Endoplasmic reticulum (ER) stress-induced apoptosis may arise from multiple environmental and pharmacologic causes but the precise mechanism(s) involved are not completely known. Members of Bcl-2 protein family are important regulators of apoptosis. Here we report that, in a process dependent on the pro-apoptotic Bcl-2 members Bax and Bak, exogenously expressed fluorescent protein localized to the ER lumen is released into the cytosol in cells undergoing ER stress. Upon ER stress induction, endogenous ER luminal proteins are also released into the cytosol in a similar fashion accompanied by translocation and anchorage of Bax to the ER membrane. In addition, Bax and tBid mediate a global increase in ER membrane permeability to ER luminal proteins in vitro. Importantly, anti-apoptotic Bcl-XL antagonizes the effects of pro-apoptotic Bcl-2 proteins on ER membrane permeability. Consistent with Bax translocation to the ER membrane in whole apoptotic cells, there is also increased tight association of Bax with the ER membrane correlated with the increase in ER membrane permeability in vitro. Overall, these data suggest that the regulation of ER membrane permeability by Bcl-2 proteins could be an important molecular mechanism of ER stress-induced apoptosis.

Synopsis Changes in metabolic processes play a critical role in the survival or death of cells subjected to various stresses. Here, we have investigated the effects of endoplasmic reticulum (ER) stress on cellular metabolism. A major difficulty in studying metabolic responses to ER stress is that ER stress normally leads to apoptosis and metabolic changes observed in dying cells may be misleading. Therefore, we have used IL-3-dependent Bak−/− Bax−/− hematopoietic cells which do not die in the presence of the ER stress-inducing drug, tunicamycin. Tunicamycin-treated Bak−/−Bax−/− cells remain viable but cease growth, arresting in G1 and undergoing autophagy in the absence of apoptosis. In these cells we used NMR-based stable isotope resolved metabolomics (SIRM) to determine the metabolic effects of tunicamycin. Glucose was found to be the major carbon source for energy production and anabolic metabolism. Following tunicamycin exposure, glucose uptake and lactate production are greatly reduced. Decreased 13C labeling in several cellular metabolites suggests that mitochondrial function in cells undergoing ER stress is compromised. Consistent with this, mitochondrial membrane potential, oxygen consumption, and cellular ATP level are much lower compared with untreated cells. Importantly, the effects of tunicamycin on cellular metabolic processes may be related to a reduction of cell surface Glut-1 levels which, in turn, may reflect decreased Akt signaling. These results suggest that ER stress exerts profound effects on several central metabolic processes which may help explain cell death arising from ER stress in normal cells.