Maresin 1 mitigates liver steatosis in ob/ob and diet-induced obese mice.

BACKGROUND/OBJECTIVES: The aim of this study was to characterize the effects of Maresin 1 (MaR1) in obesity-related liver steatosis and the mechanisms involved. METHODS: MaR1 effects on fatty liver disease were tested in ob/ob (2-10 µg kg-1 i.p., 20 days) and in diet-induced obese (DIO) mice (2 µg kg-1, i.p., or 50 µg kg-1, oral gavage for 10 days), as well as in cultured hepatocytes. RESULTS: In ob/ob mice, MaR1 reduced liver triglycerides (TG) content, fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 protein expression, while increased acetyl-CoA carboxylase (ACC) phosphorylation and LC3II protein expression, in parallel with a drop in p62 levels. Similar effects on hepatic TG, ACC phosphorylation, p62 and LC3II were observed in DIO mice after MaR1 i.p. injection. Interestingly, oral gavage of MaR1 also decreased serum transaminases, reduced liver weight and TG content. MaR1-treated mice exhibited reduced hepatic lipogenic enzymes content (FAS) or activation (by phosphorylation of ACC), accompanied by upregulation of carnitine palmitoyltransferase (Cpt1a), acyl-coenzyme A oxidase (Acox1) and autophagy-related proteins 5 and 7 (Atg5-7) gene expression, along with increased number of autophagic vacuoles and reduced p62 protein levels. MaR1 also induced AMP-activated protein kinase (AMPK) phosphorylation in DIO mice and in primary hepatocytes, and AMPK inhibition completely blocked MaR1 effects on Cpt1a, Acox1, Atg5 and Atg7 expression. CONCLUSIONS: MaR1 ameliorates liver steatosis by decreasing lipogenic enzymes, while inducing fatty acid oxidation genes and autophagy, which could be related to AMPK activation. Thus, MaR1 may be a new therapeutic candidate for reducing fatty liver in obesity.

Pattern of orexin expression and direct biological actions of orexin-a in rat testis.

Orexins, hypothalamic neuropeptides initially involved in the control of food intake and sleep-wake cycle, have recently emerged as pleiotropic regulators of different biological systems, including the reproductive axis. Besides central actions, peripheral expression and functions of orexins have been reported, and prepro-orexin and orexin type-1 receptor mRNAs have been detected in the testis. However, the pattern of expression and biological actions of orexin in the male gonad remain mostly unexplored. In this study, we report analyses on testicular prepro-orexin mRNA expression and orexin-A immunoreactivity in different experimental settings, and on direct effects of orexin-A on seminiferous tubule functions. Expression of prepro-orexin mRNA was demonstrated in the rat testis at different stages of postnatal development, with negligible levels at early juvenile period and maximum values in adulthood. Likewise, orexin-A immunoreactivity was demonstrated along postnatal maturation, with strong peptide signal in Leydig cells and spermatocytes at specific stages of meiosis. Testicular expression of prepro-orexin mRNA appeared hormonally regulated; its levels decreased after hypophysectomy and increased after gonadotropin replacement and ghrelin stimulation. Finally, orexin-A suppressed the expression of key Sertoli cell genes, such as Müllerian-inhibiting substance and stem cell factor, and inhibited DNA synthesis in specific stages of the seminiferous epithelium. In conclusion, we provide evidence for the regulated expression of orexin in the rat testis and its potential involvement in the control of seminiferous tubule functions. Together with our recent results on the expression of orexin type-1 receptor in the rat testis, our data further document a novel testicular site of action of orexins in the control of male reproductive axis.

Detection of nitric oxide synthase (NOS) in somatostatin-producing cells of human and murine stomach and pancreas.

The aim of this study was to identify by immunocytochemistry the distribution of nitric oxide synthase (NOS) in human and murine gastric epithelium. Using two different antisera specific for neuronal NOS (nNOS), we detected nNOS immunoreactivity in endocrine cells of the epithelium of the body and pyloric regions as well as in ganglion cells of the intrinsic plexi of the stomach of the three species studied. Both immunocytochemistry of contiguous sections and double immunolabeling methods showed that the nNOS-immunoreactive cells were also immunoreactive for somatostatin. Co-localization of nNOS and somatostatin has also been found in the pancreatic islets, where strong nNOS immunoreactivity appeared in scattered cells, which were peripheral in rat and mouse islets and more randomly distributed in human. The possibility of crossreactivity between the antisera against nNOS and somatostatin was ruled out by means of absorption controls. Immunocytochemical techniques were also applied to thin sections, confirming the immunostaining of gastric D-cells, which was restricted principally to the secretory granules. The possible functional implications of these findings for gastric and pancreatic physiology are discussed.

Expression of neuronal nitric oxide synthase in several cell types of the rat gastric epithelium.

The aim of this study was to identify which cell types of the rat gastric epithelium express neuronal nitric oxide synthase (nNOS) because the results of the previous studies have been very divergent regarding this point. By the combination of immunohistochemical (IHC) and in situ hybridization (ISH) techniques, we detected expression of nNOS in chief and mucosecretory cells of the gastric epithelium. Moreover, some gastric endocrine cells were immunoreactive for nNOS, although they could not be distinguished in sections treated with ISH techniques. The strongest signal for all antibodies in IHC techniques was obtained when microwave (MW) heating was performed before the IHC procedure. Our results indicate that in the gastric epithelium a variety of cell types are able to produce NO. The NO produced by the different cell types (chief, mucous, and endocrine) may form a complex network of paracrine communication with an important role in gastric physiology.

Localization of amidating enzymes (PAM) in rat gastrointestinal tract.

We studied the distribution of the two enzymes involved in post-translational C-terminal alpha-amidation of regulatory peptides in rat digestive tract, using immunocytochemical methods and in situ hybridization techniques. The enzymes were located in most of the fibers and neurons of the myenteric and submucous plexus throughout the entire digestive tract and in endocrine cells of the stomach and colon. Staining of reverse-face serial sections demonstrated that the enzymes in endocrine cells of the stomach co-localized with gastrin in the bottom of the gastric glands. Some gastrin-immunoreactive cells near the neck of the gland were negative for PAM, suggesting that amidation takes place only in the more mature cells. In the colon all cells immunoreactive for glucagon and GLP1 were also positive for peptidylglycine alpha-hydroxylating monooxygenase (PHM) but not for peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The absence of immunoreactivity for the amidating enzymes in endocrine cells of the small intestine, known to produce C-terminally amidated peptides, suggests the existence of other amidating enzymes.

Expression of proadrenomedullin derived peptides in the mammalian pituitary: co-localization of follicle stimulating hormone and proadrenomedullin N-20 terminal peptide-like peptide in the same secretory granules of the gonadotropes.

Expression of proadrenomedullin-derived peptides in the rat, cow and human pituitary was studied by a variety of techniques. Immunocytochemical detection showed a widespread expression of adrenomedullin peptide in the adenohypophysis and the neural lobe, with low expression in the intermediate pituitary. Proadrenomedullin N-20 terminal peptide (PAMP)-immunoreactivity was also present in the anterior pituitary but showed a more marked heterogeneous distribution, with cells going from very strong to negative immunostaining. Lower levels of PAMP were found in the neural lobe. Interestingly, the distribution of adrenomedullin and PAMP immunoreactivity in the anterior pituitary did not completely overlap. In the present study, we concentrated our efforts to determine which cell type of the adenohypophysis expresses PAMP. Paraffin and semithin serial sections immunostained for PAMP and the classical pituitary hormones revealed that a subpopulation of the gonadotropes expresses high levels of PAMP-immunoreactive material. Ultrastructural analysis clearly showed PAMP-immunoreactivity in the follicle stimulating hormone (FSH)-containing large secretory granules of the gonadotropes, suggesting simultaneous secretion of PAMP and FSH by this cell type. Three mouse adenohypophysis-derived cell lines (AtT20, GH3, and alphaT3-1 derived from corticotropes, lacto/somatotropes and gonadotropes, respectively) were also analysed and showed expression of both proadrenomedullin-derived peptides and their mRNA. Functional studies in these three cell lines showed that neither adrenomedullin nor PAMP was able to stimulate cAMP production in our experimental conditions. Taken together, our results support that proadrenomedullin derived peptides are expressed in the pituitary in cell-specific and not overlapping patterns, that could be explained by differences in postranslational processing. Our data showing costorage of PAMP and FSH in the same secretory granules open a way by which PAMP could be involved in the control of reproductive physiology in a coordinated manner with FSH.

Brush-like cells within bronchial epithelia of chicken lung (Gallus gallus).

The secondary and primary (mesobronchus) bronchi of chicken lung are lined by a typical respiratory epithelium: pseudostratified columnar ciliated with goblet cells. Up to date, four constituting epithelial cell types have been identified: ciliated, mucosecretory, basal and endocrine cells. In this study a putative new epithelial cell type, the brush-like cell, is described. The avian brush-like cells have only been found in the bronchial epithelia but never in the gas-exchange areas. They are scattered among the other epithelial cells, mainly ciliated cells, and their number is extremely low. The characteristic morphological feature of these cells is an apical protruding cytoplasm with microvilli. This cell type is similar to that found in the lung of some mammalian and non-mammalian species. The functional role of these cells is not yet clear; they could carry out absorptive processes.

Immunocytochemical and ultrastructural characterization of endocrine cells and nerves in the intestine of Rana temporaria.

Endocrine cells have been identified in the intestine of the frog Rana temporaria after application of the Grimelius and Masson-Fontana techniques. These endocrine cells were examined using immunocytochemical techniques on paraffin and semithin sections for light microscopy. After testing 19 antisera, 12 immunoreactivities were identified. Numerous serotonin-, somatostatin- and GLP-1-immunoreactive cells; a moderate number of PYY-, glucagon-, VIP-, gastrin/CCK-immunoreactive cells and few human PP-, bombesin-, substance P- and neurotensin-immunoreactive cells were found. VIP- and met-enkephalin were identified in nerve fibers of the muscular layer. Using semithin-thin sections five types of endocrine cells (serotonin-, somatostatin-, gastrin/CCK-, glucagon- and bombesin-immunoreactive cells) have been characterized according to their immunocytochemical reaction and the ultrastructure of the secretory granules.

Endocrine cells and nerves in the stomach of the lizard Podarcis hispanica detected by immunocytochemistry.

The general identification of endocrine cells in the stomach of the lizard Podarcis hispanica was carried out by their response to the Grimelius and Masson-Fontana techniques. 11 immunoreactive cell-types, positive for chromogranin-, serotonin-, caerulein/gastrin/ cholecystokinin (CAER/G/CCK)-, glucagon-like-peptide-1 (GLP-1)-. glucagon-, bombesin-,somatostatin-, pancreatic polypeptide (PP)-, peptide tyrosine tyrosine (PYY)-, neurotensin-and calcitonin gene related peptide (CGRP)- antisera were detected by immunocytochemical methods. Co-existence of glucagon with GLP-1, and PP with PYY were observed in some cells. Furthermore, immunoreactivities for members of gastrin and PP families were also found to co-exist in a few cells. In the muscular layer, vasoactive intestinal peptide (VIP)- and substance P-immunoreactive nerve fibers were also found.

Relationship between epithelial and connective tissues in the stomach of the frog Rana temporaria during metamorphosis: an ultrastructural study.

In the course of metamorphosis of the stomach of Rana temporaria tadpoles there is a marked increase in the amount of active mesenchymal fibroblasts and extracellular matrix underlying the regenerating gastric epithelium. At the onset of metamorphosis, a thick PAS-positive basement membrane is developed around the epithelial component of the mucosa, formed by the apical, degenerating larval epithelium and the basal, regenerative epithelial cords. Under the electron microscope, a folded basement membrane is usually revealed under the apical degenerating epithelium while a compact basement membrane (up to 1-2 microns thick), forming both patches and more extensive areas, is frequently seen around the regenerative glandular cords. Cytoplasmic processes, extending from both the epithelial and mesenchymal fibroblastic cells, cross the basement membrane and make physical contact between the two cellular types. At mid-metamorphosis areas of thick PAS-positive basement membrane are still observed around the differentiating glandular outlines, before disappearing completely at late metamorphosis. The probable involvement of intertissue interactions between epithelium and connective elements in the morphogenesis, proliferation and differentiation of secondary, definitive frog stomach is discussed. Early contacts between epithelium and phagocytes, probably related to the invasion of epithelium by the phagocytic cells, have also been observed.

Immunocytochemical and ultrastructural characterization of endocrine cells in the larval stomach of the frog Rana temporaria tadpoles: a comparison with adult specimens.

According to immunostaining and ultrastructural patterns, Rana temporaria tadpole stomach displays a well-differentiated endocrine population comprising, at least, six cellular types: ECL, EC [serotonin], D [somatostatin] - all three of them abundant -, P [bombesin] - less numerous -, CCK-8 [cholecystokinin/gastrin] and A [glucagon/glicentin] - both very scarce. Larval endocrine cells are mainly located in the surface epithelium and show open or closed morphologies. Cellular diversity is similar in tadpoles and frogs, with the exception of immunoreactivity for gastrin-17, found in adults in numerous cells. Larval cells display mature ultrastructural traits, although with smaller secretory granules. The different distribution of endocrine cells, which in adults are preferentially located in the glands, probably refers to different functional requirements. However, the rich vascular plexus present in larval mucosa may be an efficient transport medium of surface hormones to-gastric targets. The enhancement in adults of endocrine population and correlative increase in hormonal secretion indicates a more active functional role, probably related to the shift from herbivorous to carnivorous habits. In summary, the tadpole gastric endocrine population, although not as numerous as that of adult frogs, displays histological traits that indicate a relevant (immunoreactive and ultrastructural properties, cellular diversity) and specific (surface location, relative abundance of open-type cells) role of local regulatory factors in amphibian larval gastric function.

An immunocytochemical and ultrastructural study of the larval anterior intestine of the frog Rana temporaria, with especial reference to endocrine cells.

Endocrine cells of the larval intestine of Rana temporaria tadpoles have been identified by argyrophilic, immunocytochemical and electron-microscopical techniques. Scarce endocrine cells have been found in both the short non-absorptive zone immediately following the stomach, and in the rest of the anterior intestine. Endocrine cells are frequently seen to extend a cytoplasmic process towards the lumen. Immunoreactivity for serotonin, somatostatin, bombesin and cholecystokinin-8 has been detected. According to the ultrastructural traits of the endocrine granules, three larval intestinal endocrine populations have been differentiated.

An immunocytochemical and ultrastructural study of the larval anterior intestine of the frog Rana temporaria, with especial reference to endocrine cells.

Endocrine cells of the larval intestine of Rana temporaria tadpoles have been identified by argyrophilic, immunocytochemical and electron-microscopical techniques. Scarce endocrine cells have been found in both the short non-absorptive zone immediately following the stomach, and in the rest of the anterior intestine. Endocrine cells are frequently seen to extend a cytoplasmic process towards the lumen. Immunoreactivity for serotonin, somatostatin, bombesin and cholecystokinin-8 has been detected. According to the ultrastructural traits of the endocrine granules, three larval intestinal endocrine populations have been differentiated.

Short- and long-term changes in gastric morphology and histopathology following sleeve gastrectomy in diet-induced obese rats.

BACKGROUND: Sleeve gastrectomy (SG) has been used as a multipurpose surgical procedure for the treatment of morbid obesity. The aim of the study was to analyze gastric morphology and histology at two different time points after SG in rats. METHODS: Thirty-five male Wistar rats were fed ad libitum during 3 months on a high-fat diet to induce obesity. Subsequently, 25 diet-induced obese rats underwent either SG (n = 12) or a sham operation (n = 13). The remaining ten obese animals encompassed the nonoperated control group (Co). Four weeks postoperatively, 15 rats (n = 5 rats/experimental group) were sacrificed, while the remaining 20 rats were sacrificed after 16 weeks (animals/group; Co = 5, sham = 8, SG = 7) to compare the gastric morphological and histopathological changes over time. Body weight and food intake were regularly recorded. RESULTS: For both time periods, the Co groups exhibited the highest body weight, while the rats undergoing the SG showed the lowest weight gain (P < 0.05). Initially, significant differences (P < 0.005) in food intake relative to body weight were observed between the Co rats and animals undergoing surgery, which disappeared thereafter. The actual total stomach size after both experimental periods in the SG group was similar to that of non- and sham-operated rats mainly due to a forestomach enlargement, which was more pronounced after 16 weeks. Traits of gastritis cystica profunda characterized by gastric foveolae elongation with hyperplasia and cystic dilatation of the glands were observed in the residual stomachs of the sleeve-gastrectomized rats. These findings were mostly observed after 16 weeks of performing the SG, although they were also detected occasionally following 4 weeks postoperatively. No intestinal metaplasia was observed. CONCLUSION: After SG gastric macro- and microscopic changes with functional implications in both the short and long term take place.

Evidence for the colocalization of gastrin/CCK- and PYY/PP-immunoreactive substances in the small intestine of the lizard Podarcis hispanica: immunocytochemical and ultrastructural study.

Peptide tyrosine tyrosine/pancreatic polypeptide (PYY/PP)- and C-terminal gastrin/cholecystokinin (G/CCK)-immunoreactive cells were investigated in the intestine of the lizard Podarcis hispanica, using immunocytochemistry with light and electron microscopy. Immunolabeling of consecutive semithin sections revealed coexistence of PYY/PP- and C-terminal G/CCK-like substances in some cells, while in others only PYY/PP or G/CCK immunoreactivity was found. Appropriate absorption controls excluded cross-reactivity between the antisera used. Ultrastructurally G/CCK+, PYY/PP+ cells were similar to G/CCK+, PYY/PP- cells but different from PYY/PP+, G/CCK- cells. Although virtually nothing is known concerning the physiological effects of these peptides in reptiles, their colocalization in the same cells in the intestine of Podarcis hispanica suggests a close relationship between them in the regulation of the digestive process.

The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation.

The ability to ensure continuous availability of energy despite highly variable supplies in the environment is a major determinant of the survival of all species. In higher organisms, including mammals, the capacity to efficiently store excess energy as triglycerides in adipocytes, from which stored energy could be rapidly released for use at other sites, was developed. To orchestrate the processes of energy storage and release, highly integrated systems operating on several physiological levels have evolved. The adipocyte is no longer considered a passive bystander, because fat cells actively secrete many members of the cytokine family, such as leptin, tumor necrosis factor-alpha, and interleukin-6, among other cytokine signals, which influence peripheral fuel storage, mobilization, and combustion, as well as energy homeostasis. The existence of a network of adipose tissue signaling pathways, arranged in a hierarchical fashion, constitutes a metabolic repertoire that enables the organism to adapt to a wide range of different metabolic challenges, such as starvation, stress, infection, and short periods of gross energy excess.