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Glutamine triggers and potentiates glucagon-like peptide-1 secretion by raising cytosolic Ca2+ and cAMP.

Tolhurst G, Zheng Y, Parker HE, Habib AM, Reimann F, Gribble FM - Endocrinology (2011)

Bottom Line: The greater effectiveness of glutamine as a secretagogue was paralleled by its ability to increase cAMP in GLUTag cells.Glutamine elevated intracellular cAMP to 36% of that produced by a maximal stimulus, whereas asparagine only increased intracellular cAMP by 24% and phenylalanine was without effect.Glutamine elevates both cytosolic Ca(2+) and cAMP in L cells, which may account for the effectiveness of glutamine as a GLP-1 secretagogue.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge CB2 0XY, United Kingdom.

ABSTRACT
L-glutamine stimulates glucagon-like peptide 1 (GLP-1) secretion in human subjects and cell lines. As recent advances have enabled the study of primary GLP-1-releasing L cells, this study aimed to characterize glutamine-sensing pathways in native murine L cells. L cells were identified using transgenic mice with cell-specific expression of fluorescent markers. Cells were studied in primary colonic cultures from adult mice, or purified by flow cytometry for expression analysis. Intracellular Ca(2+) was monitored in cultures loaded with Fura2, and cAMP was studied using Förster resonance energy transfer sensors expressed in GLUTag cells. Asparagine, phenylalanine, and glutamine (10 mm) triggered GLP-1 release from primary cultures, but glutamine was the most efficacious, increasing secretion 1.9-fold with an EC(50) of 0.19 mm. Several amino acids triggered Ca(2+) changes in L cells, comparable in magnitude to that induced by glutamine. Glutamine-induced Ca(2+) responses were abolished in low Na(+) solution and attenuated in Ca(2+) free solution, suggesting a role for Na(+) dependent uptake and Ca(2+) influx. The greater effectiveness of glutamine as a secretagogue was paralleled by its ability to increase cAMP in GLUTag cells. Glutamine elevated intracellular cAMP to 36% of that produced by a maximal stimulus, whereas asparagine only increased intracellular cAMP by 24% and phenylalanine was without effect. Glutamine elevates both cytosolic Ca(2+) and cAMP in L cells, which may account for the effectiveness of glutamine as a GLP-1 secretagogue. Therapeutic agents like glutamine that target synergistic pathways in L cells might play a future role in the treatment of type 2 diabetes.

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Glutamine-evoked cAMPi responses in GLUTag cells. GLUTag cells were transfected with the cAMP probe, Epac2 camps. FRET was measured as the YFP/CFP ratio by exciting the cells at 435/10 nm (A inset shows example image) A, Representative trace showing the CFP/YFP fluorescence ratio (reflecting [cAMP]i) recorded from an individual cell after addition of 10 mm glutamine and the positive control 10 μm forskolin and 10 μm IBMX (F/I). B, Mean cAMP changes in response to a vehicle control (con) and a range of L amino acids (10 mm of each), as indicated (open bars). Gln (10 mmol/liter) was also tested in the absence of extracellular Na+ (Gln, gray bars). CFP/YFP ratios in the presence of the test agent were expressed relative to the positive control performed in each experiment, as shown in A. Data represent the mean and sem. **, P < 0.01; ***, P < 0.001 compared with vehicle by Student's t test.
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Figure 4: Glutamine-evoked cAMPi responses in GLUTag cells. GLUTag cells were transfected with the cAMP probe, Epac2 camps. FRET was measured as the YFP/CFP ratio by exciting the cells at 435/10 nm (A inset shows example image) A, Representative trace showing the CFP/YFP fluorescence ratio (reflecting [cAMP]i) recorded from an individual cell after addition of 10 mm glutamine and the positive control 10 μm forskolin and 10 μm IBMX (F/I). B, Mean cAMP changes in response to a vehicle control (con) and a range of L amino acids (10 mm of each), as indicated (open bars). Gln (10 mmol/liter) was also tested in the absence of extracellular Na+ (Gln, gray bars). CFP/YFP ratios in the presence of the test agent were expressed relative to the positive control performed in each experiment, as shown in A. Data represent the mean and sem. **, P < 0.01; ***, P < 0.001 compared with vehicle by Student's t test.

Mentions: An alternative signaling pathway could involve elevation of cAMPi which is a known potent L cell stimulus (16) (7). We investigated this using a cAMP FRET sensor based on Epac2, which we validated recently for monitoring cAMP levels in single GLUTag cells (11, 17) (Fig. 4A). Gln (10 mm) evoked a significant rise in the CFP/YFP ratio in GLUTag cells compared with vehicle control, indicative of elevated cAMPi (P < 0.001, Fig 4, A and B). The response to Gln was 36 ± 3% (n = 72) of that produced by a maximal stimulus induced by 10 μm fsk/IBMX (Fig. 4, A and B). By comparison, the response to 10 mm Asn was smaller, at 24 ± 3% of maximal (P < 0.01 compared with vehicle, n = 52; Fig. 4B), whereas 10 mm Phe failed to stimulate a significant response (n = 43). In contrast to the Ca2+ mobilization experiments, 10 mm meAIB failed to elevate cAMPi (n = 44), and removing the extracellular Na+ had no effect on the Gln-evoked FRET response (42 ± 3%, n = 36; Fig. 4B).


Glutamine triggers and potentiates glucagon-like peptide-1 secretion by raising cytosolic Ca2+ and cAMP.

Tolhurst G, Zheng Y, Parker HE, Habib AM, Reimann F, Gribble FM - Endocrinology (2011)

Glutamine-evoked cAMPi responses in GLUTag cells. GLUTag cells were transfected with the cAMP probe, Epac2 camps. FRET was measured as the YFP/CFP ratio by exciting the cells at 435/10 nm (A inset shows example image) A, Representative trace showing the CFP/YFP fluorescence ratio (reflecting [cAMP]i) recorded from an individual cell after addition of 10 mm glutamine and the positive control 10 μm forskolin and 10 μm IBMX (F/I). B, Mean cAMP changes in response to a vehicle control (con) and a range of L amino acids (10 mm of each), as indicated (open bars). Gln (10 mmol/liter) was also tested in the absence of extracellular Na+ (Gln, gray bars). CFP/YFP ratios in the presence of the test agent were expressed relative to the positive control performed in each experiment, as shown in A. Data represent the mean and sem. **, P < 0.01; ***, P < 0.001 compared with vehicle by Student's t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3140224&req=5

Figure 4: Glutamine-evoked cAMPi responses in GLUTag cells. GLUTag cells were transfected with the cAMP probe, Epac2 camps. FRET was measured as the YFP/CFP ratio by exciting the cells at 435/10 nm (A inset shows example image) A, Representative trace showing the CFP/YFP fluorescence ratio (reflecting [cAMP]i) recorded from an individual cell after addition of 10 mm glutamine and the positive control 10 μm forskolin and 10 μm IBMX (F/I). B, Mean cAMP changes in response to a vehicle control (con) and a range of L amino acids (10 mm of each), as indicated (open bars). Gln (10 mmol/liter) was also tested in the absence of extracellular Na+ (Gln, gray bars). CFP/YFP ratios in the presence of the test agent were expressed relative to the positive control performed in each experiment, as shown in A. Data represent the mean and sem. **, P < 0.01; ***, P < 0.001 compared with vehicle by Student's t test.
Mentions: An alternative signaling pathway could involve elevation of cAMPi which is a known potent L cell stimulus (16) (7). We investigated this using a cAMP FRET sensor based on Epac2, which we validated recently for monitoring cAMP levels in single GLUTag cells (11, 17) (Fig. 4A). Gln (10 mm) evoked a significant rise in the CFP/YFP ratio in GLUTag cells compared with vehicle control, indicative of elevated cAMPi (P < 0.001, Fig 4, A and B). The response to Gln was 36 ± 3% (n = 72) of that produced by a maximal stimulus induced by 10 μm fsk/IBMX (Fig. 4, A and B). By comparison, the response to 10 mm Asn was smaller, at 24 ± 3% of maximal (P < 0.01 compared with vehicle, n = 52; Fig. 4B), whereas 10 mm Phe failed to stimulate a significant response (n = 43). In contrast to the Ca2+ mobilization experiments, 10 mm meAIB failed to elevate cAMPi (n = 44), and removing the extracellular Na+ had no effect on the Gln-evoked FRET response (42 ± 3%, n = 36; Fig. 4B).

Bottom Line: The greater effectiveness of glutamine as a secretagogue was paralleled by its ability to increase cAMP in GLUTag cells.Glutamine elevated intracellular cAMP to 36% of that produced by a maximal stimulus, whereas asparagine only increased intracellular cAMP by 24% and phenylalanine was without effect.Glutamine elevates both cytosolic Ca(2+) and cAMP in L cells, which may account for the effectiveness of glutamine as a GLP-1 secretagogue.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge CB2 0XY, United Kingdom.

ABSTRACT
L-glutamine stimulates glucagon-like peptide 1 (GLP-1) secretion in human subjects and cell lines. As recent advances have enabled the study of primary GLP-1-releasing L cells, this study aimed to characterize glutamine-sensing pathways in native murine L cells. L cells were identified using transgenic mice with cell-specific expression of fluorescent markers. Cells were studied in primary colonic cultures from adult mice, or purified by flow cytometry for expression analysis. Intracellular Ca(2+) was monitored in cultures loaded with Fura2, and cAMP was studied using Förster resonance energy transfer sensors expressed in GLUTag cells. Asparagine, phenylalanine, and glutamine (10 mm) triggered GLP-1 release from primary cultures, but glutamine was the most efficacious, increasing secretion 1.9-fold with an EC(50) of 0.19 mm. Several amino acids triggered Ca(2+) changes in L cells, comparable in magnitude to that induced by glutamine. Glutamine-induced Ca(2+) responses were abolished in low Na(+) solution and attenuated in Ca(2+) free solution, suggesting a role for Na(+) dependent uptake and Ca(2+) influx. The greater effectiveness of glutamine as a secretagogue was paralleled by its ability to increase cAMP in GLUTag cells. Glutamine elevated intracellular cAMP to 36% of that produced by a maximal stimulus, whereas asparagine only increased intracellular cAMP by 24% and phenylalanine was without effect. Glutamine elevates both cytosolic Ca(2+) and cAMP in L cells, which may account for the effectiveness of glutamine as a GLP-1 secretagogue. Therapeutic agents like glutamine that target synergistic pathways in L cells might play a future role in the treatment of type 2 diabetes.

Show MeSH
Related in: MedlinePlus