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
] 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 findings presented in this manuscript identify: i) that novel electron transfer sites are regulated by SIRT3; ii) that SIRT3 is downregulated in the liver in response to a high-fat diet, and iii) that the depletion of SIRT3
exacerbated lipotoxicity via the modulation of ROS.
Mitochondrial metabolism and redox stress proteins have been shown to undergo deacetylation in response to fasting in the liver [1
]. However, whether SIRT3 is the deacetylase that orchestrates these effects is predominantly unknown and the functional effect of known targets of SIRT3 deacetylation are only beginning to be explored (reviewed [17
]). The known targets of SIRT3 deacetylation modulate substrate entry into the TCA cycle and regulate proximal subunits in the ETC. Recently proteomic analysis implicates ATP synthase alpha subunit of the ETC as a probably additional target of SIRT3 deacetylation [12
]. Our functional in vivo
oxygen consumption studies support that components of the ETC downstream of complex III are modulated and subsequently activated by SIRT3. In addition, we show that numerous subunits of complex V are deacetylated in wild-type compared to SIRT3
knockout mice liver tissue, and that one or more ATP synthase subunits directly interacts with SIRT3. Additionally, we demonstrate that ATP synthase subunit α shows a higher level of acetylation in SIRT3
deficient compared to wildtype MEF cells. Although these data support the ATPase subunit α as a substrate of SIRT3, further work will be required to fully delineate the extent of the functional interaction between these two proteins Interestingly, we show that complex II of the ETC is not directly modulated by SIRT3, which although compatible with the findings of Ahn et al [4
], differs from the observations of Cimen et al [6
]. This discrepancy is currently unexplained but warrants further investigation.
A primary method of post-translational modification and signaling in mitochondria appears to be via a change in protein acetylation, and SIRT3 is one of the primary protein deacetylases. SIRT3 is activated, at least in part, by changes in the ratio of NAD+
/NADH and SIRT3 deacetylase activity may play a role in metabolic diseases such as obesity or diabetes since these conditions are associated with a diminished NAD+
/NADH ratio and lower NADH oxidase activity [18
]. Interestingly, a recent study has shown that SIRT3 activates a pivotal mitochondrial fatty acid oxidation enzyme [20
]. This observation would further suggest that SIRT3 may play an adaptive role in handling excess fat in the diet whereby it could facilitate the catabolism of these nutrients.
Chronic liver damage is also associated with a reduction in NAD levels [21
]. In this regard, our study shows that chronic high-fat feeding increases hepatosteatosis and oxidative liver damage in parallel with the downregulation of SIRT3 levels. We therefore postulated that the mitochondrial dysfunction associated with SIRT3 downregulation or depletion may exacerbate injury in response to lipotoxic insults. The plausibility of this postulate is further supported in that lipotoxicity in the liver is associated with mitochondrial dysfunction [15
] and saturated fats per se
increase mitochondrial ROS levels [22
]. In this study we demonstrate that the SIRT3
knockdown enhances ROS levels and that the ROS scavenger NAC, reverses this SIRT3
depletion effect. In parallel, SIRT3 depletion enhanced susceptibility to palmitate toxicity and this injury is reversed by both NAC and the reconstitution of SIRT3
. Taken together, these results suggest that the downregulation of SIRT3 in the liver responding to excess fatty acids would be a maladaptive response and might play a role in hepatic pathophysiology in obesity.
Interestingly, SIRT3 deficiency has previously been shown to increase susceptibility to direct oxidative stress and in facilitating ROS-dependent oncogenic transformation [23
]. Mechanisms identified through which the reduction or absence of SIRT3 mediates these ROS-dependent effects include the disruption of anti-apoptotic programs [23
] and via the attenuation of anti-oxidant defense programs [24
]. Whether the direct disruption of ETC functioning with the diminution in SIRT3 is an additional mechanism enabling excess ROS generation, resulting in increased susceptibility to biological stressors, is suggested by the palmitate-mediated adverse effects in this study. However, as additional targets of SIRT3 deacetylation are being identified, it is increasingly being recognized that the number of targets that may be operational in the control of the mitochondrial and cellular redox state are probably multi-factorial [17
Although the predominant focus of investigation into SIRT3 has been studied under conditions that mimic caloric restriction, the reduction in its function, under nutrient excess conditions, may play a similarly important biological role. Excess fat in the diet gives rise to both diabetes and obesity and both of these conditions can give rise to non-alcoholic fatty liver disease. Finally, we expand the targets of SIRT3 to include the modulation of distal ETC complexes and confirm that the reduction in SIRT3 levels results in excess ROS levels that is further accentuated in the presence of palmitate. An interesting question arising from this research is whether the retardation of ETC flux with low or absent SIRT3 levels is a significant contributor to the cytotoxic effects of excess ROS generation. The complexity of dissecting this possible pathophysiology from the myriad of additional yet, uncharacterized targets of SIRT3 is an important challenge in understanding the full scope of the functioning of SIRT3. The importance of the delineation of this biology is, however, underscored by the prevalence of nutrient excess in society, the increasing pathology associated with nutrient excesses and the potential that the regulation of SIRT3 may play a role in ameliorating these pathologies.