Role And Mechanism Of Chemerin On Insulin Resisitance In Cardiomyocyte

Diabetes mellitus (DM) is a metabolic disorder characterized by elevatedblood glucose secondary to insulin resistance that results in many seriouscomplications，such as diabetic nephropathy, diabetic retinopathy and diabeticcardiomyopathy. A large body of experimental evidence supports the notionthat adipokines have a significant influence on glucose metabolism in varioustissues. As an endocrine organ，adipose tissue could secrete a variety of fatfactors, such as adiponectin, leptin, visfatin, IL-6, and so on. Chemerin (alsoknown as retinoicacid receptor responder protein2and tazarotene-inducedgene2) is a recently discovered adipokine that is associated with inflammation,adipogenesis, and insulin resisitance. Studies have previously shown thatchemerin and its receptor, chemokine-like receptor1(CMKLR1, or ChemR23)are expressed in many tissues and particularly highly expressed in whiteadipose tissue, liver and kidney. A high level of circulating chemerin inhumans is considered to be a marker of inflammation and metabolic syndrome.These observations suggest that chemerin may be involved in insulinresistance and the development of DM.Although the evidence described above demonstrates the influence ofchemerin on glucose homeostasis, at present the precise role and significanceof chemerin is unclear in different cell types. A previous study showed thathuman skeletal muscle cells do not express chemerin but do express CMKLR1,and chemerin impairs insulin signaling and induces insulin resistance inskeletal muscle cells. However, there were conflicting results in3T3-L1adipocytes. One study showed that chemerin induces insulin resistance,whereas another study showed that high levels of chemerin enhance insulinsignaling and glucose uptake in3T3-L1adipocytes.Chemerin and CMKLR1have been shown to be expressed in rat heart tissue as well. However, no studies to date have described the presence ofchemerin in rat cardiomyocytes in vitro or the role of chemerin in insulinresistance. Diabetic cardiomyopathy is one of the serious cardiovascularcomplications of long-term DM. Therefore, we investigated the possibleinterplay between the chemerin/ChemR23system and insulin resistance in ratcardiomyocytes in vitro. The objective of this study was to clarify the role andmolecular biological mechanisms of chemerin on insulin resistance in ratcardiomyocytes. It follows by four parts:Part1The expression of chemerin in rat cardiomyocytes and cardiacfibroblastsObjectives: To explore whether cardiomyocytes and cardiac fibroblastscan express chemerin, lay the foundation for the further study.Methods: Primary cardiomyocytes and cardiac fibroblasts were isolatedfrom the ventricles of three-day-old neonatal Sprague-Dawley rats. Chemerinexpression in cardiomyocytes and cardiac fibroblasts were measured byreal-time PCR and Western-Blot.Results: The expression of chemerin mRNA were detected incardiomyocytes and cardiac fibroblasts. The expression of chemerin proteinwere detected in cardiomyocytes and cardiac fibroblasts.Conclusions: Rat cardiomyocytes and cardiac fibroblasts can expresschemerin.Part2Changes of chemerin expression in rat cardiomyocytes in highglucose and inflammatory environmentObjectives: To observe the effects of high glucose and inflammatoryfactor TNF-α on chemerin mRNA expression in rat cardiomyocytes.Methods: Ventricular cardiomyocytes were dissociated from the2to3-day old neonatal SD rats.48hours after seeding, the cardiomyocytes werecultured with the serum-free culture medium for another24hours.1To test the variation of chemerin mRNA expression after glucosestimulation in cardiomyocytes:(1)The cells were treated with increasingconcentrations of D-glucose (5.5,10,20,30, and40mmol/L) and hypertonic control (5.5mmol/L D-glucose+34.5mmol/L mannitol) for24hours. Theexpression of chemerin mRNA were measured by real-time PCR.(2)Thecardiomyocytes were then treated with high glucose (30mmol/L) for differentdurations (0,6,12,24, and48hours). The expression of chemerin mRNAwere measured by real-time PCR.2To test the variation of chemerin mRNA expression afteradministration of TNF-α:(1)The cardiomyocytes were treated with TNF-α (0,5,10,20ng/ml) for24hours. The expression of chemerin mRNA weremeasured by real-time PCR.(2)The cardiomyocytes were then treated withTNF-α (20ng/ml) for different durations (0,6,12,24, and48hours). Theexpression of chemerin mRNA were measured by real-time PCR.Results:1Chemerin mRNA expression was upregulated by administration ofhigh glucose.(1)The expression of chemerin mRNA increased in adose-dependent manner up to30mmol/L glucose (P<0.05), and chemerinmRNA expression was slightly, but not significantly, lower with40mmol/Lglucose (P>0.05). Interestingly, compared with that the control (treated with5.5mmol/L D-glucose), chemerin expression was not significantly increasedin the hypertonic control group (P>0.05).(2)Chemerin mRNA levelsincreased in a time-dependent manner peaking at24hours of treatment andthen significantly decreasing by48hours (P<0.05).2Chemerin mRNA expression was upregulated by administration ofTNF-α.(1)The expression of chemerin mRNA increased in a dose-dependentmanner compared with the control, and the increases were significantly in thegroups of10g/ml and20ng/ml (P<0.05).(2)Chemerin mRNA levels increasedin a time-dependent manner peaking at24hours of treatment and thendecreasing by48hours (P<0.05).Conclusions: Chemerin mRNA expression was upregulated by highglucose and inflammatory environment in rat cardiomyocytes.Part3Chemerin induced insulin resistance in cardiomyocytesObjectives: To study whether chemerin can impaire insulin signaling and induce insulin resistance in cardiomyocytesMethods: Ventricular cardiomyocytes were dissociated from the2to3-day old neonatal SD rats.48hours after seeding, the cardiomyocytes werecultured with the serum-free culture medium for another24hours. Afterthat,cardiomyocytes were cultured with recombinant rat chemerin (0,10, and100ng/ml) for24hours, with chemerin still in the media, cardiomyocytes wereexposed insulin (10-7mol/L) for30min. The phosphorylation of Akt, IRS-1and AMPKα were measured by Western blot analysis. Glucose uptake wasevaluated using a fluorescence microplate reader.Results: The cardiomyocytes showed a marked and dose-dependentdecrease both in basal and insulin-stimulated phosphorylation of Akt uponadministration of chemerin (P<0.05). Upstream of Akt, chemerin significantlyand dose-dependently increased the basal and insulin-stimulated serinephosphorylation of IRS-1(P<0.05).Glucose uptake and phosphorylation of AMPKα (Thr172) weresignificantly decreased compared with basal levels and that of theinsulin-stimulated control (P<0.05).Conclusions: Chemerin induced insulin resistance in cardiomyocytes.Part4The possible molecular mechanism of chemerin on insulinresistance in cardiomyocytes.Objectives: Investigating the possible molecular mechanism of chemerinon insulin resistance in cardiomyocytes.Methods: Ventricular cardiomyocytes were dissociated from the2to3-day old neonatal SD rats.48hours after seeding, the cardiomyocytes werecultured with the serum-free culture medium for another24hours.1To investigate which intracellular signaling pathways are important inchemerin-mediated insulin resistance, cardiomyocytes were pretreated with orwithout chemerin (100ng/ml) for24hours before acute stimulation withinsulin (10-7mol/l,30min). The phosphorylation of p38MAPK, ERK-1/2andJNK were measured by Western blot analysis.2To analyze the role of the ERK1/2pathway in the impairment of insulin signaling by chemerin, cardiomyocytes were pre-cultured with thespecific ERK inhibitor PD98059(50umol/l) for15min before startingadministration of chemerin (100ng/ml) for24hours. The groups were asfollows:①Blank control group;②10-7mol/l insulin stimulation for30min;③100ng/ml chemerin stimulation for24hours;④100ng/ml chemerinstimulation for24hours, then10-7mol/l insulin stimulation for30min;⑤PD98059(50umol/l) pre-stimulation for15min,100ng/ml chemerinstimulation for24hours;⑥PD98059(50umol/l) pre-stimulation for15min,100ng/ml chemerin stimulation for24hours, then10-7mol/l insulin stimulationfor30min. The phosphorylation of Akt and AMPKα were measured byWestern blot analysis. Glucose uptake was evaluated using a fluorescencemicroplate reader.Results:1Chemerin activated p38MAPK and ERK1/2signaling pathways ininsulin-stimulated cardiomyocytes:p38MAPK phosphorylation was not significantly increased in the basalstate (P>0.05) but still significantly increased in the insulin-stimulated state(P<0.05). However, chemerin increased both the basal and insulin-stimulatedphosphorylation of ERK1/2(P<0.05). Interestingly, chemerin had no effect onJNK activity (P>0.05).2ERK1/2inhibition partially restored insulin sensitivity inchemerin-treated cardiomyocytes:The phosphorylation of Akt was significantly increased compared with thosein the insulin-stimulated control with chemerin, but still significantlydecreased compared with those in the insulin-stimulated control withoutchemerin (P<0.05). Meanwhile, the phosphorylation of AMPKα and glucoseuptake had the similar changes.Conclusions: Chemerin activated p38MAPK and ERK1/2signalingpathways in insulin-stimulated cardiomyocytes, and chemerin induced insulinresistance part of by activating ERK1/2signaling pathway in cardiomyocytes.