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INTRODUCTION: Liver-expressed antimicrobial peptide-2 (LEAP2) is an endogenous ghrelin receptor antagonist, which is upregulated in the fed state and downregulated during fasting. We hypothesized that the ketone body beta-hydroxybutyrate (BHB) is involved in the downregulation of LEAP2 during conditions with high circulating levels of BHB.METHODS: Hepatic and intestinal Leap2 expression were determined in 3 groups of mice with increasing circulating levels of BHB: prolonged fasting, prolonged ketogenic diet, and oral BHB treatment. LEAP2 levels were measured in lean and obese individuals, in human individuals following endurance exercise, and in mice after BHB treatment. Lastly, we investigated Leap2 expression in isolated murine hepatocytes challenged with BHB.RESULTS: We confirmed increased circulating LEAP2 levels in individuals with obesity compared to lean individuals. The recovery period after endurance exercise was associated with increased plasma levels of BHB levels and decreased LEAP2 levels in humans. Leap2 expression was selectively decreased in the liver after fasting and after exposure to a ketogenic diet for 3 weeks. Importantly, we found that oral administration of BHB increased circulating levels of BHB in mice and decreased Leap2 expression levels and circulating LEAP2 plasma levels, as did Leap2 expression after direct exposure to BHB in isolated murine hepatocytes.CONCLUSION: From our data, we suggest that LEAP2 is downregulated during different states of energy deprivation in both humans and rodents. Furthermore, we here provide evidence that the ketone body, BHB, which is highly upregulated during fasting metabolism, directly downregulates LEAP2 levels. This may be relevant in ghrelin receptor-induced hunger signaling during energy deprivation.

Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross-linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane-bound protein that binds to multiple nAChR subtypes in the human brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine-induced hippocampal inward currents in rat brain slices and decreases nicotine-induced extracellular signal-regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit nAChR-mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post-natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx protein Lypd6 binds to nAChRs in human brain extracts, and that recombinant Lypd6 decreases nicotine-induced ERK phosphorylation and attenuates nicotine-induced hippocampal inward currents. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain.

Narcolepsy is a lifelong sleep disorder characterized by excessive daytime sleepiness, sudden loss of muscle tone (cataplexy), fragmentation of nocturnal sleep and sleep paralysis. The symptoms of the disease strongly correlate with a reduction in hypocretin levels in CSF and a reduction in hypocretin neurons in hypothalamus in post-mortem tissue. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are important for activity-dependent neuronal function and synaptic modulation and it is considered that these mechanisms are important in sleep regulation. We hypothesised that serum levels of these factors are altered in patients with narcolepsy compared to healthy controls without sleep disturbances. Polysomnography data was obtained and serum BDNF and NGF levels measured using ELISA, while hypocretin was measured using RIA. Serum BDNF levels were significantly higher in narcolepsy patients than in healthy controls (64.2±3.9ng/ml vs 47.3±2.6ng/ml, P

Prefrontal serotonin 2A receptors (5-HT2A Rs) have been linked to the pathogenesis and treatment of schizophrenia. Many antipsychotics fully occupy 5-HT2A R at clinical relevant doses, and activation of 5-HT2A receptors by lysergic acid diethylamide (LSD) and LSD-like drugs induces a schizophrenia-like psychosis in humans. Subchronic phencyclidine (PCP) administration is a well-established model for schizophrenia-like symptoms in rodents. The aim of the present study was to investigate whether subchronic PCP administration changes expression, binding, or functionality of cortical 5-HT2A Rs. As a measure of 5-HT2A R functionality, we used the 5-HT2A R agonist 2,5-dimethoxy-4-iodoamphetamine (DOI)-induced head-twitch response (HTR) and mRNA expression of the immediate-early genes (IEGs) activity-related cytoskeletal associated-protein (Arc), c-fos, and early growth response protein 2 (egr-2) in the frontal cortex. Mice were treated with PCP (10 mg/kg) or saline for 10 days, followed by a 5-day washout period. The PCP pretreatment increased the overall induction of HTR and frontal cortex IEG mRNA expression following a single challenge with DOI. These functional changes were not associated with changes in 5-HT2A R binding. Also, binding of the 5-HT1A R and the 5-HT transporter was unaffected. Finally, basal mRNA level of Arc was increased in the prefrontal cortex after subchronic PCP administration as revealed with in situ hybridization. Together these findings indicate that PCP administration produces changes in the brain that result in an increase in the absolute effect of DOI. Therefore, neurotransmission involving the 5-HT2A R could contribute to the behavioral deficits observed after PCP treatment. © 2013 Wiley Periodicals, Inc.

The recently identified G protein-coupled receptor GPRC6A is activated by dietary amino acids and expressed in multiple tissues. Although the receptor is hypothesised to exert biological impact on metabolic and endocrine-related parameters, the role of the receptor in obesity and metabolic complications is still elusive. In the present study, we investigated the impact of GPRC6A deficiency in a murine model of diet-induced obesity (DIO). Male Gprc6a knockout (KO) mice and WT littermates were subjected to a high-fat diet (HFD) for 25 weeks and exposed to comprehensive metabolic phenotyping. A significant increase in body weight, corresponding to a selective increase in body fat, was observed in Gprc6a KO mice exposed to an HFD relative to WT controls. The obese phenotype was linked to subtle perturbations in energy homoeostasis as GPRC6A deficiency resulted in chronic hyperphagia and decreased locomotor activity. Moreover, diet-induced obese Gprc6a KO mice had increased circulating insulin and leptin levels relative to WT animals, thereby demonstrating that endocrine abnormalities associate with the reported disturbances in energy balance. The phenotype was further accompanied by disruptions in glucose metabolism showing that Gprc6a KO mice on an HFD display increased susceptibility to develop metabolic-related disorders. Altogether, these data suggest that the amino acid sensing receptor GPRC6A plays an important role in resistance to DIO and metabolic complications. Future studies will illuminate the underlying molecular mechanisms mediating the herein reported findings and potentially facilitate the development of novel therapeutic compounds targeting the GPRC6A receptor.

The beneficial effect of exercise on hippocampal plasticity is possibly mediated by increased angiogenesis and neurogenesis. In angiogenesis insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor 1, alpha subunit (HIF1a) are important factors, while the induction of neurogenesis requires signaling through the VEGF receptor, Flk-1 (VEGFR-2). VEGF expression is believed to be regulated by two distinct mTOR (mammalian Target of Rapamycin)-containing multiprotein complexes mTORC1 and mTORC2, respectively. This study was initiated to investigate the effect of exercise on the expression of VEGF, cognate receptors, HIF1a, mTORC1, and mTORC2 in hippocampus and frontal cortex. To this end, we measured messenger RNA (mRNA) levels in rat brain using quantitative real-time polymerase chain reaction (real-time qPCR) after forced treadmill exercise for 1 day, 2 weeks, and 8 weeks. Rats were euthanized either immediately (0 hrs) or 24 hours after last exercise session. Here, we show that exercise affected mRNA levels of VEGF, VEGFR2, and the co-receptor neuropilin 2 (NRP2) when the rats were euthanized immediately, whereas at 24 hours only the expression of mTOR was regulated after a single bout of exercise. In conclusion, the effect of treadmill exercise on the VEGF system is acute rather than chronic and there is a transient activation of mTOR. More studies are needed to understand whether this could be beneficial in the treatment of neuropsychiatric disorders. Synapse, 2013. © 2013 Wiley Periodicals, Inc.

GPRC6A is an amino acid-sensing receptor highly expressed in the brain and in skeletal muscle. Although recent evidence suggests that genetically engineered GPRC6A receptor knockout (KO) mice are susceptible to develop subtle endocrine and metabolic disturbances, the underlying disruptions in energy metabolism are largely unexplored. Based on GPRC6A's expression pattern and ligand preferences, we hypothesize that the receptor may impact energy metabolism via regulating physical activity levels. Thus, in the present study, we exposed GPRC6A receptor KO mice and their wild-type (WT) littermates to voluntary wheel running and forced treadmill exercise. Moreover, we assessed energy expenditure in the basal state, and evaluated the effects of wheel running on food intake, body composition, and a range of exercise-induced central and peripheral biomarkers. We found that adaptation to voluntary wheel running is affected by GPRC6A, as ablation of the receptor significantly enhances wheel running in KO relative to WT mice. Both genotypes responded to voluntary exercise by increasing food intake and improving body composition to a similar degree. In conclusion, these data demonstrate that the GPRC6A receptor is involved in regulating exercise behaviour. Future studies are highly warranted to delineate the underlying molecular details and to assess if these findings hold any translational value.

Compelling evidence has shown, that neurotrophins responsible for the regulation of neuronal growth, survival, and differentiation are involved in neurodegenerative diseases. Whereas lower serum levels of brain derived neurotrophic factor (BDNF) have been observed in patients with Parkinson's disease, no studies have directly related the degree of striatal neurodegeneration of dopaminergic neurons (DA) with serum BDNF levels. In this study we examined the relationship between striatal neurodegeneration as determined with (123)I-PE2I-single photon emission computer tomography (SPECT) and serum BDNF levels in patients with parkinsonism. Twenty-one patients with abnormal in vivo striatal dopamine transporter (DAT) binding as evidenced with [(123)I]PE2I SPECT brain scanning were included. Samples for serum BDNF levels were collected at the time of the SPECT scanning, and BDNF was measured with enzyme-linked immunosorbent assay (ELISA). The striatal binding potential of non-displaceable [(123)I]PE2I was calculated. We found a positive correlation between serum BDNF levels and striatal DAT availability (p <0.01, R(2) = 0.36). We find that in patients with striatal dopaminergic neurodegeneration serum BDNF levels decrease along with loss in striatal DAT binding.

Brain-derived neurotrophic factor (BDNF) is involved in synaptic plasticity, neuronal differentiation and survival of neurons. Observations of decreased serum BDNF levels in patients with neuropsychiatric disorders have highlighted the potential of BDNF as a biomarker, but so far there have been no studies directly comparing blood BDNF levels to brain BDNF levels in different species. We examined blood, serum, plasma and brain-tissue BDNF levels in three different mammalian species: rat, pig, and mouse, using an ELISA method. As a control, we included an analysis of blood and brain tissue from conditional BDNF knockout mice and their wild-type littermates. Whereas BDNF could readily be measured in rat blood, plasma and brain tissue, it was undetectable in mouse blood. In pigs, whole-blood levels of BDNF could not be measured with a commercially available ELISA kit, but pig plasma BDNF levels (mean 994±186 pg/ml) were comparable to previously reported values in humans. We demonstrated positive correlations between whole-blood BDNF levels and hippocampal BDNF levels in rats (r2=0.44, p=0.025) and between plasma BDNF and hippocampal BDNF in pigs (r2=0.41, p=0.025). Moreover, we found a significant positive correlation between frontal cortex and hippocampal BDNF levels in mice (r2=0.81, p=0.0139). Our data support the view that measures of blood and plasma BDNF levels reflect brain-tissue BDNF levels.

Intrahippocampal injections of aggregated amyloid-beta (Abeta)1-42 in rats result in memory impairment and in reduction of hippocampal 5-HT2A receptor levels. In order to investigate how changes in 5-HT2A levels and functionality relate to the progressive accumulation of Abeta protein, we studied 5-HT2A receptor regulation in double transgenic AbetaPPswe/PS1dE9 mice which display excess production of Abeta and age-dependent increase in amyloid plaques. Three different age-groups, 4-month-old, 8- month-old, and 11-month-old were included in the study. [3H]-MDL100907, [3H]-escitalopram, and [11C]-PIB autoradiography was performed for measuring 5-HT2A receptor, serotonin transporter (SERT), and Abeta plaque levels in medial prefrontal cortex (mPFC), prefrontal cortex (PFC), frontoparietal cortex (FPC), dorsal and ventral hippocampus, and somatosensory cortex. To investigate 5-HT2A receptor functionality, animals were treated with the 5-HT2A receptor agonist DOI and head-twitch response (HTR) subsequently recorded. Expression level of the immediate early gene c-fos was measured by in situ hybridization. We found that the age-related increase in Abeta plaque burden was accompanied by a significant decrease in 5-HT2A receptor binding in mPFC in the 11-month-old group. The changes in 5-HT2A receptor binding correlated negatively with [11C]-PIB binding and were not accompanied by decreases in SERT binding. Correspondingly, 11-month-old transgenic mice showed diminished DOI-induced HTR and reduced increase in expression of c-fos mRNA in mPFC and FPC. These observations point towards a direct association between Abeta accumulation and changes in 5-HT2A receptor expression that is independent of upstream changes in the serotonergic system.

Epidemiological studies have revealed a strong genetic contribution to the risk for depression. Both reduced hippocampal serotonin neurotransmission and brain-derived neurotrophic factor (BDNF) levels have been associated with increased depression vulnerability and are also regulated during aging. Brains from young (5 months old) and old (13 months old) congenital Learned Helplessness rats (cLH), and congenital Non Learned Helplessness rats (cNLH) were immunohistochemically stained for the serotonin transporter and subsequently stereologically quantified for estimating hippocampal serotonin fiber density. Hippocampal BDNF protein levels were measured by ELISA. An exacerbated age-related loss of serotonin fiber density specific for the CA1 area was observed in the cLH animals, whereas reduced hippocampal BDNF levels were seen in young and old cLH when compared with age-matched cNLH controls. These observations indicate that aging should be taken into account when studying the neurobiological factors behind the vulnerability for depression and that understanding the effect of aging on genetically predisposed individuals may contribute to a better understanding of the pathophysiology behind depression, particularly in the elderly.