1 Introduction: 26RFa (QRFP) is a biologically active peptide that has been found to control 2 feeding behaviour by stimulating food intake, and to regulate glucose homeostasis by acting as 3 an incretin. The aim of the present study was thus to investigate the impact of 26RFa gene 4 knockout on the regulation of energy and glucose metabolism. 5Research design and methods: 26RFa mutant mice were generated by homologous 6 recombination, in which the entire coding region of prepro-26RFa was replaced by the iCre 7 sequence. Energy and glucose metabolism was evaluated through measurement of 8 complementary parameters. Morphological and physiological alterations of the pancreatic islets 9 were also investigated. 10Results: Our data do not reveal significant alteration of energy metabolism in the 26RFa-11 deficient mice except the occurrence of an increased basal metabolic rate. By contrast, 26RFa 12 mutant mice exhibit an altered glycemic phenotype with an increased hyperglycemia after a 13 glucose challenge associated with an impaired insulin production, and an elevated hepatic 14 glucose production. 2D and 3D immunohistochemical experiments indicate that the insulin 15 content of pancreatic β cells is much lower in the 26RFa -/mice as compared to the wild-type 16 littermates. 17Conclusion: Disruption of the 26RFa gene induces substantial alteration in the regulation of 18 glucose homeostasis with, in particular, a deficit in insulin production by the pancreatic islets. 19These findings further support the notion that 26RFa is an important regulator of glucose 20 homeostasis. 21 22

26RFa (QRFP) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localized in the hypothalamus. In the present study, we have investigated whether the 26RFa neurons may be involved in the hypothalamic regulation of glucose homeostasis. Our data indicate that 26RFa, i.c.v. injected, induces a robust antihyperglycemic effect associated with an increase of insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26RFa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26RFa in the lateral hypothalamic area and the ventromedial hypothalamic nucleus also express the insulin receptor and that insulin induces the expression of 26RFa in these neurons. Concurrently, we show that the central antihyperglycemic effect of insulin is abolished in presence of a 26RFa receptor (GPR103) antagonist as well as in mice deficient for 26RFa. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production. To conclude, in the present study we have identified a novel actor of the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system and we provide the evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.