Palmitic Acid Lipotoxicity in Microglia Cells Is Ameliorated by Unsaturated Fatty Acids

Abstract

Obesity and metabolic syndrome are associated with cognitive decline and dementia. Palmitic acid (PA) is increased in the cerebrospinal fluid of obese patients with cognitive impairment. This study was therefore designed to examine fatty acid (FA) lipotoxicity in BV2 microglia cells. We found that PA induced time- and dose-dependent decrease in cell viability and increase in cell death without affecting the cell cycle profile and that PA lipotoxicity did not depend on cell surface free fatty acid receptors but rather on FA uptake. Treatment with sulfosuccinimidyl oleate (SSO), an irreversible inhibitor of fatty acid translocase CD36, significantly inhibited FA uptake in BSA- and PA-treated cells and blocked PA-induced decrease in cell viability. Inhibition of ER stress or treatment with N-acetylcysteine was not able to rescue PA lipotoxicity. Our study also showed that unsaturated fatty acids (UFAs), such as linoleic acid (LA), oleic acid (OA), α-linolenic acid (ALA), and docosahexaenoic acid (DHA), were not lipotoxic but instead protected microglia against PA-induced decrease in cell viability. Co-treatment of PA with LA, OA, and DHA significantly inhibited FA uptake in PA-treated cells. All UFAs tested induced the incorporation of FAs into and the amount of neutral lipids, while PA did not significantly affect the amount of neutral lipids compared with BSA control.

Role of CD36 in Palmitic Acid Lipotoxicity in Neuro-2a Neuroblastoma Cells

“…Therefore, BODIPY TM FL C12 was deemed as a suitable fluorescent PA analog. BODIPY TM FL C12 uptake assay by flow cytometry was performed as described previously [ 10 ]. Briefly, N2a cells were incubated with 15 μM BODIPY TM FL C12 in serum-free media for 30 min at 37 °C.…”

“…To visualize FA uptake under confocal microscopy, the fluorescent PA analog BODIPY TM FL C12 was used and the uptake assay was performed as described previously [ 10 ]. Briefly, N2a cells were treated with BSA or 200 µM PA for 6 h, then incubated with 15 μM BODIPY TM FL C12 for 30 min, washed with PBS, and fixed with 4% paraformaldehyde for 20 min at 4 °C.…”

“…Therefore, BODIPY TM FL C12 was deemed as a suitable fluorescent PA analog. BODIPY TM FL C12 uptake assay by flow cytometry was performed as described previously [10]. Briefly, N2a cells were incubated with 15 µM BODIPY TM FL C12 in serumfree media for 30 min at 37 • C. Following 30 min incubation, cells were then collected by trypsinization, resuspended in ice-cold D-PBS, and immediately analyzed by flow cytometry.…”

“…To visualize FA uptake under confocal microscopy, the fluorescent PA analog BODIPY TM FL C12 was used and the uptake assay was performed as described previously [10]. Briefly, N2a cells were treated with BSA or 200 µM PA for 6 h, then incubated with 15 µM BODIPY TM FL C12 for 30 min, washed with PBS, and fixed with 4% paraformaldehyde for 20 min at 4 • C. Cells were then washed with PBS again and stained with 1 µg/mL DAPI for 10 min, and then visualized under FluoView FV1000 confocal microscope (Olympus; Center Valley, PA, USA).…”

“…Circulating levels of free fatty acids (FFAs) are reportedly elevated in obese subjects [ 1 , 2 ], overweight/obese subjects with diabetes mellitus [ 3 ], and obese non-alcoholic fatty liver disease (NAFLD) patients [ 3 , 4 ]. Excessive level of palmitic acid (PA), a long-chain saturated fatty acid (SFA), has been shown to induce lipotoxicity in many types of cells including skeletal muscles, liver cells, neuronal cells, neuroblastoma, and microglia cells [ 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. There have been studies showing that PA induces oxidative stress in rat cortical cells [ 12 ], bovine endometrial cells [ 13 ], and H9C2 cardiomyocytes [ 14 ] as well as endoplasmic reticulum (ER) stress in different cells including H9C2 cardiomyocytes [ 14 ], skeletal muscle cells [ 14 , 15 ], pancreatic β cells [ 16 ], and hypothalamic neurons [ 17 ] while N-acetyl cysteine, an antioxidant, and 4-phenylbutyrate, an inhibitor of ER stress, are not able to alleviate PA-induced decrease of cell viability in neuroblastoma Neuro-2a (N2a) cells [ 9 ].…”