Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).

Significance of DNA base excision repair in the maintenance of mitochondrial function in Saccharomyces cerevisiae /

Mitochondria have an important role in cell metabolism, being the major site of ATP production via oxidative phosphorylation (OXPHOS). Accumulation of mtDNA mutations have been linked to the development of respiratory dysfunction, apoptosis, and aging. Base excision repair (BER) is the major and the only certain repair pathway existing in mitochondria that is in responsible for removing and repairing various base modifications as well as abasic sites (AP sites). In this research, Saccharomyces cerevisiae (S. cerevisiae) BER gene knockout strains, including 3 single DNA glycosylase gene knockout strains and Ap endonuclease (Apn 1 p) knockout strain were used to examine the importance of this DNA repair pathway to the maintenance of respiratory function. Here, I show that individual DNA glycosylases are nonessential in maintenance of normal function in yeast mitochondria, corroborating with previous research in mammalian experimental models. The yeast strain lacking Apn 1 p activity exhibits respiratory deficits, including inefficient and significantly low intracellular ATP level, which maybe due to partial uncoupling of OXPHOS. Growth of this yeast strain on respiratory medium is inhibited, but no evidence was found for increased ROS level in Apn 1 p mitochondria. This strain also shows an increased cell size, and this observation combined with an uncoupled OXPHOS may indicate a premature aging in the Apnlp knockout strain, but more evidence is needed to support this hypothesis. However, the BER is necessary for maintenance of mitochondrial function in respiring S.cerevisiae.

The influence of skeletal muscle cell volume on the regulation of carbohydrate uptake and muscle metabolism

This study investigated the regulation of carbohydrate metabolism and glucose uptake through changes in skeletal muscle cell volume. Using an established invitro isolated whole muscle model, soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected from male rats and incubated in an organ bath containing Sigma medium-199 with 8 mM D-glucose altered to target osmolality (hypo-osmotic: HYPO, iso-osmotic: ISO, hyper-osmotic: HYPER; 190, 290, 400 mmol/kg). Muscles were divided into two groups; metabolite (MM) and uptake (MU). MM (N=48) were incubated for 60 minutes and were then immediately flash frozen. MU (N=24) were incubated for 30 minutes and then the extracellular fluid was exchanged for media containing ^H-glucose and ^'*C-mannitol and incubated for another 30 minutes. After the incubation, the muscles were freeze clamped. Results demonstrated a relative water decrease and increase in HYPER and HYPO, respectively. EDL and SOL glucose uptakes were found to be significantly greater in HYPER conditions. The HYPER condition resulted in significant alterations in muscle metabolite concentrations (lower glycogen, elevated lactate, and G-6-P) suggesting a catabolic cell state, and an increase in glycogen synthase transformation when compared to the HYPO group. In conclusion, skeletal muscle cell volume alters rates of glucose uptake with further alterations in muscle metabolites and glycogen synthase transformation.

The effect of extracellular osmolality on cell volume and resting skeletal muscle metabolism

The purpose of the current investigation was to establish an in-l'itro skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated. whole muscle (SOL and EDL) was dissected from Long Evans rats and incubated for 60 min in Sigma Medium-199 (resting tension (lg). bubbled with 95:5% 02:C02, 30 ± 2°C, and pH 7.4). Media osmolality was altered to simulate hypo-osmotic (190 ± 10 Osm) (HYPO) or hyper-osmotic conditions (400 ± 10 Osm) (HYPER) while an iso-osmotic condition (290± 1 0 Osm) (CON) served as a control (n= 17.19.17). Following incubation, relative muscle water content decreased with HYPER and increased with HYPO in both muscle types (p<0.05). The cross-sectional area of HYPO SOL type I and type II fibres increased (p<0.05) while the EDL type 11 fibre area decreased in HYPER and increascd from HYPO exposure. Furthermore, HYPER exposure in both muscles lead to decreased ATP and phosphocreatine (p<0.05) and increased creatine and lactate (p<0.05) compared to CON. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acutc alterations in muscle water content and resting muscle metabolism.

The influence of skeletal muscle cell volume on carbohydrate metabolism in contracting skeletal muscle

This study investigated the regulation of carbohydrate metabolism through changes in skeletal muscle cell volume immediately post contraction and during recovery. Using an established in vitro isolated muscle strip model, soleus (SOL) and extensor digitorum longus (EDL) were dissected from male rats and incubated in an organ bath (perfused with 95% O2; 5% CO2, pH 7.4, temperature 25°C) containing medium- 199 altered to a target osmotic condition (iso-, hypo- or hyper-osmotic; 290, 1 80, 400 mmol/kg). Muscles were stimulated for 10 minutes (40 Hz SOL; 30 Hz EDL) and then either immediately flash frozen or allowed to recover for 20 minutes before subsequent metabolite and enzyme analysis. Results demonstrated a relative water decrease in HYPER vs. HYPOosmotic condition (n=8/group; p<0.05) regardless of muscle type. Specifically, the SOL HYPER condition had elevated metabolite concentrations after 10 minutes of stimulation in comparison to both HYPO and ISO (p<0.05), while EDL muscle did not show any significant difTerences between the HYPER or HYPO conditions. After 20 minutes of recovery, metabolic changes occurred in both SOL and EDL with the SOL HYPER condition showing greater relative changes in metabolite concentrations versus HYPO. The results of the current study have demonstrated that osmotic imbalance induces metabolic change within the skeletal muscle cell and muscle type may influence the mechanisms utilized for cell volume regulation.

Electrogenic proton/L-glutamate symport in isolated asparagus sprengeri mesophyll cell

Medium' alkaliniiation occurred -lipon the addition of L-Glu to mechanically isolated Asparagus sprenger-i mesophyll cells suspended in 1 mM CaS04. Alkalinization resulted from the coupled entry of H+ and L-Glu anion into the cells. This H+ IL-Glu symport did not stimulate K+ efflux. K+ efflux has been observed during H~ lamino acid symport in other systems. The stimulation of K+ efflux by proton coupled symport is regarded as an indicator of a plasma membrane depolarizing electrogenic symport process. H+ IL-Glu symport in Asparagus sprengerimesophyl1 cells was investigated to determine whether or not the process was electrogenic. The rate of uptake of 0.25 11M 3H-MTPP+ ( Methyltriphenylphosphonium, methyl-3H ) is a probe for monitoring changes in the membrane potential. 3HMTPP+ uptake was reduced by K+ or CCCP, agents known to depolarize the membrane potential. Uptake of 3H-MTPP+ was also inhibited by L-Glu but not by D-Glu. Conversely, 10 mM external MTPP+ inhibited the uptake of 14C-U-LGlu. Simultaneous measurements of the rates of 14C-U-L-Glu uptake and L-Glu dependent H+ influx showed that the molar stoichiometry of H+ IL-Glu symport was 2 to 1. K+ or Na+ stimulated H+ efflux was completely inhibited by DCCD, DES, oligomycin and antimycin reagents which inhibit ATP driven H+ efflux. The H+ efflux \Vas also stimulate.d by the weak acids, butyric acid and acetic acid, which are known fo-aCidify the cytoplasm. This weak acid stimulated H+ efflux was also completely inhibited by oligomycin. It was calculated that net L-Glu dependent H+ influx increased by 100% in the presence of oligomycin and that despite net medium alkalinization H+ IL-Glu symport stimulates ATP dependent H+ efflux. 11 The data presented in this study indicate that H+ IL-Glu symport is electrogenic. The data also show that ATP dependent Ht efflux rather than K+ efflux is the- process compensating for thi~ electrogenic H+ IL-Glu symport.

Synthesis of cytochrome P450 inhibitors of vitamin E metabolism

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999 C54 O46 2007

Cellular Mechanisms of Resveratrol's Interaction with Mitochondrial Reactive Oxygen Species Metabolism

Resveratrol, a polyphenol found naturally in red wines, has attracted great interest in both the scientific community and the general public for its reported ability to protect against many of the diseases facing Western society today. While the purported health effects of resveratrol are well characterized, details of the cellular mechanisms that give rise to these observations are unclear. Here, the mitochondrial antioxidant enzyme Mn superoxide dismutase (MnSOD) was identified as a proximal target of resveratrol in vitro and in vivo. MnSOD protein and activity levels increase significantly in cultured cells treated with resveratrol, and in the brain tissue of mice given resveratrol in a high fat diet. Preventing the increase in MnSOD levels eliminates two of resveratrol’s more interesting effects in the context of human health: inhibition of proliferative cell growth and cytoprotection. Thus, the induction of MnSOD is a critical step in the molecular mechanism of resveratrol. Mitochondrial morphology is a malleable property that is capable of impeding cell cycle progression and conferring resistance against stress induced cell death. Using confocal microscopy and a novel ‘cell free’ fusion assay it was determined that concurrent with changes in MnSOD protein levels, resveratrol treatment leads to a more fused mitochondrial reticulum. This observation may be important to resveratrol’s ability to slow proliferative cell growth and confer cytoprotection. Resveratrol's biological activities, including the ability to increase MnSOD levels, are strikingly similar to what is observed with estrogen treatment. Resveratrol fails to increase MnSOD levels, slow proliferative cell growth and confer cytoprotection in the presence of an estrogen receptor antagonist. Resveratrol's effects can be replicated with the specific estrogen receptor beta agonist diarylpropionitrile, and are absent in myoblasts lacking estrogen receptor beta. Four compounds that are structurally similar to resveratrol and seven phytoestrogens predicted to bind to estrogen receptor beta were screened for their effects on MnSOD, proliferative growth rates and stress resistance in cultured mammalian cells. Several of these compounds were able to mimic the effects of resveratrol on MnSOD levels, proliferative cell growth and stress resistance in vitro. Thus, I hypothesize that resveratrol interacts with estrogen receptor beta to induce the upregulation of MnSOD, which in turn affects cell cycle progression and stress resistance. These results have important implications for the understanding of RES’s biological activities and potential applications to human health.