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>>  Thank you for joining us.  We are going to begin in a few moments.  My name is Mallory earnings and I am a program coordinator here at the national Institute of Food and agriculture.  It is my privilege to introduce to you our speaker today, Doctor Richard Bruno.  Doctor Bruno is a bio nutritionist and registered dietitian with expertise in the areas of phytochemical and anti-oxidant utilization and metabolism is in relation to free radical mediated toxicology.  Doctor Bruno is a professor of human nutrition at the Ohio State University.  Prior to joining the OSU faculty he completed a prestigious postdoctoral training at the lines poly Institute at Oregon State University.  And earned his PhD in human nutrition from the OSU [ Indiscernible -- loud coughing ] nutrition program.  Is research program is primarily focused on defining the mechanisms regulating the bioavailability and bioactivity of phytochemicals.  Particularly, green tea polyphenols and vitamin E in the mechanisms by which these bioactive components protect against oxidative stress and inflammatory responses otherwise contributed to nonalcoholic settle hepatitis.  The underlying goal of his research is to translate experimental findings into practical dietary recommendations that enhance health standing humans.  Since 2003 he has polished over 80 peer review -- published in the area of his expertise.  His research has been featured multiple times by the American Society of nutrition.  He was the recipient of the 2014 ASN me Johnson award and his published work has been recognized by the NIH office of dietary supplements as one of the top 25 dietary supplement papers published in 2005.  Doctor Bruno currently serves as an editorial board member for the Journal of nutritional biochemistry, molecular nutrition and feed were -- and food research, nutrition research, and nutrition reviews.  He is an active member of the American Society for nutrition.  This society of free radical biology and medicine, and the Academy of nutrition and dietetics.  And he has been elected to the scientific Council of the Society of experimental biology and medicine.  Doctor Bruno, thank you very much for being with us today. 
>>  Thank you, Mallory for the introduction.  It is a pleasure to be here and being able to give this webinar from afar.  I will do my best to avoid any technical glitches and I'm really pleased to share with you a story that we have developed over the last 8 - 10 years, largely with support directed to my laboratory friend USDA.  The title of my talk is anti-inflammatory activities of green tea and the lessons that we have learned from preclinical models of Nash or nonalcoholic settle hepatitis.  Check for those of you who are not familiar, nonalcoholic fatty liver disease or in a felt the represents the term given to a progressive disorder that increases liver related morbidity and mortality.  It begins as relatively benign hepatic steatosis, characterized by excess fat speculation and liver.  These livers are susceptible to liver injury induced by oxidative stress and inflammation resulting in the progression towards nonalcoholic status hepatitis, fibrosis and potentially cirrhosis and liver cancer and potentially liver related death.  
>>  Tragically, there is about 80 - 100 million Americans who are estimated to have some form of nonalcoholic fatty liver disease.  It is prevalence have largely parallel the obesity epidemic with roughly 3/4 of obese adults have some form of in Flight largely in the form of  liver steatosis.  Additionally, concerning is that the rate of liver cancer from the city -induced nonalcoholic batting liver disease is estimated to be outpacing viral hepatitis or alcohol abuse.  Therefore, strategies are desperately needed to manage the growing risks attributed to in Flight. NT, there are currently no  food and drug approved treatment for this disorder.  Despite significant efforts by the pharmaceutical industry.  Therefore, dietary approaches have been emphasized to manage early inflammatory responses implicated in Nash development and progression.  Ending, the focus of my laboratory is aimed at studying the progression towards Nash.  These represent early and reversible periods of liver injury.  
>>  The mechanisms leading to Nash are often characterized by these so-called quote, unquote, hits the drive pathogenesis towards Nash.  Under conditions of obesity and insulin resistance there are metabolic changes that result in a hit that leads to liver steatosis or excess Vatican relation of the liver.  Some of these metabolic changes result in increasing delivery of free at a assets to the liver and there steer furcation towards triglyceride, increased lipogenesis or fat synthesis, impairment in the oxidation of lipid and mitochondria, in addition, decreases in -- and export of the LDL from the liver.  The net effect is that the liver retains excess fat resulting in lever state -- liver steatosis.  Consequently, these steel topic livers are susceptible to another series of hits in the form of oxidative stress and inflammation.  That drives a liver to this more inflamed state which characterized by increased liver injury.  As well as increased reactive oxygen and reactive nitrogen species accumulation as well is greater activation of inflammatory cells of the liver.  Particularly, resident Cooper sells.  
>>  To this end, my lab as well as another -- a number of other low -- labs have been managed on dietary manages to -- lifestyle modification is considered at present the only validated therapy for Nash.  Indeed, weight management can mitigate liver steatosis and its progression to Nash.  However, complaints to maintain weight loss has a core long-term success rate of only about 15 percent.  My laboratory has been focusing on functional approaches that can manage the multiple hits contribute to obesity triggered Nash specifically, we have been focusing on green tea consistent with epidemiological evidence that green tea has a diverse number of health benefits . 
>> The fire activities of green tea have been historically attributed to his polyphenolic catechist.  For those of you who are not familiar, green tea as well as black and along to you art all derive from the same plant called Camillus and uses.  With -- the difference between green, black, and long teeth are the manner in which they are processed, excuse me, the manner in which the plant is processed post are being harvesting, the tea that is destined to become green tea undergoes rapid post harvest processing to inactivate -- to activate the polyphenolic Cadigan content of green tea.  In fact, as you can see here, on the left side of the slide, the Cadigan content of green tea is approximately 4 times higher than that of black and oolong tea.  The distribution of Cadigan's are such that e.g. CG represents approximately 50 percent of the total Cadigan content of green tea.  Indy, these Cadigan's have been touted for a number of health benefits including anti-inflammatory, hyperlipidemic and antioxidant activities.  However, there has been little to no validation, especially in Nash models.  That will be the focus on my tax just talk today, the steps we have taken using preclinical models to explore these potential benefits that can help ameliorate the risks of developing Nash.  
>>  There is strong epidemiological basis to support green tea in managing the risk of Nash.  Ending, green tea consumption has been shown to be associated with lower levels of liver injury and serum lipids in healthy, but older Japanese men.  In this perspective epidemiological study approximate 1300 Japanese men over the age of 40 were characterized for their levels of green tea on a daily basis.  It was observed that those that consumed 10 or more cups of green tea per day have lower risks of higher levels of aspartate and liver function test as well as serum lipids and the inflammatory marker ferritin.  Suggesting that green tea, not only protects against liver injury, but also has high epidemiological -- hyperlipidemic and anti-inflammatory benefits that might protect against Nash.  Unfortunately, or fortunately, depending on which camp you standing, rodent studies are necessary to define the potential benefits of green tea for humans.  This is largely because the evaluation of Nash is rather challenging and the only true way to diagnose the presence of Nash is by the use of an invasive liver biopsy.  In the absence of hundreds of people lining up on my door and volunteering for liver biopsy we have had to rely on voter -- want models to be highly invasive and study the mechanistic basis by which green tea protects against Nash.  That said, it should be recognized that there is not a single rodent model that we could -- it really capitulates all the mechanism leading to NASH in humans.  For this reason my lab has deployed a number of different models.  Regardless of the model system and despite the mechanisms being subtly different, each of these models develop steatosis and oxidative stress and do progress to NASH allowing us to study the potential benefits of green tea using the models to best understand mechanism of action at the preclinical level with the ultimate goal of taking preclinical findings and validating them in human translational models. 
>>  With that said, my lab was at the forefront of studying green tea in familiar and 80s NASH.  An early study that we conducted provided preliminary basis to support profound protective effects of green tea.  Against liver steatosis in genetically obese or leptin deficient OB/OB mice.  As you can see in these liver sections, in panel B, this is a liver section from an OB/OB mouse fed no green tea.  You can see that these livers have evidence of [ INDISCERNIBLE ].  A lien mouse fed no green tea has incredibly healthy liver that is devoid of liver steatosis.  Panel C and D show typical responses following green tea supplementation in these mice.  In particular, panel D shows the typical or average response that we observed where we get about a 50 percent reduction in liver steatosis, at least on the basis of histological observations.  
>>  Consistent with these findings we also observed that green tea, when supplemented in the diet at either 1 percent or 2 percent, on a weight for weight basis, this equates to approximately 7 - 14 cups of green tea per day consistent with the epidemiological studies I shared with you a few moments ago, you can see that serum levels as well as aspartate aminotransferase are significantly attenuated in obese mice fed green tea compared to the obese controls that had no green tea.  Moreover, you can see that there is absolutely no effect in the lien mouse, which reflects the fact that they lack liver steatosis to begin with.  It also supports that green tea is not exerting any toxicological effects that were otherwise increased with the liver function tests.  In our pre-clinical models of green tea appears to be safe at doses that are consistent with higher intake levels observed to be protective in epidemiological studies.  Consistent with these findings in leptin deficient OB/OB mice.  We have conducted similar studies using 8 diet -induced of these model in elliptic and -- they left an adequate map.  Any studies we feed the rest either a low-fat diet containing 10 percent fat or a high-fat diet containing 60 percent fat.  As you can see in the upper right histological section of the liver, these rats fed a high-fat diet, but lacking green tea supplements show histological evidence of steatosis as well as some subtle inflammatory if you look closely.  In contrast green tea at the higher dues of 2 percent reduces histological evidence of steatosis, which we were able to corroborate Rackley by measuring liver triglyceride.  In addition, these rats fed a high-fat diet and supplements of the green tea also had attenuated levels of serum ALT supporting that green tea protects against NASH and -- in left an adequate rats.  
>>  A very simple message from these early studies says that green tea can protect against genetic as well as died induced NASH as well as adiposity independent of leptin status and without any does affect.  At the bottom of my slide the leptin deficient obese mice have reduced adiposity in response to green tea at one or 2 percent green tea in the died.  Similarly speaking, adiposity was attenuated in the left an adequate rats fed green tea.  
>>  These findings were particularly exciting for us early on because we were able to demonstrate that green tea exerts profound protective effects that reduce histological and biochemical evidence in NASH at reasonable levels that can be achieved in humans, at least those in Asian cultures where green tea is consumed quite regularly.  
>>  This raises the question of how does green tea protect against NASH? We initially proposed that antioxidant activities of green tea extract might be functionally responsible for protecting against NASH.  This is based, in some regard, on Eve they took findings showing that green tea catechins exert profound free radical scavenging activities.  However, when one considers the in vivo situation it has been well documented that green tea catechins have very poor bio Dutch bioavailability.  This likely precludes the catechins from having direct scavenging activity on free radicals.  However, this does not preclude the possibility that green tea catechins may be exerting indirect antioxidant activity, either by enhancing the detoxification of ROS, by up regulating endogenous antioxidant defenses that detoxify free radicals.  Alternatively, we can insert the possibility that the anti-inflammatory act -- may be mediating its antioxidant activity  
>>  Because green tea may attenuate the formation of reactive oxygen and R and S drive from inflammatory cells that migrate to the liver and induced NASH.  
>>  In my laboratory we studied the antioxidant effects of functional foods by measuring the lipid Pro oxidation using the highly sensitive and sensitive [ INDISCERNIBLE ] method.  As you would expect, the obese controls that no green tea had a significantly elevated liver levels compared to controls that no green tea.  Green tea at a low-dose of .5 percent green tea extract in the diet were 1 percent similarly attenuates hepatic [ INDISCERNIBLE ] to levels no different than lien controls.  More ever, we show a positive correlation between liver MDA and serum ALT suggesting that dietary strategies such as green tea that attenuate liver lipid Pro oxidation can protect against liver injury.  
>>  With this in mind, we aim to understand the mechanism by which green tea lowers hepatic lipid peroxidation.  Half a dozen years ago I had a really right master student in my lab, Dana Dean Vitali, who was tasked with measuring the enzymatic activities of a number of endogenous antioxidant that detoxify reactive oxygen species.  Specifically, she measured in some activity levels of copper, zinc, manganese SOD, catalase and QuickTime proxy daters.  As you can see from my schematic these antioxidant defenses are essential for the detoxification of superoxide as well as hydrogen peroxide which would give rise to lipid Pro oxidation..  -- Peroxidation.  Green tree extract was able to restore the activities of each of these enzymes to levels no different then low-fat controls and this is important because in the obese mice fed no green tea the activities these enzymes were all suppressed.  Suggesting that obesity impairs endogenous antioxidant defenses to contribute towards lipid peroxidation during NASH.  So, part of the story is that green tea up regulates these in somatic antioxidant defenses to attenuate lipid peroxidation.  
>>  In addition to those effects we also measured liver levels of glutathione.  Glutathione is a highly abundant endogenous antioxidant.  We show that in obese mice glutathione levels are suppressed compared to lien controls and green tea in a near dose-dependent manner restores glutathione levels to levels no different than those of lien controls.  Moreover, we performed real-time PCR experiments to measure gene expression of assisting glide case, both catalytic and modulatory subunits.  The importance of this enzyme is that is responsible for the biosynthesis of glutathione.  In addition, this enzyme is under the site to protective regulation of the transcription factor.  As you can see here, green tea at higher doses upregulated the expression of GCL, both his modulatory and catalytic subunit compared to obese controls.  Suggesting that, the restoration of the Zion is at least in part driven by the greater bio the synthesis of glutathione.  -- Glutathione.  On one side of the coin we have green tea potentiating the detoxification of ROS.  We also considered the possibility that anti-inflammatory effects of green tea would attenuate the generation of reactive oxygen and reactive nitrogen species that would otherwise lead to lipid proxy peroxidation.  We redirected our attention back to liver histology and looked very closely and observed that consistent with the NASH pathology, OB/OB mice fed no green tea had greater histological evidence of inflammatory infiltrates of liver.  In contrast, those that green tea had either -- at either does has a Nick Kelly attenuated levels of inflammatory infiltrates.  Suggesting the possibility that green tea's antioxidant -- anti-inflammatory activities will help have an antioxidant effect to protects against lipid peroxidation.  
>>  With that in mind we measured the enzymatic activity of NADPH oxidase.  This enzyme is highly expressed in inflammatory cells and is part of the normal inflammatory response in which copious levels of superoxide radical are generated to have antibacterial and, killing power, I guess, IF YOU WILL.  As you can see, consistent with the NASH pathology, with the OB/OB mice having greater inflammatory infiltrates, higher activity of NADPH oxidase which was attenuated by green tea x-ray at 2 different doses.  We also observed that green tea extracts attenuated levels of reactive nitrogen species.  These 2 pieces of data are critically important because superoxide generated by NADPH oxidase readily reacts with nitric oxide to form a deleterious free radical known as proxy nitrate which can lead to protein nitration that can be evidenced by the measure of nitro tyrosine.  
>>  As you can see here, the OB/OB controls that no green tea had increased levels of nitrate tyrosine compared to lien controls and green tea does the penalty reduced protein nitration's to level no different than lien controls.  Take -- taken together green tea is attenuating ROS and R and S generation, which helps protect against liver dysfunction that is attributed to protein nitration.  We further considered studying the mechanism by which green tea mitigates protein nitration beyond what I showed you on the prior slide.  This and we measured protein expression levels of the enzyme useful [ INDISCERNIBLE ] which many of you are aware is responsible for the generation of nitric oxide from inflammatory cells.  We also measured protein levels of milo peroxidase, another pro-inflammatory enzyme expressed abundantly in macrophages and neutrophils.  As you would expect, protein expression of both of these enzymes was significantly increased in the obese controls fed no green tea.  And consistent with the earlier evidence I shared with you.  There was a significant reduction in the expression of [ INDISCERNIBLE ] and [ INDISCERNIBLE ].  Taken together these studies help us with an mechanistic understanding of how green tea limits protein nitration and lipid peroxidation because milo peroxidase utilizes hydrogen peroxide as a substrate in the presence of rack of nitrogen oxide resulting protein nitration.  In addition, I NASH generates copious levels of nitric oxide radical -- radicals can which can react with superoxide giving rise to proxy nitrate which can promote [ INDISCERNIBLE ] and also lipid peroxidation.  Thus, anti-inflammatory effects of green tea that minimize inflammatory infiltrates are helping to preserve liver injury or at least protect against liver injury associated with NASH.  
>>  The lesson to be learned from these early studies is that green tea is likely to be exerting indirect antioxidant activities to limit reactive oxygen and reactive nitrogen species mediated liver injury.  As I showed you, green tea suppresses both lipid peroxidation and protein nitration by, one, up regularly endogenous antioxidant defenses that are responsible for the detoxification of reactive option species, particularly superoxide and hydrogen peroxide.  
>>  In addition, green tea's his own to decrease the generation of reactive a oxygen species and reactive nitrogen species that are derived from enough B dependent inflammatory enzymes.  
>>  Given that we observed that green tea attenuated the expression of both IMAX and mild peroxidase which are both [ INDISCERNIBLE ] dependable enzymes we wanted to understand whether or not the antioxidant activities of green tea can protect against [ INDISCERNIBLE ] activation to limit inflammation.  The rationale for the studies is to pull.  One, activation of [ INDISCERNIBLE ] has been shown to be upregulated in the verse from both patients as well as rodents with NASH.  We know that enough B is upregulated.  The enzyme IKK is sensitive in the presence of high levels of reactive oxygen species.  IKK gets [ INDISCERNIBLE ] leading to the activation of NFkB  and its subsequent translocation to the nucleus where it up regulates the expression of a number of Pro inflammatory mediators.  Given that we observed that green tea upregulated super exit [ INDISCERNIBLE ] and [ INDISCERNIBLE ], which are all antioxidant defenses regulated by the transcription factor of interest to we hypothesized that green tea would inhibit NFkB activity  by way of its antioxidant activity.  
>>  We conducted a fairly simple study in which we fed rats either high-fat or low-fat diet and a number of them were fed a high-fat diet containing 1 percent green tea or 2 percent green tea extract.  For 12 weeks to induce NASH.  What we observed is not surprising, that OBs and most of the high-fat diet had increased P 65 binding activity, indicating that NFkB was activated in the livers from these rats  fed a high-fat diet.  Whereas, green tea at 1 percent and 2 percent significantly attenuated NFkB binding activity.   At mechanistic level we demonstrated that the reduction in NFkB activation was attributed  to an attenuation in the phosphorylation of IK be.  This is a critical protein that regulates the activation and subsequent translation of NFkB to the nucleus where it will have his transcriptional activity . 
>>  Corroborating this evidence was our observation that green tea reduced [ INDISCERNIBLE ] expression levels of TNF alpha as well S MCP-1.  In addition, attenuation at the message level was also reflected at the protein level for these 2 important clients that are NFkB  dependent.  We are confident we have a model that has increased NFkB activation and that green tea  attenuate his activation in and IK be dependent manner.  
>>  Based on our hypothesis -- hypothesis that green tea's antioxidant activities are mediating is anti-inflammatory activities, we wanted to understand the extent to which green tea exerts its protective benefits through the transcription factor in RF to.  As a mentioned a few moments ago, this transcription factor exerts a number of protective properties in that it regulates endogenous antioxidants defenses as well as [ INDISCERNIBLE ] metabolism.  In a simple cell culture study we studied -- we cultured the predominant green tea Cadigan in culture at levels from 0 to 5 micromole are, which reflects the typical levels of green tea catechins in the circulation in heavy tea drinkers..  In these hepatic types, specifically HCO for cells we observe that green tea at these physiological relevant levels increased interest to acute elation in the nucleus, suggesting that green tea extract maybe upregulated Nrf2 cited protection to mitigate liver injury.  With that in mind, we conducted the 1st of its kind study examining the extent to which green tea protects against hepatic and at -- NFkB  inflammation in and Nrf2 dependent manner.  In the studies we used Nrf2 null or knockout mice that a high fat diet containing no green tea or a high-fat diet containing 2 percent green tea.  We also had 4 controls the 57 block 6 wild type mice that were fed the same diets.  They were fed these diets for 8 weeks to induce NASH.  The underlying hypothesis is that Nerf to deficiency or Nerf to know mice would have exacerbated inflammation that would not be able to be resolved by green tea if green tea is protecting in a Nerf 2 dependent manner.  So, 1st up we validated that green tea actually upregulated Nrf2 in the wild type mice that express Nrf2.  As you can see in the green bar, wild type mice that green tea had expressed interest to as well as his downstream gene in Q01.  Suggesting that, indeed, green tea's up regulates Nrf2 dependent activities..  Based on this we expected to observe that green tea would protect the Nrf2 dependent manner.  The 1st thing we did was perform some histological experiments in which we score the severity of nonalcoholic fatty liver disease for specifically NASH.  As you can see, in the Nrf2 knockout mice they had severe liver steatosis as well as significant evidence of inflammatory infiltrates where as the wild type mice fed the high-fat diet has some evidence of steatosis, but limited inflammatory infiltrates.  No evidence of [ INDISCERNIBLE ] ballooning.  You can see on the right these effects are quantified such that the Nrf2 knockout mice have higher level of higher levels of steatosis and ballooning compared to the wild type mice fed a high-fat diet.  
>>  In contrast, those mice that green tea were observed to have an attenuation in both steatosis as well as [ INDISCERNIBLE ] regardless of Nrf2 status.  Suggesting that green tea protects against NASH independent of Nrf2 status.  Officer, this is contrary to our hypothesis.  Nonetheless, important because green tea has been touted for having Nrf2 dependent activities for quite some time.  This study provides the 1st direct evidence that it has affect on NASH that are likely independent armed Nrf2.  Nonetheless, we trekked forward.  We examined a number of genes involved in hepatic lipid uptake, esterification lipogenesis, and as we expected the Nrf2 knockout mice fed no green tea had increased expression levels of dilapidated transcription factor, sterile regulatory [ INDISCERNIBLE ] 1C, fatty acid 10 days as well as SD -- SED 1.  Each of these genes were upregulated in the Nrf2 knockout mice compared to the wild type controls fed no green tea.  However, contrary to our hypothesis green tea attenuated the expression of these leper genic genes.  Deliver lipid uptake gene, CD 36 as well as genes involving esterification of free fatty acids to triglycerides, specifically DG a T1 and DG 82.  This suggests that the anti-steatite effects of green tea are also independent of Nrf2..  We wanted to understand if green tea was also protecting against hepatic inflammation in and Nrf2 independent manner given that inflammation has an instrument to roll in provoking liver steatosis.  We measured protein expression levels of the active form of enough B, phosphorylated P 65 as well as total P 65.  What we observed was that green tea or high-fat beating, excuse me, green tea are Nrf2 status has no effect on the expression of P 65 or total in FB.  However, Nerf to deficiency exacerbates the activation of NF B compared to wild mice fed the high-fat diet.  Whereas, green tea in both wild type as well as Nrf2 knockout mice significantly attenuated NFkB activation. Suggesting that  green tea also attenuates NFkB independent of  Nrf2 status.  So, we learned some really valuable lessons from the Nrf2 mice.  Consistent with our hypothesis.  Nrf2 division mice that a high-fat diet had exacerbated evidence of NASH as well as increased NFkB inflammation. However, contrary to our hypothesis  and despite our strong evidence suggesting in earlier studies that green tea exerts hepatic protective activities in a Nrf2 dependent manner  our studies in Nrf2 knockout mice  show that -- Nrf2  -- an expression of his inflammatory mediators.  This picture on the right is pretty much what I looked at -- looked like upon completing the studies.  We were incredibly frustrated because we thought our hypothesis was well grounded.  After reflecting on the data for a while in some regards we were a bit happy that the studies turned out the way they did.  This is because NFkB  is an incredibly complicated transcription factor.  Not only can be activated by intracellular oxidative stress in and are was dependent manner, it is also activated through a number of receptor mediated signaling pathways.  So, the fact that our Nrf2 knockout studies  disproved our hypothesis was a little helpful because we were able to rule out a potential mechanism by which green tea might be functionally responsible for protecting against NASH.  We therefore focus our attention on 2 prominent targets that are implicated in NASH.  That is TNF receptor.  Both of these receptors are upregulated in NASH models and both of these receptors are implicated in inflammatory signaling that leads to NFkB  activation.  We did a somewhat simple study, although it was suddenly complicated.  To study the extent to which green tea protects against TOL 4 and TNF receptor mediated signaling.  In this study we fed wild type C 57 black 6 mice a high-fat diet for 12 weeks initially.  This was to induce NASH.  We subsequently ran the mice -- randomized these mice to either the low-fat or high-fat diet that they were already on.  However, at this time we also provided half of the mice green tea.  We were able to test the extent to which green tea exerts therapeutic anti-inflammatory activity in contrast to our earlier studies where we provided green tea in a prophylactic manner . 
>> The 1st we did was examine NFkB activation. AS WE EXPECT a,  mice fed a high-fat diet for the duration of the study had increased NFkB activation.   Quite remarkably, green tea situated phosphorylated P 65 levels to those that are no different from the low-fat controls fed no green tea.  This is an important piece of evidence because it shows that green tea him up both prophylactically in the study as well as earlier studies, excuse me, it shows a green tea provided there physically in the study as well as prophylactically as in our earlier studies has similar effects to attenuate NFkB activation. Moreover,  we performed a number of mRNA expression studies in which we measured TLR 4 as well as its adapter protein my D 88 and additional proteins that make up the receptor complex CD14 and MD 2.  As you can see, across the board in the orange bars obese controls that the high-fat diet had increased expression levels of TLR 4 and in my D 88 and CD14 and MD 2.  In green tea across the board attenuated the expression of these genes to levels no different than the low-fat controls.  Suggesting possibility that green tea is protecting in a toll 4 dependent manner.  However, we did similar studies in which we examined expression levels of TNF alpha as well as TNF receptor and proteins that make up the receptor complex TR ADD and RIP.  Expression levels of TNF alpha and TNF receptor it self are upregulated in response to a high-fat diet in green tea attenuated this.  This suggests the possibility of some involvement in which green tea is protecting against NFkB inflammation  during NASH by attenuating through the TNF receptor.  We let back to our schematic and started looking at this more closely.  We hypothesized that green tea is likely decreasing TOL 4 signaling initially to limit hepatic NF B -- NFkB activation. This is because  the like in for TNF receptor as well as a TNF receptor it self are NFkB dependent. You need  activation of NFkB to have an up regulation  in TNF alpha and TNF receptor itself.  And contract, receptor 4 is related independent of NFkB perform poorly,  it LPS as well as SFA are also NFkB independent. Thus, we embarked on studies  examining the study to which green tea protects TRL for a dependent manner.  It becomes challenging because TLR 4 combines both endotoxin as well as certain fatty acids to induce TLR 4 activation leading to [ INDISCERNIBLE ] activation.  Our studies have found observations from our own studies that wild type mice that green tea show that green tea has no effect on serum non--- non-esterified fatty acids.  In addition, and while top wild type rat [ INDISCERNIBLE ] from the fatty acid fraction are also unaffected by high-fat beating as well as green tea.  Lastly, in some recent studies that are under review in wild type mice as well as loss of function TLR 4 mice fed a high-fat diet, serum in EFA are not different by genotype, but the actual increase in concentration following supplementation of green tea.  The reason my I bring this up is because in essence these pieces of evidence suggest that if green tea is protecting in a protective manner it is not doing so by regulating saturated fatty acids that would lead to his activation.  
>>  The contrary, we examined the other like in for TLR 4.  We showed a high-fat beating increase serum endotoxin levels compared to wild type controls fed a low-fat diet.  And particularly, important was our observation that green tea attenuated serum endotoxin levels.  The reduction in serum endotoxin was associated with the restoration in the expression of several small intestinal type junction proceeds, particularly occluding and clotting one where we measured their expression in the ilium of the small intestine.  
>>  This suggests that green tea might be lowering metabolic endotoxin immune by limiting the translocation of endotoxin from the gut to this is, -- to the systemic circulation.  However, measurements of endotoxin in the serum have some limitations because even though we show that green tea attenuates serum endotoxin, we cannot rule out the possibility that green tea prevents its translocation from the gut or green tea enhances its metabolism and elimination from the body at the liver.  
>>  We therefore, conducted some recent studies in which we measured endotoxin levels directly from the portal circulation.  This is the blood that goes from the small intestine, I should say, the gastrointestinal tract, directly to the liver.  With the underlying hypothesis that if green tea lowers endotoxin in the portal vein this would suggest that green tea is preventing endotoxin translocation from the got.  Remarkably, we observed that anti-inflammatory activities of green tea at the intestine effectively reduced gut permeability to attenuate metabolic endotoxin me.  
>>  As you can see on the left, mice fed the high-fat diet had significantly increased expression levels of NFkB in the ilium  compared to low-fat controls and green tea attenuated TNF expression in this region of the small intestine.  
>>  In addition, we show that green tea attenuates portal levels of endotoxin that are otherwise increased by high-fat feedings.  And to corroborate these pieces of data we used a fluorescent sugar called FIT C dextrin to increase permeability in response to the high-fat feedings and in response to his it and waited got response ability.  The FIT C dextrin is not absorbed that much.  Therefore, following oral of Oshie when it expect to see much in circulation.  However, as you can see in the high-fat controls fed no green tea they had significantly increased levels of FIT C dextrin compared to the low-fat controls.  Green tea normalized it in the high-fat animals to those different than the low-fat -- no different than the low-fat controls, suggesting that the green tea is inhibiting got dried endotoxin translocation to the liver where I would otherwise have it lead to increased [ INDISCERNIBLE ] activation.  To test this hypothesis we used loss of function TLR for mice -- wild type controls fed the same diet.  We fed these diets for 8 weeks and then performed a series of studies in which we examined information in response to green tea to examine the extent to which green tea protects in a TLR4 manner.   Wild type mice fed a high-fat diet with green tea had attenuated levels of NFkB activation  compared to those that no green tea.  Importantly, was that the levels of NFkB activation in the wild type  mice that green tea are no different from the TLR4  muted mice that lack TLR4 signaling.   -- To the extent lowered to just TLR4 signaling.   Similarly, we observed that green tea attenuates a number of NFkB genes  to the extent to the loss of TLR4 signaling. We just reason measured liver expression levels of TLR4 itself  as well as adapter proteins and why D 88 as well as [ INDISCERNIBLE ].  In wild type mice that green tea there was a significant attenuation in TLR4.   In addition, green tea independent of genotype was able to lower the expression of and why D 88, which is important for TLR4 signaling to activate  NF B.  However, as you can see here, neither green tea nor TLR4 status express  affected expression levels of TRF -- T RIF.  This further corroborates our earlier evidence that green tea is mitigating got dried endotoxin translocation.  Indeed, we turned our attention back to the got and we observed that independent of TLR4 status green tea upregulated  the expression of CLT and one in wild type mice as well as TLR4 muted mice. In addition, clotting  -- CL DN was upregulated in the jejunum but no expression levels in the ilium.  Moreover, we observed that serum endotoxin levels were unaffected by TLR4 status as where  green tea attenuated endotoxin levels independent of TLR4 status. Moreover,  endotoxin was correlated with expression levels of clotting in both the duodenum and the jejunum and in a talk some was positively correlated with liver TLR4 expression as well as phosphorylated  NFkB suggested  -- suggesting that green tea is having hepatic protective benefits that are partly mediated at the got to limit hepatic inflammation.  
>>  Perhaps not surprising, when we redirected our attention to look at liver histology wild type mice fed a high-fat diet had significant evidence of liver steatosis and hepatic cellular ballooning.  Those that green tea were protected against these histological parameters of NASH.  To the same extent of TLR4 muted mice  regardless of those mice be fed green tea.muted mice  are resistant to high-fat induced NASH despite having metabolic endotoxin.  Green tea in wild type mice decreased hepatic NF V -- NFkB activation  to the extent that it  contributed to the loss of TLR4 signaling .  -- Suggesting it limits TLR4 MID 88  expression of TLR4 .  Green tea decreased metabolic endotoxin EMEA and increase T JP independent of intact TLR4 signaling. Therefore, when you take it together green tea is likely to inhibit  hepatic NF B -- NFkB activation in a TLR4 manner IN THE level  then -- translocation are likely independent of intact TLR4 signaling.   
>>  Some final lessons for the day, our study show that green tea when provided either prophylactically or therapeutically limit NFkB activation to mitigate the multiple hits of NASH  . 
>> Green tea apparently exerts these benefits independent of leptin status as well as independent of Seidel protective Nrf2 signaling. It's anti-inflammatory activities are likely needed  through a mechanism involving the got liver access that limits endotoxin translocation and consequent hepatic [ INDISCERNIBLE ] -- activation.  Therefore, our future studies are underway to consider that green tea might be improving macrobiotic composition of the got to limit the availability of endotoxin drive gram-negative bacteria and or alter metabolites derived from the microbiota that regulate got barrier function.  The basis of these studies are supported by recent studies published out of Asia from colleagues showing that microbial fermented green tea extract, which is relatively low in catechins, attenuates non-alcoholic that he liver disease in association with decreasing the ratios of for McCue's relative to activities as well as factoids relative to private tele-.  
>>  In addition, based on these studies using fermented green tea extract that has low levels of catechins this suggests the possibility that the predominant catechins found in green tea, e.g. CG, may not be directly responsible for the benefits of green tea during NASH.  It suggests there is some possible involvement of microbial metabolites of catechins.  Indy, it has been somewhat well-characterized over the years that parent catechins, specifically e.g. CG, epi catechins, epi catechins galley and EGC or a big a catechins are degraded by gut microbiota to form downstream and tablets including [ INDISCERNIBLE ] that have been shown to have anti-inflammatory prebiotic and antimicrobial activities at least in vitro.  Studies are needed to understand whether or not these downstream micro biotic Drive metabolites are functionally responsible for the benefits of green tea.  
>>  With that I know everybody is wondering how much green tea they need to drink in order to have some health benefits.  Since all of our studies have been conducted in mice I will leave you with some messages that are now centric or at least rodent centric.  If you are leptin deficient or elect an attic and rodent consuming a 60 percent fat containing that you need approximate 7 cups a day of green tea to reduce steatosis or 3.5 cups to reduce liver injury, Lipitor -- peroxidation and protein nitration . 
>> Our goal is to ultimately conduct some invasive studies examining green tea in NASH patients.  Today -- to date we haven't been able to conduct those because of the impracticality of -- of obtaining liver biopsies without a strong mechanism basis for the benefits of green tea.  In the last couple of years there have been 2 proof of principle -- of principal studies in  NAFLD patients showing that green tea improves  serum liver function test as well as CAT scan faced evidence of liver steatosis.  These benefits were exerted at a does have roughly half a gram to 1 gram per day of Green Tree extract which equates to about 10 cups of green tea per day.  
>>  Some final messages, green tea is safe when adjusted at reasonable levels.  I think there is a plethora of data from preclinical and clinical models to support that as well as a really elegant systematic review conducted by colleagues and future translational studies are needed to validate the mechanism of action by which green tea prevents and or resolves NASH along the got liver with potential involvement of got microbiota to advance dietary recommendations for critically needed anti-inflammatory strategy that can have profound benefits or about two thirds of Americans who are either overweight or obese and at high risk for developing NASH.  Stuck with that I will stop here and turn it back over to Mallory, who I think is going to moderate some questions.  I will be happy to answer any questions you might have picked before taking those questions I want to acknowledge USDA for its support over the years in conducting these studies.  Without that support these advancements in preclinical models would not have been possible.  In addition, I would like to thank some companies, Unilever who provided Green Tree extract that we use in our studies.  Thank you very much.  I hope you enjoyed the talk.  I will be happy to take any questions.  
>>  Thank you very much.  If there are any questions at this time please type them into your chat box.  We will begin to answer them as you enter your questions.  I'm going to go ahead and start us off.  WHAT WOULD your next steps be given your very exciting results?  
>>  Our next steps are focused on understanding the extent to which green tea improves got micro biotic composition to either, 8, and attenuate gram-negative bacteria from which endotoxin is derived.  That is based on evidence in vitro suggesting that green tea catechins have antimicrobial as well as prebiotic effects.  In addition, our studies on the gut microbiota will help us understand the extent to which the micro biotic generate anti-inflammatory metabolites themselves that may attenuate impairments in got barrier function to limit endotoxin translocation.  
>>  Will this still be in your rodent model?  
>>  We are actually, we would like to do it in parallel in both rodents and humans.  We are examining gut level benefits, translational studies in humans are feasible with some noninvasive techniques.  
>>  Fabulous.  
>>  We have our 1st question.  Was there a difference between use of caffeinated and decaffeinated green tea?  
>>  We have not that he got ourselves.  Our Green Tree extract contains caffeine.  As many of you are aware, caffeine has been shown to have some anti-obesity affects.  Or at least it can mobilize pre-fatty acids from adipose.  Radically, caffeine responses are desensitized in humans.  Suggesting that the effects are not attributed to caffeine in our chronic studies.  Nonetheless, we have not done a head-to-head comparison to caffeinated versus decaffeinated green tea.  Largely because most people drink caffeinated tea beverage.  We wanted to mimic that as closely as possible.  
>>  Thank you.  
>>  There is another question.  Could you confirm how many fluid ounces of green tea are recommended to reduce the lipoprotein gene expression? Was that 7 cups a day? Was that in your rodent example or human example?  
>>  All of my recommendations are based on my rodent daddies.  In which we are able to extrapolate the -- of rodents to humans.  To answer the question about how much he is needed in the human to reduce leverage in the gene expression, our studies point to about 7 cups per day in a human, assuming the rodent models translate to human benefits.  With that said, there were studies in Asia where imaging-based evaluation of steatosis was shown to be attenuated by half a gram to 1 gram of a Green Tree extract per day which equates to about 5 or T cups of green tea per day.  I think our mile -- mouse models are fairly reflective of the human situation.  
>>  Wonderful.  We have another question coming in.  Could you elaborate on mechanism involved in the intestinal endotoxin translocation in this model? Is it primarily in the small intestine or that the Polin which has a higher MICHAEL BOYLE -- micro something -- [ INDISCERNIBLE ] level. 
>>  I should probably clarify.  We have been limited to looking at tight protein in the small intestine.  We chose this because despite the small intestine having a lower bacterial load, in NASH there is evidence of what is called a small intestinal bacterial overgrowth.  Such that bacteria from the colon migrate up into the small intestine.  Therefore, the small intestine becomes a target for managing and the toxemia.  That say, we do need to contact studies of the colon to see if similar benefits of green tea are observed on type junction proteins.  I simply don't have the answer for you today.  Maybe next time.  
>>  Thank you very much.  
>>  Not seeing any more questions.  I'm going to go ahead and open the poll questions that we have.  Please give us your feedback.  This is how we at the national Institute of Food and agriculture are able to provide the type of seminar series and seminars on the topics that are -- that our audience appreciates.  If you would take the time to give us your feedback that would be greatly appreciated.  We always like to form presentations based on your recommendations.  If you could pull up the final slide, please.  If you have, are a registered dietitian and would like to receive your continuing education units for today's seminar, please email me at the email address shown on the screen.  If you are interested in any future Institute seminars or would like to listen to the recordings of any of our previous seminars you may find that information on our seminar series webpage shown on your screen.  With that I would like you all to join me in thanking Doctor Bruno for a wonderful presentation today.  I see we have one more question. 
>>  Sure . 
>> What was your source of the Green Tree extract?  
>>  We obtain our Green Tree extract commercially.  We have used 2 different vendors.  One was Unilever Best Foods and the other was Hyo international.  These are both aqueous extracts of Green Tree extract, commercial food grade.  
>>  You said that was Unilever and what was the 2nd company? Taiyo  
>>  
>>  Thank you for joining us today and we look forward to seeing you on future seminars. 
>>  Thank you very much, it was a pleasure. [ Event Concluded ]