 I'm going to start with the end and then show you the data that supports the overall scheme. And that is what I'm going to try and convince you is that intestinal microbes play a contributory role to the development of cardiovascular disease. What our group has found is that diets that were familiar or food sources that were familiar with increased risk of cardiovascular disease such as those abundant in fat and cholesterol and illustrated on the left there are also associated with increased levels of trimethylamine containing nutrients such as phosphorylcholine or choline shown in the bottom left and also carnitine shown in the mid-left. And what I'm going to show you is that gut flora contain enzymes that will actually cleave the trimethylamine moiety off of the nutrient generating TMA or trimethylamine. This is a gas at body temperatures. We have within our livers a cluster of enzymes called Flavin monoxygenases which are capable of then very efficiently oxidizing TMA into what we are the field used to think of as purely a nitrogenous waste product TMAO or trimethylamine an oxide. But what I hope to show you is that this actual compound has biological activity and can actually alter cholesterol and sterile metabolism at multiple different compartments and not only is it associated with cardiovascular disease outcomes in humans but in animal models will actually facilitate or accelerate the development of atherosclerosis suggesting that gut flora through this axis can actually perturb the development of cardiovascular disease. There are also a couple other take home messages that I'd like to try and discuss today. That is the first is that if you think about things coming from either genetics or environment, how we perceive that environment is influenced through the filters that are there. Our largest environmental exposure is what we eat. We literally consume kilogram quantities of foreign material if you think of food as a foreign object and it is through this filter of the intestinal microbiome and if you have different compositions and you're making slightly different metabolites at different levels from one person to another the filters can actually influence phenotype in that way. The second thing that I'd like to also suggest is that we should think of the intestinal microbiome as an endocrine organ. An endocrine organ is of course something that makes a hormone and if a hormone by definition is a biologically active compound that will diffuse in the circulation and act at a distant site. I'm going to give you examples of how compounds are being generated in the intestines and diffusing and then acting at the site of let's say a macrophage in the artery wall or a hepatocyte in the liver or an enterocyte in the intestine and influencing the biology. It's a quite plastic organ to be sure and it changes depending upon what the chronic dietary exposure is and what the acute dietary intake is but nonetheless it's functioning like an endocrine organ and then lastly, well I'm not going to show you data on this. I think what is exciting about this whole field is that this is a druggable target. If we start to understand the chemical braille of the different pathways of what the microbes are generating and how they're biologically active then these become targets for pharmacologic intervention through inhibitors or agonists. Not an antibiotic but an inhibitor and so perhaps in our future we'll look at our medicine cabinet and see let's say a statin and say oh that's our homo sapien enzyme inhibitor for blocking cholesterol synthesis and right next to it will be a tablet for blocking a specific enzyme in bacteria that doesn't kill it but actually prevents the formation of compounds that might be linked to cardiometabolic diseases or obesity or whatever or maybe even our behavior as we just recently heard. Okay so how did we actually get into this? I am not a microbiologist and I am not actually my history has not been working on microbiome. Instead I'm actually a chemist and run the mass spectrometry facility at my institution and we started with an unbiased metabolomic screening study. We were using a cohort for which I'm the PI it's called Gene Bank has over 10,000 subjects for whom we've collected their blood and then followed longitudinally over time and so from this cohort we identified a small subset who went on and experienced heart attack, stroke, and death and age and gender matched control subjects and then within each person's plasma in this small case control cohort we asked can we find small molecules that are associated with cardiovascular disease risk. So we did this in a learning set and we did it in a validation cohort. In the end we came up with a small subset of compounds which actually seem to reproducibly associate with cardiovascular disease. Well the surprising finding was is that some of these compounds fell into a common pathway and that was involved in phosphatidicoline metabolism. Now what the three main candidates in our first paper were choline butane which is the oxidation product of choline and then this small molecule that I call TMAO or trimethylamine an oxide which is shown in the structures at the bottom and so with this association study suggested is that that at least was an association between multiple metabolites in phosphatidicoline metabolism and cardiovascular disease risk. So we first wanted to actually look at mechanistic studies to see was there a causal link. So I'm sorry I'm switching the order of my slides here. So the first thing we actually wanted to also confirm was if one looks at phosphatidicoline metabolism the classic pathway that how all of us digest phosphatidicoline is we have lipases that cleave off the fatty acids and then glycerophosphicoline and the fatty acids are absorbed in our intestines. However there was a suggestion that TMA and TMAO might be ultimately generated via gut microbes because certain bacteria could cleave choline and forming TMA but this really hadn't been shown especially for phosphatidicoline to involve intestinal microbes. So we received germ-free animals from Teconic and then did the experiment where we took the germ-free animals and immediately upon opening the micro isolators, gavaged them with isotope labeled phosphatidicoline or egg yolk phosphatidicoline and then monitored in the plasma the appearance of the different isotopologs of the metabolites of phosphatidicoline such as choline, butane, and TMAO and as can be seen on the left what was found is that in the germ-free animals following ingestion of phosphatidicoline no TMAO was generated or appeared in the plasma over time but in contrast if you took the same germ-free animals and now we put them in conventional cages and two weeks later they've now assumed intestinal microbial communities they've become conventionalized and now when we gavaged them with phosphatidicoline TMAO rapidly appeared in the plasma following ingestion of the phosphatidicoline showing that TMAO was indeed formed in an obligatory way or it required the role of gut flora to be generated or gut microbiota. Now you've seen this slide shown I guess throughout this just earlier today but more recently Wilson Tang in our group kind of tried to extend these studies to humans and what we looked at we're using egg yolk or two hard-boiled eggs as the dietary source of phosphatidicoline in subjects and at baseline we would give them the hard-boiled eggs and then monitor for the appearance of this metabolite TMAO in the plasma and in the urine. We also gave them a capsule of synthetic isotope labeled phosphatidicoline where the N-methyl hydrogens were now deuteron so we could trace the and so for certain that the metabolite that we were measuring TMAO came from phosphatidicoline because there's many things inside of a hard-boiled egg besides phosphatidicoline and what we're able to show is that at baseline you could readily detect TMAO if this is six hours following ingestion of two hard-boiled eggs. If then the these are healthy volunteers replaced on a cocktail of oral antibiotics and then came back five or six days later and repeated the challenge the formation of the TMAO was virtually completely eliminated and so none was detected in both the plasma and in the urine and then after cessation of the antibiotics and going home and coming back month later repeated the challenge and now you see it again so this showed an obligatory role for intestinal microbes in the formation of this metabolite that we're measuring in the blood. Now why do we think that's important? So first of all if we actually measured the metabolite in the plasma of subjects we saw that it was strikingly associated with cardiovascular risk. What is shown here is an independent study of over 1,800 subjects where we have the baseline plasma level and we're looking at cardiovascular disease this is the risk is shown on the y-axis the odds of having cardiovascular disease after adjustment for traditional risk factors such as those in the Framingham risk factor or formula age gender diabetes hypertension smoking LDL HDL and also has the addition of other labs that are currently used for evaluating cardiac risk such as triglyceride and CRP and an estimate of renal function and if you focus your attention on the center graph what you can see is that there's a striking distribution or association between the plasma level of TMAO and the likelihood of risk of having cardiovascular disease in subjects so the line in the middle is the odd ratio and the dotted lines are the 95% confidence interval and there's quite a steep association and if one were to look at LDL cholesterol for example it would be a much more horizontal line in this cohort so this is still just associative data but nonetheless it's compelling because that was independent and above on top of traditional risk factors more recently we've extended this to an alternative independent cohort for whom we followed over time who went on to develop heart attack stroke or death and what we see is that baseline levels are predicting future risks of myocardial infarction stroke and death if one looks at the composite of this of major adverse cardiac events that's in the bottom left-hand corner the fourth quartile so the top 1,000 subjects compared to the bottom 1,000 the first quartile is the index group for comparison they have about a fourfold increased likelihood of experiencing an event in the next three-year period and the line represents the 95% confidence interval and shown on the right are cap and viral survival plot showing that your plasma TMAO level is a good independent predictor of prospective survival so to take this into a more mechanistic realm we started doing animal model studies and at first we actually fed mice phosphatidylcholine and actually saw that they got accelerated atherosclerosis but they also gained more weight they were getting more calories and there was the whole issue of the fatty acid composition maybe that was contributing to the disease so we instead started just giving choline in the diet now choline for animals for mammals has no calories we don't catabolize choline and generate calories from it and we also don't see changes in either the cholesterol or the lipoprotein or glucose or insulin levels in the animals that are on the choline diet but nonetheless what we found is that augmenting an atherosclerosis prone animals diet with choline led to accelerated atherosclerosis so shown in the open bars where the wild type conventionalized animals on a normal child diet on the control side and the choline represents a high choline diet which is approximately what would be a very high fat Western diet in terms of its choline content shown in the black bars are animals in which the intestinal microbial community was suppressed by a cocktail broad spectrum antibiotics the TMAO levels in plasma which are shown below in red were suppressed to near zero levels throughout the course of the study which is about a 20 week duration and what was seen is that the increase in atherosclerotic plaque here measured by macrophage of the major cell type in the in the macrophage in the aorta but also this is done has been done by cholesterol content or already staining that the diet induced atherosclerosis was inhibited in the animals in which the gut flora was suppressed in TMAO was not generated now importantly what was also found and I'm sorry I don't have it here what was also found is that if we fed TMAO directly to the animals and bypassed the microbes that alone was sufficient to increase an augment accelerated atherosclerosis in the mouse model now I'm working out the mechanism we started looking at a variety different places and if what was found is that the macrophage was accumulating cholesterol so we focused on cholesterol metabolism and so if one thinks of the the macrophage kind of shown in that ugly cell on the right in purple as a black box and you say it's accumulating cholesterol you can either have enhanced pathways for delivering cholesterol into the cell more cholesterol being synthesized in the cell are decreased removal of cholesterol from the cell you know increased flux in decreased flux out or more synthesis in the cell and so we kind of looked at it in this simplified black box approach and looked at candidates involved in cholesterol metabolism to try to work out the details of how was cholesterol accumulating in cells of the artery wall it turned out to be a little bit of a complex mechanism and we still don't know precisely all of the pathways that are involved but what was found was that there was both enhanced forward cholesterol transport and in particular on the macrophage there was up regulation of genes involved in recognition of modified forms of LDL such as scavenger receptor SRA1 and CD36 these were effects of directly of TMAO when fed to animals as well as when cultured with when incubated with cultured macrophages what was also observed is that there were changes in cholesterol and bile acid metabolism at the level of the hepatocyte and also the enterocyte so there's a substantial reduction in bile acid pool size and composition changes in the bile acid transport pathways such as CYPS 27a and CYPS 7a1 and then also changes in enterocyte cholesterol and bile acid transporters as well the net effect is shown on the top right hand corner there's enhanced forward cholesterol transport and if one actually directly measured the reverse cholesterol transport pathway using methods that were first developed by Dan Rader and colleagues at University of Pennsylvania we saw that there was actually about a 30% reduction in reverse cholesterol transport that was mediated by either TMAO directly in animals that were fed the TMAO or in animals that were fed the precursor choline when they had intact flora but if you suppressed the flora and blocked the TMAO formation you no longer saw the changes in cholesterol and sterol metabolism or in bile acid synthesis that was being observed now I so far kind of showed you data that was mostly done with choline but more recently we've actually started expanding these studies to alternative dietary nutrients that are similarly trimethylamine containing and so like carnitine was the one we're focusing on now the reason why we focused on carnitine is because there's substantial epidemiologic data that argues that red meat is associated with increased cardiovascular risks and in particular for example this recent study that came out by the Harvard nutrition group looking at both the health profession follow-up study and the nurses health study combined they have over almost three million follow-up years of information with almost 24,000 deaths followed with an average follow-up period of over 20 years and what was seen is that for each one portion increase per day in red meat amongst the individuals that were followed in this these cook these studies and it counts for somewhere between a 13 to a 20% increase in mortality over the course of the duration of follow-up which is as I said either a 20 or 28 year period depending on the two different studies and and by the way a portion size by the nutritionists it's always strikes me as a little bit funny that they're so small it's only three ounces is considered one portion of red meat I don't know about you but that seems to me like the snack that you eat before you have dinner as opposed to the real portion size so why with the interest in red meat well carnitine is a nutrient that's almost exclusively found in red meat certainly in meat and the structure is shown here and it has that same trimethylamine moiety on it and it actually derives its name from the Latin root word where carnivore comes from meaning flesh and that's because the chemists who first discovered carnitine structure over a century ago found that the the food substances that it was in were essentially a flesh and red meat in particular has high level and if you're wondering what red meat has the highest level kangaroo by the way has 50 fold higher level carnitine than beef so don't go out meet kangaroo patties if you want to try and cut down on your carnitine ingestion but in any way carnitine plays a role normally in fatty acid transport into mitochondria we make all the carnitine we eat from our diet it's not an essential amino acid it is made by lysine after and lysine being the single most abundant amino acid in both plant and animal proteins most individuals unless you have a genetic defect do not have carnitine deficiency or it's a very rare phenotype so we were interested in the same kind of story could carnitine generate TMAO and accelerate atherosclerosis just to speed up because I want to leave time for questions we use the germ-free animals and we're able to show that when you ingest or when animals ingest carnitine they in their germ-free they do not make TMAO but when they're convention lines they do we then wanted to translate this to human clinical studies Bob Koth a postdoc or I'm sorry is an MSTP in my lab this was his thesis research the carnitine story he did the study were actually we generated we used a literally a a grill that had that on the box cover and and used steak as our source of natural source of carnitine isotope labeled carnitine in a pill and did the same kind of experiment we're at visit one they get a carnitine challenge and over time measure the appearance and disappearance of the metabolites go on antibiotics suppress the intestinal flora etc so doing that same kind of experiment as we did with the phosphatidylcholine what we saw is that while they hadn't individuals have intact flora they readily generate TMAO from ingestion of carnitine but following suppression of the intestinal flora no TMAO is generated by ingesting carnitine suggesting that carnitine formation of TMAO has an obligatory requirement for gut flora as well so taking this to atherosclerosis animal models we use the APOE and all mouse model what we saw is there was about a two-fold increase in aortic root atherosclerosis and animals that were on a carnitine diet despite no changes in their cholesterol levels their weights or triglyceride levels if you suppress the intestinal flora and bring the TMA and TMAO levels down to near zero over the course of the duration of the study we saw and looking at the right hand panel no diet dependent increase in atherosclerosis now one of the intriguing findings that we saw during the course of these studies was that the animals that were on the chronic carnitine diet showed a tenfold increase in their synthetic capacity to make TMAO compared to normal chow mice this suggested that the chronic dietary exposure to carnitine had actually shifted the intestinal microbial composition to such that the microbes were now those that those that preferred carnitine as a substrate had become had a selective advantage and had grown more and now carnitine dependent conversion into TMA and TMAO was occurring more readily because of the shift in intestinal microbes and actually through you looking at the 16s ribosomal DNA of the feces or and also of the the sequel contents we saw that this had actually in fact happened in studies that we actually performed in collaboration with our U Penn colleagues such as Rick Bushman and Gary who are here we actually looked at omnivores versus vegetarian and vegans as well to see if this naturally occurring diversity in carnitine ingestion that is vegetarian and vegans have a very low carnitine ingestion rate compared to omnivores could we see a microbial shift if you will in the formation of TMA production in the subjects and this is data just from one omnivore who was characteristic or exemplary and one vegan who is an intrepid vegan who agreed to eat a steak as well as take the capsule with the methylated carnitine the deuterated carnitine and what was seen is that following ingestion of carnitine the vegan had an exceedingly low synthetic capacity to make TMAO compared to the omnivore and this was true with both the natural abundance as well as the isotope labeled precursor carnitine what's shown here are data with larger numbers that when we compared a larger number of vegetarians to omnivores that the omnivores had a higher level of TMAO in general and then shown on the right are for those who went through the full isotope labeled carnitine challenge there is a very dramatic difference in TMAO synthetic capacity or production rate following ingestion of carnitine seen in the vegans compared to the omnivores the vegans as was seen with the the other vegan who ate only the steak and capsule those who just had the capsule were very very poor at generating TMAO now when doing the microbial composition analysis of the from feces analysis what was seen is that there are multiple different taxonomic groupings of microbes that associated not only with the dietary pattern but also with the plasma TMAO level and shown in the red box here is an example of two different patterns the top one is illustrated as a lower proportion in vegetarian and vegans this particular taxa and associated with lower TMAO level and in contrast for example in the lack no spirit that's a proportion of a specific genus that's higher in the vegetarian and vegan and associated with lower TMAO and so the chronic dietary pattern had actually shifted the intestinal microbial composition of the subjects and this was associated with the altered TMAO level now to bring this back to humans and cardiovascular disease which is my main research interest we wanted to know how could we do this and it turns out that there have been many studies that suggests that individuals who eat more carnitine have higher plasma levels of carnitine and so we actually then one head and measured carnitine in over 2,500 subjects and asked does it track with cardiac risks and the answer was it did actually quite well if you adjusted for traditional risk factors it worked as good or better as LDL cholesterol or blood sugar or any of the current diagnostic tests that we use for and are associated with cardiac risk carnitine was independent and better if you will but what we also found is that in individuals if you then stratified by their TMAO level only those subjects who had a concurrent high TMAO level where this is above and below the median analysis so you needed to have a high carnitine and a high TMAO level to have increased cardiac risk if you have a high carnitine level but a low TMAO level that is let's say your microbiota composition was healthier you didn't you weren't a TMAO producer you're at low risk even though you had the high carnitine level presumably these were high carnitine ingesting subjects we don't we don't know the dietary patterns of these individuals unfortunately this was a study of over 2,500 subjects so to summarize I'd like to suggest that in addition to foods high in cholesterol and saturated fat there are other dietary nutrients that through their the action of gut microbes that is like choline and carnitine can generate small molecule metabolites that are biologically active acting at a distant site and influencing cholesterol and stereometabolism in the artery wall such as in the macrophage in the in the liver such as in the synthesis of bile acids and in the enterocyte in terms of cholesterol absorption and cholesterol and bile acid metabolism and collectively it's serving like a rheostat on the light switch you need cholesterol to have atherosclerosis just like you need electricity to have a light bulb turn on but you can have a rheostat and if you have a high TMAO level the dimmer switch is bright and at any given cholesterol level you have a more bigger chance of getting atherosclerosis if the dimmer switch is down you have a low TMAO level you have less chance of getting atherosclerosis now I'm going to end just by saying where else do we get carnitine well in addition to these foods it is a very common recent dietary supplement and not only in things that are shown here but also you know some of these energy drinks have an extraordinary amount of carnitine within them if you'll notice that it's the carnitine content and monster in particular is in front of the glucose so it's the most abundant additive that they have and it's one portion of a steak is approximately 160 to 180 milligrams so we're talking a lot of carnitine in the can so we were interested in asking this is the last piece of data this is unpublished if you're on a chronic vegan diet are you protected if you start taking carnitine supplements because actually many vegetarian and vegans to try to augment their carnitine will be on a daily carnitine supplement thinking that they're missing it because there are on a vegetarian or vegan diet and what's shown on the left are the carnitine challenges that were done at baseline or after one month or two month of daily carnitine supplementation and the vegans are that we've done so far shown on the left the omnivores on the top and the omnivores on the bottom and what can be seen and then on the right-hand side is just fasting plasma levels and that tells the story if you have chronic carnitine supplementation regardless of your diet you know we are walking tissue culture dishes and if you give a nutrient that the microbes like those that have the selective they get a selective advantage and now they're going to be more populous and grow and so actually chronic dieting carnitine exposure is changing the intestinal microbial composition and making the subjects more prone to making TMAO which we think is a pro-athrogenic phenotype so it suggests or makes us wonder if chronic carnitine exposure and things even like energy drinks might be a long-term adverse thing that certainly needs further studies in the future. I'll just end by thanking you for your attention and point out four main individuals in particular the four major papers that I've discussed were each co-first authors by individuals in my groups in Aang, Wang, Bob Koth and Wilson Tang, Brian Bennett working with you at UCLA with Jake Lucis was the author of the Selma tab paper and I also want to point out our colleagues at UPenn, Rick Bushman, Dr. Chen and Gary Wu as well thank you very much. Okay maybe one question while we're transitioning between speakers here we're running a little bit behind. Well here's one the over here the since a number of of lipids can be a source of the of trimethylamine that with the choline head group why is it that carnitine sticks out as being a substrate or is that the conclusion that you have or is that not the case in other words is it that making the trimethylamine is one thing but then there's some other ecology or indirect process related to L-carnitine which goes with the cardiovascular risk. Well we I think that any of the trimethylamine nutrients whether it's carnitine or phosphatylcholine or choline possibly butane, acetylcarnitine all of these can be converted into TMA and TMAO and so far in an animal model level all of them are associating with accelerated atherosclerosis so I don't think there's anything unique about carnitine I think we studied carnitine after our first study on choline so carnitine just has that trimethylamine group and is yet the gut flora can actually cleave it off and it has the same conserved group that's why we started looking at the carnitine as a so but the two most abundant trimethylamines in a Western diet are going to be carnitine and phosphatylcholine less than. Do you have a quick question? Excellent talk you measure you mentioned that carnitine can be bad if TMO levels are high but carnitine has a lot of benefit in certain diseases of interest there are diseases with low carnitine autism the work we're doing in working in other efforts to look at TMO levels in other disorders where carnitine metabolism is involved and you've made an excellent point which went for supplements what might be good for some disorder maybe very bad for another. We haven't looked at autism we actually after our paper came out I got a landslide of emails asking about acetylcarnitine because there apparently it's a common nutrient or supplement that's used with reported benefits for cognition and in various neurodegenerative disorders or dementia. We have been studying it all I can say is that we're looking at it from the cardiovascular standpoint not the dementia standpoint and I can say that you get accelerated or you get enhanced TMA and TMAO development by ingestion of acetylcarnitine whether that leads to accelerated atherosclerosis in an animal model we don't know the answer yet. But it's a good example that you know for some disorders it shows promise in specific studies as opposed to being a carp launch thing that everyone eats it's an excellent study. Thank you. Okay thank you I think we need to move on. Okay the next speaker is Christian Chabon from the University of Florida School of Medicine and he's going to be talking about the gut microbiome and colorectal cancer.