Original ContributionSIRT3 is regulated by nutrient excess and modulates hepatic susceptibility to lipotoxicity
Section snippets
Cell cultures and transfections
The HepG2 human hepatocyte cell line was from the American Type Cell Culture (Manassas, VA, USA) and was maintained in DMEM containing 25 mM glucose and 10% fetal bovine serum. Primary mouse hepatocytes and mouse embryonic fibroblasts were isolated and cultured as described previously [9], [10]. For siRNA transfection, 106 HepG2 cells were electroporated with 100 nmol of SIRT3 or control On-Target plus SMARTpool siRNA (Thermoscientific) according to the manufacturer's instructions (Amaxa). Unless
SIRT3 regulates mitochondrial respiration via modulation of multiple complexes in the electron transfer chain
To investigate the modulation of the mitochondrial ETC by SIRT3, we employed siRNA to deplete SIRT3 in HepG2 cells (Fig. 1A) and transient transfection to restore SIRT3 levels in the SIRT3 knockout primary hepatocytes (Fig. 1B). Consistent with prior studies in SIRT3−/− mouse embryonic fibroblasts cellular oxygen consumption and ATP levels were significantly reduced compared to levels in scrambled siRNA-transfected cells (Figs. 1C and D, P < 0.001). Interestingly, the reduction in oxygen
Discussion
Similar to other sirtuins, the mitochondrial enriched SIRT3 is activated by caloric restriction and fasting [5]. The functional targets of SIRT3 support its regulatory role in mitochondrial bioenergetics [4], [6], [10] and SIRT3 levels are enriched in the liver [4], an important nutrient homeostasis organ. Thus, we reasoned that the delineation of the role of SIRT3 in nutrient-excess-mediated hepatic pathology is important to further delineate nutrient-dependent SIRT3 functioning. The major
Acknowledgments
This study was funded by the NHLBI Division of Intramural Research. We acknowledge with appreciation the technical assistance obtained from the NHLBI Intramural flow cytometry and light microscopy core facilities.
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