Metformin has adenosine-monophosphate activated protein kinase (AMPK)-independent effects on LPS-stimulated rat primary microglial cultures

Abstract

The results of recent studies suggest that metformin, in addition to its efficacy in treating type 2 diabetes, may also have therapeutic potential for the treatment of neuroinflammatory diseases in which reactive microglia play an essential role. However, the molecular mechanisms by which metformin exerts its anti-inflammatory effects remain largely unknown. Adenosine-monophosphate-activated protein kinase (AMPK) activation is the most well-known mechanism of metformin action; however, some of the biological responses to metformin are not limited to AMPK activation but are mediated by AMPK-independent mechanisms. In this paper, we attempted to evaluate the effects of metformin on unstimulated and LPS-activated rat primary microglial cell cultures. The presented evidence supports the conclusion that metformin-activated AMPK participates in regulating the release of TNF-α. Furthermore, the effects of metformin on the release of IL-1β, IL-6, IL-10, TGF-β, NO, and ROS as well as on the expression of arginase I, iNOS, NF-κB p65 and PGC-1α were not AMPK-dependent, because pretreatment of LPS-activated microglia with compound C, a pharmacological inhibitor of AMPK, did not reverse the effect of metformin. Based on the present findings, we propose that the shift of microglia toward alternative activation may underlie the beneficial effects of metformin observed in animal models of neurological disorders.

Introduction

Metformin is the only drug of the biguanide class currently used for the treatment of type 2 diabetes. The results of recent clinical and experimental studies suggest that metformin, apart from its hypoglycemic action, may attenuate both peripheral and central inflammation. The anti-inflammatory potential of met-formin has been reported in particular in many experimental models of peripheral inflammation. It has been shown that metformin attenuates pro-inflammatory responses in endothelial cells [22], diminishes human aortic smooth muscle cell proliferation [30] and ameliorates macrophage activation [34].

The activation of AMP-activated protein kinase (AMPK) constitutes the best-known mechanism of metformin action [61]. AMPK is a highly conserved heterotrimeric serine/threonine kinase that is involved in the regulation of cellular metabolism and energy distribution. Phosphorylation of the specific threonine residue (Thr172) is crucial for AMPK activity [53]. AMPK is an intracellular metabolic sensor that through the reduction of ATP-consuming processes and the stimulation of ATP-generating pathways, maintains cellular energy homeostasis. The latter effect relies on the up-regulation of the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which induces mitochondrial biogenesis [25].

It seems reasonable to consider that activation of AMPK by metformin may affect the above-mentioned processes not only in peripheral tissues but also in the brain, in particular because the drug has been shown to cross the blood-brain barrier and accumulate in the rodent brain [55]. Consequently, metformin is increasingly recognized as a drug that acts directly on the central nervous system and is curently being tested in various experimental models of neurodegeneration and neuroinflammation. To date, metformin has been shown to prolong survival time in the transgenic mouse model of Huntington’s disease [33], attenuate the induction of experimental autoimmune encephalomyelitis [37], diminish the migration and invasion of U87 and LN229 glioma cells [3], and exhibit neuroprotective effects against etoposide-induced apoptosis in primary cortical neurons [13]. It is also known that some of the biological responses to metformin are not limited to the activation of AMPK but are mediated by AMPK-independent mechanisms, including the inhibition of different intracellular targets such as p70S6K1 kinase [53], p38 mitogen-activated protein kinase (p38 MAPK), and protein kinase C (PKC) [43].

AMPK activation has been shown to affect the pro-inflammatory responses of microglia, which are currently recognized as the primary components of the intrinsic brain immune system [17]. Microglia constantly control the content and evaluate the safety of the neuronal microenvironment, which reciprocally regulates these cells [20]. However, the sustained activation of microglia has been implicated in the pathogenesis of a number of neurological disorders including ischemia/reperfusion brain injury, Alzheimer’s disease, Parkinson’s disease, HIV-associated dementia and multiple sclerosis [4].

Based on the described properties of metformin, because it can cross into the brain [8, 55] and taking into account that AMPK is expressed in microglial cells [17, 26], we hypothesized that metformin may modulate the LPS-induced proinflammatory response in rat primary microglia. In the present study, the response of microglia was parameterized with the production of nitric oxide (NO), reactive oxygen species (ROS) and the release of the major classes of both pro- and anti-inflammatory cytokines. To elucidate our findings, we determined the expression of nuclear factor κB (NF-κB) p65, PGC-1α, inducible nitric oxide synthase (iNOS) and arginase I. Additionally, to assess whether the mechanism of metformin action was AMPK-dependent, we measured AMPK activity and, in parallel experiments, applied 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) as an activator of AMPK and compound C as a confirmed pharmacological inhibitor of AMPK.