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Aims To analyse the histological structure and histomorphometric characteristics of human hard palatal mucosa in order to determine the donor site of choice for connective tissue grafts from a histological point of view. Materials and Methods Palatal mucosa samples from six cadaver heads were harvested at four sites: incisal, premolar, molar and tuberosity. Histological and immunohistochemical techniques were performed, as was histomorphometric analysis. Results In the current study, we found that the density and size of cells were higher in the superficial papillary layer, whereas the thickness of the collagen bundles increased in the reticular layer. Excluding the epithelium, the mean percentage of lamina propria (LP) and submucosa (SM) was 37% and 63%, respectively (p

There are contradictory data regarding the correlation between HER2 amplification level determined by in situ hybridization and evolution after treatment with anti-HER2 therapies. The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEP17 and number of HER2 signals/nucleus) with pathological response achieved after neoadjuvant treatment with trastuzumab and chemotherapy. For this purpose, we analysed 100 consecutive HER2-positive cases of breast carcinoma treated with neoadjuvant therapy. HER2 amplification determined by FISH was found in 92 of the 100 cases studied. pCR was obtained in 58% of the patients whose tumours presented amplification. In contrast, no pCR was obtained in the 8 patients with non-amplified tumours. A significant direct correlation between HER2 high amplification (HER2/CEP17 ratio > 5 or HER2 signals/nucleus > 10) and pCR was found. In conclusion, HER2 amplification levels are clinically relevant because they provide oncologists with valuable information on the possibilities of achieving pCR after neoadjuvant treatment.

Immunohistological patterns of density and distribution of neural tissue in the human penis, including the prepuce, are not fully characterized, and effects of circumcision (partial or total removal of the penile prepuce) on penile sexual sensation are controversial. This study analyzed extra- and intracavernosal innervation patterns on the main penile axes using formalin-fixed, paraffin-embedded human adult and fetal penile tissues, single- and double-staining immunohistochemistry and a variety of neural and non-neural markers, with a special emphasis on the prepuce and potential sexual effects of circumcision. Immunohistochemical profiles of neural structures were determined and the most detailed immunohistological characterizations to date of preputial nerve supply are provided. The penile prepuce has a highly organized, dense, afferent innervation pattern that is manifest early in fetal development. Autonomically, it receives noradrenergic sympathetic and nitrergic parasympathetic innervation. Cholinergic nerves are also present. We observed cutaneous and subcutaneous neural density distribution biases across our specimens towards the ventral prepuce, including a region corresponding in the adult anatomical position (penis erect) to the distal third of the ventral penile aspect. We also describe a concept of innervation gradients across the longitudinal and transverse penile axes. Results are discussed in relation to the specialized literature. An argument is made that neuroanatomic substrates underlying unusual permanent penile sensory disturbances post-circumcision are related to heightened neural levels in the distal third of the ventral penile aspect, which could potentially be compromised by deep incisions during circumcision.

Objective: To investigate whether thermogenesis and the hypothalamus may be involved in the physiopathology of experimental arthritis (EA). Methods: EA was induced in male Lewis rats by intradermal injection of Freund's complete adjuvant (CFA). Food intake, body weight, plasma cytokines, thermographic analysis, gene and protein expression of thermogenic markers in brown adipose tissue (BAT) and white adipose tissue (WAT), and hypothalamic AMP-activated protein kinase (AMPK) were analyzed. Virogenetic activation of hypothalamic AMPK was performed. Results: We first demonstrated that EA was associated with increased BAT thermogenesis and browning of subcutaneous WAT leading to elevated energy expenditure. Moreover, rats experiencing EA showed inhibition of hypothalamic AMPK, a canonical energy sensor modulating energy homeostasis at the central level. Notably, specific genetic activation of AMPK in the ventromedial nucleus of the hypothalamus (a key site modulating energy metabolism) reversed the effect of EA on energy balance, brown fat, and browning, as well as promoting amelioration of synovial inflammation in experimental arthritis. Conclusion: Overall, these data indicate that EA promotes a central catabolic state that can be targeted and reversed by the activation of hypothalamic AMPK. This might provide new therapeutic alternatives to treat rheumatoid arthritis (RA)–associated metabolic comorbidities, improving the overall prognosis in patients with RA The Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS) is supported by the Xunta de Galicia (2016-2019, ED431G/05). The Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición (CIBERobn) is an initiative of the Instituto de Salud Carlos III. Supported by the Xunta de Galicia (grants 2016-PG057 to Dr. Nogueiras, GPC IN607B2019/10 to Dr. Gualillo, and 2016-PG068 to Dr. López), the Ministerio de Economía y Competitividad (MINECO) co-funded by the FEDER Program of the European Union (grants BFU2017-90578-REDT/Adipoplast to Drs. Fernández-Real and López, RTI2018-099413-B-I00 to Dr. Nogueiras, BFU2017-87721-P to Dr. Diéguez, and RTI2018-101840-B-I00 to Dr. López), the Instituto de Salud Carlos III (grants PI15–01934 and PI18/0102224 to Dr. Fernández-Real and PI17/00409, PI20/00902, RD21/0002/0025, and RD16/0012/0014 to Dr. Gualillo), “la Caixa” Foundation (ID 100010434) (grant LCF/PR/HR19/52160022 to Dr. López), and the European Research Council (synergy grant-2019-WATCH- 810331 to Dr. Nogueiras). Dr. Seoane-Collazo's work was supported by the Xunta de Galicia (fellowship ED481B 2018/050) and the Horizon 2020 Research and Innovation Program of the European Union under the Marie Sklodowska-Curie actions. Drs. Nogueiras and López's work was supported in part by the Atresmedia Corporación. Dr. Nogueiras's work was supported in part by Fundación BBVA and the European Foundation for the Study of Diabetes. Dr. Gualillo's work was supported in part by the Horizon 2020 Research and Innovation Program of the European Union under the Marie Sklodowska-Curie actions (project no. 734899) and by the Xunta de Galicia through a research staff contract (ISCIII/SERGAS) SI

Myostatin is a member of the transforming growth factor β (TGFβ) superfamily that has a well-established role as a mediator of muscle growth and development. However, myostatin is now emerging as a pleiotropic hormone with multiple actions in the regulation of the metabolism as well as several aspects of both cardiac and smooth muscle cells physiology. In addition, myostatin is also expressed in several nonmuscular cells where its physiological role remains to be elucidated in most cases. In this report, we have shown that both myostatin and its receptor system are expressed in blood cells and in hematopoietic cell lines. Furthermore, myostatin treatment promotes differentiation of both HL60 and K562 cells through a mechanism that involves activation of extracellular signal-regulated kinases 1/2 and p38-mitogen-activated protein kinase, thus leading to the possibility that myostatin may be a paracrine/autocrine factor involved in the control of haematopoiesis. In addition, the presence of myostatin expression in immune cells could envisage a novel role for the hormone in the pathogenesis of inflammatory diseases.

BACKGROUND This study was performed to test the therapeutic potential of obestatin, an autocrine anabolic factor regulating skeletal muscle repair, to ameliorate the Duchenne muscular dystrophy (DMD) phenotype. METHODS AND RESULTS Using a multidisciplinary approach, we characterized the ageing-related preproghrelin/GPR39 expression patterns in tibialis anterior (TA) muscles of 4-, 8-, and 18-week-old mdx mice (n = 3/group) and established the effects of obestatin administration at this level in 8-week-old mdx mice (n = 5/group). The findings were extended to in vitro effects on human immortalized DMD myotubes. An analysis of TAs revealed an age-related loss of preproghrelin expression, as precursor of obestatin, in mdx mice. Administration of obestatin resulted in a significant increase in tetanic specific force (33.0% ± 1.5%, P

Studies about head trauma are experimental or have a clinical or prognosis purpose. In this study, we used samples from human autopsies to answer common medical-legal questions.We studied 21 problem cases and 4 controls. Samples were obtained directly from the injured area, fixed in 10% formalin during 24 hours and then preserved in 70% ethanol. This procedure optimizes the immunohistochemical technique.The neurofilament antibody shows beaded axons since the first moment; over time, they increase their density and diameter as survival time also increases. These changes begin in the gray matter, 2 hours after trauma can be seen around vessels and in hemorrhagic areas. At 24 hours, beaded axons appear in the white mater, which finally loses its structure and cellular density.On the other hand, the β-amyloid precursor protein marker begins to be weakly seen 2 hours after injury. At 24 hours, a diffuse pattern can appear, suggesting primary traumatic injury. The marker reading keeps increasing until day 26, when a "Z" pattern appears in the white matter, suggesting secondary hypoxic injury.All these chronologic changes could be useful to approach the date of trauma. They let us to distinguish between long surviving cases from those whose death was immediate (within the first 30 minutes).

The coupling of energy homeostasis to thermoregulation is essential to maintain homeothermy in changing external environments. We studied the role of the cold thermoreceptor TRPM8 in this interplay in mice of both sexes. We demonstrate that TRPM8 is required for a precise thermoregulation in response to cold, in fed and fasting. Trpm8−/− mice exhibited a fall of 0.7°C in core body temperature when housed at cold temperatures, and a deep hypothermia (, This work was supported by Project SAF2009-11175 and PI12/0058 to R.S., SAF2010-14990-R and SAF2016-77233-R to F.V., (Ministry of Economy and Competitiveness). European Regional Development Fund, and Severo Ochoa Programme for Centres of Excellence in R&D SEV-2013-0317. C.F.-P. and P.O. held predoctoral fellowships of the Generalitat Valenciana (GRISOLIA/2008/025) and MINECO (BES-2011-047063), respectively. J.L.P.-V. received Human Frontier Science Program Long-Term Award LT000442/2012.

Objective Recent reports have implicated the p53 tumor suppressor in the regulation of lipid metabolism. We hypothesized that the pharmacological activation of p53 with low-dose doxorubicin, which is widely used to treat several types of cancer, may have beneficial effects on nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Methods We used long-term pharmacological activation of p53 by i.p. or oral administration of low-dose doxorubicin in different animal models of NAFLD (high fat diet containing 45% and 60% kcal fat) and NASH (methionine- and choline-deficient diet and choline deficiency combined with high fat diet). We also administered doxorubicin in mice lacking p53 in the liver and in two human hepatic cells lines (HepG2 and THLE2). Results The attenuation of liver damage was accompanied by the stimulation of fatty acid oxidation and decrease of lipogenesis, inflammation, and ER stress. The effects of doxorubicin were abrogated in mice with liver-specific ablation of p53. Finally, the effects of doxorubicin on lipid metabolism found in animal models were also present in two human hepatic cells lines, in which the drug stimulated fatty acid oxidation and inhibited de novo lipogenesis at doses that did not cause changes in apoptosis or cell viability. Conclusion These data provide new evidence for targeting p53 as a strategy to treat liver disease., Highlights • Intraperitoneal and oral low-dose doxorubicin ameliorates NAFLD and NASH in animal models. • Doxorubicin requires p53 for its hepatic actions. • Doxorubin decreases lipid content in human hepatocytes without affecting cell viability and apoptosis.

Breast cancer is a heterogeneous disease. Surrogate classification of intrinsic subtypes of invasive carcinomas by combined immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 (4-IHC) has increased steadily since the 2011 St Gallen symposium, due to its rapid subtyping of tumors at a reasonable cost. An important step in improving 4-IHC reproducibility and reliability will be to provide reference values from the routine use of standardized 4-IHC followed by image analysis. The aims of the current study were (1) to analyze invasive breast carcinomas using standardized 4-IHC and quantitative image analysis and (2) to compare the results obtained in the classification of biological subtypes using current Ki67 and PR threshold values proposed by different authors to sub-classifying the luminal A-like and the luminal B-like (HER2-negative) subtypes. Five hundred twenty-one tumors were analyzed by standardized immunohistochemistry, with automatic image analysis, and HER2 FISH technique. Positivity for ER was found in 82.7% and for PR in 70.1% of cases. Using the Allred scoring system, hormone receptor results showed a bimodal distribution, particularly for ER. HER2 positivity was found in 15.7% of cases, and the mean Ki67 score was 32.3%. Using the most recently proposed surrogate definitions for the classification of luminal breast cancer subtypes, the percentages of different subtypes that we found were similar to those published with genomic platforms: 40.7% luminal A-like, 32.4% luminal B-like/HER2-negative, 9.8% luminal B-like/HER2-positive, 6.0% HER2-positive, and 11.1% triple negative. Standardized 4-IHC with automatic image analysis constitutes a low-cost method for surrogate definitions of biological subtypes of breast cancer that delivers accurate results in a day.

International audience; Dopamine signaling is a crucial part of the brain reward system and can affect feeding behavior. Dopamine receptors are also expressed in the hypothalamus, which is known to control energy metabolism in peripheral tissues. Here we show that pharmacological or chemogenetic stimulation of dopamine receptor 2 (D2R) expressing cells in the lateral hypothalamic area (LHA) and the zona incerta (ZI) decreases body weight and stimulates brown fat activity in rodents in a feeding-independent manner. LHA/ZI D2R stimulation requires an intact sympathetic nervous system and orexin system to exert its action and involves inhibition of PI3K in the LHA/ZI. We further demonstrate that, as early as 3 months after onset of treatment, patients treated with the D2R agonist cabergoline experience an increase in energy expenditure that persists for one year, leading to total body weight and fat loss through a prolactin-independent mechanism. Our results may provide a mechanistic explanation for how clinically used D2R agonists act in the CNS to regulate energy balance.

International audience; Melanin-concentrating hormone (MCH) is an important regulator of food intake, glucose metabolism, and adiposity. However, the mechanisms mediating these actions remain largely unknown. We used pharmacological and genetic approaches to show that the sirtuin 1 (SIRT1)/FoxO1 signaling pathway in the hypothalamic arcuate nucleus (ARC) mediates MCH-induced feeding, adiposity, and glucose intolerance. MCH reduces proopiomelanocortin (POMC) neuronal activity, and the SIRT1/FoxO1 pathway regulates the inhibitory effect of MCH on POMC expression. Remarkably, the metabolic actions of MCH are compromised in mice lacking SIRT1 specifically in POMC neurons. Of note, the actions of MCH are independent of agouti-related peptide (AgRP) neurons because inhibition of γ-aminobutyric acid receptor in the ARC did not prevent the orexigenic action of MCH, and the hypophagic effect of MCH silencing was maintained after chemogenetic stimulation of AgRP neurons. Central SIRT1 is required for MCH-induced weight gain through its actions on the sympathetic nervous system. The central MCH knockdown causes hypophagia and weight loss in diet-induced obese wild-type mice; however, these effects were abolished in mice overexpressing SIRT1 fed a high-fat diet. These data reveal the neuronal basis for the effects of MCH on food intake, body weight, and glucose metabolism and highlight the relevance of SIRT1/FoxO1 pathway in obesity.

Obestatin/GPR39 signaling stimulates skeletal muscle growth and repair by inducing both G-protein-dependent and -independent mechanisms linking the activated GPR39 receptor with distinct sets of accessory and effector proteins. In this work, we describe a new level of activity where obestatin signaling plays a role in the formation, contractile properties and metabolic profile of skeletal muscle through determination of oxidative fiber type. Our data indicate that obestatin regulates Mef2 activity and PGC-1α expression. Both mechanisms result in a shift in muscle metabolism and function. The increase in Mef2 and PGC-1α signaling activates oxidative capacity, whereas Akt/mTOR signaling positively regulates myofiber growth. Taken together, these data indicate that the obestatin signaling acts on muscle fiber-type program in skeletal muscle.

The opioid system is widely known to modulate the brain reward system and thus affect the behavior of humans and other animals, including feeding. We hypothesized that the hypothalamic opioid system might also control energy metabolism in peripheral tissues. Mice lacking the kappa opioid receptor (κOR) and adenoviral vectors overexpressing or silencing κOR were stereotaxically delivered in the lateral hypothalamic area (LHA) of rats. Vagal denervation was performed to assess its effect on liver metabolism. Endoplasmic reticulum (ER) stress was inhibited by pharmacological (tauroursodeoxycholic acid) and genetic (overexpression of the chaperone glucose-regulated protein 78 kDa) approaches. The peripheral effects on lipid metabolism were assessed by histological techniques and western blot. We show that in the LHA κOR directly controls hepatic lipid metabolism through the parasympathetic nervous system, independent of changes in food intake and body weight. κOR colocalizes with melanin concentrating hormone receptor 1 (MCH-R1) in the LHA, and genetic disruption of κOR reduced melanin concentrating hormone–induced liver steatosis. The functional relevance of these findings was given by the fact that silencing of κOR in the LHA attenuated both methionine choline–deficient, diet-induced and choline-deficient, high-fat diet–induced ER stress, inflammation, steatohepatitis, and fibrosis, whereas overexpression of κOR in this area promoted liver steatosis. Overexpression of glucose-regulated protein 78 kDa in the liver abolished hypothalamic κOR-induced steatosis by reducing hepatic ER stress. Conclusions: This study reveals a novel hypothalamic–parasympathetic circuit modulating hepatic function through inflammation and ER stress independent of changes in food intake or body weight; these findings might have implications for the clinical use of opioid receptor antagonists. (Hepatology 2016;64:1086-1104)

// Begona O. Alen 1, 2 , Saul Leal-Lopez 1, 2 , Maria Otero Alen 3, 4 , Patricia Viano 3, 4 , Victoria Garcia-Castro 4 , Carlos S. Mosteiro 1, 2 , Andres Beiras 3, 4, 5 , Felipe F. Casanueva 1, 2, 6 , Rosalia Gallego 2, 3, 5 , Tomas Garcia-Caballero 3, 4, 5 , Jesus P. Camina 1, 2 , Yolanda Pazos 1, 2 1 Area de Endocrinologia Molecular y Celular, Instituto de Investigacion Sanitaria de Santiago (IDIS), Complejo Hospitalario Universitario de Santiago (CHUS), Servicio Gallego de Salud (SERGAS), Santiago de Compostela, Spain 2 CIBER Fisiopatologia de la Obesidad y Nutricion, Santiago de Compostela, Spain 3 IDIS, CHUS, Santiago de Compostela, Spain 4 Servicio de Anatomia Patologica, CHUS, SERGAS, Santiago de Compostela, Spain 5 Departamento de Ciencias Morfologicas, Universidad de Santiago de Compostela (USC), Santiago de Compostela, Spain 6 Departamento de Medicina, USC, Santiago de Compostela, Spain Correspondence to: Yolanda Pazos, e-mail: yolanda.pazos.randulfe@sergas.es Keywords: GPR39, obestatin, stomach, adenocarcinoma Received: July 29, 2015 Accepted: November 25, 2015 Published: December 22, 2015 ABSTRACT Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.

e12641 Background: HER2 targeted therapy in combination with chemotherapy have changed the aggressive natural history of HER2-positive tumors. To select patients to be treated with this type of agents, determination of HER2 status is usually carried out by immunohistochemistry, followed by in situ hybridization in equivocal cases (2+). There are contradictory data in literature regarding correlation between level of HER2 amplification determined by FISH and early and long-term treatment benefits. Objectives: The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEN17 and number of HER2 signals/nucleus) with the grade of response to neoadjuvant treatment with trastuzumab and chemotherapy. Methods: We analyzed 100 consecutive cases of stage I-III breast carcinomas treated with trastuzumab and chemotherapy in the neoadjuvant setting at A Coruña University Hospital between 2005 and 2016. At Santiago University Hospital, 4 tissue microarrays were prepared and the IQFISH (Dako-Agilent) technique was performed using pretreatment biopsies. Results: HER2 amplification was found in 92 of 100 cases studied. Pathological Complete Response (pCR) (Miller-Payne grading system) was obtained in 58% of the patients whose tumors showed amplification. No pCR was obtained in the 8 patients whose tumors were negative by FISH (not amplified). Analysis of the quantitative results demonstrated a statistical significant direct correlation between pCR and both HER2/CEN17 ratios and HER2 gene copies/nucleus (p = 0.002 and p= 0.004, respectively). We also demonstrated an association between the HER2 amplification level (both ratios and numbers of HER2 signals) and a trend toward improved disease free survival (DFS) (p = 0.451 and p= 0.619, respectively). Conclusions: HER2 amplification level determined by FISH it is a good and available predictive factor of response and must be included in pathologic reports because it can provide valuable information to oncologists on the possibilities of achieving pCR after neoadjuvant treatment in HER2-positive breast cancer. Key words: Breast cancer, FISH, HER2 amplification, trastuzumab, neoadjuvant therapy, pathological complete response.

p53 is a well-known tumor suppressor that has emerged as an important player in energy balance. However, its metabolic role in the hypothalamus remains unknown. Herein, we show that mice lacking p53 in agouti-related peptide (AgRP), but not proopiomelanocortin (POMC) or steroidogenic factor-1 (SF1) neurons, are more prone to develop diet-induced obesity and show reduced brown adipose tissue (BAT) thermogenic activity. AgRP-specific ablation of p53 resulted in increased hypothalamic c-Jun N-terminal kinase (JNK) activity before the mice developed obesity, and central inhibition of JNK reversed the obese phenotype of these mice. The overexpression of p53 in the ARC or specifically in AgRP neurons of obese mice decreased body weight and stimulated BAT thermogenesis, resulting in body weight loss. Finally, p53 in AgRP neurons regulates the ghrelin-induced food intake and body weight. Overall, our findings provide evidence that p53 in AgRP neurons is required for normal adaptations against diet-induced obesity., Emerging studies suggest that p53 is an important regulator of energy metabolism, yet there is little known about the metabolic function of this tumor suppressor in the hypothalamus. Here, authors illustrate that p53, specifically in AgRP neurons, is required for adaptation to diet-induced obesity.

Background This study was performed to test the therapeutic potential of obestatin, an autocrine anabolic factor regulating skeletal muscle repair, to ameliorate the Duchenne muscular dystrophy (DMD) phenotype. Methods and results Using a multidisciplinary approach, we characterized the ageing‐related preproghrelin/GPR39 expression patterns in tibialis anterior (TA) muscles of 4‐, 8‐, and 18‐week‐old mdx mice (n = 3/group) and established the effects of obestatin administration at this level in 8‐week‐old mdx mice (n = 5/group). The findings were extended to in vitro effects on human immortalized DMD myotubes. An analysis of TAs revealed an age‐related loss of preproghrelin expression, as precursor of obestatin, in mdx mice. Administration of obestatin resulted in a significant increase in tetanic specific force (33.0% ± 1.5%, P

Objective Glucagon receptor antagonists and humanized glucagon antibodies are currently studied as promising therapies for obesity and type II diabetes. Among its variety of actions, glucagon reduces food intake, but the molecular mechanisms mediating this effect as well as glucagon resistance are totally unknown. Methods Glucagon and adenoviral vectors were administered in specific hypothalamic nuclei of lean and diet-induced obese rats. The expression of neuropeptides controlling food intake was performed by in situ hybridization. The regulation of factors of the glucagon signaling pathway was assessed by western blot. Results The central injection of glucagon decreased feeding through a hypothalamic pathway involving protein kinase A (PKA)/Ca2+-calmodulin-dependent protein kinase kinase β (CaMKKβ)/AMP-activated protein kinase (AMPK)-dependent mechanism. More specifically, the central injection of glucagon increases PKA activity and reduces protein levels of CaMKKβ and its downstream target phosphorylated AMPK in the hypothalamic arcuate nucleus (ARC). Consistently, central glucagon significantly decreased AgRP expression. Inhibition of PKA and genetic activation of AMPK in the ARC blocked glucagon-induced anorexia in lean rats. Genetic down-regulation of glucagon receptors in the ARC stimulates fasting-induced hyperphagia. Although glucagon was unable to decrease food intake in DIO rats, glucagon sensitivity was restored after inactivation of CaMKKβ, specifically in the ARC. Thus, glucagon decreases food intake acutely via PKA/CaMKKβ/AMPK dependent pathways in the ARC, and CaMKKβ mediates its obesity-induced hypothalamic resistance. Conclusions This work reveals the molecular underpinnings by which glucagon controls feeding that may lead to a better understanding of disease states linked to anorexia and cachexia., Highlights • Glucagon stimulates PKA and inhibits CaMKKβ and AMPK in the arcuate nucleus (ARC). • Down-regulation of glucagon receptor in the ARC increases fasting-induced hyperphagia. • Glucagon is unable to decrease food intake in diet-induced obese (DIO) rats. • In DIO rats, glucagon fails to alter CaMKKβ and its downstream targets AMPK and pACC. • Down-regulation of CaMKKβ in the ARC restores glucagon sensitivity in obese rodents.

Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins β-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, β-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for β-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.

International audience; Although cell-based therapy is considered a promising method aiming at treating different muscular disorders, little clinical benefit has been reported. One of major hurdles limiting the efficiency of myoblast transfer therapy is the poor survival of the transplanted cells. Any intervention upon the donor cells focused on enhancing in vivo survival, proliferation, and expansion is essential to improve the effectiveness of such therapies in regenerative medicine. In the present work, we investigated the potential role of obestatin, an autocrine peptide factor regulating skeletal muscle growth and repair, to improve the outcome of myoblast-based therapy by xenotransplanting primary human myoblasts into immunodeficient mice. The data proved that short in vivo obestatin treatment of primary human myoblasts not only enhances the efficiency of engraftment, but also facilitates an even distribution of myoblasts in the host muscle. Moreover, this treatment leads to a hypertrophic response of the human-derived regenerating myofibers. Taken together, the activation of the obestatin/GPR39 pathway resulted in an overall improvement of the efficacy of cell engraftment within the host's skeletal muscle. These data suggest considerable potential for future therapeutic applications and highlight the importance of combinatorial therapies.

International audience; Obestatin/GPR39 signaling stimulates skeletal muscle growth and repair by inducing both G-protein-dependent and-independent mechanisms linking the activated GPR39 receptor with distinct sets of accessory and effector proteins. In this work, we describe a new level of activity where obestatin signaling plays a role in the formation, contractile properties and metabolic profile of skeletal muscle through determination of oxidative fiber type. Our data indicate that obestatin regulates Mef2 activity and PGC-1α expression. Both mechanisms result in a shift in muscle metabolism and function. The increase in Mef2 and PGC-1α signaling activates oxidative capacity, whereas Akt/mTOR signaling positively regulates myofiber growth. Taken together, these data indicate that the obestatin signaling acts on muscle fiber-type program in skeletal muscle. The heterogeneous population of muscle fibers determines the plasticity of skeletal muscle.

SUMMARY Hypothalamic endoplasmic reticulum (ER) stress is a key mechanism leading to obesity. Here, we demonstrate that ceramides induce lipotoxicity and hypothalamic ER stress, leading to sympathetic inhibition, reduced brown adipose tissue (BAT) thermogenesis, and weight gain. Genetic overexpression of the chaperone GRP78/BiP (glucose-regulated protein 78 kDa/binding immunoglobulin protein) in the ventromedial nucleus of the hypothalamus (VMH) abolishes ceramide action by reducing hypothalamic ER stress and increasing BAT thermogenesis, which leads to weight loss and improved glucose homeostasis. The pathophysiological relevance of this mechanism is demonstrated in obese Zucker rats, which show increased hypothalamic ceramide levels and ER stress. Overexpression of GRP78 in the VMH of these animals reduced body weight by increasing BAT thermogenesis as well as decreasing leptin and insulin resistance and hepatic steatosis. Overall, these data identify a triangulated signaling network involving central ceramides, hypothalamic lipotoxicity/ER stress, and BAT thermogenesis as a pathophysiological mechanism of obesity., In Brief The brain senses lipids, such as fatty acids, and modifies energy metabolism accordingly. However, it is unclear whether other lipid species may be involved. Contreras et al. now demonstrate that ceramides regulate energy balance through the induction of hypothalamic lipotoxicity and modulation of endoplasmic reticulum functionality. This leads to changes in sympathetic tone and brown adipose tissue (BAT)-induced thermogenesis, impacting body weight.

Summary Estrogens play a major role in the modulation of energy balance through central and peripheral actions. Here, we demonstrate that central action of estradiol (E2) inhibits AMP-activated protein kinase (AMPK) through estrogen receptor alpha (ERα) selectively in the ventromedial nucleus of the hypothalamus (VMH), leading to activation of thermogenesis in brown adipose tissue (BAT) through the sympathetic nervous system (SNS) in a feeding-independent manner. Genetic activation of AMPK in the VMH prevented E2-induced increase in BAT-mediated thermogenesis and weight loss. Notably, fluctuations in E2 levels during estrous cycle also modulate this integrated physiological network. Together, these findings demonstrate that E2 regulation of the VMH AMPK-SNS-BAT axis is an important determinant of energy balance and suggest that dysregulation in this axis may account for the common changes in energy homeostasis and obesity linked to dysfunction of the female gonadal axis., Graphical Abstract, Highlights • Central estradiol (E2) promotes negative energy balance • Central E2 increases thermogenic sympathetic nerve activity • Central E2 inhibits AMPK, specifically in the VMH, through ERα • The VMH AMPK-SNS-BAT axis mediates the central actions of E2 on energy balance, Estrogens play a major role in the regulation of body weight. Martínez de Morentin and colleagues show that estradiol (E2), acting through estrogen receptor alpha, selectively inhibits the energy sensor AMPK in the brain hypothalamic ventromedial nucleus, leading to activation of thermogenesis in brown adipose tissue.

Ghrelin is an endocrine peptide that has been identified in gastric oxyntic glands and that induces growth hormone secretion in the pituitary gland. This growth hormone secretagogue is expressed in many tissues such as stomach, pituitary gland, thyroid, testis, placenta and pancreas. Initial studies of ghrelin focused on its role as a circulating orexigenic signal. However, ghrelin has also been found to be involved in the modulation of glucose homeostasis. Although a number of studies have reported ghrelin expression in developing pancreas, the location of ghrelin-immunoreactive cells in adult pancreas (epsilon cells) remains controversial. In this study, we have analysed the distribution of pancreatic epsilon cells in adult human and rat islets by immunohistochemistry and in situ hybridisation. In humans, our immunohistochemical analysis has shown that ghrelin is expressed in glucagon-secreting cells, whereas in rats, it is present in insulin-secreting cells. Similar observations have been revealed by in situ hybridisation.

Summary Thermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b−/− mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain despite hypophagia. BMP8B is also expressed in the hypothalamus, and Bmp8b−/− mice display altered neuropeptide levels and reduced phosphorylation of AMP-activated protein kinase (AMPK), indicating an anorexigenic state. Central BMP8B treatment increased sympathetic activation of BAT, dependent on the status of AMPK in key hypothalamic nuclei. Our results indicate that BMP8B is a thermogenic protein that regulates energy balance in partnership with hypothalamic AMPK. BMP8B may offer a mechanism to specifically increase energy dissipation by BAT., Graphical Abstract Highlights ► BMP8B is expressed in BAT and is regulated in response to thermogenic stimuli ► BMP8B increases the thermogenic response of BAT to adrenergic stimulation ► Loss of BMP8B substantially increases susceptibility to diet-induced obesity ► BMP8B acts in the CNS to increase SNS activation of thermogenesis, Cold exposure and a high-fat diet trigger BMP8 signaling in brown adipose tissue and in the brain to boost energy expenditure.

Hyperthyroidism is characterized in rats by increased energy expenditure and marked hyperphagia. Alterations of thermogenesis linked to hyperthyroidism are associated with dysregulation of hypothalamic AMPK and fatty acid metabolism; however, the central mechanisms mediating hyperthyroidism-induced hyperphagia remain largely unclear. Here, we demonstrate that hyperthyroid rats exhibit marked up-regulation of the hypothalamic mammalian target of rapamycin (mTOR) signalling pathway associated with increased mRNA levels of agouti-related protein (AgRP) and neuropeptide Y (NPY), and decreased mRNA levels of pro-opiomelanocortin (POMC) in the arcuate nucleus of the hypothalamus (ARC), an area where mTOR co-localizes with thyroid hormone receptor-α (TRα). Central administration of thyroid hormone (T3) or genetic activation of thyroid hormone signalling in the ARC recapitulated hyperthyroidism effects on feeding and the mTOR pathway. In turn, central inhibition of mTOR signalling with rapamycin in hyperthyroid rats reversed hyperphagia and normalized the expression of ARC-derived neuropeptides, resulting in substantial body weight loss. The data indicate that in the hyperthyroid state, increased feeding is associated with thyroid hormone-induced up-regulation of mTOR signalling. Furthermore, our findings that different neuronal modulations influence food intake and energy expenditure in hyperthyroidism pave the way for a more rational design of specific and selective therapeutic compounds aimed at reversing the metabolic consequences of this disease.

This study aimed to investigate the role of preproghrelin-derived peptides in adipogenesis. Immunocytochemical analysis of 3T3-L1 adipocyte cells showed stronger preproghrelin expression compared with that observed in 3T3-L1 preadipocyte cells. Insulin promoted this expression throughout adipogenesis identifying mTORC1 as a critical downstream substrate for this profile. The role of preproghrelin-derived peptides on the differentiation process was supported by preproghrelin knockdown experiments, which revealed its contribution to adipogenesis. Neutralization of endogenous O-acyl ghrelin (acylated ghrelin), unacylated ghrelin, and obestatin by specific antibodies supported their adipogenic potential. Furthermore, a parallel increase in the expression of ghrelin-associated enzymatic machinery, prohormone convertase 1/3 (PC1/3) and membrane-bound O-acyltransferase 4 (MBOAT4), was dependent on the expression of preproghrelin in the course of insulin-induced adipogenesis. The coexpression of preproghrelin system and their receptors, GHSR1a and GPR39, during adipogenesis supports an autocrine/paracrine role for these peptides. Preproghrelin, PC1/3, and MBOAT4 exhibited dissimilar expression depending on the white fat depot, revealing their regulation in a positive energy balance situation in mice. The results underscore a key role for preproghrelin-derived peptides on adipogenesis through an autocrine/paracrine mechanism.

The aim of the present work was to study the regulation of circulating adiponectin levels and the expression of adiponectin receptor 2 (Adipo-R2) in several rat tissues in relation to fasting, leptin challenge, pregnancy, and chronic undernutrition. Using real-time PCR, we found Adipo-R2 mRNA expression in the liver, stomach, white and brown adipose tissues (WAT and BAT) of adult rats. Immunohistochemical studies confirmed protein expression in the same tissues. Adipo-R2 mRNA levels were decreased in liver after fasting, with no changes in the other tissues. Leptin decreased Adipo-R2 expression in liver and stomach, but increased its expression in WAT and BAT. Chronic caloric restriction in normal rats increased Adipo-R2 gene expression in stomach, while it decreased hepatic Adipo-R2 levels in pregnant rats. Using radioimmunoassay, we found that plasma adiponectin levels were diminished by fasting and leptin. Conversely, circulating adiponectin was increased in food-restricted rats, whereas its levels decreased in food-restricted pregnant rats by the end of gestation. In conclusion our findings provide the first evidence that (a) Adipo-R2 mRNA is regulated in a tissue-specific manner by fasting, but leptin is not responsible for those changes; (b) chronic caloric restriction in normal and pregnant rats also regulate Adipo-R2 mRNA in a tissue-specific manner; and (c) Adipo-R2 mRNA does not show a clear correlation with plasma adiponectin levels.

Thyroid hormones have widespread cellular effects; however it is unclear whether their effects on the central nervous system (CNS) contribute to global energy balance. Here we demonstrate that either whole-body hyperthyroidism or central administration of triiodothyronine (T3) decreases the activity of hypothalamic AMP-activated protein kinase (AMPK), increases sympathetic nervous system (SNS) activity and upregulates thermogenic markers in brown adipose tissue (BAT). Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus (VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism. This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and increases expression of thermogenic markers in BAT. These effects are reversed by pharmacological blockade of the SNS. Thus, thyroid hormone-induced modulation of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of whole-body energy homeostasis.

It has previously been demonstrated that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] administration, whether in cell cultures or in vivo to rats, increases glial cell line-derived neurotrophic factor (GDNF) expression levels, suggesting that this hormone may have beneficial effects in neurodegenerative disorders. This study was carried out to explore the effects of 1,25(OH)(2)D(3) administration in a 6-OHDA-lesioned rat model of Parkinson's disease on GDNF and tyrosine hydroxylase (TH) expression in substantia nigra (SN) and striatum. Two groups of animals received 1,25(OH)(2)D(3) intraperitoneally, the first group 7 days before the unilateral injection of 6-OHDA into the medial forebrain bundle (MFB) and the second group 21 days (days 21-28) after the unilateral injection of 6-OHDA. Animals of both groups were sacrificed on day 28. In addition, two other groups received a unilateral injection of either saline or 6-OHDA into the MFB. Rats were killed, and the SN and striatum were then removed for GDNF and TH determination. Striatal GDNF protein expression was increased on the ipsilateral with respect to the contralateral side after 6-OHDA injection alone as well as in 1,25(OH)(2)D(3)-treated rats before or after 6-OHDA administration. As expected, 6-OHDA injection induced an ipsilateral decrease in TH-immunopositive neuronal cell bodies and axonal terminals in the SN and striatum. However, treatment with 1,25(OH)(2)D(3) before and after 6-OHDA injection partially restored TH expression in SN. These data suggest that 1,25(OH)(2)D(3) may help to prevent dopaminergic neuron damage.

Current evidence suggests that hypothalamic fatty acid metabolism may play a role in regulating food intake; however, confirmation that it is a physiologically relevant regulatory system of feeding is still incomplete. Here, we use pharmacological and genetic approaches to demonstrate that the physiological orexigenic response to ghrelin involves specific inhibition of fatty acid biosynthesis induced by AMP-activated protein kinase (AMPK) resulting in decreased hypothalamic levels of malonyl-CoA and increased carnitine palmitoyltransferase 1 (CPT1) activity. In addition, we also demonstrate that fasting downregulates fatty acid synthase (FAS) in a region-specific manner and that this effect is mediated by an AMPK and ghrelin-dependent mechanisms. Thus, decreasing AMPK activity in the ventromedial nucleus of the hypothalamus (VMH) is sufficient to inhibit ghrelin's effects on FAS expression and feeding. Overall, our results indicate that modulation of hypothalamic fatty acid metabolism specifically in the VMH in response to ghrelin is a physiological mechanism that controls feeding. © 2008 Elsevier Inc. All rights reserved.

The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration.

Introduccion y objetivos La grasa visceral presenta una intensa actividad metabolica relacionada con el desarrollo de aterotrombosis. El tejido adiposo subcutaneo secreta en mayor cantidad las adipocitocinas leptina y adiponectina que el tejido adiposo visceral. El tejido adipose epicardico secreta mediadores inflamatorios; esta actividad proinflamatoria es mayor que la del tejido adipose subcutaneo. En este trabajo tratamos de determinar y comparar la expresion de adiponectina y leptina en el tejido adiposo epicardico y subcutaneo humano. Metodos Para el analisis se extrajo una muestra de tejido adiposo epicardico y tejido adiposo subcutaneo de 46 pacientes en los que se habia realizado cirugia cardiaca, bypass coronario o reemplazo valvular aortico/mitral. La expresion proteica y genica de la adiponectina y la leptina fue analizada mediante inmunohistoquimica y RT-PCR, respectivamente. Los niveles de expresion de ARN mensajero se analizaron mediante la tecnica comparativa de PCR en tiempo real, en ambos tejidos. Resultados Hay diferencias significativas de expression de ARN mensajero de la adiponectina y la leptina entre el tejido adiposo epicardico y subcutaneo, con una menor expresion de la adiponectina y la leptina en la grasa epicardica. En comparacion con los varones, las mujeres muestran una mayor expresion de adiponectina y leptina en tejido adiposo epicardico. Conclusiones El tejido adiposo epicardico expresa menos adiponectina y leptina que el subcutaneo. Hay una expresion diferencial entre varones y mujeres en cuanto a la expresion de adiponectina y leptina en el tejido adiposo epicardico.

During gestation, hyperphagia is necessary to cope with the metabolic demands of embryonic development. There were three main aims of this study: Firstly, to investigate the effect of pregnancy on hypothalamic fatty acid metabolism, a key pathway for the regulation of energy balance; secondly, to study whether pregnancy induces resistance to the anorectic effect of fatty acid synthase (FAS) inhibition and accumulation of malonyl-coenzyme A (CoA) in the hypothalamus; and, thirdly, to study whether changes in hypothalamic AMPK signaling are associated with brown adipose tissue (BAT) thermogenesis during pregnancy. Our data suggest that in pregnant rats, the hypothalamic fatty acid pathway shows an overall state that should lead to anorexia and elevated BAT thermogenesis: decreased activities of AMP-activated protein kinase (AMPK), FAS, and carnitine palmitoyltransferase 1, coupled with increased acetyl-CoA carboxylase function with subsequent elevation of malonyl-CoA levels. This profile seems dependent of estradiol levels but not prolactin or progesterone. Despite the apparent anorexic and thermogenic signaling in the hypothalamus, pregnant rats remain hyperphagic and display reduced temperature and BAT function. Actually, pregnant rats develop resistance to the anorectic effects of central FAS inhibition, which is associated with a reduction of proopiomelanocortin (POMC) expression and its transcription factors phospho-signal transducer and activator of transcription 3, and phospho-forkhead box O1. This evidence demonstrates that pregnancy induces a state of resistance to the anorectic and thermogenic actions of hypothalamic cellular signals of energy surplus, which, in parallel to the already known refractoriness to leptin effects, likely contributes to gestational hyperphagia and adiposity.

Context: Adiponectin is an adipocyte hormone involved in glucose and lipid metabolism. Recently, two receptors of this protein, called adiponectin receptor 1 (Adipo-R1) and Adipo-R2, have been cloned. Objective: The aim of this study was to examine whether adiponectin and its receptors are expressed in human and rat placentas and to evaluate the regulation of these factors by gestational age and nutritional status. Results: Our results demonstrate that adiponectin and Adipo-R2 are localized in both human and rat placentas. Human adiponectin and Adipo-R2 are presented in cytotrophoblast and syncytiotrophoblast cells. However, rat adiponectin and Adipo-R2 change their specific cell type immunostaining during gestation. Furthermore, placental adiponectin mRNA expression is increased during pregnancy in the rat, whereas Adipo-R2 has the contrary pattern. We also assessed the effect of food restriction (30%) during gestation, and we observed that adiponectin mRNA levels decrease after 16 d of undernutrition. In contrast, placental Adipo-R2 mRNA is unchanged by undernutrition. Finally, treatment with adiponectin during gestation decreases Adipo-R2, glucose transporter 3, lipoprotein lipase, and TGF- mRNA expression. Conclusion: Taken together, our results suggest that, at least in rodents, adiponectin may be involved in the regulation of several placental functions. (J Clin Endocrinol Metab 90: 4276–4286, 2005)

Peroxisome proliferator activated-receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily and, in addition to its relation with obesity and insulin sensitivity, it has recently been localized in human and mice pituitary, indicating a functional significance of PPARgamma in adenopituitary tumours. In the present study, we localized the PPARgamma mRNA and protein in different cell types of rat pituitary. Moreover, using the real-time polymerase chain reaction, we assessed the mRNA expression of PPARgamma in different physiological and pathological settings known to be associated with alterations in anterior pituitary cell proliferation and/or function. Our experiments have shown that PPARgamma mRNA levels were repressed by oestrogen through an oestrogen receptor-alpha effect. However, PPARgamma protein levels were only modified in males but not in females. On the other hand, PPARgamma mRNA expression was increased in dwarf rats in comparison with Lewis rats. Finally, nutritional, thyroid status or pregnancy did not change PPARgamma expression. Taken together, we provide new data regarding the regulation of pituitary PPARgamma mRNA by hormonal and metabolic status.

Objective: Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), acts on the pituitary and the hypothalamus to stimulate the release of growth hormone (GH) and promotes appetite and adiposity. It has also been reported to increase myocardial contractility, induce vasodilation, and protect against myocardial-infarction-induced heart failure. Though principally gastric in origin, it is also produced by other tissues. This work investigated whether cardiomyocytes synthesize and secrete ghrelin, and how its production in these cells responds to stress and exogenous apoptotic agents. Methods: Ghrelin and its receptor expression was studied by RT-PCR, immunohistochemistry, and competitive binding studies in mouse adult cardiomyocyte cell line HL-1, and primary cultured human cardiomyocytes. Ghrelin accumulation in cardiomyocyte culture medium was measured by radioimmunoassay. Viability and apoptosis assays were carried on by MTT and Hoechst dye vital staining, respectively. Results: RT-PCR showed that HL-1 cells produce mRNAs for both ghrelin and GHS-R, and that GHS-R1a is expressed in human cardiomyocytes; and competitive binding studies using 125I-labelled ghrelin showed efficient constitutive expression of GHS-R at the surface of HL-1 cells. Immunohistochemistry confirmed the presence of ghrelin in the cytoplasm of HL-1 cells and of isolated human cardiomyocytes in primary culture. Radioimmunoassay showed that ghrelin was secreted by HL-1 cells and human cardiomyocytes into the culture medium. Ghrelin did not modify the viability of HL-1 cells subjected to 12-h starvation, but did protect against the apoptosis inducer cytosine arabinoside (AraC). Finally, production of ghrelin mRNA in HL-1 cardiomyocytes was reduced by AraC but increased if exposure to AraC was preceded by GH treatment. Conclusions: Ghrelin is synthesized and secreted by isolated murine and human cardiomyocytes, probably with paracrine/autocrine effects, and may be involved in protecting these cells from apoptosis.

1 In this study, we investigated whether serotonin could regulate the in vitro activity of phagocytosis through 5-hydroxytryptamine or serotonin (5-HT(1A)) receptors. 2 Mouse peritoneal macrophages were cultured with serotonin and the activity of phagocytosis was assessed by the uptake of zymosan and latex particles added to the culture media. Specific binding of [(3)H]8-OH-DPAT and immunohistochemistry using an affinity-purified anti-5-HT(1A)-receptor antibody were assayed in the macrophages. In addition, we took advantage of the availability of pharmacological inhibitors of nuclear factor-kappaB (NF-kappaB) to explore its role in the regulation of the 5-HT(1A) receptor. 3 Serotonin increased the in vitro activity of phagocytosis in a dose-dependent manner. The 5-HT(1A) receptor agonist (+/-)-8-hydroxy-2-(di-n-propyl-amino)-tetralin (R(+)-8-OH-DPAT) reproduced these effects. Serotonin- or R(+)-8-OH-DPAT-induced increases in phagocytosis were blocked by the 5-HT(1A) receptor antagonist WAY100635 and the NF-kappaB inhibitor pyrrolidinedithiocarbamate. Moreover, mouse peritoneal macrophages expressed specific binding sites for [(3)H]8-OH-DPAT when cultivated in the presence of zymosan or latex beads. Immunohistochemistry confirmed the expression of the 5-HT(1A) receptor protein in the macrophages. 4 These results show that serotonin can upregulate the activity of peritoneal macrophages through 5-HT(1A) receptors.

Orexin-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and related with the regulation of the energy balance and arousal. They act on G protein receptors named orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). In the present study, we used immunohistochemical techniques to detect the distribution of OXR in normal human adrenal gland and adrenal tumours (adrenocortical adenomas and pheochromocytomas). OX1R was expressed in the cortex of the normal human adrenal gland (glomerulosa, fasciculata and reticular zones) and OX2R was located in the medulla (epinephrine and norepinephrine cells). By the double immunofluorescence techniques, we demonstrated that virtually all medullar cells (epinephrine and norepinephrine cells) expressed OX2R. As was expected, according to the results obtained in normal tissues, cortical tumours (adrenocortical adenomas) were positive for OX1R but not for OX2R and conversely, medullar tumours (pheochromocytomas) expressed only OX2R.

Krüppel-like factor 4 (KLF4) is a zinc-finger-type transcription factor expressed in a range of tissues that plays multiple functions. We report that hypothalamic KLF4 represents a new transcription factor specifically modulating agouti-related protein (AgRP) expression in vivo. Hypothalamic KLF4 colocalizes with AgRP neurons and is modulated by nutritional status and leptin. Over-expression of KLF4 in the hypothalamic arcuate nucleus (ARC) induces food intake and increases body weight through the specific stimulation of AgRP, as well as blunting leptin sensitivity in lean rats independent of forkhead box protein 01 (FoxO1). Down-regulation of KLF4 in the ARC inhibits fasting-induced food intake in both lean and diet-induced obese (DIO) rats. Silencing KLF4, however, does not, on its own, enhance peripheral leptin sensitivity in DIO rats., Graphical abstract