Melatonin Protects Against Mdivi-1-Induced Abnormal Spindle Assembly and Mitochondrial Superoxide Production During Porcine Oocyte Maturation

Mitochondrial division inhibitor 1 (Mdivi-1) reportedly provides a close connection between oocyte maturation and mitochondrial function in pigs. N-acetyl-5-methoxy-tryptamine (melatonin) is known to be a representative antioxidant with the ability to rehabilitate meiotic maturation of porcine oocytes. However, the ability of melatonin to recover Mdivi-1-mediated disruption of spindle formation during meiotic maturation of porcine oocytes during in vitro maturation (IVM) has not been studied. Here, we first investigated changes in mitochondrial length, such as fragmentation and elongation form, in mature porcine oocytes during IVM. Mature oocytes require appropriate mitochondrial fission for porcine oocyte maturation. We identified a dose-dependent reduction in meiotic maturation in porcine oocytes following Mdivi-1 treatment (50, 75, and 100 μM). We also confirmed changes in mitochondrial fission protein levels [dynamin-related protein 1 phosphorylation at serine 616 (pDRP1-Ser616) and dynamin-related protein 1 (DRP1)], mitochondrial membrane potential, and ATP production in 75 μM Mdivi-1-treated oocytes. As expected, Mdivi-1 significantly reduced mitochondrial function and DRP1 protein levels and increased spindle abnormalities in porcine oocytes. In addition, we confirmed that melatonin restores abnormal spindle assembly and reduces meiotic maturation rates by Mdivi-1 during porcine oocyte maturation. Interestingly, the expression levels of genes that reduce DNA damage and improve tubulin formation were enhanced during porcine meiotic maturation. Taken together, these results suggest that melatonin has direct beneficial effects on meiotic maturation through tubulin formation factors during porcine oocyte maturation.

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207 GANGLIOSIDE IMPROVES THE MEIOTIC MATURATION AND ANTI-APOPTOTIC EFFECTS DURING IN VITRO MATURATION OF PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab207 ◽

2016 ◽

Vol 28 (2) ◽

pp. 234

Author(s):

J.-W. Kim ◽

H.-J. Park ◽

S.-K. Chae ◽

J.-H. Ahn ◽

G.-Y. Do ◽

...

Keyword(s):

Oocyte Maturation ◽

Egf Receptor ◽

Untreated Group ◽

Meiotic Maturation ◽

Signal Molecules ◽

Developmental Potential ◽

Protein Levels ◽

Porcine Oocyte ◽

Apoptotic Effects

Gangliosides are sialic-acid-glycosphingolipids expressed in almost all vertebrate cells. These play a role in regulating cell differentiation and signal molecules of cellular signaling, including interactions with molecules of signal transduction pathways, such as receptors tyrosine kinases (RTK). Gangliosides perform critical functions through interactions with proteins as receptors in cell‐cell recognition, such as the epidermal growth factor (EGF) receptor (EGFR). Recent studies investigated functional a link between gangliosides-derived RTK signaling and embryo development until blastocyst in mice. However, there is no report on the directly relationship of GM3 or GD1a and oocyte maturation in pigs. Our studies have verified an interaction mechanism between EGF receptor signaling and gangliosides (GM3 and/or GD1a) in porcine oocyte during meiotic maturation and further development. The GM3 expression increased in mouse oocytes that were in vitro-matured (IVM) under apoptotic conditions. To induce the apoptotic conditions of oocytes, we used H2O2. We first confirmed that the expression of GM3 in oocytes of H2O2-treated groups (1 mM) was gradually increased compared with those of the control group (P < 0.05). We investigated the anti-apoptotic effect of GM3 as the inhibition apoptosis in porcine oocyte IVM by treatment of GM3 (5 or 10 μM) after pretreatment with H2O2. Next EGFR protein levels and meiotic maturation were investigated by Western blot analysis and acetic-orcerin staining, respectively, of IVM oocytes. The proportion of germinal vesicle arrested oocytes at 22 h was significantly increased (P < 0.05; 41.6 ± 1.5% v. 25.0 ± 0.0% in the untreated group) in EGF (10 ng mL–1) + GD1a (10 μM) treated group. To confirm the completion of meiotic maturation, we investigated the proportion of metaphase II (MII) oocytes at 44 h. The MII oocytes were increased (P < 0.05; 89.9 ± 3.6% v. 57.4 ± 5.3% in the untreated group) in oocytes cultured with EGF+GD1a. Interestingly, p-EGFR protein levels of matured oocytes were significantly increased (P < 0.05) by the EGF+GD1a treatment in the maturation process. In addition, EGF+GD1a treatment increased the proportion of normal pronucleus formation (2PN of 43.1 ± 5.2%) and increased (P < 0.05; 50.4 ± 6.1% v. 27.2 ± 2.7% in the untreated group) pre-implantation developmental potential until blastocyst. These results suggest that EGF+GD1a treatment improved the developmental competence of embryos via enhanced meiotic maturation of porcine oocytes by inducing EGFR activation. We confirmed that GM3 has anti-apoptotic effects during porcine oocyte maturation periods. Furthermore, these data will be helpful for better understanding the receptor mechanisms during oocyte maturation in pigs.

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Butylparaben Is Toxic to Porcine Oocyte Maturation and Subsequent Embryonic Development Following In Vitro Fertilization

International Journal of Molecular Sciences ◽

10.3390/ijms21103692 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3692 ◽

Cited By ~ 1

Author(s):

Pil-Soo Jeong ◽

Sanghoon Lee ◽

Soo-Hyun Park ◽

Min Ju Kim ◽

Hyo-Gu Kang ◽

...

Keyword(s):

Embryonic Development ◽

Oocyte Maturation ◽

Mitochondrial Function ◽

Cumulus Cell ◽

Annexin V ◽

Ros Generation ◽

Vitro Fertilization ◽

Porcine Oocyte ◽

Mitochondrial Distribution

Parabens are widely used in personal care products due to their antimicrobial effects. Although the toxicity of parabens has been reported, little information is available on the toxicity of butylparaben (BP) on oocyte maturation. Therefore, we investigated the effects of various concentrations of BP (0 μM, 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM) on the in vitro maturation of porcine oocytes. BP supplementation at a concentration greater than 300 μM significantly reduced the proportion of complete cumulus cell expansion and metaphase II oocytes compared to the control. The 300 μM BP significantly decreased fertilization, cleavage, and blastocyst formation rates with lower total cell numbers and a higher rate of apoptosis in blastocysts compared to the control. The BP-treated oocytes showed significantly higher reactive oxygen species (ROS) levels, and lower glutathione (GSH) levels than the control. BP significantly increased the aberrant mitochondrial distribution and decreased mitochondrial function compared to the control. BP-treated oocytes exhibited significantly higher percentage of γ-H2AX, annexin V-positive oocytes and expression of LC3 than the control. In conclusion, we demonstrated that BP impaired oocyte maturation and subsequent embryonic development, by inducing ROS generation and reducing GSH levels. Furthermore, BP disrupted mitochondrial function and triggered DNA damage, early apoptosis, and autophagy in oocytes.

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172 THE APOPTOTIC EFFECTS OF BISPHENOL A-INDUCED MITOCHONDRIAL-DERIVED REACTIVE OXYGEN SPECIES ON MATURATION OF PORCINE OOCYTES IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab172 ◽

2017 ◽

Vol 29 (1) ◽

pp. 194

Author(s):

S.-Y. Park ◽

H.-J. Park ◽

J.-W. Kim ◽

J.-Y. Park ◽

S.-G. Yang ◽

...

Keyword(s):

Oocyte Maturation ◽

Critical Role ◽

Cumulus Cell ◽

Cumulus Cells ◽

Meiotic Maturation ◽

Control Group ◽

Ros Production ◽

Maturation In Vitro ◽

Porcine Oocyte

Bisphenol A (BPA) is well known as oestrogen-like chemical and it is widely used in plastic products. Many studies have reported that BPA exposure has a well-known toxicity effect on reproduction function, such as reducing the number of ovulated oocytes, oocyte quality, and maturation rate. Recently, BPA induced mitochondrial-derived reactive oxygen species (mito-ROS) and disrupted mitochondrial homeostasis by increasing of superoxide anions production. In this study, we investigated how the regulation of mito-ROS production may play a critical role in meiotic maturation and expansion of cumulus cells during the in vitro maturation progression of porcine oocytes. Furthermore, we investigated the toxicity effect of BPA exposure on mitochondrial functions and mito-ROS production during porcine oocyte maturation in vitro. All results were analysed using a 1-way ANOVA followed by Bonferroni’s and Tukey’s Multiple Comparison Test and t-tests. First, porcine oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75, and 100 µM) for 44 h. Our results indicated that the rates of matured oocytes were significantly decreased by BPA exposure in a dose-dependent manner (69.4 ± 5.1, 50.9 ± 6.3, and 29.9 ± 5.8% for BPA treatments of 50, 75, and 100 μM) compared with control group (70.2 ± 7.8%; P < 0.05). Next, we confirmed the secretion functions of oocyte and cumulus cell of cumulus-oocyte complex (COC) and ROS production. Cumulus cell secretion factors (has2, tnfaip6, and cx37) mRNA expression in COC were decreased in the BPA-treated (75 µM) group. In addition, mRNA expressions of mitochondrial-specific antioxidant enzymes (sod2, P < 0.001; prdx3, P < 0.01; prdx5, P < 0.001) and mitochondrial apoptosis genes (bax and caspase-3, P < 0.01) were significantly increased in COC of the BPA-treated (75 µM) group. We measured mitochondrial membrane potential and mito-ROS production using JC-1 analysis and Mito-SOX staining, respectively. The BPA treatment caused a rapid decrease of mitochondrial membrane potential maintenance and increase of mito-ROS production in porcine COC. Moreover, mitochondrial-specific ROS scavenger, Mito-Tempo (0.1 µM) treatment was significantly increased the meiotic maturation of porcine oocytes compared with control group (78.5 ± 3.5 v. 65.8 ± 5.0%; P < 0.05). Based on these results, we first confirmed that BPA exposure reduces the meiotic maturation and cumulus cells expansion of COC by increasing mito-ROS production during porcine oocyte maturation in vitro. Therefore, controlling of mito-ROS for mitochondrial function maintenance and apoptosis plays a critical role in improving porcine oocyte maturation in vitro. This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037–04–1-HD020) through the Rural Development Administration, the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

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Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes

Animals ◽

10.3390/ani9040163 ◽

2019 ◽

Vol 9 (4) ◽

pp. 163

Author(s):

Payungsuk Intawicha ◽

Li-Kuang Tsai ◽

Shih-Ying Yen ◽

Neng-Wen Lo ◽

Jyh-Cherng Ju

Keyword(s):

Oocyte Maturation ◽

Mammalian Cells ◽

In Vitro Maturation ◽

Germinal Vesicle ◽

Mitogen Activated Protein Kinase ◽

Meiotic Maturation ◽

Germinal Vesicle Breakdown ◽

Mitogen Activated Protein ◽

Porcine Oocyte

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

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Luteolin Prevents Cardiac Dysfunction and Improves the Chemotherapeutic Efficacy of Doxorubicin in Breast Cancer

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.750186 ◽

2021 ◽

Vol 8 ◽

Author(s):

Youyang Shi ◽

Feifei Li ◽

Man Shen ◽

Chenpin Sun ◽

Wei Hao ◽

...

Keyword(s):

Breast Cancer ◽

Mitochondrial Fission ◽

Mitochondrial Morphology ◽

Protective Effects ◽

Myocardial Cells ◽

Related Protein ◽

Zebrafish Model ◽

Protein Levels

Background: Doxorubicin (Dox) is one of the most effective chemotherapy agents used in the treatment of solid tumors and hematological malignancies. However, it causes dose-related cardiotoxicity that may lead to heart failure in patients. Luteolin (Lut) is a common flavonoid that exists in many types of plants. It has been studied for treating various diseases such as hypertension, inflammatory disorders, and cancer. In this study, we evaluated the cardioprotective and anticancer effects of Lut on Dox-induced cardiomyopathy in vitro and in vivo to explore related mechanisms in alleviating dynamin-related protein (Drp1)-mediated mitochondrial apoptosis.Methods: MTT and LDH assay were used to determine the viability and toxicity of cardiomyocytes treated with Dox and Lut. Flow cytometry was used to examine ROS levels, and electron and confocal microscopy was employed to assess the mitochondrial morphology. The level of apoptosis was examined by Hoechst 33258 staining. The protein levels of myocardial fission protein and apoptosis-related protein were examined using Western blot. Transcriptome analysis of the protective effect of Lut against Dox-induced cardiac toxicity in myocardial cells was performed using RNA sequencing technology. The protective effects of Lut against cardiotoxicity mediated by Dox in zebrafish were quantified. The effect of Lut increase the antitumor activity of Dox in breast cancer both in vitro and in vivo were further employed.Results: Lut ameliorated Dox-induced toxicity in H9c2 and AC16 cells. The level of oxidative stress was downregulated by Lut after Dox treatment of myocardial cells. Lut effectively reduced the increased mitochondrial fission post Dox stimulation in cardiomyocytes. Apoptosis, fission protein Drp1, and Ser616 phosphorylation were also increased post Dox and reduced by Lut. In the zebrafish model, Lut significantly preserved the ventricular function of zebrafish after Dox treatment. Moreover, in the mouse model, Lut prevented Dox-induced cardiotoxicity and enhanced the cytotoxicity in triple-negative breast cancer by inhibiting proliferation and metastasis and inducing apoptosis.

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All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission

Cells ◽

10.3390/cells10051202 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1202

Author(s):

Bojjibabu Chidipi ◽

Syed Islamuddin Shah ◽

Michelle Reiser ◽

Manasa Kanithi ◽

Amanda Garces ◽

...

Keyword(s):

Retinoic Acid ◽

Mitochondrial Fission ◽

Normal Function ◽

Mitochondrial Network ◽

Protein Levels ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Area And Perimeter

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

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Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes

Journal of Animal Science ◽

10.1093/jas/skab072 ◽

2021 ◽

Author(s):

Sicong Yu ◽

Lepeng Gao ◽

Yang Song ◽

Xin Ma ◽

Shuang Liang ◽

...

Keyword(s):

Oxidative Stress ◽

Mitochondrial Dysfunction ◽

Oocyte Maturation ◽

Mitochondrial Function ◽

Protective Action ◽

Damage Model ◽

Developmental Competence ◽

Free Calcium ◽

Maturation In Vitro

Abstract Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which glycine affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether glycine could reverse the mitochondrial dysfunction induced by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, induced oxidative stress, which was confirmed by decreased mitochondrial membrane potential (Δ⍦m) and the expression of mitochondrial function-related genes (PGC-1α), and increased reactive oxygen species (ROS) levels and the expression of apoptosis-associated genes (Bax, caspase-3, CytC). More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca 2+]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP3R1) distribution. Nevertheless, treatment with glycine significantly ameliorated mitochondrial dysfunction, oxidative stress and apoptosis, glycine also regulated [Ca 2+]i levels and IP3R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes. Taken together, our results indicate that glycine has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.

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H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽

10.1017/s0967199414000021 ◽

2014 ◽

Vol 23 (3) ◽

pp. 416-425 ◽

Cited By ~ 13

Author(s):

Yan Yun ◽

Peng An ◽

Jing Ning ◽

Gui-Ming Zhao ◽

Wen-Lin Yang ◽

...

Keyword(s):

Oocyte Maturation ◽

Polar Body ◽

Linker Histone ◽

Meiotic Maturation ◽

Control Group ◽

Bovine Oocyte ◽

First Polar Body ◽

Effective Sirnas ◽

Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

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Melatonin Improves Oocyte Maturation and Mitochondrial Functions by Reducing Bisphenol A-Derived Superoxide in Porcine Oocytes In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms19113422 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3422 ◽

Cited By ~ 9

Author(s):

Hyo-Jin Park ◽

Soo-Yong Park ◽

Jin-Woo Kim ◽

Seul-Gi Yang ◽

Min-Ji Kim ◽

...

Keyword(s):

Bisphenol A ◽

Oocyte Maturation ◽

Cell Damage ◽

Cumulus Cell ◽

Superoxide Production ◽

Positive Effects ◽

Mitochondrial Functions ◽

Porcine Oocyte ◽

Antioxidative Effects

Bisphenol A (BPA) is synthetic organic compound that exhibits estrogen-like properties and it induces mitochondrial superoxide production. Melatonin (Mela) protects against BPA-mediated cell damage and apoptosis. However, the antioxidative effects of Mela against BPA-induced superoxide production in porcine oocytes are still not known. In this study, we investigated the antioxidative effects of Mela against BPA-derived superoxide on oocyte maturation in pigs. To investigate the effects of the superoxide specific scavenger, Mito-TEMPO, on porcine oocyte maturation in response to BPA exposure apoptosis proteins, we treated the oocytes with Mito-TEMPO (0.1 µM) after pre-treating them with BPA (75 µM) for 22 h. As expected, the reduction in meiotic maturation and cumulus cell expansion of cumulus-oocyte-complexes (COCs) in the BPA (75 µM) treated group was recovered (p < 0.01) by treatment with Mito-TEMPO (0.1 µM). An increase in the levels of mitochondrial apoptotic proteins (AIF, cleaved Cas 3 and cleaved Parp1) in response to BPA-induced damage was also reduced by Mito-TEMPO treatment in porcine COCs. Interestingly, we confirmed the positive effects of Mela with respect to superoxide production upon BPA exposure during oocyte maturation and also confirmed the reduction in mitochondrial apoptosis in Mela (0.1 µM)-treated porcine COCs. These results provide evidence for the first time that antioxidative effects of Mela on BPA-derived superoxide improve porcine oocyte maturation.

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Phytochemicals from the Cocoa Shell Modulate Mitochondrial Function, Lipid and Glucose Metabolism in Hepatocytes via Activation of FGF21/ERK, AKT, and mTOR Pathways

Antioxidants ◽

10.3390/antiox11010136 ◽

2022 ◽

Vol 11 (1) ◽

pp. 136

Author(s):

Miguel Rebollo-Hernanz ◽

Yolanda Aguilera ◽

Maria A. Martin-Cabrejas ◽

Elvira Gonzalez de Gonzalez de Mejia

Keyword(s):

Oxidative Stress ◽

Mitochondrial Function ◽

Phosphoenolpyruvate Carboxykinase ◽

Fatty Acid Synthase ◽

Cell Model ◽

Fibroblast Growth Factor 21 ◽

Alcoholic Fatty Liver ◽

Atp Production ◽

Cocoa Shell

The cocoa shell is a by-product that may be revalorized as a source of bioactive compounds to prevent chronic cardiometabolic diseases. This study aimed to investigate the phytochemicals from the cocoa shell as targeted compounds for activating fibroblast growth factor 21 (FGF21) signaling and regulating non-alcoholic fatty liver disease (NAFLD)-related biomarkers linked to oxidative stress, mitochondrial function, and metabolism in hepatocytes. HepG2 cells treated with palmitic acid (PA, 500 µmol L−1) were used in an NAFLD cell model. Phytochemicals from the cocoa shell (50 µmol L−1) and an aqueous extract (CAE, 100 µg mL−1) enhanced ERK1/2 phosphorylation (1.7- to 3.3-fold) and FGF21 release (1.4- to 3.4-fold) via PPARα activation. Oxidative stress markers were reduced though Nrf-2 regulation. Mitochondrial function (mitochondrial respiration and ATP production) was protected by the PGC-1α pathway modulation. Cocoa shell phytochemicals reduced lipid accumulation (53–115%) and fatty acid synthase activity (59–93%) and prompted CPT-1 activity. Glucose uptake and glucokinase activity were enhanced, whereas glucose production and phosphoenolpyruvate carboxykinase activity were diminished. The increase in the phosphorylation of the insulin receptor, AKT, AMPKα, mTOR, and ERK1/2 conduced to the regulation of hepatic mitochondrial function and energy metabolism. For the first time, the cocoa shell phytochemicals are proved to modulate FGF21 signaling. Results demonstrate the in vitro preventive effect of the phytochemicals from the cocoa shell on NAFLD.

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