Narrator - Dr. Abel 00:00 Welcome to HelixTalk, an educational podcast for healthcare students and providers, covering real life clinical pearls, professional pharmacy topics and drug therapy discussions. Narrator - ? 00:11 This podcast is provided by pharmacists and faculty members at Rosalind Franklin University, College of Pharmacy. Narrator - Dr. Abel 00:17 This podcast contains general information for educational purposes only. This is not professional advice and should not be used in lieu of obtaining advice from a qualified health care provider. Narrator - ? 00:27 And now on to the show. Dr. Sean Kane 00:31 Welcome to HelixTalk episode 189 I'm your co host, Dr. Kane, and I'm Dr. Patel, and the title of today's episode is mice, macrophages and metabolism – Browning keeps obesity at bay. Dr. Khyati Patel 00:45 You know, Dr. Kane I'm really excited to talk about in this episode, like early stages of drug targets and development with our very own colleague, Dr. Mohd Shahid. His research involves the IER3 gene, which is an important modulator for the body's inflammatory response, but there's action on immune cells, particularly those macrophages and T cells, which then particularly play a role in some of these chronic inflammatory condition metabolic conditions such as obesity, diabetes, atherosclerosis, and you know, I'm excited to talk about the functions of this protein, how Dr. Shahid's research is involved, and introduce him to his grant platform. Dr. Sean Kane 01:23 So why don't we start with Dr. Shahid? You kind of introduce yourself and a little bit about your background. Speaker 1 01:29 Hello everyone. Thank you for this opportunity. My name is mal shahid. I was introduced currently. I am an associate professor of pharmacology in the College of Pharmacy of Rosalind Franklin University. I did my PhD in cardiovascular pharmacology from University of Delhi, and right after that, I joined Mass General Hospital and Harvard Medical School as a postdoc. And I think after four years of my postdoc, I became a junior faculty, which is an instructor position at Harvard Medical School. After, you know, my final year of my postdoc, I secured an NIH grant, and that allowed me to accept a full time independent faculty position at Chicago State University. So I worked at Chicago State University for almost five years prior to my current Dr. Sean Kane 02:14 position, and now you're at Rosalind Franklin University, and now I'm at Rosalind Franklin University, perfect. So I think just a little bit of context to our listeners. So I think it might be nice to share with the audience, which is mostly clinical audience, why we brought you on today. So Dr. Patel, you know a lot of what we talk about are phase three human trials, right? And especially in the pharmacy practice community, I think it's really easy to forget about all of the amazing steps that happened before you get to phase three, before you get to phase two, and even before you get to phase one, which is what we're talking about today. Dr. Khyati Patel 02:47 So we're gonna go from talking about patients in the trial to talking about mice in the trials, right? And the amount of, yeah, like I said, Dr. Kane, the amount of time that goes into clinical trials, it's really immense. So we are here today to appreciate all the behind the scenes work of a researcher like Dr. shahid. You know, we're talking about, how are we identifying these drug targets? You know, are we testing further drug targets? How are we testing them? And then we want to make sure when we are working on these drug targets and we develop a drug that not only works, but that's not toxic or, you know, harmful to the subjects or the human beings. So excited to dive into talking about the ier three gene and the pathway, Dr. Sean Kane 03:28 focusing on that Dr. Shahid with this IER3 gene, can you tell us a little bit about what it does and why humans and mice have this gene? Definitely. Speaker 1 03:39 You know, I've been working with this gene for about 10 years now. You know, like any other gene, IR three also encodes for a protein, like all genes encode for proteins, and therefore it is called a gene precisely. It is located on chromosome six, and the protein size. Protein is made up of 156 amino acids. It actually belongs to a large group of genes and the family of gene that is called immediate early response genes, which means their expression rapidly increases and peaks within 15 to 20 minutes upon exposure to, you know, external signals such as stress signals such as irradiation or inflammatory signal. Interestingly, IR three is expressed abundantly in a variety of tissues that are that come in direct contact with external environments. For example, you know, epithelial cells of skin and respiratory tract, vascular endothelial cell and immune cells such as T cells and macrophages. And that's where my research comes in. My mentor at Harvard Medical School had already discovered that, you know, if you over Express ier three, if you increase the level of IR three in T cells selectively, the T cells in mice, the mice go on to develop. A lupus like syndrome due to an extended duration of immune response of T cells. That was the first indication that you know, IR three could exert an anti apoptotic action in T cells, specifically in T cells. Yes. Dr. Sean Kane 05:15 So what you're saying, Dr. Shahid, is that this is a gene that is involved in a bunch of different cells in the body, and when this gene is activated or encoded for its protein, that protein has a kind of pro inflammatory state where it revs up the immune system to hopefully fight an infection or a pathogen. And you studied this specific gene in terms of its role, as you said, in immune cells in specific macrophages, right, right? Speaker 1 05:43 Okay, and T cell was one of the immune cells where, you know, its role was discovered, and that's where, you know, my research came in. I actually took this research into completely different direction, because from the prior research, we learned that not only T cells, IR three could also play a role in macrophages. And macrophages are the primary immune cells in the body that contribute to inflammation. And speaking of macrophages, you know, macrophages can exist in amongst many states that could include resident state. Means macrophages are just sitting there, lying there and doing nothing, but they could also be triggered into an pro inflammatory state whenever there is a stress signal or inflammatory signals, and in these cells, IR three levels goes up. On the other hand, macrophages can also exist in an anti inflammatory states, and the primary function of these macrophages is to suppress inflammation and promote repairing process. And surprisingly, IR three expression is down in these cells. So what we learned from this study that a differential expression of IR three could alter macrophage phenotype that could have some meaningful implication in terms of the clinic, because we know macrophages are the primary mediator of inflammation, and inflammation plays an important role in many chronic inflammatory diseases such as atherosclerosis, obesity and diabetes. Since IR three controls macrophage phenotype, this suggests to us that it could be an attractive therapeutic target for the development of development of novel drugs for the treatment of these diseases. Dr. Sean Kane 07:29 So one thing that came to my mind as you're talking about the story of ier three gene and macrophages and T cells, it almost feels like this would be more of an autoimmune disease target versus a cardiovascular or, as we'll talk about, obesity target, Is there research being done for this as a target for rheumatoid arthritis, or, you know, other autoimmune conditions? Speaker 1 07:51 That's an excellent question. Actually, that's what it was discovered for, if I could say that, and that's what it was studied for in the beginning, in the first five years of its discovery, and my mentor one was was one of the pioneers in IR three research, and she had found that it actually did play a role in autoimmune diseases such as colitis and abnormal T cell response in many disease conditions such as lupus. I was the one who took this IR three gene into cardiovascular research because, and I think 10 years or 15 years ago, that was a time when new studies were coming out suggesting an important role of chronic inflammation in cardiovascular diseases. And that took us in that direction, because we knew IR three could alter inflammation by modulating macrophage and T cells phenotype. Dr. Khyati Patel 08:48 This is all very interesting, and it makes sense. So kind of pivoting a little bit back to some of the clinical trials of drugs and how they alter the inflammatory response, we have quick two examples of such drugs. We had the Jupiter trial where we used rosuvastatin, 20 milligrams daily versus placebo in almost like 18,000 patients, and we found that patients who were in the rosuvastatin arm had decreased C reactive protein, which is another inflammatory marker. And that makes sense, because then we also found, looking at the clinical picture, that in rosuvastatin arm ASAP, events were lowered by 44% and so there is a connection by lowering not just LDL, which obviously rosuvastatin does a pretty nice job lowering, but Having this pleiotropic effect on the inflammatory marker does impact your ascvd outcome. Dr. Sean Kane 09:44 And what was really interesting about Jupiter Dr. tell is that they only included people who normally wouldn't be indicated for a statin, but they had this high C reactive protein, this high inflammatory marker, again, showing this link, that just because your LDL isn't crazy high or. Acvd risk isn't crazy high, that if you have this, like systemic inflammation going on, the statin might be able to help calm that down a little bit, which is really interesting. Yeah, another trial that comes to my mind is the COLCOT trial. This is kind of new as well. It's also in our show notes, and this is about 5000 patients who had a recent MI, and they randomized them to either Colchicine, point five milligrams daily, or placebo. And this is a very different pathway. So this is not like a statin or blood pressure medication. Colchicine, more commonly we think about it for gout, right? And it is an anti inflammatory that helps calm down the neutrophils. Has an anti inflammatory pathway to it. Technically, it is involved in preventing and you'll have to go back to like General Biology, microtubules from polymerizing, which helps with activation of those neutrophils. And basically what they found was, in patients who had an MI who got Colchicine, their acvd risk in the future was reduced by 23% and they did a little bit of CRP testing, that C reactive protein testing, and of course, they showed that that was better than placebo. So it achieved its anti inflammatory properties, but also its reduction in ascv risk, again, highlighting this link between inflammation and cardiovascular disease. Dr. Khyati Patel 11:15 And last but not the least, we have the GLP one agonist, or GLP one gip combined agonist market drugs marketed for weight loss. And we kind of discussed this in our HelixTalk episode 186 while they are going for the weight loss, we are also seeing that some of these also have that positive cardiovascular outcome, the ASCVD risk reduction outcome. And so there is this link about, you know, decreasing the inflammatory markers and then having an impact on various inflammatory conditions like, you know, ascvd, diabetes or even weight loss. Dr. Sean Kane 11:49 So Dr. shahid, getting back to your story with ier three. So this is a gene and a protein that is associated with inflammation, and the more of it you have, the more activated and pro inflammatory your macrophages are help us understand the link between that and obesity, which is where we're going to start our story here, right? Speaker 1 12:08 Yeah, exactly. And that's what actually was a turning point in my research. 10 years ago, we had first strong cue that, you know, IR three could alter metabolic disorders such as obesity by altering inflammation, you know, but then another indication came from Pioneer study involving a pro inflammatory transcription factor, NF, kappa B. In this study, they had demonstrated that blocking this central inflammatory pathway could protect mice from insulin resistance and obesity, and we knew that IR three is a direct downstream target of NF, kappa B pathway. But that led us to hypothesize that if IR three is turned off, means, if you reduce its level, it could prevent and preventing its protein formation, thus inhibiting this component of the inflammation pathway. Does obesity still happen? Does diabetes still happen? So? Dr. Sean Kane 13:03 Dr. shahid, one technical question I have is, you've identified a gene, ier three that you're interested in. How do you literally turn off a gene and a mouse? That sounds kind of technical and kind of interesting? Speaker 1 13:14 Yeah, it is actually interesting process, and it is kind of exhaustive process as well. It takes sometimes, like, several months to knock out or knock off a gene. You can delete an entire gene from all cells in animals or in organism. The routine technique that we use in the lab is that you either remove or modify the coding regions of a gene that we call exomes. And there is another component that is called intron that does not code for the protein. So you can just alter the coding regions of a gene and in the embryonic stem cells. And then you can implant that embryonic stem cell which has this modified gene. Now, because you have modified the exome region of a gene, then you can implant it in a pseudo mom, because, since embryonic stem cells are a precursor of essentially all cells in an organism, then every cell that will be derived from this master cell will be lacking a functional IR three gene. So that's how you essentially silence the expression of a gene, Dr. Khyati Patel 14:22 and I'm assuming that this is not what you do, right? Like you don't knock out genes in mice like you. Where do you get these mice? Speaker 1 14:30 Yeah, I used to do that, and a lot of labs do that as well, and I did that as well. But then over time, we realized that the it's a very time consuming process. It takes a lot of time, and essentially we are not addressing a scientific questions by generating mutant mice. Yet, we are just generating the tool. So we thought it was not worth spending time on it. So now there are companies out there that are specialized in generating mutant mice for you, but essentially they follow the similar. Approach, like modifying the coding region of a gene, altering its translation into protein. Dr. Sean Kane 15:07 So just to highlight this for a second, you're paying someone to do IVF. Essentially, in a mouse, the cells that are being implanted have been genetically modified before the mouse even grows. So it's not like you have a mouse and you give him a drug that knocks out the gene, you're changing the embryo before it even goes into the mother mouse, right? Speaker 1 15:26 Yeah, I don't think that's feasible at this stage of science that we have reached. I think the best way is to alter the gene at the embryonic stage so that anything that is derived from the cell will have the global impact, because every cell is derived from that master cell, embryonic stem cell. Dr. Sean Kane 15:46 So this sounds very technical, yeah, I'm guessing it has a fancy price tag to get along with that technical. Yeah, it Speaker 1 15:52 takes months or years to develop mutant mice, and it costs approximately $10,000 just to generate first pair of mutant mice. And you know, that's how the company charges these reasons, Dr. Khyati Patel 16:02 and folks who are wondering where all this NIH money goes? Yeah, this is part of the answer. Speaker 1 16:08 This is part of it. Yeah. And, but these tools are essential. I mean, it allows you to ask and test your specific questions in an unambiguous manner. Dr. Sean Kane 16:17 So then, as we get into your research, we're gonna talk about this, but you have these $10,000 mice that you've ordered, they've come in, and then you have non knockout, or kind of wild type mice. And now you have the tools, as you said, for the experiment, to look at how different conditions changed. You know, the outcome or the measurements of knockout mice, those that don't have ier three gene, versus wild type that do have that gene, right? Speaker 1 16:45 So now, having these two groups side by side, you can expose them to similar experimental conditions, and then at the end of the study, make an observation, and based on the observation, you can conclude at the end of the day that whatever the differences you are seeing between the wild type and knockout mice is due to the absence of this functional gene. Dr. Khyati Patel 17:08 So, you know, we normally, Dr. Shahid talk on this, on these HelixTalk episode about human clinical trials, and we have 1000s of patients, and we're talking about, you know, four, three year interventions, and we look at data, surrogate markers, long term clinical markers, outcomes and stuff. Is that feasible in the mice study that you do Speaker 1 17:30 not, I'm not sure if it is feasible, because, you know, first of all, this is too inefficient of a model for mice, and it would be really, really expensive to have 15,000 mice on hand to mimic an RCT. And mice, you know, they have a short lifespan, and they don't normally die of, you know, atherosclerotic cardiovascular disease events and obesity. In order to mimic the clinical condition, you need to induce those condition, these diseases in order to study them. Dr. Sean Kane 18:00 So what you're saying is, one, you can't have 15,000 because the cost of this, maintaining that would be extravagant. But then two, because mice usually aren't obese and mice usually don't have heart disease, you have to make fat mice that have heart disease. So absolutely, how do you do that? Speaker 2 18:17 Dr. Shahid, she's like Ratatouille, right? Speaker 1 18:20 Exactly. I do it even better. I give them Double Whopper burger. So Well, technically, we have a, you know, typical, we have a diet that is full of fat that provides at least 45% calories in the form of fat and and, and this, this is like very high fat content diet. So we feed the mice with this diet for about three to five months, and then also compare this group with another group of mice, which has been fed with regular diet that is not very rich in fat, and that normal diet provides 13% calories in the form of fat. So by feeding them for three to five months, we essentially, you know, induce obesity and diabetes disease and in these animal models, Dr. Sean Kane 19:11 and then as the animals get obese, does that also give them cardiovascular disease just from being obese? Speaker 1 19:18 It depends upon the mouse strain. Yeah, regular mice that the most common mouse strain that we use in the lab is called C 57 black sex. And they do have tendency to develop cardiovascular diseases such as such as vascular dysfunction, hypertension and cardiac hypertrophy. And I think they also develop some sort of left ventricular end diastolic dysfunction, as you is continue to feed them with high fat diet. Dr. Sean Kane 19:49 That's true. And is that the kind of mouse that you use? Speaker 1 19:52 Yeah, exactly. I mean, our primary goal was to because we didn't. The role of i three was not known in card. Vascular disease at all. We had no other end points at that moment, except for obesity, because IR three was a direct downstream target of NF kappa B, and NF kappa B was found to play a crucial role in obesity development. So originally, we were just focusing on the weight gain due to high fat diet condition and then diabetes and other cardiovascular conditions. Dr. Sean Kane 20:23 In your hypothesis at the time was just, if you knock out the gene, they won't get fat or they won't have diabetes and insulin resistance, absolutely. Dr. Khyati Patel 20:31 Yeah. So you know, for the listeners, we have some manuscript links on the show notes. But Dr. shahid, so you had this experiment, you had the wild type mice, and then the ier three knockout mice. What did your study find? Speaker 1 20:48 So first, it was important to show that mice without IR three, that is IR three knockout mice, they breed and grow normally. It is possible that knocking this gene out could have some in could have induced some inherent defect in the mice. They could have altered the outcome of the study. So therefore, we monitor their monitored them under normal condition, and we found that these mice, in the absence, even in the absence of this gene, they grew and reproduced normally. They did not develop any condition, disease condition. So these mice, even in the absence of the gene, remain in healthy conditions. So having established that, you know, IR three mice, knockout mice, did not have any inherent defect. So we started our study. We gave these mice this triple Whopper Baconator equivalent diet, which is a high fat diet for five months, and we monitored their body weight and other metabolic functions after five months, we what we found that while the wild type mice gained approximately 92% weight increase, and they had their higher glucose and insulin levels, high cholesterol levels and very high levels of pro-inflammatory cytokines such as TNF-alpha, in contrast to wild type mice, what people have found that IR three knockout mice only gained 44% increase in their body weight. In fact, they weighed very similar to the wild type mice that were fed a regular diet, normal diet. So essentially, they did not develop obese. They did not develop obesity at all. Upon more critical examination, what we noticed that these mice were actually consuming more food rather than eating less, because that was our suspicious in the first place. Maybe they are. They are not becoming obese because they were eating less. However, we found out they were rather eating more, and therefore we also monitored their core body temperature, and we saw that their core body temperature was one degree Celsius higher than the wild type mice, suggesting that these mice were generating more heat and perhaps burning more calories because of that. Additionally, the IR three knockout mice had lower level of glucose, insulin, cholesterol and inflammatory cytokine such as TNF-alpha, even on high fat diet for five months. So overall, this data suggested that absence of IR three activity prevented diet induced obesity, impairment of glucose and lipid metabolism and systemic information. Dr. Khyati Patel 23:24 So, Dr. shahid, you mentioned an interesting finding in this IR three knockout gene mice group, that their core body temperature was one degree Celsius higher. And now I know there is some relationship between that core body temperature and metabolism. So was there a relation here. Yeah. Speaker 1 23:42 So what we found out that IR three knockout mice were resistant to obesity, but that was it. That's all we knew about that we had no clue how IR three knockout mice were protected against obesity and diabetes, except for the fact that they had higher core body temperature. And that gave us some cues, because if the animals, in our case, mice, had higher body temperature, it suggests that they are burning more calories in the form of heat. So to confirm this, we measure their total body energy expenditure using a technique called indirect calorimetry, and this allowed us to measure their total oxygen consumption and carbon dioxide release as well as their heat production, and we found that oxygen consumption and thus metabolism was, in fact, higher in knockout mice, suggesting increased energy expenditure in the absence of IR three. Dr. Sean Kane 24:34 Just to summarize, like at that point prior to doing the indirect calorimetry, you actually weren't sure how they weren't getting obese yet, right? You knew that they were eating more, but it could have been that they weren't absorbing it in their gut, or, I mean, come up with 10 other reasons why you don't gain weight when you eat food. So really, your indirect calorimetry showed that you're giving like the equivalent of hydroxy cut to these mice, where their metabolisms were just ramping up. And. That they were burning through the calories, as opposed to not ever getting the calories in their body Right, right? And it's interesting, you mentioned indirect calorimetry when I was in the burn ICU on my API rotation, we actually did this on several patients that were very severe burn patients. And we do that because we want to optimize our nutrition as their body's healing from a burn. And you don't want to over feed patients, but you definitely don't want to under feed patients that are critically ill with these burn injuries. We would literally do indirect calorimetry to verify that we were giving the right amount of calories to our critically ill ICU patients. And this is neat that the same technique can be used, even on a little mouse right to confirm that hypothesis Exactly. Speaker 1 25:38 It gives a lot of information. Just one point I want to point out here that people could argue that, you know, there might be some changes in their preference for substrate selection, for the production of energy. So we also monitored their R, E, R, using the same technique in direct calorimetry, re R is called respiratory exchange ratio, and we didn't find any difference here, suggesting there was no change in the preference for the substrate, for the energy production, Dr. Sean Kane 26:05 and you mean like breaking down carbohydrates versus fat, exactly Unknown Speaker 26:09 like carbohydrate proteins or fat. Dr. Khyati Patel 26:12 So there is a relationship between this core body temperature and then increased metabolism, and that's why they're producing heat. But the question is, how is this absence of ier three is doing Speaker 1 26:26 it right? So? So next we ask, how IR three absence increase heat production and thereby accelerated calorie consumption, because we saw that entire calorimetry technique confirmed that they had increased energy expenditure due to heat production. So to find out how IR three absence increase heat production, we focus on the two major sources of heat in the body, skeletal muscles and brown fat. The observation that total body motor activity was comparable between knockout and wild type mice from indirect calorimetry study, it ruled out an abnormal heat production from the muscles, so the only one thing was left, and that was brown fat. Therefore we next focus on mouse brown fat, which is the powerful source of heat and is rich in mitochondria. So a little background on fat. Mice and people have two kinds of fat, white fat and brown fat. White fat is located in our subcutaneous and visceral regions, and its primary role is to store energy, and it has scanty mitochondria. Means it doesn't have lot of mitochondria like other cells. On the other hand, brown adipose tissue or brown fat. It is mostly found in the neck, shoulder and spine region, and this is also called baby fat, because it is mostly found in newborns and infant and not in adults. However, it can be induced in adults. Its primary role is to generate heat, and so accordingly, it has tons of mitochondria, which is a powerhouse of the cell, and it is highly vascular. Dr. Khyati Patel 28:05 So Dr. shahid, if I kind of just simplify this, it sounds more like we don't like that visceral fat, which is the white fat. So if you assign the good and bad property, like good lipoprotein and the bad lipoprotein. So I think similar analogy here. I'm going to say the white adipose tissues around the visceral organs. It's not a healthy fat. Versus that brown fat sounds like it's a healthy fat because it provides the heat that babies are absolutely Unknown Speaker 28:29 need. Yeah, that's correct. Dr. Sean Kane 28:31 Just to clarify, adult humans tend to lose their baby fat, right, right? That brown fat? Speaker 1 28:36 Yeah, I think it's an evolutionary process, you know, as like, you know, there are so many things we have lost, you know, over the ages. This is one of the evolutionary process that happens over the span of one's lifetime. Because baby don't have a lot of muscular movements, and therefore they need a, you know, an alternate source of heat, which is your brown fat. But adults, you know, they can work out. They move a lot, they have a lot of muscular movements, and they can generate their heat. They can obtain their heat from that muscular movements. So it makes sense that you're losing your brown fat because you don't need that anymore. Dr. Sean Kane 29:08 So then the next logical question I have, Dr. Shahid, is, if I don't have much brown fat, but I have a lot of white fat, can I trade it? Can I get some brown fat for my white fat? Is that even a thing, or are they completely different types? Speaker 1 29:22 Yeah, it is doable. I think one of the ways you can do it go to the Michigan Avenue and jump in the Michigan Lake Michigan in the month of January for five minutes every day. Dr. Sean Kane 29:32 They do have polar plunges. Are you saying cold plunges will make my white fat Brown? Speaker 1 29:36 Yeah, it's there. There have been studies where they have shown that, you know, cold exposure can transform your white fat into Brown. Dr. Sean Kane 29:46 Does that have a term associated with it? This process is Speaker 1 29:50 called Browning, and I like to call it Beijing. That's what I prefer for my research. Dr. Sean Kane 29:56 And then, before we get too much more into the Beijing process. Process, if you were to, like, literally look at Brown fat versus white fat, does it literally have a different color to it? I know the mitochondria is different, but Speaker 1 30:09 yeah, yeah, yeah, definitely, brown fat is definitely, apparently looks brown. It's so obvious it looks brown. That's why the name is brown fat, and white fat looks pale and white in color, and when you induce Browning or Beijing in white fat, yes, it does indeed change its color into brown like fat. It doesn't become it doesn't look exactly like typical brown fat, but it starts Browning Dr. Sean Kane 30:36 so it sounds like Dr. sheep. Where we're going here is we knew that the metabolic rate of mice was higher that had the ier three knockout. You hypothesized that maybe it's because of the brown fat, because you looked at their muscles, and the muscles weren't really producing a lot of new energy or new heat. So then the question was, how do you prove that a mouse has more brown fat, or that the white fat turned to brown fat, or the brown fat was more active. How do you actually do that? Right? Speaker 1 31:05 So initially we thought that perhaps, you know, il three knockout mice had higher heat production due to increased brown fat activity, and therefore we monitor their brown fat activity in both wild type and il three knockout mice. And it turns out that there was no difference in brown fat activity between wild type and IR three knockout mice. So actually, and what we noticed and what was happening was that white fat, you know, was actually turning into Brown, like fat, or by a process that we call Browning or Beijing, meaning the white fat, which normally is not very thermogenic, was turning on its mitochondria and burning calories, and this was evident from an increased level of mitochondrial protein called uncoupling protein, UCP one, which is a primary driver of heat production in the mitochondria. We know UCP one plays a crucial role in generating heat in mitochondria. So it makes a lot of sense that as knockout mice had higher core temperature. Dr. Sean Kane 32:07 So then, just to verify the way that you knew that white fat was turning to brown fat, was that you looked at essentially the mitochondrial activity within what used to be the white fat, and it was behaving more like a brown fat type tissue, right? Speaker 1 32:21 This is one of the indicators, one of the markers of brown fat that UCP, one level was going up. There could be some, you know, histological markers as well. Like white fats are usually, you know, the bigger in size, and they contain a lot of fats in it. You can see it under the microscope. But when this white fat is turning into brown fat, it becomes smaller in size. It becomes multi lobular, and you can you could see the presence of multiple mitochondria in each cell. These observations suggest that somehow lack of IR three transform white fat into brown When mice are fed a high fat diet. Interestingly, one striking observation was that wild type mice completely lost their UCP one from white fat when fed a high fat diet, whereas knockout mice maintain it even when exposed to similar condition. This is an interesting finding and reveals what a high fat diet could do to our healthy fat. Dr. Khyati Patel 33:22 This is really interesting. So you're saying even even though these, these mice that are ier three knockout were given high fat diet that body, their body was converting the bad fat into good fat. Absolutely interesting. Dr. Sean Kane 33:36 And then we kind of started off our story thinking about inflammation, right? So ier three was something that we thought more about from its inflammatory pathway, as opposed to, like, its obesity pathway, even though we kind of discussed that there's some connections there. I'm assuming, Dr. Shahid, that you looked at some inflammatory markers with these AR three knockouts. Can you tell us more Speaker 1 33:56 about that? Yes, yeah, because these mice, they were showing the science of Beijing. And there was a study that was that came out right before you know, my observation in this research, the investigators reported that macrophages could play a role in Beijing process, and that led us to analyze the macrophage in our mice model. And so what we did, we use flow cytometer to look into macrophages, specifically those that are localized in white fat. We call them adipose tissue macrophages. So we what? We noticed that the number of macrophages in white fat increase when given a high fat diet, and we saw that in wild type mice, there was an increase of three to four fold in the number of macrophages, whereas in the knockout mice, only the number of macrophages increase only two folds. Dr. Sean Kane 34:53 So what you're saying is that it might not even be the fat cells in terms of their expression of IAR three but. The macrophages that are just hanging out in these white fat areas, they might be doing some of the action as well. Speaker 1 35:06 Absolutely, initially, we just want to, we just wanted to look at the percentage of macrophages that were there in the fat. We didn't know what was happening to their phenotype. This study showed that, yes, there was an increase in the macrophage and filtration into the white fat upon high fat diet feeding and IR three knockout mice were resistant to this, this phenomena. Dr. Khyati Patel 35:31 So Dr. Shahid, based on this finding, you've kind of arrived to your current research, which is looking at the ier three impact on atherosclerosis. Obviously, you know, we can have another episode to just talk about that particular research, but what are some of the main concept of that current project? Speaker 1 35:49 Absolutely, and before I go ahead and talk about that, I forgot to mention one point that we also looked at the phenotype of macrophages in our obesity study, and we found that it's not just the number of macrophages that were less in IR three knockout mice, it was the phenotype of the macrophages which was different in IR three. In the absence of IR three, we noticed that while high fat diet feeding transformed most of the anti inflammatory macrophages into pro inflammatory state in wild type mice, knockout mice were completely resistant to this phenomenon, meaning knockout mice sustain majority of their anti inflammatory macrophages. In short, we can conclude that absence of IR three essentially promoted an anti inflammatory state of macrophages rather than pro inflammatory state. And we think it could be the link of IRC with the obesity development in obesity study and speaking about talking about atherosclerosis, we know that macrophages play a central role in the pathogenesis of atherosclerosis, and we also know that it is a specific macrophage phenotype that contribute more to atherosclerosis development. We know that it is a pro inflammatory phenotype of macrophages that contribute to fatty plaque formation, and as we observe in our previous study, that marriages behave more like anti inflammatory rather than pro inflammatory, when IR three was silenced. This led us to hypothesize that perhaps IR three deficiency, or suppressing its expression, could protect mice against fatigue plaque formation, as occurs in atherosclerosis. In fact, you know, our community does suggest that IR three knockout does, in fact, prevent atherosclerosis in mice. Dr. Sean Kane 37:48 So to kind of summarize where we're at with kind of this really interesting story of your research, we identified the IR three is an important contributor when you overfeed mice and they get obese, and by blocking that, you can prevent the obesity, insulin resistance, inflammatory markers and fatty plaque formation, and you know, atherosclerosis, and a lot of this is driven just by the macrophages. And you can prevent this whole cascade by blocking or not having that IR three gene expressed Dr. Khyati Patel 38:21 so all these years of research, and it seems like we have, you know, come down to this one protein or one gene that expresses the protein, ier three, that could be a good drug target, but it seems like it's a long road ahead to make the drug and make it to The market. Can you kind of explain what happens between your research and landing to a drug target to then actually having the drug that we can use in human Speaker 1 38:50 Yeah, excellent question. And you know, most of the basic science researchers, their main goal is to discover the novel drug targets. Developing a drug that could target that, you know, gene or protein. It's a long road, but we know that a drug or therapy might, you know, ultimate goal is to develop a drug or a therapy. So we need to develop a drug or therapy that could change IR three gene expression, or, you know, reduce its protein levels. There are several approaches, and there's several steps involved. For example, recently, you know, with the advent of AI, you know, we could incorporate that So, and we can use AI based drug modeling or computer aided drug designing to come up with drug candidates. Once the candidate has been identified, then the drug has to actually make it into macrophages or adipose tissue, or whatever the tissue that we are that we intend to target. We have to ensure that it reaches here so active drug transport mechanism, or other mechanism of cellular entry, needs to be studied and developed and. Then we have to make sure that drug is not toxic, because if it, if we the drug is toxic, it can interfere with important cellular process that would cause adverse effect. And we know from our extensive studies on this protein that IR three does not play a major role in maintenance of normal homeostasis, which is a good thing, because under normal steady state, IR three doesn't seem to play a critical role in maintaining the normal physiological function. However, its expression is rapidly induced upon exposure to different kind of stress signals, for example, high peptide, high cholesterol level, ultraviolet radiation, where it plays a critical role in pathogenesis of several disorders such as obesity, atherosclerosis and diabetes. This suggests that targeting i three could be a promising strategy to treat this disorder without the risk of serious side effects. Dr. Khyati Patel 40:55 So I love that you know any any part of drug development, we want to make sure that the drug is safe, it's not harming Right? Like not doing something that we don't intend it to do. But then, aside from the efficacy that the drug works, and then the safety that it doesn't cause more harm, you have to think about the other aspect, right? Like you said, drug needs to reach the target. Needs to have a certain kinetic profile. It can't be just that you had the drug, and then the half life is like two minutes, and then the drug can't even reach the target organ to exert it. It's a fact, right? Plus, all of this is like, first we're going to test this drug into our animal models, right? And that doesn't always mean that even a drug works and animal models that it's going to work in human too, right? So then you have to make sure that the human targets and kinetics and efficacy and safety, all of that kind of is replicated Speaker 1 41:48 after having conducted the safety and, you know, therapeutic studies of the drug. We have to make sure that it has, you know, a reasonable kinetic profile, right. And then you have to prove that the drug actually works in human clinical trial, as you said, Because and vast majority of the drugs that have worked in mice model doesn't have not worked in human clinical trial. Therefore, a clinical trial would need to show prevention of atherosclerotic cardiovascular disease, obesity and or diabetes. If we were to show that any drug that targets IR three is effective, and then the drug might make it to the market, and even after that post marketing surveying, we still won't be sure that the drug is completely safe and it's going to work in real clinical scenarios. Dr. Sean Kane 42:38 So Dr. sheet, one of the reasons that Dr. Patel and I had you on was to give context to primarily the clinicians who listen to the podcast of, again, that phase three trial that we love to read and talk about all the amazing steps that happened well before that. And you know, in your example here, we haven't even found the drug yet, but you can't find the drug until you figure out where the drug target is, which is what you're focused on, but then there's a whole new road after that, once you try to figure out what is the best drug for that drug target, and then getting that all the way through there, and then getting it to human studies, is a whole nother animal, a whole nother process. You know, I would imagine that some researchers might spend their whole career doing just one of those aspects, to get a drug target, or to get a drug candidate, or to study it in humans, I would imagine that this is a really long road for multiple people to even get to a phase one clinical trial in a human. Is that correct? Speaker 1 43:34 That's correct. Actually, yeah, it takes a I mean, I have, I have seen people, you know, spending their entire life just studying one molecule or one drug target or one molecular pathway. And yes, it takes, you know, 10 to 15 years to bring the to bring a new drug into the market. Even then, we are not certain that the drug is going to work in the clinic, or whether the drug would be, you know, devoid of any serious side effects. However, the silver lining is that, for example, in our case, you know, in our IR three pathway, even though we studied its role in obesity, but it led us to find out its essential or critical role in many other disease conditions. So once we have established the safety and efficacy of this drug in one disease condition, you could translate these findings into other disease areas, expediting the discovery process. Dr. Khyati Patel 44:33 You know, in the world of instant gratification, I have a newfound respect for your patients working on this project. Like you said, for nearly 10 to 15 years, I'm just focusing on this one particular drug target, and I'm going to put on my activist hat for a little bit, and then just say, you know, to all this slashing of the NIH funding and the research funding that's going on, this is where the money goes. This is where all the efforts of. Scientist that goes behind even before we see the drug, and then we can talk about the drug costs and how to lower them thereafter. Dr. Sean Kane 45:08 Right? So Dr. sheet for the listeners, we're going to have references in our show notes. The first one is going to be one of the papers that you shared with us to kind of prep for the episode. So for the listeners, I do not consider myself a bench scientist. I've never worked with animal models. I don't know what flow cytometry even looks like, but I was able to kind of read through the paper fully understand it. It's very well written. So if you want to know what does it look like to do this kind of research and publish on it, take a look at the show notes, and you'll find that reference, plus some of the clinical references that we already talked about. So Dr. shahid, again, thank you so much for your time, your expertise, and as Dr. Patel alluded to like your commitment to a decade plus long devotion to one gene and its role in all of these different disease states. So thank you very much. Thank you so much for having me. Dr. Khyati Patel 45:56 Yeah, it's very exciting to hear the work that you're doing and and the potential it has to make a difference in human life. You know humankind, so thank you. Speaker 3 46:04 Thank you. So with that, I'm Dr. Kane. I'm Dr. Patel, and as always, study hard. Narrator - Dr. Abel 46:10 If you enjoyed the show, please help us climb the iTunes rankings for medical podcasts by giving us a five star review in the iTunes Store. Search for HelixTalk and place your review there Narrator - ? 46:21 to suggest an episode or contact us. We're online at HelixTalk.com. Thank you for listening to this episode of HelixTalk. This is an educational production copyright Rosalind Franklin University of Medicine and Science.