Proceedings of The Physiological Society
AstraZeneca (2010) Proc Physiol Soc 18, C15 and PC15
L-Glutamine stimulates the release of GLP-1 from primary murine L-cells
G. Tolhurst1, H. Parker1, Y. Zheng1, F. Reimann1, F. Gribble1
1. Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Glutamine has been shown to elevate plasma levels of the incretin hormone, GLP-1, in human subjects (1). The mechanisms behind glutamine-evoked GLP-1 release have previously been investigated using the model cell line, GLUTag (2), but remain poorly defined. This project has sought to clarify the validity of this cell model whilst further elucidating the pathways behind glutamine-induced GLP-1 secretion. Calcium imaging and static secretion experiments were performed on colonic cultures from adult transgenic mice expressing the fluorescent protein, Venus, driven by the proglucagon promoter. GLP-1 release was measured from 24hr old cultures and fura2 loaded cells were monitored in real time to track intracellular Ca2+ levels in individual identified L-cells. Real time intracellular cAMP levels were investigated in GLUTag cells transfected with a cAMP FRET sensor. A range of L-amino acids and meAIB, a specific substrate of system A transporters (SNAT), elevated Ca2+i to comparable levels in primary L-cells. The glutamine-evoked Ca2+ response was shown to be dependent on Na+o (n=8, p<0.01). However, only glutamine (Gln), asparagine (Asn) and phenylalanine (Phe) stimulated a significant release of GLP-1 from the cultures (n=9, p<0.05), with Gln elevating GLP-1 levels significantly higher than Asn and Phe (n=9, p<0.01). Gln triggered the release of GLP-1 in a dose-dependent fashion with an EC50 of 0.1 mM (n=6), comparable to that seen in GLUTag cells (2). The response was attenuated ~20% by the inhibition of either L-type voltage gated Ca2+ channels or TTX-sensitive Na+ channels (n=9, p<0.01). In addition, Gln stimulated secretion via a mechanism that was downstream or independent of membrane depolarisation-evoked Ca2+ entry (n=6, p<0.01). Antagonising components of the Gq pathway were without effect as was inhibition of a range of Gln-dependent metabolic pathways. In the GLUTag cell line, both Gln and Asn triggered a transient rise in cAMPi (n=21-66, p<0.001), which was independent of Na+o (n=15-36). Together, these data suggest a role for SNAT in the release of GLP-1. The coupled Na+ entry accompanying Gln influx, depolarises the membrane, triggering action potentials and Ca2+ entry via L-type Ca2+ channels. However, this pathway alone is not sufficient to stimulate secretion of GLP-1, given the lack of effect of meAIB. In addition, Gln elevates intracellular cAMP levels, a known and potent signalling pathway in enteroendocrine cells, thus potentiating the secretory response. The findings validate the use of GLUTag cells for the study of GLP-1 secretion in relation to the sensing of L-Gln. The ability of amino acids to modulate cAMPi suggests the involvement of an amino acid-sensitive plasma membrane receptor, the molecular identity of which is under current investigation.
Where applicable, experiments conform with Society ethical requirements