Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes

Abstract

Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, β-hydroxy-β-methyl butyrate (HMB), a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), EX527 (SIRT1 inhibitor, 25 μM), and Compound C (AMPK inhibitor, 25 μM) alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD(+), SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.

Figure 1

Leucine treatment induces mitochondrial biogenesis and SIRT1 enzymatic activity in C2C12 myotubes. (a) Mitochondrial content was quantitated with NAO dye (10 μM) 48 hours after treatment with leucine (0.5 mM), alanine (0.5 mM), and valine (0.5 mM); (b) mRNA expression levels of PGC-1α and Sirt3 with the same treatments were evaluated by quantitative RT-PCR. The relative mRNA expression was normalized to 18S and expressed as dark bars for PGC-1α and grey bars for Sirt3. (c) Cellular SIRT1 activity and (d) palmitate oxidation were measured after treatment for 48 hours. The results were normalized to cellular protein level for each sample. Data are mean ± SE (n = 4). *Significantly different from controls with P < 0.05.

Figure 2

Leucine improves mitochondrial biogenesis in C2C12 myotubes in a SIRT1-dependent manner. (a) Mitochondrial content was measured using NAO (10 μM) dye after 48-hour leucine (dark bars), leucine plus SIRT1 inhibitor (EX527 25 μM; grey bars) for 48 hours in C2C12 myotubes. (b, c) SIRT1 activity and mitochondrial biogenesis- related genes (PGC-1α, Sirt3, and COX5b) mRNA levels were measured after the same treatments. The relative SIRT1 activity was normalized to cellular protein level, and mRNA level was normalized to housekeeper gene 18S. (b) Dark bars are DMSO control, grey bars are EX527. (c) Dark bars are PGC-1α, grey bars are Sirt3; striped bars are COX5b. (d) Palmitate oxidation level was detected after the same treatment, and the results were normalized to cellular protein for each sample. Data are mean ± SE (n = 4). Different letters indicate significant differences within a given variable. Dark bars are DMSO control and grey bars are EX527. *Significantly different from controls, and **significantly different from control and EX527 groups with P < 0.05.

Figure 3

Leucine-induced phosphorylation of AMPK and ACC requires SIRT1 in C2C12 myotubes. (a) C2C12 myotubes were serum starved overnight and treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), and DMSO for 6 hours. The cell lysates were assessed by western blotting analysis with specific antibodies against phosphor-AMPKα (Thr 172), phosphor-ACC (Ser 79), total AMPKα (Thr 172), and beta-actin. Integrated density values for the p-AMPK and p-ACC were normalized to total-AMPK band density and represented as dark or gray bars. (b) C2C12 myotubes were treated with 0.2% FBS medium overnight and then treated with leucine (0.5 mM), resveratrol (100 nM), and leucine plus EX527 (25 μM) for 6 hours. Whole cell lysates were prepared and detected by western blotting with specific antibodies against phosphor-AMPKα, AMPKα, and beta-actin. Integrated density value for phosphor-AMPK was normalized to total-AMPK. *Significantly different from controls with P < 0.05.

Figure 4

Leucine stimulates SIRT1 activity, AMPK phosphorylation, and cellular NAD⁺ in a time-dependent manner. C2C12 myotubes were serum starved overnight and treated with leucine (0.5 mM). Cell lysate was collected and analyzed for cellular SIRT1 activity, western blotting of p-AMPK and cellular NAD⁺ levels at indicated certain time points. (a) SIRT1 activity. (b) Cellular NAD⁺. Both SIRT1 activity and NAD⁺ level were normalized to cellular protein for each sample. (c) Phosphorylation level of AMPK was detected using western blotting following the same time course in C2C12 cells, with resveratrol serving as positive control. Data are mean ± SE (n = 3). Different letters indicate significant differences between dark or gray bars. *Significantly different from point 0, and **significantly different from time point 1.

Figure 5

Leucine-induced mitochondrial biogenesis in C2C12 myotubes requires AMPK. (a) C2C12 myotubes were treated with leucine (0.5 mM), AICAR (20 μM), and Compound C (25 μM) for 24 hours. mtDNA levels of the cells were analyzed by the mitochondrial markers gene expression, Hspd1 and COX2, using real-time PCR. (b) Sirt1 and mitochondrial biogenesis related mRNA level of PGC-1α and COX5b were evaluated also by RT-PCR after treating with leucine and Compound C for 24 hours was measured; all the mRNA levels were normalized to 18S housekeeping gene. Data are mean ± SE (n = 4). Dark bars are vehicle control; grey bars are Compound C. *Significantly different from controls. ^#Significant Compound C effects.

Figure 6

Proposed mechanism of leucine-induced mitochondrial biogenesis. In C2C12 myotubes, leucine treatment leads to activation of SIRT1. SIRT1 then deacetylates and activates LKB1, which subsequently induces AMPK phosphorylation and activation. In turn, activated AMPK could promote SIRT1 activation via intracellular NAD⁺ level by changing expression and activity of Nampt. Activated AMPK and SIRT1 further activate PGC-1α via phosphorylation and deacetylation, resulting in elevated mitochondrial biogenesis and oxidative function.