Protein expressions of Complex I–V and Cytochrome C of the mitochondria electron transport chain in TA muscles from WT and KD mice treated with metformin or saline for two weeks.

<p>White and black bars shows results from saline treated and metformin treated mice respectively. A: Complex I protein expression. B: Comp lex II protein expression. C: Complex III protein expression. D: Cytochrome C protein expression. E: Complex IV protein expression. F: Complex V protein expression. *: Indicates significant difference between genotype (P<0.05). (*): P = 0.059. N = 12–17. Values are S.E.M.</p

Protein expression of AMPKα subunits and ACC Ser²²¹ phosphorylation.

<p>A: Representative Western blot of AMPK α₂ in AMPK WT and KD muscles. The absence of a detectable band at the expected molecular weight of ∼63 kDa in KD mice muscles indicates a strongly reduced endogenous α₂ protein expression. Instead the slightly heavier band in KD muscles is detection of the myc-tag transgenic α₂ protein. B: α₁ protein expression in WT and AMPK KD muscles. <b>*</b>: Indicates significant difference between genotype, P<0.001, n = 10. Values are means ± S.E.M. C: ACC Ser²²¹ phosporyaltion in AMPK WT and KD muscle lysates (TA) from chronic saline (white bars) and metformin (black bars) treated mice. <b>*</b>: Indicates significant difference between genotype, P<0.001, n = 11–14. Values are means ± S.E.M.</p

Figure 1

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053533#pone-0053533-g001" target="_blank">Figure 1A+B</a>: Mitochondrial respiratory capacity in TA muscles from WT and KD mice treated with metformin or saline for two weeks. A: Mitochondrial respiration (O₂ flux) related to milligram muscle. B: Mitochondrial respiration (O₂ flux) related to milligram muscle and expressed relative to mitochondrial content (CS activity). Dark grey bars shows results in TA muscles from saline treated WT mice. Light grey bars shows results in TA muscles from saline treated KD mice. Dark grey bars with slashes shows results in TA muscles from metformin treated WT mice. Light grey bars with slashes shows results in TA muscles from metformin treated KD mice. ‡: Indicate significant difference between genotype within intervention (P<0.05). #: Indicate significant difference between interventions in AMPK KD mice (P<0.05). †: Indicate significant interaction between genotype and intervention (P<0.05). *: Indicate significant main effect of genotype (P<0.05). n = 11–14. Values are means ± S.E.M. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053533#pone-0053533-g001" target="_blank">Figure 1C</a>: Substrate control ratio (SCR) is calculated as state 3 respiration with complex I and II linked substrates divided by state 3 respiration with complex I linked substrates. White bars shows results in TA muscles from saline treated mice. Black bars shows results in TA muscles from metformin treated mice. n = 11–14. Values are means ± S.E.M.</p

Representative Western blots of Complex I–V, Cytochrome C, Pyruvate Dehydrogenase (PDH) subunit E1α protein, PDH site1 and PDH site2 phosphorylation and ACC^Ser211 phosphorylation.

<p>Representative Western blots of Complex I–V, Cytochrome C, Pyruvate Dehydrogenase (PDH) subunit E1α protein, PDH site1 and PDH site2 phosphorylation and ACC^Ser211 phosphorylation.</p

HAD and CS activity in TA muscles from WT and KD mice treated with metformin or saline for two weeks.

<p>White and black bars shows results from saline treated and metformin treated mice respectively. A: HAD activity. B: CS activity. *: Indicates significant difference between genotype (P<0.05), N = 12–17. Values are S.E.M.</p

Staining of human <i>VL</i> muscle lipid and glycogen stores with an optimized protocol reveals the existence of four metabolically distinct fiber types.

<p>(A) Representative images of consecutive muscle crossections stained for lipid (top), glycogen and laminin (middle) and myosin heavy chain I (green) and II (red)(bottom) are presented. Bars; 20 µm. (B) ATPase staining of consecutive sections allowed for type IIa and IIx distinction. (C) Lipid (grey bars) and glycogen (black bars) stores were analyzed in <i>vastus lateteralis</i> muscle from 6 young healthy subjects and, the results are plotted as mean grey value ± SEM. Statistical differences <i>versus</i> I-1 are represented as *, <i>versus</i> I-2 are represented as # and, <i>versus</i> IIA are represented as ¤. Measurement of Mean Grey Value does not allow differentiating IMTG content in the 4 fiber types. A more detailed analysis of IMTG stores, as lipid droplets’ density and size (D), allows for the differentiation of 4 metabolically distinct fiber types. Two populations of type I muscle fibers, I-1 and I-2, can be differentiated by muscle fiber crossectional area (E) and lipid droplet density. Type I-2 muscle fibers are similar in fiber crossectional area and lipid content and distribution to type IIA. Quantification of lipid and glycogen content in 10 human <i>VL</i> muscles before (black bars) and after exhausting exercise (grey bars) was performed. Glycogen content is expressed as mean grey value (F), lipid droplets’ density as number of lipid droplets in 3.6 µm² (G) and, lipid droplets’ size as number of pixels (H) (Pixel size is 3.6*10⁻³ µm²). Two-way repeated Measures Analysis of Variance was performed and statistically significant differences between fiber types (C and D: *vs I-1, #vs I-2) and, between basal and exhausted muscle (F–H: *p<0.05, **p<0.01 and ***p<0.005) are represented.</p