Vanillic acid retains redox status in HepG2 cells during hyperinsulinemic shock using the mitochondrial pathway
Introduction
Alternative approaches are needed to prevent and treat metabolic diseases such as type 2 diabetes mellitus (T2DM) and associated health issues. Non-pharmacological management with the utilization of herbal dietary products has been an option and further work is needed in the search for culinary plants for prophylactic and therapeutic use. These edible biomaterials have been shown to alleviate complex disorders using nutritional intervention (Choudhury et al., 2018). Functional foods are being developed to manage chronic diseases, such as T2DM and cardiovascular diseases. Some have enhanced antioxidant, anti-inflammatory and insulin sensitivity functions.
Hyperinsulinemia is associated with health complications of diabetes. Insulin resistance (IR) is a major issue with hyperinsulinemia (Marin-Juez et al., 2014). This has been established in animal and human studies (Shanik et al., 2008). Insulin is one of the main hormones for regulating glucose metabolism (Wilcox, 2005). Circulating levels are controlled by the nutrients involved in glucose uptake, glycolysis and glycogen storage, lipogenesis, and protein synthesis (Czech et al., 2013; Fu et al., 2013). Insulin may also have some autocrine functions like the promotion of β-cell growth and influence its own production and release (Wang et al., 2013). Hyperinsulinemia could enhance the desensitization of the insulin receptor which results in IR (Templeman et al., 2017). Corkey (2012) showed that hyperinsulinemia is the root cause of IR and diabetes. Inhibition of hyperinsulinemia results in the reduction of IR without affecting glucose tolerance including in human studies (Reed et al., 2011). Thus, early recognition of hyperinsulinemia may be helpful to guide earlier intervention strategies to prevent or delay diabetes onset and related chronic diseases. Hyperinsulinemia could alter redox status (Kim et al., 2008) and induce surplus generation of superoxide anions, hydrogen peroxide and hydroxyl radicals (Ge et al., 2008; Li et al., 2015). These effects were reversed using antioxidants such as N-acetyl-cysteine, superoxide dismutase or catalase. Therefore, oxidative stress (OS) could be a potential interventional target for hyperinsulinemia induced IR and related diseases. Mitochondria are the powerhouse of the cell and involved in important functions of the cell such as regulation of ATP production, redox status and apoptosis. Mitochondrial dysfunction and associated OS are often involved at the start in the genesis of metabolic syndromes. Hyperinsulinemia associated pathologies have been associated with OS and mitochondrial dysfunction but the detailed information needed to design therapeutic strategies based on molecular mechanisms might be beneficial (Gonzalez-Franquesa & Patti, 2017).
Based on the importance of antioxidants in protecting the mitochondria from OS during hyperinsulinemia, vanillic acid (VA) was selected for this study. It is a flavoring agent mainly found in the root of the Chinese medicinal plant Angelica sinensis. It is also found in many alcoholic beverages, cereals, dried fruits, nuts and herbs. It is a strong antioxidant (Tai et al., 2012) and anti-lipid-peroxidative agent (Vinoth & Kowsalya, 2018). It is the oxidized form of vanillin and has antibacterial, antimicrobial, and chemopreventive activities (Itoh et al., 2010). Only one report showed that VA protects against hyperinsulinemia and hyperlipidemia by decreasing the serum glucose, triglycerides, and free fatty acids (Chang et al., 2015). Similarly, not much research has been done with hyperinsulinemia induced alterations in redox status associated with mitochondrial dysfunction and glycation in human hepatocellular carcinoma (HepG2) cells. In this study the effects of VA on hyperinsulinemia in HepG2 cells, an in vitro model of the hyperinsulinemic insulin resistant liver, was studied.
Section snippets
Materials and methods
Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin antibiotics (10,000 IU/mL of each) and 0.5% trypsin (porcine pancreas)- ethylenediaminetetraacetic acid (trypsin-EDTA) were from Gibco-BRL Life Technologies (Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), radioimmunoprecipitation assay buffer (RIPA buffer), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and VA were from Sigma Aldrich
Induction of IR through hyperinsulinemic shock in HepG2 cells
The results of the glucose uptake with different concentrations of insulin treated HepG2 cells are shown in Fig. 1a. The cellular uptake of 2-DG with various concentrations of insulin (50, 100 nM and 1 μM) treated cells was decreased 5.5, 35.4 and 51.9%, respectively, as compared with normal cells. Among these, 100 nM and 1 μM insulin showed significant (p ≤ 0.05) decreases in glucose uptake. The expression levels of IRS2 and Glut2 were significantly decreased with 1 μM insulin compared to
Discussion
During T2DM pancreatic cells are unable to compensate for the normal blood glucose level even by producing large amounts of insulin (Steneberg et al., 2015). This most often leads to the malfunction of insulin generation, which opens the pathway of hyperinsulinemia related IR (Schofield & Sutherland, 2012). Many studies have shown that hyperinsulinemia and IR are the independent risk factors for the development of T2DM (Facchini et al., 2001; Marin-Juez et al., 2014). Excessive insulin
Conclusions
Hyperinsulinemia altered the redox status of HepG2 cells by glycation, mitochondrial dysfunction, depletion of innate antioxidant status and lipid peroxidation. VA reduced adverse biochemical effects induced by hyperinsulinemia in HepG2 cells by the AMPK and PGC-1α signaling pathways. Its potential antioxidant and antiglycation property also contributed to its beneficial activities. Detailed in vivo and preclinical studies are needed before recommending applications for hyperinsulinemia in
Author statement
Sreelekshmi Mohan conducted the experiments, collected, analyzed and interpreted the data and she wrote the first draft of the manuscript. Dr. Genu George edited the manuscript. Dr. K.G. Raghu designed work plan, concept, interpreted the data, contributed intellectual content.
Declaration of competing interest
The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Sreelekshmi Mohan thanks the University Grants Commission (UGC, New Delhi, India) for financial assistance to conduct the research. Genu George thanks the Science and Engineering Research Board (SERB, New Delhi, India) for her financial assistance.
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