In vitro evaluation of effects of metformin on morphine and methadone tolerance through mammalian target of rapamycin signaling pathway.

The chronic use of opioids leads to tolerance, psychological, and physical dependence that limits their use as an effective long-term pain control. Several studies have shown that mammalian target of rapamycin (mTOR) plays a crucial role in the development of opioid tolerance. Metformin activates 5' adenosine monophosphate-activated protein kinase (AMPK) which directly suppresses the mTOR complex 1 signaling pathway. On the other hand, metformin can also inhibit mTOR directly and in an AMPK-independent manner. Thus, in the current study, we aimed to investigate the effects of metformin on the development of morphine and/or methadone-induced tolerance in human glioblastoma (T98G) cell line. We examined the effects of chronic treatment of morphine and/or methadone in the presence or absence of metformin with or without AMPK inhibitor (dorsomorphin hydrochloride) on levels of nitric oxide and cyclic adenosine monophosphate (cAMP), phosphorylated and dephosphorylated ribosomal protein S6 kinase β-1 (S6K1) and 4E-binding protein 1 (4E-BP1) in T98G cells. Pretreatment of cells with metformin (40 µM) with or without AMPK inhibitor (dorsomorphin hydrochloride; 1 µM) before adding of morphine (2.5 µM) or methadone (1 µM) revealed a protective effects on the development of opioid tolerance. Prior administration of metformin reversed the elevation of nitric oxide levels induced by morphine (p < 0.001) and methadone (p < 0.001) and also prevented the raise of cAMP levels induced by morphine in T98G cells (p < 0.05). Contribution of mTOR signaling pathway in metformin-induced effect was shown by the inhibition of phosphorylation of S6K1 and 4E-BP1, the downstream targets of mTOR. mTOR activation suppresses opioid-induced antinociception, and its activity has also been increased during opioid tolerance.