Metformin, besides exhibiting strong in vivo anti-inflammatory properties, increases mptp-induced damage to the nigrostriatal dopaminergic system

https://doi.org/10.1016/j.taap.2016.03.004Get rights and content

Highlights

  • Metformin treatment decreases microglial activation in the MPTP model of Parkinson's disease.

  • Metformin treatment increases the neurodegeneration in the MPTP model of Parkinson's disease, both in vivo and vitro.

  • Metformin treatment could be a risk factor for the development of Parkinson's disease.

Abstract

Metformin is a widely used oral antidiabetic drug with known anti-inflammatory properties due to its action on AMPK protein. This drug has shown a protective effect on various tissues, including cortical neurons. The aim of this study was to determine the effect of metformin on the dopaminergic neurons of the substantia nigra of mice using the animal model of Parkinson's disease based on the injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an inhibitor of the mitochondrial complex I. In vivo and in vitro experiments were used to study the activation of microglia and the damage of the dopaminergic neurons. Our results show that metformin reduced microglial activation measured both at cellular and molecular levels. Rather than protecting, metformin exacerbated dopaminergic damage in response to MPTP. Our data suggest that, contrary to other brain structures, metformin treatment could be deleterious for the dopaminergic system. Hence, metformin treatment may be considered as a risk factor for the development of Parkinson's disease.

Introduction

Parkinson's disease (PD) is a neurodegenerative disorder related to age that is characterized by the progressive degeneration of nigrostriatal dopaminergic neurons of the substantia nigra pars compacta (SNpc; Olanow et al., 2003). This leads to motor dysfunction of the extrapyramidal system, which is accompanied by cognitive dysfunction, progressive loss of autonomy and mood alterations (Di Monte and Langston, 1995, Dauer and Przedborski, 2003, Olanow et al., 2003). Although several genes have been identified as responsible for some types of early-onset familial PD (Lesage and Brice, 2009), the etiology of the disease remains unknown. Several factors, such as oxidative stress, mitochondrial dysfunction, reduced trophic factors, alterations in the ubiquitin-proteasome system and neuroinflammatory mechanisms, seems to cooperate in the progressive death of neurons in the SNpc (Dauer and Przedborski, 2003, Gao et al., 2003a, Hunot and Hirsch, 2003, Olanow et al., 2003, McGeer and McGeer, 2004, Marchetti and Abbracchio, 2005, Marchetti et al., 2005a, Marchetti et al., 2005b, Jenner, 2007).

Accumulating evidence suggests that inflammation may play a central role in the cell loss that occurs in PD. Hence, several authors have shown the existence of activated microglia in the SN of PD patients (McGeer et al., 1988, Klegeris et al., 2007, McGeer and McGeer, 2008), which is accompanied by an increased expression of inflammatory cytokines (Tansey et al., 2007, Hirsch and Hunot, 2009). Moreover, it has been shown that inflammation is present in different animal models of PD, including those produced by toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA) or rotenone (Liberatore et al., 1999, Cicchetti et al., 2002, Gao et al., 2003b, Miklossy et al., 2006). In fact, epidemiological studies have shown that the incidence of idiopathic PD is about 50% lower in chronic users of nonsteroidal anti-inflammatory agents or cyclooxygenase inhibitors with respect to non-users (Chen et al., 2003, Chen et al., 2005, Esposito et al., 2007).

Although the cause of the disease is unknown, there are various known risk factors that may increase the likelihood of developing the disease, which include age, sleep disorders, brain trauma and exposure to certain environmental factors such as pesticides and herbicides (Kieburtz and Wunderle, 2013, Postuma et al., 2013, Wong and Hazrati, 2013, Abdullah et al., 2015). In this sense, diabetes mellitus has also begun to be considered as a possible risk factor for developing this neurodegenerative disorder (Klimek et al., 2015). The association between diabetes and PD is not entirely clear. Some authors have proposed that chronic inflammation and oxidative stress produced in diabetes could lead to an increased risk of PD some years later (Xu et al., 2011). Moreover, animal and in vitro studies have shown a role for insulin in the regulation of the brain dopaminergic activity. Hence, deregulation and changes in insulin action have been studied in the pathophysiology and clinical symptoms of PD (Craft and Watson, 2004). Mitochondrial dysfunction, one of the possible causes of PD, may be also a common mechanism shared by both pathologies, since it has been described a reduced expression of certain genes involved in the impaired mitochondrial oxidation pathway in type 2 diabetes (Horan, 2009, Schernhammer et al., 2011).

One of the drugs globally accepted to treat type 2 diabetes is metformin, chemically called metformin hydrochloride; it is a member of the biguanide class that reduces hepatic glucose production and decreases insulin resistance and the levels of fasting plasma insulin through a mechanism of action that appears to be mainly mediated by activation of the adenosine monophosphate-activated protein kinase (AMPK).

Recently, various clinical and experimental studies suggest that metformin, besides their hypoglycemic actions, can attenuate inflammation both peripherally and centrally. The anti-inflammatory potential of metformin has been particularly described in numerous experimental models of peripheral inflammation, so that metformin has been shown to decrease the inflammatory response in endothelial cells (Isoda et al., 2006); it also decreases cell proliferation in the smooth muscle of human aortas (Li et al., 2005) and has antiatherogenic properties (Mamputu et al., 2003).

Given the anti-inflammatory activity of metformin and the importance of neuroinflammation in the development of PD, the aim of this study was to determine whether treatment with metformin is able to protect dopaminergic neurons in the SNpc using a model of PD based on the injection of MPTP.

Section snippets

Materials and methods

C57BL male mice (20–25 g) were used for these studies. Mice were kept at constant room temperature (22 ± 1 °C) and relative humidity (60%) with a 12-h light–dark cycle with free access to food and water. Experiments were carried out in accordance with the Guidelines of the European Union Directive (2010/63/EU) and Spanish regulations (BOE 34/11370-421, 2013) for the use of laboratory animals; the study was approved by the Scientific Committee of the University of Seville.

Animals were divided in

Effect of metformin and MPTP on microglial activation

We first evaluated activation of microglial cells based on morphological features and on the expression levels of TNF-α, IL-1β and iNOS mRNAs. Upon activation, microglial cells change their morphology from resting resident ramified microglia with two or three fine processes to round cells resembling tissue macrophages and proliferate when challenged. As expected, immunohistochemistry of Iba-1 showed that i.p. injection of saline solution in animals treated orally with tap water or metformin did

Discussion

PD is accompanied by an inflammatory reaction that may play a critical role in the degeneration of nigral dopaminergic neurons. Consequently, anti-inflammatory strategies are receiving increasing attention for their potential to prevent or delay the pathological deterioration that takes place in this disease.

Some recent clinical and experimental studies suggest that metformin, besides to its hypoglycemic actions, has anti-inflammatory properties. These anti-inflammatory effects have been

Conclusions

Since activity of complex I is decreased in parkinsonian patients, and considering metformin produces the inhibition of this multienzyme complex, our results suggest that treatment with this drug in diabetic patients might accelerate the onset and/or progression of the disease. Further studies should be carried out on the use of metformin at clinical doses.

Our results show that metformin is able to decrease the inflammatory response in the animal model of PD used in this study. However, despite

Conflict of interest statement

All authors declare that there are no conflicts of interest.

Transparency document

Transparency document.

Acknowledgements

This work was supported by grants of Junta de Andalucía P09-CTS-5244 and P10-CTS-6494, and a grant from the Spanish Ministerio de Economia y Competitividad (SAF2012-3902).

References (79)

  • V. Jackson-Lewis et al.

    Time course and morphology of dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

    Neurodegeneration

    (1995)
  • P. Jenner

    Oxidative stress and Parkinson's disease

    Handb. Clin. Neurol.

    (2007)
  • L.V. Kalia et al.

    Parkinson's disease

    Lancet

    (2015)
  • B. Kelly et al.

    Metformin Inhibits the Production of Reactive Oxygen Species from NADH:Ubiquinone Oxidoreductase to Limit Induction of Interleukin-1β (IL-1β) and Boosts Interleukin-10 (IL-10) in Lipopolysaccharide (LPS)-activated Macrophages

    J. Biol. Chem.

    (2015)
  • K. Łabuzek et al.

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

    Pharmacol. Rep.

    (2010)
  • L.J. Lawson et al.

    Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain

    Neuroscience

    (1990)
  • H.H. Liou et al.

    Interaction between nicotine and MPTP/MPP + in rat brain endothelial cells

    Life Sci.

    (2007)
  • T.C. Ma et al.

    Metformin therapy in a transgenic mouse model of Huntington's disease

    Neurosci. Lett.

    (2007)
  • B. Marchetti et al.

    To be or not to be (inflamed)—is that the question in anti-inflammatory drug therapy of neurodegenerative disorders?

    Trends Pharmacol. Sci.

    (2005)
  • B. Marchetti et al.

    Glucocorticoid receptor-nitric oxide crosstalk and vulnerability to experimental parkinsonism: pivotal role for glia-neuron interactions

    Brain Res. Brain Res. Rev.

    (2005)
  • P.L. McGeer et al.

    Inflammation and neurodegeneration in Parkinson's disease

    Parkinsonism Relat. Disord.

    (2004)
  • J. Miklossy et al.

    Role of ICAM-1 in persisting inflammation in Parkinson disease and MPTP monkeys

    Exp. Neurol.

    (2006)
  • W.J. Nicklas et al.

    Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine

    Life Sci.

    (1985)
  • S.P. Patil et al.

    Neuroprotective effect of metformin in MPTP-induced Parkinson's disease in mice

    Neuroscience

    (2014)
  • R.B. Postuma et al.

    Rapid eye movement sleep behavior disorder as a biomarker for neurodegeneration: the past 10 years

    Sleep Med.

    (2013)
  • M.G. Tansey et al.

    Neuroinflammatory mechanisms in Parkinson's disease: potential environmental triggers, pathways, and targets for early therapeutic intervention

    Exp. Neurol.

    (2007)
  • N.A. Tatton et al.

    In situ detection of apoptotic nuclei in the substantia nigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using terminal deoxynucleotidyl transferase labelling and acridine orange staining

    Neuroscience

    (1997)
  • J.L. Venero et al.

    Determination of monoamine oxidase in rat's substantia nigra during postnatal development

    Life Sci.

    (1989)
  • G. Ashabi et al.

    Pre-treatment with metformin activates Nrf2 antioxidant pathways and inhibits inflammatory responses through induction of AMPK after transient global cerebral ischemia

    Metab. Brain Dis.

    (2015)
  • C. Berry et al.

    Paraquat and Parkinson's disease

    Cell Death Differ.

    (2010)
  • E. Bezard et al.

    Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson's disease

    J. Neurosci.

    (2001)
  • J. Blesa et al.

    Parkinson's disease: animal models and dopaminergic cell vulnerability

    Front. Neuroanat.

    (2014)
  • B. Brunmair et al.

    Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions?

    Diabetes

    (2004)
  • H. Chen et al.

    Nonsteroidal anti-inflammatory drugs and the risk of Parkinson disease

    Arch. Neurol.

    (2003)
  • H. Chen et al.

    Nonsteroidal antiinflammatory drug use and the risk for Parkinson's disease

    Ann. Neurol.

    (2005)
  • L. Chen et al.

    OCT1 is a high-capacity thiamine transporter that regulates hepatic steatosis and is a target of metformin

    Proc. Natl. Acad. Sci. U. S. A.

    (2014)
  • S.H. Chen et al.

    Critical role of the Mac1/NOX2 pathway in mediating reactive microgliosis-generated chronic neuroinflammation and progressive neurodegeneration

    Curr. Opin. Pharmacol.

    (2015)
  • K. Chiba et al.

    Studies on the molecular mechanism of bioactivation of the selective nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

    Drug Metab. Dispos.

    (1985)
  • M.M. Christensen et al.

    Steady-state pharmacokinetics of metformin is independent of the OCT1 genotype in healthy volunteers

    Eur. J. Clin. Pharmacol.

    (2015)
  • Cited by (0)

    View full text