Written by Christopher Kelly
Dec. 12, 2018
Megan: Hello and welcome to the Nourish Balance Thrive podcast. My name is Megan Roberts and today I am very honored to be joined by someone who has been a great mentor to me, Dr. Jon Ramsey. Hi, Jon.
Jon: Hi, Megan.
Megan: Thank you so much for taking the time out for this podcast. I'm really looking forward to our discussion on calorie restriction. Before we start, do you want to give listeners a brief background as to who you are and what you do?
Jon: I'm a professor at University of California Davis in the Department of Molecular Biosciences, in the School of Veterinary Medicine and my research area is really related to nutrition and energy metabolism and in particular the impact of nutrition on aging. We've also done some work in relation to obesity. When I was a grad student, I primarily began looking into nutrition and calorie restriction and there I was primarily looking at its effects on weight loss.
And then as a post doc, I was introduced to questions about what role calorie restriction may play in aging and trying to look at mechanisms through which calorie restriction may or if it impacts aging. Really that's kind of an area that I've been interested in now for over 20 years. A big part of our research still is trying to understand how energy metabolism and how calorie restriction and diet macronutrient composition impact aging.
Megan: So, our topic today is largely going to be on calorie restriction in the context of aging and longevity. Just to set the stage for people, we know that calorie restriction is one of the single most effective interventions for increasing life span in animals. However, there's question about whether it is as effective in monkeys and humans.
Today, I'm hoping to dive into the science of calorie restriction and answer the question, if possible, of whether or not chronic calorie restriction is actually good for people and have you detail the evidence for and against it along the way. As far as aging is concerned, if we can just give a broad overview of the many theories -- there's the rate of living theory, there's the free radical theory, there's other theories. Can you give a general overview of what the most accepted theories are and then also, and this might be a leading question because there's so many moving parts, but does one theory necessarily try to explain why calorie restriction works to expand lifespan?
Jon: That's a really tough question because there's really hundreds of theories for what causes aging and you've hit on some of the ones that have been very popular at different times. The rate of living theory is one of the older theories for aging. The free radical theory has gone through different kind of iterations and different ideas on how free radicals and oxidative stress can affect aging.
There's also theories about cellular senescence and cell regeneration and effects of those may have on aging, and then general theories that cover a range of topics that usually are related to areas of stress response and the idea that calorie restriction or interventions that affect aging are upregulating stress response systems or allowing the organism to better respond to stresses and then be able to increase lifespan.
Those along with interest also and ability of animal to regenerate tissue or to be able to respond to energy or stress are very popular and broad theories and within those there's more specifics that people are looking at. As far as calorie restriction, I wouldn't say that there's one overriding most popular theory. For me, I think one of the things that's more interesting is that we're understanding more about metabolism. We're learning more about signaling pathways and pathways that are upregulated when an animal enters calorie restriction.
I think there's a lot of intriguing things and theories that are built around those that it's a response to an energetic stress and some of those pathways that allow an animal to survive short periods of starvation may also have benefit for aging if they're chronically exposed to these stresses. I think that's a particularly intriguing idea that's out there now but I wouldn't say that there's anywhere close to uniform agreement on a major mechanism right now for calorie restriction.
Megan: As far as the history of calorie restriction research, can you take us back to the days of Clive McCay and the beginnings of the research in the context of aging?
Jon: McCay is generally credited with at least popularizing calorie restriction. Probably about 20 years before Clive McCay there was also Osborne and Mendel that found that calorie restricted animals tended to live longer than control animals. It's been at least 100 years that people have been looking at calorie restriction. Clive McCay is the one who really -- In his studies, what he was looking at originally was they were first looking at effects of calorie restriction of stunted and growing animals.
They were looking at some of those initial studies where they were stunting growth and looking at effects on those and then they'd kept some animals around and found that they lived a long time. Clive McCay became very interested in the idea that calorie restriction increases lifespan and he devoted a big portion of his career and was very active in trying to get gerontology as a science itself and just to study mechanisms of aging.
His initial work was in the '30s and he had really promoted this work through the '40s. It's been around for quite a while. And then there was maybe a little bit of a lull. There was quite some level of research in that area. And then I would say a lot of aging research has really taken off again in the late '70s and '80s, and then major interest all this time in trying to understand why calorie restriction works.
Because up until just recently, it was the only intervention that was consistently being shown to increase lifespan in a range of animal models. And so that's why there was great interest in seeing if we can understand how this works we might be able to get a better grasp on what are the mechanisms that influence aging.
Megan: And speaking of the animal and rodent models, I know there's years and years and hundreds of publications on the topic, but can you give a broad overview of what the calorie restriction literature in animals show and maybe just focus on the rodents and then maybe also discuss some of the important study design details when it comes to animal studies such as the age at which the animals began the restriction, the strain, the sex?
Jon: The original work was with rats and then that moved to mice and, in general, what was found with most of those studies was that calorie restriction increased mean and maximum lifespan. The level of calorie restriction that was used is there's a lot of variability in approaches that people have used with calorie restriction. There had been the general idea that calorie restriction increase lifespan and that the effect of calorie restriction increased up to the point where you reached malnutrition. And then, of course, you had a dramatic decrease in lifespan.
And so the levels of calorie restriction that have been used in a lot of the mouse and rat studies were relatively large that they were often 30% to 40% calorie restriction from ad libitum. The strains of mice and rats used have been different over time with rats. There'd have been studies in Fischer 344s. There have been studies in Sprague Dawleys and Fischer Brown Norways and so a range of those in mice. There's also been a range of species but probably the C57 black 6 is the type of mouse that's been studied the most and it shows a very robust response to calorie restriction.
I think there's been the general idea that calorie restriction worked in all rodents up until recently. There were some papers that were published, I think it's about ten years ago now for a while where they had looked at very wide range of 40 different mouse strains and reported that some of them showed an increase in lifespan with calorie restriction, some didn't and some showed a decrease. That's created a lot of interest in this idea that maybe there are some strains of rodents that don't respond to calorie restriction.
I think one of the issues with those studies is they were all done at relatively high levels of calorie restriction. And so one question is: Is it really that you have animals that don't respond to calorie restriction or is it you just have to find a particular level of calorie restriction to which certain animals respond? I think that's still an open question.
The other question that's out there still is both the diet and the approach for calorie restriction and so people have used a range of diets and they get still an open question whether there's a particular diet that's better than others or particular diet composition that's better than others. And the other is the feeding approach, that really almost all calorie restriction studies are single meal feeds.
These animals definitely have a time restricted feeding component. They generally eat their food pretty rapidly and have a period of fasting before their next meal. That can be anything from a daily type feeding or these approaches where the animals are only fed three times during the week and so they have potentially these extended period of fasting between meals. I think all of those are variables that could explain at least some differences between studies and it just highlights that there isn't a standard within the field that everybody follows.
Megan: You mentioned median and maximum lifespan. Can you explain to the listeners the differences between those and then which, if either, the gerontologist and aging researchers care most about?
Jon: So, median is just the lifespan at which if you started with, say, 100 animals, it's the age at which half of those animals have died. So, which 50 of the animals, if you started with 100, have died. And there's also variance. You could also say average lifespan, which would be then just taking all life spans of all your group and just averaging that.
Maximum lifespan is technically the age at which the oldest animal lives. There'd been some kind of variations on that that have been done with animal studies because you can't use statistics on a single individual. And so there's been some attempts to also define a maximum lifespan value which is like the top 10% or the top few remaining animals so that you can actually do a statistical comparison.
What I would say is that for a long time people who are studying aging, that was the standard. If you were looking at an intervention you want to prove at work you did these lifespan curves and you prove that you increase lifespan, and the ideal thing was to show an increase in both mean and maximum lifespan. In recent years, I would say that there's been more of an appreciation or more of an interest in not necessarily the individual values within a lifespan curve but trying to say are the animals still healthy?
Are you getting an increase in median or lifespan? Are you shifting lifespan curve but in doing that are you maintaining animals that are healthy? I think a lot of the attempt has been to avoid situations where you have an intervention that increases lifespan but it increases the period of time at which an animal is experiencing health problems.
Megan: I know you mentioned different amounts of calorie restriction as far as spectrum goes. Can you give us an idea of what the minimum amount of calorie restriction is in the animal studies that have been shown to be efficacious for increasing longevity and then also at the top end where do they cross the line of malnutrition and calorie restriction as far as longevity promoting purposes are concerned?
Jon: I think on the upper end it's probably variable depending on the strain. Probably the paper that really stands out that looked at levels of calorie restriction was one that was done by Rick Weindruch and Roy Walford back around 1980 or a long time ago where they'd looked at, in mice, several levels of calorie restriction. They had promoted the idea that the greater the level of calorie restriction the greater the increase in lifespan up until malnutrition that occur.
In those cases, with those mice, and they were getting it into levels of calorie restriction that I believe were getting around 50% or so and still getting up to a lifespan effect. That's on the high end. I would say it's pretty unusual to see studies that go beyond 40% calorie restriction. I think that's, for many people, that's a pretty severe calorie restriction. I think just for safety concerns and to avoid potentially crossing the line into malnutrition, most people don't go beyond 40% calorie restriction for their studies.
For some animals, 40% calorie restriction is too severe also. That I would say, from a practical standpoint, I'm looking at the literature, it's probably the upper end of what you will typically see for calorie restriction studies. On the lower end, there's been a lot of debate about that. There's been in recent years, there's been people who have been questioning the idea that there's this relationship between level of calorie restriction and increase in lifespan and some people have brought back that maybe it isn't as simple as you increase level of calorie restriction you get greater increase in lifespan.
There's been more interest in the lower end, which you talked about, and there is a paper out just recently within the last year or two by Roger McCarter in rats that were saying that they were seeing significant increases in lifespan with only 10% calorie restriction.
Megan: That would definitely be more reasonable at least for human than 40%. In my mind, especially as, and I don't know if you see this with the animals, but in human's age, it's more important for them to get sufficient nutrition especially sufficient protein for the muscle effects and the anabolic resistance effects and whatnot. I don't know if you have thoughts on that but we can also talk about the human studies here in a second as well.
Jon: I absolutely agree. I think that's one really important thing to take into consideration because in our animal studies they are relatively sedentary models because they're living their lives in a very controlled environment. They're not asked to do a lot. Most cases, their level of exercise is just walking around the cage and not particularly asking them to do a whole lot.
That's certainly a thing that has to be taken into consideration and particularly with the high levels of calorie restriction. That's always been my concern. In those cases, you can have an animal that becomes quite thin with higher levels of calorie restriction but they're in a controlled environment where they're not exposed to disease and they're not asked to do a lot. And so that's definitely a concern when you get to humans where we know that going into old age with low muscle mass and frailty is not a good thing.
Megan: That was great. Let's move in to talking about some of the human studies. I know the big one was the CALERIE study which was a study that was conducted, I believe, at the Duke University to look at the effects of two-year long calorie restriction in healthy non-obese adults. I believe they aimed for about 25% calorie restriction overall but I think they ended up at less than that. Can you talk about the different analysis that have been conducted and maybe your opinion on the highlights of those?
Jon: The CALERIE study was an important study that is to try to see the feasibility of calorie restriction in humans. Duke was a part that I think that had part of the database for it but it was a three-center study. A big part of it occurred at Pennington Biomedical Research Center in Baton Rouge, Washington University and also at Tufts. The goal was to take non-obese humans and to do -- There was a short term and then there was a little bit longer period calorie restriction to look at the feasibility and just to look at some markers of health in these individuals.
And so I think really the highlights were they weren't using obese individuals. So, these were individuals who had BMIs that were less than 30. The goal was to do a 25% calorie restriction. I think one of the interesting things at least with the longer term studies where they did achieve the 25% calorie restriction and I think that's maybe not surprising they did achieve -- it was around 12% or somewhere between 12% and 13%, I think.
To me, that's quite interesting because it shows that people could achieve some level of calorie restriction even though it wasn't really what they targeted. Along with that, you saw a number of markers that would suggest that there was some improvement potentially in health in some of those individuals. And so a lot of just looking at markers of glucose metabolism, looking at blood markers to get an idea on potentially metabolic and cardiovascular health.
Many of these markers that would suggest that just small amount of calorie restriction and probably weight loss along with that that had showed potential to at least have benefit in these individuals. Of course, these studies were limited that the initial study was six months and then they went out to a year in that. They definitely showed proof of principle that's at least good. Of course, human studies were not going to be capable of really answering aging questions. We just don't have the time. But at least this took that first step on saying could at least a slight level of restriction be feasible and is there any evidence that it would have potential health benefits?
Megan: I think there are a couple of different analysis, at least recent analysis. The two most significant results, at least in my mind, when it comes to the aging process were the one that suggested that there was a slowing of biological age using serum biomarkers and then the other being that calorie restriction cause a metabolic slowing which was accompanied by reduced markers of oxidative stress. The others, I believe, said that this suggested or that this supported the rate of living and oxidative damage theories of aging.
Jon: Yeah. That was very interesting. That was a study at Pennington with that was involved in it. I think this rate of living theory idea has been bouncing back and forth and there are several people that say it's been disproven. This one was one of those that it was interesting because it suggested it bridged also this debate which is general weight loss, is when people go on calorie restriction as their metabolic adaptation to decrease basic energy expenditure. That's an adaptation to your attempt at weight loss.
I mean, I think there it just reopens this question that says that there's a lot we don't know about how individuals respond to calorie restriction. The effect on energy expenditure is probably something we shouldn't completely ignore and say that we've already solved everything as far as how individuals respond to calorie restriction.
I think the oxidative stress is also interesting because that's an area that for a long time was the favorite theory and then there were some really interesting work. Holly Van Remmen had a nice review article where she looked at the data from calorie restriction studies and had said that at least decreases in reactive oxygen species there's not uniform agreement, and that while you have some studies that show a decrease you have others that don't, and a lot of questions about what's going on with oxidative stress.
I think highlighting this shift that we're learning more about changes in oxidative stress and we know that many of the changes that occur with calorie restriction or when an individual go on calorie restriction there's often at least one compensation is potentially an increase in antioxidant defense systems. Another question is what impact does that have? Is it really oxidative stress that's important?
You see some studies that suggest that may be the case. The other thing may be something that's more difficult to measure is are you getting changes in redox signaling and having effects that are doing things beyond just damage that are a little bit more difficult to quantify?
Megan: Yeah. I know we've talked at length about the complexity of oxidative stress on a biological level on the podcast before and we could link to some of those episodes in the show notes. It seems that, just as many things in biology are, the link between calorie restriction and oxidative stress is far more complex than it seems. I know that there was a paper you co-authored a while back, the effects of short and medium term calorie restriction on muscle mitochondria proton leak and reactive oxidative species production.
You guys showed that a reduction in mitochondrial oxygen consumption in reactive oxygen species could be contributing to the longevity effects of calorie restriction. And recently, that was on muscle level, there was a study on [0:20:59] [Indiscernible] and it was in trout, of all things, that showed fasting, not calorie restriction but fasting, could reduce the energy metabolism but then it increased hydrogen peroxide consumption. But they were interestingly looking at the liver. Potentially, we may need to be cognizant of which organ we're also talking about as well.
Jon: Yeah, absolutely. I think that makes sense. All organs respond differently when you look at response to calorie restriction or fasting. They all have their kind of specialized role. That's one of the challenges is measurements in one tissue may be don't extrapolate to other tissues. I think that's part of the reason why it's been so challenging to try to figure out calorie restriction in part potentially what's interesting is it's complex. You're changing, entirely changing metabolism and how each tissue respond to that is going to be slightly different.
Megan: So, another thing I know that you're very well versed in is the topic of control of food intake. And so one question, when it comes to the differences, going back to mice versus people, is this question of are people truly ad libitum fed and might we need to rethink how we're setting up animal studies to reflect a more realistic human food environment?
Jon: Yeah. I think that's a good question. I think it's something that I've been thinking about more recently than in the past. I think part of it is that many humans definitely showed diurnal patterns in eating and that many humans do probably show some level of dietary restraint. With animals, that has become an issue where we've made the assumption that the ad lib fed rodent is similar to a human.
I think what I've seen recently in looking at the data for a lot of animal models is we make assumptions about mice that aren't always correct. One of them is we have this idea that they have this very clear diurnal shift in food intake and that they eat their food during the night and they're basically resting during the day. What we've seen when we've done studies looking at meal consumption with these animals is they do show a diurnal difference but I think people would be surprised the amount of food that a lot of animals eat during the life cycle also and that it's not uncommon that we'll see that mice eats 60% to 70% during the dark but they'll eat 30% to 40% some of them during the light and so they'll eat many small meals during the day time.
There's a number of animals where that's brought up questions for me on whether the true ad lib is being a good model because there's certainly animals who have these little meals all the time. They're never getting into a point where they have just a fasting really low insulin levels. They're getting these meal kind of stimulation of insulin multiple times throughout 24 hours.
Under those cases, I think that would be an unusual human that would be getting up and eating meals during the night and just maintaining getting insulin spikes around the clock. And so I think we -- I think that's something that we really need to take into consideration more and say humans generally are meal feeders and we should probably try to have our animals mimic that if we really want a true to try to get metabolism to look similar between or to have the animals model human metabolism.
Megan: And part of that, I would assume, would be the environmental factors. So, like you talked about before, mice are in their cage with ad libitum access to food whereas humans have lives, we're doing our jobs, we're exercising, we're going to sleep. I would guess that the environment would play a potential role in the differences in the responses of how restriction in humans and people as well.
Jon: Absolutely. I mean, I think the ad lib animal would be like a human in a jail cell with a buffet table set up basically all the time. I'm sure there's a fair amount of pride that says boredom eating too. And not that that doesn't occur in humans too but that humans definitely have some control on times in their life where food is not right in front of them.
Megan: Yeah. And this kind of brings to mind the recent study I think that showed that enriching the cage environment of mice actually helps with aging, which really wasn't surprising. Some of these researchers put running wheels and toys and mazes inside the cages in these mice and then did some studies and they promoted liver health, glycemic regulation, some upregulation of anti-inflammatory genes were shown, and it was a trend towards a positive impact on median life span. But at that point, quite honestly, I don't really care about the lifespan effect. I'm just I'm sure that those mice had a lot more fun than they would have otherwise.
Jon: That was a very interesting paper. I think, yeah, it does just kind of say even something else just to distract them and change their behavior is important.
Megan: Yeah. And I'm not sure if they looked at food intake in that setting but it would have been interesting to see what the food intake was like compared to the animals without the enrichment.
Jon: Yeah. I don't remember. I'd have to go back and look at the paper.
Megan: We can definitely link to it in the show notes for people who want to look at it. But I wanted to move on to some of the monkey studies because I know you're involved in one of them at least. There are many logistical and ethical problems with conducting calorie restriction longevity studies in humans. The monkey studies may also be the closest we ever get to human trial.
I know that you co-authored at least one article back in 2000, I believe, that discussed the University of Wisconsin study while it was still going on and now almost 20 years later we have the results. Can you give us an overview of the studies and compare and contrast the findings and the methods, if possible?
Jon: Both studies were started the very late '80s and they both used Rhesus macaques. The origin of the monkeys were at least slightly different between University of Wisconsin study and the study that was completed at the National Institute on Aging. The level of calorie restriction was 30% for the University of Wisconsin study. They used different diets. The University of Wisconsin study used a semi-purified diet similar to that's used in some rodent studies. And the National institute on Aging study used basically a chow type diet. It wasn't semi purified.
Both studies had lifespan and end of life pathology as an end point and then they also had some measurements that were completed kind of cross-sectional on the animals. I think the big difference that was reported was the University of Wisconsin study found a significant increase in lifespan whereas the National Institute on Aging study didn't. But both studies reported increases in markers of health span or measures of health.
They saw an increase in insulin sensitivity. They saw decreases in a number of different age related pathologies in these animals. I think there was both agreement between the two studies that their calorie restriction approaches were promoting health. The difference was is that change in health translating also to an increase in lifespan?
Part of the debate is why there were differences between the two studies and there'd been a number of ideas that had been floated but not really any definitive answer. One thing that's important too with those studies is that the control animals in the National Institute on Aging study had quite long life spans. That makes it challenging to pick out a difference in increase in lifespan when your control groups are very long lived also. And so why that was that case, I'm not sure.
The other things that they've looked at is, is it possible that diet had some effect on these? The diets were quite different in type of fiber that's in it, also differed in that at that time that the Wisconsin study was being conducted, the purified diet of choice at that time for rodent studies used sucrose as a major component of carbohydrates and so that was a relatively high sucrose diet too. And part of the question would be did that have some impact maybe on lifespan of controls or other animals?
And then also the genetics. The Rhesus monkeys came from different backgrounds and whether the genetics influenced those studies. A number of different questions but I think probably the most important thing was that both seemed in agreement that calorie restriction improved markers of health and decreased age related pathology. And so at least at that point there was agreement that it had positive impacts at least on those, markers of health in later life.
Megan: For people who aren't aware of what a semi-purified diet is, can you explain what those components are and contrast it to something like a chow diet? Just so that people realize that part of the discrepancy that people are thinking was going up between the different results of the two studies were the fact that potentially eating less of the semi-purified diet which is, obviously, more processed. So, eating less of a crappier diet is more likely to have a greater effect on health and longevity than eating lots of a healthier diet, right?
Jon: A semi-purified diet, they were developed really for nutrition studies. The idea was to create a diet where everybody could have the same ingredients so you wouldn't have variability in ingredients. And then from a nutrition standpoint, you could manipulate one component or so and you would have some guarantee that everything else was the same and if somebody wanted to replicate your study they could do it.
The idea was to keep things simple and so the standard was to use, for carbohydrates, just to use corn starch and sucrose as the carbohydrate source and to use, for protein, to use casein as protein source. For lipid, the original studies were using corn oil only. They changed that later to soy bean oil. And then to have a very defined vitamin and mineral mix. They would go along with that.
So, those were very simple diets that are nutritionally adequate if you're looking at NRC requirements. But they're simple. They're something that everybody could copy if you're doing research. Whereas in contrast, the chow type diet is something where your main ingredient is maybe whole grain maybe like ground corn or ground wheat and they have different types of meals and they're to help provide protein or other things that are not highly processed.
Megan: So, let's move on to talking about some of the mechanisms that play with carb restriction and I'll preface this by saying that the mechanistic research is largely conducted in animals. In your opinion, what are some of the main mechanisms by which most researchers believe that carb restriction is increasing life span?
Jon: Yeah. I think the issue would be the term most. I would say it's still pretty fractured as far as people have their areas. I think there are a small group who are still interested in the idea that there may be effects on energy expenditure and metabolic rate, kind of the old rate of living theory. Part of that is because of the paper you mentioned [0:32:05] [Indiscernible] with humans that also it brought that up. And just the idea that that's seen often in weight loss studies.
That is an interesting idea but it in itself isn't necessarily a mechanism. That was always a weakness of that theory is even if that occurred how does that lead to an increase in lifespan? There's still a fair amount of interest in the oxidative stress theories and a lot of people who still look at oxidative damage but I think that has also evolved, the people who are looking at redox signaling. Are there just shifts that are occurring with calorie restriction in levels of reactive oxygen species and are there changes that are occurring, that aren't occurring as damaged but are occurring as signaling pathways that are affecting self function and potentially cell response to stress?
To me, one of the areas that's most intriguing is this idea of just central metabolism sensors, that you have these pathways that are there to sense periods when you have shift in metabolism like where you have low energy and to shift how your cell is using fuels. Those would be this kind of whole area of things that have become focuses like AMP kinase, the sirtuins, mTOR pathway.
Part of this idea that you have these sensors and that it may be important with calorie restriction that you at least activate those either chronically or periodically and that those give a cue to your cell on which substrates to use as energy but also along that time it also sets up a bunch of stress response pathways and so can upregulate antioxidant defense systems and they also shift autophagy or your changes in turnover of your cell proteins and repair mechanisms and can also change how you're allocating your energy like how much you're putting to maintenance versus how much you're putting just to growth.
To me, those are the areas that I think is most intriguing. And that seems to be panning out in some ways as being shown as ways that you might be able to translate or do calorie restriction mimetics that can do part of that. That's been the focus on things like rapamyacin in this attempt to do drugs that target certain of those signaling, energy sensing signaling pathways.
I think there's also a lot of interest in areas that calorie restriction may have on just also cell maintenance and rejuvenation and interest in stem cells and effects that it may have and maybe maintaining certain cell populations to allow a tissue better to respond to periods where there's damage and potentially growth. I think those are all kind of really areas of continuing research in the calorie restriction field that at least from my standpoint, I think, the metabolic sensing pathways are really interesting and show great potential to develop potential mimetics and approaches that can at least show some of the benefit of calorie restriction.
Megan: Yeah, for sure. I definitely agree with that. I do want to talk about mimetics in a second here but one thing I want to obviously cover is what's happening at the mitochondria level with calorie restriction. So, can you talk a little bit about mitochondrial proton leak and uncoupling and maybe explain what they're doing using normal physiology and then what are happening to both during aging and calorie restriction and then the implications that might have for longevity?
Jon: So, mitochondrial proton leak is this process where in the mitochondria the mitochondrial membrane is the central component of energy metabolism in mammals. And so within the membrane are proteins that pump protons across the membrane and create electrical gradient and that electrical gradient then goes through ATPase and is what makes ATP that the animals are primarily using for its energy.
If that process were 100% efficient you would build up that membrane potential and the only way it could be dissipated would be going through the ATPase. And that doesn't occur. There is some level of inefficiency in that and where that membrane potential can be dissipated without going through the ATPase. And there's two general ways that it could do that. One is considered just a basal way. It's probably not controlled in that it's either leaking related just to a physical structure of the membrane either through the lipid bilayer interaction and lipid bilayer with the protein and that's a basal protein leak.
And then there's regulated proton leak which is with uncoupling proteins and that's kind of more the classic that at least in rodents and some animals with like brown fat in responding to need to generate heat rapidly, and you can activate these uncoupling proteins and they cause the membrane potential to be dissipated by passing through these proteins instead of ATPase.
We were interested in this process just trying to figure out if it might play a role in calorie restriction, that it might be a way that an animal tries to become efficient to adapt to calorie restriction and in the process whether that might affect reactive oxygen species. And I would say that this is an area that's still quite interesting but it's an area where I wouldn't say there's 100% agreement.
What we saw with our study is that it depended a little bit on the tissue but we often saw that mitochondria would decrease their proton leak and appeared to try to become more efficient in response to calorie restriction. And little bit of a surprising thing with this study was that, in general, at least in the short term, increase in proton leak with an uncoupling protein is a way to decrease reactive oxygen species production.
But in the long term, what we were finding was when you're just doing a basal change in proton leak, there were other changes that were occurring within the mitochondria. In our case, those mitochondria tended to decrease reactive oxygen species also. And so to us, it was more like you're having this major global change to multiple things within the mitochondria and it's an adaptation to a chronic change instead of just a short kind of response where you're upregulating or just activating a protein for a short period of time.
So, that was an area that I think we're still interested in, that this idea of is this potentially a benefit that occurs with calorie restriction and is it something that potentially negates age related changes in mitochondria? And one of them we'd seen in other studies we were looking at older animals is that some with very advanced age that animals had an increase mitochondrial proton leak which may have reflected damage to mitochondria or other issues that were occurring where they weren't able to maintain that same level of efficiency.
Megan: Very interesting. One little bit of a tangent question, which I'm sure people will be interested in, but how do you measure proton leak in a lab?
Jon: The challenge with proton leak is it's not easy to measure. There's indirect ways that you can do it and one of the indirect ones is just to measure respiration when mitochondria are phosphorylating. That's not a perfect way because you could have kind of a range of membrane potentials under that situation. The accurate way to measure it is to do a basically a proton leak kinetics curve and that's where it's challenging.
Usually, you can do it in intact cells. It's most often done in isolated mitochondria. And that's where you measure simultaneously mitochondrial respiration and membrane potential over a range of respiration rates. And then you plot them and you get this kinetics curve and then you look at those together and then you can make comparisons in between groups or whatever you're looking at.
And usually with those curves, it's basically trying to get an idea at a given membrane potential is one group able to maintain lower respiration rate or high respiration rate or at a given respiration rate is one group able to maintain a higher membrane potential? Those give you an idea then of really proton leak membrane permeability over a range of membrane potentials of respirations and it's really kind of the gold standard of determining whether you have a change in proton leak.
Megan: Well, it's fascinating. So, one last point here which will probably be a nice segue into the next topic. Originally, it seemed that a lot of people were just, all right, thought that it was just a mere restriction of calories that was more responsible for the longevity promoting effects of calorie restriction. But then came along some of the studies that you and others did in particular the thinking that you've done recently where the ones manipulating dietary fatty acids and then also the study that I participated in with the ketogenic diet and longevity.
Perhaps you're thinking around this has changed. So, maybe even if simple calorie restriction is still at the main core of behind what we see, maybe there are other related aspects that play a minor yet so important role. Would you agree with that?
Jon: Yeah, I would agree with that. I would say that's changed. I mean, it's gone from the idea that calorie restriction is the only intervention that changes lifespan to this idea that there's other manipulations that could have an effect on lifespan. I think part of the question is: Are there interactions? Are there ones that are having effects by themselves and then are there others that may be interact with calorie restriction to have effects on lifespan?
I think some of the examples like the study you are involved with, the ketogenic diet, that was part of our theory was if you could mimic the shift in metabolism that occurs with calorie restriction without inducing severe calorie restriction do you get a lifespan effect? And in our study, we did show that we had an increase in lifespan and it was an appreciable amount, over 10%. So, it wasn't a tiny change in lifespan.
I think there've been other studies too that have suggested like one example has been some of the protein restriction work and that started really in the '90s. The Weindruch studies and then recent ones where they look at either protein restriction or methionine restriction in some of those where they also have an increase in lifespan. And there's been some attempts to say that those are a mechanism through which calorie restriction works, that calorie restrictions really restriction of protein and that's why you have increase in lifespan.
I don't agree with that because there are approaches to calorie restriction in the Weindruch and Walford calorie restriction diets. They tried to match absolute levels of protein intake and restrict calories and they had very long increases in lifespan. I think those are strongly argued that it's not protein restriction that's a mechanism with calorie restriction but that these increases in lifespan that are occurring with restriction in methionine or protein are going through another mechanism.
And I think there's also just this idea on diet components and lifespan that if energy was the only thing that influenced lifespan with calorie restriction, it probably wouldn't make any difference what diet a calorie restricted animal was consuming. It would just be restricting the calories. That's something where there'd been studies, Morris Ross in the late '50s and '60s did some initial studies where he was showing that there were different responses with rats to calorie restriction, that he changed levels of carbohydrate and protein and he found that he had longer life spans on his rats that had the lower carbohydrate content in their calorie restriction diets.
We did similar studies later on looking at just manipulating diet macronutrients and we did studies where we changed the lipid composition of calorie restriction diet and got differences in lifespan. I think this opens the idea that, yes, there are things beyond energy that affect lifespan and some of those are approaches that maybe easier to develop into dietary interventions that people could follow.
But having said all of that, all of these interventions, none of them have produced lifespan increases that are as great as you see with calorie restriction. So, I think it still suggests that there's something about energy that's quite important but there's diet components other than energy also have an effect on lifespan.
Megan: I know you briefly talked or you mentioned the study that you did, the influence of dietary fat stores on the lifespan of calorie restricted mice, so I believe that part of the impetus for this study was the membrane theory of aging. Can you briefly explain what that is and then what that study showed? Because I think people will be a bit surprised of the results.
Jon: Yeah. So, we were practically following some ideas that were put forward by AJ Hulbert's group in Australia. They were looking at this membrane theory of aging more as kind of a comparative trying to understand why diverse animal species have very different life spans. This theory was that long lived animal species tend to have more saturated membranes than short lived and it was very much tied to the oxidative stress theory.
It was this idea that membranes can be damaged by oxidative stress and become peroxidized and that if you have an animal species that maintains a more saturated membrane it's less prone to oxidative damage and peroxidation and these animals can live longer. What we got interested in is there were some variants that were coming off of this where they were saying that this theory might also be relevant to calorie restriction in the idea that calorie restricted animals maybe have more saturated membranes than animals that aren't calorie restricted.
And we thought we can test that because we had been doing work anyway with mitochondrial membranes and shown that we could change the membrane composition depending on the dietary fat source that we gave the animals. And so we thought we can make calorie restricted animals that have unsaturated membranes and we can make calorie restricted animals that have more saturated membranes. And so that's what we decided to do. And so what we wanted to do was compare a range of different dietary lipids that affect -- in particular we were interested in mitochondrial membrane composition.
And so we used for it very unsaturated fish oil. We were using that as a way to create mitochondrial membranes that had a lot of very unsaturated omega-3 fatty acids. And then we used, for just our control or intermediate kind of level of unsaturation, we used soybean oil. And that's the standard oil that's used in semi-purified diets.
And then for a more saturated lipid we use lard. We were kind of interested in lard primarily because we're looking for something more saturated. One of the interesting things that we weren't originally considering is that lard contains a very large amount of monounsaturated fatty acids. And so what we were finding was we were changing these mitochondrial membrane lipid composition. We were getting the changes we mostly expected.
So, the animals on the fish oil were getting a lot of omega-3's and were getting a lot of these very long chain polyunsaturated fatty acids in the mitochondrial membrane. The animals on the soybean oil were getting a lot of linoleic acid so as two double bonds, and so a lot of those. And then what was happening with the lard is what we'd found was you really couldn't manipulate the completely saturated fatty acids in the membrane. What they were changing was level of monounsaturates.
And so we were getting this change in type of lipid that was in the membranes. And then all these animals were on calorie restriction. We looked at lifespan in those and what we saw was that it kind of followed what would have been predicted by the membrane theory of aging. The animals on the lard diet lived longer than the animals on the fish oil diet and the soybean was in between. It did show you could change lifespan in calorie restricted animals and that changing the lipids did have an impact on lifespan.
Megan: It's interesting because I know there's been periods of time where high dose fish oil consumption is very much the rage and I think the most recent at least study meta-analysis that was in JAMA for cardiovascular disease found omega-3's didn't prevent the disease progression. So, perhaps their benefits are overstated a little bit and I think -- I know they're kind of potential here is that many of the positive effects of fish oil in both people and animals are in disease populations and so it's possible in my mind that omega-3's could be acutely anti-inflammatory in specific context but then perhaps not in the context of carb restriction where we already know that inflammation is lower. What do you think about that?
Jon: I would agree with that. It's one where I think if you don't already have a problem taking very large doses of fish oil, it doesn't have a benefit, it has a potential risk with the level of polyunsaturation. I think it's also, like for our study, one of the differences is people, if you looked at that study and they've asked does that mean that I shouldn't eat fish? I haven't taken it at that way.
I've said no, not for individual meal but it would give me reason to question whether I would want to take a fish oil supplement if I didn't have a particular reason, if I wasn't particularly concerned about inflammation at that time or some issue that I was trying to address.
Megan: I completely agree with that. I wanted to circle back around to ketogenic diet and ketosis.
I don't have to go in depth on this study that we did because we've actually done a podcast where I talked about the results. A couple questions for you on the topic is, one, do you think normal people are considering the ketogenic diet or just circulating ketones through exogenous ketone production or sourcing as a calorie restriction mimetic and might periods of time spend in that metabolic state benefit people from a longevity or health span perspective? I know this is largely speculative right now but I'd like to get your thoughts on that.
Jon: I think there's no doubt that there's been an increase in interest in ketogenic diets. Along with that an increase in ketone supplements which can may be a different thing, how they're working. I think there's growing evidence that ketogenic diets, I mean, the concern always in the past was that they were unhealthy. I think there's increasing evidence that you can do a ketogenic diet and if properly followed that there isn't, that some of the concern is probably not based on data, at least in animals you can certainly formulate a healthy ketogenic diet and in humans there's number of studies now for people who have been on it not for real long periods of time but long enough to show that you can have, under certain circumstances, at least health benefits.
I think the big question with calorie restriction, and you may be brought it up when you're talking about this study, is that with a ketogenic diet you can get animals in there and you can get them to be on sustained ketosis. But that's not what a calorie restricted animal does. It goes in and out of ketosis. And so that to me is still a very much an open question like we know from animal studies and we know from some of the human studies with epilepsy and now some of the studies with diabetes and other things where people have been on ketogenic diets where they're getting some sustained ketosis, that there are some positive health effects that can occur with these diets.
But I think it's still a little bit an open question, that if these changes in ketosis that occur with calorie restriction occurred in humans would there be a benefit? Or would there even be a benefit in animals? That's still I think a bit of an open question is intermittent ketosis beneficial or does the effect of the diet have to be continuous ketosis? In the case of epilepsy, clearly, it has to be continuous ketosis likely but I think when we're talking about aging or other interventions, I think that's still an open question. Is there any benefit to intermittent ketosis?
Megan: And ketones are also elevated during fasting and we talked a little bit about time restricted eating which seems to be very popular. But it's very possible that the fasting periods that we see with the time restricted eating which are ultimately increasing ketones, the mechanisms are overlapping between the ketogenic diet and fasting or time restricted eating.
Jon: No, I think you're right, absolutely. There could be overlap on that because calorie restriction is a time restricted feeding approach also. Yeah, how much of the effect can be mimicked just by time restricted feeding? I think it gets a little bit more complicated with humans. One of the things with rodents is you can get them to go into a noticeable ketosis within a relatively short period of time, 12 to 14 hours or so, and you'll see an increase in ketones.
In humans, it takes longer to get a bigger effect on ketones. That may be one issue like on how much of how to translate animal data to humans, what level of length of time between meals would a human need to be able to get the same effect you would see in an animal model.
Megan: Right. And there was that recent, just a couple months ago, a study published in cell metabolism that showed the daily intermittent fasting improved health and survival in mice independent of the diet composition and even calories. I'm guessing there would be a lot more interest, a lot more people looking at this kind of thing in the near future.
Jon: Absolutely. I think that's really the area that's going to really take off now because it's easier for people to follow like this kind of either intermittent approach where you do time restricted feeding or even stretching at the things like Valter Longo's studies like this idea of, well, do you really just need to either undergo restriction or undergo potentially something that might put you in ketosis for just a few days out of a month potentially? Does that really have benefit?
I think all of those are really open questions but areas that people are going to be very interested in this idea of does diet change have to be continuous or can you do things with an intermittent approach? And there was even the study that just came out, David Allison's, about with mice where they were doing like an intermittent calorie restriction. It was basically this idea of weight cycling. Is there a benefit to at least try to lose weight or restrict calories?
Their conclusion was that there was at least in their animal model. And so that is all this idea that I think is quite important right now on this, are there approaches that are intermittent that are going to be effective and easier for people to follow?
Megan: I also briefly wanted to touch on exercise in the context of carb restriction and longevity because I think there's a major question and some people listening might be thinking this as well, of whether it's up the actual calorie restriction or energy restriction that's responsible for the enhanced longevity effect. So, you can be energy restricted by eating fewer calories or by eating energy deficit or being in energy deficit in a different way such as exercise. So, question for you, is can you tell us about the literature on this topic and do they have the same longevity effects?
Jon: It's a very interesting idea because it's creating basically an energy deficit in two ways. One where by restricting energy intake how the animal responds versus increasing energy output and how it responds and are they the same? I think the best studies are ones that were done by John Holloszy looking at exercise in rodent models and looking at their effects on lifespan. In this case, the exercise was wheel running.
What he reported was that exercise had an effect where it would increase mean or median lifespan but it didn't increase maximum lifespan. And so his conclusions were that exercise was having an effect on health but it wasn't itself fundamentally changing rate of aging. I know that there's people who still there hasn't been a lot of studies in this area and there does need to be more work in that area.
But I think his conclusion is pretty important. Even if he didn't see an increase in lifespan, and I think the literature completely agrees with this, that exercise has benefit for aging. Part of it is that it affects health and it decreases disease processes. In the animal models, it allowed them to have average lifespan and to increase health span and ways to be healthy later into life.
I think those are important and that's one of the things that if asked about what approaches people should do if they want to live a long and healthy lifespan, and I think exercise would absolutely be something that should be promoted, and I think because of its clear benefits and effects on helping to maintain health span and function and also maintain muscle mass going into old age, that individuals have muscle mass and are physically active and are ready to be able to handle any stresses of that that occur with advanced age and potential injury.
Megan: Before we get into some of the take home points for people, is there anything else that you'd think that we haven't covered on the topic of calorie -- Obviously, there are many things we haven't covered but any broad things that you think people would be interested in that we haven't touched on that we should cover?
Jon: I think you've mostly touched on the major points with it. I think also just touched on the recent areas and the hot topics right now which are like the time restricted feeding, intermittent fasting, ketone, ketone supplements, that kind of thing.
Megan: As far as take home points are concerned, what does this mean for people, if anything, as far as calorie restriction is concerned? I definitely have my thoughts but I'd like to hear yours. And also, if not calorie restriction, and we just talked about exercise, what would be some of the things you would recommend for people wanting to live a long healthy life?
Jon: So, I think one of the take homes with a lot of the calorie restriction approaches that have been done on rodents, I think that they're very important for trying to identify mechanisms for aging. I would not recommend somebody to follow 30% to 40% calorie restriction as an idea to increase lifespan. I think the risk with that level of calorie restriction, and I definitely have concerns about weight loss and being very thin going into aging, what I do think is based on the literature that is available because I do think exercise is important going into aging, being physically fit, being able to maintain muscle mass and going into aging, being active.
I think that's extremely important. I think the other thing that maybe come out is not so much the severe calorie restriction but probably something that a lot of people are doing for health, anyways, is watching what they eat and showing some level of restraint with their diet. Just this idea of not that you have to be on severe calorie restriction but just avoiding being overweight and trying to be active.
I think that's probably more in line with this 10% restriction that McCarter was seeing in rats or the 12 to 13% that the CALERIE study was showing. I think the other things are still more research is needed. I think it's intriguing, the idea about the time restricted feeding. I think ketones are very intriguing but right now I think those are areas that need more work before -- I think they are merging areas and I think they're areas that could have a big impact in the near future but areas that we definitely need to do more research.
Megan: Yeah. I also think that it's interesting that a lot of these calorie restriction mimetics that we talk about, they're all hormetic stressors. So, the idea that a little bit what doesn't kill you ultimately makes you stronger. So, maybe cycling some of these things with exercise, with intermittent fasting, periods of ketosis, periods of maybe higher or lower protein intake, all of these things are going to be beneficial at a cellular level as well.
Jon: Yeah. I think that's quite possible. I think those are definitely areas that need more research.
Megan: Cool. So, I do want to be respectful of your time. I have one more question for you which is: If you have all of the resources in the world, time, energy, et cetera, what study would you love to do? You cannot say longevity study in humans but anything else?
Jon: I think right now the two areas that--
Megan: You like two.
Jon: I'd do a combined study. The two areas that I'm very interested in, one, is this idea of continuous versus intermittent. I really am interested in determining is the ketosis that occurs with calorie restriction which isn't continuous, is that having an effect on aging? And then the other is my still interest in diet composition and the idea that especially in animals that aren't overweight what effect does diet macronutrient composition have on aging? What's the range?
Those are the two areas that I'm most interested in now. If I had to pick studies that I would pursue, those would be the ones, to see effects on health span and also be able to look at potential mechanisms whether they're affecting the mechanisms that I think may influence aging.
Megan: I love that. And I look forward to seeing the results of those studies in the future. Well, thank you. This has been really great. I really appreciate your time, Jon.
Jon: All right. Thanks, Megan.
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