Biochemical and Biophysical Research Communications 406 (2011) 268–272
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Retinoids synergized with insulin to induce Srebp-1c expression and activated
its promoter via the two liver X receptor binding sites that mediate insulin action
Rui Li, Wei Chen, Yang Li, Yan Zhang, Guoxun Chen ⇑
Department of Nutrition, University of Tennessee at Knoxville, Knoxville, TN 37996, USA
a r t i c l e i n f o a b s t r a c t
Article history: We have reported that the rat liver lipophilic extract (LE) synergized with insulin to induce Gck and
Received 29 January 2011 Srebp-1c in primary rat hepatocytes. After identification of retinol and retinal in LE, only their effects
Available online 18 February 2011 in the absence or presence of insulin on Gck, but not that on Srebp-1c, were investigated subsequently.
The retinoid effects on the Srebp-1c expression and the activation of its promoter were examined with
Keywords: real-time PCR and reporter gene assays, respectively. In primary hepatocytes, retinal and retinoic acid
Insulin (RA) synergized with insulin to induce Srebp-1c expression. This induction was followed by the elevation
Metabolism
of its target gene, fatty acid synthase. Activation of retinoid X receptor, but not retinoic acid receptor, was
Primary hepatocytes
Retinoids
responsible for the induction of Srebp-1c expression. RA, but not retinal, also induced Srebp-1c expression
Srebp-1c expression in a dose dependent manner in INS-1 cells. The RA responsive elements in Srebp-1c promoter were deter-
Retinoid X receptor mined as previously identified two liver X receptor elements responsible for mediating insulin action. We
conclude that retinoids regulate hepatic Srebp-1c expression through activation of retinoid X receptor.
The RA- and insulin-induced Srebp-1c expression converged at the same sites in its promoter, indicating
the roles of vitamin A in regulation of hepatic gene expression.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction by all-trans RA, and retinoid X receptors (RXRa, b and c) activated
only by 9-cis RA [7].
Elevation of hepatic vitamin A (VA, retinol) content in patients Insulin resistance, diabetes and other metabolic abnormalities
with diabetes was observed more than 70 years ago (1937) [1]. are associated with profound changes of hepatic lipid and glucose
Subsequently, depletion of hepatic glycogen content in VA defi- metabolism. These can be attributed to the altered expression of
cient (VAD) rats was reported in 1957 [2]. When isotretinoin, 13- genes involved in glucose and lipid metabolism [8]. Insulin respon-
cis retinoic acid, was used to treat patients with acne, some of them sive elements in the Srebp-1c promoter have been identified as two
developed isotretinoin-induced hypertriglyceridemia [3]. All these liver X receptor (LXR) binding sites and one sterol regulatory ele-
early observations suggested that VA status affected glucose and li- ment [9,10]. This implies that insulin regulates the expression of
pid homeostasis, a topic remained to be investigated. its responsive genes after it stimulates the synthesis of endogenous
As an essential micronutrient, VA plays crucial roles in the gen- agonists for nuclear receptor activation. When we analyzed the ef-
eral health of an individual [4]. Therefore, retinol homeostasis fects of the lipophilic extract (LE) from rat livers on insulin-
must be delicately maintained to meet optimal physiological regulated gene expression, we found that the LE synergized with
requirements. This is achieved by a network of enzymes and pro- insulin to induce glucokinase gene (Gck) and Srebp-1c expression
teins involved in the transport, production, and catabolism of ret- in primary rat hepatocytes with different induction patterns [11].
inoids [5]. The regulation of this system can be attributed to the The existence of retinol and retinal in LE was confirmed later,
control of the expression levels of some of these enzymes by the and their effects on Gck, but not Srebp-1c, were examined in that
active metabolite of retinol, retinoic acid (RA) [6]. RA exists in mul- study [11]. It has been reported that SREBP-1c mediated the reti-
tiple isomeric forms, such as all-trans RA and 9-cis RA, and RA reg- noid-dependent increase in fatty acid synthase (Fas) promoter
ulates gene expression through activation of two families of activity in HepG2 [12]. Therefore, we hypothesized that retinoids
nuclear receptors, retinoic acid receptors (RARa, b and c) activated may regulate the expression of Srebp-1c in primary hepatocytes.
In this study, we report that retinoids transiently synergized
⇑ Corresponding author. Address: 229 Jessie Harris Building, 1215 West Cum- with insulin to induce the expression of Srebp-1c in primary rat
berland Avenue, Knoxville, TN 37996, USA. Fax: +1 865 974 3491. hepatocytes via the activation of RXR, but not RAR. The retinoic
E-mail address: gchen6@utk.edu (G. Chen). acid responsive elements (RAREs) in its promoter are the
0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2011.02.031
R. Li et al. / Biochemical and Biophysical Research Communications 406 (2011) 268–272 269
previously identified two LXR responsive elements that mediated 3. Results
insulin-induced Srebp-1c transcription.
3.1. Retinal and retinoic acid synergized with insulin to induce Srebp-
1c expression
2. Materials and methods
Since we have observed that rat liver LE which contained retinol
2.1. Reagents (ROL) and retinal (RAL) synergized with insulin to induce Gck and
Srebp-1c expression with different induction patterns, we only re-
The reagents for primary hepatocyte isolation and culture have ported the effects of ROL, RAL and RA on Gck expression in the pre-
been published [13]. Reagents for cDNA synthesis and real time vious publication [11]. We decided to check the direct effects of
PCR were obtained from Applied Biosystems (Foster city, CA). retinoids on the expression level of Srebp-1c, a key transcription
Source of LG268 was reported previously [11]. All other com- factor controlling the hepatic fatty acid biosynthesis [16]. Primary
pounds or enzymes were purchased from Sigma (St. Louis, MO) un- rat hepatocytes were treated with increasing concentrations of
less described otherwise. ROL, RAL and RA in the absence or presence of insulin. As shown
in Fig. 1, retinol up to 20 lM did not induce Srebp-1c expression
without or with insulin. In the absence of insulin, RAL up to
20 lM did not affect Srebp-1c expression. RAL synergized with
2.2. Animals and diets
insulin to induce Srebp-1c expression when its concentration
reached 20 lM. Without insulin, RA at 20 lM induced Srebp-1c
Sprague–Dawley rats (for hepatocytes) were purchased from
expression. RA at 2 and 20 lM synergized with insulin to induce
Harlan Breeders (Indianapolis, IN). Rats were housed in colony
Srebp-1c expression. All these results demonstrated that RAL and
cages, and fed a standard rodent diet before isolation of primary
RA had the ability to synergize with insulin to induce Srebp-1c
hepatocytes. All procedures were approved by the Institutional
expression in primary rat hepatocytes.
Animal Care and Use Committee at the University of Tennessee
at Knoxville.
3.2. RA transiently synergized with insulin to induce the expressions of
Gck and Srebp-1c differently, and resulted in elevation of SREBP-1c
target gene, Fas
2.3. Primary hepatocytes, RNA extraction and quantitative real-time
PCR
Since RA induced the expression of both Gck and Srebp-1c, it is
important to determine whether their induction patterns are simi-
Methods for preparation of primary hepatocytes and analysis of
lar or not. The expression levels of Gck, Srebp-1c and Fas, a target
RNA were described previously [11]. The real time PCR primer sets
gene of SREBP-1c, were examined by real time PCR at 0, 3, 9, 12
for detecting Fas (from Dr. Bruce Spigelman’s group in Harvard
and 24 h after treatment of 5 lM RA in the absence or presence of
Medical School), Gck, Cyp26a1 [14], Srebp-1c [11] have been pub-
1 nM insulin. As shown in Fig. 2A, RA robustly synergized with insu-
lished. The primers for Rarb (forward 50 -GGCCTCTGGGACAAATT-
lin to induce Gck expression as early as three hours. The fold induc-
CAG-30 , and reverse 50 -GCAGACGCTTGGCGAACT-30 ) were
tion started to decline at 6 h after the stimulation, and the synergy
designed using Primer Express software (Applied Biosystems).
lasted for at least 12 h. Fig. 2B showed that hepatocytes treated
The gene expression level was normalized to that of 36B4 unless
with 5 lM RA had significantly higher levels of Srebp-1c mRNA than
described otherwise. Data were presented as the fold induction
control cells did at 3 (2.1 ± 0.5- vs 0.71 ± 0.07-fold) and 6
calculated from the DDCt values [13] using 36B4 as the invariable
(0.53 ± 0.09- vs 0.26 ± 0.02-fold) hours after treatment. RA also ro-
control gene [11].
bustly synergized with insulin to induce Srebp-1c expression at 3 h
and the fold induction began to drop at 6 h, similar to the pattern of
Gck expression. However, at 9 h after the stimulation, the synergis-
2.4. INS-1 cell culture, and reporter gene constructs and assay
tic induction of RA and insulin to Srebp-1c expression no longer
INS-1 cells (833/15) were maintained as described previously
[15]. Standard protocols (Molecular Cloning) were followed in all 70
recombinant DNA engineering procedures. The reporter gene con- C Retinol Retinal Retinoic Acid
structs reported previously [10] were transfected into INS-1 cells
60 *
Insulin - *
Fold Induction
using Fugene 6 transfection reagent (Roche, Indianapolis, IN) 50
according to the manufacture’s manual. The activation of reporter 40
Insulin + *
gene constructs were measured using dual luciferase assay as de-
30
scribed previously [10] and reported as fold induction.
20
10
2.5. Statistics
**
0
0.002
0.2
0.002
0.002
0.2
0.02
2
0.02
0.2
2
0.02
2
20
20
20
0
Data were presented as means ± SD. The number of experi-
ments represented the independent experiments using hepato- Concentrations of indicated retinoids
cytes isolated from different animals on different days. Levene’s
test was used to determined homogeneity of variance among Fig. 1. Retinal and retinoic acid synergized with insulin to induce Srebp-1c mRNA
groups using SPSS 17.0 statistical software and where necessary expression in rat primary hepatocytes. Hepatocytes were treated with indicated
natural log transformation was performed before analysis. Multi- ligands without or with 1 nM insulin for 6 h. Total RNA was isolated and subjected
to real-time PCR analysis. Srebp-1c mRNA level in vehicle control group was
ple comparisons were analyzed by one-way ANOVA. The indepen- assigned a value of 1 (mean ± SD, n = 3, Ã for comparing indicated groups with
dent sample t-test was used to compare two conditions. control in the presence of insulin; ÃÃ for comparing retinoic acid group with control
Differences were considered statistically significant at P b > d > e, a > c > e, a0 > b0 > c0 > d0 , all P < 0.05). promoter similar to the spatial and temporal recruitment of
272 R. Li et al. / Biochemical and Biophysical Research Communications 406 (2011) 268–272
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