Aims/hypothesis Increased lipid supply causes beta cell death, which may contribute to reduced beta cell mass in type 2 diabetes. We investigated whether endoplasmic reticulum (ER) stress is necessary for lipid-induced apoptosis in beta cells and also whether ER stress is present in islets of an animal model of diabetes and of humans with type 2 diabetes. Methods Expression of genes involved in ER stress was evaluated in insulin-secreting MIN6 cells exposed to elevated lipids, in islets isolated from db/db mice and in pancreas sections of humans with type 2 diabetes. Overproduction of the ER chaperone heat shock 70 kDa protein 5 (HSPA5, previously known as immunoglobulin heavy chain binding protein [BIP]) was performed to assess whether attenuation of ER stress affected lipid-induced apoptosis.Results We demonstrated that the pro-apoptotic fatty acid palmitate triggers a comprehensive ER stress response in MIN6 cells, which was virtually absent using non-apoptotic fatty acid oleate. Time-dependent increases in mRNA levels for activating transcription factor 4 (Atf4), DNA-damage inducible transcript 3 (Ddit3, previously known as C/EBP homologous protein [Chop]) and DnaJ homologue (HSP40) C3 (Dnajc3, previously known as p58) correlated with increased apoptosis in palmitate-but not in oleate-treated MIN6 cells. Attenuation of ER stress by overproduction of HSPA5 in MIN6 cells significantly protected against lipidinduced apoptosis. In islets of db/db mice, a variety of marker genes of ER stress were also upregulated. Increased processing (activation) of X-box binding protein 1 (Xbp1) mRNA was also observed, confirming the existence of ER stress. Finally, we observed increased islet protein production of HSPA5, DDIT3, DNAJC3 and BCL2-associated X protein in human pancreas sections of type 2 diabetes subjects. Conclusions/interpretation Our results provide evidence that ER stress occurs in type 2 diabetes and is required for aspects of the underlying beta cell failure.

Increased availability of fatty acids causes cell death and dysfunction in ␤-cell lines, isolated islets, and animal models of diabetes. From the MIN6 ␤-cell line, we selected two subpools that are resistant to palmitate-induced apoptosis. Protection was not universal because palmitate-resistant cells remained sensitive to cytokine-and streptozotocininduced apoptosis. Palmitate oxidation and incorporation into cholesterol ester (but not triglycerides) were significantly higher in palmitate-resistant cells than in control cells. Consistent with these findings, transcript profiling revealed increased expression in palmitate-resistant cells of several ␤-oxidation genes as well as a 2.8-fold upregulation of stearoyl-CoA desaturase 1 (SCD1). Correspondingly, the oleate-to-palmitate ratio of palmitate-resistant cells was double that of palmitate-pretreated control cells. At least some of this additional oleate in palmitate-resistant cells was incorporated into cholesterol ester stored in the form of large cytosolic lipid bodies. However, blocking cholesterol ester formation did not render palmitate-resistant cells sensitive to palmitate-induced apoptosis. On the other hand, an inhibitor of SCD1, 10,12-conjugated linoleic acid, dose dependently overcame the resistance of palmitate-resistant cells to lipoapoptosis. Our results suggest that desaturation per se is more important in protecting ␤-cells from the cytotoxic effects of palmitate than is the nature of neutral lipid storage pool thus generated.

Chronic lipid exposure is implicated in ␤-cell dysfunction in type 2 diabetes. We therefore used oligonucleotide arrays to define global alterations in gene expression in MIN6 cells after 48-h pretreatment with oleate or palmitate. Altogether, 126 genes were altered >1.9-fold by palmitate, 62 by oleate, and 46 by both lipids. Importantly, nine of the palmitate-regulated genes are known to be correspondingly changed in models of type 2 diabetes. A tendency toward ␤-cell de-differentiation was also apparent with palmitate: pyruvate carboxylase and mitochondrial glycerol 3-phosphate dehydrogenase were downregulated, whereas lactate dehydrogenase and fructose 1,6-bisphosphatases were induced. Increases in the latter (also seen with oleate), along with glucosamine-phosphate N-acetyl transferase, imply upregulation of the hexosamine biosynthesis pathway in palmitate-treated cells. However, palmitate also increased expression of calcyclin and 25-kDa synaptosomal-associated protein (SNAP25), which control distal secretory processes. Consistent with these findings, secretory responses to noncarbohydrate stimuli, especially palmitate itself, were upregulated in palmitate-treated cells (much less so with oleate). Indeed, glucose-stimulated secretion was slightly sensitized by chronic palmitate exposure but inhibited by oleate treatment, whereas both lipids enhanced basal secretion. Oleate and palmitate also induced expression of chemokines (MCP-1 and GRO1 oncogene) and genes of the acute phase response (