Good afternoon, everybody. Let's get started. My name is Mabel Hussain. I'm from the University of Michigan. I'm going to be chairing the session. I have a co-chair who has come down with some illness and has apologized. He can't be here. We're just preventing any super spreading event. So I'll be doing this alone. Can I have the first slide? That's not it. So this symposium is about novel aspects in incretin and glucagon biology. You can see the speakers here and the chairs on the other side of the slide. Next slide. I think I can handle that. I just want to give you a little introduction so that we have more time to talk about the new aspects. Every speaker is going to have about 20 minutes. So incretin, I don't think it requires a lot of introduction for this audience. Incretins are gut peptides that get secreted in response to food intake. They circulate through the blood system, perhaps also paracrine effects of these, at least GLP-1, and act on the pancreas. That's the classical paradigm. And you can see the classic experiment on the bottom right where intravenous glucose excursions do not elicit as much as oral glucose uptake on the pancreas interpretive insulin secretory response. The pro-glucagon protein, this is a pro-peptide that gets spliced in different tissues in different ways. And the products glucagon and GLP-1, there's classic information, old information, more than 50, 70, or 100 years old, that show that glucagon stimulates hepatic glucose production and GLP-1 stimulates insulin secretion. But more recently, there's new evidence that these peptides work in different ways. GLP-1 and GIP seem to have very similar effects. They have slightly different distributions of their receptors, so maybe some differences in biological activity. So we want to focus on the new aspects of these peptides and then also the new developments of these hybrid peptides that act in ways that are rather unexpected maybe. So we'll go to the first speaker, which is David D'Alessio. He's going to talk about new twists in the biology of glucagon. Thank you. Thanks for the introduction, Maybu. I actually came to this meeting thinking I was chairing this session. And in preparation, I flipped through and was looking at the titles. I thought, boy, that first talk looks really interesting. And the presenters actually used a title similar to one I used. And I scrolled through and found out that the presenter was me. I may be disorganized, but I always travel with a lot of slides. So this is what I'm going to talk about today. Maybu's done a nice introduction, and I'll speed through the incredent effect stuff. But talk about why we started looking beyond an endocrine action to explain the effects of GLP-1 signaling. And then revisit glucagon as an incredent. That may sound surprising to some. And talk about alpha to beta cell communication, which we think is an important regulatory pathway in the islet. And the implications of glucagon in therapeutics. I would say that this work's been done in collaboration with my colleague, John Campbell, at Duke. And a team of excellent postdoctoral fellows. I try to acknowledge their names on the slides where they do things. But we work as a team, mostly. And so everybody's participated. So the origin of endocrinology actually was in the gut. You could argue that the paper of Baylis and Starling, where they discovered secretin, was the first demonstration of a factor elicited in one organ. That worked through the circulation to affect other cells. Early on, duodenal extracts were used to treat diabetes in dogs. And about 100 years ago, LeBaron still coined the term, incredent, for gut factors that stimulated internal secretions. Insulin, it turns out. And this definition has been with us since. Maybe we've talked about the incredent effect, which is when you control for glycemic stimulus to insulin secretion. Glucose coming in through the gut has about a three-fold greater effect to stimulate insulin secretion. In the search for the peptides that caused this, Brown and Pedersen discovered GIP, glucose-dependent insulinotropic polypeptide, in the early 70s. And it comes mostly from the upper gut. About 10 years later, when people cloned the cDNA for proglucagon, they found downstream sequences that also looked like regulatory peptides. Called them GLP-1 and GLP-2. And GLP-1 turned out to be insulinotropic in vitro in animals and eventually in humans. The incredent acts by increasing in the circulation after meals. These are data for GIP, which goes up after a glucose protein or fat meal. The rise is pretty high and lasts pretty long. And if you give graded doses of nutrients, you get graded doses of incredence. And this is a way to link the amount of nutrients coming in through the gut to the pancreas so you get an appropriate insulin response. So the conventional model of GLP-1 action, I think I made this slide 25 years ago and I used to use it to teach medical students. Is that after a meal, GLP secreted into the portal vein, it burbles through the liver, out into the lungs, back to the heart. And then goes out into the arterial circulation where it can interact with target tissues like beta cells in the islets. And as many times as I've shown this slide, I show it now to say that I think it's incorrect and that this isn't how GLP-1 works. I think there's other players or considerations that explain the incredent effect of GLP-1 signaling. So why do I say this? GLP circulates at relatively low levels compared to GIP, which increases 10 or 15 fold after a meal. GLP doubles or triples. So it has a minimal dynamic range and it's rapidly metabolized in the vasculature. Whereas if you really cut the y-axis off here, you can see a GLP response. If you plot it on a molar basis next to GIP, you can see a big rise in GIP and a little rise in GLP-1. It doesn't go up very much. And then this peptidase DPP-4, which is ubiquitous, rapidly inactivates GLP-1. So there's a lot of DPP-4 in the liver. There's a lot in the lung. And I would argue that by the time it gets from the gut to the beta cell, most of it's inactivated. So the question then is where is this other incredent? And I would pose the question, have we been wrong when we look at glucagon? And we tend to juxtapose the two islet hormones across a line of scrimmage where they're bashing heads against each other to control the glycemic level. And again, we teach this in our textbooks and to our medical students that in the fasting state, glucagon predominates and there's hardly any insulin. In the fed state, it goes the other way where there's a lot of insulin and not much glucagon. And these are sort of a fasting hormone and a fed hormone that really have opposing actions. And I think that's been challenged in the last few years. It turns out that glucagon can stimulate insulin secretion. If you go back to literature in the 1960s, Vincent Marx and his colleagues in London and Surrey were opposing this, that glucagon might stimulate insulin secretion. And this is shown here at a perifusion of mouse islets where if you go from low to high blood sugar, you get an increase in insulin secretion. And then if you ramp in glucagon, you get this graded increase in insulin secretion. And here's GLP-1 as a comparison. And based on these doses and the insulin response, you can calculate an EC50, which is about 30-fold higher for GLP-1 on a molar basis. It's stronger. But you have to consider inside an islet, glucagon exists in about 100 or maybe 150-fold excess over GLP-1, which I think most people agree is also made by alpha cells now. And so you could argue that the alpha cell makes two peptides that can interact with receptors on beta cells to stimulate insulin secretion. And we call this alpha-to-beta cell communication. So we made a glucagon receptor knockout mouse, knocked it out of the beta cell to see how much glucagon contributed to perennial insulin secretion and glucose tolerance. And you can see here we got a very nice knockout where these are the wild-type islet glucagon receptors, and the knockouts were nil. Whereas we still had normal amounts of GLP receptors in the islet and normal amounts of glucagon receptors in the liver. When we did the GTTs, however, we saw no difference. That is, you could knock the glucagon receptor out of mouse beta cells, and it didn't have any effect. We went on, though, to explore this a little deeper. We took islets out of normal mice, and we gave them this graded dose of glucagon, and you got a nice insulin secretion. Took the islets out of the glucagon receptor knockouts and got almost the same effect. They just stacked right on top of us. Now, the medical student who was doing these studies decided to try and block the glucagon effect with Xcendin-9. And if she'd have come to me and asked, I'd have told her, don't bother. It's a GLP receptor antagonist. But she didn't come to me. She just did the experiment, and it turned out that Xcendin-9 just blocked the hell out of glucagon, decreased the amount of insulin came out by about 80%. And then she repeated this in her glucagon receptor knockouts and got even a little more. So it looked like in mouse islets that glucagon was a good insulin secretagogue, that it acted primarily through the GLP-1 receptor, and that the glucagon receptor contributed little. So when we published this, about four groups published the same thing in a year. And it turns out if you look through the literature, you could find it 20 years ago that people had blocked glucagon with Xcendin-9. So it turns out glucagon's a good insulin secretagogue in mouse islets, and that it stimulates insulin release during hyperglycemia. But when would glucagon be useful to trigger the beta cell? The story is that if you give an oral glucose load, the glucagon doesn't change very much. And that's true. And if you give IV glucose, you actually suppress glucagon. But, of course, most animals and humans don't just eat glucose. They eat meals. And so here we did oral glucose tolerance tests and meal tolerance tests in a group of mice, gave them the same amount of carbohydrate, and measured the islet hormones. And you can see here when we just gave them a gavage of PBS, there was no change in insulin, very little change in glucagon. The OGT gave us an insulin response. The meal gave us a bigger insulin response. The OGT didn't change, didn't suppress or stimulate glucagon. But the meal test did. And, in fact, when we sampled from the portal vein, you could see even bigger responses, bigger perineal responses. So, I mean, this is not new stuff. Roger Unger talked about this in the 60s. Meals, particularly protein-containing meals, stimulate glucagon. So, how might this work? Well, again, when I was in medical school, everybody talked about proteins like alanine causing a nice rise in glucagon. And you see that in this perifusion. It turns out that GIP, the other incretin, or the non-GLP incretin, also stimulates glucagon. And it turns out there's synergy. So, here's a perifusion with just glucose, and you don't see much glucagon. GIP gives you a little effect. Alanine gives you a little effect. If you put them together, you get a huge effect. Note that the Y axis here is broken. It goes way up. And so this would mimic the perineal state when you have a release of GIP, which I think is a bonafide hormone. And you also have an increase in portal circulation and peripheral circulation of amino acids coming in from the gut. We did this in vivo. So, if you give IPGIP to mice, you get a little response. IP alanine, not much. But you see this nice synergy even in vivo. And then we knocked the GIP receptor out of the alpha cell so that the alpha cells in these mice don't respond to GIP. And the effect of the synergy between amino acids and GIP, of course, goes away. So, what about the role of alpha cells and the incretin effect here? So, same mice get an IPGTT, OGT, or a meal tolerance test. Same amount of glucose or carbohydrate in each. The IPGTT gives some insulin, no glucagon change. The OGT gives you a little more insulin, no glucagon. And the meal tolerance test gives you the most insulin, but also some glucagon. When we do this with the GIP receptor null alpha cell mice, it doesn't affect the IPGTT at all. Well, there's nothing to stimulate glucagon in this setting. Similarly, with the OGT, where you don't have much of a change in glucagon, glucose tolerance is about the same in these animals. But if you give it, with a meal test, a setting where you're stimulating glucagon, and this might contribute to insulin secretion, now you unmask this glucose intolerance and start to demonstrate the effect of prandial glucagon on meal tolerance. So, what happens to insulin secretion when you get glucagon completely out of the system? This is a mouse that doesn't make proglucagon. It has a stop codon in front of proglucagon. It's got normal beta cells that express normal incretin receptors, but it doesn't make any proglucagon. So, here's a perifusion showing a glucose effect, and then you can stimulate with amino acids, glutamine and arginine. This all goes away in these nulls. So, there's no amino acid-stimulated insulin secretion in the absence of glucagon. It also knocks the hell out of the glucose response, which really surprised us. But, these animals respond to glucagon, they respond to GLP, they respond to GIP. And, when we remove the stop codon in these animals with four days culture with a CRE, you can see that we restore glucagon and restore GLP secretion in these islets, and then they look perfectly normal. So, the mice don't have messed up islets. They just don't make proglucagon, and when you take that away, that's really a critical signal. So, how does this play out in vivo? This is giving mice, fasted mice with a low blood sugar, 75, a bolus of glucagon. You can see it makes them hyperglycemic. This is glycolysis and maybe a little bit of gluconeogenesis. Again, that same data presented here. If you give the dose of glucagon to fed mice, mice with a full gut and a blood sugar of 150, you don't get this response. In fact, you get a decrease in insulin secretion, and that's because glucose goes up threefold. This just shows you that glucagon, like GLP-1, is glucose-dependent. That's shown in this perifusion, where you can give glucagon, GLP, or GIP, not get much of an effect at low glucose, but when you raise the glucose, they all become insulinotropic. So, this is a sort of a common feature of all the, what I'll call, incredins. So, what if we take away alpha to beta cell communication? So, here we made a double-receptor knockout mouse. These animals make proglucagon peptides, so they make GLP, they make glucagon in their alpha cells, but we've taken away the two receptors that we've shown to be insulinotropic. In these animals, if you study them when they're young and lean and chow-fed, they look perfectly fine. If you fatten them up, they become pretty glucose intolerant. I never know when to call a mouse diabetic, but this looks pretty bad. And what this shows is that you need alpha to beta cell communication. You need signaling through these two receptors for normal compensation for metabolic stress. So, interrupting alpha to beta cell communication seems to be really important in mice. Is it just tricks with mice, or can we see this in humans? So, these are human islets that we got from Pat McDonald in Edmonton, and you can see here we perifused them at low glucose. They have a basal insulin secretion. When we block the glucagon and GLP-1 receptor with Xcendin-9 and a glucagon receptor antagonist, we just bottom out basal insulin secretion. Again, these are islets perifused without any exogenous peptides. All the incredins, all the proglucagon peptides are coming from the islet. And then here you can see the same thing. We block the two receptors in human islets during glucose stimulation. Here's the control, and here's the about 50% or more decrease in stimulated insulin secretion. So, it looks like in human islets you need proglucagon peptides for a normal beta cell response as well. So, these studies raise a question to us. What if we just go to the whole human? Let's try one of those studies. We did the following experiment. Sarah Gray was a postdoc in the lab who knocked off this nice study. We brought people in, fasted them for a couple of hours, and then infused them with glucagon for 30 minutes. Brought them back a different week, raised the blood sugar with a glucose clamp to about 150, what we thought was prandial levels, and gave them the same glucagon infusion. What we were comparing was the insulin stimulation with and without glucagon. We had trouble. It's hard to find a lot of glucose studies in humans. We didn't want to mimic regular peripheral levels. We wanted to get the levels high like they might be inside the islet. So, we just gave them a high dose. You can see here these are humongous glucagon levels that we get during the stimulation part of the study. Here's the beta cell function. This is during the saline study. Here's the blood sugar. It's pretty steady. We turn on the glucagon. You get the typical hepatic effect. Blood sugar goes up conveniently to about 150. Then this is the C-peptides, which are stable, and then sort of chase up the hyperglycemia to about 700 picomoles. This is the study with the clamp. You can see when we raise the blood sugar, we also raise the C-peptide significantly over the control study. But then when we turn on the glucagon, instead of the lazy chase up, we get this prompt response, and the C-peptide goes way up. So, it acts just like we were infusing GLP-1. This is the basal C-peptide in the control study that goes up a little bit with glucagon, actually up to the same level as the hyperglycemic clamp alone. But when you add glucagon to the hyperglycemic clamp, you get this big effect. So, one of the things about human islets is they have a lot more alpha cells than mouse islets, where it's about 10 or 20%. In humans, it's about 30 and 40%, with a lot of the beta cells having an alpha cell right next to them. On an anatomic basis, you might say, this is a really good anatomy for alpha to beta cell communication. And we wondered if glucagon might have a bigger effect through the glucagon receptor in this setting. So again, human islets, peri-fused with glucose in black, you see the insulin response. When we give the GRA, the glucagon receptor antagonist, we can see a meaningful decrease. It's not as big as what we get with Xcendin 9, that is, when we block the GLP receptor. You can see here, it's about a 30% decrease, and with Xcendin 9, it's closer to 50%. And interestingly, when we give the blockers during basal insulin secretion, glucagon doesn't do much, but Xcendin 9 will knock down basal insulin secretion. So this is interesting, and I think we're gonna pursue this to see if maybe the alpha to beta cell communication is even higher in humans than mice. So the insulinotropic effects of glucagon in humans are potent. They're contingent on circulating glucose levels, that is, they're glucose-dependent, just like the effects of GLP and GIP, and maybe more balanced through the two receptors, although that's still something that we're working on. And so alpha to beta cell communication seems to be a key player in the response, and I would argue that with the multi-receptor agonist, this is an important consideration. So this is the dawn of the MRA era, right here, this paper by Dave, but it's Matthias Chepp and Richard DeMarchi driving this research into a co-agonist that activates the GLP and the glucagon receptor and seems to give a better weight loss and better glucose tolerance, and then a paper by Brian Finnan with a tri-agonist, where if you add glucagon activity to a dual-GIP-GLP agonist, you get better effects. And people keep arguing that you have to overcome the glucagon effect on hyperglycemia with a lot of GLP action, and I would argue that glucagon may be having an important beta cell effect in these tri-agonists, and that may be one of the reasons it works. So nutrient-stimulated peptide hormones are important in normal insulin secretion and glucose tolerance. Glucagon shares several important properties with the classic incretins. Elimination of proglucagon peptide signaling impairs glucose regulation, and glucagon stimulates insulin secretion potently in humans in a glucose-dependent fashion. So I think we gotta get these guys to take off their helmets, tap the keg, teach each other their respective school fight songs, and get along more harmoniously. And similarly, I think we have to think of the two islet peptides not as antagonists, but oftentimes as working hand-in-hand to control glucose tolerance. Thank you. Thank you very much. Thank you. I have, I'll start. So, does the beta cell need a glucagon receptor? Yeah, I mean, it's a question we talk, that's been the focus of lab meeting for four or five years in our group. So you have, I mean, why does glucagon have two receptors on the beta cell, and why are there two proglucagon ligands that seem to be insulinotropic? And that's a hard one to figure. So, we haven't come up with an answer now, but I mean, I suspect it's not there randomly or redundantly. Now, whether there's effects in development that are important are things we haven't looked at, although we've talked about. Whether one of them plays a bigger role in growth of beta cells or protection of beta cells, another hypothesis that could be pursued. We tend to see, and the literature supports, that under times of stress, there's more GLP. And so it might be that GLP would be responsive to things like beta cell damage, or inflammation, or a greater need for insulin secretion. But these are the key questions that are unanswered. I mean, but they sit there for anybody to chase down. I mean, they seem really important to me. I have another question. Okay. Is there a role for GIP receptor on beta cells? That's the classic view, but you haven't really shown it. I mean, in the studies that have come out now that there's a GIP receptor antagonist, and I'm not sure this is good an antagonist, as complete an antagonist as Xenon 9. But in those studies, mostly done by Philip Knopf and his colleagues in Denmark, they argue that GIP in a non-diabetic person is the more important incredent. And that if you block that, you get a bigger decrease in insulin secretion, a bigger glucose intolerance effect. And so I think there's no question that GIP has a role in physiology. And I think it behaves as a hormone, as a bonafide hormone. It may be the only one we know of that actually links the gut to the islet. Because as I said in the preamble to this talk, I don't think much GLP gets from the gut to the islet. Do I have a third question if nobody asks anything? So, there is, you presented some data, but there's also other experimental evidence that GLP-1 actually may never, active GLP-1 may actually never reach from the intestine to the beta or to the islet, right? And so is the GLP-1 receptor actually a glucagon receptor on the beta cells and not a GLP-1 receptor, number one? Number two, how then is GLP-1 working? Is it neuronal? Is it all just relays? Yeah, so there's a couple issues there. One is that I think in the beta cell, the GLP-1 receptor is a glucagon receptor. I don't think that's the case for the GLP-1 receptor in the brain. Although I also don't know anybody that's used that as the hypothesis to test with behavioral or other neural functions. I think, I think I forgot the second part of your question. So, the first part was, is the GLP-1 receptor actually a glucagon receptor on beta cells? And then the second part of the question essentially is, how does the intestinal GLP-1 work? So, one extreme on the intestinal GLP-1 is that it just gets secreted because the gut needs to secrete GLP-2, which has a lot of functions in the gut. GLP-1 probably has other functions in the gut as well. But it's not clear to me how much of that gut GLP-1 is important in glucose regulation for sure, but other things as well. Hi, Mike. A question? Right. So. Introduce yourself, please, and. Mike Schwartz, University of Washington. So, we know that GLP-1 agonists are pretty good insulin secretogogs in type 2 diabetes. And based on your work, you would argue that it's basically mimicking what glucagon would do. So, you give a large dose of GLP-1, you get a glucagon effect. Does that mean that the pathogenesis of beta cell dysfunction in type 2 involves deficient glucagon stimulation of the beta cell? Yeah, no, that's a good question, Mike. I mean, the one thing I would say that a lot of people miss is that if you actually look at beta cell sensitivity to GLP-1 in type 2 diabetes, it's pretty lousy, right? Just turns out that if you give enough, you can overcome that. I'm not aware of anybody that's, well, I mean, we're getting ready to study glucagon in type 2 diabetes patients, and I'd be really interested to see if they have a deficient sensitivity. We have some indirect evidence that as the beta cells fail, the glucagon secretion goes up. So, what people have typically called hyperglucagonemia in type 2 diabetes, and sort of blamed some hyperglycemia on that, may actually be the alpha cell trying to flog a failing beta cell. That's a hypothesis we're testing right now. So, again, I think the findings that we've reported and other people have reported in the last several years raise the possibility that alpha cell dysfunction contributes to diabetes, not by stimulating the liver too much, although that may play a role too, but actually in how it stimulates beta cells. Can I just add a side question? So, you did knock out glucagon receptor and GLP-1 receptor in beta cells. They're not responsive to glucagon. Do those mice have hyperglucagonemia? They do. Anytime we, well, no, they didn't. They didn't? Yeah, it's only when we've knocked it out in the liver do we see the hyperglucagonemia, although I gotta tell you, we didn't study that to the depth that we need to do, which would be isolated. Next question, please. Ido Goldstein, Hebrew University. So, what happens during fasting? Glucagon levels go up, but insulin levels do not go up, and it is known, as you mentioned, that glucagon and insulin have antagonizing effects, at least in the liver. Yeah. So, if glucagon increases during fasting, it shouldn't lead to insulin secretion from the beta cells. It should, except that it's glucose-dependent, and during a fasting state, blood glucose levels are pretty low. The other thing is, glucagon levels don't really go up. Glucagon levels in a fasting state aren't like glucagon levels during hypoglycemia, where they go up eightfold. They sneak up a little bit, and if you starve somebody for more than a couple of days, the glucagon levels come back down. Thanks. Thank you. Another question? Please introduce yourself. Yes, hi, my name's Jay Park. I'm a resident from University of Maryland. So, these results, what would this mean for patients with type 1 diabetes? And also, there has been recent studies about using GLP-1s for new-onset type 1 diabetes, who still have a little bit of insulin function. The second part of the question is, have you thought about creating mice models who don't produce glucagon and insulin? Yeah, so other people have made those mice, and they're tricky to interpret, but usable. So, Pedro Herrera's group got data on that. In type 1 diabetes, I don't think there's probably much alpha-to-beta cell communication, because there's not much beta cell to receive the communication. GLP-1 has been used, almost got approval for type 1 diabetes, but I think more for weight loss than for stimulating beta cells, because again, there's not a lot left to stimulate. Right, they've only resulted in 0.3% decrease, so I guess that makes sense. Thank you so much. Okay, thank you very much. We need to move on. The next speaker is Dr. Clemence Blouet. Is that, did I pronounce it properly? Dr. Blouet has received her training in Paris and at Albert Einstein College of Medicine and now has her own laboratory in Cambridge, at Cambridge University. And the title of her presentation is Molecular Mechanisms of Ingrid effects in the brain, how the gut regulates energy and glucose homeostasis. Dr. Blouet, would you like to start? Okay, so I'm going to talk about oligodendrocytes and how deep receptor signaling in oligodendrocytes is an interesting topic to study. But before I talk about oligodendrocytes, I'd like to start with generic slides for you to understand where I'm coming from. So we know that obesity is a disease of the brain. And this statement is mostly supported by recent GWAS data showing that genetic variants associated with high BMI are expressed and active in the brain. And many of these genes are associated with brain nutrient and metabolic sensing pathways, as well as pathways regulating brain plasticity. And also brain plasticity in adulthood can seem a bit intriguing. It's not recognized as a major mechanism through which the brain normal function occur. And it's intriguing to think about how brain plasticity might be impaired in the better physiology of obesity and metabolic diseases. But not much is known about it. In contrast, there is a lot of preclinical data supporting a crucial role of brain metabolic and nutrient sensing in the control of metabolic homeostasis. The brain receives a variety of signals about the qualitative and quantitative aspects of energy availability. Many of these signals are generated by distal sensing sites, for example, in the oral cavity or the GI tract. And these signals are important for the production of anticipatory and peri-ingestive responses. They typically produce rapid but transient changes in brain activity. And they need to be reinforced by signals that are detected directly by the brain and that provide consistent information to produce long-term and sustained changes in brain activity and eventually control energy balance. So it's very important that the brain has direct access to the energy available in the body. And this is possible through brain direct nutrient sensing and metabolic sensing. However, most of the brain is protected from circulated signals by the blood-brain barrier. And many of the hormones, many of the incretins actually don't cross the blood-brain barrier. But there are some specific sites, the circumventricular organs, where the brain can directly access circulating information. For example, the median eminence and the areopostrema. I'm not sure how the pointer works. For example, in the median eminence, we know that some of the most important neurons involved in the regulation of energy balance, the ERG-RPN pumps neurons, are located just above this circumventricular organ, the median eminence. In the median eminence, there is a population of fenestrated capillaries that can be visualized with omega-33 immunostaining. And this gives this area exclusive access to circulating signals. These signals can fully diffuse in the median eminence and the nearby arcuate nucleus. And this can be visualized after an intravenous injection of Evans Blue, which is a blood-brain barrier impermeable substance. And we can see with an Evans Blue injection that there is a territory in blue that has access to circulating signals. But this diffusion is restricted by a diffusion barrier that we know exists, but it's not clear which cells makes this barrier. So that's an interesting question because this barrier is critical for access of key neurons in the brain to circulating signals. So how does it work? We don't know. Interestingly, while the hypothalamus is gray matter, so most of it is devoid from myelin, there is a population of myelinated axons that pass through the median eminence. And intriguingly, these myelinated axons overlap with the permeability barrier, the barrier between the median eminence and the arcuate nucleus of the hypothalamus. So myelin, as you may know, is membranes, lipid membranes that unravel axons and allow circulatory condition of action potential. And myelin is made by oligodendrocytes. And we became interested in myelin and oligodendrocytes in the median eminence because we found that these cells seem to have very high nutrient sensing activity. I don't have, so this is just a video showing the nice distribution of these myelinated axons between the median eminence and the arcuate nucleus. And we've done a number of studies to study these oligodendrocytes and myelin in this area. I don't have time to go through all the data, but basically what we found is that there is continuous and rapid proliferation of oligodendrocyte progenitor cells, OPCs, in the median eminence. And the rate of proliferation and differentiation is really high compared to other brain areas. So this proliferation and differentiation of OPCs supports a continuous generation of new oligodendrocytes that form new myelin and existing oligodendrocytes at the same time so that the amount of myelin and the number of oligodendrocytes is stable in this area. So this supports the idea that there is unique plasticity of oligodendrocytes and myelin in the median eminence. In diet-induced obese mice, this plasticity is blunted. And we found using BRDU incorporation to measure proliferation of the progenitors that the progenitors no longer differentiate as much. So they don't differentiate. And this is associated with an accumulation of oligodendrocytes. Oh, there you go. And you can see nicely here after eight weeks of high-fat feeding is an accumulation of SOX10 positive cells. So it's a specific marker for oligodendrocytes. And they accumulate here just below beta-thetanocytes, which are quite well described in the median eminence. And actually, the decrease in oligodendrocyte plasticity correlates with weight gain. And this is associated with an accumulation of myelin in the median eminence. So this is just to show that diet-induced obesity blunts this plasticity. But the role of blunted plasticity of oligodendrocytes in the pathogenesis of obesity is not known. So we've looked at the role of this plasticity and we've done a number of studies, but I'm just gonna show a few of them. We've used this model where we can block the production of new oligodendrocytes in adulthood. It's an induced model by knocking out a transcription factor required for the differentiation of progenitors. And this produces rapid demyelination in the median eminence. And one of the most interesting results is that this is associated with an increase in the diffusion of Evans blue between the median eminence and the arcuate nucleus. And we also find an increase in the density of fenestrated capillaries, suggesting that some way that we don't know yet, we don't really know the mechanisms, but it seems that oligodendrocyte plasticity in the median eminence regulates the permeability barrier. So why am I talking about oligodendrocytes in this session? Well, it turns out that when we did single-site transcriptomic analysis of median eminence oligodendrocyte, we found that they're highly enriched in some interesting receptors, including G-preceptor. And in fact, G-preceptor in the median eminence is exclusively expressed in oligodendrocytes. And this is, in fact, also the case in the rest of the brain. Oligodendrocytes are highly enriched in G-preceptor. This was also found by Frank Ryman and Fiona Gribble when they used a G-preceptor reporter line when they did fact-sorting of G-preceptor-expressing cells. They also found a population of oligodendrocytes that expressed G-preceptor in the medial basal hypothalamus. And likewise, in the hindbrain, in the dorsal-vagal complex containing the aorapostrema and nucleus of the solitary tract, oligodendrocytes, as you can see, expressed G-preceptor. And it's probably one of the populations that is the most enriched in G-preceptor. So why is G-preceptor expressed? And how does it regulate oligodendrocyte biology? And one particularly interesting, again, in the median imminence. So we first confirmed expression of G-preceptor in oligodendrocytes using RNA scope. So level of G-preceptor expression seems quite low compared to what you can see with this technique in some neurons. But it's definitely there. And so, again, raising the question of what is the role of this signaling of G-preceptor signaling in oligodendrocyte biology? To ask this question, we used, again, fate mapping tools. So in this model, TD tomato, like a red fluorescent protein, is expressed specifically under the control of opaline, which is a gene only expressed in myelinating oligodendrocytes. So when we expose the mice to tamoxifen in adulthood, this induces expression of TD tomato in myelinating oligodendrocytes. And as you can see, over time, in the median imminence, the TD tomato signals goes down significantly. But the total number of oligodendrocytes does not go down. And this is specific to the median imminence. If you look in the literature, it's already described. In all white matter tracts, but also in other circumventricular organ, there is no such decrease in the TD tomato signal. So this turnover seems to be specific to the median imminence. So we can use this tool to test the effect of interventions on myelin plasticity in the median imminence. And here, what we did is that we exposed the mice to tamoxifen and then gave them two weeks of subcutaneous injection of a long-lasting G-preceptor agonist. And what we found is that G-preceptor agonist treatment increased the number of oligodendrocyte lineage cells, increased both the number of progenitors and the number of myelinating cells. And when we looked at the TD tomato signal, we found that G-preceptor agonism significantly increased the number of TD tomato expressing oligodendrocytes, suggesting that it increased the survival of existing myelinating oligodendrocytes. We also found an increase in the number of unlabeled oligodendrocytes. So these are formed after tamoxifen administration and therefore don't express TD tomato. So the intervention also increased the differentiation of progenitors. So this data supports the idea that G-preceptor signaling in oligodendrocytes increases the differentiation of progenitors and the survival of existing myelinating oligodendrocytes. And this is associated with a significant increase in myelin density in this area. So we next wanted to test whether endogenous G-preceptor signaling is also involved in the regulation of oligodendrocyte plasticity in the median eminence. And we generated a mouse model where we can delete G-preceptor specifically in oligodendrocytes, this time with a PLP1 reporter in adulthood as well. So this is inducible. And when you think about it, targeting cell populations that turns over isn't straightforward because the knockout can fade if these cells die. But at the end of our studies, we still had a significant decrease in G-preceptor expression, giving us confidence that we did achieve G-preceptor knockout. So what we found is that a bit of the reciprocal picture of the agonist studies that I've shown before is that in the knockout, we had a decrease in the number of oligodendrocyte lineage cells, with both a decrease in progenitors and myelinating oligodendrocytes. So it looks like endogenous G-preceptor signaling in oligodendrocytes regulates oligodendrocyte lineage progression in the median eminence. None of this is observed in the rest of the brain. And we've looked at CVOs and other white matter tract like the corpus callosum. So it seems to be a median eminence specific effect. So we have this working model. On one hand, we have data supporting the idea that median eminence myelin and median eminence oligodendrocytes contribute to the regulation of this diffusion barrier that is important for access of circulating signals to the target neurons. And on the other hand, we have evidence that G-preceptor signaling in oligodendrocytes regulates myelin in this area and oligodendrocyte plasticity. So we wondered if G-preceptor signaling in oligodendrocytes might regulate the median eminence accurate barrier. And we performed the following experiment. We're specifically interested in the entry of liraglutide into the brain because of looking for mechanism of action of G-preceptor GLP-1 receptor coagulantism. So we took obese mice and we treated them for seven days with either a vehicle or a long-acting G-preceptor agonist. And then at the end of the experiment, at the terminal, we did a terminal injection of a fluorescent liraglutide to access brain entry of liraglutide in these animals. And what we observed is that, first of all, liraglutide is mostly visible in CVOs. It doesn't enter the brain. So it's really highly concentrated in the median eminence and the AP. So these are full brains that were cleared and imaged with a light-shift microscopy. And in the animals pre-treated with a G-preceptor agonist, we saw a significant increase in liraglutide entry into the brain. We also tested the effect of seven days pre-treatment with a GLP-1 receptor agonist. And also, as our GLP-1 receptor agonist, we also had a perfect group. And none of these other interventions produced the same increase in arc and median eminence GLP-1 receptor agonist entry. So it seems that it's not an increasing effect. And something about G-preceptor agonism specifically increases entry of fluorescent liraglutide. So then we tested whether this could be a mechanism of action of dual GLP-1 G-preceptor coagonism. So in all wild-type mice, we administered GLP-1 receptor agonist alone or together with a GLP-1 receptor agonist. And we observed something similar to what has been described before, that adding a GLP-1 receptor agonist to GLP-1 increases the weight suppressive and food intake suppressive effect. However, in the knockouts, first of all, we observed that they were producing a bigger weight suppression in response to GLP-1. But adding GLP on top of GLP-1 receptor agonist didn't further increase weight loss, suggesting that this mechanism might contribute to the beneficial action of dual agonism. So this is to finish working model. We still have a lot of questions to answer. But it seems like in obese mice, myelin plasticity is impaired. We've shown that. And this, we think, impairs the ability of circulating factors like GLP-1 receptor agonist, if we think about these therapies, into access to the target neurons in the arcuate nucleus. However, when we treat obese mice with a G-preceptor agonist, we restore oligonucleotide plasticity. And this somehow might, seems to increase access. And maybe this is a mechanism of action of dual-increting therapies. Thank you for your attention. Thank you very much. Thank you very much. Some papers open for questions. I'll start. So we just heard about GLP-1 not circulating very well. And so if you take away the agonist that might have a different kinetic and different pharmacologic properties, physiologically, how does GLP-1 exert its effects? And how does that relate to GIP actions in the median eminence? You understand what I'm trying to get at? So maybe GLP-1. Yes, so the actual biology of endogenous GLP-1. Doesn't reach the brain through the circulation. No, so it's likely that the action of GIP on oligodendrocytes, which might still be real in normal biology, does not really affect the action of endogenous GLP-1. Yeah, we haven't looked at it. And yes, there is no reason to believe it would because we know that GLP-1 mostly acts on vagal appearance to suppress food intake. So by logical extension of that question, what is then the role of GIP receptor if it might not be modulating GLP-1 entry? Well, in general, it seems to be regulating the barrier and access of circulating signals. So we took the example of a GLP-1 receptor agonist because of the interest in how these two molecules can work together. But you can extend the observation of increased entry to other circulating signals. So, yeah. All right, next question. Really nice talk, Lamos. Thanks very much. I'm interested in taking off on that. Did you, have you looked at other signals? I mean, the one that comes to mind is leptin. Is GIP signaling in these oligodendrocytes important for hypothalamic penetrance of leptin? That would be an interesting control. I mean, the notion of whether it's specific for just GLP-1 seems a little far-fetched, but interesting. We haven't looked, and I agree it would be a very nice experiment to do. Yeah, it's, we just need to do it. Next question, please introduce yourself. Hi, Clemens. Great talk, congratulations. This is Hong Xiaoran from Indiana University. I have a question regarding the permeability. You have demonstrated very well the medial eminence permeability, or the alteration of the chemical compositions of oligodendrocytes in regarding to incretin, for example, GLP-1 and GIP accessibility to the brain. You have done excellent work about the amino acids function in the satiety and the feeding behavior regulation before. Have you looked at the permeability of essential nutrients, for example, amino acid or other metabolites? Thank you. No, we haven't, but I think these nutrients enter the brain. I mean, how the regulation of entry of amino acids is regulated is probably very different, because there are much smaller molecules, and there are specific transporters. So I don't know if this applies to small nutrients. I guess we could look. We haven't done it. Next question. Mike Schwartz, Seattle. It's a very interesting story, and very interesting to see oligodendrocytes woven into the CMS control of metabolism in this way. My question is, can you speculate about how myelination of neurons that penetrate into the median eminence is regulating the permeability barrier? I'm not really following exactly how that would work. We're not sure either, but there are a number of possibilities. One would be that although myelin is primarily known to insulate axons, we know that myelin compactness can affect permeability and diffusion in a structure, in the accessory matrix. So it's been described that mechanical properties of myelin might give it some barrier function. The other possibility is that the formation of neoligodendrocytes regulates other local processes. For example, the production of other extracellular matrix. Parallelness, and this might be indirect. Yes, so it could be an indirect mechanism. Yeah. The next question, please. Hi, I'm Nicolas Varas from Indiana University. My question is regarding other factors. Is there any other factors or hormones that could be used to protect this barrier apart from GAP, either internal from the brain or circulating factor that could be used to enhance or protect this barrier? Yes, we're actually looking into that right now because it's likely that other molecules might be able to restore plasticity in obesity. So what's really striking is that in dieting of obese mice, the barrier is impaired and plasticity of neoligodendrocytes is impaired. So we're looking at strategies that might restore plasticity and differentiation and survival of neoligodendrocytes. Thank you. By extension of that question, does the plasticity change over age? It does, yes, as well, yeah. Goes up, goes down? It goes down, yeah. Okay, that's why we can't eat that much when we get older. Okay. Any more questions? Thank you very much. Thank you. Very nice presentation. Thank you. So the next speaker in the symposium is Dr. Shweta Urba. I hope I'm pronouncing that correctly. Is that correct? Yeah, close. Yeah, very close. Okay, good enough. The title, Dr. Shweta, Dr. Urba has received her undergraduate training in Mumbai, India and her graduate training in Buffalo at the State University System of New York and is currently working with Eli Lilly and her title of her presentation is Tripling Gritten Agonists, Ready for Prime Time. Thank you. Okay. Thank you. I'm very honored to be here and present on behalf of my clinical team at Eli Lilly. I am a PKPD and pharmacometric scientist and these are my disclosures. I was told to stop here for a second to allow anyone who wants to scan to scan. Okay. So I present this work on behalf of all my non-clinical and clinical team members who are presented here. So I don't think I can describe the incretins any more or any better than Dr. DeLazio has already done and I would like to spend more time on the clinical aspects of this molecule. So basically, why do we need a multifunctional incretin agonist? Haven't we had enough of GLP-1 receptor agonist, dual receptor agonist? So why are we targeting all three molecules or what is the intent? So the basic premise here is that there is increasing incidence of obesity. We want better drugs that help in body weight loss control, help improve metabolic health. So we want to make the best of what all these three incretin pathways can offer us. Of course, like I just said, that there are GLP-1 receptor agonists that are already approved and they help in regulating appetite, have a role in increasing insulin secretion as well as sensitivity, play a role in delaying gastric emptying. When it comes to GYP, it doesn't really decrease or delay gastric emptying but may have a role in appetite regulation, improved insulin sensitivity, as well as glucagon secretion. Now these aspects of GYP and GLP, along with glucagon, which has been shown to increase energy expenditure at least in rodent models, increase insulin secretion, delay gastric emptying, there is potential that when you combine all these aspects, favorable aspects, in the right proportion, there is a potential to lead to greater weight loss and glucose control and that is the potential we are after. So what are we talking about? We are talking about LY3437943 and I will henceforth just call it LY to save two minutes of my talk time. So we already have a dual GYP or GYP and GLP receptor agonist to zepatite now approved. It has been shown to increase body weight loss and glucose lowering and the papers are published in all the reputed journals. But what do we need to do more? So the potential for LY is have a single peptide, which is based on a GYP backbone, have the triple agonist activity for GLP1 receptor and glucagon added to this GYP backbone. Now this peptide is then linked to a C20 diacid acylated peptide. The expectation is that based on this engineering, it will have high albumin binding, thus be safe from the FCR and salvage and then enable once weekly dosing. The GYP receptor activity in a molecule is expected to be seven times more potent when compared to the native GYP ligand, while the GLP and glucagon activities on this peptide are expected to be more balanced. Okay, so first what was done by our non-clinical folks was they looked at it in a in vitro system. These are HEC293 clonal cells and this pretty much presents what I said on the last slide is the activity is more balanced or more like the native for the GLP1 receptor and the glucagon receptor, while as it is more potent compared to the GYP receptor. Next, what they did was a series of non-clinical experiments in the left panel, what is shown is LY is dosed across a very wide dose range from 0.3 all the way up to 30 nanomolar per kg. And what you can see is dose dependent increases in weight loss. In the middle panel, you can see how LY reduces food intake again in a dose dependent manner. And most importantly, as a glucagon containing, I'm sorry, the mouse is too sensitive. Glucagon containing agonists, it also increases energy expenditure. We believe this is due to the glucagon involvement. Of course, we have to study this in true clinical studies when the point is right for our program. But this is what we have based on the non-clinical data. Then a series of experiments were done in the RAD IV GTD model. And what we saw was LY did stimulate insulin secretion and it also lowered fasting glucose and improved insulin sensitivity after chronic treatment. Next, of course, we are talking about all of this in comparison with things that already exist, right? There are GLP receptor monoagonists, there are dual agonists. So of course, in the non-clinical model, we then compared our LY to liraglutide and other GLP-1 glucagon coagonists or to zepatide, which is our dual GYP-GLYP receptor agonist. And what we see is, in red, you see that the most amount of weight loss in this left panel is seen with our triple agonist LY. Also, anytime we talk about weight, we wanna make sure that we're not losing too much lean mass compared to what is seen. Okay, I guess I'm showing you my entire presentation before I get there. So what is seen is that it is the fat mass that is lost and not the lean mass in a disproportionate manner. So this is, again, a test we wanna do before we take the molecule any further. So after we had all this non-clinical data, we did our first study. This was done in Singapore. It was in healthy participants. It was a single ascending-dose study. And most of the participants were males. There were two females in the study. And these people, participants, had HbA1c in the normal range. So all we are looking predominantly is for safety tolerability and the PK to be well-behaved. And we did see that. In line with expectation with other incretins, what we saw was that all the way up to 3mg LY was very well-tolerated with increasing dose levels, especially at the highest two dose levels of 4.5 and 6mg. We did see more GI events, nausea, vomiting, abdominal distension, but nothing that was severe in incidence. And most of them resolved on their own without additional treatment. There were low incidence of hypoglycemia events, and there were no injection site reactions or ALT-AST elevations or deaths in any of the LY-treated groups. Now, based on this study, we, of course, also wanted to make sure that we did indeed have a once-weekly molecule, and that is what this panel shows. Basically, we have dose-proportional increases in exposure, Cmax, and AUC after dosing it all the way from 0.1 to 6mg. And the Half-Life did support once-weekly dosing, and peak exposures were attained mostly within a day of dosing. So the left panel here, what it shows you is a dose-dependent increase in body weight loss. From 0.1 to 6mg, what you see is max weight loss of about 3.5 kgs in healthy participants after a single dose, and this weight loss was maintained all the way up to day 43. Again, a reminder that this is a single-dose study, and this is what was seen in these participants. On the right panel, you see appetite index. Highest score signifies lower appetite. So what you can see is, in a dose-dependent way, almost, you can see that soon after dosing, that is around the time of peak exposures, you have the highest values of the VASCOs that shows the appetite regulation, or lower appetite following administration of LY. So, of course, the whole point of doing the SAD study was to get to do the more relevant MAD study in the population of interest. These are type 2 patients. We had five cohorts. The first three were what we call a fixed-dose escalation, that is you gave 0.5, 1.5, or 3mg once weekly for 12 weeks. At the higher two cohorts, what we employed was a dose-escalation scheme where we started at 3mg for four weeks, followed by 6mg. In the last cohort, we dosed 3mg for two weeks, then 6mg for the next two, 9mg for the next four, and finally 12mg. Of course, it might come across that we dosed only up to 6mg in the SAD, but this is well understood now with other molecules, including terzapatide, that you can increase doses beyond what you have studied in the SAD as long as you do a stepwise escalation and you follow the right steps. Of course, it is a little bit of trial and error. There are understandings based on gastric emptying delay, everything that we employ into designing these studies. This study was conducted in the US. It was conducted at four sites. All the patients were on metformin. And unfortunately, the starting of the study was during COVID times, so when I present some of the data, the first two dose levels of 0.5 and 1.5mg had to be discontinued early on because this was in March. We didn't quite know what to do with those patients at the site, so there was a discussion with the investigators and it was thought best to avoid the patients from having to have repeated on-site visits. So if you see any gaps in the data, it is mentioned in footnotes why that's the case. But overall, irrespective of COVID-related discontinuations or decisions to discontinue the patients, what we saw was that overall, there were very few severe or serious adverse events. None of the serious adverse events were related to LY. I can list a few. There was a clavicle fracture. There was a motor accident. So these were the severe events unrelated to LY, placebo, or dulaglutide. And the treatment-related TEAEs, you can see that it increases in a dose-proportional way. There were no hypoglycemia events or injection site-related TEAEs. There were no deaths in any LY-treated group and no significant alterations in lab values. In line with all incredence, what is of most interest is the GI events. We did see them as being the most frequently reported treatment-emergent events. They were mostly of mild severity, most resolved on their own, and sometimes they resolved at a median time of about 10 days after the first initiation of the event. So in the left panel, what you see here is the effect of this incretin LY, triple agonist on blood pressure. We do see decreases in blood pressure as seen with other incretins. On the right panel, you see some increases in pulse rate. I would ask all of us to be mindful of when we look at these increases in relation to what we see in phase three studies because it's well-known with this LY and other agents that in early studies, in ClinFarm kind of studies, with additional, more monitoring than phase two or phase three, it is not very unexpected to see higher pulse rate increases, which then hopefully should show tachyphylaxis. I cannot at this point comment of it will or will not, but this is the expectation based on behavior and consistency seen with incretins across the platform. These are our efficacy results. What we see is the HbA1c decrease up to 1.9% and up to 1.6% at the highest dose group. Pretty much, we see HbA1c decreases that are, I think, profound at all dose levels, about 3mg within a 12-week period. This is the OGTT results. You can see consistent with incretin effect, there is augmentation of insulin response. Of course, OGTT was performed at multiple times throughout the 12 weeks, but what we are showing here is only the one at the endpoint. These are the efficacy results for the impact of our LY on body weight. What we see is that with increasing dose levels, there is a dose-dependent decrease in body weight, especially at all dose levels above 3mg. There was also an increase in appetite score, similar to what was shown non-clinically. The reason I have not presented here is it was, in some ways, very comparable to dulaglutide, so we speculate that it's possible that this is because of increased energy expenditure, but of course, we have not done that study, and in due time, when appropriate, we would think of doing such a study for this molecule. We did see body weight decreases all the way up to almost 8.5 kilograms. In summary, we now have a multifunctional incretin peptide, LY3437943, which is a potent agonist at the human GIP, GLP-1, and glucagon receptor. It has been shown to reduce food intake and increase energy expenditure in non-clinical models, and also improve insulin sensitivity. The phase one clinical studies, tolerability events were nothing different from what we expected based on our understanding of what is seen with the incretin class. The PK, the molecule supports a half-life of about five to six days, supports once-weekly dosing. In terms of PD, the glycemic efficacy we saw decreases up to 1.9% within a 12-week study in HbA1c, and for body weight, we saw about a 10% body weight decrease. Of course, these are only the MAD results. It is a 12-week study. While it is profound, we are currently waiting on results of our type two study in phase two, and we hope to present that in due time. Thank you. Thank you very much. Paper's open for questions. I'll start with a question. So could you possibly explain to us what the rationale is to have balanced GLP-1 and Leukogon signaling, but a much more potent GIP? So the short answer is I don't know. The long answer is many ratios have been tried, some of them empirically based on the vast experience that our non-clinical folks have. So I would say they try to preserve what we do know and what has worked with the dual agonist while trying to change one thing at a time, if I may. Okay, and then another question that I have, and then I'll let everybody else. Has anybody measured what the endogenous hormones are doing, like Leukogon, GIP, GLP-1? Like how do they look like? So I know the Leukogon levels, there is some decrease, but I cannot comment on each of them. If it is, I won't say they're not measured, but in the scheme of things, they don't play out to be something that we look at it and say, okay, now what do we do with this? I think it's come down to that. But Leukogon levels, I can definitely answer, we have looked at it, it's decreased. Hi, I'm Tahir Modarassi. I'm with Hamilton Cardiology Associates. I was just intrigued by your 12-week data with the weight loss. I know there's other compounds that are just in the dual, you know, GLP-1 Leukogon, including Lily's masdutide. Seems like some of that data from that simple dual agonism is even more potent. And I was just surprised given the, what you'd mentioned about the GIP agonism being even more than from terzapatide. So is there something that comes to your mind in terms of why, even though you're adding additional agonism, you may not be getting the same effects compared to just a straightforward GLP-1 glucagon agonist? Yeah, so if you're referring to the ADA data that was just disclosed for the GLP-1 glucagon, yes, numerically, this one looks not as equal as that. But I would say it's a little sometimes, I would say it's not unfair of you to compare, but it is not possible for us to do all the studies simultaneously at the same time. But to simply answer your question, not all of these things likely can move ahead. So it depends on what looks best and sometimes might be simplest, better, and just a dual agonist is what's needed, might be just terzapatide. But I think there are many other factors other than just glucose control. And we hope that there is greater weight loss as we have seen in this 12-week study and other factors that can help defend which one has to go ahead. Next question. Hi, Shweta, very exciting talk. Thank you, this is Hong Xiaoran from Indiana University. Two quick questions. One is, when you do your PK studies in rodent models, perhaps, have you looked at the brain enrichment of terzapatide and your LY compound? I'm sorry, this looks like I missed a key word. Which measurements? Brain enrichment of your compound. So there will be C14 studies that will be published in due time, at least in the non-clinical space, and I think that should be able to answer your questions. But it's something we are interested in. Okay, have you looked at the hepatic glucose production function, specifically with your LY compound? That's not something that I'm aware of, at least in the clinical studies. Okay, thank you. Okay, thank you very much. Thank you. Thank you. He received his training in Costa Rica and fellowship training at Hammersmith Hospital and at the University of Texas Southwest Medical Center. His title is Emerging Dual Peptide Enrichment and Receptor Agonists. How far have we gone? So that was very interesting, the triple G. That is just in the infancy, but I'm gonna go back and go back to the dual peptides, which sound very promising. And we're gonna go move in from the mice data to the human data and show you real studies that show where do we stand these days and how far we have come with it to create this. So we really are now in a position that we're moving the needle beyond the GLP-1 receptor agonist therapy. This is semi-disclosure, I've been fortunate to be involved in many of these trials and I'm gonna give you an overview of where do we stand these days with the dual peptides. So in terms of looking at the GLP-1 receptor agonist, the question is how far have we come with the GLP-1 receptor agonist? And if we looked at the head-to-head trials trying to differentiate A1C and the weight they're lowering capacity, you see multiple trials there and there's certainly the weekly GLP-1 receptor agonist are doing better than the daily ones. And if we looked at specifically in the ones that we compare the laglutide with semaglutide, clearly semaglutide tend to be, have a much greater A1C reduction. And also when we looked at the head-to-head comparisons assessing the weight loss, again semaglutide appears to have a better weight lowering the potency. This one thing that we learned looking at all these different trials is that there is a dose response effect. And as here when dulaglutide locate less weight the lowering effect, so it came a study where they say, well, we're gonna push the dose of dulaglutide instead of going 1.5 milligrams, we're gonna go up to three and 4.5 milligrams. And indeed, when that was done, the A1C lowering was greater and the body weight loss was greater. That was for dulaglutide. And so if that was good for dulaglutide, so it should be good also for semaglutide. And that's exactly what was done in this study where the maximum dose of semaglutide, one milligram, was pushed to two milligrams, and then you get there a little bit better A1C reduction, as you can see, a 2.2% A1C reduction that was pretty robust. And the weight loss was certainly enhanced by doubling the dose. Doubling the dose was good enough, and if you go even higher up with semaglutide to 2.4 milligrams, then there's this step program with semaglutide that was very successful for obesity in general, and the step two, for instance, where there was people with type two diabetes, you can appreciate there that semaglutide at 2.4 milligrams had a very robust weight loss there, and also had significant improvement of the A1C levels. So this is what we have accomplished so far with the GLP-1 receptor agonist. The question is, can we do better? And in that respect, well, before I go into that, I mean, there are also things that we know very well with the GLP-1 receptor agonist, is that when you looked at this latest meta-analysis by Navid Sattar, where they added the amplitude, oh, it was with a fake lenatide, you can see that there's a 14% reduction. So here's the GLP-1 receptor agonist, highly successful in terms of weight-lowering capacity, especially when you go to the higher dosages with semaglutide, you have a very robust A1C reductions, and again, what I said before, can we do better? And that's what was done with the dual peptides. And the rationale for that, when we looked at this is, question is, which one is gonna be the best partner? Which is gonna be the best compadre, as they say there, for the GLP-1 receptor agonist? And you have all the multiple peptides that you can choose from. And obviously, there is plenty of work for David D'Alicio to play with all these peptides to see what the mice are gonna behave with the different combinations there. But the ones that I'm actually gonna refer specifically is the amylin, the GIP, and the glucagon, where there's more data and there's more advance in terms of clinical trials. When we looked at what is the clinical evidence of the dual peptide there, the one that comes first, where there's been plenty of different data, is the dual peptide with the glucagon. Here we have one molecule that there is some emerging data that is very consistent there, is the Cotadutai from AstraZeneca. Here is the rationale for the glucagon. I'm not gonna repeat what David D'Alicio mentioned, but certainly glucagon has effect in the liver. This drug has important reductions in terms of reducing fibrosis inflammation in the liver and removing fat from the liver. That's one of the rationales that the Cotadutai was being developed for the purpose and also has effect on satiety and other aspects. So this is the data that was presented. In people with type 2 diabetes over 14 weeks, you can appreciate that the combination of the GLP-1 with the glucagon had a 5% reduction in body weight. They have also a A1C reduction of around 1.2% of the 14 weeks a pretty robust data in that respect. A percentage of people getting to the target of less than 7% with the dual peptide and also a reduction of 6.5%, a target of 6.5% with a reduction of the A1C over 14 weeks period. This was the cotaduotide. There is another dual peptide that was presented last week at the American Diabetes Association of Alt-Immune where the combination of the GLP-1 with the glucagon here, the ratio was one to one as I've mentioned before. One of the key issues in terms of these dual peptides is what is the ratio of the glucagon that is very critical. Here they have a one to one, pretty high ratio of the GLP-1 and this is what they hypothesized. This data was presented by Sam Klein there and where they basically insisting in that you are gonna have a much greater potency by this ratio. And so what they did is a very small trial. It's also a phase one trial. There was a different dosages there. One of the things that I was claiming is that this compound is gonna have less need for titration and better tolerance. This is certainly something that is to be demonstrated in the future but what this study show is a very robust reduction on weight loss. You can appreciate 10% reduction of body weight with this ratio of GLP-1 and glucagon over a very short period of time of 12 weeks and also you can appreciate that almost 90 to 100% of people lost 5% in a substantial number of people also 50% reduce 10% of body weight. Here's another interesting compound that was presented also the American Diabetes Association last week is oxyntomodulin or even has a name, Mazdutai from Eli Lilly. This is an acetylated peptide, the analog to oxyntomodulin that has a glucagon and GLP-1 receptor dual agonist and is done, the acetylated is to prolong the half-life and can be given once weekly. They did a dose range study there. Initially with the lower dosages and then they have another cohort with higher dosages and the data was very impressive. As you can see there, the A1C reduction with oxyntomodulin was 2.2% and 1.9% there and in terms of fasting plasma glucose also had a very robust reduction of glucose. Body weight also was over a very short period of time at almost 13% body weight loss and here also in waist circumference. So this is another molecule that has a potential to probably is gonna move on to a phase two B study before they decide or not to pursue the phase three study. Amylin is another interesting peptide. We know of amylin because there is available in terms of the promlytide that are all very familiar that is accepted for the treatment of type one diabetes but is not used that much because of the problems of hypoglycemia. Amylin is co-secreted by the beta cell with insulin. It has a robust effect in terms of slowing gastric emptying, reducing glucagon and also doing some satiety and what is interesting is that amylin or promlytide was actually used several years ago when it was tested in obese patients and as you can see given three times a day it had some potential in terms of having weight loss but obviously given three shots a day is not an ideal or feasible type of treatment for obesity but kagrelentide is an analog of amylin developed by Novo and what they did is a long-acting lipidated albumin binding stable amylin that allows for a weekly administration of the amylin and they call it kagrelentide. They actually did a very good study that was published in the Lancet in a dose finding study phase two trial. As you can appreciate they tested several dosages of kagrelentide and had very robust reductions of body weight and as you can appreciate that even at week 26 the weight loss does not seem to level off. So it appears that you may have continuous weight loss and you can see there the percentage of people who lost weight even up to 10 or 10, 11%. Here's the body weight loss targets. A substantial number of patients lost the 5% body weight loss which is what the regulators require for approval for obesity but here up to 50% of patients lost 10% and a quarter of a patient lost 50% which is also very robust results. This is with the long-acting amylin compound. Now what is being explored also is the combination of kagrelentide with imaglutide or kagresema that also had a very positive study published in Lancet also. This is a phase one B trial. Despite being such an early study got published in the Lancet and as you can see there was also different cohorts in those ascending fashion especially because you have to be careful with the progressive titration of imaglutide there and as you can appreciate also you have a very robust weight loss with a combination of the amylin kagrelentide there with imaglutide as I can appreciate it up to 17% weight loss there and of course this is relatively short term and the potential that more prolonged study may have even greater robust, greater weight loss. Here you can appreciate between the two studies one in obese patients, the other in type two, I'm sorry, in obese patients, the combination with kagresema or kagre alone. And so just then in the last five minutes just to review some of the data that you know very well, tercepatide from Lilly, that is the combination of the GLP-1 and GIP and I'm not gonna go over this, that is very well known. Suffice it to say that the GLP-1 receptor agonist has these multiple aspects and we already discussed the potential effect of GIP with that beautiful presentation of the effect of the CNS in terms of enhancing the satiety and different aspects that the GIP can have. But the important point is the comprehensive program for the development of tercepatide. This is the studies that have been published for SORPAS type two diabetes that was recently approved in the United States and awaiting approval in Europe. And there is also a program for obesity that is called CERMONT and there is also a program for NASH that is called CINEGY. And the tercepatide was tested in parallel fashion with progressive increments of the titration by 2.5 milligrams to enhance tolerance. And as you can see in this parallel studies, randomized control parallel studies, it tested five, 10, and 15 milligrams, testing for inferiority or superiority depending on what the control is. The data was very impressive. But I'm gonna focus in one study that we reported because I think that the real potential for this kind of compounds is to try to change the paradigm of the management of type two diabetes. Of course, this tercepatide has been tested throughout the whole range of type two diabetes at all stages of diabetes. But I wanna focus on the early treatment of type two diabetes. In this trial where we gave tercepatide in patients with early type two diabetes, four years of diabetes, obese, treatment naive, you get the very robust reductions of the A1C. So robust that you can actually get down to normal A1C levels. And that is also at the expense of getting a substantial number of patients reaching target of 7%, less than 7%, 80% getting less than 6.5%. And what was remarkable is that half of the patients actually get an A1C of less than 5.7% which is unprecedented in the management of type two diabetes. Never before we even dreamed to have patients getting A1Cs less than 6% or 5.7%. And what is important is that this also in combination with robust weight loss as you are all very familiar, 10, almost 10 kilograms weight loss, that was not a fluke. It was actually consistent throughout the whole program having anywhere from 10 to 12 or 13% body weight loss. And what is important is that when you looked at the A1C changes, and as I mentioned before, you have three arms, five, 10, and 15 milligrams. All arms are significantly positive in terms of reduction of the A1C. But if you looked at the baseline A1C in the top dose of 15 milligrams, it is clear that all terceptive five milligrams arms got A1Cs below 6% which is again unparalleled in terms of accomplishment of glucose control. And what is important also is that this target of less than 5.7% was achieving 40 to 60%. So there is the potential there to literally get people to normal glycemia and revert type two diabetes. And if you are fond of a timing range, you can see for instance in the SURPASS-III that was a sub-study where they was doing CGM. You can see there that the goal is to have a timing range of 70% which correlates with an A1C of 7%. So this was tested versus dulaglutide. Dulaglutide is what was at baseline. And you can see that they're giving a treat to target with dulaglutide, the baseline basal insulin, you get that down to 75%. Nice improvement with basal insulin. But even with five milligrams of terceptive, you get even better people get into time to range. And this is what you get with 10 and 15 milligrams. Substantial number of patients increased to timing range there. And the other important point where these drugs have a tremendous potential in the treatment of NASH, there's a sub-study where there was done MRI-PDFF which is a protein density fat fraction that very specifically measured the amount of fat in the liver. This is a patient that even was with five milligrams. This is the amount of fat that you can see in the liver. This is what happens after the patient have lost weight. And as I can say that one picture is worth a thousand words and you can see the removal of almost all the fat from the liver. The other important thing is what was published last week which is part of the Sermont trial. This was in obesity. That there was a very impressive reduction of body weight of 21% there. That was a really wow kind of result in terms of reduction of weight. Whether you do it by the treatment regime estimate and the efficacy estimate where you actually remove patients that actually when you take the analysis only on patients that are being treated, you can see that up to 22% of body weight loss was achieved. And what is really impressive in this obesity trial is that 41% of those patients obese had prediabetes and 95% of those patients reverted to normal. So here's a tool that can be used much earlier in terms of not just reversing diabetes, also preventing diabetes. And the last word that I just want to say is that treating type two diabetes and taking care of people with diabetes for more than 30 years, we always wanted to have a tool that can improve diabetes such to achieve normal glycemia, to try to revert diabetes or even prevent diabetes. And what I said that there's hepatitis in the emerging obesity medication that can attain weight loss similar to bariatric surgery, I believe that will move the goalposts for type two diabetes management toward attaining diabetes reversal or remission. And that might not be just the impossible dream that we also all thought that it was impossible to get. And now I think it's feasible. Thank you very much. Thank you. Thank you very much. I'm open for questions. So, I have a question. What is known about the relationship between energy expenditure in these subjects versus food intake? We really don't know. I think that certainly there is a reduction of food intake. I mean, the cessation of the mitigation of the, I would say that the main effect probably is in satiety. And certainly there is a reduction of caloric intake. One of the basis to use glucagon is that it is also thought that that will increase energy expenditure. I mean, the base model is what nature gave us. Glucagonoma patients are totally emaciated in terms of that. More questions? Vincent Fong from University of Cincinnati. So, for the comparison, I mean, you gave a very nice comparison of all the different agents at the beginning. Is there a comparison in terms of relative receptor activity or affinity? Is there differences due to just different levels of agonism for each of the receptors? Or is there actually something else about the molecules? Or, you know, is three times of one and two times of the other, there's different types of synergism between like the relative levels of agonism? Or is that part just happened to be whatever the drug company gives us? The truth is that there is difference in potency of the GLP-1 receptor agonist. It could be related to the molecule. It could be related to whether or not you get, for instance, one of the things that is said that's a maglutide may have a better entrance in the median eminence because of the fatty acid and so on and so forth. It's also, there's a dose response. I mean, we see the dose response that there is like a floor for the A1C, but in terms of weight loss, there is a dose response. And that's why some of these companies are pushing the dose to the max. And the problem that a limiting factor is the GI side effects. But the GI side effects tend to be more at the beginning of even with the lower dosages. And then that tends to subside. Next question. Sure. Thank you. It's Carlos from Colombia. My question is there is a systematic effect on A1C for most of the agents, but then there is a large degree of heterogeneity in terms of weight loss. Especially if you think about albiglutide or dulaglutide, which are tremendously effective in A1C, but not so much in terms of weight loss. And it's related to the last question. What could be the biological substrate for such a large heterogeneity? It's just huge. And it's not just about dose. It's something else. No, it is true. I mean, in our experience and everybody experienced, whether in clinical practice or in clinical trials, there is a big heterogeneity in terms of response. You have wonderful response in some patients and some patients do not respond that well. We don't know if it's because of lack of response or tolerance or whether or not the patients are really taking the medication. But certainly there are differences there. But there are differences also in the molecular, with some of the molecules and also in the dose. But some patients respond differently. And I think that one of the points there, it may be related to whether or not the patients are taking the medication or not. So we've run over time. I need to close the session. One more question and then we'll finish. Yeah. Hi, Allison Alvear, University of Minnesota. And I saw the one slide that showed like after the study was over that the weight went back up very quickly. More quickly than it had come down. And I'm wondering just your thoughts about that. And then, you know, considering the situation in our country where it's nearly impossible to keep most people on any drug continuously without, you know, lapses in insurance and pharmacy problems, etc. Like, what are your thoughts long term about that? The fact that all these trials for regulatory purposes, you need to stop the medication because the regulators want to see the effect on and off the drug. And in most of those patients, we're going to revert and have a regain of the weight. Last year, the ADA had a consensus or an agreement in terms of the definition of diabetes remission. And they defined diabetes remission as maintaining an A1C less than 6.5% after stopping the medication for three months. That is a definition maybe for when you are doing a clinical trial. But in terms of reality, if we, if the aspiration is to have diabetes remission, we cannot stop these medications. People are going to get a regain weight. We just recently published the step 8 trial extension where semaglutide 2.4 milligrams was compared with liraglutide 3 milligrams, both obesity drugs. The semaglutide 2.4 milligrams did better than the liraglutide 3 milligrams. But when we stopped the medications, the weight loss went down. The only thing is that those who lost 20% of the body weight after one year still have a 5% body weight loss, which is not bad. But these drugs need to, the next step is for these sponsors do studies in terms of maintenance. Is maintenance going to be a lower dose? Is maintenance going to be an intermittent dose or holidays and so on? But that's the question there. We're going to close now. You can discuss later on. Let's thank the speakers again.

brain plasticity

obesity

metabolic diseases

oligodendrocytes

myelin

GPCR signaling

permeability barrier

therapeutic agents

dual peptide therapies

GLP-1

clinical trial data

weight loss

type 2 diabetes

long-term treatment