Significance: Hormonal factors from specialized enteroendocrine cells in the gut epithelium link the availability of dietary nutrients to energy utilization and storage. Many gut hormones also affect behaviors such as appetite and foraging, conveying for example the satiating effects of food consumption. Here we identify insulin-like peptide 5 (Insl5) as a product of colonic endocrine L-cells, and show that levels were elevated in calorie-restricted mice and reduced after feeding. Consistent with this profile Insl5 administration stimulated food intake in mice, indicating it should join ghrelin as only the second identified gut hormone that enhances appetite. Modulating the Insl5 axis presents a new strategy for the treatment of metabolic disease and obesity.

Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BΔ23–27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly²³ to Arg, a truncation at the C terminus (Gly²⁴-Trp²⁷ deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BΔ23–27)R/I5 binds to human GPCR135 (IC₅₀ = 0.67 nm) and GPCR142 (IC₅₀ = 2.29 nm) with high affinity and is a potent functional GPCR135 antagonist (pA2 = 9.15) but is not a human LGR7 ligand. Furthermore, R3(BΔ23–27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC₅₀ = 0.25 nm, pA2 = 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BΔ23–27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BΔ23–27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.

Hippocampal theta rhythm is thought to underlie learning and memory, and it is well established that “pacemaker” neurons in medial septum (MS) modulate theta activity. Recent studies in the rat demonstrated that brainstem-generated theta rhythm occurs through a multisynaptic pathway via the nucleus incertus (NI), which is the primary source of the neuropeptide relaxin-3 (RLN3). Therefore, this study examined the possible contribution of RLN3 to MS activity, and associated hippocampal theta activity and spatial memory. In anesthetized and conscious rats, we identified the ability of intraseptal RLN3 signaling to modulate neuronal activity in the MS and hippocampus and promote hippocampal theta rhythm. Behavioral studies in a spontaneous alternation task indicated that endogenous RLN3 signaling within MS promoted spatial memory and exploratory activity significantly increased c-Fos immunoreactivity in RLN3-producing NI neurons. Anatomical studies demonstrated axons/terminals from NI/RLN3 neurons make close contact with septal GABAergic (and cholinergic) neurons, including those that project to the hippocampus. In summary, RLN3 neurons of the NI can modulate spatial memory and underlying hippocampal theta activity through axonal projections to pacemaker neurons of the MS. NI/RLN3 neurons are highly responsive to stress and express corticotropin-releasing factor type-1 receptors, suggesting that the effects observed could be an important component of memory processing associated with stress responses.