The International Journal of Biochemistry & Cell Biology 45 (2013) 1419–1426
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The International Journal of Biochemistry
& Cell Biology
journal h om epage: www.elsevier.com/locate/biocel
Low level of hydrogen peroxide induces lipid synthesis in BRL-3A cells
through a CAP-independent SREBP-1a activation
Anna Maria Giudetti, Fabrizio Damiano
∗
, Gabriele Vincenzo Gnoni, Luisa Siculella
Laboratory of Biochemistry and Molecular Biology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via
Prov.le Lecce-Monteroni, 73100 Lecce, Italy
a r t i c l e i n f o
Article history:
Received 22 January 2013
Received in revised form 26 March 2013
Accepted 3 April 2013
Available online 10 April 2013
Keywords:
Cell viability
Cholesterologenesis
Lipogenesis
Reactive oxygen species
SREBP
a b s t r a c t
Although H
2
O
2
is traditionally known to have cytotoxic effects, recent studies argue about its regulatory
role on lipid metabolism. However, the mechanism underlying the induction of lipid biosynthesis by
oxidative stress still remains unknown. To shed light on this aspect we investigated the H
2
O
2
-induced
lipogenesis in rat liver BRL-3A cells.
We found that a short-term exposition of cells to 35 M H
2
O
2
didn’t cause any significant sign of cell
damage measured by following diene formation and lactate dehydrogenase release from cells. How-
ever, in this stressful condition, a significant increase of [1-
14
C]acetate incorporation into fatty acids and
cholesterol, associated to an increase in the activity and expression of key enzymes of fatty acid and
cholesterol synthesis, were measured. mRNA and protein contents of the transcription factors SREBP-1
and SREBP-2, involved in the activation of lipid synthesis, increased as well. The analysis of molecular
mechanism of SREBP-1 activation revealed, in treated compared to control cells, a higher SREBP-1a mRNA
translation involving an internal ribosome entry side (IRES), present in the leader region of its mRNA.
Longer exposition to the pro-oxidant induced a progressive loss of cell viability together with an increase
of cell triacylglycerol content.
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
In mammalian, Reactive Oxygen Species (ROS) are unavoidably
by-products of mitochondrial metabolic processes (Valko et al.,
2007). Furthermore, ROS can be produced in different cellular
compartments other than mitochondria (i.e. endoplasmic reticu-
lum, peroxisomes, cytosol, plasma membrane etc.) (Brown and
Borutaite, 2012).
Under physiological steady state conditions these molecules
are eliminated by complex set of non enzymatic and enzymatic
detoxification mechanisms. When these systems are altered the
intracellular level of ROS can rapidly raise, generating a condition
called “oxidative stress” (Packer and Cadenas, 2007). Because of
their high reactivity ROS are harmful for many cellular constituents
such as lipids, proteins and nucleic acids (Packer and Cadenas,
2007). A wide range of disorders including atherosclerosis, Parkin-
son’s disease, Alzheimer’s disease, and drug-induced liver injury
have been attributed to oxidative stress (Packer and Cadenas, 2007).
Among ROS, H
2
O
2
is the most weak and stable oxidant, so that
it is present with a relatively long half-life in the tissues (Fridovich,
1998). Recent studies have demonstrated that in mammalian cells
∗
Corresponding author. Tel.: +39 0832298698; fax: +39 0832298626.
E-mail address: fabrizio.damiano@unisalento.it (F. Damiano).
H
2
O
2
may act as intracellular messenger, stimulating biological
responses and activating specific biochemical pathways (Gautam
et al., 2006; Stone and Yang, 2006).
H
2
O
2
has been shown to stimulate hexose monophosphate
shunt in insulin-responsive tissue in a manner similar to insulin
(Czech et al., 1974; May and de Haën, 1979). In particular, it
has been demonstrated H
2
O
2
stimulates glucose transport, glu-
cose C-l oxidation and glucose incorporation into glycogen in rat
adipocytes (Lawrence and Larner, 1978). In addition, H
2
O
2
inhibits
theophylline-stimulated lipolysis (Livingston et al., 1977).
Recent finding showed that high H
2
O
2
concentrations stimu-
late triacylglycerols accumulation in the hepatic cancer cells HepG2
(Sekiya et al., 2008). Bettzieche and coll. (Bettzieche et al., 2008)
measured higher mRNA levels compared to untreated cells, for sev-
eral lipogenic enzymes in HepG2 cells treated with the pro-oxidant
CuSO
4
.
Despite the interest in the topic, sparse data are available
regarding the molecular mechanism/s of the H
2
O
2
-induced effects
on lipid metabolism. Understanding of the mechanism/s of stress-
induced lipogenesis could provide new insight into the metabolic
background of correlated pathogenesis. Aim of the present study
was to deeply investigate the molecular basis of the induction of
lipid synthesis in rat liver BRL-3A cells treated with H
2
O
2
.
Here, we report that during the early phase of oxidative stress,
induced by H
2
O
2
, a remarkable increase of both cholesterol and
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http://dx.doi.org/10.1016/j.biocel.2013.04.004