Kate Petersen Shay

Alpha-lipoic acid as a dietary supplement: Molecular mechanisms and therapeutic potential

Alpha-lipoic acid (LA) has become a common ingredient in multivitamin formulas, anti-aging supplements, and even pet food. It is well-defined as a therapy for preventing diabetic polyneuropathies, and scavenges free radicals, chelates metals, and restores intracellular glutathione levels which otherwise decline with age. How do the biochemical properties of LA relate to its biological effects? Herein, we review the molecular mechanisms of LA discovered using cell and animal models, and the effects of LA on human subjects. Though LA has long been touted as an antioxidant, it has also been shown to improve glucose and ascorbate handling, increase eNOS activity, activate Phase II detoxification via the transcription factor Nrf2, and lower expression of MMP-9 and VCAM-1 through repression of NF-kappa B. LA and its reduced form, dihydrolipoic acid, may use their chemical properties as a redox couple to alter protein conformations by forming mixed disulfides. Beneficial effects are achieved with low micromolar levels of LA, suggesting that some of its therapeutic potential extends beyond the strict definition of an antioxidant. Current trials are investigating whether these beneficial properties of LA make it an appropriate treatment not just for diabetes, but also for the prevention of vascular disease, hypertension, and inflammation.

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.

Cap-independent Nrf2 translation is part of a lipoic acid-stimulated detoxification stress response.

Little is known about either the basal or stimulated homeostatic mechanisms regulating nuclear tenure of Nf-e2-related factor 2 (Nrf2), a transcription factor that mediates expression of over 200 detoxification genes. Our data show that stress-induced nuclear Nrf2 accumulation is largely from de novo protein synthesis, rather than translocation from a pre-existing cytoplasmic pool. HepG2 cells were used to monitor nuclear Nrf2 24h following treatment with the dithiol micronutrient (R)-α-lipoic acid (LA; 50μM), or vehicle. LA caused a ≥2.5-fold increase in nuclear Nrf2 within 1h. However, pretreating cells with cycloheximide (50μg/ml) inhibited LA-induced Nrf2 nuclear accumulation by 94%. Providing cells with the mTOR inhibitor, rapamycin, decreased basal Nrf2 levels by 84% after 4h, but LA overcame this inhibition. LA-mediated de novo protein translation was confirmed using HepG2 cells transfected with a bicistronic construct containing an internal ribosome entry sequence (IRES) for Nrf2, with significant (P<0.05) increase in IRES use under LA treatment. These results suggest that a dithiol stimulus mediates Nrf2 nuclear tenure via cap-independent protein translation. Thus, translational control of Nrf2 synthesis, rather than reliance solely on pre-existing protein, may mediate the rapid burst of Nrf2 nuclear accumulation following stress stimuli.

(R)-α-Lipoic Acid Treatment Restores Ceramide Balance in Aging Rat Cardiac Mitochondria

Inflammation results in heightened mitochondrial ceramide levels, which cause electron transport chain dysfunction, elevates reactive oxygen species, and increases apoptosis. As mitochondria in aged hearts also display many of these characteristics, we hypothesized that mitochondrial decay stems partly from an age-related ceramidosis that heretofore has not been recognized for the heart. Intact mitochondria or their purified inner membranes (IMM) were isolated from young (4-6 mo) and old (26-28 mo) rats and analyzed for ceramides by LC-MS/MS. Results showed that ceramide levels increased by 32% with age and three ceramide isoforms, found primarily in the IMM (e.g. C(16)-, C(18)-, and C(24:1)-ceramide), caused this increase. The ceramidosis may stem from enhanced hydrolysis of sphingomyelin, as neutral sphingomyelinase (nSMase) activity doubled with age but with no attendant change in ceramidase activity. Because (R)-α-lipoic acid (LA) improves many parameters of cardiac mitochondrial decay in aging and lowers ceramide levels in vascular endothelial cells, we hypothesized that LA may limit cardiac ceramidosis and thereby improve mitochondrial function. Feeding LA [0.2%, w/w] to old rats for two weeks prior to mitochondrial isolation reversed the age-associated decline in glutathione levels and concomitantly improved Complex IV activity. This improvement was associated with lower nSMase activity and a remediation in mitochondrial ceramide levels. In summary, LA treatment lowers ceramide levels to that seen in young rat heart mitochondria and restores Complex IV activity which otherwise declines with age.

Age-related loss of hepatic Nrf2 protein homeostasis: Potential role for heightened expression of miR-146a.

Nrf2 regulates the expression of numerous anti-oxidant, anti-inflammatory, and metabolic genes. We observed that, paradoxically, Nrf2 protein levels decline in the livers of aged rats despite the inflammatory environment evident in that organ. To examine the cause(s) of this loss, we investigated the age-related changes in Nrf2 protein homeostasis and activation in cultured hepatocytes from young (4-6 months) and old (24-28 months) Fischer 344 rats. While no age-dependent change in Nrf2 mRNA levels was observed (p>0.05), Nrf2 protein content, and the basal and anetholetrithione (A3T)-induced expression of Nrf2-dependent genes were attenuated with age. Conversely, overexpression of Nrf2 in cells from old animals reinstated gene induction. Treatment with A3T, along with bortezomib to inhibit degradation of existing protein, caused Nrf2 to accumulate significantly in cells from young animals (p<0.05), but not old, indicating a lack of new Nrf2 synthesis. We hypothesized that the loss of Nrf2 protein synthesis with age may partly stem from an age-related increase in microRNA inhibition of Nrf2 translation. Microarray analysis revealed that six microRNAs significantly increase >2-fold with age (p<0.05). One of these, miRNA-146a, is predicted to bind Nrf2 mRNA. Transfection of hepatocytes from young rats with a miRNA-146a mimic caused a 55% attenuation of Nrf2 translation that paralleled the age-related loss of Nrf2. Overall, these results provide novel insights for the age-related decline in Nrf2 and identify new targets to maintain Nrf2-dependent detoxification with age.

A rat primary hepatocyte culture model for aging studies.

The purpose of this protocol is to establish a primary hepatocyte culture system as a suitable model to examine age‐related changes in Phase II detoxication gene expression. Hepatocytes are isolated using a two‐step collagenase perfusion technique from young (3 to 6 months) and old (24 to 28 months) rats and placed in primary culture using collagen (Type I)‐coated plates as the extracellular matrix. A supplemented Williams E Medium is used as the medium. This culture system maintains hepatocyte viability from both young and old rats for ∼60 hr, as measured by lactate dehydrogenase activity, while also maintaining their respective phenotypes relative to Phase II detoxification. We thus conclude that a collagen‐based cell culture system is suitable to study age‐associated deficits in Nrf2/ARE‐mediated Phase II gene regulation provided that experiments can be conducted within 60 hr after cell isolation. Curr. Protoc. Toxicol. 37:14.7.1‐14.7.10.

Glutathione maintenance mitigates age-related susceptibility to redox cycling agents.

Isolated hepatocytes from young (4–6 mo) and old (24–26 mo) F344 rats were exposed to increasing concentrations of menadione, a vitamin K derivative and redox cycling agent, to determine whether the age-related decline in Nrf2-mediated detoxification defenses resulted in heightened susceptibility to xenobiotic insult. An LC50 for each age group was established, which showed that aging resulted in a nearly 2-fold increase in susceptibility to menadione (LC50 for young: 405 μM; LC50 for old: 275 μM). Examination of the known Nrf2-regulated pathways associated with menadione detoxification revealed, surprisingly, that NAD(P)H: quinone oxido-reductase 1 (NQO1) protein levels and activity were induced 9-fold and 4-fold with age, respectively (p=0.0019 and p=0.018; N=3), but glutathione peroxidase 4 (GPX4) declined by 70% (p=0.0043; N=3). These results indicate toxicity may stem from vulnerability to lipid peroxidation instead of inadequate reduction of menadione semi-quinone. Lipid peroxidation was 2-fold higher, and GSH declined by a 3-fold greater margin in old versus young rat cells given 300 µM menadione (p<0.05 and p≤0.01 respectively; N=3). We therefore provided 400 µM N-acetyl-cysteine (NAC) to hepatocytes from old rats before menadione exposure to alleviate limits in cysteine substrate availability for GSH synthesis during challenge. NAC pretreatment resulted in a >2-fold reduction in cell death, suggesting that the age-related increase in menadione susceptibility likely stems from attenuated GSH-dependent defenses. This data identifies cellular targets for intervention in order to limit age-related toxicological insults to menadione and potentially other redox cycling compounds.

Transcription Factor Nrf2: Examination of Nuclear Protein Levels by Immunoblotting and Promoter Response Element Binding by Chromatin Immunoprecipitation (ChIP)

Nuclear factor erythroid 2 (NF‐E2) related factor 2 (Nrf2) is a transcription factor that governs the expression of over a hundred so‐called phase II detoxification and antioxidant genes that are regulated through the antioxidant response element (ARE). Loss of Nrf2 activity has been implicated in cardiovascular disease, inflammation, aging, and cancer. Nrf2 is induced to accumulate in the nucleus when the cell encounters an oxidative stress, a fact that has been exploited experimentally to test the conditions under which ARE‐containing genes are expressed. The nuclear levels of Nrf2 give an indication of whether an experimental treatment results in Nrf2 localization and induction. mRNA levels of phase II genes may be measured as a follow‐up, but in order to show a direct link between nuclear Nrf2 accumulation and increases in gene expression, it is useful to show that Nrf2 binds to AREs in the promoters of target genes. The simplest way to do this is to employ a chromatin immunoprecipitation (ChIP) assay along with an examination of cellular Nrf2 levels by immunoblotting. Curr. Protoc. Toxicol. 45:17.13.1‐17.13.13.

Dietary resveratrol increases mid-life fecundity of female Nothobranchius guentheri

The decline of female reproductive function is an early phenotype of aging in humans, occurring only midway through the lifespan. Yet the number of women delaying pregnancy continues to rise in industrialized societies due to personal or socioeconomic circumstances, often resulting in subfertility or difficulty conceiving. There are few defined mechanisms associated with this etiology, and equally few effective therapies. To combat this problem, we used a novel emerging model, Nothobranchius guentheri, that recapitulates the age-associated spectrum of changes that adversely affect human fertility. We hypothesized that resveratrol (RSV), which activates SirT1 as an oxidative stress sensor and longevity assurance enzyme, would improve female fecundity in mid-life. RSV, a polyphenol found in grapes and red wine, has been touted as an anti-aging dietary supplement due to its ability to prolong both lifespan and health span. SirT1 is an NAD+ dependent histone deacetylase, whose activity is regulated by the nicotinamide to NAD+ salvage pathway, especially the rate-limiting enzyme NAMPT. We found that female N. guentheri fed 600μgRSV/g food into mid-life (~20weeks), beginning at sexual maturity, showed increased embryo production compared to those on Control diet. Furthermore, the RSV-fed fish had significantly increased NAMPT. This suggests that dietary RSV has a positive effect on female fertility, and that it may become an effective therapy to regulate sirtuin activity and combat reproductive senescence.