Mechanisms Mediating the Actions of Fatty Acids in the Hypothalamus

Journal Pre-proofs Review Mechanisms mediating the actions of fatty acids in the hypothalamus Nathalia R. Dragano, Milena Monfort-Pires, Licio A. Velloso PII: DOI: Reference:

S0306-4522(19)30698-0 https://doi.org/10.1016/j.neuroscience.2019.10.012 NSC 19315

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Neuroscience

Received Date: Revised Date: Accepted Date:

24 June 2019 2 October 2019 3 October 2019

Please cite this article as: N.R. Dragano, M. Monfort-Pires, L.A. Velloso, Mechanisms mediating the actions of fatty acids in the hypothalamus, Neuroscience (2019), doi: https://doi.org/10.1016/j.neuroscience.2019.10.012

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Review

Mechanisms mediating the actions of fatty acids in the hypothalamus

Nathalia R. Dragano§, Milena Monfort-Pires§, Licio A. Velloso* Laboratory of Cell Signaling – Obesity and Comorbidities Research Center, University of Campinas – 13083-8864, Campinas SP, Brazil

*Corresponding Author E-mail address: [email protected] (L. A. Velloso)

§The

authors contributed equally to the work.

Abbreviations AA: Arachidonic acid ACC: Acetyl-CoA carboxylase ACS: Long-chain fatty acid CoA synthase ADHA: Attention-deficit/hyperactivity disorder AgRP: Agouti-related peptide AMPK: AMP-activated protein kinase ARC: Arcuate nucleus BAT: Brown adipose tissue BBB: Blood-brain barrier CD1: Cluster of differentiation 1 CD36: Cluster of differentiation 36 CNS: Central nervous system CPT1: Carnitine palmitoyltransferase-1 DAG: Diacyl-glycerol DHA: Docosahexaenoic acid DIO: Diet-induced obesity DPA: Docosapentaenoic acid EPA: Eicosapentaenoic acid ER: Endoplasmic reticulum FA: Fatty acid FAS: Fatty acid synthase FATP: Fatty acid transport protein FFA: Free fatty acid GLP1: Glucagon-like peptide-1 GPR: G-protein-coupled receptors HFD: High-fat diet Icv: Intracerebroventricular iNKT: Invariant natural killer T cell LA: Linolenic acid LCFA: Long-chain fatty acid LPL: Lipoprotein lipase MCD: Malonyl-CoA decarboxylase MD: Mediterranean diet MSFD2A: Major facilitator superfamily domain-containing protein 2A

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MUFA: Monounsaturated fatty acid NPY: Neuropeptide Y OA: Oleic acid PC2 Prohormone convertase 2 POMC: Proopiomelanocortin PPAR: Proliferator of peroxisome activated receptor PUFA: Polyunsaturated fatty acid RvD2: Resolvin D2 SFA: Saturated fatty acid TLR4: Toll-like receptor 4 UFA: Unsaturated fatty acid UPR: Unfolded protein response VMH: Ventromedial hypothalamus

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Abstract Selected neurons of the hypothalamus are equipped with molecules specialized in sensing the energy status of the organism. Upon activation or inhibition by central and systemic factors, such as neurotransmitters, hormones, cytokines, and nutrients, these molecules play important roles in the regulation of neuronal responses that control whole-body energy homeostasis. Dietary fats can control hypothalamic function by acting upon distinct energy sensing systems. They can be metabolized inside neurons, producing signals that control the expression of neurotransmitters involved in energy homeostasis; moreover, excessive amounts of certain fatty acids can activate inflammatory signaling in microglia, astrocytes, and neurons, leading to functional abnormalities and, eventually, neuronal apoptosis. In addition, recent studies have identified lipid-sensing G-protein-coupled receptors in the hypothalamus, revealing their involvement in the regulation of caloric intake and energy expenditure, as well as in the hypothalamic inflammatory response that occurs in obesity. Because of advances in the generation of synthetic ligands for this class of receptors, it is expected that pharmacological modulation of selected lipid-sensing G-protein-coupled receptors in the central nervous system could provide therapeutic advances in obesity and other metabolic diseases. Here we review seminal work in this field. Key Words: G-protein-coupled receptors; lipid; hypothalamus; neuron; nutrient; obesity.

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Introduction Physiologically, dietary fats provide the second most important source of energy for humans. Due to their high caloric density, it has been proposed that evolutionary pressure provided humans and other species with various lipid-sensing mechanisms that evolved to enhance the capacity to detect and consume fats, thereby increasing the energy harvesting and storage capacity of the organism (Muskiet, 2010). These lipid-sensing mechanisms include the obvious olfactory and taste sensors but also sensors in other regions of the body that integrate a complex network of cells and systems involved in whole-body energy homeostasis. One such region is the hypothalamus. Studies dating back to the 1950’s have provided seminal evidence for the existence of selected hypothalamic neuronal populations capable of sensing the energy status of the body and responding by controlling hunger/caloric intake and energy expenditure (Kennedy, 1950; Miller et al., 1950). During the 1960’s-1970’s electrophysiological studies refined the anatomical characterization of the hypothalamic regions involved in energy homeostasis (Mendelson and Chorover, 1965; Steinbaum and Miller, 1965; Feldman et al., 1966; Shimazu et al., 1966; Weiner et al., 1971); and in the 1990’s, the identification of leptin and its receptor paved the way for the subsequent description of a number of distinct brain and systemic substances that play important roles controlling the energy status of the body (Zhang et al., 1994; Tartaglia et al., 1995). These substances include hormones, neurotransmitters, cytokines, and nutrients that impact with different potencies on distinct neuronal subpopulations to provide a complex and integrated response aimed at maintaining body mass stability (Liu and Kanoski, 2018; Rossi and Stuber, 2018). Dietary fats have emerged as important regulators of the hypothalamic neurons involved in energy homeostasis (Obici and Rossetti, 2003; Chianese et al., 2018). Evidence of scientific efforts to characterize the effects of dietary fats on the hypothalamus is the progressive increase in the number of publications in this field: using the search terms hypothalamus and fat, we found 36 publications in 1993 (the year before the identification of leptin); 107 in 2004 (10 years after the identification of leptin), and 223 in 2018 (PubMed search). Studies published during this period identified a number of fat sensors in hypothalamic cells and defined the mechanisms

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by which fatty acids (FAs) control the function of neurons involved in the regulation of caloric intake and energy expenditure. In addition to classical biochemical mechanisms by which the metabolism of fat inside neurons triggers responses that increase food intake and inhibit energy expenditure, studies have identified FA membrane receptors that act through other signaling mechanisms to control whole-body energy homeostasis (Obici and Rossetti, 2003; Milligan et al., 2017; Elizondo-Vega et al., 2019). Some of these receptors have emerged as potential targets for the treatment of obesity and metabolic diseases, further increasing the interest in this field (Christiansen et al., 2008; Wagner et al., 2013; Oh et al., 2014; Dragano et al., 2017; Pascoal et al., 2017). In this review, we show the wide range of mechanisms and outcomes related to lipid sensing in the hypothalamus.

The transport of circulating fatty acids to the brain FAs are transported from the circulation to the brain where they can be converted into longchain fatty acid-Coenzyme A (LCFA-CoA) and metabolized by β-oxidation or incorporated into phospholipids (Obici et al., 2003); however, the exact mechanism by which FAs from the periphery can cross the blood–brain barrier and enter the cells of the central nervous system (CNS) is only partially characterized (Levin et al., 2011; Bazinet and Laye, 2014; CrucianiGuglielmacci et al., 2017). The brain is rich in polyunsaturated FAs (PUFAs), but essential FAs are transported into the brain from the circulation, and there is evidence that up to 8% of longchain PUFAs undergo a daily turnover, being replaced by plasma-derived FAs (Rapoport et al., 2001; Bruce et al., 2017). There is evidence that FAs can enter by passive diffusion (Hamilton et al., 2001) or be translocated by carrier proteins, mainly cluster of differentiation 36 (CD36) and FA transport proteins (FATP)-1 (Le Foll et al., 2009), which are most likely to bind to LCFA (Fig. 1). Some authors suggested that CD36 and other carrier proteins act as trapping proteins for FAs instead of transporters (Bazinet and Laye, 2014). Recent data indicate that FATP1 and FATP4 are the most important FA transport proteins expressed on the blood brain barrier (BBB) and that CD36 has an important role in the transport of FAs across human brain microvessel endothelial cells 6

(Duca and Lam, 2014; Bruce et al., 2017) (Fig. 1). Using dissociated neurons from the ventromedial hypothalamus (VMH) and investigating the glucose- and oleic acid (OA, C18:1)induced changes in intracellular Ca2+, Le Foll (Le Foll et al., 2009) observed that these neurons express FATP-1 and CD36 mRNAs and thus, can take up FAs. The inhibition of VMH neuronal expression of CD36 using an adenovirus carrying a short hairpin RNA resulted in increased caloric intake and increased adiposity (Le Foll et al., 2015); thus the authors suggested that some of the effects of OA on neurons could occur via a mechanism independent of its intracellular metabolism (Le Foll et al., 2013; Le Foll et al., 2014). Nevertheless, it is still unclear, which VMH neuronal subpopulation is actually involved in this regulation. It is believed that at least half of the lipid sensing responses in hypothalamic neurons are mediated by the translocator/receptor CD36 (Moulle et al., 2014; Magnan et al., 2015). Recent studies have also shown that the orphan receptor MSFD2A (major facilitator superfamily domain-containing protein 2A) could act as a transporter for docosahexaenoic acid (DHA) esterified to lysophospholipids (Rodriguez-Navas et al., 2016). Furthermore, other intracellular mechanisms, including FA oxidation or synthesis of diacyl-glycerol (DAG) and ceramides, which are mediators of lipotoxicity in peripheral organs, are important in lipid sensing and energy homeostasis (Cruciani-Guglielmacci et al., 2017).

Hypothalamic lipid sensing by intracellular mechanisms A number of studies have demonstrated that hypothalamic lipid metabolism has a critical role in the regulation of energy balance. Enzymes and intermediates of FA metabolism can provide signals that inform the hypothalamus of the energy status, allowing it to adjust food intake and energy expenditure as well as regulate glucose metabolism and lipogenesis (Wolfgang and Lane, 2006; Bruce et al., 2017). This phenomenon was named lipid sensing, and the molecular mechanisms behind its regulation are still under intense investigation (Cruciani-Guglielmacci et al., 2017). The concept that lipids may be an important part of a central fuel-sensing mechanism was introduced as early as the 1950’s and became known as the lipostatic hypothesis (CrucianiGuglielmacci et al., 2017). In 1975, Oomura and coworkers provided the first evidence that 7

neurons in the lateral hypothalamus are able to respond to FAs present in the blood and that free fatty acids (FFAs) can inhibit the activity of glucoreceptor cells in the VMH (Oomura, 1983). Furthermore, it was shown that peripheral infusion of lipids suppressed spontaneous food intake in primates (Woods et al., 1984; Lopez et al., 2007). This outcome suggested that circulating FAs could act as signaling molecules that provide information to the brain on the energy status of the organism (Fig. 1). The interaction between hypothalamic FA sensing and the regulation of food intake was confirmed as studies demonstrated that central administration of LCFAs exerted a signaling role within hypothalamic energy centers (Obici et al., 2002; Morgan et al., 2004). For instance, intracerebroventricular (icv) administration of OA markedly inhibits food intake. The anorectic effects of OA are related to decreased expression of hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AgRP). Conversely, hypothalamic proopiomelanocortin (POMC) expression is not altered following icv OA treatment, indicating that the anorectic action of LCFAs is mediated by specific inhibition of orexigenic neuropeptides. Additionally, icv OA infusion results in a reduction of hepatic glucose production, thus enhancing insulin sensitivity (Obici et al., 2002; Morgan et al., 2004). To further investigate the physiological relevance of these findings, Rossetti and colleagues evaluated whether elevation of circulating levels of LCFAs could produce an increase in LCFACoA levels in the hypothalamus and consequently generate a metabolic signal of energy surplus. Indeed, upon entry into neurons, LCFAs are quickly esterified by LCFA-CoA synthase (ACS) to form LCFA-CoA. Furthermore, this increase is prevented by simultaneous icv treatment with triascin-C, a pharmacological inhibitor of ACS. Taken together, these findings suggest that an increase in the hypothalamic levels of LCFA-CoAs is important in the inhibition of food intake during systemic increases in lipid availability (Lam et al., 2005b). Considering that cellular accumulation of LCFA-CoAs within the hypothalamus is translated as a lipid signal of satiety, it is expected that increased LCFA availability or decreased cellular usage of esterified LCFAs could trigger similar anorectic effects (Lam et al., 2005b). The range of the intracellular pool of LCFA-CoAs is determined by the rate of its oxidative versus biosynthetic pathways. In metabolic tissues, intracellular LCFA-CoAs are translocated into mitochondria via 8

carnitine palmitoyltransferase-1 (CPT1) where they undergo β-oxidation. Compelling evidence has shown that the activity of CPT1 is a key determinant of the level of cytosolic LCFA-CoAs. For example, enriching LCFAs-CoAs by blocking their catabolism via molecular and pharmacological hypothalamic CPT1 inhibition is sufficient to suppress food intake and to reduce the expression of NPY and AgRP in the arcuate nucleus (ARC) (Obici et al., 2003) (Fig. 1). Moreover, mice deficient in CPT1c, the CNS-specific isoform of CPT1, have lower body weight and food intake than their wild-type littermates but gain excessive body mass when fed a high-fat diet (HFD) (Wolfgang and Lane, 2006; Wolfgang et al., 2008). In neurons of the ARC, mitochondrial CPT1c activity is regulated by the availability of malonylCoA. Under physiological conditions, CPT1 activity is inhibited by increased malonyl-CoA concentration, and hypothalamic malonyl-CoA levels correlate closely with nutritional status. Thus, when the supply of glucose is high, malonyl-CoA levels increase along with a decreased need for FA β-oxidation. Therefore, it has been hypothesized that increased levels of malonylCoA may act as a signal of energy surplus that regulates orexigenic and anorexigenic neuropeptide release to suppress food intake and increase energy expenditure (Lopez et al., 2005; Wolfgang and Lane, 2006; Wolfgang et al., 2008). Moreover, the levels of malonyl-CoA is point of integration between the signals generated by lipids and glucose as reviewed elsewhere (Blouet and Schwartz, 2010). Levels of malonyl-CoA depend on the equilibrium of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and malonyl-CoA descarboxilase (MDC). The activities of ACC and malonylCoA descarboxilase are directly regulated by phosphorylation via AMP-activated protein kinase (AMPK). When AMPK is active, it phosphorylates and inhibits ACC; decreases FAS mRNA expression, and activates MCD. Thus, as a whole, AMPK activation reduces malonyl-CoA levels and the flux of substrates through the fatty acid biosynthetic pathway. Decreased malonyl-CoA levels stimulate CPT1, which promotes access of LCFAs-CoA into the mitochondria, followed by an increase in fatty-acid oxidation (Dowell et al., 2005; Lopez et al., 2007; Lage et al., 2008; Dieguez et al., 2009). Experimental manipulation of the key enzymes involved in malonyl-CoA metabolism, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and malonylCoA decarboxylase (MCD), has been shown to have major effects on hypothalamic energy 9

homeostasis regulation (Bruce et al., 2017). For example, inhibition of hypothalamic ACC abolished leptin-mediated decreases in food intake, body weight, and expression of NPY. These findings demonstrate that energy regulation by ACC is due to a rise in its product, malonyl-CoA (Gao et al., 2007). Similarly, inhibition of FAS reduces food intake and body weight in rodents through a leptin-independent mechanism, as ob/ob mice respond similarly to wild-type mice when treated with FAS inhibitors. The biochemical outcome of FAS inhibition is the accumulation of malonyl-CoA. The increased levels of malonyl-CoA are due to the accumulation of substrate following FAS inhibition as well as the inhibition of AMP-activated protein kinase (AMPK) activity, which leads to increased synthesis of malonyl-CoA by ACC (Loftus et al., 2000; Clegg et al., 2002; Lam et al., 2005a; Lopez et al., 2007). Accordingly, malonyl-CoA, an intermediate in the fatty acid biosynthetic pathway, has emerged as a central player in the energy homeostatic signaling system of the hypothalamus. Interestingly, other pathways have been described as important in the regulation of lipid sensing. Recent data indicate that lipoprotein lipase (LPL) may facilitate the uptake of FAs into dissociated hypothalamic neurons and that mice with neuronal LPL deficiency have a defect in nutrient sensing, exhibiting a hyperphagic behavior when compared with control mice (Wang et al., 2011; Bruce et al., 2017). Contrary to physiological conditions, during increased and sustained availability of nutrients, the hypothalamic nutrient-sensing system may be deregulated and the pool of LCFA increased (Obici et al., 2002; Morgan et al., 2004), impacting on energy intake and expenditure. As short as three days of overfeeding is enough to suppress the effects of OA in reducing food intake (Karmi et al., 2010). Moreover, under pathological conditions, such as obesity and type 2 diabetes, there is an increase in circulating LCFA and increased uptake by the brain, which leads to augmented LCFA-CoA in the hypothalamus impacting on the control of caloric intake and energy expenditure (Karmi et al., 2010; Dietrich and Horvath, 2013).

Gut lipid sensing provides signals to the brain The gastrointestinal system has an important role in hypothalamic lipid sensing, and studies have uncovered bidirectional signaling between the gut and the brain, which plays an important role in the control of energy homeostasis (Sundaresan and Abumrad, 2015; Romano et al., 10

2017). Some of these signals depend on neural connections through the nucleus of the solitary tract, whereas others depend on the production of gut peptides that signal directly in hypothalamic neurons; comprehensive reviews on this theme have been recently published (Duca and Yue, 2014; Mayer et al., 2015; Andermann and Lowell, 2017; Fetissov, 2017). FATP1 and CD36 are expressed in the mouth and the gut, suggesting that lipid sensing is a complex widespread process (Magnan et al., 2015; Romano et al., 2017). Also, the G-protein-coupled receptors (GPRs) involved in medium- and long-chain FA sensing are expressed in taste receptor cells, and both GPRs and CD36 are found in the gut (Duca et al., 2013). Moreover, lipid infusion into the small intestine can suppress food intake in rodents and humans, and this effect could be mediated by sensory gut innervation and CD36, as CD36 KO mice present a blunted response to lipid infusion into the gut (Schwartz et al., 2008). The proposed lipid sensing mechanism that occurs in the gut and brain includes the presence of the GPRs 40 and 120, as well as CD36 and FATP-4. LCFAs can activate enteroendocrine cells that release gut peptides, either by intracellular pathways or electrical activity, leading to Ca2+ mobilization and peptide exocytosis. The release of cholecystokinin and glucagon-like peptide-1 (GLP1) can, in turn, signal the hindbrain to reduce food intake and control hepatic glucose production (Duca et al., 2013; Duca and Yue, 2014). In addition, studies with the GLP1 agonist liraglutide have identified GLP1-binding sites in POMC neurons and GABA-producing neurons, resulting in integrated regulation of caloric intake and energy expenditure by direct action in the energy homeostasis regulation center of the hypothalamus (Beiroa et al., 2014; Secher et al., 2014; Geloneze et al., 2017).

Inflammatory response to dietary fats in the hypothalamus The consumption of nutritionally suitable amounts of dietary fats results in the correct regulation of hypothalamic neurons involved in whole-body energy homeostasis. However, studies have shown that overconsumption of dietary fats, particularly saturated fats, impacts negatively on the hypothalamus (Velloso and Schwartz, 2011) (Fig. 2). In rodents, the transcription of inflammatory genes in the hypothalamus is triggered as early as few hours after the first exposure to a high-fat diet (HFD) (Thaler et al., 2012; Morari et al., 2014; Souza et al., 2016; 11

Carraro et al., 2018). As the consumption of a HFD continues for a prolonged period, inflammation is boosted by the activation of endoplasmic reticulum (ER) stress (Zhang et al., 2008; Milanski et al., 2009; Ozcan et al., 2009) and by the progressive development of a number of cellular abnormalities, such as defects in mitochondria (Schneeberger et al., 2013), activation of RNA stress (Araujo et al., 2014; Yan et al., 2014) and the abnormal regulation of proteostasis and autophagy (Portovedo et al., 2015), eventually leading to neuronal apoptosis (Moraes et al., 2009). ARC microglia and astrocytes provide the first-line inflammatory response to excessive dietary fats (Morari et al., 2014; Valdearcos et al., 2015; Chowen et al., 2017). As early as 6 h after the introduction of a HFD, ARC microglia produce fractalkine, which acts on hypothalamic neurons to regulate the production of neurotransmitters, thus affecting whole-body energy homeostasis. As the consumption of a HFD persists, the median eminence BBB is damaged, leading to increased permeability, which boosts the inflammatory response in the ARC (Langlet et al., 2013; Ramalho et al., 2018). Thereafter, resident microglia move progressively towards the dorsal limits of the ARC and are replaced by new cells originating from bone-marrow-derived monocytes (Morari et al., 2014; Valdearcos et al., 2015). The hypothalamic inflammatory response to dietary fats occurs in two phases, as the classical inflammatory response that takes place in other regions of the body (Jais and Bruning, 2017). In the first phase, which lasts for approximately 1 week, activation of microglia and astrocytes (Guyenet et al., 2013) leads to increased hypothalamic expression of inflammatory chemokines and cytokines (Thaler et al., 2012; Morari et al., 2014; Fuente-Martin et al., 2016; Fioravante et al., 2017; Carraro et al., 2018; Chowen et al., 2019; Fioravante et al., 2019). Inflammatory markers are significantly reduced after two weeks only to return after 3-4 weeks if the HFD is maintained (Thaler et al., 2012). This two-phase pattern of response suggests that components of the adaptive immune system could be recruited to expand the inflammatory response. In fact, it was recently shown that T lymphocytes and invariant natural killer T cells (iNKT) are recruited to the hypothalamus of mice fed a HFD, and cluster of differentiation-1 (CD1), a lipid-presenting molecule is involved in the progression of the inflammatory response, as its inhibition reduces the severity of obesity and glucose intolerance (Bombassaro et al., 2019). 12

Several findings support the concept that inflammatory mediators and cellular signals coupled to the inflammatory response induced by a HFD contribute to metabolic dysfunction in obesity (Lumeng and Saltiel, 2011). However, it is important to consider that the ability of a HFD to elicit intracellular inflammatory pathways in the hypothalamus varies greatly according to the dietary fatty acid composition. Screening for pro- and anti-inflammatory effects of fatty acids injected directly into the hypothalamus revealed that the saturated fatty acids (SFAs) arachidonic acid (C20:0) and stearic acid (C18:0) exerted the most potent inflammatory activity, inducing the increased hypothalamic expression of TNF-α and IL1-β. Conversely, the icv infusion of the unsaturated fatty acids OA and linolenic acid (LA-C18:3) induced only the expression of IL-10 and IL-6, both known to have anti-inflammatory properties (Milanski et al., 2009; Cintra et al., 2012; Araujo et al., 2016; Dragano et al., 2017). The search for mechanisms upstream of inflammatory cytokine expression identified the molecular mechanisms involved in the activation of hypothalamic inflammation. SFAs, unlike unsaturated fatty acids, are able to induce inflammatory signaling by activating toll-like receptor 4 (TLR4)-mediated pathways such as JNK and IKKβ/NF-κB. Indeed, TLR4 expression is increased in genetically obese and diet-induced obese (DIO) mice. Preventing TLR4 signaling by antibody blockade or through deletion of MyD88 and consequently reducing the inflammatory activity of saturated fats in the hypothalamus improves central leptin and insulin sensitivity and limits DIO (Kleinridders et al., 2009; Milanski et al., 2009; Dorfman and Thaler, 2015) (Fig. 2). Overnutrition is a metabolic stress condition that disrupts ER homeostasis and leads to accumulation of potentially toxic misfolded proteins in the ER. In order to restore the ER's adaptive capacity, cells activate stress-responsive signaling pathways collectively termed the unfolded protein response (UPR) (Ozcan et al., 2004; Ozcan et al., 2006). Studies indicate that ER stress and activation of the UPR lead to the development of insulin and leptin resistance in the hypothalamus of obese mice (Zhang et al., 2008; Milanski et al., 2009; Ozcan et al., 2009; Won et al., 2009). Conversely, pharmacological interventions that increase the capacity for protein folding, such as icv injection of chemical chaperones, improve central leptin resistance and reduce body mass in DIO mice (Zhang et al., 2008; Ozcan et al., 2009). In addition, genetic overexpression of the chaperone GRP78/BiP in the VMH reduces hypothalamic ER stress and 13

increases brown adipose tissue (BAT) thermogenesis, resulting in weight loss and improvement in glucose homeostasis (Contreras et al., 2014) (Fig. 2). Studies have shown that hypothalamic inflammation resulting from long-term consumption of large amounts of dietary fats leads to reactive gliosis and neuronal injury (Moraes et al., 2009; Horvath et al., 2010; Dorfman and Thaler, 2015). Reactive gliosis, characterized by recruitment, proliferation, and morphological changes in local astrocytes and microglia, develops as a response to distinct insults (Burda and Sofroniew, 2014). As in gliosis developing in other regions of the brain and under different contexts, in the hypothalamus it is also associated with increased apoptosis (Thaler et al., 2012; Dorfman and Thaler, 2015). Interestingly, apoptosis seems to affect predominantly the anorexigenic, pro-thermogenic POMC neurons (Moraes et al., 2009). In fact, there is evidence of a reduction of POMC expression and POMC neuron numbers in the hypothalamus of DIO mice (Moraes et al., 2009; Schneeberger et al., 2013). However, some investigators have challenged these data and, currently, it stands as an important unsolved question in this field (Lemus et al., 2015). If confirmed, the reduction of POMC neuron numbers could indicate that during the progression of obesity, the damage caused by chronic hypothalamic inflammation may upset the balance between orexigenic and anorexigenic neurons, which could explain the refractory nature of obesity (Fig. 2).

Unsaturated fatty acid signaling in the brain Robust evidence supports the neuroprotective role of unsaturated fatty acids (UFAs) in the brain (Orr et al., 2013; Nascimento et al., 2016; Song et al., 2016; Chang et al., 2018). Most studies in this field focused on the actions of omega-3 polyunsaturated FAs (ω-3 or n-3 PUFAs) and the monounsaturated fatty acid (MUFA) OA (Bazinet and Laye, 2014; Laye et al., 2018). However, the mechanisms by which MUFAs and PUFAs can act to reduce the damage caused by neurodegenerative diseases are still a matter of intense investigation (Orr et al., 2013; Laye et al., 2018). In contrast to other tissues, the brain has a specific FA composition, with high levels of palmitic, arachidonic (AA), and DHA acids and low levels other PUFAs, such as eicosapentaenoic (EPA) and docosapentaenoic (DPA) acids. This specific composition has stimulated the evaluation of 14

the potential involvement of UFAs in neurodegenerative and neurological diseases (Dyall, 2015; Laye et al., 2018). Studies have proposed that the beneficial neural effects of UFAs rely on the regulation of membrane dynamics (Salem et al., 2001; Hishikawa et al., 2017), as well as the activation of nuclear receptors such as proliferator of peroxisome activated receptor (PPAR)-γ, thus inhibiting nuclear factor κB (Ricote and Glass, 2007; Hishikawa et al., 2017; Laye et al., 2018). The endocannabinoid system is another target for UFAs that can bind to the CB1 and CB2 receptors in neurons, leading to a reduction of inflammation (Stella, 2009; Bazinet and Laye, 2014). In the intracellular environment UFAs can be employed as substrates to produce substances with potent immunomodulatory activity, such as resolvins, maresin, and protectin (Pascoal et al., 2017; Serhan et al., 2018). Estrogens are also known to interact with PUFAs providing a synergistic action on neuroprotection (reviewed by (Marin and Diaz, 2018)). The brain has limited capacity to produce DHA and its deficiency increases the risk of age-related cognitive disorders (Bazan et al., 2011; Salem et al., 2015; Colin et al., 2016; Sun et al., 2018). Estrogens can up-regulate both the biosynthesis and uptake of DHA to the brain, thus, playing a role in the preservation of brain integrity (Giltay et al., 2004; Alessandri et al., 2008; Fabelo et al., 2012). It was also shown that the combination of DHA-enriched diets and administration of physiological doses of estrogens in a mouse model of Alzheimer’s disease, restores the normal lipid landscape in the hippocampus, suggesting that manipulation of these factors could be therapeutically interesting in this clinical condition. A potential mechanism of action by which UFAs dampen inflammation relies on their actions on GPRs that have been deorphanized during the last 10-15 years (Oh et al., 2010; Dragano et al., 2017; Laschet et al., 2018). Two of these receptors, GPR40 and GPR120, are potently activated by UFAs and are the focus of intense investigation in the fields of obesity, inflammation, and related disorders (Itoh et al., 2003; Oh et al., 2010; Dragano et al., 2017). There is clinical and epidemiological evidence for the association between altered UFA metabolism in the brain and neurological disorders such as attention-deficit/hyperactivity disorder (ADHD), Alzheimer’s disease, and schizophrenia (Morris et al., 2003; Muskiet and Kemperman, 2006; McNamara, 2010). Indeed, two recent systematic reviews showed that patients with ADHD, depression, and mood disorder have low levels of ω-3 (Konigs and Kiliaan, 2016; Chang et al., 2018). In addition, patients with schizophrenia and autism have abnormal 15

brain phospholipid metabolism, local arachidonic acid depletion and impaired EPA-mediated signal transduction (Muskiet and Kemperman, 2006). Also, schizophrenic patients showed lower circulating levels of EPA and DHA than individuals with bipolar mood disease (Messamore et al., 2017). Despite the apparent association between UFA deficiency and neurological disorders, nutritional supplementation with PUFAs and MUFAs has produced conflicting results (Appleton et al., 2010; Messamore et al., 2017; Chang et al., 2018). In a recent crossover clinical trial that investigated the effects of the palmitic/oleic acid consumption ratio on brain activation, an increase in working memory-related brain activation in the caudate and putamen of the basal ganglia and an increase in the secretion of cytokines was observed with palmitic oil when compared with olive oil consumption (Dumas et al., 2016). Supplementation with n-3 PUFAs was shown to improve clinical symptoms and cognitive performance in children and adolescents with ADHD (Chang et al., 2018). MUFAs have also been implicated in promoting cognitive benefits. In a prospective study with a 3-year follow-up, MUFA intake was associated with a lower cognitive decline when compared with SFA, whereas adherence to a Mediterranean diet (MD) rich in MUFAs was associated with slower cognitive decline, a lower risk of Alzheimer’s disease (Lourida et al., 2013), and a decreased risk of developing mild cognitive impairment (Scarmeas et al., 2009). Furthermore, interventions with MD using either nuts (rich in n-3 PUFAs) or olive oil (rich in MUFAs) promoted benefits to cognitive function, like global cognitive composition (Valls-Pedret et al., 2015). Recent data from the PREDIMED study indicate that a MD including olive oil is better for cognitive function and mild cognitive impairment when compared with a low-fat diet (MartinezLapiscina et al., 2013). In the 3-city study a positive association between a high intake of olive oil and visual memory, but not verbal fluency, was detected (Berr et al., 2009). A recent metaanalysis (Loughrey et al., 2017) showed that MD was associated with improved global cognition, but the comparison between MUFA- and PUFA-enriched diets showed conflicting results. Nevertheless, another study (Samieri et al., 2013) found no association between MD and lower cognitive decline in older women, whereas total n-3 PUFA and DHA, but not EPA, intakes were associated with a lower risk for this neurodegenerative disease. 16

The effects of UFAs on mood disorders have also been investigated. A recent systematic review suggested that distinct PUFAs vary in their effects on the brain. EPA intake was associated with improvements in the symptoms of depression when compared with a placebo, while DHA had no effect. However, EPA intake was incapable of preventing the development of the disease (Hallahan et al., 2016). Similarly, a meta-analysis was performed to evaluate the effects of n-3 PUFAs on depressive mood disorder and observed benefits in subjects with a more severe disease expression (Appleton et al., 2016). Another study reported greater effects on mood/depression when the proportion of EPA to DHA was higher than 3/2 (Sublette et al., 2011).

Unsaturated fatty acids and hypothalamic inflammation UFAs exert potent anti-inflammatory effects on the hypothalamus of experimental models of obesity, both when used as components of the diet and when injected icv directly into the hypothalamus (Cintra et al., 2012). At least in part, the beneficial effects of UFAs rely on their actions on GPR40, GPR120, and GPR18 (Ulven and Christiansen, 2015; Nascimento et al., 2016; Dragano et al., 2017; Pascoal et al., 2017; Riddy et al., 2018). GPR40 is a former orphan membrane receptor that was deorphanized upon demonstration of its activation by fatty acids (Tazoe et al., 2008). It is present predominantly in pancreatic islets and in some regions of the brain, including the hypothalamus. DHA is the most important activator of GPR40; however, linoleic acid, -linoleic acid, and EPA also activate signal transduction through GPR40 with considerable potency (Itoh et al., 2003). Upon ligand binding, GPR40 transduces signals through at least two pathways: i) cAMP and ii) MAP kinase (Wang et al., 2015). In pancreatic islets, the activation of GPR40 result in increased insulin secretion, leading to improved glucose tolerance in both experimental models and humans (Krug et al., 2017; Syed et al., 2018). In the hypothalamus, GPR40 is expressed in Neu-N positive cells, but not in GFAP cells (Nakamoto et al., 2013); ependymal cells of the lateral ventricle were also shown to express GPR40 (Ma et al., 2010). Regarding the neuronal subpopulations, GPR40 is predominantly expressed in POMC and AgRP neurons (Dragano et al., 2017). In POMC neurons, upon ligand 17

binding, GPR40 increases the expression of prohormone convertase 2 (PC2), a calciumdependent serine protease that catalyzes POMC processing to produce its active subproducts (Nakamoto et al., 2015). In addition, the activation of hypothalamic GPR40 by PUFAs increases POMC neurogenesis with a magnitude similar to the effect obtained with BDNF (Nascimento et al., 2016) (Table 1). GRP120 is activated predominantly by DHA and EPA (Oh et al., 2010). It has a more widespread distribution than GPR40; however, in the hypothalamus it is present particularly in microglia (Dragano et al., 2017). Initially it was proposed that most of the actions of GPR120 depended on cytosolic calcium increase and activation of ERK (Hirasawa et al., 2005). In 2010, a groundbreaking study revealed that upon ligand binding, GPR120 is internalized with βarrestin 2, recruiting TAB1, which results in the inhibition of inflammatory signaling through the JNK and IKK pathways (Oh et al., 2010). In mice, the systemic activation of GPR120 with dietary PUFAs resulted in increased insulin sensitivity (Oh et al., 2010), which was recapitulated by a GPR120-specific synthetic agonist (Oh et al., 2014). In the hypothalamus, activation of GPR120 by dietary fats reduces obesity-associated inflammation, increasing the expression of POMC, which is accompanied by reduced body mass. At least in part, these effects are mediated by GPR120-dependent inactivation of inflammatory signaling through JNK and IKK (Cintra et al., 2012) (Table 1). In addition to the activation of GPR40 and GPR120, PUFAs can be employed as substrates for the production of endogenous substances, lipoxins, resolvins, protectins, and maresins, that act in the resolution phase of inflammatory processes (Serhan et al., 2008). In obesity, the hypothalamic levels of resolvin D2 (RvD2) are reduced, and upon dietary substitution of saturated fats by PUFAs, the defect is corrected (Pascoal et al., 2017). GRP18, the receptor for RvD2 (Chiang et al., 2015), is expressed in POMC and NPY neurons but not in hypothalamic microglia (Pascoal et al., 2017). Upon activation by RvD2, GPR18 transduces anti-inflammatory signals that result in increased hypothalamic leptin sensitivity and reduction of body mass (Pascoal et al., 2017). Because pharmacological targeting of UFA receptors may offer advance in the treatment of metabolic and inflammatory diseases, a number of studies have made attempts to develop and 18

characterize ligands with specificity for some of these receptors (Shimpukade et al., 2012; Urban et al., 2013; Sparks et al., 2014; Gozal et al., 2016; Hansen et al., 2016; Syed et al., 2018). Indeed, greatest advance in this field has been achieved in diabetes; a number of synthetic agonists for GPR40 are under evaluation in clinical studies, and improvement of glucose tolerance associated with increased insulin secretion has been reported in some trials (Kaku et al., 2015; Krug et al., 2017; Suckow and Briscoe, 2017); nevertheless, at least one of the agonists, fasiglifam, was recently reported to produce liver injury and the trial was terminated (Menon et al., 2018). There is also promising data for GPR120 synthetic agonists that resulted in reduced systemic inflammation and improved insulin sensitivity in experimental models (Oh et al., 2014; Azevedo et al., 2016); however, currently, there is only one clinical trial using a synthetic agonist of GPR120 to evaluate insulin resistance and the results are yet to be published (Clinical Trial NCT02444910). In the hypothalamus, only few studies have evaluated the impact of synthetic ligands for UFA receptors in experimental models. The treatment of obese mice with a GPR40/GPR120 dual agonist reduced inflammation and energy efficiency (Dragano et al., 2017); the use of a GPR120-specific agonist reduced inflammation only, whereas the use of a GPR40-specific agonist increase POMC expression and reduces body mass (Dragano et al., 2017). In addition, hypothalamic activation of GPR40 increases neurogenesis by a mechanism dependent, at least in part on BDNF (Nascimento et al., 2016) and also reduce inflammatory pain (Nakamoto et al., 2013). Table 1 presents the main finding of studies employing synthetic ligands for GPR120 and GPR40 in the hypothalamus.

Conclusions Fatty acids have a wide spectrum of action in the hypothalamus: physiologically, they can act in the intracellular compartment of neurons, modulating signaling systems that are involved in the control of whole-body energy homeostasis. They can also act through membrane receptors to control inflammation and energy homeostasis. In obesity, anomalous levels of fatty acids activate inflammation and disrupt the systems that control food intake and energy expenditure. UFAs can act through membrane receptors or by inducing the endogenous production of resolution phase substances, correcting some of the hypothalamic abnormalities generated in 19

obesity. The development of ligands specific for UFA receptors has emerged as potential pharmacological approaches to treat obesity and other metabolic and inflammatory diseases.

Competing interests The authors declare that they have no competing interests.

Acknowledgments. The review was supported by grants from the São Paulo Research Foundation (2013/07607-8) and Conselho Nacional de Pesquisa e Desenvolvimento Cientifico.

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Legends for the figures Figure 1. Fatty acid transport and action in the hypothalamus. Systemic fatty acids (FA) reach the hypothalamus via bloodstream and are transported by fatty acid transport proteins (FATP) -1 and -4 and also by cluster of differentiation 36 (CD36), therefore reaching hypothalamic cells. Whenever under energy surplus conditions, neuronal mitochondria employ glucose as preferential energy source, which generates a signal for reduction of the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP) leading to reduced caloric intake and increased energy expenditure. Conversely, under energy deficiency, FAs are -oxidized in mitochondria; this, results in a signal that increased NPY and AgRP levels resulting in increased caloric intake and reduced energy expenditure. 3V, third ventricle; ME, median eminence

Figure 2. Abnormalities in the hypothalamus of mice fed a high-fat diet. When mice are fed a high-fat diet (HFD) the 2-tanycytes that are important components of the blood-brain interface undergo structural and functional changes resulting in a leaky barrier that allows increased fatty acid (FA) entrance in the hypothalamus. Astrocytes respond to increased FA by producing transforming growth factor 2 (TGF2) and inflammatory cytokines; microglia is activated by toll-like receptor 4 (TLR4) and produce chemokines and cytokines that, in concert with the substances produced by astrocytes and also with FAs produce a number of abnormalities in the hypothalamic neurons, such as: endoplasmic reticulum (ER) stress; RNA granule stress; mitochondrial structural and functional defects; proteostasis and autophagy abnormalities, which eventually can result in neuronal apoptosis.

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Table 1. Studies employing synthetic agonists to evaluate the impact of GPR120 and/or GPR40 activation in the hypothalamus or in hypothalamic cells.

GPR

Cell type

Effect

First author/PMID

GPR120

Hypothalamic neuron cell-line

Wellhauser/24674717

GPR120

Microglia

GPR40

Neuron – undefined subpopulation

GPR40

POMC neuron

GPR40

POMC neuron

GPR40

POMC/NPY neurons

Inhibition of inflammatory signaling Inhibition of dietinduced inflammation Reduction of inflammatory pain Reduction of expression of PC2 Increased neurogenesis Reduced body mass and increased POMC

Dragano/2844241 Nakamoto/24349089 Nakamoto/26071852 Nascimento/26512023 Dragano/2844241

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Highlights

Mechanisms mediating the actions of fatty acids in the hypothalamus Nathalia R. Dragano§, Milena Monfort-Pires§, Licio A. Velloso*

Fatty acids control hypothalamic neurons involved in whole body energy homeostasis Fatty acids act through distinct mechanism to regulate hypothalamic neurons These mechanisms include subtracted metabolism membrane and nuclear receptors Fatty acid receptors are potential targets for drug therapy in metabolic conditions

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