A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy

Abstract

We demonstrate a role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. In particular, transient increased expression of Sirt1 is sufficient to stimulate basal rates of autophagy. In addition, we show that Sirt1(-/-) mouse embryonic fibroblasts do not fully activate autophagy under starved conditions. Reconstitution with wild-type but not a deacetylase-inactive mutant of Sirt1 restores autophagy in these cells. We further demonstrate that Sirt1 can form a molecular complex with several essential components of the autophagy machinery, including autophagy genes (Atg)5, Atg7, and Atg8. In vitro, Sirt1 can, in an NAD-dependent fashion, directly deacetylate these components. The absence of Sirt1 leads to markedly elevated acetylation of proteins known to be required for autophagy in both cultured cells and in embryonic and neonatal tissues. Finally, we show that Sirt1(-/-) mice partially resemble Atg5(-/-) mice, including the accumulation of damaged organelles, disruption of energy homeostasis, and early perinatal mortality. Furthermore, the in utero delivery of the metabolic substrate pyruvate extends the survival of Sirt1(-/-) pups. These results suggest that the Sirt1 deacetylase is an important in vivo regulator of autophagy and provide a link between sirtuin function and the overall cellular response to limited nutrients.

Fig. 1.

Sirt1 is necessary for autophagy. (A) Transient expression of wild-type (WT) Sirt1 but not a deacetylase-inactive (HY) point mutant of Sirt1 stimulates conversion of LC3-I to LC3-II in HCT116 cells. Quantification of the relative (rel.) levels of LC3-II/LC3-I is shown with the ratio of 1.0 being assigned to vector-transfected cells under fed conditions. (B and C) Distribution of GFP-LC3 in HCT116 cells under fed conditions when transfected along with either a control empty vector (B) or when cotransfected with wild-type Sirt1 (C). (D) The deacetylase-inactive mutant of Sirt1 acts in a dominant-negative fashion to inhibit starvation-induced conversion of LC3-I to LC3-II in HCT116 cells. (E) Quantification of GFP-LC3 punctae in wild-type (+/+) and Sirt1^−/− MEFs under fed or starved conditions. (F) Level of autophagy under fed or starved conditions in Sirt1^−/− MEFs transfected with GFP-LC3 along with an empty vector, wild-type Sirt1, or the described deacetylase-inactive mutant, Sirt1 (HY).

Fig. 2.

Sirt1 interacts with components of the autophagy machinery. (A) Immunoprecipitation (IP) from 2 mg of HeLa cell protein lysate either with an Atg7-specific antibody or a control IgG serum demonstrating the endogenous interaction between Sirt1 and Atg7. Input represents Atg7 and Sirt1 expression in 50 μg of HeLa cell lysate. WB, Western blotting. (B) HeLa cells were transfected with Myc-tagged Atg5, Atg7, or Atg8 along with a HA epitope-tagged form of Sirt1. Lysates were subsequently immunoprecipitated with an antibody against the Myc epitope or with a matched IgG isotype control serum. Coimmunoprecipitated Sirt1 was assessed by Western blotting with a HA epitope-specific antibody. (C) Levels of acetylation in lysates prepared from transiently transfected HeLa cells with the indicated epitope-tagged Atg constructs. Acetylation was assessed under either fed or starved conditions and demonstrates reduced acetylation during starvation. (D) His-tagged Atg7 was purified from transfected HeLa cells treated or untreated with 10 mM nicotinamide for 14 h before harvest. Levels of Atg7 acetylation were detected by Western blotting with an acetyllysine antibody. Total levels of Atg7 were unaffected by nicotinamide treatment. (E) In vitro Sirt1 deacetylation reactions with purified acetylated His-Atg7 as a substrate in the presence or absence of 10 mM NAD and purified wild-type (WT) or deacetylase-inactive (HY) mutant Sirt1 protein.

Fig. 3.

Sirt1 regulates acetylation of autophagy gene products. (A) Transient increased expression of Sirt1 reduces acetylation. HeLa cells were transfected with the indicated epitope-tagged Atg construct along with, where indicated, wild-type Sirt1 (+). Measurements were done under fed conditions. IP, immunoprecipitation; WB, Western blotting. (B) Acetylation of autophagy proteins in wild-type or Sirt1^−/− MEFs under fed conditions. Absence of Sirt1 dramatically increased acetylation. (C) Levels of endogenous Atg7 acetylation in various wild-type (+/+) and Sirt1^−/− neonatal tissue. Tissues were harvested several hours after birth. (D) Levels of endogenous Atg7 acetylation in the brain and liver of approximately E15.5 embryos. Western blot data represent results obtained from one of two similar littermate pairs.

Fig. 4.

Absence of Sirt1 inhibits autophagy in vivo. (A) Accumulation of the autophagy marker p62 in the myocardium of embryonic and neonatal Sirt1^−/− knockout tissue. Neonatal tissue was harvested in the first 3 h after birth. (B) Electron micrographs of neonatal wild-type myocardium. (Inset) Normal appearing mitochondria (white arrows). (C) Electron micrograph of the myocardium of a Sirt1^−/− littermate. The tissue demonstrates prominent accumulation of abnormally shaped mitochondria, examples of which are seen with higher power in the Inset. (D) Survival of Sirt1^−/− pups (in hours) for animals receiving either PBS injections or injections of ethyl pyruvate diluted in PBS while in utero. Median survival difference between groups was significant with P < 0.02. (E) Compared with wild-type littermates, phosphorylated AMPK (p-AMPK, Thr-172) levels are increased in the liver of unfed Sirt1^−/− mice when assessed 15 h after birth. Western blot data are from one representative littermate pair with similar results obtained from at least four separate littermate pairs.