Activation of the AMP-activated Protein Kinase by the Anti-diabetic Drug Metformin in Vivo

Abstract

Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Further, either depletion of mitochondria or adenoviral overexpression of superoxide dismutases, as well as inhibition of nitric-oxide synthase, abolished the metformin-enhanced phosphorylations and activities of AMPK, implicating that activation of AMPK by metformin might be mediated by the mitochondria-derived RNS. Furthermore, administration of metformin, which increased 3-nitrotyrosine staining in hearts of C57BL6, resulted in parallel activation of AMPK in the aorta and hearts of C57BL6 mice but not in those of endothelial nitric-oxide synthase (eNOS) knockout mice in which metformin had no effect on 3-nitrotyrosine staining. Because the eNOS knockout mice expressed normal levels of AMPK-␣ that was activated by 5-aminoimidazole-4-carboxamide riboside, an AMPK agonist, these data indicate that RNS generated by metformin is required for AMPK activation in vivo. In addition, metformin significantly increased the coimmunoprecipitation of AMPK and its upstream kinase, LKB1, in C57BL6 mice administered to metformin in vivo. Using pharmacological and genetic inhibitors, we found that inhibition of either c-Src or PI3K abolished AMPK that was enhanced by metformin. We conclude that activation of AMPK by metformin might be mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote AMPK activation by the LKB1 complex.

Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling

“…However, for the first time, we have demonstrated that persisting overproduction of ROS may explain this phenomenon. Also, for the first time, we have found that the inhibition of mitochondrial ROS production with ALA [13] or UCP2 transfection [17] can interrupt aspects of this 'memory' phenomenon. Moreover, inhibiting extra-mitochondrial ROS production or PARP activity also reduced several, but not all, of the persistently induced stress markers.…”

“…Blockade of NAD(P)H oxidase by apocynin [14] or of xanthine oxidoreductase by oxypurinol [15] during the glucose normalisation period interrupted the induction of several high glucose stress markers, but not all (Fig. 3), while overexpression of the mitochondrial respiratory chain uncoupling protein UCP2 [17] during the normalisation period reduced the induction of all the stress proteins (Fig. 3).…”

“…Effects of ROS inhibition on the 'memory' phenomenon in HUVECs In the last week of normal glucose in HUVECs, glucose was given with or without agents capable of inhibiting ROS production as follows: 62.5 μmol/l α-lipoic acid (ALA), an antioxidant working also at the mitochondria level [13]; 10 μmol/l apocynin [14]; 10 μmol/l oxypurinol [15]; 1 μmol/l PJ34 [16]; and 25 to 100 plaque-forming units per cell of an uncoupling protein 2 (UCP2) overexpressing adenovirus, used to specifically inhibit mitochondrial ROS production [17]. Inhibitors were replaced every other day and 24 h before the end of the experiment.…”

AMP-activated protein kinase (AMPK) activating agents cause dephosphorylation of Akt and glycogen synthase kinase-3

“…In order to investigate its physiological effects, AMPK is often experimentally activated by either of two agents, phenformin or AICAR. These two drugs activate AMPK by different mechanisms, with AICAR structurally mimicking the endogenous activator AMP, while several different mechanisms involving activation of an AMPK-phosphorylating kinase have been proposed for the effect of phenformin and its analog metformin [43][44][45][46][47][48]. In non-proliferating differentiated hippocampal neurons and in proliferating neuroblastoma SH-SY5Y cells, the present study found that both phenformin and AICAR not only activated AMPK, but also greatly reduced the phosphorylation of Akt on its regulatory Ser/Thr sites.…”

Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases

“…AMP kinase is activated by phosphorylation thus possible mechanisms of inactivation could include allosteric modification by AMP and ATP, activation of protein phosphatases, and modulation by other signaling pathways [31,32]. More recently, studies have suggested that the finely tuned mitochondrial ROS/RNS balance may also be a critical controlling factor in the activation of AMP kinase [33][34][35]. Therefore, it is possible that chronic alcohol and obesity mediated effects on mitochondrial functioning and redox signaling inhibit AMP kinase activity thereby contributing to the development and progression of fatty liver disease.…”