Methyl succinate antagonises biguanide-induced AMPK-activation and death of pancreatic β-cells through restoration of mitochondrial electron transfer

Background and purpose: Two mechanisms have been proposed to explain the insulin-sensitising properties of metformin in peripheral tissues: ( a) inhibition of electron transport chain complex I, and ( b) activation of the AMP activated protein kinase ( AMPK). However the relationship between these mechanisms and their contribution to beta-cell death and dysfunction in vitro, are currently unclear. Experimental approach: The effects of biguanides ( metformin and phenformin) were tested on MIN6 beta-cells and primary FACS-purified rat beta-cells. Cell metabolism was assessed biochemically and by FACS analysis, and correlated with AMPK phosphorylation state and cell viability, with or without fuel substrates. Key results: In MIN6 cells, metformin reduced mitochondrial complex I activity by up to 44% and a 25% net reduction in mitochondrial reducing potential. In rat beta-cells, metformin caused NAD( P) H accumulation above maximal glucose-inducible levels, mimicking the effect of rotenone. Drug exposure caused phosphorylation of AMPK on Thr(172) in MIN6 cell extracts, indicative of kinase activation. Methyl succinate, a complex II substrate, appeared to bypass metformin blockade of complex I. This resulted in reduced phosphorylation of AMPK, establishing a link between biguanide-induced mitochondrial inhibition and AMPK activation. Corresponding assessment of cell death indicated that methyl succinate decreased biguanide toxicity to beta-cells in vitro. Conclusions and implications: AMPK activation can partly be attributed to metformin's inhibitory action on mitochondrial complex I. Anaplerotic fuel metabolism via complex II rescued beta-cells from metformin-associated toxicity. We propose that utilisation of anaplerotic nutrients may reconcile in vitro and in vivo effects of metformin on the pancreatic beta-cell.