The gut hormone INSL5 activates multiple signalling pathways in the human enterocyte cell line NCI-H716

S. Y. Ang1, D. S. Hutchinson1, B. A. Evans1, M. Kocan2, R. J. Summers1. 1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, AUSTRALIA, 2The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, AUSTRALIA.

Introduction: Insulin-like peptide 5 (INSL5) is a peptide hormone synthesized and secreted from enteroendocrine L-cells together with GLP-1. The cognate receptor for INSL5 is the relaxin family peptide receptor 4 (RXFP4), a GPCR predominantly expressed in the colorectum1. Although recognised as an appetite stimulant2, other biological roles of INSL5 remain unclear. A recent study showed that INSL5 stimulated GLP-1 release from GLUTag murine L-cells3. Here, we have used the human enterocyte line, NCI-H716 and GLUTag cells, to elucidate the biological roles of INSL5.

Method: NCI-H716 or GLUTag cells plated onto multi-well plates were grown for 48h before experiments. Real-time RT-PCR was performed using Taqman gene expression assay. INSL5 time course (100 nM) and/or concentration-response (0.1-300 nM) cell signaling experiments were performed using AlphaScreen assays and Western blotting. INSL5-mediated inhibition of forskolin (3 μM)-stimulated cAMP accumulation was measured using LANCE HTRF. Effect of INSL5 on basal and forskolin (3 μM)-stimulated GLP-1 secretion (2h) was measured using a HTRF kit (Cisbio). Data points represent mean±SEM of n experiments.

Results: RXFP4 and proglucagon (GCG) but not INSL5 mRNA expression was identified in NCI-H716 cells. In these cells, INSL5 activated p-ERK1/2 with a 5 min peak response (n=5, p<0.0001) and activated AKT maximally at 15 min at both p-Ser473 (n=5, p<0.001) and p-Thr308 (n=5, p<0.001). Activation of p-S6RP occurred later at 30 min (n=5, p<0.001). In the same cells, INSL5 concentration-dependently inhibited forskolin-stimulated cAMP accumulation (n=5). Concentration-response curves revealed comparable pEC50 values for the pathways investigated (Table 1). However, INSL5 treatment affected neither basal nor forskolin-stimulated GLP-1 secretion from NCI-H716 cells. Surprisingly in GLUTag cells little to no Rxfp4 expression was detected in contrast to the previous study3, although transcripts for Gcg and Insl5 were expressed. Moreover GLUTag cells were unresponsive to INSL5 treatment within the timeframe tested in p-AKT and p-S6RP assays.

Table 1 E_max and pEC₅₀ values for signalling pathways activated by INSL5 in NCI-H716 cells

Conclusion: NCI-H716 express RXFP4 and are responsive to INSL5 although the peptide does not appear to regulate GLP-1 secretion in these cells. Importantly, NCI-H716 cells are transformed enterocytes with mixed endo/exocrine properties and may not necessarily represent L-cells. This coupled with the lack of RXFP4 expression in GLUTag cells suggest that the roles of INSL5 in enterocytes have yet to be determined.

References:

1. Uhlen M et al. (2015). Science 347: 1260419

2. Grosse J et al. (2014). PNAS 111: 11133-11138

3. Luo X et al. (2015). Biochem J 466: 467-473