Identification of a fatty acid binding protein4-UCP2 axis regulating microglial mediated neuroinflammation

https://doi.org/10.1016/j.mcn.2017.02.004Get rights and content

Highlights

  • FABP4 regulates UCP2 expression in microglia.

  • UCP2 opposes PA-induced upregulation of pro-inflammatory marker iNOS.

  • UCP2 mediates expression of the anti-inflammatory marker arginase-1.

  • Lack of hypothalamic FABP4 increases UCP2 and reduces microglial inflammation.

Abstract

Hypothalamic inflammation contributes to metabolic dysregulation and the onset of obesity. Dietary saturated fats activate microglia via a nuclear factor-kappa B (NFκB) mediated pathway to release pro-inflammatory cytokines resulting in dysfunction or death of surrounding neurons. Fatty acid binding proteins (FABPs) are lipid chaperones regulating metabolic and inflammatory pathways in response to fatty acids. Loss of FABP4 in peripheral macrophages via either molecular or pharmacologic mechanisms results in reduced obesity-induced inflammation via a UCP2-redox based mechanism. Despite the widespread appreciation for the role of FABP4 in mediating peripheral inflammation, the expression of FABP4 and a potential FABP4-UCP2 axis regulating microglial inflammatory capacity is largely uncharacterized. To that end, we hypothesized that microglial cells express FABP4 and that inhibition would upregulate UCP2 and attenuate palmitic acid (PA)-induced pro-inflammatory response. Gene expression confirmed expression of FABP4 in brain tissue lysate from C57Bl/6J mice and BV2 microglia. Treatment of microglial cells with an FABP inhibitor (HTS01037) increased expression of Ucp2 and arginase in the presence or absence of PA. Moreover, cells exposed to HTS01037 exhibited attenuated expression of inducible nitric oxide synthase (iNOS) compared to PA alone indicating reduced NFκB signaling. Hypothalamic tissue from mice lacking FABP4 exhibit increased UCP2 expression and reduced iNOS, tumor necrosis factor-alpha (TNF-α), and ionized calcium-binding adapter molecule 1 (Iba1; microglial activation marker) expression compared to wild type mice. Further, this effect is negated in microglia lacking UCP2, indicating the FABP4-UCP2 axis is pivotal in obesity induced neuroinflammation. To our knowledge, this is the first report demonstrating a FABP4-UCP2 axis with the potential to modulate the microglial inflammatory response.

Introduction

Saturated fatty acids (SFAs) such as palmitic acid (PA) contribute to the onset of metabolic inflammatory diseases, including obesity, in part through hypothalamic dysregulation and degeneration (Cai, 2013, Karmi et al., 2010, Thaler et al., 2012, Valdearcos et al., 2014). In the hypothalamus, dietary PA activates microglia (immune cells of the brain) via a nuclear factor kappa B (NFκB)-mediated pathway to release pro-inflammatory cytokines and contribute to damage of neurons responsible for regulating body weight (Thaler et al., 2013, Wang et al., 2012).

Microglia are sensitive and highly dynamic in response to changes in the surrounding microenvironment. As microglia respond to the surrounding environment, they are activated to either a pro-inflammatory (M1) or anti-inflammatory, protective (M2) phenotype, depending on external stimuli. For example, PA activates microglia via a toll like receptor (TLR)-4 and induces the release of pro-inflammatory cytokines and factors such as tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) (Duffy et al., 2015, Valdearcos et al., 2014). Conversely, microglia activated with the anti-inflammatory cytokine interleukin (IL)-4 polarize to an M2 protective state, characterized by the release of anti-inflammatory cytokines and factors such as arginase-1 (Fumagalli et al., 2011). The polarization of microglial cells is a highly energetic process and dependent upon mitochondrial integrity and activation. Uncoupling protein 2 (UCP2) has been implicated in mediating energetic processes of microglial activation states (Emre and Nubel, 2010). Microglia activated to an M1 phenotype have reduced UCP2 activity and expression, resulting in increased production of reactive oxygen species (ROS) (De Simone et al., 2015). Conversely, UCP2 activity is robustly increased following activation of an M2 protective phenotype (De Simone et al., 2015), indicating a potential target to manipulate microglial activation states.

UCP2 activity is regulated by fatty acid binding proteins (FABP), lipid chaperones regulating metabolic and inflammatory pathways in response to fatty acids (Hotamisligil and Bernlohr, 2015). Targeted deletion of the adipocyte FABP (FABP4, also known as aP2) is sufficient to prevent obesity induced insulin resistance, diabetes, atherosclerosis and asthma, (Boord et al., 2004, Furuhashi et al., 2007, Hotamisligil et al., 1996, Shum et al., 2006). Mice lacking FABP4 (also referred to as aP2 deficient mice) have been used to extensively characterize diabetes, atherosclerosis and asthma linking FABP4 signaling to important roles in metabolic homeostasis and immunometabolic diseases, as reviewed in (Hotamisligil and Bernlohr, 2015). In peripheral murine macrophages, the loss of FABP4 protects against the development of atherosclerosis and dyslipidemia (Makowski et al., 2001). The loss of FABP4 in macrophages via either molecular or pharmacologic means results in attenuated obesity-induced inflammation through a UCP2-redox based mechanism (Xu et al., 2016, Xu et al., 2015). While the role of the FABP4-UCP2 axis in peripheral macrophages has been extensively characterized, this axis has not been explored in the brain immune cells such as microglia.

We hypothesized that inhibition of FBAP4 in microglia would attenuate PA-induced pro-inflammatory response through a UCP2 mediated mechanism. Herein we demonstrate that mice lacking FABP4 have increased expression of UCP2 and reduced expression of TNF-α, iNOS, and ionized calcium-binding adapter molecule 1 (Iba1, a marker of microglial activation) in the hypothalamus. Moreover, pharmacological inhibition of FABP increases UCP2 expression and reduces PA-induced pro-inflammatory response and ROS production. Further, this effect is negated in microglia lacking UCP2, indicating the FABP4-UCP2 axis is pivotal in obesity-induced neuroinflammation.

Section snippets

Cell culture and reagents

Immortalized murine microglial cells (BV2) and UCP2 knockdown BV2 microglia (UCP2kd) were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad CA USA) supplemented with 10% fetal bovine serum and 1% penicillin, streptomycin, and neomycin and maintained at 37 °C with 5% CO2. Pan-FABP inhibitor (HTS01037; Cayman Chemical) was suspended in dimethyl sulfoxide (DMSO; Sigma Aldrich, St. Louis MO USA) and diluted to 30 μM in DMEM. Palmitic acid (PA; Sigma Aldrich) was conjugated to

Microglial cells express FABP4 and FABP5, but not FABP7

To verify that microglial cells express FABP, real-time PCR products of FABP4 (Fig. 1A), FABP5 (Fig. 1B), and FABP7 (Fig. 1C) were separated via electrophoresis and visualized on an agarose gel. Similar to macrophages, BV2 microglial cells express both FABP4 and FABP5, whereas in total brain lysates, FABP4, FABP5, and FABP7 are expressed.

Hypothalamic pro-inflammatory response is suppressed in mice lacking FABP4

In peripheral macrophages, lack of FABP4 contributes to upregulation of UCP2 (Xu et al., 2015). To determine if the absence of FABP4 leads to upregulated UCP2

Discussion

Obesity is often coupled with peripheral and central chronic low-grade inflammation (Businaro et al., 2012, Uranga et al., 2010). Prolonged overnutrition and excess intake of SFA increase the amount of fatty acids in the brain and induce neuroinflammation (Cai, 2013, Cai and Liu, 2011, Karmi et al., 2010, Thaler et al., 2012, Valdearcos et al., 2014). The onset of chronic neuroinflammation has identified as a major contributor to HFD-induced hypothalamic dysregulation (Valdearcos et al., 2014).

Acknowledgments

This work was funded by the U.S. Department of Veterans Affairs BLR&D IK2 BX001686 (to TAB), the University of Minnesota Healthy Foods, Healthy Lives Institute (to CMD, JPN and TAB), the National Institutes of Health NIH R01 DK053189 (to DAB), and the Minnesota Nutrition and Obesity Center (NIH P30 DK050456). We also thank members of the Bernlohr lab and Martha Grace for assistance with tissue collection.

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