes after the initial leptin microinjection (Table S1).

Differences in leptin receptor and PI3K mRNA in SD and ASrAOGEN rats

Relative gene expression of leptin receptor and PI3K regulatory subunit p85 alpha were measured in dorsal medullary tissue of naïve SD (n = 9) and ASrAOGEN (n = 7) rats at 15-weeks of age (Figure 3). Leptin receptor mRNA was 3 to 4-fold higher and p85 alpha mRNA was 2-fold higher in dorsal medulla of ASrAOGEN relative to SD rats. There were no differences in control 18S ribosomal Ct values between SD (16.781 ± 0.136) and ASrAOGEN (16.587 ± 0.356) rats.

Figure 3
Leptin receptor and PI3K p85 alpha mRNA in dorsal medulla of SD and ASrAOGEN rats

Discussion

In the present study, we determined the effects of exogenous leptin on blood pressure and baroreceptor reflex regulation at the level of the NTS. Our results demonstrate that NTS microinjection of leptin impairs BRS for control of HR in response to increases in AP, an index of parasympathetic activity. The leptin-mediated impairment in BRS was associated with a shift in indices of sympathovagal balance towards a decrease in parasympathetic function, with no significant acute effect on resting MAP or HR. The novel finding that exogenous leptin impairs BRS for control of HR within the NTS may have implications for understanding the contribution of elevated leptin to baroreflex dysfunction. In addition, we examined whether leptin modulation of the BRS was altered in a model of enhanced leptin sensitivity to metabolic actions associated with basal differences in the central RAS by comparing leptin responses in control SD and transgenic ASrAOGEN rats. ASrAOGEN rats were more sensitive to BRS impairment in response to NTS microinjection of leptin. The enhanced sensitivity to exogenous leptin was associated with increased leptin receptor and PI3K p85 alpha mRNA in the dorsal medulla of ASrAOGEN rats, suggesting long-term reductions in brain Ang peptides or the subsequent consequences may be associated with upregulation of leptin signaling pathways.

Evidence suggests that key hypothalamic nuclei mediate increases in SNA and AP in response to exogenous leptin,3 likely involving descending pathways to brainstem nuclei involved in cardiovascular regulation4. Independent of descending pathways, leptin receptors have been localized within the NTS and mediate both gastric21 and cardiovascular responses6. NTS microinjection of a substantially higher leptin dose than used in the present study (1 μg) increases SNA and AP at 2 hours after the injection, consistent with baroreflex modulation.6 However, these studies did not examine the effect of NTS microinjection of leptin on baroreceptor reflex regulation. Indeed, the localization of leptin receptors to vagal afferent fibers and within the NTS5,6 implicates leptin as a direct modulator of BRS for control of HR. While previous studies have shown that IV leptin does not acutely alter the sympathetically-mediated baroreflex control of renal SNA,22 the contribution of circulating leptin to BRS for control of HR, a vagally-mediated index that is often impaired in conditions with chronically elevated plasma leptin levels,7 has yet to be examined.

The results of the present study provide evidence for a direct action of exogenous leptin to modulate baroreflex function as NTS microinjection of 500 fmol leptin impaired BRS for control of HR in both SD and ASrAOGEN rats. Leptin injection selectively altered BRS measured as the bradycardic response to increases in AP with no effect on BRS measured as the tachycardic response to decreases in AP in both SD and ASrAOGEN rats, similar to actions of Ang II within the NTS.20 While there are no published studies evaluating the tachycardic BRS in anesthetized ASrAOGEN rats, the baseline tachycardic BRS values in SD rats are within the range of previously reported values using the same methods.20 NTS microinjection of 500 fmol leptin did not alter depressor and bradycardic responses to cardiac vagal chemosensitive fiber activation (CVA) induced by IV phenylbiguanide in ASrAOGEN rats (unpublished observation). The lack of alteration in CVA responses supports specificity of leptin actions as these responses are mediated by chemoreceptor fibers that converge with baroreceptor inputs within the NTS.23 Leptin modulation of the BRS was transient in SD rats, with partial recovery at 120 min after the initial leptin microinjection. In contrast, there was no evidence of recovery in ASrAOGEN rats at 120 min after the leptin injection. While the mechanism for the lack of recovery of BRS in ASrAOGEN rats is currently unknown, it may represent more sustained leptin actions within the NTS due to the upregulation of leptin receptor and signaling pathways.

We can not exclude the possibility that the spread of the leptin injection may have accessed the area postrema or dorsal motor nucleus of the vagus for effects on baroreflex function. However, injection of a 100 nL of 125I-Sar-Thr Ang II was mostly confined to the NTS.20 In addition, functional assessments show that 50 nL of an AT1 antagonist into the dorsal motor nucleus does not alter responses to NTS injection of Ang II.24 Since the injection of leptin accessed neuronal cell bodies as well as presynaptic vagal afferents within the NTS, it is not clear which elements mediate the effects on BRS. However, Ang II is thought to exhibit its action on BRS primarily through vagal afferent fibers.25

In SD rats, the 144 fmol leptin dose had no significant effect on BRS, while the 500 fmol dose impaired the BRS suggesting a dose-response relationship. It appears maximal suppression of the BRS was achieved with the lower dose of leptin in ASrAOGEN rats as both the 144 fmol and 500 fmol leptin doses impaired BRS to a similar degree. These results implicate an enhanced sensitivity of ASrAOGEN rats to exogenous leptin within the NTS. Leptin impaired the BRS to approximately 0.5 msec/mm Hg in both strains, a level often observed in hypertension.26 As a possible mechanism for the enhanced sensitivity of ASrAOGEN to exogenous leptin, we observed higher expression of leptin receptor and PI3K p85 alpha mRNA in dorsal medullary tissue of ASrAOGEN relative to SD rats at 15-weeks of age. While there are no differences in basal circulating leptin levels between strains at this age,10 sensitivity of leptin to a glucose challenge is enhanced in ASrAOGEN rats11.

The baseline values of MAP and HR were higher in anesthetized ASrOGEN relative to SD rats, possibly due to an anesthesia-induced activation of the sympathetic nervous system observed in these animals12. NTS microinjection of leptin resulted in no significant changes in resting MAP in either SD or ASrAOGEN rats, consistent with previous NTS microinjection studies using higher doses (8 - 31 pmol) of leptin.6,27 Only microinjection of 1 μg (63 pmol) leptin within the NTS results in increases in SNA and AP in SD rats.6 However, these effects are delayed, requiring 2+ hours for manifestation. Importantly, prolonged suppression of the BRS may contribute to the delayed modest increase in AP observed with NTS injection of higher doses of leptin6. In the present study differences in BRS after leptin administration are not attributable to differences in resting hemodynamics, confirming that the set-point of the baroreflex is controlled independently from the sensitivity.12,13

Spontaneous and spectral analysis methods for measurements of BRS,15 revealed no difference in spontaneous BRS values between strains, in contrast to the higher BRS in ASrAOGEN rats using the pharmacologic approach. While the classical method evokes changes in AP in an open-loop system, the spontaneous method measures changes over a smaller range (beat-to-beat) in a closed-loop model. Although a highly significant correlation exists between the two methods,15 differences have been reported in the BRS values obtained perhaps due to differences in sensitivity of the methods. Consistent with previous studies,28 baseline HRV was significantly higher in ASrAOGEN relative to SD rats, suggestive of an increased resting vagal tone in these animals. Interestingly, baseline BPV was also higher in anesthetized ASrAOGEN relative to SD rats suggesting elevated sympathetic tone. While in the conscious state there are no reported differences in BPV in ASrAOGEN rats,10,29 the state of anesthesia may result in an activation of the sympathetic nervous system in these animals12.

Similar to evoked BRS measurements, 144 fmol leptin had no effect on spontaneous BRS indices, HRV or BPV in SD rats. In ASrAOGEN rats, the 144 fmol leptin impaired Seq All, Seq Up, HFα and reduced HRV providing further evidence for increased sensitivity to exogenous leptin in these animals. In both SD and ASrAOGEN rats, the 500 fmol leptin dose decreased Seq All as well as vagal indices of spontaneous BRS (HFα and Seq Up) and HRV further suggesting that leptin modulates BRS in response to increases but not decreases in AP. In ASrAOGEN rats, the 500 fmol leptin also reduced the LFα index with no effect in SD rats. Although this index is generally used as a marker of sympathetic activity, the spectral density of AP contained within this frequency is partially controlled by vagal tone.30 The 500 fmol leptin dose increased BPV in SD rats further evidencing its role in altering cardiovascular autonomic balance. There was no effect of leptin on BPV in ASrAOGEN rats at either dose perhaps due to the already high basal level of this index under anesthesia. Collectively, leptin altered blood pressure regulation as assessed with either method and using a number of indices of autonomic function, similar to patterns observed in hypertension, obesity, and stroke,31 where the circulating peptide is often elevated.

Ang II increases leptin levels and promotes leptin production in vitro suggesting a regulatory relationship between the peptides.32 Ang converting enzyme inhibitors or Ang II AT1 receptor blockers reduce plasma leptin levels in patients with mild/moderate hypertension.33 Chronic AT1 receptor blockade prevents age-related increases in circulating leptin levels that are associated with decreases in dorsal medullary leptin receptor mRNA in Fischer 344 rats.34 Thus, chronic Ang II blockade maintains low endogenous leptin levels and increases leptin receptor mRNA. ASrAOGEN rats, with low endogenous brain Aogen, have decreased endogenous Ang II tone contributing to BRS suppression within the NTS12 and therefore may also have decreased leptin levels within the NTS. While dorsal medullary leptin levels were not assessed in this study, leptin receptors and signaling pathways in ASrAOGEN rats appear up-regulated on the basis of higher mRNA for leptin receptor and PI3K p85 alpha relative to SD rats. The enhanced BRS suppression with exogenously administered leptin in ASrAOGEN rats is functional evidence consistent with this interpretation. Enhanced sensitivity to leptin, could contribute to the overall enhanced metabolic phenotype observed in ASrAOGEN rats while maintaining low endogenous levels of leptin and thus a positive cardiovascular profile.10,12,28 Whether the upregulation of leptin receptor and PI3K p85 alpha mRNA is attributed to a direct interaction with the RAS in the ASrAOGEN rats or an indirect effect is currently unknown; either low endogenous Ang II or leptin could contribute to the increased leptin receptor and PI3K mRNA expression in dorsal medulla of ASrAOGEN rats.

We examined changes in BRS, MAP and HR in response to acute, site-specific leptin administration. Effects of chronic peripheral or central leptin administration will need to be determined to further evaluate the role of leptin-mediated BRS impairments to pathophysiologies associated with elevated circulating, CSF or brain tissue leptin. Examination of the role of leptin in concert with other known modulators of cardiovascular and metabolic function such as Ang peptides, insulin and glucose, may yield differing results as recent studies show central leptin infusion may improve glucose utilization in diabetic rats to have indirect beneficial effects on BRS and sympathovagal balance.35 Finally, examining the signaling pathways mediating the effects of leptin modulation of BRS within the NTS will help determine whether leptin utilizes different pathways for negative cardiovascular versus positive metabolic actions.

Perspectives

BRS for control of HR, a measure of vagal function, is often impaired in hypertension, obesity-related hypertension and stroke.7,31 Uncovering factors that modulate BRS may be important in understanding the predisposition to these conditions. Plasma leptin levels are elevated in obesity and independently in hypertension and stroke36,37 and exogenous leptin contributes to sympathetically-mediated elevations in AP38. The present data suggests that leptin impairs BRS for control of HR, an index believed to precede and contribute to the development of hypertension. Therefore, leptin-mediated impairments in BRS may be permissive towards increases in AP observed in populations with elevated leptin levels. Studies suggest that resistance develops to metabolic with maintenance of sensitivity to cardiovascular actions of leptin. Metabolic resistance to leptin is associated with reduced transport into brain and defects in intracellular signaling pathways.39,40 Reduction of leptin levels in patients with leptin resistance may prevent saturation of receptors to increase leptin transport as well as reduce negative regulators of leptin signaling to allow for maintenance of sensitivity to leptin’s positive metabolic effects. Concomitantly, low endogenous leptin levels may reduce activation of cardiovascular signaling pathways to prevent impairments in baroreflex function, as well as increases in AP and SNA. Understanding mechanisms to preserve leptin sensitivity, in the presence of low endogenous leptin levels, may be important for maintaining satiety effects while preventing negative cardiovascular effects of the peptide.

Supplementary Material

supp1

supp2

supp3

Acknowledgements

We thank Ellen Tommasi for technical assistance. Dr. Hossam A. Shaltout is currently a faculty member in the Department of Pharmacology and Toxicology, School of Pharmacy, University of Alexandria, Egypt.

Sources of Funding: The authors gratefully acknowledge support from the National Heart, Lung and Blood Institute Grant HL-51952, Unifi (Greensboro, NC) and the Farley-Hudson Foundation (Jacksonville, NC).

Footnotes

Conflicts of Interest/Disclosures: None

Reference List

1. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med. 1995;1:1155–1161. [PubMed]
2. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science. 1995;269:546–549. [PubMed]
3. Rahmouni K, Morgan DA. Hypothalamic arcuate nucleus mediates the sympathetic and arterial pressure responses to leptin. Hypertension. 2007;49:647–652. [PubMed]
4. Dampney RA. Functional organization of central pathways regulating the cardiovascular system. Physiol Rev. 1994;74:323–364. [PubMed]
5. Mercer JG, Moar KM, Hoggard N. Localization of leptin receptor (Ob-R) messenger ribonucleic acid in the rodent hindbrain. Endocrinology. 1998;139:29–34. [PubMed]
6. 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;53:375–380. [PMC free article] [PubMed]
7. Grassi G, Seravalle G, Dell’Oro R, Turri C, Bolla GB, Mancia G. Adrenergic and reflex abnormalities in obesity-related hypertension. Hypertension. 2000;36:538–542. [PubMed]
8. Schinke M, Baltatu O, Bohm M, Peters J, Rascher W, Bricca G, Lippoldt A, Ganten D, Bader M. Blood pressure reduction and diabetes insipidus in transgenic rats deficient in brain angiotensinogen. Proc Natl Acad Sci U S A. 1999;96:3975–3980. [PubMed]
9. Huang BS, Ganten D, Leenen FH. Responses to central Na(+) and ouabain are attenuated in transgenic rats deficient in brain angiotensinogen. Hypertension. 2001;37:683–686. [PubMed]
10. Kasper SO, Carter CS, Ferrario CM, Ganten D, Ferder LF, Sonntag WE, Gallagher PE, Diz DI. Growth, metabolism, and blood pressure disturbances during aging in transgenic rats with altered brain renin-angiotensin systems. Physiol Genomics. 2005;23:311–317. [PubMed]
11. Kasper SO, Ferrario CM, Ganten D, Diz DI. Rats with low brain angiotensinogen do not exhibit insulin resistance during early aging. Endocrine. 2006;30:167–174. [PubMed]
12. Sakima A, Averill DB, Kasper SO, Jackson L, Ganten D, Ferrario CM, Gallagher PE, Diz DI. Baroreceptor reflex regulation in anesthetized transgenic rats with low glia-derived angiotensinogen. Am J Physiol Heart Circ Physiol. 2007;292:H1412–H1419. [PubMed]
13. Arnold AC, Sakima A, Ganten D, Ferrario CM, Diz DI. Modulation of reflex function by endogenous angiotensins in older transgenic rats with low glial angiotensinogen. Hypertension. 2008;51:1326–1331. [PMC free article] [PubMed]
14. Korner PI. Cardiac baroreflex in hypertension: role of the heart and angiotensin II. Clin Exp Hypertens. 1995;17:425–439. [PubMed]
15. Shaltout HA, Abdel-Rahman AA. Mechanism of fatty acids induced suppression of cardiovascular reflexes in rats. J Pharmacol Exp Ther. 2005;314:1328–1337. [PubMed]
16. Fisher JP, Ogoh S, Junor C, Khaja A, Northrup M, Fadel PJ. Spontaneous baroreflex measures are unable to detect age-related impairments in cardiac baroreflex function during dynamic exercise in humans. Exp Physiol. 2009;94:447–458. [PMC free article] [PubMed]
17. Padley JR, Overstreet DH, Pilowsky PM, Goodchild AK. Impaired cardiac and sympathetic autonomic control in rats differing in acetylcholine receptor sensitivity. Am J Physiol Heart Circ Physiol. 2005;289:H1985–H1992. [PubMed]
18. Gerhardt U, Schafer M, Hohage H. Arterial blood pressure oscillation after active standing up in kidney transplant recipients. J Auton Nerv Syst. 2000;80:93–100. [PubMed]
19. Caligiorne SM, Santos RA, Campagnole-Santos MJ. Cardiovascular effects produced by bradykinin microinjection into the nucleus tractus solitarii of anesthetized rats. Brain Res. 1996;720:183–190. [PubMed]
20. Campagnole-Santos MJ, Diz DI, Ferrario CM. Baroreceptor reflex modulation by angiotensin II at the nucleus tractus solitarii. Hypertension. 1988;11:I167–I171. [PubMed]
21. Grill HJ, Schwartz MW, Kaplan JM, Foxhall JS, Breininger J, Baskin DG. Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake. Endocrinology. 2002;143:239–246. [PubMed]
22. Hausberg M, Morgan DA, Chapleau MA, Sivitz WI, Mark AL, Haynes WG. Differential modulation of leptin-induced sympathoexcitation by baroreflex activation. J Hypertens. 2002;20:1633–1641. [PubMed]
23. Paton JF. Convergence properties of solitary tract neurones driven synaptically by cardiac vagal afferents in the mouse. J Physiol. 1998;508(Pt 1):237–252. [PubMed]
24. Fow JE, Averill DB, Barnes KL. Mechanisms of angiotensin-induced hypotension and bradycardia in the medial solitary tract nucleus. Am J Physiol. 1994;267:H259–H266. [PubMed]
25. Diz DI, Westwood B, Averill DB. AT(1) antisense distinguishes receptors mediating angiotensin II actions in solitary tract nucleus. Hypertension. 2001;37:1292–1297. [PubMed]
26. Diz DI, Garcia-Espinosa MA, Gallagher PE, Ganten D, Ferrario CM, Averill DB. Angiotensin-(1-7) and baroreflex function in nucleus tractus solitarii of (mRen2)27 transgenic rats. J Cardiovasc Pharmacol. 2008;51:542–548. [PMC free article] [PubMed]
27. Matsumura K, Abe I, Tsuchihashi T, Fujishima M. Central effects of leptin on cardiovascular and neurohormonal responses in conscious rabbits. Am J Physiol Regul Integr Comp Physiol. 2000;278:R1314–R1320. [PubMed]
28. Caligiorne SM, Silva AQ, Fontes MA, Silva JR, Baltatu O, Bader M, Santos RA, Campagnole-Santos MJ. Baroreflex control of heart rate and renal sympathetic nerve activity in rats with low brain angiotensinogen. Neuropeptides. 2008;42:159–168. [PubMed]
29. Baltatu O, Janssen BJ, Bricca G, Plehm R, Monti J, Ganten D, Bader M. Alterations in blood pressure and heart rate variability in transgenic rats with low brain angiotensinogen. Hypertension. 2001;37:408–413. [PubMed]
30. Cerutti C, Barres C, Paultre C. Baroreflex modulation of blood pressure and heart rate variabilities in rats: assessment by spectral analysis. Am J Physiol. 1994;266:H1993–H2000. [PubMed]
31. Thayer JF, Lane RD. The role of vagal function in the risk for cardiovascular disease and mortality. Biol Psychol. 2007;74:224–242. [PubMed]
32. Kim S, Whelan J, Claycombe K, Reath DB, Moustaid-Moussa N. Angiotensin II increases leptin secretion by 3T3-L1 and human adipocytes via a prostaglandin-independent mechanism. J Nutr. 2002;132:1135–1140. [PubMed]
33. Koh KK, Quon MJ, Han SH, Lee Y, Kim SJ, Koh Y, Shin EK. Distinct vascular and metabolic effects of different classes of anti-hypertensive drugs. Int J Cardiol. 2008 In Press. [PMC free article] [PubMed]
34. Gilliam-Davis S, Payne VS, Kasper SO, Tommasi EN, Robbins ME, Diz DI. Long-term AT1 receptor blockade improves metabolic function and provides renoprotection in Fischer-344 rats. Am J Physiol Heart Circ Physiol. 2007;293:H1327–H1333. [PubMed]
35. do Carmo JM, Hall JE, da Silva AA. Chronic central leptin infusion restores cardiac sympathetic-vagal balance and baroreflex sensitivity in diabetic rats. Am J Physiol Heart Circ Physiol. 2008;295:H1974–H1981. [PubMed]
36. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med. 1996;334:292–295. [PubMed]
37. Sierra-Johnson J, Romero-Corral A, Lopez-Jimenez F, Gami AS, Kuniyoshi FH Sert, Wolk R, Somers VK. Relation of increased leptin concentrations to history of myocardial infarction and stroke in the United States population. Am J Cardiol. 2007;100:234–239. [PMC free article] [PubMed]
38. Carlyle M, Jones OB, Kuo JJ, Hall JE. Chronic cardiovascular and renal actions of leptin: role of adrenergic activity. Hypertension. 2002;39:496–501. [PubMed]
39. Correia ML, Rahmouni K. Role of leptin in the cardiovascular and endocrine complications of metabolic syndrome. Diabetes Obes Metab. 2006;8:603–610. [PubMed]
40. Hall JE, Hildebrandt DA, Kuo J. Obesity hypertension: role of leptin and sympathetic nervous system. Am J Hypertens. 2001;14:103S–115S. [PubMed]