Background: Lipid peroxidation generates unsaturated aldehydes that form conjugates with histidyl dipeptides. Results: Carnosine-aldehyde conjugates form covalent adducts with proteins and are reduced by aldose reductase. Conclusion: Detoxification of carnosine-aldehyde by aldose reductase prevents protein carnosinylation. Significance: Aldose reductase prevents tissue injury due to aldehyde-carnosine conjugates.

A well-regulated redox state is essential for normal physiological function and cellular metabolism. In most eukaryotic cells, protein cysteine thiols are most sensitive to fluctuations in the cellular redox state. Under normal physiological conditions, the cytosol has a highly reducing environment, which is due to high levels of reduced glutathione and complex system of redox enzymes that maintain glutathione in the reduced state. The reducing environment of the cytosol maintains most protein thiols in the reduced state; although some non-exposed cysteine could be present as disulfides. Upon physiological increase in cellular oxidants, such as due to growth factors, cytokines and thiol-disulfide exchange reactions, specific proteins could act as redox switches that regulate the conformation and activity of different proteins. This reversible post translational modification enables redox-sensitive dynamic changes in cell signaling and function. Physiological oxidative stress could lead to the formation of sulfenic acids, which are usually intermediate states of thiol oxidation that are converted to higher order oxidation states, intramolecular disulfides or mixed disulfides with glutathione. Such glutathiolation reactions have been found to regulate the function of several proteins involved in intracellular metabolism, signal transduction and cell structure. Excessive oxidative stress results in indiscriminate and irreversible oxidation of protein thiols, depletion of glutathione and cell death. Further elucidation of the relationship between changes in cell redox and thiol reactivity could provide a better understanding of how redox changes regulate cell function and how disruption of these relationships lead to tissue injury and dysfunction and the development of chronic diseases such as cancer and cardiovascular disease.

OBJECTIVETo examine the role of aldo-keto reductases (AKRs) in the cardiovascular metabolism of the precursors of advanced glycation end products (AGEs).RESEARCH DESIGN AND METHODSSteady-state kinetic parameters of AKRs with AGE precursors were determined using recombinant proteins expressed in bacteria. Metabolism of methylglyoxal and AGE accumulation were studied in human umbilical vein endothelial cells (HUVECs) and C57 wild-type, akr1b3 (aldose reductase)-null, cardiospecific-akr1b4 (rat aldose reductase), and akr1b8 (FR-1)-transgenic mice. AGE accumulation and atherosclerotic lesions were studied 12 weeks after streptozotocin treatment of C57, akr1b3-null, and apoE- and akr1b3-apoE–null mice.RESULTSHigher levels of AGEs were generated in the cytosol than at the external surface of HUVECs cultured in high glucose, indicating that intracellular metabolism may be an important regulator of AGE accumulation and toxicity. In vitro, AKR 1A and 1B catalyzed the reduction of AGE precursors, whereas AKR1C, AKR6, and AKR7 were relatively ineffective. Highest catalytic efficiency was observed with AKR1B1. Acetol formation in methylglyoxal-treated HUVECs was prevented by the aldose reductase inhibitor sorbinil. Acetol was generated in hearts perfused with methylglyoxal, and its formation was increased in akr1b4- or akr1b8-transgenic mice. Reduction of AGE precursors was diminished in hearts from akr1b3-null mice. Diabetic akr1b3-null mice accumulated more AGEs in the plasma and the heart than wild-type mice, and deletion of akr1b3 increased AGE accumulation and atherosclerotic lesion formation in apoE-null mice.CONCLUSIONSAldose reductase–catalyzed reduction is an important pathway in the endothelial and cardiac metabolism of AGE precursors, and it prevents AGE accumulation and atherosclerotic lesion formation.

Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone ('core' aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte-endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C(16:0-20:4) phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C(16:0-20:4) phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes.

Objectives Acrolein is a toxic chemical present in tobacco, wood and coal smoke as well as automobile exhaust. Because exposure to these pollutants is associated with an increase in cardiovascular disease risk, we studied the effects of acrolein on Flk-1+/Sca-1+ cells that are involved in vascular repair. Methods and Results In adult male C57BL/6 mice, inhalation of acrolein (1ppm, 6h/day, 4 days or 5ppm for 2 or 6h) led to the formation of protein-acrolein adducts in the bone marrow and diminished levels of plasma NOx and circulating Flk-1+/Sca-1+ but not Sca-1+ only cells. Acrolein exposure increased the number of apoptotic Flk-1+/Sca1+ cells in circulation, and increased bone marrow-derived cells with endothelial characteristics (Dil-acLDL/UE-lectin and Flk-1+/Sca-1+) in culture. Deficits in the circulating levels of Flk-1+/Sca-1+ cells were reversed after 7 days of recovery in acrolein-free air. Exposure to acrolein blocked VEGF/AMD3100-stimulated mobilization of Flk-1+/Sca-1+ but not Sca-1+ only cells and prevented VEGF-induced phosphorylation of Akt and eNOS in the aorta. Conclusions Inhalation of acrolein increases apoptosis and suppresses the circulating levels of Flk-1+/Sca-1+ cells, while increasing these cells in the bone marrow and preventing their mobilization by VEGF. Exposure to acrolein-rich pollutants could impair vascular repair capacity.

Key pointsr Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that β-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase.r The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of β-alanine, which is in turn controlled by degradation of β-alanine in liver and kidney.r Chronic oral β-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the β-alanine supplementation protocol is so inefficient, by demonstrating that exogenous β-alanine can be effectively routed toward oxidation.Abstract The metabolic fate of orally ingested β-alanine is largely unknown. Chronic β-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (β-alanyl-L-histidine) in muscle. However, only a small fraction (3-6%) of the ingested β-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two β-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate β-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on β-alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3-fold increase in circulating β-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating β-alanine levels, which are suppressed by hepatic and renal β-alanine transamination upon oral β-alanine intake.

Objective Atherosclerotic lesions are associated with the accumulation of reactive aldehydes derived from oxidized lipids. Although inhibition of aldehyde metabolism has been shown to exacerbate atherosclerosis and enhance the accumulation of aldehyde-modified proteins in atherosclerotic plaques, no therapeutic interventions have been devised to prevent aldehyde accumulation in atherosclerotic lesions. Approach and Results We examined the efficacy of carnosine, a naturally occurring β-alanyl-histidine dipeptide in preventing aldehyde toxicity and atherogenesis in apoE-null mice. In vitro, carnosine reacted rapidly with lipid peroxidation-derived unsaturated aldehydes. Gas chromatography mass-spectrometry analysis showed that carnosine inhibits the formation of free aldehydes - HNE and malonaldialdehyde in Cu2+-oxidized LDL. Preloading bone marrow-derived macrophages with cell-permeable carnosine analogs reduced HNE-induced apoptosis. Oral supplementation with octyl-D-carnosine decreased atherosclerotic lesion formation in aortic valves of apoE-null mice and attenuated the accumulation of protein-acrolein, protein-HHE and protein-HNE adducts in atherosclerotic lesions, while urinary excretion of aldehydes as carnosine conjugates was increased. Conclusions The results of this study suggest that carnosine inhibits atherogenesis by facilitating aldehyde removal from atherosclerotic lesions. Endogenous levels of carnosine may be important determinants of atherosclerotic lesion formation and treatment with carnosine or related peptides could be a useful therapy for the prevention or the treatment of atherosclerosis.

Preclinical studies of animals with risk factors, and how those risk factors contribute to the development of cardiovascular disease and cardiac dysfunction, are clearly needed. One such approach is to feed mice a diet rich in fat (i.e. 60%). Here, we determined whether a high fat diet was sufficient to induce cardiac dysfunction in mice. We subjected mice to two different high fat diets (lard or milk as fat source) and followed them for over six months and found no significant decrement in cardiac function (via echocardiography), despite robust adiposity and impaired glucose disposal. We next determined whether antecedent and concomitant exposure to high fat diet (lard) altered the murine heart’s response to infarct-induced heart failure; high fat feeding during, or before and during, heart failure did not significantly exacerbate cardiac dysfunction. Given the lack of a robust effect on cardiac dysfunction with high fat feeding, we then examined a commonly used mouse model of overt diabetes, hyperglycemia, and obesity (db/db mice). db/db mice (or STZ treated wild-type mice) subjected to pressure overload exhibited no significant exacerbation of cardiac dysfunction; however, ischemia-reperfusion injury significantly depressed cardiac function in db/db mice compared to their non-diabetic littermates. Thus, we were able to document a negative influence of a risk factor in a relevant cardiovascular disease model; however, this did not involve exposure to a high fat diet. High fat diet, obesity, or hyperglycemia does not necessarily induce cardiac dysfunction in mice. Although many investigators use such diabetes/obesity models to understand cardiac defects related to risk factors, this study, along with those from several other groups, serves as a cautionary note regarding the use of murine models of diabetes and obesity in the context of heart failure.