N-acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Systemic lupus erythematosus (SLE) patients exhibit T cell dysfunction, which can be regulated through mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione (GSH). This randomized, double-blind, placebo-controlled study was undertaken to examine the safety, tolerance, and efficacy of the GSH precursor N-acetylcysteine (NAC). METHODS: A total of 36 SLE patients received either daily placebo or 1.2 gm, 2.4 gm, or 4.8 gm of NAC. Disease activity was evaluated monthly by the British Isles Lupus Assessment Group (BILAG) index, the SLE Disease Activity Index (SLEDAI), and the Fatigue Assessment Scale (FAS) before, during, and after a 3-month treatment period. Mitochondrial transmembrane potential and mTOR were assessed by flow cytometry. Forty-two healthy subjects matched to patients for age, sex, and ethnicity were studied as controls. RESULTS: NAC up to 2.4 gm/day was tolerated by all patients, while 33% of those receiving 4.8 gm/day had reversible nausea. Placebo or NAC 1.2 gm/day did not influence disease activity. Considered together, 2.4 gm and 4.8 gm NAC reduced the SLEDAI score after 1 month (P = 0.0007), 2 months (P = 0.0009), 3 months (P = 0.0030), and 4 months (P = 0.0046); the BILAG score after 1 month (P = 0.029) and 3 months (P = 0.009); and the FAS score after 2 months (P = 0.0006) and 3 months (P = 0.005). NAC increased Δψm (P = 0.0001) in all T cells, profoundly reduced mTOR activity (P = 0.0009), enhanced apoptosis (P = 0.0004), reversed expansion of CD4-CD8- T cells (mean ± SEM 1.35 ± 0.12-fold change; P = 0.008), stimulated FoxP3 expression in CD4+CD25+ T cells (P = 0.045), and reduced anti-DNA production (P = 0.049). CONCLUSION: This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.

Comprehensive metabolome analyses reveal N-acetylcysteine-responsive accumulation of kynurenine in systemic lupus erythematosus: implications for activation of the mechanistic target of rapamycin.

Systemic lupus erythematosus (SLE) patients exhibit depletion of the intracellular antioxidant glutathione and downstream activation of the metabolic sensor, mechanistic target of rapamycin (mTOR). Since reversal of glutathione depletion by the amino acid precursor, N-acetylcysteine (NAC), is therapeutic in SLE, its mechanism of impact on the metabolome was examined within the context of a double-blind placebo-controlled trial. Quantitative metabolome profiling of peripheral blood lymphocytes (PBL) was performed in 36 SLE patients and 42 healthy controls matched for age, gender, and ethnicity of patients using mass spectrometry that covers all major metabolic pathways. mTOR activity was assessed by western blot and flow cytometry. Metabolome changes in lupus PBL affected 27 of 80 KEGG pathways at FDR p < 0.05 with most prominent impact on the pentose phosphate pathway (PPP). While cysteine was depleted, cystine, kynurenine, cytosine, and dCTP were the most increased metabolites. Area under the receiver operating characteristic curve (AUC) logistic regression approach identified kynurenine (AUC = 0.859), dCTP (AUC = 0.762), and methionine sulfoxide (AUC = 0.708), as top predictors of SLE. Kynurenine was the top predictor of NAC effect in SLE (AUC = 0.851). NAC treatment significantly reduced kynurenine levels relative to placebo in vivo (raw p = 2.8 × 10-7, FDR corrected p = 6.6 × 10-5). Kynurenine stimulated mTOR activity in healthy control PBL in vitro. Metabolome changes in lupus PBL reveal a dominant impact on the PPP that reflect greater demand for nucleotides and oxidative stress. The PPP-connected and NAC-responsive accumulation of kynurenine and its stimulation of mTOR are identified as novel metabolic checkpoints in lupus pathogenesis.

Assessment of mitochondrial dysfunction in lymphocytes of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by abnormal activation and cell death signaling within the immune system. Activation, proliferation, or death of cells of the immune system is dependent on controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mitochondrial transmembrane potential (∆ψ (m)) reflects the energy stored in the electrochemical gradient across the inner mitochondrial membrane which, in turn, is used by F(0)F(1)-ATPase to convert ADP to ATP during oxidative phosphorylation. Mitochondrial hyperpolarization (MHP) and transient ATP depletion represent early and reversible steps in T cell activation and apoptosis. By contrast, T lymphocytes of patients with SLE exhibit elevated ∆ψ (m), i.e., persistent mitochondrial hyperpolarization (MHP), cytoplasmic alkalinization, increased ROI production, as well as diminished levels of intracellular glutathione and ATP. Increased production of nitric oxide has been identified as a cause of MHP and increased mitochondrial biogenesis. Oxidative stress affects signaling through the T cell receptor as well as activity of redox--sensitive caspases. ATP depletion causes diminished activation-induced apoptosis and sensitizes lupus T cells to necrosis. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key sensor of MHP and mediator of enhanced Ca(2+) flux in lupus T cells.

Oxidative stress, inflammation and carcinogenesis are controlled through the pentose phosphate pathway by transaldolase.

Metabolism of glucose through the pentose phosphate pathway (PPP) influences the development of diverse pathologies. Hemolytic anemia due to deficiency of PPP enzyme glucose 6-phosphate dehydrogenase is the most common genetic disease in humans. Recently, inactivation of another PPP enzyme, transaldolase (TAL), has been implicated in male infertility and fatty liver progressing to steatohepatitis and cancer. Hepatocarcinogenesis was associated with activation of aldose reductase and redox-sensitive transcription factors and prevented by N-acetylcysteine. In this paper, we discuss how alternative formulations of the PPP with and without TAL reflect cell type-specific metabolic control of oxidative stress, a crucial source of inflammation and carcinogenesis. Ongoing studies of TAL deficiency will identify new molecular targets for diagnosis and treatment in clinical practice.

Prevention of hepatocarcinogenesis and increased susceptibility to acetaminophen-induced liver failure in transaldolase-deficient mice by N-acetylcysteine.

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1-/- and Taldo1+/- mice spontaneously developed HCC, and Taldo1-/- mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1-/- livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced beta-catenin phosphorylation and enhanced c-Jun expression in Taldo1-/- livers reflected adaptation to oxidative stress. Taldo1-/- hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1-/- mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.