Lester Packer

Molecular Aspects of Lipoic Acid in the Prevention of Diabetes Complications

Alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid, are powerful antioxidants. LA scavenges hydroxyl radicals, hypochlorous acid, peroxynitrite, and singlet oxygen. Dihydrolipoic acid also scavenges superoxide and peroxyl radicals and can regenerate thioredoxin, vitamin C, and glutathione, which in turn can recycle vitamin E. There are several possible sources of oxidative stress in diabetes including glycation reactions, decompartmentalization of transition metals, and a shift in the reduced-oxygen status of the diabetic cells. Diabetics have increased levels of lipid hydroperoxides, DNA adducts, and protein carbonyls. Available data strongly suggest that LA, because of its antioxidant properties, is particularly suited to the prevention and/or treatment of diabetic complications that arise from an overproduction of reactive oxygen and nitrogen species. In addition to its antioxidant properties, LA increases glucose uptake through recruitment of the glucose transporter-4 to plasma membranes, a mechanism that is shared with insulin-stimulated glucose uptake. Further, recent trials have demonstrated that LA improves glucose disposal in patients with type II diabetes. In experimental and clinical studies, LA markedly reduced the symptoms of diabetic pathologies, including cataract formation, vascular damage, and polyneuropathy. To develop a better understanding of the preventative and therapeutic potentials of LA, much of the current interest is focused on elucidating its molecular mechanisms in redox dependent gene expression.

ESR and cell culture studies on free radical-scavenging and antioxidant activities of isoflavonoids.

Isoflavonoids are thought to be the biologically active components in soy that play a role in the prevention of coronary heart disease and breast and prostate cancer. Mechanisms to explain how isoflavonoids mediate beneficial effects have not yet been clearly established. This study was undertaken to investigate the free radical-scavenging and antioxidant activities of various structure-related isoflavonoids including genistein, daidzein, biochanin A, and genistin in a cell-free and an endothelial cell model system. Electron spin resonance spectroscopy and spin trapping techniques were applied to evaluate the ability of isoflavonoids to scavenge hydroxyl, superoxide, nitric oxide, diphenylpicrylhydrazyl, galvinoxyl, and lipid-derived radicals. All isoflavonoids tested had no significant scavenging effects on the aforementioned radicals in concentrations up to 1.0 mM. However, at a physiologically achievable concentration of 5 nM, both genistein and daidzein slightly increased intracellular-reduced glutathione levels approximately by 10 and 30%, respectively, in human endothelial cells, whereas cellular alpha-tocopherol and uric acid remained unchanged by the isoflavonoid treatments. Present data indicate that free radical-scavenging activities of the isoflavonoids tested probably do not substantially contribute to their antioxidant properties. The ability of genistein and daidzein to increase cellular GSH (reduced glutathione) might be important for their action in biological system.

Lipoic acid: energy metabolism and redox regulation of transcription and cell signaling.

The role of R-α-lipoic acid as a cofactor (lipoyllysine) in mitochondrial energy metabolism is well established. Lipoic acid non-covalently bound and exogenously administered to cells or supplemented in the diet is a potent modulator of the cell’s redox status. The diversity of beneficial effects of lipoic acid in a variety of tissues can be mechanistically viewed in terms of thiol/disulfide exchange reactions that modulate the environment’s redox and energy status. Lipoic acid-driven thiol/disulfide exchange reactions appear critical for the modulation of proteins involved in cell signaling and transcription factors. This review emphasizes the effects of lipoic acid on PI3K and AMPK signaling and related transcriptional pathways that are integrated by PGC-1α, a critical regulator of energy homoestasis. The effects of lipoic acid on the neuronal energy-redox axis are largely reviewed in terms of their outcomes for aging and age-related neurodegenerative diseases.

Gene expression profile of oxidant stress and neurodegeneration in transgenic mice deficient in α-tocopherol transfer protein

Alpha-tocopherol transfer protein (TTP) regulates the retention and secretion of alpha-tocopherol (alpha-T) by the liver. Deletion of the TTP gene (Ttpa) in mice results in systemic deficiency of alpha-T and neurological dysfunctions described in patients with mutated Ttpa. We have explored genome-wide changes in mRNAs from brain cortex and liver of Ttpa-deficient (Ttpa(-/-)) mice and wild-type (Ttpa(+/+)) mice. Selective inductions of genes regulated by antioxidant response elements were detected in Ttpa(-/-) livers compared to Ttpa(+/+) livers, suggesting increased oxidant stress in Ttpa(-/-) livers. The activation of cell proliferation pathways in Ttpa(-/-) livers was indicated by the induction of genes that encode growth factor-binding proteins, mitogen-activated protein kinase kinase 3, and apoptosis inhibitor 6. The induction of synuclein-alpha and repression of synuclein-beta genes was detected in Ttpa(-/-) cortex. This may predispose Ttpa(-/-) cortex to increased formation of synuclein-alpha aggregates and Lewy body, often associated with oxidant stress. Cortex of Ttpa(-/-) mice revealed repression of genes encoding synaptic proteins, protein kinase C family members, and myelin proteins. A 13-fold decrease in the expression of retinoic acid receptor-related orphan receptor-alpha mRNA predicts staggerer-like phenotype (ataxia and deficits of motor coordination) of Ttpa(-/-) mice. The repression of specific genes that determine synaptic plasticity and neuronal development may account for suppressed electrophysiological activities of cortex and impaired behavior in Ttpa(-/-) mice.

Antioxidants and the Response of Skin to Oxidative Stress: Vitamin E as a Key Indicator

As the outermost barrier of the body, the skin is directly and frequently exposed to a prooxidative environment, including solar UVA and UVB radiation, and air pollution. The skin is equipped with an elaborate system of antioxidant substances and enzymes that includes a network of redox active antioxidants. Among these, vitamin E has been identified as the predominant antioxidant both in murine and human skin and shows a characteristic gradient with lower levels towards the outer stratum corneum layers. Skin exposure to UV and ozon alone and in combination resulted in a significant potentiation of the UV-induced vitamin E depletion. Oxidants and antioxidants play an important role in maintaining a balance between free radicals produced by metabolism or derived from environmental sources. Cellular antioxidants may change their redox state, be targeted for destruction, regulate oxidative process involved in signal transduction, affect gene expression and pathways of cell proliferation and death. Here we provide an overview of the antioxidant system with a special relevance to skin.

R-α-Lipoic acid and acetyl-l-carnitine complementarily promote mitochondrial biogenesis in murine 3T3-L1 adipocytes

Aims/hypothesisThe aim of the study was to address the importance of mitochondrial function in insulin resistance and type 2 diabetes, and also to identify effective agents for ameliorating insulin resistance in type 2 diabetes. We examined the effect of two mitochondrial nutrients, R-α-lipoic acid (LA) and acetyl-l-carnitine (ALC), as well as their combined effect, on mitochondrial biogenesis in 3T3-L1 adipocytes.MethodsMitochondrial mass and oxygen consumption were determined in 3T3-L1 adipocytes cultured in the presence of LA and/or ALC for 24xa0h. Mitochondrial DNA and mRNA from peroxisome proliferator-activated receptor gamma and alpha (Pparg and Ppara) and carnitine palmitoyl transferase 1a (Cpt1a), as well as several transcription factors involved in mitochondrial biogenesis, were evaluated by real-time PCR or electrophoretic mobility shift (EMSA) assay. Mitochondrial complexes proteins were measured by western blot and fatty acid oxidation was measured by quantifying CO2 production from [1-14C]palmitate.ResultsTreatments with the combination of LA and ALC at concentrations of 0.1, 1 and 10xa0μmol/l for 24xa0h significantly increased mitochondrial mass, expression of mitochondrial DNA, mitochondrial complexes, oxygen consumption and fatty acid oxidation in 3T3L1 adipocytes. These changes were accompanied by an increase in expression of Pparg, Ppara and Cpt1a mRNA, as well as increased expression of peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha (Ppargc1a), mitochondrial transcription factor A (Tfam) and nuclear respiratory factors 1 and 2 (Nrf1 and Nrf2). However, the treatments with LA or ALC alone at the same concentrations showed little effect on mitochondrial function and biogenesis.Conclusions/interpretationWe conclude that the combination of LA and ALC may act as PPARG/A dual ligands to complementarily promote mitochondrial synthesis and adipocyte metabolism.

Plasma C-reactive protein concentrations in active and passive smokers: Influence of antioxidant supplementation

Objective: C-reactive protein (CRP) may directly affect the progression of atherosclerosis, and therefore, may be a target for reducing disease risk. The objective was to determine whether antioxidant supplementation reduces plasma CRP in active and passive smokers. Design: Randomized, double-blind, placebo-controlled, parallel group trial with 2 months exposure to study supplements. Setting: Berkeley and Oakland, California. Subjects: Healthy adult men and women, consuming <4 daily servings of fruits and vegetables, and who were actively or passively exposed to cigarette smoke. Analysis was limited to participants with detectable baseline CRP concentrations and no evidence of inflammation associated with acute illness at baseline or follow-up as reflected in CRP elevations (≥10.0 mg/L). A total of 1393 individuals were screened, 216 randomized, 203 completed the study, and 160 were included in the analysis. Interventions: Participants were randomized to receive a placebo or vitamin C (515 mg/day) or antioxidant mixture (per day: 515 mg vitamin C, 371 mg α-tocopherol, 171 mg γ-tocopherol, 252 mg mixed tocotrienols, and 95 mg α-lipoic acid). Measures of Outcome: Change in plasma CRP concentration. Results: Vitamin C supplementation yielded a 24.0% reduction (95% confidence interval, −38.9% to −5.5%, p = 0.036 compared to control) in plasma CRP, whereas the antioxidant mixture and placebo produced a nonsignificant 4.7% reduction (−23.9% to 19.3%) and 4.3% increase (−15.1% to 28.2%), respectively. Results were adjusted for baseline body mass index and CRP concentrations. Conclusions: Plasma CRP itself may serve as a potential target for reducing the risk of atherosclerosis, and antioxidants, including vitamin C, should be investigated further to confirm their CRP-lowering and anti-inflammatory effects.

Ozone exposure activates oxidative stress responses in murine skin.

Ozone (O(3)) is among the most reactive environmental oxidant to which skin is exposed. O(3) exposure has previously been shown to induce antioxidant depletion as well as lipid and protein oxidation in the outermost skin layer, the stratum corneum (SC), but little is known regarding the potential effects of O(3) on the skin epidermis and dermis. To evaluate such skin responses to O(3), SKH-1 hairless mice were exposed for 2 h to 8.0 ppm O(3) or to ambient air. O(3) exposure caused a significant increase in skin carbonyls (28%) compared to the skin of air exposed control animals. An evident increase in 4-hydroxynonenal-protein adducts was detected after O(3) exposure. O(3) exposure caused a rapid up-regulation of HSP27 (20-fold), and more delayed induction of HSP70 (2.8-fold) and heme oxygenase-1 (5-fold). O(3) exposure also led to the induction of nitric oxide synthase (iNOS) 6-12 h following O(3) exposure. We conclude that skin exposure to high levels of O(3) not only affects antioxidant levels and oxidation markers in the SC, but also induces stress responses in the active layers of the skin, most likely by indirect mechanisms, since it is unlikely that O(3) itself penetrates the protective SC layers.

Oxidants and antioxidants revisited. New concepts of oxidative stress

Through evolution, oxygen, itself a free radical, was chosen as the terminal electron acceptor for respiration. The two unpaired electrons of oxygen spin in the same direction; thus, oxygen is a biradical. Other oxygen-derived free radicals, such as superoxide anion or hydroxyl radicals, formed during metabolism or by ionizing radiation are stronger oxidants , i.e. endowed with a higher chemical reactivity. Oxygenderived free radicals are generated during metabolism and energy production in the body and are involved in the regulation of signal transduction and gene expression, activation of receptors and nuclear transcription factors, oxidative damage to cell components, the antimicrobial and cytotoxic action inherent in immune system cells, as well as in ageing and agerelated degenerative diseases. Conversely, the cell conserves antioxidant mechanisms to counteract the effect of oxidants; these antioxidants may remove oxidants either in a highly specific manner as for example by superoxide dismutases or in a less specific manner (for example, small molecules such as vitamin E, vitamin C and glutathione).

Inhibition mechanisms of bioflavonoids extracted from the bark of Pinus maritima on the expression of proinflammatory cytokines.

Abstract: The effect of bioflavonoids extracted from the bark of Pinus maritima, Pycnogenol (PYC), on gene expression of the proinflammatory cytokines interleukin‐1β (IL‐1β) and interleukin‐2 (IL‐2) were investigated in RAW 264.7 cells and Jurkat E6.1 cells, respectively. PYC exerted strong scavenging activities against reactive oxygen species (ROS) generated by H2O2 in RAW 264.7. In situ ELISA, immunoblot analysis, and competitive RT‐PCR demonstrated that pretreatment of LPS‐stimulated RAW 264.7 cells with PYC dose‐dependently reduced both the production of IL‐1β and its mRNA levels. Furthermore, in the same cells, PYC blocked the activation of nuclear factor κB (NF‐κB) and activator protein‐1 (AP‐1), two major transcription factors centrally involved in IL‐1β gene expression. Concordantly, pretreatment of the cells with PYC abolished the LPS‐induced IκB degradation. We also investigated the effect of PYC on IL‐2 gene expression in phorbol 12‐myristate 13acetate plus ionomycin (PMA/Io)‐stimulated human T‐cell line Jurkat E6.1. PYC inhibited the PMA/Io‐induced IL‐2 mRNA expression. However, as demonstrated in a reporter gene assay system, the mechanism of IL‐2 gene transcriptional regulation by PYC was different from the regulation of IL‐1β. PYC inhibited both NF‐AT and AP‐1 chloramphenicol acetyltransferase (CAT) activities in transiently transfected Jurkat E6.1, but not NF‐κB CAT activity. We also found that PYC can destabilize PMA/Io‐induced IL‐2 mRNA by posttranscriptional regulation. All these results suggest that bioflavonids can be useful therapeutic agents in treating many inflammatory, autoimmune, and cardiovascular diseases based on its diverse action mechanisms.