In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3 0 -kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3-Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

The inositol 1,4,5-trisphosphate receptor (InsP 3 R) is an intracellular Ca 2+ channel that plays a role in the regulation of insulin secretion. In rat isolated pancreatic islets the expression of types I, II and III InsP 3 R mRNA was identified by reverse transcriptase-polymerase chain reaction and confirmed by cDNA cloning and sequencing. The islet ratios of types I, II and III InsP 3 R mRNA to -actin mRNA were 0·08 0·02, 0·08 0·03 and 0·25 0·04 respectively. Types I, II and III InsP 3 R mRNA were also expressed in rat (RINm5F) and mouse ( HC9) pancreatic -cell lines, and rat cerebellum. Type III InsP 3 R mRNA was quantitatively the most abundant form in rat islets and RINm5F cells. In HC9 cells, types II and III InsP 3 R mRNA were expressed at similar levels, and in much greater abundance than type I mRNA. Type III was the least abundant InsP 3 R mRNA in cerebellum. Culture of HC9 cells for 5 days at 2·8 and 25 mM glucose, or RINm5F cells for 7 days at 5·5 and 20 mM glucose, resulted in significantly enhanced expression of type III, but not types I and II, InsP 3 R mRNA in the cells at the higher glucose concentrations. During short-term (0·5-2 h) incubations, HC9 cell type III InsP 3 R mRNA levels increased in response to glucose in a timeand concentration-dependent manner. Actinomycin D inhibited the glucose response. -Ketoisocaproic acid also stimulated HC9 cell type III InsP 3 R mRNA expression in a concentration-dependent manner, whereas 2-deoxyglucose and 3-Omethylglucose were without effect. The different levels of expression of mRNA for three InsP 3 R isoforms in islets and insulinoma cells, and the influence of glucose and -ketoisocaproic acid on the expression of type III mRNA, suggests that nutrient metabolism plays a role in the regulation of this gene and that the function of InsP 3 R subtypes may be unique with each playing a distinct role in -cell signal transduction and insulin secretion.