Advances in Bioscience and Biotechnology, 2013, 4, 1-5 ABB Published Online October 2013 (
Inhibitory roles of protein kinase B and Peroxisome
Proliferator-activated receptor gamma coactivator on
hepatic HMG-CoA Reductase promoter activity
Gene C. Ness*, Jeffrey L. Edelman
Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, USA
Email: *
Received 20 June 2013; revised 20 July 2013; accepted 15 August 2013
Copyright © 2013 Gene C. Ness, Jeffrey L. Edelman. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Since we had previously demonstrated that siRNAs to
tristetraprolin (TTP) markedly inhibited insulin sti-
mulation of hepatic HMG-CoA reductase (HMGR)
transcription, we investigated the effects of transfect-
ing rat liver with TTP constructs. We found that trans-
fecting diabetic rats with TTP did not increase HMGR
transcription but rather led to modest inhibition. We
then investigated whether co-transfection with pro-
tein kinase B, hepatic form (AKT2), might lead to pho-
sphorylation and result in activation of HMGR tran-
scription. We found that this treatment resulted in
near complete inhibition of transcription. Transfec-
tion with peroxisome proliferator-activated receptor
coactivator (PGC-1
) also inhibited HMGR tran-
scription. These results show that although TTP is
needed for activation of HMGR transcription, it can-
not by itself activate this process. AKT2 and PGC-1
which mediate the activation of gluconeogenic genes
by insulin, exert the opposite effect on HMGR.
Keywords: In Vivo Electroporation; HMG-CoA
Reductase; Insulin; Protein Kinase B;
Peroxisome Proliferator-Activated Receptor
Coactivator; Tristetraprolin
The rate of transcription of hepatic 3-hydroxy-3-methy-
lglutaryl coenzyme A reductase (HMGR) is rapidly, within
1 hr increased in response to insulin [1]. This leads to
correspondingly rapid rises in HMGR mRNA, immu-
noreactive protein and enzyme activity levels [2-4]. The
increase in hepatic HMGR expression promotes resis-
tance to the serum cholesterol raising action of dietary
cholesterol [5,6].
One of the characteristics of rapid response genes is
the presence of AU-rich sequences in the 3’-untranslated
regions of their mRNAs [7]. These sequences generally
serve as binding sites for proteins that act either to accel-
erate the rate of degradation of the mRNA or to stabilize
it. However some of these AU-rich RNA binding pro-
teins act as translational repressors [8] or act at the level
of transcription [9,10]. In a recent study, we examined
the effects of siRNAs to several of these AU-rich RNA
binding proteins on hepatic HMGR promoter activity in
vivo by introducing an HMGR luciferase promoter con-
struct directly into localized sites in livers of rats by elec-
troporation [11]. Strikingly, siRNAs to one of these, tris-
tetraprolin (TTP), completely eliminated the transcrip-
tion of HMGR. The TTP siRNAs also prevented the sti-
mulation of HMGR transcription caused by insulin. It is
known that insulin treatment of NIH 3T3 fibroblasts over-
expressing human insulin receptors causes a dramatic in-
crease in TTP mRNA within 10 minutes [12]. We have
observed a 6-fold increase in TTP protein in liver nuclear
extracts from diabetic rats treated with insulin [11].
Thus, we sought to determine whether co-transfection
of TTP with HMGR promoter construct by in vivo elec-
troporation would stimulate HMGR transcription inde-
pendent of insulin treatment. As phosphorylation of TTP
has been reported to impair the mRNA degradation nor-
mally promoted by TTP [13], we also performed trans-
fections with and without protein kinase B/Akt2. Since
peroxisome proliferator, activated receptor-co activator 1
) which regulates hepatic metabolism during fast-
ing is phosphorylated and inactivated by Akt2 [14], we
also examined the effects of transfecting liver with PGC-
and the effects of siRNAs to PGC-1
*Corresponding author.
G. C. Ness, J. L. Edelman / Advances in Bioscience and Biotechnology 4 (2013) 1-5
2.1. Experimental Animals
Male Sprague-Dawley rats weighing 100 - 125 g were
purchased from Harlan (Madison, WI). The rats were
housed in a reversed lighting cycle room (12 h dark/12 h
light) and fed Harland Teklad 22/5 rodent chow. A single
subcutaneous injection of streptozotocin (Sigma), 65
mg/kg in 0.1 M sodium citrate, pH 5.5, was given to the
rats to induce diabetes. Clinistix (Bayer) were used to de-
tect the presence of urinary glucose to confirm the induc-
tion of diabetes in the rats. In vivo electroporation expe-
riments were preformed 4 days after induction of diabe-
tes. Insulin treatment consisted of giving a single subcu-
taneous injection of recombinant human insulin (3.0
units/100 g of Novolin 70/30 from Novo Nordisk) 2 h
prior to taking tissue samples. All procedures were car-
ried out according to protocol 3571 approved by the
University of South Florida Institutional Animal Care
and Use Committee.
2.2. Materials
The TTP clone was kindly provided by Dr. Perry J.
Blackshear [12]. FLAG-tagged TTP was provided to us
by Dr. Jens Lykke-Andersen [13]. A synthetic TTP clone
was purchased from GeneCopoeia. Dr. Denise Cooper
and N. Patel kindly provided the AKT 2 clone [15] and
Dr. Edwards A. Parks generously furnished the perox-
isomeproliferator activated receptor gamma coactivator
) [16]. siRNAs to PGC-1
were purchased
from Dharmacon.
2.3. In Vivo Electroporation
The rats are anaesthetized with 5% isoflurane and main-
tained under 3% isoflurane. The hair on the abdomen is
trimmed closely using a number 10 blade. The surgical
area is scrubbed with 70% isopropyl alcohol followed by
scrubbing with a betadine solution. A subcutaneous in-
jection of Ketoprofen (5 mg/kg) is given for analgesia.
The surgical area is drapped. Sterile instruments and
electrode are used for the in vivo electroporation. A
transverse incision over the sterum is made. The under-
lying muscle is then cut taking care to not cause any or-
gan damage. Thus, the left, right and median lobes are
exposed. The clones and siRNA in 50 l of sterile saline
are injected subcapusularly using a 26 gauge, 3/8 inch
length needle. The 0.5 cm six-needle hexagonal array
electrode is then placed over the site and six 150 ms puls-
es of 75 V with 150 ms rests between pulses are deliver-
ed. Duplicates are placed in each lobe. Ten g of the
325/+70 HMGR promoter in pGL3-Basic [1] with and
without ten g of TTP, AKT2 or PGC-1
or PGC-1
siRNA is introduced. The abdominal muscle layer is su-
tured with Surgilene. Then the skin layer is closed with
surgical staples. The rats are up in two minutes following
2.4. Luciferase Assays
One day after surgery, the sites in each liver lobe are re-
moved using a 5 mm cork-borer. The light scarring from
the six acupuncture needles helps to identify the electro-
porated sites. The liver pieces, approximately 0.1 g are
homogenized in passive lysis buffer (Promega) using a
Polytron homogenizer. The samples are processed for lu-
ciferase activity using the dual luciferase assay kit from
Promega [17]. Luciferase activity was calculated as the
ratio of firefly (reporter) to Renilla (transfection control).
3.1. Effect of Co-Transfecting TTP Clones on
Hepatic HMGR Promoter Activity
Since we have previously shown [11] that introduction of
siRNAs to TTP into liver by in vivo electroporation com-
pletely inhibits hepatic HMGR promoter activity and the
insulin mediated stimulation of promoter activity we
tested whether transfecting with a TTP clone might sti-
mulate HMGR promoter activity without insulin. Trans-
fecting livers of diabetic rats with the wild type HMGR
luciferase construct and a TTP clone did not stimulate
HMGR promoter activity; but rather produced decreases
of about 50% (Figure 1). This same result was also ob-
tained with FLAG-tagged TTP and the synthetic TTP
clones. Surprisingly, co-transfection of insulin-treated di-
abetic rats with TTP resulted in a marked decrease in
HMGR promoter activity (Figure 1).
Figure 1. Effect of transfecting TTP on hepatic HMGR pro-
moter activity in diabetic and insulin-treated diabetic rats. Dia-
betic and insulin-treated diabetic rats were transfected with
wild type HMGR with and without the TTP clone from Dr.
Blackshear. The data are expressed as means SD. p 0.01
compared with insulin-treated rats transfected with only WT
HMGR. Five rats were used in each group.
Copyright © 2013 SciRes. OPEN ACCESS
G. C. Ness, J. L. Edelman / Advances in Bioscience and Biotechnology 4 (2013) 1-5 3
3.2. Effect of Co-Transfecting with TTP and
Akt2 Clones on Hepatic HMGR Promoter
Since it is generally felt that insulin signaling is mediated
through Akt kinase phosphorylations [18] and TTP is a
phosphoprotein [13], we performed transfections with
TTP and Akt2 by in vivo electroporation. Introduction of
an Akt2 clone together with the TTP clone into livers of
diabetic rats resulted in near complete elimination of he-
patic HMGR promoter activity (Figure 2). A recent stu-
dy showed that insulin signaling was normal in mice
with hepatic deletion of both Akt1 and Akt2 and Foxo 1
[19]. The authors concluded that Akt is not an obligate
intermediate for insulin signaling.
3.3. Effects of Peroxisome Proliferator-Activated
Receptor-Coactivator 1
on Hepatic HMGR
Promoter Activity
Since Akt/PKB is known to phosphorylate and thus inac-
tivate PGC-1
[14], we decided to test the effects of co-
transfecting PGC-1
on HMGR promoter activity.
co-activates transcription of key gluconeogenic
enzymes in fasting such as PEPCK and G6Pase [20]. In-
sulin acts to repress these genes and promotes utilization
of incoming dietary carbohydrate in the fed state. Co-
transfection of liver sites in diabetic rats with PGC-1
markedly inhibited HMGR promoter activity (Figure 3).
Co-transfection with both PGC-1
and TTP also resulted
in very low levels of promoter activity. Since PGC-1
transfection acted to inhibit HMGR transcription, we
tested the effect PGC-1
siRNA. As would be expected,
this treatment increased hepatic HMGR transcription in
diabetic rats treated with insulin (Figure 4). For com-
parison, transfection with Akt2 by itself was performed.
Figure 2. Effect of transfectingwith both TTP and AKT2 on
hepatic HMGR promoter activity in diabetic rats. Diabetic rats
were transfected with wild type HMGR with TTP or with both
TTP and AKT2. The data are expressed as means S.D. for
five rats in each group. p = 0.01 as compared with diabetic rats
tansfected with only wild type HMGR.
Figure 3. Effect of transfecting with PGC-1
and TTP on he-
patic HMGR promoter activity in diabetic rats. Diabetic rats
were transfected with wild type HMGR with PGC-1
or with
both PGC-1
and TTP. The data are expressed as means S.D.
for three rats in each group. p = 0.01 as compared with dia-
betic rats transfected with only wild type HMGR.
Figure 4. Effects of PGC-1
siRNA as compared with AKT2
on hepatic HMGR promoter activity in insulin-treated diabetic
rats. Insulin treated diabetic rats transfected with wild type
HMGR were also given either AKT2 or PGC-1
siRNA. The
data are expressed as means S.D. for three rats in each group.
p 0.01 for AKT2 transfected rats as compared with insulin-
treated diabetic transfected with only wild type HMGR.
This treatment markedly inhibited HMGR transcription.
Although it has been established that the insulin medi-
ated rapid increases in hepatic HMGR mRNA and pro-
tein levels are due to activation of transcription [1] and
that this effect does not require protein synthesis [21], the
signaling pathway from insulin binding to the insulin
receptor to an increased rate of transcription has not been
established. The insulin signaling pathways that have
been investigated have focused on PEPCK and G6Pase,
(key enzymes of gluconeogenesis), glucokinase, glycol-
lysis, fatty acid oxidation, fatty acid synthesis, bile acid
synthesis, protein translation and cell growth [14,20,22,
Copyright © 2013 SciRes. OPEN ACCESS
G. C. Ness, J. L. Edelman / Advances in Bioscience and Biotechnology 4 (2013) 1-5
23]. None of these studies have addressed the signaling
pathway leading to activation of cholesterol biosynthesis
in normal liver. One study reported that activation of Akt
promoted accumulation of HMGR mRNA in tumor cells
associated with increased SREBP-1 [24].
Our recent finding that siRNAs to TTP abolished tran-
scriptional activation of hepatic HMGR by insulin [11]
raised the possibility that TTP might be involved in this
signaling pathway. However, in vivo transfection of liver
with TTP clones did not stimulate transcription in dia-
betic rats and markedly inhibited transcription of HMGR
in insulin treated diabetic rats (Figure 1). Thus, it ap-
pears that TTP is required for activation of hepatic
HMGR transcription; however TTP alone does not acti-
vate HMGR transcription. Further increases in TTP ex-
pression resulted in inhibition of HMGR transcription.
It is generally held that insulin signaling following in-
teraction with its receptor involves phosphorylation of
the insulin receptor substrate family that initiates a linear
cascade that culminates in phosphorylation of Akt kinases
[19]. These kinases then phosphorylate mediators that
activate or inactivate key proteins to decrease hepatic
glucose output, increase lipogenesis etc. However, trans-
fecting liver sites with Akt2 in addition to TTP resulted
in near complete elimination of HMGR transcription ra-
ther than stimulation (Figure 2). Also transfecting with
inhibits HMGR transcription. These results are
opposite to those found for the gluconeogenic genes [20],
which are elevated under fasting conditions while hepatic
HMGR is increased under fed conditions. Thus, this re-
sult would actually be anticipated.
It has also been reported that fenofibrate induction of
LDL receptor involves protein kinase B (Akt) and pero-
xisome proliferator-activated receptor
[25]. In many si-
tuations hepatic HMGR and LDL receptor activation go
together. However, in this case they do not.
We demonstrated in the investigation reported here that
tristetraprolin (TTP) alone cannot mediate insulin’s sti-
mulation of HMGR transcription. We also showed that
two of the leading candidates that might assist TTP, name-
ly protein kinase B and peroxisome proliferator-activated
coactivator, actually cause marked inhibition.
It is our hope that other investigators will use this know-
ledge and perhaps employ the in vivo electroporatic ap-
proach used here to study other potential regulatory fac-
tors and define the molecular mechanism by which insu-
lin acts to increase hepatic HMGR expression and there-
by confer resistance to dietary induced increases in se-
rum and tissue cholesterol levels.
This investigation was supported by grant R01DK075414 from the Na-
tional Institutes of Diabetes and Digestive and Kidney Diseases and
does not necessarily represent the official views of the NIDDK or the
National Institutes of Health.
[1] Lagor, W.R., de Groh, E.D. and Ness, G.C. (2005) Dia-
betes alters the occupancy of the hepatic 3-hydroxy-3-
methylglutaryl CoAreductase promoter. The Journal of
Biological Chemistry, 280, 36601-36608.
[2] Lakshmanan, M.R., Nepokroeff, C.M., Ness, G.C., Du-
gan, R.E. and Porter, J.W. (1973) Stimulation by insulin
of rat liver 3-hydroxy-3-methylglutaryl coenzyme A re-
ductase and cholesterol synthesizing activity. Biochemi-
cal and Biophysical Research Communications, 50, 704-
[3] Ness, G.C., Wiggins, L. and Zhao, Z. (1994) Insulin in-
creases hepatic 3-hydroxy-3-methylglutaryl coenzyme A
reductase mRNA and immunoreactive protein levels in
diabetic rats. Archives of Biochemistry and Biophysics,
309, 193-194.
[4] Ness, G.C., Zhao, Z. and Wiggins, L. (1994) Insulin and
glucagon modulate hepatic 3-hydroxy-3-methylglutaryl co-
enzyme A reductase activity by affecting immunoreactive
protein levels. The Journal of Biological Chemistry, 269,
[5] Ness, G.C. and Chambers, C.M. (2000) Feedback and hor-
monal regulation of hepatic 3-hydroxy-3-methylglutaryl
coenzyme reductase: The concept of cholesterol buffering
capacity. Proceedings of the Society for Experimental Bi-
ology and Medicine, 224, 8-19.
[6] Ness, G.C. and Gertz, K.R. (2004) Increased sensitivity
to dietary cholesterol in diabetic and hypothyroid rats as-
sociated with low levels of hepatic HMG-CoA reductase
expression. Experimental Biology and Medicine, 229,
[7] Stoecklin, G., Tenenbaum, S.A., Mayo, T., Chittur, S.V.,
George, A.D., Baroni, T.E., Blackshear, P.J. and Ander-
son, P. (2008) Genome-wide analysis identifies interleu-
kin-10 mRNA as target of tristetraprolin. The Journal of
Biological Chemistry, 283, 11689-11699.
[8] Masuda, K., Marasa, B., Martindale, J.L., Halushka, M.K.
and Gorospe, M. (2009) Tissue- and age-dependent ex-
pression of RNA-binding proteins that influence mRNA
turnover and translation. Aging, 1, 681-698.
[9] Liang, J., Lei, T., Song, Y., Yanes, N., Qi, Y. and Fu, M.
(2009) RNA-destabilizing factor tristetraprolin negatively
regulates NF-B signaling. The Journal of Biological
Chemistry, 284, 29383-29390.
[10] Schichi, Y.M., Resch, U., Hofer-Warbinek, R. and de
Martin, R. (2009) Tristetraprolin impairs NF-B nuclear
translocation. The Journal of Biological Chemistry, 284,
[11] Ness, G.C., Edelman, J.M. and Brooks, P.A. (2012) In-
Copyright © 2013 SciRes. OPEN ACCESS
G. C. Ness, J. L. Edelman / Advances in Bioscience and Biotechnology 4 (2013) 1-5
Copyright © 2013 SciRes.
volvement of tristetraprolin in transcriptional activation
of hepatic 3-hydroxy-3-methylglutaryl coenzyme A re-
ductase by insulin. Biochemical and Biophysical Re-
search Communications, 420, 178-182.
[12] Lai, W.S., Stumpo, D.J. and Blackshear, P.J. (1990) Ra-
pid insulin-stimulated accumulation of an mRNA encod-
ing a proline-rich protein. The Journal of Biological Che-
mistry, 265, 16556-16563.
[13] Clement, S.L., Scheckel, C., Stoecklin, G. and Lykke-An-
dersen, J. (2011) Phosphorylation of tristetraprolin by MK2
impairs AU-rich element mRNA decay by preventing
deadenylase recruitment. Molecular and Cellular Biology,
31, 256-266.
[14] Li, X., Monks, B., Ge, Q. and Birnbaum, M.J. (2007) Akt/
PKB regulates hepatic metabolism by directly inhibiting
transcription coactivator. Nature, 447, 1012-
[15] Kleinman, E., Carter, G., Ghansah, T., Patel, N.A. and
Cooper, D.R. (2009) Developmentally spliced PKC beta
II provides a possible link between mTORC2 and Akt-
kinase to regulate 3T3-L1 adipocyte insulin-stimulated glu-
cose transport. Biochemical and Biophysical Research
Communications, 388, 554-559.
[16] Song, S., Attia, R.R., Connaughton, S., Niesen, M.I.,
Ness, G.C., Elam, M.B., Hori, R.T., Cook, G.A. and Park,
E.A. (2010) Peroxisome proliferator activated receptor
) and PPAR gamma coactivator (PGC-1
) in-
duce carntine palmitoyltransferase IA (CPT-1A) via inde-
pendent gene elements. Molecular and Cellular Endocri-
nology, 325, 54-63.
[17] Boone, L.R., Niesen, M.I., Jaroszeski, M. and Ness, G.C.
(2009) In Vivo identification of promoter elements and
transcription factors mediating activation of hepatic HMG-
CoAreductase by T3. Biochemical and Biophysical Re-
search Communications, 385, 466-471.
[18] Saltiel, A.R. and Kahn, C.R. (2001) Insulin signaling and
the regulation of glucose and lipid metabolism. Nature,
414, 799-806.
[19] Lu, M., Wan, M., Leavens, K.F., Chu, Q., Monks, B.R.,
Fernandez, S., Ahima, R.S., Ueki, K., Kahn, C.R. and
Birnbaum, M.J. (2012) Insulin regulates liver metabolism
in vivo in the absence of hepatic Akt and Foxo1. Nature
Medicine, 18, 388-395.
[20] Yoon, J.C., Puigserver, P., Chen, G., Donovan, J., Wu, Z.,
Rhee, J., Adelmant, G., Stafford, J., Kahn, C.R., Granner,
D.K., Newgard, C.B. and Spiegelman, B.M. (2001) Con-
trol of hepatic gluconeogenesis through the transcriptio-
nal coactivator PGC-1. Nature, 413, 131-138.
[21] Ness, G.C., Wiggins, L. and Zhao, Z. (1994) Insulin in-
creases hepatic 3-hydroxy-3-methylglutaryl coenzyme A
reductase mRNA and immunoreactive protein levels in
diabetic rats. Archives of Biochemistry and Biophysics,
309, 193-194.
[22] Roth, U., Curth, K., Unterman, T.G. and Kietzmann, T.
(2004) The transcription factors HIF-1 and HNF-4 and
the coactivator p300 are involved in insulin-regulated
glucokinase gene expression via the phosphatidylinosi-
tol-3-kinase/protein kinase B pathway. The Journal of
Biological Chemistry, 279, 2623-2631.
[23] Hagiwara, A., Cornu, M., Cybulski, N., Polak, P., Betz,
C., Trapani, F., Terracciano, L., Heim, M.H., Ruegg, M.A.
and Hall, M.N. (2012) Hepatic mTORC2 activates glyco-
lysis and lipogenesis through Akt, glucokinase and
SREBP1c. Cell Metabolism, 15, 725-738.
[24] Porstmann, T., Griffths, B., Chung, Y.-L., Delpuech, O.,
Griffths, J.R., Downward, J. and Schulze, A. (2005) PKB/
Akt induces transcription of enzymes involved in choles-
terol and fatty acid biosynthesis via activation of SREBP.
Oncogene, 24, 6465-6481.
[25] Huang, Z., Zhou, X., Nicholson, A.C., Gotto Jr., A.M.,
Hajjar, D.P. and Han, J. (2008) Activation of peroxisome-
proliferator-activated receptor
in mice induces expres-
sion of the hepatic low-density lipoprotein receptor. Brit-
ish Journal of Pharmacology, 155, 596-605.