Mechanism Of Obesity - Induced Sympathetic Activation And Irisin 's Anti - Metabolic Disorder And Lipolysis Mechanism

Obesity is characterized by excessive sympathetic activation, disorders of glucose and lipid metabolism, insulin resistance, and it could cause a series of syndrome including hypertension, diabetes and metabolic syndrome. The present study was designed to dertermine the mechanisms of sympathetic activation and metabolic derangements in obesity, and we conducted intervention studies on obesity. The high fat diet induced obesity animal and 3T3L1 adipocyte were used in the study. The research is divided into three parts: ① Mechanisms of adipose afferent reflex in the sympathetic activation of obesity; ②Mchanisms of irisin in ameliorating metabolic derangements in obesity; ③Mchanisms of irisin in enhancing lipolysis in 3T3L1 adipocyte.PartⅠEnhanced Adipose Afferent Reflex Contributes to Sympathetic Activation in Diet-induced Obesity HypertensionBackgroundExcessive sympathetic activity plays a critical role in the pathogenesis and target organ complication of obesity and obesity hypertension. The mechanisms involved in the sympathetic activation of obesity hypertension remain uncertain. We recently found that WAT injection of capsaicin, bradykinin, adenosine or leptin increased renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in normal rats. Chemical stimulation of WAT induced sympatho-excitatory reflex is called AAR. WAT denervation or chemical lesion of the neurons in paraventricular nucleus (PVN) abolished the AAR induced by chemical stimulation. AAR is involved in the regulation of sympathetic activity and blood pressure. Neuroanatomical studies have shown sensory and efferent sympathetic innervations of WAT. We hypothesize that the enhanced AAR may play an important role in the sympathetic activation and hypertension in obesity. The obesity hypertension model induced by a high fat diet (HFD) in the present study was used to determine the contribution of AAR to the sympathetic overdrive and hypertension as well as the possible mechanisms of enhanced AAR and sympathetic activity.Objective1. To determine whether the AAR is enhanced in HFD-induced obesity hypertensive rats.2. To determine whether the enhanced AAR are involved in the sympathetic overdrive and hypertension in obesity hypertension.MethodsMale Sprague-Dawley rats were randomly assigned to receive a control diet (12% kcal as fat, n=55) or HFD (42% kcal as fat, n=165) for 12 weeks. The rats with weight gains equal to or less than the heaviest control rats were defined as obesity-resistant rats (OR), and the rats with greater weight gains were obesity-prone rats (OP). The OP rats with systolic blood pressure (SBP) equal to or more than 150 mm Hg were referred to as obesity hypertensive rats (OH). The OP rats with lower SBP were obesity non-hypertensive rats (ON). Acute experiments were carried out at the end of the 12th week under anesthesia with intraperitoneal administration of urethane (800 mg/kg) and a-chloralose (40 mg/kg). RSNA, MAP and heart rate (HR) were continuously recorded. AAR was evaluated by the RSNA and MAP responses to injections of capsaicin (1.0 nmol/μl) into four sites of right inguinal WAT (iWAT) at a rate of 4.0 μl/min for 2 min for each site. Plasma renin, angiotensin (Ang) Ⅱ and norepinephrine (NE) levels were determined with ELISA. The c-fos expression in the PVN was determined with immunohistochemistry.Protocol 1:Basal sympathetic tone was determined in Ctrl, OR, ON and OH rats. The plasma NE level and maximal depressor response of intravenous injection of ganglionic blockade, hexamethonium hydrochloride (30 mg/kg), were used as indexes of sympathetic activity.Protocol 2:AAR was evaluated by the RSNA, MAP and HR responses to capsaicin in Ctrl, OR, ON and OH rats.Protocol 3:Changes of plasma renin, Ang II and NE levels were determined 15 min after iWAT capsaicin injection in OH rats.Protocol 4:Effects of WAT sensory denervation with resiniferatoxin (RTX) on the baseline RSNA, MAP, HR and AAR were investigated in Ctrl, OR, ON and OH rats.Protocol 5:Long-term depressor effect of WAT sensory denervation with RTX was determined in a conscious state of OH rats.Protocol 6:WAT afferent nerve activity (WANA) was recorded for comparison of basal WANA among Ctrl, OR, ON and OH rats. WAT nerve was cut at its central end to abolish its efferent activity to the WAT. The maximal change of WANA caused by the topical application of lidocaine solution on the distal end of the nerve was used to evaluate the basal WANA level.Protocol 7:Effects of WAT injection of leptin antagonist on the RSNA, MAP and HR were investigated in OH rats.Protocol 8:Change of c-fos immunoreactivity in the PVN caused by WAT injection of capsaicin to induce AAR was investigated in Ctrl, OR, ON and OH rats.Protocol 9:Effects of inhibition of PVN neurons with lidocaine on the baseline RSNA, MAP, HR and AAR were investigated in Ctrl, OR, ON and OH rats.Results1. The plasma NE level and maximal depressor response to hexamethonium were higher in OH compared with ON, OR or Ctrl, and in ON compared with OR or Ctrl.2. The iWAT injection of capsaicin increased RSNA and MAP in all four groups. The capsaicin-induced AAR was enhanced in OH compared with ON, OR or Ctrl, and in ON compared with OR or Ctrl. There was no significant difference in the AAR between OR and Ctrl.3. Simulation of iWAT with capsaicin to induce the AAR increased the plasma renin, Ang II and NE levels in OH.4. Bilateral iWAT and retroperitoneal WAT (rWAT) injection of RTX caused immediate and short-term increases in RSNA and MAP in all groups. About 20 min later, RTX caused long-lasting decreases in RSNA and MAP in ON and OH but not in Ctrl and OR. Both excitatory and inhibitory effects of RTX were enhanced in OH compared with ON, OR or Ctrl, and in ON compared with OR or Ctrl.5. Bilateral iWAT and rWAT injection of RTX in OH caused a persistent decrease in SBP lasting at least 3 weeks. Four weeks later, RTX only had a tendency to decrease SBP, and the difference did not reach statistical significance.6. Blocking the WAT nerve at its peripheral end with lidocaine caused more decrease in OH than ON, OR or Ctrl, and in ON than OR or Ctrl, suggesting enhanced baseline WANA in OP, especially in OH.7. Bilateral iWAT and rWAT injection of leptin antagonist in OH decreased the RSNA and MAP peaking at 5 min and lasting at least 15 min.8. Injection of capsaicin into WAT increased the c-fos expression in PVN in Ctrl and OH. After injection of capsaicin, the c-fos expression in PVN was increased in OH compared with ON, Ctrl or OR, and in ON compared with Ctrl or OR.9. Bilateral PVN perfusion of lidocaine to inhibit the activity of PVN neurons attenuated baseline RSNA in both OH and ON, and decreased baseline MAP and HR in OH. The effect of lidocaine on RSNA was greater in OH than ON. Lidocaine in the PVN abolished the AAR in all four groups.ConclusionsAAR is enhanced in obesity hypertension. Enhanced AAR contributes to sympathetic activation, and PVN is involved in the enhanced AAR and sympathetic activation in obesity hypertension.Part II FNDC5/irisin ameliorates metabolic derangements in obesityBackgroundObesity is characterized by a cluster of disorders in energy metabolism, including insulin resistance, hyperlipidemia and type II diabetes. Despite recent advances in understanding the disease mechanism, effective treatments are still lacking. Irisin was firstly identified in 2012 as a muscle-derived factor that promotes the formation of brown-adipocyte-like cells in mice. Irisin is a cleaved and secreted fragment of FNDC5. Exercise stimulates PGC-la expression, which promotes FNDC5 expression, and thereby increases irisin levels. Cross-sectional studies revealed that circulating irisin levels were positively correlated with biceps circumference, body mass index (BMI), glucose, ghrelin, and IGF-1, but negatively with age, insulin, cholesterol, and adiponectin levels in humans and intrahepatic triglyceride contents in obese adults. Levels of irisin were significantly higher in participants with the metabolic syndrome than in those without the syndrome. However, whether persistent increased irisin level plays a beneficial role in obesity is completely unknown. The obesity model induced by a high fat diet (HFD), lentiviral vector which express full-length FNDC5 and irisin pump were used in the present study to determine the contribution of FNDC5/irisin to the metabolic derangements in obesity.Objective1. To determine the effects of FNDC5 overexpression on energy expenditure and metabolic derangements in obesity mice.2. To determine the effects of FNDC5 overexpression on lipolysis in obesity mice.3. To determine the effects of irisin subcutaneous perfusion on metabolic derangements in obesity mice.MethodsMale C57/BL6J mice were randomly assigned to receive a control diet (12% kcal as fat, n=24) or HFD (60% kcal as fat, n=24) for 12 weeks, starting at 6 weeks of age. The mice in Ctrl and HFD group were randomly divided into vector and FNDC5 subgroups (n=12 for each group) at the end of 6th week after the diet, which were injected with lentivirus expressing vector or FNDC5. Another group of C57/BL6J mice were given HFD starting at 6 weeks of age. The mice were divided into Irisin and Saline subgroups at the end of 8th week after the diet, which were implanted subcutaneously with micro-osmotic pump containing irisin and saline, respectively. Acute experiments were carried out at the end of the 12th week after diet. Serum FNDC5, irisin, leptin, insulin, cholesterol, triglyceride, free fatty acid (FFA), glycerol, angiotensin II and norepinephrine levels were determined with ELISA. The mRNA and protein levels of FNDC5, uncoupling protein 1 (UCP1) and hormone-sensitive lipase (HSL) were determined by real-time PCR and Western Blot.Protocol 1:Effects of FNDC5 overexpression:(1) Effectiveness of the FNDC5 gene transfer:Blood samples and the skeletal muscle, heart, liver, subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) of mice were collected to determine the protein levels of FNDC5 and irisin.(2) Changes of body weight and food intake.(3) Fat mass and 24 h energy expenditure parameters were determined with micro-CT and PhenoMaster systems, respectively.(4) Changes of fasting blood glucose, glucose tolerance test (GTT) and insulin tolerance test (ITT), blood pressure.(5) Serum leptin, insulin, cholesterol, triglyceride, FFA, glycerol, angiotensin II and norepinephrine levels.(6) The adipocyte sizes of SAT and VAT.(7) The UCP1 expression in SAT and VAT.(8) The lipolysis was evaluated by the serum FFA levels after the mice fasted for 4hor24h;(9) Changes of HSL mRNA and phosphorylated HSL (Ser563,565,660) in SAT and VAT.Protocol 2:Effects of subcutaneous perfusion with irisin:(1) Changes of body weight and food intake.(2) The fasting blood glucose, GTT and ITT.(3) Serum cholesterol, triglyceride and FFA levels.Results1. The FNDC5 and irisin protein levels in the muscle were higher in obesity mice than these in Ctrl mice, but these in the liver, SAT and VAT were not higher in obesity mice. FNDC5 gene transfer increased serum FNDC5 and irisin levels in Ctrl and HFD mice as well as in the muscle.2. The FNDC5 protein level was high in muscle, moderate in the heart and liver, and low in the SAT and VAT. Irisin level was high in muscle, moderate in SAT, VAT and heart, and very low in the liver.3. FNDC5 overexpression had no significant effects on the body weight, food intake, total fat mass in Ctrl and HFD mice.4. FNDC5 overexpression increased the O2 consumption, CO2 and heat production in HFD mice without significant effect on the total activity.5. GTT and ITT showed the improved insulin sensitivity and the blood pressure and fasting blood glucose were reduced in HFD mice after FNDC5 overexpression.6. Serum cholesterol, triglyceride, free fatty acid, insulin and norepinephrine levels were increased in HFD mice, which were reduced by FNDC5 overexpression. There was no significant change of leptin and Ang II level in HFD mice.7. FNDC5 overexpression only significantly reduced the diameter of adipocytes only in SAT not VAT in HFD mice.8. FNDC5 overexpression significantly increased UCP1 mRNA and protein expression in the SAT but not in the VAT in Ctrl and HFD mice.9. FNDC5 overexpression increased the fasting induced lipolysis in both Ctrl and HFD mice.10. FNDC5 overexpression increased the phosphorylation at HSL Ser563 or Ser660 but not at HSL Ser565 and the HSL mRNA in SAT and VAT in Ctrl and HFD mice.11. Subcutaneous perfusion with irisin had no significant effects on body weight and food intake.12. Subcutaneous perfusion with irisin improved insulin sensitivity and reduced fasting blood glucose level.13. Subcutaneous perfusion with irisin reduced serum cholesterol, triglyceride and FFA level.ConclusionsFNDC5/irisin enhances energy expenditure, ameliorates metabolic derangements, and attenuates insulin resistance in HFD-induced obesity mice. FNDC5/irisin enhances lipolysis by cAMP-PKA-mediated HSL activation and perilipin reduction. Irisin may be taken as an effectively therapeutic target for metabolic diseases.Part III Irisin enhances lipolysis via cAMP/PKA/HSL pathway in 3T3L1 adipocyteBackgroundAdipose tissue lipolysis is an important process in which triacylglycerides are mobilized, releasing fatty acids and glycerol and thus providing the body with substrates. Irisin is proteolytically processed from the extracellular portion of FNDC5, and proposed to mediate the beneficial effects of exercise on metabolism via stimulating uncoupling protein 1 (UCP1) expression and brown-fat-like development. A recent study showed that FNDC5/irisin was not only a myokine but also an adipokine. We recently found that irisin enhances energy expenditure, ameliorates metabolic derangements and enhances lipolysis in obesity mice. The present study was designed to determine the effects of irisin on 3T3L1 adipocyte in vitro as well as the possible signaling mechanisms.Objective1. To determine the effects of irisin on lipolysis in 3T3L1 adipocyte.2. To determine the mechanisms of enhanced lipolysis in 3T3L1 adipocyte.MethodsThe lipolysis was determined by the glycerol and free fatty acids (FFA) levels from the media of 3T3L1 adipocyte. The mRNA and protein levels of hormone-sensitive lipase (HSL) were determined by real-time PCR and Western Blot.Protocol 1:Time effect of irisin on lipolysis was determined in basal and ISO-stimulated condition in 3T3L1 adipocytes. The cells were treated with 1000 ng/ul of irisin for 30 min,1 h,2 h and 4 h, respectively.Protocol 2:Dose effect of irisin on lipolysis was determined in basal and ISO-stimulated condition in differentiated 3T3L1 adipocytes. The cells were treated with 0, 10,100 and 1000 ng/μl of irisin, respectively; media were then collected for glycerol and FFA level determinations.Protocol 3:To determine the effects of cAMP and PKA in irisin-induced lipolysis, the cells were pre-treated with adenylate cyclase inhibitor SQ22536 or PKA inhibitor H-89 for 12 h, and treated with irisin for 1 h in the presence or absence of ISO, media were then collected for glycerol and FFA level determinations.Protocol 4:The cAMP level and HSL mRNA were measured after 3T3L1 cells treated with 1000 ng/μl irisin for 1 h.Protocol 5:The phosphorylated HSL (Ser563,565,660) were determined in 3T3L1 adipocytes after irisin treatments.Results1. The basal lipolysis was highest at 1 h after irisin treatment. The ISO-stimulated lipolysis did not change significantly in 4 h after irisin treatment.2. Irisin dose-dependently enhanced the basal lipolysis but not the ISO-stimulated lipolysis.3. Irisin enhanced the lipolysis in 3T3L1 adipocyte, which was prevented after pre-treatment with SQ22536 or H-89.4. The cAMP level and HSL mRNA were increased after irisin treatment.5. Irisin increased the phosphorylation at HSL Ser563 or Ser660 but not at HSL Ser565 in basal condition.ConclusionsIrisin enhances the basal lipolysis but not ISO-induced lipolysis in 3T3L1 adipocyte. The increased phosphorylation at HSL Ser563 or Ser660 is involved in the enhanced lipolysis.