PALMITOLEATE PROTECTS AGAINST 3-HYDROXY FATTY ACID-INDUCED LIPOTOXICITY TO PLACENTAL AND MATERNAL LIVER DURING ACUTE FATTY LIVER OF PREGNANCY (AFLP)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1014526
• Project Start Date: Oct 26, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Nutritional & Health Sciences

Non Technical Summary
Acute fatty liver of pregnancy (AFLP) is a pregnancy-induced liver disease. AFLP causes significant perinatal and maternal mortality in the third trimester of pregnancy. AFLP is an inheritable autosomal recessive disease, and highly associated with a mutation in the enzyme, long chain hydroxy acyl CoA dehydrogenase (LCHAD). Since fetal part of placenta is the genetic makeup of the fetus, homozygous fetuses for the defect in LCHAD results in defective placental fatty acid oxidation and accumulation of 3-hydroxy fatty acids in the placenta. Accumulated 3-hydroxy fatty acids were shunted into maternal circulation and affects maternal liver. Toxic exposure of 3-hydroxy fatty acid and saturated free fatty acids to maternal liver results in the development microvesicular steatosis (fatty liver). Our long-term goal is to develop a natural food compound, palmitoleate supplementation as a therapeutic approach to mitigate placental and maternal injury in AFLP. Our preliminary data shows that arachidonic acid results in increased levels of lipid droplets, mitochondrial reactive oxygen species and caspase 3 activity in hepatocytes. We also show that treatment of 3-hydroxy fatty acids to hepatocytes and trophoblast-derived cells results in lipoapoptosis. Further, we observe that co-treatment of palmitoleate protects 3-hydroxy fatty acid-induced trophoblast and hepatocyte lipoapoptosis. In objective 1,Palmitoleate protects against 3-hydroxy fatty acid-induced mitochondrial dysfunction and oxidative stress in hepatocytes and placental trophoblast cells; objective 2, Identify the mechanisms by which palmitoleate protects against 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis and trophoblast necroptosis; and objective 3: Ascertain the protective role of palmitoleate against LCHAD deficiency induced lipotoxicity. The successful completion of these studies will demonstrate that toxic 3-hydroxy fatty acid intermediates induce liver and placental injury during AFLP and will also provide evidence for dietary palmitoleate supplementation mitigating placental and liver injury in AFLP

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
724	3840	1010	100%

Knowledge Area
724 - Healthy Lifestyle;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

mitochondrial fatty acid oxidation defect

nutrient signaling

palmitoleate

placenta and liver lipotoxicity

forkhead family of transcription factor class o 3

receptor interacting protein kinases

lipoapoptosis and necroptosis

Goals / Objectives
Our long-term goal is to develop a nutrient compound that will mitigate placental and maternal liver injury due to 3-HFA-induced lipotoxicity in AFLP patients'. Our central hypothesis for the present hatch grant application is palmitoleate supplementation will mitigate placental and maternal injury in AFLP due to lipotoxicityThe central hypothesis will be tested using following objectivesPalmitoleate protects against 3-hydroxy fatty acid-induced oxidative stress and mitochondrial dysfunction in hepatocytes and placental trophoblast cellsIdentify the mechanisms by which palmitoleate protects against 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis and trophoblast necroptosisAscertian the protective role of palmitoleate against LCHAD deficiency induced lipotoxicity

Project Methods
Objective 1: Palmitoleate protects against 3-hydroxy fatty acid-induced mitochondrial dysfunction and oxidative stress hepatocytes and placental trophoblast cells 1A: Can 3-hydroxy fatty acid induce oxidative stress in trophoblast and hepatocyte mitochondria?We had earlier demonstrated that placenta from patients with AFLP showed mitochondrial dysfunction and oxidative stress [9, 62, 77]. Here, we will determine the mechanistic role for 3-hydroxy fatty acids such as 3-hydroxy myristic acid and 3-hydroxy palmitic acid in causing oxidative stress and mitochondrial dysfunction in the hepatocytes and placental trophoblast cells. Cellular glutathione, oxidative stress markers, like lipid hydroperoxides, and malondialdehyde will be measured; protein oxidation biomarker like protein carbonyls content and cellular thiol status will be analyzed with 3-hydroxy fatty acid exposure using in vitro system. We will also analyze treat the cells with medium chain 3-hydroxy octanoic acid (C8) as control fatty acids. Further, mitochondria will be isolated using differential centrifugation from 3-hydroxy fatty acid exposed cells for the determination of oxidative stress markers. We would also measure the levels of antioxidant enzymes like catalase, mitochondrial superoxide dismutase and glutathione peroxidase with 3-hydroxy fatty acids and palmitoleate treatment conditions. We will determine the activation of EGFR via analyzing its phosphorylation status with 3-hydroxy fatty acids and palmitoleate treatment conditions.1B: Determine the causative role for the LCHAD deficiency in causing oxidative stress, and mitochondrial dysfunction and cell death. We will utilize CRISPR/Cas9 technology system and would create the pathogenic mutation in the Exon 15 of HADHA gene (LCHAD coding gene). We will test the accumulation of 3-hydroxy fatty acids and free fatty acids in placental trophoblast cells and hepatocytes. We will determine the levels of oxidative stress biomarkers, nitrosative stress markers like nitric oxide levels and antioxidant enzyme activities. We will also utilize seahorse instrument to detect the mitochondrial function in LCHAD mutated placental trophoblast and hepatocytes. We would also test the protective role of palmitoleate against 3-hydroxy fatty acid-induced oxidative and nitrosative stress. We also anticipate that knockdown of LCHAD or mutation of LCHAD would result in an accumulation of 3-hydroxy fatty acids. Lipotoxic effect of 3-hydroxy fatty acids will be measured by analyzing the percent apoptotic nuclear morphological characteristics changes and caspase3/7 activity compared to control transfected cells. The levels of 3-hydroxy fatty acids will be measured using GCMS available in NPOD core facility lab, UNL.1C: Palmitoleate protects from liver damage in pregnant wild-type mice injected with 3-hydroxy fatty acids (3-HFA): We will test the protective role for palmitoleate in preventing 3-HFA-induced liver damage in wild-type C57BL/6 pregnant mice. We will measure the biochemical markers of liver injury in serum from the animals administered with 3-HFA using Vitro chemistry analyzer available in the NPOD BORC core facility. Palmitoleate (600 mg/kg body weight) will be supplemented to wild-type mice in diet during 3-HFA injection. Further, we will establish the characteristics or symptoms of AFLP patients like hyperbilirubinemia, cholestasis, hypoglycemia, insulin sensitivity and hepatic encephalopathy in 3-HFA injected animals. We are also aware of the difference in the metabolic activity of rodents and humans. We will modify our concentration of 3-HFA administration after our pilot lipotoxicity experiments. Palmitoleate is also anticipated to modulate the pro-apoptotic mediators in maternal liver and placenta. Clarifying the mechanisms of protection from lipotoxicity will help improve the treatment of a maternal multi-organ injury that occurs due to 3-hydroxy fatty acids and free fatty acids in AFLP.Objective 2: Identify the mechanisms by which palmitoleate protects against 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis and placental trophoblast necroptosis2A: Determine the cellular fate of 3-hydroxy fatty acid metabolite such as toxic lysophosphatidyl choline and lipotoxicity? Here, we will determine the cellular fate of 3-hydroxy fatty acid and test the formation of lysophosphatidyl choline in 3-hydroxy fatty acid-induced lipotoxicity. 3-hydroxy fatty acids can be incorporated into diacylglycerol, and phosphatidyl choline can be derived from diacylglycerol. Oxidative stress has been shown to activate cytosolic phospholipase A2 (cPLA2) to metabolize phosphatidyl choline or phosphatidic acid into lysophosphatidyl choline which can enhance placental trophoblast- and hepatocyte lipotoxicity. We hypothesize that 3-hydroxy fatty acid-induced oxidative stress would activate phospholipase A2 for the increased generation of lysophosphatidic choline. We will determine the levels of lysophosphatidyl choline in 3-hydroxy fatty acid treated cells using thin-layer chromatography for the separation of neutral lipids and GCMS analysis, in vitro. Briefly, cell lysate, lipid droplets, and subcellular membrane will be isolated from the cells treated with 3-hydroxy fatty acids after 16h for lysophospholipids measurements. We will also determine the expression of cPLA2 using western blot analysis with 3-hydroxy fatty acid treatment.2B: Determine the mechanism of palmitoleate protection against 3-hydroxy fatty acid-induced placental trophoblast necroptosis.Our preliminary data demonstrate that 3-hydroxy fatty acids induce placental trophoblast necroptosis. 3-HFA induced a caspase-independent cell death and co-treatment of necrostatin-1, an inhibitor receptor interacting protein kinase (RIPK) 1 with 3-HPA abolished 3-HFA-induced placental trophoblast cell death suggesting a critical role for RIPK1. Further, we will aim to confirm 3-HFA-induced placental trophoblast necroptosis using Z-VAD fmk (pan-caspase inhibitor) and assess Annexin V+ and propidium iodide positive cell using flow cytometry available in the Nebraska Center for Virology, UNL.2C: Palmitoleate prevents 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis via Wnt3a secretion Experiments in this sub-aim will determine whether the mechanism of palmitoleate protection is via secretion of Wnt3a/β-catenin signaling and FoxO3 inactivation. Loss of β-catenin results in decreased expression of its downstream target, serum- and glucocorticoid-inducible protein kinase 1 (SGK-1) [106-115]. SGK-1 is known to phosphorylate FoxO3 and reduces the pro-apoptotic function of FoxO3 which results in enhanced cell survival. Palmitoylation of Wnt3a can secrete through exosomes and act as a morphogen in the plasma membrane in an autocrine or paracrine fashion to stabilize β-catenin in association with the LRP6 (LDL receptor-related protein 6) receptor and Frizzled protein receptor (a family of G-protein coupled receptor). Stable β-catenin could induce the expression of SGK-1, which can phosphorylate FoxO3 and decrease the nuclear accumulation of FoxO3 [106-115]. We will also test the critical role of Wnt3a, SGK-1, FoxO3 and β-catenin using stable knockdowns by CRISPR/Cas9 technology. We will also determine the role of porcupine (O-acyl transferase) in palmitoylation of Wnt3a and its secretion from the ER for palmitoleate protection against 3-HFAs lipotoxicity. Here, we hypothesize that 3-HFA-induced lipoapoptosis can result in the activation of GSK-3β and β-catenin phosphorylation which initiates the ubiquitination and proteasomal degradation of β-catenin will be elucidated

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems:The pandamic created a major challenge to my lab graduate students What opportunities for training and professional development has the project provided?Natarajan Lab trained new undergraduate student, Jenna Whitmore from the Nebraska Weselyan University with our lab techniques. Jenna was funded by the undergraduate scholarship for her research from Nebraska Weselyan University. How have the results been disseminated to communities of interest?We have submitted our work in the Nebraska Center for Prevention of Obesity Disease annual symposium and in Greenwald Symposium on Reproduction held virtually via zoom. My graduate students have also submitted abstracts to Experimental Biology 2020 Conference. Unfortunately, this conference was cancelled due to the pandemic, however their abstracts were published in Faseb J. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We will work on the mechanism of lipoapoptosis in hepatocytes and necroptosis in the placenta of 3-HFA administered pregnant mice Objective 2: We have observed increased nuclear translocation of FoxO3 with 3-HMA- and 3-HPA-induced hepatocyte lipoapoptosis. We will work on the post-translational modifications of FoxO3 such as acetylation and phosphorylation and the levels of microRNA 34a, a downstream target of FoxO3. Objective 3: We will work on elucidating role of MAPK activation in placental trophoblast cells with 3-HFA treatment and in 3-HFA administered pregnant mice's placentae.

Impacts
What was accomplished under these goals? We have established that 3-hydroxy fatty acid administration to pregnant wild type C57BL/6 mice induces hepatocyte injury, in vivo. We have also identified that 3-hydroxy fatty acids induce p38 mitogen activated protein kinase-dependent hepatocyte lipoapoptosis. Further, the activation and role of receptor interacting protein kinase and its downstream kinases were established in 3-hydroxy fatty acid-induced placental trophoblast necroptosis Progress Toward Obejctive 1: We have also observed that 3-hydroxy fatty acids induces retinal pigment epithelial cell lipoapoptosis. The mechanism of 3-hydroxy fatty acid-induced retinal pigment epithelial cell is by the activation of caspase 3/7 enzymes and inhibition of caspases using small molecule dramatically blocked 3-HFA-induced retinal lipoapoptosis. We also observed that biochemical markers of apoptosis were increased with 3-HFA treatment to retinal pigment epithelial cells, and showed increased levels of cleaved poly ADP ribose polymerase (PARP), p53 upregulated modulator of apoptosis (PUMA) and cleaved caspase 3. However, similar concentration of saturated free fatty acid (100-250 μM) treatment to retinal pigment epithelial cells did not induce lipoapoptosis. These data suggest that 3-HFA is a lipotoxic fatty acids that are elevated in patients with AFLP could result in multi-organ failure. Mechanistically, 3-HFA-induced retinal lipoapoptosis involves the activation of cJun N-terminal kinase (JNK) and small molecule inhibition of JNK using SP600125 dramatically prevents 3-HFA-induced retinal lipoapoptosis. Progress towards Aim 2: We have earlier observed that pathophysiological concentration of 3-HFA to placental trophoblasts showed caspase-independent cell death. Our data shows that 3-HFA exposure to placental trophoblast cells induces necroptosis (i.e., inflammatory cell death). 3-HFA-induced trophoblast necroptosis involves the activation of receptor interacting protein kinase (RIPK) 1 and mixed-lineage kinase domain-like protein (MLKL) via phosphorylation. We have also observed that 3-HFA induces plasma membrane translocation of phospho-MLKL suggesting its role in necrosome formation. We are currently working on immunoprecipitation of MLKL to detect its bindering partners like RIPK1 and RIPK3 with 3-HFA treatment in placental trophoblasts and in the placenta administered with 3-HFA. Interestingly, we have observed that 3-HFA treatment increases the levels of phosphorylated forms of peroxisomal proliferator-activated receptor alpha (PPARα) and increased transcriptional expression of its downstream target, carnitine palmitoyl transferase 1a (CPT1a) in the placental trophoblasts. These data suggest that activation of PPARα would protect against apoptosis in placental trophoblast with 3-HFA, however this protective function does not seem to block 3-HFA-induced trophoblast necroptosis. The critical role for RIPK1 in 3-HFA-induced placental trophoblast necroptosis will be further established using short hairpin RNA against RIPK1. Progress towards Objective 3: We have demonstrated that the administration of 3-hydroxy fatty acids (3?HFA) such as 3-hydroxy myristic acid (3-HMA) and 3-hydroxy palmitic acid (3-HPA) results in liver injury as evidenced by an increase in the circulating markers of hepatocyte injury like alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the maternal circulation. We are currently working on establishing lipoapoptosis in hepatocytes and necroptosis in the placenta of 3-HFA administered pregnant mice.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Muthuraj PG, Sahoo PK, Kraus M, Bruett T, Annamalai AS, Pattnaik A, Pattnaik AK, Byrareddy SN, Natarajan SK. Zika virus infection induces endoplasmic reticulum stress and apoptosis in placental trophoblasts. Cell Death Discovery. 2020 (in press).
• Type: Book Chapters Status: Awaiting Publication Year Published: 2020 Citation: Mohr A, Sahoo PK, Muthuraj PG, Spriet MR, Mott JL, Natarajan SK*. Epigenetics, Noncoding RNAs, and Gene Expression. Comprehensive Foodomics, 2020 Chapter 00021. Elsevier Publications. *Corresponding author. Editor/Contributor: Alejandro Cifuentes, Epigenetics & Noncoding RNA Section Editor: Juan Cui
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Muthuraj PG, Natarajan, SK. Fetal Programming in Maternal Obesity. Diabesity 2020; 6(3): 36-39. DOI:10.15562/diabesity.2020.71
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Thoene M, Van Ormer M, Lyden E, Thompson M, Yuil-Valdes A, Natarajan SK, Mukherjee M, Nordgren TM, Furtado, Anderson-Berry A, Hanson C, Snowden J. Inflammatory Compounds and Fat-Soluble Nutrients in Mother-Infant Dyads at Time of Delivery. Pediatr Res (2020). In Press.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:We have dessiminated our results in scientific community through presentations in conferences and publications Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Natarajan lab travelled to Dr. Theresa Powell lab at the University of Colorado Anschutz Medical Campus in Aurora, CO to learn the isolation of human placental trophoblast cells from term placenta. Further, we also learned to isolated placental brush border membrane and basolateral membrane from the placenta. How have the results been disseminated to communities of interest?My graduate students and lab manager presented their research work in the annual fall sympoisum and spring retreat of the Nebraska Center for the Prevenstion of Obesity and Obesity-related diseases (NPOD), in the 16th Greenwald Symposium on Reproduction and Perinatal Research held at the Kansas University Medical Center and in American Association for the Study of Liver Disease (AASLD). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We will plan on testing the lipotoxic role of 3-hydroxy fatty acids in retinal pigment epithelial cells and measure the lipoapoptosis Objective 2: We will test the protective role of palmitoleate protection against 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis Objective 3: We will inject 3-hydroxy fatty acids to pregant mice to test the oxidative stress, mitochondrial dysfunction and lipoapoptosis in the placenta, maternal liver and fetal liver

Impacts
What was accomplished under these goals? IMPACT: We have established placental trophoblast necroptosis and hepatocyte lipoapoptosis with the exposure of 3-hydroxy fatty acids. We also showed mitochondrial structural and functional damage and activation of mitogen-activated protein kinase in the hepatocytes treated with 3-hydroxy fatty acids. We also identified the activation of pro-apoptotic transcription factor class O 3 (FoxO3) in hepatocytes treated with 3-hydroxy fatty acids. Progress towards Objective 1: We have demonstrated that the treatment of 3-hydroxy fatty acids (3?HFA) such as 3-hydroxy myristic acid (3-HMA) and 3-hydroxy palmitic acid (3-HPA) results in hepatocyte lipoapoptosis. Primary hepatocytes treated with 3-HMA or 3-HPA showed increased percent apoptotic nuclei levels; similarly, we saw an increased caspase 3/7 activation with increasing concentration of 3-HMA and 3-HPA treatment suggesting hepatocyte lipoapoptosis. We went on detecting structural changes in the hepatocytes with 3-HFA treatment using electron microscope and visualized apoptotic event such as mitochondrial structural damage, nuclear fragmentation, and nuclear condensation in 3-HMA and 3-HPA treated cells as compared to vehicle treatment. We also observed that 3?hydroxy fatty acid-induced a caspase-dependent hepatocyte lipoapoptosis. Additionally, we have found the evidence for the activation of mitogen-activated protein kinases (MAPK) like JNK, ERK, and p38 in the hepatocytes treated with 3-hydroxy fatty acids (3-HFA). Both 3-HMA and 3-HPA treated cells showed an increase in the levels of phosphorylated forms of JNK, p38 and ERK in the hepatocytes suggesting MAPK activation. However, total forms of JNK, p38 and ERT were unchanged. We are currently working on whether the activation of MAPK is critical for hepatocyte lipoapoptosis. We are also working on to elucidate the MAPK activation in placental trophoblast cells with 3-hydroxy fatty acid treatment. Progress towards Objective 2: Placental trophoblast cells exposed to the pathophysiological concentrations of 3-hydroxy fatty acids showed a caspase-independent cell death. These data are in contrast to the hepatocytes, where we observed caspase activation and lipoapoptosis. Further, we also found the mechanistic evidence for necroptosis in 3-hydroxy fatty acid-induced placental trophoblast cell death. Palmitoleate, a mono-unsaturated fatty acids treatment showed protection against 3?hydroxy fatty acid-induced hepatocyte lipoapoptosis and placental trophoblast necroptosis. We will begin to elucidate the mechanism of palmitoleate protection against 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis and placental necroptosis. To look for the mechanism of hepatocyte lipoapoptosis with 3-hydroxy fatty acids, we sought to analyze FoxO3, a pro-apoptotic transcription factor with 3-HFA treatment. We observed a dramatic increase the nuclear levels of FoxO3 with 3-HMA and 3-HPA treatment, suggesting the involvement of pro-apoptotic FoxO3 in 3-HFA-induced hepatocyte lipoapoptosis. Further, FoxO3 downstream target, PUMA a pro-apoptotic BH3 containing protein levels were also increased in cells treated with 3-HMA or 3-HPA. We will begin to look for the post-translational modifications and other downstream targets of FoxO3 like microRNA 34a. We also observed that 3-hydroxy fatty acids induce significant increase in the cellular triacylglycerol levels and lipid droplets accumulation in the hepatocytes. Further, we observed microvesicular steatosis in the hepatocytes treated with 3-HMA or 3-HPA, a liver pathology observed similar to the patients with acute fatty liver of pregnancy. Progress towards Objective 3: For our initial experiments on hepatocyte lipotoxicity, we have teamed up with UNL and UNMC investigators who work on liver disease in sharing the primary hepatocytes. We have obtained primary hepatocyte from Dr. Casey, UNMC for the primary hepatocytes from rat liver. We will begin to breed our colony of mice with LCHAD deficiency to establish placental mitochondrial dysfunction, oxidative stress and lipoapoptosis. Rigor: We have maintained experimental rigor and unbiased approach to the studies conducted and reported here. The statistical analysis like ANOVA with post- hoc Bonferroni corrections were employed to avoid any false positive results.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ulu A#, Sahoo PK#, Yuil-Valdes AG, Mukherjee M, VanOrmer M, Muthuraj PG, Thompson M, Anderson-Berry A, Hanson CK, Natarajan SK*, Nordgren TM*. Omega-3 fatty acid-derived resolving D2 regulates human placental vascular smooth muscle and extravillous trophoblast activities. Int. J. Mol. Sci. 2019
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: 3-Hydroxy Fatty Acids Induce Retinal Pigment Epithelial Cell Lipoapoptosis. By Mona Hadidi, https://digitalcommons.unl.edu/nutritiondiss/83/
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2019 Citation: Abstract ID: R2385 Sahoo P, Natarajan SK Abstract Title: '3-hydroxy fatty acids Induce Placental Trophoblast Necroptosis during Acute Fatty Liver of Pregnancy' Topic Category: 1180-APS Alcoholic and nonalcoholic fatty liver diseases

Progress 10/26/17 to 09/30/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?My graduate student and postdoctoral fellow presented their research in the annual retreat of the Nebraska Center for the Prevention of Obesity and obesity-related disease (NPOD), in the annual meetings for the Society for the Study of Reproduction (SSR) and American Association for the study of Liver Disease (AASLD) What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We will also plan on testing how 3-hydroxy fatty acids induces pro-apoptotic mediators of cell death pathway and mitochondrial function using seahorse instrument in hepatocytes and placental trophoblast cells Objective 2: Experiments are underway to test whether palmitoleate can prevent placental trophoblast lipoapoptosis. Objective #3: We are in the process of signing material transfer agreement with our collaborator and will soon begin our colony of mice with LCHAD deficiency to establish placental mitochondrial dysfunction, oxidative stress and lipoapoptosis.

Impacts
What was accomplished under these goals? Patients with acute fatty liver of pregnancy (AFLP) have elevated levels of toxic 3-hydroxy-fatty acids and free fatty acids in the circulation. The fetal part of placenta has the genetic makeup of the fetus; therefore, fetuses with homozygous mutations in the enzyme long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) were highly associated with this pregnancy-induced liver disease. We had earlier demonstrated that defective placental fatty acid oxidation results in the accumulation of fatty acids in the placenta and toxic free fatty acids released from the placenta were shunted into the maternal circulation. Further, we have also shown elevated levels of myristic acid, palmitic acid, and arachidonic acid in the circulation of AFLP patients. Increased circulating levels of 3-hydroxy-fatty acids have also been reported in patients with AFLP, however the exact mechanism of 3-hydroxy-fatty acid-induced lipotoxicity to placental trophoblast and hepatocyte were unknown. This project is unique and conceptually innovative as it tests 3-hydroxy-fatty acid lipotoxicity in a cross-organ model of injury between the placenta and liver cells. Our long-term goal is to develop a nutrient compound that will mitigate placental and maternal liver injury due to 3-HFA-induced lipotoxicity in AFLP patients'. Our central hypothesis for the present hatch grant application is palmitoleate supplementation will mitigate placental and maternal injury in AFLP due to lipotoxicity. The central hypothesis will be tested using following objectives Palmitoleate protects against 3-hydroxy fatty acid-induced oxidative stress and mitochondrial dysfunction in hepatocytes and placental trophoblast cells Identify the mechanisms by which palmitoleate protects against 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis and trophoblast necroptosis Ascertian the protective role of palmitoleate against LCHAD deficiency induced lipotoxicity Progress towards Objective #1: We have demonstrated that treatment of 100-200 µM of 3-hydroxy fatty acids such as 3-hydroxy myristic acid and 3-hydroxy palmitic acid results in significant increase in lipoapoptosis in the hepatoma cell line (Huh7 cells) as evidenced by an increase in percent apoptotic nuclear morphology changes and caspase 3/7 activity. Hepatocytes exposed with 100 µM of 3-hydroxy-myristic acid plus 200 µM palmitate results in a synergistic effect on hepatocyte lipoapoptosis with a dramatic increase in caspase 3/7 activity compared to the cells treated with 3-hydroxy-myristic acid alone. Our next approach is to test the effect of 3-hydroxy fatty acids on placental trophoblast lipoapoptosis. We will also plan on testing how 3-hydroxy fatty acids induces pro-apoptotic mediators of cell death pathway and mitochondrial function using seahorse instrument in hepatocytes and placental trophoblast cells. 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis reported is a significant accomplishment of this objective. Progress towards Objective #2: Palmitoleate, a mono-unsaturated fatty acid were shown to protect against hepatocyte and cholangiocyte lipoapoptosis. Here we have found that palmitoleate significantly protects 3-hydroxy-fatty acid-induced hepatocyte lipoapoptosis. Treatment of palmitoleate was also protective against palmitate plus 3-hydroxy fatty acid-induced hepatocyte lipoapoptosis. Experiments are underway to test whether palmitoleate can prevent placental trophoblast lipoapoptosis. Progress towards Objective #3: Our goal for this objective is to show the protective role of palmitoleate against LCHAD deficiency induced lipotoxicity. We will soon begin our colony of mice with LCHAD deficiency to establish placental mitochondrial dysfunction, oxidative stress and lipoapoptosis.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: 1. Natarajan SK, Ibdah JA, Role of 3-hydroxy fatty acid-induced hepatic lipotoxicity in acute fatty liver of pregnancy. Int. J. Mol.Sci 2018,19, 322. PMID: 29361796.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: 1. Kim Y, Natarajan SK, Chung S. Gamma-tocotrienol attenuates the hepatic inflammation and fibrosis by suppressing endoplasmic reticulum stress in mice. Mol Nutr Food Res. 2018 Nov; 62(21):e1800519. Doi: 10.1002/mnfr.201800519. PMID: 30183139