Fig 1.
Light micrograph of the monkey pancreas. The Langerhans islet after the hydroxynonenal injections (HNE) shows many vacuole formation (circles), compared to the sham-operated control (Cont). Nuclear chromatin is generally more dense after the hydroxynonenal injections, compared to the control. A small number of nucleus shows diffuse dissolution or punctuate condensation (dot circle). However, neither apoptotic bodies nor membrane blebbings were seen. Acinar cells are distributed in the surrounding area. Hematoxylin-eosin staining.
Fig 2.
Electron micrograph of the Langerhans islet of the monkey before and after the hydroxynonenal injections.
The Langerhans islet cells are situated surrounding a capillary (Cap), and each cell is characterized by peculiar secretory granules. Insulin secretory granules of the β-cell (β) are the largest with a clear halo. Glucagon secretory granules of the α-cell (α) lack a clear halo, being a little bit smaller but more electron-dense than insulin granules. Somatostatin-containing granules of the δ-cell (δ) are also electron-dense like glucagon granules, showing a sparse distribution. PP-cells (PP) contain spherical or elliptical small granules, which are very heterogeneous in size. The greatest changes after the hydroxynonenal injections are a remarkable decrease of insulin and somatostatin granules as well as an increase of autophagosomes and autolysosomes. In the control β-cell (Cont), insulin granules were distributed throughout the cytoplasm. In contrast, in the β-cell after the hydroxynonenal injections (HNE), the distribution of insulin granules was restricted in the cytoplasm toward the capillary. Instead, especially β-cell showed formation of many autolysosomes (circles), compared to the control (Cont). Further, both β-cell and δ-cell showed vacuole formations (stars) and more electron-dense nuclear chromatin. N; nucleus.
Fig 3.
Electron micrograph of the β-cell in the monkey after the hydroxynonenal injections.
β-cell shows fusion of an autophagosome (AP) with a lysosome (Ly) exhibiting evidence of membrane permeabilization (arrows). This autophagosome is devoid of double membrane, whereas another one (Ap) presumably containing degenerated mitochondria, has distinct double membrane. G: insulin granule.
Fig 4.
Electron micrograph of the β-cell in the monkey after the hydroxynonenal injections.
β-cell shows fusion of an autolysosome (Al) with a peroxisome (P) exhibiting membrane disruption (arrows). The autolysosome (Al) contains mitochondria-derived debris (asterisk). The neighbouring lysosome (Ly) shows membrane permeabilization. G: insulin granule, Ap: autophagosome, m: degenerating mitochondria.
Fig 5.
Electron micrograph of the β-cell in the monkey after the hydroxynonenal injections.
β-cell shows enlargement of rough ER (ER), as seen in Fig 2. Proliferation of peroxisomes (P) is obvious, and they are intermingled with degenerating mitochondria and insulin granules in the cytoplasm toward the capillary lumen, as seen in Fig 2. Some peroxisomes with membrane disruptions (arrows) are fusing with degenerating mitochondria (m, circles) or autolysosomes presumably containing mitochondrial debris (dot circles). G: insulin granule.
Fig 6.
a) Electron micrograph of the δ-cell in the monkey after the hydroxynonenal injections. δ-cell shows fusion of an autolysosome (Al) with a peroxisome (P) exhibiting membrane disruption. G: somatostatin granule without a halo, Ly: lysosome. b) Electron micrograph of the δ-cell in the monkey after the hydroxynonenal injections. δ-cell shows peroxisomal proliferation (P) around the autolysosomes (Al). The latter are presumably in the process of fusing with peroxisomes (P). Some peroxisomes show membrane disruption (arrows). ER: rough ER.
Fig 7.
Calpain activation, Hsp70.1 cleavage, and cathepsin B leakage.
a) Immunofluorescence histochemical staining of Hsp70.1 (red) and activated μ-calpain (green). Activated μ-calpain immunoreactivity is negligible without hydroxynonenal injections (Cont), whereas μ-calpain activation occurs with hydroxynonenal injections (HNE), being consistent with the Western blotting data (c, activated μ-calpain). After hydroxynonenal injections, activated μ-calpain immunoreactivity (green) is colocalized with Hsp70.1 immunoreactivity (red), showing a merged color of yellow (HNE, yellow). The distribution pattern of granular merged colors is compatible with that of autolysosomes as seen in Fig 2. b) Immunofluorescence histochemical staining of cathepsin B (green). Cathepsin B is stained as tiny granules in the control Langerhans islet (Cont), whereas stained as coarse granules with the perigranular immunoreactivity after hydroxynonenal injections (HNE), which indicates lysosomal membrane rupture/permeabilization. The nuclei show negligible cathepsin B immunoreactivity. Cathepsin B immunoreactivity was colocalized with that of Lamp2 (data not shown here). c, d) Western blotting analyses of activated μ-calpain (c, 76 kDa), GPR109A (c, 41.8 kDa), and Hsp70.1 (d, 70 kDa). Compared to the control (c, Cont), μ-calpain is activated after hydroxynonenal injections (c, HNE). In response to the cell stress due to hydroxynonenal injections (d, HNE), not only Hsp70.1 main bands (d, rectangle) but also cleaved Hsp70.1 bands of 30 kDa (d, dot rectangle) are increased, compared to the control (d, Cont). GPR109A is expressed in the pancreas tissue, although showing no change after hydroxynonenal injections (c, GPR109A).
Fig 8.
The molecular cascade explaining degeneration of β- and δ- cells in the Langerhans islet of the monkey after hydroxynonenal injections.
Peroxisomes play a crucial role for the sustained H2O2 production, while calpain-mediated cleavage of carbonylated Hsp70.1 causes lysosomal membrane rupture/permeabilization. In both cascades, hydroxynonenal plays a central role for the β- and δ- cell degeneration.