The mammalian target of rapamycin complex 1 (mTORC1), metformin and a novel route for the regulation of opioid tolerance.

A. AL-Subaiyel¹, E. Middleton¹, S. Eldabe², I. Obara¹. ¹Newcastle University, Newcastle, United Kingdom, ²The James Cook University Hospital, Middlesbrough, United Kingdom.

Introduction: We have previously showed that a widely used anti-diabetic drug metformin blocked morphine tolerance and potentiated morphine-induced analgesia but the mechanisms underlying these effects remain unknown. Metformin is known to inhibit the mammalian target of rapamycin complex 1 (mTORC1), a kinase controlling protein synthesis, through activation of the 5\' adenosine monophosphate-activated protein kinase (AMPK). Here, by expanding on studies on the role of mTORC1 in nociception¹ and opioid efficacy², we determined the extent to which the alterations in mTORC1 activity within nociceptive pathway underlie the responsiveness to prolonged administration of morphine and its interaction with metformin.

Methods: We used the tyramide signal amplification ptotocol¹ to visualize the distribution and cellular localization of the mTORC1 downstream effector phospho-S6 ribosomal protein (Ser235/236) (P-S6 RP). Transverse section of fixed spinal cord (40 μm) from naïve adult male C57BL/6J mice (n=3-5/group) treated with morphine (20 mg/kg, i.p., twice daily at 12 h intervals) and metformin (200 mg/kg, i.p., once daily, 24 h before morning morphine injection) for 9 consecutive days, were immunolabelled with P-S6 RP (1:1000, Cell Signaling) and then incubated with NeuN (1:1000, Millipore) for neurons or glial fibrillary acidic protein (GFAP) (1:1000, Millipore) for astrocytes identification. We also verify the specificity of mTORC1 inhibition by metformin using immunoblotting¹. Samples of spinal dorsal horn (15μl, n=3-5/group) were electrophoretically transferred onto a PVDF membrane (dry transfer). Membranes were then incubated with another downstream target of mTORC1 phospho-p70 S6 kinase (P-p70 S6 K, 1:500, Cell Signaling). The signal was analysed by ImageJ and one-way ANOVA with Bonferroni’s test.

Results: Our immunohistochemistry analysis showed that our previously observed behavioural effects linked to the inhibition of morphine tolerance by metformin were attributed to the inhibition of mTORC1 activity as immunoreactivity of P-S6 RP was reduced by metformin in the dorsal horn spinal cord. This reduction was observed predominantly in neurons, although a small population of astrocytes expressing P-S6 RP was also detected. In addition, our immunoblotting analysis revealed 68.4±16.4% decrease in the phosphorylation levels of P-p70 S6 kinase in the spinal cord after metformin treatment (F_(4,14)=4.74, P=0.01).

Conclusion: By evaluating the viability of mTORC1 as a therapeutic target to improve opiate responsiveness in pain, our studies may provide a novel insight into the complexity of the adaptive molecular mechanisms that underlie opioid treatment and its side effects.

References:

(1) Obara I et al. (2011). Pain 152:2582-2596.

(2) Hu JT et al. (2014) J Clin Ivest 124:592-603.