PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation

Abstract

SIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD(+) levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD(+) availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD(+)-consuming enzyme, increases NAD(+) content and SIRT1 activity in brown adipose tissue and muscle. PARP-1(-/-) mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD(+) content and SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that could be useful in the management not only of metabolic diseases, but also of cancer.

Figure 1. Phenotyping the PARP-1^−/− mice

(A) Body weight (BW) evolution in PARP-1^+/+ and ^−/− mice (n=8/9). (B) Epidydimal white adipose tissue mass. (C) Average food consumption. (D) O₂ consumption and (E) respiratory quotient (RQ) of PARP-1^+/+ and ^−/− mice (n=9/9) determined by indirect calorimetry. (F) Body temperature after exposure to 4°C (n=6/5). (G) Oral glucose tolerance test (OGTT) (n=5/5) and the area under curve (AUC). (H) PARP-1 autoPARylation (arrow), analyzed in 100μg of protein extract from gastrocnemius muscles of 16 wk-old mice fed ad libitum or fasted (24h). PARP-1 and tubulin levels were checked using 50μg of protein extract. (I) Gastrocnemius from mice on CD or HFD (12 wk) were analyzed as in (H). (J) BW evolution in PARP-1^+/+ and ^−/− mice (n=10/10) fed a CD (circles) or HFD (squares) from 8 wk of age. (K) OGTT and (L) an insulin tolerance test in HFD fed PARP-1^+/+ and ^−/− mice at 16 wk of age (n=10/10). The AUC of the OGTT is shown on the top-right. (M-O) BAT from PARP-1^+/+ and ^−/− mice on CD was extracted and mitochondrial biogenesis was analyzed by (M) transmission electron microscopy, (N) mRNA expression of the genes indicated and (O) the abundance of mitochondrial complexes in 25μg of total protein extracts. (P) SDH staining of sections from gastrocnemius and soleus of PARP-1^+/+ and ^−/− mice on CD. (Q-R) Gastrocnemius was also used to analyze mRNA levels of the indicated genes (Q) and the abundance of mitochondrial complexes in 25μg of total protein extracts (R). White bars represent PARP-1^+/+; black bars represent PARP1^−/− mice. * indicates statistical difference vs. PARP-1^+/+ at p<0.05. For abbreviations, see Table S1.

Figure 2. PARP-1 deletion raises NAD⁺ levels and activates SIRT1

(A) Protein PARylation determined by α-PAR staining on formalin-fixed 7μm BAT and muscle tissue sections of PARP-1^+/+ and ^−/− mice. White bar = 10μm. (B) NAD⁺ and (C) NAM levels in BAT and muscle from PARP-1^+/+ (white bars) and PARP-1^−/− (black bars) mice determined by mass spectrometry. (D-E) PARP-1, SIRT1 and actin protein content in BAT (D) and muscle (E) were determined by Western blot, using 100μg of protein lysate. PGC-1α and FOXO1 acetylation were examined by immunoprecipitation. (F) Tubulin and acetylated-tubulin levels were tested in PARP-1^+/+ and ^−/− gastrocnemius. (G) The Ndufa9 subunit of mitochondrial complex I was immunoprecipitated from 400μg of total protein from gastrocnemius and acetylation levels were analyzed by Western blot. * indicates statistical difference vs. PARP-1^+/+ mice at p<0.05.

Figure 3. PARP-1 knock-down promotes SIRT1 activity and oxidative metabolism

(A-C) HEK293T cells were transfected with either scramble (control) or PARP-1 shRNA and HA-PGC-1α expression vector for 48h. Then, (A) PARP-1 protein levels and autoPARylation (arrowhead) were analyzed in total protein lysates. (B) Intracellular NAD⁺ was measured on total acid extracts. (C) PGC-1α acetylation was analyzed in HA immunoprecipitates. (D-F) HEK293T cells were transfected with either a pool of PARP-1 siRNAs, a pool of SIRT1 siRNAs, or different combinations of both using the corresponding scramble siRNAs as control (−). The cells were simultaneously transfected with HA-PGC-1α for 48h. Then, (D) relative mitochondrial DNA content, (E) mRNA levels of the genes indicated and (F) total O₂ consumption was analyzed. * indicates statistical difference vs. respective control sh/siRNA-transfected cells at p<0.05.

Figure 4. PARP-1 inhibition enhances mitochondrial function through SIRT1

(A-C) C2C12 myotubes expressing FLAG-HA-PGC-1α were treated for 6h with either PBS (vehicle (Veh)), H₂O₂ (500μM) or H₂O₂ and PJ34 (1μM). Then, (A) SIRT1, PARP-1 and unspecific IgG immunoprecipitates from 500 μg of protein extracts were used to test PARylation and the proteins indicated. (B) proteins were analyzed in total cell extracts and the arrow indicates PARP-1 autoPARylation. (C) NAD⁺ content was measured and (D) PGC-1α acetylation was tested in FLAG immunoprecipitates. Tubulin was checked on the supernatants as input. (E) C2C12 myotubes were treated with PJ34 (1μM) for the times indicated and NAD⁺ levels were evaluated in acidic extracts. (F-G) C2C12 myotubes expressing FLAG-HA-PGC-1α were treated for 24h with PBS (Veh) or with PJ34 (1 μM, unless stated). (F) PARP-1 protein and autoPARylation (arrow) were determined by Western blot and (G) NAD⁺ content and PGC-1α acetylation were measured. (H-I) 10-wk old mice received PJ34 (10mg/kg BID i.p.) or saline (Veh) for 5 days before sacrifice (n=10/10); then (H) PARP-1 autoPARylation (arrow), p-ACC and SIRT1 levels were determined in 100μg of total protein extracts from gastrocnemius and (I) NAD⁺ and PGC-1α acetylation were determined. PGC-1α was immunoprecipitated using 2mg of protein from gastrocnemius muscle and 5μg of antibody. (J-K) C2C12 myotubes expressing FLAG-HA-PGC-1α and either a control or a SIRT1 shRNA were treated with PJ34 for 48h. Then, (J) PGC-1α acetylation levels were quantified in FLAG immunoprecipitates and 50μg of total protein extracts were used to measure the other markers indicated; (K) mRNA levels of selected genes were quantified. (L) Mice were treated as in (H) and mRNA of selected genes was determined in gastrocnemius. (M) C2C12 were treated as in (J) and cellular O₂ consumption was measured. White bars represent Veh; black bars represent PJ34 treatment. * indicates statistical difference vs. Veh group at p<0.05. For abbreviations, see Table S1.