Pharmacodynamic effects of orally administered dexlipotam on endothelial function in type 2-diabetic patients.

OBJECTIVE: Diabetic endotheliopathy is the result of hyperglycemia and the production of oxygen-free radicals. In vitro and in vivo data have shown beneficial effects of dexlipotam (DEX), a tromethamine salt of R(+)-alpha-lipoic acid, on oxidative stress in hyperglycemic states, but no data are available on the effects of this agent on endothelial function. The purpose of this pilot study was to evaluate the impact of DEX on endothelial function in patients with type 2 diabetes (DM2) and to estimate the safety and tolerability of DEX. MATERIAL AND METHODS: DEX 960 mg and DEX 1,920 mg were investigated in DM2 patients over a period of 4 weeks using a randomized, placebo- (PLA) controlled, double-blinded study with 3 parallel groups. The marker of arterial function after 4-week therapy with DEX was the maximum percentage change versus baseline in the flow-mediated dilation of the brachial artery (FMD) after reperfusion. RESULTS: A total of 114 diabetic patients were randomized to the three study groups. DEX was safe and well tolerated. Dyspepsia appeared to be the most relevant side effect of DEX treatment. Systolic (p = 0.078) and diastolic blood pressure (p = 0.059) tended to be lower in patients treated with DEX at a dose of 1,920 mg. There were no significant differences in FMD between the placebo- and the DEX-treated groups. In patients with poorer glucose control (HbA1c > 6.5% Hb), FMD increased significantly after 4-week treatment with DEX: PLA -1.51 +/- 2.98%, DEX 960 mg +1.22 +/- 3.22, p = 0.027, DEX 1,920 mg +1.47 +/- 3.78, p= 0.012. The magnitude of the mean change compared to placebo was 2.73% (DEX 920) and 2.98% (DEX 1,920) in patients with HbAlc > 7.5% Hb (DEX 960, p = 0.007, DEX 1,920, p = 0.032). The effects of treatment were usually statistically significant in subgroups with more severe vascular stress (longer duration of disease, pretreatment history, higher LDL-C, higher blood pressure). CONCLUSION: DEX therapy appears to reduce endothelial dysfunction in DM2, especially in men with long history of DM2 and having poor glucose control. These findings will be useful in patient selection in future prospective clinical trials with drugs to treat vascular stress.

The roles of oxidative stress and antioxidant treatment in experimental diabetic neuropathy.

Oxidative stress is present in the diabetic state. Our work has focused on its presence in peripheral nerves. Antioxidant enzymes are reduced in peripheral nerves and are further reduced in diabetic nerves. That lipid peroxidation will cause neuropathy is supported by evidence of the development of neuropathy de novo when normal nerves are rendered alpha-tocopherol deficient and by the augmentation of the conduction deficit in diabetic nerves subjected to this insult. Oxidative stress appears to be primarily due to the processes of nerve ischemia and hyperglycemia auto-oxidation. The indexes of oxidative stress include an increase in nerve, dorsal root, and sympathetic ganglia lipid hydroperoxides and conjugated dienes. The most reliable and sensitive index, however, is a reduction in reduced glutathione. Experimental diabetic neuropathy results in myelinopathy of dorsal roots and a vacuolar neuropathy of dorsal root ganglion. The vacuoles are mitochondrial; we posit that lipid peroxidation causes mitochondrial DNA mutations that increase reduced oxygen species, causing further damage to mitochondrial respiratory chain and function and resulting in a sensory neuropathy. Alpha-lipoic acid is a potent antioxidant that prevents lipid peroxidation in vitro and in vivo. We evaluated the efficacy of the drug in doses of 20, 50, and 100 mg/kg administered intraperitoneally in preventing the biochemical, electrophysiological, and nerve blood flow deficits in the peripheral nerves of experimental diabetic neuropathy. Alpha-lipoic acid dose- and time-dependently prevented the deficits in nerve conduction and nerve blood flow and biochemical abnormalities (reductions in reduced glutathione and lipid peroxidation). The nerve blood flow deficit was 50% (P < 0.001). Supplementation dose-dependently prevented the deficit; at the highest concentration, nerve blood flow was not different from that of control nerves. Digital nerve conduction underwent a dose-dependent improvement at 1 month (P < 0.05). By 3 months, all treated groups had lost their deficit. The antioxidant drug is potentially efficacious for human diabetic sensory neuropathy.

Stimulation of glucose uptake by the natural coenzyme alpha-lipoic acid/thioctic acid: participation of elements of the insulin signaling pathway.

Thioctic acid (alpha-lipoic acid), a natural cofactor in dehydrogenase complexes, is used in Germany in the treatment of symptoms of diabetic neuropathy. Thioctic acid improves insulin-responsive glucose utilization in rat muscle preparations and during insulin clamp studies performed in diabetic individuals. The aim of this study was to determine the direct effect of thioctic acid on glucose uptake and glucose transporters. In L6 muscle cells and 3T3-L1 adipocytes in culture, glucose uptake was rapidly increased by (R)-thioctic acid. The increment was higher than that elicited by the (S)-isomer or the racemic mixture and was comparable with that caused by insulin. In parallel to insulin action, the stimulation of glucose uptake by thioctic acid was abolished by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, in both cell lines. Thioctic acid provoked an upward shift of the glucose-uptake insulin dose-response curve. The molar content of GLUT1 and GLUT4 transporters was measured in both cell lines. 3T3-L1 adipocytes were shown to have >10 times more glucose transporters but similar ratios of GLUT4:GLUT1 than L6 myotubes. The effect of (R)-thioctic acid on glucose transporters was studied in the L6 myotubes. Its stimulatory effect on glucose uptake was associated with an intracellular redistribution of GLUT1 and GLUT4 glucose transporters, similar to that caused by insulin, with minimal effects on GLUT3 transporters. In conclusion, thioctic acid stimulates basal glucose transport and has a positive effect on insulin-stimulated glucose uptake. The stimulatory effect is dependent on phosphatidylinositol 3-kinase activity and may be explained by a redistribution of glucose transporters. This is evidence that a physiologically relevant compound can stimulate glucose transport via the insulin signaling pathway.

Advanced glycation end product-induced activation of NF-kappaB is suppressed by alpha-lipoic acid in cultured endothelial cells.

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.

The antioxidant alpha-lipoic acid enhances insulin-stimulated glucose metabolism in insulin-resistant rat skeletal muscle.

Insulin resistance of muscle glucose metabolism is a hallmark of NIDDM. The obese Zucker (fa/fa) rat--an animal model of muscle insulin resistance--was used to test whether acute (100 mg/kg body wt for 1 h) and chronic (5-100 mg/kg for 10 days) parenteral treatments with a racemic mixture of the antioxidant alpha-lipoic acid (ALA) could improve glucose metabolism in insulin-resistant skeletal muscle. Glucose transport activity (assessed by net 2-deoxyglucose [2-DG] uptake), net glycogen synthesis, and glucose oxidation were determined in the isolated epitrochlearis muscles in the absence or presence of insulin (13.3 nmol/l). Severe insulin resistance of 2-DG uptake, glycogen synthesis, and glucose oxidation was observed in muscle from the vehicle-treated obese rats compared with muscle from vehicle-treated lean (Fa/-) rats. Acute and chronic treatments (30 mg.kg-1.day-1, a maximally effective dose) with ALA significantly (P < 0.05) improved insulin-mediated 2-DG uptake in epitrochlearis muscles from the obese rats by 62 and 64%, respectively. Chronic ALA treatment increased both insulin-stimulated glucose oxidation (33%) and glycogen synthesis (38%) and was associated with a significantly greater (21%) in vivo muscle glycogen concentration. These adaptive responses after chronic ALA administration were also associated with significantly lower (15-17%) plasma levels of insulin and free fatty acids. No significant effects on glucose transporter (GLUT4) protein level or on the activities of hexokinase and citrate synthase were observed. Collectively, these findings indicate that parenteral administration of the antioxidant ALA significantly enhances the capacity of the insulin-stimulatable glucose transport system and of both oxidative and nonoxidative pathways of glucose metabolism in insulin-resistant rat skeletal muscle.

Alpha-lipoic acid: effect on glucose uptake, sorbitol pathway, and energy metabolism in experimental diabetic neuropathy.

The peripheral nerve of experimental diabetic neuropathy (EDN) is reported to be ischemic and hypoxic, with an increased dependence on anaerobic metabolism, requiring increased energy substrate stores. When glucose stores become reduced, fiber degeneration has been reported. We evaluated glucose uptake, nerve energy metabolism, the polyol pathway, and protein kinase C (PKC) activity in EDN induced by streptozotocin. Control and diabetic rats received lipoic acid (0, 10, 25, 50, 100 mg/kg). Duration of diabetes was 1 month, and alpha-lipoic acid was administered intraperitoneally 5 times per week for the final week of the experiment. Nerve glucose uptake was reduced to 60, s 37, and 30% of control values in the sciatic nerve, L5 dorsal root ganglion, and superior cervical ganglion (SCG), respectively, in rats with EDN. Alpha-lipoic acid supplementation had no effect on glucose uptake in normal nerves at any dose, but reversed the deficit in EDN, with a threshold between 10 and 25 mg/kg. Endoneurial glucose, fructose, sorbitol, and myo-inositol were measured in sciatic nerve. Alpha-lipoic acid had no significant effect on either energy metabolism or polyol pathway of normal nerves. In EDN, endoneurial glucose, fructose, and sorbitol were significantly increased, while myo-inositol was significantly reduced. Alpha-lipoic acid had a biphasic effect: it dose-dependently increased fructose, glucose, and sorbitol, peaking at 25 mg/kg, and then fell beyond that dose, and it dose-dependently increased myo-inositol. Sciatic nerve cytosolic PKC was increased in EDN. ATP, creatine phosphate, and lactate were measured in sciatic nerve and SCG. Alpha-lipoic acid prevented the reduction in SCG creatine phosphate. We conclude that glucose uptake is reduced in EDN and that this deficit is dose-dependently reversed by alpha-lipoic acid, a change associated with an improvement in peripheral nerve function.

Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB.

Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has been suggested to participate in chronic disorders, such as diabetes and its complications. In contrast to the short and transient activation of NF-kappaB in vitro, we observed a long-lasting sustained activation of NF-kappaB in the absence of decreased IkappaBalpha in mononuclear cells from patients with type 1 diabetes. This was associated with increased transcription of NF-kappaBp65. A comparable increase in NF-kappaBp65 antigen and mRNA was also observed in vascular endothelial cells of diabetic rats. As a mechanism, we propose that binding of ligands such as advanced glycosylation end products (AGEs), members of the S100 family, or amyloid-beta peptide (Abeta) to the transmembrane receptor for AGE (RAGE) results in protein synthesis-dependent sustained activation of NF-kappaB both in vitro and in vivo. Infusion of AGE-albumin into mice bearing a beta-globin reporter transgene under control of NF-kappaB also resulted in prolonged expression of the reporter transgene. In vitro studies showed that RAGE-expressing cells induced sustained translocation of NF-kappaB (p50/p65) from the cytoplasm into the nucleus for >1 week. Sustained NF-kappaB activation by ligands of RAGE was mediated by initial degradation of IkappaB proteins followed by new synthesis of NF-kappaBp65 mRNA and protein in the presence of newly synthesized IkappaBalpha and IkappaBbeta. These data demonstrate that ligands of RAGE can induce sustained activation of NF-kappaB as a result of increased levels of de novo synthesized NF-kappaBp65 overriding endogenous negative feedback mechanisms and thus might contribute to the persistent NF-kappaB activation observed in hyperglycemia and possibly other chronic diseases.

Lipoic acid improves nerve blood flow, reduces oxidative stress, and improves distal nerve conduction in experimental diabetic neuropathy.

OBJECTIVE: To determine whether lipoic acid (LA) will reduce oxidative stress in diabetic peripheral nerves and improve neuropathy. RESEARCH DESIGN AND METHODS: We used the model of streptozotocin-induced diabetic neuropathy (SDN) and evaluated the efficacy of LA supplementation in improving nerve blood flow (NBF), electrophysiology, and indexes of oxidative stress in peripheral nerves affected by SDN, at 1 month after onset of diabetes and in age-matched control rats. LA, in doses of 20, 50, and 100 mg/kg, was administered intraperitoneally five times per week after onset of diabetes. RESULTS: NBF in SDN was reduced by 50%; LA did not affect the NBF of normal nerves but improved that of SDN in a dose-dependent manner. After 1 month of treatment, LA-supplemented rats (100 mg/kg) exhibited normal NBF. The most sensitive and reliable indicator of oxidative stress was reduction in reduced glutathione, which was significantly reduced in streptozotocin-induced diabetic and alpha-tocopherol-deficient nerves; it was improved in a dose-dependent manner in LA-supplemented rats. The conduction velocity of the digital nerve was reduced in SDN and was significantly improved by LA. CONCLUSIONS: These studies suggest that LA improves SDN, in significant part by reducing the effects of oxidative stress. The drug may have potential in the treatment of human diabetic neuropathy.

Hydroperoxides in plasma are reduced by intensified insulin treatment. A randomized controlled study of IDDM patients with microalbuminuria.

OBJECTIVE: An association between reactive oxygen species and diabetic micro- and macrovascular complications has been proposed. In the present study, we have examined the effect of an improved blood glucose control on plasma levels of hydroperoxides in patients with IDDM. RESEARCH DESIGN AND METHODS: Subjects included 30 young IDDM patients with microalbuminuria who were randomized to receive either continuous subcutaneous insulin infusion (CSII) by a portable insulin pump (n = 15) or conventional insulin treatment (CIT) (n = 15) for 24 months. Plasma levels of hydroperoxides were measured by the ferrous oxidation with Xylenol Orange, version 2 (FOX2) assay. This method measures total lipid hydroperoxides and, unlike other methods, does not suffer from extraction losses. RESULTS: The mean HbA1c level was lower in the CSII group at the end of the study than in the CIT group: (mean [95% CI]) 8.6 (8.1-9.1) vs. 9.6 (9.0-10.3)%, respectively (P < 0.002). The level of plasma hydroperoxides was very similar at the start of the study but was significantly lower in the CSII group compared with the CIT group at the end of the study: 2.9 (2.1-3.7) vs. 4.3 (3.2-5.4) mumol/l, respectively (P < 0.02). In the CSII group, hydroperoxides were reduced by 31% from baseline (P < 0.001), whereas there was no change in levels of hydroperoxides in the CIT group. Mean hydroperoxide levels correlated with mean HbA1c during the study (r = 0.39, P < 0.04). Hydroperoxide levels were associated with the levels of microalbuminuria (r = 0.45, P < 0.02). CONCLUSIONS: This study provides support for the hypothesis that hyperglycemia is an important factor in the generation of hydroperoxides, and, thus, reactive oxygen species, in the circulation of IDDM patients.

alpha-Lipoic acid treatment decreases serum lactate and pyruvate concentrations and improves glucose effectiveness in lean and obese patients with type 2 diabetes.

OBJECTIVE: We examined the effect of lipoic acid (LA), a cofactor of the pyruvate dehydrogenase complex (PDH), on insulin sensitivity (SI) and glucose effectiveness (SG) and on serum lactate and pyruvate levels after oral glucose tolerance tests (OGTTs) and modified frequently sampled intravenous glucose tolerance tests (FSIGTTs) in lean (n = 10) and obese (n = 10) patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: FSIGTT data were analyzed by minimal modeling technique to determine SI and SG before and after oral treatment (600 mg, twice a day, for 4 weeks). Serum lactate and pyruvate levels of diabetic patients after glucose loading were compared with those of lean (n = 10) and obese (n = 10) healthy control subjects in which SI and SG were also determined from FSIGTT data. RESULTS: Fasting lactate and pyruvate levels were significantly increased in patients with type 2 diabetes. These metabolites did not exceed elevated fasting concentrations after glucose loading in lean patients with type 2 diabetes. However, a twofold increase of lactate and pyruvate levels was measured in obese diabetic patients. LA treatment was associated with increased SG in both diabetic groups (lean 1.28 +/- 0.14 to 1.93 +/- 0.13; obese 1.07 +/- 0.11 to 1.53 +/- 0.08 x 10(-2) min-1, P < 0.05). Higher SI and lower fasting glucose were measured in lean diabetic patients only (P < 0.05). Lactate and pyruvate before and after glucose loading were approximately 45% lower in lean and obese diabetic patients after LA treatment. CONCLUSIONS: Treatment of lean and obese diabetic patients with LA prevents hyperglycemia-induced increments of serum lactate and pyruvate levels and increases SG.

Insufficient glycemic control increases nuclear factor-kappa B binding activity in peripheral blood mononuclear cells isolated from patients with type 1 diabetes.

OBJECTIVE: The redox-sensitive transcription factor nuclear factor-kappa B (NF-kappa B) is believed to contribute to late diabetic complications. It is unknown whether NF-kappa B is influenced by glycemic control. RESEARCH DESIGN AND METHODS: To determine whether NF-kappa B is activated in patients with insufficient glycemic control (HbA1c > 10%), we developed a tissue culture-independent electrophoretic mobility shift assay (EMSA)-based semiquantitative detection system that allowed us to determine NF-kappa B activation in ex vivo-isolated peripheral blood mononuclear cells (PBMCs). We included 43 patients with type 1 diabetes in this cross-sectional study. 10 of those received the antioxidant thioctic acid (600 mg/day p.o.) for 2 weeks. RESULTS: Monocytes of patients with HbA1c levels > 10% demonstrated significantly higher NF-kappa B binding activity in an EMSA and a stronger NF-kappa B staining in immunohistochemistry than monocytes of patients with HbA1c levels of 6-8%. The increase in NF-kappa B activation correlated with an increase in plasmatic markers of lipid peroxidation. Treatment with the antioxidant thioctic acid decreased NF-kappa B binding activity. CONCLUSIONS: Hyperglycemia induces activation of the transcription factor NF-kappa B in ex vivo-isolated PBMCs of patients with type 1 diabetes. NF-kappa B activation is at least partially dependent on oxidative stress, since the antioxidant thioctic acid significantly lowered the extent of NF-kappa B binding activity.

Protection against peroxynitrite-dependent tyrosine nitration and alpha 1-antiproteinase inactivation by oxidized and reduced lipoic acid.

Peroxynitrite, formed by combination of superoxide radical with nitric oxide, is a reactive tissue-damaging species apparently involved in the pathology of several human diseases. Peroxynitrite nitrates tyrosine residues and inactivates alpha 1-antiproteinase. We show that both lipoic acid and dihydrolipoic acid efficiently protect against damage by peroxynitrite. By contrast, other disulphides tested did not. The biological antioxidant effects of lipoate/dihydrolipoate may involve scavenging of reactive nitrogen species as well as reactive oxygen species.

Neuroprotection by the metabolic antioxidant alpha-lipoic acid.

Reactive oxygen species are thought to be involved in a number of types of acute and chronic pathologic conditions in the brain and neural tissue. The metabolic antioxidant alpha-lipoate (thioctic acid, 1, 2-dithiolane-3-pentanoic acid; 1, 2-dithiolane-3 valeric acid; and 6, 8-dithiooctanoic acid) is a low molecular weight substance that is absorbed from the diet and crosses the blood-brain barrier. alpha-Lipoate is taken up and reduced in cells and tissues to dihydrolipoate, which is also exported to the extracellular medium; hence, protection is afforded to both intracellular and extracellular environments. Both alpha-lipoate and especially dihydrolipoate have been shown to be potent antioxidants, to regenerate through redox cycling other antioxidants like vitamin C and vitamin E, and to raise intracellular glutathione levels. Thus, it would seem an ideal substance in the treatment of oxidative brain and neural disorders involving free radical processes. Examination of current research reveals protective effects of these compounds in cerebral ischemia-reperfusion, excitotoxic amino acid brain injury, mitochondrial dysfunction, diabetes and diabetic neuropathy, inborn errors of metabolism, and other causes of acute or chronic damage to brain or neural tissue. Very few neuropharmacological intervention strategies are currently available for the treatment of stroke and numerous other brain disorders involving free radical injury. We propose that the various metabolic antioxidant properties of alpha-lipoate relate to its possible therapeutic roles in a variety of brain and neuronal tissue pathologies: thiols are central to antioxidant defense in brain and other tissues. The most important thiol antioxidant, glutathione, cannot be directly administered, whereas alpha-lipoic acid can. In vitro, animal, and preliminary human studies indicate that alpha-lipoate may be effective in numerous neurodegenerative disorders.

alpha-Lipoic acid as a biological antioxidant.

alpha-Lipoic acid, which plays an essential role in mitochondrial dehydrogenase reactions, has recently gained considerable attention as an antioxidant. Lipoate, or its reduced form, dihydrolipoate, reacts with reactive oxygen species such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also protects membranes by interacting with vitamin C and glutathione, which may in turn recycle vitamin E. In addition to its antioxidant activities, dihydrolipoate may exert prooxidant actions through reduction of iron. alpha-Lipoic acid administration has been shown to be beneficial in a number of oxidative stress models such as ischemia-reperfusion injury, diabetes (both alpha-lipoic acid and dihydrolipoic acid exhibit hydrophobic binding to proteins such as albumin, which can prevent glycation reactions), cataract formation, HIV activation, neurodegeneration, and radiation injury. Furthermore, lipoate can function as a redox regulator of proteins such as myoglobin, prolactin, thioredoxin and NF-kappa B transcription factor. We review the properties of lipoate in terms of (1) reactions with reactive oxygen species; (2) interactions with other antioxidants; (3) beneficial effects in oxidative stress models or clinical conditions.

Alpha-lipoic acid: antioxidant potency against lipid peroxidation of neural tissues in vitro and implications for diabetic neuropathy.

Nerve lipid peroxidation is increased in experimental diabetic neuropathy, and alpha-lipoic acid will prevent the deficits in nerve blood flow, oxidative stress, and distal sensory conduction. Because these alterations can occur by mechanisms other than augmenting lipid peroxidation in vivo, and because both pro-oxidant and antioxidant effects of the agent have been reported, we undertook studies of in vitro lipid peroxidation of brain and sciatic nerve using an in vitro lipid peroxidation model with an ascorbate-iron-EDTA system. We evaluated the effectiveness of the R(+)-, S(-)-enantiomers, and racemate of alpha-lipoic acid in reducing thiobarbituric acid reactive substances (TBARS) generation in rat brain and sciatic nerve. Studies were also done in an incubation medium containing 20 mM glucose, which increased lipid peroxidation up to fourfold. A dose-dependent and statistically significant reduction in lipid peroxidation was seen with both tissues with similar potencies for both enantiomers. This effect was unassociated with any reduction in the loss of alpha-tocopherol.

Antioxidant alpha-lipoic acid and protein turnover in insulin-resistant rat muscle.

We have shown previously that the antioxidant alpha-lipoic acid (ALA) can stimulate glucose transport and can enhance the stimulation of this process by insulin in skeletal muscle from insulin-resistant obese Zucker rats. As insulin can also acutely activate general protein synthesis and inhibit net protein degradation in skeletal muscle, we hypothesized that ALA could directly affect protein turnover and also increase the effect of insulin on protein turnover in isolated skeletal muscle from developing obese Zucker rats. In epitrochlearis muscles isolated from obese Zucker rats, insulin (2 mU/ml) significantly (p < 0.05) increased in vitro protein synthesis (phenylalanine incorporation into protein) and decreased net protein degradation (tyrosine release), whereas a racemic mixture of ALA (2 mM) had no effect on either process. Interestingly, rates of protein synthesis in muscle from obese Zucker rats were substantially lower compared to those values observed in age-matched insulin-sensitive Wistar rats, whereas rates of protein degradation were comparable. Obese Zucker rats were also treated chronically with either vehicle or ALA (50 mg/kg/d for 10 d). Again, insulin significantly increased net protein synthesis and decreased net protein degradation in epitrochlearis muscles isolated from vehicle-treated obese Zucker rats; however, this stimulatory effect of insulin was not improved by prior in vivo ALA treatment. These results indicate that the previously described effect of the antioxidant ALA to increase insulin-stimulated glucose transport in skeletal muscle of obese, insulin-resistant rats does not apply to another important insulin-regulatable process, protein turnover. These findings imply that the cellular mode of action for ALA is restricted to signaling factors unique to the activation of glucose transport, and does not involve the pathway of stimulation of general protein synthesis and net protein degradation.

Cytosolic and mitochondrial systems for NADH- and NADPH-dependent reduction of alpha-lipoic acid.

In cellular, tissue, and organismal systems, exogenously supplied alpha-lipoic acid (thioctic acid) has a variety of significant effects, including direct radical scavenging, redox modulation of cell metabolism, and potential to inhibit oxidatively-induced injury. Because reduction of lipoate to dihydrolipoate is a crucial step in many of these processes, we investigated mechanisms of its reduction. The mitochondrial NADH-dependent dihydrolipoamide dehydrogenase exhibits a marked preference for R(+)-lipoate, whereas NADPH-dependent glutathione reductase shows slightly greater activity toward the S(-)-lipoate stereoisomer. Rat liver mitochondria also reduced exogenous lipoic acid. The rate of reduction was stimulated by substrates which increased the NADH content of the mitochondria, and was inhibited by methoxyindole-2-carboxylic acid, a dihydrolipoamide dehydrogenase inhibitor. In rat liver cytosol, NADPH-dependent reduction was greater than NADH, and lipoate reduction was inhibited by glutathione disulfide. In rat heart, kidney, and brain whole cell-soluble fractions, NADH contributed more to reduction (70-90%) than NADPH, whereas with liver, NADH and NADPH were about equally active. An intact organ, the isolated perfused rat heart, reduced R-lipoate six to eight times more rapidly than S-lipoate, consistent with high mitochondrial dihydrolipoamide dehydrogenase activity and results with isolated cardiac mitochondria. On the other hand, erythrocytes, which lack mitochondria, somewhat more actively reduced S- than R-lipoate. These results demonstrate differing stereospecific reduction by intact cells and tissues. Thus, mechanisms of reduction of alpha-lipoate are highly tissue-specific and effects of exogenously supplied alpha-lipoate are determined by tissue glutathione reductase and dihydrolipoamide dehydrogenase activity.

Alpha-lipoic acid in liver metabolism and disease.

R-alpha-Lipoic acid is found naturally occurring as a prosthetic group in alpha-keto acid dehydrogenase complexes of the mitochondria, and as such plays a fundamental role in metabolism. Although this has been known for decades, only recently has free supplemented alpha-lipoic acid been found to affect cellular metabolic processes in vitro, as it has the ability to alter the redox status of cells and interact with thiols and other antioxidants. Therefore, it appears that this compound has important therapeutic potential in conditions where oxidative stress is involved. Early case studies with alpha-lipoic acid were performed with little knowledge of the action of alpha-lipoic acid at a cellular level, but with the rationale that because the naturally occurring protein bound form of alpha-lipoic acid has a pivotal role in metabolism, that supplementation may have some beneficial effect. Such studies sought to evaluate the effect of supplemented alpha-lipoic acid, using low doses, on lipid or carbohydrate metabolism, but little or no effect was observed. A common response in these trials was an increase in glucose uptake, but increased plasma levels of pyruvate and lactate were also observed, suggesting that an inhibitory effect on the pyruvate dehydrogenase complex was occurring. During the same period, alpha-lipoic acid was also used as a therapeutic agent in a number of conditions relating to liver disease, including alcohol-induced damage, mushroom poisoning, metal intoxification, and CCl4 poisoning. Alpha-Lipoic acid supplementation was successful in the treatment for these conditions in many cases. Experimental studies and clinical trials in the last 5 years using high doses of alpha-lipoic acid (600 mg in humans) have provided new and consistent evidence for the therapeutic role of antioxidant alpha-lipoic acid in the treatment of insulin resistance and diabetic polyneuropathy. This new insight should encourage clinicians to use alpha-lipoic acid in diseases affecting liver in which oxidative stress is involved.

Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus.

Gas chromatography-mass spectrometry was used to measure the oxidative DNA damage in diabetic subjects and controls. Levels of multiple DNA base oxidation products, but not DNA base de-amination or chlorination products, were found to be elevated in white blood cell DNA from patients with type II diabetes as compared with age-matched controls. The chemical pattern of base damage is characteristic of that caused by an attack on DNA by hydroxyl radical. An increased formation of the highly reactive hydroxyl radical could account for many of the reports of oxidative stress in diabetic subjects. There was no evidence of an increased DNA damage by reactive nitrogen or chlorine species.

Oral administration of RAC-alpha-lipoic acid modulates insulin sensitivity in patients with type-2 diabetes mellitus: a placebo-controlled pilot trial.

Alpha-lipoic acid (ALA), a naturally occuring compound and a radical scavenger was shown to enhance glucose transport and utilization in different experimental and animal models. Clinical studies described an increase of insulin sensitivity after acute and short-term (10 d) parenteral administration of ALA. The effects of a 4-week oral treatment with alpha-lipoic acid were evaluated in a placebo-controlled, multicenter pilot study to determine see whether oral treatment also improves insulin sensitivity. Seventy-four patients with type-2 diabetes were randomized to either placebo (n = 19); or active treatment in various doses of 600 mg once daily (n = 19), twice daily (1200 mg; n = 18), or thrice daily (1800 mg; n = 18) alpha-lipoic acid. An isoglycemic glucose-clamp was done on days 0 (pre) and 29 (post). In this explorative study, analysis was done according to the number of subjects showing an improvement of insulin sensitivity after treatment. Furthermore, the effects of active vs. placebo treatment on insulin sensitivity was compared. All four groups were comparable and had a similar degree of hyperglycemia and insulin sensitivity at baseline. When compared to placebo, significantly more subjects had an increase in insulin-stimulated glucose disposal (MCR) after ALA treatment in each group. As there was no dose effect seen in the three different alpha-lipoic acid groups, all subjects receiving ALA were combined in the "active" group and then compared to placebo. This revealed significantly different changes in MCR after treatment (+27% vs. placebo; p < .01). This placebo-controlled explorative study confirms previous observations of an increase of insulin sensitivity in type-2 diabetes after acute and chronic intravenous administration of ALA. The results suggest that oral administration of alpha-lipoic acid can improve insulin sensitivity in patients with type-2 diabetes. The encouraging findings of this pilot trial need to be substantiated by further investigations.