Autonomic regulation of hepatic glucose production.

Autonomic Regulation of Hepatic Glucose Production Peter H. Bisschop1 , Eric Fliers1 , and Andries Kalsbeek*1,2 ABSTRACT: Glucose produced by the liver is a major energy source for the brain. Considering its critical dependence on glucose, it seems only natural that the brain is capable of monitoring and controlling glucose homeostasis. In addition to neuroendocrine pathways, the brain uses the autonomic nervous system to communicate with peripheral organs. Within the brain, the hypothalamus is the key region to integrate signals on energy status, including signals from lipid, glucose, and hormone sensing cells, with afferent neural signals from the internal and external milieu. In turn, the hypothalamus regulates metabolism in peripheral organs, including the liver, not only via the anterior pituitary gland but also via multiple neuropeptidergic pathways in the hypothalamus that have been identified as regulators of hepatic glucose metabolism. These pathways comprise preautonomic neurons projecting to nuclei in the brain stem and spinal cord, which relay signals from the hypothalamus to the liver via the autonomic nervous system. The neuroendocrine and neuronal outputs of the hypothalamus are not separate entities. They appear to act as a single integrated regulatory system, far more subtle, and complex than when each is viewed in isolation. Consequently, hypothalamic regulation should be viewed as a summation of both neuroendocrine and neural influences. As a result, our endocrine-based understanding of diseases such as diabetes and obesity should be expanded by integration of neural inputs into our conC 2015 American Physiological Society. Compr Physiol cept of the pathophysiological process. 5:147-165, 2015.

Introduction Since the 1970s, it has been known that circulating hormones act on hormone-sensitive neurons in the hypothalamus to regulate the endocrine output of the pituitary gland. In this way, plasma hormone concentrations in the classic neuroendocrine “axes” are kept within narrow margins. Sparked by the discovery of leptin in 1994, the past two decades have yielded a wealth of data showing that several classic, as well as novel, hormones act in the hypothalamus as well, to regulate not only (feeding) behavior but also the activity of the sympathetic and parasympathetic branch of the autonomic nervous system (ANS). The ANS is regulated via preautonomic hypothalamic neurons projecting to various brain stem nuclei. Although experimental studies demonstrated the presence of hypothalamic neurons adapting firing behavior upon changes in extracellular glucose concentrations already in the 1960s, and in vivo studies showed that lipids can activate neurons in the lateral hypothalamus (LH) already in the 1970s, the relevance and mechanistic pathways of hypothalamic nutrient sensing have begun to be recognized only in the past decade. Interestingly, hypothalamic glucose and lipid concentrations use more or less similar output pathways as hormones acting intrahypothalamically, that is, the ANS and (eating) behavior. One of the target organs of the autonomic output from the CNS is the liver, which is one of the most prominent organs in terms of glucose homeostasis.

Volume 5, January 2015

In general, glucose homeostasis is regulated through a complex interaction between hormonal and neuronal signals from and to organs involved in the regulation, production and consumption of glucose. Under physiological conditions, plasma glucose concentrations are maintained within a well-defined reference range during fasting and feeding. Under fasting conditions, glucose production by the liver represents the main source of plasma glucose. After a meal, hepatic glucose production decreases as a result of increasing insulin concentrations. In this review, we will first discuss recent insights in the structure and function of the autonomic innervation of the liver. Then we will focus on the hypothalamic control of hepatic glucose production via the ANS, highlighting several hypothalamic neuropeptides implicated in peripheral glucose homeostasis. We will also review studies showing that the autonomic innervation of the liver is an important determinant of hepatic glucose production. Next, we will discuss the role of hypothalamic sensing of * Correspondence

to [email protected] Department of Endocrinology and Metabolism, Academic Medical Center (AMC), University of Amsterdam, The Netherlands 2 Hypothalamic Integration Mechanisms, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands Published online, January 2015 (comprehensivephysiology.com) DOI: 10.1002/cphy.140009 C American Physiological Society. Copyright 1

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nutrients, especially lipids and glucose, in the hypothalamic regulation of hepatic glucose production. Finally, we will show the importance of classic glucoregulatory hormones, including insulin, glucagon, estradiol, and glucocorticoids, in the hypothalamic control of hepatic glucose homeostasis.

Autonomic Innervation of the Liver This subject has previously been reviewed by our group and others (82, 216). 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 dorsal brainstem. Unlike other visceral organs, no clear intrahepatic postganglionic neurons have been identified (14). 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 demonstrated to be present as markers for sympathetic efferent fibers (2, 40). In addition, α-adrenergic and β-adrenergic receptors for the sympathetic neurotransmitter noradrenaline are present in the hepatic artery and portal vein (51, 58). On the other hand, acetylcholinesterase (AChE) and vesicular acetylcholine transporter (VAChT) (8), located in parasympathetic postganglionic cells (14), tend to be the vagal efferent markers of choice (73, 208). 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) (59). Finally, substance P (SP) has been used as a marker for both vagal and spinal afferents (49, 72). Different combinations of these markers have been applied to hepatic tissues of human, monkey, dog, rabbit, rat, hamster, guinea pig, mouse, carp, bullfrog, and turtle. The distribution of sympathetic and parasympathetic nerves in the liver is markedly species-dependent. Some results are not firmly established as they could not be repeated in later studies, but this is probably due to nonspecific technical limitations, such as those encountered using earlier histochemical methods for the demonstration of AChE in rat parenchyma (183). 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 colocalization, 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. In rabbit liver parenchyma, TH is expressed without

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colocalization of NPY. AChE fibers were not found in human liver parenchyma, or in any other species studied (2, 35, 36, 40, 55, 180). Although NPY is sometimes also considered a neurotransmitter for postganglionic parasympathetic nerves (37, 157), no study has shown colocalization of NPY and AChE in liver tissue. A major difference between sympathetic innervation of humans and the most commonly used animal models, that is, rats and mice, is that the latter have no clear parenchymal sympathetic innervation, while in human liver (also in guinea pig), sympathetic TH-immunoreactive fibers penetrate deep into the lobule to end of hepatocytes (47). However, the functionality of the sympathetic efferent innervation of species with parenchymal sympathetic innervation could still correspond to that of species without (13) 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 (66, 167). Indeed, signal propagation through gap junctions, that is, via electrotonic coupling, can compensate for the sparse direct inputs to the hepatocytes, especially with respect to sympathetic signal transduction (79, 167). Also, there is considerable homology between the rat and human liver gap junctions (100). 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 (181). Furthermore, the sympathetic signal may be propagated via the release of prostaglandins from Ito cells (9, 64) [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 (194)]. Only few approaches are available to investigate and differentiate the intrahepatic 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 (15, 131), with the axon processes from the nodose ganglion projecting to the nucleus of the solitary tract (NTS). Sympathetic afferents from the liver enter the DRG, with the DRG axons terminating in the dorsal horn of the spinal cord (14). The remarkable lack of either vagal efferent or afferent innervation of liver parenchyma as indicated by the (immuno)histochemical studies was confirmed by early tracing studies in rats. By contrast, later studies using the transneuronal retrograde tracer PRV and/or selective hepatic denervations in combination with euglycemic, hyperinsulinemic clamp experiments showed functional efferent connections between preautonomic neurons in the hypothalamus and the liver via the sympathetic and parasympathetic branch of the ANS [reviewed in Kalsbeek et al. (85)].

Hypothalamic Neuropeptides and Hepatic Glucose Metabolism The hypothalamic control of hepatic glucose production is an evident aspect of energy homeostasis. In addition to the




control of glucose metabolism by the circadian timing system, the hypothalamus also serves as a key relay center for (humoral) feedback information from the periphery, with the important role for hypothalamic leptin receptors as striking example. Euglycemic, hyperinsulinemic clamp experiments combined with either sympathetic-, parasympathetic-, or sham-denervations of the autonomic input to the liver have further delineated the hypothalamic pathways that affect plasma glucose concentration, hepatic glucose metabolism, peripheral glucose uptake and insulin sensitivity, with the preautonomic neurons as the central hypothalamic hub. More recently it has become clear that similar pathways may be involved in the control of lipid metabolism in liver and white adipose tissue.

Nowadays, it is evident that the hypothalamic control of hepatic glucose production is an important aspect of energy homeostasis. A host of studies in the last decade has evidenced the involvement of several hypothalamic neuropeptides in the control of hepatic insulin sensitivity and glucose production (Fig. 1). Each of these peptides has its distinct physiological role in the control of food intake and energy homeostasis, while in addition affecting common signaling pathways that are involved in the control of energy homeostasis. Therefore, in the remainder of this paragraph, we will present an overview of the current knowledge on the hypothalamic control of glucose and lipid metabolism, with a special focus on neuropeptidergic pathways involved in the regulation of hepatic glucose production.

Parasympathetic P

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Figure 1

Sagittal scheme of the sympathetic and parasympathetic control of the liver. Brain areas providing first-order projections are indicated in red, brain areas containing second-order neurons are indicated in blue, and those containing third-order neurons are indicated in yellow. It is clear by comparing the parasympathetic pattern against the sympathetic pattern that far more second-order cell groups are in control of the first-order parasympathetic (i.e., DMV) than first-order sympathetic (i.e., IML) motor neurons. RVLM also includes the catecholaminergic A5, C1, and C3 areas. Abbreviations: Ace, Amygdala, central part; ARC, Arcuate nucleus; Ba, Barrington nucleus; BNST, Bed nucleus of the stria terminalis; DMH, Dorsomedial nucleus of the hypothalamus; DMV, Dorsal motor nucleus of the vagus nerve; IML, Intermediolateral column of the spinal cord; INS; Insular cortex; LH, Lateral hypothalamus; MPO, Medial preoptic area; NTS, Nucleus of the tractus solitaries; OVLT, Organum vasculosum of the lamina terminalis; P, Pineal gland; PVN, Paraventricular nucleus of the hypothalamus; vPVN, Ventral part of the PVN; dPVN, Dorsal part of the PVN; RA, Raphe nucleus; RCA, Retrochiasmatic area; RVLM, Rostroventrolateral medulla; SCN, Suprachiasmatic nucleus; SFO, Subfornical organ; VMH, Ventromedial nucleus of the hypothalamus; X, Nervus vagus; ZI, Zona incerta


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Neuropeptide Y One of the most familiar hypothalamic neuropeptidergic networks involved in the control of energy metabolism is that of the NPY-containing neurons in the ARC with their projections to several hypothalamic brain areas including the PVN. NPY is well-known for its appetite-stimulating effects and, in fact, NPY is considered the most potent orexigenic neuropeptide known in mammals to date (26). The first report on the glucoregulatory effects of the hypothalamic NPY system appeared in the mid-1990s when it was shown that intracerebroventricular (ICV) administration of NPY increases endogenous glucose production in rats, probably by decreasing hepatic insulin sensitivity (109, 110). Later on, these results were confirmed in mice (197). In view of the inhibitory effects of hypothalamic insulin receptors on hepatic glucose production (133, 135), the abundant expression of insulin receptors in the ARC (195), the inhibitory effect of insulin on NPY neuronal activity (163), and the effects of ICV NPY on sympathetic activity (39, 74, 199), we decided to test whether NPY could be the hypothalamic intermediate between the insulin receptors in the ARC and the preautonomic neurons in the PVN. Hereto we combined the euglycemic hyperinsulinemic clamp technique with the ICV administration of NPY, and performed these experiments in hepatic sympathetic-, hepatic parasympathetic-, and hepatic sham-denervated rats. Our results confirmed that ICV NPY is able to (partially) block the inhibitory effects of peripheral hyperinsulinemia on hepatic glucose production, but they also showed that selective denervation of hepatic sympathetic nerves blocks the effect of NPY on hepatic insulin sensitivity (196). 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 preautonomic neurons in the PVN and thus reduce the sympathetic stimulation of hepatic glucose production. The results of Pocai et al. (145), 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 preautonomic neurons, there is probably another neurotransmitter that is responsible for the transmission of insulin’s effects in the ARC to the parasympathetic preautonomic neurons in the PVN. Moreover, the effects of NPY also seem to be specific for glucose production as in none of the above-mentioned studies, there was a significant effect on whole body glucose disposal. Several studies have shown that chronic infusion of NPY, mimicking high levels of hypothalamic NPY in animal models of obesity, causes hypertriglyceridemia and lipogenesis in liver and white adipose tissue even when animals are pairfed (11, 114, 221). Interestingly, the effect on plasma triglycerides was abolished in adrenalectomized rats, but reappeared in adrenalectomized rats coinfused centrally with NPY and

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glucocorticoids (158, 220). Interestingly, glucocorticoids have a strong effect on the arcuate NPY neurons (213). Van den Hoek et al. (197) were the first to describe an acute effect of NPY on liver triglyceride secretion, in mice. In this study, hyperinsulinemia decreased the secretion of triglycerides in VLDL particles by the liver. This effect was abolished when the hyperinsulemic clamp was combined with the ICV administration of NPY, suggesting that in the fed state the inhibitory effect of insulin on the activity of NPY neurons is responsible for the decrease in triglyceride secretion. No effects on triglyceride secretion were found in the basal state. More recently, however, Stafford et al. (177) showed that ICV administration of NPY in rats increased triglyceride secretion in the postabsorbative state. The changes in hepatic mRNA expression suggest that the increased release of hypothalamic NPY causes a mobilization of stored triglycerides in the liver, whereas de novo fatty acid synthesis is inhibited. Moreover, after a longer fast, ICV administration of an Y1 antagonist decreased triglyceride secretion, suggesting a physiological role for the hypothalamic NPY neurons in the ARC in the control of hepatic triglyceride secretion during the transition from feeding to fasting (177). In addition, Rojas et al. (154) showed that the hypothalamic effects of NPY signaling on hepatic VLDL-TG secretion via the Y1 receptor were dissociable from its effect on feeding via the Y2 receptor. Recently, we showed that central NPY increases VLDL-TG secretion via the sympathetic branch of the ANS, as a selective hepatic sympathetic denervation completely blocked the stimulatory effect of ICV administered NPY (23). Moreover, in the same study we also showed that an intact arcuate nucleus and hepatic sympathetic innervation are necessary to maintain VLDLTG secretion during fasting. Contrary to the effects of chronic insulin, acute infusion of peripheral insulin decreases VLDLTG secretion. As this can be prevented by the ICV infusion of NPY, a central mechanism of insulin seems to be involved in the decreased VLDL-TG secretion (197). Insulin infused into the mediobasal hypothalamus (MBH) increases lipogenesis and suppresses lipolysis in WAT, which is mediated by the sympathetic nervous system, but no effects on the liver have been described so far (162). In addition, ICV administered insulin has tissue specific effects on the uptake of glucose and fatty acids (30). It is not clear at present whether these central effects of insulin are also mediated via the NPY neurons in the ARC. Previously, it has been shown that NPY differentially regulates energy intake and energy expenditure in the PVN and LH (189), but together the findings above indicate that also within one hypothalamic nucleus the function of NPY is differentiated. The increased release of NPY during fasting from arcuate nucleus neurons onto preautonomic PVN neurons stimulates the sympathetic nervous system to maintain hepatic glucose production as well as VLDL-TG secretion. Remarkably, a similar pathway has recently been described for the control of BAT activity, that is, increased release of NPY from ARC-derived terminals results in a decreased expression of



TH in preautonomic, probably dopaminergic, neurons in the PVN and subsequently downregulates uncoupling protein 1 (UCP1) expression and thermogenesis in BAT (170).

Pro-opiomelanocortin In addition to the orexigenic NPY/AGRP neurons, the ARC also contains a population of anorexigenic proopiomelanocortin (POMC)/CART-containing neurons. The most important POMC-derived peptide with respect to feeding and metabolism is alpha-MSH. The reciprocally 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 (MC3R, MC4R), for which alpha-MSH is the main endogenous agonist. MC signaling plays an important role in energy metabolism, as clearly evidenced by the fact that MC4R deficiency represents the commonest known monogenic cause of human obesity (42). Given the important and opposite effects of POMC and NPY neurons on energy homeostasis, the POMC neurons seemed to be good candidates for the missing parasympathetic link in the previous paragraph, that is, the neurons in the arcuate nucleus that are responsible for transmission of insulin’s effects to the parasympathetic preautonomic neurons in the PVN, but the experimental evidence is ambiguous. Genetic deletion or reactivation of insulin receptors specifically on POMC neurons did not alter hepatic insulin sensitivity in a way that could be separated from its impact on energy expenditure (95). On the other hand, when insulin receptors are selectively removed or reactivated specifically within AgRP neurons, hepatic insulin sensitivity is respectively impaired or improved (95). In MC4R knockout mice, plasma insulin levels are increased and central administration of the alpha-MSH agonist MTII dose-dependently inhibits basal insulin release, but ICV administration of alpha-MSH or MTII enhances the action of insulin on both glucose uptake and production and transgenic overexpression of alpha-MSH leads to improved glucose metabolism. Moreover, the phenotype of the MC4R-deficient mice and humans, that is, reduced heart rate and diastolic blood pressure in the face of severe obesity, is explained by a decreased sympathetic/parasympathic balance (43). Finally, recently Rossi and colleagues (156) used loxP-modified null Mc4r alleles (loxTB MC4R) to genetically dissect the specific role of MC signaling in sympathetic versus parasympathetic preganglionic neurons. This was achieved by either the general reexpression of MC4R in all cholinergic neurons including brainstem and spinal cord autonomic motorneurons (loxTB MC4R, ChAT-Cre), or by specifically reexpressing MC4Rs only in autonomic control neurons, including the parasympathetic motor neurons in the DMV (loxTB MC4R and Phox2b-Cre), but excluding the sympathetic motorneurons in the spinal cord. Interestingly, reactivation of MC4R signaling in cholinergic neurons improved hyperinsulinemia and hyperglycemia, while reexpression of



MC4R selectively in brainstem neurons only improved hyperinsulinemia. Specifically, they found improved efficiency of insulin-induced inhibition of hepatic glucose production following general reexpression of MC4R in cholinergic neurons, but not by specific reexpression in the vagal motor neurons. These observations nicely fit with our previous data (196) and the concept that activity of the sympathetic input to the liver is balanced by the NPY- and POMC-containing projections from the arcuate nucleus. An increased NPY input to sympathetic preautonomic hypothalamic neurons reduces hepatic insulin sensitivity, whereas a reinstatement of MC signaling (derived from the arcuate nucleus) onto sympathetic preganglionic neurons in the spinal cord increases hepatic insulin sensitivity. Surprisingly, however, alpha-MSH does not seem to be involved in the inhibitory effect of hypothalamic insulin on HGP, as coadministration of a melanocortin antagonist failed to block the decrease in HGP induced by hyperinsulinemia (61). Blocking alpha-MSH signaling via ICV infusion of the melanocortin 3/4 receptor (MC3R/MC4R) antagonist SHU9119 has no effects on glucose metabolism, but ICV infusion of alpha-MSH itself has a clear stimulatory effect on EGP via gluconeogenesis (GNG) which can be antagonized by SHU9119 (61). It has been proposed that the hypothalamic MC3R/MC4R signaling pathway mediates the effect of systemic leptin on EGP (190). Central administration of leptin has been proven to be involved in the autoregulation of hepatic glucose output, that is, an increase in GNG with a concomitant decrease in glycogenolysis without changing total glucose production. Recently, elegant experiments showed that the adenoviralinduced expression of leptin receptors in the ARC of leptin receptor knockout animals improves glucose tolerance via enhanced suppression of EGP (50). 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. Moreover, the effects of hypothalamic leptin signaling on hepatic insulin sensitivity could be blocked by a selective hepatic vagotomy, providing further support for the idea that ARC projections to preautonomic neurons (in the PVN) are important for the transmission of the effect of leptin on EGP. Further supporting the involvement of POMC neurons in these effects are the observations that reexpression of leptin receptors specifically in POMC neurons improves glucose metabolism, whereas removal of both leptin and insulin receptors specifically from POMC neurons causes severe insulin resistance and increases HGP. Evidently more experiments are needed to unravel the precise hypothalamic pathways. Contrary to the effect of chronic ICV administration of NPY, chronic blockade of the central melanocortin system does not change plasma levels of triglycerides and fatty acids, although it does increase circulating HDL cholesterol levels (144) and induce an obese phenotype. On the other hand, both chronic blockade of the melanocortin system in the

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Agouti yellow mice and infusion of a melanocortin antagonist promote lipogenesis and lipid accumulation in liver with a possible role for SREB1c and PPARgamma. MC4R knockout mice display increased levels of the lipogenic gene, fatty acid synthase (FAS), as well as hepatic steatosis. In contrast, activation of the melanocortin system with leptin, MTII or NDP-MSH reduces the expression of lipogenic genes in the liver. Thus, although the melanocortin system seems to be involved in the control of hepatic lipid metabolism, its interaction with dietary factors and hormones such as insulin is still under debate. For instance, the significance of insulin’s stimulating effect on the expression of lipogenic genes, such as SREBP1c and possibly PPARgamma, is unclear at present, as the increased FAS expression and hepatic steatosis in MC4R knockout mice is abolished in preobese mice not displaying hyperinsulinemia. Likewise, the interpretation of two other observations in these mice: (i) increased hepatic lipogenesis and fat content is largely prevented by pair-feeding and (ii) acute blockade or activation of the central melanocortin system does not significantly alter VLDL-TG secretion, remains enigmatic (177). Lam et al. (101) investigated the acute effects of ICV glucose on VLDL-TG secretion, to mimic a fed state only in the brain, while the rat is fasted. Acute ICV infusion of glucose lowered VLDL-TG secretion, which was prevented by a hepatic vagotomy. This indicates that the parasympathetic nervous system is involved in the central effects of glucose on VLDL-TG secretion. The authors proposed that the effect of ICV glucose on VLDL-TG secretion is mediated via lowered SCD1 activity and decreased oleyl-CoA levels. Recent studies in our lab have provided additional evidence for the involvement of the parasympathetic branch of the ANS in the control of VLDL-TG secretion. We found that postprandial plasma triglyceride concentrations were significantly elevated in parasympathetically denervated rats as compared to control rats, and that VLDL-TG production tended to be increased. Furthermore, in rats fed on a six-meals-a-day schedule for several weeks, a parasympathetic denervation resulted in >70% higher plasma triglycerides during the day, whereas a sympathetic denervation had no effect (22). These results show that abolishing the parasympathetic input to the liver results in increased plasma triglyceride levels during postprandial conditions.

Orexin or hypocretin 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 (27, 34). They were subsequently recognized as regulators of feeding behavior and energy metabolism because of the exclusive localization of their cell bodies in the LH, the induction of feeding upon their ICV administration, their responsiveness to peripheral metabolic cues such as leptin and glucose, and the metabolic phenotype of orexin knockout models. More recent

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studies suggested a primary role for the orexin system in the maintenance of wakefulness (90). However, our data showing that an increased availability of orexin in the central nervous system, either by ICV infusion, or by local activation via removal of GABA inhibition, increases plasma glucose concentrations through an increase in hepatic glucose production, have clearly revitalized the concept of regulation of metabolism by the orexin system. Similar to NPY, the stimulatory effect of orexin on EGP could be blocked by a hepatic sympathetic but not parasympathetic denervation (214). Moreover, Shiuchi et al. (173) demonstrated that via its action in the VMH orexin also stimulates glucose uptake in skeletal muscle. The effect of orexin on glucose uptake was also mediated via the sympathetic nervous system. These effects of central orexin on hepatic glucose metabolism are nicely in line with the increased hypothalamic orexin expression observed in mice homozygous for the tubby mutation (201). Mice carrying a tubby mutation develop retinal and cochlear degeneration as well as late-onset obesity and disturbed carbohydrate metabolism. The tubby phenotype of sensory loss coupled with obesity and insulin resistance is similar to that found in two human syndromes, Alstrom and Bardet-Biedl. The ICV infusion experiments and the presence of a pronounced orexin-containing fiber network in the PVN suggest that the main action is at the level of the sympathetic preautonomic neurons in the PVN. However, in view of the electrophysiological data of Van Den Top et al. (198), a direct effect of orexin at the level of the sympathetic preganglionic neurons in the intermediolateral column of the spinal cord cannot be excluded at this stage. Unfortunately, the selective liver denervations do not allow for a distinction between these two options. In addition, it is not clear yet what are the endogenous triggers for the stimulatory effect of orexin on EGP, but we have proposed two possible pathways. First, the orexin neurons could be an alternative target for the output from the ARC, that is, in addition to the ARC projection to the preautonomic neurons in the PVN. Second, the orexin neurons may integrate circadian information from the SCN. Our as well as other studies clearly showed that the activity of the orexin neurons is under tight control of a GABAergic input that is probably derived from the circadian system (3, 87). These data indicate that the circadian rhythm in orexin release (226) might be implicated in the genesis of the circadian rhythm in plasma glucose concentrations. To test this hypothesis, we administered the orexin antagonist SB-408124, either ICV or i.v., during the final 8 h of the light period and simultaneously measured glucose appearance (Ra) from ZT3-ZT15 with the isotope dilution technique. The ICV but not IV, administration of the orexinantagonist completely blocked the endogenous increase in Ra until the start of the dark period. Once the animals start eating, in the dark period, Ra also increases in the ICV orexinantagonist treated animals. This ICV administration of the orexin-antagonist did not inhibit food intake. Together these data strongly suggest that the perifornical orexin neurons are an important link in the circadian control of the daily



peripheral glucose rhythm. Hereby, the orexin system provides a nice example of hypothalamic integration, as the increased activity of the orexin system at the end of the light period not only initiates the wake state but at the same time ensures a sufficient supply of energy. This concept may be highly relevant for the recently discovered correlation between sleep duration and type 2 diabetes (115, 130, 176). We hypothesize that short sleep or sleep deprivation may cause an over activation of the orexin system (112, 143, 218) and thereby a disproportionate increase in EGP (89). More recently, the activation of orexin neurons has also been implicated in the metabolic side effects, such as weight gain, hyperglycemia, and insulin resistance, induced by the use of atypical antipsychotic drugs. Not only did the administration of Olanzapine, one of the AAPS with the most dramatic effects on weight gain, cause an activation of orexincontaining neurons in the LH (41, 179, 200), ICV administration of the orexin-receptor-1 antagonist SB-408124 was also able to block the Olanzapine induced increase in EGP (54). On the other hand, hypothalamic orexin reduces the hepatic insulin resistance induced by social defeat (192). Poli et al. (146) compared energy metabolism between patients displaying narcolepsy with cataplexy and patients with idiopathic hypersomnia. Lumbar punctures showed that orexin-A levels in CSF were low in narcoleptic patients. In addition, narcoleptic patients displayed higher waist circumference and higher plasma levels of triglycerides, despite eating less than hypersomnic patients. Conversely, animal experiments have shown that sleep deprivation induces higher prepro-orexin gene expression, hyperphagia, slight weight loss, and lower levels of triglycerides (112). The apparent contradiction between the hyperglycemic effect of ICV orexin described above and the increased weight circumference in narcoleptic patients might be explained by the antiobesity effect of prolonged stimulation of the orexin receptor2. Transgenic overexpression of orexin in mice made them resistant to diet-induced obesity (48), probably mediated by an increased metabolic rate and leptin sensitivity. Chronic administration of orexin-A in rats, however, did not lead to changes in body mass, plasma triglycerides, fatty acids, or cholesterol (210). More recently, Shen et al. (168) have shown in an acute experiment that low-dose ICV administration of orexin-A decreased the activity of autonomic nerves innervating white adipose tissue and lipolysis as measured by plasma FFA. This effect was blocked by systemic pretreatment with a muscarinic receptor blocker. By contrast, a high dose of orexin-A resulted in increased autonomic activity and lipolysis, an effect that was blocked by pretreatment with a beta adrenergic receptor blocker. These data further support a role for central orexin pathways in the metabolic control of adipose tissue, possibly via the ANS.

Melanin concentrating hormone Melanin concentrating hormone (MCH) is a cyclic 19-aminoacid polypeptide with an expression pattern that is limited to



the LH, zona incerta and perifornical area, that is very similar to orexin. However, despite the almost complete overlap in their distribution, the two peptides do not colocalize. The MCH neurons have been implicated as an additional important regulator of food intake, because the central administration of MCH promotes feeding, MCH mRNA levels rise as a result of starvation and leptin deficiency, knockout animals are hypophagic and lean (147, 160, 171), and MCH neurons are essential for the leptin-deficient phenotype (166). In addition, overexpression of MCH results in hyperglycemia (106). Despite these earlier observations, we found no effect on glucose metabolism of either ICV administered MCH in wild-type rats (214) nor of the MCH knockout in the MCH knockout rat (124). In fact, the reduced metabolic rate we found in the MCH knockout rats was perfectly adapted to the leaner body composition of these animals. More recently, also the chronic treatment of mice with the MCH antagonist GW803430 did not affect glucose metabolism, although it did decrease energy intake and increase energy expenditure (225). On the other hand, ablation of the MCH neurons did improve glucose tolerance, as well as improve body composition, in both normal mice (203) and adult Lepob/ob mice (206). Therefore, MCH neurons may regulate glucose homeostasis through signaling molecules other than MCH (such as GABA, nesfatin, or CART). Chronic ICV infusion of MCH in pair fed mice results in increased plasma triglycerides, with unchanged liver triglycerides. Ex vivo measurements in the MCH treated animals showed an increased lipogenic activity, as measured by the incorporation of 14C-acetate, in both liver and WAT. But as the expression of SREB1c and FAS was not changed, the increased lipogenic activity could not be explained via this pathway (77). Acute or chronic ICV infusion of a MCH antagonist did not change plasma triglyceride or FFA levels (98). In both an obese and lean steatosis model, a MCH antagonist reduced hepatic TG contents without affecting the expression levels of lipogenic genes, but the MCH antagonist clearly suppressed gene expression of CYP4A10 and CYP4A14, that is, two enzymes that are believed to have a key role in the pathophysiology of steatohepatitis (78). Additionally, MCH1r knockout mice display lower levels of triglycerides in plasma and liver compared to wild type with lower levels of SREB1c. The MCH1r knockout mice and mice infused with a MCH antagonist, also seem to be protected against hepatic steatosis induced by ovariectomy, indicating a role for MCH in the effect of estrogen on metabolism (56). The role of insulin and possible other factors in the changes caused by MCH are yet to be investigated before a direct neural connection can be determined.

Pituitary adenylate cyclase activating peptide Pituitary adenylate cyclase activating peptide (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 adenylate cyclase activity

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in rat anterior pituitary cells (117). Studies conducted in rodents have shown that PACAP exerts a wide array of biological activities both in the CNS and in peripheral organs. The results from knockout studies clearly indicated the involvement of PACAP in glucose metabolism (57, 80, 129). However, these studies did not reveal which part of the metabolic phenotype could be attributed to central signaling pathways of PACAP, although some evidence for central effects on energy metabolism was available. Among others, it has been shown that in the brain PACAP decreases food intake (118, 121) and increases plasma glucose (123). We showed that ICV administered PACAP causes a strong increase of EGP (215). Additional tracing and denervation experiments provided strong evidence that the effects of PACAP are mediated through the preautonomic neurons in the hypothalamus. Moreover, ICV administration of PACAP causes augmented sympathetic nerve activity, whereas parasympathetic nerve activity is decreased (187). Paradoxically, intrathecal administration of PACAP also causes a widespread sympathoexcitation, but it reduces plasma glucose levels (75). Contrary to the neuropeptidergic systems discussed above, PACAPproducing neurons do not show a restricted localization, but are widespread throughout the CNS. Prominent populations of PACAP neurons can be found in the ARC and the VMH, but part of the PACAP innervation in the PVN is also derived from other sources such as the brainstem and the bed nucleus of the stria terminalis (BNST) (63). Since at present only little is known about the stimuli that modulate PACAP release, it is not clear what the physiological function of PACAP could be. We speculated that it could be involved in the counterregulatory response to hypoglycemia, as PACAP knockout animals have a defective counterregulatory response (57). Recovery from hypoglycemia is believed to be mainly processed by the VMH and the sympatho-adrenal pathway (20), and the VMH contains a prominent population of PACAP containing neurons (63, 165). In line, PACAP is involved in the chronic and acute stress response via the PVN CRH neurons, the HPAaxis and the sympathetic nervous system (1, 60, 62). Thus, the PACAP system may be an important gateway to control hepatic glucose production during stressful conditions including hypoglycemia. PACAP−/− mice show no expression of PACAP in the brain and die after several days, possibly due to cardiovascular distress. They display high levels of FFA, triglycerides and cholesterol compared to wild-type animals, with high amounts of microvascular fat in liver and heart cells. Subcutaneous white adipose tissue deposits were totally depleted at time of death, possibly indicating a function of PACAP in the mobilization of fatty acids. The site of action of PACAP in these metabolic changes has yet to be determined (57). To our knowledge the only study to have addressed the possible central role of PACAP in lipid metabolism was performed in chickens. It showed that ICV injections of either PACAP or VIP increased NEFA, with a tendency to decrease triglycerides, indicating increased lipolysis (185). Mice lacking the Adycap1−/− gene, which encodes PACAP, showed a

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markedly reduced white adipose tissue mass (191), raising the possibility that PACAP signaling pathways favor energy storage.

Vasoactive intestinal polypeptide Vasoactive intestinal polypeptide (VIP) is a 28 amino acid peptide expressed at multiple sites throughout the body. It was discovered as a potent muscle relaxant with vasodilatory activity and as a stimulator of secretory activity in the gut. Mice defective in VIP signaling show dysglycemia and overtly altered daily rhythms of metabolism and feeding behavior (12, 111). With VPAC2 being one of the main receptors for VIP, unsurprisingly, similar finding were obtained in mice defective in VPAC2 signaling. However, a close comparison of these two knockout genotypes revealed that whereas daily food intake and metabolic rate were significantly reduced in the VPAC2 receptor knockout mice, no such reduction was seen in the VIP knockout mice. These results are completely in line with the fact that virtually all of the VIP projections in the hypothalamus are derived from the circadian oscillator in the SCN, but that the VPAC2 receptors (in the PVN) bind PACAP with equal affinity as VIP. The VPAC2 receptor may thus contribute to the regulation of feeding and metabolism independently from its role in the circadian clock. Therefore, our results with ICV VIP on glucose metabolism most likely reflect the influence of the clock (215), whereas the effects of PACAP probably are a reflection of the activity of PACAPcontaining neurons in the VMH as discussed above. The proposed stimulatory role of a VIP-containing projection from the SCN on plasma glucose concentrations is fully supported by an elegant series of experiments by the Nagai group (127). Preliminary evidence from the same group indicates that also this SCN-VIP effect may be mediated via the sympathetic innervations of the liver (126). Remarkably, the hereditary blind microphtalmic rat (with a greatly reduced VIP content in the SCN) shows a greatly increased fat deposition, without significant differences in body weight or naso-anal length (128).

Thyrotropin-releasing hormone The tripeptideamide pyroGlu-His-Pro-NH2, was originally isolated as the first hypothalamic hormone and named thyrotropin-releasing hormone (TRH) based on its capacity to stimulate the release of thyroid-stimulating hormone (TSH) from the anterior pituitary. Later studies showed that TRH is also a potent prolactin releasing hormone. TRH positive neurons and fibers are widely distributed throughout the brain. Although the hypothalamus contains the highest concentration, actually over 70% of total brain TRH is found in extra hypothalamic areas, such as the olfactory system, cortex, thalamus, amygdala, brainstem, spinal cord, and pineal gland. The expression of the TRH receptor type 1 (TRHR1) in the brain is very restricted and mainly confined to the hypothalamus, brainstem and spinal cord. By contrast, the



type 2 receptor (TRH-R2) is widely distributed throughout many brain areas including cerebellar and cerebral cortex and the reticular information which may explain central effects of TRH on cognitive functions and arousal (67). TRH may play a dual role in energy homeostasis, firstly by controlling the secretion of thyroid hormones that are a key determinant of metabolism, and secondly through central mechanisms that are independent of its endocrine actions. Already in the 1980s, it was found that the hypothalamic or ICV administration of TRH induces hyperglycemia through pathways involving the adrenal gland, the pancreas, and the liver (76, 84, 113). More recently, it became clear that a major part of this hyperglycemic effect of central TRH may depend on its action in the brainstem (5). Remarkably, TRH−/− mice also show a marked hyperglycemia, and this has been attributed to impaired pancreatic insulin secretion (209). In the past two decades, the physiological role of TRH in the autonomic regulation of visceral functions has been further established and seems to involve both sympathetic and parasympathetic effects (122, 151, 211, 217). The major site of action for these visceral TRH effects seems to be the brainstem and spinal cord, and may predominantly involve the TRH-R1. Indeed, also the TRH-R1 knockout mice are hyperglycemic (148, 222), on the other hand TRH-R2 knockout mice are euglycemic (182). TRH containing projections from the raphe nuclei innervate the dorsal vagal complex, the NTS, the ventrolateral medulla, and the intermediolateral column of the spinal cord (71, 107) and the same areas abundantly express the TRH-receptor-1 (108). Thus, in spite of the pronounced hypothalamic effects of TRH on glucose metabolism and the considerable number of centrally projecting TRH neurons in the PVN, the contribution of TRH-containing projections from the PVN to these brainstem and spinal cord areas is very limited (70, 103, 107, 205), although probably not completely absent (81, 86, 202). Despite the ubiquitous distribution of TRH within the central nervous system, the exact mechanism via which TRH regulates visceral and endocrine remains to be fully elucidated, probably related to the absence of reliable selective TRH agonists and antagonists. In the thyrotoxic state major changes in lipid metabolism of liver and WAT occur to supply fuel for increased energy demands, with a major role for fatty acids supplied by de novo lipogenesis and lipolysis (93, 141). Riedel et al. (153) showed that TRH injected into the cisterna magna in rabbits increased plasma FFA levels, indicating lipolysis. This increase of FFA was higher after transsection of the spinal cord at level C6-7, contrary to the increase of plasma concentrations of glucose and insulin which was significantly lower in the spinal cord transected group. After thyroidal denervation the TRH-induced rise in FT3 and FT4 levels was not present, but the increase in FFA was even higher. Abdominal vagotomy did not affect this increase in FFA levels. Therefore, despite this pronounced effect of central TRH on lipolysis its neural mechanism remains unclear, as it seems independent of thyroid hormone, insulin and autonomic nervous input.



Arginine-vasopressin Vasopressin is a neuropeptide hormone that is involved in diverse functions, including the regulation of osmotic homeostasis, coagulation, vasomotor tone, ACTH release, and circadian rhythms. Several reports indicated the involvement of arginine-vasopressin (AVP) in plasma glucose homeostasis. It has been known for a long time that circulating AVP affects glucose metabolism by promoting GNG and glycogenolysis in the liver (65, 188), and by modulating insulin and glucagon release secretion from the endocrine pancreas (38). Indeed, the vasopressin-deficient Brattleboro rat has difficulty maintaining euglycemia during restricted feeding (204). Moreover, both vasopressin V1a and V1b receptor knockout animals show an altered glucose homeostasis (6, 46). As till now, however, no clear evidence has been presented for a modulatory role of the hypothalamic vasopressin systems on glucose metabolism, despite the direct indications from an excitatory effect of vasopressin on glucose-responsive VMH neurons (97). In line, the moderate hyperglycemia we observed previously upon administration of the vasopressin V1a antagonist in the PVN, but not the DMH, may involve increased release of circulating vasopressin (88). The only direct evidence so far for a role of central AVP in glucose homeostasis is the hyperglycemia induced by local administration of AVP in the NTS (212), which may point at a role for the descending AVP-containing projection from the preautonomic neurons in the PVN. In addition to glucose metabolism, AVP also exerts effects on lipid metabolism. Vasopressin V1a receptor knockout mice exhibit a phenotype with hypermetabolism of fat, while V1b receptor knockout mice show suppressed lipolysis and enhanced lipogenesis, resulting in increased fat weight (68, 69). However, as for glucose metabolism, no clear evidence exists for a modulatory role of hypothalamic vasopressin neurons in lipid metabolism. The effects of AVP on lipid metabolism may be attributed solely to its peripheral effects (69).

Oxytocin Oxytocin (OT) is a nonapeptide hormone synthesized in neurons of the PVN and supraoptic nucleus (SON). The bulk of the peptide is transported from the magnocellular oxytocin neurons of the PVN and SON via the internal zone of the median eminence to the posterior pituitary where it is secreted into the periphery. The typical actions of peripheral oxytocin are stimulation of uteral smooth muscle contraction during labor and milk secretion during lactation. The central oxytocin-containing projections are mainly derived from the parvocellular neurons in the PVN. The parvocellular oxytocin neurons project to various hypothalamic, limbic, and brainstem regions, as well as to the spinal cord. Oxytocin receptors are abundantly expressed in the spinal cord where oxytocin excites sympathetic preganglionic neurons (10, 52). Early studies showed an inhibitory effect of centrally administered oxytocin on food intake (7, 137). It has been

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proposed that the preautonomic oxytocin neurons in the PVN are a component of the leptin-sensitive signaling circuit between the hypothalamus and the brainstem (17), with some oxytocin neurons projecting polysynaptically to brown and white adipose tissue, as well as liver or pancreas (24, 136, 169, 178). The strong decrease in the number of oxytocin expressing neurons in the PVN of Prader-Willi patients (184), and the decreased oxytocin expression in the obese Sim1 mice (99) are in total agreement with its proposed role as satiety neurons. Moreover, both the oxytocin- and the oxytocinreceptor deficient mice show an obese phenotype (25, 186). More recently, Zhang et al. (223) nicely showed that a high-fat diet resulted in an upregulation of synaptotagmin-4 (Syt4) in hypothalamic oxytocin neurons. Syt-4 is a negative regulator of neuropeptide release. Syt-4 knockdown resulted in restoration of oxytocin release and protected against the high-fat diet-induced obesity (223). Further, oxytocin may play a vital role in integrating circadian control and metabolic regulation, as oxytocin treatment could amend the obesigenic effect of circadian dysregulation (224). Part of this phenotype might be the result of a low sympathetic tone due to the absence of oxytocinergic signaling in the hypothalamic projections to the spinal cord. However, the possibility that lack of oxytocin signaling in peripheral organs, such as adipose tissue and pancreas, is contributing to the phenotype can certainly not be excluded (16, 53, 161). Oxytocin deficient mice display mild normophagic obesity, increased WAT weight, and elevated fasting plasma triglycerides (186), suggesting a role for oxytocin in lipid metabolism. But again, at present no evidence is available to directly link hypothalamic oxytocin to these effects.

Hypothalamic Nutrient Sensing and Hepatic Glucose Metabolism It is essential for the central nervous system to gather information about the body’s nutritional status to be able to trigger the appropriate metabolic and behavioral responses aimed at maintaining homeostasis. The key brain area to sense and integrate nutrient-related feedback signals is the hypothalamus, and in particular the hypothalamic arcuate nucleus at the base of the third ventricle. In the arcuate nucleus, where the bloodbrain-barrier is discontinuous, direct contact between circulating hormones and nutrients with the central nervous system takes place. It has been known for many years that metabolic hormones such as insulin, leptin, ghrelin, and thyroid hormone are sensed by neurons in the arcuate nucleus. More recently, evidence has been accumulating that in addition to these hormones, glucose, and lipids are sensed directly as well by specialized fuel-sensing neurons within the hypothalamus. The combination of input from endocrine signals (hormones) and nutrients (lipids, glucose) enables the hypothalamus to balance the activity of neuron populations in control of food intake, energy expenditure, and glucose homeostasis. This paragraph will focus on hypothalamic lipid and glucose

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sensing, and explore its role in the regulation of hepatic glucose metabolism.

Hypothalamic lipid sensing In the 1970s, it was demonstrated already that fatty acids are capable of activating neurons in the LH (138). More than 20 years later, Obici et al. (134) reported that the ICV administration of the long-chain fatty acid (LCFA) oleic acid in rats reduced food intake as well as endogenous glucose production (EGP). The anorectic effect of oleic acid was independent of leptin and was accompanied by a reduction in the hypothalamic expression of NPY. These observations showed beyond doubt that fatty acids can signal nutrient availability to the CNS directly. A short-chain fatty acid did not produce the same effect, while the effect of LCFA was abolished after high-fat feeding (119). Of interest, biochemical as well as genetic inhibition of the enzyme carnitine palmitoyltransferase (CPT1) in the hypothalamus, thus inhibiting LCFA breakdown, also decreased food intake and EGP, which highlighted the hypothalamus as an important central nutrient sensor (132). A few years later, these studies were followed by elegant experiments demonstrating that hypothalamic lipid fluxes activate KATP channels. Moreover, it appeared that parasympathetic outflow from the brain stem to the liver is needed for hypothalamic lipid fluxes to modulate EGP (145). After the recognition of hypothalamic KATP channels as a common pathway for hypothalamic fatty acid sensing in the rat, it was shown that protein kinase C (PKC) activation was required for modulation of EGP by central lipid administration (155). In sum, hypothalamic LCFA regulate peripheral glucose metabolism via complex intrahypothalamic pathways. Conversely, the central administration of insulin regulates peripheral lipid metabolism to some extent. The infusion of insulin into the rat MBH increases lipogenic protein expression in white adipose tissue (WAT), inactivates hormone-sensitive lipase, and suppresses lipolysis. In line, mice that lack the neuronal insulin receptor exhibited unrestrained lipolysis and decreased de novo lipogenesis in WAT (162). Thus, hypothalamic LCFA regulate food intake and hepatic glucose production, while hypothalamic insulin action plays a pivotal role in WAT function.

Hypothalamic metabolism of fatty acids Various hypothalamic neuron populations in the arcuate nucleus and ventromedial hypothalamus express enzymes involved in fatty acid metabolism, including acetyl-CoA carboxylase (ACC), FAS, and CPT1 (175). FAS colocalizes with NPY in arcuate nucleus neurons (91). This may explain why intrahypothalamic fatty acid metabolism is a determinant of food intake. In line, the central administration of a chemical inhibitor of FAS (C75) suppresses food intake and induces weight loss in mice. C75 rapidly and dramatically reduces food intake in lean mice, and prevents the fasting-induced upregulation of hypothalamic NPY mRNA, as well as the downregulation of CART and POMC mRNA in the arcuate



nucleus. In obese mice, C75 not only rapidly suppresses food intake, thereby reducing body weight, but it also normalizes obesity-associated hyperglycemia and hyperinsulinemia. The suppressive effect of C75 on food intake in lean mice appeared to be mediated both by NPY/AgRP and POMC/CART neurons, whereas in obese mice the effect was be mediated primarily by NPY/AgRP neurons (172). These studies highlight the possible therapeutic potential of manipulating intrahypothalamic metabolism of fatty acids in the context of human obesity and insulin resistance.

Hypothalamic glucose sensing In the 1960s, experimental studies in hypothalamic slices demonstrated the presence of glucose sensing neurons that are able to alter their firing behavior upon manipulation of extracellular glucose concentrations (4, 139). Soon it appeared that the neuronal glucose sensing system consists of at least of two types of glucose sensitive neurons: glucose-excited (GE) neurons, that increase firing rate upon elevation of extracellular glucose concentrations, and glucose-inhibited (GI) neurons, responding in the reverse way (140). Both types are distributed throughout the brain, but are most abundant in the hypothalamus. GE neurons are especially prominent in the VMH, PVN, and arcuate nucleus, while GI neurons are prevalent in the PVN, arcuate nucleus, and the LH (174). Within the arcuate nucleus, separate neuron sets are responsive to glucose either in the lower or higher concentration range (45). Moreover, electrophysiological studies demonstrated that increasing glucose concentrations inhibit NPY neurons, while exciting POMC neurons in the arcuate nucleus (reviewed in (83). Within the hypothalamus, additional glucose sensing neuron populations include orexin neurons and melanin-concentrating hormone neurons in the LH. GE neurons respond in a similar way to rising glucose as pancreatic beta cells that are activated by increasing glucose. In the latter cells, glucose uptake by GLUT2, glucose phosphorylation by glucokinase, and subsequent breakdown of glucose to form ATP are required to trigger closure of KATP channels, as well as membrane depolarization and Ca2+ entry, before insulin can finally be secreted. Many studies have addressed these mechanisms in GE neurons, and there are now many data to support the concept that—like in pancreatic beta cells—increasing glucose raises ATP levels in GE neurons, inducing closure of KATP channels and Ca2+ entry, finally increasing neuronal activity and transmitter secretion. In GI neurons, however, the link between decreasing extracellular glucose and increased neuronal activity is more enigmatic, and probably involves activation by AMPK [cf (83)]. Hypothalamic GE and GI neurons are likely players in the regulation of hepatic glucose metabolism, as they are directly informed on the glucose concentration in the blood and are well positioned neuroanatomically to send neural signals to the liver to adapt endogenous glucose production (EGP) to maintain homeostasis. Indeed, in response to hypothalamic glucose, EGP is suppressed in rats. The effect of glucose



appeared to require its conversion to lactate, followed by stimulation of pyruvate metabolism, ultimately leading to activation of KATP channels (102). Interestingly, impaired glucose tolerance was observed in mice with impaired glucose sensing by POMC neurons due to expression of a mutant Kir6.2 subunit. This genetic manipulation impaired the response to a systemic glucose load, showing a role for glucose sensing by POMC neurons in the regulation of blood glucose. As glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, a loss of glucose sensing by POMC neurons may have a role in the development of type 2 diabetes (142). In addition to glucose, the hypothalamic sensing of circulating lactate also regulates EGP (94). Interestingly, similar to EGP, hepatic lipid metabolism is modulated upon hypothalamic administration of glucose (101). Thus, hypothalamic glucose sensing is implicated in hepatic glucose and lipid production. In addition to these physiological functions, glucose sensing in the brain is important for the counterregulatory response to hypoglycemia. This counterregulation involves the stimulation of glucagon secretion from the pancreatic alpha cells (31). Studies in rats have shown that the infusion of D-glucose into the VMH inhibits the counterregulatory endocrine response during hypoglycemia, indicating that glucose sensing in the VMH is important for the response to systemic hypoglycemia (18). A similar change in the counterregulatory response was seen after VMH infusion of L-lactate, which is an alternative fuel for neurons during glucose deficiency (19).

Glucoregulatory Hormones in the Hypothalamus and Hepatic Glucose Metabolism Insulin Following a meal, plasma glucose concentration increases leading to a gradient dependent glucose influx into pancreatic beta-cells. In the beta-cell generation of ATP from glycolysis closes KATP channels inducing depolarization followed by secretion of insulin (96). After binding to the insulin receptor (IR) in targets organs insulin suppresses glucose production by the liver and stimulates glucose uptake by skeletal muscle and adipose tissue. For a long time the liver, skeletal muscle and adipose have been considered the primary target organs of insulin, but in 2000 this view changed with the report on a mouse with neuron specific deletion of the insulin receptor (NIRKO) (21). NIRKO mice were generated by crossing mice carrying a floxed allele of the IR gene with mice expressing the Cre-recombinase under control of the rat nestin promoter and enhancer. Female, but not male NIRKO mice were hyperphagic and slightly heavier than wild-types. Fasting insulin concentrations were increased by 50%, but there was only a trend toward higher glucose concentrations after intraperitoneal administration of insulin. Interpretation of the direct effects of the neural insulin receptor signaling on energy metabolism in NIRKO mice was hampered by the

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coexistence of hypogonadotrophic hypogonadism The role of insulin signaling in the brain was further elucidated by a series of experiments in which agonists and antagonists of the insulin receptor were administered in the third cerebral ventricle to modulate brain insulin signaling independently of peripheral insulin signaling (135). ICV administration of insulin or an insulin mimetic compound suppressed hepatic glucose production by 40% independently of circulating insulin and glucagon concentrations. The ability of ICV administered insulin to suppress hepatic glucose production was blunted after hepatic vagotomy (145). Inhibition of brain insulin signaling by ICV administration of insulin antibodies, insulin receptor antisense, or inhibitors of downstream insulin receptor signaling reduced the ability of peripherally administered insulin to lower hepatic glucose production, suggesting that part of the effect of insulin on hepatic glucose production is mediated via insulin signaling in the brain. Considering that insulin activates phosphoinositide 3kinase, which in turn can activate KATP in the hypothalamus, KATP was suggested as an important mediator of insulin in the brain. Sulfonylurea derivatives are potent inhibitors of KATP and are commonly used to treat diabetes because inhibition of KATP in the beta-cells stimulates insulin secretion. When sulfonylurea derivates were administered ICV suppression of hepatic glucose production by insulin was indeed reduced. Conversely ICV administration of the KATP activator diazoxide lowered plasma glucose concentrations by inhibition of hepatic glucose production (145). More recently, the relevance of KATP channels in the brain for glucose metabolism was also demonstrated in humans (92). Systemic administration of diazoxide reduced hepatic glucose production while systemic concentrations of insulin and glucagon were kept constant using a pancreatic clamp. Complementary studies in rats indicated that orally administered diazoxide effectively crossed the blood-brain-barrier and that its effect on hepatic glucose production was inhibited by simultaneous ICV administration of a KATP inhibitor. A challenging and relevant question that remains to be addressed is to what extent hypothalamic (insulin) signaling is involved in direct day-to-day, physiological control of hepatic glucose metabolism independent of the direct effects of insulin and glucagon on the liver. A limitation of most of the above-mentioned studies is that the exposure of the liver to portal insulin and glucagon has not been determined and hence additional or compensatory effects via direct insulin and glucagon signaling in the liver cannot be excluded. To address this issue a series of elegant experiment was performed in conscious dogs [reviewed in (150)] indicating that insulin also acts in the canine hypothalamus, but that its role in rapid glucose regulation is redundant and overridden by the compensatory effect of pancreatic insulin secretion. In this model, where portal insulin and glucagon concentrations were clamped to constant concentrations, brain insulin administration failed to inhibit hepatic glucose production, but increased hepatic glucose uptake instead, resulting in reduced net glucose production by the liver (149). Whether similar

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mechanisms are operative in conditions where brain-centered control of glucose metabolism is more extensively impaired remains to be elucidated and is subject to intensive investigation (164). Notwithstanding the well-known effect of insulin on the liver, mice with liver-specific deletion of key intracellular insulin signaling proteins appeared to have normal glucose metabolism, suggesting that hepatic insulin signaling is perhaps not the predominant regulator of hepatic glucose metabolism (105), whereas neuron-specific deletion of the insulin receptor resulted diet-sensitive obesity and mild insulin resistance (21).

Estradiol The above-mentioned NIRKO mice were also hypogonadal (21), which could explain at least part of the phenotype. Hyperphagia and excessive weight gain are robust and highly reproducible effects of ovariectomy in rodents. Multiple lines of evidence indicate that decreased estrogen signaling in the hypothalamus mediates many of the adverse metabolic effects of ovariectomy. Intermittent ICV administration of a low dose of estradiol for 4 weeks attenuated weight gain in ovariectomized rats and reduced the ratio of visceral to subcutaneous adipose tissue (29). Subsequent experiments have indicated that the hypothalamic estrogen receptor α (ERα) is an important mediator of these effects. Selective silencing of ERα in the VMH bilaterally with an AAV vector encoding for ERα specific shRNA resulted in obesity with reduced glucose tolerance (125). Comparable effects were observed in again only female mice with selective knockout of ERα in SF1 expressing neurons (207). These ERαlox/lox /SF1Cre mice had increased body weight, visceral adiposity and impaired glucose tolerance. The increased body weight was explained by reductions in basal metabolic rate, decreased diet-induced thermogenesis, and possibly impaired brown adipose tissue thermogenesis. ERαlox/lox /SF1-Cre mice also had lower plasma norepinephrine concentrations suggesting decreased sympathetic outflow to peripheral organs. Selective knockout of ERα in proopiomelanocortin (POMC) neurons also increased body weight (again only in females) by induction of hyperphagia, since POMC neurons are critical regulators of food intake. Collectively, these data indicate that a long-term reduction in hypothalamic estrogen signaling leads to increased food intake and decreased energy expenditure resulting in obesity and insulin resistance. The adverse effects of chronically impaired estradiol signaling on glucose metabolism are most likely secondary to obesity, because recent data indicated that acute modulation of estrogen signaling had opposite effects on glucose metabolism. One week following ovariectomy in rats plasma glucose concentrations were decreased and normalized within one hour after systemic estradiol administration (104). Normalization could be blocked by simultaneous administration of an ER antagonist in the VMH, suggesting that the acute effects of systemic estradiol were mediated via the VMH. As expected from the previous experiments, administration of estradiol in the



VMH of ovariectomized rats acutely induced hepatic insulin resistance in otherwise intact rats. In rats with sympathetic denervation of the liver estradiol in the VMH did no longer affect hepatic insulin sensitivity, indicating that estradiol in the VMH acutely induces hepatic insulin resistance via sympathetic signaling to the liver (100). The above-mentioned studies have clearly demonstrated that a chronic lack of estradiol signaling in the hypothalamus leads to insulin resistance secondary to obesity, but also that the acute, direct effects of estradiol on hepatic insulin sensitivity oppose the chronic effects.

Glucagon Glucagon is produced by the pancreatic alpha-cell and stimulates hepatic glucose production via activation of hepatic glucagon receptors and hepatic protein kinase A (PKA). Glucagon receptors are not only expressed in the liver, but also in the PVN and in AgRP neurons in the arcuate nucleus (116). Unexpectedly, administration of glucagon in the MBH decreased hepatic glucose production in pancreatic clamp conditions and improved glucose tolerance in nonclamp conditions (116). Subsequent functional studies convincingly demonstrated that the effect of glucagon in the MBH was mediated via the glucagon receptor in AgRP neurons and required activation of hypothalamic PKA. Hepatic vagotomy abolished the inhibitory effect of MBH glucagon on hepatic glucose production (116). The effect of continuous systemic glucagon administration on hepatic glucose production is transient and wears off within an hour. The effect could be sustained by simultaneous administration of a glucagon receptor antagonist in the MBH, indicating that the stimulatory effect of systemic glucagon on hepatic glucose production is transient because it is antagonized by activation of glucagon receptors in the MBH. This observation could have important implications for pathological conditions like diabetes. Chronic hyperglucagonemia has been documented in diabetes and obesity and could contribute to impaired glucose tolerance and increased hepatic glucose production if hypothalamic counterregulation is impaired. In rats after 3 days of high-fat feeding, the stimulatory effect of systemic glucagon on hepatic glucose production was more pronounced and administration of glucagon in the MBH failed to lower hepatic glucose, indicating hypothalamic glucagon resistance (116). Together these findings point to an important counterregulatory system by which glucagon antagonizes its own action on hepatic glucose production and glucose tolerance via the hypothalamus and vagal output to the liver. Although the relevance of this counterregulatory system or lack thereof for pathological conditions like diabetes or obesity needs to be established, it could contribute substantially to our understanding of the implications of chronic hyperglucogenemia.

Glucocorticoids Glucocorticoids are produced by the adrenal cortex. In rodents the active form of glucocorticoids is corticosterone and its human equivalent is cortisol. Glucocorticoids are important



regulators of energy metabolism and in particular of glucose metabolism. Clinical and experimental conditions of glucocorticoid excess are invariably associated with body weight gain and deranged glucose metabolism, which is reversible upon restoration of the glucocorticoid excess. Vice versa, glucocorticoid deficiency induces hypophagia and weight loss (193). Glucocorticoids act via glucocorticoid (GR) and mineralocorticoid receptors. Interestingly, GRs are abundantly expressed in areas of the hypothalamus that regulate food intake and hepatic glucose metabolism (28, 120), suggesting that at least part of the effects of glucocorticoids on glucose metabolism could be relayed via the hypothalamus. Indeed, ICV infusion of dexamethasone, a potent GR agonist, stimulated body weight gain (219), and impaired insulin mediated glucose uptake (32). More localized administration of dexamethasone into the arcuate nucleus did not affect basal endogenous glucose production, but completely blocked the ability of insulin to suppress hepatic glucose production at low postprandial insulin concentrations (213). Insulin action could be restored by simultaneous ICV administration of the NPY1R antagonist BIBP3226 or by sympathetic denervation of the liver, suggesting that the effect of dexamethason in the ARC on hepatic insulin sensitivity involves NPY neurons and the sympathetic nervous system.

Glucagon-like peptide GLP-1 is an incretin derived from ileal L cells. Upon feeding plasma GLP-1 concentrations increase, inducing a glucosedependent stimulation of insulin secretion, while suppressing glucagon secretion. Due to their glucose lowering capacity GLP-1 agonist are now successfully used to treat diabetes. Apart from its glycemic effects, GLP-1 also induces anorexia possibly via modulation of hypothalamic orexigenic and anorexigenic neuropeptide expression (33). This may explain why the use of GLP-1R agonists was associated with moderate weight reduction in several clinical studies. GLP-1 receptors are abundantly expressed not only in the pancreatic beta-cells, but also in the hypothalamic ARC, PVN, and ventromedial hypothalamus (152). ICV administration of GLP-1 enhanced the ability of insulin to decrease hepatic glucose production, whereas administration of a GLP-1 receptor antagonist had the opposite effect (33), suggesting that central GLP-1 signaling is involved the regulation of hepatic insulin sensitivity. In an earlier study, this effect of ICV administration of GLP-1 was not demonstrated, but more targeted administration of GLP-1 to the ARC enhanced insulin-mediated suppression of hepatic glucose production (159).

Conclusion The studies summarized in this overview have clearly identified the hypothalamus as a key regulator of hepatic glucose production. Multiple hypothalamic neuropeptides including NPY, POMC, orexin, and MCH can modulate glucose

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metabolism via autonomic projections to the liver. Not surprisingly, these neuropeptides are also well-known regulators of food intake. Since the discovery that insulin can only reach its full effect on glucose homeostasis when hypothalamic insulin signaling and autonomic projections to the liver are intact, surmounting evidence indicates that also other glucoregulatory hormones can act via the hypothalamus and in the case of glucagon can even antagonize the peripheral effect. Finally, in response to local changes in lipid or glucose concentrations, the hypothalamus can modulate glucose metabolism via autonomic projections to the liver. Despite the wealth of data indicating that hormones and neuropeptides in the hypothalamus can regulate hepatic glucose metabolism via the ANS a number of challenging questions remain to be addressed, the first being to what extent the hypothalamus and ANS are involved in direct day-to-day, physiological control of hepatic glucose metabolism independent of the direct effect of insulin and glucagon on the liver. The next challenge will be to extrapolate these observations to a relevant clinical setting and to selectively target one or more of the multiple pathways that are involved in hypothalamic and autonomic regulation of glucose metabolism as a novel therapeutic strategy. The use of peptide mediated selective delivery of hormones to the hypothalamus as reported by Finan et al. (44) seems a promising approach. In short, these authors showed that the administration of a GLP-1-estrogen conjugate corrected obesity, hyperglycemia, and dyslipidemia in mice more effectively than combined administration of the individual hormones, without the adverse off-target effects of estrogen on estrogen responsive non-metabolic tissues like the reproductive system. Finally, many of the above-mentioned studies used hyperinsulinemic euglycemic clamps to determine insulinmediated suppression of hepatic glucose production. It should be noted that results from hyperinsulinemic euglycemic clamps can be misleading. Changes in insulin mediated suppression after an intervention are commonly attributed to changes in insulin sensitivity, but could also reflect an insulin-independent effect. In conclusion, hypothalamic regulation of hepatic glucose production should be viewed as a summation of both neuroendocrine and neural influences, as integrated by the hypothalamus. As a result, our endocrine-based understanding of diseases such as diabetes and obesity should be expanded by integration of neural inputs into our concept of the pathophysiological processes.


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Metabolism: Mechanisms of hepatic glucose production revealed.

Insulin control of hepatic glucose production.

Regulation of hepatic microsomal glucose transport.

Regulation of the hepatic microsomal glucose-6-phosphatase enzyme.

Regulation of net hepatic glucose uptake in vivo.

Effect of difference in glucose infusion rate on quantification of hepatic glucose production.

Regulation of dipeptidyl peptidase 4 production in adipocytes by glucose.

Hypothalamic orexin prevents hepatic insulin resistance via daily bidirectional regulation of autonomic nervous system in mice.

Correction of glucose carbon recycling for the determination of 'true' hepatic glucose production rates by (1-13C1)glucose.

Defective blood glucose counter-regulation in diabetics is a selective form of autonomic neuropathy.

Akt interaction induced by physical exercise and its effect on the hepatic glucose production in an insulin resistance state.

Autonomic regulation in muscular dystrophy.

Autonomic regulation of cellular immune function.

The Effect of Phloroglucinol, A Component of Ecklonia cava Extract, on Hepatic Glucose Production.

Autonomic regulation of hematopoiesis and cancer.

Thyroid hormone treatment decreases hepatic glucose production and renal reabsorption of glucose in alloxan-induced diabetic Wistar rats.

Role of placental insufficiency and intrauterine growth restriction on the activation of fetal hepatic glucose production.

Effects of diabetes on glucose regulation of enzymes involved in hepatic glycogen metabolism.

Interaction between insulin and glucose-delivery route in regulation of net hepatic glucose uptake in conscious dogs.

Regulation of hepatic pyruvate dehydrogenase phosphorylation in offspring glucose intolerance induced by intrauterine hyperglycemia.

Integrated Regulation of Hepatic Lipid and Glucose Metabolism by Adipose Triacylglycerol Lipase and FoxO Proteins.

Coordinated changes in hepatic amino acid metabolism and endocrine signals support hepatic glucose production during fetal hypoglycemia.

Autonomic Regulation Therapy in Heart Failure.

Duodenal GLP-1 signaling regulates hepatic glucose production through a PKC-δ-dependent neurocircuitry.