Fibronectin type III domain-containing protein 5 (FNDC5)-like immunoreactivity and mRNA abundance in domestic animal tissues.

Irisin is a 112-amino acid peptide hormone that is cleaved from fibronectin type III domain-containing protein 5 (FNDC5), a type I transmembrane protein abundantly found in muscle tissue. Irisin is a putative mediator of the benefits of exercise, neuroprotection, bone growth, and cardiac health. However, few studies have focused on irisin in domestic animals. Further, whether processed irisin is detectable in domestic animal tissues remains uncertain. To address this, we determined FNDC5 mRNA and protein concentration in anatine (duck) and porcine (pig) skeletal muscle, and in equine (horse), swine, and anatine serum samples. RT-PCR analysis identified FNDC5 mRNA in all pig and duck skeletal muscle samples. An approximately 25 kDa band representing FNDC5 was detected in both pig and duck skeletal muscle. Fluorescence immunohistochemistry using a rabbit monoclonal FNDC5/irisin primary antibody and a goat polyclonal anti-rabbit secondary antibody localized FNDC5/irisin-like immunoreactivity in both the glandular and muscular regions of pig stomach. FNDC5/irisin-like immunoreactivity was also identified in horse, pig, and duck serum using a multispecies irisin ELISA. The average values of irisin-like immunoreactivity were 13.7 (duck), 15.4 (horse), and 7.0 (pig) ng/mL in samples tested. Our results support the presence of irisin precursor in several domestic animals. Processed irisin, however, was not detectable. Further studies are required to validate reliable tools to detect and quantify processed irisin in domestic animals.

Irisin in domestic animals.

Irisin is a 112 amino acid peptide hormone cleaved from the fibronectin type III domain-containing protein. Irisin is highly conserved across vertebrates, suggesting evolutionarily conserved common functions among domestic animals. These functions include the browning of white adipose tissue and increased energy expenditure. Irisin has been detected and studied primarily in plasma, serum, and skeletal muscle, but has also been found in adipose tissue, liver, kidney, lungs, cerebrospinal fluid, breast milk, and saliva. This wider tissue presence of irisin suggests additional functions beyond its role as a myokine in regulating energy use. We are beginning to understand irisin in domestic animals. The goal of this review is to provide an up-to-date commentary on irisin structure, tissue distribution, and functions across vertebrates, especially mammals of importance in veterinary medicine. Irisin could be explored as a potential candidate for developing therapeutic agents and biomarkers in domestic animal endocrinology.

Safety and efficacy assessment of a GLP-1 mimetic: insulin glargine combination for treatment of feline diabetes mellitus.

A commonly used therapeutic strategy for type 2 diabetes mellitus (DM) in humans involves the use of synthetic incretin hormone-based therapies including exenatide, a glucagon-like pepetide-1 hormone agonist. Glucagon-like pepetide-1 agonists can be used alone or as an ancillary therapy with other agents, including insulin and oral antihyperglycemics. Little is known about the role of these therapies for DM in cats. Therefore, the primary objective of this study was to evaluate the safety and efficacy of short-acting exenatide combined with insulin, as compared to placebo and insulin for the treatment of DM in cats. Treatment with exenatide was well tolerated; only 2 cats developed side effects requiring dose reduction. Two cats (25%) went into diabetic remission while receiving exenatide and insulin, whereas remission was not reported during placebo treatment. The average change in the daily exogenous insulin dose was significant (ß = -0.56 U/kg, 95% confidence interval, -0.96 to -0.15, P = 0.007), and the dose of insulin administered was lower during exenatide treatment. The average weight loss experienced on exenatide was significantly higher than on placebo (ß = 0.65 kg, 95% confidence interval, 0.09-1.21, P = 0.02). There was no significant difference in any of the hormone concentrations evaluated for cats on exenatide vs placebo treatments. Overall, the treatment of diabetic cats with insulin and a fixed dose of exenatide was found to be safe. The weight loss and decreased exogenous insulin requirement experienced with exenatide treatment could be a significant benefit for overweight diabetic cats and warrants further evaluation.

Tissue-specific expression and circulating concentrations of nesfatin-1 in domestic animals.

Nesfatin-1 is a naturally occurring 82-amino acid protein encoded in the precursor nucleobindin-2 (NUCB2) and has been implicated in multiple physiological functions, including food intake and blood glucose regulation. This study aimed to characterize nesfatin-1 in domestic species, especially cats (Felis catus), dogs (Canis lupus familiaris), and pigs (Sus scrofa). Our in silico analysis demonstrated that the NUCB2/nesfatin-1 amino acid sequence, especially the bioactive core region of the peptide, is very highly conserved (more than 90% identity) in domestic animals. Expression of mRNAs encoding NUCB2/nesfatin-1 was detected in the cat, dog, and pig stomach and pancreas. Immunohistochemistry revealed the presence of nesfatin-1 in the gastric mucosa of the stomach of dogs, cats, and pigs, and in the pancreatic islet ß-cells of dogs and pigs. No nesfatin-1 immunoreactivity was found in the cat pancreas. Nesfatin-1 was detected in the serum of dog, cat, pig, bison, cow, horse, sheep, and chicken. Circulating nesfatin-1 in male and female dogs remained unchanged at 60 min after glucose administration, suggesting a lack of meal responsiveness in nesfatin-1 secretion in this species. The presence of nesfatin-1 in the gastric and endocrine pancreatic tissues suggests possible roles for this peptide in the metabolism of domestic animals. Future research should focus on elucidating the species-specific functions and mechanisms of action of nesfatin-1 in health and disease of domestic animals.

Gonadotropin-releasing hormone, kisspeptin, and gonadal steroids directly modulate nucleobindin-2/nesfatin-1 in murine hypothalamic gonadotropin-releasing hormone neurons and gonadotropes.

Neuroendocrine regulation of metabolism and reproduction are tightly interlinked. Nesfatin-1 is an 82 amino acid metabolic peptide derived from nucleobindin-2 (NUCB2). NUCB2 mRNA and protein significantly increase in the hypothalamus of rats during puberty-to-adult transition. Administration of nesfatin-1 modulates circulating LH and testosterone in male rats. However, whether nesfatin-1 acts directly on neurons and gonadotropes remain unknown. In addition, whether reproductive hormones of the hypothalamo-pituitary gonadal axis modulate NUCB2/nesfatin-1 is unclear. To address these, we employed murine hypothalamic (GT1-7) and pituitary (LßT2) cells in vitro. Nucb2 expression, and NUCB2/nesfatin-1 immunoreactivity were observed in both GT1-7 and LßT2 cells, and in the hypothalamus of mice. Nesfatin-1 co-localized GnRH in GT1-7 cells, and in the hypothalamic perikarya of mice. Cells were treated with kisspeptin, GnRH, and estradiol and testosterone, as well as nesfatin-1 for 2, 6 or 24 hours. Synthetic nesfatin-1 increased Kiss1r and Gnrh expression in GT1-7 cells and Lhß in LßT2. Nesfatin-1 increased GnRH and LHß protein expression in GT1-7 and LßT2 at 6-hour post incubation respectively. Both NUCB2 mRNA and protein were increased in GT1-7 cells treated with kisspeptin. Testosterone increased NUCB2 mRNA and protein expression in GT1-7 and LßT2. 17ß-estradiol increased NUCB2 mRNA and protein expression in LßT2. Nesfatin-1 acts directly on hypothalamic neurons and gonadotropes to elicit a generally positive influence on the endocrine milieu regulating reproduction in mice. Reproductive hormones, in turn, modulate brain and pituitary NUCB2/nesfatin-1. In conclusion, we provide additional information to designate nesfatin-1 as a novel, additional factor that helps reproductive success.

Mass spectrometry-assisted confirmation of the inability of dipeptidyl peptidase-4 to cleave goldfish peptide YY(1-36) and the lack of anorexigenic effects of peptide YY(3-36) in goldfish (Carassius auratus).

Dipeptidyl peptidase-4 (DPP4) is a serine protease of great interest because it has been shown to modulate the activity of several peptidergic factors including peptide YY (PYY) and glucagon-like peptide-1/2. While PYY(1-36) is orexigenic in mammals, PYY(3-36) recently garnered interest as a potent anorexigen. In silico phylogenetic analysis found that the DPP4 cleavage sites are absent in fish PYY sequences. However, no studies were conducted to show that indeed PYY(3-36) is not produced by DPP4 in fish. If DPP4 does not cleave PYY(1-36), is PYY(3-36) an anorexigen in fish? The objectives of this research were to (1) test whether DPP4 cleaves goldfish PYY(1-36) and (2) determine whether PYY(3-36) is an anorexigen in goldfish. First, we identified the highly conserved catalytic region of DPP4 in goldfish. Abundant expression of DPP4 mRNA was found within the gastrointestinal tract. We also report the first MALDI-MS cleavage analysis of DPP4 effects on PYY(1-36) in a non-mammalian vertebrate. Our novel results indicate that DPP4 is unable to cleave goldfish PYY(1-36) to PYY(3-36) in vitro. It also confirms a previously held hypothesis that DPP4 is unable to cleave fish PYY(1-36) that contains N-terminal proline-proline residues. PYY(3-36) had no effects on food intake of goldfish. The appetite inhibitory effects of intraperitoneal and intracerebroventricular injections of 10 ng/g body weight gfPYY(1-36) were abolished by coinjections of BIBP3226, a Y1 receptor antagonist. These results are significant because it shows the lack of generation of endogenous PYY(3-36) and its anorectic effects in goldfish.

Nesfatin-1 suppresses energy intake, co-localises ghrelin in the brain and gut, and alters ghrelin, cholecystokinin and orexin mRNA expression in goldfish.

Nesfatin-1 is a novel anorectic peptide encoded in the precursor protein nucleobindin-2 (NUCB2). We recently reported the presence and appetite suppressing effects of nesfatin-1 in goldfish. Nesfatin-1 has been co-localised with ghrelin in the stomach of rats. Whether nesfatin-1 influences other appetite regulatory peptides in goldfish remains unclear. The main objectives of the present study were to investigate whether nesfatin-1 co-localises ghrelin in goldfish, and to test whether exogenous nesfatin-1 influences endogenous ghrelin, cholecystokinin (CCK) and orexin A (OXA). We found co-localisation of nesfatin-1-like and ghrelin-like immunoreactivity in the enteroendocrine cells of the goldfish anterior intestine (J-loop). Furthermore, co-localisation of ghrelin and nesfatin-1 was also observed in the posterior nucleus lateralis tuberis of the goldfish hypothalamus, a brain region implicated in the regulation of food intake. These findings suggest a functional relationship between ghrelin and nesfatin-1 in goldfish. In support of this, i.c.v. administration of goldfish (gf) nesfatin-1 [25 ng/g body weight (BW)], suppressed food intake and the expression of mRNAs encoding preproghrelin, ghrelin receptor (GHS-R 1a-1), CCK and NUCB2 in the forebrain of fed fish, as well as ghrelin and NUCB2 mRNA in the hypothalamus of unfed fish, both at 1 h post-injection. Nesfatin-1 stimulated hypothalamic CCK mRNA expression at 30 min post-injection in fed fish, and inhibited OXA mRNA in the unfed fish hypothalamus 1 h post-injection. Similarly, i.c.v. injections of gfghrelin (1 ng/g BW), although stimulating food intake, suppressed NUCB2 and preproghrelin mRNAs, but not ghrelin receptor mRNA expression in the forebrain. It is also evident that exogenous ghrelin and nesfatin-1 mRNAs encoding these peptides. Our novel results indicate interactions between nesfatin-1 and ghrelin, CCK and orexin, and show that nesfatin-1 acts on other appetite regulatory peptides in a time- and feeding status-dependent, as well as tissue-specific, manner in goldfish.

Nutrient responsive nesfatin-1 regulates energy balance and induces glucose-stimulated insulin secretion in rats.

Nesfatin-1 is a recently discovered anorexigen, and we first reported nesfatin-like immunoreactivity in the pancreatic ß-cells. The aim of this study was to characterize the effects of nesfatin-1 on whole-body energy homeostasis, insulin secretion, and glycemia. The in vivo effects of continuous peripheral delivery of nesfatin-1 using osmotic minipumps on food intake and substrate partitioning were examined in ad libitum-fed male Fischer 344 rats. The effects of nesfatin-1 on glucose-stimulated insulin secretion (GSIS) were examined in isolated pancreatic islets. L6 skeletal muscle cells and isolated rat adipocytes were used to assess the effects of nesfatin-1 on basal and insulin-mediated glucose uptake as well as on major steps of insulin signaling in these cells. Nesfatin-1 reduced cumulative food intake and increased spontaneous physical activity, whole-body fat oxidation, and carnitine palmitoyltransferase I mRNA expression in brown adipose tissue but did not affect uncoupling protein 1 mRNA in the brown adipose tissue. Nesfatin-1 significantly enhanced GSIS in vivo during an oral glucose tolerance test and improved insulin sensitivity. Although insulin-stimulated glucose uptake in L6 muscle cells was inhibited by nesfatin-1 pretreatment, basal and insulin-induced glucose uptake in adipocytes from nesfatin-1-treated rats was significantly increased. In agreement with our in vivo results, nesfatin-1 enhanced GSIS from isolated pancreatic islets at both normal (5.6 mM) and high (16.7 mM), but not at low (2 mM), glucose concentrations. Furthermore, nesfatin-1/nucleobindin 2 release from rat pancreatic islets was stimulated by glucose. Collectively, our data indicate that glucose-responsive nesfatin-1 regulates insulin secretion, glucose homeostasis, and whole-body energy balance in rats.

Effects of dipeptidyl peptidase IV on the satiety actions of peptide YY.

AIMS/HYPOTHESIS: Dipeptidyl peptidase IV (DP IV) inhibitors are currently being developed to prolong the biological activity of insulinotropic peptides as a novel approach in the treatment of diabetes. We hypothesised that DP IV inhibition could attenuate the satiety actions of peptide YY (PYY) by altering the conversion of PYY(1-36) to PYY(3-36). MATERIALS AND METHODS: The effects of PYY delivered by osmotic mini-pumps were assessed in rats treated with a DP IV inhibitor and in a rat model deficient in DP IV. RESULTS: Pharmacological levels of total PYY were found in the circulation after the exogenous administration of PYY(3-36). While both PYY(1-36) and PYY(3-36) reduced food intake in normal rats, PYY(1-36) was ineffective in rats deficient in DP IV. When re-fed after a 24-h fast, DP IV-deficient rats exhibited higher food intake and weight gain than normal rats. Moreover, unlike controls, there was no postprandial increase in PYY levels in DP IV-deficient rats. Despite these findings, administration of a DP IV inhibitor, Pro-boroPro, did not alter the acute anorectic effects of exogenous PYY(1-36) in normal rats. This could be the result of the protection of other appetite regulatory peptides or the generation of PYY(3-36) by remaining DP IV activity or other dipeptidyl peptidases. CONCLUSIONS/INTERPRETATION: Although DP IV inhibition with Pro-boroPro attenuated the generation of PYY(3-36), our results indicate that short-term DP IV inhibition does not eliminate the satiety actions of exogenously administered PYY(1-36) at the doses tested.

Neuropeptides and the control of food intake in fish.

The brain, particularly the hypothalamus, integrates input from factors that stimulate (orexigenic) and inhibit (anorexigenic) food intake. In fish, the identification of appetite regulators has been achieved by the use of both peptide injections followed by measurements of food intake, and by molecular cloning combined with gene expression studies. Neuropeptide Y (NPY) is the most potent orexigenic factor in fish. Other orexigenic peptides, orexin A and B and galanin, have been found to interact with NPY in the control of food intake in an interdependent and coordinated manner. On the other hand cholecystokinin (CCK), cocaine and amphetamine-regulated transcript (CART), and corticotropin-releasing factor (CRF) are potent anorexigenic factors in fish, the latter being involved in stress-related anorexia. CCK and CART have synergistic effects on food intake and modulate the actions of NPY and orexins. Although leptin has not yet been identified in fish, administration of mammalian leptin inhibits food intake in goldfish. Moreover, leptin induces CCK gene expression in the hypothalamus and its actions are mediated at least in part by CCK. Other orexigenic factors have been identified in teleost fish, including the agouti-related protein (AgRP) and ghrelin. Additional anorexigenic factors include bombesin (or gastrin-releasing peptide), alpha-melanocyte-stimulating hormone (alpha-MSH), tachykinins, and urotensin I. In goldfish, nutritional status can modify the expression of mRNAs encoding a number of these peptides, which provides further evidence for their roles as appetite regulators: (1) brain mRNA expression of CCK, CART, tachykinins, galanin, ghrelin, and NPY undergo peri-prandial variations; and (2) fasting increases the brain mRNA expression of NPY, AgRP, and ghrelin as well as serum ghrelin levels, and decreases the brain mRNA expression of tachykinins, CART, and CCK. This review will provide an overview of recent findings in this field.