Vanillic acid retains redox status in hepg2 cells during hyperinsulinemic shock using the mitochondrial pathway

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:

/>
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.
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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.

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K.G. Raghu designed work plan, concept, interpreted the data, contributed intellectual content.

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Vanillic acid retains redox status in HepG2 cells during

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hyperinsulinemic shock using the mitochondrial pathway.

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Running title: Vanillic acid ameliorates hyperinsulinemic complications in HepG2 cells.
Sreelekshmi Mohan a,b, Genu George a, Raghu K.G a,b *.

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a

Biochemistry and Molecular Mechanism Laboratory, Agro-Processing and Technology,
Division,

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CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram,
Kerala, India, 695019.

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*For correspondence: Dr. K. G. Raghu, Biochemistry and Molecular Mechanism Laboratory,

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Agro-Processing and Technology Division, CSIR-National Institute for Interdisciplinary Science

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and Technology, Thiruvananthapuram -695019, Kerala, India.

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Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India, 201002.

Tel: +919495902522

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Fax: +914712491712

email:

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Abstract

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Vanillic acid (VA) is a flavoring and nutritional agent found in many fruits and vegetables. It is

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an antioxidant but its nutraceutical potential has not been studied in detail. In this study, the

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potential of VA against hyperinsulinemia mediated changes on redox status and mitochondria in

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HepG2 cells were investigated. Incubation of cells with 1 μM insulin for 24 hr was found to

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induce insulin resistance using the inhibition of Glut2 and glucose uptake (51.9%).

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Hyperinsulinemia caused depletion of superoxide dismutase, glutathione, glutathione peroxidase

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and generation of reactive oxygen species (68%). It also caused overexpression of the receptor

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for advanced glycation end products (120%) and a decreases of dolichyl-diphospho-

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oligosaccharide-protein glycosyltransferase non-catalytic subunit (34%). Mitochondria were

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affected with alterations in mitochondrial transmembrane potential, aconitase activity,

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mitochondrial fission and fusion, biogenesis (AMPK, Sirt1 and PGC-1α) and bioenergetics (ATP

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and oxygen consumption). Co-treatment with VA decreased oxidative stress by reducing of

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reactive oxygen species and lipid peroxidation during hyperinsulinemia. Similarly, VA protected

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the mitochondria during insulin shock. VA also prevented glycation through the decrease of the

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receptor for advanced glycation end products expression. VA was found to act through the

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AMPK/Sirt1/PGC-1α pathway to obtain its beneficial activity. From the overall results it was

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concluded that VA is expected to be a potential nutraceutical which could be explored for the

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development of affordable nutraceuticals after detailed in vivo study.

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Key words: Hyperinsulinemia; Reactive oxygen species; Glycation; Mitochondria; HepG2

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cells, Angelica sinensis.

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Abbreviations

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∆ψm

mitochondrial membrane potential

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2-DG

2-deoxy-D-glucose

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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)

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DTT

dithiothreitol

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ECL

enhanced chemiluminescence

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dolichyl-diphospho-oligosaccharide-protein glycosyltransferase non-catalytic

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EDTA

ethylenediaminetetraacetic acid

63

EGTA

ethylene glycol tetraacetic acid

64

FBS

fetal bovine serum

65

FIS1

fission 1 protein

66

GLUT2

glucose transporter 2

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

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HI

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HRP

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IR

insulin resistance

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IRS2

insulin receptor substrate 2

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JC-1

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MDA

malondialdehyde

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MES

2-(N-morpholino)ethanesulfonic acid

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high insulin

horseradish peroxidase

5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazol carbocyanine iodide

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MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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NAC

N-acetyl-cysteine

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NADP

nicotinamide adenine dinucleotide phosphate

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NCCS

National Centre for Cell Science

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OPA1

optic atrophy 1

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OS

oxidative stress

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p-AMPK

phospho-AMP activated kinase

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PBS

phosphate-buffered saline

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PGC-1α

peroxisome proliferator activated receptor γ coactivator-1α

88

PVDF

polyvinylidene difluoride

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RAGE

90

RIPA

91

ROS

reactive oxygen species

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RT

room temperature

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SEM

standard error of the mean

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Sirt1

sirtuin 1

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SOD

superoxide dismutase

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receptor for advanced glycation end products

radioimmuno precipitation assay

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SPSS

Statistical Package for the Social Sciences

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T2DM

type 2 diabetes mellitus

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TBST

tris buffered saline-Tween 20

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VA

vanillic acid

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1. Introduction
Alternative approaches are needed to prevent and treat metabolic diseases such as type 2

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diabetes mellitus (T2DM) and associated health issues. Non-pharmacological management with

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the utilization of herbal dietary products has been an option and further work is needed in the

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search for culinary plants for prophylactic and therapeutic use. These edible biomaterials have

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been shown to alleviate complex disorders using nutritional intervention (Choudhury et al.,

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2018). Functional foods are being developed to manage chronic diseases, such as T2DM and

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cardiovascular diseases. Some have enhanced antioxidant, anti-inflammatory and insulin

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sensitivity functions.

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Hyperinsulinemia is associated with health complications of diabetes. Insulin resistance (IR) is a

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major issue with hyperinsulinemia (Marin-Juez et al., 2014). This has been established in animal

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and human studies (Shanik et al., 2008). Insulin is one of the main hormones for regulating

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glucose metabolism (Wilcox, 2005). Circulating levels are controlled by the nutrients involved in

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glucose uptake, glycolysis and glycogen storage, lipogenesis, and protein synthesis (Czech et al.,

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2013; Fu et al., 2013). Insulin may also have some autocrine functions like the promotion of β-

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cell growth and influence its own production and release (Wang et al., 2013). Hyperinsulinemia

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could enhance the desensitization of the insulin receptor which results in IR (Templeman et al.,

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2017). Corkey (2012) showed that hyperinsulinemia is the root cause of IR and diabetes.

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Inhibition of hyperinsulinemia results in the reduction of IR without affecting glucose tolerance

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including in human studies (Reed et al., 2011). Thus, early recognition of hyperinsulinemia may

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be helpful to guide earlier intervention strategies to prevent or delay diabetes onset and related

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chronic diseases. Hyperinsulinemia could alter redox status (Kim et al., 2008) and induce surplus

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generation of superoxide anions, hydrogen peroxide and hydroxyl radicals (Ge et al., 2008; Li et

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al., 2015). These effects were reversed using antioxidants such as N-acetyl-cysteine, superoxide

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dismutase or catalase. Therefore, oxidative stress (OS) could be a potential interventional target

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for hyperinsulinemia induced IR and related diseases. Mitochondria are the powerhouse of the

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cell and involved in important functions of the cell such as regulation of ATP production, redox

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status and apoptosis. Mitochondrial dysfunction and associated OS are often involved at the start

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in the genesis of metabolic syndromes. Hyperinsulinemia associated pathologies have been

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associated with OS and mitochondrial dysfunction but the detailed information needed to design

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therapeutic strategies based on molecular mechanisms might be beneficial (Gonzalez-Franquesa

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et al., 2017).

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Based on the importance of antioxidants in protecting the mitochondria from OS during

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hyperinsulinemia, vanillic acid (VA) was selected for this study. It is a flavoring agent mainly

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found in the root of the Chinese medicinal plant Angelica sinensis. It is also found in many

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alcoholic beverages, cereals, dried fruits, nuts and herbs. It is a strong antioxidant (Tai et al.,

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2012) and anti-lipid-peroxidative agent (Vinoth & Kowsalya, 2018). It is the oxidized form of

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vanillin and has antibacterial, antimicrobial, and chemopreventive activities (Itoh et al., 2010).

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Only one report showed that VA protects against hyperinsulinemia and hyperlipidemia by

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decreasing the serum glucose, triglycerides, and free fatty acids (Chang et al., 2015). Similarly,

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not much research has been done with hyperinsulinemia induced alterations in redox status

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associated with mitochondrial dysfunction and glycation in human hepatocellular carcinoma

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(HepG2) cells. In this study the effects of VA on hyperinsulinemia in HepG2 cells, an in vitro

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model of the hyperinsulinemic insulin resistant liver, was studied.

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2. Materials and methods

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Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-

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streptomycin antibiotics (10,000 IU/mL of each) and 0.5% trypsin (porcine pancreas)-

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ethylenediaminetetraacetic acid (trypsin-EDTA) were from Gibco-BRL Life Technologies

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(Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),

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dimethylsulfoxide (DMSO), radioimmunoprecipitation assay buffer (RIPA buffer), 2,7-

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dichlorodihydrofluorescein diacetate (DCFH-DA), and VA were from Sigma Aldrich Chemical

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Co. (St. Louis, MO, USA). Adenosine monophosphate activated kinase (AMPK), phospho-

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AMPK (p-AMPK), peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), sirtuin

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1 (Sirt 1), fission 1 protein (FIS 1), optic atrophy 1 (OPA 1), β-actin and all other secondary

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antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). RAGE and DDOST were

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from Abcam (Cambridge, MA, USA). Metformin, N–acetyl-cysteine (NAC) and amino

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guanidine were from SRL (Mumbai, India). Recombinant human insulin, MitoSoxTM dye and

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JC-1 dye were from Merck (Kenilworth, NJ, USA). The remaining chemicals used were of

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analytical grade from SRL.

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2.1. Cell culture

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HepG2 cell lines which were purchased from the National Centre for Cell Science (NCCS,

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Maharashtra, India) were maintained in DMEM supplemented with 10% FBS and antibiotics

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(100 IU/mL of each penicillin and streptomycin) in a humidified atmosphere with 5% CO2 at 37

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C.

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2.2. Establishment of IR through hyperinsulinemic shock of HepG2 cells

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HepG2 cells were seeded in 96-well plates at a density of 2×104 cells/well counted using a

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haemocytometer (Merck) and grown for 24 hr to reach 80% confluence. Then the cells were

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cultured in the presence or absence of different concentrations (50 or 100 nM or 1 μM) of insulin

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and parameters relevant to IR (glucose uptake, insulin receptor substrate (IRS) 2, glucose

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transporter 2 (Glut2)) were studied to confirm the development of hyperinsulinemia mediated IR

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in HepG2 cells.

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2.2.1. Glucose uptake

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Briefly, the cells were incubated with various concentrations of insulin for 24 hr. Then glucose

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uptake was assessed using a glucose uptake colorimetric assay kit (Abcam). The control and

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hyperinsulinemic group were incubated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

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(HEPES) buffered saline (20 mM) containing 10 μM 2-deoxy-D-glucose (2-DG) at room

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temperature (RT, 22 to 28 oC) for 5 min. After two washes the cells were trypsinized by adding

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100 μL of 10X trypsin-EDTA and centrifuged at 20,000 x g (AG-716 rotor, model 7780 high

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speed refrigerated centrifuge, Kubota Laboratory Centrifuges Co., Tokyo, Japan) for 15 min at 4

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o

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min at 4 oC. The supernatant was collected. Reaction mix A (10 μL) was mixed with 50 μL

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supernatant and incubated for 1 hr at RT. Extraction buffer (90 μL) was added and heated to 90

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Then the absorbance was measured every 2-3 min at 412 nm (BioTek Synergy 4, BioTek

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Instruments Corp., Winooski, VT, USA).

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C. The pellets were dissolved in neutralizing buffer (10 μL), centrifuged at 20,000 x g for 15

C for 40 min. Finally, 12 μL of neutralizing buffer and 38 μL of reaction mix B were added.

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2.2.2. Alterations of insulin signaling pathway

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The expression of IRS 2 and Glut 2 were visualized using western blotting (for details please see

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section 2.19).

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2.3. Experimental groups to check potential of VA against hyperinsulinemic shock

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The experimental groups consisted of control (C), insulin resistant (HI; 1 μM insulin) cells (IR),

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IR + 5 μM VA (VA1), IR +10 μM VA (VA2), IR+1 mM metformin (M).

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2.4. Cell viability with VA

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Cell viability was measured using the MTT assay. After 80% confluency they were treated with

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VA. After 24 hr, the medium was replaced with 100 μL of MTT (0.05 mg/mL) solution and

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incubated for 3-4 hr at 37 oC. The formazan crystals were dissolved in 100 μL of DMSO and the

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purple color was measured after 20 min at 570 nm using a microplate reader (BioTek Synergy

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4).

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2.5. Intracellular ROS

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Intracellular ROS levels were measured using DCFH-DA as a probe. DCFH-DA is oxidized by

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intracellular nonspecific esterases and high level of fluorescence occurs with ROS. Briefly, the

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cells were incubated with the DCFH-DA (20 μM) at 37 oC for 20 min. Then fluorescence was

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measured at an excitation of 488 nm and emission of 525 nm using a fluorescence microplate

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reader (BioTek Synergy 4). Fluorescent imaging was done using a bioimager system (BD

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pathway

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analyses were done with a flow cytometer, by quantifying the fluorescence produced by ROS.

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Bioimager, BD Biosciences, San Jose, CA, USA). For the conformation, ROS

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The cells after treatment were incubated with DCFH-DA at 37 oC for 20 min. Then the cells

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were trypsinized as previously and analyzed using a flow cytometer FACS Aria

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Biosciences).

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2.6. Superoxide dismutase (SOD) activity

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Total SOD activity was assayed using a SOD assay kit (Cayman Chemical, Ann Arbor, MI,

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USA). For this the cells were homogenized with a Tissue master 125-watt laboratory

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homogenizer with a 5 mm probe (Omni International, Perkin Elmer Co., Kennesaw GA, USA) in

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cold 20 mM HEPES buffer (1 mM ethylene glycol tetraacetic acid (EGTA), 210 mM mannitol,

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70 mM sucrose) and centrifuged at 1500 x g for 5 min at 4 oC. The supernatant (10 μL) and

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standards (bovine-erythrocyte SOD) were added to each well containing 200 μL of diluted

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radical detector. Then 20 μL of diluted xanthine oxidase were added quickly and incubated for

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30 min. The absorbance was measured at 450 nm using the microplate reader.

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2.7. Activity of Glutathione (GSH)

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GSH was assayed according to the manufacturer's instructions (Cayman Chemical). After

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treatments with VA, the cell pellets were dissolved in 2 mL sample buffer. Sample and standard

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of 50 μL each were added to corresponding wells. Then 150 μL of assay cocktail were added and

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kept in the dark. Then absorbance was measured every 2-3 min at 407 nm for 30 min using the

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microplate reader.

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2.8. Glutathione peroxidase (GPx) determination

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This assay measured GPx activity by a coupled reaction with glutathione reductase. The cell

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pellet was mixed in cold buffer containing 50 mM tris HCl (pH = 5.5), 5 mM

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ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT). Sample (20 μL), assay

II (BD

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buffer (100 μL) and co-substrate mix (50 μL) were added to respective wells. Then 20 μL of

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cumene hydroperoxide was added to initiate the reaction. The absorbance was measured at 340

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nm.

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2.9. Antiglycation activity assay

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The method of Riya et al. (2015) was used to measure bovine serum albumin (BSA) derived

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advanced glycation end products (AGE) with slight modifications. VA of different

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concentrations was added to BSA (10 mg/mL) and glucose (500 mM). Fluorescence of AGE at

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an excitation/emission wavelengths of 370/440 nm was obtained after 24 hr and 7 days using the

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fluorescence microplate reader. The two results were compared and the percentage inhibition of

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VA against glycation was measured.

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2.10. Analysis of AGE with hyperinsulinemia

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The method of Rani et al. (2018) was used to analyze the AGE. Cell samples and a standard

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(provided with the kit) were added to the AGE conjugate coated ELISA plate provided with the

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kit. After 10 min incubation at RT, 50 μL of anti-AGE polyclonal antibody was added and

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incubated for 1 hr. The plate was washed with 250 μL 1X wash buffer. Horseradish peroxidase

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(HRP) conjugated secondary antibody (100 μL) was added to the wells and incubated for 1hr at

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RT. Substrate solution (100 μL) was added which was followed by the addition of 100 μL of

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stop solution and measured immediately at 450 nm using the microplate reader.

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2.11. Estimation of malondialdehyde (MDA).

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Malondialdehyde levels were measured using a lipid peroxidation assay kit (Cayman Chemical).

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After treatment with VA the cells were subjected to trypsinization using 1 mL of 10X trypsin-

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EDTA as previously and the cell pellets were sonicated (Elmasonic, Elmasonic S 30 (H), 37 kHz

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Elma Schmidbauer GmbH, Gottlieb-Daimler, D-78224 Singen, Germany) for 5 sec. Sample and

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standard (100 μL each) were added to 100 μL of sodium dodecyl sulfate (SDS). After 1 hr of

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boiling, all the tubes were kept in ice for 10 min to terminate the reaction. After 10 min it was

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centrifuged at 3600 x g rpm for 10 min at 4 oC. Then 150 μL of samples were added into a 96

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well plate and then read at 530 nm.

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2.12. Mitochondrial superoxide.

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Mitochondrial superoxide production was evaluated with a MitoSoxTM (Merck) kit. The cells

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were treated with 5 mM mitosox and incubated for 20 min. After three washes with HBSS, the

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bioimages were visualized using the bioimager system at 514 and 580 nm. Fluorescence was

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measured with excitation at 514 nm and emission at 580 nm using the microplate reader.

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2.13. Aconitase activity.

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After treatments, cells were subjected to trypsinization using 1 mL of 10X trypsin-EDTA as

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previously and centrifugated at 800 x g for 10 min at 4 oC and the pellet was collected. The cell

279

pellet was resuspended in 1 mL assay buffer. The cell suspension was centrifuged at 20,000 x g

280

for 10 min at 4 oC. Sample (cell supernatant) of 50 μL was added to the respective wells. Then

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aconitase nicotinamide adenine dinucleotide phosphate (aconitase NADP) (50 μL) reagent and

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aconitase isocitrate dehydrogenase solution (50 μL) were added along with 50 μL of substrate.

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The absorbance was measured at 340 nm for 30 min at 37 oC. The change in absorbance/min was

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determined and reaction rate was calculated. The aconitase activity was calculated as nM.

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2.14. Mitochondrial membrane potential (∆ψm).

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The method of Anupama et al. (2018) was used. After treatments with VA, the medium was

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changed and the cells were stained with JC-1 stain (Merck) for 20 min at 37 oC. In normal cells,

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the JC-1 dye accumulates inside the mitochondria and forms JC-1 aggregates and gave a red

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fluorescence. Distortion of the ∆ψm prevents the dye entry into the mitochondria, as a result JC-

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1 monomers were formed and produced green fluorescence. The shift of fluorescence was

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visualized and fluorescence intensity was measured using the fluorescence microplate reader

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with an excitation 490 and emission wavelength of 530 nm for JC-1 monomers, and the

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excitation 525 and emission wavelength 590 nm for aggregates.

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2.15. Mitochondrial dynamics.

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The expression levels of fission and fusion proteins were analysed using western blotting (for

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details please see section 2.19).

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2.16. ATP content.

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ATP levels were measured using the ATP determination assay kit. After treatment with VA the

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cells were homogenized as previously described with ATP assay buffer. Then 100 μL of 1X

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somatic cell ATP releasing agent, and 50 μL of ultrapure water provided in the kit were added

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into 50 μL of sample. A 100 μL aliquot was transferred to the reaction vial with 100 μL of ATP

303

assay mix and kept at RT for 3 min. The amount of light emitted at 560 nm was measured using

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the microplate reader.

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2.17. Oxygen consumption.

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This oxygen consumption rate assay kit uses a phosphorescent oxygen probe to check oxygen

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consumption rate. Blank wells were filled with only culture medium. After treatments with VA,

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the spent medium was replaced with fresh medium. MitoXpress xtra solution (10 μL) was added

310

to all the wells except the blank. Then 100 μL of HS mineral oil (provided with the kit) was

311

added over each well. After that the fluorescence was read at 380 nm (excitation) and 650 nm

312

(emission) kinetically for 150 min using the microplate reader.

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2.18. Mitochondrial biogenesis

314

The expression of proteins like AMPK, p-AMPK, Sirt1 and PGC-1α were evaluated with

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western blotting (for details please see section 2.19).

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2.19. Western blot

317

Expression of various proteins of pharmacological and functional importance in glucose

318

transport, glycation and mitochondrial function such as IRS2, GLUT2, RAGE, DDOST, AMPK,

319

p-AMPK, PGC-1α, Sirt 1, FIS1 and OPA1 were studied using western blotting. Cells were

320

cultured in T-25 flask containing 5 mL DMEM medium and the respective treatments with VA

321

were done as described above. After that the cells were harvested and lysed in lysis buffer with a

322

protease inhibitor cocktail and Triton X 100. Then the lysate was centrifuged at 20,000 x g for 15

323

min at 4 oC. The supernatant was collected and protein content was measured and normalized

324

using a Pierce BCA protein assay kit (Thermo Fisher Scientific Co., Waltham, MA, USA) using

325

bovine serum albumin (BSA) as a standard and expressing the results as BSA equivalents.

326

Samples were then run on an SDS-PAGE gel (10%) (BioRad, Hercules, CA, USA), transferred

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at 25 V for 15 min to polyvinylidene difluoride (PVDF) membrane (a non-reactive thermoplastic

328

fluoropolymer produced using the polymerization of vinylidene difluoride) (Merck) using a trans

329

blot apparatus (BioRad). The samples (25 μL) were loaded into each well. After transfer, the

330

PVDF membrane was blocked with 3% BSA in tris buffered saline-Tween 20 (TBST, pH=8) for

331

1 hr at RT. After washing with TBST, the membrane was probed with primary antibodies (IRS2,

332

GLUT2, RAGE, DDOST, AMPK, p-AMPK, PGC-1α, Sirt 1, FIS1 and OPA1, 1:1000 dilution)

333

in TBST and incubated for 2 hr at RT with moderate shaking. The membrane was washed three

334

times with TBST for 10 min. HRP-conjugated secondary antibody (1:2000) was added and

335

agitated for 90 min at RT. After three TBST washes, membranes were incubated with enhanced

336

chemiluminescence substrate (ECL substrate) (BioRad) and the proportional thickness of bands

337

measured using Image Lab software in the Chemi Doc system (ChemiDoc MP Imaging System,

338

Bio-Rad) assuming all bands were in the Beer-Lambert law region.

339

2.20. Statistical analysis

340

All analyses were carried out with sextuplicates and data are shown as mean ± standard error of

341

the mean (SEM) for control and treated cells. The normality of the variables were tested using

342

the Kolmogorov Smirnov Z test and the variables were found to be approximately normally

343

distributed. Hence the significance difference between the groups were tested using one-way

344

analysis of variance (ANOVA) and further significantly different pairs (p≤0.05) were identified

345

using Duncan's multiple comparison test. All calculations were done using the Statistical

346

Package for the Social Sciences for Windows standard version 20 (SPSS Inc., Chicaco, IL,

347

USA).

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3. Results

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3.1. Induction of IR through hyperinsulinemic shock in HepG2 cells

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The results of the glucose uptake with different concentrations of insulin treated HepG2 cells are

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shown in Fig. 1a. The cellular uptake of 2-DG with various concentrations of insulin (50, 100

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nM and 1 µM) treated cells was decreased 5.5, 35.4 and 51.9%, respectively, as compared with

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normal cells. Among these, 100 nM and 1 µM insulin showed significant (p≤0.05) decreases in

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glucose uptake. The expression levels of IRS2 and Glut2 were significantly decreased with 1 µM

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insulin compared to normal (Fig. 1b and 1c) suggesting that HI (1 µM insulin) significantly

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affected glucose uptake, insulin sensitivity and develop IR. Insulin (1 µM) was chosen to induce

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hyperinsulinemic shock in subsequent experiments.

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3.2. Cell viability

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To select suitable concentration of VA, cell viability was evaluated with 5, 10, 20, 30, 40, 50 and

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100 μM for 24 hr of incubation. Based on the results 5 and 10 μM were selected for further

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studies (Fig. 2a).

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Effect of VA on viability of IR cells were evaluated. Incubation of HepG2 cells with HI for 24 hr

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caused 17.2% cell death that was significant compared to control (p≤0.05). Co-treatment with 5

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and 10 μM of VA and metformin (1 mM) improved (90.5, 98.1 and 92.6%, respectively)

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viability compared to IR (Fig. 2b).

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3.3. Effect of VA on ROS generation

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Determination of ROS by both spinning disk fluorescence microscopy and flow cytometry found

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a surplus generation of ROS in IR. IR showed a significant increase in ROS levels (68%) (Fig.

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3a and 3b) compared to control (Fig. 3a and 3b). Co-treatment with VA (5 and 10 μM) was

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significantly (p≤0.05) effective in preventing ROS formation by 88.2 and 99.6%, respectively,

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compared to the IR group (Fig. 3a and 3b). Co-treatment with metformin and NAC, an

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antioxidant significantly reduced the ROS generation by 96 and 108%, respectively (Fig. 3a and

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3b). This was also confirmed with cytometry data (Fig. 3c and 3d) which determined the number

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of cells that could produce ROS. The IR cells showed a significantly (p≤0.05) enhanced ROS

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levels (40.7%) compared to control cells (Fig. 3c and 3d). Co-treatment with VA (5 and 10 μM

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concentrations) significantly (p≤0.05) decreased the ROS levels by 51.3 and 58.3%, respectively,

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compared with IR cells (Fig. 3c and 3d). Co-treatment with metformin and NAC significantly

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reduced the ROS levels 63.3 and 65.5%, respectively, compared to IR treated group (Fig. 3c and

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3d).

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3.4. Effect of high insulin on SOD levels

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Compared to control cells, the activity of SOD was significantly (p≤0.05) increased in IR treated

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cells (74.7%). VA co-treatment reduced the SOD activity by 92.2% at 5 μM and by 113% at 10

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μM (p≤0.05) compared with IR (Fig. 4). But metformin significantly (p≤0.05) improved the

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SOD activity by 81.2%, respectively.

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3.5. GSH levels

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Compared to control IR caused a significant (p≤0.05) drop in GSH level by 31.4%. VA co-

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treatment at both concentrations (5 and 10 μM) caused a significant (p≤0.05) increase in GSH

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levels by 58 and 65.3%, respectively, compared to IR. Metformin significantly improved the

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GSH level by 85.3% (Fig. 5) compared to IR.

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3.6. Level of GPx

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GPx activity was decreased in IR by 19.9% compared to control. But VA co-treatment

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significantly (p≤0.05) increased the GPx activity by 29.3 (5 μM) and 35.8% (10 μM) compared

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to IR (Fig. 6). Metformin increased the GPx levels by 42.9%.

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3.7. Antiglycation capacity of VA

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VA showed a dose-response inhibition of glycation (IC50; 397 μM). The half maximal inhibitory

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concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological

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or biochemical function. The effect of VA was better than that of the aminoguanidine, as the

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positive control (IC50; 500 μM; Fig. 7).

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3.8. Production of AGE during high insulin

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IR showed an increased level of AGE (221%) as compared to control. While with VA, the

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formation of AGE was reduced by 232 and 252% for 5 and 10 μM, respectively, with IR.

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Metformin significantly reduced the AGE levels by 283%. (Fig. 8a).

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Western blotting was done for DDOST and RAGE proteins. IR caused a significant increase in

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the expression of RAGE (120%) compared to control. VA co-treatment significantly decreased

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the expression of RAGE (147 and 165% for 5 and 10 μM; p≤0.05) compared to the IR group

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(Fig. 8b and 8c). Metformin significantly (p≤0.05) decreased RAGE levels by 168%. Expression

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of DDOST was reduced by 34.5% in IR cells compared to control cells. But VA co-treatment at

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5 and 10 μM concentrations significantly (p≤0.05) increased the DDOST protein levels by 56.1

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and 63.3%, respectively, compared to IR. Metformin also increased the DDOST levels by 68.9%

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compared to IR cells (Fig. 8b and 8c).

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3.9. Lipid peroxidation

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Lipid peroxidation was also increased (p≤0.05) in IR cells by 52.8% (Fig. 9). Co-treatment with

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VA at 5 and 10 μM and metformin lowered MDA by 67.6, 78.7 and 87.4%, respectively,

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compared to IR.

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3.10. Effect of VA on mitochondrial superoxide production

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Surplus generation of ROS was observed in IR groups (Fig. 10) compared to control.

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Fluorescence detection also showed an increment of fluorescence in IR cells (143%). VA of both

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concentrations (5 and 10 μM) reduced superoxide by 161 and 193%, respectively (Fig. 10).

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Compared with metformin (187%) 10 μM of VA showed better results (Fig. 10).

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3.11. Aconitase activity

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The aconitase activity was significantly decreased in IR (34%) groups compared to the control.

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VA (5 and 10 μM) in a dose dependent manner showed a tendency to increased the enzyme level

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by 24.2 and 36.6% compared to IR. Co-treatment with metformin significantly increased the

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aconitase levels by 56.5% compared to IR (Fig. 11).

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3.12. Effect of vanillic acid on ∆ψm

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Analysis of the ∆ψm of mitochondria during IR showed significant distortion of ∆ψm compared

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with control (Fig. 12a). In control cells, the JC-1 dye forms aggregate inside the mitochondrial

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matrix and emit a red fluorescence due to the potential gradient. Alteration in ∆ψm resulted in

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the prevention of entry of JC-1 in the mitochondria and resulted in green fluorescence (JC-1

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monomers). IR cells showed depolarized ∆ψm as can be seen in Fig. 12a, which had a higher

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amount of green fluorescence (103%) compared to that of control. VA co-treatment with IR

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prevented the alteration of ∆ψm which was evident from the significant increase (p≤0.05) of red

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fluorescence by 79.9 and 93.9%, respectively, for 5 and 10 μM of VA compared to IR (Fig. 12b).

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The quantity of red fluorescence (aggregates) with metformin was improved, and the green

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fluorescence was decreased by 116% compared to IR (Fig. 12b). Valinomycin was the negative

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control, and it caused a decrease of the red fluorescence by 40.4% and improved the green

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fluorescence by 108% compared to control.

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3.13. Mitochondrial fission and fusion proteins.

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During IR condition there was a noticeable decrease in the levels of mitochondrial fusion protein

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OPA1 by 33.7% (p≤0.05; Fig. 13a and 13b) and an up-regulation of fission protein FIS1 by

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24.2% (Fig. 13a and 13b). VA co-treatment at 5 and 10 μM significantly (p≤0.05) increased the

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expression of OPA1 by 52.2 and 60.6%, respectively, and in a dose dependent manner decreased

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the FIS1 by 92.3 and 109% compared to the IR group. Metformin co-treatment also significantly

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(p≤0.05) increased the fusion protein by 58.1% and reduced the fission protein FIS 1 by 81.6%

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compared to the IR group (Fig. 13a and 13b).

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3.14. ATP levels and oxygen consumption

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There was a significant decrease in ATP levels (p≤0.05, 35.6%; Fig. 14) in IR cells compared to

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control cells. Co-treatment with VA at both concentrations (5 and 10 μM) significantly (p≤0.05)

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increased the ATP levels by 56.3 and 71.9%, respectively, compared to IR. Metformin also

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significantly increased the ATP levels by 133% compared with IR.

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Oxygen consumption in cells was evaluated by calculating the change in fluorescence for two

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and a half hr. More changes in fluorescence represent more usage of oxygen by cells. This, in

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turn, represented the good metabolic status of cells. With the IR group oxygen consumption rate

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was reduced (25%) as compared to control. Co-treatment with VA at 5 and 10 μM significantly

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(p≤0.05) improved the oxygen consumption rate by 13.1 and 26.2% compared with IR group

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(Fig. 15). VA with better oxygen utilization seems to be effective against mitochondrial

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dysfunction in IR. Metformin also increased the oxygen consumption rate by 29.6% compared to

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IR.

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3.15. Mitochondrial biogenesis regulated by VA.

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AMPK, PGC-1α and Sirt1 are the major proteins responsible for mitochondrial biogenesis. The

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active form of AMPK was determined in various groups using the ratio of p-AMPK to total

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AMPK. During IR, the expression of p-AMPK/AMPK was decreased significantly by 62.3%

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compared to control cells whereas VA in a dose dependent manner increased the ratio

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significantly (193 and 185% for 5 and 10 μM, respectively) (Fig. 16a and 16b). Also, VA (5 and

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10 μM) significantly (p≤0.05) increased the level of PGC-1α by 50.4 and 71.3%, respectively,

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compared to the IR group (Fig. 16a and 16c). In IR the level of PGC-1α was reduced to 29.5%

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compared to control as well as there was also a significant decrease in Sirt1 expression in IR

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cells (31.5%). On the other hand, VA co-treatment at 5 and 10 μM increased the expression of

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Sirt1 by 115 and 119%, respectively (Fig. 16a and 16c). Metformin significantly (p≤0.05)

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increased all three proteins (p-AMPK/AMPK, PGC-1α and Sirt1) involved in mitochondrial

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biogenesis by 300, 80.6 and 113%, respectively, compared to IR cells (Fig. 16a-16c).

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