In Europe alcohol consumption causes 6.5% of deaths and Alcoholic Liver Disease (ALD) is the predominant cause of liver disease[1]. In the pathogenesis of ALD the involvement of mitochondria is well established[2, 3], morphological alterations (megamitochondria) in the liver biopsies of patients are recognised as hallmarks of ALD[4]. However, the impact of alcohol on mitochondrial dynamics and mitochondria-shaping proteins (MSP) remains unknown. The effect of alcohol was investigated in vitro (hepatoma cells), ex vivo (human liver slices) and in 55 patients with ALD. The analysis by confocal/electron microscopy revealed an initial mitochondrial hyper-fragmentation induced by short-term ethanol treatment, preceding cell injury or mitochondrial dysfunction; while megamitochondria developed as a consequence of longer exposure. These structural modifications were associated with changes in the MSP regulating fragmentation but not fusion (gene/protein expression), in particular in Dynamin related protein-1 (Drp-1) and its receptors MiD51 and Mff. When Drp-1 was inactivated, the cells shown abrogation of ethanol-induced hyper-fragmentation and increased megamitochondria formation, suggesting that both phenomena are induced by alcohol via Drp-1. The pivotal role of Drp-1 in ALD was confirmed in liver biopsies of patients with alcoholic hepatitis, opening new perspectives in the development of therapies aimed to modulate its activity.
Quantitative indicators of mitochondria functions and dysfunctions and physiological state of the organism were estimated by the activity of dehydrogenases (DH): succinate DH (SDH), SDH + isocitrate (ISC), lactate DH ( LDH), and LDH/SDH as glycolysis-respiration ratio or Warburg еffect measure proposed by our group. Our advanced cyto-BIO-chemical method was used to detect the state of mitochondria within lymphocytes in a smear of blood [1, 2]. The typical examples for the children examined are shown in the figure below. The pronounced rise of LDH activity and decrease in SDH are observed under leucosis. The dysfunctions are even more manifested in the LDH/SDH ratio. There is little difference between DH activity under myopathy and this in healthy children. However, the LDH/SDH ratio clearly reveals the decrease. The increase in Warburg effect is a beneficial property of cancer cells, which is essential for intensive biosynthesis and proliferation [3]. In contrast, the weakening of the restorative processes is typical for myopathy. Biomarkers of glycolysis ( LDH), respiration (SDH) and Warburg effect (LDH/SDH) in young patients suffering from leucosis (L) n=22 and myopathy (M) n=9, compared with healthy(H) n=25 children of similar age, measured by nitroblue reduction (NBR).
The mitochondrion is deeply involved in ROS production through electron leak that occur in the respiratory chain. Measurement of mitochondrial ROS is useful to evaluate the consequences of MRC inhibition, electron leak, complex I dysfunction and stimulation of some oxidative pathways induced by chemicals or natural products. These parameters measured on isolated mitochondria allow identification of direct mitochondrial impairment with subsequent toxicity on skin, blood or organs. On the other hand, the mitochondrion is itself a target of ROS (vicious circle or cellular ROS) which may lead to irreversible damage of mtDNA or mitochondrial membrane lipids and proteins, resulting in mitochondrial dysfunction. For example, permeability transition pore (mPTP) susceptibility to oxidative stress is observed in various pathological cases (aging, neuronal injury, cardiac reperfusion injury...). Such damages can be reproduced on isolated mitochondria to identify mitochondrial protective molecules against oxidative stress induced-mPTP opening. In conclusion, simultaneous assessment of mitochondrial integrity, function and ROS production is a valuable toolbox to identify the risk of compound-induced liability in human, in particular regarding organ toxicity. Such approach can also be used to identify antioxidant properties of compounds in order to preserve mitochondrial integrity and cell life.
Mitochondria play an essential role in myocardial tissue homeostasis and deterioration in mitochondrial function resulting from “off target” effects of drug exposure can lead to cardiomyocyte and endothelial cell death and as a consequence cardiovascular dysfunction. This unpredicted cardiotoxicity is one of the causes of high rates of attrition during drug development, and poses and enormous financial burden on the pharmaceutical industry. For example, a number of tyrosine kinase inhibitors (TKIs), have revealed previously unknown cardiotoxicity in the later clinical phases of drug development or after their regulatory approval. Assays that able to accurately predict mitochondrial toxicity are therefore urgently required. We have compared and evaluated a number of assays to assess mitochondrial toxicity following exposure to the TKI Mubritinib; a HER2 TKI that failed clinical safety and tolerability trials. Our data have confirmed its mechanism of toxicity and we show that this drug acts as an inhibitor of the electron transport chain. In general, our data demonstrate that simple cellular assays are able to provide an accurate prediction of toxicity as well as more sophisticated assays e.g. those which employ the use of human-stem cell derived cardiomyocytes.
Mitochondrial oxidative stress is an important pathologic factor in neurodegenerative diseases, including Alzheimer’s disease. Abnormal production of reactive oxygen species (ROS), resulting from mitochondrial dysfunction, leads to neuronal cell death. Ceria (CeO2) nanoparticles less than 5 nm are known to perform as powerful and recyclable ROS scavengers by exchanging between Ce3+ and Ce4+ oxidation states. Hence, targeting ceria nanoparticles specifically to mitochondria might be a promising therapeutic approach for neurodegenerative diseases. Here, we report the design and synthesis of triphenylphosphonium-conjugated ceria nanoparticles which localize to mitochondria of subicular cells due to their small hydrodynamic diameter (22 nm) and highly positive charge (+45 mV). The triphenylphosphonium-conjugated ceria nanoparticles diminish mitochondrial oxidative stress and suppress neuronal death in a 5XFAD transgenic Alzheimer’s disease mouse model after two months. The triphenylphosphonium-conjugated ceria nanoparticles alleviate reactive gliosis and morphological mitochondria damage observed in these mice. Altogether, our data indicate that the triphenylphosphonium-conjugated ceria nanoparticles are a potential therapeutic candidate for mitochondrial oxidative stress in Alzheimer’s disease.
Introduction&Objectives Mitochondria are dynamic organelles, involved in different cellular functions, including oxidative phosphorylation, where iron is a fundamental co-factor [1]. Besides being central part of mitochondrial I-IV complexes in the electron transport system, iron also regulates the Krebs cycle by modulating mitochondrial aconitase [2, 3]. Hence, imbalances of iron homeostasis could affect mitochondrial activity and cellular metabolism [4]. Nevertheless, little is known on that. Therefore, we aimed at investigating the impact of alterations of iron homeostasis on mitochondrial function, and on peripheral blood metabolites, in order to potentially identify distinctive signatures. Materials&Methods Mitochondrial function was studied in liver samples of 10-week old FVB mice and C57BL/6N mice, receiving either normal- or high iron (25 g/kg)-diet two weeks before being sacrificed. Livers were homogenized and mitochondrial respiration was assessed by means of high resolution respirometry (OROBOROS Instruments, Austria). Peripheral blood was collected, and metabolomics analysis was performed by using liquid chromatography-mass spectrometry (LC-MS). Results&Conclusion Our ongoing experiments indicate that dietary iron supplementation affects the phosphorylation system in the mouse liver of both FVB and C57BL/6N mice. The analysis of peripheral blood metabolites is currently under investigation, and might provide useful information on changes in the overall metabolism.
Objective: We investigated the role of MRT in preventing mitochondrial diseases caused by mtDNA mutation. Method: MRT was conducted by spindle nuclear transfer (SNT) between human oocytes. Mutant mtDNA load was analyzed. Results: of 18 oocytes collected from a female carrier (24.5% mtDNA 8993T>G) of Leigh Syndrome, 7 oocytes (haplogroup I) were attempted for MRT to enucleated donor oocytes (haplogroup L2c). Of the 4 blastocysts created from the 5 reconstituted oocytes, 1 euploid embryo was achieved, carrying 5.73% mtDNA 8993T>G mutation load comparing to 3.66% in an aneuploid embryo. The calculated mtDNA 8993T>G load was about 100% in both carrier’s oocytes, from which the above two embryos were created. A boy was delivered after euploid embryo transfer to the carrier. The mutant mtDNA load was differentiated expressed among the fetal and fetal appendage tissues, ranging from 0% to 9.23%. The child is still asymptomatic of Leigh Syndrome or other diseases at 5-month old now. Conclusions: Mitochondrial disease caused by mtDNA mutation may be prevented by MTR through SNT among different haplogroups. More cases and a long term follow-up are warranted to evaluate the safety of this technique.
Mitochondria play a central role in cellular energy production and their dysfunction can trigger a compensatory increase in glycolytic flux to sustain cellular ATP levels. Here we studied the mechanism of this homeostatic phenomenon in C2C12 myoblasts. Acute (30 min) mitoenergetic dysfunction induced by the mitochondrial inhibitors piericidin A and antimycin A, stimulated Glut1-mediated glucose uptake without altering Glut1 mRNA or plasma membrane levels. The serine/threonine liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK) played a central role in this stimulation. In contrast, ataxia-telangiectasia mutated (ATM; a potential AMPK kinase), Src (previously highlighted to stimulate Glut1-mediated glucose uptake), and hydroethidium (HEt)-oxidizing reactive oxygen species (ROS; increased in piericidin A- and antimycin A-treated cells), appeared not to be involved in glucose uptake simulation. Inhibitor treatment increased NAD+ and NADH levels (leading to a lower NAD+/NADH ratio), but did not affect the level of Glut1 acetylation. Stimulation of glucose uptake was greatly reduced upon inhibition of Sirt2 or mTOR/RAPTOR. We propose that mitochondrial dysfunction triggers LKB1-mediated AMPK activation, which stimulates Sirt2 phosphorylation, leading to activation of Akt/mTOR/RAPTOR and Glut1-mediated glucose uptake.
Metastasis-related methyltransferase 1 (Merm1) is a highly conserved protein which mediates N7-methylation of G1639 on ribosomal 18S RNA (1). Here, we identify a novel role for Merm1 in modulating mitochondrial transcription and thereby function. Merm1 function was investigated by siRNA mediated knockdown followed by RNAseq analysis, revealing reduced abundance of all mitochondrially encoded transcripts. This observation was corroborated by qPCR analysis, and there was no change in mitochondrial genome copy number. The changes in mitochondrial transcript abundance induced by Merm1 knockdown were found to be functionally important, reducing basal mitochondrial respiration, spare respiratory capacity and ATP production, as measured by Seahorse™ XF analyser. Immunofluorescence imaging combined with subcellular fractionation studies in A549 cells did not co-localise Merm1 within mitochondria. We therefore propose that Merm1 is eliciting these effects from outside the mitochondria, through its role as a ribosomal modulator. Importantly, in a panel of ~400 exomes from patients with unexplained mitochondrial defects, three heterozygous exonic missense mutations were identified in the Merm1 gene. Delineating the mechanism by which Merm1 regulates mitochondrial function may inform new therapies with clinical impact.
Preclinical data suggest that chronic stress may cause cellular damage and mitochondrial dysfunction, potentially leading to the release of mitochondrial DNA (mtDNA) into the bloodstream. Major depressive disorder has been associated with an increased amount of mtDNA in leukocytes from saliva samples and blood; however, no previous studies have measured plasma levels of free-circulating mtDNA in a clinical psychiatric sample. In this study, free circulating mtDNA was quantified in plasma samples from 37 suicide attempters, who had undergone a dexamethasone suppression test (DST), and 37 healthy controls. We hypothesized that free circulating mtDNA would be elevated in the suicide attempters and would be associated with hypothalamic-pituitary-adrenal (HPA)-axis hyperactivity. Suicide attempters had significantly higher plasma levels of free-circulating mtDNA compared with healthy controls at different time points (pre- and post-DST; all P-values < 2.98E - 12, Cohen's d ranging from 2.55 to 4.01). Pre-DST plasma levels of mtDNA were positively correlated with post-DST cortisol levels (rho = 0.49, P < 0.003). Suicide attempters may have elevated plasma levels of free-circulating mtDNA, which are related to impaired HPA-axis negative feedback. This peripheral index is consistent with an increased cellular or mitochondrial damage. The specific cells and tissues contributing to plasma levels of free-circulating mtDNA are not known, as is the specificity of this finding for suicide attempters. Future studies are needed in order to better understand the relevance of increased free-circulating mtDNA in relation to the pathophysiology underlying suicidal behavior and depression.
Mitochondrial function and glycolysis play critical roles in a variety of vital cellular processes, including cellular activation, proliferation, differentiation, cell death, and disease progression. Seahorse Bioscience has developed a technology that enables the measurement of various metabolic parameters and functions using live cells, in real-time, in a microplate. Seahorse Analyzers profile cellular metabolic functions, using label-free, solid-state disposable optical sensors. The Seahorse Analyzers simultaneously measure mitochondrial respiration (oxidative phosphorylation; OXPHOS) via the oxygen consumption rate (OCR), and glycolysis via the extracellular acidification rate (ECAR). Integrated drug injection ports allow for up to 4 reagent additions (e.g. drug or substrate) that can be programmed for automated delivery into the independent cell culture wells. Assay kits and reagents provide standard methods for quantifying mitochondrial respiration, glycolytic activity, endogenous and exogenous fatty acid oxidation, substrate oxidation, and metabolic phenotype. Seahorse XF technology has been applied to multiple research areas, including cancer, obesity, diabetes, metabolic disorders, immunology, cardiovascular function, neurodegeneration, virology, and aging.
Over 2/3 of childhood cancer survivors treated with total body irradiation develop metabolic complications. Given that skeletal muscle plays a major role in insulin-stimulated glucose disposal, we aimed to investigate the role of skeletal muscle in impaired whole body metabolism following irradiation.Mice were exposed to a single dose of 5.95Gy and 5 weeks post-irradiation (baseline) were fed a high-fat diet (HFD) for 12 weeks. Energy expenditure, glucose homeostasis and insulin sensitivity were assessed throughout the HFD period and metabolic functions were measured at baseline and endpoint in skeletal muscles and muscle stem cells.Irradiated mice had increased respiratory exchange ratio on HFD despite similar food intake. Lipid metabolism and citrate synthase activity in muscle were impaired compared to the non-irradiated mice suggesting altered fat utilisation and compromised mitochondrial function. Irradiated mice showed altered fasting glucose and impaired ex-vivo insulin-stimulated glucose uptake in muscles after 12 weeks of HFD. Muscle stem cells isolated from irradiated mice showed both impaired lipid and glucose oxidation, suggesting long-term memory of the exposure to irradiation.We propose that irradiation may alter skeletal muscle mitochondrial metabolism resulting in impaired whole body metabolism and insulin resistance.
Globally, Stroke ranks as the second leading cause of death. Reperfusion of the ischemic brain also initiates pathologic intracellular cascades that contribute to post-ischemic brain injury. Mitochondrial dysfunction has been known to be a major contributor of neuronal injury during stroke. During reperfusion, [Ca2+]m overload causes mitochondrial ROS overproduction, mitochondrial permeability transition, and activation of cell death signaling cascades. The consequences of mitochondrial calcium overload during stroke has been studied, but the causal upstream mechanisms that leads to calcium overload remains unclear. In the past five years, we1-4 and others have identified the molecular components of the mitochondrial calcium uniporter. The objective of the study was to investigate the expression pattern of uniporter complex during ischemic stroke. Using in-vitro (Oxygen-Glucose deprivation) and in-vivo (murine MCAO) model systems, we investigated in detail, the changes in expression pattern of uniporter components during Hypoxia-Reoxygenation and Ischemia-Reperfusion injury respectively. Finally, we validated our in-vitro and in-vivo findings in human stroke using stroke patient derived post-mortem brain samples. Collectively, data from our study suggests that the unique changes in expression of MCU complex, during reperfusion, results in enhanced [Ca2+]m uptake. The detailed findings of our study will be presented in the conference.
Background: Fatty acids (FAs) are the predominant metabolic substrates for myocardial ATP. So far, the effects of FAs on myocyte contraction in normal and hypertensive hearts are unclear. Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) has been implicated to be essential in FA oxidation in mitochondria. Recently, we have shown that neuronal nitric oxide synthase (nNOS) is up-regulated in left ventricular (LV) myocytes from hypertensive hearts, whereas eNOS protein expression was reduced. Purpose: We aim to analyze palmitic acid (PA)-regulation of myocyte contraction and the roles of eNOS and nNOS in LV myocytes from sham and angiotensin II (Ang II)-induced hypertensive rats. Methods: Sarcomere length and Fura-2 ratio (Fura-2AM, 2 μM) were measured (field stimulation, 2Hz, IonOptix Corp, 37°C). Oxygen consumption rate (OCR) was measured (Instech). NO (nitrite content) was measured by NO assay kit (Griess Reagent System). Used whole cell patch clamp technique, was recording L-type Ca2+ current (ICa) and Na+ - Ca2+ exchanger activity (INCX). Results: Our results showed that PA (100 μM) increased the amplitude of sarcomere shortening and Ca2+ transients in LV myocytes from sham but not in hypertension. Etomoxir (10 μM), a selective carnitine palmitoyl transferase I inhibitor, blunted the inotropic effect of PA in sham, but not effect hypertension, suggesting the contribution of beta-oxidation to in PA-regulation of cardiac inotropy. PA increased basal oxygen consumption and mitochondrial OC capacity in cardiomyocytes from sham and HTN rats. Etomoxir was reversed PA induced basal OC and mitochondrial OC capacity in sham and HTN. Inhibition of eNOS and nNOS with Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mM, 30 min – 1hr) prevented PA-induced myocyte contraction and Ca2+ transients in sham; such an effect was not observed with nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC, 100 nM, 30 min – 1hr). Similarly, PA failed to increase myocyte contraction in LV myocytes from eNOS-/- mice, suggesting the critical role of eNOS in PA-induced myocyte contraction in sham. In hypertension, both L-NAME and SMTC restored PA-enhancement of myocyte contraction, suggesting the modulatory role of nNOS. PA tended to reduce eNOS-derived NO in sham but significantly increased nNOS-derived NO in hypertension. Indeed, PA increased OCR in sham and L-NAME but not SMTC reduced PA-induced DOCR. In hypertension, PA increased OCR. Importantly, L-NAME and SMTC abolished both basal and PA-induced DOCR. PA maintained Ca2+ influx via L-type Ca2+ channels, and nNOS inhibitor increased Ca2+ influx, in LV cardiomyocyte from sham. PA reduced Ca2+ influx, but nNOS inhibitor significantly increased Ca2+ influx via L-type Ca2+ channels in LV cardiomyocyte from hypertensive rats. Further experiments have shown that SMTC increased the amplitude of Ca2+ transients in hypertension. Conclusion: PA increases Ca2+ transients and myocyte contraction in normal heart, mediated by eNOS-dependent mitochondrial beta-oxidation. In hypertension, nNOS restricts PA-dependent cardiac inotropy by modulating Ca2+ handling. Nevertheless, nNOS is required for maintaining mitochondrial function in the myocytes from hypertensive hearts.
The combination of premature ovarian failure and age-associated hearing deafness, inherited in autosomal recessive manner, was named Perrault syndrome. It may be caused by mutations in HSD17B4, HARS2, LARS2, PSMC3IP, C10orf2, CLPP and in un-identified genes. CLPP is a mitochondrial matrix peptidase that is crucial for the unfolded protein response of mitochondria (mtUPR). In a study of Clpp-/- mice we found the Perrault phenotype, but also loss of spermatids, growth retardation and anti-microbial protection. An accumulation of CLPX protein and mitochondrial DNA was accompanied by widespread induction of inflammatory mRNAs in several tissues (Gispert 2013 HMG). Now we tried to elucidate the age-associated hearing loss. Preliminary analyses of DPOAE (distortion product optoacoustic emissions) showed no response above 20000 Hz. Preliminary studies of acoustic startle responses showed a maximal deficit already at an age of 12 months. To understand mtUPR-triggered neurotoxicity at molecular level, we analyzed brain tissue with global proteomics. Elevated levels of inflammatory factors downstream the transcription factor STAT1 were prominent. STAT1 induction was also documented in murine embryonal fibroblasts. Thus, the innate immune system is triggered by the mitochondrial matrix protein degradation deficit due to CLPP deletion.
The Parkinson’s disease (PD) related proteins PINK1 and Parkin act jointly as a mitochondrial quality control system, linking loss of mitochondrial import efficiency to the autophagy-dependent degradation of dysfunctional mitochondria. The aim of this study was to evaluate a possible involvement of the PINK1/Parkin system in the regulation of mitochondrial protein import. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway in living cells with a quantitative bioluminescence-based readout. Validation of this probe in HEK293T cells showed that it was appropriately targeted to mitochondria and sensitive to import impairment caused by pharmacological or genetic approaches. By using complementary tools to mimic or antagonize the activation of Parkin by PINK1 on mitochondria (siRNA-mediated downregulation of PINK1 or Parkin; overexpression of Parkin, PD-causing Parkin variants, phosphomimetic or non-phosphorylatable Parkin ; co-expression of ubiquitin, phosphomimetic, non-phosphorylatable or lysine-less ubiquitin), we show that the PINK1/Parkin system stimulates mitochondrial import in a kinase- and ubiquitin ligase-dependent manner. Use of the biosensor in primary skin fibroblasts from PD patients revealed lower levels of import compared to controls in a subset of PARK2 (Parkin) and PINK1 patients. Altogether, our results suggest that mitochondrial import defects may contribute to PD pathogenesis.
Deficiency of the mitochondrial persulfide dioxygenase ETHE1 causes proteome-wide alterations in ethylmalonic encephalopathy patient fibroblastsThe mitochondrial persulfide dioxygenase ETHE1 is essential for clearance of the gasotransmitter hydrogen sulfide, and ETHE1 deficiency causes a severe and complex inherited metabolic disorder known as ethylmalonic encephalopathy. Despite well-described clinical symptoms of the disease [1], detailed molecular characterization is still ambiguous. The objective was to obtain a better understanding of the molecular effects of deficiency of the sulfide regulating enzyme ETHE1, by applying proteomics. Materials and methods: Quantitative MS-based proteomics was performed on cultivated skin fibroblasts originating from patients and controls. Results: The cultured cells exhibited only a mild phenotype with few pronounced proteomic changes of central metabolic pathways; however, the data captured more than hundred regulated proteins indicating broad effects on cellular physiology. Functional pathway analyses exhibited overrepresentation of nucleic acid binding transcription factors, glycoproteins and translational regulators. Discussion and conclusions: These proteomics data show a complementary picture to previous metabolomics data [2], and describe how ETHE1 deficiency can trigger changes not only in mitochondria but also throughout the cell.
Fluorescent carbon quantum dots (CQDs) are considered as a good candidate for bioimaging as well as drug delivery system (DDS) due to their low cytotoxicity and high biocompatibility.1 D-α-tocopheryl polyethylene glycol succinate (TPGS), an inhibitor of P-glycoprotein (P-gp), has been used for developing a variety of TPGS-containing nanomedicines to overcome multidrug resistance (MDR) and enhance treatment efficacy in cancer chemotherapy. 2 In this work, a new DDS is constructed by using triphenylphosphine conjugated TPGS (TPP-TPGS) as a mitochondrial targeting modifier to functionalize oil-soluble CQDs and loading an anticancer drug, doxorubicin (DOX), in the bioconjugates of CQDs-TPGS-TPP. Accordingly, multi-functionalities (water-solubility, mitochondria-targeting, P-gp inhibition capability as well as imaging-trackable drug delivery property) are endowed to the as-formed DDS. Evaluations of cytotoxicity, cell imaging, and mitochondrial targeting were performed on the human breast cancer MCF-7 cell line. The TPP-modified DDS could be more uptaken by cells and preferentially distributed to the mitochondria. In DOX-resistant MCF-7 cells, an enhanced cytotoxicity was observed for the DDS loading with DOX (CQDs-TPGS-DOX-TPP) in comparison to free DOX and non-targeting conjugates (CQDs-TPGS-DOX). This work highlights promising application of the mitochondrial targeted DDS in reversing drug resistance in cancer therapy.
Several studies highlighted the strong negative correlation between obesity and active brown adipose tissue amount in adult humans. There are at least two types of thermogenic fat depots, classical brown and beige, which have different origins and tissue distribution [1]. We intended to clarify whether preadipocytes from different anatomical sites are capable of initiating a browning program in parallel with the enhancement of mitochondrial respiration in response to browning-inducers. Preadipocytes obtained from herniotomy (abdominal subcutaneous) or thyroid surgery (“deep neck” and cervical subcutaneous) and a human preadipocyte cell line (SGBS) were differentiated into white, brown (by BMP7 treatment) or beige (by irisin and clozapine administration or by a previously described cocktail) adipocytes. To assess browning, gene expression measurements and laser-scanning cytometry based morphology analysis were performed [2]. Oxygen consumption was measured using an XF96 oxymeter. Differentiating adipocytes treated with browning-inducers had smaller lipid droplets, more mitochondrial DNA, higher mitochondrial respiration and contained more Ucp1 protein than white adipocytes. Browning adipocytes utilize more fatty acids by beta-oxidation and increase their respiration by activating a futile cycle of creatine metabolism [3]. Next, we intend to identify molecular markers to characterize those preadipocytes that are capable to implement an effective browning program.
The mitochondrial ETC complex I (CI) is present in C. albicans and regulation of CI is based on nutrient availability. Our recent studies indicate that NDH51 (a component of the CI core), NUO1 and NUO2 (two fungal specific- CI proteins), and GOA1 (Candida specific- CI protein), are all required for CI integrity. Lack of any of these subunits results in an azole hypersensitivity, avirulence, failure of immunity, and a loss of chronological aging. Genomics and proteomics studies reveal that cell membrane synthesis and wall assembly in this organism are regulated in tandem with CI nDNA genes but not mtDNA-encoded genes. When synthesis of CI subunits, phospholipid and ergosterol are repressed in three CI mutants, proteins for CIV assembly and the alternative oxidase (AOX2) are upgraded. Differential proteins of mutants suggest that NUO1 participates in nucleotide synthesis, ribosomal biogenesis and negatively regulates mitochondrial ribosomal biogenesis. NUO2 plays a redundant but minor role in these cellular processes; however, vesicular trafficking and negatively regulated CI assembly via inhibition of CIA30 are more NUO2–specific. GOA1, on the other hand, appears to regulate membrane transport, to provide protection against mitochondrial ROS and to balance TCA intermediate flow between mitochondria and the cytoplasm.
