FoundMyFitness - #038 Dr. Guido Kroemer on Autophagy, Caloric Restriction Mimetics, Fasting & Protein Acetylation

Episode Date: July 31, 2017

Dr. Guido Kroemer Dr. Guido Kroemer is a professor at the University of Paris Descartes and an expert in immunology, cancer biology, aging, and autophagy. He is one of the most highly cited authors in the field of cell biology and was the most highly cited cell biologist for the period between 2007 and 2013. Especially notable among his contributions: he was the first to discover that the permeabilization of mitochondrial membranes is a concrete step towards apoptotic cell death. In this episode, you'll discover:  (00:00) Introduction (09:09) The three main nutrient signals that activate autophagy (20:55) The role of fasting and nutrient deprivation in autophagy (28:52) Exercise induces autography  (33:07) Autophagy cleans out damaged organelles (35:14) Mitophagy keeps mitochondria healthy (39:38) Autophagy clears away neurodegenerative proteins in the brain (48:29) Autophagy in cancer is a double-edged sword (54:52) Fasting mimetics (e.g., resveratrol, spermidine, hydroxycitrate) induce autophagy If you're interested in learning more, you can read the full show notes here. Join over 300,000 people and get the latest distilled information on fasting & caloric restriction straight to your inbox weekly: https://www.foundmyfitness.com/newsletter Become a FoundMyFitness premium member to get access to exclusive episodes, emails, live Q+A's with Rhonda and more: https://www.foundmyfitness.com/crowdsponsor

Welcome back, Found My Fitness podcast listeners. Today's episode features Dr. Guido Cromer, who was a professor at the University of Paris, Descartes. He is a cell biologist who has made major contributions to understanding the role of mitochondria in cell death and has published numerous studies in immunology, cancer biology, aging, and atophagy, the latter of which I'm especially interested in because if you've listened to this podcast for a while, you should be aware that autophagy is a very important process for cleaning up cellular damage, which is intrinsic to the aging process and cancer.

Etophagy may be an extremely important process that, interestingly, can be triggered through occasional periods of intense fasting. It's not only related to fasting, but that's an important connection. This podcast is a little bit of a higher level one. If you have trouble keeping up with it, it is helpful to watch the video on YouTube, where definitions of scientific terms and figures are provided. In fact, if you're wanting to learn more about atophagy, the video that accompanies, the video that accompanies this podcast is a really good resource. That said, the first 15 minutes of the podcast

is probably the most difficult. So I will give you guys a quick review on some of the trickier jargon. One of the things we talk about is the relationship of a few important cellular pathways and the triggering of cellular autophagy. The most important among these that come up again and again is a pathway known as mTOR and another one known as AMP kinase. As Dr. Kromer explains in this interview, generalized nutrient deprivation, which occurs during periods of fasting, or starvation, especially prolonged fasting, triggers a reduction in the pool of an energy maclore molecule known as acetyl-CoA. When reductions in acetyl-CoA occur, it causes a decrease in protein acetylation, and this is associated with the activation of hundreds of cellular

proteins that are responsible for scavenging cellular products for degradation. In the context of carbohydrate metabolism, acetyl-coa is formed from a product of glucose known as pyruvate. There are other paths that create acetyl-coa, including amino acid catalytic catalysis and also beta oxidation of fatty acids. When we talk about mTOR, however, it is important to keep in mind that mTOR is activated by IGF1 and IGF1 is most robustly activated by protein consumption, especially protein rich in essential amino acids. That means when we restrict protein, we reduce the activation of IGF1 and thus also reduce mTOR. MTOR is involved in protein synthesis, so it makes sense that amino acids would activate it. It is also involved in cell growth and proliferation.

AMP kinase, on the other hand, is activated when cites. cellular ATP levels decrease, which again happens in a fasted state. AMP kinase is involved in regulating cellular energy homeostasis by inhibiting the synthesis of fatty acids and triglycerides and activating fatty acid uptake and beta oxidation in the liver. If you think about it, increased beta oxidation in the liver to induce the breakdown of fats and reductions in protein synthesis sounds about right for a fasting state. And that's basically what's being described when we say the induction of a top. is associated with increases in AMP kinase, but decreases in EMTOR.

So there are really three main signals that activate atopagy, and all of them are linked to energy metabolism. The first signal is a decrease in the pool of acetyl-CoA, which again means a decrease in glucose and in fatty acids. The second signal is a decrease in mTOR and IGF-1, which means a decrease in amino acids. The third signal is activation of AMP-Kinase, which means a decrease in total cellular energy or ATP. We also talk in this podcast a little bit about specific proteins involved in fasting-induced autophagy, such as LC3, which by responding to deacetylation caused by reductions in acetyl-coa

availability in the cytoplasm of a cell, associated with cellular digestion machinery known as the autophagosome. Unfortunately, the tools needed to watch these sort of molecular events occurring in cells are not the sort you can find in the clinic just yet, and it's one of the reasons why we can't just go out and buy an atophagy test from our local lab. We also talk about the role of autophagy in normal aging, nerve degeneration, and cancer. Lastly, we talk about some of the fasting memetics, such as hydroxycitrate, spermane, and resveratrol that are able to induce atopogy by tricking the cell by modulating one or more of the three main autophagy signaling pathways we just talked about.

All right. So that said, let's dive right into the podcast. It's deep biology, but you guys have now heard the worst of it. And look at you. You're sticking with it. Go you. On to Dr. Cromer. Hello, everyone. Today I'm sitting here with Dr. Guido Cromer, who is a professor at the University of Paris, Descartes. He is a cell biologist who has made major contributions to understanding

the role mitochondria play in cell death. He has also published numerous publications in the fields of cell biology, cancer biology, immunology, aging, and autophagy. The latter of which I suspect we're probably going to talk a lot about today because it's such an important topic. So autophagy is a spectacular phenomenon in cell biology, one that you can see with your eyes because cells become vacuolated when this process is induced. So you can see it by light microscopy and better of course by electron microscopy. It is a process that consists in sequestering portions of the cytoplasm of the cell and then digesting them to recycle the material and to degrade macromolecules into micro molecules,

metabolites, and to allow rebuilding the structures that have been destroyed. So technically it works in the sense that the mouth that is involved in this self-digestion process is the autofibosome. so it will sequester portions of the cell in the cytoplasm. It can be entire organelles, including mitochondria, and the protein aggregates. It can be bacteria or viruses that invade the cell. And this autofagosome, once it closes, will fuse with the lysosome, which is the sort of stomach of the cell.

And in the lysosome, there is a... has been fusing with the autofagosome, which is then called autofagolizosome, the luminal content will then be digested. So you mentioned the digesting of these multiple, you know, organelles, amygandria, but also aggregates, protein aggregates, and viruses, bacteria, pieces of chromatin, things, all seem to be sort of things that, at least in the... In the sense, if you're looking at the aggregates and, you know, the damage that occurs in a cell seems to be something that's associated with aging in general. So it's sort of like kind of seems like it's getting rid of all these damaging, potentially damaging, not just aging, but also obviously an infection.

But this process is getting rid of these damaging, potentially damaging molecules and aggregates and mitochondria, which are defective. what is the actual goal of autophagy is like you said is to get rid of these defective things to then provide energy or well it's actually interesting to look at the history of autophagy the name comes from autos far gain in greek which means self-eating and it's actually a linguistic invitation to think about self-mutilation and self-destruction and cell death And actually, the phenomenon is observed mostly in the context of stress. So cells, when they are stressed, will often undergo an autophagic reaction, which occurs before the cells die. And so this chronology of the phenomenon has been also an invitation to think that

autophagy is a mechanism that leads to cell death until it has been understood that inhibition of autophagy, which can only be... achieved in a specific way by genetic tricks, will actually sensitize cells to cell death induction. And so this means that autophagy is a means of adaptation to stress and a technique of the cell to avoid cell death. So the primary goal of autophagy is adaptation to changing conditions. and adaptation to external stress, and at the end, avoidance of the unwarranted demise of the cell. You bring up so many important points that I'd love to touch on.

The first is the external signals that are actually causing autophagy. You mentioned it's a response, it's a generalized response to stress, like, you know, mostly probably a hormetic type of stress, but even even this is another question I'll have for you later, the differentiating between the type of stress that can cause autophagy versus actually pushing it over to cell death. But in terms of the actual external signals that, the main ones that we know about that cause autophagy,

a lot of them have to do with nutrient sensing. Exactly. So perhaps the most physiological way to induce autophagy that is phyogenetically conserved from yeast to primitive, animals to ourselves is Newton deprivation, starvation, hunger. And so the idea is that a cell that is deprived from its energetic supply, which can be the absence of neutrons or the absence of growth factors that are required for these neutrons to be transported from the outside world into the intercellular space,

or the absence of oxygen, all these factors can induce autophagy, and the cells actually will destroy its bio-energetic reserves, which are macromolecules, proteins, lipids, and ribonuclear acids, to generate energy. And in terms of the energy part, I was reading about three really major pathways that seem to lead to autophagy, one being the actual energetic charge of the cell, like ATP status, when that lowers, that activates the AMP kinase pathway. And then the amino acid sensing pathway, which then when you don't have enough amino acids, that can basically inhibit mTOR.

And then there's a third one, the protein acidilation pathway, which I'm not as familiar with how that, essentially how that activates autophagy. So it's actually extremely easy. When you think about basic biochemistry, one of the central metabolizes acidyl-Qua. And so the cytosolic point, pool of acetyl-QA determines the level of protein acetylation for the simple reason that

acetyl transferases, which use the acety of acetyl-CoA to transfer it on lysine residues in proteins, acetyl transferases are having a low affinity for acetyl-co-A as compared to kinases which have a high affinity for ATP. So if you vary the ATP concentration, you vary the ATP concentration, in the cell, it has little impact on phosphorylation reactions. But if you've vera acetyl concentrations, it has a major impact on the acetylation level of cellular proteins. So that's a major difference. And so since acetyl-cure is built in the degradation of glucose, the glycolytic pathway,

from purovator, or in the catabolism of branch. amino acids as well as a final product of beta oxidation. All major nutrients are actually supplying acetyl-QA as an end product. And taking away glucose or amino acids or fatty acids will cause a reduction in the acetyl-coa pool and which is important to note it is the cytosolic acetyl-coa pool that is a accounting for autophagy regulation. This reduction in acetyl QA in the cytosol will cause deacetylation at the end of cellular proteins, hundreds of different proteins, and hence a sort of multiprunked induction of a major sub-pathways of the apoptotic process. Autophagy is actually a very

complicated process that it involves dozens, perhaps hundreds of different proteins, and is regulated by hundreds, perhaps thousands of additional proteins. And so this common regulation by acetylation is very efficient in stimulating the autophagic pathway. As a side effect of deacetylation reactions, you usually also observe the inhibition of mTOR and the activation of AMP kinase. So, everything comes together at the end. There's no exclusivity for one or the other pathway. They are connected. Really? So changing the acidulation status of proteins affects mTOR and amygines? Yes. Indirectly, we don't know how this actually works in molecular details.

Because that was sort of my next question was, I know that if you take cultured cells in a dish and you're doing some, you know, specific nutrient withdrawal. You withdraw amino acids where you withdraw glucose or you withdraw glutamine. You can induce autophagy. But in the whole organism, for example, in mice and humans, ultimately, you know, can you just limit your protein intake for a week and induce autophagy, even in the presence of a normally caloric diet, where you're still getting enough energy?

That's a good question. We have never tested selective prepletion of one or the other nutrient. I suppose that this would work, because protein depletion may affect neuroendocrine factors, like insulin growth factor, that at the end, will, due to its depletion, decrease the transport of glucose, into the cells and hence stimulate autophagy. But it has not been tested thoroughly in mice.

What we usually do is we starve mice and sometimes human volunteers completely from any kind of caloric uptake. And in this case, we do see at the whole body level that in all major cell types, perhaps with the exception of the brain that is somehow buffered against this effect, protein deacetylation occurs mostly in the cytoplasm. And that's a biomarker of autophagy, you would say? Well, it's too early to say that it is a surrogate or proxy of autophagy. So far, we have not been able to dissociate the two phenomena,

autophagy and protein deacetylation in the response to neutrons. However, when you induce autophagy by pharmacological trick, such as a cell permeable peptides that dissociates an inhibitory interaction between a Golgi protein and Becline 1, you can induce autophagy without that protein deacetylation would occur before. And similarly, when you give chemical inhibitors of mTOR like rapamycin or the rappolochs, there's also no protein deacetylation. So you can induce autophagy without protein deacetylation. acidilation, which means that the epoxy would be imperfect.

So we do have a system to measure autophagy, which is relatively easy to be used in experimental systems, which is the study of the redistribution of LC3 and other members of the same family that are usually diffusely distributed all over the cell, mostly in the cytosol, and then we'll aggregate or redistribute towards autophagosomes and autolysosomes. So they acquire a punctate distribution, small dots in the cell in the cytoplasm, and these dots can be seen by fluorescence microscopy if LC3 is labelled. labeled by immunoflorescence or when it is fused with green fluorescent protein or similar biosensors.

And so in humans, the only accessible cell type is the circulating white blood cell, the lucosite. So we can draw blood and determine by immunoflorescence the redistribution of LC3 from a diffuse to a punctiform pattern. And this is then a sort of detection of autophagy that can be applied to human beings as well. That seems like it's kind of complicated, though, for your standard clinic to be able to use

amino fluorescence to look at some leukocytes, circulating leukocytes, right? I mean, that's more. Well, you need some technology, especially ordinary cytofluometry cannot be used for this kind of approach because the standard cytofluometer just measures in intensity of fluorescence signal per cell, not its subseller distribution. So there are cytofloreometers that take pictures of the cells that are flying in front of the detector and using these pictures and analyzing them by image.

analysis software allows them to quantitate the redistribution of L-C-3 to autophagosomes. So there's hope for a non-invasive, clinically relevant biomarker for autophagy, but there still seems like there needs to be more work done before that actually happened. Before I can go into my doctor and say,

I did a four-day fast. I'd like to see if I've activated atophagy. Can you please draw some blood, right? were not quite there yet. Yeah, it would be wonderful to have the reward of measuring autophagy as a result of fasting and to get an objective incentive as a biomarker for doing that. Right. So it kind of brings up another question I had, which related to when you started talking about

how you can fast and fasting in organisms like rodents and also in some human volunteers, doesn't do setopagy. And the question that I had for you is, like, I've talked with Dr. Walter Longo, he was on the podcast, and he talked quite a bit about his research on prolonged fasting in both rodents and also in humans and how the prolonged fast, at least in rodents, is 48 hours, which in humans is around four days, four to five days. And that was able to very robustly not only activate autophagy, but also cell death. And that was followed by, a regeneration period. But the question is, do we know what the minimum amount of fasting time is for humans or rodents that can activate autophagy? So, for example, when I'm not pregnant,

I usually followed a very time-restricted eating schedule where I like to eat all of my food within at least 10 hours. And then I fast for 14 hours every night. Some people do even more strict, they eat within eight hours and they fast for 16 hours. Does that 16-hour fast induce any autophagy in any of our tissues? Is there any evidence? Do we know? We don't know. So Craig Thompson published a paper on circumdial variations in hepatic autophagy. So you know that mice don't eat during the day and they eat during the night. So the entire cycle is inversed. And he observed that as a result of not eating during the day, there was more autophagy in the liver. So this result is intriguing.

It has not been, to my knowledge, extrapolated to other organs, and it certainly requires more profound studies. So what we did on circulating leukocytes is that we needed to wait for three or four days to see massive induction of autophagin. There's a fundamental difference between rodents and humans. So the two days that you have been alluding to cause a 20% weight loss in mice that are at this time point at the verge of death. Another day would potentially kill them. And so 20% is a lot.

Imagine this for yourself. In two days. In four days, a human being only loses. 1 to 2% of his or her weight. Is that because they have a higher metabolism, rodents do? Yeah, it's certainly linked to the change in the surface volume ratio that is classically associated with an accelerated metabolism. Yeah, okay, that's sort of, so we don't really know to what extent

autophagy can be occurring in a shorter, intermittent fast. there's some hope that it does. I mean, I know, for example, you mentioned IGF1 and how IGF1 lowering IGF1 is important for reducing autophagy because the whole mTOR pathway and so on. But I do know that the half-life of IGF-1, in serum, at least, is around 12 hours.

So the question becomes, well, okay, if you start to lower IGF1 after 12 hours, is do you still need more to occur, like more ATP depletion, more? What, you know, what is it that needs to happen, you know, to actually send a signal to the cells to go, oh, I'm stressed, I need to start eating my whatever organelle or damaged proteins or something? So that would, is that something that people are currently investigating, like the minimum amount of time that it would sort of take to induce, or for fasting at least, to induce autophagy? It's an extremely interesting question that is easy to be answered in rodents and difficult in humans because it may be easy to find a volunteer who fasts and allows for regular blood drawing, but it will be very difficult to find a volunteer who fasts and allows for liver, muscle or skin biopsis.

Right, right. It kind of reminds me there was a study I was actually reading the other day that, was done in the caloric restricted society. You know, there's a group of people that are out there practicing caloric restriction, which typically is eating around, what, 30% less food than you normally would eat or something like that. A lot more difficult for people to maintain, I think, than intermittent fasting is. But there was a study that was published, and these individuals had been doing caloric restriction for about six years, plus or minus. I'm sure you've seen this study. but they did muscle biopsies on them, and they measured LC3.

They measured some of the biomarkers of atopagy, I think Becklin, I think some other things. And then they measured heat shock proteins, which are also a stress response. And it was, you know, like in some cases, like the heat shock proteins, like HSP 70 was elevated by 12-fold compared to age-matched lean controls that eat more of a Western-type diet. Yeah. But, you know, the fact of the matter is that they did do a muscle biopsy. Autophagy was activated. You know, the stress response pathway in general was activated. But six years of doing caloric restriction is not very sustainable for the majority of the population

in the, you know, at least in the United States and Western world. So actually in mice, you can obtain exactly the same longevity extension that you would obtain with 30 percent of caloric restriction by intermittent. fasten. So it's logistically much more difficult. Imagine you have to wait for each mouse the amount of food that they would eat normally, subtract 30%, put it in the cage, individual cages because caloricotia mice tend to eat each other. Oh, wow. Yeah, they become aggressive because they are hungry. They become cannibals. They become cannibals. So it is logistically much more simple to take out the food from the KHM completely and to put back the foot on the next day.

So it's one day without any further and another day with normal nutrition. And at the end, so you have an oscillation of the weight of the mice, 10% every day. These oscillations tend to become smaller because the mice somehow adapt to this sort of stress. but the final result is that the intermittently fasted mouse has the same weight as a normally fat mouse. At difference with the caloricially restricted mouse, it weights also 20 to 30% less. And in spite of this difference in the body weight, intermittent fasting allows for lifetime expansion in the same way as does, caloric restriction. So one can also consider that this may be more amusing to have, if I was a mouse, I would probably prefer the intermittent regimen because it means satisfaction during one day

and dissatisfaction on the other day, but not permanent dissatisfaction. Most people prefer doing intermittent fasting. I mean, that's, so it'll be very interesting to see more studies come out on, you know, the translation of this to humans. And as you mentioned, you needed three days of, was it a water fast they did? Was it a complete fast or they had coffee or? Coffee, tea. Okay. So, no sugar, no milk, and water. Okay. So three days was enough to at least show signs of autophagy in circulating leukocytes and, um, and Valter's work has shown you know, four to five days and he's done, you know, he's got his fasting and then he's got the fasting-imicant diet.

Some also hints that that also isn't enough. So that's sort of encouraging. It would be more encouraging to have like a 24-hour faster or 48-hour. I mean, that's so much easier to do in general. But the other thing that induces autophagy, you were mentioning the stress response and oxidative oxygen and it sort of reminded me of exercise and how exercise also induces autophagy. I've seen some studies where in humans, they've looked at muscle, skeletal muscle, and how aerobic exercise and eccentric and concentric exercise all can activate atophagene skeletal

muscle. Do you know if it activates autophagy in multiple tissues, exercise? That's something that we have not studied. So it is known that endurance training is particularly efficient in mice to induce autophagy, and that it mediates anti-obesity and anti-diabetic effects that are depending in a way on autophagy induction. Because genetic modifications of the process that leads to autophagy induction, its inhibition specifically by exercise can prevent these anti-diabetic effects. Really? Really? Oh, I didn't know that the role of the exercise in preventing diabetes was shown to be dependent on autophagy to some degree. That's very interesting. So do you think that has to do with in the liver and the like pancreas somewhere? I mean, is it now?

Yeah, to know this in detail, it would be necessary to inhibit autophagy specifically in different tissues. To my knowledge, this has not been done yet. Okay. So do you think that? fasting while you're exercising in a fasted state. Now, that's another thing that's, do you think that would be important? Or do we, I mean, I've seen some studies in mice where they claim it is, but mice have a very high metabolism. And so there's a synergy there.

But when you look in humans, it's not so important. Like, the exercise can still induce autophageean skeletal muscle in humans even without being in a fasted state. but the question is like, will you synergize more? You can speculate, but we don't know. I'm giving you a lot of ideas here. So maybe we can kind of shift a little bit into the general role that autophagy plays in some of these age-led diseases like neurodegenerative disease, cardiovascular disease, cancer. Talk a little bit about the micro-autophagy, or is that what you call it?

when you're talking about the specific degradation of organelles like mitochondria or protein aggregates? Well, so normally when we refer to autophagy, we talk about macro atrophagy, which is the phenomenon that you can see easily by microscopy because of the formation of the orthofergosomes that are big enough to be seen by conventional microscopy, face contrast,

and especially, of course, when you enhance your solution by immunophorescence or similar technologies. So there are other types of autophagy that are less well studied, like Chaparone mediated autophagy or microautophagy, where basically proteins or portions of the cytosol are introduced directly into lysosomes. So you don't need the mouth of the process, the autofogosome, you just need the lysosome. and they are much less studied. And then there's a special case among different kinds of macro autophagy. So to be very simple in the dichotomy, there is the case that autophagy is dictated by general stress

or general absence of neutrons, which means that it is dictated by demand. So the cell needs to eat some portions of itself to adapt to Newton stress. And the other kind of autophagy is dictated by the offer. So a damaged organelle will change the composition of its surface in a way that it is decorated by signals for stimulating its engulfment by the orthophagosome. And so it's another kind of autophagy that then can be specific, specific for organelles of different types like mitochondria when it is called mitophagy or for paroxysomes, what it is called pexophagy, for the endoplasmic reticulum when it's called reticolophagy, specific for ribosomes,

ribophagy, perfect, yes, and specific for viruses when it is called virophagy. So, and the two processes may also interact in a way. So when you stimulate general autophagy by activating the neutron sensors, AMP kinase inhibition of mTOR or by provoking deacetylation, then you increase the demand, and the autophagic machinery actually prefers in a way to sequester and to destroy those organelles that are already slightly marked for destruction. The protein aggregates that are not yet harmful enough to emit a signal per se, but that they are there.

And so it's a sort of preferential cleaning of the slightly damaged and slightly aging portions of the cell. And this may actually explain why stimulation of autophagy in cells, when there are monocular organisms or at the organismal level at different organs can be a sort of device against aging. Wow, that was a very beautiful explanation that you actually answered a question I was going to ask you, which was, you know, the difference between the signal, for example, example, the neutralized topogy, when you have the nutrient sensing stress, that even that can, to some degree, selectively degrade mitochondria, for example. But the actual signal that really does activate mitophagy when you're talking about mitophagy, it's a little different, right? It's the actual mitochondrial damage, the membrane potential. Yes, so when a mitochondrial is suboptimal in its function, it will decrease its mitochondrial transmembrane potential.

And this is a signal to activate enzymes on the surface of the mitochondrial that cause ubiquitilation, recruitment of autophagic adapters, and leads at the end to autophagy because the offer of the organelles. So the organ in a way offers itself, it proclaims its sacrifice by autophagy. And so, of course, this is not an all-or-nothing phenomenon. So mitochondria can be aging in the cell, and as they age, they gradually decrease the performance and the mitochondrial transmembrate potential. So those mitochondria that are most dysfunctional, they will be eaten first if you increase the demand for autophagy. That is very cool.

And if you are selectively degrading these damaged mitochondria, which are, you know, or aged, which are damaged, do they get replaced by new mitochondria? Does that, is that a signal for mitochondrial biogenesis? Yes. So in C. elegance, this world studied that actually the whole turnover of mitochondria is regulated. So there's a sort of coupling between mitophagy and mitochondrial biogenesis. That's good to know. So it's very clever how the system has been designed. That's great.

So it's not like you're losing, you're not losing the pool of mitochondria, you're effectively losing the defective pool and you're almost making younger mitochondria. If you're going to make a new mitochondria, then it's going to be young and fresh and not damaged. So it's a very elegant way to sort of replenish your mitochondrial population, it seems. So we have to make the difference between homeostatic conditions and, for instance, cellular differentiation when cells change their metabolic programs. So the easiest example is yeast that you suddenly place in a glucose-containing medium to allow for the fermentation of glucose in wine or beer production. So these yeast cells don't need much oxidative phospholation and they essentially rely

during the process on glycolysis. So they adapt to this change. by destroying most of their mitochondria by metropathy. And this makes actually the metabolic adaptation of the yeast cell efficient. You have similar examples in the embryonic development of the retina for retinal gangion cells or the differentiation of macrophages from so-called M0 to M1 macrophages, in which the cells change from

oxidative phosphorylation, respiration, to an essentially glycolytic metabolism that is coupled to mitophagy. And so inhibition of mitophagy actually avoids the differentiation process in both examples that I just gave to you. That's really interesting. So obviously these processes are not just as a stress response. They're part of development as well. They can be used in multiple different instances. And in the case of mitophagy, it's also, it plays an important role in the prevention of neurodegenerative diseases, correct? Yes.

So most known neurodegenerative diseases are either caused by the aggregation of poorly built proteins that somehow create a protein aggregated. that are toxic for the cell, or they can also be caused by subtle deficiencies in the autophagic and lysosomal machineries that lead to the accumulation of unfolded proteins at the end. And so either the excessive production of unfolded proteins or their reduced removal causes to a slow accumulation of these toxic protein aggregates. Remember that neurogenerative diseases are slow processes in most cases that manifest with old age. And so one strategy to treat neurogeneration, at least theoretically, is to increase orthofergic turnover.

And so one technique is actually then to stimulate general autophagy by increasing the demand by starvation or by biochemical tracts that substitute for starvation and to reduce the protein aggregates that are the cause of the disease. So these protein aggregates like amyloid beta plaques and Alzheimer's disease or alpha nucleine and Parkinson's disease. So basically clearing out those protein aggregates obviously would play an important role, not only prevention, but presumably also to some degree help with treatment. Of course, that's speculation. And then the mitochondria, the one I was thinking about with mitophagy, was the role, at least some of the proteins that are

involved in that, like the pink parking and how they seem to be important for Parkinson's disease. Is that accurate? So the pink parking pathway is one pathway among others. that allows for marking mitochondia that are damaged for destruction. And so inhibition of this pathway leads to the accumulation of malfunctioning mitochondia with major consequences for the cell that harbors those mitochondia because all of a sudden, bioenergy metabolism becomes inefficient. Reactive oxygen species are produced, and as you know, mitochondria are...

latent bombs in the sense that they enclose potentially dangerous proteins that once released will activate the apoptotic machinery and cause cellular suicide. Yeah. So that's the question. What's, do we know the threshold for the stress threshold, for, you know, activating atophagy, and when that pushes the mitochondria then to permeabilize and cause cell death. Like where, for example, with Volta's work in mice, he had done 48-hour fasts, and there was both autophagy and massive apoptosis occurring. So is it just the intensity of the signal that can then say, okay, autophagy is not going to work

here? We've got to die. or do we know? Well, autophagies are used in most cell types, while apoptosis occurring in selected cell types. So what Walter has been observing, if I remember, well is destruction of leukocytes, white blood cells, which are very easily to be rebuilt.

And so the loss of 50% or 75% of leukocytes can be easily repaired in a few days. And it is a way to adapt the repertoire of immune cells to changing circumstances. It is a way also to inhibit unwarranted inflammatory reactions. So depending on the context, induction of autophagy can be actually a subtle way to avoid excessive inflammation. One example is the so-called sickness response. So a cat or a dog, a human being or a mouse that is sick that has a bacterial infection will hide away, avoid light and noise, and will not eat. It's a classical philogeneously conserved reaction in most cases of bacterial infection.

And so this phenomenon leads to changes in the metabolism, ketone and the production of ketone bodies, the reduction of glucose levels, presumably also induction of autophagy, and altogether these mechanisms then avoid excessive inflammation that may be lethal. So Rasslan Metzitov published a paper in cell last year showing that force-feeding mice or just increasing the glucose levels to a normal concentration was sufficient to make bacterial infection that otherwise would have been able to cope with lethal. Wow. So I know in humans too when we have a bacterial infection, for example, a stomach virus,

or something that's bacterial of origin, you don't eat as well. So it sounds like it's sort of a protective mechanism. It is. That's really interesting. I didn't know that. It's very interesting. I want to kind of move on to cancer, just for time purposes. So cancer is another sort of very, it's been in regards to autophagy, something that I've

always sort of been unsure about because it's very clear to me that preventing, you accumulation of damage, you know, pieces of nucleic acids and pieces of chromatin and, you know, all sorts of, you know, things that can cause inflammation by having, you know, damaged proteins around and things like that. Obviously, clearing those out would be very important for preventing cancer. But when it comes to treating cancer, it's not as clear. There seems to be, I mean, for example, you know, there's a very classic drug out there, chloroquine, right, that inhibits autophagy that's used to kill cancer cells. Well, it's not exactly true.

So chloroquine is a lysosomal inhibitor. It's a molecule that due to its charge will specifically enrich in the membranes of lysosomes and then causes lysosomal membrane damage, potentially also inhibition of autophagy, but fundamentally also liberates the potentially toxic content of lysosomes into the cytosolic space. And so there are a few reports around showing that inhibition of autophagy is not the sole mechanism by which chloroquine can mediate cytosalic.

toxic effects. Okay, well that's good to know. And the other thing that is important to note is that chloroquine and hydroxychloroquine, which are anti-malaria agents that have been used for a long period. And also actually used for the treatment of rheumatoid arthritis because they have anti-inflammatory properties are only introduced into clinical trials, mostly in combination with chemotherapy or radiotherapy to treat cancer. And those clinical trials, so far, are not convincing. Okay. So what about the fact that some cancer cells do activate autophagy?

So how's... So one relatively general mechanism may be that early during oncogenesis, the deletion of tumor suppressor genes or the activation of oncogenees leads to autophagy suppression. So there are several examples for this. And it is part of the process that leads to cellular transformation, because autophagy is a homeostatic mechanism that, if inhibited, favors genomic instability and malignant transformation of the cells.

So there are examples on the literature, also that direct inhibition of autophagy is sufficient, to cause oncogenesis, in particular in the context of leukemia. And so later on, when the cells strive and adapt to an ever more hostile microenvironment, hostile because there's too little vascularization for the expanding cancer cells, so initially there are hypoxic areas, There's no normal tissue architecture, so the cells are usually undernourished. The doctor may apply some chemotherapy agent, which is an additional stress. So there are internal and external stress pathways that the cell has to cope with, and it

is an advantage for the cancer cells to reactivate the auto-octivate the auto-of-restealth. process. And so it has been proposed that inhibition of autophagy would be a way to make the cancer cells more fragile and vulnerable to therapeutic intervention by chemotherapy, radiotherapy, targeted therapies. The problem is that nothing is simple in oncology and that can't cancer is not just a cell autonomous disease. It is more. It is not just that one cell has become wild and has been accumulating genetic and epigenetic changes that make it selfish.

No. A cancer cell will only survive if it escapes from immunosurveance. So the immune system, fortunately for us, is usually very efficient in eliminating aberrant cells. pre-malignant cells and the initial cancer cells. And actually, the inhibition of autophagy that occurs during early oncogenesis may be also a way for the cancer cells to hide from the immune system. And so it's this complex, it's immunology, multiple different players come into action. Autophagy, for instance, is required for stress cells to release ATP into the microenvironment.

You know, of course, ATP is the most important energy-rich metabolite in the cell. It's like the equivalent of the dollar for bioenergy clinics in the economy. And ATP, when it appears all of a sudden outside of the cell is considered as non-physiological, is this a dangerous signal. It is perceived by so-called puneerogic receptors that are present, among other cell types, on leukocytes, in particular myroid cells. And a cell that undergoes autophagy may, especially when this occurs, before cell death, release ATP to attract myeloid cells into its proximity and to start

an immune response against tumor antigens in the context of the initial oncogenic events. And so autophagy is required for some steps of the immunosurveillance process. And it is exactly this process that makes cancer therapy. efficient. So in contrast to the official dogma that has been on vogue for several decades, chemotherapy is not just killing the cancer cells as if we used an antibiotic that specifically paralyzes the metabolism of bacteria. No, it is true that chemotherapy, induces cancer cell death, but the important point is that chemotherapy must provoke this cell death in a way that it later leads to an immune response. And so if you have a long-term effect of

chemotherapy for years or decades that continues beyond removal of the drug, it is due to an anti-cancer immune response. And so since this is so important, the capacity of the chemotherapy agent to induce autophagy is actually required for the long-term efficacy of the treatment. I did not know that. I had no idea that the induction of autophagy would stimulate the immune system through this extracellular ATP mechanism and how that is

I mean, obviously, the immune system is extremely important for killing cancer cells, but that's very cool and probably leads to the next topic on some of your work with the fasting, so-called fasting memetics, like spermidine, hydroxycitrate that you've done. Maybe can you kind of just briefly explain, let's start with spermine? What is spermidine? What does it do? Well, I will first start to explain what are these. fasting memetics, as you say, and caloric restriction memetics, as we say.

So the CRMs, caloric restriction memetics, are actually inducing the same biochemical changes in the cells as would do starvation or fasting. So we have been discussing on the importance of acetylcoa and protein deacetylation resulting from the depletion of acetylcoye in the context of fasting. and caloric restriction memetics similarly induce deacetylation reactions to stimulate autophagy. And this can be actually achieved in three different ways. First, you simply inhibit the generation of acetylchase, the enzyme that generates acetylchore in our cells, the most important one for the cytosolic pool is ATP citrate lyase and hydroxycytrate, or,

pharmacological compounds that inhibit this enzyme cause acetyl-CoA depletion, deacetylation, and autophagy. And you can have the same effect by inhibiting the protein acetyl transferases. Some of them have been identified, like EP300, which appears extremely important for autofagy regulation. And specific inhibitors, of EP300 such as Bermidine and natural compound or C64-6, which is a pharmacological compound, specifically designed for this function, they can also cause deacetylation and autophagy.

And finally, it is possible to activate deacetylases, so enzymes that remove acetyl groups from proteins and cause hypoacetylation and autophagy. And one example that is well known, Resveratrol contained in red wine that induces autophagy through this pathway. So all these agents, caloric restriction memetics, have different molecular targets, but activate autophagy by a final common pathway. The protein acidulation seems like that. Yes, exactly. Okay.

So with some of the major ones, you've worked with, spermidine. I've read quite a bit about spermidine. I know it's found in high concentrations in Nato, the Japanese fermented soybean that doesn't taste like great. But I've seen studies about aging, you know, giving it to even, you know, aging mice or something can extend their lifespan.

Is that true? So spermidine, to come to the source of spermatine, is contained in the nuclei of all kinds of cells, so in the nucleoid of bacteria, but also in the nuclei from yeast cells or from plant cells or animal cells. So all food items that contain nuclei cells actually containing spermidine,

also there are large variations in the content. So we have to know that the spermium is volatile, and accounts for the smell of sperm. So it is frequently found in food items that have some kind of smell, like nato or durian fruit, or fermented cheese when it is generated from non-posterized sources and very rich in bacteria and fungi that are contented. to the fermentation process, which is of course smelly cheese.

And it's also quite abundant in some vegetable tables and food where this scent is more agreeable to most people because it is complex to other molecules that reduce its volatility. So spermidine has the capacity to induce it. to induce autophagy when it is taken up with food or with the drinking water when we treat mice.

It can also be injected, of course. It is produced by our microbiota. So one third of the spermidine in our body is probably produced in the intestine and you can manipulate the microbiome

to increase its production of polyamines. including spermidine. Through what probiotics? Or through... Yes. Yeah. So you know what strains of bacteria?

Yeah, there's a Japanese group that has been publishing that specific bacteria overproducing polyamines can be used to reduce the development of colon cancer or to reduce aging. Wow. Fascinating. That's very interesting. And you've shown with the spermidine, I know we have... limited time here with the spermidine that's been shown to, was it spermine or hydroxycitrate? I think that was shown to synergize with, like you were mentioning before, the chemotherapy.

Yeah, both of them actually. Both. Okay. So the mechanism is that when you combine chemotherapy with caloric asphymetic, all the caloric restriction memetics that I mentioned, including spermidine, and, hydroxycytrate in Respiratol will enhance the antacancer immune response that makes the therapy durable. So we have been able to show that inhibition of autophagy in the malignant cells or destruction of the extracellular ATP that is released as a result of autophagy

is sufficient to abolish the favorable interaction between caloric restriction memetics and chemotherapy. And similarly, actually, it is sufficient to remove T cells from the system and you will lose any kind of tumor growth reduction induced by chemotherapy combined with caloric restriction memetics as a proof that the cellaric. immune response is actually decisive for therapeutic outcome. Wow. That's really quite promising, I think, for, you know, at least in the clinic, if you can somehow then test whether or not these caloric restriction or fasting

memetics work in conjunction with some of these immunotherapies. That would be fantastic. But I want to ask you one last thing, too, about some of these fasting memetics. Like, if I were to just supplement with hydroxycitrate, for example, or if there were a spermating supplement, and I was still eating a normal, you know, healthy diet, but not caloric, and not fasting, do you think that would be sufficient to induce autophagy in?

Well, I can respond for mice. Okay, how about mice? I don't know about humans because we have no clinical studies. So in mice, it does? It does. and in mice, you actually can give a combination of high-fat diet that usually would cause obesity with spermidine to reduce weight gain through mechanisms that we don't understand, that we believe to be autophagy dependent, but have to elucidate in some molecular detail.

That's very cool. So I'm going to look up those strains of bacteria, you know, to see, you know, can I eat fermented food? to increase that population and get more spermating, things like that would be very interesting to me as useful little tools. Do you have any practices that you do? Do you, for example, do intermittent fasting or any type of fasting or time-restricted eating? So usually in my ordinary life when I'm in Paris and work,

I only have one meal per day. So I have dinner with cheese and wine. containing spermidine and resveratol. It's an ideal combination because inhibiting the acetyl transferase and activating the deacetylase will have, of course, a synergistic effect. Well, you can show this in mice, and this is an excuse for me. Yeah. Wine and cheese got great together.

From a very good combination. and I do also practice some fasting twice per year or so when I don't eat anything for five days. Oh, wow. So you do a prolonged fast twice a year? Well, it's not so prolonged. So you know that Gandhi, for instance, has been doing fasting exercises for 20 days or more, which is the time at which a normal... individual could have some long-term consequences on the health.

So 20 days is some period that usually could be easily supported by a healthy, middle-aged individual that has no underlying pathologies. I have a couple of friends that have done, one of them is used long. very prolonged fast, like 20 days. He's morbidly obese, and he's lost now like 200 pounds over the course of a year by doing just several rounds of these prolonged fast. But he's got a lot of fat, you know, to supply energy. I'm not sure I would subject myself to a 20-day water fast.

But it's cool to know that you do these 5-day water fast twice a year and eating one meal a day. Yeah, and doing exercise. And when you're eating, when you're, sorry, throughout the day, day when you're not eating, do you drink coffee? Yes. And there's polyphenols in coffee that actually... Yes, we published a study in mice, giving them a non-toxic dose of caffeinated or decaffeinated coffee with the drinking water continuously, and we could show that this was magnificently

inducing autophagy. The caffeine independent fashion. Right, totally independent caffeine, so just polyphenols. And this was like, I think I read it. study was like after four hours or something in the mice. So in humans, potentially maybe the fasting plus the coffee. Well, coffee abuse has been linked to major health-promoting effects. So most cardiovascular and neurogenerative diseases are actually reduced in heavy coffee

drinkers as an independent link between lifestyle and pathology. So, of course, it's an association that always can be criticized because it's just a study in which you find statistical correlation. It would be much more interesting to do a randomized clinical trial on coffee intake. More interesting and more expensive, yeah. Absolutely. So, great. So fasting, coffee, wine and cheese, one meal, exercise, you're doing it all. Awesome. Well, I really enjoyed this conversation, Guido. If people want to find you, you have a lab website, cromerlab.com. That's K-R-O-E-M-E-R-M-E-R-L-A-B.com. So thank you again for this wonderful, very illuminating conversation. It's a pleasure.

I hope you enjoyed learning about all things atophagy, both macro and micro from Dr. Guido Kromer. Dr. Kromer, in my mind, is the consummate expert in this field and also is a amazingly prolific with somewhere in the neighborhood of a thousand publications to his name, quite the feat to say the least, and all the more reason why it was an enormous privilege to get to talk to him. Do you love these geeky, deep biology podcasts? You can be a part of the community that nurtures the existence of this podcast and other useful creative offshoots that can be found at Found My Fitness by heading over to foundmyfitness.com forward slash crowd sponsor. About to buy a solar powered witchiper or a gargantuan vat of ghee. If you get them from Amazon, make sure to click the

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