C-Abl Inhibitor Imatinib Enhances Insulin Production by β Cells: C-Abl Negatively Regulates Insulin Production via Interfering with the Expression of NKx2.2 and GLUT-2

<div><p>Chronic myelogenous leukemia patients treated with tyrosine kinase inhibitor, Imatinib, were shown to have increased serum levels of C-peptide. Imatinib specifically inhibits the tyrosine kinase, c-Abl. However, the mechanism of how Imatinib treatment can lead to increased insulin level is unclear. Specifically, there is little investigation into whether Imatinib directly affects β cells to promote insulin production. In this study, we showed that Imatinib significantly induced insulin expression in both glucose-stimulated and resting β cells. In line with this finding, c-Abl knockdown by siRNA and overexpression of c-Abl markedly enhanced and inhibited insulin expression in β cells, respectively. Unexpectedly, high concentrations of glucose significantly induced c-Abl expression, suggesting c-Abl may play a role in balancing insulin production during glucose stimulation. Further studies demonstrated that c-Abl inhibition did not affect the major insulin gene transcription factor, pancreatic and duodenal homeobox-1 (PDX-1) expression. Of interest, inhibition of c-Abl enhanced NKx2.2 and overexpression of c-Abl in β cells markedly down-regulated NKx2.2, which is a positive regulator for insulin gene expression. Additionally, we found that c-Abl inhibition significantly enhanced the expression of glucose transporter GLUT2 on β cells. Our study demonstrates a previously unrecognized mechanism that controls insulin expression through c-Abl-regulated NKx2.2 and GLUT2. Therapeutic targeting β cell c-Abl could be employed in the treatment of diabetes or β cell tumor, insulinoma.</p></div

Inhibition of c-Abl promotes insulin production by β cells in resting state.

<p>NIT-1 cells were cultured in low glucose DMEM medium (glucose 4.5 mM) for a few days. <b>A</b>. The NIT-1 cells were harvested and then cultured in low glucose DMEM medium with different conditions: Imatinib 0 µM, 0.3 µM, 1 µM or 3 µM for 6 hrs. The supernatants from all cultures were harvested and measured for insulin production by ELISA. The results presented were from a representative of three independent experiments. Two-way ANOVA with Bonferroni post-test was performed. <b>B</b>. The NIT-1 cells were cultured in low glucose DMEM medium with Imatinib 0 µM, 0.3 µM or 1 µM for 6 hrs. The insulin gene expression was examined by real-time RT-PCR. The levels of insulin gene expression were normalized relative to β actin. The experiments were repeated 3 times with reproducible results. Two-way ANOVA with Bonferroni post-test was performed. <b>C</b>. NIT-1 cells were transfected with c-Abl siRNA or control siRNA for 24 hrs. Then the transfected cells were cultured in low glucose DMEM medium for 6 hrs. An additional group was also included by culturing c-Abl siRNA transfected NIT-1 cells with 1 µM Imatinib. The supernatants were harvested from the above cultures and the insulin concentration was examined by ELISA. One-way ANOVA with post hoc test was performed. Similar results were obtained from at least 3 independent experiments. *: p<0.05, **:p<0.01,***:p<0.001.</p

Inhibition of c-Abl significantly enhance glucose-stimulated insulin production by β cells.

<p><b>A</b>. NIT-1 cells were cultured in the low glucose media (glucose 4.5 mM) with glucose 8 mM, 16 mM, 8 mM plus Imatinib 3 µM or 16 mM plus Imatinib 3 µM for 6 hrs. The insulin concentration was measured using ELISA. The similar results were obtained in 3 independent experiments. Two-way ANOVA with Bonferroni post-test was performed. <b>B</b>. NIT-1 cells were cultured in the low glucose media (glucose 4.5 mM) with glucose 16 mM or 16 mM plus Imatinib 3 µM for 6 hrs, insulin gene expression was examined by real-time RT-PCR. The levels of insulin gene expression were normalized relative to β actin. The results were reproduced in 3 independent experiments. Student t test was performed. <b>C</b>. In the cultures of <b>B</b> above, C-peptide concentration in each incuation was examined using ELISA. Student t test was performed. <b>D</b>. NIT-1 cells were transfected with c-Abl siRNA or control siRNA for 24 hrs, then were stimulated with 16 mM glucose for 6 hrs. Insulin gene expression was examined by real-time RT-PCR, the data were calculated relative to the group with NIT-1 cells transfected with control siRNA without glucose stimulation. Three independent experiments were performed with similar results. Student t test was performed. *: p<0.05, **:p<0.01,***:p<0.001.</p

C-Abl regulates insulin gene expression via NKx2.2.

<p><b>A</b>. NIT-1 cells were tranduced with retrovirus vector encoding c-Abl gene to overexpress c-Abl or control cDNA. Twenty-four hrs later, the cells were examined for the gene expression of insulin, PDX-1, NKx6.1, NKx2.2, respectively by real-time RT-PCR. The PCR results were normalized relative to β actin for each individual gene, and the relative values of c-Abl transduced cells were compared to those of control cDNA transduced cells which were defined as 1 (the white bar). The results shown were from a representative of 3 independent experiments. One-way ANOVA with post hoc test was performed. *: p>0.05, **:p<0.01, ***:p<0.001. <b>B</b>. NIT-1 cells were cultured in the low glucose DMEM medium alone (Immatinib 0 µM), or in the presence of 0.3 µM Imatinib, or 3 µM Imatinib (3 µM) for 24 hrs. The protein levels of NKx2.2 were examined by Western blot. The densitometry was analyzed relative to the levels of β-actin, and the relative level of the cells incubated with 0 µM Imatinib was defined as 1.</p

Diagram of c-Abl regulating insulin gene expression.

<p>β cells receiving glucose signals upon glucose intracellular transportation by GLUT2 transporters up-regulate insulin gene positive regulatory factors, e.g. PDX-1 and NKx2.2 to enhance insulin gene expression. On the other hand, β cells initiate the negative regulatory pathway through up-regulating C-ABL expression, and C-ABL in turn down-regulates gene expression of NKx2.2 and its downstream insulin gene, as well as GLUT2.</p

Glucose stimulation induces up-regulation of both c-Abl and insulin expression.

<p><b>A</b> . NIT-1 cells were cultured in low glucose DMEM medium with or without glucose 16 mM for 6 hrs. Thereafter, the cells were examined for c-Abl and insulin gene expression using real-time RT-PCR. Student t test was performed. The results shown were from a representative of 3 independent experiments. *: p>0.05, **:p<0.001. <b>B.</b> NIT-1 cells were cultured in low glucose DMEM medium with or without glucose 16 mM for 24 hrs. Then, the cells were cytospun onto slides and stained with anti-c-Abl antibodies and Dappi, and visualized by a fluorescent microscope (Zeiss Axioskop). A representative image of three slides in each group is shown.</p

Relationship between c-Abl and insulin gene transcription factor PDX-1.

<p><b>A</b>. NIT-1 cells were cultured in the low glucose DMEM medium, or with glucose (16 mM) or with Imatinib (3 µM) for 6 hrs. The cells in each group were harvested and the expression of PDX-1 was examined by real-time RT-PCR. The results shown were from a representative of 3 independent experiments. <b>B</b>. NIT-1 cells were cultured in the low glucose DMEM medium with different concentrations of Imatinib (0, 0.3, 1, 3 µM) for 24 hrs. Then, the protein levels of PDX-1 and GAPDH were examined by Western blot. Relative quantity of each PDX-1 band relative to its GAPDH control was shown below the Westerblot image. These results were reproduced by additional two experiments. <b>C</b>. 293 cell line were transfected with pdx-1 promoter-driven luciferase reporter gene, together with transfection of plasmids encoding <i>c-Abl</i> gene or control plasmids. Twenty four hrs later, the cells from the above conditions were harvested and luciferase activities were measured by using the Dual Luciferase Reporter Kit. This experiment was repeated twice with similar results. The targeted gene expression levels were normalized relative to β actin. One-way ANOVA with post hoc test was performed. *: p>0.05, **:p<0.001.</p

Intra- & Inter-assay Variance for the flow cytometric analysis of STAT5Ptyr in human peripheral blood monocytes.

<p>Whole blood from T1D (#418, 237,327, and 434) and non-autoimmune healthy controls (75, 365, and 236) was analyzed for STAT5 activation as described in the Methods & Materials. Each sample aliquot was analyzed 4 to 5 times during each run of the analyses. <b>A. Intra-assay Variance</b>: There was an assay variance of <5% in between control samples and intra-assay from 0 to 6.3% for a given control sample’s analysis; whereas, there was an assay variance of <13% between T1D samples with an intra-assay 1 to 7.9% for a given T1D sample’s analysis. There was a significant (>13-fold) difference between the % STAT5Ptyr/CD14+ cells detected in the T1D samples tested (mean 65.85%, SD 12.79%, n = 4) as compared to controls analyzed in the same assays (mean 4.952%, SD 4.547%, n = 3). <b>B. </b><b>Inter-assa. </b><b>Variance:</b> Each sample analyzed 24 hr to 48 hr after shipment and storage at 4°C. An inter-assay variance of 2-6-fold between 24 hr (filled circles) and 48 hr (clear circles) assay runs of 3 control samples and a 1.1-fold variance in 3 T1D samples was seen between 24 and 48 hrs. At 24 hr, there was a significant (>13-fold, *p = 0.0017) difference between the % STAT5Ptyr/CD14+ cells of T1D samples (mean 65.85%, SD 12.79%, n = 4) as compared to controls (mean 4.952%, SD 4.547%, n = 3) in same assays. However, this significant difference decreased to 5.4-fold at 48 hr (*p = 0.013), due to the increased inter-assay variability seen in control samples after storage.</p

Characteristics of patient, control and relative samples collected for <i>CSF2</i> and <i>PTGS2</i> sequencing analysis.

*<p>Individuals with overt hyperglycemia or abnormal GTT/FPIR; mean disease duration = 8.5 years ±7.65 SD.</p

T1D Patient/Control Sequence Analysis for Upstream Regulatory Regions of the <i>CSF2</i> and <i>PTGS2</i> genes. A.

<p>Schematic representation and representative sequence data of the human promoter region upstream of <i>CSF2,</i> the gene encoding GM-CSF. The location of the amplified region is delineated by forward and reverse primers, FP1 and RP1, respectively. Also defined are the STAT5 and STAT6 binding sites (boxed) within the region. No T1D-specific polymorphisms have been detected in the 24 samples tested. <b>B.</b> Schematic representation and representative sequence data of the enhancer region upstream of the PGS2/COX2 gene, <i>PTGS2.</i> The location of the amplified region is delineated by forward and reverse primers, FP1 and RP1, respectively. Also defined are the STAT5 and STAT6 binding sites within the region (boxed). No T1D-specific polymorphisms have been detected in the 24 samples tested.</p