Recently, we reported that calcium-sensing receptor (CaSR) is a receptor for kokumi substances, which enhance the intensities of salty, sweet and umami tastes. Furthermore, we found that several γ-glutamyl peptides, which are CaSR agonists, are kokumi substances. In this study, we elucidated the receptor cells for kokumi substances, and their physiological properties. For this purpose, we used Calcium Green-1 loaded mouse taste cells in lingual tissue slices and confocal microscopy. Kokumi substances, applied focally around taste pores, induced an increase in the intracellular Ca2+ concentration ([Ca2+]i) in a subset of taste cells. These responses were inhibited by pretreatment with the CaSR inhibitor, NPS2143. However, the kokumi substance-induced responses did not require extracellular Ca2+. CaSR-expressing taste cells are a different subset of cells from the T1R3-expressing umami or sweet taste receptor cells. These observations indicate that CaSR-expressing taste cells are the primary detectors of kokumi substances, and that they are an independent population from the influenced basic taste receptor cells, at least in the case of sweet and umami.
The incretin effect is markedly reduced in patients with type 2 diabetes, mainly due to defective glucagon-like peptide-1 (GLP-1) secretion from the intestinal L cells in response to stimulation by various nutrients. 1,2) This leads to impairment of early-phase insulin secretion after food intake and consequently results in postprandial hyperglycemia and hyperlipidemia. [3][4][5][6] GLP-1 is an incretin hormone that is released by intestinal L cells following their stimulation by nutrients, 7,8) and it promotes glucose-stimulated insulin secretion by pancreatic bcells. GLP-1 has also been reported to have various other beneficial effects, such as promoting b-cell proliferation, 9) suppressing glucagon release, 10) suppressing food intake, 11,12) slowing gastric emptying, 13,14) and cardiovascular protection.15) Therefore, restoration of GLP-1 secretion by intestinal L cells could be an important new therapeutic option for the management of metabolic syndrome. Although the exact mechanism of GLP-1 secretion by intestinal L cells remains to be elucidated, the sulfonylurea receptor 1 (SUR1)/K ATP channel, 16,17) Na ϩ /glucose co-transporter 1 (SGLT1), 18) glucose transporter 2 (GLUT2), 19) sweet taste receptor, 20)TGR5 21) and G-protein coupled receptors (GPRs) [22][23][24][25] have all been reported to be involved in this process. In the present study, we investigated the effect of nateglinide on GLP-1 secretion by human intestinal L cells.Nateglinide is a short-acting insulin secretagogue with a rapid effect, which restores early-phase insulin secretion in patients with type 2 diabetes by rapidly binding to SUR1 [26][27][28][29][30] and thus suppresses postprandial hyperglycemia. It has been unclear whether or not GLP-1 is involved in these effects of nateglinide. Recently, Duffy et al. reported that nateglinide increases the plasma GLP-1 level by inhibiting dipeptidyl peptidase IV (DPP IV), 31) but the influence of nateglinide on GLP-1 secretion is still unknown. Therefore, we investigated the effect of nateglinide on GLP-1 secretion in vivo by monitoring GLP-1 levels in the portal venous blood after oral administration of nateglinide to normal Wistar rats. We also conducted an in vitro study to assess the direct effect of nateglinide on GLP-1 secretion by human intestinal L cells (NCI-H716). MATERIALS AND METHODSAnimals Male Goto-Kakizaki (GK) rats and male Wistar rats (6 weeks old) were purchased from Japan SLC Inc. (Hamamatsu, Japan). The animals were housed in individual polycarbonate cages with woodchip bedding, and were provided with food and water ad libitum. The animal room was maintained on a 12-h light/dark cycle (7 a.m.-7 p.m.: dark; 7 p.m.-7 a.m.: light), with a temperature range of 22Ϯ1°C and a relative humidity of 55Ϯ5%, throughout the experimental period. These animal experiments were performed in accordance with the Guiding Principles for the care and use of laboratory animals approved by the Japanese Pharmacological Society. In addition, this study was approved by the Animal Care and Use Committ...
Proteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio less than 0.1 (w/w), fluid phase C4b, and methylamine-treated C4 (C4ma) were degraded by factor I into C4bi: minimal Cd4 was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CR1. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell culture medium replacement is necessary to replenish nutrients and remove waste products, and perfusion and batch media exchange methods are available. The former can establish an environment similar to that in vivo, and microfluidic devices are frequently used. However, these methods are hampered by incompatibility with commercially available circular culture dishes and the difficulty in controlling liquid flow. Here, we fabricated a culture dish adapter using polydimethylsiloxane that has a small recess structure for flow control compatible with commercially available culture dishes. We designed U-shaped and I-shaped recess structure adapters and we examined the effects of groove structure on medium flow using simulation. We found that the U-shaped and I-shaped structures allowed a uniform and uneven flow of medium, respectively. We then applied these adaptors to 293T cell culture and examined the effects of recess structures on cell proliferation. As expected, cell proliferation was similar in each area of a dish in the U-shaped structure adapter, whereas in the early flow area in the I-shaped structure adapter, it was significantly higher. In summary, we succeeded in controlling liquid flow in culture dishes with the fabricated adapter, as well as in applying the modulation of culture medium flow to control cell culture.
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