Acute Regulation of Cardiac Metabolism by the Hexosamine Biosynthesis Pathway and Protein O-GlcNAcylation

OBJECTIVE: The hexosamine biosynthesis pathway (HBP) flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc) levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization. METHODS: Isolated rat hearts were perfused with glucosamine (0-10 mM) to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by (13)C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36. RESULTS: Glucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation. CONCLUSION/INTERPRETATION: These data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism

α-Lipoic acid increases cardiac glucose oxidation independent of AMP-activated protein kinase in isolated working rat hearts

α-Lipoic acid (ALA) is a naturally occurring enantiomer of lipoic acid and is a cofactor of key metabolic enzyme complexes catalyzing the decarboxylation of α-keto acids. It was recently shown that ALA increases insulin sensitivity by activating AMP-activated protein kinase (AMPK) in skeletal muscle. Also, administration of ALA to obese rats increases insulin-stimulated glucose uptake in the whole body. We investigated the metabolic effects of ALA on isolated working rat hearts. ALA (500μM) stimulated glucose oxidation (157 ± 31 nmol·dry wt-1·min-1 in control vs 315 ± 63 nmoldry wt-1·min-1 in ALA-treated, p < 0.05) without affecting glycolysis, lactate oxidation,or palmitate oxidation.Cardiac work was not affected by ALA treatment. The effect of ALA on glucose oxidation was not associated with an activation of AMPK. AMPK activity was 190 ± 14 pmol · mg protein-1·min-1 in control vs 190±16 pmol·mg protein-1·min-1 in ALAtreated hearts. This study shows that ALA stimulates glucose oxidation in isolated working rat hearts independent of AMPK activation. The beneficial effects of ALA treatment in diabetic patients may be at least in part related to its effect on glucose metabolism. © Steinkopff Verlag 2007.link_to_subscribed_fulltex

Alpha-lipoic acid increases cardiac glucose oxidation independent of AMP-activated protein kinase in isolated working rat hearts

α-Lipoic acid (ALA) is a naturally occurring enantiomer of lipoic acid and is a cofactor of key metabolic enzyme complexes catalyzing the decarboxylation of α-keto acids. It was recently shown that ALA increases insulin sensitivity by activating AMP-activated protein kinase (AMPK) in skeletal muscle. Also, administration of ALA to obese rats increases insulin-stimulated glucose uptake in the whole body. We investigated the metabolic effects of ALA on isolated working rat hearts. ALA (500μM) stimulated glucose oxidation (157 ± 31 nmol·dry wt-1·min-1 in control vs 315 ± 63 nmoldry wt-1·min-1 in ALA-treated, p < 0.05) without affecting glycolysis, lactate oxidation,or palmitate oxidation.Cardiac work was not affected by ALA treatment. The effect of ALA on glucose oxidation was not associated with an activation of AMPK. AMPK activity was 190 ± 14 pmol · mg protein-1·min-1 in control vs 190±16 pmol·mg protein-1·min-1 in ALAtreated hearts. This study shows that ALA stimulates glucose oxidation in isolated working rat hearts independent of AMPK activation. The beneficial effects of ALA treatment in diabetic patients may be at least in part related to its effect on glucose metabolism. © Steinkopff Verlag 2007.link_to_subscribed_fulltex

Skin Localization of Lipid Nanoparticles (SLN/NLC): Focusing the Influence of Formulation Parameters

In this study, fluorescein labeled SLN and NLC formulations were prepared for improving the dermal distribution of the hydrophilic active ingredients and for enhancing the skin penetration. To determine skin distribution of the lipid nanoparticles ex-vivo penetration/permeation experiments were performed using full thickness rat skin by means of Franz diffusion cells. Studies on the localization of fluorescence labeled nanoparticles were performed by confocal laser scanning microscopy (CLSM). Cellular uptake studies were performed on human keratinocyte cell line (HaCaT) and visualized by fluorescence microscope. Both tissue and cell uptake were also quantitatively determined by means of fluorimetric method in the skin extract or cell extract. Both imaging and quantification studies suggest that the dermal localization of the lipid nanoparticles depends on their dimensions and particle size distribution. The CLSM images clearly show that the Tripalmitin based lipid nanoparticles have higher accumulation in the skin. It is possible to overcome the stratum corneum barrier function with T-NLC05 coded lipid nanoparticle formulation. Additionally cellular uptake of this NLC formulation is time dependent