Rhonda: Welcome back, everyone, I'm really exited to be sitting here with Dr. Morgan Levine who is an assistant professor at Yale. Her research focuses on understanding the science of aging. She uses bioinformatics to quantify the aging process using something called epigenetic aging clocks, which we are going to talk about in great detail today. Her research really aims to understand the underlying mechanisms that drive the aging process with the hope of, potentially, testing interventions, whether they're lifestyle or pharmacologic, that can perturb the aging process. And when I say perturb, I mean maybe slow.

So, she's also a principal investigator at Altos Labs, which is a new biotech company that also aims to understand the underlying mechanisms that drive the aging process, again, with the hope of possibly having interventions that could slow aging.

So, again, I'm super excited to be here. It's really great to meet you in person. I've been following your research over the years. You did post-doctoral training in one of my favorite scientists Dr. Steve Horvath, his lab. And we've had him on the podcast, you know, talking about epigenetic aging clocks before. So, I was thinking, to start this off, we could start talking about aging more as a concept. And maybe you can explain to people how to differentiate between age as a number, so, chronological aging, versus biological aging, phenotypic aging, and functional aging.

Dr. Levine: Yeah. So, I think, even in the field of aging, there isn't a good definition for what we're actually talking about when we're saying, "We're working on aging." I think most people in the public think of aging in terms of just chronological time, so, whatever age your driver's license says or your passport. But really what we care about is this thing that we would call biological aging, or even phenotypic or functional aging, and that is all the changes that your body undergoes as a function of this time, usually.

So, aging in our society has kind of a negative connotation. But it's not the years you've been alive that's actually the problem, it's kind of how your body has changed over that time. And not everyone's body changes in the same degree or at the same pace. So that's the really important thing is can we figure out what's changed, how that's going to affect your risk of disease, your functioning ability, or any of these things.

So, as you mentioned, we can talk about this in terms of biological age, so, we can measure it in terms of molecules and cells, how those have changed in your body. We talk about maybe phenotypic or functional, which I think is kind of the stuff you can feel and see in your body. Are you able to, you know, run a mile as quickly as you used to or get up a flight of stairs? And these are the things that people actually worry about losing over their life course. And it's really important to try and quantify these, so, we can understand what drives these changes and, potentially, how you would slow that or, hopefully, like some people are interested in, even reverse it.

Rhonda: As you mentioned, you know, people do think about chronological age often. For me, when I think about aging, I often will think about functional aging. I mean, it's more personal for me, I'm worried about becoming demented or losing my cognitive function or, as you said, I'm not able to walk as well, I'm just physically declining. But as a scientist, biological aging is much more interesting because it's more fundamental, would you agree?

Dr. Levine: Yeah, and it's really where we think it all starts. So, we kind of take a perspective of what we would consider these biological levels of organization. So, you have all the kind of molecules and atoms in your body that kind of feed into, you know, cells that make up tissues that make up whole organ systems and then the whole organism. And we think the aging process, all these changes are starting at these lower levels, so, you have changes in molecules and cells, but we don't see that until you feel it at these higher levels, until you feel, you know, you have weaker muscles or you're not thinking as well as you used to. And that's really, once it's reached a certain level. But if we talk about understanding what's driving these and where to intervene, you have to do it at the lower levels if you want to affect all those things that we see and feel every day.

Rhonda: So, there's some pretty well-defined hallmarks of aging. And, as you know, these are things like genomic instability, telomere, you know, shortening, cellular senescence, mitochondrial dysfunction, epigenetic alterations, nutrient sensing problems or dysfunction, stem-cell depletion. So, there's quite a few of these hallmarks that are sort of accepted within most, you know, of the scientific community, you know, they together seem to drive the aging process in a way, right, as you mentioned, at the molecular level, cellular level. I'm sort of curious what your thoughts are on what you think some of the major drivers of aging are, caveats included, or why it's important to really understand what those drivers of aging are.

Dr. Levine: Yeah. I think, you know, there's a big push in the field to figure out what's causal in aging, like what are the things that are changing, that are really pushing this aging process and driving all the other changes? And I don't think we have a good idea about that, you know, what's actually causal versus just correlative, it's just an outcome of aging that we can observe. In my lab, we're really interested in epigenetics, and that's mostly because I think of the epigenetic system as the operating system of the cell. So, most of the cells in your body have, essentially, the same DNA, but what makes something a neuron or a, you know, brain cell or a skin cell is the epigenetic program. So, it gives the cells the ability to respond to stress, it gives them almost their kind of physical form and all of the things that they are supposed to do.

The problem is that this program gets completely rewritten with aging. And we don't know exactly why, whether it's errors or whether it's just the program kind of having glitches along the way. But we think that this then produces cells that are not adapting correctly to their environment or maybe doing things they shouldn't be doing. And potentially, this is something we think might cause aging, although there's still some debate on whether it's truly causal in the aging process. And I always say, "No one really knows yet what the true causes are."

Rhonda: Since your research really does focus on the epigenetic alterations, can you explain to people what epigenetic aging clocks are, generally speaking? And then maybe we can get into some of the differences between the first-generation epigenetic aging clocks, like corvette clock, and then second-generation like Levine, or PhenoAge it's also called, and GrimAge? So...

Dr. Levine: Yeah, absolutely. So, there are a bunch of different types of epigenetic modifications but the type that these clocks are based on is something called CpG methylation or DNA methylation. And really what that means is you can look across, you know, one strand of DNA, and we know we have A, C, G, and T, but you have these regions which we call CpG sites, and that's where you basically just have a C right next to a G. And these tend to be located in, you know, specific regulatory regions of the genome. But what happens is the CpGs can become methylated. Some of them are supposed to be methylated from the beginning. But what we find with aging is that the ones that we expect to have methylation lose methylation with aging, and the ones that shouldn't have methylation gain methylation with aging.

And the methylation in this is basically turning on or off different parts of your genome. So, when you have methylation, we can, essentially, assume that part is repressed. So that, wherever it is in the genome, it's not accessible, you're not expressing the genes in that region. Versus, when you remove the methylation, we consider this more an active region.

So, epigenetic clocks look across either hundreds or hundreds of thousands of these sites and just say, "Does the pattern of whether you have methylation or not resemble someone of a given age typically?" So, it would say, "Oh, your pattern looks like someone who is 40-years-old," even though maybe you're 50-years-old chronologically. And what we find is that that kind of difference is biologically meaningful.

Rhonda: What do you think the major advantages of using these epigenetic aging clocks are in terms of, you know, their use? I mean...

Dr. Levine: Yeah, so, people have been really interested in this idea of quantifying biological aging or estimating biological aging. Because again, chronological age is just an imperfect proxy of this process that we actually care about, that, you know, is why people get diseases when they get older, why people die when they get older. So, if you can actually quantify the process as best as possible, it's better than chronological age.

But there's been some disagreement on the types of data or the types of markers that you could use to do that. Some people think we should look at just, you know, clinical markers, and those are useful. But for me, the exciting thing about epigenetic clocks and measuring DNA methylation for this is that you can use the exact same clock across almost any tissue type and almost any cell type. So, I can compare aging in, again, skin to aging in brain using the exact same measure. And I don't think there's any other type of biomarker that you could do that with.

Rhonda: What about the differences in these clocks with respect to, you know, the original Horvath clock you hear about versus the one that you developed with your mentor Steve Horvath with respect to phenotypic aging, phenotypic age clock, GrimAge? Like what are some of the major differences in those clocks in terms of their predictive power?

Dr. Levine: Yeah. So, the original clocks were used. So, when you develop these clocks or train these clocks, we use machine learning, so, we're usually trying to predict something. So, you take all your methylation data and you say, "How can I predict whatever this is?" And the original clocks use chronological age. So, the idea is, "Can I look at methylation and predict someone's chronological age?" And so, again, that's kind of this idea of the pattern of someone who's this age.

But we know, again, chronological age is an imperfect proxy of this process we're actually trying to quantify. So, what the second-generation clocks did, the one that we published in 2018 was the first example, is we said, "Oh, can we come up with a better thing to try and kind of tune these measures to?" So, in that case, we used kind of normal lab tests that we combined into a measure that was predictive of mortality and then we trained a predictor of those lab tests.

And a similar thing was done with the GrimAge clock where they took these different proteins and they trained predictors of that and then trained the predictor of mortality. So, we think that something that captures mortality or health span or kind of physiological decline is going to be a better thing to tune these clocks to than just chronological age.

Rhonda: How do these epigenetic aging clocks, I think more specifically the second-generation ones, the PhenoAge one that you mentioned, or the GrimAge, compared to measuring biological age to like some of these clinical biomarkers, so, HbA1c, your cholesterol, you know, lung function, like the classical things that people are measuring to measure biological age? And, of course, I think a similar question would be also mortality risk as well, how do they compare in terms of their predictive power, you know, as a biomarker?

Dr. Levine: So, if you're measuring the epigenetic clock in blood, I would actually say that they're on par with not the individual clinical lab tests, so, you know, they're going to be more predictive than if I just look at HbA1c or just look at cholesterol, but we can also combine these clinical tests into a single kind of risk measure, which is what we do with PhenoAge. And then I would say they're actually on par with that. The advantage of epigenetic clocks is, again, you can get a different measure for different tissues or cells in your body. So, the blood tests, these clinical tests, you'll get one measure, one biological-age measure out of them, but for the epigenetic clocks we can measure your skin's age or, you know, if you had a biopsy, you can measure different organs' age. So, even though people usually just use them in blood, they have a lot more potential just to compare kind of how different organ systems are aging.

Rhonda: Kind of that was kind of my next question a bit is that, you know, as you know, people age at different rates but, even within an individual, their organs can be aging at different rates as well. I know there's been work from Dr. Mike Snyder, who we had on the podcast not too long ago, who I know you're familiar with his work showing that some people are metabolic agers. So, their liver and, you know, their kidneys, they may age quicker. Some people are cardiac agers where they seem to be more at risk for having heart problems. Or they could be immune agers, so, their immune system, they're more susceptible to pathogens and stuff with age. So, if you were to measure like a blood sample from a person using...you know, would the epigenetic clocks pick up that or is it measuring more of the system's level type of aging?

Dr. Levine: So, the current ones are not going to capture more of this multi-dimensionality in aging. And I'm a huge fan of Mike Snyder's work and actually it's kind of influencing some of the work that we're doing now. So, what we're actually trying to do now is to build clocks that are proxying aging in different organ systems. So, these aren't out yet but the idea is that, if you can build a clock that's going to, essentially, try and proxy what your brain aging is or your liver aging or your kidney aging, then you can have multiple kind of epigenetic-age estimates and really understand more of the profile of the person.

And so, yeah, it's kind of getting back to this idea of ageotypes. We both age at a different rate from each other but also in a different way. Right? So, I might diverge more down kind of this way and be more of a metabolic ager, or whatever it is, and someone else might be more of an immune ager. And understanding that is going to give people both, potentially, insight into what interventions might be the most helpful for them and also what they might be most at risk for.

Rhonda: Right. Mortality risk is a big one that you see with like GrimAge and even, I think, PhenoAge as well, like, how well do they predict mortality risk and even disease, you know, specific mortality, like your cancer mortality risk or your...and how do they compare to like a frailty risk or a frailty index measurement or something like that where, you know, you can also measure mortality risk?

Dr. Levine: Yeah. So, they're actually pretty powerful when it comes to mortality risk. And I would say right now GrimAge is the best in terms of predicting, what we would consider, all-cause mortality, so, basically any mortality risk all combined together. GrimAge is particularly good at cardiovascular risk mortality, which is why it does well at all-cause mortality because that's the biggest killer of people, at least in the United States. But I think, in terms of predicting more specific types of mortality that someone might be more or less at risk for, I think this is where you need more of these systems' measures. But they are pretty powerful at predicting kind of remaining life expectancy or all-cause mortality. Of course, they can't predict who's going to get hit by a bus, or whatever, but in terms of kind of population averages, are you more or less likely than someone else with the same chronological age to have early mortality, they're actually pretty good at that.

Rhonda: What about in young people? So, it's really fascinating because, you know, like, when I think about mortality risk, I think of like an older person going in, doing a battery of tests, getting all their bloodwork done and, you know, trying to do their grip strength and breathing, to do that. You know, so, I think about more of, like, things that are being measured and aggregated together to come up with this frailty index. But for someone who's younger, like in their 30s, like, and they go and do all this stuff, like, I don't know that it's really going to be a good predictor of their mortality. They're young, you know, they've got pretty good lung function. You know what I mean? So, is this where GrimAge may shine? Like, if you have a 30-year-old or 25-year-old and they do their GrimAge, does it, like, accurately predict mortality risk?

Dr. Levine: Yeah. So, we don't have these really really long follow-up studies but at least the preliminary data seems that it's going to be much better for young people. Because, exactly as you said, these functional things are going to have, what we consider, either a floor or ceiling effect. So, for most people below some certain age, they're all going to perform well on it. They're not at a level where they're seeing dysfunctional decline yet, which goes back to this idea of biological age versus functional age. Whereas the epigenetic clocks are meant to capture more this biological molecular cellular aging that we think will, eventually, feed into that. So, if you can say, "Oh, you're aging faster at a molecular level than we'd expect. We'll also expect you, down the road, probably to have these functional manifestations earlier, even if we can't see them yet." So, I totally agree. In younger people, when these things haven't really emerged, this is kind of the only way to kind of proxy who might be heading in that direction.

Rhonda: Totally. I mean you can't look in the mirror and go, "I've got more DNA damage today," like, you know...

Dr. Levine: Exactly, yeah.

Rhonda: Like, but you could be. And lifestyle factors do play a role and you could have someone who is in their, you know, 30s and they're sort of just living a hard life and perhaps they wouldn't pick up on those sort of functional declines yet but this is where something that is more measuring, something biological, you know, at the molecular level that's going to kind of open their eyes.

So, I kind of wanted to shift gears and talk a little bit about this epigenetic age acceleration. I mean, we've been sort of talking about how people age at different rates. I think you were a co-author on one of the studies like a few years back, that was one of the big ones that came out where it was like, "People age at different rates," and there was like 18 biomarkers that were looked at. And I think it was PNAS or something, a PNAS paper.

Dr. Levine: Oh yeah, the Belsky paper.

Rhonda: Yes, yes. And it was like, "Look, people are aging at different rates, and you can even look at their faces and it correlates with their, you know, biological age more than their chronological age." And so, to me, you know, clearly there's lifestyle factors, environmental factors that play a role in the way you age. Can you explain to people what epigenetic age acceleration is and what some of the most robust biological, environmental, perhaps social causes of epigenetic age acceleration are?

Dr. Levine: Yeah. So, we usually use this term "age acceleration" to just mean kind of the discordance between your chronological age, so, the age you know that you are and your predicted age based on whether it's GrimAge or PhenoAge or any of these epigenetic clocks. And that, again, we think it's biologically meaningful. So, someone who's predicted much older than they are chronologically are people who are higher risk for disease or mortality.

And so, you know, the next question is why are some people predicted older and other people are predicted younger? And a lot of people think, "Oh, it's just genetic, you know, maybe my family is just high-risk." But actually it seems to have very little impact on your epigenetic age, so, I think they estimate like 10%, maybe at the upper most 20% impact, your genes have that kind of impact on your epigenetic aging rate. And actually probably the majority of it is environment and lifestyle.

And when we look, again, these are not clinical trials, it's looking at epidemiological data, so, just saying, "In the population, the people who are predicted to be older versus people who are predicted to be younger, what are their characteristics?" We find things that are not surprising, so, socioeconomic status is a big thing in terms of differences in epigenetic age but also behavior. So, smoking really accelerates your epigenetic age. Generally, exercise will tend to decrease epigenetic age. Eating we think probably plant-based diet is going to decrease epigenetic age. And then, yeah, a lot of the things, don't drink heavily, get, you know, good-quality sleep, minimize stress, all the things that everyone's mother and grandmother told them to do in life.

Rhonda: How does being a male affect epigenetic age? Because males live on average, what, four years...like, their life span's like four or so years shorter than females', right? Is that reflected in...

Dr. Levine: Yeah, it is reflected in epigenetic. So, on average, again, not across the board but, if you look at the distributions, females on average will have lower epigenetic age than same chronological age males.

Rhonda: Similar question, females undergo menopause when they reach like 50s or something like that, plus or minus, I don't know how many years, but how does menopause affect epigenetic aging?

Dr. Levine: Yeah, so, this is actually a study I did while I was in Steve Horvath's Lab. So, we looked at women who undergone menopause and how long since they'd undergone menopause. And it seems to be that menopause is actually an epigenetic-aging-accelerated event. So, before menopause, women are doing pretty well and then, when they go through menopause, it seems to accelerate their epigenetic age. And we didn't have the kind of data you would want where we'd have the same women pre and post but we can even look at surgical menopause and that seems to also show this kind of accelerated epigenetic aging kind of manifestation.

Rhonda: That kind of leads me into another question, which is do the changes in these methylation patterns, that you and others are measuring, is it pretty stable over the lifespan or are they, like, you know, like, once you hit mid-life...because, like, there's functional aging that really starts to hit you, you start to get, like, late to midlife and then it starts to go down, right? So, does the epigenetic clock mirror that or is it pretty stable?

Dr. Levine: So, it's not stable but it actually doesn't mirror what we think of in terms of functional aging. So, if you think of a frailty index or even mortality risk, it increases exponentially after, let's say, age 30. The epigenetic clocks show a totally different pattern, it's still not linear but actually most of the changes happen during development. So, you have this huge increase in epigenetic age between...we can even measure it in fetal samples...and then it kind of starts becoming more linear and steady around age 20 and then, interestingly, actually slows down again, you know, very late, so, after, let's say, age 80. We don't know why these patterns look this way but, yeah, it's not perfectly stable across the life course.

Rhonda: Okay, this leads to a couple of other questions that, you know, sort of came in my mind. One is then what about, when people get, you know, disease states, so, like, they do get type-2 diabetes or cardiovascular disease, you know, does that then, being in that disease state, in that functional decline, like, state, does that accelerate the aging clock?

Dr. Levine: So, I don't know if we actually know that because we don't have very good...the problem with epigenetic data right now is we don't have good kind of time course data. We're not following people longitudinally, there are very few studies that do this. There are more studies that are starting to but I don't think we've reached the point to say, "I can look at someone's epigenetic aging pre-disease state and then see what happens after they've developed some disease," but I would imagine that it would probably kind of snowball and accelerate. And we do know, not looking at epigenetics, that, once you get a disease, it's actually shorter time to each subsequent disease. So, there does seem to be this kind of accelerating event in aging that occurs.

Rhonda: Biobank data might be a good source, they do a lot of, you know, like, they've got just tons and tons of samples, you know, because they have people come in for, like, routine...

Dr. Levine: Yeah. So, we've talked to them, the problem is that the epigenetic data is not super cheap. So, you know, to do that many people that many times, yeah, you have to come up with quite a bit of funding to be able to do that.

Rhonda: It would be interesting, it would definitely be...

Dr. Levine: Yeah, I know. I'm all for this. The more data samples we can get, I think the better we'll be able to figure this all out.

Rhonda: Right. You also mentioned that the most of the changes are happening during development. And this is also really interesting, mostly...Steve, when Dr. Steve Horvath was on the podcast last time, and this kind of gets into the next section, which is, like, underlying mechanisms causing these changes in the epigenetic patterns, but he had mentioned that, like, he had developed at least, you know, and maybe there's something new now, but back then, a few years ago, there was a few clocks that he had used that could really beautifully measure gestational age.

Which was interesting because measuring the aging process during gestation where you've got this really coordinated program that has very little background noise, right, inflammatory processes aren't going off and all this damage and, you know, this stuff, I mean, it's just a very clean place to, like, you know, measure aging.

So, and then he's also...I think there's a preprint I saw pretty recently where he had developed a universal aging clock.

Dr. Levine: Pan-mammalian.

Rhonda: Yeah, there was like I don't know how many different mammalian samples that were, you know, used to generate the clock, and I don't understand everything that goes into generating it. But I just looked, you know, skimmed it and it was really talking about...this universal clock was also really coordinated with development. And so, you're mentioning development, it poses the question, like, do you think that the epigenetic aging could be sort of like a program, like a program that's regulating aging? Like, is that a possibility?

Dr. Levine: Yeah, I don't think it was a program designed...you know, some people would argue that it is, you know, a program designed to drive aging. Because I think, you know, for species selection, you need things to age and die so that the species [inaudible 00:28:16]. I think it's a developmental program that just doesn't really get turned off and maybe goes a little bit awry as all these other changes start to accumulate in our bodies. But yes, the epigenetic clocks are really tracking something very central to development because most of these changes we can see during development and a lot of the genes that seem to be involved are these developmental genes. We're still, again, not sure what this means or what this program actually is but it definitely is tied to development. But people would also argue that aging is very tied to development. So, there are beautiful experiments in flies where, if you can extend kind of the developmental period, it extends the lifespan of these animals.

And so, development and aging are kind of two dichotomous things that we usually think of, right, that you're going through development, you hit age 20 and...okay, maybe age 20 or 30 is when your aging starts. But there's a lot of great work, even one of my colleagues at Harvard, Vadim Gladyshev, showing kind of when he thinks this ground zero when aging starts, which is, according to him, day eight of gestation. So, there's...

Rhonda: In humans?

Dr. Levine: In humans, yes. So...

Rhonda: When you said, "In flies and fruit flies," Drosophila probably...

Dr. Levine: Yeah, yeah.

Rhonda: "When they extend the development," can you explain that, what do you mean by that?

Dr. Levine: Yeah, so, I think, in this case, actually, the study I'm thinking of, they extended kind of the reproductive kind of age of the fly. So, they can push flies to, what we would consider, like a late fecundity, so, they can develop a little bit longer and don't reproduce until slightly later.

Rhonda: And how do they do that?

Dr. Levine: It was more like selection. So, they're selecting for flies over generations that are going to be these later-fecundity flies, and they show that they also live longer in the end.

Rhonda: Like, is it a genetic...genes that are controlling it?

Dr. Levine: Yeah.

Rhonda: Okay. So, a couple of things here, then back to this...it's very interesting, the development thing. And another thing came to mind from the conversation I had with Steve was he had mentioned, like, if you take a cell that has not been immortalized in tissue culture and then you immortalize it with a component of telomerase, TERT, and you, essentially, overcome cellular senescence, which is one of the hallmarks of aging. Right? When a cell undergoes senescence, I mean, it's pretty much not...I mean, it's still metabolically active but, you know, it's considered sort of the end, right?

Dr. Levine: Yeah.

Rhonda: And these cells, if you continue culturing them and tissue culture, their epigenetic age just keeps going and going and going.

Dr. Levine: Yeah, we're actually doing this exact thing in my lab right now where we use hTERT to immortalize cells and we are just seeing how long...like, there has to be some...like, eventually, can it just continue to change forever? I think two people have looked at things like HeLa cells which have just been changing, you know, they've been evolving for decades. And like at what point does the epigenetic age kind of reach a saturation point? And again, I don't think we know but, yeah, definitely with these immortalized cells, every time you passage them, their epigenetic age keeps...at least it seems to continue to increase over time.

Rhonda: It's interesting. And sort of, on the flip side of that, would be, like you mentioned, like, are the epigenetic aging clocks biomarking something, like, something else that's causing aging? And a study that you were a co-author on, as I was preparing for this podcast, I was thinking about it and I was, you know, in my mind, I was like, "Well, how could you, like, cause something like that would be massive damage to accelerate aging?" And so, I googled "cancer chemotherapy epigenetic clock" and, like, the paper you were a co-author came up on it. I was like, "Oh, this is Morgan's."

Okay, so, I was reading the paper and these patients that had head and neck cancer and they were getting treated for it, radiotherapy, chemotherapy, you know, which causes massive damage, inflammation, these patients, their epigenetic age was measured before the treatment, after the treatment, and then six months later and a year later. And it was so interesting to me because they had aged...like, their epigenetic age had accelerated by 4.9 years right after the treatment. But then, six months later and a year later, like, their epigenetic age had, like, normalized back to baseline. And sub-analysis then showed actually not only did the epigenetic age acceleration of almost five years correlate with inflammatory biomarkers but people that had extremely high inflammatory biomarkers one year later did still experience the age acceleration. So, I'm curious as to what your thoughts are on what that means? Like, to me, I look at that and I go, "Wow, inflammation is causing epigenetic age acceleration," because you see this like graph, right, I mean... So...

Dr. Levine: Yeah. I think, definitely, when we measure aging in blood, we need to think, you know, what is, you know, probably driving these signals that we see. And I would guess that epigenetic age acceleration in blood is mostly reflective of inflammation. Unless, again, you're developing a clock that's specifically tuned to some other thing. Although inflammation seems so, you know, vast and systemic it affects so many different things.

But I don't think everything that epigenetic clocks are capturing is inflammation. Because again, when you look at immortalized cells, it's not because they're becoming more inflammatory every time you're passaging them per se but definitely, I think, epigenetic aging measured in blood is very much tied to inflammation. Which again, is probably why it's highly predictive of a number of diseases which we know inflammation can be a major driver of.

Rhonda: Is that where the extrinsic and intrinsic aging clock...or, I don't know exactly, one of them considers the external factors in blood and one doesn't or something, is inflammation calculated in that or not really? Is it sort of...

Dr. Levine: Yeah. So, these are two of the first-generation clocks, so, I think, you know, Steve kind of called them intrinsic-extrinsic aging. I think he called the original Horvath pan-issue clock was the intrinsic aging, it wasn't that tuned to differences in kind of cell turnover or inflammation. Whereas a clock, that was developed by [inaudible 00:35:08], he kind of added these different kind of cell composition measures that actually ended up picking up inflammation a little bit better. But this was before these second-generation clocks came into being. And then I think, once they came into being, they're probably picking up inflammation a lot more than even the first-generation clocks.

And again, we can make these kind of systems clocks and one of our systems is inflammation, and we can show that it's highly predictive of outcomes, it's definitely capturing things related to inflammation. Preliminarily, I can say we have data from individuals with COVID, and we can look at the inflammation measure and we find that people with severe symptoms have much more accelerated inflammation, epigenetic clock, than people with basically asymptomatic or mild symptoms.

Rhonda: It'd be interesting to see when those symptoms resolve and how long it takes for a person to like go back to more of their baseline ever, hopefully. But so, that's definitely...are you guys going to continue looking at that or...

Dr. Levine: Yeah, I mean, we don't have the ability to track the same people over time but I think this is an important thing. And I think this is important when people start to look at applications of the clocks for intervention testing. Because you can do an intervention that's going to change kind of your blood cell composition and it might be reflective of inflammation but, you know, it could be this acute event. Right? And whether that really means you change your aging I think still needs to be kind of considered.

Rhonda: Yeah. And I think that was the big eye-opener for me when I read this study, I don't know, a couple days ago. And it wasn't a new study but, you know, it was like, "Oh, well, this changed really dramatically." But then it wasn't, like, a permanent thing, I mean, it went back. And so, yeah, it's almost like you're saying with interventions, it's like, "Well, I mean, make sure you didn't get sick," or, like, you know, sick, like, too early before, you know, measuring. And we can talk about that in a little bit, a little bit later, but to get sort of just back into the cause and effect of aging and if the epigenetic clock changes are really causal, I mean, of course, you're, obviously, trying to figure that out, but, like, even if it was, let's say, downstream of something, if it was biomarking aging, what...the epigenetic changes that are happening with aging, you kind of mentioned this early in the podcast about how, you know, they're clustering in gene regulatory regions, and, so, they're changing the way genes are turned on or turned off, is there like a feed-forward loop in aging where it's like, "Okay, now these epigenetic changes are turning off genes that we want on to repair damage and they're turning on genes that are cellular senescence?" you know, so, it's accelerating this feed forward loop...

Dr. Levine: Yeah. I mean it's definitely possible. I think, yeah, it's really hard to figure out causality here, right? Like, and it could be that, you know...I mean my perspective is not, "There's a cause of aging," right, and, you know, "it's this thing and, once you fix that, everything else will go away." I mean so many things go wrong and your system can change, it can diverge in some...going back to kind of Mike Snyder saying, "Even if you bring that down to the molecular level, there's so many different ways that someone's system can kind of change over time." And I don't think it's just this one thing that's going to then drive all of aging.

And yeah, you know, our systems are responsive, right, so, one thing changes, something else is going to respond. And that can be maladaptive, which would, you know, snowball things. So yeah, I think it's going to be hard to figure out like what's causal, what's correlative, but I would say, even if it's not what some people might consider the central driver, as long as it's picking up things that are critical to aging and you can use that to track aging or understand it a little bit better, I think it still has utility. I don't know if it needs to be kind of the central cause of aging for it to be useful.

Rhonda: Right, exactly. If you can track it and/or use it for basic science, you know, to understand the processes better...but with some of these genes, I'm just curious, do they know, has research shown, your research and others' shown that, as the epigenetic clock ages, we are seeing more genes that are regulating NF-kappaB turn on causing more inflammation? So, it's not necessarily the cause of aging but it's helping accelerate it as you start to accumulate these epigenetic changes, as they start to shift.

Dr. Levine: Yeah. So, the hard thing has actually been looking at the genes that these CpGs are assigned to or that they collocate with. And actually that's been a little bit less clear because the methylation patterns are not, as we might expect, correlating with the expression patterns. And there's a number of reasons that could be, it's because we're looking at lots of cells in the population, you need to look within an individual cell to actually be able to see this. It could be, you know, there are other epigenetic modifiers that could also be important in this, so, it's not a one-to-one. But we can just look at general gene expression that epigenetic clocks are associated with. And, you know, you can kind of find certain pathways that seem to be important. And some of these are inflammatory, or other aging pathways that we'd kind of expect.

But yeah, we still don't know exactly what the CpGs that are in these clocks are functionally doing. Even though we say, "Oh..." I actually said it in the beginning of this talk, when you have methylation, it's repressive, when you don't, it's active. But it seems like it's actually a lot more complicated. And one thing that I always come back to is I can make a clock out of a few hundred CpGs that are, you know, they're in specific genes, I can remove all of those genes and remake a new clock and I can get the same clock from a totally different set of genes. I don't know what that means in terms of understanding the functionality of what we're trying to capture but I think it just suggests it's not as simple as, you know, these 20 genes are turned on and these 20 genes are turned off.

Rhonda: Right, right, a lot more to learn. Epigenetic age reversal, that's a big interest, of course. And I'm sort of curious about, like, some of your thoughts on some of the...there's been some recent studies. So, we were talking about programming, right, we were talking about, in a way right, with the developmental program and the epigenetic clock sort of really tracking that well, being part of that, in some way connected. I don't exactly understand why [inaudible 00:42:11]. But I don't think anyone knows. Okay. So, some of this work with interrupted cellular reprogramming, or the partial reprogramming, as it's called, a lot of the works come from Juan Carlos Belmonte's Group where they can...maybe you can explain like what this is to people and how that affects epigenetic aging or what's known or not known?

Dr. Levine: Yeah. So, this really came out of work, originally, from Shinya Yamanaka who discovered, what we call, these Yamanaka factors, which are four transcription factors, we just call them OSKM, which, when expressed, you can actually take a somatic, so, an adult cell, and convert it back into what looks like an embryonic stem cell. So, we call these induced pluripotent stem cells. And then you can use those to make a number of different types of cells.

But the interesting thing and why aging researchers got really invested in this science is that, not only are you making it embryonic-like in terms of its stem-cell properties, but the epigenetic clocks seem to be almost completely reversed. And we've actually shown recently they're not completely reversed but you can take a skin cell that has an epigenetic age of 40 and do this, it takes, you know, a few weeks to do, and basically get back to an epigenetic age of zero in those cells.

Rhonda: And you keep it the skin cell, it doesn't lose its identity?

Dr. Levine: Yeah, so, it loses its identity...

Rhonda: Okay, when [inaudible 00:43:45] back.

Dr. Levine: Yeah, so, this is considered kind of this full epigenetic reprogramming. And then what Juan Carlos Belmonte and others have done is look at this idea of partial reprogramming. So, can we push the cell back a little bit? Because actually what we find is that this age reversal happens first prior to the cell losing its identity. So, can you do that part without pushing it all the way back, what we consider up or down the landscape, to this pluripotent stem cell? So, can I just make an old skin cell a young skin cell but still a skin cell? So, that's the goal.

Rhonda: And with some of the recent work, at least out of his lab, they're using a premature aging mouse model, a progeria model, and have shown...I know there's a new publication I haven't read, just came out, but the older one, the first one, 2016 or something, cell paper, I remember they showed, in multiple different organs, it seemed to reverse some of the hallmarks of aging, you know, and the organs were performing functionally a little bit, you know, younger than you would imagine, at least in this premature aging mouse model, and I think even health span of this mouse model that's prematurely aging, it seemed to be improved. I mean, what that means for humans, that are not mice, with premature aging syndromes is to be determined but the epigenetic clock was also reversed as well. Right?

Dr. Levine: Yeah. And I think the new publication, which is done in more of a wild type, not a progeroid mouse, does show kind of some reversal of the epigenetic clock. And you can do this just cells in a dish, we can partially reprogram them and show reversal of the epigenetic clock and other functional improvements in the cells.

Rhonda: So, to you, what does that mean, like, that you can do that?

Dr. Levine: Yeah. No, I mean, I think this is the most fascinating thing. Again, I don't know in terms of translation, like actually making this a therapeutic, and I don't even think people were at the point where [inaudible 00:45:41]. But yeah, I just think it's so amazing. I mean even the original thing, that you can take, you know, a skin cell and turn it into an embryonic stem cell. And just we always think of, you know, like, this is one direction, cells are going to only move, you know, what we consider this landscape, in terms of their states, and they can only go from this state to that state. The idea that it can go back I think is amazing, and I think just understanding how that process works...

And then the other thing we're really interested in is what are the features of this programmed cell? Like, does it truly look like a young cell or is it a totally different type of cell that, in nature, maybe we haven't even seen? And what does that mean for how it's going to function?

Rhonda: Totally. The questions I have on my mind are, "Okay, well, you take this, you know, 40-year-old skin cell," as you mentioned, "and let's say you're going to completely reprogram it to a stem cell and your epigenetic age goes back but, like, what happens to all the damaged mitochondria? Are they still there? Like, what about the pieces of DNA that, you know..." Like, is that stuff still there? Like, where does it go? How does it go away if it does?

Dr. Levine: The exciting thing is actually the mitochondria seems to also be kind of rejuvenated. I mean, I don't really like that term, rejuvenated, but it seems to be kind of set back to a better functioning state.

Rhonda: Oh, really?

Dr. Levine: Yeah, again, it's not clear how all these things are linked to each other. I think the other question though is, you know, cells also build up kind of these aggregates and other, you know, nasty kind of byproducts that accumulate. What happens to them? I don't think we know that. But it's an important thing I think to figure out.

Rhonda: So, I mean, if the epigenetic clock, because it's controlling gene expression, it's like, well, maybe the nuclear coded mitochondrial proteins, maybe everything's just bouncing back to how it was and, so, you're building better mitochondria. Right? I mean...

Dr. Levine: Yeah, I have some colleagues who argue that it starts with the mitochondria, getting rejuvenated, and then that's how everything else...but yeah, I think...

Rhonda: Yeah, it's back to that hallmarks of aging, you know, mitochondrial dysfunction...I mean or, as you mentioned, it's probably not just one thing, it's a combination of all these factors together combined where your proteins are misfolding and your mitochondria is functioning and, you know, your DNA damage is accumulating. And so, yeah, I mean it's a fascinating area. And the programming part, like, I was just curious what your thoughts are in terms of the basic science? Like, to me, it goes back to, again, that program. Like, there's something going on, we don't quite understand, but it's something.

Dr. Levine: Yeah, this comes back to this whole idea that I don't think what we see with aging is just random stochastic damage or errors. I think we've always thought of aging as just accumulation of errors but it really might just be a program that kind of goes wrong and there's nothing evolutionarily that needs to prevent it from doing that because it doesn't benefit, you know, fitness to prevent that program from going wrong. But the idea that it can be reprogrammed, again, using the operating system kind of analogy, that you can just take an, you know, operating system that's not doing well and do an update and take it back to this better state...and again, we need to figure out exactly what that means, but I think it's really exciting.

Rhonda: It is. And like there's no doubt that accumulation of damage does play a role in aging but like maybe it's not the cause or the only thing, maybe it's just the feed forward loop accelerating it. Who knows, right? I mean, it's also interesting.

Another really interesting area is the plasma exchange. I'd love, like, you know, for you to kind of explain to people what some of this interesting research is in the aging field, plasma exchange. You can start back to, you know, their original parabiosis studies maybe.

Dr. Levine: Yeah. Well, and actually I think this relates exactly to what you just said is there is accumulation of damage and there are, you know, these things that are accumulating in our systems. And it could just be the program responding to that damage in a way that, you know, it was set up to do. So, this idea of parabiosis, I mean, this is, what, a century...really old. Like, people were doing this in like the early 20th century, where basically you can take two mice and connect their circulatory systems. It's not a pleasant procedure if you're one of the mice but they'll do, what's called, heterochronic where they take one young mouse and one old mouse and connect them and then just say what happens to the aging. The young mouse, you know, is now having some influence from the old mouse and vice versa.

And what we find is that the young mice has accelerated aging compared to one that's paired with another young mouse, and the old mouse is somewhat rejuvenated compared to an old mouse, compared to an old mouse. And then, more recently, people said, "Okay, well, maybe you don't have to connect them, you can just do this whole plasma-exchange method where you can put young plasma into an old mouse," and it seems to, in some ways, again, rejuvenate them. Not to use that kind of snake-oily term but that's the best we have.

And actually, we've been doing this with cells in a dish. So, we actually buy serum from older individuals versus younger individuals and we can grow our cells in these two different conditions. And we, again, can age even fetal cells using old serum, the young serum seems to be not as problematic.

Rhonda: Very interesting. I know some of the recent work out of Irina Conboy's lab at UC Berkeley, what was interesting to me about her research, or her recent research, was that they were able to take this plasma and, you know, basically, it was just the saline and albumin...they took old mice and, like, it was essentially diluting out their old plasma and it rejuvenated these mice. Whereas they did it with the young mice, there's really no effect. Yeah. So, it really indicates, like, there is, as you mentioned, there's something accumulating, at least in the bloodstream, with age that may, in some way, be accelerating the aging process. So, what do you think the epigenetic age would do, like, if that was measured? Is that going to be measured?

Dr. Levine: Yeah. So, this is what we're doing in the cells. So, actually the Conboy's were the first ones who did this in-vitro experiment as well, so, that's how we knew that it would work. And now we're looking at the epigenetics of those cells and also the RNA. And people have started to do this too in terms of the mice. I don't know if they've done it in terms of just saline-albumin exchange but, in the normal kind of parabiosis context, it does change the epigenetic clock. And so, the question again is, "Is the methylation patterns that were capturing the clocks just a response to the accumulation of these kind of problematic factors?" Because people always wondered, "Oh, is there something magical in young blood that's rejuvenating?" versus, "is it just this problematic things that accumulate in old blood?" and it seems to be more of that. And yeah, the idea that you can just dilute it out and get the whole program kind of responds and rejuvenates itself I think is really amazing.

Rhonda: So, I guess the main questions would be, you know, at least if I remember from the Conboy study, like, even the brain, like, I think it was the hypothalamus or something, I don't know how significant it was in terms of like they were measuring, whatever biomarkers they're measuring for marking aging, it seemed to be better, even in the brain. The question would be for the epigenetics is like, "Well, is it again one of those things with the cancer chemotherapy experiment where there's just inflammation, there's something causing damage, and it's a transient thing, like then you have to keep getting these plasma exchanges, you know, which isn't yeah sustainable really." Well, I don't know, at least I think I don't think it is." You know, or is there something that does it long-term effect the other organs and stuff? Like is it something that's going to be a permanent thing, you know?

Dr. Levine: I mean I think we don't know. Actually, I'm stealing this from my husband, so, I'll give him credit for it. He talks about...it's kind of the like climate change in the body. Right? So, the cells are in a problematic climate and they're going to not behave the way that, you know, they should be. And then, if you remove that, you know, everything kind of gets better. But if it's not sustained, how quickly is it going to return? And I think we don't know that. My guess would be about as transient as kind of the effect...you know, if you could dilute and that's maintained for a while, it would probably be maintained in terms of the cells' kind of features and epigenetic measures. But yeah, if it returned really quickly, then I think it would be more transient.

Rhonda: I'm sure people are trying to figure out is there a factor in the old blood causing...

Dr. Levine: Yeah, people [inaudible 00:55:07] a factor.

Rhonda: Or is it, like, you know...you're only as good as the sensitivity of your assay. And, you know, like, I always go back to this inflammation thing, you know, because there's a lot of data out there with it, right?

Dr. Levine: Oh, yeah.

Rhonda: I mean, and we have biomarkers for inflammation. Like I've gotten my high sensitivity [inaudible 00:55:26], it's like 0.2. Like, does that mean I don't have inflammation going on? No, like, inflammation is happening, right, it's happening all the time, it's happening every day, every second. Like it just means that assay is only as sensitive to pick up, you know, that much inflammation. And so, maybe, like, we just haven't gotten the right tools yet to, like, pick up, like, even that. You know what I mean? So, there's always the sensitivity question and the tools that you're...which is why you, guys, are developing these great, you know, tools to measure aging, quantify it.

But to kind of shift into, you mentioned the exercise, and we're talking about age reversal and kind of slowing...like, so, exercise is also associated with the slowing of epigenetic age. And then I think the other thing you kind of alluded to for a moment was genetics, and I had a question here because you were saying, "Genetics," it seems as though...10% to 20% you mentioned?

Dr. Levine: Yeah.

Rhonda: Pretty small, pretty small in terms of epigenetic aging. Like...

Dr. Levine: But even in terms of lifespan, it seems to be on par with that.

Rhonda: So, only a small percentage of the way you age is controlled by genetics?

Dr. Levine: Yeah.

Rhonda: Now, this is my caveat, or my question, what if you are a super centenarian or a semi super centenarian, like there's obviously you're an outlier, right, like, that's an outlier but it exists and it's thought to be under genetic control, I think...

Dr. Levine: Yeah, yeah. So, they're probably not just randomly making it to that. So, for most of us, our aging is going to be less under genetic control but there are definitely people you might think win the genetic lottery. Right? So, they're very unique and they have the perfect combination. It's probably not one gene, they just have the perfect combination of different gene variants and that somehow enables them to live much longer than the rest of us, it seems even despite having bad health behaviors. So, these super centenarians don't necessarily smoke less or eat better or exercise more than people in the general public but they're somehow able to overcome that and survive to extreme ages. And that's probably more under genetic control.

But for most people, unless you have, you know, a string of grandparents that all survived to 110, you're probably not going to be able to rely on your genes to get you there. And actually, my PhD dissertation was on long-lived smokers and thinking, you know, smoking decreases people's life expectancy by about 10 years but you have these people who survive to 100 or beyond still smoking, and, you know, what is it about their genetics that allows them to kind of overcome that?

Rhonda: And what was it?

Dr. Levine: I mean, the stuff that came up was major aging pathways like insulin IGF-1 pathway, but again, we haven't proven this out causally. But yeah...

Rhonda: Did you read that Japanese semi or super-centenarian study that came out, I don't know, a few years ago?

Dr. Levine: I don't know if I...

Rhonda: It was a study where they looked at...

Dr. Levine: Was that the men, where they looked at...

Rhonda: Yes, men, and they looked at elderly and then they looked at, you know, going from elderly to a centenarian, from a centenarian to a semi-super centenarian, which is 105, and then to a super centenarian, which is like 110. And they looked at all battery of biomarkers, I don't think epigenetic clock was in there, but they looked at, you know, like, telomere length, immunosenescence, all the bloodwork stuff, the metabolic and, you know, lipids and stuff. And then they looked at inflammatory biomarkers.

And it was fun, it was interesting because the suppression of inflammation was the only thing that could predict going to the next age group or age...I don't know what it's called but transitioning to surviving to the next...right, just being able to like low inflammation, basically. So, that was sort of interesting as well.

And also it kind of goes back...you mentioned smoking and, of course, genetic control there, typical pathways, it kind of brings to mind we recently had Dr. Bill Harris on the podcast, and he's probably one of the world experts on omega-3 fatty acids and just been doing decades and decades of research. And he has all this interesting data. He does a lot of work using the omega-3 index, which he co-developed, where they measure omega-3 and red blood cells. It's a long-term marker of omega-3 rather than, like, what you had the night before, you know And, you know, he's done all sorts of studies using Framingham data and has found that...so, a typical American diet is they have about a 4% omega-3 index. And that's kind of low, especially if you compare it to other countries like Japan where their average omega-3 index is like 10% or 11% or something, much higher.

Anyway, so, he did studies with Framingham data and he stratified people based on their omega-3 index. So, low was like lower than 4% and high was about 8%. And people with an 8% omega-3 index had a 5-year increased life expectancy compared to people with the 4% omega-3 index. Interesting. But what was also really interesting from this data was he looked at smokers. And smokers, as you would imagine, had a much lower life expectancy. But smokers that took omega-3, that had high omega-3 indexes did not have that same low life expectancy. But here's the really interesting thing is that the smokers that took high omega-3 had the same life expectancy as the non-smokers with low omega-3. So, in a way, low omega-3 was like smoking, you know, for your life expectancy. Right? I mean, to me, it was very interesting data. You should pair up with him. And I would be so interested to know the epigenetic age as it correlates with the omega-3 index.

Dr. Levine: I mean, yeah, depending on which samples, in Framingham, they do have methylation already measured.

Rhonda: Okay, there was also a really interesting study. This kind of gets into interventions as well, I kind of wanted that you touched on that for a moment, and there was an interesting study...and I don't know if I really have a question but I also just like to seed ideas, you know. The study was, like, women that were genetically predisposed to breast cancer, they were given 5 grams a day of fish oil. So, it was EPA, DHA omega-3, the marine omega-3 fatty acids, and this was like 6 months treatment. And, like, they had done some sort of methylation profiling, not epigenetic clock but profiling of their blood, PBMC's, their peripheral blood mononuclear cells. And there was like hypomethylation in I think it was like TNF-alpha or one of the major controllers of inflammation where it was like decreasing a lot of the pro-inflammatory pathways. So, you know, at the level of methylation, I thought that was so interesting.

I'm not sure what's going on there because there's lots of ways omega-3 regulate inflammation, they suppress it, they resolve it. You know, and so, I was like, "Wow, they're changing methylation patterns, like, how is that happening?" So, just sort of interesting, you know, potential research ideas there.

Dr. Levine: Yeah. No, I think there's a ton of things to start connecting and, you know, all these environmental things and just physiologically how are these things connected when you go up in this, how does that affect these other things downstream. And yeah, I think, as more and more data gets collected and we actually have good measures of all these different things, we can start doing that. But yeah, it's...

Rhonda: What do you think about some of the...so, there's consumer-available epigenetic, you know, aging clocks out there, like, what are your thoughts? Like, are they accurate? I mean, what's...and, of course, I know you're an advisor for Elysium, which makes one of them. So...

Dr. Levine: Yeah, no, this is an important question. And I think, you know, the hard thing with epigenetic age is, again, this is something people can't...you don't know the answer, right? So, you can take one of these tests and you get a number back and there's no way for the consumer to verify whether that's, number one, correct, I mean, if there is a correct answer, or if it's even meaningful. And I think, basically, there are two things that I think are really important when you're talking about epigenetic tests being used by individuals or even clinically. This comes back to the reliability of these tests. So, what I mean by that is, if I were to take the exact same test twice on the same day, will I get the same answer? And unfortunately, what we found is actually, if you use the original epigenetic clocks, you do not get the same, you get wildly different answers. They're actually highly unreliable and very noisy. So, we've taken blood samples. You can split them, like, the same sample, run it twice, and you can get upwards of eight-years difference in your epigenetic age, using traditional clocks.

So, actually, a few years earlier, and this actually came out from my work with Elysium because they saw this in their data first and then we went back and looked at it more. Because I thought, "Oh, this is the end of epigenetic clocks." So, if this is what's actually happening, they can't be useful for anything, unless you have thousands of people. And so, we actually developed a statistical method that completely removes all this technical noise, and I won't go into the math for people on the podcast, but basically, we can get this down to you can split the sample and now you're getting only about one-year difference at max. Most people are predicted exactly the same age on their two tests.

And so, what I would say...so, Elysium, as you mentioned, I'm no longer an advisor for them because I'm doing other stuff with Altos but I was an advisor for them, and they actually did care a lot about this reliability thing, this is why I was helping them to try and sort this out. So, at least for their tests they felt like, if someone took it twice, they would get the same answer. You know, assuming you're taking it twice within a short period of time. But most tests, I would say, on the market are using the old methods that are highly unreliable. And I don't know that but I would suggest to consumers to, you know, find out if there is data on the reliability of tests.

The second thing that's really important for epigenetic tests is something that statisticians call construct validity, which is just this idea of biological age is, what we call, latent. You can't see it, you can't truly measure it. It's not CRP where I know I'm trying to measure something very specific. So, it's can we try and approximate something that's not really measurable? And then the way we evaluate if we did that well is does it predict or track with things we would expect? So again, with epigenetic tests, it should predict things like mortality risk after you adjust out the chronological age. So, being higher/lower than your chronological age should be very predictive of mortality risk or disease risk. And people who are using these first-generation clocks, the ones trained to predict chronological age, are not as good at that.

So yes, there's a lot of tests on the market but I think it's really important to make sure you're using ones trained more like the second-generation clock, so, things like GrimAge or PhenoAge, but again, using these methods that make them more kind of reliable and take out the noise.

Rhonda: Are there that many epigenetic clocks that are consumer-available now, are there?

Dr. Levine: I think, I mean, I actually don't know the number but I constantly see different companies launching epigenetic age tests. There's at least, I would say, probably close to 10 on the market.

Rhonda: Wow. Is the one that was developed by Elysium, when you were advising for them, that was more for biological age, that was the...

Dr. Levine: Yeah. So, it was similar to the PhenoAge one but with this additional statistical method that removes the technical noise. So, that one we've shown is predictive of mortality above and beyond chronological age. And actually Elysium I licensing these systems' measures, so, I think they'll be putting those out too where you can get kind of approximation of aging in different systems and get your kind of ageotype kind of thing.

Rhonda: My questions kind of got many layers to it and it has to do with like, if a person is, you know, trying to measure their epigenetic age and they want to do a lifestyle intervention and then measure it again...so, there was a very very small study, an extremely small study, it was published by a gal that reached out to me, her name was Kara Fitzgerald. And she and her colleagues had taken a small sample of people and they underwent like an extreme dietary change where, you know, they were eating a lot of leafy greens, cruciferous vegetables, blueberries, but they also were eating animal meat, liver, like, organ meat, and eggs. No refined sugar. Meditation, exercise, probiotics. I mean it was just a kitchen sink, it was a lot.

And this was like...I think it was pretty short treatment, like it wasn't like six months or anything. I can't remember off the top of my head, maybe two months or something like that or three months, I don't know. But their epigenetic age, according to the clock they used, I think it was maybe the original...

Dr. Levine: [inaudible 01:09:18] the original.

Rhonda: So, I guess you kind of answered the question. But it had reversed by like 3 years or something. So, I guess the question is, you know, like, can you track interventions accurately? Can you, you know, make assumptions based on it, or what are your thoughts? Like...

Dr. Levine: Yeah, and I'm not saying this to speak badly about any study but I mean the great thing about that study is they made their data public. And we were actually able to go back in and show that the entire effect was noise. So, actually, once you do the statistical method that removes the noise, there was actually no effect of the intervention. And, of course, you know, these methods weren't available originally and they were using an original clock like they thought they should.

But I think this shows us that we need to be a little bit careful how we interpret the results from these things and the epigenetic clocks are powerful tools to kind of...I think they are good at giving you an idea of your health status, same as if I go into my doctor's office for, you know, a kind of lab test and do a metabolic panel or whatever. They can give you a good idea of your status. But when we think of how they're applied to interventions, I think we need to be careful that we don't take any change at just face value, we need to really think about, you know, "If I measure it again, was that sustained?" like things we talked about earlier. Because I would argue that we haven't proven that a change in the epigenetic clock is a change truly in your biological aging process. And like we said, you can have changes in kind of inflammatory markers that are acute and not truly capturing the aging process but that'll change what you see in terms of your clock.

So, I think epigenetic clocks are really helpful for like a wake-up call. So, you know, a lot of young people are kind of going about their life, we don't know, like we said, till you see these dysfunctional things start happening, if we're doing well or not. And I think they can be used to kind of inform. And as we move forward and develop better more sophisticated ones and we look at actually linking changes in epigenetic aging to changes in other kind of health parameters, they will be good for kind of people testing interventions, either personally or in clinical studies. But I think right now we're in the really early days and these are really high-potential tools but we're not at the point yet where you can just, you know, take it and believe everything that you get back.

Rhonda: Right. So, you're better off also doing all the classical biomarker tests too and measure lots of things?

Dr. Levine: Yeah.

Rhonda: It's always good to have more data. Before we wrap this up, two questions. What are you most excited about with respect to the aging field in general, like what's being researched right now, what's coming out of the pipeline? And then what are you most excited about coming out of your lab? Or I guess they could be the same answer.

Dr. Levine: Yeah. No, they're related. So, the thing in the aging field, again, is this kind of transient or partial reprogramming thing. And just, again, not necessarily for an intervention but just figuring out what it is. To me, it's just so magical that you can change the state of a cell. And what does that mean for the cell, and does it now function better, and do populations of cells work better together, and how does that happen? Can you figure out even better ways to do this? You know, we've used kind of the Yamanaka factors but there could be, you know, tons of other ways that you could actually change this and just not knowing that that's possible. So, that kind of basic science I'm the most excited about.

And then, in my lab, I'm just really excited to figure out what the epigenetic clocks are. We have no idea, we apply these measures to a bunch of different things, we have no idea what drives these changes or, functionally, what they even mean. So, why is your epigenetic clock related to your mortality risk? It's like what is the pathway that links those two things? And I think that'll keep us busy for a really long time, that's something I'm excited to kind of start working on.

Rhonda: Awesome. I'd like to ask this last question to a lot of podcast guests, and that is, you know, what lifestyle changes, like, do you incorporate the most, you know, into your life based on science?

Dr. Levine: Yeah. I mean, for me, there's kind of two things, diet and exercise I try to pay a lot of attention to. So, exercise I think is such a simple thing that so few people...you know, everyone's waiting for the magic pill. I mean, if they could bottle the effects of exercise, it would be the biggest thing in aging research that, you know, exists. It's probably the most powerful tool we have to actually intervene in our aging process, or at least to slow it. Or they've even shown, you know, you can reverse diabetes through exercise or any of these things.

Rhonda: Better than Metformin.

Dr. Levine: Yeah, I know. So, again, exercise is amazing, it makes you feel good. So, for me, trying to maintain an active lifestyle as much as possible, you know, I sit at a desk a lot, so...

Rhonda: What's your favorite kind of exercise that you do?

Dr. Levine: I mean I like to do things that just are fun. So, hiking or anything like that or, you know, any of these classes. But probably, if I were thinking the most beneficial, probably things like HIIT, just try to do that as much as I can. And then the other thing is just diet. I eat, I would say, 90% to 95% plant-based, I do eat some fish, try to keep kind of like a Japanese type diet. And then I do intermittent fasting where I don't eat until usually like 1:00. Again, that I think, we don't know for sure whether that's beneficial, but for now, just sticking to it, seeing kind of what happens.

Rhonda: Yeah. We had Mark Mattson on the podcast, not long ago, and he definitely convinced me that there are some benefits for sure in intermittent fasting with respect to, you know, keeping in ketosis and the metabolic switch. But so, did you see that paper that came out of Yale from Vishva Dixit's lab?

Dr. Levine: Oh, yeah, yeah. Yeah.

Rhonda: Like, I didn't in-depth read it, I just sort of glanced at it, but like there was a two-year caloric-restriction study.

Dr. Levine: Yeah, that's from the calorie study. Yeah.

Rhonda: Okay. So, and thymic aging was like slowed, which was kind of...

Dr. Levine: Yeah. Yeah, I think, you know, calorie restriction has been going around for quite a while. I think the issue is most people can't actually sustain...

Rhonda: Right. No, I mean I think there's, you bring up a really good point, like there's been a lot, it's like the classical intervention that's been done in rodents, it's been done in non-human primates, and it's been shown to...you know, it does improve health span of those animals that are not humans. And in some cases, they really restrict it, like 30%, like they eat 30% less calories or fewer calories. Whereas I think this study the calorie restriction was like 15% or 14%.

Dr. Levine: Yeah, so, I think it's like 12% or...yeah.

Rhonda: I don't know what that translates. Like we don't know if caloric restriction actually is how beneficial it is for humans. And it always comes back to, again, that like...well, overeating is not good, you know, overeating is not good, we know that, and, you know, there's many ways that you can affect calories in, calories out, and one of them is exercise too. You know, so, there's a lot of ways that you can sort of get to a similar, you know, end point where you're not just eating as many calories. Right?

Dr. Levine: Yeah.

Rhonda: It doesn't have to be like you're...and you certainly don't want to starve yourself. Like, calorie restriction, like, you know, there's a problem with muscle wasting and frailty with age. Right? So...

Dr. Levine: Yeah, especially if you're getting older.

Rhonda: Right, exactly. And getting enough protein. So again, yeah, all these little nuances, especially when you start to translate the research. But epigenetic age was, if I remember correctly, shown to be slowed in the rodent, in...

Dr. Levine: Oh yeah, so, in mice, epigenetic age is affected by calorie restriction and substantially slowed. And the longer the animals are on it, the kind of slower the increase in epigenetic age over time becomes. But again, I think, you know, how much of this is the absence of excess food? And there are studies in rodents showing that different genetic backgrounds have different responses. Some actually do worse with caloric restriction. So, that amount of restriction I think is probably going to, you know, depend on you personally, not just preference but, you know, some of your genetics.

And the other thing, the reason I don't do caloric restriction personally is because there's studies, even in mice, that, if you stop it, you lose the benefit. And I can't imagine spending my entire life on caloric restriction to continue to get this benefit. So, I always say to people, "Do whatever you can stick to." Right? You don't have to be perfect in your diet or exercise but, as long as I think you have the knowledge of how things are affecting, you can make just an informed decision on, "This part's worth like, potentially, an extra four years of healthy life. Or maybe something else isn't worth it to you, and just having that kind of information and feedback I think it's going to be critical.

Rhonda: Right, yeah. And there's also the aspect of confounding it with time-restricted feeding in mice. I know [inaudible 01:18:57] has talked about this many times on our podcast about how the people, like postdocs and graduate students that are feeding these mice, they come, they're there for like eight hours, you know, so, there's a component of these calorie-restriction diets that actually...

Dr. Levine: That is a time-restricted diet.

Rhonda: Yeah, how much is it that they're just not eating for 16 hours? Because that's when they did all at once and then that's it. Right, yeah. So, anyways. Awesome, Morgan. Well, really that was really interesting information. I know people are going to love it. I certainly was super excited to talk about, you know, all things aging with you, and I will continue to follow your research. I'm excited that you'll be close to me so that we can hang out sometime.

Dr. Levine: Exactly. Go [inaudible 01:19:42]...

Rhonda: Yeah, totally, go hiking, I love hiking. I'll show you all the great trails. So, people that want to learn more about your research, follow you, you're very active on Twitter, I follow you on Twitter. Your Twitter handle is DrMorganLevine.

Dr. Levine: Sounds right.

Rhonda: Yeah. And your Instagram, you're also on Instagram, dr.morganlevine. You have a new book coming out, it's available for pre-order right now, it'll be out in May.

Dr. Levine: Yeah, I think May 3rd.

Rhonda: May 3rd? Wow, okay. And it's called "True Age."

Dr. Levine: Yeah, so, it's a basically about all the stuff we talked about, about measuring what biological age is, how can we approximate it and, potentially, use that information to inform our lifestyle decisions.

Rhonda: Awesome. Well, I'll look forward. I'm going to pre-order it. Anything else? Any other...

Dr. Levine: No, just thank you for having me. This was really fun discussing...

Rhonda: Thanks so much, Morgan, I'm excitedly.

Dr. Levine: Yeah, absolutely. All right. Perfect. Bye.