Proximal tubular dysfunction in pregnant women receiving tenofovir disoproxil fumarate to prevent mother-to-child transmission of hepatitis B virus.

BACKGROUND: Data evaluating the risk of proximal tubular dysfunction in women receiving tenofovir disoproxil fumarate for the prevention of mother-to-child transmission (PMTCT) of HBV are scarce. OBJECTIVES: To assess the risk of proximal tubulopathy in pregnant women receiving tenofovir disoproxil fumarate for PMTCT of HBV. PATIENTS AND METHODS: We used urine samples collected from HBV monoinfected pregnant women who participated in a Phase III, multicentre, randomized, double-blind, placebo-controlled clinical trial assessing a tenofovir disoproxil fumarate short course from 28 weeks gestational age (28-wk-GA) to 2 months post-partum (2-months-PP) for PMTCT of HBV in Thailand. Markers of tubular dysfunction, including retinol binding protein, kidney injury molecule-1, α1-microglobuin and ß2-microglobulin, were assayed at 28- and 32-wk-GA and 2-months-PP visits. Proximal tubulopathy was defined as the presence of ≥2 of the following: tubular proteinuria, euglycaemic glycosuria and increased urinary phosphate. RESULTS: A total of 291 women participated in the study. No kidney-related adverse events were severe, and none led to tenofovir disoproxil fumarate discontinuation. At 2-months-PP, 3 of the 120 (3%) evaluated women in the tenofovir disoproxil fumarate group experienced proximal tubulopathy versus 3 of 125 (2%) in the placebo group (P = 1.00). None of the six women met the criteria for proximal tubulopathy at 12-months-PP but proteinuria persisted in three of them. No growth abnormalities were found at 1 year of age in infants born to mothers with proximal tubulopathy at 2-months-PP. CONCLUSIONS: In these HBV-infected pregnant and breastfeeding women, tenofovir disoproxil fumarate administered from 28-wk-GA to 2-months-PP was not associated with a higher risk of proximal tubulopathy.

Effects of ascorbic acid supplementation on oxidative stress markers in healthy women following a single bout of exercise.

BACKGROUND: Ascorbic acid is a water-soluble chain breaking antioxidant. It scavenges free radicals and reactive oxygen species (ROS), which are produced during metabolic pathways. Exercise can produce an imbalance between ROS and antioxidants, leading to oxidative stress-related tissue damages. This study was designed to determine the effects of ascorbic acid supplementation on circulating biomarkers of oxidative stress and muscle damage following a single bout of exercise. METHODS: In a crossover design with a 1 wk. wash-out period, 19 healthy women performed 30 min moderate-intensity cycling after ingesting 1000 mg of ascorbic acid (AA) or placebo. Blood samples were taken immediately before, immediately after and 30 min post-exercise to determine plasma albumin, total protein, glucose, oxidative stress and muscle damage markers. RESULTS: Plasma albumin and total protein levels increased immediately after exercise in placebo alongside slight reductions in glucose (p = 0.001). These effects were absent in AA cohort. Ferric reducing ability of plasma and vitamin C levels in AA cohort significantly increased after exercise (p < 0.05). Superoxide dismutase activity was significantly elevated after exercise (p = 0.002) in placebo but not AA. Plasma malondialdehyde did not change after exercise in placebo but was significantly decreased in AA (p < 0.05). The exercise protocol promoted slight muscle damage, reflected in significant increases in total creatine kinase in all subjects after exercise. On the other hand, plasma C-reactive protein and lactate dehydrogenase remained unchanged. CONCLUSION: Supplementation with ascorbic acid prior exercise improves antioxidant power but does not prevent muscle damage.

Ascorbic acid supplementation does not alter oxidative stress markers in healthy volunteers engaged in a supervised exercise program.

The purpose of this study was to investigate the impact of ascorbic acid (AA) consumption on the oxidative stress status of untrained volunteers participating in a supervised exercise program. The study included 46 young adults (average age, 23.5 ± 0.59 years; 37 females, 9 males) who remained sedentary (n = 16) or participated in 30 min of outdoor aerobic running (n = 30) at an intensity corresponding to 65%-75% of maximum heart rate for 3 times per week for 12 weeks. Exercised subjects were randomly assigned to an exercise group without AA supplementation (control; n = 10) or received either 250 mg (n = 10) or 500 mg (n = 10) of AA supplementation previous to each exercise session. Blood samples were taken on day 0 and day 84 to evaluate metabolic profiles and antioxidant status. Sedentary subjects underwent in a single bout of aerobic running to determine total antioxidant status (TAS) and malondiadehyde (MDA) at pre- and postexercise with or without AA supplementation. No significant change in TAS was observed. Plasma MDA significantly increased at postexercise (P < 0.05), and AA supplementation decreased MDA level significantly (P < 0.05). After 3 months of exercise, there was no significant change in blood glucose, lipid profile, MDA, TAS, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase activities amongst groups. Supplementation of AA was associated with minor and inconsistent reductions in SOD, GPx, and catalase activities (P < 0.05). These findings indicate that pre-exercise supplementation of ascorbic acid does not alter oxidative stress markers in the plasma and erythrocytes of young adults engaged in a supervised exercise program.

General control nonderepressible 2 (GCN2) kinase protects oligodendrocytes and white matter during branched-chain amino acid deficiency in mice.

Branched-chain amino acid (BCAA) catabolism is regulated by branched-chain α-keto acid dehydrogenase, an enzyme complex that is inhibited when phosphorylated by its kinase (BDK). Loss of BDK function in mice and humans causes BCAA deficiency and epilepsy with autistic features. In response to amino acid deficiency, phosphorylation of eukaryotic initiation factor 2α (eIF2∼P) by general control nonderepressible 2 (GCN2) activates the amino acid stress response. We hypothesized that GCN2 functions to protect the brain during chronic BCAA deficiency. To test this idea, we generated mice lacking both Gcn2 and Bdk (GBDK) and examined the development of progeny. GBDK mice appeared normal at birth, but they soon stopped growing, developed severe ataxia, tremor, and anorexia, and died by postnatal day 15. BCAA levels in brain were diminished in both Bdk(-/-) and GBDK pups. Brains from Bdk(-/-) pups exhibited robust eIF2∼P and amino acid stress response induction, whereas these responses were absent in GBDK mouse brains. Instead, myelin deficiency and diminished expression of myelin basic protein were noted in GBDK brains. Genetic markers of oligodendrocytes and astrocytes were also reduced in GBDK brains in association with apoptotic cell death in white matter regions of the brain. GBDK brains further demonstrated reduced Sod2 and Cat mRNA and increased Tnfα mRNA expression. The data are consistent with the idea that loss of GCN2 during BCAA deficiency compromises glial cell defenses to oxidative and inflammatory stress. We conclude that GCN2 protects the brain from developing a lethal leukodystrophy in response to amino acid deficiencies.

The eukaryotic initiation factor 2 kinase GCN2 protects against hepatotoxicity during asparaginase treatment.

Asparaginase is an important drug in the treatment regimen for acute lymphoblastic leukemia. Asparaginase depletes circulating asparagine and glutamine, activating an amino acid stress response (AAR) involving phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible kinase 2 (GCN2). We hypothesized that GCN2 functions to mitigate hepatic stress during asparaginase therapy by activating the AAR. To test this idea, C57BL/6J wild-type mice (Gcn2(+/+)) and those deleted for Gcn2 (Gcn2(-/-)) were injected with asparaginase or saline excipient one time daily for 1 or 6 days. In liver, increased phosphorylation of eIF2 and mRNA expression of AAR target genes activating transcription factor 4, asparagine synthetase, eIF4E-binding protein 1, and CAAT enhancer-binding protein homologous protein were significantly blunted or blocked in the liver of Gcn2(-/-) mice. Loss of AAR during asparaginase coincided with increases in mammalian target of rapamycin signaling, hepatic triglyceride accumulation, and DNA damage in association with genetic markers of oxidative stress (glutathione peroxidase) and inflammation (tumor necrosis factor alpha-α). Although asparaginase depleted circulating asparagine in both Gcn2(+/+) and Gcn2(-/-) mice, all other amino acids, including plasma glutamine, were elevated in the plasma of Gcn2(-/-) mice. This study shows that loss of GCN2 promotes oxidative stress and inflammatory-mediated DNA damage during asparaginase therapy, suggesting that patients with reduced or dysfunctional AAR may be at risk of developing hepatic complications during asparaginase treatment.

The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress.

Disruptions of the endoplasmic reticulum (ER) that perturb protein folding cause ER stress and elicit an unfolded protein response (UPR) that involves translational and transcriptional changes in gene expression aimed at expanding the ER processing capacity and alleviating cellular injury. Three ER stress sensors (PERK, ATF6, and IRE1) implement the UPR. PERK phosphorylation of the α subunit of eIF2 during ER stress represses protein synthesis, which prevents further influx of ER client proteins. Phosphorylation of eIF2α (eIF2α~P) also induces preferential translation of ATF4, a transcription activator of the integrated stress response. In this study we show that the PERK/eIF2α~P/ATF4 pathway is required not only for translational control, but also for activation of ATF6 and its target genes. The PERK pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi for intramembrane proteolysis and activation of ATF6. As a consequence, liver-specific depletion of PERK significantly reduces both the translational and transcriptional phases of the UPR, leading to reduced protein chaperone expression, disruptions of lipid metabolism, and enhanced apoptosis. These findings show that the regulatory networks of the UPR are fully integrated and help explain the diverse biological defects associated with loss of PERK.

The eIF2 kinase GCN2 is essential for the murine immune system to adapt to amino acid deprivation by asparaginase.

Amino acid starvation by asparaginase (ASNase) enhances phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible 2 (GCN2) kinase, leading to reduced global mRNA translation rates. This conserves energy and allows cells time to reprogram stress-related gene expression to alleviate cell injury. This study addressed the importance of GCN2 for the immune system to adapt to amino acid starvation by ASNase. GCN2(+/+) and GCN2(-/-) mice were injected once daily with ASNase or saline for up to 7 d. In both thymus and spleen, activation of amino acid stress response genes to ASNase, such as asparagine synthetase and CAAT enhancer binding protein homologous protein, required GCN2. ASNase reduced food intake and body weight in both genotypes, but spleen and thymus wet weights and total cell numbers in thymus, spleen, bone marrow, and mesenteric lymph nodes were less in GCN2(-/-) mice treated with ASNase (genotype x ASNase, P < 0.05). In the thymus, GCN2(-/-) mice treated with ASNase demonstrated enhanced apoptosis and fewer cells in all subpopulations examined (CD3+, CD4-8-, CD4+8+, CD4+8-, CD4-8+) compared with GCN2(+/+) mice treated with ASNase (genotype x ASNase, P < 0.05). In the spleen, GCN2 deletion magnified ASNase-induced reductions in CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+ leukocytes (genotype x ASNase, P < 0.05). These results indicate that loss of GCN2 enhances immunosuppression by ASNase and that this eIF2 kinase is broadly required for amino acid stress management in the immune system.

GCN2 protein kinase is required to activate amino acid deprivation responses in mice treated with the anti-cancer agent L-asparaginase.

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein beta homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.

The leucine content of a complete meal directs peak activation but not duration of skeletal muscle protein synthesis and mammalian target of rapamycin signaling in rats.

This study examined the impact of leucine (Leu) derived from complete meals on stimulation of skeletal muscle protein synthesis (MPS). Expt. 1 examined time course changes in translation initiation and MPS after a meal. Male rats ( approximately 300 g) were trained for 5 d to eat 3 meals/d providing 20, 50, and 30% of energy from whey protein, carbohydrates, and fats, respectively. Plasma and skeletal muscle were collected at time 0 (baseline) after 12 h of food deprivation and then at 45, 90, 135, 180, and 300 min after a 4-g meal. Plasma Leu increased at 45 min and remained elevated through 180 min. MPS peaked at 45-90 min and returned to baseline by 180 min. Plasma Leu correlated with phosphorylation of ribosomal protein p70 S6 kinase (r = 0.723; P < 0.05), eukaryotic initiation factor 4E binding protein-1 (r = 0.773; P < 0.05), and MPS (r = 0.608; P < 0.05) over time. Expt. 2 examined 3 levels of protein intake (10, 20, and 30% of energy) from 2 sources (wheat and whey) with different Leu contents ( approximately 6.8 and approximately 10.9%, respectively) on stimulation of initiation and MPS. Rats were trained to eat 3 meals/d providing 14, 56, and 30% of energy from protein, carbohydrates, and fats. On d 6, MPS was evaluated at 90 min after rats consumed 1 of the 6 test meals. Whey protein stimulated initiation and MPS more than wheat and the differential response related to greater plasma Leu responses in the whey groups. These studies demonstrate that peak activation but not duration of MPS is proportional to the Leu content of a meal.

Alanyl-glutamine consumption modifies the suppressive effect of L-asparaginase on lymphocyte populations in mice.

Asparaginase (Elspar) is used in the treatment of acute lymphoblastic leukemia. It depletes plasma asparagine and glutamine, killing leukemic lymphoblasts but also causing immunosuppression. The objective of this work was to assess whether supplementing the diet with glutamine modifies the effect of asparaginase on normal lymphocyte populations in the spleen, thymus, and bone marrow. Mice consuming water ad libitum with or without alanyl-glutamine dipeptide (AlaGln; 0.05 mol/L) were injected once daily with 0 or 3 international units/g body weight Escherichia coli L-asparaginase for 7 d. Tissue expression of specific immune cell surface markers was analyzed by flow cytometry. Asparaginase reduced B220+ and sIgM+ cells in the bone marrow (P < 0.05) and diminished total cell numbers in thymus (-42%) and spleen (-53%) (P < 0.05). In thymus, asparaginase depleted double positive (CD4+ CD8+) and single positive (CD4+ CD8-, CD4-CD8+) thymocytes by over 40% (P < 0.05). In spleen, asparaginase reduced CD19+ B cells to 33% of controls and substantially depleted the CD4+ and CD8+ T cell populations. CD11b-expressing leukocytes were reduced by 50% (P < 0.05). Consumption of AlaGln did not lessen the effects of asparaginase in bone marrow or thymus but mitigated cellular losses in the CD4+, CD8+, and CD11b+ populations in spleen. AlaGln also blunted the increase in eukaryotic initiation factor 2 (eIF2) phosphorylation by asparaginase in spleen, whereas eIF2 phosphorylation did not change in thymus in response to asparaginase or AlaGln. In conclusion, asparaginase reduces maturing populations of normal B and T cells in thymus, bone marrow, and spleen. Oral consumption of AlaGln mitigates metabolic stress in spleen, supporting the peripheral immune system and cell-mediated immunity during asparaginase chemotherapy.

Feeding meals containing soy or whey protein after exercise stimulates protein synthesis and translation initiation in the skeletal muscle of male rats.

The purpose of this investigation was to compare the early response of skeletal muscle protein synthesis and translation initiation following the ingestion of different protein sources after endurance exercise. Treadmill-acclimated rats were designated as either nonexercised controls (NEX) or treadmill exercised for 2 h at 26 m/min (approximately 75% VO2max) and then fed either carbohydrate only (EC), carbohydrate plus soy protein (ES), or carbohydrate plus whey protein (EW). One hour after exercise, serum insulin concentrations in EC, ES, and EW were greater than in NEX (P<0.05); the concentration in EW was greater than in EC, with that in ES intermediate. Serum concentrations of branched-chain amino acids in ES and EW were higher than in EC, but serum leucine and isoleucine in EW were higher than in ES (P<0.05). Nevertheless, both ES and EW promoted the fractional rate of skeletal muscle protein synthesis significantly more than EC. Likewise, compared with EC, both ES and EW increased formation of the mRNA cap binding complex eIF4F and stimulated phosphorylation of the translational repressor, 4E-BP1, the 70kD ribosomal protein S6 kinase (S6K1), and the mammalian target of rapamycin (mTOR) kinase at serine 2448. On the other hand, phosphorylation of S6K1 and mTOR was greater in EW than in ES (P<0.05). In conclusion, general protein synthesis and the mRNA cap binding step are promoted comparably by soy protein and whey protein in the skeletal muscle of exercised rats. Furthermore, the data suggest that mTOR signaling in skeletal muscle is acutely responsive to physiological variations in dietary amino acids.

Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase.

L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.

Inhibitory effects of asiatic acid and CPT-11 on growth of HT-29 cells.

Asiatic acid is a pentacyclic triterpene contained in medicinal plants. The cytotoxic effect of this compound and its augmentative effect on the anticancer drug irinotecan hydrochloride (CPT-11) were investigated in the human colon adenocarcinoma cell line HT-29. Asiatic acid dose-dependently showed cytotoxicity in HT-29 cells. DNA fragmentation, annexin-positive apoptotic cells, and caspase-3 activation were observed in a dose-dependent manner. A caspase-3 inhibitor suppressed the DNA ladder formation in a concentration-dependent manner. Bcl-2 and Bcl-XL proteins were decreased by asiatic acid treatment. These results indicate that asiatic acid induced apoptosis in HT-29 cells via caspase-3 activation. Cytotoxic effects of combined treatment with CPT-11 and asiatic acid on HT-29 cells were further examined. Simultaneous treatment or sequential exposure first to asiatic acid and then to CPT-11 showed an additive effect. Synergism was observed when cells were first exposed to CPT-11 and then to asiatic acid. These results suggest that asiatic acid can be used as an agent for increasing sensitivity of colon cancer cells to treatment with CPT-11 or as an agent for reducing adverse effects of CPT-11.