Loss of cGMP-dependent protein kinase II alters ultrasonic vocalizations in mice, a model for speech impairment in human microdeletion 4q21 syndrome.

Chromosome 4q21 microdeletion leads to a human syndrome that exhibits restricted growth, facial dysmorphisms, mental retardation, and absent or delayed speech. One of the key genes in the affected region of the chromosome is PRKG2, which encodes cGMP-dependent protein kinase II (cGKII). Mice lacking cGKII exhibit restricted growth and deficits in learning and memory, as seen in the human syndrome. However, vocalization impairments in these mice have not been determined. The molecular pathway underlying vocalization impairment in humans is not fully understood. Here, we employed cGKII knockout (KO) mice as a model for the human microdeletion syndrome to test whether vocalizations are affected by loss of the PRKG2 gene. Mice emit ultrasonic vocalizations (USVs) to communicate in social situations, stress, and isolation. We thus recorded ultrasonic vocalizations as a model for human speech. We isolated postnatal day 5-7 pups from the nest to record and analyze USVs and found significant differences in vocalizations of KO mice relative to wild-type and heterozygous mutant mice. KO mice produced fewer calls that were shorter duration and higher frequency. Because neuronal activation in the arcuate nucleus in the hypothalamus is important for the production of animal USVs following isolation from the nest, we assessed neuronal activity in the arcuate nucleus of KO pups following isolation. We found significant reduction of neuronal activation in cGKII KO pups after isolation. Taken together, our studies indicate that cGKII is important for neuronal activation in the arcuate nucleus, which significantly contributes to the production of USVs in neonatal mice. We further suggest cGKII KO mice can be a valuable animal model to investigate pathophysiology of human microdeletion 4q21 syndrome.

Increased NADPH-diaphorase reactivity in the hypothalamic paraventricular nucleus and tanycytes following systemic administration of leptin in rats.

Leptin, a hormone mainly produced by adipocytes in proportion to fat mass, is a key component in the regulation of energy homeostasis and reproductive, neuroendocrine, immune, and metabolic functions. Leptin binds to the leptin receptor, which is expressed throughout the central nervous system but particularly in neurons of several nuclei of the hypothalamus, such as the arcuate nucleus (ARC) and paraventricular nucleus (PVN). It has been found that nitric oxide (NO) plays an important role in mediating effects of leptin. Since PVN and ARC neurons are known to express leptin receptors, we investigated the effects of leptin on nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in the PVN and ARC of male Wistar rats. Our results have shown that systemic administration of leptin resulted in increased NADPH-d positive cell number in the PVN and ARC, suggesting that both the PVN and ARC may be important centers in the hypothalamus for the leptin action, mediated by increased NO production. In addition, we have also observed that hypothalamic tanycytes in the ventral portion of the third ventricle were NADPH-d positive. We speculate that leptin may affect the release of neurohormones and hypothalamic neurogenesis by activating nitric oxide synthase in hypothalamic tanycytes.

Hypothalamic arcuate nucleus glucokinase regulates insulin secretion and glucose homeostasis.

AIMS: To investigate the role of arcuate glucokinase (GK) in the regulation of glucose homeostasis. MATERIALS AND METHODS: A recombinant adeno-associated virus expressing either GK or an antisense GK construct was used to alter GK activity specifically in the hypothalamic arcuate nucleus (arc). GK activity in this nucleus was also increased by stereotactic injection of the GK activator, compound A. The effect of altered arc GK activity on glucose homeostasis was subsequently investigated using glucose and insulin tolerance tests. RESULTS: Increased GK activity specifically within the arc increased insulin secretion and improved glucose tolerance in rats during oral glucose tolerance tests. Decreased GK activity in this nucleus reduced insulin secretion and increased glucose levels during the same tests. Insulin sensitivity was not affected in either case. The effect of arc GK was maintained in a model of type 2 diabetes. CONCLUSIONS: These results demonstrate a role for arc GK in systemic glucose homeostasis.

Role of PTP1B in POMC neurons during chronic high-fat diet: sex differences in regulation of liver lipids and glucose tolerance.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of leptin receptor signaling and may contribute to leptin resistance in diet-induced obesity. Although PTP1B inhibition has been suggested as a potential weight loss therapy, the role of specific neuronal PTP1B signaling in cardiovascular and metabolic regulation and the importance of sex differences in this regulation are still unclear. In this study, we investigated the impact of proopiomelanocortin (POMC) neuronal PTP1B deficiency in cardiometabolic regulation in male and female mice fed a high-fat diet (HFD). When compared with control mice (PTP1B flox/flox), male and female mice deficient in POMC neuronal PTP1B (PTP1B flox/flox/POMC-Cre) had attenuated body weight gain (males: -18%; females: -16%) and fat mass (males: -33%; female: -29%) in response to HFD. Glucose tolerance was improved by 40%, and liver lipid accumulation was reduced by 40% in PTP1B/POMC-Cre males but not in females. When compared with control mice, deficiency of POMC neuronal PTP1B did not alter mean arterial pressure (MAP) in male or female mice (males: 112 ± 1 vs. 112 ± 1 mmHg in controls; females: 106 ± 3 vs. 109 ± 3 mmHg in controls). Deficiency of POMC neuronal PTP1B also did not alter MAP response to acute stress in males or females compared with control mice (males: Δ32 ± 0 vs. Δ29 ± 4 mmHg; females: Δ22 ± 2 vs. Δ27 ± 4 mmHg). These data demonstrate that POMC-specific PTP1B deficiency improved glucose tolerance and attenuated diet-induced fatty liver only in male mice and attenuated weight gain in males and females but did not enhance the MAP and HR responses to a HFD or to acute stress.

Hypothalamic AMPK-induced autophagy increases food intake by regulating NPY and POMC expression.

Hypothalamic AMP-activated protein kinase (AMPK) plays important roles in the regulation of food intake by altering the expression of orexigenic or anorexigenic neuropeptides. However, little is known about the mechanisms of this regulation. Here, we report that hypothalamic AMPK modulates the expression of NPY (neuropeptide Y), an orexigenic neuropeptide, and POMC (pro-opiomelanocortin-α), an anorexigenic neuropeptide, by regulating autophagic activity in vitro and in vivo. In hypothalamic cell lines subjected to low glucose availability such as 2-deoxy-d-glucose (2DG)-induced glucoprivation or glucose deprivation, autophagy was induced via the activation of AMPK, which regulates ULK1 and MTOR complex 1 followed by increased Npy and decreased Pomc expression. Pharmacological or genetic inhibition of autophagy diminished the effect of AMPK on neuropeptide expression in hypothalamic cell lines. Moreover, AMPK knockdown in the arcuate nucleus of the hypothalamus decreased autophagic activity and changed Npy and Pomc expression, leading to a reduction in food intake and body weight. AMPK knockdown abolished the orexigenic effects of intraperitoneal 2DG injection by decreasing autophagy and changing Npy and Pomc expression in mice fed a high-fat diet. We suggest that the induction of autophagy is a possible mechanism of AMPK-mediated regulation of neuropeptide expression and control of feeding in response to low glucose availability.

Effects of Age and Estradiol on Gene Expression in the Rhesus Macaque Hypothalamus.

BACKGROUND: The hypothalamus plays a key role in mediating the effects of estrogen on many physiological functions, including reproduction, metabolism, and thermoregulation. We have previously observed marked estrogen-dependent gene expression changes within the hypothalamus of rhesus macaques during aging, especially in the KNDy neurons of the arcuate-median eminence (ARC-ME) that produce kisspeptin, neurokinin B, and dynorphin A. Little is known, however, about the mechanisms involved in mediating the feedback from estrogen onto these neurons. METHODS: We used quantitative real-time PCR to profile age- and estrogen-dependent gene expression changes in the rhesus macaque hypothalamus. Our focus was on genes that encode steroid receptors (ESR1, ESR2, PGR, and AR) and on enzymes that contribute to the local synthesis of 17ß-estradiol (E2; STS, HSD3B1/2, HSD17B5, and CYP19A). In addition, we used RT(2) Profiler™ PCR Arrays to profile a larger set of genes that are integral to hypothalamic function. RESULTS: KISS1, KISS1R, TAC3, and NPY2R mRNA levels increased in surgically menopausal (ovariectomized) old females relative to age-matched ovariectomized animals that received E2 hormone therapy. In contrast, PGR, HSD17B, GNRH2, SLC6A3, KISS1, TAC3, and NPY2R mRNA levels increased after E2 supplementation. CONCLUSION: The rhesus macaque ARC-ME expresses many genes that are responsive to changes in circulating estrogen levels, even during old age, and these may contribute to causing the normal and pathophysiological changes that occur during menopause.

Glucokinase activity in the arcuate nucleus regulates glucose intake.

The brain relies on a constant supply of glucose, its primary fuel, for optimal function. A taste-independent mechanism within the CNS that promotes glucose delivery to the brain has been postulated to maintain glucose homeostasis; however, evidence for such a mechanism is lacking. Here, we determined that glucokinase activity within the hypothalamic arcuate nucleus is involved in regulation of dietary glucose intake. In fasted rats, glucokinase activity was specifically increased in the arcuate nucleus but not other regions of the hypothalamus. Moreover, pharmacologic and genetic activation of glucokinase in the arcuate nucleus of rodent models increased glucose ingestion, while decreased arcuate nucleus glucokinase activity reduced glucose intake. Pharmacologic targeting of potential downstream glucokinase effectors revealed that ATP-sensitive potassium channel and P/Q calcium channel activity are required for glucokinase-mediated glucose intake. Additionally, altered glucokinase activity affected release of the orexigenic neurotransmitter neuropeptide Y in response to glucose. Together, our results suggest that glucokinase activity in the arcuate nucleus specifically regulates glucose intake and that appetite for glucose is an important driver of overall food intake. Arcuate nucleus glucokinase activation may represent a CNS mechanism that underlies the oft-described phenomena of the "sweet tooth" and carbohydrate craving.

Olanzapine depot formulation in rat: a step forward in modelling antipsychotic-induced metabolic adverse effects.

Rats are used as animal models in the study of antipsychotic-induced metabolic adverse effects, with oral drug administration yielding hyperphagia, weight gain and, in some cases, lipogenic effects. However, the rapid half-life of these drugs in rats, in combination with development of drug tolerance after a few weeks of treatment, has limited the validity of the model. In order to prevent fluctuating drug serum concentrations seen with daily repeated administrations, we injected female rats with a single intramuscular dose of long-acting olanzapine formulation. The olanzapine depot injection yielded plasma olanzapine concentrations in the range of those achieved in patients, and induced changes in metabolic parameters similar to those previously observed with oral administration, including increased food intake, weight gain and elevated plasma triglycerides. Moreover, the sensitivity to olanzapine was maintained beyond the 2-3 wk of weight gain observed with oral administration. In a separate olanzapine depot experiment, we aimed to clarify the role of hypothalamic AMP-activated protein kinase (AMPK) in olanzapine-induced weight gain, which has been subject to debate. Adenovirus-mediated inhibition of AMPK was performed in the arcuate (ARC) or the ventromedial hypothalamic (VMH) nuclei in female rats, with subsequent injection of olanzapine depot solution. Inhibition of AMPK in the ARC, but not in the VMH, attenuated the weight-inducing effect of olanzapine, suggesting an important role for ARC-specific AMPK activation in mediating the orexigenic potential of olanzapine. Taken together, olanzapine depot formulation provides an improved mode of drug administration, preventing fluctuating plasma concentrations, reducing handling stress and opening up possibilities to perform complex mechanistic studies.

Arcuate Src activation-induced phosphorylation of NR2B NMDA subunit contributes to inflammatory pain in rats.

The tyrosine kinases of Src family play an important role in the central sensitization following peripheral inflammation. However, whether the Src family in the arcuate nucleus (ARC) of mediobasal hypothalamus is involved in central sensitization remains unknown. The aim of this study was to investigate the role and mechanisms of tyrosine kinases of Src family in N-methyl-d-aspartate (NMDA) receptor activity in the ARC following peripheral inflammation. Peripheral inflammation was induced by unilateral injection of complete Freund's adjuvant (CFA) into rat hindpaw. The neuronal activities of the ARC were recorded using electrophysiological field recording from the in vitro mediobasal hypothalamic slices from control and CFA rats. Expression of total and phosphorylated Src and NR2B subunit protein was analyzed by Western blot and immuoprecipitation. Our results showed that CFA injection resulted in an increase in mechanical and thermal sensitivity, which was partially blocked by neonatal monosodium glutamate treatment. CFA injection also enhanced spontaneous firings of ARC neurons, which were reversed by the NMDA receptor NR2B subunit specific antagonist Ro25-6981 and by PP2, an Src family tyrosine kinase inhibitor. In addition, peripheral inflammation enhanced Src phosphorylation and NMDA receptor NR2B subunit phosphorylation without alteration of total NR2B subunit expression in the ARC. Peripheral inflammation also increased the association of NR2B protein with p-Src protein in the ARC. Administration of PP2 blocked the upregulation of NR2B phosphorylation induced by CFA injection. Taken together, our present results suggest that the arcuate Src activation-induced tyrosine phosphorylation of NR2B NMDA subunit may contribute to inflammatory pain.

Hypothalamic glycogen synthase kinase 3ß has a central role in the regulation of food intake and glucose metabolism.

GSK3ß (glycogen synthase kinase 3ß) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3ß activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3ß in leptin-deficient Lep(ob/ob) mice and show that intracerebroventricular injection of a GSK3ß inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3ß inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3ß in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3ß signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.

Inositol polyphosphate multikinase: an emerging player for the central action of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an essential enzyme indispensable for energy sensing and metabolic homeostasis at both the cellular and whole-body levels. Phosphorylation of AMPK, a key step for its activation, is known to be regulated by upstream kinases such as liver kinase B1 (LKB1) and calmodulin-dependent protein kinase kinase-beta (CaMKKß). Recent evidence shows that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid inositol kinase activities, can physiologically regulate AMPK signaling in cultured cells and in the arcuate nucleus. IPMK-mediated regulation of AMPK occurs through the dynamic protein interactions of IPMK with AMPK in response to glucose availability. Here we review and discuss a novel role for the hypothalamic IPMK signaling in the control of AMPK and central energy homeostasis.

Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3ß inhibition.

AIM: To identify the mechanisms underlying the elevation of intracellular Ca(2+) level ([Ca(2+)](i)) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons. METHODS: Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca(2+)](i) was measured using fura-2 AM. Ca(2+) current was recorded using whole-cell patch clamp recording. AMPK and GSK3ß levels were measured using Western blot assay. RESULTS: Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca(2+)](i) in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca(2+)](i) in ARC neurons depended on extracellular Ca(2+), and was blocked by P/Q-type Ca(2+)channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca(2+) channel blocker nifedipine (10 µmol/L) or N-type Ca(2+)channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca(2+) current in ARC neurons. The low-glucose induced elevation of [Ca(2+)](i) in ARC neurons was blocked by the AMPK inhibitor compound C (20 µmol/L), and enhanced by the GSK3ß inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3ß, which was inhibited by compound C (20 µmol/L). CONCLUSION: Lowering glucose level enhances the activity of P/Q type Ca(2+)channels and elevates [Ca(2+)](i) level in hypothalamic arcuate nucleus neurons via inhibition of GSK3ß.

Mechanism of programmed obesity in intrauterine fetal growth restricted offspring: paradoxically enhanced appetite stimulation in fed and fasting states.

We have shown that intrauterine fetal growth restriction (IUGR) newborn rats exhibit hyperphagia, reduced satiety, and adult obesity. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a principal metabolic regulator that specifically regulates appetite in the hypothalamic arcuate nucleus (ARC). In response to fasting, upregulated AMPK activity increases the expression of orexigenic (neuropeptide Y [NPY] and agouti-related protein [AgRP]) and decreases anorexigenic (proopiomelanocortin [POMC]) peptides. We hypothesized that IUGR offspring would exhibit upregulated hypothalamic AMPK, contributing to hyperphagia and obesity. We determined AMPK activity and appetite-modulating peptides (NPY and POMC) during fasting and fed conditions in the ARC of adult IUGR and control females. Pregnant rats were fed ad libitum diet (control) or were 50% food restricted from gestation day 10 to 21 to produce IUGR newborns. At 10 months of age, hypothalamic ARC was dissected from fasted (48 hours) and fed control and IUGR females. Arcuate nucleus messenger RNA ([mRNA] NPY, AgRP, and POMC) and protein expression (total and phosphorylated AMPK, Akt) was determined by quantitative reverse transcriptase-polymerase chain reaction and Western Blot, respectively. In the fed state, IUGR adult females demonstrated evidence of persistent appetite stimulation with significantly upregulated phospho (Thr(172))-AMPKα/AMPK (1.3-fold), NPY/AgRP (2.3/1.8-fold) and decreased pAkt/Akt (0.6-fold) and POMC (0.7-fold) as compared to fed controls. In controls though not IUGR adult females, fasting significantly increased pAMPK/AMPK, NPY, and AgRP and decreased pAkt/Akt and POMC. Despite obesity, fed IUGR adult females exhibit upregulated AMPK activity and appetite stimulatory factors, similar to that exhibited by fasting controls. These results suggest that an enhanced appetite drive in both fed and fasting states contributes to hyperphagia and obesity in IUGR offspring.

Importance of mitochondrial dynamin-related protein 1 in hypothalamic glucose sensitivity in rats.

AIMS: Hypothalamic mitochondrial reactive oxygen species (mROS)-mediated signaling has been recently shown to be involved in the regulation of energy homeostasis. However, the upstream signals that control this mechanism have not yet been determined. Here, we hypothesize that glucose-induced mitochondrial fission plays a significant role in mROS-dependent hypothalamic glucose sensing. RESULTS: Glucose-triggered translocation of the fission protein dynamin-related protein 1 (DRP1) to mitochondria was first investigated in vivo in hypothalamus. Thus, we show that intracarotid glucose injection induces the recruitment of DRP1 to VMH mitochondria in vivo. Then, expression was transiently knocked down by intra-ventromedial hypothalamus (VMH) DRP1 siRNA (siDRP1) injection. 72 h post siRNA injection, brain intracarotid glucose induced insulin secretion, and VMH glucose infusion-induced refeeding decrease were measured, as well as mROS production. The SiDRP1 rats decreased mROS and impaired intracarotid glucose injection-induced insulin secretion. In addition, the VMH glucose infusion-induced refeeding decrease was lost in siDRP1 rats. Finally, mitochondrial function was evaluated by oxygen consumption measurements after DRP1 knock down. Although hypothalamic mitochondrial respiration was not modified in the resting state, substrate-driven respiration was impaired in siDRP1 rats and associated with an alteration of the coupling mechanism. INNOVATION AND CONCLUSION: Collectively, our results suggest that glucose-induced DRP1-dependent mitochondrial fission is an upstream regulator for mROS signaling, and consequently, a key mechanism in hypothalamic glucose sensing. Thus, for the first time, we demonstrate the involvement of DRP1 in physiological regulation of brain glucose-induced insulin secretion and food intake inhibition. Such involvement implies DRP1-dependent mROS production.

AMP-activated protein kinase activates neuropeptide Y neurons in the hypothalamic arcuate nucleus to increase food intake in rats.

AMP-activated protein kinase (AMPK) is an energy sensor that is activated by the increase of intracellular AMP:ATP ratio. AMPK in the hypothalamic arcuate nucleus (ARC) is activated during fasting and the activation of AMPK stimulates food intake. To clarify the pathway underlying AMPK-induced feeding, we monitored the activity of single ARC neurons by measuring cytosolic Ca(2+) concentration ([Ca(2+)](i)) with fura-2 fluorescence imaging. An AMPK activator, AICA-riboside (AICAR), at 200 µM increased [Ca(2+)](i) in 24% of ARC neurons. AMPK and acetyl CoA carboxylase were phosphorylated in the neurons with [Ca(2+)](i) responses to AICAR. AICAR-induced [Ca(2+)](i) increases were inhibited by Ca(2+)-free condition but not by thapsigargin, suggesting that AICAR increases [Ca(2+)](i) through Ca(2+) influx from extracellular space. Among AICAR-responding ARC neurons, 38% were neuropeptide Y (NPY)-immunoreactive neurons while no proopiomelanocortin (POMC)-immunoreactive neuron was observed. Intracerebroventricular administration of AICAR increased food intake, and the AICAR-induced food intake was abolished by the co-administration of NPY Y1 receptor antagonist, 1229U91. These results indicate that the activation of AMPK leads to the activation of ARC NPY neurons through Ca(2+) influx, thereby causing NPY-dependent food intake. These mechanisms could be implicated in the stimulation of food intake by physiological orexigenic substances.

Fasting and high-fat diet alter histone deacetylase expression in the medial hypothalamus.

Increasing attention is now being given to the epigenetic regulation of animal and human behaviors including the stress response and drug addiction. Epigenetic factors also influence feeding behavior and metabolic phenotypes, such as obesity and insulin sensitivity. In response to fasting and high-fat diets, the medial hypothalamus changes the expression of neuropeptides regulating feeding, metabolism, and reproductive behaviors. Histone deacetylases (HDACs) are involved in the epigenetic control of gene expression and alter behavior in response to a variety of environmental factors. Here, we examined the expression of HDAC family members in the medial hypothalamus of mice in response to either fasting or a high-fat diet. In response to fasting, HDAC3 and -4 expression levels increased while HDAC10 and -11 levels decreased. Four weeks on a high-fat diet resulted in the increased expression of HDAC5 and -8. Moreover, fasting decreased the number of acetylated histone H3- and acetylated histone H4-positive cells in the ventrolateral subdivision of the ventromedial hypothalamus. Therefore, HDACs may be implicated in altered gene expression profiles in the medial hypothalamus under different metabolic states.

The role of neuronal nitric oxide synthase on hypobaric hypoxia-induced antinociception in writhing test.

It has been reported that hypobaric hypoxia exposure by high altitude is responsible for neuropsychological impairment. In the present study, we examined an effect of hypobaric hypoxia on the writhing test. The ICR mice were exposed in hypobaric chamber with several altitudes (5000, 10,000 or 20,000 ft) for 1 or 2 h, and then immediately injected intraperitoneally (i.p.) with 1% acetic acid for writhing test. Our results show that both 10,000 ft and 20,000 ft exposure induce antinociceptive effect in writhing test, but 5,000 ft does not. In addition, this antinociceptive effect was abolished by L-NAME (nitric oxide synthase inhibitor) pre-treated intraperitoneally, but not naloxone (non-specific opioid receptor antagonist). Furthermore, we examined that neuronal NOS immunoreactivities in the hypothalamus (paraventricular nucleus and arcuate nucleus) were increased by hypobaric hypoxic exposure (10,000ft). These results suggest that hypobaric hypoxic-induced antinociception may be associated with neuronal NOS IR in the hypothalamus.

Critical role of arcuate Y4 receptors and the melanocortin system in pancreatic polypeptide-induced reduction in food intake in mice.

BACKGROUND: Pancreatic polypeptide (PP) is a potent anti-obesity agent known to inhibit food intake in the absence of nausea, but the mechanism behind this process is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that in response to i.p. injection of PP in wild type but not in Y4 receptor knockout mice, immunostaining for the neuronal activation marker c-Fos is induced specifically in neurons of the nucleus tractus solitarius and the area postrema in the brainstem, notably in cells also showing immunostaining for tyrosine hydroxylase. Importantly, strong c-Fos activation is also detected in the arcuate nucleus of the hypothalamus (ARC), particularly in neurons that co-express alpha melanocyte stimulating hormone (alpha-MSH), the anorexigenic product of the proopiomelanocortin (POMC) gene. Interestingly, other hypothalamic regions such as the paraventricular nucleus, the ventromedial nucleus and the lateral hypothalamic area also show c-Fos induction after PP injection. In addition to c-Fos activation, PP injection up-regulates POMC mRNA expression in the ARC as detected by in situ hybridization. These effects are a direct consequence of local Y4 signaling, since hypothalamus-specific conditional Y4 receptor knockout abolishes PP-induced ARC c-Fos activation and blocks the PP-induced increase in POMC mRNA expression. Additionally, the hypophagic effect of i.p. PP seen in wild type mice is completely absent in melanocortin 4 receptor knockout mice. CONCLUSIONS/SIGNIFICANCE: Taken together, these findings show that PP reduces food intake predominantly via stimulation of the anorexigenic alpha-MSH signaling pathway, and that this effect is mediated by direct action on local Y4 receptors within the ARC, highlighting a potential novel avenue for the treatment of obesity.

[Monoamine synthesis by non-monoaminergic neurons: illusion of reality].

Apart from monoaminergic neurons possessing the whole set of enzymes of monoamine synthesis from the precursor amino acid, the neurons expressing individual enzymes of monoamine synthesis have been discovered in the mid-eighties. Most numerous monoenzymatic neurons express individual enzymes of dopamine (DA), thyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC). Functional characteristics and the functional significance of the monoenzymatic neurons have been evaluated in a series of our studies, mainly of the hypothalamic arcuate nucleus (AN), one of the most important DA-ergic centers of the brain. It has been demonstrated that the AN of rats contains numerous monoenzymatic neurons. Their portion among the neurons expressing enzymes of DA synthesis exceeded 99 % whereas it decreased continuously in postnatal period still reaching 50 % in adulthood. It was shown that the monoenzymatic neurons expressing complementary enzymes of DNA synthesis produce this neurotransmitter in cooperation. In this case, L-tyrosine is transformed to L-DOPA in TH containing neurons that is followed by L-DOPA release and uptake to AADC containing neurons with a semi-specific membrane transporter of large neutral amino acids for DA synthesis. Turning on the expression of enzymes of DA synthesis in non-dopaminergic neurons is an adaptive reaction under the functional insufficiency of DA-ergic neurons. So, hyperprolactinemia that is developed under the degeneration of DA-ergic neurons of the AN and the deficiency of DA, the prolactin-inhibiting neurohormone, was compensated in due time to increase in number of monoenzymatic neurons and the strengthening of cooperative synthesis of DA in the nucleus. The same compensatory cooperative synthesis if DA is supposed to be turned on under the degeneration of DA-ergic neurons of the nigrostriatal system that was manifested by appearance of the neurons expressing enzymes of DA synthesis in the deafferentiated striatum in rats. The expression of enzymes of DA synthesis in non-dopaminergic neurons is under the control by intercellular signals. e.g., catecholamines. Thus, numerous non-monoaminergic neurons in the brain expressing individual complementary enzymes of monoamine synthesis produce monoamines in cooperation that is a compensatory reaction to a functional insufficiency of monoaminergic neurons.