Figure 1.
Apoptosis-inducing saturated FFA, palmitate upregulated DRP-1 in INS-1 cells and rat islets.
(A) Dose-response and (B) time-course of palmitate-induced apoptosis in INS-1 cells. To study dose-response, cells were cultured with the indicated doses (0 mM, 0.2 mM, 0.4 mM, and 0.8 mM) of palmitate for 24 h. To study the time-course, cells were cultured in a medium containing 0.2 mM palmitate for the indicated amounts of time (0 h, 24 h, 48 h, and 96 h). Analysis of apoptosis in INS-1 cells was done by annexin V staining. The data are presented as mean percentage ± S.E. of annexin V-positive cells. The experiments were carried out for three independent times using triplicate samples (*p<0.05, **p<0.01, ANOVA/Tukey test). (C) Dose-response and (D) time-course of palmitate on DRP-1 expression in INS-1 cells. To study the dose-response, cells were cultured with the indicated doses of palmitate for 24 h. To study the time-course, cells were cultured in a medium containing 0.2 mM palmitate for the indicated amounts of time (E) DRP-1 was augmented in isolated islets treated with 0.5 mM palmitate for 24 h. (F) Serum FFA levels were increased after palmitate stimulation. C57BL/6 mice were i.p. injected with 0.5 ml of 5 mM palmitate once daily for 5 days. Their serum FFA levels 2 h after injection at the fifth day were determined utilizing the nonesterified fatty acid assay kit. Samples were tested in duplicate. Mean ± S.D. of the palmitate injected groups and control groups (BSA injected) are shown (n = 6 for each group). The difference is statistically significant (**p<0.01, unpaired two-tailed t test). (G, H) Increased FFA levels upregulated islet DRP-1 expression. After i.p. injection of palmitate once daily for 5 days, immunohistochemistry was performed to evaluated islet DRP-1 expression in (G)control group and (H) palmitate injected group (n = 6). Scale bar, 50 µm.
Figure 2.
Characterization of the DRP-1WT and DRP-1 K38A inducible beta cell lines.
(A) Western blot analysis for Dox-induced DRP-1 protein expression in DRP-1 WT and DRP-1 K38A cells. (B) DRP-1 WT and DRP-1 K38A proteins were induced by Dox. Phase-contrast images (top) and immunofluorescence staining (bottom). Scale bar, 100 µm.
Figure 3.
Analysis of apoptosis in DRP-1 WT and DRP-1 K38A cells.
DRP-1 WT and DRP-1 K38A cells were cultured in the presence (black bars) or absence (white bars) of 500 ng/ml Dox for 48 h, and then treated with 0.2 mM palmitate for 24 h. (A) TUNEL staining of apoptosis in DRP-1 WT cells and DRP-1 K38A cells. Columns represent mean ± S.E. of three different experiments conducted in triplicate (*p<0.05, **p<0.01, ANOVA/Tukey test). Scale bar, 50 µm. (B) Flow cytometric analysis of apoptosis in DRP-1 WT cells and DRP-1 K38A cells. This assay could discriminate between intact cells (FITC−/PI−), early apoptotic cells (FITC+/PI−), late apoptotic cells (FITC+/PI+), and necrotic cells (FITC−/PI+). The bar graph shows the average apoptosis rate (early apoptosis and late apoptosis) of INS-1-derived cells with different Dox and palmitate treatments. Columns represent mean ± S.E. of three different experiments conducted in triplicate (*p<0.05, **p<0.01, ANOVA/Tukey test).
Figure 4.
DRP-1 mediated FFA-induced INS-1-derived cell apoptosis in vivo.
(A) Subrenal capsule implantation surgery. Left kidney of a nu/nu mouse was exposed. DRP-1 WT cells or DRP-1 K38A cells were injected under the capsule membrane. (B) Average blood glucose levels in fasting animals of the eight experimental groups. Data are expressed as mean ± S.E. (n = 6, **p<0.01, ANOVA/Tukey test). (C) Average blood insulin levels in fasting animals of the eight experimental groups. Data are expressed as mean ± S.E. (n = 6, *p<0.05, ANOVA/Tukey test). (D) Average blood insulin levels in fed animals of the eight experimental groups. Data are expressed as mean ± S.E. (n = 6, **p<0.01, ANOVA/Tukey test). (E) The mouse kidneys of each group at day 33. In each group, the left kidneys were transplanted with DRP-1 WT cells or DRP-1 K38A cells. (F) H&E staining showed DRP-1 WT cells can form tumors after subrenal capsules transplantation. DRP-1 K38A cells also can form tumors similar to DRP-1 WT cells (data not shown). (G) Example case of insulin expression in DRP-1 WT cell xenografts observed by immunohistochemical staining. Insulin is also expressed in DRP-1 K38A cell xenografts (data not shown). (H) Example case of DRP-1 expression in DRP-1 WT cell or DRP-1 K38A cell xenografts of each group observed by immunohistochemical staining. (I) The TUNEL staining was employed to detect the apoptosis in DRP-1 WT cell or DRP-1 K38A cell xenografts. Data are expressed as mean ± S.E. (n = 6, *p<0.05, **p<0.01, ANOVA/Tukey test). Scale bar, 50 µm.
Figure 5.
Effect of DRP-1 over-expression on mitochondrial membrane potential and mitochondrial morphology in DRP-1 WT cells and DRP-1 K38A cells.
(A, B) Effects of (A) DRP-1 WT and (B) DRP-1 K38A over-expression on mitochondrial membrane potential. Data are presented as mean ± S.E. of three different experiments conducted in triplicate (*p<0.05, ANOVA/Tukey test). (C, D) Effects of DRP-1 WT and DRP-1 K38A over-expression on mitochondrial morphology. Transmission electron microscopy of mitochondrial morphology in (C) DRP-1WT cells and (D) DRP-1K38A cells in different palmitate and Dox conditions. Black arrows point to the typical fused mitochondria (elongated mitochondria). White arrows point to the typical punctiform mitochondria. The bar graph shows the average ratio of fused mitochondria vs. individual mitochondria (elongated & small punctiform mitochondria) for each of the experimental conditions. Data are presented as mean ± S.E. of three different experiments conducted in triplicate (*p<0.05, **p<0.01, ANOVA/Tukey test).
Figure 6.
The effect of DRP-1 on cytochrome c release in INS-1-derived cells.
(A) A representative western blot of DRP-1 and cytoplasmic cytochrome c expression in DRP-1 WT and DRP-1 K38A cells with different Dox and palmitate treatments. (B) A bar graph showing the relative cytochrome c release in INS-1-derived cells with different Dox and palmitate treaments. The relative intensity of each band was determined as a ratio to its corresponding beta-actin band. Data are presented as mean ± S.E. of three different experiments (*p<0.05, **p<0.01, ANOVA/Tukey test).
Figure 7.
The effect of DRP-1 on ROS production and caspase-3 activity in DRP-1 WT and DRP-1 K38A cells.
Data are presented as mean ± S.E. of three different experiments conducted in triplicate (A, B) The effect of DRP-1 on ROS production in DRP-1 WT and DRP-1 K38A cells (*p<0.05, **p<0.01, ANOVA/Tukey test). (C, D) The effect of DRP-1 expression on caspase-3 activity in DRP-1 WT cells and DRP-1 K38A cells (*p<0.05, **p<0.01, ANOVA/Tukey test).