Impact of silencing hepatic SREBP-1 on insulin signaling
Impact of silencing hepatic SREBP-1 on insulin signaling
Victoria Jideonwo 0 1
Yongyong Hou 0 1
Miwon Ahn 0 1
Sneha Surendran 0 1
NuÂ ria Morral 0 1
0 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , Indianapolis, Indiana , United States of America
1 Editor: Raul M. Luque, University of Cordoba , SPAIN
Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that plays a central role in regulating expression of genes of carbohydrate and fatty acid metabolism in the liver. SREBP-1 activity is essential for the control of insulin-induced anabolic processes during the fed state. In addition, SREBP-1 regulates expression of key molecules in the insulin signaling pathway, including insulin receptor substrate 2 (IRS2) and a subunit of the phosphatidylinositol 3-kinase (PI3K) complex, PIK3R3, suggesting that feedback mechanisms exist between SREBP-1 and this pathway. Nevertheless, the overall contribution of SREBP-1 activity to maintain insulin signal transduction is unknown. Furthermore, Akt is a known activator of mTORC1, a sensor of energy availability that plays a fundamental role in metabolism, cellular growth and survival. We have silenced SREBP-1 and explored the impact on insulin signaling and mTOR in mice under fed, fasted and refed conditions. No alterations in circulating levels of insulin were observed. The studies revealed that depletion of SREBP-1 had no impact on IRS1Y612, AktS473, and downstream effectors GSK3αS21 and FoxO1S256 during the fed state. Nevertheless, reduced levels of these molecules were observed under fasting conditions. These effects were not associated with changes in phosphorylation of mTOR. Overall, our data indicate that the contribution of SREBP-1 to maintain insulin signal transduction in liver is modest.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by grants from
the National Institute of Diabetes and Digestive and
Kidney Diseases (R01-DK078595 and
P30DK097512), by the American Diabetes Association
(1-08-RA-135), and by a Biomedical Research
Grant from Indiana University School of Medicine.
This research was conducted in a facility
constructed with support from Research Facilities
Improvement Program Grant Number C06
A complex network of molecules senses nutrient availability, activating anabolic processes
during periods of abundance, and shutting down biosynthetic programs that consume energy
when nutrients are scarce. In the liver, insulin signaling plays a pivotal role at promoting
anabolic responses during fed conditions, including synthesis of glycogen and fatty acids from
glucose, as well as inhibiting hepatic glucose production. Insulin binding to the insulin receptor/
insulin-like growth factor (IGF-1) receptor elicits phosphorylation of insulin receptor substrates
1 and 2 (IRS1/2) on tyrosine residues, sending a downstream signal that activates
phosphatidylinositol 3-kinase (PI3K) [
]. A main outcome of PI3K activation is phosphorylation of protein
RR020128-01 from the National Center for
Research Resources, National Institutes of Health.
Victoria Jideonwo was supported by grant
R01DK078595-05S1 and by an Indiana University
President's Diversity Dissertation Fellowship;
Sneha Surendran was supported by an American
Heart Association pre-doctoral fellowship. Support
for article processing charges provided by the
IUPUI Open Access Fund. The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
kinase B (Akt) [
]. Akt activation prompts the phosphorylation and nuclear exclusion of
forkhead box O1 (FKHR or FoxO1), a key transcription factor that activates expression of
gluconeogenesis genes. FoxO1 phosphorylation by Akt is a fundamental process to downregulate
gluconeogenesis gene expression during the fed state. In addition, Akt inactivates glycogen
synthase kinase 3 (GSK3), thereby activating the enzyme glycogen synthase and promoting
glycogen biosynthesis [
]. Conversely, low insulin levels during fasting conditions lead to
activation of the gluconeogenesis and glycogenolysis pathways, increasing hepatic glucose
Insulin signaling and Akt activity are interconnected with the mechanistic target of
rapamycin (mTOR) pathway. TOR is a highly conserved protein from yeast to mammals, and plays a
key role at orchestrating fundamental aspects of metabolism, cellular growth, proliferation and
survival. Two complexes exist, TORC1 and TORC2, with distinct functions. In mammals,
mTORC1 regulates ribosome biogenesis and protein synthesis [
], while mTORC2 regulates
actin cytoskeleton organization [
]. Unique protein subunits participate in the specific
functions of each complex. The major partner of mTORC1 is regulatory-associated protein of
mTOR (Raptor) [
]. mTORC2 associates with rapamycin-insensitive companion of mTOR
]. Insulin activates mTORC1 through Akt-mediated inhibition of GTPase-activating
protein heterodimer tuberous sclerosis 1/2 (TSC1/2), an inhibitor of mTORC1 [
addition, Akt directly phosphorylates mTOR at serine 2448 [
]. Insulin also activates mTORC2,
which in turn, phosphorylates Akt at serine 473 residue, a necessary step for full activation of
Importantly, insulin-induced mTORC1 activity upregulates expression of Sterol Regulatory
Element Binding Protein-1 (SREBP-1) [11±14], a conserved transcription factor of the basic
helix-loop-helix leucine zipper family (bHLH-Zip) that primarily controls expression of
glycolysis and de novo lipogenesis (DNL) enzymes. SREBP-1a and SREBP-1c are isoforms of the
same gene, and both regulate L-pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase,
stearoyl-CoA desaturase 1, and mitochondrial glycerol-3-phosphate acyltransferase 1, among
other genes in the lipogenesis and glycolysis pathways [
]. SREBPs are synthesized as
precursors that are bound to the endoplasmic reticulum membrane. In response to specific signals,
SREBPs transition to the Golgi, where they are cleaved, releasing the mature form, which
translocates to the nucleus and activates expression of target genes [
]. mTORC1 activity is
necessary for activation of SREBP-1 gene expression and for its processing from precursor
to the mature form [11±14]. Thus, activation of SREBP-1 is a critical function of the Akt/
mTORC1 signaling axis. Actually, multiple studies have provided evidence that SREBP-1
coordinates a variety of responses needed for cell survival and growth, including lipogenesis [
]; glycogen synthesis [
]; phagocytosis and membrane biosynthesis [
]; as well as
insulin signaling molecules [
]. Despite evidence indicating that SREBP-1 regulates IRS2 and
], the overall contribution of SREBP-1 activity on hepatic insulin signaling is
unknown. Here, we explored the impact of knocking-down SREBP-1 on the insulin signaling
pathway and mTOR.
Materials and methods
All animal studies were in accordance with the National Institutes of Health guidelines and
were approved by the Indiana University School of Medicine Institutional Animal Care and
Use Committee. Male eight-week old C57BLKS/J mice were obtained from The Jackson
Laboratory (Bar Harbor, ME), and allowed to acclimate for at least a week before
experimentation. A standard 12 h light/12 h dark cycle (7 AM/7 PM) was maintained throughout the
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experiments. Mice were maintained in a BSL2-certified room and were fed rodent chow ad
libitum and allowed free access of water. Mice (n = 5±6) were given 1x1011 viral particles (vp)
by tail vein injection, and euthanized 8 days after adenovirus vector administration under ad
libitum fed conditions, 24-h fasted or 24-h fasted followed by a 4.5-h refeeding period. Tissues
were collected and snap frozen in liquid nitrogen and kept at -80ÊC.
Male C57BL/6J mice (The Jackson Laboratory) were used for isolation of primary
Adenoviral vector production
Helper-dependent or `gutless' adenoviral (HD-Ad) vectors are the most advanced type of
adenoviral vector, and are devoid of viral coding sequences, only retaining the inverted
terminal repeats and packaging signal. The lack of viral genes virtually eliminates inflammatory
responses and toxicity in mice and non-human primates [22±24]. Helper-dependent
adenoviral vectors have identical tropism to first generation (E1-deleted) adenoviral vectors, and
predominantly transduce the liver [
]. HD-Ad vectors were generated in HEK293Cre cells,
using a Cre-loxP system developed by Merck Laboratories and Microbix (Toronto, Canada)
]. The production of HD-Ad vectors expressing an shRNA to target SREBP-1 or a
scrambled sequenced has been previously described [
]. After production, vectors were
stored at -80ÊC in 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 150 mM NaCl, 10% glycerol. Total
particle counts were determined spectrophotometrically, as described [
For overexpression of SREBP-1c in primary hepatocytes, a first generation (E1-deleted)
vector expressing the N-terminal form (amino acids 1~436) of human SREBP-1c was used
(Eton Bioscience, San Diego, CA). An adenovirus without expression cassette (Null) was used
as control. Both adenoviral vectors were grown in HEK293 cells [
] and stored as described
for helper-dependent adenoviral vectors. Titers were determined by plaque assay in HEK293
Primary hepatocyte isolation and culture
Primary hepatocytes were isolated from C57BL/6J mice using a two-step collagenase
procedure followed by Percoll gradient centrifugation to separate hepatocytes from
non-parenchymal cells, as previously described [
]. Cell viability was assessed by trypan blue staining
exclusion (>80% viability). Cells were seeded at a density of 4-6x105 cells per well in 6-well
plates, and incubated in a humidified 5% CO2 incubator at 37ÊC. Cells were allowed to attach
for 4 hours, and medium was then replaced with fresh medium.
To address the impact of overexpressing SREBP-1c on insulin signaling, primary
hepatocytes were infected with an adenovirus expressing human SREBP-1c or a control vector at
MOI 20, 40, 60, 80 or 100. Medium was changed the next day. Cells were cultured in DMEM
containing with 5 mM glucose, 10% FBS and 100 IU/ml penicillin/100 μg/ml streptomycin,
100 nM dexamethasone, and washed twice with 1x PBS, prior to harvesting.
Liver tissue or primary hepatocytes were lysed in RIPA buffer (Thermo Scientific, Rockford,
IL) containing protease and phosphatase inhibitors (Roche, Indianapolis, IN). Protein
concentration was determined using the BCA kit from Pierce (Rockford, IL). Proteins (7±30 μg)
were separated in 10% or 4±20% Tris-HCl SDS PAGE Criterion gel (Bio-Rad, Hercules, CA)
and transferred to 0.2-mm PVDF membrane (Bio-Rad). Membranes were blocked with 5%
BSA-TBST or 5% dry milk-TBST for 1±2 h and incubated with the following antibodies:
ACACA/B (aka ACC1/2), IRS1, PDK-1S241, PDK-1, AktS473, Akt (Pan), Foxo1S256, Foxo1,
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GSK3αS21, GSK3βS9, mTORS2448, mTOR, Rictor, Raptor (Cell Signaling, Danvers, MA);
αtubulin, SREBP-1 MS-1207 (Thermo Scientific, Waltham, MA); β-actin, SREBP-1 H-160,
glucokinase H-88 (Santa Cruz Biotechnology, Dallas, TX); IRS2 (EMD Millipore, Billerica,
Massachusetts); IRS1Y612, GSK3 (Invitrogen, Life Technologies). HRP-conjugated secondary
antibody was added and incubated at room temperature for 1 hour. Blots were developed with
Pierce ECL kit (Thermo Scientific) and exposed to enhanced chemiluminescence (ECL) film
(GE Healthcare, Piscataway, NJ). Bands on blots were quantified by densitometry using ImageJ
v1.48s, and results were normalized to control protein, as specified in the figure legends.
Blood glucose was measured with an Ascensia Elite XL meter (Bayer, Tarrytown, NY), from a
drop collected from the tail vein. β-hydroxybutyrate was analyzed with a kit from Pointe
Scientific (Canton, MI). Insulin was analyzed by the Translation Core of the Center for Diabetes
and Metabolic Diseases using a Rodent Insulin Chemiluminescence ELISA assay (ALPCO,
Salem, NH). All reactions were carried out in duplicate, following the manufacturer's
Numerical values represent mean ± SD. P values were calculated using unpaired two-tailed
Student's t-tests. A P value of less than 0.05 was considered statistically significant.
Results and discussion
Silencing SREBP-1 decreases insulin signaling in mouse liver
In addition to its role in controlling expression of genes of glycolysis and fatty acid metabolism
], SREBP-1 inhibits IRS2  and activates expression of a subunit of the PI3 kinase
complex, phosphatidylinositol-3 kinase regulatory subunit p55γ (PIK3R3) [
], two essential
molecules in the insulin signaling pathway. To investigate the relative contribution of
SREBP1 to regulate hepatic insulin signaling, mice received a helper-dependent adenoviral vector
expressing a short hairpin RNA (shRNA) to knock-down SREBP-1, or a control vector
expressing a scrambled sequence. Mice were studied a week later under fed conditions,
24-hour fasted, or 24-hour fasted followed by a 4.5-hour refeeding period. SREBP-1 silencing
did not significantly affect body weight or insulin levels relative to the shSCR control group
(Table 1). Blood glucose was only slightly decreased under fasting conditions (Table 1). As
expected, ACACA and ACACB were decreased, confirming that silencing resulted in
decreased SREBP-1 target gene expression. Remarkably, loss of SREBP-1 did not lead to
changes in IRS2, contrary to what would have been anticipated, based on the known inhibitory
effects on expression of this molecule [
]. SREBP-1 deficiency did not affect insulin signaling
under the fed state, when insulin levels are high (Fig 1). However, a reduction in the levels of
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Fig 1. Silencing SREBP-1 in vivo reduces hepatic insulin signaling. Mice were administered 1x1011 viral particles of HD-Ad.shSREBP1 or HD-Ad.shSCR, and
euthanized after 8 days under fed, 24-hour fasted or 24-hour fasted followed by a 4.5 hour refeeding period. (A) Tissue lysates were subjected to immunoblotting
analysis using the antibodies shown on the left. (B) Densitometry analysis of insulin signaling molecules. Values represent mean ± SD (n = 4); p<0.05 shSCR vs
IRS1Y612 phosphorylation was observed under fasting conditions (i.e., low insulin), and a
decrease was also evident in downstream molecules, including PDK-1S241, AktS473, GSK3αS21,
and FoxO1S256, suggesting that depletion of SREBP-1 had a negative impact on this pathway.
Nevertheless, the decrease was moderate and was distinct only under fasting conditions,
suggesting that nutrients and/or high insulin attained under fed conditions are sufficient to
maintain normal insulin signal transduction. Interestingly, SREBP-1 deficiency resulted in changes
in glucokinase expression following the same trends (i.e. lower under the fasted, but not the
fed state). This indicates that, similar to IRS2, SREBP-1 is permissive for glucokinase
expression, and other factors may be more relevant for its regulation. Even though some studies
point at SREBP-1 as the mediator of insulin-induced glucokinase expression, other studies
have argued against a major role for this transcription factor [
]. Indeed, multiple
transcription factors have been shown to directly activate glucokinase gene expression, including
Kruppel-like factor 6 [
], peroxisome proliferator-activated receptor gamma (PPARγ) [
receptor homolog 1 (LRH-1) [
], hepatocyte nuclear factor 4 (HNF4) [
hypoxiainducible factor 1 alpha (HIF1α) [
]. In addition, glucokinase protein is stabilized by other
proteins, including glucokinase regulatory protein (GKRP), Bcl-2-associated agonist of cell
death (BAD), and 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK2/FBP2), in
response to nutritional signals [
]. Thus, overall levels of glucokinase in liver may be
influenced by multiple factors at the transcriptional and post-translational level.
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Fig 2. SREBP-1 expression increases insulin signaling in primary hepatocytes. (A) Mouse primary hepatocytes were
cultured in DMEM containing 5 mM glucose, 10% FBS and 100 IU/ml penicillin/100 μg/ml streptomycin, 100 nM
dexamethasone. Cells were transduced with an adenovirus expressing SREBP-1c or a control vector (Null) at the
multiplicity of infection (MOI) indicated on the top. Cells were harvested 48 hours later. IRS2 levels decreased while
ACACA/B increased in hepatocytes treated with SREBP-1c, as expected. (B) Insulin signaling in primary hepatocytes
transduced with an adenovirus expressing the mature form of human SREBP-1c or a control vector (Null) at
multiplicity of infection (MOI) 20, 40, or 100. Data are representative of 2 separate experiments.
To further evaluate the influence of SREBP-1 activity on insulin signaling, primary
hepatocytes were transduced with an adenoviral vector expressing the mature form of human
SREBP-1c or with a control vector, at multiplicity of infection (MOI) 20, 40, 60, 80 and 100. As
expected, SREBP-1 targets IRS2 and ACACA/B (aka ACC1/2) were downregulated and
upregulated, respectively (Fig 2A). Overexpression of SREBP-1c resulted in opposite effects to
those observed by silencing SREBP-1 in vivo, i.e., increased IRS1Y612 and AktS473
phosphorylation, as well as the downstream Akt target FoxO1S256 (Fig 2B and S1 Fig). These data support
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the concept that, in hepatocytes, short-term overexpression of SREBP-1 is associated with
enhanced insulin signal transduction, and are in agreement with recent data in human
hepatocellular carcinoma (HCC). Overexpressing SREBP-1 in HCC cell lines, accelerated their
growth and reduced apoptosis. Remarkably, this was accompanied by increased levels of
phosphorylated AktS473 [
]. Conversely, in HCC cell lines expressing high SREBP-1 levels,
knocking down SREBP-1 reduced AktS473 activity [
]. Of note, chronic SREBP-1 overexpression in
liver has been associated with hepatic steatosis and insulin resistance [
], due to
accumulation of lipid molecules that interfere with the insulin signaling pathway [
increased SREBP-1 activity may be perceived at the short-term as a signal of nutrient
abundance and cell growth, but its prolonged overexpression leads to negative effects due to
buildup of lipid classes that hinder insulin signaling.
Glucokinase protein levels were not affected by overexpression of SREBP-1 in primary
hepatocytes (S2 Fig). This could be due to the fact that primary hepatocytes do not exhibit
the same expression profiles of the intact liver, and transcription factors/cofactors that are
involved in the insulin-induced response, may not be present in the primary hepatocytes.
Alternatively, it could be due to changes in levels of glucose metabolites, known to
downregulate glucokinase expression [
Altogether, the data in vivo and in vitro suggest that SREBP-1 influences insulin signaling,
although only to a moderate extent. Short-term (1 week) SREBP-1 depletion was associated
with lower IRS1Y612, AktS473 and downstream molecules, GSK3αS21 and FoxO1S256, under
fasting conditions. Thus, in the fed/refed state, nutrients (e.g., fatty acids, cholesterol) and/or high
insulin, are sufficient to restore normal signaling. Remarkably, loss of SREBP-1 did not have
consequences for IRS2, even though its overexpression in primary hepatocytes resulted in
decreased IRS2. Our data suggest that other factors may be more important at maintaining
basal IRS2 levels than SREBP-1. Contrary to a previous report [
], expressing SREBP-1 in
primary hepatocytes had no effect on total levels of IRS1. Furthermore, IRS1 did not change upon
SREBP-1 depletion. Thus, it is possible that the alterations in AktS473 activity and downstream
Akt effectors such as GSK3α and FoxO1, were mostly influenced by IRS1 activity, and not
Although the molecular mechanisms guiding the changes in insulin signal transduction are
unknown, it is possible that multiple intracellular factors are coordinated to sense energy
availability and elicit this response. First, it has been shown that silencing SREBPs triggers changes
in the composition of lipid rafts [
]. Lipid rafts are membrane microdomains with unique
lipid composition, and are essential for numerous cellular functions, including signaling events
]. Lipid rafts are rich in sphingolipids and cholesterol phospholipids. Saturated fatty acids
are the main components of the side chains of phospholipids [
], and SREBP-1 plays a central
role in their synthesis. Indeed, the PI3K-Akt-mTORC1 axis is vital to upregulate SREBP-1 and
2 to attain appropriate cellular levels of fatty acids and cholesterol, as well as for the integrity of
lipid rafts [
]. Furthermore, it has been shown that the ratio of monounsaturated to saturated
fat in total lipids is critical for Akt signaling and AktS473 phosphorylation [
]. In addition,
changes in the rate of glycolysis and lipogenesis (whose genes are regulated by SREBP-1) are
likely to be detected by energy sensors like AMPK [
], a master regulator of energy
homeostasis, leading to changes in insulin signaling to control overall cellular homeostasis.
Silencing SREBP-1 is not associated with decreased mTOR activity
The mTOR pathway functions as the hub for sensing nutrient abundance and changes in
energy supplies [
]. The Akt/mTORC1 axis has emerged as a critical regulatory point in the
control of cell growth and cellular proliferation, and both molecules are targets for drug
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Fig 3. SREBP-1 depletion has no impact on mTOR. (A) Mice were treated as described in Fig 1. Tissue lysates were
subjected to immunoblotting analysis using the antibodies shown on the left. (B) Densitometry analysis of insulin
signaling molecules. Values represent mean ± SD (n = 4).
development in cancer treatment [48±52]. Two main mechanisms activate mTOR in response
to insulin signaling, both mediated by Akt: (i) direct phosphorylation of mTOR at residue
]; (ii) phosphorylation of the tuberous sclerosis complex 2 (TSC2), thereby activating
mTORC1 . Given the dependence of mTOR on Akt activity, and that loss of SREBP-1
activity reduced Akt phosphorylation under fasting conditions, we questioned whether the
mTOR pathway would be affected. Depleting SREBP-1 in vivo did not induce changes in
mTOR phosphorylation at S2448, or in total levels of mTOR and the subunits of mTORC1
and mTORC2, Rictor and Raptor (Fig 3). Thus, the lower insulin sensitivity resulting from
silencing SREBP-1 did not have an impact on mTOR. It is possible that the moderate decrease
in insulin signal transduction during fasting was not sufficient to influence mTOR.
Alternatively, mTOR activity is most relevant during nutrient abundance (fed state) [
], and insulin
signaling was not affected in SREBP-1-depleted animals.
SREBP-1 is a transcription factor that controls important aspects of hepatic function, including
carbohydrate and lipid metabolism, in response to insulin [
15, 17, 18
]. SREBP-1 positively
correlates with mTOR activity, and both are upregulated in animal models of type 2 diabetes and
in cancer [11, 40, 55±59]. The existing evidence that SREBP-1 controls expression of molecules
in the insulin signaling pathway, including IRS2 and PIK3R3 [
], suggests that feedback
mechanisms exist between SREBP-1 and this pathway. Our data indicates that SREBP-1
activity is dispensable for normal insulin signal transduction under fed conditions. Depleting
SREBP-1 in the fasted state results in a modest decrease in insulin signaling, without
influencing mTOR activity. Even though the specific molecular event/s leading to this decrease remain
to be determined, it is possible that they are linked to a reduction in metabolites generated in
the de novo lipogenesis pathway. The presence of these metabolites in the diet and/or the
higher level of insulin during the fed state are sufficient to maintain normal signal transduction
levels. Overall, the contribution of SREBP-1 to sustain insulin signaling is modest.
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S1 Fig. SREBP-1c expression increases insulin signaling.
S2 Fig. Glucokinase is not upregulated in primary hepatocytes overexpressing SREBP-1c.
Conceptualization: NuÂria Morral.
Data curation: Victoria Jideonwo, Yongyong Hou, Miwon Ahn, Sneha Surendran.
Formal analysis: Victoria Jideonwo, NuÂria Morral.
Project administration: NuÂria Morral.
Supervision: NuÂria Morral.
Writing ± original draft: Victoria Jideonwo.
Writing ± review & editing: Victoria Jideonwo, Yongyong Hou, Miwon Ahn, Sneha
Surendran, NuÂria Morral.
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