Hello. Welcome to today's webinar. I'm Ryan Russell, Communications Director of the Exercise Physiology Interest Group Leadership Team. On behalf of our team, we're excited to welcome our expert presenters to discuss exercise-derived signaling molecules, namely appellin and growth differentiation factor 15, and their role in modulating metabolic health. Here's a glance at today's agenda. There will be a few announcements, and we'll introduce our experts in a few moments. 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, but you can send in your questions at any time, so please don't wait until the end of the session. Instead, type it into the question and answer box in your control panel. Be sure to use the question and answer box and not the chat function for questions. We're going to use the chat box to send you important links during this announcement segment. So as I said, I'm the Communications Director for the Exercise Physiology Interest Group Leadership Team, the team that coordinated this webinar. I'd like to take a moment to thank all of the members of the leadership team for their work throughout the year to provide the opportunities to the interest group members. If you want to learn more about the interest groups, look at the link in the chat. You can access over 70 free continuing education programs, webinars, podcasts, self-assessments, and more through the Institute of Learning. Be sure to catch the link that's in the chat box. The Exercise Physiology Interest Group has an upcoming hands-on webinar The Exercise Physiology Interest Group has an upcoming hands-on webinar as part of the Hensley's hands-on webinar series. This webinar will be exclusive and free to all ADA members, and attendance provides the opportunity to earn one continuing education credit. You can register using the link in the chat now. Finally, I'd like to introduce today's first presenter, Dr. Minh Du, who's a Regents Professor for Nutrigenomics and Growth Biology at Washington State University. His research focuses on the impacts of maternal nutrition and exercise on fetal placental development, particularly epigenetic changes associated with the early differentiation of mesenchymal progenitor cells. Today, he will discuss how obesity in expecting mothers impairs placental and fetal development, and how the exocrine, a pill, can improve fetal and placental development and offspring's metabolic health. Thank you, Dr. Du. Now I'd like to turn the presentation over to you. Okay, so thanks for introduction. So today I'm going to talk about the impact of maternal exercise and obesity could have on the fetal development and its long-term consequence on the offspring health, but focusing more on the placental side. So this basically shows the effects of placental importance. So maternal obesity right now accounts for more than 40% of all pregnancies in the United States. So it's a serious problem. We know that could predispose offspring for the metabolic diseases and these changes are associated with placentals which are associated with reduced vascular density and the nutrient delivery, and also due to the increased glucose levels of fetus usually are overgrowth. There's higher insulin resistance, but all these changes actually can be reversed or at least in the hypothesis side, they can be reversed by maternal exercise. So to test this, we did a mice study where we use the mice, female mice. We first fed 60% hyperdiet to make them obese relatively faster. Then two weeks before mating, we fed the mice with a 45% hyperdiet. So since the 45% diet is really maintained during the whole pregnancy and lactation period, then the hyperdiet group continue to separate into two further groups when we see no exercise and when have a daily exercise. So for the control group. So this resulted in four groups and the mice will be sacrificed and 18.5, so that's late gestation to analyze the change in placental and some fetal changes. So we can see that the placental weight actually are slightly increased by maternal exercise for both control groups and those are hyperdiet groups. And this is naturally correlated actually with the fetal weight. Fetal weight was slightly reduced due to exercise, especially in the hyperdiet group. So hyperdiet increased fetal weight, but it was reduced or corrected by exercise during the pregnancy. And there's no effect in the number of fetuses, but the glucose levels was increased due to hyperdiet. So this is consistent with the human changes and actually exercise reduced, suppresses the increased glucose level for both male and female and male fetuses. And this is associated with the insulin changes. So similar change, exercise was reduced while hyperdiet increased. And there's a calculated insulin resistance was recovered by exercise, which was induced by hyperdiet. Okay, so we next check the placental vascular density. So we can see that the density was slightly reduced by the maternal hyperdiet in the placental, but exercise can recover, so as shown here. So hyperdiet decreased, exercise recovered the vascular density of the placental. And so next question, we asked what causes those beneficial changes associated with maternal exercise. So we thought about pollen. We're interested in the pollen because it is stimulated by exercise. And there's quite a lot of reports shows that the pollen actually can enhance the placental development, including increased vascular density. So we just want to check the role of pollen. To test this, we first use immunohistochemistry to check the expression of pollen in the placental. We can see that it's expression mainly focused or concentrated on the labris region of placental. This region have dense blood vessels. And when we magnify it, we can see that the expression is more focused on the blood vessels of the labris of the placental. And if we check the expression, we can see that maternal exercise can significantly increase the pollen expression in the placental, brown fat, different tissues of the late gestation fetus. So the placental level is especially high compared with some other tissues, but the BAT and the muscles are also relatively high. So for the intestine. And we measure the saline of pollen levels. They are increased by maternal exercise in the muscle size, although they are increased in fetus due to maternal exercise. So we want to examine whether pollen has a major role in mediating the beneficial effects of maternal exercise on the placental and fetal development. So we did a further study where the half-fat group or the maternal obesity group mice was injected with either PBS as a control or pollen. So this just to show whether pollen alone have the beneficial effects because maternal exercise could have many other effects. So this is what we use. We use daily injections between the after mating until day 16.5, so daily injection of pollen. So this injection result in increase in the circulation of pollen levels in the fetus. And 18.5 where we sacrifice the mice and get the fetus and the placental. So we can see the placental structures. There is not much change in area of the junctional zones. So as you'll hear, those are junctional zones. Their major function is more focusing on the front side, but the livery sections where the blood vessel is dense. So those regions actually we see maternal obesity or half-fat diet reduces the blood vessel density. So consistent with the previous data present, but since the vascular density was recovered by the pollen injection. So this shows that pollen alone can generalize beneficial effects or similar effects as maternal exercise. And we also check the blood vessel density. We can see that the density was dramatically increased by the pollen injection. So again, this mimics the beneficial effect of maternal exercise on the placental vascularization. So we further measured some markers of angiogenesis. So all of these are recovered by the pollen injection, but reduced due to obesity. So this again shows pollen is a major mediator in the improvement of vascular density in the placental detail maternal exercise. And this also associated with the improved mitochondrial biogenesis, as shown by the markers and mitochondrial DNA density. And this PRDM16 is a major transcription factor regulating oxidative metabolism and mitochondrial biogenesis, which also become dramatically enhanced by a pollen injection. So all of these are very consistent with the improvement we are observing in maternal exercise in the placental. Okay? So the next we question whether these emphases have a major role. We're supposed to see impairment or abolishment of the beneficial effects by using a pollen receptor not compromised. So since the receptor is deficient, so we did show that by removing the receptors, so beneficial effect of maternal exercise dramatically suppressed. So again, conforming the beneficial effects of the maternal exercise is mainly mediated by pollen signaling, okay? Which involves enhanced oxidative metabolism and increased vascularization. Okay? So we also wanted to check changes in the offspring due to the development due to maternal obesity and the exercise. So we get the offspring during the lactation and then after weaning, we did two treatment. One is a cold exposure, which can stimulate brown adipogenesis and the result, half a diet challenge, right? So we can see that under the late fetal stage, so in the brown fat, UCP1 and the source markers of oxidative metabolism and BAT markers was reduced due to, actually it was improved due to maternal exercise when compared with the controls showing the beneficial effect of maternal exercise could have on the brown adipogenesis. And for the offspring mice and the weaning, we subject them to the cold stimulus. You can see that the body temperature is different cold exposure, reduced body temperature, surface temperature of the mice, but the surface temperature was higher in exercise group, maternal exercise group, showing that maternal exercise can enhance the heat generation of the offspring, right? So that's very good for the metabolic health of the offspring. And this shows that this is a protective effect against the half a diet induced obesity. So since two mice we challenged with a half a diet, we can see that the exercise group was a male, female and the male offsprings, their feed intake actually are slightly higher, but they are leaner. So the body weight actually is an increase for the females was significantly less for the exercise group. Males, no difference, but considering the intake more, so that also indicates they are more resistant to half a diet induced obesity. And when we checked about the fat pack weight, so we know fat pack, so we can see the size was smaller in the exercise offspring. So all of these shows maternal exercise can protect offspring get obese when challenged by half a diet. We also measured actually some metabolic parameters, which I did not report here. And we basically was all analyzed some offspring muscle functions, all of these are improved and the weaning improved by maternal exercise and the weaning challenged by half a diet, the improvement maintains. So really maternal exercise could have a major effects on the offspring health. And of course, offspring health has been well demonstrated to be impaired by maternal obesity. So otherwise, this is the last slide I have. So it appears maternal exercise can enhance fetal development or improve fetal development. Both the muscle development and the bone fat development and the social improvement appears to mainly mediated by pollen, which are synthesized or secreted by the placental, especially the library's section of the placental. So I think that's all I'm going to say. Thanks for attending. Thank you, Dr. Du. Now I'd like to introduce the second speaker of the day. Christopher Axelrod is the Director of Integrated Physiology in Molecular Medicine and the Mitochondrial Phenotyping Facility at the Pennington Biomedical Research Center in Baton Rouge, Louisiana. His research applies a bioenergetic lens to understand the pathogenesis of complex metabolic diseases such as insulin resistance, obesity, type 2 diabetes, non-alcoholic fatty liver disease, and cancer. Today, he will be providing an overview of how exercise positively influences glucose-stimulated insulin secretion through direct inter-organ communication between the skeletal muscle and the pancreas, and will discuss the mechanistic role of skeletal muscle-derived growth differentiation factor 15, or GDF-15, in enhancing glucose-stimulated insulin secretion in beta cell function in type 2 diabetes. I don't know why my slide, can you hear me? All right, good afternoon everyone. My name is Chris Axelrod as Ryan mentioned and thank you all for tuning in today. I look forward to speaking about an ongoing work in our lab related to the role of GDF-15 in exercise induced muscle contraction and specifically how it pertains to insulin secretion. So first I wanted to start off by talking a little bit about the glucoregulatory system. So we all know that in response to elevated blood glucose, there are a number of triggers that serve and are coordinated to clear blood glucose, including the release of insulin, which then acts on peripheral metabolic tissues. We also know that diabetes in essence is a disease of decompensated insulin secretion, whereby the amount of insulin secretion that is required no longer adequately supports the ability to clear glucose from the bloodstream, which can be a function of impaired hepatic insulin action or decreased glucose uptake. We also know that exercise is extremely beneficial for the management of type two diabetes. And depending on the mode of exercise, there can be dramatic improvements in insulin secretion, insulin sensitivity, hepatic glucose production and glucose effectiveness. And one point that I'd like to highlight is one of the classical studies in this area, where even in the short term, in this case, this is seven days of exercise training in humans with diabetes, that there are improvements in glucose disposal and in the ability of insulin to suppress hepatic glucose production, indicating that these effects are not strictly mediated by full body improvements in fat or muscle composition, but that there are likely some specific molecules or signals that are produced by exercise that improve the secretion and or action of insulin. So over the past 20 years, there's been a revolution in this idea of how these exercise derived proteins or molecules cue stress and ultimately mediate multi-organ adaptation through action of an endocrine organ. So I think the basic working model is that from exercise, you produce these proteins, which then travel through the circulation and act on distal organs. And some of the most well-studied include the skeletal muscle, adipose tissue in the liver. However, our question today really is gonna be focused on the mechanisms whereby contracting skeletal muscle enhanced pancreatic beta cell function. So to do this, we first established exercise in a dish model, so that we could produce and isolate conditioned media from contracted cells. And we do this through essentially exposing cells to either a sedentary condition where they do not get exposed to a pulse stimulus or to an exercise condition where they get exposed to a pulse stimulus for some period of time. And as you can see from this video on the right, if you look at the red arrows, you can see that as we increase the number of events, you can increase the contraction and the contractility of the muscle fibers. So we know that this is a valid model for muscle contraction. And to answer the question related to how muscle communicates with the pancreas, what we first did is we basically took non-EPS or EPS stimulated myotubes, and we collected the conditioned medium, which was then used to pre-treat beta cells. And from that, we then measured the ability of the cells to secrete insulin. And in a parallel experiment, we also collected the cell lysate for bulk sequencing, which we then filtered through a secretome database to identify potential biological mediators. And what you can see in these panels B and C is that the EPS does indeed enhance the glucose-stimulated insulin secretion rate in two different beta cell lines, as well as in panel C that you can see that between these three different secretome databases, we were able to identify 71 lead targets that potentially mediate muscle pancreas crosstalk. So looking at the heat map in the upper left corner, you can see that GDF15 is one of the top differentially regulated genes. And it's also evidence from this volcano plot. We then went on and validated this by PCR, and we also measured the conditioned media directly of non-EPS and EPS-stimulated myotubes, and the GDF15 level was increased. Furthermore, we also measured the mRNA in response to a 12-week exercise intervention in human skeletal muscle. So these are patients that completed 12 weeks of training at 85% of heart rate max for 60 minutes a day. And you can see that there's a nice change in the GDF15 mRNA. And all of this together led us to feel very confident that GDF15 was indeed a bona fide myokine. So next, just a little bit about the GDF15 protein. So the protein itself has this signal peptide region, propeptide region, and also the mature GDF15 peptide. And it's ultimately this mature GDF15 peptide, which is dimerized and secreted into the circulation. And we know, and what, in some ways, accelerated our interest in this area is that around the time that we discovered its secretory function in skeletal muscle, there were also major landmark studies showing that GDF15 was a potent regulator of body weight and food intake, and that it was very localized to this receptor in the brain called GFRAL, which has very specific binding specificity for GDF15. So next, we wanted to see whether GDF15 itself enhanced insulin secretion. So how we approach this is that in human islets, beta TC cells, and in vivo in a C57 black six mouse, we basically pretreated cells with control or GDF15, or in the case of mice with recombinant GDF15 or a vehicle solution, and then measured the insulin secretion. And you can see in all three conditions, in the human islets and the beta TC cells, and in the mice, that GDF15 did indeed increase the insulin secretion rate, and it did so without altering the insulin content. So from this, we felt very comfortable concluding that GDF15 does indeed enhance glucose-stimulated insulin secretion, but it requires the presence of glucose to do so. And to us, this seemed relatively unproblematic, but as we found out from the scientific community and from some of the reviewers of this work, there was a lot of concern that GDF15 was able to act on islets because it does not, or is believed to largely have low expression of GFRAL, which is the predominant known receptor. But if you look at this upper panel, you can see that in, at least in these mouse islets, that GDF15 largely co-localizes with insulin. So even if GFRAL is not present, the GDF15 certainly is, and that it is present to some extent in beta cells. However, we could not ignore the fact that the GFRAL receptor is really most expressed in the brain. And this is where you see large co-localization. In this case, they used a GFRAL-CRE where you could really identify the regions of the brain that expressed GDF15. So we went about to address this in two ways. The first, to show that it was, GDF15 was required to mediate the effects. We used a neutralizing antibody that essentially is added to the conditioned medium to deplete all of the exogenous GDF15 produced by exercise. So the beta cells still have normal GDF15 function, but it's entirely depleted from the conditioned medium. And what you can see here is that, again, we see that with exercise, you see this nice increase in insulin secretion, which is reversed to the level of the non-EPS condition when we neutralize GDF15. We then went on to isolate primary mouse islets from GFRAL-competent and GFRAL-deficient mice. And what we saw that in the presence or absence of GFRAL, that there was entirely no influence on the ability of GDF15 to stimulate insulin secretion, suggesting to us that GFRAL, the effects of GDF15 on glucose-stimulated insulin secretion are GFRAL-independent. And again, as I showed in the previous slide, this was not surprising to us because we know that GFRAL is largely unexpressed in the pancreas. But it was surprising to the reviewers because it does show that these are not secondary effects to changes in brain function. So next we wanted to know what the potential mechanisms of action were. And the most likely starting point for us was the insulin release pathway. So we know that in response to an increase in glucose, that glycolysis is activated, which ultimately increases the ATP-ADP ratio, causing closure of the potassium channel and ultimately increase in cytosolic calcium. So what we did is we first did a bioenergetic study where we measured the extracellular acidification rate, which is a readout of basically the glycolytic flux of the cell. And you can see in this red line that there's an increase of glycolysis in the presence of glucose and oligomyosin, indicating that the glycolytic rate was increased. We also measured under basal and glucose conditions, the ATP-ADP ratio, which was increased after GDF15 treatment relative to vehicle. Furthermore, we traced the calcium cycling in the presence or absence of KCL. And what we found was that in the presence of GDF15, that the calcium release was increased. Now, as a proof of concept experiment, we returned to our neutralizing antibody model, and we showed that when we pre-treat the cells with this neutralizing antibody, that we completely reversed the effects of GDF15 on the glycolytic flux, which is the most upstream component of the insulin release pathway, telling us that GDF15 indeed does stimulate the insulin secretion pathway. And then as a proof of concept, we carried out a couple of investigations using available datasets in our lab, where we looked at GDF15 across the insulin sensitivity spectrum. And what we found is that compared to control patients that patients with pre-diabetes and patients with diabetes have increasingly elevated GDF15, which occurs in the parallel to the rise in C-peptide. And you can see here that the C-peptide levels correlate very nicely with GDF15, suggesting that the rise in GDF15 is essentially to enhance the insulin secretion rate, which we know is a compensatory mechanism to the glucose becoming elevated. And then lastly, we used a dataset where we had a high intensity interval training in patients with type two diabetes. And what we found is that from pre to post, there was an increase in the GDF15 levels with correlated nicely with the change in the disposition index, suggesting that GDF15 is related to the change in insulin secretion in humans. So the question is, does this model hold? And I think that the future is largely unwritten here, but at least some preliminary evidence that was published recently suggests that when you silence GDF15 in beta cells, you can reverse the effect of what we observed. So you see that the GDF15 levels decline, which leads to a decline in protein expression, which decreases the insulin secretion rate in the presence of glucose without affecting the insulin content. So this is essentially the opposite of what we saw with exercise and the exact same thing we saw with the neutralization of GDF15. And in terms of the larger landscape of diabetes treatment, we think that there is potentially promise for GDF15 as a pharmacotherapy, both to reduce body weight, but also to treat diabetes directly by enhancing insulin secretion. And this is just one example of a long-acting GDF15 analog that was developed by Lilly that really nicely reduced body weight and also changed food intake. Now, this program was discontinued by Lilly, but there are other programs that I'm aware of that are being pursued relative to GDF15 long-acting compounds to act on metabolic function in patients with diabetes. So we will see how that plays out. And just to summarize briefly, we know that exercise training now increases GDF15, which of one of the targets is the pancreas, where it acts on beta cells in islets. And it does so largely by stimulating the glycolytic rate, which in turn increases the ADP-ATP ratio, causing closure of the potassium channel, depolarization of the membrane, and a rise in cytosolic calcium, which ultimately increases the release of insulin. So I thank you all for your time today. And I briefly just want to acknowledge some folks from the team here. So first, John Kerwin, who largely funded these studies and oversaw many of the PhD and postdoc students that worked on this. Jason Collier, who helped with a lot of the insulin secretion studies. Kui Hanna-Zhang, who was one of the project leaders and really saw this from beginning to end. And Randy Celia and Nadia Kramer, who helped us with some of the revision studies and also provided us with the G for all deficient mouse. So at this point, I'm happy to answer any questions that you may have. Thank you, Christopher. With our remaining time, I will ask questions from the Q&A box. Please make sure to put your questions in the Q&A box, not the chat function. We have a question for Dr. Du. Okay, how was the dose of appellant determined? And is this dosing protocol similar to what would be released in response to exercise in mice? Yes. So we basically measure this pollen concentration in the mice circulation of the exercise. So the dose is corresponding to the level in the feeders of the exercise. And a question for Christopher. So we know from previous experiments that concomitant increases in glucose and insulin can cause acute insulin resistance. Do you have any hypothesis on the downstream or long-term effects of an intervention such as GDF-15 since you said it would need higher glucose in order to stimulate the insulin? Yeah, that's a great question. We had this same question ourselves and I think now several groups have shown that GDF-15 also serves to improve insulin action. So the pancreas is not the only organ that it's acting on. It does act on other organs that affect the whole body glucose maintenance. So yeah, we don't think that prolonged treatment as an example with GDF-15 would cause any issues to insulin sensitivity. In fact, we think it would have the opposite effect. And I believe there was a recent cell metabolism paper that actually showed this and they demonstrated that it had some effects on energy expenditure as well as a putative mechanism of action. Thank you for your answer. Just remind people to put your questions in the Q&A box. So we'll have a last call for questions in case there's any remaining questions. For Christopher, were there any sex differences analyzed? In the human studies, yes. In the animal studies, no. And in the human studies, there do appear to be some sex differences with GDF-15 regulation. Yes, there are. So the expression appears to be higher in women, which is interesting, and there may be some explanation there related to sexual dimorphism and diabetes prevalence, but we haven't really pursued that further than that. And for Dr. Du, you mentioned some changes to liver function with apellin. Are there any differences that you know of between endogenous apellin and exogenous apellin on liver function? So I'm not sure what's the meaning of endogenous. So in apolin we inject is apolin-13. So apolin basically there are several forms, and the 13 is the most active form which we injected. I'm not sure about the question. Well, if I recall from your slides, there were some systemic changes that you talked about, including liver, and I believe there was increased liver fibrosis, a couple other things with the apelin, and I didn't know if that was in response to maternal exercise or if it's externally administrated. Is there a difference between what happens with the liver between those different scenarios? Yes, on liver side, I did not present here, but we did a chat, and the improvement I would say is we think it's mediated by apolin. So when we inject it, there's apolin to the maternal circulation daily, and it does improve fetal development. Actually, we also have some data shows that there's improvement in offspring liver, including reduced lipid content in the liver and fibrosis. We do show there is some effect by maternal injection of apolin. So another question for you. There's been some work by the Boschel group that shows that maternal exercise can confer benefits up to two generations. Do you think that your work might have an impact on explaining the second generation? Yes. We did not specifically examine the maternal exercise, but for the maternal obesity, we did show that they can affect the F2 generations through changes in oocytes during the fetal development. So yes, I will say it should be. We did not directly test the maternal exercise. And Christopher, you mentioned that the GDF15 also seems to have an insulin sensitizing effect. What kind of mechanism or tissue has that been noted in? Because we know that not all tissue displays insulin resistance at the same time. So for example, myocyte, which is downstream from vascular insulin resistance. Yeah. It's been showed at least in adipose tissue. I think the sort of autocrine or local action of GDF15 in muscle is still hotly disputed. There were some studies by Eric Richter's group showing that the effects appear to largely not be on the muscle. So I think the jury's still out there, but I think long-term it will. The question is whether it's primary or secondary to the systemic effects. So as an example with GLP-1 analogs and the like, there are lots of secondary effects that occur due to the appetite restriction, weight loss, and enhancement of insulin secretion. So I think the real question is, where are the primary effects? And I think, yeah, the fat is one place that we know for sure that there are primary effects. Well, this has been informative and entertaining. If there are no more questions, I just want to thank the speakers for their time and call it an end.

webinar

signaling molecules

metabolic health

maternal obesity

Apelin

Growth Differentiation Factor 15

insulin secretion

exercise