Regulation of Hepatic Gluconeogenesis by an ER-Bound Transcription Factor, CREBH

Article · April 2010with85 Reads
DOI: 10.1016/j.cmet.2010.02.016 · Source: PubMed
Abstract
Endoplasmic reticulum (ER)-bound transcription factor families are shown to be involved in the control of various metabolic pathways. Here, we report a critical function of ER-bound transcription factor, CREBH, in the regulation of hepatic gluconeogenesis. Expression of CREBH is markedly induced by fasting or in the insulin-resistant state in rodents in a dexamethasone- and PGC-1alpha-dependent manner, which results in the accumulation of active nuclear form of CREBH (CREBH-N). Overexpression of constitutively active CREBH activates transcription of PEPCK-C or G6Pase by binding to its enhancer site that is distinct from the well-characterized CREB/CRTC2 regulatory sequences in vivo. Of interest, knockdown of CREBH in liver significantly reduces blood glucose levels without altering expression of genes involved in the ER stress signaling cascades in mice. These data suggest a crucial role for CREBH in the regulation of hepatic glucose metabolism in mammals.
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Cell Metabolism
Short Article
Regulation of Hepatic Gluconeogenesis
by an ER-Bound Transcription Factor, CREBH
Min-Woo Lee,
1,9
Dipanjan Chanda,
2,9
Jianqi Yang,
4
Hyunhee Oh,
5
Su Sung Kim,
5
Young-Sil Yoon,
1
Sungpyo Hong,
1
Keun-Gyu Park,
8
In-Kyu Lee,
7
Cheol Soo Choi,
5,6
Richard W. Hanson,
4
Hueng-Sik Choi,
2,3,
*and Seung-Hoi Koo
1,
*
1
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, 300 Chunchun-dong, Jangan-gu, Suwon,
Gyeonggi-do 440-746, Korea
2
Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Korea
3
Research Institute of Medical Sciences, Department of Biomedical Sciences, Chonnam National University Medical School,
Gwangju 501-746, Korea
4
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4935, USA
5
Lee Gil Ya Cancer and Diabetes Institute
6
Division of Endocrinology
Gil Medical Center, Gachon University of Medicine and Science, Incheon 405-760, Korea
7
Departments of Internal Medicine and Biochemistry and World Class University Program, Research Institute of Aging and Metabolism,
Kyungpook National University School of Medicine, Daegu 700-422, Korea
8
Department of Internal Medicine, Keimyung University School of Medicine, Daegu, Korea
9
These authors contributed equally to this work
*Correspondence: hsc@chonnam.ac.kr (H.-S.C.), shkoo@med.skku.ac.kr (S.-H.K.)
DOI 10.1016/j.cmet.2010.02.016
SUMMARY
Endoplasmic reticulum (ER)-bound transcription fac-
tor families are shown to be involved in the control
of various metabolic pathways. Here, we report a
critical function of ER-bound transcription factor,
CREBH, in the regulation of hepatic gluconeogen-
esis. Expression of CREBH is markedly induced by
fasting or in the insulin-resistant state in rodents in
a dexamethasone- and PGC-1a-dependent manner,
which results in the accumulation of active nuclear
form of CREBH (CREBH-N). Overexpression of con-
stitutively active CREBH activates transcription of
PEPCK-C or G6Pase by binding to its enhancer site
that is distinct from the well-characterized CREB/
CRTC2 regulatory sequences in vivo. Of interest,
knockdown of CREBH in liver significantly reduces
blood glucose levels without altering expression of
genes involved in the ER stress signaling cascades
in mice. These data suggest a crucial role for CREBH
in the regulation of hepatic glucose metabolism in
mammals.
INTRODUCTION
Glucose homeostasis is tightly regulated to meet the fuel
requirement in mammals. Under fasting, secretion of pancreatic
hormone glucagon and adrenal hormone glucocorticoid is
induced to enhance hepatic glucose production (Pilkis et al.,
1988a, 1988b). This process is accomplished, in part, via activa-
tion of gluconeogenesis, resulting from the transcriptional acti-
vation of gluconeogenic genes such as cytosolic isoform of
phosphoenolpyruvate carboxykinase (PEPCK-C) or glucose 6
phosphatase (G6Pase)(Hall and Granner, 1999; Hanson and
Reshef, 1997). Activation of cAMP-dependent transcriptional
program is mainly mediated by CREB/CREB-regulated tran-
scriptional coactivator 2 (CRTC2)-dependent transcriptional
machinery, whereas glucocorticoid signal is conveyed via the
action of glucocorticoid receptor (GR), a member of nuclear hor-
mone receptor (NR) superfamilies (Herzig et al., 2001; Koo et al.,
2005; van Schaftingen and Gerin, 2002). Indeed, both cAMP
response element (CRE) and GR response element (GRE) have
been found in gluconeogenic gene promoters, underscoring
the importance of these transcriptional machineries in this path-
way (Hanson and Reshef, 1997; van Schaftingen and Gerin,
2002). As demonstrated in Cushing’s syndrome, excessive glu-
cocorticoid could promote insulin resistance by opposing the
action of insulin in peripheral tissues, including liver, suggesting
that the increased GR activity might be responsible for the pro-
gression of type II diabetes (Andrews and Walker, 1999; Ross and
Linch, 1982; Zinker et al., 2007). Of interest, both GR and CREB/
CRTC2 were shown to induce the NR coactivator peroxisome
proliferator-activated receptor gcoactivator 1 alpha (PGC-1a),
which regulates activities of various transcription factors, includ-
ing GR, hepatic nuclear factor 4 (HNF4), or FOXO1a (Herzig et al.,
2001; Koo et al., 2005; Puigserver et al., 2003; Yoon et al., 2001).
Elevation of PGC-1aexpression is displayed in mouse models of
type II diabetes, and its liver-specific ablation markedly affects
glycemic profiles in mice, showing the importance of this factor
in the regulation of hepatic glucose metabolism (Herzig et al.,
2001; Koo et al., 2004; Leone et al., 2005; Lin et al., 2004; Puig-
server et al., 2003).
Recently, regulated intramembrane proteolysis (RIP) has
emerged as a new mechanism to influence energy metabolism,
differentiation, and endoplasmic reticulum (ER) stress response/
unfolded protein response (UPR) (Brou et al., 2000; Brown et al.,
2000; Haze et al., 1999). Members of the ER membrane-bound
basic leucine zipper (bZIP) transcription factor family, such as
ATF6, Luman, and OASIS, constitute a novel class of factors
that are regulated by ER stress-dependent mechanisms (Haze
et al., 1999; Kondo et al., 2005; Raggo et al., 2002). ATF6,
Cell Metabolism 11, 331–339, April 7, 2010 ª2010 Elsevier Inc. 331
a founding member of this family, is an ER resident transcription
factor and is activated following UPR-dependent translocation
to Golgi, where the proteolytic cleavage of this factor releases
its N-terminal transcription factor moiety into the nucleus.
Activated ATF6 is responsible for UPR-mediated activation of
target genes such as GRP78/Bip,CHOP, and XBP-1 (Chen
et al., 2002; Shen et al., 2002; Ye et al., 2000).
cAMP response element-binding protein H (CREBH), a liver-
specific bZIP transcription factor that belongs to this family, is
shown to be regulated by UPR-dependent proteolytic cleavage
(Chin et al., 2005; Omori et al., 2001) and regulates the transcrip-
tional process of genes such as serum amyloid P-component
(SAP) and C-reactive protein (CRP) in response to systemic
inflammatory signals in liver (Zhang et al., 2006). Of interest,
CREBH was also shown to transcriptionally activate PEPCK-C
promoter, suggesting a potential link between this factor and
the hepatic glucose metabolism (Chin et al., 2005). In this study,
we show that CREBH expression is induced during fasting
or insulin-resistant state, which, in turn, results in the accumula-
tion of the active nuclear form of CREBH (CREBH-N). Nuclear
CREBH enhances hepatic gluconeogenesis by activating
transcription of PEPCK-C or G6Pase via a unique regulatory
sequence in a CRTC2-dependent manner. Furthermore, acute
depletion of hepatic CREBH results in the reduction of blood
glucose levels both in wild-type and diabetic mice. These data
support that CREBH is an important physiological regulator of
hepatic gluconeogenesis.
RESULTS
CREBH Expression Is Induced during Fasting
and by Insulin Resistance
Previously, nuclear CREBH (CREBH-N) was shown to enhance
PEPCK-C promoter activity in hepatic cells without further delin-
eation in its physiological relevance in gluconeogenesis (Chin
et al., 2005). To assess the potential involvement of CREBH in
hepatic gluconeogenesis, we measured its expression levels in
mouse liver. Of interest, CREBH expression was significantly
induced during fasting conditions and was reduced upon
refeeding, a characteristic regulatory pattern known for genes
in the gluconeogenesis (Figure 1A). Furthermore, mRNA levels
for hepatic CREBH were also induced in mouse models of diet-
induced or genetic insulin resistance (Figure 1B and Figure S1A
available online), showing a strong correlation between CREBH
expression and gluconeogenic potential in liver. Indeed, we
observed increased appearance of both full-length and nuclear
CREBH under fasting or by insulin resistance (1.9-fold [FL] or
4.9-fold [N] induction under fasting over refeeding, 6.3-fold [FL]
or 1.8-fold [N] induction in db/db over WT mice; Figures 1A
and 1B), suggesting that CREBH could be involved in the tran-
scriptional process of hepatic gluconeogenesis.
To identify a mechanism for CREBH regulation, we treated
hepatocytes with stimuli known to mimic fasting signals. Treat-
ment of cells with dexamethasone, but not with cAMP agonist
forskolin, enhanced CREBH expression significantly (Figure 1C
and data not shown), suggesting an involvement of GR in the
process. Indeed, transfection analysis, as well as chromatin
immunoprecipitation (ChIP) assay, revealed a functional involve-
ment of GR in the regulation of CREBH transcription (Figures
S1B and S1C). The involvement of GR in the regulation of
CREBH was further supported by recent microarray analysis
showing that GR knockout mice displayed reduced dexametha-
sone-dependent expression of CREBH (ArrayExpress: http://
www.ebi.ac.uk; ID: E-MEXP-1816). PGC-1afunctions as a tran-
scriptional coactivator for NRs (Rhee et al., 2003; Yoon et al.,
2001). We thus tested whether PGC-1awas also involved in
the transcriptional regulation of hepatic CREBH. Expression of
PGC-1asignificantly enhanced expression of CREBH in hepato-
cytes (Figure 1D). Conversely, knockdown of PGC-1ain the
mouse liver resulted in reduction of mRNA and protein levels
for CREBH (Figure 1E). Dexamethasone-dependent activation
of CREBH promoter activity was induced synergistically with
cotransfection of PGC-1aexpression vector; the effect was
blunted by coexpression of either constitutively active AKT or
small heterodimer partner (SHP), a known inhibitor for PGC-
1a-NR interaction (Chanda et al., 2008)(Figures S1D–S1F).
These data suggest the presence of a mechanism for PGC-1a/
GR-dependent activation of CREBH that may contribute to the
regulation of hepatic glucose metabolism.
CREBH-N Promotes Hepatic Gluconeogenesis
To test the functional significance of CREBH expression, we
prepared adenovirus for nuclear CREBH (Ad CREBH-N). Trans-
duction of hepatocytes with Ad CREBH-N increased mRNA
levels for gluconeogenic genes (Figure 2A). In addition, hepatic
glucose production was significantly induced by CREBH-N
expression in hepatocytes, suggesting that CREBH-N indeed
promotes hepatic gluconeogenesis (Figure 2B). To verify the
effect of CREBH-N on glucose homeostasis in vivo, we injected
Ad CREBH-N or Ad GFP control virus into the wild-type mice. Ad
CREBH-N infection led to elevations in hepatic mRNA levels of
PEPCK-C and G6Pase (Figures 2C and 2D). Furthermore, blood
glucose levels were significantly higher in mice with Ad CREBH-
N compared with those in control mice (Figure 2E), showing that
acute overexpression of CREBH promoted hepatic gluconeo-
genic gene expression in vivo, without altering body weight
(Figure S2A). The insulin levels were generally higher in mice
with Ad CREBH-N without reaching the statistical significance
(Figure S2B). No changes were shown for the phosphorylation
status of CRTC2, excluding a potential indirect regulation of glu-
coneogenic genes by CREBH-N (Figure S2B). CREBH was
shown to regulate genes involved in the acute phase response
(APR) such as CRP and SAP (Zhang et al., 2006). We thus
measured the mRNA levels for such genes in livers with Ad
CREBH-N. Though CRP expression was slightly elevated in
animals infected with Ad-CREBH-N, we were not able to observe
any changes in SAP expression (Figure 2F). Furthermore, no
detectable differences were noted in GRP78 or ATF6 expression
between two groups of mice. We therefore speculated that,
whereas expression of CREBH alone was sufficient to induce
expression of gluconeogenic program, additional components
such as ATF6 might be required for the complete activation of
hepatic genes in the APR.
CREBH Activates Transcription of Gluconeogenic
Genes via a CRTC2-Dependent Manner
Next, we wanted to delineate the mechanism for CREBH-medi-
ated transcriptional process. A previous study suggested that
Cell Metabolism
Role of CREBH in Hepatic Gluconeogenesis in Mammals
332 Cell Metabolism 11, 331–339, April 7, 2010 ª2010 Elsevier Inc.
Figure 1. Induction of CREBH Expression during Fasting or by Insulin Resistance
(A) (Top) Q-PCR analysis showing hepatic CREBH or gluconeogenic gene expression in mice less under 16 hr fasted or 16 hr fasted/4 hr refed conditions
(**p < 0.01, t test; n = 5). (Bottom) Western blot analysis showing hepatic CREBH protein levels in mice as indicated above (FL, full-length; N, nuclear).
(B) (Top) Q-PCR analysis showing hepatic CREBH or gluconeogenic gene expression in either wild-type or db/db mice under 16 hr fasted condition (**p < 0.01,
*p < 0.05, t test; n = 3). (Bottom) Western blot analysis showing hepatic CREBH protein levels in either wild-type or db/db mice as indicated above.
(C) Effects of dexamethasone on CREBH expression. Northern blot (top) or Q-PCR (middle) analysis of G6Pase,PEPCK-C, and CREBH expression using rat
primary hepatocytes treated with 100 nM dexamethasone for an indicated time period (*p < 0.05 compared to 0 hr, t test; n = 3). (Bottom) Western blot analysis
showing hepatic CREBH-N protein levels by dexamethasone treatment.
(D)Effects of Ad-PGC-1aon CREBHexpression.(Top)Q-PCR analysisof G6Pase,PEPCK-C,andCREBHexpressionusing RNAsfrom rat primaryhepatocytes infected
witheither Ad-GFPor Ad-PGC-1a(**p< 0.01,*p < 0.05,t test; n = 3). (Bottom) Westernblot analysisshowinghepatic CREBH-Nprotein levelsby Ad-GFP or Ad-PGC-1a.
(E) Effects of PGC-1aknockdownon CREBH expression. (Top)Q-PCR analysis of G6Pase,PEPCK-C,andCREBH expressionusing RNAs from livers of mice infected
with either Ad-USor Ad-PGC-1aRNAi (**p < 0.01,*p < 0.05,t test; n = 5). (Bottom) Western blot analysis showing hepatic CREBH-Nprotein levelsby Ad-US or Ad-PGC-
1aRNAi.
Data represent the mean ± SD.
Cell Metabolism
Role of CREBH in Hepatic Gluconeogenesis in Mammals
Cell Metabolism 11, 331–339, April 7, 2010 ª2010 Elsevier Inc. 333
CREBH could enhance PEPCK-C promoter activity by binding
to the well-characterized CRE at 125 from the transcriptional
start site (Chin et al., 2005). Contrary to the previous report, our
mapping studies revealed a unique putative CREBH response
element (CREBHRE) in the promoters of PEPCK-C and G6Pase
that are distinct from CRE (452 versus 125 for PEPCK-C,
91 versus 161/136 for G6Pase)(Figure 3A); the occupancy
of CREBH over putative CREBHRE on each promoter was
Figure 2. CREBH Promotes Hepatic Gluconeogenesis
(A) Effects of CREBH on gluconeogenic gene expression. Northern blot (top, left) or Q-PCR (bottom) analysis of G6Pase,PEPCK-C, and CR EBH expression using
RNAs from rat primary hepatocytes infected with either Ad-GFP or Ad-CREBH-N (*p < 0.05, t test; n = 3). A representative western blot analysis showing hepatic
nuclear CREBH (CREBH-N) protein levels was also shown (top, right).
(B) (Top) Glucose output assay showing effects of CREBH on glucose production in rat primary hepatocytes. Forskolin (10 mM) was used as a positive control.
(Bottom) Western blot assay shows the expression level of Flag-tagged CREBH-N (*p < 0.05, t test; n = 3).
(C and D) Effects of Ad-CREBH-N infection on hepatic gene expression. Q-PCR analysis and western blot analysis of CREBH (C) and Q-PCR analysis of gluco-
neogenic genes (D) from mouse liver infected with either Ad-GFP or Ad-CREBH-N (**p < 0.01, t test; n = 5).
(E) Sixteen hour fasting glucose levels in mice expressing Ad-GFP or Ad-CREBH-N (*p < 0.05; t test; n = 5).
(F) Effects of Ad-CREBH-N infection on ER stress genes using RNAs from mouse liver infected with either Ad-GFP or Ad-CREBH-N (**p < 0.01, t test; n = 5).
Data represent the mean ± SD.
Cell Metabolism
Role of CREBH in Hepatic Gluconeogenesis in Mammals
334 Cell Metabolism 11, 331–339, April 7, 2010 ª2010 Elsevier Inc.
Figure 3. CREBH Regulates Gluconeogenic Genes via a CREBH Response Element
(A) Luciferase assay using HepG2 cells transiently transfected with PEPCK-C (left) or G6Pase (right) luciferase construct together with expression vector for
CREBH-N. Representative data were shown from three independent experiments.
(B) Live imaging of hepatic G6Pase-luciferase (Ad-WT G6Pase [231/+57]-Luc or Ad- CREBHRE mutant G6Pase [231/+57]-Luc) activity in response to
CREBH-N in C57BL/6 mice.
(C) (Top) Amino acid sequence comparison among mouse CREB, ATF6, and CREBH showing conservation at Arg314 in CREB, Arg324 in ATF6, and Arg270 in
CREBH. (Bottom) Coimmunoprecipitation assay showing endogenous interaction betwee n CRTC2 and CREB or CREBH in rat primary hepatocytes. A represen-
tative western blot analysis was shown.
(D) (Left) Luciferase assay using HepG2 cells transiently transfected with basal pGL4 PK or pGL4 PK 3XCREBHRE construct together with expression vector for
CREBH-N or wild-type CRTC2. Representative data were shown from three independent experiments with triplicate conditions. (Right) Q-PCR analysis of chro-
matin immunoprecipitation experiments using anti-CRTC2 antibody showing specific occupancy of CRTC2 over CRE (between 213 and 13, top) or CREBHRE
(between 549 and 339, bottom) on rat PEPCK promoter.
(E) Sixteen hour fasting glucose levels from wild-type mice (**p < 0.01, *p < 0.05, t test; n = 6–7) injected with Ad-GFP + Ad-US, Ad-CREBH-N + Ad-US, or
Ad-CREBH-N + Ad-CRTC2 RNAi.
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Role of CREBH in Hepatic Gluconeogenesis in Mammals
Cell Metabolism 11, 331–339, April 7, 2010 ª2010 Elsevier Inc. 335
confirmed by ChIP assay and electrophoretic mobility shift
assay (Figures S2C and S2D). Furthermore, CREBHRE muta-
tions significantly blunted cAMP/DEX-mediated induction of
PEPCK-C/G6Pase promoter activity, suggesting that CREBH
mediates fasting signals to induce gluconeogenic genes (Fig-
ure S2E). To confirm the transcriptional regulation of G6Pase
by CREBH in vivo, we generated reporter adenovirus bearing
either wild-type or CREBHRE mutant G6Pase promoter and
injected it into the wild-type mice. Optical in vivo imaging anal-
ysis revealed a normal fasting-dependent induction of wild-
type G6Pase promoter (Figure S3A), which was ablated in mice
with CREBHRE mutant promoter (data not shown). Coinfection
of Ad CREBH-N or Ad CREBH-FL enhanced wild-type G6Pase
promoter activity (more than 3- to 4-fold) compared with Ad
GFP control (Figure S3B). However, the stimulatory effect of
CREBH-N was largely blunted in mice with CREBHRE mutant
promoter, showing that, indeed, CREBHRE is critical in medi-
ating CREBH-dependent G6Pase transcription in vivo (Figures
3B and S3C).
Recently, ATF6, a closely related bZIP factor of CREBH, was
shown to interact with CRTC2 and inhibit CREB/CRTC2-depen-
dent transcriptional regulation (Wang et al., 2009). Sequence
comparison revealed a conservation of critical amino acids for
CRTC2 interaction among CREB, ATF6, and CREBH, prompting
us to investigate whether CREBH would interact with CRTC2
(Figure 3C, top). Indeed, Flag-tagged CRTC2 interacted with
either HA-tagged CREB or HA-tagged CREBH similarly in a
coimmunoprecipitation study (Figure S3D). Furthermore, we
observed the physical interaction between endogenous CREBH
and CRTC2 in primary hepatocytes as well as in db/db mouse
liver (Figures 3C, bottom, and S3E), which would synergistically
activate CREBHRE-dependent transcription (Figure 3D, left).
The occupancy of CRTC2 over both CREBHRE and CRE was
enhanced upon FSK treatment, showing that both sites could
be under the further control by cAMP-dependent transcriptional
mechanism (Figure 3D, right). On the other hand, a closely
related bZIP factor ATF6 did not alter CREBH-dependent
activation of G6Pase promoter activity (Figure S3F). Instead,
we observed that nuclear ATF6 repressed FSK-dependent
G6Pase promoter activity in a CRE-dependent manner, in
accordance with the recent report regarding the inhibitory role
of ATF6 on CREB/CRTC2-dependent transcription (Wang
et al., 2009).
To further confirm the dependency of CRTC2 on CREBH-
mediated induction of gluconeogenic program, we monitored
the effects of Ad CREBH-N on blood glucose levels upon
CRTC2 knockdown. As shown in Figure 2E, mice with CREBH-N
expression displayed higher glucose levels compared with
control groups. The CREBH-N-dependent elevations in blood
glucose levels were largely blunted by knockdown of CRTC2
by Ad CRTC2 RNAi coinjection, indicating that CRTC2 is
required for the CREBH-dependent process in vivo (Figures 3E
and S4A). Pyruvate challenge test demonstrated that CREBH-
dependent activation of hepatic gluconeogenesis appeared to
be impaired upon CRTC2 knockdown (Figure S4B). Indeed,
acute depletion of CRTC2 significantly reduced CREBH-depen-
dent activation of PEPCK-C and G6Pase mRNA levels in mouse
liver (Figure 3F). Similar results were obtained with experiments
performed in primary hepatocytes (Figures S4C and S4D). These
data support our hypothesis that CRTC2 could function as a
coactivator for CREBH.
Knockdown of CREBH Improves Fasting Hyperglycemia
in Diabetic Mice
To verify the physiological role of CREBH in glucose homeo-
stasis, we produced adenovirus-expressing small hairpin RNA
for CREBH (Ad CREBH RNAi). Transduction of hepatocytes
with Ad CREBH RNAi significantly reduced expression of gluco-
neogenic genes, as well as glucose production (Figure S4E).
Indeed, mice with reduced hepatic CREBH expression displayed
lower fasting blood glucose levels compared with controls in the
normal context (Figure 4A, left). Hepatic mRNA levels for gluco-
neogenic genes were reduced accordingly upon depletion of
CREBH (Figure 4B, left). We then wanted to investigate whether
acute reduction of hepatic CREBH would also affect glucose
metabolism in pathological conditions. Of interest, hepatic
CREBH knockdown significantly lowered blood glucose levels
and reduced gluconeogenic gene expression in db/db mice
(Figures 4A, right, and 4B, right). In both wild-type and db/db
mice, no significant changes in plasma insulin levels or CRTC2
phosphorylation status were observed (data not shown). Unlike
the changes in gluconeogenic genes, no significant changes
were shown for mRNA levels of known ER stress regulators
such as ATF6 or GRP78 upon CREBH knockdown (Fig-
ure S4F). Although no changes were shown in SAP expression,
mRNA levels for CRP was significantly reduced by depletion of
CREBH in mouse liver, in accordance with its increase by Ad
CREBH-N (Figure 2F for comparison) or the previous report
describing the role of this factor in the regulation of CRP tran-
scription (Zhang et al., 2006). Finally, in order to further verify
whether CREBH is involved in the hepatic glucose production
in vivo, we performed euglycemic-hyperinsulinemic clamp
studies. Indeed, hepatic glucose production was significantly
reduced upon CREBH knockdown both at the basal condition
and during the clamp period, without changes in glucose
disposal rate, showing that reduced blood glucose levels by
CREBH knockdown were largely due to the decreased glucose
production from liver in vivo (Figure 4C).
DISCUSSION
Previously, CREBH was shown to be activated by APR via
enhanced expression and increased proteolytic processing to
produce active nuclear moiety (CREBH-N) (Zhang et al., 2006).
Following its activation, CREBH works in conjunction with
ATF6, another ER resident bZIP factor that is regulated similarly,
to induce transcription of genes in the APR pathway such as SAP,
CRP,orAPOB. In our hands, acute knockdown or overexpres-
sion of CREBH in liver only affected expression of CRP, but not
of SAP or APOB (Figures 2F and S4F and data not shown). The
(F) Q-PCR analysis showing effect of adenoviruses as in (E) on hepatic expression of gluconeogenic genes in wild-type mice fasted for 16 hr (**p < 0.01, t test;
n = 5–7).
Data in (A) and (D–F) represent the mean ± SD.
Cell Metabolism
Role of CREBH in Hepatic Gluconeogenesis in Mammals
336 Cell Metabolism 11, 331–339, April 7, 2010 ª2010 Elsevier Inc.
discrepancy might stem from the fact that we utilized a transient
system, whereas Zhang et al. employed chronic CREBH-knock-
down animals. Alternatively, regulation of APR genes by CREBH
requires additional factors such as ATF6 that are also induced by
proinflammatory signals. Because we studied the role of CREBH
in more physiological settings, we may not be able to recapitulate
Figure 4. Knockdown of CREBH Relieves Hyperglycemia
(A) Sixteen hour fasting glucose levels from wild-type mice (left; **p < 0.01, t test; n = 5) or db/db mice (right; *p < 0.05, t test; n = 5) injected with either Ad-US or
Ad-CREBH RNAi.
(B) Q-PCR analysis showing effect of Ad-US or Ad-CREBH RNAi infection on hepatic expression of gluconeogenic genes in wild-type mice (top-left; **p < 0.01,
*p < 0.05, t test; n = 5) or db/db mice (top-right; **p < 0.01, t test; n = 5) fasted for 16 hr. A representative western blot analysis of CREBH-FL and CREBH-N protein
levels from mouse liver infected with either Ad-US or Ad-CREBH RNAi (bottom-left, wild-type mice; bottom-right, db/db mice).
(C) Peripheral and hepatic insulin sensitivity were assessed by means of hyperinsulinemic-euglycemic clamps. From left to right, basal hepatic glucose produc-
tion, clamp hepatic glucose production, whole-body glucose infusion rate, and percent inhibition of insulin-dependent hepatic glucose production are shown
(*p < 0.05; n = 5–7).
(D) A proposed model for the role of fasting-dependent activation of CREBH in hepatic gluconeogenesis.
Data in (A–C) represent the mean ± SD.
Cell Metabolism
Role of CREBH in Hepatic Gluconeogenesis in Mammals
Cell Metabolism 11, 331–339, April 7, 2010 ª2010 Elsevier Inc. 337
such conditions that would fully activate CREBH-containing
complex required for the induction of APR pathway.
We noticed that hepatic CREBH was also induced during fast-
ing via a cortisol-dependent mechanism at the transcriptional
level. It will be of great interest to verify whether there is an
involvement of a yet-to-be defined mechanism to activate
proteolytic cleavage of CREBH in response to fasting. We did
not observe changes in blood glucose levels upon CREBH-N
expression or CREBH knockdown during feeding conditions,
underscoring the potential importance of CREBH in the regula-
tion of fasting metabolism in vivo (data not shown). We are
currently investigating whether specific fasting signals such as
cAMP or glucocorticoid are involved in the regulated processing
of CREBH in liver.
In summary, our data provide an alternative fasting-mediated
transcriptional route to modulate hepatic gluconeogenesis (Fig-
ure 4D). PGC-1a, a major regulator of hepatic glucose metabo-
lism (Herzig et al., 2001; Yoon et al., 2001), was also involved
in the regulation of CREBH by inducing its expression. Unlike
the previous report (Chin et al., 2005), we identified that CREBH
utilized a unique CREBHRE in the promoter of PEPCK-C or
G6Pase to transcriptionally modulate hepatic gluconeogenesis
in a CRTC2-dependent manner (Figures S3A–S3F). It will be
interesting to delineate the relative contribution between pre-
existing mechanisms and the new transcriptional machinery
proposed in this report to hepatic glucose metabolism. Regula-
tion of hepatic glucose production is an important therapeutic
strategy to alleviate hyperglycemia in type II diabetes. Identifica-
tion of CREBH as a critical regulator for hepatic gluconeogenesis
would expand our knowledge to understand the intricate mech-
anisms for proper glycemic control and help to develop a poten-
tial treatment for such disease.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures
and four figures and can be found with this article online at doi:10.1016/
j.cmet.2010.02.016.
ACKNOWLEDGMENTS
We would like to thank Sun Myung Park and Yo-Na Kim for technical assis-
tance. We would also like to thank Dr. Seok-Yong Choi for critical reading.
This work was supported by a grant of the Korea Healthcare technology
R&D Project, Ministry for Health, Welfare, and Family Affairs, Republic of Korea
(A080150) (S.-H.K.) and by the NRF through the National Research Laboratory
program (NRL-ROA-2005-000-10047-0) (H.-S.C.).
Received: June 29, 2009
Revised: December 1, 2009
Accepted: February 26, 2010
Published: April 6, 2010
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    • CREB-H has now been shown to play a key role in lipid and triglyceride metabolism (Lee et al., 2010a;Lee et al., 2011;Lee, 2012;Zhang et al., 2012;Barbosa et al., 2013;Xu et al., 2014). Several distinct physiological stimuli including fasting, high-fat diet, saturated fatty acids and insulin have been reported to increase transcription of CREB-H and promote its activation.
    [Show abstract] [Hide abstract] ABSTRACT: CREB-H, an ER-anchored transcription factor plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases releasing the N-terminal product which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif, termed the P-motif located in the N-terminus between serines 73 to 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation promoting ER-retention while mutation of target serines or drug inhibition of GSK-3 activity, co-ordinately induces both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations which regulate stability by targeting the protein to the SCF(Fbw1a) E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilisation of the active form in the nucleus.
    Full-text · Article · Apr 2017
    • However, depending on which UPR pathway is activated, gluconeogenesis could be enhanced or compromised. For example, activation of CREBH, an ATF6 homolog upon ER stress primarily in the liver increases the expression of the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6 phosphatase (G6pase), along with inflammatory markers such as C-reactive protein [31]. By contrast, generating XBP1s by ER stress-mediated IRE1 pathway induces ubiquitin-mediated proteasomal degradation of forkhead box O1 (FoxO1), resulting in reduced glu- coneogenesis [32].
    [Show abstract] [Hide abstract] ABSTRACT: Chronic endoplasmic reticulum (ER) stress culminating in proteotoxicity contributes to the development of insulin resistance and progression to type 2 diabetes mellitus. Pharmacologic interventions targeting several different nuclear receptors have emerged as potential treatments for insulin resistance. The mechanistic basis for these antidiabetic effects has primarily been attributed to multiple metabolic and inflammatory functions. Here we review recent advances in our understanding of the association of ER stress with insulin resistance and the role of nuclear receptors in promoting ER stress resolution and improving insulin resistance in the liver.
    Full-text · Article · Feb 2017
    • Another ER-bound bZIP factor CREBH was also shown to interact with CRTC2, using it as a transcriptional coactivator in the liver (Lee et al., 2010b). As a member of CREB3 subfamily of bZIP proteins, CREBH is mainly expressed in the liver, and its expression and activity is enhanced under fasting or by insulin resistance.
    Full-text · Article · Oct 2016 · PLoS ONE
    • We transfected isogenic constructs expressing full-length versions of the wt and mutants and analyzed relative levels of the cleaved products (Figure 9c). Consistent with our previous results (Bailey et al., 2007; Bailey and O'Hare, 2007; Llarena et al., 2010) and those of other laboratories (Danno et al., 2010; Lee et al., 2010; Xu et al., 2014), the full-length wt protein produces some but minor amounts of the cleaved product, again almost all of which is in the N 1 form (Figure 9c, lane 1). Each of the mutants 3S+DSG, DSG, and FIGURE 8: The GSK-3 inhibitor CHIR99021 increases the abundance of CREB-HΔTMC.
    [Show abstract] [Hide abstract] ABSTRACT: CREB‑H, an ER-anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identify a motif around S87 to S90 with homology to DSG type phosphodegrons. We show that this region is subject to multiple phosphorylations which regulate CREB-H stability by targeting it to the SCF(Fbw1a) E3 ubiquitin ligase. Data from phosphatase treatment, use of phosopho-specific antibody and substitution of serine residues, demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant negative Cul1 enhances steady state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally mutations within the phosphodegron when incorporated into the full length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Jun 2015
    • The data also allowed us to conclude that during fasting, only a small quantity of pyruvate is directed toward the generation of GLY-TAG (1/20 to glucose) in the liver of both the C and LPHC animals. Other studies (Cassuto et al. 2005; Hall et al. 2007; Lee et al. 2010) have indicated that corticosterone, a glucocorticoid hormone found in high levels in the blood of LPHC animals (Dos Santos et al. 2012), inhibits the transcription of the PEPCK gene in adipose tissue but stimulates it in the liver and renal cortex (Meisner et al. Fig. 4. Protein levels of PEPCK and GyK in the livers of fasted rats (15 h) previously fed a control diet (C) or a low-protein, high-carbohydrate diet (LPHC) for 15 days.
    [Show abstract] [Hide abstract] ABSTRACT: The our objective was to investigate the adaptations induced by a low-protein, high-carbohydrate (LPHC) diet in growing rats, which by comparison with the rats fed a control (C) diet at displayed lower fasting glycemia and similar fasting insulinemia, despite impairment in insulin signaling in adipose tissues. In the insulin tolerance test the LPHC rats showed higher rates of glucose disappearance (30%) and higher tolerance to overload of glucose than C rats. The glucose uptake by the soleus muscle, evaluated in vivo by administration of 2-deoxy-[(14)C]glucose, increased by 81%. The phosphoenolpyruvate carboxykinase content and the incorporation of [1-(14)C]pyruvate into glucose was also higher in the slices of liver from the LPHC rats than in those from C rats. The LPHC rats showed increases in l-lactate as well as in other gluconeogenic precursors in the blood. These rats also had a higher hepatic production of glucose, evaluated by in situ perfusion. The data obtained indicate that the main substrates for gluconeogenesis in the LPHC rats are l-lactate and glycerol. Thus, we concluded that the fasting glycemia in the LPHC animals was maintained mainly by increases in the hepatic gluconeogenesis from glycerol and l-lactate, compensating, at least in part, for the higher glucose uptake by the tissues.
    Full-text · Article · Apr 2014
    • In addition, CREBH is a master regulator of the lipin 1 gene, a key regulator of lipid metabolism [17]. Both CREBH and ERRγ gene expression is enhanced in a diabetic mouse model to increase blood glucose level [15], [6]. Regulation of both factors is important to regulate gluconeogenesis in response to ER stress, and diabetes.
    [Show abstract] [Hide abstract] ABSTRACT: The orphan nuclear receptor estrogen-related receptor-γ (ERRγ) is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER) stress response. cAMP responsive element-binding protein H (CREBH) is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERRγ directly regulates CREBH gene expression in response to ER stress. ERRγ bound to the ERRγ response element (ERRE) in the CREBH promoter. Overexpression of ERRγ by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP), whereas either knockdown of ERRγ or inhibition of ERRγ by ERRγ specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1α was required for ERRγ mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP) assay showing binding of both ERRγ and PGC1α on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERRγ and PGC1α binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERRγ directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions.
    Full-text · Article · Jan 2014
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