The Role Of Interleukin-18 In Adipose Tissue On Development Of Metabolic Syndrome: Involvement Of Signalling Pathway

BackgroundMetabolic syndrome(MS) has become one of the major public health challenges worldwide accompanied with changes in human lifestyle and dietary habits.MS consists of the combined presentation of multiple cardiovascular risk factors that include abdominal obesity,glucose intolerance,dyslipidemia,hypertension and insulin resistance.In recent half a century,a great of studies on MS have been conducted,but development mechanisms of MS remains largely unclear.Recently,MS has been considered to be usually due to the interaction between genetic and environmental factors,and insulin resistance has been discussed as major pathophysiological mechanism for the development of MS.However,the molecular mechanisms triggering insulin resistance are not fully understood.A growing number of studies indicate that low-grade chronic inflammation is involved in the pathogenesis of MS.Adipose tissue is now viewed as an active endocrine organ that plays a crucial role in the pathophysiological process of inflammation-insulin resistance-metabolic syndrome.MS often accompanies the elevated inflammatory factors such as TNF-a,Interleukin (IL)-6,Interleukin(IL)-18 and so on.IL-18,a particularly important cytokines is recently thought as a risk predictor for MS.Circulating levels of IL-18 are elevated in obesity and reduced by weight loss.Increased plasma IL-18 is a marker of insulin resistance in type 2 diabetic and non-diabetic humans.Elevated IL-18 levels are associated with the MS independent of obesity and insulin resistance.New data show that IL-18 is expressed in human adipose tissue and strongly upregulated by TNFa in human adipocytes,and IL-18 release from adipocytes from obese donors was about 3-fold higher compared to adipocytes from non-obese donors.Adittionally,Adipose tissue(AT) IL-18 mRNA content was higher in the obese group than in the non-obese group and positively correlated with IR.Previous studies on the relationship between MS and IL-18 were limited in clinical trials,and animal experiments were never reported.There is a great significance to search effective drugs of preventing and treating MS due to its risk of cardiovascular disease.A growing number of studies indicate that low-grade chronic inflammation is involved in the pathogenesis of MS,and suppression of inflammation has already become a novel strategy to improve MS. There is some evidence that these drugs have anti-inflammatory actions,including hypoglycemic agent,lipid regulating agent,large dose aspirin,angiotensin-converting enzyme inhibitor(ACEI) and angiotensin receptor antagonist(ARB).Compared with these drugs,the effects of CCB on subjects with insulin resistance and MS were not thought highly.Several large-scale clinical tests showed that calcium-channel blocker (CCB) were safe and effective in reducing most types of cardiovascular morbidity and mortality in diabetic hypertensive patients,and thus have cardiovascular protection besides reducing blood pressure(BP).Besides reducing BP,amlodipine seemed to improve insulin resistance and decrease tumor necrosis factor-alpha(TNF-a) levels in obese hypertensive type 2 diabetic patients.Whether CCB can inhibit and delay MS by anti-inflammatory actions is not still conclusive.Objective1.To investigate the expression of IL-18 at both the mRNA and protein levels in adipose tissue of Rats with Fructose-Induced MS.2.To evaluate the correlation between the adipose tissue expression of IL-18 and MS in Fructose-fed rats.3.To investigate the effects of Filodipine on a cluster of metabolic abnormalities and adipose tissue expression of IL-18 in fructose-fed rats.MethodsThirty three male Wistar rats were divided randomly into two groups:a control group (n=12) in which the rats were fed the normal Rodent Diet and water,and a fructose-fed group(n=21) in which the rats were fed the normal Rodent Diet plus fructose in the drinking water as a 10%(w/v) solution.At the end of the 32th weeks, the fructose-fed rats were subdivided randomly into two groups:fructose group(n= 9) was continued with a fructose diet,and filodipine(5mg/kg/d) was administered by gavage daily for 6 weeks to filodipine group(n=9) with a fructose diet.All animals were sacrificed by cervical decapitation and the epididymal fat pads were stored at -80℃for analysis.The follwing parameters were measured during the study:(1) All the rats have their body weight and tail blood pressure measured once per 2 week.(2) Blood was collected from jugular vein at 0 week,the end of 32 weeks,and 6 weeks after treatment respectively.Plasm lipid,glucose,insulin and IL-18 were determined using routine method,and the HOMA-IR expressed as an index of insulin resistance was calculated.(3) Pathological study of adipose tissue.(4) Immunohistochemistry for the location of IL-18 in adipose tissue.(5) The mRNA expression of IL-18 was measured by quantification real-time RT-PCR.(6) The protein expression of IL-18 was analyzed by western blot.Results1.The experimental animalsThree rats of fructose group died in the entire experiment,none of control group died. A total of 30 rats finished the study,12 rats in control group,9 rats in fructose group and 9 rats infelodipine group.2.Comparison of metabolic variables between control and fructose group after fructose treatmentThere is no significant difference in terms of body weight,tail blood pressure,glucose, lipids and insulin before fructose treatment.Chronic administration of fructose during 32 weeks induced a cluster of metabolic abnormalities.Fructose-fed rats had higher body weight,systolic BP,fasting levels of plasma triglycerides,plasma insulin,and HOMA-R than the Con rats.There were significant differences in total cholesterol and glucose,both of which were although slightly increased.3.Effect of felodipine on metabolic variables in ratsAfter treatment with felodipine for 6 weeks,systolic BP,insulin and HOMA were significantly decreased in felodipine group compared with fructose group,other parameters showed no significant difference.Systolic BP,insulin and HOMA were significantly decreased in felodipine group after 6 weeks treatment compared with those before treatment;other parameters showed no significant difference.4.Comparison of Serum IL-18 levels between pro-treatment and post-treatment with felodipineSerum IL-18 levels were significantly higher in fructose and filodipine group than those in control group.6 weeks treatment with filodipine significantly decreased serum IL-18 levels compared with those before treatment.5.Pathological findingsHE staining slides under optical microscopy showed that in control group,cell size of adipocytes was smaller,uniform,and array was regular;In fructose group,adipocytes size was greater,not uniform,cell form was irregular,and cell arrange was disorder; Cell wall was not clear and cell fusion was observed at intersection of cell. Adipocytes infelodipine group lied between control and fructose group.6.Location of IL-18 by immunohistochemistryThe positive reaction of IL-18 protein was stained brown,and localized in intercellular substance and adipocyte cytoplasm.Intercellular substance was stained weak brown and weak brown granules were seldom distributed in cytoplasm in control group,whereas intercellular substance was stained deep brown and deep brown granules were thickly distributed in cytoplasm in fructose group.Staining in felodipine group lied between control and fructose group.7.IL-18 mRNA expression by RT-PCRIn the fructose-fed groups,the mRNA expression levels of IL-18 in adipose tissue were significantly increased compared with those in the control group.IL-18 mRNA levels in felodipine group were markedly lower than in fructose group.In addition, IL-18 mRNA levels showed a significant positive correlation with HOMA-IR in the fructose group(r=0.74,P＜0.01).8.IL-18 protein expression by westein blotCompared to control group,the groups fed on a fructose diet showed a significant rise in IL-18 protein content in adipose tissue.IL-18 protein content in the felodipine group was significantly lower than in fructose group.Moreover,IL-18 protein content showed a significant positive correlation with HOMA-IR in the fructose group (r=0.82,P＜0.01).Conclusions1.Rats model mimicked human MS can be achieved by kepting rats on a high-fructose diet for up to 32 week.This lays the foundation for further studying the mechanism of MS.2.IL-18 is confirmed to be expressed in asipose tissue at both the mRNA and protein levels.3.IL-18 expression is increased in serum and adipose tissue and showed a significant positive correlation with HOMA-IR in fructose group,which suggests that IL-18 is involved in the pathogenesis of MS via adipose tissue.4.Filodipine down regulates serum and adipose tissue expression of IL-18,with blood pressure reduced,and insulin resistance improved,showing that Filodipine may improve insulin resistance,prevent and delay MS by anti-inflammatory effect. BackgroundThe mechanisms of metabolic syndrome(MS) are very complex,usually due to the interaction between genetic and environmental factors.Insulin resistance has been discussed as major pathophysiological mechanism for the development of MS.Insulin resistance,the inability of target tissues to adequately increase glucose transport in response to a physiological level of insulin,results from defects of insulin signaling behind insulin receptor.Of the total,It is crucial for impaired IRS-1,PI-3K and PKB/Akt.A growing number of studies indicate that low-grade chronic inflammation is involved in the pathogenesis of MS.There exist intersections between signaling pathway behind insulin receptor and signaling transduction of inflammatory cytokines. Inflammatory cytokines are involved in the molecular mechanism that inflammation leads to insulin resistance by interfering with IRS-1/PI-3K insulin signaling pathway. Studies demonstrate that adipose tissue is considered as dominant sites affecting systemic insulin resistance and especially visceral adipose tissue plays an important role in metabolic syndrome.Adipose tissue is not only an energy storage organ,but an active endocrine organ.Abdominal obesity results in changes of adipocytokines which are involved in the development of insulin resistance by blocking insulin receptor signaling transduction in adipose tissue and other insulin-sensitive tissues. In part 1 of our study,results showed adipose tissue expression of IL-18 was higher in fructose-induced MS group than in the control group and positively correlated with IR, which suggests that IL-18 is involved in the pathogenesis of MS via adipose tissue. Recent studies indicate that IL-18 regutes inflammatory reaction through NF-κB -dependent signal transduction.Additionally,there exist intersections between signaling pathway behind insulin receptor and signaling transduction of inflammatory cytokines.Thereby we suggested that IL-18 promoted IR via defects of IRS-1/PI-3K/Akt insulin signaling pathway,and inducing the development of MS. On the other hand,Akt stands as the furthest known downstream component of the insulin-signaling pathway that leads to metabolic regulation for the pivotal role of this kinase in the linkage between the insulin signal and the control of glucose uptake.At present,much interest has recently been focused on Ttibble3(TRB3) because it is involved in insulin resistance.The current studies suggest that TRB3 disrupts insulin signaling by binding directly to Akt and blocking activation of the kinase,thereby inducing insulin resistance in liver.Furthermore,the expression of TRB3 mRNA level can be upregulated by various cellular stresses such as inflammatory stimulation.For the above reasons,we suggested that IL-18 promotes insulin resistance through TRB3 upregulation and TRB3-mediated inhibition of Akt in adipose tissue.Taken together,the hypothesis is proposed that IL-18 promotes insulin resistance through suppression of Akt activation via IRS/PI-3K and TRB3 signaling pathway in adipose tissue,thus leading to the development of MS.Objective1.To investigate the protein expression of key signaling molecule in IRS/PI-3K/Akt and TRB3/Akt pathway in adipose tissue of Rats with Fructose-Induced MS.2.To investigate the effects of IL-18 on a cluster of metabolic abnormalities in fructose-fed rats used IL-18 adenovirus administered.3.To investigate the role of IL-18 on development of IR:involvement of signalling pathway in adipose tissue.MethodsForty-eight male Wistar ratswere divided randomly into two groups:the control group (Con,n=12),which received a standard rodent chow and water,and the fructose-fed group(Fru,n=36) which received the standard rodent chow plus fructose in the drinking water as a 10%(w/v) solution.At the end of the 32th weeks,the fructose-fed rats were subdivided randomly into three groups:the fructose-fed group(Fru,n=9), the adenovirus-GFP control group(Ad-Con,n=9),and the adenovirus-IL-18 group (Ad-IL-18,n=12).The three groups were treated with PBS,Ad-GFP(1×1010pfu in 0.4ml PBS) and Ad-IL-18(1×1010pfu in 0.4ml PBS) via tail vein injection, respectively,and continued on a fructose diet for 6 weeks.All animals were sacrificed by cervical decapitation and the epididymal fat pads were stored at -80℃for analysis. The follwing parameters were measured during the study:(1) All the rats have their body weight and tail blood pressure measured once per 2 week.(2) Blood was collected fi'om jugular vein at 0 week,the end of 32 weeks,and 6 weeks after adenovirus transfection respectively.Plasm lipid,glucose,insulin were determined using routine method,and the HOMA-IR expressed as an index of insulin resistance was calculated.(3) Serum IL-18 levels of four groups were determined by ELISA at the end of 1st,2nd,3rd,4th,and 6th weeks after adenovirus injection.(4) The mRNA expression of IL-18 and TRB3 were measured by quantification real-time RT-PCR.(6) The protein expression of IL-18 and IL-18-induced signaling pathway in adipose tissue were analyzed by western blot.Results1.The experimental animalsThree rats of fructose group died in the entire experiment,none of control group died. A total of 42 rats finished the study,12 rats in Con group,9 rats in Fru group,9 rats in Ad-Con group and 12 rats in Ad-IL-18 group.2.Comparison of metabolic variables between Con and fructose-fed group before adenovirus transfectionChronic administration of fructose as a 10%drinking solution during 32 weeks induced a cluster of metabolic abnormalities.Fructose-fed rats had higher body weight and systolic BP than the Con rats.Compared with the Con group,three other variables including fasting levels of plasma triglycerides,plasma insulin,and HOMA-R were significantly increased in fructose-fed group,without significant differences in total cholesterol and glucose,both of which were although slightly increased.3.Changes of serum IL-18 after adenoviral transfectionAd-IL-18 group showed a significant change with time,and the other three groups had no changes in serum IL-18 levels after adenovirus transfection.Fructose-fed rats had higher serum IL-18 levels than Con rats.Serum IL-18 levels were higher in Ad-IL-18 group than in Ad-Con group at 1st week, without significant difference at 0 week after transfection.Serum IL-18 levels,which reached to peak value at 2nd week,were about 4-fold higher in the Ad-IL-18 group than in the Ad-Con group.At 3rd week,Ad-IL-18 group showed a significant decline tendency,but a significant increase in serum IL-18 levels compared with the Ad-Con group.There was no significant difference in IL-18 levels between the two groups at 4th and 6th week.4.Effect of IL-18 on metabolic parameters in ratsAfter IL-18 transfection for 6 weeks,insulin and HOMA were significantly increased in IL-18 vector group compared to vehicle group;other parameters showed no significant difference.Insulin and HOMA-IR were significantly increased in Ad-IL-18 group after transfection for 6 weeks compared to the levels of insulin and HOMA-IR before transfection;other parameters showed no significant difference.5.IL-18 and TRB3 mRNA expression by RT-PCRIn the fructose-fed groups,the mRNA expression levels of IL-18 and TRB3 in adipose tissue were significantly increased compared with those in the Con group.IL-18 and TRB3 mRNA levels in Ad-IL-18 group were markedly higher than in both Fru group and Ad-Con group,without significant differences between the Fru and Ad-Con groups6.Protein expression of IL-18 and IL-18 induced signaling pathway by westein blot(1) Comparison of IL-18 protein expression in adipose tissue of rats Compared to Con group,the three fructose-fed groups showed a significant rise in IL-18 protein content in adipose tissue.IL-18 protein content in the Ad-IL-18 group was significantly higher than in both the Fru group and the Ad-Con group.IL-18 protein content in the Fru group was similar to that in the Ad-Con group.(2) Comparison of IRS-1 protein expression in adipose tissue of ratsWestern blot analysis showed that IRS-1 protein expression was significantly decreased in adipose tissue of fructose-fed rats compared with the Con rats.The rats treated with Ad-IL-18 had significantly lower IRS-1 protein content in comparison with Fru and Ad-Con rats.But IRS-1 protein had no significant difference between the Fru group and the Ad-Con group.(3) Comparison of p85/P-p85 protein expression in adipose tissue of ratsIn adipose tissue,there was no significant difference among the 4 groups in the p85 regulatory subunit of PI3K(T-p85) protein expression.Phospho-p85(P-p85) protein expression was markedly reduced in adipose tissue of fructose-fed rats compared with that of Con rats.The P-p85 protein expression of the Ad-IL-18 group was significantly reduced compared with that of the Fru and the Ad-Con groups,without significant difference between Fru group and Ad-Con group.(4) Comparison of Akt/P-Akt protein expression in adipose tissue of ratsIn adipose tissue,total-Akt(T-Akt) protein expression was similar among the 4 groups.In adipose tissue,phospho-Akt(Ser473) protein expression was markedly lower in fructose-fed groups than Con group.Rats treated with Ad-IL-18 had markedly lower P-Akt protein expression than both the Fru and Ad-Con rats.P-Akt protein expression in the Fru group was similar to that in the Ad-Con group.(5) Comparison of TRB3 protein expression in adipose tissue of rats In the fructose-fed groups,the mRNA expression levels of TRB3 in adipose tissue were significantly increased compared with those in the Con group.TRB3 mRNA levels in the Ad-IL-18 group were markedly higher than in both Fru group and Ad-Con group.However,there were no significant differences in mRNA levels of TRB3 between the Fru and the Ad-Con group.Conclusion 1.Inhibition of IRS-1/PI-3K signal and upregulation of TRB3 expression induce suppression of Akt activation in adipose tissue of fructose-fed rats,suggesting defects in IRS-1/PI-3K/Akt and TRB3/Akt pathway in adipose tissue are involved in the development of insulin resistance.2.After IL-18-adenovirus was administered,serum and adipose tissue expression of IL-18 were high,and serum insulin and HOMA-IR were further increased,which suggests that IL-18 promotes insulin resistance in fructose-induced MS rats.3.Ad-IL-18 group showed marked inhibition of IRS-1/PI-3K signal and upregulation of TRB3 expression and more significant suppression of Akt activation in adipose tissue,which suggests that IL-18 promotes insulin resistance through suppression of Akt activation via suppression of PI-3K signaling pathway and upregulation of TRB3 in adipose tissue.