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

<div><p>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 <i>in vitro</i> 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.</p></div

A comparison of the biosynthetic metabolites between the control and IRKD αTC1-6 cells, as determined by CE-TOFMS.

<p>Control, shRNA control αTC1-6 cells; IRKD, shRNA insulin receptor knockdown αTC1-6 cells; −1.5, treated with 1.5 mM glucose; −5.6, treated with 5.6 mM glucose; −30, treated with 30 mM glucose.</p><p>*<i>P</i><0.05,</p><p>**<i>P</i><0.01,</p><p>***<i>P</i><0.001 (Control vs. IRKD).</p>¶<p>Control/IRKD ratio.</p><p>A comparison of the biosynthetic metabolites between the control and IRKD αTC1-6 cells, as determined by CE-TOFMS.</p

Exaggerated glucose-stimulated glucagon secretion in IRKD αTC1-6 cells and islets in the presence of taurine.

<p>αTC1-6 cells (<b>A, B</b>) or islets (<b>C, D</b>) were preincubated for 1 h with KRB containing 5.6 mM glucose, and were subsequently stimulated for 2 h with 1.5, 5.6 or 30 mM glucose with or without 10 mM taurine. <i>n</i> = 6 (<b>A, B</b>) or 3 (<b>C, D</b>) in each group. The bars represent the means ± SEM; *<i>P</i><0.05, taurine-treated vs. vehicle-treated cells or islets.</p

Results of an analysis of the effect of stable IR knockdown in αTC1-6 cells and IR knockdown in mouse islets.

<p>(<b>A</b>) 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 “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113254#s2" target="_blank">Materials and Methods</a>”), 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 <i>top</i>, and the relative amount of IRβ protein normalized to the β-actin level is shown on the <i>bottom</i> (<i>n</i> = 3. The bars represent the means± SEM; *<i>P</i><0.05, control vs. IRKD). (<b>B</b>) The relative expression of the <i>Insr</i> gene in isolated mouse islets infected with lentiviruses expressing control and IRKD (<i>n</i> = 6. The bars represent the means± SEM; *<i>P</i><0.05, control vs. IRKD). (<b>C</b>) 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. (<b>D–F</b>) 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. <i>n</i> = 3 in each group. The data are expressed as the means ± SEM; *<i>P</i><0.05, **<i>P</i><0.01, control vs. IRKD. (<b>G</b>) The time course of BrdU incorporation in the control and IRKD cells. <i>n</i> = 3 in each group. The data are expressed as the means ± SEM; *<i>P</i><0.05, **<i>P</i><0.01, control vs. IRKD.</p

Time course of [³H] taurine and amino acid uptake in control and IRKD αTC1-6 cells.

<p><i>n</i> = 4 in each group. The data are expressed as the means ± SEM; *<i>P</i><0.05, control vs. IRKD.</p

Altered gene expression of the major enzymes required for taurine, arginine and glutamine synthesis in IRKD αTC1-6 cells.

<p>The relative expression of <i>Cdo1</i>, <i>Csad</i>, <i>Ass1</i>, <i>Asl</i> and <i>Glul</i> genes in the control and IRKD αTC1-6 cells incubated for 3 h with KRB containing 5.6 mM glucose was measured by quantitative real-time PCR and normalized to the expression of the reference gene, <i>Actb</i>. <i>n</i> = 4 in each group. The bars represent the means ± SEM; *<i>P</i><0.05, control vs. IRKD.</p

Comparison of the metabolic profile of control and IRKD αTC1-6 cells, as determined by CE-TOFMS.

<p>A principal component analysis (PCA) score plot (<b>A</b>) and heat map (<b>B</b>) from the metabolic data of the control and IRKD αTC1-6 cells treated with three different concentrations of glucose (1.5, 5.6 or 30 mM) (<i>n</i> = 3 in each group).</p

Taurine-stimulated glucagon secretion and cell contents in control αTC1-6 cells with or without insulin.

<p>(<b>A</b>) Cells were preincubated for 1 h with KRB containing 5.6 mM glucose, and were subsequently stimulated for 2 h with 0, 1, 10 or 100 mM taurine and treated with or without insulin (100 nM). <i>n</i> = 6 in each group. (<b>B</b>) The total protein content and total glucagon content. <i>n</i> = 6 in each group. The bars represent the means ± SEM; *<i>P</i><0.05, vs. vehicle-treated cells.</p

PET/CT scores in patients with PMR and EORA.

<p>The PET/CT scores were significantly higher in the patients with polymyalgia rheumatica (PMR) than in the patients with elderly-onset rheumatoid arthritis (EORA) (<i>P</i> = 0.0003).</p

Sites of abnormal FDG accumulation in patients with PMR and EORA.

<p>Sites of abnormal FDG accumulation in patients with PMR and EORA.</p