all right so this is going to be the outline of my talk I'm gonna introduce ray Doc's biology hopefully begin to introduce what the other speakers are talking about a little bit going to talk about mitochondria as the major source of an endogenous oxidative stress in cells talk a little bit about mitochondrial function and biochemistry associated with feed efficiency and I've got this one in a different color because a week ago Thursday I found a recent paper on reverse electron flow so I basically jettisoned a whole bunch of stuff out of the talk so I could introduce this because this is relatively new in mitochondrial biology proton leak is used it attenuates mitochondrial ross production some studies that we've done now showing that progesterone may be involved in feed efficiency a summary and then if I have time I've got what I'm putting down here is a mitochondrial surprise so rate ox biology so my perspective on antioxidants include a silent for many years was this basically glutathione was the center of the antioxidant worked universe but there I've gotten a greater appreciation and I've become a full fledged mitochondria so a little bit different view you have non enzymatic antioxidants this includes vitamin E vitamin C vitamin A uric acid and glutathione and in enzymatic antioxidants but in my view glutathione is still at the center of the table so if you look at this table you've got detoxification of formation of Epoque sides that are radicals by p450 metabolism there's radicals generated through inflammation there's radicals being generated in muscle differentiation minor Condor isn't the center because they're producing lots of radicals as a consequence or in addition to rate oxidative phosphorylation you can also have proc cyl and nitro sir radicals over here on the right are a bunch of cofactors that are used for many of the enzymatic a antioxidants so I've got those as forks knives and spoons metallo thymine is very important in sequestering ions you don't want free metal ions roaming around because this will cause oxidation so it's an antioxidant table with an oxidant feast but if they don't clean them all up then then you get into situation where you've got DNA damage protein oxidation lipid peroxidation a toughie gee my Tophet ji apoptosis etc you have to have a whole lotta ATP invested in and ordered to clean this up so it's a very expensive process so glutathione is at the center of a whole lot of radix coupling glutathione is in milli molar concentrations and virtually all cells and so it's a very central to this and I know that subsequent speakers are going to be talking about selenium with glutathione peroxidase so there's there's many of these radix coupled reactions and it should be recognized that glutathione is also very beneficial in regenerating vitamin E or took off Rob from oxidized form dehydroascorbic back to vitamin C so it's very central to too much of the antioxidant protection so this is electron micrograph of mitochondria the electron transport chain is on the inner mitochondrial membrane but the mitochondria it sort of gets a false sense of what the mitochondria look like so this is a scanning electron tomography and you get more of a sense of this 3-dimensional structure so these are the Christy rather than seeing just the nice little Christian electron micrograph there they're three-dimensional the endoplasmic reticulum surrounds the mitochondria so this facilitates a lot of the transport things in and out of the mitochondria and the mitochondria of sausage-shaped and they they extend throughout the cell and they're not static they don't just sit there in one place they divide they fuse and they can move around in the cells so this is kind of a going to be a quick overview of mitochondrial biology just noting this glutathione is synthesized in the cytosol and it's imported into the mitochondria the mitochondria can't make their own glutathione so it participates in the glutathione recycling reaction for deep antioxidant protection now on the top and the bottom of this or electron transport chains and I show it with the different complexes substrates can either enter in from succinate fadh2 int on the top or NADH linked substrates on the bottom these are multi protein complexes and so they they consist of nine nuclear encoded proteins as well as mitochondrial DNA encoded proteins and so they have to be constructed in in order to get optimal activity of the complexes then as oxidative phosphorylation occurs electrons move down the electron transport chain you get proton pumping across into the inner membrane space and then when the protons flow back through ATP synthase this is what generates the energy for ATP production but 80 ATP and ADP they have to shuttle in and out of the mitochondria and they do this with a combination of a couple of proteins it's a and T is adenine nucleotide translocase and then v-tach is voltage dependent activated channel what happens with oxidative phosphorylation is that electrons leak out of the electron transport chain they do you know valent reduction of oxygen to form superoxide and the superoxide can then form additional reactive oxygen species the white shark of the reactive but oxygen species is hydroxyl radical and a lot of it like metallo theanine and some of these other things the reason that they're there is to sequester iron and copper so you don't get hydroxyl radical formation one thing that also goes on in the mitochondria is a thing called proton leak and proton leak dissipates the membrane potential a higher membrane potential results in higher Ross production and so the proton leak it's a self-limiting system within the mitochondria to try to lower the amount of electrons that are leaking out if oxidation continues then you can have formation of oxidized glutathione the GS s G this gets to where you can have protein dive sulfides forming these are damaged so you end up with protein damaged so they're not doing what they need to be doing and then the reactive oxygen species they can cause damage specially hydroxyl radical it it nails anything that it bumps into but you can have oxidation going on of all the structures around it and then finally you get signal transduction so you can modulate gene expression through Ross production so this is this is a quick little paradigm we've been working on feed efficiency for some time so this was done in conjunction with Cobb mantris in a pedigree oil male broiler line so it's six weeks of age they took a hundred Birds put them in individual cages did individual phenotyping and then what we did is we took the top and ate and the bottom eight from this and this is basically what we've been using in our studies just to put this down and there's a big difference in feed efficiency so it's point eight in the hyphy deficient group and point six one and a loafie deficient group so very large difference in feed efficiency so this is oxidative phosphorylation electrons inner as I said from a malate or nadh linked or succinate and the electrons flow from complex 1 2 3 or complex 2 to 3 and this shuttling is carried out by Cohen's own Q and then it flows down until the it gets to reducing oxygen to water so that's in a quick summary along the way hydrogen ions gets pumped sets a proton motive force and that's what's driving ATP synthesis but what's electron leak basically electron leak is these it get fumbled that all of these proteins it's not a hundred percent that they're able to transmit the electrons down the transport chain so they get fumbled and when they do they react with oxygen to form superoxide that's converted to hydrogen peroxide by superoxide dismutase and formation of reactive oxygen species to magnify this or to locate where these defects are occurring you can put in chemical inhibitors rotenone is used to block complex one and then an ax minus an A or mix it dies off can block it complex three now in the when this started this started in the 70s and pretty much through that to thousands it was basically all Ross were bad they were causing damage and that was the end of the story and I'm gonna show you in a little bit that that's not always the case so this is a study we did this is in breast muscle mitochondria we had a basal condition no inhibitor or we treated isolated mitochondria of various inhibitors and so the low fee deficient which are in the open bars we saw increase in reactive oxygen species from complex one in complex 3 compared to the hyphy deficient phenotype so there were defects and electron transport in the loafie deficient mitochondria we did a number of tissues long way and so this is looking at basal radical production and in in breast muscle duodenum and liver and in all cases except in leg muscle mitochondria we saw higher basal radical production in the loafie deficient mitochondria the increased radical production resulted in increased protein carbonyl formation protein carbonyls are oxidized proteins so the first bar up there is for breast muscle mitochondria big difference in protein oxidation and then the others are other tissues all of them had higher protein oxidation in these homogeneous than in loafie deficient compared to - efficient so there's basically in a pervasive oxidative stress going on in the low feet efficient broilers this just a reminder all of these the electron transport chain they're all enzyme complexes and so you can measure these with various enzymatic acids this is just one example this was in droite no mitochondria activities were higher in high feat efficient for all of the complexes except for complex for in this particular case glutathione it's got a very important Thyle group in it and it helps stabilize complexes and so we looked at relationships between mitochondrial glutathione and activity and with complex 2 4 & 5 we had higher we had correlations between glutathione in the mitochondria and complex activity so it helps maintain the complex activity and then we found an inverse correlation between activity and protein carbonyl formation some more protein carbonyls lower activity of the enzyme complex when we looked across several tissues this is the the dotted line that's across there this is being compared that's the level set for high feet efficient and then we compared it to a percentage and low feet efficient in almost all cases complex activity was lower in low feet efficient compared to hide feet efficiently and so we think that that was possibly due to higher protein oxidation all right so now I want to get to this mitochondrial reverse electron flow and so this is what I found this paper we could go Thursday on mitochondrial reverse electron transport flow I knew this talk was coming up and I thought well I got to find out what the heck that is so I and also I had this reaction reverse what because the dogma had been that reverse electron flow was an artifact of in vitro preparations it didn't happen in the real world this paper is loaded with things where it's happening in the real world so this is showing this is a diagram showing forward electron transport this is how oxidation oxidation for elation is occurring and you and it's showing a certain amount of radical production going on at complex one this is reverse electron flow and what they're looking at it's sensed by the reduction state of coenzyme q and what happens is in this particular case electrons are flowing back from complex to to complex one and so it enhances radical production and so they say you get radical production occurring at two different sites in complex one this is going back we did this is intestinal mitochondria and we'd been looking at route ross production and when at the time that we had this and we had this data I thought this was could be an artifact showing the big increase with inhibition at complex two with the t TF a there was a big increase snuck the paper in it got accepted so it's like okay that was cool but whoa what I wish we would have done in this case is that if we had done this study with rotenone we could have we might have found and been able to conclusively say this was due to elect our verse electron flow but possibly in duardo mitochondria reverse electron flow could be occurring and we expected a big increase from complex 3 when we were feeding these mitochondrial succinate when it's dead we thought it complex too and that's why the question is could reverse electron transport flow be going on so from the from the paper this is something as I find extremely interesting because there's a topology that's involved in how and where the electrons are leaking out of mitochondria so from complex one which is at the bottom there is Ross product electron leak resulting in Ross production and that this electron leak goes primarily into the mitochondrial make matrix and it's primarily associated with oxidative damage but from complex 3 the third one up there it gets released the electrons get released mostly into the Christie space in the inner membrane space and it has it's predominantly involved in signal transduction it has some oxidative component but when these are separated out complex one was primarily earth yeah complex one was primarily involved in oxidative stress complex 3 is where signal transduction is being induced and this is a really interesting paper we're all aging as we're sitting here every one of us are aging and they've they've there's a long been held a free radical reactive oxygen species involvement in aging but this this paper talks about the abundance of this matrix arm that I've circled there of involved in radical for maisha and so basically in in a case where there's disruption of complex one so there's lower for example lower expression of proteins in complex one the the lower abundance results in higher radical formation and they tied this to cell senescence as well as longevity and mice so the shorter that matrix arm is in complex one more radicals being produced and this can stimulate aging so here's another one we have inflammation and all kinds of different models different things we're looking at this is looking at macrophages and that reverse electron flow from complex 2 to complex 1 according to this paper is saying this is where the radical reactive oxygen species are being produced so in a pro-inflammatory macrophage you end up with and for example in response to bacterial infection there's this reorganization of the electron transport chain and so it decreases complex 1 but it enhances complex 2 and in the process of doing this it shifts ATP from oxidative phosphorylation to the glycolytic pathway and it leads to a high mitochondrial membrane potential and when there's a higher mitochondrial membrane potential you get more radicals being produced so there's a buildup of succinate and it does reverse electron transport flow and this is where the electrons are leaking form it forming superoxide and high levels of a hydrogen peroxide if you treat these macrophages with rotenone it basically puts it into anti-inflammatory Meck Meck Rivage oh and the other thing on the inflammatory macro file just this stimulates inflammatory cytokines with rotenone you get you get inhibition of the reverse electron flow this decreases Ross production and instead of inflammatory cytokines Briand produce you now get any inflammatory cytokines so it's a it's a different it's a different cell it's a different beast at this point and it's shifted it and the shift is based on reverse electron flow here's another one ischemia reperfusion injury a schema this is in heart attack so with ischemia you have low blood flow low oxygen levels the top is ischemia in the bottom is norm oxy and so in norm oxy in heart muscle and in other muscle you'll have electron transport oxidative phosphorylation going on but in ischemia there's a buildup of a MP and succinate when blood flow Reece is restored to the tissue now you have a buildup of succinate that's in the in that tissue and it's going to run into the electron transport chain the other way so that you get now you've got reverse electron flow you get Ross production and this is what they are saying that the great amount of damage is occurring is from reverse electron flow once nor masya is restored I teach undergraduate physiology course idea we talked about sensing of oxygen in the animal from the carotid body chemoreceptors in this one if there are low levels of reactive oxygen species now let me let me back up when there's hypoxia what happens is there's a buildup of reactive oxygen species in NADH and this buildup stimulates the potassium channels in the Glomus cell which is the sensing cell of hypoxia and it increases activity from the sensory nerve so oxygen sensing comes down to another case where there's reverse electron flow resulting in stimulation or respiration so for all of us are dealing with muscle in one way shape or form because we're always wanting to eat chicken etc so this is this has to do with muscle cell differentiation and in the conclusion from this paper is that during that reverse electron transport is running to produce cytokines I meant to produce Ross that end up stimulating muscle cell differentiation so reverse electron flows is in that transition in muscle development how many exercise in here on a regular basis one two there's a ought to be more ought to be more of you because this shows ok an exercise you in during exercise you lower mitochondrial membrane potential you have less write a reactive oxygen species being produced when that's occurring you get mitochondrial biogenesis and you can get fusion going on in the mitochondria and you get these various genes that are being involved in this activity get greater antioxidant capacity mitochondrial biogenesis you improve mitochondrial function there's greater cell survival and muscle adaptation if you're a couch potato what ends up happening you can have a higher membrane potential you have high levels of Ross that are coming out and you stimulate something completely different and this can lead to mitochondria that are not functioning optimally and you have my top a G and a lot of things that are going on in and cell turnover so it's two different things and it's related to reverse electron flow so now I'm just going to touch on proton leak we did some studies with proton leak kinetics and the bottom line with this is what we found is that in all the different situations where we're looking at proton leak kinetics that proton leak was less than or equal to it was in the hyphy deficient mitochondria proton leak was less than or equal to but never greater than in the low ficient mitochondria and so what this seems to be saying is that this proton leak was being stimulated by the hier raus production that's going on in lo of feed efficient mitochondria so now jump forward a little bit we've been doing some pro do genomics genomics studies and we've done this looking at breast muscle samples we did see da see DNA microarray we've done RNA seek most recently as well as shotgun proteomics all of these studies were done on the same tissue samples so we're looking at the same they came from birds that we either had high feet efficient phenotype or low feet efficient phenotype and I have to take a second and just thank some people I would never have even thought of doing this stuff that they hadn't been for dr. Kong bioinformatics kind of he's got four screens there because he's got to do this stuff all the time I have admired that Nick Hudson contacted me a couple years ago and we we ended up getting funded by USDA and we we had Tony reverter who's a statistician and what they said with with the RNA seek data rather than using a cut-off that most people are using they said give me all the data I said you're nuts but they wanted all the data so we sent it to him and they came up with what they did they had developed this thing called regulatory impact factor analysis I'm not going to go into this I died basically they're described in these papers and it has to do with a differential wiring analysis you can have a gene that could be having a big effect on the phenotype through its interconnectedness but you don't see it if you're doing the cut off with a fold or a difference or a p-value so they just took all of the data woops good so they did the regulatory impact factor analysis and and through this analysis they came up with progesterone signaling is playing an important role in feed efficiency and we just published this a couple months ago and BMC systems biology so five or four out of the top ten regulatory impact factors had to do something with with progesterone we in the proteomics paper that we did we also found that progesterone was predicted to be activated in the hyphy deficient phenotype and i want to point out here that also there's peers to be insulin and triiodothyronine that are predicted to be activated so why these are immature male broilers why progesterone why and the muscle so I started looking and found a couple of papers that talked about hormonal involvement in mitochondrial function so when we compared what we had found with mitochondrial function in the comparison high and low feed efficiency to what they found with progesterone in a couple of these different models and it had - the progesterone was protective of mitochondrial function in ischemic in traumatic brain injury so they in feed efficiency they had a higher coupling higher respiratory control ratio lower state four we had lower Ross production that was also seen lower electron leak in the brain studies lower oxidative stress and there was a higher complex for activity we saw a number of complexes all of the complexes were basically lower or higher and high feed efficiency so progesterone seems to be having effect in the central nervous system similar to what we were seeing it with the mitochondria in high and low feed efficiency and then this led to a whole bunch of papers they've known this in mammals for some time we looked in the literature but we didn't see anything in a VM cells that this had been documented it doesn't mean it's not out there we just haven't found it yet so can't do Laster and I can't say enough about what he's been doing he's been working with me for a long time thank goodness don't go anywhere otherwise I'm out work anyway so he did these studies this is in qm7 cells looking at expression of receptors on the mitochondria so this is progesterone receptor using immunofluorescence and the merged figure at the bottom is indicating the presence of my two these receptors on mitochondria so who got it in these qm7 cells with progesterone glucocorticoid thyroid hormone and insulin receptor and he presented on Tuesday most recent findings which was estrogen also being located in these qm7 cells and importantly he was he'd been trying to identify through Western blot analysis the the presence of these hormones he had a lot of nonspecific binding so it wasn't clear he got a monoclonal and now we have a very clear indication that estrogen receptor is located in on the mitochondria because this is the mitochondrial fraction so now I want to return to ray about redox biology and nuclear respiratory factor is it orchestrates a cell responds to oxidative stress so it stimulates the antioxidant response element in the nucleus to help with preparing the cell or conditioning the cell to oxidative stress this is real simple diagram but in a normal situation nerf to nfe2l2 is bound to a couple of regulatory molecules keep one and Cullen three it's bound if no oxidative stress is present it turns over every ten to fifteen minutes through proteasome 'el degradation if oxidative stress is occurring it breaks these bonds that are holding on to nfe2l2 it then goes into the nucleus stimulates antioxidant response element that stimulates production antioxidant enzymes and antioxidants in the cell to respond to the oxidative stress so in the shotgun proteomics we found that they was predicted to be activated in the high feat efficient the orange is indicative of activation and high food efficient phenotype and nan-zhao who did a masters with bento mabashi they found this with RNA seek in a broiler commercial broiler model so they also found that they were it was predicted to be activated in high feed efficiency so these were these predictions were made but but the molecules the downstream molecules upon which the prediction was made were different between the two sets so there's a slight difference there but the prediction was the same so possibly this activation or up regulation of nurse to in a high feed efficient broilers helps orchestrate or respond it prepares these high feed efficient cells to handle oxidative stress better than the ones than the low feed efficient it's a hypothesis at this point nerve to is very transient it's very hard to measure these things because it's it's changing very rapidly over time so here's kind of a summary that I've got I hope I should sum radix biology and oxidative stress talked a little bit about electron transport mitochondrial Ross production and how that's attenuated by proton Lee mitochondrial reverse electron transport I really think that these papers in the last three or four years are highlighting something new that it has fundamental importance to what every one of us are doing we see that there's a possible role of progesterone that could be involved in feed efficiency and then it could be the antioxidant response may be coordinated by nfe2l2 so now I want to these are a couple other things that we've published recently how am i doing on time almost up I'm getting that he's getting the hook out I'm gonna skip this this is some other stuff that we've we've published Martin brand recently published this there's not just three sites of electron leak there's now 10 sites so now these are different sites that are within the mitochondria they designed a couple of Ross suppressors that are targeted these are antioxidants that are tardy targeting specific parts of the electron transport chain but they the cool thing about this it does not prevent normal electron flow one last thing the mitochondria may be playing a role in fixing damaged proteins and this is a paper that came out in March they named this mitochondria as garden Guardian in cytosol or magic so just leaving you with a little bit of mitochondrial magic thank you [Music]