Tuesday, September 19, 2023
8:00 am PT / 9:00 am MT / 10:00 am CT / 11:00 am ET
Please join Sable Systems and our partner, Inside Scientific, for the upcoming webinar “Exploring Estrogen’s Role in Metabolism and the Use of 13C-Labeled Nutrients for Advanced Animal Physiology and Nutrition Research.” The webinar will take place on Tuesday, September 19 at 8:00 Pacific Time. The registration link is below.
Reilly Enos, PhD – Harnessing the Power of Estrogen to Regulate Metabolic Processes: Dr. Enos’ research focuses on the role that sex steroids and their receptors play in regulating metabolic processes, particularly in the setting of obesity. He will discuss his research on tissue-specific fluctuations of sex steroids throughout the estrous cycle in mice, provide insights into the importance of the quantity of estrogen necessary to impact physiological processes, as well as an understanding of the central versus peripheral effects of estrogen action.
Eran Levin, PhD – Unlocking Insights: Utilizing 13C Labeled Nutrients for Cutting-Edge Physiology and Nutrition Research: Dr. Levin will discuss the potential of using 13C-labeled nutrients in physiology and nutrition research in animal models. Specifically, he will share practical tips for designing and conducting experiments using isotopic labeling techniques and demonstrate how they can provide unprecedented insights into metabolic pathways, nutrient utilization, and behaviors in both vertebrate and invertebrate models including insects, reptiles, and mammals.
There will be a short Q&A session following each presentation.
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Exploring Estrogens Role in Metabolism and the Use of 13C-Labeled Nutrients for Advanced Animal Physiology and Nutrition Research
Liam Sanio: Good morning, good afternoon and good evening, everyone, and welcome to this Inside Scientific webinar titled “Exploring Estrogens Role in Metabolism and the use of 13C-Labeled Nutrients for Advanced Animal Physiology and Nutrition Research.” This is Liam Sanio from the events team here at Scientist.com, and I’ll be the host today. This webinar has been sponsored by Sable Systems International, so a big thanks to them for helping to make this webinar possible. We’re very fortunate to be joined by Doctor Riley Enos, who’s Assistant Professor in the Department of Pathology, Microbiology and Immunology at the University of South Carolina School of Medicine, and doctor Eran Levin, head of the Nutritional Ecology Lab at the Tel Aviv University School of Zoology.
And now just a few housekeeping notes to help you get the most out of your experience. So first, this webinar is being recorded and will be available to watch on demand shortly after the live event. Next, if you take a look at the interactivity panel on the right-hand side of your screen, you’ll see four tabs corresponding to chat, Q&A, polls, and handouts. If you happen to experience any technical issues, please first try refreshing your browsers. This tends to fix most issues, but if you’re still having trouble, you can reach out to us using the Q&A tab or using the private chat function, and we’ll help you to troubleshoot. We’d also love to hear from you, so please send us any questions, thoughts or comments using the Q&A tab. And we’ll address as many of your questions as we can in a Q&A session following the presentations. And finally, you can also explore a few relevant resources in the handouts tab to learn more about some of the content presented today. And just before I welcome the first speaker, we just have just one quick audience poll. And it is, which of the following research models do you use? So here you can let us know which of these models you use, whether that’s most models, rats, insects, other small animals or large animals, if you use human participants in your research, or you can click that last option if you don’t use any research models for your work. Seeing the results come in, it looks like, yeah, most people are using most models, a few using rats. But yeah, we have a bit of, you know, some people using all of these. So yeah, it’s cool to see the diverse range. All right. And without any further ado, I’m very pleased to welcome the first speaker, Doctor Riley Enos. Riley, thanks so much for joining us today, and the floor is yours whenever you’re ready.
Dr. Riley Enos: Thank you. Liam. Okay, so today, with respect to the webinar, I’m going to give you a brief outline of what I’m going to talk about. I’m going to talk about sex steroids in general, including androgens and estrogens, clinical morbidities linked to estrogen deficiency, and then I’m also going to give a brief overview of some of the questions that elude scientists with respect to estrogen and how we, as a group, a lab group, are trying to shed light on these unanswered questions using basic science in animal models. And then finally I’m going to talk about the translation or relevance of our research, just because we want to understand from a basic perspective how we can help patients.
So, jumping straight into it, we are well aware that there are a number of steroids; we’re going to focus on the sex steroids – the androgens and the estrogens in particular. We’re going to focus on these estrogens, estradiol to be specific. I want to bring your attention to aromatase – CYP19A1 is also known as aromatase. It is important because it is necessary to convert androgens into estrogens. Now when we talk about sites of steroid synthesis, the predominant sites are the gonads. So, when we think of a female, we’ll think of ovaries. When we think of a male, we’ll think of testes, right? And then the adrenal glands will also synthesize steroids, in particular the adrenal cortex. Now there are also secondary sites where androgens, for example, can be metabolized into estrogens -adipose tissue, brain, skeletal muscle, kidney. These are examples of tissues which do metabolize androgens. And to give you a brief snippet of that, for example, you can have androstenedione or testosterone that goes into the tissue of interest and gets metabolized through that aromatase enzyme into estrogen, for example. Now when we talk about androgens and estrogens, there are primary androgens here. And there are also estrogens – E1, E2, E3, and E4. The primary estrogen that we’ll be discussing is 17-beta estradiol, also known as E2. And this is important because it is the primary estrogen during pre-menopause okay. In humans for example, estrone is the primary estrogen during post menopause. And that’s primarily synthesized, for example, by the adipose tissue. So, when we talk about estrogen throughout the lifespan and we look at the y axis over here, this is the percentage of maximum through an individual’s life. So, let’s look at estradiol for example. And this is in red, okay, the straight line here. And as you see as women transition into the menopausal transition, you see this steep drop off. Now the question then becomes is what is this drop off associated with? What is the problem with this drop off? And when we look at clinical morbidities, it’s already established that there’s a higher risk for diabetes in females during that age. And there was a clinical paper that was published that followed females through the post-menopausal or the menopausal transition. And they found that, yes, estradiol drops, follicle stimulating hormone increased, but they also found that visceral adipose tissue increases and activity decreases.
So given this baseline, okay, that of information that I’ve given you, what are some of the questions that elude scientists? How is E2 protective against obesity and associated metabolic dysfunction? Are these effects central or peripheral? Meaning, is the effect of estradiol in the brain driving the outcome, or is it the effect of E2 in the periphery, for example? And then one thing that we’re very much interested in is what levels of E2 are necessary to elicit certain physiological effects. Now, why is this important? Well, we know that E2 deficiency, for example, is also associated with osteoporosis. So, for example, do we need a minimal amount of E2 to rescue bone mineral density loss? Is there a certain threshold that we need to reach to impact E2’s effects in the brain, or affect inflammation, or affect glucose metabolism? These are questions that we’d like to answer. And in order to do that, we must understand the natural cyclicity of E2, which I’ll discuss later in a few slides.
So, before I talk about these or give answers to these questions or thoughts to these questions, we need to understand what animal models we can use to answer these questions? So, the two models that we primarily use in our experiments are the ovariectomy or aromatase knockout mouse model. Now the ovariectomy mouse model is very straightforward. You simply do a bilateral removal of the ovaries. And this is very quick and easy to do. And it produces non-detectable or very low levels of plasma estradiol. The drawback of this particular model is the fact that there’s no perimenopause, right, you just basically lose estrogen almost immediately. And depending on the age at which you OVX, the duration of sexual maturity can be a factor. So, we typically wait until the mice are sexually mature, you know, weeks of age. And then we induce that surgery. The other model is a genetic model, which is the aromatase knockout model. And if you remember in our previous slides, we know that aromatase is necessary to convert androgens to estrogens. So, if we have an aromatase normal mouse, we should theoretically have no estrogen.
So, this brings us to the first question is how is E2 protective against obesity and associated metabolic dysfunction? Are the effects central or peripheral? Now we know centrally from previous research from established investigators that removal of E2 from the brain leads to increased food intake and decreases physical activity. So, we know that estradiol impacts food intake and physical activity, and peripherally, primarily through deletion or blockade of the primary estrogen receptor ER-alpha, we’ve been able, or scientists have been able, to assess the impact the signaling has on peripheral tissues.
Now we will talk about data that is not published, but here is an example. So here are two models of estrogen deficiency. And what we have here is female wildtype mice – C57BL6, all 10 to 11 weeks of age. And here they are on a low-fat diet. We put these mice on a high fat diet and sure enough, they gain weight. And if you ovariectomize and put them on the high fat diet, they also gain a significant amount of weight, more so than the female wild type alone. Now what is interesting, if you look at the female aromatase knockout mice, so these are both low fat diet and high fat diet, they start baseline at a much heavier weight than these other mice. However, when we normalize to delta or body weight gain, we can see that the OVX and the aromatase knockout mice gain a similar amount of weight over time. So, what is driving this difference in baseline? Well, these mice don’t move as much, and they eat a little more. And because of that, they already at ten weeks of age show an increase in body weight gain.
So, we have access to the Promethion metabolic cages from Sable Systems. And this is established, but we show that sure enough, if you look at just meters moved or physical activities, general physical activity in the cage, both the ovariectomized mice and the female aromatase knockout mice, they move less. Now, if you give them access to running wheels – and if you don’t know this, mice like to run, especially during the dark cycle – and we show that we are running is dramatically decreased both in the OVX and the aromatase knockout mice, suggesting that there is a central effect at play. Also, food intake is also increased in the knockout mice, okay? So given these central effects, how do you tease out central versus peripheral effects? Because the argument can be made that sure enough, if you have these central effects which leads to increased food intake, decreased physical activity and weight gain, it may be that the central effects truly are the predominant player in regulating the metabolic dysfunction And it’s not necessarily the peripheral effects or the peripheral action of E2 that really is important for the beneficial effects of E2 therapy.
So, what we did is we designed a mouse model that allows us to inducible turn on aromatase in skeletal muscle. And this is the Doxycycline induced model, so we treat the mice with Doxycycline. When Doxycycline is given, it leads to skeletal muscle aromatase overexpression. And we did two different studies – a prevention study and an intervention study. And we did both gonadally-intact and ovariectomized female mice. And the purpose of the prevention study was to say, hey, let’s give a high fat diet and Doxycycline, turn on skeletal muscle aromatase at the initiation of the experiment. For the intervention study, we wanted to get the mice fat and obese and then turn on skeletal muscle aromatase to see if that would be beneficial as a therapeutic option. The idea being that if you turn on or increased skeletal muscle aromatase, you should then have increased E2 or estradiol in skeletal muscle.
So, what we found is that both in the intact and ovariectomized mice models, we did not find any benefit with respect to differences in body weight or body fat percentage between the genotypes. There was obviously a diet effect, but there was no genotype effect okay, neither for visceral fat in both the OVX and intake models. Interestingly, however, we did see an increase in uterus in both the OVX and intake models and bone mineral density. Now I know the question is going to be “What are the E2 levels”, and we’ll get to that in a second. Now with respect to glucose metabolism, we do not see any benefit. But with respect to adipose tissue inflammation, we looked at markers, macrophage markers in particular CD68 which is a pan macrophage marker in CD11c which is a pro-inflammatory macrophage marker in adipose tissue, and we saw reductions. Now if you’re not familiar with inflammation in adipose tissue, inflammation in adipose tissue is a hallmark of obesity in the primary immune cells – and driving that inflammation are macrophages. So, this suggests that in our mouse models we did reduce adipose tissue inflammation.
Now for the intervention study, again, remember that we got the mice fat first and then initiated overexpression of aromatase. We did not similar to the prevention study, see any benefit with respect to body composition changes. We didn’t even see bone mineral density changes. We did not see adipose tissue inflammation changes, nor did we see any metabolic changes. We did, however, see a change in uterus size. So, this leads us to what about the estrogen levels. And the number one issue that plagues sex steroid researchers is the ability to accurately, at very low levels, measure androgens and estrogens in particular. So, we have developed a methodology – or we didn’t develop it, but we’re using a previously published methodology – where we can derivatize E2 and measure it at this limit of quantification. So, with that being said, we assessed the androgen and estrogen levels of testosterone and E2. But I really want you to focus on the E2 levels in these two models. So, in the prevention model, we can clearly see that E2 was increased in both the intact and ovariectomized mice. Also, when you just look at wild type mice you can see the difference between intact mice and ovariectomized mice. So, there’s a big decrease in E2 in skeletal muscle upon ovariectomy. Surprisingly, we found that this E2 leaked into plasma, leaked into circulation, as you can see here. Now for the intervention study, we did increase E2 in skeletal muscle, and it did leak in the circulation, but it was less than what we found in the prevention study.
Now, we didn’t publish this in our in our publication, but what we did do is after we gained the ability to assess adipose tissue steroid levels, we then did a follow up analysis. And what we found is if you look at female high fat diet, wild type in adipose tissue, this is the level of E2 that you would find. Here’s the OVX, it’s about 2.5 picograms or lower per gram of tissue. And here’s our prevention level, and here is our intervention level in the adipose tissue. So, this suggests that this level of E2 was sufficient to reduce inflammation. But this level was not. Also, based upon the fact that in the prevention experiment we were able to increase bone mineral density, right, this level of plasma E2 is probably sufficient to impact bone mineral density, but this was not. Now, granted, it also may be the time or the timing of how long the bone was exposed to this level of E2, but it just goes to show that level of E2 is important to probably impact physiological outcomes.
So, to go further into this point, we did a follow up study in males. And so again we did an intervention experiment gave Doxycycline to overexpress aromatase in skeletal muscle. And surprisingly – or maybe not surprisingly – what we found is a reduction in body weight, improved body composition change, decreased fat mass, decreased visceral fat liver weight, hepatic limit accumulation, and if you look at this is actually a handout that you have access to, but we show reduced adipose tissue inflammation and improved glucose metabolism. So why did the males respond better than the females? We have to remember that for this particular model testosterone is converted to E2. So, these are males, and they have higher testosterone levels. And because they have higher testosterone levels, you would think they have higher E2 levels. And sure enough, what you found is in the skeletal muscle, we had significantly higher E2 levels in both the skeletal muscle plasma and adipose tissue. And if we go back to our previous slide, you can appreciate the fact that in the adipose tissue, you’re only getting about 15 picograms here per gram. And in plasma you’re only getting about 26 picograms in the prevention study. And here we’re getting a drastically higher amount. And it’s likely that this higher amount leads to an increase in the brain which impacts this.
And to follow up on that point, we obviously put these mice or not obviously, we put these mice in the metabolic cages. And what we found is there was a difference in respiratory exchange ratio and food intake, which led to a decrease in energy balance. Now of interest was the fact that the RER was lower within about 72 hours. So, we saw changes in RER very early on, where food intake was not impacted until after 72 hours, suggesting that whatever happened, the leakage of E2 into circulation promoted fat oxidation or promoted a lower RER, which suggests that there’s increased fat oxidation. We would obviously have to perform follow-up studies to conclude that. But interestingly, we saw the decrease in total food consumed, which led to a negative energy balance. This did not impact physical activity, but it impacted food intake, suggesting that there was a central effect of E2.
So, different levels of E2 seem to impact different physiological processes, whether that be in the periphery, bone mineral density, for example, adipose tissue inflammation or centrally when it comes to food intake. So why is this important for hormone replacement therapy? Well, I will discuss that in the next couple of slides. So, what levels of E2 are necessary to elicit certain physiological effects? And I pose this question, but in order to do that, we must understand the natural cyclicity of E2. And if we want to understand the natural simplicity of E2, we have to look at our models. So, when we look at humans and we look at mice, we conclude we clearly know in humans that females go through a menstrual cycle and where we see variations in the level of E2, for example. Mice are different than humans in that their cycle is about five days through different stages. And the general consensus is that during proestrus is when you see the highest levels of estrogen.
So, this, I’d like to say ahead of time, this is debatable because there is uncertainty in the field. For example, David Handelsman, who is a leader in this field, published a paper where he looked at E2 levels and he found during proestrus this was found to be the highest levels of E2. However, a follow up study in 2023, Handelsman was on the paper. But interestingly, they found that E2 is highest during diestrus. I encourage you to look at both these papers. I can’t explain the discrepancies, but this is the knowledge we had going into our study. So, what we did, this is an unpublished study, but we monitored mice, okay. We assessed the stage of the estrus cycle, and we focused on proestrus, estrus and diestrus 1. On a daily basis, we did vaginal lavage and looked at the cytology of the cells in order to determine the stage of the estrus cycle. And then after three weeks of confirming that the mice were cycling, we killed them (or sacrificed them) between 9 to 10:30 a.m. Mice were sacrificed, we collected their blood, and then we perfused the whole body with mass-spec grade water, 50 mils. And then we collected these 16 tissues and examined sex steroids. Now the uterus weight of these mice followed as expected; proestrus had the highest wet weight, followed by estrus, diestrus and OVX. And then serum E2 followed a similar pattern.
Now I will show you this data to share with you, it’s rather interesting looking at the different tissues. Here you will see that proestrus, estrus, diestrus and OVX – you can see the levels of E2 for all these different tissues. And what’s striking is the fact that proestrus over the course of all the tissues has the highest levels of E2 in every single one. OVX is almost non-detectable in every single tissue. And what was also interesting was the fact that we saw differences with respect to pancreas, for example, has very high levels of E2 during the proestrus, where this falls off during estrus and diestrus. Also, adipose tissue is a rich source of E2 in terms of content. Now we’ve also analyzed other steroids, but I’m not going to share this data. We’ve also started to analyze the brain, which is also very exciting. Again, I’m not going to show you this data because we’re focusing on E2 for example.
So, what’s the clinical relevance of this? Well think of hormone replacement therapy. There are different modes of hormone replacement – nasal pills, skin gels, skin patches, vaginal creams, vaginal rings, there’s combinational therapies. So, all of these therapies in different modes of deliveries will affect the bioavailability of E2. So, I know that there is weariness in the potential side effects due to whole body effects. So potentially the ideal goal would be to get tissue-specific delivery of E2. But the question then becomes is if we want to do tissue specific delivery, what level of E2 do we need to reach in order to have physiological benefits? And that’s basically what my lab or what our group is trying to figure out, because hormone replacement therapy in its current state is a whole-body treatment in most cases. And we don’t truly understand or don’t understand well enough the bioavailability – how the mode of delivery affects bioavailability.
So, in conclusion, we believe that brain is king just by the aromatase knockout we clearly show, or it’s established, that if you don’t have E2 in the whole body you’re essentially going to be impacted in the sense that physical activity dramatically decreases, and increased food intake happens. And this seems to occur, although the periphery is involved, it seems that brain is the key target and then inducible overexpression of aromatase and other tissues. This is what we’re trying to do. We’re trying to look at specific tissues to figure out the role of estrogen in those tissues. In the level of E2 we need to provide a therapeutic benefit. So, targeted estrogen therapies in the future — what are the needs of the patient? We need to think about motor delivery, type of estrogen, dose of delivery, when to administer and combinational therapies. These are all important for developing therapies that are effective and do not provide any off-target effects.
So, thank you very much. I’d like to acknowledge my current and former lab team, the funding faculty, mentors, and collaborators, especially Priscilla Furth and John McCarthy, providing some of the unique animal models that were described today. I’d also like to thank my mentors, Angela Murphy and then Deborah Clegg, who’s been a mentor from afar. And then, thank you very much. And more than anything, I’d like to say that we are happy to collaborate – we are one of the few labs in the United States to be able to measure E2 in multiple tissues. So, if you’d like to measure E2 or other steroids, please contact us. And then lastly, I’d like to thank Sable Systems. Although they have a great product, more than anything their service is second to none and we thank them very much. So, thank you. And please feel free to ask me any questions at the end of the talk.
Liam Sanio: All right, Riley. Well, thank you for a really fantastic presentation. Just before welcome the next speaker, we just have another quick audience poll. We would just like to know what your level of experience or expertise is with metabolic and or behavioral measurement. And so here you can let us know whether you have, you know, zero experience or if you’re anywhere up to very highly experienced. Already. And again, it looks like we have a really wide range of answers. We do have a few people that are really highly experienced, so that’s really cool to see already. And with that, I will invite Doctor Eran Levin here. Eran, welcome, thanks so much for being here. And yeah, you can take it away whenever you’re ready.
Dr. Eran Levin: Thank you. Yeah, so I’m a zoologist, it’s a kind of extinct animal, and I believe that the truth is out there. I believe that science today is focused on very specific 4 or 5 animal models. And that nature is full of other animals with extreme adaptation developed during millions of years of evolution. And I usually pick my models as their physiology or nutrition is special. And we work with shrews, the smallest mammals, with bats, with hawk moth, snakes and lizards, and hornets. And I will show you some of our work with these animals. And I will show you the work that we do when we use stable carbon isotopes to study nutrition physiology.
So first I have to say that during the last decades, the stable carbon isotope analyzer became very compact, very affordable, and easy to work with. And you can use them as classical respirometry to measure ratio of carbon in exhaled breath and the ratio of isotope in exhaled breath and also to measure in specific tissues the ratio between the stable isotopes. And I’ll show you some implications of it. And in addition, there are tons of enriched carbon and enriched nutrients that are commercially available. Almost any nutrient you can think about, you can buy when it’s labeled specifically on one carbon or all the carbon, and I will show you some examples. To the ones that don’t know much about respiratory, so classical respirometry, you put the animal inside the closed chamber, air comes in, air comes out. And when it comes out, you can measure how much carbon dioxide was exhaled by the animal and how much oxygen consumed by the animal. And you can calculate what metabolic fuel that the animal has used, which is the RER. And you can calculate, of course, the metabolic rate. And in addition, today you can add the stable carbon analyzer in parallel. And you can measure specific nutrients that you fed to the animal. You can measure how they go out in the breath of the animal. A more simple setting of this system is to get rid of all the analyzers of oxygen and carbon dioxide and work only with the stable carbon analyzer as also a carbon dioxide analyzer, and assume that you know the metabolic fuel that the animal is burning. And I will give more details about it in my talk.
And to work with tissues, you have to transform them into gas. So, you have to burn them in a combustion chamber in an oxygen rich environment, and then you analyze the gas from the tissue. And by the analyzer today, you need a very small amount of tissue for this kind of analysis – one milligram of dry matter, it’s like the brain of a bumblebee. So, we can work with very small animals in very small tissues and still have a signal from them for your work.
And now I’ll give you examples I use in my lab – macronutrients. I’ll talk about carbohydrates, fatty acids and amino acids, and I’ll give you some examples. Carbohydrates, there are many different carbohydrates that you can buy labeled. You can buy them labeled on specific carbon or labeled like glucose with six carbon labels. You can feed them to the animal and then track them when they come out in their breathing or track their specific metabolic pathways, and I’ll show you an example of something that we did. Here you see the RER for animals that feed on nectar. Nectar is very rich in sugar. These animals usually have a very high metabolic rate. On the left, you see the hummingbird, songbird (it’s the African version of the hummingbird) and nectar feeding bit on the right. And you can see the RER for these animals, and you can see that after they feed, the RER is above 1. To the ones that are not familiar with RER, RER is usually between one – when animals burn only carbohydrates – to 0.7 when they burn lipids. And in the middle is a mixture. And for nectar-feeding animals, you see that RER goes above one. It’s known for more than hundreds of years. And in the literature, it was written that it’s because of the synthesized lipids. But when you go to the metabolic pathway of lipid synthesis, there is no extra carbon dioxide when you synthesize lipids.
So, we worked with hawk moths, I like them because they have the highest metabolic rate known in nature. And when you feed the hawk moths and you test for the RER, you get the very high value above one. And in the beginning, we saw that we have a problem when we measure this very high RER. We also tested the monarch butterfly, again, very high RER. And to be sure that we are doing the measurement right, we tested bees. Bees always burn carbohydrates, and they should have high RER. So, when we measured it for a bee, we found that our system worked properly and was calibrated, and we got very high RER for the other animals. So, what can bring us extra carbon dioxide? We started going to the biochemistry, and we found that when you activate the pentose phosphate pathway in the oxidative phase of the pentose phosphate pathway, when glucose turns into ribulose, you get the carbon number from the glucose, goes out as carbon dioxide without consuming any oxygen. So, it’s a potential pathway that can explain the very high RER that we get in our feeding animals.
So, what we did next was feed the hawk moth with two different labeled sugars. First, we fed them with glucose-labeled carbon number one. So, we expect that if the RER goes down, there will be less labeled carbon, the delta 13C would be lower. And if we feed the hawk moth glucose-labeled on carbon number two, when the RER goes down, we will have more labeled sugar because we will have less unlabeled carbon from carbon one. And I will show you the results which will help. Here you see in black is the RER, and you see that when it goes down, also the delta C13 goes down like we expected when we labeled and fed them glucose labeled on carbon one. When we fed them with glucose labeled carbon two, we got exactly what we expected. When the RER goes down, there is more labeled carbon from carbon number two. And we can say that the high RER is because of the use of pentose phosphate pathway that we see in these animals, and in our paper, we explain why it’s important for nectar feeding animals, but this is for another talk.
We can also test complex sugars, the most common polymers in nature – cellulose and chitin. Cellulose, you can buy them labeled online. Chitin, we cannot find them anywhere to buy labeled chitin. And we wanted to know if hornets can break chitin. Hornets are very interesting animals because they hunt for other animals or eat carrion. They bring it to the nest, and they feed it to the larvae, and the larvae break the protein and the skeleton of the insect and feed it back to the adult. And to test if hornets can digest chitin, we reared beetles on labeled glucose for one month. Then we extracted the chitin from their skeleton, and we fed it to hornets without larvae, and to larvae. And then we tested their breath in the carbon analyzer. You can see that for the adult hornet, they couldn’t break any of the chitin. But you can see that for the larvae, they could break the chitin and they can easily digest it. Later, we tested the microbiome of the larvae, and we found three bacteria that are suspected to be able to break the chitin for these insects, and they’re absent from the adult, and it might explain how they can break the chitin, which is a polymer that most animals cannot break.
We also tested ethanol, which is very popular now in the study of insects and in mammals. Five percent of all deaths around the world are because of abuse of ethanol use. And we test if hornets can break ethanol because it’s very known that hornets are attracted to ethanol. And there are a lot of funny news about ethanol, crazy hornets in Europe especially. And in this experiment, we fed ethanol-labeled carbon one to a hornet and to a bee and put them in a metabolic chamber. And you can see the rate of oxidation of the ethanol by the hornet, which is extremely fast, while the bee very slowly metabolizes the ethanol. And this might explain why bees get poisoned by ethanol and get addicted while hornets are not. And it’s a project that we are working on right now.
I really like fatty acids. I think they have a lot of implications in ecology and in physiology of animals. And as you know, there are saturated fatty acids that usually you find in plants in warm areas, and unsaturated fatty acids that are more common in a cold area. And a lot of people think about good fat and bad fat. I don’t believe in this kind of thing, but there is a very big importance to understand what the ability is to assimilate this fat. And to test the stimulation of this fat, we tested three fatty acids, palmitic acids, linoleic acid, and oleic acid. These are the three most common fatty acids in our diet and in animal diet. Palmitic acid is saturated, oleic acid is monounsaturated, and linoleic acid is poly unsaturated with two double bonds. We tested two insect-eating vertebrates. First, it’s the pygmy shrew – it’s the smallest mammal in the world with the highest metabolic rate fed on insects. The second is a gecko – the same size as the shrew, but a reptile and insectivore. And the third one is a bumblebee – an herbivore that feeds on pollen. We fed this animal with one of the three labeled fatty acids and for the gecko and the shrew, you can see that the maximum delta C13 was more or less the same for the unsaturated fatty acids, but there was no oxidation of the saturated fatty acid. And when we test their feces on the right, you can see that most of the palmitic acid was excreted in their feces, so there was no absorbance of the saturated fatty acids. And we found more or less the same for the bee, you see on the right, in the respiration, bees oxidize the oleic acid and the linoleic acid at a high rate, but not palmitic acid. And when we tested their tissues later, we found the linoleic acid in the fat body and oleic acid at a lower rate in the fat body than other parts of the body. A very interesting finding that we found now, in preparation, we found that the linoleic acid that’s considered essential when the bees feed on this fatty acid, they saturated it back and accumulated it in the body as saturated fatty acid.
Amino acids and proteins are also available, especially amino acids. And we did some experiments using these amino acids. I’ll show you something interesting that we did with hornets again. As I told you, the common knowledge is that hornets cannot break protein. They have to bring it to the nest, feed it to their sister, that there are larvae that are sessile, the larvae by gluconeogenesis make the protein into sugar. And they give back to the adult hornets a drop of liquid that is very rich in sugar and in amino acids, which is especially proline, lutein, and glycine. A few years ago, somebody took this composition, made it into an energy drink and everybody is using it today. It’s very efficient, but nobody ever tested what is the efficiency – why so efficient? So, we tried to see what happened with these amino acids in the hornets. We made artificial larvae secretion, but each time we labeled one of them amino acids proline and glycine, we fed it to the hornets. And then we tested in reverse what was coming out. And – in their brain, muscle, and fat body – what is the accumulation of the amino acids? You see during on the left, during rest, the excitation of the amino acids, on the right during flight. So, you can see that the three amino acids are oxidized during rest, but during flight only a proline oxidized at a very high rate in this hornet. And when we test in their tissue later, we see that there is no incorporation of proline at all into this issue, only incorporation of the leucine and glycine into the tissues, in every tissue that we test. And we can say that proline is oxidized as metabolic fuel. And probably this is what gives the extra energy through proline in the body of these insects and gives them more efficient performance of the mitochondria.
We also challenge the common knowledge that I told you about the possibility of hornets to break protein. We fed bumblebees with leucine labeled on carbon number one. Any modification of the leucine will throw away the labeled carbon. So, we can be sure that every labeled carbon that we find in the body of the bee later is related to a labeled protein. Then we fed these bees into hornets with or without their larvae. And we tested the muscles of these hornets after ten days. And what we found is that hornets can break protein. Here you see for queens, workers, but males cannot. And when we test for the hornets with the larvae, we can see that males depend on the larvae to break the protein and workers break more efficiently the protein when they have the worker, but they can also break the protein by themselves.
And the last part I’ll show is about a free flight metabolic rate. You know that you can measure metabolic rate in a metabolic chamber, and you can measure metabolic rate by more energetic expenditure, by doubly labeled water. But this is for a few days, usually, or more than a few days. But if you want to measure the metabolic rate during free flight of an animal or during a sprint, it’s very difficult because you can’t make them run inside the metabolic chamber or only on a treadmill. And it’s not mimicking real life. So, we modified a method to inject labeled bicarbonate into the insect. Here on the left, you can see the curve that you get of the Delta C13 after injecting the sodium bicarbonate. So, after a few minutes, it’s getting even in all the body, and you get the peak and then you get the depletion of the bicarbonate as it’s coming from the body of the insect. You can calculate the rate of the depletion of the bicarbonate and plot it against the known metabolic rates of the insect and calculate the rate of metabolic rate.
So, what we did, we injected these beetles with labeled bicarbonate. We put them in a metabolic chamber, we got the slope of the elimination of the metabolic rate. Then we took them out of the metabolic cage, put them in a wind tunnel, let them fly for a few minutes, put them back in the metabolic chamber, and measured again the rate of depletion of carbon dioxide. You can see here the missing parts. You can calculate the slope between before the animals start flying and when it’s in flight. And from the slope you can calculate the elimination rate of carbon dioxide and plot it against the curve and get the metabolic rate during flight of the animal. You can do it with birds, with bats, and with any other animal.
So, to sum up all these examples that I showed you, there is endless application for the use of delta C13 labeled macronutrients, where you really just start to discover all the potential of these methods and you can study all organism physiology or specific tissue. You can take it out to the field or do it in the lab, and you can answer a question in physiology, metabolism, behavior, and nutrient flow. And it’s very easy and simple to operate. And with that, I want to thank first my great lab members that did most of the work that we showed you, and Sable Systems for the equipment that we use, and for you for listening.
Liam Sanio [00:48:11] Fantastic. Well, thank you so much, Eran, for the great presentation. We’ll move on to the Q&A in just a moment. But first, we just have one last audience poll. We just wanted to know what your field of research is. So here you can select any and all that apply, the options being aging, diabetes, exercise, biology, ingested behavior, nutrition, obesity, physiology, neuroscience, or if there’s something else you can select that “other” option. And while your answers come in, we’ll move on right to the Q&A. So, Riley, we’ll invite you back onto the audio and video. Then we can get started here. So Riley, maybe we’ll start with a question for you. Could you maybe expand upon the differences between human and mouse estrogen deficiency?
Dr. Riley Enos: Can you hear me?
Liam Sanio: I can, yeah. Loud and clear.
Dr. Riley Enos: So, one of the things – and I’ve been looking at the questions that have been coming in – a major difference…can you still hear me? Sorry.
Liam Sanio: It doesn’t look like it, but it should be okay.
Dr. Riley Enos: Okay. I mean, a major difference between humans and mice, is the fact that mice, in terms of peripheral aromatization, do not aromatize. They don’t have a large expression of aromatase as do humans. So, one of the questions was with respect to brain aromatization. So, the mouse model we presented was a whole-body aromatase knockout. So, it’s very difficult to compare mice and humans with respect to peripheral aromatization because they don’t have that much expression.
Liam Sanio: Excellent. Great answer. Eran, next question here. So obviously, insects have pretty different physiology from humans. Can we really learn something from studying their physiology, from studying these insect models?
Dr. Eran Levin: Actually, I think that we have a lot in common. We have the same metabolic pathways. For example, for understanding the pentose phosphate pathway in nectar feeding animals, we can learn about and understand people that have deficiency in this, possibly in their body. And there are millions of people around the world. It can help us to understand why they suffer from this because of oxidative stress, that nectar feeding animals develop these to protect from oxidative stress. And there is much in common, but I think that science today is not very integrative. And we work in different fields, and usually we don’t do this integration. And I think this is a big problem in science today.
Liam Sanio: Yeah, definitely. I mean, I have seen a bit of, you know, trends lately where there are more and more systems biology approaches where there is a bit more integration. But I think there’s still a long way to go. And I think there’s a lot more that we can learn from these kinds of approaches. All right, Riley, maybe one for you. What other thoughts or insights can you give us regarding hormone replacement therapy?
Dr. Riley Enos: Yeah, that’s a great question. So, some of my slides were cut off at the end, but I talk about the fact that there’s many different modes and methods of delivery for estradiol, you know, skin patches, orally, injections, gels, creams, all these things are going to have different bioavailability. In our research, we find that if you give E2 chronically, we don’t see an effect with respect to physical activity that we do, but we do see an effect with food intake. So, whether you’re giving a chronic E2 dose or a pulsatile dose, similar to what we see in physiology, I think these are going to have ramifications for hormone replacement therapy.
Liam Sanio: Fantastic. Eran, and we could probably actually question Riley as well, but how complicated is it to operate these analyzers?
Dr. Eran Levin: Actually, I really like this analyzer because I need, like, two hours to get the undergrad student, the professional working with this analyzer, so that is very simple. It’s very hard to damage the analyzer. You can take it out in the field, very sturdy.
Liam Sanio: Excellent. And really, what is your experience with them?
Dr. Riley Enos: They’re very straightforward, very easy to use. More than anything, the service I mean, if we ever have issues, we’ve had issues with mice chewing things sometimes, that’s just the way mice are. Sable Systems is on the ball, they’re very helpful, and their technical support is second to none.
Liam Sanio: Awesome. Yeah. So great to hear. An interesting question here is “can changes in the E2 levels in tissue plasma be correlated by body weight or change in body weight?” And if so, how can that be accounted for to ensure the changes in the E2 aren’t due to the weight. So Riley, that would be for you?
Dr. Riley Enos: Yeah, that’s a great question. So, one of the issues I alluded to in my talk is the fact that it’s very difficult to measure E2 over the course of a lifespan, for example. And obviously we’d like more human data, but with respect to a mouse, because of the fact that there’s very little peripheral aromatization, it’s very difficult to examine E2 changes over the course of the lifespan and over the course of weight gain. We’ve done that a little bit in our work, showing that sure enough, there’s changes. But also, a paper just came out suggesting that what you eat or your diet, the dietary component with respect to progesterone, can actually impact steroid physiology. So, there’s a lot of factors that go into that.
Liam Sanio: Excellent. Great answer. Now Eran, one for you. The insect measurements, were they done with a continuous or a stopped flow setup?
Dr. Eran Levin: Usually I prefer to work with continuous, but sometimes, for example, now I’m working with termites, and they are very, very small and their metabolic rate is low, and I have to work with stop flow. But you can work both in both ways.
Liam Sanio: Excellent. Riley, one for you. So, since E2 is lower over time, does the expression of the ER alpha beta receptors follow? And if not, why?
Dr. Riley Enos: Another great question. So, Dr. Hevener from UCLA published a paper looking at adipose tissue expression of ER alpha, and she found that it was lower, I believe this is correct, it was lower in females that have poor metabolic outcomes. The difficulty again with this field is the fact that measuring tissue levels of estradiol or estrogen and associating that with the specific expression of the receptor, because usually we can get the receptor, but due to limitations, I mean, there’s only a few laboratories in the world that measure actual tissue levels of estradiol.
Liam Sanio: Excellent. Great answer. All right, one for Dr. Levin. So, the extremely high respiratory exchange ratio of the hawk moth is really impressive. Can you discuss the evidence that this is caused through the PPP? And are there PPP inhibitors that that can normalize RER? And are these toxic to these insects?
Dr. Eran Levin: It’s a good question. Actually, we only did the very strict experiment with labeled glucose. And I think it’s proved beyond any question that this is what caused the high RER. Why they have this high RER, it’s the other part of our work that’s published, and we suggest that it’s to build antioxidant potential in the muscles of the mouse. And we showed it in another method. But we never tried to give blockers, and there are tons of things that can be done, but we just show that this is what gives this high RER.
Liam Sanio: Excellent. Another question on RER for you Dr. Enos, so looking at the variation of RER under E2 treatment, can we conclude that we have a higher metabolic flexibility?
Dr. Riley Enos: I don’t know if we can conclude that. We’re still doing ongoing studies right now where we’re actually pair-feeding mice to determine if there is an improvement in metabolism independent of a body weight change. Now, we didn’t show a body weight change in that data set. But the short answer is we cannot confirm that, we’re still trying to figure that out.
Liam Sanio: Excellent. Alright, Doctor Levin, are there any other types of instrumentation necessary to measure the levels of accumulated 13C from carbon dioxide in these insects?
Dr. Eran Levin: For solids, you must use a combustion chamber, which is quite simple and straightforward to operate. It does burn the sample, it uses very high temperature with oxygen, pure oxygen. And then it goes through the analyzer.
Liam Sanio: Fantastic. I think in the interest of time, I’ll just ask one last question. and maybe Riley, we can start with you, but what kinds of exciting research do you think are coming up? What is the sort of big developments that are coming up, maybe in the next five years in this field?
Dr. Riley Enos: I’m hoping that we get more. First, there’s the Women’s Health Initiative that really hurt people, or hurt hormone replacement therapy, with respect to, it gave it a bad rap, because I think the data was misunderstood. I think that, moving forward, is better assessments of E2 levels and then hopefully in the future, targeted E2 delivery therapies. That would be the goal.
Liam Sanio: Fantastic. And Eran, from your perspective, what do you think are the really exciting developments coming up in the next few years?
Dr. Eran Levin: I think that people will start to realize how simple and cool this method is, and you can apply it on almost every field in zoology, behavior, and physiology, and it’s really easy to use and it will give you clearer results.
Liam Sanio: Yeah, very cool. All right, thanks. Well, Eran and Riley, thanks so much for joining us. It’s really been a pleasure having you with us. Big thanks also, of course, to the audience for joining us today. And last but not least, we’d also like to thank Sable Systems for sponsoring this event. We now invite you to take a moment to complete the survey, which should pop up on your screen in just a moment. We do read all your comments and really appreciate any input you can provide us with. If you still have a question, it’s not too late. You can still submit it using the Q&A panel, and we’ll forward it along to the speakers following the event. If you’re interested in learning more about the research, we have added a number of handouts to the handouts tab, so be sure to check those out if you’d like to learn more. And in closing, we hope you enjoyed this Inside Scientific webinar, and we’ll see you again next time. Have a great day everyone.