Effect of glucosamine on A) glucose; B) pyruvate; C) lactate and D) palmitate oxidation.

<p>^* P<0.05 vs. 0 mM glucosamine, one-way ANOVA with Dunnett's posthoc test. 0 mM (n = 6), 0.05 mM (n = 4), 0.1 mM (n = 5), 5 mM (n = 5), 10 mM (n = 4).</p

Effect of glucosamine on A, B) Overall cardiac O-GlcNAc levels; C) UDP-HexNAc concentrations and D) ATP concentrations.

<p>^* P<0.05 vs. 0 mM, one-way ANOVA with Dunnett's posthoc test. Western blots: 0 mM (n = 8), 0.05 mM (n = 5), 0.1 mM (n = 9), 1 mM (n = 4), 5 mM (n = 8), 10 mM (n = 7). HPLC: 0 mM (n = 4), 0.05 mM (n = 5), 0.1 mM (n = 5), 1 mM (n = 4), 5 mM (n = 3), 10 mM (n = 3). 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 [3-¹³C]lactate uptake ^* P<0.05 vs. 0 mM, Student's t-test.

<p>0 mM (n = 4), 0.1 mM (n = 5).</p

Cardiac function of isolated rat hearts perfused with 0 (n = 8), 0.05 (n = 5), 0.1 (n = 10), 1 (n = 4), 5 (n = 8) and 10 mM (n = 7) 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 product = left ventricular developed pressure×heart 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>n = 2 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 acute exposure to fatty acids on human BMMSC energy metabolism.

<p>A) Glycolysis, B) glucose oxidation, C) palmitate oxidation, D) palmitate uptake, E) oleate oxidation, and F) oleate uptake were measured in untreated human bone marrow mesenchymal stem cells (BMMSCs). n = 5–7 During each assay Krebs Henseleit buffer was supplemented with 5 mM glucose and, as indicated in each graph, either 0.55 mM albumin (BSA group) or 0.55 mM albumin bound to 0.4 mM palmitate and/or 0.4 mM oleate. In addition, the Krebs Henseleit buffer was supplemented with either [U-¹⁴C]glucose, [1–¹⁴C]palmitate, [1–¹⁴C]oleate, or [5–³H]glucose for the measurement of glucose oxidation, palmitate oxidation and uptake, oleate oxidation and uptake, or glycolysis, respectively. Since these experiments assessed the acute effect of palmitate and oleate, BMMSCs were maintained in cell culture media used to culture these immediately up to the start of each assay when the media was switched to Krebs Henseleit buffer supplemented with fatty acids. The levels and type of fatty acid BMMSCs were exposed to is indicated on the x-axis of the figures. * Significantly different from all groups. Values are shown as the mean ± SEM.</p

Effect of 24 hour exposure to fatty acids on human BMMSC energy metabolism.

<p>A) Palmitate oxidation, B) palmitate uptake, C) glycolysis, and D) glucose oxidation were measured in human BMMSCs that had been treated for 24 hr with either 0.55 mM albumin (BSA group) or 0.55 mM albumin and 0.4 mM palmitate and/or 0.4 mM oleate prior to these metabolism measurements being made. n = 5–8 The graphs indicate which groups were exposed to these different treatments for the 24 hr prior to the metabolism measurements. During each assay all groups were given Krebs buffer supplemented with 5 mM glucose and 0.4 mM palmitate bound to 0.55 mM albumin. In addition, the Krebs buffer was supplemented with either [U-¹⁴C]glucose, [1–¹⁴C]palmitate, or [5–³H]glucose for the measurement of glucose oxidation, palmitate oxidation and uptake, or glycolysis, respectively. E) The contribution of metabolic pathways to ATP production were calculated from the metabolic rate results. * Significantly different from all groups. Values are shown as mean ± SEM.</p

Effect of 24 hr exposure to palmitate and oleate on human BMMSC mitochondrial membrane potential.

<p>A) Images of tetramethylrhodamine methyl ester (TMRM) stained bone marrow mesenchymal stem cells (BMMSCs) treated for 24 hr with indicated treatments. B) Relative TMRM levels. BMMSCs were treated for 24 hr with 0.55 mM bovine serum albumin (BSA) alone or palmitate and/or oleate bound to 0.55 mM albumin. TMRM and Hoechst stain were added to the medium to measure mitochondrial membrane potential and stain nuclei, respectively, and images were taken. All fatty acid treated groups were also treated with media supplemented with 0.55 mM albumin in addition to the type and amount of fatty acid indicated in the figures. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120257#pone.0120257.g004" target="_blank">4</a> separate observations. Values are shown as the mean ± SEM. * Significantly different from the BSA group.</p

Oleate prevents palmitate-induced human BMMSC apoptosis and reduction in proliferation.

<p>A) Caspase activity after 48 hr treatment with indicated treatments. B) Images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Ki67 staining of bone marrow mesenchymal stem cells (BMMSCs) treated for 24 hr with indicated treatments. Image is 28X. C) % nuclei positive for TUNEL. D) % nuclei positive for Ki67. n = 5–7 The BSA group was treated with media supplemented with 0.55 mM albumin. All fatty acid treated groups were also treated with media supplemented with 0.55 mM albumin in addition to the type and amount of fatty acid indicated in the figures. * Significantly different from BSA group. # Significantly different from 0.2 mM Palmitate and 0.4 mM Palmitate groups. ** Significantly different from all groups. Values are shown as the mean ± SEM.</p

Contribution of energy metabolism pathways to ATP production in human BMMSCs.

<p>Contribution of energy metabolism pathways to ATP production in human BMMSCs.</p