The Regulation And Mechanism Of Adipose Afferent Reflex On Sympathetic Activity And Lipolysis

Adipose afferent reflex(AAR)is a sympatho-excitatory reflex induced by chemical stimulation of white adiposetissue(WAT).Paraventricular nucleus(PVN)of the hypothalamus is an integrative site in the control of AAR.The interaction of the WAT and brain may be important in promoting lipolysis and energy expenditure,and is proposed to be helpful for keeping total body fat and body weight stable and preventing obesity via its reflex control of sympathetic outflow.AAR was enhanced in obese rats.The sympatho-excitatory effect of AAR was greater but lipolysis effect was weaker in obesity than those in control rats.The high fat diet induced obese animal and primary adipocyte were used in the study.The research is divided into three parts:(1)the role and mechanism of superoxide anions in PVN in modulating the AAR.(2)the effects of AAR on lipolysis(3)the mechanisms of attenuated lipolysis response to enhanced AAR in obesity.Part ? Superoxide Anions in Paraventricular Nucleus Modulate Adipose Afferent Reflex and Sympathetic Activity in RatsBackgroundAdipose afferent reflex(AAR)is a sympatho-excitatory reflex induced by chemical stimulation of white adipose tissue(WAT).Ionotropic glutamate receptors including NMDA receptors(NMDAR)and non-NMDA receptors(non-NMDAR)in paraventricular nucleus(PVN)mediate the AAR.Enhanced AAR contributes to sympathetic activation and hypertension in obese rats.Objective1.To investigate the role and mechanism of superoxide anions in PVN in modulating the AAR.2.To determine whether activation of ionotropic glutamate receptors are involved in increased superoxide anion level of AARMethodsExperiments were carried out on male Sprague–Dawley rats weighing between 350 and 400 g,The rat was anesthetized with intraperitoneal injection of urethane(800 mg/kg,)and ?-chloralose(40 mg/kg).right inguinal WAT(i WAT)was exposed through an inguinal area incision.The AAR was induced by the injections of capsaicin(1.0 nmol ?l-1)into four sites of the right i WAT at a rate of 4.0 ?l min-1 for 2 min for each site.AAR was evaluated by the RSNA and MAP responses to injections of capsaicin.Stereotaxic coordinates for PVN were 1.8 mm caudal from bregma,0.4 mm lateral to the midline and 7.9 mm ventral to the dorsal surface.Microinjection volume for each side of the PVN was 50 nl,Specific fluorogenic probe dihydroethidim(DHE)and lucigenin-derived chemiluminescence method was used to detect in situ superoxide anions in the PVN.Results1.PVN microinjection of moderate or high dose of PEG-SOD(1 or 5 units),superoxide anion scavenger tempol or NAD(P)H oxidase inhibitor apocynin to scavenge superoxide anions in the PVN significantly decreased baseline RSNA and MAP,and attenuated the capsaicin-induced AAR2.Injection of capsaicin into the right i WAT increased the superoxide anions in the bilateral PVN compared with injection of saline.3.Compared with saline,injection of capsaicin into the right i WAT increased the superoxide anion level and NAD(P)H oxidase activity in the PVN.4.Microinjection of NMDAR agonist NMDA or non-NMDAR agonist AMPA into the PVN increased superoxide anion level and NAD(P)H oxidase activity in the PVN,combined AP5 and CNQX reduced superoxide anion level and NAD(P)H oxidase activity in the PVN.Microinjection of AP5 or CNQX,the differences did not reach statistical significance.5.Microinjection of NMDAR antagonist AP5,non-NMDAR antagonist CNQX and combined NMDAR antagonist AP5 and non-NMDAR antagonist CNQX into the PVN prevented the increases in superoxide anion level and NAD(P)H oxidase activity in the PVN caused by i WAT injection of capsaicin.AP5 alone also significantly attenuated the effects of the i WAT injection of capsaicin on the superoxide anion level and NAD(P)H oxidase activity in the PVN.However,CNQX alone only showed a tendency in attenuating the effects of capsaicin and the differences did not reach statistical significance.ConclusionsNAD(P)H oxidase-derived superoxide anions in the PVN contributes to the tonic modulation of AAR.Activation of ionotropic glutamate receptors in the PVN is involved in the AAR-induced production of superoxide anions in the PVN.Part ? Reduced lipolysis versus enhanced adipose afferent reflex in obesity: impaired activation of adrenoceptor-cAMPhormone sensitive lipase pathwayBackgroundAccumulated evidence indicate the sympathetic and sensory innervations of white adipose tissue(WAT).We found that chemical stimulation of WAT with bradykinin,adenosine or capsaicin caused a similar sympathetic activation and a mild pressor response in normal rats,and the sympatho-excitatory reflex was called adipose afferent reflex(AAR).The AAR was mediated by ionotropic glutamate receptors and modulated by superoxide anions and melanocortin 4 receptors in paraventricular nucleus(PVN)of hypothalamus.The interaction of the WAT and brain may be important in promoting lipolysis and energy expenditure,and is proposed to be helpful for keeping total body fat and body weight stable and preventing obesity via its reflex control of sympathetic outflow.We recently found that the AAR was enhanced in highfat diet(HFD)-induced obese rats,which greatly contributes to the excessive sympathetic activation and hypertension in obesity Sympathetic activity is known to increase lipolysis via adrenergic receptor-medicated activation of hormone sensitive lipase(HSL).However,it is unknown whether the enhanced AAR induces lipolysis.A more interesting question is why enhanced AAR and sympathetic outflow in obese rats fails to prevent obesity.The present study is designed to investigate the effects of AAR on lipolysis and the mechanisms of the attenuated lipolysis response to the enhanced AAR in obese rats.Objective1.To investigate the effects of AAR on lipolysis.2.To investigate the mechanisms of attenuated lipolysis response to enhanced AAR in obesity..MethodsMale Sprague-Dawley rats(n=90)weighing between 300-350 g were randomly divided into three groups.The rats in one group received a control diet(Ctrl,12% kcal as fat,n=30),and the rats in another group received a high-fat diet(HFD,42% kcal as fat,n=60)for 12 weeks to induce obese models After 12 weeks,rats were ranked based on their body weight gain.The HFD rats with a greater weight gain than the Ctrl rat with the greatest weight gain were defined as obese rats(OB).Rats were anesthetized with intraperitoneal injection.The renal sympathetic nerve activity(RSNA)and mean arterial pressure(MAP)were simultaneously recorded by a Power Lab data acquisition system.The AAR were induced by simultaneous injections of capsaicin into the four sites of right i WAT(1.0 nmol/?l,4.0 ?l/min for each site,lasting for 2 min).The right epididymal WAT(e WAT)nerve was isolated and cut distally.A pair of silver stimulating electrodes was placed on the central end of this nerve.Stimulus was delivered with a stimulator.Adipocytes were isolated as previously described.ISO induced lipolysis was determined by assaying FFA and glycerol released with Commercial acyl-Co A oxidase-based colorimetric kits and Free Glycerol Determination Kits.Serum NE levels and WAT cAMP levels were measured with commercial ELISA kits for NE and cAMP.The HSL,?1 receptor,?2 receptor and ?3 receptor m RNA levels in the WAT were determined with Real-time quantitative PCR.HSL phosphorylation and adrenergic receptor protein expression were determined with Western blot methodResults1.The right i WAT injection of capsaicin increased RSNA and MAP in both Ctrl and OB rats.The effects were much greater in OB rats than those in Ctrl rats.Capsaicin increased serum NE level in both Ctrl and OB rats,and serum FFA in Ctrl rats.However,capsaicin only induced a tendency in increasing serum FFA in OB rats,but differences did not reach statistical significance.the effects of capsaicin in the right i WAT in normal rats were abolished by the i WAT denervation2.Electrical stimulation of right eWAT nerve increased RSNA and MAP in both Ctrl and OB rats.The effects were much greater in OB rats than those in Ctrl rats.The stimulation-induced increase in serum NE in OB rats was more than that in Ctrl rats,while the increase in serum FFA in OB rats was much less than that in Ctrl rats.electrical stimulation of adjacent subcutaneous tissue in normal rats failed to cause sympathetic activation and lipolysis3.Capsaicin-induced AAR in both Ctrl and OB rats increased the HSL phosphorylation at Ser660 and Ser563 in VAT and SAT.However,the phosphorylation effect in OB rats was greatly reduced in both VAT and SAT compared with those in Ctrl rats.In addition,capsaicin-induced AAR in Ctrl rats caused a greater increase in the HSL phosphorylation at Ser563 in VAT than that in SAT4.Adrenergic ?1 and ?3 receptor m RNA and protein expression in both VAT and SAT were down-regulated in OB rats.The ?1 and ?3 receptor protein levels in Ctrl rats were lower in SAT than that in VAT,but not in OB rats.There was no significant difference in the ?2 receptor m RNA and protein expression between VAT and SAT or between Ctrl and OB rats5.ISO stimulated lipolysis of primary VAT and SAT adipocytes derived from both Ctrl and OB rats.ISO-induced lipolysis effect was greatly reduced in VAT and SAT adipocytes from OB rats compared those from Ctrl rats.The lipolysis effect was greater in VAT adipocytes than that in SAT adipocytes from Ctrl rat.6.The cAMP levels were lower in VAT and SAT adipocytes from OB rats than those from Ctrl rats.ISO increased cAMP levels,but the effect of ISO was much less in VAT and SAT adipocytes from OB rats than those from Ctrl rats.Moreover,the ISO-induced increase in cAMP level was less in SAT adipocytes than that in VAT adipocytes from Ctrl rats7.A cAMP analogue dbcAMP was used to rectify the insufficient cAMP in the VAT adipocytes from OB rats.Although the ISO-induced lipolysis effect was greatly reduced in the adipocytes from OB rats compared with those from Ctrl rats,dbcAMP in the adipocytes from OB rats induced a similar degree of lipolysis effect to the ISO or dbcAMP-induced effect in the adipocytes from Ctrl rats.Combined incubation of ISO and dbcAMP failed to cause greater lipolysis effect than dbcAMP alone.8.The basal phosphorylated HSL(at Ser563 and Ser660)levels were very low in the adipocytes from OB rats compared with those from Ctrl rats.The ISO-induced HSL phosphorylation effect was much weaker in the adipocytes from OB rats than those from Ctrl rats.The dbcAMP in the adipocytes from OB rats induced a similar degree of HSL phosphorylation effect to the ISO-or dbcAMP-induced effect in the adipocytes from Ctrl rats.Combined incubation of ISO and dbcAMP failed to cause greater HSL phosphorylation effect than dbcAMP aloneConclusionsReduced lipolysis response to the enhanced AAR in obesity is attributed to the impaired activation of ?-adrenoceptor-cAMP-HSL pathway.Increased cAMP level in the WAT rectifies the attenuated lipolysis in obesity.