Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mTOR complex 2 (mTORC2).
OBJECTIVE
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 rapamycin-insensitive companion of target of rapamycin (RICTOR), mimicked the toxic effects of chronic rapamycin treatment.


CONCLUSIONS
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.
Department of Cell Physiology and Pharmacology, University of Leicester, The Henry Wellcome Building, University Road, Leicester LE1 9HN, UK.
A D
AD
Barlow
J
J
Xie
C E
CE
Moore
S C
SC
Campbell
J A M
JA
Shaw
M L
ML
Nicholson
T P
TP
Herbert
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000009:adverse effects*; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000009:adverse effects*; D015534:Trans-Activators / Q000037:antagonists & inhibitors*; D014157:Transcription Factors / Q000037:antagonists & inhibitors*
C493245:CRTC2 protein, human; C545700:CRTC2 protein, rat; D007166:Immunosuppressive Agents; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus