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Abstract

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on β-cell function and survival. The present study sought to determine the mechanism by which palmitate affects intracellular Ca²⁺ in pancreatic β-cells, and in particular the role of the endoplasmic reticulum (ER). In the MIN6 β-cell line, palmitate rapidly increased cytosolic Ca²⁺ through a combination of Ca²⁺ store release and extracellular Ca²⁺ influx. Palmitate caused a reversible lowering of ER Ca²⁺, measured directly with the fluorescent protein-based ER Ca²⁺ sensor, D1ER. Using another genetically encoded indicator, long-lasting oscillations of cytosolic Ca²⁺ in palmitate-treated cells were observed. The kinetics of pharmacological SERCA inhibition on the β-cell ER stress response were characterized, and the ER calcium sensor PERK was found to be rapidly activated in response to irreversible ER calcium depletion. ER calcium depletion in palmitate-treated cells also induced rapid phosphorylation of PERK, as well as other subsequent downstream ER stress signals. In summary, the effects of the free fatty acid palmitate on pancreatic β-cell Ca²⁺ homeostasis were characterized in this thesis. This study provides the first direct evidence that free fatty acids reduce ER Ca²⁺ and sheds light on pathways involved in β-cell ER stress, lipotoxicity and the pathogenesis of type 2 diabetes.