Chun-Xia Yi

Susanne E La Fleur

Eric Fliers

Andries Kalsbeek
email: a.kalsbeek@amc.uva.nl

The role of the autonomic nervous liver innervation in the control of energy metabolism

Despite a longstanding research interest ever since the early work by Claude Bernard, the functional significance of autonomic liver innervation, either sympathetic or parasympathetic, is still ill defined. This scarcity of information not only holds for the brain control of hepatic metabolism, but also for the metabolic sensing function of the liver and the way in which this metabolic information from the liver affects the brain.

Clinical information from the bedside suggests that successful human liver transplantation (implying a complete autonomic liver denervation) causes no life threatening metabolic derangements, at least in the absence of severe metabolic challenges such as hypoglycemia. However, from the benchside, data are accumulating that interference with the neuronal brain-liver connection does cause pronounced changes in liver metabolism. This review provides an extensive overview how metabolic information is sensed by the liver, and how this information is processed via neuronal pathways to the brain. With this information the brain controls liver metabolism and that of other organs and tissues. We will pay special attention to the hypothalamic pathways involved in these liver -brain -liver circuits. At this stage, we still do not know the final destination and processing of the metabolic information that is transferred from the liver to the brain. On the other hand, in recent years, there has been a considerable increase in the understanding which brain areas are involved in the control of liver metabolism via its autonomic innervation. However, in view of the ever rising prevalence of type 2 diabetes, this potentially highly relevant knowledge is still by far too limited. Thus the autonomic innervation of the liver and its role in the control of metabolism needs our continued and devoted attention.

Efferent pathway and the control of hepatic metabolism

Neuronal signals in control of liver glucose and lipid metabolism Brain areas involved in the control of liver glucose metabolism Hypothalamic neuropeptides involved in the control of liver glucose metabolism Liver innervation and the disarrangement between glycogenolysis and gluconeogenesis

Metabolic integration in liver and brain

Intrahepatic interaction of neuronal and humoral signals

The brain as an intermediate for liver control of other organs CNS integration of liver and blood born metabolic information

Discussion and Perspective

What can be learned from "gain of function" and "loss of function" studies? Perspective A C C E P T E D M A N U S C R I P T

1-Introduction

The autonomic nervous system is the unconscious part of the peripheral nervous system and comprises the motor system for viscera, smooth muscles and exocrine glands. The scientific interest for the innervation of the liver by the autonomic nervous system and its function in the control of energy metabolism can be traced back to a time when the need to understand the pathophysiology of metabolic diseases like type 2 diabetes mellitus may have been less urgent than in our modern affluent society. The great French physiologist Claude Bernard was the first to discover and describe that the liver can store and release glucose, and he named this stored form of glucose glycogen. At that time in history the general assumption was that the body could not synthesize glucose de novo (gluconeogenesis). However, Claude Bernard believed that hepatic glucose production was under the control of autonomic nerves and carried out the earliest study on the autonomic innervation of the liver and glucose metabolism in 1848. He observed a decrease in hepatic glucose output after peripheral vagotomy, but failed to show increased glucose output by electrically stimulating the vagal nerves. He then tried to punch the floor of the fourth ventricle, i.e., the place of origin of the vagal nerves. The short-term "artificial diabetes" he induced in this way (the well-known piqûre diabetique)

was not blocked by cutting the vagal nerves, but it could be blocked by transection of the spinal cord, i.e., the origin of the sympathetic nerves that innervate the liver. Although this earliest functional study did not mean he could distinguish the glucoregulatory function of the liver from that of other visceral organs, it was the first indication of a brain mechanism controlling peripheral glucose homeostasis and of the involvement of the autonomic nervous system. Despite this inspiring starting point, the role liver innervation plays in the control of energy metabolism is still not fully uncovered today, especially regarding metabolic sensing by the liver.

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After insulin was discovered by Banting and Macleod in the 1920s, physiologists and biochemists focused their attention on the control of liver metabolism by hormones, and in subsequent years only little attention was paid to the involvement of neuronal signaling in liver metabolism. In the 1950s Mayer put forward the "glucostatic theory", the "lipostatic theory" and the "glucoreceptor" conception [START_REF] Mayer | Glucostatic mechanism of regulation of food intake[END_REF][START_REF] Mayer | Genetic, traumatic and environmental factors in the etiology of obesity[END_REF][START_REF] Mayer | Regulation of energy intake and the body weight: the glucostatic theory and the lipostatic hypothesis[END_REF] as explanatory brain mechanisms involved in the control of feeding behavior and glucose metabolism.

Although these theories renewed interest in neuronal sensing mechanisms involved in the control of liver function, the "glucostatic theory" itself was soon rejected based on experimental studies by others [START_REF] Bernstein | An experimental test of the glucostatic theory of regulation of food intake[END_REF]. In the early sixties the existence of glucose sensitive neurons in the ventromedial hypothalamus (VMH) and lateral hypothalamus (LH) was proven by Anand [START_REF] Anand | Effect of glucose on the activity of hypothalamic "feeding centers[END_REF] and Oomura [START_REF] Oomura | reciprocal activities of the ventromedial and lateral hypothalamic areas of cats[END_REF]. Russek then proposed the existence of glucose receptors in the liver [START_REF] Russek | Participation of hepatic glucoreceptors in the control of intake of food[END_REF]. Soon after, a change of vagal activity was recorded by Niijima upon glucose infusions into the portal vein [START_REF] Niijima | Afferent impulse discharges from glucoreceptors in the liver of the guinea pig[END_REF].

The term "glucoreceptor" was gradually replaced by "glucose sennsing". Glucokinase (hexokinase) (GK) [START_REF] Matschinsky | Metabolism of glucose in the islets of Langerhans[END_REF] and glucose transporters (GLUT) [START_REF] Mueckler | Sequence and structure of a human glucose transporter[END_REF][START_REF] Kayano | Evidence for a family of human glucose transporter-like proteins. Sequence and gene localization of a protein expressed in fetal skeletal muscle and other tissues[END_REF] are considered to be the main molecular components of neuronal glucose sensing [START_REF] Levin | Neuronal glucosensing: what do we know after 50 years?[END_REF], other mechanism such as sodium/glucose cotransporter type 3 (SGLT3) [START_REF] Diaz-Munoz | Anticipatory changes in liver metabolism and entrainment of insulin, glucagon, and corticosterone in food-restricted rats[END_REF] and twik1-related acid-sensitive K + channel subunit (TASK) 1 and 3 [START_REF] Guyon | Glucose inhibition persists in hypothalamic neurons lacking tandem-pore K+ channels[END_REF][START_REF] Gonzalez | Deletion of TASK1 and TASK3 channels disrupts intrinsic excitability but does not abolish glucose or pH responses of orexin/hypocretin neurons[END_REF] could be also involved.

In contrast to glucose sensing, a mechanism for lipid sensing by the liver was strongly doubted at the beginning of last century. It was thought that, of the three macronutrients, only hydrophilic glucose and amino acids could enter the liver via the portal vein after absorption from the intestine into the mesenteric veins. Fatty acids were thought to be absorbed in the intestinal lymph and to enter the heart through the thoracic duct and the superior vena cava. Compared with other organs and tissues, the liver was thus thought to have only nutritional input via glucose and amino acids, but not via lipids. It was

Frazer [START_REF] Frazer | Differentiation in the absorption of olive oil and oleic acid in the rat[END_REF] who discovered that indeed neutral fat globules will only enter the systemic A C C E P T E D M A N U S C R I P T circulation, but that fatty acids do pass into the portal capillaries and enter the liver.

Thereafter, short-and medium-chain fatty acids were confirmed to be transported into the portal vein [START_REF] Bloom | Intestinal lymph as pathway for transport of absorbed fatty acids of different chain lengths[END_REF], and this was later confirmed for long-chain fatty acids and triglycerides as well [START_REF] Mcdonald | Portal venous transport of long-chain fatty acids absorbed from rat intestine[END_REF].

As was the case with glucose, lipid was proven to be a metabolic signal that can be sensed by vagal nerves as well [START_REF] Randich | Jejunal or portal vein infusions of lipids increase hepatic vagal afferent activity[END_REF]. More importantly, however, it became clear that the brain too, is an important metabolic sensing organ. Today, there is no doubt that the brain (especially the hypothalamus) and liver, as well as the gastrointestinal tract, can independently detect glucose, lipids and other metabolic information. The relative importance, however, of each of these components separately has only recently become the subject of both animal and human studies.

This review will start with an overview of the anatomical and physiological approaches used to identify the pattern of autonomic liver innervation. Then we describe the different roles played by the spinal and vagal afferents and efferents in liver metabolic sensing as well as in liver glucose and lipid production. The review ends with a description of the different levels of integration of metabolic information followed by a discussion of the current status of the known connections and missing links between brain and liver in the control of energy metabolism.

-Neuroanatomy of the hepatic innervation

The liver is innervated by sympathetic and parasympathetic nerves, both containing afferent as well as efferent fibers. The sympathetic splanchnic nerves innervating the liver originate from neurons in the celiac and superior mesenteric ganglia, which are innervated by preganglionic neurons located in the intermediolateral column of the spinal cord (T7-T12). The parasympathetic nerves innervating the liver originate from preganglionic neurons in the dorsal motor nucleus of the vagus (DMV) located in the A C C E P T E D M A N U S C R I P T dorsal brainstem. Unlike other visceral organs, no clear intrahepatic postganglionic neurons have been identified [START_REF] Berthoud | Anatomy and function of sensory hepatic nerves[END_REF].

The distribution of sympathetic and parasympathetic nerves in the liver is markedly species-dependent. Histochemical and immunochemical studies have been performed using a variety of markers for autonomic liver innervation. For instance, vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY) have been used as markers for sympathetic efferent fibers [START_REF] Akiyoshi | A comparative histochemical and immunohistochemical study of aminergic, cholinergic and peptidergic innervation in rat, hamster, guinea pig, dog and human livers[END_REF][START_REF] El-Salhy | Peptidergic innervation and endocrine cells in the human liver[END_REF]. In addition, α-adrenergic and βadrenergic receptors for the sympathetic neurotransmitter noradrenaline are present in the hepatic artery and portal vein [START_REF] Geumei | The presence of beta-adrenergic receptors in the hepatic vasculature[END_REF][START_REF] Green | Autonomic vasomotor responses in the canine hepatic arterial and venous beds[END_REF]. On the other hand, acetylcholinesterase (AChE) and vesicular acetylcholine transporter (VAChT) [START_REF] Arvidsson | Vesicular acetylcholine transporter (VAChT) protein: a novel and unique marker for cholinergic neurons in the central and peripheral nervous systems[END_REF], located in parasympathetic postganglionic cells [START_REF] Berthoud | Anatomy and function of sensory hepatic nerves[END_REF], tend to be the vagal efferent markers of choice [START_REF] Hoover | Localization and acetylcholinesterase content of vagal efferent neurons[END_REF][START_REF] Xue | Isolated hepatic cholinergic denervation impairs glucose and glycogen metabolism[END_REF].

Calcitonin gene-related peptide (CGRP) has been used as a spinal afferent marker, since the nodose ganglia express much less CGRP than the dorsal root ganglia (DRG) [START_REF] Green | Calcitonin gene-related peptide and substance P in afferents to the upper gastrointestinal tract in the rat[END_REF]. Finally, substance P (SP) has been used as a marker for both vagal and spinal afferents [START_REF] Hokfelt | Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons[END_REF][START_REF] Gamse | Substance P in the vagus nerve. Immunochemical and immunohistochemical evidence for axoplasmic transport[END_REF]. Different combinations of these markers have been applied to the hepatic tissue of human, monkey, dog, rabbit, rat, hamster, guinea pig, mouse, carp, bullfrog, and turtle. Some results are not firmly established as they could not be repeated in later studies, but this is probably due to non-specific technical limitations, such as those encountered using earlier histochemical methods for the demonstration of acetylcholinesterase (AChE) in rat parenchyma [START_REF] Sutherland | An evaluation of cholinesterase techniques in the study of the intrinsic innervation of the liver[END_REF]. Generally, in most of the examined species, sympathetic and parasympathetic markers (either neurotransmitters or synthesizing enzymes) could be detected in the hepatic artery, the portal vein region and the area around the bile ducts. Differences mainly exist in the interlobular area and parenchyma. In several studies, TH and NPY showed co-localization, and both kinds of terminals have been observed in the connective tissue of the interlobular septum and parenchyma from human, monkey, and guinea pig, but not from rat, hamster and mouse.

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In rabbit liver parenchyma, TH is expressed without co-localization of NPY. AChE fibers were not found in human liver parenchyma, nor in any other species studied [START_REF] Akiyoshi | A comparative histochemical and immunohistochemical study of aminergic, cholinergic and peptidergic innervation in rat, hamster, guinea pig, dog and human livers[END_REF][START_REF] El-Salhy | Peptidergic innervation and endocrine cells in the human liver[END_REF][START_REF] Burt | Localization of adrenergic and neuropeptide tyrosine-containing nerves in the mammalian liver[END_REF][START_REF] Goehler | Neuropeptide Y immunoreactivity in the mammalian liver: pattern of innervation and coexistence with tyrosine hydroxylase immunoreactivity[END_REF][START_REF] Stoyanova | Peptidergic nerve fibres in the human liver[END_REF][START_REF] Ding | Development of neuropeptide Y innervation in the liver[END_REF][START_REF] Ding | Phylogenetic and ontogenetic study of neuropeptide Y-containing nerves in the liver[END_REF]. Although NPY is sometimes also considered a neurotransmitter for post-ganglionic parasympathetic nerves [START_REF] Domeij | Enkephalin-like immunoreactivity in ganglionic cells in the larynx and superior cervical ganglion of the rat[END_REF][START_REF] Rudnicki | Vagal cooling blocks circulating neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) release[END_REF], no study has shown co-localization of NPY and AChE in liver tissue. A major difference between sympathetic innervation of humans and the most commonly used animal models, i.e., rats and mice, is that the latter have no clear parenchymal sympathetic innervation, while in human liver (also in guinea pig), sympathetic noradrenaline-immunoreactive fibes penetrate deep into the lobule to end on hepatocytes [START_REF] Fukuda | Demonstration of noradrenaline-immunoreactive nerve fibres in the liver[END_REF]. However, the functionality of the sympathetic efferent innervation of species with parenchymal sympathetic innervation could still correspond to that of species without [START_REF] Beckh | Activation of glycogenolysis by stimulation of the hepatic nerves in perfused livers of guinea pig and tree shrew as compared to rat: differences in the mode of action[END_REF] as information from aminergic and peptidergic nerve terminals can be relayed electrically to individual cells by structures such as cell-to-cell connecting gap junctions [START_REF] Hertzberg | Isolation and characterization of gap junctions from rat liver[END_REF][START_REF] Seseke | Signal propagation via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves[END_REF]. Indeed, signal propagation through gap junctions, i.e., via electrotonic coupling, can compensate for the sparse direct inputs to the hepatocytes, especially with respect to sympathetic signal transduction [START_REF] Seseke | Signal propagation via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves[END_REF][START_REF] Iwai | Inhibition of glucose production during hepatic nerve stimulation in regenerating rat liver perfused in situ. Possible involvement of gap junctions in the action of sympathetic nerves[END_REF]. Also, there is considerable homology between the rat and human liver gap junctions [START_REF] Kumar | Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein[END_REF]. This brought about the idea of additional functions of the gap junctions, such as the relay of hormonal signals from the periportal area to the hepatocytes [START_REF] Stumpel | Connexin 32 gap junctions enhance stimulation of glucose output by glucagon and noradrenaline in mouse liver[END_REF]. Furthermore, the sympathetic signal may be propagated via the release of prostaglandins from Ito cells [START_REF] Haussinger | Prostaglandin responses in isolated perfused rat liver: Ca2+ and K+ fluxes, hemodynamic and metabolic effects[END_REF][START_REF] Athari | Prostaglandin F2 alpha and D2 release from primary Ito cell cultures after stimulation with noradrenaline and ATP but not adenosine[END_REF] (the Ito or stellate cells are located in the space of Disse, which is separated from the lumen by the fenestrated endothelium, while Kupffer cell and dendritic cell face the sinusoidal lumen [START_REF] Unanue | Ito cells, stellate cells, and myofibroblasts: new actors in antigen presentation[END_REF]).

Only few approaches are available to investigate and differentiate the intra-hepatic neuroanatomy of the autonomic innervation, such as neuronal tracing or neuronal denervation in combination with physiological interventions. In rats, liver vagal afferents mainly ascend to the left nodose ganglion [START_REF] Berthoud | An anterograde tracing study of the vagal innervation of rat liver, portal vein and biliary system[END_REF][START_REF] Norgren | Central distribution of subdiaphragmatic vagal branches in the rat[END_REF], with the axon processes from the nodose ganglion projecting to the nucleus of the solitary tract (NTS). Sympathetic

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afferents from the liver enter the dorsal root ganglion (DRG), with the DRG axons terminating in the dorsal horn of the spinal cord [START_REF] Berthoud | Anatomy and function of sensory hepatic nerves[END_REF]. The remarkable lack of either vagal efferent or afferent innervation of liver parenchyma as indicated by the (immuno)histochemical studies was confirmed by neuronal tracing studies in rats, as no anterograde labeling was found in the parenchymal liver tissue from anterograde tracer injections in either the nodose ganglion or the dorsal motor nuclei [START_REF] Berthoud | An anterograde tracing study of the vagal innervation of rat liver, portal vein and biliary system[END_REF]. In addition, it was reported that the hepatic vagal nerve is only a sub-branch from the common hepatic branch that terminates in the upper duodenum, pancreas, pyloric sphincter, and antral stomach [START_REF] Berthoud | An anterograde tracing study of the vagal innervation of rat liver, portal vein and biliary system[END_REF] (Fig. 1). This observation was not in line with the assumption that vagal nerves fibers derived from the common hepatic branch and the periarterial plexus of the hepatic artery proper exclusively innervate the liver. The anatomical observation that the gastroduodenal vagal branch joins the hepatic vagal branch to form the so-called "common hepatic branch" complicates the interpretation of data from experiments in which the common hepatic vagal branch is disrupted or its neural activity is recorded. This is well evidenced by the separation of the gastroduodenal branch from the common hepatic branch by means of denervation, as it turns out that an electrical signal of the common hepatic branch that is sensitive to serotonin is blocked by transection of the gastroduodenal branch [START_REF] Horn | Separation of hepatic and gastrointestinal signals from the common "hepatic" branch of the vagus[END_REF]. In other words, stimulation of the intestinal tract may affect recordings of the hepatic branch, and "hepatic parasympathetic denervation" will in fact denervate the common hepatic branch.

Retrograde tracing from liver parenchyma close to the hilus (i.e., where all nerve bundles enter the liver) only results in a limited amount of DRG labeling from Th7-11, indicating that only very few DRG afferents innervate the hepatoportal system [START_REF] Berthoud | Anatomy and function of sensory hepatic nerves[END_REF].

As just mentioned, the classic anterograde and retrograde tracers are only able to reveal the location of first order sensory and motor neurons. The location of second order motor neurons in the central nervous system can be revealed with the multi-synaptic viral A C C E P T E D M A N U S C R I P T tracing technique. After injection of the pseudo-rabies virus in rat liver, several hypothalamic areas are highlighted (in addition to the brainstem) as containing secondorder neurons, such as the lateral hypothalamus (LH), the paraventricular nucleus (PVN)

and the retrochiasmatic area [START_REF] La Fleur | Polysynaptic neural pathways between the hypothalamus, including the suprachiasmatic nucleus, and the liver[END_REF]. Combining the viral tracing technique with selective denervations of either sympathetic or parasympathetic liver innervation clearly demonstrated the presence of separate populations of, respectively, parasympathetic and sympathetic motor neurons, both first and second order [START_REF] Kalsbeek | Suprachiasmatic GABAergic inputs to the paraventricular nucleus control plasma glucose concentrations in the rat via sympathetic innervation of the liver[END_REF].

Human liver nerve fibers show a strong plasticity in their distribution. Pathological challenges related to autonomic hyperactivity, such as (pre)cirrhosis and noncirrhotic portal hypertension, cause a decreased parenchymal innervation [START_REF] Lee | Disappearance of hepatic parenchymal nerves in human liver cirrhosis[END_REF], whereas nerves are proliferating in the portal region [START_REF] Jaskiewicz | Toxic hepatic injury is associated with proliferation of portal nerve fibers[END_REF][START_REF] Jaskiewicz | Distribution of hepatic nerve fibers in liver diseases[END_REF] as indicated by an increased number of NPY fibers [START_REF] Miyazawa | Immunohistochemical studies on the distribution of nerve fibers in chronic liver diseases[END_REF]. Whether this redistribution is related to a change in liver metabolism is not clear.

Summarizing it is evident that, despite the clear evidence for both a sympathetic and parasympathetic innervation of the liver, many details still have to be filled in and a lot is still to be learned about the autonomic innervation of the liver. Technical limitations still prevent a clear mapping of some parts of the liver neuronal network, such as, for instance, the location of parasympathetic postganglionic cell bodies. This lack of more detailed knowledge is a major obstacle for functional studies on the role of the autonomic innervation in the control of liver metabolism as they very much depend on the existing knowledge on its neuroanatomy. Also the difficulty of separating the sensory and motor branches still provides a profound obstacle to differentiate the various functional aspects. Despite these neuroanatomical difficulties, the evidence presented below clearly shows that the liver is a powerful metabolic sensor, and a crucial link in the brain control of energy metabolism.

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-Afferent pathways and metabolic sensing

All macronutrients absorbed from intestinal digestion will be "sensed" first by the gastrointestinal tract [START_REF] Dockray | Making sense of gut contents[END_REF], before entering the liver via the portal vein. Cephalic, postprandial and post-absorptive gastro-intestinal secretions will also reach the liver via the portal vein. With the large variety of chemoreceptors located in the hepatoportal system, it is more than likely that these metabolic signals will also be sensed by the liver and reported to the brain. For a long time, metabolic sensing by the liver (or hepatoportal system) was therefore considered to be the primary source of information for the brain to regulate metabolism. However, in the last two decades, the prominent role of liver metabolic sensing has been challenged, since it has become clear that the brain is able to sense metabolic information from many more sources than just the liver. For instance, the presence of insulin [START_REF] Spanswick | Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats[END_REF], leptin [START_REF] Pelleymounter | Effects of the obese gene product on body weight regulation in ob/ob mice[END_REF][START_REF] Campfield | Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks[END_REF], adiponectin [START_REF] Qi | Adiponectin acts in the brain to decrease body weight[END_REF], ghrelin [START_REF] Tschop | Ghrelin induces adiposity in rodents[END_REF] and neuropeptide Y [START_REF] Morley | Peptide YY (PYY), a potent orexigenic agent[END_REF] receptors in the brain makes it very likely that the brain can also sense metabolic information coming from organs such as the pancreas, adipose tissue, stomach and gut directly. The liver releases a number of factors that function as paracrine and endocrine regulators of glucose and lipid metabolism, such as adropin [START_REF] Kumar | Identification of adropin as a secreted factor linking dietary macronutrient intake with energy homeostasis and lipid metabolism[END_REF] and fibroblast growth factor 21 (FGF21) [START_REF] Badman | Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states[END_REF][START_REF] Inagaki | Endocrine regulation of the fasting response by PPARalpha-mediated induction of fibroblast growth factor 21[END_REF]. Remarkably, to date, none of these circulating factors have been reported to convey metabolic information from the liver to the brain. Therefore, brain and liver are probably specialized in collecting different types of information, and this information is converging into integration centers, such as the nucleus of the solitary tract (NTS) in the hindbrain [START_REF] Berthoud | Brainstem mechanisms integrating gut-derived satiety signals and descending forebrain information in the control of meal size[END_REF] and the paraventricular nucleus (PVN) in the hypothalamus [START_REF] Swanson | Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms[END_REF]. The studies on glucose and lipid sensing provide some of the best examples to illustrate this complex network. In addition, a different type of metabolic sensing is relayed within the afferent fibers from intra-hepatic metabolic sensors such as the liver X receptor (LXR) [START_REF] Mitro | The nuclear receptor LXR is a glucose sensor[END_REF] and the carbohydrate response element binding protein (ChREBP) [START_REF] Yamashita | A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver[END_REF], as these intra-hepatic sensors mainly work as

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coordinators between glucose sensing and lipid synthesis at the intracellular level.

Recently, the peptide adropin, secreted by liver and brain, was identified as a downstream signal regulated by LXR. Adropin mainly functions as a paracrine factor, but it also works as an endocrine factor to regulate glucose homeostasis and lipid metabolism [START_REF] Kumar | Identification of adropin as a secreted factor linking dietary macronutrient intake with energy homeostasis and lipid metabolism[END_REF]. Whether adropin acts locally on its targets or via an autonomic neuronal connection has not been elucidated yet. Finally, there is the hepatic insulin sensitizing substance (HISS), a substance released from the liver by parasympathetic stimulation, but not yet identified at the molecular level, that seems to be involved in the remote control of skeletal muscle glucose uptake [START_REF] Lautt | Insulin sensitivity in skeletal muscle regulated by a hepatic hormone, HISS, Can[END_REF].

Glucose sensing

The brain is unable to either synthesize or store the amount of glucose required for its normal cellular function, therefore it accurately keeps track of the whole process of glucose metabolism, from the initial stages of glucose intake, through absorption, storage, production and ultimately utilization. The mechanism of glucose detection, or glucose sensing, takes place in different areas of the brain [START_REF] Anand | Effect of glucose on the activity of hypothalamic "feeding centers[END_REF][START_REF] Oomura | reciprocal activities of the ventromedial and lateral hypothalamic areas of cats[END_REF][START_REF] Levin | Neuronal glucosensing: what do we know after 50 years?[END_REF][START_REF] Horvath | Fuel utilization by hypothalamic neurons: roles for ROS[END_REF][START_REF] Levin | Brain glucose sensing and body energy homeostasis: role in obesity and diabetes[END_REF][START_REF] Levin | Glucosensing neurons do more than just sense glucose[END_REF], as well as in the periphery. Moreover, in the brain, not only neurons can detect glucose levels, but glial cells expressing glucose transporter type 2 (glut2) are also involved, especially in the hypoglycemia-stimulated glucagon secretion mechanism [START_REF] Magistretti | Energy on demand[END_REF][START_REF] Marty | Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors[END_REF]. The primary places of peripheral glucose sensing are located in the taste bud cells [START_REF] Yamamoto | Brain mechanisms of sweetness and palatability of sugars[END_REF], the intestinal lumen [START_REF] Sjovall | On the mode of action of the sympathetic fibres on intestinal fluid transport: evidence for the existence of a glucose-stimulated secretory nervous pathway in the intestinal wall[END_REF][START_REF] Raybould | Sensing of glucose in the gastrointestinal tract[END_REF], the carotid body [START_REF] Pardal | Low glucose-sensing cells in the carotid body[END_REF], and the best studied locus, the hepatoportal area [START_REF] Hevener | Hypoglycemic detection does not occur in the hepatic artery or liver: findings consistent with a portal vein glucosensor locus[END_REF][START_REF] Hevener | Novel glucosensor for hypoglycemic detection localized to the portal vein[END_REF][START_REF] Hevener | Portal vein afferents are critical for the sympathoadrenal response to hypoglycemia[END_REF][START_REF] Novin | Infusion of 2-deoxy-D-glucose into the hepatic-portal system causes eating: evidence for peripheral glucoreceptors[END_REF].

For an up to date review on this issue the reader is referred to the recent review by

Watts & Donovan [START_REF] Watts | Sweet talk in the brain: Glucosensing, neural networks, and hypoglycemic counterregulation[END_REF].

In the earliest study of liver glucose sensing, using fairly high concentrations of glucose, Niijima [START_REF] Niijima | Afferent impulse discharges from glucoreceptors in the liver of the guinea pig[END_REF] proposed three different locations for the "glucose receptor": 1) the wall of the portal vein, 2) the parenchyma of the liver, and 3) the wall of the hepatic vein. Nowadays it is generally accepted that glucosensors are mainly located in the portal area [START_REF] Hevener | Hypoglycemic detection does not occur in the hepatic artery or liver: findings consistent with a portal vein glucosensor locus[END_REF][START_REF] Hevener | Novel glucosensor for hypoglycemic detection localized to the portal vein[END_REF][START_REF] Hevener | Portal vein afferents are critical for the sympathoadrenal response to hypoglycemia[END_REF],

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which is innervated by both sympathetic and vagal afferents [START_REF] Barja | Sensory innervation of the rat portal vein and the hepatic artery[END_REF][START_REF] Ody | Postnatal development of the rat portal vein: correlation with occurrence of peptidergic innervation[END_REF]. The functional meaning of glucosensing by these two types of afferents is still under investigation. High concentrations of glucose presented in the portal vein by a positive porto-arterial glucose gradient are mainly sensed by the vagus nerve and inhibit its activity [START_REF] Niijima | Afferent impulse discharges from glucoreceptors in the liver of the guinea pig[END_REF]. The signal transduction from portal vein glucose infusion to the NTS can be blocked by hepatic vagotomy [START_REF] Adachi | Convergence of hepatoportal glucose-sensitive afferent signals to glucose-sensitive units within the nucleus of the solitary tract[END_REF][START_REF] Niijima | Reflex control of the autonomic nervous system activity from the glucose sensors in the liver in normal and midpontine-transected animals[END_REF].

One hypothesis is that hepatic glucosensors participate in the control of food intake, by sensing the flow of digested glucose when its concentration is high. However, total liver denervation does not seem to affect feeding behavior in any significant way [START_REF] Louis-Sylvestre | Effect of liver denervation on the feeding pattern of rats[END_REF][START_REF] Bellinger | Failure of portal glucose and adrenaline infusions or liver denervation to affect food intake in dogs[END_REF].

Possibly the portal-peripheral glucose gradient alone is not sufficient to inhibit feeding, but when joined with brain glucose sensing it may be able to reduce food intake. On the other hand, decreasing the portal-peripheral glucose gradient will activate vagal afferent activity [START_REF] Niijima | The effect of D-glucose on the firing rate of glucose-sensitive vagal afferents in the liver in comparison with the effect of 2-deoxy-D-glucose[END_REF], and this activation has been proven to be essential for the initiation of food intake [START_REF] Novin | Infusion of 2-deoxy-D-glucose into the hepatic-portal system causes eating: evidence for peripheral glucoreceptors[END_REF][START_REF] Smith | Increased feeding in response to decreased glucose utilization in the rat and monkey[END_REF][START_REF] Likuski | Inhibition of gold thioglucose-induced hypothalamic obesity by glucose analogues[END_REF][START_REF] Friedman | Food intake and peripheral factors after recovery from insulin-induced hypoglycemia[END_REF]. Therefore, in the control of feeding behavior, hepatic glucosensing might play a more important role in triggering the initiation of feeding than in its termination.

In more recent studies, hypoglycemia sensed by the portal vein has received increased attention especially in the setting of insulin therapy in view of its essential role in tight glycemic control in diabetes patients. The occurrence of frequent and severe hypoglycemia may partially originate from defective glucose counterregulation [START_REF] Cryer | Mechanisms of hypoglycemia-associated autonomic failure and its component syndromes in diabetes[END_REF].

Hypoglycemia detection is not mediated by vagal afferents [START_REF] Fujita | Celiac-superior mesenteric ganglionectomy, but not vagotomy, suppresses the sympathoadrenal response to insulin-induced hypoglycemia[END_REF][START_REF] Jackson | Effects of vagal blockade on the counterregulatory response to insulin-induced hypoglycemia in the dog[END_REF], but does involve the capsaicin-sensitive primary spinal afferent nerves [START_REF] Fujita | Hypoglycemic detection at the portal vein is mediated by capsaicin-sensitive primary sensory neurons[END_REF]. It has also been suggested that portal vein glucose sensors only play a key role in the response to slow-onset hypoglycemia [START_REF] Matveyenko | Portal vein hypoglycemia is essential for full induction of hypoglycemia-associated autonomic failure with slow-onset hypoglycemia[END_REF], but that the primary place of detection shifts to other loci such as the brain when hypoglycemia develops rapidly [START_REF] Saberi | The locus for hypoglycemic detection shifts with the rate of fall in glycemia: the role of portal-superior mesenteric vein glucose sensing[END_REF][START_REF] Cherrington | Central versus peripheral glucose sensing and the response to hypoglycemia[END_REF].

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In animal studies, a selective sympathetic or parasympathetic hepatic denervation is not sufficient to prevent the counterregulatory response resulting from an insulin-induced hypoglycemia [START_REF] Jackson | Effects of vagal blockade on the counterregulatory response to insulin-induced hypoglycemia in the dog[END_REF][START_REF] Dolnikoff | Neural mechanisms involved in the recovery from insulin hypoglycemia in dogs[END_REF][START_REF] Mikines | Glucose turnover during insulin-induced hypoglycemia in liver-denervated rats[END_REF]. On the other hand, contradictory results were found when a total denervation was applied [START_REF] Jackson | Effects of vagal blockade on the counterregulatory response to insulin-induced hypoglycemia in the dog[END_REF][START_REF] Dolnikoff | Neural mechanisms involved in the recovery from insulin hypoglycemia in dogs[END_REF], apparently the results are very much depending on the denervation method.

The hypoglycemia counterregulatory response has also been investigated in patients who received a liver transplantation. In this "model", all direct neural connections but not the neurohumoral factors are lacking. In clinical studies carefully excluding bias from immunosuppressive treatment such as prednisone [START_REF] Pagano | An in vivo and in vitro study of the mechanism of prednisone-induced insulin resistance in healthy subjects[END_REF] data from chronic uveitis patients are compared with data from liver transplant patients. During a hypoglycemic clamp, liver transplanted patients appeared to have impaired hypoglycemic counterregulation as shown by less epinephrine and a cortisol release and a blunt recovery of endogenous glucose production (EGP) to basal levels [START_REF] Perseghin | Regulation of glucose homeostasis in humans with denervated livers[END_REF]. These defects indicate that neuronal inputs from liver to brain (and/or brain to liver) are important to elicit the appropriate hypoglycemic counterregulation. The exact intrahepatic mechanism of glucose sensing is not clear yet, but it has similarities with the pancreatic β-cells, as it needs activation of the glucagon-like peptide-1 (GLP-1) receptor [START_REF] Nakagawa | Receptor gene expression of glucagon-like peptide-1, but not glucose-dependent insulinotropic polypeptide, in rat nodose ganglion cells[END_REF][START_REF] Nishizawa | Effect of intraportal glucagon-like peptide-1 on glucose metabolism in conscious dogs[END_REF][START_REF] Nishizawa | The hepatic vagal reception of intraportal GLP-1 is via receptor different from the pancreatic GLP-1 receptor[END_REF][START_REF] Burcelin | Glucose competence of the hepatoportal vein sensor requires the presence of an activated glucagon-like peptide-1 receptor[END_REF][START_REF] Nakabayashi | Vagal hepatopancreatic reflex effect evoked by intraportal appearance of tGLP-1[END_REF][START_REF] Burcelin | Portal glucose infusion in the mouse induces hypoglycemia: evidence that the hepatoportal glucose sensor stimulates glucose utilization[END_REF], and is GLUT-2 dependent [START_REF] Burcelin | Glucose sensing by the hepatoportal sensor is GLUT2-dependent: in vivo analysis in GLUT2-null mice[END_REF].

Lipid sensing

Although normal feeding behavior does not require intact liver innervation, overingestion of lipids can affect food intake through the autonomic innervation of the liver.

Experimentally administered free fatty acids (FFAs) in the small intestine can be directly transported to the portal vein to enter the liver. Gastrointestinal primary sensory nerves have been reported to be involved in mediating the suppressive effect of intestinal lipid on feeding behavior [START_REF] Yox | Vagotomy attenuates suppression of sham feeding induced by intestinal nutrients[END_REF][START_REF] Yox | Capsaicin attenuates suppression of sham feeding induced by intestinal nutrients[END_REF]. Further studies specifically differentiating general

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abdominal vagotomy from hepatic vagotomy suggested that the hepatic vagus has a major involvement in this mechanism [START_REF] Cox | Truncal and hepatic vagotomy reduce suppression of feeding by jejunal lipid infusions[END_REF]. Hepatic portal infusions of lipids significantly increase hepatic vagal afferent activity [START_REF] Randich | Jejunal or portal vein infusions of lipids increase hepatic vagal afferent activity[END_REF]. The inhibitory effect on feeding behavior induced by hepatic lipid sensing has also been confirmed under diabetic conditions in experimental animals. A high-fat diet normalizes food intake in STZ-diabetic rats [START_REF] Chavez | Effect of a high-fat diet on food intake and hypothalamic neuropeptide gene expression in streptozotocin diabetes[END_REF][START_REF] Warne | Afferent signalling through the common hepatic branch of the vagus inhibits voluntary lard intake and modifies plasma metabolite levels in rats[END_REF][START_REF] La Fleur | The hepatic vagus mediates fat-induced inhibition of diabetic hyperphagia[END_REF], but common branch hepatic vagotomy can completely block both the fat-induced decrease in caloric intake as well as normalize the increased hypothalamic NPY and CRF mRNA expression [START_REF] Warne | Afferent signalling through the common hepatic branch of the vagus inhibits voluntary lard intake and modifies plasma metabolite levels in rats[END_REF][START_REF] La Fleur | Hepatic vagotomy alters limbic and hypothalamic neuropeptide responses to insulindependent diabetes and voluntary lard ingestion[END_REF].

Still, in some studies, the inhibitory effects of FFAs on food intake cannot be simply blocked by subdiaphragmatic bilateral vagotomy [START_REF] Ogawa | The vagal afferent pathway does not play a major role in the induction of satiety by intestinal fatty acid in rats[END_REF]. Thus, in addition to FFA, high fat diets might contain information that affects feeding. Alternatively, FFAs may be able to manipulate feeding via other mechanisms such as the hypothalamus [START_REF] Lam | Hypothalamic sensing of circulating fatty acids is required for glucose homeostasis[END_REF].

Under physiological conditions not only food-derived lipids will enter the liver. Fatty acids are also the metabolically most important product of adipose tissue lipolysis, and especially triglycerides stored in the abdominal adipose tissue will release FFAs into the portal vein. The hepatoportal vagal sensing of lipids may therefore not only be important for the "reflexive" regulation of feeding behavior, but it may also play a role in the pathophysiology of metabolic abnormalities such as hepatic insulin resistance.

Physiologically, abdominal adipose tissue only contributes a small part of the total amount of FFAs entering the portal vein, but an increased release of FFAs from the adipose tissue can be triggered by stress, anger, frustration, and other factors, such as smoking [START_REF] Meisner | Regulation of lipolysis in adipose tissue[END_REF]. Elevated levels of FFAs in the portal vein have been suggested to be the cause of insulin resistance, as they might directly reduce insulin clearance by the liver [START_REF] Kabir | Molecular evidence supporting the portal theory: a causative link between visceral adiposity and hepatic insulin resistance[END_REF][START_REF] Bergman | Central role of the adipocyte in the metabolic syndrome[END_REF][START_REF] Bergman | Non-esterified fatty acids and the liver: why is insulin secreted into the portal vein?[END_REF]. Furthermore, since the anti-lipolytic effect of insulin on omental adipose tissue is minor, due to the low insulin receptor number [START_REF] Bolinder | Differences at the receptor and postreceptor levels between human omental and subcutaneous adipose tissue in the action of insulin on lipolysis[END_REF], insulin resistance will result in a further increase of the amount of FFAs released. The overloading of the liver by FFAs As with glucose sensing, a specific lipid receptor has not yet been identified, but lipids are natural ligands for the peroxisome-proliferator-activated receptors (PPARs) [START_REF] Fu | Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha[END_REF] that are also involved in glucose metabolism. A knock-out of the lipogenic enzyme fatty-acid synthase (FAS) will not only induce hypoglycemia and a fatty liver, but also a defective expression of PPARα. Furthermore, PPARα deficiency prevents glucose intolerance caused by diet-induced obesity [START_REF] Guerre-Millo | PPAR-alpha-null mice are protected from high-fat diet-induced insulin resistance[END_REF][START_REF] Tordjman | PPARalpha deficiency reduces insulin resistance and atherosclerosis in apoE-null mice[END_REF]. Unlike glucose sensing, splanchnic-nerve afferent activity is not affected by jejunal lipid administration [START_REF] Cox | Truncal and hepatic vagotomy reduce suppression of feeding by jejunal lipid infusions[END_REF], and no study so far has shown spinal afferent responses to any other kind of adiposity information.

Protein sensing

Protein digestion will elevate portal vein amino acid levels and high protein feeding has been reported to stimulate glucagon release independently of amino acids blood levels [START_REF] Eisenstein | Increased glucagon secretion in protein-fed rats: lack of relationship to plasma amino acids[END_REF]. It is hard to say whether this is due to a special portal amino acid sensor, because surprisingly, only few studies on hepatoportal protein sensing are available. A high protein diet is able to inhibit food intake [START_REF] Jean | Metabolic evidence for adaptation to a high protein diet in rats[END_REF][START_REF] Booth | Relative effectiveness of protein in the late stages of appetite suppression in man[END_REF] but this effect might not be due to a direct hepatoportal protein sensing mechanism, but rather to the stimulation of intestinal gluconeogenesis [START_REF] Rajas | The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats[END_REF][START_REF] Rajas | Induction of PEPCK gene expression in insulinopenia in rat small intestine[END_REF][START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF] which may consequently activate portal glucose sensing [START_REF] Mithieux | Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein[END_REF]. This high glucose loading may then eventually result in inhibited feeding.

Hormone sensing

Not only nutrition itself can be sensed by the liver, but the nutrition and absorption process will elicit the release of a variety of hormones (so-called incretins), that can also Besides its brain origin and its involvement in the control of food intake by a central mechanism [START_REF] Han | Cellular localization of proglucagon/glucagon-like peptide I messenger RNAs in rat brain[END_REF][START_REF] Sandoval | CNS GLP-1 regulation of peripheral glucose homeostasis[END_REF], GLP-1 is also secreted from the intestines during meal absorption.

Its stimulatory effect on insulin secretion and its depressive effect on glucagon secretion implicate a remote control on the pancreas [START_REF] Orskov | Complete sequences of glucagon-like peptide-1 from human and pig small intestine[END_REF][START_REF] Orskov | Proglucagon products in plasma of noninsulin-dependent diabetics and nondiabetic controls in the fasting state and after oral glucose and intravenous arginine[END_REF]. Because of its low postprandial plasma levels and its rapid degradation, the action of GLP-1 is probably close to its site of release [START_REF] Ahren | Sensory nerves contribute to insulin secretion by glucagon-like peptide-1 in mice[END_REF]. The action of GLP-1 most likely also involves a neuronal pathway.

Indeed, GLP-1 receptors are expressed in the hepatoportal area [START_REF] Vahl | Glucagon-like peptide-1 (GLP-1) receptors expressed on nerve terminals in the portal vein mediate the effects of endogenous GLP-1 on glucose tolerance in rats[END_REF]. Infusion of a truncated form of GLP-1(7-36)amide (tGLP-I) into the portal vein increases hepatic vagal afferent activity and pancreatic vagal efferent activity [START_REF] Nakabayashi | Vagal hepatopancreatic reflex effect evoked by intraportal appearance of tGLP-1[END_REF], while it augments glucosestimulated insulin secretion [START_REF] Balkan | Portal GLP-1 administration in rats augments the insulin response to glucose via neuronal mechanisms[END_REF]. Peripheral GLP-1 is also able to influence feeding behavior. Its effects on meal size are mediated by the vagal nerve, but this effect does not involve the hepatoportal mechanism [START_REF] Ruttimann | Intrameal hepatic portal and intraperitoneal infusions of glucagon-like peptide-1 reduce spontaneous meal size in the rat via different mechanisms[END_REF], which we will discuss later.

The brain has been proven to be the main sensor of the adipokine leptin, affecting hepatic vagal afferent activity directly in a dose-dependent manner, with low doses inhibiting its activity and high doses increasing it. In addition, the activity of glucosesensitive units is inhibited by low concentrations of leptin [START_REF] Shiraishi | Leptin effects on feeding-related hypothalamic and peripheral neuronal activities in normal and obese rats[END_REF]. The gastrointestinal peptide cholecystokinin (CCK) released from the duodenal mucosa also has satiety effects, which are mediated by the gastric branch of the vagus [START_REF] Smith | Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat[END_REF]. CCK can be sensed by hepatoportal regional terminals [START_REF] Horn | Separation of hepatic and gastrointestinal signals from the common "hepatic" branch of the vagus[END_REF], but whether its inhibitory effect on feeding is mediated by the hepatoportal vagal branch or via the gastric vagal branch is not clear. The fact that lesions of the termination centers of the common vagal afferents, i.e., area postrema and NTS, do not impair the satiety induced by CCK [START_REF] Weatherford | Lesion of vagal afferent terminals impairs glucagon-induced suppression of food intake[END_REF], further complicates the understanding of the central processing of peripheral CCK information.

Portal infusion of somatostatin, yet another one of the gastroenteropancreatic hormones, A C C E P T E D M A N U S C R I P T also increases hepatic vagal afferent activity [START_REF] Nakabayashi | Neural monitoring system for circulating somatostatin in the hepatoportal area[END_REF]. How this signal is processed further, within the brain, is not yet known.

The inhibitory effect of illness on feeding activity is thought to be mediated by inflammatory factors [START_REF] Kent | Mechanisms of sicknessinduced decreases in food-motivated behavior[END_REF]. The cytokine interleukin-1 (IL-1) released during immune activation, has been studied in great detail. Intraperitoneal administration of IL-1β inhibits food intake [START_REF] Kent | Different receptor mechanisms mediate the pyrogenic and behavioral effects of interleukin 1[END_REF], and this effect is proposed to involve hepatic vagal sensing since its afferent activity is increased by IL-1β stimulation [START_REF] Niijima | The afferent discharges from sensors for interleukin 1 beta in the hepatoportal system in the anesthetized rat[END_REF]. Peripheral administration of IL-1α has similar anorexic effects [START_REF] Moldawer | Regulation of food intake and hepatic protein synthesis by recombinant-derived cytokines[END_REF]. However, hepatic vagotomy is unable to block this effect [START_REF] Laviano | Hepatic vagus does not mediate IL-1 alpha induced anorexia[END_REF].

In conclusion, the contribution of metabolic sensing in the liver to the overall whole body energy balancing network is still under discussion. Unlike the brain, where the glucose concentration is sensed by glucose excitatory and inhibitory neurons (and glia), in the liver the glucose concentration is more likely to be monitered by the sympathetic and parasympathetic nerves. The glucose sensing mechanisms in brain and liver clearly interact, but many details still need further clarification. Unfortunately, very little is still known about the cellular mechanism of glucose sensing in the liver.

-Efferent pathway and the control of hepatic metabolism

Neuronal signals in control of liver glucose and lipid metabolism

After the initial findings of Bernard, numerous efforts have been made to understand in more detail the brain mechanisms responsible for the control of liver metabolism, be it with some delay. First of all, Bernard's findings on the control of liver glucose output by the autonomic nerves were confirmed and extended. For instance, liver glycogen content can be changed by neuronal inputs independent of influences from circulating glucoregulatory hormones, such as the catecholamines, insulin and glucagon. Follow-up

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studies focused on specific brain-liver connections, such as the information from different brain regions and their output pathways to the liver via specific autonomic nerves.

The early studies often used surgical removal of other metabolic organs or a total isolation of the liver. For instance, it was shown that in adrenalectomized and/or pancreatectomized animals, electrical stimulation of the splanchnic nerve decreases liver glycogen content and causes an increased release of glucose by increasing the activity of the liver glycogen phosphorylase and glucose-6-phosphatase enzymes [START_REF] Shimazu | Increased activities of glycogenolytic enzymes in liver after splanchnic-nerve stimulation[END_REF][START_REF] Edwards | The glycogenolytic response to stimulation of the splanchnic nerves in adrenalectomized calves[END_REF], as well as a partial inactivation of phosphorylase phosphatase activity [START_REF] Shimazu | Regulation of glycogen metabolism in liver by the autonomic nervous system. VI. Possible mechanism of phosphorylase activation by the splanchnic nerve[END_REF]. Stimulation of the vagus nerve, alternatively, accelerates glycogen synthesis, and this effect could be completely counteracted by a simultaneous stimulation of the splanchnic nerve [START_REF] Shimazu | Glycogen synthetase activity in liver: regulation by the autonomic nerves[END_REF][START_REF] Shimazu | Regulation of glycogen metabolism in liver by the autonomic nervous system. V. Activation of glycogen synthetase by vagal stimulation[END_REF].

Later on, the independence of this neuronal pathway in the control of liver glucose production was also confirmed in an eviscerated animal model. In these experiments the authors showed that the reduction of blood glucose concentrations induced by insulin administration to the brain (via the carotid artery) was conserved after a total pancreatectomy together with the removal of the entire gastrointestinal tract and the spleen [START_REF] Szabo | Influence of the insulin sensitive central nervous system glucoregulator receptor on hepatic glucose metabolism[END_REF]. Recently, the original data on the inhibitory effects of central insulin on plasma glucose concentrations were confirmed when insulin was applied directly into the cerebral ventricle. In addition, it was shown that the inhibitory effect of central insulin on hepatic glucose production could be blocked by both hepatic vagotomy and sympathectomy, and that this central insulin effect only needs vagal efferents (not afferents) without affecting glucose uptake [START_REF] Pocai | Hypothalamic K(ATP) channels control hepatic glucose production[END_REF][START_REF] Van Den Hoek | Intracerebroventricular administration of neuropeptide Y induces hepatic insulin resistance via sympathetic innervation[END_REF]. Very recently, it became clear that the vagal efferents to the liver are vital for the CCK signal generated in the gut to regulate hepatic glucose production and maintain glucose homeostasis. In this study, it was shown that the intraduodenal administration of CCK-8 can enhance hepatic insulin sensitivity and inhibit glucose production, and that these effects can be negated by hepatic vagotomy [START_REF] Cheung | Intestinal cholecystokinin controls glucose production through a neuronal network[END_REF].

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Lipid metabolism in the liver mainly includes the synthesis and secretion of VLDL, ketone bodies and fatty acid oxidation. Reducing liver noradrenaline by phenol-induced hepatic sympathetic denervation can decrease carnitine palmitoyltransferase (CPT), which is responsible for transferring long-chain fatty acid into the mitochondria [START_REF] Carreno | Liver denervation affects hepatocyte mitochondrial fatty acid transport capacity[END_REF]. The hepatic sympathetic input is also involved in regulating the secretion of apoB-containing lipoproteins, including VLDL [START_REF] Eaton | Radioimmunoassay of beta lipoprotein-protein of rat serum[END_REF]. In the perfused liver model, noradrenaline is able to inhibit the secretion of triglyceride and apoB as well as the release of VLDL at a posttranscriptional level [START_REF] Yamauchi | Noradrenaline and ATP decrease the secretion of triglyceride and apoprotein B from perfused rat liver[END_REF][START_REF] Brindle | Alpha-adrenergic suppression of very-low-densitylipoprotein triacylglycerol secretion by isolated rat hepatocytes[END_REF]. In line with this, hepatic sympathetic denervation results in lower VLDL secretion, and higher concentrations of plasma cholesterol and VLDLcholesterol [START_REF] Tavares | Hepatic denervation impairs the assembly and secretion of VLDL-TAG[END_REF]. The sympathetic innervation to the liver also influences ketone body metabolism. In the perfused liver model, sympathetic stimulation inhibits hepatic ketogenesis [START_REF] Beuers | Control of ketogenesis in the perfused rat liver by the sympathetic innervation[END_REF], resulting in a reduced ketone-body output from the liver [START_REF] Yamamoto | The mechanism of action of hepatic sympathetic nerves on ketone-body output from perfused rat liver. The effect of the interaction of noradrenaline with ATP on the release of betahydroxybutyrate[END_REF].

Perfusion of isolated liver has also been used to highlight the importance of the autonomic innervation of the liver and eliminate the possibility of extra-liver humoral inputs. In the perfusion model, splanchnic nerve stimulation caused an increased glucose and lactate output, whereas decreased urea, glutamine formation and ketone body production were observed [START_REF] Niijima | Release of glucose from perfused liver preparation in response to stimulation of the splanchnic nerves in the toad[END_REF][START_REF] Jungermann | Regulation of liver metabolism by the hepatic nerves[END_REF]. These effects mainly involved activation of the α1-adrenergic receptors [START_REF] Ulken | Increase in glucose and lactate output and perfusion resistance by stimulation of hepatic nerves in isolated perfused rat liver: role of alpha 1-, alpha 2-, beta 1-and beta 2-receptors[END_REF], and to a much lesser degree that of the β-receptors [START_REF] Seydoux | alpha-Sympathetic control of glucose output of mouse liver perfused in situ[END_REF].

However, the physiological significance of the sympathetic innervation for hepatic glucose production is constantly being questiones. For instance, in systemic α1adrenergic receptors deficient mice higher liver glycogen content was found together with a higher parasympathetic tone [START_REF] Burcelin | Impaired glucose homeostasis in mice lacking the alpha1b-adrenergic receptor subtype[END_REF]. However, blocking the portal α1 and βreceptors during heavy exercise does not change the hepatic glucose production [START_REF] Coker | Role of hepatic alpha-and beta-adrenergic receptor stimulation on hepatic glucose production during heavy exercise[END_REF].

In the perfusion model, effects of neuronal manipulation on liver hemodynamics are inevitable. For instance, acetylcholine (Ach) applied into such an isolated model causes

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vasoconstriction and a reduction of blood flow and oxygen supply to the liver [START_REF] Vatamaniuk | Acetylcholine affects rat liver metabolism via type 3 muscarinic receptors in hepatocytes[END_REF].

However, the early liver perfusion studies provided convincing evidence that the metabolic changes observed in the liver were not caused by hemodynamic changes in the liver or an overflow of noradrenalin into the hepatic vein [START_REF] Beckh | Mechanism of action of sympathetic hepatic nerves on carbohydrate metabolism in perfused rat liver[END_REF][START_REF] Hartmann | Direct control of glycogen metabolism in the perfused rat liver by the sympathetic innervation[END_REF].

Brain areas involved in liver control of glucose metabolism

Stimulation of several brain areas has been shown to induce similar changes in liver glucose metabolism as produced by direct stimulation of sympathetic or parasympathetic nerves. These brain areas are either proposed or proven to be able to affect liver glucose metabolism via their effect on autonomic neuronal output. For instance, electrical stimulation of the VMH causes an increased activity of the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and a marked suppression of hepatic pyruvate kinase (PK) activity, a key glycolytic enzyme [START_REF] Shimazu | Effects of hypothalamic stimulation on gluconeogenesis and glycolysis in rat liver[END_REF]. These responses could not be abolished by adrenalectomy [START_REF] Takahashi | Effects of ventromedial hypothalamus stimulation on glycogenolysis in rat liver using in vivo microdialysis[END_REF]. Stimulation of the LH, on the other hand, resulted in a decrease in PEPCK activity but did not alter PK activity [START_REF] Shimazu | Effects of hypothalamic stimulation on gluconeogenesis and glycolysis in rat liver[END_REF].

Physiologically, it seems that cholinergic, but not noradrenergic, dopaminergic or serotonergic receptors in the LH are selectively involved in the stimulation of liver glycogen synthesis [START_REF] Shimazu | Cholinergic stimulation of the rat hypothalamus: effects of liver glycogen synthesis[END_REF]. These early proposals for putative direct neuronal pathways were only recently supported by evidence from more detailed studies on the effects of brain insulin signaling on liver glucose metabolism. Brain insulin signaling inhibits liver gluconeogenesis (GNG) via neurons in the mediobasal hypothalamus that contain ATPsensitive potassium (KATP) channels [START_REF] Obici | Hypothalamic insulin signaling is required for inhibition of glucose production[END_REF], an effect that can be largely blocked by hepatic vagotomy [START_REF] Pocai | Hypothalamic K(ATP) channels control hepatic glucose production[END_REF], but also involves NPY signaling to sympathetic pre-autonomic neurons [START_REF] Van Den Hoek | Intracerebroventricular administration of neuropeptide Y induces hepatic insulin resistance via sympathetic innervation[END_REF].

Electrical stimulation of the suprachiasmatic nucleus (SCN) has also been reported to induce hyperglycemia [START_REF] Fujii | Involvement of adrenergic mechanism in hyperglycemia due to SCN stimulation[END_REF]. This hyperglycemia might involve both direct hepatic and A C C E P T E D M A N U S C R I P T indirect (i.e., pancreatic) stimulatory effects on glycogenolysis [START_REF] Nagai | Electrical stimulation of the suprachiasmatic nucleus of the hypothalamus causes hyperglycemia[END_REF]. Blocking the GABAcontaining projections from the SCN to the PVN by bicuculline also induces hyperglycemia, and this hyperglycemia could be eliminated by hepatic sympathetic but not parasympathetic denervation [START_REF] Kalsbeek | Suprachiasmatic GABAergic inputs to the paraventricular nucleus control plasma glucose concentrations in the rat via sympathetic innervation of the liver[END_REF]. The autonomic innervation of the liver is also involved in the hepatic insulin resistance and in increased hepatic glucose production induced by thyrotoxicosis, again an effect that seems to be mediated via a PVN mechanism and is independent of circulating glucoregulatory hormones, including the thyroid hormone itself [START_REF] Klieverik | Effects of thyrotoxicosis and selective hepatic autonomic denervation on hepatic glucose metabolism in rats[END_REF][START_REF] Klieverik | Thyroid hormone modulates glucose production via a sympathetic pathway from the hypothalamic paraventricular nucleus to the liver[END_REF]. Among the aforementioned hypothalamic peptidergic systems, NPY is well defined as a target of several circulating factors, due to its location in the arcuate nucleus (ARC), i.e., the "metabolic window of the brain". For instance, the adipokine resistin can modulate lipid metabolism and increase glucose production via its action on NPY neurons [START_REF] Vazquez | Central resistin regulates hypothalamic and peripheral lipid metabolism in a nutritionaldependent fashion[END_REF][START_REF] Muse | Hypothalamic resistin induces hepatic insulin resistance[END_REF][START_REF] Singhal | Central resistin induces hepatic insulin resistance via neuropeptide Y[END_REF]. Considering the well-established role of NPY in the control of hepatic insulin sensitivity via the hepatic sympathetic innervation [START_REF] Van Den Hoek | Intracerebroventricular administration of neuropeptide Y induces hepatic insulin resistance via sympathetic innervation[END_REF][START_REF] Van Den Hoek | Intracerebroventricular neuropeptide Y infusion precludes inhibition of glucose and VLDL production by insulin[END_REF], it is very likely that autonomic nerves will also mediate the central effects of resistin on liver metabolism. The involvement of NPY and other hypothalamic neuropeptides in control of glucose homeostasis will be discussed in more details in the next paragraph.

Virtually nothing is known about brain areas that are in control of hepatic lipid metabolism. Two recent papers have described effects of the i.c.v. administration of glucose and NPY on hepatic lipid metabolism [START_REF] Van Den Hoek | Intracerebroventricular neuropeptide Y infusion precludes inhibition of glucose and VLDL production by insulin[END_REF][START_REF] Lam | Brain glucose metabolism controls the hepatic secretion of triglyceriderich lipoproteins[END_REF], but no information is available about the possible location of these effects.

Hypothalamic neuropeptides involved in the control of liver glucose metabolism -Neuropeptide Y (NPY)

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Best known among the hypothalamic neuropeptidergic networks are the NPY-containing neurons in the ARC with their projections to several hypothalamic brain areas including the PVN. The first report on the glucoregulatory effects of the hypothalamic NPY system appeared in the mid nineties when it was shown that i.c.v. administration of NPY increases endogenous glucose production in rats, probably by decreasing hepatic insulin sensitivity [START_REF] Marks | Intracerebroventricular neuropeptide Y acutely influences glucose metabolism and insulin sensitivity in the rat[END_REF][START_REF] Marks | Some acute effects of intracerebroventricular neuropeptide Y on insulin secretion and glucose metabolism in the rat[END_REF]. Later on these results were confirmed in mice [START_REF] Van Den Hoek | Intracerebroventricular neuropeptide Y infusion precludes inhibition of glucose and VLDL production by insulin[END_REF]. In view of the inhibitory effects of hypothalamic insulin receptors on hepatic glucose production [START_REF] Obici | Hypothalamic insulin signaling is required for inhibition of glucose production[END_REF][START_REF] Obici | Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats[END_REF], the abundant expression of insulin receptors in the ARC [START_REF] Unger | Distribution of insulin receptor-like immunoreactivity in the rat forebrain[END_REF], the inhibitory effect of insulin on NPY neuronal activity [START_REF] Schwartz | Central insulin administration reduces neuropeptide Y mRNA expression in the arcuate nucleus of food-deprived lean (Fa/Fa) but not obese (fa/fa) Zucker rats[END_REF], and the effects of i.c.v. NPY on sympathetic activity [START_REF] Hu | Intracerebroventricular administration of NPY increases sympathetic tone selectively in vascular beds[END_REF][START_REF] Van | Hormonal and metabolic effects of paraventricular hypothalamic administration of neuropeptide Y during rest and feeding[END_REF][START_REF] Egawa | Neuropeptide Y suppresses sympathetic activity to interscapular brown adipose tissue in rats[END_REF], studies in our group by combining the euglycemic hyperinsulinemic clamp technique with the i.c.v. administration of NPY, and hepatic sympathetic-, parasympathetic-and sham-denervated in rats, confirmed once again that i.c.v. NPY is able to block (partially) the inhibitory effects of hyperinsulinemia on hepatic glucose production, but it also showed that a specific denervation of hepatic sympathetic nerves blocks the effect of NPY. Therefore, the brain-mediated inhibitory effect of insulin on hepatic glucose production is probably effectuated via an inhibition of NPY neuronal activity in the ARC. Subsequently, the resulting diminished release of NPY will decrease the stimulatory input to the sympathetic pre-autonomic neurons in the PVN and thus reduce the sympathetic stimulation of hepatic glucose production. The results of Pocai et al. [START_REF] Pocai | Hypothalamic K(ATP) channels control hepatic glucose production[END_REF], however, show that also the parasympathetic innervation of the liver is involved in the inhibitory effect of insulin on hepatic glucose production. This means that in addition to the effect of NPY on the sympathetic pre-autonomic neurons there is probably another neurotransmitter that is responsible for the transmission of insulin's effects in the ARC to the parasympathetic pre-autonomic neurons in the PVN. Moreover, the effects of NPY also seem to be specific for glucose production as in none of the above experiments there was a significant effect on whole body glucose disposal.

A C C E P T E D M A N U S C R I P T -Pro-opiomelanocortin (POMC)

Next to the orexigenic NPY/AGRP neurons, the ARC also contains a population of anorexigenic POMC/CART-containing neurons. The most important POMC-derived peptide with respect to feeding and metabolism is alpha-MSH. The antagonistic function of the NPY/AGRP and POMC/CART cell populations is most clearly illustrated by the fact that AGRP acts as an endogenous antagonist of the melanocortin receptors 3 and 4 [START_REF] Ollmann | Antagonism of central melanocortin receptors in vitro and in vivo by agoutirelated protein[END_REF]. The antagonizing mechanism of these neuropeptides are extremely important to adapt the hypothalamic-pituitary-thyroid (HPT) axis to the prevailing food/energy status, i.e., the fasting-induced suppression of TRH mRNA in the PVN needs the reduction in alpha-MSH and the increase in AGRP [START_REF] Lechan | Role of melanocortin signaling in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis[END_REF]. Surprisingly, this antagonistic cell population does not seem to be involved in the inhibitory effect of hypothalamic insulin on glucose production, as co-administration of a melanocortin antagonist failed to block the decrease in glucose production induced by central insulin [START_REF] Gutierrez-Juarez | Melanocortin-independent effects of leptin on hepatic glucose fluxes[END_REF]. Blocking alpha-MSH signaling via i.c.v. infusion of the melanocortin 3/4 receptor (MC3R/MC4R) antagonist SHU9119 has no effects on glucose metabolism, but i.c.v. infusion of alpha-MSH itself has a clear stimulatory effect on glucose production via GNG which can be antagonized by SHU9119. In the liver, the stimulation of GNG is confirmed by the increased expression of glucose 6-phosphatase (G6Pase) and PEPCK. In addition, all these central manipulations had no effects on peripheral glucose uptake. It has been proposed

that the hypothalamic MC3R/MC4R signaling pathway mediates the effects of systemic leptin on glucose production. Central administration of leptin has been proven to be involved in liver GNG regulation, by redistribution of intrahepatic glucose fluxes, in which GNG is increased and glycogenolysis was suppressed. However, unlike the central action of insulin to inhibit glucose production [START_REF] Obici | Hypothalamic insulin signaling is required for inhibition of glucose production[END_REF], this redistribution action of leptin does not change total glucose production by the liver [START_REF] Liu | Intracerebroventricular leptin regulates hepatic but not peripheral glucose fluxes[END_REF][START_REF] Rossetti | Short term effects of leptin on hepatic gluconeogenesis and in vivo insulin action[END_REF]. Although systemic leptin

A C C E P T E D M A N U S C R I P T

alone did not alter hepatic insulin sensitivity, it can enhance the hepatic insulin sensitivity by the i.c.v. co-infusion of SHU9119 [START_REF] Gutierrez-Juarez | Melanocortin-independent effects of leptin on hepatic glucose fluxes[END_REF]. Indeed, just like insulin, also the effects of systemic leptin on glucose production are mediated via a hypothalamic mechanism.

Recently, it was nicely shown that the adenoviral-induced expression of leptin receptors in the ARC of leptin receptor knock-out animals improves peripheral insulin sensitivity via enhanced suppression of glucose production [START_REF] German | Hypothalamic Leptin Signaling Regulates Hepatic Insulin Sensitivity via a Neurocircuit Involving the Vagus Nerve[END_REF]. The ARC-induced expression of the leptin receptor was associated with a reduced hepatic expression of G6Pase and PEPCK, but again, no significant changes in the insulin-stimulated whole body glucose utilization were apparent, which is different from the leptin action in the VMH on stimulatioing glucose uptake via an insulin-independent way [START_REF] Minokoshi | Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats[END_REF]. Moreover, the effects of hypothalamic leptin signaling on hepatic insulin sensitivity could be blocked by a selective hepatic vagotomy, providing further supportive evidence for the idea that ARC projections to pre-autonomic neurons (in the PVN) are important for the transmission of the effect of leptin on glucose production.

-Orexin

The neuropeptides orexin-A and orexin-B (also known as hypocretin-1 and hypocretin-2) were initially identified as the endogenous ligands for orphan receptors involved in the pathogenesis of narcolepsy [START_REF] Chemelli | Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation[END_REF][START_REF] De Lecea | The hypocretins: hypothalamusspecific peptides with neuroexcitatory activity[END_REF]. They were recognized as regulators of feeding behavior and energy metabolism because of the exclusive localization of their cell bodies in the lateral hypothalamus (LH), the induction of feeding upon their i.c.v. administration, their responsiveness to peripheral metabolic cues such as leptin and glucose, and the metabolic phenotype of the knock-out animals. More recent studies suggest that the orexin system is particularly important for the maintenance of wakefulness. However, recent data from our group also clearly revitalize the metabolic function of the orexin system. We showed that an increased availability of orexin in the A C C E P T E D M A N U S C R I P T central nervous system, either by i.c.v. infusion, or by local activation via removal of GABA inhibition, increases plasma glucose concentrations through an increase in hepatic glucose production. As with NPY also the stimulatory effect of orexin on glucose production could be blocked by a hepatic sympathetic but not parasympathetic denervation. Although it is not entirely clear yet where in the brain orexin is acting to stimulate glucose production, the i.c.v. infusion experiments and the presence of a pronounced orexin-containing fiber network in the PVN suggest that its main action is again at the level of the sympathetic pre-autonomic neurons in the PVN, but in view of the electrophysiological data of Van Den Top et al [START_REF] Van Den Top | Orexins induce increased excitability and synchronisation of rat sympathetic preganglionic neurones[END_REF], a direct effect of orexin at the level of the sympathetic pre-ganglionic neurons in the intermediolateral column of the spinal cord can also not be excluded.

-Pituitary adenylate cyclase activating peptide (PACAP)

PACAP is a 38-amino acid, C-terminally α-amidated neuropeptide that was originally isolated from the ovine hypothalamus on the basis of its ability to stimulate adenyl cyclase activity in rat anterior pituitary cells [START_REF] Miyata | Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells[END_REF]. Studies conducted in rodents have

shown that PACAP exerts a wide array of biological activities both in the CNS and in peripheral organs. Again the results from knock-out studies indicated the involvement of PACAP in glucose metabolism. However, these studies did not reveal which part of the metabolic phenotype should be attributed to central signaling pathways of PACAP, although some evidence for central effects on energy metabolism was available.

Amongst others it has been shown that in the brain PACAP decreases food intake [START_REF] Morley | Pituitary adenylate cyclase activating polypeptide (PACAP) reduces food intake in mice[END_REF][START_REF] Mizuno | Anorectic effect of pituitary adenylate cyclase activating polypeptide (PACAP) in rats: lack of evidence for involvement of hypothalamic neuropeptide gene expression[END_REF] and increases plasma glucose [START_REF] Mounien | Pituitary adenylate cyclaseactivating polypeptide inhibits food intake in mice through activation of the hypothalamic melanocortin system[END_REF]. Data obtained in our lab clearly show that i.c.v. administered PACAP has a strong effect on glucose production [START_REF] Yi | Pituitary Adenylate Cyclase-Activating Polypeptide Stimulates Glucose Production Through Glycogenolysis and Via the Hepatic Sympathetic Innervation in Rats[END_REF]. This effect is very likely mediated through the pre-autonomic neurons in the hypothalamus,

-Glucagon-like peptide 1 (GLP-1)

Besides the aboved mentioned effects of GLP-1 as a metabolic signal sensed by liver portal vein nervous system, GLP-1 is also known to decrease food intake in rodents and humans [218,[START_REF] Gutzwiller | Glucagon-like peptide-1: a potent regulator of food intake in humans[END_REF]. The anorectic effects of GLP-1 are probably mediated through both peripheral and central mechanisms, as a population of GLP-1 positive neurons is located in the brainstem and projects to hypothalamic and brainstem areas important in the control of energy homeostasis [START_REF] Jin | Distribution of glucagonlike peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study[END_REF][START_REF] Larsen | Distribution of glucagon-like peptide-1 and other preproglucagon-derived peptides in the rat hypothalamus and brainstem[END_REF]. GLP-1 is also involved in glucose metabolism and may lower plasma glucose levels through multiple mechanisms [START_REF] Heijboer | Gut-brain axis: regulation of glucose metabolism[END_REF], including central mechanisms. First, Knauf et al [START_REF] Knauf | Brain glucagon-like peptide-1 increases insulin secretion and muscle insulin resistance to favor hepatic glycogen storage[END_REF] demonstrated that during hyperglycemia i.c.v. administered GLP-1 decreases non-insulin-dependent muscle glucose uptake.

Although the hypothalamic GLP-1 projections from the brainstem target both the PVN and the ARC [START_REF] Jin | Distribution of glucagonlike peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study[END_REF], it was shown that direct administration of GLP-1 in the ARC, but not in the PVN, reduces glucose production [START_REF] Sandoval | CNS GLP-1 regulation of peripheral glucose homeostasis[END_REF]. On the other hand, GLP-1 administration in the PVN causes a decrease in food intake. Interestingly, GLP-1 receptor mRNA is also expressed in ~70% of the ARC-POMC neurons. These data seem to suggest that both central and peripheral GLP-1 can regulate food intake and glucose homeostasis, and provides a nice example of how the peripheral and central effects of a hormone/neurotransmitter can work synergistically to regulate glucose homeostasis.

Most of the above mentioned neuropeptides share a common effective hypothalamic area i.e., the PVN, but not for all of them it has been fully proven yet that they regulate specifically glucose metabolism via the PVN. In our studies and studies of others, central administration of orexin-A, PACAP-38, NPY [START_REF] Pomonis | Interaction of the hypothalamic paraventricular nucleus and central nucleus of the amygdala in naloxone blockade of neuropeptide Y-induced feeding revealed by c-fos expression[END_REF] and synthetic MC3R and MC4R

A C C E P T E D M A N U S C R I P T agonist [START_REF] Beckman | Amygdalar opioids modulate hypothalamic melanocortin-induced anorexia[END_REF] are all associated with Fos immunoreactivity in this nucleus. Moreover, for the PACAP-38 induced Fos-ir neurons in the PVN we have shown that they project to the sympathetic pre-ganglionic neurons in the spinal cord. Since some of these peptides are orexigenic (orexin, NPY, MCH), while others (POMC, PACAP) are anorexigenic, this suggests that the mechanism of feeding regulation is separated from that of glucoregulation. Secondly, sympathetic and parasympathetic pre-autonomic neurons in the PVN are separated [START_REF] Buijs | The suprachiasmatic nucleus balances sympathetic and parasympathetic output to peripheral organs through separate preautonomic neurons[END_REF]; this brings about the question, whether the neuropeptidergic effects we just described can be categorized into two groups, depending on their specific effects on either sympathetic or parasympathetic output pathways. In our studies, the orexin-A and PACAP-38 induced hyperglycemia can only be blocked by hepatic sympathetic but not parasympathetic denervation. Also in the case of NPY, although it does not influence basal glucose turnover, the suppressive effect on hepatic insulin sensitivity is only blocked by hepatic sympathetic denervation.

On the other hand, it has been shown that insulin and leptin signaling in ARC influence hepatic insulin sensitivity also via the vagal nerves, supposedly via another type of neurotransmission from the ARC (to the PVN) to influence pre-autonomic neurons in the hypothalamus. Clearly, further studies combining neuroanatomy and physiology are necessary to reveal this "parasympathetic pathway".

Liver innervation and the balance between glycogenolysis and gluconeogenesis

Sympathetic stimulation of glycogenolysis is enhanced further via Ca 2+ release from mitochondria, the stimulation of phosphorylaseβ kinase activity, and the subsequent activation of phosphorylase activity [START_REF] Assimacopoulos-Jeannet | Studies on alphaadrenergic activation of hepatic glucose output. Studies on role of calcium in alphaadrenergic activation of phosphorylase[END_REF][START_REF] Blackmore | Studies on alpha-adrenergic activation of hepatic glucose output. The role of mitochondrial calcium release in alpha-adrenergic activation of phosphorylase in perfused rat liver[END_REF][START_REF] Khoo | Stimulation of rat liver phosphorylase kinase by micromolar concentrations of Ca2+[END_REF][START_REF] Taylor | Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with alpha-adrenergic agonists and with glucagon[END_REF]. Vagal efferent activity is thought to regulate gluconeogenesis (GNG) [START_REF] Pocai | Hypothalamic K(ATP) channels control hepatic glucose production[END_REF] by targeting liver IL-6 and STAT3 signaling [START_REF] Inoue | Role of hepatic STAT3 in brain-insulin action on hepatic glucose production[END_REF] A C C E P T E D M A N U S C R I P T via SirT1 [START_REF] Nie | STAT3 inhibition of gluconeogenesis is downregulated by SirT1[END_REF]. One important issue in liver glucose metabolism is how the balance between glycogenolysis and GNG is regulated, or under which conditions GNG can be augmented. Clarification of this issue is of direct importance for understanding the mechanisms that underlie the development of the metabolic syndrome and, maybe more importantly, how obesity can trigger the development of type 2 diabetes. One of the hallmarks of type 2 diabetes is the excessive hepatic glucose production. Hepatic insulin resistance has been considered to be central to the pathophysiology of the excessive glucose production, especially via an increased GNG through the increased activity of PEPCK and G6Pase [START_REF] Valera | Transgenic mice overexpressing phosphoenolpyruvate carboxykinase develop non-insulin-dependent diabetes mellitus[END_REF]. However, this change in GNG is not explained by an alteration in glucoregulatory hormones. An acute increase or decrease in the insulin concentration mainly influences the rate of glycogenolysis but has little effect on GNG in nondiabetic subjects and/or in type 2 diabetic patients [START_REF] Gastaldelli | Effect of physiological hyperinsulinemia on gluconeogenesis in nondiabetic subjects and in type 2 diabetic patients[END_REF][START_REF] Boden | FFA cause hepatic insulin resistance by inhibiting insulin suppression of glycogenolysis[END_REF][START_REF] Boden | Effects of acute insulin excess and deficiency on gluconeogenesis and glycogenolysis in type 1 diabetes[END_REF], and infusion of glucagon in non-diabetic subjects does not increase the rate of GNG either [START_REF] Allick | Measurement of gluconeogenesis by deuterated water: the effect of equilibration time and fasting period[END_REF]. In addition, a clear organization of GNG based on zone can be observed in the liver, with higher rates of GNG being found in the periportal zone of the rat as compared to the pericentral zone, both under fed [START_REF] Tosh | Glucagon regulation of gluconeogenesis and ketogenesis in periportal and perivenous rat hepatocytes. Heterogeneity of hormone action and of the mitochondrial redox state[END_REF] and fasting conditions [START_REF] Quistorff | Periportal and perivenous hepatocytes retain their zonal characteristics in primary culture[END_REF].

The involvement of brain signaling was thus considered and most of the evidence highlights the importance of hypothalamic mechanisms. Electrical stimulation of the VMH and LH changes the activity of the gluconeogenic enzyme PEPCK [START_REF] Klieverik | Effects of thyrotoxicosis and selective hepatic autonomic denervation on hepatic glucose metabolism in rats[END_REF], whereas increased insulin signaling in the mediobasal hypothalamus inhibits liver GNG via the hepatic vagal innervation [START_REF] Pocai | Hypothalamic K(ATP) channels control hepatic glucose production[END_REF]. In addition, central administration of leptin has been shown to redistribute intrahepatic glucose fluxes, in such a way that GNG is increased and glycogenolysis suppressed. However, unlike the inhibitory action of central insulin on total glucose production [START_REF] Muse | Hypothalamic resistin induces hepatic insulin resistance[END_REF], leptin does not change total liver glucose production [START_REF] Van Den Top | Orexins induce increased excitability and synchronisation of rat sympathetic preganglionic neurones[END_REF][START_REF] Miyata | Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells[END_REF]. When one of the downstream signaling components of the leptin -the melanocortin pathway -is interrupted, total glucose production is decreased due to A C C E P T E D M A N U S C R I P T abolishment of GNG. In addition, activation of melanocortin receptors can stimulate GNG [START_REF] De Lecea | The hypocretins: hypothalamusspecific peptides with neuroexcitatory activity[END_REF].

In addition to leptin and insulin, evidence is accumulating that FFAs are also sensed by the hypothalamus directly. It has been shown that fatty acids within the hypothalamus stimulate liver GNG [START_REF] Lam | Hypothalamic sensing of circulating fatty acids is required for glucose homeostasis[END_REF]. However, it remains to be elucidated where and at what level FFAs elicit this effect.

Although there are indications that K(ATP) channels in the mediobasal hypothalamus are involved, the (neuronal) pathways from brain to liver that control GNG are still poorly defined. In the hypothalamus, ARC NPY/AGRP neurons, LH orexin neurons and PVN neurons (in the lateral magnocellular subdivision) are all activated during fasting [START_REF] Morikawa | Fasting-induced activation of mitogenactivated protein kinases (ERK/p38) in the mouse hypothalamus[END_REF][START_REF] Yamanaka | Hypothalamic orexin neurons regulate arousal according to energy balance in mice[END_REF]. Activation of the parvocellular division of the PVN including the sympathetic pre-autonomic neurons by the neuropeptide PACAP-38 [START_REF] Knauf | Brain glucagon-like peptide-1 increases insulin secretion and muscle insulin resistance to favor hepatic glycogen storage[END_REF] mainly stimulates glycogenolysis and hardly changes GNG. Therefore, either there is a cellular differentiation within the PVN with respect to the control of the balance between GNG and glycogenolysis, or other signaling pathways than the PVN have to be considered.

In summary, in recent years major progress has been made in the unraveling of the hypothalamic circuitry that is involved in the control of hepatic glucose production. This progress involves both the awareness that many peripheral nutritional and metabolic signals may "use" the central nervous system as an intermediate to affect hepatic glucose metabolism, as well as more detailed information on neural pathways and neurotransmitters able to affect hepatic glucose production. Main challenge in the years to come will be to connect all these peripheral signals to the appropriate hypothalamic pathways.

-Metabolic integration in liver and brain

Intrahepatic interaction of neuronal and humoral signals that control metabolism

Sympathetic and parasympathetic liver efferent nerves contain the classic aminergic (epinephrine and norepinephrine) and cholinergic neurotransmitters, as well as peptidergic components such as NPY which can be co-released with the classic neurotransmitters from both sympathetic and parasympathetic terminals [START_REF] Lundberg | High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man[END_REF][START_REF] Taborsky | Canine liver releases neuropeptide Y during sympathetic nerve stimulation[END_REF]. At the same time, the liver also receives a large amount of humoral information via the portal vein or arterial inputs. One of the unsettled issues is the interaction between the humoral signals and the neuronal signals that the liver receives during different energetic situations. First of all, insulin has an antagonizing effect on sympathetic (adrenergic) stimulation of glucose production [START_REF] Beckh | Modulation by insulin and glucagon of the activation of glycogenolysis by perivascular nerve stimulation in the perfused rat liver[END_REF], probably by interrupting the Ca2+ flux into the mitochondria of the hepatocytes [START_REF] Dehaye | Insulin inhibition of alpha-adrenergic actions in liver[END_REF]. Secondly, sympathetic nerve stimulation in the presence of glucagon increases the output of glucose further and reduces the enhanced lactate uptake by glucagon [START_REF] Beckh | Modulation by insulin and glucagon of the activation of glycogenolysis by perivascular nerve stimulation in the perfused rat liver[END_REF]. These data implicate that the stimulatory effects of hepatic nerves on glucose output can be modulated by circulating glucoregulatory hormones. The opposite pathway, i.e., neuronal signals modulating a hormonal signal, does not seem to exist since in dogs' complete liver denervation has no clear effects on the glucose production-increasing properties of glucagon [START_REF] Wada | Hepatic denervation does not significantly change the response of the liver to glucagon in conscious dogs[END_REF].

NPY may interact with both humoral and neuronal signaling in the liver. In dogs, NPY infusion into the portal vein stimulates hepatic glucose uptake without significantly altering whole body glucose disposal [START_REF] Nishizawa | Intraportal administration of neuropeptide Y and hepatic glucose metabolism[END_REF]. On the other hand, although NPY per se is not able to directly regulate hepatic glucose production, it can inhibit the stimulatory effects of glucagon and noradrenaline on glucose production [START_REF] Luers | Neuropeptide Y and peptide YY, but not pancreatic polypeptide, substance P, cholecystokinin and gastric inhibitory polypeptide, inhibit the glucagon-and noradrenaline-dependent increase in glucose output in rat liver[END_REF]. In contrast to NPY, galanin, the other neuropeptide released from the celiac ganglion as a sympathetic A C C E P T E D M A N U S C R I P T neurotransmitter, has no effects on its own when infused into the portal vein, but it does potentiate the action of norepinephrine to stimulate hepatic glucose production [START_REF] Taborsky | The canine sympathetic neuropeptide galanin: a neurotransmitter in pancreas, a neuromodulator in liver[END_REF].

It has been hypothesized that insulin induces liver glucose uptake and storage into glycogen when the glucose level is higher in the portal vein than in the hepatic artery (portal-artery gradient). Thus this gradient sensing is involved in the stimulatory action of insulin on liver glucose uptake [START_REF] Gardemann | A portal-arterial glucose concentration gradient as a signal for an insulin-dependent net glucose uptake in perfused rat liver[END_REF]. This process can be augmented by vagal stimulation as mimicked by an intraportal infusion of acetylcholine [START_REF] Shiota | Combined intraportal infusion of acetylcholine and adrenergic blockers augments net hepatic glucose uptake[END_REF]. Type 3 muscarinic receptors in the hepatocyte possibly mediate these effects [START_REF] Vatamaniuk | Acetylcholine affects rat liver metabolism via type 3 muscarinic receptors in hepatocytes[END_REF]. In contrast, blocking vagal efferent signals by atropine can inhibit insulin-mediated glucose uptake under the portal-artery gradient situation [START_REF] Stumpel | Sensing by intrahepatic muscarinic nerves of a portal-arterial glucose concentration gradient as a signal for insulin-dependent glucose uptake in the perfused rat liver[END_REF]. Not surprisingly, the effects of vagal activation on the effects of glucagon are opposite to its synergistic action on insulin, i.e., the glucagon-stimulated glucose release is antagonized by either vagal nerve stimulation or Ach [START_REF] Gardemann | Control of glucose balance in the perfused rat liver by the parasympathetic innervation[END_REF]. Whether these interactions take place at the level of receptor sensitivity or are due to a direct influence on the fractional extraction of these hormones is not clear.

The brain as an intermediary for the outflow of metabolic liver information to other organs

The increased PPAR activity in the liver needs intact vagal afferents to the brain in order to result in the expected systemic effects [START_REF] Uno | Neuronal pathway from the liver modulates energy expenditure and systemic insulin sensitivity[END_REF]. Interestingly, vagal inputs seem to be mediating strikingly different signals, since on the one hand disruption of vagal afferent fibers can prevent PPARα mediated glucocorticoid-induced metabolic derangements including insulin resistance [START_REF] Bernal-Mizrachi | An afferent vagal nerve pathway links hepatic PPARalpha activation to glucocorticoid-induced insulin resistance and hypertension[END_REF], whereas on the other hand they are also essential for hepatically expressed PPARγ2 to increase insulin sensitivity [START_REF] Uno | Neuronal pathway from the liver modulates energy expenditure and systemic insulin sensitivity[END_REF]. Again, this brings back the topic of the location of vagal sensing: due to the apparent lack of vagal afferent innervation in parenchyma, the above-described mechanisms seem to depend mainly on A C C E P T E D M A N U S C R I P T sensors located in the portal region. Within the brain-liver circuit, hepatoportal afferents and efferents form an autonomic feedback loop. This circuit could be "built" as a simple sensory-motor reflex, but most likely involves a complex brain mechanism [START_REF] Watts | Sweet talk in the brain: Glucosensing, neural networks, and hypoglycemic counterregulation[END_REF].

Net hepatic glucose uptake is more pronounced when glucose is loaded directly into the portal vein instead of into the general circulation, despite controlled insulin and glucagon levels [START_REF] Adkins-Marshall | Role of hepatic nerves in response of liver to intraportal glucose delivery in dogs[END_REF][START_REF] Abumrad | Absorption and disposition of a glucose load in the conscious dog[END_REF][START_REF] Ishida | Differential effects of oral, peripheral intravenous, and intraportal glucose on hepatic glucose uptake and insulin and glucagon extraction in conscious dogs[END_REF][START_REF] Defronzo | Influence of hyperinsulinemia, hyperglycemia, and the route of glucose administration on splanchnic glucose exchange[END_REF]. This means that the hepatoportal signal evoked by glucose has to be sensed by a specific pathway. Indeed, it was reported that an intact nerve supply to the liver is vital for this response. First, after a complete liver denervation the extra uptake by the overload of glucose into the portal vein was blocked [START_REF] Adkins-Marshall | Role of hepatic nerves in response of liver to intraportal glucose delivery in dogs[END_REF]; second, after hepatic vagotomy liver glucose uptake was decreased by approximately 40% under euglycemichyperinsulinemic conditions during portal glucose infusions [START_REF] Matsuhisa | Important role of the hepatic vagus nerve in glucose uptake and production by the liver[END_REF]. It is therefore logical to expect that the storage of glucose in the liver will be changed by interruption of liver innervation. However, a complete denervation only changes the variability of both glycogen synthesis and GNG but not its absolute level [START_REF] Moore | Effect of hepatic nerves on disposition of an intraduodenal glucose load[END_REF][START_REF] Wasserman | Hepatic nerves are not essential to the increase in hepatic glucose production during muscular work[END_REF]. The central pathway can thus influence these processes, but the liver also contains a strong autoregulatory mechanism. Interestingly, under hyperinsulinemic conditions, total hepatic glucose production can be increased during portal glucose loading [START_REF] Matsuhisa | Important role of the hepatic vagus nerve in glucose uptake and production by the liver[END_REF]. This raises the possibility that the autonomic nerves interact with insulin signaling as discussed before.

Stimulation of the hepatoportal glucose sensors by glucose infusions can stimulate glucose uptake in heart, brown adipose tissue, and muscle. These portal glucoseinduced changes in glucose utilization involve both an insulin-dependent and an insulinindependent mechanism [START_REF] Burcelin | Portal glucose infusion in the mouse induces hypoglycemia: evidence that the hepatoportal glucose sensor stimulates glucose utilization[END_REF]. The insulin-independent mechanism possibly relates to GLUT-4 and AMP-activated protein kinase (AMPK) signaling in muscle [START_REF] Burcelin | GLUT4, AMP kinase, but not the insulin receptor, are required for hepatoportal glucose sensor-stimulated muscle glucose utilization[END_REF]. Besides glucose, high physiological doses of GLP-1 infusion in the portal vein are also able to stimulate glucose clearance in non-hepatic tissues [START_REF] Nishizawa | Effect of intraportal glucagon-like peptide-1 on glucose metabolism in conscious dogs[END_REF].

A C C E P T E D M A N U S C R I P T

LH responses to glucose, whereas vagal denervation exaggerates this response [START_REF] Schmitt | Influences of hepatic portal receptors on hypothalamic feeding and satiety centers[END_REF].

These data suggest that the LH receives both spinal and vagal liver glucose sensing information and thus may be one of the main hypothalamic integration areas for these neuronal inputs.

Regarding the integration of neuronal and humoral sensing information, the arcuate nucleus, which is the unique metabolic sensor for almost all circulating metabolites and their relatives, is a strong candidate for the integration of humoral and vagal inputs via the NTS-ARC projection [START_REF] Yi | Ventromedial arcuate nucleus communicates peripheral metabolic information to the suprachiasmatic nucleus[END_REF]. Another major candidate is the PVN, as both sympathetic and parasympathetic metabolic sensing information reaches the PVN by direct and indirect pathways [START_REF] Swanson | Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms[END_REF]. Moreover, other forebrain areas, such as the bed nucleus of the stria terminalis and a number of hypothalamic nuclei also innervate the PVN. This unique position allows the PVN to integrate a very broad spectrum of neuronal and humoral metabolic sensing information and to organize its autonomic and neuroendocrine outflow consequently according to these different inputs.

Finally, at the level of the hypothalamus probably also the integration of information from the metabolic-homeostatic system and the cognitive-hedonic processes involved in the control of energy balance and food intake take place, as information from the limbic system and cortex also reaches the PVN via projections to the BNST and subPVN [START_REF] Berthoud | The brain, appetite, and obesity[END_REF].

Vice versa it is clear that hepatic autonomic afferents transmit glucosensing information to higher brain areas, but how the many different brain regions are involved in accepting and integrating this information is still very ill defined. However, a very recently published anatomical study on the communication within the hypothalamic, cortical and mesolimbic circuitries involved in the regulation of energy balance, provides a clear clue that indeed neuronal pathways do transfer energy sensory information from the hypothalamus to cerebral cortex [START_REF] Kampe | An anatomic basis for the communication of hypothalamic, cortical and mesolimbic circuitry in the regulation of energy balance[END_REF].

A C C E P T E D M A N U S C R I P T

-Discussion and Perspectives

What can be learned from "gain of function" and "loss of function" studies?

Despite all the evidence for manipulation-induced effects of neuronal elements on liver glucose and lipid metabolism, the physiological significance of the afferent and efferent liver innervations is still not clear.

Liver transplanted humans and animals are a suitable model to study the consequences of a complete denervation on liver metabolism. In liver transplanted patients, no reinnervation of either hepatic sympathetic efferent or afferent nerves has been observed until 30 months [START_REF] Kjaer | No reinnervation of hepatic sympathetic nerves after liver transplantation in human subjects[END_REF]. In transplanted rats, by using growth-associated protein (GAP)-43 as an axonal marker, regenerating axons possibly originating from parasympathetic ganglion cells in the hepatic hilus of liver allografts have been observed [START_REF] Sakamoto | Experimental study on hepatic reinnervation after orthotopic liver transplantation in rats[END_REF][START_REF] Takahashi | Immunohistochemical and electron microscopic study of extrinsic hepatic reinnervation following orthotopic liver transplantation in rats[END_REF], but a centrally derived reinnervation has never been claimed.

Liver transplanted patients have relatively normal insulin-dependent glucose metabolism, and after a long term recovery from the initial effects of the immunosuppressive treatment, also protein and FFA metabolism return to nearly normal [START_REF] Perseghin | Regulation of glucose homeostasis in humans with denervated livers[END_REF][START_REF] Luzi | Metabolic effects of liver transplantation in cirrhotic patients[END_REF]. When comparing results from matched healthy subjects and kidney-transplant patients who received immunosuppressive medication similar to that received by the liver-transplant patients, exercise data showed glucose production increased to a similar extent in control subjects as in liver transplanted patients [START_REF] Kjaer | Glucose homeostasis during exercise in humans with a liver or kidney transplant[END_REF].

In mice, 9 months after orthotopic liver transplantation, body weight and other hepatic and general metabolic parameters are in the normal range, despite an increase in LDL cholesterol, LDL/HDL ratio and in hepatic glucose production. In short-term (2 weeks) completely liver denervated dogs, liver glycogenolysis response to a physiological increase in glucagon is unaltered [START_REF] Wada | Hepatic denervation does not significantly change the response of the liver to glucagon in conscious dogs[END_REF], despite the fact that in the intact situation vagal A C C E P T E D M A N U S C R I P T activity will antagonize the effects of glucagon. A similar observation has been made in other experimental animals [START_REF] Wasserman | Hepatic nerves are not essential to the increase in hepatic glucose production during muscular work[END_REF].

Taken together, these data indicate that the direct efferent liver innervation may not be essential for maintaining normal liver glucose, amino acid and lipid metabolism, and that autoregulatory mechanisms inside the liver are fully able to control liver metabolism in basal (i.e., non-stimulated) conditions. However, the defective hypoglycemic counterregulation observed in liver transplanted individuals, as discussed before, suggests that the autonomic innervation of the liver might be more essential during "fight or flight" responses, by triggering catabolic responses such as stimulation of glucose output via a fast neuronal pathway, preparing the individual for emergency conditions.

Interestingly, if only a sympathetic or parasympathetic liver denervation is performed, the normal daily rhythm in plasma glucose concentrations is eliminated but a complete denervation of both branches simultaneously does not result in such disturbed glucose rhythmicity [START_REF] Cailotto | Daily rhythms in metabolic liver enzymes and plasma glucose require a balance in the autonomic output to the liver[END_REF]. Apparently for the remaining humoral and autoregulatory control systems it is easier to maintain a balanced glucose output without any autonomic innervation, than with an unbalanced autonomic input. These findings support the idea that disorders of (hepatic) glucose metabolism are more likely to be derived from an unbalanced autonomic input to the liver than from a complete absence of such inputs.

Perspective

From the viewpoint of whole-body harmonization, afferent liver signaling is important for allowing the brain to synchronize liver physiology with that of other parts of the body. The liver can be considered as a primary nutritional sensor, while the brain serves as an overall energy sensor. Metabolic sensing in the liver is organized in such a way that most of the metabolic signals that indicate "plentifulness" are sensed by the vagal nerves, i.e., high concentrations of glucose and lipid absorbed from food, leptin released from A C C E P T E D M A N U S C R I P T adipose tissue, and lipid-activated PPARs activity (Fig. 2). Continuous high levels of lipid sensed by the portal vein nerves may lead this sensing system to increase its threshold; consequently more lipids are needed to generate the appropriate afferent signal. In a chronic situation this may mean that the brain integration center loses this piece of peripheral information. However, whether this eventually results in a profound loss of feedback about the metabolic situation from periphery to brain needs to be investigated.

Probably, parallel running humoral factors such as the newly defined lipid sensor ghrelin O-acyl transferase (Goat)-ghrelin can also signal to the brain about the availability of energy [START_REF] Kirchner | GOAT links dietary lipids with the endocrine control of energy balance[END_REF], and this pathway may either complete the neuronal metabolic sensing from liver or even take over when neuronal sensing from the liver is not available, for instance after liver transplantation. Checking the metabolic profile after blocking the (Goat)-ghrelin signaling with hepatic vagal denervation could test this possibility.

In contrast to vagal innervation, spinal nerves predominantly sense the "inadequate" metabolic information, especially the lack of glucose. These opposing roles might also be involved in a dysbalance during pathophysiological states. One of the hypotheses is that a hypoglycemia counterregulation pathway may be activated inappropriately during a euglycemic or hyperglycemic condition, driving the liver continuously to produce glucose, and overriding the inhibition from insulin.

  A C C E P T E DM A N U S C R I P T may finally cause an increased synthesis of triglycerides and an excess secretion of very low density lipoprotein (VLDL).

  A C C E P T E DM A N U S C R I P T be sensed by the liver. Glucagon-like peptide-1 (GLP-1) is one of the best studied signals in this class of hormones.

  since i.c.v. infusion of PACAP-38 induced c-Fos is expressed by sympathetic A C C E P T E D M A N U S C R I P T preautonomic neurons in PVN, and sympathetic but not parasympathetic hepatic denervation can eliminate the hyperglycemic response to i.c.v. infusion of PACAP-38.

Figure. 1

 1 Figure legends

Figure. 2 Figure 1 A

 21 Figure.2 Summary of the hepatic metabolic sensing and the neuronal pathways

CNS integration of liver and blood borne metabolic information

The brain controls energy metabolism by balancing energy expenditure and energy intake and/or production. In order to maintain the right balance, the brain, liver, gastrointestinal and other metabolic sensing mechanisms, either via the neuronal or the humoral pathway, cannot be separated from each other.

From the information provided above it is clear that a multilevel network is dedicated to receiving and relaying the liver and other visceral metabolic information to the brain via both hormonal and neuronal routes. The first-order of convergence for the neural information from the metabolic sensing from the different visceral organs is found at the level of the nodose and dorsal root ganglia. Hereafter, the second level for convergence of information is in the NTS and the dorsal horn of spinal cord. Although this has never been investigated specifically for the liver, there is the possibility of an ultrashort feedback loop via projections from the dorsal horn to IML, or from NTS to the dorsal motor nucleus of the vagus (DMV). Interestingly, although spinal ascending pathways and the NTS both project into the parabrachial nuclei (PB), the topographic separation of spinal and NTS inputs into PB [START_REF] Herbert | Organization of medullary adrenergic and noradrenergic projections to the periaqueductal gray matter in the rat[END_REF] does not make this area a likely integration place for spinal and vagal inputs. One of the possible integration sites in the midbrain is the periaqueductal gray matter (PAG), which receives inputs from both the general-visceral recipient part of the NTS and the thoracic spinal cord [START_REF] Keay | Spinal afferents to functionally distinct periaqueductal gray columns in the rat: an anterograde and retrograde tracing study[END_REF]. How this sympatheticparasympathetic integration overlaps or "interferes" with the proposed columnar organization of the PAG [START_REF] Bandler | Columnar organization in the midbrain periaqueductal gray: modules for emotional expression?[END_REF] in the control of liver metabolism is not yet known.

Within the hypothalamus, the lateral hypothalamus (LH) has been reported to respond to portal vein infusions of glucose or CCK [START_REF] Niijima | Visceral afferents and metabolic function[END_REF][START_REF] Shimizu | Functional correlations between lateral hypothalamic glucose-sensitive neurons and hepatic portal glucose-sensitive units in rat[END_REF]. The responses to glucose were not found in other hypothalamic areas like the supraoptic nucleus, PVN or ventromedial hypothalamus [START_REF] Schmitt | Influences of hepatic portal receptors on hypothalamic feeding and satiety centers[END_REF]. Furthermore, the excitability of LH neurons seems to follow the rhythmy of the day/night cycle [START_REF] Schmitt | Circadian rhythmicity in responses of cells in the lateral hypothalamus[END_REF]. Interestingly, splanchnic denervation eliminates the