Increase In Mitochondrial Activity Research Articles

Expression of estrogen receptor α in human breast cancer cells regulates mitochondrial oxidative stress under simulated microgravity

This study investigated intracellular oxidative stress and its underlying mechanisms in a rotary cell culture system used to achieve a simulated microgravity (SMG) environment. Experiments were conducted with human breast cancer cell lines MCF-7 (an estrogen receptor (ER) α positive cell line) and MDA-MB-231 (an ERα negative cell line) encapsulated in alginate/collagen carriers. After 48h, SMG led to oxidative stress and DNA damage in the MDA-MB-231 cells but a significant increase in mitochondrial activity and minimal DNA damage in the MCF-7 cells. The activity of superoxide dismutase (SOD) significantly increased in the MCF-7 cells and decreased in MDA-MB-231 cells in the SMG environment compared with a standard gravity control. Moreover, SMG promoted expression of ERα and protein kinase C (PKC) epsilon in MCF-7 cells treated with PKC inhibitor Gö6983. Overall, exposure to SMG increased mitochondrial activity in ERα positive cells but induced cellular oxidative damage in ERα negative cells. Thus, ERα may play an important role in protecting cells from oxidative stress damage under simulated microgravity.

199 PREMATURATION OF BOVINE OOCYTES WITH BUTYROLACTONE I AND/OR CILOSTAMIDE: EFFECTS ON MEIOSIS PROGRESSION, CYTOPLASMIC MATURATION AND GENE EXPRESSION

Meiotic block during a prematuration culture (pre-IVM) before in vitro maturation (IVM) is suggested as a way to provide additional time to synchronize oocyte-somatic cell communication, leading to improved cytoplasmic maturation and nuclear meiotic competence and the acquisition of critical cellular functions necessary for developmental competence. This study was designed to evaluate the effects of the inhibitors butyrolactone I (Bl-I) and cilostamide on oocyte nuclear and cytoplasmic maturation. For that, 2 well-established methods of pre-IVM and IVM for cilostamide (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011) or Bl-I (Hashimoto et al. 2002 Biol Reprod. 66, 1696–1701) were used. Abattoir-collected oocytes were IVM in maturation medium (MM: mSOF with 0.8% BSA and hormones) for 24 h, without a previous pre-IVM culture (control). Cilostamide-group oocytes were treated for the first 2 h in vitro (pre-IVM) with 100 μM of the adenylate cyclase activator forskolin and 500 μM IBMX (nonspecific phosphodiesterase inhibitor) and then oocytes underwent extended IVM for 30 h in the presence of 20 μM cilostamide (type 3 phosphodiesterase inhibitor; pre-IVM 2 h + IVM 30 h). The Bl-I-group oocytes were pre-IVM for 24 h with 100 μM Bl-I diluted in TCM-199 supplemented with 0.2 mM pyruvate and then were IVM in MM for 20 h (pre-IVM 24 h + IVM 20 h). The Bl-I + Cilost group was a combination of both procedures: oocytes were first pre-IVM with Bl-I for 24 h and then cultured as described for the cilostamide group (pre-IVM 24 h + IVM 30 h). Cultures were carried out at 38.5°C in 5% CO2 in humidified air. After IVM, oocytes were stained with 500 nM mitotracker red to assess the mitochondrial membrane potential (Δψm) and with 10 μg mL–1 of Hoescht 33342 to evaluate the nuclear maturation (n = 207). Images were captured by an Olympus confocal microscope and analyzed with Fluoview software. The TUNEL assay was used to detect oocyte DNA fragmentation (n = 74). Relative amounts of mRNA for apoptotic-related genes were quantified after IVM in individual oocytes using real-time PCR (Kameyama et al. 2007 Reproduction 133, 423–32). Means were compared by ANOVA and Tukey's test or by chi-square (P ≤ 0.05). The percentage of oocytes reaching metaphase II after IVM did not differ among groups (73.8 to 90.4%; P ≥ 0.05), indicating that in vitro meiotic resumption was normal. The Δψm, expressed in arbitrary units of fluorescence, was 1.0 ± 0.1a (control), 2.7 ± 0.4b (Bl-I), 3.2 ± 0.5b (Cilost) and 2.1 ± 0.3ab (Bl-I + Cilost). The percentage of TUNEL-positive oocytes (18.8–41.3%) did not differ among groups (P ≥ 0.05). The relative abundance of BAX (1.0 ± 0.4 to 2.3 ± 0.4) and BCL-XL (1.0 ± 0.3 to 0.3 ± 0.1) transcripts was unaffected by pre-IVM and IVM (P ≥ 0.05). In conclusion, except for an increase in mitochondrial activity, pre-IVM with cilostamide and/or Bl-I did not affect cytoplasmic and nuclear oocyte maturation. However, oocyte developmental potential needs to be better evaluated in a future study through assessment of embryonic development. We acknowledge FAPESP and NSERC.

Human cytomegalovirus infection increases mitochondrial biogenesis

Fibroblasts infected by Human Cytomegalovirus (CMV) undergo a robust increase in mitochondrial biogenesis with a corresponding increase in mitochondrial activity that is partly dependent on the viral anti-apoptotic pUL37x1 protein (vMIA). The increased respiration activity is blocked by the mitochondrial translation inhibitor chloramphenicol, which additionally suppresses viral production. Intriguingly, chloramphenicol and pUL37x1 depletion have different effects on respiration capacity but similar effects on CMV production, suggesting that pUL37x1 promotes viral replication by efficient utilization of new mitochondria. These results argue for a role of pUL37x1 beyond controlling apoptosis.

Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide, driving the Warburg effect

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a “weapon” to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis, and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the “reverse Warburg effect.” To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system, and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake, and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose, and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity, and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity, and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress, and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.

Transcranial Laser Therapy Attenuates Amyloid-β Peptide Neuropathology in Amyloid-β Protein Precursor Transgenic Mice

Transcranial laser therapy (TLT) was tested for efficacy in a mouse model of Alzheimer's disease (AD) using a near-infrared energy laser system. TLT is thought to stimulate ATP production, increase mitochondrial activity, and help maintain neuronal function. Studies were performed to determine the effect of TLT in an amyloid-β protein precursor (AβPP) transgenic mouse model. TLT was administered 3 times/week at various doses, starting at 3 months of age, and was compared to a control group (no laser treatment). Treatment was continued for a total of six months. Animals were examined for amyloid load, inflammatory markers, brain amyloid-β (Aβ) levels, plasma Aβ levels, cerebrospinal fluid Aβ levels, soluble AβPP (sAβPP) levels, and behavioral changes. The numbers of Aβ plaques were significantly reduced in the brain with administration of TLT in a dose-dependent fashion. Administration of TLT was associated with a dose-dependent reduction in amyloid load. All TLT doses mitigated the behavioral effects seen with advanced amyloid deposition and reduce the expression of inflammatory markers in the AβPP transgenic mice. All TLT doses produced an increase in sAβPPα and a decrease in CTFβ levels consistent with inhibition of the β-secretase activity. In addition, TLT showed an increase in ATP levels, mitochondrial function, and c-fos suggesting an overall improvement in neurological function. These studies suggest that TLT is a potential candidate for treatment of AD.

‘In Vitro’ Capacitation and Acrosome Reaction are Concomitant with Specific Changes in Mitochondrial Activity in Boar Sperm: Evidence for a Nucleated Mitochondrial Activation and for the Existence of a Capacitation‐Sensitive Subpopulational Structure

The main scope of this manuscript is to analyse the dynamics of mitochondrial activity in boar sperm subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome reaction (IVAR). This was determined after analysis of the rhythm of O(2) consumption and concomitant changes in the mitochondria activity-specific JC-1 staining. Results showed that IVC, and especially IVAR, was concomitant with a peak in O(2) consumption (from 1.61 ± 0.08 nmol O(2)/min/10(7) viable sperm at 0 h of incubation to 2.62 ± 0.12 nmol O(2) /min/10(7) viable sperm after 5 min of IVAR induction). These results were accompanied by parallel changes in the mean intensity of JC-1 staining. Based on JC-1, mitochondrial activation followed a nucleated pattern, with specific, activation starting points at the midpiece from which mitochondrial activation was spread. Moreover, four separate sperm subpopulations were detected following the JC-1 orange-red/green ratio, and the observed changes in the mean JC-1 staining during IVC and IVAR were related to concomitant changes in both the orange-red/green JC-1 ratio and the percentage of sperm included in each subpopulation. All of these results indicate that IVC and the first minutes of IVAR are accompanied by a progressive increase in mitochondrial activity, which reached a peak coincidental with the achievement of IVAR. Moreover, results suggest the presence of separate sperm subpopulations, which show a different mitochondrial sensitivity to IVC and IVAR. Finally, mitochondrial activation, at least under JC-1 staining, seems to originate in concrete nucleation points at the midpiece, thus suggesting thus a well-coordinated pattern in boar-sperm mitochondrial activity modulation.

A gene network switch enhances the oxidative capacity of ovine skeletal muscle during late fetal development

BackgroundThe developmental transition between the late fetus and a newborn animal is associated with profound changes in skeletal muscle function as it adapts to the new physiological demands of locomotion and postural support against gravity. The mechanisms underpinning this adaption process are unclear but are likely to be initiated by changes in hormone levels. We tested the hypothesis that this developmental transition is associated with large coordinated changes in the transcription of skeletal muscle genes.ResultsUsing an ovine model, transcriptional profiling was performed on Longissimus dorsi skeletal muscle taken at three fetal developmental time points (80, 100 and 120 d of fetal development) and two postnatal time points, one approximately 3 days postpartum and a second at 3 months of age. The developmental time course was dominated by large changes in expression of 2,471 genes during the interval between late fetal development (120 d fetal development) and 1-3 days postpartum. Analysis of the functions of genes that were uniquely up-regulated in this interval showed strong enrichment for oxidative metabolism and the tricarboxylic acid cycle indicating enhanced mitochondrial activity. Histological examination of tissues from these developmental time points directly confirmed a marked increase in mitochondrial activity between the late fetal and early postnatal samples. The promoters of genes that were up-regulated during this fetal to neonatal transition were enriched for estrogen receptor 1 and estrogen related receptor alpha cis-regulatory motifs. The genes down-regulated during this interval highlighted de-emphasis of an array of functions including Wnt signaling, cell adhesion and differentiation. There were also changes in gene expression prior to this late fetal - postnatal transition and between the two postnatal time points. The former genes were enriched for functions involving the extracellular matrix and immune response while the latter principally involved functions associated with transcriptional regulation of metabolic processes.ConclusionsIt is concluded that during late skeletal muscle development there are substantial and coordinated changes in the transcription of a large number of genes many of which are probably triggered by increased estrogen levels. These changes probably underpin the adaption of muscle to new physiological demands in the postnatal environment.

Abstract 39: Specific amino acid restriction affects glucose metabolism and induces oxidative damage of mitochondria in prostate cancer cells

Abstract The purpose of this study was to examine how specific amino acid restriction targets mitochondria to induce apoptosis in DU145 and PC3 prostate cancer cells. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), and methionine (Met) differentially modulates glucose metabolism, the activity of pyruvate dehydrogenase (PDH) and antioxidant status in the mitochondria of these two cell lines. In DU145 cells, Gln and Met restriction but not Tyr/Phe restriction, increase glucose consumption and decrease lactate production. All the examined restrictions increase mitochondrial PDH activity and reactive oxygen species (ROS). Gln and Met restriction increase mitochondrial activity of glutathione peroxidase (GPx). Tyr/Phe and Met restriction increase mitochondrial activity of manganese superoxide dismutase (SOD) in the first 2 days of the restriction, and the activity returns to basal level on day 4 of restriction. Moreover, all the examined restrictions decrease reduced glutathione (GSH) and cause mitochondrial DNA damage. In PC3 cells, all of the examined restrictions reduce glucose consumption and lactate production; however, they do not alter mitochondrial PDH activity. Gln restriction increases accumulation of ROS and elevates mitochondrial GPx activity. Tyr/Phe restriction increases mitochondrial activity of SOD during the entire restriction period. Although all the restrictions decrease GSH in PC3 cells, they do not cause mitochondrial DNA damage. The data indicate that specific amino acid dependency differentially regulates glucose metabolism, oxidation-reduction reactions of mitochondria, and oxidative damage in mitochondria of these two cell lines. This partially explains how selective amino acid restriction targets mitochondria to induce apoptosis. (The project described was supported by Award Number R01CA101035 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 39.

Neuronal expression of a single‐subunit yeast NADH–ubiquinone oxidoreductase (Ndi1) extends Drosophila lifespan

The 'rate of living' theory predicts that longevity should be inversely correlated with the rate of mitochondrial respiration. However, recent studies in a number of model organisms, including mice, have reported that interventions that retard the aging process are, in fact, associated with an increase in mitochondrial activity. To better understand the relationship between energy metabolism and longevity, we supplemented the endogenous respiratory chain machinery of the fruit fly Drosophila melanogaster with the alternative single-subunit NADH-ubiquinone oxidoreductase (Ndi1) of the baker's yeast Saccharomyces cerevisiae. Here, we report that expression of Ndi1 in fly mitochondria leads to an increase in NADH-ubiquinone oxidoreductase activity, oxygen consumption, and ATP levels. In addition, exogenous Ndi1 expression results in increased CO2 production in living flies. Using an inducible gene-expression system, we expressed Ndi1 in different cells and tissues and examined the impact on longevity. In doing so, we discovered that targeted expression of Ndi1 in fly neurons significantly increases lifespan without compromising fertility or physical activity. These findings are consistent with the idea that enhanced respiratory chain activity in neuronal tissue can prolong fly lifespan.

Ascorbic acid accelerates osteoclast formation and death

Ascorbic acid (AA) plays a key role in bone formation. However controversy remains about the effect of AA on cells responsible for bone destruction, osteoclasts. We investigated the effect of AA on osteoclastogenesis using primary mouse bone marrow cultures and monocytic RAW 264.7 cells treated with osteoclastogenic factors RANKL and MCSF. Treatment with AA resulted in significant increase in osteoclast number, size and nucleation. To assess osteoclast oxidative stress level, a ratio of reduced (GSH) to oxidized (GSSG) glutathione and the total glutathione content (GSHt) were evaluated. Osteoclast differentiation was associated with a decrease in GSH/GSSG and GSHt. AA induced further decrease in both parameters, and resulted in significant production of H2O2, indicating its pro-oxidant action. At low concentration, H2O2 induced similar effects to AA, although less potently, and catalase partially inhibited AA-induced osteoclastogenesis. To assess the modification in osteoclast metabolism, the mitochondrial activity was evaluated using JC-1 and the ATP levels were assessed. Osteoclast formation was associated with the increase in mitochondrial activity and ATP concentration, which were further increased in the presence of AA. Importantly, the stimulatory effect of AA was only evident at early phase of osteoclastogenesis, whereas at the late stage AA significantly accelerated osteoclast death. Thus, during osteoclastogenesis AA acts as an oxidant, first stimulating osteoclast formation, but later limiting osteoclast lifespan. This duality of AA action allows reconciling the stimulatory action of AA on osteoclastogenesis observed in vitro with an overall attenuation of bone resorption in the presence of AA observed in vivo.

Building a new bridge between metabolism, free radicals and longevity

Building a new bridge between metabolism, free radicals and longevity

Increase in mitochondrial density within axons and supporting cells in response to demyelination in the Plp1 mouse model

We used the Plp1-overexpressing transgenic mouse model to investigate whether progressive demyelination of axons results in adaptive changes involving mitochondria within the axons. These models have myelinated axons from birth but gradually lose myelin and develop axonal loss associated with progressive neurological disability analogous to patients with secondary progressive mulltiple sclerosis (SPMS). At 1 and 2 months, electron microscopy demonstrated a significant increase in intraaxonal mitochondrial density in the homozygous line 72 Plp1-overexpressing mice compared with wild type (1.43 +/- 0.31 vs. 0.84 +/- 0.16 microm(-3), P = 0.031; 1.66 +/- 0.11 vs. 0.92 +/- 0.43 microm(-3), P = 0.02) and a significant increase at 1 and 4 months in the density of mitochondria in the surrounding cells in the same mice (1.86 +/- 0.31 vs. 0.81 +/- 0.30 microm(-3), P = 0.006; 2.77 +/- 0.44 vs. 1.37 +/- 0.42 microm(-3), P = 0.016). At both 1 and 4 months, COX histochemistry and time-lapse histochemistry demonstrated a significant increase in mitochondrial activity and rate of mitochondrial activity in the homozygous Plp1-overexpressing mouse optic nerve compared with the wild type (112.37 +/- 11.9 vs. 136.89 +/- 9.1 MeanD, P = 0.006; 128.02 +/- 3.0 vs. 188.77 +/- 9.7 MeanD P < 0.001; Rate -0.78 +/- 0.25 vs. -0.58 +/- 0.15 MeanD min(-1), P < 0.001; -1.48 +/- 0.15 vs. 0.51 +/- 0.17 MeanD min(-1), P < 0.001, respectively). We propose that adaptive changes involving mitochondria occur within CNS axons in Plp1-overexpressing mice, which may be detrimental to long-term viability. Analogous changes occurring in chronically demyelinated axons in MS lesions would be one mechanism increasing axonal vulnerability in SPMS.

Variation in yeast mitochondrial activity associated with asci

An increase in mitochondrial membrane potential (DeltaPsim) and mitochondrially produced 3-hydroxy (3-OH) oxylipins was experienced in asci of the nonfermentative yeasts Galactomyces reessii and Lipomyces starkeyi and the fermentative yeasts Pichia farinosa and Schizosaccharomyces octosporus. Strikingly, asci of Zygosaccharomyces bailii showed no increase in mitochondrial activity (DeltaPsim and oxylipin production). As expected, oxygen deprivation only inhibited ascus formation in those yeasts with increased ascus mitochondrial activity. We conclude that ascus formation in yeasts is not always dependent on mitochondrial activity. In this case, fermentation may provide enough energy for ascus formation in Z. bailii.

Mitochondria—A Nexus for Aging, Calorie Restriction, and Sirtuins?

Recent studies of calorie restriction in several organisms demonstrate an increase in mitochondrial activity that is associated with the salutary effects of this dietary restriction regimen. In this Essay, I speculate on how an increase in mitochondrial activity might provide benefit and discuss how diet, mitochondria, and sirtuins might interact in a pathway to slow aging and associated diseases.

Increased axonal mitochondrial activity as an adaptation to myelin deficiency in the Shiverer mouse

Axonal pathology in multiple sclerosis (MS) has been described for over a century, but new insights into axonal loss and disability have refocused interest in this area. There is evidence of oxidative damage to mitochondrial DNA in chronic MS plaques, suggesting that mitochondrial failure may play a role in MS pathology. We propose that in the chronic absence of myelin the maintenance of conduction relies partially on an increase in mitochondria to provide energy. This increased energy requirement also promotes reactive oxygen species (ROS), because most intraaxonal ROS are generated by mitochondria. If antioxidant defenses are overwhelmed by an excess of ROS, this may result in damage to the axon. Our aim was to investigate whether a chronic lack of myelin results in adaptive changes involving mitochondria within the axon. We investigated this in the shiverer mouse. This myelin basic protein gene mutant provides a model of how adult central nervous system (CNS) axons cope with the chronic absence of a compact myelin sheath. Cytochrome c histochemistry demonstrated a twofold increase in mitochondrial activity in white matter tracts of shiverer, and electron microscopy confirmed a significantly higher number of mitochondria within the dysmyelinated axons. Our data demonstrate that there are adaptive changes involving mitochondria occurring within CNS axons in shiverer mice in response to a lack of myelin. This work contributes to our understanding of the adaptive changes occurring in response to a lack of myelin in a noninflammatory environment similar to the situation seen in chronically demyelinated MS plaques.

Stimulation of bovine sperm mitochondrial activity by homeopathic dilutions of monensin

Mitochondrial activity is an important viability parameter of spermatozoa and is linked to sperm motility. Monensin is commonly used as an inhibitor for sperm mitochondrial activity in the laboratory. This study was conducted to evaluate the influence of some homeopathic dilutions of monensin on sperm mitochondrial activity. Fresh ejaculates from 6 mature bulls were used in the study. Samples of the semen were tested using a flow cytometer for mitochondrial activity and sperm viability using Rhodamine 123 and SYBR-14, respectively. The 9× dilution of monensin resulted in very highly significant ( P<0.001 ) stimulation of mitochondrial activity. Monensin 5×, 7×, 8× and 13× caused highly significant ( P<0.01 ) stimulation of the sperm mitochondrial activity. Other homeopathic dilutions of monensin (6×, 10×, 11×, 12× and 14×) also had a significant ( P<0.05 ) stimulatory effect. The use of monensin did not have any negative effect on sperm viability. We conclude that some homeopathic dilutions of monensin increase mitochondrial activity of bovine spermatozoa without negative effect on sperm viability, the 9× dilution was the most effective. Further in vivo studies are required to estimate the effect of homeopathic dilutions of monensin on semen quality.

Mitochondrial-dependent regulation of myoblast proliferation

The aim of the present study was to determine whether mitochondrial activity could regulate myoblast proliferation. We demonstrate that an increase in mitochondrial activity of L6E9 myoblasts can be easily obtained by simply raising extracellular pyruvate concentration in the culture dish. Under this condition, L6E9 myoblasts underwent a rapid growth arrest in G1 + S phases concomitant to a marked cellular hypertrophy. No sign of myoblast fusion was evident. This was accompanied by the down-regulation of proliferating cell nuclear antigen expression and an increase in p21 expression. Mitochondrial biogenesis was also stimulated, as indicated by a twofold increase in mitochondrial content. These cells exhibited a large increase in the production of reactive oxygen species that could contribute to the observed phenotypic alterations. However, exposure of pyruvate-treated cells to antioxidants did not reverse growth arrest. Similarly, exposure of control cells to oxidants did not induce growth arrest. Our observations suggest that mitochondrial activity appears to play a central role in regulating myoblast proliferation. They also argue strongly in favor of a retrograde communication establishing a mitochondrial control of nuclear gene expression that could be modulated by mitochondrial activity.

L-Glutamate decreases glucose utilization by rat cortical astrocytes

Microenvironmental changes including elevated levels of l-glutamate, ionic homeostasis, acidification, and oxygen level are associated with brain insults. Their effects on energy metabolism were studied in cultured astrocytes. l-Glutamate caused a decrease in lactate accumulation through the activation of transporter in astrocytes in a concentration- and time-dependent manner. Decreases in glucose uptake, lactate synthesis and accumulation as well as increases in mitochondrial activity indicated a switch of the astrocytic metabolism from glycolytic to oxidative. Environmental changes co-operated with l-glutamate to regulate the metabolic strategy, e.g. KCl and oxygen deprivation reversed but acidification exacerbated the l-glutamate-mediated decrease in lactate accumulation. Taken together, during chronic exposure, oxidation of non-glucose substrates such as l-glutamate fuels the active transport of l-glutamate into astrocytes.

Induction of DNA synthesis by ligation of the CD53 tetraspanin antigen in primary cultures of mesangial cells

The interaction of mesangial cells with the extracellular matrix plays a major role in kidney biology. Tetraspanin proteins modulate cell interaction with the extracellular matrix. Tetraspanins form supramolecular structures on the cell membrane that send signals after engagement by unknown ligands, modulate different signaling processes, and regulate cell adhesion and motility. CD53 was determined by immunohistochemistry, and on the cell surface of cultured rat mesangial cells by flow cytometry. Mesangial cell cultures were stimulated with MRC OX-44 antibody. DNA synthesis was measured by thymidine incorporation. Extracellular signal-regulated kinase (ERK) activation was determined by Western blot. CD53 was present in mesangial cells in vivo and in culture. Ligation of CD53 antigen with a monoclonal antibody triggered the induction of DNA synthesis, which was not sensitive to inhibitors of signaling pathways that use phosphatidylinositol 3-kinase (PI3K) and protein kinase C, or to calcium channel inhibitors, such as thapsigargin and verapamil. The DNA synthesis was inhibited by PD98059, a specific inhibitor of MEK that prevents ERK1/ERK2 activation. In addition, ERK1 and ERK2 activation by phosphorylation occurred following CD53 antigen ligation. The DNA synthesis was due to de novo synthesis and not to DNA repair as a consequence of the initiation of apoptosis, determined by flow cytometry, and lack of proteolytic activation of PARP by caspase 3. CD53 antigen ligation also induced an increase in mitochondrial activity. To our knowledge this is the first identification of a tetraspanin protein in mesangial cells. CD53 antigen delivers a signal that initiates DNA synthesis. This signal is mediated by ERK1/ERK2 activation, but it is not sufficient to complete the cell cycle.

Effect of dexamethasone on mitochondrial maturation in the fetal rat brain

OBJECTIVE: The objective of the present study was to explore whether prenatal dexamethasone treatment influences mitochondrial maturation in the fetal rat brain. STUDY DESIGN: Mitochondrial respiration was measured polarographically with homogenates of fetal cerebral cortical tissues on day 16 (with saline solution, n = 8; with dexamethasone, n = 8), day 18 (with saline solution, n = 8; with dexamethasone, n = 8), and day 20 (with saline solution, n = 8; with dexamethasone,n = 8) of gestation. Four doses of dexamethasone (0.1 mg · kg) or vehicle (saline solution) were given, withan interval of 12 hours, until 12 hours before each measurement. RESULTS: In the vehicle-treated animals, mitochondrial respiratory activity was increased significantly after day 18 of gestation. Dexamethasone-treated animals showed a significant increase in mitochondrial activity at day 16 of gestation compared with vehicle-treated animals. CONCLUSION: The results indicate that prenatal dexamethasone treatment contributes to the precocious maturation of mitochondrial activity in the fetal rat brain. Because acceleration in cerebral mitochondrial activities is required immediately after birth to maintain high-energy phosphate levels, the precocious maturation may be crucial for the successful outcome of the preterm infant. (Am J Obstet Gynecol 2002;186:574-8.)