Učinek sulfasalazina na inzulinsko signalno pot v skeletni mišici

Vidović, Anja (author), Pirkmajer, Sergej (mentor), Miš, Katarina (co-mentor)

Uvod: Skeletne mišice so pomembno tarčno tkivo pri zdravljenju sladkorne bolezni tipa 2, saj predstavljajo glavno mesto z inzulinom stimuliranega privzema glukoze v postprandialnem stanju. Pri preučevanju delovanja signalnih poti, ki vplivajo na inzulinsko rezistenco, lahko izhajamo iz salicilne kisline. Njeni derivati imajo poleg protivnetnih učinkov, ugodne učinke na delovanje inzulina in presnovo glukoze, tudi zaradi aktivacije z AMP-aktivirane protein kinaze. Namen: Sulfasalazin je predzdravilo iz 5-aminosalicilne kisline in sulfapiridina. Klinični primeri in živalski modeli so nakazali, da bi lahko vplival na koncentracijo glukoze ter zaviral nastanek zapletov sladkorne bolezni. Ker njegov mehanizem na molekularni ravni pri diabetesu ni raziskan, smo želeli ugotoviti, ali učinkuje na inzulinsko signalno pot prek kinaze Akt. Hipoteze: 1) Sulfasalazin poveča bazalno in z inzulinom ali rastnim dejavnikom iz trombocitov (PDGF) spodbujeno fosforilacijo kinaze Akt. 2) Sulfasalazin poveča bazalno in z inzulinom ali s PDGF spodbujeno fosforilacijo AS160. 3) Sulfasalazin ne vpliva na z inzulinom ali s PDGF spodbujeno fosforilacijo kinaze ERK1/2. Metode: Učinke sulfasalazina na znotrajcelično signalizacijo smo preučili na in vitro modelu linije podganjih skeletnomišičnih celic L6. Aktivnost signalnih poti smo ovrednotili z merjenjem fosforilacije proteinov: Akt (Ser473), AS160 (Ser588), ERK1/2 (Thr202/Tyr204), 4E-BP1 (Thr37/Thr46) in acetil-CoA karboksilaze (Ser79). Uporabili smo metodo gelske elektroforeze, metodo prenosa western in zaznali proteine z metodo ojačane kemiluminiscence. Rezultati: Sulfasalazin je zmanjšal z inzulinom spodbujeno fosforilacijo Akt, ni pa vplival na bazalno niti s PDGF spodbujeno fosforilacijo Akt. Sulfasalazin je inhibiral fosforilacijo AS160, tudi ob prisotnosti inzulina ali PDGF. S tem smo zavrgli prvo in drugo hipotezo. Sulfasalazin je zmanjšal s PDGF spodbujeno fosforilacijo ERK1/2, s čimer smo delno podprli tretjo hipotezo. Zaključki: Ugotovili smo, da sulfasalazin zavira z inzulinom in s PDGF spodbujeno fosforilacijo AS160. Kljub temu da naj bi sulfasalazin deloval kot farmakološki aktivator z AMP-aktivirane protein kinaze, predvidevamo, da do translokacije GLUT4 na površino mišičnih celic ne bi prišlo, saj je fosforilacija AS160 pri tem ključen dogodek. Potrebne bi bile dodatne raziskave, s katerimi bi ugotovili, zakaj sulfasalazin zmanjšuje fosforilacijo AS160.

• Language: Slovenian
• Keywords: sulfasalazin, sladkorna bolezen tipa 2, inzulin, PDGF, skeletna mišica
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-109492
• Publication date in RUL: 04.09.2019

Secondary language

• Language: English
• Title: Effect of sulfasalazine on the insulin signalling pathway in skeletal muscle

Abstract

Background: Skeletal muscles are important target tissues for treatment of type 2 diabetes, as they represent a main site of insulin stimulated glucose uptake in the postprandial state. When examining the signalling pathways related to insulin resistance, we can turn our focus on salicylic acid. Its derivatives not only have anti-inflammatory properties, but also beneficial effects on insulin and metabolism of glucose also because of activation of AMP-activated protein kinase. Aim: Sulfasalazine is a prodrug from 5-aminosalicylic acid and sulfapyridine. Some clinical reports and animal studies suggested that sulfasalazine could have beneficial effect on concentration of glucose in plasma and prevent the complications of diabetes. Our aim was to determine whether sulfasalazine modulates insulin signalling via Akt kinase. Hypotheses: 1) Sulfasalazine promotes basal and insulin- or PDGF-stimulated phosphorylation of Akt. 2) Sulfasalazine promotes basal and insulin- or PDGF-stimulated phosphorylation of AS160. 3) Sulfasalazine does not alter insulin- or PDGF-stimulated phosphorylation of ERK1/2. Methods: We examined the effects of sulfasalazine by using in vitro model of L6 rat skeletal muscle cells. Activation of signalling pathways was assessed by measuring phosphorylation of kinase Akt (Ser473), AS160 (Ser588), ERK1/2 (Thr202/Tyr204), 4E-BP1 (Thr37/Thr46), acetyl-CoA carboxylase (Ser79), using polyacrylamide gel electrophoresis, western blotting and detection of specific proteins with chemiluminescent imaging method. Results: Sulfasalazine inhibited the insulin-stimulated phosphorylation of Akt, but it did not alter basal or PDGF-stimulated phosphorylation. Sulfasalazine inhibited the phosphorylation of AS160, also in the presence of insulin or PDGF. These results do not support our first and second hypothesis. Sulfasalazine inhibited PDGF-stimulated phosphorylation of ERK1/2, what partially support the third hypothesis. Conclusions: We found out that sulfasalazine inhibits insulin- and PDGF-stimulated phosphorylation of AS160. Even though sulfasalazine should act as an activator of AMP-activated protein kinase, our results suggest that there is no translocation of GLUT4 on the surface of muscle cells, because phosphorylation of AS160 is the key event in the insulin signalling pathway leading to increased glucose uptake. Additional research should be done to uncover molecular mechanisms by which sulfasalazine suppresses phosphorylation of AS160.

• Keywords: sulfasalazine, type 2 diabetes, insulin, PDGF, skeletal muscle