Investigation of novel functions of RXFP4-INSL5 and potential effects of gene variants identified in Polynesian populations

Abstract

Insulin-family peptide 5 (INSL5) and relaxin-family peptide receptor 4 (RXFP4) are an endogenous hormone-receptor pair expressed primarily in the distal gastrointestinal tract. The exact functions of INSL5-RXFP4 are not clear, but published reports indicate roles relating to appetite, diet preference, colon motility, energy, and glucose metabolism. In this thesis we sought to uncover novel functions of INSL5-RXFP4 and evaluate phenotypic changes associated with gene variants identified in Polynesian populations. rs76232257 in RXFP4 results in Thr6Asn amino acid change and is prevalent in 15% of Polynesian males. In two independent cohorts of Polynesian males, rs76232257 was associated with a significant increase in systolic blood pressure (SBP). Additionally, in the cohort analysed in this thesis, rs76232257 was associated with trends towards increased adiposity represented by phenotypes such as body mass index, fat mass, visceral fat, waist circumference, and neck circumference. The large effect size on SBP prompted an investigation into RXFP4 effects on the renin-angiotensin system in the kidney, which regulates blood pressure. RXFP4 was found to be expressed in juxtaglomerular cells in the kidney, the site of renin secretion, however, activation of RXFP4 did not affect synthesis or secretion of renin. By analysing publicly available single cell RNA sequencing datasets pertaining to human gut epithelium, RXFP4 expression was identified in a subset of serotonin-producing TPH1-positive enterochromaffin cells. Therefore, the effects of RXFP4 on serotonin synthesis and secretion were investigated by overexpressing RXFP4 in a human colorectal cancer cell line Colo320. Overexpression of RXFP4 caused an inherent suppression of cAMP, TPH1 expression and protein levels in Colo320 cells. RXFP4 agonists were able to further suppress cAMP levels, TPH1 expression, and serotonin secretion indicating activation of RXFP4 leads to downstream inhibition of serotonin synthesis and secretion. rs1034742236 in INSL5 is present in <5% of Polynesian individuals and results in a Arg114Ser substitution at an essential peptide cleavage site. By overexpressing reference and variant versions of INSL5, differences in peptide cleavage efficiencies and patterns were observed, suggesting rs1034742236 may have physiological implications. Together the findings provide evidence for the site of RXFP4 expression within the gut and a novel role for INSL5-RXFP4 in serotonin synthesis and would help uncover the pathophysiology underlying the altered phenotypes in order to provide better therapeutic options for people carrying the variants.