Ghrelin neutralization during fasting-refeeding cycle impairs the recuperation of body weight and alters hepatic energy metabolism.

Ghrelin, a hormone whose levels increase during food deprivation, plays a pivotal role in the regulation of food intake, energy metabolism and storage, as well as in insulin sensitivity. Here, we investigated the effects of acyl-ghrelin neutralization with the acyl-ghrelin-binding compound NOX-B11(2) during the fasting-refeeding cycle. Our data demonstrate that ghrelin neutralization with NOX-B11(2) impairs recuperation of lost body weight after food deprivation. Analysis of enzymes involved in glucose and lipid metabolism in liver of fed, fasted and refed rats revealed that neutralization of acyl-ghrelin resulted in minor decreases in the enzymes of glycolytic and lipogenic pathways during fasting. However, during refeeding these enzymes as well as glycogen levels recovered more slowly when acyl-ghrelin was blocked. The high levels of ghrelin in response to food deprivation may contribute to an adequate decrease in hepatic glycolytic and lipogenic enzymes and aid in the recovery of body weight and energetic reserves once food becomes available after the fasting period.

The promise of ghrelin antagonism in obesity treatment.

According to the World Health Organization, 300 million people are clinically obese worldwide. As a major risk factor in the development of life-threatening diseases such as diabetes, cardiovascular disease and certain cancers, obesity is quickly evolving into a serious public health threat on a global scale. This alarming situation calls for the development of effective treatments, including pharmacological intervention. Many biotechnology and pharmaceutical companies have embarked on the endeavor to develop safe new therapeutics for weight loss and durable weight management. Much progress has been made to improve our understanding of the regulation of energy homeostasis, but this knowledge has not yet translated into new medicines. However, it has led to the identification of molecules that promise to be highly interesting targets for therapeutic intervention. One such molecule is the enteric hormone ghrelin. Ghrelin was identified in 1999 as the endogenous ligand for the growth hormone secretagogue-receptor 1a (GHS-R1a). Soon after its discovery ghrelin was shown to increase food intake, downregulate energy expenditure and conserve body fat, causing weight gain and adipogenesis. Unsurprisingly, these findings placed ghrelin and its receptor on the radar screens of many medical researchers in academia and the pharmaceutical industry. The resulting attention has led to a steadily growing body of evidence in support of ghrelin antagonism as a potential means to ameliorate obesity. But the causes for obesity are manifold, and skepticism about the utility of this approach remains. The current review summarizes the arguments for and against ghrelin as a potential antiobesity target and discusses recent pharmaceutical developments to interfere with this exciting pathway.

Ghrelin neutralization by a ribonucleic acid-SPM ameliorates obesity in diet-induced obese mice.

Ghrelin, an acylated peptide secreted from the stomach, acts as a short-term signal of nutrient depletion. Ghrelin is an endogenous ligand for the GH secretagogue receptor 1a, a G protein-coupled receptor expressed in the hypothalamus and pituitary. We used a synthetic oligonucleotide, NOX-B11-2, capable of specific high-affinity binding to bioactive ghrelin to determine whether ghrelin neutralization would alter indices of energy balance in vivo. This novel type of ghrelin-blocking agent, called an RNA Spiegelmer (SPM), is a polyethylene glycol-modified l-RNA oligonucleotide, the nonnatural configuration of which confers in vivo stability. NOX-B11-2 blocked ghrelin mediated activation of GH secretagogue receptor 1a in cell culture (IC50 approximately 5 nm). We explored the effects of acute NOX-B11-2 administration on ghrelin-induced feeding in mice. NOX-B11-2 (66 mg/kg, sc) blocked ghrelin-induced feeding and was without effect on feeding evoked by an orally active nonpeptide ghrelin receptor agonist. We demonstrated that selective ghrelin blockade effectively promoted weight loss in diet-induced obese (DIO) mice. Chronic infusion of NOX-B11-2 (33 mg/kg.d, sc) to DIO mice evoked body weight loss for 13 d and reduced food intake and fat mass relative to control SPM-infused mice. In a 7-d study, DIO mice infused with NOX-B11-2 (33 mg/kg.d, sc) showed body weight loss, compared with animals receiving control SPM. This effect was directly mediated by SPM neutralization of ghrelin because NOX-B11-2 administration to ghrelin-deficient mice resulted in no weight loss. The decreased obesity observed in SPM-treated DIO mice provides validation for ghrelin neutralization as a potential antiobesity therapy.

Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer.

Employing in vitro selection techniques, we have generated biostable RNA-based compounds, so-called Spiegelmers, that specifically bind n-octanoyl ghrelin, the recently discovered endogenous ligand for the type 1a growth hormone secretagogue (GHS) receptor. Ghrelin is a potent stimulant of growth hormone release, food intake, and adiposity. We demonstrate that our lead compound, L-NOX-B11, binds ghrelin with low-nanomolar affinity and inhibits ghrelin-mediated GHS-receptor activation in cell culture with an IC(50) of 5 nM. l-NOX-B11 is highly specific for the bioactive, n-octanoylated form of ghrelin. Like the GHS receptor, it does not recognize the inactive unmodified peptide and requires only the N-terminal five amino acids for the interaction. The i.v. administration of polyethylene glycol modified l-NOX-B11 efficiently suppresses ghrelin-induced growth hormone release in rats. These results demonstrate that the neutralization of circulating bioactive ghrelin leads to inhibition of ghrelin's secretory effects in the CNS.

Mutations in the middle domain of yeast poly(A) polymerase affect interactions with RNA but not ATP.

The eukaryotic poly(A) polymerase (PAP) is responsible for the posttranscriptional extension of mRNA 3' ends by the addition of a poly(A) tract. The recently published three-dimensional structures of yeast and bovine PAPs have made a more directed biochemical analysis of this enzyme possible. Based on these structures, the middle domain of PAP was predicted to interact with ATP. However, in this study, we show that mutations of conserved residues in this domain of yeast PAP, Pap1, do not affect interaction with ATP, but instead disrupt the interaction with RNA and affect the enzyme's ability to process substrate lacking 2' hydroxyls at the 3' end. These results are most consistent with a model in which the middle domain of PAP interacts directly with the recently extended RNA and pyrophosphate byproduct.

Functional dissection of the zinc finger and flanking domains of the Yth1 cleavage/polyadenylation factor.

Yth1, a subunit of yeast Cleavage Polyadenylation Factor (CPF), contains five CCCH zinc fingers. Yth1 was previously shown to interact with pre-mRNA and with two CPF subunits, Brr5/Ysh1 and the polyadenylation-specific Fip1, and to act in both steps of mRNA 3' end processing. In the present study, we have identified new domains involved in each interaction and have analyzed the consequences of mutating these regions on Yth1 function in vivo and in vitro. We have found that the essential fourth zinc finger (ZF4) of Yth1 is critical for interaction with Fip1 and RNA, but not for cleavage, and a single point mutation in ZF4 impairs only polyadenylation. Deletion of the essential N-terminal region that includes the ZF1 or deletion of ZF4 weakened the interaction with Brr5 in vitro. In vitro assays showed that the N-terminus is necessary for both processing steps. Of particular importance, we find that the binding of Fip1 to Yth1 blocks the RNA-Yth1 interaction, and that this inhibition requires the Yth1-interacting domain on Fip1. Our results suggest a role for Yth1 not only in the execution of cleavage and poly(A) addition, but also in the transition from one step to the other.