Increase In Number Of Mitochondria Research Articles

Elucidation of the mechanism of changes in the antioxidant function with the aging in the liver of the senescence-accelerated mouse P10 (SAMP10)

Senescence-accelerated mice are known to display a variety of deficits and signs of accelerated aging, but the specific mechanisms involved in this process are still unclear. In this study, we examined the expression levels of antioxidant enzymes, transcription factors responsible for the regulation of expression of these enzymes, and mitochondrial proteins in the liver of SAMP10 and SAMR1 mice at 3 and 12 months of age using western blotting analysis. To investigate the amount of oxidative damage to DNA, levels of 8-OHdG were measured in the liver of these mice. At 3 months of age, the levels of catalase, Mn-SOD, GPx, UQCRC2 and COXIV were significantly upregulated in SAMP10 mice compared with that in SAMR1 mice. However, NDUFS3 levels were not significantly different at this young age. In contrast, the expression level of catalase was significantly lower, and the levels of phosphorylated FoxO-1a and UQCRC2 were significantly higher in SAMP10 mice compared to those in SAMR1 mice; however, at 12 months of age, there were no significant differences in Mn-SOD, GPx, total -FoxO-1a, COXIV, and NDUFS3 expression between the two groups of mice. The levels of 8-OHdG in the liver were markedly higher in 12-month-old SAMP10 mice than those in 3-month-old SAMP10 and SAMR1 mice. These results suggest that an increase in number of mitochondria or a collapse in the balance between the levels of complexes I and III results in an increase in the amount of ROS and induces the expression of antioxidant enzymes in the liver of SAMP10 mice at 3 months of age. Although young SAMP10 mice produce a large amount of ROS, they also produce suitable levels of antioxidant enzymes that decompose ROS; consequently accelerated aging does not occur in young SAMP10 mice. In addition to excessive ROS production which is an important cause of aging, the level of catalase was significantly lower in SAMP10 than that in SAMR1 mice. These results suggested that overexpression of ROS and a decrease in the levels of catalase resulted in the accelerated aging observed in older SAMP10 mice. Moreover, the level of phosphorylated FoxO-1a was increased in SAMP10 compared to that in SAMR1 mice though the total amount of FoxO-1a was not significantly different between the two groups in old age. These results suggest that some impairment in the regulation mechanism of FoxO-1a phosphorylation is responsible for abnormal catalase expression and that a significant decrease in the level of catalase with aging decisively affects the metabolic balance of ROS; thus, ROS that cannot be metabolized contributes to the accelerated aging of SAMP10 mice.

Resistance exercise improves cardiac function and mitochondrial efficiency in diabetic rat hearts.

Metabolic disturbance and mitochondrial dysfunction are a hallmark of diabetic cardiomyopathy (DC). Resistance exercise (RE) not only enhances the condition of healthy individuals but could also improve the status of those with disease. However, the beneficial effects of RE in the prevention of DC and mitochondrial dysfunction are uncertain. Therefore, this study investigated whether RE attenuates DC by improving mitochondrial function using an in vivo rat model of diabetes. Fourteen Otsuka Long-Evans Tokushima Fatty rats were assigned to sedentary control (SC, n=7) and RE (n=7) groups at 28weeks of age. Long-Evans Tokushima Otsuka rats were used as the non-diabetic control. The RE rats were trained by 20 repetitions of climbing a ladder 5days per week. RE rats exhibited higher glucose uptake and lower lipid profiles, indicating changes in energy metabolism. RE rats significantly increased the ejection fraction and fractional shortening compared with the SC rats. Isolated mitochondria in RE rats showed increase in mitochondrial numbers, which were accompanied by higher expression of mitochondrial biogenesis proteins such as proliferator-activated receptor-γ coactivator-1α and TFAM. Moreover, RE rats reduced proton leakage and reactive oxygen species production, with higher membrane potential. These results were accompanied by higher superoxide dismutase 2 and lower uncoupling protein 2 (UCP2) and UCP3 levels in RE rats. These data suggest that RE is effective at ameliorating DC by improving mitochondrial function, which may contribute to the maintenance of diabetic cardiac contractility.

Ursolic acid induces mitochondrial biogenesis through the activation of AMPK and PGC-1 in C2C12 myotubes: a possible mechanism underlying its beneficial effect on exercise endurance.

Mitochondrial biogenesis, which involves an increase in mitochondrial number and the overall capacity of oxidative phosphorylation, is a critical determinant of skeletal muscle function. Recent findings have shown that some natural products can enhance mitochondrial adaptation to aerobic exercise, which in turn improves exercise performance, presumably by delaying muscle fatigue. Ursolic acid (UA), a natural triterpene, is commonly found in various vegetables and fruits. In the current study, UA was shown to increase mitochondrial mass and ATP generation capacity, with a concomitant production of a low level of mitochondrial reactive oxygen species (ROS) in C2C12 myotubes. Mitochondrial ROS, in turn, activated the redox sensitive adenosine monophosphate-dependent protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1(PGC-1) pathway. The activation of AMPK/PGC-1 further increased the expression of cytochrome c oxidase (COX) and uncoupling protein 3. Animal studies showed that UA can also dose-dependently increase the endurance exercise capacity in mice, as assessed by a weight-loaded swimming test and a hanging wire test. Our findings suggest that UA may induce mitochondrial biogenesis through the activation of AMPK and PGC-1 pathways in skeletal muscle, thereby offering a promising prospect for its use to enhance exercise endurance and alleviating fatigue in humans.

Fish oil prevents colon cancer by modulation of structure and function of mitochondria.

Cancer cells are more susceptible to metabolic perturbations due to impaired electron transport chain (ETC) that promote uncontrolled proliferation. Mitochondria play a pivotal role in bioenergetics and apoptosis, hence are considered as a promising target in tumor cell eradication. Therefore, the present study is designed to elucidate chemopreventive action of fish oil (FO) in combination with corn oil (CO) on mitochondria in colorectal cancer (CRC). Male Wistar rats were divided into groups depending on dietary regimen-Control group, FO+CO(1:1) and FO+CO(2.5:1). These groups were further subdivided depending on whether these received a weekly intraperitoneal injection of ethylenediamine tetra-acetic acid (EDTA) or N,N-dimethylhydrazine dihydrochloride (DMH) for a period of 4 weeks. The animals sacrificed 48h and 16 weeks after EDTA/DMH treatment constituted initiation and post-initiation phase respectively. The structural and functional alterations in mitochondria were evaluated using transmission electron microscopy (TEM) and by assaying electron transport chain (ETC) enzymes. Mitochondrial lipid composition and cholesterol levels were also assessed. DMH treatment led to mitochondrial degeneration, disrupted cristae and a significant decrease in ETC complexes suggestive of metabolic reprogramming. Moreover, an increase in cholesterol and cardiolipin (CL) levels in post-initiation phase led to evasion of apoptosis. FO in both the ratios resulted in stabilization and increase in number of mitochondria, however, FO+CO(2.5:1)+DMH group also exhibited mitophagy and crystolysis alongwith altered dynamics in ETC which facilitated apoptosis. It also decreased cholesterol and CL levels to increase apoptosis. Fish oil targets mitochondria in a dose dependent manner that augments apoptosis and hence attenuates carcinogenesis.

Effects of Two Sublethal Concentrations of Mercury Chloride on the Morphology and Metallothionein Activity in the Liver of Zebrafish (Danio rerio).

Mercury (Hg) is a highly hazardous pollutant widely used in industrial, pharmaceutical and agricultural fields. Mercury is found in the environment in several forms, elemental, inorganic (iHg) and organic, all of which are toxic. Considering that the liver is the organ primarily involved in the regulation of metabolic pathways, homeostasis and detoxification we investigated the morphological and ultrastructural effects in Danio rerio liver after 96 h exposure to two low HgCl2 concentrations (7.7 and 38.5 μg/L). We showed that a short-term exposure to very low concentrations of iHg severely affects liver morphology and ultrastructure. The main effects recorded in this work were: cytoplasm vacuolization, decrease in both lipid droplets and glycogen granules, increase in number of mitochondria, increase of rough endoplasmic reticulum and pyknotic nuclei. Pathological alterations observed were dose dependent. Trough immunohistochemistry, in situ hybridization and real-time PCR analysis, the induction of metallothionein (MT) under stressor conditions was also evaluated. Some of observed alterations could be considered as a general response of tissue to heavy metals, whereas others (such as increased number of mitochondria and increase of RER) may be considered as an adaptive response to mercury.

2015 AHA Late-Breaking Basic Science Abstracts.

### 23060 ### Validation Of Circulating MicroRnas As Biomarkers In Heart Failure In Two Large Independent Cohorts Antoni Bayes-Genis1, David Lanfear2, Maurice de Ronde3, Josep Lupon1, Joost Leenders4, Elisabet Zamora1, L. Keoki Williams2, De Antonio Marta5, Zhen Liu4, Koos Zwinderman3, Sara-Joan Pinto-Sietsma3, Yigal Pinto3; 1Universitat Autonoma de Barcelona, Barcelona, Spain; 2Henry Ford Hospital, Detroit, MI; 3Academic Med Cntr, Univ of Amsterdam, Amsterdam, Netherlands; 4Mirnext BV, Amsterdam, Netherlands; 5Germans Trias i Pujol Hosp, Badalona, Spain Background: Small studies suggested circulating microRNAs (circmiRs) as biomarkers for Heart Failure (HF). However, standardized approaches and quality assessment are not established, and results have been inconsistent, with little replication between studies. We aimed to implement quality standards to enable comparison between cohorts and assess which circmiRs may add prognostic information in HF. Methods: We measured 15 circmiRs in two independent cohorts totaling >2000 subjects. Cohort I (Barcelona) comprised of n=843 chronic HFrEF patients. Cohort II from Detroit comprised n= 1384 chronic HF patients (892 HFrEF, 492 HFpEF). Each sample was measured in duplicate, and normalized to an abundant and stable circmiR (miR-486-5p). Algorithms were installed to define each circmiR measurement as “valid”, “unmeasurable” or “invalid”. This allowed inclusion of valid low-level circmiR measurements while reducing noise from false amplification signals. Results: In general, between 20–40% of measurements were “invalid”, while miR-499a\_5p and -208a were “unmeasurable” in the majority of patients in both cohorts. Higher levels of circmiRs-133b, -1254, -622, -208a and -499a\_5p were significantly associated with risk of death in both cohorts, with hazard ratios ranging from 1.103 to 1.365 per log increase (p-values 0.001 to 0.05). However, adding these circmiRs to established predictors (age, renal function and NTproBNP) did not further augment the c-stat beyond 0.71 (cohort I) or 0.78 (cohort II). …

Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway

The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6h before aluminium (10mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits – ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases.

Post‐weaning western style diet leads to increased activity of Complex IV of non‐human primates when exposed to western style diet in utero.

Adult offspring of insulin resistant mothers show increased susceptibility to type 2 diabetes and obesity. Previous studies in this population have found reduced mitochondrial function and decreased lipid oxidation in skeletal muscle consistent with insulin resistance. It is not known how maternal diet affects functioning of the individual electron transport chain complexes. We were specifically interested in investigating whether exposure to maternal western style diet (WSD), or post‐weaning (PW) exposure to WSD, would impact functioning of Complex IV (CIV) when compared across treatment groups. Gastrocnemius muscle homogenates taken from 1 year old juvenile Japanese Macaque of dams exposed either to a control (CTR) or WSD prenatal (PN), and a CTR or WSD PW diet, were assayed for activity in Cytochrome C oxidation, reporting on the activity of CIV in these tissues. We hypothesized that in‐utero exposure to maternal WSD would lead to functional changes in complex IV of the electron transport chain. Our results showed no direct effect of maternal diet on CIV activity, but an increase in CIV activity when prenatally exposed offspring were fed a PW WSD. When analyzed by sex, males showed an increase in CIV activity when exposed to PN WSD, while females showed an increase in CIV with exposure to PW WSD, regardless of PN exposure. Experiments assessing citrate synthase activity shows no change with exposure to PN WSD, indicating that there is no increase in mitochondrial number. Taken together, these data show that while there is no increase in mitochondrial number, there is an increase in CIV activity with exposure to both PN and PW WSD in males and females, and increased activity in females regardless of PN exposure.

Deficiency in DNA Repair Enzyme Ogg1 in Mice Leads to Impaired Mitochondrial Function and Cardiomyopathy

Postnatal cardiac development in rodents is associated with robust mitochondrial biogenesis, which is accompanied by oxidative mitochondrial (mt) DNA damage and repair (Pohjoismaki et al, NAR, 2012). Our previous experiments in knock-out mice deficient in Ogg1 (KO) showed higher mtDNA oxidation in cardiac tissue and increased heart-to-body mass ratio when compared to wild type (WT) mice. Conversely, over-expression of mitochondria-targeted Ogg1 in KO mice decreased mtDNA oxidation and blunted elevations in cardiac mass. In the present study we tested the hypothesis that Ogg1 deficiency impairs cardiac and mitochondrial functions. Echocardiography confirmed our previous results showing increased cardiac mass in KO mice. Importantly, we did not find significant changes in cardiac functions in 3-week old animals, although a trend towards decrease of cardiac contractility was observed in Ogg1 deficient mice. Assessment of mitochondrial respiration using an oxygraph revealed significant increase in mitochondrial oxygen flux during state II and III with concurrent decrease in respiratory ratio in KO mice compared to WT animals. These results indicate either an increased mitochondria number or a constitutive uncoupling in the cardiac tissue. Over-expression of mitochondria-targeted Ogg1 in KO mice blunted changes in mitochondrial bioenergetics. Our results suggest that alteration in mtDNA repair may lead to impaired mitochondrial function and cardiomyopathy. Supported by NIH.

Activating HSP72 in rodent skeletal muscle increases mitochondrial number and oxidative capacity and decreases insulin resistance.

Induction of heat shock protein (HSP)72 protects against obesity-induced insulin resistance, but the underlying mechanisms are unknown. Here, we show that HSP72 plays a pivotal role in increasing skeletal muscle mitochondrial number and oxidative metabolism. Mice overexpressing HSP72 in skeletal muscle (HSP72Tg) and control wild-type (WT) mice were fed either a chow or high-fat diet (HFD). Despite a similar energy intake when HSP72Tg mice were compared with WT mice, the HFD increased body weight, intramuscular lipid accumulation (triacylglycerol and diacylglycerol but not ceramide), and severe glucose intolerance in WT mice alone. Whole-body VO2, fatty acid oxidation, and endurance running capacity were markedly increased in HSP72Tg mice. Moreover, HSP72Tg mice exhibited an increase in mitochondrial number. In addition, the HSP72 coinducer BGP-15, currently in human clinical trials for type 2 diabetes, also increased mitochondrial number and insulin sensitivity in a rat model of type 2 diabetes. Together, these data identify a novel role for activation of HSP72 in skeletal muscle. Thus, the increased oxidative metabolism associated with activation of HSP72 has potential clinical implications not only for type 2 diabetes but also for other disorders where mitochondrial function is compromised.

Myelin Loss Does Not Lead to Axonal Degeneration in a Long-Lived Model of Chronic Demyelination

Current dogma suggests that chronically demyelinated axons are at risk for degeneration, with axonal loss resulting in permanent disability in myelin disease. However, the trophic role of the myelin sheath in long-term axonal survival is incompletely understood. Previous observations of the effect of dysmyelination or demyelination on axonal survival in the myelin mutants has been limited because of their short life span. In this study, we used the Long-Evans shaker (les) rat, which can live up to 9 months, to study axonal health and survival after chronic demyelination. At 2 weeks, ∼29% of medium and ∼47% of large fiber axons are myelinated in les spinal cord. However, by 3 months, no medium and ∼<1% of large-diameter axons retain myelin. After demyelination, axons have a reduced-caliber, abnormal neurofilament distribution and an increase in mitochondrial number. However, there are no signs of axonal degeneration in les rats up to 9 months. Instead, there is a profound increase in oligodendrocytes, which were found to express BDNF, NT-3, and IGF-1. Importantly, this study provides in vivo evidence that mature glial cells produce various neurotrophic factors that may aid in the survival of axons after chronic demyelination.

Continuous exposure to low amplitude extremely low frequency electrical fields characterizing the vascular streaming potential alters elastin accumulation in vascular smooth muscle cells

In normal development and pathology, the vascular system depends on complex interactions between cellular elements, biochemical molecules, and physical forces. The electrokinetic vascular streaming potential (EVSP) is an endogenous extremely low frequency (ELF) electrical field resulting from blood flowing past the vessel wall. While generally unrecognized, it is a ubiquitous electrical biophysical force to which the vascular tree is exposed. Extracellular matrix elastin plays a central role in normal blood vessel function and in the development of atherosclerosis. It was hypothesized that ELF fields of low amplitude would alter elastin accumulation, supporting a link between the EVSP and the biology of vascular smooth muscle cells. Neonatal rat aortic smooth muscle cell cultures were exposed chronically to electrical fields characteristic of the EVSP. Extracellular protein accumulation, DNA content, and electron microscopic (EM) evaluation were performed after 2 weeks of exposure. Stimulated cultures showed no significant change in cellular proliferation as measured by the DNA concentration. The per-DNA normalized protein in the extracellular matrix was unchanged while extracellular elastin accumulation decreased 38% on average. EM analysis showed that the stimulated cells had a 2.85-fold increase in mitochondrial number. These results support the formulation that ELF fields are a potential factor in both normal vessel biology and in the pathogenesis of atherosclerotic diseases including heart disease, stroke, and peripheral vascular disease.

Arabidopsis thaliana organellar DNA polymerase IB mutants exhibit reduced mtDNA levels with a decrease in mitochondrial area density

Plant organelle genomes are complex and the mechanisms for their replication and maintenance remain unclear. Arabidopsis thaliana has two DNA polymerase genes, DNA polymerase IA (polIA) and polIB, that are dual targeted to mitochondria and chloroplasts and are differentially expressed in primary plant tissues. PolIB gene expression occurs at higher levels in tissues not primary for photosynthesis. Arabidopsis T-DNA polIB mutants have a 30% reduction in relative mitochondrial DNA (mtDNA) levels, but also exhibit a 70% increase in polIA gene expression. The polIB mutant shows an increase in mitochondrial numbers but a significant decrease in mitochondrial area density within the hypocotyl epidermis, shoot apex and root tips. Chloroplast numbers are not significantly different in mesophyll protoplasts. These mutants do not have a significant difference in total dark mitorespiration levels but exhibit a difference in light respiration levels and photosynthesis capacity. Organelle-encoded genes for components of respiration and photosynthesis are upregulated in polIB mutants. The mutants exhibited slow growth in conjunction with a decreased rate of cell expansion and other secondary phenotypic effects. Evidence suggests that early plastid development and DNA levels are directly affected by a polIB mutation but are resolved to wild-type levels over time. However, mitochondria numbers and DNA levels never reach wild-type levels in the polIB mutant. We propose that both polIA and polIB are required for mtDNA replication. The results suggest that polIB mutants undergo an adjustment in cell homeostasis, enabling them to maintain functional mitochondria at the cost of normal cell expansion and plant growth.

Toxic Interaction Between Fumonisin B1and Moniliformin for Cardiac Lesions in Japanese Quail

This study examined the effects of fumonisin B1 (FB1) and moniliformin (M) on the heart of Japanese quail (Coturnix coturnix japonica). Three hundred and ninety day-old Japanese quail were randomly divided into four groups: 1) FB1 alone (FX), 2) M alone (MX), 3) FB1 and M (FM), and 4) chick mash alone (CX). We used three pen replicates of 35 quail per pen in groups FX, MX, and FM and three pen replicates of 25 quail per pen in group CX. Gross and microscopic changes in the heart were studied in nine birds (three birds per replicate) from each group at weekly intervals up to 28 days postfeeding (DPF). Ultrastructural changes were studied in the heart of three birds (one bird per replicate) from each group at 21 DPF. Thinning of the heart was the only significant gross lesion in group FX. In contrast, mild-to-severe cardiomegaly was a significant finding in groups MX and FM throughout the study. Microscopically, thinning of cardiomyocytes was evident at 7 DPF in group FX. In addition to the hypertrophy of cardiomyocytes evident as early as 7 DPF, myocardial karyomegaly, nuclear hyperchromasia, and myofibril disarray exhibiting a wavy pattern were more pronounced at 28 DPF in group MX. Similar but more severe lesions were observed in the FM combination group that included myocardial hemorrhages, vacuolar changes, hypertrophy of cardiomyocytes, focal myocarditis, and loss of myofibrils cross-striations. Via transmission electron microscopy, the maximum effect of FB1 toxicity was observed on mitochondria. In addition to an increase in the number of mitochondria, the mitochondria seemed invariably swollen and pleomorphic, although the outer membrane was intact, and the membrane cristae were usually distinct. Myofibrils seemed thinner, without much disruption in their architecture. Large numbers of vacuolar bodies of irregular size, both in the sarcoplasm and in between the myofibrils, were conspicuous in group FX. In contrast to group FX, the increase in number of mitochondria resulted in widespread separation of muscle fibers in group MX. In addition, the mitochondria were swollen and varied from round to oval to slightly elongated and occasionally forked, and vacuolation was rarely noticed in group MX. In the FM combination group, a significant increase in the number of mitochondria caused muscle fibers to look much thinner and assume a wavy pattern. We conclude that the effect of M on the heart is exaggerated in the presence of FB1. Although the overall interactive effect of FB1 and M was less than additive, the interactive effects between the two toxins for cardiac lesions were greater than additive to synergistic up to the second week, raising serious concerns on early age exposure to a combination of these two mycotoxins.

Divergent skeletal muscle respiratory capacities in rats artificially selected for high and low running ability: a role for Nor1?

Inactivity-related diseases are becoming a huge burden on Western society. While there is a major environmental contribution to metabolic health, the intrinsic properties that predispose or protect against particular health traits are harder to define. We used rat models of inborn high running capacity (HCR) and low running capacity (LCR) to determine inherent differences in mitochondrial volume and function, hypothesizing that HCR rats would have greater skeletal muscle respiratory capacity due to an increase in mitochondrial number. Additionally, we sought to determine if there was a link between the expression of the orphan nuclear receptor neuron-derived orphan receptor (Nor)1, a regulator of oxidative metabolism, and inherent skeletal muscle respiratory capacity. LCR rats were 28% heavier (P < 0.0001), and fasting serum insulin concentrations were 62% greater than in HCR rats (P = 0.02). In contrast, HCR rats had better glucose tolerance and reduced adiposity. In the primarily oxidative soleus muscle, maximal respiratory capacity was 21% greater in HCR rats (P = 0.001), for which the relative contribution of fat oxidation was 20% higher than in LCR rats (P = 0.02). This was associated with increased citrate synthase (CS; 33%, P = 0.009) and β-hydroxyacyl-CoA (β-HAD; 33%, P = 0.0003) activities. In the primarily glycolytic extensor digitum longus muscle, CS activity was 29% greater (P = 0.01) and β-HAD activity was 41% (P = 0.0004) greater in HCR rats compared with LCR rats. Mitochondrial DNA copy numbers were also elevated in the extensor digitum longus muscles of HCR rats (35%, P = 0.049) and in soleus muscles (44%, P = 0.16). Additionally, HCR rats had increased protein expression of individual mitochondrial respiratory complexes, CS, and uncoupling protein 3 in both muscle types (all P < 0.05). In both muscles, Nor1 protein was greater in HCR rats compared with LCR rats (P < 0.05). We propose that the differential expression of Nor1 may contribute to the differences in metabolic regulation between LCR and HCR phenotypes.

PKCε promotes cardiac mitochondrial and metabolic adaptation to chronic hypobaric hypoxia by GSK3β inhibition.

PKCε is central to cardioprotection. Sub-proteome analysis demonstrated co-localization of activated cardiac PKCε (aPKCε) with metabolic, mitochondrial, and cardioprotective modulators like hypoxia-inducible factor 1α (HIF-1α). aPKCε relocates to the mitochondrion, inactivating glycogen synthase kinase 3β (GSK3β) to modulate glycogen metabolism, hypertrophy and HIF-1α. However, there is no established mechanistic link between PKCε, p-GSK3β and HIF1-α. Here we hypothesized that cardiac-restricted aPKCε improves mitochondrial response to hypobaric hypoxia by altered substrate fuel selection via a GSK3β/HIF-1α-dependent mechanism. aPKCε and wild-type (WT) mice were exposed to 14 days of hypobaric hypoxia (45 kPa, 11% O(2)) and cardiac metabolism, functional parameters, p-GSK3β/HIF-1α expression, mitochondrial function and ultrastructure analyzed versus normoxic controls. Mitochondrial ADP-dependent respiration, ATP production and membrane potential were attenuated in hypoxic WT but maintained in hypoxic aPKCε mitochondria (P < 0.005, n = 8). Electron microscopy revealed a hypoxia-associated increase in mitochondrial number with ultrastructural disarray in WT versus aPKCε hearts. Concordantly, left ventricular work was diminished in hypoxic WT but not aPKCε mice (glucose only perfusions). However, addition of palmitate abrogated this (P < 0.05 vs. WT). aPKCε hearts displayed increased glucose utilization at baseline and with hypoxia. In parallel, p-GSK3β and HIF1-α peptide levels were increased in hypoxic aPKCε hearts versus WT. Our study demonstrates that modest, sustained PKCε activation blunts cardiac pathophysiologic responses usually observed in response to chronic hypoxia. Moreover, we propose that preferential glucose utilization by PKCε hearts is orchestrated by a p-GSK3β/HIF-1α-mediated mechanism, playing a crucial role to sustain contractile function in response to chronic hypobaric hypoxia.

Paradigms in multiple sclerosis: time for a change, time for a unifying concept

It has recently been suggested that, rather than being an autoimmune disease, multiple sclerosis (MS) is an example of a neurocristopathy, a pathological process resulting from a faulty development of the neural crest. Whilst several characteristics of the disease suggest a neurocristopathy, other aetiological factors require consideration, including hygiene-related factors that alter the immune responses to common pathogens resulting in an eclipse of immune reactivity that could protect against MS, the possible role of human endogenous retroviruses (HERVs) in pathogenesis and autoimmune phenomena, HLA polymorphism, vitamin D levels before and after birth and immune repair mechanisms. A postulated aetiological factor in MS, associated with altered vitamin D metabolism and abnormal HERV expression, is a long-lasting disturbed redox regulation in the biosynthesis of a melanoma-like melanin pigment. Although intensive further studies on melanin pigments in nerve tissue in MS are required, the known properties of a pathological form of such pigments in melanoma could explain a number of observations in MS, including the impact of light, UV-light, and vitamin D, and could explain the clinical manifestations of MS on the basis of an oscillating process of oxidative charge and discharge of the pigments and a threshold phenomenon with a change of the quasi-catalytic function of the pigment from destroying reactive oxygen radicals or species to transforming them to more harmful long-persisting highly reactive species. Taken together with the consequences of an adaptive process in partly demyelinated neurons, resulting in an increase in number of mitochondria, and the impact of stressful life events, these conditions are necessary and sufficient to explain the disease process of MS with its spatial (plaques) and temporal (attacks and remissions) characteristics. This suggested unifying concept of the pathogenesis of MS may open perspectives for prevention, diagnosis and therapy. In particular, prevention may be achieved by vaccinating against Epstein-Barr virus in early childhood.

Abstract 965: The KISS1 metastasis suppressor appears to reverse the ‘Warburg Effect’

Abstract In 1924, Otto Warburg described the preference of cancer cells for glycolytic metabolism, even under normoxic conditions and that these metabolic changes directly correlate with malignant potential of several cancers. Although its purpose remains unclear, the “Warburg Effect” is thought to confer proliferative and survival advantages by increasing uptake of nutrients into biomass. The KISS1 metastasis suppressor protein is secreted and proteolytically cleaved into so-called kisspeptins (KP) that block the colonization of metastatic C8161.9 human melanoma cells at secondary sites. We asked whether secreted KISS1 mediates its inhibitory effects on metastatic growth through regulation of the “Warburg Effect.” Comparing multiple bioenergetic and metabolic aspects of glucose metabolism in C8161.9 ± KISS1 showed that all KISS1-secreting clones had significantly (P&amp;lt;0.05) reduced invasion (60%) and reduced lactate production (100 mg/dL vs. 128 mg/dL). Irrespective of cell density, KISS1-expressing cells had a significantly higher extracellular pH (pHe=7.2) compared to cells transfected with empty vector or cells transfected with KISS1 harboring a deleted signal sequence (ΔSS; pHe=6.7). Utilizing a Seahorse® bioanalyzer, reduced extracellular acidification by KISS1 cells was verified concomitant with increased O2 consumption. Interestingly, mitochondrial reserve capacity, an indicator thought to reflect a cell's ability to cope with oxidative stresses, was also elevated in KISS1-expressing cells. Using mitochondrial-selective fluorescent probes, C8161.9KISS1 melanoma and MDA-MB-435KISS1 breast carcinoma cells have ∼30% more mitochondria compared to empty vector or KISS1ΔSS-expressing cells. Increased mitochondrial number in KISS1-expressing cells was correlated with higher levels of PGC-1α, a major mitochondrial biogenesis regulatory molecule, which was confirmed using siRNA to KISS1. Expression of KISS1 also protected C8161.9 cells from dichloroacetate-induced cell death. Unexpectedly, addition of KP10 to C8161.9 cells did not alter pHe, raising questions regarding the mechanism by which KISS1/KP alter PGC-1α in the absence of KISS1 receptor expression in the tumor cells. Nonetheless, these data appear to directly connect changes in mitochondrial number, metabolic pathway regulation and the metastatic process. Future studies will determine whether the increase in mitochondrial number is directly responsible for the change in glycolytic metabolism and whether these changes are necessary for KISS1's effects on metastatic growth. Support: RO1-CA134981, the National Foundation for Cancer Research, METAvivor, and UAB Med-into-Grad Fellowship. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2011-965

The influence of brachial plexus compression on the ultrastractures of cervical sympathetic ganglion

Objective To investigate the changes of the ultrastructures of cervical sympathetic ganglia (CSG) in brachial plexus compression and the influence of nerve decompression on these ultrastructural changes. Methods Twenty-four SD rats were divided into 4 groups with 6 each: upper trunk compression group, lower trunk compression group, upper trunk decompression group and lower trunk decompression group. The CSG were harvested 3 months after brachial plexus compression in the upper trunk compression group and lower trunk compression group. For the two decompression groups, the CSG were harvested 1 month later following the 3 months compression period. infrastructures of the CSG were observed using electron microscope. Results Ultrastructural changes in the upper and lower trunk compression groups were represented by swelling mitochondria, missing mitochondria cristae and mitochondria vacuolation in the CSG. The major ultrastructural changes of CSG in the upper and lower trunk decompression groups were endoplasmic reticulum enlargement and the decrease in volume and increase in number of mitochondria. Conclusion Following brachial plexus compression, the ultrastructural changes in CSG are related to metabolism of the neurons. After nerve decompression the changes in CSG are related to neuron secretion and functional recovery of mitochondria. Key words: Brachial plexus; Ganglia, sympathetic; Nerve entrapments; Ultrastructrue

Baicalin reduces mitochondrial damage in streptozotocin‐induced diabetic Wistar rats

Hyperglycemia-induced superoxide production in the mitochondria is known to be the primary cause of diabetic micro- and macro-vascular complications and mitochondrial membranal damage. This study in streptozotocin-induced diabetic Wistar rats investigated the anti-hyperglycemic and mitochondrial membrane protection effects of baicalin, a flavonoid known for its radical scavenging activity. The following oral treatments were given to diabetic rats for 30 days: (1) metformin 500 mg/kg, (2) baicalin 120 mg/kg, and (3) metformin 500 mg/kg & baicalin 120 mg/kg, with vehicle-treated diabetic and non-diabetic groups serving as controls. Transmission electron microscopy imaging of pancreatic beta-cells revealed loss of integrity of the inner membrane of the mitochondria in the diabetic rats, which was not observed in the baicalin-treated group. In addition, baicalin and the combined treatment of metformin and baicalin had significantly reduced (p < 0.05) the number of mitochondria with a damaged membrane compared to the diabetic control as well as the metformin-treated group in the hepatic tissues. Baicalin had also increased the plasma leptin content (p < 0.05) versus the diabetic control, which in turn had effected the total expression of hepatic mitochondria per cell indicating its effects in SIRT1 activity. The increase in mitochondrial number was further complemented with similar trends in the hepatic citrate synthase activity. Baicalin had reduced the hyperglycemia-induced mitochondrial membrane damage, as well as enhanced the effects of metformin, as was observed in the results from the metformin and baicalin treated groups.