Faculty Bibliography

Objective: In diabetes, hyperglycemia causes the accumulation of advanced glycation end products (AGEs) that trigger reactive oxygen species (ROS) generation through binding the receptor for AGEs (RAGE). Because exogenous growth factors have had little success in enhancing chronic wound healing, we investigated whether hyperglycemia-induced AGEs interfere with cellular responses to extracellular signals. We used stromal cell-derived factor-1 (SDF-1), an angiogenic chemokine also known to promote stem cell recruitment in skin wounds. Approach: Human leukemia-60 (HL-60) cells and mouse peripheral blood mononuclear cells (PBMCs), which express the SDF-1 receptor CXCR-4, were incubated for 24 h in medium supplemented with 25 mM d-glucose. Soluble RAGE (sRAGE) was used to block RAGE activation. Response to SDF-1 was measured in cellular migration and ROS assays. A diabetic murine excisional wound model measured SDF-1 liposome and sRAGE activity in vivo. Results: Hyperglycemia led to significant accumulation of AGEs, decreased SDF-1-directed migration, and elevated baseline ROS levels; it suppressed the ROS spike normally triggered by SDF-1. sRAGE decreased the ROS baseline and restored both the SDF-1-mediated spike and cell migration. Topically applied sRAGE alone promoted healing and enhanced the effect of exogenous SDF-1 on diabetic murine wounds. Innovation: While there is interest in using growth factors to improve wound healing, this strategy is largely ineffective in diabetic wounds. We show that sRAGE may restore signaling, thus potentiating the effect of exogenously applied growth factors. Conclusion: Blocking RAGE with sRAGE restores SDF-1-mediated cellular responses in hyperglycemic environments and may potentiate the effectiveness of SDF-1 applied in vivo.

Acetylation of proteins as a post-translational modification is gaining rapid acceptance as a cellular control mechanism on par with other protein modification mechanisms such as phosphorylation and ubiquitination. Through genetic manipulations and evolving proteomic technologies, identification and consequences of transcription factor acetylation is beginning to emerge. In this review, we summarize the field and discuss newly unfolding mechanisms and consequences of transcription factor acetylation in normal and stressed hearts. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.

Post-translational modification of proteins imparts diversity to protein functions. The process of glycation represents a complex set of pathways that mediates advanced glycation endproduct (AGE) formation, detoxification, intracellular disposition, extracellular release, and induction of signal transduction. These processes modulate the response to hyperglycemia, obesity, aging, inflammation, and renal failure, in which AGE formation and accumulation is facilitated. It has been shown that endogenous anti-AGE protective mechanisms are thwarted in chronic disease, thereby amplifying accumulation and detrimental cellular actions of these species. Atop these considerations, receptor for advanced glycation endproducts (RAGE)-mediated pathways downregulate expression and activity of the key anti-AGE detoxification enzyme, glyoxalase-1 (GLO1), thereby setting in motion an interminable feed-forward loop in which AGE-mediated cellular perturbation is not readily extinguished. In this review, we consider recent work in the field highlighting roles for glycation in obesity and atherosclerosis and discuss emerging strategies to block the adverse consequences of AGEs. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.

Introduction Brown adipose tissue (BAT) is one of the primary tissues responsible for adaptive non-shivering thermogenesis, mediated by mitochondria uncoupling protein-1 (Ucp1), and contributes to energy expenditure. BAT has been shown to play a role in adiposity, insulin resistance and hyperlipidaemia. We and others^1-2 have shown that genetic deficiency of RAGE (receptor for advanced glycation end products) prevented the effects of a high-fat diet (HFD) on energy expenditure, weight gain, adipose tissue inflammation, and insulin resistance. The aim of this study is to compare and quantitate the BAT activity and BAT volume in RAGE null (Ager ^-/-) and wild type (WT) mice in response to HFD and low-fat diets (LFD) using in vivo PET imaging and ex vivo autoradiography (ARG) studies with [¹⁸F]FDG. Since BAT is not readily visible with [¹⁸F] FDG at room temperature based on our previous BAT imaging study³, all comparative studies were conducted under identical mild- cold stimulation conditions with [¹⁸F]FDG. Methods PET/CT imaging (Siemens, Inveon) of 4 groups of male C57/BL6 mice were compared (Ager ^-/--LFD, Ager ^-/--HFD, WT-LFD and WT-HFD). Before each scan, all mice were fasted for at least 4 h and then exposed to a cold-pack (0- 4degreeC) for 30 min. For each study, a dynamic PET scan was carried out for 1 h, followed by a CT scan (6 min) for the purpose of attenuation correction and co-registration. SUVmean (mean standard uptake values), SUVR (ratio of SUV BAT/muscle), and %ID/g for the interscapular BAT were determined. Mice were dissected after the scan to obtain the BAT tissue for further quantification of BAT via ex-vivo autoradiography (ARG) study. Results Preliminary results are summarized here: 1) For LFD mice, the uptake was significantly higher in WT-LFD (SUVR: 11.7) than that in Ager ^-/--LFD (SUVR: 2.63); 2) The SUVR were similar for both WT-HFD and Ager ^-/--HFD (2.53 and 2.44, respectively) (Fig.1). 3) Among Ager ^-/- types, the uptake was similar in both Ager ^-/--HFD (SUVR: 2.44) and Ager ^-/--LFD (SUVR: 2.63), and the %ID/g values were similar in both as well (Fig.1). These findings corroborate our previous findings on Ucp1²; that is, (1) Ucp1-mRNA transcripts in BAT are almost halved in Ager ^-/--LFD than in WTLFD; (2) in HFD, Ucp1-mRNA transcripts did not differ in BAT between WT and Ager ^-/- mice; (3) a bigger change was observed in Ucp1 between WT-LFD and WT-HFD, while a smaller change was seen between Ager ^-/--LFD and Ager ^-/- -HFD. Results from ex vivo ARG studies were consistent with the PET imaging data. Conclusion In WT mice, BAT activity was significantly reduced after HFD as compared to LFD; however, BAT activity was not affected by HFD in Ager ^-/- mice. The consistent findings between the current in vivo PET imaging study of BAT and our previous study of Ucp1 further support our hypothesis that RAGE may contribute to altered energy expenditure, and provide a protective effect against HFD by Ager deletion. [TABLE PRESENTED]

SCOPE: Nvarepsilon -Carboxymethyl-lysine (CML) is a prominent advanced glycation end-product which is not only found in vivo but also in food. It is known that a percentage of the dietary CML (dCML) is absorbed into the circulation and only partly excreted in the urine. Several studies have tried to measure how much dCML remains in tissues. However obstacles to interpreting the data have been found. METHODS AND RESULTS: A new protocol which discriminates dCML from native CML (nCML) has been developed. Three CML isotopes with different mass-to-charge ratios were used: nCML Nepsilon -carboxymethyl-L-lysine, dCML Nepsilon -[13 C]carboxy[13 C]methyl-L-lysine and internal standard Nepsilon -carboxymethyl-L-[4,4,5,5-2 H4 ]lysine. Wild-type (n = 7) and RAGE-/- (n = 8) mice were fed for 30 days with either a control, or a BSA-bound dCML-enriched diet. Organs were analyzed for nCML and dCML using liquid chromatography-tandem mass spectrometry. Mice exposed to dCML showed an accumulation in all tissues tested except fat. The rate of deposition was high (81-320 mugdCML /g dry matter) in kidneys, intestine, and lungs and low (<5 mug/g) in heart, muscle, and liver. This accumulation was not RAGE dependent. CONCLUSION: The kidney is not the only organ affected by the accumulation of dCML. Its high accumulation in other tissues and organs may also, however, have important physiological consequences.