Acetyl-L-carnitine supplementation reverses the age-related decline in carnitine palmitoyltransferase 1 (CPT1) activity in interfibrillar mitochondria without changing the L-carnitine content in the rat heart.

The aging heart displays a loss of bioenergetic reserve capacity partially mediated through lower fatty acid utilization. We investigated whether the age-related impairment of cardiac fatty acid catabolism occurs, at least partially, through diminished levels of L-carnitine, which would adversely affect carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme for fatty acyl-CoA uptake into mitochondria for ß-oxidation. Old (24-28 mos) Fischer 344 rats were fed±acetyl-L-carnitine (ALCAR; 1.5% [w/v]) for up to four weeks prior to sacrifice and isolation of cardiac interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria. IFM displayed a 28% (p<0.05) age-related loss of CPT1 activity, which correlated with a decline (41%, p<0.05) in palmitoyl-CoA-driven state 3 respiration. Interestingly, SSM had preserved enzyme function and efficiently utilized palmitate. Analysis of IFM CPT1 kinetics showed both diminished V(max) and K(m) (60% and 49% respectively, p<0.05) when palmitoyl-CoA was the substrate. However, no age-related changes in enzyme kinetics were evident with respect to L-carnitine. ALCAR supplementation restored CPT1 activity in heart IFM, but not apparently through remediation of L-carnitine levels. Rather, ALCAR influenced enzyme activity over time, potentially by modulating conditions in the aging heart that ultimately affect palmitoyl-CoA binding and CPT1 kinetics.

Assessment of endoplasmic reticulum glutathione redox status is confounded by extensive ex vivo oxidation.

Glutathione (GSH) and glutathione disulfide (GSSG) form the principal thiol redox couple in the endoplasmic reticulum (ER); however, few studies have attempted to quantify GSH redox status in this organelle. To address this gap, GSH and GSSG levels and the extent of protein glutathionylation were analyzed in rat liver microsomes. Because of the likelihood of artifactual GSH oxidation during the lengthy microsomal isolation procedure, iodoacetic acid (IAA) was used to preserve the physiological thiol redox state. Non-IAA-treated microsomes exhibited a GSH:GSSG ratio between 0.7:1 to 1.2:1 compared to IAA-treated microsomes that yielded a GSH:GSSG redox ratio between 4.7:1 and 5.5:1. The majority of artifactual oxidation occurred within the first 2 h of isolation. Thus, the ER GSH redox ratio is subject to extensive ex vivo oxidation and when controlled, the microsomal GSH redox state is significantly higher than previously believed. Moreover, in vitro studies showed that PDI reductase activity was markedly increased at this higher thiol redox ratio versus previously reported GSH:GSSG ratios for the ER. Lastly, we show by both HPLC and Western blot analysis that ER proteins are highly resistant to glutathionylation. Together, these results may necessitate a re-evaluation of GSH and its role in ER function.

Dietary supplementation with (R)-alpha-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex.

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24-28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-alpha-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.

Age-related compensatory activation of pyruvate dehydrogenase complex in rat heart.

Mitochondrial uptake and beta-oxidation of long-chain fatty acids are markedly impaired in the aging rat heart. While these alterations would be expected to adversely affect overall pyridine nucleotides, NADH levels do not change significantly with age. This conundrum suggests that specific compensatory mechanisms occur in the aging heart. The comparison of cardiac pyruvate dehydrogenase complex (PDC) kinetics in 4- and 24- to 28-month-old F344 rats revealed a 60% significant increase in V(max) with no change in PDC expression, and a 1.6-fold decrease in the Michaelis constant (K(m)) in old compared to young rats. The observed kinetic adjustments were selective to PDC, as neither the V(max) nor K(m) of citrate synthase changed with age. PDC kinase-4 mRNA levels decreased by 57% in old vs young rat hearts and correlated with a 45% decrease in PDC phosphorylation. We conclude that PDC from old rat hearts catabolizes pyruvate more efficiently due to an adaptive change in phosphorylation.

Age-related increase in 4-hydroxynonenal adduction to rat heart alpha-ketoglutarate dehydrogenase does not cause loss of its catalytic activity.

4-hydroxynonenal (HNE), a product of omega-6 polyunsaturated fatty acid peroxidation, impairs mitochondrial respiration in vitro by adducting the alpha-ketoglutarate dehydrogenase complex (KGDC) and inhibiting its activity. The present study seeks to define whether aging increases HNE adduction to rat heart KGDC, and whether such adduction impacts KGDC activity. We found that hearts from old rats exhibit significantly (p< or =0.01) higher HNE-modified mitochondrial proteins when compared with those from young rats. Among these proteins, dihydrolipoamide succinyltransferase, the E2k component of KGDC, was most markedly modified (p< or =0.01) by HNE with age. As opposed to that seen in vitro, no significant change in electrophoretic mobility or impairment in enzyme activity was observed. On the contrary, KGDC activity increased onefold (p< or =0.01) in old rats, suggesting that the aging myocardium is not affected by HNE adduction or compensates for such damage. Further analysis revealed that heightened KGDC activity was not due to increased protein content or gene expression, but correlates with a lower Km for alpha-ketoglutarate. Thus, contrary to that observed in vitro, the measurement of HNE-KGDC adduct in rat heart is more relevant as a marker of age-related protein oxidation than a factor of mitochondrial dysfunction.