Microglia energy metabolism in metabolic disorder

Accepted Manuscript Microglia energy metabolism in metabolic disorder Martin J.T. Kalsbeek, Laurie Mulder, Chun-Xia Yi PII:

S0303-7207(16)30402-6

DOI:

10.1016/j.mce.2016.09.028

Reference:

MCE 9672

To appear in:

Molecular and Cellular Endocrinology

Received Date: 20 May 2016 Revised Date:

23 September 2016

Accepted Date: 26 September 2016

Please cite this article as: Kalsbeek, M.J.T., Mulder, L., Yi, C.-X., Microglia energy metabolism in metabolic disorder, Molecular and Cellular Endocrinology (2016), doi: 10.1016/j.mce.2016.09.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Microglia energy metabolism in metabolic disorder 1

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Martin J.T. Kalsbeek *, Laurie Mulder , Chun-Xia Yi

1) Department of Endocrinology and Metabolism, 
 Academic Medical Center (AMC), University of Amsterdam (UvA), 
 Meibergdreef 9, 
 1105 AZ, Amsterdam, 
 The Netherlands;

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Abstract Microglia are the resident macrophages of the CNS, and are in charge of maintaining a healthy microenvironment to ensure neuronal survival. Microglia carry out a non-stop patrol of the CNS, make contact with neurons and look for abnormalities, all of which requires a vast amount of energy. This non-signaling energy demand increases after activation by pathogens, neuronal damage or other kinds of stimulation. Of the three major energy substrates – glucose, fatty acids and glutamine – glucose is crucial for microglia survival and several glucose transporters are expressed to supply sufficient glucose influx. Fatty acids are another source of energy for microglia and have also been shown to strongly influence microglial immune activity. Glutamine, although possibly suitable for use as an energy substrate by microglia, has been shown to have neurotoxic effects when overloaded. Microglial fuel metabolism might be associated with microglial reactivity under different pathophysiological conditions and a microglial fuel switch may thus be the underlying cause of hypothalamic dysregulation, which is associated with obesity.

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Corresponding author, [email protected] Department of Endocrinology and Metabolism, 
 Academic Medical Center, University of Amsterdam(UvA),
Meibergdreef 9,
1105 AZ, Amsterdam,
The Netherlands

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Abbreviations: 2-DG, deoxyglucose; ATP, adenosine triphosphate; CNS, central nervous system; GLUT, glucose transporter; BBB, blood brain barrier;  IFNγ, interferon γ; HK2, hexokinase 2; ICU, intensive care unit; LPL, lipoprotein lipase; ACS, acyl-CoA synthetase; ACSL, acyl-CoA synthetase long chain; ROS, reactive oxygen species; ICV, intracerebroventricular; GPR120, G protein-coupled receptor 120; PUFA, polyunsaturated fatty acid; LPS, Lipopolysaccharide; TLR4, toll-like receptor 4; LCFA, long chain fatty acid; SCFA, short chain fatty acid; FFAR2, free fatty acid receptor 2; GLS, glutaminase; GLUD1, glutamate dehydrogenase 1; SLC1A5, solute carrier family 1 member 5; GABABR, gammaaminobutyric acid B receptor; HBMG1, high-mobility group box 1; IL-1β, interleukin-1β; TNF-α tumor necrosis factor-α; GLP-1, glucagon-like peptide-1; CX3CL1, chemokine (C-X3-C motif) ligand 1

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1. Introduction Neurons in the central nervous system require a healthy microenvironment to survive, the maintenance of which critically depends on the innate immune cells, the microglia. Microglia can initiate immune protection under physiological or pathological conditions to efficiently clear away metabolic wastes, cellular debris and pathogens (Hughes 2012). When microglial activity cannot match the demands of the immune defense, the homeostatic and “clean” microenvironment will not be maintained properly (Kreutzberg 1996), and various aspects of neuronal function will be impaired. Under physiological conditions, the debris production in the microenvironment is largely derived from active neurons, especially at the synaptic level (so-called synaptic debris) (Tremblay 2011). Several studies suggest that microglia play an important role in the regulation of synaptic plasticity (Eyo and Dailey 2013, Parkhurst et al. 2013). Plasticity of synaptic activity is an essential part of neurophysiology in a variety of neural circuits, such as those in the hypothalamus that are involved in the control of energy homeostasis (Yang et al. 2011). Thus properly functioning microglia play an important role in the maintenance of hypothalamic neural circuits. Microglia are heterogeneously distributed throughout the brain and account for about 10–15% of cells in the brain (Lawson et al. 1990, Yang et al. 2013). Microglia function is usually considered to be a “double-edged sword”: under different circumstances microglia can be either beneficial or detrimental to the brain. During development, microglia control the wiring of the brain by regulating processes such as programmed cell death, synaptic pruning and synapse maturation (Bilimoria and Stevens 2015). In the adult brain, microglia maintain a healthy microenvironment by actively surveying the brain parenchyma, scanning for abnormalities and making contact with synapses and neuronal soma (Wake et al. 2011). As the brains innate immune cells, microglia can phagocytose invading pathogens and clear away debris, thereby restoring homeostasis in the CNS (Mariani and Kielian 2009). Multiple studies have shown that microglia act as neuroprotectors in a number of ways, including synaptic stripping, promoting neurogenesis and suppressing destructive inflammation by the release of anti-inflammatory cytokines such as Il-10 and TGFβ (Chen and Trapp 2016). But microglia have also been shown to have beneficial effects on the brain. Activation of microglia has been implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis, Huntington disease, multiple sclerosis and Alzheimer’s disease (Cartier et al. 2014). Activated microglia are also found in the hypothalamus in diet-induced obesity (Thaler et al. 2012). Whether activation of microglia is an effect or a cause of this disease-related neurodegeneration, or whether microglia activity actually limit neurodegeneration is currently an area of intense study. Microglia are highly dynamic cells and the protrusions of so-called “resting” microglia are continuously moving around, scanning the brain (Davalos et al. 2005, Nimmerjahn et al. 2005). After activation, e.g. by damaged neurons or invading microorganisms, microglia rapidly change shape by extending processes towards the damaged site and retracting processes facing the other way (Koizumi et al. 2007). These processes are employed to

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phagocytize cellular debris and damaged neurons (Fu et al. 2014). Microglia are also able to replace their scanning processes with new, highly motile processes so they can translocate their complete cell body to an injured site (Stence et al. 2001). Morphogenesis, phagocytosis and translocation all require dynamic reorganization of the actin cytoskeleton, for which vast amounts of adenosine triphosphate (ATP) are necessary (Engl and Attwell 2015). In order to meet this demand of non-signaling energy, microglia express transporters for the three main energy substrates, indicating a flexible use of available energy resources (Zhang et al. 2014). Prolonged hypercaloric challenge results in hypothalamic dysregulation, which coincides with an activation of hypothalamic microglia (Thaler et al. 2012). After hypercaloric challenge, mediobasal hypothalamic microglia enter a reactive stage, which is initially transient, suggesting a beneficial response. Continued hypercaloric challenge reestablishes the reactive stage of microglia, with possible detrimental effects to surrounding neurons (Thaler et al. 2012). How a hypercaloric environment is associated with hypothalamic microglial activation is unclear. Saturated fat is supposed to be one of the main triggers, but the driving force behind the uptake of saturated fat into the microglia is unclear, especially regarding the brain fuel dogma, which states that glucose is the main energy resource. So far, very little is known about the hypothalamic microglial energy influx mechanism. This review will summarize microglial energy metabolism in general and discuss the possible links between hypothalamic microglial reactivity and its energy metabolism in metabolic disorders.

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2 Microglial energy metabolism Cellular energy demand determines the energy production and energy substrate influx into the cell. Under physiological conditions each cell type has a specific metabolic profile for the energy substrates it uses, but changes in the microenvironment or cellular activity can induce a switch of this metabolic profile. For instance, it is known that activation induces peripheral macrophages to switch from oxidative phosphorylation to anaerobic glycolysis, in order to increase ATP production (Kelly and O'Neill 2015). A similar metabolic switch, known as the Warburg effect, has been well-established in specific tumor cells and serves to maintain their increased energy demand (Griffin and Shockcor 2004). Furthermore, it has been shown in a human liver cell line that glutamine deprivation results in upregulation of lipid metabolism (Long et al. 2016). These studies show that changes in energy demand can induce different pathways to metabolize energy substrates, or promote the use of a different substrate. As microglia are in constant need of ATP, particularly after activation, and glucose availability in the brain is not infinite, one would speculate that microglia are capable of a flexible use of energy substrates as well. It is therefore not surprising that microglia show high expression of genes involved in the metabolism of the three major energy substrates. A metabolic switch might be necessary to accommodate the beneficial role of microglia in certain pathophysiological conditions. However, it is also conceivable that a metabolic switch of microglia might be accompanied by the generation of metabolites that alter neuronal health. However, very little is known yet about how microglial cellular metabolism is integrated in their functionality. 2.1 Glucose Glucose is the main energy source for the brain. The concentration of glucose in the brain is around 20% of that of the glucose concentration in the blood (Dunn-Meynell et al. 2009). Five main glucose transporters (GLUT 1-5) mediate glucose uptake in the brain and various peripheral tissues (Thorens and Mueckler 2010). GLUTs 1 and 3 are expressed in many tissues at variable levels and are thought to regulate basal glucose uptake. Once in the brain, glucose can be taken up by neurons with their high affinity glucose transporters 3 (GLUT3), ensuring a constant influx of glucose even when glucose levels are low (Mergenthaler et al. 2013). Similar to neurons, microglia also express GLUT3 (figure 1), but apart from that, microglia can also express other GLUTs under different circumstances. Indeed, previous

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studies have indicated different GLUTs to be expressed on microglia, in order to ensure sufficient glucose influx to meet the energy demand. Microglial survival vitally depends on sufficient glucose supply. Culturing primary mice microglia in glucose-free balanced salt solution resulted in 74% cell death within the first 30 hours and even 98% after 48 hours (Yenari and Giffard 2001). Supplementing the balanced salt solution with 5.5 mM glucose decreased microglial death to less than 20% the first 30 hours but a delay in cell death occurred up to 73% after 48 hours (Yenari and Giffard 2001). This suggests that glucose is essential for microglial survival, although survival does not solely depend on glucose in vitro. The effect of glucose deprivation on microglia in vivo is less clear. Early studies in rats show neurodegeneration with ensuing gliosis one week after a 30 minute period of insulin-induced hypoglycemia (Auer et al. 1985). A more recent study showed significant activation of CD11b positive microglia together with neurodegeneration, one week after 30 minutes of insulin-induced hypoglycemia (Kim et al. 2015). In this study, both microglia activation and neurodegeneration could be attenuated by injection of melatonin right after the hypoglycemic period. Melatonin has been shown to impair NADPH oxidase assembly in microglia, thereby effectively decreasing microglial ROS production (Zhou et al. 2008). Researchers imply that the degenerative effect of insulin-induced hypoglycemia on neurons is, at least partly, driven by microglia activation. On the other hand, several studies have reported neuroprotective effects when glucose utilization was inhibited using a different method. Treatment with 2-deoxy-D-glucose (2-DG), a glucose analogue blocking glycolysis by inhibiting hexokinase activity, reduces cellular glucose utilization and shows a neuroprotective effect in pathologies such as epilepsy and Parkinson’s disease (Duan and Mattson 1999, Garriga-Canut et al. 2006). Different (non-glial) mechanisms underlying the neuroprotective effect of 2-DG were suggested, but one study showed a reduction of activated microglia in an Alzheimer’s disease mouse model treated with 2-DG (Yao et al. 2011). In this study the reduction in activated microglia is clear, but whether this is a cause or consequence is unclear. Some in vitro studies show evidence that 2-DG can inhibit microglia, which decreases neurodegeneration. Using neuronal-glial co-cultures, amyloid beta or trauma-induced neuronal loss could be prevented by depleting microglia from culture using 2DG under normoxic conditions (Vilalta and Brown 2014). 2-DG mainly inhibits anaerobic glycolytic glucose utilization and to a lesser extent oxidative phosphorylation of glucose, which suggests that microglia are more dependent on anaerobic glycolysis than neurons are. Under hypoxic conditions 2-DG treatment not only depleted microglia but also aggravated neuronal loss, probably because during hypoxia neurons rely more on anaerobic glycolysis for ATP production than when oxygen is present (Vilalta and Brown 2014). These studies suggest that microglia can be activated and cause neuronal damage when glucose levels are low. On the other hand, when glycolytic glucose utilization is blocked, neurons do not seem to be affected, but microglia become necrotic, at least in vitro. A similar dependency of glycolytic utilization of glucose is seen in high energy demanding cancer cells, and inhibition of glycolysis has been considered as anti-cancer treatment (Pelicano et al. 2006). In fact, 2-DG has reached the stage of clinical trials as a combination therapy in the treatment of advanced solid tumors (Raez et al. 2013). Whether the 2-DG-induced microglia depletion seen in vitro plays a role in the neuroprotective effects of 2-DG in vivo remains unclear. The increased energy demand of activated microglia can possibly be met by increasing the glycolytic use of glucose, which is also seen in activated macrophages (Kelly and O'Neill 2015). Similar to macrophages, quiescent microglia are likely to rely on oxidative phosphorylation of glucose for their ATP production. When stimulated, microglia switch from oxidative phosphorylation to glycolytic metabolism of glucose (Voloboueva et al. 2013). Activation with Lipopolysaccharide (LPS) not only increased lactate production but also decreased mitochondrial oxygen consumption and mitochondrial ATP production, indicative of increased glycolysis and decreased oxidative phosphorylation (Voloboueva et al. 2013). Another study confirmed increased lactate production by activated microglia when stimulated

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with both LPS and IFNγ (Gimeno-Bayon et al. 2014). This study also showed an upregulation of the glucose transporters GLUT1 and GLUT4 and of hexokinase 2 (HK2) (Gimeno-Bayon et al. 2014), a key enzyme in glycolysis (figure 1). The glucose transporters were proposed to increase glucose influx, whereas HK2 is thought to be a key promotor of aerobic glycolysis and has been shown to be upregulated in high energy demanding brain tumors (Wolf et al. 2011). Recent transcriptome studies on both human and mouse brain tissue show high expression of HK2 in microglia (Hickman et al. 2013, Lavin et al. 2014, Zhang et al. 2014). Together, these studies suggest that microglia adjust their glucose metabolic profile after a change in activity state, possibly to meet an increased energy demand. Low glucose levels induce microglial death, but high glucose levels have been shown to augment microglial activation. In postmortem brain tissue of critically ill septic patients treated in the ICU, an attenuation of microglial activation was found in patients who had controlled glucose levels before death (Sonneville et al. 2012). Insulin controlled euglycemic patients showed less microglial activity and neuronal damage compared to untreated hyperglycemic patients (Sonneville et al. 2012). In the same study, a hyper-inflammatory response was induced by applying third-degree burn injuries to rabbits. In this setting, uncontrolled hyperglycemic rabbits showed increased microglial activation, which could be prevented by maintaining insulin-induced euglycemia (Sonneville et al. 2012). However, whether this hyperglycemia-induced microglial activation needs inflammatory pre-conditioning is not clear. A number of in vitro studies showed comparable effects of glucose on microglial activation. One study, using primary rat microglia, showed that increasing the glucose concentration from 10 mM to 25 mM resulted in a 4-fold increased secretion of the proinflammatory cytokine TNFα (Quan et al. 2011). A second study, using primary rat microglia cultures, showed that TNFα secretion doubled when glucose concentration was raised from 25 mM to 50 mM (Zhang et al. 2015). In the same study, the presence of low levels of LPS in combination with high levels of glucose (35 mM) showed a dramatic amplification of TNFα secretion by microglia, which was not seen in the 25 mM glucose condition. This indicates that high glucose levels can enhance the LPS-induced inflammatory response in microglia cultures. Interestingly, microglia appear to be the only cells in the CNS expressing GLUT5 (Horikoshi et al. 2003). The exact function of GLUT5 in microglia in relation to glucose metabolism is not known. What is known is that GLUT5 has a low affinity for glucose, similar to GLUT2 (Manolescu et al. 2007). This could mean that GLUT5 has a function that is similar to that of GLUT2, e.g. to enable neurons and pancreatic β-cells to sense fluctuating glucose concentrations (Elizondo-Vega et al. 2015, Leloup et al. 2016). In contrast to the low glucose affinity of GLUT5, its affinity for fructose is much higher. Fructose is a monosaccharide closely related to glucose and a major component of our modern diet. Accumulating evidence from both human and animal studies shows that an excess of fructose causes an increased risk of metabolic disorders such as type II diabetes and insulin resistance, as well as an increased risk of neuroinflammation (Montonen et al. 2007, Lin et al. 2016, Xu et al. 2016). It has been shown that fructose fed rats have activated microglia and that primary rat microglia cultured under high fructose conditions showed upregulation of inflammatory pathways (Li et al. 2015). Thus, microglia could play a key role in these fructose-induced metabolic disorders, possibly via a hypothalamic mechanism. Most of the data we summarized about microglial glucose metabolism are from in vitro studies. However, the interpretation of data from in vitro studies needs to be done with caution when they contradict those from in vivo studies. Humans are considered to be diabetic with a fasting plasma glucose concentration above 7 mM or a 2-hour postprandial concentration above 11.1 mM (Giugliano et al. 2008). In diabetic mice, continuous measurements of blood glucose showed a mean concentration of approximately 20 mM, with peak concentrations up to 30 mM. These are the plasma concentrations; brain glucose concentrations are thought to be 5-fold lower than that of plasma (Dunn-Meynell et al. 2009). Yet, in vitro studies usually culture microglia under 25 mM glucose conditions (Dai et al. 2015,

ACCEPTED MANUSCRIPT Zhang et al. 2015, Beins et al. 2016, Vinoth Kumar et al. 2016), a concentration rarely seen in the brain during physiological or pathophysiological conditions. In leptin receptor mutant db/db mice, which are hyperglycemic, no microglial activation in the hypothalamus was observed (Gao et al. 2014), indicating that brain glucose may indeed be kept at a lower concentration.

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2.2 Fatty acids Fatty acids can be categorized into short, medium, long and very long-chain fatty acids and are called saturated when they contain no double bond, and unsaturated when they contain at least one double bond. Once taken up by the digestive system, fatty acids either reach the circulation directly or are released into the lymphatic system as chylomicrons. Peripheral tissue can take up fatty acids by releasing them from the chylomicrons by lipoprotein lipase (LPL) bound to endothelial cells (Niot et al. 2009). Once inside the cell, fatty acids can be used by mitochondria to generate ATP through a process called β-oxidation. In the brain, fatty acids are not generally considered to be a major substrate for ATP production. Nevertheless, fatty acids are taken up by the brain, and some studies report that fatty acid oxidation constitutes 20% of the total brain energy requirement (Ebert et al. 2003). Additionally, proteins involved in fatty acid metabolism, such as carnitine, are present in the brain, suggesting that fatty acid metabolism takes place in the brain (Jones et al. 2010). There are still two popular theories to explain the transport of fatty acids across the blood brain barrier (BBB), one theory proposes passive diffusion (Mashek and Coleman 2006, Hamilton and Brunaldi 2007) and the other proposes a protein-mediated transport mechanism (Mitchell and Hatch 2011). Cellular uptake of fatty acids appears to be mainly regulated by trapping them inside the cell by their β-oxidation into downstream metabolic intermediates like acyl-CoA. The β-oxidation of fatty acids into acyl-CoA is regulated by acyl-CoA synthetases (ACSs). Indeed, overexpression of the long-chain family members 4 and 5 of ACS (ACSL4 and ACSL5) in cell cultures, increased fatty acid uptake by 67% and 25–30%, respectively (Heimli et al. 2003, Mashek et al. 2006). In the brain, astrocytes have been considered to be the only cells capable of β-oxidizing fatty acids, but very recent transcriptome data show that microglia also express ACSs such as ACSL1, 3, 4 and 5 (Zhang et al. 2014) (figure 1). Furthermore, microglia express relatively high levels of LPL (Zhang et al. 2014) (figure 1), necessary for the release of fatty acids from triglycerides. This could indicate that also microglia are able to β-oxidize fatty acids and use them as an energy source. Utilization of fatty acids as an energy source can, in part, sustain the increased energy demand of activated microglia. Fatty acids have been shown to increase ROS production in a wide variety of cell types, including brain cells (Schonfeld and Wojtczak 2008). The production of ROS is the main contributor to cellular oxidative stress, a pathological feature found in a wide range of neurological diseases (Chen et al. 2012, Carvalho et al. 2016). Brain tissue is prone to be damaged by ROS due to the relatively low levels of anti-oxidants and its high content of unsaturated lipids (i.e., containing double bonds), which are especially vulnerable to damaging modifications by ROS (Uttara et al. 2009). In phagocytic neutrophils, fatty acids are known to stimulate ROS production by interfering with the plasma membrane NADPH oxidase system (Schonfeld and Wojtczak 2008). Complex assembly of the two phox phox membrane subunits p22 and gp91 is regulated by three regulatory cytosolic phox phox phox components p40 , p47 and p67 , and a small GTPase, either Rac1 or Rac2 (Gonzalez et al. 2007). Fatty acids are assumed to interfere with these components, resulting in an increased production of ROS (Schonfeld and Wojtczak 2008). Whether fatty acids induce a similar overproduction of ROS by microglia is not known. However, microglia highly express all the components of the NADPH oxidase system (Zhang et al. 2014), and the NADPH oxidase system has been shown to be the source of LPS-stimulated extracellular superoxide production by microglia (Bordt and Polster 2014). Furthermore, compared to

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glucose, fatty acid metabolism requires ~15% more oxygen to generate the same amount of ATP (Schönfeld and Reiser 2013). This fatty acid driven increase in oxygen demand is well tolerated by well-oxygenated organs, such as the heart, but could possibly cause problems in the brain, where oxygen concentrations are lower (Schönfeld and Reiser 2013). Apart from their possible role as an energy substrate for microglia, fatty acids are also thought to serve as signaling molecules and to influence microglial activity. In general, unsaturated fatty acids are considered to have a beneficial effect on human health, for instance through their anti-inflammatory effects. Intracerebroventricular (ICV) injections of ω-3 linolenic and ω9 oleic fatty acid significantly reduced hypothalamic inflammation, not only shown by a reduction of inflammatory markers but also by a reduction of F4/80 positive microglia in the arcuate nucleus (Cintra et al. 2012). Both these fatty acids are supposed to activate the unsaturated fatty acid receptor (GPR120) found on hypothalamic neurons (Cintra et al. 2012). However, recent transcriptome studies in human brain showed expression of GPR120 not only in neuronal cells but also in microglia (Zhang et al. 2014), indicating that these fatty acids might also activate microglia directly. More evidence of this direct effect was provided by a different study, which showed that pre-treatment of primary microglia with polyunsaturated fatty acids (PUFAs) decreased their inflammatory response to the inflammatory stimulators, myelin and IFNγ (Chen et al. 2014). A different in vitro study showed that a mixture of ω-3 PUFAs protected against LPS-mediated cytotoxicity in a microglia cell line (BV2) (Corsi et al. 2015). Incubation with PUFAs also inhibited ROS and nitric oxide production by LPSactivated BV2 cells (Corsi et al. 2015). Together these data indicate that unsaturated fatty acids have an inflammatory-inhibiting effect on activated microglia, but whether this is through the GPR120 receptor remains to be resolved. Unlike unsaturated fatty acids, saturated fatty acids are generally considered as unhealthy fatty acids. Although the inflammatory effect of saturated fatty acids on adipose tissue and macrophages has been well documented and seems to be mediated through the toll-like receptor 4 (TLR4) (Chait and Kim 2010), not much is known about their effect on microglial activation. An in vivo study showed microglial activation in the hypothalamus when mice were given a diet rich in saturated fatty acids (Valdearcos et al. 2014). The same study showed a direct effect of saturated fatty acids, especially the long chain fatty acids (LCFA), on cultured mice microglia (Valdearcos et al. 2014). A different study investigated the direct effect of both saturated and unsaturated LCFAs on hypothalamic microglia in vivo. Rats were treated with ICV injections containing different LCFAs, such as palmitic, stearic, linoleic, linolenic, arachidic or behenic acids (Milanski et al. 2009). Only the saturated LCFAs - stearic, arachidic and behenic - induced an inflammatory response (Milanski et al. 2009). It turned out that this inflammatory response could be attenuated by ICV administration of TLR4 inhibitors, which suggests a TLR4 mediated response. Microglia might be involved in the saturated fatty acid-induced inflammation, as they are one of the TLR4 expressing cells in the brain (Milanski et al. 2009). This research suggests that saturated fatty acids possibly induce a microglialinduced inflammatory response in the hypothalamus, but more research is needed to clarify the precise mechanism. A different kind of saturated fatty acids are the short chain fatty acids (SCFAs), exclusively produced by the microbiota in the colon, the main ones being acetate, propionate and butyrate (den Besten et al. 2013). SFCAs have been shown to have beneficial effects on appetite regulation and energy homeostasis (Byrne et al. 2015), and several mechanisms involved in metabolism have been implicated, including the hypothalamic arcuate nucleus (Frost et al. 2014). Here they show that intraperitoneal administered acetate accumulated in the hypothalamus and also resulted in reduced food intake and induction of an anorexigenic neuropeptide expression profile in the hypothalamus (Frost et al. 2014). Whether microglia play a role in any of these processes that underlie the beneficial effect of SFCAs is not known. However, SCFAs do seem to be of critical importance for microglia functioning in general. Studies using germ free mice, which do not have SCFAs because they lack a

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microbiota, showed impaired maturation and function of microglia (Erny et al. 2015). Microglial impairment could be restored not only by the introduction of a complex microbiota, but also by supplementing SCFAs via the drinking water (Erny et al. 2015). A similar -/microglial impairment was found in SCFA receptor knockout mice (FFAR2 ), although thus far in mice no FFAR2 expression has been found in microglia or any other cells of the CNS. Instead, expression was only found on peripheral myeloid cells (Erny et al. 2015). This suggests that SCFAs indirectly alter microglia functioning (Erny et al. 2015). A recent transcriptome study corroborated the lack of FFAR2 expression in the murine CNS, but did find FFAR2 expression in human microglia (Zhang et al. 2014). The FFAR2 expression in human microglia could be an interspecies difference, but expression is very low so whether this has any relevance is uncertain. Another study showed that the SCFA butyrate protected against an LPS-induced inflammatory response in rat primary microglia, hippocampal slice and co-cultured microglia, astrocytes and cerebellar granule neurons (Huuskonen et al. 2004). In the same study, the opposite effect of SFCAs was seen on a cell line of immortalized microglia (N9), where an enhanced LPS response was found (Huuskonen et al. 2004). The majority of these studies thus suggest that SCFAs can influence microglial functioning in an anti-inflammatory fashion, but the role of the FFAR2 receptor remains unclear. Whether the anti-inflammatory effect of SFCAs on microglia found in-vitro possibly plays a role in the beneficial effect on appetite regulation in vivo might be interesting avenue of investigation.

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2.3 Glutamine Glutamine is the most abundant amino acid in plasma and has been considered to be unable to freely pass the BBB. Multiple glutamine transporters are present in the BBB and these are mainly responsible for moving glutamine from the brain into the circulation. However, some might also play a role in glutamine uptake into the brain (Xiang et al. 2003). In the brain, astrocytes produce glutamine from excess glutamate and ammonia in order to protect neurons against excitotoxicity (Suarez et al. 2002). Both glutamate and ammonia have been shown to be neurotoxic (Manev et al. 1989, Felipo and Butterworth 2002, Suarez et al. 2002), and subcutaneous glutamate injections resulted in an obese phenotype (Olney 1969). Although glutamine is, first and foremost, the end product of glutamate and ammonia metabolism, it can also be used as an energy source in the brain. Cellular uptake of glutamine is accomplished by several amino acid transporters (Pochini et al. 2014). The amino acid transporter mainly responsible for glutamine uptake is solute linked carrier family 1 member A5 (SLC1A5), which has a high affinity for glutamine and has been shown to be upregulated in several high-energy demanding tumors (De Vitto et al. 2016). When glutamine is taken up it can be broken down to α-ketoglutarate via glutaminase (GLS) and glutamate dehydrogenase (GLUD1). GLS, GLUD1 as well as SLC1A5 are highly expressed in human and mouse microglia (figure 1). The majority of amino acid transporters listed in (Pochini et al. 2014) are also expressed in human and mouse microglia (figure 1), indicating microglia have an active glutamine metabolism pathway. Several studies have shown that microglial glutamate production is neurotoxic. A mouse model with upregulated microglial levels of the glutamine transporter SLC38A1 (figure 1) constitutively produced and excreted glutamate into the interstitial space, which resulted in low levels of neurotoxicity (Jin et al. 2015). Research using primary cultures of human microglia showed that infection with HIV-1 increased the extracellular glutamate levels and amplified neurotoxicity (Huang et al. 2011). Cultured microglia infected with Japanese encephalitis virus showed elevated release of glutamate into the medium, and this conditioned medium could thus provoke significant neuronal damage in neuronal cultures (Chen et al. 2012). The role of hypothalamic microglial glutamine metabolism and its interactions with other energy resources is still completely unknown. More studies are needed to clarify the degree

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3.1 Microglia, astrocyte and neuron interaction Microglial energy consumption is dependent on the degree of activity of the microglia. Healthy neurons and astrocytes keep them in the relatively low energy demanding “resting state” (Shih et al. 2006, Eyo and Wu 2013). Detection of neuronal activity is important for microglia, indicated by the great variety of neurotransmitter receptors they express, including glutamate, GABABR, dopamine, purinergic, cholinergic and opioid receptors (Pocock and Kettenmann 2007). Possibly, the amount of extra synaptic neurotransmitters released during normal brain function can suppress microglial activation (Pocock and Kettenmann 2007). Astrocyte-conditioned medium has been shown to keep microglia in a quiescent state (Shih et al. 2006). Activation of both microglia and astrocytes has been associated with various neurodegenerative diseases (Mandybur , Beach et al. 1989, Bates et al. 2002, AlfonsoLoeches et al. 2010, Howell et al. 2010, Popescu et al. 2013). Whether astrocytes can activate microglia or if microglia are activated by astrocytes first, has been a long-standing debate (Liu et al. 2011). One study has shown that cultured astrocytes could be activated with LPS, indicating astrocytes can be activated without microglia (Brahmachari et al. 2006). However, astrocyte cultures frequently contain small amounts of microglia, which renders interpretation of these results difficult. Research using astrocyte cultures with depleted residual microglia showed a decreased LPS response, but when these astrocyte cultures were reconstituted with microglia, the LPS response returned, and even exceeded the LPS response of microglia alone (Barbierato et al. 2013). Furthermore, research focusing on highmobility group box 1 (HBMG1)-induced edema, showed that astrocyte cultures only showed a response to HBMG1 when microglia were present (Ohnishi et al. 2014). A different study showed that the LPS-induced astrocyte activation needed functional receptors for IL-1β and TNF-α, the ligands of which are produced by microglia (Abudara et al. 2015). Together these data show that astrocytes can interact with microglia and keep them in a quiescent state. Whether astrocytes can also activate microglia remains obscure.

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3.2 Microglial reactivity and hypothalamic dysfunction in control of energy homeostasis In diet-induced obese mice, activated microglia are found specifically in the mediobasal hypothalamus (Thaler et al. 2012). It is not surprising that microglia in the mediobasal hypothalamus are activated by certain blood borne factors, because this part of the brain is in close proximity to the median eminence, a region known for having a locally decreased bloodbrain barrier (BBB) function (Johnson and Gross 1993). Because of this, circulating fatty acids and other blood borne factors can diffuse more directly into this region (Jastroch et al. 2014). Hypothalamic inflammation in this part of the brain has been associated with metabolic disorders (Thaler et al. 2012, Dorfman and Thaler 2015). Two key questions raised by the association of hypothalamic microglial activation and metabolic disorders are: 1) are there fuel switches between fatty acids, glucose and/or glutamine in activated microglia and 2) which stimulus is linked to which microglial fuel preference and is thus responsible for the different microglial metabolic phenotypes? Clarifying microglial fuel preference might prove to be vital for understanding microglial pathogenesis and might become of interest as a cellspecific therapeutic strategy to normalize microglial function. 3.3 Therapeutic strategy targeting microglia in obesity treatment Most anti-obesity treatments target the neurons that are responsible for the regulation of energy metabolism by either inhibiting the orexigenic neurons or stimulating the anorexigenic neurons. Due to a lack of specificity, anti-obesity drugs targeting these regulatory neurons either show little weight reduction or have serious side effects, as exemplified by the drugs

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targeting neuropeptide Y, serotonin and endocannabinoids (Di Marzo 2008, Sargent and Moore 2009, Rodgers et al. 2012, Berglund et al. 2013). New targets are needed for the treatment of obesity and these might be found in microglia, in view of their newly identified roles in hypothalamic dysregulation associated with obesity. In DIO mice, depletion of microglia in the mediobasal hypothalamus abolished diet-induced inflammation and reduced food intake (Valdearcos et al. 2014). Treatment of microglianeuron co-cultures with 2-DG has shown that inhibition specifically of microglia increases neuronal survival (Vilalta and Brown 2014). Anti-inflammatory drugs like corticosteroid analogues would be a possible treatment to dampen the microglial inflammation during obesity in humans, but these drugs have been shown to have serious side effects, such as osteoporosis, aseptic joint necrosis and adrenal insufficiency (Buchman 2001). A more specific effect, i.e., only inhibiting microglia, is desired when using drugs like corticosteroids. A new and promising strategy seems to be the cell specific pharmacotherapy as introduced by Dimarchi & Tschöp (Finan et al. 2012). They successfully targeted specific tissues, while at the same time preventing adverse effects in other tissues, by conjugating estrogen to glucagon-like peptide-1 (GLP-1). Only cells expressing both estrogen and GLP-1 receptors were affected by this conjugate (Finan et al. 2012). A similar strategy could be used to specifically inhibit microglial activation during obesity. In the CNS, the receptor for chemokine (C-X3-C motif) ligand 1 (CX3CL1) is exclusively expressed by microglia. Conjugation of a corticosteroid analogue to CX3CL1 would only target cells expressing the CX3CL1 receptor, without affecting other cells in the CNS (Kalin et al. 2015).

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4. Conclusion An increased energy demand by activated microglia may be accompanied by a change in their metabolic profile. A switch towards increased fatty acids and glutamine metabolism might result in the generation of metabolites, which are harmful for neuronal survival. Furthermore, different fatty acids have been shown to either have proinflammatory or antiinflammatory effects on microglia. As the hypothalamus is in close contact with the circulation, this region is specifically prone to be influenced by a change in circulating blood borne factors. Also, obesity has been associated with hypothalamic inflammation, possibly caused by activated microglia. If activated microglia create an unhealthy microenvironment for neuronal survival, and cause hypothalamic dysfunction, eventually leading to obesity needs to be answered. Research into the cellular metabolism of how activated microglia meet their energy demands and which blood borne factors can induce such an activation might shed some light onto the underlying causes of obesity.

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Acknowledgements We thank Wilma Verweij for her help during the editing process.

ACCEPTED MANUSCRIPT glucose

fructose

fatty acids

glutamine

2-DG LPL

GPR120

fatty acids

glucose

ACSL

fatty acyl CoA

glycolysis

acetyl-CoA

TCA

pyruvate

anaerobic respiration

SLC38A1

glutamine

fatty acyl CoA + carnitine

ATP

HK2

SLC1A5

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GLUT4 GLUT5

GLS

Glu

NH4+

GLUD1

mitochondrion mitochondrion

ETC

lactate

fatty acids

microglia lactate

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NADPH oxidase complex

α-KG

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GLUT1 GLUT3

ROS

glutamate + NH4+

possibly neurotoxic

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Figure 1. Overview of metabolic pathways for the three major energy substrates in microglia. Glucose can enter microglia via glucose transporters GLUT1, GLUT3, GLUT4 and GLUT5. GLUT5 has also been shown to facilitate the passage of fructose across the plasma membrane. Once inside the cell, glucose undergoes glycolysis which, in part, is mediated by hexokinase2 (HK2). The generated pyruvate can either enter the mitochondria where it is further metabolized with the use of oxygen by the tricarboxylic acid (TCA) and electron transport chain (ETC), or pyruvate can be further metabolized in the cytosol without the use of oxygen, resulting in the formation and secretion of lactate. 2-Deoxy-D-glucose (2-DG), a glucose analogue, uses the glucose transporters to enter the cell where it inhibits glycolysis. Lipoprotein lipase (LPL) mediates the cellular uptake of fatty acids. Long chain fatty acyl CoA synthetase (ACSL) catalyzes the formation of fatty acyl CoA, which can only enter the mitochondria together with the carrier protein carnitine. Once inside the mitochondria, fatty acyl CoA is β-oxidized into acetyl-CoA, which enters the TCA cycle and subsequently the ETC in order to generate ATP. GPR120 is known to bind unsaturated fatty acids and is possibly involved in the anti-inflammatory effects of unsaturated fatty acids. Microglia highly express all the components of the NAPDH oxidase complex, which has been shown, in macrophages, to be stimulated by fatty acids to increase its production of reactive oxygen species (ROS). The glutamine receptors SLC1A5 and SLC38A1 are expressed with microglia and enable microglia to take up glutamine. Inside the mitochondria glutamine is converted to glutamate and ammonia (NH4+) by the enzyme glutaminase (GLS), glutamate is further metabolized by the enzyme glutamate dehydrogenase 1 (GLUD1) to α-ketoglutarate, which can enter the TCA cycle. Both microglial-produced glutamate and ammonia have been shown to have neurotoxic effects.

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Highlights Microglia express proteins involved in metabolism of the main energy substrates.

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Activated microglia might meet their increased energy demand by a metabolic switch.

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Such a switch could generate metabolites harmful to neighboring neurons.

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