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Possible contribution of taurine to distorted glucagon secretion in intra-islet insulin deficiency: a metabolome analysis using a novel α-cell model of insulin-deficient diabetes.

Bessho M, Murase-Mishiba Y, Imagawa A, Terasaki J, Hanafusa T - PLoS ONE (2014)

Bottom Line: A comprehensive metabolomic analysis of the IRKD αTC1-6 cells (IRKD cells) revealed some candidate metabolites whose levels differed markedly compared to those in control αTC1-6 cells, but also which could affect the glucagon release in IRKD cells.Of these candidates, taurine was remarkably increased in the IRKD cells and was identified as a stimulator of glucagon in αTC1-6 cells.These results indicate that the metabolic alterations induced by IRKD in α-cells, especially the increase of taurine, may lead to the distorted glucagon response in IRKD cells, suggesting the importance of taurine in the paradoxical glucagon response and the resultant glucose instability in insulin-deficient diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine (I), Osaka Medical College, Osaka, Japan.

ABSTRACT
Glycemic instability is a serious problem in patients with insulin-deficient diabetes, and it may be due in part to abnormal endogenous glucagon secretion. However, the intracellular metabolic mechanism(s) involved in the aberrant glucagon response under the condition of insulin deficiency has not yet been elucidated. To investigate the metabolic traits that underlie the distortion of glucagon secretion under insulin deficient conditions, we generated an αTC1-6 cell line with stable knockdown of the insulin receptor (IRKD), i.e., an in vitro α-cell model for insulin-deficient diabetes, which exhibits an abnormal glucagon response to glucose. A comprehensive metabolomic analysis of the IRKD αTC1-6 cells (IRKD cells) revealed some candidate metabolites whose levels differed markedly compared to those in control αTC1-6 cells, but also which could affect the glucagon release in IRKD cells. Of these candidates, taurine was remarkably increased in the IRKD cells and was identified as a stimulator of glucagon in αTC1-6 cells. Taurine also paradoxically exaggerated the glucagon secretion at a high glucose concentration in IRKD cells and islets with IRKD. These results indicate that the metabolic alterations induced by IRKD in α-cells, especially the increase of taurine, may lead to the distorted glucagon response in IRKD cells, suggesting the importance of taurine in the paradoxical glucagon response and the resultant glucose instability in insulin-deficient diabetes.

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Results of an analysis of the effect of stable IR knockdown in αTC1-6 cells and IR knockdown in mouse islets.(A) An immunoblot analysis of the insulin receptor (IR) expression in αTC1-6 cells after the transduction with lentiviruses expressing control shRNA (control) and shRNA-IR (IRKD). αTC1-6 cells were infected with lentiviruses (see details in the “Materials and Methods”), and were cultured in medium containing 1 µg/ml puromycin for one month to generate homogenous stable cell lines. Cells were then analyzed by a Western blot analysis using specific polyclonal antibodies against the β-subunit of the IR (IR-β). Stable αTC1-6 cells maintained in culture for two months were also examined. Blots are shown on the top, and the relative amount of IRβ protein normalized to the β-actin level is shown on the bottom (n = 3. The bars represent the means± SEM; *P<0.05, control vs. IRKD). (B) The relative expression of the Insr gene in isolated mouse islets infected with lentiviruses expressing control and IRKD (n = 6. The bars represent the means± SEM; *P<0.05, control vs. IRKD). (C) The results of the immunoblot analysis of the activation of insulin signal transduction pathways in control and IRKD αTC1-6 cells. Cells were serum-starved for 24 h in DMEM and were subsequently stimulated with 100 nM of insulin for 15 min. Samples were analyzed by a Western blot analysis using specific antibodies against phospho- and non-phospho-proteins, as indicated. (D–F) The time course of the changes in cell viability, cytotoxicity and apoptosis assessed using the ApoTox-Glo Triplex Assay (Promega) in the control and IRKD cells. n = 3 in each group. The data are expressed as the means ± SEM; *P<0.05, **P<0.01, control vs. IRKD. (G) The time course of BrdU incorporation in the control and IRKD cells. n = 3 in each group. The data are expressed as the means ± SEM; *P<0.05, **P<0.01, control vs. IRKD.
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pone-0113254-g001: Results of an analysis of the effect of stable IR knockdown in αTC1-6 cells and IR knockdown in mouse islets.(A) An immunoblot analysis of the insulin receptor (IR) expression in αTC1-6 cells after the transduction with lentiviruses expressing control shRNA (control) and shRNA-IR (IRKD). αTC1-6 cells were infected with lentiviruses (see details in the “Materials and Methods”), and were cultured in medium containing 1 µg/ml puromycin for one month to generate homogenous stable cell lines. Cells were then analyzed by a Western blot analysis using specific polyclonal antibodies against the β-subunit of the IR (IR-β). Stable αTC1-6 cells maintained in culture for two months were also examined. Blots are shown on the top, and the relative amount of IRβ protein normalized to the β-actin level is shown on the bottom (n = 3. The bars represent the means± SEM; *P<0.05, control vs. IRKD). (B) The relative expression of the Insr gene in isolated mouse islets infected with lentiviruses expressing control and IRKD (n = 6. The bars represent the means± SEM; *P<0.05, control vs. IRKD). (C) The results of the immunoblot analysis of the activation of insulin signal transduction pathways in control and IRKD αTC1-6 cells. Cells were serum-starved for 24 h in DMEM and were subsequently stimulated with 100 nM of insulin for 15 min. Samples were analyzed by a Western blot analysis using specific antibodies against phospho- and non-phospho-proteins, as indicated. (D–F) The time course of the changes in cell viability, cytotoxicity and apoptosis assessed using the ApoTox-Glo Triplex Assay (Promega) in the control and IRKD cells. n = 3 in each group. The data are expressed as the means ± SEM; *P<0.05, **P<0.01, control vs. IRKD. (G) The time course of BrdU incorporation in the control and IRKD cells. n = 3 in each group. The data are expressed as the means ± SEM; *P<0.05, **P<0.01, control vs. IRKD.

Mentions: To achieve stable expression of shRNA in cultured cell lines, a lentiviral vector was used to transduce the αTC1-6 cells. The effectiveness of knockdown in the stable cell lines was confirmed by a Western blot analysis using cells at six and 12 passages post-infection (one month and two months), and a maximum of 80% IR-knockdown at the protein level was observed in the cells at both of these passages (Fig. 1A). Similarly, isolated islets infected with a shRNA lentivirus targeting the IR showed an 82.1% decrease in Insr expression (Fig. 1B).


Possible contribution of taurine to distorted glucagon secretion in intra-islet insulin deficiency: a metabolome analysis using a novel α-cell model of insulin-deficient diabetes.

Bessho M, Murase-Mishiba Y, Imagawa A, Terasaki J, Hanafusa T - PLoS ONE (2014)

Results of an analysis of the effect of stable IR knockdown in αTC1-6 cells and IR knockdown in mouse islets.(A) An immunoblot analysis of the insulin receptor (IR) expression in αTC1-6 cells after the transduction with lentiviruses expressing control shRNA (control) and shRNA-IR (IRKD). αTC1-6 cells were infected with lentiviruses (see details in the “Materials and Methods”), and were cultured in medium containing 1 µg/ml puromycin for one month to generate homogenous stable cell lines. Cells were then analyzed by a Western blot analysis using specific polyclonal antibodies against the β-subunit of the IR (IR-β). Stable αTC1-6 cells maintained in culture for two months were also examined. Blots are shown on the top, and the relative amount of IRβ protein normalized to the β-actin level is shown on the bottom (n = 3. The bars represent the means± SEM; *P<0.05, control vs. IRKD). (B) The relative expression of the Insr gene in isolated mouse islets infected with lentiviruses expressing control and IRKD (n = 6. The bars represent the means± SEM; *P<0.05, control vs. IRKD). (C) The results of the immunoblot analysis of the activation of insulin signal transduction pathways in control and IRKD αTC1-6 cells. Cells were serum-starved for 24 h in DMEM and were subsequently stimulated with 100 nM of insulin for 15 min. Samples were analyzed by a Western blot analysis using specific antibodies against phospho- and non-phospho-proteins, as indicated. (D–F) The time course of the changes in cell viability, cytotoxicity and apoptosis assessed using the ApoTox-Glo Triplex Assay (Promega) in the control and IRKD cells. n = 3 in each group. The data are expressed as the means ± SEM; *P<0.05, **P<0.01, control vs. IRKD. (G) The time course of BrdU incorporation in the control and IRKD cells. n = 3 in each group. The data are expressed as the means ± SEM; *P<0.05, **P<0.01, control vs. IRKD.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231115&req=5

pone-0113254-g001: Results of an analysis of the effect of stable IR knockdown in αTC1-6 cells and IR knockdown in mouse islets.(A) An immunoblot analysis of the insulin receptor (IR) expression in αTC1-6 cells after the transduction with lentiviruses expressing control shRNA (control) and shRNA-IR (IRKD). αTC1-6 cells were infected with lentiviruses (see details in the “Materials and Methods”), and were cultured in medium containing 1 µg/ml puromycin for one month to generate homogenous stable cell lines. Cells were then analyzed by a Western blot analysis using specific polyclonal antibodies against the β-subunit of the IR (IR-β). Stable αTC1-6 cells maintained in culture for two months were also examined. Blots are shown on the top, and the relative amount of IRβ protein normalized to the β-actin level is shown on the bottom (n = 3. The bars represent the means± SEM; *P<0.05, control vs. IRKD). (B) The relative expression of the Insr gene in isolated mouse islets infected with lentiviruses expressing control and IRKD (n = 6. The bars represent the means± SEM; *P<0.05, control vs. IRKD). (C) The results of the immunoblot analysis of the activation of insulin signal transduction pathways in control and IRKD αTC1-6 cells. Cells were serum-starved for 24 h in DMEM and were subsequently stimulated with 100 nM of insulin for 15 min. Samples were analyzed by a Western blot analysis using specific antibodies against phospho- and non-phospho-proteins, as indicated. (D–F) The time course of the changes in cell viability, cytotoxicity and apoptosis assessed using the ApoTox-Glo Triplex Assay (Promega) in the control and IRKD cells. n = 3 in each group. The data are expressed as the means ± SEM; *P<0.05, **P<0.01, control vs. IRKD. (G) The time course of BrdU incorporation in the control and IRKD cells. n = 3 in each group. The data are expressed as the means ± SEM; *P<0.05, **P<0.01, control vs. IRKD.
Mentions: To achieve stable expression of shRNA in cultured cell lines, a lentiviral vector was used to transduce the αTC1-6 cells. The effectiveness of knockdown in the stable cell lines was confirmed by a Western blot analysis using cells at six and 12 passages post-infection (one month and two months), and a maximum of 80% IR-knockdown at the protein level was observed in the cells at both of these passages (Fig. 1A). Similarly, isolated islets infected with a shRNA lentivirus targeting the IR showed an 82.1% decrease in Insr expression (Fig. 1B).

Bottom Line: A comprehensive metabolomic analysis of the IRKD αTC1-6 cells (IRKD cells) revealed some candidate metabolites whose levels differed markedly compared to those in control αTC1-6 cells, but also which could affect the glucagon release in IRKD cells.Of these candidates, taurine was remarkably increased in the IRKD cells and was identified as a stimulator of glucagon in αTC1-6 cells.These results indicate that the metabolic alterations induced by IRKD in α-cells, especially the increase of taurine, may lead to the distorted glucagon response in IRKD cells, suggesting the importance of taurine in the paradoxical glucagon response and the resultant glucose instability in insulin-deficient diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine (I), Osaka Medical College, Osaka, Japan.

ABSTRACT
Glycemic instability is a serious problem in patients with insulin-deficient diabetes, and it may be due in part to abnormal endogenous glucagon secretion. However, the intracellular metabolic mechanism(s) involved in the aberrant glucagon response under the condition of insulin deficiency has not yet been elucidated. To investigate the metabolic traits that underlie the distortion of glucagon secretion under insulin deficient conditions, we generated an αTC1-6 cell line with stable knockdown of the insulin receptor (IRKD), i.e., an in vitro α-cell model for insulin-deficient diabetes, which exhibits an abnormal glucagon response to glucose. A comprehensive metabolomic analysis of the IRKD αTC1-6 cells (IRKD cells) revealed some candidate metabolites whose levels differed markedly compared to those in control αTC1-6 cells, but also which could affect the glucagon release in IRKD cells. Of these candidates, taurine was remarkably increased in the IRKD cells and was identified as a stimulator of glucagon in αTC1-6 cells. Taurine also paradoxically exaggerated the glucagon secretion at a high glucose concentration in IRKD cells and islets with IRKD. These results indicate that the metabolic alterations induced by IRKD in α-cells, especially the increase of taurine, may lead to the distorted glucagon response in IRKD cells, suggesting the importance of taurine in the paradoxical glucagon response and the resultant glucose instability in insulin-deficient diabetes.

Show MeSH
Related in: MedlinePlus