Stimulation of Insulin Secretion by Denatonium, One of the Most Bitter-Tasting Substances Known

Abstract

Denatonium, one of the most bitter-tasting substances known, stimulated insulin secretion in clonal HIT-T15 ␤-cells and rat pancreatic islets. Stimulation of release began promptly after exposure of the ␤-cells to denatonium, reached peak rates after 4 -5 min, and then declined to near basal values after 20 -30 min. In islets, no effect was observed at 2.8 mmol/l glucose, whereas a marked stimulation was observed at 8. M ultiple signal transduction mechanisms in taste cells detect sweet, sour, bitter, salty, and umami tastes and involve both receptormediated second messenger pathways and direct interactions with ion channels (1-3). Four mechanisms have been identified for signal transduction by bitter compounds. Two mechanisms result from bitter substances interacting with receptors of the T2R/TRB family and activation of gustducin (4 -10). In one, the ␣-subunit of gustducin activates cyclic nucleotide phosphodiesterase activity to decrease cyclic nucleotide monophosphate (cyclic NMP) levels and de-inhibit the activity of a cyclic NMP-inhibited cation channel leading to depolarization and increased [Ca 2ϩ ] i (11,12). In the second, the ␤␥-subunits of gustducin (probably ␤1␥13 and/or ␤3␥13) activate PLC␤2 and increase IP 3 and DAG levels (13,14). IP 3 increases [Ca 2ϩ ] i via IP 3 R3 activation (14 -16). A third mechanism involves a direct interaction and inhibition of K ϩ channels resulting in depolarization and increased [Ca 2ϩ ] i (17). The fourth known mechanism is the direct gating of nonselective cation channels and depolarization, again leading to increased [Ca 2ϩ ] i (18 -20). In all cases, increased [Ca 2ϩ ] i leads to increased neurotransmitter release and activation of the afferent gustatory nerves that transmit the bitter signal to the brain. There is evidence for the activation of multiple second messenger pathways in individual taste cells (4). There is also evidence that some bitter stimuli activate only subpopulations of bitter-sensitive taste cells (4 -6). Also, individual taste cells can respond selectively to different bitter substances (7). However, there is still much that is unknown about individual taste cell regulation.Stimulation of taste cell membranes with denatonium activates not only gustducin but also transducin (9), with which it shares high sequence homology (21). Because transducin mRNA is reported to be present in pancreatic islets at approximately one-fifth the level of that in the retina (22), we were interested to determine the effects of denatonium on insulin secretion in ␤-cells as a tool to study the potential involvement of gustducin or transducin in stimulus-secretion coupling. The experiments described here were performed on clonal HIT-T15 cells and rat pancreatic islets. RESEARCH DESIGN AND METHODSCell culture. Clonal HIT-T15 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 g/ml streptomycin, and 100 units/ml penicillin in an atmosphere of 95% air and 5% CO 2 . Cells of passage numbers 72-85 were used for the expe...

Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels

“…10. In agreement with this model, a previous study (69) indicated that DB does not exert any direct effect on L-type VSCC activity. It is interesting that the signaling cascade proposed in Fig.…”

“…Furthermore, we also verified that pretreatment of STC-1 cells with glybenclamide (1 to 50 M) for 1 h had no effect on the subsequent increase in [Ca 2ϩ ] i induced by DB (data not shown). In contrast, a concentration of glybenclamide of 1 M was sufficient to mimic DB effects in HIT-T15 ␤-cells (69).…”

“…It has been reported that DB stimulated insulin secretion in clonal HIT-T15 ␤-cells and rat pancreatic islets through the closure of the K ϩ channel Kir6.2, causing depolarization of the membrane of these cells and thereby triggering L-type VSCC activation (69). To test whether the Ca 2ϩ response elicited by RT-PCR was carried out as described in MATERIALS AND METHODS using the primers described in Table 1.…”

Genomic organization, expression, and function of bitter taste receptors (T2R) in mouse and rat

“…The most conserved T2Rs (e.g., T2R1, T2R4, and T2R38) between human and rodents have known bitter trait or ligands [6-n-propylthiouracil (PROP)/sucrose octaacetate (SOA)/PTC]. DB is regarded as the bitterest substances perceived by human and is widely used in taste warning (4,25). Potential T2Rs that recognize DB include hT2R4 and mT2R108 and hT2R44 and mT2R120, as demonstrated by their sensitivity to DB in a heterologous expression system (5,22).…”

Taste Receptor Gene Expression Outside the Gustatory System

“…A good example for the fact that GI responses must not necessarily involve (bitter) taste-related signaling elements has been obtained in rat pancreatic tissue. Here, Straub et al [150] used the bitter substance denatonium benzoate to stimulate insulin release from clonal HIT-T15 β-cells as well as from isolated rat pancreatic islets. In the presence of denatonium, islets bathed in glucose solution start secreting insulin through a mechanism that does not involve gustducin or transducin as assessed in a trypsin digestion assay.…”