Different neuropeptides, all containing a common carboxy-terminal RFamide sequence, have been characterized as ligands of the RFamide peptide receptor family. Currently, five subgroups have been characterized with respect to their N-terminal sequence and hence cover a wide pattern of biological functions, like important neuroendocrine, behavioral, sensory and automatic functions. The RFamide peptide receptor family represents a multiligand/multireceptor system, as many ligands are recognized by several GPCR subtypes within one family. Multireceptor systems are often susceptible to cross-reactions, as their numerous ligands are frequently closely related. In this review we focus on recent results in the field of structure-activity studies as well as mutational exploration of crucial positions within this GPCR system. The review summarizes the reported peptide analogs and recently developed small molecule ligands (agonists and antagonists) to highlight the current understanding of the pharmacophoric elements, required for affinity and activity at the receptor family. Furthermore, we address the biological functions of the ligands and give an overview on their involvement in physiological processes. We provide insights in the knowledge for the design of highly selective ligands for single receptor subtypes to minimize cross-talk and to eliminate effects from interactions within the GPCR system. This will support the drug development of members of the RFamide family.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.Type 2 diabetes (T2D) is highly prevalent, with an estimated 370 million affected individuals worldwide, and this is predicted to double by 2030 1,2 . Despite the presence of several well-established drug classes for treating T2D, there is still a considerable unmet need for a drug that halts or reverses disease progression. The gp130 receptor cytokines IL-6 and CNTF modify food intake and body weight and improve insulin resistance in mice and humans [3][4][5][6] . Axokine, the human variant of CNTF, underwent human clinical trials for the treatment of amyotrophic lateral sclerosis, but the drug was repurposed to treat obesity and T2D 7-9 . After showing promise, the clinical development of Axokine was discontinued when some treated patients developed antibodies 9 , because of the fear that this could interfere with the neuroprotective action of endogenous CNTF. Although IL-6 protects against obesity and insulin resistance 10 , it is also pro-inflammatory owing-in part-to its 'trans-signalling' effects 11,12 , which limits its therapeutic utility.The gp130 cytokines signal by binding to the cytokine α-receptors, which are the IL-6 receptor (IL-6R) and CNTF receptor (CNTFR) for IL-6 and CNTF, respectively. Binding initiates the recruitment and dimerization of two transmembrane β-receptors: the IL-6-IL-6R complex binds two gp130 receptors as a homodimer, whereas the CNTF-CNTFR complex binds gp130 receptor and the LIF receptor (LIFR) as a heterodimer 13 . By transferring the LIFR-binding module from CNTF to IL-6, we engineered the chimeric protein IC7 14 , which predominantly consists of IL-6 residues, rendering it far less likely to induce an immune response compared with CNTF, because IL-6 circulates freely whereas CNTF is intracellular, and lacks a signal sequence peptide. Because IC7 is a chimaera with a unique sequence

It has been suggested that interleukin-6 (IL-6) produced by adipocytes in obesity leads to liver insulin resistance, although this hypothesis has never been definitively tested. Accordingly, we did so by generating adipocyte-specific IL-6-deficient (AdipoIL-6−/−) mice and studying them in the context of diet-induced and genetic obesity. Mice carrying two floxed alleles of IL-6 (C57Bl/6J) were crossed with Cre recombinase-overexpressing mice driven by the adiponectin promoter to generate AdipoIL-6−/− mice. AdipoIL-6−/− and floxed littermate controls were fed a standard chow or high-fat diet (HFD) for 16 wk and comprehensively metabolically phenotyped. In addition to a diet-induced obesity model, we also examined the role of adipocyte-derived IL-6 in a genetic model of obesity and insulin resistance by crossing the AdipoIL-6−/− mice with leptin-deficient ( ob/ob) mice. As expected, mice on HFD and ob/ob mice displayed marked weight gain and increased fat mass compared with chow-fed and ob/+ (littermate control) animals, respectively. However, deletion of IL-6 from adipocytes in either model had no effect on glucose tolerance or fasting hyperinsulinemia. We concluded that adipocyte-specific IL-6 does not contribute to whole body glucose intolerance in obese mice.

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