Báo cáo khoa học: Tumour necrosis factor-a attenuates insulin action on phosphoenolpyruvate carboxykinase gene expression and gluconeogenesis by altering the cellular localization of Foxa2 in HepG2 cells pptx

Tumour necrosis factor-a attenuates insulin action on
phosphoenolpyruvate carboxykinase gene expression and
gluconeogenesis by altering the cellular localization of
Foxa2 in HepG2 cells
Amit K. Pandey, Vikash Bhardwaj* and Malabika Datta
Institute of Genomics and Integrative Biology (CSIR), Delhi, India
Introduction
Type 2 diabetes, which accounts for almost 90% of
the total diabetic population, stems from the decreased
responsiveness of the body to insulin (insulin resis-
tance), accompanied by the failure of pancreatic b-cells
to secrete insulin to counteract this insulin-resistant
state. Obesity is invariably associated with diabetes
Keywords
diabetes; Foxa2; insulin;
phosphoenolpyruvate carboxykinase
(PEPCK); tumour necrosis factor-a (TNFa)
Correspondence
M. Datta, Institute of Genomics and
Integrative Biology, Mall Road,
Delhi-110 007, India
Fax: +91 11 27667471
Tel: +91 11 27667439, 27667602, ext. 135
E-mail:
*Present address
Special Centre for Molecular Medicine,
Jawaharlal Nehru University, New Delhi,
India
(Received 19 January 2009, revised 18
March 2009, accepted 12 May 2009)
doi:10.1111/j.1742-4658.2009.07091.x

Circulating tumour necrosis factor-a (TNFa) levels, which are elevated in
obesity-associated insulin resistance and diabetes, inhibit insulin signalling at
several points in the signalling cascade. The liver is critical in maintaining cir-
culating glucose levels and, in a preliminary investigation using the human
hepatoma (HepG2) cell line in this study, we demonstrated the role of TNFa
in the regulation of this phenomenon and determined the underlying
molecular mechanisms. As the transcription factor Foxa2 has been impli-
cated, in part, in the regulation of gluconeogenic genes, we studied the effects
of TNFa and ⁄ or insulin on its cellular status in hepatocytes, followed by an
assessment of its occupancy on the phosphoenolpyruvate carboxykinase
(PEPCK) promoter. Preincubation of cells with TNFa, followed by insulin,
significantly prevented insulin-mediated nuclear exclusion of Foxa2 and sub-
stantially increased its nuclear concentration. Foxa2 was subsequently found
to occupy its binding element on the PEPCK promoter. TNFa alone, how-
ever, did not alter the status of cellular Foxa2 or its occupancy on the PEP-
CK promoter. TNFa preincubation also significantly attenuated insulin-
induced inhibition of the expression of gluconeogenic enzymes and hepatic
glucose production. Insulin inhibition of PEPCK expression and the preven-
tive effect of TNFa could be partially but significantly restored in the pres-
ence of Foxa2 siRNA. Several other well-known mediators of insulin action
in the liver in general and of gluconeogenic genes in particular include Foxo1,
PGC-1 and SREBP-1c. Our results indicate that another transcription factor,
Foxa2, is at least partly responsible for the attenuating effect of TNFa on
insulin action on PEPCK expression and glucose production in HepG2 cells.
Structured digital abstract
l
MINT-7040448: TBP (uniprotkb:P20226), FOXA2 (uniprotkb:Q9Y261) and FOXA1 (uni-
protkb:
P55317) colocalize (MI:0403)bycosedimentation (MI:0027)
Abbreviations

F1,6bpase, fructose-1,6-bisphosphatase; G6Pase, glucose-6-phosphatase; Hnf-3, hepatocyte nuclear factor 3; PEPCK, phosphoenolpyruvate
carboxykinase; TBP, TATA box-binding protein; TNFa, tumour necrosis factor-a.
FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS 3757
and a parallel increase in the occurrence of both is
evident across all populations [1,2]. Obesity-induced
insulin resistance is thereby characterized by a loss of
insulin sensitivity mediated by factors released from
adipocytes, mainly free fatty acids and proteins,
termed adipocytokines, which act to control various
metabolic functions [3–6] with well-described physio-
logical effects [7]. One such adipocytokine is tumour
necrosis factor-a (TNFa), which has been identified as
a significant contributor to insulin resistance, and its
levels have been reported to be increased significantly
in obese diabetic individuals and in several animal
models of obesity [8–12].
The liver is a major insulin target tissue and plays a
significant role in glucose homeostasis, as it can
alternate between cycles of glucose output and its
inhibition to maintain normal circulating glucose levels
[13]; it is this precisely regulated cycle that is disturbed
under conditions of insulin resistance and type 2 diabe-
tes. Nuclear transcription factors that are crucial in
governing this metabolic switch are regulated by
circulating levels of insulin and glucagon [14]. Insulin
triggers the activation of a series of phosphorylation
cascades that are lost in insulin-resistant states, thereby
preventing insulin from correctly regulating glucose
and fat metabolism [15].
The hepatocyte nuclear factor 3 (Hnf-3) forkhead

family of nuclear transcription factors, which includes
three members designated as Foxa-1 (Hnf-3a), Foxa-2
(Hnf-3b) and Foxa-3 (Hnf-3c) [16–18], play an impor-
tant regulatory role in the maintenance of normal glu-
cose homeostasis; they do so by regulating the gene
expression of rate-limiting enzymes of gluconeogenesis
and glycogenolysis, including phosphoenolpyruvate
carboxykinase (PEPCK) and glucose-6-phosphatase
(G6Pase), and by regulating glucagon and Pdx-1 gene
expression in the pancreas [17,19–22]. In addition,
although some reports have shown the regulation of
gluconeogenic enzymes by another forkhead transcrip-
tion factor, Foxo1 [23,24], others have reported that
the overexpression of Foxo1 carries the message to
G6Pase only and that PEPCK levels remain unaffected
[25,26]. Thus, the mechanisms involved in the regula-
tion of gluconeogenic enzymes are very controversial,
and it is thereby hypothesized that both of these fac-
tors contribute to insulin action on glucose production
by regulating the expression of different gluconeogenic
enzymes [17], and ⁄ or synchronize with other transcrip-
tion factors to regulate the same.
In view of these controversial reports, we sought to
decipher the role of Foxa2 (HNF-3b), if any, in the
regulation of gluconeogenesis in HepG2 cells, and the
effects of TNFa pretreatment on this phenomenon,
with an objective to decode its regulation in obesity
and insulin resistance. Although HepG2 cells are hepa-
toma cells, they retain several normal human liver
properties, including the synthesis of albumin, lipopro-

tein and several other liver-specific functions, and,
most importantly, these functions are stable through
passages. They are therefore valuable in the study of
several hepatic functions and other aspects of metabo-
lism, and have been recognized as an in vitro human
model system [27,28]. In this article, we report that
Foxa2, in part, is critical in the attenuating effects of
TNFa on insulin-mediated Foxa2 localization in
HepG2 cells, and the ensuing effect on gluconeogenesis
and glucose output. Our results show that, in the pres-
ence of TNFa, insulin-induced inhibition of gluconeo-
genesis and glucose output is attenuated and Foxa2, at
least in part, plays an important role in this effect. The
results presented here require subsequent validation in
primary cells and animal models but, as a preliminary
step, they unravel one of the mechanisms of TNFa-
mediated withdrawal of insulin action in HepG2 cells.
Results
Incubation of HepG2 cells with TNFa attenuates
insulin-stimulated Akt phosphorylation and
nuclear exclusion of Foxa2 in HepG2 cells
Initially, we determined whether the cells were insulin
unresponsive in our study at the dose and period of
TNFa preincubation prior to insulin treatment used.
Akt is one of the most important insulin signalling
intermediates, and is well known to be activated by
insulin, an effect that is equally well known to be pre-
vented in cells preincubated with TNFa prior to insu-
lin incubation. Gupta et al. [29] and Gupta and
Khandelwal [30] have demonstrated previously that

insulin-stimulated Akt phosphorylation is significantly
prevented in HepG2 cells preincubated with TNFa
prior to insulin incubation. Interestingly, HepG2 cells
overexpressing a constitutively active form of Akt
demonstrated restoration of this preventative effect of
TNFa on insulin action [30]. As our study was direc-
ted towards the underlying mechanisms of insulin and
TNFa pretreatment on gluconeogenesis within the
hepatocyte, we started by studying the status of Akt
under the aforesaid conditions. Insulin significantly
stimulated Akt phosphorylation relative to the control
(P < 0.001), and this effect was decreased significantly
on TNFa pretreatment (P < 0.01) (Fig. 1A,B).
The inhibition of insulin signalling in the liver is pri-
marily reflected by the attenuation of the insulin-medi-
ated inhibition of gluconeogenic gene expression. As
Effect of TNFa on insulin action on PEPCK expression A. K. Pandey et al.
3758 FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS
the forkhead protein, Foxa2, has been suggested to
regulate, at least in part, the expression of gluconeo-
genic genes [17,31], we studied its status within the cell
in the given experimental conditions. Foxo1, another
member of the forkhead family of transcription
factors, is a very well-established mediator of the
effects of insulin on gluconeogenic gene expression
[24], and has also been implicated in several cellular
effects of TNFa [32,33]. Together with Foxa2, we also
assessed its cellular status in the presence and absence
of insulin and ⁄ or TNFa. Figure 2A, B shows the
effects of TNFa pretreatment on insulin action on the

localization of Foxa2 and Foxo1 within the cell. Incu-
bation with 50 nm insulin resulted in relative nuclear
exclusion of Foxa2, with significant localization in the
cytosol (P < 0.01 relative to control). An identical but
more pronounced trend was observed for Foxo1,
implying that it is a much stronger candidate for insu-
lin action. Surprisingly, in cells pretreated with TNFa
(1 nm, 24 h), followed by insulin incubation, Foxa2
was found to be mainly localized in the nucleus
(P < 0.05) and was significantly ( P < 0.01) less
detected in the cytosol, relative to cells incubated in
the presence of insulin alone. Foxo1 also showed an
almost complete nuclear localization in cells pretreated
with TNFa prior to insulin incubation, whereas, in
cells incubated in the presence of insulin alone, it was
exclusively localized in the cytosol. As Foxo1 is
already known to mediate the effects of insulin on glu-
coneogenic genes, we carried out further experiments
to decipher the role of Foxa2 only, if any, on these
series of events. There was no significant alteration in
Foxa2 localization in cells treated with TNFa alone
relative to cells incubated in the absence of any of
these reagents (Control) (Fig. 2A–C). These results
imply that, in the presence of TNFa, wherein cells are
rendered insulin insensitive, insulin-mediated nuclear
exclusion and inactivation of Foxa2 are prevented,
with the result that it is primarily localized in the
nucleus. Thus, although TNFa alone does not alter
the status of Foxa2 within the cell, it attenuates insu-
lin-stimulated Foxa2 nuclear exclusion, possibly by

blunting insulin signalling within the cell. The subcellu-
lar distribution of Foxa2 under the conditions stated
above was also checked by immunofluoresence staining
with anti-Foxa2 IgG. In cells incubated with 50 nm
insulin, Foxa2 was fairly strongly detected in the cyto-
sol, when compared with cells incubated in the absence
of insulin. Although Foxa2 was not completely
excluded from the nucleus by treatment with insulin, it
was strongly detected in the cytosol of insulin-treated
cells, but was largely absent in control cells. However,
when cells were pretreated with TNFa (1 nm,24h)
prior to insulin incubation, this nuclear extrusion of
Foxa2 and its localization in the cytosol were signifi-
cantly attenuated, with the result that, in these TNFa-
pretreated cells, Foxa2 was very weakly detected in the
cytosol with the major fraction being in the nucleus
(Fig. 2C).
TNFa pretreatment increases Foxa2 occupancy
on the PEPCK promoter
As we observed a predominant localization of Foxa2
in the nuclei of cells pretreated with TNFa prior to
insulin incubation, and considering its possible involve-
ment in the regulation of gluconeogenic enzymes, we
analysed the Foxa2 occupancy of the promoter of
gluconeogenic genes, mainly PEPCK, it being the
rate-limiting enzyme, to categorically determine
whether Foxa2 can exert its effects on the transcrip-
tional regulation of its targets in the absence and
presence of TNFa and ⁄ or insulin. Foxa2 occupancy of
p-AKT

AKT
TNFα
α
(1 nM)
+–
––
–+
++Ins (50 n
M)
Con
TNF
α
TNF
α
+ Ins
Ins
400
350
300
250
200
150
100
50
*
p-Akt
Arbitrary units
**
0
A

B
Fig. 1. Effect of TNFa on insulin-stimulated Akt activation in HepG2
cells. Serum-starved HepG2 cells were incubated in the absence or
presence of TNFa (1 n
M, 24 h) and then with or without insulin
(50 n
M, 15 min). Cellular lysate (50 lg) from each group was
resolved by SDS-PAGE, transferred to poly(vinylidene difluoride)
membranes and probed by western blotting with p-Akt and Akt
(total) antibodies. Each band is a representative of three indepen-
dent blots (A). Signals were scanned, analysed densitometrically
and intensities are expressed as arbitrary units (B). Values are the
means ± SEM of three experiments. *P < 0.001 when compared
with control; **P < 0.01 when compared with incubation with
insulin alone.
A. K. Pandey et al. Effect of TNFa on insulin action on PEPCK expression
FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS 3759
the PEPCK promoter was determined by semiquantita-
tive (Fig. 3A,B) and quantitative (Fig. 3C) RT-PCR.
When compared with the control, insulin caused a
significant marginal (P < 0.01) decrease in Foxa2
occupancy of the PEPCK promoter. This decrease was
significantly (P < 0.01) attenuated in cells preincubat-
ed in the presence of TNFa prior to insulin incubation.
In cells incubated in the presence of TNFa alone,
Foxa2 did not show any significant change in its occu-
pancy on the PEPCK promoter after normalization
with the input DNA and comparison with the control.
All of these results indicate that preincubation with
TNFa significantly abrogates the insulin-mediated

decrease in Foxa2 occupancy of the PEPCK promoter,
with the result being that, under these conditions,
Foxa2 significantly occupies its binding element on the
PEPCK promoter which, however, is not observed in
cells incubated in the presence of TNFa alone.
Effect of TNFa pretreatment on PEPCK and
G6Pase mRNA in HepG2 cells
Gluconeogenesis is a very significant phenomenon in the
liver, and gluconeogenic enzymes, namely PEPCK,
Foxa2
Foxa2
Foxo1
Foxo1
TBP
β
-actin
TBP
β
-actin
Nuclear pellet
Cytosol
Con
TNFα + Ins
TNFα
Ins
Con
TNFα + Ins
TNFα
Ins
A

C
120
60
80
100
Foxo1
Foxa2
Nuclear pellet
**
b
0
20
40
*
a
Con
TNFα + Ins
TNFα
Ins
160
200
240
Foxa2
Foxo1
*
a
Relative arbitrary units
Cytosol
40
80

120
***
c
0
B
DAPI FITC
Merge
Control
TNF
α
+ Insulin
Insulin
TNF
α
Fig. 2. Effect of TNFa on Foxa2 and Foxo1 localization. HepG2 cells were incubated with TNFa (1 nM) or insulin (50 nM), or pretreated with
TNFa (1 n
M, 24 h) followed by insulin treatment (50 nM, 15 min). Cells incubated in the absence of any of these were taken as the control
(Con). On termination of incubation, cells were lysed and the nuclear (50 lg) and cytosolic (40 lg) protein extracts were assessed for the
presence of Foxa2 or Foxo1 by western blotting. Each band is a representative of three separate blots from three independent experiments.
Blots were probed with TBP and b-actin antibodies and taken as nuclear and cytosolic loading controls, respectively, and also used to ascer-
tain the purity of the nuclear and cytosolic preparations (A). Bands were scanned, quantified densitometrically and are expressed as arbitrary
units (a.u). Values depicted are the means ± SEM of three values obtained from three independent blots (B). (C) HepG2 cells were incu-
bated as described in (A) and Foxa2 localization was detected by incubation with anti-Foxa2 IgG and fluorescein isothiocyanate-linked sec-
ondary antibody. Cells were visualized in a fluorescence microscope at a magnification of ·40. DAPI, 4¢,6-diamidino-2-phenylindole; FITC,
fluorescein isothiocyanate. *P < 0.01 when compared with control; **P < 0.05 when compared with insulin incubation (nuclear pellet);
***P < 0.01 when compared with insulin incubation (cytosol).
a
P < 0.001 when compared with control;
b
P < 0.01 and

c
P < 0.001 when
compared with incubation with insulin alone.
Effect of TNFa on insulin action on PEPCK expression A. K. Pandey et al.
3760 FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS
fructose-1,6-bisphosphatase (F1,6bpase) and G6Pase,
are critical in determining the rate of gluconeogenesis
and hepatic glucose production. Considering these
phenomena, which are elevated under diabetic condi-
tions, and also the fact that, in cells that are rendered
insulin insensitive by TNFa, there is a relatively
increased nuclear translocation of Foxa2, we studied the
resulting effects of TNFa pretreatment on the effect of
insulin on the expression of PEPCK and another glu-
coneogenic enzyme, G6Pase. Compared with the con-
trol, insulin incubation caused a significant inhibition of
PEPCK and G6Pase gene expression (P < 0.001,
Fig. 4B). However, TNFa pretreatment prior to insulin
incubation considerably attenuated this inhibitory effect
(PEPCK, P < 0.01; G6Pase, P < 0.05; when compared
with insulin alone). This indicates that, in the presence
of TNFa, HepG2 cells do not respond to insulin and the
subsequent enhanced occupation of Foxa2 on its bind-
ing element (as observed in the case of PEPCK) leads to
elevated levels of these gene transcripts. When compared
with the control, TNFa alone caused a significant
(P < 0.05) inhibition of PEPCK and G6Pase tran-
scripts. However, as described in the earlier results,
Foxa2 localization and occupancy on the PEPCK pro-
moter in cells incubated in the presence of TNFa alone

were not altered significantly from those of the control;
these results indicate that, although PEPCK and
G6Pase transcripts are decreased in cells incubated in
the presence of TNFa and insulin alone, the upstream
events facilitating this are possibly different, with
Foxa2, at least in part, mediating the insulin effect.
Real-time PCR data also depicted an identical pattern,
in which PEPCK and G6Pase mRNA were significantly
(P < 0.001) inhibited in the presence of insulin; how-
ever, this was not observed when the cells were pretreat-
ed with TNFa prior to insulin treatment (P < 0.001;
Fig. 4C). TNFa also inhibited significantly the levels of
PEPCK and G6Pase gene transcripts (P < 0.01). The
specificity of Foxa2 was checked with the use of Foxa2
siRNA that could knock down Foxa2 protein levels by
almost 70% (data not shown). Incubation with Foxa2
siRNA prior to insulin treatment could only partially
withdraw insulin-mediated inhibition of PEPCK gene
expression (P < 0.05, Fig. 4D), and a complete restora-
tion was not observed, indicating that Foxa2 is critical,
but not the sole mediator, of insulin effects. The preven-
tative effect of TNFa on insulin-mediated inhibition of
PEPCK expression was also partially reversed by Foxa2
siRNA in cells pretreated with TNFa prior to insulin
incubation (P < 0.05).
TNFa attenuates insulin-induced inhibition of
hepatic glucose output in HepG2 cells
As we had observed, so far, an increase in gluconeogenic
gene transcript levels in TNFa-pretreated cells as a
2.5

1
1.5
2
*
**
0
0.5
Foxa2 binding normalized
to input (arbitary units)
Con
Ins
TNF
α
+ Ins
TNF
α
***
*
Foxa2 binding to PEPCK promoter
(normalized to input)
Con
Ins
TNF
α
+ Ins
TNF
α
Foxa2
Input
IgG

Con
Ins
TNF
α
+ Ins
TNF
α
A
B
C
2.5
1
1.5
2
0
0.5
Fig. 3. Effect of TNFa on PEPCK promoter occupancy by Foxa2 in
HepG2 cells. Cells were pretreated with TNFa (1 n
M) followed by insu-
lin incubation (50 n
M), or incubated with TNFa or insulin alone. On
termination of incubation, nuclear chromatin was isolated and immu-
noprecipitated with either normal IgG or anti-Foxa2 IgG. The chroma-
tin–antibody aggregates were pulled down with protein A-Sepharose
and the occupancy of Foxa2 on the PEPCK promoter was determined
by semiquantitative (A, B) and real-time quantitative (C) PCR using
primers enclosing the Foxa2 binding sites on the PEPCK promoter.
The relative quantity of Foxa2 occupancy was determined by the rela-
tive standard curve method. Each value presented has been normal-
ized with that of input DNA and is the mean ± SEM of three

independent values. *P < 0.01 when compared with control;
**P < 0.01 and ***P < 0.05 when compared with insulin incubation.
A. K. Pandey et al. Effect of TNFa on insulin action on PEPCK expression
FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS 3761
result of a decrease in the effects of insulin, mediated in
part, by the transcription factor, Foxa2, we sought to
determine the effect(s) of this on glucose production
from HepG2 cells, the ultimate phenotype that, together
with glucose uptake, regulates the circulating glucose
level within the body. The incubation of HepG2 cells
with insulin inhibited glucose release by almost threefold
when compared with the control (P < 0.01); pretreat-
ment with TNFa prior to insulin incubation significantly
attenuated this inhibition (P < 0.001), i.e. in the pres-
ence of TNFa, the extent of inhibition of hepatic glucose
output by insulin was markedly attenuated (Fig. 5).
Discussion
TNFa, which is widely implicated in obesity-associated
insulin resistance, impairs the insulin signalling
pathway [4–6,29,30,34,35]; however, its role in hepatic
gluconeogenesis during insulin resistance and the com-
plex underlying mechanisms are not well understood.
Impaired glucose tolerance and insulin resistance are
early metabolic disturbances in the development of
type 2 diabetes. Glucose homeostasis in the body is
largely controlled by the liver, and hyperglycemia, as
observed in type 2 diabetes, reflects increased hepatic
glucose production [36,37], as well as reduced glucose
uptake [38]. Indeed, the onset of hepatic insulin resis-
tance typically precedes peripheral insulin resistance in

humans [39]. The stimulation of gluconeogenesis
occurs invariably as a result of increased activity of
PEPCK, G6Pase and F1,6bpase, and the targeted
overexpression or knockouts of these enzymes play a
major regulatory role in glucose homeostasis [40,41].
As far as the regulation of these genes is con-
cerned, the Foxa family of transcription factors acts
synergistically with other hepatocyte nuclear factors to
TNFα
α
(1 n
M
)
Ins (50 n
M
)
+–
––
–+
++
PEPCK
18S
G6Pase
0.9
1.2
1.5
PEPCK/18S
G6Pase/18S
**
***

***
***
**
0
0.3
0.6
Arbitrary units
*
Con TNF
α
TNF
α
+
Ins
Ins
0.8
1
1.2
PEPCK/18S
G6Pase/18S
*
*
**
**
0.4
0.6
*
*
0
0.2

Con TNF
α
TNF
α
+
Ins
Ins
Relative mRNA levels
0.8
1
1.2
Control
Insulin
TNFα
TNFα + Insulin
0.2
0.4
0.6
a
b,c
0
Control siRNA Foxa2 siRNA
Relative PEPCK mRNA levels
normalized to 18S rRNA
A
B
DC
Fig. 4. Analysis of PEPCK and G6Pase expression following TNFa treatment. HepG2 cells were pretreated with TNFa (1 nM, 24 h), followed
by insulin incubation (50 n
M, 4 h), or incubated with TNFa or insulin alone for the respective indicated times. Cells in the absence of any of

these were taken as the control (Con). Two micrograms of total RNA were reverse transcribed with random primers and the levels of PEP-
CK and G6Pase mRNA were measured by RT-PCR using gene-specific primers. 18S rRNA was taken as the internal loading control (A). Each
band was analysed densitometrically and the values are depicted after normalization of PEPCK and G6Pase bands with those of 18S rRNA
(B). Each point is the mean ± SEM of three sets of experiments. (C) Real-time PCR quantification of PEPCK and G6Pase mRNA in cells incu-
bated as described in (A). Values were normalized to those of 18S rRNA and are the means ± SEM of three independent experiments. (D)
Real-time PCR quantification of PEPCK in cells transfected with either control or Foxa2 siRNA prior to incubation as described in (A) above.
Values are the means ± SEM of three independent experiments after normalization with 18S rRNA. *P < 0.001 when compared with control
(B) and control vs. insulin and TNFa plus insulin vs. insulin alone (C); **P < 0.01 when compared with insulin alone (B) and TNFa incubation
when compared with control (C); ***P < 0.05, TNFa plus insulin vs. insulin alone and TNFa alone compared with control (B).
a,b
P < 0.05
when compared with insulin alone and TNFa plus insulin, respectively, in the presence of control siRNA;
c
P < 0.05 when compared with
insulin alone in the presence of Foxa2 siRNA (D).
Effect of TNFa on insulin action on PEPCK expression A. K. Pandey et al.
3762 FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS
coordinately regulate liver-specific gene expression [42].
Their transcriptional regulation, particularly that of
PEPCK by insulin, is protein synthesis independent,
but involves the participation of several transcription
factors, including Foxo1, Foxo3, PGC-1a, SREBP
etc., although none can be singled out to mediate the
effect of insulin. The PEPCK promoter is undoubtedly
complex and possesses the binding elements of several
transcription factor complexes [43]. The regulation by
the Foxa group of transcription factors, which possess
considerably identical DNA-binding domains and bind
to the promoters of target genes as monomers, is even
more controversial. Foxa2 plays a significant regula-

tory role in hepatic and ⁄ or pancreatic physiology [16–
22,44–47]. It is excluded from the nucleus as a result
of its phosphorylation at Thr156 by Akt, resulting in
its inactivation and subsequent repression of the tran-
scriptional response of key gluconeogenic enzymes
[17]. Zhang et al. [31] have also demonstrated that
Foxa2 is required for hepatic gluconeogenesis, the acti-
vation of PEPCK is significantly downregulated in the
absence of Foxa2, and a clear enrichment of its pro-
moter by Foxa2 antibody has been reported [31,48].
Similar results in relation to the identification of a
Foxa2-binding site within the PEPCK promoter have
also been reported by others [20,22,49,50], and Wolf-
rum et al. [17] suggested that Foxa2 may contribute to
hepatic insulin resistance in Akt) ⁄ ) mice as a result of
an inability to phosphorylate Foxa2 and suppress the
transcription of gluconeogenic enzymes. Based on their
results, O’Brien et al. [20] reported that insulin
mediates its negative effect on glucocorticoid-induced
PEPCK gene transcription by inhibiting the binding of
Hnf-3 proteins. However, Hall et al. [51] reported that
insulin response sequences themselves are not sufficient
for the complete effect of insulin on its targets. They
found insulin-mediated dissociation of glucocorticoid-
induced accumulation of several transcription factors,
including Foxa2, from the PEPCK promoter. Taken
together, several transcription factors act in tandem to
regulate PEPCK gene transcription in response to
insulin, and none has been definitively established as
physiologically mediating the basal, as well as hor-

mone-mediated, alterations in PEPCK gene expression.
In this study, we found Foxa2 to be predominantly
localized in the nuclei of HepG2 cells incubated with
TNFa prior to insulin incubation. As reported earlier,
insulin incubation resulted in a relative increase in the
nuclear exclusion of Foxa2, with it being strongly
localized in the cytosol. TNFa alone, however, did not
alter the status of Foxa2 localization when compared
with the control. These results imply that, in a TNFa-
mediated insulin-resistant cell, insulin-induced nuclear
exclusion of Foxa2 is reasonably prevented, with the
result that the majority is localized in the nucleus. Pre-
treatment with TNF a prior to insulin also led to
enhanced binding to the PEPCK promoter by Foxa2.
In our study, Foxa2 localization and its subsequent
effects therefore appear to be modest, but steady,
which points to the fact that other mechanisms and
factors are also crucial in mediating the effects of insu-
lin [51]. That this is so corroborates well, considering
the complexity of the PEPCK promoter, which
harbours the binding elements of several transcription
factors [43]. Another such transcription factor and a
strong regulator of gluconeogenesis is the protein,
Foxo1 [24]. This is a very well-studied transcription
factor regulating insulin action on gluconeogenic
enzymes. Our results also show an increased nuclear
extrusion of Foxo1 in the presence of insulin. How-
ever, some reports have stated that insulin-mediated
phosphorylation inactivates Foxo1, but, surprisingly,
the message is carried only onto G6Pase and not to

PEPCK, as evident from studies on epithelial kidney
cells which lack Foxa2 but express Foxo1 [25]. Along
similar lines, Barthel et al. [26] reported that the over-
expression of Foxo1 in rat hepatoma cells increased
G6Pase transcript levels without affecting those of
PEPCK. In the light of this, our results identify Foxa2
as a crucial mediator which, at least in part, plays a
significant role in TNFa-mediated abrogation of insu-
lin signalling within hepatocytes.
1.4
1
.
6

P < 0.001
0.8
1
1.2
**
Hepatic glucose release
(µg·mg
–1
protein)
0
0.2
Con
TNF
α
TNF
α

+ Ins
Ins
0.4
0.6
*
Fig. 5. TNFa attenuates insulin-induced inhibition of hepatic glu-
cose output. HepG2 cells were serum starved overnight and incu-
bated for 24 h in the presence of TNFa (1 n
M) or insulin (50 nM)
alone, or pretreated with TNFa followed by insulin for these time
periods. Control cells were incubated in the absence of any of
these agents. On termination of incubation, the glucose released in
the medium was assayed as described in Materials and methods,
and the values were normalized to the total cellular protein content.
Each value is the mean ± SEM of three independent incubations.
*P < 0.01 when compared with control; **P < 0.001 when com-
pared with insulin incubation.
A. K. Pandey et al. Effect of TNFa on insulin action on PEPCK expression
FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS 3763
Consequent to the increased presence of Foxa2 in
the nuclei of cells pretreated with TNFa, insulin inhibi-
tion of both PEPCK and G6Pase was significantly
prevented in such cells. Experiments with Foxa2 siR-
NA showed that decreased levels of the Foxa2 protein
marginally but significantly restored both insulin inhi-
bition of PEPCK expression and the prevention of this
by TNFa. This probably contributes towards the
observed hyperglycaemic status in obese diabetics. In
cells incubated in the presence of TNFa alone,
although there was a significant inhibition of gluconeo-

genic gene transcription, we did not observe any alter-
ation of Foxa2 localization, probably meaning that,
although both insulin and TNFa alone decrease the
transcription of gluconeogenic genes, Foxa2 may not
be involved in the TNFa effect. This could be a possi-
bility considering the complex promoter regulation of
PEPCK [52,53]. It has been shown recently that the
nuclear corepressor is required in the TNFa-mediated
inhibition of PEPCK [54]. Therefore, in cells preincu-
bated with TNFa prior to insulin, insulin signalling is
prevented, resulting in abrogation of this inhibitory
effect on PEPCK expression. PEPCK overexpression,
in turn, has been shown to attenuate insulin signalling
and hepatic insulin sensitivity in transgenic mice
[41,55]. Interestingly, adipose selective overexpression
of PEPCK led to increased glyceroneogenesis,
increased fat mass and adipose size, increased body
weight and severe susceptibility to diet-induced insulin
resistance [56,57].
Circulating TNFa levels, which are elevated in obese
diabetic individuals [8], inhibit several mediators of the
insulin signalling cascade [4–6,29,30,34,35], and this
leads to the prevention of insulin-mediated inhibition
of hepatic glucose output. Indeed, whole-body infusion
with TNFa is associated with a significant increase in
hepatic glucose output as a result of an impaired abil-
ity of insulin to suppress hepatic glucose production
[58,59]. In this article, we have demonstrated that
TNFa pretreatment prevents insulin-induced inhibition
of hepatic glucose output, indicating that, in such con-

ditions, cells become insulin insensitive; this is in agree-
ment with studies in which the overexpression of
IKKb, a downstream mediator of TNFa signalling,
leads to local and systemic insulin resistance, whereas
mice lacking this enzyme in the liver retain liver insulin
responsiveness [60,61].
In summary, our results have unfolded a series of
events beginning with the TNFa-mediated prevention
of the effect of insulin on Foxa2 localization and lead-
ing to the abrogation of insulin inhibition of gluconeo-
genesis and glucose output in HepG2 cells. Although
TNFa-mediated inhibition of insulin signalling has
been known for some time, the focus has primarily
been on glucose uptake in the skeletal muscle and
adipocytes. Although the results presented here need
to be validated in primary cells and in in vivo models,
they provide a preliminary picture of the consequent
effects of this inhibition on hepatic gluconeogenesis
and, in part, the mechanisms involved. As TNFa is a
major adipocytokine associated with obesity and type
2 diabetes, this pathway of impairment of insulin
action, as observed in HepG2 cells mediated by Foxa2,
possibly explains one of the contributory mechanisms
for the observed hyperglycaemia in obese diabetics.
Materials and methods
Materials
DMEM, antibiotic–antimycotic, protein A-Sepharose,
human insulin and TNFa were purchased from Sigma
(St. Louis, MO, USA). The glucose assay, protein estimation
and RNeasy kits were obtained from Merck (Darmstadt,

Germany), Biorad Laboratories (Hercules, CA, USA) and
Qiagen (Hilden, Germany), respectively. SYBR Green Real
Time PCR Master Mix was purchased from Applied Biosys-
tems (Foster City, CA, USA). Foxa2, Foxo1, TATA
box-binding protein (TBP) and b -actin primary antibodies
were obtained from Santa Cruz Biotechnology Inc. (Santa
Cruz, CA, USA), whereas those of p-Akt and total Akt were
purchased from Cell Signaling Technology (Danvers, MA,
USA). All secondary antibodies used were obtained from
Bangalore Genei, India. All other chemicals and reagents
used were purchased from Sigma. Control and Foxa2
siRNA was obtained from Santa Cruz Biotechnology Inc.
Cell culture
All experiments were performed in HepG2 (human hepato-
cellular carcinoma) cells obtained from the National Centre
for Cell Science, Pune, India. HepG2 cells have been
reported to confer many hepatocyte functions [27] and
thereby to serve as a resource for metabolic studies [28].
These cells are extensively used for the study of insulin sig-
nalling and hepatic glucose output [62–65]. Cells were main-
tained in DMEM supplemented with 10% fetal calf serum
and 1% antibiotic–antimycotic (100 unitsÆmL
)1
penicillin,
0.1 mgÆmL
)1
streptomycin and 0.25 lgÆmL
)1
amphotericin
B) at 37 °C in a humidified atmosphere of 5% CO

2
.
All incubations were carried out after overnight serum
starvation.
Western blotting
HepG2 cells were plated in six-well plates and incubated
with TNFa (1 nm, 24 h) or insulin (50 nm, 15 min), or
Effect of TNFa on insulin action on PEPCK expression A. K. Pandey et al.
3764 FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS
preincubated with TNFa followed by insulin treatment. On
termination of incubation, cells were lysed in ice-cold lysis
buffer [10 mm Tris, 50 mm NaCl, 1% Triton X-100, 5 mm
EDTA, 20 mm sodium pyrophosphate, 50 mm NaF,
100 lm Na
3
VO
4
,5lgÆmL
)1
each of leupeptin, aprotinin
and pepstatin, and 1 m m phenylmethylsulphonyl fluoride
(pH 7.4)]. Lysates were centrifuged at 10 000 g for 10 min
at 4 °C, and the supernatant was used as the cytosolic prep-
aration. To the pellet, 50 lLof10mm Tris (pH 7.5) con-
taining 10% v ⁄ v glycerol, 0.1 m KCl, 0.2 mm EDTA,
20 mm sodium pyrophosphate, 50 mm NaF, 100 lm
Na
3
VO
4

,5lgÆmL
)1
each of leupeptin, aprotinin and pepst-
atin, and 1 mm phenylmethylsulphonyl fluoride was added
and stirred at 4 °C for 30 min. These nuclear extracts were
centrifuged at 15 000 g for 20 min at 4 ° C, and the super-
natant was used as the nuclear fraction. Equal amounts of
nuclear and cytosolic proteins were resolved by
SDS-PAGE, transferred to poly(vinylidene difluoride) mem-
branes and probed with p-Akt, Akt, Foxa2 and Foxo1
antibodies. Blots were probed identically for b-actin or
TBP, and taken as the loading controls, and also to assess
the purity of nuclear and cytosolic preparations. Bands
were analysed densitometrically as described below.
Immunofluoresence microscopy
HepG2 cells were treated as described above with TNFa
(1 nm) and ⁄ or insulin (50 nm), or in the absence of any of
these (control). On termination of incubation, cells were
fixed for 15 min at room temperature with 3.5% parafor-
maldehyde. The cells were then permeabilized with 0.5%
Triton X-100 and incubated with anti-Foxa2 IgG (1 : 50)
for 2 h at room temperature. After washing, the cells were
treated with anti-goat secondary IgG linked to fluorescein
isothiocyanate (1 : 100) for 2 h at room temperature. The
cells were then washed thoroughly, 4¢,6-diamidino-2-pheny-
lindole (DAPI) was added to a final concentration of
1 lgÆmL
)1
and the cells were visualized in a fluorescent
microscope (Carl Zeiss Inc., New York, NY, USA).

Chromatin immunoprecipitation assay
Cells were treated with either TNFa (1 nm, 24 h) or insulin
(50 nm, 15 min) alone, or pretreated with TNFa followed
by insulin incubation. On termination of incubation, chro-
matin was isolated according to the method of Buser et al.
[66]. Twenty per cent of the chromatin preparation was
reserved as the total input control and the remainder was
incubated overnight at 4 °C in the presence of either nor-
mal IgG or anti-Foxa2 IgG (5 lg). Immune complexes
were reverse crosslinked and the Foxa2 enrichment of the
target DNA fragments in the immunoprecipitated DNA
was checked by PCR and quantified by real-time PCR. In
both cases, the sequences of sense and antisense primers
used were 5¢-GCCTGTGTGTCCTCAAAACC-3¢ and
5¢-GCAACTGTCCCTTGTCAAAA-3¢, respectively, which
were specific to the Foxa2 binding site within the human
PEPCK promoter. PCRs were performed in the presence of
0.25 mm dNTPs, 1.5 mm MgCl
2
, 10 pmol of each primer
and 0.5 U Taq polymerase, and consisted of 35 cycles of
denaturation at 94 °C for 45 s, annealing at 58 °C for 30 s
and extension at 72 °C for 30 s (10 min last cycle; Gene-
Amp PCR System 9700, Applied Biosystems). PCR prod-
ucts were separated on a 1.0% agarose gel, photographed
with the Alpha Innotech gel documentation system and the
intensity of each band was analysed densitometrically and
plotted after normalization to that of the input DNA. For
real-time PCR, reaction components were put together
using the SYBR Green PCR Master Mix (Applied Biosys-

tems), and the reactions were performed according to the
manufacturer’s instructions (ABI 7500, Applied Biosys-
tems). Reactions were performed in triplicate and the rela-
tive quantity was determined by the relative standard curve
method. Values were normalized to those of input DNA
and the control was arbitrarily assigned a value of unity.
RNA isolation, RT-PCR and quantitative
real-time PCR
The subsequent effects of TNFa incubation prior to insulin
treatment, or insulin or TNFa treatments alone, on the
transcript levels of gluconeogenic genes were examined as
described by Gabbay et al. [67]. Cells were incubated either
in the presence of TNFa (1 nm) for 24 h, followed by insu-
lin (50 nm) for 4 h, or with insulin or TNFa alone, or in
the absence of any of these agents. Total RNA was
extracted using the RNeasy kit (Qiagen), reverse tran-
scribed and amplified (GeneAmp PCR System 9700,
Applied Biosystems) with gene-specific primers (PEPCK:
sense, 5¢-GGTTCCCAGGGTGCATGAAA-3¢; antisense,
5¢-CACGTAGGGTGAATCCGTCAG-3¢; G6Pase: sense,
5¢-ATGAGTCTGGTTACTACAGCCA-3¢; antisense, 5¢-
AAGACAGGGCCGTCATTATGG-3¢). All reactions were
performed in triplicate and expression levels were normal-
ized to those of 18S rRNA. Real-time PCR for quantifica-
tion was performed as described above, according to the
manufacturer’s instructions (ABI 7500, Applied Biosys-
tems). Reactions were performed in triplicate and the
expression of each transcript was quantified by the relative
standard curve method and normalized to that of 18S
rRNA. The transcript value for the control obtained after

normalization was arbitrarily assigned a value of unity. To
further validate the role of Foxa2, HepG2 cells were trans-
fected with 100 nm of either control or Foxa2 siRNA
(Santa Cruz Biotechnology Inc.), according to the manufac-
turer’s instructions. After allowing the cells to grow in fresh
DMEM for 48 h, they were incubated with insulin or
TNFa, or pretreated with TNFa prior to insulin, as men-
tioned above. Cells incubated in the absence of any of these
were taken as the control. On termination of incubation,
A. K. Pandey et al. Effect of TNFa on insulin action on PEPCK expression
FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS 3765
RNA was isolated and the status of PEPCK was deter-
mined by real-time PCR, as described previously.
Glucose production assay
Glucose production was carried out essentially as described
previously [63] with slight modifications. Briefly, after over-
night serum starvation, HepG2 cells were incubated with
TNFa (1 nm, 24 h) or insulin (50 nm, 24 h), or pretreated
with TNFa prior to insulin incubation. Glucose released
into the medium was assayed by subsequent incubation in
glucose production medium [glucose- and phenol red-free
DMEM containing the gluconeogenic substrates, sodium
lactate (20 mm) and sodium pyruvate (2 mm)] and measure-
ment of the glucose concentration using the glucose assay
kit (Merckotest Glucose kit, Merck). This was normalized
with total cellular protein measured using the protein assay
kit (Biorad Laboratories).
Densitometric analysis
Each band, when mentioned, was analysed by alpha digi-
doc 1201 software (Alpha Innotech Corporation, San

Leandro, CA, USA). The same sized rectangular box was
drawn surrounding each band and the intensity of each was
analysed by the program after subtraction of the back-
ground intensity.
Statistical analysis
All experiments were performed in triplicate and the data
are presented as the mean ± standard error of the mean
(SEM). Student’s t-test was used for statistical analysis and
P < 0.05 was taken to be statistically significant.
Acknowledgements
This work was supported by an Indian National Sci-
ence Academy (INSA) Young Scientist Project grant
(INSA, New Delhi, India; SPYSP-51 ⁄ 2006 ⁄ 3705).
A.K.P. acknowledges the receipt of a fellowship from
the Council of Scientific and Industrial Research, New
Delhi, India (NWP0036). The authors also thank
Dr S. Chandna (Institute of Nuclear Medicine and
Allied Sciences, DRDO, India) for the fluorescent
microscopic images.
References
1 Mokdad AH, Ford ES, Bowman BA, Dietz WH,
Vinicor F, Bales VS & Marks JS (2003) Prevalence of
obesity, diabetes, and obesity-related health risk factors,
2001. J Am Med Assoc 289, 76–79.
2 Zimmet P, Alberti KG & Shaw J (2001) Global and
societal implications of the diabetes epidemic. Nature
414, 782–787.
3 Pittas AG, Joseph NA & Greenberg AS (2004) Adipo-
cytokines and insulin resistance. J Clin Endocrinol
Metab 89, 447–452.

4 Hotamisligil GS, Murray DL, Choy LN & Spiegelman
BM (1994) Tumor necrosis factor alpha inhibits signal-
ing from the insulin receptor. Proc Natl Acad Sci USA
91, 4854–4858.
5 Cheung AT, Ree D, Kolls JK, Fuselier J, Coy DH &
Bryer-Ash M (1998) An in vivo model for elucidation
of the mechanism of tumor necrosis factor-alpha (TNF-
alpha)-induced insulin resistance: evidence for differen-
tial regulation of insulin signaling by TNF-alpha.
Endocrinology 139, 4928–4935.
6 Liu LS, Spelleken M, Rohrig K, Hauner H & Eckel J
(1998) Tumor necrosis factor-alpha acutely inhibits
insulin signaling in human adipocytes: implication of
the p80 tumor necrosis factor receptor. Diabetes 47,
515–522.
7 Boden G & Shulman GI (2002) Free fatty acids in obes-
ity and type 2 diabetes: defining their role in the devel-
opment of insulin resistance and beta-cell dysfunction.
Eur J Clin Invest 32(Suppl 3), 14–23.
8 Katsuki A, Sumida Y, Murashima S, Murata K,
Takarada Y, Ito K, Fujii M, Tsuchihashi K, Goto H,
Nakatani K et al. (1998) Serum levels of tumor necrosis
factor-alpha are increased in obese patients with
noninsulin-dependent diabetes mellitus. J Clin Endocri-
nol Metab 83, 859–862.
9 Hotamisligil GS, Shargill NS & Spiegelman BM (1993)
Adipose expression of tumor necrosis factor-alpha:
direct role in obesity-linked insulin resistance. Science
259, 87–91.
10 Hamann A, Benecke H, Le Marchand-Brustel Y, Susu-

lic VS, Lowell BB & Flier JS (1995) Characterization of
insulin resistance and NIDDM in transgenic mice with
reduced brown fat. Diabetes 44, 1266–1273.
11 Hotamisligil GS, Arner P, Caro JF, Atkinson RL &
Spiegelman BM (1995) Increased adipose tissue expres-
sion of tumor necrosis factor-alpha in human obesity
and insulin resistance. J Clin Invest 95, 2409–2415.
12 Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R
& Simsolo RB (1995) The expression of tumor necrosis
factor in human adipose tissue. Regulation by obesity,
weight loss, and relationship to lipoprotein lipase.
J Clin Invest 95, 2111–2119.
13 Granner D, Andreone T, Sasaki K & Beale E (1983)
Inhibition of transcription of the phosphoenolpyruvate
carboxykinase gene by insulin. Nature 305, 549–551.
14 Spiegelman BM & Heinrich R (2004) Biological control
through regulated transcriptional coactivators. Cell 119,
157–167.
Effect of TNFa on insulin action on PEPCK expression A. K. Pandey et al.
3766 FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS
15 Saltiel AR & Kahn CR (2001) Insulin signaling and the
regulation of glucose and lipid metabolism. Nature 414,
799–806.
16 Kaestner KH, Hiemisch H, Luckow B & Schutz G
(1994) The HNF-3 gene family of transcription factors
in mice: gene structure, cDNA sequence, and mRNA
distribution. Genomics 20, 377–385.
17 Wolfrum C, Besser D, Luca E & Stoffel M (2003) Insu-
lin regulates the activity of forkhead transcription factor
Hnf-3beta ⁄ Foxa-2 by Akt-mediated phosphorylation

and nuclear ⁄ cytosolic localization. Proc Natl Acad Sci
USA 100, 11624–11629.
18 Brennan RG (1993) The winged-helix DNA-binding
motif: another helix-turn-helix takeoff. Cell 74, 773–
776.
19 Gerrish K, Gannon M, Shih D, Henderson E, Stoffel
M, Wright CV & Stein R (2000) Pancreatic beta cell-
specific transcription of the pdx-1 gene. The role of con-
served upstream control regions and their hepatic
nuclear factor 3beta sites. J Biol Chem 275, 3485–3492.
20 O’Brien RM, Noisin EL, Suwanichkul A, Yamasaki T,
Lucas PC, Wang JC, Powell DR & Granner DK (1995)
Hepatic nuclear factor 3- and hormone-regulated
expression of the phosphoenolpyruvate carboxykinase
and insulin-like growth factor-binding protein 1 genes.
Mol Cell Biol 15, 1747–1758.
21 Shih DQ, Navas MA, Kuwajima S, Duncan SA &
Stoffel M (1999) Impaired glucose homeostasis and
neonatal mortality in hepatocyte nuclear factor 3alpha-
deficient mice. Proc Natl Acad Sci USA 96,
10152–10157.
22 Wang JC, Stafford JM, Scott DK, Sutherland C &
Granner DK (2000) The molecular physiology of hepa-
tic nuclear factor 3 in the regulation of gluconeogenesis.
J Biol Chem 275, 14717–14721.
23 Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu
LS, Anderson MJ, Arden KC, Blenis J & Greenberg
ME (1999) Akt promotes cell survival by phosphorylat-
ing and inhibiting a Forkhead transcription factor. Cell
96, 857–868.

24 Zhang W, Patil S, Chauhan B, Guo S, Powell DR, Le
J, Klotsas A, Matika R, Xiao X, Franks R et al. (2006)
Foxo1 regulates multiple metabolic pathways in the
liver: effects on gluconeogenic, glycolytic, and lipogenic
gene expression. J Biol Chem 281, 10105–10117.
25 Nakae J, Kitamura T, Silver DL & Accili D (2001) The
forkhead transcription factor Foxo1 (Fkhr) confers
insulin sensitivity onto glucose-6-phosphatase expres-
sion. J Clin Invest 108, 1359–1367.
26 Barthel A, Schmoll D, Kruger KD, Bahrenberg G,
Walther R, Roth RA & Joost HG (2001) Differential
regulation of endogenous glucose-6-phosphatase and
phosphoenolpyruvate carboxykinase gene expression by
the forkhead transcription factor FKHR in H4IIE-hepa-
toma cells. Biochem Biophys Res Commun 285, 897–902.
27 Mavri-Damelin D, Eaton S, Damelin LH, Rees M,
Hodgson HJ & Selden C (2007) Ornithine transcarbamy-
lase and arginase I deficiency are responsible for dimin-
ished urea cycle function in the human hepatoblastoma
cell line HepG2. Int J Biochem Cell Biol 39, 555–564.
28 Javitt BN (1990) HepG2 cells as a resource for meta-
bolic studies: lipoprotein, cholesterol, and bile acids.
FASEB J 4, 161–168.
29 Gupta D, Varma S & Khandelwal RL (2007) Long-
term effects of tumor necrosis factor-alpha treatment on
insulin signaling pathway in HepG2 cells and HepG2
cells overexpressing constitutively active Akt ⁄ PKB.
J Cell Biochem 100, 593–607.
30 Gupta D & Khandelwal RL (2004) Modulation of insu-
lin effects on phosphorylation of protein kinase B and

glycogen synthesis by tumor necrosis factor a in HepG2
cells. Biochem Biophys Acta 1671, 51–58.
31 Zhang L, Rubins NE, Ahima RS, Greenbaum LE &
Kaestner KH (2005) Foxa2 integrates the transcrip-
tional response of the hepatocyte to fasting. Cell Metab
2, 141–148.
32 Alikhani M, Alikhani Z & Graves DT (2005) Foxo1
functions as a master switch that regulates gene expres-
sion necessary for tumor necrosis factor-induced fibro-
blast apoptosis. J Biol Chem 280, 12096–12102.
33 Ito Y, Daitoku H & Fukamizu A (2009) Foxo1
increases pro-inflammatory gene expression by inducing
C ⁄ EBPbeta in TNF-alpha-treated adipocytes. Biochem
Biophys Res Commun 9, 290–295.
34 Rui L, Aguirre V, Kim JK, Shulman GI, Lee A, Cor-
bould A, Dunaif A & White MF (2001) Insulin ⁄ IGF-1
and TNF-alpha stimulate phosphorylation of IRS-1 at
inhibitory Ser307 via distinct pathways. J Clin Invest
107, 181–189.
35 de Alavaro C, Teruel T, Hernandez R & Lorenzo M
(2004) Tumor necrosis factor alpha produces insulin
resistance in skeletal muscle by activation of inhibitor
kappaB kinase in a p38 MAPK-dependent manner.
J Biol Chem 279, 17070–17078.
36 Meyer C, Stumvoll M, Nadkarni V, Dostou J, Mit-
rakou A & Gerich J (1998) Abnormal renal and hepatic
glucose metabolism in type 2 diabetes mellitus. J Clin
Invest 102, 619–624.
37 Roden M, Petersen KF & Shulman GI (2001) Nuclear
magnetic resonance studies of hepatic glucose metabo-

lism in humans. Recent Prog Horm Res 56, 219–237.
38 Nielsen MF, Basu R, Wise S, Caumo A, Cobelli C &
Rizza RA (1998) Normal glucose-induced suppression
of glucose production but impaired stimulation of
glucose disposal in type 2 diabetes: evidence for a con-
centration-dependent defect in uptake. Diabetes 47,
1735–1747.
39 Clore JN, Helm ST & Blackard WG (1995) Loss of
hepatic autoregulation after carbohydrate overfeeding
in normal man. J Clin Invest 96, 1967–1972.
A. K. Pandey et al. Effect of TNFa on insulin action on PEPCK expression
FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS 3767
40 Niswender KD, Shiota M, Postic C, Cherrington AD &
Magnuson MA (1997) Effects of increased glucokinase
gene copy number on glucose homeostasis and hepatic
glucose metabolism. J Biol Chem 272, 22570–22575.
41 Sun Y, Liu S, Ferguson S, Wang L, Klepcyk P, Yun JS
& Friedman JE (2002) Phosphoenolpyruvate carboxy-
kinase overexpression selectively attenuates insulin sig-
naling and hepatic insulin sensitivity in transgenic mice.
J Biol Chem 277, 23301–23307.
42 Hughes DE, Stolz DB, Yu S, Tan Y, Reddy JK,
Watkins SC, Diehl AM & Costa RH (2003) Elevated
hepatocyte levels of the Forkhead box A2 (HNF-3beta)
transcription factor cause postnatal steatosis and mito-
chondrial damage. Hepatology 37, 1414–1424.
43 Chakravarty K, Wu SY, Chiang CM, Samols D &
Hanson RW (2004) SREBP-1c and Sp1 interact to regu-
late transcription of the gene for phosphoenolpyruvate
carboxykinase (GTP) in the liver. J Biol Chem 279,

15385–15395.
44 Kaestner KH, Katz J, Liu Y, Drucker DJ & Schutz G
(1999) Inactivation of the winged helix transcription
factor HNF3alpha affects glucose homeostasis and
islet glucagon gene expression in vivo. Genes Dev 13,
495–504.
45 Rausa FM, Tan Y, Zhou H, Yoo KW, Stolz DB, Wat-
kins SC, Franks RR, Unterman TG & Costa RH
(2000) Elevated levels of hepatocyte nuclear factor 3bS
in mouse hepatocytes influence expression of genes
involved in bile acid and glucose homeostasis. Mol Cell
Biol 20, 8264–8282.
46 Sund NJ, Ang SL, Sackett SD, Shen W, Daigle N,
Magnuson MA & Kaestner KH (2000) Hepatocyte
nuclear factor 3beta (Foxa2) is dispensable for main-
taining the differentiated state of the adult hepatocyte.
Mol Cell Biol 20, 5175–5183.
47 Vallet V, Antoine B, Chafey P, Vandewalle A & Kahn A
(1995) Overproduction of a truncated hepatocyte nuclear
factor 3 protein inhibits expression of liver-specific genes
in hepatoma cells. Mol Cell Biol 15, 5453–5460.
48 Steneberg P, Rubins N, Bartoov-Shifman R, Walker
MD & Edlund H (2005) The FFA receptor GPR40
links hyperinsulinemia, hepatic steatosis, and impaired
glucose homeostasis in mouse. Cell Metab 1, 245–258.
49 Wang JC, Stromstedt PE, O’Brien RM & Granner DK
(1996) Hepatic nuclear factor 3 is an accessory factor
required for the stimulation of phosphoenolpyruvate
carboxykinase gene transcription by glucocorticoids.
Mol Endocrinol 10, 794–800.

50 Wang JC, Stromstedt PE, Sugiyama T & Granner DK
(1999) The phosphoenolpyruvate carboxykinase gene
glucocorticoid response unit: identification of the func-
tional domains of accessory factors HNF3 beta (hepatic
nuclear factor-3 beta) and HNF4 and the necessity of
proper alignment of their cognate binding sites. Mol
Endocrinol 13, 604–618.
51 Hall RK, Wang XL, George L, Koch SR & Granner
DK (2007) Insulin represses phosphoenolpyruvate
carboxykinase gene transcription by causing the rapid
disruption of an active transcription complex: a poten-
tial epigenetic effect. Mol Endocrinol 21, 550–563.
52 Hill MR & McCallum RE (1992) Identification of
tumor necrosis factor a as a transcriptional regulator of
phosphoenolpyruvate carboxykinase gene following
endotoxin treatment in mice. Infect Immun 60, 4040–
4050.
53 Chang CK, Gatan M & Schumer W (1996) Efficacy of
anti-tumor necrosis factor polyclonal antibody on phos-
phoenolpyruvate carboxykinase expression in septic and
endotoxemic rats. Shock 6, 57–60.
54 Yan J, Gao Z, Yu G, He Q, Weng J & Ye J (2007)
Nuclear corepressor is required for inhibition of phos-
phoenolpyruvate carboxykinase expression by tumor
necrosis factor-alpha. Mol Endocrinol 21, 1630–1641.
55 Valera A, Pujol A, Pelegrin M & Bosch F (1994) Trans-
genic mice overexpressing phosphoenolpyruvate carb-
oxykinase develop non-insulin-dependent diabetes
mellitus. Proc Natl Acad Sci USA 91, 9151–9154.
56 Franckhauser S, Munoz S, Pujol A, Casellas A, Riu E,

Otaegui P, Su B & Bosch F (2002) Increased fatty acid
re-esterification by PEPCK overexpression in adipose
tissue leads to obesity without insulin resistance. Diabe-
tes 51, 624–630.
57 Franckhauser S, Munoz S, Elias I, Ferre T & Bosch F
(2006) Adipose overexpression of phosphoenolpyruvate
carboxykinase leads to high susceptibility to diet-
induced insulin resistance and obesity. Diabetes 55,
273–280.
58 Ling PR, Bistrian BR, Mendez B & Istfan NW (1994)
Effects of systemic infusions of endotoxin, tumor necro-
sis factor, and interleukin-1 on glucose metabolism in
the rat: relationship to endogenous glucose production
and peripheral tissue glucose uptake. Metabolism 43,
279–284.
59 Lang CH, Dobrescu C & Bagby GJ (1992) Tumor
necrosis factor impairs insulin action on peripheral glu-
cose disposal and hepatic glucose output. Endocrinology
130, 43–52.
60 Arkan MC, Hevener AL, Greten FR, Maeda S, Li ZW,
Long JM, Wynshaw-Boris A, Poli G, Olefsky J &
Karin M (2005) IKK-beta links inflammation to obes-
ity-induced insulin resistance. Nat Med 11, 191–198.
61 Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L,
Lee J & Shoelson SE (2005) Local and systemic insulin
resistance resulting from hepatic activation of IKK-beta
and NF-kappaB. Nat Med 11, 183–190.
62 Bae EJ, Yang YM, Kim JW & Kim SG (2007) Identifi-
cation of a novel class of dithiolethiones that prevent
hepatic insulin resistance via the adenosine monophos-

phate-activated protein kinase-p70 ribosomal S6
kinase-1 pathway. Hepatology 46, 730–739.
Effect of TNFa on insulin action on PEPCK expression A. K. Pandey et al.
3768 FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS
63 Choi JH, Park MJ, Kim KW, Choi YH, Park SH,
An WG, Yang US & Cheong J (2005) Molecular
mechanism of hypoxia-mediated hepatic
gluconeogenesis by transcriptional regulation. FEBS
Lett 579, 2795–2801.
64 Lam NT, Lewis JT, Cheung AT, Luk CT, Tse J,
Wang J, Bryer-Ash M, Kolls JK & Kieffer TJ (2004)
Leptin increases hepatic insulin sensitivity and protein
tyrosine phosphatase 1B expression. Mol Endocrinol 18,
1333–1345.
65 Senn JJ, Klover PJ, Nowak IA & Mooney RA (2002)
Interleukin-6 induces cellular insulin resistance in
hepatocytes. Diabetes 51, 3391–3399.
66 Buser AC, Gass-Handel EK, Wyszomierski SL, Dopp-
ler W, Leonhardt SA, Schaack J, Rosen JM,
Watkin H, Anderson SM & Edwards DP (2007)
Progesterone receptor repression of prolactin ⁄ signal
transducer and activator of transcription 5-mediated
transcription of the beta-casein gene in mammary
epithelial cells. Mol Endocrinol 21, 106–125.
67 Gabbay A, Sutherland C, Gnudi L, Kahn BB, O’Brien
RM, Granner DK & Flier JS (1996) Insulin regulation
of phosphoenolpyruvate carboxykinase gene expression
does not require activation of the Ras ⁄ mitogen-acti-
vated protein kinase signaling pathway. J Biol Chem
271, 1890–1897.

A. K. Pandey et al. Effect of TNFa on insulin action on PEPCK expression
FEBS Journal 276 (2009) 3757–3769 ª 2009 The Authors Journal compilation ª 2009 FEBS 3769