A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis

Abstract

Here, we demonstrate a role for the mitochondrial NAD-dependent deacetylase Sirt3 in the maintenance of basal ATP levels and as a regulator of mitochondrial electron transport. We note that Sirt3(-/-) mouse embryonic fibroblasts have a reduction in basal ATP levels. Reconstitution with wild-type but not a deacetylase-deficient form of Sirt3 restored ATP levels in these cells. Furthermore in wild-type mice, the resting level of ATP correlates with organ-specific Sirt3 protein expression. Remarkably, in mice lacking Sirt3, basal levels of ATP in the heart, kidney, and liver were reduced >50%. We further demonstrate that mitochondrial protein acetylation is markedly elevated in Sirt3(-/-) tissues. In addition, in the absence of Sirt3, multiple components of Complex I of the electron transport chain demonstrate increased acetylation. Sirt3 can also physically interact with at least one of the known subunits of Complex I, the 39-kDa protein NDUFA9. Functional studies demonstrate that mitochondria from Sirt3(-/-) animals display a selective inhibition of Complex I activity. Furthermore, incubation of exogenous Sirt3 with mitochondria can augment Complex I activity. These results implicate protein acetylation as an important regulator of Complex I activity and demonstrate that Sirt3 functions in vivo to regulate and maintain basal ATP levels.

Fig. 1.

Sirt3 regulates basal ATP levels. (A) Levels of basal ATP in five independent isolates of primary wild-type MEFs and a similar number of independent Sirt3^−/− MEF cell isolates. (B) Sirt3^−/− MEFs were transfected with an expression vector encoding GFP along with an empty vector, epitope-tagged wild type, or a deacetylase inactive (HY) Sirt3. Thirty-six hours after transfection, GFP-positive cells were sorted by FACS and ATP determined. ATP levels are expressed relative to vector-transfected Sirt3^−/− cells. Shown is the average of three independent experiments each performed in triplicate. (C) HeLa cells were transfected with an empty vector, epitope-tagged wild type, or Sirt3(HY) and levels of ATP determined 48 h after transfection. Shown is the mean ± SD of four independent experiments. (D) Absolute level of ATP in various tissues and organs of wild-type mice (n = 3; mean ± SD). Shown is the corresponding level of expressed Sirt3 within each tissue as well as the 70-kDa complex II-associated protein Fp (CII-Fp) as a general measure of mitochondrial number and GAPDH for protein loading. (E) Normalized levels of ATP in wild-type (black bars) and Sirt3^−/− mice (white bars) in various organs with known high Sirt3 expression (heart, liver, and kidney) as well as an organ (pancreas) with low or absent endogenous Sirt3 expression (n = 4 animals per group). *, P < 0.01.

Fig. 2.

Sirt3 regulates mitochondrial protein acetylation. (A) Western blot (WB) analysis for internal acetyl-lysine residues using total liver mitochondrial protein extracts from two age-matched wild-type (+/+) or Sirt3^−/− mice. The mitochondrial protein VDAC1 is used as a loading control, and Sirt3 expression is also shown. (B) Levels of acetylation from immunocaptured hepatic Complex I in a wild-type versus Sirt3^−/− mouse. Subunit 9 (NDUFA9) of Complex I was used as a loading control for the immunocapture. (C) Similar acetylation analysis for immunocaptured Complex II. The 70-kDa Fp subunit of Complex II (C II-Fp) was used as a loading control. (D) Sirt3 deacetylates Complex I in vitro. Complex I was isolated from nicotinamide-treated HeLa cells and incubated for 2 hours in vitro with deacteylase reaction buffer only (−) or with reaction buffer containing purified Sirt3 or Sirt4. The level of the Complex I protein NDUFA9 is shown as a loading control as is the level of exogenously added Flag-tagged Sirt3 and Sirt4. (E) Level of in vivo Complex I acetylation in HeLa cells transfected with empty vector (V), wild-type Sirt3 (WT), or a deacetylase inactive form of Sirt3 (HY). IP, immunoprecipitation; MW, molecular mass.

Fig. 3.

Sirt3 associates with Complex I of the ETC. (A) Sirt3 associates with Complex I. Equal amounts of HeLa cell lysate was used to immunocapture either Complex I or Complex II. These ETC components were then resolved on SDS/PAGE and probed for association with Sirt3. Both the short and long form of human Sirt3 associates with Complex I. The purity of the immunocapture complexes are demonstrated by probing the stripped blot for the Complex I component NDUFA9 and the 70-kDa Fp subunit of Complex II. (B) Reversible association of endogenous Sirt3 with Complex I. Immunocaptured Complex I was probed for associated Sirt3 under fed conditions (−), after 2 or 6 h of starvation, after hydrogen peroxide (0.5 mM, 30 min) treatment, or after rotenone (10 μM, 30 min) treatment. The arrows indicate the short and long form of human Sirt3. Below, total levels of Sirt3 or the Complex I component NDUFA9 were assessed for each condition by using 30 μg of mitochondrial protein lysate. (C) ATP levels in wild-type (+/+) or Sirt3^−/− MEFs under basal conditions (black bars), or after a 30-min exposure to rotenone (50 μM; white bars), cyanide (20 μM; hatched bars), or hydrogen peroxide (0.5 mM; gray bars). Basal levels of ATP are reduced in the Sirt3^−/− cells and are relatively resistant to rotenone or hydrogen peroxide challenge but exhibit normal ATP sensitivity to cyanide. (D and E) Levels of acetylation of NDUFA9 in MEFs (D) or liver protein lysates from wild type (+/+) or Sirt3^−/− mice (E). (F) HeLa cells were transfected with a myc-tagged empty vector, wild-type Sirt3, or a deacetylase-inactive Sirt3(HY) and equal amounts of lysate immunoprecipitated with a myc-epitope antibody or an irrelevant Flag-epitope antibody. Sirt3 immunoprecipitation reveals the presence of coprecipitated endogenous NDUFA9. IP, immunoprecipitation; WB, Western blotting.

Fig. 4.

The absence of Sirt3 selectively affects Complex I activity. (A and B) Representative rate of oxygen consumption using Complex I-dependent substrates for liver mitochondria obtained from a wild-type (A) or Sirt3^−/− (B) mouse. (C) Calculated State 3 respiration rate for Complex I from intact liver mitochondria of four wild-type and four Sirt3^−/− mice (mean ± SD; *, P < 0.02). (D) Calculated State 3 respiration rate for Complex II-dependent substrate (succinate + rotenone) in wild-type and knockout mice. NS, not significant. (E) Rates of NADH consumption for purified HeLa cell mitochondria after in vitro incubation with deacetylase buffer containing purified Flag-Sirt4 (green), Flag-Sirt3 (red), or Flag-vector (purple). Normalized NADH absorbance was monitored at 340 nm. The arrow indicates the time of addition of rotenone (4 μM final concentration). Shown is the mean rate (±SD) of NADH consumption of triplicate determinations from one of two similar experiments.