Schematic model for 2-AA mediated molecular mechanisms of mitochondrial dysfunction in skeletal muscle.

<p>2-AA triggers mitochondrial dysfunction via the down-regulation of peroxisome proliferative activated receptor, gamma, coactivator (PGC)-1β, peroxisome proliferator activated receptor (PPAR)-γ, sirtuin (Sirt)-1, the insulin signaling pathway, overall energy metabolism, and increased lipid accumulation.</p

NMR spectra from ¹H-NMR HRMAS analysis of gastrocnemius skeletal muscle from 2-AA treated versus control mice.

<p>The spectra were acquired from 2-AA treated mice at 4 d versus control mice, and scaled to the phosphocreatine plus creatine peak (3.02 ppm). Resonance signals of lipids correspond to: 1) terminal methyl CH₃ protons (0.9 ppm); 2) acyl chain methylene protons (CH₂)_n of intramyocellular lipids (IMCLs) (1.3 ppm); 3) methylene protons CH₂C-CO (1.6 ppm); 4) allylic methylene protons C = C-CH₂-C of monounsaturated fatty acyl moieties (MUFAs) (2.05 ppm); 5) α methylene protons CH₂CO (2.25 ppm); 6) diallylic methylene protons  = C-CH₂-C =  of polyunsaturated fatty acyl moieties (PUFAs); and 7) N-methyl protons of phosphocreatine and creatine (3.0 ppm), respectively. The NMR spectra demonstrate increased biomarkers of insulin resistance IMCLs.</p

Differential expression of genes involved in fatty acid oxidation in mouse skeletal muscle at 4-AA treatment versus control muscle.

<p>(+) Upregulation of genes compared with control untreated muscle.</p><p>(−) Downregulation of genes compared with control untreated muscle.</p

2-AA treatment down-regulates muscle function.

<p>Black bars indicate the number of down-regulated genes; gray bars indicate the number of up-regulated genes, in the skeletal muscle of mice 4 days post 2-AA treatment versus control mice (left vertical axis). The negative log10 of p-values represented by gray triangles are indicated on the right vertical axis.</p

Downregulation of muscle contraction genes in mouse skeletal muscle at 4-AA treatment versus control muscle.

<p>(−) Downregulation of genes compared with control untreated muscle.</p

2-AA treatment dampens key metabolic protein levels in mouse skeletal muscle cells.

<p>Western blotting of cellular extracts with specific antibodies of PGC-1β, PPAR-γ and Sirt-1 in 2-AA treated cells at the indicated time points. One representative experiment (out of three) is shown. Loading was normalized relative to mouse α-tubulin. Densitometric data are the average of three replicate experiments and are expressed as mean ± SD (vertical bars).*p<0.05 vs. naïve. DU, densitometric units.</p

Effects of 2-AA on viability of mouse muscle cells.

<p>MTT assay measuring cell viability in mouse skeletal muscle cells (C2C12) after treatment with 0.8 mM 2-AA over time, as indicated in the figure. SDs (vertical bars) were calculated from three replicate experiments.</p

NMR spectra of <i>in vivo</i>³¹P NMR saturation-transfer on mouse hind limb skeletal muscle.

<p>Representative summed ³¹P-NMR spectra acquired from control and 2-AA treated mice at day 4, before (A) and after (B) saturation of the γ-ATP resonance, with the difference spectrum between the two shown below (A–B). The arrow on γ-ATP indicates the position of saturation (sat) by rf irradiation (−2.4 ppm). ppm, chemical shift in parts per million.</p

Differential expression of key metabolic genes in skeletal muscle from 2-AA treated mice versus untreated control mice.

<p>Values are the relative expression intensity of the 2-AA treated versus control mice after 4 d Gene annotations for biological functions are from the Gene Ontology Consortium and the Ingenuity database.</p><p>(+) Upregulation of genes compared with control untreated muscle.</p><p>(−) Downregulation of genes compared with control untreated muscle.</p

<i>In vivo</i>³¹P-NMR saturation transfer analysis of limb skeletal muscle from control and 2-AA treated mice.

<p>Values are means ± SEM; ΔM/M₀ is the fractional change in P_i or PCr magnetization as a result of saturation transfer; T_1obs is the observed spin lattice relaxation time of P_i or PCr during γ-ATP saturation in seconds; <i>Κ_f</i> is the rate constant for the reactions P_i → γ-ATP and PCr → γ-ATP, calculated as (1/T_1obs) × (Δ<i>M/M₀</i>). ATP synthesis is calculated as P_i or PCr × <i>Κ_f</i>. NS: not significant. Unpaired one-tailed Student's t-test was used for the comparisons.</p