Effect of relaxin on semen quality variables of cryopreserved stallion semen.

The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 106 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferred into 0.5 mL straws, equilibrated at 5 °C for 30 min, and placed in liquid nitrogen (LN2) vapour for 15 min before being plunged into LN2. After thawing, sperm samples were evaluated for motility and velocity variables, mitochondrial membrane potential, apoptosis, and plasma membrane and DNA integrities. For sperm motility variables, there were dose- and time-dependent effects, with the largest values recorded when 12.5 and 25 ng/mL relaxin were used for 0-120 min of incubation. Furthermore, at all of the concentrations at which there were evaluations, relaxin additions to semen diluent led to a marked improvement in sperm mitochondrial membrane potential and a lesser percentage of apoptotic cells compared to the control group. Plasma membranes and DNA integrities were not affected by relaxin supplementations to the diluent. In conclusion, supplementation of relaxin in extender before semen cryopreservation, especially at 12.5 and 25 ng/mL, had a positive effect on the sperm quality variables.

GH-Releasing Hormone Promotes Survival and Prevents TNF-α-Induced Apoptosis and Atrophy in C2C12 Myotubes.

Skeletal muscle atrophy is a consequence of different chronic diseases, including cancer, heart failure, and diabetes, and also occurs in aging and genetic myopathies. It results from an imbalance between anabolic and catabolic processes, and inflammatory cytokines, such as TNF-α, have been found elevated in muscle atrophy and implicated in its pathogenesis. GHRH, in addition to stimulating GH secretion from the pituitary, exerts survival and antiapoptotic effects in different cell types. Moreover, we and others have recently shown that GHRH displays antiapoptotic effects in isolated cardiac myocytes and protects the isolated heart from ischemia/reperfusion injury and myocardial infarction in vivo. On these bases, we investigated the effects of GHRH on survival and apoptosis of TNF-α-treated C2C12 myotubes along with the underlying mechanisms. GHRH increased myotube survival and prevented TNF-α-induced apoptosis through GHRH receptor-mediated mechanisms. These effects involved activation of phosphoinositide 3-kinase/Akt pathway and inactivation of glycogen synthase kinase-3ß, whereas mammalian target of rapamycin was unaffected. GHRH also increased the expression of myosin heavy chain and the myogenic transcription factor myogenin, which were both reduced by the cytokine. Furthermore, GHRH inhibited TNF-α-induced expression of nuclear factor-κB, calpain, and muscle ring finger1, which are all involved in muscle protein degradation. In summary, these results indicate that GHRH exerts survival and antiapoptotic effects in skeletal muscle cells through the activation of anabolic pathways and the inhibition of proteolytic routes. Overall, our findings suggest a novel therapeutic role for GHRH in the treatment of muscle atrophy-associated diseases.

Obestatin: is it really doing something?

Obestatin was identified in 2005 by Zhang and colleagues as a ghrelin-associated peptide, derived from posttranslational processing of the prepro-ghrelin gene. Initially, obestatin was reported to activate the G-protein-coupled receptor GPR39 and to reduce food intake and gastric emptying. However, obestatin remains a controversial peptide, as these findings have been questioned and its receptor is still a matter of debate, as well as its effects on feeding behavior. Recently, interaction with the glucagon-like peptide 1 receptor has been suggested, in line with obestatin-positive effects on glucose and lipid metabolism. In addition, obestatin displays a variety of cellular effects, by regulating metabolic cell functions, increasing cell survival and proliferation, and inhibiting apoptosis and inflammation in different cell types. Finally, like ghrelin, obestatin is produced in the gastrointestinal tract, including the pancreas and adipose tissue, and exerts both local actions in peripheral tissues, and distant effects at the central level. Therefore, obestatin may indeed be considered a hormone, although additional studies are required to clarify its physiopathological role and to definitely identify its receptor.

RFamide peptides 43RFa and 26RFa both promote survival of pancreatic ß-cells and human pancreatic islets but exert opposite effects on insulin secretion.

RFamide peptides 43RFa and 26RFa have been shown to promote food intake and to exert different peripheral actions through G-protein-coupled receptor 103 (GPR103) binding. Moreover, 26RFa was found to inhibit pancreatic insulin secretion, whereas the role of 43RFa on ß-cell function is unknown, as well as the effects of both peptides on ß-cell survival. Herein, we investigated the effects of 43RFa and 26RFa on survival and apoptosis of pancreatic ß-cells and human pancreatic islets. In addition, we explored the role of these peptides on insulin secretion and the underlying signaling mechanisms. Our results show that in INS-1E ß-cells and human pancreatic islets both 43RFa and 26RFa prevented cell death and apoptosis induced by serum starvation, cytokine synergism, and glucolipotoxicity, through phosphatidylinositol 3-kinase/Akt- and extracellular signal-related kinase 1/2-mediated signaling. Moreover, 43RFa promoted, whereas 26RFa inhibited, glucose- and exendin-4-induced insulin secretion, through Gαs and Gαi/o proteins, respectively. Inhibition of GPR103 expression by small interfering RNA blocked 43RFa insulinotropic effect, but not the insulinostatic action of 26RFa. Finally, 43RFa, but not 26RFa, induced cAMP increase and glucose uptake. In conclusion, because of their survival effects along with the effects on insulin secretion, these findings suggest potential for 43RFa and 26RFa as therapeutic targets in the treatment of diabetes.

Human recombinant lysozyme downregulates advanced glycation endproduct-induced interleukin-6 production and release in an in-vitro model of human proximal tubular epithelial cells.

Diabetic nephropathy is the leading cause of chronic renal disease and one of the major causes of cardiovascular mortality. Evidence suggests that its progression is due to the chronic hyperglycemia consequent to the production and accumulation of advanced glycation endproducts (AGEs). Lysozyme was shown to posses AGE-sequestering properties and the capacity to reduce the severity of the early stage manifestations of the diabetic nephropathy. This study was aimed to contribute to the understanding the molecular mechanisms of lysozyme effectiveness in the diabetic nephropathy, using an in-vitro cellular model, represented by the HK-2 cells, human proximal tubular epithelial cells. Lysozyme significantly reduced the AGE-induced IL-6 mRNA and an ELISA assay showed also a decreased release of the functional protein with a dose-dependent trend. In addition, lysozyme prevented macrophage recruitment, suggesting its capacity to elicit an anti-inflammatory action. We may conclude that the protective action of lysozyme on the nephrotoxic effects of AGE may depend, at least in part, on its ability to prevent the production and release of inflammatory mediators, such as IL-6 and to reduce macrophage recruitment in the inflammatory sites.

Pyroglutamylated RF-amide peptide (QRFP) gene is regulated by metabolic endotoxemia.

Pyroglutamylated RF-amide peptide (QRFP) is involved in the regulation of food intake, thermogenesis, adipogenesis, and lipolysis. The expression of QRFP in adipose tissue is reduced in diet-induced obesity, a mouse model in which plasma concentrations of endotoxins are slightly elevated. The present study investigated the role of metabolic endotoxemia (ME) on QRFP gene regulation. Our results uncovered the expression of QRFP in murine macrophages and cell lines. This expression has been found to be decreased in mice with ME. Low doses of lipopolysaccharide (LPS) transiently down-regulated QRFP by 59% in RAW264.7 macrophages but not in 3T3-L1 adipocytes. The effect of LPS on QRFP expression in macrophages was dependent on the inhibitor of kB kinase and TIR-domain-containing adapter-inducing interferon (IFN)-ß (TRIF) but not myeloid differentiation primary response gene 88. IFN-ß was induced by ME in macrophages. IFN-ß sustainably reduced QRFP expression in macrophages (64%) and adipocytes (49%). IFN-γ down-regulated QRFP (74%) in macrophages only. Both IFNs inhibited QRFP secretion from macrophages. LPS-stimulated macrophage-conditioned medium reduced QRFP expression in adipocytes, an effect blocked by IFN-ß neutralizing antibody. The effect of IFN-ß on QRFP expression was dependent on phosphoinositide 3-kinase, p38 MAPK, and histone deacetylases. The effect of IFN-γ was dependent on MAPK/ERK kinase 1/2 and histone deacetylases. Macrophage-conditioned medium containing increased amounts of QRFP preserved adipogenesis in adipocytes. In conclusion, LPS induces IFN-ß release from macrophages, which reduces QRFP expression in both macrophages and adipocytes in an autocrine/paracrine-dependent manner, suggesting QRFP as a potential biomarker in ME.

Obestatin: a new metabolic player in the pancreas and white adipose tissue.

Obestatin is a 23 amino acid amidated peptide, member of the preproghrelin gene-derived peptides. Initially, obestatin was reported to exert opposite effects to those of ghrelin on food intake and body weight gain, through interaction with GPR39; however, these findings are still strongly debated and obestatin biological role remains largely unknown. Interestingly, binding of obestatin to the glucagon-like peptide 1 receptor has been recently suggested. Despite being a controversial peptide, recent findings have clearly indicated that obestatin is indeed a multifunctional peptide, exerting a variety of effects, such as stimulation of cell proliferation, survival and differentiation, influence on glucose and lipid metabolism, as well as anti-inflammatory and cardioprotective actions. Its positive effects on glucose and lipid metabolism candidate this peptide as a potential therapeutic tool in pathological conditions such as insulin resistance and diabetes.

Obestatin enhances in vitro generation of pancreatic islets through regulation of developmental pathways.

Availability of large amounts of in vitro generated ß-cells may support replacement therapy in diabetes. However, methods to obtain ß-cells from stem/progenitor cells are limited by inefficient endocrine differentiation. We have recently shown that the ghrelin gene product obestatin displays beneficial effects on pancreatic ß-cell survival and function. Obestatin prevents ß-cell apoptosis, preserves ß-cell mass and stimulates insulin secretion in vitro and in vivo, in both normal and diabetic conditions. In the present study, we investigated whether obestatin may promote in vitro ß-cell generation from mouse pancreatic islet-derived precursor cells. Treatment of cultured islets of Langerhans with obestatin (i) enriched cells expressing the mesenchymal/neuronal marker nestin, which is associated with pancreatic precursors; (ii) increased cell survival and reduced apoptosis during precursor selection; (iii) promoted the generation of islet-like cell clusters (ICCs) with increased insulin gene expression and C-peptide secretion. Furthermore, obestatin modulated the expression of fibroblast growth factor receptors (FGFRs), Notch receptors and neurogenin 3 (Ngn3) during islet-derived precursor cell selection and endocrine differentiation. These results indicate that obestatin improves the generation of functional ß-cells/ICCs in vitro, suggesting implications for cell-based replacement therapy in diabetes. Moreover, obestatin may play a role in regulating pathways involved in pancreas development and regeneration.

Microencapsulation of bioactive principles with an airless spray-gun suitable for processing high viscous solutions.

PURPOSE: to design, assemble and test a prototype of a novel production plant, suitable for producing microparticles (MPs) by processing highly viscous feed solutions (FSs). METHODS: the prototype has been built using a commercial air compressor, a piston pump, an airless spray-gun, a customized air-treatment section, a timer, a rotating base, and a filtration section. Preliminary prototype parameter setting was carried out to individuate the best performing nozzle's dimension, the nebulization timing, and the CaCl2 concentration in the gelation fluid. In addition, prototype throughput (1 L to 5 L) and the range of practicable feed solution (FS) viscosities were assayed. A set of four batches was prepared in order to characterize the MPs, in terms of mean particle size and distribution, flow properties, swelling, encapsulation efficiency and release. RESULTS: according to a qualitative scoring, the large nozzle was suitable to nebulize FSs at a higher alginate concentration. Conversely, the small nozzle performed better in the processing of FSs with an alginate concentration up to 2% w/v. Only at the highest degree of viscosity, corresponding to 5% w/v of alginate, the FS processing was not technically possible. Among the CaCl2 concentrations considered, 15% w/v was recognized as the most versatile. The prototype appears to be convenient and suitable to grant a high yield starting from 2 L of FS. The flow behavior of the FSs assayed can be satisfactorily described with the Carreau-Yasuda equation and the throughput begins to slightly decrease for FSs at alginate concentrations exceeding 3% w/v. MP morphology was irregular with crumpled shape. The angle of repose indicates a good flowability and the release studies showed gastro-resistance and potential prolonged release applications. CONCLUSIONS: the novel prototype of production plant is suitable to process large amounts (2 L or more) of FSs, characterized by a high viscosity, to produce MPs suitable for bioactive principle delivery.

Obestatin regulates adipocyte function and protects against diet-induced insulin resistance and inflammation.

The metabolic actions of the ghrelin gene-derived peptide obestatin are still unclear. We investigated obestatin effects in vitro, on adipocyte function, and in vivo, on insulin resistance and inflammation in mice fed a high-fat diet (HFD). Obestatin effects on apoptosis, differentiation, lipolysis, and glucose uptake were determined in vitro in mouse 3T3-L1 and in human subcutaneous (hSC) and omental (hOM) adipocytes. In vivo, the influence of obestatin on glucose metabolism was assessed in mice fed an HFD for 8 wk. 3T3-L1, hSC, and hOM preadipocytes and adipocytes secreted obestatin and showed specific binding for the hormone. Obestatin prevented apoptosis in 3T3-L1 preadipocytes by increasing phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 signaling. In both mice and human adipocytes, obestatin inhibited isoproterenol-induced lipolysis, promoted AMP-activated protein kinase phosphorylation, induced adiponectin, and reduced leptin secretion. Obestatin also enhanced glucose uptake in either the absence or presence of insulin, promoted GLUT4 translocation, and increased Akt phosphorylation and sirtuin 1 (SIRT1) protein expression. Inhibition of SIRT1 by small interfering RNA reduced obestatin-induced glucose uptake. In HFD-fed mice, obestatin reduced insulin resistance, increased insulin secretion from pancreatic islets, and reduced adipocyte apoptosis and inflammation in metabolic tissues. These results provide evidence of a novel role for obestatin in adipocyte function and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.

Des-acyl ghrelin fragments and analogues promote survival of pancreatic ß-cells and human pancreatic islets and prevent diabetes in streptozotocin-treated rats.

Des-acyl ghrelin, although devoid of binding to ghrelin receptor (GRLN), exerts many biological effects, including regulation of glucose and lipid metabolism. Indeed, des-acyl ghrelin promotes pancreatic ß-cell and human islet cell survival and prevents diabetes in streptozotocin (STZ) treated rats. We investigated whether des-acyl ghrelin fragments excluding serine(3), which is essential for binding to GRLN, would display similar actions. Among the different compounds tested, des-acyl ghrelin((6-13)) and des-acyl ghrelin((6-13)) with alanine substitutions or cyclization, but not with d-amino acid substitutions, showed the best survival effect, similar to des-acyl ghrelin. Des-acyl ghrelin((6-13)) even prevented diabetes in STZ-treated rats and protected human circulating angiogenic cells from oxidative stress and senescence, similar to des-acyl ghrelin. These results suggest that not only full-length des-acyl ghrelin but also short des-acyl ghrelin fragments have clear beneficial effects on several tissues in vitro and in vivo.

GPR103b functions in the peripheral regulation of adipogenesis.

The activation of G protein-coupled receptor 103 (GPR103) by its endogenous peptidic ligands, QRFPs, is involved in the central regulation of feeding by increasing food intake, body weight, and fat mass after intracerebroventricular injection in mice. However, the role of GPR103 in regulating peripheral metabolic pathways has not yet been explored. The present study aimed to investigate the role of GPR103 in adipogenesis and lipid metabolism using 3T3-L1 adipocyte cells. Our results show that differentiated 3T3-L1 cells expressed the GPR103b subtype mRNA and protein, as well as QRFP mRNA. QRFP-43 and -26 induced an increase in triglyceride accumulation of 50 and 41%, respectively, and elicited a dose-dependent increase in fatty acid uptake, by up to approximately 60% at the highest concentration, in 3T3-L1-differentiated cells. QRFP-43 and -26 inhibited isoproterenol (ISO)-induced lipolysis in a dose-dependent manner, with IC(50)s of 2.3 +/- 1.2 and 1.1 +/- 1.0 nm, respectively. The expression of genes involved in lipid uptake (FATP1, CD36, LPL, ACSL1, PPAR-gamma, and C/EBP-alpha), was increased by 2- to 3-fold after treatment with QRFP. The effects of QRFP on ISO-induced lipolysis and fatty acid uptake were abolished when GPR103b was silenced. In a mouse model of diet-induced obesity, the expression of GPR103b in epididymal fat pads was elevated by 16-fold whereas that of QRFP was reduced by 46% compared to lean mice. Furthermore, QRFP was bioactive in omental adipocytes from obese individuals, inhibiting ISO-induced lipolysis in these cells. Our results suggest that GPR103b and QRFP work in an autocrine/paracrine manner to regulate adipogenesis.

Obestatin promotes survival of pancreatic beta-cells and human islets and induces expression of genes involved in the regulation of beta-cell mass and function.

OBJECTIVE: Obestatin is a newly discovered peptide encoded by the ghrelin gene whose biological functions are poorly understood. We investigated obestatin effect on survival of beta-cells and human pancreatic islets and the underlying signaling pathways. RESEARCH DESIGN AND METHODS: beta-Cells and human islets were used to assess obestatin effect on cell proliferation, survival, apoptosis, intracellular signaling, and gene expression. RESULTS: Obestatin showed specific binding on HIT-T15 and INS-1E beta-cells, bound to glucagon-like peptide-1 receptor (GLP-1R), and recognized ghrelin binding sites. Obestatin exerted proliferative, survival, and antiapoptotic effects under serum-deprived conditions and interferon-gamma/tumor necrosis factor-alpha/interleukin-1 beta treatment, particularly at pharmacological concentrations. Ghrelin receptor antagonist [D-Lys(3)]-growth hormone releasing peptide-6 and anti-ghrelin antibody prevented obestatin-induced survival in beta-cells and human islets. beta-Cells and islet cells released obestatin, and addition of anti-obestatin antibody reduced their viability. Obestatin increased beta-cell cAMP and activated extracellular signal-related kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt; its antiapoptotic effect was blocked by inhibition of adenylyl cyclase/cAMP/protein kinase A (PKA), PI 3-kinase/Akt, and ERK1/2 signaling. Moreover, obestatin upregulated GLP-1R mRNA and insulin receptor substrate-2 (IRS-2) expression and phosphorylation. The GLP-1R antagonist exendin-(9-39) reduced obestatin effect on beta-cell survival. In human islets, obestatin, whose immunoreactivity colocalized with that of ghrelin, promoted cell survival and blocked cytokine-induced apoptosis through cAMP increase and involvement of adenylyl cyclase/cAMP/PKA signaling. Moreover, obestatin 1) induced PI 3-kinase/Akt, ERK1/2, and also cAMP response element-binding protein phosphorylation; 2) stimulated insulin secretion and gene expression; and 3) upregulated GLP-1R, IRS-2, pancreatic and duodenal homeobox-1, and glucokinase mRNA. CONCLUSIONS: These results indicate that obestatin promotes beta-cell and human islet cell survival and stimulates the expression of main regulatory beta-cell genes, identifying a new role for this peptide within the endocrine pancreas.

Reversible acute fetal renal failure due to maternal exposure to angiotensin receptor blocker.

We report on a case of fetal toxicity due to maternal treatment with olmesartan medoxomil. At 29 weeks' gestation, oligohydramnios was found, although the fetus had normal kidneys on ultrasound evaluation. Withdrawal of olmesartan medoxomil, maternal rehydration, and a single dose of furosemide reversed the renal impairment. After term delivery, the neonate was confirmed to have normal renal function. The case suggested that fetal renal impairment due to olmesartan medoxomil may be reversible.