Scientist Stories: Alan Saghatelian, Small Molecules in Heath and Disease

Thank you, Rebecca, and welcome everybody. I see a lot of friendly, familiar faces, and I see a few new faces. So it's nice to officially kick off the school year here at Salk with this special nighttime event. So I want to give just a few words. I could, you know, certainly if I had another Louis Kahn out there, I could say more than a few words about our speaker, but I Let me just say a few words about Alan Sagetilian. So I first met Alan a couple of years ago when I had the good fortune to be invited to a special small meeting that's held annually on cancer, which is a relationship between Harvard Medical School and a city in Italy known as Trento, where there is a historical connection between the universities on certain cancer topics. That particular year, the topic was on cancer metabolism, and there was a relatively young man, younger than I, who was a rising star in the field. And I knew something about this young man because he in fact had gotten his PhD at Scripps Research Institute, a phenomenal chemistry and chemical biology institute just up the street from Salk. And he had been one of the first STAR postdocs out of one of the young faculty there's lab, a man named Ben Cravatt was his postdoc advisor, who's one of the leaders of the topic of chemical biology and particularly chemistry and metabolism. And so Alan had left, had gotten his PhD and postdoc and gone on to Harvard. What I did not know was that Alan was actually a native of Southern California. And in fact had gone to UCLA as an undergrad like all good native Southern Californians should, other than UCSD, of course. Or USC. Let me actually just stop. You can tell I'm not from Southern California originally, so I'll just stop there and say, so, you know, we should all be proud of Alan. He is a native son of Southern California, but Alan got up there, was the youngest person on this slate of esteemed speakers. And blew the room away with the depth of what he has done. He can only touch on a fraction of that today, but after he finished talking, like partway in, I thought, you know, we've really been trying to recruit a senior faculty in this space of metabolism and cancer and metabolism and diabetes. And I thought the problem is like we're always looking to a 70-year-old person who is the department chair of this and This guy is like a superstar, but he's younger than most of the people on the faculty, and that's going to be a challenge. Because how are we going to sell that this young man actually has the depth of experience? But then after I saw him talk, I thought, no, he can sell it. He can be the one to do it. So there's only one challenge left. I was like, this is all fantastic if I could encourage him to move back to Southern California. The only problem is it looks like he's actually a professor at Harvard. So let's see what we can do. So I'll leave the rest of the story to many other drinks, or maybe just to myth and mythology. But suffice to say, we have the incredible fortune to have been able to convince Alan to move back to his native Southern California and to grace us. His discovery this past week is just emblematic of the kind of research. He's made many discoveries to get to this point. As Rebecca said, the Salk has only allowed— in the past year, 10 years, we've only ever hired one person with tenure as a senior fully tenured faculty, and that is Alan. In the decade before that, they maybe did two in the 10 years before that. So this is almost never done and has to be someone of that extreme caliber. So I'm honored to have him here. I don't want to build him up too much, but he is going to No pressure, Alan. No pressure at all. He's going to tell us about small molecules and metabolism and disease and how to fight it. Thank you.

Great. So first of all, I'd like to start by thanking you all for coming out. I used to teach a lot, and those kids all had to be there. To get people to come out on a weeknight and actually show up by their own accord is actually saying a lot, and I'm really humbled that you all would come out to see this. Second, I'd also really like to take a moment to just thank Reuben, because really it was his effort to spearhead bringing me out here. And I've been here since July, and it's just been wonderful. And we're already talking about starting projects together. So I'm looking forward to— not to make this sound like too much of a bromance growing old together, but someday soon. —back on to the science. So my goal today is to tell you about one project in the lab that we're focusing on that has to do with small molecules. But I think the best way to do that is I've actually broken the talk down into kind of 3 discrete areas. The first is that I'll tell you a little bit about the disease that we're working on and give you a little bit of the background so that you can have some understanding of really what it is and what area of that disease we actually have made an impact on. The second part, I'll then come back and tell you a little bit about what the lab does specifically. So every research lab in science does something unique or tries to do something unique. If we were all doing the same thing, it'd be a little bit of a waste of time and money. And as a result, there's different ways to go at it. Some people study different biological problems. Some people study cancer. Some people study diabetes. And there's also others of us that develop technologies in many ways. Majority of what we've done up until this point has really been developing new techniques, as Ruben kind of mentioned, to analyze a class of molecules that are important. We know they're important, but we really haven't had the ability to measure and detect them in biological samples. And so I'll describe how we do that. And then in the last part, what I'll do is I'll bring those two together by essentially outlining how we can take that technology and apply it and how it can reveal something new and something that we're we're very excited about. So the story today is gonna really focus in the area of diabetes. And as this line says here, diabetes is really a number of diseases that are related to having high blood sugar levels. Now, for short periods of time, having high blood sugar is not a bad thing. After we eat, for example, our blood sugar will spike, but then it goes back down. But over prolonged periods, this can cause a lot of problems. So some of the symptoms include, early on, frequent urination, for example. But then later, you really develop serious complications. So for example, you can have kidney failure. There's vision problems. And in some severe cases, you can even have such poor circulation that you'll need to amputate limbs and other things like that. And so really, the goal in diabetes research and diabetes treatment is to try and figure out ways to lower blood glucose levels, and that comes— or blood sugar levels. And that comes really through in research towards understanding what controls the levels of sugar and really trying to use that to try and control levels. So if any of you know people that have diabetes, you'll often find that they're pricking their fingers and measuring basically blood glucose levels using these, these tools called glucometers. And really the idea is there that if they can measure it, they can then do something about it. If their blood sugar is too high, stop eating, for example. Or if they're a type 1 diabetic, which I'll get into in a second, they'll need to take an insulin shot. OK, so as a disease and a problem, I'm going to show you some data that's, I would say, a little bit disheartening, but really exemplifies the problem that we're dealing with. So this is essentially— diabetic adults greater than 20 years old in 2004, and essentially darker here is, is bad. And so as you can see, in a matter of 5 years, so in 2009, you can see a wave coming through here. There's really a growing epidemic. And, you know, I've seen Ruben's accelerator talk actually where he, you know, mentions the fact that there are lifestyle changes you can make. For example, you can exercise more and improve your diet. But with this many people kind of moving forward with the disease, that's really not going to be enough. And plus, if we take into account the fact that most professions are now being fairly sedentary— I spend 8 to 10 hours a day behind a computer— this is only going to get worse and worse. Now one thing I want to mention that I'll bring up a little bit later is that right now there's a great correlation between diabetes and obesity. So if we look at the same pattern and we're looking at obese adults 20 years old or older, you can see that you get a very similar pattern here as people are essentially gaining weight or they're changing whatever it means to be an obese adult and the bar's moving lower. But the truth is that essentially the two are linked. And I'll try and get into that because that plays an important role in what we try to do, or at least what led us to our discovery, in trying to understand how basically fat and insulin resistance, or having high blood glucose levels, are connected. So we're talking about sugar, or the chemical name glucose here, metabolism. And really, when you talk about diabetes, you kind of vilify glucose, if you will, as something that's terrible and that you want to control it and lower it. But in reality, glucose is a molecule that we all need for everyday function of ourselves. So glucose is basically the fuel our cells use to basically make other molecules. So it's the material that is then converted into other molecules that make up the cell. It is also used to basically— the energy and the bonds of glucose is used to essentially fuel a cell. And so as a result, basically the body was really trying to maintain very steady glucose levels because your cells have to be bathed bathed in the right amount of glucose for them to function normally. You don't want, for example, glucose levels to drop too low. Cells would die and other things like that. But there's a challenge here. So it's very similar. You want to keep your blood oxygen levels about the same, but you're constantly breathing. With glucose, you get basically a very pulsatile system. So when you eat, or as scientists will call it, you're eating, you're going to have an influx of glucose into your system. So now your body has to deal with basically a very high amount of sugar coming in a very short amount of time. And then when you're not eating, which we call fasting, essentially your body has to deal with that fact that there's no sugar coming in from the diet, and it's got to figure out ways to basically either make glucose or take some of the glucose that was introduced here, store it, and then release it again here so that you can maintain that, that steady state level. So when we're eating, basically it's coming from food, and then when we were fasting, basically the liver plays a major role in making sugar and also releasing some of the sugar that it stored up while you were feeding here. Okay, so what I'd like to do now, before we can talk about diabetes and what's going wrong in diabetes, we really have to take a step back and understand what's going on in normal glucose metabolism. Metabolism, like when things are working fine. And so we're gonna take an example of a redheaded kid here who's eating an apple, and we're gonna walk through what's gonna happen in his body, first at the physiological level, then I'll introduce some of the molecules that are important players in this. So what happens as you eat, obviously food hits the stomach, and that's where it gets broken down, and it gets, gets broken down into different nutrients— carbohydrates, lipids, fats. And sugars are carbohydrates. And with sugars specifically, what they do is they leave your stomach, they enter the bloodstream. And when they hit the bloodstream, what they'll do is they'll target the pancreas. And the pancreas is somehow kind of— it evolved to be the major player, the regulator of kind of traffic here. And what it does is it receives the signal that there's sugar in your bloodstream and that we need to get a message out to the rest of the tissues that We're in a state of high, high sugar. So what it does is it releases a molecule, a hormone that I'll show you in one second called insulin. And insulin goes to the muscle, the liver, and the fat where it can actually stimulate glucose uptake. Now in the muscle, it can use that glucose for energy. It can do the same thing in the liver, but in the liver it can also store a lot of that glucose in the form of— that can be used later when you're fasting. And then the fat, oftentimes that that glucose is actually converted to fat and stored. Because fat can also be used under certain conditions. So when you exercise, some people say you're burning fat. Basically, it's the idea here is that fat is also an energy source, a way for us to store energy. So essentially, it's the body's way of dealing with having a meal. And it wants to put some of it away for later. And it wants to use some of it now for energy. So what's going on at the molecular level? So this is a molecule of glucose. And just not to get too simplistic, but basically each of these dots here corresponds to a carbon. And this is an oxygen and hydrogen. So this is what we refer to— the name carbohydrate comes from the fact that these things have carbon and hydrogen and oxygen in them. And when it hits the pancreas, it releases another molecule that's what's called the peptide insulin. And a peptide is nothing but a string of amino acids. Is the same thing that you would see in a protein but smaller. So while most proteins have maybe 150 amino acids or more, peptides simply are 50 or less, for example. Now, in the next few slides, we'll go through a little bit of what's happening when these molecules are released. And the way I want you to think about insulin is that insulin is going to be a key. And it's actually going to have a lock that we're going to call on the surface of cells that's called the insulin receptor. So this is a protein. And scientists give things funny names like receptors. But what it's going to do is it's going to bind insulin, and that's going to send a signal into the cell. And that signal is going to then regulate or control the movement of a protein that's called the glucose transporter. You could call it something else if you'd like. But basically, you'll see in a second that the job of this protein is to shuttle glucose from outside of the cell, which is shown here, to inside of the cell. Oops, sorry. Okay, so when insulin binds here, basically the key unlocks the door. Those glucose transporters move from inside the cell to the cell membrane, as it's called, or the cell surface. And what happens is at that point, the blood glucose— the glucose that's in the blood that's outside the cell can then travel through here and inside the cell where it can be used for energy, be converted or stored for, for later use, or be converted into fats or metabolized into other things. Okay, so that's a very basic model of how this whole thing is working, the key players that are going on in here. And now what I'd like to do is kind of talk about what happens in the case of diabetes. And the first place I'll start is with type 1 diabetes. So as I'll tell you, there's two major forms of diabetes. Type 1 diabetes and Type 2 diabetes. Type 1 diabetes makes up about 10% of all diabetes cases. And the two are fairly different in how they work. They both result in high blood glucose levels, but they're actually very different. So in Type 1 diabetes, what you have is called— it's an autoimmune disease. And so your own immune system that's normally there to fight off bacteria, fight off viruses, or what have you, for whatever reason recognizes attacks the cells that produce insulin in your pancreas as being formed, and it attacks them. And so basically, a person with type 1 diabetes has lost the cells in their body that are necessary for making insulin. So everything looks the same from the outside, but their pancreas just cannot make insulin. And so what happens in that situation is basically glucose comes in, but there's no insulin there to signal. And what ends up happening for from that is that basically the glucose doesn't have anywhere to go and you get high blood glucose levels. Now in some situations, in some sense, this is actually a fairly easy case to treat because what you can do now is if you're a type 1 diabetic, you haven't lost any of the machinery outside of just losing insulin. So if you want to, if you measure and you notice that your blood glucose levels are going higher, you inject yourself with a little bit of insulin From the outside, that comes in, it binds to this receptor, and your sugar starts getting taken up into cells. And so you essentially repair the system with an outside dose of the molecule. Now, type 2 diabetes is a little bit different, and I would say in some sense not as well understood mechanistically. But everything for the most part looks the same. Often it's tied with obesity, so there is a little bit more fat. Sometimes you get a little bit of fat in the liver. But what I'm going to introduce to you is a term that we refer to as insulin resistance, which means that somehow in type 2 diabetes, the tissues that normally respond to insulin have lost their sensitivity to it. And so what's going on here is you still have glucose signaling. You still have insulin signaling. It still binds to the receptor., but for whatever reason that key isn't turning anymore. That lock isn't opening up. And so you're having a problem and the glucose transporter isn't coming up to the cell surface at the levels that it's actually needed. And so again, you result in high blood glucose levels. So one thing you could be thinking is, okay, well, what we can do is we can, we can raise insulin levels if we'd like. But actually the problem with insulin resistance is that the body actually does this. It realizes that the glucose metabolism isn't working right. So in a type 2 diabetic, you actually find that they have higher insulin levels than in people with normal metabolism. So the body's really trying to do that. It's saying that this signal's not getting across. I'm going to turn this up even more and more. But the problem is it's the reception that's the problem. It's just the signal's not being received properly. And so as a result, dosing more insulin isn't really a viable solution in this case. And so people have really started to work, and scientists as a whole have tried to understand really what's behind this so that we can identify what's actually broken so we can try and fix it. OK, so for example, we'll focus now on— move from talking about type 1 and type 2 diabetes to specifically this insulin resistance here. And so somehow, we know it's linked with obesity. And this happens in both humans and rodents as well. So if we feed our mice a high-fat diet, they will also become insulin resistant as they gain weight. And so big questions are, what's going on and what's actually causing this? So one thing that has been found, for example, is that these molecules that are called fatty acids— so you've heard about these things. People argue about whether trans fatty acids are good or bad for you. They're essentially a class of molecules that look exactly like this. And one of these is a molecule called palmitic acid. It's probably in almost everything you eat with any fat in it— oil, butter, what have you. But if the levels of this get too high, it's known that basically for whatever reason— and again, the mechanisms aren't 100% clear— it'll cause insulin resistance. The thing about this that got me interested in this problem is that essentially it's dealing with two things that I'm going to refer to as small molecules. So these fatty acids here are what we refer to as metabolites. And so in the body, the carbon-based compounds that the body makes, uses for energy, or actually transfers around are called metabolites. And then these peptides like insulin are also considered as small molecules. And so one of the things that I'm gonna have to convince you of is basically the first question you're gonna ask is, aren't all small molecules small? And the simple answer to that is yes, of course. Compared to us, we can't see any of these molecules. We're, you know, this— I was doing the math and I was gonna do a funny analogy, but I don't really do very well with the spatial stuff, so I was thinking you guys didn't either. But, you know, that compared to a molecule would be basically like comparing one person to the size of the Earth. So we're talking about really big differences here. But within the group of molecules, we actually have a range of sizes. So you can have something like a chromosome, which is made up of DNA that is fairly large. You can see it under a microscope. You can have things like proteins, which can vary in size as well. And then you have peptides and metabolites, which are technically much smaller, both in size than these other counterparts. Now, for most of the history of molecular biology is really focused on proteins, DNA, and RNA. And as a result, have built a great set of tools to measure these molecules. And what we do when we measure these molecules is oftentimes we'll do comparisons. So we look at a disease model or disease human, and we ask what genes or what proteins are changing. But this has been done far less with peptides and metabolites because the tools just haven't been available as long. So the color's not showing up great, but this should be light blue and red here. But essentially, I would say if you look historically, probably 99% of all biological research is kind of published in this area, whereas 1%— and I'm just making these numbers up, but just roll with me here— focus on peptides and metabolites. —So my background, this is where it comes a little bit personal. I came to biology much more from a chemical background. As chemists, this is the type of stuff, as when I was an undergrad at UCLA, we would make. We would just buy some chemicals, we'd mix them together, and we would make things like this. And so I got very good at basically being able to analyze these types of molecules. And then in grad school, I started learning how to analyze those molecules. So one of the things is that the idea was that, OK, so there's these tools that chemists use to basically be able to study and make these molecules. How about starting to bring those over into biology? And so the first question I had to ask when we were getting into this was that if you bring a tool, you want to make sure that there's a lot of room to learn things and a lot of opportunity to make new discoveries. And so one of the questions— I asked, and maybe you guys are asking right now, is really how many metabolites are there in the human body? And I will tell you that this number is probably the least confident number I'll show you in a while. So this is— and I don't expect you to read any of this. I'll read it for you. This is something called the Human Metabolite Database. It's maybe been set up 10 years. This is version 1, version 2, and version 3. And from version 1, they go from 2,000 to 6,000 to about 37,000. I'm not really sure where they're getting all those molecules from to throw in there. And I would say that this is probably on the high side because we only have about 24,000 genes. So I doubt that we have more metabolites in our body than genes. But I would put the number based on the types of experiments we do at about 10,000 molecules. Now, the thing about it is we don't know what all of these things are. We don't have tools to go in and just basically comprehensively tell us what everything is in a cellular tissue. And so really what we have to rely on is developing tools to do that and also developing tools that can identify the ones that might be the most interesting things to look at in the first place. So the main question we come out with now is, let's say we're on the order of thousands. Let's not worry about the exact number. But how do we go about measuring that many molecules in a cell or tissue or what have you. Okay, so the first thing you might want to look at is physical properties. So I put caffeine up here. So caffeine is obviously not a molecule that our body normally makes, but if you drink as much caffeine as I do or as much Rockstar as Rubin does, it's pretty much an endogenous molecule at this point. You can't live without it. So we're going to consider it an honorary metabolite. At this point. So what you wanna do when you look at these things, you can see that their structures are different. Each of these again is a carbon, so this one's made up of mostly carbon, oxygen, and hydrogen. This one has some nitrogen in it. And so you get a sense that they're physically different, but what is it that we can take advantage of? And people have tried many, many different things to separate these things, but the one thing that's worked very well has been that you can actually separate things, or identify things based on their molecular weight. So depending on how many carbons are there or how many nitrogens are there, you add all of those up. So each carbon weighs 12. There's 3 hydrogens here. You just add all those things up and you get to 255. If you add up all the atoms in here, you get to 194. And that's a pretty distinguishing feature of these molecules that you can use as kind of an ID. So the numbers I have up here are called atomic mass units.. And if you want to look at it, 1 pound is about 3 times 10 to the— so 26 zeros after that. So it's pretty big. So when you want to weigh these things, it's not like you can go to a scale and put one of these things on, because it's going to be very, very light. The other thing is that you can't actually manipulate it at all. And so really what's— one of the things about it— as you come to appreciate as a scientist kind of the times you're born into— is that I happen to be born at a time where really the tools to be able to do these kinds of measurements and measure these kinds of molecules have just been coming online. And so we use a technique that's called mass spectrometry that really allows us to use the molecular weights of molecules to measure thousands of these things from very complex mixtures, so blood, tissue extracts, whatever we can think of. So we've done most of our work in mice, but we can do human samples too. Right before, there's actually two machines that are hooked up in tandem. One is an instrument that is designed to separate molecules based on their physical properties, how greasy they are or how big they are. Any way you can think of, you can separate these. And the idea for— the reason you want to separate it is because what's going to happen next is after it gets separated, it's going to go into the mass spectrometer. And the mass spectrometer is going to give you a few bits of information. One is it's going to tell you the molecular weight of a compound. So you're going to be able to ID it based on a molecular weight. The other thing is it's going to tell you how much is in there. So when we start these experiments, what we do is we'll compare a disease sample to a normal sample.. And if we find, for example, something like 165.0790 is up in the disease sample versus the normal sample, that'll be a calling card that this is basically the levels of this compound are changing. And oftentimes, we have to do a lot more work to figure out what they are. But that's basically how the experiment works. Now, one thing I did want to show you guys, just so you can see that scientists are a little bit geeker than we probably even look, is that just like you'd go to a car company and they'd show you video of their car, you can get videos of mass spectrometers. And so this is the latest, greatest mass spectrometer, which we're actually going to install in the next 2 weeks here at the Salk, which will be fantastic because we'll be able to do some really cool experiments. And I've turned off the German techno music so you guys don't have to be exposed exposed to that. I know Ruben really wants to hear it. But the reason I'm actually putting this up is it gives you a unique opportunity to kind of see— they do a good job in terms of drawing how these things work. So what you'll see is they've taken the mass spec apart here. And these are the actual molecules that are coming in here. And what you're doing is, as these molecules are traveling through here, you're basically guiding them to a point where you can detect their actual molecular weight. So let's see if I can— oops. OK, so sorry. So basically, you can generate those things and you can get molecular weight. So that's the methodology we use. And that's— there are other labs that do it, but not many. And so that's what distinguishes the type of research that we do from, I think, other labs. So we're able to look at these pools of molecules that aren't normally studied as frequently. But might be just as important as other pools. So now the story I'll tell you about starts with a mouse that are engineered. And if you guys have seen Ratatouille, you know these are rats, but you're going to have to let me slide with the fact that they're mice for my talk. And this was all work done in collaboration with Dr. Barbara Kahn at Beth Israel Medical Deaconess Center in Boston. And so what had happened is that Barbara had actually engineered a mouse. To be resistant to diabetes. Okay, and the way she did that is that she took the mouse and that glucose transporter protein I was telling you about earlier, she made it so that the mouse had much, much higher levels of that protein in their fat. Okay, so no matter how much glucose you dump into this mouse, it never has high blood glucose levels because it always has so much glucose transporter. Its fat's just sucking it up like a sponge. Well, one problem with that is its fat sucking— as its fat is sucking up the glucose, it's also growing. So these mice get actually obese. But the interesting thing about them is unlike the other cases where we get obese mice, this guy here happens to be insulin sensitive. It doesn't develop insulin resistance that we see in kind of normal mice that are obese. And so one question before we started working together is Barbara asked was— or actually the reason we started working together was if these mice are obese, We know, are they— is it because— but they're insulin sensitive. Is it because things like fatty acid levels are actually lower? And so we did a measurement and we measured the fatty acid levels in these mice, and what we found is that the levels were actually higher. Okay, so that's actually a mystery. It's a little bit of a paradox. And, you know, in some ways, that's— as scientists, that's kind of what we live for, a situation where the data doesn't actually fit our predictions. So basically You would normally equate these things with causing insulin resistance. They're up in this mouse, but it's insulin sensitive. So there's something going on that we don't understand. And so that's when we decided to start to dig a little bit deeper and ask what's going on in the fat of these animals. And so the experiment is actually fairly simple. So we take fat from basically a normal mouse, and then we take fat from these mice that have higher levels of these glucose transporters. And then we look without any kind of predisposition towards what's in there at all the molecules we can actually detect in these samples by mass spec. And what we're looking for is we're looking for things that are changing that might give us a clue as to why these animals have actually improved insulin sensitivity. So this is about the only data slide I think I'm showing. Maybe there's another bar graph coming along later. But let me walk you through this. Each one of these points here corresponds to metabolite. This is what we call a volcano plot for obvious reasons. It looks like a volcano. Essentially on this x-axis here, you're looking at changes between the insulin-resistant mouse and a wild-type mouse. So things to the right here are higher in the fat of the insulin-sensitive mouse. Things to the left are lower. This is basically how much confidence we have in the data. So above this line, is essentially a cutoff for where we're very confident in there. So you can see here that there's a lot of things changing, except what we found in her mice were this group of molecules here that clustered together. And it was very interesting because you can take it, and I told you that there was a human metabolite database, for example. And you can plug that mass in there and go ask, what metabolites could these possibly be? And you get no hits. And there's several other databases you can do the same thing on. We get no hits. And so at that point, we were excited/scared because we didn't know how long it would take to figure out the structures, but we knew we had something new. And so the idea was we had to figure out what these molecules are. And again, this is kind of where the chemistry comes in really handy because that's what chemists do a lot of times. Even though you set up reactions knowing what you want to make, a lot of times you get mystery gunk, and you have to figure out what's going on. So it becomes a game of working it out. And this was kind of the same problem here. We had something. We knew it was changing. We just didn't know what it was physically. And through a series of experiments that I'm not going to go into, we identified the molecules as these new fats. So when I say new, what I mean is that you can search for these in any possible way you want. Nobody had ever reported these present in any organism that we could find. Before. So it was a bit of a shock in terms of we found these things, we had them, and the question is like, where do we go from here? And so sometimes when you make these discoveries, again, it's exciting. But Barbara, to her credit, was very focused. And she'd studied these mice since 1993, so she knew them pretty well. And she was convinced that these molecules were actually involved in the actual insulin sensitivity of these animals. And so to give ourselves a little bit more confidence, what we wanted to do before we did a lot of work is we now have the tools. We now knew what we were looking for. We could look for these molecules anywhere. We could look for them in food. We could look for them in people. We could do whatever we want. And that's really where we went next. We asked the question about what would happen if we looked in people. And we looked at people basically that had diabetes or didn't have diabetes. Could we find that these molecules were changing in a way that might suggest that they're part of the disease itself. And so in collaboration with a group in Sweden— and because of their nationalized medicine, they actually have pretty great sample collection and sample retrieval ability. So even if a patient has given blood and we're running low on the blood, they'll call the patient up and they'll come back in and give more sample. So it's kind of like a never-ending source. What we did is we took people that were matched in almost every characteristic possible, body weight, BMI, all those things. The only difference was that one group of those people were insulin sensitive and the other were insulin resistant. Okay, and we asked the question of what was happening with this lipid, with their levels in blood, and we even got fat biopsies from these people and looked at it. And what we found is that in people that were insulin resistant, the levels of these lipids were significantly lower in both cases. So here we're looking at insulin sensitive in black, insulin resistant in red, and essentially the y-axis is how we quantitated these. OK, so now we've got kind of the same correlation, but in the other direction. So in the mice, what we see is that the levels of these are up, and we see that the mice are insulin sensitive. In people, what we're finding is when the levels are down, they're insulin resistant. So it kind of says that this molecule could be a sort of rheostat. And if you turn it up, you get insulin sensitive. If you turn it down, you get insulin resistant. But it also gave us the idea that we should actually test the molecule. So my lab made a bunch of the molecule. And the idea was that we're going to feed these to mice and see— sorry. We're going to feed these to mice and see what happens. And the results have been actually fairly exciting and very clear, actually. So if we give these compounds to mice, just normal mice or mice that have been put put on a high-fat diet so they have a little bit of insulin resistance. We can see that their insulin resistance improves. They do a much better job of handling glucose after a meal. It also reduces their inflammation and fat. And so that's all data that's being published. But what's happened since then is we've now gone and actually fed this for much longer periods. So the initial studies in the paper that we're publishing has given these compounds to mice for about one day, but now we've gone out and given it to them for 2 or 3 weeks, and the data keeps getting even better. They keep getting more insulin sensitive. Their blood glucose levels drop. And so we really think we've kind of stumbled upon something really exciting. Now there's a ton of questions to ask about these things. They're new metabolites. We have no idea how they're made in the body, how they're broken down, what percentage of them comes from What percentage of them are made in the tissues? But we're kind of tackling it one at a time. And one of the things we're really kind of excited about and getting geared up about is trying to figure out how to actually get these compounds and have people with diabetes try them to see if it actually works to reverse their insulin resistance. And so the group in Sweden, they're convinced that they can actually do a small-scale like 20-people clinical trial. And I've been on the phone with a bunch of contract chemical companies to see how you can actually make these compounds at high enough purity to do those trials. So we're really excited about that from a kind of a societal aspect that these things could eventually be used to maybe treat disease. But also from a scientific aspect, there's so many interesting questions that come up when you just find something new like this that we're excited to explore as we move forward. So with that, I'd really like to thank you for your time and attention. I really appreciate for you guys coming out. I know I've met some of you. I'd like to meet all of you at some point and get to know you better. And hopefully I'll be coming to more of these in the future. Thanks so much.