Fig 1.
Hypoxia induces mitochondrial fragmentation in pancreatic beta INS-1E cells.
A. Representative confocal microscope images of the mitochondrial in pancreatic beta INS-1E cellsafter treatment with hypoxia at different oxygen levels (3% or 1% O2 for 24 h) as indicated. Scale bars, 10 μm.B.The proportion of pancreatic beta INS-1E cellswith tubulated, intermediate, and fragmented mitochondria was quantified. C. Representative transmission electron microscopy (TEM) images of the mitochondrialin INS-1Ecells treated as indicated. Scale bars, 0.5 μm. D.Image J was used for mitochondrial length quantification in INS-1Ecells treated as indicated.All experiments were repeated three times.*P< 0.05.
Fig 2.
HIF-1α activation and subsequently DRP1(S616) phosphorylationis involved in hypoxia-induced mitochondrial fragmentation in pancreatic beta cells.
A-B. Expression levels of MFN1, MFN2, OPA1, DRP1, Fis1, and MFF were detected by RT–PCR (A) and western blot (B)analysis in pancreatic beta INS-1E cellswith treatment as indicated. (C)Top: Levels of HIF-1α and phosphorylated DRP1 (S637)or(S616)were detected by western blot analysis in pancreatic beta INS-1E cellswith treatment as indicated.Bottom: Western blot scanning densitometry for three independent experiments.Blots were probed for β-actin to ensure equal proteinloading. *P<0.05.(D)Top: Levels of DRP1 in total lysates and mitochondrial fraction were examined by western blot analysis in pancreatic beta INS-1E cellswith treatment as indicated. VDACserves as loading controls. Mito: mitochondria.Bottom: Western blot scanning densitometry for three independent experiments.Blots were probed for β-actinor VDAC respectively to ensure equal proteinloading. *P<0.05.All experiments were repeated three times.
Fig 3.
Mitochondrial fragmentation is involved inpancreatic beta cell death in hypoxia conditions.
A.Detection of DRP1 (S616) and mitochondrial-fractional DRP1by western blot analysis in pancreatic beta INS-1E cellswith treatment as indicated. B.Detection of apoptosis by flow cytometry in pancreatic beta INS-1E cellswith treatment as indicated. C.Western blot analysis for levels of cytochrome c inpancreatic beta INS-1E cellswith treatment as indicated. β-actin and VDAC were used as loading controls for cytoplasm and mitochondria, respectively. Cyto, cytoplasm; Mito, mitochondrial. D.Western blot analysis for levels ofcleaved Caspase-9 and Caspase-3 inpancreatic beta INS-1E cellswith treatment as indicated. E.TUNEL staining in pancreatic beta INS-1E cells from type 2 diabetes animal models. Blue: Hoechst 33342; Green: TUNEL positive nucleus. All experiments were repeated three times.*P< 0.05.
Fig 4.
Hypoxia-induced mitochondrial fragmentation facilitates cristae remodeling in pancreatic beta cells.
A.Representative transmission electron microscopy (TEM) images of mitochondria in pancreatic beta INS-1E cells treatedas indicated.Scale bars, 0.1 μm.B-D. Mitochondrial length (B), cristae width (C) and cristae density(number/mitochondrial length)(D)were quantitatively analyzedin pancreatic beta INS-1E cells treatedas indicated. Over twenty mitochondria were randomly selected for quantitative analysis in each treatment group.All experiments were repeated three times.*P< 0.05.
Fig 5.
Mdivi-1 exhibits a therapeutic effect on hypoxia-induced cell death of pancreatic beta cells.
A.Mitochondrial morphologyanalysis by confocal microscopy in pancreatic beta INS-1E cells treated with Mdivi-1 (50 μM) or DMSO for 12 h as indicated.Scale bars, 10 μm.B.Cell viability analysis by MTS assay in pancreatic beta INS-1E cells treated with Mdivi-1or DMSO as indicated. C.Detection of apoptosis by flow cytometry in pancreatic beta INS-1E cellstreated with Mdivi-1 or DMSO as indicated. D. TUNEL staining in pancreatic beta INS-1E cells treated with Mdivi-1 or DMSO as indicated.Scale bars, 50 μm.All experiments were repeated three times.*P< 0.05.