Please join Christopher Axelrod and Sander Kooijman, PhD for a discussion on their research into combatting obesity using various new therapies.
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Sarah: Good morning, good afternoon, and good evening, everyone, and welcome to our webinar titled “Rodent Models of Pharmacotherapy and Chronotherapy for Obesity and Cardiometabolic Disease.” This webinar has been sponsored by Sable Systems International, so a big thank you to them for helping to make this event possible. Joining us today, we’re very fortunate to have Christopher Axelrod from Pennington Biomedical Research Center and Dr. Sander Kooijman, an associate professor at Leiden University Medical Center. Their presentations will discuss their research into combating obesity using various new therapies. And with that, I’m very pleased to welcome our first presenter, Christopher Axelrod. Chris, thanks so much for joining us today and the floor is yours whenever you’re ready.
Dr. Axelrod: Thank you, Sarah. Good morning, good afternoon, good evening to everyone in attendance today. Today I’m going to discuss some ongoing work in our lab addressing the need for improved pharmacological strategies to treat obesity-related diseases, with a particular emphasis on a recently identified small molecule mitochondrial uncoupler named BAM15. I’ll then show some data to establish the safety, tolerability, and efficacy of BAM15 for the treatment of obesity with a secondary focus on glycemic control. Finally, I will show evidence pertaining to an adipose tissue specific mechanism whereby BAM15 mediated mitochondrial uncoupling lowers body weight and improves glycemic control.
It should come as no surprise to individuals in the audience that obesity is a widespread and uncontrolled non-communicable disease. As shown on this figure on the left, the global prevalence of obesity increases by approximately 1% or 78 million people annually, with the most aggressive growth occurring in the Americas, Africa, and Southeast Asia. More than $2 billion are spent annually addressing medical and societal causes and consequences of obesity. Outside of excess weight gain, obesity is a primary risk factor for the development of many other non-communicable diseases, such as type 2 diabetes, hepatosteatosis, hypertension, sleep apnea, and certain types of cancer. However, unlike other diseases, patients with obesity encounter intense stigmatization, where societal blame is placed on the person, not the disease. As such, many patients opt out of or do not receive adequate medical treatment, which in turn exacerbates the cost burden, morbidity, and overall mortality of disease.
For those patients who opt for treatment, the overarching goal is to reduce and sustain body weight. There are three overarching paradigms used to achieve and sustain weight loss. Behavioral therapies such as exercise, calorie restriction, and/or the combination are widely used and generally effective for short-term weight reduction. However, burden is high, adherence is low, and long-term relapse is common. Surgical treatments for obesity emerged over 70 years ago and are now considered by many to be the gold standard for durable weight reduction. The most common surgical approaches are sleeve gastrectomy, which reduces the size of the stomach by approximately 75 percent, and Roux-en-Y gastric bypass, where a smaller stomach pouch is made and connected to the distal intestine by a Roux limb. Both approaches yield superior weight loss to behavioral interventions and are durable for at least 10 years from the time of surgery. However, many patients are not eligible for surgery and for those who are, only approximately 1% elect to have the procedure. Additionally, procedures such as RUE and Y have greater complication rates than behavioral interventions and can result in lifelong mineral deficiencies that detract some patients from treatment. Lastly, there are a limited number of pharmacological approaches, including oral and injectable medications, that alter satiety, appetite, and or absorption. The drugs that are available tend to have limited efficacy and are less effective than surgical approaches for weight management, and thus is the focus of our discussion today.
Currently, there are four approved drugs used in obesity management. Orlistat, which prevents absorption of lipids by enzymatically blocking lipases, is safe for long-term use and produces approximately 5% weight loss. Phentermine-topiramate, an appetite suppressant, is commonly prescribed for short-term weight reduction and can produce approximately 10% weight loss. Similarly, Naltrexone-bupropion treatment suppresses appetite and is prescribed as a combination therapy to exercise and calorie restriction to produce approximately 10% weight loss. More recently, Liraglutide, the GLP-1 agonist, was approved for obesity management and achieves approximately 10% weight loss sustained outwards of three years. Like Phentermine-topiramate and Naltrexone-bupropion, Liraglutide largely reduces weight by suppressing appetite, but some metabolic effects have been observed, such as enhanced insulin secretory function. However, most of the currently approved medications cause mild to severe gastrointestinal discomfort and fall dramatically short of producing the weight loss required to improve quality of life in patients with moderate to severe obesity. Furthermore, pharmacotherapy typically requires combination therapy with behavioral and or surgical interventions to achieve the degree of weight loss required for health benefit. As such, there is a critical need for medications that, at minimum, manage obesity-related disease or, at best, can reverse the causes and consequences of obesity.
A major limitation to therapies that reduce energy intake is that, over time, metabolic adaptation occurs, which necessitates greater suppression of intake. As such, at a certain point, the patient will be eating significantly less food to maintain body weight had they not lost weight in the first place. For those reasons, increasing energy expenditure has long been desired as a target for obesity management. Energy expenditure agonists work by increasing metabolic output in one of two ways. The first involves direct stimulation of thermogenic processes or pathways, as is observed with adrenergic receptor agonists or drugs that mimic sympathetic stimulation. No such drugs have been used for the clinical management of obesity, but there’s a renewed interest centered around thermogenic activation of adipose tissue, as observed with cold exposure or specific types of beta-3 adrenergic receptors. Unlike sympathetic stimulation, uncoupling agents such as 2,4-Dinitrophenol (also known as DNP), FCCP, or Niclosamide work by restricting energy efficiency and will be the focus for the remainder of our discussion.
To briefly outline how uncoupling works, under homeostatic conditions, Oxidation of substrates such as pyruvate, malate, succinate, and or glycerol 3-phosphate are tightly coupled to production of ATP through the generation of high-energy electron carriers that are oxidized by the electron transport chain. This series of stepwise redox reactions results in a proton efflux into the intermembrane space, which establishes the electrochemical potential to drive ATP synthase. To overcome the limitation of efficiency, electron flow is subsequently increased in an attempt to restore ATP concentrations, and in turn, substrate oxidation is also increased.
DNP is one of the earliest and certainly best-known compounds that induce mitochondrial uncoupling. DNP was first used by the French during the First World War in the production of ammunition. At this time, factory workers reported abnormal side effects, including sweating and unintentional weight loss, that later was attributed to DNP poisoning. In the early 1930s, Tainter and colleagues conducted landmark first in human studies where patients with moderate obesity received DNP orally and were followed over a three-month period. DNP appeared to be well tolerated and produced marked weight loss in most patients, sometimes upwards of 1.5 kg per week. Over 100,000 patients were reportedly treated with DNP in the mid-1930s. However, at very high doses, DNP can cause acute toxicity, overdose, hyperthermia, cataracts, fever, and fatigue. After the formation of the FDA in 1938, DNP was banned federally. However, black market use and distribution persists today, primarily in fitness athletes.
It would take more than 30 years after the work of Tainter and colleagues for Peter Mitchell to elucidate the mechanism of action for DNP and other compounds that exhibit uncoupling properties. Since then, dozens of such compounds have been discovered, many of which are used routinely in mitochondrial research. However, prototype uncouplers such as DNP or FCCP have narrow therapeutic windows and off-target effects such as depolarization of the plasma membrane, which entirely limits pharmacological development in the native form. Recently, a weakly acidic lipophilic cation named BAM15 was identified by a small molecule screen to have potent uncoupling activity in vitro, and it selectively accumulates in the mitochondria. However, until recently, the therapeutic potential of BAM15 for treatment of obesity-related diseases remained entirely unknown. The data I am showing you today address three primary research questions. Our first question was simply, is BAM15 safe, available, and or tolerable? Our second question focused on the ability of BAM15 to prevent weight gain in a preclinical model of obesity. We performed secondary experiments to establish the ability of BAM15 to improve other comorbidities, such as glycemic control and steatosis. Based on our efficacy data, we then sought to determine the weight-lowering mechanisms of BAM15.
One of the essential parameters for safe delivery of an uncoupler is body temperature. DNP, among other prototype uncouplers, are noted for inducing hyperthermia-related symptoms, which drastically limits the therapeutic window and value. We first tested this by IP-injecting mice with variable concentrations of BAM15 in measuring whole body temperature over the course of a 24-hour period. IP injection of BAM15 did not alter body temperature at any dose. We then provided BAM15 orally, which allowed for greater delivery of compound, and similarly, we observed that BAM15 had no effect on body temperature. For these experiments, we conducted extensive safety monitoring and observed no unfavorable effects of BAM15 on these mice. Based on these outcomes, we then measured BAM15 in the blood and tissues and found that BAM15 was orally available, the half-life was relatively short (no more than three to four hours), and that the primary distribution occurred into adipose tissue.
We additionally performed histopathology of all major organ systems following three-week exposure to oral administration of BAM15. And consistent with our other in vivo data, BAM15 did not appear to negatively impact organ structure or function in key tissues such as brain, liver, kidney, muscle, or intestines. In fact, in some circumstances, BAM15 appeared to improve histological parameters, as was noted in the liver, with a marked reduction in lipid deposition, a point which will be addressed briefly later in this presentation. Based on our preliminary studies, we felt comfortable in asserting that BAM15 was indeed orally available, appeared to be relatively safe, and accumulated into lipid-rich tissues, a desirable feature of an obesity drug.
We then shifted our focus on establishing preclinical efficacy for obesity treatment. To achieve this, two types of trials were conducted. In the first trial, wild type C57 Black 6J mice were placed on high-fat diet to induce obesity, and were subsequently randomized to receive either placebo treatment which was high-fat diet alone, or 0.1% BAM15 supplemented into high-fat diet for three weeks. In the second trial, we aim to dissect the weight-lowering contributions of BAM15 to enhanced metabolic parameters by minimizing mice to either high-fat diet alone, 0.1% BAM15 in high-fat diet, or calorie restriction of high-fat diet to match the body weight of BAM15-treated animals.
In trial one, mice treated with BAM15 were protected from diet-induced obesity compared to control animals. Notably, there was a reduction in fat mass accompanied by sustained lean mass compared to control animals. In trial two, mice treated with BAM15 or calorie restriction were protected from diet-induced obesity compared to control animals. However, BAM15 treated animals displayed significantly lower fat mass compared to both control in calorie-restricted animals. Furthermore, lean mass was entirely maintained in BAM15 treated animals, whereas calorie restriction resulted in a modest reduction in lean mass. Taken together, we concluded from these experiments that BAM15 prevents fat gain.
After three weeks of treatment in trial one and two, we conducted IPGTTs to determine if BAM15 improved glycemic control. In trial one, BAM15 lowered fasting glucose and insulin compared to control animals. During the IPGTT, BAM15 treated animals displayed significantly lower area under the curve glucose, driven primarily by rapid decline in glucose from the 30- to 120-minute time point, which is more routinely observed in otherwise healthy mice. In trial number two, BAM15 and calorie restriction lowered fasting glucose and insulin compared to control animals, consistent with trial one. Notably, both BAM15 and calorie restriction improved area under the curve glucose relative to controls. However, BAM15 treated animals displayed enhanced glycemic control compared to calorie restriction as well. Taken together, we concluded from these experiments that BAM15 enhances glycemic control independent of body weight.
We then briefly focused on other clinical parameters related to obesity and glycemic control through histological analysis. As you can see on the left panel, BAM15 essentially reversed hepatosteostosis and prevented beta cell hypertrophy, which is quantified on the right. Overall, these data combined with the obesity and glycemic control studies gave us the confidence that BAM15 was favorably improving obesity-related disease that was not strictly a function of weight reduction. Since BAM15 elicited a more favorable metabolic phenotype than weight-matched calorie restriction, we then turned to understand possible mechanisms whereby BAM15 lowers body weight and improves glycemic control. Though clinically significant, the idea that uncouplers can reduce body weight is not new. As illustrated earlier in the talk, DNP was one of the first drugs used to treat obesity and several other compounds such as NEN and PPC1 lower body weight in mice. Despite these findings, the mechanism remains unclear. Furthermore, since these drugs are functionally but not structurally related, it’s entirely possible that the mechanisms of weight regulation are not entirely shared.
Since food intake is the major driver of weight control, we first evaluated changes that may have occurred in animals from trial one and trial two. As you can see on the left and middle panel, BAM15 did not alter daily food intake whatsoever compared to control mice. This data was further confirmed in trial two, where the food intake was similar between BAM15 and control animals but was significantly lower by design in calorie-restricted animals. Based on the known effects of uncouplers on thermogenesis, we then conducted an experiment where animals were placed into a Promethion system for seven days following three weeks of treatment to evaluate potential changes in energy expenditure and substrate utilization. Interestingly, despite no change in body temperature as displayed earlier in this talk, the BAM15 treated animals exhibited significantly increased daily energy expenditure, which was driven entirely by changes during the dark phase. Since BAM15 was administered through the diet, the drug has a relatively short half-life, and the animals eat primarily during the dark phase, these results appear to be consistent with our pharmacodynamic studies. Notably, BAM15 treated animals also displayed a lower respiratory quotient compared to controls, indicative of greater whole-body fat utilization. Taken together, these data demonstrate that BAM15 prevents weight gain in part by increasing whole-body energy expenditure.
Since mitochondrial encouplers work by inducing mitochondrial stress in the form of lowering energy efficiency, we shifted our focus on identifying signaling nodes that link uncoupling-mediated processes to alterations in substrate utilization. For example, it has previously been demonstrated that uncouplers such as nucleosamide and SR4 activate AMPK in target tissues, which has been associated with enhanced metabolic parameters. To achieve this, we first conducted an untargeted RNA sequencing on cells treated overnight with a vehicle solution, or BAM15. The resulting transcriptome was filtered to identify lead targets of interest and piped into Ingenuity software for pathway analysis. From this screen, we generated an enormous amount of data, which is simplified in the following figure. To overview briefly, BAM15 activates AMPK both as a direct effect of uncoupling and through indirect stimulation of the insulin receptor signaling pathway. A number of downstream targets are then activated, such as SIRT1/FOXO signaling and GLUT4 translocation, to enhance cellular uptake of nutrients. Since BAM15 primarily accumulated into adipose tissue, we then sought to identify whether AMPK was indeed activated. Consistent with our sequencing data, AMPK was markedly elevated in white adipose tissue. Other tissues such as brain, liver, and heart were evaluated, and no AMPK activity was observed.
In white adipose tissue, AMPK can serve multiple roles including influencing the rate of lipolysis and lipogenesis. This finding led us to believe that maybe BAM15-mediated AMPK activation in white adipose tissue was enhancing lipolysis, which in turn was increasing the pool of fatty acids available for oxidation. As it turned out, BAM15 had no effect on lipolysis in any of the white adipose tissue depots. However, palmitate oxidation rates in skeletal muscle were markedly increased. We had difficulty for some time conceptualizing where the fat was coming from to support increased nutrient oxidation.
We then turned back to our histological analysis and observed some dramatic effects in white adipose tissue. As you can see in the left panel, BAM15 almost entirely prevented adipose tissue expansion, a routine function and response to excess lipids. On the right, you can see that there are considerably more small, immature adipocytes in BAM15 treated animals compared to control. These data indicated to us that rather than enhancing lipolysis, BAM15 was more likely limiting adipogenesis, which frees the pool of fatty acids to be oxidized in more metabolically demanding tissues, such as skeletal muscle. To address this issue, we performed expression analysis of genes essential to lipogenic processes in white adipose tissue. As you can see, nearly all genes required for lipogenesis and synthesis of adipocytes were dramatically inhibited. Some genes, such as PPAR-gamma and the adipose triglyceride lipase, had no expression in BAM15 treated animals. We are currently working more extensively to understand how bioenergetics efficiency may regulate lipogenic processes and hope to have more data to show you all in the future.
To briefly summarize, BAM15 is an orally available mitochondrial uncoupler that prevents diet-induced obesity and improves glycemic control in C57 black 6 mice. BAM15 results in a more favorable body composition and glycemia phenotype compared to calorie restriction, indicating they are mechanistically dissimilar. BAM15 appears to reduce body weight primarily through increasing energy expenditure in a way that does not alter body temperature. Mechanistically, BAM15 appears to favorably alter systemic energy homeostasis through AMPK-mediated suppression of lipogenic processes in adipose tissue. Overall, these data provide pre-clinical efficacy for BAM15 and related uncouplers for application in the treatment of obesity and related diseases.
Importantly, I would like to acknowledge the individuals who contributed to this work. This work could not have happened without the countless support from Pennington Biomedical and our external collaborators. Briefly, I would like to thank John Kirwan for his mentorship throughout this project and commitment both physical and financial resources to seeing it through. Additionally, I would like to thank Will King and Kathryn Pergola from my team who remained open and optimistic despite the numerous challenges we faced during this project. I would also like to briefly acknowledge our funders and am open to any questions you may have. Thank you for your time today.
Sarah: And without further delay, I’m very pleased to welcome our second presenter, Dr. Sander Kooijman. Sander, the floor is yours whenever you’re ready.
Dr. Kooijman: Okay. Thank you, Sarah. So, I’m going to talk about circadian rhythms. And having said that, our body is designed to be active and to eat during the day and to sleep during the night. And the energy demand has to be adapted accordingly. And that’s probably why if most, if not all metabolic processes are under circadian or diurnal control, and this rhythm has to be synchronized and that’s done by a light exposure mostly but also a temperature, physical activity, food intake, and then there are hormones in force that involve that prolongated signal. So, as is the autonomic nervous system. But there is a strong circadian rhythm in many metabolic processes. And that’s probably why any small problem in alignment of one of these factors will result in an increased risk for cardiometabolic diseases. And that’s particularly true for shift workers who have an increased risk for obesity, diabetes, and cardiovascular disease. However, the problem with those diseases is that they are chronic and progressive. So, it can be both difficult and very time consuming to study these associations in humans, let alone the effect of interventions. For that reason, we actually need animal models to study the underlying problems and to determine the most effective interventions to prevent cardiometabolic diseases caused by shift work or other metabolic disturbances.
For the first part of my presentation, I’ll focus on circadian rhythms in brown adipose tissue, and brown adipose tissue is an organ that uses lipids and glucose for the production of heat. But from an energetic perspective, it doesn’t make a lot of sense for brown fat to be active all day. It actually makes much more sense if brow fat is only active when we don’t have our muscles, when we’re not physically active, so during the night when we’re asleep, and when we don’t have our muscles to produce heat, or during the cold winter months. And the fact that brown fat takes up glucose and lipids for the production of heat can also be used to monitor its activity. So, in humans, we use the glucose as a proxy for its activity. So, the uptake of glucose by the tissue in the left side, you see the glucose uptake during thermoneutral conditions. And on the right side in this image, there’s a market uptake of this radiolabel, this radiolabeled glucose by a lot of brown fat deposed under cold conditions. And we can actually do the same in mice. But what we’ve shown before is that probably the provincial substrate for thermogenesis in brown fat is our lipids. So, what we do actually in our lab is we take radio-labeled triglycerides. So, the fatty acids within the triglycerides are radio-labeled and we incorporate those triglycerides into lipoprotein-like particles and we inject those particles into the mice and monitor the uptake of the radio-labeled fatty acids by the metabolic organs, including brown adipose tissue. And just to show you what it looks like, I have this example of this specific experiment and I chose this experiment because here we were interested in the effects of salsalate and salsalate, its suspected mode of action was actually true AMPK activation. And when we give salsalate to the high-fat diet fat mice, we actually see quite a remarkable weight loss in these animals, which was due to a decrease in fat mass and accompanied by a decrease in circulating triglyceride levels. And when we inject those mice with the radio-labeled fatty acids, then we see in the animals that were treated with this salsalate, we see quite a remarkable increase in the uptake of these fatty acids by brown fat. And further molecular analysis actually showed that it was brown fat that was responsible for the metabolic phenotype.
You can also take this one step further and look into the consequences for cardiovascular disease. And what we’ve done in this experiment is inject APOE3-Leiden.CETP mice, which is a model for human-like lipoprotein metabolism and atherosclerosis development. We injected them with a specific beta-3 adrenergic receptor agonist, CL316243 compound, and when we give those mice again the radio-labeled lipoproteins, we see a rapid clearance in the treatment group from the circulation, so the lipids are rapidly clear from the circulation. And in the right-hand panel, you can see that they actually all end up in the brown fat deposed, which was good because this beta-3 adrenergic receptor is almost selectively present on brown fat in the mouse. So, this activation of brown fat and the uptake of fatty acids resulted in a marked decrease in plasma triglyceride levels, but not only triglyceride levels were decreased, but also cholesterol levels are lower in the animals treated with the CL compound. And that’s because delipidated lipoproteins are more prone for uptake by the liver. So, you get enhanced uptake of cholesterol enriched lipoproteins by the liver. And that’s why cholesterol levels also decrease. And the consequence of the combination of these effects of the lower triglycerides and lower cholesterol levels is decreased at those sclerosis formation. So, we can actually protect ourselves from cardiovascular disease by brown fat activation.
This is the current working model on the protection of cardiovascular disease through activated brown fat and I already explained part of it. So, in the middle, you see the triglyceride rich lipoprotein and fatty acids are extracted in the presence of lipoprotein lipase by the brown adipose tissue, and that results in delipidation of the lipoproteins. And those lipoproteins can rapidly be taken up by the LDL receptor on the liver. And they don’t end up or form a risk for atherosclerotic plaque development. And on the right side of the figure, you see that there are also phospholipids budding off these lipoproteins. So, when the lipoproteins become smaller, the phospholipids in the shell become redundant and they actually bud off and intercalate with the HDL pool and the HDL becomes more functionally active because of this intercalation with the phospholipids. And the HDL can therefore take up more cholesterol from, among others, foam cells in the atherosclerotic plex, which can then, and the HDL then removes, and the liver then via SRB1 actually removes cholesterol from the HDL, thereby reducing cholesterol (atrogenic cholesterol) in the circulation and also arterioscleroticplaque development.
All right, but now back to the circadian rhythms. So, in this experiment, we looked at the effects of prolonged artificial light exposure in mice, in regular black six mice on a chow diet, and we exposed the mice to either 12 hours of light per day, 16 hours of light per day, or even continuous light. And what we noticed was that with prolonged light exposure, the mice gain more fat mass. And when we then inject those mice with the radio-labeled lipoprotein-like particles and monitor the fatty acid uptake by the various organs, we only found pronounced differences in the uptake by the brown fat, which is quite diminished in the prolonged light exposure conditions. So, we actually interpreted this result as brown fat is taking up less lipids, so the lipids become available for other tissues to store them. And that’s why the animals gain in fat mass. But this experiment also suggested to us that there must be a circadian rhythm in brown fat. And that’s what we investigated in this next experiment.
So, here we injected those radio-labeled glycerides over the course of today in different light conditions. So, we have a normal light dark condition in the middle and a prolonged light dark condition, prolonged light condition on the right side, and actually a short day also, and that’s what we also included in this experiment on the left side. And what you can appreciate is that there is hardly any rhythm in the fatty acid uptake by the liver, independent of the light cycle. But if we look at the uptake of these fatty acids by brown fat, we see quite a pronounced rhythm here. So, there’s a gradual increase in the uptake of fatty acids by brown fat during the light phase, so when the mice are actually asleep. And as soon as they wake up at the start of the dark phase, brown fat is probably not needed anymore for heat production, so the brown fat is shut off again, so the uptake of the fatty acids also drops again. And what’s quite striking here is that we also see changes of differences between the different light dark conditions. So, in the long summer like conditions on the right side, you see as there is a day-night rhythm in the uptake of fatty acids by brown fat, but it’s not as pronounced as in the shorter days on the left side. So, it’s very tempting to speculate that this mechanism prepares the body for temperature changes that are coming up with the seasons.
So, I told you on my first slide that hormones and the nervous system are responsible for transmitting the signal of the light-dark conditions among others. And in this experiment, we actually followed up on the observation that corticosterone rhythms nicely follow the circadian fatty acid uptake by the brown fat. So, what we did in this experiment is experimentally flatten corticosterone levels by implanting a pellet containing a very low dose of corticosterone in the mice and thereby this flattening of the corticosterone rhythms as you can see in this panel. This is confirmation that experiment works well. And what we then see on the various adipose, the adipose is first of all, in the vehicle-treated mice confirmation that we have a circadian rhythm in the fatty acids update by brown fat. But this rhythm is completely ablated in the corticosterone-treated animals. So, corticosterone is an important signaling molecule in the circadian regulation of brown fat.
All right, I’ll take you back to the previous study that I showed you on the circadian regulation, the circadian uptake of fatty acids by brown fat. And what we could show was that this uptake in the fatty acids by brown fat translated – or were actually reflected – in the circulating triglyceride levels in these animals. So, when brown fat is highly active, triglycerides are low, but not only baseline triglycerides, but also post perennial triglycerides are dependent on the activity of brown fat. So, maybe given olive oil bolus, when brown fat is most active in this case, at time eight, so where the green arrow is located, then the triglycerides do not get the chance to accumulate in the plasma, while at other time points they are. And as postprandial triglycerides are an independent risk factor for cardiovascular disease, this may suggest that timing of food intake can be an important factor in the risk of cardiovascular disease.
But this is of course in mice, so how about humans? Well, actually it’s about the same. So, in humans, we suspect that brown fat is most active at wakening, so at 9 AM in this case. And in this study, we gave volunteers an isocaloric meal, so the same amount of calories during breakfast and lunch and dinner. And what you can appreciate from this figure is when you give this meal at breakfast, lipids do not accumulate a lot in the blood, but during lunchtime and during dinner, those lipids do accumulate in blood, similar to what we observed in mice. So, actually it’s an old saying, so eat breakfast like a king, lunch like a prince, and dinner like a pauper may be true.
So, what are the implications of eating at the wrong time, which is actually happening during shift work for cardiovascular disease? In this study, we went on with a slightly different model, but this is a model for shift work in mice, and where we expose mice to weekly changes in the light-dark cycle. So, either controlled light-dark conditions or exposure to this weekly change, so the complete shift in light-dark cycle, a weekly six hour advance or a weekly six hour delay, as you can see in these activity grams, actograms on the top of the slide. And when we then look at atherosclerosis formation, so these are stained slides from the heart, so the aortic valve area of the heart, and the arrows indicate atherosclerosis formation, so thickening of the vascular wall. We can clearly see that these circadian disturbances, the shift work model leads to enhanced atherosclerosis formation, and this is the quantification of it. So, with these more extreme full shifts in the light-dark cycle, so the weekly alternating light dark-cycles, we see more than a doubling of the atherosclerotic lesion area and not only the lesion size was increased, but also there was a shift from more mild lesions to more severe lesions. So, that’s really not good. And this was actually the first study where we showed in a non-genetic model that circadian disturbances can accelerate and so sclerosis formation, thereby increasing the risk for cardiovascular disease.
But, to our surprise, we did not see any effect in the study on brown fat function or on plasma lipid levels or on weight gain. And also, there was no effect on circulating immune cell number or activation status. So, what was going on? We did quite a lot of studies, quite a lot of other measurements to finally figure out that there was an increased number of macrophages within the lesion and that was accompanied by increased signs of oxidative stress and therefore enhanced immune attraction to the vasculature. So, here in the bottom figure you see 4-HNE staining as a measure for metabolic oxidative stress and that was quite pronounced, so where’s this oxidative stress coming from? That’s probably related to a difference in food intake. I’ll come back to that in one of my later slides. But for now, I’d like to focus on another difference we had during our experiments, and that’s related to the difference in brown fat function and the difference in weight gain that we observed throughout our experiment. And this probably relates to sexual dimorphism because all the studies on weight gain were performed in male mice and the adverse sclerosis study was performed in female mice.
So, we were interested in this potential sexual dimorphism and that’s why in one study we compared males exposed to a jet lag to females exposed to jet lag. In this particular study, the mice received a jet lag of six hours every three days. And what you can see is that while the males are coping pretty well with the jet lag, so there’s a slight shift in their activity as you would expect, because you’re giving them a jet lag, so they’re adapting to it. The males actually showed quite a strong reduction in their daily activity, so the rhythmic activity was really attenuated. So, the males and the females really respond differently to sort of shift work model. When we then look at fat mass over time, we see again, as shown before, an increase in fat mass in the male mice, but not in the female mice. This increase in fat mass in the male mice was accompanied by a decrease in fatty acid uptake by brown fat, like we’ve seen with the prolonged light exposure, but again in males only and not in females. But in females, we saw something else happening. So, I haven’t talked about glucose. I only talked about triglycerides right before. But when we look at glucose levels in the males, they are not affected, plus my insulin levels are increased. So, there’s some form of insulin resistance in the males that are exposed to jet lag. But in females, the females do not respond very well to this jet lag, at least not by increasing their plasma insulin levels. So, we see a rather increase in glucose but also in triglycerides levels in these female mice.
So, this is the hypothetical model that we have so far on the metabolic risks of shift work. And so what I suspect is that a misalignment between food intake and the body’s ability to process the nutrients and leads to accumulation of triglycerides and glucose in the circulation. And in male mice, these triglycerides are taken up by the adipose tissue deposition, so you get fat mass accumulation, possibly ectopic fatty position as well. While in the females, these triglycerides and glucose remain in the circulation and thereby forming a risk for the vasculature. So, glucose may lead to glycosylation of proteins in the vasculature, it can disturb endothelial, this function leads to oxidative stress, therefore local inflammation, a little bit of insulin resistance as well. And in females, that’s why we see in females increased atherosclerosis formation, while in males we mostly see increase in fat mass.
Okay, so there are a lot of outstanding questions that I still intend to answer during the coming years, but I will highlight at least three of them. So, one of the obvious questions is “how to prevent cardiometabolic problems caused by circadian disruption?” One of the obvious things you can do is adjust the eating time or take away the fat and the glucose from the diet, and that’s actually what we’re currently investigating. But one other question you might ask is “do we have to take circadian rhythms into account when measuring metabolic activity” and I’ll give you one example of that. In this study that was already published in 2015, we were interested in the metabolic phenotype of angiopoietin-like-4 knockout mice, and angiopoietin-like-4 is an inhibitor of LPL activity. So, if you don’t have angiopoietin-like-4, you have active lipoprotein lipase and therefore low plasma lipid levels. At least, if you measure at the right time point, because this study was actually performed only last year, in wild-type mice, we see a nice circadian rhythm in the activity of lipoprotein lipase, and this rhythm is ablated in the angiopoietin-like-4 knockout mice, and is translated in a rhythm in fatty acid uptake by the brown fat in wild-type mice and the ablated rhythm in the knockup mice, but actually effects of angiopoietin of the knockouts are only apparent when you would measure the activity in the light phase, and that’s what we did in this previous publication. We were unaware of the circadian rhythm, but just for practical reasons, we performed the measurements in the light phase. But if you would have performed the same measurements in the dark phase, we would have come to a completely different conclusion and probably have concluded that angiopoietin-like-4 plays only a minor role or no role in regulation of brown fat activity, which is absolutely not true.
Okay, so my third question is “should we consider circadian cycles in metabolic tissues germ therapy?”. So, this is known as chronotherapy. And I’ll give you an example here as well. So, what we did in this experiment is expose mice that are housed at 21 degrees. We exposed them to cold for four hours per day at four degrees. So, it’s four degrees of Celsius for four hours per day while the animals were fasted. And for this experiment, you have access to a 32-cage Promethion system that does all this protocol automatically for us, which was very helpful. And when we then look at energy expenditure in these different groups that are exposed to cold at different times of the day, we actually see in them a red dotted line that cold exposure in the early light phase may actually be more effective than cold exposure at the early dark phase. So, when brown fat is not so active, we can promote its activity much more in the early light phase.
However, when we look at activity levels in these mice, and that’s a bit difficult to see maybe, but what we can see is that if you focus on the red lines, so the early, the mice that were cold exposed during the early dark phase, you can see that the activity drops as soon as the cold exposure starts. So, any changes in the effect of on energy expenditure may have been masked by changes in physical activity. So, mice may not be a very good model to study the effects of cold exposure or timing of cold exposure on energy metabolism.
So, that’s why in this next experiment we moved on to a pharmacological treatment. In this case, Mirabegron is a beta-3 adrenergic receptor agonist. And here are sort of similar to the cold exposure, we see that by looking at the black lines and the black dots, we see that when treating mice with Mirabegron at the early light phase, we may have a slightly larger effect on energy expenditure, and this time it was not accompanied by changes in activity, which we also expected. And when we then look at brown fat activity, so in all cases, we see in the fatty acid uptake by the brown fat depot in the right panel, in all cases, we see that Mirabegron increases the uptake of fatty acids by brown fat, which we expected. And if any but the effect is not so pronounced, the effect during the early light phase is bigger than the effect of Mirabegron at the early, when given at the early dark phase. So, again, so how about humans? Here I exposed myself to cold. I would not recommend you to do that, but everything for science. So here I exposed myself to cold for two hours. And what you can see on the right graph is that my energy expenditure increases quite, quite substantial in the early morning, Well, it doesn’t at the end of the day, and this is just me, but we have repeated this now, repeated this in 10 more individuals, and except for one, they all showed the same phenomenon. So, at the cold exposure in the morning leads to a larger increase in energy expenditure than in the evening. And this is accompanied by a change in temperature of the supraclavicular region, so where brown fat is located.
Okay, so to summarize, metabolic activity in brown fat protects from cardiometabolic diseases. Brown fat activity display shows a pronounced diurnal rhythm with the highest activity at the onset of wakening and is reflected in post perineal lipid levels, so it matters when you eat, circadian disruption leads to reduced brown fat activity and weight gain in male mice and accelerates atherosclerosis development in female mice and circadian rhythms in brown fat and probably also in other metabolic tissues should be considered by measuring its activity or when therapeutically targeting this tissue.
With that, I would to thank all my collaborators and due for your attention and I will be happy to take any questions.
Sarah: All right, so this question is for you, Chris. Have studies been conducted to determine the long-term safety and efficacy of BAM15?
Dr. Axelrod: No, not formally. They have not. But we do have some studies in older mice currently, where we are evaluating efficacy and safety, these are over 20 to 30 weeks of treatment, whereas in the previous studies that I showed today, they were three to four weeks of treatment. And the tolerability looks similar, at least in our hands.
Sarah: Okay, great. And, Sander, this question is for you. Are female shift workers more susceptible to cardiovascular disease compared to male shift workers?
Dr. Kooijman: Yeah, that’s a very good question. And, well, the very short answer is that I don’t know. That’s because all the studies that have been performed so far are typically in people with specific occupations. So, the female shift workers are often nurses while males are often factory workers. So, it’s very difficult to compare those. But for obesity, there is a little bit more data and it seems that like in the general population, that shift workers with obesity are often males.
Sarah: Okay. We have another question here for you, Chris. Has your team investigated whether BAM15 alters differential mobilization of different fatty acids? In short, might BAM15 treatment alter the fatty acid composition of the body?
Dr. Axelrod: That’s a good question, and we have to a limited extent. We haven’t done lipidomics or anything far reaching, but the analysis that we have done shows that they’re all similarly reduced, short, medium, and long chain fatty acids. So, it doesn’t appear on the surface that, as an example, certain fatty acids are being elevated, whereas others are being depressed. It appears that short, medium, and long are all being similarly oxidized and utilized.
Sarah: Okay, that makes sense. All right, before we continue with our Q&A, because I know we are just a minute over already, if you do have to go now, we would love to hear from you in the survey. So, that will come up on your screen right now. And then if you can stick around for a couple more minutes, we’d love to answer some more of your questions. So, I will continue here. Sander, this question is for you. Do you have any breathing testing data that shows the rates at which the animals are oxidizing endogenous lipids?
Dr. Kooijman: No, that’s actually a question that we would love to answer. We’re actually planning to do a study with stable isotopes measuring how fast the brown fat responds in terms increasing energy expenditure when injecting lipids during cold or during thermoneutrality. So, it’s a very good, very interesting question.
Sarah: Yes. Okay. Chris, this question is for you. Did BAM15 alter brown adipose tissue function for thermogenesis? And did you observe UCP1 activation in white or brown adipose tissue?
Dr. Axelrod: Thanks, Sarah. And I guess to briefly answer that question, no. brown adipose tissue function did not seem to be altered at all and there’s no UCP1 activation or you know beiging or browning of white adipose tissue and it’s not changed in the brown adipose tissue, so this is a UCP1 independent mechanism which makes sense since we are exogenously uncoupling the mitochondria, at least to me.
Sarah: Okay great. We’ve got another question here I’m not quite sure who it’s for, but maybe you can both take a crack at it. This person has asked, have you measured the energy expenditure at thermo-neutral conditions?
Dr. Axelrod: In our models, we didn’t publish that, but we have, and the phenotype is exacerbated, interestingly. So, whatever the mild cold stress that’s being induced at human thermo-neutrality is slightly depressing the phenotype which is much more rapid in terms of onset and large in terms of magnitude when we do it nearing thermo-neutral conditions.
Dr. Kooijman: Yeah and we also we also have done experiments at thermoneutrality and there we see actually in some aspects a more pronounced circadian rhythm in brown fat and that’s because with this at thermoneutrality of course, you’re taking away a very dominant driver of brown fat function. So, the sympathetic nervous system is not active anymore. So that’s why you see much more pronounced rhythm in certain aspects of brown fat function.
Sarah: Fantastic. Okay. Just in interest of time, this is going to be our last question. So, this question is for you, Chris. This person has asked, do you plan to take this research into humans, either in vitro or in vivo?
Dr. Axelrod: That’s a great question. We do not have immediate plans to do that. The IND process is very long and painful, but we are in discussions right now to do more formal pre-clinical testing and in larger animal models that would be required to obtain an IND to do either early phase one or phase one type studies. But other labs and ours included think that this is likely going to require some type of structural modification to the native compound for it to be more suitable for humans since the half-life is fairly short and mouse metabolism is very rapid. So, it’s entirely possible that the drug will be structurally modified down the road to make it more suitable for humans.
Sarah: Okay. Fantastic. All right. Well, I just want to take a moment here to thank you both for your fantastic insights today.
Dr. Axelrod: Thank you, Sarah.
Dr. Kooijman: Thanks.
Sarah: I want to send one last shout out to our sponsor, Sable Systems. Thank you so much for making this event possible, and we look forward to having everyone with us again soon.