 Okay, our next speaker is Stefan Guine, also really needs no introduction, but he's one of the reasons I got into the Ancestral Health field in the first place through his whole health source blog, and he's been a source of inspiration ever since. Thank you. Thank you. It's good to be here at my fourth consecutive Ancestral Health Symposium. I'm glad to see some familiar faces in the audience. Today I'm going to talk about a subject that I think has very profound implications for our current epidemics of obesity and metabolic dysfunction, and that is a condition called leptin resistance. So to give you an overview of what the talk is going to be like, first I'm going to go over the history and the importance of leptin, then I'm going to talk about the cellular causes of leptin resistance that have been identified, and then I'm going to talk about some potential diet and lifestyle contributors to leptin resistance. So let's start with the history and importance of leptin. Weight regain is a major barrier to obesity therapy, and to illustrate that what I have up here is a 10-year follow-up of one of the longest followed up and most successful weight loss studies, which is a diabetes prevention program study. What you can see is that in a diet and lifestyle weight loss group, patients lost about 7 kilos. They maintained it at a year, but over the course of the next 10 years they steadily regained that lost weight until after 10 years had elapsed, there was virtually no difference between the diet and lifestyle group and the group that received no intervention at all. And this is pretty typical of a variety of diet and lifestyle weight loss interventions. Weight regain is the rule rather than the exception in these types of interventions. And that's thought to relate to a couple different things, but one of the main factors is that the body actively resists weight loss, and this resistance to weight loss is related to the leptin signaling pathway. So one of the first signs that body fat mass is regulated came from a physician named Bernard Moore, who was practicing in the German city of Würzburg. Dr. Moore had a patient named Eliza Moser, 57 years old, who came to his hospital with a condition of mental disruption which disappeared with a few weeks of bloodletting and cathartic therapy. I'm not sure what cathartic therapy is. It doesn't sound good though. Rather the patient had become uncommonly obese over the last few years. Present status, uncommonly extreme obesity. This patient died two months after being admitted to the hospital and Dr. Moore performed an autopsy on her. As he removed her brain, he turned it over to the underside and he noted the presence of a tumor that had destroyed a part of her brain called the hypothalamus, and I've circled that here on this brain. The year was 1839. At the time, and for many decades thereafter, researchers believed that this was due to damage to the pituitary gland which lies directly below the hypothalamus, but it was demonstrated in 1921 in dogs that lesions to the hypothalamus and not to the pituitary gland create obesity. And this work was replicated in rats shortly thereafter and you can see on the left a normal rat and on the right a rat that has sustained a lesion to its ventromedial hypothalamus. They become extremely obese. The field of genetics began making major contributions in this area only a couple of decades later. So in 1949, researchers from Jackson Labs identified the genetically obese OB-OB mouse. This animal has a single gene mutation. You can see on the left, extremely obese compared to normal mice. And at the time, they had no idea what this mutation did. And over the course of the next few decades, they identified a number of other single gene mutations that cause extreme obesity and rodents. So naturally, researchers were interested in understanding the mechanisms by which these lesions and these genetic mutations produced obesity. And so they turned to a gruesome technique called parabiosis. And parabiosis, you literally sew together two animals such that their circulatory systems communicate in a limited way. And what they were able to demonstrate is that when you parabyose an OB-OB obese animal to a normal animal, it causes it to lose weight. The OB-OB loses weight, its insulin levels normalized, blood sugar levels normalized. And through a series of related experiments, they were able to determine about a half century ago that OB-OB, or excuse me, that there was a circulating factor that powerfully controls body fatness, that OB-OB mice lacked this factor, and that animals with ventromedial hypothalamic lesions were not able to respond to that very same factor. So this led in 1959 to the researcher Romain Hervey proposing the following model of body fat regulation. What he proposed is that fat tissue secretes a factor that they called the satiety factor in proportion to its size and that that factor travels through the circulation is detected by the hypothalamus and that the hypothalamus then regulated food intake to maintain body fat stores within a relatively stable level. So it wasn't until 1994 that researchers identified what the satiety factor was. And what they did was they identified what the genetic mutation was in OB-OB mice. And it was a gene that indeed coded for a circulating factor that they named leptin. And so the OB-OB mouse is a leptin deficient mouse, and it turns out that all of these other single gene mutations that have been identified spontaneously in rodents that produced obesity were all in the leptin signaling pathway. And in fact, it didn't take long for researchers to identify leptin deficient humans. So you can see on the left that's a three year old boy weighing 42 kilos, and on the right is the same boy at seven years old weighing 32 kilos after having been treated with pharmacological leptin. So since 1994, there have been 26,000 studies published on leptin, and it's widely accepted as the most important known regulator of body fatness in mammals. This is roughly how it works. It's very similar to the model that Romain Hervey proposed, although not identical. Body fat tissue produces leptin in proportion to its size, and that leptin enters the circulation, travels to the brain, and acts primarily in the hypothalamus, although not exclusively, to regulate a variety of variables that relate to the size of body fat stores. This includes food intake and energy expenditure. So the way this works, if you try to lose weight, let's say you restrict your calorie intake, your level of body fatness goes down, your leptin levels go down, and your brain receives a signal and it enacts a coordinated physiological and psychological program designed to restore the lost fat, and that includes increasing your level of hunger, your interest in food, it includes reducing your physical activity, energy expenditure, reducing your, in some cases, basal energy expenditure, and it continues doing this until your fat comes back, and then your leptin comes back, and those responses normalize. So how does this work on a cellular level? We're going to have to go cellular here for the purposes of this talk. So here's a schematic of a cell. This would be a neuron in the hypothalamus. We have our leptin receptor here sitting on the membrane, and we have some little leptins floating around. So when leptin binds to that receptor, you get cascaded events happening in the cell that activates a protein called stat3. Stat3 goes into the nucleus and it has effects on gene expression that mediate leptin's effects on just about everything related to the body's energy availability, including appetite, metabolic rate, physical activity, reproduction, and growth. In addition, the leptin receptor activates another pathway called the PI3 kinase pathway. This is the same pathway that's activated by insulin, and it has similar effects in part through transcription of genes in the nucleus as well. So if we have this system that regulates body fat levels and tries to keep them at a stable level, how is it that obesity can occur? How can obesity develop if you have this system that's regulating body fatness? Well that's thought to relate to a phenomenon called leptin resistance. So leptin resistance is very similar to insulin resistance. So in insulin resistance, your liver or your muscles can't respond very well to that insulin. It takes more insulin for those biological effects to occur. And so in leptin resistance, it takes more leptin for your brain to feel like you have sufficient fat stores to feel like you're not starving. So what you observe in leptin resistant obese people, and obese people in general, is you have a new regulated steady state in which body fat levels are higher, leptin levels are higher, and food intake levels are higher. And this is actually a state that's defended against changes just like a lean person's level of fat mass is defended against changes. And just to show some of the more compelling data that have been produced to support this idea, this concept has extensive support in animal models of obesity, but I wanted to show you some human data as well to bolster this. These data are from the basis for what I'm going to show you is from Rudy Lible's lab. And he's shown, as have many other groups, that there's a fairly tight relationship between a person's lean mass, that is their total mass minus their fat mass, and their total daily energy expenditure. So the higher your lean mass, the higher your total energy expenditure. And you can draw a pretty good line through this if you get a bunch of people together. However, what he's also shown, and by the way, this holds true of both lean and obese people. Doesn't matter what your level of body fatness is, you still fall on this line approximately. What he's also shown is that when he weight reduces people by 10%, doesn't matter if they're lean or obese, they fall off this line. So their energy expenditure per unit lean mass goes down. So that's part of an energy conservation strategy that the body initiates to resist weight loss. And it's part of a suite of things that the brain initiates, including increased hunger, increased interest in food, increased muscular efficiency, which means you burn fewer calories for the same contraction, and reduced levels of thyroid hormone. But what he did next was what was really, really interesting. So he took weight-reduced obese people and he replaced their leptin levels up to the pre-weight loss level, just to the level that they were at before they lost weight. And what he found is that this largely eliminated this metabolic adaptation, psychological and physiological adaptation to weight loss, and it brought their energy expenditure per unit lean mass back onto this line. So what this shows is two things. One, that obese people actively defend their higher weight, and that's one of the main reasons why it's difficult to lose weight sustainably. And two, that this relates to changes in the leptin signal. Okay, so let's move on to cellular causes of leptin resistance. Let's come back to this schematic here, the simplified schematic of leptin signaling. So we have the leptin signal going through the STAT3 and PI3 kinase pathway. So what cellular signals can disrupt these signaling pathways? Well, one thing, ironically, is elevated leptin. So if your leptin goes very high, there's actually a feedback mechanism through this protein, SOX3, that dampens that leptin signal and dampens its effects in neurons. But in fact, there's another thing that can activate SOX3 besides high levels of leptin, and that is inflammatory signaling in the cell. Inflammatory signaling can also activate a second protein called PTP1B that also negatively regulates leptin signaling through the STAT3 pathway. And then a third mechanism is the buildup of lipid metabolites within the cell, and we'll get into more detail on this in a moment, but that activates the protein PKC theta, which has been shown to dampen the PI3 kinase arm of leptin signaling. So together, these cellular mechanisms can greatly diminish the ability of leptin outside the cell to change the activity of neurons that mediate leptin's effects on physiology and psychology. And just to give you some of the studies that support the relevance of some of what I'm talking about here, if you take regular mice and you knock out important inflammatory pathways specifically in the brain or in the hypothalamus, and then you put those animals on a fattening diet, what you see is that they are resistant to fat gain, they are more leptin sensitive than regular mice, and they experience less weight gain and less fat gain. Furthermore, the same applies to those factors that dampen leptin signaling that I just showed you, SOX3, PTP1B, and PKC theta. When you get rid of them in the hypothalamus, you get animals that are increasingly leptin sensitive show reduced weight gain and reduced fat gain in response to a fattening diet. So these things are causally related to the development of leptin resistance and obesity in animal models of dietary obesity. Okay, I'm going to skip these next couple slides here, we don't have time for them. So now we arrive at the most speculative portion of this talk. This is the possible diet and lifestyle contributors to leptin resistance. I want to emphasize that research is ongoing on this, and what I've done is to do my best to collect plausible mechanisms from the literature. These are by no means established to cause leptin resistance and obesity in humans. So I just want to start with that caveat. So the three mechanisms of cellular leptin resistance that we discussed so far are inflammatory signaling, accumulation of lipid metabolites, and excessive leptin exposure. So what diet and lifestyle factors can trigger those? Well one of the most important inflammatory triggers in the body is these cell surface receptors called toll-like receptors. These are receptors that have been crafted by evolution to specifically respond to molecules associated with bacteria and viruses. So they're basically detectors, cell surface detectors for bacterial and viral infection. And there are a variety of these TLR proteins and they each have their own special molecule that they respond to. But once these receptors are activated, they activate an innate immune response in the cell. In other words, they trigger an inflammatory response. And one of these that's received a particularly large amount of attention in the obesity and metabolism field is TLR4, toll-like receptor 4. This responds to a bacterial cell wall component called lipopolysaccharide or LPS. So there's tons of bacteria in your digestive tract. All of us have tons of LPS in there. Normally your gut barrier does a good job of excluding almost all of it from your circulation. But if some gets in, it's highly inflammatory. So it's been shown by Remy Bursalin's group and his colleagues in France that when you take animals and you put them on a fattening diet, compared to animals not on a fattening diet, they experience much higher levels of circulating LPS. Endotoxin is basically LPS. These animals also experience altered gut bacteria, heightened intestinal permeability, and visceral obesity. But what they did next is really interesting. They actually infused LPS into mice not on a fattening diet. And they found that when they brought their LPS levels up to the same level as the animals on the fattening diet, these animals exhibited a very similar syndrome, including visceral obesity. And they did the same thing in animals that were deficient in this TLR4 signaling pathway that LPS signals through, the animals were just fine. And it's also been shown that if you induce gut dysfunction, for example, colitis in animals, you get an increase in visceral fat mass, and in humans, intestinal permeability is associated with higher levels of fat mass, or visceral fat mass. So the Burel-Sinan group went further. So they showed, as I said, that these fattening diets promote gut dysfunction, visceral obesity, inflammation, and insulin resistance. However, since that involved this alteration of the microbial community and gut health, they wanted to see if they could block it by adding fermentable fiber, which is a substrate that's basically food for these gut bacteria. And what they found is that, indeed, the addition of fermentable fiber to these fattening diets made them less fattening and less metabolically damaging. So the point is that gut bacteria need healthy food, too, and that food is fermentable fiber. Okay, so to summarize the effects of LPS on leptin and insulin signaling, you have a bunch of bacteria in your gut, and those bacteria, many of those species are full of LPS. Normally, this LPS is almost completely excluded from the circulation by the gut barrier. However, when you eat an unhealthy diet, or perhaps for other reasons as well, and your microbial community is disrupted and your gut health declines, that can allow some of this LPS both directly through and between the cells of your intestinal barrier. And this promotes inflammation and leptin and insulin resistance. Okay, another thing that is inflammatory is excess fat mass. And to illustrate that, here's a schematic of fat tissue over the course of increasing body fatness. So in a lean person, you have small fat cells, you have a few immune cells that are not activated in an inflammatory way. As a person gains fat, the fat cells increase in size, you get the recruitment of activated immune cells to that tissue, and you get the secretion of inflammatory factors. And that situation basically just continues to become exacerbated the more fat a person gains and the larger their fat cells become. And now it has not been established that inflammation in fat tissue causes inflammation in the hypothalamus. We still don't know what the connection is there, but it's logical to speculate that there might be some kind of connection. Refined processed food is also inflammatory. To illustrate that, I'm going to show the results of a study on the effects of diet on post-meal inflammation, or post-prandial inflammation. So they compared two diets. One they described as a high-fat, high-carbohydrate diet. And this is eggs, sausage, muffins, sandwiches, and hash browns. It's basically processed meat, refined carbohydrates, fried potatoes, versus an American Heart Association approved diet, which although it may not be viewed by some people in this audience as the ideal diet, is composed primarily of unrefined foods. So oatmeal, milk, orange juice, raisins, peanut butter, and I don't know why they had an English muffin in there. It seems kind of like the sore thumb there, but in any case, they were both identical in calories although the high-fat, high-carb diet was higher in fat. And they tracked the levels of glucose and blood fats over the course of the next three hours and found that both were higher after the high-fat, high-carbohydrate diet, which is not that surprising considering it's a more rapidly, easily digestible meal. Furthermore they showed that the levels of LPS, which is that inflammatory bacterial compound, went much higher on the high-fat, high-carbohydrate diet over those three hours after the meal. And it was continuing to increase even after those three hours were over. And associated with that, when they looked in the people circulating immune cells, they found higher levels of oxidative stress, higher levels of inflammatory activation, and higher levels of SOX3 activation, which is that inflammatory activated protein that dampens leptin signaling. So again, we don't know exactly what the connection is between this type of inflammation and inflammation in the hypothalamus. However, the fact that LPS levels are much higher and SOX3 levels are going up makes it highly plausible that this would have inflammatory effects in the hypothalamus. And by the way, this postprandial inflammatory response is known to relate to certain components of the diet, in particular the concentration of plant phytochemicals such as polyphenols. So having more polyphenols reduces this response. Physical activity is anti-inflammatory to the brain. We know that physical activity protects against obesity in animal models, and we know that it's associated with less weight gain over time in humans. And we have pretty good reasons to believe that that's more than just an association. The mechanism for this is pretty well worked out in animal models. So when you exercise, the stress, the muscular cellular stress of exercise causes that muscle to secrete an inflammatory, or what's typically considered an inflammatory cytokine called interleukin 6. Interleukin 6 goes through the circulation to the brain, and paradoxically in the brain it actually has anti-inflammatory effects. So I think the brain is basically recognizing this as a signal that you've engaged in physical activity. And this causes inflammation to decrease, and leptin sensitivity, and insulin sensitivity to increase. So the second factor after inflammation is lipid metabolite accumulation. So normally you have your neurons, and you have fatty acids hanging out outside the neurons, and these enter the cell. Some of them become acyl coase, some of them become diacylglycerols, which are lipid metabolites, and typically these end up in the mitochondria getting burned for ATP, which is the cell's energy currency. However, if you have more fatty acids coming into the cell than the cell is able to handle, then you get an accumulation of lipid metabolites in the cell. These activate that protein PKC theta, which dampens insulin and leptin sensitivity. So how do you get cellular fatty acid overload? Well, one way is to gain body fat. So what this graph shows is the rate of release of fatty acids from fat tissue on the vertical axis versus total fat mass on the horizontal axis. Each one of these represents one person. What you can see is that there's a pretty good relationship between the amount of fat a person has and the rate at which that person's fat releases fatty acids. The more fat you have, the more fatty acids your fat is releasing, and the higher the rate of fatty acid exposure to your lean tissues. Another mechanism that's received some attention in the research community relates more specifically to diet. What this group has shown is that when you feed animals a diet that's enriched in polymitic acid, which is the main saturated fatty acid in body fat and in the diet, versus oleic acid, which is the main mono-unsaturated fatty acid in the body and in the diet, you get higher levels of acyl coase and diacyl glycerols in neurons with the palmitate than you do with the oleate. That translates to higher levels of PKC theta activation, leptin resistance, and higher body fat gain, at least in the conditions of this particular experiment. Now, I don't want to take this too far because it's been shown that dietary fats with very little saturated fat content are able to produce obesity just fine and rodent. I'm not sure exactly how relevant this mechanism is, but I wanted to mention it because it has received a lot of attention in the literature and there are some studies supporting this mechanism. The third factor is elevated leptin levels itself. If you're looking through the literature on this, it can be pretty confusing because there are some studies showing that elevating leptin levels experimentally in animal models doesn't really cause durable leptin resistance or obesity. So if you elevate leptin levels, animals become very, very lean as you would expect and they develop a moderate level of leptin resistance, but when you take it away, they don't retain the leptin resistance and they don't gain fat. However, what's also been shown, this is what I think is the most interesting thing, is that if you do that in the context of a fattening unhealthy diet, that excess leptin paradoxically accelerates the fat gain, which is the opposite of what it should be doing. So on the left, we have two groups of animals on a healthy normal diet. One group is receiving leptin. You can see it's gaining less weight than the group that's not receiving leptin and it has a lot less body fat if you do body composition. However, on a fattening unhealthy diet, you get accelerated weight gain, really remarkably accelerated weight gain and the development of leptin resistance. So this suggests that when in the presence of other inflammatory or harmful aspects of a diet, elevated leptin can actually exacerbate and contribute to leptin resistance and fat gain. So how do you get elevated leptin? Well, obviously you can gain body fat, but there's one detail of leptin signaling that I didn't mention in the schematic that I showed you earlier. And that's that leptin responds not only to your level of body fat but also to the amount of food that you've been eating recently. So it's both, even before your body fat changes, it can respond to your calorie intake. So it's both body fat mass and recent calorie intake. So if you eat more calories than usual, your leptin level will increase. And normally that's a signal to your brain that says, hey, we ate a little too much. I'm going to feel a little less hungry in the next few days. And it's been shown that that does occur in humans. A few days after we eat too much, we tend to eat a bit less. However, in the presence of an unhealthy diet, that may be able to, the increase in leptin plus the other effects from the unhealthy diet may be able to dampen that leptin signal and create or contribute to a state of leptin resistance. And I think this is really important. I think this is really relevant. And one of the studies I keep coming back to over and over again, a very, very simple study is a study of holiday weight gain in the United States. In this particular study, researchers, in their particular cohort, Americans gained just over 0.6 kilos per year over the course of the entire year. So each year these people were gaining just over 0.6 kilos. However, what they also showed is that during the six-week holiday period, people gained almost half of that much. So almost half of this annual weight gain, 52% of it, to be exact, occurs over only 12% of the year corresponding to our annual period of feasting primarily on unhealthy foods. And of course, it won't be any surprise to you to learn that people's calorie intake goes up. They tend to eat more unhealthy foods. There's a lot of sweets and snacks around. And this weight doesn't go away. It's retained. So holiday weight gain, most of that is retained. People say they're going to lose it in January. It turns out they don't, at least not on average. And it just builds up. The next year it gets added to, the next year it gets added to, et cetera, et cetera. And we gradually, through these periods of overeating unhealthy food, ratchet up yearly our level of leptin resistance and body fatness. So to summarize what I've been talking about today, first of all, I just want, I have this little, little word here down in the bottom corner, genetics. And I didn't talk about genetics today, but I just want to acknowledge that it is very important and it influences everything we talked about today. How much time do we have left? Okay. Influences everything we talked about today. So I just want to acknowledge that. So we know that on a cellular level, SOX3, PTP1B, and PKC theta activity can suppress leptin signaling. And we know that those can be activated by high levels of leptin, by inflammatory signaling, and by the accumulation of lipid metabolites. And we know that those factors can be driven by overeating, by obesity itself, by low quality food, and by physical inactivity. So that's it. I want to acknowledge my former mentor, Mike Schwartz, my colleagues, Josh Thaler and Catherine Berkseth who contributed data that unfortunately I had to skip over in my haste and all the other researchers who made this talk possible. If you enjoyed this talk, check out my website at www.wholehealthsource.org. Thank you. Yeah. So we have time for some questions. Great talk, Stefan. Thank you. I don't know if you care. So the story about leptin is very compelling. I guess the other hormone and the other kind of resistance that might play in here is insulin resistance. How do you, what context do you put that in? Is that part of the same story or is there some differentiation? Is there some evidence that subcutaneous fat versus central adipose tissue might give off those hormones differentially? And are there any differences for the type of fat that you have, any implications for the types of foods you should avoid or is it basically boiled down to the same story for both hormones? So are you talking about insulin resistance in the brain or in the periphery? Both. Both. Okay. So insulin in the brain is kind of like leptin's kid brother. It serves a relatively similar function. And so many of the same things that I discussed that are relevant to leptin are also relevant to insulin signaling specifically in the brain. In the periphery, the mechanisms are somewhat overlapping. There's definitely a lot of overlap. PKC-theta was originally identified as a factor that contributes to insulin resistance in the periphery in response to fatty acid metabolite accumulation in the cell. In terms of the association between insulin resistance and body fat distribution, I really don't know whether that's... I don't know which one is the cause and which one is the effect, but I would love to know the answer to that question. The other question was, depending on your distribution, does that have any implications for the kinds of foods you should avoid or does it come down to the same kinds of dietary prescription? No matter which. Yeah. I'd have to think about that a little bit. The only thing I'll say off the top of my head is I've seen some suggestion that people who have a more central distribution of body fat and insulin-resistant phenotype benefit more from carbohydrate restriction than people who don't. That's the only thing off the top of my head that I can think of. Do you have a view on cold thermogenesis and leptin resistance? From an ancestral perspective, well, not specifically for leptin resistance, but from an ancestral perspective, I think cold thermogenesis is probably something that's a good idea to do from time to time. It's another form of physiological stress that we evolve to tolerate and thrive on like exercise. It's something that you can do just like you can build your muscles through exercise. You can build your thermogenic capacity through cold exposure. I've done the polar bear plunge for the last two years in Lake Washington just for kicks. Last year I took cold showers in anticipation of it and definitely made it easier. As far as leptin resistance, I really don't know. So a number of your slides made use of the terms like unhealthy diet, low quality diet, fattening diet. Would you mind defining what you mean by that? Yeah, yeah. That's a good question. I deliberately left that fairly non-specific. The reason is because we use these refined fattening diets in our rodent studies that differ in a number of different ways from the healthier comparison diet that we use. Based on these studies, we can't make any specific claims about what dietary elements are responsible for the effect. What I can tell you is the fattening diet, typical one that we use is 60% fat. That's primarily lard-based. And then the remainder is refined carbohydrates with a little bit of sugar, a little bit of cellulose for fiber, and then some vitamins and minerals. So we're not sure exactly what it is about that diet that's fattening and unhealthy, but I could speculate another time, but I'll go on to the next question. Yes? I've read about a study that purported to show that if you have metabolic syndrome or diabetes, wheat, even whole grain wheat, doesn't suppress appetite in the same way it would in a normal person and relating that to leptin. Wheat specifically? Wheat specifically. It was comparing wheat and oats. Okay. People who had metabolic syndrome and those who didn't, and showing that they responded normally fullness to oats, but sort of didn't register they'd eaten to the wheat. Yeah, I'm not familiar with that, but it sounds interesting. Are there any lab tests that you would recommend to show levels of leptin and or leptin resistance? Well, the only test that I would recommend would be to look in the mirror because it's highly correlated with body fat levels. So I mean, if someone is obese or has formerly been obese, chances are very high, but no, there's not really any lab tests that is currently available. And this is actually a major limitation of research in humans because in animals you can directly test leptin resistance by putting leptin into the circulation or into the brain and watching their food intake and body fat levels decrease. You can't do that in humans, or at least it's very difficult to do. And so it's a lot harder to study leptin resistance in humans. Linda. Yeah. So in Stefan Lindberg's Ketavan people who all weighed like 25 kilos less than the Swedish control group, they had extremely low leptin levels. So any theories as to the sort of paradoxically low leptin? Well, I'm not sure they were lower than they should have been given their body composition. I don't know. Yeah. I think they may have been similar to what they would be expected to be for that low level of body fatness that they exhibited. Last question. So you said that if someone was obese, even if they lose the weight, can the leptin resistance stick around? Is there a way to reverse that? How do you judge if you've maybe beat it? Yeah, that's a really good question. If you ask Rudy Lible, who's one of the top researchers in this area, he'll say we've had people on that have been weight reduced for five years and they never get over this. However, I'm not so sure about that. I mean, certainly under his conditions, that's true. I tend to think that certainly the defended level of body weight and possibly also leptin signaling depends on other factors. It's not really a rigid number that you set like in your thermostat. It really depends on the dietary and environmental context. There's not a lot of evidence to support that in humans, not a lot of direct evidence, but I think there's enough indirect evidence to make it a plausible speculation. So I believe that it's possible, and I mean, certainly in rodents you can reverse leptin resistance. We just published a paper actually showing that you can by using a drastic dietary shift back to a healthy diet. It does actually eliminate essentially their leptin resistance and hypothalamic inflammation, but until further research is done, we won't have a very clear idea of whether or how that's possible in humans. Thank you very much, Stefan.