Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mTOR complex 2 (mTORC2)

A. D. Barlow, J. Xie, C. E. Moore, S. C. Campbell, J. A.M. Shaw, M. L. Nicholson, T. P. Herbert

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Abstract

Aims/hypothesis Rapamycin (sirolimus) is one of the primary immunosuppressants for islet transplantation. Yet there is evidence that the long-term treatment of islet-transplant patients with rapamycin may be responsible for subsequent loss of islet graft function and viability. Therefore, the primary objective of this study was to elucidate the molecular mechanism of rapamycin toxicity in beta cells. Methods Experiments were performed on isolated rat and human islets of Langerhans and MIN6 cells. The effects of rapamycin and the roles of mammalian target of rapamycin complex 2 (mTORC2)/protein kinase B (PKB) on beta cell signalling, function and viability were investigated using cell viability assays, insulin ELISA assays, kinase assays, western blotting, pharmacological inhibitors, small interfering (si)RNA and through the overproduction of a constitutively active mutant of PKB. Results Rapamycin treatment of MIN6 cells and islets of Langerhans resulted in a loss of cell function and viability. Although rapamycin acutely inhibited mTOR complex 1 (mTORC1), the toxic effects of rapamycin were more closely correlated to the dissociation and inactivation of mTORC2 and the inhibition of PKB. Indeed, the overproduction of constitutively active PKB protected islets from rapamycin toxicity whereas the inhibition of PKB led to a loss of cell viability. Moreover, the selective inactivation of mTORC2 using siRNA directed towards rapamycininsensitive companion of target of rapamycin (RICTOR), mimicked the toxic effects of chronic rapamycin treatment. Conclusions/interpretation This report provides evidence that rapamycin toxicity is mediated by the inactivation of mTORC2 and the inhibition of PKB and thus reveals the molecular basis of rapamycin toxicity and the essential role of mTORC2 in maintaining beta cell function and survival.

Original languageEnglish
Pages (from-to)1355-1365
Number of pages11
JournalDiabetologia
Volume55
Issue number5
DOIs
Publication statusPublished - May 2012
Externally publishedYes

Bibliographical note

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Keywords

  • Apoptosis
  • Beta cell
  • Diabetes mellitus
  • GSIS
  • Islet
  • Islet transplantation
  • MTOR
  • MTORC2
  • PKB
  • Rapamycin
  • RICTOR

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