I want to thank adizero for giving me this opportunity to come here and interact with experts in the field and one good thing about being one of the last speakers is the speakers before me I've done most of the job so I probably have to just go very quickly I was joking with somebody in the afternoon that I should just pack my bag and go to the airport because they've spoken for me but a few things which I want to emphasize which is some of the work we've done on the gut but also outside of God like I said a lot of things about bitrate sauce as we said we do know that bitrate it's a product of fiber fermentation but it also can be produced endogenously in fact it's one of the things that contributes to the smell of body odor so if you see somebody that has a very strong body odor maybe did actually buy a lot of Italy so it might not be a bad thing after all considering all the good things we've said about BT rates today it's also been said that bitrate can be produced by several bacterial groups that have the various enzymes mostly from a subtract acetylcholine that that has been discussed very well today already but there's a lot of interest about butyrate being applied not only in nutritional research but a neuroscience research as well I'm going to be presenting some data on that as far as animal nutrition is concerned bitrate is being supplied as sodium bitrate but also as tributary as also as tributary which can be broken down into three bitrate units so bit rate we can get supplement bitrate for so many reasons also in India frame forms and you know butyrate is very important as far as God health is concerned hypocrite is said all diseases all diseases originates in the God and that just shows us clearly the role of the God as an interface for health and disease but also for for performance okay because the GI tract is integrated for regulation of various body functions and if you look at all these functions butyrate as been shown to interact with virtually all these functions okay so bitrate is a very key molecule in regulating many of these functions okay if we know we've talked about it today already is an energy source okay it's implicated in obesity regulation of show some data especially in a rodent model in immune system regulation in cancer and of course in brain function and the question people always ask is do we get enough bit rate in circulation to justify some of the attributes that we are claiming the bit rate is responsible for and the question is yeah we may not get the same level of bit rate that we see in the gut in peripheral circulation but this bit rate is in circulation okay this particular work is a human study and what they have done is to feed different kinds of fiber to people and then they measured the amount of a bit rate okay in circulation so in whether you feed barley bread or or white bread it's associated with increased level of bitrate and circulation in humans and that's also especially with lower level of glucose and low level of low circulating concentration of insulin so clearly you can pick up increase butyrate in circulation when you take fermentable fiber even humans okay so bit rate responds to diet even in humans so it's not just that his level is increasing they got by even peripheral circulation so he must be doing something if it's in peripheral circulation okay and one of the things he did was to reduce the level of inflammatory cytokines such as intelligence 6 and TNF alpha so clearly bitter easily we reduce peripheral circulating inflammatory cytokines even humans okay but but neatly that we know it's an expert on fiber also showed that if you give RIE to pick and you measure potent mesenteric artery blood you also have increased circulating bit rate concentration so the same thing I humans and pigs of coursing in chickens and other animals that eat a lot of fermentable fiber ok but like I said we do know that bit reacts first in the gut because the cells of the God the epithelial cells have the first tab ok at the bee tree that is being produced especially in the colon so most of the effects of bee tree there have been characterized have been in the gut when it serves as energy source helping you know an improved very same maturation of the tight junctions but also in immune response as well in Trinity mewn system ok so I'm going to be talking briefly on some of the things that happen in the gut in the nervous system and peripheral tissues as well so there's only 3 things I'll be I'll be talking about today so let's look at some of the effects in the God and it will repeat some of the things that have been said today but a few instances I'll be mentioning some things that have not been emphasized and in this particular paper I added for my PhD student I use the Ipek j2 cells these are porcine intestinal epithelial cells to try to study some of the mechanisms of butyrate in that cell model again in trying to understand very detailed action I'll since and this is very appropriate for this meeting because we're trying to understand the mechanism of butyrate effect again some of the things have been mentioned earlier this morning but we do know that maintaining a proper tight Junction is very important for animals again when you compromise the that tight Junction you break up the you mess up the integrity then you allow a lot of antigens or microorganisms and toxins to pass through into the body and so maintaining those tight Junction is very very important and that was why if we focus on that in this in this particular study most of the tight junctions that we focus on are the cloudy's or the occluding again that they help to maintain that tight Junction preventing a lot of perhaps cellular flocks of toxins and things that may be dangerous for the body if they get through into circulation so the question we asked was can butyrate protect Ipek cells or these cells from LPS in this damage on tight Junction expression one thing we've emphasized today at this meeting is that maybe on the condition of stress okay what is inflammatory stress or even temperature stress maybe that's when you see a beneficial effect of Batory and that was why we use this model of stress to see if bitter it can can help mitigate that okay and we saw when we look at the expression of some cytokines especially in turkey 8 we saw a very slight but significant induction of that in the presence of vitriol you consider level is very small but this is not too surprising because we know that germ-free animals actually have impaired immune response okay so and then when you when you when you colonize those germ-free animals with with with bacteria with their you have the enhanced immune response so betrayed may be helping to train the immune immune system as well okay not to be you know inflammatory but to make it very effective okay and and then we began to look at the interested a parasail eula optic you see the co-culture system and we measured the optic of this fixie dextran which is such a big molecule it cannot be taken via the transcellular pathway cells can absorb it it can only go through the para cellular pathway and we saw that with with lipopolysaccharide again indicating or represented bacterial infection or activation of Tola receptor you see increased 50 dextran transport which is suppressed by by butyrate so clearly betrayed can help suppress para cellular transport of bacteria that is not supposed to go through and then we began to probe the molecules ok the proteins the tie Junction proteins that involved and we we saw that with bacterial polysaccharide you have suppression of tight Junction proteins like clothing tree and cladding for but betrayed especially at the 1 millimolar concentration reversed this so pressure so happy to preserve tight junction protein expression but this effect was selective in that we saw it with cloud nutrient calling for but not with the one so it's not a generalized just you know on regulated effect this shows that b2 is actually targeting specific pathways to regulate the expression of these of these genes so again it's betrayed the title of our talk today you know it's a message as a signaling molecule okay so that this kind of results confirms that the bit rate target specific pathways okay and for for bacterial endotoxin to activate inflammation they have to bind to totally receptor for in particular for gram-negative bacteria what we saw was that with bit rate you have decreased expression of to lie receptor for again that might explain some of the anti-inflammatory effect of bit rate okay decreasing the expression of this receptor and so that that ligand receptor interaction is reduced and then we began to look at the mechanism for protein synthesis okay if bit oracle effect this mechanism as well we do know that our tie junction proteins which we measured they were they were increased the question is can be T rate also regulate protein synthesis machinery and that's exactly what we saw I don't have a pointer here but clearly activation of a KT by insulin stimulates protein synthesis okay that's our protein synthesis is stimulated so that's our protein synthesis a stimulated when you activate a KT and then this is the system that leads to stimulation of that protein synthesis initiation complex you have these four EBP for eukaryotic binding protein and we could see that this Lippo polysaccharide suppress the activation of that protein by phosphorylation and bit rate reversed okay bit rate increased the phosphorylation of a KT the ratting the bit rate did was to increase the phosphorylation of these four BP okay again first increase phosphorylation is associated with activation of protein synthesis so butyrate activated protein synthesis so that and that was partly responsible for why betrayal restored the expression of Claudine for and cloudy three okay so again more insight into the mechanism of betrayed at the cellular level but beauty rates also a source of energy and ATP is the energy currency of the cell and what we saw was the bit rate also increased inside intracellular concentration of bit rate and is in this epithelial cell so bit rate is working via different mechanisms increasing the energy level of the cell then regulating inflammation increasing our protein synthesis of tight Junction proteins to ultimately enhance integrity of the tight junctions okay so that is the summary of what we how we think bit rate is acting you know to suppress inflammation cytokine the other ones which addition was interleukin 6 and TNF Ave which world but clearly deregulated but bit rate is increasing aka signaling helping to increase protein synthesis happy to operate away maintain the integrity of the tight junctions so I but I fell a bit rate in the brain like I said Beauty rate exist in a peripheral circulation okay we do know that of course that amount is not as high as in the gut but it is there okay it is there evil in the brain you can find bit rate even very small amount and that's why a lot of labs all over the world are trying to investigate whether Beauty rate is implicated in this god brain axis ok the communication between the gut and the brain and clearly this is an area with a lot of of with a lot of interest and how could bitter rate be implicated you know play a role in this God brain access communication well we know that betrayed can lead to secretion of things like pyy like glp-1 he was mentioned earlier this morning those those hormones act as endocrine hormones okay they are in distant places whether than where they are produced in the brain pyy suppresses appetite for instance but betray doesn't have to just release endocrine hormones it can actually stimulate virgos nerve dependent activation of the brain as well and we do know it can regulate the immune system there's a lot of data on that but also it can act as a signaling molecule in the brain in other words it binding to its receptor in the brain or audience other peripheral tissues it can act via that mechanism mechanism away so multiple mechanisms could explain why betrayed to activate or regulate that god brain access communication okay that's evidence that butyrate may be able to cross this is the blood-brain barrier okay that's evidence that yes that could actually happen and that's because betray and this is a dose dependent increasing glia neuron out there these are macrophages in the brain nuclei histone acetylation okay with higher-level betrayed we know that bitter is a histone deacetylase inhibitor okay that's one of the mechanism of betray so so betrayed actually you know little higher level acetylation in the brain even though it's given orally okay so that means is making its way into the brain to affect to affect the level of saturation in the brain and as shown from studies that Karolinska Institute it was shown clearly that germ-free mice have decreased increased permeability the blood-brain barrier okay and when you give betrayed you restore that blood-brain barrier so clearly bit rate is also linked with maintenance of blood-brain barrier inner integrity okay so clearly betray can act in the brain okay however the interesting thing is that betrayed does not necessarily need to enter the brain okay but can also indirectly influence processes in the brain in order to attack may affect brain function okay doesn't have to make it way to the brain but even hand distally even from the god leading to changes in the brain okay again making such as release of serotonin okay i can can be bitter II can lead to serotonin production and then we know what serotonin does it can activate vagus nerve via ds5 hd3 receptor of course vagus nerve we know that can affect the brain I can also stimulate people away and glp-1 production from internal endocrine cells so again and distally and yet affect the brain for instance with bit rate and acetate you have increased expression of the gene trip to tryptophan hydroxylase one that leads to serotonin production yeah you can see clearly betrayed leads to increased expression of the gene and like I mentioned the vagus nerve can can go straight to the brain to affect processes in the brain so a bit rate stimulates dialed nerve it can will to affect distally in the brain and like the the other thing the bit rate does is the regulation of inflammation okay this again microglia there there are the macrophages in the brain and it's shown that germ-free mice they are an immature are less active microglia okay so there the cell the immune system are not active they they are immature and this is normalized by adding a short chain fatty acid cocktail consisting of acetate propionate and butyrate to the drinking water okay so bitter helps to normalize microglia activation stay on maturity but butyrate certain fires they not only inhibit microglia but we do know that this support precise tuning to ensure necessary functioning on that non inflammatory condition and that that is what we need to understand more about Batory how it affects this precise tuning of the immune system so that is well prepared well activated but we learn a lot of these from germ-free mice experiments but the other thing that there's a lab labs around the world are trying to study is this concept of inflammation okay which is aging the results from inflammation okay because we do know that inflammation affects so many organs and we know that butyrate because of its anti-inflammatory effect you know in brain resident macrophages can reduce this nf-kb signaling induce apoptosis and niro protection happy to you know dampen excessive inflammation in the brain and again derrida helps to reverse aging or slow down the aging aging process this this particular paper and i know we've talked about t-rex this morning as well it shows that this regulatory t cells which are the you know they help to dampen immune activation they express force Fox p3 and we sodium betrayed you have an increase in their expression you have an increase in this force Petri expressing cells so again indicated that bit rate and aunt I cannot aunt I can have anti-inflammatory effect by regulating this dreg differentiation a lot of work of my colleagues at University of Illinois looking at how bateria can regulate inflammation in the brain both of these were done in the mouse model but clearly what they have shown in microglia again these are macrophages or in the hippocampus is specific section of the brain if you look at the expression of interleukin-1 beta ok very Pro inflammatory inflammatory gene again with sodium bit rate in the presence of LPS sodium betrayed leads to reduction in those in those in that inflammatory gene but you can see that these are the age eight miles dawa oh they had more interleukin-1 beta and B to reverse that I also did that in adult mice but most significantly in the in the old miles something in the epoch ampoules reduced level of interleukin-1 beta in age eight mind so bit rate you know can be a source of reversing the aging in the brain okay as really as expressed in this model in the mice and that's why there's a lot of interest in that some potential mechanism I did mention about a KT in the epithelial cell system in this particular model they also they used vascular occlusion okay to cause dementia in mice so vascular dementia okay again another form of brain disease and what you see is that brain-derived neurotropic factor is increased where you add betrayed okay these arbitrary producing bacteria now so betray producing bacteria increased the expression of that brain derived neurotrophic factor increased the expression of forceful akt okay which which I also mentioned with epithelial cell and so clearly clearly betrayed can target brain or brain cells and this this bacteria of course Clostridium Bertram can measure the measure the amount of bit rate in the brain it's very small but compared to the the control which were not given this bacteria there was slightly increased expression of a level of bit rate in the brain so clearly this material topic bacteria arbitral Jennie but you know clearly affecting the amount of bit rate in the brain and as having a positive effect on brain function under this vascular occlusion model okay we just concluded this experiment in pigs where we fed pigs inulin and cellulose and at the end of a four-week study we measure the expression of interleukin-1 beta and superoxide dismutase in the epithalamus of the brain of course we saw a reduction in the abundance of interleukin-1 beta compared to cellulose which is poorly fermented but also increased expression of superoxide dismutase in only fed pigs potentially via a bit rate mediated effect okay so clearly this process is also applicable in the pig model as well perhaps in the chicken as well okay but but before I go to a fellow bit rate another peripheral tissues like muscle adipose tissue we do know that for instance when you regulate the expression of Cerro Tony or the level of serotonin okay this is associated with depression in humans but in lifestyle since it's a livestock livestock conference in a low level of serotonin is miageta which increased aggression in anonymous okay so beautiful for instance increasing the expression the level of serotonin can help dampen or reduce the the level of aggression in animals and there's a lot of work in this areas where so we we talk about a peachy case maybe maybe they're happy you know we we may not be - turbo we can tell that they're better if they are less aggressive maybe by increasing the level of serotonin production by giving them a bit rate of betrayal genic bacteria but work has also been done in peripheral tissues and the first one in mice was what was done to study further betrayed on insulin sensitivity and energy expenditure and clearly it was shown that betrayal would increase the body director temperature of the mice but also increase the amount of brown fat or do you consider the size of the cells here these are fat cells the control on the high-fat diet well a large do with betrayed it was smaller so clearly betrayed is programming the fat tissue and we we talked about bitterly being a ligand for papers one of the effects of that is increased people gamma co-activator that we can see in this particular western blood so clearly betrayed stimulated metabolism okay and the consequence of that is the reduced weight gain of the of the abdomen eyes okay some of the work I've done I've used a commercial but also a biomedical Pig model called the also by pigs and for those from Spain is a causing of the Iberia pigs the Spanish brought them to the US about three hundred years ago they put them on the island of the Oba Island so we and they are very they have metabolic syndrome on the high-fat diets and some of the work with though we investigated in fact of in early and clearly what we saw that it only reduce the level of adiposity in this in these pigs but also increased expression of genes related of oxidation again it agrees with findings in mice perhaps the same thing is applicable in humans as well some of the other you know potential impact of butyrate comes from a well study we deal with braces times time so amylose versus amylopectin con amylose is highly fermentable and what we did was to look at the quality of milk for instance okay on the high amylose scone you have increased milk fat percentage you have increased milk total solids okay so again youuuu pigs are very low bit rate in make concentration but that betrayed can actually serve as a precursor okay for things that going to make arrived the fatty acids or some other things we can enrich the the energy value of the milk this yeah so clearly a lot of work needs to be done on feeding bit rate to gestating sows okay we just hug there was a question about at the bitrate in eggs okay so again utilized a histone deacetylase inhibitor and that is associated with epigenetic regulation of gene expression okay so maybe again the earlier we feed is betrayed in agreement with what we said today the earlier we expose animals to be tree the more effect we'll see and that's clearly shown in even regulation of milk composition but some of the work we've also done directly and in fact cells again trying to understand the mechanism is to see what happens in the in the cell as far as differentiation is concerned again when when fast cells differentiate they go from flat cells to to round and lipid fuel cells a lot of the genes that are involved in incorrect differentiation you know appropriate differentiation why induced in the presence of bitter aid especially people gamma you know highly significantly induced again in this in these cells but we were also interested in the expression of adiponectin adiponectin a protein that stimulates or sensitizes to insulin so insulin sensitivity increased energy expenditure with beauty we saw that there was increased adiponectin in those so happy to promote insulin sensitivity again could be a mechanism by which betrayed could oppose a type type 2 diabetes for instance and again in it to increase the phosphorylation of akt in this in this cell so it did did it in the in epithelial cells we saw it did that in the brain but clearly also did it in adipose I so activation of a Katie you know phosphorylation of a Katie appears to be a common mechanism of butyrate in different tissue types and of course it leads to increased optic of of glucose again helping to prevent insulin resistance by increasing insulin sensitivity again clearly happy to incorporate lipids into the cell regulating genes that are involved in the lipid synthesis pathway but also helping promoting healthy adipocyte differentiation okay because what happens on the sudden circumstances you have impaired adipocyte differentiation and obliterate helps to stimulate that and healthy cells well differentiated cells are sensitive to insulin but we also look at what betrayed does to release of lipids okay to release of lipids again very complicated pathway which I am NOT going to go into too much detail but we know that bit rate binds to these receptors FFA are 2 3 and of course GPR 109a and this is pathways associated with relation of release of lipids one of the problems in the Brazilian diabetes is excessive release of lipids into circulation which makes people this lipid emic they have too much lipid in circulation and a lipid goes into liver people there was a question about fatty liver in chickens but clearly butyrate can help promote oxidation or the storage of lipid in adipose tissue where I should be long helping to beat with more bit rate you have reduced release of blister or a marker of lipolysis you have that but we think that affect my becoming through FFA are three because butyrate led to increased expression of FFA our tree and fat cells but delegated FFA are two so differential regulation of these of these receptors so if we if we look at what butyrate does in in adipose tissue we know that it enhances insulin sensitivity perhaps by because it leads to increased expression of adiponectin but that could actually be because it's a histone deacetylase inhibitor which is associated with increased gene expression so within bitter air could be leading to increased adiponectin via histone deacetylase inhibition but eventually leads to suppression of lipolysis increased glucose uptake increased triglyceride synthesis increased with de novo synthesis helping to enhance insulin sensitivity in adipose science so again the level of butyrate that we see in circulation may not be as high as what you see in the in the hindgut but I do believe it doesn't necessarily need to be the high okay now you can bind to this receptor okay and activate this gene expression that we were talking about we just haven't studied or understood the system well enough to fully characterize it and I think there's a lot more work that needs to be done in this area against is talking about the peg we we saw increased activation of this PDC one in primary adipocyte in a muscle explains as well again this is an agreement with increased oxidation fatty acid oxidation by in vitro supplemented sales and in this particular study where we fed piglets bitterly you get point three percent betrayed orally given okay this by or agave at one by one pig pile by one by one we saw increased a bit body weight at their 84 in those in those piglets clearly one of the things we've talked about these conferences also know how early should you give bit rate in this case we gave them even after day three of birth you know so after they have taken colostrum we began to supplement them with bit rate orally and we saw the effect and their body weight when we measure it at a date for so clearly very early on you see some benefits so to put everything in perspective the things I've talked about today we do see that booty rate coming from fiber or assistance that all supplemented ok all supplemented can lead to in in I mean actually with resistant starch can lead to increase microbial diversity have a lot of data that which have no able to present today but bit rate can help serve as energy source for muscle and you know her program adipose tissue for increased oxidation but more importantly can stimulate protein synthesis helping to repair that tight Junction okay happy to repair the tight Junction perhaps by increasing also helping in adipose tissue increasing adiponectin and helping to enhance insulin sensitivity and and glucose uptake so it's it's you know producing the God but it has effect in peripheral tissues as well so bitter rate at okay to under God we know that also connects the gut to the brain okay via the gut brain access you know either through the vagus nerve or even stimulation of neuro peptides and Sarah Tony can directly regulate what is happening in the brain of course as I've shown there I'd also regulate peripheral tissues muscle and adipose tissue which is where I've done a lot of the of the work but it definitely needs to promotion of integrity of the tight Junction proteins stimulates new peptides and hormones stimulates metabolism suppresses inflammation at the entry level beta and that suitable yes we know the bitterest of places suppresses inflammation perhaps acting as a histone deacetylase inhibitor and as a ligand for this GPR 4143 or also called F F F 2 and F F F 3 and GPR 109 so multiple mechanisms can explain bit rate effects I just wanted to leave some of these questions or practical considerations for us we've talked about some of them even during the workshop we had this afternoon question on age when should we supplement butyrate you know easy it's between fare related to the maturity or behind god these are things we need to really determine because you know to really promote a consistent result of using battery we need to understand its mechanism but also when and how best and what level to use especially related to the age of maturity and behind gout level of stress the stress level effect bit rate effect we know what stress does to animals disease wina stress about the level of fiber in the diet the type of fiber whatever used enzyme or not holidays and things like that probiotic use do they interfere with bitrate effectiveness even antibiotic use of course you can you know affect the the percentage of some of these microbes but zinc zinc leads to direct programming direct of microbial community structure how does that Febuary production we don't know and of course several alternatives to antibiotics that are out there we still have a lot to learn about our they affect betray production or even the environment even the housing design now all these can affect local local bacterial population you know and oldest and eventually affect betray that is being produced what is the optimal concentration and form coated versus on coated what a solute was also mixed in feed pigs versus poetry you know this they still learn of questions that we need to answer and I believe trying to understand some of the fundamental mechanism would be one way to go in try to answer some of these questions I've raised here so thank you so much for your attention [Music]