Progress 07/01/15 to 06/30/20
Outputs
Target Audience:The target audience for this work is food scientists, dairy scientists and nutrition science professionals. Changes/Problems:The first aim was to develop a method to measure CML with a GC-MS in Dr. Ward's lab. However, despite following published protocols we could not get reliable data. The second aim was to measure CML with the method from the first aim. Since this was not possible we moved to aims 3 and 4. In completing these aims, we found that previous studies had not differentiated what heat-damaged products may promote insulin resistance. We switched to characterizing the lipid oxidation in the rodent diets and developing methods to determine the mechanism of action of the heated protein/sugars in improving glucose metabolism. What opportunities for training and professional development has the project provided?One MS student conducted the mouse study and one BS student has conducted the in vitro work. How have the results been disseminated to communities of interest?One presentation was given at the Experimental Biology meeting in 2016. A second presentation on this material was given in the Nutrition, Dietetics and Food Sciences seminar in 2017. Last, the final data was presented to an international meeting via Zoom in October of 2020. What do you plan to do during the next reporting period to accomplish the goals?While this project is finished, we have two manuscripts in preparation detailing the mouse study, and one on the in vitro study.
Impacts
What was accomplished under these goals?
This project started with four goals: a) develop a method to measure carbosymethylysine with GCMS, b) estimate the amount of CML in the US diet, c) conduct a feeding study with mice with biologically relevant levels of CML in the diets, and d) determine the effects of CML on insulin sensitivity. Due to technical problems described below, we could not complete the first two goals, and shifted to the third in years 1 and 2. Prior work has shown that heat processed diets cause insulin resistance. Using a more precise design, we found that diets that contained only heat damaged protein (as measured with CML) promoted insulin sensitivity. We then determined that in previous studies the whole diet pellets had been baked. While this promotes the Maillard Browning reaction and formation of CML, it also destroys viatmins and oxidizes lipids. We subsequently analyzed baked diets of several formulas and determined that lipid oxidation was significant. We have continually sought funding to continue these studies with mice. We would like to disentangle the relative effects of oxidized lipids and Maillard Reaction products on glucose metabolism. However, we have not received it, and then shifted to work that could be accomplished with a more modest budget. Our hypothesis regarding the effects of heat processed proteins is that they are acting as a sort of dietary fiber and affecting the microbiome composition and metabolic activity. In the last year we have focused on preparing protein/sugar mixes that have similar levels of CML with four major commodity proteins (casein, whey, egg white protein and soy protein isolate). We had planned to subject the heat processed protein/sugar mixes to an in vitro digestion and then investigate their effects on the microbiome in a fecal slurry system. However this was stopped due to the COVID pandemic.
Publications
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience for this reporting period is fellow agricultural and food scientists. Changes/Problems:We have adjusted the aims of this project based on our difficulty with measuring CML via GCMS. We have worked a bit with Casey Simons of Chemistry on NMR and LC-MS, but have not been confident with the methods. Based on this, we shifted to working to establish how MRPs affect the microbiome. If the ADA grant is funded, we will continue with the rodent studies. What opportunities for training and professional development has the project provided?One MS student, who had conducted the second feeding study, finished and defended her MS thesis. An undergraduate student developed a new method of producing MRPs in protein/sugar blends. How have the results been disseminated to communities of interest?Two manuscripts are being edited that describe the rodent studies. Data from these studies has been used in grant applications to the USU Research Office, NIFA and the American Diabetes Association. The USU grant and the NIFA grants were not funded, but we received news in November that the ADA grant had passed the initial screen and we will be notified of the decision in January. What do you plan to do during the next reporting period to accomplish the goals?In the spring of 2020, we will investigate the effects of MRPs on the microbiome in a series of in vitro trials. Using the method developed in 2019, we will produce MRPs from casein/sugar, whey protein/sugar, egg white protein/sugar and soy protein/sugar blends, and subject them to an in vitro digesting. Next, we will purify the indigestible components and incubate them with fecal slurries to determine how they affect the microbiome composition.
Impacts
What was accomplished under these goals?
The overall goal of this project is to determine how heat processing foods affects health. In the literature, it has been repeatedly shown that baking rodent diet pellets will negatively affect health, and the 'culprit' is assumed to be Maillard Reaction Products (MRPs). However, these studies have a fundamental design flaw in that baking the diets will also promote lipid oxidation and thermal destruction of heat-labile vitamins. We designed diets wherein the MRPs were produced only in the protein/sugar component, and the lipids and vitamins were added later. In our studies, mice have become MORE insulin sensitive, but it is not clear why. In related studies, we have shown that baking the pellets, as done in previous studies, causes significant lipid oxidation, which can easily explain the results of others. To date, we have only seen this effect when the milk protein casein was used as the protein source in the diets. Thus, we are not sure if it is an MRP-specific effect, or a casein-MRP effect. In addition, when we created the MRPs in previous studies, we microwaved the casein/sugar mixtures and the process was not well controlled. Last, we have no idea on what the mechanism of action is, but believe it has to do with the microbiome. In the last 2 years we used data from this project in NIFA applications, and sought to conduct further animal studies. We have not received funding. We also requested funds in a Research Catalyst grant to investigate the mechanism of benefit of MRPs, but were not funded. Consequently, in 2019 we focused on aspects of this question that could be completed with fewer resources. An undergraduate student in the laboratory developed a standardized method to create MRPs in protein/sugar mixtures. In this work we used the major protein sources that are used in rodent diets, namely casein, whey protein, egg whites, and soy protein. We successfully developed a standardized, repeatable method of producting MRPs and are now working to characterize the products.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience for this reporting period is fellow agricultural and food scientists. Changes/Problems:The main student working on this project took her oral exam for her PhD in December. She did not pass, and will retake the exam in January. If she does not pass the second time, she will write a Master's thesis. What opportunities for training and professional development has the project provided?Two PhD students and one visiting scientist conducted the work on this project in 2018. One PhD student in the lab of Dr. Sulaiman Martarneh conducted the protein analysis of liver tissues. A second PhD student subjected mouse diets to heat treatment and characterized the effects on lipid oxidation. In addition, this student also developed a method to create Maillard Reaction Products with other protein sources. The visiting scholar developed a method to determine the digestibility of the oxidized protein-sugar complexes. How have the results been disseminated to communities of interest?Data resulting from this project was used in a USDA grant submitted in July of 2018. What do you plan to do during the next reporting period to accomplish the goals?In the coming year, we will submit a manuscript (or possibly two) detailing the work conducted on this project thus far. In addition, grants will be submitted to solicit funds for further animal experiments.
Impacts
What was accomplished under these goals?
The focus of this work has been to determine the effects of cooking on food chemistry and nutrition. We have been producing rodent diets that are high in Maillard Reaction Products (MRPs), and we have used the levels of carboxymethyllysine (CML) as an index of oxidation. Many previous publications have shown that baking rodent diets promotes insulin resistance. We previously reported that we found MRPs actually promote insulin sensitivity, which is in contrast to all previous studies in the literature, and in 2018 we focused on possible mechanisms. First, we have been trying to determine how our diets differ from previous studies, and have been characterizing our diets compared with those previously used in the literature. One major difference between our research design and those of others is that we did not bake the whole diets, but rather produced MRPs by heating only the sugar and protein components. Baking rodent diets will produce MRPs and CML, but it will also oxidize lipids and vitamins. In 2017, we developed a method to oxidize the oils the fats in these rodent diets, and to characterize them. In 2018, we evaluated how baking the diets affects lipid oxidation. We found that the procedures used to produce MRPs and CML in rodent diets also add a substantial load of oxidized fats. In 2018 we also worked to understand how the diets are improving metabolism, and we measured the levels of two mitochondrial proteins (succinate dehydrogenase and myosin heavy chain 1) in muscle tissue of the mice by Western blot. We hypothesized that the increase in insulin sensitivity was mediated via gut production of short chain fatty acids, which then may have increased mitochondria biogenesis. However, there were no differences between the treatments in these mitochondria markers. In both of the mouse feeding studies we have conducted, we have noted the MRPs affect the composition and metabolic activity of the microbiome. Thus, we have hypothesized that diets high in MRPs may contain some indigestible components. To study this, we developed a method to chemically digest the diets in order to determine if heating the sugar-protein mixtures reduces digestibility. We found that this is the case, but have not characterized the material that resists digestion. In our mouse studies, we have only used casein as the protein source. It is not clear if heating the protein-sugar mixes to produce MRPs will be different with different protein sources. In 2018 we determined how to produce MRPs with sugars and the following proteins; egg white, soy and whey.
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience for this reporting period is fellow agricultural and food scientists. Changes/Problems:None What opportunities for training and professional development has the project provided?Three students conducted the work on this project in 2017. One PhD student was responsible for conducting the mouse study. In the summer of 2017, one undergraduate student helped on the project for 10 weeks. He was from the Summer Undergraduate Agricultural Biotechnology Research Experience (SURE) program. All students attended the Laboratory Safety training through Environmental Health and Safety. Starting in the Fall of 2017, an MS student began working on the project to help isolate melanoidins from the diets. How have the results been disseminated to communities of interest?Data from this project was disseminated at Experimental Biology, an international meeting that includes nutrition research. In addition, data from the study was used for a USDA grant submission in July of 2017. What do you plan to do during the next reporting period to accomplish the goals?In the first part of 2018, gene expression analysis will be conducted in liver and adipose tissues to determine if dietary CML has an effect on mitochondria biogenesis. In addition, in 2018 a third feeding study will be conducted with different levels of oxidized dietary fat. The ultimate goal is to determine whether oxidized dietary fats can explain the disagreement between our results and those of others that have been previously published.
Impacts
What was accomplished under these goals?
In 2017, work was continued on evaluating the effects of dietary carboxymethyllysine (CML) on metabolic and gut health parameters in mice. Thus far, two studies with identical designs have been completed and the data was compared between studies. It was determined that increased dietary CML promotes insulin sensitivity in a dose-dependent manner, and that the effect is repeatable. This effect appears to be associated with lower systemic inflammation. In addition, gene expression in liver and adipose suggest dietary CML may be upregulating the production of mitochondria. This effect is consistent with effects on the microbiome and the increase in fecal short chain fatty acids (SCFA) in mice fed higher CML diets. Our studies are not in agreement with previous work, which suggests diets high in CML should predispose mice to the development of type 2 diabetes. However, there is one critical aspect of our design that may explain the disagreement. In previous work, CML has been produced in diets by heating all the macronutrients (i.e. protein, carbohydrate and fats) to induce the browning reaction. This process will not only cause Maillard Browning, but will also oxidize lipids. Prior studies have shown that oxidized dietary fat may cause deterioration in insulin sensitivity. In our design, the fats were added to the diets after heating, and therefore there was no increase in oxidized lipids. We believe previous studies have mistakenly attributed effects to CML which are actually caused by oxidized fats. Based on the data we have collected, we hypothesize that heating the diets to produce CML may also produce melanoidins, which are the brown colors associated with roasted foods. We believe the melanoidins in the diet may be acting as a type of dietary fiber, which increases the production of short chain fatty acids, such as butyrate and propionate.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Target Audience The target audience for this reporting period is fellow agricultural and food scientists. Changes/Problems:Changes/Problems As described above, we still lack a reliable method to measure CML in foods and biological tissues. Thus, we plan to continue to look for an LCMS we can use. Once we can measure CML (Objective 1), we can complete objective 2. What opportunities for training and professional development has the project provided?Opportunities Three students conducted the work on this project in 2016. One PhD student was responsible for conducting the mouse study. In the summer of 2016, two undergraduate students helped on the project for 10 weeks. They were both from the Summer Undergraduate Agricultural Biotechnology Research Experience (SURE) program. One undergraduate student has continued to work in Dr. Ward's lab. All students attended the Laboratory Safety training through Environmental Health and Safety. How have the results been disseminated to communities of interest?Dissemination Data from this project was disseminated at Experimental Biology, an international meeting that includes nutrition research. In addition, data from the study was used for a USDA grant submission in July of 2016. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work In 2017, a second mouse feeding study will be conducted. This study will test the hypothesis that the changes in the gut microbiome mediated by CML will be associated with increased susceptibility to gut mucosal damage mediated by dextran sodium sulfate (DSS). DSS is a chemical that is used to mimic inflammatory bowel disease (IBD), and previous studies have implicated changes in the microbiome with increased IBD incidence. These same microbiome changes appear to be promoted by DSS. In addition, Dr. Ward will try again to see if the technician in the CIB will respond to inquiries about using the LC-MS to measure CML.
Impacts
What was accomplished under these goals?
Accomplishments In 2016, work on Objective 1 was suspended, and work on Objective 2 was conducted. As work on Objective 2 required a method from Objective 1, a work-around was implemented. As described in the last report, Objective 1 was to develop a method to measure carboxymethyllysine (CML) in foods using GCMS. The preferred method for this analysis is LC-MS, but that is not available in the Ward Lab. After extensive work to develop and validate a method was conducted, the procedure was abandoned. While there is an LC-MS in the Center for Integrated BioSystems at USU, the technician would not respond to emails, and the PI was in a bind to make progress on the project. As a workaround, effort was shifted to Objective 3. The first goal of this objective was to create a mouse diet with different levels of CML. This was done by heating a combination of casein (protein source) and sugars (glucose, fructose and sucrose). CML was measured in the mixture by a collaborators lab in France. Diets were subsequently prepared with these substrates to create three levels in the diets. Our original intent was to tie the levels in the mouse diets to those we could measure in foods typical of the US diet. However, since we did not have a reliable method to measure CML, we instead tried to match levels used in previous human and rodent studies. The background diet was the Total Western Diet developed by Dr. Ward and collaborators at USU. A first mouse feeding study was completed at the beginning of 2015, and it was determined that dietary CML affects several metabolic parameters, including fasting glucose and glucose tolerance. There were three major findings from this feeding study. First, CML appears to increase populations of the microbiome that are associated with gut dysbiosis. Next, the metabolites of the microbiome (i.e. short chain fatty acids) were significantly affected by the CML content of the diet. Last, dietary CML caused a dose-dependent increase in several plasma metabolites, all of which are associated with decreases in metabolic health. Data is still being collected on a few parameters, and this new data should help us to determine how dietary CML is affecting metabolism.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Presentations
Ward, R. E., Hintze, K. J., Xiao, S., Lefevre, M., Experimental Biology, "Effect of Dietary Carboxymethyllysine on glucose and insulin sensitivity and the plasma metabolome in mice fed the Total Western Diet," American Society of Nutrition, San Diego, CA. (April 5, 2016)
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Progress 07/01/15 to 09/30/15
Outputs
Target Audience:Target Audience The target audience for this reporting period is fellow agricultural and food scientists. Changes/Problems:Changes/Problems At this point we are not able to make the method in Objective 1 work. We will spend a few more months trying before moving on to another strategy. What opportunities for training and professional development has the project provided?Opportunities This project provided an opportunity for an MS student in Dr. Ward's lab. This student became proficient at running a GCMS and at preparing standard curves for the analyte of interest. The student was not able to overcome methodological issues with the method, which is beyond the scope of training for an MS degree. As discussed above, the student was removed from the project when it became clear that it could not be completed in a timeframe that would allow her to finish her MS in a timely manner. How have the results been disseminated to communities of interest?Dissemination At this point we do not have any significant results to disseminate. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work My plan is to spend some time working on the method this winter to see if I can figure out the problems. I was teaching two classes in the Fall and did not have sufficient time to spend with the student to troubleshoot the method. I plan to spend several months on Objective 1. If I cannot make the method work by then, I will change strategies.
Impacts
What was accomplished under these goals?
Accomplishments This project started on July 1, 2015, and thus far work has been focused on the first objective. The first objective was to develop a method using GCMS to measure the carboxymethyllysine levels in diets used in a mouse feeding study. All materials were acquired and a MS students worked for several months on the method. Despite success in producing linear calibration curves with the analytes of interest, we were not able to successfully apply the method to actual food products. Dr. Ward worked with the student to troubleshoot various aspects of the method, but was not able to identify what step was not working. We knew the method was not working as we had the same samples analyzed by a collaborators lab in France. The major difference in the methods is that the lab in France uses LC-MS/MS, which is an instrument Dr. Ward does not have. In October the MS student was switched to another project as she had been at USU for 1.5 years and it was necessary to have her work on a project that could be completed in a reasonable timeframe. Although we planned to be finished with Objective 1 in 2016, Dr. Ward will spend some time reviewing the method in an effort to get it to work. If it is not possible to measure CML with GCMS, alternative plans will be made.
Publications
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