Vpliv alopurinola na delovanje z AMP aktivirane protein kinaze in inzulina v kulturi skeletnomišičnih celic L6

Božič, Meta (author), Pirkmajer, Sergej (mentor), Miš, Katarina (comentor)

Uvod: Z AMP aktivirana protein kinaza (AMPK) ima pomembno vlogo pri uravnavanju presnovnih procesov in ohranjanju energijske homeostaze v skeletnih mišicah. Aktivacija skeletnomišične AMPK spodbudi privzem glukoze neodvisno od inzulina, s čimer zaobide okvare v inzulinski signalizaciji pri sladkorni bolezni tipa 2. Inzulinska rezistenca v skeletnih mišicah znatno prispeva k razvoju sladkorne bolezni tipa 2, zato predstavljajo farmakološki aktivatorji skeletnomišične AMPK obetavni pristop zdravljenja presnovnih obolenj. Metotreksat in druge zdravilne učinkovine s prijemališčem delovanja v presnovnih poteh purinskih nukleotidov so se izkazale učinkovite pri aktivaciji AMPK. Namen: AICAR, farmakološki aktivator AMPK, se presnavlja v sečno kislino, ki je neodvisni dejavnik tveganja nastanka inzulinske rezistence, sladkorne bolezni tipa 2 in številnih drugih obolenj. Sočasna aplikacija alopurinola, zaviralca ksantin oksidoreduktaze, bi zmanjšala obremenitev pacientov s sečno kislino. Namen magistrske naloge je bil raziskati vpliv alopurinola na delovanje AMPK in inzulina v skeletnomišičnih celicah L6. Hipoteze: 1) Alopurinol ne aktivira AMPK, ampak ojača z AICAR spodbujeno aktivacijo AMPK v skeletnomišičnih celicah L6; 2) Alopurinol v prisotnosti metotreksata ne vpliva na z AICAR spodbujeno aktivacijo AMPK v skeletnomišičnih celicah L6; 3) Alopurinol ne vpliva na z inzulinom spodbujeno aktivacijo Akt in ERK1/2 v skeletnomišičnih celicah L6; 4) Alopurinol zveča z AICAR spodbujen privzem glukoze, a ne vpliva na z inzulinom spodbujen privzem glukoze v skeletnomišičnih celicah L6. Metode: Poskuse smo opravili na kulturi skeletnomišičnih celic L6. Aktivacijo AMPK smo spremljali z metodo prenos western, tako da smo merili fosforilacijo AMPK (Thr172) in acetil-CoA karboksilaze (ACC, Ser79). Določili smo tudi fosforilacijo kinaz Akt (Ser473) in ERK1/2 (Thr202/Tyr204). Presnovne učinke smo ovrednotili z merjenjem privzema 3H-2-deoksiglukoze. Izražanje ksantin oksidoreduktaze v vzorcih skeletnomišičnih celic in tkiv smo ocenili s kvantitativno verižno reakcijo s polimerazo (PCR) v realnem času. Rezultati: Alopurinol (100 µM) samostojno ali v kombinaciji z AICAR ni aktiviral skeletnomišične AMPK. Prav tako alopurinol ni vplival na z AICAR spodbujeno fosforilacijo AMPK ob prisotnosti metotreksata. Alopurinol je zmanjšal z inzulinom spodbujeno fosforilacijo Akt, na z inzulinom spodbujeno fosforilacijo ERK1/2 pa ni imel vpliva. Alopurinol ni vplival na z AICAR ali z inzulinom spodbujen privzem glukoze v skeletnomišične celice. Zaključek: 1) Alopurinol ni aktiviral AMPK ali ojačal z AICAR spodbujene aktivacije AMPK. 2) Alopurinol ob prisotnosti metotreksata ni vplival na z AICAR spodbujeno aktivacijo AMPK. 3) Alopurinol je zavrl z inzulinom spodbujeno aktivacijo Akt, na z inzulinom spodbujeno aktivacijo ERK1/2 pa ni vplival. 4) Alopurinol ni vplival na z AICAR ali z inzulinom spodbujen privzem glukoze v skeletnomišične celice. Naši rezultati torej podpirajo drugo hipotezo. Prva, tretja in četrta hipoteza pa so le delno skladne s pridobljenimi rezultati.

• Language: Slovenian
• Keywords: alopurinol, hiperurikemija, AMPK, sladkorna bolezen tipa 2, skeletne mišice.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-136302
• Publication date in RUL: 23.04.2022

Secondary language

• Language: English
• Title: The influence of alopurinol on the action of AMP-activated protein kinase and insulin in cultured L6 skeletal muscle cells

Abstract

Background: AMP-activated protein kinase (AMPK) has a significant role in regulating metabolic processes and maintaining energy homeostasis in skeletal muscle. AMPK activation in skeletal muscle stimulates glucose uptake independently of insulin action thereby bypassing defects in insulin signaling. Insulin resistance in skeletal muscle considerably contributes to development of type 2 diabetes, therefore pharmacological activators of AMPK in skeletal muscle present a promising strategy for its treatment. Methotrexate and various other compounds that target purine metabolic pathways were shown to promote AMPK activation. Aim: AICAR, a pharmacological activator of AMPK, is metabolized to uric acid, which is an independent risk factor of development insulin resistance, type 2 diabetes and numerous other diseases. AICAR application combined with alopurinol, inhibitor of xanthine oxidoreductase, reduces the plasma levels of uric acid. The aim of the master thesis was to investigate the effect of alopurinol on the action of AMPK and insulin in L6 skeletal muscle cells. Hypotheses: 1) Alopurinol does not activate AMPK, but enhances the AICAR-induced AMPK activation in L6 skeletal muscle cells. 2) In the presence of methotrexate alopurinol does not affect the AICAR-induced AMPK activation in L6 skeletal muscle cells. 3) Alopurinol does not affect the insulin-induced Akt and ERK1/2 phosphorylation in L6 skeletal muscle cells. 4) Alopurinol enhances the AICAR-induced glucose uptake, but does not affect the insulin-induced glucose uptake in L6 skeletal muscle cells. Methods: To investigate our hypotheses we used cultured L6 skeletal muscle cells. AMPK activation was monitored with western blot by measuring the phosphorylation of AMPK (Thr172) and acetyl-CoA carboxylase (ACC, Ser79). We also determined the phosphorylation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204). Metabolic effects were evaluated by estimating the upake of 3H-2-deoxyglucose. The mRNA expression of xanthine oxidoreductase was estimated with quantitative real-time PCR. Results: Alopurinol itself (100 µM) or in combination with AICAR did not promote the activation of AMPK in skeletal muscle cells. Alopurinol also did not affect the AICAR-induced AMPK phosphorylation in the presence of methotrexate. In addition, alopurinol reduced the insulin-induced phosphorylation of Akt, but did not affect the phosphorylation of ERK1/2. Finally, alopurinol did not affect the AICAR or insulin-induced glucose uptake in skeletal muscle cells. Conclusion: 1) Alopurinol did not activate AMPK or enhanced AICAR-induced AMPK activation. 2) In the presence of methotrexate alopurinol did not affect the AICAR-induced AMPK activation. 3) Alopurinol reduced the insulin-induced Akt activation, but did not affect the ERK1/2 activation. 4) Alopurinol did not affect the AICAR or insulin-induced glucose uptake in skeletal muscle cells. Our results therefore, support the second hypothesis. The first, third and fourth hypothesis are only partially consistent with the obtained results.

• Keywords: alopurinol, hyperuricemia, AMPK, type 2 diabetes, skeletal muscles.