Jiankang Liu

The development of type 2 diabetes is accompanied by decreased immune function and the mechanisms are unclear. We hypothesize that oxidative damage and mitochondrial dysfunction may play an important role in the immune dysfunction in diabetes. In the present study, we investigated this hypothesis in diabetic Goto-Kakizaki rats by treatment with a combination of four mitochondrial-targeting nutrients, namely, R-alpha-lipoic acid, acetyl-L-carnitine, nicotinamide and biotin. We first studied the effects of the combination of these four nutrients on immune function by examining cell proliferation in immune organs (spleen and thymus) and immunomodulating factors in the plasma. We then examined, in the plasma and thymus, oxidative damage biomarkers, including lipid peroxidation, protein oxidation, reactive oxygen species, calcium and antioxidant defence systems, mitochondrial potential and apoptosis-inducing factors (caspase 3, p53 and p21). We found that immune dysfunction in these animals is associated with increased oxidative damage and mitochondrial dysfunction and that the nutrient treatment effectively elevated immune function, decreased oxidative damage, enhanced mitochondrial function and inhibited the elevation of apoptosis factors. These effects are comparable to, or greater than, those of the anti-diabetic drug pioglitazone. These data suggest that a rational combination of mitochondrial-targeting nutrients may be effective in improving immune function in type 2 diabetes through enhancement of mitochondrial function, decreased oxidative damage, and delayed cell death in the immune organs and blood.

Recent evidence indicates that insulin resistance in skeletal muscle may be related to reduce mitochondrial number and oxidation capacity. However, it is not known whether increasing mitochondrial number and function improves insulin resistance. In the present study, we investigated the effects of a combination of nutrients on insulin resistance and mitochondrial biogenesis/function in skeletal muscle of type 2 diabetic Goto–Kakizaki rats. We demonstrated that defect of glucose and lipid metabolism is associated with low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle of the diabetic Goto-Kakizaki rats. The treatment of combination of R-α-lipoic acid, acetyl-L-carnitine, nicotinamide, and biotin effectively improved glucose tolerance, decreased the basal insulin secretion and the level of circulating free fatty acid (FFA), and prevented the reduction of mitochondrial biogenesis in skeletal muscle. The nutrients treatment also significantly increased mRNA levels of genes involved in lipid metabolism, including peroxisome proliferator–activated receptor-α (Pparα), peroxisome proliferator–activated receptor-δ (Pparδ), and carnitine palmitoyl transferase-1 (Mcpt-1) and activity of mitochondrial complex I and II in skeletal muscle. All of these effects of mitochondrial nutrients are comparable to that of the antidiabetic drug, pioglitazone. In addition, the treatment with nutrients, unlike pioglitazone, did not cause body weight gain. These data suggest that a combination of mitochondrial targeting nutrients may improve skeletal mitochondrial dysfunction and exert hypoglycemic effects, without causing weight gain.

Nicotinamide, the principal form of niacin (vitamin B3), has been proposed to be neuroprotective in Parkinson's disease. However, the effects and mechanisms of nicotinamide on motor function in animals and on mitochondrial function in cellular systems have not been well studied. We hypothesized that niacin‐derived NAD(P)H as antioxidants and enzyme cofactors could inhibit oxidative damage and improve mitochondrial function and thus protect neurodegeneration and improve motor function. In the present study, the effects of nicotinamide on mitochondrial function and oxidative stress were studied in a 1‐methyl‐4‐phenylpyridinium (MPP⁺)–induced cellular model of Parkinson's disease, and the effects of improving motor dysfunction were studied in an α‐synuclein transgenic Drosophila Parkinson's model. Mitochondrial function was tested by measuring the activity of mitochondrial complex I and α‐ketoglutarate dehydrogenase, and oxidative damage was tested by measuring reactive oxygen species, DNA damage (8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and Comet assay), and protein oxidation (protein carbonyls) levels. Nicotinamide at a relatively higher concentration, that is, 100‐fold of the level in the cell culture medium (101 mg/L), significantly protected SK‐N‐MC human neuroblastoma cells from an MPP⁺‐induced decrease in cell viability, complex I and α‐ketoglutarate dehydrogenase activity, and an increase in oxidant generation, DNA damage, and protein oxidation. In the Drosophila model, nicotinamide at 15 and 30 mg/100 g diet significantly improved climbing ability. These results suggest that nutritional supplementation of nicotinamide at high doses decreases oxidative stress and improves mitochondrial and motor function in cellular and/or Drosophila models and may be an effective strategy for preventing and ameliorating Parkinson's disease.

Mitochondrial dysfunction due to oxidative stress and concomitant impaired β‐cell function may play a key role in type 2 diabetes. Preventing and/or ameliorating oxidative mitochondrial dysfunction with mitochondria‐specific nutrients may have preventive or therapeutic potential. In the present study, the oxidative mechanism of mitochondrial dysfunction in pancreatic β‐cells exposed to sublethal levels of oleic acid (OA) and the protective effects of mitochondrial nutrients [R‐alpha‐lipoic acid (LA) and acetyl‐L‐carnitine (ALC)] were investigated. Chronic exposure (72 h) of insulinoma MIN6 cells to OA (0.2–0.8 mM) increased intracellular oxidant formation, decreased mitochondrial membrane potential (MMP), enhanced uncoupling protein‐2 (UCP‐2) mRNA and protein expression, and consequently, decreased glucose‐induced ATP production and suppressed glucose‐stimulated insulin secretion. Pretreatment with LA and/or ALC reduced oxidant formation, increased MMP, regulated UCP‐2 mRNA and protein expression, increased glucose‐induced ATP production, and restored glucose‐stimulated insulin secretion. The key findings on ATP production and insulin secretion were verified with isolated rat islets. These results suggest that mitochondrial dysfunction is involved in OA‐induced pancreatic β‐cell dysfunction and that pretreatment with mitochondrial protective nutrients could be an effective strategy to prevent β‐cell dysfunction. J. Cell. Biochem. 104: 1232–1243, 2008.

The 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.

Hydroxytyrosol (HTS) is a natural polyphenol abundant in olive oil. Increasing evidence indicates HTS has beneficial effect on human health for preventing various diseases. In the present study, we investigated the protective effects of HTS on acrolein‐induced toxicity in human retinal pigment epithelial cell line, ARPE‐19, a cellular model of smoking‐ and age‐related macular degeneration. Acrolein, a major component of the gas phase cigarette smoke and also a product of lipid peroxidation in vivo, at 75 μmol/L for 24 h caused significant loss of cell viability, oxidative damage (increase in oxidant generation and oxidative damage to proteins and DNA, decrease in antioxidants and antioxidant enzymes, and also inactivation of the Keap1/Nrf2 pathway), and mitochondrial dysfunction (decrease in membrane potential, activities of mitochondrial complexes, viable mitochondria, oxygen consumption, and factors for mitochondrial biogenesis, and increase in calcium). Pre‐treatment with HTS dose dependently and also time dependently protected the ARPE‐19 cells from acrolein‐induced oxidative damage and mitochondrial dysfunction. A short‐term pre‐treatment with HTS (48 h) required > 75 μmol/L for showing protection while a long‐term pre‐treatment (7 days) showed protective effect from 5 μmol/L on. The protective effect of HTS in this model was as potent as that of established mitochondria‐targeting antioxidant nutrients. These results suggest that HTS is also a mitochondrial‐targeting antioxidant nutrient and that dietary administration of HTS may be an effective measure in reducing and or preventing cigarette smoke‐induced or age‐related retinal pigment epithelial degeneration, such as age‐associated macular degeneration.

We have identified a group of nutrients that can directly or indirectly protect mitochondria from oxidative damage and improve mitochondrial function and named them “mitochondrial nutrients”. The direct protection includes preventing the generation of oxidants, scavenging free radicals or inhibiting oxidant reactivity, and elevating cofactors of defective mitochondrial enzymes with increased Michaelis–Menten constant to stimulate enzyme activity, and also protect enzymes from further oxidation, and the indirect protection includes repairing oxidative damage by enhancing antioxidant defense systems either through activation of phase 2 enzymes or through increase in mitochondrial biogenesis. In this review, we take α-lipoic acid (LA) as an example of mitochondrial nutrients by summarizing the protective effects and possible mechanisms of LA and its derivatives on age-associated cognitive and mitochondrial dysfunction of the brain. LA and its derivatives improve the age-associated decline of memory, improve mitochondrial structure and function, inhibit the age-associated increase of oxidative damage, elevate the levels of antioxidants, and restore the activity of key enzymes. In addition, co-administration of LA with other mitochondrial nutrients, such as acetyl-l-carnitine and coenzyme Q10, appears more effective in improving cognitive dysfunction and reducing oxidative mitochondrial dysfunction. Therefore, administrating mitochondrial nutrients, such as LA and its derivatives in combination with other mitochondrial nutrients to aged people and patients suffering from neurodegenerative diseases, may be an effective strategy for improving mitochondrial and cognitive dysfunction.

D-Galactose (d-gal) -induced aging models in Drosophila, houseflies, mice and rats have been widely used; however, the underlying mechanisms are poorly understood. To investigate the involvement of mitochondrial dysfunction of d-gal, mitochondrial function was examined in the brain and liver of C57BL/6J mice, subjected to a treatment of d-gal with or without a concomitant treatment with a mitochondrial nutrient, R-alpha-lipoic acid (LA). d-Gal treatment induced a significant decrease in succinate-linked respiratory control ratio (RCR) and ADP/O ratio in the liver and brain, and also a significant increase in the maximum velocity (Vmax) and substrate binding affinity (Km) of complex II in the liver. LA treatment to d-gal-injected animals restored mitochondrial RCR in both brain and liver, ADP/O and Km of complex II in the liver. These results suggest LA is effective in delaying d-gal toxicity by ameliorating mitochondrial dysfunction.

Purpose. To understand better the cell and molecular basis for the epidemiologic association between cigarette smoke, oxidant injury, and age-associated macular degeneration, the authors examined the effects of acrolein, a major toxicant in cigarette smoke, on oxidative mitochondrial damage in retinal pigment epithelial (RPE) cells and the reduction of this damage by lipoic acid. methods. Cultured human ARPE19 cells and primary cultures of human fetal (hf)RPE were treated with acrolein. The toxicity of acrolein and the protective effects of R-α-lipoic acid were examined with a variety of previously described techniques. results. Acute acrolein exposure exceeding 50 μM (24 hours) in ARPR19 cells caused toxicity, including decreases in cell viability, mitochondrial potential, GSH, antioxidant capacity, Nrf2 expression, enzyme activity (mitochondrial complexes I, II, III; superoxide dismutase; and glutathione peroxidase). Acute exposure also increased oxidant levels, protein carbonyls, and calcium. Continuous acrolein exposure over 8 or 32 days caused similar toxicity but from 10- to 100-fold lower doses (0.1–5 μM). Pretreatment with R-α-lipoic acid effectively protected ARPE-19 cells from acrolein toxicity. Primary hfRPE cells were comparable to the ARPE-19 cells in sensitivity to acrolein toxicity and lipoic acid protection. conclusions. These results show that acrolein is a mitochondrial toxicant in RPE cells and that acrolein-induced oxidative mitochondrial dysfunction is reduced by lipoic acid. The similar sensitivity of the ARPE-19 and hfRPE cells suggests that both models are useful for studying RPE toxicity and protection. These experiments indicate that mitochondria-targeted antioxidants such as lipoic acid may be an effective strategy for reducing or preventing chronic oxidant-induced RPE degeneration in vivo from a variety of sources, including cigarette smoke.