Essential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes

doi:10.1016/j.freeradbiomed.2015.03.019

Free Radical Biology and Medicine

Volume 84, July 2015, Pages 263-278

https://doi.org/10.1016/j.freeradbiomed.2015.03.019 Get rights and content

Highlights

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LA reduced PA-mediated ER stress, ROS levels, and lipoapoptosis in hepatocytes.
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LA induced early nuclear translocation of Nrf2 in response to PA in hepatocytes.
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Lipophagy is involved in the positive effects of LA on lipid storage in hepatocytes.
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Silencing Nrf2 in hepatocytes by siRNA abolished the effects of LA on lipoapoptosis.
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LA prevents and reverses apoptosis and ER stress and activates Nrf2 in rats with NAFLD.

Abstract

Excess of saturated free fatty acids, such as palmitic acid (PA), in hepatocytes has been implicated in nonalcoholic fatty liver disease. α-Lipoic acid (LA) is an antioxidant that protects against oxidative stress conditions. We have investigated the effects of LA in the early activation of oxidative and endoplasmic reticulum stress, lipid accumulation, and Nrf2-mediated antioxidant defenses in hepatocytes treated with PA or in rats fed a high-fat diet. In primary human hepatocytes, a lipotoxic concentration of PA triggered endoplasmic reticulum stress, induced the apoptotic transcription factor CHOP, and increased the percentage of apoptotic cells. Cotreatment with LA prevented these effects. Similar results were found in mouse hepatocytes in which LA attenuated PA-mediated activation of caspase 3 and reduced lipid accumulation by decreasing PA uptake and increasing fatty acid oxidation and lipophagy, thereby preventing lipoapoptosis. Moreover, LA augmented the proliferation capacity of hepatocytes after PA challenge. Antioxidant effects of LA ameliorated reactive oxygen species production and endoplasmic reticulum stress and protected against mitochondrial apoptosis in hepatocytes treated with PA. Cotreatment with PA and LA induced an early nuclear translocation of Nrf2 and activated antioxidant enzymes, whereas reduction of Nrf2 by siRNA abolished the benefit of LA on PA-induced lipoapoptosis. Importantly, posttreatment with LA reversed the established damage induced by PA in hepatocytes, as well as preventing obesity-induced oxidative stress and lipoapoptosis in rat liver. In conclusion, our work has revealed that in hepatocytes, Nrf2 is an essential early player in the rescue of oxidative stress by LA leading to protection against PA-mediated lipoapoptosis.

Graphical abstract

Section snippets

Reagents and antibodies

Fetal bovine serum (FBS) and culture media were obtained from Invitrogen (Carlsbad, CA, USA). PA and LA were from Sigma–Aldrich (St. Louis, MO, USA). Anti-cleaved (Asp175) caspase 3 (9661), anti-phospho-JNK (Thr183/Tyr185) (4668), anti-phospho-p38 MAPK (Thr180/Tyr182) (9211), anti-phospho-PERK (Thr980) (3179), anti-phospho-eIF2α (Ser51) (9721), anti-phospho-p53 (Ser15) (9286), anti-Bax (2772), anti-Puma (7467), and anti-Bim (2919) antibodies were purchased from Cell Signaling Technology

LA prevents the effects of PA in the activation of stress kinases, ER stress, and lipoapoptosis and increases the expression of Nrf2-related proteins in primary human hepatocytes

We first analyzed the effects of LA on PA-induced lipoapoptosis in primary human hepatocytes treated with PA (800 μM) in the absence or presence of LA (250 μM). Quantification of TUNEL-positive hepatocytes revealed a significant increase after PA treatment for 16 h, and this effect was not observed in cells cotreated with LA (Fig. 1A). At the molecular level, phosphorylation of stress kinases such as JNK and p38 MAPK, as well as the ER stress kinase eIF2α, was observed at 8 h in PA-treated

Discussion

Nonalcoholic fatty liver disease may result in progressive liver disease with risks for cirrhosis and HCC [53]. Intracellular lipid accumulation in hepatocytes (first hit) followed by enhancement of oxidative stress (second hit) is required for the progression from simple fatty liver to NASH [54], [55], [56], [57]. Moreover, several lines of evidence suggest that toxic ROS can cause ER stress responses such as the UPR [58]. Among therapies suggested for slowing disease progression, we have

Conclusion

In conclusion and as summarized in the graphical abstract, our results strongly suggest that in hepatocytes, Nrf2 is an essential early player in the rescue of oxidative stress by LA leading to protection against PA-mediated lipoapoptosis. Importantly, we have demonstrated for the first time by in vitro and in vivo approaches that an intervention protocol based on the administration of LA after PA overload is able to substantially decrease hepatocyte lipoapoptosis.

Acknowledgments

We acknowledge the following grant support: SAF2012-33283 (Ministry of Economy and Competitiveness, MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant, and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (ISCIII, Spain) to A.M.V.; SAF2010-17822 (MINECO, Spain) and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (ISCIII, Spain) to A.C.; PI09/0185 (ISCIII, Spain) and Centro de

References (83)

C Garcia-Monzon et al.
Hepatic insulin resistance is associated with increased apoptosis and fibrogenesis in nonalcoholic steatohepatitis and chronic hepatitis C
J. Hepatol
(2011)
E Vanni et al.
From the metabolic syndrome to NAFLD or vice versa?
Dig Liver Dis
(2010)
AE Feldstein et al.
Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis
Gastroenterology
(2003)
RH Unger et al.
Lipoapoptosis: its mechanism and its diseases
Biochim. Biophys. Acta
(2002)
K Begriche et al.
Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it
Mitochondrion
(2006)
D Pessayre et al.
NASH: a mitochondrial disease
J. Hepatol.
(2005)
H Kamata et al.
Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases
Cell
(2005)
Y Akazawa et al.
Palmitoleate attenuates palmitate-induced Bim and PUMA up-regulation and hepatocyte lipoapoptosis
J. Hepatol.
(2010)
SC Cazanave et al.
JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis
J. Biol. Chem.
(2009)
S Nakamura et al.
Palmitate induces insulin resistance in H4IIEC3 hepatocytes through reactive oxygen species produced by mitochondria
J. Biol. Chem.
(2009)

MW Greene et al.

PKC{delta} is activated in a dietary model of steatohepatitis and regulates endoplasmic reticulum stress and cell death

J. Biol. Chem.

(2010)

P Puri et al.

Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease

Gastroenterology

(2008)

NR Kitteringham et al.

Proteomic analysis of Nrf2 deficient transgenic mice reveals cellular defence and lipid metabolism as primary Nrf2-dependent pathways in the liver

J. Proteomics

(2010)

SG Huerta-Olvera et al.

Alpha-lipoic acid regulates heme oxygenase gene expression and nuclear Nrf2 activation as a mechanism of protection against arsenic exposure in HepG2 cells

Environ. Toxicol. Pharmacol.

(2010)

SM Elshazly et al.

Insights in the mechanism underlying the protective effect of alpha-lipoic acid against acetaminophen-hepatotoxicity

Eur. J. Pharmacol.

(2014)

SD Wollin et al.

Effects of a medium chain triglyceride oil mixture and alpha-lipoic acid diet on body composition, antioxidant status, and plasma lipid levels in the Golden Syrian hamster

J. Nutr. Biochem.

(2004)

AK Min et al.

Alpha-lipoic acid attenuates methionine choline deficient diet-induced steatohepatitis in C57BL/6 mice

Life Sci.

(2012)

MP Valdecantos et al.

Lipoic acid administration prevents nonalcoholic steatosis linked to long-term high-fat feeding by modulating mitochondrial function

J. Nutr. Biochem.

(2012)

R Singh et al.

Autophagy in the cellular energetic balance

Cell Metab.

(2011)

EM. Brunt

Pathology of nonalcoholic steatohepatitis

Hepatol. Res.

(2005)

EM. Brunt

Histopathology of non-alcoholic fatty liver disease

Clin. Liver Dis

(2009)

MM Richardson et al.

Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction

Gastroenterology

(2007)

MD Lewis et al.

Role of CYP2E1 activity in endoplasmic reticulum ubiquitination, proteasome association, and the unfolded protein response

Arch. Biochem. Biophys.

(2005)

Y Yang et al.

Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway

J. Nutr. Biochem.

(2014)

F Kaya-Dagistanli et al.

The effects of alpha lipoic acid on liver cells damages and apoptosis induced by polyunsaturated fatty acids

Food Chem. Toxicol.

(2013)

M Fernandez-Galilea et al.

J. alpha-Lipoic acid treatment increases mitochondrial biogenesis and promotes beige adipose features in subcutaneous adipocytes from overweight/obese subjects

Biochim. Biophys. Acta

(2015)

V Gianturco et al.

Impact of therapy with alpha-lipoic acid (ALA) on the oxidative stress in the controlled NIDDM: a possible preventive way against the organ dysfunction?

Arch. Gerontol. Geriatr

(2009)

KP Shay et al.

Cap-independent Nrf2 translation is part of a lipoic acid-stimulated detoxification stress response

Biochim. Biophys. Acta

(2012)

M McMahon et al.

Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression

J. Biol. Chem.

(2003)

AJ Chia et al.

Differential effect of covalent protein modification and glutathione depletion on the transcriptional response of Nrf2 and NF-kappaB

Biochem. Pharmacol.

(2010)

AC Brewer et al.

Nox4 regulates Nrf2 and glutathione redox in cardiomyocytes in vivo

Free Radic. Biol. Med.

(2011)

YT Kuo et al.

Alpha-lipoic acid induces adipose triglyceride lipase expression and decreases intracellular lipid accumulation in HepG2 cells

Eur. J. Pharmacol.

(2012)

RA Egnatchik et al.

Palmitate-induced activation of mitochondrial metabolism promotes oxidative stress and apoptosis in H4IIEC3 rat hepatocytes

Metabolism

(2014)

S Fu et al.

The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling

Cell Metab.

(2012)

NJ Hancer et al.

Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation

J. Biol. Chem.

(2014)

A Berlanga et al.

Molecular pathways in non-alcoholic fatty liver disease

Clin. Exp. Gastroenterol

(2014)

CP Day et al.

Non-alcoholic steatohepatitis: definitions and pathogenesis

J. Gastroenterol. Hepatol.

(2002)

SC Cazanave et al.

Mechanisms and clinical implications of hepatocyte lipoapoptosis

Clin. Lipidol

(2010)

L Yuzefovych et al.

Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

Am. J. Physiol. Endocrinol. Metab.

(2010)

LV Yuzefovych et al.

Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress, mitochondrial dysfunction, apoptosis, and impaired insulin signaling in rat L6 skeletal muscle cells

Endocrinology

(2012)

DW Jun et al.

Prevention of free fatty acid-induced hepatic lipotoxicity by carnitine via reversal of mitochondrial dysfunction

Liver Int.

(2011)

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Original ContributionEssential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes

Highlights

Abstract

Graphical abstract

Section snippets

Reagents and antibodies

LA prevents the effects of PA in the activation of stress kinases, ER stress, and lipoapoptosis and increases the expression of Nrf2-related proteins in primary human hepatocytes

Discussion

Conclusion

Acknowledgments

J. Hepatol

Dig Liver Dis

Gastroenterology

Biochim. Biophys. Acta

Mitochondrion

J. Hepatol.

Cell

J. Hepatol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Gastroenterology

J. Proteomics

Environ. Toxicol. Pharmacol.

Eur. J. Pharmacol.

J. Nutr. Biochem.

Life Sci.

J. Nutr. Biochem.

Cell Metab.

Hepatol. Res.

Clin. Liver Dis

Gastroenterology

Arch. Biochem. Biophys.

J. Nutr. Biochem.

Food Chem. Toxicol.

Biochim. Biophys. Acta

Arch. Gerontol. Geriatr

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Pharmacol.

Free Radic. Biol. Med.

Eur. J. Pharmacol.

Metabolism

Cell Metab.

J. Biol. Chem.

Molecular pathways in non-alcoholic fatty liver disease

Clin. Exp. Gastroenterol

Non-alcoholic steatohepatitis: definitions and pathogenesis

J. Gastroenterol. Hepatol.

Mechanisms and clinical implications of hepatocyte lipoapoptosis

Clin. Lipidol

Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress

Am. J. Physiol. Endocrinol. Metab.

Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress, mitochondrial dysfunction, apoptosis, and impaired insulin signaling in rat L6 skeletal muscle cells

Endocrinology

Prevention of free fatty acid-induced hepatic lipotoxicity by carnitine via reversal of mitochondrial dysfunction

Liver Int.

Original Contribution
Essential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes