19 research outputs found
Effect of glucosamine on A, B) Overall cardiac O-GlcNAc levels; C) UDP-HexNAc concentrations and D) ATP concentrations.
<p><sup>*</sup> P<0.05 vs. 0 mM, one-way ANOVA with Dunnett's posthoc test. Western blots: 0 mM (nâ=â8), 0.05 mM (nâ=â5), 0.1 mM (nâ=â9), 1 mM (nâ=â4), 5 mM (nâ=â8), 10 mM (nâ=â7). HPLC: 0 mM (nâ=â4), 0.05 mM (nâ=â5), 0.1 mM (nâ=â5), 1 mM (nâ=â4), 5 mM (nâ=â3), 10 mM (nâ=â3). Note that equal protein loading for the O-GlcNAc immunoblots was assessed by Sypro staining and overall O-GlcNAc levels were normalized to untreated control group.</p
Effect of 0.1 mM glucosamine on A) unlabeled glycolytic lactate efflux and B) exogenous [3-<sup>13</sup>C]lactate uptake <sup>*</sup> P<0.05 vs. 0 mM, Student's t-test.
<p>0 mM (nâ=â4), 0.1 mM (nâ=â5).</p
Effect of glucosamine on A) glucose; B) pyruvate; C) lactate and D) palmitate oxidation.
<p><sup>*</sup> P<0.05 vs. 0 mM glucosamine, one-way ANOVA with Dunnett's posthoc test. 0 mM (nâ=â6), 0.05 mM (nâ=â4), 0.1 mM (nâ=â5), 5 mM (nâ=â5), 10 mM (nâ=â4).</p
Cardiac function of isolated rat hearts perfused with 0 (nâ=â8), 0.05 (nâ=â5), 0.1 (nâ=â10), 1 (nâ=â4), 5 (nâ=â8) and 10 mM (nâ=â7) glucosamine for 60 minutes (hearts were paced at 320 beats/min rate).
<p>Glucosamine had no effect either on cardiac or on coronary flow. (RPP: rate pressure productâ=âleft ventricular developed pressureĂheart rate; dp/dt: change of pressure over time).</p
A) Immunoblots of Plasma membrane fraction for FAT/CD36 following 60 min perfusion with 0, 0.05, 0.1, 1, 5 and 10 mM; pan-cadherin included as a plasma membrane marker and protein loading control; B) Densitometric analysis of FAT/CD36 immunoblots normalized to 0 mM glucosamine; P<0.05 vs. 0 mM, one-way ANOVA with Dunnett's posthoc test; <i>nâ=â2 in each group</i>; C) Immunoprecipitation of FAT/CD36 from whole tissue and plasma membrane lysates, followed by O-GlcNAc and OGT immunoblots.
<p>Specificity of O-GlcNAc antibody was confirmed by co-incubation with 10 mM N-acetylglucosamine (GlcNAc).</p
Effect of the LRPPRC A354V mutation on basal mitochondrial network morphology and functions.
<p>Representative live cell images of MTG-loaded control <b>(A)</b> and LSFC <b>(B)</b> fibroblasts used for quantitative analysis of mitochondrial network morphology. <b>(C)</b> Form Factor (FF) values calculated using the equation FF = 4Ï*Area/perimeter2 (n = 6). Representative live cell images of control <b>(D)</b> and LSFC <b>(E)</b> fibroblasts labeled with TMRE (red) and MTG (green). <b>(F)</b> Mitochondrial membrane (ÎΚ) potential expressed as the ratio of TMRE to MTG (n = 5). Lower values are indicative of reduced ÎΚ. <b>(G)</b> Mean fluorescence intensity of the mitochondria-specific superoxide probe MitoSOX in control and LSFC fibroblasts (n = 5). <b>(H)</b> Maximal ADP-driven respiration in digitonin-permeabilized fibroblasts energized with complex I (5 mM glutamateâ2.5 mM malate; Glut-Mal; n = 15) or complex II substrates in presence of the complex I inhibitor rotenone (5 mM succinate + 1 ÎŒM rotenone; Succ+Rot; n = 14). Inset shows representative respirometry traces confirming that respiratory rates increased promptly in response to the addition of respiratory substrates, and were potently inhibited by complex I (rotenone), and complex II (malonate) blockers. <b>(I)</b> Mitochondrial calcium retention capacity (CRC) in control and LSFC fibroblasts exposed to progressive Ca2+ loading (n = 8). Inset shows representative Ca2+ kinetic tracings observed in control and LSFC fibroblasts. Tracings show progressive Ca2+ accumulation followed by PTP-induced release of accumulated Ca2+. Each spike indicates the addition of a calcium pulse of 83 nmoles. All experiments were performed in one control (EBS-4) and one LSFC (AL-006) cell line, except for the determination of ÎΚ, which was performed in EBS-3 and AL-002. Data are expressed as means ± S.E. Difference between control and LSFC cells was assessed with a paired t-test. Significantly different from the control group: * <i>p</i> < 0.05, ** <i>p</i> †0.01. Statistical power: C: 92%; F: 85%; G: 80%; H: Glut-Mal 80%; Succ+Rot: 96%; I: 73%.</p
Stress-induced cell death in control and LSFC fibroblasts.
<p><b>(A)</b> Lactate dehydrogenase (LDH) release in control and LSFC fibroblasts exposed for 48 h to factors relevant to acidotic crises. <b>(B)</b> Mean LDH release (<i>n</i> = 5), <b>(C)</b> Caspase 3/7 activity (<i>n</i> = 7), <b>(D)</b> Cellular ATP content (<i>n</i> = 7) and <b>(E)</b> COX/CS activity ratio (<i>n</i> = 3) assessed at baseline and after exposure to PL (palmitate 1 mM; lactate 10 mM) for 34 h (LDH release) or 24 h (other parameters). All experiments were performed in one control (EBS-4) and one LSFC (AL-006) cell line. Data are expressed as means ± S.E. Statistical significance of differences between groups or conditions was assessed with a two-way ANOVA for repeated measures followed by a Bonferroni post hoc test. Significantly different from the control cells in the same experimental condition: * <i>p</i> < 0.05, ** <i>p</i> < 0.01. Significantly different from baseline within the same experimental group: <sup>Ï</sup><i>p</i> < 0.05, <sup>ÏÏ</sup><i>p</i> < 0.01, <sup>ÏÏÏ</sup><i>p</i> < 0.001.</p
Control and Leigh Syndrome French Canadian (LSFC) skin fibroblast cell lines.
<p>Control and Leigh Syndrome French Canadian (LSFC) skin fibroblast cell lines.</p
Effect of the LRPPRC A354V mutation on LRPPRC content, mitochondrial content and cellular ATP levels.
<p><b>(A)</b> Immunoblot and densitometric analysis of LRPPRC content in whole cell lysates from control and LSFC fibroblasts (n = 4). Representative immunofluorescence images of control <b>(B;C)</b> and LSFC fibroblasts <b>(D;E)</b> labeled with anti-LRPPRC (green) and anti-pyruvate dehydrogenase (red) antibodies. Overlay images (yellow), and line scan analysis show the cellular distribution of LRPPRC in the mitochondrial compartment. <b>(F)</b> Cytochrome c oxidase (COX) enzyme activity in whole cell lysates from control and LSFC fibroblasts was normalized to that of the mitochondrial marker citrate synthase (CS) to take into account possible differences in mitochondrial content (n = 4). <b>(G)</b> Immunoblot and densitometric analysis of VDAC, a mitochondrial marker protein, in whole cell lysates from control and LSFC fibroblasts (n = 3). <b>(H)</b> Citrate synthase activity in whole cell lysates from control and LSFC fibroblasts (n = 4). <b>(I)</b> Cellular ATP content in control and LSFC fibroblasts (n = 4). Data are expressed as means ± S.E. Experiments were performed in one control (EBS-4) and one LSFC cell line (AL-006). Difference between control and LSFC cells was assessed with a paired t-test. ** Significantly different from the control group <i>p</i> †0.01. Statistical power for LRPPRC and COX was 97%.</p
Mitochondrial Respiration.
<p>Data are the mean ± SEM. CTRL, nâ=â14. DHA, nâ=â13. ARA, nâ=â13. DHA+ARA, nâ=â14. All Rates are expressed in ng atoms O·mg<sup>â1</sup>·min<sup>â1</sup>. The RCR, defined as the ratio of State 3 to State 4 respiration rate, was calculated from the State 4 rate with oligomycin.</p