Cysteine-rich protein reverses weight loss in lung cancer patients receiving chemotherapy or radiotherapy.

Oxidative stress plays a role in the tumor-cytotoxic effect of cancer chemotherapy and radiotherapy and also in certain adverse events. In view of these conflicting aspects, a double-blind trial over a 6-month period was performed to determine whether a cysteine-rich protein (IMN1207) may have a positive or negative effect on the clinical outcome if compared with casein, a widely used protein supplement low in cysteine. Sixty-six patients with stage IIIB-IV non-small cell lung cancer were randomly assigned to IMN1207 or casein. Included were patients with a previous involuntary weight loss of > or =3%, Karnofsky status > or =70, and an estimated survival of >3 months. Thirty-five lung cancer patients remained on study at 6 weeks. Overall compliance was not different between treatment arms (42-44% or 13 g/day). The patients treated with the cysteine-rich protein had a mean increase of 2.5% body weight, whereas casein-treated patients lost 2.6% (p = 0.049). Differences in secondary endpoints included an increase in survival, hand-grip force, and quality of life. Adverse events were mild or moderate. Further studies will have to show whether the positive clinical effects can be confirmed and related to specific parameters of oxidative stress in the host.

Aberrant insulin receptor signaling and amino acid homeostasis as a major cause of oxidative stress in aging.

The mechanisms leading to the increase in free radical-derived oxidative stress in "normal aging" remains obscure. Here we present our perspective on studies from different fields that reveal a previously unnoticed vicious cycle of oxidative stress. The plasma cysteine concentrations during starvation in the night and early morning hours (the postabsorptive state) decreases with age. This decrease is associated with a decrease in tissue concentrations of the cysteine derivative and quantitatively important antioxidant glutathione. The decrease in cysteine reflects changes in the autophagic protein catabolism that normally ensures free amino acid homeostasis during starvation. Autophagy is negatively regulated by the insulin receptor signaling cascade that is enhanced by oxidative stress in the absence of insulin. This synopsis of seemingly unrelated processes reveals a novel mechanism of progressive oxidative stress in which decreasing antioxidant concentrations and increasing basal (postabsorptive) insulin receptor signaling activity compromise not only the autophagic protein catabolism but also the activity of FOXO transcription factors (i.e., two functions that were found to have an impact on lifespan in several animal models of aging). In addition, the aging-related decrease in glutathione levels is likely to facilitate certain "secondary" disease-related mechanisms of oxidative stress. Studies on cysteine supplementation show therapeutic promise.

Oxidative stress and aberrant signaling in aging and cognitive decline.

Brain aging is associated with a progressive imbalance between antioxidant defenses and intracellular concentrations of reactive oxygen species (ROS) as exemplified by increases in products of lipid peroxidation, protein oxidation, and DNA oxidation. Oxidative conditions cause not only structural damage but also changes in the set points of redox-sensitive signaling processes including the insulin receptor signaling pathway. In the absence of insulin, the otherwise low insulin receptor signaling is strongly enhanced by oxidative conditions. Autophagic proteolysis and sirtuin activity, in turn, are downregulated by the insulin signaling pathway, and impaired autophagic activity has been associated with neurodegeneration. In genetic studies, impairment of insulin receptor signaling causes spectacular lifespan extension in nematodes, fruit flies, and mice. The predicted effects of age-related oxidative stress on sirtuins and autophagic activity and the corresponding effects of antioxidants remain to be tested experimentally. However, several correlates of aging have been shown to be ameliorated by antioxidants. Oxidative damage to mitochondrial DNA and the electron transport chain, perturbations in brain iron and calcium homeostasis, and changes in plasma cysteine homeostasis may altogether represent causes and consequences of increased oxidative stress. Aging and cognitive decline thus appear to involve changes at multiple nodes within a complex regulatory network.

Low postabsorptive net protein degradation in male cancer patients: lack of sensitivity to regulatory amino acids?

Autophagic (lysosomal) and proteasomic protein degradation are important regulatory mechanisms in the homeostasis of muscle mass, that may be profoundly disturbed in cancer and other wasting syndromes. Due to the inhibiting effect of amino acids and insulin, net proteolysis is restricted to the fasted state, and in autophagy certain amino acids have been identified as 'regulatory' in the rat, including leucine, tyrosine, phenylalanine, methionine, and histidine (i.e. LYFMH). The present cross-sectional study assessed postabsorptive net protein catabolism in male cancer patients as well as in healthy male volunteers, to analyse its relation to such 'regulatory amino acids'. Postabsorptive amino acid exchange rates across the leg were determined in patients with gastrointestinal cancer (GIC, n=47) or renal cell carcinoma (RCC, n=15), age-matched (n=33), and young male control subjects (n=42). Both groups of cancer patients revealed a significantly lower postabsorptive net protein catabolism than control subjects. Furthermore, in the control subjects, the postabsorptive net protein catabolism was found to be inversely and significantly correlated with the arterial concentrations of the 8 amino acids YSHMFGI and L which include 5 of the 'regulatory amino acids'. Cancer patients, in contrast, revealed no such significant correlations. These results may indicate i) that postabsorptive net protein catabolism in skeletal muscle of healthy subjects may be sensitive to amino acids which reportedly regulate autophagy and ii) that such amino acid-sensitive mechanism of protein catabolism may be disturbed in cancer patients.

Increased gene expression of scavenger receptors and proinflammatory markers in peripheral blood mononuclear cells of hyperlipidemic males.

Interactions between peripheral blood mononuclear cells (PBMCs) and those within plaques are suggested to be pathophysiologically relevant to lipid-induced arteriosclerosis. In this study, gene expressions of scavenger receptors (CD36, CD68), LPS receptor (CD14), proinflammatory (tumor necrosis factor alpha [TNFalpha], CD40, interleukin-1 beta [IL-1beta]) and oxidative stress-related (manganese superoxide dismutase [MnSOD]) markers were analyzed in PBMCs of clinically asymptomatic males with classical proatherogenic risk factors such as smoking and/or hyperlipidemia. PBMCs were isolated from venous blood of normolipidemic non-smokers (n = 10) and smokers (n = 8), and hyperlipidemic non-smokers (n = 9) and smokers (n = 8). RNA from PBMCs was used for PCR analyses. Plasma concentrations of oxidized low-density lipoproteins (oxLDL) were measured by ELISA. The gene expressions of CD36, CD68, CD40, TNFalpha, and MnSOD were significantly higher in PBMCs of hyperlipidemics than in normolipidemics, irrespective of whether they were smoking or not. The individual expression of these genes showed significant positive correlations with each other but also with serum cholesterol or plasma oxLDL concentrations. The higher expressions of scavenger receptors, proinflammatory and oxidative stress-related genes of PBMCs are suggested to result mainly from hyperlipidemia and the accompanied increase of oxLDL concentrations.

Low intestinal glutamine level and low glutaminase activity in Crohn's disease: a rational for glutamine supplementation?

Intestinal glutamine utilization is integral to mucosal regeneration. We analyzed the systemic and intestinal glutamine status in Crohn's disease (CD) and evaluated the therapeutic effect of glutamine supplementation in an animal model of ileitis. In CD, glutamine concentrations were decreased systemically and in noninflamed and inflamed ileal/colonic mucosa. Mucosal glutaminase activities were depressed in the ileum independent of inflammation but were not different from controls in the colon. In experimental ileitis, oral glutamine feeding prevented macroscopic inflammation, enhanced ileal and colonic glutaminase activities above controls, and normalized the intestinal glutathione redox status. However, glutamine supplementation enhanced myeloperoxidase activity along the gastrointestinal tract and potentiated lipid peroxidation in the colon. In conclusion, glutamine metabolism is impaired in CD. In experimental ileitis, glutamine supplementation prevents inflammatory tissue damage. In the colon, however, which does not use glutamine as its principal energy source, immune enhancement of inflammatory cells by glutamine increases oxidative tissue injury.

The deficit in low molecular weight thiols as a target for antiageing therapy.

The popular use of antioxidative vitamins illustrates the growing awareness of oxidative stress as an important hazard to our health and as an important factor in the ageing process. Superoxide radicals and superoxide-derived reactive oxygen species (ROS) are constantly formed in most cells and tissues. To ensure that ROS can function as biological signaling molecules without excessive tissue damage, ROS are typically scavenged by antioxidants such as glutathione and the vitamins A, C, and E. "Oxidative stress" occurs if the production of ROS is abnormally increased or antioxidant concentrations are decreased. Genetic studies in mice, Drosophila, and C.elegans suggested that ageing may be mechanistically linked to oxidative stress. Several manifestations of oxidative stress were shown to increase with age, whereas tissue levels of vitamin E, plasma concentrations of vitamin C, and intracellular glutathione concentrations decrease with age. In at least two independent studies, cysteine supplementation on top of the normal protein diet has shown significant beneficial effects on each of several different parameters relevant to ageing, including skeletal muscle functions. As the quality of life in old age is severely compromised by the loss of skeletal muscle function, and as muscle function can be measured with satisfactory precision, loss of muscle function is one of the most attractive surrogate parameters of ageing. The mechanisms by which a deficit in glutathione and its precursor cysteine contributes to various ageing-related degenerative processes appears to be related largely but not exclusively to the dysregulation of redox-regulated biological signaling cascades.

Hydrogen peroxide inhibits activity of the IGF-1 receptor kinase.

IGF-1 receptor (IGF1R) is a transmembrane tyrosine kinase, which is indispensable for cellular growth and differentiation. Using a recombinant GST-tagged cytosolic fragment of IGF1R (GST-IGFK), we now show that oxidation by low doses (50 muM) of hydrogen peroxide markedly inhibits maximum phosphate incorporation in autophosphorylation and substrate phosphorylation assays. A similar inhibition was observed on the activity of intact IGF1R after treatment of T-47D cells. These results are in sharp contrast to the positive influence of hydrogen peroxide on the highly homologous insulin receptor kinase, which was assayed for comparison. This reciprocal influence of physiologically relevant doses of hydrogen peroxide may have important implications on signal transduction of the closely related receptors for insulin and IGF-1.

Redox regulation in anabolic and catabolic processes.

PURPOSE OF REVIEW: Muscle wasting as it typically occurs in old age and in certain diseases is poorly understood. This review summarizes recent findings suggesting a role for redox-sensitive signaling cascades in catabolic processes. RECENT FINDINGS: The redox-sensitive transcription factors nuclear factor kappaB and activator protein 1 facilitate ubiquitin-proteasome-dependent proteolysis. Nuclear factor kappaB also plays a role in induced expression of tumor necrosis factor alpha and other inflammatory cytokines that have been implicated in catabolic processes. The activities of nuclear factor kappaB and activator protein 1 are stimulated not only by hydrogen peroxide, which is produced in tissues by regulated enzymatic processes, but also by an oxidative shift in thiol-disulfide redox status. The oxidative shift that is typically seen in old age and certain catabolic conditions may thus play a causative role in catabolic processes. Another prominent case in point is insulin-independent 'basal' insulin receptor kinase activity, which is strongly enhanced by hydrogen peroxide or by an oxidative shift in redox status. The insulin receptor signaling cascade induces anabolic and anticatabolic effects, but its abnormal upregulation under starving conditions potentially compromises glucose and amino acid homeostasis. In genetic animal studies, impairment of insulin receptor signaling was shown to increase life span. SUMMARY: These findings may provide a rationale for cysteine supplementation in catabolic conditions.

Oxidative stress and ageing: is ageing a cysteine deficiency syndrome?

Reactive oxygen species (ROS) are constantly produced in biological tissues and play a role in various signalling pathways. Abnormally high ROS concentrations cause oxidative stress associated with tissue damage and dysregulation of physiological signals. There is growing evidence that oxidative stress increases with age. It has also been shown that the life span of worms, flies and mice can be significantly increased by mutations which impede the insulin receptor signalling cascade. Molecular studies revealed that the insulin-independent basal activity of the insulin receptor is increased by ROS and downregulated by certain antioxidants. Complementary clinical studies confirmed that supplementation of the glutathione precursor cysteine decreases insulin responsiveness in the fasted state. In several clinical trials, cysteine supplementation improved skeletal muscle functions, decreased the body fat/lean body mass ratio, decreased plasma levels of the inflammatory cytokine tumour necrosis factor alpha (TNF-alpha), improved immune functions, and increased plasma albumin levels. As all these parameters degenerate with age, these findings suggest: (i) that loss of youth, health and quality of life may be partly explained by a deficit in cysteine and (ii) that the dietary consumption of cysteine is generally suboptimal and everybody is likely to have a cysteine deficiency sooner or later.

Oxidative aging and insulin receptor signaling.

The life span of nematodes, fruit flies, and mice can be significantly increased (and aging-related changes decreased) by mutations affecting insulin receptor signaling. This effect involves several cellular functions which are negatively regulated by the insulin receptor and thus typically expressed under fasting conditions. This involvement raises the question of whether the insulin-independent basal receptor kinase activity in the postabsorptive state can be decreased without compromising the physiologically important response to insulin in the postprandial state. Recent studies have shown that (a) the basal human insulin receptor kinase activity is increased under oxidative conditions in the absence of insulin and (b) insulin signaling in the fasted state can be decreased by cysteine supplementation. Cysteine supplementation has also been shown to improve certain aging-related parameters, suggesting that the average dietary cysteine consumption in Western countries may be suboptimal. These findings provide a conceptual framework that extends the "free radical theory of aging."

Oxidative enhancement of insulin receptor signaling: experimental findings and clinical implications.

Signaling through the insulin receptor and several other receptor tyrosine kinases is subject to redox regulation. Prolonged exposure to hydrogen peroxide impairs the action of insulin, and may account to some extent for the decreased insulin responsiveness in hyperglycemic diabetic patients. However, insulin receptor kinase (IRK) autophosphorylation and/or kinase activity were found to be markedly enhanced by a more limited exposure to hydrogen peroxide or by an oxidative shift in the thiol/disulfide redox status. Oxidative enhancement of IRK function may be mediated by two different mechanisms with similar effects, i.e., by direct oxidative activation of IRK activity or by oxidative inactivation of a protein tyrosine phosphatase, which otherwise down-regulates IRK-mediated signaling. As both mechanisms enhance IRK activity in the absence of insulin, there is a strong possibility that the background IRK activity in the postabsorptive period may be abnormally increased in certain oxidative conditions and thereby disturb the metabolism of glucose and other energy substrates. This remains to be tested. In line with the oxidative enhancement of IRK activity, clinical studies have shown that treatment with a thiol-containing antioxidant increases the postabsorptive glucose and/or insulin concentrations (i.e., the HOMA-R index) at least under certain conditions. This effect may have therapeutic implications.

Autophagy and aging: the importance of maintaining "clean" cells.

A decrease in the turnover of cellular components and the intracellular accumulation of altered macromolecules and organelles are features common to all aged cells. Diminished autophagic activity plays a major role in these age-related manifestations. In this work we review the molecular defects responsible for the malfunctioning of two forms of autophagy, macroautophagy and chaperone-mediated autophagy, in old mammals, and highlight general and cell-type specific consequences of dysfunction of the autophagic system with age. Dietary caloric restriction and antilipolytic agents have been proven to efficiently stimulate autophagy in old rodents. These and other possible experimental restorative efforts are discussed.

Interdependent regulation of insulin receptor kinase activity by ADP and hydrogen peroxide.

Insulin signaling requires autophosphorylation of the insulin receptor kinase (IRK) domain. Using purified recombinant IRK fragments and the isolated intact insulin receptor, we show here that autophosphorylation is inhibited by ADP and that this effect is essentially reversed by hydrogen peroxide. Autophosphorylation was inhibited by hydrogen peroxide (60 microM) in the absence of ADP but enhanced in the presence of inhibitory concentrations of ADP (67 microM). Enhancement by hydrogen peroxide required direct interaction of hydrogen peroxide with the kinase domain and was not seen in insulin receptor mutants C1245A and C1308A. A similar enhancement was obtained in intact cells in the absence of insulin upon treatment with 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea, indicating that IRK activity can be alternatively enhanced by a shift in the thiol/disulfide redox status. Molecular modeling of the IRK domain indicated that the ATP-binding site becomes distorted after releasing the nucleotide unless the IRK domain is oxidatively derivatized at Cys1245. Recent clinical studies suggest that these effects may play a role in obesity due to the fact that cytoplasmic creatine kinase in combination with phosphocreatine normally ensures rapid removal of ADP in muscle cells but not in fat cells.

Increased cyclooxygenase-2 expression in peripheral blood mononuclear cells of smokers and hyperlipidemic subjects.

Cyclooxygenase (COX)-2 is expressed in macrophages of arteriosclerotic lesions and promotes inflammation. We investigated whether COX-2 is already expressed in peripheral blood mononuclear cells (PBMCs) of subjects possessing risk-related factors, such as in smokers and hyperlipidemics. PBMCs were isolated from the venous blood of normolipidemic nonsmokers (NL-NSM; n = 15), normolipidemic smokers (NL-SM; n = 12), hyperlipidemic nonsmokers (HL-NSM; n = 10), and hyperlipidemic smokers (HL-SM; n = 10). RNA from PBMCs was used for RT-PCR. Plasma concentrations of oxidized low-density lipoproteins (oxLDL) were measured by ELISA, those of glutamate and cystine by HPLC. The results show that COX-2 expression in PBMCs was significantly increased in the groups with cardiovascular risk factors (NL-SM, HL-SM, HL-NSM) compared with NL-NSM. COX-2 expression in PBMCs was positively correlated with concentrations of total serum cholesterol, oxLDL, glutamate, or cystine. We suggest that the elevated COX-2 expression indicates a priming of PBMCs as a response to a systemic pro-oxidative and proinflammatory shift in subjects with cardiovascular risk factors, which might also contribute to growth and instability of arteriosclerotic lesions.

Autophagy and aging--importance of amino acid levels.

Melendez et al. [Science 301 (2003) 1387] have recently shown that the increased longevity of Caenorhabditis elegans mutants with defective Daf-2 protein, i.e. an insulin receptor analog, involves increased autophagy. Autophagy increases the free amino acid pool and is in certain cells essential for survival at times of limited amino acid availability. In addition, autophagy plays an important role in the turnover of proteins and organelles including mitochondria. The autophagic activity is sensitive to changes in physiological conditions, i.e. it is strongly inhibited by an increase in amino acid concentrations or in insulin receptor signaling. In line with this fact, clinical studies indicate that autophagy mainly occurs at times of low plasma amino acid and insulin concentrations in the post-absorptive (fasted) state, and that the post-absorptive amino acid-sensitive protein catabolism may be taken as a bona fide indicator of autophagic activity. The increased longevity of insulin receptor mutants or of organisms subjected to calorie restriction may, therefore, be attributed to an increase in autophagic activity. Importantly, the autophagic activity decreases with age. Recent studies suggest that this decrease may result from an age-related increase in post-absorptive amino acid levels and/or from an increase in baseline insulin receptor signaling. If so, it is potentially reversible.

Effect of thiol antioxidant on body fat and insulin reactivity.

Insulin signaling is enhanced by moderate concentrations of reactive oxygen species (ROS) and suppressed by persistent exposure to ROS. Diabetic patients show abnormally high ROS levels and a decrease in insulin reactivity which is ameliorated by antioxidants, such as N-acetylcysteine (NAC). A similar effect of NAC has not been reported for non-diabetic subjects. We now show that the insulin receptor (IR) kinase is inhibited in cell culture by physiologic concentrations of cysteine. In two double-blind trials involving a total of 140 non-diabetic subjects we found furthermore that NAC increased the HOMA-R index (derived from the fasting insulin and glucose concentrations) in smokers and obese patients, but not in nonobese non-smokers. In obese patients NAC also caused a decrease in glucose tolerance and body fat mass. Simultaneous treatment with creatine, a metabolite utilized by skeletal muscle and brain for the interconversion of ADP and ATP, reversed the NAC-mediated increase in HOMA-R index and the decrease in glucose tolerance without preventing the decrease in body fat. As the obese and hyperlipidemic patients had lower plasma thiol concentrations than the normolipidemic subjects, our results suggest that low thiol levels facilitate the development of obesity. Supplementation of thiols plus creatine may reduce body fat without compromising glucose tolerance.

Sp100 is important for the stimulatory effect of homeodomain-interacting protein kinase-2 on p53-dependent gene expression.

HIPK2 shows overlapping localization with p53 in promyelocytic leukemia (PML) nuclear bodies (PML-NBs) and functionally interacts with p53 to increase gene expression. Here we demonstrate that HIPK2 and the PML-NB resident protein Sp100 synergize for the activation of p53-dependent gene expression. Sp100 and HIPK2 interact and partially colocalize in PML-NBs. The cooperation of HIPK2 and Sp100 for the induction of p21(Waf1) is completely dependent on the presence of p53 and the kinase function of HIPK2. Downregulation of Sp100 levels by expression of siRNA does not interfere with p53-mediated transcription, but obviates the enhancing effect of HIPK2. In summary, these experiments reveal a novel function for Sp100 as a coactivator for HIPK2-mediated p53 activation.

Cholesterol levels linked to abnormal plasma thiol concentrations and thiol/disulfide redox status in hyperlipidemic subjects.

Treatment of hyperlipidemic patients with the thiol compound N-acetylcysteine (NAC) was previously shown to cause a significant dose-related increase in the high-density lipoprotein (HDL)-cholesterol serum level, suggesting the possibility that its disease-related decrease may result from a diminished thiol concentration and/or thiol/disulfide redox status (REDST) in the plasma. We therefore investigated plasma thiol levels and REDST in normo-/hyperlipidemic subjects with and without coronary heart disease (CHD). The thiol level, REDST, and amino acid concentrations in the plasma and intracellular REDST of peripheral blood mononuclear cells (PBMC) have been determined in 62 normo- and hyperlipidemic subjects. Thirty-three of these subjects underwent coronary angiography, because of clinical symptoms of CHD. All groups of hyperlipidemic patients under test and those normolipidemic individuals with documented coronary stenoses showed a marked decrease in plasma thiol concentrations, plasma and intracellular REDST of PBMCs, and a marked increase in plasma taurine levels. Individual plasma thiol concentrations and plasma REDST were strongly negatively correlated with the serum LDL-cholesterol and positively correlated with the serum HDL-cholesterol level. Together with the earlier report about the effect of NAC on the HDL-cholesterol serum level, our findings suggest strongly that lower HDL-cholesterol serum levels may result from a decrease in plasma thiol level and/or REDST possibly through an excessive cysteine catabolism into taurine.

PML is required for homeodomain-interacting protein kinase 2 (HIPK2)-mediated p53 phosphorylation and cell cycle arrest but is dispensable for the formation of HIPK domains.

Here we demonstrate that endogenous human homeodomain-interacting protein kinase (HIPK) 2 and the highly homologous kinase HIPK3 are found in a novel subnuclear domain, the HIPK domains. These are distinct from other subnuclear structures such as Cajal bodies and nucleoli and show only a partial colocalization with promyelocytic leukemia (PML) nuclear bodies (PML-NBs). A kinase inactive HIPK2 point mutant is localized in the nucleoplasm. The occurrence of HIPK domains in PML-/- fibroblasts reveals their independence from the PML protein. HIPK2 can be almost completely recruited to PML-NBs by the PML isoform PML IV, but not by PML-III. PML IV-mediated recruitment of HIPK2 does not rely on its kinase function and also occurs in PML-/- fibroblasts, showing that this PML isoform is sufficient for recruitment of HIPK2. Whereas the architecture of HIPK domains is PML independent, HIPK2-mediated enhancement of p53-dependent transcription, p53 serine 46 phosphorylation and the antiproliferative function of HIPK2 strictly rely on the presence of PML.