Biomedical subjects

H Van Remmen

Publications and source records attributed to H Van Remmen.

At least 19 recordsLinked to original sources

PubMed2006

Genetic modification of the manganese superoxide dismutase/glutathione peroxidase 1 pathway influences intracellular ROS generation in quiescent, but not contracting, skeletal muscle cells.

Increased amounts of reactive oxygen species (ROS) are generated by skeletal muscle during contractile activity, but their intracellular source is unclear. The oxidation of 2',7'-dichlorodihydrofluorescein (DCFH) was examined as an intracellular probe for reactive oxygen species in skeletal muscle myotubes derived from muscles of wild-type mice and mice that were heterozygous knockout for manganese superoxide dismutase (Sod2(+/-)), homozygous knockout for glutathione peroxidase 1 (GPx1(-/-)), or MnSOD transgenic overexpressors (Sod2-Tg). Myoblasts were stimulated to fuse and loaded with DCFH 5-7 days later. Intracellular DCF epifluorescence was measured and myotubes were electrically stimulated to contract for 15 min. Quiescent myotubes with decreased MnSOD or GPx1 showed a significant increase in the rate of DCFH oxidation whereas those with increased MnSOD did not differ from wild type. Following contractions, myotubes from all groups showed an equivalent increase in DCF fluorescence. Thus the oxidation of DCFH in quiescent skeletal muscle myotubes is influenced by the content of enzymes that regulate mitochondrial superoxide and hydrogen peroxide content. In contrast, the increase in DCFH oxidation following contractions was unaffected by reduced or enhanced MnSOD or absent GPx1, indicating that reactive oxygen species produced by contractions were predominantly generated by nonmitochondrial sources.

Animals↗

PubMed2006

Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity.

Oxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction-induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction-induced ROS generation. Hind limb muscles of adult and old mice underwent a 15-min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with pre-contraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age-related exacerbation of ROS generation.

Aging↗

PubMed2005

Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.

Mutations in mitochondrial DNA (mtDNA) accumulate in tissues of mammalian species and have been hypothesized to contribute to aging. We show that mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase g (POLG) accumulate mtDNA mutations and display features of accelerated aging. Accumulation of mtDNA mutations was not associated with increased markers of oxidative stress or a defect in cellular proliferation, but was correlated with the induction of apoptotic markers, particularly in tissues characterized by rapid cellular turnover. The levels of apoptotic markers were also found to increase during aging in normal mice. Thus, accumulation of mtDNA mutations that promote apoptosis may be a central mechanism driving mammalian aging.

Aging↗

PubMed2004

Role of mitochondrial superoxide dismutase in contraction-induced generation of reactive oxygen species in skeletal muscle extracellular space.

Contractions of skeletal muscles produce increases in concentrations of superoxide anions and activity of hydroxyl radicals in the extracellular space. The sources of these reactive oxygen species are not clear. We tested the hypothesis that, after a demanding isometric contraction protocol, the major source of superoxide and hydroxyl radical activity in the extracellular space of muscles is mitochondrial generation of superoxide anions and that, with a reduction in MnSOD activity, concentration of superoxide anions in the extracellular space is unchanged but concentration of hydroxyl radicals is decreased. For gastrocnemius muscles from adult (6-8 mo old) wild-type (Sod2(+/+)) mice and knockout mice heterozygous for the MnSOD gene (Sod2(+/-)), concentrations of superoxide anions and hydroxyl radical activity were measured in the extracellular space by microdialysis. A 15-min protocol of 180 isometric contractions induced a rapid, equivalent increase in reduction of cytochrome c as an index of superoxide anion concentrations in the extracellular space of Sod2(+/+) and Sod2(+/-) mice, whereas hydroxyl radical activity measured by formation of 2,3-dihydroxybenzoate from salicylate increased only in the extracellular space of muscles of Sod2(+/+) mice. The lack of a difference in increase in superoxide anion concentration in the extracellular space of Sod2(+/+) and Sod2(+/-) mice after the contraction protocol supported the hypothesis that superoxide anions were not directly derived from mitochondria. In contrast, the data obtained suggest that the increase in hydroxyl radical concentration in the extracellular space of muscles from wild-type mice after the contraction protocol most likely results from degradation of hydrogen peroxide generated by MnSOD activity.

Animals↗

PubMed2001

Does oxidative damage to DNA increase with age?

The levels of 8-oxo-2-deoxyguanosine (oxo8dG) in DNA isolated from tissues of rodents (male F344 rats, male B6D2F1 mice, male C57BL/6 mice, and female C57BL/6 mice) of various ages were measured using sodium iodide to prevent oxidative damage to DNA during DNA isolation. Oxo8dG was measured in nuclear DNA (nDNA) isolated from liver, heart, brain, kidney, skeletal muscle, and spleen and in mitochondrial DNA (mtDNA) isolated from liver. We observed a significant increase in oxo8dG levels in nDNA with age in all tissues and strains of rodents studied. The age-related increase in oxo8dG in nDNA from old mice was shown not to the result of the tissue's reduced ability to remove the oxo8dG lesion. Rather, the increase in oxo8dG levels appears to arise from an age-related increase in the sensitivity of these tissues to oxidative stress. We also observed an age-related increase in oxo8dG in mtDNA isolated from the livers of the rats and mice. Dietary restriction, which is known to retard aging and increase the lifespan of rodents, was shown to significantly reduce the age-related accumulation of oxo8dG levels in nDNA in all tissues of male B6D23F1 mice and in most tissues of male F344 rats. Our study also showed that dietary restriction prevented the age-related increase in oxo8dG levels in mtDNA isolated from the livers of both rats and mice.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA.

A major controversy in the area of DNA biochemistry concerns the actual in vivo levels of oxidative damage in DNA. We show here that 8-oxo-2-deoxyguanosine (oxo8dG) generation during DNA isolation is eliminated using the sodium iodide (NaI) isolation method and that the level of oxo8dG in nuclear DNA (nDNA) is almost one-hundredth of the level obtained using the classical phenol method. We found using NaI that the ratio of oxo8dG/10(5 )deoxyguanosine (dG) in nDNA isolated from mouse tissues ranged from 0.032 +/- 0.002 for liver to 0.015 +/- 0.003 for brain. We observed a significant increase (10-fold) in oxo8dG in nDNA isolated from liver tissue after 2 Gy of gamma-irradiation when NaI was used to isolate DNA. The turnover of oxo8dG in nDNA was rapid, e.g. disappearance of oxo8dG in the mouse liver in vivo after gamma-irradiation had a half-life of 11 min. The levels of oxo8dG in mitochondrial DNA isolated from liver, heart and brain were 6-, 16- and 23-fold higher than nDNA from these tissues. Thus, our results showed that the steady-state levels of oxo8dG in mouse tissues range from 180 to 360 lesions in the nuclear genome and from one to two lesions in 100 mitochondrial genomes.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

The anti-ageing action of dietary restriction.

Over 60 years ago, McCay's laboratory showed that dietary or calorie-restriction dramatically increased the lifespan of rats. Since then, numerous laboratories with a variety of strains of rats and mice have confirmed this initial observation and have shown that reducing calorie intake (without malnutrition) significantly increases both the mean and maximum survival of rodents. Currently, dietary restriction is the only experimental manipulation that has been shown to retard ageing of mammals. Although mechanism whereby dietary restriction retards ageing is currently unknown, much of the emerging data suggest that the calorie-restricted rodents live longer and age more slowly because they are more resistant to stress and have an enhanced ability to protect cells against damaging agents.

Aging↗

PubMed2001

Oxidative damage to mitochondria and aging.

Oxidative damage has been implicated to be a major factor in the decline in physiologic function that occurs during the aging process. Because mitochondria are a primary site of generation of reactive oxygen species, they have become a major focus of research in this area. Increased oxidative damage to mitochondrial proteins, lipid and DNA has been reported to occur with age in several tissues in a variety of organisms. Decreased activity of electron transport chain complexes and increased release of reactive oxygen species from the mitochondria with age suggest that alterations in mitochondrial function occur with age as a consequence of increased oxidative damage. In addition, age-related alterations in the mitochondrial pathway of apoptosis, which could have profound affects on the physiological function of a tissue, could arise from oxidative damage to mitochondria. Alterations in mitochondrial turnover with age could also contribute to an increase in the number of dysfunctional mitochondria with age.

Aging↗

PubMed2001

Knockout mice heterozygous for Sod2 show alterations in cardiac mitochondrial function and apoptosis.

Heart mitochondria from heterozygous (Sod2(-/+)) knockout mice have a 50% reduction in manganese superoxide dismutase (MnSOD) activity. The decrease in MnSOD activity was associated with increased mitochondrial oxidative damage as demonstrated by a decrease in the activities of iron sulfhydryl proteins sensitive to oxygen stress (aconitase and reduced nicotinamide adenine dinucleotide-oxidoreductase). Mitochondrial function was altered in the Sod2(-/+) mice, as shown by decreased respiration by complex I and an increase in the sensitivity of the permeability transition to induction by calcium and t-butylhydroperoxide. The increased induction of the permeability transition in heart mitochondria from Sod2(-/+.)mice was associated with increased release of cytochrome c and an increase in DNA fragmentation. Cardiomyocytes isolated from neonatal Sod2(-/+) and Sod2(-/-) mice were more sensitive to cell death than cardiomyocytes from Sod2(+/+) mice after t-butylhydroperoxide treatment, and this increased sensitivity was prevented by inhibiting the permeability transition with cyclosporin A. These experiments demonstrate that MnSOD may play an important role in the induction of the mitochondrial pathway of apoptosis in the heart, and this appears to occur primarily through the permeability transition.

Aconitate Hydratase↗

PubMed2001

Changes in expression of antioxidant enzymes affect cell-mediated LDL oxidation and oxidized LDL-induced apoptosis in mouse aortic cells.

Transgenic mice overexpressing Cu/Zn superoxide dismutase (hSod1Tg(+/0)) or catalase (hCatTg(+/0)) and knockout mice underexpressing manganese superoxide dismutase (Sod2(+/)(-)) or glutathione peroxidase-1 (Gpx1(-/-)) were used to study the effect of antioxidant enzymes on cell-mediated low density lipoprotein (LDL) oxidation and oxidized LDL (oxLDL)-induced apoptosis. Incubation of LDL with mouse aortic segments or smooth muscle cells (SMCs) resulted in a significant increase in LDL oxidation. However, LDL oxidation was significantly reduced when LDL was incubated with aortic segments and SMCs obtained from hSod1Tg(+/0) and hCatTg(+/0) mice compared with those obtained from wild-type mice. In contrast, LDL oxidation was significantly increased when LDL was incubated with aortic segments and SMCs obtained from Sod2(+/)(-) and Gpx1(-/-) mice. CuSO(4)-oxidized LDL increased DNA fragmentation and caspase activities in the primary cultures of mouse aortic SMCs. However, oxLDL-induced DNA fragmentation and caspase activities were reduced 50% in SMCs obtained from hSod1Tg(+/0) and hCatTg(+/0) mice compared with wild-type control mice. In contrast, oxLDL-induced DNA fragmentation and caspase activities were significantly increased in SMCs obtained from Sod2(+/)(-) and Gpx1(-/-) mice. These findings suggest that overexpression of Cu/Zn superoxide dismutase or catalase reduces cell-mediated LDL oxidation and oxLDL-induced apoptosis, whereas underexpression of manganese superoxide dismutase or glutathione peroxidase-1 increases cell-mediated LDL oxidation and oxLDL-induced apoptosis.

Animals↗

PubMed1999

Characterization of the antioxidant status of the heterozygous manganese superoxide dismutase knockout mouse.

The antioxidant status of several tissues (liver, kidney, lung, brain, heart, muscle, stomach, and spleen) from heterozygous manganese superoxide dismutase (MnSOD) mutant mice (Sod2-/+) was characterized. The activity of MnSOD was decreased (30 to 80%) in all tissues examined. The levels of mRNA coding for the major antioxidant enzymes (CuZnSOD, catalase, and glutathione peroxidase) were not significantly altered in liver, kidney, heart, lung, or brain in the Sod2-/+ mice. The activities of the enzymes were not altered in any of these tissues, with the exception of a decrease in glutathione peroxidase activity in muscle in the Sod2-/+ mice compared to the Sod2+/+ mice. Thus, there was no up-regulation of the activities of the major antioxidant enzymes to compensate for the decrease in MnSOD activity. Reduced glutathione levels were 30 to 50% lower in the lung, brain, and muscle of the Sod2-/+ mice compared to the wild-type Sod2+/+ mice. In addition, the ratio of GSH/GSSG was decreased approximately 50% in Sod2-/+ muscle, indicating that the decrease in MnSOD activity in the Sod2-/+ mice results in some degree of oxidative stress in this tissue.

Animals↗

PubMed1998

Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice.

This study characterizes mitochondria isolated from livers of Sod2(-/+) and Sod2(+/+) mice. A 50% decrease in manganese superoxide dismutase (MnSOD) activity was observed in mitochondria isolated from Sod2(-/+) mice compared with Sod2(+/+) mice, with no change in the activities of either glutathione peroxidase or copper/zinc superoxide dismutase. However, the level of total glutathione was 30% less in liver mitochondria of the Sod2(-/+) mice. The reduction in MnSOD activity in Sod2(-/+) mice was correlated to an increase in oxidative damage to mitochondria: decreased activities of the Fe-S proteins (aconitase and NADH oxidoreductase), increased carbonyl groups in proteins, and increased levels of 8-hydroxydeoxyguanosine in mitochondrial DNA. In contrast, there were no significant changes in oxidative damage in the cytosolic proteins or nuclear DNA. The increase in oxidative damage in mitochondria was correlated to altered mitochondrial function. A significant decrease in the respiratory control ratio was observed in mitochondria isolated from Sod2(-/+) mice compared with Sod2(+/+) mice for substrates metabolized by complexes I, II, and III. In addition, mitochondria isolated from Sod2(-/+) mice showed an increased rate of induction of the permeability transition. Therefore, this study provides direct evidence correlating reduced MnSOD activity in vivo to increased oxidative damage in mitochondria and alterations in mitochondrial function.

Aconitate Hydratase↗

PubMed1998

Effect of cAMP-induced transcription on the repair of the phosphoenolpyruvate carboxykinase gene by hepatocytes isolated from young and old rats.

The repair of UV-induced DNA damage in the phosphoenolpyruvate carboxykinase (PEPCK) gene was studied in primary cultures of hepatocytes isolated from young (6-month-old) and old (24-month-old) rats fed ad libitum and old rats fed a calorie-restricted diet. Incubation of the hepatocytes with cAMP rapidly induced PEPCK transcription and mRNA levels 4- to 5-fold. In absence of cAMP, the repair of the PEPCK fragment was similar in cultured hepatocytes isolated from young and old rats fed ad libitum. However, cAMP significantly increased the percentage of cyclobutane pyrimidine dimers (CPDs) removed from the PEPCK fragment 12 h after UV-irradiation in cultured hepatocytes isolated from young rats fed ad libitum. This increase was due to an increase in the repair of the transcribed strand of the PEPCK fragment. In contrast, cAMP did not increase the repair of the PEPCK fragment in cultured hepatocytes isolated from old rats fed ad libitum in spite of an increase in PEPCK transcription. Thus, it appears that the coupling of transcription and DNA repair is compromised in cultured hepatocytes isolated from old rats fed ad libitum. However, cultured hepatocytes isolated from old rats fed a calorie-restricted diet showed an induction in the rate of repair of the transcribed strand of the PEPCK fragment by cAMP that was similar to hepatocytes isolated from young rats fed ad libitum.

Aging↗

PubMed1998

Effect of dietary restriction on hepatic and renal phosphoenolpyruvate carboxykinase induction in young and old Fischer 344 rats.

Food restriction is known to ameliorate many of the adverse physiologic effects of age. In this study, we have examined the effect of food restriction on the induction of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK) in liver and kidney following a 12-h fasting period in young (6 month) and old (24 month) ad libitum-fed and food-restricted male Fischer 344 rats. In the liver, following the 12-h fast, the activity of PEPCK increased approximately 2-fold in the young ad libitum fed rats and 3-fold in the young restricted animals. However, PEPCK activity remained unchanged in response to the 12-h fast in the 24 month old ad libitum fed rats. In the old restricted rats, the induction of PEPCK mimicked that of the young rats (PEPCK activity increased 2-fold within the 12-h fasting period). Therefore, dietary restriction not only enhanced the induction response in the liver in young rats, but also restored the induction of hepatic PEPCK in the old animals. In the kidney, there was no effect of age or dietary restriction on the induction of PEPCK as the activity of renal PEPCK did not change in response to the 12-h fast in any of the four groups of rats.

Aging↗

PubMed1998

Analysis of the transcriptional activity of the 5'-flanking region of the rat catalase gene in transiently transfected cells and in transgenic mice.

Transiently transfected cell lines and transgenic mice were used to study the transcriptional activity of the 5'-flanking region of the catalase gene. Fragments of the 5'-flanking region of the rat catalase gene ranging in length from 3,421 base pairs (bp) to 69 bp were fused to the chloramphenicol acetyltransferase (CAT) reporter gene, and the transcriptional activity of the reporter gene was measured following transient transfection in three cell lines: a human hepatoma cell line (HepG2), a porcine kidney epithelial cell line (LLCPK1), and a human glioma cell line (U-138 MG). The 3,421-bp fragment of the 5'-flanking region resulted in a high level of expression of the reporter gene in all three cell lines. Shorter fragments of the 5'-flanking region resulted in a decrease in the level of CAT reporter expression that varied among the three cell lines, implying the presence of tissue-specific regulatory sites. To study the tissue-specific regulation of the catalase promoter, transgenic mice containing the 3,421-bp 5'-flanking sequence attached to the CAT reporter gene were produced, and CAT expression was measured in various tissues of three independent transgenic lines. CAT activity was consistently high in muscle tissue (heart, skeletal muscle, and diaphragm) and low in most other tissues studied, particularly in liver and kidney. In contrast, the endogenous expression of catalase is low in muscle and high in liver and kidney; thus, the tissue-specific expression of the reporter gene driven by the 3,421-bp fragment of the 5'-flanking region of the catalase gene was not similar to the expression of the endogenous catalase gene.

Animals↗

PubMed1997

Overexpression of superoxide dismutase and catalase in immortalized neural cells: toxic effects of hydrogen peroxide.

Hydrogen peroxide (H2O2) is a known toxicant which causes its damage via the production of hydroxyl radicals. It has been reported to cause both necrotic and apoptotic cell death. The present study was undertaken to evaluate the mode of H2O2-induced cell death and to assess if overexpression of catalase could protect against its toxicity. H2O2 causes cell death of immortalized CSM 14.1 neural cells in a dose-dependent manner. H2O2-induced death was associated with DNA laddering as shown by agarose gel electrophoresis. Stable overexpression of catalase by transfection of a vector containing human cDNA into these cells markedly attenuated H2O2-induced toxic effects. Transfection of a vector containing a SOD cDNA afforded no protection. These results indicate that H2O2 can lead to the activation of endonuclease enzyme that breaks DNA into oligosomes. These cells which overexpress catalase or SOD will help to determine the specific role of H2O2 or O2- in the deleterious effects of a number of toxins.

Catalase↗

PubMed1997

Effect of age on the activity of phosphoenolpyruvate carboxykinase in the kidney following fasting and refeeding.

The activity of renal phosphoenolpyruvate carboxykinase (PEPCK) was measured in 3-18 month old male Fischer 344 rats after alternate periods of fasting and refeeding. To compare the induction of renal PEPCK activity in response to fasting in young and old rats, 3, 6, 12 and 18 month old animals were fasted for 30 h followed by a 24 h ad libitum refeeding period to reduce PEPCK activity toward basal levels. The refeeding period was followed by a second 24 h fasting period during which the time course of PEPCK induction was monitored in the young and old animals. The first fast resulted in over a 20% increase in renal PEPCK activity in the 3 month old and slightly over a 70% increase in the 6 month old animals. In contrast, the activity did not increase significantly in the 12 or 18 month old animals during this fasting period. Therefore the induction of PEPCK in the kidney in response to fasting appears to be altered in the older animals. Refeeding for 24 h resulted in a decrease in PEPCK activity in all four age groups; therefore there was no indication of an age-related impairment in the response of renal PEPCK to refeeding. After the refeeding period, the food was removed again and the activity was measured at short intervals over the next 24 h to determine the time course of the induction in PEPCK activity. Interestingly, during the second fast, the activity of renal PEPCK was not significantly induced in either the young or the older animals. However, the activity measured in the older 18 month rats was consistently lower during the first 12 h of the second fast as compared to the activity in the 6 month old rats. In summary, the induction of PEPCK activity in the kidney is altered with age during an initial fast; in addition, PEPCK activity is not induced in either young or old rats during a second fasting period.

Age Factors↗

PubMed1996

Expression of genes coding for antioxidant enzymes and heat shock proteins is altered in primary cultures of rat hepatocytes.

The expression of genes for heat shock proteins in the HSP70 family and genes for antioxidant enzymes was studied in rat hepatocytes cultured in either L-15 or Williams E media on a collagen matrix for up to 48 hours. The mRNA transcripts for the heat shock proteins hsp70, hsc70, and grp78 were induced dramatically when hepatocytes were cultured in L-15, and to a lesser extent when cultured in Williams E. The increase in hsp70 and hsc70 mRNA levels in the cultured hepatocytes was correlated with an increase in the nuclear transcription of these two genes and the binding activity of the heat shock transcription factor to the heat shock element. Culturing rat hepatocytes in either L-15 or Williams E resulted in a decrease in the levels of the mRNA transcripts for catalase and glutathione peroxidase and the activities of these two enzymes. However, the expression of Cu/Zn-superoxide dismutase, i.e., the level of the mRNA transcript or the enzymatic activity, did not change appreciably when hepatocytes were cultured for up to 48 hours. The decline in catalase and glutathione peroxidase expression in the cultured hepatocytes was correlated with a decrease in the GSH/GSSG ratio and an increase in lipid peroxidation. These data show that the expression of several genes involved in cellular protection change when hepatocytes are placed in primary cultures. Therefore, one must be careful in extrapolating from primary cultures to the liver in vivo, especially when studying processes that might be affected by heat shock proteins or antioxidant enzymes.

Animals↗