The effects of glutamine in differentiation of leukemia cell lines

Agonisti AMP-ovisne kinaze (AMPK), bigvanid metformin i 5-aminoimidazol-4-karboksamid ribonukleozid (AICAr), koče proliferaciju stanica leukemijskih linija, ali samo AICAr potiče izražaj diferencijacijskih biljega u monocitnoj liniji U937. Mehanizam diferencijacije potaknute AICAr-om nije u potpunosti razjašnjen, ali je znano da ne ovisi o AMPK-u. Cilj ovog istraživanja bio je utvrditi ulogu metabolizma glukoze i glutamina u učincima AICAr-a na stanice leukemijskih linija. Koristili smo komercijalno dostupne setove za mjerenje glukoze, laktata i amonijaka u odgovoru na agoniste AMPK-a i sve-trans-retinsku kiselinu (ATRA) koja potiče granulocitnu diferencijaciju. Rezultati su pokazali da ATRA smanjuje, metformin povećava, a AICAr nema učinaka na potrošnju glukoze i proizvodnju laktata. Međutim, AICAr povećava proizvodnju amonijaka, a to povećanje izostaje ako se stanice uzgajaju bez glutamina ili uz dodatak bis-2-(5-fenilacetamido-1,3,4-tiadiazol-2-il)etil sulfida (BPTES), farmakološkog inhibitora glutaminaze 1 (GLS1). Analize protočnom citometrijom pokazale su da manjak glutamina i inhibicija GLS1 koče izražaj diferencijacijskih biljega u odgovoru na AICAr, ali nemaju učinaka na diferencijaciju potaknutu ATRA-om. Ti rezultati upućuju da je metabolizam glutamina povezan s diferencijacijom stanica U937 u odgovoru na AICAr.
AMP-dependent kinase (AMPK) agonists, the biguanide metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) inhibit proliferation of leukemia cell lines, but only AICAr induces expression of differentiation markers in monocytic U937 cell line. The mechanism of AICAr-induced differentiation is not fully understood, but it is known to be AMPK-independent. In this study, we investigated the role of glucose and glutamine metabolism in AICAr-mediated effects in leukemia cell lines. We used commercially available kits for measuring glucose, lactate and ammonia in response to AMPK-agonists and all-trans-retinoic acid (ATRA) as an inducer of granulocytic differentiation. The results showed that ATRA decreased, metformin increased, and AICAr had no effects on glucose consumption and lactate production. However, AICAr increased ammonia production, and the increase was abolished when U937 cells were grown in the absence of glutamine or in the presence of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)-ethyl sulphide (BPTES), a pharmacological inhibitor of glutaminase 1 (GLS1). Flow cytometry analyses revealed that the lack of glutamine and the inhibition of GLS1 inhibited the expression of differentiation markers in response to AICAr, but had no effects on ATRA-mediated differentiation. These results show that glutamine metabolism is associated with AICAr-mediated differentiation of U937 cells.