Dr. Guido Kroemer on Autophagy, Caloric Restriction Mimetics, Fasting & Protein Acetylation

Hello, everyone. Today I’m sitting here with Dr. Guido Kroemer, 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 micromolecules, 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 autophagosome. So, it will sequester portions of the cell in the cytoplasm It can be an entire organelles including mitochondria, it can be protein aggregates, it can be bacteria or viruses that invade the cell, and this autophagosome, once it closes, will fuse with the lysosomes, which is the sort of stomach of the cell. And in the lysosome, it has been fusing with the autophagosome, which is then called Autophagolysosome, the luminal content will then be digested So you mentioned the digesting of these multiple organelles or mitochondria, but also protein aggregates, and viruses, bacteria, pieces of chromatin, and things all seem to be sort of things that at least in the sense, if you’re looking at the aggregates, and you know, 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? Well, it’s actually interesting to look at the history of autophagy. The name comes from “autos phagy” 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…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 phylogenetically conserved from yeast to primitive animals to ourselves is nutrient deprivation, starvation, hunger. And so, the idea is that a cell that is deprived from its energetic supply, which can be the absence of nutrients or the absence of growth factors that are required for these nutrients 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 bioenergetic reserves, which are macromolecules, proteins, lipids, and ribonucleic acids to generate energy In terms of the energy part, I was reading about like 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 assimilation pathway, which I’m not as familiar with essentially how that activates autophagy So, it’s actually extremely easy. When you think about basic biochemistry, one of the central metabolites is Acetyl-CoA. And so, the cytosolic pool of acetyl-CoA determines the level of protein acetylation for the simple reason that acetyltransferases, which you see acetyl, moiety of Acetyl-CoA, to transfer it on lysine residues in proteins Acetyltransferases are having a low affinity for Acetyl-CoA as compared to kinases which have a high affinity for ATP. So if you vary the ATP concentration in the cell, it has little impact on phosphorylation reactions But if you vary acetyl concentrations, it has a major impact on the acetylation level of cellular proteins. So that’s a major difference. And so, since Acetyl-CoA is built in the degradation of glucose, a glycolytic pathway, from pure beta or in the catabolism of branched amino acids, as well as a final product of beta-oxidation. All major nutrients are actually supplying Acetyl-CoA as an end product. And taking away glucose or amino acids or fatty acids will cause a reduction in the Acetyl-CoA pool which is important to note, it is a cytosolic acid Acetyl-Co-A pool that is accounting for autophagy regulation. And this reduction of Acetyl-CoA in the cytosol will cause deacetylation at the end of cellular proteins, hundreds of different proteins, and hence, a sort of multi punked induction of major subpathways of the apoptotic process. Autophagy is actually a very complicated process that 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 from 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 acetylation status of proteins affects mTOR and AMP Kinase? 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 just…you’re doing some, you know, specific nutrient withdraw, you withdraw amino acids when you withdraw glucose, or you withdraw glutamine, you can induce autophagy. But in the whole organism, for example, in mice and in 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 to selective completion of one or the other nutrient, I suppose that this would work, because protein depletion may affect a newer endocrine 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 phenomenon, autophagy and protein deacetylation, in the response to nutrients However, when you induce autophagy by pharmacological tricks, such as cell permeable peptides that dissociates an inhibitory interaction between a Golgi protein and Beclin 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 rapalogs, there’s also no protein deacetylation. So, you can induce autophagy without protein deacetylation which means that the proxy 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 cytoplasm and these dots can be seen by fluorescence microscopy if LC3 is labeled by immunofluorescence 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 leucocyte. So we can draw blood and determine by immunofluorescence the redistribution of LC3 from a diffuse to a punctate from a 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 immunofluorescence to look at some leukocytes, circulating leukocytes, right? I mean that’s more Well, you need some technology. Especially, ordinary cytofluorometry cannot be used for this kind of approach, because the standard cytofluorometer just measures an intensity of fluorescence signal per cell not its subcellular distribution. So there are cytofluorometers 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 LC3 to autophagosomes So there is hope for a non-invasive clinically relevant biomarker for autophagy but there still, it seems like there needs to be more work done before that actually happen…before I can go to my doctor and say, “I did a four-day fast. I’d like to see if I’ve activated autophagy Can you please draw some blood.” Right? We’re 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 does induce autophagy And the question that I had for you is, like, I’ve talked with Dr. Valter 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 4 days, 4 to 5 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 8 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 circadian variations in hepatic autophagy So you know that mice don’t eat during the day and they eat during the night, and 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 still certainly requires more profound studies Okay When you say during So what we did on circulating leukocytes is that we needed to wait for three or four days to see a massive induction of autophagy There’s a fundamental difference between rodents and humans, and 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, so imagine this for yourself In two days In four days, a human being only loses one to two percent of his or her weight Is that because they have a higher metabolism, rodents do, or…? Yeah, it’s certainly linked to the change in the surface volume ratio that is classically associated with an accelerated metabolism Yeah, okay. That 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 IGF-1 and how IGF-1…lowering IGF-1 is important for inducing autophagy because in the whole mTOR pathway and so on. But I do know that the half-life of IGF-1 is, in a serum at least, is around 12 hours. So, the question becomes well, okay, if you start to lower IGF-1, 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 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 is that something that people are currently investigating? Like, the minimum amount of time that it would sort of take to induce, 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 biopsies 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 the study. But they did muscle biopsies on them and they measured LC3. They measured some of the biomarkers of autophagy, I think Beclin and 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, the heat shock protein, HSP70, was elevated by 12 fold compared to age-matched, lean controls that eat more of a Western-type diet 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 in Western world Actually, in mice, you can obtain exactly the same longevity extension that you would obtain, the 30% of caloric restriction by intermittent fasting. So, it’s logistically much more difficult. Imagine, you have to weigh, for each mouse, the amount of food that they would eat normally, subtract 30%, put it in the cage, individual cages because calorically restricted mice tend to eat each other Oh, wow Yeah, they become aggressive because they are hungry They become cannibals Yeah, they become cannibals. So, it is logistically much more simple to take out the food from the cage completely, and to put back the food on the next day. So it’s one day without any food, 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. A difference with the calorically restricted mouse, it weighs also 20% to 30% less. And in spite of this difference in the body weight, intermittent fasting allows for a 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, 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. No food No sugar, no milk. And water Okay. So three days was enough to, at least, show signs of autophagy in circulating leukocytes. 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-mimicking diet into… Some also hints that that also is enough So, that’s sort of encouraging. It would be more encouraging to have like a 24-hour fast or 48-hour. I mean, that’s so much easier to do in general But the other thing that induces autophagy, you’re 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 autophagy in 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, it’s inhibition, specifically by exercise, can prevent these anti-diabetic effects Really? Yeah 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 the liver, and the like pancreas, somewhere… I mean, is it known? To know this in detail, it would be necessary to inhibit autophagy specifically in different tissues and, to my knowledge, this has not been done yet Okay. So do you think fasting while you’re like 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 autophagy in skeletal muscle in humans even without being in a fasted state, but the question is like will you synergize more and You can speculate, but we don’t know I’m giving you a lot of ideas here. So maybe we can kind of talk, shift a little bit into the general role that autophagy plays in some of these age-related diseases, like neurodegenerative disease, cardiovascular disease, and cancer. Talk a little bit about the microautophagy, or is that what you call it, like, when you’re talking about the specific degradation of organelles like mitochondria or protein aggregates? So normally, when we refer to autophagy, we talk about macroautophagy, which is the phenomenon that you can see easily by microscopy Because of the formation of the autophagosomes that are big enough to be seen by conventional microscopy, face contrast, and especially of course when you enhance a solution by immunofluorescence or similar technologies. So, there are other types of autophagy that are less well studied Like, chaperone-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 or autophagosome, you just need the lysosome. And they are much less studied And then there’s a special case among different kinds of macroautophagy. 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 nutrient stress, and the other kind of autophagy is dictated by the offers. 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 autophagosome And so, it’s another kind of autophagy that then can be specific Specific for organelles of different types like mitochondria, and it is called mitophagy, or for peroxisomes, and it is called pexophagy, for the endoplasmic reticulum, and it’s called reticulophagy, specific for ribosomes, ribophagy. Perfect, yes. And specific for viruses, and it is called virophagy And the two processes may also interact in a way. So when you stimulate general autophagy by activating the nutrient sensors, AMP kinase, inhibition of mTOR, or by provoking deacetylation, then you increase the demand, and the autophagy 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 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 they are monocellular organisms or at the organismal level at different organs, can be a sort of device against aging Wow, that was very beautiful explanation. It actually answered a question I was going to ask you which was, you know, the difference between the signal, for example, nutrient generalized autophagy, 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 mitochondrion 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 mitochondria that cause ubiquity relation, recruitment of autophagy adapters, and leads at the end to autophagy because of the organelles or the organelle 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 transmembrane 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? Is that a signal for mitochondrial biogenesis? Yes. So in C.elegans, this was a study 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 It’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 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 program. So the easiest example is yeast that you suddenly place in the glucose-containing medium to allow for the fermentation of glucose in wine or beer production. So these yeast cells don’t eat much oxidative phosphorylation, and they essentially rely during the process on glycolysis. So they adapt to this change by destroying most of their mitochondria, by mitophagy And this makes actually a metabolic adaptation of the yeast cell efficient Do you have similar examples in the embryonic development of the retina for retinal ganglion 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 Very interesting. And in the case of mitophagy here, 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 protein cell that somehow create protein aggregates that are toxic for the cell Or they can also be caused by septal 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 neurodegenerative diseases are slow processes in most cases that manifests with old age. And so, one strategy to treat neurodegeneration, at least theoretically, is to increase autophagic turnover. And so, one technique is actually then to stimulate general autophagy by increasing the demand, by starvation, or by biochemical trickster 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 in Alzheimer’s disease or alpha-synuclein in Parkinson’s disease. So, basically, the clearing out of those protein aggregates obviously would play an important role not only in prevention but presumably also, to some degree, in help with the treatment. Of course, that’s you know, an speculation, but… 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/Parkin, and how they seem to be important for Parkinson’s disease Is that accurate? Yeah. So the PINK/Parkin pathway is one pathway among others, that allows for marking mitochondria that are damaged for destruction. And so, inhibition of this pathway leads to the accumulation of malfunctioning mitochondria with major consequences for the cell that harbours cells’ mitochondria because all of a sudden bioenergetic 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 Do we know the threshold for the stress threshold for, you know, activating autophagy, and when that pushes the mitochondria then to permeabilize and cause cell death? Like, where, for example, with Valter’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 got to die.” Or do we know? Well, autophagy in used in most cell types, while apoptosis is occurring in selected cell types So what Volter has been observing, if I remember well, is destruction of leukocytes, right, 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 or 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 phylogenetically conserved reaction in most cases of bacterial infection. And so this phenomenon leads to changes in the metabolism. Ketone in the production of ketone bodies, the reduction of glucose levels, presumably also induction of autophagy, and altogether these mechanisms avoid excessive inflammation that may be lethal. So Aslan Medzhitov 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 And 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 the accumulation of damage, you know, pieces of nucleic acid and pieces of chromatin and all sorts of things that can cause inflammation by having 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. But Well, it’s not exactly [inaudible] or 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 it 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 such a toxic effects Okay. Well, that’s good to know The other thing that is important to notice is that chloroquine and hydroxychloroquine, which are antimalarial 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. Only introduced into clinical trials, most 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 one relatively general mechanism may be that early during oncogenesis, the deletion of tumor suppressor genes or the activation of oncogenes 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 dived 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 chemotherapeutic 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 autophagic 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 cancer is not just a cell-autonomous disease It is more. It is not just that one cell has become wild term and has been accumulating genetic and epigenetic changes that make it selfish No, a cancer cell will only survive if it escapes from amino surveillance So the immune system, fortunately for us, is usually very efficient in eliminating aberrant sells, premalignant cells, and the initial cancer cells And actually, the inhibition of autophagy that occurs during early oncogenesis maybe also a way for the cancer cells to hide from the immune system And so, it is complex. It’s immunology, multiple different players come into action. Autophagy, for instance, is required for stressed 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. It is a dangerous signal. It is perceived by so-called purinergic receptors that are present, among other cell types, on leukocytes, and particularly myeloid 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 therapies efficient So, in contrast to the official dogma that has been en 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 chemotherapeutic 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 mimetics like spermidine, hydroxycitrate that you’ve done. Maybe can you kind of just briefly explain… I’ll start with spermidine What is spermidine? What does it do? Well, it was first start to explain what are these fasting mimetics is as you say and caloric restriction mimetics as we say So the CRM’s, caloric restriction mimetics, 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 acetyl-CoA and protein deacetylation resulting from the inhibition of Acetyl-CoA in the context of fasting. And caloric restriction mimetics, similar induced deacetylation reactions to stimulate autophagy. And this can be actually achieved in three different ways First, you simply inhibit the generation of Acetyl-CoA. The enzyme that generates Acetyl-CoA in our cells, the most important one for the cytosolic pool, is ATP citrate lyase and hydroxycitrate 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 acetyltransferases, some of them have been identified. Like, EP300 which appears extremely important for autophagy regulation, and specific inhibitors of EP300 such as spermidine and natural compound or C646 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 or enzymes that remove acetyl groups from proteins and cause hypoacetylation and autophagy. And one example that is well known is resveratrol contained in red wine that induces autophagy through this pathway. So, all these agents, caloric restriction mimetics, have different molecular targets, but it activate autophagy by a final common pathway The protein acetylation seems like that Yes, exactly Okay. So, with some of the major ones that you’ve worked with, spermidine. I’ve read quite a bit about spermidine. I know it’s found in high concentrations in natto, the Japanese fermented soybean that doesn’t taste like great. But I’ve seen studies about aging, you know, giving it to even aging mice or something, can then extend their lifespan Is that true? So spermidine, to come to the source of spermidine is contained in the nuclei of all kind of cells. So, in the nucleoid of bacteria but also the nuclei from yeast cells or from plant cells or animal cells. So, all food items that contain nuclei cells, are actually containing spermidine Also, there are large variations in the content. So we have to know, on this spermidine, it also is volatile and accounts for the smell of sperm So, it is frequently found in food items that have some kind of smell like natto or durian fruit or fermented cheese, when it is generated from non pasteurized sources and very rich in bacteria and fungi that are contributing to the fermentation process, which is, of course, smelly cheese. And it’s also quite abundant in some vegetables and food where the 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 autophagy when it is taken up with fruit or with the drinking water when we take 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 a microbiome to increase its production of polyamines, including spermidine Through what? Probiotics? Or through Yes So you know what strains of bacteria… ? Yeah, there is 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 a limited time here, with the spermidine that’s been shown to… Was it spermidine or hydroxycitric, I think, that was shown to synergize with, like you were mentioning before, the chemotherapeutic Yeah, both of them actually Both? Okay So the mechanism is that when you combine chemotherapy with caloric restriction mimetics, all the caloric restriction mimetics that I mentioned, including spermidine and hydroxycitrate and resveratrol, will enhance the anti-cancer 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 mimetics 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 mimetics as it proves that the cellular 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 test whether or not this caloric restriction or fasting mimetics 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 mimetics. Like, if I were to just supplement with hydroxycitric, for example, or if there were spermidine supplement, and I was still eating a normal, you know, healthy diet, but not caloric restricted and not fasting Do you think that would be sufficient to induce autophagy? Well, I can respond for mice cell Okay How about in mice? that this is certainly the case. I don’t know about humans because we have no clinical studies in this field So in mice, it does? It does. And in mice, you actually can give a combination of high-fat diets, that usually would cause obesity, with spermidine to reduce weight gain through mechanisms that we don’t understand and 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 that can I eat fermented foods to increase that population and get more spermidine. You know, 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 working, I only have one meal per day So I have dinner with cheese and wine containing spermidine resveratrol. It’s an ideal combination because inhibiting the acetyltransferase and activating the deacetylase will have, of course, a synergistic effect Oh, you do? Well, if you can show this in mice and this is an excuse for me to

Yeah, wine and cheese go great together profit from a banquet combination. And I do also practice some fasting twice per year also 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 is 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 to 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 still subject myself to a 20-day water fast, but it’s cool to know that you do these five-day water fast, twice a year, and eating one meal a day Yeah, and doing exercise And doing exercise And when you’re eating… Sorry, throughout the 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 a drinking water continuously And we could show that this was magnificently inducing autophagy Like caffeine independent fasting Right, totally independent caffeine, so just the polyphenols And this was like, I think, I read your study, it 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 neurodegenerative diseases are actually reduced in heavy coffee drinkers as an independent link between lifestyle and pathology. So, of course, it’s an association that obviously can be criticized because it’s just a study in which you find a statistical correlation It would be much more interesting to do a randomized clinical trial on coffee intake More interesting and more expensive, yeah Yeah Absolutely Great. So fasting, coffee, wine and cheese, one meal, exercise, you’re doing it all Awesome Well, really enjoyed this conversation, Guido. If people want to find you, you have a lab website, kroemerlab.com That’s K-R-O-E-M-E-R lab, 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 autophagy, both macro and micro from Dr. Guido Kroemer. Dr. Kroemer in my mind is the consummate expert in this field, and also is amazingly prolific with somewhere in the neighborhood of a thousand publications to his name. Quite the feat to say the least, and all of 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 FoundMyFitness by heading over to foundmyfitness.com/crowdsponsor. About to buy a solar-powered wood chipper or a gargantuan vat of gee? If you get them from Amazon, make sure to click the FoundMyFitness affiliate link first Any time you click our Amazon affiliate link, it actually makes sure the podcast will get a small cut for any orders you make in the next 24 hours. It literally doesn’t affect your transaction at all And the best part is you probably already shop at Amazon anyway No big deal, right? You can find the Amazon affiliate link, as well as other great ways to support the podcast, such as the Pay What You Can subscription, either direct or through Patreon, by heading over to foundmyfitness.com/crowdsponsor That’s foundmyfitness.com/ C-R-O-W-D-S-P-O-N-S-O-R Crowd Sponsor. Finally, are you a genetics geek? If you’ve done your 23andMe, and already have your raw genetic data available to you, you can run it through the genome analysis tool on my website at foundmyfitness.com/genetics That’s foundmyfitness.com/genetics. What’s that? You may ask. This tool is a way for

me to tell you about interesting single nucleotide polymorphisms, that’s SNIPs for short, and to quickly check your raw data for them Maybe you’ve heard me talk about a polymorphism that affects how you metabolize saturated fat or vitamin D or any number of other polymorphisms that I’ve talked about that usually share the common theme of being diet or lifestyle modifiable. Since I’m still learning, my report is still learning, and I’ll be adding more and more interesting genetic polymorphisms to the report as time goes on. Definitely worth checking out if you have your 23andMe data or, in the near future, ancestry.com or any of the other genetic providers We’ll be adding those soon Learn more by heading over to foundmyfitness.com/genetics Thanks so much for listening and I’ll catch you next time