Please note that this presentation aims at summarizing currently available data only without the claim of comprehensively representing established knowledge. Thus, information and conclusions presented in this course may be subject to change in the future due to the rapid evolution of the field of extracellular vesicles. The current MOOC chapter is entitled EVs in Metabolism. We would like to thank the coordinators and contributors to this module shown here. My name is Kenneth [inaudible] , the executive chair of science and meetings for ESO, and I will be narrating this module. Now let's begin. Metabolism is a concept that encompasses all reactions that take place in an organism. For example, making new building blocks by a process known as anabolism, reusing existing micro-molecules by a process known as catabolism, inactivating toxic foreign compounds by a set of reactions known as xenobiotic metabolism, and of course, generating energy for all of these processes. In this scheme, enzymatic reactions are summarized and indicated by the connecting lines. The dots indicate molecular substrates and products of the enzymatic reactions. The enzymatic reaction is organized into metabolic pathways such as the pentose phosphate pathway, one carbon metabolism, beta oxidation, the methionine cycle, and so on. Metabolomics is one of the most powerful techniques for understanding biological processes and also the main technology for studying metabolism. Its major focus is the analysis of metabolites, defined as small molecules of less than 2,000 daltons. This category is estimated to include more than 40,000 molecules of different chemical natures, such as lipids, carbohydrates, amino acids, vitamins, nucleotides, and more. Looking at disease, metabolomics is an unbiased technology that allows the identification of final products of a pathological process. This identification can in turn guide investigations into the actual causes of pathology, a so-called bottom-up strategy. Currently, the technology is based on ultra-high performance chromatography coupled with mass spectrometry. Metabolomics can detect thousands of metabolites in small sample volumes. Slightly different methodological approaches may be needed to cover the entire range of metabolites which can have different chemical natures. Over the last few years, it has been shown that extracellular vesicles or EVs, contain a variety of biomolecules including nucleic acids, proteins, lipids, and metabolites. Several questions arise from the presence of these molecules in EVs traveling through the body fluids and captured by different cells. First, are these biomolecules functional? Second, do these cargo carrying EVs have a metabolic impact on a cell that takes them in or on the surrounding environment? In other words, is there a role for EVs in metabolism? To convey the importance of EVs in metabolism, we will next present different studies that involve vesicles and changes in metabolism and metabolic profiles. Also, we will highlight the importance of EVs in the pathology of metabolic syndrome, and alterations to the microbiome. EVs released by hepatocytes harbor active arginase, which transforms the metabolome profile serum. EV arginase activity increases after liver damage. Indeed, various studies have observed enzymatic activity associated with extracellular vesicles. This suggests that cells export their machinery in order to perform certain functions in the extracellular space. One of the earliest described phenomenon was that EVs carry metalloproteinases that help tumor cells degrade the extracellular matrix and thus contribute to cancer cell migration. However, EVs released by non-tumor primary cells such as hepatocytes can also modify the extracellular environment through enzymatic reactions. These observations have field speculation that EVs transport specific cellular machinery towards other areas of the organism, where it could be locally required, either in the extracellular matrix or in acceptor cells. As noted earlier, hepatocyte-derived EVs are physiologically secreted into the bloodstream, and this phenomenon increases after liver damage. Potentially, this circulating EVs may alter the serum metabolome. One of the most important such changes is the transformation of arginine into ornithine by arginase. The depletion of arginine from serum could have consequences for vascular dynamics. Why? In the vascular endothelium, the enzyme nitric oxide synthase acts on arginine to produce nitric oxide. Nitric oxide in turn is necessary for normal vasoconstriction of blood vessels. In vitro, it has been observed that EVs released from damaged hepatocytes can impair vascular relaxation of pulmonary arteries. Quite possibly, depletion of arginine by EV born arginase may contribute to the pulmonary hypertension observed in patients with liver diseases. In another example, stem cells are known to pack asparaginase into EVs. This asparaginase is even more active in the EV than in the cytosol of the parent cell, indicating that the vesicles may become independent metabolizing units that modify critical metabolites in their environment. In the classical experiment depicted here, an effort was made to ascertain the presence of metabolic activities in EVs. A consumption release assay was conducted with commercial media exposed to EVs prepared from neural stem cells or NSCs. Relative levels of small metabolites before and after incubation were determined by LCMS based metabolimics. This crude assay revealed an unexpected set of multiple metabolic activities in the EVs, leading overall to consumption of asparagine and production of aspartate and glutamate among other metabolites detected. Note that this early experiment was conducted using commercial media with glutamine being 50 times more concentrated than asparagine. In extensive biochemical characterization of the extracellular distribution of Asrgl1 or L, asparaginase demonstrated first, that this gene is indeed trafficked into EVs. Second, that there is a strong enrichment of the enzyme into EV fractions floating at the same density as bonafide exosomes and other EVs. Third, there is no evidence of asparaginase in fractions enrich for protein aggregates. Finally, enzymatic activity was compared between free recombinant asparaginase, cytoplasmic extracts from NSCs, and NSC EVs indicating that EV loaded asparaginase is as much as 100 times more active than controls. That is, the parent stem cells or EV free protein. This result suggests that enzymes associated with membranes could be more active than when they are soluble. This discovery of functional asparaginase being trafficked into stem cell EVs anticipates a new mechanism of cellular signaling mediated by EVs. Specifically, EVs behave as functional, fully independent small metabolic units that communicate with the micro-environment, and modify critical amounts of small metabolites that affect cell behavior. Metabolic syndrome is a condition that affects millions of people. In patients, increased circulating EV levels have been observed including EVs derived from several different cell types. Here, EV cargo could serve as important biomarkers of disease. But more importantly, EVs might transmit signals between different tissues triggering symptoms of metabolic syndrome. What is metabolic syndrome? This syndrome is a cluster of interrelated risk factors for cardiovascular disease and diabetes. These factors include metabolic abnormalities such as elevated glucose and triglyceride levels, low high-density lipoprotein cholesterol levels, high blood pressure, and obesity mainly abdominal adiposity. Circulating blood levels of EVs are increased in metabolic disorders such as type 2 diabetes, dyslipidemia, obesity, and hypertension. Frequently, after treatment of each pathology levels of EVs returned to a basal threshold. In metabolic syndrome patients, the cells that contribute to increased circulating levels of EVs include platelets, red blood cells, and endothelial cells expressing CD41, CD235 and CD146 respectively. Also, a correlation is seen between components of metabolic syndrome and the levels of EVs carrying tissue factor, a molecule involved in the clotting process. Finally, it has been shown that in patients with clinical manifestation of vascular disease, levels of EVs containing cystatin C correlate with metabolic complications of obesity. Altogether, these data suggest that EVs are biomarkers of metabolic syndrome. But EVs are not just biomarkers, they also contribute to pathogenesis. In the next few slides, we will examine the effects of EVs on several tissue types as depicted in this cartoon. Let's start with the endothelium, during metabolic alterations Evs reduce nitric oxide bioavailability and increase reactive oxygen species production in endothelial cells. This leads to endothelial dysfunction. This is an early event in atherosclerotic plaque formation and calcification and it increases the risk of cardiovascular diseases. Next is the liver, acting on the liver Evs can evoke insulin resistance as well as fibrosis resulting in the accumulation of fat and thus progression to nonalcoholic steatohepatitis. Lastly, when EVs interact with adipocytes they increase insulin resistance, secretion of proinflammatory adipokines, and lipid synthesis. All of these effects are involved in the generation of a chronic inflammatory environment in the adipose tissue. Evs can also signal from adipocytes. During excess caloric intake, white adipose tissue expands, this creates a hypoxic environment that is only low levels of oxygen are available to adipocytes. Macrophages also accumulate within the tissue and release proinflammatory cytokines. These conditions generate insulin resistance locally characterized by impaired insulin signaling. Adipose tissue becomes dysfunctional releasing not only [inaudible] but also EVs that in turn have autocrine and paracrine effects on other cells and tissue. This slide shows how EVs produced and released by adipocytes under conditions of hypoxia alter insulin responsiveness in recipient cells. Panel A at the top left, depicts glucose uptake in response to insulin after cells are left untreated labeled as none or treated with EVs produced by adipose cells that are cultured in normoxic labeled as Control or hypoxic conditions labeled as Hypoxia and how these compare with normal cells that have not been treated with exogenous vesicles. We see that while the basal glucose uptake is not affected, the response to insulin is decreased by approximately 25 percent in cells that have been treated with EVs from hypoxic adipocytes. Panel B, glucose uptake rates are measured for cells treated with EVs from adipocytes that have been exposed to proinflammatory cytokines. There is no difference when the treatment is no EVs labeled as none, Evs from Control adipocytes labeled as Control, or EVs from cytokine exposed adipocytes labeled MCM. In panel C, we see that EVs isolated from plasma of obese individuals also inhibit insulin stimulated glucose transport in cultured cells as compared with EVs from lean individuals. In panel D, cells treated with EVs obtained from hypoxic adipocytes reduce the activation of the PI3 kinase cascade by insulin as seen by a reduction in the phosphorylation levels of the protein AKT. Taken together these data and there are shown earlier underlying the EVs can transfer deleterious signals and actively participate in the development of metabolic syndrome. We now come to a final example of the role of EVs in the transport and metabolites. Evs may carry metabolites of the intestinal microbiota into the host organism. In this way, changes to the microbiota are translated into alterations of EV cargo and lead toward overall metabolite alteration in the host organism. Bacteria of the gut microbiome release EVs known as outer membrane vesicles or OMVs. Just like eukaryotic EVs, OMVs have a complex cargo including enzymes capable of transforming their environment. They can modify the intestinal metabolome producing new metabolites that will be absorbed by the intestine. In normal physiology, OMVs may interact with the intestinal epithelium and even help to maintain gut barrier integrity. However, changes in the microbiota may increase permeability of the epithelium. For example during such conditions as inflammatory bowel disease or chronic ulcerative colitis. In these scenarios, OMVs may transgress the intestinal barriers and enter the bloodstream, there they will contribute to the inflammatory status of the host organism. Reactive enzymes carried by OMVs may indeed be virulence factors. For instance, a pathogenic stream of bacteroides fragilis produces EVs with more active enzymes than those of the nonpathogenic counterpart leading to more OMV persistence and disruption of the epithelium. Changes in the microbiome due to diet also lead to changes in metabolism. It has been observed that OMVs can induce diabetic phenotypes in mouse muscle. The dissemination of OMVs through the blood leads to systemic inflammation in mice. The cargo of OMV is can also affect the liver inducing changes in metabolism as well as transcription via epigenetic modifications. It has been proposed that a connection between the microbiome in the brain in pathologies such as depression or chronic pain maybe mediated by circulating bacteria derived vesicles. To wrap up this section, we offer these statements remarking on the role of EVs at three levels. First, EVs can serve as enzymatic tools. Second, EVs serve as both biomarkers and effectors of disease conditions. Third, EVs reflect and transmit alterations in the microbiome posts relationship. We now offer several slides with complete references from this chapter.
