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. 2009 Apr 23;458(7241):1056-60.
doi: 10.1038/nature07813.

AMPK Regulates Energy Expenditure by Modulating NAD+ Metabolism and SIRT1 Activity

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Free PMC article

AMPK Regulates Energy Expenditure by Modulating NAD+ Metabolism and SIRT1 Activity

Carles Cantó et al. Nature. .
Free PMC article

Abstract

AMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes in the intracellular AMP/ATP ratio. Recent evidence indicated an important role for AMPK in the therapeutic benefits of metformin, thiazolidinediones and exercise, which form the cornerstones of the clinical management of type 2 diabetes and associated metabolic disorders. In general, activation of AMPK acts to maintain cellular energy stores, switching on catabolic pathways that produce ATP, mostly by enhancing oxidative metabolism and mitochondrial biogenesis, while switching off anabolic pathways that consume ATP. This regulation can take place acutely, through the regulation of fast post-translational events, but also by transcriptionally reprogramming the cell to meet energetic needs. Here we demonstrate that AMPK controls the expression of genes involved in energy metabolism in mouse skeletal muscle by acting in coordination with another metabolic sensor, the NAD+-dependent type III deacetylase SIRT1. AMPK enhances SIRT1 activity by increasing cellular NAD+ levels, resulting in the deacetylation and modulation of the activity of downstream SIRT1 targets that include the peroxisome proliferator-activated receptor-gamma coactivator 1alpha and the forkhead box O1 (FOXO1) and O3 (FOXO3a) transcription factors. The AMPK-induced SIRT1-mediated deacetylation of these targets explains many of the convergent biological effects of AMPK and SIRT1 on energy metabolism.

Figures

Figure 1
Figure 1. Activation of AMPK triggers PGC-1α deacetylation in C2C12 myotubes and skeletal muscle
(A-B) C2C12 myotubes infected with adenoviruses for GFP or FLAG-HA-tagged PGC-1α (PGC-1α) were treated with vehicle (−), AICAR (0.5mM, 2-8hrs) or nicotinamide (NAM; 5mM; 12hrs). Then, acetyl-lysine levels were checked on PGC-1α immunoprecipitates (IP). The supernatant (SN) was blotted against actin as input control. Relative quantification of PGC-1α acetylation is shown on the right (B) As in (A), but myotubes were treated for 8hrs with vehicle (−), Resveratrol (Rsv; 50μM), AICAR (AIC), Metformin (Metf; 1mM), DNP (0.5mM) or NAM. (C) C2C12 were infected with adenoviruses encoding GFP, PGC-1α, and wild-type (WT), constitutively active (CA) or dominant negative (DN) forms of AMPKα1. After AICAR treatment, total lysates were analyzed as in (A). (D) PGC-1α acetylation was measured on total protein (soleus and EDL) or nuclear extracts (gastrocnemius) from muscles of mice treated with AICAR or saline. Relative acetylation levels are shown on top of the panels (E) Soleus, EDL and gastrocnemius were obtained from non-exercised or exercised mice at 0, 3 or 6hrs after cessation of exercise, and analysed as in (D). Values are expressed as mean +/− S.E.M. * indicates statistical difference vs. corresponding vehicle, saline or non-exercised group at P < 0.05.
Figure 2
Figure 2. SIRT1 mediates AMPK-induced PGC-1α deacetylation
(A and C) C2C12 myocytes were infected with adenoviruses encoding GFP, PGC-1α, and either control or SIRT1 shRNAs. After 8hrs (A) or 1hr (C) of AICAR treatment, PGC-1α acetylation and AMPK phosphorylation were analyzed (D-E) SIRT1+/+ and SIRT1−/− MEFs were infected with GFP and FLAG-HA-PGC-1α and treated for 8hrs (D) or 1hr (E) with AICAR to test PGC-1α acetylation and AMPK phosphorylation.
Figure 3
Figure 3. AICAR modulates PGC-1α-dependent transcriptional activity, mitochondrial gene expression and oxygen consumption through SIRT1 and NAD+ metabolism
(A) C2C12 myocytes were transfected with a 2-kb mPGC-1α promoter luciferase reporter, a plasmid for mPGC-1α and simultaneously infected with adenovirus encoding control or SIRT1 shRNA. 36hrs later, cells were treated with AICAR (12hrs) and reporter activity was determined. (B) SIRT1+/+ MEFs were analyzed as in (A) (C) SIRT1−/− MEFs were transfected with the 2-kb mPGC-1α reporter and expression plasmids for PGC-1α, SIRT1 or the corresponding empty vectors. Then, cells were treated and analyzed as in (A) (D) C2C12 myocytes were infected with adenoviruses for GFP, PGC-1α and either control or SIRT1 shRNAs. After AICAR treatment, target mRNAs were analyzed by Q-RT-PCR. (E) O2 consumption in C2C12 myotubes infected with PGC-1α, and either control or SIRT1 shRNAs. Total length of the bar equals total O2 consumption. The white part of the bar is the O2 consumption in each group when treated with etomoxir (1mM). Therefore, the grey part represents lipid oxidation-derived O2 consumption. Values for O2 consumption due to the oxidation of lipids and other substrates are indicated on the right. (F) NAD+ and NADH content in C2C12 myotubes treated with AICAR for the times indicated. (G) Whole tibialis anterior muscles from mice treated with saline or AICAR were used for the measurement of NAD+ and NADH. (H-I) C2C12 myotubes preincubated with vehicle or etomoxir (50μM) for 1hr were treated with either vehicle (−) or AICAR (AIC). Then, NAD+ and NADH (H) or PGC-1α acetylation levels (I) were measured. (J-K) As in (H-I), but using FK866 (10nM) instead of etomoxir. All values are expressed as mean +/− S.E.M. * indicates statistical difference vs. vehicle/saline group at p < 0.05. # indicates statistical difference vs. respective control shRNA group at p < 0.05.
Figure 4
Figure 4. The PGC-1α phosphorylation mutant is resistant to deacetylation
(A) C2C12 myocytes were transfected with the wild-type or the 2A mutant form of PGC-1α, using empty vector as control. After 36hrs, cells were treated with AICAR and total lysates were used to test PGC-1α acetylation. Relative acetylation levels of PGC-1α are shown on the right. (B) Cells were treated as in (A), and, after AICAR treatment, target mRNA levels were analyzed by RT-Q-PCR. (C) Cells were treated as in (A), and acidic or alkali lysates were obtained to measure NAD+ and NADH. (D) C2C12 myotubes were treated with AICAR for the times indicated. Then, total protein lysates were used for immunoprecipitation of FOXO1. Relative FOXO1 acetylation is shown on the right (E) As in (A), but immunoprecipitations were performed against FOXO1. (F) Scheme illustrating the convergent actions of AMPK and SIRT1 on PGC-1α. Pharmacological (metformin) and physiological (fasting or exercise) activation of AMPK in muscle triggers an increase in the NAD+/NADH ratio, which activate SIRT1. AMPK also induces the phosphorylation of PGC-1α and primes it for subsequent deacetylation by SIRT1. The impact of AMPK and SIRT1 on the acetylation status of PGC-1α and other transcriptional regulators, such as the FOXO family of transcription factors, will then modulate mitochondrial function and lipid metabolism. All values are presented as mean+/-SE. * indicates statistical difference vs. corresponding vehicle group at P<0.05.

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