Abstract 122: The Anti-Oxidant Function Of Apolipoprotein A-I Rescues Pancreatic β-Cells From Cholesterol-Induced Mitochondrial Dysfunction

Introduction: Apolipoprotein (apo) A-I, the main apolipoprotein constituent of high-density lipoproteins, increases glucose-stimulated insulin secretion (GSIS) and is internalised by β-cells. Cholesterol accumulation in β-cells causes oxidative stress, reduces islet mass and decreases GSIS. ApoA-I increases GSIS in β-cells with high cholesterol levels, but it is not known if this is dependent on apoA-I internalisation. Hypothesis: Internalised apoA-I increases GSIS in β-cells with high cholesterol levels by lowering oxidative stress. Methods: Cholesterol-loaded Ins-1E insulinoma cells were incubated with unlabelled and/or Alexa Fluor488-labelled apoA-I. Alexa488-labelled apoA-I internalisation was quantified by flow cytometry and its intracellular location was determined by confocal microscopy and western blotting. ApoA-I binding partners on the Ins-1E surface were identified by mass spectroscopy. Generation of reactive oxygen species (ROS) in mitochondria was measured using MitoSOX. Results: ApoA-I was internalised by 43.4±3.6% of the Ins-1E cells (apoA-I + cells) in a dose-, time-, temperature- and cholesterol-dependent manner. ApoA-I internalisation was comparable under basal (2.8 mM) and high (25 mM) glucose conditions and mediated by an F 1 -ATPase β-subunit on the Ins-1E cell surface. Cell surface F 1 -ATPase β-subunit and cholesterol levels in apoA-I + Ins-1E cells were 1.7±0.3- and 1.3±0.1-fold higher, respectively, than in Ins-1E cells without internalised apoA-I (apoA-I - cells). Differentially expressed genes in the apoA-I + and apoA-I - Ins-1E cells were related to protein synthesis, the ER stress-related unfolded protein response, insulin secretion and mitochondrial function. Internalised apoA-I localised to mitochondria, lowered the cholesterol-mediated increase in ROS levels, and improved insulin secretion in cholesterol-loaded Ins-1E cells. The ATPase inhibitory factor 1, IF 1 , inhibited apoA-I internalisation and attenuated the apoA-I-mediated decrease of ROS levels in Ins-1E cells and isolated mouse islets. Conclusions: These results establish that internalised apoA-I restores insulin secretion in β-cells with high cholesterol levels by improving mitochondrial redox balance.