Hyperglycemia Induces Cellular Hypoxia through Production of Mitochondrial ROS Followed by Suppression of Aquaporin-1.

We previously proposed that hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) generation is a key event in the development of diabetic complications. Interestingly, some common aspects exist between hyperglycemia and hypoxia-induced phenomena. Thus, hyperglycemia may induce cellular hypoxia, and this phenomenon may also be involved in the pathogenesis of diabetic complications. In endothelial cells (ECs), cellular hypoxia increased after incubation with high glucose (HG). A similar phenomenon was observed in glomeruli of diabetic mice. HG-induced cellular hypoxia was suppressed by mitochondria blockades or manganese superoxide dismutase (MnSOD) overexpression, which is a specific SOD for mtROS. Overexpression of MnSOD also increased the expression of aquaporin-1 (AQP1), a water and oxygen channel. AQP1 overexpression in ECs suppressed hyperglycemia-induced cellular hypoxia, endothelin-1 and fibronectin overproduction, and apoptosis. Therefore, hyperglycemia-induced cellular hypoxia and mtROS generation may promote hyperglycemic damage in a coordinated manner

Cellular hypoxia was attenuated by inhibiting mitochondrial respiration and MnSOD overexpression <i>in vitro and vivo</i>.

<p>(A) Effect of mitochondrial respiratory inhibitors on cellular hypoxia. Cells were incubated for 3 h with indicated reagents (5 μM rotenone, 10 μM antimycin A) and 10 μM pimonidazole. Relative intensity of pimonidazole staining was measured. *P < 0.05 compared with 21% O₂ and 5.5 mM glucose; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and 5 μM rotenone; †P < 0.05 compared with 21% O₂, 5.5 mM glucose and 10 μM antimycin A. Rote, rotenone; anti, antimycin A. Data are 10 independent experiments in duplicate ± SEM. (B) Effect of manganese superoxide dismutase (MnSOD) overexpression on pimonidazole staining. Cells transduced with MnSOD or control adenovirus were incubated for 3 h under indicated conditions and 10 μM pimonidazole, and relative intensity of pimonidazole staining was measured. Black bars = control adenovirus; white bars = MnSOD adenovirus. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and MnSOD adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and MnSOD adenovirus. Data from B and C are eight independent experiments in duplicate ± SEM. (C) Effect of MnSOD overexpression on LOX-1 phosphorescence. Cells transduced with MnSOD or control adenovirus were incubated for 24 h under indicated conditions and 2 μM LOX-1, and the relative intensity of LOX-1 phosphorescence was measured. Black bars = control adenovirus; white bars = MnSOD adenovirus. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and MnSOD adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and MnSOD adenovirus. (D-G) Immunofluorescence for 8-OHdG (8-hydroxy-2'-deoxyguanosine, D and E) and pimonidazole (F and G) in mice glomeruli (<i>blue</i>: 4′,6-diamidino-2-phenylindole; <i>red</i>: 8-OHdG; <i>green</i>: pimonidazole). Diabetes mellitus (DM) was induced in C57Bl/6 mice (8–10 weeks old) by streptozotocin injection. Immunohistochemistry was performed at 4 weeks after the onset of DM. Scale bars represent 20 μm. *P < 0.05 compared with non-DM control mice; #P < 0.05 compared with non-DM MnSOD-Tg mice; †P < 0.05 compared with diabetic MnSOD-Tg mice. n = 6/group</p

AQP1 is involved in high glucose-induced cellular hypoxia and is affected by mitochondrial ROS generation.

<p>(A) Expression levels of membrane aquaporin-1 (AQP1) protein. Cells transduced with manganese superoxide dismutase (MnSOD) or control adenovirus were incubated under indicated conditions for 24 h, and membrane proteins were examined by western blotting for detection of membrane AQP1 expression. The experiments were repeated at least four times. *P < 0.05 compared with 5.5 mM glucose and control adenovirus; #P < 0.05 compared with 5.5 mM glucose, 19.5 mM L-glucose, and control adenovirus; †P < 0.05 compared with 5.5 mM glucose, MnSOD adenovirus. Data from A and B are four independent experiments in duplicate ± SEM. (B) Hydrogen peroxide attenuated membrane AQP1 expression. Cells were incubated under indicated conditions for 30 min, and membrane proteins were subjected to western blotting. The experiments were repeated at least four times. *P < 0.05 compared with no agent. (C and D) Immunofluorescence for AQP1 in mouse glomeruli (<i>red</i>: AQP1). C57Bl/6 mice (8 weeks old) were made diabetes mellitus (DM) by streptozotocin injection. Immunohistochemistry was performed at 4 weeks after the onset of DM. Scale bars represent 20 μm. *P < 0.05 compared with non-DM control mice. n = 5/group. (E and F) Effect of AQP1 overexpression on pimonidazole staining. Cells infected with AQP1 or control adenovirus were incubated for 3 h, and relative intensity of pimonidazole staining (<i>green</i>) was measured. Black bars = control adenovirus; white bars = AQP1 adenovirus. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus. Data are eight independent experiments in duplicate ± SEM.</p

Proposed model of the pathogenesis of diabetic complications.

<p>High glucose increases mitochondrial reactive oxygen species (mtROS) generation. High glucose also induces cellular hypoxia through increased O₂ consumption in mitochondria. Cellular hypoxia may also be affected through suppressed aquaporin-1 (AQP1) expression induced by mtROS generation. Hyperglycemia-induced cellular hypoxia and mtROS generation may simultaneously promote hyperglycemic damage including overproduction of endothelin-1 and fibronectin, and induction of apoptosis, which leading to diabetic vascular complications.</p

AQP1 overexpression decreased high glucose-induced mitochondrial ROS generation and high glucose-induced phenomena.

<p>(A) Mitochondrial reactive oxygen species (mtROS) generation in aquaporin-1 (AQP1) overexpression cells. Cells were incubated under indicated conditions, and treated with 300 nM CM-H₂XRos for 15 min. Relative intensity of fluorescence of CM-H₂XRos was measured. Black bars = control adenovirus; white bars = AQP1 adenovirus. *P < 0.05 compared with 5.5 mM glucose and control adenovirus; #P < 0.05 compared with 5.5 mM glucose and AQP1 adenovirus; †P < 0.05 compared with 25 mM glucose and AQP1 adenovirus. Data are eight independent experiments in duplicate ± SEM. (B) Effect of AQP1 overexpression on endothelin-1 mRNA expression. Cells were incubated under indicated conditions for 24 h. The expression levels of endothelin-1 mRNA were measured by quantitative RT-PCR analysis. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus. Data are five independent experiments in duplicate ± SEM. (C) Effect of AQP1 overexpression on endothelin-1 secretion. Cells were incubated under indicated conditions for 24 h, and endothelin-1 secretion was measured by ELISA assay. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus. Data are six independent experiments in duplicate ± SEM. (D) Effect of AQP1 overexpression on fibronectin mRNA expression. Cells were incubated under indicated conditions for 24 h. The expression levels of fibronectin mRNA were measured by quantitative RT-PCR analysis. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus. Data are five independent experiments in duplicate ± SEM. (E) Effect of AQP1 overexpression on cell apoptosis. Cells were incubated under indicated conditions for 168 hours. Data are expressed as the mean number of positive cells/section of 10 independent sections ± SEM. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus.</p

High-glucose induced cellular hypoxia was detected by pimonidazole and LOX-1 in BAECs and mice glomeruli.

<p>(A and B) Pimonidazole immunofluorescence of bovine aortic endothelial cells (BAECs). BAECs were incubated with the indicated conditions for 3 h in the presence of 10 μM pimonidazole (<i>green</i>). Relative intensities of pimonidazole staining were measured. *P < 0.05 compared with 21% O₂ and 5.5 mM glucose; #P < 0.05 compared with 21% O₂, 5.5 mM glucose and 19.5 mM L-glucose; †P < 0.05 compared with 21% O₂ and 25 mM glucose. Data are five independent experiments in duplicate ± SEM. (C) Relative intensity of pimonidazole staining in cells incubated for various times at 21% O₂. *P < 0.05 compared with 21% O₂ and 5.5 mM glucose at each incubation time. Data from B and C are eight independent experiments in duplicate ± SEM. (D and E) Phosphorescence of hypoxia probe LOX-1. Cells were incubated with indicated conditions in the presence of 2 μM LOX-1 (<i>red</i>). Intensity of LOX-1 phosphorescence was measured. *P < 0.05 compared with 21% O₂, 5.5 mM glucose. (F and G) Pimonidazole immunofluorescence of mouse glomeruli. Diabetes mellitus (DM) was induced by streptozotocin injection (120 mg/kg, intraperitoneally). Mice were studied at 4 weeks after DM induction. Intensity of pimonidazole staining (<i>green</i>) in glomeruli was measured. Scale bars represent 20 μm. *P < 0.05 compared with non-DM, control mice. n = 6/group.</p