Breed and Feeding System Impact the Bioactive Anti-Inflammatory Properties of Bovine Milk.

In the present study, we aimed at assessing the influence of breed and feeding system on the bovine milk profile of betaines and carnitines and milk capacity in counteracting the inflammatory endothelial cell (EC) damage induced by interleukin (IL)-6. In the first experimental design, two breeds were chosen (Holstein vs. Modicana) to investigate the biomolecule content and antioxidant capacity in milk and dairy products. In the second experimental design, two feeding systems (pasture vs. total mixed ratio) were tested only in Holstein to evaluate the possible effect on the functional profile of milk and dairy products. Finally, the bulk milk from the two experimental designs was used to evaluate the efficacy of preventing IL-6-induced endothelial inflammatory damage. Results showed that Modicana milk and whey had higher biomolecule content and antioxidant activity compared to Holstein milk (p < 0.01). Milk from Holstein fed TMR showed higher concentration of γ-butyrobetaine, δ-valerobetaine (p < 0.01), and l-carnitine (p < 0.05). Similarly, whey from Holstein fed TMR also showed higher content of δ-valerobetaine, glycine betaine, l-carnitine, and acetyl-l-carnitine (p < 0.01) compared to the Holstein fed pasture. Conversely, the antioxidant activity of milk and dairy products was not affected by the feeding system. In ECs, all milk samples reduced the IL-6-induced cytokine release, as well as the accumulation of reactive oxygen species (ROS) and the induction of cell death, with the most robust effect elicited by Modicana milk (p < 0.01). Overall, Modicana milk showed a higher content of biomolecules and antioxidant activity compared to Holstein, suggesting that the breed, more than the feeding system, can positively affect the health-promoting profile of dairy cattle milk.

Short-term exposure to the simple polyphenolic compound gallic acid induces neuronal hyperactivity in zebrafish larvae.

A growing body of evidence suggests that the biological effects of polyphenols are not restricted to antioxidant activity, but they exert a wide range of modulatory effects on metabolic pathways, cellular signaling and gene expression. In this study, we tested the minimum safe concentration of gallic acid (GA) in 72 hpf zebrafish larvae in order to evaluate the effects on the central nervous system and the behavioral response. We showed that a short exposure (30 min) induces the depletion of the two main excitatory and inhibitory neurotransmitters, Glu and GABA, respectively, in the larval nervous system. The acute impairment of GABAergic-glutamatergic balance was paralleled by an increase of the fosab neuronal activity marker in specific brain areas, such as the forebrain, olfactory bulbs, pallial area, ventral midbrain, tegmentum, and the medulla oblongata ventral area. The neuronal excitation was mirrored by the increased cumulative motor response. The inhibition of the olfactory epithelium with brief cadmium exposition suggests a direct involvement of olfaction in the larvae response to GA. Our results demonstrate that a brief exposure to GA induces motoneuronal hyperexcitability in zebrafish. The behavioral response was probably elicited through the activation of an odorous, or chemical, stimulus. The specificity of the activated neuronal territories suggests the involvement of additional signaling pathways. Although the underlying molecular mechanisms remain to be elucidated, our data support the hypothesis that GA acts as an excitatory molecule, capable of inducing a specific nerve response. These results offer a new vision on potential effects of GA.

CARD14/CARMA2sh and TANK differentially regulate poly(I:C)-induced inflammatory reaction in keratinocytes.

CARD14/CARMA2sh (CARMA2sh) is a scaffold protein whose mutations are associated with the onset of human genetic psoriasis and other inflammatory skin disorders. Here we show that the immunomodulatory adapter protein TRAF family member-associated NF-κB activator (TANK) forms a complex with CARMA2sh and MALT1 in a human keratinocytic cell line. We also show that CARMA2 and TANK are individually required to activate the nuclear factor κB (NF-κB) response following exposure to polyinosinic-polycytidylic (poly [I:C]), an agonist of toll-like receptor 3. Finally, we present data indicating that TANK is essential for activation of the TBK1/IRF3 pathway following poly (I:C) stimulation, whereas CARMA2sh functions as a repressor of it. More important, we report that two CARMA2sh mutants associated with psoriasis bind less efficiently to TANK and are therefore less effective in suppressing the TBK1/IRF3 pathway. Overall, our data indicate that TANK and CARMA2sh regulate TLR3 signaling in human keratinocytes, which could play a role in the pathophysiology of psoriasis.

A cross-talk between leptin and 17ß-estradiol in vitellogenin synthesis in rainbow trout Oncorhynchus mykiss liver.

The existence of nutritional and energy reserves is fundamental for fish female fertility, so that the existence of a correlation between metabolic reserves and reproductive capacity is suggested. Leptin regulates body weight and energy homeostasis. Estradiol induces the synthesis of vitellogenin, a phospholipoglycoprotein produced by the liver and taken up by the growing oocytes. The objective of this study was to investigate the possible existence of a crosstalk between 17ß-estradiol (E2) and leptin in the modulation of E2-induced vtg in the rainbow trout Oncorhynchus mykiss. Liver slices were incubated with recombinant trout leptin (rt-lep) at three different concentrations (1-10-100 ng/ml). rt-lep brought about the decrease of E2-induced vtg secretion in the medium and the down-regulation of vtg mRNA expression. Moreover, rt-lep stimulated the lipase activity and diminished the liver fatty acid content. The combined employment of signal transduction inhibitors and the analysis of signal transduction phosphorylated factors revealed that rt-lep effect on E2-induced vtg occurred through the activation of phosphodiesterase, protein kinase C, MAP kinases, and protein kinase A. In conclusion, our study suggests that leptin influences E2-induced vtg synthesis in the rainbow trout Oncorhynchus mykiss by modifying both the protein and the lipid components.

Effects of dietary supplementation with polyphenols on meat quality in Saanen goat kids.

BACKGROUND: Diet supplementation with polyphenols is a novel strategy to improve meat quality in livestock, by preventing oxidative deterioration of lipids and protein. Polyphenols have beneficial effects on both human and animal health and can be obtained from several sources, such as olive mill wastewaters (OMWW). These are severe environmental pollutants and therefore may be recycled and utilized in other sectors. The aim of this study was to evaluate growth performance, meat characteristics, fatty acid composition, antioxidant status, different forms of myoglobin and malondialdehyde formation in kids who received a diet supplemented with polyphenols obtained from OMWW. Weaned goat kids (n = 18) were divided into two homogenous groups: control (C) group (n = 9) received a fattening standard diet while the other group (n = 9) received the same diet, supplemented with 3.2 mg/day of polyphenols powder extract (PE group). Average daily gain (ADG) was calculated 10 days apart throughout the study. After 78 days, the kids were slaughtered and pH and carcass yield were evaluated. Longissimus thoracis et lumborum muscle was collected and utilized for chemical analysis, meat quality evaluation and oxidative stability. RESULTS: No differences were recorded in ADG, carcass weight, pH and dressing between the two groups. Furthermore a similar meat proximate composition, texture and color was observed. Dietary polyphenols supplementation significantly (P < 0.01) decreased short chains (

Effect of Polyethylene Glycol-Simulated Drought Stress on Stomatal Opening in "Modern" and "Ancient" Wheat Varieties.

Climate change is leading to an increase in the intensity, duration, and frequency of severe droughts, especially in southern and southeastern Europe, thus aggravating water scarcity problems. Water deficit stress harms the growth, physiology, and yield of crops like durum wheat. Hence, studying ancient wheat varieties' stress responses could help identify genetic traits to enhance crop tolerance to environmental stresses. In this background, this study aimed to investigate the effects of PEG 6000-stimulated drought stress in the ancient wheat variety Saragolla and the modern one Svevo by analyzing various biochemical and molecular parameters that can especially condition the stomatal movement. Our data revealed that drought stress caused a significant increase in the levels of total soluble sugars, ABA, and IAA in both selected cultivars to a greater extent in the Saragolla than in the Svevo. We demonstrated that, under water deficit stress, calcium dynamics as well as the expression of ERF109, MAPK3/6, MYB60, and TaTPC1, involved in the activation of drought-related calcium-sensitive pathways, display significant differences between the two varieties. Therefore, our study provided further evidence regarding the ability of the ancient wheat variety Saragolla to better cope with drought stress compared to the modern variety Svevo.

The physiology and anatomy study in leaves of Saragolla and Svevo wheat cultivars under polyethylene glycol-simulated drought stress.

Drought stress is increasing in frequency and severity with the progression of global climate change, thereby becoming a major concern for the growth and yield of crop plants, including wheat. The current challenge is to explore different ways of developing wheat genotypes with increased tolerance to drought. Therefore, we renewed interest in 'ancient' varieties expected to be more tolerant to environmental stress than the few elite varieties nowadays cultivated. This study aimed to perform comparative analysis of the effect of drought-simulating polyethylene glycol (PEG-6000) treatment on morpho-anatomical and physiological foliar traits of two durum wheat seedlings cultivars, Saragolla and Svevo, as these can reflect the adaptability of the plant to the environment to a certain extent. Results demonstrated that drought-stressed Saragolla leaves exhibited a greater reduction of stomatal density, a minor reduction of stomatal pore width, a wider xylem vessel mean area, greater compactness of mesophyll cells, a minor loss of chlorophyll content, as well as better photosynthetic and growth performance compared to the other variety. From such behaviours, we consider the Saragolla cultivar more drought tolerant than Svevo and therefore probably very promising for cultivation in dry areas.

Quercetin's Dual Mode of Action to Counteract the Sp1-miR-27a Axis in Colorectal Cancer Cells.

Quercetin (Qc) inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The molecular mechanism of action has not been fully elucidated; however, interplay with some miRNAs has been reported, specifically with miR-27a, an onco-miRNA overexpressed in several malignancies. Here, we show that Qc reduces cell viability and induces apoptosis in HCT116 and HT-29 colon cancer cells, by upregulating negative modulators of proliferation pathways such as Sprouty2, PTEN and SFRP1. These are targets of miR-27a whose high expression is reduced by Qc. Moreover, miR-23a, and miR-24-2, the two other components of the unique gene cluster, and the pri-miRNA transcript are reduced, evoking a transcriptional regulation of the entire cluster by Sp1. Mechanistically, we show that Qc is rapidly internalized and localizes in the nucleus, where it likely interacts with Sp1, inducing its proteasomal degradation. Sp1 is further repressed by ZBTB10, an Sp1 competitor for DNA binding that is an miR-27a target and whose levels increase following Qc. SP1 mRNA is also reduced, supporting the regulation of its own gene transcription. Finally, Sp1 knockdown elicits the impaired transcription of the entire cluster and the upregulation of the miR-27a targets, phenocopying the effects of Qc. Through this dual mode of action, Qc counteracts the protumoral Sp1-miR-27a axis, opening the way for novel therapies based on its association as neoadjuvant with known anticancer treatments.

Effects of Olea europaea L. Polyphenols on the Animal Welfare and Milk Quality in Dairy Cows.

Here, we evaluated the effect of dietary supplementation with an Olea europaea L. extract on the animal welfare and milk quality of dairy cows. Thirty Italian Holstein-Friesian dairy cows in the mid-lactation phase (90 to 210 days) were blocked into experimental groups based on parity class (namely, primiparous (P) (n = 10), secondiparous (S) (n = 10) and pluriparous (PL) (n = 10)) and received, for 60 days, Phenofeed Dry® at 500 mg/cow/day. Milk and blood samples were collected before the start of the treatment (T0), subsequently every 15 days (T1-T4) and at 45 days after the end of treatment (T5). In the serum, glucose and triglycerides, stress, the thyroid, lactation and sex hormones were measured; in the milk, lysozyme content as well as the fatty acid profile were assessed. In the whole animal, the enriched feed helped to maintain hormonal parameters in the physiological range while producing hypoglycemic (T4 vs. T0, for P and PL p < 0.001) and hypolipidemic effects (T4 vs. T0, for P p < 0.001 and for PL p < 0.01). At the milk level, it resulted in a reduction in total fat (T5 vs. T0, for P, S and PL p < 0.001) and in the saturated fatty acids (SFAs)/monounsaturated fatty acids (MUFAs) ratio paralleled by an increase in polyunsaturated fatty acids (PUFAs) (T5 vs. T0, for P, S and PL p < 0.001), protein content (lysozyme (T4 vs. T0, for P and PL p < 0.001)) and lactose (T5 vs. T0, for P, S and PL p < 0.001). Thus, the inclusion of natural bioactive molecules such as O. europaea L. polyphenols in the dairy cow diet may help to improve animal welfare and milk quality.

Plasma fatty acid profile in Italian Holstein-Friesian dairy cows supplemented with natural polyphenols from the olive plant Olea Europaea L.

This study evaluated the effects of supplementing with natural functional feed on the plasma fatty acid profile of lactating Italian Holstein-Friesian dairy cows. Thirty cows in mid-lactation received the natural olive extract PHENOFEED DRY (500 mg/cow/day) which mainly comprises hydroxytyrosol, tyrosol and verbascoside. The total content of polyphenols and the antioxidant power of standard feed, enriched feed and pure extract was evaluated respectively by Folin-Ciocalteu and DPPH assay, and a characterization in HPLC-UV (High-Performance Liquid Chromatography-Ultraviolet) of bioactive molecules present in the extract PHENOFEED DRY was performed. PHENOFEED DRY was provided for 60 days, and the plasma profile of fatty acids was determined by Gas Chromatography. The administration of enriched feed resulted in an increase in the ratio of Omega-6 to Omega-3 polyunsaturated fatty acids from 3:1 to 4:1 (p<0.001). This was not influenced by the calving order. The addition of polyphenols helped to keep monounsaturated (MUFA) and saturated (SFA) levels constant and results in a significant increase in polyunsaturated (PUFA) fatty acid after 15 days of administration. The Omega-6/Omega-3 ratio was in the optimal range. The findings show that inclusion of natural functional food such as plant polyphenols helps to maintain a healthy blood fatty acid profile in lactating dairy cows.

Chestnut Shell Tannins: Effects on Intestinal Inflammation and Dysbiosis in Zebrafish.

The aim of the present study was to test the possible ameliorative efficacy of phytochemicals such as tannins on intestinal inflammation and dysbiosis. The effect of a chestnut shell (Castanea sativa) extract (CSE) rich in polyphenols, mainly represented by tannins, on k-carrageenan-induced intestinal inflammation in adult zebrafish (Danio rerio) was tested in a feeding trial. Intestinal inflammation was induced by 0.1% k-carrageenan added to the diet for 10 days. CSE was administered for 10 days after k-carrageenan induced inflammation. The intestinal morphology and histopathology, cytokine expression, and microbiota were analyzed. The k-carrageenan treatment led to gut lumen expansion, reduction of intestinal folds, and increase of the goblet cells number, accompanied by the upregulation of pro-inflammatory factors (TNFα, COX2) and alteration in the number and ratio of taxonomic groups of bacteria. CSE counteracted the inflammatory status enhancing the growth of health helpful bacteria (Enterobacteriaceae and Pseudomonas), decreasing the pro-inflammatory factors, and activating the anti-inflammatory cytokine IL-10. In conclusion, CSE acted as a prebiotic on zebrafish gut microbiota, sustaining the use of tannins as food additives to ameliorate the intestinal inflammation. Our results may be relevant for both aquaculture and medical clinic fields.

Generation and functional characterization of a BCL10-inhibitory peptide that represses NF-kappaB activation.

The molecular complex containing BCL10 and CARMA [CARD (caspase recruitment domain)-containing MAGUK (membrane-associated guanylate kinase)] proteins has recently been identified as a key component in the signal transduction pathways that regulate activation of the transcription factor NF-kappaB (nuclear factor kappaB) in lymphoid and non-lymphoid cells. Assembly of complexes containing BCL10 and CARMA proteins relies on homophilic interactions established between the CARDs of these proteins. In order to identify BCL10-inhibitory peptides, we have established a method of assaying peptides derived from the CARD of BCL10 in binding competition assays of CARD-CARD self-association. By this procedure, a short peptide corresponding to amino acid residues 91-98 of BCL10 has been selected as an effective inhibitor of protein self-association. When tested in cell assays for its capacity to block NF-kappaB activation, this peptide represses activation of NF-kappaB mediated by BCL10, CARMA3 and PMA/ionomycin stimulation. Collectively, these results indicate that residues 91-98 of BCL10 are involved in BCL10 self-association and also participate in the interaction with external partners. We also show that blocking of the CARD of BCL10 may potentially be used for the treatment of pathological conditions associated with inappropriate NF-kappaB activation.

In vitro Synergy of Polyphenolic Extracts From Honey, Myrtle and Pomegranate Against Oral Pathogens, S. mutans and R. dentocariosa.

The increasing incidence rate of oral diseases, the wide spread of antimicrobial resistance, and the adverse effects of conventional antibiotics mean alternative prevention and treatment options are needed to counteract oral pathogens. In this regard, our study aims to evaluate the antibacterial activity of polyphenolic extracts prepared from acacia honey, myrtle leaves, and pomegranate peel against cariogenic bacteria, such as Streptococcus mutans and Rothia dentocariosa. The chemical-physical parameters of acacia honey and the RP-HPLC polyphenolic profile of pomegranate peel extract have been previously described in our studies, while the characterization of myrtle extract, performed by HPLC analysis, is reported here. All the extracts were used singly and in binary combinations to highlight any synergistic effects. Moreover, the extracts were tested in association with amoxicillin to evaluate their ability to reduce the effective dose of this drug in vitro. The values of minimal inhibitory concentrations and minimal bactericidal concentrations have been used to quantitatively measure the antibacterial activity of the single extracts, while the fractional inhibitory concentration index has been considered as predictor of in vitro anticariogenic synergistic effects. Finally, a time-kill curve method allowed for the evaluation of the bactericidal efficacy of the combined extracts. The microbiological tests suggest that acacia honey, myrtle, and pomegranate extracts are able to inhibit the cariogenic bacteria, also with synergistic effects. This study provides useful and encouraging results for the use of natural extract combinations alone or in association with antibiotics (adjuvant therapy) as a valid alternative for the prevention and treatment of oral infectious diseases.

Phytocompounds vs. Dental Plaque Bacteria: In vitro Effects of Myrtle and Pomegranate Polyphenolic Extracts Against Single-Species and Multispecies Oral Biofilms.

In the last decades, resistant microbial infection rate has dramatically increased, especially infections due to biofilm-producing strains that require increasingly complex treatments and are responsible for the increased mortality percentages compared with other infectious diseases. Considering that biofilms represent a key factor for a wide range of chronic infections with high drug tolerance, the treatment of biofilm-causing bacterial infections represents a great challenge for the future. Among new alternative strategies to conventional antimicrobial agents, the scientific interest has shifted to the study of biologically active compounds from plant-related extracts with known antimicrobial properties, in order to also evaluate their antibiofilm activity. In this regard, the aim of this study has been to assess the antibiofilm activity of polyphenolic extracts from myrtle leaf and pomegranate peel against oral pathogens of dental plaque, an excellent polymicrobial biofilm model. In particular, the in vitro antibiofilm properties of myrtle and pomegranate extracts, also in binary combination, were highlighted. In addition to inhibiting the biofilm formation, the tested polyphenolic extracts have been proven to destroy both preformed single-species and multispecies biofilms formed by Streptococcus mutans, Streptococcus oralis, Streptococcus mitis, and Rothia dentocariosa oral isolates, suggesting that the new natural sources are rich in promising compounds able to counteract biofilm-related infections.

Immunohistochemical Analysis of Intestinal and Central Nervous System Morphology in an Obese Animal Model (Danio rerio) Treated with 3,5-T2: A Possible Farm Management Practice?

The 3,5-diiodo-L-thyronine (3,5-T2) is an endogenous metabolite of thyroid hormones, whose administration to rodents fed high-fat diet (HFD) prevents body weight increase and reverts the expression pattern of pro-inflammatory factors associated to HFD. The diet-induced obese (D.I.O.) zebrafish (Danio rerio) has been recently used as an experimental model to investigate fundamental processes underlying central and peripheral obesity-driven inflammation. Herein, we aim to understand the role of 3,5-T2 in regulating central and peripheral inflammation in D.I.O. model of zebrafish. 3,5-T2 (10 nM and 100 nM) was administered with the obesity-inducing diet (D.I.O. with 3,5-T2) or after 4 weeks of obesity-inducing diet (D.I.O. flw 3,5-T2). 3,5-T2 significantly increased the body weight and serum triglyceride levels in D.I.O. zebrafish in both conditions. Moreover, 3,5-T2 sustained or increased inflammation in the anterior (AI) and mid (MI) intestine when administered with the obesity-inducing diet, as indicated by the immunoexpression of the inflammatory markers tumor-necrosis factor-α (TNFα), cyclooxygenase 2 (COX2), calnexin, caspase 3, and proliferating cell nuclear antigen (PCNA). On the contrary, when 3,5-T2 was administered after the obesity-inducing diet, partly reverted the intestinal alteration induced by D.I.O. In addition, brain inflammation, as indicated by the increase in the activation of microglia, was detected in D.I.O. zebrafish and D.I.O. treated with 3,5-T2. These findings reveal that the effects of 3,5-T2 on fish intestine and brain can deviate from those shown in obese mammals, opening new avenues to the investigation of the potential impact of this thyroid metabolite in different diseases including obesity.

Leptin effects on testis and epididymis in the lizard Podarcis sicula, during summer regression.

In this study we assessed the effect of leptin treatment on testicular morphology, spermatogenesis, Peroxisome Proliferator Activated Receptor (PPAR) alpha, 17beta-hydroxysteroide dehydrogenase, 17beta-estradiol and testosterone levels in the testis and blood of the lizard Podarcis sicula at the beginning of summer regression before entering the refractory period, when lizards no longer respond to hormonal and environmental stimuli. Lizards treated with five injections of leptin showed seminiferous tubules with germinal cells at all stages and wider lumina with respect to the controls. After 10 injections, the diameter of the lumina increased compared to the controls and 5 injection-group. After 10 injections plus 20 days before the sacrifice, the seminiferous tubules with open lumina and germinal cells were less abundant than in the 5 and 10 injection-groups. In all groups, the epididymis epithelium was higher than in the controls, with mitosis and binucleated cells. In both the control and treated animals secondary spermatocytes and spermatids were immunoreactive to leptin receptor and PPARalpha. In treated animals the interstitial cells and peritubular fibrocytes were also leptin receptor immunoreactive, while PPARalpha immunoreactivity translocated from the cytoplasm to the nucleus. 17beta-HSD immunoreactivity was present in the spermatids and interstitial cells of control lizards and in secondary spermatocytes and spermatids of treated lizards. Leptin treatment had no statistically significant effect on testicular and circulating 17beta-estradiol and testosterone levels. These observations indicate that leptin brings about a delay in testis summer regression in Podarcis sicula, playing a regulatory role in reproduction in this species as already hypothesized for mammals.

Comparative proteomic analysis of durum wheat shoots from modern and ancient cultivars.

Durum wheat is widespread cultivated in the Mediterranean basin, where it is used to produce high-quality semolina for pasta. Although over the years local and ancient wheat cultivars have been replaced by new ones, better suited to intensive cultivation, the increasing demand of consumers for nutritional and sensory qualities, as well as their attention to sustainable agronomic practices, renewed the interest toward traditional varieties. In order to fully exploit their agronomical and nutritional potential, a systematic analysis of molecular traits would be desirable. Nowadays, this examination is greatly facilitated by the current availability of high-throughput genomic and proteomic methods, which are integrated with classical measurements on plant physiology. To this purpose, we performed a comparative study on germination performances, hormone level variations, and differential protein representations of three-days germinated shoots of two traditional wheat cultivars from Southern Italy, namely Senatore Cappelli and Saragolla, and the commercial elite variety Svevo. Two-dimensional electrophoresis- and nanoLC-ESI-LIT-MS/MS-based proteomic analysis revealed 45 differentially represented spots, which were associated with 32 non-redundant protein species grouping into storage, stress/defense and metabolism/energy production functional categories. Major differences in the traditional varieties concerned over-representation of glutenins, gamma-gliadin and some enzymes of glycolysis and TCA cycle, as well as a down-representation of proteins involved in the response to stress conditions. These features were here discussed in relation to the hormone profile and the known agronomic features of traditional varieties, as compared to the commercial one.

Evidence for leptin receptor immunoreactivity in the gastrointestinal tract and gastric leptin regulation in the rainbow trout (Oncorhynchus mykiss).

In this study, evidence for leptin receptor (LR) and gastric leptin immunoreactivity along the digestive tract of the rainbow trout (Oncorhynchus mykiss), is reported. Besides this, the regulation of gastric leptin and its transcript by fatty acids was analyzed in vitro. LR was detected mainly in the cells of the stomach gastric glands and in the brush border of the epithelium of the anterior, middle and distal intestine. In the stomach LR was co-distributed with leptin. The regulation of gastric leptin and its transcript by fatty acids was analyzed by in vitro incubations. Rabbit polyclonal antibodies anti rainbow trout leptin were developed and employed to detect leptin concentration in the stomach and in the incubation medium. Stomach slices were incubated with butyric (4:0), oleic (18:1n-9), α-linolenic (18:3n-3) and arachidonic fatty acids (20:4n-6). All fatty acids caused an increase in the protein in both the stomach and culture medium, while leptin transcript was not modified. Overall, the results confirm the gastric leptin release upon nutritional modulation.

Overlapping Distribution of Orexin and Endocannabinoid Receptors and Their Functional Interaction in the Brain of Adult Zebrafish.

Hypocretins/Orexins neuropeptides are known to regulate numerous physiological functions, such as energy homeostasis, food intake, sleep/wake cycle, arousal and wakefulness, in vertebrates. Previous studies on mice have revealed an intriguing orexins/endocannabinoids (ECs) signaling interaction at both structural and functional levels, with OX-A behaving as a strong enhancer of 2-arachydonoyl-glycerol (2-AG) biosynthesis. In this study, we describe, for the first time in the brain of zebrafish, the anatomical distribution and co-expression of orexin (OX-2R) and endocannabinoid (CB1R) receptors, suggesting a functional interaction. The immunohistochemical colocalization of these receptors by confocal imaging in the dorsal and ventral telencephalon, suprachiasmatic nucleus (SC), thalamus, hypothalamus, preoptic area (PO) and cerebellum, is reported. Moreover, biochemical quantification of 2-AG levels by LC-MS supports the occurrence of OX-A-induced 2-AG biosynthesis in the zebrafish brain after 3 h of OX-A intraperitoneal (i.p.; 3 pmol/g) or intracerebroventricular (i.c.v.; 0.3 pmol/g) injection. This effect is likely mediated by OX-2R as it is counteracted by i.p./i.c.v administration of OX-2R antagonist (SB334867, 10 pmol/g). This study provides compelling morphological and functional evidence of an OX-2R/CB1R signaling interaction in the brain of adult zebrafish, suggesting the use of this well-established vertebrate animal model for the study of complex and phylogenetically conserved physiological functions.

The Case Study of Nesfatin-1 in the Pancreas of Tursiops truncatus.

Nesfatin-1 (Nesf-1) is an anorexigenic peptide involved in the regulation of homeostatic feeding. Nesf-1 is expressed in the central nervous system and other organs, including pancreas, where it promotes the release of insulin from ß-cells. This raises the possibility that Nesf-1 dysfunction could be involved in metabolic disorders, particularly in type 2 diabetes mellitus (T2D). Recently, it has been discovered that dolphins can be a natural animal model that fully replicates human T2D, due to its prolonged glucose tolerance curve and maintenance of a state of hyperglycemia similar to human T2D during fasting. This correspondence suggests that dolphins may be a suitable model for investigating physiological and pathological metabolic disorders. Here, we have characterized Nesf-1 distribution in the pancreas of the common bottlenose dolphin (Tursiops truncatus) and measured plasmatic levels of Nesf-1 and glucose during fasting and post-prandial states. The Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padova provided us with pancreas samples, derived from four animals, and plasma samples, collected before and after the main meal. Interestingly, our results showed that Nesf-1-immunoreactive cells were distributed in Langerhans islets, co-localized with glucagon in α-cells. Similar to humans, dolphin plasma Nesf-1 concentration doesn't show a statistically significant difference when comparing fasting and post-prandial states. On the other hand, blood glucose levels were significantly higher before than after the main meal. Our data provide a comparative analysis for further studies on the involvement of Nesf-1 in mammalian metabolic disorders.