High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types

Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.</p

The cold-induced lipokine 12,13-diHOME promotes fatty acid transport into brown adipose tissue

Brown adipose tissue (BAT) and beige adipose tissue combust fuels for heat production in adult humans, and so constitute an appealing target for the treatment of metabolic disorders such as obesity, diabetes and hyperlipidemia1,2. Cold exposure can enhance energy expenditure by activating BAT, and it has been shown to improve nutrient metabolism3–5. These therapies, however, are time consuming and uncomfortable, demonstrating the need for pharmacological interventions. Recently, lipids have been identified that are released from tissues and act locally or systemically to promote insulin sensitivity and glucose tolerance; as a class, these lipids are referred to as ‘lipokines’6–8. Because BAT is a specialized metabolic tissue that takes up and burns lipids and is linked to systemic metabolic homeostasis, we hypothesized that there might be thermogenic lipokines that activate BAT in response to cold. Here we show that the lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) is a stimulator of BAT activity, and that its levels are negatively correlated with body-mass index and insulin sensitivity. Using a global lipidomic analysis, we found that 12,13-diHOME was increased in the circulation of humans and mice exposed to cold. Furthermore, we found that the enzymes that produce 12,13-diHOME were uniquely induced in BAT by cold stimulation. The injection of 12,13-diHOME acutely activated BAT fuel uptake and enhanced cold tolerance, which resulted in decreased levels of serum triglycerides. Mechanistically, 12,13-diHOME increased fatty acid (FA) uptake into brown adipocytes by promoting the translocation of the FA transporters FATP1 and CD36 to the cell membrane. These data suggest that 12,13-diHOME, or a functional analog, could be developed as a treatment for metabolic disorders

Abstract 990: Metabolic shift in cancer by re-programming of mitochondrial machinery: Novel insight into Warburg-dependent mechanism

Abstract Otto Warburg described his seminal observation that cancer cells negotiated a switch towards anaerobic respiration from oxidative phosphorylation in return for sustained growth and evasion of normal cellular processes such as cell cycle control and apoptosis. The underlying mechanism that governs this phenomenon has yet to be firmly established. Given the centrality of mitochondria to apoptosis and bioenergetics, we proposed experiments that employed ubidecarenone as an intracellular energy ambassador wherein high-throughput genomic, metabolomic, and proteomic analyses were employed to capture the energetic and molecular signature of cancer cells. Analyses were performed on oncogenic breast, prostate, liver, pancreatic, skin (melanoma, squamous cell carcinoma) and were compared to normal fibroblasts, keratinocytes, hepatocytes, kidney, adipocytes, and human aortic and endothelial cells. The results suggested a hallmark discovery that clearly delineated a differential effect of cancer vs. normal cells under hyperglycemic, hypoxic, and lactate-stressed conditions. A decreased activation of the Pentose Phosphate Shunt was significantly downregulated in cancer cells suggestive of a shift towards utilization of oxygen and glycolysis while Bcl-2 dependent cell death mechanisms were restored. Most notably, cross-talk mechanisms related to p53 and Vegf in the cancer cells and tumor markers and pathology in animal models of melanoma and pancreatic cancer demonstrated efficacy in a topical and intravenous form of Cytotech Labs API 31510, a ubidecarenone based technology that targets the Bcl-2 protein family downstream of a cellular metabolic shift. Conversely, normal cells did not exhibit the aforementioned characteristic indicative of a cancer-specific mechanism. We propose a novel underlying mechanism for the Warburg Hypothesis and demonstrate that pre-clinical work on API 31510 in cancer suggest that it may be a viable agent for control of cancer metabolism and serve as safe, effective anti-cancer agent. 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 990. doi:10.1158/1538-7445.AM2011-990</jats:p

Current Status of Metabolomic Biomarker Discovery: Impact of Study Design and Demographic Characteristics

Widespread application of omic technologies is evolving our understanding of population health and holds promise in providing precise guidance for selection of therapeutic interventions based on patient biology. The opportunity to use hundreds of analytes for diagnostic assessment of human health compared to the current use of 10&ndash;20 analytes will provide greater accuracy in deconstructing the complexity of human biology in disease states. Conventional biochemical measurements like cholesterol, creatinine, and urea nitrogen are currently used to assess health status; however, metabolomics captures a comprehensive set of analytes characterizing the human phenotype and its complex metabolic processes in real-time. Unlike conventional clinical analytes, metabolomic profiles are dramatically influenced by demographic and environmental factors that affect the range of normal values and increase the risk of false biomarker discovery. This review addresses the challenges and opportunities created by the evolving field of clinical metabolomics and highlights features of study design and bioinformatics necessary to maximize the utility of metabolomics data across demographic groups

Transplantation of Brown Adipose Tissue with the Ability of Converting Omega-6 to Omega-3 Polyunsaturated Fatty Acids Counteracts High-Fat-Induced Metabolic Abnormalities in Mice

A balanced omega (ω)-6/ω-3 polyunsaturated fatty acids (PUFAs) ratio has been linked to metabolic health and the prevention of chronic diseases. Brown adipose tissue (BAT) specializes in energy expenditure and secretes signaling molecules that regulate metabolism via inter-organ crosstalk. Recent studies have uncovered that BAT produces different PUFA species and circulating oxylipin levels are correlated with BAT-mediated energy expenditure in mice and humans. However, the impact of BAT ω-6/ω-3 PUFAs on metabolic phenotype has not been fully elucidated. The Fat-1 transgenic mice can convert ω-6 to ω-3 PUFAs. Here, we demonstrated that mice receiving Fat-1 BAT transplants displayed better glucose tolerance and higher energy expenditure. Expression of genes involved in thermogenesis and nutrient utilization was increased in the endogenous BAT of mice receiving Fat-1 BAT, suggesting that the transplants may activate recipients’ BAT. Using targeted lipidomic analysis, we found that the levels of several ω-6 oxylipins were significantly reduced in the circulation of mice receiving Fat-1 BAT transplants than in mice with wild-type BAT transplants. The major altered oxylipins between the WT and Fat-1 BAT transplantation were ω-6 arachidonic acid-derived oxylipins via the lipoxygenase pathway. Taken together, these findings suggest an important role of BAT-derived oxylipins in combating obesity-related metabolic disorders