Source: UNIV OF WISCONSIN submitted to NRP
DEFINING A PROTEOMIC SIGNATURE FOR SOY-INDUCED METABOLIC CHANGES IN MICE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1016165
Grant No.
2018-67001-28266
Cumulative Award Amt.
$1,625,000.00
Proposal No.
2018-03094
Multistate No.
(N/A)
Project Start Date
Jul 15, 2018
Project End Date
Jul 14, 2024
Grant Year
2018
Program Code
[A1342]- Food Specific Molecular Profiles and Biomarkers of Food and Nutrient Intake, and Dietary Exposure
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Neurology
Non Technical Summary
SummaryChildhood obesity is an epidemic in the United States where 32% of children and adolescents ages 2-19 are overweight (BMI<85th percentile) and 16.9% are obese (BMI>95th percentile). Ten percent of infants and toddlers have high weight-for-recumbent length measurements putting them at risk developing childhood obesity. Obese children are more likely to exhibit attention deficit/hyperactivity disorder (ADHD), depression, learning disability and developmental delay. It has been proposed that a common underlying dysfunction, the oversupply of information in the form of nutritional or sensory content, may independently predispose children to both obesity and ADHD. A critical gap in the literature is the identification of food-specific biomarkers associated with obesity. Our preliminary data indicate that the consumption of single-source, soy-based diets is associated with increased seizure susceptibility and body mass index (BMI). Increased BMI is a risk factor for the development of obesity, metabolic syndrome and type 2 diabetes mellitus (T2DM). We hypothesize that high consumption of soy-based infant formula, particularly in babies that are genetically predisposed to developmental disabilities, is an environmental exposure that increases the risk of developing obesity. As many as 25% of infants consume soy-based infant formula during their first year of life, but there is a paucity of information regarding potential health effects. Through this R01 application in response to PAR-15-024, "Food Specific Molecular Profiles and Biomarkers of Nutrient Intake, and Dietary Exposure," we expect to identify metabolic and proteomic biomarkers, in both control and disease model mice, which are altered in response to the consumption of soy protein. Positive findings could have powerful translational implications in terms of reducing the incidence of childhood obesity through dietary restriction of soy-based infant formulas.This contribution utilizes state-of-the-art mass spectrometry techniques to identify novel dietary biomarkers under rigorous scientific methods. The tangible benefits of this contribution are manifold. First, there is high potential to identify dietary biomarkers for clinically monitoring metabolic changes. Often studies of human disorders lack an ideal proxy tissue that could aid personalized medicine for diagnosis and treatment. In this proposal, we have the opportunity to test plasma, brain and peripheral tissue from the same mice in both wild type and a disease model. This provides the opportunity to identity nutritional biomarkers in an accessible proxy tissue and in response to disease status. Biomarkers found here can be directly tested in future clinical studies. Moreover, the proteins identified in this study can be interrogated in future studies for psychiatric- and obesity-related genetic variants in at-risk patients. Thus, this proposal may help bridge the gap between basic and clinical research in the field of nutritional biomarkers. Second, the findings will inform subsequent thinking and research in the field of pediatric nutrition, particularly in regard to infants with developmental disabilities that are comorbid with obesity such as Prader-Willi syndrome, autism spectrum disorders, Down syndrome, and fragile X syndrome. And third, positive findings would support increased dissemination regarding the benefits of breast-feeding, and in cases where formula is required, a dietary intervention (soy restriction) to improve medical outcomes. The American Academy of Pediatrics recommends infants be breastfed exclusively for the first several months and that breastfeeding continue through the first year of life. While 83% of mothers initiate breastfeeding, only 50% are breastfeeding at 6 months and 24% at 12 months. Over half of newborns receive formula in the hospital. Approximately 20-25% of infants receive some soy-based formula during their first year, but there is no data regarding how many are exclusively fed soy-based formula. Because dietary restriction of soy, like sugar or wheat, is not regulated by the FDA and poses no health hazards in an otherwise balanced diet, this type of medical intervention could be rapidly implemented.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30518201010100%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Goals / ObjectivesOur long-term goal is to implement a dietary intervention (restriction of soy-based infant formula in vulnerable populations) to reduce the incidence of childhood autism and obesity. The overall objective of this grant application is to identify nutritional biomarkers that are correlated with the consumption of a soy protein and obesity.1. Determine the Effects of Soy Protein Consumption on Metabolic Phenotypes. The experimental approach will include monitoring anthropometrics, energy expenditure, biochemistry, activity, hematology and necropsy phenotypes in response to consumption of soy- versus nonsoy-based diets in both wild type and disease-model (fragile X syndrome) mice. Our working hypothesis is that metabolic phenotypes will be altered in response to soy.2. Identify & Quantitate Proteomic Profiles in Response to Soy Protein Consumption. Tissue from soy- and nonsoy-treated cohorts will undergo proteomic mass spectrometry analysis. Identified targets will be quantitated by in situ labeling of MALDI tissue sampling. We postulate that a proteomic signature can be generated that identifies molecular targets that are responsive to soy.3. Validate Soy Protein Responsive Molecular Biomarkers. Molecular biomarkers will be validated in plasma and other tissues by western blot and/or ELISA and expression correlated with altered metabolic phenotypes identified in Aim 1. Our working hypothesis is that a molecular signature can be generated that reflects the effects of soy consumption on weight gain. With respect to outcomes, the proposed work is expected to identify and validate biological indicators of dietary soy protein intake. The results are expected to have positive translational impact because the identified nutritional biomarkers will likely provide new targets for preventive and therapeutic interventions.
Project Methods
MethodsAim 1 The experimental approach includes monitoring anthropometric, energy expenditure, biochemistry, activity, hematology and necropsy phenotypes in response to consumption of soy- versus casein-based diets. Mouse Cohorts: Heterozygous (Fmr1HET) females will be mated with WT male mice to generate WT and Fmr1KO male and WT female littermates and with Fmr1KO male mice to generate WT and Fmr1KO male and Fmr1KO female littermates. We will test both sexes at juvenile (P21) and two adult ages (P56, P180). Power analysis indicates18 subjects per cohort are required to achieve a power of 0.80 with a medium effect size of (f2=0.25) when the alpha factor for ANOVA is set at 0.05. Thus, starting cohorts will consist of 24 mice. At P21 and P56, 3 mice per cohort will be removed for hematology/necropsy resulting in 21 mice per cohort at P56 and 18 mice per cohort at P180.Diet Formulation: AIN-93G will serve as the "casein-based diet". "Soy-based diet" will be formulated by Teklad/Envigo by swapping the casein protein in AIN-93G gram for gram dry protein weight with isolated soy protein (Solae SUPRO® 661). In addition, the soy-based diet will be supplemented with calcium to match the AIN-93G. Thus, the diets will be exactly matched for macro- and micronutrient content albeit the differing proteins (soy versus casein). SUPRO® 661 will be purchased in bulk to ensure the same lot is used for all studies. The phytoestrogen content of SUPRO® 661 will be measured by LC-MS/MS.Anthropometric Measures: Neonates will be individually identified and measured to the nearest 0.01g (weight) and the nearest 0.1cm (length) at ages P3, P6, P9, P12, P15, P18, P21 and then once per week on the same day each week until the end of the study or age P180. Litter size and pup mortality will be monitored. Body adiposity of live animals will be assessed by NMR in an EchoMRI, which provides adiposity as a measure of whole body fat, lean, free water and total water masses.Energy Balance: Energy balance will be measured using the TSE Systems metabolic phenotyping cage system. Food and water intake, oxygen consumption (VO2) and carbon dioxide production (VCO2) will be continuously measured over the 3-day period, and the respiratory exchange ratio (RER, VCO2/VO2) calculated. Indirect calorimetry measurements (VO2 and VCO2) and the RER will be averaged and expressed per light/dark circadian phases for each mouse. Data will be analyzed by ANCOVA with a correction for body weight.Biochemistry: At P21, P56 and P180, blood samples will be withdrawn from the retro-orbital plexus of fasting mice. Total cholesterol, high density lipoprotein (HDL), triglycerides and glucose levels in the blood plasma will be measured by commercially available colorimetric or fluorometric assay kits. Blood samples to assess insulin will be collected from non-fasted animals and assessed by ELISA. Values will be compared with Jackson Laboratories phenotype data for C57BL/6J. In addition, glycosuria (excess glucose in the urine) will be tested twice per month.Behavior Testing: Cohorts will be assessed at P56 and P180 for grip strength, locomotion and anxiety in the rotarod and open field tests, respectively. These are standard rodent behavioral tests to asses drug toxicity. Length of time on the rotarod will assess grip strength, and total distance traveled in the center of the arena versus the entire open field will be used to assess anxiety and hyperactivity, respectively. Both measures will be analyzed via one-way ANOVA and Bonferroni posthoc tests with Prism 5.Hematology: At P21 and P56, 3 mice from each cohort will be anesthetized with isoflurane and the blood removed by venipuncture of the abdominal aortic artery. Remaining animals will undergo this procedure at P180. Blood aliquots (250 mL each) will be submitted for hematology analyses of red, white and platelet blood cell counts and hemoglobin levels. Remaining blood will be used to quantitate bioactive compounds. Mice will undergo phenotype analysis as described below.Bioactive Compound Quantitation: The concentration of 11 dietary phytoestrogens (genistein, daidzein, dihydrodaidzein (DHD), equol,O-desmethylangolesin (O-DMA), glycitein, biochanin A, coumestrol, enterodiol, enterolactone, and chrysin) in blood plasma will be determined by LC-MS/MS. Bioactive compound levels will be normalized to internal controls and correlated with outcomes from the Metabolic Test Panel.Phenotype Analysis: Necropsy will include pathological analysis of organs with particular attention to the liver, kidney and adipose tissue. Prior to dissection, bone mineral density will be determined by DEXA body scans.Aim 2 will use a discovery-based approach to identify protein biomarkers in response to soy-based diet in mice by combined MALDI-MSI. Tissue: Brain samples will be prepared from male WT and Fmr1KO mice maintained on the casein- versus soy-based diets (biological replicates: n=3 mice per cohort, age P21).Proteome Data Collection: MALDI profiling spectra of manually spotted tissue sections and imaging data of 14 intact brain sections in brain from mice fed casein- and soy-based diets will be acquired on an UltrafleXtreme MALDI-TOF/TOF mass spectrometer. The profiling spectra will be compared region-wise to evaluate the sensitivity of cells in various brain regions to respond to diet. Four serial sections will be collected and profiled for 4 animals, with their intensities averaged for each group. Data will be analyzed with the MATLAB-supported software ClinProTools 2.2. Statistical analysis will include t-test/ANOVA (PTTA) of individual peaks. Models of casein and soy groups will be generated using available algorithm, genetic algorithm, where the minimum number of peaks was set at 10 and the maximum set as ''default''. For image processing, the Bruker proprietary software flexImaging 3.0 will be utilized to generate MS images and overlay the optical image with the MS images for good visualization.Liquid-phase protein extraction and fractionation: Mouse brain hemispheres will be manually homogenized in acidified methanol at a 1:5 ratio on ice. Extracts will be centrifuged at supernatants collected and vacuum dried. Samples will be resuspended in water containing 0.1% formic acid and separated on a Phenomenex Kinetex 2.6 uM particle C18 RP HPLC column.RP-HPLC and Top-Down MS: Fractions containing peaks of interest will be analyzed by on-line top-down MS on an Ultimate 3000 RSLCnano system coupled to an Orbitrap Elite mass spectrometer. Data will be deisotoped with Xtract using the cRAWler algorithm and searched with a custom 168-core ProSightPC 3.0 cluster using an iterative search tree.Sample Size Justification: We will initially construct baseline molecular profiles for control mice. Based on our previous experience with MALDI-MSI and LC-MS/MS proteomic analysis, we will perform biological triplicates and technical quadruplicates to ensure strict adherence to rigor and reproducibility guidelines.Aim 3 The experimental approach will include a combination of ELISA and western blotting to assess protein expression in hypothalamus, adipose (intra-abdominal omental fat deposits), liver and plasma from WT and Fmr1KO mice as a function of sex, age (P21, P56, P180) and diet (casein- versus soy). Comparisons will be made to identify sex-, age- and genotype-specific proteins.Efforts: Results will be published in peer-reviewed scientific journals, presented at conferences and seminars, and used as part of curriculum development for a new course on Diet and Brain Disorders that the P.I. is developing for 3rd year medical students to cause a change in knowledge and infant feeding practices. Outputs will be evaluated for impact based on feedback from the intended audiences. We will collect peer review from manuscript submissions to journals and course evaluations. Key milestones will be publication and presentation of results.

Progress 07/15/18 to 07/14/24

Outputs
Target Audience:During the awardperiod the P.I. presented several seminars and posters on the work with the target audiences of junior high, high school, undergraduate, graduate students, residents, faculty, researchers and parents/caregivers. Venues included: lab tours, Society for Neuroscience conference, Gladstone Institute invited seminar, the 5 Big Questions Conference, Pediatric Neurology Residents' Seminar, and Diet & Neurological Disorders course lectures. Changes/Problems:In Aim 3, instead of quantitating biomarker expression by western blotting and ELISA, we submitted samples to RayBiotech for microarray analysis. This allowed testing of 640 samples versus a few dozen. This was cost effective and necessaryas the graduate student who was supposed to do the westerns and ELISAs decided to graduate early with a Masters and there was no one to do the bench work. The data has been organized by an undergraduate researcher in the lab and the P.I. will prepare the data for publication in a special issue on FXS for IJMS for publication in late 2024/early 2025. What opportunities for training and professional development has the project provided?Two graduate students, fifteen undergraduate students, twohigh school students and a post-bac research internworking in the lab have received training in molecular biology and mouse husbandry. The P.I. has participated and presented at several conferences. How have the results been disseminated to communities of interest?The P.I. has made presentations at multiple venues: lab tours for junior high school students, Society for Neuroscience conference, Gladstone Institute invited seminar, the 5 Big Questions Conference, Pediatric Neurology Residents' Seminar, Neurology Department Research Day, and Diet & Neurological Disorders course lecture. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Under Aim 1, a manuscript was published in January of 2024 that contained anthropometric, behavior, DEXA and blood biomarker data in mice in response to matched casein and soy-based diets in wild type and fragile X model mice. There were significant differences in outcome measures as a function of diet. Under Aim 2, a manuscript was published in January of 2024 describing the mass spectrometry techniques and proteomic profiles in wild type and fragile X model mice as a function of matched casein and soy-based diets. Under Aim 3, blood and tissue samples were sent for analysis of biomarker expression using RayBiotech arrays quantitating 640 protein targets as a function of genotype and diet. Data analysis is in progress.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Effects of Soy Protein Isolate on Fragile X Phenotypes in Mice Pamela R Westmark, Greg Lyon, Alejandra Gutierrez, Brynne Boeck, Olivia Van Hammond, Nathan Ripp, Nicole Arianne Pagan-Torres, James Brower, Patrice K Held, Cameron Scarlett, Cara J Westmark. Nutrients 2024 Jan 18;16(2):284. doi: 10.3390/nu16020284.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: On-Tissue Spatial Proteomics Integrating MALDI-MS Imaging with Shotgun Proteomics Reveals Soy Consumption-Induced Protein Changes in a Fragile X Syndrome Mouse Model. Ma M, Yu Q, Delafield DG, Cui Y, Li Z, Li M, Wu W, Shi X, Westmark PR, Gutierrez A, Ma G, Gao A, Xu M, Xu W, Westmark CJ, Li L. ACS Chem Neurosci. 2024 Jan 3;15(1):119-133. doi: 10.1021/acschemneuro.3c00497. Epub 2023 Dec 18.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Bibliometric Analysis and a Call for Increased Rigor in Citing Scientific Literature: Folic Acid Fortification and Neural Tube Defect Risk as an Example Nutrients 2024, 16(15), 2503; https://doi.org/10.3390/nu16152503 by Brynne Boeck andCara J. Westmark


Progress 07/15/22 to 07/14/23

Outputs
Target Audience:During the reporting period the P.I. presented several seminars and posters on the work with the target audiences of junior high, high school, undergraduate, graduate students, residents, faculty, researchers and parents/caregivers. Venues included: lab tours, Society for Neuroscience conference, Gladstone Institute invited seminar,the 5 Big Questions Conference, Pediatric Neurology Residents' Seminar, and Diet & Neurological Disorders course lecture. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five undergraduate students and one high school studentworking in the lab have received training in molecular biology and mouse husbandry.The P.I. has participated and presented at several conferences. How have the results been disseminated to communities of interest?The P.I. has made presentations at multiple venues:lab tours for junior high school students, Society for Neuroscience conference, Gladstone Institute invited seminar,the 5 Big Questions Conference, Pediatric Neurology Residents' Seminar, and Diet & Neurological Disorders course lecture. What do you plan to do during the next reporting period to accomplish the goals?Publish 3papers. Finish biomarker analysis. Submit grants to continue project.

Impacts
What was accomplished under these goals? Under Aim 1, Data on growth, activity, behavior, blood biomarkers and organ weightswereanalyzed includingstatistics. Substantial progress was made on the manuscript with an anticipatedsubmissionin the fall of 2023. Under Aim 2, Manuscript was submitted and is undergoing revisions. Additional tissue was collected to support future steps for mass spectrometry experiments. Under Aim 3, Tisse was collected and banked for upcoming biomarker validation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Diet in treatment of autism spectrum disorders Sabiha Alam, Cara J. Westmark and Elizabeth A. McCullagh Frontiers, Review, published 10 July 2023 doi: 10.3389/fnins.2022.1031016
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Improving Reproducibility to Enhance Scientific Rigor through Consideration of Mouse Diet. Westmark CJ, Brower J, Held PK. Animals (Basel). 2022 Dec 7;12(24):3448. doi: 10.3390/ani12243448.


Progress 07/15/21 to 07/14/22

Outputs
Target Audience:During the reporting period the P.I. presented several seminars and posters on the work with the target audiences of undergraduate students, graduate students, residents, faculty, researchers and parents/caregivers. Venues included: the 5 Big Questions Conference, ASN 2022, Department of Neurology Epilepsy Research Seminar, SfN virtual conference, ASENT conference, Lily Grace Grant Committee,and in herteaching Pediatric Neurology Residents' Seminar, multiple ZOO-500 undergraduate seminars, and Neurobiolgy of Disease class for neuroscience graduate students. Changes/Problems:No major changes in the approach for this reporting period. What opportunities for training and professional development has the project provided?Three undergraduate students working in the lab have received training in molecular biology, DEXA scans, and mouse husbandry. One graduate student has received training in proteomics. The P.I. has participated and presentedatseveral conferences. How have the results been disseminated to communities of interest?The P.I. has made presentations at multiple venues: the 5 Big Questions Conference, ASN 2022, Department of Neurology Epilepsy Research Seminar, SfN virtual conference, ASENT conference, Lily Grace Grant Committee,and in herteaching Pediatric Neurology Residents' Seminar, multiple ZOO-500 undergraduate seminars, and Neurobiolgy of Disease class for neuroscience graduate students. What do you plan to do during the next reporting period to accomplish the goals?We plan to finish all data analysis, submit/publish 3 papers that are in progress, and finish biomarker analysis.

Impacts
What was accomplished under these goals? Under Aim 1: Data is being analyzed and papers writtenfor submission of two manuscripts by the end of summer and fall 2022, respectively. Breeding was completed for mice to repeat the phytoestrogen analysis that was ruined by the COVID lockdown. Those animals weresacrificedand samples submitted for analysismid-May 2022. Under Aim 2: Data was analyzed, submitted for publication, and is currently under revision for re-submission in June2022. Under Aim 3: We commenced validation of biomarkers and preliminary data is included in the manuscript that will be re-submitted in May 2022. Tissue is being collected to validate further biomarkers over the next 6 months.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Parental Reports on Early Autism Behaviors in Their Children with Fragile X Syndrome as a Function of Infant Feeding. Westmark CJ. Nutrients. 2021 Aug 22;13(8):2888. doi: 10.3390/nu13082888. PMID: 34445048 Free PMC article.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Effects of Soy-Based Infant Formula on Weight Gain and Neurodevelopment in an Autism Mouse Model by Cara J. Westmark,Mikolaj J. Filon,Patricia Maina,Lauren I. Steinberg,Chrysanthy Ikonomidou andPamela R. Westmark Cells 2022, 11(8), 1350; https://doi.org/10.3390/cells11081350 - 15 Apr 2022
  • Type: Other Status: Published Year Published: 2022 Citation: Increased Incidence of Epilepsy in Response to Soy-Based Infant Formula in a National Korean Cohort Study. Journal of Nutrition, 2022. Letter to the Editor.
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: On-tissue spatial proteomics integrating MALDI-MS imaging with shotgun proteomics reveals soy consumption-induced biomarkers in a fragile X syndrome mouse model Min Ma, Qinying Yu, Daniel G. Delafield, Yusi Cui, Zihui Li, Wenxin Wu, Xudong Shi, Alejandra Gutierrez, Pamela R. Westmark, Meng Xu, Cara J. Westmark, Lingjun Li doi: https://doi.org/10.1101/2021.11.09.467989
  • Type: Other Status: Published Year Published: 2021 Citation: Diet in the Treatment of Epilepsy. Westmark CJ. Nutrients. 2021 Mar 12;13(3):917. doi: 10.3390/nu13030917.


Progress 07/15/20 to 07/14/21

Outputs
Target Audience:During the reporting period the P.I. presented several seminars and posters on the work with the target audiences of undergraduate students, graduate students, residents, faculty, researchers and parents/caregivers. Venues included: the NFXF conference, the RARE Disease Showcase, ASN Conference, FindACure, SfN Global Connectome, and her in her teaching Neurol914 Diet & Neurological Disorders, Obgyn 956 Seminar, Pediatric Neurology Residents' Seminar, ZOO-500 undergraduate seminar, and Neuroscience Training Program sub-group. Changes/Problems:With COVID lockdown rules, the mass spec testing lab that was scheduled to measure phytoestrogen samples in our mouse blood samples was shut down in the spring 2020. 80 of our samples sat in the freezer for months and many were degraded when they were finally tested. We hope to repeat the sample collection and quantitation. What opportunities for training and professional development has the project provided?The project has provided training for 2 undergraduates and 3graduate students. The students help with mouse husbandry, genotyping, behavior studies, DEXA scans, proteomics and data analysis. In terms of professional development, the P.I. has presented the work at multiple conferences and course lectures. How have the results been disseminated to communities of interest?The P.I. has made presentations for the NFXF and FindACure communties. What do you plan to do during the next reporting period to accomplish the goals?We plan to publish 2 large papers on the behavior and proteomics work. Our collaborator received funding for new indirect calorimtry equipment, and we plan to test our mice. We received funding from the Univ. WI to purchase a DEXA scanner for an add-on study to this grant, and we are writing up that data for publication during the next reporting period. We plan to start validation of select targets identified by proteomics.

Impacts
What was accomplished under these goals? Under Aim 1, data has been collected for all behavior testing and is currently being analyzed and prepared for publication. Under Aim 2, proteomics data has been collected and analyzed and the manuscript is in preparation. We will start Aim 3 after further evaluation of the proteomics results.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Westmark, P.R., Gutierrez, A., and Westmark C.J. (2021) A Simple, Reliable, and Inexpensive Method to Individually Identify Neonate Mice. Laboratory Animal Science Professional Jan/Feb: 46-48.
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Westmark, C.J. Consumption of Breast Milk is Associated with Decreased Prevalence of Autism in Fragile X. Nutrients (submitted, 2021).
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Westmark, C.J. Exploratory Associations between Infant Feeding and Autistic Behaviors in Fragile X Syndrome. Nutrients (submitted, 2021).


Progress 07/15/19 to 07/14/20

Outputs
Target Audience:p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica} During this reporting period, the P.I. Dr. Cara Westmark presented several seminars & posters on the soy diet and proteomics with the target audience of undergraduate students, graduate students, and faculty at the University of Wisconsin-Madison. Venues included: the graduate studentsin Nutritional Sciences, graduate students in Neuroscience, graduate students in Molecular & Environmental Toxicology, undergraduates in the CHARS program, the Midwest Fragile X Exchange, and the Fall Neurology Faculty Research Meeting. Changes/Problems:A problem that has slowed progress on the energy expenditure study in Aim 1 is that Dr. Yen's indirect calorimetry equipment brokeand his scientist David Nelson hadto discontinue testing our animals until it is repaired or replaced. Dr. Yen applied for several grants to replace the set-up, received funding and will be ready to commence new studies for us the Fall of 2020. Consequent to the equipment failure, Dr. Nelson delayed his percent effort on this grant until he resumes the experiments. What opportunities for training and professional development has the project provided?p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica} The project has involved training for 5undergraduate students (Westmark lab), 2graduate students (1 Westmark Lab & Li lab), 1 post-doctoral researcher (Li lab), and 2scientists (1 Westmark Lab & Yen lab). The project has involved professional development for a Scientist who participated in the UW-Madison Summer Mass Spectrometry School. How have the results been disseminated to communities of interest?p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica} The results have been disseminated in the form of research seminars and poster sessions to interested groups at the UW Madison Molecular Environmental Toxicology, Nutritional Sciences, and Metabolism groups. What do you plan to do during the next reporting period to accomplish the goals?p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica} We plan to continue with the anthropometric, behavior, biochemistry andproteomic data analysis under Aims 1 & 2. In addition, we will continue data collection for indirect calorimtery and hematology and necropsy analyses under Aim 1.

Impacts
What was accomplished under these goals? Under Goal 1: we have collected anthropometric, biochemistry and/or behavior data on over 400 mice in response to consumption of soy- versus casein-based matched diets in male and female wild type and Fmr1 mutant mice. We are in the midst of analyzing the data and preparing several manuscripts.We have collected energy expenditure data on several litters of mice and plan to resume studies in the Fall when our collaborator re-opens his lab after COVID-19. Under Goal 2: we collectedmass spec dataon wild type and Fmr1KO mice and are preparing the manuscript.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Folic Acid Fortification and Neural Tube Defect Risk: Analysis of the Food Fortification Initiative Dataset. Murphy ME, Westmark CJ.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Reply to "The Fallacy of Using Administrative Data in Assessing the Effectiveness of Food Fortification. Comment on: Folic Acid Fortification and Neural Tube Defect Risk: Analysis of the Food Fortification Initiative Dataset. Nutrients 2020, 12, 247". Westmark CJ, Murphy ME.
  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: FMRP Regulates the Nuclear Export of Adam9 and Psen1 mRNAs:: Secondary analysis of an N6-methyladenosine dataset.


Progress 07/15/18 to 07/14/19

Outputs
Target Audience:During this reporting period, the P.I. Dr. Cara Westmark has presented several seminars & posters on the soy diet and proteomics with the target audience of undergraduate students, graduate students, and faculty at the University of Wisconsin-Madison. Venues include: the Interdepartment Graduate Program in Nutritional Sciences, the Midwest Fragile X Exchange, the Fall Neurology Faculty Research Meeting adn the Morgridge Metabolism Symposium. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has involved training for 4undergraduate students (Westmark lab), 1 graduate student (Li lab),1 post-doctoral researcher(Li lab), 1 scientist (Yen lab), and 1 scientist (Westmark lab). The project has involved professional development for the PI who participated in the UW-Madison Summer Mass Spectrometry School. The scientist in her lab will participate this year. The PI is also participating in the UW-Madison Institute for Clinical & Translational Sciences (ICTR) Team Science Program. How have the results been disseminated to communities of interest?The results have been disseminated in the form of research seminars and poster sessions to interested groups at the UW-Madison: Molecular Environmental Toxicology, Nutritional Sciences, and Metabolism groups. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue with the anthropometric, behavior, indirect calorimetry and proteomic data collection under Aims 1 & 2. In addition, we will commence the biochemistry, hematology and necropsy analyses under Aim 1.

Impacts
What was accomplished under these goals? Childhood obesity is an epidemic in the United States where 32% of children and adolescents ages 2-19 are overweight (BMI<85th percentile) and 16.9% are obese (BMI>95th percentile). Ten percent of infants and toddlers have high weight-for-recumbent length measurements putting them at risk developing childhood obesity. Obese children are more likely to exhibit attention deficit/hyperactivity disorder (ADHD), depression, learning disability and developmental delay. It has been proposed that a common underlying dysfunction, the oversupply of information in the form of nutritional or sensory content, may independently predispose children to both obesity and ADHD. A critical gap in the literature is the identification of food-specific biomarkers associated with obesity. Our preliminary data indicate that the consumption of single-source, soy-based diets is associated with increased seizure susceptibility and body mass index (BMI). Increased BMI is a risk factor for the development of obesity, metabolic syndrome and type 2 diabetes mellitus (T2DM). We hypothesize that high consumption of soy-based infant formula, particularly in babies that are genetically predisposed to developmental disabilities, is an environmental exposure that increases the risk of developing obesity. As many as 25% of infants consume soy-based infant formula during their first year of life, but there is a paucity of information regarding potential health effects. Through this R01 application in response to NIH/USDA PAR-15-024, "Food Specific Molecular Profiles and Biomarkers of Nutrient Intake, and Dietary Exposure," we expect to identify metabolic and proteomic biomarkers, in both control and disease model mice, which are altered in response to the consumption of soy protein. Positive findings could have powerful translational implications in terms of reducing the incidence of childhood obesity through dietary restriction of soy-based infant formulas. Our goal in this grant application is to identify nutritional biomarkers of soy consumption. The prevailing view is that soy products are healthy; however, our preliminary data indicate adverse neurological and metabolic phenotypes in both mouse and human models. Successful outcomes from these studies will identify altered metabolic phenotypes and define a proteomic signature in mice in response to soy consumption. Our long-term goal is to implement a dietary intervention (restriction of soy-based infant formula in vulnerable populations) to reduce the incidence of childhood autism and obesity. The overall objective of this grant application is to identify nutritional biomarkers that are correlated with the consumption of a soy protein and obesity. 1. Determine the Effects of Soy Protein Consumption on Metabolic Phenotypes. The experimental approach will include monitoring anthropometrics, energy expenditure, biochemistry, activity, hematology and necropsy phenotypes in response to consumption of soy- versus nonsoy-based diets in both wild type and disease-model (fragile X syndrome) mice. Our working hypothesis is that metabolic phenotypes will be altered in response to soy. Extensive breeding on casein and soy-based diets was accomplished as well as monitoring anthropometrics and conducting behavior assessmentson over 120 mice. Energy expenditure was tested on over 2 dozen mice. Samples were collected/banked for biochemistry assays and proteomics. 2. Identify & Quantitate Proteomic Profiles in Response to Soy Protein Consumption. Tissue from soy- and nonsoy-treated cohorts will undergo proteomic mass spectrometry analysis. Identified targets will be quantitated by in situ labeling of MALDI tissue sampling. We postulate that a proteomic signature can be generated that identifies molecular targets that are responsive to soy. Optimization of proteomics methods is in progress. 3. Validate Soy Protein Responsive Molecular Biomarkers. Molecular biomarkers will be validated in plasma and other tissues by western blot and/or ELISA and expression correlated with altered metabolic phenotypes identified in Aim 1. Our working hypothesis is that a molecular signature can be generated that reflects the effects of soy consumption on weight gain. With respect to outcomes, the proposed work is expected to identify and validate biological indicators of dietary soy protein intake. The results are expected to have positive translational impact because the identified nutritional biomarkers will likely provide new targets for preventive and therapeutic interventions. Plasma and tissue are banked for future analysis.

Publications