Study Of Fucosylated Chondroitin Sulfate Isolated From Sea Cucumber On Improving Insulin Resistance Effects And Mechanism

Sulphated polysaccharide from sea cucumber is one of the significant bioactivesubstances in the bodywall of sea cucumber with a content of7%-10%. They contain twocompoents: fucan from sea cucumber (SC-FUC) and fucosylated chondroitin fulfate fromsea cucumber (SC-CHS). SC-CHS is a kinde of acid mucopolysaccharides with achondroitin sulfate-E backbone with branches of sulfated fucose. It exhibitedanticoagulation, antithrombosis, and antiumor. The reports about its effects on insulinresistant intervention were few. Therefore, this paper investigated the effects andmechanism of SC-CHS isolated form Cusumaria frondosa and Acaudina molpadioides oninsulin resistant improvement in vitro and in vito. The main results of these studies are asfollows:The chemical composition of SC-CHS consisted mainly of glucuronic acid,galactosamine, and fucose. The structures of SC-CHS isolated from different sea cucumberare significantly different. Cf-CHS from Cusumaria frondosa consisted mainly of thesethree monosaccharide in a molar ratio of1︰1.5︰1.16with sulphate contents at30.07%and14.76kDa average molecular weitht, while they are1︰1.14︰1.55in Am-CHS fromAcaudina molpadioides with sulphate contents at27.81%and21.53kDa averagemolecular weitht.The effects and mechanism of Cf-CHS and Am-CHS were studied, using a3T3-L1insulin resistant adipocyte model established by TNF-α introduction in vitro. Resultsshowed that Cf-CHS and Am-CHS could both increase glucose transportation in a basicand insulin stimulated states, and enhance the effects of RSG. The effects of Am-CHS were better than Cf-CHS. The key genes protein expression in PI3K/PKB insulin signalingpathway and ERK protein expression and phosphorylation were analysed using Westernblot. The results showed that Cf-CHS and Am-CHS unchanged the total protein expressionof PI3K, PKB, ERK, and GLUT4. However, phosphorylated p85regulatory subunit ofPI3K, serine473and threonine308loci of PKB, and increased GLUT4expression in cellsurface were increased by Cf-CHS and Am-CHS treatment. In addition, Cf-CHS andAm-CHS also significantly increased phosphorylation of ERK1/2. It indicated that Cf-CHSand Am-CHS could promote glucose transopt and improve insulin resistance viaup-regulation PI3K/PKB signling and activation ERK in insulin resistant model cells.The effects and mechanisms of Cf-CHS and Am-CHS on improvement insulinresistance were verified using PI3K inhibitor wortmannin intervened, PKB inhibitorMK2206intervened, or ERK inhibitor U0126intervention, respectively. Results showedthat Cf-CHS and Am-CHS could both reverse the inhibition of the three inhibitors onglucose transport. Western blot showed that Cf-CHS and Am-CHS significantly increasedp-Ser473-PKB, p-Thr308-PKB, p-ERK1/2, and m-GLUT4expressions in insulin resistantmodel cells under wortmannin intervention, through p-p85PI3K expression was unchanged.These indicated that SC-CHS can promote GLUT4transolation via activation PKB andERK, ranther than via activation PI3K. The expression of p-Ser473-PKB, p-Thr308-PKB,and m-GLUT4were increased under MK2206intervention when treated with Cf-CHS andAm-CHS. It indicated that SC-CHS can increase GLUT4translocation throughup-regulating PI3K/PKB signling. The expressions of p-ERK1/2and m-GLUT4underU0126were also increased by Cf-CHS and Am-CHS treatment. It indicated that SC-CHSalso increase GLUT4translocation through activating ERK. These results suggested thatCf-CHS and Am-CHS increased GLUT4translocation and improved insulin resistance viaup-regulation PI3K/PKB signling and activation ERK. SC-CHS play the role throughactivating PKB and ERK directly without PI3K, that is said their target genes are PKB andERK, ranther than PI3K.This paper further verified the improvement insulin resistance effects of Cf-CHS and Am-CHS in vivo. The insulin resistant model was established by feeding a high-fathigh-sucrose diet using male C57BL/6J mice. After feeding19w, Cf-CHS and Am-CHScould both significantly reduce fasting blood glucose level, improve glucose tolerance,decrease insulin level and HOMA-IR value, increase QUICKI score, lower body weightand adipose weight, increase serum adiponectin level, reduce serum resistin, leptin, andTNF-α contents. SC-CHS could weaken the side effects of RSG on body weight gain andadipopexis, and synergistically enhance the other effects. The effects of Am-CHS werebetter than Cf-CHS. These indicated that Cf-CHS and Am-CHS could remarkably improveinsulin resistance intervention in vivo.The relatively insufficient insulin secreted by pancreatic islets cells is the maincharacteristic of insulin resistance. The most direct reason is pancreatic islets cellsapoptosis induced by high concentration of glucose. In mammal, mitochondria pathway isthe primary pathway of cells apoptosis.In order to study the effects of SC-CHS on protection pancreatic islets in insulinresistant mice, the microstructure changes of pancreatic islets were observed using anoptical microscope. Results demonstrated that Cf-CHS and Am-CHS could lower HFSDinduced pancreatic islets injure, and its morphology was nearly normal level with the clearbasement-membrane and connective tissue in the combination group mice. It indicated thatCf-CHS and Am-CHS could singificantly inhibited pancreatic islets apoptosis. Themechanism of SC-CHS protection pancreatic islets was further investigated. RT-PCRresults showed that Cf-CHS and Am-CHS could significantly down-regulate Bid, Bax,cytochrome c, caspase9, and caspase3mRNA expressions, and up-regulate Bcl-2andBcl-xL mRNA expressions. Western blot results showed that Cf-CHS and Am-CHS couldmarkedly increase t-Bid, Bax, caspase9, and caspase3protein expressions, enhancecytochrome c release form mitochondria, inhibit Bcl-2and Bcl-xL protein expressions.These effects were strengthened when SC-CHS combined with RSG. These indicated thatCf-CHS and Am-CHS could inhibit pancreatic islets apoptosis and improve insulinresistance via blocking mitochondrial pathway at transcriptional and post-transcriptional levels in insulin resistant mice.Skeletal muscle and adipose tissues are the main target organs to dispose glucose inbodies, and skeletal muscle occupies75%. PI3K/PKB singling pathway is the keyapproach of glucose transport into cells. In order to discuss the mechanism of insulinresistance intervention and glucose translocation in vivo, the paper further analyzed thekey genes mRNA and protein expressions in PI3K/PKB signaling mediated by insulin inskeletal muscle and adipose tissues using qRT-PCR and western blot. Results demonstratedthat Cf-CHS and Am-CHS could remarkably increase serum adiponectin level, and lowerresistin, leptin, and TNF-α levels. qRT-PCR results showed that Cf-CHS and Am-CHScould significantly increase IR, IRS-1, PI3K, PKB, GLUT4mRNA expressions in skeletalmuscle and adipose tissues in model mice. Western blot results showed that Cf-CHS andAm-CHS could significantly promote p-Tyr-IR-β, p-Tyr612-IRS-1, p-p85-PI3K,p-Ser473-PKB, p-Thr308-PKB, and m-GLUT4protein expression in skeletal muscle. Theyalso increased p-Tyr612-IRS-1, p-p85-PI3K, p-Ser473-PKB, and m-GLUT4proteinexpressions, and decreased p-Ser308-IRS-1protein expression. The combination couldsynergistically enhance these effects, and Am-CHS were better than Cf-CHS. Theseindicated that Cf-CHS and Am-CHS could activate PI3K/PKB signaling at transcriptionaland translational levels, enhance GLUT4translocation, reduce blood glucose level, andimprove insulin resistance.Glucose metabolism is closely related to glucose homeostasis. Glycogen synthesis isanother approach of insulin to dispose glucose. For studying glucose metabolism andglycogen synthesis effects of SC-CHS, on the basis of previous research, hepatic glycogencontent, glycogen synthesis-related enzymes hexokinase (HK) and pyruvate kinase (PK),gluconeogenesis-related enzymes glycogen phosphorylase (GP) and glucose-6-phosphatase(G6Pase) were detected, and the pivotal genes mRNA expressions in PKB/GSK-3βsignaling were investigated usig qRT-PCR in the liver of insulin resistant mice. Resultsshowed that Cf-CHS and Am-CHS could significantly increase hepatic glycogen content,enhance HK and PK activities, and decrease PK and G6Pase activities. qRT-PCR results showed that SC-CHS could markedly increase IR, IRS-2, PI3K, PKB, and GS mRNAexpressions, lower GSK-3β mRNA expression, which negatively correlated to glycogensynthesis, in the liver of model mice. These effects were further enhanced when SC-CHScombined with RSG, and the effects of Am-CHS were better. These indicated that Cf-CHSand Am-CHS could promote glucose metabolism and glycogen synthesis via regulation onhepatic glucose metabolism-related enzymes and activation on PKB/GSK-3β signaling attranscriptional level.Firstly, this paper systematically studied insulin resistant intervention in vitro and invivo, clarified the mechanisms of SC-CHS on inhibiting pancreatic islets apoptosis,promoting glucose translocation, enhancing glucose metabolism and glycogen synthesis.For the first time, the paper also reported SC-CHS not only synergistically strengthened theeffects of RSG, but also weakened the side effects of RSG. These provide theoretical basisfor use of SC-CHS on functional food, high-value processing and utilization of seacucumber, or replacement RSG with SC-CHS partly.