The effect of obesity on protein carbonylation, antioxidant response and mitochondrial function in the absence of Haptoglobin.

<p>Each panel shows WT (white bars) and Hp^-/- (black bars) skeletal muscle under regular Chow Food Diet Feeding (the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g001" target="_blank">figures 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g003" target="_blank">3</a>, referred to as “CFD”, n = 6–8) and in the obesity condition following 12 weeks of High Fat Diet (referred to as “HFD”, n = 6). A) Oxyblot reveals an obesity dependent increase in carbonylated proteins in the <i>EDL</i> and <i>Soleus</i> muscle of Hp^-/- mice. B) mRNA analysis for anti-oxidant response (names of genes as indicated) in <i>Tibialis anterior</i> muscle. C) mRNA analysis for mitochondrial biogenesis (names of genes as indicated) in the skeletal muscle: obesity dependent down regulation of <i>PGC1α</i> in HFD Hp^-/- mice, and <i>Tfam</i> upregulation in HFD WT animals. D) Mitochondrial response to oligomycin in single myofibers isolated from <i>flexor digitorum brevis</i> (FDB) skeletal muscles of WT and Hp^-/- mice. Where indicated, 6 µM oligomycin (arrow) or 4 µM of the protonophore carbonylcyanide-<i>p</i>-trifluoromethoxyphenyl hydrazone (FCCP) (arrowhead) were added. Traces represent tetramethylrhodamine methyl ester (TMRM) fluorescence as the mean of all the fibers of a group (n≥12). Fibers are considered as depolarized when they lose more than 10% of initial value of TMRM fluorescence after oligomycin addition. mRNA analysis and histological assessment are performed on <i>Tibialis anterior</i> skeletal muscle. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; §P<0.05; §§P<0.01; §§§P<0.001.</p

Haptoglobin deficiency induces activation of the ubiquitin-proteasome system and inhibition of protein synthesis in the skeletal muscle.

<p>A) <i>Atrogin-1</i> and <i>Murf1</i> mRNA transcripts are increased in Hp^-/- as compared to WT skeletal muscle. B) mRNA expression levels of autophagy related genes (<i>names of genes as indicated</i>) in the skeletal muscle of WT and Hp^-/- mice. (C-I) Western blots analysis on protein isolated from skeletal muscle of WT and Hp^-/- mice showing: C) Beclin-1 protein levels, D) LC3 lipidation, E) p62 protein levels, F) Akt phosphorylation (ratio between signal on Ser 473 and total Akt), G) 4EBP1 phosphorylation (ratio between signal on Thr 37/46 and total 4EBP1), H) S6 phosphorylation (ratio between signal on Ser 235/236 and total p70S6), I) AMPK phosphorylation (ratio between Thr172 and total AMPK). The bar graphs represent the quantification of the sum of experiments; data are expressed as percentage of “WT”, where WT is considered 100%. J) mRNA expression levels of <i>4EBP</i>1. Real time PCR and Western Blot analyses were respectively performed on <i>Tibialis anterior</i> cDNA (n = 8) and <i>Gastrocnemius</i> muscle lysates (n = 6). GAPDH is used as control of equal loading in both cases. Muscles were dissected from 5 months old mice. Data are expressed as mean ± SEM. Student's t-test: *P<0.05; **P<0.01.</p

Haptoglobin deficiency affects muscle performance.

<p>A) and B) Forelimbs strength assessment by grip test. A) WT and Hp^-/- mice show overlapping basal strength; following rotarod exercise (end of task) Hp^-/- undergo a more pronounced strength reduction as compared to WT. B) residual strength, expressed as percentage of basal strength before the exercise (dashed line) is lower in Hp^-/- as compared to WT mice. C) Number of falls/hour from rotarod is higher in Hp^-/- mice (RUN Hp^-/-) as compared to controls (RUN WT). D) The average consecutive time that mice spent on rotarod is significantly shorter in RUN Hp^-/- as compared to controls (RUN WT) (n = 8). Data are expressed as mean ± SEM. Student's t-test: *P<0.05; **P<0.01; ***P<0.001. Paired Student's t-test: ∧∧∧P<0.001.</p

The effects of obesity on skeletal muscle protein balance in the absence of Haptoglobin.

<p>Each panel shows WT (white bars) and Hp^-/- (black bars) skeletal muscle under regular Chow Food Diet Feeding (the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g001" target="_blank">figures 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g003" target="_blank">3</a>, referred to as “CFD”, n = 6–8) and in the obesity condition following 12 weeks of High Fat Diet (referred to as “HFD”, n = 6). A) Obesity results in <i>Atrogin-1</i> transcript upregulation in HFD WT mice and in <i>MuRF1</i> transcript upregulation both in HFD WT and HFD Hp^-/- mice. B) mRNA levels of autophagy related genes (names as indicated) in CFD and HFD WT and Hp^-/- skeletal muscle. (C-H) Quantification of Western blot analysis on skeletal muscle of CFD and HFD WT and Hp^-/- mice; data are expressed as percentage of “CFD WT”, where CFD WT is considered 100%. C) LC3 lipidation, D) p62 protein levels, E) Beclin-1 protein levels, F) Akt phosphorylation (ratio between signal on Ser 473 and total AKT), G) S6 phosphorylation (ratio between signal on Ser 240/244 and total p70S6), H) 4EBP1 phosphorylation (ratio between signal on Thr 37/46 and total 4EBP1). I) <i>4EBP1</i> mRNA level is increased in both obese WT and HFD Hp^-/- skeletal muscle with respect to relative CFD. Real Time PCR and western blot analysis were respectively performed on <i>Tibialis anterior</i> cDNA and <i>Gastrocnemius</i> muscles protein lysates. GAPDH is used as control of equal loading in both cases. Muscles were dissected from 5 months old mice. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; §P<0.05; §§P<0.01; §§§P<0.001.</p

The effects of obesity on skeletal muscle size and performance in the absence of Haptoglobin.

<p>Each panel shows WT (white bars) and Hp^-/- (black bars) skeletal muscle under regular Chow Food Diet Feeding (the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g001" target="_blank">figures 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g003" target="_blank">3</a>, referred to as “CFD”, n = 6–8) and in the obesity condition following 12 weeks of High Fat Diet (referred to as “HFD”, n = 6). A) Hp deficiency exacerbates the obesity dependent reduction of <i>Tibialis anterior</i> cross sectional area (CSA) (n≥5000 fibers). B) Cross sectional area (CSA) frequency distribution of <i>Tibialis anterior</i> in obese Hp^-/- and WT mice. The bar graph indicates relative frequencies as percent. Red-dotted lines indicates the median for each distribution (n≥5000 fibers). C) Grip test assessment of forelimbs strength reveals an obesity dependent reduction only in Hp^-/- mice. D) mRNA analysis for inflammatory response (names of genes as indicated) in <i>Tibialis anterior</i> muscle. Data are expressed as mean ± SEM. *P<0.05; ***P<0.001, §P<0.05; §§§P<0.001.</p

Haptoglobin deficiency induces muscle atrophy in skeletal muscle.

<p>A) Hematoxylin and Eosin staining of WT and Hp^-/-<i>Tibialis anterior</i> muscle shows no sign of fiber degeneration and/or inflammation. B) Succinate Dehydrogenase (SDH) staining of WT and Hp^-/-<i>Tibialis anterior</i> muscle shows no major differences of fiber types. C) mRNA expression levels of <i>PGC1α</i>, <i>Nrf1</i> and <i>Tfam</i> in <i>Tibialis anterior</i> of WT and Hp^-/- mice (n = 8). D) Cross sectional area (CSA) of <i>Tibialis anterior</i> is reduced in Hp^-/- (10%) as compared to WT mice (n≥2500 fibers). Muscles were dissected from 5 months old mice. E) Cross sectional area (CSA) frequency distribution of <i>Tibialis anterior</i> in Hp^-/- and WT mice. The bar graph represents relative frequencies as percent. Red-dotted lines indicates the median for each distribution (n≥2500 fibers). Data are expressed as mean ± SEM. Student's t-test: **P<0.01; ***P<0.001.</p

Following acute exercise Haptoglobin deficiency results in exacerbated oxidative stress and impaired antioxidant response.

<p>Each panel shows WT (white bars) and Hp^-/- (black bars) skeletal muscle in resting conditions (the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g001" target="_blank">figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100745#pone-0100745-g002" target="_blank">2</a>, referred to as control, n = 6–8) and following 3 hours of rotarod exercise (referred to as “RUN”, n = 6). A) The exercise dependent increase in carbonylated proteins is more pronounced in the <i>EDL</i> and <i>Soleus</i> muscle of Hp^-/- as compared to WT mice. B) mRNA analysis for anti-oxidant response (names of genes as indicated) in <i>Tibialis anterior</i> muscle. C) mRNA analysis for inflammatory response (names of genes as indicated) in <i>Tibialis anterior</i> muscle. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; §P<0.05; §§P<0.01; §§§P<0.001.</p

Haptoglobin deficiency effect on protein carbonylation, antioxidant response and inflammatory response in the skeletal muscle.

<p>A) Protein carbonylation is similar in <i>EDL</i> and <i>soleus</i> muscle of WT and Hp^-/- mice (n = 11). B) mRNA analysis for anti-oxidant response (names of genes as indicated) in <i>Tibialis anterior</i> muscle of WT and Hp^-/- mice (n = 8). C) mRNA analysis for inflammatory response (names of genes as indicated) in <i>Tibialis anterior</i> muscle of WT and Hp^-/- mice (n = 8). Data are expressed as mean ± SEM. Student's t-test: *P<0.05.</p

Following acute exercise Haptoglobin deficiency results in altered mitochondrial signaling in skeletal muscle.

<p>A) mRNA analysis for mitochondrial biogenesis (names of genes as indicated): <i>PGC1α</i> transcript is induced by exercise in WT mice and not in Hp^-/-; <i>Tfam</i> and <i>Nrf1</i> are downregulated by exercise in Hp^-/- skeletal muscle. B) Exercise does not affect <i>Atrogin-1</i> and <i>MuRF1</i> mRNA levels in any of the two genotypes. C) mRNA amount of autophagy related genes (names as indicated) in control and RUN WT and Hp^-/- skeletal muscle. (D-I) Quantification of Western blots analysis on protein isolated from skeletal muscle of control and RUN WT and Hp^-/- mice; data are expressed as percentage of “control WT”, where control WT is considered 100%. D) Beclin-1 protein levels, E) LC3 lipidation (ANOVA treatment effect P<0.05), F) p62 protein levels, G) Akt phosphorylation (ratio between signal on Ser 473 and total Akt), H) S6 phosphorylation (ratio between signal on Ser 235/236 and total p70S6), I) 4EBP1 phosphorylation (ratio between signal on Thr 37/46 and total 4EBP1). J) <i>4EBP1</i> mRNA level. Real Time PCR and western blot analysis were respectively performed on <i>Tibialis anterior</i> cDNA and <i>Gastrocnemius</i> muscles protein lysates. GAPDH is used as control of equal loading in both cases. Muscles were dissected from 5 months old mice. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; §§P<0.01; §§§P<0.001.</p

Antibodies listed in alphabetical order.

<p>Antibodies listed in alphabetical order.</p