Apigenin and Quercetin Ameliorate Mitochondrial Alterations by Tunicamycin-Induced ER Stress in 3T3-L1 Adipocytes
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- Nisha, V.M., Anusree, S.S., Priyanka, A. et al. Appl Biochem Biotechnol (2014) 174: 1365. doi:10.1007/s12010-014-1129-2
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Endoplasmic reticulum (ER) is an important organelle with functions like protein synthesis, folding, and calcium homeostasis. ER stress, a condition that dramatically affects protein folding homeostasis in cells, has been associated with a number of metabolic disorders. Emerging clinical and preclinical evidence support the notion that pharmacological modulators of ER stress have therapeutic potential as a novel target for treating metabolic diseases. ER is in physical contact with mitochondria, and there is a strong cross talk between these organelles at functional level. The present investigation was aimed to check the mitochondrial alterations in adipocytes with tunicamycin-induced ER stress and modulation by apigenin and quercetin. For this, differentiated adipocytes were incubated with tunicamycin (2 μg/ml) for 18 h, and changes in mitochondrial membrane potential, biogenesis, reactive oxygen species production, and adiponectin secretion were seen. Tunicamycin-induced ER stress altered reactive oxygen species (ROS) (6.34-fold↑), membrane potential (4.1-fold↑), mitochondrial biogenesis (2.4-fold↓), and adiponectin secretion (3.5-fold↓). Apigenin and quercetin ameliorated alterations in mitochondria. From results, we conclude that ER stress significantly alters mitochondrial functions and both the bioactives significantly protected mitochondrial alterations during ER stressand reestablished adiponectin secretion.
The growing epidemic of obesity and other metabolic disorders, associated with insulin resistance, cardiovascular diseases, and cancer, has made adipose tissue an important subject of scientific study and a target of therapeutic intervention. In addition to its function as storage depot of excess energy, adipose tissue is an active endocrine organ. It secretes proteins known as adipokines . Adipose tissue from obese mice shows signs of endoplasmic reticulum (ER) stress . However, the exact physiology of ER stress in obese adipose tissue is unknown; it may result from various physiological conditions like nutrient overload , high demand for protein synthesis , low glucose in the setting of insulin resistance, and decreased adipose tissue revascularization.
ER is an important organelle of eukaryotic cells, with variety of functions including protein biosynthesis, lipid metabolism, and calcium homeostasis . They are also involved in drug detoxification, attachment of receptors on cell membrane proteins, and steroid metabolism . Most of the newly synthesized proteins are transported to the lumen of the ER, for proper folding and transport to their target places in the cell . ER stress can be triggered by N-glycosylation inhibition, hypoxia, nutrient deprivation, perturbation of redox status, aberrant Ca2+ regulation, viral infection, failure of posttranslational modifications, and increased protein synthesis and/or accumulation of unfolded or misfolded proteins in the ER [8, 9]. Maintenance of ER homeostasis is critical to cell survival.
Mitochondrial dysfunction and ER stress interact to perpetuate one another and facilitate disease development. The mitochondria-associated ER membrane (MAM) plays critical role in cellular physiology and homeostasis, including lipid transport, Ca2+ signaling, and apoptosis. A number of proteins bound to the mitochondria or ER serve as important mediators of communication between the two organelles at the MAM [10, 11]. The ER of adipocytes plays a major role in the assembly and secretion of adipokines. ER stress significantly decreases the adiponectin mRNA expression . ER stress also induces leptin resistance .
The use of natural products is on continuous rise, for the management of lifestyle-related metabolic disorders. These agents are being used both as medicine and food supplements. Most of these preparations possess antioxidant and anti-inflammatory properties, due to the presence of high polyphenolic content . So, it would be logical to explore these products on management of ER stress. However, little is known about the mechanisms by which bioactives ameliorate ER-stress-induced alterations in adipocytes. Apigenin (4′,5,7-trihydroxyflavone) and quercetin are found in many fruits and vegetables and belong to the class of flavanoids. They have been reported with many beneficial health effects including antioxidant, antiobesity, anti-inflammatory, and antidiabetic properties[15–17]. Tunicamycin is a mixture of homologous nucleoside antibiotics that inhibit the UDP-HexNAc: polyprenol-P HexNAc-1-P family of enzymes, produced by several bacteria, including Streptomyces clavuligerus and Streptomyces lysosuperficus. Tunicamycin blocks N-linked glycosylation (N-glycans) and causes cell cycle arrest in G1 phase. It is used as an experimental tool in biology, e.g., to induce unfolded protein response via ER stress.
However, no report is available on apigenin and quercetin for its behavior against ER-stress-induced alterations in mitochondria and endocrine function of adipocytes. Therefore, we investigated the effect of two common, biologically active natural products, apigenin and quercetin, against adipocyte mitochondrial alterations and adiponectin secretion in tunicamycin-induced ER stress model.
Materials and Methods
Cell Culture and Treatment
3T3-L1 preadipocytes (American Type Culture Collection, USA) were cultured in complete growth medium, high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10 % FBS (Himedia, India) and 100 units/ml penicillin and 100 g/ml streptomycin (Himedia). Two days postconfluence, the cells were induced to differentiate with standard cocktail consisting of growth medium with 1 μM dexamethasone, 10 μg/ml bovine insulin, and 0.5 mM isobutyl-1-methylxanthine (Sigma, USA). After 2 days of differentiation, medium was replaced with growth medium containing 10 μg/ml bovine insulin for 2 days and then maintained in growth medium alone. Matured adipocytes on 8th day post induction of differentiation were treated with tunicamycin.
Cytotoxicity of tunicamycin, quercetin (5, 10, 20, 50, 100 μM) and apigenin (5, 10, 20, 50, 100 μM) were tested for indicated concentrations by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Ideal dose of tunicamycin for the study was chosen after incubating the cells with varying doses (1, 2, 5 μg/ml) for different time intervals (12, 18, 24 h). The cells were incubated with and without quercetin or apigenin in the absence and presence of tunicamycin (2 μg/ml) for 18 h. The reduction of yellow MTT by mitochondrial succinate dehydrogenase is measured in this assay. The MTT enters the cells and reaches the mitochondria, where it forms reduced, insoluble, colored (dark purple) formazan crystals. The cells are then solubilized with isopropanol, and the released solubilized formazan crystals are measured spectrophotometrically at 570 nm.
Determination of Intracellular Reactive Oxygen Species Generation
Intracellular reactive oxygen species (ROS) generation was assessed using 6-carboxy-2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA). For this, cells were washed two times in Krebs Ringer buffer (KRB) and then incubated in prewarmed KRB containing 5 μM DCFDA at 37 °C. After 20 min, the cells were washed two times with KRB, and the images were captured using confocal microscope. The fluorescence intensity was also measured in each group for comparative quantitative measurements.
Determination of Mitochondrial Membrane Potential
Changes in mitochondrial membrane potential was assessed using (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetra-ethylbenzimidazolcarbocyanine iodide (JC-1; Sigma, USA). This is a mitochondrial dye that normally exists in solution as a monomer emitting a green fluorescence, but which assumes a dimeric configuration emitting red fluorescence, in a reaction driven by membrane potential. The dye was added to the cells which were exposed to tunicamycin and phytochemicals, and fluorescence was measured using multimode reader (Synergy 4, Biotek Instruments, VT, USA) and imaged using confocal microscope (BD Pathway, BD Biosciences, USA).
Mitochondrial Mass Analysis
Relative mitochondrial mass was measured using MitoTracker Green FM (Molecular Probes, Invitrogen, USA). MitoTracker is a fluorescent dye that localizes to the mitochondrial matrix regardless of the mitochondrial membrane potential and covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Fluorescence was considered relative to mitochondrial number. For this, cells were exposed as described previously and incubated for 18 h after exposure. Growth medium was removed from cells and was rinsed with phosphate-buffered saline (PBS). MitoTracker was added to each well and incubated for 30 min. Then, the cells were rinsed three times in PBS and imaged using confocal microscope. Fluorescence intensity was measured in a multiplate reader using excitation and emission wavelengths of 485 and 535 nm, respectively.
Measurement of Adiponectin
On day 10, after induced differentiation, 3T3-L1 adipocytes were treated with 2 μg/ml tunicamycin for 18 h along with and without quercetin or apigenin. Adiponectin concentration in the medium was measured by enzyme-linked immunosorbent assay (ELISA) using mouse adiponectin immunoassay kit (Cayman, USA) according to the manufacturer’s protocol.
Effect of Tunicamycin, Apigenin, and Quercetin on Cell Viability
Effect of Quercetin and Apigenin on Production of ROS in 3T3-L1 Adipocytes
Effect of Quercetin and Apigenin on Tunicamycin-Induced Mitochondrial Depolarization
Effect of Quercetin and Apigenin on Mitochondrial Biogenesis
Effect of Quercetin and Apigenin on Adiponectin Secretion
Adipose tissue regulates metabolic homeostasis, energy storage, and redistribution, as well as secretion of adipokines like adiponectin, leptin, and resistin. Adipose ER stress during obesity has been observed in rodents and humans. Free fatty acids is one of the metabolic signals that can induce ER stress in adipocytes which then leads to increased lipolysis, altered secretion of adipokines, induction of proinflammatory cytokines, and insulin resistance in adipose tissue. However, underlying mechanism is poorly understood.
Generation of oxidative stress seems to play important role in mitochondrial dysfunction. This can further exacerbate stress signals and reduce ATP production. The pathways leading to insulin resistance may be synergistic, and mitochondrial dysfunction can create a feedback loop adding to the oxidative stress. Several studies have shown that mitochondrial content and transmembrane potential are altered in insulin resistance, obesity, and type 2 diabetes. This suggests that mitochondrial dysfunction might play a role in the physiology of insulin resistance. There is strong possibility that ER stress by any means induces mitochondrial dysfunction in adipocytes and may contribute in the physiology of metabolic disorders like obesity and diabetes.
We used tunicamycin-induced ER stress model in 3T3-L1 adipocyte to assess the alterations in mitochondria and adiponectin secretion, and modulation of these changes by biologically active natural products quercetin and apigenin. It was clear from our studies that exposure of cells to 2 μg/ml tunicamycin for 18 h was an ideal experimental condition to study the various alterations relevant to adipocyte biology. Our investigation revealed that ER stress via inhibition of N-glycosylation significantly alter various features of mitochondria and adiponectin secretion. There was significant hyperpolarization of mitochondria, depletion of mitochondrial biogenesis, and surplus production of the ROS. In addition, significant depletion in adiponectin secretion was also seen.
Alterations in mitochondria by ER stress are mainly through calcium (Ca2+) signaling. ER is known for its importance in calcium homeostasis in addition to its role in protein synthesis, folding, transport, etc. The release of Ca2+ from the ER lumen during ER stress leads to increased Ca2+ uptake by mitochondrial matrix. It causes an imbalance between mitochondrial Ca2+ load and the buffer concentrations of the matrix. Such imbalance causes a prolonged Ca2+ accumulation, alteration in transmembrane potential, and opening of mitochondrial permeability transition pore . It finally ends in mitochondrial swelling, breaking of the outer mitochondrial membrane, and release of proapoptotic factors . Altered metabolic signals can induce ER stress in adipocytes leading to altered secretion of adipokines including adiponectin . An increased succination of adiponectin under ER stress reduces the level of adiponectin in type 2 diabetes . ER of adipocytes is known for its role in the assembly and secretion of adipokines. So, it is assumed that the levels of serum adiponectin secreted by adipocytes are decreased in insulin resistance and obesity via ER stress . There are studies available to reveal that inhibition of ER stress may be an effective approach to reduce the risk of obesity and its complications . In other words, ER stress and its response offer novel target for metabolic syndrome.
Treatment with both bioactives (quercetin or apigenin) ameliorated tunicamycin-induced major alterations in mitochondria and reestablished the secretion of normal amount of adiponectin by adipocytes. Quercetin is a polyphenolic compound with potent pleiotropic bioactivities including antiproliferative, anti-inflammatory, antioxidant, and immune system effects. Studies have demonstrated that quercetin can enhance the expression of PGC-1α, a master regulator of the transcriptional network that regulates mitochondrial biogenesis, in HepG2 cells. It was reported that quercetin treatment induced expression of mitochondrial biogenesis activators (PGC-1α, NRF-1, TFAM), mitochondrial DNA (mtDNA), and proteins (COX IV) in HepG2 cells . Apigenin is an anti-inflammatory dietary flavonoid, associated with lower prevalence of cardiovascular diseases . Apigenin exerts anti-inflammatory activity in vitro and in vivo by modulating NF-κB activity and reducing inflammatory cytokine production in LPS-treated mice . Finally, we conclude that antioxidant and mitochondrial protective properties of natural products may be helpful in developing therapeutic strategies to enhance cell survival under ER stress.
We thank Council for Scientific and Industrial Research, Govt. of India, for financial assistance in the form of research fellowship. We are also thankful to the CSIR 12th 5-year plan project “NaPAHA” for financial support. We thank the Director, CSIR-NIIST, and Head, Agroprocessing and Natural Products Division, for providing necessary facilities.
Conflict of Interest
There is no conflict of interest existing between the authors.