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


METHODS
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.


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
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.
Hinke
S A
SA
Diabetes Research Center and Juvenile Diabetes Research Center for Beta Cell Therapy in Europe, Brussels Free University VUB, Brussels, Belgium.
Martens
G A
GA
Cai
Y
Y
Finsi
J
J
Heimberg
H
H
Pipeleers
D
D
Van de Casteele
M
M
D055372:AMP-Activated Protein Kinases; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001645:Biguanides / Q000037:antagonists & inhibitors / Q000633:toxicity*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport / Q000187:drug effects; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000378:metabolism; D042963:Electron Transport Complex II / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000037:antagonists & inhibitors / Q000633:toxicity*; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism; D008687:Metformin / Q000633:toxicity; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010629:Phenformin / Q000633:toxicity; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D013386:Succinates / Q000378:metabolism / Q000494:pharmacology*; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors
D001645:Biguanides; D007004:Hypoglycemic Agents; D009097:Multienzyme Complexes; D013386:Succinates; D013778:Tetrazolium Salts; D013844:Thiazoles; D009249:NADP; D008687:Metformin; D010629:Phenformin; D042963:Electron Transport Complex II; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D042967:Electron Transport Complex I; C022616:thiazolyl blue; D005947:Glucose; C041105:monomethyl succinate