Source: UNIVERSITY OF NEBRASKA submitted to NRP
BIOACTIVE COMPONENTS OF MACADAMIA NUT PROTECTS AGAINST MATERNAL OBESITY-INDUCED COMPLICATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030755
Grant No.
2023-67017-40223
Cumulative Award Amt.
$638,000.00
Proposal No.
2022-09465
Multistate No.
(N/A)
Project Start Date
Jun 1, 2023
Project End Date
May 31, 2028
Grant Year
2023
Program Code
[A1343]- Food and Human Health
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
(N/A)
Non Technical Summary
Maternal obesity (MO) associated with the development of maternal gut dysbiosis with increased gram negative bacteria, intestinal barrier dysfunction resulting release of lipopolysaccharide (LPS) from a leaky gut, which can cause maternal systemic inflammation and enhance lipotoxicity to placenta and fetal liver. Our preliminary data showed increased lipid droplets and trophoblast apoptosis in the placenta of animals fed with high fat/high sucrose (HFHS) diet-induced MO. Supplementation of macadamia nut-containing HFHS diet to pregnant mice showed decreased proinflammatory cytokine levels in the placenta and fetal liver compared to HFHS diet alone fed obese pregnant mice. We also observed that co-treatment of trophoblast with palmitate and LPS exacerbated palmitate-induced trophoblast lipoapoptosis. Treatment of trophoblasts with monounsaturated fatty acids (MUFA) dramatically prevented palmitate-induced trophoblast lipoapoptosis. MUFAs also prevented LPS-induced inflammation by blocking inflammasome activation. We hypothesize that supplementation of macadamia nuts (contains high MUFAs, Phytosterol, and insoluble fiber), as a whole food approach, prevents MO-induced maternal intestinal barrier dysfunction, and gut dysbiosis; lipotoxicity and inflammation to the placenta and fetal liver. Proposed hypothesis will be tested in the following objectives: 1) determine the protective role of dietary macadamia nut against placental lipotoxicity and inflammation during MO; 2) ascertain the protective role of macadamia nut supplementation against MO-induced fetal fatty liver and inflammation; 3) determine the preventive role of macadamia nut against maternal intestinal barrier dysfunction and gut dysbiosis during MO. The proposed objectives will contribute to the mechanistic insights on the whole food-derived bioactive components present in the macadamia nut to prevent MO-induced complications.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70112191010100%
Knowledge Area
701 - Nutrient Composition of Food;

Subject Of Investigation
1219 - Edible tree nuts, general/other;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Objective 1 - Determine the protective role of dietary macadamia nut against placental lipotoxicity and inflammation during maternal obesity.Objective 2 - Ascertain the protective role of macadamia nut supplementation against maternal obesity-induced fetal fatty liver and inflammationObjective 3 - Determine the preventive role of macadamia nut against maternal intestinal barrier dysfunction and gut dysbiosis during maternal obesity.
Project Methods
Animal model for MO: C57BL/6J mice at 6 weeks of age will be randomly divided into the following groups: 1) an isocaloric control diet feeding (D12450K); 2) a high fat/high sucrose with 45 kcal% fat with 0.2% cholesterol containing diet or HFHS diet (D00071401, open source diets from Research Diets); 3) HFHS diet with 2% macadamia nut containing diet (D2240802); 4) HFHS diet with 5% macadamia nut containing diet (D22040802); 5) HFHS diet with 10% macadamia nut containing diet (D22040803); 6) 2% macadamia nut containing control diet; 7) 5% macadamia nut containing control diet; 8) 10% macadamia nut containing control diet; 9) HFHS diet + MUFAs; 10) HFHS diet + phytosterols; and 11) HFHS diet + MUFA + phytosterol supplementations. The mouse colony will be housed in a 25°C room and food and water will be given ad libitum. Additionally, HFHS diet-fed mice will be supplemented with 20% sucrose-containing drinking water to mimic human phenotype of large for gestation age offspring99, 100. The source of macadamia nuts obtained for the present study is from Hawaii. This MO animal model is already established as described in the preliminary data. Obese and lean animals will then be mated with control diet fed male mice and the vaginal plug will be considered as gestational day 0.5. Placenta will be collected 18.5 days post coitus. The amount of macadamia nut in 5% macadamia nut supplementation, physiologically relevant is equivalent to 1.5-3 ounces of nut consumption in humans. We have planned to use 2% macadamia containing diet (low) and 10% macadamia nut containing HFHS diet as outlined in table 1. Our whole food approach of using macadamia nut replaces saturated fatty acids with MUFA-enriched diets. We have maintained isocaloric diet with similar levels of protein, fat,carbohydrate, and fiber in HFHS diet and in macadamia nut containing HFHS diets (Table 1), were custom made in collaboration with Dr. Pellizzon, Research Diets, Inc .Placental inflammation: We will measure mRNA expression profiles of proinflammatory cytokines like monocyte chemoattractant protein (MCP1), interleukin (IL)-1β, IL-6, IL-18, and TNFα as well as the mRNA levels of IL-10, an anti-inflammatory cytokine in placental tissues, relative to mRNAs expression of acidic ribosomal phosphoprotein P0 (36B4), as an internal control. We will also measure protein levels of proinflammatory cytokines in the maternal circulation and in placental tissues using the multiplex cytokine ELISA.Inflammasome activation: We will measure the activation of nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by measuring two downstream products of its activation: placental IL-1β levels will be measured using ELISA, and placental caspase-1 activity will be measured using caspase 1 specific fluorescent probe (FAM-YVAD-FMK); protein expression of absent in melanoma (AIM)2, NLRP3, and NLR family CARD domain containing 4 receptors and apoptosis-associated speck-like protein containing a CARD (ASC) adaptor; and the levels of auto-cleaved product of caspase 1 p10 subunit using immunoblot analysis as described 101, 102.Circulating markers of endotoxemia: Portal and systemic blood will be used to measure LPS and LPS-binding proteins (LBP) levels, using Aviva Systems Biology Endotoxin ELISA kits.Measurement of FFA and SCFA: We will measure the maternal circulating levels of FFAs from obese dams by solvent extraction of lipids, and fatty acid methylation and quantitated using GCMS 89, 103, 104. Cecum SCFA and circulating SCFA will be measured using GCMS as described105.Terminal deoxyUTP nick-end transferase labelling (TUNEL) and immunohistochemistry (IHC): We will examine fetal placental tissue sections from maternal obese dams with HFHS diet, HFHS with different macadamia nuts containing diets compared to control, low fat diet control and macadamia nut fed-control pregnant mice by co-staining of placental trophoblast markers like keratin 7 with TUNEL for measuring the extent of apoptosis in placental tissue sections. IHC analysis of biochemical markers like cleaved PARP and cleaved caspase 3 will also be detected to measure trophoblast lipoapoptosis in the placental tissue's sections from control, HFHS diet and different macadamia nut containing HFHS diet supplemented animal groups.Experimental methods: The amount of bioactive components will be similar to the diet used in our pilot studies. Measurement of intestinal barrier function: We will measure the FITC-dextran permeability assay, and the expression levels of tight junction proteins like zonula occludens 1 (ZO-1), and claudin?2 will be measured in the intestinal tissue sections of maternal obese mice and HFHS diet with different macadamia nut supplementation and bioactive nutrient supplementation as described 72, 75, 110. We will also stain for mucin-2 proteins and periodic acid-Schiff staining to determine the intestinal villi barrier integrity. Altered glycosylation is associated with increased bacterial adherence to the maternal intestine and can help bacterial translocation79.Microbial composition of 16sRNA sequencing analysis: Fecal samples collected from pregnant female mice for microbial composition analysis at 6.5-, 12.5-, and 18.5-days of gestation to mimic first, second, and third trimesters of pregnancy. Bacterial DNA will be extracted from fecal samples using the QIAamp DNA stool mini kit (Qiagen, CA), Deep amplicon sequencing will be performed using universal primers at Cosmos ID with guidance from the Ramer-Tait lab94. We will employ shallow shotgun metagenome sequencing technology and analysis of microbial composition will performed using the Cosmos ID analysis tool.Expected results and Data analysis: We expect to establish that dietary supplementation of macadamia nut and its bioactive components protects against gut microbiome compositional changes during MO and prevents intestinal barrier dysfunction. Supplementation of bioactive nutrients would also decrease inflammation in the intestine. Non-parametric Kruskal-Wallis test will be employed for groups of three or more, and we will also employ sex as a biological variance and will use SRY gene expression to determine fetal sex. For the in vivo gut dysbiosis and maternal intestine dysfunction study, assuming a coefficient of variation of 30 percent, a power analysis based on the non-parametric Mann-Whitney U test showed that a sample size of n=8 in each group (total n=96 female mice for this objective) would be able to detect a 50 percent difference between lean and obese animals with 80 percent power to compare control pregnant and HFHS diet-fed pregnant mice. In addition, we also plan on using 20 control male mice for mating purpose Data will be presented as mean± SEM and P<0.05 as statistically significant.

Progress 06/01/24 to 05/31/25

Outputs
Target Audience:Our target audience was members of Nebraska Center for Prevention of Obesity Diseases (NPOD) to get feedback about our work and then dissemination of our work in the form of presentation in NPOD symposiums and retreat, UNL Research Days, Society for Reproductive Investigations (SRI), US DoHAD society, Perinatal Biology Symposium, Midlands Society for Physiological Sciencces: a local chapter for American Physiological Sciences in the form of poster and oral presentation. We have also desseminated our finding through scientific journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students attended professional development training workshop in the annual meeting for American Society for Nutrition in 2024. How have the results been disseminated to communities of interest?Deekshika Sekar, graduate student presented her research outcomes in Midlands Society of Physiological Sciences held at University of South Dakota, Vermillion, SD and in the annual meeting of American Soceity for Nutrition. PD will present the outcome and progress report in A1343 PD meeting will be held on June 10-13, 2025 via zoom virtual meeting. What do you plan to do during the next reporting period to accomplish the goals?We are currently working on completing our objective 2 and progress on this will be in the next reporting period.

Impacts
What was accomplished under these goals? Objective 1 - Determine the protective role of dietary macadamia nut against placental lipotoxicity and inflammation during maternal obesity. We established that palmitoleate, an omega-7 monounsaturated fatty acid exerts anti-inflammatory properties in response to lipopolysaccharide (LPS)-mediated inflammation. Exposure of bone marrow-derived macrophages (BMDMs) to LPS or TNFα induces robust increase in the expression of proinflammatory cytokines and supplementation of palmitoleate inhibited LPS-mediated upregulation of proinflammatory cytokines. We also observed that palmitoleate was able to block LPS + ATP-induced inflammasome activation mediated cleavage of procaspase 1 and prointerleukin-1β. Further, treatment of palmitoleate protects against LPS-induced inflammation in human THP-1-derived macrophages and trophoblasts. Coexposure of LPS and palmitate (saturated free fatty acid) induces inflammasome and cell death in BMDMs, however, treatment of palmitoleate blocked LPS and palmitate-induced cell death in BMDMs. Further, LPS and palmitate together results in the activation of mitogen-activated protein kinases and pretreatment of palmitoleate inhibited the activation of mitogen-activated protein kinases and nuclear translocation of nuclear factor kappa B in BMDMs. In conclusion, palmitoleate shows anti-inflammatory properties against LPS-induced inflammation and LPS + palmitate/ATP-induced inflammasome activity and cell death. Objective 2 - Ascertain the protective role of macadamia nut supplementation against maternal obesity-induced fetal fatty liver and inflammation Work in progress Objective 3 - Determine the preventive role of macadamia nut against maternal intestinal barrier dysfunction and gut dysbiosis during maternal obesity. Maternal macadamia-rich diet supplementation (5-10%) significantly enriched beneficial taxa like Akkermansia muciniphila and Bifidobacterium pseudolongum, which are associated with improved gut barrier integrity and metabolic health. The western diet (WD) group exhibited an elevated Firmicutes/Bacteroidetes ratio, indicative of an obesogenic microbiota profile. Metabolically, macadamia supplementation enhanced SCFA-producing pathways (glycolysis, butanoate fermentation) and folate biosynthesis, driven by Bifidobacterium pseudolongum. In contrast, the WD group showed increased activity in pro-inflammatory pathways, such as lipopolysaccharide biosynthesis and branched-chain amino acid metabolism. IMPACT: A macadamia-rich diet effectively remodels maternal gut microbiota, enhances SCFA and folate production, and may mitigate adverse fetal programming linked to maternal obesity. Our findings highlight its potential as a microbiota-targeted dietary intervention.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Liu B, Nguyen PL, Yu H, Li X, Wang H, Nguyen TGB, Sahoo PK, Sur M, Reddy J, Sillman S, Kachman SD, Altartouri B, Lu G, Natarajan SK, Pattabiraman M, Yu J. Honey vesicle-like nanoparticles protect aged liver from non-alcoholic steatohepatitis. Acta Pharm Sin B. 2024 Aug;14(8):3661-3679. doi: 10.1016/j.apsb.2024.05.002. Epub 2024 May 10. PMID: 39220874; PMCID: PMC11365403.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Sahoo PK, Ravi A, Liu B, Yu J, Natarajan SK. Palmitoleate protects against lipopolysaccharide-induced inflammation and inflammasome activity. J Lipid Res. 2024 Nov;65(11):100672. doi: 10.1016/j.jlr.2024.100672. Epub 2024 Oct 11. PMID: 39396700; PMCID: PMC11585775.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Hahka T, Sekar D, Sahoo PK, Ravi A, Freel C, Krishnamoorthy C, Ramamurthy S, Rapoza R, Drakowski R, Akbar A, VanOrmer M, Thoene M, Hanson CK, Nordgren T, Natarajan SK, Anderson Berry A. RvD2 mitigates TNF?-Induced mitochondrial reactive oxygen species through NRF2 signaling in placental trophoblasts. Front Physiol. 2025 Apr 2;16:1547940. doi: 10.3389/fphys.2025.1547940. PMID: 40241717; PMCID: PMC12000658.
  • Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2025 Citation: Jackson MK, Wang B, Rasmussen H, Natarajan SK, Bilek LD, Ehlers DK, Graeff-Armas L, DAngelo C, Cochran T, Harp K, Hanson C. Urinary metabolites as biomarkers of dietary intake: a systematic review. Frontiers in Nutrition, Volume 12 - 2025
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Investigating the Pathogenesis of Hypertensive Disorders of Pregnancy: Role of Placental Inflammation and Mitochondria. Hahka, Taija M et al. Current Developments in Nutrition, Volume 8, 103615


Progress 06/01/23 to 05/31/24

Outputs
Target Audience:Target Audience in Scientific community, Maternal Obese mom (public) and macadamia nut tree farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant provided our lab and my postdoc with a new training opportunity to establish and learn Flow Cytometry from UNL flow cytometry core facility. This flow cytometry expertise that Prakash gained helped him to get his next job as Post Doc at the School of Medicine, Indiana University. How have the results been disseminated to communities of interest?My postdoc, Prakash K Sahoo presented the research outcomes in the Midlands Soceity for Physiological Sciences held at University of Nebraska Medical Center. Prakash also presented the work in the Nebraska Center for Prevention of Obesity Diseases Symposia held at University of Nebraska-Lincoln Natarajan, PD, presented the outcome and progress reporte in the A1343 PD meeting held at Purdue University on May 23, 2024. What do you plan to do during the next reporting period to accomplish the goals?We are currently feeding western diet and western diet containing macadamia nut and will establish our animal model for the successful completion of objective 1-3

Impacts
What was accomplished under these goals? Objective 1 - Determine the protective role of dietary macadamia nut against placental lipotoxicity and inflammation during maternal obesity. We have established that saturated free fatty acids induce trophoblast lipoapoptosis in a concentration dependent manner. We have also demonstrated that caspase activation is crucial for free fatty acid-induced trophblast lipotoxicity. Further, activation of pro-apoptotic BH3 domain containing proteins like p53 upregulated modulator of apoptosis (PUMA) were increased in placental trophoblast lipotoxicity. In addition, we have also shown activation of mitogen activated protein kinases like JNK and ERK. To our surprise JNK activation is critical for lipotoxicity and ERK activation is cell survival. Together we concluded that palmitate induces integrated stress response and lipoapoptosis in trophoblasts. We have also preliminary data to show that monounsaturated fatty acids like Palmitoleate and oleate works against placental lipotoxicity. Further studies on the molecular mechanisms of monounsaturated fatty acid protection against saturated free fatty acid-induced lipotoxicity is currently underway.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Sahoo PK, Krishnamoorthy C, Wood JR, Hanson C, Anderson-Berry A, Mott JL, Natarajan SK. Palmitate induces integrated stress response and lipoapoptosis in trophoblasts. Cell Death Dis. 2024 Jan 11;15(1):31. doi: 10.1038/s41419-023-06415-6. PMID: 38212315. https://www.nature.com/articles/s41419-023-06415-6