Eur J Nutr DOI 10.1007/s00394-014-0703-2

ORIGINAL CONTRIBUTION

GSH protects against oxidative stress and toxicity in VL-17A cells exposed to high glucose S. Mathan Kumar • Kavitha Swaminathan Dahn L. Clemens • Aparajita Dey



Received: 30 January 2014 / Accepted: 10 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Purpose The deficiency of glutathione (GSH) has been linked to several diseases. The study investigated the role of GSH as a protective factor against hyperglycemiamediated injury in VL-17A cells treated with 50 mM glucose. Methods The cell viability and different oxidative stress parameters including glyoxalase I activity were measured. Results GSH supplementation with 2 mM N-acetyl cysteine (NAC) or 0.1 mM ursodeoxycholic acid (UDCA) increased the viability, GSH level and the GSH-dependent glyoxalase I activity in 50 mM glucose-treated VL-17A cells. Further, pretreatment of 50 mM glucose-treated VL17A cells with NAC or UDCA decreased oxidative stress (levels of reactive oxygen species and protein carbonylation), apoptosis (caspase 3 activity and annexin V–propidium iodide positive cells) and glutathionylated protein formation, a measure of oxidative stress. GSH depletion with 0.4 mM buthionine sulfoximine (BSO) or 1 mM diethyl maleate (DEM) potentiated the decrease in viability, glyoxalase I activity and increase in oxidative stress

S. M. Kumar  K. Swaminathan  A. Dey (&) Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai 600044, India e-mail: [email protected]; [email protected]; [email protected]

and apoptosis, with decreased GSH levels in 50 mM glucose-treated VL-17A cells. Conclusion Thus, changes in GSH levels with exogenous agents such as NAC, UDCA, BSO or DEM modulate hyperglycemia-mediated injury in a cell model of VL-17A liver cells. Keywords Glutathione  Hyperglycemia  Liver  Reactive oxygen species  Injury Abbreviations Ac-DEVD-pNA ADH AGEs BSO CYP2E1 DEM DCF-DA DAS GSH MTT NAC PI pNA PYR ROS UDCA

Ac-DEVD-p-nitroanilide Alcohol dehydrogenase Advanced glycated end products Buthionine sulfoximine Cytochrome P4502E1 Diethyl maleate 20 ,70 -dichlorofluorescein diacetate Diallyl sulfide Glutathione Thiazolyl blue tetrazolium bromide N-Acetyl cysteine Propidium iodide p-Nitroaniline Pyrazole Reactive oxygen species Ursodeoxycholic acid

D. L. Clemens Nebraska and Western Iowa Veterans Administration Medical Center, University of Nebraska Medical Center, Omaha, NE, USA

Introduction

D. L. Clemens Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA

Oxidative stress is a prominent mechanism for diabetic complications [1, 2]. Hyperglycemia-mediated induction

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of cytochrome P4502E1 (CYP2E1) promotes oxidative stress and consequent impairment of cellular processes due to its ability to generate reactive oxygen species (ROS) [3–5]. Glutathione (GSH) is a major cellular antioxidant with higher concentrations being expressed in liver [6, 7]. The liver plays a major role in interorgan GSH homeostasis [8, 9]. Apart from acting as an antioxidant, GSH serves as a reservoir of cellular cysteine, maintains intracellular redox state and modulates cell growth [10]. Buthionine sulfoximine (BSO) has been found to be the most specific and potent inhibitor of gamma-glutamyl cysteine synthetase among several other inhibitors as the Salkyl moiety of the sulfoximine binds at its active site that normally binds the acceptor amino acid [11, 12]. BSO besides being involved in depletion of GSH increases oxidative stress in the cells through increased sensitivity of the cells to H2O2 and several other oxidative stressinducing agents [13]. Another mode of GSH depletion occurs through GSH transferase-mediated reactions where diethyl maleate (DEM) conjugates with GSH to form hydrophilic GSH conjugates and causes rapid depletion of GSH [12, 14, 15]. Similarly, N-acetyl cysteine (NAC) and ursodeoxycholic acid (UDCA) increase GSH levels in the cells and differ in their modes of actions. While NAC is a cysteine donor that is a structural component of GSH [16], UDCA increases the transcription of gamma-glutamyl cysteine synthetase [17]. In VL-17A cells over-expressing alcohol dehydrogenase (ADH) and CYP2E1 [18], studies from our laboratory have shown that a high concentration of glucose (50 mM) causes oxidative stress and toxicity [5]. This in vitro model of liver cells effectively replicates the in vivo diabetic models (15–20 mM glucose) due to similar increases in glucose concentration from the normoglycemic condition (5 mM glucose) since HepG2 cells can grow well in medium containing 24.5 mM glucose (threefold to fourfold increase vs. twofold increase). It was interesting to investigate the role of GSH as a protective mechanism against ADH or CYP2E1-mediated toxicity which potentiates hyperglycemia-mediated injury in liver cells, utilizing the exogenous agents for altering intracellular GSH level-NAC, UDCA, BSO or DEM.

Materials and methods This study was performed using HepG2 cells devoid of ADH and CYP2E1 expression and VL-17A cells, which are HepG2 cells constitutively expressing ADH and CYP2E1 [18]. Liver cells were cultured using routine

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culture protocol. Experiments were performed with 2 mM NAC [19] or 0.1 mM UDCA [17] as GSH-supplementing agents or 0.4 mM BSO [20] or 1 mM DEM [21] as GSHdepleting agents. The cells were incubated for 72 h. Both HepG2 and VL-17A cells were each divided into eight experimental groups and were treated with the different agents as indicated: group 1: untreated HepG2/VL-17A cells (Control); group 2: 5 mM D-glucose (normoglycemic control); group 3: 5 mM glucose ? 45 mM D-mannitol (osmotic control); group 4: 50 mM D-glucose (hyperglycemia); group 5: 2 mM NAC ? 50 mM D-glucose; group 6: 0.1 mM UDCA ? 50 mM D-glucose; group 7: 0.4 mM BSO ? 50 mM D-glucose; and group 8: 1 mM DEM (removed after 2 h) ? 50 mM D-glucose. Experiments were also performed with specific inhibitors for CYP2E110 lM diallyl sulfide (DAS) [18, 22] and ADH- 2 mM pyrazole (PYR) [23]. The cell viability was assayed by MTT assay, using MTT cell proliferation assay kit according to the manufacturer’s instructions. Fluorescence spectrometry was used to measure the intracellular levels of ROS with 20 ,70 -dichlorofluorescein diacetate (DCF-DA) as described previously [24]. Protein carbonyl content in cells was analyzed spectrophotometrically as described by Palmeira [25]. GSH content was estimated as previously described in liver cells [26]. Glyoxalase I activity in liver cells was measured using a modified method of Di Ilio et al. [27] and Baba et al. [28]. The formation of glutathionylated protein adducts was detected through immunostaining by a slight modification of the method described by Palanisamy et al. [29]. Caspase 3 activity was measured in the supernatant by detecting the hydrolysis of Ac-DEVD-p-nitroanilide (Ac-DEVD-pNA) as previously described [24]. Apoptosis or necrosis was determined using an annexin V–FITC apoptosis detection kit (Sigma-Aldrich, India) as previously described [24]. The formation of advanced glycated end (AGE) product was measured by following a previously described method [30]. Results are expressed as mean ± SE of three experiments. For photomicrographs and Western blot experiments, 1 representative experiment of 3 is shown. One way ANOVA followed by Bonferroni posthoc test was performed to analyze results between the different groups. p \ 0.05 was considered to be statistically significant. *p \ 0.05 compared with HepG2 or VL-17A cells in group 1, -tp \ 0.05 compared with HepG2 or VL-17A cells in group 2, § p \ 0.05 compared with HepG2 or VL-17A cells in group 3, àp \ 0.05 compared with HepG2 cells in group 4, (only group 4 is compared with groups 1–3, and groups 5–8 are not compared with groups 1–3, instead they are compared with group 4 only), and #p \ 0.05 compared with HepG2 or VL-17A cells in group 4.

Eur J Nutr Table 1 Effect of GSH supplementation or depletion on cell viability of HepG2 and VL-17A cells Groups

HepG2 cells

VL-17A cells

Group 1

100.0 ± 1.5

100.0 ± 1.8

Group 2

97.5 ± 2.1

95.9 ± 0.2

Group 3

98.6 ± 0.2

93.2 ± 0.8

Group 4

71.1 ± 0.6*,t-,§

60.8 ± 1.2*,t-,§

101.1 ± 7.0

#

83.6 ± 1.9#

Group 6

114.0 ± 5.9

#

92.0 ± 1.4#

Group 7 Group 8

73.1 ± 0.6 68.7 ± 5.5

Group 5

44.0 ± 5.2# 48.0 ± 3.1#

Results are expressed as % viability * p \ 0.05 compared with HepG2 or VL-17A cells in group 1 -t

p \ 0.05 compared with HepG2 or VL-17A cells in group 2

§

p \ 0.05 compared with HepG2 or VL-17A cells in group 3

#

p \ 0.05 compared with HepG2 or VL-17A cells in group 4

Results Effect of GSH supplementation or depletion on cell viability of HepG2 and VL-17A cells The viability data between the different groups of HepG2 and VL-17A cells showed significant differences (p \ 0.02) as assessed by one way ANOVA wherever indicated significant. Further, Bonferroni posthoc analysis revealed that the viability of HepG2 and VL-17A cells in group 4 was significantly decreased to 71 % (p \ 0.05) and 61 % (p B 0.0002) when compared with HepG2 and VL17A cells, respectively, in groups 1–3 (Table 1). The viability of HepG2 and VL-17A cells in group 5 was significantly increased to 101 % (p B 0.0002) and 83 % (p B 0.0002) when compared with HepG2 and VL-17A cells in group 4. The viability of HepG2 and VL-17A cells in group 6 was significantly increased to 114 % (p B 0.0002) and 92 % (p B 0.0002) when compared with HepG2 and VL-17A cells in group 4. The viability of HepG2 cells in groups 7 and 8 was not significantly decreased (69–71 % viability) (p [ 0.05) when compared with HepG2 cells in group 4. However, VL-17A cells in groups 7 and 8 exhibited 44 and 48 % viability (p B 0.0002), respectively, when compared with VL-17A cells in group 4. Effect of GSH supplementation or depletion on ROS level in HepG2 and VL-17A cells The level of ROS between the different groups of HepG2 and VL-17A cells showed significant differences (p B 0.01) wherever indicated significant. HepG2 cells in group 4 exhibited 1.1-fold (p \ 0.05) significantly higher

ROS levels than HepG2 cells in groups 1–3 (Fig. 1a). Similarly, VL-17A cells in group 4 exhibited 1.8- to 1.9fold (p B 0.0002) significantly higher ROS levels than VL17A cells in groups 1–3. HepG2 cells in groups 5 and 6 exhibited 1.3- and 1.5-fold (p B 0.0002) significantly decreased ROS level when compared with HepG2 cells in group 4. VL-17A cells in groups 5 and 6 showed 1.7- and 1.3-fold (p B 0.0002) significantly decreased ROS level when compared with VL-17A cells in group 4. HepG2 cells in groups 7 and 8 (p [ 0.05) did not exhibit significant changes in ROS level when compared with HepG2 cells in group 4. VL-17A cells in groups 7 and 8 exhibited 1.2- and 1.5-fold (p B 0.0002) significantly increased ROS level when compared with VL-17A cells in group 4. Effect of GSH supplementation or depletion on protein carbonyl formation of HepG2 and VL-17A cells The protein carbonyl formation between the different groups of HepG2 and VL-17A cells showed significant differences (p \ 0.0005) wherever indicated significant. HepG2 cells in group 4 exhibited 1.0- to 1.1-fold (p B 0.0002) significantly increased protein carbonyl formation, respectively, than HepG2 cells in groups 1–3 (Fig. 1b). VL-17A cells in group 4 exhibited 1.5- to 1.6fold (p B 0.0002) significantly increased protein carbonyl formation, respectively, than VL-17A cells in groups 1–3. Further, protein carbonyl formation was 1.1-fold (p \ 0.001) significantly higher in VL-17A cells in group 4 when compared with HepG2 cells in group 4. HepG2 cells in groups 5 and 6 exhibited 1.1- to 1.2-fold (p B 0.0002) significantly decreased protein carbonyl formation when compared with HepG2 cells in group 4. Similarly, significant decreases of 1.1- to 1.2-fold (p \ 0.001) in protein carbonyl formation were observed in VL-17A cells in group 5 and 6 when compared with VL-17A cells in group 4. However, no significant changes in protein carbonyl formation were observed in HepG2 cells in groups 7 and 8 (p [ 0.05). VL-17A cells in groups 7 and 8 showed 1.4and 1.8-fold (p B 0.0002) significantly increased protein carbonyl formation when compared with VL-17A cells in group 4. Effect of GSH supplementation or depletion on caspase 3 activity of HepG2 and VL-17A cells The activity of caspase 3 between the different groups of HepG2 and VL-17A cells showed significant differences (p \ 0.05) wherever indicated significant. HepG2 cells in group 4 exhibited 1.1- to 1.2-fold (p \ 0.05) significantly increased caspase 3 activity when compared with HepG2 cells in groups 1–3 (Fig. 2a). VL-17A cells in group 4 exhibited 1.5- to 1.6-fold (p \ 0.01) significantly

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Eur J Nutr

(a) DCF-DA fluorescence (Arbitrary units/10 4 cells

150

HepG2 VL-17A

#

ठ75







#

*

§

#

#

*

#

#

Group 5

Group 6

0 Group 1

Group 2

Group 3

Group 4

Group 7

Group 8

Protein Carbonyl (n moles/mg protein)

(b)

# 100

#

‡§ § *

HepG2 VL- 17A

# #

*

50

#

#

0 Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group 7

Group 8

Fig. 1 Effect of GSH supplementation or depletion on a intracellular ROS level and b protein carbonyl formation in HepG2 and VL-17A cells

increased caspase 3 activity when compared with VL-17A cells in groups 1–3. Further, caspase 3 activity was 1.2fold (p \ 0.05) significantly higher in VL-17A cells in group 4 when compared with HepG2 cells in group 4. HepG2 cells in groups 5 and 6 exhibited 1.1- to 1.2-fold (p \ 0.05) significant decreases in caspase 3 activity when compared with HepG2 cells in group 4. VL-17A cells in groups 5 and 6 exhibited 1.4- and 1.7-fold (p B 0.002) significantly decreased caspase 3 activity when compared with VL-17A cells in group 4. However, no significant changes in caspase 3 activity were observed in HepG2 cells in groups 7 and 8 (p [ 0.05). VL-17A cells in groups 7 and 8 showed 1.2- to 1.3-fold (p B 0.02) significantly increased caspase 3 activity when compared with VL-17A cells in group 4. Effect of GSH supplementation or depletion on apoptosis in HepG2 and VL-17A cells HepG2 cells in groups 1–3 were characterized by the presence of a very few number of annexin V–propidium iodide (PI)-stained cells (?) (Fig. 2b). Similar trends were observed with the corresponding groups of VL-17A cells (??). An increase in the number of annexin V–PI-stained cells was observed in HepG2 (??) or VL-17A cells (????) in group 4. HepG2 and VL-17A cells in groups 5 and 6 showed decreased number of annexin V–PI-stained cells (? and ??, respectively) when compared with HepG2 and VL-17A cells in group 4. HepG2 cells in groups 7 and 8 did not show increased number of cells stained positively for annexin V–PI (??) when compared

123

with HepG2 cells in group 4. In contrast, VL-17A cells in groups 7 and 8 exhibited an increase in annexin V–PIstained cells (?????). Effect of GSH supplementation or depletion on GSH level in HepG2 and VL-17A cells The levels of GSH between the different groups of HepG2 and VL-17A cells showed significant differences (p B 0.05) wherever indicated significant. HepG2 cells in group 4 exhibited 1.1-fold (p \ 0.05) significantly higher GSH level when compared with HepG2 cells in groups 1–3 (Fig. 3a). VL-17A cells in group 4 exhibited 1.5-fold (p B 0.0002) significantly higher GSH level when compared with VL-17A cells in groups 1–3. Further, GSH level was 2.2-fold (p B 0.002) significantly higher in VL-17A cells in group 4 when compared with HepG2 cells in group 4. HepG2 and VL-17A cells in group 5 exhibited 1.1- to 1.3-fold significantly higher GSH levels when compared with HepG2 (p B 0.01) and VL-17A cells (p B 0.02) in group 4. Similarly, HepG2 and VL-17A cells in group 6 exhibited 1.2- to 1.3-fold significantly higher GSH levels when compared with HepG2 (p B 0.02) and VL-17A cells (p \ 0.0005) in group 4. HepG2 and VL-17A cells in group 7 exhibited 1.4- and 1.6-fold significantly decreased GSH levels when compared with HepG2 (p \ 0.01) and VL-17A cells (p \ 0.0005) in group 4, respectively. Similarly, significant decreases of 1.4- and 1.6-fold in GSH levels were observed in HepG2 (p \ 0.01) and VL-17A cells (p B 0.0002) in group 8 when compared with HepG2 and VL-17A cells in group 4, respectively.

Eur J Nutr

pNA (n moles/min/mg protein)

(a)

(b)

0.15

#

‡§

HepG2 VL-17A

§ * * 0.08

#

#

#

#

#

0.00 Group 1

Group 2

Group 3

Group 4

VL-17A

HepG2

Group 1

Group 3

Group 5

Group 6

Group 5

Group 6

Group 7

Group 8

VL-17A

HepG2

Group 2

Group 4

Group 7

Group 8

Fig. 2 Effect of GSH supplementation or depletion on a caspase-3 activity and b annexin V–PI staining in HepG2 and VL-17A cells

Effect of GSH supplementation or depletion on glyoxalase I activity in HepG2 and VL-17A cells The glyoxalase I (glyoxal) activity between the different groups of HepG2 and VL-17A cells showed significant differences (p B 0.02) wherever indicated significant. HepG2 cells in groups 1–3 exhibited 1.2- to 1.4-fold

(p \ 0.05) significantly increased glyoxalase I (glyoxal) activity when compared with HepG2 cells in group 4 (Fig. 3a (i)). VL-17A cells in groups 1–3 exhibited 1.6- to 1.8-fold (p B 0.002) significantly increased glyoxalase I (glyoxal) activity when compared with VL-17A cells in group 4. Further, glyoxalase I (glyoxal) activity was 1.5fold (p \ 0.01) significantly higher in VL-17A cells in

123

Eur J Nutr

Glyoxalase I activity (S-glycolyglutathione mM/ml) Glyoxalase I activity (S-D-lactoylglutathione mM/ml)

(b)

GSH (µ moles/min/mg protein)

# (a)





#

ठ*

0.010



§

0.005

HepG2 VL-17A

#

#

#

#

#

*

#

0.000 Group 1

7.0



Group 2



Group 3

Group 4



‡§ § * *

3.5

Group 5

#

Group 6

Group 7

Group 8

HepG2 VL-17A

# #

#

0.0 Group 1

7.0



Group 2



Group 3

Group 4

Group 5

Group 6

Group 7

Group 8

HepG2 VL-17A



§ §

3.5

*

*

#

#

# #

# #

0.0 Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group 7

Group 8

Fig. 3 Effect of GSH supplementation or depletion on a GSH level; b (i) glyoxalase I activity (Glyoxal); (ii) glyoxalase I activity (Methyl glyoxal); and c glutathionylated protein adduct formation of HepG2 and VL-17A cells

group 4 when compared with HepG2 cells in group 4. VL17A cells in groups 5 and 6 exhibited 1.2- and 1.5-fold (p B 0.002) significantly increased glyoxalase I (glyoxal) activity, whereas no significant changes (p [ 0.05) in glyoxalase I (glyoxal) activity were observed in HepG2 cells in groups 5 and 6 when compared with HepG2 cells in group 4. No significant changes (p [ 0.05) in glyoxalase I (glyoxal) activity were observed in HepG2 cells in groups 7 and 8, a 1.2-fold (p \ 0.01) significant decrease in glyoxalase I (glyoxal) activity was observed in VL-17A cells in groups 7 and 8 when compared with VL-17A cells in group 4. The glyoxalase I (methyl glyoxal) activity between the different groups of HepG2 and VL-17A cells showed significant differences (p \ 0.05) wherever indicated significant. HepG2 cells in group 4 did not exhibit a significant decrease (p [ 0.05) in glyoxalase I (methylglyoxal) activity when compared with HepG2 cells in groups 1–3 (Fig. 3b (ii)). VL-17A cells in group 4 exhibited 2.5- to

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2.8-fold (p B 0.002) significantly decreased glyoxalase I (methylglyoxal) activity, respectively, when compared with VL-17A cells in groups 1–3. Glyoxalase I (methylglyoxal) activity was not significantly higher (p [ 0.05) in VL-17A cells in group 4 when compared with HepG2 cells in group 4. While no significant changes in glyoxalase I (methylglyoxal) activity were observed in HepG2 cells in groups 5 and 6 (p [ 0.05), 1.2- to 1.3-fold (p B 0.02) significant increases in glyoxalase I (methylglyoxal) activity were observed in VL-17A cells in groups 5 and 6 when compared with VL-17A cells in group 4. While 1.7to 2.1-fold (p \ 0.01) significant decreases in glyoxalase I (methylglyoxal) activity were observed in HepG2 cells in groups 7 and 8 when compared with HepG2 cells in group 4, 1.0- to 1.5-fold (p \ 0.05) significant decreases in glyoxalase I (methylglyoxal) activity were observed in VL17A cells in groups 7 and 8 when compared with VL-17A cells in group 4.

Eur J Nutr

(c)

VL-17A

HepG2

Group 1

Group 3

Group 5

Group 6

VL-17A

HepG2

Group 2

Group 4

Group 7

Group 8

Fig. 3 continued

Effect of GSH supplementation or depletion on glutathionylated protein adduct formation in HepG2 and VL-17A cells Glutathionylated protein adduct formation was evident in HepG2 (?) or VL-17A (???) cells in group 4 (Fig. 3c). HepG2 and VL-17A cells in groups 5 and 6 exhibited decreased glutathionylated protein adduct formation (-) and (??), respectively, observed as decrease in the intensity of the staining when compared with HepG2 and VL-17A cells in group 4. Besides, HepG2 and VL-17A cells in groups 5 and 6 showed greater number of clusterforming cells. HepG2 cells in groups 7 and 8 did not exhibit increased glutathionylated protein adduct formation (?) when compared with HepG2 cells in group 4. VL-17A cells in groups 7 and 8 exhibited an increased glutathionylated protein adduct formation (???? and ?????), respectively,

along with the presence of few scattered cells when compared with VL-17A cells in group 4. Effect of CYP2E1 or ADH or CYP2E1 and ADH inhibition and GSH supplementation on cell viability in VL-17A cells The viability data between the different groups of HepG2 and VL-17A cells showed significant differences (p \ 0.05) wherever indicated significant. VL-17A cells in group 4 exhibited significantly decreased viability (62–63 %) (p B 0.0002) (Fig. 4a, b) and treatment with 10 lM DAS increased the viability to 86 % (p B 0.0002) (Fig. 4a). VL-17A cells in group 4 treated with 2 mM PYR showed 80 % viability (p B 0.0002) (Fig. 4b). VL-17A cells in groups 5 and 6 exhibited 100–102 % and 91–94 % viability (p B 0.0002), respectively (Fig. 4a, b). VL-17A cells in group 5 and 6 treated with 10 lM DAS showed 97

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Eur J Nutr VL-17A

(a) 150.0 % Viability

#

#

#

#

#

*

75.0

0.0 Group 1

% Viabilbity

(b)

Group 4

Group 5

Group 6

Group 4 + 10 µM DAS Group 5 + 10 µM DAS Group 6 + 10 µM DAS

150

VL-17A

#

#

#

#

#

*

75

0 Group 1

Group 4

Group 5

Group 6

Group 4 + 2 mM PY Group 5 + 2mM PY

Group 6 + 2mM PY

Fig. 4 Effect of CYP2E1 or ADH inhibition and GSH supplementation on cell viability in VL-17A cells

and 94 % viability (p B 0.0002), respectively, (Fig. 4a) and VL-17A cells in groups 5 and 6 treated with 2 mM PYR showed 96 % viability (p \ 0.01) (Fig. 4b).

Table 2 Effect of GSH supplementation or depletion on advanced glycated end product formation in HepG2 and VL-17A cells Groups

HepG2

VL-17A

Effect of GSH supplementation or depletion on AGE formation in HepG2 and VL-17A cells

Group 1 Group 2

2.2 ± 0.19 2.1 ± 0.33

1.1 ± 0.22= 1.2 ± 0.13=

Group 3

2.1 ± 0.24

1.1 ± 0.08=

Group 4

3.1 ± 0.45*,t-,§

AGE receptors comprise of specific and non-specific receptors. The ratios of specific/non-specific AGE formation between the different groups of HepG2 and VL-17A cells showed significant differences (p B 0.01) wherever indicated significant. The ratio of specific/non-specific AGE formation was significantly increased 1.2- to 1.4-fold (p \ 0.005) in HepG2 cells in group 4 when compared with HepG2 cells in groups 1–3 (Table 2). The ratio of specific/non-specific AGE formation was significantly increased 1.3- to 1.5-fold (p \ 0.05) in VL-17A cells in group 4 when compared with VL-17A cells in groups 1–3. Further, the ratio of specific/non-specific AGE formation was 1.7-fold (p \ 0.001) significantly higher in HepG2 cells in group 4 than VL-17A cells in group 4. HepG2 cells in groups 5 and 6 exhibited 1.8- and 2.2-fold (p \ 0.05) significantly decreased AGE formation when compared with HepG2 cells in group 4. VL-17A cells in groups 5 and 6 exhibited significantly decreased AGE formation 1.5- and 1.8-fold (p \ 0.05) when compared with VL-17A cells in group 4. HepG2 cells in groups 7 and 8 showed 1.0- to 1.2fold (p \ 0.05) significantly increased AGE formation when compared with HepG2 cells in group 4. However,

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Group 5 Group 6 Group 7 Group 8

1.8 ± 0.49*,t-,§,à

1.7 ± 0.40

#

1.0 ± 0.27#

1.4 ± 0.30

#

1.2 ± 0.44#

3.6 ± 0.31

#

3.0 ± 0.34#

3.2 ± 0.16

#

3.1 ± 0.25#

Results are expressed as OD/mg protein =

p \ 0.05 compared with the corresponding group of HepG2 cells

* p \ 0.05 compared with HepG2 or VL-17A cells in group 1 -t

p \ 0.05 compared with HepG2 or VL-17A cells in group 2

§

p \ 0.05 compared with HepG2 or VL-17A cells in group 3 p \ 0.05 compared with HepG2 cells in group 4

à #

p \ 0.05 compared with HepG2 or VL-17A cells in group 4

VL-17A cells in groups 7 and 8 exhibited 1.7-fold (p \ 0.001) significantly increased AGE formation when compared with VL-17A cells in group 4. Discussion Oxidative stress is a major mechanism for hyperglycemiamediated injury in both in vivo and in vitro conditions [4,

Eur J Nutr

5, 20, 31]. Regulation of blood glucose concentration through insulin is an essential mechanism to maintain normoglycemic condition, and insulin has been shown to increase GSH synthesis in cultured primary rat hepatocytes [32]. Studies in our laboratory have led to conclusive evidences for CYP2E1 to be a key player in hyperglycemia-induced liver cell toxicity [33]. The present study addresses both aspects of GSH i.e., its supplementation and depletion with various agents in protecting or promoting high glucose-mediated oxidative stress and toxicity in VL17A cells. The study utilized an in vitro model of VL-17A liver cells and although the findings cannot be extrapolated to human studies and direct comparisons cannot be made between cellular and human studies, several in vivo studies have reported similar findings which have been discussed here. The increase in viability and decrease in ROS level in 50 mM glucose-treated HepG2 and VL-17A cells with NAC or UDCA and similarly the decreased viability and increased ROS level in 50 mM glucose-treated VL-17A cells with BSO or DEM suggest that GSH plays a crucial role in protecting liver cells against hyperglycemia-mediated oxidative stress and toxicity in vitro. Increased levels of GSH can protect cells against oxidative stress by directly acting as an antioxidant or by serving as a substrate for the removal of ROS or reactive metabolites by antioxidant enzymes [20]. Neither BSO nor DEM increased ROS level and protein carbonyl formation in 50 mM glucose-treated HepG2 cells. In contrast, significant increases in ROS levels with BSO or DEM were observed in 50 mM glucose-treated VL-17A cells, due to severe oxidative stress— a net effect of the three factors involved: hyperglycemiamediated ROS, hyperglycemia inducible CYP2E1-mediated ROS and depletion of GSH. The accumulation of ROS due to GSH depletion by BSO [34] may account for the effects observed with BSO treatment in VL-17A cells. A similar trend was observed for the mode of cell death in 50 mM glucose-treated liver cells and the modifying effects due to GSH supplementation with NAC or UDCA and GSH depletion with BSO or DEM. Apoptosis in 50 mM glucose-treated HepG2 and VL-17A cells was decreased with NAC or UDCA, and the anti-apoptotic actions of NAC or UDCA have been reported previously [35]. Although BSO or DEM increased the apoptotic mode of cell death in 50 mM glucose-treated VL-17A cells, GSH depletion with these agents did not increase apoptosis in HepG2 cells further. The increased incidence of apoptosis in VL-17A cells links CYP2E1-mediated oxidative stress under hyperglycemic condition and GSH depletion as additive factors in potentiating apoptosis in vitro. Apoptotic agents have been shown to be either oxidants or stimulate oxidative metabolism, while many inhibitors of apoptosis are antioxidants or mediators of cellular

antioxidant defenses [36]. Depletion of cellular antioxidant defenses leads to the generation of significant quantities of ROS, which may act as a signal for the induction of apoptosis [37]. However, antioxidant depletion (e.g., GSH), rather than production of ROS, may cause apoptosis [38].The pro-apoptotic actions of BSO or DEM have been well documented [39, 40]. Treatment of liver cells with 50 mM glucose increased GSH levels, which reflected an adaptive response to hyperglycemia-mediated oxidative stress in HepG2 cells and hyperglycemia-mediated oxidative stress plus CYP2E1-mediated oxidative stress in VL-17A cells. The status of GSH levels in hyperglycemic models varies: some studies have reported decrease in levels of GSH in diabetes [41], while others have reported elevated GSH levels under hyperglycemic conditions [42, 43]. Both NAC and UDCA increased the GSH levels and BSO and DEM decreased the GSH levels in 50 mM glucose-treated HepG2 and VL-17A cells. Advanced glycated end products (AGEs) induce hyperglycemic injury and are derived from products generated from auto-oxidation of glucose and fructose which include deoxyglucosone, methyl glyoxal and glyoxal [28]. Methyl glyoxal and glyoxal are metabolized and detoxified by the glyoxalase system consisting of glyoxalase I and II enzymes [28]. A recent study has indicated that glyoxalase I catalyzes a large percentage of methyl glyoxal formed (60 %) and only 15 % of glyoxal is metabolized at low concentrations of glyoxal (1–100 mM) and methylglyoxal (1–20 mM) [44]. Glyoxalase I activity using glyoxal as substrate was higher in untreated VL-17A cells when compared with HepG2 cells, consistent with the earlier observations that VL-17A cells have higher GSH levels to counteract the increased oxidative stress [45]. Glyoxalase I (glyoxal) activity was decreased in both 50 mM glucose-treated HepG2 cells and VL-17A cells, but to a greater extent in VL-17A cells, and glyoxalase I (methylglyoxal) activity was decreased only in 50 mM glucose-treated VL-17A cells, reflecting the overwhelming oxidative stress due to hyperglycemia per se and hyperglycemia inducible CYP2E1 in vitro. While NAC or UDCA increased glyoxalase I activity in 50 mM glucose-treated VL-17A cells, the glyoxalase I activity in 50 mM glucose-treated HepG2 cells was unchanged. We speculate that the increased glyoxalase I activity due to NAC or UDCA in 50 mM glucose-treated VL-17A cells reflects the beneficial effects of the antioxidants in a severe oxidative stress environment in vitro. Both BSO and DEM caused significant decreases in glyoxalase I activity in 50 mM glucose-treated VL-17A cells, due to severe oxidative stress, a consequence of GSH depletion, which was not observed in HepG2 cells.

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Glyoxalase I activity was found to be significantly reduced in patients with type 1 and type 2 diabetes mellitus and painful diabetic neuropathy compared with patients with painless diabetic neuropathy, and the increase in Glo1 activity was significantly associated with reduced occurrence of painful diabetic neuropathy [46]. Further, glyoxalase I and glyoxalase II activities were found to be decreased in the liver of diabetic rats compared with normal controls [47]. Consistent with the higher AGE formation in untreated or 5 mM glucose or 45 mM mannitol plus 5 mM glucosetreated HepG2 cells than corresponding groups of VL-17A cells, lower glyoxalase I activity using methylglyoxal as substrate was observed in HepG2 cells. Further, consistent with the decreases in glyoxalase I (methylglyoxal) activity, increases in ratio of specific/non-specific AGE formation were observed in 50 mM glucose-treated VL-17A cells. Although glyoxalase I (methylglyoxal) activity did not exhibit significant changes in NAC or UDCA plus 50 mM glucose-treated HepG2 cells, AGE formation was decreased and the decrease in AGE formation was greater than that observed with VL-17A cells reflecting the absence of a severe oxidative stress in HepG2 cells devoid of ADH and CYP2E1 and hence the greater beneficial effects of NAC or UDCA. The increase in glyoxalase I (methylglyoxal) activity is consistent with decreased AGE formation observed in NAC or UDCA plus 50 mM glucose-treated VL-17A cells. Although a greater decrease in glyoxalase I (methylglyoxal) activity was observed in BSO or DEM plus 50 mM glucose-treated HepG2 cells (1.7- to 2.1-fold) when compared with VL-17A cells (1.0- to 1.5-fold), the increase in AGE formation was greater in VL-17A cells (1.7-fold) than HepG2 cells (1.0- to 1.2-fold). GSH is higher in 50 mM glucose-treated VL-17A cells than HepG2 cells, glyoxalase I is a GSH-dependent enzyme and with higher levels of GSH in untreated and 50 mM glucose-treated VL-17A cells leads to greater glyoxalase I activity and hence inhibition or decrease with BSO or DEM is lesser in VL-17A cells than HepG2 cells. However, the increased oxidative stress due to high glucose plus BSO or DEM favors the greater formation of AGE in VL17A cells. S-glutathionylation of proteins occurs through the specific posttranslational modification of protein cysteine residues by the addition of GSH [48]. Oxidative stress promotes the S-glutathionylation of proteins, and the formation of glutathionylated protein adducts may reflect a protective response toward oxidative stress [48]. Glutathionylated protein adduct formation was increased to a much greater extent in 50 mM glucose-treated VL-17A cells than HepG2 cells. NAC or UDCA were completely effective in decreasing the glutathionylated protein adduct

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formation in 50 mM glucose-treated HepG2 cells. The effects of NAC or UDCA were observed to a lesser extent in decreasing the glutathionylated protein adduct formation in 50 mM glucose-treated VL-17A cells, suggesting increased oxidative stress in 50 mM glucose-treated VL17A cells and hence the upregulation of glutathionylated protein adduct formation. While BSO or DEM did not increase the glutathionylated protein adduct formation in 50 mM glucose-treated HepG2 cells, it was greatly increased in BSO or DEM plus 50 mM glucose-treated VL-17A cells. Glutathionylated hemoglobin levels have been found to be markedly increased in diabetic subjects with microangiopathy, and the authors speculate that since diabetic subjects also exhibited increased lipid peroxidation and decreased GSH levels, it appears that enhanced oxidative stress may account for the increased glutathionylated hemoglobin concentrations and altered redox signaling [49]. The effects of the CYP2E1 inhibitor DAS, ADH inhibitor PYR and 4-methyl PYR were studied in different combinations and permutations with the GSH-contributing agents— NAC or UDCA in VL-17A cells. NAC or UDCA in the presence of DAS increased the viability of 50 mM glucose-treated VL-17A cells by 8–11 % versus DAS alone, suggesting that upregulation of GSH due to exogenous GSH supplementation is an additional factor contributing to the increased viability of 50 mM glucosetreated VL-17A cells apart from inhibition of CYP2E1mediated oxidative stress and toxicity. NAC or UDCA in the presence of PYR increased the viability of 50 mM glucose-treated VL-17A cells by 16 % versus PYR alone, suggesting that upregulation of GSH due to exogenous GSH supplementation and hence decreased oxidative stress leads to increased viability of 50 mM glucose-treated VL17A cells.

Conclusions In conclusion, GSH plays a crucial role in decreasing oxidative stress and toxicity in VL-17A liver cells. Pretreatment of 50 mM glucose-treated VL-17A cells with 2 mM NAC or 0.1 mM UDCA reversed the decrease in viability and increased ROS and protein carbonyl levels and 0.4 mM BSO or 1 mM DEM potentiated oxidative stress and toxicity. Likewise, the increased apoptosis observed in 50 mM glucose-treated VL-17A cells was abrogated with NAC or UDCA, but BSO or DEM promoted apoptotic injury. The elevated GSH level observed in 50 mM glucose-treated VL-17A cells was further increased with NAC or UDCA, but decreased with BSO or DEM. The activity of the detoxification enzyme—glyoxalase I was decreased in 50 mM glucose-treated VL-17A

Eur J Nutr

cells. NAC or UDCA increased the glyoxalase I activity, and BSO or DEM were characterized by their inhibitory effects on glyoxalase I activity. Likewise, glutathionylated protein adduct formation observed in VL-17A cells was accentuated with BSO or DEM and lowered with NAC or UDCA treatment. Further, the inhibition of CYP2E1 and ADH complemented the protective effects of NAC or UDCA in increasing the viability of 50 mM glucose-treated VL-17A cells. The findings of the study suggest that GSH or derivatives of GSH may protect against hyperglycemia inducible ADH and CYP2E1-mediated oxidative stress and toxicity in an in vitro model of VL-17A cells.

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Acknowledgments AD acknowledges the financial support received from Department of Biotechnology, New Delhi, India (Rapid Grant for Young Investigators). DLC was supported by a grant from the Veterans Administration. KS is grateful to Council of Scientific and Industrial Research (CSIR), New Delhi, India for awarding the Junior and Senior Research Fellowships. We thank Dr. BM Jaffar Ali, Life Sciences Division, AU-KBC Research Centre, Chennai, India for his kind suggestions as a Co-PI in the DBT sponsored project. We thank Dr. Suvro Chatterjee, Life Sciences Division, AU-KBC Research Centre, Chennai, India for generously sharing his laboratory facilities for fluorescence microscopy.

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The authors declare that there are no conflicts

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Conflict of interest of interest.

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GSH protects against oxidative stress and toxicity in VL-17A cells exposed to high glucose.

The deficiency of glutathione (GSH) has been linked to several diseases. The study investigated the role of GSH as a protective factor against hypergl...
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