Gábor Wittmann

The Orexigenic Effect of Ghrelin Is Mediated through Central Activation of the Endogenous Cannabinoid System

Introduction Ghrelin and cannabinoids stimulate appetite, this effect possibly being mediated by the activation of hypothalamic AMP-activated protein kinase (AMPK), a key enzyme in appetite and metabolism regulation. The cannabinoid receptor type 1 (CB1) antagonist rimonabant can block the orexigenic effect of ghrelin. In this study, we have elucidated the mechanism of the putative ghrelin-cannabinoid interaction. Methods The effects of ghrelin and CB1 antagonist rimonabant in wild-type mice, and the effect of ghrelin in CB1-knockout animals, were studied on food intake, hypothalamic AMPK activity and endogenous cannabinoid content. In patch-clamp electrophysiology experiments the effect of ghrelin was assessed on the synaptic inputs in parvocellular neurons of the hypothalamic paraventricular nucleus, with or without the pre-administration of a CB1 antagonist or of cannabinoid synthesis inhibitors. Results and Conclusions Ghrelin did not induce an orexigenic effect in CB1-knockout mice. Correspondingly, both the genetic lack of CB1 and the pharmacological blockade of CB1 inhibited the effect of ghrelin on AMPK activity. Ghrelin increased the endocannabinoid content of the hypothalamus in wild-type mice and this effect was abolished by rimonabant pre-treatment, while no effect was observed in CB1-KO animals. Electrophysiology studies showed that ghrelin can inhibit the excitatory inputs on the parvocellular neurons of the paraventricular nucleus, and that this effect is abolished by administration of a CB1 antagonist or an inhibitor of the DAG lipase, the enzyme responsible for 2-AG synthesis. The effect is also lost in the presence of BAPTA, an intracellular calcium chelator, which inhibits endocannabinoid synthesis in the recorded parvocellular neuron and therefore blocks the retrograde signaling exerted by endocannabinoids. In summary, an intact cannabinoid signaling pathway is necessary for the stimulatory effects of ghrelin on AMPK activity and food intake, and for the inhibitory effect of ghrelin on paraventricular neurons.

Peripheral, but Not Central, CB1 Antagonism Provides Food Intake–Independent Metabolic Benefits in Diet-Induced Obese Rats

OBJECTIVE—Blockade of the CB1 receptor is one of the promising strategies for the treatment of obesity. Although antagonists suppress food intake and reduce body weight, the role of central versus peripheral CB1 activation on weight loss and related metabolic parameters remains to be elucidated. We therefore specifically assessed and compared the respective potential relevance of central nervous system (CNS) versus peripheral CB1 receptors in the regulation of energy homeostasis and lipid and glucose metabolism in diet-induced obese (DIO) rats. RESEARCH DESIGN AND METHODS—Both lean and DIO rats were used for our experiments. The expression of key enzymes involved in lipid metabolism was measured by real-time PCR, and euglycemic-hyperinsulinemic clamps were used for insulin sensitivity and glucose metabolism studies. RESULTS—Specific CNS-CB1 blockade decreased body weight and food intake but, independent of those effects, had no beneficial influence on peripheral lipid and glucose metabolism. Peripheral treatment with CB1 antagonist (Rimonabant) also reduced food intake and body weight but, in addition, independently triggered lipid mobilization pathways in white adipose tissue and cellular glucose uptake. Insulin sensitivity and skeletal muscle glucose uptake were enhanced, while hepatic glucose production was decreased during peripheral infusion of the CB1 antagonist. However, these effects depended on the antagonist-elicited reduction of food intake. CONCLUSIONS—Several relevant metabolic processes appear to independently benefit from peripheral blockade of CB1, while CNS-CB1 blockade alone predominantly affects food intake and body weight.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing’s syndrome

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushings syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate‐activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid‐excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroidinduced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid‐induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.— Christ‐Crain M., Kola, B., Lolli F., Fekete, C., Seboek, D., Wittmann, G., Feltrin, D., Igreja, S. C., Ajodha, S., Harvey‐White, J., Kunos, G., Müller B., Pralong, F., Aubert, G., Arnaldi, G., Giacchetti, G., Boscaro, M., Grossman, A. B., Korbonits M. AMP‐activated protein kinase mediates glucocorticoidinduced metabolic changes: a novel mechanism in Cushings syndrome. FASEB J. 22, 1672–1683 (2008)

Distribution of type 1 cannabinoid receptor (CB1)-immunoreactive axons in the mouse hypothalamus

Type 1 cannabinoid receptor (CB1) is the principal receptor for endocannabinoids in the brain; it mainly occurs in preterminal/terminal axons and mediates retrograde neuronal signaling mechanisms. A large body of physiological and electrophysiological evidence indicates the critical role of CB1 in the regulation of hypothalamic functions. Conversely, the distribution of CB1‐containing axons in the hypothalamus is essentially unknown. Therefore, we have analyzed the distribution and the ultrastructural characteristics of the CB1‐immunoreactive (IR) axons in the mouse hypothalamus by using an antiserum against the C‐terminal 31 amino acids of the mouse CB1. We found that CB1‐IR axons innervated densely the majority of hypothalamic nuclei, except for the suprachiasmatic and lateral mammillary nuclei, in which only scattered CB1‐IR fibers occurred. CB1‐IR innervation of the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the external zone of the median eminence corroborated the important role of CB1 in the regulation of energy homeostasis and neuroendocrine functions. Ultrastructural studies to characterize the phenotype of CB1‐IR fibers established that most CB1 immunoreactivity appeared in the preterminal and terminal portions of axons. The CB1‐IR boutons formed axospinous, axodendritic, and axosomatic synapses. Analysis of labeled synapses in the paraventricular and arcuate nuclei detected approximately equal numbers of symmetric and asymmetric specializations. In conclusion, the study revealed the dense and differential CB1‐IR innervation of most hypothalamic nuclei and the median eminence of the mouse brain. At the ultrastructural level, CB1‐IR axons established communication with hypothalamic neurons via symmetric and asymmetric synapses indicating the occurrence of retrograde signaling by endocannabinoids in hypothalamic neuronal networks. J. Comp. Neurol. 503:270–279, 2007.

The E3 ubiquitin ligase TEB4 mediates degradation of type 2 iodothyronine deiodinase

ABSTRACT The endoplasmic reticulum resident thyroid hormone-activating type 2 deiodinase (D2) is inactivated by ubiquitination via the hedgehog-inducible WSB-1. Ubiquitinated D2 can then be subsequently taken up by the proteasomal system or be reactivated by USP-33/20-mediated deubiquitination. Given that heterologously expressed D2 accumulates in Saccharomyces cerevisiae lacking the E3 ligase Doa10, we tested whether the human Doa10 ortholog, TEB4, plays a role in D2 ubiquitination and degradation. In a setting of transient coexpression in HEK-293 cells, TEB4 and D2 could be coimmunoprecipitated, and additional TEB4 expression decreased D2 activity by ∼50% (P < 0.05). A highly efficient TEB4 knockdown (>90% reduction in mRNA and protein levels) decreased D2 ubiquitination and increased D2 activity and protein levels by about fourfold. The other activating deiodinase, D1, or a truncated D2 molecule (Δ18-D2) that lacks a critical instability domain was not affected by TEB4 knockdown. Furthermore, TEB4 knockdown prolonged D2 activity half-life at least fourfold, even under conditions known to promote D2 ubiquitination. Neither exposure to 1 μM of the proteasomal inhibitor MG132 for 24 h nor RNA interference WSB-1 knockdown resulted in additive effects on D2 expression when combined with TEB4 knockdown. Similar results were obtained with MSTO-211 cells, which endogenously express D2, after TEB4 knockdown using a lentivirus-based transduction strategy. While TEB4 expression predominates in the hematopoietic lineage, both WSB-1 and TEB4 are coexpressed with D2 in a number of tissues and cell types, except the thyroid and brown adipose tissue, where TEB4 expression is minimal. We conclude that TEB4 interacts with and mediates loss of D2 activity, indicating that D2 ubiquitination and degradation can be tissue specific, depending on WSB-1 and TEB4 expression levels.

Inhibition of the Type 2 Iodothyronine Deiodinase Underlies the Elevated Plasma TSH Associated with Amiodarone Treatment

The widely prescribed cardiac antiarrhythmic drug amiodarone (AMIO) and its main metabolite, desethylamiodarone (DEA), have multiple side effects on thyroid economy, including an elevation in serum TSH levels. To study the AMIO effect on TSH, mice with targeted disruption of the type 2 deiodinase gene (D2KO) were treated with 80 mg/kg AMIO for 4 wk. Only wild-type (WT) mice controls developed the expected approximate twofold rise in plasma TSH, illustrating a critical role for D2 in this mechanism. A disruption in the D2 pathway caused by AMIO could interfere with the transduction of the T4 signal, generating less T3 and softening the TSH feedback mechanism. When added directly to sonicates of HEK-293 cells transiently expressing D2, both AMIO and DEA behaved as noncompetitive inhibitors of D2 [IC(50) of >100 μm and ∼5 μm, respectively]. Accordingly, D2 activity was significantly decreased in the median eminence and anterior pituitary sonicates of AMIO-treated mice. However, the underlying effect on TSH is likely to be at the pituitary gland given that in AMIO-treated mice the paraventricular TRH mRNA levels (which are negatively regulated by D2-generated T3) were decreased. In contrast, AMIO and DEA both exhibited dose-dependent inhibition of D2 activity and elevation of TSH secretion in intact TαT1 cells, a pituitary thyrotroph cell line used to model the TSH feedback mechanism. In conclusion, AMIO and DEA are noncompetitive inhibitors of D2, with DEA being much more potent, and this inhibition at the level of the pituitary gland contributes to the rise in TSH seen in patients taking AMIO.

Origin of cocaine- and amphetamine-regulated transcript (CART)-immunoreactive innervation of the hypothalamic paraventricular nucleus.

Axons containing cocaine‐ and amphetamine‐regulated transcript (CART) densely innervate the hypothalamic paraventricular nucleus (PVN). Recent data from our laboratory demonstrated that CART‐immunoreactive (IR) neurons of arcuate nucleus origin innervate the PVN, but comprise only a portion of the total CART‐IR input to this region of the brain. To identify sources other than the arcuate nucleus, retrograde transport studies were performed with cholera toxin B subunit (CTB), focally delivered into the PVN of adult rats. Neurons double‐labeled for CTB and CART were visualized by immunofluorescence. The most prominent groups of double‐labeled cells were identified in the retrochiasmatic area, arcuate nucleus, lateral hypothalamus, perifornical area, zona incerta, C1–3 regions, and the medial subnucleus of the nucleus tractus solitarius (NTS). In addition, scattered retrogradely labeled CART‐IR neurons were found in the parabrachial nucleus. In the diencephalon, the majority of double‐labeled neurons were localized ipsilateral to the injection site; however, in the medulla the CART/CTB‐containing neurons were found bilaterally. By triple‐labeling immunofluorescence, CART/CTB neurons in the perifornical area, zona incerta complex, and more medial portions of the lateral hypothalamus were found to co‐contain melanin concentrating hormone (MCH), whereas CART/CTB neurons of the C1–3 regions of the brainstem but not medial subnucleus of the NTS were observed to express phenylethanolamine N‐methyltransferase (PNMT). We conclude that the CART innervation of the PVN derives from multiple neuronal sources of the hypothalamus and medulla. These observations raise the possibility that CART serves multiple functions in the PVN and is utilized to transmit diverse physiological signals that contribute to the complex regulation of homeostatic functions of the PVN. J. Comp. Neurol. 469:340–350, 2004.

Efferent projections of thyrotropin-releasing hormone-synthesizing neurons residing in the anterior parvocellular subdivision of the hypothalamic paraventricular nucleus

The anterior parvocellular subdivision of the PVN (aPVN) contains nonhypophysiotropic thyrotropin‐releasing hormone (TRH) neurons that are densely innervated by feeding‐related neuronal groups of the hypothalamic arcuate nucleus. To determine how these TRH neurons are integrated within the brain, the major projection fields of this cell group were studied by anterograde and retrograde tract‐tracing methods. Projection sites were identified by injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) into the aPVN, and subsequent double immunofluorescent staining was used to visualize axons containing both PHAL and pro‐TRH. To distinguish between the projection sites of TRH neurons residing in the aPVN and the closely situated perifornical area, the retrograde tracer cholera toxin B subunit (CTB) was injected into regions where PHAL/pro‐TRH‐containing axons were densely accumulated. TRH neurons in the aPVN were found to project to the hypothalamic arcuate, dorsomedial and ventral premammillary nuclei, medial preoptic region, tuber cinereum area, paraventricular thalamic nucleus, bed nucleus of the stria terminalis, lateral septal nucleus, and central amygdaloid nucleus. Projection fields of perifornical TRH neurons were in partial overlap with those of the aPVN TRH cells. In addition, these neurons also innervated the hypothalamic ventromedial nucleus, the medial amygdaloid nucleus, and the amygdalohippocampal area. The data suggest that, through its efferent connections, aPVN TRH neurons may be involved in the regulation of energy homeostasis coordinately with effects on behavior, locomotor activity, and thermogenesis. In addition, the major differences in the projection fields of aPVN and perifornical TRH neurons suggest that these two TRH‐synthesizing neuronal groups are functionally different. J. Comp. Neurol. 515:313–330, 2009.

Improved method for combination of immunocytochemistry and Nissl staining

Nissl staining is a widely used method to study morphology and pathology of neural tissue. After standard immunocytochemistry, the Nissl staining labels only the nucleus of neurons and the characteristic staining of the neuronal perikarya is absent or very weak. We hypothesized that the RNA degradation during the immunocytochemical treatment results in the loss of cytoplasmic staining with Nissl-dyes. To test this hypothesis, we used RNAse-free conditions for all steps of immunostaining. To further prevent the RNA-degradation by RNAse contaminations, the RNAse inhibitor heparin was added to all antibody-containing solutions. The efficiency of Nissl staining after standard and RNAse-free double-labeling immunocytochemistry was compared using antibodies against c-Fos and neuropeptide Y (NPY) on tissues of rats refed after 3 days of fasting. After standard immunocytochemistry, the Nissl-staining labeled the nuclei of neurons and only very faintly the cytoplasm of these cells. The RNAse-free treatment did not alter the distribution of immunoreaction signal, but preserved the staining of neuronal perikarya by the Nissl-dyes. In conclusion, the RNAse-free conditions during immunocytochemistry allow the labeling of neuronal perikarya by Nissl-dyes. The described method facilitates the mapping of immunocytochemical signals and makes possible the light microscopic examination of the innervation of neurons identified by their nuclear protein content.

Distinct glutamatergic and GABAergic subsets of hypothalamic pro-opiomelanocortin neurons revealed by in situ hybridization in male rats and mice

Pro‐opiomelanocortin (POMC) and agouti‐related protein (AGRP) neurons in the hypothalamus regulate various aspects of energy homeostasis and metabolism. POMC and AGRP neurons, respectively, agonize and antagonize melanocortin receptors on their common downstream neurons. However, it is unknown whether they also reciprocally stimulate and inhibit the same neurons by amino acid transmitters. Whereas AGRP neurons are mostly GABAergic, surprisingly, only a small population of POMC neurons has been found to be glutamatergic, and a significantly larger subpopulation to be GABAergic. To further examine amino acid phenotypes of POMC neurons, we studied mRNA expression for the glutamatergic marker, type 2 vesicular glutamate transporter (VGLUT2), and the GABA synthetic enzyme, glutamic acid decarboxylase 67 (GAD67), in POMC neurons of both rats and mice by using in situ hybridization techniques. In rats, approximately 58% of POMC neurons were labeled for VGLUT2 and 37% for GAD67 mRNA. In mice, approximately 43% of POMC neurons contained VGLUT2, and 54% contained GAD67 mRNA. In both species, a prominent mediolateral distribution pattern was observed at rostral and mid levels of the POMC cell group with VGLUT2–POMC neurons dominating in lateral portions and GAD67–POMC neurons in medial portions. These data demonstrate that both glutamatergic and GABAergic cells are present in comparably significant numbers among POMC neurons. Their glutamatergic or GABAergic phenotype may represent a major functional division within the POMC cell group. J. Comp. Neurol. 521:3287–3302, 2013.