Xianwen Yi

Endogenous Production of Lipoic Acid Is Essential for Mouse Development

ABSTRACT α-Lipoic acid (LA) is a cofactor for mitochondrial α-ketoacid dehydrogenase complexes and is one of the most potent, natural antioxidants. Reduction of oxidative stress by LA supplementation has been demonstrated in patients with diabetic neuropathy and in animal models. To determine how normal development or pathological conditions are affected by genetic alterations in the ability of mammalian cells to synthesize LA and whether dietary LA can circumvent its endogenous absence, we have generated mice deficient in lipoic acid synthase (Lias). Mice heterozygous for disruption of the Lias gene develop normally, and their plasma levels of thiobarbituric acid-reactive substances do not differ from those of wild-type mice. However, the heterozygotes have significantly reduced erythrocyte glutathione levels, indicating that their endogenous antioxidant capacity is lower than those of wild-type mice. Homozygous embryos lacking Lias appear healthy at the blastocyst stage, but their development is retarded globally by 7.5 days postcoitum (dpc), and all the null embryos die before 9.5 dpc. Supplementing the diet of heterozygous mothers with LA (1.65 g/kg of body weight) during pregnancy fails to prevent the prenatal deaths of homozygous embryos. Thus, endogenous LA synthesis is essential for developmental survival and cannot be replaced by LA in maternal tissues and blood.

α-Lipoic Acid Prevents the Increase in Atherosclerosis Induced by Diabetes in Apolipoprotein E–Deficient Mice Fed High-Fat/Low-Cholesterol Diet

Considerable evidence indicates that hyperglycemia increases oxidative stress and contributes to the increased incidence of atherosclerosis and cardiovascular complications in diabetic patients. To examine the effect of α-lipoic acid, a potent natural antioxidant, on atherosclerosis in diabetic mice, 3-month-old apolipoprotein (apo) E-deficient (apoE−/−) mice were made diabetic by administering streptozotocin (STZ). At 4 weeks after starting the STZ administration, a high-fat diet with or without α-lipoic acid (1.65 g/kg) was given to the mice and to nondiabetic apoE−/− controls. At 20 weeks, markers of oxidative stress were significantly lower in both the diabetic apoE−/− mice and their nondiabetic apoE−/− controls with α-lipoic acid supplement than in those without it. Remarkably, α-lipoic acid completely prevented the increase in plasma total cholesterol, atherosclerotic lesions, and the general deterioration of health caused by diabetes. These protective effects of α-lipoic acid were accompanied by a reduction of plasma glucose and an accelerated recovery of insulin-producing cells in the pancreas, suggesting that part of its effects are attributable to protecting pancreatic β-cells from damage. Our results suggest that dietary α-lipoic acid is a promising protective agent for reducing cardiovascular complications of diabetes.

α-Lipoic acid protects diabetic apolipoprotein E-deficient mice from nephropathy☆☆☆

AIM Both hyperglycemia and hyperlipidemia increase oxidative stress and contribute to the development of diabetic nephropathy (DN). We investigated the effects of α-lipoic acid, a natural antioxidant and a cofactor in the multienzyme complexes, on the development of DN in diabetic apolipoprotein E-deficient mice. METHODS Twelve-week-old male apoE-/- mice on C57BL/6J genetic background were made diabetic with injections of streptozotocin (STZ). STZ-treated diabetic apoE-/- mice and non-diabetic control were fed with a synthetic high-fat (HF) diet with or without lipoic acid (LA) supplementation. Multiple parameters including plasma glucose, cholesterol, oxidative stress markers, cytokines, and kidney cortex gene expression, and glomerular morphology were evaluated. RESULTS LA supplementation markedly protected the β cells, reduced cholesterol levels, and attenuated albuminuria and glomerular mesangial expansion in the diabetic mice. Renoprotection by LA was equally effective regardless of whether the dietary supplementation was started 4 weeks before, simultaneously with, or 4 weeks after the induction of diabetes by STZ. LA supplementation significantly improved DN and oxidative stress in the diabetic mice. Severity of albuminuria was positively correlated with level of thiobarbituric acid reactive substances (TBARs) in the kidney (r(2)=0.62, P<.05). Diabetes significantly changed the kidney expression of Rage, Sod2, Tgfb1 and Ctgf, Pdp2, nephrin, and Lias. LA supplementation corrected these changes except that it further suppressed the expression of the Lias gene coding for lipoic acid synthase. CONCLUSIONS Our data indicate that LA supplementation effectively attenuates the development and progression of DN through its antioxidant effect as well as enhances glucose oxidation.

Reduced Expression of Lipoic Acid Synthase Accelerates Diabetic Nephropathy

Oxidative stress contributes to the pathogenesis of diabetic nephropathy. In mitochondria, lipoic acid synthase produces α-lipoic acid, an antioxidant and an essential cofactor in α-ketoacid dehydrogenase complexes, which participate in glucose oxidation and ATP generation. Administration of lipoic acid abrogates diabetic nephropathy in animal models, but whether lower production of endogenous lipoic acid promotes diabetic nephropathy is unknown. Here, we crossed mice heterozygous for lipoic acid synthase deficiency (Lias(+/-)) with Ins2(Akita/+) mice, a well characterized model of type 1 diabetes. Double mutant mice had more overt diabetic nephropathy, including microalbuminuria, glomerular basement thickening, mesangial matrix expansion, and hypertension, compared with Lias(+/+)Ins2(Akita/+) controls. We also identified proximal tubules as a major site for generation of superoxide anions during diabetic nephropathy. Mitochondria in proximal tubular cells were particularly sensitive to damage in diabetic mice with reduced lipoic acid production. These results suggest that lipoic acid synthase deficiency increases oxidative stress and accelerates the development of diabetic nephropathy.

Diabetic atherosclerosis in APOE*4 mice: synergy between lipoprotein metabolism and vascular inflammation

Diabetes is a major risk factor for cardiovascular disease. To examine how diabetes interacts with a mildly compromised lipid metabolism, we introduced the diabetogenic Ins2C96Y/+ (Akita) mutation into mice expressing human apoE4 (E4) combined with either an overexpressing human LDL receptor gene (hLDLR) or the wild-type mouse gene. The hLDLR allele caused 2-fold reductions in plasma HDL-cholesterol, plasma apoA1, and hepatic triglyceride secretion. Diabetes increased plasma total cholesterol 1.3-fold and increased apoB48 secretion 3-fold, while reducing triglyceride secretion 2-fold. Consequently, diabetic E4 mice with hLDLR secrete increased numbers of small, cholesterol-enriched, apoB48-containing VLDL, although they have near normal plasma cholesterol (<120 mg/dl). Small foam cell lesions were present in the aortic roots of all diabetic E4 mice with hLDLR that we analyzed at six months of age. None were present in nondiabetic mice or in diabetic mice without hLDLR. Aortic expression of genes affecting leukocyte recruitment and adhesion was enhanced by diabetes. ApoA1 levels, but not diabetes, were strongly correlated with the ability of plasma to efflux cholesterol from macrophages. We conclude that the diabetes-induced proinflammatory changes in the vasculature and the hLDLR-mediated cholesterol accumulation in macrophages synergistically trigger atherosclerosis in mice with human apoE4, although neither alone is sufficient.

Genetic reduction of lipoic acid synthase expression modestly increases atherosclerosis in male, but not in female, apolipoprotein E-deficient mice

OBJECTIVES To evaluate the effects of a genetic reduction of Lias gene expression on atherosclerosis development. METHODS AND RESULTS Heterozygous knockout mice for the lipoid acid synthase gene (Lias(+/-)) were crossed with apolipoprotein E-deficient (ApoE(-/-)) mice, and the plaque size in aortic sinuses of Lias(+/-)ApoE(-/-)mice was evaluated at 6 months of age. Lesions at the aortic sinus in Lias(+/-)ApoE(-/-) males were significantly larger (1.5x) than those in Lias(+/+) ApoE(-/-) littermate males. The lesion size was inversely correlated with an increased erythrocyte reduced glutathione/oxidized glutathione (GSH/GSSH) ratio, a systemic index of body redox balance. Lias(+/-)ApoE(-/-)males also had significantly increased plasma cholesterol and reduced pyruvate dehydrogenase complex activity in the liver. Significant reductions in the expression of genes for antioxidant enzymes, including superoxide dismutase 1 (SOD1) and SOD2, were observed in aortas of Lias(+/-)ApoE(-/-)males. Female Lias(+/-)ApoE(-/-)also exhibited changes in these parameters, parallel to those observed in males. However, the Lias gene effects for the majority of these factors, including atherosclerotic lesion size, were not significant in females. CONCLUSIONS Our data provide evidence that Lias deficiency enhances atherosclerosis in male mice, at least in part due to reduced antioxidant capacity. The notable absence of such effects in females leaves open the possibility of a gender-specific protection mechanism.

Mice with heterozygous deficiency of lipoic acid synthase have an increased sensitivity to lipopolysaccharide-induced tissue injury

α‐Lipoic acid (1, 2‐dithiolane‐3‐pentanoic acid; LA), synthesized in mitochondria by LA synthase (Lias), is a potent antioxidant and a cofactor for metabolic enzyme complexes. In this study, we examined the effect of genetic reduction of LA synthesis on its antioxidant and anti‐inflammatory properties using a model of LPS‐induced inflammation in Lias+/– mice. The increase of plasma proinflammatory cytokine, TNF‐α, and NF‐κB at an early phase following LPS injection was greater in Lias+/– mice compared with Lias+/+ mice. The circulating blood white blood cell (WBC) and platelet counts dropped continuously during the initial 4 h. The counts subsequently recovered partially in Lias+/+ mice, but the recovery was impaired totally in Lias+/– mice. Administration of exogenous LA normalized the recovery of WBC counts in Lias+/– mice but not platelets. Enhanced neutrophil sequestration in the livers of Lias+/– mice was associated with increased hepatocyte injury and increased gene expression of growth‐related oncogene, E‐selectin, and VCAM‐1 in the liver and/or lung. Lias gene expression in tissues was 50% of normal expression in Lias+/– mice and reduced further by LPS treatment. Decreased Lias expression was associated with diminished hepatic LA and tissue oxidative stress. Finally, Lias+/– mice displayed enhanced mortality when exposed to LPS‐induced sepsis. These data demonstrate the importance of endogenously produced LA for preventing leukocyte accumulation and tissue injury that result from LPS‐induced inflammation.

Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress

BACKGROUND S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. METHODS In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. RESULTS We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. CONCLUSIONS Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. GENERAL SIGNIFICANCE Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

α-Lipoic acid protects mitochondrial enzymes and attenuates lipopolysaccharide-induced hypothermia in mice

Hypothermia is a key symptom of sepsis, but the mechanism(s) leading to hypothermia during sepsis is largely unknown and thus no effective therapy is available for hypothermia. Therefore, it is important to investigate the mechanism and develop effective therapeutic methods. Lipopolysaccharide (LPS)-induced hypothermia accompanied by excess nitric oxide (NO) production leads to a reduction in energy production in wild-type mice. However, mice lacking inducible nitric oxide synthase did not suffer from LPS-induced hypothermia, suggesting that hypothermia is associated with excess NO production during sepsis. This observation is supported by the treatment of wild-type mice with α-lipoic acid (LA) in that it effectively attenuates LPS-induced hypothermia with decreased NO production. We also found that LA partially restored ATP production, and activities of the mitochondrial enzymes involved in energy metabolism, which were inhibited during sepsis. These data suggest that hypothermia is related to mitochondrial dysfunction, which is probably compromised by excess NO production and that LA administration attenuates hypothermia mainly by protecting mitochondrial enzymes from NO damage.

Reduced alpha-lipoic acid synthase gene expression exacerbates atherosclerosis in diabetic apolipoprotein E-deficient mice.

OBJECTIVES To study the effects of reduced lipoic acid gene expression on diabetic atherosclerosis in apolipoprotein E null mice (Apoe(-/-)). METHODS AND RESULTS Heterozygous lipoic acid synthase gene knockout mice (Lias(+/-)) crossed with Apoe(-/-) mice were used to evaluate the diabetic effect induced by streptozotocin on atherosclerosis in the aortic sinus of the heart. While diabetes markedly increased atherosclerotic plaque size in Apoe(-/-) mice, a small but significant effect of reduced expression of lipoic acid gene was observed in diabetic Lias(+/-)Apoe(-/-) mice. In the aortic lesion area, the Lias(+/-)Apoe(-/-) mice exhibited significantly increased macrophage accumulation and cellular apoptosis than diabetic Lias(+/+)Apoe(-/-) littermates. Plasma glucose, cholesterol, and interleukin-6 were also higher. These abnormalities were accompanied with increased oxidative stress including a decreased ratio of reduced glutathione/oxidized glutathione in erythrocytes, increased systemic lipid peroxidation, and increased Gpx1 and MCP1 gene expression in the aorta. CONCLUSIONS Decreased endogenous lipoic acid gene expression plays a role in development of diabetic atherosclerosis. These findings extend our understanding of the role of antioxidant in diabetic atherosclerosis.