Good afternoon, everyone. Welcome to today's webinar. I'm Dr. Kristen Stanford, and I'll be the moderator for today's webinar. On behalf of the Exercise Physiology Interest Group leadership team, I'm excited to welcome our expert presenters to discuss exercises impact on cardiometabolic function with topics specifically including the impact of acute exercise on fasting and insulin-stimulated extracellular vesicles and the effects of exercise on adipose tissue remodeling. Here's a glance at today's agenda. We will provide a few announcements, and we will introduce our experts in just a moment. They will each present lectures and will be followed by a panel discussion. The presenters will be taking questions from the audience at the end of the event. Please don't wait until the end of the session to send in your questions. Instead, please go ahead and type it into the Q&A box in your control panel. Please be sure to use the Q&A box and not the chat function for questions. We will be using the chat box to send you important links during this announcement. The Exercise Physiology Interest Group leadership team coordinated this webinar. I want to take a moment to thank all the members of the leadership team for their work throughout the year and to provide opportunities to the interest group members. You can learn more about ADA's interest groups at the link in the chat. ADA's Institute of Learning provides access to over 70 free continuing education programs, webinars, podcasts, self-assessments, and more. Please catch the link that's in the chat box. The Behavioral Medicine and Psychology Interest Group has an upcoming hands-on webinar as a part of the Helmsley hands-on webinar series entitled Empowering Adults with Diabetes Strategies to Support CGM Use in Diabetes Self-Management. This webinar will be exclusive and free to all ADA members, and attendance provides an opportunity for one CE credit. This webinar is tomorrow afternoon, so please register using the link in the chat now. Finally, I'd like to introduce today's first presenter. Dr. Stephen Mallon is an associate professor with dual appointments in the Department of Kinesiology and Health, as well as the Department of Medicine, Division of Endocrinology, Metabolism and Nutrition at Rutgers University. He's the director of the Applied Metabolism and Physiology Laboratory in the School of Arts and Sciences. His lab focuses on investigating insulin resistance as a key factor underlying chronic diseases, including metabolic syndrome, type 2 diabetes, and cardiovascular disease. Dr. Mallon's group currently investigates the interaction of exercise with diet and medication to optimize well-being. Today, Dr. Mallon will discuss the impact of insulin on extracellular vesicle-mediated changes in vascular function, and will highlight the effect of exercise on extracellular vesicles during fasted and insulin-stimulated states in adults. Dr. Mallon, you can go ahead. Thank you very much for that kind introduction, Kristen. It's a pleasure to be here. I thank everyone on the leadership team for inviting us. Well, you know, as Kristen eloquently shared, we're really interested right now in how insulin impacts the body, in particular to glucose regulation, and a part of that interest lied in this potential mechanism, extracellular vesicles, in a journal article that we had published not too long ago. And to share, we have no conflicts of interest. When thinking about putting this lecture together, I thought it was perhaps prudent to just start with the general idea that we all recognized, which is that many individuals in the United States have obesity. And this is quite problematic, given that obesity is tied to many vascular-related diseases, including heart disease, stroke, to name a few, potentially even Alzheimer's, as some have pointed out. Within that, though, excess fat may be linked particularly to insulin resistance, in addition to factors like endothelial dysfunction and arterial stiffness. Now, while we think obesity is quite problematic, it's worth perhaps pointing out that maybe obesity isn't the entire issue. Something to recognize within this situation is that many adults in the United States have metabolic syndrome. And as I'm sure many of you are familiar with, metabolic syndrome is a host of ailments that I'll name in a second. But I'd like to point off initially that with metabolic syndrome is usually an aggregate of different vascular risk factors. And within this combination of risk factors, we can appreciate on this graph here from the American Heart Association, that as the number of risk factors increase, so does the impact on coronary artery disease. In particular, what I find to be quite interesting is this notion of when you have metabolic syndrome defined by three risk factors, you may have upwards of close to three times the hazard risk ratio for coronary artery disease. But notice what happens if you have diabetes in the far right on the blue bar. This increases perhaps exponentially, suggesting there may be some underlying issues within diabetes that really augments this risk of vascular dysfunction. Now within that idea, metabolic syndrome is defined by a host of factors. At the center of this though, is insulin resistance tied together tightly with visceral adiposity. We find that to be quite important because of the intersections of adipose tissue with insulin resistance on promoting overall cardiometabolic health. So when considering something like this, it may be that individuals don't necessarily have to have frank obesity per se to begin having underlying pathological issues related to diabetes and other ailments. And that way, when thinking about glucose regulation itself, we can appreciate that glucose regulation is probably a combination of factors. Most notably is metabolism. When we think about glucose uptake within the transport process, and then as glucose is taken up into the myocyte, it's metabolized to various metabolites such as glucose 6-phosphate and so forth. Preceding that, it's important to recognize delivery is a critical factor. Without actually delivering that glucose to the myocyte, those mechanisms may or may not be as priority. Case in point, when thinking about this phenomenon, we can appreciate on levels here that by Eugene Barrett, past president of the American Diabetes Association, that insulin actually plays an important role within affecting the vasculature. And this was a cross-sectional study done about eight years ago, looking at how individuals with metabolic syndrome actually compare to control groups. And this was done using a euglycemic clamp, so insulin was infused over a period of time. And there was a healthy control group compared to this group of people with metabolic syndrome. And they characterized the vasculature in different ways. First, they looked at the large conduit artery through a technique known as flow-mediated dilation. This approach demonstrated that in lean, healthy individuals, that insulin raised endothelial function at the large conduit artery, where this phenomenon was completely blunted in participants with metabolic syndrome. When looking at post-ischemic flow velocity, a measure of resistance arterial function, where about 80% of our blood pressure is managed, there's an indication that healthy controls responded to insulin by actually showing elevation, suggesting that the resistant arterials were dilating and allowing more blood to flow. Metabolic syndrome patients had no response. And lastly here, using a technique known as CEU, or contrast-enhanced ultrasound through DFINITY microbubble infusion, during the clamp, there is notice of a rise again in microvascular blood flow, suggesting the capillaries are being perfused, whereas patients with metabolic syndrome had no responses. And what's really striking to me is when looking at these data, amongst others, fasting values were not different. So by looking at the fasting state alone, it may trick us in some ways into appreciating what are some of the underlying pathologies of individuals. And in this case here, it seems that individuals with metabolic syndrome really had challenges in the face of insulin stimulation. Now thinking about how to combat that insulin resistance is a challenge, but one possibility is lifestyle through exercise. And nearly almost 45 years ago, John Halazi had demonstrated quite nicely in a study that with about one year of exercise training, participants with diabetes and prediabetes were able to nearly normalize their insulin responses. And many of these individuals had reversal of their abnormal glucose levels, highlighting that exercise is a powerful medicine and can actually reverse diabetes. Perhaps more recently, although still about 20 years ago, one of the really elegant studies done by Joe Homard and Bill Krauss had shown through the STRIDE trials that the dose of exercise may be really important for insulin sensitivity benefit. While there's intense debates on whether high or low intensity exercise is more beneficial, these data would point towards time. That is, the amount of time spent exercise, independent of the intensity, was really important. And this is a busy graph, but the highlight here, the comparison of the two 12-mile groups you'll notice has a difference in time. The difference is because groups were matched on how many calories were expended. So the group exercising for less time actually exercised at a higher intensity than the group with greater time at 170 minutes. And you'll notice for the same amount of distance covered through running, it was the lower intensity group that actually performed better for insulin sensitivity. And strikingly, when this group had performed the same amount of time, but high intensity exercise, thereby covering greater distances, it didn't yield greater results. So in some ways, this points towards time spent exercising is really important. And I bring this up not to necessarily debate the optimal dose today, but just to say exercise doesn't always have to be done in large blocks of time. This can be done throughout the day, as we've been learning more recently over the years through various groups around the country and world. But to say here that exercise is a powerful treatment against insulin resistance. Now training is clearly important, but this is a classic study done out of John Halazi's lab back in the 1980s. And what I like about it is that it took trained individuals and it wanted to look at the effects of detraining to see how quickly some of these benefits of insulin sensitivity from exercise can be lost. And it was noticed within about 10 days, you could see here at the top lines, insulin levels rose quite dramatically, again, just 10 days of no exercise. But to say here, what's impressive to me is that a single bout of exercise nearly rescued the effect such that it began to restore these values near to train levels. And it didn't completely return them. But to say here, this may be some of the early evidence we have as a field showing that single bouts of exercise are incredibly important for improving health and stringing together single bouts of exercise over the course of the week is vital to maintaining insulin sensitivity. Now, I bring this up because thinking about how exercise impacts the vasculature and insulin is an area we're really interested in. And we know that insulin as shown by Dr. Barrett study most recently, as I described, impacts the vasculature. And it does though mechanistically believe through largely a nitric oxide mediated pathway. So people with insulin resistance tend to have blunted enols or endothelial nitric oxide synthase, which down regulates the bioavailability of nitric oxide to allow vasodilation of the smooth muscle near the endothelium. That said, this is going to drastically reduce the delivery of blood to that myocyte for metabolism. Now I raise this because insulin resistance here can be thought to be quite problematic for glucose control. Exercise though can bypass this mechanism through a variety of factors. One of which is sheer stress that can promote nitric oxide. So in this way, we can almost think of exercise as a non-insulin dependent pathway. Exercise itself can drive blood flow and that's beneficial, but of course people don't exercise all day long. So thinking about the benefits of exercise in this post-exercise period over the course of days is quite important and thereby understanding how exercise can improve blood flow independent of insulin resistance is great for supporting glucose uptake, but we wanted to understand really how does exercise impact the vasculature. And to say here generally in the field of exercise, most work is done using flow media dilation to assess large conduit artery function. The effects of exercise are quite variable on the endothelium. These responses can take many forms as shown here. The important thing I'd like to take away from this is exercise does seem to have benefits on the fasting endothelium and it may take periods of time. Perhaps in the immediate post-exercise period as shown in the graph, there are slight dips to be seen, but within 12, 16, 24 hours, the benefits are quite prominent. The trick with all that though is that's all mostly fasted state data. We don't have a lot of information in the fed state, nor in particular during the insulin stimulated states to characterize vascular insulin sensitivity. So with that, we undertook a study and this was a study done by Dr. Emily Heaston who was conducting her PhD studies with us at the time. And we had individuals arrive to the lab where we had provided a control condition where they were sitting or they performed exercise at a moderate intensity of about 65% of their VO2 max. Individuals were then fed a dinner that was standardized in the lab such that they received the same food, whether they were in their control condition or exercise condition. And we gave them a snack such that they can consume it at home if they wished. And people then repeated that, of course, in subsequent conditions. They then arrived in our clinical research unit the following day in which we underwent a series of tests, including indirect calorimetry, blood collections, and vascular assessments using flow-mediated dilation and contrast-enhanced ultrasound before the euglycemic clamp. This was to establish a baseline condition. We then performed a euglycemic clamp for two hours in which we repeated those measures to understand how insulin impacted the vasculature in line with glucose metabolism. Now to summarize these results, these results, importantly, too, were conducted in men and women who were about 50 years of age and had a BMI of nearly 34. The results showed, as others, that a single bout of exercise improved the metabolic insulin sensitivity by about 15%. When looking at the vasculature, interestingly, we noted that exercise also raised microcirculation function, suggesting, again, enhanced capillary perfusions during this state. And what was also fascinating was that insulin raised the brachial artery diameter, suggesting insulin is also promoting large conduit artery blood flow, highlighting that different sections of the arterial tree are benefited from exercise. Now, as we talked about before, nitric oxide was and is thought to be a major dominant factor in how insulin and exercise work to improve dilation. But we began to wonder, is that the only way? Shown here is Dr. Uta Oerteburg, a great friend and colleague who studies extracellular vesicles. And we joined together to discuss how EVs, as they're known as more generally, can impact vascular health. And a part of the working hypothesis we developed nearly seven years ago was perhaps this is a way in which insulin, diabetes somehow work together to alter health. In this picture here, we recently published a review article showing that EVs may carry different content or cargo, as they're known as. And this cargo can combine with cells in some way that's still unknown entirely, but can release content cargo to the cell such that it can influence insulin signaling, as well as perhaps how insulin acts on the vasculature to influence vasodilation. With this in mind, extracellular vesicles, or EVs again, are a wide ranging topic. Just in case some may not be familiar with this, I'd like to take a moment to share that there are different subcategories of EVs. Generally, they're known as apoptotic bodies, which result from a beveling process in which the plasma membrane fuses together and can release apoptotic bodies. There's also known as microparticles or microvesicles. These result from a shedding of the plasma membrane. And then there's also exosomes, the smallest form of EVs. And these are thought to result from membrane fusions and exocytosis processes. Collectively here, throughout our talk, I'll refer to EVs. But just to say, based on the technology we have, we're most confident in that these are more of the microparticle to microvesicle size. Nonetheless, EVs is what we'll refer to them as. And generally speaking, they're elevated in individuals with obesity and type 2 diabetes. EVs can be detected in a range of cells throughout the body. Some are listed here, but they also include muscle cells and adipocytes. Again, generally, they're elevated in people with obesity and diabetes, and this is thought to occur in part due to stress responses of the system, such as oxidative stress or inflammation. Now, this elevation is concerning on levels given the hypothesis might be that they contain different cargo that can influence health. So circling back to Emily's work, we began to wonder, does insulin do anything to these EVs and can exercise impact them? Given the model of study design, we thought we might be able to address some of these basic questions. By performing the clamp, we were able to look at the EV content within. And we focused in particular on the vascular endothelial-related EVs, although we did study platelet and leukocyte as well, given the immune system and platelets play roles in adhesion and atherosclerosis processes. I apologize for the busy slide, but to highlight here, the zero minutes would refer to fasting, whereas the 120 minutes would refer to insulin stimulation. In this way, what we noticed is that upon insulin stimulation across both large EVs and medium-sized EVs, that there were reductions in a number of EVs related to total EVs, as well as endothelial, platelet, and leukocyte. In this way, it began to make us think on levels. What was interesting is that particular sizes, the medium EVs, this is the microparticles and microvesicles, were responsive to insulin in going down within the blood. Now, we thought that was interesting because in vitro data would show that EVs actually reduce GLUT4 translocation by inhibiting AKT and adipocytes, suggesting EVs may directly impair metabolic insulin resistance. And that suggestion of the cargo means there could be something going on within the system as a whole, but really there hasn't been much work on the vasculature. So in collaboration with Brent Isaacson from the University of Virginia, we wanted to study how EVs from humans may actually impact arterial function. In this way, we had EVs collected from healthy participants as well as EVs collected from patients with metabolic syndrome. And we applied these to mesenteric arteries obtained from rodents. And with insulin stimulation, as shown here in the dark gray bar, insulin itself at various doses of concentration increased the percent of vasodilation. That would be suggested and that served as our control. What was then interesting is we looked at healthy participants and healthy EVs to see how they would influence the insulin response. And what you'll notice is generally across doses, EVs from healthy participants actually potentiated elevated the insulin effect on vasodilation, suggesting this was a good phenomenon. But if you look here on the far right side with metabolic syndrome, this effect was nearly lost. In fact, you could barely see the bars across the doses. This highlighted to us that patients with metabolic syndrome may have different EVs. Something about the EV physiology here is quite different from the healthy participant, such that they no longer responding to insulin. And we don't know what that is right now. It could be something related to nitric oxide or perhaps micro RNA has been some of the leading thoughts. But I raise this to say, there seems to be something going on in how EVs and insulin interact to influence dilation. When looking at the effect of exercise on EVs, what became really interesting to us is that a single bout of exercise, again, done at moderate intensity and expending about 400 calories actually promoted greater reductions in EVs both in the facet state, but you'll notice here on the graphs, also in the insulin stimulated state. Suggesting to us exercise may lower EVs in patients with obesity through unique mechanisms, whether this is in the facet or insulin stimulated state. And we thought, you know, does this even matter? Is that clinically relevant at all? Well, to share some of the data from the paper, we think it might be. In these patients, not only did we see improved metabolic insulin sensitivity, but these data point towards various peripheral and central hemodynamic effects related to aortic waveforms. And generally speaking here, we found that exercise improved aortic waveforms such that people might have less arterial stiffness and improved load on the heart such that the heart did not have to work as hard to provide blood throughout the system. Furthermore, we had shown that as people elevated their insulin sensitivity after exercise, this was directly correlated with reductions in EVs during the insulin stimulated state, giving us some confidence that perhaps a mechanism by which exercise is improving insulin sensitivity effects in the body is through an EV related process. Certainly correlation is not causation. We have to do more work on that, but we were excited that these data served as pilot work. And we're now starting up in this summer, a five-year project where we're gonna further try and understand some of these processes related to how EVs impact vascular function and insulin sensitivity in different phenotypes of individuals with diabetes or obesity compared to lean healthy controls. And furthermore, trying to understand how exercise over time may be able to change EV physiology in relation to some of these classic improvements in cardiometabolic health and further delineate what is potentially some of that content and cargo within the EV that could influence this functionality. So with that as a conclusion, I just like to say, it seems to us that EVs may be a unique way in which metabolic and vascular insulin sensitivity is impacted. And exercise of course, is a complex drug in of itself. And we need to think about how these EVs from multiple tissues, not just the vasculature, but perhaps muscle and adipose and others could influence the health of the system. Such that by studying these combinations of sleep, diet, alcohol ingestion, and other health style behaviors, we can begin to optimize the response for wellbeing. And with that, I'd just like to thank all the members of the lab for, without this work could not be possible. Thank you. All right, thank you very much, Dr. Mallon. That was a really interesting presentation. As a reminder, we're gonna hold all questions at the end of the next speaker, but please continue to add your questions to the Q&A box on the screen. Now I would like to introduce the second speaker today. Dr. Pasquale Nibro is a research associate at the Jocelyn Diabetes Center and is an instructor of medicine at Harvard Medical School with expertise in discovering molecular mechanisms of adipose tissue, neuronal remodeling and metabolic disease. With a specific emphasis on lipid metabolism and tissue remodeling in response to exercise training. Today, he will be presenting on the effects of exercise training on inclinal white adipose tissue remodeling, including changes in the extracellular matrix, vascularization and innervation. He will also explore how exercise training can promote a change from an unhealthy to a healthy adipocyte phenotype, ultimately resulting in healthier adipose tissue. Dr. Nibro, go ahead. Thank you, Christine, for a kind introduction and thanks for giving me an opportunity to present my data. So the topic that I will introduce today is this exercise training remodels adipo-specific extracellular matrix proteins and alter innervation and vascularization in inclinal white adipose tissue. So this is the outline for my presentation. I would like to show how exercise training reduce the ECM deposition in inclinal white adipose tissue and show how exercise increase innervation in the same tissue. And then talk about the effects of exercise training about this novel exercise-induced protein that is involved in the inguinal neuronal refinement, NAGR1 or neuronal growth regulatory one, and how PPR gamma and PRN16 are involved in the NAGR1 expression. So just briefly background, we know that white adipose tissue is pretty high dynamic and heterogeneous in the green organ. And we know that with obesity, this adipose tissue go through an expansion with the appears of hypertrophic mature adipocyte that promote hypoxia. And at the same time, we have an increase of inflammation. But what most important, we see an increase of these fibrosis. Fibrosis that we can define has an expansion of extracellular matrix. What extracellular matrix is, is a complex meshwork of highly cross-linked proteins that control organ and tissue organization and mechanical properties. And also this protein provide biochemical signals that control various cellular process, such as proliferation, survival, and differentiation. And then the ECM remodeling affect important cellular function, regulate tissue homeostasis, as it's closely linked with fundamental tissue processes, including vascularization and innervation. Almost 10 years ago, Koch Institute with Brook Institute released this matrixome project. It's a kind of consortium that was able to annotate all the genes, all the protein that were annotated for extracellular matrix. And this represent important resources for our research. What they define is how the matrixome is divided. So generally is divided into main categories of genes, the matrixome associated genes and the core matrixome genes. And what they show that in the matrixome associated genes, we can find secreted factor, regulators, and ECM affiliated. And then almost a third of these matrixome proteins are also called core because they are the constitutive aspect of the ECM. And then with them, we found glycoproteins, the 44 collagen species, and the protoglycans. And interestingly, there is no such differences when we compare the species between human and mouse. Another division for the matrixome is the compartments. We know that core matrixome can be divided in interstitial matrix, is the ECM that is present in between the cells. And then we have another ECM called basement membrane. That is a kind of envelope. Here, I'm showing in a mature adipocyte with his basement membrane net, where it's present species of collagen four and collagen six, for example. So our hypothesis is that ECM is essential for the beneficial effect of training on OVAT metabolism. But which kind of effect exercise promote in adipose tissue? We know that exercise reduce body fat, increase energy expenditure, and then adipose tissue level reduce the cell size, reducing tissue inflammatory markers, increase insulin-stimulated glucose uptake, increase adipocyte mitochondrial activity, and promote aging in rodents. So to study the effect of exercise training on extracellular matrix, we have to start from circutaneous white adipose tissue, in particular, the inguinal white adipose tissue. We explain this tissue from a cohort of black six male mice in sedentary state from another cohort of exercise training mice for 11 days. They perform 11 days, sorry, of voluntary wheel running. So they have free access to the wheel. What we do with this adipose tissue, we decide to perform a genomics analysis and a proteomics analysis. At the same time, we perform some imaging. Let's start with the first macroscopic result that we can find from this effect of exercise. First of all, here I'm presenting HNA staining on inguinal white adipose tissue. And clearly we can see that after 11 days of exercise, this tissue appear smaller and also lighter and also darker because we can easily see the presence of beige adipocyte. So this is a specific effect that exercise promote in mouse, but we'd not see in human. But most interestingly, we see also in a reduction of what is called connective tissue, the pink layer, that is more visible when we perform a serious PCROS-RED. The serious PCROS-RED is able to stain specifically the extracellular matrix that in this staining appear red. As we can see in sedentary, it's very thick and also very well distributed across the tissue with exercise disappear. We are able even to quantify this using an imaging tool. We see clearly that exercise reduce DCM in this tissue. But like I mentioned before, the omics approach represented the best strategy approach to study the extracellular matrix. We decide to perform first an analysis of the microarray and then generate a new data set with the global proteomics and secretome. Secretome analysis was conducted from conditional media derivated by adipose tissue organ culture. This media was in contact with the tissue for 48 hours. So this is one of the best approach to quantify protein that represented the extracellular matrix. So look at that, the gene expression. We see clearly that 11 days of exercise impact both the two categories of matrix genes, the associated one and the core matrix in using promoting the expression or reduce some of them. But most importantly, we look at the protein effect, the protein of this ECM. And on the left Vulcano plot, we show that exercise training surprisingly increase some of the ECM affiliated or regulated proteins and reduce some of the collagen species. Most evident and most robust effect we can appreciate in the secretome where we can see a dramatic reduction of all the collagen species or all the core matrix on proteins. We can also annotate in which kind of compartment this core matrix were present. We see that most of the down-regulated protein are mainly present in the basement membrane compartment and some of them in interstitial matrix. Like we can see the collagen one species. And then to summarize just this first aspect of my research, we show that exercise training dramatically reduce inguinal VAT ECM content and exercise alter the gene expression of interstitial matrix and basement membrane proteins. And the secretome analysis of inguinal VAT conditioning media confirm that exercise training specifically reduce the amount of basement membrane proteins and collagens involved in ECM organization. But we have another research question. We know that the adipose tissue is a heterogeneous adipose tissue with several cell types. And we ask which cell types are responsible for the ECM deposition in inguinal white adipose tissue. To answer this question, we decide to perform a spatial transcriptomics. It's a analysis, it's a gene analysis that is able to maintain the spatial distribution of the cells. And we analyze what is called clusters of cells because what the technique will show us is the dots of 15 micrometer where it's possible to identify several types of cells that we define clusters. So the first evidence is that here the composition of the adipose tissue in sedentary and exercise. We clearly see that with exercise training, we see an increase of the cluster for white and beige adipocyte, an increase of endothelial and perivascular microglia cells, an increase of beige adipocyte, and then a dramatic reduction of fibroblasts. Here is the collagens species that are mainly expressed interstitial matrix. And we can see how the beige adipocyte, for example, that are the main cell types are now present with exercise have the lowest value of collagen expression. So clearly we see that these cell types, they don't contribute at all to the ECM deposition. And on the other side, we see clearly that the fibroblasts are the main cell types involved in the ECM deposition. But looking back to the distribution of the tissue composition, was surprisingly we didn't observe any differences in white adipocyte subpopulation. So then we decide to even dissect better this proportion of adipocyte. Referring to a previous paper that was published from Susan Mandrell group, they identify that in epigonal vulva there are three different subpopulation of mature adipocyte. The insulin sensitive adipocyte also called lipogenic adipocyte, LGA, and then two subpopulation of hypertrophic adipocyte called LSA and SLSA. We decide to discriminate these three subpopulation in our subcutaneous white adipose tissue. And what we observe is that with exercise training, we have a shift of the hypertrophic adipocyte LSA and then a reduction of the LSA and then a dramatically increase of the LGA or the insulin sensitive adipocyte was barely present the third subpopulation that stressed adipocytes. Then we take a look at the gene expression level for the collagen species. And clearly we observe that LSA show the highest values for the collagen species and LGA has a lower level. To summarize also these other aspects, we know that exercise induced ACM reduction is related to change in eye water cell composition and heterogeneity. So we have an reduction of fibroblasts that is responsible for the interstitial matrix deposition. At the same times, we have this kind of switch of mature adipocyte that with LGA, they have a lower content of collagen against the LSA that has higher level of collagen species. Then the other part of my talk regarding the innervation of the inguinal white adipose tissue. What we observe that look at again at the tissue composition, we have this increase of endothelial and perivascular microglia cells. The perivascular microglia cells are just cells that accessory cells along the blood vessels that help to keep and maintaining the endothelial cells, but also the neurons. Neurons that cannot be detected using transcriptomics. So what we did that computationally, we were able to identify that we have an increase of this cluster of endothelium perivascular cells and these perivascular cells are closest to the beige and insulin sensitive cells. So what we start to speculate that probably these changes in cell types integration are also translated in changing cellular process such as innervation. So to look at the innervation level and how exercise training modified these in the tissue, we decided to perform the adipoclear is an old tissue technique that allow us to identify the innervation in the tissue. And after 11 days of exercise, we clearly see an increase of neurites across the inguinal white adipose tissue. So the microscopic and molecular data underscore the ability of exercise to promote tissue remodeling, increasing vascularization and innervation, and the refinement of the neuronal compartment in inguinal VAT is due to neuritogenesis in a specific neurogenesis process. So the other question was, if there is some protein that is responsible for this neurite outgrow and which, how the best way to identify these cells is this, sorry, this protein was to combine the gene expression microarray and the global proteomics. What we look at is here, the correlation plot between trascutomics and proteome to two distinct data set on the same inguinal white adipose tissue. And we found that proteins that were annotated for neurogenesis were upregulated with exercise and the one that were downregulated. And the one that they show in a strongest correlation was NHGRI-1. NHGRI-1, it's an immunoglobulin superfamily cell adhesion molecules that is highly expressed in the brain. And then also was recently identified be expressed in a secretaneous adipose tissue. And there are several genetic studies that reveal how these molecules is also associated with human obesity and high BMI. So looking back to our special trascutomics data, we show that NHGRI-1 is mainly expressed in the cluster of white and beige adipocyte. And like expected with exercise, we see also an increase of this protein in the beige adipocyte. Interestingly, we can see here by the adipoclear analysis that there is not overlap with the nerves. So this molecule is mainly and exclusively expressed by mature adipocyte and not on the nerve ends. And we look at also a secretaneous white adipocyte in human from two different cohort of subjects. Subjects that perform 12 weeks of treadmill training. And we here look at the abdominal secretaneous white adipocyte and the gluteal secretaneous adipocyte of young adult women. And they performs a moderate intensive exercise and we see an increase of NHGRI-1 in the secretaneous adipose tissue. At the same time, we see in lean subjects that perform 12 weeks of exercise using cycling and increase also of these genes in the tissue. But how does exercise training to promote NHGRI-1 expression? We decide to take a look at the promoter analysis of the genes. And we discovered that PPR-gamma is one of the transcription factor that regulate the expression of these genes. And a clear experiment was to use a mouse embryonic fibroblast knocked down for PPR-gamma and treated with one of the powerful agonist of PPR-gamma, rosiglitazone. What we can see here is that there is a nice dose response after rosiglitazone treatment in these cells. And this is, we are looking at the NHGRI-1 expression in the cells. When is PPR-gamma completely absent, there is no effect on NHGRI-1 expression. But I would like just to remind that this PPR-gamma generate a complex with other two transcription factor. One is the RXR and the other one is PRNT16. And then Kajimura was showing that all the time that a PPR-gamma ligand is able to recognize PPR-gamma, it starts to increase the junction between these transcription factor and PRNT16 guarantee what is called stabilization of the transcription factor. And most importantly, there are two other papers that show how PRNT16 in saccutaneous white adipose tissue is responsible to inhibit the fibrosis or ECM deposition at the same time is responsible for the sympathetic peripheral innervation in the same tissue. So this show that PRNT16 is a very nice candidate to knock down and look at the effect of exercise on ECM and innervation. So at the baseline, we use, we generate these adipocripyrin-16 knockout mice, and we show that NHGRI-1, it's dramatically absent in the inguinal vat of these mice. And then not only has mRNA, but also has protein. When we move these mice in the wheel cage, we see no effect of exercise on NHGRI-1 expression. And then most importantly, we observe that we don't see any reduction of extracellular matrix deposition. Actually, we see an increase of ECM deposition with exercise training in these animals. And the reason why was because look at the spatial transcriptomics on these knockout animals, PRNT16 knockout animals, we observe an increase of fibroblast. And again, there is no dramatically change in terms of white adipocyte population that looks pretty similar when we compare the white adipocyte in sedentary and exercise. And then most important, these subpopulation of adipocytes show similar level of collagen species across them. So to summarize, we will say that NHGRI-1 and neuronal growth regulatory one is an exercise training induced cells adhesion molecules may be involved in neurogenesis of inguinal white adipocyte tissue. Human studies show an increase in NHGRI-1 with exercise training, regardless of training period and fat depot. NHGRI-1 expression is modulated by PPR-gamma and PRNT16. And we highlighted the critical role of the PRNT16 transcriptional complex in mediating the exercise induced ECM remodeling. In conclusion, this is the take-off message that when the sedentary male mice perform an exercise training for 11 days, we observe an increase of basal adipocyte, an increase of vascularization and a reduction of extracellular matrix and increase of innervation. And this is also what we also observe. We have with exercise, an increase of what is called insulin-sensitive adipocyte. They carry less collagens. And then PRNT16 is a critical transcription factor that inhibit the fibrotic genes deposition and also promote the expression of NHGRI-1. With this, I would say thank you, my collaborators and Lori Goudia, my mentor and happy to receive any questions. Great. Thank you so much for that wonderful talk. So I'm going to start asking questions. Just as a reminder, please put any questions you have for either of our presenters today in the chat box. Dr. Mallon, I'll start with you. So one of the first questions we had was, do you think exercise or insulin sensitivity influences EV content or site of secretion? And how can you identify that EVs are from specific tissue sources? Yeah, great question. I wish I knew the exact answer, but to say, maybe starting with the last one first. So you're able to, there's a few different techniques, one of which is using Western blots to identify with different antibodies, specific cells. So in our cases, that's what we were attempting to do by using Western blots and chiro-microscopy to identify specific endothelial, platelet, leukocyte-derived cells. I know colleagues around the country are looking at neuronal-based cells in this way too, in addition to skeletal muscle. For the first one, I do hypothesize that EVs contain content or cargo that could influence various tissues' function and insulin sensitivity, whether that be skeletal muscle, liver, adipocyte, or vasculature. I think if anything, we have most evidence right now in adipocytes and hepatocytes for this, followed by skeletal muscle with probably the least amount in the vasculature, somewhat then of our interest in trying to fill that knowledge gap. In that way, what the content is, what the cargo is, again, I think there are lots of different hypotheses. MicroRNA has been one of the leading hypotheses. What can influence the release of them or the influence of the docking and uptake into the cell, not known. I think this is actually an area we're trying to understand better ourselves, but when combing through the literature in that, that's really just an unknown territory of how these EVs may release the content to a given cell. It's really just not known. But the microRNA is a leading factor. And then in the vasculature, I would just add that my esteemed colleague, Dr. Udo Uderberger has also shown some hypertensive rodent models that nitric oxide seems to be able to be released. So from the vascular perspective, we're also thinking that there might be something in that way. Given that these things influence insulin signaling themselves, we think that could be a potential mechanism, but some people have also postulated that they can carry other hormones like adiponectin and leptin. So those have been recognized as insulin sensitizing or insulin insensitizing that way. Lipid species like ceramides, diacylglycerols have also been identified within them. So is it possible they're delivering lipid content in a cargo? I would say absolutely, but how exactly, I don't know. But I think it's fair at this time in the literature to hypothesize these things, but we need more work to really identify what's going on there. And I would just add one other caveat. With a lot of this is, I was coming from the story of uptake into the cell, but I think it's fair just to recognize it's also possible that maybe exercise on some ways is minimizing the release of these cells into the blood and transfer. So that would be the other end sort of thinking in metabolism or turnover. Certainly, I think there is this possibility of the uptake story, but I can't dismiss that it could also be not releasing them. So they're not shedding off the cells and maybe in that in of itself is a mechanism by which exercise is helping the insulin sensitivity story we're talking about. Great, thanks. The next question for you, Dr. Mallon, was how do non-steady state exercise and resistance training impact EVs? Another really good question. So, yeah, I guess that's a double edge. From the non-steady state, I suppose on some levels, you could define that through, say, interval training models. Interestingly enough, we had done a study with interval training for two weeks in older adults with obesity and prediabetes and compared head-to-head interval training, which was three minutes, 90%, and then three minutes, 50%, to sort of average out the 70%, such that the caloric expenditure from that was then matched to continuous moderate intensity exercise. So they did the entire session at one hour for 70%. So calories were matched, the best we understand. This was all done on the cycle ergometer. And what we found were actually divergent responses of the EVs. So endothelial-derived, like CD105, which is a classic indicator, actually was going down with interval exercise, but continuous exercise, perhaps paradoxically, elevated it. And what was striking in this study is that that was directly correlated with gains in VO2 max after just two weeks. So that was really interesting to us. I think some of the resistance exercise has also shown influences, but to my recollection, they showed reductions. So I think an important thing in the exercise literature with EVs right now, to say generally, is time course may matter. Martin Wilhelm, one of the first studies showing that acute exercise in the immediate post-exercise period, and it was continuous, mind you, but I think the time course is important in how some of these non-steady states could influence or just exercise in general, is that immediately post-exercise, you'll see some elevations. So greater stresses may promote elevations, but over time, that immediate post-exercise response can be quite different than, say, 16, 24 hours later, whereby most of the literature in that case shows that they go down. So perhaps some of the differences there on how blood flow is perturbing the system during these steady-state, non-steady-state exercises, and then the impact of how they release these EVs potentially or take them up and whatnot into the circulation for measures. All right, thank you. Dr. Negar, I'm going to switch to you for a second. So one question for you is that you stated that the exercise training increases the cell proximity between basodipocytes and endothelial cells. What role do you hypothesize the closer proximity would play in terms of vascular function, such as endothelial reactivity or smooth muscle relaxation? Thanks for the question. Yeah, we observe clearly that these endothelial cells, they are more approximately at least very close, close to less than 50 micrometers. So then these cells means that indicate that the presence of capillaries are close to the maturadipocytes. And then at the same time, along these capillaries, we have the nerves. So this means that the epinephrine or the norepinephrine can reach much more easily the maturadipocyte and activate, for example, the lipolysis rate on these maturadipocytes. And this will make these cells smaller and also at the same time, the epinephrine can also be responsible for the PKA activation. So the mitochondrial biogenesis and the production of increase of the UCP1 expression, this is responsible for the beige. Clearly the better vascularization and closer innovation promote this new phenotype for the maturadipocyte. It makes also the cells much more insulin sensitive. All right. Thank you. One more for you, Dr. Negro. Along with the changes in IWAT remodeling, do you have any data demonstrating changes in metabolic functions such as mitochondrial respiration or citrate synthase activity? On the citrate synthase, we have a previous experiment that showed there is an increase in activity. And also we perform a couple of C-ORS experiments that clearly show that there is an increase of the mitochondrial respiration in these cells. So clearly there is also a functional effect. Great. Thank you, Dr. Malou. I've got a few more for you. How do you think exercise training, rather than a single bout, could impact insulin-stimulated EVs in adults with obesity or metabolic syndrome? And what do you think might be different about the EV cargo with regard to acute or chronic exercise? Yeah, another really good question. I think if we consider training more with regards to, say, 10, 12, 16 weeks, or perhaps even longer, it's possible that they could differ from, say, the single bout session or even up to two weeks. I don't necessarily have data from myself or from our team that has seen that. If anything, I can say, with looking at a single bout of exercise versus two weeks, we actually see similar phenomenon occur. So whether, again, that's short-term, whether that's training, I don't know. We've seen some of the relations with VO2max this way. Even in cross-sectional analysis, that leads me to think that there could be a lot of overlap. But nonetheless, it doesn't mean that there's going to be differential effects. One of the challenging topics, too, within the EV field is the size or types of EVs here, as we're talking about a little bit, these apoptotic bodies, microvesicles versus exosomes. Most of what we've studied have really been in the microparticle, microvesicle ranges. So to answer this question, is it possible that the exosomes, for example, more of the smaller fragments could be differentially expressed? The answer is yes. I just don't know. I haven't seen necessarily literature either comparing the sizes this way to a given training model. And I think some of that comes to just limitations in our technology right now, or at least the diversity of the technology that's being used. So some of these things are, I think, confounders in just our overall ability to understand them. The content itself, too, I really don't know. I haven't actually seen much on, from an exercise perspective, how the content is changing with single versus, say, short-term training or long-term training. So I'm not even really able to speak towards, I think, in fairness, if I think they'd be differentially expressed. I think one of the exciting things for us, perhaps, is that with this recent grant, we're going to be looking at some of these things for the first time. And at least to our knowledge, the first time in this population where we're looking at across EV types, we're looking at exosomes, we're looking at the microparticle sizes, we're looking at the content, specifically focused on microRNA and NO, nitric oxide in these pathways, to try and understand that. We don't have plans to do the acute exercise models as of right now, given the scope and breadth and multi-trial perspectives. We're collaborating with University of Virginia and Mount Sinai to really get at some of the proteomics and other experiments. So I'm sorry, I'm just not able to say. I don't know. No, that's great. I think those data will be really exciting to see as you guys start pulling it out. And one last question for you. How do you think peripheral arterial stiffness would compare to the results of aortic stiffness and augmentation index? Well, I certainly think, yeah, so for us, the data we had was more on the peripheral side. So that's a great question because for the central side we had, so an indicator being pulse wave velocity. We did see trends in reductions, but we had some difficulty with the techniques so we weren't able to really get appropriate data to confidently say one way or another, whether it was an effect. I don't know if there would necessarily be a differential. Certainly, just drawing on the findings we had with a single bout of exercise is that effects both at the large chondroid artery and the microcirculation were identified with exercise. So in other words, thinking about the large arterial tree that we have, the bigger the vessel to the smallest level of the vessel that we were able to detect and measure, there were differentials. Certainly, I appreciate pulsatile pressures are going to be different across that tree. So is it possible that something within the aorta itself is going to be differential? Could be it's a thicker tissue, so it's more dense. It's harder to manipulate in terms of the dilation factors. This is one of the benefits to withstand the pressures. So it's possible that way. I think using some of the literature this way, in vitro studies have been assessed with EVs and have applied hyperglycemic models to try and understand how they play roles in the aorta. And they do see that EVs respond to these hyperglycemics in a pro-coagulant way, oxidative stress-induced way. So certainly, there are mechanisms, I think, in place that could be impacting them deleteriously. And I think exercise has been shown to improve arterial stiffness in the aorta. So I think it's reasonable to hypothesize that. But certainly, we just don't have the data specifically to address that. So it's a good question. Great. Thank you. I think we are right at our minute or two past 2 o'clock. But I want to thank our presenters again for some really excellent presentations of their data. And thank you all for attending and to the leadership team for putting this together. So thank you all very much.

insulin sensitivity

exercise

extracellular vesicles

obesity

metabolic syndrome

vascular function

IWAT remodeling

ECM deposition

NHGRI-1

PRDM16