Christian J. Madson

Induction of endoplasmic reticulum stress in retinal pericytes by glucose deprivation

Diabetic retinopathy is one of the major microvascular complications associated with diabetes mellitus, and the selective degeneration of retinal capillary pericytes is considered to be a hallmark of early retinopathy. Because glucose fluctuations commonly occur in diabetes, we hypothesized that these fluctuations will increase the endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR) in retinal pericytes. To study whether ER stress and the UPR can be induced in retinal pericytes, rat retinal capillary pericytes were cultured in different concentrations of glucose. Hypoglycemia but not hyperglycemia was found to activate UPR-specific enzymes in pericytes. Strong UPR activation leading to apoptosis was also observed when pericytes were cultured in glucose concentrations that were reduced from high to low or no glucose. These results indicate that induction of UPR is related not only to absolute concentrations but also to a shifting from higher to lower concentrations of glucose.

Age-related cataracts: homocysteine coupled endoplasmic reticulum stress and suppression of Nrf2-dependent antioxidant protection.

To determine whether high levels of homocysteine (Hcy) induce endoplasmic reticulum (ER) stress with suppression of the nuclear factor-erythroid-2-related factor 2 (Nrf2)-dependent antioxidant protection in lens epithelial cells (LECs). ER stress was acutely induced by exposure of LECs to 100 μM Hcy without FCS and also by exposure to 5 mM Hcy with 10% FCS. After exposure to Hcy, significant changes were found in P-PERK, P-eIF2α, XBP1, Nrf2, and Keap1 within 24 h. The production of reactive oxygen species (ROS) was increased after Hcy exposure. The downstream enzymes of Nrf2 like, catalase, and glutathione reductase, were significantly decreased. These results suggested that the Hcy-induced ER stress suppressed the Nrf2-dependent antioxidant protection and simultaneously generated ROS which resulted in further oxidation and death of LECs. The loss of Nrf2 is mainly due to proteosomal degradation and m-calpain activation by the increased levels of cytoplasmic Ca(++). The caspases also play a role in the degradation of Nrf2. Our findings demonstrated that high levels of Hcy induce ER stress, chronic UPR, alter the levels of UPR specific proteins, increase the production of ROS, degrade Nrf2 and block the Nrf2-dependent antioxidant defense protection in LECs. Thus, the upregulation of ROS might exceed the Nrf2 dependent antioxidant defense protection in the LECs and result in the highly oxidized lenses and resulted in ARCs.

Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells

Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions.