15 research outputs found
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
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
Time course of [<sup>3</sup>H] 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
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
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
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
Antibody to CMRF35-Like Molecule 2, CD300e A Novel Biomarker Detected in Patients with Fulminant Type 1 Diabetes
<div><p>Aims/Hypothesis</p><p>Fulminant type 1 diabetes (FT1D) is a distinct subtype of type 1 diabetes and is fatal without immediate diagnosis and treatment. At present, there are no biomarkers for early and predictive detection of FT1D.</p><p>Methods</p><p>First, we analyzed a total of 6 serum samples from 3 patients with FT1D (1 sample in the acute and 1 in the sub-acute phases from each patient) by seromic analysis. Second, titres of the antibody were measured by ELISA in sera from 30 patients with FT1D (both in the acute and sub-acute phases), 13 patients with FT1D in the chronic phase, 32 patients with autoimmune type 1 (type 1A) diabetes (T1AD), 30 patients with type 2 diabetes (T2D), 23 patients with autoimmune thyroid disease (AITD) and 31 healthy control subjects (HC).</p><p>Results</p><p>Seromic analysis revealed 9 antibodies which showed high signals from all 3 patients with FT1D in the acute phase. Among them, the titre of anti-CD300e antibody was significantly higher in FT1D patients in the acute phase than that in T1AD, T2D, AITD patients and HC, as determined by ELISA (P<0.01, respectively). The titre of anti-CD300e antibody was also higher in FT1D in the acute phase than that in the sub-acute phase (P = 0.0018, Wilcoxon signed-rank test). The titre of anti-LGALS3 antibody in FT1D patients in the acute phase did not differ from that in patients with FT1D in the sub-acute phase, T1AD, T2D, AITD and HC.</p><p>Conclusion/Interpretation</p><p>The titre of a novel antibody, anti-CD300e, was high in sera from patients with FT1D. This antibody might be a diagnostic marker and provide new insight into the pathogenesis of FT1D.</p></div
Clinical characteristics of the subjects.
<p>Clinical characteristics of the subjects.</p