Journal Pre-proof Vanillic acid retains redox status in HepG2 cells during hyperinsulinemic shock using the mitochondrial pathway Sreelekshmi Mohan, Genu George, K.G Raghu PII: S2212-4292(21)00141-3 DOI: https://doi.org/10.1016/j.fbio.2021.101016 Reference: FBIO 101016 To appear in: Food Bioscience Received Date: 15 December 2019 Revised Date: 17 March 2021 Accepted Date: 18 March 2021 Please cite this article as: Mohan S., George G & Raghu K.G, Vanillic acid retains redox status in HepG2 cells during hyperinsulinemic shock using the mitochondrial pathway, Food Bioscience, https:// doi.org/10.1016/j.fbio.2021.101016 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain © 2021 Elsevier Ltd All rights reserved 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 Jo ur na lP re -p ro of K.G Raghu designed work plan, concept, interpreted the data, contributed intellectual content Vanillic acid retains redox status in HepG2 cells during hyperinsulinemic shock using the mitochondrial pathway Running title: Vanillic acid ameliorates hyperinsulinemic complications in HepG2 cells Sreelekshmi Mohan a,b, Genu George a, Raghu K.G a,b * a Biochemistry and Molecular Mechanism Laboratory, Agro-Processing and Technology, Division, 10 CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India, 695019 11 *For correspondence: Dr K G Raghu, Biochemistry and Molecular Mechanism Laboratory, 12 Agro-Processing and Technology Division, CSIR-National Institute for Interdisciplinary Science 13 and Technology, Thiruvananthapuram -695019, Kerala, India ro -p na lP re Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India, 201002 Tel: +919495902522 15 ur b 14 Fax: +914712491712 email: raghukgopal@niist.res.in Jo 16 of 17 18 19 20 21 22 Abstract 24 Vanillic acid (VA) is a flavoring and nutritional agent found in many fruits and vegetables It is 25 an antioxidant but its nutraceutical potential has not been studied in detail In this study, the 26 potential of VA against hyperinsulinemia mediated changes on redox status and mitochondria in 27 HepG2 cells were investigated Incubation of cells with μM insulin for 24 hr was found to 28 induce insulin resistance using the inhibition of Glut2 and glucose uptake (51.9%) 29 Hyperinsulinemia caused depletion of superoxide dismutase, glutathione, glutathione peroxidase 30 and generation of reactive oxygen species (68%) It also caused overexpression of the receptor 31 for advanced glycation end products (120%) and a decreases of dolichyl-diphospho- 32 oligosaccharide-protein glycosyltransferase non-catalytic subunit (34%) Mitochondria were 33 affected with alterations in mitochondrial transmembrane potential, aconitase activity, 34 mitochondrial fission and fusion, biogenesis (AMPK, Sirt1 and PGC-1α) and bioenergetics (ATP 35 and oxygen consumption) Co-treatment with VA decreased oxidative stress by reducing of 36 reactive oxygen species and lipid peroxidation during hyperinsulinemia Similarly, VA protected 37 the mitochondria during insulin shock VA also prevented glycation through the decrease of the 38 receptor for advanced glycation end products expression VA was found to act through the 39 AMPK/Sirt1/PGC-1α pathway to obtain its beneficial activity From the overall results it was 40 concluded that VA is expected to be a potential nutraceutical which could be explored for the 41 development of affordable nutraceuticals after detailed in vivo study 42 Key words: Hyperinsulinemia; Reactive oxygen species; Glycation; Mitochondria; HepG2 43 cells, Angelica sinensis Jo ur na lP re -p ro of 23 44 Abbreviations 46 ∆ψm mitochondrial membrane potential 47 2-DG 2-deoxy-D-glucose 48 AGE advanced glycation end products 49 ALE advanced lipoxidation end products 50 AMPK adenosine monophosphate activated kinase 51 ANOVA one-way analysis of variance 52 ATP adenosine triphosphate 53 BSA bovine serum albumin 54 DCFH-DA 2,7-dichlorodihydrofluorescein diacetate 55 DDOST 56 subunit 57 DMEM Dulbecco’s modified eagle’s medium 58 DMSO dimethyl sulfoxide 59 DTNB 5,5’-dithio-bis (2-nitrobenzoic acid) 60 DTT dithiothreitol 61 ECL enhanced chemiluminescence ur na lP re -p ro of 45 Jo dolichyl-diphospho-oligosaccharide-protein glycosyltransferase non-catalytic EDTA ethylenediaminetetraacetic acid 63 EGTA ethylene glycol tetraacetic acid 64 FBS fetal bovine serum 65 FIS1 fission protein 66 GLUT2 glucose transporter 67 GPx glutathione peroxidase 68 GSH glutathione 69 HBSS Hanks balanced saline solution 70 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 71 HepG2 human hepatocellular carcinoma cells 72 HI 73 HRP 74 IR insulin resistance 75 IRS2 insulin receptor substrate 76 JC-1 77 MDA malondialdehyde 78 MES 2-(N-morpholino)ethanesulfonic acid ur na lP re -p ro of 62 Jo high insulin horseradish peroxidase 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazol carbocyanine iodide MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 80 NAC N-acetyl-cysteine 81 NADP nicotinamide adenine dinucleotide phosphate 82 NCCS National Centre for Cell Science 83 OPA1 optic atrophy 84 OS oxidative stress 85 p-AMPK phospho-AMP activated kinase 86 PBS phosphate-buffered saline 87 PGC-1α peroxisome proliferator activated receptor γ coactivator-1α 88 PVDF polyvinylidene difluoride 89 RAGE 90 RIPA 91 ROS reactive oxygen species 92 RT room temperature 93 SEM standard error of the mean 94 Sirt1 sirtuin 95 SOD superoxide dismutase ur na lP re -p ro of 79 Jo receptor for advanced glycation end products radioimmuno precipitation assay 96 SPSS Statistical Package for the Social Sciences 97 T2DM type diabetes mellitus 98 TBST tris buffered saline-Tween 20 99 VA vanillic acid of 100 ro 101 re -p 102 lP 103 107 ur 106 Jo 105 na 104 108 109 110 111 112 113 Introduction Alternative approaches are needed to prevent and treat metabolic diseases such as type 115 diabetes mellitus (T2DM) and associated health issues Non-pharmacological management with 116 the utilization of herbal dietary products has been an option and further work is needed in the 117 search for culinary plants for prophylactic and therapeutic use These edible biomaterials have 118 been shown to alleviate complex disorders using nutritional intervention (Choudhury et al., 119 2018) Functional foods are being developed to manage chronic diseases, such as T2DM and 120 cardiovascular diseases Some have enhanced antioxidant, anti-inflammatory and insulin 121 sensitivity functions 122 Hyperinsulinemia is associated with health complications of diabetes Insulin resistance (IR) is a 123 major issue with hyperinsulinemia (Marin-Juez et al., 2014) This has been established in animal 124 and human studies (Shanik et al., 2008) Insulin is one of the main hormones for regulating 125 glucose metabolism (Wilcox, 2005) Circulating levels are controlled by the nutrients involved in 126 glucose uptake, glycolysis and glycogen storage, lipogenesis, and protein synthesis (Czech et al., 127 2013; Fu et al., 2013) Insulin may also have some autocrine functions like the promotion of β- 128 cell growth and influence its own production and release (Wang et al., 2013) Hyperinsulinemia 129 could enhance the desensitization of the insulin receptor which results in IR (Templeman et al., 130 2017) Corkey (2012) showed that hyperinsulinemia is the root cause of IR and diabetes 131 Inhibition of hyperinsulinemia results in the reduction of IR without affecting glucose tolerance 132 including in human studies (Reed et al., 2011) Thus, early recognition of hyperinsulinemia may 133 be helpful to guide earlier intervention strategies to prevent or delay diabetes onset and related 134 chronic diseases Hyperinsulinemia could alter redox status (Kim et al., 2008) and induce surplus 135 generation of superoxide anions, hydrogen peroxide and hydroxyl radicals (Ge et al., 2008; Li et Jo ur na lP re -p ro of 114 al., 2015) These effects were reversed using antioxidants such as N-acetyl-cysteine, superoxide 137 dismutase or catalase Therefore, oxidative stress (OS) could be a potential interventional target 138 for hyperinsulinemia induced IR and related diseases Mitochondria are the powerhouse of the 139 cell and involved in important functions of the cell such as regulation of ATP production, redox 140 status and apoptosis Mitochondrial dysfunction and associated OS are often involved at the start 141 in the genesis of metabolic syndromes Hyperinsulinemia associated pathologies have been 142 associated with OS and mitochondrial dysfunction but the detailed information needed to design 143 therapeutic strategies based on molecular mechanisms might be beneficial (Gonzalez-Franquesa 144 et al., 2017) 145 Based on the importance of antioxidants in protecting the mitochondria from OS during 146 hyperinsulinemia, vanillic acid (VA) was selected for this study It is a flavoring agent mainly 147 found in the root of the Chinese medicinal plant Angelica sinensis It is also found in many 148 alcoholic beverages, cereals, dried fruits, nuts and herbs It is a strong antioxidant (Tai et al., 149 2012) and anti-lipid-peroxidative agent (Vinoth & Kowsalya, 2018) It is the oxidized form of 150 vanillin and has antibacterial, antimicrobial, and chemopreventive activities (Itoh et al., 2010) 151 Only one report showed that VA protects against hyperinsulinemia and hyperlipidemia by 152 decreasing the serum glucose, triglycerides, and free fatty acids (Chang et al., 2015) Similarly, 153 not much research has been done with hyperinsulinemia induced alterations in redox status 154 associated with mitochondrial dysfunction and glycation in human hepatocellular carcinoma 155 (HepG2) cells In this study the effects of VA on hyperinsulinemia in HepG2 cells, an in vitro 156 model of the hyperinsulinemic insulin resistant liver, was studied Jo ur na lP re -p ro of 136 157 158 Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Jo na ur re lP ro -p of Highlights ● Hyperinsulinemia led to oxidative stress, glycation and mitochondrial dysfunction in HepG2 cells ● Vanillic acid protected cells during hyperinsulinemic shock ● The beneficial action of vanillic acid used the AMPK/Sirt1/PGC-1α activation pathway Jo ur na lP re -p ro of and its potential antiglycation property Conflict of interest The authors confirm that they have no conflicts of interest with respect to the work described in Jo ur na lP re -p ro of this manuscript ... concept, interpreted the data, contributed intellectual content 1 Vanillic acid retains redox status in HepG2 cells during hyperinsulinemic shock using the mitochondrial pathway Running title: Vanillic. .. suggesting the impairment of the insulin signaling pathway Besides, with 504 the hyperinsulinemic condition, the OS was developed by increasing the levels of ROS 505 Hyperinsulinemic shock could... IR in HepG2 cells The effect of hyperinsulinemia 506 on the redox status of HepG2 cells which is essential for maintaining the normal functional status 507 of cells was the primary focus In addition,