Vpliv kortizola in deksametazona na delovanje inzulina ter z AMP aktivirane protein kinaze v kulturi skeletnomišičnih celic L6

Tratnik, Rahela (author), Pirkmajer, Sergej (mentor), Dolinar, Klemen (co-mentor)

Uvod: Sladkorna bolezen zajema več presnovnih motenj z različno etiopatogenezo. Vsem je skupna hiperglikemija, ki je posledica motenega izločanja inzulina in/ali inzulinske rezistence v na inzulin odzivnih tkivih kot so jetra, maščevje in skeletne mišice. Presežek endogenih ali eksogenih glukokortikoidov, kot sta na primer kortizol in deksametazon, lahko vodi v inzulinsko rezistenco, moteno toleranco za glukozo in sladkorno bolezen (t.i. steroidni diabetes). Skeletne mišice so pomembno mesto z inzulinom spodbujenega privzema glukoze in eden izmed najpomembnejših organov, ki jih prizadene inzulinska rezistenca. Farmakološka aktivacija z AMP-aktivirane protein kinaze (AMPK), celičnega energijskega senzorja, v skeletnih mišicah poviša občutljivost na inzulin in s tem pripomore k izboljšanju homeostaze glukoze na ravni celega telesa. Namen: Natančen mehanizem z glukokortikoidi povzročene inzulinske rezistence v skeletnih mišicah ni poznan. Preveriti smo želeli, ali bi k inzulinski rezistenci, ki jo povzročijo glukokortikoidi, lahko prispevala zavora delovanja AMPK. Hipoteze: 1) Kortizol in deksametazon zmanjšata aktivnost inzulinske signalne poti v skeletnomišičnih celicah L6. 2) Kortizol in deksametazon zmanjšata z inzulinom spodbujeni privzem glukoze v skeletnomišičnih celicah L6 kljub povišanju izražanja prenašalcev GLUT1 in GLUT4. 3) Kortizol in deksametazon zmanjšata z A769662 spodbujeno fosforilacijo AMPK in acetil-CoA karboksilaze (ACC) v skeletnomišičnih celicah L6. 4) Kortizol in deksametazon zmanjšata z A769662 spodbujeni privzem glukoze v skeletnomišičnih celicah L6 kljub povišanju izražanja prenašalcev GLUT1 in GLUT4. Metode: Za poskusni model smo uporabili podganje skeletnomišične celice L6. Aktivnost inzulinske signalne poti in AMPK smo ovrednotili s prenosom western, s katerim smo merili fosforilacijo AKT (Thr308 in Ser473), AS160 (Thr642), ERK1/2 (Thr202/Tyr204), ribosomskega proteina S6 (Ser235/236, S6RP), AMPK (Thr172) in ACC (Ser79). Privzem glukoze v skeletnomišične celice L6 smo ocenili z merjenjem privzema 3H-2-deoksiglukoze. Izražanje prenašalcev GLUT1 in GLUT4 smo določili s kvantitativno reakcijo verižne polimeraze v realnem času. Za farmakološko aktivacijo AMPK smo uporabili učinkovino A769662. Rezultati: Kortizol in deksametazonom (0,1 µM in 1 µM) sta v prisotnosti inzulina (12 in 120 nM) povzročila porast fosforilacije AKT (Thr308 in Ser473). V inzulinsko spodbujenih vzorcih kortizol in deksametazon na fosforilacijo AS160 (Thr642) nista vplivala (24-urno tretiranje) oziroma sta jo zmanjšala (72-urno tretiranje). V vzorcih z inzulinom (12 in 120 nM) sta kljub temu povzročila porast privzema glukoze. Fosforilacija ERK1/2 (Thr202/Tyr204) in S6RP (Ser235//236) se je pod vplivom kortizola in deksametazona zmanjšala v prisotnosti inzulina. Izražanje mRNA za prenašalce GLUT1 in GLUT4 se je pod vplivom kortizola in deksametazona povišalo. Kortizol in deksametazon nista spremenila fosforilacije AMPK (Thr172). Z A769662 spodbujena fosforilacija ACC (Ser79) se je ob dodatku kortizola in deksametazona zmanjšala. Kortizol in deksametazon sta v prisotnosti A769662 zvečala privzem glukoze. Zaključek: 1) Kortizol in deksametazon nista zmanjšala aktivnosti inzulinske signalne poti v skeletnomišičnih celicah L6. 2) Kortizol in deksametazon sta zvečala tako izražanje prenašalcev GLUT1 in GLUT4 kot z inzulinom spodbujeni privzem glukoze. 3) Kortizol in deksametazon sta zmanjšala z A769662 spodbujeno fosforilacijo ACC, nista pa vplivala na fosforilacijo AMPK. 4) Kortizol in deksametazon sta zvečala z A769662 spodbujeni privzem glukoze.

• Language: Slovenian
• Keywords: kortizol, deksametazon, inzulin, sladkorna bolezen, skeletna mišičnina, AMPK
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-135917
• Publication date in RUL: 01.04.2022

Secondary language

• Language: English
• Title: The influence of cortisol and dexamethasone on insulin and AMP-activated protein kinase activity in cultured skeletal muscle cells L6

Abstract

Introduction: Diabetes mellitus is comprised of several metabolic disorders with different aetiopathogenesis. They are characterized by hyperglycaemia, which results from impaired insulin secretion and/or insulin resistance in insulin-responsive tissues, such as liver, fat and skeletal muscle. Excess endogenous or exogenous glucocorticoids, such as cortisol and dexamethasone, can lead to insulin resistance, impaired glucose tolerance and diabetes mellitus (so-called steroid diabetes). Skeletal muscle is an important site of insulin-stimulated glucose uptake and one of the most important organs affected by insulin resistance. Pharmacological activation of AMP-activated protein kinase (AMPK), a cellular energy sensor, in skeletal muscle increases insulin sensitivity and thus helps to improve glucose homeostasis of the whole body. Aim: The exact mechanism of glucocorticoid induced insulin resistance in skeletal muscle is unknown. The aim of our study was to test whether inhibition of AMPK action could contribute to glucocorticoid-induced insulin resistance. Hypotheses: 1) Cortisol and dexamethasone suppress insulin signalling in L6 skeletal muscle cells. 2) Cortisol and dexamethasone suppress insulin-stimulated glucose uptake in L6 skeletal muscle cells despite an increase in the expression of GLUT1 and GLUT4 transporters. 3) Cortisol and dexamethasone decrease A769662-induced AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in L6 skeletal muscle cells. 4) Cortisol and dexamethasone decrease A769662-stimulated glucose uptake in L6 skeletal muscle cells despite an increase in the expression of GLUT1 and GLUT4 transporters. Methods: The experiments were performed on rat L6 skeletal muscle cells. Insulin signalling pathway and AMPK activity were evaluated by western blotting, which assessed phosphorylation of AKT (Thr308 and Ser473), AS160 (Thr642), ERK1/2 (Thr202 and Tyr204), S6 ribosomal protein (Ser235/236, S6RP), AMPK (Thr172) and ACC (Ser79). The glucose uptake in cultured cells was estimated by 3H-2-deoxyglucose uptake assay. The expression of GLUT1 and GLUT4 transporters was evaluated with real time quantitative polymerase chain reaction. A769662 was used for pharmacological activation of AMPK. Results: In the presence of insulin (12 and 120 nM), cortisol and dexamethasone (0.1 µM and 1 µM) increased the AKT (Thr308 and Ser473) phosphorylation. In insulin stimulated samples, cortisol and dexamethasone did not affect (24-hour treatment) or reduced (72-hour treatment) phosphorylation of AS160 (Thr642), while they increased GLUT1 and GLUT4 mRNA expression and enhanced the insulin-stimulated (12 and 120 nM) glucose uptake. In the presence of insulin, phosphorylation of ERK1/2 (Thr202 and Tyr204) and S6RP (Ser235/236) was reduced by the treatment with glucocorticoids. While cortisol and dexamethasone did not alter the phosphorylation of AMPK (Thr172), they suppressed the A769662-induced phosphorylation of ACC (Ser79) and enhanced the A769662-stimulated glucose uptake. Conclusions: 1) Cortisol and dexamethasone did not decrease the activity of insulin signaling in L6 skeletal muscle cells. 2) Cortisol and dexamethasone increased both the expression of GLUT1 and GLUT4 transporters and insulin-stimulated glucose uptake. 3) Cortisol and dexamethasone diminished A769662-stimulated phosphorylation of ACC but not AMPK. 4) Cortisol and dexamethasone elevated A769662-stimulated glucose uptake.

• Keywords: cortisol, dexamethasone, insulin, diabetes, skeletal muscle, AMPK