Systematic Investigation of Dose-Dependent Protein Thermal Stability Changes to Uncover the Mechanisms of the Pleiotropic Effects of Metformin.

Metformin is a widely used drug to treat type II diabetes. Beyond lowering blood sugar, it has been reported to have pleiotropic effects such as suppressing cancer growth and attenuating cell oxidative stress and inflammation. However, the underlying mechanisms of these effects remain to be explored. Here, we systematically study the thermal stability changes of proteins in liver cells (HepG2) induced by a wide dosage range of metformin by using the proteome integral solubility alteration (PISA) assay. The current results demonstrate that, besides the most accepted target of metformin (complex I), low concentrations of metformin (such as 0.2 μM) stabilize the complex IV subunits, suggesting its important role in the sugar-lowering effect. Low-dose metformin also results in stability alterations of ribosomal proteins, correlating with its inhibitive effect on cell proliferation. We further find that low-concentration metformin impacts mitochondrial cargo and vesicle transport, while high-concentration metformin affects cell redox responses and cell membrane protein sorting. This study provides mechanistic insights into the molecular mechanisms of lowering blood sugar and the pleiotropic effects of metformin.