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Cited by 134Mitochondrial Function and PathologyMetabolism, Diabetes, and CancerDiet, Metabolism, and Disease

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Augmentation of Glucotoxicity, Oxidative Stress, Apoptosis and Mitochondrial Dysfunction in HepG2 Cells by Palmitic Acid

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Alnahdi, Arwa, John, Annie, Raza, Haider·2019·Nutrients
DOI 10.3390/nu11091979PMC PMC6770774
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Abstract

Hyperglycemia and hyperlipidemia are the hallmarks of diabetes and obesity. Experimental and epidemiological studies have suggested that dietary management and caloric restriction are beneficial in reducing the complications of diabesity. Studies have suggested that increased availability of energy metabolites like glucose and saturated fatty acids induces metabolic, oxidative, and mitochondrial stress, accompanied by inflammation that may lead to chronic complications in diabetes. In the present study, we used human hepatoma HepG2 cells to investigate the effects of high glucose (25 mM) and high palmitic acid (up to 0.3 mM) on metabolic-, inflammatory-, and redox-stress-associated alterations in these cells. Our results showed increased lipid, protein, and DNA damage, leading to caspase-dependent apoptosis and mitochondrial dysfunction. Glucolipotoxicity increased ROS production and redox stress appeared to alter mitochondrial membrane potential and bioenergetics. Our results also demonstrate the enhanced ability of cytochrome P450s-dependent drug metabolism and antioxidant adaptation in HepG2 cells treated with palmitic acid, which was further augmented with high glucose. Altered NF-kB/AMPK/mTOR-dependent cell signaling and inflammatory (IL6/TNF-α) responses were also observed. Our results suggest that the presence of high-energy metabolites enhances apoptosis while suppressing autophagy by inducing inflammatory and oxidative stress responses that may be responsible for alterations in cell signaling and metabolism.

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Figure 1 Effect of high glucose/high palmitic acid (HG/HPA) on cell viability. HepG2 cells (~2 × 104) were treated with MTT as described in the Materials and Methods section, and viability was measured by formazan formation as a mitochondrial function of viable cells and measured at 570 nm. ‘C’ represents untreated control cells. The results are expressed as mean +/− SEM of three independent experiments. The asterisks indicate significant difference (* p ≤ 0.05, ** p ≤ 0.005,) from the control untreated cells.
Figure 1 Effect of high glucose/high palmitic acid (HG/HPA) on cell viability. HepG2 cells (~2 × 104) were treated with MTT as described in the Materials and Methods section, and viability was measured by formazan formation as a mitochondrial function of viable cells and measured at 570 nm. ‘C’ represents untreated control cells. The results are expressed as mean +/− SEM of three independent experiments. The asterisks indicate significant difference (* p ≤ 0.05, ** p ≤ 0.005,) from the control untreated cells.CC BY · PubMed Central
Figure 2 Effect of high glucose/high palmitic acid (HG/HPA) on reactive oxygen species (ROS) production. HepG2 cells were treated with high glucose/high palmitic acid, as described in the Materials and Methods section, and ROS were measured fluorimetrically (A) using the cell permeable probe 2′,7′-dichlorofluorescein diacetate (DCFDA), and by flow cytometry (B) using the FACSDiva software, as described previously. Production of ROS was also measured microscopically, in which cells grown on cover slips were incubated with DCFDA after high glucose/high palmitic acid treatment and fluorescence immediately visualized using an Olympus fluorescence microscope (C). Original magnification ×200. The results are expressed as mean +/− SEM of three independent experiments. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).
Figure 2 Effect of high glucose/high palmitic acid (HG/HPA) on reactive oxygen species (ROS) production. HepG2 cells were treated with high glucose/high palmitic acid, as described in the Materials and Methods section, and ROS were measured fluorimetrically (A) using the cell permeable probe 2′,7′-dichlorofluorescein diacetate (DCFDA), and by flow cytometry (B) using the FACSDiva software, as described previously. Production of ROS was also measured microscopically, in which cells grown on cover slips were incubated with DCFDA after high glucose/high palmitic acid treatment and fluorescence immediately visualized using an Olympus fluorescence microscope (C). Original magnification ×200. The results are expressed as mean +/− SEM of three independent experiments. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 3 Effect of high glucose/high palmitic acid (HG/HPA) on lipids, proteins and DNA degradation. Lipid peroxidation (LPO) was measured as the total amount of malonedialdehyde (A), as per the vendor’s protocol (Oxis Research, Inc.). Protein carbonylation (B) was assayed by the dinitrophenylhydrazine (DNPH) method, as described in the Materials and Methods section. DNA fragmentation (C) was analyzed by agarose gel (2%) electrophoresis and ethidium bromide staining. DNA fragmentation was also analyzed microscopically (D) by staining the treated cells with Hoechst 33,342 dye, as described in the Materials and Methods section. Asterisks indicate significant differences (** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).
Figure 3 Effect of high glucose/high palmitic acid (HG/HPA) on lipids, proteins and DNA degradation. Lipid peroxidation (LPO) was measured as the total amount of malonedialdehyde (A), as per the vendor’s protocol (Oxis Research, Inc.). Protein carbonylation (B) was assayed by the dinitrophenylhydrazine (DNPH) method, as described in the Materials and Methods section. DNA fragmentation (C) was analyzed by agarose gel (2%) electrophoresis and ethidium bromide staining. DNA fragmentation was also analyzed microscopically (D) by staining the treated cells with Hoechst 33,342 dye, as described in the Materials and Methods section. Asterisks indicate significant differences (** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 4 Effect of high glucose/high palmitic acid (HG/HPA) on apoptosis. Apoptosis was measured by FACS analysis, as described previously [25,26,27,28,29]. Histogram shows percentage of viable, early apoptotic, late apoptotic, and dead cells. Representative dot plots from three experiments are shown. The results are mean +/− SEM of three independent experiments.
Figure 4 Effect of high glucose/high palmitic acid (HG/HPA) on apoptosis. Apoptosis was measured by FACS analysis, as described previously [25,26,27,28,29]. Histogram shows percentage of viable, early apoptotic, late apoptotic, and dead cells. Representative dot plots from three experiments are shown. The results are mean +/− SEM of three independent experiments.CC BY · PubMed Central
Figure 5 Effect of high glucose/high palmitic acid (HG/HPA) on glutathione (GSH) metabolism. HepG2 cells were treated with different doses of palmitic acid under normal and high glucose conditions. GSH/GSSG (reduced/oxidized glutathione) ratio (A), GSH-Px (glutathione peroxidase) (B), and GSH reductase (glutathione reductase) (C) were measured. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), triangles indicate significant differences (Δ p ≤ 0.05, ΔΔ p ≤ 0.005, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).
Figure 5 Effect of high glucose/high palmitic acid (HG/HPA) on glutathione (GSH) metabolism. HepG2 cells were treated with different doses of palmitic acid under normal and high glucose conditions. GSH/GSSG (reduced/oxidized glutathione) ratio (A), GSH-Px (glutathione peroxidase) (B), and GSH reductase (glutathione reductase) (C) were measured. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), triangles indicate significant differences (Δ p ≤ 0.05, ΔΔ p ≤ 0.005, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 6 Effect of high glucose/high palmitic acid (HG/HPA) on the activities of antioxidant enzymes, superoxide dismutase (SOD), and catalase. HepG2 cells were treated with high glucose and high fatty acids, as described in the Materials and Methods section. SOD (A) was measured as percentage conversion of nitroblue tetrazolium (NBT) to NBT-diformazan according to the vendor’s protocol. Catalase measurement (B) was based on the formaldehyde produced, which was measured colorimetrically. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).
Figure 6 Effect of high glucose/high palmitic acid (HG/HPA) on the activities of antioxidant enzymes, superoxide dismutase (SOD), and catalase. HepG2 cells were treated with high glucose and high fatty acids, as described in the Materials and Methods section. SOD (A) was measured as percentage conversion of nitroblue tetrazolium (NBT) to NBT-diformazan according to the vendor’s protocol. Catalase measurement (B) was based on the formaldehyde produced, which was measured colorimetrically. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 7 Effect of HG/HPA on CYP450 and GST activities. HepG2 cells were treated with high glucose and high fatty acids, and the activities of different CYP450 enzymes (A–D) and GST (E) were measured using the appropriate substrates, as described in the Materials and Methods section. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).
Figure 7 Effect of HG/HPA on CYP450 and GST activities. HepG2 cells were treated with high glucose and high fatty acids, and the activities of different CYP450 enzymes (A–D) and GST (E) were measured using the appropriate substrates, as described in the Materials and Methods section. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 8 Effect of high glucose/high palmitic acid (HG/HPA) on mitochondrial membrane potential. HepG2 cells were treated with high glucose and high fatty acids, and mitochondrial membrane potential was measured by flow cytometry using a fluorescent cationic dye according to the vendor’s protocol (DePsipherTM, R &D System Inc.). A typical histogram representing the percentage loss of mitochondrial membrane potential is shown. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).
Figure 8 Effect of high glucose/high palmitic acid (HG/HPA) on mitochondrial membrane potential. HepG2 cells were treated with high glucose and high fatty acids, and mitochondrial membrane potential was measured by flow cytometry using a fluorescent cationic dye according to the vendor’s protocol (DePsipherTM, R &D System Inc.). A typical histogram representing the percentage loss of mitochondrial membrane potential is shown. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 9 Effect of high glucose/high palmitic acid (HG/HPA) on mitochondrial enzymes and bioenergetics. HepG2 cells were treated with different doses of palmitic acid in the presence of normal and high glucose. Mitochondrial enzyme complexes, Complex I (A) and Complex IV (B), as well as ATP levels (C) and a ROS-sensitive enzyme, aconitase (D), were measured as described in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (Δ p ≤ 0.05) relative to untreated control cells under high glucose condition (HG-C).
Figure 9 Effect of high glucose/high palmitic acid (HG/HPA) on mitochondrial enzymes and bioenergetics. HepG2 cells were treated with different doses of palmitic acid in the presence of normal and high glucose. Mitochondrial enzyme complexes, Complex I (A) and Complex IV (B), as well as ATP levels (C) and a ROS-sensitive enzyme, aconitase (D), were measured as described in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (Δ p ≤ 0.05) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 10 Effect of high glucose/high palmitic acid (HG/HPA) on cytosolic energy metabolizing enzymes. HepG2 cells were treated with high glucose and high palmitic acid, and the activities of hexokinase (A), glucose-6-phosphate dehydrogenase (G6PDH) (B), and glutamate dehydrogenase (GDH) (C) were measured as described in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).
Figure 10 Effect of high glucose/high palmitic acid (HG/HPA) on cytosolic energy metabolizing enzymes. HepG2 cells were treated with high glucose and high palmitic acid, and the activities of hexokinase (A), glucose-6-phosphate dehydrogenase (G6PDH) (B), and glutamate dehydrogenase (GDH) (C) were measured as described in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005, ΔΔΔ p ≤ 0.001) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 11 Effect of high glucose/high palmitic acid (HG/HPA) on lipid accumulation. HepG2 cells were grown on coverslips, treated with different doses of palmitic acid under normal and high glucose conditions, and stained with Oil Red O dye, as described in the Materials and Methods section. Hematoxylin was used for nuclear staining and viewed using the EVOS XL Core Imaging system (40× magnification). Lipid droplets appear red and nuclei appear blue.
Figure 11 Effect of high glucose/high palmitic acid (HG/HPA) on lipid accumulation. HepG2 cells were grown on coverslips, treated with different doses of palmitic acid under normal and high glucose conditions, and stained with Oil Red O dye, as described in the Materials and Methods section. Hematoxylin was used for nuclear staining and viewed using the EVOS XL Core Imaging system (40× magnification). Lipid droplets appear red and nuclei appear blue.CC BY · PubMed Central
Figure 12 Effect of high glucose/high palmitic acid (HG/HPA) on inflammatory markers. HepG2 cells were treated with different doses of palmitic acid in normal/high glucose media, and inflammatory markers, TNF-α (A), IL6 (B), and NO levels (C) were measured in the media supernatants, as described in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).
Figure 12 Effect of high glucose/high palmitic acid (HG/HPA) on inflammatory markers. HepG2 cells were treated with different doses of palmitic acid in normal/high glucose media, and inflammatory markers, TNF-α (A), IL6 (B), and NO levels (C) were measured in the media supernatants, as described in the Materials and Methods section. Results are expressed as mean +/− SEM of three experiments. Asterisks indicate significant differences (** p ≤ 0.005, *** p ≤ 0.001) relative to untreated control cells under normal glucose condition (NG-C), and triangles indicate significant differences (ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central
Figure 13 Effect of HG/HPA on protein expression. Samples of 50 ug protein from control and HG/HPA treated cellular extracts were separated on 12% SDS-PAGE gels and electroblotted onto nitrocellulose by western blot technique. The proteins were then detected using specific antibodies for PARP, caspase-3, mTOR, p-mTOR, AMPK, p-AMPK (A), and NFkB, CYP 2E1, and CYP 3A4 (B). Beta-actin was used as loading control. The quantitation of the protein bands is expressed as relative ratios normalized against actin or other specific proteins as appropriate. The figures are representative of three experiments. Asterisks indicate significant difference (* p ≤ 0.05, ** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), (Δ p ≤ 0.05, ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).
Figure 13 Effect of HG/HPA on protein expression. Samples of 50 ug protein from control and HG/HPA treated cellular extracts were separated on 12% SDS-PAGE gels and electroblotted onto nitrocellulose by western blot technique. The proteins were then detected using specific antibodies for PARP, caspase-3, mTOR, p-mTOR, AMPK, p-AMPK (A), and NFkB, CYP 2E1, and CYP 3A4 (B). Beta-actin was used as loading control. The quantitation of the protein bands is expressed as relative ratios normalized against actin or other specific proteins as appropriate. The figures are representative of three experiments. Asterisks indicate significant difference (* p ≤ 0.05, ** p ≤ 0.005) relative to untreated control cells under normal glucose condition (NG-C), (Δ p ≤ 0.05, ΔΔ p ≤ 0.005) relative to untreated control cells under high glucose condition (HG-C).CC BY · PubMed Central

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Metadata

Authors
Alnahdi, A., John, A., & Raza, H.
Affiliations
United Arab Emirates University (AE)
Corresponding author
Raza, Haider
ORCID
Venue
Nutrients
Publisher
MDPI AG
Published
2019-08-22
Volume
11
Issue
9
Pages
1979
ISSN
2072-6643 (ISSN-L)
DOI
10.3390/nu11091979
PMC
PMC6770774
OpenAlex
W2969456983
Cited by
134
References
60
Citations by year
2026: 13 · 2025: 24 · 2024: 26 · 2023: 16 · 2022: 26 · 2021: 22 · 2020: 6 · 2019: 1
FWCI
4.55
Type
journal article
Language
en
Open access
Licence
Locations
  • Nutrients (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Sheikh Hamdan Bin Rashid Al Maktoum Award for Medical Sciences (MRG-18/2013-2014)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Diet, Metabolism, and Disease
Keywords
palmitic acid, glucolipotoxicity, high glucose, oxidative stress, apoptosis, mitochondrial dysfunction, HepG2 cells, hepatocytes, redox stress, mitochondrial membrane potential, antioxidant adaptation, autophagy, diabesity, saturated fatty acids
MeSH terms
Autophagy, Energy Metabolism, Glucose, Humans, Mitochondria, Liver, Signal Transduction, NF-kappa B, Apoptosis, Oxidative Stress, Inflammation Mediators, Palmitic Acid, Hepatocytes, AMP-Activated Protein Kinases, Hep G2 Cells, TOR Serine-Threonine Kinases

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