Figure 1.
Inhibition of c-Abl significantly enhance glucose-stimulated insulin production by β cells.
A. 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. 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. C. In the cultures of B above, C-peptide concentration in each incuation was examined using ELISA. Student t test was performed. D. 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.
Figure 2.
Inhibition of c-Abl promotes insulin production by β cells in resting state.
NIT-1 cells were cultured in low glucose DMEM medium (glucose 4.5 mM) for a few days. A. 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. 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. C. 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.
Figure 3.
Glucose stimulation induces up-regulation of both c-Abl and insulin expression.
A . 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. 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.
Figure 4.
Relationship between c-Abl and insulin gene transcription factor PDX-1.
A. 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. 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. C. 293 cell line were transfected with pdx-1 promoter-driven luciferase reporter gene, together with transfection of plasmids encoding c-Abl 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.
Figure 5.
C-Abl regulates insulin gene expression via NKx2.2.
A. 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. 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.
Figure 6.
c-Abl inhibition up-reguates GLUT2 expression on β cells.
A. NIT-1 cells were cultured in low glucose DMEM alone or with 8 mM glucose, 16 mM glucose, 3 µM Imatinib, 8 mM glucose+3 µM Imatinib, or 16 mM glucose+3 µM Imatinib for 24 hrs. The GLUT2 protein levels were evaluated using Western blot. The expression levels were calculated relative to β actin. The similar results were obtained in additional two independent experiments. B. NIT-1 cells cultured in low glucose DMEM media alone, or with Imatinib 3 µM for 6 hours. The cultures were triplicated for each condition. Then the cells were harvested and RNA was extracted. The GLUT2 mRNA levels were examined by Real-time RT-PCR. Student t test was performed. ***:p<0.001. The data shown were from one representative of three independent experiments.
Figure 7.
Diagram of c-Abl regulating insulin gene expression.
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.