Tory M. Hagen

Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress.

alpha-Lipoic acid (LA), a naturally occurring dithiol compound, has long been known as an essential cofactor for mitochondrial bioenergetic enzymes. Aside from its enzymatic role, in vitro and in vivo studies suggest that LA also acts as a powerful micronutrient with diverse pharmacologic and antioxidant properties. Pharmacologically, LA improves glycemic control, polyneuropathies associated with diabetes mellitus, and effectively mitigates toxicities associated with heavy metal poisoning. As an antioxidant, LA directly terminates free radicals, chelates transition metal ions (e.g. iron and copper), increases cytosolic glutathione and vitamin C levels and prevents toxicities associated with their loss. These diverse actions suggest that LA acts by multiple mechanisms both physiologically and pharmacologically, many of which are only now being explored. Herein, we review the known biochemical properties of LA with particular reference to how LA may be an effective agent to ameliorate certain pathophysiologies of many chronic diseases.

Glutathione Metabolism during Aging and in Alzheimer Disease

Abstract: The concentration of glutathione (GSH), the most abundant intracellular nonprotein thiol and important antioxidant, declines with age and in some age‐related diseases. The underlying mechanism, however, is not clear. The previous studies from our laboratory showed that the age‐dependent decline in GSH content in Fisher 344 rats was associated with a downregulation of glutamate cysteine ligase (GCL), the rate‐limiting enzyme in de novo GSH synthesis. Our recent studies further indicated that the activity and mRNA content of glutathione synthase (GS), which catalyzes the second reaction in de novo GSH synthesis, were also decreased with age in some tissues. No age‐associated change was observed in glutathione reductase or γ‐glutamyl transpeptidase activities. Also, although GSH content declined with age in both male and female mice, male mice experienced more dramatic age‐associated decline in many tissues/organs than female mice. Furthermore, we found that GSH content was significantly decreased in the red blood cells from male Alzheimer disease patients, which was associated with decreases in GCL and GS activities. Finally, we showed that estrogen increased GSH content, GS and GR activities, and GCL gene expression in the liver of both male and female mice. Taken together, our results suggest that (1) GCL plays a critical role in maintaining GSH homeostasis under both physiological and pathological conditions; (2) decreased GSH content may be involved in AD pathology in humans; and (3) estrogen increases GSH content in mice by multiple mechanisms.

Mitochondrial Decay in the Aging Rat Heart

Mitochondrial decay has been postulated to be a significant underlying part of the aging process. Decline in mitochondrial function may lead to cellular energy deficits, especially in times of greater energy demand, and compromise vital ATP‐dependent cellular operations, including detoxification, repair systems, DNA replication, and osmotic balance. Mitochondrial decay may also lead to enhanced oxidant production and thus render the cell more prone to oxidative insult. In particular, the heart may be especially susceptible to mitochondrial dysfunction due to myocardial dependency on β‐oxidation of fatty acids for energy and the postmitotic nature of cardiac myocytes, which would allow for greater accumulation of mitochondrial mutations and deletions. Thus, maintenance of mitochondrial function may be important to maintain overall myocardial function. Herein, we review the major age‐related changes that occur to mitochondria in the aging heart and the evidence that two such supplements, acetyl‐l‐carnitine (ALCAR) and (R)‐α‐lipoic acid, may improve myocardial bioenergetics and lower the increased oxidative stress associated with aging. We and others have shown that feeding old rats ALCAR reverses the age‐related decline in carnitine levels and improves mitochondrial β‐oxidation in a number of tissues studied. However, ALCAR supplementation does not appear to reverse the age‐related decline in cardiac antioxidant status and thus may not substantially alter indices of oxidative stress. Lipoic acid, a potent thiol antioxidant and mitochondrial metabolite, appears to increase low molecular weight antioxidant status and thereby decreases age‐associated oxidative insult. Thus, ALCAR along with lipoic acid may be effective supplemental regimens to maintain myocardial function.

Is α‐lipoic acid a scavenger of reactive oxygen species in vivo? Evidence for its initiation of stress signaling pathways that promote endogenous antioxidant capacity

The chemical reduction and oxidation (redox) properties of α‐lipoic acid (LA) suggest that it may have potent antioxidant potential. A significant number of studies now show that LA and its reduced form, dihydrolipoic acid (DHLA), directly scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) species and protect cells against a host of insults where oxidative stress is part of the underlying etiology. However, owing to its limited and transient accumulation in tissues following oral intake, the efficacy of nonprotein‐bound LA to function as a physiological antioxidant has been questioned. Herein, we review the evidence that the micronutrient functions of LA may be more as an effector of important cellular stress response pathways that ultimately influence endogenous cellular antioxidant levels and reduce proinflammatory mechanisms. This would promote a sustained improvement in cellular resistance to pathologies where oxidative stress is involved, which would not be forthcoming if LA solely acted as a transient ROS scavenger.

Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress

Cardiac mitochondria are composed of two distinct subpopulations: one beneath the sarcolemma (subsarcolemmal mitochondria: SSM), and another along the myofilaments (interfibrillary mitochondria: IFM). Previous studies suggest a preferential loss of IFM function with age; however, the age-related changes in oxidative stress in these mitochondrial subpopulations have not been examined. To this end, the changes in mitochondrial antioxidant capacity, oxidant output, and oxidative damage to Complex IV in IFM and SSM from young and old rats were studied. Results show no apparent differences in any parameters examined between IFM and SSM from young rats. However, relative to young, only IFM from old rats had a significantly higher rate of oxidant production and a decline in mitochondrial ascorbate levels and GSH redox status. The age-related decline in mitochondrial antioxidant capacity in IFM was accompanied by a marked loss in glutaredoxin and GSSG reductase activities, suggesting a diminished reductive capacity in IFM with age. Moreover, the loss in Complex IV activity was limited to the IFM of old rats, which was accompanied by a 4-fold increase in 4-hydroxynonenal-modified Complex IV. Thus, mitochondrial decay is not uniform and further indicates that myofibrils may be uniquely under oxidative stress in the aging heart.

Altered Mitochondrial Membrane Potential, Mass, and Morphology in the Mononuclear Cells of Humans with Type 2 Diabetes

Mitochondrial membrane hyperpolarization and morphologic changes are important in inflammatory cell activation. Despite the pathophysiologic relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is available currently. The purpose of this study was to define and validate reproducible methods for measuring relevant mitochondrial perturbations and to determine whether these methods could discern mitochondrial perturbations in type 2 diabetes mellitus (T2DM), which is a condition associated with altered mitochondrial homeostasis. We employed 5,5,6,6-tetrachloro-1,13,3-tetraethylbenzamidazol-carboncyanine (JC-1) to estimate mitochondrial membrane potential (Psi(m)) and acridine orange 10-nonyl bromide (NAO) to assess mitochondrial mass in human mononuclear cells isolated from blood. Both assays were reproducible. We validated our findings by electron microscopy and pharmacologic manipulation of Psi(m). We measured JC-1 and NAO fluorescence in the mononuclear cells of 27 T2DM patients and 32 controls. Mitochondria were more polarized (P = 0.02) and mitochondrial mass was lower in T2DM (P = 0.008). Electron microscopy demonstrated diabetic mitochondria were smaller, were more spherical, and occupied less cellular area in T2DM. Mitochondrial superoxide production was higher in T2DM (P = 0.01). Valid and reproducible measurements of mitochondrial homeostasis can be made in human mononuclear cells using these fluorophores. Furthermore, potentially clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected.

Age-related changes in endothelial nitric oxide synthase phosphorylation and nitric oxide dependent vasodilation: evidence for a novel mechanism involving sphingomyelinase and ceramide-activated phosphatase 2A

Aging is the single most important risk factor for cardiovascular diseases (CVD), which are the leading cause of morbidity and mortality in the elderly. The underlying etiologies that elevate CVD risk are unknown, but increased vessel rigidity appears to be a major hallmark of cardiovascular aging. We hypothesized that post‐translational signaling pathways become disrupted with age and adversely affect endothelial nitric oxide synthase (eNOS) activity and endothelial‐derived nitric oxide (NO) production. Using arterial vessels and isolated endothelia from old (33‐month) vs. young (3‐month) F344XBrN rats, we show a loss of vasomotor function with age that is attributable to a decline in eNOS activity and NO bioavailability. An altered eNOS phosphorylation pattern consistent with its inactivation was observed: phosphorylation at the inhibitory threonine 494 site increased while phosphorylation at the activating serine 1176 site declined by 50%. Loss of phosphorylation on serine 1176 was related to higher ceramide‐activated protein phosphatase 2 A activity, which was driven by a 125% increase in ceramide in aged endothelia. Elevated ceramide levels were attributable to chronic activation of neutral sphingomyelinases without a concomitant increase in ceramidase activity. This imbalance may stem from an observed 33% decline in endothelial glutathione (GSH) levels, a loss known to differentially induce neutral sphingomyelinases. Pretreating aged vessel rings with the neutral sphingomyelinase inhibitor, GW4869, significantly reversed the age‐dependent loss of vasomotor function. Taken together, these results suggest a novel mechanism that at least partly explains the persistent loss of eNOS activity and endothelial‐derived NO availability in aging conduit arteries.

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

Abstract 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)-α-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.

Supercomplexes of the Mitochondrial Electron Transport Chain Decline in the Aging Rat Heart

Accumulation of mitochondrial electron transport chain (ETC) defects is a recognized hallmark of the age-associated decline in cardiac bioenergetics; however, the molecular events involved are only poorly understood. In the present work, we hypothesized that age-related ETC deterioration stemmed partly from disassociation of large solid-state macromolecular assemblies termed supercomplexes. Mitochondrial proteins from young and old rat hearts were separated by blue native-PAGE, protein bands analyzed by LC-MALDI-MS/MS, and protein levels quantified by densitometry. Results showed that supercomplexes comprised of various stoichiometries of complexes I, III and IV were observed, and declined significantly (p<0.05, n=4) with age. Supercomplexes displaying the highest molecular masses were the most severely affected. Considering that certain diseases (e.g. Barth Syndrome) display similar supercomplex destabilization as our results for aging, the deterioration in ETC supercomplexes may be an important underlying factor for both impaired mitochondrial function and loss of cardiac bioenergetics with age.

Effect of Combined Treatment with Alpha Lipoic Acid and Acetyl- L-Carnitine on Vascular Function and Blood Pressure in Coronary Artery Disease Patients

Mitochondria produce reactive oxygen species that may contribute to vascular dysfunction. α‐Lipoic acid and acetyl‐L‐carnitine reduce oxidative stress and improve mitochondrial function. In a double‐blind crossover study, the authors examined the effects of combined α‐lipoic acid/acetyl‐L‐carnitine treatment and placebo (8 weeks per treatment) on vasodilator function and blood pressure in 36 subjects with coronary artery disease. Active treatment increased brachial artery diameter by 2.3% (P=.008), consistent with reduced arterial tone. Active treatment tended to decrease systolic blood pressure for the whole group (P=.07) and had a significant effect in the subgroup with blood pressure above the median (151±20 to 142±18 mm Hg; P=.03) and in the subgroup with the metabolic syndrome (139±21 to 130±18 mm Hg; P=.03). Thus, mitochondrial dysfunction may contribute to the regulation of blood pressure and vascular tone. Further studies are needed to confirm these findings and determine the clinical utility of α‐lipoic acid/acetyl‐L‐carnitine as antihypertensive therapy.