Elsevier

Free Radical Biology and Medicine

Volume 49, Issue 7, 15 October 2010, Pages 1230-1237
Free Radical Biology and Medicine

Original Contribution
SIRT3 is regulated by nutrient excess and modulates hepatic susceptibility to lipotoxicity

https://doi.org/10.1016/j.freeradbiomed.2010.07.009Get rights and content

Abstract

SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways. However, its role in response to nutrient excess remains unknown. Thus, we investigated SIRT3 regulation of the electron transfer chain and evaluated the role of SIRT3 in hepatic lipotoxic stress. SIRT3-depleted HepG2 cells show diffuse disruption in mitochondrial electron transfer chain functioning, a concurrent reduction in the mitochondrial membrane potential, and excess basal reactive oxygen species levels. As this phenotype may predispose to increased lipotoxic hepatic susceptibility we evaluated the expression of SIRT3 in murine liver after chronic high-fat feeding. In this nutrient-excess model SIRT3 transcript and protein levels are downregulated in parallel with increased hepatic fat storage and oxidative stress. Palmitate was used to investigate lipotoxic susceptibility in SIRT3 knockout mouse primary hepatocytes and SIRT3-siRNA-transfected HepG2 cells. Under SIRT3-deficient conditions palmitate enhances reactive oxygen species and increases hepatocyte death. Reconstitution of SIRT3 levels and/or treatment with N-acetylcysteine ameliorates these adverse effects. In conclusion SIRT3 functions to ameliorate hepatic lipotoxicity, although paradoxically, exposure to high fat downregulates this adaptive program in the liver. This SIRT3-dependent lipotoxic susceptibility is possibly modulated, in part, by SIRT3-mediated control of electron transfer chain flux.

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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