**Differential Effect of Fatty Acids in Nervous Control of Energy Balance**

Christophe Magnan, Hervé Le Stunff and Stéphanie Migrenne *Université Paris Diderot, Sorbonne Paris Cité, Biologie Fonctionnelle et Adaptative, Equipe d'accueil conventionnée Centre National de la Recherche Scientifique, Paris, France* 

#### **1. Introduction**

396 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

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Energy homeostasis is kept through a complex interplay of nutritional, neuronal and hormonal inputs that are integrated at the level of the central nervous system (CNS). A disruption of this regulation gives rise to life-threatening conditions that include obesity and type-2 diabetes, pathologies that are strongly linked epidemiologically and experimentally. The hypothalamus is a key integrator of nutrient-induced signals of hunger and satiety, crucial for processing information regarding energy stores and food availability. Much effort has been focused on the identification of hypothalamic pathways that control food intake but, until now, little attention has been given to a potential role for the hypothalamus in direct control of glucose homeostasis and nergy balance. Recent studies have cast a new light on the role of the CNS in regulating peripheral glucose via a hypothalamic fatty acid (FA)-sensing device that detects nutrient availability and relays, through the autonomic nervous system, a negative feedback signal on food intake, insulin sensitivity and insulin secretion. Indeed, accumulating evidences suggest that FA are used in specific areas of CNS not as nutrients, but as cellular messengers which inform "FA sensitive neurons" about the energy status of the whole body (Blouet & Schwartz, 2010; Migrenne et al., 2006; Migrenne et al., 2011). Thus it has been described that up to 70% of hypothalamic arcuate nucleus (ARC) and ventromedian nucleus (VMN) neurons are either excited or inhibited by long chain fatty acids such as oleic acid (Jo et al., 2009; Le Foll et al., 2009; Migrenne et al., 2011). Within the VMN, 90% of the glucosensing neurons also have their activity altered by FA. In a large percentage of these neurons, glucose and FA have opposing effects on neuronal activity, much as they do on intracellular metabolism in many other cells (Randle et al., 1994). Neuronal FA sensing mechanisms include activation of the KATP channel by long chain fatty acid acyl CoA (Gribble et al., 1998) or inactivation by generation of ATP or reactive oxygen species during mitochondrial β-oxidation (Jo et al., 2009; Le Foll et al., 2009; Migrenne et al., 2011; Wang et al., 2006). Many fatty acid sensing neurons are activated by interaction of long chain fatty acids with the fatty acid transporter/receptor, FAT/CD36, presumably by activation of store-operated calcium channels by a mechanism that is independent of fatty acid metabolism (Jo et al., 2009). Importantly, most neurons utilize FA primarily for membrane production rather than as a metabolic substrate (Rapoport et al., 2001; Smith & Nagura, 2001) and only nanomolar concentrations of fatty acid are required to

Differential Effect of Fatty Acids in Nervous Control of Energy Balance 399

of whether brain FA uptake occurs by passive diffusion or involves a protein which facilitates the transport is still matter of debate. However, once across the BBB, it is likely that neurons can take up FA since some neurons do appear to have FA transporters. For example, dissociated neurons from the VMN of rats express mRNA's for FA transport proteins (FATP)-1 and 4 and the FA transporter/receptor FAT/CD36 (Le Foll et al., 2009). Also, while it is unlikely that neurons derive much of their energy supply from FA, these same neurons do express mRNA's for the intracellular metabolism of FA such as long chain acyl-CoA synthetase, carnitine palmitoyltransferase-1a and 1c and uncoupling protein-2 (Le Foll et al., 2009). They also express enzymes for de novo FA synthesis such as FA synthetase (Le Foll et al., 2009). But, it seems likely that much of the reported oxidation of FA such as palmitate in the brain probably occurs in astrocytes (Escartin et al., 2007) whereas other FA such as arachidonate are largely incorporated into phospholipids (Rapoport et al., 2001).

The presence of neurons sensitive to variations in extracellular glucose levels is clearly demonstrated in the brain (Gilbert et al., 2003) and in particular in the hypothalamus (review in (Luquet & Magnan, 2009; Migrenne et al., 2011; Penicaud et al., 2002). Thirty-five years ago Oomura and colleagues first showed that FA activated lateral hypothalamic neurons which suggested a role for FA as neuronal signaling molecules (Oomura et al., 1975). As shown in Figure 1, FA also modify neuronal firing rate in hypothalamic arcuate nucleus (ARC) (Wang et al., 2006). Both FA "excited" (around 20% of arcuate neurons) and "inhibited" neurons (about 12%) are detected in arcuate nucleus of rat using this patch clamp technique (Wang et al., 2006). These FA sensitive neurons are also detected *in vivo* using multi-unit recording approaches (Wang et al., 2006). Therefore we demonstrated that single injection of oleic acid (OA) through carotid artery induced either increased or decreased neuronal activity depending on location of microelectrode in hypothalamus. It seems that some areas are mainly composed with FA "excited" neurons whereas others are mainly composed with FA "inhibited" neurons. Such data also suggest that physiological variations of plasma FA concentrations (reflecting the metabolic state and energy availability) can be detected and integrated by FA sensing neurons in critical brain areas involved in the regulation of feeding behaviour, glucose and lipid metabolism (Clement et al., 2002; Obici et al., 2002). Indeed increased plasma FA concentration during fasted or starvation may be detected by FA excited neurons which in turn may have an impact on nervous control of energy balance. On the contrary decreased plasma FA concentration during a meal could be also detected by these sensitive neurons which may act a satiety signal like insulin and glucose do during a meal

The physiological relevance of brain FA sensing is supported by various studies showing that local increases in brain and hypothalamic FA levels are associated with changes in insulin secretion and hepatic glucose output with variable effects on food intake (Clement et al., 2002; Obici et al., 2002; Ross et al., 2010; Schwinkendorf et al., 2010). For example, a 6 hour intracerebroventricular (icv) infusion of the monounsaturated FA, oleic acid (OA), reduced food intake as well as hepatic glucose production (HGP) (Obici et al., 2002). Reducing hypothalamic FA oxidation by inhibition of carnitine palmitoyl transferase -1 (CPT1), the enzyme that promotes β-oxidation by facilitating transport of medium- and

**2.2 Some hypothalamic neurons are lipid responsive** 

when acting on hypothalamus sensitive neurons (Gilbert et al., 2003).

alter the activity of fatty acid sensing neurons in the absence of astrocytes (Jo et al., 2009). While cerebral lipids are both produced in the brain and transported into it from the periphery (Rapoport et al., 2001; Smith & Nagura, 2001), the mechanism of this transport and the actual levels of various FA in the extracellular space in the brain remains largely unknown. As mentioned above, hypothalamic FA sensing may be involved in the control of feeding behaviour, hepatic glucose production and insulin secretion. It seems also that intracellular FA metabolism is important to relay their effects (β oxidation has been showed to be involved in oleate effect in hypothalamus) (Cruciani-Guglielmacci et al., 2004; Obici et al., 2003). In addition differential effect of FA in regard to feeding behaviour or glucose production may be related to their chain length and degree of saturation. For exemple, it has been showed in rodents that oleate both inhibits food intake and hepatic glucose production whereas octanoate has no effect on these parameters (Obici et al., 2002). In another study we showed that intracerebroventricular infusion of palmitate induced an hepatic insulin resistance and an impaired insulin signaling in hypothalamus (Benoit et al., 2009). In contrast oleate has no deleterious effect in this parameter (Benoit et al., 2009). Poly-unsaturated fatty acids (PUFA) such as n-3 or n-6 may have also different effects in neuronal activity and cognitive function such as memorization. The present work was aimed at studying differential effect of FA or triglycerides emulsion infused in rats in glucose homeostasis. In addition, in order to identify molecular mechanisms involved in specific effects of FA, mRNA expression of key genes involved in FA metabolism as well as ceramides and diacylglycerol (DAG) content have been measured in hypothalamus. Regarding physiopathology aspects it must be pointed out that dysfunction of central FA sensing could be a contributing factor to the early development of type 2 diabetes mellitus and/or obesity which leads to further dysfunction in predisposed subjects. A better understanding of these mechanisms, as well as further characterization of FA sensitive neurons and their role in physiological and pathological processes, might lead to identification of novel pharmacological targets for the prevention and treatment of diabetes and obesity.

#### **2. FA sensing in hypothalamus**

There is now growing amount of evidence suggesting, at least in rodents models, that some neurons located in hypothalamus (and brainstem) are sensitive to FA, ie their electrical activity is either increased or decreased in presence of variations of FA concentration. This has been evidenced both in vivo and in vitro. A key point is the transport of FA across the blood brain barrier (BBB). It cannot be excluded that FA may be produce directly in neurons from hydrolysis of intracellular troglycerides (TG).

#### **2.1 Transport of FA uptake into the brain and neurons**

Cerebral lipids are an essential component of both membranes and intracellular signalling pathways. They represent 50% of brain dry weight; the highest organ lipid content after adipose tissue (Edmond, 2001; Watkins et al., 2001). However, the mechanism by which FA are transported into the brain remains poorly understood. A growing body of evidence suggests that cerebral lipids are derived both from local synthesis and uptake from the blood (Rapoport et al., 2001). Several studies show that some poly-unsaturated FA (PUFA) have the ability to cross the BBB (Rapoport et al., 2001; Smith & Nagura, 2001). The question

alter the activity of fatty acid sensing neurons in the absence of astrocytes (Jo et al., 2009). While cerebral lipids are both produced in the brain and transported into it from the periphery (Rapoport et al., 2001; Smith & Nagura, 2001), the mechanism of this transport and the actual levels of various FA in the extracellular space in the brain remains largely unknown. As mentioned above, hypothalamic FA sensing may be involved in the control of feeding behaviour, hepatic glucose production and insulin secretion. It seems also that intracellular FA metabolism is important to relay their effects (β oxidation has been showed to be involved in oleate effect in hypothalamus) (Cruciani-Guglielmacci et al., 2004; Obici et al., 2003). In addition differential effect of FA in regard to feeding behaviour or glucose production may be related to their chain length and degree of saturation. For exemple, it has been showed in rodents that oleate both inhibits food intake and hepatic glucose production whereas octanoate has no effect on these parameters (Obici et al., 2002). In another study we showed that intracerebroventricular infusion of palmitate induced an hepatic insulin resistance and an impaired insulin signaling in hypothalamus (Benoit et al., 2009). In contrast oleate has no deleterious effect in this parameter (Benoit et al., 2009). Poly-unsaturated fatty acids (PUFA) such as n-3 or n-6 may have also different effects in neuronal activity and cognitive function such as memorization. The present work was aimed at studying differential effect of FA or triglycerides emulsion infused in rats in glucose homeostasis. In addition, in order to identify molecular mechanisms involved in specific effects of FA, mRNA expression of key genes involved in FA metabolism as well as ceramides and diacylglycerol (DAG) content have been measured in hypothalamus. Regarding physiopathology aspects it must be pointed out that dysfunction of central FA sensing could be a contributing factor to the early development of type 2 diabetes mellitus and/or obesity which leads to further dysfunction in predisposed subjects. A better understanding of these mechanisms, as well as further characterization of FA sensitive neurons and their role in physiological and pathological processes, might lead to identification of novel pharmacological targets for the prevention and

There is now growing amount of evidence suggesting, at least in rodents models, that some neurons located in hypothalamus (and brainstem) are sensitive to FA, ie their electrical activity is either increased or decreased in presence of variations of FA concentration. This has been evidenced both in vivo and in vitro. A key point is the transport of FA across the blood brain barrier (BBB). It cannot be excluded that FA may be produce directly in neurons

Cerebral lipids are an essential component of both membranes and intracellular signalling pathways. They represent 50% of brain dry weight; the highest organ lipid content after adipose tissue (Edmond, 2001; Watkins et al., 2001). However, the mechanism by which FA are transported into the brain remains poorly understood. A growing body of evidence suggests that cerebral lipids are derived both from local synthesis and uptake from the blood (Rapoport et al., 2001). Several studies show that some poly-unsaturated FA (PUFA) have the ability to cross the BBB (Rapoport et al., 2001; Smith & Nagura, 2001). The question

treatment of diabetes and obesity.

**2. FA sensing in hypothalamus** 

from hydrolysis of intracellular troglycerides (TG).

**2.1 Transport of FA uptake into the brain and neurons** 

of whether brain FA uptake occurs by passive diffusion or involves a protein which facilitates the transport is still matter of debate. However, once across the BBB, it is likely that neurons can take up FA since some neurons do appear to have FA transporters. For example, dissociated neurons from the VMN of rats express mRNA's for FA transport proteins (FATP)-1 and 4 and the FA transporter/receptor FAT/CD36 (Le Foll et al., 2009). Also, while it is unlikely that neurons derive much of their energy supply from FA, these same neurons do express mRNA's for the intracellular metabolism of FA such as long chain acyl-CoA synthetase, carnitine palmitoyltransferase-1a and 1c and uncoupling protein-2 (Le Foll et al., 2009). They also express enzymes for de novo FA synthesis such as FA synthetase (Le Foll et al., 2009). But, it seems likely that much of the reported oxidation of FA such as palmitate in the brain probably occurs in astrocytes (Escartin et al., 2007) whereas other FA such as arachidonate are largely incorporated into phospholipids (Rapoport et al., 2001).

### **2.2 Some hypothalamic neurons are lipid responsive**

The presence of neurons sensitive to variations in extracellular glucose levels is clearly demonstrated in the brain (Gilbert et al., 2003) and in particular in the hypothalamus (review in (Luquet & Magnan, 2009; Migrenne et al., 2011; Penicaud et al., 2002). Thirty-five years ago Oomura and colleagues first showed that FA activated lateral hypothalamic neurons which suggested a role for FA as neuronal signaling molecules (Oomura et al., 1975). As shown in Figure 1, FA also modify neuronal firing rate in hypothalamic arcuate nucleus (ARC) (Wang et al., 2006). Both FA "excited" (around 20% of arcuate neurons) and "inhibited" neurons (about 12%) are detected in arcuate nucleus of rat using this patch clamp technique (Wang et al., 2006). These FA sensitive neurons are also detected *in vivo* using multi-unit recording approaches (Wang et al., 2006). Therefore we demonstrated that single injection of oleic acid (OA) through carotid artery induced either increased or decreased neuronal activity depending on location of microelectrode in hypothalamus. It seems that some areas are mainly composed with FA "excited" neurons whereas others are mainly composed with FA "inhibited" neurons. Such data also suggest that physiological variations of plasma FA concentrations (reflecting the metabolic state and energy availability) can be detected and integrated by FA sensing neurons in critical brain areas involved in the regulation of feeding behaviour, glucose and lipid metabolism (Clement et al., 2002; Obici et al., 2002). Indeed increased plasma FA concentration during fasted or starvation may be detected by FA excited neurons which in turn may have an impact on nervous control of energy balance. On the contrary decreased plasma FA concentration during a meal could be also detected by these sensitive neurons which may act a satiety signal like insulin and glucose do during a meal when acting on hypothalamus sensitive neurons (Gilbert et al., 2003).

The physiological relevance of brain FA sensing is supported by various studies showing that local increases in brain and hypothalamic FA levels are associated with changes in insulin secretion and hepatic glucose output with variable effects on food intake (Clement et al., 2002; Obici et al., 2002; Ross et al., 2010; Schwinkendorf et al., 2010). For example, a 6 hour intracerebroventricular (icv) infusion of the monounsaturated FA, oleic acid (OA), reduced food intake as well as hepatic glucose production (HGP) (Obici et al., 2002). Reducing hypothalamic FA oxidation by inhibition of carnitine palmitoyl transferase -1 (CPT1), the enzyme that promotes β-oxidation by facilitating transport of medium- and

Differential Effect of Fatty Acids in Nervous Control of Energy Balance 401

More physiological routes include elevating systemic levels of FA or infusing them directly into the carotid arteries, the major route by which FA reach the forebrain. For example, a twofold increase in plasma triglycerides produced by a two day systemic infusion of triglycerides was associated with decreased sympathetic activity. This reduced sympathetic tone, which is also produced by central FA infusions (Magnan et al., 1999), might contribute to the associated FA-induced exaggeration of glucose-induced insulin secretion (GIIS), a condition which is similar to what occurs in the prediabetic state (Magnan et al., 1999). Also, this exaggerated GIIS and a reduction in HGP were mimicked by infusing triglycerides into the carotid artery (Cruciani-Guglielmacci et al., 2004). These exaggerated responses were reduced by central inhibition CPT1 (Magnan et al., 1999). Similarly, central CPT1 inhibition was associated with an increase in the acyl CoA intracellular pool which was postulated to be the "final" satiety

signal rather than FA themselves (review in (Lam et al., 2005; Luquet & Magnan, 2009).

enriched in ω3, ω6 FA or saturated FA (lard oil) have been also tested.

All animal care and experimental procedures were approved by the animal ethics committee of the university Paris-Diderot. Four weeks-old male wistar rats were purchased from Charles Rivers (Lyon, France) and housed at 21°C with normal light/dark cycle and free

Rats received an intracerebroventricular (icv) infusion of FA during 3 days. Briefly, rats anesthetized with isoflurane were stereotactically implanted with a chronic stainless steel

**3. Methods** 

access to water and food.

**3.1 First serie of experiments** 

However, there are at least two potential problems involved in the interpretation of such in vivo data. First, the idea that increases in brain FA levels act as a satiety signal to inhibit feeding (Obici et al., 2003) is counterintuitive given the fact that plasma FA levels do not rise substantially after food ingestion, but do rise significantly during fasting (Ruge et al., 2009). Second, the vast majority of FA oxidation in the brain occurs in astrocytes rather than neurons (Escartin et al., 2007). While a select group of neurons in the hypothalamus clearly responds directly to changes in ambient FA levels by altering their activity (Le Foll et al., 2009; Oomura et al., 1975), only a relatively small percentage of these responses depend upon neuronal FA metabolism (Le Foll et al., 2009). Furthermore, although β-oxidation and formation of malonyl-CoA and FA metabolites such as acyl-CoA may be mediators of the in vivo effects produced by FA infusions (Dowell et al., 2005; Migrenne et al., 2011) it is likely that most of these occur at the level of the astrocyte. If so, then there must be a mechanism by which alterations in astrocyte FA metabolism can provide a signal to those neurons which regulate HGP and food intake. We suggest that this communication between astrocyte FA metabolism and neuronal FA sensing involves the production and export of ketone bodies from astrocytes (Escartin et al., 2007) and subsequent uptake by neurons. Finally another important issue is the nature of the FA and its effect on sensitive neurons. As previously mentioned OA and octanoate have differential effect regarding food intake or hepatic glucose production, suggesting that medium or long chain fatty acids may have different effects (Obici et al., 2002). Thus, the aim of the present study was to test whether different FA may have different effect on glucose homeostasis when infused in rats brains through carotid artery. Triglyceride emulsion either

long-chain FA into mitochondria, mimicked these effects on food intake and HGP induced by icv infusion of OA (Obici et al., 2003). In another study a direct bilateral infusion of OA into the mediobasal hypothalamus decreased hepatic glucose production (Ross et al., 2010). In addition, it seems that the hypothalamus differentially senses FA. For example, icv infusions of OA or docosahexanoic acid, but not palmitic acid, reduce food intake and body weight (Schwinkendorf et al., 2010). However, icv and direct infusions of FA into the brain are not physiological. Thus, they might produce non-specific effects by evoking an inflammatory response by irritating ependymocytes and tanycytes lining the ventricles or by exciting microglia and astrocytes in the brain parenchyma.

Fig. 1. Fragments of whole cell current clamp recordings of oleic acid (OA) excited (A) and inhibited (B) neuron in arcuate nucleus of rat (Adapted from Wang et al, 2006 et Migrenne et al, 2006).The inhibitory effect of OA on neuronal activity is inhibited by tolbutamide (C), suggesting involvement of KATP channels in OA effect.

More physiological routes include elevating systemic levels of FA or infusing them directly into the carotid arteries, the major route by which FA reach the forebrain. For example, a twofold increase in plasma triglycerides produced by a two day systemic infusion of triglycerides was associated with decreased sympathetic activity. This reduced sympathetic tone, which is also produced by central FA infusions (Magnan et al., 1999), might contribute to the associated FA-induced exaggeration of glucose-induced insulin secretion (GIIS), a condition which is similar to what occurs in the prediabetic state (Magnan et al., 1999). Also, this exaggerated GIIS and a reduction in HGP were mimicked by infusing triglycerides into the carotid artery (Cruciani-Guglielmacci et al., 2004). These exaggerated responses were reduced by central inhibition CPT1 (Magnan et al., 1999). Similarly, central CPT1 inhibition was associated with an increase in the acyl CoA intracellular pool which was postulated to be the "final" satiety signal rather than FA themselves (review in (Lam et al., 2005; Luquet & Magnan, 2009).

However, there are at least two potential problems involved in the interpretation of such in vivo data. First, the idea that increases in brain FA levels act as a satiety signal to inhibit feeding (Obici et al., 2003) is counterintuitive given the fact that plasma FA levels do not rise substantially after food ingestion, but do rise significantly during fasting (Ruge et al., 2009). Second, the vast majority of FA oxidation in the brain occurs in astrocytes rather than neurons (Escartin et al., 2007). While a select group of neurons in the hypothalamus clearly responds directly to changes in ambient FA levels by altering their activity (Le Foll et al., 2009; Oomura et al., 1975), only a relatively small percentage of these responses depend upon neuronal FA metabolism (Le Foll et al., 2009). Furthermore, although β-oxidation and formation of malonyl-CoA and FA metabolites such as acyl-CoA may be mediators of the in vivo effects produced by FA infusions (Dowell et al., 2005; Migrenne et al., 2011) it is likely that most of these occur at the level of the astrocyte. If so, then there must be a mechanism by which alterations in astrocyte FA metabolism can provide a signal to those neurons which regulate HGP and food intake. We suggest that this communication between astrocyte FA metabolism and neuronal FA sensing involves the production and export of ketone bodies from astrocytes (Escartin et al., 2007) and subsequent uptake by neurons. Finally another important issue is the nature of the FA and its effect on sensitive neurons. As previously mentioned OA and octanoate have differential effect regarding food intake or hepatic glucose production, suggesting that medium or long chain fatty acids may have different effects (Obici et al., 2002). Thus, the aim of the present study was to test whether different FA may have different effect on glucose homeostasis when infused in rats brains through carotid artery. Triglyceride emulsion either enriched in ω3, ω6 FA or saturated FA (lard oil) have been also tested.
