Neural Mechanism Of Control Of Regulation Of Breathing By Leptin

The respiratory chemoreflex maintains oxygen supplies and acid-base balance via excretion of excessive CO2,and is therefore a critical homeostatic mechanism.The respiratory chemoreceptors are distributed peripherally and centrally.Peripheral chemoreceptors consist of carotid body(CB)and aortic body,both of which sense changes in PaO2,PaCO2 and H+.Central chemoreceptors are mainly located in brainstem,at least consisting of retrotrapezoid nucleus(RTN)and nucleus tractus solitorii(NTS),dorsal motor nucleus of the vagus(DMNV),medullary raphe and locus coeruleus.Candidate central respiratory chemoreceptors should present at least the following requisite properties:(1)intrinsic sensitivity to physiologically relevant changes in CO2/H+;(2)sensitivity to changes in CO2 that are readily demonstrable in vivo;(3)functional connectivity to respiratory centers,such that their selective activation,inhibition,or elimination produces the appropriate effect on the respiratory chemoreflex.Neural circuits controlling breathing in mammals are organized within serially arrayed and functionally interacting brainstem compartments extending from the pons to the lower medulla.The rhythmic center and central pattern generator are controlled by multiple drives from more rostral brainstem components,including the brainstem chemoreceptors and pons.Either peripheral or central chemoreceptors are intrinsically sensitive to CO2 but only the former is activated by hypoxia.Prior evidence has demonstrated that lack of leptin in mice produced impaired hypercapnic ventilatory response(HCVR)but exogenous administration of leptin rescued HCVR.Perturbation of leptin signaling pathway is correlated with obese-related breathing disorders.However,it remains to be ascertained concerning central targets and signaling pathway involved in regulation of breathing by leptin.The presence of leptin receptor(ob-R)has been implicated in the CBs,the main peripheral chemoreceptor.Intermittent hypoxia has been shown to raise blood leptin,with the resultant activation of pSTAT3 in the CBs.However,it remains poor understood with regard to effect of leptin on chemosensitivity of CBs.Evidence obtained in vivo and in vitro demonstrates that the NTS contributes to HCVR and some NTS neurons possess inherent pH sensitivity.Nevertheless,the cellular and molecular mechanism underlying such pH sensitivity remains unknown in the NTS.It is reminiscent of TWIK-related acid-sensitive K+ channel(TASK),acid sensitive ion channel(ASIC),G-protein coupled receptor 4(GPR4),voltage-gated K+ channel(Kv),which have been implicated previously elsewhere.Interestingly,injection of leptin into the NTS in anesthetized rats increased basal renal sympathetic nerve activity,suggesting an involvement of ob-Rs in regulation of autonomic activity.Likewise,basic minute ventilation was increased by injection of leptin into NTS in favor of facilitation of central chemoreflex by leptin signaling pathway.It is not yet unknown regarding whether the facilitated chemoreflex is due to activation of pH-sensitive channels in the NTS by leptin.Collectively,using multidisciplinary approaches including plethysmography,molecular techniques and immunohistology,the aim of this project is sought to determine whether leptin facilitates central chemoreflex,to determine the mechanism by which leptin signaling pathway contribute to HCVR,to determine whether leptin facilitates hypoxic ventilatory response(HVR)in the CBs.Part I: Facilitation of central chemoreflex by leptinObjective: To study effects of either activation or inhibition of leptin signaling pathway on HCVR in carotid body denervated rats.Methods: Two protocols were used.In the first protocol,the animals were divided into four groups,including Lean Zucker rat(Lean Group,used as control),Lean rats with high fat diet(Lean-HFD Group,used as simpleobese),Obese Zucker rat(Obese Group,deficiency of ob-R)and Obese Zucker rat with high fat diet(Obese-HFD,deficiency of ob-R).Ventilation was measured using whole body plethysmography and assessed by breathing parameters including minute ventilation(MV),tidal volume(TV)and breathing frequency(BF).In the second protocol,the animals were also divided into four groups: Lean+saline,Lean+leptin,Obese+saline and Obese+leptin.For leptin-treated groups,the rat was subjected to subcutaneous injection of leptin(60?g/kg)once daily for 7 days.Likewise,breathing parameters were measured as mentioned above.To determine effect of leptin on HCVR,expression of pSTAT3/STAT3,the downstream molecules of ob-Rs,was quantified using Western blot in four groups.Results:1 The averaged body weight was larger in the three obese groups than Lean Group(P<0.01),accompanying with hypercapnia(P<0.01).No significant difference in blood leptin and in expression of pSTAT3 has been found between Lean and Lean-HFD groups(P>0.05).Higher level of blood leptin and lower expression of pSTAT3 in Obese and Obese-HFD groups were comparable to the Lean Group(P<0.05~0.01).2 During exposure to hyperoxia,MV and TV were lower but BF was higher in three obese groups compared with the Lean Group(P<0.01),suggesting a manifestation of obese-related hypoventilation with breathing pattern of fast and shallow in three obese groups.3 Although Lean-HFD rats exhibited resting hypoventilation,no significant difference in increase in MV was found between Lean and Lean-HFD rats when challenged by 5% and 8% CO2 in inspired air.A remarkably impaired HCVR occurred in Obese and Obese-HFD groups as compared to Lean Group(P<0.01).There was no marked difference in breathing parameters between Obese and Obese-HFD groups(P>0.05).4 Chronic administration of leptin(subcutaneous injection)produced no detectable increase in body weight(P>0.05)and higher blood leptin(4-8?g/L,P<0.05~0.01).5 Injection of leptin for 7 days produced an increase in expression of pSTAT3/STAT3 in the Lean Group(P<0.01)but not in the Obese Group and vehicle controls(P>0.05).6 Exogenous leptin caused no significant changes in PaCO2 and blood pH in relative to vehicle controls(P>0.05).7 Resting breathing parameters retained normal between each group during normocapnia in response to injection of leptin(P>0.05).8 Chronic administration of leptin considerably increased CO2-stimulatory MV compared with the Lean+Saline group(P<0.01),suggesting facilitation of HCVR by leptin effect.Summary: Deficiency of ob-Rs(leptin resistance-like)impairs but activation of ob-Rs enhances HCVR.These effects are correlated with downstream pSTAT3/STAT3 signaling.Part II: Mechanisms of facilitation of central chemoreflex by leptinObjective : To study whether the NTS neurons contribute to leptin-falicitated central chemoreflex.Methods: The animal was divided into three groups,similar to Part 1protocol as depicted hereinabove,excluding the Obese-HFD group.To determine whether CO2 sensitivity of NTS neurons was affected by leptin,we counted CO2-activated neurons and quantitatively analyzed some pH-sensitive channels(TASK,ASIC,Kir2.3 channels)or receptors(GPR4)in the NTS.CO2-activated neurons were represented by c-fos immunoreactivity(c-fos+)and Western blot was used for quantitative analysis of expression level of pH-sensitive channels or receptors.Results:1 In the Obese Group(deficiency of ob-R),downregulation of TASK-1,TASK-2,TASK-3,ASIC1,ASIC2,Kir2.3 and GPR4 occurred compared with lean rats(P<0.01).2 Chronic administration of leptin produced upregulation of TASK-1,TASK-2,TASK-3,ASIC1,ASIC2,Kir2.3 and GPR4 in lean rats(P<0.01),with no significant change in obese rats(P>0.05).3 In the Obese Group,the number of CO2-activated NTS neurons was less compared with lean rats(P<0.01).4 Chronic administration of leptin increased remarkably the number of CO2-activated NTS neurons in lean(P<0.01)but not obese rats.Summary: Facilitation of HCVR by leptin signaling pathway is most likely due to upregulation of pH-sensitive channels or receptors in the NTS.Part III: Effect of leptin on hypoxic chemoreceptor reflexObjective: To determine the role of leptin in the peripheral chemoreceptor reflex and to reveal the underlying mechanism.Methods: Animal group and measurement of breathing parameters are depicted as above in Part Two.Hypoxia was achieved by exposure to 10% O2 in inspired air.Western blot was employed for quantitative analysis of O2-sensitive channels including TASK-1,TASK-2 TASK-3 and Kv1.5 and of expression level of c-fos.Results:1 Under baseline conditions,expression of ob-Rb and pSTAT3/STAT3 in CBs of obese rats was less than lean rats(P<0.01).2 During exposure to 10% O2,MV increased in all three groups.But the change in increase in MV was less in Obese and Lean-HFD group than that in Lean Group(P<0.01).3 Hypoxia-elicited increases in MV,TV and BF were significantly attenuated following sectioning carotid sinus nerves in all three groups.4 Chronic administration of leptin for 7 days produced greater upregulation of ob-Rs and of pSTAT3 compared to vehicle controls(P<0.01)and produced no marked changes in expression of ob-Rs and of pSTAT3 in obese rats.5 Addition of leptin for 7 days increased considerably MV,TV and BF during inhaling 10% O2 in relative to vehicle controls(P<0.05~0.01),an effect without ocurrence in obese rats.6 The leptin stimulatory effect on breathing parameters during hypoxia retained for 2 weeks and then subsided and finally dispeared 4 weeks afterinjection.7 The leptin stimulatory effect on HVR was impaired after sectioning carotid sinus nerves in support of the role of CBs.8 Under normoxic conditions,the expression of in glomus cells of Obese group was lower than that in Lean and Lean-HFD Groups(P<0.01).9 Chronic administration of leptin for 7 days upregulated expression of TASK-1,TASK-2,TASK-3 and Kv1.5(P<0.01).10 Hypoxia-induced expression of c-fos in CBs increased in all three groups but the change in increase was lower in obese rats compared with other two groups(P<0.01).11 Chronic administration of leptin for 7 days produced a significant increase in c-fos in all four groups.But the change in increase was greater in leptin-treated lean rats compared with other groups(P<0.01).Summary: Activation of leptin signaling facilitates but disruption of leptin signaling attenuates HVR.These effects are associated with changes in expression of TASK-1,TASK-2,TASK-3 and Kv1.5 channels.Part ?: Facilitation of central chemoreflex by chronic intermittent hypobaric hypoxia(CIHH)though leptinObjective: To determine the role of CIHH in the central chemoreceptor reflex and to reveal the underlying mechanism.Methods: The animals were divided into four groups,including control group,HFD Group,CIHH group(28day CIHH treatment)and CIHHwith HFD Group.Ventilation was measured using whole body plethysmography and assessed by breathing parameters including minute ventilation(MV),tidal volume(TV)and breathing frequency(BF).Results:1 In conscious state,MV and TV were lower in HFD group than control.However,the breathing parameters were not significantly differently between CIHH+HFD and control groups.Breathing rate were similar in each group.2 Hypercapnia increased MV in all four groups.The change in MV was lower in HFD group compared with control but such a inhibitory response wasimproved following CIHH.3 Likewise,the change in TV in HFD group was lower than that in control group when exposed to high concentrations of CO2.CIHH pretreatment remarkably antagonized such inhibition.4 During exposure to 5% and 8% CO2,breathing rate in HFD group was greater than control group.However,CIHH significantly improved such respiratory pattern.Summary: CIHH modulates the sensitivity to hypoxia and hypercapnia in fructose-fed obese rats via leptin signaling pathway.Conclusion: Leptin facilitate control of regulation of breathing.Facilitation of HCVR by leptin signaling pathway is most likely due to upregulation of p H-sensitive channels or receptors in the NTS.Activation of leptin signaling facilitates HVR.The effect is associated with changes in expression of TASK-1,TASK-2,TASK-3 and Kv1.5 channels.