Metformin, a commonly prescribed first-line drug for the treatment of type 2 diabetes mellitus, has been shown to reduce cardiac injury under diabetic and other pathological conditions. However, the mechanisms that mediate the cardioprotective effects of metformin remain controversial. One prevailing hypothesis suggests that metformin activates AMPK-autophagy pathway which eliminates protein aggregates and damaged mitochondria, thus protecting the heart from injury. In the present study, we tested this hypothesis using both in vivo and in vitro models. FVB/N mice were administered 200 mg/kg of metformin through gavage once a day for two days. As expected, metformin activated AMPK signaling in the heart as shown by the increased phosphorylation of AMPK alpha at Thr172 and its downstream effecter Acetyl-CoA Carboxylase at Ser79. Autophagy and mitophagy were determined by Western blot analysis of LC3-II, a well-established marker of autophagic vesicles. Surprisingly, however, metformin did not affect LC3-II levels in total tissue lysates and significantly reduced LC3-II in mitochondrial fractions, suggesting that metformin actually inhibited rather stimulated mitophagy, contrary to the current belief. This was confirmed by measuring mitophagy flux using lysosomal inhibitors pepstatin A and E64d. The ability of metformin to inhibit mitophagy was also corroborated by a novel dual fluorescent mitophagy reporter showing reduced degradation rate of mitochondria in the lysosomes. All these results were reproduced in H9c2 rat cardiac myoblast cells. Collectively, our results show that metformin is able to activate AMPK signaling but paradoxically inhibits mitophagy in the heart, suggesting that the cardioprotective benefit of metformin observed in many different models may be mediated through mechanisms other than autophagy and mitophagy. Future studies will explore the mechanisms by which metformin inhibits mitophagy and protects the heart.
Y. Zhang: None. M.M. Chang: None. P.R. Pinkhasova: None. F. Zhao: None. T. Kobayashi: None. Y. Huang: None. S. Kobayashi: None. Q. Liang: None.