Interdependence of AMPK and SIRT1 for metabolic adaptation to fasting and exercise in skeletal muscle

Abstract

During fasting and after exercise, skeletal muscle efficiently switches from carbohydrate to lipid as the main energy source to preserve glycogen stores and blood glucose levels for glucose-dependent tissues. Skeletal muscle cells sense this limitation in glucose availability and transform this information into transcriptional and metabolic adaptations. Here we demonstrate that AMPK acts as the prime initial sensor that translates this information into SIRT1-dependent deacetylation of the transcriptional regulators PGC-1alpha and FOXO1, culminating in the transcriptional modulation of mitochondrial and lipid utilization genes. Deficient AMPK activity compromises SIRT1-dependent responses to exercise and fasting, resulting in impaired PGC-1alpha deacetylation and blunted induction of mitochondrial gene expression. Thus, we conclude that AMPK acts as the primordial trigger for fasting- and exercise-induced adaptations in skeletal muscle and that activation of SIRT1 and its downstream signaling pathways are improperly triggered in AMPK-deficient states.

Figure 1. Glucose restriction increases PGC-1α-dependent gene expression

(A-C) C2C12 myotubes were infected with adenoviruses encoding FLAG-HA-PGC-1α. Then, cells were left for 48hrs in medium containing either 25mM glucose (HG) or 5mM glucose (LG). A third group was treated by adding 25mM glucose medium for 6hrs after 48hrs in LG (LG + 6hrs HG). (A) Total protein was obtained and 100μg were used for Western blot analysis to test the markers indicated. (B) 500μg of total protein extracts were used for immunoprecipitation (IP) against FLAG antibody or against FOXO to determine the acetylation levels of PGC-1α or FOXO1, respectively. (C) Acidic lysates were obtained for measurement of NAD⁺ levels. Data are presented as mean±S.E. from 6 different experiments. * Indicates statistical difference vs. HG group. (D) 300μg of nuclear extracts from gastrocnemius muscle from fed, fasted or re-fed C57BL/6J mice were used for IP against PGC-1α or FOXO1 to measure acetylation. (E-G) After infection with adenoviruses encoding FLAG-HA-PGC-1α C2C12 myotubes were left in 5mM glucose medium and samples were obtained at the times indicated. (E) 100μg of total protein extracts were used for Western analysis to test the markers indicated. (F) 500μg of total protein extracts were used for immunoprecipitation to detect the acetylation levels of PGC-1α (G) Acidic lysates were obtained for measurement of NAD⁺ levels. Data are presented as mean±S.E. from 5 different experiments. * indicates statistical difference vs. t=0. All images are representative of 3-6 independent experiments.

Figure 2. Disruption of AMPK activity renders PGC-1α activity and metabolic adaptations unresponsive to glucose restriction

(A) C2C12 myotubes were infected with adenovirus encoding FLAG-HA-PGC-1α and either LacZ, a wild-type (WT) or a dominant negative (DN) form of AMPKα₁. After infection cells were left for 48hrs in 25 or 5mM glucose medium. 100μg of total extracts were used for Western analysis. (B) As in (A), but 500 μg from total extracts were used for immunoprecipitation against FLAG to test PGC-1α acetylation levels. (C) C2C12 myoblasts were transfected with lacZ and either an empty vector (not shown) or a luciferase reporter construct linked to the mouse PGC-1α promoter. Simultaneously, cells were infected with adenovirus encoding for GFP (−), FLAG-HA-PGC-1α, FLAG-HA-PGC-1α R13, WT-AMPKα₁ or DN-AMPKα₁ as indicated. 36hrs later, cells were incubated for 48hrs in medium containing either 25 or 5mM glucose and luciferase activity was measured. A representative assay of 4 independent experiments is shown. Values are expressed as mean±SE. * indicates statistical difference vs. corresponding 25mM glucose group. (D) Similar to (A), but total mRNA was obtained for qRT-PCR analysis. Relative mRNA levels are shown as mean±SE from 4 experiments. * indicates statistical difference vs. corresponding 25mM glucose group. # indicates statistical difference vs. lacZ-infected 25mM group. (E) C2C12 myotubes were infected as in (A). Then, cells were left in 25 or 5mM glucose medium. At the indicated time points, O₂ consumption, oleate and glucose oxidation rates were measured. Results are shown as mean±SE from 12, 6 and 4 independent experiments, respectively. * indicates statistical difference vs. time=0hrs.

Figure 3. Impaired PGC-1α deacetylation and transcriptional response to fasting in AMPKγ₃ KOs

Wild type and AMPKγ₃ KO mice in fed or fasted (20hrs) state were sacrificed and muscles were extracted and frozen (A) 2mg of total protein extracts from EDL muscles were used to immunoprecipitate FOXO1 and PGC-1α and check their acetylation. Relative quantifications of FOXO1 and PGC-1α acetylation levels are shown on the right as mean±SE of 6 muscles/group. (B) Acidic extracts from 50mg of gastrocnemius muscle were used to measure NAD⁺ content. Results are shown as mean±S.E. from 6 muscles measured in duplicate. (C) 20mg of quadriceps muscle were used to measure glycogen content. Results are shown as mean±S.E. from 5 muscles. # indicates statistical difference between the groups indicated (D) Total mRNA was extracted from quadriceps muscles and used for qPCR analysis. Results are shown as mean±SE from 10 muscles/group. Through the figure, * indicates statistical difference vs. respective fed group.

Figure 4. Impaired PGC-1α deacetylation in response to exercise in AMPKγ₃ KOs

(A) Wild-type and AMPKγ₃ KO mice were sacrificed at rest or 2.5hrs after swimming (see methods) and muscles were frozen. 300μg of nuclear extracts from gastrocnemius or 2mg of total proteins from EDL muscles were used to immunoprecipitate PGC-1α and check its acetylation levels. (B) Acidic extracts from 50mg of gastrocnemius were used to measure NAD⁺. Results are shown as mean±S.E. from 3-4 muscles per group measured in duplicate. (C) 20mg of quadriceps were used to measure Nampt expression. (D) 20mg of quadriceps muscle were used to measure glycogen content. Results are shown as mean±S.E. from 3-4 muscles per group measured in duplicate. # indicates statistical difference between the groups indicated (E) Proposed scheme for the coordinated regulation of lipid utilization during energy stress. Upon low nutrient availability or increased energy demand, as during exercise, there is an increase in the AMP/ATP ratio, which activates AMPK, enhancing lipid oxidation in the mitochondria and inducing Nampt levels. These events will raise intracellular NAD⁺ levels, triggering SIRT1 activation, which deacetylates PGC-1α and FOXOs, both of which regulate genes that further favour mitochondrial respiration and lipid mobilization. Through the figure, * indicates statistical difference vs. respective fed group.