Effects of palmitate on ER and cytosolic [Ca.sup.2+] homeostasis in [beta]-cells

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on [beta]-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular [Ca.sup.2+], and in particular the role of the endoplasmic reticulum (ER). In human [beta]-cells and MIN6 cells, palmitate rapidly increased cytosolic [Ca.sup.2+] through a combination of [Ca.sup.2+] store release and extracellular [Ca.sup.2+] influx. Palmitate caused a reversible lowering of ER [Ca.sup.2+], measured directly with the fluorescent protein-based ER [Ca.sup.2+] sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic [Ca.sup.2+] in palmitate-treated cells. In keeping with this observed ER [Ca.sup.2+] depletion, palmitate induced rapid phosphorylation of the ER [Ca.sup.2+] sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and [beta]-cell death. We detected little palmitate-induced insulin secretion, suggesting that these [Ca.sup.2+] signals are poorly coupled to exocytosis. In summary, we have characterized [Ca.sup.2+]-dependent mechanisms involved in altered [beta]-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER [Ca.sup.2+] and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes. diabetes; free fatty acids; fluorescence resonance energy transfer; calcium homeostasis; endoplasmic reticulum