Presented by: Sharon Ladyman, PhD, and Vicki Vieira-Potter, PhD
During a live 60-minute webinar on October 1, 2020, Dr. Sharon Ladyman and Dr. Victoria Vieira-Potter shared their current research on rodent metabolic phenotyping with a focus on the effects of hormones and pregnancy on daily activity in mice. To watch the full archived webinar, click here.
Sharon Ladyman, PhD, is Senior Research Fellow at the Centre for Neuroendocrinology at the University of Otago, New Zealand. Dr. Ladyman research “A reduction in voluntary physical activity during pregnancy in mice is mediated by prolactin” demonstrates a key role for prolactin in suppressing voluntary physical activity during early pregnancy and highlights a biological basis for lower activity levels during pregnancy.
Vicki Vieira-Potter, PhD, is Associate Professor in the Department of Nutrition and Exercise Physiology at the University of Missouri, Columbia (MU). Dr. Vieira-Potter’s research “Neuronal and Metabolic Pathways Influenced by Sex Hormones” demonstrates that estrogen’s neural regulation of adipose tissue metabolism and physical activity may drive menopause-associated metabolic dysfunction.
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Sarah: Good morning, good afternoon, and good evening everyone, and welcome to our webinar titled “From Pregnancy to Menopause: Studies of Physical Activity, Behavior, and Energy Balance in Mice.” 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 fortunate to have Dr. Sharon Ladyman, a Senior Research Fellow at the University of Otago, and Dr. Victoria Vieira-Potter, an Associate Professor at University of Missouri-Columbia. Their presentations will discuss applications of rodent metabolic phenotyping with a focus on the effects of hormones on daily activity levels in mice. I’m Sarah McFarlane from the events team here at Inside Scientific, and I’m very pleased to be your host for today’s event. Now, before we get started, I would just like to share a few housekeeping notes to help you get the most out of today’s webinar. First, this webinar is being recorded and resources will be made available following the event. Next up, if the webinar panels look too big or too small, you can zoom in or zoom out on your internet browser to adjust the viewing area. You can also resize some of these panels or make the media panel full screen. Please send us questions, thoughts, and comments using the Ask a Question box next to the media panel at any time. You can also take a look at the resources panel where you’ll find a bunch of links and documents that have been provided to you by our wonderful speakers. We will also be running a number of audience polls during the webinar and a survey at the end, so please chime in and share your perspectives with us. And finally, if you do experience any technical issues during the event, the easiest fix tends to be a simple refresh of the webinar auditorium by refreshing your browser. This should successfully re-establish your connection. But if you still are having issues, use the Ask a Question box to communicate your issue with our team, and we’ll be happy to help you get back up and running. So with that, I’m pleased to welcome our first presenter, Dr. Sharon Ladyman. Sharon, thank you so much for joining us today, and the floor is yours whenever you’re ready.
Dr. Ladyman: Thanks, Sarah. First, I’d just like to begin by thanking Sable Systems for sponsoring this seminar and also for inviting me to come and share some of my work today. And also I’d just like to thank the Inside Scientific team for doing such a great job organizing this webinar. And today I’m going to talk to you about some of our work looking at changes in physical activity during pregnancy. So, pregnancy and lactation are highly challenging metabolic states for the mother to undergo. She must provide all the fuel for growth and development to the fetus, and then during the period of lactation, she has to provide milk for her offspring, and of course, that requires a lot of energy. And during pregnancy, we know that the maternal body itself undergoes a number of adaptations such as growth and development of the mammary gland and increase in adipose tissue. And this requires, you know, extra energy on top of what she needs to direct towards the fetus. So, we know that pregnancy is associated with an increase in food intake to try and meet these increased metabolic demands. But we became interested in the idea that there might be a change in the partitioning of energy usage during pregnancy, such that there may be a reduction in functions that are not essential to pregnancy to conserve energy for those processes of fetal and maternal development.
And so to start with, we wanted to look at energy expenditure during pregnancy. And what we know from human data is really sort of summarized in this diagram from a review a couple of years ago on the left, showing that as pregnancy advances, energy expenditure in humans increases. And this is mostly due to increases in basal metabolic rate and, of course, that energy used due to synthesis of new tissue. So we wanted to know what happens in the mouse and see does this reflect what we see in humans. So we housed our standard C57 black 6 mice in our Promethion metabolic and behavioral phenotyping cages. And we did that before they got pregnant and then throughout pregnancy. And I’ll just point out that mice have a gestation period of about 20 days. So, on this graph where I say early, mid, and late, that represents about a week of pregnancy for each of those three phases. And so, what you can see is that as pregnancy advances in a mouse, energy expenditure increases, mimicking what we see in humans. But what we were really interested in was what happened in early pregnancy, because if you look at the graph on the far right, this shows the change of energy expenditure in the virgin state to those first few days of pregnancy in a mouse. And you can see that all mice reduce their total daily energy expenditure in early pregnancy. We thought that this data sort of supports that idea that there might be a suppression in energy usage by functions not essential to pregnancy, and it may indicate a sort of change in partitioning of energy during pregnancy. And so what we decided to investigate to sort of probe this idea was physical activity, and we thought of this as sort of an example that is a function where you might see a reduction to conserve energy for other processes in pregnancy.
So, we looked at how much our mice were basically walking around the cages, from the difference between the virgin state and early pregnancy. So, this is total daily ambulation. And you can see in those mice where there’s a decrease in energy expenditure, they all reduce their physical activity in the home cage basically as soon as they become pregnant. Now, we found this really interesting, but we wanted to look at other aspects of physical activity as well. So we decided to look at voluntary physical activity by housing mice with a running wheel. So, if you house a mouse with a running wheel, it can run as much or as little as it wants, but mostly mice will run on a wheel. And so, this data just shows an average of what the mice run on four days in the non-pregnant state, and then what happens as soon as we mate them and they become pregnant. And you can see, as soon as they become pregnant, from day one of pregnancy, there is a reduction in running wheel activity. And these levels are maintained until late in pregnancy where they start to drop even further. And this graph on the right shows that individual change for each mouse in this experiment where we have what they run in the non-pregnant state and then what they run on the first day of pregnancy. And I like to show this graph because it shows the variation in how much running wheel activity each individual mouse would do. So, each mouse will run a pretty consistent amount each day in the non-pregnant state, but there’s huge variation about the level of running between mice. But you can see whether our mice are high runners or low runners, they all reduce their running wheel activity from day one of pregnancy. And this graph down the bottom just shows running wheel activity from one mouse throughout a whole entire reproductive cycle. And you can see that reduction during pregnancy, it’s pretty much totally suppressed during lactation, and then as soon as you take her pups away and wean them, she is back running at her non-pregnant level or her virgin level.
So, we were fascinated by the sudden change in activity as soon as mice became pregnant, and we really wanted to investigate what was going on here and what was driving this. And so we thought about what are some of the key changes that take place early on in a mouse pregnancy. And what we thought of was the hormone prolactin because rodents, mice, and rats, as soon as they mate, they start getting these twice daily surges in prolactin. Now prolactin is a hormone that is most commonly known for its role in milk production. So, it’s high throughout lactation to drive milk production is also high during gestation. That’s due to these early surges of prolactin and then as the placenta develops, it secretes placental lactogen, which is a prolactin homolog and also acts on the prolactin receptor. So, you have this entire gestation is high levels of prolactin receptor activation. And due to this, the knowledge we have that this changes, you know, is one of the first changes we see in pregnancy, we decided to investigate whether prolactin was driving this reduction in running wheel activity and physical activity as soon as mice became pregnant. However, there’s not really any data out there linking prolactin and physical activity, but we know that prolactin will drive increases in body weight and drive food intake. And a suppression of physical activity by prolactin, you know, does fit into the idea that prolactin promotes a state of positive energy balance.
So, we looked at the acute effects of prolactin of various aspects of physical activity. And here is our data looking at running wheel activity after prolactin treatment. So we give an IP injection of prolactin or vehicle to mice just before the start of the dark phase, because it’s during the dark phase when mice do all their running, and then we just looked at how much they ran overnight. And we saw a significant reduction in the animals treated with prolactin, and this was particularly evident in the first few hours after the injection. However, when we looked at the acute effects of prolactin on various other aspects of physical activity, we really didn’t see any major effects. And I don’t want to go through all of these graphs, they’re just all our data showing really that we only see very subtle effects of prolactin on other aspects of physical activity such as home cage ambulation or distance travelled in a novel environment. So for the rest of the talk, I’m really just going to focus on voluntary physical activities such as running wheel activity, since we know it has this major change in pattern as soon as mice become pregnant and we know that prolactin can influence it.
So, we asked the question, what happens? Is prolactin required to drive this behavior in early pregnancy? And to do this, we made a transgenic mouse where we deleted prolactin receptors from four brain neurons. So we knew this effect was most likely going to be prolactin action in the brain. And to generate this mouse, we use CreLox technology. So, for anyone who’s not familiar with this technique, what we have is we have this mouse which we call the prolactin receptor flox animal. It has these lox P sites around exon 5 of the prolactin receptor gene, but it normally produces a normal prolactin receptor. However, in the presence of Cre recombinase, it’ll undergo an inversion and you’ll no longer get a functional prolactin receptor. And instead we turn on the expression of green fluorescent protein or GFP and we can use that as a marker to see what cells have undergone this recombination and deleted the prolactin receptor.
So, we crossed our floxed prolactin receptor mouse with a mouse that has CRE in all forebrain neurons to generate this mouse where there’s a forebrain neuron-specific deletion of the prolactin receptor, then we look to see what happened to running wheel activity during pregnancy. And what we see here is that throughout pregnancy, our knockout animals run more than our control animals. And then this graph on the right really just looks at that behavioral change. How much are you running during pregnancy? How much are you running in the first few days of pregnancy? And you can see in our control animals, which are just our floxed animals without Cre, that all our mice reduced their running wheel activity in early pregnancy. However, we saw no significant effect in our knockout animals, indicating that central prolactin receptors are required for this pregnancy-induced suppression of running wheel activity. However, we wanted to replicate this work in a slightly different transgenic animal, so instead of knocking prolactin receptors out of all forebrain neurons, we just knocked it out of all GABA neurons. Prolactin is quite widely expressed in GABA neurons, so we thought this would be a good way to sort of replicate our own data. And when we did this, we saw a very similar result. We saw increased running wheel activity during pregnancy in these knockout mice, and we saw no significant reduction in early running wheel activity in our animals with the prolactin receptor deleted from GABA neurons.
So, we really think now that prolactin action that in the brain is required for this change in running wheel activity during pregnancy. However, we wanted to know what are the neuron populations mediating this effect. And our first thought was, well, running wheel activity is very rewarding for a mouse. Maybe prolactin is influencing running the reward pathways in the brain, but prolactin receptor it’s not really expressed in those particular regions of the brain. So, we had to look a bit further afield, and what we came up with was criteria for areas we wanted to investigate that had to be implicated in running wheel activity, and they also had to have prolactin receptors.
So the first population of neurons I’m going to talk about is the arcuate nucleus kisspeptin neurons. Now, these neurons are most well-known for their role in reproduction and driving GnRH secretion and then on to LH secretion. But what we know from our work, and work of our colleagues, is that these neurons are prolactin responsive. We can inject animals with prolactin and see prolactin-induced intracellular signaling pathways activated in kisspeptin neurons, and that’s what that immunohistochemistry picture shows. The brown is kisspeptin neurons, and the black nucleus staining is prolactin-induced PSTAT5. We also have more recently shown that kisspeptin neurons and prolactin receptor messenger RNA co-localize using in-situ hybridization. And work from Dr. Stephanie Padilla has shown that if you destroy these accurate nucleus kisspeptin neurons, you get a reduction in running wheel activity. So, we have the link there with running wheel activity.
So, what we did is we deleted prolactin receptors specifically from these neurons and looked at running wheel behavior, and really we didn’t see too much of an effect. When you look at that change in behavior in early pregnancy, we saw that both our knockout and our controls had a decrease in running wheel activity, but this was slightly attenuated in our knockout animals, suggesting that these neurons, the prolactin action on these neurons, may play a small role in this behavior. But when we compare it to what we saw in the forebrain-specific prolactin receptor knockout, it really couldn’t account for the whole effect. So we continued to look at other neuron populations. And one of the areas that I decided I really wanted to look at was the medial preoptic area of the hypothalamus. And what we have shown recently is that this area has prolactin-sensitive projections to the VTA. So, we have a link from this area to the reward pathway that is prolactin sensitive. We know that you need prolactin receptors in this area for mice to show maternal behavior. We also know that if you take a standard C56 black 6 mice or any mouse line, sometimes the mums will abandon their pups for no reason and not look after them or show maternal behavior. And we’ve done some work trying to investigate what’s going on here. And one of the things we see is that mice that go on to abandon their pups run more in late pregnancy. So, we’ve got this link between this area and prolactin and running wheel and rewarding activity.
So I decided to delete prolactin from this area and see, prolactin receptors I should say, and see if it was involved in the effects of prolactin on running wheel activity and early pregnancy. Now to do this, I used a slightly different technique. At this time, we injected Cree directly into the medial pre-optic area of our prolactin receptor floxed animals. And, of course, we also had animals that had control injections. And then we can see how effective this is by looking for GFP in this area. So if you look at these top two immunohistochemistry pictures, you can see in the control area, we see none of those black dots, so we don’t see any GFP staining, but we see lots of black, purplish dots in the MPOA of these animals that have been injected with CRE, indicating that recombination has gone on and the prolactin receptor gene has been deleted. We can also look at the responsivity of these neurons to prolactin by injecting prolactin and then looking for PSAT5, because that’s what our signaling pathway that prolactin acts through. And you can see lots of the black dots of PSTAT5 in our control injected, but that’s greatly attenuated when we have injected CRE and knocked out the prolactin receptor. We can also repeat our work, showing that without prolactin receptors in this area, the mothers abandon their pups and have none left alive on day 3 of lactation.
So when we look at running wheel activity in these animals you can see that they run significantly more during pregnancy than our control animals and they don’t show that significant reduction in early running wheel behavior that our controls are showing. So, what this tells us is that there’s a population of neurons within the medial pre-optic area that plays a key role in the prolactin effects of prolactin-driven suppression of running wheel activity during pregnancy.
So we see this rapid change in a biological system as soon as mice become pregnant. We know it requires prolactin. It appears that it needs prolactin receptor activation in the medial preoptic area during pregnancy. But really, you know, what does this tell us about pregnancy? And I think to understand this, we really need to think about, well, what does running wheel behavior in a mouse… what does it mean? What is it reflecting? And so, we know that this behavior isn’t just something a mouse in a lab will show because it’s stuck in a cage and it’s just got a wheel. There was some work done a few years ago that has demonstrated that in the wild, if mice and actually other small creatures as well are given the opportunity and access to a running wheel, they will voluntarily engage in running wheel activity. So it is a behavior that’s even shown in the wild.
But we really don’t have a full understanding yet what this behavior means and what this change in pregnancy specifically means. And we’ve got some ideas that we’re working on at the moment. I mean, thinking back to my introduction, you know, our data really shows that there’s a suppression, there’s a pathway in the brain that is suppressing voluntary physical activity during pregnancy. And maybe this is all about a conservation of energy during pregnancy. But that’s not the only possibility. We know running wheel activity is rewarding for rodents and maybe this change in behavior more reflects a change in pregnancy-induced changes in reward and what the pregnant mouse or mammal really finds rewarding during pregnancy. Or another possibility is this has to do with changes in thermoregulation during pregnancy. We know physical activity increases core body temperature and core increases in body temperature are not optimal for fetal development. So, this change in running wheel behavior might really just reflect the changes in the processes regulating thermoregulation during pregnancy. So these are the types of things we are really working on now.
And with that, I would just like to thank all the funding agencies that have sponsored this work, and of course the various people who have been involved in this work or who are now involved in moving forward in this project. And of course, thank you everyone for tuning into this webinar to hear the talk.
Sarah: Awesome, thank you so much for that wonderful presentation, Sharon. Before we move on to our next speaker, we are going to run an audience poll. So, this question, you can select one of the following answers. Which of the following animal models do you most often use? And so go ahead and make your selections, and then once we have almost everyone answered, maybe about five more seconds, then we’ll move on to our next speaker. Okay, it looks like there’s only a couple of people left. So, thank you so much for answering those questions. And with that, I’m pleased to welcome our next speaker, Dr. Victoria Vieira-Potter. Victoria, thank you for joining us today, and the floor is yours whenever you’re ready.
Dr. Vieira-Potter: Well, thank you, Sarah. And hi, everyone. Thank you for listening, thank you to Sable, and thank you too Inside Scientific. So, to start us out, I would like to just first identify the major sex differences that exist in obesity and metabolism. So, Sharon, at this point, has convinced you all that female sex hormones absolutely affect running behavior and metabolism, and I am going to continue on that discussion by first taking a step back and just reminding you about kind of some major sex differences that exist in metabolism and obesity. And the major difference that I want to point out is that there’s really a sex difference in fat partitioning, such that females store fat more in a gynoid manner, whereas males store fat more viscerally, and it turns out that this protects women metabolically.
And so, what’s really interesting though is despite the fact that women are protected metabolically, now, I will say that I’m talking about premenopausal women in the non-pregnant state, but so even though women are protected metabolically, obesity prevalence is actually higher among women, and this is true across ages, among adults. And so, what this graph is showing you are some NHANES data indicating the obesity prevalence in these various age groups. And what you can appreciate is that women display more obesity than do men. Yet the risk is really much lower among young women compared to young men in terms of metabolic comorbidities of obesity. So why might that be the case? Well, what happens during menopause is we know that women develop an increase in risk for many of these metabolic diseases that are higher among males in the pre-menopause age group. And so, the main change that happens is post-menopausal women develop more abdominal obesity, and so they become kind of more like males in that way. And so, what might be protecting premenopausal women from this metabolic phenotype? Well, we think it involves estrogen and estrogen has effects in many different cells and tissues of the body, but our lab really focuses on two metabolic tissues in particular, and then as brain and adipose tissue. And we use rodent models to investigate these questions.
So for those of you who may not be familiar with these basic sex differences that exist in rodents, I’m just going to highlight some sort of generic data that we generate in the lab, identifying these major differences. And so, what you can appreciate here is that male mice weigh more and they have more lean mass than do female mice. But even when the mice are at equal adiposity, there is a sex difference in their metabolic health, such that the male mice are more insulin resistant. And that’s shown here in two different surrogate indices of insulin resistance. One is the HOMA-IR index, which tells you about systemic insulin resistance, and the other is specific to adipose tissue insulin resistance. And those are both higher among males compared to females.
So, let’s look at the adipose tissue and look at some basic sex differences in that tissue. The reason I’m interested in adipose tissue and the reason I’m telling you about these differences in adipose tissue is because adipose tissue inflammation, at least in obesity, is known to be a causative factor for insulin resistance, which sets up the stage for those metabolic comorbidities of obesity that postmenopausal women are at risk for. And so, what you can appreciate here, which are gene expression data collected from white adipose tissue in the abdominal visceral region. So that kind of intra-abdominal fat that I mentioned. What you can see here is that the levels of these inflammatory markers are significantly lower among those female mice. And these are the same mice that I showed you in the previous slide. And so, what you can also appreciate here is that this thermogenic protein called UCP1 or uncoupling protein one, which is the signature protein that is expressed in brown fat and it allows brown fat to be thermogenic. Well, it’s also expressed in white fat. And we and others have shown that this protein has really powerful insulin sensitizing effects. And we’ve done some work showing that when mice don’t have UCP1, like genetic knockout mice, they actually develop insulin resistance, even when they’re equally as fat as their wild type litter mates. So female mice, in addition to being protected from adipose tissue inflammation, also have higher levels of UCP1 in their white fat. In fact, this protein isn’t even detected in male fat. And so, exercise is another way we know to mitigate inflammation. So there are data to show that exercise and physical activity, lower adipose tissue inflammation. And so, we asked the question, what role might physical activity play in these sex differences? And so what you weren’t able to appreciate from Sharon’s talk, where she described the wheel running changes that happen when female mice become pregnant, what you couldn’t appreciate from that was that male mice compared to female mice in the young age group are considerably more active. So female mice are considerably more active in their cage than our male mice.
And so, what role might ovarian hormones play here is kind of the question that we are trying to answer in our lab. So this is sort of the overarching hypothesis is that ovarian hormone loss leads to physical inactivity, which leads to obesity and adipose tissue inflammation and ultimately insulin resistance. So, what happens to a mouse when you remove her ovaries? Well, when you oophorectomize a mouse, she actually responds much like a post-menopausal woman does and that is she gains weight, she develops insulin resistance, she develops adipose tissue inflammation. And what’s really important to note here is that the physical activity level of those mice that are generally really active, again, females are really a lot more active than males, but when you oophorectomize that female, she becomes significantly less active in her cage.
And so several years ago, I was introduced to these really interesting divergent rat lines that are bred to be either really high runners, the high-fit HCR rats, or they’re bred to be really low runners, these LCR low-fit rats. And so I wanted to take the opportunity to look at how rats that are really bred for high levels of activity, I wanted to look at how they might be protected in the menopause transition, or if we remove the ovaries from these high running animals, might they be protected from the metabolic comorbidities of oophorectomy in these mice? So, what we did was we looked at voluntary wheel running in the HCR rats in blue and the LCR rats in red, and then we oophorectomize them. And what you can appreciate here is that as expected, the HCR rats are significantly more active on their voluntary running wheels than are the LCR rats. But what happens when following oophorectomy is that there is a significant reduction in this voluntary wheel running, and we were really surprised to see that this was true in both the HCR rats and the LCR rats. So even the rats who were bred for really high levels of physical activity still experienced this reduction in voluntary wheel running following oophorectomy, which really, we think speaks to the neuroendocrine mechanisms really driving this estrogen loss mediated reduction in physical activity.
So, the million dollar question though, right, is does this happen in humans? So what is menopause look like in humans in terms of its effect on physical activity? Well, it turns out that there have been some human studies in this area. And one study in particular was conducted using over 100 women who they followed over the menopause transition. And it was a really nicely controlled study where they assessed energy expenditure in these women yearly, much like in the same manner that we do in our mice using the Promethion metabolic cage system. Now, what these authors did was they assessed the energy expenditure of these women, they assessed their physical activity levels using pedometers, and they also assessed their dietary intake. And what they found was that the weight gain that was observed in these women following menopause or during the menopause transition was really explained by their reduction in energy expenditure and their physical activity, and it really wasn’t explained by any dietary changes. In fact, if anything, they reduced, these women reduced their dietary intake throughout that menopause transition, yet they were still gaining weight. So why might this happen? Well, to answer this question, we began to become interested in a region of the brain that drives motivated behavior, and that is the nucleus accumbens brain region. And as Sharon indicated earlier, wheel running in rodents is actually a really rewarding behavior, and we think physical activity is a rewarding behavior in humans as well. But we used our rats to kind of explore this a little bit, how this particular brain region might play a role in dictating these running differences. And so, this was work that was led by my former PhD student, Young-Min Park. And what Young-Min did was he compared the HCR and the LCR rats for their gene expression differences in this nucleus accumbens brain region, which by the way is a key region known to drive motivated behavior. And others have actually shown that this region in particular drives motivation for physical activity. So Young-Min wanted to compare these different rat lines for gene expression changes in that area of the brain, as well as determine how oophorectomy affected that area of the brain. And here’s what he found. So first, not surprisingly, he found that the wheel running distance was much greater in the HCR rats compared to the LCR rats. But when he went on to look at gene expression changes in the nucleus accumbens brain region, what he found really quite remarkably was that there was a significant suppression in genes related to dopamine signaling. And dopamine is the major neurotransmitter that drives motivated behavior in the nucleus accumbens brain region. So this was really an exciting finding. And we then looked at how those changes in the nucleus accumbens brain region of those dopamine genes, how those changes related to changes in voluntary wheel running. And in fact, he found a highly significant relationship between those two things, meaning the higher the expression of positive dopamine signaling genes, the higher the wheel running distance. And that was true in both the oophorectomized and the sham operated rats of both of those different lines.
So, the hypothesis became, does perhaps a reduction in estrogen due to menopause cause a reduction in dopamine signaling in the nucleus accumbens, which would then lead to a reduction in motivated physical activity, which of course then would set the stage up for obesity and metabolic dysfunction. And so to do this, to answer this, to begin to answer this or address this hypothesis, we went to an aromatase knockout mouse. And the reason is that aromatase is the enzyme that is responsible for estradiol production. And so, this was the work led by my current doctoral student, Dusti Shay, who used these rats, these aromatase knockout and wild type rats, to determine how their changes in the nucleus accumbens brain region might relate to differences in physical activity and energy expenditure. And so, what she did was she first assessed the physical activity and energy expenditure levels using the Promethion system. She also assessed body composition via echo MRI. And then of course we assessed the brain expression changes this time using RNA-seq to really get sort of a broad perspective on gene expression changes. And we also did some pathway analyses to see the major pathways that were affected. And we looked at the adipose tissue as well.
So here are the results from that study. First, we found that aromatase deletion increases adiposity, and this was true in both sexes. And this is expressed here in this percent body graph, percent body fat graph, that is. And these data are not unique. So, others have published on this model before, and it is known that the aromatase deletion does cause obesity in mice. It also causes an increase in visceral fat and an increase in insulin resistance. I’m not showing you those data here, but that’s been published. So, the question is, what is responsible for this increase in adiposity in these mice? Well, to answer that question, we assessed all aspects of energy expenditure and food intake, and what we found was that very similar to what happens in postmenopausal women, we saw a significant reduction in energy expenditure, both in the rested state and total energy expenditure. And of course, we want to ask the question, is energy intake different? And so because that’s going to play a role into any body weight changes, and in fact, very similar to the postmenopausal women, is that there wasn’t an increase in energy intake in the aromatase knockout mice or the estrogen absent mice. In fact, there was a reduction in relative energy intake. And but what was significantly affected was the spontaneous physical activity levels in those mice.
And so of course, let’s go to look at what’s happening in the brain because we think that is probably where, what is driving these differences in physical activity and energy expenditure. So here are those RNA sequencing data that were generated. And what I’m showing you here in this Venn diagram are the differentially regulated genes that were found in males in blue between the knockout and the wild types. And then in pink, the differentially affected genes, in the differentially expressed genes rather in the females. But what we’re really interested in was the overlapping genes. So what genes were consistently dysregulated or differentially regulated in both sexes? And when we looked at those five genes, the gene not shown here is aromatase because that was indicative of the model. But with the number one gene that was differentially regulated was this gene PTS, which is an enzyme, which encodes an enzyme that catalyzes the second and irreversible step in the formation of BH4, which is an essential cofactor in catecholamine biosynthesis. So, what that means is this enzyme is necessary for dopamine synthesis in the nucleus accumbens brain region. And what we found was that mice that lack aromatase have significantly lower levels of this enzyme.
So, we were pretty excited about that finding because it certainly supports the hypothesis that estrogen mediated dopamine synthesis might be related to estrogen’s effect on voluntary wheel running. So after that finding, we began to just explore what genes were associated with this PTS gene. And we surveyed many different genes and the one that came up as really tightly correlated with PTS was this gene that encodes the circadian regulator, which is PER3. And as you can see here, there was quite a strong correlation that was highly significant in those two genes. And so that got us thinking about sleep, because sleep is something that certainly is regulated by circadian cycles, and its patterns are affected by menopause. And so that’s another reason we are really sort of excited to see the circadian regulator gene relating to PTS, because we know that one of the things that happens in menopause, which I haven’t yet described to you, but that is that sleep patterns become dysregulated. Women develop more problems with sleep.
So we went back to our data, and we were able to assess sleep patterns in these animals, again, using the Promethion system, which allows us to assess sleep using custom designed macros. And we were able to then associate those differences in sleep as well as many of our other physiological variables with the genes that were differentially expressed in the nucleus accumbens brain region. So to kind of take you through this, what I’m highlighting here are the sleep, the hours of sleep in the different groups of animals and how that sleep pattern was affected by aromatase deletion. And so, because mice are nocturnal, they’re actually, they’re meant to be sleeping in the light cycle and not in the dark cycle. So, an increase in sleep, which we’re seeing here in the dark cycle, we’re seeing a little bit too in the light cycle, but it’s really more pronounced in the dark cycle, which indicates to us that there’s something dysregulated about their circadian clock. And so, we then took that data and we related it to all of the different differentially expressed genes. And what I really just want to highlight are the correlations that we found with the PTS gene, because that was our number one gene that was differentially regulated between our genotypes. And when you look at how that gene relates to sleep, there was a highly significant inverse correlation between sleep in the dark cycle and expression of that gene at 0.87, so really strong inverse correlation. We also looked at visceral fat, because that’s another thing we’re really interested in, given that what we know about estrogen deletion and menopause adversely affecting abdominal fat. And so, we looked at what things were correlated both with abdominal fat and as well as with cage activity. So those are some major outcomes that we’re interested in. And what I want to bring your attention to here in the physical activity data is that we found remarkably a very strong correlation between this enzyme that, you know, as I mentioned is responsible for dopamine synthesis in the nucleus accumbens brain region, we found a significant correlation between expression of that gene in physical activity in the cage. In addition, we found that that gene was inversely correlated with visceral fat. So, we were really excited to find that finding as well. And that made us sort of become interested in the relationship between adipose tissue and PTS expression. And so, there’s that correlation that we found in our animals.
And after identifying that relationship, we went to the literature to see if others have identified any relationship between this expression of this enzyme and adiposity. And in fact, we found this one paper where these authors downregulated the expression of this enzyme, and lo and behold, they documented an unusual body fat distribution pattern exemplified by excess abdominal fat in mice. So, this was really exciting and quite interesting to us, given that we saw a similar relationship in our animals with visceral fat.
So, to bring this kind of full circle for you, what might be explaining this relationship? Why would there be a connection between fat and brain? And might that connection have something to do with this protein UCP1 that I told you is significantly higher in women, in females compared to males. And so, we looked at that relationship between PTS expression and UCP1, and remarkably we found a positive correlation. So, we found that nucleus accumbens PTS gene expression was positively correlated with white adipose tissue UCP1.
So, to leave you with some takeaway points, prior to menopause, females are metabolically protected, but in the mechanism may involve estrogen’s protective effects on both adipose tissue and brain. Females also have fit fat, meaning they have more relative brown adipose tissue. Actually, I didn’t show you that data, but that is true. And I did show you data that females have higher expression of UCP1 in their white fat. They also have less inflammation in their adipose tissue, and their adipose tissue is more insulin sensitive. Prior to estrogen loss, females are also more physically active, yet estrogen loss causes weight gain, reduces energy expenditure, and this leads to insulin resistance. And finally, sex differences and pathways associated with fat metabolism, dopamine signaling, and circadian regulation in the nucleus accumbens brain region may help explain some sex differences in behavior and metabolism.
And with that, I would like to acknowledge my lab, my lab manager, Rebecca Welly, who does most of the things in my lab, my former PhD student, Young-Min Park, who did work with the HCR-LCR rats, and my current student, Dusti Shay, who’s leading the work with the aromatase knockout mice. And many collaborators I would like to acknowledge. And again, I want to thank Sable Systems and I would be happy to take any questions that anyone has.
Sarah: Great. Thank you so much for that, Victoria, and for the fantastic presentation. We are going to run one more quick poll before we dive into our Q&A with our speakers. So please answer the following question. Do you currently use metabolic phenotyping in your research, yes or no? And pretty much most of everyone has answered, so thank you so much for doing that, and we’ll give you a couple more seconds for everyone who hasn’t. Great, okay, so let’s move to our Q&A. So I’m going to welcome back Sharon and Victoria, you’re still on with us. And if you have any questions for our speakers, please use the Q&A box beside the media panel to submit any questions that you have. And to start us off, we have a question for Sharon. So, Sharon, someone has asked, what accounts for the spike in running just after giving birth in these mice?
Dr. Ladyman: Right. So, yeah, on that figure early on across the whole reproductive cycle, yeah, there was that little spike after birth. And this has to do, we think, with mice and rats actually ovulate after giving birth, so they can potentially mate there. So, we think they have a spike in estrogen, which might be driving that running wheel activity, because estrogen can drive running will activity. So that’s what we think that little spike is, but we see it on most mice. But yeah, so we think it has to do with that postpartum ovulation.
Sarah: Okay, fantastic. That’s really cool. And then we have another question here for you, Sharon. Which factors most explained the reduction in running behavior? For example, did mice run more slowly or did the mice just spend less time running or was it a little bit of both?
Dr. Ladyman: So when we look at the speed that the mice are running across pregnancy, it really doesn’t change from the virgin levels until late pregnancy. And we think that that is due to the fact that these mice have put on 15 grams of weight over three weeks, and they’re just physically incapable of going faster. But the early-on change, there’s no change in speed. It’s a decrease in the time that they are running, and what they’re mostly doing instead is sleeping. So, we see an increase in sleeping behavior. What we don’t know yet is whether they are having, like, so mice run in, like, bursts of running, they don’t get on and run for an hour. They’re just on and off, on and off, and we don’t know if it’s less bouts of running or if it is shorter bouts of running. So that’s something that we’re quite interested in looking at in the future.
Sarah: Oh, that’s really cool. So potentially a future study plan for you guys. Awesome. Okay, so now we have a question for Victoria. So, someone has asked, are there alternatives to estrogen therapy to improve metabolism following menopause?
Dr. Vieira-Potter: So yeah, that’s an excellent question because although we know that estrogen really does rescue many of the metabolic effects that we see following hormone loss, both in humans and in rodents, there are definitely risks involved with estrogen replacement. Namely there’s an increase in breast cancer risk, there may be some cardiovascular risks, and so it’s not recommended for all women to get estrogen replacement. So, in terms of alternatives to estrogen replacement, we’re actually, in the lab, we’re becoming really interested in selective receptor ligands. So, I wasn’t able to share these data with you all, but so estrogen mediates its effects through two main receptors, alpha and beta. And it turns out that most of the risks associated with estrogen therapy are due to it signaling through the alpha receptor. And what we’re finding in the lab is that there are benefits through the beta receptor that would bypass any risks. And so those aren’t clinically available, but I think that’s an area of research that’s kind of up and coming, is looking at selective ligands. But as far as now, there are dietary approaches, like soy-based diets are used by some women because phytoestrogens in soy kind of look like estrogen. There’s not great data on the efficacy of those, meaning there’s not a lot of harm in using them, but they’re certainly not as powerful as estrogen in terms of having a benefit metabolically.
Sarah: Okay. Really cool. We have another question for Sharon here. Somebody has asked, how soon in early pregnancy does the timing of food consumption change, so i.e. to eat more around the clock or eat during the dark period, and is it connected to the change in running wheel activity?
Dr. Ladyman: So, in terms of food intake in mice, we don’t really see like significantly increase until maybe day 14 or 15 of pregnancy and you can appreciate that a mouse eats quite a little amount and to see a significant increase requires, yeah, it requires time during pregnancy. So, we don’t see a significant increase in food intake until about the middle to late pregnancy. What we do see with that is that, in terms of how they’re eating, they have not more meals over the 24 hours, but it’s bigger meals. Yeah, and I think our data doesn’t really show that the percentage-wise what they eat during the night and during the day doesn’t change, even with increasing food intakes, they still eat most of their food during the night. Although other people, you know, see slightly different things during pregnancy where maybe they start increasing a bit more during the light phase. So there is a little bit of variation between different people’s mice. But so like during lactation food intake increases just in the light phase as well, which we don’t really see too much of it during pregnancy. So in terms of how that reflects running wheel activity, we don’t really see too much of a connection. I mean, we see those really early changes in pregnancy, but the food intake is not till much later in the second half of pregnancy
Sarah: Really cool. Okay, so we have another question here for Victoria. So somebody has asked, did you appreciate regionality differences in the visceral adipose depot when measuring UCP1 expression levels?
Dr. Vieira-Potter: Oh, that’s a really great question. So, we didn’t really assess that. And so there is this kind of, it’s called beiging or browning of white adipose tissue for people who aren’t really familiar with this. But what happens is, yeah, white adipose tissue kind of increases its expression of UCP1. And that really does seem to happen kind of regionally. Like it’ll happen little pockets of the tissue, but maybe not kind of globally. We only looked at a very, you know, small section of tissue. So, we didn’t do a thorough analysis of the entire depot. And so in our little itty bitty section of the window that we looked, we really didn’t see regionality, but that’s not to say that that didn’t happen. And also we’re not, so in the studies that I was just sharing with you, those animals weren’t manipulated in any way where they would have an upregulation of UCP1, whereas we’ve done other studies where we use a drug to activate UCP1. Exercise is also another way where you can increase UCP1 and white fat. And in those studies, you really see a robust increase in UCP1. And you can really kind of appreciate in looking at the tissue, little pockets of where that browning is happening. But in this particular study, we were just looking, yeah, more and really small kind of a window of tissue and we didn’t see those regional differences.
Sarah: Okay, and then we have another question for you, Victoria. Someone has asked, what did your sham for your oophorectomy entail in your mice?
Dr. Vieira-Potter: Yeah, so we essentially open up the mouse and we take out, we basically, we make the same incision, and we sort of pull the ovary out and then we just put it back in, we leave it intact and we sew the animal back up. And we’ve done these so many times that, the study that I just showed you, we did actual sham surgeries on those rats. So, it was the rats that I showed you that were OVX’d. But we’ve done that so many times now we don’t see differences. We’ve gone to just, in some studies, we just leave the, we leave the animals intact to kind of save them from that surgery. But, but yeah, that’s generally what it looks like. We make the incision, we expose the ovary, we put it back in and we sew it up.
Sarah: Okay. That makes sense. All right. So, we have another question here, and it’s going to be our last question just to respect everybody’s time, because we’re already a minute over, but this last question is for Sharon. Regarding your findings of the importance of the MPOA-specific expression of prolactin receptors, are there changes in prolactin receptors in these neurons with aging or prior breeding?
Dr. Ladyman: I don’t know about aging. I do know that we get a lot more prolactin responsive neurons during pregnancy and lactation in this area, particularly in lactation, it lights up more if we look at prolactin-induced P step 5. We haven’t looked in animals that have gone through many cycles of breeding, but that’s something that we’ve talked about because we know that, you know, rodents become better mothers, there’s this idea of maternal memory, which we think is likely to involve that area and prolactin action in that area. So, we do have plans to actually look specifically at that, but we haven’t done it yet.
Sarah: Okay. So another future study plan for you. Alrighty. Well, that is the last question for the Q&A, all of the questions that have been submitted. We thank you for them. If you have any more questions, you can use the Ask a Question panel still for the next couple of minutes to submit any more questions that you have for our speakers, and they’ll be answered in a report in a couple of weeks. So, I just want to thank Sharon and Victoria again so much for all of your fantastic insights today, both in your presentations as well as the Q&A session, and thank you again to everybody who joined us today for the webinar. The slides and a recording of today’s webinar will be available soon, so look out for an email in the next day or two giving you access to the video recording. Before you go, we invite you to take a moment to provide your feedback on a survey that will show up, thank you, and let us know what webinar topics you’d like to see in the future. And finally, if you still have any questions like I mentioned, you can submit them in the Q&A box. And in closing, thank you again for taking part in this webinar and thank you again to Sable Systems for making this event possible, and we look forward to having you with us again soon.