Iacopo Gesmundo

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.—Granata, R., Gallo, D., Luque, R. M., Baragli, A., Scarlatti, F., Grande, C., Gesmundo, I., Córdoba‐Chacón, J., Bergandi, L., Settanni, F., Togliatto, G., Volante, M., Garetto, S., Annunziata, M., Chanclón, B., Gargantini, E., Rocchietto, S., Matera, L., Datta, G., Morino, M., Brizzi, M. F., Ong, H., Camussi, G., Castaño, J. P., Papotti, M., Ghigo, E. Obestatin regulates adipocyte function and protects against diet‐induced insulin resistance and inflammation. FASEB J. 26, 3393–3411 (2012). www.fasebj.org

Unacylated ghrelin and obestatin increase islet cell mass and prevent diabetes in streptozotocin-treated newborn rats

The ghrelin gene products, namely acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob), were shown to prevent pancreatic beta-cell death and to improve beta-cell function under treatment with cytokines, which are major cause of beta-cell destruction in diabetes. Moreover, AG had been described previously to prevent streptozotocin (STZ)-induced diabetes in rats; however, the effect of either UAG or Ob has never been examined in this context. In the present study, we investigated the potential of UAG and Ob to increase islet beta-cell mass and to reduce diabetes at adult age in STZ-treated neonatal rats. One-day-old rats were injected with STZ and subsequently administered with either AG, UAG or Ob for 7 days. On day 70, plasma glucose levels, plasma and pancreatic insulin levels, pancreatic islet area and number, insulin and pancreatic/duodenal homeobox-1 (Pdx1) gene expression, and antiapoptotic BCL2 protein expression were determined. Similarly to AG, both UAG and Ob counteracted STZ-induced high glucose levels and improved plasma and pancreatic insulin levels, which were reduced by the diabetogenic compound. UAG and Ob increased islet area, islet number, and beta-cell mass with respect to STZ treatment alone. Finally, in STZ-treated animals, UAG and Ob up-regulated insulin and Pdx1 mRNA and increased the expression of BCL2 similarly to AG. Taken together, our results suggest that in STZ-treated newborn rats, UAG and Ob improve glucose metabolism and preserve islet cell mass, granting a therapeutic potential in medical conditions associated with impaired beta-cell function.

RFamide Peptides 43RFa and 26RFa Both Promote Survival of Pancreatic β-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 β-cell function is unknown, as well as the effects of both peptides on β-cell survival. Herein, we investigated the effects of 43RFa and 26RFa on survival and apoptosis of pancreatic β-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 β-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.

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.

Obestatin Enhances In Vitro Generation of Pancreatic Islets through Regulation of Developmental Pathways

Availability of large amounts of in vitro generated β-cells may support replacement therapy in diabetes. However, methods to obtain β-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 β-cell survival and function. Obestatin prevents β-cell apoptosis, preserves β-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 β-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 β-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.

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 Plays an Opposite Role in the Regulation of Pituitary Somatotrope and Corticotrope Function in Female Primates and Male/Female Mice

Obestatin is a 23-amino-acid amidated peptide that is encoded by the ghrelin gene. Previous studies have shown obestatin can modulate the hypothalamic neuronal circuitry that regulates pituitary function, perhaps by modulating the actions of ghrelin. However, the direct actions of obestatin on pituitary function remain controversial. Here, primary pituitary cell cultures from a nonhuman primate (baboon) and mice were used to test the effects of obestatin on pituitary hormone expression and secretion. In pituitary cultures from both species, obestatin had no effect on prolactin, LH, FSH, or TSH expression/release. Conversely, obestatin stimulated proopiomelanocortin expression and ACTH release and inhibited GH expression/release in vitro, actions that were also observed in vivo in mice treated with obestatin. In vitro, obestatin inhibited the stimulatory actions of ghrelin on GH but not ACTH release. The inhibitory effect of obestatin on somatotrope function was associated with an overall reduction in pituitary transcription factor-1 and GHRH receptor mRNA levels in vitro and in vivo as well as a reduction in hypothalamic GHRH and ghrelin expression in vivo. The stimulatory effect of obestatin on ACTH was associated with an increase in pituitary CRF receptors. Obestatin also reduced the expression of pituitary somatostatin receptors (sst1/sst2), which could serve to modify its impact on hormone secretion. The in vitro actions of obestatin on both GH and ACTH release required the adenylyl cyclase and MAPK routes. Taken together, our results provide evidence that obestatin can act directly at the pituitary to control somatotrope and corticotrope function, and these effects are conserved across species.

Obestatin promotes proliferation and survival of adult hippocampal progenitors and reduces amyloid-β-induced toxicity.

The ghrelin gene-derived peptide obestatin promotes survival in different cell types through a yet undefined receptor; however, its potential neuroprotective activities are still unknown. Here, obestatin effects were investigated on proliferation and survival of adult rat hippocampal progenitor cells (AHPs). Obestatin immunoreactivity was found in AHPs; moreover, obestatin binding to AHPs was displaced by the GLP-1R agonist Ex-4 and antagonist Ex-9. Furthermore, obestatin increased cell proliferation and survival in growth factor deprived medium and inhibited apoptosis; these effects were blocked by Ex-9. The underlying mechanisms involved Gαs/cAMP/PKA/CREB signaling, phosphorylation of ERK1/2 and PI3K/Akt, and the PI3K targets GSK-3β/β-catenin and mTOR. Obestatin also counteracted Aβ1-42-induced detrimental effects through inhibition of GSK-3β activity and Tau hyperphosphorylation, main hallmarks of neuronal death in Alzheimers disease. These findings indicate a novel protective role for obestatin in AHPs and candidate this peptide as potential therapeutic target for increasing neurogenesis and for approaching neurodegenerative disorders.

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-3β, 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.

The Mineralocorticoid Agonist Fludrocortisone Promotes Survival and Proliferation of Adult Hippocampal Progenitors.

Glucocorticoid receptor (GR) activation has been shown to reduce adult hippocampal progenitor cell proliferation and neurogenesis. By contrast, mineralocorticoid receptor (MR) signaling is associated with neuronal survival in the dentate gyrus of the hippocampus, and impairment of hippocampal MR has been linked to pathological conditions, such as depression or neurodegenerative disorders. Here, we aimed to further clarify the protective role of MR in adult hippocampal neurons by studying the survival and proliferative effects of the highly potent MR agonist fludrocortisone (Fludro) in adult rat hippocampal progenitor cells (AHPs), along with the associated signaling mechanisms. Fludro, which upregulated MR but not GR expression, increased survival and proliferation and prevented apoptosis in AHPs cultured in growth factor-deprived medium. These effects were blunted by the MR antagonist spironolactone and by high doses of the GR agonist dexamethasone. Moreover, they involved signaling through cAMP/protein kinase A (PKA)/cAMP response element-binding protein, phosphoinositide 3-kinase (PI3K)/Akt and its downstream targets glycogen synthase kinase-3β (GSK-3β) and mammalian target of rapamycin. Furthermore, Fludro attenuated the detrimental effects of amyloid-β peptide 1–42 (Aβ1–42) on cell survival, proliferation, and apoptosis in AHPs, and increased the phosphorylation of both PI3K/Akt and GSK-3β, which was reduced by Aβ1–42. Finally, Fludro blocked Aβ1–42-induced hyperphosphorylation of Tau protein, which is a main feature of Alzheimer’s disease. Overall, these results are the first to show the protective and proliferative role of Fludro in AHPs, suggesting the potential therapeutic importance of targeting MR for increasing hippocampal neurogenesis and for treating neurodegenerative diseases.