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

Abstract

Aims/hypothesisRapamycin (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.MethodsExperiments 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.ResultsRapamycin 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/interpretationThis 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2475-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

mTORC1 and mTORC2 regulate insulin secretion through Akt in INS-1 cells

“…mTORC1 overactivation might also be deleterious for b-cell development and function (Shigeyama et al 2008, Rachdi et al 2012. Rapamycin treatment has been shown to have deleterious effects on the function and survival of the murine b-cell line (Bell et al 2003) and isolated islets from mouse, rat, or humans (Bell et al 2003, Hui et al 2005, Zhang et al 2006, Johnson et al 2009, Barlow et al 2012. The results we obtained with a moderate reduction of mTORC1 activity by raptor siRNA are in discrepancy with these data.…”

“…However, in these studies, mTORC1 hyperactivation was driven by the expression of constitutively active form of AKT (Balcazar et al 2009), RHEB (Hamada et al 2009), or deletion of TSC2 (Rachdi et al 2008, Mori et al 2009. None of these studies investigated the direct role of raptor, except one where raptor knockdown did not alter insulin secretion in dispersed islets (Barlow et al 2012). This latter result is in discrepancy with our data and might result from the biological material used for the experiment: INS-1 cell line vs islet.…”

“…In agreement, transduction of islets with the antiapoptotic gene XIAP prevents the negative effects of sirolimus (a rapamycin analog) on synthesis and secretion of insulin (Hui et al 2005). Recently, it was demonstrated that loss of MIN6 cell function and viability induced by rapamycin was associated with the dissociation of the mTORC2 complex, leading to the complete inhibition of Akt (Barlow et al 2012), a key modulator of cell viability. Altogether, these results suggest the deleterious effects of rapamycin on islet and b-cell function are mainly mediated through impaired cell viability.…”

“…The longterm toxic effects of rapamycin in b-cells are closely related to the dissociation and inactivation of mTORC2 and the inhibition of Akt1 activity (Barlow et al 2012). Furthermore, in the mouse, specific deletion of rictor in b-cells leads to hyperglycemia and glucose intolerance due to reduced b-cell mass and proliferation (Gu et al 2011).…”

Exendin-4 stimulates islet cell replication via the IGF1 receptor activation of mTORC1/S6K1

“…As anticipated, the addition of rapamycin inhibited exendin-4 plus glucose-stimulated rpS6 phosphorylation and caused an increase in the hypo-phosphorylated (a) form of 4EBP1. However, rapamycin treatment also led to a decrease in PKB phosphorylation on S473, presumably via the inactivation of mTORC2 (Barlow et al 2012).…”