Vpliv inzulina in adrenalina na uravnavanje z AMP aktivirane protein kinaze (AMPK) v kulturi skeletnomišičnih celic

Požun, Tina (avtor), Pirkmajer, Sergej (mentor)

Uvod: Pri uravnavanju normalne koncentracije glukoze v krvi sodelujejo skeletne mišice, presnovno eno najbolj aktivnih tkiv in pomembno mesto od inzulina odvisnega privzema glukoze po obroku. Za ohranjanje energijske homeostaze je pomembna z AMP aktivirana kinaza (AMPK). AMPK je tudi zanimiva tarča za razvoj novih oblik zdravljenja inzulinske rezistence in sladkorne bolezni tipa 2, saj njena aktivacija izboljša homeostazo glukoze. Namen: AMPK se aktivira alosterično in s fosforilacijo Thr172 na katalitični podenoti α, medtem ko fosforilacija Ser485 njeno aktivnost zmanjša. Namen naloge je bil preveriti, kako inzulin in adrenalin vplivata na delovanje AMPK oziroma farmakoloških aktivatorjev AMPK v skeletnomišičnih celicah. Hipoteze: 1) Inzulin zmanjša aktivnost AMPK v skeletnomišičnih celicah L6 tako, da zavre fosforilacijo AMPKα Thr172 in spodbudi fosforilacijo AMPKα Ser485. 2) Adrenalin poveča aktivnost AMPK v skeletnomišičnih celicah L6, saj spodbudi fosforilacijo AMPKα Thr172 in zavre fosforilacijo AMPKα Ser485. 3) Inzulin zmanjša, adrenalin pa poveča učinke aktivatorja AMPK A-769662 na fosforilacijo AMPKα Thr172 in Ser485. 4) Neposredni in posredni farmakološki aktivatorji AMPK v skeletnomišičnih celicah L6 povečajo fosforilacijo Thr172 in hkrati zmanjšajo fosforilacijo Ser485 na podenoti AMPKα. 5) Adrenalin kljub aktivaciji AMPK zmanjša bazalni in z inzulinom spodbujen privzem glukoze v skeletnomišičnih celicah L6. Metode: Uravnavanje AMPK smo raziskovali na podganji skeletnomišični celični liniji L6. Z metodo prenosa western smo analizirali fosforilacijo AMPKα Thr172 in Ser485. Aktivnost AMPK smo določili posredno, z analizo fosforilacije acetil-CoA karboksilaze (Ser79), ki je neposredna tarča AMPK. Privzem glukoze v celice smo izmerili s [3H]-2-deoksiglukozo. Rezultati: Potrdili smo prvo hipotezo, saj je inzulin spodbudil fosforilacijo AMPKα Ser485, zavrl fosforilacijo AMPKα Thr172 in zmanjšal aktivnost AMPK. V nasprotju z drugo hipotezo je imel adrenalin na fosforilacijo in aktivnost AMPK podobne učinke kot inzulin. Tretjo hipotezo smo podprli le delno, saj je predhodna stimulacija z inzulinom in adrenalinom zmanjšala učinke A-769662 na fosforilacijo AMPKα Thr172, vendar povečala učinke A-769662 na fosforilacijo AMPKα Ser485. Večina testiranih farmakoloških aktivatorjev je stimulirala fosforilacijo AMPKα Thr172, a na fosforilacijo AMPKα Ser485 niso imeli vpliva oziroma so jo celo povečali, kar je v nasprotju s četrto hipotezo. Peto hipotezo smo zavrgli, saj je adrenalin povečal bazalni in z inzulinom spodbujen privzem glukoze. Zaključek: Naši rezultati kažejo, da adrenalin in inzulin na uravnavanje AMPK in privzem glukoze v skeletnomišičnih celicah L6 delujeta podobno in da nekateri farmakološki aktivatorji hkrati povečajo fosforilacijo AMPKα Thr172 in AMPKα Ser485, kar kaže na obstoj številnih regulacijskih signalnih poti, ki lahko predstavljajo nove možnosti za razvoj novih oblik zdravljenja sladkorne bolezni tipa 2.

• Jezik: Slovenski jezik
• Ključne besede: inzulin, adrenalin, AMPK, skeletne mišice, sladkorna bolezen tipa 2
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-119302
• COBISS.SI-ID: 28725507
• Datum objave v RUL: 07.09.2020

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Effect of insulin and adrenaline on regulation of AMP activated protein kinase (AMPK) in cultured skeletal muscle cells

Izvleček

Background: Skeletal muscles, metabolically one of the most active tissues, and an important site of insulin-dependent postprandial glucose uptake, are involved in maintaining normoglycaemia. For maintaining energy homeostasis is important AMP activated protein kinase (AMPK). AMPK is also a promising target for the development of new therapies for insulin resistance and type 2 diabetes, as activated enhances glucose homeostasis Aim: AMPK is activated allosterically and by phosphorylation of Thr172 on the catalytic α subunit, while phosphorylation of the Ser485 decreases its activity. Our aim was to determine how insulin and adrenaline affect the function of AMPK or AMPK pharmacological activators in skeletal muscle cells. Hypotheses: 1) Insulin impairs AMPK activity in skeletal muscle cells by suppressing AMPKα Thr172 phosphorylation and promoting AMPKα Ser485 phosphorylation. 2) Adrenaline increases AMPK activity in skeletal muscle cells, as it stimulates AMPKα Thr172 phosphorylation and inhibits AMPKα Ser485 phosphorylation. 3) Insulin decreases while adrenaline increases the effects of AMPK activator A-769662 on phosphorylation of AMPKα Thr172 and Ser485. 4) Direct and indirect AMPK pharmacological activators increase the phosphorylation of AMPKα Thr172 and decrease the phosphorylation of AMPKα Ser485 in skeletal muscle cells. 5) Adrenaline, despite AMPK activation, reduces basal and insulin-induced glucose uptake by L6 skeletal muscle cells. Methods: We investigated the regulation of AMPK in the rat skeletal muscle cell line L6. The phosphorylation sites of AMPKα Thr172 and Ser485 were analyzed with the Western blot method. Activation of AMPK was estimated by measuring phosphorylation of acetyl-CoA carboxylase (Ser79), a direct downstream target of AMPK. Glucose uptake in cells was assessed with the [3H]-2-deoxyglucose uptake assay. Results: We confirmed the first hypothesis, as insulin stimulated AMPKα Ser485 phosphorylation, inhibited AMPKα Thr172 phosphorylation and decreased AMPK activity. In contrast to the second hypothesis, adrenaline had similar effects on phosphorylation and AMPK activity as insulin. The third hypothesis was only partially supported, as prior stimulation with insulin and adrenaline reduced the effects of A-769662 on AMPKα Thr172 phosphorylation but increased the effects of A-769662 on AMPKα Ser485 phosphorylation. Most of the tested pharmacological activators stimulated the phosphorylation of AMPKα Thr172, but they did not affect or even incrased the AMPKα phosphorylation, which is contrary to the fourth hypothesis. We rejected the fifth hypothesis because adrenaline increased basal and insulin-induced glucose uptake in L6 skeletal muscle cells. Conclusion: Our results highlight that adrenaline and insulin act similarly on regulation of AMPK and glucose uptake in L6 skeletal muscle cells and that some pharmacological activators simultaneously increase phosphorylation of AMPKα Thr172 and AMPKα Ser485, indicating existence of multiple regulatory signalling pathways that may present new opportunities for the development of new strategies for treatment of type 2 diabetes.

• Ključne besede: insulin, adrenaline, AMPK, skeletal muscle, type 2 diabetes